CN120025459A

CN120025459A - Human papillomavirus mRNA vaccine

Info

Publication number: CN120025459A
Application number: CN202311558440.4A
Authority: CN
Inventors: 张伟; 李航文
Original assignee: Siwei Shanghai Biotechnology Co ltd
Current assignee: Siwei Shanghai Biotechnology Co ltd
Priority date: 2023-11-21
Filing date: 2023-11-21
Publication date: 2025-05-23

Abstract

The invention relates to the field of biological medicine, in particular to mRNA (messenger ribonucleic acid), composition, vaccine preparation and application thereof, which are designed aiming at human papilloma virus type 16 and type 18. The mRNA, the composition and the vaccine preparation can induce strong and long-acting cellular immune response, induce protective T cell memory, resist re-attack and relapse of viruses, and slow down the growth rate of human papilloma virus positive tumors or mediate the regression of human papilloma virus positive tumors. The mRNA, the composition and the vaccine preparation of the invention provide a new technical means for treating human papillomavirus 16 type infection, human papillomavirus 16 type positive cancer or precancerous lesion, human papillomavirus 18 type infection, human papillomavirus 18 type positive cancer or precancerous lesion.

Description

Human papillomavirus mRNA vaccine

Technical Field

The invention relates to the field of biological medicine, in particular to an mRNA vaccine for treating human papillomavirus positive tumor or precancerous lesions. More specifically, the invention relates to mRNA vaccines for treating human papillomavirus type 16 and/or human papillomavirus type 18 positive tumors or precancerous lesions.

Background

Human Papillomaviruses (HPV), which belong to the papillomaviridae family, are small, non-enveloped, double-stranded circular DNA viruses, of which more than 200 types have been identified to date. Among them, HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82 are considered to be 15 high-risk HPV types, and persistent infection of these high-risk HPV types may lead to HPV-related cancers such as cervical cancer, head and Neck Squamous Cell Carcinoma (HNSCC), anogenital cancer, and penile cancer.

Currently, commercially available vaccines against HPV viruses are prophylactic vaccines, such as the greek (Cervarix) of the gram company, merck company, jia da 4 (Gardasil 4) and jia da 9 (Gardasil), but no HPV therapeutic vaccine has been approved for use in humans. While prophylactic HPV vaccines can prevent most HPV-associated cancers, they are not able to treat existing HPV infections. Moreover, insufficient vaccination coverage greatly affects the prevention of global cancer morbidity. Thus, there is a great need in the art for effective new therapies, including therapeutic HPV vaccines, to help treat those already suffering from HPV-related malignancies.

Although 15 high-risk types of HPV are known, of which HPV type 16 is the most common and most oncogenic type, about 60% of cervical cancer patients are positive for HPV type 16. At present, most types of developed HPV therapeutic vaccines are HPV16 type, and some types of developed HPV therapeutic vaccines are 18 type, wherein vaccine types comprise subunit vaccines, nucleic acid vaccines (DNA vaccines and RNA vaccines), live vector vaccines (bacterial vector vaccines and viral vector vaccines) and cell vaccines, but different therapeutic HPV vaccines have advantages and disadvantages, and the clinical effect of the vaccine is particularly excellent, so that breakthrough is not yet heard, and the field still needs to be truly strong, long-acting and high-safety therapeutic vaccines aiming at high-risk types such as HPV 16. The application designs a novel therapeutic HPV16 mRNA vaccine and evaluates the preclinical tumor inhibition effect, which provides a novel technical means for treating HPV16 positive cancers and precancerous lesions and extends the vaccine design to other high-risk types such as HPV 18 on the basis of the novel therapeutic HPV16 mRNA vaccine.

Disclosure of Invention

In a first aspect, the invention provides a polypeptide or a combination of polypeptides, wherein the polypeptide or the combination of polypeptides comprises a signal peptide sequence at the N-terminus of the polypeptide and an MHC-I transmembrane-intracellular domain sequence at the C-terminus of the polypeptide and the polypeptide or the combination of polypeptides comprises the amino acid sequence of human papillomavirus HPV 16E 6 protein or an immunogenic fragment thereof and/or the amino acid sequence of human papillomavirus HPV 16E 7 protein or an immunogenic fragment thereof, or the polypeptide or the combination of polypeptides comprises the amino acid sequence of human papillomavirus HPV 18E 6 protein or an immunogenic fragment thereof and/or the amino acid sequence of human papillomavirus HPV 18E 7 protein or an immunogenic fragment thereof, wherein the human papillomavirus HPV 16E 6 protein comprises the amino acid sequence shown in SEQ ID NO. 105 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 105, the human HPV 16E 7 protein comprises the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 6, and/or an amino acid sequence shown in SEQ ID NO. 95 to the human papillomavirus HPV 18E 7 protein or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 6.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 106 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 106, and/or the amino acid sequence shown in SEQ ID NO. 5 or 78 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 5 or 78, or the amino acid sequence shown in SEQ ID NO. 132 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 132, and/or the amino acid sequence shown in SEQ ID NO. 80 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 80.

In a second aspect, the invention provides a nucleic acid or nucleic acid combination comprising a polynucleotide encoding a human papillomavirus HPV 16E 6 protein or immunogenic fragment thereof of the invention, wherein the polynucleotide is RNA, the polynucleotide comprises the nucleotide sequence shown as one of SEQ ID NOS 124, 125 and 126 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown as one of SEQ ID NOS 124, 125 and 126, and/or the nucleic acid or nucleic acid combination comprises a polynucleotide encoding a human papillomavirus HPV 16E 7 protein or immunogenic fragment thereof of the invention, wherein the polynucleotide is RNA, the polynucleotide comprises the nucleotide sequence shown as one of SEQ ID NOS 17, 20, 63, 64, 65, 66, 67, 68 and 69 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown as one of SEQ ID NOS 17, 20, 63, 64, 65, 66, 67, 68 and 69, or the nucleic acid or nucleic acid combination comprises a polynucleotide encoding human papillomavirus 18E 6 protein or immunogenic fragment thereof of the invention, wherein the polynucleotide is RNA, the polynucleotide comprises the nucleotide sequence shown as one of SEQ ID NOS 150, 152, 151, 65, 67, 68 and 69, or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown as one of SEQ ID NOS 17, 63, 64, 65, 68, 67, 68 and 69, or a nucleotide sequence of the nucleotide sequence shown as one of SEQ ID NOS 100 or 98, or a nucleotide sequence of the nucleotide sequence shown as one of nucleotide sequence of SEQ ID 46 and 98 99 and 100 has a nucleotide sequence having at least 85% identity.

In a third aspect, the invention provides a nucleic acid or nucleic acid combination comprising a polynucleotide encoding a human papillomavirus HPV 16E 6 protein or immunogenic fragment thereof of the invention, wherein the polynucleotide is DNA comprising a nucleotide sequence shown as one of SEQ ID NOS: 128, 129 and 130 or a nucleotide sequence having at least 85% identity to a nucleotide sequence shown as one of SEQ ID NOS: 128, 129 and 130, and/or a nucleic acid or nucleic acid combination comprising a polynucleotide encoding a human papillomavirus HPV 16E 7 protein or immunogenic fragment thereof of the invention, wherein the polynucleotide is DNA comprising a nucleotide sequence shown as one of SEQ ID NOS: 18, 42, 70, 71, 72, 73, 74, 75 and 76 or a nucleotide sequence having at least 85% identity to a nucleotide sequence shown as one of SEQ ID NOS: 18, 42, 70, 71, 72, 73, 74, 75 and 76, or a nucleic acid combination comprising a polynucleotide encoding a human papillomavirus 18E 6 protein or immunogenic fragment thereof of the invention, wherein the polynucleotide is DNA, wherein the polynucleotide comprises a nucleotide sequence shown as one of SEQ ID NOS: 103, 155 and 155, and 156, or a nucleotide sequence shown as one of SEQ ID NOS: 102, or a nucleotide sequence having at least 85% identity to a nucleotide sequence shown as one of SEQ ID NOS: 18, 42, 70, 71, 72, 73, 74 and 75 and 76, or a polynucleotide comprising a nucleotide sequence shown as one of SEQ ID NOS.102 and 102 or an immunogenic fragment thereof 103 and 104 has a nucleotide sequence having at least 85% identity.

In a fourth aspect, the invention provides a nucleic acid or combination of nucleic acids comprising a polynucleotide encoding a polypeptide or combination of polypeptides of the invention.

In one embodiment, the polynucleotide is RNA, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS 107, 108, 109 and 110 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS 107, 108, 109 and 110, and/or the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS 1, 21, 22, 23, 24, 25, 26, 27, 28 and 29 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS 1, 21, 22, 23, 24, 25, 26, 27, 28 and 29, or the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS 133, 134, 135 and 136 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS 133, 134, 135 and 136, and/or the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS 81, 82, 83 and 84 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS 81, 82, 83 and 84.

In one embodiment, the polynucleotide is RNA, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS: 111, 112, 113 and 114 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS: 111, 112, 113 and 114, and/or the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS: 2, 32, 33, 34, 35, 36, 37, 38, 39 and 40 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS: 2, 32, 33, 34, 35, 36, 37, 38, 39 and 40, or the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS: 137, 138, 139 and 140 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS: 137, 138, 139 and 140, and/or the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS: 85, 86, 87 and 88 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS: 85, 86, 87 and 88.

In one embodiment, the polynucleotide is DNA, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 115, 116, 117 and 118 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown in SEQ ID NO. 115, 116, 117 and 118, and/or the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO. 3, 43, 44, 45, 46, 47, 48, 49, 50 and 51 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NO. 3, 43, 44, 45, 46, 47, 48, 49, 50 and 51, or the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO. 141, 142, 143 and 144 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NO. 141, 142, 143 and 144, and/or the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO. 89, 90, 91 and 92 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NO. 89, 90, 91 and 92.

In one embodiment, the polynucleotide is DNA, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS 119, 120, 121 and 122 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS 119, 120, 121 and 122, and/or the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS 4, 54, 55, 56, 57, 58, 59, 60, 61 and 62 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS 4, 54, 55, 56, 57, 58, 59, 60, 61 and 62, or the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS 145, 146, 147 and 148 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS 145, 146, 147 and 148, and/or the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS 93, 94, 95 and 96 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS 93, 94, 95 and 96.

In a fifth aspect, the invention provides a composition comprising a polypeptide or combination of polypeptides of the invention.

In a sixth aspect, the invention provides a composition comprising a nucleic acid or combination of nucleic acids of the invention. In some embodiments, the composition comprises a lipid encapsulating a nucleic acid or a combination of nucleic acids. In some embodiments, the composition comprises a lipid nanoparticle or a lipid-multimeric complex. In some embodiments, the lipid encapsulating the polynucleotide comprises a cationic lipid, a phospholipid, a steroid, and a polyethylene glycol modified lipid, and optionally the composition further comprises a cationic polymer, wherein the cationic polymer associates with the nucleic acid or nucleic acid combination as a complex, and is co-encapsulated in the lipid to form a lipopolysaccharide complex. In some preferred embodiments, the cationic polymer is protamine. In some embodiments, the cationic lipid comprises a lipid compound of formula (I), or a pharmaceutically acceptable salt thereof, as defined herein. In a preferred embodiment, the cationic lipid is SW-II-140-2.

In a preferred embodiment, the composition comprises 40 mole% SW-II-140-2, 15 mole% DOPE, 43.5 mole% cholesterol and 1.5 mole% DMG-PEG.

In a seventh aspect, the invention provides a vaccine formulation comprising a polypeptide or combination of polypeptides of the invention or a composition of the invention.

In an eighth aspect, the invention provides a vaccine formulation comprising a nucleic acid or combination of nucleic acids of the invention or a composition of the invention.

In a ninth aspect, the invention provides an expression vector comprising a nucleic acid or combination of nucleic acids of the invention.

In a tenth aspect, the invention provides a host cell comprising a nucleic acid or combination of nucleic acids of the invention or an expression vector of the invention.

In an eleventh aspect, the invention provides a kit comprising a polypeptide or combination of polypeptides of the invention, a nucleic acid or combination of nucleic acids of the invention, a composition of the invention or a vaccine formulation of the invention, and one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors and tumor antigen targeting drugs. In a preferred embodiment, the therapeutic agent is an anti-PD-L1 antibody or antigen-binding fragment thereof.

In a twelfth aspect, the invention provides the use of a polypeptide or combination of polypeptides of the invention, a nucleic acid or combination of nucleic acids of the invention, a composition of the invention, a vaccine formulation of the invention or a kit of the invention for the manufacture of a medicament for treating a human papillomavirus type 16 infection, a human papillomavirus type 16 positive cancer or a precancerous condition, a human papillomavirus type 18 infection, a human papillomavirus type 18 positive cancer or a precancerous condition in a subject.

Drawings

FIG. 1 shows the results of in vitro expression assays for mRNA # 8, 10#, 18#, 19#, 22#, 24#, 29#, 31#, 33#, 47#, 50# and 51# prepared.

Figures 2A-2D show that SW0128LPP vaccine formulations induce a cellular immune response. FIG. 2A shows the C57BL/6NTac mice immunization program, FIG. 2B and FIG. 2C show the results of cellular immune responses detected by ELISPot after administration of SW0128LPP vaccine formulations comprising 3 μg, 10 μg, 30 μg gmRNA, respectively, to each group of mice on day 0, mice spleens on day 6, and FIG. 2D shows the results of cellular immune responses detected by ELISPot after administration of SW0128LPP vaccine formulations comprising 3 μg, 10 μg, 30 μ gmRNA, respectively, to each group of mice on day 0 and day 7, mice spleens on day 13.

Figures 3A-3B show that low doses of SW0128 LPP vaccine formulation induce a cellular immune response. FIG. 3A shows the C57BL/6NTac mouse immunization program and FIG. 3B shows the results of the cellular immune response induced by the SW0128 LPP vaccine formulation under different immunization programs tested by ELISPot, where P is positive control well and N is negative control well.

FIGS. 4A-4C show SW0128 induces regression of HPV-16 positive TC-1 tumors in C57BL/6NTac mice. FIG. 4A shows the C57BL/6NTac mouse immunization program, FIG. 4B shows the effect of SW0128 LPP vaccine preparation immunization on tumor volume under different immunization programs, and FIG. 4C shows the effect of SW0128 LPP vaccine preparation immunization on body weight under different immunization programs.

FIG. 5 shows that SW0128 LPP vaccine formulation induces a reduction in tumor growth rate in C57BL/6J mice.

Figures 6A-6C show that SW0128 LPP vaccine formulations promote proliferation of memory T cells. FIG. 6A shows the results of flow assays of E7 _49-57dextramer⁺CD8⁺ T cells from C57BL/6NTac mice with complete tumor regression after SW0128 LPP vaccine formulation and C57BL/6NTac mice without SW0128 LPP vaccine formulation after the initial TC-1 tumor cell challenge, FIG. 6B shows the results of ELISPot assays of C57BL/6NTac mice with complete tumor regression after SW0128 LPP vaccine formulation and C57BL/6NTac mice without SW0128 LPP vaccine formulation after the initial TC-1 tumor cell challenge after the subcutaneous inoculation (D59).

Figures 7A-7D show the tumor inhibiting effect of co-administration of SW0128LPP vaccine formulation and anti-PD-L1 antibody. FIG. 7A shows the C57BL/6J mouse dosing regimen, FIG. 7B shows the tumor volume detection results, FIG. 7C shows the survival rate of the C57BL/6J mice, and FIG. 7D shows the analysis results of tumor regression.

FIG. 8 shows the results of in vitro expression assays of prepared mRNA-LPP preparations 1801, 1802, 1803 and 1804.

Figures 9A-9C show that LPP vaccine formulations 1801, 1802, 1803, and 1804 induce a cellular immune response. FIG. 9A shows the C57BL/6J mouse immunization program, and FIGS. 9B and 9C show the results of cellular immune responses detected by ELISPot after administration of LPP vaccine formulations 1801, 1802, 1803 and 1804 containing 10. Mu.g mRNA, respectively, to each group of mice on day 0, taking the spleens of the mice on day 7.

Detailed description of the preferred embodiments

General definitions and terms

All patents, patent applications, scientific publications, manufacturer's instructions and guidelines, and the like, cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure.

Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology related terms as used herein are terms that are widely used in the corresponding field (see, e.g., ,MolecularCloning：A Laboratory Manual,2n^dEdition,J.Sambrook et al.eds.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor 1989)., while, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

As used herein, the terms "comprises," "comprising," "includes," "including," "having" and "containing" are open-ended, meaning the inclusion of the stated elements, steps or components, but not the exclusion of other non-recited elements, steps or components. The expression "consisting of" does not include any element not specified steps or components. The expression "consisting essentially of means that the scope is limited to the specified elements, steps or components, plus any optional elements, steps or components that do not significantly affect the basic and novel properties of the claimed subject matter. It is to be understood that the expression "consisting essentially of the expression" comprising "and" consisting of the expression "comprising" are encompassed within the meaning of the expression "comprising".

As used herein, the term "and/or" in connection with a plurality of recited elements should be understood to include both individual and combined options. In other words, "and/or" includes "and" as well as "or". For example, a and/or B includes A, B and a+b. A. B and/or C include A, B, C and any combination thereof, such as A+ B, A + C, B +C and A+B+C. Further elements defined by "and/or" are to be understood in a similar manner and include any one of, and any combination of, these.

As used herein, the singular forms "a," "an," or "the" include plural referents unless the context clearly dictates otherwise. The term "one or more" or "at least one" encompasses 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. Unless specifically indicated to the contrary, the numerical values or ranges set forth herein are modified by "about" to mean the enumerated or claimed values or ranges are + -20%, + -10%, + -5%, or + -3%.

All methods described herein can be performed in any suitable order unless otherwise indicated.

As used herein, "nucleic acid combination" may refer to a combination or collection comprising more than one nucleic acid. Such nucleic acids may be present separately, for example in different parts of different compositions or kits or in different vectors, or may be present simultaneously in the same part of the same composition or kit or in the same vector. The nucleic acids in the nucleic acid combination may be identical or different, may each encode the same polypeptide (portion) or may encode different polypeptides (portions).

As used herein, "polypeptide combination" may refer to a combination or collection comprising more than one polypeptide. Such polypeptides may be present separately, e.g., in different compositions or in different parts of a kit, or may be present simultaneously in the same composition or in the same part of a kit. The polypeptides in the polypeptide combination may be identical or different from each other, may each comprise the same amino acid sequence (part) or may comprise different amino acid sequences (parts).

As used herein, the term "polypeptide" refers to a polymer comprising two or more amino acids covalently linked by peptide bonds. A "protein" may comprise one or more polypeptides, wherein the polypeptides interact with each other by covalent or non-covalent means. Unless otherwise indicated, "polypeptide" and "protein" may be used interchangeably.

As used herein, the term "wild-type" means that the sequence is naturally occurring and not artificially modified, including naturally occurring mutants.

As used herein, the term "% identity" with respect to sequences refers to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences to be compared. The difference between the two sequences may be distributed over a local area (section) or the entire length of the sequences to be compared. The identity between two sequences is typically determined after optimal alignment of the segments or "comparison windows". The optimal alignment may be performed manually or by means of algorithms known in the art, including but not limited to the local homology algorithms described by SMITH AND WATERMAN,1981,ADS APP.MATH.2,482 and NEDDLEMAN AND Wunsch,1970, j.mol. Biol.48,443, the similarity search method described by Pearson AND LIPMAN,1988,Proc.Natl Acad.Sci.USA88,2444, or using a computer program, such as GAP, BESTFIT, FASTA, BLAST P, BLAST N and tfast a in Wisconsin Genetics Software Package, genetics Computer Group,575Science Drive,Madison,Wis. For example, the percent identity of two sequences may be determined using the BLASTN or BLASTP algorithm commonly available at the National Center for Biotechnology Information (NCBI) website.

The% identity is obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100. In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the regions give a degree of identity. In some embodiments, the degree of identity is given to the entire length of the reference sequence. Alignment for determining sequence identity can be performed using tools known in the art, preferably using optimal sequence alignment, e.g., using Align, using standard settings, preferably EMBOSS:: needle, matrix: blosum62, gap Open 10.0, gap Extend0.5.

Herein, "nucleotide" includes deoxyribonucleotides and ribonucleotides and derivatives thereof. As used herein, a "ribonucleotide" is a constituent material of ribonucleic acid (RNA) and consists of one molecule of base, one molecule of pentose, and one molecule of phosphate, which refers to a nucleotide having a hydroxyl group at the 2' -position of the β -D-ribofuranose (β -D-ribofuranosyl) group. The "deoxyribonucleotide" is a constituent substance of deoxyribonucleic acid (DNA), and also comprises one molecule of base, one molecule of pentose and one molecule of phosphoric acid, and refers to a nucleotide in which the hydroxyl group at the 2' -position of the beta-D-ribofuranose (beta-D-ribofuranosyl) group is replaced by hydrogen, and is a main chemical component of a chromosome. "nucleotide" is generally referred to by a single letter representing a base therein, "A (a)" means deoxyadenylate or adenylate containing adenine, "C (C)" means deoxycytidylate or cytidylate containing cytosine, "G (G)" means deoxyguanylate or guanylate containing guanine, "U (U)" means uridylate containing uracil, and "T (T)" means deoxythymidylate containing thymine.

As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA). "Polynucleotide sequence", "nucleic acid sequence" and "nucleotide sequence" are used interchangeably to refer to the ordering of nucleotides in a polynucleotide. It will be appreciated by those skilled in the art that the coding strand (sense strand) of DNA can be considered to have the same nucleotide sequence as the RNA it encodes, with deoxythymidylate in the sequence of the coding strand of DNA corresponding to uridylate in the sequence of the RNA it encodes.

As used herein, the term "expression" includes transcription and/or translation of a nucleotide sequence. Thus, expression may involve the production of transcripts and/or polypeptides. The term "transcription" relates to the process of transcribing the genetic code in a DNA sequence into RNA (transcript). The term "in vitro transcription" refers to the synthesis of RNA, in particular mRNA, in vitro in a cell-free system (e.g. in a suitable cell extract). Vectors that can be used to produce transcripts are also referred to as "transcription vectors" which contain the regulatory sequences required for transcription. The term "transcription" encompasses "in vitro transcription".

As used herein, "encoding" refers to the inherent properties of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, that can be used as a template to synthesize polymers and macromolecules in other biological processes, provided that there is a defined nucleotide sequence or a defined amino acid sequence. Thus, a gene encodes a protein, meaning that mRNA of the gene is transcribed and translated to produce the protein in a cell or other biological system.

As used herein, the term "untranslated region (UTR)" generally refers to a region in RNA (e.g., mRNA) that is not translated into an amino acid sequence (non-coding region), or a corresponding region in DNA.

As used herein, the term "coding region" refers to a portion of DNA that is capable of transcribing messenger RNA, which is capable of synthesizing a corresponding protein, or a corresponding region in RNA.

As used herein, the term "host cell" refers to a cell that is used to receive, hold, replicate, express a polynucleotide (e.g., a nucleic acid or combination of nucleic acids herein) or a vector. In some embodiments, the host cell may be a cell in which the polypeptide or combination of polypeptides of the invention is expressed.

As used herein, "antigen" refers to a molecule that upon entry into the body can elicit an immune response that is acquired by the body and that can be directed to the production of antibodies, or to specific immunogenically active cells, or both. It will be appreciated by those skilled in the art that any macromolecule, including almost all proteins or peptide fragments, may act as an antigen. Still further, the antigen may be from recombinant or genomic DNA or RNA. It will be appreciated by those skilled in the art that any of the DNA or RNA herein, the nucleotide sequence or portions thereof, may encode a protein capable of eliciting an acquired immunity in the body. Still further, it will be understood by those skilled in the art that an antigen need not solely encode the full length nucleotide sequence of only one gene. It will be apparent that the invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences form different mixtures to induce the onset of a response. Still further, it will be appreciated by those skilled in the art that the antigen need not be encoded entirely by one gene. Obviously, the antigen may be synthetically produced or may be derived from a biological sample. Biological samples include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

As used herein, "antibody" refers to a protein that has a protective effect by the body as a result of stimulation by an antigen. It is an immunoglobulin produced by B lymphocytes. The monomer of the antibody is a Y-shaped molecule and consists of 4 polypeptide chains. The chain comprises two identical heavy chains and two identical light chains, which are connected together by disulfide bonds. Each heavy chain is 50kDa, each light chain is 25kDa, and disulfide bonds exist between the light and heavy chains. It is unique in high affinity and specificity for binding partners.

As used herein, "vaccine" refers to a composition comprising an active ingredient (e.g., a polypeptide or polynucleotide of the invention) that is capable of eliciting an immune response in an vaccinated subject upon vaccination. As used herein, "mRNA cancer vaccine" provides a unique therapeutic alternative to peptide-based vaccines or DNA vaccines. When an mRNA cancer vaccine is delivered to a cell, the mRNA will be processed within the cell into a polypeptide, which is further processed into an immunosensitive fragment capable of stimulating an immune response against the tumor. The cancer vaccines described herein include at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one cancer antigenic polypeptide or immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response to cancer).

As used herein, an "aliphatic" group is a non-aromatic group in which carbon atoms are linked in a chain, and may be saturated or unsaturated.

As used herein, the term "alkyl" refers to an optionally substituted straight or branched chain saturated hydrocarbon comprising one or more carbon atoms. The term "C ₁-C₁₂ alkyl" or "C _1-12 alkyl" refers to optionally substituted straight or branched chain saturated hydrocarbons comprising 1 to 12 carbon atoms. As used herein, the term "alkoxy" refers to an alkyl group as described herein that is attached to the remainder of the molecule through an oxygen atom. The term "alkylene" refers to a divalent group formed by the corresponding alkyl group losing one hydrogen atom. The term "C ₁-C₁₂ alkylene" or "C _1-12 alkylene" refers to an optionally substituted straight or branched chain alkylene group comprising 1 to 12 carbon atoms.

As used herein, the term "alkenyl" refers to an optionally substituted straight or branched chain hydrocarbon comprising two or more carbon atoms and at least one double bond. The term "C ₂-C₁₂ alkenyl" or "C _2-12 alkenyl" refers to optionally substituted straight or branched chain hydrocarbons comprising 2 to 12 carbon atoms and at least one carbon-carbon double bond. Alkenyl groups may include one, two, three, four or more carbon-carbon double bonds.

As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine.

As used herein, the term "carbocyclic" refers to a monocyclic or polycyclic non-aromatic system comprising one or more rings of carbon atoms. The term "C _3-8 carbocycle" means a carbocycle comprising 3 to 8 carbon atoms. Carbocycles may include one or more carbon-carbon double or triple bonds. Examples of carbocycles include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, and the like. As used herein, when a carbocycle is saturated (i.e., free of unsaturation), the corresponding cycloalkyl group may also be referred to. Unless specifically stated otherwise, carbocycles as described herein refer to unsubstituted and substituted, i.e., optionally substituted carbocycles.

As used herein, the term "heterocycle" refers to a single or multiple ring system comprising one or more rings and including at least one heteroatom. The heteroatom may be, for example, a nitrogen, oxygen, phosphorus or sulfur atom. The heterocycle may include one or more double or triple bonds and may be non-aromatic. Examples of heterocycles include, but are not limited to, imidazolidinyl, oxazolidinyl, thiazolidinyl, pyrazolidinyl, isoxazolidinyl, isothiazolidinyl, morpholinyl, pyrrolidinyl, tetrahydrofuranyl, and piperidinyl. The heterocyclic ring may comprise, for example, 3-10 atoms (other than hydrogen), i.e., a 3-10 membered heterocyclic ring (e.g., 3,4, 5, 6, 7, 8, 9, or 10 membered), wherein one or more of the atoms is a heteroatom (e.g., N, O, S or P). When the heterocycle is saturated (i.e., does not contain an unsaturated bond), the corresponding heterocycloalkyl group may also be referred to. Unless specifically stated otherwise, a heterocycle as described herein refers to both unsubstituted and substituted heterocyclic groups, i.e., an optionally substituted heterocycle.

As used herein, the term "aryl" refers to an all-carbon monocyclic or fused-polycyclic aromatic ring radical having a conjugated pi-electron system. For example, a C ₆-C₁₀ alkylaryl group can have from 6 to 10 carbon atoms, such as 6, 7, 8, 9, 10 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and the like.

As used herein, the term "heteroaryl" refers to a monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, S, the remaining ring atoms being C, and having at least one aromatic ring. Heteroaryl groups may have 5 to 10 ring atoms (5 to 10 membered heteroaryl groups) including 5, 6,7, 8,9 or 10 membered, especially 5 or 6 membered heteroaryl. Examples of heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazolyl, triazinyl, benzofuranyl, benzothienyl, indolyl, isoindolyl, and the like.

As used herein, the term "interrupted by one or more groups" means that the one or more groups are present on the carbon chain and the remainder of the carbon chain is attached to both ends of the one or more groups.

Unless specifically stated otherwise, a group described herein (e.g., any of R ₁-R₇, such as alkyl, alkylene, alkenyl, aryl, amino, etc.) may be optionally substituted. The optional substituents may be selected from, but are not limited to, halogen atoms (e.g., chloro, bromo, fluoro, OR iodo), carboxylic acids (e.g., -C (O) OH), alcohols (e.g., hydroxy, -OH), esters (e.g., -C (O) OR OR-OC (O) R), aldehydes (e.g., -C (O) H), carbonyl groups (e.g., -C (O) R, OR represented by C=O), acyl halides (e.g., -C (O) X, where X is a halogen selected from bromo, fluoro, chloro, and iodo), carbonate groups (e.g., -OC (O) OR), alkoxy groups (e.g., -OR), acetals (e.g., -C (OR) ₂ R ', where each OR is the same OR different alkoxy group and R' "is alkyl OR alkenyl), acyl halides (e.g., -C (O) X, wherein X is a halogen selected from bromo, fluoro, chloro, and iodo), Phosphate (e.g., P (O) ₄ ^3-), thiol (e.g., -SH), sulfoxide (e.g., -S (O) R), sulfinic acid (e.g., -S (O) OH), sulfonic acid (e.g., -S (O) ₂ OH), thioaldehyde (e.g., -C (S) H), thiol (e.g., thiol) and thiol (e.g., thiol and thiol), Sulfate (e.g., S (O) ₄ ^2-), sulfonyl (e.g., -S (O) ₂ -), amide (e.g., -C (O) NR ₂ or-N (R) C (O) R), Azido (e.g., -N ₃), nitro (e.g., -NO ₂), cyano (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC (O) R), amino (e.g., -NR ₂, NRH or-NH ₂), amino, carbamoyl (e.g., -OC (O) NR ₂, -OC (O) NRH or-OC (O) NH ₂), sulfonamide (e.g., -S(O)₂NR₂、-S(O)₂NRH、-S(O)₂NH₂、-N(R)S(O)₂R、-N(H)S(O)₂R、-N(R)S(O)₂H、-N(H)S(O)₂H)、C₁-C₁₂ alkyl, C ₂-C₁₂ alkenyl), C ₆-C₁₀ aryl, 5-10 membered heteroaryl, or 3-10 membered heterocycle. In any of the foregoing, each R independently can be a substituent as defined herein, such as alkyl, alkoxy, alkylene, halo, carbocycle, heterocycle, aryl, heteroaryl, alkenyl. In some embodiments, the substituents themselves may be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein. For example, an alkyl group may be further substituted with one, two, three, four, five, or six substituents as described herein.

As used herein, the term "compound" is intended to include isotopic compounds of the depicted structure. "isotope" refers to an atom having the same atomic number but different mass numbers due to the number of neutrons in the core, such as a deuterium isotope. Isotopes of hydrogen include, for example, tritium and deuterium. In addition, the compounds, salts or complexes of the invention may be prepared in combination with solvents or water molecules to form solvates and hydrates by conventional methods.

The term "optionally" or "optionally" (e.g., optionally substituted) means that the subsequently described event may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and the description includes both substituted and unsubstituted alkyl radicals.

It is understood that when chemical groups are written in a particular order, the reverse order is also contemplated unless otherwise indicated. For example, in the general formula- (R) _i-(M1)_k-(R)_m - (i.e., - (R) _i-C(O)-NH-(R)_m -) where M ₁ is defined as-C (O) NH-, compounds where M ₁ is-NHC (O) -, i.e., - (R) _i-NHC(O)-(R)_m -, are also contemplated unless otherwise indicated.

As used herein, a "lipid component" is a component of a composition that includes one or more lipids. For example, the lipid component may include one or more cationic lipids, pegylated lipids, structural lipids, or helper lipids.

The phrase "pharmaceutically acceptable" is used herein to refer to compounds, salts, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is altered by converting the existing acid or base moiety to its salt form (e.g., by reacting a free basic group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, alkali metal or organic salts of acidic residues such as carboxylic acids, and the like. Representative acid addition salts include, but are not limited to, acetates, adipates, alginates, ascorbates, aspartate, benzenesulfonates, benzoates, bisulphates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptonates, glycerophosphate, hemisulfates, heptanoates, caprates, hydrobromides, hydrochlorides, hydroiodides, 2-hydroxy-ethane sulfonates, lactobionic, lactates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalene sulfonates, nicotinates, nitrates, oleates, oxalates, palmates, pamonates, pectates, persulfates, 3-phenylpropionates, phosphates, bitrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, toluenesulfonates, undecanoates, valerates, and the like. Representative alkali or alkaline earth metal salts include, but are not limited to, sodium, lithium, potassium, calcium, magnesium salts, and the like, and non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to, ammonium, tetramethyl ammonium, tetraethyl ammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts of the invention include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the invention can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. In general, these salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two, with non-aqueous media such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile being generally preferred.

Signal peptide sequence and transmembrane-intracellular domain sequence of MHC-I

As used herein, the term "signal peptide (SIGNAL PEPTIDE, SP) sequence" refers to a signal peptide fragment of MHC-I that aids in the distribution of an antigen of interest to cellular vesicle structures. As used herein, the term "transmembrane-intracellular segment domain (MHC CLASS I TRAFFICKING domain, MITD) sequence of MHC-I" refers to the amino acid sequence of the transmembrane and cytoplasmic domains of MHC-I. It has been reported that the addition of SP sequences to the N-terminus of antigen and MITD sequences to the C-terminus helps to improve presentation of MHC class I and class II epitopes in human and mouse Dendritic Cells (DCs), enhancing antigen presentation efficiency (see, e.g. Kreiter S,et al.,Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking signals.J Immunol.2008Jan 1;180(1):309-18.).

As used herein, an "epitope (also referred to as an antigenic determinant)" is a portion of an antigen that is recognized by the immune system (particularly by antibodies, B cells or T cells) in a suitable context. Epitopes include B cell epitopes and T cell epitopes. B cell epitopes are peptide sequences necessary for recognition by B cells producing specific antibodies. B cell epitopes refer to specific regions of an antigen that are recognized by an antibody. The portion of the antibody that binds to this epitope is referred to as the paratope. Epitopes can be conformational epitopes or linear epitopes based on structure and interaction with paratopes. A linear or continuous epitope is defined by the primary amino acid sequence of a particular region of a protein. The sequences that interact with the antibody are located successively next to each other on the protein, and the epitope can be generally mimicked by a single peptide. Conformational epitopes are epitopes defined by the conformational structure of the native protein. These epitopes can be contiguous or discontinuous, i.e., the components of the epitope can be located on different parts of the protein that are in proximity to each other in the folded native protein structure.

The term "domain" or "region" relates to a specific part of an amino acid sequence, which may preferably be linked to a specific function or structure. The polypeptides of MHC-II molecules have two domains, α1, α2 and β1, β2, respectively a transmembrane region and a cytoplasmic region. The alpha chain of an MHC-I molecule has three domains, α1, α2 and α3, a transmembrane region and a cytoplasmic region. The term "transmembrane region" relates to a portion of a protein which substantially occupies the portion present in the cell membrane and is preferably used to anchor the protein in the membrane.

The term "Major Histocompatibility Complex (MHC)" relates to the gene complex that occurs in all vertebrates. MHC proteins or molecules play a role in the signaling between lymphocytes and antigen presenting cells in a normal immune response. Human MHC, also known as HLA, a human leukocyte antigen, is located on chromosome 6, including MHC-I and MHC-II.

The term "MHC-I" or "MHC class I" refers to a major histocompatibility complex class I protein or gene. Within the human MHC-I region are HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, CD1a, CD1b and CD1c sub-regions. MHC class I proteins are present on almost all cell surfaces, including most tumor cells. MHC-I proteins are loaded with antigens that are typically derived from endogenous proteins or pathogens present within the cell and then presented to Cytotoxic T Lymphocytes (CTLs). T cell receptors are capable of recognizing and binding peptides complexed with MHC class I molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor, capable of binding to a specific MHC/peptide complex. MHC class I molecules mediate primarily the presentation of endogenous antigens.

The alpha chain of MHC-I is a glycoprotein with a molecular weight of about 44 kDa. It can be divided into three functional regions, an outer region, a transmembrane region and a cytoplasmic region. The outer region is divided into three domains, α1, α2 and α3. The transmembrane region spans the lipid bilayer of the plasma membrane. It consists of 23 generally hydrophobic amino acid residues arranged in an alpha helix. The cytoplasmic region, the portion facing the cytoplasm and attached to the transmembrane region, is typically 32 amino acid residues in length and is capable of interacting with elements of the cytoskeleton.

The term "MHC-II" or "MHC class II" refers to a major histocompatibility complex class II protein or gene. MHC class II proteins are mainly expressed on antigen presenting cells such as B cells, monocytes, macrophages and dendritic cells. MHC class II molecules mediate primarily the presentation of exogenous antigens, which present exogenous antigen polypeptide molecules to Th cells (helper T cells).

The exact number of amino acids in different MHC molecule domains or regions depends on the differences between mammalian species and the genetic class within the species. The skilled artisan will appreciate that function may also be maintained if a complete amino acid sequence is used that is not the domain or region of choice.

The term "MHC/peptide complex" relates to a non-covalent complex of a binding domain of an MHC class I or MHC class II molecule and an MHC class I or MHC class II binding peptide.

Human papilloma virus

As used herein, the term "human papillomavirus (Human Papillomavirus, HPV)" belongs to the papillomaviridae family, and is a small, non-enveloped, double-stranded circular DNA virus, 52-55nm in diameter, commonly infecting humans. More than 200 types have been identified to date. Among them, HPVs 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82 are considered as high-risk HPVs. The DNA virus is approximately 8kb and is structurally divided into mainly 3 regions, an early protein coding region (E region), a late protein coding region (L region) and an upstream regulatory region (i.e., non-coding region). Early proteins E1, E2, E4, E5, E6, E7 and the like encoded by the E region play an important role in the processes of virus replication, transcription, translation, cell transformation and the like, wherein the E6 and E7 proteins are the most important oncogenic proteins. Although both high-risk and low-risk HPV contain E6 and E7 proteins, the activity of E6 and E7 proteins in low-risk HPV is insufficient to trigger preneoplastic lesions and the development of cancer, whereas the activity of E6 and E7 proteins in high-risk HPV is closely related to the development of cancer, e.g., E6 protein of HPV16 inhibits apoptosis by degrading p53 and E7 protein inhibits cell cycle progression by degrading pRB (see, e.g. Ki,E.Y.,Park,J.S.Natural History of Human Papillomavirus Infection.Curr Obstet Gynecol Rep 3,123–127(2014).).

HPV type 16 is the most common and most oncogenic mucosal high risk HPV, and about 60% of cervical cancer patients are infected with HPV type 16. Currently, most of the HPV therapeutic vaccines studied are preferentially designed to target HPV type 16, followed by the second most abundant HPV type 18 in HPV-associated tumor patients, and the primary targets are their E6 or E7 proteins. As used herein, HPV 16 type E6 protein is shown as NCBI accession number AAA46939.1, HPV 16 type E7 protein is shown as NCBI accession number AAA46940.1, HPV18 type E6 protein is shown as GenBank accession number CAA28664.1, and HPV18 type E7 protein is shown as GenBank accession number CAA 28665.1.

Polypeptides or polypeptide combinations

In a general aspect, the invention provides a polypeptide or combination of polypeptides.

In some embodiments, the polypeptide or combination of polypeptides comprises the amino acid sequence of a human papillomavirus E6 protein or immunogenic fragment thereof. In some embodiments, the polypeptide or combination of polypeptides comprises the amino acid sequence of a human papillomavirus E7 protein or immunogenic fragment thereof. In some embodiments, the polypeptide or polypeptide combination comprises the amino acid sequence of a human papillomavirus E6 protein or immunogenic fragment thereof and the amino acid sequence of a human papillomavirus E7 protein or immunogenic fragment thereof.

In some embodiments, the polypeptide or combination of polypeptides comprises the amino acid sequence of a human papillomavirus HPV 16E 6 protein or immunogenic fragment thereof. In some embodiments, the polypeptide or combination of polypeptides comprises the amino acid sequence of a human papillomavirus HPV 16E 7 protein or immunogenic fragment thereof. In some embodiments, the polypeptide or polypeptide combination comprises the amino acid sequence of a human papillomavirus HPV 16E 6 protein or immunogenic fragment thereof and the amino acid sequence of a human papillomavirus HPV 16E 7 protein or immunogenic fragment thereof.

In some embodiments, the polypeptide or combination of polypeptides comprises the amino acid sequence of a human papillomavirus HPV 18E 6 protein or immunogenic fragment thereof. In some embodiments, the polypeptide or combination of polypeptides comprises the amino acid sequence of a human papillomavirus HPV 18E 7 protein or immunogenic fragment thereof. In some embodiments, the polypeptide or polypeptide combination comprises the amino acid sequence of a human papillomavirus HPV 18E 6 protein or immunogenic fragment thereof and the amino acid sequence of a human papillomavirus HPV 18E 7 protein or immunogenic fragment thereof.

As used herein, "immunogenicity" refers primarily to the ability of the body to elicit an immune response to itself or a related protein (e.g., a therapeutic protein) or to cause an immune-related event, i.e., the ability of an antigen to stimulate a particular immune cell, to activate, proliferate, differentiate the immune cell, ultimately producing immune effector antibodies and sensitized lymphocytes. An "immunogenic fragment" refers to any portion of a full-length polypeptide or polypeptide antigen that is less than full length, but retains the ability to elicit an immune response in an organism against the polypeptide or polypeptide antigen.

In some embodiments, the human papillomavirus HPV 16E 6 protein comprises the amino acid sequence shown in SEQ ID NO. 105 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO. 105. In some embodiments, the human papillomavirus HPV 16E 7 protein comprises the amino acid sequence shown in SEQ ID No. 6 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID No. 6. In some embodiments, the human papillomavirus HPV 16E 6 protein comprises the amino acid sequence shown in SEQ ID No. 105 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID No. 105, and the human papillomavirus HPV 16E 7 protein comprises the amino acid sequence shown in SEQ ID No. 6 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID No. 6.

In some embodiments, the human papillomavirus HPV 18E 6 protein comprises the amino acid sequence shown in SEQ ID NO. 131 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO. 131. In some embodiments, the human papillomavirus HPV 18E 7 protein comprises the amino acid sequence shown in SEQ ID NO:79 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO: 79. In some embodiments, the human papillomavirus HPV 18E 6 protein comprises the amino acid sequence shown in SEQ ID No. 131 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID No. 131, and the human papillomavirus HPV 18E 7 protein comprises the amino acid sequence shown in SEQ ID No. 79 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID No. 79.

In some embodiments, the N-terminus of the polypeptide or combination of polypeptides comprises a signal peptide sequence. In some embodiments, the C-terminal end of the polypeptide or polypeptide combination comprises a transmembrane-intracellular segment domain sequence of MHC-I. In some embodiments, the N-terminus of the polypeptide or combination of polypeptides comprises a signal peptide sequence and the C-terminus comprises a transmembrane-intracellular domain sequence of MHC-I.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of a human papillomavirus HPV 16E 6 protein or immunogenic fragment thereof, wherein the N-terminus of the polypeptide or combination of polypeptides comprises a signal peptide sequence and the C-terminus comprises a transmembrane-intracellular domain sequence of MHC-I, and wherein the human papillomavirus HPV 16E 6 protein comprises the amino acid sequence depicted in SEQ ID No. 105 or an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID No. 105.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of a human papillomavirus HPV 18E 6 protein or immunogenic fragment thereof, wherein the N-terminus of the polypeptide or combination of polypeptides comprises a signal peptide sequence and the C-terminus comprises a transmembrane-intracellular domain sequence of MHC-I, and wherein the human papillomavirus HPV 18E 6 protein comprises the amino acid sequence depicted in SEQ ID No. 131 or an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID No. 131.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of a human papillomavirus HPV 16E 6 protein or immunogenic fragment thereof and the amino acid sequence of a human papillomavirus HPV 16E 7 protein or immunogenic fragment thereof, wherein the N-terminus of the polypeptide or combination of polypeptides comprises a signal peptide sequence and the C-terminus comprises the transmembrane-intracellular domain sequence of MHC-I, and wherein the human papillomavirus HPV 16E 6 protein comprises the amino acid sequence shown in SEQ ID NO. 105 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 105, and the human papillomavirus HPV 16E 7 protein comprises the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 6.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of a human papillomavirus HPV 18E 6 protein or immunogenic fragment thereof and the amino acid sequence of a human papillomavirus HPV 18E 7 protein or immunogenic fragment thereof, wherein the N-terminus of the polypeptide or combination of polypeptides comprises a signal peptide sequence and the C-terminus comprises the transmembrane-intracellular domain sequence of MHC-I, and wherein the human papillomavirus HPV 18E 6 protein comprises the amino acid sequence shown in SEQ ID NO:131 or an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:131, and the human papillomavirus HPV 18E 7 protein comprises the amino acid sequence shown in SEQ ID NO:79 or an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 79.

In a preferred embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of the human papillomavirus HPV 16E 7 protein or immunogenic fragment thereof, the N-terminus of the polypeptide or combination of polypeptides comprises a signal peptide sequence, the C-terminus comprises the transmembrane-intracellular domain sequence of MHC-I, and wherein the human papillomavirus HPV 16E 7 protein comprises the amino acid sequence depicted in SEQ ID No. 6 or an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID No. 6.

In a preferred embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence of the human papillomavirus HPV 18E 7 protein or immunogenic fragment thereof, wherein the N-terminus of the polypeptide or combination of polypeptides comprises a signal peptide sequence and the C-terminus comprises the transmembrane-intracellular domain sequence of MHC-I, and wherein the human papillomavirus HPV 18E 7 protein comprises the amino acid sequence shown in SEQ ID NO:79 or an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 79.

In some embodiments, the signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO. 7 (this signal peptide sequence is also referred to herein as the SP sequence or sec 1.0). In some embodiments, the signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO. 77 (this signal peptide sequence is also referred to herein as sec 2.0). In some embodiments, the transmembrane-intracellular domain sequence of MHC-I comprises the amino acid sequence shown in SEQ ID NO. 8. In some embodiments, the signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO. 7 or 77, and the transmembrane-intracellular domain sequence of MHC-I comprises the amino acid sequence shown in SEQ ID NO. 8. In a preferred embodiment, the signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO. 7, and the transmembrane-intracellular domain sequence of MHC-I comprises the amino acid sequence shown in SEQ ID NO. 8.

In some embodiments, the polypeptide further comprises one or more linkers.

The linker may comprise an amino acid sequence of any length, in particular an amino acid sequence of 1 to 50, preferably 1 to 30, for example 1 to 10 amino acid residues. Exemplary linkers may include, but are not limited to, poly glycine (G), poly alanine (a), poly serine (S), or combinations thereof, such as GGAS, GGGS, GGGSG or (G4S) n, where n is an integer from 1 to 30, preferably from 1 to 10. The joint may also be a hinge region or a functional equivalent thereof. Other suitable linkers may be organic compounds or polymers generally suitable for use in pharmaceutical proteins, including but not limited to polyethylene glycol.

In a preferred embodiment, the linker is a GS linker. In one embodiment, the linker comprises the amino acid sequence shown in SEQ ID NO 9 or 10.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 106 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO. 106.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5 or 78 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO. 5 or 78.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 106 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO. 106, and the amino acid sequence shown in SEQ ID NO. 5 or 78 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO. 5 or 78

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 132 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO. 132.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 80 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO. 80.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 132 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO. 132, and the amino acid sequence shown in SEQ ID NO. 80 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO. 80

In one embodiment, the polypeptide or polypeptides of the combination of polypeptides comprises the amino acid sequences shown in SEQ ID NO. 7, 9, 105, 10 and 8 in sequence from the N-terminus to the C-terminus. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 106.

In one embodiment, the polypeptide or polypeptides of the combination of polypeptides comprises the amino acid sequences shown in SEQ ID NO. 7, 9, 6, 10 and 8 in sequence from the N-terminus to the C-terminus. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5.

In one embodiment, the polypeptide or polypeptides of the combination of polypeptides comprises the amino acid sequences shown in SEQ ID NO 77, 9, 6, 10 and 8 in sequence from the N-terminus to the C-terminus. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 78.

In one embodiment, the polypeptide or polypeptides of the combination of polypeptides comprises the amino acid sequences shown in SEQ ID NO. 7, 9, 131, 10 and 8 in sequence from the N-terminus to the C-terminus. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 132.

In one embodiment, the polypeptide or polypeptides of the combination of polypeptides comprises the amino acid sequences shown in SEQ ID NO. 7, 9, 79, 10 and 8 in sequence from the N-terminus to the C-terminus. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 80.

In some embodiments, a polypeptide or combination of polypeptides encoded by a polynucleotide of the invention comprises the amino acid sequence set forth in SEQ ID NO. 105 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO. 105. The amino acid sequence shown in SEQ ID NO. 105 is the amino acid sequence of E6 protein of human papillomavirus type 16.

In some embodiments, a polypeptide or combination of polypeptides encoded by a polynucleotide of the invention comprises the amino acid sequence set forth in SEQ ID NO. 6 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO. 6. The amino acid sequence shown in SEQ ID NO. 6 is the amino acid sequence of E7 protein of human papillomavirus type 16.

In some embodiments, a polypeptide or combination of polypeptides encoded by a polynucleotide of the invention comprises the amino acid sequence set forth in SEQ ID NO. 131 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO. 131. The amino acid sequence shown in SEQ ID NO. 131 is the amino acid sequence of E6 protein of human papillomavirus type 18.

In some embodiments, a polypeptide or combination of polypeptides encoded by a polynucleotide of the invention comprises the amino acid sequence set forth in SEQ ID NO. 79 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO. 79. The amino acid sequence shown in SEQ ID NO. 79 is the amino acid sequence of E7 protein of human papillomavirus type 18.

In some embodiments, a polypeptide or combination of polypeptides encoded by a polynucleotide of the invention comprises the amino acid sequence set forth in SEQ ID NO. 106 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO. 106. The amino acid sequence shown in SEQ ID NO. 106 is the amino acid sequence of E6 protein of human papillomavirus type 16 comprising a signal peptide sequence (sec 1.0) at the N-terminus and an MHC-I transmembrane-intracellular domain sequence at the C-terminus.

In some embodiments, a polypeptide or combination of polypeptides encoded by a polynucleotide of the invention comprises the amino acid sequence set forth in SEQ ID NO. 5 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO. 5. The amino acid sequence shown in SEQ ID NO. 5 is the amino acid sequence of E7 protein of human papillomavirus type 16, the N-terminal of which comprises a signal peptide sequence (sec 1.0) and the C-terminal of which comprises the transmembrane-intracellular domain sequence of MHC-I.

In some embodiments, a polypeptide or combination of polypeptides encoded by a polynucleotide of the invention comprises the amino acid sequence set forth in SEQ ID NO:78 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 78. The amino acid sequence shown in SEQ ID NO. 78 is the amino acid sequence of E7 protein of human papillomavirus type 16, the N-terminus of which comprises a signal peptide sequence (sec 2.0) and the C-terminus of which comprises the transmembrane-intracellular domain sequence of MHC-I.

In some embodiments, a polypeptide or combination of polypeptides encoded by a polynucleotide of the invention comprises the amino acid sequence set forth in SEQ ID NO. 132 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO. 132. The amino acid sequence shown in SEQ ID NO. 132 is the amino acid sequence of E6 protein of human papillomavirus type 18, the N-terminal of which comprises a signal peptide sequence (sec 1.0) and the C-terminal of which comprises a transmembrane-intracellular domain sequence of MHC-I.

In some embodiments, a polypeptide or combination of polypeptides encoded by a polynucleotide of the invention comprises the amino acid sequence set forth in SEQ ID NO. 80 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO. 80. The amino acid sequence shown in SEQ ID NO. 80 is the amino acid sequence of E7 protein of human papillomavirus type 18, the N-terminal of which comprises a signal peptide sequence (sec 1.0) and the C-terminal of which comprises the transmembrane-intracellular domain sequence of MHC-I.

In some embodiments, a polypeptide or combination of polypeptides encoded by a polynucleotide of the invention comprises an amino acid sequence set forth in SEQ ID NO 105 or 106 and an amino acid sequence set forth in one of SEQ ID NO 5, 6 and 78.

In some embodiments, a polypeptide or combination of polypeptides encoded by a polynucleotide of the invention comprises the amino acid sequence set forth in SEQ ID NO. 131 or 132 and the amino acid sequence set forth in SEQ ID NO. 79 or 80.

The polypeptides or combinations of polypeptides encoded by the polynucleotides of the invention may be used as polypeptide antigens for inducing a protective immune response against human papillomavirus infection in a subject.

Nucleic acid or nucleic acid combination

In another aspect, the invention provides a nucleic acid or combination of nucleic acids encoding a polypeptide or combination of polypeptides described herein. In some embodiments, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a polypeptide or combination of polypeptides described herein. The nucleic acid (or polynucleotide) may be single-stranded or double-stranded. Nucleic acids (or polynucleotides) include, but are not limited to DNA, cDNA, RNA (e.g., mRNA), recombinantly produced, and chemically synthesized nucleic acids (or polynucleotides). The nucleic acid (or polynucleotide) or nucleic acid combination of the invention may be contained in a vector. The nucleic acids (or polynucleotides) or nucleic acid combinations of the invention may include naturally occurring, synthetic, and modified nucleotides. In some embodiments, a nucleic acid (or polynucleotide) or combination of nucleic acids of the invention can be used to express a polypeptide or combination of polypeptides described herein in a cell to provide a polypeptide antigen. In some embodiments, the polypeptide antigen may induce an immune response against human papilloma virus type 16 in a suitable subject. In some embodiments, the polypeptide antigen may induce an immune response against human papilloma virus type 18 in a suitable subject.

A nucleic acid (or polynucleotide) or combination of nucleic acids may comprise one or more segments (nucleotide fragments) (e.g., 1, 2, 3, 4, 5, 6, 7, 8 segments). The nucleic acid (or polynucleotide) or combination of nucleic acids may comprise segments encoding polypeptides of interest (e.g., polypeptides and polypeptide antigens described herein). In particular embodiments, the nucleic acid (or polynucleotide) or combination of nucleic acids may comprise a coding sequence for a polypeptide of interest as well as regulatory sequences (including but not limited to transcriptional and translational regulatory sequences). In some embodiments, the regulatory sequences comprise one or more of a promoter sequence, a 5 'untranslated region (5' UTR) sequence, a3 'untranslated region (3' UTR) sequence, and a poly (A) sequence.

Coding sequence

As used herein, "coding sequence" or "coding region sequence" refers to a nucleotide sequence in a polynucleotide that can be used as a template for synthesis of a polypeptide having a defined nucleotide sequence (e.g., tRNA and mRNA) or a defined amino acid sequence in a biological process. The coding sequence may be a DNA sequence or an RNA sequence. If an mRNA corresponding to a DNA sequence (including the same coding strand as the mRNA sequence and the template strand complementary thereto) is translated into a polypeptide in a biological process, the DNA sequence or mRNA sequence may be considered to encode the polypeptide.

As used herein, "codon" refers to three consecutive nucleotide sequences (also known as triplet codes) in a polynucleotide that encode a particular amino acid. Synonymous codons (codons encoding the same amino acid) are used differently in different species, termed "codon bias". It is generally believed that for a given species, coding sequences using codons that are favored by it can have higher translational efficiency and accuracy in the expression system of that species. Thus, a polynucleotide may be "codon optimized," i.e., codons in the polynucleotide are altered to reflect codons favored by the host cell, preferably without altering the amino acid sequence it encodes. One of skill in the art will appreciate that due to the degeneracy of the codons, a polynucleotide of the invention may comprise a coding sequence which differs from (e.g., has about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to) a coding sequence described herein but encodes the same amino acid sequence.

In some embodiments, a nucleic acid or combination of nucleic acids of the invention comprises a coding sequence for a polypeptide antigen as described herein. In some embodiments, a nucleic acid or combination of nucleic acids of the invention comprises a coding sequence for a polypeptide as described herein. In some embodiments, a nucleic acid or combination of nucleic acids of the invention comprises a nucleotide sequence that is complementary to a coding sequence for a polypeptide antigen described herein. In some embodiments, a nucleic acid or combination of nucleic acids of the invention comprises a nucleotide sequence that is complementary to a coding sequence for a polypeptide described herein. In some embodiments, the coding sequence comprises an initiation codon at its 5 'end and a termination codon at its 3' end. In some embodiments, the coding sequence comprises an Open Reading Frame (ORF) as described herein.

In some embodiments, the coding sequences of the invention encode any of the polypeptide antigens described above.

In one embodiment, the coding sequence of the invention encodes a polypeptide antigen comprising (1) an amino acid sequence of SEQ ID NO. 105, (2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 105, (3) an immunogenic fragment of the amino acid sequence of SEQ ID NO. 105, or (4) an immunogenic fragment of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 105.

In one embodiment, the coding sequence of the invention encodes a polypeptide antigen, which further comprises an SP sequence (SEQ ID NO: 7) and MITD sequences (SEQ ID NO: 8), which polypeptide antigen comprises (1) an amino acid sequence shown as SEQ ID NO:106, (2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown as SEQ ID NO:106, (3) an immunogenic fragment of the amino acid sequence shown as SEQ ID NO:106, or (4) an immunogenic fragment of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown as SEQ ID NO: 106.

In one embodiment, the coding sequence of the invention encodes a polypeptide or a combination of polypeptides, the polypeptide or combination of polypeptides comprising the amino acid sequence of a polypeptide antigen as described above.

In one embodiment, the coding sequence (region) of a polypeptide or combination of polypeptides described herein comprises a nucleotide sequence comprising (1) a nucleotide sequence of one of SEQ ID NOs 124, 125, 126, 107, 108, 109 and 110, (2) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to a nucleotide sequence of one of SEQ ID NOs 124, 125, 126, 107, 108, 109 and 110, (3) a nucleotide sequence of one of SEQ ID NOs 128, 129, 130, 115, 116, 117 and 118, or (4) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to a nucleotide sequence of one of SEQ ID NOs 128, 129, 130, 115, 116, 117 and 118.

In one embodiment, the coding sequence of the invention encodes a polypeptide antigen comprising (1) an amino acid sequence of SEQ ID NO. 6, (2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 6, (3) an immunogenic fragment of the amino acid sequence of SEQ ID NO. 6, or (4) an immunogenic fragment of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 6.

In one embodiment, the coding sequence of the present invention encodes a polypeptide antigen, which further comprises an SP sequence (SEQ ID NO: 7) and MITD sequence (SEQ ID NO: 8), which polypeptide antigen comprises (1) an amino acid sequence shown as SEQ ID NO:5, (2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown as SEQ ID NO:5, (3) an immunogenic fragment of the amino acid sequence shown as SEQ ID NO:5, or (4) an immunogenic fragment of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown as SEQ ID NO: 5.

In one embodiment, the coding sequence (region) of a polypeptide or combination of polypeptides described herein comprises a nucleotide sequence comprising (1) the nucleotide sequence set forth in one of SEQ ID NOs 17, 20, 63, 65, 66, 1, 22, 23, 25 and 26, (2) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the nucleotide sequence set forth in one of SEQ ID NOs 17, 20, 63, 65, 66, 1, 22, 23, 25 and 26, (3) a nucleotide sequence set forth in one of SEQ ID NOs 18, 42, 70, 72, 73, 3, 44, 45, 47 and 48, or (4) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the nucleotide sequence set forth in one of SEQ ID NOs 18, 42, 70, 72, 73, 3, 44, 45, 47 and 48.

In one embodiment, the coding sequence of the present invention encodes a polypeptide antigen that further comprises the sec2.0 sequence (SEQ ID NO: 77) and MITD sequence (SEQ ID NO: 8), the polypeptide antigen comprising (1) the amino acid sequence shown in SEQ ID NO:78, (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:78, (3) an immunogenic fragment of the amino acid sequence shown in SEQ ID NO:78, or (4) an immunogenic fragment of the amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 78.

In one embodiment, the coding sequence (region) of a polypeptide or combination of polypeptides described herein comprises a nucleotide sequence comprising (1) a nucleotide sequence set forth in one of SEQ ID NOs 64, 67, 68, 69, 21, 24, 27, 28 and 29, (2) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to a nucleotide sequence set forth in one of SEQ ID NOs 64, 67, 68, 69, 21, 24, 27, 28 and 29, (3) a nucleotide sequence set forth in one of SEQ ID NOs 71, 74, 75, 76, 43, 46, 49, 50 and 51, or (4) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to a nucleotide sequence set forth in one of SEQ ID NOs 71, 74, 75, 76, 43, 46, 49, 50 and 51.

In a preferred embodiment, the coding sequence for a polypeptide or combination of polypeptides described herein comprises a nucleotide sequence comprising (1) the nucleotide sequence of SEQ ID NO:1, (2) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the nucleotide sequence of SEQ ID NO:1, (3) the nucleotide sequence of SEQ ID NO:3, or (4) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the nucleotide sequence of SEQ ID NO: 3.

In one embodiment, the coding sequence of the invention encodes a polypeptide antigen comprising (1) an amino acid sequence shown as SEQ ID NO. 131, (2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown as SEQ ID NO. 131, (3) an immunogenic fragment of the amino acid sequence shown as SEQ ID NO. 131, or (4) an immunogenic fragment of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown as SEQ ID NO. 131.

In one embodiment, the coding sequence of the present invention encodes a polypeptide antigen, which further comprises an SP sequence (SEQ ID NO: 7) and MITD sequences (SEQ ID NO: 8), which polypeptide antigen comprises (1) an amino acid sequence shown as SEQ ID NO:132, (2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown as SEQ ID NO:132, (3) an immunogenic fragment of the amino acid sequence shown as SEQ ID NO:132, or (4) an immunogenic fragment of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown as SEQ ID NO: 132.

In one embodiment, the coding sequence (region) of a polypeptide or combination of polypeptides described herein comprises a nucleotide sequence comprising (1) a nucleotide sequence set forth in one of SEQ ID NOs 150, 151, 152, 133, 134, 135 and 136, (2) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to a nucleotide sequence set forth in one of SEQ ID NOs 150, 151, 152, 133, 134, 135 and 136, (3) a nucleotide sequence set forth in one of SEQ ID NOs 154, 155, 156, 141, 142, 143 and 144, or (4) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to a nucleotide sequence set forth in one of SEQ ID NOs 154, 155, 156, 141, 142, 143 and 144.

In one embodiment, the coding sequence of the invention encodes a polypeptide antigen comprising (1) an amino acid sequence shown as SEQ ID NO:79, (2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown as SEQ ID NO:79, (3) an immunogenic fragment of the amino acid sequence shown as SEQ ID NO:79, or (4) an immunogenic fragment of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown as SEQ ID NO: 79.

In one embodiment, the coding sequence of the present invention encodes a polypeptide antigen, which further comprises an SP sequence (SEQ ID NO: 7) and MITD sequences (SEQ ID NO: 8), which polypeptide antigen comprises (1) an amino acid sequence shown as SEQ ID NO:80, (2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown as SEQ ID NO:80, (3) an immunogenic fragment of the amino acid sequence shown as SEQ ID NO:80, or (4) an immunogenic fragment of the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown as SEQ ID NO: 80.

In one embodiment, the coding sequence (region) of a polypeptide or combination of polypeptides described herein comprises a nucleotide sequence comprising (1) a nucleotide sequence set forth in one of SEQ ID NOs 98, 99, 100, 81, 82, 83 and 84, (2) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to a nucleotide sequence set forth in one of SEQ ID NOs 98, 99, 100, 81, 82, 83 and 84, (3) a nucleotide sequence set forth in one of SEQ ID NOs 102, 103, 104, 89, 90, 91 and 92, or (4) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to a nucleotide sequence set forth in one of SEQ ID NOs 102, 103, 104, 89, 90, 91 and 92. In one embodiment, the coding sequence (region) for a polypeptide or combination of polypeptides described herein comprises a nucleotide sequence comprising the nucleotide sequence set forth in one of SEQ ID NOS: 124, 125, 126, 107, 108, 109 and 110 and the nucleotide sequence set forth in one of SEQ ID NOS: 17, 20, 63, 64, 65, 66, 67, 68, 69, 1, 21, 22, 23, 24, 25, 26, 27, 28 and 29, or the nucleotide sequence set forth in one of SEQ ID NOS: 128, 129, 130, 115, 116, 117 and 118 and the nucleotide sequence set forth in one of SEQ ID NOS: 18, 42, 70, 71, 72, 73, 74, 75, 76, 3, 43, 44, 45, 46, 47, 48, 49, 50 and 51.

In one embodiment, the coding sequence (region) for a polypeptide or combination of polypeptides described herein comprises a nucleotide sequence comprising one of the nucleotide sequences set forth in SEQ ID NOS: 150, 151, 152, 133, 134, 135 and 136 and one of the nucleotide sequences set forth in SEQ ID NOS: 98, 99, 100, 81, 82, 83 and 84, or one of the nucleotide sequences set forth in SEQ ID NOS: 154, 155, 156, 141, 142, 143 and 144 and one of the nucleotide sequences set forth in SEQ ID NOS: 102, 103, 104, 89, 90, 91 and 92.

RNA

In some embodiments, the nucleic acid or combination of nucleic acids of the invention is RNA. In some embodiments, the polynucleotides of the invention are RNA. As used herein, the definition of "RNA" encompasses single-stranded, double-stranded, linear, and circular RNAs. The RNA of the invention may be RNA produced by chemical synthesis, recombination and in vitro transcription. In one embodiment, the RNA of the invention is used to express a polypeptide or combination of polypeptides of the invention in a host cell. In one embodiment, the RNA of the invention is single stranded RNA. In one embodiment, the RNA of the invention is in vitro transcribed RNA (IVT-RNA). IVT-RNA can be obtained by in vitro transcription with a DNA template by RNA polymerase (e.g., as described herein).

In some embodiments, the RNA of the invention is messenger RNA (mRNA). In general, an mRNA can comprise a 5'-UTR sequence, a coding sequence for a polypeptide (e.g., a polypeptide or combination of polypeptides of the invention), a 3' -UTR sequence, and optionally a poly (a) sequence. mRNA can be produced, for example, by in vitro transcription or chemical synthesis. In one embodiment, the mRNA of the invention is obtained by in vitro transcription by RNA polymerase (e.g., T7 RNA polymerase) using a DNA template. In one embodiment, the mRNA of the invention comprises (1) a 5'-UTR, (2) a coding sequence, (3) a 3' -UTR, and (4) optionally, a poly (A) sequence. The 5'-UTR, coding sequence, 3' -UTR and poly (A) sequences are as described herein. In one embodiment, the mRNA of the present invention is a nucleoside modified mRNA. In one embodiment, the mRNA of the present invention comprises an optional 5' cap.

In some embodiments, the RNA of the invention comprises a coding sequence for a polypeptide antigen as described herein. In some embodiments, the RNA of the invention comprises a coding sequence for a polypeptide as described herein. In some embodiments, the RNA of the invention comprises a coding sequence for a combination of polypeptides as described herein.

In some embodiments, the RNAs of the invention further comprise structural elements that help to improve stability and/or translation efficiency of the RNAs, including, but not limited to, 5' caps, 5' -UTRs, 3' -UTRs, and poly (a) sequences.

As used herein, the term "untranslated region (UTR)" generally refers to a region in RNA (e.g., mRNA) that is not translated into an amino acid sequence (non-coding region), or a corresponding region in DNA. In general, UTRs located 5' to the open reading frame (start codon) (upstream) may be referred to as 5' -UTRs of the 5' untranslated region and UTRs located 3' to the open reading frame (stop codon) (downstream) may be referred to as 3' -UTRs. In the presence of a 5 'cap, the 5' -UTR is located downstream of the 5 'cap, e.g., immediately adjacent to the 5' cap. In particular embodiments, an optimized "Kozak sequence" may be included in the 5' -UTR, e.g., adjacent to the start codon, to increase translation efficiency. In the presence of a poly (A) sequence, the 3' -UTR is located upstream of the poly (A) sequence, e.g., immediately adjacent to the poly (A) sequence.

In some embodiments, the RNA comprises a 5' -UTR. In a preferred embodiment, the 5' -UTR comprises the nucleotide sequence shown as SEQ ID NO. 11. In some embodiments, the RNA comprises a 3' -UTR. In a preferred embodiment, the 3' -UTR comprises the nucleotide sequence shown in SEQ ID NO. 12. In some embodiments, the RNA comprises a 5'-UTR and a 3' -UTR. In a specific embodiment, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO. 11 and the 3' -UTR comprises the nucleotide sequence of SEQ ID NO. 12.

As used herein, the term "poly (a) sequence" or "poly (a) tail" refers to a nucleotide sequence comprising continuous or discontinuous adenylates. The poly (A) sequence is typically located at the 3' end of the RNA, e.g., 3' end (downstream) of the 3' -UTR. In some embodiments, the poly (a) sequence does not comprise nucleotides other than adenylate at its 3' end. Poly (A) sequences can be transcribed from the coding sequence of a DNA template by a DNA-dependent RNA polymerase during the preparation of IVT-RNA or can be linked to the free 3' end of IVT-RNA, e.g., the 3' end of the 3' -UTR, by a DNA-independent RNA polymerase (Poly (A) polymerase).

In some embodiments, the RNA comprises a poly (a) sequence. In one embodiment, the poly (A) sequence comprises contiguous adenylates. In one embodiment, the poly (a) sequence can comprise at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 95, or 100 and up to 120, 150, 180, 200, 300 adenylates. In one embodiment, the poly (a) sequence comprises at least 50 nucleotides. In one embodiment, the poly (a) sequence comprises at least 80 nucleotides. In one embodiment, the poly (a) sequence comprises at least 100 nucleotides. In some embodiments, the poly (a) sequence comprises about 70, 80, 90, 100, 120, or 150 nucleotides. In one embodiment, the contiguous adenylate sequence in the poly (a) sequence is interrupted by a sequence comprising U, C or G nucleotides. In a specific embodiment, the poly (A) sequence comprises the nucleotide sequence set forth in SEQ ID NO. 15.

As used herein, the term "5 'cap" generally refers to an N7-methylguanosine structure (also known as "m7G cap", "m7 Gppp-") attached to the 5' end of an mRNA by a 5 'to 5' triphosphate bond. The 5' cap may be co-transcribed into the RNA in vitro transcription (e.g., using an anti-reverse cap analogue "ARCA") or may be post-transcriptionally linked to the RNA using a capping enzyme.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding human papillomavirus HPV 16E 6 protein or an immunogenic fragment thereof, wherein the polynucleotide is RNA and the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOs 124, 125 and 126. In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding human papillomavirus HPV 16E 6 protein or an immunogenic fragment thereof, wherein the polynucleotide is RNA and the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOS 124, 125 and 126.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding human papillomavirus HPV 16E 7 protein or an immunogenic fragment thereof, wherein the polynucleotide is RNA and the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOs 17, 20, 63, 64, 65, 66, 67, 68 and 69. In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a human papillomavirus HPV 16E 7 protein or immunogenic fragment thereof, wherein the polynucleotide is RNA and the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOs 17, 20, 63, 64, 65, 66, 67, 68 and 69.

In one embodiment, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS 124, 125 and 126 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOS 124, 125 and 126, and the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS 17, 20, 63, 64, 65, 66, 67, 68 and 69 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOS 17, 20, 63, 64, 65, 66, 67, 68 and 69.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a polypeptide or combination of polypeptides of the invention, the polypeptide or combination of polypeptides comprising the human papillomavirus HPV 16E 6 protein or immunogenic fragment thereof, the N-terminus of the polypeptide or combination of polypeptides comprising a signal peptide sequence, the C-terminus comprising the amino acid sequence of the transmembrane-intracellular segment domain sequence of MHC-I. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 106 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 106, wherein the polynucleotide is RNA and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO. 107, 108, 109, 110, 111, 112, 113 and 114 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in one of SEQ ID NO. 107, 108, 109, 110, 111, 112, 113 and 114.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 106 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 107 or 111. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 106 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 108 or 112. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 106 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 109 or 113. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 106 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 110 or 114.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a polypeptide or combination of polypeptides of the invention, the polypeptide or combination of polypeptides comprising the human papillomavirus HPV 16E 7 protein or immunogenic fragment thereof, the N-terminus of the polypeptide or combination of polypeptides comprising a signal peptide sequence, the C-terminus comprising the amino acid sequence of the transmembrane-intracellular segment domain sequence of MHC-I. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 5, wherein the polynucleotide is RNA and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO. 1, 22, 23, 25, 26, 2, 33, 34, 36 and 37 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in one of SEQ ID NO. 1, 22, 23, 25, 26, 2, 33, 34, 36 and 37.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO.1 or 2. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 22 or 33. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 23 or 34. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 25 or 36. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 26 or 37.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a polypeptide or combination of polypeptides of the invention, the polypeptide or combination of polypeptides comprising the human papillomavirus HPV 16E 7 protein or immunogenic fragment thereof, the N-terminus of the polypeptide or combination of polypeptides comprising a signal peptide sequence, the C-terminus comprising the amino acid sequence of the transmembrane-intracellular segment domain sequence of MHC-I. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:78 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO:78, wherein the polynucleotide is RNA and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO:21, 24, 27, 28, 29, 32, 35, 38, 39 and 40 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in one of SEQ ID NO:21, 24, 27, 28, 29, 32, 35, 38, 39 and 40.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 78 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 21 or 32. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 78 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 24 or 35. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 78 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 27 or 38. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 78 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 28 or 39. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 78 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 29 or 40.

In a preferred embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 1 or 2.

In one embodiment, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NO 107, 108, 109, 110, 111, 112, 113 and 114 or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence set forth in one of SEQ ID NO 107, 108, 109, 110, 111, 112, 113 and 114, and the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NO 1, 21, 22, 23, 24, 25, 26, 27, 28, 29, 2, 32, 33, 34, 35, 36, 37, 38, 39 and 40 or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence set forth in one of SEQ ID NO 1, 21, 22, 23, 24, 25, 26, 27, 28, 29, 2, 32, 33, 34, 35, 36, 37, 38, 39 and 40.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding human papillomavirus HPV 18E 6 protein or an immunogenic fragment thereof, wherein the polynucleotide is RNA and the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOs 150, 151 and 152. In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a human papillomavirus HPV 18E 6 protein or immunogenic fragment thereof, wherein the polynucleotide is RNA and the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOs 150, 151 and 152.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding human papillomavirus HPV 18E 7 protein or an immunogenic fragment thereof, wherein the polynucleotide is RNA and the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOs 98, 99 and 100. In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a human papillomavirus HPV 18E 7 protein, or an immunogenic fragment thereof, wherein the polynucleotide is RNA and the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOs 98, 99 and 100.

In one embodiment, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS: 150, 151 and 152 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOS: 150, 151 and 152, and the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS: 98, 99 and 100 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOS: 98, 99 and 100.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a polypeptide or combination of polypeptides of the invention, the polypeptide or combination of polypeptides comprising the human papillomavirus HPV 18E 6 protein or immunogenic fragment thereof, the N-terminus of the polypeptide or combination of polypeptides comprising a signal peptide sequence, the C-terminus comprising the amino acid sequence of the transmembrane-intracellular segment domain sequence of MHC-I. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 132 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 132, wherein the polynucleotide is RNA and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO. 133, 134, 135, 136, 137, 138, 139 and 140 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in one of SEQ ID NO. 133, 134, 135, 136, 137, 138, 139 and 140.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 132 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 133 or 137. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 132 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 134 or 138. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 132 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 135 or 139. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 132 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 136 or 140.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a polypeptide or combination of polypeptides of the invention, the polypeptide or combination of polypeptides comprising the human papillomavirus HPV 18E 7 protein or immunogenic fragment thereof, the N-terminus of the polypeptide or combination of polypeptides comprising a signal peptide sequence, the C-terminus comprising the amino acid sequence of the transmembrane-intracellular segment domain sequence of MHC-I. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 80 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 80, wherein the polynucleotide is RNA and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO. 81, 82, 83, 84, 85, 87 and 88 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in one of SEQ ID NO. 81, 82, 83, 84, 85, 86, 87 and 88.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 80 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 81 or 85. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 80 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 83 or 87. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 80 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 84 or 88.

In a preferred embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 80 and the polynucleotide RNA comprises the nucleotide sequence shown in SEQ ID NO. 82 or 86.

In one embodiment, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS: 133, 134, 135, 136, 137, 138, 139 and 140 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOS: 133, 134, 135, 136, 137, 138, 139 and 140, and the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS: 81, 82, 83, 84, 85, 86, 87 and 88 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOS: 81, 82, 83, 84, 85, 86, 87 and 88.

Modified nucleotides

In some embodiments, the nucleotides in the RNAs (e.g., mrnas) of the invention can be naturally occurring nucleotides (e.g., naturally occurring ribonucleotides) and modified nucleotides. The modified nucleotide may be, for example, a nucleotide that is not present in the naturally occurring RNA, such as a nonstandard nucleotide or a deoxynucleotide. Modification of the nucleotide may occur on the nucleoside, for example on the ribose moiety and/or nucleobase moiety. The modified nucleotides may be incorporated during transcription (e.g., in vitro transcription) or may be added during RNA chemical synthesis.

In one embodiment, the RNA is modified by including one or more modified nucleosides. In one embodiment, the RNA is modified by replacing one or more uracils with a modified uridine. In one embodiment, the modified uridine comprises 1-methyl pseudouracil, 5-methyl-uracil, or a combination thereof.

Examples of modified uridine may include, but are not limited to: 1-methyluridine, 1-methyl-pseudouridine, 3-methyl-uridine, 3-methyl-pseudouridine, 2-methoxy-uridine, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5-aminoallyl-uridine, 5-halo-uridine, uridine 5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine 5-carboxyhydroxymethyl-uridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5-methoxycarbonylmethyl-2-thio-uridine, 5-aminomethyl-2-thio-uridine, 5-methylaminomethyl-uridine, 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine, 5-carbamoylmethyl-uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurine methyl-uridine, 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine, 1-taurine methyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine, 1-methyl-4-thio-pseudouridine, 4-thio-1-methyl-pseudouridine, 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 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 3- (3-amino-3-carboxypropyl) uridine, 5- (isopentenyl-aminomethyl) -2-thio-uridine, alpha-thio-uridine, 2 '-O-methyl-uridine, 5,2' -O-dimethyl-uridine, 2 '-O-methyl-pseudouridine, 2-thio-2' -O-methyl-uridine, 5-methoxycarbonylmethyl-2 ' -O-methyl-uridine, 5-carbamoylmethyl-2 ' -O-methyl-uridine, 5-carboxymethylaminomethyl-2 ' -O-methyl-uridine, 3,2' -O-dimethyl-uridine, 5- (isopentenylaminomethyl) -2' -O-methyl-uridine, 1-thio-uridine, 5- (2-methoxycarbonylvinyl) uridine and 5- [3- (1-E-propenyl amino) uridine.

In one embodiment, the RNA (e.g., mRNA) of the present invention is modified by inclusion of one or more modified nucleobases. In one embodiment, the modified nucleobase comprises a modified cytosine, a modified uracil, or a combination thereof. In one embodiment, the modified uracil is independently selected from pseudouracil, 1-methyl-pseudouracil, 5-methyl-uracil, or a combination thereof. In one embodiment, the modified cytosine is independently selected from 5-methylcytosine, 5-hydroxymethylcytosine, or a combination thereof. In one embodiment, the proportion of modified nucleobases in the RNA of the invention is from 10% to 100%, i.e.the RNA of the invention can be modified by replacing 10% to 100% of the nucleobases with modified nucleobases.

In some embodiments, the RNA (e.g., mRNA) of the invention is modified by replacing one or more uracils with a modified uracil. In one embodiment, the modified uracil comprises 1-methyl pseudouracil, 5-methyl-uracil, or a combination thereof. In one embodiment, the modified uracil comprises pseudouracil. In one embodiment, the modified uracil comprises 5-methyl-uracil. In one embodiment, the modified uracil comprises 1-methyl-pseudouracil.

In one embodiment, the RNA is modified by replacing at least one uracil with a modified uracil. In one embodiment, the RNA is modified by replacing all uracils with modified uracils. In one embodiment, the proportion of modified uracil in RNA is 10% -100%, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In one embodiment, the proportion of modified uracil in RNA is 20% to 100%. In one embodiment, 20% to 100% of uracil in the RNA is replaced with 1-methyl pseudouracil. In a preferred embodiment, 100% of uracil in RNA is replaced with 1-methyl pseudouracil.

1-Methyl-pseudouridine has the following structure:

In a specific embodiment, the mRNA of the present invention comprises the nucleotide sequence of any of SEQ ID NOs 2, 32, 33, 34, 35, 36, 37, 38, 39, 40, 85, 86, 87, 88, 111, 112, 113, 114, 137, 138, 139 and 140, and wherein 100% of the uracil is replaced with 1-methyl pseudouracil.

DNA

In some embodiments, the nucleic acid or combination of nucleic acids of the invention is DNA. In some embodiments, the polynucleotides of the invention are DNA. Such DNA may be, for example, a DNA template for in vitro transcription of the RNA of the invention or a DNA vaccine for expression of a polypeptide antigen in a host cell. The DNA may be double-stranded, single-stranded, linear and circular.

The DNA template may be provided in a suitable transcription vector. In general, a DNA template may be a double-stranded complex comprising a nucleotide sequence (coding strand) identical to a coding sequence described herein and a nucleotide sequence (template strand) complementary to a coding sequence described herein. As known to those skilled in the art, a DNA template may comprise a promoter, a 5'-UTR, a coding sequence, a 3' -UTR, and optionally a DNA sequence corresponding to a poly (a) sequence. Promoters may be available to suitable RNA polymerases (particularly DNA-dependent RNA polymerases) known to those skilled in the art, including but not limited to promoters of SP6, T3 and T7 RNA polymerases. In some embodiments, the DNA comprises a T7 promoter. In some embodiments, the T7 promoter comprises the nucleotide sequence of SEQ ID NO. 157. In some embodiments, the 5'-UTR, coding sequence, 3' -UTR, and poly (a) sequences in the DNA templates are or are complementary to the corresponding sequences contained in the RNAs described herein. Polynucleotides as DNA vaccines may be provided in plasmid vectors (e.g., circular plasmid vectors).

In some embodiments, a DNA polynucleotide of the invention comprises a coding sequence for a polypeptide antigen as described herein. In some embodiments, a DNA polynucleotide of the invention comprises a coding sequence for a polypeptide as described herein. In some embodiments, a DNA polynucleotide of the invention comprises coding sequences for a combination of polypeptides as described herein. In some embodiments, a DNA polynucleotide of the invention comprises a DNA sequence corresponding to (1) a T7 promoter, (2) a 5'-UTR, (3) a coding sequence, (4) a 3' -UTR, and (5) an optionally present poly (a) sequence as described herein.

In some embodiments, the DNA polynucleotide comprises a DNA sequence corresponding to the 5' -UTR. In a preferred embodiment, the DNA sequence corresponding to the 5' -UTR comprises the nucleotide sequence of SEQ ID NO. 13. In some embodiments, the DNA polynucleotide comprises a DNA sequence corresponding to the 3' -UTR. In a preferred embodiment, the DNA sequence corresponding to the 3' -UTR comprises the nucleotide sequence of SEQ ID NO. 14. In some embodiments, the DNA polynucleotide comprises DNA sequences corresponding to 5'-UTR and 3' -UTR. In a specific embodiment, the DNA sequence corresponding to the 5'-UTR comprises the nucleotide sequence of SEQ ID NO. 13 and the DNA sequence corresponding to the 3' -UTR comprises the nucleotide sequence of SEQ ID NO. 14.

In some embodiments, the DNA polynucleotide comprises a DNA sequence corresponding to a poly (a) sequence. In one embodiment, the DNA sequence corresponding to the poly (A) sequence comprises contiguous deoxyadenosine. In one embodiment, the DNA sequence corresponding to the poly (a) sequence may comprise at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 95, or 100 and up to 120, 150, 180, 200, 300 deoxyadenylates. In one embodiment, the continuous deoxyadenosine sequence in the DNA sequence corresponding to the poly (a) sequence is interrupted by a sequence comprising T, C or G nucleotides. In one embodiment, the DNA sequence corresponding to the poly (A) sequence comprises the nucleotide sequence of SEQ ID NO. 16.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding human papillomavirus HPV 16E 6 protein or an immunogenic fragment thereof, wherein the polynucleotide is DNA and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NOs 128, 129 and 130. In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding human papillomavirus HPV 16E 6 protein or an immunogenic fragment thereof, wherein the polynucleotide is DNA and the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOS 128, 129 and 130.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding human papillomavirus HPV 16E 7 protein or an immunogenic fragment thereof, wherein the polynucleotide is DNA and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NOs 18, 42, 70, 71, 72, 73, 74, 75 and 76. In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding human papillomavirus HPV 16E 7 protein or an immunogenic fragment thereof, wherein the polynucleotide is DNA, the polynucleotide comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOs 18, 42, 70, 71, 72, 73, 74, 75 and 76.

In one embodiment, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS 128, 129 and 130 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOS 128, 129 and 130, and the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS 18, 42, 70, 71, 72, 73, 74, 75 and 76 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOS 18, 42, 70, 71, 72, 73, 74, 75 and 76.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a polypeptide or combination of polypeptides of the invention, the polypeptide or combination of polypeptides comprising the human papillomavirus HPV 16E 6 protein or immunogenic fragment thereof, the N-terminus of the polypeptide or combination of polypeptides comprising a signal peptide sequence, the C-terminus comprising the amino acid sequence of the transmembrane-intracellular segment domain sequence of MHC-I. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 106 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 106, wherein the polynucleotide is DNA and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO. 115, 116, 117, 118, 119, 120, 121 and 122 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in one of SEQ ID NO. 115, 116, 117, 118, 119, 120, 121 and 122.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 106 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 115 or 119. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 106 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 116 or 120. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 106 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 117 or 121. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 106 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 118 or 122.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a polypeptide or combination of polypeptides of the invention, the polypeptide or combination of polypeptides comprising the human papillomavirus HPV 16E 7 protein or immunogenic fragment thereof, the N-terminus of the polypeptide or combination of polypeptides comprising a signal peptide sequence, the C-terminus comprising the amino acid sequence of the transmembrane-intracellular segment domain sequence of MHC-I. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 5, wherein the polynucleotide is DNA and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO. 3, 44, 45, 47, 48, 4, 55, 56, 58 and 59 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in one of SEQ ID NO. 3, 44, 45, 47, 48, 4, 55, 56, 58 and 59.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO.3 or 4. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 44 or 55. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 45 or 56. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 47 or 58. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 48 or 59.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a polypeptide or combination of polypeptides of the invention, the polypeptide or combination of polypeptides comprising the human papillomavirus HPV 16E 7 protein or immunogenic fragment thereof, the N-terminus of the polypeptide or combination of polypeptides comprising a signal peptide sequence, the C-terminus comprising the amino acid sequence of the transmembrane-intracellular segment domain sequence of MHC-I. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO:78 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO:78, wherein the polynucleotide is DNA and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO:43, 46, 49, 50, 51, 54, 57, 60, 61 and 62 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in one of SEQ ID NO:43, 46, 49, 50, 51, 54, 57, 60, 61 and 62.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 78 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 43 or 54. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 78 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 46 or 57. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 78 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 49 or 60. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 78 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 50 or 61. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 78 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 51 or 62.

In a preferred embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 5 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 3 or 4.

In one embodiment, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NO 115, 116, 117, 118, 119, 120, 121 and 122 or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence set forth in one of SEQ ID NO 115, 116, 117, 118, 119, 120, 121 and 122, and the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NO 3, 43, 44, 45, 46, 47, 48, 49, 50, 51, 4, 54, 55, 56, 57, 58, 59, 60, 61 and 62 or a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence set forth in one of SEQ ID NO 3, 43, 44, 45, 46, 47, 48, 49, 50, 51, 4, 54, 55, 56, 57, 59, 60, 61 and 62.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding human papillomavirus HPV 18E 6 protein or an immunogenic fragment thereof, wherein the polynucleotide is DNA and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NOs 154, 155 and 156. In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding human papillomavirus HPV 18E 6 protein or an immunogenic fragment thereof, wherein the polynucleotide is DNA and the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOs 154, 155 and 156.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding human papillomavirus HPV 18E 7 protein or an immunogenic fragment thereof, wherein the polynucleotide is DNA and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NOs 102, 103 and 104. In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a human papillomavirus HPV 18E 7 protein or an immunogenic fragment thereof, wherein the polynucleotide is DNA and the polynucleotide comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOs 102, 103 and 104.

In one embodiment, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS: 154, 155 and 156 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOS: 154, 155 and 156, and the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS: 102, 103 and 104 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOS: 102, 103 and 104.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a polypeptide or combination of polypeptides of the invention, the polypeptide or combination of polypeptides comprising the human papillomavirus HPV 18E 6 protein or immunogenic fragment thereof, the N-terminus of the polypeptide or combination of polypeptides comprising a signal peptide sequence, the C-terminus comprising the amino acid sequence of the transmembrane-intracellular segment domain sequence of MHC-I. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 132 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 132 wherein the polynucleotide is DNA and the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO. 141, 142, 143, 144, 145, 146, 147 and 148 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in one of SEQ ID NO. 141, 142, 143, 144, 145, 146, 147 and 148.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 132 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 141 or 145. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 132 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 142 or 146. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 132 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 143 or 147. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 132 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 144 or 148.

In one embodiment, the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a polypeptide or combination of polypeptides of the invention, the polypeptide or combination of polypeptides comprising the human papillomavirus HPV 18E 7 protein or immunogenic fragment thereof, the N-terminus of the polypeptide or combination of polypeptides comprising a signal peptide sequence, the C-terminus comprising the amino acid sequence of the transmembrane-intracellular segment domain sequence of MHC-I. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 80 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 80, wherein the polynucleotide is DNA and the polynucleotide comprises the nucleotide sequences shown in SEQ ID NO. 89, 90, 91, 92, 93, 94, 95 and 96 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95, 96%, 97%, 98% or 99% identity to the nucleotide sequence shown in SEQ ID NO. 89, 90, 91, 92, 93, 94, 95 and 96.

In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 80 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 89 or 93. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 80 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 91 or 95. In one embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 80 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 92 or 96.

In a preferred embodiment, the polypeptide or combination of polypeptides comprises the amino acid sequence shown in SEQ ID NO. 80 and the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO. 90 or 94.

In one embodiment, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS 141, 142, 143, 144, 145, 146, 147 and 148 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOS 141, 142, 143, 144, 145, 146, 147 and 148, and the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS 89, 90, 91, 92, 93, 94, 95 and 96 or a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in one of SEQ ID NOS 89, 90, 91, 92, 93, 94, 95 and 96.

Composition and method for producing the same

In one aspect, the invention provides a composition comprising a polypeptide or combination of polypeptides of the invention.

In yet another aspect, the invention also provides a composition comprising a nucleic acid or combination of nucleic acids of the invention. In one embodiment, the composition is used to provide therapeutic immunity against a human papillomavirus infection or a human papillomavirus positive cancer in a subject. In some embodiments, the composition comprises a DNA polynucleotide of the invention. In some embodiments, the composition comprises an RNA polynucleotide of the invention. In one embodiment, the RNA is in vitro transcribed RNA. In one embodiment, the RNA is mRNA.

In some embodiments, the composition comprises a nucleic acid or combination of nucleic acids as described herein, and a lipid encapsulating the nucleic acid or combination of nucleic acids. The composition is a lipid delivery vehicle, the lipid can encapsulate the nucleic acid or nucleic acid combination of the invention to form a nanoparticle for delivery into an organism.

As used herein, the term "lipid" refers to an organic compound comprising a hydrophobic moiety and optionally also a hydrophilic moiety. Lipids are generally poorly soluble in water but soluble in many organic solvents. Generally, amphiphilic lipids comprising a hydrophobic portion and a hydrophilic portion may be organized in an aqueous environment as a lipid bilayer structure, for example in the form of vesicles. Lipids may include, but are not limited to, fatty acids, glycerides, phospholipids, sphingolipids, glycolipids, and steroids, cholesterol esters, and the like.

As used herein, "lipid nanoparticle" or "LNP" refers to a lipid vesicle with a homogeneous lipid core, which is a particle formed from lipids, the lipid components undergoing intermolecular interactions to form a nanostructure entity. The nucleic acid or nucleic acid combination (e.g., mRNA or mRNA combination) is encapsulated in a lipid.

Particularly preferred nucleic acid compositions can be, for example, lipid Nanoparticles (LNPs) and lipid multimeric complexes (LPPs) as described herein. Methods of preparing such compositions may be as described herein. LPP is a particle having a core-shell structure in which nucleic acids are contained in a multimeric complex, which itself is encapsulated in a biocompatible lipid bilayer shell to constitute the lipid nanoparticle of the present invention. In some embodiments, the compositions of the invention comprise Lipid Nanoparticles (LNPs) or lipid multimeric complexes (LPPs). In some embodiments, the compositions of the invention are Lipid Nanoparticles (LNPs) or lipid multimeric complexes (LPPs) comprising the nucleic acids or nucleic acid combinations of the invention.

In some embodiments, the lipid encapsulating the nucleic acid or nucleic acid combination of the invention is selected from one or more of a cationic lipid, a phospholipid, a steroid and/or a polyethylene glycol modified lipid. In a preferred embodiment, the cationic lipid is an ionizable cationic lipid.

The composition of the invention comprises a nucleic acid or combination of nucleic acids of the invention and a lipid encapsulating the nucleic acid or combination of nucleic acids. Lipids encapsulating nucleic acids or nucleic acid combinations include cationic lipids, phospholipids, steroids, and polyethylene glycol modified lipids.

In some embodiments, the composition comprises a cationic lipid. In some embodiments, the cationic lipid comprises a compound of formula (I) as described herein, or a combination thereof. In some preferred embodiments, the cationic lipid comprises SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2. In a preferred embodiment, the cationic lipid comprises SW-II-140-2.

In some embodiments, the composition comprises a phospholipid and/or a steroid. In some embodiments, the composition comprises a phospholipid as described herein. In some embodiments, the phospholipid comprises 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or a combination thereof. In some embodiments, the composition comprises a steroid as described herein. In some embodiments, the steroid comprises cholesterol. In some embodiments, the composition comprises a phospholipid and a steroid as described herein. In one embodiment, the composition comprises DOPE. In one embodiment, the composition comprises DSPC. In one embodiment, the composition comprises cholesterol. In one embodiment, the composition comprises DOPE and cholesterol. In one embodiment, the composition comprises DSPC and cholesterol.

In one embodiment, the composition comprises cationic lipids SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, phospholipid DOPE and cholesterol. In one embodiment, the composition comprises cationic lipids SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, phospholipid DSPC and cholesterol.

In some embodiments, the lipid encapsulating the nucleic acid or nucleic acid combination of the invention further comprises a polyethylene glycol modified lipid. In one embodiment, the polyethylene glycol modified lipid comprises DMG-PEG (e.g., DMG-PEG 2000), DOG-PEG, and DSPE-PEG, or a combination thereof. In one embodiment, the polyethylene glycol modified lipid is DSPE-PEG. In one embodiment, the polyethylene glycol modified lipid is DMG-PEG (e.g., DMG-PEG 2000).

In one embodiment, the composition comprises a cationic lipid, DOPE, cholesterol, and DSPE-PEG. In one embodiment, the composition comprises a cationic lipid, DSPC, cholesterol, and DSPE-PEG. In one embodiment, the composition comprises a cationic lipid, DSPC, cholesterol, and DMG-PEG. In a preferred embodiment, the composition comprises a cationic lipid, DOPE, cholesterol, and DMG-PEG.

In a preferred embodiment, the composition comprises the cationic lipids SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, DOPE, cholesterol and DMG-PEG.

In some embodiments, the compositions of the invention further comprise a cationic polymer associated with the polynucleic acid or nucleic acid combination as a complex, co-encapsulated in the lipid. In one embodiment, the cationic polymer comprises poly-L-lysine, protamine, polyethylenimine (PEI), or a combination thereof. In one embodiment, the cationic polymer is protamine.

In some embodiments, the amount of lipid in the composition is calculated as mole percent (mole%) based on the total moles of lipid in the composition. The sum of the amounts (mole%) of the respective lipids in the composition is 100 mole%, i.e. the sum of the amounts (mole%) of the cationic lipid, the phospholipid, the steroid and the polyethylene glycol modified lipid is 100 mole%, unless otherwise specified.

In some embodiments, the amount of cationic lipid in the composition is from about 10 to about 70 mole%. In some embodiments, the amount of cationic lipid in the composition is from about 20 to about 60 mole%, from about 30 to about 50 mole%, from about 35 to about 45 mole%, from about 38 to about 45 mole%, from about 40 to about 50 mole%, or from about 45 to about 50 mole%. For example, the amount of cationic lipid may be about 10, 15, 20, 25, 30, 32.5, 35, 37.5, 38, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 65, 70 mole%.

In some embodiments, the amount of phospholipid in the composition is from about 10 to about 70 mole%. In one embodiment, the amount of phospholipid in the composition is from about 20 to about 60 mole%, from about 30 to about 50 mole%, from about 10 to about 30 mole%, from about 10 to about 20 mole%, or from about 10 to about 15 mole%. For example, the amount of phospholipid may be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 mole%.

In some embodiments, the amount of cholesterol in the composition is from about 10 to about 70 mole%. In one embodiment, the amount of cholesterol in the composition is from about 20 to about 60 mole%, from about 24 to about 44 mole%, from about 30 to about 50 mole%, from about 35 to about 40 mole%, from about 35 to about 45 mole%, from about 40 to about 45 mole%, or from about 45 to about 50 mole%. For example, the amount of cholesterol may be about 10、15、17.5、18.75、20、22.5、24、25、27.5、28.75、30、32.5、33.75、35、40、42.5、44、45、46.25、47.5、48.75、50、52.5、53.75、55、60、62.5、63.75、65 or 70 mole%.

In some embodiments, the amount of polyethylene glycol modified lipid in the composition is from about 0.05 to about 20 mole%. In one embodiment, the amount of polyethylene glycol modified lipid in the composition is from about 0.5 to about 15 mole%, from about 1 to about 10 mole%, from about 5 to about 15 mole%, from about 1 to about 5 mole%, from about 1 to about 1.5 mole%, from about 1.5 to about 3 mole%, or from about 2 to 5 mole%. For example, the amount of polyethylene glycol modified lipid may be about 0.05, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, or 20 mole%.

In one embodiment, the composition comprises 10-70 mole% cationic lipid, 10-70 mole% phospholipid, 10-70 mole% steroid, and 0.05-20 mole% polyethylene glycol modified lipid. In one embodiment, the composition comprises 35 to 50 mole% cationic lipid, 10 to 30 mole% phospholipid, 24 to 44 mole% fusoid alcohol, and 1 to 1.5 mole% polyethylene glycol modified lipid.

In one embodiment, the LPP comprises a nucleic acid or combination of nucleic acids of the present invention and a lipid encapsulating the nucleic acid or combination of nucleic acids, wherein the lipid encapsulating the nucleic acid or combination of nucleic acids comprises a cationic lipid, a phospholipid, a steroid, and a polyethylene glycol modified lipid, and further comprises a cationic polymer, wherein the cationic polymer associates with the nucleic acid or combination of nucleic acids as a complex. In one embodiment, the composition of the invention comprises a nucleic acid or combination of nucleic acids of the invention and a lipid encapsulating the nucleic acid or combination of nucleic acids, wherein the lipid encapsulating the nucleic acid or combination of nucleic acids comprises a cationic lipid, a phospholipid, a steroid, and a polyethylene glycol modified lipid, and a cationic polymer, wherein the cationic polymer associates with the nucleic acid or combination of nucleic acids as a complex, and is co-encapsulated in the lipid to form a lipid-multimeric complex. In one embodiment, the phospholipid is selected from 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoyl phosphatidylcholine (DSPC), or a combination thereof. In one embodiment, the steroid is cholesterol. In one embodiment, the cationic polymer is protamine. In one embodiment, the polyethylene glycol modified lipid is selected from 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (DMG-PEG), 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-poly (ethylene glycol) (DSPE-PEG), or a combination thereof. In one embodiment, the cationic lipid is selected from SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2. In a preferred embodiment, the cationic lipid is SW-II-140-2.

In one embodiment, the lipid encapsulating the complex comprises 50 mole% SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2,10 mole% DOPE,38.5 mole% cholesterol and 1.5 mole% DMG-PEG. In one embodiment, the lipid encapsulating the complex comprises 40 mole% SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2,15 mole% DOPE,43.5 mole% cholesterol and 1.5 mole% DMG-PEG. In a preferred embodiment, the lipid encapsulating the complex comprises 40 mole% SW-II-140-2,15 mole% DOPE,43.5 mole% cholesterol and 1.5 mole% DMG-PEG.

In one embodiment, the LNP comprises a nucleic acid or combination of nucleic acids of the invention and a lipid encapsulating the nucleic acid or combination of nucleic acids, wherein the lipid encapsulating the nucleic acid or combination of nucleic acids comprises a cationic lipid, a phospholipid, a steroid, and a polyethylene glycol modified lipid. In one embodiment, the cationic lipid is SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2. In one embodiment, the phospholipid is DSPC. In one embodiment, the polyethylene glycol modified lipid is DMG-PEG 2000. In one embodiment, the cationic lipid is SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, the phospholipid is DSPC, and the polyethylene glycol modified lipid is DMG-PEG 2000.

In one embodiment the lipid encapsulating a nucleic acid or combination of nucleic acids comprises 50 mole% SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, 10 mole% DSPC, 38.5 mole% cholesterol and 1.5 mole% DMG-PEG 2000.

In one aspect, the invention provides a vaccine formulation comprising a polypeptide or combination of polypeptides as described herein or a composition comprising said polypeptide or combination of polypeptides.

In yet another aspect, the invention also provides a vaccine formulation comprising a nucleic acid or combination of nucleic acids as described herein or a composition comprising said nucleic acid or combination of nucleic acids.

In some embodiments, the vaccine formulations (also referred to as "vaccine agents") of the present invention comprise a nucleic acid or combination of nucleic acids described herein.

In some embodiments, the vaccine formulations (also referred to as "vaccine agents") of the invention comprise a composition described herein, wherein the lipid comprises 10-70 mole% SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, 10-70 mole% DOPE, 10-70 mole% cholesterol, and 0.05-20 mole% DMG-PEG 2000, wherein the nucleic acid or combination of nucleic acids comprises a polynucleotide encoding a polypeptide or combination of polypeptides described herein.

In some embodiments, the polynucleotide comprises a nucleotide sequence set forth in one of SEQ ID NOs 124, 125, 126, 128, 129, 130, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, and 122. In some embodiments, the polynucleotide comprises a nucleotide sequence set forth in one of SEQ ID NO:17、20、63、64、65、66、67、68、69、18、42、70、71、72、73、74、75、76、1、21、22、23、24、25、26、27、28、29、2、32、33、34、35、36、37、38、39、40、3、43、44、45、46、47、48、49、50、51、4、54、55、56、57、58、59、60、61 and 62. In some embodiments, the polynucleotide comprises a nucleotide sequence set forth in one of SEQ ID NOs 150, 151, 152, 154, 155, 156, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, and 148. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOs 98, 99, 100, 102, 103, 104, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 94, 95 and 96. In a preferred embodiment, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOs 1,2, 3 and 4.

In one embodiment, the vaccine formulation comprises a nucleic acid or combination of nucleic acids described herein, the nucleic acid or combination of nucleic acids comprising a polynucleotide encoding a polypeptide or combination of polypeptides described herein, and the vaccine formulation comprises a lipid encapsulating the nucleic acid or combination of nucleic acids, the lipid comprising 10 to 70 mole% SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, 10 to 70 mole% DOPE, 10 to 70 mole% cholesterol, and 0.05 to 20 mole% DMG-PEG2000. In a preferred embodiment, the lipid comprises 40 mole% SW-II-140-2, 15 mole% DOPE, 43.5 mole% cholesterol and 1.5 mole% DMG-PEG2000. In some embodiments, the polynucleotide comprises a nucleotide sequence set forth in one of SEQ ID NOs 124, 125, 126, 128, 129, 130, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, and 122. In some embodiments, the polynucleotide comprises a nucleotide sequence set forth in one of SEQ ID NO：17、20、63、64、65、66、67、68、69、18、42、70、71、72、73、74、75、76、1、21、22、23、24、25、26、27、28、29、2、32、33、34、35、36、37、38、39、40、3、43、44、45、46、47、48、49、50、51、4、54、55、56、57、58、59、60、61 and 62. In some embodiments, the polynucleotide comprises a nucleotide sequence set forth in one of SEQ ID NOs 150, 151, 152, 154, 155, 156, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, and 148. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOs 98, 99, 100, 102, 103, 104, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 94, 95 and 96. In a preferred embodiment, the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOs 1,2, 3 and 4. Optionally, the vaccine formulation further comprises a cationic polymer, wherein the cationic polymer associates with the nucleic acid or nucleic acid combination as a complex, and is co-encapsulated in the lipid to form a lipid-multimeric complex.

Cationic lipids

Cationic lipids are lipids that can carry a net positive charge at a given pH. Lipids with a net positive charge can associate with nucleic acids through electrostatic interactions.

Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecenyl-3-trimethylammonium propane (1, 2-di-O-octadecenyl-3-trimethylammonium-propane, DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (1, 2-dioleoyl-3-trimethylammonium-propane, DOTAP), bisdecanyl dimethylammonium bromide (Didecyldimethylammonium bromide, DDAB), 2, 3-Dioleoyloxy-N- [2 (spermine carboxamide) ethyl ] -N, N-dimethyl-1-propylamine onium trifluoroacetate (2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate,DOSPA)、 dioctadecyl dimethyl ammonium chloride (dioctadecyldimethyl ammonium chloride, DODAC), 1,2-dioleoyl-3-dimethyl ammonium-propane (1, 2-dioleoyl-3-dimethylammonium-propane, DODAP), 3- (N (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol (3- (N (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol, DC-Chol), 2,3-di (tetradecyloxy) propyl- (2-hydroxyethyl) -dimethylaminoonium (2, 3-di (tetradecoxy) propyl- (2-hydroxyethyl) -dimethylazanium, DMRIE), N-dimethyl-3, 4-dioleoxybenzylamine (N, N-dimethyl-3,4-dioleyloxybenzylamine, DMOBA), 1, 2-Dialkenyloxy-N, N-dimethylaminopropane (1, 2-dilinoleyloxy-N, N-dimethylaminopropane, DLinDMA), 1, 2-Dialkenyloxy-N, N-dimethylaminopropane (1, 2-dilinolenyloxy-N, N-dimethylaminopropane, DLinDMA), 3-dimethylamino-2- (cholest-5-en-3- β -oxybutan-4-oxy) -1- (cis, cis-9, 12-octadecadienyloxy) propane (3-dimethylamino-2-(cholest-5-eh-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane,CLinDMA)、N-(2- aminoethyl) -N, N-dimethyl-2,3-bis (tetradecyloxy) propane-1-aminium bromide (N- (2-aminoethyl) -N, N-dimethyl-2,3-bis (tetradecyloxy) propan-1-aminium bromide, DMORIE), N, N-dimethyl-2,3-bis (dodecyloxy) propan-1-amine (N, N-dimethyl-2,3-bis (dodecyloxy) propan-1-amine, DLDMA), N-dimethyl-2,3-bis (tetradecyloxy) propan-1-amine (N, N-dimethyl-2,3-bis (tetradecyloxy) propan-1-amine, DMDMA), dioctadecyl amidoglycyl spermine (dioctadecylamidoglycyl spermine, DOGS), N4-cholesteryl-spermine (N4-cholesteryl-spermine), 2-diiodol-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (2, 2-dilinoleyl-4- (2-dimethylaminoethyl) - [1,3] -dioxane, DLin-KC 2-DMA), triacontan-6,9,28,31-tetraen-19-yl-4- (dimethylamino) butanoate (heptatriaconta-6,9,28,31-tetraen-19-yl-4- (dimethylamino) butanoate, DLin-MC 3-DMA), A compound of formula (I) as described herein or a combination thereof.

In some embodiments, the cationic lipid is preferably an ionizable cationic lipid. The ionizable cationic lipid carries a net positive charge at, for example, an acidic pH, and is neutral at a higher pH (e.g., physiological pH). Examples of ionizable cationic lipids include, but are not limited to, dioctadecyl amidoglycyl spermine (dioctadecylamidoglycyl spermine, DOGS), N4-cholesteryl-spermine (N4-cholesteryl-spermine), 2-diiodol-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (2, 2-dilinoleyl-4- (2-dimethylaminoethyl) - [1,3] -diolane, DLin-KC 2-DMA), triacontanyl-6,9,28,31-tetraen-19-yl-4- (dimethylamino) butyrate (heptatriaconta-6,9,28,31-tetraen-19-yl-4- (dimethyllamino) butanoate, DLin-MC 3-DMA), compounds of formula (I) as described herein, or combinations thereof.

In one embodiment, the cationic lipid comprises a compound of formula (I):

Wherein,

R ₁ and R ₂ are each independently selected from the group consisting of a bond, C ₁-C₁₂ alkyl and C ₂-C₁₂ alkenyl;

R ₃ and R ₄ are each independently selected from C ₁-C₁₂ alkyl, C ₂-C₁₂ alkenyl, C ₆-C₁₀ aryl, and 5-10 membered heteroaryl, and R ₃ and R ₄ are each independently optionally substituted with t R ₆, t being an integer selected from 1-5;

Each R ₆ is independently selected from C ₁-C₁₂ alkyl and C ₂-C₁₂ alkenyl;

M ₁ and M ₂ are each independently selected from the group consisting of bond, H, -O-, -S-, -C (O) -, -OC (O) -, -C (O) O-, -OC (O) O-, -SC (S) -, -C (S) S-, 3-10 membered heterocycle, -NR ₇ -, or

R ₅ together with one of M ₁ and M ₂ together with the N atom to which they are attached form a 3-to 10-membered heterocyclic ring, and the corresponding R ₁/R₃ or R ₂/R₄ is not

The heterocyclic ring is optionally substituted with R ₇;

R ₅ is selected from C _3-8 carbocycle, -C _1-12 alkylene-Q, Q is selected from H、-OR₇、-SR₇、-OC(O)R₇、-C(O)OR₇、-N(R₇)C(O)R₇、-N(R₇)S(O)₂R₇、-N(R₇)C(S)R₇、-N(R₇)₂、 cyano, C _3-8 carbocycle, 3-10 membered heterocycle, C ₆-C₁₀ aryl, each optionally substituted with one or more C ₁-C₁₂ alkyl, C ₂-C₁₂ alkenyl, C ₁-C₁₂ alkoxy, C ₆-C₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O);

m and n are each independently an integer selected from 0 to 12;

The alkyl, alkenyl and alkylene groups each optionally being independently interrupted by one or more groups selected from the group consisting of-O-, -S-, -NR ₇-、-C(O)-、-OC(O)-、-C(O)O-、-SC(S)-、-C(S)S-、C_3-8 carbocycle, and the alkyl, alkenyl and alkylene groups each optionally being substituted by one or more R ₇;

R ₇ is each independently selected from H, C ₁-C₁₂ alkyl, C ₂-C₁₂ alkenyl, C ₁-C₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C ₆-C₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, C _3-8 carbocycle, each of which is optionally substituted with one or more C ₁-C₁₂ alkyl, C ₂-C₁₂ alkenyl, C ₁-C₁₂ alkoxy, C ₆-C₁₀ aryl, 5-10 membered heteroaryl, 3-10 membered heterocycle, halogen, hydroxy, oxo (=O).

In one embodiment, the cationic lipid comprises a compound of formula (I):

R ₁ and R ₂ are each independently selected from C ₁-C₁₂ alkyl and C ₂-C₁₂ alkenyl;

R ₃ and R ₄ are each independently selected from the group consisting of C ₁-C₁₂ alkyl, C ₂-C₁₂ alkenyl, C ₆-C₁₀ aryl, and 5-to 10-membered heteroaryl;

Provided that at least one of R ₃ and R ₄ is a C ₆-C₁₀ aryl or a 5-10 membered heteroaryl, and R ₃ and R ₄ are each independently optionally substituted with t R ₆, t is an integer selected from 1-5, R ₆ are each independently selected from C ₁-C₁₂ alkyl and C ₂-C₁₂ alkenyl;

m ₁ and M ₂ are each independently selected from the group consisting of-OC (O) -, -C (O) O-, -OC (O) O-, -SC (S) -and-C (S) S-;

R ₅ is selected from-C _1-12 alkylene-Q, Q is selected from-OR ₇ and-SR ₇,R₇ is independently selected from H, C ₁-C₁₂ alkyl, C ₂-C₁₂ alkenyl, C ₁-C₁₂ alkoxy, carboxylic acid, sulfinic acid, sulfonic acid, sulfonyl, nitro, cyano, amino, carbamoyl, sulfonamide, C ₆-C₁₀ aryl and 5-10 membered heteroaryl;

m and n are each independently an integer selected from 1 to 12.

In a preferred embodiment, the cationic lipid comprises a lipid compound having the structure shown below, or a pharmaceutically acceptable salt thereof:

in some embodiments, the cationic lipid comprises a lipid compound of SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2. In a preferred embodiment, the cationic lipid comprises SW-II-140-2.

Phospholipid

The compositions of the present invention comprise phospholipids which assist in cell penetration of the compositions.

Examples of phospholipids include, but are not limited to, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-2-cholesteryl-sn-glycero-3-phosphorylcholine (18:DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphorylcholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphorylcholine (C48), 1, 2-dioleoyl-2-oleoyl-glycero-sn-3-phosphorylcholine, 2-glycero-phosphorylcholine, hexa-glycero-3-phosphorylcholine (Lyso PC) 1, 2-dioleoyl-sn-glycero-3-phosphate ethanolamine (DOPE), 1, 2-di-phytanoyl-sn-glycero-3-phosphate ethanolamine (ME 16.0 PE), 1, 2-di-stearoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-oleoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-linolenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-docosahexaenoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPG) dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoyl ethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl-based phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) or a combination thereof.

Steroid compounds

The compositions of the present invention comprise a steroid which may act as a structural component of the composition.

Examples of steroids include, but are not limited to, for example, cholesterol, fecal sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassinosteroids, lycorine, ursolic acid, alpha-tocopherol, and derivatives thereof.

Polyethylene glycol modified lipids

As used herein, the term "polyethylene glycol modified lipid" or "PEG lipid" refers to a molecule comprising a polyethylene glycol moiety and a lipid moiety, which is a lipid modified with polyethylene glycol. The PEG lipid may be selected from the non-limiting group consisting of PEG modified phosphatidylethanolamine, PEG modified phosphatidic acid, PEG modified ceramide (PEG-CER), PEG modified dialkylamine, PEG modified diacylglycerol (PEG-DEG), PEG modified dialkylglycerol, or combinations thereof. For example, examples of polyethylene glycol modified lipids include, but are not limited to, 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol, DMG-PEG), 1,2-dioleoyl-rac-glycerol, methoxy-polyethylene glycol (1, 2-Dioleoyl-rac-glycol, methoxypolyethylene Glycol, DOGPEG) and 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-poly (ethylene glycol) (1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly (ethylene glycol), DSPE-PEG).

In one embodiment, the polyethylene glycol modified lipid is DMG-PEG, such as DMG-PEG2000. In one embodiment, the DMG-PEG2000 has the following structure:

wherein n has an average value of 44.

Cationic polymers

As used herein, the term "cationic polymer" refers to any ionic polymer capable of carrying a net positive charge at a specified pH to electrostatically bind nucleic acids. Examples of cationic polymers include, but are not limited to, poly-L-lysine, protamine, polyethylenimine (PEI), or combinations thereof. The polyethyleneimine may be a linear or branched polyethyleneimine.

The term "protamine" refers to arginine-rich low molecular weight basic proteins that are present in sperm cells of various animals (particularly fish) and bind to DNA in place of histones. In a preferred embodiment, the cationic polymer is protamine (e.g., protamine sulfate).

Vectors and host cells

In a further aspect, the invention also provides an expression vector comprising a nucleic acid or combination of nucleic acids of the invention. The expression vector may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The nucleic acid or combination of nucleic acids of the invention may be present in one or more expression vectors.

The invention also provides a host cell comprising a nucleic acid or combination of nucleic acids or expression vector of the invention. The nucleic acids or nucleic acid combinations or expression vectors of the invention may be introduced into a suitable host cell using a variety of methods known in the art. Such methods include, but are not limited to, liposome transfection, electroporation, viral transduction, and calcium phosphate transfection, among others.

In a preferred embodiment, the host cell is used to express the human papillomavirus E6 protein and/or E7 protein of the invention. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, e.g., E.coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Mammalian host cells suitable for expression include, but are not limited to, external human cervical cancer cells (HeLa cells), human embryonic kidney cells (HEK cells, e.g., HEK293 cells), chinese Hamster Ovary (CHO) cells, DC2.4 cells (mouse bone marrow derived dendritic cells), caSki cells (human cervical cancer transfer cells), and other mammalian cells suitable for expression.

Therapeutic application

The present invention provides a polypeptide or combination of polypeptides, nucleic acid or combination of nucleic acids (in particular RNA), composition or vaccine formulation of the invention for use in the treatment of human papillomavirus type 16 infection, human papillomavirus type 16 positive cancer or precancerous lesion, human papillomavirus type 18 infection, human papillomavirus type 18 positive cancer or precancerous lesion in a subject.

The invention provides the use of a polypeptide or a combination of polypeptides, nucleic acids or a combination of nucleic acids (in particular RNA), a composition or a vaccine formulation of the invention for the manufacture of a medicament for the treatment of a human papillomavirus type 16 infection, a human papillomavirus type 16 positive cancer or a precancerous lesion, a human papillomavirus type 18 infection, a human papillomavirus type 18 positive cancer or a precancerous lesion in a subject.

The present invention provides a method for treating a human papillomavirus type 16 infection, a human papillomavirus type 16 positive cancer or a precancerous condition, a human papillomavirus type 18 infection, a human papillomavirus type 18 positive cancer or a precancerous condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide or combination of polypeptides, nucleic acid or combination of nucleic acids (particularly RNA), composition or vaccine formulation of the invention. In one embodiment, the method comprises administering a therapeutically effective amount of a composition comprising a polypeptide or combination of polypeptides, or a nucleic acid or combination of nucleic acids (particularly RNA), of the invention, particularly a composition comprising LNP or LPP as described herein.

In some embodiments, human papillomavirus type 16 positive cancers include, but are not limited to, cervical cancer, anogenital cancer, penile cancer, and head and neck cancers, such as genital cancer, head and neck squamous cell carcinoma.

In some embodiments, human papillomavirus type 18 positive cancers include, but are not limited to, cervical cancer, anogenital cancer, penile cancer, and head and neck cancers, such as genital cancer, head and neck squamous cell carcinoma.

The term "therapeutically effective amount" refers to an amount sufficient to inhibit the occurrence of a disease or symptom and/or to slow, alleviate, delay the progression or severity of a disease or symptom. The therapeutically effective amount is affected by factors including, but not limited to, the rate and severity of the disease or condition development, the age, sex, weight and physiological condition of the subject, the duration of the treatment, and the particular route of administration. The therapeutically effective amount may be administered in one or more doses. A therapeutically effective amount may be achieved by continuous or intermittent administration.

In some embodiments, the therapeutically effective amount is provided in one or more administrations. In some embodiments, the therapeutically effective amount is provided in two administrations. In some embodiments, the therapeutically effective amount is provided in three administrations. In some embodiments, the therapeutically effective amount is provided in four administrations. In some embodiments, the therapeutically effective amount is provided in five administrations. In some embodiments, the therapeutically effective amount is provided in six administrations. In some embodiments, the therapeutically effective amount is provided in seven administrations. In some embodiments, the therapeutically effective amount is provided in eight administrations. In some embodiments, the therapeutically effective amount is provided in more than eight administrations.

In some embodiments, the polypeptide or combination of polypeptides, nucleic acid or combination of nucleic acids (in particular RNA), composition or vaccine formulation of the invention may be administered to a subject by any method known to those skilled in the art, such as parenterally, orally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously or intraperitoneally. Preferably, the composition or vaccine formulation of the invention is administered by subcutaneous injection.

As used herein, the term "subject" describes an organism, e.g., a mammal, to which treatment of immunity using a polypeptide or combination of polypeptides, nucleic acid or combination of nucleic acids (particularly RNA), composition or vaccine formulation of the present invention may be provided. In some embodiments, the subject is a mouse. In a preferred embodiment, the subject is a human.

In some embodiments, the uses and methods of the invention further comprise administering an anti-PD-L1 antibody or antigen-binding fragment thereof.

Combination therapy

For human papillomavirus type 16 positive cancers or in human papillomavirus type 18 positive cancer treatment, the polypeptides or polypeptide combinations, nucleic acids or nucleic acid combinations (particularly RNA), compositions or vaccine formulations of the invention may be used in combination with other therapeutic methods including, but not limited to, surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, angiogenesis inhibition and palliative therapy.

The polypeptide or combination of polypeptides, nucleic acid or combination of nucleic acids (in particular RNA), composition or vaccine formulation of the invention may also be administered in combination with at least one or more therapeutic agents described herein. The mode of administration of the combination is not limited. For example, the therapeutic agents described below may be administered all at once or separately. When administered separately (in the case of using mutually different administration regimens), they can be administered continuously without interruption or at predetermined intervals.

In certain embodiments, the polypeptide or combination of polypeptides, nucleic acid or combination of nucleic acids (particularly RNA), composition or vaccine formulation of the invention is further administered in combination with one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors and tumor antigen targeting drugs. In a preferred embodiment, the polypeptide or combination of polypeptides, nucleic acid or combination of nucleic acids (in particular RNA), composition or vaccine formulation of the invention is administered in combination with an immune checkpoint inhibitor.

In one embodiment, the polypeptide or combination of polypeptides, nucleic acid or combination of nucleic acids (in particular RNA), composition or vaccine formulation of the invention is administered in combination with an anti-PD-L1 antibody or antigen-binding fragment thereof, thereby mediating a more excellent tumor inhibiting effect.

Kit for detecting a substance in a sample

The invention also provides a kit comprising a polypeptide or combination of polypeptides, a nucleic acid or combination of nucleic acids (particularly RNA), a composition or vaccine formulation of the invention, and instructions for use. The kit may also comprise a suitable container. In certain embodiments, the kit further comprises a device for administering the drug. Kits generally include a label that indicates the intended use and/or method of use of the kit contents. The term "label" includes any written or recorded material provided on or with or otherwise with the kit.

In one embodiment, the kit comprises a polypeptide or combination of polypeptides, a nucleic acid or combination of nucleic acids (particularly RNA), a composition or vaccine formulation, and one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, and tumor antigen targeting drugs.

In one embodiment, the kit comprises a polypeptide or combination of polypeptides, a nucleic acid or combination of nucleic acids (particularly RNA), a composition or vaccine formulation, and an immune checkpoint inhibitor.

In one embodiment, the kit comprises a polypeptide or combination of polypeptides, a nucleic acid or combination of nucleic acids (particularly RNA), a composition or vaccine formulation, and an anti-PD-L1 antibody or antigen-binding fragment thereof.

The present invention provides a kit of the invention for treating a papillomavirus type 16 infection, a human papillomavirus type 16 positive cancer or a precancerous lesion, a human papillomavirus type 18 infection, a human papillomavirus type 18 positive cancer or a precancerous lesion in a subject in need thereof.

The invention provides the use of a kit of the invention in the manufacture of a medicament for treating a papillomavirus type 16 infection, a human papillomavirus type 16 positive cancer or a precancerous lesion, a human papillomavirus type 18 infection, a human papillomavirus type 18 positive cancer or a precancerous lesion in a subject in need thereof.

The present invention provides a method for treating papillomavirus type 16 infection, human papillomavirus type 16 positive cancer or precancerous condition, human papillomavirus type 18 infection, human papillomavirus type 18 positive cancer or precancerous condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent (e.g., a polypeptide or combination of polypeptides, nucleic acid or combination of nucleic acids (particularly RNA), composition or vaccine formulation) in a kit of the invention.

In some embodiments, the human papillomavirus type 16 positive cancer or human papillomavirus type 18 positive cancer includes, but is not limited to, cervical cancer, anogenital cancer, penile cancer, and head and neck cancer, such as genital cancer, head and neck squamous cell carcinoma.

Advantageous effects

The polypeptide or polypeptide combination, nucleic acid or nucleic acid combination, composition, vaccine preparation and kit aiming at HPV type 16 can show excellent effects, such as but not limited to (1) inducing a powerful cellular immune response, and showing excellent immunogenicity even at low dosage, (2) maintaining the immune efficacy for 3 months or longer, (3) inducing protective memory T cells, helping to clear tumor cells positive by HPV related antigens of the corresponding type encountered again in the body, thereby preventing tumor recurrence, helping the body maintain a long-term tumor-free state, (4) slowing down the growth rate of HPV-16 positive tumors, (5) mediating complete regression of HPV-16 positive tumors, (6) improving survival rate, (7) having excellent safety, (8) the polypeptide or polypeptide combination, nucleic acid or nucleic acid combination, composition, vaccine preparation and immune checkpoint inhibitor can further improve tumor suppression effect and survival rate by combined administration, (9) can be used with chemotherapy, radiotherapy, targeted therapy, immune checkpoint inhibitor and the like, so as to enhance therapeutic effect.

The polypeptide, polynucleotide, composition, vaccine preparation and kit aiming at HPV18 type can show excellent effects, such as but not limited to (1) high expression in cells, (2) induction of strong cellular immune response, (3) induction of protective memory T cells, help to clear tumor cells positive with corresponding HPV related antigens encountered again in a body, thereby preventing tumor recurrence, helping the body maintain a long-term tumor-free state, (4) slowing down growth rate of HPV-18 positive tumors, (5) mediating complete regression of HPV-18 positive tumors, (6) improving survival rate, (7) having good safety, and (8) combined administration of the polypeptide, polynucleotide, composition, vaccine preparation and immune checkpoint inhibitor can further improve tumor inhibition effect and survival rate, and (9) can be used with chemotherapy, radiotherapy, targeted therapy, immunotherapy and the like to enhance therapeutic effect.

Exemplary embodiments

Embodiment 1. A polypeptide or combination of polypeptides wherein the polypeptide or combination of polypeptides comprises a signal peptide sequence at the N-terminus and an MHC-I transmembrane-intracellular domain sequence at the C-terminus, and the polypeptide or combination of polypeptides comprises the amino acid sequence of a human papillomavirus HPV 16E 6 protein or immunogenic fragment thereof, and/or the amino acid sequence of a human papillomavirus HPV 16E 7 protein or immunogenic fragment thereof, or

The polypeptide or polypeptide combination comprises the amino acid sequence of human papillomavirus HPV 18E 6 protein or an immunogenic fragment thereof and/or the amino acid sequence of human papillomavirus HPV 18E 7 protein or an immunogenic fragment thereof,

The human papillomavirus HPV 16E 6 protein comprises an amino acid sequence shown in SEQ ID NO. 105 or an amino acid sequence with at least 95% of the same as the amino acid sequence shown in SEQ ID NO. 105, the human papillomavirus HPV 16E 7 protein comprises an amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence with at least 95% of the same as the amino acid sequence shown in SEQ ID NO. 6, the human papillomavirus HPV 18E 6 protein comprises an amino acid sequence shown in SEQ ID NO. 131 or an amino acid sequence with at least 95% of the same as the amino acid sequence shown in SEQ ID NO. 131, and the human papillomavirus HPV 18E 7 protein comprises an amino acid sequence shown in SEQ ID NO. 79 or an amino acid sequence with at least 95% of the same as the amino acid sequence shown in SEQ ID NO. 79.

Embodiment 2. The polypeptide or combination of polypeptides of embodiment 1, wherein the signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO. 7 or 77, and/or wherein the transmembrane-intracellular domain sequence of MHC-I comprises the amino acid sequence shown in SEQ ID NO. 8.

Embodiment 3. The polypeptide or combination of polypeptides of embodiments 1 or 2 further comprises one or more linkers, preferably a GS linker, more preferably the linker comprises the amino acid sequence shown in SEQ ID NO 9 or 10.

Embodiment 4. The polypeptide or polypeptide combination of embodiment 1, comprising the amino acid sequence shown in SEQ ID NO. 106 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 106, and/or the amino acid sequence shown in SEQ ID NO. 5 or 78 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 5 or 78, or the amino acid sequence shown in SEQ ID NO. 132 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 132, and/or the amino acid sequence shown in SEQ ID NO. 80 or an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO. 80.

Embodiment 5. A nucleic acid or combination of nucleic acids comprising a polynucleotide encoding a human papillomavirus HPV 16E 6 protein or an immunogenic fragment thereof as defined in any one of embodiments 1 to 4, wherein said polynucleotide is RNA comprising the nucleotide sequence shown in one of SEQ ID NOS 124, 125 and 126 or having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NOS 124, 125 and 126, and/or a polynucleotide encoding a human papillomavirus HPV 16E 7 protein or an immunogenic fragment thereof as defined in any one of embodiments 1 to 4, wherein said polynucleotide is RNA comprising the nucleotide sequence shown in one of SEQ ID NOS 17, 20, 63, 64, 65, 66, 67, 68 and 69 or having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NOS 17, 20, 63, 64, 65, 66, 67, 68 and 69, or

The nucleic acid or combination of nucleic acids comprising a polynucleotide encoding a human papillomavirus HPV 18E 6 protein or an immunogenic fragment thereof as defined in any one of embodiments 1 to 4 wherein the polynucleotide is RNA comprising the nucleotide sequence shown in one of SEQ ID NOS: 150, 151 and 152 or having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NOS: 150, 151 and 152 and/or the nucleic acid or combination of nucleic acids comprising a polynucleotide encoding a human papillomavirus HPV 18E 7 protein or an immunogenic fragment thereof as defined in any one of embodiments 1 to 4 wherein the polynucleotide is RNA comprising the nucleotide sequence shown in one of SEQ ID NOS: 98, 99 and 100 or having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NOS: 98, 99 and 100.

Embodiment 6. A nucleic acid or combination of nucleic acids comprising a nucleotide sequence encoding a human papillomavirus HPV 16E 6 protein or an immunogenic fragment thereof as defined in any one of embodiments 1to 4, wherein the polynucleotide is DNA comprising the nucleotide sequence shown in one of SEQ ID NOS: 128, 129 and 130 or having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NOS: 128, 129 and 130, and/or a polynucleotide encoding a human papillomavirus HPV 16E 7 protein or an immunogenic fragment thereof as defined in any one of embodiments 1to 4, wherein the polynucleotide is DNA comprising the nucleotide sequence shown in one of SEQ ID NOS: 18, 42, 70, 71, 72, 73, 74, 75 and 76 or having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NOS: 18, 42, 70, 71, 72, 73, 74, 75 and 76, or

The nucleic acid or combination of nucleic acids comprising a polynucleotide encoding a human papillomavirus HPV 18E 6 protein or an immunogenic fragment thereof as defined in any one of embodiments 1 to 4, wherein the polynucleotide is DNA comprising the nucleotide sequence shown in one of SEQ ID NOS: 154, 155 and 156 or having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NOS: 154, 155 and 156, and/or the nucleic acid or combination of nucleic acids comprising a polynucleotide encoding a human papillomavirus HPV 18E 7 protein or an immunogenic fragment thereof as defined in any one of embodiments 1 to 4, wherein the polynucleotide is DNA comprising the nucleotide sequence shown in one of SEQ ID NOS: 102, 103 and 104 or having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NOS: 102, 103 and 104.

Embodiment 7. A nucleic acid or combination of nucleic acids comprising a polynucleotide encoding a polypeptide or combination of polypeptides of any one of embodiments 1-4.

Embodiment 8 the nucleic acid or nucleic acid combination of embodiment 7 wherein the polynucleotide is RNA comprising the nucleotide sequence set forth in one of SEQ ID NOS: 107, 108, 109 and 110 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS: 107, 108, 109 and 110 and/or the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS: 1, 21, 22, 23, 24, 25, 26, 27, 28 and 29 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS: 1, 21, 22, 23, 24, 25, 26, 27, 28 and 29, or

The polynucleotide comprises the nucleotide sequence shown as one of SEQ ID NO. 133, 134, 135 and 136 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown as one of SEQ ID NO. 133, 134, 135 and 136, and/or the polynucleotide comprises the nucleotide sequence shown as one of SEQ ID NO. 81, 82, 83 and 84 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown as one of SEQ ID NO. 81, 82, 83 and 84.

Embodiment 9. The nucleic acid or nucleic acid combination of embodiment 8, wherein the polynucleotide further comprises a 5'-UTR sequence, preferably the 5' -UTR sequence comprises the nucleotide sequence of SEQ ID NO:11, the polynucleotide further comprises a 3'-UTR sequence, preferably the 3' -UTR sequence comprises the nucleotide sequence of SEQ ID NO:12, and/or the polynucleotide further comprises a poly (A) sequence, preferably the poly (A) sequence comprises the nucleotide sequence of SEQ ID NO: 15.

Embodiment 10. The nucleic acid or nucleic acid combination of embodiment 7 wherein the polynucleotide is RNA and the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOs 111, 112, 113 and 114 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOs 111, 112, 113 and 114 and/or the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOs 2, 32, 34, 35, 36, 37, 38, 39 and 40 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOs 2, 32, 33, 34, 35, 36, 37, 38, 39 and 40, or

The polynucleotide comprises the nucleotide sequence shown as one of SEQ ID NO. 137, 138, 139 and 140 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown as one of SEQ ID NO. 137, 138, 139 and 140, and/or the polynucleotide comprises the nucleotide sequence shown as one of SEQ ID NO. 85, 86, 87 and 88 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown as one of SEQ ID NO. 85, 86, 87 and 88.

Embodiment 11. The nucleic acid or nucleic acid combination of embodiment 7 wherein the polynucleotide is DNA, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NOS 115, 116, 117 and 118 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown in SEQ ID NOS 115, 116, 117 and 118, and/or the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NOS 3, 43, 44, 45, 46, 47, 48, 49, 50 and 51 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NOS 3, 43, 44, 45, 46, 47, 48, 49, 50 and 51, or

The polynucleotide comprises the nucleotide sequence shown as one of SEQ ID NO 141, 142, 143 and 144 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown as one of SEQ ID NO 141, 142, 143 and 144, and/or the polynucleotide comprises the nucleotide sequence shown as one of SEQ ID NO 89, 90, 91 and 92 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown as one of SEQ ID NO 89, 90, 91 and 92.

Embodiment 12. The nucleic acid or nucleic acid combination of embodiment 11, the polynucleotide further comprises a DNA sequence corresponding to a 5'-UTR sequence, preferably the DNA sequence corresponding to the 5' -UTR sequence comprises the nucleotide sequence of SEQ ID NO:13, the polynucleotide further comprises a DNA sequence corresponding to a 3'-UTR sequence, preferably the DNA sequence corresponding to the 3' -UTR sequence comprises the nucleotide sequence of SEQ ID NO:14, and/or the polynucleotide further comprises a DNA sequence corresponding to a poly (A) sequence, preferably the DNA sequence corresponding to the poly (A) sequence comprises the nucleotide sequence of SEQ ID NO: 16.

Embodiment 13 the polynucleotide of embodiment 7 wherein the polynucleotide is DNA comprising the nucleotide sequence set forth in one of SEQ ID NOS 119, 120, 121 and 122 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS 119, 120, 121 and 122 and/or the polynucleotide comprises the nucleotide sequence set forth in one of SEQ ID NOS 4, 54, 55, 56, 57, 58, 59, 60, 61 and 62 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOS 4, 54, 55, 56, 57, 58, 59, 60, 61 and 62, or

The polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO. 145, 146, 147 and 148 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NO. 145, 146, 147 and 148 and/or the polynucleotide comprises the nucleotide sequence shown in one of SEQ ID NO. 93, 94, 95 and 96 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NO. 93, 94, 95 and 96.

Embodiment 14. A composition comprising the polypeptide or combination of polypeptides of any one of embodiments 1-4.

Embodiment 15. A composition comprising the nucleic acid or combination of nucleic acids of any one of embodiments 5-13.

Embodiment 16. The composition of embodiment 15, comprising a lipid encapsulating the nucleic acid or nucleic acid combination.

Embodiment 17 the composition of embodiment 15 or 16 comprising a lipid nanoparticle or a lipid multimeric complex.

Embodiment 18. The composition of embodiment 17, wherein the lipid encapsulating the nucleic acid or nucleic acid combination comprises a cationic lipid, a phospholipid, a steroid, and a polyethylene glycol modified lipid, optionally, the composition further comprises a cationic polymer, wherein the cationic polymer associates with the nucleic acid or nucleic acid combination as a complex, and is co-encapsulated in the lipid to form a lipid-multimeric complex, preferably, the cationic polymer is protamine.

Embodiment 19 the composition of embodiment 18 wherein the cationic lipid comprises a compound of formula (I), or a pharmaceutically acceptable salt thereof

Wherein,

The heterocyclic ring is optionally substituted with R ₇;

m and n are each independently an integer selected from 0 to 12;

Embodiment 20 the composition of embodiment 19, wherein the cationic lipid comprises a lipid compound having the structure shown below, or a pharmaceutically acceptable salt thereof:

Preferably, the cationic lipid is SW-II-115, SW-II-121, SW-II-122, SW-II-134-3, SW-II-138-2, SW-II-139-2 or SW-II-140-2, more preferably, the cationic lipid is SW-II-140-2.

Embodiment 21. The composition of any of embodiments 15-20, comprising 40 mole% SW-II-140-2, 15 mole% DOPE, 43.5 mole% cholesterol, and 1.5 mole% DMG-PEG.

Embodiment 22. A vaccine formulation comprising the polypeptide or combination of polypeptides of any one of embodiments 1-4 or the composition of embodiment 14.

Embodiment 23 a vaccine formulation comprising the nucleic acid or combination of nucleic acids of any one of embodiments 5-13 or the composition of any one of embodiments 15-21.

Embodiment 24. An expression vector comprising the nucleic acid or combination of nucleic acids of any one of embodiments 5-13.

Embodiment 25 a host cell comprising the nucleic acid or combination of nucleic acids of any one of embodiments 5-13 or the expression vector of embodiment 24.

Embodiment 26. A kit comprising the polypeptide or combination of polypeptides of any of embodiments 1-4, the nucleic acid or combination of nucleic acids of any of embodiments 5-13, the composition of any of embodiments 14-21 or the vaccine formulation of embodiments 22 or 23, and one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors, and tumor antigen targeting drugs, preferably the therapeutic agent is an anti-PD-L1 antibody or antigen binding fragment thereof.

Use of the polypeptide or combination of polypeptides of any of embodiments 1-4, the nucleic acid or combination of nucleic acids of any of embodiments 5-13, the composition of any of embodiments 14-21, the vaccine formulation of embodiments 22 or 23, or the kit of embodiment 26 in the manufacture of a medicament for treating a human papillomavirus type 16 infection, a human papillomavirus type 16 positive cancer or a precancerous condition, a human papillomavirus type 18 infection, a human papillomavirus type 18 positive cancer or a precancerous condition in a subject.

Embodiment 28 the use of embodiment 27, wherein said human papillomavirus type 16 positive cancer or said human papillomavirus type 18 positive cancer comprises cervical cancer, anogenital cancer, penile cancer, head and neck cancer, e.g., genital cancer, head and neck squamous cell carcinoma.

Examples

The invention is further described by reference to the following examples. It should be understood that these embodiments are by way of example only and are not limiting of the invention. The following materials and instruments are commercially available or prepared according to methods well known in the art. The following experiments were performed according to the manufacturer's instructions or according to methods and procedures well known in the art.

Example 1 preparation of mRNA

Design and Synthesis of DNA templates

The applicant designed and synthesized a codon optimized DNA Open Reading Frame (ORF) sequence of E7 protein or E6 protein of human papillomavirus HPV16 comprising a signal peptide sequence sec1.0 (SEQ ID NO: 7) or sec2.0 (SEQ ID NO: 77) and a GS linker (SEQ ID NO: 9) at the N-terminus and a transmembrane-intracellular domain (MITD) sequence of the GS linker (SEQ ID NO: 10) and MHC-I (SEQ ID NO: 8) at the C-terminus. The nucleotide sequences of nucleic acids (designated as 22#, 29#, 31#, 33#, 24#, 47#, 50#, 51#, respectively) encoding HPV 16E 7 proteins (SEQ ID NO: 106) comprising signal peptide sequences and MITD at both ends, respectively, and nucleic acids (designated as E6-2, E6-3, E6-4, respectively) encoding the same form of HPV 16E 6 protein (SEQ ID NO: 106) are shown in Table 1.

Meanwhile, the applicant designed and synthesized the original, non-optimized, HPV16E 7-encoding protein alone (SEQ ID NO: 6), a codon-optimized DNA ORF sequence (designated 8#) of the HPV16E7 protein encoding only HPV16E7 protein (designated 10#), an original, non-optimized DNA sequence (designated 19) of the HPV16E7 protein comprising the signal peptide sequence sec2.0 (SEQ ID NO: 77) and the GS linker (SEQ ID NO: 9) at the N-terminus and the transmembrane-intracellular domain (MITD) sequence (SEQ ID NO: 8) of the GS linker (SEQ ID NO: 10) and an original, non-optimized DNA ORF sequence (designated 18#) of the HPV16E7 protein encoding only the HPV16E7 protein, an original, non-optimized DNA sequence (designated 18#) comprising the signal peptide sequence sec1.0 (SEQ ID NO: 7) and the GS linker (SEQ ID NO: 9) and the transmembrane-intracellular domain (MITD) sequence (SEQ ID NO: 8) of the GS 16E7 protein comprising the GS linker (SEQ ID NO: 10) and the MHC-I as a control sequence (SEQ ID NO: 34) and the transmembrane-intracellular domain (SEQ ID NO: 8) of the original, non-optimized DNA sequence (designated 18#) encoding the HPV16E7 protein comprising the signal peptide sequence sec1.0 (SEQ ID NO: 9) and the GS linker (SEQ ID NO: 8) and the transmembrane-intracellular domain (MITD) of the DNA sequence (SEQ ID NO: 8), the corresponding nucleotide sequences are also shown in Table 1.

The applicant has also designed and synthesized a codon optimized DNA Open Reading Frame (ORF) sequence encoding the E7 protein of human papillomavirus HPV18 comprising the N-terminal comprising the signal peptide sequence sec1.0 (SEQ ID NO: 7) and the GS linker (SEQ ID NO: 9) and the C-terminal comprising the transmembrane-intracellular domain (MITD) sequence of the GS linker (SEQ ID NO: 10) and MHC-I (SEQ ID NO: 8). The nucleotide sequences of nucleic acids (designated 18-2, 18-3 and 18-4, respectively) encoding HPV 18E7 proteins (SEQ ID NO: 80) comprising SP and MITD at both ends are shown in Table 2. Meanwhile, the applicant designed and synthesized the original, non-optimized DNA ORF sequence (designated 18-1) of HPV 18E7 protein comprising the signal peptide sequence sec1.0 (SEQ ID NO: 7) and the GS linker (SEQ ID NO: 9) at the N-terminus and the transmembrane-intracellular domain (MITD) domain sequence (SEQ ID NO: 8) of MHC-I at the C-terminus as a control, the corresponding nucleotide sequences of which are also shown in Table 2.

Furthermore, the applicant designed and synthesized a codon optimized DNA ORF sequence encoding HPV 18E6 protein comprising the signal peptide sequence sec1.0 (SEQ ID NO: 7) and the GS linker (SEQ ID NO: 9) at the N-terminus and the transmembrane-intracellular domain (MITD) sequence of the GS linker (SEQ ID NO: 10) and MHC-I (SEQ ID NO: 8) at the C-terminus. The nucleotide sequences of nucleic acids (designated 18E6-2, 18E6-3, 18E6-4, respectively) encoding HPV 18E6 protein (SEQ ID NO: 132) comprising a signal peptide sequence and MITD at both ends, respectively, are shown in Table 2. Meanwhile, the applicant designed and synthesized the original, non-optimized DNA ORF sequence (designated 18E 6-1) of HPV 18E6 protein comprising the signal peptide sequence sec1.0 (SEQ ID NO: 7) and the GS linker (SEQ ID NO: 9) at the N-terminus and the transmembrane-intracellular domain (MITD) domain sequence (SEQ ID NO: 8) of MHC-I at the C-terminus as a control, the corresponding nucleotide sequences of which are also shown in Table 2.

T7 promoter sequences (TAATACGACTCACTATA, SEQ ID NO: 157), 5'-UTR sequences (SEQ ID NO: 13) and 3' -UTR sequences (SEQ ID NO: 14) were also designed. The Kozak sequence "GCCACC" is contained in the 5' -UTR sequence (SEQ ID NO: 13).

Then, the DNA was ligated in the order of T7 promoter sequence, 5'-UTR, DNA ORF, 3' -UTR and PolyA, and total gene synthesis (Nanjing Jinsri Biotechnology Co., ltd.) was carried out using pUC57 as a vector, to obtain a plasmid DNA template.

The plasmid was linearized using restriction endonucleases and purified (Takara purification kit) to obtain the linearized plasmid, i.e., the DNA template required for in vitro transcription of mRNA.

1.2. In vitro transcription of mRNA from DNA templates

Co-transcription capping reactions were performed by T7 RNA polymerase, in vitro transcription of RNA was performed, resulting in Cap1 mRNA. In vitro transcription 1-methyl-pseudouridine triphosphate was used instead of Uridine Triphosphate (UTP), and therefore the modification ratio of 1-methyl-pseudouracil in vitro transcribed Cap1 mRNA was 100%. After transcription, the DNA template was digested with dnaseli (sameil technologies limited) to reduce the risk of residual DNA template.

MRNA was purified using DynabeadsMyone (Semer Feishul technologies Co., ltd.). Purified mRNA was dissolved in 1mM sodium citrate buffer (pH 6.5+/-0.1), sterile filtered, and cryopreserved at-80℃until use. The mRNA sequences obtained are shown in tables 1 and 2.

TABLE 1 nucleic acids encoding the E6 and E7 proteins of human papillomavirus HPV 16

TABLE 2 nucleic acids encoding E6 and E7 proteins of human papillomavirus HPV 18

EXAMPLE 2 expression of candidate HPV16 mRNA in cells

To further examine the expression of designed and synthesized candidate mRNA in cells, applicant validated the expression of mRNA for HPV type 16 prepared as in example 1.2 in DC2.4 cells. The specific method is that dendritic cells DC2.4 cells (Shanghai cell bank) derived from mouse bone marrow are seeded in 6-well plates at 1.2X10 ⁶ cells/well. After 18 hours of seeding the cells, the corresponding mRNA stock solution was transfected into DC2.4 cells using transfection reagent LipofectamineMessengerMax (Invitrogen, LMRNA 015), 2 μ gmRNA per well of cells. The transfected cells were placed in a cell incubator and cultured for an additional 24 hours at 37℃with 5% CO ₂. Then, the cells were collected, the collected cells were treated with 6xloading buffer (whole gold, DL 101-02) and heated at 100 ℃ for 10min, and the treated cell samples were subjected to polyacrylamide gel electrophoresis and transferred onto PVDF membrane, which was sealed with 5% nonfat milk powder at room temperature for 1h. HPV 16E7 antibody (HPV Type 16E7 Antibody,Invitrogen#28-0006) diluted 1:200 was used as primary antibody, incubated overnight at 4℃with shaking, washed 5 times with PBST (PBS solution containing 0.5% Tween 20), incubated 1h at room temperature as secondary antibody diluted 1:5000 (Goat anti-Mouse IgG1Cross-Adsorbed Secondary Antibody, HRP Invitrogen#A10551), washed 5 times with PBST, and developed and imaged with ECL luminescence (Pierce ^TM ECL Plus Western Blotting Substrate,Thermo Scientific^TM #32132X3). Wherein DC CTRL is a DC2.4 cell that was not transfected with mRNA (as a negative control).

As a result, as shown in FIG. 1, the expression of mRNA (10#, 22#, 24#, 29#, 31#, 33#, 47#, 50# and 51# mRNA) after sequence optimization was significantly increased compared to wild-type mRNA 8#, which encodes HPV 16E 7 protein. Under the same conditions, the 29#mRNA with optimized sequence expresses the most E7 protein with the highest expression efficiency, so that the method is used as a preferable scheme and is further tested.

EXAMPLE 3 preparation of mRNA vaccine formulations

This example uses a lipopolymer Lipopolyplex (LPP) delivery system to deliver mRNA. LPP is a nano-delivery system of "core-shell" structure. The negatively charged mRNA is first microfluidic mixed with the positively charged polymer to form a core structure of uniform particle size. The lipid molecule mixture (core ionizable lipid molecule SW-II-140-2, helper phospholipid DOPE, pegylated lipid molecule PEG-DMG2K, and cholesterol) dissolved in the organic phase is then subjected to a second step of microfluidic mixing with the core structure to form uniform LPP-mRNA nanoparticles.

3.1. Experimental materials

Cationic lipid SW-II-140-2 was synthesized by Sterculia (see WO2022/233291 for specific methods of synthesis), helper phospholipid (DOPE) purchased from CordenPharma, cholesterol purchased from Sigma-Aldrich, mPEG2000-DMG (i.e., DMG-PEG 2000 or PEG-DMG 2K) purchased from AvantiPolarLipids, inc., PBS purchased from Invitrogen, and protamine sulfate purchased from Beijing-S Lian pharmaceutical Co.

Preparation of lipid multimeric complexes (LPP) of mRNA

Preparation of aqueous mRNA solution mRNA prepared in example 1.2 was diluted to 0.2mg/mL of mRNA solution with 10mM citric acid-sodium citrate buffer (pH 4.0).

Preparation of lipid solution cationic lipid (SW-II-140-2) DOPE cholesterol mPEG2000-DMG was dissolved in absolute ethanol at a molar ratio of 40:15:43.5:1.5 to prepare a lipid solution of 10 mg/mL.

The preparation of the protamine sulfate solution comprises the step of dissolving protamine sulfate in water without a nuclease to prepare the protamine sulfate solution with the working concentration of 0.25 mg/mL.

Preparation of a core nanoparticle (core nanoparticle) solution core nanoparticle solution formed from protamine and mRNA was obtained by mixing a solution of protamine sulfate with a solution of mRNA using microfluidic technology (model: inanoD, available from maianan technologies, inc.) under the following conditions: volume=4.0 mL, flow rate ratio=5 (mRNA): 1 (protamine solution), total flow rate=12 mL/min, pre-waste (START WASTE) =0.35 mL, post-waste (end waste) =0.1 mL, room temperature.

Preparation of LPP the core nanoparticle solution was mixed twice with the lipid solution at volume=4.0 mL, flow rate ratio=1 (lipid solution): 3 (core nanoparticle solution), total flow rate=12 mL/min, front waste=0.35 mL, rear waste=0.1 mL, room temperature, diluted with PBS to obtain LPP solution.

Centrifugal ultrafiltration the LPP solution was centrifuged by ultrafiltration to remove ethanol (rotation speed 3000g, centrifugation time 60min, temperature 4 ℃) to obtain an LPP preparation containing mRNA prepared in example 1.2. Among them, the LPP preparation containing 29#mRNAs was designated SW0128, and the LPP preparations containing mRNAs 18-1, 18-2, 18-3, 18-4 were designated 1801, 1802, 1803, 1804, respectively.

Example 4SW0128 HPV16 LPP vaccine formulations induce cellular immune responses

4.1 Laboratory mice

Female, 6 week old C57BL/6NTac mice from Tacouc corporation and C57BL/6J mice from Vetong Lihua were used. C57BL/6NTac and C57BL/6J mice were kept and cared for at Shanghai model biological center Co.Ltd. Animal studies were strictly performed according to the recommendations in Shanghai laboratory animal feeding administration and use guidelines.

4.2 Experimental procedures and results

To evaluate the immunogenicity of the LPP formulation SW0128 prepared in example 3.2, this example immunized C57BL/6NTac mice with SW0128 and evaluated the level of activation of specific CD8 ⁺ T cell immune responses in the mice.

Mice were randomly divided into 4 groups (n=10 mice per group). On day 0 (D0), the mice in each group were given PBS, SW0128 mRNA LPP vaccine containing 3 μg, 10 μg, 30 μg gmRNA, and 5 mice were sacrificed on day 6 (D6), spleen lymphocytes from the mice were collected, and the IFN-. Gamma.ELISPot effect after one immunization was measured, on day 7 (D7), the remaining mice were given a second immunization by subcutaneous two-point injection, the remaining mice were sacrificed on day 13 (D13), the spleen cells were collected, and the IFN-. Gamma.ELISPot effect after two-immunization was measured.

ELISA spot (ELISPot) assay

The mouse IFN-. Gamma.ELISpot assay was performed using the IFN-. Gamma. ELISpotPLUS kit (Mabtech, 3321-4 APT-10) according to the manufacturer's instructions. Briefly, plates were blocked in RPMI 1640 medium (supplemented with 10% fbs) and incubated for 30 min. Spleen cells were plated at 3×10 ⁵ cells/well and stimulated in vitro with 2 μg/mlHPV E7 polypeptide (RAHYNIVTF, gill biochemistry), unrelated 2 μg/mL vesicular stomatitis virus VSV polypeptide (RGYVYQGL, gill biochemistry), positive stimulation 500ng/mL phorbol ester (PMA) +10 μg/mL Ionomycin (Ionomycin) (daceae, 2030421) and PBS only (negative control), incubated for 20 hours at 37 ℃,5% co 2. After that, with biotinylated IFN-. Gamma. -detection antibody and streptavidin-alkaline phosphatase (ALP), BCIP/NBT-plus (5-bromo-4-chloro-3-indole-phosphate/nitro blue tetrazolium-plus) substrate was added for color development and counted with an ELISPOT reader (ImmunoSpot S6 Core Analyzer (CTL)).

As shown in fig. 2B and 2C, SW0128 was effective in activating E7 polypeptide specific CD8 ⁺ T cells 6 days after a (D0) challenge (D6), and this cellular immune response exhibited a dose-dependent, with increased IFN- γ -spots, i.e., an increased cellular immune response, as the SW0128 immune dose was increased. Meanwhile, as shown in fig. 2D, activation of this specific CD8 ⁺ T cell immunity was further enhanced 6 days after the second (D7) (D13).

To further investigate whether immunization with lower doses of SW0128 also activated specific CD8 ⁺ T cell immunization, applicant immunized and followed mice with SW0128 containing 0.3 μg, 1 μ gmRNA for a long period of time.

In this experiment, mice were randomly divided into 5 groups of 20 mice each. On day 0 (D0), priming (one-shot) was performed by subcutaneous two-spot immunization, PBS, SW0128 mRNA LPP vaccine containing 0.3 μg, 1 μg, 3 μg gmRNA, or LPP preparation containing 3 μ GEGFPMRNA as a control was injected into each group of mice, 5 mice were sacrificed on day 6 (D6), spleen lymphocytes of the mice were collected, IFN-gamma ELISPOT effect after one-shot was tested, on day 7 (D7), the remaining mice were subjected to second-shot immunization by subcutaneous two-spot injection, 5 mice were sacrificed on day 13 (D13), spleen lymphocytes of the second group of mice were collected, IFN-gamma ELISPOT effect after the second-shot was measured, three-shot was performed by subcutaneous two-spot injection on the remaining mice on day 14 (D14), 5 mice were sacrificed on day 20, three mice were collected by subcutaneous two-spot injection, and three mice were continuously killed on day 90. Mouse by three-month of the three-shot mice were measured, and the mouse-gamma ELISPOT effect after the third-shot was measured.

The experimental results are shown in fig. 3B, and at 6 days after priming (D6), specific CD8 ⁺ T cell immunity was activated effectively even at a low dose of 0.3 μg SW0128, and the induced T cell immune response was further enhanced after both the two and three priming. This induced immune response was sustained for a longer period of time and activation of specific CD8 ⁺ T cell immunity was observed 90 days after priming.

Taken together, these data indicate that SW0128 can induce a sensitive, potent and sustained specific T cell immune response against HPV in mice.

Example 5 tumor inhibiting Effect of SW0128 LPP vaccine preparation

5.1SW0128 LPP vaccine preparation induces regression of C57BL/6NTac mouse HPV-16 positive TC-1 tumor

Applicants further examined the tumor-inhibiting effect of SW0128 in a syngeneic transplanted mouse TC-1 tumor model. Wherein, the TC-1 tumor model is constructed by TC-1 cells, and the TC-1 cells are primary lung cells of C57BL/6 mice containing HPV16 type E6/E7 genes, which are obtained by transduction and immortalization of HPV 16E 6/E7 retroviruses. The specific modeling and detection process is as follows.

C57BL/6NTac mice were subcutaneously injected with 2X10 ^s TC-1 tumor cells and periodically examined for tumor growth. When the TC-1 tumor tissue size was about 1000mm ³, it was cut to about 30mm ³ and implanted into the flank subcutaneous space of C57BL/6NTac mice (n=15 per group).

Immunization procedure as shown in fig. 4A, mice implanted with tumors were administered SW0128 comprising 3 μg, 10 μg, 30 μg gmRNA, respectively, at a frequency of 1 time per week for 3 weeks (QW, immunization at days 4, 11, 18, respectively) or 2 times per week for 3 weeks (BIW, immunization at days 4, 7, 11, 14, 18, 21, respectively) after tumor implantation (D4), and PBS and EGFP were administered as controls to tumor implanted mice at the same time points. Tumor volumes were measured blindly every 3 or 4 days and the tumor volume calculation formula was (a ² x b)/2 (a represents tumor width; b represents tumor length). The end point of the experiment was to kill the mice when the tumor volume reached 2000mm ³, the tumor developed ulceration or severe tissue necrosis, or the mice lost >20% of their body weight.

Tumor volume measurements as shown in fig. 4B, tumors of most control mice grew rapidly within 18-30 days after tumor implantation, resulting in reaching the experimental endpoint (each broken line represents one mouse). In contrast, most mice immunized with SW0128 containing 3 μg, 10 μg, or 30 μg gmRNA exhibited complete tumor regression, and no significant difference in the rate of complete regression (complete remission, CR) was found between the QW and BIW dosed groups.

The above results demonstrate that SW0128 under different immunization programs (QW and BIW) can induce complete regression of HPV-16 positive tumors. And as shown in fig. 4C, the body weight of the mice administered with SW0128 was not significantly changed or abnormal (each broken line represents one mouse), further illustrating the excellent tumor-inhibiting effect and safety of SW 0128.

5.2SW0128 LPP vaccine preparation induces a reduction in the tumor growth rate of C57BL/6J mice

In addition, the applicant tested the tumor inhibiting effect of SW0128 on the C57BL/6J mouse tumor model.

C57BL/6J mice (female 6 weeks old, taconic Biosciences) were subcutaneously injected with 2X10 ⁵ TC-1 tumor cells and periodically examined for tumor growth. When the TC-1 tumor tissue size was about 1000mm ³, it was cut to about 30mm ³ and implanted into the flank subcutaneous space of C57BL/6J mice (n=15 per group).

Immunization procedure as shown in fig. 5, after 7 days (D7) of tumor implantation, SW0128 containing 0.3 μg,1 μg, 3 μg gmRNA was administered to tumor-implanted mice at a frequency of 1 time per week for 3 weeks (immunization on days 7, 14, 21, respectively) or 1 time per week for 2 weeks (immunization on days 7, 14, respectively), respectively, and PBS and EGFP were administered to tumor-implanted mice at the same time point as controls. Tumor volumes were measured blindly every 3 or 4 days. Tumor volume calculation formula and experimental endpoint are described above.

Tumor volume detection results as shown in fig. 5, C57BL/6J mice immunized with SW0128 containing 0.3 μg, 1 μg, or 3 μg gmRNA did not show complete tumor regression, but exhibited a significant decrease in tumor growth rate. This slowing of tumor growth rate was dose dependent, with C57BL/6J mice immunized with SW0128 containing 3 μ gmRNA, exhibiting minimal tumor growth rate.

The applicant also noted that C57BL/6NTac mice immunized with SW0128 QW containing 3 μ gmRNA exhibited a significant decrease in tumor volume, 10 out of 15 mice exhibited complete regression, while C57BL/6J mice immunized with SW0128 QW containing 3 μ gmRNA exhibited only a significant decrease in tumor growth rate and did not induce tumor regression, as shown in fig. 4B. These differences in data are likely due to differences in mice, and it is generally believed that C57BL/6NTac mice are the mouse strain more suitable for tumor syngeneic transplantation mice experiments.

EXAMPLE 6SW0128 LPP vaccine preparation promotes proliferation of memory T cells

To evaluate the protective efficacy of SW0128, the applicant continued to apply C57BL/6NTac mice in this example 5 that reached complete tumor regression by treatment with3 μg SW 0128. These mice were first placed in a tumor-free physiological condition for at least 4 weeks and then randomized into a new study group (5 mice per group). After grouping, C57BL/6NTac mice were inoculated subcutaneously with 5x10 ⁵ TC-1 tumor cells and the preparation of the mouse tumor model was again performed as described in example 5. Meanwhile, mice inoculated subcutaneously with the same amount of TC-1 cells and not treated with SW0128 were used as a control group.

As a result, as shown in FIG. 6A, all mice treated with SW0128 of 3 μg before the mice were still in a tumor-free state for a long period after being challenged again with tumor cells, and the survival rate was 100%. The control group was sacrificed 21-25 days after TC-1 implantation because the rapid growth of the tumor reached the end of the experiment. These results demonstrate the sustainability of SW0128 protective efficacy, effectively inhibiting tumor growth and regressing tumors after re-challenge with tumor cells 4 months after immunization.

Further, the applicant analyzed the reason why the in vivo protective efficacy of mice with complete tumor regression after SW0128 treatment was durable. Flow detection analysis of E7 _49-57dextramer⁺CD8⁺ T cells was performed on blood lymphocytes from C57BL/6NTac mice (n=5 per group) or C57BL/6NTac mice (n=5 per group) without SW0128 treatment in example 5 with 3 μg SW0128 treatment, and the reagents used were MHC IH-2Db(RAHYNIVTF),IMMUDEX,#JA2195。

Results as shown in fig. 6B, on day 59 (D59) after the initial TC-1 tumor cell subcutaneous inoculation, up to 20% of E7 _49-57 -specific CD8 ⁺ T cells were present in the blood of SW 0128-treated mice. It follows that SW0128 immunization induces a potent antigen-specific CD8 ⁺ T cell response in mice. The above experimental results demonstrate that immunization with SW0128 is effective in eliciting and promoting proliferation of protective memory CD8 ⁺ T cells specific for the E7 _49-57 epitope, enabling killing of tumor cells when HPV-16E7 antigen positive tumor cells are re-encountered.

In addition, antigen-specific-T cell responses in C57BL/6NTac mice (6) and C57BL/6NTac mice (3) without SW0128 treatment, which had completely resolved tumors after 3 μ gSW0128 treatment, were also examined by ELISPot 93 days after the initial TC-1 tumor cell inoculation (D93). The specific experimental procedure is as described in example 4.

The results are shown in FIG. 6C, which clearly detected IFN-. Gamma.spots of splenic lymphocytes from SW 0128-treated mice 3 months after vaccination, again demonstrating the persistence of SW0128 protective efficacy, and the antigen-specific CD8 ⁺ T cell response was induced 3 months after immunization.

Taken together, these data indicate that HPV-16mRNA vaccine SW0128 is capable of inducing regression of TC-1 tumors and of inducing strong protective cellular immune memory to HPV antigens for at least 3 months, and possibly longer, to resist re-challenge and recurrence of the virus.

Example 7 tumor inhibiting Effect of Co-administration of SW0128 LPP vaccine formulation and anti-PD-L1 antibody

It has been reported that HPVmRNA vaccine up-regulates immune checkpoint molecules PD-1 and PD-L1(Grunwitz C et al.,HPV 16RNA-LPX vaccine mediates complete regression of aggressively growing HPV-positivemouse tumors and establishes protective T cell memory.Oncoimmunology.201 9Jul 11;8(9)∶e 1629259.). in Tumor Microenvironment (TME) in order to further increase the immunopotency of SW0128 LPP vaccine formulations, applicant examined the co-therapeutic effect of SW0128 with checkpoint inhibitor anti-PD-L1 antibodies in TC-1 mouse tumor model. The specific detection method is as follows.

C57BL/6J mice (female 6 weeks old Taconic Biosciences) were subcutaneously injected with 5X10 ⁵ TC-1 tumor cells on day 0 (D0) and periodically examined for tumor growth. When the TC-1 tumor tissue size was about 1000mm ³, it was cut to about 30mm ³ and implanted into the lateral subcutaneous space of the C57BL/6J mice.

Dosing regimen as shown in fig. 7A, mice were randomly divided into 7 groups (n=15). Wherein, on day 6 (D6), day 13 (D13) and day 20 (D20), SW0128 containing 3. Mu.g of mRNA was administered to mice of groups 1 and2, respectively, SW0128 containing 10. Mu.g of mRNA was administered to mice of groups 3 and 4, respectively, LPP preparation containing 10. Mu.g of GFP mRNA was administered to mice of groups 5 and 6, respectively, and PBS was administered to mice of group 7 as a negative control group at the same time point. Also, 200 μg of PD-L1 antibody (InVivoMAb anti-mouse PD-L1 (B7-H1), bio X Cell, # BE 0101) or IgG2B isotype control antibody (InVivoMAb rat IgG2B isotype control, anti-keyhole limpet hemocyanin, bio X Cell, # BE 0090) was intraperitoneally injected every 3-4 days, i.e., 200 μg of PD-L1 antibody was administered to group 1, group 3 and group 5 mice on day 9 (D9), day 13 (D13), day 16 (D16), day 20 (D20) and day 23 (D23), respectively, and IgG2B isotype control antibody was administered to group 2, group 4 and group 6 mice at the same time point.

Tumor volumes were measured blindly every 3 or 4 days and the tumor volume calculation formula was (a ² x b)/2 (a represents tumor width; b represents tumor length). The end point of the experiment was to kill the mice when the tumor volume reached 2000mm ³, the tumor developed ulceration or severe tissue necrosis, or the mice lost >20% of their body weight.

Tumor volume detection results are shown in fig. 7B, where the combined administration of SW0128 and PD-L1 antibodies enhanced tumor growth inhibition compared to administration of SW0128 alone or PD-L1 antibody alone. Wherein, the tumor growth inhibition rate of the group 2 mice with 3 mug SW0128 and IgG2b reaches 45%, the tumor growth inhibition rate of the group 1 mice with 3 mug SW0128 and PD-L1 antibodies reaches 78%, the tumor growth inhibition rate of the group 4 mice with 10 mug SW0128 and IgG2b reaches 71%, and the tumor growth inhibition rate of the group 3 mice with 10 mug SW0128 and PD-L1 antibodies reaches 87%. It can be seen that the use of the immune checkpoint inhibitor PD-L1 antibody can further help reduce TC-1 tumor burden.

Survival of mice as shown in fig. 7C, PBS group, group 5 mice administered LPP preparation comprising GFPmRNA and PD-L1 antibody, and group 6 mice administered LPP preparation comprising GFP mRNA and IgG2b were sacrificed within 33 days after TC-1 implantation because rapid growth of tumor reached the experimental endpoint. Whereas the survival rate of mice administered with 3 doses of SW0128 and 5 doses of PD-L1 antibody was significantly increased, group 3 mice administered with 10 μg of SW0128 and PD-L1 antibody had the highest survival rate.

As shown in fig. 7D, the tumor regressions were analyzed, with complete regressions of tumors in 4 mice in the group 4 mice administered with 10 μg SW0128 alone, complete regressions of tumors in no mice in the group 2 mice administered with 3 μg SW0128 alone, and complete regressions of tumors in 8 mice in the group 3 mice administered with 10 μg SW0128 and PD-L1 antibody in combination, and complete regressions of tumors in 5 mice in the group 1 mice administered with 3 μg SW0128 and PD-L1 antibody in combination. It can be seen that the combined administration of SW0128 and PD-L1 antibodies significantly increases the tumor complete regression rate,

EXAMPLE 8 expression of HPV18 E7 mRNA-LPP preparation in cells

In this example, the expression of 4 mRNA-LPP preparations for HPV18 as prepared in example 3 was verified. The specific assay was performed by seeding human kidney epithelial cells 293 (Shanghai cell bank) in 6 well plates at 1.2X10 ⁶ cells/well. After 18 hours of seeding of the cells, HPV18 mRNA LPP preparations 1801, 1802, 1803 and 1804 containing 2 μg mRNA were diluted with 250 μl Opti-MEM reduced serum medium (Gibco, # 31985070) and added dropwise to 293 cells for mixing. The transfected cells were placed in a cell incubator and cultured for an additional 24 hours at 37℃with 5% CO ₂. Then, the cells were collected, the collected cells were treated with 6× loadingbuffer (whole gold, DL 101-02) and heated at 100 ℃ for 10min, and the treated cell samples were subjected to polyacrylamide gel electrophoresis and transferred onto PVDF membrane, which was sealed with 5% nonfat milk powder at room temperature for 1h. HPV 18E 7 antibody (Anti-HPV 18E 7 Anti-body [8E2] (ab 100953)) diluted 1:1000 was used as primary antibody, incubated overnight at 4℃with shaking, washed 5 times with PBST (PBS solution containing 0.5% Tween 20), incubated 1h at room temperature as secondary antibody diluted 1:5000 (Goat Anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, HRP Invitrogen#A10551), washed 5 times with PBST, and developed and imaged with ECL luminescence (Pierce ^TMECLPlus Western Blotting Substrate,Thermo Scientific^TM #32132X3).

As a result, as shown in fig. 8, the LPP preparations 1802 and 1804, which contained the sequence-optimized mRNA, were significantly increased in intracellular expression compared to 1801, which contained the non-optimized mRNA.

EXAMPLE 9HPV18 E7 mRNA-LPP vaccine preparation induces cellular immune response

9.1 Laboratory mice

Female, 6 week old C57BL/6J mice were used. C57BL/6J mice were kept and cared for at Shanghai model biological center Co.Ltd. Animal studies were strictly performed according to the recommendations in Shanghai laboratory animal feeding administration and use guidelines.

9.2 Experimental procedures and results

To evaluate the immunogenicity of LPP formulations 1801, 1802, 1803 and 1804 prepared in example 3.2, this example uses HPV18E7mRNA-LPP vaccine formulations 1801, 1802, 1803 and 1804 to immunize C57BL/6J mice and evaluate their level of activation of specific cd8+ T cell immune responses in the mice.

Mice were randomly divided into 5 groups (n=5 mice per group). On day 0 (D0), priming (one-shot) was performed by means of subcutaneous two-spot immunization, and each group of mice was administered PBS, LPP formulation 1801, 1802, 1803, 1804 containing 10 μg mRNA 18-1, 18-2, 18-3, 18-4, respectively. On day 7 post-immunization (D7), mice were sacrificed, spleen lymphocytes from the mice were collected, and post-immunization IFN-. Gamma.ELISPOT effects were tested.

ELISA spot (ELISPot) assay

The mouse IFN-. Gamma.ELISpot assay was performed using the IFN-. Gamma. ELISpotPLUS kit (Mabtech, 3321-4 APT-10) according to the manufacturer's instructions. Briefly, plates were blocked in RPMI 1640 medium (supplemented with 10% fbs) and incubated for 30 min. Spleen cells were plated at 3×10 ⁵ cells/well and stimulated in vitro with 2 μg/mlHPV E7 polypeptide (Gill Biochemical, sequences shown in Table 3 below), unrelated 2 μg/mL vesicular stomatitis virus VSV polypeptide (RGYVYQGL, gill Biochemical), positive stimulation 500ng/mL phorbol ester (PMA) +10 μg/mL Ionomycin (Ionomycin) (Darcy 2030421) and PBS only (negative control), incubated for 20 hours at 37 ℃,5% CO 2. After that, with biotinylated IFN-. Gamma. -detection antibody and streptavidin-alkaline phosphatase (ALP), BCIP/NBT-plus (5-bromo-4-chloro-3-indole-phosphate/nitro blue tetrazolium-plus) substrate was added for color development and counted with an ELISPOT reader (ImmunoSpot S6 Core Analyzer (CTL)).

TABLE 3E 7 and E6 polypeptide libraries of HPV 18

The results of the assays are shown in FIGS. 9B and 9C, where 1801, 1802, 1803 and 1804 all induced HPV 18-specific T cell immune responses in non-tumor bearing mice. Among them, 1802 induced the highest level of HPV 18-specific T cell immune response in non-tumor bearing mice.

In summary, HPV18-2 vaccine molecules perform optimally in both in vitro protein expression results and in the results of HPV 18-specific T cell immune responses in non-tumor bearing mice.

It will be apparent to those skilled in the art that many modifications and variations of the present invention can be made without departing from its spirit and scope. The specific embodiments described herein are offered by way of example only and are not meant to be limiting in any way. The true scope and spirit of the invention is indicated by the following claims, which are exemplary only.

Claims

1. A polypeptide or combination of polypeptides, wherein the polypeptide or combination of polypeptides comprises a signal peptide sequence at the N-terminus, an MHC-I transmembrane-intracellular domain sequence at the C-terminus, and

The polypeptide or combination of polypeptides comprises

Amino acid sequence of human papillomavirus HPV 16E 6 protein or immunogenic fragment thereof, and/or

Amino acid sequence of human papillomavirus HPV 16E 7 protein or an immunogenic fragment thereof;

Or alternatively

The polypeptide or combination of polypeptides comprises

Amino acid sequence of human papillomavirus HPV 18E 6 protein or immunogenic fragment thereof, and/or

An amino acid sequence of a human papillomavirus HPV 18E 7 protein or an immunogenic fragment thereof;

Wherein,

The human papillomavirus HPV 16E 6 protein comprises an amino acid sequence shown in SEQ ID NO. 105 or an amino acid sequence with at least 95% identity with the amino acid sequence shown in SEQ ID NO. 105;

the human papillomavirus HPV 16E 7 protein comprises an amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence with at least 95% identity with the amino acid sequence shown in SEQ ID NO. 6;

the human papillomavirus HPV 18E 6 protein comprises an amino acid sequence shown in SEQ ID NO. 131 or an amino acid sequence with at least 95% identity with the amino acid sequence shown in SEQ ID NO. 131;

The human papillomavirus HPV 18E 7 protein comprises an amino acid sequence shown in SEQ ID NO. 79 or an amino acid sequence with at least 95% identity with the amino acid sequence shown in SEQ ID NO. 79.

2.A nucleic acid or a combination of nucleic acids,

The nucleic acid or combination of nucleic acids comprising a polynucleotide encoding the human papillomavirus HPV 16E 6 protein or immunogenic fragment thereof as defined in claim 1 wherein said polynucleotide is RNA comprising the nucleotide sequence shown in one of SEQ ID NOS 124, 125 and 126 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NOS 124, 125 and 126, and/or

The nucleic acid or combination of nucleic acids comprising a polynucleotide encoding a human papillomavirus HPV 16E 7 protein or immunogenic fragment thereof as defined in claim 1, wherein the polynucleotide is RNA, the polynucleotide comprising a nucleotide sequence set forth in one of SEQ ID NOs 17, 20, 63, 64, 65, 66, 67, 68 and 69 or a nucleotide sequence having at least 85% identity to a nucleotide sequence set forth in one of SEQ ID NOs 17, 20, 63, 64, 65, 66, 67, 68 and 69;

Or alternatively

The nucleic acid or combination of nucleic acids comprising a polynucleotide encoding the human papillomavirus HPV 18E 6 protein or immunogenic fragment thereof as defined in claim 1 wherein said polynucleotide is RNA comprising a nucleotide sequence represented by one of SEQ ID NOS 150, 151 and 152 or a nucleotide sequence having at least 85% identity to a nucleotide sequence represented by one of SEQ ID NOS 150, 151 and 152, and/or

The nucleic acid or combination of nucleic acids comprises a polynucleotide encoding the human papillomavirus HPV 18E 7 protein or immunogenic fragment thereof as defined in claim 1, wherein the polynucleotide is RNA, the polynucleotide comprising a nucleotide sequence set forth in one of SEQ ID NOs 98, 99 and 100 or a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOs 98, 99 and 100.

3. A nucleic acid or a combination of nucleic acids,

The nucleic acid or combination of nucleic acids comprising a nucleotide sequence encoding the human papillomavirus HPV 16E 6 protein or immunogenic fragment thereof as defined in claim 1 wherein the polynucleotide is DNA comprising one of the nucleotide sequences shown in SEQ ID NOS 128, 129 and 130 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NOS 128, 129 and 130, and/or

The nucleic acid or combination of nucleic acids comprising a polynucleotide encoding a human papillomavirus HPV 16E 7 protein or immunogenic fragment thereof as defined in claim 1, wherein the polynucleotide is DNA comprising the nucleotide sequence set forth in one of SEQ ID NOs 18, 42, 70, 71, 72, 73, 74, 75 and 76 or having at least 85% identity to the nucleotide sequence set forth in one of SEQ ID NOs 18, 42, 70, 71, 72, 73, 74, 75 and 76;

Or alternatively

The nucleic acid or combination of nucleic acids comprising a polynucleotide encoding the human papillomavirus HPV 18E 6 protein or immunogenic fragment thereof as defined in claim 1 wherein said polynucleotide is DNA comprising the nucleotide sequence shown in one of SEQ ID NOS 154, 155 and 156 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NOS 154, 155 and 156, and/or

The nucleic acid or combination of nucleic acids comprises a polynucleotide encoding the human papillomavirus HPV 18E 7 protein or immunogenic fragment thereof as defined in claim 1, wherein the polynucleotide is DNA comprising the nucleotide sequence shown in one of SEQ ID NOs 102, 103 and 104 or a nucleotide sequence having at least 85% identity to the nucleotide sequence shown in one of SEQ ID NOs 102, 103 and 104.

4. A nucleic acid or combination of nucleic acids comprising a polynucleotide encoding the polypeptide or combination of polypeptides of claim 1.

5. A composition comprising the polypeptide or combination of polypeptides of claim 1, or the nucleic acid or combination of nucleic acids of any one of claims 2-4.

6. A vaccine formulation comprising the polypeptide or combination of polypeptides of claim 1, the nucleic acid or combination of nucleic acids of any one of claims 2-4 or the composition of claim 5.

7. An expression vector comprising the nucleic acid or combination of nucleic acids of any one of claims 2-4.

8. A host cell comprising the nucleic acid or combination of nucleic acids of any one of claims 2-4 or the expression vector of claim 7.

9. A kit comprising the polypeptide or combination of polypeptides of claim 1, the nucleic acid or combination of nucleic acids of any one of claims 2-4, the composition of claim 5 or the vaccine formulation of claim 6 and one or more therapeutic agents selected from the group consisting of chemotherapeutic agents, radioisotopes, immune checkpoint inhibitors and tumor antigen targeting drugs, preferably the therapeutic agent is an anti-PD-L1 antibody or antigen binding fragment thereof.

10. Use of the polypeptide or combination of polypeptides of claim 1, the nucleic acid or combination of nucleic acids of any one of claims 2-4, the composition of claim 5, the vaccine formulation of claim 6 or the kit of claim 9 for the manufacture of a medicament for treating a human papillomavirus type 16 infection, a human papillomavirus type 16 positive cancer or precancerous lesion, a human papillomavirus type 18 infection, a human papillomavirus type 18 positive cancer or precancerous lesion in a subject.