CN118922194A - Pharmaceutical compositions for delivery of herpes simplex virus antigens and related methods - Google Patents

Abstract

The present disclosure provides pharmaceutical compositions (eg, HSV vaccines) for delivering HSV antigens and related technologies (eg, components thereof and/or methods related thereto).

Description

Pharmaceutical compositions for delivery of herpes simplex virus antigens and related methods

Background

Herpes Simplex Virus (HSV), commonly known only as herpes, is divided into two types: herpes simplex virus type 1 (HSV-1 or oral herpes) and herpes simplex virus type 2 (HSV-2 or genital herpes). According to the world health organization, there are estimated 37 million people under 50 years of age worldwide (67% of the world population) who are infected with HSV-1. HSV-1 prevalence is known to be highest in africa and lowest in america. It is estimated that 4.91 million people 15-49 years old (13% of the global population) worldwide are infected with HSV-2. Women infected with HSV-2 are more efficient than men because the sexually transmitted HSV is more efficient from male to female than from female to male. The prevalence of HSV-2 infection is estimated to be highest in Africa, followed by America. The prevalence of HSV-2 has also been shown to increase with age, although historically the most recently infected people are adolescents. Both HSV-1 and HSV-2 infections are life-long.

Disclosure of Invention

The present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) and related techniques (e.g., methods) for delivering a particular Herpes Simplex Virus (HSV) antigen construct (e.g., an HSV-1 antigen construct, an HSV-2 antigen construct, or a combination thereof) to a subject (e.g., a patient). In particular, the present disclosure provides HSV (e.g., HSV-1, HSV-2, or both) vaccine compositions and related techniques (e.g., methods). The present disclosure includes the unexpected discovery that the HSV antigens and antigenic fragments thereof provided in tables 3-5 below are particularly advantageous for use in the prevention or treatment of HSV, for example in HSV antigen constructs and/or HSV vaccines, as further disclosed herein.

The present disclosure provides, for example, polyribonucleotides that encode one or more HSV antigens (e.g., HSV-1 antigen, HSV-2 antigen, or a combination thereof) or antigenic fragments thereof. In some embodiments, such polyribonucleotides may be part of an RNA construct. In some embodiments, a polyribonucleotide or RNA construct as described herein can be part of a composition (e.g., a pharmaceutical composition, e.g., an immunogenic composition, e.g., a vaccine).

The present disclosure provides polyribonucleotides encoding polypeptides. In some embodiments, the polypeptide comprises one or more Herpes Simplex Virus (HSV) antigens or antigenic fragments thereof.

In some embodiments, the one or more HSV antigens or antigenic fragments thereof comprise: (i) an HSV-1 antigen or an antigenic fragment thereof, (ii) an HSV-2 antigen or an antigenic fragment thereof, or (iii) a combination thereof.

In some embodiments, the polypeptide comprises a single HSV antigen or an antigenic fragment thereof. In some embodiments, the polypeptide comprises a single HSV antigen. In some embodiments, the polypeptide comprises a single HSV antigenic fragment.

In some embodiments, the polypeptide comprises two or more HSV antigens or antigenic fragments thereof. In some embodiments, the polypeptide comprises two or more HSV antigens. In some embodiments, the polypeptide comprises two or more HSV antigenic fragments, wherein each of the two or more HSV antigenic fragments is a fragment of a different HSV antigen. In some embodiments, the polypeptide comprises two or more HSV antigenic fragments, wherein at least two of the HSV antigenic fragments are fragments from the same HSV antigen. In some embodiments, the polypeptide comprises three or more HSV antigens or antigenic fragments thereof. In some embodiments, the polypeptide comprises four or more HSV antigens or antigenic fragments thereof.

In some embodiments, the polypeptide does not comprise a full length HSV antigen.

In some embodiments, the one or more HSV antigens or antigenic fragments thereof comprise one or more T cell antigens or antigenic fragments thereof. In some embodiments, the one or more HSV antigens or antigenic fragments thereof comprise one or more B cell antigens or antigenic fragments thereof.

In some embodiments, one or more HSV antigens or antigenic fragments thereof have at least 80% sequence identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity, to one or more sequences selected from SEQ ID NOs 1-74 or antigenic fragments thereof. In some embodiments, the polypeptide comprises or consists of one or more HSV-2 antigens or antigenic fragments thereof, which HSV-2 antigen or antigenic fragment thereof comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence selected from SEQ ID NOS: 174-196.

In some embodiments, the one or more HSV antigens or antigenic fragments thereof comprise: (i) one or more HSV RS1 polypeptides or antigenic fragments thereof, (ii) one or more HSV RL2 polypeptides or antigenic fragments thereof, (iii) one or more HSV UL1 polypeptides or antigenic fragments thereof, (iv) one or more HSV UL5 polypeptides or antigenic fragments thereof, (v) one or more HSV UL9 polypeptides or antigenic fragments thereof, (vi) one or more HSV UL19 polypeptides or antigenic fragments thereof, (vii) one or more HSV UL21 polypeptides or antigenic fragments thereof, (viii) One or more HSV UL25 polypeptides or antigenic fragments thereof, (ix) one or more HSV UL27 polypeptides or antigenic fragments thereof, (x) one or more HSV UL29 polypeptides or antigenic fragments thereof, (xi) one or more HSV UL30 polypeptides or antigenic fragments thereof, (xii) one or more HSV UL39 polypeptides or antigenic fragments thereof, (xiii) one or more HSV UL40 polypeptides or antigenic fragments thereof, (xiv) one or more HSV UL46 polypeptides or antigenic fragments thereof, (xv) one or more HSV UL47 polypeptides or antigenic fragments thereof, (xvi) One or more HSV UL48 polypeptides or antigenic fragments thereof, (xvii) one or more HSV UL49 polypeptides or antigenic fragments thereof, (xviii) one or more HSV UL52 polypeptides or antigenic fragments thereof, (xix) one or more HSV UL54 polypeptides or antigenic fragments thereof, or (xx) combinations thereof. in some embodiments, the polypeptide comprises one or more HSV antigenic fragments, and the one or more HSV antigenic fragments comprise: (i) one or more HSV RS1 polypeptide antigenic fragments, (ii) one or more HSV RL2 polypeptide antigenic fragments, (iii) one or more HSV UL1 polypeptide antigenic fragments, (iv) one or more HSV UL5 polypeptide antigenic fragments, (v) one or more HSV UL9 polypeptide antigenic fragments, (vi) one or more HSV UL19 polypeptide antigenic fragments, (vii) one or more HSV UL21 polypeptide antigenic fragments, (viii) One or more HSV UL25 polypeptide antigenic fragments, (ix) one or more HSV UL27 polypeptide antigenic fragments, (x) one or more HSV UL29 polypeptide antigenic fragments, (xi) one or more HSV UL30 polypeptide antigenic fragments, (xii) one or more HSV UL39 polypeptide antigenic fragments, (xiii) one or more HSV UL40 polypeptide antigenic fragments, (xiv) one or more HSV UL46 polypeptide antigenic fragments, (xv) one or more HSV UL47 polypeptide antigenic fragments, (xvi) One or more HSV UL48 polypeptide antigenic fragments, (xvii) one or more HSV UL49 polypeptide antigenic fragments, (xviii) one or more HSV UL52 polypeptide antigenic fragments, (xix) one or more HSV UL54 polypeptide antigenic fragments, or (xx) combinations thereof.

In some embodiments, the polypeptides comprise one or more HSV RL2 polypeptides or antigenic fragments thereof, one or more HSV RS1 polypeptides or antigenic fragments thereof, and one or more HSV UL54 polypeptides or antigenic fragments thereof. In some embodiments, the polypeptide comprises an HSV-1gD secretion signal, one or more RL2 polypeptides or antigenic fragments thereof, one or more RS1 polypeptides or antigenic fragments thereof, one or more UL54 polypeptides or antigenic fragments thereof, and MITD.

In some embodiments, the polypeptide comprises, in N-terminal to C-terminal order, a nucleotide sequence encoding an HSV-1gD secretion signal, an RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, and MITD. In some embodiments, the polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO 197.

In some embodiments, the polypeptide comprises, in N-terminal to C-terminal order, a nucleotide sequence encoding an HSV-1gD secretion signal, a UL54 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or antigenic fragment thereof, a linker, and MITD. In some embodiments, the polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence according to SEQ ID NO. 201.

In some embodiments, the polypeptide comprises, in N-terminal to C-terminal order, a nucleotide sequence encoding an HSV-2gD secretion signal, an RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, and MITD. In some embodiments, the polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence according to SEQ ID NO. 205.

In some embodiments, the polypeptide comprises one or more HSV UL29 polypeptides or antigenic fragments thereof, one or more HSV UL39 polypeptides or antigenic fragments thereof, one or more HSV UL49 polypeptides or antigenic fragments thereof, and one or more HSV UL9 polypeptides or antigenic fragments thereof. In some embodiments, the polypeptide comprises an HSV-1gD secretion signal, one or more HSV UL29 polypeptides or antigenic fragments thereof, one or more HSV UL39 polypeptides or antigenic fragments thereof, one or more HSV UL49 polypeptides or antigenic fragments thereof, one or more HSV UL9 polypeptides or antigenic fragments thereof, and MITD.

In some embodiments, the polypeptide comprises, in N-terminal to C-terminal order, a nucleotide sequence encoding an HSV-1gD secretion signal, a UL29 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL49 polypeptide or antigenic fragment thereof, a linker, a UL9 polypeptide or antigenic fragment thereof, a linker, and MITD. In some embodiments, the polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence according to SEQ ID NO. 198.

In some embodiments, the polypeptide comprises, in N-terminal to C-terminal order, a nucleotide sequence encoding an HSV-1gD secretion signal, an UL9 polypeptide or antigenic fragment thereof, a linker, an UL49 polypeptide or antigenic fragment thereof, a linker, an UL39 polypeptide or antigenic fragment thereof, a linker, an UL29 polypeptide or antigenic fragment thereof, a linker, and MITD. In some embodiments, the polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence according to SEQ ID NO 202.

In some embodiments, the polypeptide comprises one or more HSV UL30 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, one or more HSV UL5 polypeptides or antigenic fragments thereof, and one or more HSV UL52 polypeptides or antigenic fragments thereof. In some embodiments, the polypeptide comprises an HSV-1gD secretion signal, one or more HSV UL30 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, one or more HSV UL5 polypeptides or antigenic fragments thereof, one or more HSV UL52 polypeptides or antigenic fragments thereof, and MITD.

In some embodiments, the polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL30 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL52 polypeptide or antigenic fragment thereof, a linker, and MITD. In some embodiments, the polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence according to SEQ ID NO 199.

In some embodiments, the polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL52 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or antigenic fragment thereof, a linker, a UL30 polypeptide or antigenic fragment thereof, a linker, and MITD. In some embodiments, the polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence according to SEQ ID NO. 203.

In some embodiments, the polypeptide comprises one or more HSV UL1 polypeptides or antigenic fragments thereof, one or more HSV UL19 polypeptides or antigenic fragments thereof, one or more HSV UL21 polypeptides or antigenic fragments thereof, one or more HSV UL27 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more UL48 polypeptides or antigenic fragments thereof, and one or more HSV UL25 polypeptides or antigenic fragments thereof. In some embodiments, the polypeptide comprises an HSV-1gD secretion signal, one or more HSV UL1 polypeptides or antigenic fragments thereof, one or more HSV UL19 polypeptides or antigenic fragments thereof, one or more HSV UL21 polypeptides or antigenic fragments thereof, one or more HSV UL27 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more UL48 polypeptides or antigenic fragments thereof, one or more HSV UL25 polypeptides or antigenic fragments thereof, and MITD.

In some embodiments, the polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, an HSV UL1 polypeptide or an antigenic fragment thereof, a linker, an HSV UL19 polypeptide or an antigenic fragment thereof, a linker, an HSV UL21 polypeptide or an antigenic fragment thereof, a linker, an HSV UL27 polypeptide or an antigenic fragment thereof, a linker, an HSV UL46 polypeptide or an antigenic fragment thereof, a linker, an HSV UL47 polypeptide or an antigenic fragment thereof, a linker, an HSV UL25 polypeptide or an antigenic fragment thereof, a linker, an HSV UL48 polypeptide or an antigenic fragment thereof, a linker, and MITD. In some embodiments, the polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence according to SEQ ID NO. 200.

In some embodiments, the polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, an HSV UL48 polypeptide or antigenic fragment thereof, a linker, an HSV UL25 polypeptide or antigenic fragment thereof, a linker, an HSV UL47 polypeptide or antigenic fragment thereof, a linker, an HSV UL46 polypeptide or antigenic fragment thereof, a linker, an HSV UL27 polypeptide or antigenic fragment thereof, a linker, an HSV UL21 polypeptide or antigenic fragment thereof, a linker, an HSV UL19 polypeptide or antigenic fragment thereof, a linker, an HSV UL1 polypeptide or antigenic fragment thereof, a linker, and MITD. In some embodiments, the polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence according to SEQ ID NO. 204.

In some embodiments, the one or more HSV antigens or antigenic fragments thereof comprise one or more HSV glycoproteins. In some embodiments, the one or more HSV glycoproteins comprises HSV glycoprotein B (gB), HSV glycoprotein E (gE), HSV glycoprotein G (gG), HSV glycoprotein H (gH), HSV glycoprotein I (gL), HSV glycoprotein L (gL), or a combination thereof.

In some embodiments, the polypeptide comprises a single HSV antigen. In some embodiments, the single HSV antigen is an HSV glycoprotein. In some embodiments, the HSV glycoprotein is a full length HSV glycoprotein. In some embodiments, the HSV glycoprotein is HSV gB, HSV gE, HSV gG, HSV gH, HSV gI, and HSV gL.

In some embodiments, the HSV glycoprotein is HSV-2gB. In some embodiments, HSV-2gB is or comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 7, 8, 9 or 74. In some embodiments, HSV-2gB consists of or comprises an amino acid sequence according to SEQ ID NO. 7, 8, 9 or 74.

In some embodiments, the HSV glycoprotein is HSV-2gE. In some embodiments, HSV-2gE is or comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 66, 67, 68 or 69. In some embodiments, HSV-2gE consists of or comprises an amino acid sequence according to SEQ ID NO:66, 67, 68 or 69. In some embodiments, a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO 80, 81, 82, 83, or 84.

In some embodiments, the HSV glycoprotein is HSV-2gH. In some embodiments, HSV-2gH is or comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 70, 71, 72 or 74. In some embodiments, HSV-2gH consists of or comprises an amino acid sequence according to SEQ ID NO. 70, 71, 72 or 74.

In some embodiments, the HSV glycoprotein is HSV-2gI. In some embodiments, HSV-2gI is or comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 62, 63, 64 or 65. In some embodiments, HSV-2gI consists of or comprises an amino acid sequence according to SEQ ID NO. 62, 63, 64 or 65. In some embodiments, the sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 75, 76, 77, 78 or 79.

In some embodiments, the HSV glycoprotein is HSV-2gL. In some embodiments, HSV-2gL is or comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 58, 59, 60 or 61. In some embodiments, HSV-2gL consists of or comprises an amino acid sequence according to SEQ ID NO:58, 59, 60 or 61.

In some embodiments, the polypeptide comprises a secretion signal. In some embodiments, the secretion signal comprises or consists of a viral secretion signal. In some embodiments, the viral secretion signal comprises or consists of an HSV secretion signal. In some embodiments, the secretion signal is a heterologous secretion signal. In some embodiments, the HSV secretion signal comprises or consists of an HSV-1 or HSV-2 secretion signal.

In some embodiments, the HSV secretion signal is selected from: a) gD2 secretion signal; b) gD1 secretion signal; c) gB1 secretion signal; d) gI2 secretion signal; e) gE2 secretion signal; f) gC2 secretion signal; g) Eboz secretion signals; h) An IL2 secretion signal; and i) HLA-DR secretion signals.

In some embodiments, the HSV secretion signal comprises or consists of an HSV gD secretion signal. In some embodiments, the HSV gD secretion signal comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO. In some embodiments, the HSV gD secretion signal comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 88. In some embodiments, the HSV gD secretion signal consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 110. In some embodiments, the HSV gD secretion signal consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 111.

In some embodiments, the secretion signal is located at the N-terminus of the polypeptide.

In some embodiments, the HSV secretion signal comprises or consists of an HSV-2 glycoprotein I (gI) secretion signal.

In some embodiments, the HSV-2gI secretion signal comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO. 107.

In some embodiments, the HSV-2gI secretion signal comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 108.

In some embodiments, the polypeptide comprises a transmembrane region. In some embodiments, the transmembrane region comprises or consists of a viral transmembrane region. In some embodiments, the transmembrane region comprises or consists of an HSV transmembrane region. In some embodiments, the HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region. In some embodiments, the HSV transmembrane region comprises or consists of an HSV gD transmembrane region. In some embodiments, the HSV gD transmembrane region consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 160.

In some embodiments, the polypeptide does not comprise a transmembrane region.

In some embodiments, the polypeptide comprises a multimerization domain.

In some embodiments, the polypeptide comprises one or more linkers. The polyribonucleotide of claim 215, wherein one or more linkers comprise one or more glycine (G) residues and/or one or more serine (S) residues. In some embodiments, one or more linkers comprise or consist of an amino acid sequence according to SEQ ID NO. 163. In some embodiments, one or more linkers comprise or consist of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No. 165. In some embodiments, one or more linkers comprise or consist of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No. 168. In some embodiments, one or more linkers comprise or consist of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID No. 217.

In some embodiments, the polyribonucleotide is an isolated polyribonucleotide.

In some embodiments, the polyribonucleotide is an engineered polyribonucleotide.

In some embodiments, the polyribonucleotide is a codon-optimized polyribonucleotide.

The present disclosure also provides RNA constructs.

In some embodiments, the RNA construct comprises in 5 'to 3' order: (i) a 5' UTR; (ii) any polyribonucleotide according to the present disclosure; (iv) a 3' UTR; and (v) a poly A tail sequence. In some embodiments, the RNA construct comprises (i) a 5'UTR comprising or consisting of a modified human α -globulin 5' -UTR; (ii) A 3' utr comprising or consisting of a first sequence from a split amino-terminal enhancer (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA; or (iii) both.

In some embodiments, the 5' UTR comprises or consists of a ribonucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 208. In some embodiments, the 5' UTR comprises or consists of a ribonucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 209. In some embodiments, the 3' UTR comprises or consists of a ribonucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 215. In some embodiments, the 3' UTR comprises or consists of a ribonucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 216. In some embodiments, the poly a tail sequence is a split poly a tail sequence. In some embodiments, the split poly A tail sequence consists of a ribonucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a ribonucleic acid sequence selected from SEQ ID NOs 210, 212 or 213. In some embodiments, the RNA construct further comprises a 5' cap. In some embodiments, the RNA construct comprises a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the polyribonucleotide. In some embodiments, the 5' cap comprises or consists of m7 (3 ' ome G) (5 ') ppp (5 ') (2 ' ome a ₁)pG₂, wherein a ₁ is position +1 of the polyribonucleotide and G ₂ is position +2 of the polyribonucleotide. In some embodiments, the Cap proximal sequence comprises A ₁ and G ₂ of the Cap1 structure and a sequence comprising A ₃A₄U₅ (SEQ ID NO: 207) at positions +3, +4, and +5 of the polyribonucleotide, respectively.

In some embodiments, the polyribonucleotide includes a modified uridine that replaces all uridine, optionally wherein the modified uridine is each N1-methyl-pseudouridine.

The present disclosure also provides compositions.

In some embodiments, the composition comprises one or more polyribonucleotides according to the present disclosure. In some embodiments, the composition comprises one or more RNA constructs of any one of items 224 to 236. In some embodiments, the composition further comprises a lipid nanoparticle, a Polyplex (PLX), a Lipidated Polyplex (LPLX), or a liposome, wherein the one or more polyribonucleotides are wholly or partially encapsulated within the lipid nanoparticle, the Polyplex (PLX), the Lipidated Polyplex (LPLX), or the liposome. In some embodiments, the composition further comprises a lipid nanoparticle, wherein the one or more polyribonucleotides are encapsulated within the lipid nanoparticle.

The present disclosure also provides pharmaceutical compositions.

In some embodiments, the pharmaceutical composition comprises a composition according to the present disclosure and at least one pharmaceutically acceptable excipient. In some embodiments, the medicament comprises a cryoprotectant, optionally wherein the cryoprotectant is sucrose. In some embodiments, the medicament comprises an aqueous buffer solution, optionally wherein the aqueous buffer solution comprises one or more of Tris base, tris HCl, naCl, KCl, na ₂HPO₄, and KH ₂PO₄.

The present disclosure also provides combinations.

In some embodiments, the combination comprises a first polynucleic acid according to the disclosure; and a second polyribonucleotide according to the present disclosure, wherein the first polyribonucleotide and the second polyribonucleotide are different.

In some embodiments, the combination comprises a first pharmaceutical composition comprising a first polynucleotide, wherein the first polynucleotide is a polynucleotide according to the disclosure, and a second pharmaceutical composition; the second pharmaceutical composition comprises a second polynucleotide, wherein the second polynucleotide is a polynucleotide according to the disclosure, wherein the first and second polynucleotide are different.

In some embodiments, the combination comprises a first polynucleic acid according to the disclosure; and a second polyribonucleotide encoding a second polypeptide, wherein the second polypeptide comprises one or more HSV RL2 polypeptides or antigenic fragments thereof, one or more HSV RS1 polypeptides or antigenic fragments thereof, and one or more HSV UL54 polypeptides or antigenic fragments thereof. In some embodiments, the combination comprises: a first pharmaceutical composition comprising a first polyribonucleotide, wherein the first polyribonucleotide is a polyribonucleotide according to the present disclosure; and a second pharmaceutical composition comprising a second polyribonucleotide, wherein the second polyribonucleotide comprises one or more HSV RL2 polypeptides or antigenic fragments thereof, one or more HSV RS1 polypeptides or antigenic fragments thereof, and one or more HSV UL54 polypeptides or antigenic fragments thereof. In some embodiments, the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, an RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, and MITD. In some embodiments, the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 197. In some embodiments, the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, a UL54 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or antigenic fragment thereof, a linker, and MITD. In some embodiments, the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID No. 201. In some embodiments, the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-2gD secretion signal, an RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker, and MITD. In some embodiments, the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID No. 205.

In some embodiments, the combination comprises a first polynucleic acid according to the disclosure; and a second polyribonucleotide encoding a second polypeptide, wherein the second polypeptide comprises one or more HSV UL29 polypeptides or antigenic fragments thereof, one or more HSV UL39 polypeptides or antigenic fragments thereof, one or more HSV UL49 polypeptides or antigenic fragments thereof, and one or more HSV UL9 polypeptides or antigenic fragments thereof. In some embodiments, the combination comprises a first pharmaceutical composition comprising a first polynucleotide, wherein the first polynucleotide is a polynucleotide according to the disclosure, and a second pharmaceutical composition; the second pharmaceutical composition comprises a second polyribonucleotide, wherein the second polypeptide comprises one or more HSV UL29 polypeptides or antigenic fragments thereof, one or more HSV UL39 polypeptides or antigenic fragments thereof, one or more HSV UL49 polypeptides or antigenic fragments thereof, and one or more HSV UL9 polypeptides or antigenic fragments thereof. In some embodiments, the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, a UL29 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL49 polypeptide or antigenic fragment thereof, a linker, a UL9 polypeptide or antigenic fragment thereof, a linker, and MITD. In some embodiments, the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 198. In some embodiments, the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, a UL9 polypeptide or antigenic fragment thereof, a linker, a UL49 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL29 polypeptide or antigenic fragment thereof, a linker, and MITD. In some embodiments, the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID No. 202.

In some embodiments, the combination comprises a first polynucleic acid according to the disclosure; and a second polyribonucleotide encoding a second polypeptide, wherein the second polypeptide comprises one or more HSV UL30 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, one or more HSV UL5 polypeptides or antigenic fragments thereof, and one or more HSV UL52 polypeptides or antigenic fragments thereof. In some embodiments, the combination comprises a first pharmaceutical composition comprising a first polynucleotide, wherein the first polynucleotide is a polynucleotide according to the disclosure, and a second pharmaceutical composition; the second pharmaceutical composition comprises a second polyribonucleotide, wherein the second polypeptide comprises one or more HSV UL30 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, one or more HSV UL5 polypeptides or antigenic fragments thereof, and one or more HSV UL52 polypeptides or antigenic fragments thereof. In some embodiments, the second polypeptide comprises, in order from N-terminus to C-terminus, an HSV-1gD secretion signal, a UL30 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL52 polypeptide or antigenic fragment thereof, a linker, and MITD. In some embodiments, the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO 199. In some embodiments, the second polypeptide comprises, in order from N-terminus to C-terminus, an HSV-1gD secretion signal, a UL52 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or antigenic fragment thereof, a linker, a UL30 polypeptide or antigenic fragment thereof, a linker, and MITD. In some embodiments, the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID No. 203.

In some embodiments, the combination comprises a first polynucleic acid according to the disclosure; And a second polyribonucleotide encoding a second polypeptide, wherein the second polypeptide comprises one or more HSV UL1 polypeptides or antigenic fragments thereof, one or more HSV UL19 polypeptides or antigenic fragments thereof, one or more HSV UL21 polypeptides or antigenic fragments thereof, one or more HSV UL27 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more UL48 polypeptides or antigenic fragments thereof, and one or more HSV UL25 polypeptides or antigenic fragments thereof. in some embodiments, the combination comprises a first pharmaceutical composition comprising a first polynucleotide, wherein the first polynucleotide is a polynucleotide according to the disclosure, and a second pharmaceutical composition; The second pharmaceutical composition comprises a second polyribonucleotide, wherein the second polypeptide comprises one or more HSV UL1 polypeptides or antigenic fragments thereof, one or more HSV UL19 polypeptides or antigenic fragments thereof, one or more HSV UL21 polypeptides or antigenic fragments thereof, one or more HSV UL27 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more UL48 polypeptides or antigenic fragments thereof, and one or more HSV UL25 polypeptides or antigenic fragments thereof. In some embodiments of the present invention, in some embodiments, the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, an HSV UL1 polypeptide or antigenic fragment thereof, a linker, an HSV UL19 polypeptide or antigenic fragment thereof, a linker, an HSV UL21 polypeptide or antigenic fragment thereof, a linker, an HSV UL27 polypeptide or antigenic fragment thereof, a linker, an HSV UL46 polypeptide or antigenic fragment thereof, a linker, an HSV UL47 polypeptide or antigenic fragment thereof, a linker, an HSV UL25 polypeptide or antigenic fragment thereof, a polypeptide comprising a polypeptide of HSV UL27 or antigenic fragment thereof, a polypeptide of HSV UL46, or antigenic fragment thereof, a polypeptide of HSV UL21 or antigenic fragment thereof, a polypeptide of HSV UL27, a polypeptide of HSV UL, A linker, an HSV UL48 polypeptide or an antigenic fragment thereof, a linker, and MITD. In some embodiments, the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID No. 200. In some embodiments of the present invention, in some embodiments, the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, an HSV UL48 polypeptide or antigenic fragment thereof, a linker, an HSV UL25 polypeptide or antigenic fragment thereof, a linker, an HSV UL47 polypeptide or antigenic fragment thereof, a linker an HSV UL46 polypeptide or antigenic fragment thereof, a linker, an HSV UL27 polypeptide or antigenic fragment thereof, a linker, an HSV UL21 polypeptide or antigenic fragment thereof, a linker, an HSV UL19 polypeptide or antigenic fragment thereof, a linker, an HSV UL1 polypeptide or an antigenic fragment thereof, a linker, and MITD. In some embodiments, the second polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID No. 204. In some embodiments, the second polypeptide is HSV gB. In some embodiments, the second polypeptide consists of or comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID No. 7, 8, 9 or 74.

The present disclosure also provides a method comprising administering to a subject a polyribonucleotide according to the present disclosure or an RNA construct according to the present disclosure.

The present disclosure also provides a method comprising administering to a subject a composition according to the present disclosure.

The present disclosure also provides a method comprising administering one or more doses of a composition according to the present disclosure or a pharmaceutical composition according to the present disclosure to a subject.

The present disclosure also provides a method comprising administering to a subject a combination according to the present disclosure.

The present disclosure also provides pharmaceutical compositions according to the present disclosure for use in treating HSV infection comprising administering one or more doses of the pharmaceutical composition to a subject.

The present disclosure also provides pharmaceutical compositions according to the present disclosure for use in preventing HSV infection comprising administering one or more doses of the pharmaceutical composition to a subject.

In some embodiments, the use of a method according to the present disclosure or a pharmaceutical composition according to the present disclosure comprises administering two or more doses of the pharmaceutical composition to a subject.

In some embodiments, the use of a method according to the present disclosure or a pharmaceutical composition according to the present disclosure comprises administering three or more doses of the pharmaceutical composition to a subject.

The present disclosure also provides a method comprising administering to a subject a combination according to the present disclosure. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered on the same day. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered on different dates. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered to the subject at different locations on the subject's body. In some embodiments, the method is a method of treating HSV infection. In some embodiments, the method is a method of preventing HSV infection. In some embodiments, the subject has or is at risk of having an HSV infection.

In some embodiments, the subject is a human.

In some embodiments, administration induces an anti-HSV immune response in a subject. In some embodiments, the anti-HSV immune response in the subject comprises an adaptive immune response. In some embodiments, the anti-HSV immune response in the subject comprises a T cell response. In some embodiments, the T cell response is or includes a cd4+ T cell response. In some embodiments, the T cell response is or includes a cd8+ T cell response. In some embodiments, the anti-HSV immune system response comprises a B cell response. In some embodiments, the anti-HSV immune system response comprises producing antibodies against one or more HSV antigens or antigenic fragments thereof that are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to one or more sequences selected from SEQ ID NOS: 1-74 or antigenic fragments thereof.

The present disclosure also provides the use of a pharmaceutical composition according to the present disclosure in the treatment of herpes simplex virus infection.

The present disclosure also provides the use of a pharmaceutical composition according to the present disclosure in the prevention of herpes simplex virus infection.

The present disclosure also provides the use of a pharmaceutical composition according to the invention for inducing an anti-herpes simplex immune virus response in a subject.

The present disclosure also provides polypeptides encoded by the polyribonucleotides according to the present disclosure.

The disclosure also provides a polypeptide encoded by the RNA construct of any one of claims 224 to 236.

The present disclosure also provides host cells comprising polyribonucleotides according to the present disclosure.

The present disclosure also provides host cells comprising RNA constructs according to the present disclosure.

The present disclosure also provides host cells comprising a polypeptide according to the present disclosure.

Drawings

Fig. 1 is a schematic view of HSV particles.

Fig. 2 is a schematic overview of an HSV lifecycle. Fig. 2 has been modified according to Ibanez, f.j. Et al ,"Experimental Dissection of the Lytic Replication Cycles of Herpes Simplex Virus in vitro,"Front Microbiol.2018;9:2406, which is incorporated herein by reference in its entirety.

Figure 3 is a schematic representation of a model of HSV latent infection. Fig. 3 has been modified according to Knipe, d.m. et al, "Clues to mechanisms of HERPESVIRAL LATENT infection and potential cures," PNAS2015, 9, 29, 112 (39) 11993-11994, which is incorporated herein by reference in its entirety.

Figure 4 is a summary of the results of clinical trials of HSV candidate vaccines. The table has been modified according to Aschner, c.b. and herld, b.c. (2021), alphaherpesvirus vaccines.current Issues in Molecular Biology,41, 469-508, which is incorporated herein by reference in its entirety.

Figure 5 is a summary of HSV-2 candidate vaccines in preclinical development. The table has been modified according to Aschner, c.b. and herld, b.c. (2021), alphaherpesvirus vaccines.current Issues in Molecular Biology,41, 469-508, which is incorporated herein by reference in its entirety.

FIG. 6 is a heat map evaluating phylogenetic and homology of HSV-1 and HSV-2 genes. As shown, the HSV-1 and HSV-2 genes are homologous, having about 75% sequence identity. HSV-2 exhibits minimal cross-strain variability.

Fig. 7 includes line graphs depicting the time when intermediate early, early and late genes are expressed following HSV infection.

Fig. 8 is a table showing certain features of data analyzed by Hosken 2006, sting 2012, and Long 2014, including HSV category, number of subjects, number of genes assayed, experimental methods, and symptomatic status of the subjects.

Fig. 9 is a graph showing the percentage of subjects determined from the data analyzed by Hosken 2006, each of which had T cells targeting the products of each of the 48 analyzed HSV genes at levels above the indicated threshold (greater than 20SFU/10 ⁶). Data is taken from pictures of Hosken 2006.

Fig. 10 is a graph showing the percentage of subjects with T cells and/or CD 4T cells targeting the products of each of the 75 analyzed HSV genes, respectively, as determined in the data analyzed by Long 2014. Data is taken from a picture of Long 2014.

Fig. 11 is a set of three graphs showing the correlation of T cells detected as targeting each of a series of individual HSV genes between pairs of datasets analyzed by the literature, particularly Hosken between 2006 and sting 2012, hosken between 2006 and Long 2014, or sting 2012 and Long 2014. The R value for each graph is shown. Hosken 2006/Jing 2012 was observed to be relevant (Jing 2012HSV-1, hosken 2006 HSV-2), although of different kinds. No correlation was observed between the data of Long 2014 and the data of Hosken 2006 or of jin 2012.

Fig. 12 is a graph showing the expression levels of each of a series of HSV genes determined by analysis of multiple data sets from different sources, including human cells, minced meat, and DRG from latent infected tree shrew. The horizontal dashed line shows the determined median expression.

Fig. 13 is a graph plotting data from Hosken 2006 on the% of T cells targeting HSV gene products and the median expression of each of the various HSV genes (see fig. 12). The threshold indicates a gene that is immunogenic and well expressed (the upper right quadrant based on the dashed line indicates the threshold).

Fig. 14 is a graph plotting data from jin 2012 for% of T cells targeting HSV gene products and median expression of each of the various HSV genes (see fig. 12). The threshold indicates a gene that is immunogenic and well expressed (the upper right quadrant based on the dashed line indicates the threshold).

FIG. 15 depicts conservation scores determined for amino acids located at positions along the RL2 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

FIG. 16 depicts conservation scores determined for amino acids located at positions along the RS1 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Fig. 17 depicts conservation scores determined for amino acids located at positions along the UL19 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Fig. 18 depicts conservation scores determined for amino acids located at positions along the UL1 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Fig. 19 depicts conservation scores determined for amino acids located at positions along the UL21 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Fig. 20 depicts conservation scores determined for amino acids located at positions along the UL25 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Figure 21 depicts conservation scores determined for amino acids located at positions along the UL27 consensus sequence. UL27 encodes HSV gB. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Fig. 22 depicts conservation scores determined for amino acids located at positions along the UL29 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Fig. 23 depicts conservation scores determined for amino acids located at positions along the UL30 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Figure 24 depicts conservation scores determined for amino acids located at positions along the UL39 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Fig. 25 depicts conservation scores determined for amino acids located at positions along the UL40 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Fig. 26 depicts conservation scores determined for amino acids located at positions along the UL46 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Fig. 27 depicts conservation scores determined for amino acids located at positions along the UL47 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Fig. 28 depicts conservation scores determined for amino acids located at positions along the UL48 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Fig. 29 depicts conservation scores determined for amino acids located at positions along the UL49 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Fig. 30 depicts conservation scores determined for amino acids located at positions along the UL52 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Fig. 31 depicts conservation scores determined for amino acids located at positions along the UL54 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Fig. 32 depicts conservation scores determined for amino acids located at positions along the UL5 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Fig. 33 depicts conservation scores determined for amino acids located at positions along the UL9 consensus sequence. To perform this analysis, the complete HSV-1 and HSV-2 genomes were downloaded from the VIPR database, and HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HSV-2, respectively.

Figure 34 depicts the conservation scores for HSV strains determined for amino acids located at positions along the RL2 consensus sequence.

Figure 35 depicts conservation scores for HSV strains determined for amino acids located at positions along the RS1 consensus sequence.

Figure 36 depicts conservation scores for HSV strains determined for amino acids located at positions along the UL19 consensus sequence.

Figure 37 depicts conservation scores for HSV strains determined for amino acids located at positions along the UL1 consensus sequence.

Figure 38 depicts the conservation scores for HSV strains determined for amino acids located at positions along the UL21 consensus sequence.

Figure 39 depicts a conservation score for HSV strains determined for amino acids located at positions along the UL25 consensus sequence.

Figure 40 depicts conservation scores for HSV strains determined for amino acids located at positions along the UL27 consensus sequence.

Figure 41 depicts the conservation scores for HSV strains determined for amino acids located at positions along the UL29 consensus sequence.

Fig. 42 depicts conservation scores for HSV strains determined for amino acids located at positions along the UL30 consensus sequence.

Figure 43 depicts conservation scores for HSV strains determined for amino acids located at positions along the UL39 consensus sequence.

Figure 44 depicts conservation scores for HSV strains determined for amino acids located at positions along the UL40 consensus sequence.

Figure 45 depicts a conservation score for HSV strains determined for amino acids located at positions along the UL46 consensus sequence.

Figure 46 depicts conservation scores for HSV strains determined for amino acids located at positions along the UL47 consensus sequence.

Figure 47 depicts a conservation score for HSV strains determined for amino acids located at positions along the UL48 consensus sequence.

Figure 48 depicts a conservation score for HSV strains determined for amino acids located at positions along the UL49 consensus sequence.

Figure 49 depicts a conservation score for HSV strains determined for amino acids located at positions along the UL52 consensus sequence.

Figure 50 depicts conservation scores for HSV strains determined for amino acids located at positions along the UL54 consensus sequence.

Figure 51 depicts the conservation scores for HSV strains determined for amino acids located at positions along the UL5 consensus sequence.

Figure 52 depicts a conservation score for HSV strains determined for amino acids located at positions along the UL9 consensus sequence.

Figure 53 depicts four HSV antigen constructs A, B, C and D. Construct a included RL2, RS1 and UL 54T cell antigens. Construct B included UL29, UL39, UL49 and UL 9T cell antigens. Construct C includes UL30, UL40, UL5 and UL52T cell antigens. Construct D included UL1, UL19, UL21, UL27, UL46, UL47, UL25 and UL 48T cell antigens.

Certain definitions

Generally, terms used herein are consistent with their meaning as understood in the art, unless explicitly indicated otherwise. The following provides a clear definition of certain terms; the meaning of these and other terms in the specific context throughout this specification will be apparent to those skilled in the art from the context.

In order that the invention may be more readily understood, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.

About: the term "about" as used herein to refer to a value refers to a value in a context similar to the value referred to. In general, those skilled in the art who are familiar with the context will recognize the relative degree of variation that is covered by "about" in this context. For example, in some embodiments, the term "about" may encompass a range of values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the referenced value.

The preparation method comprises the following steps: as used herein, the term "agent" may refer to a physical entity or phenomenon. In some embodiments, the agent may be characterized by a particular characteristic and/or effect. In some embodiments, the agent may be any chemical class of compound, molecule, or entity, including, for example, a small molecule, polypeptide, nucleic acid, sugar, lipid, metal, or a combination or complex thereof. In some embodiments, the term "agent" may refer to a compound, molecule, or entity comprising a polymer. In some embodiments, the term may refer to a compound or entity comprising one or more polymeric moieties. In some embodiments, the term "agent" may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymeric moiety. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymeric or polymeric moiety.

Amino acid: in the broadest sense, the term "amino acid" as used herein refers to a compound and/or substance that may be, is, or has been incorporated into a polypeptide chain, for example by forming one or more peptide bonds. In some embodiments, the amino acid has the general structure H ₂ N-C (H) (R) -COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a non-natural amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is an L-amino acid. "Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. "non-standard amino acid" refers to any amino acid other than a standard amino acid, whether synthetically prepared or obtained from natural sources. In some embodiments, the amino acids (including carboxyl and/or amino terminal amino acids) in the polypeptide may contain structural modifications as compared to the general structures described above. For example, in some embodiments, amino acids may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., substitution of amino groups, carboxylic acid groups, one or more protons, and/or hydroxyl groups) as compared to the general structure. In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide containing a modified amino acid as compared to a polypeptide containing an otherwise identical unmodified amino acid. In some embodiments, such modifications do not significantly alter the activity associated with a polypeptide containing a modified amino acid compared to a polypeptide containing an otherwise identical unmodified amino acid. As will be clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments, it may be used to refer to an amino acid residue of a polypeptide.

Antibody preparation: as used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses polypeptides or polypeptide complexes comprising immunoglobulin structural elements sufficient to confer specific binding. For example, in some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as Complementarity Determining Regions (CDRs); in some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to a CDR found in a reference antibody. In some embodiments, the CDRs included are substantially identical to the reference CDRs because they are either identical in sequence or contain between 1-5 amino acid substitutions as compared to the reference CDRs. In some embodiments, the CDRs included are substantially identical to the reference CDRs in that they exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference CDRs. In some embodiments, the CDRs included are substantially identical to the reference CDRs in that they exhibit at least 96%, 97%, 98%, 99% or 100% sequence identity to the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid is deleted, added, or substituted within the included CDRs as compared to the reference CDRs, but the included CDRs have an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs because 1-5 amino acids are deleted, added, or substituted within the included CDRs as compared to the reference CDRs, but the included CDRs have an amino acid sequence that is otherwise identical to the reference CDRs. In some embodiments, the included CDR is substantially identical to the reference CDR in that at least one amino acid is substituted within the included CDR as compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs because 1-5 amino acids are deleted, added, or substituted within the included CDRs as compared to the reference CDRs, but the included CDRs have an amino acid sequence that is otherwise identical to the reference CDRs. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as immunoglobulin variable domains. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence comprises structural elements recognized by one of skill in the art as corresponding to CDRs 1,2 and 3 of the antibody variable domain; In some such embodiments, the antibody agent is or comprises a polypeptide or group of polypeptides whose amino acid sequences together comprise structural elements recognized by those of skill in the art as corresponding to heavy and light chain variable region CDRs (e.g., heavy chain CDRs 1,2 and/or 3 and light chain CDRs 1,2 and/or 3). In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain. In some embodiments, the antibody agent may be or comprise a polyclonal antibody preparation. In some embodiments, the antibody agent may be or comprise a monoclonal antibody preparation. In some embodiments, an antibody agent may include one or more constant region sequences specific for a particular organism, such as camel, human, mouse, primate, rabbit, rat; in many embodiments, an antibody agent may include one or more constant region sequences that are characteristic of humans. In some embodiments, an antibody agent may include one or more sequence elements that one of skill in the art would recognize as a humanized sequence, a primatized sequence, a chimeric sequence, or the like. In some embodiments, the antibody agent may be a canonical antibody (e.g., may comprise two heavy chains and two light chains). In some embodiments, the antibody agent may be in a form selected from, but not limited to, the following: intact IgA, igG, igE or IgM antibodies; Bispecific or multispecific antibodies (e.g.,Etc.); antibody fragments, such as Fab fragments, fab ' fragments, F (ab ') 2 fragments, fd ' fragments, fd fragments, and isolated CDRs, or a collection thereof; a single chain Fv; a polypeptide-Fc fusion; single domain antibodies (e.g., shark single domain antibodies, such as IgNAR or fragments thereof); camelid antibodies; the masking antibody (e.g.,) ; Small modular immunopharmaceuticals ("SMIPs ^TM"); single-chain or tandem diabodiesVHH；A minibody; Ankyrin repeat protein or DARTs; TCR-like antibodies;MicroProteins； And In some embodiments, an antibody may lack a covalent modification (e.g., a linkage of glycans) that it would have in the case of naturally occurring. In some embodiments, the antibodies can contain a covalent modification (e.g., attachment of a glycan, payload (e.g., detectable moiety, therapeutic moiety, catalytic moiety, etc.) or other pendent group (e.g., polyethylene glycol, etc.).

Antigen: those skilled in the art who review this specification will appreciate that the term "antigen" refers to a molecule that is recognized by the immune system (e.g., in particular embodiments, the adaptive immune system) such that it elicits an antigen-specific immune response. In some embodiments, the antigen-specific immune response may be or include the production of antibodies and/or antigen-specific T cells. In some embodiments, the antigen is a peptide or polypeptide comprising at least one epitope against which an immune response can be generated. In one embodiment, the antigen is presented by cells of the immune system, such as antigen presenting cells, such as dendritic cells or macrophages. In one embodiment, the antigen or a processed product thereof, such as a T cell antigen, is bound by a T or B cell receptor or by an immunoglobulin molecule, such as an antibody. Thus, the antigen or processed product thereof may specifically react with an antibody or a T lymphocyte (T cell). In one embodiment, the antigen is a parasite antigen. According to the present disclosure, in some embodiments, the antigen may be delivered by an RNA molecule described herein. In some embodiments, the peptide or polypeptide antigen may be 2-100 amino acids in length, including, for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids. In some embodiments, the peptide or polypeptide antigen may be more than 50 amino acids. In some embodiments, the peptide or polypeptide antigen may be more than 100 amino acids. In some embodiments, the antigen is recognized by immune effector cells. In some embodiments, an antigen, if recognized by an immune effector cell, is capable of inducing stimulation, priming, and/or expansion of an immune effector cell carrying an antigen receptor that recognizes the antigen in the presence of an appropriate co-stimulatory signal. In the context of embodiments of the present disclosure, in some embodiments, the antigen may be presented or present on the surface of a cell (e.g., an antigen presenting cell). In one embodiment, the antigen is presented by a diseased cell, such as a virus-infected cell. In one embodiment, the antigen receptor is a TCR that binds to an epitope of an antigen presented in the context of MHC. In one embodiment, when the TCR is expressed by and/or presented on a T cell, its binding to an antigen presented by the cell (e.g., an antigen presenting cell) results in stimulation, priming, and/or expansion of the T cell. In one embodiment, when the TCR is expressed by and/or presented on T cells, its binding to antigen presented on the diseased cells results in cytolysis and/or apoptosis of the diseased cells, wherein the T cells preferably release cytotoxic factors such as perforin and granzyme.

And (3) association: as the term is used herein, two events or entities are "associated with" each other if the presence, level, degree, type, and/or form of one event or entity is correlated with the presence, level, degree, type, and/or form of another event or entity. For example, a particular entity (e.g., polypeptide, genetic marker, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder or condition if its presence, level, and/or form is associated with the disease, disorder or condition's incidence, susceptibility, severity, stage, etc. (e.g., among the relevant populations). In some embodiments, two or more entities are physically "associated" with each other if they interact directly or indirectly such that they are and/or remain physically proximate to each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other, but are non-covalently associated, for example, by means of hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic forces, and combinations thereof.

Combining: those skilled in the art who review this description will appreciate that the term "binding" generally refers to non-covalent association between entities or moieties or with each other. In some embodiments, the binding data is represented as an "IC 50". As understood in the art, IC50 is the concentration of an agent evaluated in a binding assay at which 50% inhibition of binding of a reference agent known to bind to the relevant binding partner is observed. In some embodiments, the assay is performed under conditions in which the assay is performed (e.g., limiting binding of the target and reference concentration), these values are near the K _D value. Assays for determining binding are well known in the art, e.g., as described in detail in PCT publications WO 94/20127 and WO 94/03205, and other publications, such as Sidney et al, current Protocols in Immunology 18.3.1 (1998); sidney et al, J.Immunol.154:247 (1995); and Sette et al mol. Immunol.31:813 (1994). Or binding may be expressed relative to the binding of the referenced standard peptide. For example, IC ₅₀ may be based on its IC ₅₀ relative to a reference standard peptide. Other assay systems may also be used to determine binding, including assay systems using: living cells (e.g., CEPPELLINI et al, nature 339:392 (1989); CHRISTNICK et al, nature 352:67 (1991); busch et al, int. Immunol.2:443 (1990); hill et al, J.Immunol.147:189 (1991); del Guercio et al, J.Immunol.154:685 (1995)), cell-free systems using detergent lysates (e.g., cerundolo et al, J.Immunol 21:2069 (1991)), immobilized purified MHC (e.g., hill et al, J.Immunol.152,2890 (1994); marshall et al, J.Immunol.152:4946 (1994)), ELISA systems (e.g., reay et al, EMBO J.11:2829 (1992)), surface plasmon resonance (e.g., khilko et al, J.biol. Chem.268:15425 (1993)); high throughput soluble phase assays (Hammer et al, J.Exp. Med.180:2353 (1994)), and measurement of class I MHC stabilization or assembly (e.g., ljunggren et al, nature 346:476 (1990); Schumacher et al, cell 62:563 (1990); townsend et al, cell 62:285 (1990); parker et al, J.Immunol.149:1896 (1992)).

Cap: as used herein, the term "cap" refers to a structure comprising or consisting essentially of nucleoside-5 ' -triphosphates, which are typically attached to the 5' end of uncapped RNA (e.g., uncapped RNA with 5' -diphosphate). In some embodiments, the cap is or comprises a guanine nucleotide. In some embodiments, the cap is or comprises a naturally occurring RNA 5' cap, including for example, but not limited to, a 7-methylguanosine cap having a structure designated "m 7G". In some embodiments, the cap is or comprises a synthetic cap analogue that is similar to an RNA cap structure and has the ability to stabilize RNA (if linked thereto), including for example, but not limited to, anti-reverse cap analogues (ARCA) known in the art. Those skilled in the art will appreciate that methods of ligating caps to the 5' end of RNA are known in the art. For example, in some embodiments, the capped RNA can be obtained by in vitro capping of RNA having a 5 'triphosphate group or RNA having a 5' diphosphate group with a capping enzyme system, including, for example, but not limited to, a vaccinia capping enzyme system or a saccharomyces cerevisiae capping enzyme system. Alternatively, the capped RNA may be obtained by In Vitro Transcription (IVT) of a single stranded DNA template in the presence of a dinucleotide or trinucleotide cap analogue.

Cell-mediated immunity: "cell-mediated immunity", "cellular immune response" or similar terms are intended to include a cellular response to a cell characterized by expression of an antigen, in particular by presentation of an antigen with class I or class II MHC. Cellular responses involve immune effector cells, in particular T cells or T lymphocytes that act as "helper" or "killer". Helper T cells (also known as CD4 ⁺ T cells or CD 4T cells) play a central role by modulating the immune response, and killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8 ⁺ T cells, CD 8T cells or CTLs) kill diseased cells, such as virally infected cells, thereby preventing the production of more diseased cells.

Co-administration: as used herein, the term "co-administration" refers to the use of a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) and an additional therapeutic agent as described herein. The combined use of a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) and an additional therapeutic agent described herein can be performed simultaneously or separately (e.g., sequentially in any order). In some embodiments, the pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) and additional therapeutic agents described herein can be combined in one pharmaceutically acceptable carrier, or they can be placed in separate carriers and delivered to the target cells or administered to the subject at different times. Each of these cases is considered to be within the meaning of "co-administration" or "combination" as long as the pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) and additional therapeutic agent described herein are delivered or administered sufficiently close in time that each has at least some temporal overlap in biological effects on the target cells or subject being treated.

Codon optimized: as used herein, the term "codon optimized" refers to altering codons in the coding region of a nucleic acid molecule to reflect typical codon usage of the host organism, and not preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, in some embodiments, the coding region is codon optimized for optimal expression in a subject to be treated with an RNA molecule described herein. In some embodiments, codon optimization can be performed such that codons available to insert frequently occurring tRNAs replace "rare codons". In some embodiments, codon optimization may include increasing the guanosine/cytosine (G/C) content of the coding region of an RNA described herein as compared to the corresponding coding sequence of a wild-type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified as compared to the amino acid sequence.

Combination therapy: as used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of the first regimen are administered, followed by any doses of the second regimen); in some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, "administering" of a combination therapy may involve administering one or more agents or modes in combination to a subject receiving the other agents or modes. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even necessarily simultaneously), but in some embodiments, two or more agents or active portions thereof may be administered together in combination in a composition.

Comparable: as used herein, the term "comparable" refers to a collection of two or more agents, entities, conditions, etc., that may be different from each other, but sufficiently similar to allow comparison therebetween, such that one skilled in the art will appreciate that a conclusion may be reasonably drawn based on the observed differences or similarities. In some embodiments, a set, condition, individual, or population of comparable conditions is characterized by a plurality of substantially identical features and one or a small number of different features. Those of ordinary skill in the art will understand how the degree of consistency required for a collection of two or more such agents, entities, situations, conditions, etc., in any given instance, is to be considered comparable. For example, one of ordinary skill in the art will understand that when a set, individual, or population of conditions is characterized by a sufficient number and type of substantially identical features, they are comparable to one another to ensure that the conclusion is reasonable, i.e., the results obtained under or observed with the different set, individual, or population of conditions is caused by or indicative of a change in those features that are changed.

Corresponding to: as used herein, the term "corresponding to" refers to a relationship between two or more entities. For example, the term "corresponding to" may be used to denote the location/identity of a structural element in a compound or composition relative to another compound or composition (e.g., relative to an appropriate reference compound or composition). For example, in some embodiments, a monomer residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as "corresponding to" a residue in an appropriate reference polymer. For example, one of ordinary skill in the art will appreciate that for simplicity, residues in a polypeptide are often denominated using a canonical numbering system based on the reference to the relevant polypeptide, so that, for example, an amino acid "corresponding to" a residue at position 190 need not actually be the 190 th amino acid in a particular amino acid chain, but rather corresponds to a residue found at 190 in the reference polypeptide; one of ordinary skill in the art will readily understand how to identify "corresponding" amino acids. For example, one of skill in the art will be aware of various sequence alignment strategies, including software programs, such as BLAST、CS-BLAST、CUSASW++、DIAMOND、FASTA、GGSEARCH/GLSEARCH、Genoogle、HMMER、HHpred/HHsearch、IDF、Infernal、KLAST、USEARCH、parasail、PSI-BLAST、PSI-Search、ScalaBLAST、Sequilab、SAM、SSEARCH、SWAPHI、SWAPHI-LS、SWIMM or SWIPE, which can be used, for example, to identify "corresponding" residues in polypeptides and/or nucleic acids according to the present disclosure. Those skilled in the art will also appreciate that in some cases the term "corresponding to" may be used to describe an event or entity that shares a related similarity with another event or entity (e.g., an appropriate reference event or entity). By way of example only, a gene or protein in one organism may be described as "corresponding to" a gene or protein from another organism to indicate that it performs a similar function or performs a similar function and/or that it exhibits a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.

The sources are as follows: in the context of an amino acid sequence (peptide or polypeptide) that is "derived from" the specified amino acid sequence (peptide or polypeptide), it refers to a structural analog of the specified amino acid sequence. In some embodiments, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, substantially identical, or homologous to the particular sequence or fragment thereof. The amino acid sequence derived from a particular amino acid sequence may be a variant of that particular sequence or fragment thereof. For example, one of ordinary skill in the art will appreciate that the antigens useful herein may be altered such that they differ in sequence from the naturally occurring or native sequence from which they are derived, while retaining the desired activity of the native sequence.

The design is as follows: as used herein, the term "engineered" refers to an agent that (i) has its structure or has been selected manually; (ii) it is produced by a method requiring manual work; and/or (iii) it is different from natural substances and other known agents.

Dosing regimen: it will be appreciated by those skilled in the art that the term "dosing regimen" may be used to refer to a set of unit doses (typically more than one), which are typically administered individually to a subject at intervals of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, the dosing regimen comprises a plurality of doses, each dose being spaced apart in time from the other doses. In some embodiments, the doses are spaced from each other by a period of the same length; in some embodiments, the dosing regimen comprises a plurality of doses, and each dose is separated by at least two different time periods. In some embodiments, all doses within a dosing regimen are in the same unit dose amount. In some embodiments, different doses within a dosing regimen have different amounts. In some embodiments, the dosing regimen comprises a first dose in an amount of the first dose followed by one or more additional doses in an amount of the second dose that is different from the amount of the first dose. In some embodiments, the dosing regimen comprises a first dose in an amount of the first dose followed by one or more additional doses in an amount of the second dose that is the same as the amount of the first dose. In some embodiments, the dosing regimen is associated with a desired or beneficial outcome when administered among the relevant populations (i.e., is a therapeutic dosing regimen).

Encoding: as used herein, the term "encoding" refers to sequence information of a first molecule that directs the production of a second molecule having a defined nucleotide sequence (e.g., mRNA) or a defined amino acid sequence. For example, a DNA molecule may encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase). RNA molecules can encode polypeptides (e.g., by a translation process). Thus, if transcription and translation of an mRNA corresponding to a gene produces a polypeptide in a cell or other biological system, the gene, cDNA, or RNA molecule (e.g., mRNA) encodes the polypeptide. In some embodiments, the coding region of an RNA molecule encoding a target antigen refers to the coding strand, the nucleotide sequence of which is identical to the mRNA sequence of such target antigen. In some embodiments, the coding region of an RNA molecule encoding a target antigen refers to a non-coding strand of such target antigen, which can serve as a template for transcription of a gene or cDNA.

Engineering: in general, the term "engineered" refers to an aspect that has been manually manipulated. For example, a polynucleotide is considered "engineered" when two or more sequences that are not linked together in the order in nature are directly linked to each other in an engineered polynucleotide by manual manipulation and/or when particular residues in the polynucleotide are non-naturally occurring and/or are caused to be linked to entities or portions to which they are not naturally linked by manual manipulation.

Epitope: as used herein, the term "epitope" refers to a moiety specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. For example, an epitope may be recognized by a T cell, B cell, or antibody. In some embodiments, an epitope consists of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface exposed when the antigen adopts the relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are in spatial physical proximity to each other when the antigen adopts such a conformation. In some embodiments, at least some of such chemical atoms and groups are physically separated from each other when the antigen adopts an alternative conformation (e.g., is linearized). Thus, in some embodiments, an epitope of an antigen may comprise a continuous or discontinuous fragment of the antigen. In some embodiments, the epitope is or comprises a T cell epitope. In some embodiments, an epitope may have a length of about 5 to about 30 amino acids or about 10 to about 25 amino acids or about 5 to about 15 amino acids or about 5 to 12 amino acids or about 6 to about 9 amino acids.

Expression: as used herein, the term "expression" of a nucleic acid sequence refers to the production of a gene product from the nucleic acid sequence. In some embodiments, the gene product may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, expression of the nucleic acid sequence involves one or more of the following: (1) Generating an RNA template from the DNA sequence (e.g., by transcription); (2) Processing of the RNA transcript (e.g., by splicing, editing, etc.); (3) translating the RNA into a polypeptide or protein; and/or (4) post-translational modification of the polypeptide or protein.

Five-terminal untranslated region: as used herein, the term "five-terminal untranslated region" or "5' utr" refers to the sequence of an mRNA molecule between the transcription initiation site and the initiation codon of the coding region of RNA. In some embodiments, "5' utr" refers to the sequence of an mRNA molecule, for example, in its natural context, that starts at a transcription initiation site and ends one nucleotide (nt) before the initiation codon (typically AUG) of the coding region of the RNA molecule.

Fragments: the term "fragment" as used herein in the context of a nucleic acid sequence (e.g., an RNA sequence) or an amino acid sequence may generally be a fragment of a reference sequence. In some embodiments, the reference sequence is a full length sequence, e.g., a nucleic acid sequence or an amino acid sequence. Thus, a fragment generally refers to a sequence that is identical to a corresponding segment within a reference sequence. In some embodiments, a fragment comprises a contiguous stretch of nucleotide or amino acid residues that corresponds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the total length of the reference sequence from which the fragment is derived. In some embodiments, the term "fragment" with respect to an amino acid sequence (peptide or polypeptide) refers to a portion of the amino acid sequence, e.g., a sequence representing an amino acid sequence that is shortened at the N-terminus and/or C-terminus. In some embodiments, a fragment of an amino acid sequence comprises at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence.

Homology: as used herein, the term "homology" or "homolog" refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered "homologous" to each other if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., contain chemically related residues at the corresponding positions). For example, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids that are similar to one another, and/or have "polar" or "nonpolar" side chains, as is well known to those of ordinary skill in the art. Substitution of one amino acid with another amino acid of the same type is generally considered a "homologous" substitution.

Humoral immunity: as used herein, the term "humoral immunity" or "humoral immune response" refers to antibody production and accompanying processes, including: th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell production. It also refers to effector functions of antibodies, including pathogen neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.

Identity: as used herein, the term "identity" refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered "substantially identical" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. The calculation of the percent identity of two nucleic acid or polypeptide sequences may be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences may be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of the reference sequence. The nucleotides at the corresponding positions are then compared. When a position in a first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in a second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. A mathematical algorithm may be employed to accomplish a comparison of sequences and a determination of percent identity between two sequences. For example, the percentage identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller,1989, which has incorporated the ALIGN program (version 2.0). In some exemplary embodiments, the nucleic acid sequence comparison performed with the ALIGN program uses PAM120 weight residue table, gap length penalty 12, and gap penalty 4. Alternatively, the percentage identity between two nucleotide sequences can be determined using the nwsgapdna.cmp matrix using the GAP program in the GCG software package.

Immunological equivalence: the term "immunologically equivalent" means that an immunologically equivalent molecule, such as an immunologically equivalent amino acid sequence, exhibits the same or substantially the same immunological properties and/or exerts the same or substantially the same immunological effect, e.g., in terms of the type of immunological effect. In the context of the present disclosure, in some embodiments, the term "immunological equivalent" is used in relation to the immunological effect or properties of an antigen or antigen variant for immunization. For example, an amino acid sequence is immunologically equivalent if it induces an immune response with specificity that reacts with a reference amino acid sequence when the amino acid sequence is exposed to the immune system of a subject.

In one embodiment, the antigen receptor is an antibody or B cell receptor that binds to an epitope of an antigen. In one embodiment, the antibody or B cell receptor binds to a native epitope of the antigen.

Increased, induced or decreased: as used herein, these terms or grammatically comparable comparison terms refer to values measured relative to a comparable reference. For example, in some embodiments, the evaluation value obtained with a provided pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) may be "increased" relative to the evaluation value obtained with a comparable reference pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine). Alternatively or additionally, in some embodiments, the evaluation value obtained in a subject may be "increased" relative to an evaluation value obtained in the same subject under different conditions (e.g., before or after an event; or in the presence or absence of an event, such as administration of a pharmaceutical composition as described herein (e.g., an immunogenic composition, e.g., a vaccine)) or in a different comparable subject (e.g., in a different comparable subject than a subject of interest previously exposed to conditions such as the absence of administration of a pharmaceutical composition as described herein (e.g., an immunogenic composition, e.g., a vaccine)). In some embodiments, the comparative term refers to a statistically relevant difference (e.g., a prevalence and/or magnitude sufficient to achieve a statistical correlation). Those skilled in the art will recognize or be readily able to determine the degree of or sufficient variability and/or prevalence required to achieve such statistical significance in a given context. In some embodiments, the term "reduced" or equivalent terms refer to a reduction in the level of an assessment value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or more as compared to a comparable reference. In some embodiments, the term "reduced" or equivalent terms refer to complete or substantially complete inhibition, i.e., reduced to zero or substantially reduced to zero. In some embodiments, the term "increased" or "induced" refers to an increase in the level of an assessment value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500% or more as compared to a comparable reference.

Ionizable: the term "ionizable" refers to a compound or group or atom that is charged at a certain pH. In the context of ionizable amino lipids, such lipids or functional groups or atoms thereof are positively charged at a certain pH. In some embodiments, the ionizable amino lipid is positively charged at an acidic pH. In some embodiments, the ionizable amino lipid is predominantly neutral at physiological pH values, e.g., about 7.0-7.4 in some embodiments, but becomes positively charged at lower pH values. In some embodiments, the ionizable amino lipid may have a pKa in the range of about 5 to about 7.

Separating: the term "isolated" means altered from or out of a natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide, partially or completely separated from coexisting materials in its natural state, is "isolated. The isolated nucleic acid or protein may be present in a substantially purified form, or may be present in a non-natural environment, such as a host cell, for example.

Lipid: as used herein, the terms "lipid" and "lipid-like substance" are broadly defined as molecules comprising one or more hydrophobic moieties or groups and optionally also comprising one or more hydrophilic moieties or groups. Molecules comprising a hydrophobic portion and a hydrophilic portion are also commonly referred to as amphiphilic molecules.

RNA lipid nanoparticles: as used herein, the term "RNA lipid nanoparticle" refers to a nanoparticle comprising at least one lipid and an RNA molecule. In some embodiments, the RNA lipid nanoparticle comprises at least one ionizable amino lipid. In some embodiments, the RNA lipid nanoparticle comprises at least one ionizable amino lipid, at least one helper lipid, and at least one polymer conjugated lipid (e.g., a PEG conjugated lipid). In various embodiments, RNA lipid nanoparticles as described herein can have an average size (e.g., Z-average) of about 100nm to 1000nm or about 200nm to 900nm or about 200nm to 800nm or about 250nm to about 700 nm. In some embodiments of the present disclosure, the RNA lipid nanoparticle may have a particle size (e.g., Z-average) of about 30nm to about 200nm or about 30nm to about 150nm, about 40nm to about 150nm, about 50nm to about 150nm, about 60nm to about 130nm, about 70nm to about 110nm, about 70nm to about 100nm, about 80nm to about 100nm, about 90nm to about 100nm, about 70nm to about 90nm, about 80nm to about 90nm, or about 70nm to about 80 nm. In some embodiments, the average size of the lipid nanoparticle is determined by measuring the particle size. In some embodiments, RNA lipid nanoparticles can be prepared by mixing a lipid with an RNA molecule described herein.

Lipid: as used herein, "lipid" refers to a lipid-like molecule. In some embodiments, the lipid is an amphipathic molecule having one or more lipid-like physical properties. In the context of the present disclosure, the term lipid is considered to encompass lipids.

Nanoparticles: as used herein, the term "nanoparticle" refers to particles having an average size suitable for parenteral administration. In some embodiments, the longest dimension (e.g., diameter) of the nanoparticle is less than 1,000 nanometers (nm). In some embodiments, the nanoparticle may be characterized by a longest dimension (e.g., diameter) of less than 300 nm. In some embodiments, the nanoparticle may be characterized by a longest dimension (e.g., diameter) of less than 100 nm. In many embodiments, the nanoparticle may be characterized by a longest dimension between about 1nm and about 100nm, or between about 1 μm and about 500nm, or between about 1nm and 1,000 nm. In many embodiments, the population of nanoparticles is characterized by an average size (e.g., longest dimension) of about 1,000nm, about 500nm, about 100nm, about 50nm, about 40nm, about 30nm, about 20nm, or about 10nm or less, and typically above about 1 nm. In many embodiments, the nanoparticle may be substantially spherical such that its longest dimension may be its diameter. In some embodiments, the nanoparticle has a diameter of less than 100nm as defined by the national institutes of health.

Naturally occurring: the term "naturally occurring" as used herein refers to an entity that can be found in nature. For example, peptides or nucleic acids that are present in organisms (including viruses) and can be isolated from natural sources and that have not been intentionally modified by man in the laboratory are naturally occurring.

And (3) neutralization: as used herein, the term "neutralization" refers to an event in which a binding agent (e.g., an antibody) binds to a biologically active site (e.g., a receptor binding protein) of a virus, thereby inhibiting parasitic infection of a cell. In some embodiments, the term "neutralization" refers to an event in which the binding agent eliminates or significantly reduces the ability to infect cells.

Nucleic acid particles: "nucleic acid particles" can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, etc.). The nucleic acid particles may comprise at least one cationic or cationically ionizable lipid or lipid-like substance, at least one cationic polymer such as protamine or a mixture thereof, and a nucleic acid. In some embodiments, the nucleic acid particle is a lipid nanoparticle. In some embodiments, the nucleic acid particle is a liposome complex (lipoplex) particle.

Nucleic acid/polynucleotide: as used herein, the term "nucleic acid" refers to a polymer of at least 10 or more nucleotides. In some embodiments, the nucleic acid is or comprises DNA. In some embodiments, the nucleic acid is or comprises RNA. In some embodiments, the nucleic acid is or comprises a Peptide Nucleic Acid (PNA). In some embodiments, the nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, the nucleic acid is or comprises a double stranded nucleic acid. In some embodiments, the nucleic acid comprises both single-stranded and double-stranded fragments. In some embodiments, the nucleic acid comprises a backbone comprising one or more phosphodiester linkages. In some embodiments, the nucleic acid comprises a backbone comprising both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, the nucleic acid may comprise a backbone containing one or more phosphorothioate or 5' -N-phosphoramidite linkages and/or one or more peptide linkages, e.g., in a "peptide nucleic acid". In some embodiments, the nucleic acid comprises one or more or all of the natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, the nucleic acid comprises one or more or all of the non-natural residues. In some embodiments, the unnatural residues include nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deadenosine, 7-deazaguanosine, 8-oxo-adenosine, 8-oxo-guanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, inserted bases, and combinations thereof). In some embodiments, the non-natural residues comprise one or more modified sugars (e.g., 2 '-fluoro ribose, 2' -deoxyribose, arabinose, and hexose) as compared to the sugars in the natural residues. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product, such as RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence comprising one or more introns. In some embodiments, the nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by complementary template-based polymerization, e.g., in vivo or in vitro), replication in a recombinant cell or system, or chemical synthesis. In some embodiments, the nucleic acid is at least 3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、110、120、130、140、150、160、170、180、190、20、225、250、275、300、325、350、375、400、425、450、475、500、600、700、800、900、1000、1500、2000、2500、3000、3500、4000、4500、5000、5500、6000、6500、7000、7500、8000、8500、9000、9500、10,000、10,500、11,000、11,500、12,000、12,500、13,000、13,500、14,000、14,500、15,000、15,500、16,000、16,500、17,000、17,500、18,000、18,500、19,000、19,500 or 20,000 residues or nucleotides in length.

Nucleotide: as used herein, the term "nucleotide" refers to its art-recognized meaning. When the number of nucleotides is used as an indication of, for example, the size of a polynucleotide, a certain number of nucleotides refers to, for example, the number of nucleotides on a single strand of the polynucleotide.

Patient: as used herein, the term "patient" refers to any organism having or at risk of having a disease or disorder or condition. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, the patient is a human. In some embodiments, the patient is suffering from or susceptible to one or more diseases or conditions or disorders. In some embodiments, the patient exhibits one or more symptoms of the disease or disorder or condition. In some embodiments, the patient has been diagnosed with one or more diseases or conditions or disorders. In some embodiments, the disease or disorder or condition suitable for the provided technology is or includes HSV infection. In some embodiments, the patient is receiving or has received a therapy to diagnose and/or treat a disease, disorder, or condition. In some embodiments, the patient is a patient suffering from or susceptible to HSV infection.

PEG conjugated lipids: the term "PEG conjugated lipid" refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety.

Pharmaceutical composition: as used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in an amount suitable for administration in a unit dose in a treatment regimen that, when administered to a relevant population, exhibits a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition may be specifically formulated for parenteral administration, e.g., by subcutaneous, intramuscular, or intravenous injection, e.g., as a sterile solution or suspension formulation.

Pharmaceutically effective amount of: the term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount that alone or in combination with a further dose achieves a desired response or desired effect. In the case of treating a particular disease, the desired response involves, in some embodiments, inhibiting the course of the disease. In some embodiments, such inhibition may include slowing the progression of the disease and/or interrupting or reversing the progression of the disease. In some embodiments, the desired response in the treatment of a disease may be or include delaying or preventing the onset of the disease or condition. The effective amount of a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) described herein will depend, for example, on the disease or disorder to be treated, the severity of such disease or disorder, the individual parameters of the patient (including, for example, age, physiological condition, body size, and weight), the duration of treatment, the type of concomitant therapy (if any), the particular route of administration, and similar factors. Thus, the dosage of the pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) described herein can depend on a variety of such parameters. In cases where the patient's response to the initial dose is inadequate, higher doses (or effectively higher doses obtained by different more localized routes of administration) may be used.

Poly (a) sequence: as used herein, the term "poly (a) sequence" or "poly a tail" refers to an uninterrupted or intermittent sequence of adenylate residues typically located at the 3' end of an RNA molecule. Poly (a) sequences are known to those skilled in the art and may follow the 3' -UTR in the RNAs described herein. The uninterrupted poly (A) sequence is characterized by consecutive adenylate residues. In nature, uninterrupted poly (A) sequences are typical. The RNAs disclosed herein may have a poly (a) sequence that is linked to the free 3' end of the RNA by a template-independent RNA polymerase after transcription or a poly (a) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

Polypeptide: as used herein, the term "polypeptide" refers to a polymeric chain of amino acids. In some embodiments, the polypeptide has an amino acid sequence that occurs in nature. In some embodiments, the polypeptide has an amino acid sequence that is not found in nature. In some embodiments, the polypeptide has an engineered amino acid sequence in that it is designed and/or produced by artificial action. In some embodiments, the polypeptide may comprise, or consist of, a natural amino acid, an unnatural amino acid, or both. In some embodiments, the polypeptide may comprise or consist of only natural amino acids, or consist of only unnatural amino acids. In some embodiments, the polypeptide may comprise a D-amino acid, an L-amino acid, or both. In some embodiments, the polypeptide may comprise only D-amino acids. In some embodiments, the polypeptide may comprise only L-amino acids. In some embodiments, the polypeptide may include one or more pendant groups or other modifications, such as modification at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or any combination thereof, or attached to one or more amino acid side chains. In some embodiments, such pendant groups or modifications include acetylation, amidation, lipidation, methylation, pegylation, and the like, including combinations thereof. In some embodiments, the polypeptide may be cyclic, and/or may comprise a cyclic moiety. In some embodiments, the polypeptide is not cyclic, and/or does not comprise any cyclic moiety. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide may be or include a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to the name, activity or structure of a reference polypeptide; in this case, it is used herein to refer to polypeptides that share a related activity or structure and thus may be considered members of the same class or family of polypeptides. For each such class, the present description provides and/or those skilled in the art will know exemplary polypeptides within that class for which the amino acid sequence and/or function is known; In some embodiments, such exemplary polypeptides are reference polypeptides of the class or family of polypeptides. In some embodiments, members of a class or family of polypeptides exhibit significant sequence homology or identity to a reference polypeptide of the class, share a common sequence motif (e.g., a characteristic sequence element) with a reference polypeptide of the class, and/or share a common activity (in some embodiments within a comparable level or specified range) with a reference polypeptide of the class; in some embodiments, to all polypeptides within the class). For example, in some embodiments, a member polypeptide exhibits an overall sequence homology or identity to a reference polypeptide to a degree of at least about 30-40%, and typically greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and/or includes at least one region (e.g., a conserved region that may be or comprise a characteristic sequence element in some embodiments) that exhibits very high sequence identity, typically greater than 90% or even 95%, 96%, 97%, 98% or 99%. Such conserved regions typically encompass at least 3-4, and typically up to 20 or more amino acids; in some embodiments, the conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, the related polypeptide may comprise or consist of a fragment of the parent polypeptide.

Prevention of: as used herein, the term "prevent/prevention" when used in connection with the occurrence of a disease, disorder, and/or condition refers to reducing the risk of developing the disease, disorder, and/or condition and/or delaying the onset of one or more features or symptoms of the disease, disorder, or condition. Prevention may be considered complete when the onset of a disease, disorder or condition has been delayed for a predefined period of time.

Recombinant: the term "recombinant" in the context of the present disclosure means "made by genetic engineering". In some embodiments, a "recombinant" entity (e.g., a recombinant nucleic acid) in the context of the present disclosure is not naturally occurring.

Reference is made to: as used herein, the term "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the test and/or assay reference or control is substantially simultaneous with the test or assay of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as will be appreciated by those skilled in the art, the reference or control is determined or characterized under conditions or circumstances that are comparable to the conditions or circumstances under evaluation. Those skilled in the art will know when sufficient similarity exists to justify reliance on and/or comparison with a particular possible reference or control.

Ribonucleic acid (RNA): as used herein, the term "RNA" refers to a polymer of ribonucleotides. In some embodiments, the RNA is single stranded. In some embodiments, the RNA is double stranded. In some embodiments, the RNA comprises both single-stranded and double-stranded fragments. In some embodiments, the RNA can comprise a backbone structure as described in the definition of "nucleic acid/polynucleotide" above. The RNA can be a regulatory RNA (e.g., siRNA, microrna, etc.) or messenger RNA (mRNA). In some embodiments, wherein the RNA is mRNA. In some embodiments wherein the RNA is mRNA, the RNA typically comprises a poly (a) region at its 3' end. In some embodiments wherein the RNA is mRNA, the RNA typically comprises a cap structure at its 5' end that is recognized in the art, for example, to recognize the mRNA and attach it to the ribosome to initiate translation. In some embodiments, the RNA is synthetic RNA. Synthetic RNAs include RNAs synthesized in vitro (e.g., by enzymatic synthesis methods and/or by chemical synthesis methods).

Ribonucleotides: as used herein, the term "ribonucleotide" encompasses both unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G) and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides can include one or more modifications including, but not limited to, for example, (a) end modifications such as 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, reverse linkage, etc.), (b) base modifications such as substitutions with modified bases, stabilized bases, destabilized bases, or bases base paired with extended partner library (repertoire) or conjugated bases, (c) sugar modifications (e.g., at the 2' position or the 4' position) or sugar substitutions, and (d) internucleoside linkage modifications including phosphodiester linkage modifications or substitutions. The term "ribonucleotide" also encompasses ribonucleotides that are triphosphate, including modified and unmodified ribonucleotides.

Risk: it will be understood from the context that the "risk" of a disease, disorder and/or condition refers to the likelihood that a particular individual will suffer from the disease, disorder and/or condition. In some embodiments, the risk is expressed in percent. In some embodiments, the risk is 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% up to 100%. In some embodiments, the risk is expressed as a risk relative to a risk associated with a reference sample or a reference sample set. In some embodiments, the reference sample or group of reference samples is at risk for a known disease, disorder, condition, and/or event. In some embodiments, the reference sample or set of reference samples is from an individual that is comparable to a particular individual. In some embodiments, the relative risk is 0, 1,2, 3,4,5,6, 7, 8, 9, 10, or higher. In some embodiments, the risk may reflect one or more genetic attributes, such as, for example, that may predispose an individual to (or not) a particular disease, disorder, and/or condition. In some embodiments, the risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes.

RNA liposome complex particles: as used herein, the term "RNA liposome complex particles" refers to complexes comprising liposomes (particularly cationic liposomes) and RNA molecules. Without wishing to be bound by a particular theory, the electrostatic interaction between positively charged liposomes and negatively charged RNAs results in complexation and spontaneous formation of RNA liposome complex particles. In some embodiments, the positively charged liposome may comprise a cationic lipid, such as DOTMA in some embodiments, and an additional lipid, such as DOPE in some embodiments. In one embodiment, the RNA liposome complex particles are nanoparticles.

Selectivity or specificity: the term "selective" or "specific" when used herein to refer to an agent that is active is understood by those of skill in the art to mean that the agent will distinguish between potential target entities, states, or cells. For example, in some embodiments, an agent is considered to "specifically" bind to its target if it preferentially binds to its target in the presence of one or more competing surrogate targets. In many embodiments, the specific interaction depends on the presence of specific structural features (e.g., epitopes, clefts, binding sites) of the target entity. It is to be understood that the specificity is not necessarily absolute. In some embodiments, specificity can be assessed relative to the specificity of a target binding moiety of one or more other potential target entities (e.g., competitors). In some embodiments, specificity is assessed relative to the specificity of a reference specific binding member. In some embodiments, specificity is assessed relative to the specificity of a reference non-specific binding member.

Stable: as used herein, the term "stable" in the context of the present disclosure means that the pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) as a whole and/or components thereof meets or exceeds predetermined acceptance criteria. For example, in some embodiments, a stable pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) exhibits no unacceptable levels of microbial growth and substantially no or no degradation or degradation of the active biomolecule components. In some embodiments, a stable pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) refers to an RNA molecule whose integrity is maintained above at least 90% or higher. In some embodiments, a stable pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) means that at least 90% or more (including, e.g., at least 95%, at least 96%, at least 97% or more) of the RNA molecules remain encapsulated within the lipid nanoparticle. In some embodiments, a stable pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) refers to a formulation that is still capable of eliciting a desired immunological response when administered to a subject. In some embodiments, the pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) will remain stable for a specified period of time under certain conditions.

The subject: as used herein, the term "subject" refers to an organism to which a composition described herein is to be administered, e.g., for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, the subject is a human subject. In some embodiments, the subject has a disease, disorder, or condition (e.g., HSV infection). In some embodiments, the subject is susceptible to a disease, disorder, or condition (e.g., HSV infection). In some embodiments, the subject exhibits one or more symptoms or features of a disease, disorder, or condition (e.g., HSV infection). In some embodiments, the subject exhibits one or more non-specific symptoms of a disease, disorder, or condition (e.g., HSV infection). In some embodiments, the subject does not exhibit any symptoms or features of the disease, disorder, or condition (e.g., HSV infection). In some embodiments, the subject is a person having a predisposition to a disease, disorder, or condition (e.g., HSV infection) or having one or more characteristics characteristic of the risk described above. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who has been and/or has been subjected to diagnosis and/or treatment.

Is provided with: individuals suffering from a disease, disorder, and/or condition have been diagnosed with and/or exhibiting one or more symptoms of the disease, disorder, and/or condition.

Is easy to suffer from: an individual who is "susceptible to" a disease, disorder and/or condition is an individual who is at a higher risk of suffering from the disease, disorder and/or condition than a member of the general public. In some embodiments, an individual susceptible to a disease, disorder, and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition will suffer from the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition will not suffer from the disease, disorder, and/or condition.

And (3) synthesis: as used herein, the term "synthetic" refers to an entity that is made by man-made or human intervention or that is synthetically produced rather than naturally occurring. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a chemically synthesized nucleic acid molecule, e.g., by solid phase synthesis in some embodiments. In some embodiments, the term "synthetic" refers to an entity made outside of a biological cell. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g., RNA) produced by in vitro transcription using a template.

Treatment: the term "therapy" refers to the administration or delivery of an agent or intervention that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect (e.g., that has been demonstrated to be statistically likely to have such an effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to reduce, ameliorate, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and/or reduce the incidence of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to reduce, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and/or reduce the incidence of one or more symptoms or features of a disease, disorder, and/or condition.

Three terminal untranslated region: as used herein, the term "three terminal untranslated region" or "3' utr" refers to a sequence of an mRNA molecule that follows a stop codon that begins in the coding region of an open reading frame sequence. In some embodiments, the 3' utr begins immediately after the stop codon of the coding region of the open reading frame sequence, e.g., in its natural context. In other embodiments, the 3' utr does not begin immediately after the stop codon of the coding region of the open reading frame sequence, e.g., in its natural context.

Threshold level (e.g., acceptance criteria): as used herein, the term "threshold level" refers to a level that is used as a reference to obtain information about and/or classify the result of a measurement, such as the result of a measurement obtained in an assay. For example, in some embodiments, the threshold level means a value measured in an assay that defines a demarcation line between two subsets of a population (e.g., a lot that meets quality control criteria and a lot that does not meet quality control criteria). Thus, values at or above the threshold level define one subset of the population, and values below the threshold level define another subset of the population. The threshold level may be determined based on one or more control samples or among a population of control samples. The threshold level may be determined before, simultaneously with, or after the measurement of interest is made. In some embodiments, the threshold level may be a series of values.

Treatment: as used herein, the term "treatment" refers to any method for partially or completely alleviating, ameliorating, alleviating, inhibiting, preventing, delaying the onset of, reducing the severity of, and/or reducing the incidence of one or more symptoms or features of a disease, disorder, and/or condition. Treatment may be administered to a subject that does not exhibit signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject that exhibits only early signs of a disease, disorder, and/or condition, e.g., for the purpose of reducing the risk of developing a pathology associated with the disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject at an advanced stage of a disease, disorder, and/or condition.

Vaccination: as used herein, the term "vaccination" refers to the administration of a composition intended to produce an immune response, e.g., against a disease-associated (e.g., pathogenic) agent. In some embodiments, vaccination may be performed before, during, and/or after exposure to a disease-related factor, and in some embodiments, vaccination may be performed before, during, and/or shortly after exposure to the factor. In some embodiments, vaccination comprises multiple administrations of the vaccine composition at appropriate intervals in time. In some embodiments, vaccination produces an immune response to an infectious agent.

Vaccine: as used herein, the term "vaccine" refers to a composition that induces an immune response upon administration to a subject. In some embodiments, the induced immune response provides protective immunity.

Variants: as used herein in the context of a molecule (e.g., a nucleic acid, protein, or small molecule), the term "variant" refers to a molecule that exhibits significant structural identity to a reference molecule but is structurally different from the reference molecule, e.g., the presence or absence or level of one or more chemical moieties is different as compared to the reference entity. In some embodiments, the variant is also functionally different from its reference molecule. In general, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. Variants are defined as different molecules that share one or more of such characteristic structural elements with a reference molecule, but differ in at least one aspect. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid due to one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., attached to the polypeptide or nucleic acid backbone). In some embodiments, the variant polypeptide or nucleic acid exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with a reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, the variant polypeptide or nucleic acid shares one or more biological activities of a reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid lacks one or more biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities as compared to a reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a "variant" of a reference polypeptide or nucleic acid if it has the same amino acid or nucleotide sequence as the reference except for a small sequence change at a particular position. Typically, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in the variant are substituted, inserted, or deleted as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Typically, a variant polypeptide or nucleic acid comprises a very small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (i.e., residues involved in a particular biological activity) relative to a reference. In some embodiments, the variant polypeptide or nucleic acid comprises no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions as compared to the reference, and in some embodiments does not comprise an addition or deletion. In some embodiments, a variant polypeptide or nucleic acid comprises less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically less than about 5, about 4, about 3, or about 2 additions or deletions as compared to a reference. In some embodiments, the reference polypeptide or nucleic acid is a polypeptide or nucleic acid found in nature.

And (3) a carrier: as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". In some embodiments, known techniques may be used, for example, for the production or manipulation of recombinant DNA, for oligonucleotide synthesis, and for tissue culture and transformation (e.g., electroporation, lipofection). The enzymatic reaction and purification techniques may be performed according to manufacturer's instructions, or accomplished as is conventional in the art, or as described herein. The foregoing techniques and procedures may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the present specification. See, for example, sambrook et al Molecular Cloning: ALaboratory Manual (4 th edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (2012)), which is incorporated herein by reference for any purpose.

All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, treatises, and web pages, are expressly incorporated by reference in their entirety, regardless of the format of such documents and similar materials. In the event that one or more of the incorporated documents and similar materials differs from or contradicts the present application (including, but not limited to, the defined terms, term usage, described techniques, etc.), the present application controls. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

Detailed description of certain embodiments

As discussed above, the present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) and related techniques (e.g., methods) for delivering a particular Herpes Simplex Virus (HSV) antigen construct (e.g., an HSV-1 antigen construct, an HSV-2 antigen construct, or a combination thereof) to a subject (e.g., a patient). In particular, the present disclosure provides HSV (e.g., HSV-1, HSV-2, or both) vaccine compositions and related techniques (e.g., methods).

The present disclosure provides, for example, polyribonucleotides that encode one or more HSV antigens. In some embodiments, such polyribonucleotides may be part of an RNA construct. In some embodiments, a polyribonucleotide or RNA construct as described herein can be part of a composition (e.g., a pharmaceutical composition, e.g., an immunogenic composition, e.g., a vaccine).

In some embodiments, the techniques provided herein are directed to HSV. The following is a description of HSV and certain exemplary features.

I. Herpes Simplex Virus (HSV)

Herpes Simplex Virus (HSV) belongs to the alpha subfamily of the human herpesvirus family and includes HSV-1 and HSV-2. The structure of HSV-1 and HSV-2 mainly comprises (from inside to outside) a DNA core, a capsid, a capsule and an envelope. HSV-1 and HSV-2 each have a double stranded DNA genome of about 153kb encoding at least 80 genes. The DNA core is surrounded by a twenty-five-sided (icosapentahedral) capsid consisting of 162 capsomers, 150 hexons, and 12 pentons, consisting of six different viral proteins. The DNA is surrounded by at least 20 different viral envelope proteins, which have structural and regulatory roles. Some of them are involved in the transport of capsids to the nucleus and other organelles, entry of viral DNA into the nucleus, activation of early gene transcription, inhibition of cellular protein biosynthesis and mRNA degradation. The viral envelopes surrounding the envelope have at least 12 different glycoproteins (B-N) on their surface. Glycoproteins may exist as heterodimers (H/L and E/I), mostly as monomers.

HSV-1 and HSV-2 are responsible for many mild, moderate and severe pathologies, including ulcers of the oral and genital organs, virus-induced blindness, viral encephalitis and disseminated infection of newborns. HSV-1 and HSV-2 typically travel through different pathways and affect different parts of the body, but the signs and symptoms they cause may overlap. Infection by HSV-1 is one of the more common infections of the orofacial area and commonly causes herpes labialis, herpetic stomatitis and keratitis. HSV-2 generally causes genital herpes and is transmitted primarily through direct sexual contact with lesions. Most genital HSV infections are caused by HSV-2, however more and more genital HSV infections have been attributed to HSV-1. Genital HSV-1 infection is generally less severe than genital HSV-2 infection and is also less likely to occur.

HSV infection is transmitted by contact with herpes lesions, mucosal surfaces, genital secretions or oral secretions. The average latency period after exposure is typically 4 days, but may range between 2 and 12 days. HSV particles can infect neuronal extensions, thereby weakening peripheral tissues, and establishing a latency period in these cells, i.e., in the trigeminal ganglion and dorsal root ganglion of the sacral region from which they can be sporadically reactivated. In addition, similar to other herpes viruses, HSV infections are life-long and generally asymptomatic. Without wishing to be bound by any particular theory, it is understood that HSV particles may shed from an infected individual, irrespective of the occurrence of clinical manifestations.

HSV infection is rarely fatal, but is characterized by the possibility of blisters breaking and causing pain. There is little apparent difference in clinical presentation depending on the type of infectious virus. However, as discussed above, HSV-1 infection is often less severe than HSV-2 infection, and patients infected with HSV-2 typically outbreak more.

A. life cycle

As described herein, to initiate infection, HSV (HSV-1 or HSV-2) particles utilize viral glycoproteins to bind to the cell surface and fuse their envelope to the plasma membrane (see, e.g., fig. 2, step 1). After membrane fusion, the viral capsid and envelope proteins are internalized in the cytoplasm (see, e.g., fig. 2, step 2). Once in the cytoplasm, the viral capsid accumulates in the nucleus and releases viral DNA into the nucleus (see, e.g., fig. 2, step 3). HSV replicates through three rounds of transcription, yielding: alpha (immediate early) proteins that regulate primarily viral replication; synthesizing and packaging beta (early) protein of DNA; and gamma (late) proteins, most of which are virosome proteins (see Whitley et al, lancet 2001, 5, 12; 357 (9267), taylor et al, front biosci.2002, 1; 7:d752-64; andEt al, front microbiol 2018, 10, 11; 9:2406; each of the above documents is incorporated herein by reference in its entirety) (see, e.g., fig. 2, steps 4-6).

HSV capsids assemble within the nucleus of infected cells (see, e.g., fig. 2, step 7). Once the viral capsids have been assembled in the nucleus, these particles will continue their maturation process in the same compartment by taking the envelope proteins. After leaving the nucleus, additional envelope proteins will be added to the capsid. At the same time, glycoproteins are translated and glycosylated in the endoplasmic reticulum and processed in the trans-golgi network (TGN) and then directed to the multivesicular body (see, e.g., fig. 2, step 8). They are then transported into plasma membrane glycoproteins in the early endosomes (see, e.g., fig. 2, step 9). The viral capsid in the cytoplasm will then fuse with the endosome containing the HSV-glycoprotein to form an infectious virion within the vesicle (see, e.g., fig. 2, steps 10-12).

HSV (HSV-1 or HSV-2) is capable of establishing latent infection. After primary infection, HSV either replicates in large numbers in epithelial cells or enters sensory neuron axons and moves into the neuronal nucleus. There, the viral DNA is still circular extrachromosomal DNA and does not have any soluble gene expression; however, latency-associated transcripts are expressed and then spliced to produce mRNA. This general transcriptional silencing may allow the virus to remain hidden in the cell by circumventing immune surveillance. In some aspects, provided herein are techniques (e.g., compositions and methods) for enhancing, inducing, promoting, enhancing, and/or improving an immune response against HSV (e.g., HSV-1 and/or HSV-2) or a component thereof (e.g., a protein or a fragment thereof). In some embodiments, the techniques provided herein aim to enhance, induce, promote, enhance, and/or improve immune memory against HSV or a component thereof (e.g., a protein or fragment thereof). In some embodiments, the techniques described herein are intended for use as immunopotentiators for primary vaccines, such as vaccines against antigens and/or epitopes of HSV (e.g., HSV-1 and/or HSV-2).

The virus remains in this state throughout the life of the host, or until an appropriate signal reactivates the virus and produces new progeny. The progeny virus then passes through the neuronal axis to the primary infection site to restart the lytic replication cycle.

HSV genome

The genome of HSV-1 and the genome of HSV-2 are double-stranded DNA approximately 150kb long, with slight differences between subtypes and strains. The genome encodes more than 80 genes and has a high GC content: 67% and 69%, respectively, for HSV-1 and HSV-2 (see Whitley et al, lancet 2001, 12. 5, 357 (9267), taylor et al, front biosci.2002, 1. 3, 7:d752-64, and Jiao et al, microbiol Resour Announc.2019, 9, 8 (39): e00993-19, which are incorporated herein by reference in their entirety).

The genome is organized into distinct long regions (UL) and distinct short regions (US). The UL is generally bounded by terminal long (TRL) repeats and internal long (IRL) repeats. US is generally bounded by terminal short (IRS) repeats and internal short (TRS) repeats. The genes found in the unique region are present in the genome as a single copy, but the genes encoded in the repeat region are present in the genome in two copies (see Whitley et al, lancet 2001, month 5, 12; 357 (9267); taylor et al, front biosci.2002, month 3, 1; 7:d752-64; and Jiao et al, microbiol Resour Announc.2019, 8 (39): e00993-19, incorporated herein by reference in their entirety).

HSV contains three origins of replication within the genome, named according to their location in the long (oriL) or short (oriS) regions of the genome. OriL are seen as single copies in the UL segment, but oriS is located in the repeat region of the short segment; thus, it is present in the genome in two copies. oriL and oriS are palindromic sequences consisting of AT-rich central regions flanked by inverted repeats containing multiple binding sites of differing affinities for a viral-derived binding polypeptide (UL 9). oriL or oriS sequences are sufficient for viral replication (see Whitley et al, lancet 2001, month 12; 357 (9267), taylor et al, front biosci.2002, month 3, 1; 7: d752-64; and Jiao et al, microbiol Resour Announc.2019, month 9; 8 (39): e00993-19, which are incorporated herein by reference in their entirety).

The viral genome also contains signals that coordinate the proper processing of the newly synthesized genome for packaging into preformed capsids. The progeny genome is produced in long concatemers that need to be cut into monomers of unit length. For this purpose, the viral genome contains two DNA sequence elements, pac1 and pac2, which ensure the correct cleavage and packaging of the progeny genome per unit length. These elements are located within the Direct Repeat (DR) sequence seen in the inverted repeat region at the ends of the viral genome (see Whitley et al, lancet 2001, month 5, 12; 357 (9267), taylor et al, front biosci.2002, month 3, 1; 7:d752-64; and Jiao et al, microbiol Resour Announc.2019, 8 (39): e00993-19, which are incorporated herein by reference in their entirety).

C. Certain HSV proteins

ICP0

Infected cell protein 0 (ICP 0) of herpes simplex virus 1 (HSV-1) is the alpha (immediate early) protein of herpes simplex virus 1 and is capable of activating HSV-1 gene expression, disrupting the Nuclear Domain (ND) 10 structure, mediating degradation of the cell protein and evading the intrinsic and innate antiviral defenses of the host cell (see Smith et al, future virol.2011, 4; 6 (4): 421-429).

ICP22

The infected cell protein 22 (ICP 22) is expressed by the Immediate Early (IE) genes during the replication cycle of HSV-1 and HSV-2. ICP22 can regulate viral and host gene transcription, generally, by altering the phosphorylation state of host RNA polymerase II (RNA pol II), and can also promote accurate localization of Nuclear Export Complexes (NECs) to nuclear membranes to promote nuclear budding (see Wu et al, front Microbiol.2021, 6, 7; 12:668761).

VP16

The UL48 gene encodes VP16 or alpha gene transactivator (alpha-TIF). VP16 is an important transactivator that can activate transcription of viral immediate early genes, as well as late stages of viral replication. In addition, VP16 is involved in viral assembly as a membrane (see Fan et al, front Microbiol 2020; 11:1910).

In the early stages of viral infection, VP16 released by the invading virion binds to the Immediate Early (IE) gene promoter to stimulate transcription of the IE gene as a transactivator acting specifically on the IE gene (see Fan et al, front Microbiol 2020; 11:1910). In the late stage VP16 assembles into the envelope to participate in the assembly of the virions and promote their maturation (see Fan et al, front Microbiol 2020; 11:1910).

Glycoprotein proteins

For replication, enveloped HSV must be able to fuse with the membrane of living cells and deliver their genetic material into their cytoplasm. The envelope-surrounding HSV viral envelopes have at least 12 different glycoproteins (gB-gN) on their surface. Glycoproteins may exist as heterodimers (gH/gL and gE/gI), mostly as monomers. HSV gC, gB, gD, gH and gL are involved in the process of viral cell entry. The initial attachment is mediated by gC, followed by gD. The gH/gL then pulls the virus and cell membrane together, and the gB then triggers membrane fusion. (Reske et al, rev Med Virol.2007, 5-6 months; and Arii et al, adv Exp Med biol.2018; 1045:3-21).

The present disclosure provides HSV glycoprotein (e.g., gB, gC, gD, gE, gG, gH, gI and/or gL) antigens and antigenic fragments thereof, which are useful for preventing or treating HSV, e.g., in HSV antigen constructs and/or HSV compositions (e.g., immunogenic compositions, e.g., vaccines) as further disclosed herein.

Glycoprotein C (gC)

Mature HSV glycoprotein C (gC) is a 56kDa protein that plays a role in initial cell attachment. Glycoprotein C is a type I membrane glycoprotein and is considered an important attachment protein and a major viral ligand for binding Heparan Sulfate Proteoglycans (HSPG) on the cell surface. This binding can occur through interaction of gC with HSPG-rich regions seen on the F-actin-rich membrane processes known as filopodia.

Glycoprotein C is also shown to be involved in regulating cell entry and infection by increasing the pH threshold of the acid-induced conformational change of gB. The low pH induces reversible conformational changes of gB domains I and V, which are functional regions containing hydrophobic loops important in cell fusion. By positively regulating the conformational change of low pH induced gB, gC can enhance the ability of HSV to invade cell types such as epithelial cells that require a low pH invasion mechanism.

Glycoprotein C has been shown to play a role in immune evasion in addition to its role in attachment. Glycoprotein C is a target of lymphocyte cytotoxicity in certain cell types and is capable of binding complement component C3b to inhibit complement activation. Further, neutralizing epitopes present on other HSV glycoproteins, such as gB, may be protected by gC, thereby preventing immune response blocking fusion.

Glycoprotein D (gD)

HSV glycoprotein D (gD) is a 46kDA type I membrane glycoprotein. The N-terminal extracellular domain consists of 316 amino acids. Glycoprotein D promotes invasion by interacting with several cell surface receptors including Herpes Virus Entry Mediators (HVEM), connexin-1 or-2 and heparin sulfate containing specific modifications. These cellular receptors do not function as co-receptors because each glycoprotein interacts with the cellular receptor independently of the other. Binding of gD to one of these cellular receptors results in a conformational change that converts the self-inhibiting closed state of gD into an active state that transmits one of two signals believed to be required for gH/gL complex activation. The first gD receptor HVEM identified belongs to the tumor necrosis factor receptor family and is commonly found on T cells, B cells, dendritic cells, natural killer cells, macrophages, and non-immune cell types such as neurons and epithelial cells. Within the N-terminus of gD there is a 37 residue hairpin structure that forms the entire site of binding to HVEM. Specifically, residues 1-32 of the N-terminal domain of gD bind to cysteine-rich domain 1 of HVEM. This N-terminal extension adopts an extended and flexible conformation when not in contact with HVEM.

Clinical strains of HSV enter cells using connexin-1; however, several mutants of HSV utilize connexin-2. Further, heparin sulfate is utilized by HSV-1 instead of HSV-2. The interaction of glycoprotein D with net-1 has been shown to be necessary in some cell types, such as neurons, even when other receptors are present on the cell surface.

Glycoprotein H (gH)/glycoprotein L (gL) complexes

Glycoprotein H (gH) is an essential 56kD protein that exists as a heterodimeric complex with 25kDa glycoprotein L (gL) (the complex is referred to herein as gH/gL). The gH/gL complex is required for cell fusion and entry. gH/gL does not share any structural similarity with the fusion proteins described in the literature and may not function as a fusion-promoting protein with gB (cofusogen). Instead, gH/gL can act as a regulatory factor for fusion and an important component in stabilizing contact between HSV and cells. Glycoprotein H receives a signal from gD through its H1 domain and transmits this signal to the membrane proximal H3 domain, which in turn propagates the signal to the cytoplasmic tail of gH. Once received by the cytoplasmic tail of the gH, it releases strain on the pre-fusion conformation of the gB, which facilitates attachment of the gB fusion loop to the cell surface, thereby facilitating gB-mediated membrane fusion. Mutations in the C-terminal tail of gH have been shown to reduce fusion activity. Further, antibody responses against gH have been shown to inhibit the fusion process mediated by gB-gH-gL. In addition to this basic role, gH contains the arginyl glycyl aspartic acid (RGD) motif that can bind to integrin receptors seen on cells. Interaction of gH with integrins is believed to trigger intracellular signals that facilitate capsid transport.

Glycoprotein B (gB)

Glycoprotein B is a protein with an apparent molecular weight of about 95-100kDa and consists of an extended rod or spike-like extracellular domain, a hydrophobic Membrane Proximal Region (MPR), a transmembrane region (TMR) and a C-terminal domain (CTD). Extracellular domains are well characterized as actively involved in fusion, whereas MPR, TMR and CTD may play a role in regulatory fusion. Glycoprotein B is a class III fusion protein (fusogen). Glycoprotein B extracellular domain architecture shares conformational similarity with fusion proteins from viruses not belonging to the herpesviridae family. Glycoprotein B is activated by its interaction with gH/gL, but HSV cannot fuse with target cells by activating gB alone, requiring interaction of gB with specific receptors to accomplish the fusion. One well-known receptor target for gB is cell-surface heparan sulfate, a non-essential interaction for HSV fusion, but is known to promote viral adhesion to the cell surface. Glycoprotein B can also interact with paired immunoglobulin-like type 2 receptors most commonly found on monocytes, macrophages and dendritic cells.

HSV gB exists in two forms, a pre-fusion form and a post-fusion form. Several changes in the pre-fusion form of gB are thought to result in its activity and post-fusion state. The first change occurs in domain V or MPR, which directs the fusion loop towards the cell membrane and away from the viral membrane. This change can result in a compact intermediate conformation 1 that has not yet been attached to the cell membrane surface. The next change occurs in domain III and involves the use of gB in the extended intermediate conformation 2, which allows its fusion loop to attach to the cell membrane surface. Finally, the change in domain V converts gB into its post-fusion conformation, which facilitates membrane fusion.

The post-fusion form of HSV-1gB has an extracellular domain that exists as three protomers that interact to produce a rod-like trimeric structure. Each promoter consists of five distinct domains, with the linker regions alone forming a hairpin shape. Each domain of a single protomer interacts with the same domain of an adjacent protomer to form the described trimeric structure. Domain I houses important fusion loops and is commonly referred to as a fusion domain. Domain II facilitates interaction with gH/gL and is referred to as the gH/gL domain. Domain III consists of an alpha helix that helps form the trimeric loop-to-loop central core of this protein. Domain IV is called the coronal domain and sits on top of the fused form; it is believed to bind to cellular receptors. Antibodies that bind to the crown domain can disrupt the binding of gB to cellular receptors. Domain V consists of long stretches and links the protomers together.

Glycoprotein E and glycoprotein I (gE/gI)

Glycoprotein E is about 53kDa and glycoprotein I is about 141kDa. These two proteins interact to form heterodimeric complexes (referred to herein as gE/gI complexes) that play a role in intercellular diffusion and virus-induced fusion. Unlike gB, gD and gH/gL, the gE/gI complex is not necessary for fusion and entry into cells, but is important for intercellular diffusion. Disruption of gE/gI formation has an effect on HSV proliferation, as the lytic cycle of this virus is dependent on intercellular diffusion. The mechanism by which gE/gI promotes intercellular diffusion is thought to depend on several envelope polypeptides. The synergistic effects of the envelope polypeptides UL11, UL16 and UL21 may play a role in the processing, transport and bioactivity of gE.

Glycoprotein G

Glycoprotein G (gG) from HSV-1 (gG 1) and HSV-1 (gG 2) is the first viral chemokine binding protein that has been shown to enhance chemokine function of cells. Glycoprotein G varies greatly in size between HSV-1 and HSV-2, with sizes of 76kDa and 43kDa, respectively. Glycoprotein G is unique in that its soluble form (SgG 2) can be immunoregulatory by virtue of its extracellular activity. Once extracellular, sgG binds to a chemokine through its glycosaminoglycan (GAG) binding domain without interfering with the G Protein Coupled Receptor (GPCR) binding site of the chemokine. The interaction of SgG with GAG-containing proteins allows initiation of lipid raft formation and accumulation, which results in aggregation of chemokine receptors into this microstructure domain. Aggregation of chemokine receptors in turn increases the local concentration of chemokines on the extracellular surface of host cells and allows these chemokines to interact with GPCRs. This interaction may lead to an increased immune signaling response and chemokine stimulation. This combination of receptor relocation and presentation of the chemokine complex with SgG provides a molecular principle of chemokine function enhancement during HSV infection. This immunomodulation is in contrast to other viruses that inhibit chemokine function, as in this case chemokine function is enhanced by SgG. Without being bound by any particular theory, it is believed that overall manipulation of endogenous immune signaling may be beneficial for HSV as a whole.

ICP47

The infectious cellular protein 47 (ICP 47) encoded by gene US12 is a polymorphic protein and can block RNA splicing early in infection and then shuttle viral mRNA from the nucleus to the cytoplasm in late infection. ICP47 binds directly to the antigen dependent Transporter (TAP), limiting antigen transport, leading to the appearance of empty MHC-I (Cheng et al, virol J.2020, 7/10; 17 (1): 101). Binding of ICP47 to TAP stabilizes the inward conformation and thus blocks translocation pathways directed to the lumen of the Endoplasmic Reticulum (ER). HSV can avoid attack by cytotoxic T lymphocytes by blocking viral antigen entry into the ER, which can lead to immune escape of HSV and establishment of life-long infection in host cells (Cheng et al, virol J.2020, 7, 10; 17 (1): 101).

VHS

Virosome-host shut-down (VHS) proteins are viral proteins synthesized with late kinetics and packaged into mature virosome particles. Functionally, VHS is a viral rnase that preferentially degrades both host and viral mRNA species. VHS is reported to interfere with Dendritic Cell (DC) activation during productive and non-productive HSV infection (Cotter et al, J Virol.2011, month 12; 85 (23): 12662-12672.).

US3

All members of the subfamily of alphaherpesvirus encode a serine/threonine kinase designated US 3. US3 is an important virulence factor for herpes simplex virus type 1 (HSV-1) and is a multifunctional polypeptide that plays various roles in the viral life cycle by phosphorylating many viruses and cell substrates (Kato et al, adv Exp Med biol.2018; 1045:45-62.).

HSV vaccine

Several HSV vaccines have been developed that target mainly HSV-2 and focus mainly on the generation of neutralizing antibodies (nabs) targeting viral envelope glycoprotein D as a relevant immune-protective agent and evaluated in human clinical trials, see table 1 below. Although these vaccines have demonstrated protective effects against HSV in preclinical and in some cases phase 2 studies, none of these vaccines has proven to have sufficient efficacy for further development or commercialization.

The present disclosure provides insight that many existing strategies for developing pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) for the treatment and/or prevention of HSV infections have focused primarily or even almost exclusively on developing neutralizing antibodies that target surface glycoproteins. The present disclosure identifies issues with such strategies, including, for example, that they may not be aware of the value or even criticality of ensuring that the induced immune response includes significant T cell activity (in some embodiments CD 4T cell activity, in some embodiments CD 8T cell activity, in some embodiments both of the foregoing). In some embodiments, pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) comprising or delivering CD4 and CD8 epitopes of one or more HSV antigens (e.g., HSV-1 antigen, HSV-2 antigen, or a combination thereof) in addition to one or more B cell antigens and/or epitopes, for example, can be used to treat and/or prevent HSV infection.

Table 1: certain HSV vaccines in clinical development

Antiviral treatment of HSV

The present disclosure provides the recognition that the constructs and/or compositions described herein can be administered as part of a regimen along with other therapeutic agents. The present disclosure also recognizes that a subject administered the constructs and/or compositions described herein may have previously been administered other therapeutic agents.

In some embodiments, for example, the subject may be receiving or has previously received an antiviral agent against HSV. In some embodiments, an antiviral agent may be administered to treat HSV-1 or HSV-2 infection or recurrent episodes. In some embodiments, the antiviral agent is or includes acyclovir, valacyclovir, famciclovir, or a combination thereof. Table 2 below provides some information about the selection of antiviral agents.

Table 2: antiviral medicament for treating HSV

Constructs

HSV antigen

The present disclosure provides HSV (e.g., HSV-1, HSV-2, or both) antigens and antigenic fragments thereof that are useful for preventing or treating HSV, for example, in HSV antigen constructs and/or HSV compositions (e.g., immunogenic compositions, such as vaccines) as further disclosed herein.

In some embodiments, HSV antigenic fragments may be used in HSV T cell antigen constructs. In some embodiments, HSV antigens (e.g., full length HSV antigens) may be used in HSV glycoprotein constructs.

In some embodiments, the polyribonucleotide encodes one or more HSV antigens or antigenic fragments thereof. In some embodiments, the polyribonucleotide encodes an HSV glycoprotein construct.

A variety of HSV antigens are known. The present disclosure provides polyribonucleotides encoding HSV antigens or antigenic fragments thereof as described herein. Tables 3-5 below provide an overview of exemplary amino acid sequences of certain HSV (HSV-1, HSV-2, or both) proteins. Table 6 below provides exemplary amino acid sequences for certain HSV (HSV-1, HSV-2, or both) proteins.

Table 3: exemplary antigens for HSV

Table 4: exemplary antigens selected from systematic analysis of Source data

Table 5: exemplary antigens

Table 6: exemplary amino acid antigen sequences

In some embodiments, the present disclosure provides certain HSV antigen constructs (e.g., HSV-1 antigen constructs, HSV-2 antigen constructs, or combinations thereof) that are particularly useful in effective vaccination.

In various embodiments, the HSV antigen construct comprises and/or encodes a plurality of HSV antigens (e.g., a plurality of HSV antigens that are or comprise one or more T cell and/or B cell antigens of HSV). As disclosed herein, T cell antigens include, for example, CD 4T cell antigens and/or CD 8T cells. In some embodiments, the HSV antigen is a T cell antigen. In some embodiments, the HSV antigen is a B cell antigen.

In certain embodiments, the HSV antigen construct may include and/or encode at least one of UL1、UL21、UL27、UL29、UL39、UL40、UL46、UL47、UL48、UL49、RS1、RL2、UL5、UL9、UL19、UL25、UL30、UL52、US1、US7、US8、UL22 and/or UL54, or a fragment thereof. In certain embodiments, the HSV antigen construct may include and/or encode at least one of UL1, UL21, UL27, UL29, UL39, UL40, UL46, UL47, UL48, and/or UL49, or a fragment thereof. In certain embodiments, the HSV antigen construct may include and/or encode at least one of RS1, RL2, UL5, UL9, UL19, UL25, UL30, UL52, US1, US7, US8, UL22, and/or UL54, or a fragment thereof.

In certain embodiments, an HSV antigen construct may include and/or encode UL1、UL21、UL27、UL29、UL39、UL40、UL46、UL47、UL48、UL49、RS1、RL2、UL5、UL9、UL19、UL25、UL30、UL52、US1、US7、US8、UL22 and/or multiple ones of UL54 (e.g., 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, or 19) or fragments thereof. In certain embodiments, an HSV antigen construct may include and/or encode multiple (e.g., 1,2,3, 4, 5, 6, 7, 8, 9, or 10) of UL1, UL21, UL27, UL29, UL39, UL40, UL46, UL47, UL48, and/or UL49, or fragments thereof. In certain embodiments, an HSV antigen construct may include and/or encode multiple (e.g., 1,2,3, 4, 5, 6, 7, 8, or 9) of RS1, RL2, UL5, UL9, UL19, UL25, UL30, UL52, US1, US7, US8, UL22, and/or UL54, or fragments thereof.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL1 polypeptide or fragment thereof. In various embodiments, the UL1 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL1 polypeptides known in the art include UL1 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL1 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to the amino acid sequence as set forth in SEQ ID nos. 1, 2 and/or 3.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL21 polypeptide or fragment thereof. In various embodiments, the UL21 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL21 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL21 polypeptides known in the art include UL21 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL21 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 4, 5 and/or 6.

The UL27 open reading frame encodes HSV gB (also referred to herein as UL27 polypeptide). In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL27 polypeptide or fragment thereof. In various embodiments, the UL27 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL27 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL27 polypeptides known in the art include UL27 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL27 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 7, 8, 9 and/or 74.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL29 polypeptide or fragment thereof. In various embodiments, the UL29 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL29 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL29 polypeptides known in the art include UL29 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL29 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 10, 11 and/or 12.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL39 polypeptide or fragment thereof. In various embodiments, the UL39 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL39 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL39 polypeptides known in the art include UL39 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL39 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 13, 14 and/or 15.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL40 polypeptide or fragment thereof. In various embodiments, the UL40 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL40 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL40 polypeptides known in the art include UL40 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL40 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 16, 17 and/or 18.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL46 polypeptide or fragment thereof. In various embodiments, the UL46 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL46 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL46 polypeptides known in the art include UL46 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL46 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 19, 20 and/or 21.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL47 polypeptide or fragment thereof. In various embodiments, the UL47 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL47 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL47 polypeptides known in the art include UL47 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL47 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 22, 23 and/or 24.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL48 polypeptide or fragment thereof. In various embodiments, the UL48 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL48 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL48 polypeptides known in the art include UL48 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL48 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 25, 26 and/or 27.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL49 polypeptide or fragment thereof. In various embodiments, the UL49 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL49 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL49 polypeptides known in the art include UL49 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL49 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 28, 29 and/or 30.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes an RS1 polypeptide or fragment thereof. In various embodiments, the RS1 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to an RS1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of RS1 polypeptides known in the art include RS1 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the RS1 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs.31, 32 and/or 33.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a RL2 polypeptide or fragment thereof. In various embodiments, the RL2 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to the RL2 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of RL2 polypeptides known in the art include RL2 polypeptides encoded by known strains of HSV, such as (but not limited to) HG52, G, 333, and MS. In some embodiments, the RL2 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to the amino acid sequence as set forth in SEQ ID NOS.34, 35 and/or 36.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL5 polypeptide or fragment thereof. In various embodiments, the UL5 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL5 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL5 polypeptides known in the art include UL5 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL5 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 37, 38 and/or 39.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL9 polypeptide or fragment thereof. In various embodiments, the UL9 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL9 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL9 polypeptides known in the art include UL9 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL9 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 40, 41 and/or 42.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL19 polypeptide or fragment thereof. In various embodiments, the UL19 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL19 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL19 polypeptides known in the art include UL19 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL19 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 43, 44 and/or 45.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL25 polypeptide or fragment thereof. In various embodiments, the UL25 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL25 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL25 polypeptides known in the art include UL25 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL25 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 46, 47 and/or 48.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL30 polypeptide or fragment thereof. In various embodiments, the UL30 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL30 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL30 polypeptides known in the art include UL30 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL30 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 49, 50 and/or 51.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL52 polypeptide or fragment thereof. In various embodiments, the UL52 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL52 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL52 polypeptides known in the art include UL52 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL52 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 52, 53 and/or 54.

The US1 open reading frame encodes HSV gL (also referred to herein as a US1 polypeptide). In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a US1 polypeptide or fragment thereof. In various embodiments, the US1 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a US1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of known US1 polypeptides in the art include US1 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the US1 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 58, 59, 60 and/or 61.

The US7 open reading frame encodes HSV gI (also referred to herein as a US7 polypeptide). In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a US7 polypeptide or fragment thereof. In various embodiments, the US7 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a US7 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of US7 polypeptides known in the art include US7 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the US7 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 62, 63, 64 and/or 65.

The US8 open reading frame encodes HSV gE (also referred to herein as a US8 polypeptide). In some embodiments, the HSV antigen is (e.g., a T cell or B cell antigen of HSV) or comprises a US8 polypeptide or fragment thereof. In various embodiments, the US8 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a US8 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of known US8 polypeptides in the art include US8 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the US8 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 66, 67, 68 and/or 69.

The UL22 open reading frame encodes HSV gH (also referred to herein as UL22 polypeptide). In some embodiments, the HSV antigen is (e.g., a T cell or B cell antigen of HSV) or comprises a UL22 polypeptide or fragment thereof. In various embodiments, the UL22 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to a UL22 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL22 polypeptides known in the art include UL22 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL22 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 70, 71, 72 and/or 73.

In some embodiments, the HSV antigen (e.g., a T cell or B cell antigen of HSV) is or includes a UL54 polypeptide or fragment thereof. In various embodiments, the UL54 polypeptide or fragment thereof has at least 80% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to the UL54 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL54 polypeptides known in the art include UL54 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL54 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 55, 56 and/or 57.

In certain embodiments, an HSV antigen construct may include and/or encode one or more HSV antigens, including one or more T cell antigens of the HSV of the present disclosure (e.g., CD4 and/or CD 8T cell antigens) and one or more HSV antigens that are not T cell antigens of the present disclosure. In certain embodiments, the HSV antigen construct may include and/or encode one or more HSV antigens, including one or more B cell antigens of the HSV of the present disclosure and one or more HSV antigens that are not B cell antigens of the present disclosure. In certain embodiments, an HSV antigen construct may include and/or encode one or more HSV antigens, including one or more T cell antigens of the HSV of the present disclosure and one or more HSV antigens that are B cell antigens of HSV (e.g., antigens that are or include B cell epitopes disclosed herein or otherwise known in the art). In certain embodiments, the HSV antigen construct may include and/or encode one or more HSV antigens, including one or more T cell antigens of the HSV of the present disclosure and one or more HSV antigens selected from the group consisting of HSV glycoproteins or fragments thereof. In certain embodiments, the HSV antigen construct may include and/or encode one or more HSV antigens, including one or more T cell antigens of the HSV of the present disclosure and one or more HSV antigens selected from the group consisting of an HSV gD protein or antigenic fragment thereof, an HSV gB protein or antigenic fragment thereof, an HSV gE protein or antigenic fragment thereof, an HSV gL protein or antigenic fragment thereof, an HSV gH protein or antigenic fragment thereof, an HSV gL protein or antigenic fragment thereof, an HSV ICP4 protein or antigenic fragment thereof, or an ICP8 protein or antigenic fragment thereof.

In various embodiments, the HSV antigen construct may be present in a composition for delivering the HSV antigen construct to a subject. In various embodiments, the HSV antigen construct may be present in a composition for delivering one or more HSV antigens and/or epitopes to a subject. In various embodiments, the HSV antigen construct may be or include an RNA molecule encoding one or more antigens and/or epitopes.

Compositions for delivery of HSV antigen constructs and/or HSV antigen constructs may advantageously include, in certain embodiments, for example, one or more B cell antigens of HSV and one or more T cell antigens of HSV (e.g., CD4 and/or CD 8T cell antigens). Without wishing to be bound by any particular scientific theory, and without implying that other embodiments are also advantageous, the combination of B cell antigens and T cell antigens may be advantageous in promoting immune system protection against HSV at multiple lifecycle points, including, but not limited to, before and after cell entry.

Among other things, the present disclosure provides insight that many existing strategies for developing pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) for the treatment and/or prevention of viral infections have focused primarily or even almost exclusively on developing neutralizing antibodies that target surface glycoproteins. The present disclosure identifies issues with such strategies, including, for example, that they may not be aware of the value or even criticality of ensuring that the induced immune response includes significant T cell activity (in some embodiments CD 4T cell activity, in some embodiments CD 8T cell activity, in some embodiments both of the foregoing).

Alternatively or additionally, the present disclosure provides insight that expression of HSV proteins (e.g., at a particular period of the HSV lifecycle and/or in a particular tissue or compartment of an infected subject) may improve vaccine effectiveness.

In some embodiments, the present disclosure provides techniques for identifying, selecting, and/or characterizing HSV protein sequences (e.g., HSV-1 protein sequences, HSV-2 protein sequences, or combinations thereof) and combinations thereof, which are particularly suitable for inclusion in a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) as described herein.

In some embodiments, the pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) comprises or delivers, for example, CD4 and CD8 antigens of one or more HSV proteins (e.g., HSV-1 protein, HSV-2 protein, or a combination thereof) in addition to one or more B cell antigens. Among other aspects, the present disclosure provides HSV antigen constructs (e.g., HSV-1 antigen constructs, HSV-2 antigen constructs, or combinations thereof) and compositions (e.g., pharmaceutical compositions, such as immunogenic compositions, such as vaccines) comprising and/or delivering antigen constructs that induce both neutralizing antibodies and T cells (e.g., CD4 and/or CD 8T cells). Such neutralizing antibodies and T cells (e.g., CD4 and/or CD 8T cells) can target, for example, HSV glycoproteins, and in some embodiments, one or more additional HSV proteins. In some embodiments, the present disclosure provides such constructs and compositions that induce particularly strong neutralizing antibody responses and/or particularly diverse T cell responses (e.g., targeting multiple T cell antigens).

In some embodiments, the present disclosure provides such constructs and compositions that induce robust B cell responses. In some embodiments, the B cell response comprises the generation of a diverse pool of specific antibodies.

In some embodiments, the present disclosure provides such constructs and compositions that induce T-cell and B-cell responses to HSV antigens and/or epitopes.

The present disclosure provides insight, for example, that constructs and compositions comprising RNA molecules as described herein (e.g., encoding one or more HSV (e.g., HSV-1 and/or HSV-2) antigens and/or epitopes) present a higher degree of antigen presentation to various immune system components and/or pathways. In some embodiments, administration of such constructs or compositions may induce T cell and/or B cell responses. The present disclosure provides insight that, for example, in some embodiments of inducing T cell and B cell responses in a subject, the subject may have a more sustained, long-term immune response. Such an immune response may be beneficial, for example, for preventing reactivation of HSV (e.g., HSV-1 and/or HSV-2) in a single administration, which may improve vaccination rate and subject compliance as compared to currently available vaccines that require administration every few years. In some embodiments, constructs and compositions comprising RNA molecules as described herein (e.g., encoding one or more HSV (e.g., HSV-1, HSV-2, or a combination thereof) antigens and/or epitopes) may provide more varied protection (e.g., protection against HSV (e.g., HSV-1 and/or HSV-2) variants) because the constructs and compositions may induce a variety of immune system responses without wishing to be bound by any particular theory.

The present disclosure also provides insight that by administering constructs and compositions encoding HSV (e.g., HSV-1 and/or HSV-2) antigens and/or epitopes, the constructs and compositions described herein avoid administering HSV (e.g., HSV-1 and/or HSV-2) virions that may infect a subject, enter a latent phase, and reactivate to cause a disease.

Still further, the present disclosure provides insight (and also identifies the root cause of problems in certain prior art HSV vaccination strategies) that, in some embodiments, particularly effective pharmaceutical compositions (e.g., immunogenic compositions such as vaccines) alter one or more characteristics of the innate immune system. The present disclosure provides certain such compositions, including, for example, compositions comprising RNA constructs encoding HSV (e.g., HSV-1 and/or HSV-2) proteins (e.g., HSV antigens or HSV epitopes) as described herein.

Separately, in some embodiments, the present disclosure provides for the formation of specific pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines), including, for example, RNA pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) comprising specific elements and/or sequences useful for vaccination. +

The present disclosure provides various insights and techniques related to such HSV (e.g., HSV-1 and/or HSV-2) antigen constructs and vaccine (e.g., RNA vaccine) compositions.

As described herein, in many embodiments, provided compositions (e.g., pharmaceutical compositions, e.g., immunogenic compositions, e.g., vaccines) include RNA actives encoding one or more HSV (e.g., HSV-1 and/or HSV-2) polypeptides or antigenic fragments thereof; in some embodiments, such RNA actives are modified RNA forms in that their uridine residues are replaced with uridine analogs such as pseudouridine; alternatively or additionally, in some embodiments, such RNA actives include specific elements (e.g., caps, 5 'utrs, 3' utrs, poly-a tails, etc.) and/or features (e.g., codon optimization) that are identified, selected, characterized, and/or demonstrated to achieve significant (e.g., improved) translatability (e.g., in vitro) and/or expression (i.e., in a subject to whom they have been administered) of the encoded protein. Still further alternatively or additionally, in some embodiments, such RNA actives include specific elements and/or features that are identified, selected, characterized, and/or demonstrated to achieve significant RNA stability and/or efficient manufacture, particularly in large scale efficient manufacture (e.g., 0.1-10g, 10-500g, 500g-1kg, 750g-1.5kg; it will be appreciated by those skilled in the art that different products may be manufactured on different scales, e.g., depending on patient population size). In some embodiments, such RNA manufacturing scale may be in the range of about 0.01g/hr RNA to about 1g/hr RNA, 1g/hr RNA to about 100g/hr RNA, about 1g RNA/hr to about 20g RNA/hr, or about 100g RNA/hr to about 10,000g RNA/hr. In some embodiments, such RNA can be produced on a scale of tens or hundreds of milligrams to tens or hundreds of grams (or more) of RNA per batch. In some embodiments, such RNA production scale may allow batch sizes in the range of about 0.01g to about 500g RNA, about 0.01g to about 10g RNA, about 1g to about 10g RNA, about 10g to about 500g RNA, about 10g to about 300g RNA, about 10g to about 200g RNA, or about 30g to about 60g RNA.

Still further, in many embodiments, provided compositions (e.g., pharmaceutical compositions, e.g., immunogenic compositions, e.g., vaccines) comprising RNA actives are prepared, formulated, and/or utilized in a particular LNP composition, as described herein.

Among other things, the present disclosure provides techniques for rapidly developing pharmaceutical compositions (e.g., immunogenic compositions, such as HSV vaccines) for delivering specific HSV (e.g., HSV-1 and/or HSV-2) antigen constructs to a subject.

In addition, the present disclosure provides, for example, nucleic acid constructs encoding HSV (e.g., HSV-1 and/or HSV-2) antigens as described herein, expressed HSV (e.g., HSV-1 and/or HSV-2) proteins and various methods of production and/or use associated therewith, as well as compositions developed therewith and methods associated therewith.

For example, the present disclosure provides techniques for preventing, characterizing, treating, and/or monitoring HSV (e.g., HSV-1 and/or HSV-2) outbreaks and/or infections, including the various nucleic acid constructs and encoding proteins as well as agents (e.g., antibodies) that bind to such proteins, and compositions comprising and/or delivering them, as described previously.

In some aspects, provided herein are techniques (e.g., compositions and methods) for enhancing, inducing, promoting, enhancing, and/or improving an immune response against HSV (e.g., HSV-1 and/or HSV-2) or a component thereof (e.g., a protein or a fragment thereof). In some embodiments, the techniques provided herein aim to enhance, induce, promote, enhance, and/or improve immune memory against HSV (e.g., HSV-1 and/or HSV-2) or a component thereof (e.g., a protein or fragment thereof). In some embodiments, the techniques described herein are intended for use as immunopotentiators for primary vaccines, such as vaccines against antigens and/or epitopes of HSV (e.g., HSV-1 and/or HSV-2). In some embodiments, the compositions of the present disclosure comprise one or more polynucleotide constructs (e.g., one or more string constructs) encoding one or more antigens from HSV (e.g., HSV-1 and/or HSV-2). In some embodiments, the present disclosure provides vaccines or other compositions comprising nucleic acids encoding such HSV (e.g., HSV-1 and/or HSV-2) antigens; those of skill in the art will appreciate from the context that when referring to a particular polynucleotide (e.g., DNA or RNA) "encodes" such an antigen, it actually refers to the coding strand or complement thereof.

The present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) and related techniques (e.g., methods) for delivering a particular HSV antigen construct to a subject (e.g., a patient). In some embodiments, the present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) and related techniques (e.g., methods) for delivering a particular HSV-1 antigen construct to a subject (e.g., a patient). In some embodiments, the present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) and related techniques (e.g., methods) for delivering a particular HSV-2 antigen construct to a subject (e.g., a patient). In some embodiments, the present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) and related techniques (e.g., methods) for delivering specific HSV-1 and HSV-2 antigen constructs to a subject (e.g., a patient).

The present disclosure further provides the recognition that some HSV antigens are common to both HSV-1 and HSV-2. The present disclosure also provides the recognition that some HSV antigens include sequences that are conserved between HSV-1 and HSV-2. Furthermore, the present disclosure recognizes that some HSV-1 antigens have, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with comparable HSV-2 antigens.

In some embodiments, the present disclosure provides certain HSV antigen constructs that are particularly useful for effective vaccination. In some embodiments, the HSV antigen construct is an HSV-1 antigen construct, an HSV-2 antigen construct, or a combination thereof.

Antigens utilized in accordance with the present disclosure are or include HSV (e.g., HSV-1 and/or HSV-2) components (e.g., antigenic fragments thereof, including epitopes that may comprise non-amino acids, such as carbohydrate moieties) that induce an immune response when administered to humans (or other animals susceptible to infection by HSV (e.g., HSV-1 and/or HSV-2), such as rodents and non-human primates).

In many embodiments, as described herein, antigens utilized in the provided pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) include B-cell and T-cell antigens and/or epitopes. In some particular embodiments, the antigen delivered comprises B cell and T cell (e.g., CD4 and/or CD 8T cell) antigens and/or epitopes, optionally together in a single antigen polypeptide. In some embodiments, the antigen utilized in the provided pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) comprises a T cell antigen and/or epitope. In some embodiments, antigens utilized in the provided pharmaceutical compositions (e.g., immunogenic compositions such as vaccines) together include B-cell, CD 4T-cell, and CD 8T-cell epitopes. Indeed, in some embodiments, the present disclosure defines epitopes that are particularly useful for inclusion in HSV (e.g., HSV-1 and/or HSV-2) vaccines, and/or provides antigens that include them.

Exemplary antigens and/or epitopes for use in the compositions described herein include, for example, those provided in tables 3-5 herein, and antigenic fragments thereof. In some embodiments, the exemplary antigens and/or fragments and/or epitopes thereof disclosed in tables 3-5 may be used in the compositions described herein.

In some embodiments, provided pharmaceutical compositions (e.g., immunogenic compositions, e.g., HSV (e.g., HSV-1 and/or HSV-2) vaccines) comprise or deliver (e.g., cause expression in a recipient organism, e.g., by administering a nucleic acid construct, such as an RNA construct, encoding the same as described herein) an antigen that is or comprises one or more epitopes (e.g., one or more B-cell and/or one or more T-cell antigens and/or epitopes) of an HSV (e.g., HSV-1 and/or HSV-2) protein. In some embodiments, the pharmaceutical compositions described herein induce an immune response associated with an effective against HSV (e.g., by targeting HSV-1 protein, HSV-2 protein, or a combination thereof).

In some embodiments, provided pharmaceutical compositions (e.g., immunogenic compositions, such as HSV (e.g., HSV-1 and/or HSV-2) vaccines) comprise or deliver antigens that are or comprise full length HSV (e.g., HSV-1 and/or HSV-2) proteins. In some embodiments, provided pharmaceutical compositions (e.g., immunogenic compositions, such as HSV (e.g., HSV-1 and/or HSV-2) vaccines) comprise or deliver antigens that are or comprise fragments of HSV (e.g., HSV-1 and/or HSV-2) proteins that are less than full-length HSV (e.g., HSV-1 and/or HSV-2) proteins. In some embodiments, provided pharmaceutical compositions (e.g., immunogenic compositions, such as HSV (e.g., HSV-1 and/or HSV-2) vaccines) comprise or deliver chimeric polypeptides that are or comprise a portion or all of an HSV (e.g., HSV-1 and/or HSV-2) protein and one or more heterologous polypeptide elements.

In some embodiments, the antigen included in and/or delivered by the provided pharmaceutical composition (e.g., an immunogenic composition, such as an HSV (e.g., HSV-1 and/or HSV-2) vaccine) is or comprises one or more peptide fragments of an HSV (e.g., HSV-1 and/or HSV-2) antigen; in some such embodiments, each of the one or more peptide fragments includes at least one epitope (e.g., one or more B cell epitopes and/or one or more T cell epitopes), e.g., can be predicted, selected, evaluated, and/or characterized as described herein.

In some embodiments, the antigen included in and/or delivered by the provided pharmaceutical composition (e.g., an immunogenic composition, such as an HSV (e.g., HSV-1 and/or HSV-2) vaccine) is or comprises a plurality of peptide fragments of one or more HSV (e.g., HSV-1 and/or HSV-2) antigens. In some embodiments, a single polypeptide antigen may comprise a plurality of such fragments, e.g., as a series of antigens as described herein or fragments thereof (e.g., because a single polypeptide comprises a plurality of amino acid sequences derived from different HSV antigens or fragments thereof, optionally separated by or otherwise associated with an amino acid linker or other intervening or terminal amino acid sequence). In some embodiments, a single RNA antigen construct may comprise multiple sequences encoding HSV antigens, e.g., as a series of antigens encoding sequences as described herein (e.g., because a single RNA molecule comprises multiple nucleic acid sequences encoding different HSV antigens or fragments thereof, optionally separated by or otherwise associated with a nucleic acid linker or other intervening or terminal nucleic acid sequence).

In some embodiments, one or more HSV (e.g., HSV-1 and/or HSV-2) antigens or antigenic fragments thereof may be linked to one or more sequences to which they are linked in nature. In some such embodiments, such sequences may be or comprise one or more heterologous elements (e.g., one or more elements not naturally found in the associated HSV (e.g., HSV-1 and/or HSV-2), such as polypeptides or antigenic fragments thereof not naturally found directly linked to the associated HSV (e.g., HSV-1 and/or HSV-2) antigen). For example, in some embodiments, an antigenic peptide provided and/or utilized in accordance with the present disclosure may include one or more linker elements and/or one or more membrane associated elements and/or one or more secretion elements, and the like. In some embodiments, an antigenic polypeptide may comprise a plurality of HSV (e.g., HSV-1 and/or HSV-2) protein fragments or epitopes separated from one another by linkers.

In some embodiments, an HSV (e.g., HSV-1 and/or HSV-2) polypeptide or fragment or epitope thereof utilized in a construct as described herein (or encoded by a polyribonucleotide as described herein) may include one or more sequence alterations relative to a particular reference HSV (e.g., HSV-1 and/or HSV-2) polypeptide or fragment or epitope thereof. For example, in some embodiments, the antigens utilized may include one or more sequence variations seen or predicted to occur in circulating strains, e.g., based on an assessment of sequence conservation and/or evolution of HSV (e.g., HSV-1 and/or HSV-2) polypeptides over time and/or across strains. Alternatively or additionally, in some embodiments, the antigen utilized may include one or more sequence variations selected, for example, to affect the stability, folding, processing, and/or display of the antigen or any epitope thereof.

In some embodiments, an HSV (e.g., HSV-1 and/or HSV-2) polypeptide or fragment or epitope thereof utilized in an antigen as described herein exhibits at least 70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％ or 100% amino acid sequence identity to a related corresponding reference (e.g., wild-type) polypeptide, fragment or epitope. In some embodiments, an HSV (e.g., HSV-1 and/or HSV-2) polypeptide or fragment or epitope thereof utilized in an antigen as described herein exhibits at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology (i.e., identity or conservative substitutions as understood in the art), amino acid sequence identity to a related corresponding reference (e.g., wild-type) protein, fragment or epitope. Furthermore, in some embodiments, an HSV (e.g., HSV-1 and/or HSV-2) polypeptide or fragment or epitope thereof utilized in an antigen as described herein shares conserved amino acid residues (e.g., at respective positions) with a related respective reference (e.g., wild-type) polypeptide, fragment or epitope. One skilled in the art will appreciate that, in general, a lower percentage of identity or homology can be tolerated for shorter peptides, as a single change will have a greater effect on the percentage of identity or homology by definition when considered with respect to a smaller number of residues. For example, one of skill in the art will appreciate that for sequences longer than about 20 amino acids, the percent identity or homology is typically greater than about 80%; for sequences longer than about 50 amino acids, the percent identity or homology is typically greater than about 90%.

In some embodiments, the degree of conservation can be assessed by considering the physicochemical differences between the two amino acids, as described, for example, in WO2014/180569, which is incorporated herein by reference in its entirety. It is well known in molecular evolution that frequently exchanged amino acids may have chemical and physical similarities, whereas rarely exchanged amino acids may have different physicochemical properties. The likelihood of such a substitution occurring in nature can be measured by a logarithmic probability matrix as compared to the likelihood of a given substitution occurring by chance. The pattern observed in the natural selection applied log-probability matrix "reflects the similarity in function of amino acid residues in the three-dimensional conformation of the protein in their weak interactions with each other" (see Dayhoff et al Atlas of protein sequence and structure 5:345, 1978180569, incorporated herein by reference in its entirety). In some embodiments, an evolution-based logarithmic probability matrix (which may be referred to as a "T-score") may be used to reflect the extent to which sequence variations may affect T-cell recognition. Substitutions with a positive T-score (i.e., log probability) are likely to occur in nature and thus correspond to two amino acids with similar physicochemical properties. Substitutions with a positive T-score are less likely to alter immunogenicity. In contrast, substitutions with a negative T-score reflect substitutions that are unlikely to occur in nature and thus correspond to two amino acids with significantly different physicochemical properties. Such substitutions are more likely to alter immunogenicity. In some embodiments, substitution of a negative T-score within the sequence, even if it is otherwise highly conserved, may indicate that it is relatively less useful in vaccine antigens as described herein.

In some embodiments, the antigen utilized induces an immune response that targets HSV envelope glycoproteins. In some embodiments, one or more antigens induce an immune response that targets HSV envelope glycoproteins. In some embodiments, the one or more antigens comprise one or more HSV protein sequences (e.g., a conserved sequence and/or a sequence comprising one or more B cell epitopes and/or one or more CD4 epitopes and/or one or more CD8 epitopes) of an antigen or epitope of an HSV envelope glycoprotein. In some embodiments, the one or more antigens are or comprise HSV gD proteins or fragments or epitopes thereof. In some embodiments, the one or more antigens are or comprise HSV gB proteins or fragments or epitopes thereof. In some embodiments, the one or more antigens are or comprise HSV gE protein or a fragment or epitope thereof. In some embodiments, the one or more antigens are or comprise HSV gG proteins or fragments or epitopes thereof. In some embodiments, the one or more antigens are or comprise HSV gI proteins or fragments or epitopes thereof. In some embodiments, the one or more antigens are or comprise HSV gE protein or a fragment or epitope thereof. In some embodiments, the one or more antigens are or comprise HSV gH protein or a fragment or epitope thereof. In some embodiments, the one or more antigens are or comprise HSV gL proteins or fragments or epitopes thereof. In some embodiments, the one or more antigens are or comprise HSV ICP4 proteins or fragments or epitopes thereof. In some embodiments, the one or more antigens are or comprise ICP8 polypeptides, fragments or epitopes thereof.

In various embodiments, the HSV antigen construct comprises and/or encodes a plurality of HSV antigens provided in table 3 (e.g., a plurality of HSV antigens that are or comprise one or more T cell antigens of HSV) or fragments thereof. In certain embodiments, the HSV antigen construct may include and/or encode at least one HSV antigen or fragment thereof provided in table 3. In certain embodiments, the HSV antigen construct may include and/or encode at least one HSV antigen or fragment thereof provided in table 4. In certain embodiments, the HSV antigen construct may include and/or encode at least one HSV antigen or fragment thereof provided in table 5.

In certain embodiments, the HSV antigen construct may include and/or encode at least one T cell antigen of HSV provided in table 3, or a fragment thereof. In certain embodiments, the HSV antigen construct may include and/or encode at least one T cell antigen of HSV provided in table 4 or a fragment thereof. In certain embodiments, the HSV antigen construct may include and/or encode at least one T cell antigen of HSV provided in table 5, or a fragment thereof.

In certain embodiments, an HSV antigen construct may include and/or encode a plurality (e.g., 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of HSV antigens provided in table 3, or fragments thereof. In certain embodiments, an HSV antigen construct may include and/or encode a plurality (e.g., 1,2,3, 4, 5, 6, 7, 8, 9, or 10) of HSV antigens provided in table 4, or fragments thereof. In certain embodiments, an HSV antigen construct may include and/or encode a plurality (e.g., 1,2,3, 4, 5, 6, 7, 8, or 9) of HSV antigens provided in table 5, or fragments thereof.

In certain embodiments, the HSV antigen construct may include and/or encode a T cell antigen or fragment thereof selected from a plurality (e.g., 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) HSV antigens provided in table 3. In certain embodiments, an HSV antigen construct may include and/or encode a T cell antigen or fragment thereof selected from a plurality (e.g., 1,2,3,4, 5, 6, 7, 8, 9, or 10) HSVs of the antigens provided in table 4. In certain embodiments, an HSV antigen construct may include and/or encode a T cell antigen or fragment thereof selected from a plurality (e.g., 1,2,3,4, 5, 6, 7, 8, or 9) HSV of antigens provided in table 5.

In some embodiments, antigens utilized in accordance with the present disclosure include HSV (e.g., HSV-1 and/or HSV-2) protein sequences identified and/or characterized by one or more of:

1) HLA-I or HLA-II binding (e.g., to HLA alleles present in a relevant population)

2) HLA ligand histology data, optionally confirmed by mass spectrometry

3) Relatively high expression

4) Sequence conservation

5) Surface exposure

6) Serum reactivity

7) Immunogenicity (e.g., the presence of one or more B cell and/or T cell antigens and/or epitopes; evidence of the ability to induce sterility protection in model systems including, for example, humans, non-human primates, and/or mice).

8) Absence of sequences overlapping the human proteome

In some embodiments, such features are evaluated experimentally or computationally. In some embodiments, such features are assessed by review of published reports.

For example, in some embodiments, HLA-I and/or HLA-II binding is experimentally assessed; in some embodiments, it is predicted.

In some embodiments, predicted HLA-I or HLA-II binding is assessed using an algorithm, such as neonmhc 1 and/or neonmhc2, which predicts and/or characterizes the likelihood of MHC class I and MHC class II binding, respectively. Alternatively or additionally, in some embodiments, the MHC-peptide presentation predictive algorithm or MHC-peptide presentation predictor is or includes NETMHCPAN or NETMHCIIPAN. In some embodiments, MHC-peptide presentation predictions and/or characterizations may be made using a hidden markov model approach. In some embodiments, a peptide prediction model MARIA may be utilized. In some embodiments, NETMHCPAN is not utilized to predict or characterize the likelihood of MHC binding of a peptide as described herein. In some embodiments, a peptide prediction model MARIA may be utilized. In some embodiments, NETMHCIIPAN is not utilized to predict or characterize the likelihood of MHC binding of a peptide as described herein. In some embodiments, neither NETMHCPAN nor NETMHCIIPAN are utilized to predict or characterize the likelihood of MHC binding of a peptide as described herein. In some embodiments, the MHC-peptide presentation predictive algorithm or MHC-peptide presentation predictive factor is or includes(Real-time epitope calculation in oncology) that provides high quality predictions of MHC-peptide presentation based on expression, processing and binding capacity. See, e.g., abelin et al, immunity 21:315, 2017; abelin et al, immunity 15:766, 2019.

In some embodiments, HLA binding and/or ligand histology assessment will take into account the geographic region of the subject to be immunized. For example, in some embodiments, HLA allele diversity will be considered. In some embodiments, the antigen included in a provided pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) will be or comprise a peptide (e.g., an epitope) that when considered together is expected or determined to bind to a significant percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more) of HLA alleles that are expected or known to be present in the relevant region or population. In some embodiments, the antigen included in a provided pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) will be or comprise a peptide that, when considered together, is expected or determined to bind to the most common (e.g., 1,2, 3,4, 5,6, 7, 8, 9, or 10 most common, or at least 1,2, 3,4, or5 most common, etc.) HLA alleles that are expected or known to be present in the relevant region or population.

In some embodiments, the expression level is determined experimentally (e.g., in a model system or in an infected person). In some embodiments, the expression level is a reported level (e.g., in a published or submitted report). In some embodiments, the expression level is assessed with RNA (e.g., via RNASeq). In some embodiments (and often preferred), expression levels are assessed as proteins.

In some embodiments, sequence conservation is assessed, for example, using publicly available sequence assessment software (e.g., multiplex sequence alignment programs MAFFT, clustal Omega, etc.). In some embodiments, sequence conservation is determined by consulting published resources (e.g., sequences). In some embodiments, sequence conservation includes consideration of the current or recently detected strain (e.g., in an active burst).

In some embodiments, surface exposure is assessed by reference to publicly available databases and/or software.

In some embodiments, serum reactivity is assessed by contacting a serum sample from an infected individual with a polypeptide comprising a sequence of interest (e.g., can be displayed via, e.g., phage display or peptide array, etc., see, e.g., whittemore et al ,"A General Me thod to Discover Epitopes from Sera"PlosOne,2016;https://doi.org/10.1371/journal.pone.0157462). in some embodiments, by review of the database data reported in the literature and/or indicative of serum recognition sequences).

In some embodiments, the assessment of the presence of an immunoreactivity and/or epitope may be or include consulting an Immune Epitope Database (IEDB), which one of skill in the art would know is a free resource funded by NIAID, which enrolls in the classification of experimental data regarding antibodies and T cell epitopes (see IEDB. Org).

In some embodiments, the antigens utilized in accordance with the present disclosure are characterized by dendritic cell presentation, which in turn may be indicative of HLA binding and/or immunogenicity.

In some embodiments, the antigen utilized in accordance with the present disclosure is or comprises a sequence (e.g., epitope, fragment, complete protein) of an HSV protein found in the HSV envelope. In some embodiments, the antigen utilized in accordance with the present disclosure is or comprises a sequence (e.g., epitope, fragment, complete protein) of an HSV protein found in an HSV envelope.

Among other things, the present disclosure provides insight that in some embodiments, it may be desirable to include two or more different epitopes, optionally from two or more different HSV (e.g., HSV-1 and/or HSV-2) proteins, in the composition of a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) useful for treating HSV.

Table 7: exemplary antigen fragments

1. Exemplary antigenic forms

In some embodiments, the antigen utilized as described herein is or comprises a full-length viral protein. In some embodiments, the antigen utilized as described herein is or comprises a fragment or domain of a viral polypeptide or an antigenic fragment thereof. In some embodiments, the antigen utilized as described herein is a membrane-based antigen (e.g., an antigenic fragment thereof fused to a membrane-associated portion, e.g., a transmembrane portion). In some embodiments, provided pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) comprise or deliver antigen sequences that are or comprise one or more antibody epitopes and/or one or more CD 4T cells and/or CD 8T cell epitopes.

In some embodiments, the antigen utilized as described herein includes one or more variant sequences relative to a related reference antigen. For example, in some embodiments, the protease cleavage site is removed or blocked; alternatively or additionally, in some embodiments, terminally truncated antigens are utilized.

In some embodiments, the antigen utilized as described herein includes a multimerization element (e.g., a heteromultimerization element).

In some embodiments, the antigen utilized as described herein includes a membrane-associated element (e.g., a heterologous membrane-associated element), such as a transmembrane domain.

In some embodiments, the antigen utilized as described herein includes a secretion signal (e.g., a heterologous secretion signal).

In some embodiments, the sequences utilized may be longer than the viral proteins found in nature (and thus may include more epitopes, for example).

In some embodiments, the sequences utilized may be from different strains or strains (e.g., may be circulating in and/or otherwise associated with a population to which a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) is administered).

In some embodiments, an antigen utilized as described herein can include a plurality of epitopes (e.g., B cell and/or T cell antigens and/or epitopes) arranged in a non-native configuration (e.g., in a string construct as described herein). In some embodiments, an antigen utilized as described herein can include multiple epitopes that are predicted or demonstrated to bind to HLA alleles that reflect a population of components of a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) to be administered as described herein.

In some embodiments, provided pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) can comprise or deliver a variety of antigens. One or more antigens comprising B cell epitopes and one or more antigens comprising T cell epitopes. In some embodiments, provided pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) can comprise or deliver one antigen comprising both B cells and CD4 epitopes and a separate antigen comprising a CD8 epitope.

As described herein, in some embodiments, the provided technology involves administering multiple antigens to the same subject. In some embodiments, multiple antigens are administered simultaneously (e.g., in a single dose). In some embodiments, different antigens may be administered at different times (e.g., at different doses-e.g., a primary dose versus a booster dose). In some embodiments, multiple antigens are administered via the same composition.

For clarity, a single "antigen" polypeptide may include multiple "epitopes," which in turn may be linked or not linked to each other in nature. For example, a single chain construct antigen includes multiple epitopes that may be from different portions of the same HSV (e.g., HSV-1 and/or HSV-2) protein, and/or from different HSV (e.g., HSV-1 and/or HSV-2) proteins linked together as described herein in a single polypeptide.

Thus, a single pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) as described herein may comprise or deliver (e.g., because the pharmaceutical composition comprises a nucleic acid, such as RNA, encoding an antigen and expressed upon administration) itself may comprise a single antigen of multiple epitopes (in their natural arrangement relative to each other or in an engineered or constructed arrangement as described herein), or may comprise or deliver multiple antigens, each of which may similarly be or comprise a single epitope or multiple epitopes (in their natural arrangement relative to each other or in an engineered or constructed arrangement as described herein). Still further, a single pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) may, for example, comprise multiple different nucleic acids (e.g., RNAs) each encoding a different antigen, or in some embodiments, may comprise a single nucleic acid encoding (and expressing) multiple antigens. Still further, in some embodiments, a single pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) comprising a plurality of different nucleic acids (e.g., RNAs) encoding antigens may be prepared by: the RNA is mixed and the mixture is then incorporated into the LNP, or by formulating the RNA alone into the LNP and then mixing the LNP. In some embodiments, the mixture (whether of pre-LNP preparation RNA or LNP) can include the relevant RNA (e.g., to achieve the desired relative presentation of antigen or epitope) at a ratio of 1:1 or at other ratios that may be preferred in the subject to whom the composition is administered.

In some particular embodiments, a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) can comprise or deliver a combination ：UL1、UL21、UL27、UL29、UL39、UL40、UL46、UL47、UL48、UL49、RS1、RL2、UL5、UL9、UL19、UL25、UL30、UL52、US1、US7、US8、UL22 comprising a polypeptide or fragment thereof encoded by all or a portion of and/or UL54 or fragment thereof.

In some embodiments, provided compositions include or deliver an HSV (e.g., HSV-1 and/or HSV-2) envelope glycoprotein antigen (e.g., full length HSV (e.g., HSV-1 and/or HSV-2) envelope glycoprotein, a fragment thereof, or one or more epitopes thereof, e.g., in a string construct). In some embodiments, provided compositions include or deliver such HSV (e.g., HSV-1 and/or HSV-2) envelope glycoprotein antigens and one or more B cell targets (e.g., epitopes) that may be, for example, or comprise, one or more other HSV (e.g., HSV-1 and/or HSV-2) proteins (or fragments or epitopes thereof). In some embodiments, such B cell targets are or comprise HSV (e.g., HSV-1 and/or HSV-2) proteins (or fragments or epitopes thereof) that are predicted or known to induce a B cell response in an infected human. For example, in some embodiments, the B cell target is or comprises an HSV (e.g., HSV-1 and/or HSV-2) protein (or a fragment thereof or a B cell epitope) against which serum from an infected individual is reactive. In some particular embodiments, the B cell target is or comprises an HSV (e.g., HSV-1 and/or HSV-2) envelope glycoprotein or other related HSV (e.g., HSV-1 and/or HSV-2) protein or fragment or epitope thereof.

In some embodiments, provided compositions comprise or deliver a string construct antigen comprising a plurality of T cell epitopes optionally from more than one HSV (e.g., HSV-1 and/or HSV-2) protein. In some such embodiments, provided compositions further comprise or deliver one or more B cell targets. Alternatively or additionally, in some embodiments, the string construct antigen so utilized includes an HSV (e.g., HSV-1 and/or HSV-2) sequence (e.g., one or more fragments or epitopes, such as a T cell epitope and/or a B cell epitope, but in some embodiments specifically a T cell epitope).

In some embodiments, the string construct antigen includes both B cell epitopes and T cell epitopes (optionally from the same HSV (e.g., HSV-1 and/or HSV-2) protein or from different HSV (e.g., HSV-1 and/or HSV-2) proteins).

In some embodiments, different antigens may be delivered by administering different compositions, which in turn may be administered simultaneously (e.g., as a mixture, or otherwise substantially simultaneously) in some embodiments, and in some embodiments, at different times. By way of example only, in some embodiments, a particular antigen or antigens may be delivered via an initial dose of a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine), and one or more other antigens may be delivered via one or more booster doses.

2. Exemplary Multi-epitope antigen

In some embodiments, the antigen utilized (i.e., included and/or otherwise delivered) by the pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) described herein comprises, for example, a single HSV (e.g., HSV-1 and/or HSV-2) protein or multiple epitopes of multiple proteins.

In some embodiments, an antigen may comprise two or more epitopes from the same HSV (e.g., HSV-1 and/or HSV-2) protein and be in their native configuration (e.g., in fragments if the protein of interest) relative to each other. However, in some embodiments, an antigen may comprise at least two epitopes configured in a non-native relationship relative to each other (e.g., included in a string construct as described herein).

Among other things, the present disclosure provides insight that string construct antigens may be particularly useful or effective for vaccination against HSV (e.g., HSV-1 and/or HSV-2) infection. Without wishing to be bound by any particular theory, the present disclosure suggests that the ability of ligation to predict or determine a single epitope that has a particular property-e.g., binding to an associated HLA allele, expressed at the relevant time of infection, representing a particularly conserved sequence, potentially spanning multiple different HSV (e.g., HSV-1 and/or HSV-2) proteins-may prove uniquely beneficial or indeed critical for effective vaccination against HSV (e.g., HSV-1 and/or HSV-2), with more traditional vaccination methods heretofore providing only limited protection.

In some embodiments, the multi-epitope antigen (e.g., a string construct antigen or a multi-epitope antigen) can be administered as a polypeptide and/or as a collection of peptides. Alternatively or additionally, the multi-epitope antigen may be administered as a preparation of cells that contain (e.g., express) the antigen. However, the present disclosure further provides the insight that in some embodiments, delivery by administration of nucleic acids (and in particular RNAs) encoding multi-epitope antigens may be particularly useful and/or effective.

As noted elsewhere herein, experience with SARS-CoV-2 has demonstrated that RNA administration may be a particularly effective way of delivering infectious disease antigens. Furthermore, the present disclosure provides insight that various features of nucleic acid forms, including, for example, their flexibility and adaptability to rapid design and modification, including integration of various insights (e.g., bioinformatic inputs, etc.), make them particularly attractive for use in HSV (e.g., HSV-1 and/or HSV-2) vaccines. Among other things, the present disclosure provides the insight that, in some embodiments, administration of RNA encoding a string construct antigen as described herein may be a particularly desirable and/or effective method of immunizing against HSV (e.g., HSV-1 and/or HSV-2) infection.

In some embodiments, a "string" polynucleotide sequence encodes a plurality of antigens and/or epitopes in tandem. In some embodiments, the strings encode about 2 to about 100, about 2 to about 75, about 2 to about 50, about 2 to about 25, about 2 to about 20, about 2 to about 15, about 2 to about 10, or about 2 to about 5 antigens and/or epitopes. In some embodiments, the strings encode about 5 to about 100, about 5 to about 75, about 5 to about 50, about 5 to about 25, about 5 to about 20, about 5 to about 15, or about 5 to about 10 antigens and/or epitopes. In some embodiments, a "string" polynucleotide sequence encodes multiple epitopes in tandem. In some embodiments, the strings encode about 2 to about 1000 or about 2 to about 10,000 antigens and/or epitopes. In some embodiments, about 2-5,000 antigens and/or epitopes are encoded in one polynucleotide string. In some embodiments, about 2-4,000 antigens and/or epitopes are encoded in one polynucleotide string. In some embodiments, about 2-3,000 antigens and/or epitopes are encoded in one polynucleotide string. In some embodiments, about 2-2,000 antigens and/or epitopes are encoded in one polynucleotide string. In some embodiments, about 2-1,000 antigens and/or epitopes are encoded in one polynucleotide string. In some embodiments, about 10-500 antigens and/or epitopes are encoded in one polynucleotide string. In some embodiments, about 10-200 antigens and/or epitopes are encoded in one polynucleotide string. In some embodiments, about 20-100 antigens and/or epitopes are encoded in one polynucleotide string.

In some embodiments, the epitope encoded by the string construct comprises an epitope predicted by HLA binding and presentation prediction software to be highly likely to be presented by HLA encoded proteins to T cells to elicit an immune response. In some embodiments, the epitope predicted to be most likely presented by the HLA-encoded protein is selected from any one of the proteins or peptides described in tables 3-5. In some embodiments, for example, at relevant times during the HSV (e.g., HSV-1 and/or HSV-2) lifecycle, the epitopes in the string construct include membrane-associated or otherwise accessible epitopes.

In some embodiments, the antigen utilized according to the present disclosure is or comprises UL1、UL21、UL27、UL29、UL39、UL40、UL46、UL47、UL48、UL49、RS1、RL2、UL5、UL9、UL19、UL25、UL30、UL52、US1、US7、US8、UL22 and/or UL54 or a fragment thereof and variants thereof and/or a fragment or epitope of any of the foregoing, as well as combinations of any of the foregoing. In some embodiments, the antigen utilized according to the present disclosure is or comprises an HSV protein, is or comprises an HSV envelope protein, an HSV membrane protein, and variants thereof, and/or fragments or epitopes of any of the foregoing, and combinations of any of the foregoing. In some embodiments, the string construct may comprise a number of epitopes from 2, 3, 4, or more HSV proteins. In some embodiments, the string construct comprises one or more features described herein (including examples and tables). In some embodiments, the string construct comprises or is encoded by a sequence as described in tables 3-5.

Alternatively or additionally, in some embodiments, one or more string constructs may include one or more other epitopes (e.g., as may be predicted or demonstrated, for example, in the literature). In some embodiments, the string construct may comprise sequences encoding features such as linkers and cleavage sites (e.g., auto-cleavage sites, e.g., T2A or P2A sequences). In some embodiments, linkers rich in G and S residues may be used. In some embodiments, exemplary linkers may have the sequence GGGGSGGGGS (SEQ ID NO: 167) or GGSGGGGS GG (SEQ ID NO: 165).

In some embodiments, the string construct comprises two or more overlapping epitope sequences.

In some embodiments, the string construct comprises or is encoded by a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences in tables 3-5. As noted above, where the sequences being compared are longer than about 20 amino acids, the percent identity or homology is typically greater than about 80%; for sequences longer than about 50 amino acids, the percent identity or homology is typically greater than about 90%.

In some embodiments, the epitopes are arranged on the string to maximize the immunogenicity of the string, e.g., by maximizing recognition by the subject's HLA allele pool. In some embodiments, the same string encodes an epitope that can bind to and/or be predicted to bind to different HLA alleles. For example, as fully exemplified in the sequence listing, e.g., at least in tables 3-5, the string may encode an epitope comprising: (a) A first epitope that binds to or is predicted to bind to a first MHC peptide encoded by a first HLA allele; (b) A second epitope that binds to or is predicted to bind to a second MHC peptide encoded by a second HLA allele; (c) A third epitope that binds to or is predicted to bind to a third MHC peptide encoded by a third HLA allele-and further such epitopes may be added, for example in a string sequence as provided herein; wherein the first, second and third epitopes are epitopes from the same HSV (e.g., HSV-1 and/or HSV-2) protein or from different HSV (e.g., HSV-1 and/or HSV-2) proteins. In this way, epitope distribution encoded by a single string is maximized to hit presentation to T cells based on different MHC, thereby maximizing the likelihood of generating a desired immune response from a wider population of patients and the robustness of each patient's response.

In some embodiments, the epitopes included in the string construct are selected based on a high scoring prediction of HLA binding by a reliable predictive algorithm or system (e.g., a RECON predictive algorithm). In some embodiments, the present disclosure provides insight that particularly successful strings can be provided in the layout of epitopes encoded by the strings by selecting epitopes based on highly reliable and efficient predictive algorithms with or without non-epitope sequences or sequences flanking the epitopes, and that the immunogenicity of the strings is verified in ex vivo cell culture models or in animal models, particularly in the display of epitope-specific T cell responses in T cell induction following vaccination with the string constructs or polypeptides encoded by the string constructs. In some embodiments, validation may be from use in human patients, and T cells obtained from post-vaccinated patients were found to exhibit epitope-specific potent and durable T cell responses. In some embodiments, the efficiency of the string as a vaccine is affected by its design, which is dependent in part on the strength of the bioinformatics information used in the design's discretion to perform, the reliability of the MHC presentation predictive model, the efficiency of epitope processing when the string vaccine is expressed in cells, and the like.

In some embodiments, a multi-epitope RNA (e.g., mRNA) construct as described above comprises 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more antigens and/or epitopes. In some embodiments, the pharmaceutical composition comprises 2,3, 4, 5, 6, 7, 8, 9, or 10 or more strings. In some embodiments, the pharmaceutical composition comprises 6 strings. In some embodiments, the pharmaceutical composition comprises 7 strings. In some embodiments, the pharmaceutical composition comprises 8 strings. In some embodiments, the pharmaceutical composition comprises 9 strings. In some embodiments, the pharmaceutical composition comprises 10 strings.

In some embodiments, the epitope-encoding sequences in the string construct are flanked by one or more sequences selected for achieving higher immunogenicity, better cleavage of the peptide presentation to MHC, better expression, and/or improved translation in the cells of the subject. In some embodiments, the flanking sequences comprise a linker having a specific cleavable sequence. In some embodiments, the epitope-encoding sequences in the string construct flank the secreted protein sequence.

In some embodiments, the epitope encoded by the string sequence may comprise or be otherwise linked to a signal sequence, such as those listed in table 7, or at least a sequence having a 1, 2, 3, 4, or 5 amino acid difference relative thereto. In some embodiments, the epitope encoded by the string sequence may comprise or be otherwise linked to a signal sequence, such as MFVFLVLLPLVSSQCVNL T (SEQ ID NO: 90), or at least a sequence having a 1, 2, 3, 4, or up to 5 amino acid difference relative thereto. In some embodiments, the epitope encoded by the string sequence may be linked at the N-terminus by a sequence rich in G and S residues or a sequence having a difference of 1, 2, 3, 4, or up to 5 amino acids relative thereto. In some embodiments, an exemplary linker useful for linking epitopes has the sequence GGSGGGGSGG (SEQ ID NO: 165).

In some embodiments, the linked sequences may comprise a linker having a cleavage sequence, e.g., a specific cleavable sequence.

In some embodiments, the string construct is linked to a transmembrane domain (TM) or other membrane associated element. In some embodiments, the length of the linker may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In some embodiments, a linker of no more than about 30, 25, 20, 15, 10, or fewer amino acids is used. In some embodiments, the linker sequence is not limited to comprising any particular amino acid; in some embodiments, the linker sequence comprises any amino acid. In some embodiments, the linker or cleavage sequence comprises glycine (G). In some embodiments, the linker or cleavage sequence comprises serine (S). In some embodiments, the linker is designed to comprise cleavage predictor-based amino acids to produce a highly cleavable sequence peptide sequence, and is a novel and efficient way to deliver immunogenic T cell epitopes in a T cell vaccine environment.

In some embodiments, the distribution of epitopes encoded in the string construct and their juxtaposition are designed to favor cleavage sequences contributed by the amino acid sequence and/or flanking or linking residues of the epitope, thereby using minimal linker sequences. Some exemplary cleavage sequences may be, but are not limited to, one or more of FRAC, KRCF, KKRY, ARMA, RRSG, MRAC, KMCG, ARCA, KKQG, YRSY, SFMN, FKAA, KRNG, YNSF, KKNG, RRRG, KRYS and ARYA (SEQ ID NOS: 62-79, respectively).

In some embodiments, the string construct is RNA (e.g., mRNA). In some embodiments, the pharmaceutical composition comprises one or more RNA (e.g., mRNA) string constructs, each construct comprising a sequence encoding a plurality of epitopes as described herein. In some embodiments, the one or more RNAs (e.g., mrnas) comprise a plurality of epitopes, wherein each of the plurality of epitopes predicts that a protein encoded by HLA is likely to be presented to T cells by an HLA binding and presentation prediction algorithm to elicit an immune response.

In some embodiments, one or more RNAs (e.g., mRNA) utilized in a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) as described herein encodes a plurality of epitopes (e.g., including one or more or two or more antigens provided in table 4, table 5, or table 6, or fragments thereof), optionally wherein each of the plurality predicts, by HLA binding and presentation prediction algorithms, that a protein encoded by HLA is most likely presented to T cells to elicit an immune response. In some embodiments, the plurality of epitopes includes epitopes from a single HSV (e.g., HSV-1 and/or HSV-2) protein. In some embodiments, the plurality of epitopes includes epitopes from a plurality of HSV (e.g., HSV-1 and/or HSV-2) proteins.

In some embodiments, the one or more RNAs (e.g., mRNA) utilized in a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) as described herein comprises a first RNA encoding an HSV (e.g., HSV-1, HSV-2, or both) antigen expressed prior to cellular infiltration or infection, and comprises one or more fragments that are expected or known to interface with the host cytoplasm. In some embodiments, the HSV antigen encoded by the first RNA is or comprises an HSV antigen, fragment or epitope, such as UL1、UL21、UL27、UL29、UL39、UL40、UL46、UL47、UL48、UL49、RS1、RL2、UL5、UL9、UL19、UL25、UL30、UL52、US1、US7、US8、UL22 and/or UL54 or a fragment thereof, an epitope thereof, and/or a combination thereof. In some embodiments, the one or more RNAs (e.g., mRNA) utilized in a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) as described herein comprises a second antigenic RNA encoding a multi-epitope (e.g., multi-epitope) antigen. In some embodiments, the multi-epitope antigen comprises two or more antigens or fragments thereof or epitopes thereof as seen in tables 3-5 herein. In some embodiments, the multi-epitope antigen comprises two or more antigens listed in tables 3-5 and/or fragments and/or epitopes thereof.

In some embodiments, one or more RNAs (e.g., mrnas) utilized in a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) as described herein include a plurality of epitopes that are predicted by HLA binding and presentation prediction algorithms to be highly likely to be presented by HLA-encoded proteins to T cells to elicit an immune response. In some embodiments, the plurality of epitopes includes epitopes from a single HSV (e.g., HSV-1, HSV-2, or both) protein. In some embodiments, the plurality of epitopes includes epitopes from a plurality of HSV (e.g., HSV-1 and/or HSV-2) proteins.

In some embodiments, the RNA (e.g., mRNA) comprises a 5'utr and a 3' utr. In some embodiments, the UTR comprises a poly a sequence. In some embodiments, the poly a sequence comprises between 50 and 200 nucleotides.

In some embodiments, an epitope encoded in the string construct may flank a signal peptide sequence, such as an SP1 sequence (HSV-1 gD signal peptide/secretion domain).

In some embodiments, the polynucleotide comprises a dEarI-hAg sequence.

In some embodiments, the poly a tail of the string construct may comprise about 150 a residues. In some embodiments, the poly a tail may comprise 120 residues or fewer. In some embodiments, the poly a tail of the string construct may comprise about 120 a residues. In some embodiments, the poly a tail of the string construct may comprise about 100 residues. In some embodiments, the poly-A tail of the string comprises a "split" or "interrupted" poly-A tail (e.g., as described in WO 2016/005324).

In some embodiments, the multi-epitope antigen encodes a polypeptide with a supermotif or with a motif, as well as helper epitopes (e.g., heterologous helper epitopes) and endoplasmic reticulum translocation signal sequences. See, e.g., an and Whitton J.Virol.71:2292, 1997; thomson et al, J.Immunol.157:822, 1996; whitton et al, J.Virol 67:348, 1993; hanke et al, vaccine 16:426, 1998.

T cell antigens and related constructs

In certain embodiments, the HSV antigen construct may include and/or encode at least one T cell antigen of HSV (e.g., at least one CD4 and/or CD 8T cell antigen) selected from UL1、UL21、UL27、UL29、UL39、UL40、UL46、UL47、UL48、UL49、RS1、RL2、UL5、UL9、UL19、UL25、UL30、UL52、US1、US7、US8、UL22 and/or UL54 or a fragment thereof. In certain embodiments, the HSV antigen construct may include and/or encode at least one T cell antigen of HSV selected from UL1, UL21, UL27, UL29, UL39, UL40, UL46, UL47, UL48, and/or UL49 or a fragment thereof. In certain embodiments, the HSV antigen construct may include and/or encode at least one T cell antigen of HSV selected from RS1, RL2, UL5, UL9, UL19, UL25, UL30, UL52, US1, US7, US8, UL22, and/or UL54, or a fragment thereof.

In certain embodiments, the HSV antigen construct may include and/or encode a plurality (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of T cell antigens (e.g., CD4 and/or CD 8T cell antigens) of HSV selected from UL1、UL21、UL27、UL29、UL39、UL40、UL46、UL47、UL48、UL49、RS1、RL2、UL5、UL9、UL19、UL25、UL30、UL52、US1、US7、US8、UL22 and/or UL54 or fragments thereof. In certain embodiments, an HSV antigen construct may include and/or encode a plurality (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10) of T cell antigens of HSV selected from UL1, UL21, UL27, UL29, UL39, UL40, UL46, UL47, UL48, and/or UL49, or fragments thereof. In certain embodiments, the HSV antigen construct may include and/or encode a plurality (e.g., 1,2, 3, 4, 5, 6, 7, 8, or 9) of T cell antigens of HSV selected from RS1, RL2, UL5, UL9, UL19, UL25, UL30, UL52, US1, US7, US8, UL22, and/or UL54, or fragments thereof.

In some embodiments, a polyribonucleotide according to the present disclosure encodes a polypeptide comprising two or more HSV antigens or antigenic fragments. In some embodiments, the two or more HSV antigenic fragments are each fragments of a different HSV antigen. In some embodiments, the at least two HSV antigenic fragments are fragments from the same HSV antigen.

In some embodiments, the polyribonucleotides encoding a polypeptide, wherein the polypeptide comprises three or more HSV antigens or antigenic fragments thereof. In some embodiments, each of the three or more HSV antigenic fragments is a fragment of a different HSV antigen. In some embodiments, at least two of the three HSV antigenic fragments are fragments from the same HSV antigen.

In some embodiments, the polyribonucleotides encoding a polypeptide, wherein the polypeptide comprises four or more HSV antigens or antigenic fragments thereof. In some embodiments, each of the four or more HSV antigenic fragments is a fragment of a different HSV antigen. In some embodiments, at least two of the four HSV antigenic fragments are fragments from the same HSV antigen.

In some embodiments, the polyribonucleotides encoding a polypeptide, wherein the polypeptide comprises five or more HSV antigens or antigenic fragments thereof. In some embodiments, each of the five or more HSV antigenic fragments is a fragment of a different HSV antigen. In some embodiments, at least two of the five HSV antigenic fragments are fragments from the same HSV antigen.

In some embodiments, the polyribonucleotide encoding a polypeptide, wherein the polypeptide comprises six or more HSV antigens or antigenic fragments thereof. In some embodiments, each of the six or more HSV antigenic fragments is a fragment of a different HSV antigen. In some embodiments, at least two of the six HSV antigenic fragments are fragments from the same HSV antigen.

In some embodiments, the polypeptide according to the present disclosure does not comprise a full length HSV antigen.

In some embodiments, a polypeptide according to the present disclosure comprises one or more HSV antigens or antigenic fragments thereof that comprise one or more T cell antigens.

In some embodiments, one or more HSV antigens or antigenic fragments thereof have at least 80% sequence identity to one or more sequences selected from SEQ ID NOS: 1-74 or corresponding fragments thereof.

In some embodiments, the HSV T cell antigen is or includes a UL1 polypeptide or fragment thereof. In various embodiments, the UL1 polypeptide or fragment thereof has at least 80% sequence identity to the UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL1 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to a UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL1 polypeptides known in the art include UL1 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL1 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to the amino acid sequence as set forth in SEQ ID nos. 1, 2 and/or 3.

In some embodiments, the HSV T cell antigen is or includes a UL21 polypeptide or fragment thereof. In various embodiments, the UL21 polypeptide or fragment thereof has at least 80% sequence identity to the UL21 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL21 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to a UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL21 polypeptides known in the art include UL21 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL21 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 4,5 and/or 6.

The UL27 open reading frame encodes HSV gB (also referred to herein as UL27 polypeptide). In some embodiments, the HSV T cell antigen is or includes a UL27 polypeptide or fragment thereof. In various embodiments, the UL27 polypeptide or fragment thereof has at least 80% sequence identity to the UL27 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL27 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to a UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL27 polypeptides known in the art include UL27 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL27 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 7, 8,9 and/or 74.

In some embodiments, the HSV T cell antigen is or includes a UL29 polypeptide or fragment thereof. In various embodiments, the UL29 polypeptide or fragment thereof has at least 80% sequence identity to the UL29 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL29 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to a UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL29 polypeptides known in the art include UL29 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL29 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 10, 11 and/or 12.

In some embodiments, the HSV T cell antigen is or includes a UL39 polypeptide or fragment thereof. In various embodiments, the UL39 polypeptide or fragment thereof has at least 80% sequence identity to the UL39 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL39 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to a UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL39 polypeptides known in the art include UL39 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL39 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 13, 14 and/or 15.

In some embodiments, the HSV T cell antigen is or includes a UL40 polypeptide or fragment thereof. In various embodiments, the UL40 polypeptide or fragment thereof has at least 80% sequence identity to the UL40 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL1 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to a UL40 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL40 polypeptides known in the art include UL40 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL40 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 16, 17 and/or 18.

In some embodiments, the HSV T cell antigen is or includes a UL46 polypeptide or fragment thereof. In various embodiments, the UL46 polypeptide or fragment thereof has at least 80% sequence identity to the UL46 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL46 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to a UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL46 polypeptides known in the art include UL46 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL46 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 19, 20 and/or 21.

In some embodiments, the HSV T cell antigen is or includes a UL47 polypeptide or fragment thereof. In various embodiments, the UL47 polypeptide or fragment thereof has at least 80% sequence identity to the UL47 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL47 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to a UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL47 polypeptides known in the art include UL47 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL47 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 22, 23 and/or 24.

In some embodiments, the HSV T cell antigen is or includes a UL48 polypeptide or fragment thereof. In various embodiments, the UL48 polypeptide or fragment thereof has at least 80% sequence identity to the UL48 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL48 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to a UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL48 polypeptides known in the art include UL48 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL48 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 25, 26 and/or 27.

In some embodiments, the HSV T cell antigen is or includes a UL49 polypeptide or fragment thereof. In various embodiments, the UL49 polypeptide or fragment thereof has at least 80% sequence identity to the UL49 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL49 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to a UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL49 polypeptides known in the art include UL49 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL49 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 28, 29 and/or 30.

In some embodiments, the HSV T cell antigen is or includes an RS1 polypeptide or fragment thereof. In various embodiments, the RS1 polypeptide or fragment thereof has at least 80% sequence identity to an RS1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL1 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to a UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of RS1 polypeptides known in the art include RS1 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the RS1 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs.31, 32 and/or 33.

In some embodiments, the HSV T cell antigen is or includes a RL2 polypeptide or fragment thereof. In various embodiments, the RL2 polypeptide or fragment thereof has at least 80% sequence identity to the RL2 amino acid sequence shown in table 3 or otherwise known in the art or to the corresponding fragment. In some embodiments, the RL2 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to a UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of RL2 polypeptides known in the art include RL2 polypeptides encoded by known strains of HSV, such as (but not limited to) HG52, G, 333, and MS. In some embodiments, the RL2 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to the amino acid sequence as set forth in SEQ ID NOS.34, 35 and/or 36.

In some embodiments, the HSV T cell antigen is or includes a UL5 polypeptide or fragment thereof. In various embodiments, the UL5 polypeptide or fragment thereof has at least 80% sequence identity to the UL5 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL5 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity to the UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL5 polypeptides known in the art include UL5 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL5 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 37, 38 and/or 39.

In some embodiments, the HSV T cell antigen is or includes a UL9 polypeptide or fragment thereof. In various embodiments, the UL9 polypeptide or fragment thereof has at least 80% sequence identity to the UL9 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL9 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to the UL1 amino acid sequence shown in table 3 or otherwise known in the art or to a corresponding fragment thereof. Examples of UL9 polypeptides known in the art include UL9 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL9 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 40, 41 and/or 42.

In some embodiments, the HSV T cell antigen is or includes a UL19 polypeptide or fragment thereof. In various embodiments, the UL19 polypeptide or fragment thereof has at least 80% sequence identity to the UL19 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL19 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to a UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL19 polypeptides known in the art include UL19 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL19 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 43, 44 and/or 45.

In some embodiments, the HSV T cell antigen is or includes a UL25 polypeptide or fragment thereof. In various embodiments, the UL25 polypeptide or fragment thereof has at least 80% sequence identity to the UL25 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL25 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to a UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL25 polypeptides known in the art include UL25 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL25 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 46, 47 and/or 48.

In some embodiments, the HSV T cell antigen is or includes a UL30 polypeptide or fragment thereof. In various embodiments, the UL30 polypeptide or fragment thereof has at least 80% sequence identity to the UL30 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL30 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity to the UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL30 polypeptides known in the art include UL30 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL30 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 49, 50 and/or 51.

In some embodiments, the HSV T cell antigen is or includes a UL52 polypeptide or fragment thereof. In various embodiments, the UL52 polypeptide or fragment thereof has at least 80% sequence identity to the UL52 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL52 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL52 polypeptides known in the art include UL52 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL52 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID nos. 52, 53 and/or 54.

The US1 open reading frame encodes HSV gL (also referred to herein as UL1 polypeptide). In some embodiments, the HSV T cell antigen is or includes a US1 polypeptide or fragment thereof. In various embodiments, the US1 polypeptide or fragment thereof has at least 80% sequence identity to a US1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, a Us1 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to a UL1 amino acid sequence shown in table 3 or otherwise known in the art, or a corresponding fragment thereof. Examples of known US1 polypeptides in the art include US1 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the US1 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 58, 59, 60 and/or 61.

The US7 open reading frame encodes HSV gI (also referred to herein as a US7 polypeptide). In some embodiments, the HSV T cell antigen is or includes a US7 polypeptide or fragment thereof. In various embodiments, the US7 polypeptide or fragment thereof has at least 80% sequence identity to a US7 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL1 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, to a US7 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of US7 polypeptides known in the art include US7 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the US7 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 62, 63, 64 and/or 65.

The US8 open reading frame encodes HSV gE (also referred to herein as a US8 polypeptide). In some embodiments, the HSV T cell antigen is or includes a US8 polypeptide or fragment thereof. In various embodiments, the US8 polypeptide or fragment thereof has at least 80% sequence identity to a US8 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL1 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the US8 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of known US8 polypeptides in the art include US8 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the US8 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 66, 67, 68 and/or 69.

The UL22 open reading frame encodes HSV gH (also referred to herein as UL22 polypeptide). In some embodiments, the HSV T cell antigen is or includes a UL22 polypeptide or fragment thereof. In various embodiments, the UL22 polypeptide or fragment thereof has at least 80% sequence identity to the UL22 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL22 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the UL1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL22 polypeptides known in the art include UL22 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL22 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 70, 71, 72 and/or 73.

In some embodiments, the HSV T cell antigen is or includes a UL54 polypeptide or fragment thereof. In various embodiments, the UL54 polypeptide or fragment thereof has at least 80% sequence identity to the UL54 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the UL1 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the UL54 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of UL54 polypeptides known in the art include UL54 polypeptides encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the UL54 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 55, 56 and/or 57.

In some embodiments, an HSV antigen used according to the present disclosure comprises an RL2 polypeptide or antigenic fragment thereof, an RS1 polypeptide or antigenic fragment thereof, an UL54 polypeptide or antigenic fragment thereof, an UL29 polypeptide or antigenic fragment thereof, an UL39 polypeptide or antigenic fragment thereof, an UL49 polypeptide or antigenic fragment thereof, an UL9 polypeptide or antigenic fragment thereof, an UL30 polypeptide or antigenic fragment thereof, an UL40 polypeptide or antigenic fragment thereof, an UL5 polypeptide or antigenic fragment thereof, an UL52 polypeptide or antigenic fragment thereof, an UL1 polypeptide or antigenic fragment thereof, an UL19 polypeptide or antigenic fragment thereof, an UL21 polypeptide or antigenic fragment thereof, an UL27 polypeptide or antigenic fragment thereof, an UL46 polypeptide or antigenic fragment thereof, an UL47 polypeptide or antigenic fragment thereof, an UL48 polypeptide or antigenic fragment thereof, an UL25 polypeptide or antigenic fragment thereof, or a combination thereof.

In some embodiments, a polyribonucleotide provided herein encodes one or more of a RL2 polypeptide or antigenic fragment thereof, a RS1 polypeptide or antigenic fragment thereof, a UL54 polypeptide or antigenic fragment thereof, a UL29 polypeptide or antigenic fragment thereof, a UL39 polypeptide or antigenic fragment thereof, a UL49 polypeptide or antigenic fragment thereof, a UL9 polypeptide or antigenic fragment thereof, a UL30 polypeptide or antigenic fragment thereof, a UL40 polypeptide or antigenic fragment thereof, a UL5 polypeptide or antigenic fragment thereof, a UL52 polypeptide or antigenic fragment thereof, a UL1 polypeptide or antigenic fragment thereof, a UL19 polypeptide or antigenic fragment thereof, a UL21 polypeptide or antigenic fragment thereof, a UL27 polypeptide or antigenic fragment thereof, a UL46 polypeptide or antigenic fragment thereof, a UL47 polypeptide or antigenic fragment thereof, a UL48 polypeptide or antigenic fragment thereof, a UL25 polypeptide or antigenic fragment thereof, a US1 polypeptide or antigenic fragment thereof, a US7 polypeptide or antigenic fragment thereof, a US8 polypeptide or antigenic fragment thereof, and a UL22 polypeptide or antigenic fragment thereof.

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a RL2 polypeptide or antigenic fragment thereof. In some embodiments, the RL2 polypeptide or antigenic fragment thereof comprises or consists of the following amino acid sequences ：CTDEIAPPLRCQSFPCL HPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDP RTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLS(SEQ ID NO:174)., in some embodiments, the RL2 polypeptide or antigenic fragment thereof comprises or consists of the following amino acid sequences ：LPIAGVSSVVALAPYVNKTVTGDCLP VLDMETGHIGAYVVLVDQTGNVADLLRAAAPAWSRRTLLPEHA RNCVRPPDYPTPPASEWNSLWMTPVGNMLFDQGTLVG(SEQ ID NO:175).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is an RS1 polypeptide or an antigenic fragment thereof. In some embodiments, the RS1 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence ：RAAAWMRQVPDPEDV RVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAAS(SEQ ID NO:176).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL54 polypeptide or antigenic fragment thereof. In some embodiments, the UL54 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence ：ETLVAHGPSLYRT FAANPRAASTAKAMRDCVLRQENLIEALASADETLAWCKMCIHHNLPLRPQDPIIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVSEIDYTTVGVGAGETMHFYIPGACMAGLIEILDTHRQECSSRVCELTASHTIAPLYVHGKYFYCNSLF(SEQ ID NO:177).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL29 polypeptide or antigenic fragment thereof. In some embodiments, the UL29 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence ：REDIETIAFIKRFSL DYGAINFINLAPNNVSELAMYYMANQILRYCDHSTYFINTLTAVIAGSRRPPSVQAAAAWAPQGGAGLEAGARALMDSLDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQAGNWASLLGGKNACPLLIFDRTRKFVL(SEQ ID NO:178).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL39 polypeptide or antigenic fragment thereof. In some embodiments, the UL39 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence ：RTFGSAPRLTEDDF GLLNYALAEMRRLCLDLPPVPPNAYTPYHLREYATRLVNGFKPLVRRSARLYRILGVLVHLRIRTREASFEEWMRSKEVDLDFGLTERLREHEAQLMILAQALNPYDCLIHSTPNTLVERGLQSALKYEEFYLKRFGGHYMESVFQMYTRIAGFLA(SEQ ID NO:179).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL49 polypeptide or antigenic fragment thereof. In some embodiments, the UL49 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence ：KMTRGAPKASATP ATDPARGRRPAQADSAVLLDAPAPTASGRTKTPAQGLAKKLHFS TAPPSPTAPWTPRVAGFNKRVFCAAVG(SEQ ID NO:180).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL9 polypeptide or antigenic fragment thereof. In some embodiments, the UL9 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence ：LLNNYDVLVLDEVM STLGQLYSPTMQQLGRVDALMLRLLRTCPRIIAMDATANAQLVDFLCSLRGEKNVHVVIGEYAMPGFSARRCLFLPRLGPEVLQAALRRRGPAGGAPPPDAPPDATFFGELEARLAGGDNVCIFSSTVSFAEVVARFCRQFTDRVLLLHSLTPPGDVTTWGRYRVVIYTTVVTVGLSFDPPHFDSMFAYVKPMNYGPDMVSVYQSLGRVRTLRKGELLIYMDGSGARSEPV(SEQ ID NO:181).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL30 polypeptide or antigenic fragment thereof. In some embodiments, the UL30 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence ：ISCLLYDLSTTALE HILLFSLGSCDLPESHLSDLASRGLPAPVVLEFDSEFEMLLAFMTF VKQYGPEFVTGYNIINFDWPFVLTKLTEIYKVPLDGYGRMNGRG VFRVWDIGQSHF(SEQ ID NO:182). in some embodiments, the UL30 polypeptide or antigenic fragment thereof comprises the amino acid sequence GLLPCLHVAATVTTIGREML LATRAYVHARWAEFDQLLADFPEAAGMRAPGPYSM (SEQ ID NO: 183).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL40 polypeptide or antigenic fragment thereof. In some embodiments, the UL40 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence ：TSQCPDINHLRSLSI LNRWLETELVFVGDEEDVSKLSEGELGFYRFLFAFLSAADDLVTENLGGLSGLFEQKDILHYYVEQECIEVVHSRVYNIIQLVLFHNNDQARRAYVARTINHPAIRVKVDWLEARVRECDSIPEKFILMILIEGVFFAASFAAIAYLRTNNLLR(SEQ ID NO:184).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL5 polypeptide or antigenic fragment thereof. In some embodiments, the UL5 polypeptide or antigenic fragment thereof comprises or consists of the following amino acid sequences ：HEFGNLMKVLEYGL PITEEHMQFVDRFVVPESYITNPANLPGWTRLFSSHKEVSAYMAK LHAYLKVTREGEFVVFTLPVLTFVSVKEFDEYRRL(SEQ ID NO:185)., in some embodiments, the UL5 polypeptide or antigenic fragment thereof comprises or consists of the following amino acid sequences ：ELFGEVFESAPFSTYVDNVIFRG CELLTGSPRGGLMSVALQTDNYTLMGYTYTRVFAFAEELRRRHA TAGVAEFLEESPLPYIVLRDQHGFMSVVNTNI(SEQ ID NO:186).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL52 polypeptide or antigenic fragment thereof. In some embodiments, the UL52 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence ：SVAAPVEVTALYA TDGCVITSSLALLTNCLLGAEPLYIFSYDAYRSDAPNGPTGAPTEQERFEGSRALYRDAGGLNGDSFRVTFCLLGTEVGVTHHPKGRTRPMFVCRFERADDVAVLQDALGRGTPLLPAHVTATLDLEATFALHANIIMALTVAIVHNAPARIGSGSTAPLYEPGESMRSVV(SEQ ID NO:187).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL1 polypeptide or antigenic fragment thereof. In some embodiments, the UL1 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence: RTPADDVSWRYEAP SVIDYARIDGIFLRYHCPGLDTFLWDRHAQRAYLVNPFLFAAGFL EDLSHSVFPADTQETT (SEQ ID NO: 188).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL19 polypeptide or antigenic fragment thereof. In some embodiments, the UL19 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence ：DGRLLHNTQARAA DAADDRPHRPADWTVHHKIYYYVLVPAFSRGRCCTAGVRFDRV YATLQNMVVPEIAPGEECPSDPVTDPAHPLHPANLVANTVKRMF HN(SEQ ID NO:189).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL21 polypeptide or antigenic fragment thereof. In some embodiments, the UL21 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence: SPTQKLAVYYYLI HRERRMSPFPALVRLVGRYIQRHGLYVPAPDEPTLADAMNGL (SEQ ID NO: 190).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL27 polypeptide or antigenic fragment thereof. In some embodiments, the UL27 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence: NYTEGIAVVFKENI APYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEV (SEQ ID NO: 191). In some embodiments, the UL27 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence: SVYPYDEFVLATG DFVYMSPFYGYREGSH (SEQ ID NO: 192).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL46 polypeptide or antigenic fragment thereof. In some embodiments, the UL46 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence ：GLASDPHYDYIRHYASAAKQALGEVELSGGQLSRAILAQYWKYLQTVVPSGLDIPDDPAGDCDPSLHVLLRPTLLPKLLVRAPFKSGAAAAKYAAAVAGLRDAAHRLQQYMFFMRPADPSRPSTDTALRLSELLAYVSVLYHWASWMLWTADKYV(SEQ ID NO:193).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL47 polypeptide or antigenic fragment thereof. In some embodiments, the UL47 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence: GPDAAVFRSSLGS LLYWPGVRALLGRDCRVAARYAGRMTYIATGALLARFNPGAVK CVLPREAAFAGRVL (SEQ ID NO: 194).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL48 polypeptide or antigenic fragment thereof. In some embodiments, the UL48 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence ：ALFNRLLDDLGFS AGPALCTMLDTWNEDLFSGFPTNADMYRECKFLSTLPSDVIDWG DAHVPERSPIDIRAHGDVAFPTLPATRDELPSYYEAMAQFFRGELR A(SEQ ID NO:195).

In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one antigen is a UL25 polypeptide or antigenic fragment thereof. In some embodiments, the UL25 polypeptide or antigenic fragment thereof comprises or consists of the amino acid sequence ：FLWEDQTLLRATA NTITALAVLRRLLANGNVYADRLDNRLQLGMLIPGAVPAEAIARG ASGLDSGAIKSGDNNLEALCVNYVLPLYQADPTVELTQLFPGLAA LCL(SEQ ID NO:196).

In some embodiments, the HSV (e.g., HSV-1 and/or HSV-2) antigen used in accordance with the present disclosure is an intermediate early protein or antigenic fragment thereof. In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one HSV antigen comprises an intermediate early protein or an antigenic fragment thereof. In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein each HSV antigen comprises an intermediate early protein or an antigenic fragment thereof.

In some embodiments, the HSV-2 antigen used according to the present disclosure is an intermediate early protein or an antigenic fragment thereof. In some embodiments, the polyribonucleotides provided herein encode one or more HSV-2 antigens, wherein at least one HSV-2 antigen comprises an intermediate early protein or an antigenic fragment thereof. In some embodiments, the polyribonucleotides provided herein encode one or more HSV-2 antigens, wherein each HSV-2 antigen comprises an intermediate early protein or an antigenic fragment thereof.

In some embodiments, an HSV-2 antigen used according to the present disclosure comprises an RL2 polypeptide or antigenic fragment thereof, an RS1 polypeptide or antigenic fragment thereof, an UL54 polypeptide or antigenic fragment thereof, or a combination thereof.

In some embodiments, the polyribonucleotides provided herein encode one or more of a RL2 polypeptide or antigenic fragment thereof, an RS1 protein or antigenic fragment thereof, and a UL54 protein or antigenic fragment thereof.

In some embodiments, the HSV (e.g., HSV-1 and/or HSV-2) antigen used in accordance with the present disclosure is an early protein or antigenic fragment thereof. In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one HSV antigen comprises an early protein or an antigenic fragment thereof. In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein each HSV antigen comprises an early protein or an antigenic fragment thereof.

In some embodiments, the HSV-2 antigen used according to the present disclosure is an early protein or an antigenic fragment thereof. In some embodiments, the polyribonucleotides provided herein encode one or more HSV-2 antigens, wherein at least one HSV-2 antigen comprises an early protein or an antigenic fragment thereof. In some embodiments, the polyribonucleotides provided herein encode one or more HSV-2 antigens, wherein each HSV-2 antigen comprises an early protein or an antigenic fragment thereof.

In some embodiments, an HSV-2 antigen used according to the present disclosure comprises an UL29 polypeptide or antigenic fragment thereof, an UL39 polypeptide or antigenic fragment thereof, an UL49 polypeptide or antigenic fragment thereof, an UL9 polypeptide or antigenic fragment thereof, an UL30 polypeptide or antigenic fragment thereof, an UL40 polypeptide or antigenic fragment thereof, an UL5 polypeptide or antigenic fragment thereof, an UL52 polypeptide or antigenic fragment thereof, or a combination thereof.

In some embodiments, the polyribonucleotides provided herein encode one or more of a UL29 polypeptide or antigenic fragment thereof, a UL39 polypeptide or antigenic fragment thereof, a UL49 polypeptide or antigenic fragment thereof, a UL9 polypeptide or antigenic fragment thereof, a UL30 polypeptide or antigenic fragment thereof, a UL40 polypeptide or antigenic fragment thereof, a UL5 polypeptide or antigenic fragment thereof, and a UL52 polypeptide or antigenic fragment thereof.

In some embodiments, an HSV-2 antigen used according to the present disclosure comprises an UL29 polypeptide or antigenic fragment thereof, an UL39 polypeptide or antigenic fragment thereof, an UL49 polypeptide or antigenic fragment thereof, an UL9 polypeptide or antigenic fragment thereof, or a combination thereof.

In some embodiments, the UL29 polypeptide or antigenic fragment thereof, the UL39 polypeptide or antigenic fragment thereof, the UL49 polypeptide or antigenic fragment thereof, and the UL9 polypeptide or antigenic fragment thereof.

In some embodiments, an HSV-2 antigen used according to the present disclosure comprises a UL30 polypeptide or antigenic fragment thereof, a UL40 polypeptide or antigenic fragment thereof, a UL5 polypeptide or antigenic fragment thereof, a UL52 polypeptide or antigenic fragment thereof, or a combination thereof.

In some embodiments, the polyribonucleotides provided herein encode one or more of a UL30 polypeptide or antigenic fragment thereof, a UL40 polypeptide or antigenic fragment thereof, a UL5 polypeptide or antigenic fragment thereof, and a UL52 polypeptide or antigenic fragment thereof.

In some embodiments, the HSV (e.g., HSV-1 and/or HSV-2) antigen used in accordance with the present disclosure is a late protein or antigenic fragment thereof. In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein at least one HSV antigen comprises an advanced protein or an antigenic fragment thereof. In some embodiments, the polyribonucleotides provided herein encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, wherein each HSV antigen comprises an advanced protein or an antigenic fragment thereof.

In some embodiments, the HSV-2 antigen used according to the present disclosure is a late protein or an antigenic fragment thereof. In some embodiments, the polyribonucleotides provided herein encode one or more HSV-2 antigens, wherein at least one HSV-2 antigen comprises an advanced protein or an antigenic fragment thereof. In some embodiments, the polyribonucleotides provided herein encode one or more HSV-2 antigens, wherein each HSV-2 antigen comprises an advanced protein or an antigenic fragment thereof.

In some embodiments, an HSV-2 antigen used according to the present disclosure comprises an UL1 polypeptide or an antigenic fragment thereof, an UL19 polypeptide or an antigenic fragment thereof, an UL21 polypeptide or an antigenic fragment thereof, an UL27 polypeptide or an antigenic fragment thereof, an UL46 polypeptide or an antigenic fragment thereof, an UL47 polypeptide or an antigenic fragment thereof, an UL48 polypeptide or an antigenic fragment thereof, an UL25 polypeptide or an antigenic fragment thereof, or a combination thereof.

In some embodiments, the polyribonucleotides provided herein encode one or more of a UL1 polypeptide or antigenic fragment thereof, a UL19 polypeptide or antigenic fragment thereof, a UL21 polypeptide or antigenic fragment thereof, a UL27 polypeptide or antigenic fragment thereof, a UL46 polypeptide or antigenic fragment thereof, a UL47 polypeptide or antigenic fragment thereof, a UL48 polypeptide or antigenic fragment thereof, and a UL25 polypeptide or antigenic fragment thereof.

An exemplary payload selected according to the present disclosure encodes the polypeptide shown in fig. 53. For example, in some embodiments, the polyribonucleotides provided herein may include, in 5 'to 3' order, a nucleotide sequence encoding an HSV-1gD secretion signal, a RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, a UL54 polypeptide or fragment thereof, a linker, and MITD (see fig. 53A). In some embodiments, such polyribonucleotides encode polypeptides having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRGCTDEIA PPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDPRTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGLPIAGVSSVVALAPYVNKTVTGDCLPVLDMETGHIGAYVVLVDQTGNVADLLRAAAPAWSRRTLLPEHARNCVRPPDYPTPPASEWNSLWMTPVGNMLFDQGTLVGGGSGGGGSGGRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGGSGGGGSGGETLVAHGPSLYRTFAANPRAASTAKAMRDCVLRQENLIEALASADETLAWCKMCIHHNLPLRPQDPIIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVSEIDYTTVGVGAGETMHFYIPGACMAGLIEILDTHRQECSSRVCELTASHTIAPLYVHGKYFYCNSLFGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:197).

In some embodiments, the polyribonucleotides provided herein may include in 5 'to 3' order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL29 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL49 polypeptide or antigenic fragment thereof, a linker, a UL9 polypeptide or fragment thereof, a linker and MITD (see fig. 53B). In some embodiments, such polyribonucleotides encode polypeptides having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRGREDIETI AFIKRFSLDYGAINFINLAPNNVSELAMYYMANQILRYCDHSTYFINTLTAVIAGSRRPPSVQAAAAWAPQGGAGLEAGARALMDSLDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQAGNWASLLGGKNACPLLIFDRTRKFVLGGSGGGGSGGRTFGSAPRLTEDDFGLLNYALAEMRRLCLDLPPVPPNAYTPYHLREYATRLVNGFKPLVRRSARLYRILGVLVHLRIRTREASFEEWMRSKEVDLDFGLTERLREHEAQLMILAQALNPYDCLIHSTPNTLVERGLQSALKYEEFYLKRFGGHYMESVFQMYTRIAGFLAGGSGGGGSGGKMTRGAPKASATPATDPARGRRPAQADSAVLLDAPAPTASGRTKTPAQGLAKKLHFSTAPPSPTAPWTPRVAGFNKRVFCAAVGGGSGGGGSGGLLNNYDVLVLDEVMSTLGQLYSPTMQQLGRVDALMLRLLRTCPRIIAMDATANAQLVDFLCSLRGEKNVHVVIGEYAMPGFSARRCLFLPRLGPEVLQAALRRRGPAGGAPPPDAPPDATFFGELEARLAGGDNVCIFSSTVSFAEVVARFCRQFTDRVLLLHSLTPPGDVTTWGRYRVVIYTTVVTVGLSFDPPHFDSMFAYVKPMNYGPDMVSVYQSLGRVRTLRKGELLIYMDGSGARSEPVGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:198).

In some embodiments, the polyribonucleotides provided herein may include in 5 'to 3' order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL30 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or fragment thereof, a linker, a UL52 polypeptide or fragment thereof, a linker, and MITD (see fig. 53C). In some embodiments, such polyribonucleotides encode polypeptides having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRGISCLLYDLSTTALE HILLFSLGSCDLPESHLSDLASRGLPAPVVLEFDSEFEMLLAFMTFVKQYGPEFVTGYNIINFDWPFVLTKLTEIYKVPLDGYGRMNGRGVFRVWDIGQSHFGGSGGGGSGGGLLPCLHVAATVTTIGREMLLATRAYVHARWAEFDQLLADFPEAAGMRAPGPYSMGGSGGGGSGGTSQCPDINHLRSLSILNRWLETELVFVGDEEDVSKLSEGELGFYRFLFAFLSAADDLVTENLGGLSGLFEQKDILHYYVEQECIEVVHSRVYNIIQLVLFHNNDQARRAYVARTINHPAIRVKVDWLEARVRECDSIPEKFILMILIEGVFFAASFAAIAYLRTNNLLRGGSGGGGSGGHEFGNLMKVLEYGLPITEEHMQFVDRFVVPESYITNPANLPGWTRLFSSHKEVSAYMAKLHAYLKVTREGEFVVFTLPVLTFVSVKEFDEYRRLGGSGGGGSGGELFGEVFESAPFSTYVDNVIFRGCELLTGSPRGGLMSVALQTDNYTLMGYTYTRVFAFAEELRRRHATAGVAEFLEESPLPYIVLRDQHGFMSVVNTNIGGSGGGGSGGSVAAPVEVTALYATDGCVITSSLALLTNCLLGAEPLYIFSYDAYRSDAPNGPTGAPTEQERFEGSRALYRDAGGLNGDSFRVTFCLLGTEVGVTHHPKGRTRPMFVCRFERADDVAVLQDALGRGTPLLPAHVTATLDLEATFALHANIIMALTVAIVHNAPARIGSGSTAPLYEPGESMRSVVGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:199).

In some embodiments, the polyribonucleotides provided herein may include in 5 'to 3' order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL1 polypeptide or antigenic fragment thereof, a linker, a UL19 polypeptide or antigenic fragment thereof, a linker, a UL21 polypeptide or antigenic fragment thereof, a linker, a UL27 polypeptide or fragment thereof, a linker, a UL46 polypeptide or fragment thereof, a linker, a UL47 polypeptide or fragment thereof, a linker, a UL25 polypeptide or fragment thereof, a linker, a UL48 polypeptide or fragment thereof, a linker, and MITD (see fig. 53D). In some embodiments, such polyribonucleotides encode polypeptides having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRG RTPADDVSWRYEAPSVIDYARIDGIFLRYHCPGLDTFLWDRHAQRAYLVNPFLFAAGFLEDLSHSVFPADTQETTGGSGGGGSGGDGRLLHNTQARAADAADDRPHRPADWTVHHKIYYYVLVPAFSRGRCCTAGVRFDRVYATLQNMVVPEIAPGEECPSDPVTDPAHPLHPANLVANTVKRMFHNGGSGGGGSGGSPTQKLAVYYYLIHRERRMSPFPALVRLVGRYIQRHGLYVPAPDEPTLADAMNGLGGSGGGGSGGNYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEVGGSGGGGSGGSVYPYDEFVLATGDFVYMSPFYGYREGSHGGSGGGGSGGGLASDPHYDYIRHYASAAKQALGEVELSGGQLSRAILAQYWKYLQTVVPSGLDIPDDPAGDCDPSLHVLLRPTLLPKLLVRAPFKSGAAAAKYAAAVAGLRDAAHRLQQYMFFMRPADPSRPSTDTALRLSELLAYVSVLYHWASWMLWTADKYVGGSGGGGSGGGPDAAVFRSSLGSLLYWPGVRALLGRDCRVAARYAGRMTYIATGALLARFNPGAVKCVLPREAAFAGRVLGGSGGGGSGGFLWEDQTLLRATANTITALAVLRRLLANGNVYADRLDNRLQLGMLIPGAVPAEAIARGASGLDSGAIKSGDNNLEALCVNYVLPLYQADPTVELTQLFPGLAALCLGGSGGGGSGGALFNRLLDDLGFSAGPALCTMLDTWNEDLFSGFPTNADMYRECKFLSTLPSDVIDWGDAHVPERSPIDIRAHGDVAFPTLPATRDELPSYYEAMAQFFRGELRAGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:200).

In some embodiments, the polyribonucleotides provided herein may include in 5 'to 3' order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL54 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or fragment thereof, a linker, and MITD. In some embodiments, such polyribonucleotides encode polypeptides having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRGETLVAHGPSLYRTFA ANPRAASTAKAMRDCVLRQENLIEALASADETLAWCKMCIHHNLPLRPQDPIIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVSEIDYTTVGVGAGETMHFYIPGACMAGLIEILDTHRQECSSRVCELTASHTIAPLYVHGKYFYCNSLFGGSGGGGSGGRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGGSGGGGSGGCTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDPRTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGLPIAGVSSVVALAPYVNKTVTGDCLPVLDMETGHIGAYVVLVDQTGNVADLLRAAAPAWSRRTLLPEHARNCVRPPDYPTPPASEWNSLWMTPVGNMLFDQGTLVGGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:201).

In some embodiments, the polyribonucleotides provided herein may include in 5 'to 3' order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL9 polypeptide or antigenic fragment thereof, a linker, a UL49 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL29 polypeptide or fragment thereof, a linker, and MITD. In some embodiments, such polyribonucleotides encode polypeptides having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRGLLNNYDVLVLDEV MSTLGQLYSPTMQQLGRVDALMLRLLRTCPRIIAMDATANAQLVDFLCSLRGEKNVHVVIGEYAMPGFSARRCLFLPRLGPEVLQAALRRRGPAGGAPPPDAPPDATFFGELEARLAGGDNVCIFSSTVSFAEVVARFCRQFTDRVLLLHSLTPPGDVTTWGRYRVVIYTTVVTVGLSFDPPHFDSMFAYVKPMNYGPDMVSVYQSLGRVRTLRKGELLIYMDGSGARSEPVGGSGGGGSGGKMTRGAPKASATPATDPARGRRPAQADSAVLLDAPAPTASGRTKTPAQGLAKKLHFSTAPPSPTAPWTPRVAGFNKRVFCAAVGGGSGGGGSGGRTFGSAPRLTEDDFGLLNYALAEMRRLCLDLPPVPPNAYTPYHLREYATRLVNGFKPLVRRSARLYRILGVLVHLRIRTREASFEEWMRSKEVDLDFGLTERLREHEAQLMILAQALNPYDCLIHSTPNTLVERGLQSALKYEEFYLKRFGGHYMESVFQMYTRIAGFLAGGSGGGGSGGREDIETIAFIKRFSLDYGAINFINLAPNNVSELAMYYMANQILRYCDHSTYFINTLTAVIAGSRRPPSVQAAAAWAPQGGAGLEAGARALMDSLDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQAGNWASLLGGKNACPLLIFDRTRKFVLGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:202).

In some embodiments, the polyribonucleotides provided herein may include in 5 'to 3' order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL52 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or fragment thereof, a linker, a UL30 polypeptide or fragment thereof, a linker, and MITD. In some embodiments, such polyribonucleotides encode polypeptides having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRGSVAAPVEVTALYATDGCVITSSLALLTNCLLGAEPLYIFSYDAYRSDAPNGPTGAPTEQERFEGSRALYRDAGGLNGDSFRVTFCLLGTEVGVTHHPKGRTRPMFVCRFERADDVAVLQDALGRGTPLLPAHVTATLDLEATFALHANIIMALTVAIVHNAPARIGSGSTAPLYEPGESMRSVVGGSGGGGSGGHEFGNLMKVLEYGLPITEEHMQFVDRFVVPESYITNPANLPGWTRLFSSHKEVSAYMAKLHAYLKVTREGEFVVFTLPVLTFVSVKEFDEYRRLGGSGGGGSGGELFGEVFESAPFSTYVDNVIFRGCELLTGSPRGGLMSVALQTDNYTLMGYTYTRVFAFAEELRRRHATAGVAEFLEESPLPYIVLRDQHGFMSVVNTNIGGSGGGGSGGTSQCPDINHLRSLSILNRWLETELVFVGDEEDVSKLSEGELGFYRFLFAFLSAADDLVTENLGGLSGLFEQKDILHYYVEQECIEVVHSRVYNIIQLVLFHNNDQARRAYVARTINHPAIRVKVDWLEARVRECDSIPEKFILMILIEGVFFAASFAAIAYLRTNNLLRGGSGGGGSGGISCLLYDLSTTALEHILLFSLGSCDLPESHLSDLASRGLPAPVVLEFDSEFEMLLAFMTFVKQYGPEFVTGYNIINFDWPFVLTKLTEIYKVPLDGYGRMNGRGVFRVWDIGQSHFGGSGGGGSGGGLLPCLHVAATVTTIGREMLLATRAYVHARWAEFDQLLADFPEAAGMRAPGPYSMGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:203).

In some embodiments, the polyribonucleotides provided herein may include in 5 'to 3' order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL48 polypeptide or antigenic fragment thereof, a linker, a UL25 polypeptide or antigenic fragment thereof, a linker, a UL47 polypeptide or antigenic fragment thereof, a linker, a UL46 polypeptide or fragment thereof, a linker, a UL27 polypeptide or fragment thereof, a linker, a UL21 polypeptide or fragment thereof, a linker, a UL19 polypeptide or fragment thereof, a linker, a UL1 polypeptide or fragment thereof, a linker, and MITD. In some embodiments, such polyribonucleotides encode polypeptides having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRGALFNRLLDDL GFSAGPALCTMLDTWNEDLFSGFPTNADMYRECKFLSTLPSDVIDWGDAHVPERSPIDIRAHGDVAFPTLPATRDELPSYYEAMAQFFRGELRAGGSGGGGSGGFLWEDQTLLRATANTITALAVLRRLLANGNVYADRLDNRLQLGMLIPGAVPAEAIARGASGLDSGAIKSGDNNLEALCVNYVLPLYQADPTVELTQLFPGLAALCLGGSGGGGSGGGPDAAVFRSSLGSLLYWPGVRALLGRDCRVAARYAGRMTYIATGALLARFNPGAVKCVLPREAAFAGRVLGGSGGGGSGGGLASDPHYDYIRHYASAAKQALGEVELSGGQLSRAILAQYWKYLQTVVPSGLDIPDDPAGDCDPSLHVLLRPTLLPKLLVRAPFKSGAAAAKYAAAVAGLRDAAHRLQQYMFFMRPADPSRPSTDTALRLSELLAYVSVLYHWASWMLWTADKYVGGSGGGGSGGNYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEVGGSGGGGSGGSVYPYDEFVLATGDFVYMSPFYGYREGSHGGSGGGGSGGSPTQKLAVYYYLIHRERRMSPFPALVRLVGRYIQRHGLYVPAPDEPTLADAMNGLGGSGGGGSGGDGRLLHNTQARAADAADDRPHRPADWTVHHKIYYYVLVPAFSRGRCCTAGVRFDRVYATLQNMVVPEIAPGEECPSDPVTDPAHPLHPANLVANTVKRMFHNGGSGGGGSGGRTPADDVSWRYEAPSVIDYARIDGIFLRYHCPGLDTFLWDRHAQRAYLVNPFLFAAGFLEDLSHSVFPADTQETTGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:204).

In some embodiments, the polyribonucleotides provided herein may include in 5 'to 3' order a nucleotide sequence encoding an HSV-2gD secretion signal, a RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, a UL54 polypeptide or fragment thereof, a linker, and MITD. In some embodiments, such polyribonucleotides encode polypeptides having an amino acid sequence comprising or consisting of ：MGRLTSGVGTAALLVVAVGLRVVCACTDEIAPPLRCQSFP CLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDPRTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGLPIAGVSSVVALAPYVNKTVTGDCLPVLDMETGHIGAYVVLVDQTGNVADLLRAAAPAWSRRTLLPEHARNCVRPPDYPTPPASEWNSLWMTPVGNMLFDQGTLVGGGSGGGGSGGRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGGSGGGGSGGETLVAHGPSLYRTFAANPRAASTAKAMRDCVLRQENLIEALASADETLAWCKMCIHHNLPLRPQDPIIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVSEIDYTTVGVGAGETMHFYIPGACMAGLIEILDTHRQECSSRVCELTASHTIAPLYVHGKYFYCNSLFGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:205).

In some embodiments, the polyribonucleotides provided herein may include in 5 'to 3' order a nucleotide sequence encoding an HSV-2gD secretion signal, a UL54 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or fragment thereof, a linker, and MITD. In some embodiments, such polyribonucleotides encode polypeptides having an amino acid sequence comprising or consisting of ：MGRLTSGVGTAALLVVAVGLRVVCAETLVAHGPSLYRTFA ANPRAASTAKAMRDCVLRQENLIEALASADETLAWCKMCIHHNLPLRPQDPIIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVSEIDYTTVGVGAGETMHFYIPGACMAGLIEILDTHRQECSSRVCELTASHTIAPLYVHGKYFYCNSLFGGSGGGGSGGRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGGSGGGGSGGCTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDPRTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGLPIAGVSSVVALAPYVNKTVTGDCLPVLDMETGHIGAYVVLVDQTGNVADLLRAAAPAWSRRTLLPEHARNCVRPPDYPTPPASEWNSLWMTPVGNMLFDQGTLVGGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:206).

4. Glycoprotein antigens and related constructs

Provided herein are HSV antigens as HSV glycoproteins. In some embodiments, the HSV glycoprotein is an HSV envelope glycoprotein. In some embodiments, the HSV glycoprotein is HSV gB, HSV gD, HSV gH, HSV gL, HSV gI, HSV gE, or HSV gC.

Further, provided herein are polyribonucleotides encoding HSV glycoproteins or antigenic fragments thereof. In some embodiments, the polyribonucleotides described herein encode HSV glycoproteins. In some embodiments, a polyribonucleotide described herein encodes an HSV envelope glycoprotein. In some embodiments, a polyribonucleotide described herein encodes HSVgB, HSV gD, HSV gH, HSV gL, HSV gI, HSV gE, or HSV gC.

In some embodiments, a polyribonucleotide according to the present disclosure encodes a polypeptide that comprises one or more HSV antigens or antigenic fragments. In some embodiments, the polypeptide comprises a single HSV antigen or antigenic fragment. In some embodiments, the polypeptide comprises a single HSV antigen. In some embodiments, the HSV antigen is a full length antigen (e.g., a full length glycoprotein). In some embodiments, the HSV antigen is an HSV B cell antigen. In some embodiments, the one or more HSV antigens or antigenic fragments thereof comprise one or more HSV glycoproteins. In some embodiments, the one or more HSV glycoproteins comprises HSV glycoprotein B (gB), HSV glycoprotein E (gE), HSV glycoprotein G (gG), HSV glycoprotein H (gH), HSV glycoprotein I (gL), HSV glycoprotein L (gL), or a combination thereof.

UL27 open reading frame encodes HSV gB. In some embodiments, the HSV antigen (e.g., a B cell antigen of HSV) is or includes HSV gB or a fragment thereof. In various embodiments, the HSV gB or fragment thereof has at least 80% sequence identity to an HSV gB amino acid sequence shown in table 3 or otherwise known in the art, or a corresponding fragment thereof. In some embodiments, the HSV gB polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an HSV gB amino acid sequence shown in table 3 or otherwise known in the art, or a corresponding fragment thereof. Examples of HSV gB known in the art include HSV gB encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, HSV gB or a fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NO. 7, 8, 9 and/or 74. In some embodiments, the HSV gB polypeptide or fragment thereof comprises or consists of the amino acid sequence set forth in SEQ ID NO 7, 8, 9 and/or 74. In some embodiments, the HSV glycoprotein is a full length gB glycoprotein.

US6 open reading frame encodes HSV gD. In some embodiments, the HSV antigen (e.g., a B cell antigen of HSV) is or includes HSV gD or a fragment thereof. In various embodiments, the HSV gD or fragment thereof has at least 80% sequence identity to an HSV gD amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the HSV gD or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an HSV gD amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of HSV gD known in the art include HSV gD encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, HSV gD or a fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid sequence as set forth in SEQ ID NO. 221. In some embodiments, HSV gD or a fragment thereof comprises or consists of the amino acid sequence set forth in SEQ ID NO: 221. In some embodiments, the HSV glycoprotein is a full length gD glycoprotein.

UL44 open reading frame encodes HSV gC. In some embodiments, the HSV antigen (e.g., a B cell antigen of HSV) is or includes HSV gC or a fragment thereof. In various embodiments, the HSV gC or fragment thereof has at least 80% sequence identity to an HSV gC amino acid sequence shown in table 3 or otherwise known in the art. In some embodiments, the HSV gC or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to a US6 amino acid sequence shown in table 3 or otherwise known in the art, or a corresponding fragment thereof. Examples of HSV gcs known in the art include HSV gcs encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, HSV gC or a fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NO: 222. In some embodiments, HSV gC or a fragment thereof comprises or consists of the amino acid sequence set forth in SEQ ID NO: 222. In some embodiments, the HSV glycoprotein is a full length gC glycoprotein.

US1 open reading frame encodes HSV gL. In some embodiments, the HSV antigen (e.g., a B cell antigen of HSV) is or includes HSV gL or a fragment thereof. In various embodiments, the HSV gL or fragment thereof has at least 80% sequence identity to the US1 amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the HSV gL or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an HSV gL amino acid sequence shown in table 3 or otherwise known in the art, or a corresponding fragment thereof. Examples of HSV gL known in the art include HSV gL encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, HSV gL or a fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 58, 59, 60 and/or 61. In some embodiments, HSV gL or a fragment thereof comprises or consists of the amino acid sequences set forth in SEQ ID NO 58, 59, 60 and/or 61. In some embodiments, the HSV glycoprotein is a full length gL glycoprotein.

US7 open reading frame encodes HSV gI. In some embodiments, the HSV antigen (e.g., a B cell antigen of HSV) is or includes HSV gI or a fragment thereof. In various embodiments, the HSV gI or fragment thereof has at least 80% sequence identity to an HSV gI amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the HSV gI or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an HSV gL amino acid sequence shown in table 3 or otherwise known in the art, or a corresponding fragment thereof. Examples of HSV gI known in the art include HSV gI encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, HSV gI or a fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 62, 63, 64 and/or 65. In some embodiments, HSV gI or a fragment thereof comprises or consists of the amino acid sequences set forth in SEQ ID NO:62, 63, 64 and/or 65. In some embodiments, the HSV glycoprotein is a full length gI glycoprotein.

US8 open reading frame encodes HSV gE. In some embodiments, the HSV antigen (e.g., a B cell antigen of HSV) is or includes HSV gE or a fragment thereof. In various embodiments, the HSV gE or fragment thereof has at least 80% sequence identity to an HSV gE amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. In some embodiments, the HSV gI or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an HSV gE amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of HSV ges known in the art include HSV ges encoded by known HSV strains such as (but not limited to) HG52, G, 333, and MS strains. In some embodiments, the US8 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 66, 67, 68 and/or 69. In some embodiments, the HSV gE or fragment thereof comprises or consists of the amino acid sequences set forth in SEQ ID NO:66, 67, 68 and/or 69. In some embodiments, the HSV glycoprotein is a full length gE glycoprotein.

UL22 open reading frame encodes HSV gH. In some embodiments, the HSV antigen (e.g., a B cell antigen of HSV) is or includes HSV gH or a fragment thereof. In various embodiments, the HSV gH, or fragment thereof, has at least 80% sequence identity to an HSV gH amino acid sequence shown in table 3 or otherwise known in the art, or a corresponding fragment thereof. In some embodiments, the HSV gH or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an HSV gH amino acid sequence shown in table 3 or otherwise known in the art or a corresponding fragment thereof. Examples of HSV gH known in the art include HSV gH encoded by known HSV strains such as (but not limited to) HG52, G, 333 and MS strains. In some embodiments, the UL22 polypeptide or fragment thereof has at least 80% sequence identity, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, to an amino acid sequence as set forth in SEQ ID NOs 70, 71, 72 and/or 73. In some embodiments, the HSV gH or fragment thereof comprises or consists of the amino acid sequence set forth in SEQ ID NO 70, 71, 72 and/or 73. In some embodiments, the HSV glycoprotein is a full length gH glycoprotein.

B. secretion signal

In some embodiments, an HSV (e.g., HSV-1 and/or HSV-2) antigen construct described herein comprises a secretion signal that is functional, for example, in a mammalian cell. In some embodiments, the secretion signal utilized is a heterologous secretion signal. In some embodiments, the heterologous secretion signal comprises or consists of a non-human secretion signal. In some embodiments, the heterologous secretion signal comprises or consists of a viral secretion signal. In some embodiments, the viral secretion signal comprises or consists of an HSV secretion signal (e.g., an HSV-1 or HSV-2 secretion signal).

In some embodiments, the secretion signal comprises or consists of an ebola virus secretion signal. In some embodiments, the ebola virus secretion signal comprises or consists of an ebola virus Spike Glycoprotein (SGP) secretion signal.

In some embodiments, the secretion signal is characterized by a length of about 15 to 30 amino acids.

In many embodiments, the secretion signal is located at the N-terminus of an HSV (e.g., HSV-1 and/or HSV-2) antigen construct as described herein. In some embodiments, the secretion signal preferably allows transport of an HSV (e.g., HSV-1 and/or HSV-2) antigen construct associated therewith into a defined cell compartment, preferably a cell surface, endoplasmic Reticulum (ER) or endosomal-lysosomal compartment.

In some embodiments, the secretion signal is selected from the group consisting of an S1S2 signal peptide (e.g., aa 1-19), an immunoglobulin secretion signal (e.g., aa 1-22), an HSV-1gD signal peptide (MGGAAARLGAVILFVVIVGLHGVRSKY; SEQ ID NO: 85), an HSV-2gD signal peptide (MGRLTSGVGTAALLVVAVGLRVVCA; SEQ ID NO: 87); human SPARC signal peptide, human insulin isoform 1 signal, human albumin signal peptide, and the like. Those skilled in the art will be aware of other secretion signals, for example as disclosed in WO2017/081082, which is incorporated herein by reference in its entirety (e.g. SEQ ID NOs: 1-1115 and 1728 or fragment variants thereof).

In some embodiments, an HSV (e.g., HSV-1 and/or HSV-2) antigen construct described herein does not comprise a secretion signal.

In certain embodiments, the signal peptide is an IgG signal peptide, such as an IgG kappa signal peptide.

In some embodiments, the HSV secretion signal comprises or consists of an HSV glycoprotein D (gD) secretion signal.

In some embodiments, the antigen encoded by the string construct sequence may comprise or otherwise be linked to a signal sequence (e.g., a secretion signal), such as those listed in table 7, or at least a sequence having a1, 2,3, 4, or 5 amino acid difference relative thereto. In some embodiments, secretion signals such as MFVFLVLLPLVSSQCVNLT (SEQ ID NO: 90), or at least sequences with 1,2,3, 4 or up to 5 amino acid differences relative thereto, are utilized.

In some embodiments, the secretion signal is selected from the group consisting of a gI signal peptide. In some embodiments, secretion signals such as MPGRSLQGLAILGLWVCATGLVVR (SEQ ID NO: 107), or at least sequences with 1,2, 3, 4 or up to 5 amino acid differences relative thereto, are utilized. In some embodiments, secretion signals such as MPGRSLQGLAILGLWVCATGL (SEQ ID NO: 108), or at least sequences with 1,2, 3, 4 or up to 5 amino acid differences relative thereto, are utilized.

In some embodiments, the secretion signal is a secretion signal listed in table 7 and/or table 8, or a secretion signal having a1, 2, 3, 4, or 5 amino acid difference relative thereto. In some embodiments, the secretion signal is selected from those included in table 7 below and/or those encoded by the sequences in table 8 below.

Table 8: exemplary secretion Signal

Table 9: exemplary Polynucleotide sequences encoding secretion signals

C. transmembrane region

In some embodiments, an HSV (e.g., HSV-1, HSV-2, or both) antigen construct as described herein comprises a transmembrane region. In some embodiments, the transmembrane region is located at the N-terminus of an HSV (e.g., HSV-1, HSV-2, or both) construct. In some embodiments, the transmembrane region is located at the C-terminus of an HSV (e.g., HSV-1, HSV-2, or both) construct. In some embodiments, the transmembrane region is not located at the N-terminus or C-terminus of an HSV (e.g., HSV-1, HSV-2, or both) construct.

The transmembrane region is known in the art, any of which may be used in the HSV (e.g., HSV-1, HSV-2, or both) constructs described herein. In some embodiments, the transmembrane region comprises or is the transmembrane domain of the Hemagglutinin (HA) of influenza virus, env of HIV-1, equine Infectious Anemia Virus (EIAV), murine Leukemia Virus (MLV), murine mammary tumor virus, G protein of Vesicular Stomatitis Virus (VSV), rabies virus, or seven transmembrane domain receptor.

In some embodiments, the heterologous transmembrane region does not include a hemagglutinin transmembrane region. In some embodiments, the heterologous transmembrane region comprises or consists of a non-human transmembrane region. In some embodiments, the heterologous transmembrane region comprises or consists of a viral transmembrane region. In some embodiments, the heterologous transmembrane region comprises or consists of an HSV transmembrane region, such as an HSV-1 or HSV-2 transmembrane region. In some embodiments, the HSV transmembrane region comprises or consists of an HSV gD transmembrane region, e.g., comprising or consisting of amino acid sequence GLIAGAVGGSLLAALVICGIVYWMRRHTQKAPKRIRLPHIR (SEQ ID NO: 160).

In some embodiments, the heterologous transmembrane region comprises or consists of a human transmembrane region. In some embodiments, the human transmembrane region comprises or consists of a human decay accelerating factor glycosyl-phosphatidylinositol (hDAF-GPI) anchor region. In some embodiments, the hDAF-GPI anchor region comprises or consists of the following amino acid sequence: PNKGSGTTSGTTRLLSGHTCFTL TGLLGTLVTMGLLT (SEQ ID NO: 161).

In some embodiments, the transmembrane region utilized is a heterologous transmembrane region.

In some embodiments, an HSV (e.g., HSV-1, HSV-2, or both) construct described herein does not comprise a transmembrane region.

Exemplary transmembrane membranes are provided in table 9 below:

Table 10: exemplary transmembrane region

D. Multimerization domains

In some embodiments, an HSV (e.g., HSV-1, HSV-2, or both) construct as described herein includes one or more multimerization regions (e.g., heteromultimerization regions).

In some embodiments, the heteromultimerization region comprises a dimerization, trimerization, or tetramerization region.

In some embodiments, the multimerization region is that described in WO2017/081082, which is incorporated herein by reference in its entirety (e.g., SEQ ID NOS: 1116-1167, or fragments or variants thereof). Exemplary trimerization and tetramerization regions include, but are not limited to, engineered leucine zippers, fibronectin folding domains from enterophage T4, GCN4pll, GCN4-pll, and p53.

In some embodiments, an HSV (e.g., HSV-1, HSV-2, or both) construct provided herein is capable of forming a trimeric complex. For example, a provided HSV (e.g., HSV-1, HSV-2, or both) construct may include a multimerization region that allows for the formation of multimeric complexes, such as trimeric complexes of an HSV (e.g., HSV-1, HSV-2, or both) construct as described herein. In some embodiments, the multimerization regions that allow for the formation of multimeric complexes include trimerization regions, such as the trimerization regions described herein. In some embodiments, an HSV (e.g., HSV-1, HSV-2, or both) construct includes a T4-fibronectin-derived "foldon" trimerization region, e.g., to increase its immunogenicity. In some embodiments, an HSV (e.g., HSV-1, HSV-2, or both) construct comprises a multimerization region comprising or consisting of: GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 162).

E. joint

In some embodiments, an HSV (e.g., HSV-1, HSV-2, or both) construct described herein comprises one or more linkers. In some embodiments, the linker is or comprises 2, 3,4, 5, 6, 7, 8, 9, 10 or more amino acids. In some embodiments, the linker is or comprises no more than about 30, 25, 20, 15, 10 or fewer amino acids. The linker may comprise any amino acid sequence and is not limited to any particular amino acid. In some embodiments, the linker comprises one or more glycine (G) amino acids. In some embodiments, the linker comprises one or more serine (S) amino acids. In some embodiments, the linker comprises an amino acid selected based on a cleavage predictor to produce a highly cleavable linker.

In some embodiments, the linker is or comprises S-G ₄-S-G₄ -S (SEQ ID NO: 163). In some embodiments, the linker is or comprises GSPGSGSGS (SEQ ID NO: 164). In some embodiments, the linker is or comprises GGSGGGGSGG (SEQ ID NO: 165). In some embodiments, the linker is a linker as set forth in table 10. In some embodiments, the linker is or comprises a sequence as shown in WO2017/081082, which is incorporated herein by reference in its entirety (see SEQ ID NOS: 1509-1565, or fragments or variants thereof).

In some embodiments, an HSV (e.g., HSV-1, HSV-2, or both) construct described herein comprises a linker between the C-terminal region or fragment thereof and the transmembrane region. In some embodiments, an HSV (e.g., HSV-1, HSV-2, or both) construct described herein comprises a linker after the smaller repeat sequence.

Exemplary joints are provided in table 10 below:

Table 11: exemplary Joint

Joint	Sequence (amino acid)	SEQ ID NO
			GSS	SGGGGSGGGGS	163
GSP	GSPGSGSGS	164
			GS	GSGSGS	217
GGS	GGSGGGGSGG	165
			From GGSL	GGSLGGGGSG	166
	GGGGSGGGGS	167
				GGS	168
	GGGS	169
				GGGGSGGGGSGGGGS	170
Furin protease	AGNRVRRSVG	171
			SGG	SGG	172

MHC class I transport signal (MITD)

In some embodiments, an HSV (e.g., HSV-1 and/or HSV-2) antigen used in accordance with the present disclosure includes a transport signal. In some embodiments, the polyribonucleotides encoding one or more HSV (e.g., HSV-1 and/or HSV-2) antigens provided herein include a transport signal. For example, in some embodiments, the transport signal is an MHC class I transport signal (MITD). In some embodiments MITD comprises or consists of the following amino acid sequences: IVGIVAGLAVLAVVVIGAVVATVMCRRKSSG GKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 173). In some embodiments MITD comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to IVGIVAGLAVLAVVVIGAVVATVMCRRK SSGGKGGSYSQAASSDSAQGSDVSLTA (SEQ ID NO: 173).

G. Helper antigens

In some embodiments, an HSV (e.g., HSV-1 and/or HSV-2) antigen used in accordance with the present disclosure includes one or more helper antigens. In some embodiments, the polyribonucleotides provided herein that encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens include one or more helper antigens. Those skilled in the art know that an effective B cell response typically requires assistance from helper T cells. As noted herein, it has been proposed that an effective HSV (e.g., HSV-1 and/or HSV-2) vaccine, particularly if it targets HSV (e.g., HSV-1 and/or HSV-2) proteins (e.g., envelope proteins, membrane proteins, or combinations thereof) expressed prior to cellular infection, may benefit from or require the ability to induce particularly robust antibody responses. In some embodiments, it may be desirable to provide a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) that includes or delivers one or more "helper antigens" (i.e., CD 4T cell antigens) in addition to, for example, one or more B cell antigens and/or epitopes and/or one or more T cell antigens and/or epitopes.

The present disclosure suggests that helper antigens may be particularly useful when the pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) includes or delivers an antigen or antigenic fragment thereof that includes a repeat element. In some embodiments, the provided pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) do not include or deliver antigens that include such repeat elements. Regardless, in some embodiments, the provided pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) do not include or deliver any helper antigen, or at least do not include or deliver helper antigens (e.g., heterologous helper elements) specifically engineered into the antigen.

Where a helper antigen is required, the skilled person is aware of a variety of potentially useful sequences including, for example, those discussed in WO2020128031, which include helper antigens derived from, for example, P2 tetanus toxin, PADRE helper epitopes, hepatitis b surface antigen (HBsAg).

Polyribonucleotides

In many embodiments, the provided pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) deliver an antigen as described herein by delivering a nucleic acid construct, e.g., in many embodiments, an RNA construct encoding one or more antigens as described herein and expressed in a subject upon administration of the pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine).

Among other things, the present disclosure encompasses the recognition that administration of nucleic acids, and particularly RNAs, to achieve delivery of encoded antigens (e.g., by expression) may provide a variety of benefits relative to other strategies for immunization against HSV (e.g., HSV-1 and/or HSV-2) infection.

Among other things, the present disclosure provides insight that, for a variety of reasons, particularly including that RNA may have inherent adjuvanticity, RNA may be particularly useful and/or effective as an active agent in a pharmaceutical composition (e.g., an immunogenic composition such as an HSV (e.g., HSV-1 and/or HSV-2) vaccine). As noted herein, very high antibody titers against HSV (e.g., HSV-1 and/or HSV-2) proteins can be induced, such as those that are expressed and/or targeted, particularly prior to cell invasion.

Further, experience with SARS-CoV-2 vaccine has demonstrated that RNA actives can also elicit significant and diverse T cell responses, particularly when combined with strong antibody responses, representing a combination of immune features that are believed to be likely to maximize the likelihood of protection.

Table 12: exemplary polyribonucleotide antigen sequences

A. exemplary polyribonucleotide features

The polyribonucleotides described herein encode one or more HSV (e.g., HSV-1, HSV-2, or both) constructs described herein. In some embodiments, the polyribonucleotides described herein may comprise a nucleotide sequence encoding a 5'utr of interest and/or a 3' utr of interest. In some embodiments, a polynucleotide described herein may comprise a nucleotide sequence encoding a poly a tail. In some embodiments, the polyribonucleotides described herein can comprise a 5' cap that can be incorporated during transcription or linked to the polyribonucleotide post-transcriptionally.

1.5' Cap

One structural feature of mRNA is the cap structure at the five-terminal end (5'). The natural eukaryotic mRNA contains a 7-methylguanosine cap linked to the mRNA via a 5 'to 5' -triphosphate bridge, resulting in a cap0 structure (m 7 GpppN). In most eukaryotic and some viral mrnas, further modification may occur at the 2 '-hydroxy-group (2' -OH) of the first and subsequent nucleotides (e.g., the 2 '-hydroxy group may be methylated to form 2' -O-Me), resulting in "cap1" and "cap2" five-terminal ends, respectively. Diamond et al, (2014) Cytokine & growth Factor Reviews,25:543-550, incorporated herein by reference in its entirety, report that cap0-mRNA cannot be translated as efficiently as cap1-mRNA, in which the role of 2'-O-Me at the penultimate position of the 5' end of mRNA is decisive. The lack of 2' -O-met has been shown to trigger innate immunity and activate IFN responses. Daffis et al, (2010) Nature,468:452-456; and Tust et al, (2011) Nature Immunology,12:137-143, each of which is incorporated herein by reference in its entirety.

RNA capping has been well studied and is described, for example, in Decroly E et al, (2012) Nature Reviews 10:51-65; and ramamathan a. Et al, (2016) Nucleic Acids Res;44 (16) 7511-7526, the entire contents of each of the above documents are hereby incorporated by reference. For example, in some embodiments, a 5 '-cap structure that may be suitable in the context of the present invention is cap0 (methylation of the first nucleobase, e.g., m7 GpppN), cap1 (additional methylation of ribose of the adjacent nucleotide of m7 GpppN), cap2 (additional methylation of ribose of the 2 nd nucleotide downstream of m7 GpppN), cap3 (additional methylation of ribose of the 3 rd nucleotide downstream of m7 GpppN), cap4 (additional methylation of ribose of the 4 th nucleotide downstream of m7 GpppN), ARCA ("anti-reverse cap analogue"), modified ARCA (e.g., phosphorothioate modified ARCA), inosine, N1-methylguanosine, 2' -fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-aza-guanosine.

The term "5 '-cap" as used herein refers to a structure found at the 5' end of an RNA (e.g., mRNA) and generally includes guanosine nucleotides (also referred to as Gppp or G (5 ') ppp (5')) linked to the RNA (e.g., mRNA) via 5 '-to 5' -triphosphate bonds. In some embodiments, guanosine included in the 5' cap can be modified, e.g., by methylation at one or more positions (e.g., at the 7-position) on the base (guanine) and/or by methylation at one or more positions of the ribose. In some embodiments, the guanosine included in the 5' cap comprises a 3' o methylation at ribose (3 ' ome g). In some embodiments, the guanosine included in the 5' cap comprises a methylation (m 7G) at the 7-position of guanine. In some embodiments, guanosine included in the 5' cap comprises methylation at the 7-position of guanine and 3' o methylation at ribose (m 7 (3 ' ome)). It will be appreciated that the symbols used in the preceding paragraphs, such as "(m ₂ ^7,3'-O) G" or "m7 (3' ome)" apply to other structures described herein.

In some embodiments, providing RNA with a 5 '-cap as disclosed herein can be accomplished by in vitro transcription, wherein the 5' -cap is co-transcriptionally expressed into the RNA strand, or can be post-transcribed using a capping enzyme attached to the RNA. In some embodiments, co-transcription capping with the disclosed caps increases the capping efficiency of RNA compared to co-transcription capping with an appropriate reference comparator. In some embodiments, increasing capping efficiency may increase the translation efficiency and/or translation rate of the RNA, and/or increase expression of the encoded polypeptide. In some embodiments, the alteration of the polynucleotide produces a non-hydrolyzable cap structure that can, for example, prevent uncapping and increase RNA half-life.

In some embodiments, the 5' cap utilized is a cap0, cap1, or cap2 structure. See, for example, FIG. 1 of RAMANATHAN A et al and FIG. 1 of Decroly E et al, each of which is incorporated herein by reference in its entirety. See, for example, FIG. 1 of RAMANATHAN A et al and FIG. 1 of Decroly E et al, each of which is incorporated herein by reference in its entirety. In some embodiments, the RNAs described herein comprise cap1 structures. In some embodiments, the RNA described herein comprises cap2.

In some embodiments, the RNAs described herein comprise cap0 structures. In some embodiments, the cap0 structure comprises a methylated guanosine ((m ⁷) G) at the 7-position of guanine. In some embodiments, such cap0 structure is linked to RNA via a5 '-to 5' -triphosphate bond, and is also referred to herein as (m ⁷) Gppp. In some embodiments, the cap0 structure comprises a methylated guanosine nucleoside at the 2' -position of the ribose of guanosine. In some embodiments, the cap0 structure comprises a methylated guanosine nucleoside at the 3' -position of the ribose of guanosine. In some embodiments, the guanosine included in the 5 'cap comprises methylation ((m ₂ ^7,2'-O) G) at the 7-position of guanine and the 2' -position of ribose. In some embodiments, the guanosine included in the 5 'cap comprises methylation ((m ₂ ^7,3'-O) G) at the 7-position of guanine and the 2' -position of ribose.

In some embodiments, the cap1 structure comprises a guanosine methylated at the 7-position of guanine ((m ⁷) G) and optionally methylated at the 2' or 3' position of ribose and a first nucleotide methylated at the 2' o in RNA ((m ^2'-O)N₁). In some embodiments, the cap1 structure comprises a guanosine methylated at the 7-position of guanine ((m ⁷) G) and methylated at the 3' position of ribose and a first nucleotide methylated at the 2' o in RNA ((m ^2'-O)N₁). In some embodiments, the cap1 structure is linked to RNA via a 5' -to 5' -triphosphate linkage and is also referred to herein as, for example ((m ⁷)Gppp(^2'-O)N₁) or (m ₂ ^7,3'-O)Gppp(^2'-O)N₁)), wherein N ₁ is as defined and described herein.

In some embodiments, cap2 structure comprises guanosine methylated at the 7-position of guanine ((m ⁷) G) and optionally methylated at the 2' or 3' position of ribose and first and second nucleotides methylated at the 2' o in RNA ((m ^2'-O)N₁p(m^2'-O)N₂). In some embodiments, cap2 comprises guanosine methylated at the 7-position of guanine ((m ⁷) G) and 3' position of ribose and first and second nucleotides methylated at the 2' o in RNA.

In some embodiments, the 5' cap is a dinucleotide cap structure. In some embodiments, the 5' cap is a dinucleotide cap structure comprising N ₁, wherein N ₁ is as defined and described herein. In some embodiments, the 5' cap is a dinucleotide cap G x N ₁, wherein N ₁ is as defined above and herein, and:

G comprises the structure of formula (I):

Or a salt thereof,

Wherein the method comprises the steps of

Each of R ² and R ³ is-OH or-OCH ₃; and is also provided with

X is O or S.

In some embodiments, R ² is —oh. In some embodiments, R ² is-OCH ₃. In some embodiments, R ³ is —oh. In some embodiments, R ³ is-OCH ₃. In some embodiments, R ² is-OH and R ³ is-OH. In some embodiments, R ² is-OH and R ³ is-CH ₃. In some embodiments, R ² is-CH ₃ and R ³ is-OH. In some embodiments, R ² is-CH ₃ and R ³ is-CH ₃.

In some embodiments, X is O. In some embodiments, X is S.

In some embodiments, the 5 'cap is a dinucleotide cap0 structure (e.g., ,(m⁷)GpppN₁,(m₂ ^7,2'-O)GpppN₁、(m₂ ^7,3'-O)GpppN₁、(m⁷)GppSpN₁、(m₂ ^7,2'-O)GppSpN₁ or (m ₂ ^7,3'-O)GppSpN₁), wherein N ₁ is as defined and described herein, in some embodiments, the 5' cap is a dinucleotide cap0 structure (e.g., ,(m⁷)GpppN₁、(m₂ ^7,2'-O)GpppN₁、(m₂ ^7,3'-O)GpppN₁、(m⁷)GppSpN₁、(m₂ ^7,2'-O)GppSpN₁ or (m ₂ ^7,3'-O)GppSpN₁), wherein N ₁ is g., in some embodiments, the 5 'cap is a dinucleotide cap0 structure (e.g., ,(m⁷)GpppN₁、(m₂ ^7,2'-O)GpppN₁、(m₂ ^7,3'-O)GpppN₁、(m⁷)GppSpN₁、(m₂ ^7,2'-O)GppSpN₁ or (m ₂ ^7,3'-O)GppSpN₁), wherein N ₁ is A, U or c., in some embodiments, the 5' cap is a dinucleotide cap1 structure (e.g., ,(m⁷)Gppp(m^2'-O)N₁、(m₂ ^7,2'-O)Gppp(m^2'-O)N₁、(m₂ ^7,3'-O)Gppp(m^2'-O)N₁、(m⁷)GppSp(m^2'-O)N₁、(m₂ ^7,2'-O)GppSp(m^2'-O)N₁ or (m ₂ ^7,3'-O)GppSp(m^2'-O)N₁), wherein N ₁ is as defined and described herein, in some embodiments, the 5 'cap is selected from the group ：(m⁷)GpppG("Ecap0")、(m⁷)Gppp(m^2'-O)G("Ecap1")、(m₂ ^7,3'-O)GpppG("ARCA" or "D1") and (m ₂ ^7,2'-O) GppSpG ("β -S-ARCA"). In some embodiments, the 5' cap is (m ⁷) gppppg ("eca 0") having the following structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ⁷)Gppp(m^2'-O) G ("Ecap 1") having the structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ₂ ^7,3'-O) GpppG ("ARCA" or "D1") having the structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ₂ ^7,2'-O) GppSpG ("β -S-ARCA") having the structure:

Or a salt thereof.

In some embodiments, the 5' cap is a trinucleotide cap structure. In some embodiments, the 5' cap is a trinucleotide cap structure comprising N ₁pN₂, wherein N ₁ and N ₂ are as defined and described herein. In some embodiments, the 5' cap is a dinucleotide cap G x N ₁pN₂, wherein N ₁ and N ₂ are as defined above and herein, and:

G comprises the structure of formula (I):

Or a salt thereof, wherein R ²、R³ and X are as defined and described herein.

In some embodiments, the 5 'cap is a trinucleotide cap0 structure (e.g., (m ⁷)GpppN₁pN₂、(m₂ ^7,2'-O)GpppN₁pN₂ or (m ₂ ^7,3'-O)GpppN₁pN₂) where N ₁ and N ₂ are as defined and described herein). In some embodiments, the 5' cap is a trinucleotide cap1 structure (e.g., ,(m⁷)Gppp(m^2'-O)N₁pN₂、(m₂ ^7,2'-O)Gppp(m^2'-O)N₁pN₂、(m₂ ^7,3'-O)Gppp(m^2'-O)N₁pN₂), where N ₁ and N ₂ are as defined and described herein). In some embodiments, the 5 'cap is a trinucleotide cap2 structure (e.g., ,(m⁷)Gppp(m^2'-O)N₁p(m^2'-O)N₂、(m₂ ^7,2'-O)Gppp(m^2'-O)N₁p(m^2'-O)N₂、(m₂ ^7,3'-O)Gppp(m^2'-O)N₁p(m^2'-O)N₂), where N ₁ and N ₂ are as defined and described herein). In some embodiments, the 5' cap is selected from the group consisting of ：(m₂ ^7,3'-O)Gppp(m^2'-O)ApG("CleanCap AG","CC413")、(m₂ ^7,3'-O)Gppp(m^2'-O)GpG("CleanCap GG")、(m⁷)Gppp(m^2'-O)ApG、(m⁷)Gppp(m^2'-O)GpG、(m₂ ^7,3'-O)Gppp(m₂ ^6,2'-O)ApG and (m ⁷)Gppp(m^2'-O) ApU.

In some embodiments, the 5' cap is (m ₂ ^7,3'-O)Gppp(m^2'-O) ApG ("CLEANCAP AG", "CC 413") having the following structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ₂ ^7,3'-O)Gppp(m^2'-O) GpG ("CLEANCAP GG") having the structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ⁷)Gppp(m^2'-O) ApG, having the following structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ⁷)Gppp(m^2'-O) GpG, having the structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ₂ ^7,3'-O)Gppp(m₂ ^6,2'-O) ApG, having the following structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ⁷)Gppp(m^2'-O) ApU, having the following structure:

Or a salt thereof.

In some embodiments, the 5' cap is a tetranucleotide cap structure. In some embodiments, the 5' cap is a tetranucleotide cap structure comprising N ₁pN₂pN₃, wherein N ₁、N₂ and N ₃ are as defined and described herein. In some embodiments, the 5' cap is a tetranucleotide cap g×n ₁pN₂pN₃, wherein N ₁、N₂ and N ₃ are as defined above and herein, and:

G comprises the structure of formula (I):

Or a salt thereof, wherein R ²、R³ and X are as defined and described herein.

In some embodiments, the 5 'Cap is a tetranucleotide Cap0 structure (e.g., (m⁷)GpppN₁pN₂pN₃、(m₂ ^7,2'-O)GpppN₁pN₂pN₃ or (m ₂ ^7,3'-O)GpppN₁N₂pN₃, where N ₁、N₂ and N ₃ are as defined and described herein). In some embodiments, the 5' Cap is a tetranucleotide Cap1 structure (e.g., ,(m⁷)Gppp(m^2'-O)N₁pN₂pN₃、(m₂ ^7,2'-O)Gppp(m^2'-O)N₁pN₂pN₃、(m₂ ^7,3'-O)Gppp(m^2'-O)N₁pN₂N₃),, where N ₁、N₂ and N ₃ are as defined and described herein; in some embodiments, the 5 'Cap is a tetranucleotide Cap2 structure (e.g., ,(m⁷)Gppp(m^2'-O)N₁p(m^2'-O)N₂pN₃、(m₂ ^7,2'-O)Gppp(m^2'-O)N₁p(m^2'-O)N₂pN₃、(m₂ ^7,3'-O)Gppp(m^2'-O)N₁p(m^2'-O)N₂pN₃),, where N ₁、N₂ and N ₃ are as defined and described herein; in some embodiments, the 5' Cap is selected from the group ：(m₂ ^7,3'-O)Gppp(m^2'-O)Ap(m^2'-O)GpG、(m₂ ^7,3'-O)Gppp(m^2'-O)Gp(m^2'-O)GpC、(m⁷)Gppp(m^2'-O)Ap(m^2'-O)UpA and (m ⁷)Gppp(m^2'-O)Ap(m^2'-O) consisting of.

In some embodiments, the 5' cap is (m ₂ ^7,3'-O)Gppp(m^2'-O)Ap(m^2'-O) GpG, having the structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ₂ ^7,3'-O)Gppp(m^2'-O)Gp(m^2'-O) GpC, having the structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ⁷)Gppp(m^2'-O)Ap(m^2'-O) UpA, having the following structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ⁷)Gppp(m^2'-O)Ap(m^2'-O) GpG, having the structure:

Or a salt thereof.

2. Cap proximal sequence

In some embodiments, the 5' utr utilized in accordance with the present disclosure comprises a cap proximal sequence, e.g., as disclosed herein. In some embodiments, the cap proximal sequence comprises a sequence adjacent to a 5' cap. In some embodiments, the cap proximal sequence comprises nucleotides at positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide.

In some embodiments, the cap structure comprises one or more polynucleotides of the cap proximal sequence. In some embodiments, the cap structure comprises nucleotide +1 (N ₁) of the m ⁷ guanosine cap and the RNA polynucleotide. In some embodiments, the cap structure comprises nucleotide +2 (N ₂) of the m ⁷ guanosine cap and the RNA polynucleotide. In some embodiments, the cap structure comprises the m ⁷ guanosine cap and nucleotides +1 and +2 (N ₁ and N ₂) of the RNA polynucleotide. In some embodiments, the cap structure comprises the m ⁷ guanosine cap and nucleotides +1, +2, and +3 (N ₁、N₂ and N ₃) of the RNA polynucleotide.

Those of skill in the art reading this disclosure will appreciate that in some embodiments, one or more residues of the cap proximal sequence (e.g., one or more of residues +1, +2, +3, +4, and/or +5) may be included in the RNA as a result of having been included in the cap entity (e.g., cap1 or cap2 structure, etc.); or in some embodiments, at least some of the residues in the cap proximal sequence may be added enzymatically (e.g., by a polymerase, such as a T7 polymerase). For example, in certain exemplary embodiments in which m ₂ ^7,3'-OGppp(m₁ ^2'-O) ApG caps are utilized, +1 (i.e., N ₁) and +2 (i.e., N ₂) are capped (m ₁ ^2'-O) a and G residues, and +3, +4, and +5 are added by a polymerase (e.g., T7 polymerase).

In some embodiments, the 5 'cap is a dinucleotide cap structure, wherein the cap proximal sequence comprises N ₁ of the 5' cap, wherein N ₁ is any nucleotide, e.g., A, C, G or U. In some embodiments, the 5 'cap is a trinucleotide cap structure (e.g., a trinucleotide cap structure described above and herein), wherein the cap proximal sequence comprises N ₁ and N ₂ of the 5' cap, wherein N ₁ and N ₂ are independently any nucleotide, e.g., A, C, G or U. In some embodiments, the 5 'cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure as described above and herein), wherein the cap proximal sequence comprises N ₁、N₂ and N ₃ of the 5' cap, wherein N ₁、N₂ and N ₃ are any nucleotide, e.g., A, C, G or U.

In some embodiments, for example where the 5 'cap is a dinucleotide cap structure, the cap proximal sequence comprises N ₁ of the 5' cap, and N ₂、N₃、N₄ and N ₅, wherein N ₁ to N ₅ correspond to positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide. In some embodiments, for example where the 5 'cap is a trinucleotide cap structure, the cap proximal sequence comprises N ₁ and N ₂, and N ₃、N₄ and N ₅ of the 5' cap, wherein N ₁ to N ₅ correspond to positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide. In some embodiments, for example where the 5 'cap is a tetranucleotide cap structure, the cap proximal sequence comprises N ₁、N₂ and N ₃, and N ₄ and N ₅ of the 5' cap, wherein N ₁ to N ₅ correspond to positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide.

In some embodiments, N ₁ is a. In some embodiments, N ₁ is C. In some embodiments, N ₁ is G. In some embodiments, N ₁ is U. In some embodiments, N ₂ is a. In some embodiments, N ₂ is C. In some embodiments, N ₂ is G. In some embodiments, N ₂ is U. in some embodiments, N ₃ is a. In some embodiments, N ₃ is C. In some embodiments, N ₃ is G. In some embodiments, N ₃ is U. In some embodiments, N ₄ is a. In some embodiments, N ₄ is C. In some embodiments, N ₄ is G. In some embodiments, N ₄ is U. In some embodiments, N ₅ is a. In some embodiments, N ₅ is C. In some embodiments, N ₅ is G. In some embodiments, N ₅ is U. it will be appreciated that each of the embodiments described above and herein (e.g., for N ₁ to N ₅) may be employed alone or in combination, and/or may be combined with other embodiments of the variables (e.g., 5' caps) described above and herein.

In some embodiments, the Cap proximal sequence comprises a ₁ and G ₂ of the Cap1 structure and comprises at positions +3, +4, and +5 of the polyribonucleotide, respectively: a ₃A₄U₅ (SEQ ID NO: 207).

3.5’UTR

In some embodiments, nucleic acids (e.g., DNA, RNA) utilized in accordance with the present disclosure comprise a 5' -UTR. In some embodiments, the 5' -UTR may comprise a plurality of different sequence elements; in some embodiments, such multiple may be or comprise multiple copies of one or more particular sequence elements (e.g., may be from a particular source or otherwise referred to as functional or characteristic sequence elements). In some embodiments, the 5' utr comprises a plurality of different sequence elements.

The term "untranslated region" or "UTR" is commonly used in the art for a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA polynucleotide, such as an mRNA molecule. The untranslated region (UTR) may be present 5 '(upstream) of the open reading frame (5' -UTR) and/or 3 '(downstream) of the open reading frame (3' -UTR). As used herein, the term "five-terminal untranslated region" or "5'utr" refers to a sequence of a polyribonucleotide between the 5' end of the polyribonucleotide (e.g., transcription initiation site) and the start codon of the coding region of the polyribonucleotide. In some embodiments, a "5'utr" refers to a sequence of a polyribonucleotide that begins at the 5' end of the polyribonucleotide (e.g., transcription start site) and ends one nucleotide (nt) before the start codon of the coding region of the polyribonucleotide (typically AUG), e.g., in its natural context. In some embodiments, the 5' utr comprises a Kozak sequence. The 5' -UTR is downstream of the 5' -cap (if present), e.g. directly adjacent to the 5' -cap. In some embodiments, the 5' utrs disclosed herein comprise cap proximal sequences, e.g., as defined and described herein. In some embodiments, the cap proximal sequence comprises a sequence adjacent to a 5' cap.

Exemplary 5' UTRs include human alpha globulin (hAg) 5' UTR or fragments thereof, TEV 5' UTR or fragments thereof, HSP70 5' UTR or fragments thereof, or c-Jun 5' UTR or fragments thereof. In some embodiments, the RNAs disclosed herein comprise hAg' utrs or fragments thereof.

In some embodiments, the RNAs disclosed herein comprise a 5'utr having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 5' utr having sequence AGAAT AAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCG CCACC (SEQ ID NO: 208). In some embodiments, the RNAs disclosed herein comprise a 5' UTR having the sequence AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAG AGAACCCGCCACC (SEQ ID NO: 208).

In some embodiments, the RNAs disclosed herein comprise 5' utrs having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 5' utr having sequence AACUA GUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 209) (hAg-Kozak/5 ' utr). In some embodiments, the RNA disclosed herein comprises a 5'UTR having the sequence AACUAGUAUUCUUCUGGUCCCCACAGACUCAGA GAGAACCCGCCACC (SEQ ID NO: 209) (hAg-Kozak/5' UTR).

4. Poly A tail

In some embodiments, a polynucleotide (e.g., DNA, RNA) disclosed herein comprises a poly-a sequence, e.g., as described herein. In some embodiments, the poly a sequence is located downstream of the 3'-UTR, e.g., adjacent to the 3' -UTR.

As used herein, the term "poly (a) sequence" or "poly-a tail" refers to an uninterrupted or intermittent sequence of adenylate residues typically located at the 3' end of an RNA polynucleotide. Poly (a) sequences are known to those skilled in the art and may follow the 3' -UTR in the RNAs described herein. The uninterrupted poly (A) sequence is characterized by consecutive adenylate residues. In nature, uninterrupted poly (A) sequences are typical. In some embodiments, a polynucleotide disclosed herein comprises an uninterrupted poly (a) sequence. In some embodiments, a polynucleotide disclosed herein comprises a discontinuous poly (a) sequence. In some embodiments, the RNAs disclosed herein can have a poly (a) sequence that is linked to the free 3' end of the RNA by a template-independent RNA polymerase after transcription or a poly (a) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

A poly (a) sequence of about 120 a nucleotides has been shown to have a beneficial effect on RNA levels in transfected eukaryotic cells as well as on the levels of proteins translated by the open reading frame present upstream (5') of the poly (a) sequence (Holtkamp et al, 2006, blood, volume 108, pages 4009-4017, incorporated herein by reference).

In some embodiments, poly (a) sequences according to the present disclosure are not limited to a particular length; in some embodiments, the poly (a) sequence is of any length. In some embodiments, the poly (a) sequence comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and at most 500, at most 400, at most 300, at most 200, or at most 150 a nucleotides, and in particular about 120 a nucleotides. In this context, "consisting essentially of" means that most of the nucleotides in the poly (a) sequence are typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% a nucleotides by number of nucleotides in the poly (a) sequence, but the remaining nucleotides are allowed to be nucleotides other than a nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid) or C nucleotides (cytidylic acid). In this context, "consisting of" means that all nucleotides in the poly (a) sequence, i.e., 100% of the nucleotides in the poly (a) sequence are a nucleotides. The term "a nucleotide" or "a" refers to an adenylate.

In some embodiments, the poly (a) sequence is linked during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylates) in the strand complementary to the coding strand. The DNA sequence encoding the poly (A) sequence (coding strand) is referred to as the poly (A) cassette.

In some embodiments, the poly (a) cassette present in the DNA coding strand consists essentially of dA nucleotides, but is interrupted by random sequences of four nucleotides (dA, dC, dG, and dT). Such random sequences may be 5 to 50, 10 to 30 or 10 to 20 nucleotides in length. Such a cartridge is disclosed in WO 2016/005324A1, which is hereby incorporated by reference. Any of the poly (A) cassettes disclosed in WO 2016/005324A1, incorporated herein by reference, may be used in accordance with the present disclosure. Poly (a) cassettes consisting essentially of dA nucleotides but interrupted by a random sequence of four nucleotides (dA, dC, dG, dT) equally distributed and for example 5 to 50 nucleotides in length show constant propagation of plasmid DNA in e.coli at the DNA level and are still related to beneficial properties in terms of supporting RNA stability and translation efficiency, covering at the RNA level. In some embodiments, the poly (a) sequence contained in the RNA polynucleotides described herein consists essentially of a nucleotides, but is interrupted by a random sequence of four nucleotides (A, C, G, U). Such random sequences may be 5 to 50, 10 to 30 or 10 to 20 nucleotides in length.

In some embodiments, no nucleotide other than the a nucleotide is flanking the 3 'end of the poly (a) sequence, i.e., the poly (a) sequence is not masked or followed at its 3' end by a nucleotide other than a.

In some embodiments, the poly (a) sequence can comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and at most 500, at most 400, at most 300, at most 200, or at most 150 nucleotides. In some embodiments, the poly (a) sequence may consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly (a) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and at most 500, at most 400, at most 300, at most 200, or at most 150 nucleotides. In some embodiments, the poly (a) sequence comprises at least 100 nucleotides. In some embodiments, the poly (a) sequence comprises about 150 nucleotides. In some embodiments, the poly (a) sequence comprises about 120 nucleotides.

In some embodiments, the poly-a tail comprises a specific number of adenosines, such as about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 120 or about 150 or about 200. In some embodiments, the poly a tail of the string construct may comprise 200 or fewer a residues. In some embodiments, the poly a tail of the string construct may comprise about 200 a residues. In some embodiments, the poly a tail of the string construct may comprise 180 or fewer a residues. In some embodiments, the poly a tail of the string construct may comprise about 180 a residues. In some embodiments, the poly a tail may comprise 150 residues or less.

In some embodiments, the RNA comprises: a poly (a) sequence comprising nucleotide sequence AAAAAAAA AAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAA(SEQ ID NO:210) or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to nucleotide sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACT AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:210). In some embodiments, the poly (a) tail comprises a plurality of a residues interrupted by a linker. In some embodiments, the linker comprises nucleotide sequence GCATA TGAC (SEQ ID NO: 211).

In some embodiments, the RNA comprises: a poly (a) sequence comprising nucleotide sequence AAAAAAAA AAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAA(SEQ ID NO:212) or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to nucleotide sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGAC UAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:213). In some embodiments, the poly (a) tail comprises a plurality of a residues interrupted by a linker. In some embodiments, the linker comprises nucleotide sequence GCA UAUGAC (SEQ ID NO: 214).

5.3’UTR

In some embodiments, the RNA utilized according to the present disclosure comprises a 3' -UTR.

As used herein, the term "three terminal untranslated region", "3 'untranslated region" or "3' utr" refers to a sequence of an mRNA molecule that follows a stop codon that begins with the coding region of an open reading frame sequence. In some embodiments, the 3' utr begins immediately after the stop codon of the coding region of the open reading frame sequence, e.g., in its natural context. In other embodiments, the 3' utr does not begin immediately after the stop codon of the coding region of the open reading frame sequence, e.g., in its natural context. The term "3' -UTR" preferably excludes poly (A) sequences. Thus, the 3' -UTR is upstream of the poly (a) sequence (if present), e.g. immediately adjacent to the poly (a) sequence.

In some embodiments, the RNAs disclosed herein comprise a 3' utr comprising an F element and/or an I element. In some embodiments, the 3' utr or proximal sequence thereof comprises a restriction site. In some embodiments, the restriction site is a BamHI site. In some embodiments, the restriction site is an XhoI site.

In some embodiments, the RNA construct comprises an F element. In some embodiments, the F element sequence is the 3' -UTR of a split amino terminal enhancer (AES).

In some embodiments, the RNAs disclosed herein comprise a 3'utr having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 3' utr having sequence CTGGTA CTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACC(SEQ ID NO:215). In some embodiments, the RNAs disclosed herein comprise a 3' utr having sequence CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACC(SEQ ID NO:215).

In some embodiments, the RNA disclosed herein comprises the 3' UTR provided in SEQ ID NO. 215.

In some embodiments, the RNAs disclosed herein comprise a 3'utr having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 3' utr having sequence CUGGU ACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACC(SEQ ID NO:216). In some embodiments, the RNAs disclosed herein comprise a 3' utr having sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACC(SEQ ID NO:216).

In some embodiments, the RNA disclosed herein comprises the 3' UTR provided in SEQ ID NO. 216.

In some embodiments, the 3' utr is an FI element as described in WO2017/060314, which patent document is incorporated herein by reference in its entirety.

RNA forms

At least three different forms have been developed that can be used in RNA compositions (e.g., pharmaceutical compositions), namely unmodified uridine-containing mRNA (uRNA), nucleoside modified mRNA (modRNA), and self-amplified mRNA (saRNA). These platforms are each featured. These platforms are each featured. In general, in all three forms, the RNA is capped, contains an Open Reading Frame (ORF) flanking an untranslated region (UTR), and has a poly-A tail at the 3' end. The ORF of uRNA and modRNA vectors encodes an antibody agent or fragment thereof. saRNA has multiple ORFs.

In some embodiments, the RNAs described herein can have modified nucleosides. In some embodiments, the RNA comprises a modified nucleoside in place of at least one (e.g., each) uridine.

The term "uracil" as used herein describes one of the nucleobases that may occur in a nucleic acid of an RNA. The uracil has the structure:

the term "uridine" as used herein describes one of the nucleosides that may occur in RNA. The structure of uridine is:

UTP (5' -uridine triphosphate) has the following structure:

pseudo-UTP (pseudouridine 5' -triphosphate) has the following structure:

"pseudouridine" is an example of a modified nucleoside that is an isomer of uridine in which uracil is linked to the pentose ring via a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond.

Another exemplary modified nucleoside is N1-methyl-pseudouridine (m 1 ψ), which has the structure:

N1-methyl-pseudo-UTP has the following structure:

Another exemplary modified nucleoside is 5-methyl-uridine (m 5U), which has the structure:

In some embodiments, one or more uridine in the RNAs described herein is replaced with a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

In some embodiments, the RNA comprises a modified nucleoside that replaces at least one uridine. In some embodiments, the RNA comprises a modified nucleoside in place of each uridine.

In some embodiments, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m 5U). In some embodiments, the RNA may comprise more than one type of modified nucleoside, and the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleosides include pseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleosides include pseudouridine (ψ) and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleosides include N1-methyl-pseudouridine (m 1 ψ) and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleosides include pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U).

In some embodiments, the modified nucleoside that replaces one or more (e.g., all) uridine in the RNA can be any one or more of the following: 3-methyl-uridine (m 3U), 5-methoxy-uridine (mo 5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2U), 4-thio-uridine (s 4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), Uridine 5-glycollic acid (cmo 5U), uridine 5-glycollic acid methyl ester (mcmo U), 5-carboxymethyl-uridine (cm 5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm U), 5-methoxycarbonylmethyl-uridine (mcm 5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5s 2U), 5-aminomethyl-2-thio-uridine (nm 5s 2U), 5-methylaminomethyl-uridine (mcm 5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mcm 5s 2U), 5-methyl-2-seleno-uridine (mnm 5se 2U), 5-carbamoylmethyl-uridine (ncm U), 5-carboxymethyl aminomethyl-uridine (cmnm U), 5-carboxymethyl aminomethyl-2-thio-uridine (cmnm s 2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s 2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m 5s 2U), 1-methyl-4-thio-pseudouridine (m 1s 4. Phi.), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3. Phi.), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5, 6-dihydrouridine, 5-methyl-dihydrouridine (m 5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 2-thio-pseudouridine, N1-methyl-pseudouridine, 3- (3-amino-3-carboxypropyl) uridine (acp 3U), 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine (acp 3. Phi.), 5- (isopentenyl aminomethyl) uridine (mm 5U), 5- (isopentenyl aminomethyl) -2-thio-uridine (mm 5s 2U), alpha-thio-uridine, 2' -O-methyl-uridine (Um), 5,2' -O-dimethyl-uridine (m 5 Um), 2' -O-methyl-pseudouridine (. Phi. ', 2-thio-2 ' -O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2 ' -O-methyl-uridine (mcm 5 Um), alpha-thio-uridine, 2' -O-methyl-uridine (mm 5 Um), 5-carbamoylmethyl-2 ' -O-methyl-uridine (ncm Um), 5-carboxymethylaminomethyl-2 ' -O-methyl-uridine (cmnm Um), 3,2' -O-dimethyl-uridine (m 3 Um), 5- (isopentenylaminomethyl) -2' -O-methyl-uridine (mm 5 Um), 1-thio-uridine, deoxythymidine, 2' -F-arabino-uridine, 2' -F-uridine, 2' -OH-arabino-uridine, 5- (2-carboxymethoxyvinyl) uridine, 5- [3- (1-E-propenyl) amino) uridine or any other modified uridine known in the art.

In some embodiments, the RNA comprises other modified nucleosides or comprises further modified nucleosides, such as modified cytidine. For example, in some embodiments, 5-methylcytidine is partially or fully substituted, preferably fully substituted, in RNA. In some embodiments, the RNA comprises 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U). In some embodiments, the RNA comprises 5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA comprises 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine (m1ψ) in place of each uridine.

In some embodiments of the disclosure, the RNA is a "replicon RNA" or simply "replicon", particularly a "self-replicating RNA" or a "self-amplifying RNA". In a particularly preferred embodiment, the replicon or self-replicating RNA is derived from or comprises elements derived from a single stranded (ss) RNA virus, in particular a positive stranded ssRNA virus such as alphavirus. Alphaviruses are typically representative of positive strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for reviews of the life cycle of alphaviruses, see Jos e et al, future microbiol.,2009, volume 4, pages 837-856, incorporated herein by reference in its entirety). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and genomic RNAs typically have a 5 'cap and a 3' poly (a) tail. The genome of alphaviruses encodes nonstructural proteins (involved in transcription, modification and replication of viral RNA, and protein modification) and structural proteins (forming viral particles). There are typically two Open Reading Frames (ORFs) in the genome. Four nonstructural proteins (nsP 1-nsP 4) are usually encoded together by a first ORF starting near the 5 'end of the genome, while the alphavirus structural proteins are encoded together by a second ORF found downstream of the first ORF and extending near the 3' end of the genome. Typically, the first ORF is larger than the second ORF in a ratio of about 2:1. In cells infected with an alphavirus, only the nucleic acid sequence encoding the nonstructural protein is translated from genomic RNA, while the genetic information encoding the structural protein can be translated from subgenomic transcripts, which are RNA molecules resembling eukaryotic messenger RNA (mRNA; gould et al, 2010,Antiviral Res, vol. 87, pages 111-124, incorporated herein by reference in its entirety). After infection, i.e., at an early stage of the viral life cycle, (+) strand genomic RNA acts directly like messenger RNA for translation of the open reading frame encoding the nonstructural polyprotein (nsP 1234).

Alphavirus-derived vectors have been proposed for delivering foreign genetic information into target cells or organisms. In a simple approach, the first ORF encodes an RNA-dependent RNA polymerase (replicase) of alphavirus origin, which mediates self-amplification of RNA after translation. The second ORF encoding the alphavirus structural protein is replaced with an open reading frame encoding an HSV (HSV-1 and/or HSV-2) construct as described herein. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase and the other nucleic acid molecule is capable of trans-replication by the replicase (hence the name trans-replication system). Trans-replication requires the simultaneous presence of both nucleic acid molecules in a given host cell. Nucleic acid molecules capable of trans-replication by replicase enzymes must contain certain alphavirus sequence elements to allow recognition and RNA synthesis by the alphavirus replicase enzymes.

Characteristics of the unmodified uridine platform may include, for example, one or more of intrinsic adjuvant effects and good tolerability and safety. Characteristics of modified uridine (e.g., pseudouridine) platforms can include reduced adjuvant effects, inactivated immune innate immunity sensor activation capability, and thus good tolerability and safety. Features of the self-amplifying platform may include, for example, long duration protein expression, good tolerance and safety, higher likelihood of achieving efficacy at very low vaccine doses.

The present disclosure provides specific RNA constructs that are optimized, for example, for improved manufacturability, encapsulation, expression levels (and/or timing), and the like. Certain components are discussed below and certain preferred embodiments are exemplified herein.

C. Codon optimization and GC enrichment

As used herein, the term "codon optimization" refers to altering codons in the coding region of a nucleic acid molecule (e.g., a polyribonucleotide) to reflect typical codon usage of a host organism (e.g., a subject receiving the nucleic acid molecule (e.g., a polyribonucleotide), rather than preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, in some embodiments, the coding region is codon optimized for optimal expression in a subject to be treated with an RNA molecule described herein. In some embodiments, codon optimization can be performed such that codons available to insert frequently occurring tRNAs replace "rare codons". In some embodiments, codon optimization may include increasing the guanosine/cytosine (G/C) content of the coding region of an RNA described herein as compared to the corresponding coding sequence of a wild-type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified as compared to the amino acid sequence.

In some embodiments, the coding sequence (also referred to as a "coding region") is codon optimized for expression in a subject (e.g., a human) to whom the composition (e.g., a pharmaceutical composition) is to be administered. Thus, in some embodiments, the sequence in such a polynucleotide (e.g., a polyribonucleotide) may differ from the wild-type sequence encoding the antigen of interest or an antigenic fragment or epitope thereof, even when the amino acid sequence of the antigen or an antigenic fragment or epitope thereof is wild-type.

In some embodiments, the codon-optimized strategy is for expression in a subject of interest (e.g., a human), and even in some cases in a particular cell or tissue.

In general, as understood, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a subject or a cell of interest thereof by: replacing at least one codon (e.g., about or more than about 1,2,3, 4,5, 10, 15, 20, 25, 50 or more codons) of the native sequence with a more or most frequently used codon in the gene of the subject or cell thereof, while maintaining the native amino acid sequence.

Different species exhibit specific bias for certain codons of a particular amino acid. Without wishing to be bound by any one theory, codon bias (the difference in codon usage between organisms) is generally related to the efficiency of translation of messenger RNA (mRNA), which in turn is believed to depend, among other things, on the nature of the codons being translated and the availability of a particular transfer RNA (tRNA) molecule. The dominance of the selected tRNA in the cell can generally reflect codons that are most frequently used in peptide synthesis. Thus, genes can be tailored based on codon optimization to achieve optimal gene expression in a given organism. The codon usage tables may be obtained, for example, in the "codon usage database" available at www.kazusa.orjp/codon/and may be adjusted in a number of ways. Computer algorithms are also available for codon optimization of specific sequences for expression in a specific subject or cell thereof, such as Gene force (Aptagen; jacobus, pa.).

In some embodiments, the polynucleotides (e.g., polyribonucleotides) of the present disclosure are codon-optimized, wherein the codons in the polynucleotide (e.g., polyribonucleotides) accommodate the use of human codons (referred to herein as "human codon-optimized polynucleotides"). Codons encoding the same amino acid occur at different frequencies in a subject (e.g., a human). Thus, in some embodiments, the coding sequences of the polynucleotides of the present disclosure are modified such that the frequency of codons encoding the same amino acid corresponds to the frequency with which the codons naturally occur according to human codon usage, e.g., as shown in table 12. For example, in the case of amino acid Ala, it is preferable to adjust the wild-type coding sequence such that the frequency of use of codon "GCC" is 0.40, the frequency of use of codon "GCT" is 0.28, the frequency of use of codon "GCA" is 0.22, and the frequency of use of codon "GCG" is 0.10 or the like (see table 12). Thus, in some embodiments, this procedure (as exemplified for Ala) is applied to each amino acid encoded by the coding sequence of the polynucleotide to obtain a sequence that is adapted for human codon usage.

Table 13: human codon usage indicating the frequency of each amino acid

Certain strategies for codon optimization and/or G/C enrichment for human expression are described in WO2002/098443, which is incorporated herein by reference in its entirety. In some embodiments, a multiparameter optimization strategy may be employed to optimize the coding sequence. In some embodiments, the optimization parameters may include parameters that affect protein expression, which may be affected, for example, at the transcriptional level, the mRNA level, and/or the translational level. In some embodiments, exemplary optimization parameters include, but are not limited to, transcriptional level parameters (including, for example, GC content, consensus splice site, cryptic splice site, SD sequence, TATA box, termination signal, artificial recombination site, and combinations thereof); mRNA level parameters (including, for example, RNA instability motifs, ribosome entry sites, repeat sequences, and combinations thereof); translation level parameters (including, for example, codon usage, premature poly (a) sites, ribosome entry sites, secondary structure, and combinations thereof); or a combination thereof. In some embodiments, the coding sequence may be optimized by the GeneOptimizer algorithm as described in Fath et al ,"Multiparameter RNA and Codon Optimization:A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression"PLoS ONE 6(3):e17596;Rabb et al ,"The GeneOptimizer Algorithm:using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization"Systems and Synthetic Biology(2010)4:215-225; and Graft et al ,"Codon-optimized genes that enable increased heterologous expression in mammalian cells and elicit efficient immune responses in mice after vaccination of naked DNA"Methods Mol Med(2004)94:197-210, the entire contents of each of which are incorporated herein for the purposes described herein. In some embodiments, the coding sequence may be optimized by adaptation of Eurofins and optimization algorithm "GENEius" as described in Eurofins'Application Notes:Eurofins'adaption and optimization software"GENEius"in comparison to other optimization algorithms, the entire contents of which are incorporated by reference for the purposes described herein.

In some embodiments, coding sequences utilized in accordance with the present disclosure have increased G/C content as compared to coding sequences of HSV (e.g., HSV-1 and/or HSV-2 polypeptides or fragments thereof) constructs described herein. In some embodiments, the guanosine/cytidine (G/C) content of the coding region is altered relative to a comparable coding sequence of an HSV (e.g., HSV-1 and/or HSV-2 polypeptide or fragment thereof) construct described herein, but the amino acid sequence encoded by the polyribonucleotide is not altered.

Without wishing to be bound by any particular theory, it is proposed that GC enrichment may improve translation of the payload sequence. In general, sequences with increased G (guanosine)/C (cytidine) content are more stable than sequences with increased A (adenosine)/U (uridine) content. Regarding the fact that several codons encode the same amino acid (so-called degeneracy of the genetic code), the most advantageous codons for stability (so-called substitution codon usage) can be determined. Depending on the amino acid to be encoded by the polyribonucleotide, modifications of the ribonucleic acid sequence compared with the wild-type sequence are possible in various ways. In particular, codons containing a and/or U nucleosides can be modified by replacing these codons with other codons encoding the same amino acid but not containing a and/or U or containing a lower content of a and/or U nucleosides.

In some embodiments, the G/C content of the coding region of a polyribonucleotide described herein is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6% or even more compared to the G/C content of the coding region prior to codon optimization of, for example, wild-type RNA. In some embodiments, the G/C content of the coding region of a polyribonucleotide described herein is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6% or even more compared to the G/C content of the coding region prior to codon optimization of, for example, wild-type RNA.

In some embodiments, to increase the stability and translation efficiency of a polyribonucleotide, one or more elements can be added that determine the stability and/or translation efficiency that contribute to the polyribonucleotide; exemplary such elements are described, for example, in PCT/EP2006/009448, which is incorporated herein by reference. In some embodiments, to increase expression of a polyribonucleotide used according to the present disclosure, the polyribonucleotide may be modified within the coding region (i.e., the sequence encoding the expressed peptide or protein) without altering the sequence of the expressed peptide or protein, e.g., in order to increase GC content, to increase mRNA stability and/or to perform codon optimization, thereby enhancing translation in the cell.

RNA delivery techniques

The provided polyribonucleotides can be delivered using any suitable method known in the art for therapeutic applications described herein, including, for example, as naked RNA delivery, or delivery mediated by viral and/or non-viral vectors, polymer-based vectors, lipid compositions, nanoparticles (e.g., lipid nanoparticles, polymer nanoparticles, lipid-polymer hybrid nanoparticles, etc.), and/or peptide-based vectors. See, e.g., wadhwa et al, "Opportuni TIES AND CHALLENGES IN THE DELIVERY of mRNA-Based Vaccines" Pha rmaceutics (page 2020) 102 (page 27), which is incorporated herein by reference for the content of information about the various methods that can be used to deliver the polyribonucleotides described herein.

In some embodiments, one or more polyribonucleotides can be formulated for delivery (e.g., administration) with a lipid nanoparticle.

In some embodiments, the lipid nanoparticle may be designed to protect the polyribonucleotide from extracellular rnase and/or engineered to deliver RNA systemically to target cells. In some embodiments, such lipid nanoparticles are particularly useful for delivering polyribonucleotides when the polyribonucleotides are administered intravenously or intramuscularly to a subject.

A. Lipid composition

1. Lipid and lipid-like substance

The terms "lipid" and "lipid-like substance" are defined herein broadly as molecules comprising one or more hydrophobic moieties or groups and optionally also comprising one or more hydrophilic moieties or groups. Molecules comprising a hydrophobic portion and a hydrophilic portion are also often referred to as amphiphilic molecules. Lipids are generally poorly soluble in water. In an aqueous environment, amphiphilic properties enable molecules to self-assemble into organized structures and distinct phases. One of these phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes or membranes in an aqueous environment. Hydrophobicity may be imparted by the inclusion of polar groups including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups, as well as such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups may include polar and/or charged groups and include carbohydrate, phosphate, carboxyl, sulfate, amino, mercapto, nitro, hydroxyl, and the like.

Typically amphiphilic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar segment is soluble in water, while the non-polar segment is insoluble in water. Furthermore, the polar moiety may have a formal positive or formal negative charge. Or the polar moiety may have both formal positive and negative charges and be a zwitterionic or an inner salt. For the purposes of this disclosure, amphiphilic compounds may be, but are not limited to, one or more natural or non-natural lipids and lipid-like compounds.

A "lipid-like substance" is a substance that is structurally and/or functionally related to a lipid, but cannot be considered to be a lipid in a strict sense. For example, the term includes compounds capable of forming amphiphilic layers, as they are present in vesicles, multilamellar/unilamellar liposomes or membranes in an aqueous environment, and include surfactants or synthetic compounds having hydrophilic and hydrophobic portions. In general, the term refers to molecules comprising hydrophilic and hydrophobic portions having different structural organization, which may or may not be similar to lipids.

Specific examples of amphiphilic compounds that may be included in the amphiphilic layer include, but are not limited to, phospholipids, amino lipids, and sphingolipids.

In general, lipids can be divided into eight classes: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from condensation of ketoacyl subunits), sterols, and isopentenol lipids (derived from condensation of isoprene subunits). While the term "lipid" is sometimes used as a synonym for fat, fat is a subset of lipids, known as triglycerides. Lipids also encompass molecules such as fatty acids and derivatives thereof (including triglycerides, diglycerides, monoglycerides and phospholipids) and metabolites containing sterols such as cholesterol.

Fatty acids are a diverse group of molecules consisting of hydrocarbon chains ending in carboxylic acid groups; this arrangement imparts a polar hydrophilic end and a water-insoluble non-polar hydrophobic end to the molecule. The carbon chain length, typically between four carbons and 24 carbons, may be saturated or unsaturated and may be linked to functional groups containing oxygen, halogen, nitrogen and sulfur. If the fatty acid contains a double bond, there is a possibility of cis or trans geometric isomerism, which can significantly affect the configuration of the molecule. Cis double bonds cause bending of the fatty acid chains, and more double bonds in the chain exacerbate this effect. Other major classes of lipids in the fatty acid class are fatty acid esters and fatty amides.

Glycerolipids consist of mono-, di-and trisubstituted glycerins, most notably fatty acid triesters of glycerol, known as triglycerides. The term "triacylglycerols" is sometimes used synonymously with "triglycerides". In these compounds, the three hydroxyl groups of glycerol are typically each esterified with a different fatty acid. Another subclass of glycerolipids is represented by glycosylglycerols, characterized by the presence of one or more sugar residues linked to glycerol via glycosidic linkages.

Glycerophospholipids are amphiphilic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" via an ester linkage and to one "head" group via a phosphate linkage. Examples of glycerophospholipids commonly referred to as phospholipids (although sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).

Sphingolipids are members of a complex family of compounds that share a common structural feature (i.e., a sphingoid base backbone). The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramide (N-acyl-sphingoid base) is a major subclass of sphingoid base derivatives with amide linked fatty acids. Fatty acids are generally saturated or monounsaturated, with chain lengths of 16 to 26 carbon atoms. The main sphingomyelin of mammals is sphingomyelin (ceramide phosphorylcholine), whereas insects mainly contain ceramide phosphorylethanolamine, and fungi have phytoceramide phosphorylinositol and mannose-containing head groups. Glycosphingolipids are a diverse family of molecules consisting of one or more sugar residues linked via glycosidic linkages to a sphingoid base. Examples of these are simple and complex glycosphingolipids, such as cerebrosides and gangliosides.

Sterols such as cholesterol and its derivatives or tocopherol and its derivatives together with glycerophospholipids and sphingomyelins are important components of membrane lipids.

Glycolipids are compounds in which fatty acids are directly linked to the sugar backbone, thereby forming a structure compatible with the membrane bilayer. In glycolipids, monosaccharides replace the glycerol backbone in glycerolipids and glycerophospholipids. The most common glycolipids are acylated glucosamine precursors of the lipid a component of lipopolysaccharide in gram-negative bacteria. A typical lipid a molecule is a disaccharide of glucosamine, which is derivatized with up to seven fatty acyl chains. The smallest lipopolysaccharide required for growth in E.coli is Kdo 2-lipid A, which is the hexaacylated disaccharide of glucosamine, glycosylated with two 3-deoxy-D-mannose-octanoonic acid (Kdo) residues.

Polyketides are synthesized by classical enzymes and iterative and multi-modular enzymes that polymerize acetyl and propionyl subunits sharing the mechanism features common to fatty acid synthases. They include a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have a great structural diversity. Many polyketides are cyclic molecules whose backbone is typically further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

Lipids and lipid-like substances may be cationic, anionic or neutral. Neutral lipids or lipid-like substances exist in uncharged or neutral zwitterionic forms at a selected pH.

In some embodiments, suitable lipids or lipid-like substances in the present disclosure include those described in WO2020/128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

2. Cationic or cationically ionizable lipids or lipid-like substances

In some embodiments, the cationic or cationically ionizable lipids or lipid-like substances contemplated for use herein include any cationic or cationically ionizable lipid or lipid-like substance capable of electrostatically binding nucleic acids. In one embodiment, a cationic or cationically ionizable lipid or lipid-like substance as contemplated herein may be associated with a nucleic acid, for example, by forming a complex with the nucleic acid or forming a vesicle in which the nucleic acid is occluded or encapsulated.

Cationic lipids or lipid-like substances are characterized in that they have a net positive charge (e.g., at an associated pH). Cationic lipids or lipid-like substances bind negatively charged nucleic acids by electrostatic interactions. Generally, cationic lipids have a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries a positive charge.

In certain embodiments, the cationic lipid or lipid-like substance has a net positive charge only at a certain pH, in particular an acidic pH, whereas it preferably has no net positive charge at a different, preferably higher pH (e.g. physiological pH), preferably has no charge, i.e. it is neutral at a different, preferably higher pH such as physiological pH. This ionizable behavior is believed to enhance efficacy by helping endosomal escape and reducing toxicity compared to particles that remain cationic at physiological pH.

In some embodiments, the cationic or cationically ionizable lipid or lipid-like substance comprises a head group comprising at least one positively charged or capable of being protonated nitrogen atom (N).

Examples of cationic lipids include, but are not limited to, 1, 2-dioleoyl-3-trimethylammoniopropane (DOTAP); n, N-dimethyl-2, 3-dioleoyloxypropylamine (DODMA), 1, 2-di-O-octadecenyl-3-trimethylammoniopropane (DOTMA), 3- (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol (DC-Chol), dimethyl Dioctadecyl Ammonium (DDAB); 1, 2-dioleoyl-3-dimethylammonium-propane (DODAP); 1, 2-diacyloxy-3-dimethylammonium propane; 1, 2-dialkoxy-3-dimethylammonium propane; Dioctadecyl dimethyl ammonium chloride (DODAC), 1, 2-distearyloxy-N, N-dimethyl-3-aminopropane (DSDMA), 2, 3-di (tetradecyloxy) propyl- (2-hydroxyethyl) -dimethyl azonia (DMRIE), 1, 2-dimyristoyl-sn-glycero-3-ethyl choline phosphate (DMEPC), l, 2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1, 2-dioleoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE) and 2, 3-dioleoyloxy-N- [2 (spermidine) ethyl ] -N, N-dimethyl-l-propylammonium trifluoroacetate (DOSPA), 1, 2-Dioleoyloxy-N, N-dimethylaminopropane (DLinDMA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLenDMA), dioctadecylamido Gan Xianji spermine (DOGS), 3-dimethylamino-2- (cholest-5-en-3- β -oxobutane-4-yloxy) -1- (cis, cis-9, 12-octadecadienyloxy) propane (CLinDMA), 2- [5'- (cholest-5-en-3- β -oxy) -3' -oxapentoxy) -3-dimethyl-1- (cis, cis-9 ',12' -octadecadienyloxy) propane (CpLinDMA), N, N-dimethyl-3, 4-Dioleyloxybenzylamine (DMOBA), 1,2-N, N '-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 2, 3-dioleoyloxy-N, N-dimethylpropylamine (DLinDAP), 1,2-N, N' -dioleylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), 1, 2-dioleylcarbamoyl-3-dimethylaminopropane (DLinCDAP), 2-dioleylidene-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA), 2, 2-Di-lino-4-dimethylaminoethyl- [1,3] -dioxolane (DLin-K-XTC 2-DMA), 2-Di-lino-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin-KC 2-DMA), thirty-seven carbon-6,9,28,31-tetraen-19-yl-4- (dimethylamino) butanoate (DLin-MC 3-DMA), N- (2-hydroxyethyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) -1-propanammonium bromide (DMRIE), (+ -) -N- (3-aminopropyl) -N, N-dimethyl-2, 3-bis (cis-9-tetradecyloxy) -1-propanammonium bromide (GAP-DMO), (±) -N- (3-aminopropyl) -N, N-dimethyl-2, 3-bis (dodecyloxy) -1-propanaminium bromide (GAP-DLRIE), (±) -N- (3-aminopropyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) -1-propanaminium bromide (GAP-dmriie), N- (2-aminoethyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) -1-propanaminium bromide (βae-dmriie), N- (4-carboxybenzyl) -N, N-dimethyl-2, 3-bis (oleoyloxy) propane-1-ammonium (DOBAQ), 2- ({ 8- [ (3β) -cholest-5-en-3-yloxy ] Octyl } oxy) -N, N-dimethyl-3- [ (9 z,12 z) -octadeca-9, 12-dien-1-yloxy ] propan-1-amine (Octyl-CLinDMA), 1, 2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1, 2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1- [2- ((1S) -1- [ (3-aminopropyl) amino ] -4- [ bis (3-aminopropyl) amino ] butylcarboxamido) ethyl ] -3, 4-bis [ oleoyloxy ] -benzamide (MVL 5), 1, 2-dioleoyl-sn-glycero-3-ethyl phosphorylcholine (DOEPC), 2, 3-bis (dodecyloxy) -N- (2-hydroxyethyl) -N, N-dimethylpropan-1-aminium bromide (DLRIE), N- (2-aminoethyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) propan-1-aminium bromide (DMORIE), di ((Z) -non-2-en-1-yl) 8,8' - ((((2 (dimethylamino) ethyl) thio) carbonyl) Azanedioctanoate (ATX), N-dimethyl-2, 3-bis (dodecyloxy) propan-1-amine (DLDMA), N, N-dimethyl-2, 3-bis (tetradecyloxy) propan-1-amine (DMDMA), di ((Z) -non-2-en-1-yl) -9- ((4- (dimethylaminobutyryl) oxy) heptadecanedioate (L319), N-dodecyl-3- ((2-dodecylcarbamoyl-ethyl) - {2- [ (2-dodecylcarbamoyl-ethyl) -2- { (2-dodecylcarbamoyl-ethyl) - [2- (2-dodecylcarbamoyl-ethylamino) -ethyl ] -amino } -ethylamino) propanamide (lipid 98N 12-5), 1- [2- [ bis (2-hydroxydodecyl) amino ] ethyl- [2- [4- [2- [ bis (2 hydroxydodecyl) amino ] ethyl ] piperazin-1-yl ] ethyl ] amino ] dodecyl-2-ol (lipid C12-200),(Commercially available cationic liposomes comprising DOTMA and 1, 2-dioleoyl-sn-3 phosphoethanolamine (DOPE), from GIBCO/BRL, grand Island, N.Y.); (commercially available cationic liposomes comprising N- (1- (2, 3 dioleyloxy) propyl) -N- (2- (spermidine carboxamido) ethyl) -N, N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and (Commercially available cationic lipids comprising dioctadecyl amido Gan Xianji carboxy spermine (DOGS) in ethanol from Promega Corp., madison, wis.) or any combination of any of the foregoing. Cationic lipids further suitable for use in the present disclosure include those described in WO2020/128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein. Cationic lipids further suitable for use in the present disclosure include those described in WO2010/053572 (including Cl 2-200 described in paragraph [00225 ]) and WO2012/170930, both of which are incorporated herein by reference for the purposes described herein. Additional suitable cationic lipids for use in the present disclosure include HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see US20150140070A1, which is incorporated herein by reference in its entirety).

In some embodiments, a formulation useful in a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) as described herein may comprise at least one cationic lipid. Representative cationic lipids include, but are not limited to, 1, 2-dioleoyl-3- (dimethylamino) acetoxypropane (DLin-DAC), 1, 2-dioleoyl-3-morpholinopropane (DLin-MA), 1, 2-dioleoyl-3-dimethylaminopropane (DLinDAP), 1, 2-dioleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1, 2-dioleyloxy-3-trimethylaminopropane chloride salt (DLin-tma. Ci), 1, 2-dioleoyl-3-trimethylaminopropane chloride salt (DLin-tap. Ci), 1, 2-dioleoyl-3- (N-methylpiperazino) propane (DLin-MPZ), 3- (N, N-diileyloxy) -1, 2-propanediol (DLinAP), 1- (N, 2-dioleyloxy) -3-dimethylaminopropane (DLin-2-DMAP), 1, 2-dioleoyl-3-trimethylaminopropane chloride salt (DLin-tap. Ci), 1, 2-dioleoyl-3- (N-methylpiperazino) propane (DLin-MPZ), 3- (N, N-diileoyl-3-propanediol) and 2- [ 2-dioleoyl-3-trimethylaminopropane chloride salt (DLin-tap. Ci) 2, 2-diiodo-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin-KC 2-DMA); diiodoyl-methyl-4-dimethylaminobutyrate (DLin-MC 3-DMA); MC3 (US 20100324120, incorporated herein by reference in its entirety).

In some embodiments, amino or cationic lipids useful according to the present disclosure have at least one protonatable or deprotonated group such that the lipid is positively charged at a pH equal to or below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably equal to or above physiological pH. Of course, it will be understood that the addition or removal of protons is an equilibrium process, depending on the pH, and that reference to charged or neutral lipids refers to the nature of the main species and does not require that all lipids must be present in charged or neutral form. Lipids having more than one protonatable or deprotonated group or lipids that are zwitterionic are not excluded and are equally suitable in the context of the present invention.

In some embodiments, the protonatable groups of the protonatable lipids have a pKa in the range of about 4 to about 11, for example, a pKa of about 5 to about 7.

In some embodiments, the cationic lipids can comprise from about 10 mole% to about 100 mole%, from about 20 mole% to about 100 mole%, from about 30 mole% to about 100 mole%, from about 40 mole% to about 100 mole%, or from about 50 mole% to about 100 mole% of the total lipids present in the lipid compositions utilized in accordance with the present disclosure.

3. Additional lipid or lipid-like substances

In some embodiments, the formulations utilized in accordance with the present disclosure may comprise lipids or lipid-like substances other than cationic or cationically ionizable lipids or lipid-like substances, i.e., non-cationic lipids or lipid-like substances (including non-cationically ionizable lipids or lipid-like substances). Anionic and neutral lipids or lipid-like substances are collectively referred to herein as non-cationic lipids or lipid-like substances. In some embodiments, optimizing the formulation of nucleic acid particles by adding other hydrophobic moieties such as cholesterol and lipids in addition to ionizable/cationic lipids or lipid-like substances may, for example, enhance particle stability and efficacy of nucleic acid delivery.

In some embodiments, lipids or lipid-like substances may be incorporated that may or may not affect the overall charge of the particle. In certain embodiments, such lipid or lipid-like substance is a non-cationic lipid or lipid-like substance.

In some embodiments, the non-cationic lipid may include, for example, one or more anionic lipids and/or neutral lipids. An "anionic lipid" is negatively charged (e.g., at a selected pH).

"Neutral lipids" exist in uncharged or neutral zwitterionic forms (e.g., at a selected pH). In some embodiments, the formulation comprises one of the following neutral lipid components: (1) phospholipids, (2) cholesterol or derivatives thereof; or (3) a mixture of phospholipids and cholesterol or derivatives thereof. Examples of cholesterol derivatives include, but are not limited to, cholesterol, cholestanone, cholestenone, faecal alkanol, cholesteryl-2 '-hydroxyethyl ether, cholesteryl-4' -hydroxybutyl ether, tocopherols and derivatives thereof, and mixtures thereof.

Specific exemplary phospholipids that may be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. Such phospholipids include inter alia diacyl phosphatidyl choline, such as distearoyl phosphatidyl choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dimyristoyl phosphatidyl choline (DMPC), dipentadecyl phosphatidyl choline, dilauroyl phosphatidyl choline (DPPC), ditolybdyl phosphatidyl choline (DAPC), dicabehenyl phosphatidyl choline (DBPC), ditridecyl phosphatidyl choline (DTPC), dimolyl phosphatidyl choline (DLPC), palmitoyl Oleoyl Phosphatidyl Choline (POPC), 1, 2-di-O-octadecenyl-sn-glycero-3-phosphoryl choline (18:0 diether PC), 1-oleoyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphoryl choline (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphoryl choline (C16 Lyso PC) and phosphatidylethanolamine, in particular diacyl phosphatidyl ethanolamine, such as distearoyl phosphatidyl ethanolamine (DOPE), distearoyl phosphatidyl ethanolamine (DSPE), dicarboxyl phosphatidylethanolamine (DPPE), and phosphatidyl ethanolamine (DPPE) have the same hydrophobicity as the phosphatidyl choline (dpp), the phosphatidyl ethanolamine (DPPE), the phosphatidyl ethanolamine has more hydrophobic chains.

In certain embodiments, the formulation utilized in accordance with the present disclosure includes DSPC or DSPC and cholesterol.

In certain embodiments, the formulations utilized in accordance with the present disclosure include both cationic lipids and additional (non-cationic) lipids.

In some embodiments, the formulations herein include a polymer conjugated lipid, such as a pegylated lipid. "PEGylated lipids" comprise a lipid moiety and a polyethylene glycol moiety. Pegylated lipids are known in the art.

Without wishing to be bound by theory, the amount of (total) cationic lipids may affect important properties such as charge, particle size, stability, tissue selectivity and biological activity of the nucleic acid compared to the amount of other lipids in the formulation. In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.

In some embodiments, the non-cationic lipid, particularly the neutral lipid (e.g., one or more phospholipids and/or cholesterol), may comprise from about 0 mol% to about 90 mol%, from about 0 mol% to about 80 mol%, from about 0 mol% to about 70 mol%, from about 0 mol% to about 60 mol%, or from about 0 mol% to about 50 mol% of the total lipid present in the formulation.

4. Liposome complex particles

In certain embodiments of the present disclosure, the RNA described herein may be present in RNA liposome complex particles.

The "RNA liposome complex particles" contain lipids (particularly cationic lipids) and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNAs lead to complexation and spontaneous formation of RNA liposome complex particles. Positively charged liposomes can generally be synthesized using cationic lipids such as DOTMA and additional lipids such as DOPE. In one embodiment, the RNA liposome complex particles are nanoparticles.

In certain embodiments, the RNA liposome complex particles comprise both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In particular embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.

In some embodiments, the RNA liposome complex particles have an average diameter in one embodiment ranging from about 200nm to about 1000nm, from about 200nm to about 800nm, from about 250nm to about 700nm, from about 400nm to about 600nm, from about 300nm to about 500nm, or from about 350nm to about 400nm. In specific embodiments, the RNA liposome complex particles have an average diameter of about 200nm, about 225nm, about 250nm, about 275nm, about 300nm, about 325nm, about 350nm, about 375nm, about 400nm, about 425nm, about 450nm, about 475nm, about 500nm, about 525nm, about 550nm, about 575nm, about 600nm, about 625nm, about 650nm, about 700nm, about 725nm, about 750nm, about 775nm, about 800nm, about 825nm, about 850nm, about 875nm, about 900nm, about 925nm, about 950nm, about 975nm, or about 1000 nm. In one embodiment, the average diameter of the RNA liposome complex particles ranges from about 250nm to about 700nm. In another embodiment, the average diameter of the RNA liposome complex particles ranges from about 300nm to about 500nm. In an exemplary embodiment, the average diameter of the RNA liposome complex particles is about 400nm.

The RNA liposome complex particles and compositions comprising the RNA liposome complex particles described herein can be used to deliver RNA to a target tissue following parenteral administration, particularly following intravenous administration. RNA liposome complex particles can be prepared using liposomes, which can be obtained by injecting a solution of lipids in ethanol into water or a suitable aqueous phase. In one embodiment, the aqueous phase has an acidic pH. In one embodiment, the aqueous phase comprises acetic acid, for example in an amount of about 5mM. Liposomes can be used to prepare RNA liposome complex particles by mixing the liposomes with RNA. In one embodiment, the liposome and RNA liposome complex particles comprise at least one cationic lipid and at least one additional lipid. In one embodiment, the at least one cationic lipid comprises 1, 2-di-O-octadecenyl-3-trimethylammoniopropane (DOTMA) and/or 1, 2-dioleoyl-3-trimethylammoniopropane (DOTAP). In one embodiment, the at least one additional lipid comprises 1, 2-di- (9Z-octadecenoyl) -sn-glycerol-3-phosphate ethanolamine (DOPE), cholesterol (Chol), and/or 1, 2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC). In one embodiment, the at least one cationic lipid comprises 1, 2-di-O-octadecenyl-3-trimethylammoniopropane (DOTMA), and the at least one additional lipid comprises 1, 2-di- (9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the liposome and RNA liposome complex particles comprise 1, 2-di-O-octadecenyl-3-trimethylammoniopropane (DOTMA) and 1, 2-di- (9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine (DOPE).

Spleen-targeting RNA liposome complex particles are described in WO 2013/143683, which is incorporated herein by reference. RNA liposome complex particles with a net negative charge have been found to be useful for preferentially targeting spleen tissue or spleen cells, such as antigen presenting cells, particularly dendritic cells. Thus, after administration of the RNA liposome complex particles, RNA accumulation and/or RNA expression occurs in the spleen. Thus, the RNA liposome complex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, no or substantially no RNA accumulation and/or RNA expression occurs in the lung and/or liver following administration of the RNA liposome complex particles. In one embodiment, RNA accumulation and/or RNA expression occurs in antigen presenting cells (e.g., professional antigen presenting cells in the spleen) after administration of the RNA liposome complex particles. Thus, the RNA liposome complex particles of the present disclosure can be used to express RNA in such antigen presenting cells. In one embodiment, the antigen presenting cells are dendritic cells and/or macrophages.

5. Lipid Nanoparticles (LNP)

In some embodiments, the nucleic acids described herein, such as RNA, are administered in the form of Lipid Nanoparticles (LNPs). In some embodiments, the LNP may comprise any lipid capable of forming a particle, one or more nucleic acid molecules attached to the particle, or one or more nucleic acid molecules encapsulated in the particle.

In some embodiments, the LNP comprises one or more cationic lipids and one or more stabilized lipids. Stabilized lipids include neutral lipids and pegylated lipids.

In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, a sterol, a polymer conjugated lipid; and RNA encapsulated within or associated with the lipid nanoparticle.

In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.

In some embodiments, the sterol is cholesterol.

In some embodiments, the polymer conjugated lipid is a pegylated lipid. In some embodiments, the pegylated lipid has the following structure:

or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

R ¹² and R ¹³ are each independently a linear or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester linkages; and w has an average value in the range of 30 to 60. In some embodiments, R ¹² and R ¹³ are each independently a straight saturated alkyl chain containing from 12 to 16 carbon atoms. In some embodiments, w has an average value in the range of 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R ¹² and R ¹³ are each independently a straight saturated alkyl chain containing about 14 carbon atoms, and w has an average value of about 45.

In some embodiments, the pegylated lipid is DMG-PEG 2000, e.g., having the following structure:

in some embodiments, the cationic lipid component of the LNP has the structure of formula (III):

Or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

One of L ¹ or L ² is –O(C＝O)-、-(C＝O)O-、-C(＝O)-、-O-、-S(O)_x-、-S-S-、-C(＝O)S-、SC(＝O)-、-NR^aC(＝O)-、-C(＝O)NR^a-、NR^aC(＝O)NR^a-、-OC(＝O)NR^a- or-NR ^a C (=o) O-, and the other of L ¹ or L ² is –O(C＝O)-、-(C＝O)O-、-C(＝O)-、-O-、-S(O)_x-、-S-S-、-C(＝O)S-、SC(＝O)-、-NR^aC(＝O)-、-C(＝O)NR^a-、NR^aC(＝O)NR^a-、-OC(＝O)NR^a- or-NR ^a C (=o) O-, or a direct bond;

Each of G ¹ and G ² is independently unsubstituted C ₁-C₁₂ alkylene or C ₁-C₁₂ alkenylene;

G ³ is C ₁-C₂₄ alkylene, C ₁-C₂₄ alkenylene, C ₃-C₈ cycloalkylene, C ₃-C₈ cycloalkenyl;

R ^a is H or C ₁-C₁₂ alkyl;

R ¹ and R ² are each independently C ₆-C₂₄ alkyl or C ₆-C₂₄ alkenyl;

R ³ is H, OR ⁵、CN、-C(＝O)OR⁴、-OC(＝O)R⁴ or-NR ⁵C(＝O)R⁴;

R ⁴ is C ₁-C₁₂ alkyl;

r ⁵ is H or C ₁-C₆ alkyl; and is also provided with

X is 0, 1 or 2.

In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

Wherein:

a is a3 to 8 membered cycloalkyl or cycloalkylene ring;

R ⁶ is independently at each occurrence H, OH or C ₁-C₂₄ alkyl; and is also provided with

N is an integer from 1 to 15.

In some of the foregoing embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

In other embodiments of formula (III), the lipid has one of the following structures (IIIC) or (IIID):

wherein y and z are each independently integers in the range of 1 to 12.

In any of the foregoing embodiments of formula (III), one of L ¹ or L ² is-O (c=o) -. For example, in some embodiments, each of L ¹ and L ² is-O (c=o) -. In some different embodiments of any of the foregoing, L ¹ and L ² are each independently- (c=o) O-or-O (c=o) -. For example, in some embodiments, each of L ¹ and L ² is- (c=o) O-.

In some different embodiments of formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):

In some of the foregoing embodiments of formula (III), n is an integer in the range of 2 to 12, for example 2 to 8 or 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

In some other of the foregoing embodiments of formula (III), y and z are each independently an integer in the range of 2 to 10. For example, in some embodiments, y and z are each independently integers in the range of 4 to 9 or 4 to 6.

In some of the foregoing embodiments of formula (III), R ⁶ is H. In other preceding embodiments, R ⁶ is C ₁-C₂₄ alkyl. In other embodiments, R ⁶ is OH.

In some embodiments of formula (III), G ³ is unsubstituted. In other embodiments, G3 is substituted. In various embodiments, G ³ is a linear C ₁-C₂₄ alkylene or linear C ₁-C₂₄ alkenylene.

In some other of the foregoing embodiments of formula (III), R ¹ or R ², or both, are C ₆-C₂₄ alkenyl. For example, in some embodiments, R ¹ and R ² each independently have the following structure:

Wherein:

R ^7a and R ^7b are independently at each occurrence H or C ₁-C₁₂ alkyl; and is also provided with

A is an integer from 2 to 12, and

Wherein R ^7a、R^7b and a are each selected such that R ¹ and R ² each independently contain from 6 to 20 carbon atoms. For example, in some embodiments, a is an integer in the range of 5 to 9 or 8 to 12.

In some of the foregoing embodiments of formula (III), R ^7a is H in at least one occurrence. For example, in some embodiments, R ^7a is H at each occurrence. In various other embodiments of the foregoing, R ^7b in at least one occurrence is C ₁-C₈ alkyl. For example, in some embodiments, the C ₁-C₈ alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

In various embodiments of formula (III), R ¹ or R ², or both, have one of the following structures:

In some of the foregoing embodiments of formula (III), R ³ is OH, CN, -C (=o) OR ⁴、-OC(＝O)R⁴, OR-NHC (=o) R ⁴. In some embodiments, R ⁴ is methyl or ethyl.

In various embodiments, the cationic lipid of formula (III) has one of the structures shown in table 13 below.

Table 14: exemplary compounds of formula (III).

In various embodiments, the cationic lipid has one of the structures shown in table 14 below.

Table 15: exemplary cationic lipid Structure

In some embodiments, the LNP comprises a cationic lipid as an ionizable lipid-like substance (lipid). In some embodiments, the cationic lipid has the following structure:

In some embodiments, the lipid nanoparticle may have an average size (e.g., average diameter) of about 30nm to about 150nm, about 40nm to about 150nm, about 50nm to about 150nm, about 60nm to about 130nm, about 70nm to about 110nm, about 70nm to about 100nm, about 70nm to about 90nm, or about 70nm to about 80 nm. In some embodiments, lipid nanoparticles according to the present disclosure may have an average size (e.g., average diameter) of about 50nm to about 100 nm. In some embodiments, the lipid nanoparticle may have an average size (e.g., average diameter) of about 50nm to about 150 nm. In some embodiments, the lipid nanoparticle may have an average size (e.g., average diameter) of about 60nm to about 120 nm. In some embodiments, lipid nanoparticles according to the present disclosure may have an average size (e.g., average diameter) of about 30nm、35nm、40nm、45nm、50nm、55nm、60nm、65nm、70nm、75nm、80nm、85nm、90nm、95nm、100nm、105nm、110nm、115nm、120nm、125nm、130nm、135nm、140nm、145nm or 150 nm. The term "average diameter" or "median diameter" refers to the average hydrodynamic diameter of particles measured by dynamic laser light scattering (DLS), and data analysis uses a so-called cumulant algorithm which as a result provides a so-called Z-average with length dimensions and a dimensionless Polydispersity Index (PI) (Koppel, d., j.chem. Phys.57,1972, pages 4814-4820, ISO 13321, incorporated herein by reference). The terms "average diameter", "median diameter", "diameter" or "size" of the particles are used synonymously herein with the mean value of Z.

In some embodiments, the lipid nanoparticles described herein can exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. For example, the lipid nanoparticle may exhibit a polydispersity index in the range of about 0.1 to about 0.3 or about 0.2 to about 0.3. The "polydispersity index" is preferably calculated on the basis of dynamic light scattering measurements by so-called cumulant analysis as mentioned in the definition of "average diameter". Under certain preconditions, it may be considered a measure of the size distribution of the ribonucleic acid nanoparticle (e.g., ribonucleic acid nanoparticle) as a whole.

The lipid nanoparticles described herein can be characterized by an "N/P ratio", which is the molar ratio of cationic (nitrogen) groups (N "in N/P) to anionic (phosphate) groups (P" in N/P) in RNA in a cationic polymer. It is understood that a cationic group is a group in the cationic form (e.g., N ⁺) or a group that can be ionized into a cation. The use of a single number in the N/P ratio (e.g., an N/P ratio of about 5) means that the number is greater than 1, e.g., an N/P ratio of about 5 is intended to represent 5:1. In some embodiments, the lipid nanoparticles described herein have an N/P ratio of greater than or equal to 5. In some embodiments, the lipid nanoparticle described herein has an N/P ratio of about 5, 6, 7, 8, 9, or 10. In some embodiments, the lipid nanoparticle described herein has an N/P ratio of about 10 to about 50. In some embodiments, the lipid nanoparticle described herein has an N/P ratio of about 10 to about 70. In some embodiments, the lipid nanoparticle described herein has an N/P ratio of about 10 to about 120.

B. Exemplary methods of preparing lipid nanoparticles

Lipids and lipid nanoparticles comprising nucleic acids and methods of making the same are known in the art and include, for example, those described in the following patent documents: U.S. patent nos. 8,569,256, 5,965,542 and 2016/0199485、2016/0009637、2015/0273068、2015/0265708、2015/0203446、2015/0005363、2014/0308304、2014/0200257、2013/086373、2013/0338210、2013/0323269、2013/0245107、2013/0195920、2013/0123338、2013/0022649、2013/0017223、2012/0295832、2012/0183581、2012/0172411、2012/0027803、2012/0058188、2011/0311583、2011/0311582、2011/0262527、2011/0216622、2011/0117125、2011/0091525、2011/0076335、2011/0060032、2010/0130588、2007/0042031、2006/0240093、2006/0083780、2006/0008910、2005/0175682、2005/017054、2005/0118253、2005/0064595、2004/0142025、2007/0042031、1999/009076 and PCT publication nos. WO 99/39741、WO 2018/081480、WO 2017/004143、WO 2017/075531、WO 2015/199952、WO 2014/008334、WO 2013/086373、WO 2013/086322、WO 2013/016058、WO 2013/086373、W02011/141705 and WO 2001/07548, the entire disclosures of each of which are incorporated herein by reference in their entirety for the purposes described herein.

For example, in some embodiments, the cationic lipid, neutral lipid (e.g., DSPC and/or cholesterol), and polymer conjugated lipid may be dissolved in ethanol at a predetermined molar ratio (e.g., the molar ratios described herein). In some embodiments, the lipid nanoparticle (lipid nanoparticle) is prepared with a total lipid to polyribonucleotide weight ratio of about 10:1 to 30:1. In some embodiments, such polyribonucleotides can be diluted to 0.2mg/mL in acetate buffer.

In some embodiments, using ethanol injection techniques, a colloidal lipid dispersion comprising polyribonucleotides can be formed as follows: an ethanol solution comprising lipids (such as cationic lipids, neutral lipids, and polymer conjugated lipids) is injected into an aqueous solution comprising polyribonucleotides (e.g., polyribonucleotides described herein).

In some embodiments, the lipid and polyribonucleotide solutions can be mixed at room temperature by pumping each solution into a mixing unit at a controlled flow rate (e.g., using a piston pump). In some embodiments, the flow rates of the lipid solution and the RNA solution into the mixing unit are maintained at a ratio of 1:3. After mixing, nucleic acid-lipid particles are formed when the alcoholic lipid solution is diluted with aqueous polyribonucleotides. Lipid solubility decreases, while positively charged cationic lipids interact with negatively charged RNAs.

In some embodiments, the solution comprising RNA-encapsulated lipid nanoparticles may be treated by one or more of concentration adjustment, buffer exchange, formulation, and/or filtration.

In some embodiments, the RNA-encapsulated lipid nanoparticle may be treated by filtration.

In some embodiments, the particle size and/or internal structure of the lipid nanoparticle (with or without RNA) may be monitored by suitable techniques, such as small angle X-ray scattering (SAXS) and/or transmission electron cryomicroscopy (CryoTEM), for example.

V. pharmaceutical composition

The present disclosure provides compositions, e.g., pharmaceutical compositions, comprising one or more polyribonucleotides as described herein.

In some embodiments, the pharmaceutical formulation comprises an active agent and one or more excipients or carriers.

In some embodiments, the active agent may be or comprise an HSV (e.g., HSV-1 and/or HSV-2) antigen-e.g., it is or comprises an HSV (e.g., HSV-1 and/or HSV-2) protein or antigen or an antigenic fragment or epitope thereof, as described herein. Thus, in some embodiments, the active agent is a polypeptide or polypeptides. In some embodiments, the polypeptide active agent comprises a plurality of HSV (e.g., HSV-1 and/or HSV-2) antigens (e.g., from a single HSV (e.g., HSV-1 and/or HSV-2) protein or from a plurality of different HSV (e.g., HSV-1 and/or HSV-2) proteins). In some embodiments, the polypeptide active agent is or comprises at least one peptide representative of a different HSV (e.g., HSV-1 and/or HSV-2) antigen.

In some embodiments, the active agent may be or comprise a population of cells-e.g., a population of cells expressing (e.g., internally, on the surface and/or secreting) at least one antigen as described herein. Alternatively or additionally, a population of cells (e.g., antigen presenting cells, such as dendritic cells) is loaded (e.g., bound in an MHC complex) with an HSV (e.g., HSV-1 and/or HSV-2) antigenic peptide as described herein.

In some embodiments, the active agent is a polynucleotide encoding an HSV (e.g., HSV-1 and/or HSV-2) antigen as described herein (or is complementary to a polynucleotide encoding an HSV (e.g., HSV-1 and/or HSV-2) antigen as described herein). In some such embodiments, the polynucleotide is single stranded; in other embodiments, the polynucleotide is double stranded. In some embodiments, the polynucleotide agent is DNA (e.g., a DNA viral vector, such as an adenovirus, adeno-associated virus, baculovirus, poxvirus [ e.g., vaccinia virus ] vector); in some embodiments, the polynucleotide active agent is RNA (e.g., a lentiviral vector, or more preferably, an mRNA construct as described herein).

In many embodiments, the polynucleotide active agent is RNA and is provided and/or utilized in a lipid composition, such as a liposome complex formulation or preferably an LNP formulation.

In some embodiments, the provided formulation is a liquid formulation. In some embodiments, the provided formulation is a solid (e.g., a frozen formulation). In some embodiments, the provided formulation is a dry formulation.

The pharmaceutical formulation may additionally comprise pharmaceutically acceptable excipients, which as used herein include any and all solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like suitable for the particular dosage form desired. Remington' S THE SCIENCE AND PRACTICE of Pharmacy, 21 st edition, A.R. Gennaro (Lippincott, williams & Wilkins, baltimore, MD,2006; incorporated herein by reference) discloses various excipients for formulating pharmaceutical compositions and known techniques for their preparation. Unless any conventional excipient medium is incompatible with a substance or derivative thereof, such as by producing any undesirable biological effect or otherwise interacting in an adverse manner with any other component of the pharmaceutical composition, its use is considered to be within the scope of the present disclosure.

In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the U.S. food and drug administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopeia (USP), the European Pharmacopeia (EP), the british pharmacopeia, and/or the international pharmacopeia.

Pharmaceutically acceptable excipients for the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surfactants and/or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening agents, flavoring agents and/or perfuming agents may be present in the composition at the discretion of the formulator.

General considerations regarding the formulation and/or manufacture of pharmaceutical agents can be found in Remington, THE SCIENCE AND PRACTICE of Pharmacy 21 st edition, lippincott Williams & Wilkins,2005 (incorporated herein by reference).

In some embodiments, the pharmaceutical compositions provided herein may be formulated according to conventional techniques, such as those disclosed in Remington: THE SCIENCE AND PRACTICE of Pharmacy 21 st edition, lippincott Williams & Wilkins,2005 (incorporated herein by reference), with one or more pharmaceutically acceptable carriers or diluents, and any other known adjuvants and excipients.

The pharmaceutical compositions described herein may be administered by any suitable method known in the art. The skilled artisan will appreciate that the route and/or mode of administration may depend on a number of factors including, for example, but not limited to, the stability and/or pharmacokinetics and/or pharmacodynamics of the pharmaceutical compositions described herein.

In some embodiments, the pharmaceutical compositions described herein are formulated for parenteral administration, including modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, subcuticular, or intra-articular injection and infusion. In preferred embodiments, the pharmaceutical compositions described herein are formulated for intravenous, intramuscular, or subcutaneous administration. In particularly preferred embodiments, the pharmaceutical compositions described herein are formulated for intramuscular administration.

In some embodiments, the pharmaceutical compositions described herein are formulated for intravenous administration. In some embodiments, pharmaceutically acceptable excipients useful for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, lipid nanoparticle, or other ordered structure suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of surfactants. In many cases, it is preferred to include an isotonic agent, for example, a sugar, a polyalcohol (e.g., mannitol, sorbitol) or sodium chloride in the composition. In some embodiments, absorption of the injectable composition may be prolonged by including agents (e.g., monostearates and gelatins) in the composition that delay absorption.

Sterile injectable solutions can be prepared by: the desired amount of active compound is incorporated, if desired, with one or a combination of the ingredients listed above, into a suitable solvent, followed by sterilization and/or microfiltration. In some embodiments, the pharmaceutical compositions may be prepared as described herein and/or according to methods known in the art. In some embodiments, the pharmaceutical composition comprises ALC-0315; ALC-0159; DSPC; cholesterol; sucrose; naCl; KCl; na ₂HPO₄;KH₂PO₄; water for injection. In some embodiments, physiological saline (isotonic 0.9% nacl) is used as a diluent.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents. Prevention of the presence of microorganisms can be ensured by sterilization procedures as well as by inclusion of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, and the like). It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the pharmaceutical compositions described herein. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known in the pharmacological arts or hereafter developed. Generally, such preparation methods comprise the following steps: the active ingredient is admixed with a diluent or another excipient and/or one or more additional ingredients, and the product is then shaped and/or packaged as desired in single or multiple dose units, if necessary and/or desired.

Pharmaceutical compositions according to the present disclosure may be prepared, packaged and/or sold in bulk as single unit doses and/or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using the systems and/or methods described herein.

The relative amounts of the polynucleic acids encapsulated in the lipid nanoparticle, the pharmaceutically acceptable excipients and/or any additional ingredients in the pharmaceutical composition may vary, depending on the subject, target cell, disease or disorder to be treated, and may further depend on the route by which the composition is to be administered.

In some embodiments, the pharmaceutical compositions described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. The actual dosage level of the active ingredient (e.g., the polyribonucleotides encapsulated in the lipid nanoparticles) in the pharmaceutical compositions described herein can be varied in order to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, and that is non-toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors including the activity of the particular compositions employed in the present disclosure, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or substances used in combination with the particular composition being employed, the age, sex, weight, condition, general health and past medical history of the patient being treated, and like factors well known in the medical arts.

The effective amount of the desired pharmaceutical composition can be readily determined and prescribed by a physician of ordinary skill in the art. For example, a physician may begin administration of the active ingredient employed in the pharmaceutical composition (e.g., the polyribonucleotides encapsulated in the lipid nanoparticle) at a level below that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

In some embodiments, the pharmaceutical composition (e.g., without limitation, for intravenous, intramuscular, or subcutaneous administration) is formulated to deliver a dose of about 5mg RNA/kg.

In some embodiments, the pharmaceutical compositions described herein may further comprise one or more additives, such as additives that may, in some embodiments, enhance the stability of such compositions under certain conditions. Examples of additives may include, but are not limited to, salts, buffer substances, preservatives, and carriers. For example, in some embodiments, the pharmaceutical composition may further comprise a cryoprotectant (e.g., sucrose) and/or an aqueous buffer, which in some embodiments may comprise one or more salts, including, for example, alkali metal salts or alkaline earth metal salts, such as, for example, sodium, potassium, and/or calcium salts.

In some embodiments, the pharmaceutical compositions provided herein are sterile RNA-lipid nanoparticle dispersions without preservatives in aqueous buffer for intravenous or intramuscular administration.

While the description of the pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, the skilled artisan will appreciate that such compositions are generally suitable for administration to all kinds of animals. Variations are well understood in order to adapt pharmaceutical compositions suitable for administration to humans to a variety of animals, and such variations can be designed and/or made by a ordinarily skilled veterinary pharmacologist using only routine experimentation, if any.

VI characterization of

Without wishing to be bound by any particular theory, it is proposed that the ability to induce CD8 ⁺ T cells may be important for the effectiveness of a composition (e.g., a pharmaceutical composition, an immunogenic composition, or a vaccine) for treating HSV (e.g., HSV-1 and/or HSV-2) infection. Alternatively or additionally, in some embodiments, the effectiveness may require a strong antibody response. In some embodiments, both may be desirable or useful.

In some embodiments, the provided techniques (e.g., compositions and/or dosing regimens, etc.) are characterized by the ability to induce (e.g., when administered to a model system and/or human, e.g., by parenteral administration, such as by intramuscular administration) an immune response characterized by CD8 ⁺ T cells that target one or more HSV (e.g., HSV-1 and/or HSV-2) antigens described herein. That is, in some embodiments, the provided techniques are characterized in that when administered (e.g., by parenteral administration, such as by intramuscular administration) to an organism (e.g., a model organism or an animal or human organism in need of protection), the provided techniques induce cd8+ T cells that target one or more HSV (e.g., HSV-1 and/or HSV-2) antigens. In some embodiments, the provided techniques are characterized in that they induce a greater CD8 ⁺ T cell response against one or more HSV (e.g., HSV-1 and/or HSV-2) antigens, and/or induce a more widely diverse (e.g., detectable and/or significant binding to a greater number of different T cell antigens) CD8 ⁺ T cell responses than are observed, for example, in table 1 or another suitable reference.

In some embodiments, the provided techniques are characterized in that they induce γδ T cells. As will be appreciated by those skilled in the art, γδ T cells typically represent only a small fraction (e.g., up to about 5%) of the total T cell population in an organism. γδ T cells express TCR chains encoded by the γ and δ loci; a subset of γδ T cells is defined by the inclusion of the invariant V- (D) -J segment of the TCR and is tissue or environment specific. γδ T cells secrete specific effector cytokines in a subtype and environment specific manner. Typically, γδ T cells express certain markers (e.g., fcγriii/CD16 and Toll-like receptors, which are typically associated with natural killer cells and/or antigen presenting cells). γδ T cells are generally devoid of CD4 and CD8.

In some embodiments, the provided techniques are characterized in that they induce polyclonal high affinity antibodies.

In some embodiments, the provided techniques are characterized in that they induce antibody titers to a level that provides a sufficient protective response against HSV when administered to a relevant population.

In some embodiments, the provided techniques are characterized in that they induce sterility protection, e.g., when evaluated in a model system such as a mouse model.

In some embodiments, the provided techniques are characterized in that they induce a cd4+ T helper cell response and/or a cd8+ T cell memory response (e.g., promote the development and/or expansion of memory cd8+ T cells).

In some embodiments, the provided compositions are evaluated as described herein, e.g., to assess RNA integrity, stability, level, capping efficiency, translatable, etc., of the RNA, and/or to assess one or more characteristics of the composition (e.g., LNP formulation), e.g., the ability to induce an antibody response, a T cell response with specific characteristics (e.g., antibody level against one or more antigens, persistence of such level, diversity of elicited antibodies, type and/or diversity of T cell response, etc.).

In some embodiments, provided formulations are identified and/or characterized for one or more activities or characteristics, including, for example, expression levels, nature of immune response, protection levels (e.g., against challenge, effect on viral load, effect on health and/or survival), immunogenicity (e.g., assessment of cytokine response, phenotypic analysis of immune response, T cell depletion and/or protection), serology, and/or functional antibody response. In some embodiments, the provided compositions can be evaluated in animal models. In many embodiments, it is desirable to evaluate the provided compositions in the human system. In some embodiments, the in vitro assessment is performed in the human system. In some embodiments, the human dendritic cells are evaluated in vitro for presentation of the provided antigen or antigenic fragment or epitope thereof to stimulate human T cells, to name a few. Alternatively or additionally, in some embodiments, serum from an infected person is assessed for in vitro binding to the provided antigen.

In some embodiments, the in vivo assessment is performed in the human system. For example, in some embodiments, one or more human trials are performed. In such assays, healthy humans (e.g., volunteers who have typically undergone screening and/or consent procedures) are treated with a provided composition (e.g., an immunostimulatory composition, such as a vaccine composition), subsequently vaccinated with HSV (e.g., HSV-1 and/or HSV-2), and clinically monitored to assess a level of protection against, for example, established infection and/or symptomatic or severe disease. Alternatively or additionally, in some embodiments, the subject's responsiveness (e.g., increased responsiveness) to a particular known or potential anti-HSV (e.g., anti-HSV-1 and/or anti-HSV-2) therapy is monitored.

In some embodiments, the provided compositions (e.g., immunostimulatory compositions, such as vaccine compositions) provide significant (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90% or more) protection against one or more established infections, symptomatic diseases, and/or severe diseases. In some embodiments, provided compositions (e.g., immunostimulatory compositions, such as vaccine compositions) provide significantly increased responsiveness to treatment, e.g., as assessed by delayed onset, reduced severity, and/or faster regression of one or more symptoms or features of the infection.

Patient population

In some aspects, the techniques of the present disclosure are used for therapeutic and/or prophylactic purposes. In some embodiments, the techniques of the present disclosure are used to treat and/or prevent HSV infection (e.g., HSV-1 and/or HSV-2 infection). The prevention purposes of the present disclosure include pre-exposure prevention and/or post-exposure prevention.

In some embodiments, the techniques of the present disclosure are used to treat and/or prevent disorders associated with such HSV (e.g., HSV-1 and/or HSV-2) infections. Disorders associated with such HSV (e.g., HSV-1 and/or HSV-2) infections include, for example, typical symptoms and/or complications of HSV (e.g., HSV-1 and/or HSV-2) infections.

In some embodiments, provided compositions (e.g., compositions that are or comprise HSV (e.g., HSV-1 and/or HSV-2) antigens) are useful for detecting and/or characterizing one or more characteristics of an anti-HSV (e.g., anti-HSV-1 and/or anti-HSV-2) immune response (e.g., detecting binding to provided antigens by serum from an infected subject).

In certain embodiments, provided compositions (e.g., compositions that are or comprise HSV (e.g., HSV-1 and/or HSV-2) antigens) are useful for producing antibodies to one or more antigens included therein; such antibodies themselves may be useful, for example, in the detection or treatment of HSV (e.g., HSV-1 and/or HSV-2) or infections caused thereby.

The present disclosure provides the use of a coding nucleic acid (e.g., DNA or RNA) to produce a coding antigen and/or the use of a DNA construct to produce RNA.

In some embodiments, the techniques of the disclosure are utilized in an unrestricted subject population; in some embodiments, the techniques of the disclosure are utilized in a particular population of subjects.

In some embodiments, the subject population comprises an adult population. In some embodiments, the adult population comprises subjects between about 19 years of age and about 60 years of age (e.g., about 20, 25, 30, 35, 40, 45, 50, 55, or 60 years of age).

In some embodiments, the subject population comprises an elderly population. In some embodiments, the elderly population comprises subjects aged about 60 years, about 70 years, or older (e.g., about 65, 70, 75, 80, 85, 90, 95, or 100 years old).

In some embodiments, the subject population comprises a pediatric population. In some embodiments, the pediatric population comprises subjects about 18 years of age or less. In some such embodiments, the pediatric population includes subjects between about 1 year of age and about 18 years of age (e.g., ages 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 years of age).

In some embodiments, the subject population comprises a neonatal population. In some embodiments, the neonatal population includes subjects of about 12 months or less (e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 month or less). In some embodiments, the population of subjects to be treated using the techniques described herein includes infants (e.g., about 12 months or less) for whom the mother did not receive such techniques described herein during pregnancy. In some embodiments, the population of subjects to be treated using the techniques described herein may include pregnant women; in some embodiments, an infant treated (e.g., receiving at least one dose, or receiving only two doses) during pregnancy with the disclosed technology is not vaccinated a few weeks, months, or even years (e.g., 1,2,3,4, 5,6,7, 8 weeks, or more, or 1,2,3,4, 5,6,7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months, or more, or 1,2,3,4, 5 years, or more) before birth. Alternatively or additionally, in some embodiments, an infant that is mother treated with the disclosed techniques during pregnancy (e.g., receives at least one dose, or receives only two doses) is reduced from receiving treatment with the disclosed techniques (e.g., lower doses and/or fewer administrations-e.g., lower total exposure over a given period of time-or may require reduced vaccination (e.g., lower doses and/or fewer administrations-e.g., over a given period of time-of a booster) after, e.g., weeks, months, or even years, 1,2,3, 7,8, 4,5,6,7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months, or more) of birth, or 1,2,3,4, 5 years or more) of birth.

In some embodiments, the population of subjects is or includes children aged 6 weeks up to an age of 17 months.

In some embodiments, the provided pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) can be administered in combination with other pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) or therapeutic interventions (i.e., such that the subject is exposed to both simultaneously), e.g., to treat or prevent HSV (e.g., HSV-1 and/or HSV-2) infection or other disease, disorder, or condition.

In some embodiments, the provided pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) can be administered with protein vaccines, DNA vaccines, RNA vaccines, cell vaccines, conjugate vaccines, and the like. In some embodiments, one or more doses of a provided pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) can be co-administered with another vaccine or other therapy (e.g., in a single visit).

In some embodiments, the provided pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) may be administered to a subject that has been exposed to or is expected to have been exposed to HSV (e.g., HSV-1 and/or HSV-2). In some embodiments, the provided pharmaceutical compositions (e.g., immunogenic compositions, such as vaccines) may be administered to subjects without symptoms of HSV (e.g., HSV-1 and/or HSV-2) infection.

VIII method of treatment

In some embodiments, the techniques of the disclosure may be administered to a subject according to a particular dosing regimen. In some embodiments, the dosing regimen may involve a single administration; in some embodiments, the dosing regimen may include one or more "booster" administrations following the initial administration. In some embodiments, the initial dose and the booster dose are the same amount; in some embodiments, they are different. In some embodiments, two or more booster doses are administered. In some embodiments, multiple doses are administered at regular intervals. In some embodiments, the time period between doses becomes longer. In some embodiments, one or more subsequent doses are administered if a particular clinical condition (e.g., reduced neutralizing antibody levels) or condition (e.g., local appearance of a new strain) even occurs or is detected.

In some embodiments, the administered pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) comprising an RNA construct encoding an HSV (e.g., HSV-1 and/or HSV-2) antigen is administered at an RNA dose of about 0.1 μg to about 300 μg, about 0.5 μg to about 200 μg, or about 1 μg to about 100 μg (e.g., about 1 μg, about 3 μg, about 10 μg, about 30 μg, about 50 μg, or about 100 μg). In some embodiments, the saRNA construct is administered at a lower dose (e.g., 1/2, 1/4, 1/5, 1/10, or lower) than the modRNA or uRNA construct.

In some embodiments, the first booster dose is administered within about six months of the initial dose and preferably within about 5, 4, 3, 2, or1 month. In some embodiments, the first booster dose is administered starting about 1, 2,3, or 4 weeks after the first dose and ending at a time period of about 2,3,4, 5,6, 7, 8, 9, 10, 11, 12 weeks after the first dose (e.g., between about 1 week and about 12 weeks after the first dose, or between about 2 or 3 weeks and about 5 and 6 weeks after the first dose, or about 3 weeks or about 4 weeks after the first dose).

In some embodiments, multiple booster doses (e.g., 2, 3, or 4) are administered within 6 months of the first dose or within 12 months of the first dose.

In some embodiments, 3 doses or less are required to achieve effective vaccination (e.g., greater than 60%, and in some embodiments greater than about 70%, about 75%, about 80%, about 85%, about 90% or more), reducing the risk of infection or serious disease. In some embodiments, no more than two doses are required. In some embodiments, a single dose is sufficient. In some embodiments, the RNA dose is about 60 μg or less, 50 μg or less, 40 μg or less, 30 μg or less, 20 μg or less, 10 μg or less, 5 μg or less, 2.5 μg or less, or 1 μg or less. In some embodiments, the RNA dose is about 0.25 μg, at least 0.5 μg, at least 1 μg, at least 2 μg, at least 3 μg, at least 4 μg, at least 5 μg, at least 10 μg, at least 20 μg, at least 30 μg, or at least 40 μg. In some embodiments, the RNA dose is about 0.25 μg to 60 μg, 0.5 μg to 55 μg,1 μg to 50 μg, 5 μg to 40 μg, or 10 μg to 30 μg per dose may be administered. In some embodiments, the RNA dose is about 30 μg. In some embodiments, at least two such doses are administered. For example, the second dose may be administered about 21 days after the first dose. In some embodiments, the first booster dose is administered about one month after the initial dose. In some such embodiments, the further enhancer is administered at least once at one month intervals. In some embodiments, longer intervals are introduced after 2 or 3 boosters, and no further boosters are administered for at least 6, 9, 12, 18, 24 months or more. In some embodiments, the further enhancer is administered once after about 18 months. In some embodiments, no further strengthening agent is required unless, for example, a substantial change in clinical or environmental conditions is observed.

IX. manufacturing method

The individual polyribonucleotides can be produced by methods known in the art. For example, in some embodiments, the polyribonucleotides may be produced by in vitro transcription, e.g., using a DNA template. Plasmid DNA that is used as a template for in vitro transcription to produce the polyribonucleotides described herein is also within the scope of the present disclosure.

The DNA templates are used for in vitro RNA synthesis in the presence of an appropriate RNA polymerase (e.g., a recombinant RNA polymerase such as T7 RNA polymerase) and a ribonucleotide triphosphate (e.g., ATP, CTP, GTP, UTP). In some embodiments, a polyribonucleotide (e.g., a polyribonucleotide as described herein) can be synthesized in the presence of a modified ribonucleotide triphosphate. For example only, in some embodiments, uridine Triphosphate (UTP) may be replaced with pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), or 5-methyl-uridine (m 5U). In some embodiments, uridine Triphosphate (UTP) may be replaced with pseudouridine (ψ). In some embodiments, N1-methyl-pseudouridine (m1ψ) may be used in place of Uridine Triphosphate (UTP). In some embodiments, 5-methyl-uridine (m 5U) may be used in place of Uridine Triphosphate (UTP).

It will be apparent to those of skill in the art that during in vitro transcription, RNA polymerase (e.g., as described and/or utilized herein) typically traverses at least a fragment of a single stranded DNA template in a 3 '. Fwdarw.5' direction to produce single stranded complementary RNA in a 5 '. Fwdarw.3' direction.

In some embodiments in which the polyribonucleotides comprise a poly-a tail, those skilled in the art will appreciate that such a poly-a tail may be encoded in a DNA template, for example by using suitable tailed PCR primers, or may be added to the polyribonucleotide after in vitro transcription, for example by enzymatic treatment (for example using a poly (a) polymerase, such as e.g. an e.coli poly (a) polymerase). Suitable poly (a) tails are described above. For example, in some embodiments, the poly (a) tail comprises the nucleotide sequence AAAAAAAAAAAAAAAAAAAAAAAAAA AAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AA(SEQ ID NO:210)., and in some embodiments, the poly (a) tail comprises a plurality of a residues that are interrupted by a linker. In some embodiments, the linker comprises nucleotide sequence GCATA TGAC (SEQ ID NO: 211).

In some embodiments, one of skill in the art will appreciate that adding a 5' cap to RNA (e.g., mRNA) can facilitate RNA recognition and ligation to ribosomes to initiate translation and increase translation efficiency. It is also appreciated by those skilled in the art that the 5 'cap may also protect the RN a product from 5' exonuclease mediated degradation, thereby extending half-life. Capping methods are known in the art; one of ordinary skill in the art will appreciate that in some embodiments, capping may be performed post-in vitro transcription in the presence of a capping system (e.g., an enzyme-based capping system, such as a capping enzyme of vaccinia virus). In some embodiments, the cap may be introduced during in vitro transcription, along with multiple ribo-triphosphates, such that the cap is incorporated into the polyribonucleotide during transcription (also referred to as co-transcriptional capping). In some embodiments, a multiple addition GTP fed-batch procedure may be employed during the reaction to maintain a low concentration of GTP in order to effectively cap the RNA. Suitable 5' caps are described above. For example, in some embodiments, the 5' cap comprises m7 (3 ' ome g) (5 ') ppp (5 ') (2 ' ome a) pG.

After transcription of the RNA, the DNA template is digested. In some embodiments, digestion may be achieved using dnase I under appropriate conditions.

In some embodiments, the in vitro transcribed polyribonucleotides may be provided in a buffer solution, for example in a buffer such as HEPES, phosphate buffer, citrate buffer, acetate buffer; in some embodiments, such solutions may be buffered to a pH in the range of, for example, about 6.5 to about 7.5; and in some embodiments about 7.0. In some embodiments, the production of the polyribonucleotide may further comprise one or more of the following steps: purification, mixing, filtration and/or packing.

In some embodiments, the polyribonucleotides (e.g., in some embodiments, after an in vitro transcription reaction) can be purified, e.g., to remove components utilized or formed during production, such as, for example, proteins, DNA fragments, and/or nucleotides. Various nucleic acid purifications known in the art may be employed in accordance with the present disclosure. Some purification steps may be or include, for example, precipitation, column chromatography (including, for example, but not limited to, anion, cation, hydrophobic Interaction Chromatography (HIC)), solid matrix-based purification (e.g., magnetic bead-based purification). In some embodiments, the polyribonucleotides may be purified using magnetic bead-based purification, which in some embodiments may be or include magnetic bead-based chromatography. In some embodiments, the polyribonucleotides may be purified using Hydrophobic Interaction Chromatography (HIC) and/or diafiltration. In some embodiments, the polyribonucleotides may be purified using HIC followed by diafiltration.

In some embodiments, the dsRNA may be obtained as a byproduct during in vitro transcription. In some such embodiments, a second purification step may be performed to remove dsRNA contamination. For example, in some embodiments, a cellulosic material (e.g., microcrystalline cellulose) may be used to remove dsRNA contamination, e.g., in some embodiments in the form of chromatography. In some embodiments, cellulosic material (e.g., microcrystalline cellulose) may be pretreated to inactivate potential rnase contamination, for example by autoclaving in some embodiments, followed by incubation with an aqueous alkaline solution (e.g., naOH). In some embodiments, cellulosic material may be used to purify polyribonucleotides according to the method described in WO 2017/182524, the entire contents of which are incorporated herein by reference.

In some embodiments, a batch of polyribonucleotides can be further processed by one or more filtration and/or concentration steps. For example, in some embodiments, the polynucleic acid may be further diafiltered (e.g., by tangential flow filtration in some embodiments), e.g., after removal of dsRNA contamination, e.g., to adjust the concentration of polynucleic acid to a desired RNA concentration and/or to exchange the buffer for a drug substance buffer.

In some embodiments, the polyribonucleotides may be treated by 0.2 μm filtration before they are filled into a suitable container.

In some embodiments, the polyribonucleotides and compositions thereof can be manufactured according to methods as described herein or otherwise known in the art.

In some embodiments, the polyribonucleotides and combinations thereof can be manufactured in large scale. For example, in some embodiments, a batch of polynucleic acids can be manufactured on a scale of greater than 1g, greater than 2g, greater than 3g, greater than 4g, greater than 5g, greater than 6g, greater than 7g, greater than 8g, greater than 9g, greater than 10g, greater than 15g, greater than 20g, or greater.

In some embodiments, RNA quality control can be performed and/or monitored at any time during the production process of the polyribonucleotides and/or the composition comprising the polyribonucleotides. For example, in some embodiments, RNA quality control parameters, including one or more of RNA characteristics (e.g., sequence, length, and/or RNA properties), RNA integrity, RNA concentration, residual DNA template, and residual dsRNA, may be assessed and/or monitored after each or certain steps of the polyribonucleotide manufacturing process (e.g., after in vitro transcription and/or each purification step).

In some embodiments, the stability of a polyribonucleotide (e.g., produced by in vitro transcription) and/or a composition comprising a polyribonucleotide can be evaluated under various test storage conditions, e.g., at room temperature and refrigerator or sub-zero temperature over a period of time (e.g., at least 3 months, at least 6 months, at least 9 months, at least 12 months, or longer). In some embodiments, a polyribonucleotide (e.g., a polyribonucleotide described herein) and/or a composition thereof can be stably stored at refrigerator temperature (e.g., about 4 ℃ to about 10 ℃) for at least 1 month or more, including at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or more. In some embodiments, a polyribonucleotide (e.g., a polyribonucleotide described herein) and/or a composition thereof can be stably stored at subzero temperatures (e.g., -20 ℃ or less) for at least 1 month or more, including at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or more. In some embodiments, the polyribonucleotides (e.g., the polyribonucleotides described herein) and/or compositions thereof can be stable for storage at room temperature (e.g., at about 25 ℃) for at least 1 month or more.

In some embodiments, one or more assessments (e.g., as a release test) may be utilized during manufacture or other preparation or use of the polyribonucleotides.

In some embodiments, one or more quality control parameters may be evaluated to determine whether the polyribonucleotides described herein meet or exceed acceptance criteria (e.g., release for subsequent formulation and/or dispensing). In some embodiments, such quality control parameters may include, but are not limited to, RNA integrity, RNA concentration, residual DNA template, and/or residual dsRNA. Certain methods of assessing RNA quality are known in the art; for example, one of skill in the art will recognize that in some embodiments, one or more analytical tests may be used for RNA quality assessment. Examples of such certain analytical tests may include, but are not limited to, gel electrophoresis, UV absorbance, and/or PCR assays.

In some embodiments, one or more characteristics of a batch of polynucleic acids as described herein may be evaluated to determine the next step of action. For example, if the RNA quality assessment indicates that a batch of polynucleotides meets or exceeds relevant acceptance criteria, such a batch of polynucleotides may be designated for one or more further steps of manufacture and/or formulation and/or distribution. Otherwise, if such a batch of polyribonucleotides does not meet or exceed acceptance criteria, then an alternative measure (e.g., discarding the batch) may be taken.

In some embodiments, a batch of polyribonucleotides that meet the evaluation result can be used in one or more further steps of manufacture and/or formulation and/or distribution.

RNA production

Those of skill in the art are aware of a variety of techniques that can be used to produce RNA as described herein, including chemical or enzymatic (e.g., by polymerization) synthesis. In many embodiments, the RNA is produced by transcription, for example by in vivo or in vitro transcription. Indeed, one advantage of using RNA as an active agent in pharmaceutical compositions (e.g., immunogenic compositions such as vaccines) or other therapeutic settings is that it is readily produced by in vitro transcription. The present disclosure teaches that RNA morphology is particularly desirable for use as an active agent in pharmaceutical compositions (e.g., immunogenic compositions such as vaccines), particularly given that relatively modest adjustments to the manufacturing process can generally optimize the production of relevant sequences. Furthermore, the present disclosure provides the specific insight that RNA is particularly useful as an active agent in HSV (e.g., HSV-1 and/or HSV-2) vaccines, as it allows for easy adaptation (e.g., sequence change) to emerging or locally related strains and/or antigens (e.g., allows for tailoring of antigen sequences according to, for example, circulating strains and/or HLA allele diversity within a relevant population (e.g., within a specific geographic/regional). Further, since RNA production requires only a single development and manufacturing platform, independent of the encoded pathogen antigen. Thus, RNA has the potential for rapid, cost-effective, mass production and flexible storage (long-term storage of frozen plasmids and low-volume libraries of unfocused RNA, which can be rapidly formulated and distributed). Particularly for HSV (e.g., HSV-1 and/or HSV-2) infections, the timing of administration (e.g., vaccine administration) relative to the incidence of seasons and/or outbreaks can substantially affect effectiveness, and the ability to store and rapidly reconstitute can prove an important advantage with critical benefits relative to alternative strategies.

Typically, RNA is transcribed in vitro from linearized (e.g., by restriction digestion) or amplified (e.g., PCR amplified) DNA templates. Those skilled in the art know that a variety of promoters may be used to direct RNA synthesis by transcription of a DNA template, for example by a DNA-dependent RNA polymerase, such as, for example, T7, T3, SP6 or Syn5 RNA polymerase.

A typical in vitro transcription reaction will include a DNA template, rtps of four bases (i.e., adenine, cytosine, guanine and uracil), optionally a cap analogue, a related RNA polymerase, and appropriate buffers and/or salts. In some embodiments, one or more ribonuclease (rnase) inhibitors and/or pyrophosphatase may be included.

In some embodiments, the rtp utilized in the in vitro transcription reaction comprises one or more nucleotide analogs. In some embodiments, the nucleotide analog is 2-amino-6-chloropurine riboside-5 '-triphosphate, 2-aminopurine-riboside-5' -triphosphate; 2-Aminoadenosine-5 '-triphosphate, 2' -amino-2 '-deoxycytidine-triphosphate, 2-thiocytidine-5' -triphosphate, 2-thiouridine-5 '-triphosphate, 2' -fluorothymidine-5 '-triphosphate, 2' -O-methyl-inosine-5 '-triphosphate, 4-thiouridine-5' -triphosphate, 5-aminoallyl cytidine-5 '-triphosphate, 5-aminoallyl uridine-5' -triphosphate, 5-bromocytidine-5 '-triphosphate, 5-bromouridine-5' -triphosphate, 5-bromo-2 '-deoxycytidine-5' -triphosphate, 5-bromo-2 '-deoxyuridine-5' -triphosphate, 5-iodocytidine-5 ' -triphosphate, 5-iodo-2 ' -deoxycytidine-5 ' -triphosphate, 5-iodouridine-5 ' -triphosphate, 5-iodo-2 ' -deoxyuridine-5 ' -triphosphate, 5-methylcytidine-5 ' -triphosphate, 5-methyluridine-5 ' -triphosphate, 5-propynyl-2 ' -deoxycytidine-5 ' -triphosphate, 5-propynyl-2 ' -deoxyuridine-5 ' -triphosphate, 6-azacytidine-5 ' -triphosphate, 6-azauridine-5 ' -triphosphate, 6-chloropurine riboside-5 ' -triphosphate, 7-deadenosine-5 ' -triphosphate, 7-deazaguanosine-5 ' -triphosphate, 8-azaadenosine-5 '-triphosphate, 8-azidoadenosine-5' -triphosphate, benzimidazole-riboside-5 '-triphosphate, N1-methyladenosine-5' -triphosphate, N1-methylguanosine-5 '-triphosphate, N6-methyladenosine-5' -triphosphate, O6-methylguanosine-5 '-triphosphate, pseudouridine-5' -triphosphate or puromycin-5 '-triphosphate, xanthosine-5' -triphosphate. Particularly preferred are base modified nucleotides for use in a base modified nucleotide set selected from the group consisting of: 5-methylcytidine-5 '-triphosphate, 7-deazaguanosine-5' -triphosphate, 5-bromocytidine-5 '-triphosphate and pseudouridine-5' -triphosphate, pyridin-4-one ribonucleoside, 5-azauridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propargyl-uridine, 1-propargyl-pseudouridine, 5-taurine methyluridine, 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine, 1-taurine methyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydro-uridine, dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-dihydro-pseudouridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytosine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, zebulin, 5-aza-zebulin, 5-methyl-zebulin, 5-aza-2-thio-zebulin, 2-thio-zebulin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine and 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6-isopentenyl adenosine, N6- (cis-hydroxyisopentenyl) adenosine, 2-methylsulfanyl-N6- (cis-hydroxyisopentenyl) adenosine, N6-glycylcarbamoyladenosine, N6-threoniylcarbamoyladenosine, 2-methylsulfanyl-N6-threoniylcarbamoyladenosine, N6-dimethyl-adenosine, 7-methyladenine, 2-methylsulfanyl-adenine and 2-methoxy-adenine, inosine, 1-methyl-inosine, hudroside, huai Dinggan, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine and N2, N2-dimethyl-6-thioguanosine, 5' -0- (1-thiophosphoric acid) -adenosine, 5' -0- (1-thiophosphoric acid) -cytidine, 5' -0- (1-thiophosphoric acid) -guanosine, 5' -0- (1-thiophosphoric acid) -uridine, 5' -0- (1-thiophosphoric acid) -pseudouridine, 6-aza-cytidine, 2-thio-cytidine, alpha-thio-cytidine, pseudoisocytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5, 6-dihydro-uridine, alpha-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxythymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudoisocytidine, 6-chloro-purine, n6-methyl-adenosine, alpha-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4' -thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2' -0-methyluridine or combinations thereof.

In some embodiments, uridine analogs are utilized. In some embodiments, natural uridine is not utilized. Thus, in some embodiments, 100% of uracil in a coding sequence has a chemical modification (relative to uridine); in many embodiments, in the 5-position of uracil. In particular embodiments, pseudouridine is utilized.

In particular embodiments, nucleotide analogs utilized include pseudouridine, N1-methyl pseudouridine, 5-methylcytosine, -methoxyuridine, and combinations thereof.

In some embodiments, four rtps are utilized in an equimolar concentration in an in vitro transcription reaction; in some embodiments, they are not equimolar. For example, in some embodiments, the concentration of one or more rtps is relatively low, while in some embodiments, replenishment can be performed over time during the reaction by one or more "feeds. In some particular embodiments, the rtpp is fed over time (thus the IVT reaction is a "G fed-batch" process). Alternatively or additionally, in some particular embodiments, the rUTP is fed over time (thus the IVT reaction is a "Ufed batch" or "G/Ufed batch" process).

In some embodiments, one or more of rtp concentration, salt concentration, metal concentration, pH, temperature, etc. are adjusted to produce a particular RNA construct in order to optimize, for example, one or more of RNA integrity, capping efficiency, contaminant (e.g., dsRNA) levels, intact transcript levels (e.g., relative to template DNA concentration in the reaction), etc.

In some embodiments, exemplary reagents for use in RNA in vitro transcription include: a DNA template (linearized plasmid DNA or PCR product) having a promoter sequence with high binding affinity for its respective RNA polymerase, such as phage-encoded RNA polymerase (T7, T3, SP6 or Syn 5); ribonucleoside triphosphates (NTPs) for four bases (adenine, cytosine, guanine and uracil); optionally, a cap analogue as defined herein (e.g., m7G (5 ') ppp (5') G (m 7G)); optionally, a further modified nucleotide as defined herein; a DNA-dependent RNA polymerase (e.g., T7, T3, SP6, or Syn5 RNA polymerase) capable of binding to a promoter sequence within a DNA template; optionally, a ribonuclease (rnase) inhibitor to inactivate any potentially contaminating rnase; optionally, pyrophosphatase to degrade pyrophosphatase, which can inhibit RNA in vitro transcription; mgCl2, which provides Mg2+ ions as cofactors for the polymerase; buffers (TRIS or HEPES) to maintain a suitable pH value, which may also contain an optimal concentration of antioxidants (e.g. OTT) and/or polyamines such as spermidine, e.g. a buffer system comprising TRIS-citrate as disclosed in W02017/109161.

In the context of RNA production, in some embodiments, it may be desirable to provide GMP-grade RNA. In some embodiments, GMP-grade RNA can be produced using regulatory agency-approved manufacturing methods. In some embodiments, RNA production is performed under current Good Manufacturing Practice (GMP), and various quality control steps are performed at the DNA and/or RNA level, e.g., in some embodiments, according to the quality steps described in WO 2016/180130. In some embodiments, the RNA of the present disclosure is GMP-grade RNA.

X.DNA construct

The present disclosure provides, among other things, DNA constructs that can encode, for example, one or more antibody agents or components thereof as described herein. In some embodiments, DNA constructs provided by and/or utilized in accordance with the present disclosure are contained in a vector.

Non-limiting examples of vectors include plasmid vectors, cosmid vectors, phage vectors (e.g., lambda phage), viral vectors (e.g., retrovirus, adenovirus, or baculovirus vectors), or artificial chromosome vectors (e.g., bacterial Artificial Chromosome (BAC), yeast Artificial Chromosome (YAC), or P1 Artificial Chromosome (PAC)). In some embodiments, the vector is an expression vector. In some embodiments, the vector is a cloning vector. In general, a vector is a nucleic acid construct (e.g., a construct that is or encodes a payload, or imparts a particular functionality, etc.) that can receive or otherwise become linked to a nucleic acid element of interest.

Expression vectors, which may be plasmids or viruses or other vectors, typically include an expressible sequence of interest (e.g., a coding sequence) that is functionally linked to one or more control elements (e.g., promoters, enhancers, transcription terminators, etc.). Typically, such control elements are selected for expression in the system of interest. In some embodiments, the system is ex vivo (e.g., an in vitro transcription system); in some embodiments, the system is in vivo (e.g., bacteria, yeast, plants, insects, fish, vertebrates, mammalian cells or tissues, etc.).

Cloning vectors are typically used for modification, engineering and/or replication (e.g., by replication in vivo, e.g., in a simple system such as bacteria or yeast, or in vitro, e.g., by amplification, e.g., polymerase chain reaction or other amplification process). In some embodiments, the cloning vector may lack an expression signal.

In many embodiments, the vector may include replication elements, such as primer binding sites and/or origins of replication. In many embodiments, the vector may include insertion or modification sites, such as restriction endonuclease recognition sites and/or guide RNA binding sites, and the like.

In some embodiments, the vector is a viral vector (e.g., an AAV vector). In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a plasmid.

Those of skill in the art are aware of a variety of techniques that can be used to produce recombinant polynucleotides (e.g., DNA or RNA) as described herein. For example, restriction digestion, reverse transcription, amplification (e.g., via polymerase chain reaction), gibson assembly, and the like are widely recognized and useful tools and techniques. Alternatively or additionally, certain nucleic acids may be prepared or assembled by chemical and/or enzymatic synthesis. In some embodiments, the recombinant polynucleotide is prepared using a combination of known methods.

In some embodiments, polynucleotides of the present disclosure are included in DNA constructs (e.g., vectors) that are susceptible to transcription and/or translation.

In some embodiments, the construct of the expression vector comprises a polynucleotide encoding a protein and/or polypeptide of the disclosure operably linked to one or more sequences that control expression (e.g., a promoter, initiation signal, termination signal, polyadenylation signal, activator, repressor, etc.). In some embodiments, one or more sequences that control expression are selected to achieve a desired level of expression. In some embodiments, more than one sequence (e.g., a promoter) that controls expression is utilized. In some embodiments, more than one sequence (e.g., a promoter) that controls expression is utilized to achieve a desired level of expression of a plurality of polynucleotides encoding a plurality of proteins and/or polypeptides. In some embodiments, the plurality of recombinant proteins and/or polypeptides are expressed from the same vector (e.g., a bicistronic vector, a tricistronic vector, a polycistronic vector). In some embodiments, multiple polypeptides are expressed, each expressed by a separate vector.

In some embodiments, expression vectors comprising the polynucleotides of the present disclosure are used to produce RNA and/or proteins and/or polypeptides in a host cell. In some embodiments, the host cell may be in vitro (e.g., a cell line) -e.g., a cell or cell line (e.g., a human embryonic kidney (HEK cell), chinese hamster ovary cell, etc.) suitable for producing the polynucleotides of the disclosure and the proteins and/or polypeptides encoded by the polynucleotides.

In some embodiments, the expression vector is an RNA expression vector. In some embodiments, the RNA expression vector comprises a polynucleotide template for producing RNA in a cell-free enzyme mixture. In some embodiments, the RNA expression vector comprising the polynucleotide template is enzymatically linearized prior to in vitro transcription. In some embodiments, the polynucleotide template is generated by PCR as a linear polynucleotide template. In some embodiments, the linearized polynucleotide is mixed with an enzyme suitable for RNA synthesis, RNA capping, and/or purification. In some embodiments, the resulting RNA is suitable for producing a protein encoded by the RNA.

Various methods for introducing expression vectors into host cells are known in the art. In some embodiments, transfection may be used to introduce the vector into a host cell. In some embodiments, the transfection is accomplished, for example, using calcium phosphate transfection, lipofection, or polyethyleneimine mediated transfection. In some embodiments, transduction may be employed to introduce the vector into a host cell.

In some embodiments, the transformed host cell is cultured after introducing the vector into the host cell to allow expression of the recombinant polynucleotide. In some embodiments, the transformed host cell is cultured for at least 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, or more. The transformed host cells are cultured under growth conditions (e.g., temperature, carbon dioxide level, growth medium) that meet the requirements of the selected host cell. The skilled artisan will recognize that the culture conditions for the selected host cells are well known in the art.

Example(s)

Example 1: confirmation of immunogenic T cell antigens

Among other things, the present embodiments include results of a systematic analysis that yields an unexpected list or collection of T cell antigens (e.g., CD4 and/or CD 8T cell antigens) of HSV. The T cell antigens of the present embodiments may be used alone or in combination to produce HSV antigen constructs (e.g., for HSV vaccine agents). Among other things, the present embodiment provides a discussion of the systematic analysis applied, including selection of source data and stepwise evaluation of source data, to produce an unexpected list of T cell antigens for HSV.

The first step in systematically validating T cell antigens of HSV involves analysis of publicly available datasets related to T cell induction in HSV infected individuals. The T cell antigen of HSV preferably exhibits immunogenicity as a target of T cells in HSV infected human subjects, and still more preferably elicits a T cell response in a majority of infected human subjects in a cohort study. Many studies of T cell responses to HSV have been performed. However, the inventors have appreciated that many studies are narrow (e.g., exploring immune responses against one or a few antigens at a time in animal models or human subjects) or biased. It is difficult to obtain a comprehensive assessment of each HSV gene with minimal bias by combining data from independent studies narrowly focused on a few targets. Furthermore, since different groups use unique methods, reagents and patient cohorts, systematic analysis of data from different studies is challenging. The analysis of this example therefore includes a study that selects for simultaneous measurement of T cell responses (e.g., CD 8T cell responses) to most HSV proteins in multiple subjects. Three publications met these criteria and were further evaluated as a source of pan-genome immunogenicity data for HSV T cell targets: hosken (J Virol, 2006, 6; 80 (11): 5509-152006-15; PMID:16699031; hereinafter "Hosken" or "Hosken 2006"), jing (J CLIN INVEST, 2012, 2; 122 (2): 654-73; PMID:22214845; hereinafter "Jing" or "Jing 2012"), and Long (Virology, 2014, 9; 464-465:296-311; PMID:25108380; hereinafter "Long" or "Long 2014").

Having identified the above sources of pan-genome immunogenicity data for HSV T cell targets, the next step is to determine how to weight these three studies. To this end, we first determined whether the frequency of individual reports of T cell responses to each antigen (percent of responders) correlated between studies. We hypothesize that the general trend in immunogenicity should correspond between studies, and the lack of correlation would indicate that there may be a methodological defect. A strong correlation (r=0.70, spearman correlation) was observed between the percentages of respondents reported in Jing 2012 and Hosken 2006, but no significant correlation was observed between the results in Long 2014 and the other data sets. We therefore consider Long 2014 to be a low reliability dataset and exclude it from further analysis.

Based on the studies of Hosken 2006 and jin 2012, selection of T cell antigens of HSV from a collection of proteins encoded by genes of the HSV genome was performed in two steps. Each step is intended to reflect the form of data available in the corresponding publication. First, all HSV proteins below the median T cell responder frequency percentage in Hosken 2006 were excluded from consideration as T cell antigens (but genes for which no data was available were not excluded from consideration since some HSV genes were not represented in the data from Hosken 2006). Second, genes in sting 2012 that have a measurable T cell response in less than 3/7 subjects are excluded from consideration as T cell antigens. A total of 14 genes remained after these exclusion steps, which were further refined based on the expression levels as described below.

Further analysis was performed to identify candidate T cell antigens characterized by expression above the median level. In this further analysis, RNA and protein expression studies were evaluated to confirm HSV genes encoding the highest expressed T cell antigens during early viral replication. To conduct this analysis, research data is derived from a variety of sources.

Some data sources include scRNA-Seq data. For these studies, gene expression profiles of individual cells were analyzed and cells were arranged in a general chronological order from early to late activation by quantifying the expression of well-characterized Immediate Early (IE) genes (US 1, UL54, UL50, UL23, UL 30) and late genes (UL 48, UL45, UL44, US11, UL 36). Specifically, the late gene expression of each single cell is averaged (referred to as "late gene score") and the cells are arranged in order of increasing late gene score. The cells were then divided into 5 equal bins and the expression profiles of cells belonging to the same bin were averaged to produce 5 different expression profiles representing different time points along the activation continuum.

All expression profiles (from batch RNA-Seq, scRNA-Seq and proteomics) were normalized by scaling each sample/stage such that the maximum expression was 1 and the minimum expression was 0. The resulting normalized spectra are used to visualize the overall expression and the spearman correlation coefficient between the pairs of expression spectra is calculated. The expression profiles were also classified according to the expression of well-characterized Immediate Early (IE) genes (US 1, UL54, UL50, UL23, UL 30) and late genes (UL 48, UL45, UL44, US11, UL 36) to help determine which profiles are more likely to represent the early stages of activation. Specifically, the late gene expression for each single cell was averaged (referred to as "late gene score") and the cells were ranked in order of increasing late gene score (in the exact manner used to rank the single cells in the scRNA-Seq data analysis). By combining analysis of correlation coefficients and "late gene scores", we selected 12 expression profiles from 11 studies that appear to reliably represent expression during early activation. For each gene, median expression levels were determined in 12 studies. Finally, the median expression was calculated among all genes and genes with expression above the median were considered for inclusion in the vaccine. Based on these analyses, a panel of 10T cell antigens (e.g., CD4 and/or CD 8T cell antigens) of HSV were identified (shown in table 4).

The source literature is listed below and a brief review of the process is made:

fox (mBio.2017, 5-6 months; 8 (3): e00745-17; PMID: 28611249)

Data (MOI 5) relating to HSV1 KOS in MRC5 fibroblasts from the expression table (supplementary file in GSE 109420) containing the original reads for each gene were normalized by the number of reads per sample and by the gene length. We took a 4hpi sample for downstream analysis.

Krenn (Cell Stem cell.2021, 8.5/8; 28 (8): 1362-1379.e7; PMID: 33838105)

The omicron data relate to RN a-seq on HSV 1/hsipsc-derived 3D brain organoids mimicking infection, with or without IFNa2 treatment (which rescue HSV1 infection). A subset of the condition-specific samples (specified by their SRR IDs and condition tags in the table) was obtained from GEO (accession ID: GSE 145496). These samples were aligned with the GRCh38 and HSV-1 strain F genomes in tandem using STAR alignment (- -runMode alignReads- -outSAMatt ributes All- -outSAMtype BAM- -outMultimapperOrder Random). Reads of overlapping HSV-1 genes were then aggregated using the HTSeq package and HSV1 strain F reference annotation (HTSeq parameter: -tCDS-i gene). TP M normalization was performed on gene-specific feature counts.

Wang (Virol J.2020, 7/8; 17 (1): 95; PMID: 32641145)

The omicron data relates to the bulk RNA-seq with HSV-1 on the trigeminal ganglion of mice and tree shrew. The alignment reads from the BAM format of HSV-1 were obtained from the BIG Data center (accession number CRA 001750) and quantified using the featureCount function from the Rsubread R package. Reads were normalized by gene length. We took 7dpi time point samples for mice and tree shrews.

·Walter(2021.Herpesviral induction of germline transcription factor DUX4 is critical for viral gene expression.bioRxiv doi:10.1101/2021.03.24.436599)

The omicron data relate to RNA-seq from HSV-1 infected 293T cells with and without DUX4 KO. HSV-1 gene level counts were cross-WT and DUX4KO conditions when 18hpi was downloaded using GEO (GEO Login: GSE 174759). The genes RS1-r have similar count spectra in two rows in the sample and are de-duplicated. Counts were then normalized by the total library size of the samples and scaled by 10A 4. We took the 18hpi time point for downstream analysis.

·Tokuyama(2021.Endogenous retroviruses mediate IFN-in dependent protection against HSV-2infection.Query DataSets fo r GSE185281available at NCBI Gene Expression Ominibus(GE O))

The data relate to RNA-seq from HSV-2 infected mice (vaginal samples). Original read counts of two HSV-2 infected WT B6 mice were downloaded from PRJNA768446 of the SRA archive. Reads were aligned with tandem human (hg 38) and HSV-2 (HGS 2) genomes using STAR software, and HSV-2 gene expression was quantified using htseq-count. Data are shown as the mean of log2 (count/total library size 1e4+ 1) values from 2 replicates.

Khoury-Hanold (Cell Host microbe.2016, 6/8; 19 (6): 788-99; PMID: 27281569)

Data on omicron with ex vivo HSV-1 infection: the vaginal route, DRG (dorsal root ganglion) and LIM (large intestine muscle) sequences. The treated and standardized expression table was obtained from GSE74215. For downstream analysis, the expression is additionally scaled to account for gene length bias.

Tombicz (Front Microbiol.2017, month 6, 20; 8:1079; PMID: 28676792)

The omicron data are related to Pacbio seq of kidney epithelial cells infected with HSV1 (moi=1). Expression data is extracted from the supplemental files in GEO record GSE97785 and the gene names are unified using NCBI nomenclature. The data is in the form of a percentage of HSV2 gene reads in each sample, and those mapped to the same gene are aggregated.

Wyler (Nature Communications, volume 10, article number 4878 (2019); PMID: 31653857)

The omicron data is related to the scRNA-seq (host+virus) of HSV-1 infected fibroblast GSE 123782. Reads mapped to host and viral genes were counted from the supplementary files in this publication. To generate expression profiles for samples from each time point, readings from cells belonging to the same time point are aggregated and per million Transcript (TPM) values for each HSV-1 gene are calculated.

Drayman (elife.2019, 5, 15; 8:e46339.doi:10.7554/eLife.46339; PMID 31090537)

The omicron data was related to scRNA-seq from HSV-1 infected fibroblasts, MOI2,5 hpi. The read counts were obtained from GSE126042. The readings were normalized by the depth of each sample, multiplied by 10000 and log2 normalized. Cells that were aligned with only the HSV genome for more than 50 reads were considered hsv1+ and used for analysis.

Kulej (Mol Cell proteomics.2017, month 4; 16 (4 suppl 1): S92-S107; PMID: 28179408)

The proteomic expression data generated by primary Human Foreskin Fibroblasts (HFF) infected with HSV-1 strain 17syn+ were extracted from Kulej et al, MCP 2017;16 (4 suppl 1) S92-S107, doi 10.1074/mcp.M116.065987. We took a 6hpi time point for downstream analysis.

Soh (Cell Rep.2020, 10/6; 33 (1): 108235; PMID: 33027661)

The proteomic expression data generated by human keratinocyte line (HaCaT) cells infected with HSV-1KOS strain was extracted from Soh et al, cell Reports 2020;33 (1), supplementary Table S1 of doi.org/10.1016/j.celep.2020.108235. We took a 4hpi time point for downstream analysis.

Saiz-Ros (cells.2019, 2, 3; 8 (2): 120; PMID: 30717447)

The proteomic expression data generated by HeLa cells infected with HSV-1 strain 17+ were extracted from Saiz-Ros et al, cells.2019feb 3;8 (2) 120, doi:10.3390/cells 8020120. We took 8hpi time points for downstream analysis.

Additional T cell antigens of HSV were identified by examination of the source document and may be used alone or in combination with other antigens described herein. A further set of antigens was selected from subunit vaccines that had entered the clinical trial (Krishnan and Stuart. Front microbiol.2021, 12, 7; 12:798927; PMID: 3495527). Another group of additional antigens is selected from the promising prophylactic vaccination data in small animal models of HSV-2 infection (Morello et al, J Virol.2011, month 4; 85 (7): 3461-72; PMID:21270160; platt et al, cells.2013, month 1, day 4; 2 (1): 19-42; PMID: 24709642). The third set of additional antigens was identified by Hosken 2006 and Jing 2012 (see above in this example) as candidate T cell antigens for HSV, but did not meet the expression threshold discussed above. Analysis of these exam-source documents produced a panel of 9 additional T cell antigens (e.g., CD4 and/or CD 8T cell antigens) of HSV (shown in table 5).

Example 2: exemplary RNA constructs encoding HSV antigen

This example describes certain exemplary HSV antigens and sequences encoding them, which may be used in certain embodiments of the present disclosure. Exemplary HSV (e.g., HSV-1 and/or HSV-2) antigens can be found in tables 3-5.

In some specific embodiments, the administered RNA has the following structure:

Structure 1: m ₂ ^7,3'-OGppp(m₁ ^2'-O) ApG-hAg-Kozak-SEC-immunogen-FI-a 30L70, wherein m ₂ ^{7,3'- O}Gppp(m₁ ^2'-O) ApG = 5' cap; hAg = 5' utr human α -globulin; SEC = Signal Peptide (SP); immunogen = a nucleotide sequence comprising a sequence encoding an antigen described herein; fi=3 '-UTR, which is or comprises a sequence (e.g., 3' UTR) from a "split amino terminal enhancer" (AES) messenger RNA and a sequence (e.g., non-coding region) from a mitochondrially encoded 12S ribosomal RNA (MT-RNR 1); and a30l70=a poly a sequence comprising 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.

In some embodiments, the administered RNA has the following structure:

Structure 2: m ₂ ^7,3'-OGppp(m₁ ^2'-O) ApG-hAg-Kozak-SEC-immunogen-MITD-FI-a 30L70, wherein m ₂ ^{7 ,3'-O}Gppp(m₁ ^2'-O) ApG = 5' cap; hAg = 5' utr human α -globulin; SEC = Signal Peptide (SP); immunogen = a nucleotide sequence comprising a sequence encoding one or more antigens described herein; MITD = MHC class I transport signal (MITD); fi=3 '-UTR, which is or comprises a sequence (e.g., 3' UTR) from a "split amino terminal enhancer" (AES) messenger RNA and a sequence (e.g., non-coding region) from a mitochondrially encoded 12S ribosomal RNA (MT-RNR 1); and a30l70=a poly a sequence comprising 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.

In some embodiments, the immunogen sequence encodes a plurality of immunogenic fragments (e.g., comprising epitopes) from an antigen. In some embodiments, the immunogen sequence encodes multiple immunogenic fragments (e.g., epitopes) from two or more antigens. In some embodiments. In some embodiments, such immunogenic fragments are joined together by a linker (e.g., in some embodiments, a linker enriched in G and/or S amino acid residues) to form an immunogenic sequence. In some embodiments, the linker may be or comprise amino acid sequence GGSGGGGSGG (SEQ ID NO: 165). In some embodiments, the linker may be or comprise amino acid sequence GGSLGGGGSG (SEQ ID NO: 166).

Example 3: exemplary RNA constructs encoding Multi-epitope HSV antigen

This example describes certain exemplary HSV multi-epitope antigens and sequences encoding them, which may be used in certain embodiments of the present disclosure.

Exemplary constructs encoding HSV polyepitope polypeptides:

structure m ₂ ^7,3'-OGppp(m₁ ^2'-O) ApG-hAg-Kozak-SEC-CD8 string-MITD-FI-A30L 70

In some embodiments, the T cell antigen string may include at least 2 (including, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, or more) T cell epitopes and/or HLA-I epitopes as listed in tables 3-5.

Example 4: exemplary prediction and/or characterization of MHC presentation

This example describes exemplary methods to assess MHC presentation, which methods can be used in accordance with the present disclosure to select and/or characterize antigenic peptides as described herein.

In some embodiments, the antigenic peptide is selected and/or characterized by analyzing its amino acid sequence using an MHC-peptide presentation predictive algorithm or MHC-peptide presentation predictive factor, e.g., implemented in a computer processor (e.g., a computer processor that has been trained by machine learning software), which determines the likelihood of binding and presenting the peptide by an MHC class I or MHC class II antigen.

In some embodiments, the MHC-peptide presentation predictive algorithm or MHC-peptide presentation predictor is or includes neonmhc 1 and/or neonmhc2, which predict and/or characterize the likelihood of MHC class I and MHC class II binding, respectively. Alternatively or additionally, in some embodiments, the MHC-peptide presentation predictive algorithm or MHC-peptide presentation predictor is or includes NETMHCPAN or NETMHCIIPAN. In some embodiments, MHC-peptide presentation predictions and/or characterizations may be made using a hidden markov model approach. In some embodiments, a peptide prediction model MARIA may be utilized. In some embodiments, NETMHCPAN is not utilized to predict or characterize the likelihood of MHC binding of a peptide as described herein. In some embodiments, a peptide prediction model MARIA may be utilized. In some embodiments, NETMHCIIPAN is not utilized to predict or characterize the likelihood of MHC binding of a peptide as described herein. In some embodiments, neither NETMHCPAN nor NETMHCIIPAN are utilized to predict or characterize the likelihood of MHC binding of a peptide as described herein. In some embodiments, the MHC-peptide presentation predictive algorithm or MHC-peptide presentation predictor is or includes RECON, which provides high quality MHC-peptide presentation predictions based on expression, processing, and binding capacity.

In some embodiments, a variety of MHC-peptide presentation predictive algorithms or MHC-peptide presentation predictors may be utilized; in some such embodiments, the results obtained with different strategies are compared to each other. In some embodiments, determining that a particular peptide is likely or most likely to be presented by MHC class I or MHC class II may be considered more definitive if two or more algorithms or predictors are consistent.

Alternatively or additionally, confirmation and/or characterization of MHC binding (e.g., MHC class I and/or MHC class II binding) may involve experimental assessment or reporting thereof, which may involve presentation in one or more in vitro systems and/or in one or more organisms. In some embodiments, such assessment utilizes mammalian cells or systems; in some embodiments, such assessment utilizes primate (e.g., in some embodiments, human, and/or in some embodiments, non-human primate) cells or systems.

Example 5: exemplary HLA class I and class II binding assays

This example describes exemplary techniques for assessing binding of peptides to HLA molecules. In some embodiments, the exemplified techniques can determine and/or characterize (e.g., quantify) the binding affinities of HLA class I and HLA class II.

In general, binding assays can be performed with peptides with or without motifs. A detailed description of exemplary protocols that can be used to measure binding of peptides to class I and class II MHC has been disclosed (Sette et al, mol. Immunol.31:813,1994; sidney et al, current Protocols in Immunology, margulies et al, john Wiley & Sons, new York, section 18.3, 1998). Briefly, purified MHC molecules (5 to 500 nM) were incubated with various unlabeled peptide inhibitors and 1-10nM ¹²⁵ -radiolabeled probe peptide in PBS containing 0.05% Nonidet P40 (NP 40) (or 20% w/v digitonin for H-2IA assay) for 48H in the presence of a protease inhibitor mixture. The assay is typically performed at a pH of 7.0, although in some embodiments may be performed at a lower pH (typically above about pH 4.0).

After incubation, the MHC-peptide complex is separated from the free peptide, for example by gel filtration, for example on a 7.8mm x 15cm TSK200 column (TosoHaas 16215, montgomeryville, pa), although one skilled in the art will appreciate that column dimensions may be adjusted if desired, for example to improve separation of bound from unbound peptide of a particular size or character. The eluate from the TSK column was passed through a Beckman 170 radioisotope detector, radioactivity mapped and integrated using a Hewlett-Packard 3396A integrator and the fraction of bound peptide was determined.

Radiolabeled peptides may be iodinated using the chloramine-T method. Typically, in a preliminary experiment, each MHC preparation is titrated in the presence of a fixed amount of radiolabeled peptide to determine the concentration of HLA molecules required to bind 10-20% of the total radioactivity. Subsequent inhibition and direct binding assays can be performed using these HLA concentrations.

Since under these conditions [ tag ] < [ HLA ] and IC ₅₀ > [ HLA ], the measured IC ₅₀ values are generally a reasonable approximation of the true K _D values. Peptide inhibitors are typically tested in a concentration range of 120 μg/ml to 1.2ng/ml and in two to four completely independent experiments. To allow comparison of the data obtained in the different experiments, the relative binding value of each peptide is typically calculated by dividing the IC ₅₀ of the inhibited positive control by the IC ₅₀ of each test peptide (typically the unlabeled form of the radiolabeled probe peptide). The relative binding values may be compiled for database purposes and for comparison between experiments. Such values can then be converted back to IC ₅₀ nM values, for example by dividing IC ₅₀ nM of the inhibited positive control by the relative binding of the peptide of interest. This method of data compilation has been demonstrated to provide accurate and consistent comparisons of peptides tested on different dates or with different batches of purified MHC.

Alternatively or additionally, assays based on living cells/flow cytometry may also be employed. This is a maturation assay using the TAP-deficient hybridoma cell line T2 (American type culture Collection (ATCC accession No. CRL-1992), manassas, va.). TAP defects in this cell line result in insufficient mhc i loading and excessive empty mhc i in the ER. Salter and CRESSWELL, EMBO J.5:94349 (1986); salter, immunogenetics21:235-46 (1985). The empty MHCI is extremely unstable and thus has a short lifetime. When T2 cells are cultured at reduced temperature, empty mhc is transiently present on the cell surface where they can be stabilized by exogenously adding mhc i binding peptides. To perform this binding assay, peptide-receptive MHCI was induced by culturing 10 ⁷ aliquots of T2 cells at 26℃overnight in serum-free AIM-V medium alone or in medium containing peptide at progressively higher concentrations (0.1 to 100. Mu.M). Cells were then washed twice with PBS and then incubated with a fluorescently labeled HLA allele-specific monoclonal antibody (e.g., HLA-a02: 01-specific monoclonal antibody BB 7.2) to quantify cell surface expression. Samples were taken on a FACS Calibur instrument (Becton Dickinson) and Mean Fluorescence Intensity (MFI) was determined using the accompanying Cellquest software.

Example 6: confirmation of immunogenicity

This example describes an exemplary method of confirming immunogenicity, particularly by testing the ability of one or more antigens or peptides to expand cd8+ T cells using an in vitro amplification (IVE) assay.

Mature professional APCs for these assays were prepared as follows. 80-90x10 ⁶ PBMCs from healthy human donors were plated in 20ml RPMI medium containing 2% human AB serum and incubated for 2 hours at 37 ℃ to allow for monocyte adhesion to plastics. Non-adherent cells were removed and adherent cells were cultured in RPMI, 2% human AB serum, 800IU/ml GM-CSF, and 500IU/ml IL-4. After 6 days, TNF-. Alpha.was added to a final concentration of 10ng/ml. Dendritic Cells (DCs) were matured by addition of 12.5mg/ml polyI: C or 0.3. Mu.g/ml CD4OL on day 7. Mature dendritic cells (mdcs) were harvested on day 8, washed, and either used directly or cryopreserved for future use.

For IVE of CD8+ T cells, aliquots of 2X10 ⁵ mDC were pulsed with each peptide at a final concentration of 100. Mu. Moles, incubated for 4 hours at 37℃and then irradiated (2500 rad). Peptide pulsed mdcs were washed twice in RPMI with 2% human AB serum. 2x10 ⁵ mDC and 2x10 ⁶ autologous CD8+ cells were plated in each well of a 24-well plate in 2ml RPMI containing 2% human AB, 20ng/ml IL-7 and 100pg/ml IL-12 and incubated for 12 days. Cd8+ T cells were then re-stimulated with peptide pulsed, irradiated mdcs. Two to three days later, 20IU/ml IL-2 and 20ng/IL7 were added. Expanded cd8+ T cells were re-stimulated every 8-10 days and maintained in media containing IL-2 and IL-7. The peptide-specific T cells of the culture are monitored using a combination of functional assays and/or tetramer staining. Parallel IVES with the modification and parent peptide allows comparison of the relative efficiencies of peptide expansion of peptide-specific T cells.

Example 7: quantification and functional assessment of cd8+ and cd4+ T cells

Tetramer staining

MHC tetramers are purchased or manufactured in situ and are used to measure peptide-specific T cell expansion in IVE assays. For evaluation, tetramers were added to lx10 ⁵ cells in PBS containing 1% FCS and 0.1% sodium azide (FACS buffer) according to the manufacturer's instructions. Cells were incubated for 20min at room temperature in the dark. Antibodies specific for T cell markers (such as CD 8) were then added to the final concentrations recommended by the manufacturer and the cells incubated in the dark for 20 minutes at 4 ℃. Cells were washed with cold FACS buffer and resuspended in buffer containing 1% formaldehyde. Cells were harvested on a FACS Calibur (Becton Dickinson) instrument and analyzed by using Cellquest software (Becton Dickinson). To analyze tetramer-positive cells, lymphocyte gates were taken from forward and lateral scatter plots. The data are reported as a percentage of cells that are cd8+/tetramer+.

An ex vivo induction protocol may be used to test CD4 ⁺ T cell responses against an antigen or peptide. In this example, the CD4 ⁺ T cell response was confirmed by monitoring ifnγ and/or tnfα production in an antigen-specific manner.

Assessment of antigen presentation:

for a subset of antigens or peptides (e.g., being or comprising an epitope predicted or selected as described herein), affinity for and/or stability with a given HLA allele can be determined.

An exemplary detailed description of a protocol that can be used to measure binding affinity of peptides to MHC class I has been disclosed (Sette et al, mol. Immunol.31 (11): 813-22, 1994). Briefly, MHCI complexes were prepared and conjugated to radiolabeled reference peptides. Different concentrations of peptide were incubated with these complexes for 2 days and the amount of remaining radiolabeled peptide bound to mhc i was measured using size exclusion gel filtration. The lower the concentration of test peptide required to replace the reference radiolabeled peptide, the greater the affinity of the peptide for mhc i. Peptides with affinity <50nM to mhc i are generally considered strong binders, while peptides with affinity <150nM are considered medium binders, and those <500nM are considered weak binders (Fritsch et al, 2014).

An exemplary detailed description of a protocol that can be used to measure the binding stability of peptides to class I MHC has been disclosed (Harndahl et al, J Immunol methods.374:5-12,2011). Briefly, synthetic genes encoding biotinylated MHC-I heavy and light chains were expressed in E.coli and purified from inclusion bodies using standard methods. The light chain (. Beta.2m) was radiolabeled with iodine (125I) and combined with purified MHC-I heavy chain and peptide of interest at 18℃to initiate pMHC-I complex formation. These reactions were performed in streptavidin coated microwells to bind biotinylated MHC-I heavy chains to the surface and allow measurement of radiolabeled light chains to monitor complex formation. Dissociation was initiated by adding a higher concentration of unlabeled light chains and incubating at 37 ℃. Stability is defined as the length of time it takes for half of the complex to dissociate, in hours, as measured by scintillation counting. The binding affinity of MHC-II to peptides was measured following the same general procedure as for MHCI-peptide binding affinity. Described herein are predictive algorithms that are utilized to predict binding of MHCII alleles to a given peptide. Alternatively or additionally, NETMHCIIPAN may be utilized to predict binding.

To assess whether a particular antigen or peptide or epitope can be processed and presented from a larger polypeptide environment, peptides eluted from HLA (class I or class II) molecules isolated from cells expressing the gene of interest can be analyzed by tandem mass spectrometry (MS/MS).

ELISPOT

Peptide-specific T cells are functionally counted, for example using the ELISPOT assay (BD Biosciences) which measures ifnγ release in T cells on a single cell basis. Target cells (T2 or specific HLA transfected C1R (e.g., HLA-a0201 transfected C1R)) were pulsed with 10uM peptide at 37 ℃ for 1 hour and washed three times. Targets of 1x105 peptide pulses were co-cultured with different concentrations of T cells (5 x102 to 2x 103) taken from IVE cultures in ELISPOT plate wells. The plates were developed according to the manufacturer's protocol and analyzed on an ELISPOT reader (Cellular Technology ltd.) with accompanying software. Spots corresponding to the number of ifnγ -producing T cells are reported as absolute spot numbers per number of T cells plated. For T cells expanded on modified peptides, they were tested not only for their ability to recognize targets pulsed with the modified peptide, but also for their ability to recognize targets pulsed with the parent peptide.

CD107 staining

CD107a and b are expressed on the cell surface of cd8+ T cells after activation with cognate peptides. The soluble particles of T cells have a lipid bilayer containing lysosomal associated membrane glycoproteins ("LAMP") including molecules CD107a and b. Without wishing to be bound by any one theory, it is proposed that when cytotoxic T cells are activated by T cell receptors, the membrane of these soluble particles moves and fuses with the plasma membrane of the T cells. The particle content is released, which results in the death of the target cells. Since the particle membrane fuses with the plasma membrane, C107a and b are exposed on the cell surface and are therefore markers of degranulation. Since degranulation as measured by CD107a and b staining was reported on a single cell basis, the exemplary assay was used to functionally enumerate peptide-specific T cells. For the assay, peptides were added to specific HLA transfected cells C1R (e.g. HLA-a0201 transfected C1R) to a final concentration of 20 μm, cells were incubated for 1 hour at 37 ℃ and washed three times. 1x105 peptide pulsed C1R cells were aliquoted into tubes and antibodies specific for CD107a and b were added to the final concentrations recommended by the manufacturer (Becton Dickinson). Antibodies are added prior to T cell addition in order to "capture" CD107 molecules, as they appear briefly on the surface during the assay process. Next 1x 105T cells from the culture were added and the samples were incubated for 4 hours at 37 ℃. Additional cell surface molecules such as CD8 were further stained for T cells and obtained on a FACS Calibur instrument (Becton Dickinson). The data were analyzed using the accompanying Cellquest software and the results reported as percentages of cd8+cd107 a and b+ cells.

CTL dissolution

Cytotoxic activity can be measured, for example, using a chromium release assay. Target T2 cells were labeled with Na ⁵¹ Cr for 1 hour at 37 ℃ and then washed 5x10 ³ target T2 cells were added to different numbers of T cells from IVE cultures. After incubation at 37 ℃ for 4 hours, chromium release was measured in the harvested supernatant. The percentage of specific lysis was calculated as:

Experimental release-spontaneous release/total release-spontaneous release x100.

Example 8: administration of multiple epitope compositions

This example describes exemplary administration of compositions comprising or delivering multiple epitopes.

For example, a multi-epitope vaccine (e.g., that comprises or delivers a collection of epitopes-e.g., as separate discrete peptides or as one or more multi-epitope peptides, such as one or more string constructs as described herein).

In some embodiments, the multi-epitope vaccine comprises or delivers a plurality of Cytotoxic T Lymphocyte (CTL) and/or Helper T Lymphocyte (HTL) epitopes. In some embodiments, such vaccines are administered to subjects at risk of infection or who have undergone exposure to infection.

In some embodiments, a multi-epitope vaccine as described herein comprises or delivers one or more polypeptides, each polypeptide encompassing multiple epitopes. In some embodiments, one or more mono-or multi-epitope antigens are delivered to a subject by administration of one or more nucleic acid (e.g., DNA or and RNA) constructs. In some embodiments, a single nucleic acid construct (e.g., DNA or RNA encoding a multi-epitope antigen) is administered. In some embodiments, multiple nucleic acids are administered (e.g., each encoding a different mono-or multi-epitope antigen). In some embodiments, the nucleic acid administered is RNA (e.g., mRNA); in some embodiments, the nucleic acid (e.g., RNA) is administered in an LNP composition.

In some embodiments, the applied composition includes an aqueous carrier and/or alum.

In some embodiments, the initial administration is followed by one or more booster doses. In some embodiments, the booster dose comprises the same amount of the multi-epitope construct as the initial dose. In some embodiments, the boost dose comprises more or less of the polyepitope construct than provided in the initial dose. In some embodiments, the booster dose is administered at least 1,2, 3, 4,5,6,7, 8, 9, 12, 11, 12, 13, 14, 15, 16, 17, 18, 29, 20, 21, 22, 23, 24 weeks or more after the initial dose. In some embodiments, multiple booster doses are administered. In some embodiments, the interval between each subsequent booster administration is the same as or longer than the interval between its immediately preceding dose and the dose preceding that dose. In some embodiments, 2,3, 4,5,6 or more boosters are administered. In some embodiments, no more than 4 doses are administered in total. In some embodiments, no more than 3 doses are administered in total. In some embodiments, no more than two doses are administered in total. In some embodiments, no more than 1,2, 3, or 4 doses are administered over a specified 12 month period. In some embodiments, no more than 3, no more than 2, or no more than 1 doses are administered over a specified 12 month period (e.g., within 12 months of the initial dose).

In some embodiments, the induced immune response (e.g., the size, character and/or diversity of the immune response, such as an antibody and/or T cell response) is assessed before and/or after one or more doses (e.g., 1,2, 3, 4 or more weeks after administration of a particular dose, and/or within 6, 5, 4, 3, 2 or 1 month of administration of a particular dose), and may be considered, for example, in determining whether one or more booster doses should be administered and/or the timing of such booster dose administration. In some embodiments, the assessment of immune response may utilize techniques such as determining the presence and/or level of a population of epitope-specific CTLs in a PBMC sample.

EXAMPLE 9 administration of dendritic cells

This example describes exemplary dendritic cell compositions comprising or delivering an antigen as described herein.

In this embodiment, a peptide comprising an epitope as described herein (e.g., identified, designed, selected, and/or characterized as described herein) is loaded onto a dendritic cell. Peptide pulsed dendritic cells can be administered to a subject. In some embodiments, such administration may stimulate a CTL response in vivo.

In this particular embodiment, dendritic cells (e.g., autologous dendritic cells) are isolated, expanded, and pulsed with peptide CTL and/or HTL epitopes as described herein. The dendritic cells can then be returned to the patient. Such infusion may elicit CTL and/or HTL responses in vivo. The induced CTLs and HTLs then destroy (CTLs) or promote destruction (HTLs) of target cells (e.g., hepatocytes) bearing the protein from which the epitope in the vaccine was derived.

An ex vivo CTL or HTL response to a particular antigen can be induced by incubating patient or genetically compatible CTL or HTL precursor cells together with a source of antigen presenting cells (e.g., dendritic cells) and appropriate immunogenic peptides in tissue culture.

After a suitable incubation time (typically about 7-28 days) in which the precursor cells are activated and expanded into effector cells, the cells are returned to the patient where they will destroy (CTL) or promote destruction (HTL) of their specific target cells, i.e. cells displaying the relevant epitope.

Example 10: administration of epitope binding agents

This example describes the administration of epitope binding agents as an alternative or in addition to the vaccination strategies described herein.

For example, the present disclosure provides, among other things, techniques for identifying and/or characterizing HSV antigens that are particularly susceptible to targeting in order to destroy one or more characteristics of an infection. In many embodiments described herein, the present disclosure provides techniques involving administration or delivery of antigens that are or comprise such epitopes, e.g., to induce an immune response in a recipient that targets such epitopes.

Alternatively or additionally, the present disclosure provides and encompasses techniques for developing, characterizing, and/or administering agents that bind to such epitopes. In some embodiments, such strategies may provide or represent therapeutic intervention, e.g., may be used as a supplement or replacement to vaccination strategies.

Epitope binding agents, such as antibody agents, TCR agents, CAR agents, and/or cells expressing any of the foregoing, may be administered according to methods known in the art and/or described herein.

In some embodiments, the relevant epitope binding agent may be delivered by administering a composition that is or comprises a polypeptide binding agent. Alternatively or additionally, in some embodiments, the relevant epitope binding agent may be delivered by administering a composition that is or comprises a polynucleotide (e.g., DNA or RNA, and in many advantageous embodiments RNA) encoding a polypeptide binding agent. In some embodiments, the polypeptide binding agent is delivered by administering a cell (or population thereof) comprising or expressing the polypeptide and/or polynucleotide encoding the polypeptide.

Example 11: exemplary confirmation and/or characterization of variant sequences with immunogenic potential

This example describes techniques for identifying and/or characterizing peptide sequences that differ from the relevant references for assessing immunogenic potential.

The full-length amino acid sequence of the variant protein is obtained (e.g., as observed in circulating strains or developed by predictive models).

The constitutive 9-mer and 10-mer peptide fragments of the variant proteins are each scored for binding potential on a common HLA allele (including, for example, but not limited to, HLA-A01:01, HLA-A02:01.HLA-A03:01, HLA-A24:02, HLA-B07:02, and HLA-B08: 01) using available algorithms. Peptides with scores of better than 1000nM are indicated as potential candidates.

Alternatively or additionally, constitutive 9-mer or 10-mer peptide sequences not found in the reference protein sequence are labeled using available algorithms and scored for binding potential on common HLA alleles (including, for example, but not limited to HLA-A01:01, HLA-A02:01.HLA-A03:01, HLA-A24:02, HLA-B07:02, and HLA-B08:01).

Example 12: exemplary HSV peptide string construct design

This example illustrates certain constructs (referred to herein as "strings") of various HSV antigens and/or epitopes present in a vaccine composition and/or linked to each other or otherwise as described herein.

The strings described in this embodiment are designed to contain specific epitopes of HSV, each of which is disclosed herein, and are predicted and/or selected, e.g., as described herein, e.g., by using an MHC binding algorithm as described herein. The strings presented in this example are designed for therapeutic use in the prevention and/or treatment of HSV infection and may be administered as a polynucleotide construct, such as mRNA encapsulated in lipid nanoparticles.

In some embodiments, the strings described throughout this disclosure are encoded in RNAs that include 5'-UTR and 3' -UTR. Epitopes are linked to each other by peptide linkers and are encoded by their respective polynucleotide sequences. In some embodiments, one or more linkers may have a specific cleavage site.

Example 13: exemplary antigen validation, selection and/or characterization

This example describes the identification, selection and/or characterization of certain HSV protein sequences that may be used as or in (i.e., as part of) an antigen as described herein (see example 1).

The degree of conservation of candidate proteins among related HSV strains (e.g., in a relevant geographic region) may be considered, for example, in the selection of sequences and/or epitopes included or encoded in the vaccine and/or antigen construct. Various laboratory and field isolates can be considered to evaluate conserved proteins and T cell epitopes. To assess conservation of HSV genes identified as T cell epitopes of HSV, the HSV1 and HSV2 genomes were downloaded from VIPR databases using "Genome search->Herpesviridae family->Alphaherpesvirinae subfamily->Simplexvirus Genus->Human Alp haherpesvirus 1&2species", with only complete genomes. It is checked whether all downloaded genomes have identical gene names. HSV-1 strain 17 and HSV-2 strain HG52 were used as reference strains for HSV-1 and HS V-2, respectively. The results of this conservation analysis are depicted in FIGS. 15-33. All sequences belonging to each of the HSV proteins were subjected to Multiple Sequence Alignment (MSA) using the mafft-linsi program with default parameters. Two measures of conservation were calculated: 1) Protein level conservation was calculated as a percentage of sequence similarity to the reference strains (HSV-1 strain 17 and HSV-2 strain HG 52) from the MS a profile, and 2) conservation at each position along each protein was quantified as the frequency of dominant 9-mers starting at each amino acid position. The results of this HSV strain conservation analysis are depicted in FIGS. 34-52.

Immunogenicity of conserved proteins may also be considered, for example, by review of the literature and/or application of predictive algorithms as described herein.

According to the present examples, in some embodiments, the antigen may be or comprise one or more different fragments (e.g., epitope-containing fragments) of one or more of these proteins, and in particular may comprise a plurality of different fragments, e.g., in a string construct as described herein.

In some embodiments, the secretion signal ("Sec") or Signal Peptide (SP) domain present in an exemplary candidate string described herein (e.g., a CD 8T cell antigen string and/or a CD 4T cell antigen string as described herein) may be from HSV-2gD SP MGRLTSGVGTAALLVVAVGLRVVCA (SEQ ID NO: 87); in alternative embodiments, a different secretion signal is used, such as from HSV-1gD SP. In some embodiments, the secretion signal peptide may be or include an IgE signal peptide. In some embodiments, the secretion signal peptide may be or include an IgE HC (Ig heavy chain ε -1) secretion signal peptide. In some embodiments, a secretion signal peptide useful according to the present disclosure may comprise one of the following sequences:

MDSKGSSQKGSRLLLLLVVSNLLLPQGVVG(SEQ ID NO:118)；

MDWTWILFLVAAATRVHS(SEQ ID NO:93)；

METPAQLLFLLLLWLPDTTG(SEQ ID NO:92)；

MLGSNSGQRVVFTILLLLVAPAYS (Japanese encephalitis PRM signal sequence; SEQ ID NO: 94);

MKCLLYLAFLFIGVNCA (VSVg protein signal sequence; SEQ ID NO: 95);

MWLVSLAIVTACAGA (Japanese encephalitis JEV signal sequence; SEQ ID NO: 119); or (b)

MFVFLVLLPLVSSQC(SEQ ID NO:120)。

In some embodiments, certain chunk boundary considerations are included in the string construct, such as establishing chunk boundaries to minimize the presence of sequences (e.g., epitopes) that may overlap with the human proteome.

Example 14: exemplary vaccine compositions

This example describes certain exemplary vaccine compositions:

In some embodiments, the provided candidate vaccine will contain at least 2 RNAs, at least one of which encodes an HSV antigen described herein (e.g., a full-length HSV protein or one or more fragments or epitopes thereof, as in a string construct described herein), and optionally at least one of which encodes at least one other conserved HSV protein (or fragment or epitope thereof, as in a string construct described herein; in some embodiments, such a string construct may comprise fragments or epitopes from two or more different HSV proteins).

In some embodiments, two or more (e.g., 3, e.g., 2 of which are/encode HSV antigen string constructs, and one of which is/encode a plurality of CD8 and/or CD4 epitopes from other conserved HSV proteins) are formulated together in a single LNP formulation; in other embodiments, separate RNAs may be formulated separately in the (same or different) LNP formulation, and they may be mixed together (e.g., each RNA to "other" RNA at a 1:1 ratio, or HSV antigen-encoding RNA to "other" RNA at a 1:1 ratio, such that for a composition comprising 2 HSV antigen-encoding RNAs and one other RNA, the ratio would be 0.5:0.5:1).

Example 15: exemplary LNP formulation

This example describes certain preferred LNP formulations that can be used in vaccine compositions as described herein.

In some embodiments, LNP formulations useful in vaccine compositions as described herein can comprise at least one ionizable amino lipid. In some embodiments, LNP formulations useful in vaccine compositions as described herein may further comprise a helper lipid, which in some embodiments may be or include a neutral helper lipid. In some embodiments, LNP formulations useful in vaccine compositions as described herein may further comprise a polymer conjugated lipid, for example, in some embodiments, a PEG conjugated lipid. In some embodiments, LNP formulations useful in vaccine compositions as described herein can comprise at least one ionizable amino lipid, at least one helper lipid (e.g., a neutral helper lipid, which in some embodiments can comprise a phospholipid, a steroid, or a combination thereof), and at least one polymer conjugated lipid (e.g., a PEG conjugated lipid). In some embodiments, exemplary LNP formulations can comprise ionizable amino lipids, phospholipids, steroids, and PEG conjugated lipids.

In some embodiments, the ionizable amino lipid may be present in the LNP formulation in a range of 45 to 55 mole percent, 40 to 50 mole percent, 41 to 49 mole percent, 41 to 48 mole percent, 42 to 48 mole percent, 43 to 48 mole percent, 44 to 48 mole percent of the total lipid. In some embodiments, exemplary ionizable amino lipids are or include ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate) (also known as 6- [ N-6- (2-hexyldecanoyloxy) hexyl-N- (4-hydroxybutyl) amino ] hexyl 2-hexyl decanoate). In some embodiments, exemplary ionizable amino lipids are or include SM-102 (heptadec-9-yl 8 ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) octanoate) or amino lipids as described in Sabnis et al ,"A Novel Amino Lipid Series for mRNA Delivery:Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates"Mol.Ther.(2018)26:1509-1519. In some embodiments, exemplary ionizable amino lipids are or include ionizable amino lipids as disclosed in US2020/0163878 or WO2018/078053, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

In some embodiments, the phospholipid may be present in the LNP formulation in the range of 5 to 15 mole percent, 7 to 13 mole percent, or 9 to 11 mole percent of the total lipid. In some embodiments, an exemplary phospholipid is or includes 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC).

In some embodiments, the sterol may be present in the LNP formulation in the range of 30 to 50 mole percent, 35 to 45 mole percent, or 38 to 43 mole percent of the total lipid. In some embodiments, the exemplary sterol is or includes cholesterol.

In some embodiments, the polymer conjugated lipid (e.g., PEG conjugated lipid) can be present in the LNP formulation in a range of 1 to 10 mole percent, 1 to 5 mole percent, or 1 to 2.5 mole percent of the total lipid. In some embodiments, the exemplary PEG conjugated lipid is or includes 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide (also known as 2- [2- (ω -methoxy (polyethylene glycol 2000) ethoxy ] -N, N-bitetradecylamide). In some embodiments, the exemplary phospholipid is or includes PEG2000-DMG (1-monomethoxypolyethylene glycol-2, 3-dimyristoylglycerol, wherein the average molecular weight of the polyethylene glycol is 2000). In some embodiments, the exemplary PEG conjugated lipid is or includes a PEG lipid as disclosed in US2020/0163878 or WO2018/078053, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

In some embodiments, exemplary LNP formulations comprise (i) an ionizable amino lipid in the range of 45 to 55 mole percent of total lipid; (ii) A phospholipid in the range of 8 to 12 mole percent of total lipid; (iii) A steroid in the range of 35 to 45 mole percent of total lipid; and (iv) polymer conjugation (e.g., PEG conjugated polymer) in the range of 1 to 2 mole percent of total lipid; and RNA molecules as described herein, encapsulated within or associated with the lipid nanoparticle.

In some embodiments, exemplary LNP formulations comprise (i) an ionizable amino lipid in the range of 45 to 55 mole percent of total lipid; (ii) DSPC in the range of 5 to 15 mole percent of total lipid; (iii) Cholesterol in the range of 35 to 45 mole percent of total lipid; and (iv) PEG conjugated lipid in the range of 1 to 2 mole percent of total lipid; and RNA molecules as described herein, encapsulated within or associated with the lipid nanoparticle.

In some embodiments, exemplary LNP formulations comprise (i) an ionizable amino lipid in the range of 40 to 50 mole percent of total lipid; (ii) A phospholipid in the range of 5 to 15 mole percent of total lipid; (iii) A steroid in the range of 35 to 45 mole percent of total lipid; and (iv) polymer conjugation (e.g., PEG conjugated polymer) in the range of 1 to 10 mole percent of total lipid; and RNA molecules as described herein, encapsulated within or associated with the lipid nanoparticle. In some such embodiments, the ionizable amino lipid is or comprises ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (also known as 6- [ N-6- (2-hexyldecanoyloxy) hexyl-N- (4-hydroxybutyl) amino ] hexyl 2-hexyldecanoate). In some such embodiments, the phospholipid is or comprises 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC). In some such embodiments, the steroid is or includes cholesterol. In some such embodiments, the polymer conjugated polymer is or includes 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide (also known as 2- [2- (ω -methoxy (polyethylene glycol 2000) ethoxy ] -N, N-bitetradecylamide).

In one embodiment, an exemplary LNP formulation comprises the following lipids and RNA molecules as described herein, included in table 15 below.

Table 16: exemplary LNP formulation

In some embodiments, an exemplary LNP formulation comprises an ionizable amino lipid, DSPC, cholesterol, and PEG conjugated lipid in a molar ratio of about 50:10:38.5:1.5 or 47.5:10:40.8:1.7. In some embodiments, the ionizable amino lipid is or comprises ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (also known as 6- [ N-6- (2-hexyldecanoyloxy) hexyl-N- (4-hydroxybutyl) amino ] hexyl 2-hexyldecanoate).

In some embodiments, exemplary LNP formulations comprise (i) SM-102 (heptadec-9-yl 8 ((2 hydroxyethyl) (6 oxo 6- (undecoxy) hexyl) amino) octanoate) in the range of 45 to 55 mole percent of total lipids; (ii) DSPC in the range of 5 to 15 mole percent of total lipid; (iii) Cholesterol in the range of 35 to 45 mole percent of total lipid; and (iv) PEG2000-DMG in the range of 1 to 2 mole percent of total lipid; and RNA molecules as described herein, encapsulated within or associated with the lipid nanoparticle.

In some embodiments, exemplary LNP formulations comprise (i) in the range of 45 to 55 mole percent of total lipid ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (also known as 6- [ N-6- (2-hexyldecanoyloxy) hexyl-N- (4-hydroxybutyl) amino ] hexyl 2-hexyldecanoate); (ii) DSPC in the range of 5 to 15 mole percent of total lipid; (iii) Cholesterol in the range of 35 to 45 mole percent of total lipid; and (iv) PEG conjugated lipid in the range of 1 to 2 mole percent of total lipid; and RNA molecules as described herein, encapsulated within or associated with the lipid nanoparticle.

Example 16: exemplary preclinical evaluation

This example describes certain preclinical evaluations that can be performed on certain RNA vaccine compositions described herein:

In some embodiments, a candidate vaccine is evaluated. In some embodiments, more than one different candidate vaccine may be evaluated. In some such embodiments, the different candidates may differ, for example, in the following:

RNA platform (e.g., unmodified RNA, modified RNA, saRNA);

An encoded antigen;

The amount of RNA;

elements of the RNA construct (e.g., cap and/or cap proximity sequences, 5'-UTR, 3' -UTR, and/or poly a tail); and/or

Lipid composition of LNP.

In some embodiments, preclinical evaluation of certain RNA vaccine compositions (e.g., LNP formulated mRNA-based HSV vaccines) includes one or more of evaluation in challenge experiments, evaluation of protection levels, evaluation of immunogenicity, and/or evaluation of functional antibody responses.

LNP formulated mRNA-based HSV vaccines were tested in challenge models. For non-human primate models such as rhesus and cynomolgus, and/or rodent models such as C57/B16 mice, balb/C mice or NODscidIL rγnull mice vaccinated with HSV; and/or guinea pig model, and may be administered a first vaccination, and additional vaccinations (e.g., 1,2, 3,4, 5, 6, 7, 8, 9, or 10 additional vaccinations) may be administered after the first vaccination. In the case of more than one vaccination, the vaccinations are performed at 1,2, 3,4, 5, 6, 7 or 8 week intervals. Following vaccination, animals are challenged by HSV. Alternatively or additionally, animals are challenged by intravenous, subcutaneous and/or intramuscular injection of virus-infected lymphocytes. Lymphocytes may be infected with any suitable strain of HSV. The animals were then assessed for reduced neuronal infection. In some embodiments, the animal model is challenged in a variety of circumstances (e.g., at any point in time between or after vaccination prior to the first vaccination and/or where additional vaccinations are administered). Following challenge, the animals under study may be evaluated according to any method known in the art, including, for example, serological evaluation, immunogenicity, protection levels, and the like.

In some embodiments, serum antibody characterization and/or serum transfer experiments (e.g., from one vaccinated species to a different unvaccinated species, e.g., from vaccinated non-human primates to unvaccinated mice) are performed (e.g., to assess protective antibody responses).

In some embodiments, the level of protection of certain RNA vaccine compositions of the present disclosure is assessed. The level of protection may be assessed according to any suitable method known in the art.

In some embodiments, the immunogenicity of certain RNA vaccine compositions of the present disclosure is assessed. For example, ELISA may be used to determine IgG specific (and subclasses thereof) titers and/or avidity of antibodies raised against HSV antigens in response to certain RNA vaccine compositions of the present disclosure. In some embodiments, serum antibody titers against HSV glycoproteins (e.g., gH and/or gL glycoproteins, etc.) are determined by ELISA using standard methods. In some embodiments, evaluation of, for example, ELISpot (e.g., for cd8+, cd4+ T cells and/or ifnγ) and pro-inflammatory cytokine responses from spleen cells of immunized and/or challenged animal models, as well as peptide collections derived from vaccine targets, can also be evaluated. In some embodiments, a phenotypic analysis such as an immune response is assessed (e.g., by flow cytometry). In some embodiments, for example, T cell depletion and/or protection assays are performed to assess immunogenicity (e.g., according to any suitable known method in the art).

In some embodiments, one or more functional responses of antibodies produced in response to certain RNA vaccine compositions of the present disclosure are assessed. Functional antibody responses may be assessed, for example, using an HSV neutralization assay. In some embodiments, an HSV in vitro neutralization assay is performed to evaluate neutralizing one or more anti-HSV glycoprotein (e.g., HSV gB, gDgH, gL) antibodies in HSV. For example, anti-HSV glycoprotein antibodies are obtained by collecting serum from animals (e.g., mice) vaccinated with HSV mRNA. HSV virus was added to the diluted serum and neutralization was allowed to continue for 1 hour at room temperature. One day in advance 3T3 cells were seeded in 96 wells and virus/serum mixtures were added to 3T3 monolayers. Cells were fixed the next day and HSV-specific staining was performed. The plates were scanned and analyzed. Neutralization titers were expressed as the highest serum dilution required to reduce plaque number by 50%.

In some embodiments, functional antibody responses can be assessed, for example, using passive transfer studies of serum from immunized animals to non-challenged animals and assessing the level of protection.

Example 17: exemplary characterization study

This example describes certain potential characterization studies that may be utilized, for example, to identify, select and/or characterize candidate vaccines or vaccine compositions (e.g., manufacturing lots thereof) or components thereof as described herein.

Immunization protocols may be used to assess the ability of a candidate vaccine comprising or delivering an antigen as described herein to induce B cells and/or T cells, for example after intramuscular immunization against the antigen and/or epitope thereof. In some embodiments, the level and/or diversity of the reaction is determined. In some embodiments, the presence and/or level of neutralizing antibodies is determined. In some embodiments, the immune subjects are assessed for protection from HSV challenge.

Alternatively or additionally, in some embodiments, one or more in vitro evaluations may be performed, for example:

(1) In vitro expression of antigens encoded by RNAs included in the vaccine composition; and/or

(2) In vitro potency of antigens expressed by RNAs included in vaccine compositions as described herein.

Example 18: exemplary clinical study of RNA vaccine compositions

This example describes certain clinical evaluations that may be performed on certain RNA vaccine compositions described herein.

In some embodiments, more than one different candidate vaccine may be evaluated. In some such embodiments, the different candidates may differ, for example, in the following:

(1) RNA platforms (e.g., unmodified RNA, nucleoside modified RNA, self-amplified RNA (saRNA), trans-amplified RNA);

(2) The antigen to be encoded-for example,

Which HSV (HSV-1 and/or HSV-2) proteins are utilised

Fusion of a fragment pair of a full-length protein antigen pair with one or more heterologous sequences (e.g., membrane tether, secretion, linker)

Epitopes from different (and/or multiple) phases of the HSV lifecycle

(3) Quantity of RNA

(4) Elements of RNA constructs

-Cap and/or cap proximity sequence

-5’UTR

-3’UTR

Poly A tail

(5) Lipid composition of LNP.

In a particular embodiment, up to three candidate vaccines having only 1 mRNA encoding an HSV glycoprotein (e.g., HSV gB, gD gH, gL) or an HSV protein or variant are evaluated, and/or up to three candidates containing 2 mrnas are evaluated, one mRNA encoding an HSV glycoprotein (e.g., HSV gB, gD gH, gL) or an HSV protein or variant, and another mRNA encoding a CD8 and/or CD4 epitope from a conserved antigen (and optionally considering conserved T cell epitopes from various stages of the HSV lifecycle). In this particular exemplary embodiment, the candidate vaccine may be evaluated by intramuscular administration, e.g., based on a dose escalation regimen.

Example 19: exemplary Generation, characterization and/or use of certain Multi-epitope vaccine compositions

In some embodiments, the immunogenicity of the multi-epitope RNA or polypeptide is tested in rodents (e.g., mice, e.g., transgenic mice) and/or large animals (e.g., non-human primates) to assess the magnitude of the immune response induced against the tested epitope. In some embodiments, the immunogenicity of an epitope encoded in vivo can be correlated with the in vitro response of a specific CTL line against a target cell expressing a multi-epitope polypeptide. Thus, in some embodiments, such exemplary experiments may show that the multi-epitope construct is used to: 1) Producing a cell-mediated and/or humoral response, and 2) inducing immune cell recognition of cells expressing the encoded epitope.

In some embodiments, for example, to generate a DNA sequence encoding a selected multi-epitope construct (e.g., DNA or RNA) for expression in a human cell, the amino acid sequence of the epitope to be included may be back translated. The human codon usage table may be used to guide the codon usage of each amino acid.

In some embodiments, the DNA sequences encoding the epitopes are directly contiguous such that when transcribed and translated, a contiguous polypeptide sequence is produced.

In some embodiments, expression and/or immunogenicity is optimized. In some such embodiments, expression and/or immunogenicity is optimized by incorporating additional elements into the coding construct. Examples of amino acid sequences that can be reverse translated and included in the multi-epitope construct sequence include, without limitation, for example: HLA class I epitopes, HLA class II epitopes, ubiquitination signal sequences and/or endoplasmic reticulum targeting signals. In some embodiments, HLA presentation of CTL and HTL epitopes can be improved by including synthetic (e.g., polyalanine) or naturally occurring flanking sequences adjacent to the CTL or HTL epitope; larger peptides comprising the epitope are within the scope of the present disclosure.

In some embodiments, the DNA sequence encoding the multiple epitopes may be generated by assembling oligonucleotides encoding the positive and negative strands of the construct. In some embodiments, overlapping oligonucleotides (e.g., 30-100 bases long) can be synthesized, phosphorylated, purified, and annealed under appropriate conditions using suitable techniques known in the art. In some embodiments, the ends of the oligonucleotides utilized may be ligated, for example, using ligation (e.g., T4 DNA ligation). In some embodiments, the synthetic construct encoding the polyepitope may then be cloned into a desired expression vector (e.g., using suitable cloning techniques known in the art).

In some embodiments, standard regulatory sequences (e.g., promoters, enhancers, etc.) well known to those of skill in the art may be included to ensure expression of the multi-epitope construct in the target cell. In some embodiments, for example, a promoter with a downstream cloning site for insertion of a multi-epitope construct coding sequence; polyadenylation signals for efficient transcription termination; an E.coli origin of replication; and an E.coli selectable marker (e.g., ampicillin or kanamycin resistance). In some embodiments, the one or more promoters utilized are not limited and may be used for this purpose, such as the human herpes simplex virus (hHSV) promoter. See, for example, U.S. Pat. nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

In some embodiments, vector modifications are used to optimize expression and/or immunogenicity. In some embodiments, introns are utilized for efficient gene expression and one or more synthetic or naturally occurring introns are incorporated into the transcribed region. In some embodiments, a stabilizing sequence (e.g., an mRNA stabilizing sequence) and/or a sequence for replication in a mammalian cell is included for increased expression.

In some embodiments, once the expression vector is selected, the multi-epitope construct coding sequence is cloned into a polylinker region downstream of the promoter (e.g., to generate a "plasmid"). In some embodiments, this plasmid is transformed into an appropriate E.coli strain and DNA is prepared using any suitable technique known in the art. In some embodiments, orientation of the multi-epitope coding sequences and DNA sequences, as well as all other elements included in the vector, are confirmed using, for example, restriction mapping and/or DNA sequence analysis. In some embodiments, bacterial cells comprising the desired plasmid may be stored, for example, as a master cell bank and/or a working cell bank.

In some embodiments, the immune modulatory sequences contribute to immunogenicity, e.g., immunogenicity of the nucleic acid vaccine construct. In some embodiments, such sequences are included in the vector, outside the coding sequence, if enhanced immunogenicity is desired. In some embodiments, such sequences are immunostimulatory. In some embodiments, such sequences are ISS or cpgs.

In some embodiments, a bicistronic expression vector is used that allows for the production of the multi-epitope construct and the second protein (e.g., including to increase or decrease immunogenicity). Without limitation, examples of proteins or polypeptides that may enhance an immune response if co-expressed with a multi-epitope construct include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., leIF), co-stimulatory molecules, or pan DR binding proteins for HTL responses. In some embodiments, the Helper (HTL) epitope is fused and/or linked to an intracellular targeting signal and expressed separately from the expressed CTL epitope; this allows HTL epitopes to be directed to a different cell compartment than CTL epitopes. This may facilitate more efficient entry of HTL epitopes into HLA class II pathways, if desired, thereby improving HTL induction. In contrast to HTL or CTL induction, specific reduction of immune responses by co-expression of immunosuppressive molecules (e.g., TGF- β) may be beneficial for certain diseases.

In some embodiments, commercially relevant amounts of plasmid DNA (e.g., for administration or for producing RNA and/or protein for administration) can be produced by fermentation, for example, in e.coli, followed by purification. In some embodiments, aliquots from the working cell bank are used to inoculate a growth medium and grown to a predetermined level (e.g., saturation) in a flask (e.g., shake flask) or bioreactor according to well known techniques. In some embodiments, plasmid DNA is purified using standard bioseparation techniques, such as, for example, solid phase anion exchange resins as provided by QIAGEN, inc. (Valencia, california). In some embodiments, supercoiled DNA is separated from open and linear forms using gel electrophoresis or other suitable methods known in the art.

In some embodiments, purified plasmid DNA is prepared for injection into a subject using a variety of formulations. In some embodiments, the lyophilized DNA is reconstituted in sterile Phosphate Buffered Saline (PBS). This method is known as "naked DNA" and is currently being used in clinical trials for Intramuscular (IM) administration. In some embodiments, to maximize the immunotherapeutic effect of the multi-epitope vaccine composition, alternative methods of formulating nucleic acids (e.g., purified plasmid DNA, in vitro transcribed RNA, etc.) may be employed. Various methods have been described and new techniques are also available. In some embodiments, cationic lipids are used in the formulation (see, e.g., as described in WO 93/24640; mannino & Gould-Fogerite, bioTechniques 6 (7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06209; and Felgner et al, proc. Nat' l Acad. Sci. USA 84:7413 (1987); in some embodiments, glycolipids, fusogenic liposomes, peptides, and compounds collectively referred to as protective interactive non-condensed compounds (PINCs) are complexed with purified plasmid DNA to affect variables such as stability, intramuscular dispersion, or transport to a particular organ or cell type.

In some embodiments, the polynucleotide is introduced into the cell by utilizing high-speed cell deformation. During high-speed deformation, the cells are squeezed, causing temporary rupture of the cell membrane, allowing the nucleic acid to enter the cells. In some embodiments, the polypeptide is produced by an expression vector, e.g., in a bacterial expression vector, and then the protein may be delivered to the cell.

In some embodiments, target cell sensitization is used as a functional assay for expression of the encoded CTL epitope and HLA class I presentation. For example, in some embodiments, the polynucleotide is introduced into a mammalian cell line suitable as a target for a standard CTL chromium release assay. In some embodiments, the transfection method employed depends on the final formulation. In some embodiments, electroporation is used, for example, for "naked" polynucleotides (e.g., DNA). In some embodiments in which cationic lipids are used, direct in vitro transfection is utilized as a transfection method. In some embodiments, plasmids expressing a marker protein or polypeptide (e.g., green Fluorescent Protein (GFP)) are co-transfected to allow enrichment of transfected cells (e.g., using Fluorescence Activated Cell Sorting (FACS)). In some embodiments, the cells are then labeled with chromium-51 (^51- Cr) and used as target cells for a epitope-specific CTL cell line; cytolysis detected by ⁵¹ Cr release indicates the production of the encoded CTL epitope and HLA presentation. In some such embodiments, the expression of HTL epitopes may be assessed in a similar manner using an assay that assesses HTL activity.

In some embodiments, functional testing is performed using in vivo immunogenicity. In some embodiments, rodents (e.g., mice, such as transgenic mice expressing appropriate human HLA proteins) are immunized with a multi-epitope vaccine composition (e.g., comprising a DNA or RNA active agent). In some embodiments, the dose and route of administration are formulation dependent (e.g., IM for DNA in PBS or LNP formulated DNA or RNA, intraperitoneal (IP) for lipid-complexed DNA). In some embodiments, for example twenty days after immunization, spleen cells are harvested and restimulated for 1 week in the presence of a peptide encoding each epitope tested. Thereafter, for CTL effector cells, cytolytic assays were performed on peptide-loaded ⁵¹ Cr-labeled target cells using standard techniques. Lysis of target cells sensitized with HLA loaded with peptide epitopes (corresponding to the epitopes encoded by the minigenes) demonstrated vaccine function in vivo to induce CTLs. The immunogenicity of HTL epitopes was assessed in a similar manner in transgenic mice.

Example 20: exemplary guinea pig T cell assay-1 phase uninfected guinea pigs

Step 1. CD4 and CD 8T cells were measured in uninfected guinea pigs (n=10).

Uninfected guinea pigs will be used for normalization agents. Two animals will be used for each normalization attempt. PMA will be used as positive control. Pan T cells (CD 3), CD4, CD8, ifnγ, tnfα, CD45 (pan white blood cells), CD1B3 (B cell markers excluding B cells) will be normalized.

Step 2. CD4 and CD 8T cells were measured in immunized guinea pigs (n=16).

Three groups of guinea pigs will be tested: PBS (n=4), gE2 mRNA-LNP315 (n=4), test "T cell string" according to the present disclosure (n=4). PBS was used as a control. Guinea pigs were immunized 2 times with gE2 mRNA (15 ug) at 4 weeks apart and CD4 and CD 8T cell responses were measured 10-14 days after the 2 nd immunization (n=4). Guinea pigs were immunized 2 times at 4 weeks intervals with a test "T cell string" (15 ug) according to the present disclosure, and CD4 and CD 8T cells were measured 10-14 days after the 2 nd immunization (n=4). All spleen cells will be harvested simultaneously. If desired, two additional animals in group 2 will be immunized and 2 additional animals in group 3 will be used for additional studies (n=4).

Numbered embodiments

1.A composition comprising one or more RNA molecules that collectively encode one or more HSV (e.g., HSV-1 and/or HSV-2) antigens or fragments thereof.

2. A composition for delivering one or more HSV (e.g., HSV-1 and/or HSV-2) antigens or fragments thereof to a subject, optionally wherein the composition comprises one or more RNA molecules that collectively encode one or more HSV antigens, and/or wherein the composition comprises one or more polypeptides comprising one or more HSV antigens.

3. The composition of claim 1 or 2, wherein the one or more HSV antigens or fragments thereof have at least 80% sequence identity to one or more sequences selected from SEQ ID NOs 1-74 (table 1) or corresponding fragments thereof.

4. The composition of any one of claims 1-3, wherein the one or more HSV antigens or fragments thereof have at least 85%, at least 90%, at least 95% or 100% sequence identity to one or more sequences selected from SEQ ID NOs 1-74 (table 1) or corresponding fragments thereof.

5. The composition of any one of claims 1-4, wherein said one or more HSV antigens or fragments thereof have at least 80% sequence identity to one or more sequences selected from SEQ ID NOs 1-30 and 74 (table 2) or corresponding fragments thereof.

6. The composition of any one of claims 1-4, wherein said one or more HSV antigens or fragments thereof have at least 85%, at least 90%, at least 95% or 100% sequence identity to one or more sequences selected from SEQ ID NOs 1-30 and 74 (table 2) or corresponding fragments thereof.

7. The composition of any one of claims 1-4, wherein said one or more HSV antigens or fragments thereof have at least 80% sequence identity to one or more sequences selected from SEQ ID NOs 31-73 (table 3) or corresponding fragments thereof.

8. The composition of any one of claims 1-4, wherein the one or more HSV antigens or fragments thereof have at least 85%, at least 90%, at least 95% or 100% sequence identity to one or more sequences selected from SEQ ID NOs 31-73 (table 3) or corresponding fragments thereof.

9. The composition of any one of claims 1-8, wherein the one or more RNA molecules comprise a sequence seen in table 11.

10. The composition of any one of claims 1-9, wherein at least one of the one or more RNA molecules encodes a plurality of HSV (e.g., HSV-1 and/or HSV-2) antigens or fragments thereof.

11. The composition of any one of claims 1-10, wherein all of the one or more RNA molecules encode a plurality of HSV (e.g., HSV-1 and/or HSV-2) antigens or fragments thereof.

12. The composition of any one of claims 1-10, wherein at least one of the one or more RNA molecules encodes a single HSV (e.g., HSV-1 and/or HSV-2) antigen or fragment thereof.

13. The composition of any one of claims 1-12, wherein two or more HSV antigens or fragments thereof are present as a single polypeptide in or encoded by one or more RNA molecules.

14. The composition of claim 13, wherein the single polypeptide further comprises a linker, optionally wherein the linker has a sequence comprising one or more glycine (G) residues and/or one or more serine (S) residues.

15. The composition of item 13 or 14, wherein the linker is a cleavable linker.

16. The composition of item 13 or 14, wherein the linker has the sequence seen in table 10 herein.

17. The composition of any one of claims 1-16, wherein the one or more RNA molecules comprise a sequence encoding one or more HSV antigens that are codon optimized for expression in a subject, optionally wherein the subject is a human.

18. The composition of any one of claims 1-17, wherein the one or more RNA molecules comprise a5 'cap or 5' cap analogue.

19. The composition of claim 18, wherein the 5' Cap analog is or comprises Cap0, cap1, or Cap2.

20. The composition of claim 18 or 19, wherein the 5' -cap analogue is or comprises m ₂ ^7,3'-OGppp(m₁ ^{2 '-O}) ApG.

21. The composition of any one of claims 1-20, wherein the one or more RNA molecules comprise a sequence encoding a signal peptide.

22. The composition of claim 21, wherein the signal peptide is or comprises the sequence found in table 7 herein.

23. The composition of claim 21, wherein the one or more RNA molecules comprise a sequence encoding a signal peptide as seen in table 8 herein.

24. The composition of any one of claims 1-23, wherein the one or more RNA molecules comprise at least one non-coding regulatory element.

25. The composition of any one of claims 1-24, wherein the one or more RNA molecules comprise a poly adenine tail.

26. The composition of claim 25, wherein the polyadenylation tail is or comprises a modified adenine sequence.

27. The composition of claim 25 or 26, wherein the polyadenylation tail comprises at least 100 a nucleotides.

28. The composition of any one of claims 25-27, wherein the polyadenylation tail is a discontinuous sequence of a nucleotides.

29. The composition of claim 28, wherein the poly adenine tail comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.

30. The composition of any one of claims 1-29, wherein the one or more RNA molecules comprise at least one 5 'untranslated region (UTR) and/or at least one 3' UTR.

31. The composition of claim 30, wherein the at least one 5'-UTR is or comprises a modified human α -globulin 5' -UTR.

32. The composition of claim 30 or 31, wherein the at least one 3' -UTR is or comprises a first sequence from a "split amino terminal enhancer" (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA.

33. The composition of any one of claims 1-32, wherein the RNA is a modified RNA that is modified by substitution of some or all uridine residues with modified uridine residues.

34. The composition of any one of claims 1-33, wherein the RNA is modified by substitution of some or all uridine residues with N1-methyl-pseudouridine.

35. The composition of any one of claims 1-34, wherein the RNA is formulated in a lipid nanoparticle comprising a cationic or cationically ionizable lipid, sterol, neutral lipid, and lipid conjugate.

36. The composition of any one of claims 1-35, wherein the RNA is formulated in a lipid nanoparticle comprising a cationically ionizable lipid, a phospholipid, cholesterol, and a polyethylene glycol (PEG) -lipid.

37. The composition of claim 35 or 36, wherein the cationic lipid or cationically ionizable lipid is or comprises ((4-hydroxybutyl) azetidinyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate), the sterol is or comprises cholesterol, the neutral lipid is or comprises a phospholipid, and the lipid conjugate is or comprises a polyethylene glycol (PEG) -lipid.

38. The composition of any one of claims 35-37, wherein the one or more lipid nanoparticles comprise:

a. ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate);

b. cholesterol;

c. Distearoyl phosphatidylcholine (DSPC); and

D.2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide.

39. The composition of claim 35 or 36, wherein the cationically ionizable lipid has the structure:

or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

Each of G ¹ and G ² is independently unsubstituted C ₁-C₁₂ alkylene or C ₁-C₁₂ alkenylene;

G ³ is C ₁-C₂₄ alkylene, C ₁-C₂₄ alkenylene, C ₃-C₈ cycloalkylene, C ₃-C₈ cycloalkenyl;

R ¹ and R ² are each independently C ₆-C₂₄ alkyl or C ₆-C₂₄ alkenyl;

R ³ is H, OR ⁵、CN、-C(＝O)OR⁴、-OC(＝O)R⁴ or-NR ⁵C(＝O)R⁴;

R ⁴ is C ₁-C₁₂ alkyl;

r ⁵ is H or C ₁-C₆ alkyl.

40. The composition of claim 39, wherein R ¹ or R ², or both, have one of the following structures:

41. the composition of claim 39, wherein the cationically ionizable lipid has the structure:

42. The composition of any one of claims 36-41, wherein the phospholipid is or comprises distearoyl phosphatidylcholine (DSPC).

43. The composition of any one of claims 36-42, wherein the (PEG) -lipid is or comprises 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylamide.

44. The composition of any one of claims 36-43, wherein the phospholipid is present at a concentration in the range of 5 to 15 mole percent of total lipid.

45. The composition of any one of claims 35-44, wherein the cationically ionizable lipid is present at a concentration in the range of 40 to 55 mole percent of total lipid.

46. The composition of any one of claims 36-45, wherein the cholesterol is present at a concentration in the range of 30 to 50 mole percent of total lipid.

47. The composition of any one of claims 36-46, wherein the (PEG) -lipid is present at a concentration in the range of 1 to 10 mole percent of total lipid.

48. The composition of any one of claims 36-47, wherein the lipid nanoparticle comprises 40 to 55 mole percent of a cationically ionizable lipid; 5 to 15 mole percent of a phospholipid; 30 to 50 mole percent cholesterol; and 1 to 10 mole percent PEG-lipid.

49. The composition of claim 38 wherein ((4-hydroxybutyl) azetidine diyl) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate) is in the range of about 40 to about 55 mole percent, cholesterol is in the range of about 30 to about 50 mole percent, distearoyl phosphatidylcholine (DSPC) is in the range of about 5 to about 15 mole percent, and 2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide is in the range of about 1 to about 10 mole percent.

50. The composition of any one of claims 1-49, further comprising at least one salt and/or a cryoprotectant, wherein the cryoprotectant is or comprises sucrose.

51. The composition of any one of claims 1-50, wherein total RNA is present in the composition in an amount ranging from 1ug to 100ug per dose.

52. A pharmaceutical composition comprising the composition of any one of claims 1-51.

53. The pharmaceutical composition of claim 52, which is in the form of a liquid formulation.

54. The pharmaceutical composition of claim 52, which is in the form of a frozen formulation.

55. The pharmaceutical composition of claim 54, wherein the frozen formulation comprises PBS.

56. The pharmaceutical composition of any one of claims 52-55, wherein the composition is formulated for intramuscular administration.

57. The pharmaceutical composition of any one of claims 52-55, wherein the composition is formulated for intravenous administration.

58. The pharmaceutical composition of any one of claims 52-55, wherein the composition is formulated for subcutaneous administration.

59. A method comprising administering at least one dose of the pharmaceutical composition of any one of claims 52-58 to a subject.

60. The method of claim 59, wherein the subject is a human.

61. The method of claim 59 or 60, wherein the subject is suffering from an HSV (e.g., HSV-1 and/or HSV-2) infection.

62. The method of claim 59 or 60, wherein the subject is intended to appear within a geographic area with high prevalence of HSV (e.g., HSV-1 and/or HSV-2) within the next three months.

63. The method of claim 62, wherein the prevalence of high HSV (e.g., HSV-1 and/or HSV-2) is greater than 10% of the population.

64. The method of any one of claims 59-63, wherein the subject has previously been treated with a different pharmaceutical composition for an HSV (e.g., HSV-1 and/or HSV-2) infection.

65. The method of any one of claims 59-64, further comprising administering a second dose of the pharmaceutical composition to the patient.

66. The method of any one of claims 59-65, further comprising administering at least two doses of the pharmaceutical composition to the patient.

67. The method of any one of claims 59-66, further comprising administering at least three doses of the pharmaceutical composition to the patient.

68. The method of any one of claims 59-67, wherein the method is a method of inducing an anti-HSV (e.g., anti-HSV-1 and/or anti-HSV-2) immune response in a subject.

69. The method of claim 68, wherein the immune response in the subject comprises an adaptive immune response.

70. The method of claim 68 or 69, wherein the immune response in the subject comprises a T cell response.

71. The method of claim 70, wherein the T cell response is or comprises a cd4+ T cell response.

72. The method of claim 70 or 71, wherein the T cell response is or comprises a cd8+ T cell response.

73. The method of any one of claims 68-72, wherein the immune system response comprises a B cell response.

74. The method of any one of claims 68-73, wherein the immune system response comprises production of antibodies to one or more HSV (e.g., HSV-1 and/or HSV-2) antigens or fragments thereof.

75. The pharmaceutical composition of any one of claims 52-58 for use in treating HSV (e.g., HSV-1 and/or HSV-2) infection.

76. The pharmaceutical composition of any one of claims 52-58, for use in inducing an anti-HSV (e.g., anti-HSV-1 and/or anti-HSV-2) immune response in a subject.

77. Use of the pharmaceutical composition of any one of claims 52-58 in the treatment of HSV (e.g., HSV-1 and/or HSV-2) infection.

78. Use of the pharmaceutical composition of any one of claims 52-58 for inducing an anti-HSV (e.g., anti-HSV-1 and/or anti-HSV-2) immune response in a subject.

79. A polypeptide comprising one or more HSV (e.g., HSV-1 and/or HSV-2) antigens or fragments thereof.

80. The polypeptide of claim 79, wherein the one or more HSV antigens or fragments thereof have at least 80% sequence identity to one or more sequences selected from SEQ ID NOs 1-74 (table 1) or corresponding fragments thereof.

81. The polypeptide of claim 79 or 80, wherein the one or more HSV antigens or fragments thereof have at least 85%, 90%, 95% or 100% sequence identity to one or more sequences selected from SEQ ID NOs 1-74 (table 1) or corresponding fragments thereof.

82. The polypeptide of any one of claims 79-81, wherein the one or more HSV antigens or fragments thereof have at least 80% sequence identity to one or more sequences selected from SEQ ID NOs 1-30 and 74 (table 2) or corresponding fragments thereof.

83. The polypeptide of any one of claims 79-81, wherein the one or more HSV antigens or fragments thereof have at least 85%, 90%, 95% or 100% sequence identity to one or more sequences selected from SEQ ID NOs 1-30 and 74 (table 2) or a corresponding fragment thereof.

84. The polypeptide of any one of claims 79-81, wherein the one or more HSV antigens or fragments thereof have at least 80% sequence identity to one or more sequences selected from SEQ ID NOs 31-73 (table 3) or corresponding fragments thereof.

85. The polypeptide of any one of claims 79-81, wherein the one or more HSV antigens or fragments thereof have at least 85%, 90%, 95% or 100% sequence identity to one or more sequences selected from SEQ ID NOs 31-73 (table 3) or corresponding fragments thereof.

86. A polynucleotide encoding one or more HSV (e.g., HSV-1 and/or HSV-2) antigens or fragments thereof.

87. The polynucleotide of claim 86, wherein said one or more HSV antigens or fragments thereof have at least 80% sequence identity to one or more sequences selected from SEQ ID NOs 1-74 (table 1) or corresponding fragments thereof.

88. The polynucleotide of claim 86 or 87, wherein said one or more HSV antigens or fragments thereof have at least 85%, 90%, 95% or 100% sequence identity to one or more sequences selected from SEQ ID NOs 1-74 (table 1) or corresponding fragments thereof.

89. The polynucleotide of any one of claims 86-88, wherein said one or more HSV antigens or fragments thereof have at least 80% sequence identity to one or more sequences selected from SEQ ID NOs 1-30 and 74 (table 2) or corresponding fragments thereof.

90. The polynucleotide of any one of claims 86-88, wherein said one or more HSV antigens or fragments thereof have at least 85%, 90%, 95% or 100% sequence identity to one or more sequences selected from SEQ ID NOs 1-30 and 74 (table 2) or a corresponding fragment thereof.

91. The polynucleotide of any one of claims 86-88, wherein said one or more HSV antigens or fragments thereof have at least 80% sequence identity to one or more sequences selected from SEQ ID NOs 31-73 (table 3) or corresponding fragments thereof.

92. The polynucleotide of any one of claims 86-88, wherein said one or more HSV antigens or fragments thereof have at least 85%, 90%, 95% or 100% sequence identity to one or more sequences selected from SEQ ID NOs 31-73 (table 3) or corresponding fragments thereof.

93. The polynucleotide of any one of claims 86-92, wherein said polynucleotide is DNA or RNA.

94. A cell comprising the polypeptide of any one of claims 79-85 and/or the polynucleotide of any one of claims 86-93.

95. A cell comprising the polynucleotide of any one of claims 86-93.

96. The cell of claim 95, wherein the cell expresses one or more HSV (e.g., HSV-1 and/or HSV-2) antigens or fragments thereof encoded by the polynucleotide.

97. A polyribonucleotide encoding a polypeptide comprising one or more HSV-2 antigens, wherein the one or more HSV-2 antigens comprise a RL2 polypeptide or antigenic fragment thereof, a RS1 polypeptide or antigenic fragment thereof, a UL54 polypeptide or antigenic fragment thereof, a UL29 polypeptide or antigenic fragment thereof, a UL39 polypeptide or antigenic fragment thereof, a UL49 polypeptide or antigenic fragment thereof, a UL9 polypeptide or antigenic fragment thereof, a UL30 polypeptide or antigenic fragment thereof, a UL40 polypeptide or antigenic fragment thereof, a UL5 polypeptide or antigenic fragment thereof, a UL52 polypeptide or antigenic fragment thereof, a UL1 polypeptide or antigenic fragment thereof, a UL19 polypeptide or antigenic fragment thereof, a UL21 polypeptide or antigenic fragment thereof, a UL27 polypeptide or antigenic fragment thereof, a UL46 polypeptide or antigenic fragment thereof, a UL47 polypeptide or antigenic fragment thereof, a UL48 polypeptide or antigenic fragment thereof, a UL25 polypeptide or antigenic fragment thereof, or a combination thereof.

98. A polyribonucleotide encoding a polypeptide comprising one or more HSV-2 antigens, wherein at least one HSV-2 antigen comprises an intermediate early protein or an antigenic fragment thereof.

99. A polyribonucleotide encoding a polypeptide comprising one or more HSV-2 antigens, wherein each of the one or more HSV-2 antigens comprises an intermediate early protein or an antigenic fragment thereof.

100. A polyribonucleotide encoding a polypeptide comprising one or more HSV-2 antigens, wherein at least one HSV-2 antigen comprises an early protein or an antigenic fragment thereof.

101. A polyribonucleotide encoding a polypeptide comprising one or more HSV-2 antigens, wherein each of the one or more HSV-2 antigens comprises an early protein or an antigenic fragment thereof.

102. A polyribonucleotide encoding a polypeptide comprising one or more HSV-2 antigens, wherein at least one HSV-2 antigen comprises an advanced protein or an antigenic fragment thereof.

103. A polyribonucleotide encoding a polypeptide comprising one or more HSV-2 antigens, wherein each of the one or more HSV-2 antigens comprises an advanced protein or an antigenic fragment thereof.

104. A polyribonucleotide comprising in 5 'to 3' order a nucleotide sequence encoding an HSV-1gD secretion signal, an RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or fragment thereof, a linker and MITD.

105. A polyribonucleotide encoding a polypeptide having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRGC TDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDPRTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGLPIAGVSSVVALAPYVNKTVTGDCLPVLDMETGHIGAYVVLVDQTGNVADLLRAAAPAWSRRTLLPEHARNCVRPPDYPTPPASEWNSLWMTPVGNMLFDQGTLVGGGSGGGGSGGRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGGSGGGGSGGETLVAHGPSLYRTFAANPRAASTAKAMRDCVLRQENLIEALASADETLAWCKMCIHHNLPLRPQDPIIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVSEIDYTTVGVGAGETMHFYIPGACMAGLIEILDTHRQECSSRVCELTASHTIAPLYVHGKYFYCNSLFGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:197).

106. A polyribonucleotide comprising in 5 'to 3' order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL29 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL49 polypeptide or antigenic fragment thereof, a linker, a UL9 polypeptide or fragment thereof, a linker and MITD.

107. A polyribonucleotide encoding a polypeptide having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRGR EDIETIAFIKRFSLDYGAINFINLAPNNVSELAMYYMANQILRYCDHSTYFINTLTAVIAGSRRPPSVQAAAAWAPQGGAGLEAGARALMDSLDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQAGNWASLLGGKNACPLLIFDRTRKFVLGGSGGGGSGGRTFGSAPRLTEDDFGLLNYALAEMRRLCLDLPPVPPNAYTPYHLREYATRLVNGFKPLVRRSARLYRILGVLVHLRIRTREASFEEWMRSKEVDLDFGLTERLREHEAQLMILAQALNPYDCLIHSTPNTLVERGLQSALKYEEFYLKRFGGHYMESVFQMYTRIAGFLAGGSGGGGSGGKMTRGAPKASATPATDPARGRRPAQADSAVLLDAPAPTASGRTKTPAQGLAKKLHFSTAPPSPTAPWTPRVAGFNKRVFCAAVGGGSGGGGSGGLLNNYDVLVLDEVMSTLGQLYSPTMQQLGRVDALMLRLLRTCPRIIAMDATANAQLVDFLCSLRGEKNVHVVIGEYAMPGFSARRCLFLPRLGPEVLQAALRRRGPAGGAPPPDAPPDATFFGELEARLAGGDNVCIFSSTVSFAEVVARFCRQFTDRVLLLHSLTPPGDVTTWGRYRVVIYTTVVTVGLSFDPPHFDSMFAYVKPMNYGPDMVSVYQSLGRVRTLRKGELLIYMDGSGARSEPVGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:198).

108. A polyribonucleotide comprising in 5 'to 3' order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL30 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or fragment thereof, a linker, a UL52 polypeptide or fragment thereof, a linker and MITD.

109. A polyribonucleotide encoding a polypeptide having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRGIS CLLYDLSTTALEHILLFSLGSCDLPESHLSDLASRGLPAPVVLEFDSEFEMLLAFMTFVKQYGPEFVTGYNIINFDWPFVLTKLTEIYKVPLDGYGRMNGRGVFRVWDIGQSHFGGSGGGGSGGGLLPCLHVAATVTTIGREMLLATRAYVHARWAEFDQLLADFPEAAGMRAPGPYSMGGSGGGGSGGTSQCPDINHLRSLSILNRWLETELVFVGDEEDVSKLSEGELGFYRFLFAFLSAADDLVTENLGGLSGLFEQKDILHYYVEQECIEVVHSRVYNIIQLVLFHNNDQARRAYVARTINHPAIRVKVDWLEARVRECDSIPEKFILMILIEGVFFAASFAAIAYLRTNNLLRGGSGGGGSGGHEFGNLMKVLEYGLPITEEHMQFVDRFVVPESYITNPANLPGWTRLFSSHKEVSAYMAKLHAYLKVTREGEFVVFTLPVLTFVSVKEFDEYRRLGGSGGGGSGGELFGEVFESAPFSTYVDNVIFRGCELLTGSPRGGLMSVALQTDNYTLMGYTYTRVFAFAEELRRRHATAGVAEFLEESPLPYIVLRDQHGFMSVVNTNIGGSGGGGSGGSVAAPVEVTALYATDGCVITSSLALLTNCLLGAEPLYIFSYDAYRSDAPNGPTGAPTEQERFEGSRALYRDAGGLNGDSFRVTFCLLGTEVGVTHHPKGRTRPMFVCRFERADDVAVLQDALGRGTPLLPAHVTATLDLEATFALHANIIMALTVAIVHNAPARIGSGSTAPLYEPGESMRSVVGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:199).

110. A polyribonucleotide comprising in 5 'to 3' order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL1 polypeptide or antigenic fragment thereof, a linker, a UL19 polypeptide or antigenic fragment thereof, a linker, a UL21 polypeptide or antigenic fragment thereof, a linker, a UL27 polypeptide or fragment thereof, a linker, a UL46 polypeptide or fragment thereof, a linker, a UL47 polypeptide or fragment thereof, a linker, a UL25 polypeptide or fragment thereof, a linker, a UL48 polypeptide or fragment thereof, a linker and MITD.

111. A polyribonucleotide encoding a polypeptide having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRGR TPADDVSWRYEAPSVIDYARIDGIFLRYHCPGLDTFLWDRHAQRAYLVNPFLFAAGFLEDLSHSVFPADTQETTGGSGGGGSGGDGRLLHNTQARAADAADDRPHRPADWTVHHKIYYYVLVPAFSRGRCCTAGVRFDRVYATLQNMVVPEIAPGEECPSDPVTDPAHPLHPANLVANTVKRMFHNGGSGGGGSGGSPTQKLAVYYYLIHRERRMSPFPALVRLVGRYIQRHGLYVPAPDEPTLADAMNGLGGSGGGGSGGNYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEVGGSGGGGSGGSVYPYDEFVLATGDFVYMSPFYGYREGSHGGSGGGGSGGGLASDPHYDYIRHYASAAKQALGEVELSGGQLSRAILAQYWKYLQTVVPSGLDIPDDPAGDCDPSLHVLLRPTLLPKLLVRAPFKSGAAAAKYAAAVAGLRDAAHRLQQYMFFMRPADPSRPSTDTALRLSELLAYVSVLYHWASWMLWTADKYVGGSGGGGSGGGPDAAVFRSSLGSLLYWPGVRALLGRDCRVAARYAGRMTYIATGALLARFNPGAVKCVLPREAAFAGRVLGGSGGGGSGGFLWEDQTLLRATANTITALAVLRRLLANGNVYADRLDNRLQLGMLIPGAVPAEAIARGASGLDSGAIKSGDNNLEALCVNYVLPLYQADPTVELTQLFPGLAALCLGGSGGGGSGGALFNRLLDDLGFSAGPALCTMLDTWNEDLFSGFPTNADMYRECKFLSTLPSDVIDWGDAHVPERSPIDIRAHGDVAFPTLPATRDELPSYYEAMAQFFRGELRAGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:200).

112. A polyribonucleotide comprising in 5 'to 3' order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL54 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or fragment thereof, a linker and MITD.

113. A polyribonucleotide encoding a polypeptide having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRGE TLVAHGPSLYRTFAANPRAASTAKAMRDCVLRQENLIEALASADETLAWCKMCIHHNLPLRPQDPIIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVSEIDYTTVGVGAGETMHFYIPGACMAGLIEILDTHRQECSSRVCELTASHTIAPLYVHGKYFYCNSLFGGSGGGGSGGRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGGSGGGGSGGCTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDPRTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGLPIAGVSSVVALAPYVNKTVTGDCLPVLDMETGHIGAYVVLVDQTGNVADLLRAAAPAWSRRTLLPEHARNCVRPPDYPTPPASEWNSLWMTPVGNMLFDQGTLVGGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:201).

114. A polyribonucleotide comprising in 5 'to 3' order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL9 polypeptide or antigenic fragment thereof, a linker, a UL49 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL29 polypeptide or fragment thereof, a linker and MITD.

115. A polyribonucleotide encoding a polypeptide having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRGL LNNYDVLVLDEVMSTLGQLYSPTMQQLGRVDALMLRLLRTCPRIIAMDATANAQLVDFLCSLRGEKNVHVVIGEYAMPGFSARRCLFLPRLGPEVLQAALRRRGPAGGAPPPDAPPDATFFGELEARLAGGDNVCIFSSTVSFAEVVARFCRQFTDRVLLLHSLTPPGDVTTWGRYRVVIYTTVVTVGLSFDPPHFDSMFAYVKPMNYGPDMVSVYQSLGRVRTLRKGELLIYMDGSGARSEPVGGSGGGGSGGKMTRGAPKASATPATDPARGRRPAQADSAVLLDAPAPTASGRTKTPAQGLAKKLHFSTAPPSPTAPWTPRVAGFNKRVFCAAVGGGSGGGGSGGRTFGSAPRLTEDDFGLLNYALAEMRRLCLDLPPVPPNAYTPYHLREYATRLVNGFKPLVRRSARLYRILGVLVHLRIRTREASFEEWMRSKEVDLDFGLTERLREHEAQLMILAQALNPYDCLIHSTPNTLVERGLQSALKYEEFYLKRFGGHYMESVFQMYTRIAGFLAGGSGGGGSGGREDIETIAFIKRFSLDYGAINFINLAPNNVSELAMYYMANQILRYCDHSTYFINTLTAVIAGSRRPPSVQAAAAWAPQGGAGLEAGARALMDSLDAHPGAWTSMFASCNLLRPVMAARPMVVLGLSISKYYGMAGNDRVFQAGNWASLLGGKNACPLLIFDRTRKFVLGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:202).

116. A polyribonucleotide comprising in 5 'to 3' order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL52 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or fragment thereof, a linker, a UL30 polypeptide or fragment thereof, a linker and MITD.

117. A polyribonucleotide encoding a polypeptide having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRGS VAAPVEVTALYATDGCVITSSLALLTNCLLGAEPLYIFSYDAYRSDAPNGPTGAPTEQERFEGSRALYRDAGGLNGDSFRVTFCLLGTEVGVTHHPKGRTRPMFVCRFERADDVAVLQDALGRGTPLLPAHVTATLDLEATFALHANIIMALTVAIVHNAPARIGSGSTAPLYEPGESMRSVVGGSGGGGSGGHEFGNLMKVLEYGLPITEEHMQFVDRFVVPESYITNPANLPGWTRLFSSHKEVSAYMAKLHAYLKVTREGEFVVFTLPVLTFVSVKEFDEYRRLGGSGGGGSGGELFGEVFESAPFSTYVDNVIFRGCELLTGSPRGGLMSVALQTDNYTLMGYTYTRVFAFAEELRRRHATAGVAEFLEESPLPYIVLRDQHGFMSVVNTNIGGSGGGGSGGTSQCPDINHLRSLSILNRWLETELVFVGDEEDVSKLSEGELGFYRFLFAFLSAADDLVTENLGGLSGLFEQKDILHYYVEQECIEVVHSRVYNIIQLVLFHNNDQARRAYVARTINHPAIRVKVDWLEARVRECDSIPEKFILMILIEGVFFAASFAAIAYLRTNNLLRGGSGGGGSGGISCLLYDLSTTALEHILLFSLGSCDLPESHLSDLASRGLPAPVVLEFDSEFEMLLAFMTFVKQYGPEFVTGYNIINFDWPFVLTKLTEIYKVPLDGYGRMNGRGVFRVWDIGQSHFGGSGGGGSGGGLLPCLHVAATVTTIGREMLLATRAYVHARWAEFDQLLADFPEAAGMRAPGPYSMGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:203).

118. A polyribonucleotide comprising in 5 'to 3' order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL48 polypeptide or antigenic fragment thereof, a linker, a UL25 polypeptide or antigenic fragment thereof, a linker, a UL47 polypeptide or antigenic fragment thereof, a linker, a UL46 polypeptide or fragment thereof, a linker, a UL27 polypeptide or fragment thereof, a linker, a UL21 polypeptide or fragment thereof, a linker, a UL19 polypeptide or fragment thereof, a linker, a UL1 polypeptide or fragment thereof, a linker and MITD.

119. A polyribonucleotide encoding a polypeptide having an amino acid sequence comprising or consisting of ：MGGAAARLGAVILFVVIVGLHGVRGA LFNRLLDDLGFSAGPALCTMLDTWNEDLFSGFPTNADMYRECKFLSTLPSDVIDWGDAHVPERSPIDIRAHGDVAFPTLPATRDELPSYYEAMAQFFRGELRAGGSGGGGSGGFLWEDQTLLRATANTITALAVLRRLLANGNVYADRLDNRLQLGMLIPGAVPAEAIARGASGLDSGAIKSGDNNLEALCVNYVLPLYQADPTVELTQLFPGLAALCLGGSGGGGSGGGPDAAVFRSSLGSLLYWPGVRALLGRDCRVAARYAGRMTYIATGALLARFNPGAVKCVLPREAAFAGRVLGGSGGGGSGGGLASDPHYDYIRHYASAAKQALGEVELSGGQLSRAILAQYWKYLQTVVPSGLDIPDDPAGDCDPSLHVLLRPTLLPKLLVRAPFKSGAAAAKYAAAVAGLRDAAHRLQQYMFFMRPADPSRPSTDTALRLSELLAYVSVLYHWASWMLWTADKYVGGSGGGGSGGNYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEVGGSGGGGSGGSVYPYDEFVLATGDFVYMSPFYGYREGSHGGSGGGGSGGSPTQKLAVYYYLIHRERRMSPFPALVRLVGRYIQRHGLYVPAPDEPTLADAMNGLGGSGGGGSGGDGRLLHNTQARAADAADDRPHRPADWTVHHKIYYYVLVPAFSRGRCCTAGVRFDRVYATLQNMVVPEIAPGEECPSDPVTDPAHPLHPANLVANTVKRMFHNGGSGGGGSGGRTPADDVSWRYEAPSVIDYARIDGIFLRYHCPGLDTFLWDRHAQRAYLVNPFLFAAGFLEDLSHSVFPADTQETTGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:204).

120. A polyribonucleotide comprising in 5 'to 3' order a nucleotide sequence encoding an HSV-2gD secretion signal, an RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or fragment thereof, a linker and MITD.

121. A polyribonucleotide encoding a polypeptide having an amino acid sequence comprising or consisting of ：MGRLTSGVGTAALLVVAVGLRVVCAC TDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDPRTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGLPIAGVSSVVALAPYVNKTVTGDCLPVLDMETGHIGAYVVLVDQTGNVADLLRAAAPAWSRRTLLPEHARNCVRPPDYPTPPASEWNSLWMTPVGNMLFDQGTLVGGGSGGGGSGGRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGGSGGGGSGGETLVAHGPSLYRTFAANPRAASTAKAMRDCVLRQENLIEALASADETLAWCKMCIHHNLPLRPQDPIIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVSEIDYTTVGVGAGETMHFYIPGACMAGLIEILDTHRQECSSRVCELTASHTIAPLYVHGKYFYCNSLFGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:205).

122. A polyribonucleotide comprising in 5 'to 3' order a nucleotide sequence encoding an HSV-2gD secretion signal, a UL54 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or fragment thereof, a linker and MITD.

123. A polyribonucleotide encoding a polypeptide having an amino acid sequence comprising or consisting of ：MGRLTSGVGTAALLVVAVGLRVVCAE TLVAHGPSLYRTFAANPRAASTAKAMRDCVLRQENLIEALASADETLAWCKMCIHHNLPLRPQDPIIGTAAAVLENLATRLRPFLQCYLKARGLCGLDDLCSRRRLSDIKDIASFVLVILARLANRVERGVSEIDYTTVGVGAGETMHFYIPGACMAGLIEILDTHRQECSSRVCELTASHTIAPLYVHGKYFYCNSLFGGSGGGGSGGRAAAWMRQVPDPEDVRVVILYSPLPGEDLAAGRAGGGPPPEWSAERGGLSCLLAALGNRLCGPATAAWAGNWTGAPDVSALGAQGVLLLSTRDLAFAGAVEFLGLLAGACDRRLIVVNAVRAADWPADGPVVSRQHAYLACEVLPAVQCAVRWPAARDLRRTVLASGRVFGPGVFARVEAAHARLYPDAPPLRLCRGANVRYRVRTRFGPDTLVPMSPREYRRAVLPALDGRAAASGGSGGGGSGGCTDEIAPPLRCQSFPCLHPFCIPCMKTWIPLRNTCPLCNTPVAYLIVGVTASGSFSTIPIVNDPRTRVEAEAAVRAGTAVDFIWTGNPRTAPRSLSGGSGGGGSGGLPIAGVSSVVALAPYVNKTVTGDCLPVLDMETGHIGAYVVLVDQTGNVADLLRAAAPAWSRRTLLPEHARNCVRPPDYPTPPASEWNSLWMTPVGNMLFDQGTLVGGGSGGGGSGGIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA(SEQ ID NO:206).

124. A polyribonucleotide encoding a polypeptide, wherein said polypeptide comprises one or more Herpes Simplex Virus (HSV) antigens or antigenic fragments thereof.

125. The polyribonucleotide of claim 124, wherein said one or more HSV antigens or antigenic fragments thereof comprise:

(i) HSV-1 antigen or an antigenic fragment thereof,

(Ii) HSV-2 antigen or antigenic fragment thereof, or

(Iii) A combination thereof.

126. The polyribonucleotide of any of claims 124-125, wherein said polypeptide comprises a single HSV antigen or antigenic fragment thereof.

127. The polyribonucleotide of any of claims 124-126, wherein said polypeptide comprises a single HSV antigen.

128. The polyribonucleotide of claim 126, wherein said polypeptide comprises a single HSV antigenic fragment.

129. The polyribonucleotide of any of claims 124-125, wherein said polypeptide comprises two or more HSV antigens or antigenic fragments thereof.

130. The polyribonucleotide of claim 129, wherein said polypeptide comprises two or more HSV antigens.

131. The polyribonucleotide of claim 129, wherein said polypeptide comprises two or more HSV antigenic fragments, wherein each of said two or more HSV antigenic fragments is a fragment of a different HSV antigen.

132. The polyribonucleotide of claim 129 or 131, wherein the polypeptide comprises two or more HSV antigenic fragments, wherein at least two of the HSV antigenic fragments are fragments from the same HSV antigen.

133. The polyribonucleotide of any of claims 124-125, wherein said polypeptide comprises three or more HSV antigens or antigenic fragments thereof.

134. The polyribonucleotide of any of claims 124-125, wherein said polypeptide comprises four or more HSV antigens or antigenic fragments thereof.

135. The polyribonucleotide of any of claims 124-126, wherein said polypeptide does not comprise full-length HSV antigen.

136. The polyribonucleotide of any of claims 124-135, wherein said one or more HSV antigens or antigenic fragments thereof comprises one or more T cell antigens or antigenic fragments thereof.

137. The polyribonucleotide of any of claims 124-135, wherein said one or more HSV antigens or antigenic fragments thereof comprises one or more B cell antigens or antigenic fragments thereof.

138. The polyribonucleotide of any of claims 124-136, wherein said one or more HSV antigens or antigenic fragments thereof have at least 80% sequence identity to one or more sequences selected from SEQ ID NOs 1-74 or antigenic fragments thereof.

139. The polyribonucleotide of claim 138, wherein said one or more HSV antigens or antigenic fragments thereof have at least 85%, 90%, 95% or 100% sequence identity with one or more sequences selected from SEQ ID NOs 1-74 or antigenic fragments thereof.

140. The polyribonucleotide of any of claims 124-135, wherein said polypeptide comprises one or more HSV-2 antigens or antigenic fragments thereof comprising or consisting of an amino acid sequence selected from SEQ ID NOs 174-196.

141. The polyribonucleotide of any of claims 138-140, wherein said one or more HSV antigens or antigenic fragments thereof comprise:

(i) One or more HSV RS1 polypeptides or antigenic fragments thereof,

(Ii) One or more HSV RL2 polypeptides or antigenic fragments thereof,

(Iii) One or more HSV UL1 polypeptides or antigenic fragments thereof,

(Iv) One or more HSV UL5 polypeptides or antigenic fragments thereof,

(V) One or more HSV UL9 polypeptides or antigenic fragments thereof,

(Vi) One or more HSV UL19 polypeptides or antigenic fragments thereof,

(Vii) One or more HSV UL21 polypeptides or antigenic fragments thereof,

(Viii) One or more HSV UL25 polypeptides or antigenic fragments thereof,

(Ix) One or more HSV UL27 polypeptides or antigenic fragments thereof,

(X) One or more HSV UL29 polypeptides or antigenic fragments thereof,

(Xi) One or more HSV UL30 polypeptides or antigenic fragments thereof,

(Xii) One or more HSV UL39 polypeptides or antigenic fragments thereof,

(Xiii) One or more HSV UL40 polypeptides or antigenic fragments thereof,

(Xiv) One or more HSV UL46 polypeptides or antigenic fragments thereof,

(Xv) One or more HSV UL47 polypeptides or antigenic fragments thereof,

(Xvi) One or more HSV UL48 polypeptides or antigenic fragments thereof,

(Xvii) One or more HSV UL49 polypeptides or antigenic fragments thereof,

(Xviii) One or more HSV UL52 polypeptides or antigenic fragments thereof,

(Xix) One or more HSV UL54 polypeptides or antigenic fragments thereof, or

(Xx) A combination thereof.

142. The polyribonucleotide of any of claims 138-141, wherein said polypeptide comprises one or more HSV antigenic fragments and said one or more HSV antigenic fragments comprise:

(i) One or more antigenic fragments of HSV RS1 polypeptides,

(Ii) One or more antigenic fragments of an HSV RL2 polypeptide,

(Iii) One or more antigenic fragments of an HSV UL1 polypeptide,

(Iv) One or more antigenic fragments of an HSV UL5 polypeptide,

(V) One or more antigenic fragments of an HSV UL9 polypeptide,

(Vi) One or more antigenic fragments of an HSV UL19 polypeptide,

(Vii) One or more antigenic fragments of an HSV UL21 polypeptide,

(Viii) One or more antigenic fragments of an HSV UL25 polypeptide,

(Ix) One or more antigenic fragments of an HSV UL27 polypeptide,

(X) One or more antigenic fragments of an HSV UL29 polypeptide,

(Xi) One or more antigenic fragments of an HSV UL30 polypeptide,

(Xii) One or more antigenic fragments of an HSV UL39 polypeptide,

(Xiii) One or more antigenic fragments of an HSV UL40 polypeptide,

(Xiv) One or more antigenic fragments of an HSV UL46 polypeptide,

(Xv) One or more antigenic fragments of an HSV UL47 polypeptide,

(Xvi) One or more antigenic fragments of an HSV UL48 polypeptide,

(Xvii) One or more antigenic fragments of an HSV UL49 polypeptide,

(Xviii) One or more antigenic fragments of an HSV UL52 polypeptide,

(Xix) One or more antigenic fragments of HSV UL54 polypeptide, or

(Xx) A combination thereof.

143. The polyribonucleotide of any of claims 138 to 142, wherein said polypeptide comprises one or more HSV RL2 polypeptides or antigenic fragments thereof, one or more HSV RS1 polypeptides or antigenic fragments thereof, and one or more HSV UL54 polypeptides or antigenic fragments thereof.

144. The polyribonucleotide of claim 143, wherein said polypeptide comprises an HSV-1gD secretion signal, one or more RL2 polypeptides or antigenic fragments thereof, one or more RS1 polypeptides or antigenic fragments thereof, one or more UL54 polypeptides or antigenic fragments thereof, and MITD.

145. The polyribonucleotide of claim 143 or 144, wherein said polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, a RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, a UL54 polypeptide or antigenic fragment thereof, a linker and MITD.

146. The polyribonucleotide of claim 145, wherein said polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 197.

147. The polyribonucleotide of claim 143 or 144, wherein said polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL54 polypeptide or an antigenic fragment thereof, a linker, an RS1 polypeptide or an antigenic fragment thereof, a linker, an RL2 polypeptide or an antigenic fragment thereof, a linker and MITD.

148. The polyribonucleotide of claim 147, wherein said polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 201.

149. The polyribonucleotide of claim 143 or 144, wherein said polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-2gD secretion signal, a RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, a UL54 polypeptide or antigenic fragment thereof, a linker and MITD.

150. The polyribonucleotide of claim 149, wherein said polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 205.

151. The polyribonucleotide of any of claims 138 to 142, wherein said polypeptide comprises one or more HSV UL29 polypeptides or antigenic fragments thereof, one or more HSV UL39 polypeptides or antigenic fragments thereof, one or more HSV UL49 polypeptides or antigenic fragments thereof, and one or more HSV UL9 polypeptides or antigenic fragments thereof.

152. The polyribonucleotide of claim 151, wherein said polypeptide comprises an HSV-1gD secretion signal, one or more HSV UL29 polypeptides or antigenic fragments thereof, one or more HSV UL39 polypeptides or antigenic fragments thereof, one or more HSV UL49 polypeptides or antigenic fragments thereof, one or more HSV UL9 polypeptides or antigenic fragments thereof, and MITD.

153. The polyribonucleotide of claim 151 or 152, wherein said polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL29 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL49 polypeptide or antigenic fragment thereof, a linker, a UL9 polypeptide or antigenic fragment thereof, a linker and MITD.

154. The polyribonucleotide of claim 153, wherein said polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 198.

155. The polyribonucleotide of claim 151 or 152, wherein said polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL9 polypeptide or antigenic fragment thereof, a linker, a UL49 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL29 polypeptide or antigenic fragment thereof, a linker and MITD.

156. The polyribonucleotide of claim 155, wherein said polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 202.

157. The polyribonucleotide of any of claims 138 to 142, wherein said polypeptide comprises one or more HSV UL30 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, one or more HSV UL5 polypeptides or antigenic fragments thereof, and one or more HSV UL52 polypeptides or antigenic fragments thereof.

158. The polyribonucleotide of claim 157, wherein said polypeptide comprises an HSV-1gD secretion signal, one or more HSV UL30 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, one or more HSV UL5 polypeptides or antigenic fragments thereof, one or more HSV UL52 polypeptides or antigenic fragments thereof, and MITD.

159. The polyribonucleotide of claim 157 or 158, wherein said polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL30 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL52 polypeptide or antigenic fragment thereof, a linker and MITD.

160. The polyribonucleotide of claim 159, wherein said polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 199.

161. The polyribonucleotide of claim 157 or 158, wherein said polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL52 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or antigenic fragment thereof, a linker, a UL30 polypeptide or antigenic fragment thereof, a linker and MITD.

162. The polyribonucleotide of claim 161, wherein said polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 203.

163. The polyribonucleotide of any of claims 138 to 142, wherein said polypeptide comprises one or more HSV UL1 polypeptides or antigenic fragments thereof, one or more HSV UL19 polypeptides or antigenic fragments thereof, one or more HSV UL21 polypeptides or antigenic fragments thereof, one or more HSV UL27 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more UL48 polypeptides or antigenic fragments thereof, and one or more HSV UL25 polypeptides or antigenic fragments thereof.

164. The polyribonucleotide of claim 163, wherein said polypeptide comprises an HSV-1gD secretion signal, one or more HSV UL1 polypeptides or antigenic fragments thereof, one or more HSV UL19 polypeptides or antigenic fragments thereof, one or more HSV UL21 polypeptides or antigenic fragments thereof, one or more HSV UL27 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more UL48 polypeptides or antigenic fragments thereof, one or more HSV UL25 polypeptides or antigenic fragments thereof, and MITD.

165. The polyribonucleotide of claim 163 or 164, wherein said polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, an HSV UL1 polypeptide or antigenic fragment thereof, a linker, an HSV UL19 polypeptide or antigenic fragment thereof, a linker, an HSV UL21 polypeptide or antigenic fragment thereof, a linker, an HSV UL27 polypeptide or antigenic fragment thereof, a linker, an HSV UL46 polypeptide or antigenic fragment thereof, a linker, an HSV UL47 polypeptide or antigenic fragment thereof, a linker, an HSV UL25 polypeptide or antigenic fragment thereof, a linker, an HSV UL48 polypeptide or antigenic fragment thereof, a linker, and MITD.

166. The polyribonucleotide of claim 165, wherein said polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 200.

167. The polyribonucleotide of claim 163 or 164, wherein said polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, an HSV UL48 polypeptide or antigenic fragment thereof, a linker, an HSV UL25 polypeptide or antigenic fragment thereof, a linker, an HSV UL47 polypeptide or antigenic fragment thereof, a linker, an HSV UL46 polypeptide or antigenic fragment thereof, a linker, an HSV UL27 polypeptide or antigenic fragment thereof, a linker, an HSV UL21 polypeptide or antigenic fragment thereof, a linker, an HSV UL19 polypeptide or antigenic fragment thereof, a linker, an HSV UL1 polypeptide or antigenic fragment thereof, a linker, and MITD.

168. The polyribonucleotide of claim 167, wherein said polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 204.

169. The polyribonucleotide of any of claims 124-137, wherein said one or more HSV antigens or antigenic fragments thereof comprise one or more HSV glycoproteins.

170. The polyribonucleotide of any of claim 169, wherein the one or more HSV glycoproteins comprises HSV glycoprotein B (gB), HSV glycoprotein E (gE), HSV glycoprotein G (gG), HSV glycoprotein H (gH), HSV glycoprotein I (gL), HSV glycoprotein L (gL), or a combination thereof.

171. The polyribonucleotide of any of claims 169 to 170, wherein said polypeptide comprises a single HSV antigen.

172. The polyribonucleotide of any of claims 169-171, wherein said single HSV antigen is an HSV glycoprotein.

173. The polyribonucleotide of any of claims 169-172, wherein said HSV glycoprotein is a full-length HSV glycoprotein.

174. The polyribonucleotide of any of claims 169 to 173, wherein said HSV glycoprotein is HSV gB, HSV gE, HSV gG, HSV gH, HSV gI, and HSV gL.

175. The polyribonucleotide of any of claims 170-174, wherein said HSV glycoprotein is HSV-2gB.

176. The polyribonucleotide of claim 175, wherein said HSV-2gB is or comprises an amino acid sequence that is at least 80%,85%,90%, 91%, 92%, 93%, 94%, 95%,96%,97%,98% or 99% identical to SEQ ID No. 7, 8, 9 or 74.

177. The polyribonucleotide of any of claims 175 to 176, wherein said HSV-2gB consists of or comprises the amino acid sequence according to SEQ ID No. 7, 8, 9 or 74.

178. The polyribonucleotide of any of claims 170 to 174, wherein said HSV glycoprotein is HSV-2gE.

179. The polyribonucleotide of claim 178, wherein said HSV-2gE is or comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 66, 67, 68 or 69.

180. The polyribonucleotide of any of claims 178 to 179, wherein said HSV-2gE consists of or comprises the amino acid sequence according to SEQ ID NO:66, 67, 68 or 69.

181. The polyribonucleotide of any of claims 178 to 180, wherein said sequence is at least 80% identical to SEQ ID No. 80, 81, 82, 83 or 84.

182. The polyribonucleotide of any of claims 170 to 174, wherein said HSV glycoprotein is HSV-2gH.

183. The polyribonucleotide of claim 182, wherein said HSV-2gH is or comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 70, 71, 72 or 74.

184. The polyribonucleotide of any of claims 182 to 183, wherein said HSV-2gH consists of or comprises the amino acid sequence according to SEQ ID No. 70, 71, 72 or 74.

185. The polyribonucleotide of any of claims 170 to 174, wherein said HSV glycoprotein is HSV-2gI.

186. The polyribonucleotide of claim 185, wherein said HSV-2gI is or comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 62, 63, 64 or 65.

187. The polyribonucleotide of any of claims 185 to 186, wherein said HSV-2gI consists of or comprises the amino acid sequence according to SEQ ID No. 62, 63, 64 or 65.

188. The polyribonucleotide of any one of claims 185 to 187, wherein said sequence is at least 80% identical to SEQ ID No. 75, 76, 77, 78 or 79.

189. The polyribonucleotide of any of claims 170-174, wherein said HSV glycoprotein is HSV-2gL.

190. The polyribonucleotide of claim 190, wherein said HSV-2gL is or comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 58, 59, 60 or 61.

191. The polyribonucleotide of any of claims 190 to 191, wherein said HSV-2gL consists of or comprises the amino acid sequence according to SEQ ID NO:58, 59, 60 or 61.

192. The polyribonucleotide of any one of claims 1 to 192, wherein said polypeptide comprises a secretion signal.

193. The polyribonucleotide of claim 192, wherein said secretion signal comprises or consists of a viral secretion signal.

194. The polyribonucleotide of claim 193, wherein said viral secretion signal comprises or consists of an HSV secretion signal.

195. The polyribonucleotide of any of claims 192 to 194, wherein said secretion signal is a heterologous secretion signal.

196. The polyribonucleotide of any of claims 194 to 195, wherein said HSV secretion signal comprises or consists of an HSV-1 or HSV-2 secretion signal.

197. The polyribonucleotide of any of claims 194 to 196, wherein said HSV secretion signal is selected from the group consisting of:

a) gD2 secretion signal;

b) gD1 secretion signal;

c) gB1 secretion signal;

d) gI2 secretion signal;

e) gE2 secretion signal;

f) gC2 secretion signal:

g) Eboz secretion signals;

h) An IL2 secretion signal; and

I) HLA-DR secretion signal.

198. The polyribonucleotide of any of claims 194 to 197, wherein said HSV secretion signal comprises or consists of an HSV gD secretion signal.

199. The polyribonucleotide of claim 198, wherein the HSV gD secretion signal comprises an amino acid sequence that is at least 80% identical to SEQ ID No. 87.

200. The polyribonucleotide of claim 198, wherein the HSV gD secretion signal consists of the amino acid sequence according to SEQ ID No. 88.

201. The polyribonucleotide of claim 198, wherein the HSV gD secretion signal consists of the amino acid sequence according to SEQ ID No. 110.

202. The polyribonucleotide of claim 198, wherein the HSV gD secretion signal consists of the amino acid sequence according to SEQ ID No. 111.

203. The polyribonucleotide of any of claims 198 to 202, wherein said secretion signal is positioned at the N-terminus of the polypeptide.

204. The polyribonucleotide of any of claims 194 to 197, wherein said HSV secretion signal comprises or consists of an HSV-2 glycoprotein I (gI) secretion signal.

205. The polyribonucleotide of claim 204, wherein said HSV-2gI secretion signal comprises an amino acid sequence that is at least 80% identical to SEQ ID No. 107.

206. The polyribonucleotide of claim 204, wherein said HSV-2gI secretion signal comprises an amino acid sequence that is at least 80% identical to SEQ ID No. 108.

207. The polyribonucleotide of any one of claims 124 to 206, wherein said polypeptide comprises a transmembrane region.

208. The polyribonucleotide of claim 207, wherein said transmembrane region comprises or consists of a viral transmembrane region.

209. The polyribonucleotide of any of claims 207 to 208, wherein said transmembrane region comprises or consists of an HSV transmembrane region.

210. The polyribonucleotide of any of claims 207 to 209, wherein said HSV transmembrane region comprises or consists of an HSV-1 or HSV-2 transmembrane region.

211. The polyribonucleotide of any of claims 207 to 210, wherein said HSV transmembrane region comprises or consists of an HSV gD transmembrane region.

212. The polyribonucleotide of claim 211, wherein said HSV gD transmembrane region consists of the amino acid sequence according to SEQ ID No. 160.

213. The polyribonucleotide of any of claims 124-206, wherein said polypeptide does not comprise a transmembrane region.

214. The polyribonucleotide of any one of claims 124 to 213, wherein said polypeptide comprises a multimerization domain.

215. The polyribonucleotide of any one of claims 124-214, wherein said polypeptide comprises one or more linkers.

216. The polyribonucleotide of claim 215, wherein said one or more linkers comprise one or more glycine (G) residues and/or one or more serine (S) residues.

217. The polyribonucleotide of any one of claims 215 to 216, wherein said one or more linkers comprise a nucleotide sequence according to SEQ ID NO:163 or consists of the amino acid sequence according to SEQ ID NO: 163.

218. The polyribonucleotide of any of claims 215 to 217, wherein said one or more linkers comprise or consist of an amino acid sequence according to SEQ ID No. 165.

219. The polyribonucleotide of any of claims 215 to 217, wherein said one or more linkers comprise or consist of an amino acid sequence according to SEQ ID No. 168.

220. The polyribonucleotide of any of claims 215 to 217, wherein said one or more linkers comprise or consist of an amino acid sequence according to SEQ ID No. 217.

221. The polyribonucleotide of any one of claims 124-220, wherein said polyribonucleotide is an isolated polyribonucleotide.

222. The polyribonucleotide of any one of claims 124-221, wherein said polyribonucleotide is an engineered polyribonucleotide.

223. The polyribonucleotide of any of claims 124-222, wherein said polyribonucleotide is a codon-optimized polyribonucleotide.

224. An RNA construct comprising, in 5 'to 3' order:

(i)5'UTR；

(ii) The polyribonucleotide of any one of claims 124 to 223;

(iv) 3' UTR; and

(V) Poly a tail sequence.

225. The RNA construct of claim 224, wherein:

(i) The 5'UTR comprises or consists of a modified human α -globulin 5' -UTR;

(ii) A 3' utr comprising or consisting of a first sequence from a split amino-terminal enhancer (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA; or (b)

(Iii) Both of the above.

226. The RNA construct of claim 224 or 225, wherein the 5' utr comprises or consists of a ribonucleic acid sequence according to SEQ ID No. 208.

227. The RNA construct of claim 224 or 225, wherein the 5' utr comprises or consists of a ribonucleic acid sequence according to SEQ ID No. 209.

228. The RNA construct of any one of claims 224 to 227, wherein the 3' utr comprises or consists of a ribonucleic acid sequence according to SEQ ID No. 215.

229. The RNA construct of any one of claims 224 to 227, wherein the 3' utr comprises or consists of a ribonucleic acid sequence according to SEQ ID No. 216.

230. The RNA construct of any one of claims 224 to 229, wherein the poly-a tail sequence is a split poly-a tail sequence.

231. The RNA construct of claim 230, wherein the split poly a tail sequence consists of a ribonucleic acid sequence selected from the group consisting of SEQ ID NOs 210, 212, or 213.

232. The RNA construct of any one of claims 224 to 231, further comprising a 5' cap.

233. The RNA construct of claim 232, further comprising a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of a polyribonucleotide.

234. The RNA construct of claim 232 or 233, wherein the 5' cap comprises or consists of m7 (3 ' ome G) (5 ') ppp (5 ') (2 ' ome a ₁)pG₂, wherein a ₁ is position +1 of the polyribonucleotide and G ₂ is position +2 of the polyribonucleotide.

235. The RNA construct of claim 233 or 234, wherein the Cap proximal sequence comprises a ₁ and G ₂ of Cap1 structure and comprises at positions +3, +4, and +5 of the polyribonucleotide, respectively: a ₃A₄U₅ (SEQ ID NO: 207).

236. The RNA construct of any one of claims 224 to 235, wherein the polyribonucleotide comprises a modified uridine that replaces all uridine, optionally wherein the modified uridine is each N1-methyl-pseudouridine.

237. A composition comprising one or more polynucleic acids according to any one of claims 124 to 223.

238. A composition comprising one or more RNA constructs of any one of claims 224-236.

239. The composition of claim 237 or 238, wherein the composition further comprises a lipid nanoparticle, a multimeric complex (PLX), a lipidated multimeric complex (LPLX), or a liposome,

Wherein the one or more polyribonucleotides are wholly or partially encapsulated within a lipid nanoparticle, a multimeric complex (PLX), a lipidated multimeric complex (LPLX), or a liposome.

240. The composition of any one of claim 237 to 239, wherein the composition further comprises lipid nanoparticles,

Wherein the one or more polyribonucleotides are encapsulated within a lipid nanoparticle.

241. A pharmaceutical composition comprising the composition of any one of claims 237 to 240 and at least one pharmaceutically acceptable excipient.

242. The pharmaceutical composition of claim 241, wherein the drug comprises a cryoprotectant, optionally wherein the cryoprotectant is sucrose.

243. The pharmaceutical composition of claim 241 or 242, wherein the drug comprises a buffered aqueous solution, optionally wherein the buffered aqueous solution comprises one or more of Tris base, tris HCl, naCl, KCl, na ₂HPO₄, and KH ₂PO₄.

244. A combination, comprising:

the first polynucleic acid of any of claims 124 to 223; and

The second polynucleic acid according to any of claims 124 to 223,

Wherein the first is polyribonucleotides and methods of making same the second polyribonucleotide is different.

245. A combination, comprising:

a first pharmaceutical composition comprising a first polynucleic acid, wherein the first polynucleic acid is a polynucleic acid according to any of items 124 to 223; and

A second pharmaceutical composition comprising a second polynucleic acid, wherein the second polynucleic acid is a polynucleic acid according to any of items 124 to 223,

Wherein the first is polyribonucleotides and methods of making same the second polyribonucleotide is different.

246. A combination, comprising:

The first polynucleic acid of any of claims 169 to 191; and

The second polynucleic acid according to any of claims 138 to 168.

247. A combination, comprising:

The first polynucleic acid of any of claims 169 to 191; and

A second polyribonucleotide encoding a second polypeptide, wherein said second polypeptide comprises one or more HSV RL2 polypeptides or antigenic fragments thereof, one or more HSV RS1 polypeptides or antigenic fragments thereof, and one or more HSV UL54 polypeptides or antigenic fragments thereof.

248. The combination of claim 247, wherein the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, a RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, a UL54 polypeptide or antigenic fragment thereof, a linker, and MITD.

249. The combination of claim 247 or 248, wherein the second polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 197.

250. The combination of claim 247, wherein the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, a UL54 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or antigenic fragment thereof, a linker, and MITD.

251. The combination of claim 247 or 250, wherein the second polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 201.

252. The combination of claim 247, wherein the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-2gD secretion signal, a RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, a UL54 polypeptide or antigenic fragment thereof, a linker, and MITD.

253. The combination of claim 247 or 252, wherein the second polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 205.

254. A combination, comprising:

The first polynucleic acid of any of claims 169 to 191; and

A second polyribonucleotide encoding a second polypeptide, wherein the second polypeptide comprises one or more HSV UL29 polypeptides or antigenic fragments thereof, one or more HSV UL39 polypeptides or antigenic fragments thereof, one or more HSV UL49 polypeptides or antigenic fragments thereof, and one or more HSV UL9 polypeptides or antigenic fragments thereof.

255. The combination of claim 254, wherein the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, a UL29 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL49 polypeptide or antigenic fragment thereof, a linker, a UL9 polypeptide or antigenic fragment thereof, a linker and MITD.

256. The combination of claim 254 or 255, wherein said second polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 198.

257. The combination of claim 254, wherein the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, a UL9 polypeptide or antigenic fragment thereof, a linker, a UL49 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL29 polypeptide or antigenic fragment thereof, a linker and MITD.

258. The combination of claim 254 or 257, wherein said second polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 202.

259. A combination, comprising:

The first polynucleic acid of any of claims 169 to 191; and

A second polyribonucleotide encoding a second polypeptide, wherein the second polypeptide comprises one or more HSV UL30 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, one or more HSV UL5 polypeptides or antigenic fragments thereof, and one or more HSV UL52 polypeptides or antigenic fragments thereof.

260. The combination of claim 259, wherein the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, a UL30 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL52 polypeptide or antigenic fragment thereof, a linker, and MITD.

261. The combination of claim 259 or 260, wherein the second polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 199.

262. The combination of claim 259, wherein the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, a UL52 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or antigenic fragment thereof, a linker, a UL30 polypeptide or antigenic fragment thereof, a linker, and MITD.

263. The combination of claim 259 or 262, wherein the second polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 203.

264. A combination, comprising:

The first polynucleic acid of any of claims 169 to 191; and

A second polyribonucleotide encoding a second polypeptide, wherein the second polypeptide comprises one or more HSV UL1 polypeptides or antigenic fragments thereof, one or more HSV UL19 polypeptides or antigenic fragments thereof, one or more HSV UL21 polypeptides or antigenic fragments thereof, one or more HSV UL27 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more UL48 polypeptides or antigenic fragments thereof, and one or more HSV UL25 polypeptides or antigenic fragments thereof.

265. The combination of claim 264, wherein the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, an HSV UL1 polypeptide or antigenic fragment thereof, a linker, an HSV UL19 polypeptide or antigenic fragment thereof, a linker, an HSV UL21 polypeptide or antigenic fragment thereof, a linker, an HSV UL27 polypeptide or antigenic fragment thereof, a linker, an HSV UL46 polypeptide or antigenic fragment thereof, a linker, an HSV UL47 polypeptide or antigenic fragment thereof, a linker, an HSV UL25 polypeptide or antigenic fragment thereof, a linker, an HSV UL48 polypeptide or antigenic fragment thereof, a linker, and MITD.

266. The combination of claim 264 or 265, wherein said second polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 200.

267. The combination of claim 264, wherein the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, an HSV UL48 polypeptide or antigenic fragment thereof, a linker, an HSV UL25 polypeptide or antigenic fragment thereof, a linker, an HSV UL47 polypeptide or antigenic fragment thereof, a linker, an HSV UL46 polypeptide or antigenic fragment thereof, a linker, an HSV UL27 polypeptide or antigenic fragment thereof, a linker, an HSV UL21 polypeptide or antigenic fragment thereof, a linker, an HSV UL19 polypeptide or antigenic fragment thereof, a linker, an HSV UL1 polypeptide or antigenic fragment thereof, a linker, and MITD.

268. The combination of claim 264 or 267, wherein the second polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 204.

269. The combination of any one of claims 264-268, wherein the second polypeptide is HSV gB.

270. The combination of any one of claims 264-269, wherein the second polypeptide consists of or comprises an amino acid sequence according to SEQ ID No. 7, 8, 9 or 74.

271. A combination, comprising:

A first pharmaceutical composition comprising a first polyribonucleotide, wherein said first polyribonucleotide is a polyribonucleotide according to any of items 169 to 191; and

A second pharmaceutical composition comprising a second polyribonucleotide, wherein said second polyribonucleotide is a polyribonucleotide according to any of items 138 to 168,

272. A combination, comprising:

A first pharmaceutical composition comprising a first polyribonucleotide, wherein said first polyribonucleotide is a polyribonucleotide according to any of items 169 to 191; and

A second pharmaceutical composition comprising a second polyribonucleotide, wherein said second polyribonucleotide comprises one or more HSV RL2 polypeptides or antigenic fragments thereof, one or more HSV RS1 polypeptides or antigenic fragments thereof, and one or more HSV UL54 polypeptides or antigenic fragments thereof.

273. The combination of claim 272, wherein the second polypeptide comprises, in order from N-terminus to C-terminus, an HSV-1gD secretion signal, a RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, a UL54 polypeptide or antigenic fragment thereof, a linker and MITD.

274. The combination of claim 272 or 273, wherein the second polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 197.

275. The combination of claim 272, wherein the second polypeptide comprises, in order from N-terminus to C-terminus, an HSV-1gD secretion signal, a UL54 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an RL2 polypeptide or antigenic fragment thereof, a linker and MITD.

276. The combination of clauses 272 or 275, wherein the second polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 201.

277. The combination of claim 272, wherein the second polypeptide comprises, in order from N-terminus to C-terminus, an HSV-2gD secretion signal, a RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, a UL54 polypeptide or antigenic fragment thereof, a linker and MITD.

278. The combination of claim 272 or 277, wherein said second polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 205.

279. A combination, comprising:

A first pharmaceutical composition comprising a first polyribonucleotide, wherein said first polyribonucleotide is a polyribonucleotide according to any of items 169 to 191; and

A second pharmaceutical composition comprising a second polyribonucleotide, wherein said second polypeptide comprises one or more HSV UL29 polypeptides or antigenic fragments thereof, one or more HSV UL39 polypeptides or antigenic fragments thereof, one or more HSV UL49 polypeptides or antigenic fragments thereof, and one or more HSV UL9 polypeptides or antigenic fragments thereof.

280. The combination of claim 279, wherein the second polypeptide comprises, in order from N-terminus to C-terminus, an HSV-1gD secretion signal, a UL29 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL49 polypeptide or antigenic fragment thereof, a linker, a UL9 polypeptide or antigenic fragment thereof, a linker and MITD.

281. The combination of claim 279 or 280, wherein the second polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 198.

282. The combination of claim 279, wherein the second polypeptide comprises, in order from N-terminus to C-terminus, an HSV-1gD secretion signal, a UL9 polypeptide or antigenic fragment thereof, a linker, a UL49 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL29 polypeptide or antigenic fragment thereof, a linker and MITD.

283. The combination of claim 279 or 282, wherein the second polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 202.

284. A combination, comprising:

A first pharmaceutical composition comprising a first polyribonucleotide, wherein said first polyribonucleotide is a polyribonucleotide according to any of items 169 to 191; and

A second pharmaceutical composition comprising a second polyribonucleotide, wherein said second polypeptide comprises one or more HSV UL30 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, one or more HSV UL5 polypeptides or antigenic fragments thereof, and one or more HSV UL52 polypeptides or antigenic fragments thereof.

285. The combination of claim 284, wherein the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, a UL30 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL52 polypeptide or antigenic fragment thereof, a linker, and MITD.

286. The combination of claim 284 or 285, wherein the second polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 199.

287. The combination of claim 284, wherein the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, a UL52 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or antigenic fragment thereof, a linker, a UL30 polypeptide or antigenic fragment thereof, a linker, and MITD.

288. The combination of claim 284 or 287, wherein the second polypeptide comprises or consists of an amino acid sequence according to SEQ ID No. 203.

289. A combination, comprising:

A first pharmaceutical composition comprising a first polyribonucleotide, wherein said first polyribonucleotide is a polyribonucleotide according to any of items 169 to 191; and

A second pharmaceutical composition comprising a second polyribonucleotide, wherein said second polypeptide comprises one or more HSV UL1 polypeptides or antigenic fragments thereof, one or more HSV UL19 polypeptides or antigenic fragments thereof, one or more HSV UL21 polypeptides or antigenic fragments thereof, one or more HSV UL27 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more UL48 polypeptides or antigenic fragments thereof, and one or more HSV UL25 polypeptides or antigenic fragments thereof.

290. The combination of claim 289, wherein the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, an HSV UL1 polypeptide or antigenic fragment thereof, a linker, an HSV UL19 polypeptide or antigenic fragment thereof, a linker, an HSV UL21 polypeptide or antigenic fragment thereof, a linker, an HSV UL27 polypeptide or antigenic fragment thereof, a linker, an HSV UL46 polypeptide or antigenic fragment thereof, a linker, an HSV UL47 polypeptide or antigenic fragment thereof, a linker, an HSV UL25 polypeptide or antigenic fragment thereof, a linker, an HSV UL48 polypeptide or antigenic fragment thereof, a linker, and MITD.

291. The combination of claim 289 or 290, wherein the second polypeptide comprises or consists of an amino acid sequence according to SEQ ID No. 200.

292. The combination of claim 289, wherein the second polypeptide comprises, in N-terminal to C-terminal order, an HSV-1gD secretion signal, an HSV UL48 polypeptide or antigenic fragment thereof, a linker, an HSV UL25 polypeptide or antigenic fragment thereof, a linker, an HSV UL47 polypeptide or antigenic fragment thereof, a linker, an HSV UL46 polypeptide or antigenic fragment thereof, a linker, an HSV UL27 polypeptide or antigenic fragment thereof, a linker, an HSV UL21 polypeptide or antigenic fragment thereof, a linker, an HSV UL19 polypeptide or antigenic fragment thereof, a linker, an HSV UL1 polypeptide or antigenic fragment thereof, a linker, and MITD.

293. The combination of claim 289 or 292, wherein the second polypeptide comprises or consists of an amino acid sequence according to SEQ ID No. 204.

294. The combination of any one of claims 289-293, wherein the second polypeptide is HSV gB.

295. The combination of any one of claims 289-294, wherein the second polypeptide consists of or comprises an amino acid sequence according to SEQ ID No. 7, 8, 9 or 74.

296. A method comprising administering to a subject the polyribonucleotide according to any one of claims 124-223 or the RNA construct according to any one of claims 224-236.

297. A method comprising administering to a subject a composition according to any one of claims 237 to 240.

298. A method comprising administering to a subject one or more doses of the composition of any one of claims 237 to 240 or the pharmaceutical composition of any one of claims 241 to 243.

299. A method comprising administering to a subject the combination of any one of claims 244-294.

300. The pharmaceutical composition of any one of claims 241 to 243, for use in treating an HSV infection, comprising administering to a subject one or more doses of the pharmaceutical composition.

301. The pharmaceutical composition of any one of claims 241 to 243, for use in preventing an HSV infection, comprising administering to a subject one or more doses of the pharmaceutical composition.

302. The method of claim 299 or the pharmaceutical composition for use of claim 300 or 301, comprising administering two or more doses of the pharmaceutical composition to a subject.

303. The method of claim 299 or the pharmaceutical composition for use of claim 300 or 301, comprising administering three or more doses of the pharmaceutical composition to a subject.

304. A method comprising administering to a subject a combination of any one of claims 271-294.

305. The method of claim 304, wherein the first pharmaceutical composition and the second pharmaceutical composition are administered on the same day.

306. The method of claims 304 and 305 wherein the first pharmaceutical composition and the second pharmaceutical composition are administered on different dates.

307. The method of any one of claims 304-306, wherein the first pharmaceutical composition and the second pharmaceutical composition are administered to the subject at different locations of the subject's body.

308. The method of any one of claims 304-307, wherein the method is a method of treating an HSV infection.

309. The method of any one of claims 304-308, wherein the method is a method of preventing an HSV infection.

310. The method of any one of claims 304-309, wherein the subject has or is at risk of having an HSV infection.

311. The method of any one of claims 304-310, wherein the subject is a human.

312. The method of any one of claims 304-311, wherein administration induces an anti-HSV immune response in the subject.

313. The method of claim 312, wherein the anti-HSV immune response in the subject comprises an adaptive immune response.

314. The method of claim 312 or 313, wherein the anti-HSV immune response in the subject comprises a T cell response.

315. The method of claim 314, wherein the T cell response is or comprises a cd4+ T cell response.

316. The method of claim 314, wherein the T cell response is or comprises a cd8+ T cell response.

317. The method of claim 312, wherein the anti-HSV immune system response comprises a B-cell response.

318. The method of any one of claims 312 to 317, wherein said anti-HSV immune system response comprises producing antibodies to one or more HSV antigens or antigenic fragments thereof having at least 80% sequence identity to one or more sequences selected from SEQ ID NOs 1-74 or antigenic fragments thereof.

319. The use of the pharmaceutical composition of any one of claims 241 to 243 in the treatment of a herpes simplex virus infection.

320. Use of the pharmaceutical composition of any one of claims 241 to 243 for the prevention of herpes simplex virus infection.

321. The use of the pharmaceutical composition of any one of claims 241 to 243 for inducing an anti-herpes simplex immune virus response in a subject.

322. A polypeptide encoded by the polyribonucleotide of any one of claims 124-223.

323. A polypeptide encoded by the RNA construct of any one of claims 224 to 236.

324. A host cell comprising the polyribonucleotide of any of claims 124-223.

325. A host cell comprising the RNA construct of any one of claims 224 to 236.

326. A host cell comprising the polypeptide of claim 322 or 323.

Equivalent scheme

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the technology described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as shown in the following claims.

Claims (47)

1. A polyribonucleotide encoding a polypeptide, wherein said polypeptide comprises one or more Herpes Simplex Virus (HSV) antigens or antigenic fragments thereof, and wherein said one or more HSV antigens or antigenic fragments thereof comprise:

(i) One or more HSV RS1 polypeptides or antigenic fragments thereof,

(Ii) One or more HSV RL2 polypeptides or antigenic fragments thereof,

(Iii) One or more HSV UL1 polypeptides or antigenic fragments thereof,

(Iv) One or more HSV UL5 polypeptides or antigenic fragments thereof,

(V) One or more HSV UL9 polypeptides or antigenic fragments thereof,

(Vi) One or more HSV UL19 polypeptides or antigenic fragments thereof,

(Vii) One or more HSV UL21 polypeptides or antigenic fragments thereof,

(Viii) One or more HSV UL25 polypeptides or antigenic fragments thereof,

(Ix) One or more HSV UL27 polypeptides or antigenic fragments thereof,

(X) One or more HSV UL29 polypeptides or antigenic fragments thereof,

(Xi) One or more HSV UL30 polypeptides or antigenic fragments thereof,

(Xii) One or more HSV UL39 polypeptides or antigenic fragments thereof,

(Xiii) One or more HSV UL40 polypeptides or antigenic fragments thereof,

(Xiv) One or more HSV UL46 polypeptides or antigenic fragments thereof,

(Xv) One or more HSV UL47 polypeptides or antigenic fragments thereof,

(Xvi) One or more HSV UL48 polypeptides or antigenic fragments thereof,

(Xvii) One or more HSV UL49 polypeptides or antigenic fragments thereof,

(Xviii) One or more HSV UL52 polypeptides or antigenic fragments thereof,

(Xix) One or more HSV UL54 polypeptides or antigenic fragments thereof, or

(Xx) A combination thereof.

2. The polyribonucleotide of claim 1, wherein the polypeptide comprises one or more HSV antigenic fragments and the one or more HSV antigenic fragments comprise:

(i) One or more antigenic fragments of HSV RS1 polypeptides,

(Ii) One or more antigenic fragments of an HSV RL2 polypeptide,

(Iii) One or more antigenic fragments of an HSV UL1 polypeptide,

(Iv) One or more antigenic fragments of an HSV UL5 polypeptide,

(V) One or more antigenic fragments of an HSV UL9 polypeptide,

(Vi) One or more antigenic fragments of an HSV UL19 polypeptide,

(Vii) One or more antigenic fragments of an HSV UL21 polypeptide,

(Viii) One or more antigenic fragments of an HSV UL25 polypeptide,

(Ix) One or more antigenic fragments of an HSV UL27 polypeptide,

(X) One or more antigenic fragments of an HSV UL29 polypeptide,

(Xi) One or more antigenic fragments of an HSV UL30 polypeptide,

(Xii) One or more antigenic fragments of an HSV UL39 polypeptide,

(Xiii) One or more antigenic fragments of an HSV UL40 polypeptide,

(Xiv) One or more antigenic fragments of an HSV UL46 polypeptide,

(Xv) One or more antigenic fragments of an HSV UL47 polypeptide,

(Xvi) One or more antigenic fragments of an HSV UL48 polypeptide,

(Xvii) One or more antigenic fragments of an HSV UL49 polypeptide,

(Xviii) One or more antigenic fragments of an HSV UL52 polypeptide,

(Xix) One or more antigenic fragments of HSV UL54 polypeptide, or

(Xx) A combination thereof.

3. The polyribonucleotide of claim 1 or 2, wherein the polypeptide comprises one or more HSV RL2 polypeptides or antigenic fragments thereof, one or more HSV RS1 polypeptides or antigenic fragments thereof, and one or more HSV UL54 polypeptides or antigenic fragments thereof.

4. The polyribonucleotide according to any of claims 1 to 3, wherein the polypeptide comprises an HSV-1gD secretion signal, one or more RL2 polypeptides or antigenic fragments thereof, one or more RS1 polypeptides or antigenic fragments thereof, one or more UL54 polypeptides or antigenic fragments thereof and MITD.

5. The polyribonucleotide according to any one of claims 1 to 4, wherein the polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, a RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, a UL54 polypeptide or antigenic fragment thereof, a linker and MITD.

6. The polyribonucleotide according to claim 5, wherein the polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 197.

7. The polyribonucleotide according to any one of claims 1 to 4, wherein the polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL54 polypeptide or an antigenic fragment thereof, a linker, an RS1 polypeptide or an antigenic fragment thereof, a linker, a RL2 polypeptide or an antigenic fragment thereof, a linker and MITD.

8. The polyribonucleotide according to claim 7, wherein the polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 201.

9. The polyribonucleotide according to any one of claims 1 to 4, wherein the polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-2gD secretion signal, an RL2 polypeptide or antigenic fragment thereof, a linker, an RS1 polypeptide or antigenic fragment thereof, a linker, an UL54 polypeptide or antigenic fragment thereof, a linker and MITD.

10. The polyribonucleotide according to claim 9, wherein said polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 205.

11. The polyribonucleotide of claim 1 or 2, wherein the polypeptide comprises one or more HSV UL29 polypeptides or antigenic fragments thereof, one or more HSV UL39 polypeptides or antigenic fragments thereof, one or more HSV UL49 polypeptides or antigenic fragments thereof, and one or more HSV UL9 polypeptides or antigenic fragments thereof.

12. The polyribonucleotide of any of claims 1,2 and 11, wherein the polypeptide comprises an HSV-1gD secretion signal, one or more HSV UL29 polypeptides or antigenic fragments thereof, one or more HSV UL39 polypeptides or antigenic fragments thereof, one or more HSV UL49 polypeptides or antigenic fragments thereof, one or more HSV UL9 polypeptides or antigenic fragments thereof, and MITD.

13. The polyribonucleotide of any one of claims 1, 2, 11 and 12, wherein said polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL29 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL49 polypeptide or antigenic fragment thereof, a linker, a UL9 polypeptide or antigenic fragment thereof, a linker and MITD.

14. The polyribonucleotide according to claim 13, wherein the polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 198.

15. The polyribonucleotide of any one of claims 1, 2, 11 and 12, wherein said polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL9 polypeptide or antigenic fragment thereof, a linker, a UL49 polypeptide or antigenic fragment thereof, a linker, a UL39 polypeptide or antigenic fragment thereof, a linker, a UL29 polypeptide or antigenic fragment thereof, a linker and MITD.

16. The polyribonucleotide according to claim 15, wherein the polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 202.

17. The polyribonucleotide of claim 1 or 2, wherein the polypeptide comprises one or more HSV UL30 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, one or more HSV UL5 polypeptides or antigenic fragments thereof, and one or more HSV UL52 polypeptides or antigenic fragments thereof.

18. The polyribonucleotide of any of claims 1,2 and 17, wherein the polypeptide comprises an HSV-1gD secretion signal, one or more HSV UL30 polypeptides or antigenic fragments thereof, one or more HSV UL40 polypeptides or antigenic fragments thereof, one or more HSV UL5 polypeptides or antigenic fragments thereof, one or more HSV UL52 polypeptides or antigenic fragments thereof, and MITD.

19. The polyribonucleotide of any one of claims 1, 2, 17 and 18, wherein said polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL30 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL52 polypeptide or antigenic fragment thereof, a linker and MITD.

20. The polyribonucleotide according to claim 19, wherein the polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 199.

21. The polyribonucleotide of any one of claims 1, 2, 17 and 18, wherein said polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, a UL52 polypeptide or antigenic fragment thereof, a linker, a UL5 polypeptide or antigenic fragment thereof, a linker, a UL40 polypeptide or antigenic fragment thereof, a linker, a UL30 polypeptide or antigenic fragment thereof, a linker and MITD.

22. The polyribonucleotide according to claim 21, wherein the polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 203.

23. The polyribonucleotide of claim 1 or 2, wherein the polypeptide comprises one or more HSV UL1 polypeptides or antigenic fragments thereof, one or more HSV UL19 polypeptides or antigenic fragments thereof, one or more HSV UL21 polypeptides or antigenic fragments thereof, one or more HSV UL27 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more UL48 polypeptides or antigenic fragments thereof, and one or more HSV UL25 polypeptides or antigenic fragments thereof.

24. The polyribonucleotide of any of claims 1,2 and 23, wherein the polypeptide comprises an HSV-1gD secretion signal, one or more HSV UL1 polypeptides or antigenic fragments thereof, one or more HSV UL19 polypeptides or antigenic fragments thereof, one or more HSV UL21 polypeptides or antigenic fragments thereof, one or more HSV UL27 polypeptides or antigenic fragments thereof, one or more HSV UL46 polypeptides or antigenic fragments thereof, one or more HSV UL47 polypeptides or antigenic fragments thereof, one or more UL48 polypeptides or antigenic fragments thereof, one or more HSV UL25 polypeptides or antigenic fragments thereof, and MITD.

25. The polyribonucleotide of any one of claims 1,2, 23 and 24, wherein said polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, an HSV UL1 polypeptide or antigenic fragment thereof, a linker, an HSV UL19 polypeptide or antigenic fragment thereof, a linker, an HSV UL21 polypeptide or antigenic fragment thereof, a linker, an HSV UL27 polypeptide or antigenic fragment thereof, a linker, an HSV UL46 polypeptide or antigenic fragment thereof, a linker, an HSV UL47 polypeptide or antigenic fragment thereof, a linker, an HSV UL25 polypeptide or antigenic fragment thereof, a linker, an HSV UL48 polypeptide or antigenic fragment thereof, a linker and MITD.

26. The polyribonucleotide according to claim 25, wherein the polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 200.

27. The polyribonucleotide of claims 1,2, 23 and 24, wherein said polypeptide comprises in N-terminal to C-terminal order a nucleotide sequence encoding an HSV-1gD secretion signal, an HSV UL48 polypeptide or antigenic fragment thereof, a linker, an HSV UL25 polypeptide or antigenic fragment thereof, a linker, an HSV UL47 polypeptide or antigenic fragment thereof, a linker, an HSV UL46 polypeptide or antigenic fragment thereof, a linker, an HSV UL27 polypeptide or antigenic fragment thereof, a linker, an HSV UL21 polypeptide or antigenic fragment thereof, a linker, an HSV UL19 polypeptide or antigenic fragment thereof, a linker, an HSV UL1 polypeptide or antigenic fragment thereof, a linker and MITD.

28. The polyribonucleotide according to claim 26, wherein the polypeptide comprises or consists of the amino acid sequence according to SEQ ID No. 204.

29. The polyribonucleotide of any one of claims 1-28, wherein said polyribonucleotide is an isolated polyribonucleotide.

30. The polyribonucleotide of any of claims 1-28, wherein said polyribonucleotide is an engineered polyribonucleotide.

31. The polyribonucleotide of any of claims 1-28, wherein said polyribonucleotide is a codon-optimized polyribonucleotide.

32. A combination, comprising:

the first polynucleic acid according to any of claims 1 to 31, and

A second polyribonucleotide, wherein said second polyribonucleotide encodes a second polypeptide, wherein said second polypeptide comprises one or more Herpes Simplex Virus (HSV) antigens or antigenic fragments thereof, and

Wherein the first is polyribonucleotides and methods of making same the second polyribonucleotide is different.

33. A combination, comprising:

A first pharmaceutical composition comprising a first polyribonucleotide, wherein said first polyribonucleotide is a polyribonucleotide according to any of claims 1-31; and

A second pharmaceutical composition comprising a second polyribonucleotide, wherein said polyribonucleotide encodes a second polypeptide, wherein said second polypeptide comprises one or more Herpes Simplex Virus (HSV) antigens or antigenic fragments thereof,

Wherein the first is polyribonucleotides and methods of making same the second polyribonucleotide is different.

34. The combination of claim 32 or 33, wherein the one or more HSV antigens or antigenic fragments thereof of the second polypeptide comprises one or more HSV glycoproteins.

35. The combination of claim 34, wherein the one or more HSV glycoproteins comprises HSV glycoprotein B (gB), HSV glycoprotein E (gE), HSV glycoprotein G (gG), HSV glycoprotein H (gH), HSV glycoprotein I (gL), HSV glycoprotein L (gL), or a combination thereof.

36. The combination of claim 32 or 33, wherein the second polypeptide comprises a single HSV antigen.

37. The combination of claim 36, wherein the single HSV antigen is an HSV glycoprotein.

38. The combination of claim 37, wherein the HSV glycoprotein is a full length HSV glycoprotein.

39. The combination of claim 37 or 38, wherein the HSV glycoprotein is HSV gB, HSV gE, HSV gG, HSV gH, HSV gI, and HSV gL.

40. An RNA construct comprising, in 5 'to 3' order:

(i)5'UTR；

(ii) The polyribonucleotide of any one of claims 1-31;

(iv) 3' UTR; and

(V) Poly a tail sequence.

41. The RNA construct of claim 40, further comprising a 5' cap.

42. A composition comprising:

(i) One or more polynucleic acids according to any one of claims 1 to 31, and

(Ii) Lipid nanoparticles, multimeric complexes (PLX), lipidated multimeric complexes (LPLX) or liposomes,

Wherein the one or more polyribonucleotides are wholly or partially encapsulated within the lipid nanoparticle, multimeric complex (PLX), lipidated multimeric complex (LPLX), or liposome.

43. A pharmaceutical composition comprising one or more polyribonucleotides according to any one of claims 1-31 and at least one pharmaceutically acceptable excipient.

44. A method comprising administering to a subject the polyribonucleotide according to any of claims 1-31.

45. A method comprising administering the RNA construct of claim 40 or 41 to a subject.

46. A method comprising administering to a subject a pharmaceutical composition according to claim 43.

47. A method comprising administering to a subject a combination according to any one of claims 32 to 39.