Human metapneumovirus vaccine

Abstract

The present invention relates to a vaccine composition for preventing and / or treating a respiratory infection, such as a human metapneumovirus infection of the respiratory system, comprising one, two or more modified human metapneumovirus (hMPV) F proteins or variants thereof, provided in a prefusion fusion conformation.

Description

[Technical field]

[0001] The present invention relates to a vaccine composition for preventing and / or treating a respiratory infection, such as a respiratory human metapneumovirus infection, comprising i) one or II) two or more modified human metapneumovirus (hMPV) F proteins or variants thereof, provided in a prefusion conformation form. [Background technology]

[0002] Currently, there are no vaccines or specific antiviral drugs available to prevent or treat hMPV infection in subjects such as humans or animals. Infants, some categories of children under 5 years of age, elderly people over 65 years of age, and immunocompromised patients are at particular risk of developing severe bronchiolitis or pneumonia due to hMPV infection. However, vaccine development is difficult because, for example, the neutralizing antibody response induced by natural hMPV infection is unfortunately short-lived and declines over time, and specific memory B cell responses are weak (Falsey, AR; Hennessey, PA; Formica, MA; Criddle, MM; Biear, JM; Walsh, EE Humoral immunity to human metapneumovirus infection in adults. Vaccine 2009, 28, 1477-1480).

[0003] Phylogenetic analysis of genome sequences from various hMPV strains and clinical isolates has revealed two major genotypes (lineages), namely A and B, which are divided into subgroups (five sublineages or subgroups), A1 / A2a / A2b and B1 / B2 (van den Hoogen BG, Herfst S, Sprong L, Cane PA, Forleo-Neto E, Swart RL de, Osterhaus ADME, Fouchier RAM. (2004) Antigenic and genetic variability of human metapneumoviruses. Emerging infectious diseases 10(4):658-66).

[0004] Protection against hMPV is primarily conferred by neutralizing antibodies against the fusion (F) glycoprotein (Williams et al. (2007) A Recombinant Human Monoclonal Antibody to Human Metapneumovirus Fusion Protein That Neutralizes Virus In Vitro and Is Effective Therapeutically In Vivo. J Virol 81(15):8315-8324; Battles et al. (2017) Nat Commun. 16;8(1):1528). The F protein is immunodominant and highly conserved among hMPV strains. Rare mutations in the F protein do not result in unstable loss of neutralizing epitopes, so hMPV genotypes and subgroups are genetically very stable over time (Yang CF, Wang CK, Tollefson SJ, Piyaratna R, Lintao LD, Chu M, Liem A, Mark M, Spaete RR, Crowe JE, Jr, Williams JV. (2009) Genetic diversity and evolution of human metapneumovirus fusion protein over twenty years. Virol J 6:138). Cross-protection has been obtained between genotypes (A and B) and subgroups (A1, A2a, A2b, B1 and B2) in several animal models, but the data are controversial. For example, the induction of cross-protective immunity upon immunization with soluble F proteins isolated from genotypes A or B was demonstrated in hamsters (Nerfst et al. 2007. Journal of General Virology (2007), 88, 2702-2709). Conversely, in vitro studies performed with serum from ferrets infected with one genotype did not neutralize the virus of the other genotype (Kahn JS (2006) Epidemiology of human metapneumovirus. Clin Microbiol 19(3):546-557).The immunogenic response to different hMPV genotypes in humans is still poorly understood (Rahman et al. (2018) Epidemiological studies in Bangladesh. J Med Virology 2018:1-6). Thus, the large number of circulating hMPV variants may pose complications in developing a broad-spectrum vaccine.

[0005] The hMPV F protein mediates fusion of the viral and cellular membranes, allowing viral ribonucleoproteins to enter the cytoplasm and initiate viral replication (Cox RG, Livesay SB, Johnson M, Ohi MD, Williams JV (2012) The human metapneumovirus fusion protein mediates entry via an interaction with RGD-binding integrins. J Virol 86:12148-12160). The F protein is a type I integral membrane protein that contains a hydrophobic transmembrane (TM) domain at its C-terminus that anchors the protein to the viral membrane and a short cytoplasmic tail. The native F protein is synthesized as an inactive single-chain precursor, F0, which is activated after cleavage by cellular proteases to generate two polypeptide chains, F1 and F2 (see FIG. 1). The biologically active hMPV F protein exists in two conformations: a metastable prefusion form and a highly stable postfusion form (see Figure 2). Published crystal structures of the prefusion and postfusion forms revealed essential differences between the two conformations that may affect the immunogenic and antigenic properties of the F protein (Melero JA & Mas V. (2015) The Pneumovirinae fusion (F) protein: A common target for vaccines and antivirals. Virus Research 209: 128-135).

[0006] Several studies have shown that both pre-fusion and post-fusion F protein forms have antigenic epitopes and can elicit neutralizing antibodies (Wen et al. (2012) Structure of the Human Metapneumovirus Fusion Protein with Neutralizing Antibody Identifies a Pneumovirus Antigenic Site. Nat Struct Mol Biol. 19(4): 461-463; Battles et al. (2017) Nat Commun. 16, 8(1): 1528; Huang et al. (2019) Antibody Epitopes of Pneumovirus Fusion Proteins. Front Immunol. 10, 2778, review). For example, Melero's group demonstrated that recombinant pre-fusion F protein induces neutralizing antibodies and immunogenicity studies in animals (Melero JA & Mas V. 2015) The Pneumovirinae fusion (F) protein: A common target for vaccines and antivirals. Virus Research 209:128-135, Michael B Battles, Vicente Mas, Eduardo Olmedillas, Olga Cano, Monica Vazquez, Laura Rodriguez, Jose A Melero, Jason S McLellan. Nat Commun. 2017 Nov 16;8(1):1528. doi:10.1038 / s41467-017-01708-9).Another study showed that recombinant post-fusion F protein was able to deplete hMPV neutralizing antibodies from seropositive human sera (Mas V, Rodriguez L, Olmedillas E, Cano O, Palomo C, Terron MC, Luque D, Melero JA, McLellan JS. (2016) Engineering, Structure and Immunogenicity of the Human Metapneumovirus F Protein in the Postfusion Conformation. PLoS pathogens. 12(9)). Another group disclosed modifications of the F protein leading to stabilization of the prefusion conformation and their applicability for vaccine development (see US Pat. No. 1,0420,834).

[0007] Previously, we demonstrated the induction of high titer neutralizing antibodies and protection of mice upon immunization with a stabilized pre-fusion form of the hMPV F protein (see WO2020 / 234300A1). In this study, five F protein candidates formulated as a single immunogen showed promising protective effects in lung colonization / infection models, MNA, FFA, and / or RT-qPCR models, preferably when the appropriate subgroup candidate A1, preferably L7F_A1_23 (SEQ ID NO: 11), or a combination of one of the five F protein candidates, A1 and B1 subgroups, preferably L7F_A1_23 (SEQ ID NO: 11) and L7F_B1_23 (SEQ ID NO: 12). These improved vaccines in simple formats (only monovalent or bivalent) that are more effective against multiple hMPV strains and clinical isolates are important. To date, no attempt to combine pre-fusion conformations of the F protein in a vaccine formulation (or to test them in a monovalent format) has been described in clinical trials. Alternatively, post-fusion sF_A1_MFur is also a preferred candidate in the immunogenic composition of the present invention. Summary of the Invention

[0008] The present invention provides compositions comprising i) one or more modified recombinant hMPV F proteins or variants thereof provided in a pre-fusion conformation, or ii) a combination of one or more modified recombinant hMPV F proteins or variants thereof provided in a pre-fusion conformation. These modified recombinant proteins are derived from different hMPV genotypes A and B, or from the same genotype, but different subgroups, or both, preferably monovalent vaccines or immunogenic compositions thereof (i.e., in particular L7F_A1_23 (SEQ ID NO: 11)) and bivalent vaccines or immunogenic compositions thereof (i.e., in particular L7F_A1_23 (SEQ ID NO: 11) and L7F_B1_23 (SEQ ID NO: 12). The present invention further provides protein constructs and expression vectors for producing said modified recombinant proteins. The present invention also provides compositions capable of inducing a specific immune response, and The present invention also provides immunogenic compositions (such as vaccines) that are capable of providing protection against hMPV infection and / or cross-neutralizing and protecting against, in particular, other subgroups and / or genotypes of hMPV. Furthermore, the use of specific combinations of two or more, preferably two, pre-fusion F proteins makes it possible to achieve protection against homologous and heterologous hMPV strains. The present invention also relates to methods of producing the disclosed recombinant proteins and immunogenic compositions and to methods of using them to treat and / or prevent human or animal subjects with mild, moderate or severe hMPV infection.

[0009] The problem underlying the present invention is to develop an immunogenic composition (vaccine) that enhances a stronger and longer-lasting immune response and provides better protection against various hMPV strains and clinical isolates than known immunogenic compositions, e.g., containing a single hMPV F protein present in a pre-fusion conformation that cross-neutralizes and / or cross-protects against subgroups and / or other genotypes of the hMPV family (A1, A2a, A2b, B1, B2 subgroups, A and B of hMPV, respectively), thereby allowing simple antigen design and therefore very reasonable production costs (simpler production, simpler quality assessment, etc.).

[0010] The problem underlying the present invention is solved by providing a composition comprising only one or two different (different subgroups and / or genotypes) F proteins or variants thereof provided in pre-fusion conformation form. Moreover, such a solution comprises two or more F proteins formulated in one composition derived from different hMPV strains belonging to the same or different genotypes, but still offers a simpler design than adding all of the different subgroups in a vaccine / immunogenic composition.

[0011] To solve this problem, several F protein candidates from different hMPV genogroups and their subgroups were produced as modified (i.e. stabilized in the pre-fusion conformation) recombinant proteins and tested in several combinations with each other for immunogenicity and protective efficacy in mouse challenge models or other functional models. Specifically, mice immunized with a combination of pre-fusion F proteins or a single pre-fusion F protein from subgroups A1 and B1 were challenged with viruses of subgroups A2a, A2b and / or B1 to examine the induction of neutralizing antibodies and viral load. Alternatively, mice immunized with a combination of pre-fusion F proteins or a single pre-fusion F protein from subgroups A1 and / or B1 can be challenged with viruses of subgroups A1, A2a, A2b and / or B1. Otherwise, the protection of mice immunized with a combination of pre-fusion F proteins or a single pre-fusion F protein from subgroups A1 and / or B1 can be evaluated after challenge with hMPV subgroups A2a, A2b or B2 or other iterations. As a result, cross-protection between the two genotypes A and B and between the different subgroups is observed.

[0012] According to one embodiment, the modified (stabilized) F protein of the composition is in a pre-fusion conformation. The pre-fusion F protein consists of a single polypeptide chain similar to the F ectodomain, but lacks a protease cleavage site and a fusion peptide (FP) between the F1 and F2 domains. Instead, the single-chain F protein contains a heterologous peptide linker between the F1 and F2 domains, which contains at least one cysteine ​​residue that forms a non-native disulfide (SS) bond with another cysteine ​​residue in the F1 domain, thus stabilizing the pre-fusion conformation. Alternatively, the pre-fusion hMPV F protein may contain two polypeptide chains, i.e., the F1 and F2 domains, covalently linked by two or more SS bonds. Such proteins may contain mutations that stabilize the pre-fusion conformation.

[0013] According to yet another embodiment, the further second F protein of the composition is a modified (stabilized) F protein that also exists in a pre-fusion conformation. The pre-fusion F protein consists of a single polypeptide chain similar to the F ectodomain, but lacks a protease cleavage site and a fusion peptide (FP) between the F1 and F2 domains. Instead, the single chain F protein contains a heterologous peptide linker between the F1 and F2 domains, which contains at least one cysteine ​​residue that forms a non-native disulfide (SS) bond with another cysteine ​​residue in the F1 domain, thus stabilizing the pre-fusion conformation. Alternatively, the pre-fusion hMPV F protein may contain two polypeptide chains, i.e., the F1 and F2 domains, covalently linked by two or more SS bonds. Such proteins may contain mutations that stabilize the pre-fusion conformation. According to another embodiment of the present invention, the pre-fusion F protein may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of the parent F protein. Modified F proteins with high sequence identity to the reference parent F protein are also referred to as variants herein. Generally, protein homologs or variants have a relatively high degree of sequence identity when aligned using standard methods well known in the art (global alignment of the sequence being investigated when compared to other sequences, e.g., the Needleman-Wunsch algorithm using standard settings, is preferred). Importantly, the homologous F protein or variant is as immunogenic and protective as the parent F protein, for example, as measured using in vitro assays herein, e.g., MNA, FFA or PCR as described elsewhere herein.

[0014] Furthermore, the pre-fusion F protein of the present invention is a recombinant protein without a transmembrane domain (also referred to herein as "TM") and / or a cytoplasmic tail that is produced as a homotrimer, or preferably as a hetero- or homotrimer, in a heterologous host cell. To facilitate the trimerization process, one or more specific modifications or trimerization helper domains may be introduced into the C-terminal portion of the F protein.

[0015] According to yet another embodiment, a pre-fusion F protein is formulated in a single composition further comprising a pharma- ceutically correct carrier and / or excipient. In addition to the F protein, such a composition may contain one or more additional antigens, e.g., another hMPV protein or another antigen against another pathogen causing an infection of the respiratory system.

[0016] Typically, the compositions of the invention are immunogenic compositions (vaccines) capable of inducing hMPV neutralizing antibodies and specific T cell responses against hMPV. Optionally, the immunogenic compositions may further comprise an adjuvant to enhance such immune responses and / or to shift the immune response towards a desired Th1 type. In general, the immune response (neutralizing antibody titer) induced by the immunogenic compositions of the invention is sufficient to protect against hMPV infection. Furthermore, immunogenic compositions comprising both conformational forms of the F protein or variants thereof induce an immune response (neutralizing antibody titer) superior to that induced by an equivalent amount of a single F protein present in either of the pre-fusion conformations.

[0017] Furthermore, the immunogenic compositions of the invention may provide protection against more than one hMPV strain, particularly strains belonging to different genotypes or different subgroups of one genotype. For example, the immunogenic compositions may provide protection against the A1 and / or A2a, A2b subgroups, alternatively the B1 and / or B2 subgroups, or both genotypes A and B. In particular, cross-protection between genotype A and genotype B is desirable.

[0018] According to the present invention, the immunogenic compositions (vaccines) of the present invention are useful for the treatment and / or prevention of human and / or animal subjects against hMPV infection, although other indications such as the treatment and / or prevention of mild, severe, hospitalization or death caused by hMPV infection are also potential target indications for the compositions of the present invention.

[0019] In a further embodiment, the invention provides a method for generating an immune response using an engineered F protein or variants thereof (including combinations) available in a pre-fusion conformation, comprising administering to a subject a therapeutically effective amount of an immunogenic composition containing the pre-fusion form of the F protein.

[0020] In a further embodiment, the present invention provides a method for treating and / or preventing a subject against hMPV infection or associated disease. Thus, the immunogenic composition (vaccine) is administered to the subject via parenteral (e.g., intramuscular, intradermal, or subcutaneous) or mucosal (e.g., intranasal, oral) routes. This results in high titers of anti-F protein neutralizing antibodies that ensure protection of the immunized subject against hMPV. In a preferred embodiment, the vaccine of the present invention induces a protective immune response against more than one hMPV strain, more preferably hMPV strains of the same genotype, most preferably both genotypes A and B. In a further embodiment, the dose of the vaccine is sufficient to provide a robust anti-hMPV protection against hMPV infection. Furthermore, the method may include prime-boost immunization with the same or different immunogenic compositions comprising modified F proteins or variants thereof from different hMPV subgroups and / or genotypes. For example, the prime immunization may be with a vaccine comprising an F protein of genotype A of the invention, while the boost immunization may be with a vaccine comprising an F protein of genotype B of the invention. In this way, better cross-protection between genotype A and genotype B can be achieved. Furthermore, the method may only comprise boost immunization with the same or a different immunogenic composition comprising a modified F protein or variants thereof from a different hMPV subgroup and / or genotype, especially for elderly or adults (e.g., adults at risk), since most of these populations have already been exposed.

[0021] Furthermore, the present invention provides methods for producing recombinant modified F proteins present in a stabilized pre-fusion conformation, and immunogenic compositions comprising these proteins, comprising the steps of: i) expressing the recombinant modified F protein from a corresponding nucleic acid molecule inserted into an expression vector in a host cell, ii) purifying said recombinant F protein, and iii) combining said purified recombinant protein with a pharma- ceutically acceptable carrier and / or excipient, and optionally an adjuvant in a pharmaceutical composition or vaccine.

[0022] More specifically, the following embodiments are provided: 1. An immunogenic composition consisting essentially of a stabilized pre-fusion conformation form of human metapneumovirus (hMPV) F protein or a fragment thereof as the sole hMPV antigen, and optionally one or more adjuvants and / or at least one pharma- ceutically correct carrier or excipient, wherein said hMPV protein is from one subgroup of genotype A or B, and said immunogenic composition cross-neutralizes hMPV from another subgroup and / or genotype. 2. The composition of embodiment 1, wherein the stabilized pre-fusion conformation form of the human metapneumovirus (hMPV) F protein or fragment thereof is of the A1 subgroup. 3. The composition according to embodiments 1-2, wherein the composition consists essentially of i) a stabilized pre-fusion conformation form of a human metapneumovirus (hMPV) F protein or a fragment thereof of genotype A, and ii) a stabilized pre-fusion conformation form of a human metapneumovirus (hMPV) F protein or a fragment thereof of genotype B, and optionally one or more adjuvants and / or at least one pharma- ceutically correct carrier or excipient, wherein said immunogenic composition cross-neutralizes other subgroups and / or others. 4. The composition of embodiment 3, wherein the stabilized pre-fusion conformation form of the human metapneumovirus (hMPV) F protein or fragment thereof of genotype A is of the A1 subgroup and the stabilized pre-fusion conformation form of the human metapneumovirus (hMPV) F protein or fragment thereof of genotype B is of the B1 subgroup. 5. The immunogenic composition of any of the preceding embodiments, wherein the pre-fusion F protein is a recombinant protein. 6. The immunogenic composition of any of the previous embodiments, wherein the pre-fusion F protein lacks a cytoplasmic tail and / or a transmembrane domain. 7. The immunogenic composition of any of the previous embodiments, wherein the pre-fusion F protein has an amino acid sequence that is a modified amino acid sequence of a native F protein derived from an hMPV strain or clinical isolate. 8. The immunogenic composition of embodiment 9, wherein the native F protein sequence is selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1-10 from hMPV strains NL / 1 / 00, NL / 17 / 00, TN / 94-49, NCL174, CAN97-83, NL / 1 / 9, NDL00-1, C1-334, CAN97-82, and TN / 89-515. 9. The immunogenic composition of any of the preceding embodiments, wherein the pre-fusion F protein comprises at least one mutation (substitution or deletion), preferably up to 10 mutations, relative to the native F protein sequence of SEQ ID NOs: 1-10. 10. The immunogenic composition of any of the preceding embodiments, wherein the pre-fusion F protein comprises one or more amino acid substitutions to cysteines that introduce one or more non-native disulfide bonds that stabilize the pre-fusion conformation. 11. Cysteine ​​substitutions are Any one of positions 103 to 120 and any one of positions 335 to 345; Any one of positions 107 to 118 and any one of positions 335 to 342; Any one of positions 117 to 129 and any one of positions 256 to 261; Any one of positions 87 to 102 and any one of positions 117 to 127; Any one of positions 102 to 113 and any one of positions 117 to 127; Any one of positions 102 to 113 and any one of positions 87 to 102; Any one of positions 337 to 341 and any one of positions 421 to 426; Any one of positions 112 to 120 and any one of positions 424 to 432; Any one of positions 150 to 156 and any one of positions 392 to 400; Any one of positions 112 to 120 and any one of positions 370 to 377; Any one of positions 365 to 375 and any one of positions 455 to 465; any one of positions 365 to 375 and any one of positions 105 to 115; or introduced at any one of positions 60-70 and any one of positions 175-185; The immunogenic composition of embodiment 10, wherein the positions correspond to amino acids of the native F protein sequence of SEQ ID NOs: 1-10 and 49. 12. The immunogenic composition of any of the previous embodiments, wherein the pre-fusion F protein consists of a single polypeptide chain stabilized by at least one non-native disulfide bond. 13. The immunogenic composition of embodiment 12, wherein the single-chain pre-fusion F protein lacks a protease cleavage site between the F1 and F2 domains compared to a native F protein. 14. The immunogenic composition of embodiments 12 and 13, wherein the single-chain pre-fusion F protein comprises a substitution of arginine with another amino acid, preferably glycine, at position 102 relative to the amino acid position of the native F protein. 15. The immunogenic composition according to embodiments 12 to 14, wherein the amino acid residues at positions 103 to 118 of the native F protein are replaced with a heterologous linker consisting of 1 to 5 amino acid residues including a cysteine ​​residue, and said cysteine ​​residue forms a disulfide bond with a cysteine ​​residue in the F1 domain. 16. The immunogenic composition according to embodiment 15, wherein the heterologous linker comprises at least one alanine, glycine or valine residue, preferably the linker has the sequence CGAGA or CGAGV. 17. The immunogenic composition of embodiments 12-16, wherein the pre-fusion F protein comprises one or more substitutions at positions corresponding to positions 49, 51, 67, 80, 137, 147, 159, 160, 161, 166, 177, 258, 266, 480, and / or 481 of the native hMPV F protein. 18. The immunogenic composition of embodiment 17, wherein the substitutions are selected from the group consisting of T49M, E80N, I137W, A147V, A159V, T160F, A161M, I67L, I177L, F258I, S266D, I480C, and / or L481C. 19. The immunogenic composition of embodiments 12-18, wherein the single-chain pre-fusion F protein comprises one of the following substitution combinations: N97Q, R102G and G294E; N97Q, R102G, T160F, I177L and G294E; N97Q, R102G, T49M, I67L, A161M, E80N, F258I and G294E; N97Q, R102G, T49M, I67L, A161M, E51C, K166C, S266D, G294E, I480C, and L481C; or N97Q, R102G, T49M, A161M, I137W, A159V, A147V, I177L, and G294E. 20. The immunogenic composition according to any of embodiments 12 to 19, wherein the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 11 (L7F_A1_23). 21. The immunogenic composition according to any of embodiments 12 to 19, wherein the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 12 (L7F_B1_23). 22. The immunogenic composition according to any of embodiments 12 to 19, wherein the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 13 (L7F_A1_23.2). 23. The immunogenic composition according to any of embodiments 12 to 19, wherein the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 14 (L7F_B1_23.2). 24. The immunogenic composition according to any of embodiments 12 to 19, wherein the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 15 (sF_A1_K_L7). 25. The immunogenic composition according to any of embodiments 12 to 19, wherein the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 16 (L7F_A1_31). 26. The immunogenic composition according to any of embodiments 12 to 19, wherein the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 17 (L7F_A1_33). 27. The immunogenic composition according to any of embodiments 12 to 19, wherein the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 18 (construct L7F_A1_4.2). 28. The immunogenic composition according to any of embodiments 1 to 11, wherein the pre-fusion F protein is a protein having two polypeptide chains and comprises or consists of the amino acid sequence of SEQ ID NO: 19. 29. The immunogenic composition according to any of embodiments 1 to 11, wherein the pre-fusion F protein is a protein having two polypeptide chains and comprises or consists of the amino acid sequence of SEQ ID NO: 20. 30. The immunogenic composition according to any one of embodiments 1 to 11, wherein the stabilized post-fusion F protein comprises, compared to the amino acid positions of the native F protein, a deletion of amino acid residues at positions 103 to 111, a replacement of R102 with the linker KKRKRR, and a substitution G294E. 31. The immunogenic composition of any of embodiments 1 to 30, wherein the pre-fusion F protein i) comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20, and ii) has immunogenicity similar to the immunogenicity of the parent F protein of SEQ ID NOs: 1 to 20. 32. The immunogenic composition of any of embodiments 1 to 30, wherein the pre-fusion F protein i) comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20, and ii) has an immunogenicity equal to or similar to the immunogenicity of the parent F protein of SEQ ID NOs: 1 to 20. 33. The immunogenic composition according to any one of embodiments 1 to 30, wherein the pre-fusion F protein i) comprises an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20, and ii) its immunogenicity is equal to or similar to the immunogenicity of the parent F protein of SEQ ID NOs: 1 to 20. 34. The immunogenic composition according to any of the preceding embodiments, wherein the pre-fusion and post-fusion hMPV F proteins comprise a trimerization helper domain (foldon) having a sequence of SEQ ID NO: 23-28 or a variant thereof. 35. The immunogenic composition of any of the previous embodiments, wherein the F protein is produced as a homo- or heterotrimer. 36. The immunogenic composition of any of the previous embodiments, wherein the composition comprises an additional non-hMPV antigen. 37. The immunogenic composition of any of the previous embodiments, wherein the adjuvant is selected from the group consisting of alum, CpG, e.g., CpG1018, ODN, I-ODN, IC31®, MF59®, AddaVax™, AS03, AS01, QS21, MPL, GLA-SE, GLA-3M-052-LS, 3M-052-Alum, or combinations thereof. 38. The immunogenic composition of any of the previous embodiments, wherein the adjuvant consists of two or more adjuvants selected from the group consisting of alum, CpG, e.g., CpG1018, ODN, I-ODN, IC31®, MF59®, AddaVax™, AS03, AS01, QS21, MPL, GLA-SE, GLA-3M-052-LS and 3M-052-Alum. 39. The immunogenic composition of any of the previous embodiments, wherein the adjuvant is alum. 40. The immunogenic composition of any of the previous embodiments, wherein the adjuvant is IC31®. 41. The immunogenic composition of any of the previous embodiments, wherein the adjuvant is GLA-SE. 42. The immunogenic composition of any of the previous embodiments, wherein the adjuvant is 3M-052-alum. 43. The immunogenic composition of any of the previous embodiments, wherein the adjuvant is GLA-3M-052-LS. 44. The immunogenic composition of any of the previous embodiments, wherein the adjuvant consists of alum and CpG1018. 45. The immunogenic composition of any of the previous embodiments, wherein the adjuvant consists of alum and MPL. 46. ​​The immunogenic composition of any of the previous embodiments, wherein the adjuvant consists of alum and IC31®. 47. The immunogenic composition of any of the previous embodiments, wherein the adjuvant is AddaVax™. 48. The immunogenic composition of any of the preceding embodiments, wherein the composition is capable of eliciting neutralizing antibodies against the pre-fusion F protein. 49. The immunogenic composition of any of the preceding embodiments, wherein a composition comprising a pre-fusion protein or a combination of pre-fusion proteins provides a superior immune response (neutralizing antibody titer) compared to the immune response (neutralizing antibody titer) elicited by a composition comprising a post-fusion F protein used at the same total protein amount. 50. The immunogenic composition of any of the preceding embodiments, wherein the composition provides protection against more than one hMPV strain. 51. The immunogenic composition of any of the previous embodiments, wherein the composition provides protection against hMPV strains of genotype A. 52. The immunogenic composition of any of the previous embodiments, wherein the composition provides protection against hMPV strains of genotype B. 53. The immunogenic composition of any of the previous embodiments, wherein the composition provides protection against genotype A and genotype B hMPV strains. 54. The immunogenic composition of any of the previous embodiments, wherein the composition is a vaccine. 55. An immunogenic composition according to any of the previous embodiments for use as a medicament. 56. An immunogenic composition according to any of the previous embodiments for treating and / or preventing hMPV infection and associated diseases in a subject. 57. A method for generating an immune response against an hMPV F protein in a subject, comprising administering to the subject an effective amount of an immunogenic composition described in any of the preceding embodiments 1 to 48. 58. The method of embodiment 56, wherein the immunogenic composition is administered intramuscularly, intradermally, subcutaneously, mucosally, rectally, or orally. 59. The method according to embodiments 56 and 57, wherein the method comprises prime-boost administration of an immunogenic composition according to any one of embodiments 1 to 55, the prime-boost being performed with the same immunogenic composition. 60. The method according to embodiments 56 and 58, wherein the method comprises a prime-boost administration of the immunogenic composition according to any one of embodiments 1 to 55, wherein the prime administration is with a composition comprising an F protein of genotype A and the boost administration is with a composition comprising an F protein of genotype B, or vice versa; or the method according to embodiments 56 and 58, wherein the method comprises a boost administration of the immunogenic composition according to any one of embodiments 1 to 55, wherein the prime administration is with an immunogenic composition comprising an F protein of genotype A and the boost administration is with a composition comprising an F protein of genotype B, or vice versa. 61. A method for treating and / or preventing hMPV infection in a subject, comprising administering to the subject a therapeutically effective amount of an immunogenic composition described in any of embodiments 1 to 55 to generate neutralizing antibodies against the pre-fusion hMPV F protein and provide protection against at least one hMPV strain of genotype A or B, preferably both. 62. A method for producing an immunogenic composition according to any of embodiments 1 to 55, the method comprising: i) expressing a recombinant pre-fusion F protein from a corresponding nucleic acid molecule inserted into an expression vector in a host cell; ii) purifying the expressed recombinant F protein; and iii) combining the purified recombinant protein with a pharma- ceutically acceptable carrier and / or excipient, and optionally with an adjuvant. 63. An immunogenic composition consisting essentially of a stabilized post-fusion conformation form of human metapneumovirus (hMPV) F protein or a fragment thereof as the sole hMPV antigen, wherein said hMPV protein is from one subgroup of genotype A or B, and wherein said immunogenic composition cross-neutralizes hMPV from another subgroup and / or genotype. 64. The composition of embodiment 63, wherein the stabilized post-fusion conformational form of the human metapneumovirus (hMPV) F protein or a fragment thereof is of the A1 subgroup. 65. The composition according to embodiments 63-64, wherein the composition consists essentially of i) a stabilized post-fusion conformation form of a human metapneumovirus (hMPV) F protein or a fragment thereof of genotype A, and ii) a stabilized pre-fusion conformation form of a human metapneumovirus (hMPV) F protein or a fragment thereof of genotype B, and optionally one or more adjuvants and / or at least one pharma- ceutically correct carrier or excipient, wherein said immunogenic composition cross-neutralizes other subgroups and / or others. 66. The composition of embodiment 65, wherein the stabilized post-fusion conformational form of the human metapneumovirus (hMPV) F protein or a fragment thereof of genotype A is of the A1 subgroup, and the stabilized post-fusion conformational form of the human metapneumovirus (hMPV) F protein or a fragment thereof of genotype B is of the B1 subgroup. 67. The immunogenic composition of any of the previous embodiments, wherein the post-fusion F protein is a recombinant protein. 68. The immunogenic composition of any of the previous embodiments, wherein the post-fusion F protein lacks a cytoplasmic tail and / or a transmembrane domain. 69. The immunogenic composition of any of the previous embodiments, wherein the post-fusion F protein has an amino acid sequence that is a modified amino acid sequence of a native F protein derived from an hMPV strain or clinical isolate. 70. The immunogenic composition of embodiment 69, wherein the native F protein sequence is selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 10 from hMPV strains NL / 1 / 00, NL / 17 / 00, TN / 94-49, NCL174, CAN97-83, NL / 1 / 9, NDL00-1, C1-334, CAN97-82, and TN / 89-515. 71. The immunogenic composition of any preceding embodiment, wherein the post-fusion F protein has the amino acid sequence of SEQ ID NO: 21 (SF_A1_MFur) or SEQ ID NO: 22 (SF_B1_MFur). [Brief description of the drawings]

[0023] [Figure 1] Schematic diagram of native hMPV F protein with domains indicated: F0 - precursor protein; F1 and F2 domains; SP - signal peptide; FP - fusion peptide; HRA, HRB - heptad repeat domains A and B; TM - transmembrane domain; CYT - cytoplasmic tail; SS - disulfide bond. [Diagram 2] The three-dimensional structures (ribbon diagrams) of the F protein in (A) the pre-fusion conformation and (B) the post-fusion conformation are shown. [Diagram 3] Serum neutralizing antibody titers in mice raised against combinations of pre-fusion and post-fusion F proteins containing (A) 0.6 μg, (B) 0.2 μg, (C) 0.02 μg antigen doses per F protein. Note that the combination of pre-fusion and post-fusion F proteins contains twice the amount of antigen. Antibodies raised against the post-fusion F protein may primarily cross-protect against the pre-fusion format. Therefore, addition of the post-fusion format F protein may not be necessary. [Figure 4]Neutralization titers (challenge with A2a subgroup) induced against F protein candidates (0.02 μg per antigen) from A1 or B1 subgroups, or combinations thereof (dose per F protein). Note that the combination of pre-fusion and post-fusion F proteins contains twice the amount of antigen. Antibodies raised against the post-fusion F protein may primarily cross-protect against the pre-fusion format. Therefore, addition of a post-fusion format F protein may not be necessary. [Diagram 5] Protection of mice upon challenge with hMPV A2a subgroup: (A) FFA, (B) RT-qPCR. Note that the combination of pre-fusion and post-fusion F proteins contains twice the amount of antigen. Antibodies raised against the post-fusion F protein may primarily cross-protect against the pre-fusion format. Thus, addition of the post-fusion format F protein may not be necessary. [Figure 6] Neutralization titers induced against F protein candidates (40, 120 and 400 ng per antigen) from groups A and B in pre-fusion and post-fusion formats. (A) MNA against hMPV A1 strain; (B) MNA against hMPV B1 strain. In both assays, a dose response is observed in all groups. When mice were immunized with the pre-fusion A1 candidate, good neutralization and cross-neutralization was observed against hMPV A1 (A) and B1 (B) strains, respectively. The pre-fusion A1 candidate may also induce higher immunogenicity, thus resulting in better neutralization and cross-neutralization. Cross-neutralization against hMPV A1 strain was less effective when immunized with the pre-fusion B1 candidate. The same observation can be made for the post-fusion candidates. However, it is possible that the post-fusion candidates produce similar antibodies (i.e., neutralizing antibodies against the pre-fusion format and additional or mainly non-neutralizing antibodies against the post-fusion format part). Therefore, our interpretation at this stage is that the pre-fusion format is probably still preferred. [Figure 7]Neutralization and cross-neutralization. Overall, the neutralization titers of the pre-fusion-post-fusion combinations are weaker than those obtained with the single candidates. As previously observed, immunization with only the A1 candidate appears to show higher cross-neutralization and / or result in higher immunogenicity. In that experiment, the best combinations were pre-fusion + post-fusion A1 or pre-fusion B1 + post-fusion A1, but the neutralization titers were still lower than those in immunization with the single candidates. [Figure 8] Adjuvant effect on induction of hMPV neutralizing antibodies. Immunization of mice with vaccine L7-A1-23+sF-A1-MFur (0.2 μg per antigen) formulated with different adjuvants or without adjuvant. The combination vaccine formulated without adjuvant did not induce neutralizing antibodies. The combination vaccine formulated with different adjuvants induced neutralizing antibodies. From this experiment, all the adjuvants tested are valuable options for the formulation of hMPV vaccines based on the F protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] It is an object of the present invention to provide a hMPV subunit vaccine for treating and / or preventing subjects against multiple hMPV strains. The subunit vaccine is based on a modified hMPV F protein stabilized in one of the pre-fusion conformations by various stabilization approaches (see FIG. 1).

[0025] hMPV strains are classified into two genotypes, A and B, which are divided into subgroups A1, A2a, A2b, B1 and B2, respectively. The modified F proteins or fragments thereof disclosed herein can be derived from any hMPV strain or clinical isolate. Preferably, the two F proteins in one composition (or vaccine) belong to different subgroups of the same genotype, and even more preferably, to different genotypes. Examples of native F protein sequences from different strains are shown in Table 1. [Table 1] JPEG2024522385000002.jpg252170JPEG2024522385000003.jpg252170JPEG2024522385000004.jpg65170

[0026] In one aspect, the present invention relates to a soluble F protein that mediates the fusion of the virus with the cell membrane during the infection process. The F protein is an integral membrane protein that spans the viral membrane once and contains a cleavable signal sequence at the N-terminus, a hydrophobic TM domain at the C-terminus that anchors the protein to the membrane, and a short cytoplasmic tail (see FIG. 1). The native F protein exists in two conformational forms, pre-fusion and post-fusion (see FIG. 2). Outside the cell, the viral F protein is in an unstable globular pre-fusion conformation and refolds into an elongated post-fusion form upon contact with the cell membrane. Both F protein conformations are antigenic and share some epitopes, although some epitopes are unique to each conformation. Antibodies raised against the F protein have previously been shown to be neutralizing and play an important role in combating hMPV infection.

[0027] To produce the F protein in a stabilized pre-fusion conformation, the native F protein was modified by recombinant techniques (genetic engineering) and the DNA construct was expressed in a recombinant host.

[0028] According to one embodiment, the recombinant pre-fusion F protein was produced as a single chain polypeptide. The single chain F polypeptide has an amino acid sequence similar to that of the F ectodomain, but lacks the fusion peptide (FP) spanning amino acid residues 103-118 of the native F protein, specifically the native F protein sequence of SEQ ID NOs: 1-10 and 49. Furthermore, the single chain F polypeptide lacks a protease cleavage site between the F1 and F2 domains, which is preferably eliminated by introducing a mutation at position 102 relative to the amino acid sequence of the native F protein. More preferably, this mutation is a substitution of an arginine residue with a glycine (R102G). Furthermore, the pre-fusion F protein comprises at least one additional amino acid modification (such as a substitution, deletion or insertion), in particular at least one substitution with a cysteine. This additional cysteine ​​residue can form a non-native disulfide (SS) bond with another cysteine ​​residue that further stabilizes the pre-fusion conformation.

[0029] According to yet another embodiment, in the single chain F protein, the F1 and F2 domains are linked by a heterologous peptide linker replacing amino acids 103-118 of the native F protein. The linker comprises up to five amino acids including alanine, glycine and / or valine, and at least one cysteine. Preferably, the cysteine ​​residue is at a position corresponding to position 103 of the native F protein. Most preferably, the linker has the sequence CGAGA or CGAGV with a C at position 103. This cysteine ​​can form a disulfide bond with a cysteine ​​residue of the F1 domain.

[0030] According to a further embodiment, the cysteine ​​residue is Any one of positions 103 to 120 and any one of positions 335 to 345; Any one of positions 107 to 118 and any one of positions 335 to 342; Any one of positions 117 to 129 and any one of positions 256 to 261; Any one of positions 87 to 102 and any one of positions 117 to 127; Any one of positions 102 to 113 and any one of positions 117 to 127; Any one of positions 102 to 113 and any one of positions 87 to 102; Any one of positions 337 to 341 and any one of positions 421 to 426; Any one of positions 112 to 120 and any one of positions 424 to 432; Any one of positions 150 to 156 and any one of positions 392 to 400; Any one of positions 112 to 120 and any one of positions 370 to 377; Any one of positions 365 to 375 and any one of positions 455 to 465; any one of positions 365 to 375 and any one of positions 105 to 115; or introduced at any one of positions 60-70 and any one of positions 175-185; The positions correspond to amino acids of the native F protein sequences, in particular the native F protein sequences of SEQ ID NOs: 1-10 and 49.

[0031] According to yet another embodiment, the pre-fusion F protein comprises one or more substitutions corresponding to amino acid positions 49, 51, 67, 80, 137, 147, 159, 160, 161, 166, 177, 258, 266, 480 and / or 481 relative to the native F protein sequence, specifically the native F protein sequence of SEQ ID NOs: 1 to 10. Preferred substitutions are selected from the group consisting of T49M, E80N, I137W, A147V, A159V, T160F, A161M, I67L, I177L, F258I, S266D, I480C and / or L481C.

[0032] More preferably, the single chain pre-fusion F protein comprises one of the following combinations: N97Q, R102G and G294E; N97Q, R102G, T160F, I177L and G294E; N97Q, R102G, T49M, I67L, A161M, E80N, F258I and G294E; N97Q, R102G, T49M, I67L, A161M, E51C, K166C, S266D, G294E, I480C, and L481C; or N97Q, R102G, T49M, A161M, I137W, A159V, A147V, I177L, and G294E.

[0033] In some embodiments, the pre-fusion single chain F protein may be selected from the group consisting of, but not limited to, the following protein constructs: L7F_A1_23 (SEQ ID NO: 11), L7F_B1_23 (SEQ ID NO: 12), L7F_A1_23.2 (SEQ ID NO: 13), L7F_B1_23.2 (SEQ ID NO: 14), sF_A1_K_L7 (SEQ ID NO: 15), L7F_A1_31 (SEQ ID NO: 16), L7F_A1_33 (SEQ ID NO: 17) and / or L7F_A1_4.2 (SEQ ID NO: 18).

[0034] In particular, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 11 (L7F_A1_23 construct). In particular, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 13 (L7F_A1_23.2 construct). In particular, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 15 (sF_A1_K_L7 construct). In particular, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 16 (L7F_A1_31 construct). In particular, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 17 (L7F_A1_33 construct). In particular, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 18 (construct L7F_A1_4.2 construct). Specifically, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 12 (L7_B1_23 construct). Specifically, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 14 (L7_B1_23.2 construct).

[0035] According to another embodiment, the pre-fusion F protein consists of two polypeptide chains, i.e., separate F1 and F2 domains linked by two or more S-S bonds, preferably further comprising at least one stabilizing mutation in the F1 domain. Exemplary two-chain pre-fusion F proteins are the sF_A1_K-E294 construct (SEQ ID NO: 19) and the sF_B1_K-E294 construct (SEQ ID NO: 20).

[0036] According to yet another embodiment, the second protein of the composition disclosed herein is a stabilized modified F protein in a pre-fusion configuration. The pre-fusion F protein comprises one or more stabilizing mutations. Specifically, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 11 (L7F_A1_23 construct). Specifically, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 13 (L7F_A1_23.2 construct). Specifically, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 15 (sF_A1_K_L7 construct). Specifically, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 16 (L7F_A1_31 construct). Specifically, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 17 (L7F_A1_33 construct). Specifically, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 18 (construct L7F_A1_4.2 construct). Specifically, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 12 (construct L7_B1_23). Specifically, the pre-fusion F protein comprises or consists of the amino acid sequence of SEQ ID NO: 14 (construct L7_B1_23.2).

[0037] According to another embodiment, the pre-fusion F protein consists of two polypeptide chains, i.e., separate F1 and F2 domains linked by two or more S-S bonds, preferably further comprising at least one stabilizing mutation in the F1 domain. Exemplary two-chain pre-fusion F proteins are the sF_A1_K-E294 construct (SEQ ID NO: 19) and the sF_B1_K-E294 construct (SEQ ID NO: 20).

[0038] In yet another embodiment, the present invention provides a post-fusion F protein composition. In particular, the stabilized post-fusion F protein comprises a deletion of amino acid residues at positions 103-111, a replacement of R102 with a linker KKRKRR, and a substitution G294E, compared to the amino acid positions of the native F protein of SEQ ID NOs: 1-9. Examples of post-fusion F protein constructs are sF_A1_Mfur (SEQ ID NO: 21) and sF_B1_Mfur (SEQ ID NO: 22). Alternatively, the post-fusion constructs are sF_A2_Mfur and sF_B2_Mfur.

[0039] According to yet another embodiment, the pre-fusion F protein may comprise or consist of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 11-22, the percentage sequence identity being determined over the entire length of the parent sequence by using the Needleman-Wunsch algorithm (Needleman&Wunsch.(1970) A general method applicable to the search for similarities in the amino acid sequence of two proteins.J Mol.Biol.48:443-453). Otherwise, the sequence identity percentage is determined by dividing the number of matches by the length of the sequence shown in the identified sequence or by a distinct delimited length (e.g., 100 consecutive nucleotides or amino acid residues from a sequence shown in the identified sequence) and then multiplying the resulting value by 100. Preferably, the sequence identity percentage is determined over the entire length of the sequence. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0% identical to the test sequence (1166÷1554×100=75.0). The sequence identity percentage value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, and 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. Protein homologs and variants are typically characterized as having at least about 75% sequence identity, counted over at least 50, 100, 150, 250, 500 amino acid residues of the reference sequence, over the entire length of the reference sequence, or over the entire length alignment with the reference amino acid sequence. Importantly, such homologous proteins or protein variants have immunogenicity and protective efficacy comparable to that of the parent F protein having any of the sequences of SEQ ID NOs: 11 to 22, and comparable immunogenicity is demonstrated by ELISA (IC 50 value) and / or neutralization assay (PRNT50 value) with the readout within a + / - 50% margin, preferably + / - 40%, more preferably + / - 30%, 20% or 10% margin.

[0040] In a further embodiment, the pre-fusion F protein of the invention does not have a transmembrane domain and a cytoplasmic tail. Nevertheless, it can be produced as a homo- or heterotrimer. Trimerization can occur due to the sequence spanning residues 480-495 of the native F protomer, but trimerization can be promoted by introducing modifications in this region. One modification involves the substitution of the nearby residues I480 and L481 with cysteines, which allows the introduction of three disulfide bonds across the three protomers in the form of a covalent ring. Another modification is the insertion of a trimerization helper, the so-called foldon domain. The addition of the trimerization helper supports the formation of stable and soluble protein trimers. The availability of a cysteine ​​ring in the foldon domain allows the formation of disulfide bonds that create a covalent bond between the three protomers. In one embodiment, the foldon domain has the sequence of SEQ ID NO: 23 derived from fibritin of the T4 bacteriophage, or a modified sequence containing one or more N-glycosylation sites (motif NxT / S, where "x" is any amino acid residue except proline) that helps to hide hMPV non-specific epitopes. Examples of such modified foldon sequences are those of SEQ ID NOs: 24-28. Alternatively, variants of the foldon domain may contain structural elements from the GCN4 leucine zipper (Harbury et al. 1993. Science 262:1401) or monomers of self-assembling nanoparticles, such as ferritin or lumacine synthase. Furthermore, linkers may be used in combination with a cleavage site introduced, for example, by replacement of the A496 residue. Non-limiting examples of short linkers are GG, SG, GS, GGG, GGA, GGS, SGG, SSG, SGS, SGA, GGA, SSA and SGGS.

[0041] In yet another embodiment, the foldon domain is attached to the C-terminus of the F protein, replacing the transmembrane and cytoplasmic domains. In this case, the N-terminal glycine residue of the foldon domain is attached to the C-terminus of the F1 domain, either directly or via a peptide linker which may contain at least one protease site. For example, the foldon domain can be attached via a "VSL" (SEQ ID NO: 29) or "VSA" (SEQ ID NO: 30) linker. Such linkers may be used in combination with a protease cleavage site, such as a thrombin cleavage site, a TEV (tobacco etch virus protease) or a factor Xa cleavage site. Such foldons may have the sequences of SEQ ID NOs: 42-47.

[0042] In some embodiments, to facilitate purification of recombinant proteins, the single chain polypeptide may contain any purification tag sequence known in the prior art. Examples of polypeptides that aid in purification include, but are not limited to, His tag, myc tag, S peptide tag, MBP tag, GST tag, FLAG tag, thioredoxin tag, GFP tag, BCCP, calmodulin tag, streptavidin tag, HSV-epitope tag, V5-epitope tag, and CBP tag. Preferably, the F protein of the present invention comprises His and / or streptavidin tag.

[0043] In yet another embodiment, the present invention provides an isolated nucleic acid molecule encoding a recombinant hMPV F protein of SEQ ID NO: 11-22 disclosed herein. In certain embodiments, the nucleic acid encoding the protein of the present invention comprises or consists of a sequence of SEQ ID NO: 31-40. In another embodiment, the nucleic acid encoding the hMPV F protein may include one or more modifications, such as substitutions, deletions, or insertions. In some embodiments, the present application also encompasses nucleic acid molecules encoding proteins having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 11-22. Preferably, the nucleic acid sequence exhibits about 80-100% (or any value therebetween) sequence identity to the polynucleotide sequence of SEQ ID NO: 31-40. Sequence identity can be determined by sequence alignment programs and parameters well known to those of skill in the art. Such tools include the BLAST suite for local alignment (Altschul SF, et al. 1997. "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402). General global alignment can be performed by using the Needleman-Wunsch algorithm (Needleman & Wunsch. 1970. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol. Biol. 48: 443-453).

[0044] In further embodiments, the nucleic acids described herein may contain additional nucleotide sequences that code for segments that can be used to enhance the formation of protein trimers (so-called foldon domains) or segments that can be used to enhance the purification of expressed proteins (purification tags). In some embodiments, the nucleic acids disclosed herein may have codon-optimized sequences. The procedure known as "codon optimization" is described, for example, in U.S. Pat. No. 5,547,871. The degeneracy of the genetic code allows for the modification of the nucleotide sequence of the F protein while still producing a polypeptide with the same amino acid sequence as the polypeptide encoded by the native polynucleotide sequence.

[0045] Further, according to one embodiment, the pre-fusion and post-fusion F proteins disclosed herein are recombinant proteins produced in a heterologous host cell. The production of recombinant proteins can be achieved by any suitable method, including, but not limited to, transient and / or stable expression of protein coding sequences in prokaryotic or eukaryotic cell culture. Protein-encoding (polynucleotide) constructs are conveniently prepared using standard recombinant techniques (see, for example, Sambrook et al., supra). The polynucleotide sequences encoding the proteins disclosed herein can be included in one or more vectors that are introduced into the host cell where the recombinant protein is expressed. Non-limiting examples of vectors that can be used to express sequences encoding the proteins of the invention include viral vectors (e.g., retrovirus, adenovirus, alphavirus, baculovirus or vaccinia virus), plasmid vectors, yeast vectors, insect vectors, mammalian vectors or artificial vectors. Many suitable expression systems are commercially available. An expression vector typically includes a coding sequence and expression control elements that allow expression of the coding sequence in a suitable host cell. The present invention provides an expression system designed to aid in the expression and presentation of isolated polypeptides. The present application also provides host cells for the expression of recombinant hMPV proteins. In one embodiment, the host cell may be a prokaryote, such as E. coli. In another embodiment, the host cell may be a eukaryotic cell, such as, but not limited to, selected from the group consisting of EB66® (Valneva SE), Vero, MDCK, BHK, MRC-5, WI-38, HT1080, CHO, COS-7, HEK293, Jurkat, CEM, CEMX174, and myeloma cells (e.g., SB20 cells), many of which are available from the ATCC. Particularly preferred cell lines for the production of the pre-fusion F proteins of the present invention are CHO cell lines. Cell lines expressing one or more of the above proteins can be readily generated by stably integrating one or more expression vectors encoding the proteins into a constitutive or inducible promoter.Selection of appropriate growth conditions and media is within the skill of the art.

[0046] Methods for producing the recombinant proteins disclosed herein or isolated nucleic acid (DNA or RNA) molecules encoding those proteins are incorporated into the present disclosure. In particular, methods for purifying recombinant proteins are included. Non-limiting examples of suitable purification from cell culture medium procedures include centrifugation and / or density gradient centrifugation (e.g., sucrose gradients), filtration, pelleting, and / or column or batch chromatography, including ion exchange, affinity, size exclusion and / or hydrophobic interaction chemistry, tangential filtration, etc. Such methods are known to those skilled in the art and are described, for example, in Protein Purification Applications: A Practical Approach (ELV Harris and S. Angal., Eds., 1990).

[0047] In a further embodiment, the F protein of the invention may be derived from any hMPV strain or clinical isolate belonging to any one of the two genotypes A and B, or to subgroups A1, A2, B1 or B2.

[0048] In a further embodiment, the invention provides a composition comprising one F protein, in particular a composition comprising an F protein present in a pre-fusion conformation. In general, the F protein may be from any hMPV strain or clinical isolate. In one embodiment, the composition of the invention comprises an F protein from genotype A or B, i.e., subgroups A1 and A2a, A2b (alternatively, B1 and B2). In another preferred embodiment, the composition of the invention comprises an F protein from subgroup A1 (see also Table 2).

[0049] In a further embodiment, the invention provides a composition comprising a combination of at least two F proteins, in particular compositions comprising F proteins present in a pre-fusion conformation. In general, the F proteins may be from any hMPV strain or clinical isolate. In one embodiment, the composition of the invention comprises F proteins from the same genotype A or B, but from different subgroups, in particular subgroups A1 and A2a, A2b (alternatively B1 and B2). In another embodiment, the composition of the invention comprises F proteins from different genotypes A and B, for example subgroups A1 (or A2a, A2b) and B1 (or B2). Preferably, the combination is an F protein of subgroup A1 with an F protein of B1 or B2.

[0050] In one particular embodiment, the combination comprises a pre-fusion F protein from genotype A, in particular from subgroup A1 or subgroup A2a, A2b, alternatively from both subgroups A1 and A2. In another embodiment, the combination comprises a pre-fusion F protein from genotype B, in particular from subgroup B1 or subgroup B2, alternatively from both subgroups B1 and B2. In yet another embodiment, the combination comprises pre-fusion F proteins from different genotypes A and B. In particular, the pre-fusion F protein is from subgroup A1 (or A2a, A2b) and the pre-fusion F protein is from subgroup B1 (or B2). Alternatively, the pre-fusion F protein is from subgroup B1 (or B2) and the pre-fusion F protein is from subgroup A1 (or A2a, A2b). More particularly, compositions that are part of the invention, comprising combinations of pre-fusion F proteins, are listed in Table 2. [Table 2]

[0051] In further embodiments, the immunogenic compositions of the invention may provide protection against more than one hMPV strain, particularly strains belonging to different genotypes or different subgroups of one genotype. For example, the immunogenic compositions may provide protection against the A1 and / or A2a, A2b subgroups, alternatively the B1 and / or B2 subgroups, or both genotypes A and B. In particular, cross-protection between genotype A and genotype B is desirable.

[0052] In a further embodiment, the present invention provides a pharmaceutical composition comprising a combination of two recombinant F proteins available in pre-fusion conformation form. Typically, the pharmaceutical composition further comprises at least one pharma- ceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers are used to formulate hMPV F proteins for clinical administration. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of immunogens. In general, the nature of the carrier will depend on the particular mode of administration to be used. For example, parenteral formulations usually contain an injectable, physiologically acceptable fluid, such as water, saline, balanced salt solution, aqueous dextrose, glycerol, or the like, as a vehicle. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, e.g., sodium acetate or sorbitan monolaurate. In certain embodiments, carriers suitable for administration to a subject are sterile and / or suspended or otherwise contained in a unit dosage form containing one or more measured doses of a composition suitable for inducing the desired anti-hMPV immune response. The unit dosage form may be, for example, in a sealed vial or syringe for injection, or lyophilized for subsequent solubilization and administration, or may be a solid or controlled release dosage form.

[0053] In some embodiments, the immunogenic composition (or vaccine) may further comprise an adjuvant, which refers to any substance used to specifically or non-specifically enhance an antigen-specific immune response, possibly through the activation of antigen-presenting cells.Non-limiting examples of adjuvants include aluminum salts (often referred to as "alum") such as aluminum hydroxide or aluminum phosphate (described in WO2013 / 083726), oil emulsions (such as complete or incomplete Freund's adjuvant), Montanide incomplete adjuvants from Seppic such as ISA51, squalene-based oil-in-water emulsion adjuvants such as MF59® (Seqirus) (Ott G. et al. 1995. Pharm Biotechnol 6:277-96), AddaVax™ (InvivoGen), monophosphoryl lipid A (MPL) (Cluff CW. 2010. Adv Exp Med Biol 667:111-23), glucopyranosyl lipid adjuvant (GLA) (Coler RN et al. Development and characterization of synthetic glucopyranosyl lipid adjuvant system as a vaccine adjuvant. PLoS One. 2011, 6(1):e16333), Toll-like receptor 7 / 8 agonists such as 3M-052 (described in Zhao BG, et al. Combination therapy targeting toll like receptors 7,8 and 9 eliminates large established tumors. J Immunother Cancer. 2014 May 13;2:12), polycationic peptides such as polyarginine (polyR) or peptides containing at least two LysLeuLys motifs, in particular KLKLLLLLKLK (described in WO02 / 32451), immunostimulatory oligodeoxynucleotides (CpG ODN) containing unmethylated cytosine-guanine dinucleotides, e.g., CpG 1018 (Dynavax) (described, for example, in WO 96 / 02555), or inosine and cytidine-based ODNs (I-ODNs) such as poly IC (described, for example, in WO 01 / 93903), or deoxynucleic acids containing deoxy-inosine and / or deoxyuridine residues (described in WO 02 / 95027), in particular oligo(dIdC). 13system adjuvant IC31® (Valneva SE) (described in WO 2004 / 084938 and Olafsdottir et al. 2009. Scand J Immunol. 69(3):194-202), neuroactive compounds, in particular human growth hormone (described in WO 01 / 24822), chemokines (e.g. defensin 1 or 2, RANTES, MIP1-α, MIP-2, interleukin-8, or cytokines (e.g. interleukin-1β, -2, -6, -10 or 12; interferon-γ; tumor necrosis factor-α; or granulocyte-monocyte-colony stimulating factor), muramyl dipeptide (MDP) variants, non-toxic variants of bacterial toxins, QS-21 (Antigenics Inc.), Quill A, Sarkar et al. Adjuvant systems include MTP-PE and others described in Giudice et al. (2019), as well as AF03, AS01, AS03 and AS04 (Giudice et al. 2018. Seminars in Immunology 39:14-21). The selection of an appropriate adjuvant usually depends on the type of B or T cell immune response desired for a particular vaccine (Sarkar et al. (2019) Selection of adjuvants for vaccines targeting specific pathogens. Expert Rev Vaccines 18(5):505-521). In general, adjuvants that deliver immunological signals through TLR3, TLR4, TLR7, TLR8, and TLR9 receptors promote Th1-biased immunity, whereas signaling through TLR2 / TLR1, TLR2 / TLR6, and TLR5 promotes Th2-biased immunity. For example, CpG Adjuvants such as ODN, Poly IC and MPL induce predominantly Th1 responses, alum is a strong inducer of Th2 responses, while MF59®, AddaVax™ and IC31® induce mixed Th1 and Th2 responses.Preferred adjuvants useful in the vaccines of the invention may be selected from alum, CpG ODN, such as, but not limited to, CpG1018 (Dynavax), Poly IC, IC31® (Valneva), MF59® (Seqirus), AddaVax™, AS03 (GSK), AS01 (GSK) or QS21 (Pfizer), or combinations thereof. Aluminum adjuvants particularly useful in the invention contain less than 350 ppb of heavy metals (such as Cu, Ni, W, Co, Os, Ru, Cd, Ag, Fe, V, Cr, Pb, Rb and Mo), particularly less than 1.25 ppb of copper (particularly Cu), based on the weight of the aqueous immunogenic composition. + or Cu 2+ In some embodiments, aluminum adjuvants, particularly aluminum adjuvants containing more than 1.25 ppb Cooper or more than 350 ppb heavy metal, may be used in combination with a radical quenching compound such as L-methionine, present in sufficient amounts, particularly in a concentration of at least 10 mmol / l in the immunogenic composition. In some embodiments, the immunogenic composition comprising an aluminum adjuvant may further comprise a reactive compound selected from the group consisting of redox-active compounds, radical building compounds, stabilizing compounds, and any combination thereof, in particular the reactive compound is selected from the group consisting of formaldehyde, ethanol, chloroform, trichloroethylene, acetone, Triton X-100, Triton X-114, deoxycholate, diethylpyrocarbonate, sulfite, Na2S2O5, beta-propiolactone, polysorbates such as Tween 20®, Tween 80®, O2, phenol, pluronic type copolymers, and any combination thereof. The adjuvant may be formulated together with the antigen in one immunogenic composition or may be administered separately from the antigen, either by the same route or a different route.

[0054] In some embodiments, the immunogenic compositions (or vaccines) disclosed herein may contain one or more additional antigens, preferably viral proteins derived from hMPV, such as another F protein or a different hMPV protein. Possibly, the inclusion of additional hMPV proteins in F protein-based vaccines can result in improved (more balanced and robust) immune responses. Among the different hMPV proteins, the M protein has been described as being able to modulate humoral and cellular immune responses (especially the Th1 / Th2 balance), thereby providing an adjuvant effect in mice when the M protein is combined with the F protein (Aerts et al. 2015. Adjuvant effect of the human metapneumovirus (HMPV) matrix protein in HMPV subunit vaccines. J Gen Virol. 96(Pt4):767-774). Thus, in one embodiment, the immunogenic compositions described herein contain a recombinant hMPV M protein to enhance the protection conferred by the vaccine. The recombinant M protein may comprise the amino acid sequence of SEQ ID NO:41 or a fragment thereof, or a variant thereof having at least 80% sequence identity to the parent M protein. Preferably, the recombinant M protein of the present invention consists of the amino acid sequence of SEQ ID NO:41.

[0055] Additional hMPV proteins could be the surface glycoprotein G or the small hydrophobic protein SH. Despite the fact that antibodies induced against the G and SH proteins do not protect against hMPV infection in animal models (Skidopoulus et al. (2006) Individual contributions of the human metapneumovirus F, G, and SH surface glycoproteins to the induction of neutralizing antibodies and protective immunity. Virology 345: 492-501; Ryder et al., (2010) Soluble recombinant human metapneumovirus G protein is immunogenic but not protective. Vaccine 28(25): 4145-4152), it could be suggested that these antigens could contribute to protection in humans. Furthermore, the high genetic diversity between genotype A and genotype B of these proteins could be important for immunological prevention, so both genotypes need to be represented in the vaccine.

[0056] In some embodiments, the additional antigen may be derived from another virus that causes a respiratory infection, such as RSV (respiratory syncytial virus), PIV3 (parainfluenza virus type 3), influenza virus, or coronavirus (SARS-CoV, SARS-CoV-2, MERS, etc.). For example, the additional antigen may be RSV F protein, PIV3F protein, influenza hemagglutinin, or coronavirus S protein. Such immunogenic compositions (vaccines) are protective against more than one virus, representing combination vaccines against respiratory infections.

[0057] In a further embodiment, the composition of the invention is an immunogenic composition or vaccine comprising at least two immunogenic hMPV F proteins, in particular a combination of two F proteins available in the pre-fusion conformation. Typically, the immunogenic composition or vaccine is capable of eliciting an antigen-specific immune response against the immunogenic proteins. The immune response can be humoral, cellular, or both. The humoral response results in the production of F protein-specific antibodies by the mammalian host upon exposure to the immunogenic composition. The F protein-specific antibodies are produced by activated B cells. The production of neutralizing antibodies depends on the activation of specific CD4+ T cells. In addition, protection against hMPV infection is achieved by CD8 T cells that cooperate synergistically with CD4+ T cells. + There is evidence that T cells (CTL response) may be used (Kolli et al. (2008) T Lymphocytes Contribute to Antiviral Immunity and Pathogenesis in Experimental Human Metapneumovirus Infection. JOURNAL OF VIROLOGY, Sept. 2008, p. 8560-8569). Thus, the immunogenic composition or vaccine of the present invention, when administered to a subject, induces a measurable B cell response (such as production of antibodies) against the hMPV F protein and / or a measurable CTL response against the hMPV virus.

[0058] According to the present invention, the immunogenic composition is capable of eliciting antibodies against both conformations of the F protein, i.e. pre-fusion and fusion. Preferably, the anti-F protein antibodies are neutralizing antibodies capable of interfering with the native F antigen present in any (or both) conformation and inactivating the virus. Most preferably, the neutralizing antibody response induced in the immunized subject is sufficient to combat hMPV infection. The neutralizing antibody response can be measured in serum by ELISA and / or PRNT and / or MNA methods or any other method known in the art.

[0059] Furthermore, the immune response (e.g., neutralizing antibody titers) generated against a composition comprising two F proteins in pre-fusion and post-fusion conformations is superior to the immune response (neutralizing antibody titers) elicited by a composition comprising a single (pre-fusion) F protein used in the same amount as a composition comprising the combination disclosed herein. Furthermore, the synergistic effect of combining two immunogenic F proteins in one composition can make the immunogenic composition (or vaccine) more potent than a single F protein composition (or vaccine), allowing for reduced therapeutic or prophylactic doses.

[0060] In one embodiment, the immunogenic composition or vaccine can reduce the severity of symptoms and / or reduce the viral load associated with hMPV infection in a subject at the time of administration compared to a control. In another embodiment, the immunogenic composition or vaccine can reduce or prevent hMPV infection. In a preferred embodiment, the immunogenic composition or vaccine of the invention can protect an immunized mammalian subject against hMPV infection.

[0061] Furthermore, the immunogenic compositions of the invention can provide protection against more than one hMPV strain, particularly different hMPV subgroups or genotypes. In one embodiment, the immunogenic compositions can provide protection against viruses of genotype A. In yet another embodiment, the immunogenic compositions can provide protection against viruses of genotype B. In preferred embodiments, the immunogenic compositions described herein are protective against both genotypes A and B. In certain embodiments, the immunogenic compositions can provide protection against the A1 and / or A2a / A2b subgroups, alternatively against the B1 and / or B2 subgroups, or against both genotypes A and B. In preferred embodiments, cross-protection between genotypes A and B is feasible.

[0062] In further embodiments, the present invention includes a combination of an immunogenic composition or vaccine disclosed herein with a different hMPV vaccine or another respiratory vaccine, such as an anti-RSV, PIV3, influenza or coronavirus (e.g., SARS-CoV, SARS-CoV-2, MERS, etc.) vaccine. In particular, the combination may include an hMPV vaccine comprising a recombinant hMPV pre-fusion / post-fusion F protein and an alternative subunit hMPV vaccine, or an hMPV vaccine based on whole virus or VLP particles. Furthermore, the combination may include a recombinant hMPV F protein vaccine and an RSV vaccine, or a recombinant hMPV F protein vaccine and a PIV3 vaccine, or a recombinant hMPV F protein vaccine and an influenza vaccine, or a recombinant hMPV F protein vaccine and a coronavirus (especially an anti-SARS-CoV-2) vaccine, as disclosed herein. Preferably, the combination includes a recombinant hMPV F protein vaccine and a recombinant RSV F protein vaccine as disclosed herein. In one embodiment, a combination is understood as a combination of separate vaccine preparations administered simultaneously or subsequently by the same or different routes. In another embodiment, the two vaccines are combined in a single formulation.

[0063] In another embodiment, the immunogenic compositions disclosed herein may be used as drugs or vaccines, particularly for treatment and / or prevention in mammalian subjects, particularly in the context of diseases associated or related to hMPV infection. Thus, the immunogenic compositions (or vaccines) described herein are administered to a subject in a therapeutically effective amount. A therapeutically effective amount is an amount of the disclosed immunogen or immunogenic composition sufficient to prevent, treat (including prevent), reduce and / or alleviate symptoms and / or underlying causes of a disorder or disease, e.g., sufficient to prevent, inhibit and / or treat hMPV infection. In some embodiments, a therapeutically effective amount is sufficient to reduce or eliminate symptoms of a disease, such as hMPV infection. For example, this may be an amount necessary to inhibit or prevent viral replication or to measurably change the outward symptoms of a viral infection. Generally, this amount is an amount sufficient to measurably inhibit viral replication or infectivity. Typically, the desired immune response inhibits, reduces or prevents hMPV infection. In one embodiment, the infection does not need to be completely eliminated, reduced or prevented for the method to be effective. For example, administration of a therapeutically effective amount of an agent can reduce infection (as measured by infection of cells or the number or percentage of infected subjects) by, for example, at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100% as compared to a suitable control. In another embodiment, complete elimination or prevention of detectable hMPV infection is desired. Further target indications are selected from the group consisting of mild respiratory disease, severe respiratory disease, hospitalization, and / or death caused by hMPV infection.

[0064] The pharmaceutical compositions (or vaccines) disclosed herein may be administered by any means and route known to those skilled in the art. In some embodiments, the compositions (vaccines) may be formulated for parenteral administration by injection. As used herein, "parenteral" administration includes, but is not limited to, subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrathecal, or by infusion. In some embodiments, the compositions may be formulated for mucosal (intranasal or oral) administration. Injectable formulations may be presented in unit dosage form, e.g., in ampoules or multi-dose containers, with added preservatives.

[0065] It should be understood that multiple administrations of the immunogenic composition may be necessary to obtain a protective immune response against hMPV. Thus, a therapeutically effective amount encompasses fractional doses that, in combination with previous or subsequent administrations, contribute to achieving a protective immune response. For example, a therapeutically effective amount of the agent can be administered in a single dose or in several doses, e.g., daily, during a course of treatment (such as a prime-boost vaccination regimen). However, the therapeutically effective amount may depend on the subject being treated, the severity and type of the condition being treated, and the mode of administration. Unit dosage forms of the agent can be packaged in a therapeutic amount or in a multiple of the therapeutic amount, e.g., in a vial (e.g., with a pierceable cap) or a syringe with a sterile component.

[0066] According to the present invention, the administration regimen needs to be adjusted to provide the optimal desired response. In general, the effective dose of the compositions disclosed herein for prophylactic and / or therapeutic treatment can vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, age, whether the patient is human or non-human, other drugs administered, whether the treatment is prophylactic or therapeutic, etc. According to the present invention, the amount of F protein in a unit dose can be anywhere within a broad range of about 0.01 μg to about 100 mg, and in particular, the compositions of the present invention can be administered in an amount ranging from about 1 μg to about 10 mg, in particular from about 10 μg to about 1 mg. Preferably, the dose of the antigen preparation needs to be titrated to optimize safety and efficacy.

[0067] In further embodiments, the invention provides a method for generating an anti-hMPV immune response in a subject comprising administering to a subject in need thereof a therapeutically effective amount of an immunogenic composition. The method comprises stimulating B cells to produce F protein-specific antibodies and cytokine-producing T helper cells to protect said subject from hMPV infection or related disease. In some cases, such methods may comprise prime-boost administration of an immunogenic composition. In other cases, such methods may comprise boosting administration of an immunogenic composition of the invention. The booster effect refers to an increase in the immune response to an immunogenic composition when the mammalian host is subsequently exposed to the same or similar immunogenic composition. For example, priming comprises administration of a composition comprising an F protein of genotype A and boosting comprises administration of a composition comprising an F protein of genotype B, or vice versa. Alternatively, prime-boost immunization uses the same composition (homologous boosting), in particular a mixed composition comprising F proteins of both genotypes A and B.

[0068] In a further embodiment, the present disclosure provides a method for treating and / or preventing hMPV infection in a subject, comprising administering to the subject a therapeutically effective amount of an immunogenic composition to generate neutralizing antibodies and provide protection against hMPV of one genotype A or B, preferably against both genotypes A and B of hMPV.

[0069] In further embodiments, the disclosure provides methods for producing pharmaceutical (immunogenic) compositions, including vaccines, for use in the invention, comprising: i) expressing a recombinant pre-fusion or post-fusion F protein from a corresponding nucleic acid molecule inserted into an expression vector in a host cell, ii) purifying the recombinant F protein, and iii) combining the purified recombinant protein with a pharma- ceutically acceptable carrier and / or excipient, and optionally an adjuvant.

[0070] The pharmaceutical (immunogenic) compositions of the present invention, including vaccines, can be produced according to methods well known and routinely practiced in the art (see, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co. 20th ed. 2000, and fact sheets on vaccine ingredients, e.g., adjuvants, enhancers, preservatives, and stabilizers, from the Centers for Disease Control and Prevention). The compositions disclosed herein are preferably manufactured under GMP conditions. The compositions of the present invention, including vaccines, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0071] The invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or carried out in various ways. Moreover, the phraseology and terminology used herein are for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," "having," "containing," "involving," and variations thereof herein means the inclusion of the items listed thereafter and equivalents thereof, as well as additional items.

[0072] The methods and techniques of this disclosure are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are described herein.

[0073] The present invention is further illustrated by the following examples, figures, tables, and sequence listing from which additional features, embodiments, and advantages may be taken, but which should in no way be construed as further limiting. EXAMPLES

[0074] Example 1: Production of recombinant pre-fusion and post-fusion F proteins KK The native hMPV F protein can be selected from any hMPV strain and any serotype represented by the sequences of SEQ ID NOs: 1-10, or fragments or variants thereof. In certain embodiments, the hMPV F protein is derived from strain NL / 1 / 00, genotype A, subgroup A1, represented by SEQ ID NO: 1, strain TN / 94-49, genotype A, subgroup A2a, represented by SEQ ID NO: 2, strain NCL174, genotype A, subgroup A2b, represented by SEQ ID NO: 4, strain C1-334, genotype B, subgroup B1, represented by SEQ ID NO: 9, or strain CAN97 / 82, genotype B, subgroup B1, represented by SEQ ID NO: 49, and strain TN / 98-515, genotype B, subgroup B2, represented by SEQ ID NO: 10.

[0075] Expression vector The plasmid used for cloning, pVVS 1371, contains: - HS4 insulator sequence from the chicken β-globin locus, - 2 CMV promoters, - Two chimeric introns downstream of the CMV promoter, consisting of a 5' donor site from the first intron of the human β-globin gene and a branch point and 3' acceptor site from an intron of the immunoglobulin gene heavy chain variable region. The sequences of the donor and acceptor sites, together with the branch point site, were adapted to match the consensus sequence for splicing. The introns are located upstream of the cDNA insert to prevent the use of possible cryptic 5' donor splice sites within the cDNA sequence. - bovine growth hormone polyadenylation signal sequence (bGH A), - the neomycin phosphotransferase gene from Tn5 under the control of the SV40 enhancer and early promoter region, - The HSV TK polyadenylation signal of the thymidine kinase gene of herpes simplex virus is located downstream of the neomycin phosphotransferase gene. - a kanamycin resistance gene under the control of a bacterial promoter, and - pUC origin of replication.

[0076] The coding sequence for the wild-type F protein was isolated from hMPV strain NL / 1 / 00, subgroup A1, and codon-optimized for expression in CHO cells. The coding sequences for the wild-type and modified F proteins were cloned into the pVVS1371 plasmid for transient or stable protein expression in CHO cells.

[0077] Briefly, the coding sequence was cloned between the chimeric intron and the polyadenylation site bGH of the pVVS1371 vector using the restriction sites SalI and PacI. The vector and the synthesized coding sequence (synthesis was performed by GeneArt) were purified on an agarose gel after digestion with SalI and PacI. The fragments were ligated with T4 DNA ligase and the ligation products were used to transform MAX Efficiency DH5α competent cells. Selected clones were checked for the designed mutations by sequence analysis.

[0078] Expression in CHO cells Protein expression was based on transient transfection of CHO cells using the MaxCyte® STX Scalable Transfection System device and following the supplier's experimental recommendations. Briefly, prior to electroporation, CHO cells were pelleted, suspended in MaxCyte® electroporation buffer and mixed with the corresponding expression plasmid DNA. The cell-DNA mixture was transferred to the cassette handling assembly and loaded into the MaxCyte® STX Scalable Transfection System. Cells were electroporated using the "CHO" protocol preloaded in the device, immediately transferred to culture flasks and incubated at 37 °C with 8% CO2 for 30-40 min. After a recovery period, cells were resuspended at high density in EX-CELL ACF CHO medium (Sigma-Aldrich). Cell culture after electroporation was performed at 37 °C with 8% CO2 and orbital shaking.

[0079] The production kinetics consisted of lowering the culture temperature to 32 °C and feeding the transfected cells daily with a fed-batch medium developed for transient protein expression in CHO cells (CHO CD EfficientFeed™ A (ThermoFischer Scientific), supplemented with yeast acetate, glucose and glutaMax). After approximately 7-14 days of cultivation, cell viability was checked and the conditioned medium was harvested after cell clarification, corresponding to two centrifugations for 10 min at maximum speed. The clarified product was filtered through a 0.22 μm sterile membrane and stored at -80 °C until protein purification.

[0080] Protein detection by intracellular immunostaining Seven days after transfection, cells were washed once in PBS and fixed for 10 min in 4% paraformaldehyde at room temperature. Fixed cells were permeabilized in BD Perm wash solution for 15 min at room temperature and incubated with primary antibodies diluted in BD Perm wash solution for 1 h at 4 °C. Finally, secondary antibodies conjugated to fluorescent markers were added for 1 h at 4 °C and stored in PBS at 4 °C until analysis by flow cytometry (MacsQuant Analyzer, Miltenyi Biotec). As primary antibodies, MPE8 N113S antibody (PRO-2015-026-01), which specifically recognizes the pre-fusion conformation of hMPV F protein, or DS7 IgG1 antibody (PRO-2016-003), which recognizes both pre- and post-fusion hMPV F proteins, were used. Fluorescent FITC-labeled secondary antibody was goat anti-mouse IgG+IgM (JIR 115-096-068).

[0081] Protein purification The frozen supernatant was brought to room temperature and dialyzed against PBS using a standard grade regenerated cellulose dialysis membrane Spectra / Por® 1-7CR (MWCO: 3.5 kDa) (Spectrum), then equilibrated with 50 mM Na2HPO4 buffer (pH 8.0), 300 mM NaCl, and the protein was purified using immobilized metal ion affinity chromatography (IMAC) followed by gel filtration chromatography.

[0082] For IMAC, Ni 2+Agarose resin containing (His GraviTrap) was packed into a chromatography column by the manufacturer (GE Healthcare). The resin was washed with 2 volumes of deionized water and equilibrated with 3 volumes of equilibration and wash buffer (20 mM sodium phosphate, pH 7.4, containing 0.5 M sodium chloride and 20 mM imidazole) as suggested by the manufacturer. After loading the sample, the column was washed with 10 mL of wash buffer. His-tagged proteins were eluted from the column using 3-10 column volumes of elution buffer (50 mM sodium phosphate, pH 8.0, containing 0.5 M sodium chloride and 500 mM imidazole) as indicated by the manufacturer. The eluate was then filtered through a 0.22 μm filter and dialyzed twice against storage buffer (50 mM Na2HPO4, 300 mM NaCl, 5 mM EDTA, pH 8.0) in a Slide-A-Lyzer™ cassette before being divided into aliquots and stored at -20 °C. Purity, size and aggregation analysis of the recombinant proteins was performed by size-exclusion chromatography (SE-HPLC) and SDS-PAGE SE-HPLC (Shimadzu) run on a SUPERDEX200 (GE Healthcare) column.

[0083] Example 2: Conformation of recombinant hMPV F protein Determination of conformational profiles by sandwich ELISA Medium binging plates (Greiner) were coated with 200 ng / well of human IgG1 DS7 capture antibody (Williams et al., 2007) and incubated overnight at 4°C. After washing three times with water, the plates were saturated with PBS, 0.05% Tween 20 and 5% nonfat dry milk (saturation buffer) for 2 h at 37°C under agitation. After removing the liquid from the wells and washing three times with water, the plates were incubated for 1 h at 37°C with purified protein of interest (2.5 ng / well) diluted in saturation buffer. After washing, five-fold serial dilutions in saturation buffer of mouse antibody MPE8 N113S against pre-fusion hMPV F protein (Corti et al., 2013) or mouse antibody MF1 against post-fusion hMPV F protein (Melero, personal communication) were incubated for 1 h at 37°C. Immune complexes were then detected using a secondary α-Ig species-specific antibody conjugated with peroxidase-HRP goat anti-mouse IgG (Covalab, experiment no. 0252) followed by 50 μL of peroxidase substrate (TMB, Sigma) by incubation for 1 h at 37° C. The colorimetric reaction was stopped by adding 3N H2SO4 and the absorbance of each well was measured at 490 nm using a spectrophotometer (MultiSkan).

[0084] Example 3: Immunogenicity studies Immunogenicity in mice Groups of 5-10 BALB / c mice were immunized subcutaneously with recombinant pre- and post-fusion F proteins, used alone or in different combinations, in amounts of 0.02-6.0 μg per mouse, with or without adjuvant, at 2- or 3-week intervals (e.g., days 0, 14, 21, and 28 or 42). One to four weeks after the last immunization, blood was collected by retro-orbital bleeding and serum was prepared. Evaluation of immune responses was performed by indirect ELISA as described below.

[0085] Subclass IgG ELISA Recombinant F protein was diluted in carbonate / bicarbonate buffer (pH 9.6) and 50 ng of protein per well was added to a 96-well high-binding plate (50 μL / well, Greiner). The plate was incubated overnight at 4° C. The wells were saturated with 150 μL of PBS, 0.05% Tween 20 and 5% dry skim milk (saturation buffer) for 30 min at room temperature. The liquid was removed from the wells and the plate was incubated for 1 h at room temperature with different dilutions (5-fold serial dilutions) of immunized mouse serum (50 μL / well) in saturation buffer. After 3 washes with PBS, 0.05% Tween 20, 50 μl of secondary anti-IgG1 or IgG labeled with peroxidase was added. 2a Immune complexes were detected by incubation with mouse-specific antibodies followed by 50 μL of peroxidase substrate (TMB, Sigma) for 1 h at room temperature. The colorimetric reaction was stopped by adding orthophosphoric acid and the absorbance of each well was measured at 490 nm using a spectrophotometer (MultiSkan). As a readout, IC was used to evaluate the specific antibody titer. 50 The value was calculated.

[0086] Example 4: Induction of neutralizing antibodies Neutralization assay Briefly, a microneutralization assay (MNA) was used to determine the serum / antibody titers of immunized subjects required to reduce the number of hMPV viral plaques by 50% compared to control serum / antibody (MNA 50 ) by using a monolayer of cells that can be infected with hMPV. 50 was performed. Serum from subjects was diluted and incubated with live hMPV virus. Viral infection was determined using an HRP-labeled anti-F protein specific monoclonal antibody. 50 / MNA 50A neutralizing antibody threshold of 1:10 dilution of serum in immunized patients was generally accepted as evidence of protection (Hombach et al. 2005. Vaccine 23:5205-5211). Neutralizing antibodies provide the best evidence that protective immunity has been established, and biological assays of neutralization show a correlation with protection (Hombach et al., 2005).

[0087] Immunization and challenge protocol hMPV viruses of the A1 (NL / 1 / 00 strain), A2 (TN / 94-49 strain), B1 (C1-334 strain), or B2 (TN / 89-515 strain) subgroups grown in LLC-MK2 cells (ATCC CCL-7) were used for animal challenge experiments as previously described (Williams et al. 2005. The cotton rat (Sigmodon hispidus) is a permissive small animal model of human metapneumovirus infection, pathogenesis, and protective immunity. Journal of virology 79:10944-10951).

[0088] BALB / c mice were immunized three times at 2-week intervals with adjuvanted recombinant F protein as described above, and 2 weeks after immunization, approximately 1 × 10 6 Intranasal challenge with pfu of hMPV. After 4-5 days, animals were sacrificed and individual serum samples were collected and frozen. To measure viral load, lung tissue samples were collected, weighed, and homogenized in 1 mL of medium. Viral load in lung tissue was determined by immunostaining of viral foci as described below. Alternatively or additionally, RT-qPCR was used to determine lung viral load.

[0089] MNA Protocol On day 1, LLC MK2 cells grown in OptiMEM containing 2% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (Anti-Anti) were cultured at 2 × 10 5Cells were seeded at a density of 100 μL / well in flat-bottom 96-well plates and incubated overnight at 37°C / 5% CO2. On day 0, serum samples were diluted in OptiMEM containing 100 μM CaCl2 and 1% Anti-Anti in U-bottom 96-well plates. Sample dilutions were mixed 1:1 with the virus later, so dilutions at 2x concentration should be prepared. To control wells without virus, medium was added instead of 2x virus dilutions. Dilutions of hMPV A1 virus, a trypsin-independent strain, were prepared in OptiMEM containing 100 μM CaCl2 and 1% Anti-Anti in U-bottom 96-well plates according to the experimental setup. Virus dilutions were mixed 1:1 with the diluted serum samples later, so virus samples were prepared at 2x concentration (e.g., 120 pfu / 60 μL). Blank wells were filled with medium. For hMPV B1 virus and all other trypsin-dependent hMPV strains, trypsin (i.e., TrypLE) is added to the medium to aid infection, ranging from 8 to 50 mrPu / mL depending on serum concentration. For neutralization, equal volumes (60 μL) of serum and virus dilutions were mixed (final concentration: 120 pfu / 120 μL) and the samples were incubated for 1 h at room temperature. Flat-bottom 96-well plates containing LLC MK2 cells were washed once with 150 μL / well of PBS. After removing the PBS, 100 μL of the pre-incubated serum:virus mixture was transferred to the plates containing LLC MK2 cells and incubated at 37 °C / 5% CO2 for 5 days. On the fifth day, 150 μL of neutral buffered formalin solution was added per well and the plates were incubated for 1 h at room temperature. The plates were washed twice with 300 μL / well of PBS and aspirated. 100 μL / well of permeabilization buffer (PBS containing 0.5% Tween® 20) was added and the plate was incubated for 30 minutes at 4° C. After aspiration of the permeabilization buffer, 100 μL / well of blocking buffer (PBS containing 0.5% Tween® 20 and 10% nonfat milk) was added and the plate was incubated for 1 hour at 4° C.HRP-labeled antibody (DS7 mIgG2a) was diluted to a concentration of 0.5 μg / mL in blocking buffer (see above) and 50 μL of antibody solution was added per well after aspiration of blocking buffer. The plate was then incubated for 1 h at 37° C. / 5% CO2 before being washed 6 times with 200 μL / well of PBS using an ELISA washer. 100 μL of TMB substrate was added per well and incubated for about 10 min at room temperature. The reaction was stopped with 50 μL of 1 M sulfuric acid per well and the absorbance was measured at 450 nm.

[0090] To test combinations of pre-fusion / post-fusion F proteins, pre-fusion L7F_A1_23 or L7F_B1_23 candidates and post-fusion sF_A1_Mfur or sF_B1_Mfur candidates were selected. The following compositions (combinations) of pre-fusion and post-fusion F proteins were examined for induction of hMPV neutralizing antibodies (see Table 3). [Table 3]

[0091] In six experiments performed in mice (see Table 4), each mouse was immunized with either a single F protein or the combination vaccine. Mouse sera were used to test for neutralizing antibody titers, performed by microneutralization assay (MNA) as described above. The results of these experiments are shown in Figures 3(A-C), 4, and 6(A). [Table 4]

[0092] The data shown in Figures 3 and 4 demonstrate that the combination of the pre-fusion construct L7F_A1_23 and the post-fusion construct sF_A1_Mfur used at an amount of 0.02 μg per antigen per dose showed an approximately 5-fold improvement in neutralization titers compared to the single F proteins (see Figures 3C and 4). At higher antigen doses of 0.2 μg and 0.6 μg, the synergistic effect of the pre-fusion and post-fusion F proteins was less pronounced (see Figures 3A and 3B). It is also clear from these experiments that the combination of two F proteins from the A1 subgroup is protective against challenge with viruses of the same genotype A, especially the A2 subgroup.

[0093] The data shown in Figure 6 (experiment 4763) demonstrated a dose response for all groups in both assays. Good neutralization and cross-neutralization against hMPV A1 (panel A) and B1 (panel B) was observed when mice were immunized with the pre-fusion A1 candidate, but cross-neutralization against the hMPV A1 strain was less effective when immunized with the pre-fusion B1 candidate. The pre-fusion A1 candidate may also induce higher immunogenicity.

[0094] The data shown in Figure 7 (experiment 4775) demonstrated that, overall, neutralization titers from combination candidates were weaker than those obtained with single candidates. As previously observed, immunization with the A1 candidate alone appears to show higher cross-neutralization (very weak neutralization against A1 in mice immunized with the B1 candidate). In this experiment, the best combination was pre-fusion-post-fusion A1, again suggesting that the A1 candidate is preferred for cross-neutralization. However, as previously mentioned, neutralization titers were still lower than immunization with single candidates.

[0095] Example 5: Protection in mice Protection of mice upon immunization with different pre-fusion / post-fusion F protein compositions was evaluated in a mouse lung infection model.

[0096] Immunization and challenge protocol BALB / c mice were immunized three times at 2-week intervals with adjuvanted recombinant F protein as described above, and 2 weeks after immunization, approximately 1 × 10 6 An intranasal challenge is performed with pfu of hMPV. After 4-5 days, animals are sacrificed and lungs are harvested and frozen. To determine viral load, lung tissue samples are collected, weighed, and homogenized in 1 mL of medium. Viral load in lung tissue is determined by immunostaining of viral foci, as described below. Additionally, RT-qPCR is used to determine lung viral load.

[0097] Immunostaining of viral plaques (foci) The assay for quantification of hMPV foci was developed based on methods published in Williams et al., 2005. J Virology 79(17):10944-51, Williams et al., 2007. J Virology 81(15):8315-24, and Cox et al., 2012. J. Virology 86(22):12148-60. Briefly, confluent cultures of Vero or LLC-MK2 cells in 24-well plates are infected with 125 μL / well of lung homogenate diluted in medium. After 1 h of incubation at 37°C / 5% CO2, an overlay containing 1.5% methylcellulose in medium is added. On day 6 post-infection, the supernatant is removed and the cells are washed twice with PBS. The cell monolayers are fixed and stained with DS7 antibody (mouse IgG 2a ) Count the foci and acquire cell images on a Zeiss microscope using a 2.5x or 10x objective or using a BioReader 6000. Immunostaining results are expressed as focus forming units per milliliter, or FFU / mL.

[0098] RT-qPCR protocol RNA is extracted from 140 μL of lung homogenate using the QIAamp Viral RNA Mini Kit according to the manufacturer's instructions, and the RNA is eluted in 60 μL. RT-qPCR is performed using the iTaq™ Universal Probes One-Step Kit (Bio-Rad). For amplification of the N gene, the following primers (e.g., forward 5'-CATATAAGCATGCTATATTAAAAGAGTCTC-3' and reverse 5'-CCTATTTCTGCAGCATATTTGTAATCAG-3') and probe (e.g., FAM-TGYAATGATGAGGGTGTCACTGCGGTTG-BHQ1) are used. The reaction volume for RT-qPCR is 20 μL using 400 nM of each primer, 200 nM of probe, and 4 μL of RNA. Reverse transcription and amplification are performed using the CFX96 Touch Deep Well Real-Time PCR System (Bio-Rad) with the conditions listed in Table 5. [Table 5]

[0099] The amount of hMPV RNA is calculated using Bio-Rad's CFX maestro program against known full-length hMPV RNA standards with known concentrations included in each run.

[0100] As used herein, clearance or reduction of hMPV infection can be determined by any method known in the art. In some embodiments, the level of hMPV infection in a subject is determined by detecting the presence of the virus, for example, by real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR).

[0101] The first question addressed in this study is to compare the protective efficacy after vaccination of a composition comprising a recombinant single F protein used in either pre- or post-fusion form with a composition comprising a combination of pre- and post-fusion F proteins. The second question addressed is to evaluate the optimal antigen dose of a composition containing a combination of pre-fusion / post-fusion F proteins. The third question to be addressed herein is to establish cross-protection between different hMPV genotypes and / or subgroups.

[0102] To assess protective efficacy, mice immunized with any of the compositions shown in Table 4 were challenged with strains TN / 94-49 (A2 subgroup) or C1-334 (B1 subgroup).

[0103] To assess the level of protection, lung infection was assessed by FFA and RT-qPCR. The results are shown in Figure 5 (A, B), Figure 6 (B, C), and Figure 7 (A, B). Notably, Figure 5A shows that mice immunized with a combination of pre- and post-fusion F proteins from the A1 subgroup and challenged with the A2 strain developed the lowest levels of foci indicative of lung infection. Unfortunately, no such significant effect was observed when using PT-qPCR (see Figure 5B), with FFA measuring live virus and RT-qPCR most likely measuring viral RNA (live and dead virus) detectable even in the absence of live virus, at a time when the dead virus has not yet been cleared from the lungs.

[0104] The different combinations shown in Table 4 at doses of 40 ng, 120 ng and 400 ng were tested in challenge experiments with either the A1, A2 or B1 strains, the results of which are shown in Figures 6 (B, C) and 7 (A, B). Very similar results are seen between the different combinations in terms of pulmonary infection with TN94-49 (subgroup A2) and C1-334 (subgroup B1) as determined by focus formation assay (Figures 6B and 7B) and RT-qPCR (data not shown).

[0105] Example 6: Effects of immunostimulation BALB / c mice were immunized three times at 2-week intervals with the adjuvanted recombinant F protein vaccine as described above. Two weeks after the last immunization, blood was collected by retro-orbital bleeding and serum was prepared. The immune response was evaluated by microneutralization assay (MNA) as described above.

[0106] To examine the impact of immunostimulation on hMPV vaccine efficacy, one exemplary combination of pre-fusion / post-fusion F proteins L7F_A1_23 and sF_A1_Mfur was tested. [Table 6]

[0107] Mice were immunized with three doses of the compositions as shown in Table 5. Mice were then challenged with hMPV strain, genotype subgroup A1. Serum was collected and used in the MNA assay for evaluation of neutralizing antibody titers.

[0108] As a result, all tested adjuvants showed enhanced production of neutralizing antibodies against the homologous hMPV in mice. At the same time, no neutralizing antibodies were detected in the absence of adjuvants. The adjuvants alum + MPL and IC31 high + The combination of pre-fusion and post-fusion F proteins formulated with Alum produced the greatest amount of neutralizing antibodies. A slightly weaker effect was observed with the 3M-052-Alum and AddaVax formulation. The results are shown in Figure 8.

[0109] From these experiments, none of the adjuvants tested can be excluded from further testing in other animal species and in humans. array SEQ ID NO:11 Purified tagged L7F_A1_23 protein sequence MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIEQPRQSGCGATAGVAIAKTIRLESEVTAIKNAL KKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLIGVYGSSVIYMVQ LPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQL NKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSAGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK L7F_A1_23 mature protein sequences MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIEQPRQSGCGAGATAGVAIAKTIRLESEVT AIKNALKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLI GVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACY KGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSAGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO:12 Purified tagged L7F_B1_23 protein sequence MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVSADQLAREEQIEQPRQSGCGATAGIAIAKTIRLESEVNAIKGAL KQTNEAVSTLGNGVRVLATAVRELKEFVSKNLTSAINRNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGILIWGYGSSVIYMVQ LPIFGVIDTPCWIIKAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNWVGIIKQL PKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRVPSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNAGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK L7F_B1_23 mature protein sequences MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVSADQLAREEQIEQPRQSGCGAGATAGIAIAKTIRLESEVN AIKGALKQTNEAVSTLGNGVRVLATAVRELKEFVSKNLTSAINRNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGILI GVYGSSVIYMVQLPIFGVIDTPCWIIKAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACY KGVSCSIGSNWVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNAGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO:13 Purified tagged L7F_A1_23.2 protein sequence MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIEQPRQSGCGAGVTAGVAIAKTIRLESEVTAIKNAL KKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLIGVYGSSVIYMVQ LPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQL NKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSAGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK L7F_A1_23.2 mature protein sequence MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIEQPRQSGCGAGVTAGVAIAKTIRLESEVT AIKNALKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLI GVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACY KGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSAGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO:14 Purified tagged L7F_B1_23.2 protein sequence * L7F_B1_23.2 mature protein sequence MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVSADQLAREEQIEQPRQSGCGAGVTAGIAIAAKTIRLESEVN AIKGALKQTNEAVSTLGNGVRVLATAVRELKEFVSKNLTSAINRNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGILI GVYGSSVIYMVQLPIFGVIDTPCWIIKAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACY KGVSCSIGSNWVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNAGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO:15 Purified tagged sF_A1_K_L7 protein sequence MSWKVVIIFSLLITPQHGLKESYLEESSCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIEQPRQSGCGAGATAGVAIAKTIRLESEVTAIKNALKK TNEAVSTLGNGVRVLAFAVRELKDFVSKNLTRALNKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAQQIKLMLENRAMVRRKGFGFGLIGVYGSSVIYMVQLPI FGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGC SYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAESAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK sF_A1_K_L7 mature protein sequence LKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIEQPRQSGCGAGATAGVAIAKTIRLESEVTAIKNALKKTNEAVST LGNGVRVLAFAVRELKDFVSKNLTRALNKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLIGVYGSSVIYMVQ LPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGS NRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAESAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO:16 Purified tagged L7F_A1_31 protein sequence MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFMLEVGDVENLTCADGPSLLKTELDLTKSALRNLRTVSADQLAREEQIEQPRQSGCGAGATAGVAIAKTIRLESEVTAIKNAL KKTNEAVSTLGNGVRVLATMVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQ LPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQL NKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSAGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK L7F_A1_31 mature protein sequence without purification tag LKESYLEESCSTITEGYLSVLRTGWYTNVFMLEVGDVENLTCADGPSLLKTELDLTKSALRNLRTVSADQLAREEQIEQPRQSGCGAGATAGVAIAKTIRLESEVTAIKNALKKTNEAVS TLGNGVRVLATMVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIY MVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVS CSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSAGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO:17 Purified tagged L7F_A1_33 protein sequence MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFMLCVGDVENLTCADGPSLLKTELDLTKSALRELRTVSADQLAREEQIEQPRQSGCGAGATAGVAIAKTIRLESEVTAIKNAL KKTNEAVSTLGNGVRVLATMVRELCDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLIGVYGSDVIYMVQ LPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQL NKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRCCSAGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK L7F_A1_33 mature protein sequence without purification tag LKESYLEESCSTITEGYLSVLRTGWYTNVFMLCVGDVENLTCADGPSLLKTELDLTKSALRELRTVSADQLAREEQIEQPRQSGCGAGATAGVAIAKTIRLESEVTAIKNALKKTNEAVS TLGNGVRVLATMVRELCDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLIGVYGSDVIY MVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVS CSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRCCSAGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO:18 Purified tagged L7F_A1_4.2 protein sequence MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFMLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIEQPRQSGCGAGATAGVAIAKTIRLESEVTAWKNALKK TNEVVSTLGNGVRVLVTMVRELKDFVSKNLTRALNKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLIGVYGSSVIYMVQLPI FGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGC SYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAESAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK L7F_A1_4.2 mature protein sequence without purification tag LKESYLEESCSTITEGYLSVLRTGWYTNVFMLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIEQPRQSGCGAGATAGVAIAKTIRLESEVTAWKNALKKTNEVVST LGNGVRVLVTMVRELKDFVSKNLTRALNKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLIGVYGSSVIYMVQ LPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGS NRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAESAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO:19 sF_A1_K-E294 Two polypeptide chain protein sequences with trimerization helper KLL and purification tag MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIENPRQSRFVLGAIALGVCTAAAVTAGVAIAKTIRLESEVT AIKNALKTNEAVSTLGNGVRVLAFAVRELKDFVSKNLTRALNKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLIGVYGSSVIYM VQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTACGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNK GCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAESAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK SEQ ID NO:20 sF_B1_K-E294 Two polypeptide chain protein sequences with trimerization helper KLL and purification tag MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSRFVLGAIALGVCTAAAVTAGIAIAKTIRLESEVN AIKGALKQTNEAVSTLGNGVRVLAFAVRELKEFVSKNLTSALNRNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYM VQLPIFGVIDTPCWIIKAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTACGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNWVGIIKQLPK GCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAESAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK Purification tag-free SF_B1_K-E294 MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSRFVLGAIALGVCTAAAVTAGIAIAAKTIR LESEVNAIKGALKQTNEAVSTLGNGVRVLAFAVRELKEFVSKNLTSALNRNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGI LIGVYGSSVIYMVQLPIFGVIDTPCWIIKAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTACGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNWVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAESAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO:21 Purified tagged sF_A1_MFur protein sequence stabilized in the post-fusion conformation MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIENPRQSKKRKRRVATAAAVTAGVAIAKTIRLESEVTAIK NALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLIGVYGSSVIYMVQL PIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGC SYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTSGRENLYFQGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGRHHHHHH sF_A1_MFur without purification tag MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIENPRQSKKRKRRVATAAAVTAGVAIAKTIRLESEVT AIKNALKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGFLIGVYGSSV IYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVG IIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTSGRENLYFQGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO:22 Purified tagged sF_B1_MFur protein sequence stabilized in the post-fusion conformation MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSKKRKRRVATAAAVTAGIAIAKTIRLESEVNAIKGAL KQTNEAVSTLGNGVRVLATAVRELKEFVSKNLTSAINRNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVI DTPCWIIKAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNWVGIIKQLPKGCSYITNQDAD TVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAEKGNTSGRENLYFQGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK SF_B1_MFur without purification tag MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSKKRKRRVATAAAVTAGIAIAKTIRLESEVN AIKGALKQTNEAVSTLGNGVRVLATAVRELKEFVSKNLTSAINRNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSV IYMVQLPIFGVIDTPCWIIKAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNWVG IIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAEKGNTSGRENLYFQGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO:23 Trimerization helper domain (foldon) from fibritin of bacteriophage T4. GYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO:24 Foldon-glyc-1 GYIPEAPRNGTAYVRKDGEWVLLSTFL SEQ ID NO:25 Foldon-glyc-2 GYIPEAPRDGQAYVRKNGTWVLLSTFL SEQ ID NO:26 Foldon-glyc-3 GYIPEAPRDGQAYVRKDGNWTLLSTFL SEQ ID NO:27 Foldon-glyc-4 GYIPEAPRNGTAYVRKNGTWVLLSTFL SEQ ID NO:28 Foldon-glyc-5 GYIPEAPRNGTAYVRKDGNWTLLSTFL SEQ ID NO:29 Trimerization helper VSL motif ILSA SEQ ID NO:30 Trimerization helper VSA motif CCSA SEQ ID NO:31 Codon-optimized L7F_A1_23 coding nucleotide sequence SEQ ID NO:32 Codon-optimized L7F_B1_23 coding nucleotide sequence SEQ ID NO:33 Codon-optimized L7F_A1_23.2 coding nucleotide sequence SEQ ID NO:34 Codon-optimized L7F_B1_23.2 coding nucleotide sequence SEQ ID NO:35 Codon-optimized sF_A1_K_L7 coding nucleotide sequence SEQ ID NO:36 Codon-optimized L7F_A1_31 coding nucleotide sequence SEQ ID NO:37 Codon-optimized L7F_A1_33 coding nucleotide sequence SEQ ID NO:38 Codon-optimized L7F_A1_4.2 coding nucleotide sequence SEQ ID NO:39 Codon-optimized sF_A1_K-E294 coding nucleotide sequence SEQ ID NO:40 Codon-optimized sF_A1_MFur coding nucleotide sequence SEQ ID NO:41 M protein sequence from strain CAN97-83 (accession no. Q6WB99) with purification tag MGHHHHHHHHHHHSSGHIDDDDKQESYLVDTYQGIPYTAAVQVDLVEKDLLPASLTIWFPLFQANTPPAVLLDQLKTLTITTLYAASQSGPILKVNASAQGAAMSVLPKKFEVNATVALDEYSKLEFDKLTVCEVKTVY LTTMKPYGMVSKFVSSAKPVGKKTHDLIALCDFMDLEKNTPVTIPAFIKSVSIKESESATVEAAISSEADQALTQAKIAPYAGLIMIMTMNNPKGIFKKLGAGTQVIVELGAYVQAESISKICKTWSHQGTRYVLKSR SEQ ID NO:42 CCKQTNECCKNLERAVSA SEQ ID NO:43 CCRELKECCKNLENAVSA SEQ ID NO:44 CCRELKDCCKNLENAVSA SEQ ID NO:45 CCRELKDCCKNLERAVSA SEQ ID NO:46 CCRELKDCCKQLNKAVSA SEQ ID NO:47 CCRELKECCKQLNKAVSA SEQ ID NO:48 Codon-optimized sF_B1_M coding nucleotide sequence SEQ ID NO:50 >sF_B1_K-L7 MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVSADQLAREEQIEQPRQSGCGAGATAGIAIAKTIRLESEVNAIKGALKQ TNEAVSTLGNGVRVLAFAVRELKEFVSKNLTSALNRNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPI FGVIDTPCWIIKAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNWVGIIKQLPKGC SYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAESAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK SEQ ID NO:51 >L7F_B1_31 MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFMLEVGDVENLTCTDGPSLLKTELDLTKSALRNLKTVSADQLAREEQIEQPRQSGCGAGATAGIAIAKTIRLESEVNAIKGAL KQTNEAVSTLGNGVRVLATMVRELKEFVSKNLTSAINRNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQ LPIFGVIDTPCWIIKAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNWVGIIKQL PKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNAGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK SEQ ID NO:52 >L7F_B1_33 MSWKVMIIISLLITPQHGLKESYLEESSCSTITEGYLSVLRTGWYTNVFMLCVGDVENLTCTDGPSLLKTELDLTKSALRELKTVSADQLAREEQIEQPRQSGCGAGATAGIAIAKTIRLESEVNAIKGAL KQTNEAVSTLGNGVRVLATMVRELCEFVSKNLTSAINRNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGILIWGYGSDVIYMVQ LPIFGVIDTPCWIIKAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNWVGIIKQL PKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRVPSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKCCNAGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK sequence number 53 >L7F_B1_4.2 MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFMLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVSADQLAREEQIEQPRQSGCGAGATAGIAIAKTIRLESEVNAWKGALKQTNEVVSTLGNGVRVLVTMVRELKEFVSKNLTSALNRNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIIKAAPSCSEKNGNYACLLREDQGWYCKNAGSTVYYPNEKDCETRGDHVFCDTCAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNWVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAESAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK

Claims

1. An immunogenic composition consisting essentially of a stabilized pre-fusion conformation form of human metapneumovirus (hMPV) F protein or a fragment thereof as the sole hMPV antigen, and optionally one or more adjuvants and / or at least one pharma- ceutically acceptable carrier or excipient, wherein the hMPV protein is from one subgroup of genotype A or B, and the immunogenic composition cross-neutralizes hMPV from another subgroup and / or genotype.

2. the stabilized prefusion conformational form of the human metapneumovirus (hMPV) F protein or fragment thereof is of the A1 subgroup, and / or the composition consists essentially of i) a stabilized pre-fusion conformation form of the human metapneumovirus (hMPV) F protein or a fragment thereof of the genotype A, and ii) a stabilized pre-fusion conformation form of the human metapneumovirus (hMPV) F protein or a fragment thereof of the genotype B, and optionally one or more adjuvants and / or at least one pharma- ceutically acceptable carrier or excipient, wherein the immunogenic composition cross-neutralizes other subgroups and / or other genotypes, and / or the stabilized pre-fusion conformational form of the human metapneumovirus (hMPV) F protein or fragment thereof of genotype A is of the A1 subgroup and the stabilized pre-fusion conformational form of the human metapneumovirus (hMPV) F protein or fragment thereof of genotype B is of the B1 subgroup; and / or The pre-fusion F protein is a recombinant protein. The immunogenic composition of claim 1.

3. The pre-fusion F protein comprises: lacking a cytoplasmic tail and / or a transmembrane domain, and / or and / or has an amino acid sequence which is a modified amino acid sequence of a native F protein derived from said hMPV strain or clinical isolate; and / or derived from a native F protein sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 10 derived from hMPV strains NL / 1 / 00, NL / 17 / 00, TN / 94-49, NCL174, CAN97-83, NL / 1 / 9, NDL00-1, C1-334, CAN97-82, and TN / 89-515; and / or and / or comprises at least one mutation (substitution or deletion), preferably up to 10 mutations, relative to the native F protein sequence of SEQ ID NO: 1-10; the pre-fusion F protein contains one or more amino acid substitutions to cysteines that introduce one or more non-native disulfide bonds that stabilize the pre-fusion conformation; and / or Cysteine ​​substitutions: Any one of positions 103-120 and any one of positions 335-345; Any one of positions 107-118 and any one of positions 335-342; Any one of positions 117-129 and any one of positions 256-261; Any one of positions 87-102 and any one of positions 117-127; Any one of positions 102-113 and any one of positions 117-127; Any one of positions 102-113 and any one of positions 87-102; Any one of positions 337-341 and any one of positions 421-426; Any one of positions 112-120 and any one of positions 424-432; Any one of positions 150-156 and any one of positions 392-400; Any one of positions 112-120 and any one of positions 370-377; Any one of positions 365-375 and any one of positions 455-465; Any one of positions 365-375 and any one of positions 105-115, or introduced at any one of positions 60-70 and any one of positions 175-185; said positions correspond to amino acids of said native F protein sequence of SEQ ID NOs: 1-10; The immunogenic composition of claim 1.

4. the pre-fusion F protein consists of a single polypeptide chain stabilized by at least one non-native disulfide bond; and / or the pre-fusion F protein comprises a single peptide stabilized by at least one introduced disulfide bond and lacks a protease cleavage site between the F1 and F2 domains compared to the native F protein; and / or the single chain pre-fusion F protein comprises a substitution of arginine with another amino acid, preferably glycine, at position 102 relative to the amino acid position of the native F protein; and / or and / or wherein amino acid residues 103 to 118 of the native F protein are replaced with a heterologous linker of 1 to 5 amino acid residues including at least one cysteine ​​residue that forms a disulfide bond with a cysteine ​​residue in the F1 domain; and / or the heterologous linker comprises at least one alanine, glycine or valine residue, preferably the linker has the sequence CGAGA or CGAGV; and / or the pre-fusion F protein comprises one or more substitutions at positions corresponding to positions 49, 51, 67, 80, 137, 147, 159, 160, 161, 166, 177, 258, 266, 480, and / or 481 of the native hMPV F protein; and / or said substitutions are selected from the group consisting of T49M, E80N, I137W, A147V, A159V, T160F, A161M, I67L, I177L, F258I, S266D, I480C, and / or L481C; and / or The single chain pre-fusion F protein comprises the following substitution combination: N97Q, R102G and G294E; N97Q, R102G, T160F, I177L and G294E; N97Q, R102G, T49M, I67L, A161M, E80N, F258I and G294E; N97Q, R102G, T49M, I67L, A161M, E51C, K166C, S266D, G294E, I480C, and L481C; or N97Q, R102G, T49M, A161M, I137W, A159V, A147V, I177L, and G294E and / or wherein the pre-fusion F protein comprises or consists of an amino acid sequence selected from SEQ ID NO: 11 (L7F_A1_23), SEQ ID NO: 12 (L7F_B1_23), SEQ ID NO: 13 (L7F_A1_23.2), SEQ ID NO: 14 (L7F_B1_23.2), SEQ ID NO: 15 (sF_A1_K_L7), SEQ ID NO: 16 (L7F_A1_31), SEQ ID NO: 17 (L7F_A1_33), SEQ ID NO: 18 (construct L7F_A1_4.2), SEQ ID NO: 50 (construct sF_B1_K_L7), SEQ ID NO: 51 (construct L7F_B1_31), SEQ ID NO: 52 (construct L7F_B1_33), and SEQ ID NO: 53 (construct L7F_B1_4.2), The immunogenic composition of claim 1.

5. the pre-fusion F protein is a protein having two polypeptide chains and comprises or consists of the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20; and / or The stabilized post-fusion F protein comprises, compared to the amino acid positions of the native F protein, a deletion of amino acid residues 103 to 111, a replacement of R102 with a linker KKRKRR, and the substitution G294E. The immunogenic composition of claim 1.

6. the pre-fusion F protein i) comprises an amino acid sequence having at least 80%, at least 90%, at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-20, and ii) its immunogenicity is similar to that of the parent F protein of SEQ ID NOs: 1-20, and / or The pre-fusion hMPV F protein comprises a trimerization helper domain (foldon) having a sequence of SEQ ID NO: 23-28 or a variant thereof; and / or the F protein is produced as a homo- or heterotrimer, and / or the composition comprises a further non-hMPV antigen, and / or the adjuvant is selected from the group consisting of alum, CpG, e.g., CpG1018, ODN, I-ODN, IC31®, MF59®, AddaVax™, AS03, AS01, QS21, MPL, GLA-SE, GLA-3M-052-LS, 3M-052-Alum, or a combination thereof; and / or The adjuvant is composed of two or more adjuvants selected from the group consisting of alum, CpG, e.g., CpG1018, ODN, I-ODN, IC31®, MF59®, AddaVax™, AS03, AS01, QS21, MPL, GLA-SE, GLA-3M-052-LS and 3M-052-alum; The immunogenic composition of claim 1.

7. the immunogenic composition is capable of eliciting neutralizing antibodies against the pre-fusion F protein; and / or said immunogenic composition comprising said pre-fusion protein or pre-fusion and post-fusion proteins provides a superior immune response (neutralizing antibody titer) compared to the immune response (neutralizing antibody titer) elicited by a composition comprising said post-fusion F protein used in the same total protein amount; and / or the immunogenic composition provides protection against more than one hMPV strain, and / or the immunogenic composition provides protection against hMPV strains of genotype A, and / or the immunogenic composition provides protection against hMPV strains of genotype B, and / or the immunogenic composition provides protection against genotype A and genotype B hMPV strains; and / or the immunogenic composition is a vaccine; The immunogenic composition of claim 1.

8. The immunogenic composition according to any one of claims 1 to 7 for use as a medicament.

9. 8. An immunogenic composition according to any one of claims 1 to 7 for use in the treatment and / or prevention of hMPV infection and / or associated diseases in a subject, comprising: The method comprises administering to the subject an effective amount of an immunogenic composition according to any one of claims 1 to 7; and / or the immunogenic composition is administered intramuscularly, intradermally, subcutaneously, mucosally, rectally, or orally; and / or The method comprises a prime-boost administration of an immunogenic composition according to any one of claims 1 to 7, the prime-boost being performed with the same immunogenic composition; and / or The method comprises a prime-boost administration of an immunogenic composition according to any one of claims 1 to 7, wherein the prime administration is with a composition comprising an F protein of genotype A and the boost administration is with a composition comprising an F protein of genotype B, or vice versa; and / or The method comprises administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of claims 1 to 7 to generate neutralizing antibodies against the pre-fusion hMPV F protein and provide protection against at least one hMPV strain of genotype A or B, preferably both. Immunogenic composition.

10. 8. A method for producing the immunogenic composition of any one of claims 1 to 7, comprising: i) expressing a recombinant pre-fusion F protein from a corresponding nucleic acid molecule inserted into an expression vector in a host cell; ii) purifying the expressed recombinant F protein; and iii) combining the purified recombinant protein with a pharma- ceutically acceptable carrier and / or excipient, and optionally an adjuvant.

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