RSV f protein mutants

Abstract

To provide immunogens derived from a RSV F protein that have improved properties, such as enhanced immunogenicity or improved stability of the pre-fusion form, as compared with the corresponding native RSV F protein, as well as compositions comprising such an immunogen.SOLUTION: The present disclosure relates to RSV F protein mutants, nucleic acids or vectors encoding a RSV F protein mutant, compositions comprising a RSV F protein mutant or nucleic acid, and uses of the RSV F protein mutants, nucleic acids or vectors, and compositions.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 387,270, filed December 23, 2015, and U.S. Provisional Patent Application No. 62 / 421,184, filed November 11, 2016. The entire contents of each of the foregoing applications are incorporated herein by reference.

[0002] The present invention relates generally to vaccines, and specifically to vaccines against respiratory syncytial virus. [Background technology]

[0003] Respiratory syncytial virus, or RSV, is a respiratory virus that infects the lungs and airways. RSV is the leading cause of severe viral lower respiratory tract disease in infants worldwide and an important cause of respiratory disease in older adults. However, no vaccine is approved to prevent RSV infection.

[0004] RSV is a member of the Paramyxoviridae family. Its genome consists of a single-stranded, negative-sense RNA molecule that encodes 11 proteins, including nine structural proteins (three glycoproteins and six internal proteins) and two nonstructural proteins. The structural proteins include three transmembrane surface glycoproteins: the attachment protein G, the fusion protein F, and a small hydrophobic SH protein. There are two subtypes of RSV, A and B, which differ primarily in the G glycoprotein, while the sequence of the F glycoprotein is more conserved between the two subtypes.

[0005] The mature F glycoprotein has three general domains: the extracellular domain (ED), the transmembrane domain (TM), and the cytoplasmic tail (CT). The CT contains a single palmitoylated cysteine ​​residue.

[0006] The F glycoprotein of human RSV is initially translated from mRNA as a single polypeptide precursor of 574 amino acids (referred to as "F0" or "F0 precursor") that contains a signal peptide sequence (amino acids 1-25) at its N-terminus. During translation, the signal peptide is removed by a signal peptidase in the endoplasmic reticulum. The remaining portion of the F0 precursor (i.e., residues 26-574) can be further cleaved by cellular proteases (specifically, furin) at two polybasic sites (amino acids 109 / 110 and 136 / 137) to remove a 27 amino acid intervening sequence (amino acids 110-136) called pep27, resulting in two linked fragments called F1 (C-terminal portion, amino acids 137-574) and F2 (N-terminal portion, amino acids 26-109). F1 contains a hydrophobic fusion peptide at its N-terminus and also contains two heptad repeat regions (HRA and HRB). HRA is located near the fusion peptide, and HRB is located near the TM domain. The F1 and F2 fragments are linked to each other via two disulfide bonds. Either the uncleaved F0 protein or the F1-F2 heterodimer without the signal peptide sequence can form the RSV F promoter. Three such promoters combine to form the final RSV F protein complex, which is a homotrimer of three promoters.

[0007] The F proteins of subtypes A and B are about 90 percent identical in amino acid sequence. An example of the sequence of the F0 precursor polypeptide of the A subtype is shown in SEQ ID NO:1 (strain A2, GenBank GI:138251, Swiss Prot P03420), and an example of the sequence of the F0 precursor polypeptide of the B subtype is shown in SEQ ID NO:2 (strain 18537, GenBank GI:138250, Swiss Prot P13843). SEQ ID NO:1 and SEQ ID NO:2 are both sequences of 574 amino acids. The signal peptide sequences of SEQ ID NO:1 and SEQ ID NO:2 are also reported as amino acids 1-25 (GenBank and UniProt). In both sequences, the TM domain is approximately amino acids 530 to 550, although some reports have said it is 525-548. The cytoplasmic tail starts at amino acid 548 or 550 and ends at amino acid 574, with a palmitoylated cysteine ​​residue located at amino acid 550.

[0008] One of the main antigens investigated for RSV subunit vaccines is the F protein. The RSV F protein trimer mediates fusion between the virion membrane and the host cell membrane and also promotes the formation of syncytia. In the virion before fusion with the host cell membrane, the largest population of F molecules forms a lollipop-shaped structure with the TM domain anchored within the viral envelope [Dormitzer, P.R., Grandi, G., Rappuoli, R., Nature Reviews Microbiol, 10, 807, 2012]. This conformation is called the prefusion conformation. Prefusion RSV F is recognized by monoclonal antibodies (mAbs) D25, AM22, and MPE8 without distinguishing between oligomeric states. The prefusion F trimer is specifically recognized by mAb AM14 [Gilman MS, Moin SM, Mas V, et al., Characterization of a prefusion-specific antibody that recognizes a quaternary, cleavage-dependent epitope on the RSV fusion glycoprotein. PLoS Pathogens, 11(7), 2015]. During RSV entry into cells, the F protein rearranges from a prefusion state (herein referred to as "prefusion F"), through an intermediate extended structure, to a postfusion state ("postfusion F"). During this rearrangement, the C-terminal coiled coil of the prefusion molecule dissociates into its three component chains, which then wrap around the globular head and combine with three additional helices to form a postfusion six-helix bundle. The prefusion RSV F trimer undergoes a conformational change when subjected to increasingly harsh chemical or physical conditions, such as increasing temperature. First, the trimeric structure is lost (at least locally within the molecule), then it rearranges into the post-fusion form, and then the domains denature. Summary of the Invention [Problem to be solved by the invention]

[0009] To prevent viral entry, F-specific neutralizing antibodies must bind to the prefusion conformation of F on the virion, possibly on a spread intermediate, before the viral envelope fuses with the cellular membrane. Thus, the prefusion form of the F protein is considered to be the preferred conformation for the desired vaccine antigen [Ngwuta, JO, Chen, M., Modjarrad, K., Joyce, MG, Kanekiyo, M., Kumar, A., Yassine, HM, Moin, SM, Killelikelly, AM, Chuang, GY, Druz, A., Georgiev, IS, Rundlet, EJ, Sastry, M., Stewart-Jones, GB, Yang. Y., Zhang, B., Nason, MC, Capella, C., Peeples, M., Ledgerwood, JE, Mcllan, JS, Kwong, PD, Graham, BS, Science Translat. Med., 14, 7, 309 (2015)].The F glycoprotein is readily converted to the post-fusion form upon extraction from membranes with detergents such as Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octylglucoside, octylthioglucoside, SDS, CHAPS, CHAPSO, or upon expression as an extracellular domain, physical or chemical stress, or storage [McLellan JS, Chen M, Leung S et al. Structure of RSV fusion glycoprotein trimer bound to a pre-fusion-specific neutralizing antibody. Science 340, 1113-1117 (2013); Chaiwatpongsakorn, S., Epand, RF, Collins, PL, Epand RM, Peeples, ME, J Virol. 85(8):3968-77 (2011); Yunus, AS, Jackson TP, Crisafi, K., Burimski, I., Kilgore, NR, Zoumplis, D., Allaway, GP, Wild, CT, Salzwedel, K. Virology. 2010 January 20, 396(2):226-37]. Thus, preparation of prefusion F as a vaccine antigen remains challenging. Because neutralizing and protective antibodies function by interfering with viral entry, it is hypothesized that F antigens that elicit only postfusion-specific antibodies would be expected to be less effective as F antigens that elicit prefusion-specific antibodies. Thus, it would be more desirable to utilize an F vaccine that contains the F protein immunogen in a prefusion form (or possibly a spread intermediate form). Attempts to date have not resulted in an RSV vaccine that has been clinically demonstrated to elicit a sufficient level of protection to support approval of an RSV vaccine. Thus, there is a need for immunogens derived from RSV F proteins that have improved properties, such as enhanced immunogenicity or improved stability of the pre-fusion form compared to the corresponding native RSV F protein, and compositions, e.g., vaccines, that contain such immunogens. [Means for solving the problem]

[0010] In some embodiments, the present invention provides mutants of wild-type RSV F proteins that exhibit mutations in their amino acid sequences relative to the corresponding amino acid sequences of the wild-type RSV F protein and are immunogenic to the wild-type RSV F protein or to viruses that contain the wild-type F protein. The amino acid mutations in the mutants include substitutions, deletions, or additions of amino acids relative to the wild-type RSV F protein.

[0011] In some embodiments, the present disclosure provides mutants of wild-type RSV F protein in which the introduced amino acid mutation is a pair of amino acid residues in the wild-type RSV F protein mutated to a pair of cysteines ("engineered disulfide mutations"). The introduced cysteine ​​residue pair allows for the formation of disulfide bonds between cysteine ​​residues that stabilize the conformation or oligomeric state of the protein, such as the prefusion conformation. Examples of specific pairs of such mutations include 55C and 188C, 155C and 290C, 103C and 148C, and 142C and 371C, such as S55C and L188C, S155C and S290C, T103C and I148C, and L142C and N371C.

[0012] In yet another embodiment, the RSV F protein mutant comprises one or more amino acid mutations that are gap-filling mutations. Examples of amino acids that can be replaced for gap-filling purposes include small aliphatic amino acids (e.g., Gly, Ala, and Val) or small polar amino acids (e.g., Ser and Thr), and amino acids that are buried in the pre-fusion conformation but exposed to the solvent in the post-fusion conformation. Examples of replacement amino acids include large aliphatic amino acids (Ile, Leu, and Met) or large aromatic amino acids (His, Phe, Tyr, and Trp). In some specific embodiments, the RSV F protein mutant comprises (1) Substitution of S at position 55, 62, 155, 190, or 290 with I, Y, L, H, or M; (2) substitution of T at position 54, 58, 189, 219, or 397 with I, Y, L, H, or M; (3) substitution of G at position 151 with A or H; (4) substitution of A at position 147 or 298 with I, L, H, or M; (5) Substitution of V at position 164, 187, 192, 207, 220, 296, 300, or 495 with I, Y, or H; and (6) Substitution of R at position 106 with W The gap filling mutations are selected from the group consisting of:

[0013] In some specific embodiments, the RSV F protein mutant comprises at least one gap-filling mutation selected from the group consisting of T54H, S190I, and V296I.

[0014] In yet another embodiment, the present disclosure provides RSV F protein mutants that include electrostatic mutations that reduce ionic repulsion or increase ionic attraction between residues in the protein that are adjacent to each other in the folded structure. In some embodiments, the RSV F protein mutants include electrostatic substitutions that reduce repulsive ionic interactions or increase attractive ionic interactions with acidic residues Glu487 and Asp489 of another promoter of the RSV F trimer. In some specific embodiments, the RSV F protein mutants include (1) Substitution of E at position 82, 92, or 487 by D, F, Q, T, S, L, or H; (2) Substitution of K at position 315, 394, or 399 by F, M, R, S, L, I, Q, or T; (3) Substitution of D at position 392, 486, or 489 by H, S, N, T, or P; and (4) Substitution of R at position 106 or 339 by F, Q, N, or W The electrostatic mutations include those selected from the group consisting of:

[0015] In yet another embodiment, the present disclosure provides RSV F protein mutants that include a combination of two or more different types of mutations selected from engineered disulfide mutations, gap-filling mutations, and electrostatic mutations. In some specific embodiments, the present invention provides mutants of wild-type RSV F proteins that include a combination of mutations relative to the corresponding wild-type RSV F protein, wherein the combination of mutations is: (1) A combination of T103C, I148C, S190I, and D486S, (2) A combination of T54H, S55C, L188C, and D486S. (3) A combination of T54H, T103C, I148C, S190I, V296I, and D486S, (4) A combination of T54H, S55C, L142C, L188C, V296I, and N371C, (5) A combination of S55C, L188C, and D486S, (6) A combination of T54H, S55C, L188C, and S190I, (7) A combination of S55C, L188C, S190I, and D486S, (8) A combination of T54H, S55C, L188C, S190I, and D486S, (9) A combination of S155C, S190I, S290C, and D486S, (10) A combination of T54H, S55C, L142C, L188C, V296I, N371C, D486S, E487Q, and D489S, and (11) Combination of T54H, S155C, S190I, S290C, and V296I The present invention provides a mutant, wherein the mutant is selected from the group consisting of:

[0016] In another aspect, the present invention provides nucleic acid molecules encoding the RSV F protein mutants described herein. In some other specific embodiments, the present disclosure provides: (1) the nucleotide sequence of SEQ ID NO:8; (2) the nucleotide sequence of SEQ ID NO:9; (3) the nucleotide sequence of SEQ ID NO: 10; (4) the nucleotide sequence of SEQ ID NO:11; (5) the nucleotide sequence of SEQ ID NO: 12; (6) the nucleotide sequence of SEQ ID NO: 13; (7) The nucleotide sequence of SEQ ID NO: 14, (8) the nucleotide sequence of SEQ ID NO: 15; (9) The nucleotide sequence of SEQ ID NO: 16; (10) the nucleotide sequence of SEQ ID NO: 17, and (11) Nucleotide sequence of SEQ ID NO: 18 The present invention provides a nucleic acid molecule encoding a RSV F protein mutant, comprising a nucleotide sequence selected from the group consisting of:

[0017] In another aspect, the present invention provides a composition comprising (1) a RSV F protein mutant described in this disclosure, or (2) a nucleic acid molecule or vector encoding such a RSV F protein mutant. In some specific embodiments, the composition is a pharmaceutical composition comprising a RSV F protein mutant provided by this disclosure and a pharma- ceutical acceptable carrier. In yet other specific embodiments, the pharmaceutical composition is a vaccine.

[0018] The present disclosure also relates to the use of RSV F protein mutants, nucleic acids encoding such RSV F protein mutants, or vectors for expressing such RSV F protein mutants, or compositions comprising RSV F protein mutants or nucleic acids. In some embodiments, the present disclosure provides a method for preventing RSV infection in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition, such as a vaccine, comprising a RSV F protein mutant, a nucleic acid encoding a RSV F protein mutant, or a vector expressing a RSV F protein mutant. [Brief description of the drawings]

[0019] [Figure 1] 3 shows the amino acid sequence of a precursor polypeptide template used for the construction of some of the RSV F protein mutants described in the Examples (SEQ ID NO:3). The precursor polypeptide contains a signal sequence (residues 1-25), an F2 polypeptide (residues 26-109), a pep27 sequence (residues 110-136), an F1 polypeptide (residues 137-513), a T4 fibritin-derived trimerization domain (foldon, residues 518-544), a thrombin recognition sequence (residues 547-552), a histidine tag (residues 553-558), a Streptag II (561-568), and a linker sequence (residues 514-517, 545-546, and 559-560). It also contains three natural substitutions (P102A, I379V, and M447V) relative to the native RSV F sequence shown in SEQ ID NO:1. The cleavage sites of Furin are indicated as RARR and KKRKRR. [Figure 2A] FIG. 1 shows sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of selected pre-fusion F mutants (pXCS847, pXCS851, and pXCS852) under non-reducing conditions. [Figure 2B] 1 is a graph showing the sedimentation coefficient distribution of selected mutants (pXCS847, pXCS851, and pXCS852) calculated from sedimentation velocity experiments using an analytical ultracentrifuge. [Figure 3A] 1 is a graph showing the circular dichroism (CD) spectra of exemplary modified RSV F proteins with specific site mutations. The far-ultraviolet (UV) CD spectra of the designed mutants confirm the integrity of the secondary structure, and the near-UV CD spectra confirm the integrity of the tertiary structure. [Figure 3B]1 is a graph showing the circular dichroism (CD) spectra of exemplary modified RSV F proteins with specific site mutations. The far ultraviolet (UV) CD spectra of the designed mutants confirm the integrity of the secondary structure, and the near UV CD spectra confirm the integrity of the tertiary structure. [Figure 4] Graph showing time-dependent stress testing of purified DS-Cav1 using two different monoclonal antibodies (mAbs) D25 and AM14 at two temperatures (50° C. and 60° C.). [Diagram 5] Graph showing differential scanning calorimetry (DSC) experiment (Example 8) with purified DS-Cav1. The experiment was performed as described for the designed pre-fusion F mutant. Solid line - first DSC scan of the sample, dashed line - repeat scan of the same sample used in the first scan. The DSC peaks largely recovered during the repeat scan, indicating that the conformational transition detected by DSC is reversible. [Figure 6A] 1 is a graph showing the far-UV CD spectrum (Example 8) of DS-Cav1 stressed at 60 ° C. The CD spectrum was recorded as described above (Example 6) for the designed pre-fusion RSV F mutant. DS-Cav1 retained the same far-UV CD spectrum after incubation at 60 ° C for up to 2 hours, indicating that no global protein unfolding occurred during that time. [Figure 6B] Graph showing the near-UV CD spectrum (Example 8) of DS-Cav1 stressed at 60 ° C. CD spectra were recorded as described above (Example 6) for the designed pre-fusion RSV F mutants. [Figure 7A] Graph showing protein concentration dependence of heat stress resistance as determined by preservation of the pre-fusion F trimer-specific AM14 epitope (Example 8). [Figure 7B]Graph showing protein concentration dependence of heat stress resistance as determined by preservation of pre-fusion F specific D25 epitope (Example 8). Protein samples were serially diluted and subjected to stress at 50° C. for 1 hour. Reactivity of D25 remaining after stress compared to control (unstressed) samples was assessed by ELISA assay. [Figure 8] Graph showing neutralizing antibody responses of mice immunized with DS-Cav1, wild-type F, or mutants pXCS852, pXCS855, pXCS830, pXCS853, pXCS780, pXCS898, pXCS851, pXCS874, pXCS881, pXCS738, or pXCS847 with or without aluminum phosphate as an adjuvant. Results are reported as 50% geometric mean titers (GMT) from 10 mice per group. Each scatter plot reflects the response of an individual mouse, with a total of 10 mice per group. The line within each group indicates the geometric mean 50% neutralizing antibody titer. "Wild-type F" refers to wild-type F extracellular domain recombinant construct. [Figure 9A] 1 is a graph illustrating the correlation between neutralizing antibody titers elicited by engineered pre-fusion F protein mutants and the stability of the engineered pre-fusion F protein mutants. Y-axis - neutralizing antibody titers elicited by immunization of mice with 0.25 μg antigen and no adjuvant or 0.025 μg antigen and 0.1 mg / ml AlPO4 adjuvant (data shown in Table 12). X-axis - stability of engineered mutants as defined by remaining AM14 reactivity after heat stress (data shown in Table 8B). [Figure 9B] 1 is a graph illustrating the correlation between neutralizing antibody titers elicited by engineered pre-fusion F protein mutants and the stability of the engineered pre-fusion F protein mutants. Y-axis - neutralizing antibody titers elicited by immunization of mice with 0.25 μg antigen and no adjuvant or 0.025 μg antigen and 0.1 mg / ml AlPO4 adjuvant (data shown in Table 12). X-axis - stability of engineered mutants as defined by remaining AM14 reactivity after heat stress (data shown in Table 8B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present disclosure relates to RSV F protein mutants, immunogenic compositions comprising RSV F protein mutants, methods for producing RSV F protein mutants, compositions comprising RSV F protein mutants, and nucleic acids encoding RSV F protein mutants.

[0021] A.Definition The term "101F" refers to the antibody described in US2006 / 0159695A1 having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:31.

[0022] As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless the context clearly indicates otherwise. For example, the term "antigen" includes singular or multiple antigens and can be considered equivalent to the phrase "at least one antigen."

[0023] The term "adjuvant" refers to a substance that can enhance, accelerate, or prolong the body's immune response to an antigen in a vaccine (even though it is not the target antigen of the vaccine itself). Adjuvants can be included in the vaccine composition or can be administered separately from the vaccine.

[0024] The term "administration" refers to the introduction of a substance or composition into a subject by a selected route. Administration can be local or systemic. For example, if the selected route is intramuscular, the composition (such as the composition comprising the disclosed immunogen) is administered by introducing the composition into the subject's muscle.

[0025] The term "AM14" refers to the antibody described in WO2008 / 147196A2 having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:24 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:25.

[0026] The term "AM22" refers to the antibody described in WO2011 / 043643A1 having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:26 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:27.

[0027] The term "antigen" refers to a molecule that can be recognized by an antibody. Examples of antigens include polypeptides, peptides, lipids, polysaccharides, and nucleic acids that contain antigenic determinants, such as those recognized by immune cells.

[0028] The term "conservative substitution" refers to the replacement of an amino acid with a chemically similar amino acid. Conservative amino acid substitutions that result in functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Serine (S), Threonine (T), 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), Glutamine (Q), 4) Arginine (R), Lysine (K), 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0029] The term "D25" refers to the antibody described in WO2008 / 147196A2 having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:22 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:23.

[0030] The term "degenerate variant" of a reference polynucleotide refers to a polynucleotide that differs in nucleotide sequence from the reference polynucleotide but encodes the same polypeptide sequence as that encoded by the reference polynucleotide. There are 20 natural amino acids, most of which are specified by more than one codon. For example, the codons CGU, CGC, CGA, CGG, AGA, and AGG all code for the amino acid arginine. Thus, at all positions where arginine is specified in a protein coding sequence, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Due to the degeneracy of the genetic code, many functionally identical nucleic acids code for any given polypeptide.

[0031] The term "DS-Cav1" refers to a certain RSV F protein having the amino acid sequence described in McLellan et al., Science, 342(6158), 592-598, 2013.

[0032] The term "effective amount" refers to an amount of an agent sufficient to produce a desired response, such as the amount necessary to inhibit viral replication or to modify to some extent the overt symptoms of a viral infection.

[0033] The term "epitope" (or "antigenic determinant" or "antigenic site") refers to the region of an antigen to which an antibody, B cell receptor, or T cell receptor binds or responds. Epitopes can be formed from contiguous or non-contiguous amino acids juxtaposed by secondary, tertiary, or quaternary protein folds. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by higher order folding are typically lost on treatment with denaturing solvents.

[0034] The term "F0 polypeptide" (F0) refers to the precursor polypeptide of the RSV F protein, which is composed of a signal polypeptide sequence, an F1 polypeptide sequence, a pep27 polypeptide sequence, and an F2 polypeptide sequence. With rare exceptions, the F0 polypeptide of known RSV strains consists of 574 amino acids.

[0035] The term "F1 polypeptide" (F1) refers to the polypeptide chain of the mature RSV F protein. Native F1 contains approximately residues 137-574 of the RSV F0 precursor and is composed (from the N-terminus to the C-terminus) of an extracellular domain (approximately residues 137-524), a transmembrane domain (approximately residues 525-550), and a cytoplasmic domain (approximately residues 551-574). As used herein, the term encompasses both native F1 polypeptides and F1 polypeptides that contain modifications (e.g., amino acid substitutions, insertions, or deletions) from the native sequence, such as modifications designed to stabilize the F mutant or enhance the immunogenicity of the F mutant.

[0036] The term "F2 polypeptide" (F2) refers to the polypeptide chain of the mature RSV F protein. Native F2 comprises approximately residues 26-109 of the RSV F0 precursor. As used herein, the term encompasses both native F2 polypeptides and F2 polypeptides that contain modifications from the native sequence (e.g., amino acid substitutions, insertions, or deletions), such as modifications designed to stabilize the F mutant or enhance the immunogenicity of the F mutant. In the native RSV F protein, the F2 polypeptide is linked to the F1 polypeptide by two disulfide bonds to form an F2-F1 heterodimer. The term "foldon" or "foldon domain" refers to an amino acid sequence that can form a trimer. One example of such a foldon domain is a peptide sequence derived from bacteriophage T4 fibritin having the sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 40). The term "mammal" refers to any animal species of the Mammalia class. Examples of mammals include humans, non-human primates such as monkeys, laboratory animals such as rats, mice and guinea pigs, farm animals such as cats, dogs, rabbits, cows, sheep, goats, horses and pigs, and captive wild animals such as lions, tigers and elephants.

[0037] The term "glycoprotein" refers to a protein that contains oligosaccharide chains (glycans) covalently attached to the polypeptide side chains. Carbohydrates are added to proteins in a co- or post-translational modification known as glycosylation. The term "glycosylation site" refers to an amino acid sequence on the surface of a polypeptide, such as a protein, that receives the addition of a glycan. An N-linked glycosylation site is a triplet sequence of NX(S / T), where N is asparagine, X is any residue except proline, and (S / T) is a serine or threonine residue. A glycan is a polysaccharide or an oligosaccharide. Glycans can also be used to refer to the carbohydrate moiety of a glycoconjugate, such as a glycoprotein, a glycolipid, or a proteoglycan.

[0038] The term "host cell" refers to cells in which a vector can be propagated and its DNA or RNA expressed. The cells can be prokaryotic or eukaryotic.

[0039] The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are identical or have a certain percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence. Methods for aligning sequences for comparison are well known in the art. Once aligned, the number of matches is determined by counting the number of positions where identical nucleotides or amino acid residues are present in both sequences. The percent sequence identity is determined by dividing the number of matches by the length of the sequence represented by the identified sequence, or by the segmental length (such as 100 consecutive nucleotides or amino acid residues from the sequence represented by the identified sequence), and then multiplying the resulting value by 100. For example, a peptide sequence with 1166 matches is 75.0 percent identical to the test sequence when aligned with a test sequence with 1554 amino acids (1166÷1554×100=75.0).

[0040] Optimal alignment of sequences for comparison can be determined, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443, 1970, by the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor, New York, 2012) and Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, 2003). This can be done by the method of Wiley and Sons, New York, through supplement 104, 2013).

[0041] The term "immunogenic" refers to the ability of a substance, with or without an adjuvant, to generate, elicit, stimulate, or induce an immune response in an animal against a particular antigen.

[0042] The term "immune response" refers to any detectable response of one or more cells of the immune system of a host mammal to a stimulus (such as an immunogen), including, but not limited to, innate immune responses (e.g., activation of the Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells, such as antigen-specific T cells, and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells, such as the production and secretion of antibodies into plasma, lymph, and / or tissue fluids). Examples of immune responses include Toll-like receptor activation, expression or secretion of lymphokines (e.g., cytokines (e.g., Th1, Th2, or Th17-type cytokines) or chemokines), macrophage activation, dendritic cell activation, T cell (e.g., CD4+ or CD8+ T cell) activation, NK cell activation, B cell activation (e.g., production and / or secretion of antibodies), binding of an immunogen (e.g., an antigen (e.g., an immunogenic polypeptide)) to an MHC molecule, elicitation of a cytotoxic T lymphocyte ("CTL") response, elicitation of a B cell response (e.g., antibody production), and changes (increases) in immune system cells (e.g., T cells and B cells) (e.g., proliferation of cell populations), as well as increased processing and presentation of antigens by antigen-presenting cells. The term "immune response" also encompasses any detectable response to a particular substance (such as an antigen or immunogen) in vitro by one or more components of a vertebrate's immune system. The term "immunogen" refers to an immunogenic compound, composition, or substance, as defined herein below.

[0043] The term "immunogenic composition" refers to a composition that includes an immunogen.

[0044] The term "MPE8" refers to an antibody described in Corti et al. [Corti, D., Bianchi, S., Vanzetta, F., Minola, A., Perez, L., Agatic, G., Lanzavecchia, A. Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature, 501(7467), 439-443 (2013)] having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 28 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 29. The terms "mutant" of the wild-type RSV F protein, "mutant" of the RSV F protein, "RSV F protein mutant," or "modified RSV F protein" refer to a polypeptide that exhibits an introduced mutation to the wild-type F protein and is immunogenic relative to the wild-type F protein.

[0045] The term "mutation" refers to the deletion, addition, or substitution of an amino acid residue in the amino acid sequence of a protein or polypeptide compared to the amino acid sequence of a reference protein or polypeptide. Throughout this specification and claims, an amino acid substitution at one particular position in a protein sequence is referred to using the notation "(amino acid residue in wild type protein) (amino acid position) (amino acid residue in engineered protein)". For example, the notation Y75A refers to the substitution of a tyrosine (Y) residue at position 75 of the amino acid sequence of the reference protein with an alanine (A) residue (in a mutant of the reference protein). In cases where the amino acid residue at the same position varies between different wild type sequences, the amino acid code preceding the position number may be omitted, such as "75A".

[0046] The term "native" or "wild-type" protein, sequence, or polypeptide refers to a naturally occurring protein, sequence, or polypeptide that has not been artificially modified by selective mutation.

[0047] The term "pep27 polypeptide" or "pep27" refers to a 27 amino acid polypeptide that is excised from the F0 precursor during maturation of the RSV F protein. The pep27 sequence is flanked by two furin cleavage sites that are cleaved by cellular proteases during maturation of the F protein to produce F1 and F2 polypeptides.

[0048] The term "pharmaceutical acceptable carrier" refers to a material or composition that, when combined with an active ingredient, is compatible with the active ingredient and does not produce toxic or otherwise undesirable reactions when administered to a subject, particularly a mammal. Examples of pharmaceutical acceptable carriers include solvents, surfactants, suspending agents, buffers, lubricants, emulsifying agents, absorbents, dispersion media, coatings, and stabilizers.

[0049] The term "pre-fusion specific antibody" refers to an antibody that specifically binds to the RSV F glycoprotein in the pre-fusion conformation but does not bind to the RSV F protein in the post-fusion conformation. Exemplary pre-fusion specific antibodies include D25, AM22, 5C4, MPE8, and AM14 antibodies.

[0050] The term "pre-fusion trimer-specific antibody" refers to an antibody that specifically binds to the RSV F glycoprotein in a pre-fusion trimer conformation, but does not bind to the RSV F protein in a post-fusion conformation or a pre-fusion conformation that is not a trimer. An exemplary pre-fusion trimer-specific antibody is the AM14 antibody. "Pre-fusion trimer-specific antibodies" are a subset of "pre-fusion specific antibodies."

[0051] The term "prime-boost vaccination" refers to an immunotherapy regimen that includes administering a first immunogenic composition (prime vaccine) to a subject, followed by administration of a second immunogenic composition (boost vaccine) to induce an immune response. The primary vaccine and the boost vaccine typically contain the same immunogen and are in the same or similar format. However, they can also be in different formats, for example, in the form of a vector on the one hand and a naked DNA plasmid on the other. Those skilled in the art will understand the appropriate time interval between administration of the primary vaccine and administration of the boost vaccine. In addition, the primary vaccine, the boost vaccine, or both the primary vaccine and the boost vaccine further comprise an adjuvant.

[0052] The term "pre-fusion conformation" refers to a conformation adopted by a RSV F protein or mutant that can be specifically bound by (i) antibody D25 when the RSV F protein or mutant is in a monomeric or trimer form, or (ii) antibody AM14 when the RSV F protein mutant is in a trimer form. The pre-fusion trimer conformation is a subset of the pre-fusion conformation.

[0053] The term "post-fusion conformation" refers to a conformation that the RSV F protein adopts in which D25, AM22, or AM14 does not specifically bind. The native F protein adopts a post-fusion conformation after the viral envelope and the host cell membrane fuse. The RSV F protein can also adopt a post-fusion conformation in the context of a fusion event, for example, when extracted from the membrane, expressed as an extracellular domain, or under stress conditions such as heat and low osmolality, such as during storage.

[0054] The term "soluble protein" refers to a protein that can dissolve and remain dissolved in an aqueous solution. The solubility of a protein can vary depending on the concentration of the protein in a water-based liquid, the buffer conditions of the liquid, the concentrations of other solutes in the liquid, such as salts and protein concentrations, and the temperature of the liquid.

[0055] The term "specifically binds" refers to the binding of an antibody to a given target molecule with a higher affinity than its binding to other tested substances. For example, an antibody that specifically binds to the RSV F protein in a pre-fusion conformation is an antibody that binds to the RSV F protein in a pre-fusion conformation with a higher affinity than it binds to the RSV F protein in a post-fusion conformation.

[0056] The term "therapeutically effective amount" refers to an amount of an agent sufficient to prevent, treat (including prevent), reduce and / or ameliorate the symptoms and / or underlying causes of a disorder.

[0057] The term "vaccine" refers to a pharmaceutical composition that includes an immunogen capable of eliciting a prophylactic or therapeutic immune response in a subject. Typically, a vaccine elicits an antigen-specific immune response against an antigen of a pathogen, such as a viral pathogen.

[0058] The term "vector" refers to a nucleic acid molecule that can transport or transfer a foreign nucleic acid molecule. This term encompasses both expression vectors and transcription vectors. The term "expression vector" refers to a vector that can express an insert in a target cell and generally contains control sequences, such as enhancer, promoter, and terminator sequences that drive the expression of the insert. The term "transcription vector" refers to a vector that can be transcribed but not translated. Transcription vectors are used to amplify their inserts. The foreign nucleic acid molecule is called an "insert" or a "transgene." A vector generally consists of an insert and a larger sequence that serves as the backbone of the vector. Based on the structure or origin of the vector, the main types of vectors include plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, such as adenovirus (Ad) vectors, and artificial chromosomes.

[0059] B. RSV F Protein Mutants In some aspects, the present invention provides mutants of wild-type RSV F proteins that exhibit mutations in the amino acid sequence relative to the amino acid sequence of the corresponding wild-type RSV F protein and are immunogenic against the wild-type RSV F protein or against viruses containing the wild-type F protein. In certain embodiments, the RSV F mutants have certain advantageous characteristics, such as increased immunogenic properties compared to the corresponding wild-type F protein, or improved stability in the mutant pre-fusion conformation or mutant pre-fusion trimer conformation. In yet other embodiments, the present disclosure provides RSV F mutants that exhibit one or more of the mutations described herein and bind to a pre-fusion specific antibody selected from antibody D25 or antibody AM14.

[0060] The amino acid mutations introduced into the RSV F protein mutant include amino acid substitutions, deletions, or additions. In some embodiments, the only mutation in the amino acid sequence of the mutant is an amino acid substitution relative to the wild-type RSV F protein.

[0061] The amino acid sequences of many native RSV F proteins from different RSV subtypes, as well as the nucleic acid sequences encoding such proteins, are known in the art. For example, the sequences of several subtype A, B, and bovine RSV F0 precursor proteins are set forth in SEQ ID NOs: 1, 2, 4, 6, and 81-270.

[0062] Native RSV F protein shows remarkable sequence conservation across RSV subtypes. For example, RSV subtypes A and B share 90% sequence identity, and RSV subtypes A and B each have 81% sequence identity with the bovine RSV F protein across the F0 precursor molecule. Within RSV subtypes, the identity of the F0 sequence is even higher, for example, within each of RSV A, B, and bovine subtypes, the RSV F0 precursor protein has about 98% sequence identity. Nearly all identified RSV F0 precursor sequences are 574 amino acids long, although they vary slightly in length, typically due to the length of the C-terminal cytoplasmic tail. Sequence identity across various native RSV F proteins is known in the art (see, for example, WO2014 / 160463). To further illustrate the level of sequence conservation of the F protein, non-consensus amino acid residues between the F0 precursor polypeptide sequences of representative RSV A and RSV B strains are shown in Tables 17 and 18, respectively (non-consensus amino acids were identified by aligning selected F protein sequences of RSV A strains with ClustalX (version 2)).

[0063] Given that the RSV F sequence is highly conserved, one skilled in the art can easily compare the amino acid positions between different native RSV F sequences to identify the corresponding RSV F amino acid positions between different RSV strains and subtypes. For example, the furin cleavage site is at the same amino acid position across almost all identified native RSV F0 precursor proteins. Thus, conservation of the native RSV F protein sequence across strains and subtypes allows the use of a reference RSV F sequence for comparison of amino acids at specific positions within the RSV F protein. For purposes of this disclosure (unless otherwise indicated by context), the amino acid positions of the RSV F protein are indicated with reference to the sequence of the F0 precursor polypeptide set forth in SEQ ID NO: 1 (the amino acid sequence of the full-length native F precursor polypeptide of the RSV A2 strain, corresponding to GenInfo identifier GI 138251 and Swiss Prot identifier P03420). However, it should be noted, and one skilled in the art would understand, that different RSV F0 sequences may have different numbering systems, for example, when additional amino acid residues are added or removed compared to SEQ ID NO: 1. Thus, when specific amino acid residues are referred to by their numbers, it should be understood that the description is not limited to only the amino acid that is located at that exact numbered position counting from the beginning of a given amino acid sequence, but also includes the equivalent / corresponding amino acid residues in any and all RSV F sequences, even if these residues are not at the same exact numbered positions, e.g., the RSV sequence is shorter or longer than SEQ ID NO:1, or has insertions or deletions compared to SEQ ID NO:1.

[0064] B-1. Structure of RSV F protein mutants The RSV F protein mutants provided by the present disclosure include an F1 polypeptide and an F2 polypeptide. In some embodiments, the mutant further comprises a trimerization domain. In some embodiments, either the F1 polypeptide or the F2 polypeptide comprises at least one introduced modification (e.g., amino acid substitution) as described herein below. In some other embodiments, each of the F1 polypeptide and the F2 polypeptide comprises at least one introduced modification (e.g., amino acid substitution) as described herein below.

[0065] B-1(a). F1 and F2 Polypeptides of RSV F Mutants In some embodiments, the mutant is a mature form of the RSV F protein that includes two separate polypeptide chains, i.e., an F1 polypeptide and an F2 polypeptide. In some other embodiments, the F2 polypeptide is linked to the F1 polypeptide by one or two disulfide bonds to form an F2-F1 polypeptide heterodimer. In yet other embodiments, the RSV F mutant is in the form of a single chain protein in which the F2 polypeptide is linked to the F1 polypeptide by a peptide bond or peptide linker. Any suitable peptide linker can be used to link the two polypeptide chains together. Examples of such linkers include G, GG, GGG, GS, and SAIG ​​linker sequences. The linker can also be a full-length pep27 sequence or a fragment thereof.

[0066] The F1 polypeptide chain of the mutant can be the same length as the full-length F1 polypeptide of the corresponding wild-type RSV F protein, but can also have deletions, such as deletions of 1 to 60 amino acid residues from the C-terminus of the full-length F1 polypeptide. The full-length F1 polypeptide of the RSV F mutant corresponds to amino acid positions 137-574 of the native RSV F0 precursor and includes (N- to C-terminally) the extracellular domain (residues 137-524), the transmembrane domain (residues 525-550), and the cytoplasmic domain (residues 551-574). Note that amino acid residues 514 onwards in the native F1 polypeptide sequence may be any sequence in the F1 polypeptides of the RSV F mutants provided herein and therefore may not be present in the mutant F1 polypeptides.

[0067] In some embodiments, the F1 polypeptide of the RSV F mutant lacks the entire cytoplasmic domain. In other embodiments, the F1 polypeptide lacks a portion or the entire cytoplasmic domain and the entire transmembrane domain. In some specific embodiments, the mutant comprises an F1 polypeptide in which amino acid residues at positions 510, 511, 512, 513, 514, 515, 520, 525, or 530 to 574 are absent. Typically, in mutants linked to trimerization domains such as foldon, amino acids 514 to 754 may be absent. Thus, in some specific embodiments, amino acid residues 514 to 574 are absent in the mutant F1 polypeptide. In yet other specific embodiments, the F1 polypeptide of the RSV F mutant comprises or consists of amino acid residues 137 to 513 of the native F0 polypeptide sequence, e.g., any of the F0 precursor sequences set forth in SEQ ID NOs: 1, 2, 4, 6, and 81 to 270.

[0068] On the other hand, the F1 polypeptide of the RSV F mutant may include a C-terminal linkage to a trimerization domain such as a foldon. Many of the sequences of the RSV F mutants disclosed herein include sequences of protease cleavage sites, such as thrombin cleavage sites (LVPRGS), protein tags, such as 6xHis tags (HHHHHH) and Streptag II (WSHPGFEK), or linker sequences (e.g., GG and GS) (see FIG. 1), which are not essential for the function of the RSV F protein, such as the induction of an immune response. Those skilled in the art will recognize such sequences and will understand that, where appropriate, these sequences are not included in the disclosed RSV F mutants.

[0069] In the RSV F mutants provided by the present disclosure, the F2 polypeptide chain can be the same length as the full-length F2 polypeptide of the corresponding wild-type RSV F protein, and can also have deletions, such as 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues from the N-terminus or C-terminus of the F2 polypeptide.

[0070] The mutants of the F0 form (i.e., single-chain polypeptides comprising an F2 polypeptide linked to an F1 polypeptide with or without partial or full-length pep27) or the F1-F2 heterodimeric form may form protomers. The mutants may also be in the form of trimers, comprising three of the same protomers. Furthermore, the mutants may be glycosylated proteins (i.e., glycoproteins) or non-glycosylated proteins. The mutants of the F0 form may contain or lack a signal peptide sequence.

[0071] The F1 and F2 polypeptides of the RSV F protein mutants having one or more mutations can be in the form of any wild-type RSV F protein known in the art or discovered in the future, including, but not limited to, the F protein amino acid sequences of RSV subtype A and subtype B strains, including A2 Ontario and Buenos Aires, or any other subtype. In some embodiments, the RSV F mutant comprises an F1 and / or F2 polypeptide of a RSV A virus, such as an F1 and / or F2 polypeptide of a RSV F0 precursor protein set forth in any one of SEQ ID NOs: 1, 2, 4, 6, and 81-270, in which one or more mutations have been introduced. In some other embodiments, the RSV F mutant comprises an F1 and / or F2 polypeptide of a RSV B virus, such as an F1 and / or F2 polypeptide of a RSV F0 precursor protein set forth in any one of SEQ ID NOs: 2 and 211-263, in which one or more mutations have been introduced. In yet another embodiment, the RSV F mutant comprises an F1 and / or F2 polypeptide of a RSV bovine virus, for example, an F1 and / or F2 polypeptide of a RSV F0 precursor protein set forth in any one of SEQ ID NOs: 264-270, wherein one or more mutations have been introduced.

[0072] In some embodiments, the RSV F protein mutant comprises an F1 polypeptide, an F2 polypeptide, and one or more introduced amino acid mutations described herein below, wherein the F1 polypeptide comprises 350 contiguous amino acids and is at least 90, 95, 98, or 99 percent identical to amino acids 137-513 of any of SEQ ID NOs: 1, 4, and 81-210; the F2 polypeptide comprises 70 contiguous amino acids and is at least 90, 95, 98, or 99 percent identical to amino acids 26-109 of any of SEQ ID NOs: 1, 4, and 81-210; and the RSV F protein mutant is stabilized in a prefusion conformation as a monomer or trimer. In some embodiments, the F1 polypeptide comprises 350 contiguous amino acids and is at least 90, 95, 98, or 99 percent identical to amino acids 137-513 of any of the sequences set forth in SEQ ID NOs: 2, 6, and 211-263; the F2 polypeptide comprises 70 contiguous amino acids and is at least 90, 95, 98, or 99 percent identical to amino acids 26-109 of any of the sequences set forth in SEQ ID NOs: 2, 6, and 211-263; and in some other embodiments, the RSV F protein mutant is stabilized in a pre-fusion trimer conformation.

[0073] B-1(b) trimerization domain In some embodiments, the RSV F mutants provided by the present disclosure are linked to a trimerization domain, in some embodiments, the trimerization domain promotes the formation of trimers of three F1 / F2 heterodimers.

[0074] Some exogenous multimerization domains that promote the formation of stable trimers of soluble proteins are known in the art. Examples of such multimerization domains that can be linked to the mutants provided by the present disclosure include (1) GCN4 leucine zipper (Harbury et al. 1993 Science 262:1401-1407), (2) trimerization motif of pulmonary surfactant protein (Hoppe et al. 1994, FEB S Lett 344:191-195), (3) collagen (McAlinden et al. 2003, Biol Chem 278:42200-42207), and (4) phage T4 fibritin foldon (Miroshnikov et al. 1998, Protein Eng 11:329-414). In some embodiments, the foldon domain is linked to the F mutant at the C-terminus of the F1 polypeptide. In a specific embodiment, the foldon domain is a T4 fibritin foldon domain, such as the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 40).

[0075] Typically, the multimerization domain is located at the C-terminus relative to the F1 polypeptide. It may be directly connected to the F1 polypeptide chain. The multimerization domain may be connected to the F1 polypeptide via a linker, such as an amino acid linker, for example, the sequence GG, GS, or SAIG. The linker may also be a longer linker (for example, including the repeat sequence GG). Many conformationally neutral linkers are known in the art that can be used in the mutants provided by the present disclosure. In some embodiments, the F mutants that include a foldon domain include a protease cleavage site for removing the foldon domain from the F1 polypeptide, for example, a thrombin site between the F1 polypeptide and the foldon domain.

[0076] B-2. Introduced mutations in RSV F protein mutants The RSV F mutants provided by the present disclosure include F1 and F2 polypeptides, where either (1) the F1 or (2) the F2 polypeptide, or (3) both the F1 and F2 polypeptides contain one or more introduced amino acid mutations to the amino acid sequence of the corresponding native F protein. The introduction of such amino acid mutations into the RSV F mutants confers advantageous properties to the mutants, such as enhanced immunogenicity, improved stability, or the formation or improved stability of a particular desired physical form or conformation of the mutant. Such introduced amino acid mutations are referred to as "engineered disulfide bond mutations," "gap-filling mutations," or "electrostatic mutations," and are described in detail herein below. RSV F mutants containing any additional mutations are also encompassed by the present invention, so long as the immunogenic properties of the mutant are not substantially adversely affected by the additional mutations.

[0077] B-2(a) Engineered disulfide bond mutations In some embodiments, the RSV F mutants provided by the present disclosure include one or more engineered disulfide bond mutations. The term "engineered disulfide bond mutation" refers to the mutation of a pair of amino acid residues in the wild-type RSV F protein to a pair of cysteine ​​residues. The introduced cysteine ​​residue pair allows the formation of a disulfide bond between the introduced cysteine ​​residues, which serves to stabilize the conformation or oligomeric state of the protein, such as the pre-fusion conformation. To stabilize the pre-fusion conformation of the mutant, the residue pair for mutation to cysteine ​​should be adjacent in the pre-fusion conformation but distal in the post-fusion conformation. Such residues can be identified by suitable methods known in the art, such as visual inspection of the crystal structure of RSV F in the pre-fusion conformation, or by more quantitative selection using computational protein design software (such as BioLuminate™ [BioLuminate, Schrodinger LLC, New York, 2015], Discovery Studio™ [Discovery Studio Modeling Environment, Accelrys, San Diego, 2015], MOE™ [Molecular Operating Environment, Chemical Computing Group Inc., Montreal, 2015], and Rosetta™ [Rosetta, University of Washington, Seattle, 2015]). Preferably, the distance between residue pairs (e.g., beta carbons) is less than 8 Å in the pre-fusion conformation, but greater than 20 Å in the post-fusion conformation.

[0078] In some embodiments, the RSV F protein mutant contains only one engineered disulfide mutation ("single engineered disulfide mutation"). In some other embodiments, the RSV F protein mutant contains at least two engineered disulfide mutations, where each pair of cysteine ​​residues of the engineered disulfide mutations are appropriately positioned when the RSV F protein mutant is in a prefusion conformation ("double engineered disulfide mutation").

[0079] In some specific embodiments, the present disclosure provides RSV F mutants comprising at least one engineered disulfide bond mutation comprising the same introduced mutation in any of the exemplary mutants shown in Tables 1 and 4-6. The exemplary RSV F mutants shown in Tables 1 and 4-6 are based on the same native F0 sequence of the RSV A2 strain (SEQ ID NO: 3) with three natural substitutions at positions 102, 379, and 447. The same introduced mutations in each of the mutants can be made to the native F0 polypeptide sequence of any other RSV subtype or strain to obtain a different RSV F mutant, such as the native F0 polypeptide sequence set forth in any of SEQ ID NOs: 1, 2, 4, 6, and 81-270. RSV F mutants based on the native F0 polypeptide sequence of any other RSV subtype or strain and comprising any of the engineered disulfide mutations are also within the scope of the present invention. In some specific embodiments, the RSV F protein mutant comprises at least one engineered disulfide mutation selected from the group consisting of 55C and 188C, 155C and 290C, 103C and 148C, and 142C and 371C, such as S55C and L188C, S155C and S290C, T103C and I148C, or L142C and N371C.

[0080] In some embodiments, the disclosure provides a RSV F protein mutant in which the amino acid mutation is a mutation of a pair of amino acid residues in the HRB region (approximately amino acids 476-524) of the RSV F protein to a pair of cysteines. The introduced cysteine ​​residue pair allows the formation of a disulfide bond between the cysteine ​​residues of two adjacent F2-F1 mutant promoters of the trimer. The disulfides linking the two promoters in the trimer serve to stabilize the mutant in the trimer state. Examples of specific pairs of such mutations include 508C and 509C, 515C and 516C, 522C and 523C, such as K508C and S509C, N515C and V516C, or T522C and T523C. In some embodiments, the RSV F mutant comprises (1) at least one pair of cysteine ​​mutations in the HRB region, and (2) at least one introduced mutation outside the HRB region selected from the engineered disulfide bond mutations described herein above, the gap-filling mutations described herein below, the electrostatic mutations described herein below, or any combination of these mutations.

[0081] B-2(b) gap-filling mutation. In another embodiment, the present disclosure provides a RSV F mutant comprising one or more gap-filling mutations. The term "gap-filling mutation" refers to the replacement of an amino acid residue of a wild-type RSV F protein with an amino acid that is expected to fill an internal gap of the mature RSV F protein. In one application, such a gap-filling mutation contributes to stabilizing the pre-fusion conformation of the RSV F protein mutant. The gap in the pre-fusion conformation of the RSV F protein can be identified by methods known in the art, for example, by visual inspection of the crystal structure of RSV F in the pre-fusion conformation, or by using computational protein design software (such as BioLuminate™ [BioLuminate, Schrodinger LLC, New York, 2015], Discovery Studio™ [Discovery Studio Modeling Environment, Accelrys, San Diego, 2015], MOE™ [Molecular Operating Environment, Chemical Computing Group Inc., Montreal, 2015], and Rosetta™ [Rosetta, University of Washington, Seattle, 2015]). Amino acids replaced for gap-filling mutations typically include small aliphatic amino acids (e.g., Gly, Ala, and Val) or small polar amino acids (e.g., Ser and Thr). These may also include amino acids that are buried in the prefusion conformation but exposed to the solvent in the postfusion conformation. The replacement amino acids may be large aliphatic amino acids (Ile, Leu, and Met) or large aromatic amino acids (His, Phe, Tyr, and Trp). For example, in some embodiments, the RSV F protein mutant comprises a T54H mutation.

[0082] In some specific embodiments, the RSV F protein mutant is (1) Substitution of S at position 55, 62, 155, 190, or 290 with I, Y, L, H, or M; (2) substitution of T at position 54, 58, 189, 219, or 397 with I, Y, L, H, or M; (3) substitution of G at position 151 with A or H; (4) substitution of A at position 147 or 298 with I, L, H, or M; (5) Substitution of V at position 164, 187, 192, 207, 220, 296, 300, or 495 with I, Y, or H; and (6) Substitution of R at position 106 with W The nucleotide sequence of the present invention comprises one or more gap-filling mutations selected from the group consisting of:

[0083] In some specific embodiments, the present disclosure provides RSV F mutants comprising one or more gap-filling mutations, including any of the gap-filling mutations in any of the mutations set forth in Tables 2, 4, and 6. The RSV F mutants set forth in Tables 2, 4, and 6 are based on the same native F0 sequence of the RSV A2 strain (SEQ ID NO: 3) with three natural substitutions at positions 102, 379, and 447. The same introduced mutations in each of the mutants can also be made to the native F0 polypeptide sequence of any other RSV subtype or strain to obtain different RSV F mutants, such as the native F0 polypeptide sequence set forth in any of SEQ ID NOs: 1, 2, 4, 6, and 81-270. RSV F mutants based on the native F0 polypeptide sequence of any other RSV subtype or strain and comprising any of one or more gap-filling mutations are also within the scope of the present invention. In some specific embodiments, the RSV F protein mutants provided by the present disclosure comprise at least one gap-filling mutation selected from the group consisting of T54H, S190I, and V296I.

[0084] B-2(c) Electrostatic mutation. In yet another embodiment, the disclosure provides an RSV F protein mutant comprising one or more electrostatic mutations. The term "electrostatic mutation" refers to an amino acid mutation introduced into a wild-type RSV F protein that reduces ionic repulsion or increases ionic attraction between residues in the protein that are adjacent to each other in the folded structure. Hydrogen bonds are a special case of ionic attraction, so electrostatic mutations can increase hydrogen bonds between such adjacent residues. In one example, electrostatic mutations can be introduced to improve trimer stability. In some embodiments, electrostatic mutations are introduced to reduce repulsive ionic interactions or increase attractive ionic interactions (potentially including hydrogen bonds) between residues that are adjacent to the RSV F glycoprotein in its prefusion form but not adjacent in its postfusion form. For example, in the prefusion form, the acidic side chain Asp486 of one promoter of the RSV F glycoprotein trimer is located at the interface of the trimer and is structurally sandwiched between two other acidic side chains Glu487 and Asp489 of another promoter. On the other hand, in the post-fusion conformation, the acidic side chain Asp486 is located on the surface of the trimer and is exposed to the solvent. In some embodiments, the RSV F protein mutant comprises an electrostatic D486S substitution that reduces the repulsive ionic interaction or increases the attractive ionic interaction with the acidic residues Glu487 and Asp489 of another promoter of the RSV F trimer. Typically, the introduction of electrostatic mutations increases the melting temperature (Tm) of the pre-fusion conformation or pre-fusion trimer conformation of the RSV F protein.

[0085] Unfavorable electrostatic interactions in the pre-fusion or pre-fusion trimer conformation can be identified by methods known in the art, such as by visual inspection of the crystal structure of RSV F in the pre-fusion or pre-fusion trimer conformation, or by using computational protein design software (such as BioLuminate™ [BioLuminate, Schrodinger LLC, New York, 2015], Discovery Studio™ [Discovery Studio Modeling Environment, Accelrys, San Diego, 2015], MOE™ [Molecular Operating Environment, Chemical Computing Group Inc., Montreal, 2015.], and Rosetta™ [Rosetta, University of Washington, Seattle, 2015]).

[0086] In some specific embodiments, the RSV F protein mutant is (1) Substitution of E at position 82, 92, or 487 by D, F, Q, T, S, L, or H; (2) Substitution of K at position 315, 394, or 399 by F, M, R, S, L, I, Q, or T; (3) Substitution of D at position 392, 486, or 489 by H, S, N, T, or P; and (4) Substitution of R at position 106 or 339 by F, Q, N, or W The at least one electrostatic mutation is selected from the group consisting of:

[0087] In some specific embodiments, the present disclosure provides RSV F mutants comprising one or more electrostatic mutations, including electrostatic mutations in any of the mutants shown in Tables 3, 5, and 6. The RSV F mutants shown in Tables 3, 5, and 6 are based on the same native F0 sequence of the RSV A2 strain (SEQ ID NO: 3) with three natural substitutions at positions 102, 379, and 447. The same introduced mutations in each of the mutants can also be made to the native F0 polypeptide sequence of any other RSV subtype or strain to obtain different RSV F mutants, such as the native F0 polypeptide sequence shown in any of SEQ ID NOs: 1, 2, 4, 6, and 81-270. RSV F mutants based on the native F0 polypeptide sequence of any other RSV subtype or strain and comprising any of one or more electrostatic mutations are also within the scope of the present invention. In some specific embodiments, the RSV F protein mutant comprises the mutation D486S.

[0088] B-2(d) A combination of engineered disulfide bond, gap-filling, and electrostatic mutations. In another aspect, the present disclosure provides RSV F protein mutants that include a combination of two or more different types of mutations selected from engineered disulfide bond mutations, gap-filling mutations, and electrostatic mutations, each of which is described herein above.

[0089] In some embodiments, the mutant comprises at least one engineered disulfide bond mutation and at least one gap-filling mutation. In some specific embodiments, the RSV F mutant comprises a combination of mutations shown in Table 4.

[0090] In some further embodiments, the RSV F protein mutant comprises at least one engineered disulfide mutation and at least one electrostatic mutation. In some specific embodiments, the RSV F mutant comprises a combination of mutations shown in Table 5.

[0091] In yet another embodiment, the RSV F protein mutant comprises at least one engineered disulfide mutation, at least one gap-filling mutation, and at least one electrostatic mutation. In some specific embodiments, the RSV F mutant comprises a combination of mutations shown in Table 6.

[0092] In some specific embodiments, the present invention provides (1) A combination of T103C, I148C, S190I, and D486S, (2) A combination of T54H, S55C, L188C, and D486S. (3) A combination of T54H, T103C, I148C, S190I, V296I, and D486S, (4) A combination of T54H, S55C, L142C, L188C, V296I, and N371C, (5) A combination of S55C, L188C, and D486S, (6) A combination of T54H, S55C, L188C, and S190I, (7) A combination of S55C, L188C, S190I, and D486S, (8) A combination of T54H, S55C, L188C, S190I, and D486S, (9) A combination of S155C, S190I, S290C, and D486S, (10) A combination of T54H, S55C, L142C, L188C, V296I, N371C, D486S, E487Q, and D489S, and (11) Combination of T54H, S155C, S190I, S290C, and V296I The present invention provides an RSV F mutant comprising a combination of mutations selected from the group consisting of:

[0093] In some specific embodiments, the present disclosure provides RSV F mutants that include a combination of introduced mutations, including a combination of mutations in any of the mutants shown in Tables 4, 5, and 6. The RSV F mutants shown in Tables 4, 5, and 6 are based on the same native F0 sequence of the RSV A2 strain (SEQ ID NO: 3) with three natural substitutions at positions 102, 379, and 447. The same introduced mutations in each of the mutants can also be made to the native F0 polypeptide sequence of any other RSV subtype or strain to obtain different RSV F mutants, such as the native F0 polypeptide sequence shown in any of SEQ ID NOs: 1, 2, 4, 6, and 81-270. RSV F mutants based on the native F0 polypeptide sequence of any other RSV subtype or strain and including any combination of mutations are also within the scope of the present invention.

[0094] In some other specific embodiments, the present invention provides an antibody that comprises cysteine ​​(C) at position 103 (103C) and position 148 (148C), isoleucine (I) at position 190 (190I), and serine (S) at position 486 (486S), and (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 41, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 42; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 41, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 42; (3) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 43, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 44; (4) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 43, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 44; (5) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 45, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 46; (6) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 45, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 46; (7) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 47, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 48; (8) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 47, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 48; (9) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 49, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 50; (10) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 49, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 50; (11) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 279, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 280; (12) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 279, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 280; (13) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 281, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 282; (14) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 281, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 282; (15) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 283, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 284; (16) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 283, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 284; (17) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 285, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 286; (18) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 285, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 286; (19) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 287, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 288; (20) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 287, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 288; (21) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 289, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 290, and (22) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 289, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 290. The present invention provides an RSV F mutant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0095] In some other specific embodiments, the present invention provides a peptide comprising a histidine (H) at position 54, a cysteine ​​(C) at positions 103 and 148, an isoleucine (I) at positions 190 and 296, and a serine (S) at position 486, and (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO:51, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO:52; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:51, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:52; (3) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 53, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 54; (4) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:53, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:54; (5) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 55, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 56; (6) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:55, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:56; (7) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 57, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 58; (8) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:57, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:58; (9) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 59, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 60; (10) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:59, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:60; (11) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 291, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 292; (12) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 291, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 292; (13) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 293, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 294; (14) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 293, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 294; (15) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 295, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 296; (16) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 295, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 296; (17) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 297, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 298; (18) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 297, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 298; (19) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 299, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 300; (20) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 299, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 300; (21) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 301, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 302, and (22) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 301, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 302. The present invention provides an RSV F mutant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0096] In certain other specific embodiments, the present invention provides a peptide comprising a histidine (H) at position 54, a cysteine ​​(C) at positions 55 and 188, and a serine (S) at position 486, and (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 61, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 62; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:61, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:62; (3) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 63, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 64; (4) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 63, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 64; (5) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 65, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 66; (6) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 65, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 66; (7) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 67, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 68; (8) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 67, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 68; (9) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 69, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 70; (10) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 69, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 70; (11) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 303, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 304; (12) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 303, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 304; (13) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 305, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 306; (14) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 305, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 306; (15) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 307, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 308; (16) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 307, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 308; (17) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 309, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 310; (18) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 309, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 310; (19) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 311, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 312; (20) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 311, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 312; (21) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 313, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 314, and (22) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 313, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 314. The present invention provides an RSV F mutant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0097] In some other specific embodiments, the present invention provides a peptide that contains a histidine (H) at position 54, a cysteine ​​(C) at positions 55 and 188, an isoleucine (I) at position 190 (190I), and a serine (S) at position 486, and (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 71, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 72; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 71, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 72; (3) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 73, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 74; (4) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 73, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 74; (5) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 75, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 76; (6) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 75, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 76; (7) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 77, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 78; (8) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 77, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 78; (9) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 79, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 80; (10) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 79, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 80; (11) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 315, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 316; (12) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 315, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 316; (13) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 317, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 318; (14) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 317, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 318; (15) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 319, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 320; (16) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 319, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 320; (17) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 321, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 322; (18) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 321, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 322; (19) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 323, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 324; (20) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 323, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 324; (21) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 325, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 326, and (22) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 325, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 326. The present invention provides an RSV F mutant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0098] The amino acid sequences of the F2 and F1 polypeptides of exemplary RSV F mutants provided by the present disclosure are shown in Tables 19-22.

[0099] In some embodiments, the foldon domain is linked to the RSV F mutant described herein above, wherein the foldon domain is linked to the C-terminus of the F1 polypeptide and comprises the amino acid sequence of SEQ ID NO:40.

[0100] The RSV F protein mutants provided by the present disclosure can be prepared by conventional methods known in the art, for example, by expression in a recombinant host system using a suitable vector. Suitable recombinant host cells include, for example, insect cells, mammalian cells, avian cells, bacteria, and yeast cells. Examples of suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (clonal isolates derived from the parent Trichoplusia ni BTI-TN-5B1-4 cell line (Invitrogen)). Examples of suitable mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 cells or Expi293 cells, typically transformed with sheared adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Suitable avian cells include, for example, chicken embryonic stem cells (e.g., EBx.RTM. cells), chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts (e.g., ELL-O), and duck cells. Suitable insect cell expression systems, such as baculovirus-vectored systems, are known to those skilled in the art and are described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form, inter alia, from Invitrogen, San Diego Calif. Avian cell expression systems are also known to those skilled in the art and are described, for example, in U.S. Pat. No. 5,340,740, U.S. Pat. No. 5,656,479, U.S. Pat. No. 5,830,510, U.S. Pat. No. 6,114,168, and U.S. Pat. No. 6,500,668. Similarly, bacterial and mammalian cell expression systems are also known in the art and are described, for example, in Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths, London.

[0101] Many suitable vectors for the expression of recombinant proteins in insect or mammalian cells are well known and conventional in the art. Suitable vectors may contain many components, including but not limited to one or more of the following: an origin of replication, a selectable marker gene, one or more expression control elements, such as transcription control elements (e.g., promoters, enhancers, terminators), and / or one or more translation signals, as well as a signal sequence or leader sequence for targeting the secretory pathway in a selected host cell (e.g., from a mammalian source or from a heterologous mammalian or non-mammalian species). For example, in expression in insect cells, a suitable baculovirus expression vector, such as pFastBac (Invitrogen), is used to produce recombinant baculovirus particles. The baculovirus particles are amplified and used to infect insect cells to express recombinant proteins. In expression in mammalian cells, a vector is used that drives the expression of the construct in the desired mammalian host cell (e.g., Chinese hamster ovary cells).

[0102] RSV F protein mutant polypeptides can be purified using any suitable method. For example, methods for purifying RSV F protein mutant polypeptides by immunoaffinity chromatography are known in the art. Ruiz-Arguello et al., J. Gen. Virol., 85:3677-3687 (2004). Suitable methods for purifying desired proteins are well known in the art, including precipitation and various types of chromatography, such as hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, chelation chromatography, and size exclusion chromatography. A suitable purification scheme can be made using two or more of these or other suitable methods. Optionally, the RSV F protein mutant polypeptide can include a "tag" that facilitates purification, such as an epitope tag or a histidine (HIS) tag. Such tagged polypeptides can be conveniently purified, for example, from conditioned medium by chelation or affinity chromatography.

[0103] C. Nucleic acids encoding RSV F protein mutants In another aspect, the present invention provides nucleic acid molecules encoding the RSV F protein mutants described hereinabove.These nucleic acid molecules include DNA sequences, cDNA sequences, and RNA sequences.Nucleic acid molecules encoding only the F2 polypeptide or only the F1 polypeptide of the RSV F mutant are also included in the present invention.The nucleic acid molecule can be incorporated into a vector, such as an expression vector.

[0104] In some embodiments, the nucleic acid molecule encodes a precursor F0 polypeptide that is processed into the disclosed RSV F mutants when expressed in a suitable cell. In some embodiments, the nucleic acid molecule encodes a precursor F0 polypeptide that is processed into the disclosed RSV F mutants when expressed in a suitable cell, where the precursor F0 polypeptide comprises, from the N-terminus to the C-terminus, a signal peptide, an F2 polypeptide, a Pep27 polypeptide, and an F1 polypeptide. In some embodiments, the Pep27 polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 4, 6, and 81-270, where the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the signal peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 4, 6, and 81-270, where the amino acid positions correspond to the reference amino acid sequence of SEQ ID NO: 1.

[0105] In some embodiments, the nucleic acid molecule is (1) A mutant containing at least one engineered disulfide mutation; (2) a mutant containing at least one gap-filling mutation; (3) mutants containing at least one electrostatic mutation; (4) A mutant comprising at least one engineered disulfide mutation and at least one gap-filling mutation; (5) A mutant comprising at least one engineered disulfide mutation and at least one electrostatic mutation; (6) A mutant comprising at least one gap-filling mutation and at least one electrostatic mutation; and (7) Mutants containing at least one engineered disulfide mutation, as well as at least one electrostatic mutation, at least one gap-filling mutation, and at least one electrostatic mutation. The compound encodes a mutant selected from the group consisting of:

[0106] In some specific embodiments, the present disclosure provides: (1) A mutant containing a combination of substitutions 103C, 148C, 190I, and 486S; (2) a mutant containing a combination of substitutions 54H, 55C, 188C, and 486S; (3) a mutant containing a combination of substitutions 54H, 103C, 148C, 190I, 296I, and 486S; (4) mutants containing a combination of substitutions 54H, 55C, 142C, 188C, 296I, and 371C; (5) A mutant containing a combination of amino acid substitutions 55C, 188C, and 486S; (6) A mutant containing a combination of amino acid substitutions 54H, 55C, 188C, and 190I; (7) A mutant containing a combination of amino acid substitutions 55C, 188C, 190I, and 486S; (8) A mutant containing a combination of amino acid substitutions 54H, 55C, 188C, 190I, and 486S; (9) A mutant containing a combination of amino acid substitutions 155C, 190I, 290C, and 486S; (10) Mutants containing a combination of amino acid substitutions 54H, 55C, 142C, 188C, 296I, 371C, 486S, 487Q, and 489S, and (11) A mutant containing a combination of amino acid substitutions 54H, 155C, 190I, 290C, and 296I. The present invention provides a nucleic acid molecule encoding a mutant selected from the group consisting of:

[0107] In certain embodiments, the nucleic acid molecule comprises cysteine ​​(C) at position 103 (103C) and position 148 (148C), an isoleucine (I) at position 190 (190I), and a serine (S) at position 486 (486S), and (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 41, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 42; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 41, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 42; (3) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 43, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 44; (4) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 43, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 44; (5) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 45, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 46; (6) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 45, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 46; (7) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 47, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 48; (8) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 47, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 48; (9) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 49, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 50; (10) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 49, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 50; (11) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 279, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 280; (12) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 279, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 280; (13) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 281, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 282; (14) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 281, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 282; (15) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 283, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 284; (16) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 283, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 284; (17) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 285, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 286; (18) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 285, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 286; (19) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 287, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 288; (20) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 287, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 288; (21) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 289, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 290, and (22) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 289, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 290. The RSV F mutant encodes an F1 polypeptide comprising an F2 polypeptide selected from the group consisting of:

[0108] In certain other specific embodiments, the nucleic acid molecule comprises a histidine (H) at position 54, a cysteine ​​(C) at positions 103 and 148, an isoleucine (I) at positions 190 and 296, and a serine (S) at position 486, and (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO:51, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO:52; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:51, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:52; (3) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 53, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 54; (4) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:53, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:54; (5) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 55, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 56; (6) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:55, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:56; (7) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 57, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 58; (8) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:57, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:58; (9) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 59, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 60; (10) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:59, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:60; (11) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 291, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 292; (12) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 291, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 292; (13) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 293, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 294; (14) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 293, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 294; (15) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 295, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 296; (16) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 295, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 296; (17) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 297, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 298; (18) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 297, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 298; (19) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 299, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 300; (20) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 299, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 300; (21) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 301, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 302, and (22) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 301, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 302. The RSV F mutant encodes an F1 polypeptide comprising an F2 polypeptide selected from the group consisting of:

[0109] In certain other specific embodiments, the nucleic acid molecule comprises a histidine (H) at position 54, a cysteine ​​(C) at positions 55 and 188, and a serine (S) at position 486, and (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 61, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 62; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:61, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:62; (3) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 63, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 64; (4) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 63, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 64; (5) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 65, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 66; (6) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 65, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 66; (7) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 67, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 68; (8) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 67, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 68; (9) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 69, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 70; (10) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 69, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 70; (11) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 303, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 304; (12) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 303, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 304; (13) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 305, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 306; (14) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 305, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 306; (15) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 307, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 308; (16) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 307, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 308; (17) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 309, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 310; (18) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 309, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 310; (19) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 311, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 312; (20) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 311, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 312; (21) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 313, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 314, and (22) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 313, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 314. The RSV F mutant encodes an F1 polypeptide comprising an F2 polypeptide selected from the group consisting of:

[0110] In certain other specific embodiments, the nucleic acid molecule comprises a histidine (H) at position 54, a cysteine ​​(C) at positions 55 and 188, an isoleucine (I) at position 190 (190I), and a serine (S) at position 486, and (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 71, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 72; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 71, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 72; (3) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 73, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 74; (4) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 73, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 74; (5) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 75, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 76; (6) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 75, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 76; (7) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 77, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 78; (8) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 77, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 78; (9) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 79, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 80; (10) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 79, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 80; (11) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 315, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 316; (12) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 315, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 316; (13) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 317, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 318; (14) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 317, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 318; (15) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 319, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 320; (16) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 319, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 320; (17) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 321, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 322; (18) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 321, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 322; (19) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 323, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 324; (20) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 323, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 324; (21) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 325, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 326, and (22) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 325, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 326. The RSV F mutant encodes an F1 polypeptide comprising an F2 polypeptide selected from the group consisting of:

[0111] In some specific embodiments, the present disclosure provides: (1) A mutant comprising amino acids 26 to 513 of SEQ ID NO: 19; (2) A mutant comprising amino acids 26 to 513 of SEQ ID NO: 20, and (3) A mutant containing amino acids 26 to 513 of SEQ ID NO: 21 The present invention provides a nucleic acid molecule encoding a mutant selected from the group consisting of:

[0112] In some other specific embodiments, the present disclosure provides: (1) a nucleotide sequence comprising nucleotides 76 to 1539 of SEQ ID NO:8; (2) a nucleotide sequence comprising nucleotides 76 to 1539 of SEQ ID NO:9; (3) a nucleotide sequence comprising nucleotides 76 to 1539 of SEQ ID NO: 10; (4) A nucleotide sequence comprising nucleotides 76 to 1539 of SEQ ID NO:11; (5) A nucleotide sequence comprising nucleotides 76 to 1539 of SEQ ID NO: 12; (6) A nucleotide sequence comprising nucleotides 76 to 1539 of SEQ ID NO: 13; (7) A nucleotide sequence comprising nucleotides 76 to 1539 of SEQ ID NO: 14; (8) A nucleotide sequence comprising nucleotides 76 to 1539 of SEQ ID NO: 15; (9) A nucleotide sequence comprising nucleotides 76 to 1539 of SEQ ID NO: 16; (10) A nucleotide sequence comprising nucleotides 76 to 1539 of SEQ ID NO: 17, and (11) A nucleotide sequence comprising nucleotides 76 to 1539 of SEQ ID NO: 18 The present invention provides a nucleic acid molecule encoding a RSV F protein mutant comprising a nucleotide sequence selected from the group consisting of:

[0113] D. Compositions Comprising RSV F Protein Mutants, Compositions Comprising Nucleic Acids Encoding RSV F Protein Mutants In another aspect, the present invention provides a composition comprising (1) a RSV F protein mutant described in this disclosure, or (2) a nucleic acid molecule or vector encoding such a RSV F protein mutant.

[0114] In some embodiments, the composition is an immunogenic composition capable of inducing an immune response to the F protein of RSV in a subject. In some specific embodiments, the immunogenic composition is a pharmaceutical composition comprising a RSV F protein mutant provided by the present disclosure and a pharma- ceutical acceptable carrier.

[0115] In yet another embodiment, the pharmaceutical composition is a vaccine. The immunogenic component in the vaccine may be (1) a RSV F protein mutant described in this disclosure, (2) a nucleic acid encoding such a RSV F protein mutant, or (3) a vector for expressing such a RSV F protein mutant.

[0116] In certain embodiments, the vaccine comprises cysteine ​​(C) at positions 103 (103C) and 148 (148C), isoleucine (I) at position 190 (190I), and serine (S) at position 486 (486S), and (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 41, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 42; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 41, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 42; (3) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 43, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 44; (4) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 43, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 44; (5) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 45, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 46; (6) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 45, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 46; (7) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 47, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 48; (8) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 47, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 48; (9) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 49, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 50; (10) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 49, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 50; (11) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 279, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 280; (12) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 279, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 280; (13) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 281, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 282; (14) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 281, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 282; (15) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 283, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 284; (16) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 283, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 284; (17) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 285, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 286; (18) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 285, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 286; (19) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 287, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 288; (20) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 287, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 288; (21) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 289, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 290, and (22) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 289, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 290. The RSV F mutant comprises an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0117] In certain other embodiments, the vaccine comprises a histidine (H) at position 54, a cysteine ​​(C) at positions 103 and 148, an isoleucine (I) at positions 190 and 296, and a serine (S) at position 486, and (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO:51, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO:52; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:51, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:52; (3) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 53, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 54; (4) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:53, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:54; (5) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 55, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 56; (6) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:55, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:56; (7) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 57, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 58; (8) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:57, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:58; (9) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 59, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 60; (10) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:59, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:60; (11) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 291, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 292; (12) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 291, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 292; (13) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 293, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 294; (14) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 293, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 294; (15) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 295, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 296; (16) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 295, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 296; (17) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 297, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 298; (18) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 297, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 298; (19) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 299, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 300; (20) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 299, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 300; (21) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 301, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 302, and (22) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 301, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 302. The RSV F mutant comprises an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0118] In certain other embodiments, the vaccine comprises a histidine (H) at position 54, a cysteine ​​(C) at positions 55 and 188, and a serine (S) at position 486, and (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 61, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 62; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:61, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:62; (3) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 63, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 64; (4) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 63, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 64; (5) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 65, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 66; (6) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 65, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 66; (7) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 67, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 68; (8) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 67, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 68; (9) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 69, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 70; (10) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 69, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 70; (11) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 303, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 304; (12) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 303, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 304; (13) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 305, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 306; (14) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 305, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 306; (15) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 307, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 308; (16) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 307, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 308; (17) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 309, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 310; (18) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 309, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 310; (19) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 311, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 312; (20) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 311, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 312; (21) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 313, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 314, and (22) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 313, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 314. The RSV F mutant comprises an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0119] In some other specific embodiments, the vaccine comprises a histidine (H) at position 54, a cysteine ​​(C) at positions 55 and 188, an isoleucine (I) at position 190 (190I), and a serine (S) at position 486, and (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 71, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 72; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 71, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 72; (3) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 73, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 74; (4) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 73, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 74; (5) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 75, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 76; (6) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 75, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 76; (7) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 77, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 78; (8) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 77, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 78; (9) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 79, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 80; (10) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 79, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 80; (11) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 315, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 316; (12) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 315, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 316; (13) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 317, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 318; (14) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 317, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 318; (15) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 319, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 320; (16) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 319, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 320; (17) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 321, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 322; (18) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 321, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 322; (19) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 323, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 324; (20) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 323, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 324; (21) An F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 325, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 326, and (22) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 325, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 326. The RSV F mutant comprises an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0120] In some embodiments, a composition, such as a pharmaceutical composition, or vaccine comprises two or more different RSV F mutants. The two or more different RSV F mutants may contain the same introduced amino acid mutation, but comprise F1 and F2 polypeptides of different RSV strains or subtypes. The two or more different RSV F mutants may contain different introduced amino acid mutations.

[0121] In some embodiments, the composition comprises two different mutants that contain the same introduced amino acid mutations, where one of the mutants comprises the F1 and F2 polypeptides of RSV subtype A and the other mutant comprises the F1 and F2 polypeptides of RSV subtype B. In some specific embodiments, the two different mutants are (1) a combination of amino acid substitutions 103C, 148C, 190I, and 486S; (2) a combination of amino acid substitutions 54H, 55C, 188C, and 486S; (3) a combination of amino acid substitutions 54H, 103C, 148C, 190I, 296I, and 486S; (4) a combination of amino acid substitutions 54H, 55C, 142C, 188C, 296I, and 371C; (5) a combination of amino acid substitutions 55C, 188C, and 486S; (6) a combination of amino acid substitutions 54H, 55C, 188C, and 190I; (7) a combination of amino acid substitutions 55C, 188C, 190I, and 486S; (8) a combination of amino acid substitutions 54H, 55C, 188C, 190I, and 486S; (9) a combination of amino acid substitutions 155C, 190I, 290C, and 486S; (10) A combination of amino acid substitutions 54H, 55C, 142C, 188C, 296I, 371C, 486S, 487Q, and 489S, and (11) A combination of amino acid substitutions 54H, 155C, 190I, 290C, and 296I. The amino acid substitutions are selected from the group consisting of:

[0122] In addition to the immunogenic components, the vaccine may further comprise immunomodulatory agents, such as adjuvants. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide and / or aluminum phosphate, oil emulsion compositions (or oil-in-water compositions) including squalene-water emulsions such as MF59 (see, e.g., WO 90 / 14837), saponin preparations such as QS21, and immune stimulating complexes (ISCOMS) (see, e.g., U.S. Pat. Nos. 5,057,540, WO 90 / 03184, WO 96 / 11711, WO 2004 / 004762, WO 2005 / 002620), bacterial or microbial derivatives, including, for example, monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motif-containing oligonucleotides, ADP-ribosylating bacterial toxins, or mutants thereof, such as E. coli heat-labile enterotoxin LT, cholera toxin CT, and the like. It is also possible to use vector-encoded adjuvants, for example by using heterologous nucleic acids encoding a fusion of the oligomerization domain of C4 binding protein (C4bp) to an antigen of interest (e.g., Solabomi et al., 2008, Infect Immun 76:3817-23). ​​In certain embodiments, the composition contains aluminum as an adjuvant, e.g., in the form of aluminum hydroxide, aluminum phosphate, aluminum potassium phosphate, or combinations thereof, at a concentration of 0.05-5 mg, e.g., an aluminum content of 0.075-1.0 mg per dose.

[0123] E. Uses of RSV F protein mutants, nucleic acid molecules, and compositions The present disclosure also relates to the use of RSV F protein mutants, nucleic acids encoding RSV F protein mutants, or vectors for expressing RSV F protein mutants, or compositions comprising RSV F protein mutants or nucleic acids.

[0124] In some embodiments, the present disclosure provides a method for eliciting an immune response against RSV in a subject, comprising administering to the subject an effective amount of a RSV F protein mutant, a nucleic acid molecule encoding a RSV F protein mutant, or a composition comprising a RSV F protein mutant or nucleic acid molecule.

[0125] In some specific embodiments, the present disclosure provides a method for preventing RSV infection in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition, such as a vaccine, comprising a RSV F protein mutant, a nucleic acid encoding a RSV F protein mutant, or a vector for expressing a RSV F protein mutant.In some embodiments, the subject is a human.In some specific embodiments, the human is a child, such as an infant.In some other specific embodiments, the human is a woman, particularly a pregnant woman.

[0126] The composition can be administered to a subject with or without administration of an adjuvant. The effective amount administered to a subject is sufficient to induce an immune response to a RSV antigen, such as the RSV F protein, in the subject. Subjects that can be selected for treatment include those at risk of developing RSV infection due to exposure or potential exposure to RSV. Since nearly all humans are infected with RSV by the age of 2 years, the entire birth cohort is included as a relevant population for immunization. This can be done, for example, by starting an immunization regimen at any time from birth to 6 months of age, from 6 months to 5 years of age, in pregnant women (or women of childbearing age) to protect their infants by passive transfer of antibodies, in families of newborns or fetuses still in utero, and in subjects over 50 years of age. Subjects at highest risk of RSV infection with severe symptoms (e.g., requiring hospitalization) include premature infants, children with bronchopulmonary dysplasia, and children with congenital heart disease.

[0127] Administration of compositions provided by the present disclosure, such as pharmaceutical compositions, can be carried out using standard routes of administration. Non-limiting embodiments include parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal, mucosal, or oral administration.

[0128] The total dose of the composition given to a subject during a single administration can vary, as will be known to the skilled practitioner.

[0129] It is also possible to provide one or more booster doses of the vaccine composition. When booster vaccinations are administered, such booster vaccinations are typically administered to the same subject one week to ten years, preferably two weeks to six months, after the subject is first administered the composition (which in such cases is referred to as a "primary vaccination"). In alternative booster regimens, a different vector, such as one or more adenoviruses, or other vectors, such as modified vaccinia virus of Ankara (MVA), or DNA, or protein, can be administered to the subject after the primary vaccination. For example, a recombinant viral vector of the present invention can be administered as a primary dose, and a composition comprising the RSV F protein can be administered as a booster dose.

[0130] In certain embodiments, the administration comprises one priming dose and at least one booster dose. In certain other embodiments, the administration occurs annually. In yet other embodiments, the administration occurs annually together with an influenza vaccine.

[0131] The vaccine provided by the present disclosure can be used together with one or more other vaccines.For example, in adults, the vaccine can be used together with influenza vaccine, Prevenar, tetanus vaccine, diphtheria vaccine, and whooping cough vaccine.For use in children, the vaccine provided by the present disclosure can be used together with any other vaccines that are indicated for pediatric patients.

[0132]

Table 1-1

[0133]

Table 1-2

[0134]

Table 2

[0135]

Table 3-1

[0136]

Table 3-2

[0137]

Table 3-3

[0138]

Table 3-4

[0139]

Table 4-1

[0140]

Table 4-2

[0141]

Table 4-3

[0142]

Table 4-4

[0143]

Table 5-1

[0144]

Table 5-2

[0145]

Table 5-3

[0146]

Table 5-4

[0147]

Table 5-5

[0148]

Table 5-6

[0149]

Table 6-1

[0150]

Table 6-2

[0151]

Table 6-3

[0152]

Table 6-4

[0153] [Table 7] EXAMPLES

[0154] F. Working Example The present invention will be further described by the following illustrative examples, which are not intended to limit the invention in any way and are intended only to illustrate the invention.

[0155] Example 1 Design and preparation of RSV F protein mutants 1A: RSV F mutants with foldon domains This example describes the design and preparation of various RSV F protein mutants, including fibritin foldon trimerization domains and introduced amino acid mutations, such as engineered disulfide bond mutations, gap-filling mutations, electrostatic mutations, or combinations thereof. Exemplary RSV F mutants, each identified by a unique identifier, such as pXCS501, pXCS601, are shown in Tables 1-6. Each of these mutants was designed and prepared based on the amino acid sequence set forth in SEQ ID NO: 3, which is also described in FIG. 1. Amino acid residues 1-513 of the sequence of SEQ ID NO: 3 are identical to amino acid residues 1-513 of the native RSV A2 F0 precursor polypeptide set forth in SEQ ID NO: 1, except for three naturally occurring substitutions, P102A, I379V, and M447V, in the sequence of SEQ ID NO: 3. Thus, the amino acid sequences of these exemplary F mutants are identical except for the introduced amino acid mutations described for each mutant listed in Tables 1-6. Each of these RSV F protein mutants includes two separate polypeptide chains. One polypeptide chain, the F2 polypeptide, comprises amino acids 26-109 of SEQ ID NO:3, except for the mutations introduced as described. The other polypeptide chain comprises an F1 polypeptide (residues 137-513) linked to a foldon trimerization domain (residues 518-544) via a SAIG ​​linker (residues 514-517). The signal peptide (residues 1-25) and pep27 (residues 110-136) of SEQ ID NO:3 were cleaved from the F0 precursor during the expression process. Processes for expression and purification of these exemplary RSV F mutants are described in Examples 2 and 3.

[0156] 1B: RSV F mutants without the foldon domain The RSV F mutant lacking the foldon domain, pXCS899, was prepared in the same manner as described in Example 1A above, except that amino acids 514-544 of the F0 precursor sequence of SEQ ID NO: 3 were deleted. The amino acid sequence of the precursor polypeptide of pXCS899 is set forth in SEQ ID NO:271.

[0157]

Table 8-1

[0158]

Table 8-2

[0159]

Table 8-3

[0160]

Table 8-4

[0161]

Table 9-1

[0162]

Table 9-2

[0163]

Table 10-1

[0164]

Table 10-2

[0165]

Table 11-1

[0166]

Table 11-2

[0167] [Table 11-3]

[0168] [Table 11-4]

[0169] [Table 12]

[0170] [Table 13-1]

[0171] [Table 13-2]

[0172] [Table 13-3]

[0173] Example 2 Construction of RSV F mutant expression vectors A nucleic acid molecule encoding a native RSV A2 F0 polypeptide, as set forth in SEQ ID NO:1, having natural substitutions P102A, I379V, and M447V, was mutated using standard molecular biology techniques to encode a precursor polypeptide of an RSV F mutant having the desired introduced amino acid mutations. The structure and components of the precursor polypeptide are shown in FIG. 1 and SEQ ID NO:3. The precursor polypeptide includes a signal peptide (residues 1-25), an F2 polypeptide (residues 26-109), a pep27 polypeptide (residues 110-136), an F1 polypeptide (residues 137-513), a T4 fibritin foldon (residues 518-544), a thrombin recognition sequence (547-552), a purification tag (HIS tag (residues 553-558)), a Strep tag II (residues 561-568), and a linker sequence (residues 514-517, 545, 546, 559, and 560).

[0174] The protein sequence of SEQ ID NO:3 was subjected to mammalian codon optimization and synthesis by DNA2.0 (Menlo Park, CA). The synthesized gene product was introduced into the commercially available expression vector pcDNA3.1 / Zeo(+) (ThermoFisher Scientific, Waltham, MA), which had been modified to encode kanamycin resistance instead of ampicillin resistance and the CAG promoter instead of the CMV promoter [Niwa, H., Yamamura, K., and Miyazaki, J., Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene, 108(2), 193-199, 1991]. Mutagenic oligonucleotides were designed with the QuikChange Primer Design algorithm (Agilent Technologies, Santa Clara, CA), and all oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). Nucleotide substitutions, insertions, and deletions were incorporated with the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies). After digestion of the original plasmid template with DpnI, the mutagenized F alleles were reamplified by polymerase chain reaction (PCR) using high-fidelity Q5 DNA polymerase (New England Biolabs, Ipswich, MA) or PrimeSTAR HS (Premix) DNA polymerase (Takara / Clontech, Mountain View, CA), and the resulting products were inserted into mammalian expression vectors using the NEBuilder HiFi DNA Assembly Kit (New England Biolabs) or Gibson Assembly Master Mix (New England Biolabs). The presence of the desired sequences was confirmed by DNA sequencing.Plasmid DNA for transfection into Expi293 cells was purified with the QIAprep Spin MiniPrep Kit (Qiagen, Valencia, CA) or EndoFree Plasmid Mega Kit (Qiagen). For all commercially available kits or reagents, procedures were performed according to the manufacturer's protocols.

[0175] Example 3 Expression and purification of RSV F protein mutants Proteins for evaluation of RSV F protein mutants were produced by transient transfection into Expi293F cells (ThermoFisher, Waltham, Mass.) of DNA constructs assembled and prepared as described in Example 2. Transient transfections were performed according to the manufacturer's protocol.

[0176] The clarified cell culture was concentrated 5-10 times using tangential flow filtration and then buffer exchanged into a buffer suitable for capture on a Ni-IMAC column. Conditioned cell culture medium containing soluble F protein was loaded onto the Ni-IMAC column. The product was eluted using increasing concentrations of imidazole. Fractions containing the product were pooled and then loaded onto a Strep-Tactin column (IBA Life Sciences, Goettingen, Germany). The product was eluted from the Strep-Tactin column using increasing concentrations of desthiobiotin. Fractions containing the product were pooled and dialyzed into the final storage buffer. Crude culture supernatant and purified protein were used for the in vitro and in vivo assays described herein.

[0177] Example 4 Stability of RSV F protein mutants The stability of the designed RSV F protein mutants was evaluated by stress tests and storage stability experiments. During the heat stress test, the crude culture supernatants of the designed mutants were incubated at 50 ° C or 60 ° C for 1 hour and examined with the pre-fusion specific monoclonal antibody D25 and the pre-fusion trimer specific antibody AM14 in an ELISA assay. The ratio of antibody reactivity of the stressed and unstressed samples is defined as the stress resistance parameter. The higher the stability of the mutant, the higher the stress resistance is expected. During the storage stability assay, the reactivity of the pre-fusion antibody in the crude culture supernatant after 1 week of storage at 4 ° C was compared with the reactivity of the fresh culture supernatant. The activity ratio is defined as the storage stability of the mutant.

[0178] The results are presented in Tables 7A-7C and 8A-8C. Stress resistance was calculated as the fractional reactivity of prefusion-specific mAbs remaining after stress ("NR" - no reactivity detected, "ND" - not determined). The most stabilizing amino acid substitutions (prefusion stability defined by the remaining reactivity of D25 after heat stress), identified from the screening of individual engineered disulfide mutants, gap-filling mutants, and electrostatic mutants, were combined into combinatorial mutants. These combinatorial mutants were also subjected to heat stress and probed with two monoclonal antibodies, D25 (prefusion-specific) and AM14 (prefusion trimer-specific). The prefusion trimer-specific quaternary epitope recognized by the AM14 antibody is significantly more sensitive to heat stress than the D25 epitope (Table 8B). Significant AM14 reactivity was not retained by any of the combination mutants after stress at 60° C., but most of the mutants retained D25 reactivity after heat stress at 60° C. This observation provides important evidence that the AM14 antibody is a much more accurate indicator of loss of prefusion structure, and in particular, loss of the prefusion trimeric state.

[0179] [Table 14-1]

[0180] [Table 14-2]

[0181] [Table 15]

[0182] [Table 16]

[0183] [Table 17]

[0184] [Table 18-1]

[0185] [Table 18-2]

[0186] [Table 19]

[0187] Example 5 Conformational integrity of RSV F protein mutants assessed with a panel of monoclonal antibodies. The goal of this study was to identify RSV F protein mutants that maintain the structural integrity of the RSV-F line fusion conformation and association, including the line fusion trimer conformation. Each mutant was tested against a panel of reference mAbs, including two site Φ-specific and line fusion specific mAbs (AM22 and D25), one mAb that binds to an epitope close to site II that is also line fusion specific (MPE8), one site II-specific mAb that binds to both pre-fusion F and post-fusion F (Palivizumab, Synagis®), one pre-fusion trimer specific mAb (AM14), and a site IV-specific antibody (101F) that binds to both pre-fusion F and post-fusion F. The RSV F protein mutants that were maintained in the line fusion conformation were expected to bind to all of the reference antibodies tested.

[0188] Antibody reactivity for each mutant was assessed using an OCTET HTX (ForteBio, Pall Corporation, Port Washington, NY) instrument that measures real-time biomolecular interaction kinetics. Experiments were performed in 96-well black plates (Greiner Bio-One, Monroe, NC) with a final volume of 200 μL per well at a temperature of 30° C. with stirring at 1000 rpm. Anti-HIS biosensor chips were equilibrated for 10 min in phosphate buffered solution (PBS), 2% bovine serum albumin (BSA), 0.05% Tween 20 (PBT) before binding measurements were started. HIS-tagged mutant proteins were captured by the anti-HIS biosensor for 5 min. A baseline was established for 3 min in PBT, followed by an association step with 20 nM antibody in PBT for 10 min. Dissociation of all antibodies was performed for 20 min in the same wells used to establish the baseline. OCTET data analysis software (version 8.2, Pall Corp.) was used for kinetic analysis, based on curve fitting for association and dissociation steps, and the assumption of a 1:1 reversible binding interaction. The binding response (nm shift) of each of the mutants with various reference monoclonal antibodies is presented in Tables 9A and 9B. Response values ​​<0.10 were considered negative and are the limit of detection (LOD) of this assay. Values ​​≥0.10 were considered positive and indicated antibody binding to the individual mutants. Varying degrees of binding were observed across mutants and for each antibody. However, 101F and Synagis bound to the majority of combination mutants, while mutants such as pXCS735 and pXCS776 showed loss of binding to at least one prefusion-specific mAb. Loss of binding indicated loss of intact prefusion conformation.

[0189] [Table 20-1]

[0190] [Table 20-2]

[0191] [Table 21-1]

[0192] [Table 21-2]

[0193] Example 6 Analysis of molecular weight and size distribution of selected prefusion RSV F mutants. The stabilized prefusion F mutants were analyzed by SDS-PAGE followed by Western blot analysis using the RSV F-specific monoclonal antibody L4 [Walsh EE, Cote PT, Fernie BF et al. Analysis of the Respiratory Syncytial Virus Fusion Protein Using Monoclonal and Polyclonal Antibodies. J. Gen. Virol. 76:505-513, 1986.]. Figure 2A shows the SDS-PAGE mobility profiles of representative mutants pXCS847, pXCS851, and pXCS852, as well as DS-Cav1. In all cases, a major band with an apparent molecular weight between 55 and 60 kDa, as expected for a monomeric RSV F mutant, was present under non-reducing conditions. The slight changes in mobility observed between DS-Cav1 and mutants pXCS847, pXCS851, and pXCS852 could be due to the nature of the individual disulfide bonds and the resulting effects on the overall compactness of the protein in the unfolded state and its accessibility to SDS.

[0194] Figure 2B illustrates the molecular weight and size distribution of mutants pXCS847, pXCS851, pXCS852, and DS-Cav1 in solution under native conditions. Molecular weight and size distribution were estimated from sedimentation velocity analysis using an analytical ultracentrifuge. The purified proteins were centrifuged at 35,000 rpm at 20 ° C, and the UV absorbance across the sample cell was monitored at 280 nm. The data was fitted to a continuous c(s) distribution assuming that the friction ratio is the same for all of the sedimenting species in the cell. All proteins sediment with a sedimentation coefficient of about 7.6S and an apparent molecular weight of about 180 kDa, indicating that the purified proteins are trimers in solution. The predicted molecular weight of the RSV F trimer calculated from its amino acid composition is 171 kDa.

[0195] Example 7 Circular dichroism spectroscopy to characterize the secondary and tertiary structural integrity of engineered RSV F protein mutants. Both far- and near-UV CD spectra were recorded on a Jasco J-810 autorecording spectropolarimeter equipped with a Peltier-type 6-position temperature-controlled cell holder. Far-UV CD spectra were recorded in 1 mm rectangular quartz cells at a protein concentration of 0.10-0.12 mg / ml in 1× PBS, pH 7.4, between 200 and 260 nm at 100 nm / min with 0.1 nm intervals and a bandwidth of 3 nm. Five spectra were collected for each sample and averaged. Near-UV CD spectra were recorded in 1 cm rectangular quartz cells at a protein concentration of 0.4-0.5 mg / ml in 1× PBS, pH 7.4, between 250 and 320 nm at 100 nm / min with 0.1 nm intervals and a bandwidth of 3 nm. Five spectra were also collected for each sample and averaged. Data were corrected for the contribution of the buffer baseline and normalized to either the mean residue ellipticity (far-UV CD) or molar ellipticity (near-UV CD) using established relationships.

[0196] The results are shown in Figures 3A and 3B. Both the far- and near-UV CD data indicate that all proteins retain well-defined secondary and tertiary structures. Furthermore, the clear similarity of the far- and near-UV CD spectra indicates that the overall secondary and tertiary structures of the mutants are similar, preserving the structural integrity of the proteins.

[0197] Example 8 Structural stability of designed RSV F protein mutants The structural stability of the purified RSV F protein mutants was characterized using differential scanning calorimetry (DSC). DSC experiments were performed on a VP-DSC microcalorimeter (MicroCal, Northampton, MA). Protein concentrations were determined by spectrophotometry and corrected for light scattering contributions. Protein samples of 0.2-0.5 mg / mL (1.0-2.4 micromolar trimer concentrations) in 1x PBS, pH 7.4 were scanned from 10°C to 80°C at 90°C / h with an 8 second response time and 5 minutes of prescan equilibration. Depending on the number of transitions seen in the thermogram, the heat capacity profiles were fitted to a two- or three-state unfolding model using Origin 7.0 software provided by the DSC manufacturer. The melting temperature of the first observable transition was taken as the melting temperature of each mutant.

[0198] DSC data shows that almost all of the designed mutants are more stable than DS-Cav1 (Table 10). The melting temperatures (defined as the DSC maximum of the first observable DSC peak in each experiment, Table 10) of all mutants (except pXCS738) are higher than DS-Cav1, up to 18 ° C. higher. The DSC data show that the computational protein design described in Example 1 successfully produced RSV F mutants that are significantly more stable and retain the prefusion conformation (Octet data, Example 5).

[0199] [Table 22]

[0200] Example 9 Mechanism of loss of prefusion trimer conformation. To characterize the specific structural pathways that lead to the loss of the prefusion conformation, we subjected purified DS-Cav1 to a heat stress test. The purified glycoprotein (0.5 mg / ml in 1x PBS, pH 7.4) was incubated at 50°C and 60°C for 30, 60, and 120 min. Binding of the prefusion-specific mAb D25 and the prefusion trimer-specific mAb AM14 to the stressed protein was assessed via ELISA experiments as described in Example 4. The structural integrity of the protein was characterized via CD and DSC as described in Examples 7 and 8, respectively. The results are shown in Figures 4-6.

[0201] The relative AM14 and D25 reactivities of the stressed samples are shown in Figure 4. Both D25 and AM14 reactivities of DS-Cav1 remain almost unchanged after up to 2 hours of incubation at 50°C. Conversely, reactivity to both prefusion-specific antibodies is gradually lost during treatment at 60°C. Moreover, AM14 reactivity is lost faster than D25 reactivity, indicating that the quaternary prefusion AM14 epitope decays faster than the D25 epitope. The results highlight the advantage of the AM14 antibody as a probe to detect loss of the prefusion trimer conformation.

[0202] DSC evaluation of unstressed DS-Cav1 (Figure 5) shows that the protein undergoes a reversible conformational transition between 50 and 60 °C. This transition does not correspond to an irreversible loss of the prefusion conformation. Moreover, this transition is not due to a global unfolding of the protein, since DS-Cav1 retains the defined far- and near-UV CD spectra (Figures 6A and 6B), indicating that the protein remains folded under these conditions. The most likely explanation for the observed DSC transition is a reversible loss of the protein's quaternary structure, i.e., at least a local dissociation of the prefusion trimer. This dissociation is necessary for the first step towards the loss of the prefusion conformation, since almost no loss of AM14 or D25 reactivity occurs before the transition occurs (Figure 4, ELISA data). These data emphasize the importance of trimer integrity for the stability of the prefusion conformation. The integrity of the trimer could only be confirmed by reactivity to the quaternary epitope-specific antibody AM14, but not to the site Φ-specific antibody D25.

[0203] These data suggest that loss of the prefusion conformation occurs via the following pathway:

[0204]

number

[0205] They slowly and irreversibly lose their prefusion conformation (3U) and eventually aggregate to form higher molecular weight species (U nNative trimers (N3) reversibly dissociate into native monomers (3N) forming 3N (Figure 7A). The dissociation of trimers then implies that DS-Cav1 would exhibit a protein concentration-dependent resistance to heat stress. That is, a decrease in total protein concentration promotes the dissociation of trimers, which in turn accelerates the loss of the prefusion conformation. Conversely, stabilized prefusion F mutants (e.g., 851) show little to no concentration-dependence of their stress resistance, if they are sufficiently stabilized. Figures 7A and 7B show further experimental evidence supporting this hypothesis. Protein samples were serially diluted and exposed to a 50°C stress for 1 h. The reactivity of AM14 and D25 remaining after stress was evaluated with ELISA assays with respect to control (unstressed) samples. The stress resistance of DS-Cav1, as determined by either AM14 antibody reactivity (Figure 7A) or D25 antibody reactivity (Figure 7B), shows a clear dependence on protein concentration. Conversely, the stress resistance of the stabilized mutants pXCS851 and pXCS898 remains broadly unchanged over the same range of protein concentrations.

[0206] Example 10 RSV F protein mutants stabilized in the prefusion conformation elicit neutralizing antibody responses in mice. Female Balb / c mice were immunized with 0.025 μg or 0.25 μg of either DS-Cav1, wild type F, F mutant pXCS852, pXCS855, pXCS830, pXCS853, pXCS780, pXCS898, pXCS851, pXCS874, pXCS881, pXCS738, or pXCS847, with or without 0.1 mg of aluminum phosphate per dose as an adjuvant. Immunizations were performed intramuscularly at weeks 0 and 3 (Table 11). Pre-dose (week 0) and post-dose 2 (PD2, week 5) sera were evaluated in an RSV subfamily A neutralization assay as described in [Eyles JE, Johnson JE, Megati S et al. Nonreplicating vaccines can protect african green monkeys from the Memphis 37 strain of respiratory syncytial virus. J Inf Dis. 208(2):319-29, 2013.] with slight modifications. Briefly, neutralizing antibody titers were determined as the serum dilution factor that reduced infectious units by 50%. Results are reported as geometric mean titers obtained from 10 mice per group. Sera with undetectable virus neutralization were assigned a titer of 20. The 50% geometric mean titer fold increase is reported as the ratio of post-dose 2 (PD2) titers to pre-immunization titers within each group.

[0207] [Table 23]

[0208] All mutants tested induced neutralizing antibody responses in mice after two immunizations (Table 12). Overall, antibody titers were consistently higher for mutants 852, 830, 851, and 847 at both antigen doses, demonstrating that these mutants were more immunogenic forms of the stabilized RSV pre-fusion F glycoprotein (Tables 12 and 13, Figure 8).

[0209] [Table 24]

[0210] [Table 25]

[0211] Comparison of PD2 50% neutralizing antibody titers with in vitro characterization data of their corresponding mutants shows a correlation between PD2 neutralizing antibody titers and AM14 heat stress resistance (Figure 9), suggesting that AM14 binding specific to the prefusion trimer state correlates with mutant immunogenicity.

[0212] Example 11 RSV F mutants containing introduced cysteine ​​mutations in the HRB region 11A. Preparation of RSV F mutants containing introduced mutations in the HRB region Representative RSV F mutants containing cysteine ​​mutations introduced in the HRB region (approximately amino acids 476-524 of the F0 polypeptide) are shown in Table 14, where the specific mutations in this region within each mutant are shown. In addition to the mutations in the HRB region, each of these mutants also contains the introduced mutations S55C, L188C, T54H, and D486S. These mutants were prepared by methods similar to those described in Examples 1-3. Briefly, a precursor polypeptide consisting of 545 amino acids was prepared for each mutant containing (1) amino acids 1-529 of the sequence of SEQ ID NO:1, except that 41 amino acids between residues 104 and 144 are deleted, (2) introduced mutations outside the HRB region (S55C, L188C, T54H, and D486S), (3) a thrombin protease recognition sequence, (4) a foldon domain, (5) a HIS tag, (6) a Streptag II, (7) a linker sequence, and (8) introduced cysteine ​​mutations as described. The signal peptide, which contains amino acids 1-25, was cleaved from the precursor during the expression process. The foldon domain was also cleaved from the mutants, which was achieved after the expression process by digestion with 500ug / ml bovine alpha-thrombin (HTI) overnight at room temperature.

[0213] [Table 26]

[0214] 11B. Stability of RSV F mutants containing introduced cysteine ​​mutations in the HRB region The stability of the RSV F mutants shown in Table 14 was evaluated according to the methods described in Examples 8 and 4. The results are presented in Tables 15 and 16, respectively.

[0215] [Table 27]

[0216] [Table 28]

[0217] Raw sequences table SEQ ID NO:1. Amino acid sequence of full-length F0 of native RSV A2 (GenBank GI:138251, Swiss Prot P03420) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYS IMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0218] SEQ ID NO:2. Amino acid sequence of full-length F0 of native RSV B (strain 18537, GenBank GI:138250, Swiss Prot P13843) MELLIHRSSAIFLTLAVNALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLG VGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNRLLPIVNQQSCRISNIETVIEFQQMNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYS IMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITTIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK

[0219] SEQ ID NO:3. RSV A2 F extracellular domain with foldon MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK

[0220] Accession number 4: RSV RSVA / Homo sapiens / USA / LA2_21 / 2013 F (Ontario) native amino acid sequence (GenBank GI: AHX57185): MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANSRARRELPRFMNYTLNNTKNTNVTLSKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0221] Accession No. 5: RSV RSVA / Homo sapiens / USA / LA2_21 / 2013 F cell extracellular domain with Foldon: MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANSRARRELPRFMNYTLNNTKNTNVTLSKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK

[0222] Accession No. 6: RSV RSVB / Homo sapiens / PER / FPP00592 / 2011F (Buenos Aires) Unmodified Amino Acid Sequence (GenBank GI: AHV80758): MELLIHRSSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLG VGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYS IMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITAIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK

[0223] SEQ ID NO: 7: RSV RSVB / Homo sapiens / PER / FPP00592 / 2011F extracellular domain with foldon: MELLIHRSSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLL GVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQ SYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASN KNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK

[0224] SEQ ID NO:8: Nucleotide sequence encoding the precursor polypeptide of pXCS738: [Related components (bp coordinates): signal sequence: 1-75, pep27: 328-408, F1: 409-1539, F2: 76-327, foldon: 1552-1632, thrombin recognition sequence: 1639-1656, His tag: 1657-1674, Streptag II: 1681-1704, linker sequence: 1540-1551, 1633-1638, 1675-1680, P102A (natural substitution): 304-306, I379V (natural substitution): 1135-1137, M447V (natural substitution): 1339-1341, T54H: 160-162, S55C: 163-165, L142C: 424-426, L188C: 562-564, V296I: 886-888, N371C: 1111-1113]

[0225] SEQ ID NO:9: Nucleotide sequence encoding the precursor polypeptide of pXCS780: [Related components (bp coordinates): signal sequence: 1-75, pep27: 328-408, F1: 409-1539, F2: 76-327, foldon: 1552-1632, thrombin recognition sequence: 1639-1656, His tag: 1657-1674, Streptag II: 1681-1704, linker sequence: 1540-1551, 1633-1638, 1675-1680, P102A (natural substitution): 304-306, I379V (natural substitution): 1135-1137, M447V (natural substitution): 1339-1341, S55C: 163-165, L188C: 562-564, D486S: 1456-1458]

[0226] SEQ ID NO:10: Nucleotide sequence encoding the precursor polypeptide of pXCS830: [Related components (bp coordinates): signal sequence: 1-75, pep27: 328-408, F1: 409-1539, F2: 76-327, foldon: 1552-1632, thrombin recognition sequence: 1639-1656, His tag: 1657-1674, Streptag II: 1681-1704, linker sequence: 1540-1551, 1633-1638, 1675-1680, P102A (natural substitution): 304-306, I379V (natural substitution): 1135-1137, M447V (natural substitution): 1339-1341, T54H: 160-162, S55C: 163-165, L188C: 562-564, S190I: 568-570]

[0227] SEQ ID NO:11: Nucleotide sequence encoding the precursor polypeptide of pXCS847: [Related components (bp coordinates): signal sequence: 1-75, pep27: 328-408, F1: 409-1539, F2: 76-327, foldon: 1552-1632, thrombin recognition sequence: 1639-1656, His tag: 1657-1674, Streptag II: 1681-1704, linker sequence: 1540-1551, 1633-1638, 1675-1680, P102A (natural substitution): 304-306, I379V (natural substitution): 1135-1137, M447V (natural substitution): 1339-1341, T103C: 307-309, I148C: 442-444, S190I: 568-570, D486S: 1456-1458]

[0228] SEQ ID NO:12: Nucleotide sequence encoding the precursor polypeptide of pXCS851: [Related components (bp coordinates): signal sequence: 1-75, pep27: 328-408, F1: 409-1539, F2: 76-327, foldon: 1552-1632, thrombin recognition sequence: 1639-1656, His tag: 1657-1674, Streptag II: 1681-1704, linker sequence: 1540-1551, 1633-1638, 1675-1680, P102A (natural substitution): 304-306, I379V (natural substitution): 1135-1137, M447V (natural substitution): 1339-1341, T54H: 160-162, T103C: 307-309, I148C: 442-444, S190I: 568-570, V296I: 886-888, D486S: 1456-1458]

[0229] SEQ ID NO:13: Nucleotide sequence encoding the precursor polypeptide of pXCS852: [Relevant components (bp coordinates): signal sequence: 1-75, pep27: 328-408, F1: 409-1539 (containing only the native RSV F sequence), F2: 76-327, foldon: 1552-1632, thrombin recognition sequence: 1639-1656, His tag: 1657-1674, Streptag II: 1681-1704, linker sequence: 1540-1551, 1633-1638, 1675-1680, P102A (natural substitution): 304-306, I379V (natural substitution): 1135-1137, M447V (natural substitution): 1339-1341, T54H: 160-162, S55C: 163-165, L188C: 562-564, D486S: 1456-1458]

[0230] SEQ ID NO:14: Nucleotide sequence encoding the precursor polypeptide of pXCS853: [Related components (bp coordinates): signal sequence: 1-75, pep27: 328-408, F1: 409-1539, F2: 76-327, foldon: 1552-1632, thrombin recognition sequence: 1639-1656, His tag: 1657-1674, Streptag II: 1681-1704, linker sequence: 1540-1551, 1633-1638, 1675-1680, P102A (natural substitution): 304-306, I379V (natural substitution): 1135-1137, M447V (natural substitution): 1339-1341, S55C: 163-165, L188C: 562-564, S190I: 568-570, D486S: 1456-1458]

[0231] SEQ ID NO: 15: Nucleotide sequence encoding the precursor polypeptide of pXCS855: [Related features (bp coordinates): signal sequence: 1-75, pep27: 328-408, F1: 409-1539, F2: 76-327, foldon: 1552-1632, thrombin recognition sequence: 1639-1656, His tag: 1657-1674, Streptag II: 1681-1704, linker sequence: 1540-1551, 1633-1638, 1675-1680, P102A (natural substitution): 304-306, I379V (natural substitution): 1135-1137, M447V (natural substitution): 1339-1341, T54H: 160-162, S55C: 163-165, L188C: 562-564, S190I: 568-570, D486S: 1456-1458]

[0232] SEQ ID NO: 16: Nucleotide sequence encoding the precursor polypeptide of pXCS874: [Related features (bp coordinates): signal sequence: 1-75, pep27: 328-408, F1: 409-1539, F2: 76-327, foldon: 1552-1632, thrombin recognition sequence: 1639-1656, His tag: 1657-1674, Streptag II: 1681-1704, linker sequence: 1540-1551, 1633-1638, 1675-1680, P102A (natural substitution): 304-306, I379V (natural substitution): 1135-1137, M447V (natural substitution): 1339-1341, S155C: 463-465, S190I: 568-570, S290C: 868-870, D486S: 1456-1458]

[0233] SEQ ID NO:17: Nucleotide sequence encoding the precursor polypeptide of pXCS881: [Related features (bp coordinates): signal sequence: 1-75, pep27: 328-408, F1: 409-1539, F2: 76-327, foldon: 1552-1632, thrombin recognition sequence: 1639-1656, His tag: 1657-1674, Streptag II: 1681-1704, linker sequence: 1540-1551, 1633-1638, 1675-1680, P102A (natural substitution): 304-306, I379V (natural substitution): 1135-1137, M447V (natural substitution): 1339-1341, T54H: 160-162, S55C: 163-165, L142C: 424-426, L188C: 562-564, V296I: 886-888, N371C: 1111-1113, D486S: 1456-1458, E487Q: 1459-1461, D489S: 1465-1467]

[0234] SEQ ID NO:18: Nucleotide sequence encoding the precursor polypeptide of pXCS898: [Related features (bp coordinates): signal sequence: 1-75, pep27: 328-408, F1: 409-1539, F2: 76-327, foldon: 1552-1632, thrombin recognition sequence: 1639-1656, His tag: 1657-1674, Streptag II: 1681-1704, linker sequence: 1540-1551, 1633-1638, 1675-1680, P102A (natural substitution): 304-306, I379V (natural substitution): 1135-1137, M447V (natural substitution): 1339-1341, T54H: 160-162, S155C: 463-465, S190I: 568-570, S290C: 868-870, V296I: 886-888]

[0235] SEQ ID NO: 19: Amino acid sequence of the precursor polypeptide of pXCS847: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPACNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLL GVGSACASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQ SYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASN KNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK [Relevant features (amino acid residue coordinates): signal sequence (absent in final product): 1-25, pep27 (absent in final product): 110-136, F1: 137-513, F2: 26-109, foldon: 518-544, thrombin recognition sequence: 547-552, His tag: 553-558, Streptag II: 561-568, P102A (natural substitution), I379V (natural substitution), M447V (natural substitution), T103C, I148C, S190I, D486S]

[0236] SEQ ID NO:20: Amino acid sequence of the precursor polypeptide of pXCS851: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPACNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLL GVGSACASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQ SYSIMSIIKEEILAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASN KNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK [Relevant features (amino acid residue coordinates): signal sequence (absent in final product): 1-25, pep27 (absent in final product): 110-136, F1: 137-513, F2: 26-109, foldon: 518-544, thrombin recognition sequence: 547-552, His tag: 553-558, Streptag II: 561-568, P102A (natural substitution), I379V (natural substitution), M447V (natural substitution), T54H, T103C, I148C, S190I, V296I, D486S]

[0237] SEQ ID NO:21: Amino acid sequence of the precursor polypeptide of pXCS852: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLL GVGSAIAGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQ SYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASN KNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK [Relevant features (amino acid residue coordinates): signal sequence (absent in final product): 1-25, pep27 (absent in final product): 110-136, F1: 137-513 (contains only the native RSV F sequence), F2: 26-109, foldon: 518-544, thrombin recognition sequence: 547-552, His tag: 553-558, Streptag II: 561-568, P102A (natural substitution), I379V (natural substitution), M447V (natural substitution), T54H (introduced mutation), S55C (introduced mutation), L188C (introduced mutation), D486S (introduced mutation)]

[0238] SEQ ID NO: 22: Amino acid sequence of the heavy chain variable domain of antibody D25: QVQLVQSGAEVKKPGSSVMVSCQASGGPLRNYIINWLRQAPGQGPEWMGGIIPVLGTVHYAPKFQGRVTITADESTDTAYIHLISLRSEDTAMYYCATETALVVSTTYLPHYFDNWGQGTLVTVSS

[0239] SEQ ID NO: 23: Amino acid sequence of the light chain variable domain of antibody D25: DIQMTQSPSSLSAAVGDRVTITCQASQDIVNYLNWYQQKPGKAPKLLIYVASNLETGVPSRFSGSGSGTDFSLTISSLQPEDVATYYCQQYDNLPLTFGGGTKVEIKR

[0240] SEQ ID NO: 24: Amino acid sequence of the heavy chain variable domain of antibody AM14: EVQLVESGGGVVQPGRSLRLSCAASGFSFSHYAMHWVRQAPGKGLEWVAVISYDGENTYYADSVKGRFSISRDNSKNTVSLQMNSLRPEDTALYYCARDRIVDDYYYYGMDVWGQGATVTVSS

[0241] SEQ ID NO: 25: Amino acid sequence of the light chain variable domain of antibody AM14: DIQMTQSPSSLSASVGDRVTITCQASQDIKKYLNWYHQKPGKVPELLMHDASNLETGVPSRFSGRGSGTDFTLTISSLQPEDIGTYYCQQYDNLPPLTFGGGTKVEIKRTV

[0242] SEQ ID NO: 26: Amino acid sequence of the heavy chain variable domain of antibody AM22 QVQLVQSGAEVKKPGATVKVSCKISGHTLIKLSIHWVRQAPGKGLEWMGGYEGEVDEIFYAQKFQHRLTVIADTATDTVYMELGRLTSDDTAVYFCGTLGVTVTEAGLGIDDYWGQGTLVTVSS

[0243] SEQ ID NO: 27: Amino acid sequence of the light chain variable domain of antibody AM22 EIVLTQSPGTLSLSPGERATLSCRASQIVSRNHLAWYQQKPGQAPRLLIFGASSRATGIPVRFSGSGSGTDFTLTINGLAPEDFAVYYCLSSDSIFTFGPGTKVDFK

[0244] SEQ ID NO: 28: Amino acid sequence of the heavy chain variable domain of antibody MPE8: EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISASSSYSDYADSAKGRFTISRDNAKTSLFLQMNSLRAEDTAIYFCARRATGYSSITPYFDIWGQGTLVTVSS

[0245] SEQ ID NO: 29: Amino acid sequence of the light chain variable domain of antibody MPE8: QSVVTQTPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYDNNNRPSGVPDRFSASKSGTSASLAITGLQAEDEADYYCQSYDRNLSGVFGTGTKVTVL

[0246] SEQ ID NO: 30: Amino acid sequence of the heavy chain variable domain of antibody 101F: QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCARLYGFTYGFAYWGQGTLVTVSA

[0247] SEQ ID NO: 31: Amino acid sequence of the light chain variable domain of antibody 101F: DIVLTQSPASLAVSLGQRATIFCRASQSVDYNGISYMHWFQQKPGQPPKLLIYAASNPESGIPARFTGSGSGTDFTLNIHPVEEEDAATYYCQQIIEDPWTFGGGTKLEIK

[0248] SEQ ID NO:32: Amino acid sequence of the precursor polypeptide of pXCS738: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLC GVGSAIAGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQ SYSIMSIIKEEILAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMCSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASN KNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK [Relevant features (amino acid residue coordinates): signal sequence (absent in final product): 1-25, pep27 (absent in final product): 110-136, F1: 137, F2: 26-109, foldon: 518-544, thrombin recognition sequence: 547-552, His tag: 553-558, Streptag II: 561-568, linker sequence: 514-517, 545-546, 559-560, P102A (natural substitution), I379V (natural substitution), M447V (natural substitution), T54H (introduced mutation), S55C (introduced mutation), L142C (introduced mutation), L188C (introduced mutation), V296I (introduced mutation), N371C (introduced mutation)]

[0249] SEQ ID NO:33: Amino acid sequence of the precursor polypeptide of pXCS780: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLL GVGSAIAGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQ SYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASN KNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK [Relevant features (amino acid residue coordinates): signal sequence (absent in final product): 1-25, pep27 (absent in final product): 110-136, F1: 137-513, F2: 26-109, foldon: 518-544, thrombin recognition sequence: 547-552, His tag: 553-558, Streptag II: 561-568, linker sequence: 514-517, 545-546, 559-560, P102A (natural substitution), I379V (natural substitution), M447V (natural substitution), S55C (introduced mutation), L188C (introduced mutation), D486S (introduced mutation)]

[0250] SEQ ID NO:34: Amino acid sequence of the precursor polypeptide of pXCS830: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLL GVGSAIAGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQ SYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASN KNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK [Relevant features (amino acid residue coordinates): signal sequence (absent in final product): 1-25, pep27 (absent in final product): 110-136, F1: 137-513, F2: 26-109, foldon: 518-544, thrombin recognition sequence: 547-552, His tag: 553-558, Streptag II: 561-568, linker sequence: 514-517, 545-546, 559-560, P102A (natural substitution), I379V (natural substitution), M447V (natural substitution), T54H (introduced mutation), S55C (introduced mutation), L188C (introduced mutation), S190I (introduced mutation)]

[0251] SEQ ID NO:35: Amino acid sequence of the precursor polypeptide of pXCS853: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLL GVGSAIAGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQ SYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASN KNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK [Relevant features (amino acid residue coordinates): signal sequence (absent in final product): 1-25, pep27 (absent in final product): 110-136, F1: 137-513, F2: 26-109, foldon: 518-544, thrombin recognition sequence: 547-552, His tag: 553-558, Streptag II: 561-568, linker sequence: 514-517, 545-546, 559-560, P102A (natural substitution), I379V (natural substitution), M447V (natural substitution), S55C (introduced mutation), L188C (introduced mutation), S190I (introduced mutation), D486S (introduced mutation)]

[0252] SEQ ID NO:36: Amino acid sequence of pXCS855 precursor polypeptide: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLL GVGSAIAGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQ SYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASN KNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK [Relevant features (amino acid residue coordinates): signal sequence (absent in final product): 1-25, pep27 (absent in final product): 110-136, F1: 137-513, F2: 26-109, foldon: 518-544, thrombin recognition sequence: 547-552, His tag: 553-558, Streptag II: 561-568, linker sequence: 514-517, 545-546, 559-560, P102A (natural substitution), I379V (natural substitution), M447V (natural substitution), T54H (introduced mutation), S55C (introduced mutation), L188C (introduced mutation), S190I (introduced mutation), D486S (introduced mutation)]

[0253] SEQ ID NO:37: Amino acid sequence of the precursor polypeptide of pXCS874: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLL GVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQ SYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASN KNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK [Relevant features (amino acid residue coordinates): signal sequence (absent in final product): 1-25, pep27 (absent in final product): 110-136, F1: 137-513, F2: 26-109, foldon: 518-544, thrombin recognition sequence: 547-552, His tag: 553-558, Streptag II: 561-568, linker sequence: 514-517, 545-546, 559-560, P102A (natural substitution), I379V (natural substitution), M447V (natural substitution), S155C (introduced mutation), S190I (introduced mutation), S290C (introduced mutation), D486S (introduced mutation)]

[0254] SEQ ID NO:38: Amino acid sequence of the precursor polypeptide of pXCS881: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLC GVGSAIAGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQ SYSIMSIIKEEILAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMCSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASN KNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSQFSASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK [Relevant features (amino acid residue coordinates): signal sequence (absent in final product): 1-25, pep27 (absent in final product): 110-136, F1: 137-513, F2: 26-109, foldon: 518-544, thrombin recognition sequence: 547-552, His tag: 553-558, Streptag II: 561-568, linker sequence: 514-517, 545-546, 559-560, P102A (natural substitution), I379V (natural substitution), M447V (natural substitution), T54H (introduced mutation), S55C (introduced mutation), L142C (introduced mutation), L188C (introduced mutation), V296I (introduced mutation), N371C (introduced mutation), D486S (introduced mutation), E487Q (introduced mutation), D489S (introduced mutation)]

[0255] SEQ ID NO:39: Amino acid sequence of the precursor polypeptide of pXCS898: MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLL GVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTIKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQ SYSIMCIIKEEILAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASN KNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHGSWSHPQFEK [Relevant features (amino acid residue coordinates): signal sequence (absent in final product): 1-25, pep27 (absent in final product): 110-136, F1: 137-513, F2: 26-109, foldon: 518-544, thrombin recognition sequence: 547-552, His tag: 553-558, Streptag II: 561-568, linker sequence: 514-517, 545-546, 559-560, P102A (natural substitution), I379V (natural substitution), M447V (natural substitution), T54H (introduced mutation), S155C (introduced mutation), S190I (introduced mutation), S290C (introduced mutation), V296I (introduced mutation)]

[0256] SEQ ID NO: 40: Amino acid sequence of T4 fibritin foldon: GYIPEAPRDGQAYVRKDGEWVLLSTFL

[0257] SEQ ID NO:271. Amino acid sequence of the precursor polypeptide of pXCS899 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRK RRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITN DQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDV SSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLGGLVPRGSHHHHHHGSWSHPQFEK

[0258] SEQ ID NO:272. Amino acid sequence of the precursor polypeptide of pXCS1106 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKA VVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPC WKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDT VSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRCCDELLHNVNAGKSTTNIMITTLVPRGSGGSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGHHHHHHGSWSHPQFEK

[0259] SEQ ID NO:273. Amino acid sequence of the precursor polypeptide of pXCS1107 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKA VVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPC WKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDT VSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLHCCNAGKSTTNIMITTLVPRGSGGSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGHHHHHHGSWSHPQFEK

[0260] SEQ ID NO:274. Amino acid sequence of the precursor polypeptide of pXCS1108 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKA VVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPC WKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDT VSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSCCNIMITTLVPRGSGGSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGHHHHHHGSWSHPQFEK

[0261] SEQ ID NO:275. Amino acid sequence of the precursor polypeptide of pXCS1109 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKA VVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPC WKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDT VSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRCCDELLHCCNAGKSTTNIMITTLVPRGSGGSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGHHHHHHGSWSHPQFEK

[0262] SEQ ID NO:276. Amino acid sequence of the precursor polypeptide of pXCS1110 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKA VVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPC WKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDT VSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRCCDELLHNVNAGKSCCNIMITTLVPRGSGGSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGHHHHHHGSWSHPQFEK

[0263] SEQ ID NO:277. Amino acid sequence of the precursor polypeptide of pXCS1111 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKA VVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPC WKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDT VSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRKSDELLHCCNAGKSCCNIMITTLVPRGSGGSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGHHHHHHGSWSHPQFEK

[0264] SEQ ID NO:278. Amino acid sequence of the precursor polypeptide of pXCS1112 MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYHCVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVCTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSSEFDASISQVNEKINQSLAFIRCCDELLHCCNAGKSCCNIMITTLVPRGSGGSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGHHHHHHGSWSHPQFEK

Claims

[Claim 1] A mutant of a wild-type RSV F protein comprising an F1 polypeptide and an F2 polypeptide, the mutant comprising at least one amino acid mutation relative to the amino acid sequence of the wild-type RSV F protein, the amino acid mutation being: (1) Engineered disulfide bond mutations; (2) gap-filling mutations, (3) electrostatic mutation, (4) a combination of at least one engineered disulfide mutation and at least one gap-filling mutation; (5) A combination of at least one engineered disulfide mutation and at least one electrostatic mutation; (6) a combination of at least one gap-filling mutation and at least one electrostatic mutation; and (7) A combination of at least one engineered disulfide mutation, at least one gap-filling mutation, and at least one electrostatic mutation. A mutant selected from the group consisting of:

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