CN118994329A - Pre-fusion RSV F protein and uses thereof - Google Patents

Abstract

The present invention discloses pre-fusion RSV F proteins comprising one or more amino acid mutations to stabilize the RSV F protein in a pre-fusion conformation, and uses thereof. Nucleic acid molecules, vectors, host cells encoding the proteins and immune compositions comprising the proteins are also disclosed.

Description

Pre-fusion RSV F protein and uses thereof

Technical Field

The invention belongs to the field of biological medicine, and relates to a pre-fusion RSVF protein and application thereof.

Background

Human respiratory syncytial virus (human respiratory syncytial virus, RSV) is a non-segmented, single-stranded negative-strand RNA enveloped virus belonging to the pneumoviridae family. RSV is the major viral pathogen causing respiratory illness in infants and elderly, causing severe lower respiratory tract infections in children under 3 thousands of 5 years of age and death in more than 6 tens of thousands of children each year. RSV has been found to be over 60 years ago, but to date there is still no effective vaccine available for preventing viral infection. In the sixties of the twentieth century, vaccines with formalin inactivated virus (FI-RSV) supplemented with aluminum adjuvant were first used in the study of infant RSV vaccines. Unfortunately, this vaccine resulted in severe respiratory disease enhancement (enhancedrespiratory disease, ERD) and extremely high hospitalization rates in vaccinated infants and two children died due to bronchiolar monocyte infiltration and eosinophilia. FI-RSV causes ERD for two reasons, on the one hand, the neutralizing antibody has low activity and cannot effectively neutralize viruses; on the other hand, vaccine-induced Th2 biased immune responses can lead to severe lung inflammation after challenge. The low neutralizing antibody activity is mainly due to the deletion of the F epitope prior to fusion on the FI-RSV surface, whereas the Th2 biased immune response is one of the properties of formalin inactivated virus vaccines. Thus, a safe and effective RSV candidate vaccine should have both good neutralizing activity and induce Thl-biased immune responses.

At present, no effective preventive vaccine is marketed. RSVF protein is the most important immunogen and induces the body to produce protective neutralizing antibodies. The F protein has two conformations before and after playing the membrane fusion function, namely a pre-fusion conformation (preF) and a post-fusion conformation (postF), wherein the preF protein can induce more efficient neutralizing antibodies. But due to its instability, the pre-fusion conformation has a tendency to refold prematurely into a post-fusion conformation in solution and on the surface of the virion. RSV F protein with high expression levels and maintaining stable pre-fusion conformation would be a promising candidate for vaccine against RSV.

Disclosure of Invention

In order to develop RSV F protein with high expression level and stable pre-fusion conformation, the present invention provides the following technical scheme:

the first aspect of the present invention provides a mutant RSVF protein comprising at least one amino acid mutation which stabilizes the recombinant RSV F protein in a pre-fusion conformation, the RSV F protein being resistant to recognition of an antigenic site And/or antibody binding of V.

In some embodiments, the amino acid mutation of the RSV F protein comprises: cavity-filling amino acid mutations, disulfide bond-introducing amino acid mutations, chemical bond-introducing amino acid mutations, amino acid mutations that induce rotation of the polypeptide backbone.

In some embodiments, the amino acid mutation comprises mutating an amino acid in close proximity to the alpha helix and beta sheet to prevent refolding into a post-fusion conformation.

In some embodiments, the RSVF protein comprises a mutation at amino acid residue 55, 186-193, 210-217, 254, 255-264, 323, 394, 461, 475, or 491, or at an amino acid corresponding to residue 55, 186-193, 210-217, 254, 255-264, 323, 394, 461, 475, or 491 as determined by alignment with SEQ ID No. 1. Wherein 255-264 and 186-193 are amino acids with alpha helices and beta sheet positions in close proximity.

In some embodiments, the RSVF protein comprises a mutation at amino acid residue 55, 188, 190, 210-214, 217, 254, 260, 263, 323, 394, 461, 475, or 491.

In some embodiments, the RSVF protein comprises a cavity-filling amino acid mutation at position 55, preferably the cavity-filling amino acid mutation comprises a F, L, W, V, Y, H or M substitution. In a preferred embodiment, the amino acid filling the 55 th cavity is mutated to S55V.

In some embodiments, the RSVF protein comprises an amino acid mutation that introduces a disulfide bond at positions 188, 190, 260, 263, 394, 491, 323, 475, and in preferred embodiments, the amino acid mutation that introduces a disulfide bond at positions 188, 190, 260, 263, 394, 491, 323, 475 is a substitution of cysteine. In a preferred embodiment, the disulfide bond introducing amino acid mutation is selected from one or more of (a) - (d):

(a) 188C and 263C substitutions;

(b) 190C and 260C substitutions;

(c) 394C and 491C substitutions; and

(D) 323C and 475C.

In specific embodiments, the 188C and 263C substitutions are L188C and D263C substitutions;

the 190C and 260C substitutions are S190C and L260C substitutions;

the 394C and 491C substitutions are K394C and S491C substitutions;

The 323C and 475C substitutions are T323C and I475C substitutions.

In some embodiments, the RSV F protein comprises an amino acid mutation that introduces a chemical bond at position 254, and in a preferred embodiment, the amino acid mutation that introduces a chemical bond at position 254 is N254R.

In some embodiments, the RSVF protein comprises amino acid mutations at positions 210-214, 217, 461 which induce rotation of the polypeptide backbone. In one embodiment, the amino acid mutation introduced to induce rotation of the polypeptide backbone comprises a proline substitution. In a specific embodiment of the invention, the amino acid introduced at positions 210-214, 217, 461 induces rotation of the polypeptide backbone to be mutated to Q210P, C212P, S213P, I214P, I217P or K461P.

In some embodiments, the RSV F protein includes a cavity-filling amino acid mutation, a chemical bond-introducing amino acid mutation, and at least one disulfide bond-introducing amino acid mutation. In some embodiments, the RSVF protein includes the following mutations: S55V, N254,254, 254R, S190C and L260C.

In other embodiments, the RSV F protein includes a mutation at position 461 and at least one disulfide bond introducing amino acid mutation. In a preferred embodiment, the RSVF protein includes the following mutations:

K461P, K394C and S491C; or (b)

K461P, T323C and I475C.

In some embodiments, the RSV F protein comprises any of the aforementioned RSV F muteins at position 215 in combination with a mutation at position 215. In a preferred embodiment, the mutation at position 215 is S215P.

In some embodiments, the RSV F protein comprises an F ₁ polypeptide and an F ₂ polypeptide, and optionally does not comprise a pep27 polypeptide or portion thereof. In some embodiments that do not include a pep27 polypeptide or a portion thereof, the C-terminal residue of the F ₂ polypeptide and the N-terminal residue of the F ₁ polypeptide comprise RSVF positions 97 and 137, 97 and 145, 97 and 150, 102 and 144, 102 and 145, 102 and 146, 102 and 147, 103 and 144, 103 and 145, 103 and 146, 103 and 147, 104 and 144, 104 and 145, 104 and 146, 104 and 147, 105 and 144, 105 and 145, 105 and 146, 105 and 147, or 105 and 150, respectively.

In embodiments of the invention, the F ₂ and F ₁ polypeptides are linked by a heterologous peptide linker or directly. Such linkers include, but are not limited to G, S, GG, GS, SG, PS, GP, GGG or GSG. In one embodiment, position 103 of the F ₂ polypeptide is linked to position 145 of the F ₁ polypeptide by a GS linker. In one embodiment, position 103 of the F ₂ polypeptide is directly linked to position 145 of the F ₁ polypeptide. In one embodiment, position 103 of the F ₂ polypeptide is linked to position 145 of the F ₁ polypeptide by a PS linker. In another embodiment, position 103 of the F ₂ polypeptide is linked to position 147 of the F ₁ polypeptide by a GP linker.

In some embodiments of the invention, the RSV F protein comprises the following amino acid mutations: deletion of S215P, pep27 polypeptide or portion thereof. In a preferred embodiment, the F ₂ and F ₁ polypeptides of the RSV F protein are linked by a heterologous peptide linker. In a specific embodiment, position 103 of the F ₂ polypeptide is linked to position 145 of the F ₁ polypeptide by a GS linker.

In some embodiments of the invention, the RSV F protein comprises the following amino acid mutations: S55V, S190C, L260C, N254R and S215P. In a preferred embodiment, the RSV F protein does not comprise a pep27 polypeptide or portion thereof. In a preferred embodiment, the F ₂ and F ₁ polypeptides of the RSV F protein are linked by a heterologous peptide linker. Such linkers include, but are not limited to, the aforementioned linkers, including, but not limited to, the positions of the C-terminal residue of the F ₂ polypeptide and the N-terminal residue of the F ₁ polypeptide described previously. In a specific embodiment position 103 of the F ₂ polypeptide is linked to position 145 of the F ₁ polypeptide by a GS linker. In another specific embodiment, position 103 of the F ₂ polypeptide is linked to position 145 of the F ₁ polypeptide by a PS linker. In another specific embodiment, position 103 of the F ₂ polypeptide is linked to position 147 of the F ₁ polypeptide by a GP linker.

In some embodiments of the invention, the RSV F protein comprises the following amino acid mutations: S55V, S190C, L C and N254R, and Q210P, S211P, C212P, S213P, I214P, S P or I217P. Optionally, the RSV F protein does not comprise a pep27 polypeptide or portion thereof. In a preferred embodiment, the F ₂ and F ₁ polypeptides of the RSV F protein are linked by a heterologous peptide linker. Such linkers include, but are not limited to, the aforementioned linkers, including, but not limited to, the positions of the C-terminal residue of the F ₂ polypeptide and the N-terminal residue of the F ₁ polypeptide described previously.

In a specific embodiment, the RSV F protein comprises the following amino acid mutations: S55V, S190C, L260C, N254R and S215P, and position 103 of the F ₂ polypeptide is linked to position 145 of the F ₁ polypeptide by a GS linker. In a specific embodiment, the RSVF protein comprises the following amino acid mutations: S55V, S190C, L260C, N R and S215P, and position 103 of the F ₂ polypeptide is linked to position 145 of the F ₁ polypeptide by a PS linker.

In a specific embodiment, the RSV F protein comprises the following amino acid mutations: S55V, S190C, L260C, N254R and S215P, and position 103 of the F ₂ polypeptide is linked to position 147 of the F ₁ polypeptide by a GP linker.

In some embodiments of the invention, the RSVF protein comprises the following amino acid mutations: K461P, K394C, S491C and S215P, optionally, said RSVF protein does not comprise a pep27 polypeptide or portion thereof. In a preferred embodiment, the F ₂ and F ₁ polypeptides of the RSV F protein are linked by a heterologous peptide linker. Such linkers include, but are not limited to, the aforementioned linkers, including, but not limited to, the positions of the C-terminal residue of the F ₂ polypeptide and the N-terminal residue of the F ₁ polypeptide described previously.

In a specific embodiment, the RSV F protein comprises the following amino acid mutations: K461P, K394C, S491C and S215P, and position 103 of the F ₂ polypeptide is linked to position 145 of the F ₁ polypeptide by a GS linker.

In some embodiments of the invention, the RSVF protein comprises the following amino acid mutations: K461P, T323C, I475C and S215P. Optionally, the RSVF protein does not comprise a pep27 polypeptide or portion thereof. In a preferred embodiment, the F ₂ and F ₁ polypeptides of the RSV F protein are linked by a heterologous peptide linker. Such linkers include, but are not limited to, the aforementioned linkers, including, but not limited to, the positions of the C-terminal residue of the F ₂ polypeptide and the N-terminal residue of the F ₁ polypeptide described previously.

In a specific embodiment, the RSV F protein comprises the following amino acid mutations: K461P, T323C, I475C and S215P, and position 103 of the F ₂ polypeptide is linked to position 145 of the F ₁ polypeptide by a GS linker.

In a specific embodiment, the RSV F protein comprises the following amino acid mutations: K461P, T323C, I475C and S215P, and position 103 of the F ₂ polypeptide is linked to position 147 of the F ₁ polypeptide by a GP linker.

In some embodiments, the RSVF protein is a full-length RSVF protein.

In some embodiments, the RSVF protein is a soluble RSVF protein.

In some embodiments, any of the foregoing RSV F muteins further comprise one or more tags that can be used to detect and/or purify the RSV F protein. In one embodiment, the RSVF protein comprises one or more tags for detecting the RSV F molecule. In another embodiment, the RSV F protein comprises one or more tags for purifying the RSV F molecule. Wherein the tag includes, but is not limited to, a Strep tag, strep II tag, FLAG tag, glutathione S-transferase (GST) tag, green Fluorescent Protein (GFP) tag, hemagglutinin A (HA) tag, histidine (His) tag, luciferase tag, maltose Binding Protein (MBP) tag, c-Myc tag, protein A tag, or protein G tag. In a preferred embodiment, the tag is a proteolytically cleavable tag.

In some embodiments, any of the foregoing RSV F muteins can be proteins of different origins and different subtypes, such as RSV a-type or RSV B-type RSV F proteins.

In a second aspect, the invention provides an immunogen comprising the RSV F protein of the first aspect of the invention.

In some embodiments, the immunogen comprises a multimer of the RSV F protein. In a preferred embodiment, the multimer is a trimer.

In a third aspect the invention provides a virus-like particle comprising the RSV F protein according to the first aspect of the invention or the immunogen according to the second aspect of the invention.

In a fourth aspect, the invention provides a protein nanoparticle comprising an RSVF protein according to the first aspect of the invention or an immunogen according to the second aspect of the invention.

In some embodiments, the protein nanoparticle is a ferritin nanoparticle, a package element nanoparticle, a Sulfur Oxidoreductase (SOR) nanoparticle, a2, 4-dioxytetrahydropteridine synthase nanoparticle, or a pyruvate dehydrogenase nanoparticle.

In a fifth aspect the invention provides a nucleic acid molecule encoding an RSVF protein according to the first aspect of the invention, an immunogen according to the second aspect of the invention, a virus-like particle according to the third aspect of the invention or a protein nanoparticle according to the fourth aspect of the invention.

In some embodiments, the nucleic acid molecule encodes a precursor protein of the immunogen, virus-like particle, or protein nanoparticle. The precursor protein comprises a signal peptide, an F2 polypeptide, a Pep27 polypeptide and an F1 polypeptide from the N end to the C end.

In some embodiments, the nucleic acid molecule is codon optimized for expression in a human or non-human mammalian cell.

Regardless of source, the nucleic acid molecule encoding any of the foregoing RSVF muteins may be cloned into any suitable vector, such as a vector for nucleic acid molecule proliferation or a vector for nucleic acid molecule expression. In embodiments where expression is desired, the nucleic acid may be operably linked to a promoter suitable for targeted expression in a desired cell type, e.g., mammalian cells or insect cells, and may be incorporated into any suitable expression vector, e.g., mammalian or insect expression vectors.

In a sixth aspect the invention provides a vector comprising a nucleic acid molecule according to the fifth aspect of the invention.

In some embodiments, the vector is a viral vector. Such viral vectors include, but are not limited to, bovine parainfluenza viral vectors, human parainfluenza viral vectors, newcastle disease viral vectors, sendai viral vectors, measles viral vectors, attenuated RSV vectors, paramyxovirus vectors, adenovirus vectors, alphavirus vectors, venezuelan equine encephalitis vectors, semliki forest viral vectors, sindbis viral vectors, adeno-associated viral vectors, poxvirus vectors, rhabdovirus vectors, vesicular stomatitis viral vectors, picornaviral vectors, or herpesvirus vectors. In a preferred embodiment, the viral vector is selected from adenovirus vectors. The adenovirus is selected from the group consisting of human adenovirus and non-human adenovirus. The non-human adenovirus includes, but is not limited to, simian, chimpanzee, gorilla, avian, canine, ovine, or bovine adenovirus. In a preferred embodiment, the adenovirus is selected from chimpanzee adenoviruses. In a more preferred embodiment, the chimpanzee adenovirus is an adenovirus with at least the E1 gene deleted.

In a seventh aspect, the invention provides a host cell comprising a nucleic acid molecule according to the fifth aspect of the invention or a vector according to the sixth aspect of the invention.

According to an eighth aspect of the present invention there is provided an immunogenic composition comprising an RSV F protein according to the first aspect of the invention, an immunogen according to the second aspect of the invention, a virus-like particle according to the third aspect of the invention, a protein nanoparticle according to the fourth aspect of the invention, a nucleic acid molecule according to the fifth aspect of the invention, a vector according to the sixth aspect of the invention or a host cell according to the seventh aspect of the invention; and a pharmaceutically acceptable carrier.

In some embodiments, the immunogenic composition further comprises an adjuvant. The adjuvant includes, but is not limited to, alum, an oil-in-water composition, MF59, AS01, AS03, AS04, MPL, QS21, a TLR9 agonist, a TLR7 agonist, a TLR4 agonist, a TLR3 agonist, or a combination of two or more thereof.

The ninth aspect of the invention provides a method as defined in any one of the following:

1) A method for detecting or isolating RSVF-binding antibodies in a subject, comprising: providing an effective amount of an RSVF protein according to the first aspect of the invention, an immunogen according to the second aspect of the invention, a virus-like particle according to the third aspect of the invention, a protein nanoparticle according to the fourth aspect of the invention;

Contacting a biological sample from the subject with the recombinant RSVF protein or the protein nanoparticle under conditions sufficient to form an immune complex between the recombinant RSVF protein or the protein nanoparticle and the RSVF-binding antibody; and

Detecting the immune complex, thereby detecting or isolating the RSVF-binding antibodies in the subject.

In some embodiments, the subject is at risk of RSV infection. In some embodiments, the subject has an RSV infection. RSV infection includes RSV infection of various origins or subtypes, including but not limited to human RSV subtype a, human RSV subtype B, or bovine RSV infection.

2) A method for stabilizing the pre-fusion conformation of an RSV fusion polypeptide, introducing a mutation as described in the first aspect of the invention;

3) A method of producing an antibody to RSV F by immunizing a non-human animal with an RSV F protein according to the first aspect of the invention or an immunogen according to the second aspect of the invention;

In some embodiments, the non-human animal is a vertebrate. The vertebrate includes, but is not limited to, a mouse, a rat, a guinea pig, a rabbit, a sheep, and a non-human primate.

In some embodiments, the antibody comprises a chimeric, humanized antibody.

The tenth aspect of the invention provides any one of the following applications:

1) Use of an RSV F protein according to the first aspect of the invention, an immunogen according to the second aspect of the invention, a virus-like particle according to the third aspect of the invention, a protein nanoparticle according to the fourth aspect of the invention, a nucleic acid molecule according to the fifth aspect of the invention, a vector according to the sixth aspect of the invention, a host cell according to the seventh aspect of the invention or an immunogenic composition according to the eighth aspect of the invention for inhibiting or preventing an RSV infection;

2) Use of an RSVF protein according to the first aspect of the invention, an immunogen according to the second aspect of the invention, a virus-like particle according to the third aspect of the invention, a protein nanoparticle according to the fourth aspect of the invention, a nucleic acid molecule according to the fifth aspect of the invention, a vector according to the sixth aspect of the invention, a host cell according to the seventh aspect of the invention or an immunogenic composition according to the eighth aspect of the invention for the preparation of a product for the treatment or prophylaxis of a disease associated with RSV infection;

3) Use of an RSVF protein according to the first aspect of the invention, an immunogen according to the second aspect of the invention, a virus-like particle according to the third aspect of the invention, a protein nanoparticle according to the fourth aspect of the invention, a nucleic acid molecule according to the fifth aspect of the invention, a vector according to the sixth aspect of the invention, a host cell according to the seventh aspect of the invention or an immunogenic composition according to the eighth aspect of the invention for the preparation of a product for inducing an immune response in a subject against an RSVF protein;

4) Use of an RSVF protein according to the first aspect of the invention, an immunogen according to the second aspect of the invention, a virus-like particle according to the third aspect of the invention, a protein nanoparticle according to the fourth aspect of the invention, a nucleic acid molecule according to the fifth aspect of the invention, a vector according to the sixth aspect of the invention, a host cell according to the seventh aspect of the invention or an immunogenic composition according to the eighth aspect of the invention for the preparation of an antibody against an RSVF protein;

5) Use of an RSV F protein according to the first aspect of the invention, an immunogen according to the second aspect of the invention, a virus-like particle according to the third aspect of the invention, a protein nanoparticle according to the fourth aspect of the invention, a nucleic acid molecule according to the fifth aspect of the invention, a vector according to the sixth aspect of the invention, a host cell according to the seventh aspect of the invention or an immunogenic composition according to the eighth aspect of the invention for the preparation of a vaccine;

6) Use of an RSV F protein according to the first aspect of the invention, an immunogen according to the second aspect of the invention, a virus-like particle according to the third aspect of the invention, a protein nanoparticle according to the fourth aspect of the invention, a nucleic acid molecule according to the fifth aspect of the invention, a vector according to the sixth aspect of the invention, a host cell according to the seventh aspect of the invention or an immunogenic composition according to the eighth aspect of the invention in the preparation of a kit.

Drawings

FIG. 1 is a graph of the results of flow cytometry detection after Ad-MF42 and Ad-wtF infection of 293T cells.

FIG. 2 is a graph showing the detection results of preF specific antibodies in serum of mice immunized with Ad-MF42 and Ad-wtF.

FIG. 3 is a graph showing the results of detection of the titer of the neutralizing antibody.

FIG. 4 is a graph showing ELISA detection results of soluble RSV F protein.

Detailed Description

The present invention has been extensively and intensively studied to find some new mutant RSV F proteins with improved properties by mutating different amino acids. As shown in the examples section of the present invention, these novel mutant RSV F molecules have a preF conformation with higher expression levels, as well as being highly immunogenic.

In the present invention, the use of alternatives (e.g., "or") is understood to mean any one, all, or any combination of the alternatives. The terms "comprising," "having," "including," and "containing" are used synonymously in the present invention, and these terms and variations thereof are intended to be interpreted as non-limiting.

In the present invention, the term "RSVF protein" includes all forms of the protein, including F1 polypeptides and F2 polypeptides, i.e., including F0 precursor polypeptides, trimers of F0 precursor polypeptides, pre-RSVF polymers and mature RSV F trimers-whether film-bound or soluble. As used herein, the term "mutant RSV F protein" refers to an RSV F protein that contains one or more artificial introduced/mutations.

In the present invention, the "F1 polypeptide" refers to a polypeptide comprising amino acid residues 137 to 513 of the RSV F0 precursor sequence. Amino acid residues 137-513 do not include the RSVF transmembrane and cytoplasmic domains. In some embodiments, the F1 polypeptide may also include RSV F transmembrane and cytoplasmic domains (located within residues 514-574 of the RSV F0 precursor sequence).

"F2 polypeptide" refers to a polypeptide comprising amino acid residues 26-109 of the RSVF 0 precursor sequence.

In the present invention, the terms "protein" and "polypeptide" are used interchangeably.

In the present invention, the terms "nucleic acid molecule", "nucleic acid sequence" and "nucleotide sequence" are used interchangeably.

In the present invention, the term "adjuvant" refers to a substance that is capable of enhancing, accelerating or prolonging the immune response of the body to an immunogen or an immunogenic composition (e.g., a vaccine).

RSVF proteins

RSV is known to exist as a single serotype with two antigenic subtypes a and B. The amino acid sequences of the two types of mature F proteins are approximately 93% identical. As in the present application, the amino acid positions are given with reference to the RSV F protein sequence (AC 083301.1, SEQ ID NO. 1) from the A2 strain. The term "amino acid at position" x "of the RSVF protein" therefore means an amino acid corresponding to the amino acid at position "x" in the RSVF protein of the RSVA2 virus strain of SEQ ID NO. 1. Note that in the numbering system used in the present application, 1 refers to the N-terminal amino acid (SEQ ID NO. 1) of one immature F0 protein. When using one RSV strain instead of the A2 strain, the amino acid position of the F protein will be numbered by inserting a gap if necessary to align the sequences of the other RSV strains with the F protein of SEQ ID No.1, referring to the numbering of the F protein of the A2 strain of SEQ ID No. 1. Sequence alignment is performed using methods well known in the art, for example, by CLUSTALW, bioedit or CLC Workbench.

Mutations in the invention may be introduced into any suitable RSV F sequence such that it is in the preF conformation. Suitable RSVF sequences are sequences of WT/native RSVF molecules. Suitable RSVF sequences also include sequences of mutant RSV F molecules, i.e., sequences comprising one or more artificially introduced mutations as compared to the WT/natural RSVF molecule.

Those skilled in the art will appreciate that the protein may be mutated by conventional molecular biological procedures. The pre-fusion RSVF polypeptides in the compositions according to the invention are stable, i.e. do not readily change to a post-fusion conformation upon processing of the polypeptide (e.g. like purification, freeze-thaw cycles, and/or storage etc.).

In certain embodiments, the RSV F protein is derived from an RSV a strain. In certain embodiments, the RSV F protein is derived from the RSVA2 strain of SEQ ID No. 1.

In certain embodiments, the polypeptide according to the invention further comprises a leader sequence, also known as a signal sequence or signal peptide, corresponding to amino acids 1-26 of SEQ ID NO. 1. It is a short (typically 5-30 amino acids long) peptide that is present at the N-terminus of most newly synthesized proteins destined for the secretory pathway. In certain embodiments, the polypeptide according to the invention does not comprise a leader sequence.

In the present invention, variant forms equivalent to the aforementioned amino acid sequences may also be used in embodiments involving specific exemplary amino acid sequences of the aforementioned mutations, or embodiments involving specific regions of these sequences (e.g., the F ₁ and/or F ₂ regions thereof). In some embodiments, amino acid sequences having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the RSV F amino acid sequences of the invention may be used, spanning their F ₁ and F ₂ regions, particularly spanning amino acid residues from 26 to 109 (F2) and 137 to 513 (portions of F ₁ not including transmembrane and cytoplasmic domains). These variant forms may have amino acids added, deleted or substituted as compared to one or more specific amino acid sequences provided herein. Thus, their length may be longer or shorter than a particular sequence.

Similarly, amino acid residues subsequent to position 514 of the amino acid sequence of each exemplary RSV F provided herein can be removed or altered. For example, in several amino acid sequences provided herein, the amino acid residues following position 514 include the natural RSVF transmembrane and cytoplasmic domains. In some embodiments, the transmembrane and cytoplasmic domains of these natural RSV F can be removed to produce soluble (i.e., non-membrane bound) versions of the RSVF molecule, or can be replaced with different transmembrane and/or cytoplasmic domains. Similarly, among the several amino acid sequences provided herein, the amino acid residues following position 514 include various artificially added C-terminal sequences, including but not limited to a folding subdomain, thrombin cleavage site, histidine tag, strep II tag, or various linkers.

The mutant RSV F protein can be obtained and/or isolated and/or maintained in preF conformations using any suitable method known in the art, including, but not limited to, standard protein purification methods, such as ion exchange chromatography, size exclusion chromatography, and/or affinity chromatography.

In some embodiments, mutant RSV F in the preF conformation can be obtained, isolated, or maintained by controlling the ionic strength of the medium/buffer in which the protein is present (e.g., by using a medium of high or low ionic strength). In some embodiments, the mutant RSVF protein may be obtained, isolated, or maintained at one or more temperatures that facilitate preservation of the preF conformation. In some embodiments, the mutant RSV F protein can be obtained, isolated, or maintained for a period of time that reduces the extent of preF conformational loss. The extent to which a protein preparation retains one or more desired conformations (e.g., preF conformations) can be determined by any suitable method known in the art, including, but not limited to, biochemical, biophysical, immunological, and virologic assays. Such assays include, but are not limited to, for example, immunoprecipitation, enzyme-linked immunosorbent assay or enzyme-linked immunosorbent spot assay, crystallographic analysis (including co-crystallization with antibodies), sedimentation, analytical ultracentrifugation, dynamic Light Scattering (DLS), electron Microscopy (EM), cryo-electron microscopy tomography, calorimetry, surface Plasmon Resonance (SPR), fluorescence Resonance Energy Transfer (FRET), circular dichroism analysis, small angle x-ray scattering, neutralization assay, antibody-dependent cytotoxicity assay, and/or in vivo toxicity studies.

Virus-like particles

In some embodiments, provided are virus-like particles (VLPs) comprising mutant RSV F proteins as previously described stabilized in a pre-fusion conformation. VLPs lack the viral components required for viral replication and thus present a highly attenuated form of the virus. When administered to a subject, VLPs may present polypeptides (e.g., recombinant RSVF proteins stabilized in a pre-fusion conformation) capable of inducing an immune response against RSV. Virus-like particles and methods of making the same are known and well known to those of ordinary skill in the art, and viral proteins from several viruses are known to form VLPs, including human papilloma virus, HIV, human polyomavirus, rotadisease, parvovirus, canine parvovirus, hepatitis e virus, and newcastle disease virus. For example, chimeric VLPs containing RSV antigens and may be newcastle disease virus-based VLPs. Newcastle disease-based VLPs have previously been demonstrated to induce a neutralizing immune response against RSV in mice. The formation of these VLPs may be detected by any suitable technique. Examples of techniques known in the art suitable for detecting VLPs in a culture medium include, for example, electron microscopy techniques, dynamic Light Scattering (DLS), selective chromatographic separation (e.g., ion exchange, hydrophobic interaction, and/or size exclusion chromatographic separation of VLPs), and density gradient centrifugation.

Protein nanoparticles

In some embodiments, protein nanoparticles comprising one or more of any of the disclosed mutant RSV F proteins stabilized in a pre-fusion conformation are provided, wherein the protein nanoparticles are specifically bound by a pre-fusion specific antibody (e.g., D25 or CR9501 antibody), and/or comprise a pre-fusion specific conformation of RSVF (e.g., an antigenic siteOr V). Non-limiting examples of nanoparticles include ferritin nanoparticles, encapsulated nanoparticles, and Sulfur Oxidoreductase (SOR) nanoparticles, which are composed of an assembly of monomeric subunits comprising ferritin, encapsulated protein, and SOR protein, respectively. To construct protein nanoparticles comprising the disclosed recombinant RSVF proteins stabilized in a pre-fusion conformation, the antigen is linked to a subunit of a protein nanoparticle (e.g., ferritin, a packaging protein, or a SOR protein). The fusion proteins self-assemble into nanoparticles under appropriate conditions. The RSV F protein included on the protein nanoparticle may be a human subtype a, a human subtype B or a bovine RSV F protein, which includes the substitutions disclosed herein with respect to pre-fusion stabilization.

Nucleic acid molecules

In some embodiments, the invention provides nucleic acid molecules encoding the mutant RSV F proteins of the invention, as well as vectors comprising the nucleic acid molecules. In view of the generally known and understood nature of the genetic code of those of ordinary skill in the art, one of ordinary skill in the art can readily determine the nucleic acid sequence of a nucleic acid molecule encoding any of the mutant RSVF proteins described herein.

Nucleic acid molecules encoding the mutant RSVF molecules of the invention may be obtained or prepared using any suitable method known in the art. For example, nucleic acid molecules encoding mutant RSV F molecules can be obtained from cloned DNA or prepared by chemical synthesis. In some embodiments, the nucleic acid molecule may be obtained by reverse transcription of RNA, wherein the RNA is prepared by any method known to one of ordinary skill in the art. Point mutations, or any other modification described herein (e.g., removal of the C-terminal sequence, substitution of the C-terminal sequence, etc.), can be accomplished by standard recombinant DNA procedures well known and understood by those of ordinary skill in the art.

In certain embodiments, nucleic acid molecules encoding proteins according to the invention are codon optimized for expression in mammalian cells, preferably human cells. A sequence is considered codon optimized if at least one non-preferred codon is replaced with a more preferred codon as compared to the wild-type sequence. Here, a non-preferred codon is a codon that is not used frequently in an organism as another codon encoding the same amino acid, and a more preferred codon is a codon that is used more frequently in an organism than a non-preferred codon. Preferably more than one non-preferred codon, preferably the most or all non-preferred codons, are replaced by more preferred codons. Preferably, codons most frequently used in an organism are used for a codon optimized sequence. Substitution by preferred codons generally results in higher expression.

Those of skill in the art will appreciate that many different polynucleotides and nucleic acid molecules may encode the same polypeptide due to the degeneracy of the genetic code. It will also be appreciated that the skilled artisan can use conventional techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the nucleic acid molecule to reflect codon usage of any particular host organism in which the polypeptide is to be expressed. Thus, unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The nucleotide sequences encoding proteins and RNAs may or may not include introns.

Viral vectors

Nucleic acid molecules encoding recombinant RSV F proteins stabilized in a pre-fusion conformation can be included in viral vectors, e.g., to express an antigen in a host cell, or for immunization of a subject as disclosed herein. In some embodiments, the viral vector is administered to the subject as part of a prime-boost vaccination. In some embodiments, the viral vector is included in a vaccine, such as a prime vaccine or a boost vaccine, for prime-boost vaccination.

In several examples, the viral vector encoding the recombinant RSVF protein that stabilizes in a pre-fusion conformation may have replication capabilities. For example, the viral vector may have a mutation in the viral genome (e.g., insertion of a nucleic acid encoding a PreF antigen) that does not inhibit viral replication in the host cell. The viral vector may also have conditional replication capacity. In other examples, the viral vector is replication defective in a host cell.

In some embodiments, the recombinant RSVF protein that is stable in the pre-fusion conformation is expressed by a viral vector that can be delivered via the respiratory tract. For example, a Paramyxovirus (PIV) vector such as a bovine parainfluenza virus (BPIV) vector (e.g., BPIV-1, BPIV-2, or BPV-3 vector) or a human PIV vector, metapneumovirus (MPV) vector, sendai virus vector, or measles virus vector is used to express the disclosed antigens. BPIV3 viral vectors expressing RSV F and hPIV F proteins (MEDI-534) are currently in clinical trials as RSV vaccines. In another example, newcastle disease virus vectors are used to express the disclosed antigens. In another example, sendai virus vectors are used to express the disclosed antigens. Other viral vectors may also be used to express the disclosed antigens, including polyomaviruses, i.e., SV40, adenovirus, adeno-associated virus, vaccinia virus, herpes viruses including HSV and EBV and CMV, sindbis virus, alphavirus, and retroviruses. Baculovirus (Medicago sativa noctuid polynuclear polyhedra; acMN PV) vectors are also known in the art and are available from commercial sources. Other viral vectors are well known to those of ordinary skill in the art.

In some embodiments, the methods and compositions disclosed herein include adenovirus vectors expressing recombinant RSVF proteins stabilized in a pre-fusion conformation. Adenoviruses from various sources, subtypes or mixtures of subtypes may be used as a source of viral genome for an adenovirus vector. Non-human adenoviruses (e.g., simian, chimpanzee, gorilla, avian, canine, ovine, or bovine adenoviruses) can be used to produce an adenovirus vector. For example, simian adenoviruses can be used as a source of viral genome for adenovirus vectors. The simian adenovirus may have serotypes 1, 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, 39, 48, 49, 50 or any other simian adenovirus serotype. Simian adenoviruses may be mentioned by using any suitable abbreviation known in the art, such as SV, SAdV, SAV or sAV. In some examples, the simian adenovirus vector is a simian adenovirus vector having, but not limited to, serotypes 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, or 39. In one example, a chimpanzee serotype Adc68 vector is used. Human adenoviruses can be used as a source of viral genome for adenovirus vectors. Human adenoviruses can have various subgroups or serotypes. For example, an adenovirus may have subgroup a (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), an unclassified serogroup (e.g., serotypes 49 and 51), or any other adenovirus serotype. Replication-competent and replication-defective adenovirus vectors (including single and multiple replication-defective adenovirus vectors) are well known to those of ordinary skill in the art.

Host cells

In the present invention, the host may include microorganisms, yeasts, insects and mammalian organisms. Methods for expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Non-limiting examples of suitable host cells include bacteria, archaea, insects, fungi (e.g., yeast), plant and animal cells (e.g., mammalian cells, such as humans). Exemplary cells used include E.coli, B.subtilis, saccharomyces cerevisiae, salmonella typhimurium, SF9 cells, C129 cells, 293 cells, neurospora, and immortalized mammalian bone marrow and lymphocyte cell lines. Techniques for proliferation of mammalian cells in culture are well known. Examples of commonly used mammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK and COS cell lines, but cell lines such as cells designed to provide higher expression, desired glycosylation patterns, or other characteristics may be used. In some embodiments, the host cell comprises a HEK293 cell or a derivative thereof, such as a GnTI ^-/- cell.

The host cell may be transformed with the recombinant DNA by conventional techniques as are well known to those skilled in the art. When the host is prokaryotic (e.g., without limitation, E.coli), competent cells capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by CaCl ₂ method using procedures well known in the art. Alternatively, mgCl ₂ or RbCl may be used. Transformation can also be carried out after formation of protoplasts of the host cell or if necessary by electroporation.

When the host is eukaryotic, these DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical procedures such as microinjection, electroporation, insertion of plasmids entrapped in liposomes, or viral vectors may be used. Eukaryotic cells may also be co-transformed with a polynucleotide sequence encoding the disclosed antigens and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another approach is to use eukaryotic viral vectors such as simian virus 40 (SV 40) or bovine papilloma virus to transiently infect or transform eukaryotic cells and express the proteins.

Composition and method for producing the same

The mutant RSVF proteins, virus-like particles, protein nanoparticles, nucleic acid molecules, host cells or various adjuvants of the present disclosure are combined with optionally other therapeutic ingredients such as antiviral drugs. In some embodiments, the composition comprising one or more of the disclosed RSV F proteins, virus-like particles, protein nanoparticles, nucleic acid molecules, or host cells is an immunogenic composition. The composition may comprise any RSV F protein comprising a recombinant RSV F protein as disclosed herein (e.g., a protein nanoparticle comprising any recombinant RSV F protein as disclosed herein), a virus-like particle comprising any recombinant RSV F protein as disclosed herein, a nucleic acid molecule encoding any recombinant RSV F protein as disclosed herein, or a vector encoding or comprising any recombinant RSV F protein as disclosed herein.

These pharmaceutical compositions can be administered to a subject by a variety of modes of administration known to those of ordinary skill in the art, for example, nasal, pulmonary, intramuscular, subcutaneous, intravenous, intraperitoneal, or parenteral routes.

To formulate the composition, the disclosed RSVF proteins, virus-like particles, protein nanoparticles, viral vectors, or nucleic acid molecules may be combined with various pharmaceutically acceptable additives as well as a matrix or vehicle for dispersing the conjugate. Desirable additives include, but are not limited to, pH control agents such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like. In addition, local anesthetics (e.g., benzyl alcohol), isotonic agents (e.g., sodium chloride, mannitol, sorbitol), adsorption inhibitors (e.g.,80 A) a solubilizing agent (e.g., cyclodextrin and derivatives thereof), a stabilizing agent (e.g., serum albumin), and a reducing agent (e.g., glutathione). The composition may include adjuvants such as aluminum hydroxide, freund's adjuvant, MPL ^TM, IL-12, a TLR agonist (e.g., a TLR-9 agonist), and many other suitable adjuvants well known in the art.

The disclosed RSV F proteins, virus-like particles, protein nanoparticles, viral vectors, or nucleic acid molecules can be dispersed in a matrix or vehicle, which can include a hydrophilic compound capable of dispersing an antigen, and any desired additives. The matrix may be selected from a wide range of suitable compounds including, but not limited to, polycarboxylic acids or salts thereof, copolymers of carboxylic anhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl (meth) acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethyl cellulose, hydroxypropyl cellulose, etc., and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and non-toxic metal salts thereof. Typically, biodegradable polymers are selected as a matrix or vehicle, for example, polylactic acid, poly (lactic-co-glycolic acid), polyhydroxybutyric acid, poly (hydroxybutyrate-co-glycolic acid), and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters, and the like may be used as vehicles. Hydrophilic polymers and other vehicles may be used alone or in combination, and may impart enhanced structural integrity to the vehicle by partial crystallization, ionic bonding, crosslinking, and the like. The vehicle may be provided in a variety of forms including fluid or viscous solutions, gels, pastes, powders, microspheres and films, for example for direct application to mucosal surfaces.

The disclosed RSV F proteins, virus-like particles, protein nanoparticles, viral vectors, or nucleic acid molecules can be combined with a matrix or vehicle according to a variety of methods, and release of antigen can be through diffusion, disintegration of the vehicle, or related formation of water channels. In some cases, the disclosed antigens or nucleic acids or viral vectors encoding, expressing or comprising the antigens are dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) prepared from a suitable polymer (e.g., isobutyl 2-cyanoacrylate) and dispersed in a biocompatible dispersion medium that provides sustained delivery and bioactivity over an extended period of time.

The pharmaceutical compositions may contain as pharmaceutically acceptable vehicles substances required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate and triethanolamine oleate. For solid compositions, conventional non-toxic pharmaceutically acceptable vehicles may be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

Pharmaceutical compositions for administration of the disclosed RSV F protein antigens, virus-like particles, protein nanoparticles, viral vectors or nucleic acid molecules can also be formulated as solutions, microemulsions or other ordered structures suitable for high concentrations of active ingredient. The vehicle may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity of the solution may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersible formulations and by the use of surfactants. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of the disclosed antigens can be brought about by including agents in the composition that delay absorption, such as monostearates and gelatin.

In certain embodiments, the disclosed RSV F proteins, virus-like particles, protein nanoparticles, viral vectors, or nucleic acid molecules can be administered in a time-release formulation, such as a composition comprising a slow-release polymer. These compositions may be prepared with a vehicle that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system or bioadhesive gel. Prolonged delivery in the various compositions of the invention can be brought about by including agents in the composition that delay absorption (e.g., aluminum monostearate hydrogels and gelatin). Where controlled release formulations are desired, controlled release binders suitable for use in accordance with the present invention include any biocompatible controlled release material that is inert to the active agent and capable of incorporating the disclosed antigens and/or other bioactive agents. Numerous such materials are known in the art. Useful controlled release adhesives are materials that slowly metabolize under physiological conditions after their delivery (e.g., at a mucosal surface, or in the presence of body fluids). Suitable binders include, but are not limited to, biocompatible polymers and copolymers well known in the art for use in sustained release formulations. These biocompatible compounds are non-toxic and inert to surrounding tissues and do not trigger significant adverse side effects such as nasal irritation, immune response, inflammation, and the like. They are metabolized into metabolites that are also biocompatible and easily removed from the body.

Exemplary polymeric materials for use include, but are not limited to, polymeric matrices derived from copolymerized and homopolymerized polyesters having hydrolyzable ester linkages. Many of these are known in the art to be biodegradable and produce degradation products that are non-toxic or have low toxicity. Exemplary polymers include polyglycolic acid and polylactic acid, poly (DL-lactic-co-glycolic acid), poly (D-lactic-co-glycolic acid), and poly (L-lactic-co-glycolic acid). Other useful biodegradable or bioerodible polymers include, but are not limited to, polymers such as: poly (epsilon-caprolactone), poly (epsilon-caprolactone-co-lactic acid), poly (epsilon-caprolactone-co-glycolic acid), poly (beta-hydroxybutyric acid), poly (alkyl 2-cyanoacrylates), hydrogels such as poly (hydroxyethyl methacrylate), polyamides, poly (amino acids) (e.g., L-leucine, glutamic acid, L-aspartic acid, etc.), poly (ester urea), poly (2-hydroxyethyl DL-asparagine), polyacetal polymers, polyorthoesters, polycarbonates, polymaleimides, glycans, and copolymers thereof. Many methods for preparing these formulations are well known to those skilled in the art. Other useful formulations include controlled release microcapsules, lactic acid-glycolic acid copolymers useful in making microcapsules and other formulations, and sustained release compositions for water-soluble peptides.

Pharmaceutical compositions are generally sterile and stable under conditions of manufacture, storage, and use. Sterile solutions can be prepared by incorporating the disclosed RSV F proteins, virus-like particles, protein nanoparticles, viral vectors or nucleic acid molecules in the required amounts in an appropriate solvent with one or a combination of the ingredients listed in the invention, followed by filter sterilization if desired. Generally, dispersions are prepared by incorporating the disclosed antigens and/or other bioactive agents into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders, the methods of preparation include vacuum drying and freeze-drying, which yields a powder of the disclosed antigen plus any additional desired ingredient from a previously sterile-filtered solution thereof. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and the like. The actual method used to prepare the administrable composition will be known or apparent to those skilled in the art.

In some embodiments, the composition comprises an adjuvant. Adjuvants are well known to those of ordinary skill in the art, for example, those that may be included in immunogenic compositions. In some embodiments, the adjuvant is selected to induce a Th 1-biased immune response in a subject administered an immunogenic composition comprising the adjuvant and the disclosed antigen, or a nucleic acid or viral vector encoding, expressing, or comprising the antigen.

The amount of RSV F protein, viral vector or nucleic acid molecule disclosed can vary depending upon, for example, the particular antigen, route of administration and regimen used, and the population of interest. The amount utilized in the immunogenic composition is selected based on the population of subjects (e.g., infants or elderly). The optimal amount of a particular composition may be determined by standard studies involving observation of antibody titers and other responses in subjects. It will be appreciated that a therapeutically effective amount of antigen in an immunogenic composition may include an amount that is not able to induce an immune response by administration of a single dose, but is effective after administration of multiple doses (e.g., in a prime-boost administration regimen).

In several examples, a pharmaceutical composition for inducing an immune response to RSV in a human includes a therapeutically effective amount of a disclosed RSV F protein, viral vector, or nucleic acid molecule for administration to an infant (e.g., an infant with an initial dose age of from birth to 1 year, e.g., 0 to 6 months) or an elderly patient subject (e.g., a subject with an age greater than 65 years). It will be appreciated that the choice of adjuvant may vary in these different applications and that the skilled person can empirically determine the optimal adjuvant and concentration for each situation.

Therapeutic method

In some embodiments, the disclosed RSV F proteins, or nucleic acids, viral vectors, virus-like particles, or protein nanoparticles encoding, expressing, or comprising RSV F proteins, are used in a subject to induce an immune response against RSV. Thus, in some embodiments, a therapeutically effective amount of an immunogenic composition comprising one or more of the disclosed RSV F proteins, or nucleic acids or viral vectors encoding, expressing, or comprising the antigens, may be administered to a subject to generate an immune response against RSV.

In the present invention, an immunogenic composition comprising RSV F protein, or a nucleic acid encoding, expressing or comprising said antigen, a viral vector, a virus-like particle or a protein nanoparticle, is administered to a subject in need of treatment in a prophylactically or therapeutically effective amount for a time and under conditions sufficient to prevent, inhibit and/or ameliorate RSV infection in the subject. The immunogenic composition is administered in an amount sufficient to induce an immune response in the subject against an RSV antigen, such as an RSV f protein.

In some embodiments, a subject having or at risk of developing an RSV infection, e.g., due to exposure to RSV or the likelihood of exposure to RSV, is selected for treatment. Following administration of a therapeutically effective amount of the disclosed therapeutic compositions, the subject may be monitored for RSV infection, symptoms associated with RSV infection, or both. Because almost all humans are infected with RSV by 3 years of age, the entire birth population is included as an immune related population. This can be done, for example, by starting an immunization regimen at any time from birth to 6 months of age, from 6 months of age to 5 years of age, in pregnant women (or women of childbearing age) to protect their infants, family members of newborns or fetuses by passive transfer of antibodies, and subjects older than 50 years of age.

The immunogenic compositions may be used in a collaborative vaccination regimen or in a combined preparation. In certain embodiments, the combined immunogenic composition and the co-immunization regimen employ separate immunogens or formulations, each directed against inducing an immune response to an RSV antigen, such as an immune response to an RSV f protein. The separate immunogenic compositions that induce an immune response to RSV antigen may be combined in a multivalent immunogenic composition that is administered to the subject in a single immunization step, or they may be administered independently (in a monovalent immunogenic composition) in a collaborative immunization regimen.

Administration of the immunogenic composition may be for prophylactic or therapeutic purposes. When provided prophylactically, the immunogenic composition is provided prior to any symptoms, e.g., prior to infection. The prophylactic administration of the immunogenic composition is used to prevent or ameliorate any subsequent infection. When provided therapeutically, the immunogenic composition is provided at or after the onset of symptoms of the disease or infection, for example, after the development of symptoms of RSV infection, or after diagnosis of RSV infection. The immunogenic composition may thus be provided prior to the intended exposure to RSV to attenuate the intended severity, duration, or extent of symptoms of the infection and/or associated disease, or after exposure or suspected exposure to virus, or after the actual initiation of the infection.

For prophylactic and therapeutic purposes, the immunogenic composition can be delivered in a single bolus, via continuous delivery (e.g., continuous transdermal, mucosal, or intravenous delivery) over an extended period of time, or administered to the subject in a repeated administration regimen (e.g., by a repeated administration regimen of once per hour, day, or week). The therapeutically effective dose of the immunogenic composition may be provided in repeated doses within an extended prophylactic or therapeutic regimen that will produce clinically significant results to alleviate one or more symptoms or detectable conditions associated with the targeted disease or condition as described herein. The determination of effective dosages in the present invention is typically based on animal model studies, followed by human clinical trials, and is guided by an administration regimen that significantly reduces the occurrence or severity of a targeted disease symptom or condition in a subject. Suitable models in this regard include, for example, murine, rat, porcine, feline, ferret, non-human primate, and other recognized animal model subjects known in the art. Alternatively, in vitro models (e.g., immunological and histopathological assays) can be used to determine effective dosages. Using these models, only ordinary calculations and adjustments are required to determine the appropriate concentrations and dosages to administer a therapeutically effective amount of the immunogenic composition (e.g., an amount effective to elicit a desired immune response or to alleviate one or more symptoms of the targeted disease). In alternative embodiments, an effective amount or dose of an immunogenic composition may simply inhibit or enhance one or more selected biological activities associated with a disease or condition as described herein for therapeutic or diagnostic purposes.

In one embodiment, a suitable immunization regimen comprises at least three separate vaccinations with one or more immunogenic compositions, wherein the second vaccination is administered greater than about two weeks, about three weeks to eight weeks, or about four weeks after the first vaccination. Typically, the third inoculation is administered several months after the second inoculation, and in particular embodiments, greater than about five months after the first inoculation, greater than about six months to about two years after the first inoculation, or about eight months to about one year after the first inoculation. Periodic vaccinations beyond the third time are also required to enhance the subject's "immune memory". The suitability of selected vaccination parameters, e.g., formulation, dose, regimen, etc., can be determined by taking an aliquot of serum from the subject and determining the antibody titer during the immunization program. If such detection indicates that vaccination is suboptimal, the subject may be boosted with additional doses of the immunogenic composition and the vaccination parameters may be adjusted in a manner that is expected to enhance the immune response. It is contemplated that there may be several reinforcements, and that each reinforcement may include the same or different RSVF proteins.

For prime-boost regimens, priming may be administered in a single dose or multiple doses, e.g., two doses, three doses, four doses, five doses, or six doses or more may be administered to a subject over days, weeks, or months. Boost may be administered in a single dose or multiple doses, e.g., two to six doses or more may be administered to a subject over a day, week, or month. Multiple enhancements, such as one to five or more, may also be given. Different doses may be used in a series of consecutive inoculations. For example, in a primary vaccination with a relatively large dose followed by a relatively small dose boost. An immune response against a selected antigen surface can be generated by one or more vaccinations of a subject with an immunogenic composition disclosed herein.

Use of mutated RSVF

In some embodiments, the mutated RSV F of the invention can be used as a research tool, a diagnostic tool, a therapeutic agent, for the production of antibody agents or as a target for therapeutic antibodies, and/or as a component of a vaccine or vaccine composition. For example, in some embodiments, the mutated RSV F of the invention can be used as a vaccine immunogen in an animal subject (e.g., a mammalian subject, including a human). Those skilled in the art will appreciate that the mutant RSV F of the invention can also be used in a variety of other applications, and all such applications and uses are intended to fall within the scope of the invention.

Investigation of anti-RSVF antibodies

In one embodiment, the mutated RSV F proteins of the invention can be used as analytes for testing and/or measuring the binding and/or titer of anti-RSVF antibodies, e.g., in ELISA assays, biacore/SPR binding assays, and/or any other antibody binding assays known in the art. For example, the mutant RSVF proteins of the invention can be used to analyze and/or compare the effects of anti-RSVF antibodies.

Antibody production

The mutant RSV F proteins or substances comprising mutant RSV F proteins of the invention can also be used to produce therapeutic antibodies, and/or antibodies that can be used as research tools, or for any other desired use. For example, the mutant RSVF proteins of the invention may be used in immunization to obtain RSV F protein antibodies useful as research tools and/or therapeutics. In some embodiments, to produce antibodies, the mutant RSV F proteins of the invention can be used to immunize non-human animals, such as vertebrates, including, but not limited to, mice, rats, guinea pigs, rabbits, goats, non-human primates, and the like. Such antibodies may then be obtained from animals, which may be monoclonal or polyclonal, and/or cells producing such antibodies. For example, in some embodiments, the mutant RSV F proteins of the invention can be used to immunize mice to produce and obtain monoclonal antibodies, and/or hybridomas that produce such monoclonal antibodies. These methods can be performed using standard methods known in the art for producing mouse monoclonal antibodies, including standard methods for producing hybridomas. In some embodiments, the mutant RSVF proteins of the invention can be used to produce chimeric (e.g., partially humanized), humanized or fully human antibodies, for example, using any method currently known in the art for producing chimeric, humanized and fully human antibodies, including but not limited to CDR grafting methods, phage display methods, transgenic mouse methods (e.g., using mice genetically engineered to be capable of producing fully human antibodies, e.g., transgenic mice), and/or any other suitable method known in the art. Antibodies raised against the mutant RSV F proteins of the invention using such a system can be antigenically characterized using one or a set of multiple antigens, preferably including the mutant RSV F proteins of the invention themselves. Such antibodies may be additionally characterized by any standard method known to those of ordinary skill in the art, including, but not limited to, ELISA-based methods, SPR-based methods, biochemical methods (e.g., without limitation, isoelectric point assays), and methods known in the art for studying the biodistribution, safety, and effectiveness of antibodies (e.g., in preclinical and clinical studies).

A subject

In the present invention, subjects include any animal species, including, inter alia, those susceptible to RSV infection or capable of providing a model animal system for RSV infection studies. In some embodiments, the subject is a mammalian species. In some embodiments, the subject is an avian species. Mammalian subjects include, but are not limited to, humans, non-human primates, rodents, rabbits, and ferrets. Avian subjects include, but are not limited to, chickens, such as chickens in a poultry farm. In a preferred embodiment, the subject is a human.

Kit for detecting a substance in a sample

The invention also provides kits comprising the mutant RSV F proteins of the invention, or compositions comprising the mutant RSV F proteins. To facilitate use of the methods and compositions of the present invention, any of the components and/or compositions described herein, as well as other components used for experimental or therapeutic or vaccination purposes, may be packaged in kit form. Typically, the kit will contain, in addition to the components described above, other materials which may include, for example, instructions for use of the components, packaging materials, containers, and/or delivery devices.

The label or package insert will also typically include instructions for using the RSVF protein, or a nucleic acid or viral vector encoding, expressing, or including the antigen, for example, in a method of treating or preventing an RSV infection. The package insert typically includes instructions, as usual, included in the commercial package of therapeutic products containing information about the indication, use, dosage, administration, contraindications and/or warnings regarding the use of such therapeutic products. The instructional material may be written in electronic form (e.g., a computer diskette or optical disk) or may be visual (e.g., a video file). The kit may also include other components to facilitate specific applications regarding the design of the kit. The kit may additionally include buffers and other reagents commonly used to carry out particular methods. Such kits and suitable contents are well known to those skilled in the art.

Detailed Description

The invention will now be described in further detail with reference to the drawings and examples. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention.

Example 1 engineering and screening of full Length Cross model F proteins

1.1 First round of protein engineering

The F protein (GeneBank AC083301.1, SEQ ID NO. 1) of the reference sequence RSV A2 strain is used as a template and is modified as follows: mutating the 55 th amino acid from S to V and mutating the 210 th amino acid from Q to P; the extracellular region of the F protein contains a deletion of 104-144 and adds a dipeptide linkage of glycine-serine between 103 and 145 (i.e., sc 9-10); the 215 th amino acid is mutated from S to P (S215P), a mutant MF (sc 9-10, S215P, SEQ ID NO. 2) is obtained, a plurality of point mutations (shown in table 1) are added to the extracellular region of the MF, 293T cells are transfected after constructing corresponding plasmids, F protein antibody is utilized to mark F protein on the cell surface, and the properties of the F protein mutant are analyzed by a flow cytometry method.

TABLE 1 mutation sites

The F protein gene of codon optimized RSV A2 strain was cloned into pcDNA3.1 vector, namely pcDNA-wtF. The codon optimized MF containing the above deletions and mutations was synthesized and cloned into pcDNA3.1 vector, pcDNA-MF.

Constructing plasmids containing the corresponding mutations in Table 1, designing primers overlapping with the carrier sequence at the 5 'end and the 3' end of the MF ORF, respectively designing forward and reverse primers overlapping with each other at the point mutation position, and PCR-amplifying an upstream fragment from the 5 'end to the mutation site and a downstream fragment from the mutation site to the 3' end of the MF by using pcDNA-MF as a template and using high-fidelity KOD enzyme. Cloning the two fragments to linearized pcDNA-MF by utilizing a homologous recombination method, transforming site-directed mutagenesis plasmid, and carrying out gene sequencing to identify whether recombination is successful. Clones with correct sequencing are extracted from the plasmid, and then sequencing is carried out again to identify the accuracy of the plasmid sequence, and a spectrophotometer analyzes the DNA concentration and then the DNA is frozen in a refrigerator at the temperature of minus 80 ℃ for storage.

The mutant plasmids were transfected into 293T cells, and F protein mutants expressed on the surface of 293T cells were bound using a plurality of antibodies directed against different epitopes (table 2), including D25 (McLellan JS et al, 2013), CR9501 (Gilman, m.s.a. et al, 2019), motavizumab (McLellan JS et al, 2010), 4D7 (Espeseth, a.s. et al, 2020). And then the F protein antibody combined on the cell surface is marked by using a secondary Anti-human IgG Fc PE. And finally analyzing F protein on the cell surface by a flow cytometry method. The method comprises the following specific steps:

On the first day, 293T cells were passaged into 6-well plates, 1.7X10 ⁶/well. The following day, when the cell confluence reached 90%, cell transfection was performed, 88. Mu.l Opti MEM, 2. Mu.g plasmid, 8. Mu.l PEI were added sequentially to a 1.5ml centrifuge tube, mixed with gentle shaking, and left at room temperature for 15min before hanging drop onto 293T cells in one well of a 6-well plate. Thirdly, discarding the cell culture solution, washing the cells with PBS, then adding pancreatin for digestion, sucking pancreatin after the cells are circularly contracted and dispersed, and blowing the cells by using PBS containing 2% of serum; cells were plated into 96-well plates (2×10 ⁵/well); centrifuging at 350g for 3min, removing supernatant, adding 100 μl of primary antibody with corresponding concentration in table 2, and dyeing at 4deg.C for 30min in dark place; centrifuging at 350g for 3min, removing supernatant, adding washing liquid, washing for 3 times, centrifuging at 350g for 3min, removing supernatant, adding 100 μl of 200-fold diluted secondary antibody anti-human IgG Fc PE, and dyeing at 4deg.C for 30min in dark place; 350g was centrifuged for 3min, the supernatant removed, wash solution was added 3 times and the cells were finally resuspended in wash solution and analyzed by flow cytometry.

Table 2 detection antibodies

The binding activity of the F mutant to each antibody was assessed by normalizing the total fluorescence intensity to MwtF or MF values, wherein staining of antibody 4D7 was used to characterize post conformation, staining of antibodies D25 and CR9501 was used to characterize pre conformation, and antibody Motavizumab was used to express the expression level of the F mutant. The ratio results are shown in tables 3 and 4 below, where Table 3 is the results of the flow assay of mutants relative to wild type MwtF and Table 4 is the results of the flow assay of mutants relative to MF. The results in table 3 show that S55V, Q210P significantly reduced the post conformational epitope while significantly improved the pre conformation; N254R significantly reduced post conformational epitopes. The results in Table 4 show that the mutation L188C-D263C, S190C-L260C, K394C-S491C, I475C-T323C, N254R, S V significantly reduced the post conformational epitope; N254R, K461P, S190C-L260C, L C-D263C significantly improves pre-conformation; K461P, N254R, S190C-L260C, I475C-T323C significantly improves the expression level.

TABLE 3 stream test results

TABLE 4 stream test results

1.2 Second round of protein engineering

Based on the first round of engineered flow results, dominant mutations were combined to obtain F mutants with a more pre-conformation. Thus the additive combinatorial mutations were designed on the basis of MF. After constructing the corresponding plasmid, the 293T cells are transfected, F protein antibody is utilized to mark F protein on the cell surface, and the properties of the F protein mutant are analyzed by a flow cytometry method.

Plasmid extraction and sequencing identification are carried out after plasmid containing corresponding mutation is constructed according to the method of 1.1, 293T cells are transfected, cells are harvested after 1 day, F protein mutants expressed on the surface of the 293T cells are combined by antibodies aiming at different epitopes and used in the first transformation, and F protein antibodies combined on the surface of the cells are marked by secondary Anti-human IgG Fc PE. And finally analyzing F protein on the cell surface by a flow cytometry method. The binding activity of the F mutant to each antibody was evaluated by normalizing the total fluorescence intensity to MF values, and the ratio results are shown in table 5. MF42 (mf+f190C/L260C, N254R, S55V), MF45 (mf+k461P, K394C/S491C), MF46 (mf+k461P, I475C/T323C) significantly reduced post conformation and increased pre-conformation F protein mutants to some extent.

TABLE 5 stream test results

1.3 Adenovirus packaging

The adenovirus vector vaccine can carry exogenous genes for expressing antigens into human bodies, and has the advantages of high expression capacity, strong immunogenicity, stable production and storage, diversified immune pathways, wide immune response excitation by single-needle immunization, and the like. The adenovirus vector used in the invention is a rare serotype chimpanzee adenovirus vector AdC68 with extremely low immunity prestored in the population, and the E1 gene is deleted from the vector and replaced by an expression cassette for expressing RSV F protein.

The genes of the cross-model F protein mutants MF42 and wtF are recombined to the E1 gene position of the AdC68 vector respectively, and the recombinant adenovirus expressing F proteins, namely Ad-MF42 and Ad-wtF, is obtained after transfection of 293A cells. Amplification and purification are carried out after obtaining adenovirus virus seed. Adding the virus seed into 293A cells with 80-90% confluency, collecting all cells and supernatant after typical lesions appear on the cells, repeatedly freezing and thawing, and centrifugally collecting supernatant, namely adenovirus amplification solution. The adenovirus supernatant was settled by PEG8000, the precipitate was collected after centrifugation, the adenovirus was purified by cesium chloride density gradient centrifugation after PBS reconstitution, the adenovirus-containing strip was removed, and the buffer (Tris buffer system containing 5% sucrose) was dialyzed.

After purification of the recombinant adenovirus, 293T cells were infected and analyzed for F protein expression. 293T cells were passaged into 6-well plates, and when confluence was 80% -90%, adenovirus purified by 1X 10 ⁹ VP was added to each 293T cell, and after 48 hours, the cells were harvested, and antigen expression was analyzed by the above-described flow method.

As shown in FIG. 1, the results of the flow pattern show that Ad-MF42 has significant pre-conformational antibody staining (D25 and CR 9501), while post conformational antibody (4D 7) has a lower staining level, indicating that Ad-MF42 expresses F protein with pre conformation.

1.4 Mouse immunization experiments

Recombinant adenoviruses Ad-wtF and Ad-MF42 of 1X 10 ¹⁰ VP were immunized against SPF-grade BALB/c mice of about 7 weeks of age, and boosted three weeks after priming. The immunization mode is intramuscular injection. Blood was collected three weeks after the second immunization, serum was isolated and inactivated, and the titer of preF specific antibodies and the titer of neutralizing antibodies in the serum were measured.

Coating preF protein DS2 protein (M Gordon Joyce et al, 2016) on ELISA plates overnight; the next day, the gradient diluted mouse serum was added; adding an HPR marked Anti-mouse IgG secondary antibody; and finally adding a chromogenic substrate for chromogenic reaction.

ELISA results are shown in FIG. 2, and immunization of Ad-MF42 induced higher titers of preF-specific antibodies than Ad-wtF. The same method is used for carrying out an immune mouse experiment on other mutations, and other mutations are found to be capable of inducing preF specific antibodies with higher titer compared with Ad-wtF.

The titrated RSV virus was diluted to 100TCID50 and the diluted virus was added to a 96 well cell culture plate at 50 μl per well. The serum to be tested was then diluted in a double ratio, 50. Mu.l each well, 4 wells per dilution and incubated for 2 hours at 37 ℃. HEp-2 cells were trypsinized, passaged to 96-well cell culture plates incubated with virus and serum, 100 μl per well (about 3e+5 cells/well). Viral and cellular controls were established simultaneously. Cytopathic effect was observed microscopically for the fifth day and 50% effective concentration (IC 50) was calculated for each serum.

The results of the neutralizing antibody titer assays are shown in FIG. 3, where Ad-MF42 induced a higher titer of RSV neutralizing antibody than Ad-wtF.

Example 2 engineering of F protein

F190C/L260C, N254R and S55V are taken as mutation main bodies, S215 and sc9-10 are respectively replaced, 293T cells are transfected after plasmids containing corresponding mutations are constructed, analysis is carried out by a flow method, total fluorescence intensity is taken as a reference value, WT values are taken as normalization, and the binding activity of F mutants and antibodies is evaluated, and the results are shown in Table 6.

TABLE 6 stream test results

Note that: the amino acid of the leading part is linker between F2 and F1, the first M corresponds to methionine at position 97 of F protein, and the last A corresponds to alanine at position 149.

The K461P, I475C/T323C are taken as mutation main bodies, S215 and sc9-10 are respectively replaced, 293T cells are transfected after plasmids containing corresponding mutations are constructed, analysis is carried out by a flow method, the total fluorescence intensity is taken as a reference value, the WT value is taken as normalization, and the binding activity of the F mutant and each antibody is evaluated, and the results are shown in Table 7.

TABLE 7 stream test results

Note that: the amino acid of the leading part is linker between F2 and F1, the first M corresponds to methionine at position 97 of F protein, and the last A corresponds to alanine at position 149.

Example 3 detection of soluble F protein

Deleting the F protein transmembrane region, only preserving the extracellular region, expressing the secreted F protein mutant and wtF containing trimerization Foldon tag, his and strep tag, and detecting the F protein secreted into the culture medium supernatant by using a sandwich ELISA method.

The expressed soluble protein F mutations include ：SF(sc9-10、S215P)、SM42(sc9-10,S215P,F190C/L260C,N254R,S55V)、SM46(sc9-10,S215P,K461P,I475C/T323C)、SM59(sc9-10,F190C/L260C,N254R,S55V,I214P)、SM60(sc9-10,F190C/L260C,N254R,S55V,S213P)、SM61(sc9-10,F190C/L260C,N254R,S55V,N216P)、SM65(S215P,F190C/L260C,N254R,S55V,"MQSTPATGPAIA")、SM91(S215P,K461P,I475C/T323C,"MQSTPATGPAIA")、SM95(sc9-10,F190C/L260C,N254R,S55V,Q210P)、SM96(sc9-10,F190C/L260C,N254R,S55V,S211P)、SM97(sc9-10,F190C/L260C,N254R,S55V,C212P)、SM98(sc9-10,F190C/L260C,N254R,S55V,I217P).

Designing a primer to amplify N-terminal to 513 amino acids of extracellular region of cross-model F protein, fusing foldon, his, strep tag at C-terminal, connecting to pcDNA3.1 vector, sequencing and identifying correctly, carrying out large extraction of plasmid, measuring DNA concentration by spectrophotometer, and freezing in-80 deg.C refrigerator.

The plasmids were transfected into 293F cells and the supernatant was collected for ELISA analysis. After 293F was grown to 2.5X10 ⁶/ml, the cells were aliquoted into disposable filter centrifuge tubes at 10 ml/tube. Taking a 1.5ml centrifuge tube, adding 500 mu l of blank culture medium, adding 10 mu g of plasmid, and uniformly mixing; into another 1.5ml centrifuge tube, 450. Mu.l of blank medium and 40. Mu.l PEI were added and mixed well. The solutions in the two centrifuge tubes were mixed, allowed to stand at room temperature for 15min, and then added dropwise to 293F cells, and fixed in a shaker for culture. After 4 days, the supernatant was collected after centrifugation at 3000rpm for 15 min.

Coating ELISA plates with Anti-Strep antibodies overnight; adding culture supernatant containing F mutant and wtF the next day, and capturing F protein in the supernatant; adding gradient diluted F protein antibodies D25 and 4D7; adding an HPR marked Anti-human IgG secondary antibody; adding a chromogenic substrate for chromogenic reaction.

The ELISA results are shown in FIG. 4, and demonstrate that all engineered clones improved pre conformation compared to wt and SF, while some clones reduced post conformation.

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention. Reference is made to:

McLellan JS,Chen M,Leung S,Graepel KW,Du X,YangY,Zhou T,Baxa U,Yasuda E,Beaumont T,Kumar A,Modjarrad K,Zheng Z,Zhao M,Xia N,Kwong PD,Graham BS.Structure of RSV fusion glycoprotein trimer bound to aprefusion-specific neutralizing antibody.Science.2013May 31;340(6136):1113-7.doi:10.1126/science.1234914.Epub 2013Apr 25.PMID:23618766;PMCID:PMC4459498.

Gilman,M.S.A.,Furmanova-Hollenstein,P.,Pascual,G.et al.Transient opening of trimeric prefusion RSV F proteins.Nat Commun 10,2105(2019).https://doi.org/10.1038/s41467-019-09807-5.

McLellan JS,Chen M,Kim A,Yang Y,Graham BS,Kwong PD.Structural basis of respiratory syncytial virus neutralization by motavizumab.Nat Struct Mol Biol.2010Feb;17(2):248-50.doi:10.1038/nsmb.1723.Epub 2010Jan 24.PMID:20098425;PMCID:PMC3050594.

Espeseth,A.S.,Cejas,P.J.,Citron,M.P.et al.Modified mRNA/lipidnanoparticle-based vaccines expressing respiratory syncytial virus F protein variantsare immunogenic and protective in rodent models of RSV infection.npj Vaccines 5,16(2020).https://doi.org/10.1038/s41541-020-0163-z.

Joyce,M.,Zhang,B.,Ou,L.et al.Iterative structure-based improvement of afusion-glycoprotein vaccine against RSV.Nat Struct Mol Biol 23,811–820(2016).

Claims (10)

1. A mutant RSV F protein comprising at least one amino acid mutation that stabilizes the recombinant RSV F protein in a pre-fusion conformation, the molecule being associated with a recognition antigenic siteAnd/or antibody binding of V;

Preferably, the RSVF protein comprises a mutation at amino acid residue 55, 186-193, 210-217, 254, 255-264, 323, 394, 461, 475 or 491, or at an amino acid corresponding to residue 55, 186-193, 210-217, 254, 255-264, 323, 394, 461, 475 or 491 as determined by alignment with SEQ ID NO. 1.

2. The RSV F protein according to claim 1, wherein said RSV F protein comprises a mutation at amino acid residue 55, 188, 190, 210-214, 217, 254, 260, 263, 323, 394, 461, 475 or 491;

Preferably, the RSV F protein comprises an amino acid mutation filling the cavity at position 55, or an amino acid mutation introducing a disulfide bond at positions 188, 190, 260, 263, 394, 491, 323, 475, or an amino acid mutation introducing a chemical bond at position 254, or an amino acid mutation introducing an induced rotation of the polypeptide backbone at positions 210-214, 217, 461;

Preferably, the cavity-filling amino acid mutation comprises a F, L, W, V, Y, H or M substitution;

Preferably, the amino acid filled in the 55 th cavity is mutated to S55V;

preferably, the disulfide bond introduced amino acid mutations at positions 188, 190, 260, 263, 394, 491, 323, 475 are cysteine substitutions;

Preferably, the disulfide bond introducing amino acid mutation is selected from one or more of (a) - (d):

(a) 188C and 263C substitutions;

(b) 190C and 260C substitutions;

(c) 394C and 491C substitutions; and

(D) 323C and 475C substitutions;

preferably, the 188C and 263C substitutions are L188C and D263C substitutions;

the 190C and 260C substitutions are S190C and L260C substitutions;

the 394C and 491C substitutions are K394C and S491C substitutions;

the 323C and 475C substitutions are T323C and I475C substitutions;

preferably, the amino acid introduced into the chemical bond at position 254 is mutated to N254R;

preferably, the introduction of an amino acid mutation that induces rotation of the polypeptide backbone comprises a proline substitution;

Preferably, the amino acid mutation introduced at the 210-214, 217, 461 positions for inducing the rotation of the polypeptide skeleton is Q210P, C212,212P, S213P, I214P, I217P or K461P;

preferably, the RSV F protein comprises cavity-filling amino acid mutations, chemical bond-introducing amino acid mutations, and at least one disulfide bond-introducing amino acid mutation;

preferably, the RSV F protein comprises the following mutations: S55V, N254R, S C190 and L260C;

preferably, the RSV F protein comprises a mutation at position 461 and at least one disulfide bond introducing amino acid mutation;

Preferably, the RSV F protein comprises the following mutations: K461P, K394C and S491C; or K461P, T C and I475C;

preferably, the RSVF protein also contains a mutation at position 215;

preferably, the mutation at position 215 is S215P;

preferably, the RSVF protein is rsvp a-type or RSV B-type RSVF protein;

Preferably, the RSVF protein comprises an F ₁ polypeptide and an F ₂ polypeptide, and optionally does not comprise a pep27 polypeptide or a portion thereof;

Preferably, the C-terminal residue of the F ₂ polypeptide and the N-terminal residue of the F ₁ polypeptide comprise RSV F positions 97 and 137, 97 and 145, 97 and 150, 102 and 144, 102 and 145, 102 and 146, 102 and 147, 103 and 144, 103 and 145, 103 and 146, 103 and 147, 104 and 144, 104 and 145, 104 and 146, 104 and 147, 105 and 144, 105 and 145, 105 and 146, 105 and 147, or 105 and 150, respectively;

Preferably, the F ₂ and F ₁ polypeptides are linked by a heterologous peptide linker or directly;

Preferably, the linker is selected from G, S, GG, GS, SG, PS, GP, GGG or GSG;

Preferably, position 103 of the F ₂ polypeptide is linked to position 145 of the F ₁ polypeptide by a GS, PS linker;

Preferably, position 103 of the F ₂ polypeptide is directly linked to position 145 of the F ₁ polypeptide;

Preferably, position 103 of the F ₂ polypeptide is linked to position 147 of the F ₁ polypeptide by a GP linker;

Preferably, the RSVF protein amino acid mutation S215P does not comprise a pep27 polypeptide or a portion thereof; preferably, the F ₂ and F ₁ polypeptides of the RSV F protein are linked by a heterologous peptide linker; preferably, position 103 of the F ₂ polypeptide is linked to position 145 of the F ₁ polypeptide by a GS linker;

Preferably, the RSV F protein comprises the following amino acid mutations: S55V, S190C, L C and N254R, and Q210P, S211P, C212P, S213P, I214P, S P or one or more of I217P; preferably, the RSV F protein does not comprise a pep27 polypeptide or portion thereof; preferably, the F ₂ and F ₁ polypeptides of the RSV F protein are linked by a heterologous peptide linker; preferably, the linker is selected from GS, PS, GP;

Preferably, the RSVF protein comprises the following amino acid mutations: K461P, K394C, S491C and S215P, preferably the RSV F protein does not comprise a pep27 polypeptide or portion thereof; preferably, the F ₂ and F ₁ polypeptides of the RSV F protein are linked by a heterologous peptide linker; preferably, position 103 of the F ₂ polypeptide is linked to position 145 of the F ₁ polypeptide by a GS linker;

Preferably, the RSVF protein comprises the following amino acid mutations: K461P, T323C, I475C and S215P; preferably, the RSV F protein does not comprise a pep27 polypeptide or portion thereof; preferably, the F ₂ and F ₁ polypeptides of the RSV F protein are linked by a heterologous peptide linker; preferably, position 103 of the F ₂ polypeptide is linked to position 145 of the F ₁ polypeptide by a GS linker or position 103 of the F ₂ polypeptide is linked to position 147 of the F ₁ polypeptide by a GP linker;

preferably, the RSVF protein is a full-length RSVF protein;

preferably, the RSVF protein is a soluble RSVF protein;

preferably, the RSVF protein comprises one or more tags for detecting RSVF molecules;

Preferably, the RSVF protein comprises one or more tags for purifying RSVF molecules;

Preferably, the tag is selected from the group consisting of a Strep tag, strep II tag, FLAG tag, glutathione S-transferase (GST) tag, green Fluorescent Protein (GFP) tag, hemagglutinin A (HA) tag, histidine (His) tag, luciferase tag, maltose Binding Protein (MBP) tag, c-Myc tag, protein a tag, or protein G tag;

Preferably, the tag is a proteolytically cleavable tag.

3. An immunogen comprising the RSVF protein of claim 1;

Preferably, the immunogen comprises a multimer of the RSVF protein;

preferably, the multimer is a trimer.

4. A virus-like particle or protein nanoparticle comprising an RSVF protein according to any one of claims 1-2 or an immunogen according to claim 3.

Preferably, the protein nanoparticle is a ferritin nanoparticle, a package element nanoparticle, a Sulphur Oxidoreductase (SOR) nanoparticle, a2, 4-dioxytetrahydropteridine synthase nanoparticle or a pyruvate dehydrogenase nanoparticle.

5. Nucleic acid molecule encoding an RSVF protein according to any one of claims 1-2, an immunogen according to claim 3, a virus-like particle according to claim 4 or a protein nanoparticle;

Preferably, the nucleic acid molecule encodes a precursor protein of the immunogen, virus-like particle or protein nanoparticle;

Preferably, the precursor protein comprises a signal peptide, an F2 polypeptide, a Pep27 polypeptide and an F1 polypeptide from the N-terminus to the C-terminus;

preferably, the nucleic acid molecule is codon optimized for expression in a human or bovine cell;

Preferably, the nucleic acid molecule is operably linked to a promoter.

6. A vector comprising the nucleic acid molecule of claim 5;

preferably, the vector is a viral vector;

preferably, the viral vector comprises a bovine parainfluenza viral vector, a human parainfluenza viral vector, a newcastle disease viral vector, a sendai viral vector, a measles viral vector, an attenuated RSV vector, a paramyxovirus vector, an adenovirus vector, an alphavirus vector, a venezuelan equine encephalitis vector, a semliki forest viral vector, a sindbis viral vector, an adeno-associated viral vector, a poxvirus vector, a rhabdovirus vector, a vesicular stomatitis viral vector, a picornaviral vector, or a herpesvirus vector;

Preferably, the viral vector is an adenovirus vector;

Preferably, the adenovirus is selected from the group consisting of human adenovirus and non-human adenovirus;

preferably, the non-human adenovirus comprises a simian, chimpanzee, gorilla, avian, canine, ovine or bovine adenovirus;

Preferably, the non-human adenovirus is selected from chimpanzee adenoviruses;

Preferably, the chimpanzee adenovirus is an adenovirus with at least the E1 gene deleted.

7. A host cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6.

8. An immunogenic composition comprising the RSV F protein of any one of claims 1-2, the immunogen of claim 3, the virus-like particle or protein nanoparticle of claim 4, the nucleic acid molecule of claim 5, the vector of claim 6, or the host cell of claim 7; and a pharmaceutically acceptable carrier;

preferably, the immunogenic composition further comprises an adjuvant;

Preferably, the adjuvant comprises an alum, an oil-in-water composition, MF59, AS01, AS03, AS04, MPL, QS21, a TLR9 agonist, a TLR7 agonist, a TLR4 agonist, a TLR3 agonist, or a combination of two or more thereof.

9. The method comprises the following steps:

1) A method for detecting or isolating RSVF-binding antibodies in a subject, comprising: providing an effective amount of an RSVF protein according to any one of claims 1-2, an immunogen according to claim 3, a virus-like particle according to claim 4 or a protein nanoparticle;

Contacting a biological sample from the subject with the recombinant RSVF protein or the protein nanoparticle under conditions sufficient to form an immune complex between the recombinant RSVF protein or the protein nanoparticle and the RSVF-binding antibody; and

Detecting the immune complex, thereby detecting or isolating the RSVF-binding antibodies in the subject; preferably, the subject is at risk of or has an RSV infection;

preferably, the RSV infection is a human RSV subtype a, human RSV subtype B or bovine RSV infection; preferably, the subject is a human subject;

2) A method for stabilizing the pre-fusion conformation of an RSV fusion polypeptide, characterized in that a mutation as defined in any one of claims 1-2 is introduced;

3) A method for preparing an antibody to RSVF, wherein the antibody is obtained by immunization of a non-human animal with the RSVF protein according to any one of claims 1-2 or the immunogen according to claim 3;

preferably, the non-human animal is a vertebrate;

Preferably, the vertebrate comprises a mouse, rat, guinea pig, rabbit, sheep, non-human primate;

preferably, the antibodies comprise chimeric, humanized antibodies.

10. Any of the following applications:

1) Use of the RSV F protein according to claims 1-2, the immunogen according to claim 3, the virus-like particle or protein nanoparticle according to claim 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, the host cell according to claim 7 or the immunogenic composition according to claim 8 for inhibiting or preventing RSV infection;

2) Use of the RSV F protein according to claims 1-2, the immunogen according to claim 3, the virus-like particle or protein nanoparticle according to claim 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, the host cell according to claim 7 or the immunogenic composition according to claim 8 for the preparation of a product for the treatment or prevention of diseases associated with RSV infection;

3) Use of the RSV F protein according to claims 1-2, the immunogen according to claim 3, the virus-like particle or protein nanoparticle according to claim 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, the host cell according to claim 7, the immunogenic composition according to claim 8 for the preparation of a product for inducing an immune response against an RSV F protein in a subject;

4) Use of the RSV F protein according to claims 1-2, the immunogen according to claim 3, the virus-like particle or protein nanoparticle according to claim 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, the host cell according to claim 7 or the immunogenic composition according to claim 8 for the preparation of an anti-RSV F protein antibody;

5) Use of the RSV F protein according to claims 1-2, the immunogen according to claim 3, the virus-like particle or protein nanoparticle according to claim 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, the host cell according to claim 7 or the immunogenic composition according to claim 8 for the preparation of a vaccine;

6) Use of the RSV F protein according to claims 1-2, the immunogen according to claim 3, the virus-like particle or protein nanoparticle according to claim 4, the nucleic acid molecule according to claim 5, the vector according to claim 6, the host cell according to claim 7 or the immunogenic composition according to claim 8 for the preparation of a kit.