Vaccines against respiratory diseases

Abstract

The present disclosure relates to hMPV F, PIV3 F, and PIV1 F protein variants, nucleic acids or vectors encoding hMPV F, PIV3 F, and PIV1 F protein variants, compositions comprising hMPV F, PIV3 F, and PIV1 F protein variants or nucleic acids, and uses of the hMPV F, PIV3 F, and PIV1 F protein variants, nucleic acids or vectors, and compositions. [Figure 1] TIFF2026504862000166.tif94170

Description

[Technical Field]

[0001] The present invention relates to vaccines in general and to vaccines against respiratory viruses such as hMPV A, hMPV B, PIV1, and PIV3. [Background technology]

[0002] Human paramyxoviruses and pneumoviruses are widespread pathogens that cause a considerable disease burden and include measles virus (MeV), mumps virus (MuV), respiratory syncytial virus (RSV), metapneumovirus (MPV), and parainfluenza virus types 1–4 (PIV1–4).

[0003] Human metapneumovirus (hMPV) is a respiratory virus that infects the lungs and airways. hMPV is a clinically important respiratory virus that causes a significant disease burden in children and is responsible for substantial pediatric hospitalizations.

[0004] There is near-universal infection by age 5 years, and reinfection continues to be a lifelong burden (van den Hoogen et al., 2001). However, infants (6-12 months), the elderly, and immunocompromised populations have more severe disease, including pneumonia and bronchiolitis, and are at increased risk of hospitalization (Deffrasnes et al., 2007). Despite the disease burden caused by hMPV, there are no approved vaccines or therapies for prevention or treatment.

[0005] hMPV is a member of the Pneumoviridae family, and its genome contains three transmembrane surface glycoproteins: the attachment protein G, the fusion protein F, and a small hydrophobic SH protein. There are two subtypes of hMPV, A and B, which differ primarily in the G glycoprotein, with the sequence of the F glycoprotein being more conserved between the two subtypes.

[0006] The mature F glycoprotein has three general domains: the ectodomain (ED), the transmembrane domain (TM), and the cytoplasmic tail (CT).

[0007] The hMPV F glycoprotein is initially translated from mRNA as a single 539 amino acid polypeptide precursor (termed "F0" or "F0 precursor"), which contains a signal peptide sequence (amino acids 1-18) at the N-terminus. Upon translation, the signal peptide is removed by a signal peptidase in the endoplasmic reticulum.

[0008] The remaining portion of the F0 precursor (i.e., residues 18-539) can be further cleaved at positions 102 / 103 by an intracellular protease to generate two linked fragments designated F1 (C-terminal portion; amino acids 103-539) and F2 (N-terminal portion; amino acids 19-102). F1 contains a hydrophobic fusion peptide at its N-terminus and two heptad repeat regions (HRA and HRB). HRA is located near the fusion peptide, and HRB is located near the TM domain. The F1 and F2 fragments are linked together through two disulfide bonds. Either the uncleaved F0 protein without the signal peptide sequence or the F1-F2 heterodimer can form the hMPV F protomer. Three such protomers associate to form the final hMPV F protein complex, which is a homotrimer of three protomers.

[0009] The F proteins of subtypes A and B are well conserved, and an example sequence of the F precursor polypeptide for the A subtype is provided in SEQ ID NO: 1 (A2b lineage (TN / 95 / 3-54) GenBank GI: ACJ53569.1)) and for the B subtype is provided in SEQ ID NO: 4 (consensus sequence). Both SEQ ID NO: 1 and SEQ ID NO: 4 are 539 amino acid sequences. The signal peptide sequence for SEQ ID NO: 1 and SEQ ID NO: 4 consists of amino acids 1-18.

[0010] One of the major antigens sought for hMPV subunit vaccines is the F protein. The hMPV F protein trimer mediates fusion between the virion membrane and the host cell membrane and also promotes syncytia formation. In the virion prior to fusion with the host cell membrane, the largest population of F molecules forms a lollipop-shaped structure, with the TM domain anchored to the viral envelope. This conformation is referred to as the pre-fusion conformation. Pre-fusion hMPV AF is recognized by, for example, the monoclonal antibody (mAb) MPE8, without distinguishing between oligomeric states. During hMPV entry into cells, the F protein rearranges from the pre-fusion state (referred to herein as "pre-F"), through an intermediate extended structure, to the post-fusion state ("post-F"). During this rearrangement, the C-terminal coiled coil of the pre-fusion molecule dissociates into its three constituent chains, which then wrap around the globular head and join three additional helices to form the post-fusion six-helix bundle. When the prefusion hMPV F trimer is subjected to increasingly harsh chemical or physical conditions, such as increasing temperature, it undergoes conformational changes: first, loss of trimeric structure (at least locally within the molecule), then rearrangement to the postfusion form, and then domain denaturation.

[0011] To block viral entry, F-specific neutralizing antibodies must likely bind to the prefusion conformation of F on the virion, or potentially to an elongation intermediate, before the viral envelope fuses with the cellular membrane. Therefore, the prefusion form of the F protein is thought to be the preferred conformation for a desired vaccine antigen (Stewart Jones et al., PNAS 2021, Vol. 118, No. 39, and Hsieh et al., Nature Communications, Vol. 13, Article No. 1299 (2022)). However, the precise role of the hMPV F prefusion form in inducing immunogenicity is less well established than that of RSV F. Upon extraction from the membrane using detergents, expression as an ectodomain, physical or chemical stress, or storage, the F glycoprotein is readily converted to a postfusion form (Mas et al., 2016 PLoS Pathog 12(9):e1005859).

[0012] PIV1 and PIV3 (genus Respirovirus) are also important pediatric pathogens within the family Paramyxoviridae, with lower incidence or disease severity caused by Paramyxoviridae members PIV2 and PIV4. While live attenuated virus vaccines can induce effective responses against measles and mumps, licensed vaccines against PIV1 and PIV3 have not been obtained using the same approach. Entry of these viruses also utilizes the viral fusion (F) glycoprotein, as disclosed above for hMPV. Summary of the Invention [Problem to be solved by the invention]

[0013] Preparation of hMPV, PIV1, or PIV3 prefusion F as a vaccine antigen remains a challenge. Because neutralizing and protective antibodies function by interfering with virus entry, it is hypothesized that F antigens that elicit only postfusion-specific antibodies would be expected to be less effective than F antigens that elicit prefusion-specific antibodies. Therefore, utilizing an F vaccine containing a prefusion form of the F protein immunogen would be more desirable. Previous efforts have not produced an hMPV, PIV1, or PIV3 vaccine that has been clinically demonstrated to elicit a sufficient level of protection to support the licensure of an hMPV, PIV1, or PIV3 vaccine. Therefore, there is a need for immunogens derived from hMPV, PIV1, and PIV3 F proteins, and compositions containing such immunogens, such as vaccines, that have improved properties compared to the corresponding native hMPV, PIV1, or PIV3 F proteins, such as increased expression when recombinantly expressed in mammalian cells, enhanced immunogenicity, or improved stability of the prefusion form.

[0014] There is also a need for a respiratory vaccine that includes a combination of hMPV, PIV1, and / or PIV3 F protein antigens to provide protection against several viruses that cause respiratory disease in a single vaccine. [Means for solving the problem]

[0015] In some aspects, the present invention provides mutants of wild-type hMPV F proteins, which exhibit introduced mutations in their amino acid sequences compared to the corresponding amino acid sequences of the wild-type hMPV F protein, and which are immunogenic against the wild-type hMPV F protein in the prefusion conformation or against viruses containing the wild-type hMPV F protein. The amino acid mutations in the mutants include amino acid substitutions, deletions, or additions compared to the wild-type hMPV F protein.

[0016] In some embodiments, the present disclosure provides mutants of wild-type hMPV F protein, wherein the introduced amino acid mutation is a mutation of a pair of amino acid residues in the wild-type hMPV F protein to a pair of cysteines (an "engineered disulfide mutation"). The introduced pair of cysteine ​​residues allows for the formation of a disulfide bond between the cysteine ​​residues, which stabilizes a conformation or oligomeric state of the protein, such as the pre-fusion conformation. Examples of specific pairs of such mutations include G366C and D454C, T411C and Q434C, I137C and A159C, A140C and S149C, L141C and A159C, L141C and A161C, E146C and T160C, V148C and L158C, and T150C and R156C, etc., 366C and 454C, 411C and 434C, 137C and 159C, 140C and 149C, 141C and 159C, 141C and 161C, 146C and 160C, 148C and 158C, and 150C and 156C.

[0017] In yet other embodiments, the hMPV F protein mutant comprises one or more amino acid mutations that are cavity-filling mutations. Examples of amino acids that can be replaced for cavity-filling purposes include small aliphatic (e.g., Gly, Ala, and Val) or small polar amino acids (e.g., Ser and Thr), and amino acids that are buried in the pre-fusion conformation but exposed to solvent in the post-fusion conformation. Examples of replacement amino acids include aliphatic amino acids (Val, Ile, Leu, and Met), aromatic amino acids (His, Phe, Tyr, and Trp), and polar amino acids (Thr) that are larger in size than the amino acid being replaced. In some specific embodiments, the hMPV F protein mutant comprises a cavity-filling mutation at one or more positions, preferably one, two, or three positions selected from 49, 149, 159, 291, 365, and 473. In some specific embodiments, the hMPV F protein mutant comprises (1) substitution of an amino acid at position 49, 291, or 365 with I, V, L, M, F, Y, or H; (2) substitution of the amino acid at position 149 with T, V, or I; (3) substitution of the amino acid at position 159 with V, I, or L; (4) substitution of amino acid at position 473 with F or W The cavity-filling mutation is selected from the group consisting of:

[0018] In certain embodiments, the hMPV F protein mutant comprises at least one cavity-filling mutation selected from the group consisting of T49I, S149T, A159V, S291I, T365I, and L473F.

[0019] In certain embodiments, the hMPV F protein mutant comprises one, two, or three cavity-filling mutations selected from the group consisting of T49I, S149T, A159V, S291I, T365I, and L473F.

[0020] In yet another embodiment, the present disclosure provides an hMPV F protein mutant, wherein the mutant comprises a proline substitution mutation that prevents structural refolding that occurs during the transition from the pre-fusion to the post-fusion conformation. In some specific embodiments, the hMPV F protein mutant comprises a proline substitution mutation selected from the group consisting of 66P, 110P, 132P, 145P, 187P, 449P, and 459P, such as L66P, ​​L110P, S132P, N145P, L187P, V449P, and A459P. In a preferred embodiment, the hMPV F protein mutant comprises the proline substitution mutation A459P.

[0021] In yet another embodiment, the present disclosure provides an hMPV F protein mutant, wherein the mutant comprises a glycine replacement mutation that removes a glycine residue in the center of the α-helix to improve protein stability.

[0022] In some specific embodiments, the hMPV F protein mutant comprises a glycine replacement mutation selected from the group consisting of G106A, G121A, and G239A.

[0023] In a preferred embodiment, the hMPV F protein mutant contains the glycine replacement mutation G239A.

[0024] In still other embodiments, the present disclosure provides hMPV F protein variants that comprise a combination of two or more different types of mutations selected from engineered disulfide mutations, cavity-filling mutations, proline substitution mutations, and glycine replacement mutations. In certain embodiments, the present invention provides variants of wild-type hMPV F proteins that comprise a combination of mutations compared to the corresponding wild-type hMPV F protein, wherein the combination of mutations is: (1) A combination of 140C and 149C; (2) a combination of 140C, 149C, 411C, and 434C; (3) combinations of 140C, 149C, 411C, 434C, and 459P; (4) combination of 140C, 149C, 411C, 434C, and 365I; (5) combination of 140C, 149C, 411C, 434C, and G239A; (6) combinations of 140C, 149C, 411C, 434C, 459P, G239A, 49I, and 365I; (7) Combination of 411C, 434C, 141C, and 161C; (8) combination of 411C, 434C, 141C, 161C, and 459P; (9) combination of 411C, 434C, 141C, 161C, and 49I; (10) combination of 411C, 434C, 141C, 161C, and 365I; (11) combination of 411C, 434C, 141C, 161C, and G239A; (12) combinations of 411C, 434C, 141C, 161C, and 149T; (13) A combination of 411C, 434C, 141C, 161C, 459P, G239A, 49I, 149T, and 365I; and (14) 411C, 434C, 146C, 160C, 459P, G239A, 49I, 149T, and 365I The present invention provides a mutant of a wild-type hMPV F protein selected from the group consisting of:

[0025] In certain embodiments, the present invention provides mutants of wild-type hMPV F proteins that comprise a combination of mutations compared to a corresponding wild-type hMPV F protein, wherein the combination of mutations is: (1) A140C and S149C combination; (2) combination of A140C, S149C, T411C, and Q434C; (3) a combination of A140C, S149C, T411C, Q434C, and A459P; (4) combination of A140C, S149C, T411C, Q434C, and T365I; (5) combination of A140C, S149C, T411C, Q434C, and G239A; (6) combination of A140C, S149C, T411C, Q434C, A459P, G239A, T49I, and T365I; (7) combination of T411C, Q434C, L141C, and A161C; (8) combination of T411C, Q434C, L141C, A161C, and A459P; (9) combination of T411C, Q434C, L141C, A161C, and T49I; (10) combination of T411C, Q434C, L141C, A161C, and T365I; (11) combination of T411C, Q434C, L141C, A161C, and G239A; (12) combination of T411C, Q434C, L141C, A161C, and S149T; (13) a combination of T411C, Q434C, L141C, A161C, A459P, G239A, T49I, S149T, and T365I; and (14) Combinations of T411C, Q434C, E146C, T160C, A459P, G239A, T49I, S149T, and T365I The present invention provides a mutant of a wild-type hMPV F protein selected from the group consisting of:

[0026] In certain embodiments, the invention provides mutants of wild-type hMPV AF proteins that comprise a combination of mutations relative to the corresponding wild-type hMPV AF protein, wherein the combination of mutations is: (1) A140C and S149C combination; (2) combination of A140C, S149C, T411C, and Q434C; (3) a combination of A140C, S149C, T411C, Q434C, and A459P; (4) combination of A140C, S149C, T411C, Q434C, and T365I; (5) combination of A140C, S149C, T411C, Q434C, and G239; (6) combination of A140C, S149C, T411C, Q434C, A459P, G239A, T49I, and T365I; (7) combination of T411C, Q434C, L141C, and A161C; (8) combination of T411C, Q434C, L141C, A161C, and A459P; (9) combination of T411C, Q434C, L141C, A161C, and T49I; (10) combination of T411C, Q434C, L141C, A161C, and T365I; (11) combination of T411C, Q434C, L141C, A161C, and G239A; (12) combination of T411C, Q434C, L141C, A161C, and S149T; (13) a combination of T411C, Q434C, L141C, A161C, A459P, G239A, T49I, S149T, and T365I; and (14) Combinations of T411C, Q434C, E146C, T160C, A459P, G239A, T49I, S149T, and T365I The present invention provides a mutant of the wild-type hMPV AF protein selected from the group consisting of:

[0027] In certain embodiments, the present invention provides mutants of wild-type hMPV F proteins that comprise a combination of mutations compared to a corresponding wild-type hMPV F protein, wherein the combination of mutations is: (1) T150C, R156C and A459P; (2) T150C, R156C and T49I; (3) T150C, R156C, T49I and A459P; (4) A140C, S149C, T411C, and Q434C; (5) L141C, A161C, T411C, and Q434C; (6) A140C, S149C, T411C, Q434C, and A459P; (7) A140C, S149C, G239A, T411C, and Q434C; (8) L141C, A161C, T411C, Q434C and A459P; (9) L141C, A161C, G239A, T411C, and Q434C; (10) T49I, T150C, R156C, G239A, and A459P; (11) A140C, S149C, G239A, T411C, Q434C, and A459P; (12) T49I, A140C, S149C, G239A, T411C, Q434C, and A459P; (13) T49I, A140C, S149C, G239A, T365I, T411C, Q434C, and A459P; (14) L141C, A161C, G239A, T411C, Q434C, and A459P; (15) T49I, L141C, A161C, G239A, T411C, Q434C, and A459P; (16) L141C, A161C, S149T, G239A, T411C, Q434C, and A459P; (17) T49I, L141C, A161C, S149T, G239A, T411C, Q434C, and A459P; (18) T49I, L141C, A161C, S149T, G239A, T365I, T411C, Q434C, and A459P; (19) T49I, S149T and A459P; (20) A140C, S149C and A459P; (21) T49I, A140C and S149C; (22) T49I, A140C, S149C and A459P; (23) T49I, L141C, A161C, T411C, and Q434C; (24) T49I, L141C, A161C, T411C, Q434C, and A459P; (25) L141C, A161C and S149T; (26) L141C, A161C, S149T, and A459P; (27) T49I, L141C, A161C and S149T, and (28)T49I, L141C, A161C, S149T, and A459P The present invention provides a mutant of a wild-type hMPV F protein selected from the group consisting of:

[0028] In certain embodiments, the present invention provides mutants of wild-type hMPV F proteins that comprise a combination of mutations compared to a corresponding wild-type hMPV F protein, wherein the combination of mutations is: (1) L66P; (2) L187P; (4) A140C, S149C and L187P; (5) T49I; (6) T365I; and (7) T49I and T365I The present invention provides a mutant of a wild-type hMPV F protein selected from the group consisting of:

[0029] In certain embodiments, the present invention provides mutants of wild-type hMPV F proteins that comprise a combination of mutations compared to a corresponding wild-type hMPV F protein, wherein the combination of mutations is: (1) L187P, Q100R, and S101R; and (2) A140C, S149C, L187P, Q100R, and S101R The present invention provides a mutant of a wild-type hMPV F protein selected from the group consisting of:

[0030] In certain embodiments, the present invention provides mutants of wild-type hMPV BF proteins that comprise a combination of mutations relative to the corresponding wild-type hMPV BF protein, wherein the combination of mutations is: (1) T150C, R156C and A459P; (2) T150C, R156C and T49I; (3) T150C, R156C, T49I and A459P; (4) A140C, S149C, T411C, and Q434C; (5) L141C, A161C, T411C, and Q434C; (6) A140C, S149C, T411C, Q434C, and A459P; (7) A140C, S149C, G239A, T411C, and Q434C; (8) L141C, A161C, T411C, Q434C and A459P; (9) L141C, A161C, G239A, T411C, and Q434C; (10) T49I, T150C, R156C, G239A, and A459P; (11) A140C, S149C, G239A, T411C, Q434C, and A459P; (12) T49I, A140C, S149C, G239A, T411C, Q434C, and A459P; (13) T49I, A140C, S149C, G239A, T365I, T411C, Q434C, and A459P; (14) L141C, A161C, G239A, T411C, Q434C, and A459P; (15) T49I, L141C, A161C, G239A, T411C, Q434C, and A459P; (16) L141C, A161C, S149T, G239A, T411C, Q434C, and A459P; (17) T49I, L141C, A161C, S149T, G239A, T411C, Q434C, and A459P; (18) T49I, L141C, A161C, S149T, G239A, T365I, T411C, Q434C, and A459P; (19) T49I, S149T and A459P; (20) A140C, S149C and A459P; (21) T49I, A140C and S149C; (22) T49I, A140C, S149C and A459P; (23) T49I, L141C, A161C, T411C, and Q434C; (24) T49I, L141C, A161C, T411C, Q434C, and A459P; (25) L141C, A161C and S149T; (26) L141C, A161C, S149T, and A459P; (27) T49I, L141C, A161C and S149T, and (28)T49I, L141C, A161C, S149T, and A459P The present invention provides a mutant of the wild-type hMPV BF protein selected from the group consisting of:

[0031] In certain embodiments, the present invention provides mutants of wild-type hMPV BF proteins that comprise a combination of mutations relative to the corresponding wild-type hMPV BF protein, wherein the combination of mutations is: (1) L66P; (2) L187P; (4) A140C, S149C and L187P; (5) T49I; (6) T365I; and (7) T49I and T365I The present invention provides a mutant of the wild-type hMPV BF protein selected from the group consisting of:

[0032] In certain embodiments, the present invention provides mutants of wild-type hMPV BF proteins that comprise a combination of mutations relative to the corresponding wild-type hMPV BF protein, wherein the combination of mutations is: (1) L187P, Q100R, and S101R; and (2) A140C, S149C, L187P, Q100R, and S101R The present invention provides a mutant of the wild-type hMPV BF protein selected from the group consisting of:

[0033] In some aspects, the present invention provides mutants of wild-type PIV1 F protein, wherein the mutants exhibit introduced mutations in their amino acid sequences compared to the corresponding amino acid sequences of the wild-type PIV1 F protein, and are immunogenic against the wild-type PIV1 F protein in a pre-fusion conformation or against viruses containing the wild-type PIV1 F protein. The amino acid mutations in the mutants include amino acid substitutions, deletions, or additions compared to the wild-type PIV1 F protein.

[0034] In some embodiments, the present disclosure provides a mutant of a wild-type PIV1 F protein, wherein the introduced amino acid mutations include at least one engineered disulfide mutation. An example of a specific pair of such mutations includes Q92C-G134C.

[0035] In still other embodiments, the PIV1 F protein variant comprises one or more amino acid mutations that are cavity-filling mutations. In some specific embodiments, the PIV1 F protein variant comprises a cavity-filling mutation at one or more positions, preferably at one, two, or three positions selected from 198, 92, 466, 473, or 480. In some specific embodiments, the PIV1 F protein variant comprises at least one cavity-filling mutation selected from the group consisting of T198A, Q92A, Q92L, A466L, A466V, A466I, S473V, S473L, S473I, S473A, A480L, and A480V.

[0036] In yet another embodiment, the present disclosure provides a PIV1 F protein variant, wherein the variant comprises a proline substitution mutation that prevents structural refolding that occurs during the transition from the pre-fusion to the post-fusion conformation.

[0037] In some specific embodiments, the PIV1 F protein variant comprises a proline substitution mutation A128P.

[0038] In still other embodiments, the present disclosure provides a PIV1 F protein variant, wherein the variant comprises a glycine replacement mutation that removes a glycine residue in the center of the α-helix to improve protein stability. In some specific embodiments, the PIV1 F protein variant comprises a glycine replacement mutation selected from the group consisting of G134A or G134L.

[0039] In yet another embodiment, the present disclosure provides a PIV1 F protein mutant, wherein the mutant comprises a cleavage site mutation that prevents cleavage of the PIV1 F protein. In such cases, the F1 and F2 polypeptides form a single polypeptide rather than two separate polypeptides linked by a disulfide bond. In some specific embodiments, the PIV1 F protein mutant comprises cleavage site mutations F113G and F114S.

[0040] In still other embodiments, the present disclosure provides PIV1 F protein variants that include a combination of two or more different types of mutations selected from engineered disulfide mutations, cavity-filling mutations, proline substitution mutations, glycine replacement mutations, and cleavage site mutations. In certain embodiments, the present invention provides variants of wild-type PIV1 F protein that include a combination of mutations compared to the corresponding wild-type PIV1 F protein, wherein the combination of mutations is: (1) Q92C and G134C; (2) A466L; (3) A466V; (4) S473V; (5) S473L; (6) A480L; (7) A466L and S473A; (8) A466L and S473L; (9) T198A; (10) G134A; (11) A128P; (12) F113G, F114S, Q92C, G134C, A466L, S473L, and A480L; (13) Q92C, G134C, A466L, S473L, and A480L; (14) Q92C, G134C, A466L, and S473L; (15) F113G, F114S, Q92C, G134C, A466V, S473V and A480V; (16) Q92C, G134C, A466V, S473V and A480V; (17) Q92C, G134C, A466V and S473V; (18) F113G, F114S, A466L, S473L, A480L, and G134A; (19) A466L, S473L, A480L and G134A; (20) A466L, S473L and G134A; (21) F113G, F114S, A466L, S473L, A480L, Q92A, and G134A; (22) F113G, F114S, A466L, S473L, and G134A; (23) A466L, S473L, A480L, Q92A, G134A; (24) A466L, S473L, Q92A, G134A; (25) F113G, F114S, Q92L, G134A; (26) A466L, S473L, A480L, Q92L, and G134A; (27) A466L, S473L, Q92L, and G134A; (28) F113G, F114S, A466L, S473L, A480L, Q92A, and G134L; (29) A466L, S473L, A480L, Q92A, and G134L; (30) F113G, F114S, Q92C, G134C, A466I, S473I, and A480L; (31) F113G, F114S, Q92C, G134C, A466I, and S473I; and (32) A466I, S473I, A480L, Q92L, and G134A The present invention provides a mutant of the wild-type PIV1 F protein selected from the group consisting of:

[0041] In some aspects, the present invention provides mutants of wild-type PIV3 F proteins, wherein the mutants exhibit introduced mutations in their amino acid sequences compared to the corresponding amino acid sequences of the wild-type PIV3 F proteins, and the mutants are immunogenic against the wild-type PIV3 F protein in a prefusion conformation or against viruses containing the wild-type PIV3 F protein. The amino acid mutations in the mutants include amino acid substitutions, deletions, or additions compared to the wild-type PIV3 F protein.

[0042] In some embodiments, the present disclosure provides mutants of wild-type PIV3 F protein, wherein the introduced amino acid mutations include at least one engineered disulfide mutation. Examples of specific pairs of such mutations include V175C-A202C, S160C-V170C, E209C-L234C, E209C-S233C, G85C-E209C, and Q162C-L168C.

[0043] In still other embodiments, the PIV3 F protein variant comprises one or more amino acid mutations that are cavity-filling mutations. In some specific embodiments, the PIV3 F protein variant comprises a cavity-filling mutation at one or more positions, preferably at one, two, or three positions selected from 277, 470, 477, 463, and 474. In some specific embodiments, the PIV3 F protein variant comprises at least one cavity-filling mutation selected from the group consisting of T277V, S470A, S470L, S477A, ​​A463L, I474F, and I474Y.

[0044] In yet another embodiment, the present disclosure provides a PIV3 F protein variant, wherein the variant comprises a proline substitution mutation that prevents structural refolding that occurs during the transition from the pre-fusion to the post-fusion conformation.

[0045] In some specific embodiments, the PIV3 F protein variant comprises the proline substitution mutations S164P and / or G219P.

[0046] In yet another embodiment, the present disclosure provides a PIV3 F protein variant, wherein the variant comprises a glycine replacement mutation that removes a glycine residue in the center of the α-helix to improve protein stability. In some specific embodiments, the PIV3 F protein variant comprises a glycine replacement mutation selected from the group consisting of G196A or G230A.

[0047] In still other embodiments, the present disclosure provides PIV3 F protein variants, wherein the variants comprise electrostatic mutations that reduce ionic repulsion or increase ionic attraction between residues in the protein that are close to each other in the folded structure. In some specific embodiments, the PIV3 F protein variants comprise the electrostatic mutations E182L and / or D455S.

[0048] In still other embodiments, the present disclosure provides PIV3 F protein variants that include a combination of two or more different types of mutations selected from engineered disulfide mutations, cavity-filling mutations, proline substitution mutations, glycine replacement mutations, and electrostatic mutations. In certain embodiments, the present invention provides variants of wild-type PIV3 F protein that include a combination of mutations compared to the corresponding wild-type PIV3 F protein, wherein the combination of mutations is: (1) V175C and A202C; (2) S160C and V170C; (3) S164P; (4) G196A; (5) G219P; (6) G230A; (7) E182L; (8) S470A; (9) S477A; (10) S470A and S477A; (11) D455S; (12) A463L; (13) Q162C, L168C, S470A and S477A; (14) S160C, V170C, S470A and S477A; (15) G230A, S470A, and S477A; (16) A463L, S470A and S477A; (17) E209C and L234C, (18) A463L and S470L, (19) S160C, V170C, E209C-L234C, A463L and S470L; (20) S160C, V170C, E209C-L234C, A463L and I474F; (21) S160C, V170C, E209C-L234C, A463L, I474F, F110G, F111S; (22) S160C, V170C, A463L and S470L; (23) Q162C, L168C, G230A, A463V, and I474Y; (24) Q162C, L168C, G230A, S470A, and S477A; (25) Q162C, L168C, G230A, and A463L; (26) Q162C, L168C, G230A, A463L, S470A, and S477A; (27) S160C, V170C, G230A, A463V and I474Y; (28) S160C, V170C, G230A, S470A and S477A; (29) S160C, V170C, G230A and A463L; (30) S160C, V170C, G230A, A463L, S470A, and S477A; and (31) S160C, V170C, and A463L; (32) E209C and S233C; (33) G85C and E209C; (34) T277V; (35) A463L and I474F; (36) A463I, S470I (37) S160C, V170C, E209C, S233C, A463L, and S470L; (38) S160C, V170C, E209C, S233C, A463L and I474F; (39) S160C, V170C, G85C, E209C, A463L and S470L; (40) S160C, V170C, G85C, E209C, A463L and I474F; (41) S160C, V170C, E209C, L234C, T277V, A463L and S470L; (42) S160C, V170C, E209C, L234C, T277V, A463L and I474F; (43) S160C, V170C, E209C, S233C, T277V, A463L and S470L; (44) S160C, V170C, E209C, S233C, T277V, A463L and I474F; (45) S160C, V170C, G85C, E209C, T277V, A463L and I474F; (46) S160C, V170C, E209C, L234C, D455S, A463L and S470L; (47) S160C, V170C, E209C, S233C, D455S, A463L, and S470L; (48) S160C, V170C, G85C, E209C, D455S, A463L and S470L; (49) S160C, V170C, E209C, L234C, T277V, D455S, A463L and S470L; (50) S160C, V170C, E209C, S233C, T277V, D455S, A463L and S470L; (51) S160C, V170C, G85C, E209C, T277V, D455S, A463L and S470L; (52) S160C, V170C and S470L; (53) R106G, T107S, E108A, R109S, S160C, V170C, E209C, L234C, A463L, and S470L; (54) R106G, T107S, E108A, R109S, S160C, V170C, E209C, S233C, A463L and S470L; (55) R106G, T107S, E108A, R109S, S160C, V170C, G85C, E209C, A463L and S470L; (56) F110G, F111S, S160C, V170C, E209C, L234C, A463L, and S470L; (57) F110G, F111S, S160C, V170C, E209C, S233C, A463L, and S470L; (58) F110G, F111S, S160C, V170C, A463L, and S470L; (59) F110G, F111S, S160C, V170C, and S470L; (60) S160C, V170C, A463L and S477L; (61) S160C, V170C, E209C, L234C, A463L and S470L; and (62) S160C, V170C, and S470L The present invention provides a mutant of the wild-type PIV3 F protein selected from the group consisting of:

[0049] In certain embodiments, the present invention provides a variant of a wild-type PIV3 F protein that comprises a combination of mutations compared to a corresponding wild-type PIV3 F protein, wherein the combination of mutations is: (1) G230A, S470A and S477A; (2) S160C, V170C, G230A and A463L; (3) S160C, V170C, S470A and S477A; (4) S160C, V170C, G230A, S470A and S477A; (5) S160C, V170C, G230A, A463L, S470A and S477A; (6) S160C, V170C, E209C, L234C, A463L and S470L; (7) S160C, V170C, E209C, L234C, A463L and I474F; (8) S160C, V170C, E209C, L234C, A463L, S470L, F110G, F111S; and (9) S160C, V170C, A463L, and S470L, and (10) E209C and L234C The present invention provides a mutant of the wild-type PIV3 F protein selected from the group consisting of:

[0050] In another aspect, the present invention provides nucleic acid molecules encoding the hMPV A, hMPV B, PIV1, or PIV3 F protein variants described herein. In one embodiment, the present invention provides nucleic acid molecules encoding the hMPV A, hMPV B, PIV1, or PIV3 F protein variants described herein. In a preferred embodiment, the nucleic acid is RNA, more preferably mRNA. In a preferred embodiment, the mRNA, when expressed in an appropriate cell, encodes a precursor F0 polypeptide that is processed into a full-length hMPV A, hMPV B, PIV1, or PIV3 F protein variant disclosed herein (e.g., an F1 polypeptide comprising one or more mutations, an ectodomain, a transmembrane domain, and a cytoplasmic domain, and an F2 polypeptide). In a preferred embodiment, the nucleic acid is an mRNA comprising chemically modified nucleotides. In a preferred embodiment, the nucleic acid is an mRNA comprising chemically modified nucleotides, preferably 1-methylpseudouridine. Preferably, all uridines in the RNA are replaced by 1-methylpseudouridine.

[0051] In another aspect, the present invention provides immunogenic compositions comprising (1) an hMPV A, hMPV B, PIV1, or PIV3 F protein variant described in this disclosure, and / or (2) a nucleic acid, preferably mRNA or modRNA, or vector encoding such an hMPV A, hMPV B, PIV1, or PIV3 F protein variant described in this disclosure.

[0052] In some embodiments, the immunogenic composition comprises: (1) an hMPV AF protein variant described in this disclosure, or a nucleic acid, preferably an mRNA, encoding such a variant; (2) an hMPV BF protein variant described in this disclosure, or a nucleic acid, preferably an mRNA, encoding such a variant; (3) a PIV1 F protein variant described in this disclosure, or a nucleic acid, preferably an mRNA, encoding such a variant; and PIV3 F protein variants described in this disclosure, or nucleic acids, preferably mRNA, encoding such variants. The present invention includes one, two, three, or four variants selected from the group consisting of:

[0053] The present disclosure also relates to the use of hMPV A, hMPV B, PIV1, or PIV3 F protein variants, nucleic acids encoding hMPV A, hMPV B, PIV1, or PIV3 F protein variants, vectors for expressing hMPV A, hMPV B, PIV1, or PIV3 F protein variants, or compositions comprising hMPV A, hMPV B, PIV1, or PIV3 F protein variants or nucleic acids.

[0054] In some embodiments, the present disclosure provides methods for inducing an immune response to hMPV A, hMPV B, PIV1, and / or PIV3 in a subject, comprising administering to the subject an effective amount of an hMPV A, hMPV B, PIV1, and / or PIV3 F protein variant, a nucleic acid encoding an hMPV A, hMPV B, PIV1, and / or PIV3 F protein variant, or a composition comprising an hMPV A, hMPV B, PIV1, and / or PIV3 F protein variant or a nucleic acid encoding such a variant. [Brief explanation of the drawings]

[0055] [Figure 1] 1A is a schematic diagram of the hMPV precursor polypeptide F0 (FIG. 1A), the PIV1 precursor polypeptide F0 (FIG. 1B), and the PIV3 precursor polypeptide F0 (FIG. 1C). [Figure 2] Schematic diagram of hMPV F modRNA (FIG. 2A), PIV3 F modRNA (FIG. 2B), and PIV1 F modRNA (FIG. 2C). [Figure 3A]1 provides the 50% neutralization titers in PD2 mouse sera raised against various recombinant hMPV F protein mutants using 3.0 μg of F protein. The dotted line represents the limit of detection at 20. [Figure 3B] 1 provides the 50% neutralization titers in PD2 mouse sera raised against various recombinant hMPV F protein mutants using 1.0 μg of F protein. The dotted line represents the limit of detection at 20. [Figure 3C] 1 provides 50% neutralization titers in PD2 mouse sera raised against various recombinant hMPV F protein mutants using 1.0 μg of F protein and LiNA-2 adjuvant. The dotted line represents the limit of detection at 20. [Figure 4] 10 provides 50% neutralization titers in PD2 mouse sera raised against various hMPV F protein mutants using 0.5 μg of LNP-formulated modRNA. The dotted line represents the limit of detection at 20. [Figure 5A] 1 provides the 50% neutralization titers in PD2 mouse sera raised against various recombinant PIV3 F protein variants using 0.25 μg of F protein. The dotted line represents the limit of detection at 20. [Figure 5B] 1 provides the 50% neutralization titers in PD2 mouse sera raised against various recombinant PIV3 F protein variants using 1.0 μg of F protein. The dotted line represents the limit of detection at 20. [Figure 5C] 1 provides 50% neutralization titers in PD2 mouse sera raised against various recombinant PIV3 F protein variants using 1.0 μg of F protein and LiNA-2 adjuvant. The dotted line represents the limit of detection at 20. [Figure 6A]

[0033] Figure 10 provides 50% neutralization titers in PD2 mouse sera generated against various PIV3 F protein designs using 0.05 μg of LNP-formulated modRNA. The dotted line represents the limit of detection at 20. [Figure 6B]

[0023] Figure 10 provides 50% neutralization titers in PD2 mouse sera generated against various PIV3 F protein designs using 0.2 μg of LNP-formulated modRNA. The dotted line represents the limit of detection at 20. [Figure 7A] 1 provides the 50% neutralization titers in PD2 mouse sera raised against various recombinant PIV1 F protein variants using 2.0 μg of F protein. The dotted line represents the limit of detection at 20. [Figure 7B] 5 provides 50% neutralization titers in PD2 mouse sera raised against various recombinant PIV1 F protein variants using 0.5 μg of F protein and LiNA-2 adjuvant. The dotted line represents the limit of detection at 20. [Figure 8]

[0023] Figure 10 provides 50% neutralization titers in PD2 mouse sera generated against various PIV1 F protein designs using 0.2 μg of LNP-formulated modRNA. The dotted line represents the limit of detection at 20. [Figure 9A] 1 provides the 50% neutralization titers in PD2 mouse sera raised against various recombinant PIV3 F protein variants using 1.0 μg of F protein. The dotted line represents the limit of detection at 20. [Figure 9B] 5 provides 50% neutralization titers in PD2 mouse sera raised against various recombinant PIV3 F protein variants using 0.5 μg of F protein and LiNA-2 adjuvant. The dotted line represents the limit of detection at 20. [Figure 10A]

[0033] Figure 10 provides 50% neutralization titers in PD2 mouse sera generated against various PIV3 F protein designs using 0.05 μg of LNP-formulated modRNA. The dotted line represents the limit of detection at 20. [Figure 10B]

[0023] Figure 10 provides 50% neutralization titers in PD2 mouse sera generated against various PIV3 F protein designs using 0.2 μg of LNP-formulated modRNA. The dotted line represents the limit of detection at 20. DETAILED DESCRIPTION OF THE INVENTION

[0056] A.Definition As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly indicates otherwise. For example, the term "an antigen" includes singular or plural antigens and can be considered equivalent to the phrase "at least one antigen."

[0057] The term "adjuvant" refers to a substance that can enhance, accelerate, or prolong the body's immune response to an antigen in a vaccine (even though it is not the target antigen of the vaccine itself). Adjuvants can be included in vaccine compositions or can be administered separately from the vaccine.

[0058] The term "administration" refers to the introduction of a substance or composition into a subject by a selected route. Administration can be local or systemic. For example, if the selected route is intramuscular, a composition (such as a composition containing a disclosed immunogen) is administered by introducing the composition into the muscle of the subject.

[0059] "Antibody" refers to an immunoglobulin molecule capable of specifically binding to a target, such as a polypeptide, carbohydrate, polynucleotide, or lipid, through at least one antigen-binding site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" can encompass any type of antibody (e.g., monospecific, bispecific), including portions of intact antibodies (e.g., "antigen-binding fragments") that retain the ability to bind to a given antigen, and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding site. Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or subclasses thereof), and antibodies need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of their heavy chains (HC), immunoglobulins can be assigned to various classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. Examples of antibody antigen-binding fragments and modified configurations include: (i) Fab fragments (monovalent fragments consisting of the VL, VH, CL, and CH1 domains); (ii) F(ab')2 fragments (bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region); and (iii) Fv fragments consisting of the VL and VH domains of a single arm of an antibody.Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv)); see, e.g., Bird et al., Science 1988;242:423-426 and Huston et al., Proc. Natl. Acad. Sci. 1988 USA 85:5879-5883. Other forms of single-chain antibodies, such as diabodies, are also encompassed.

[0060] Additionally, antibodies lacking the C-terminal lysine (K) amino acid residue on the heavy chain polypeptide are further encompassed (e.g., human IgG1 heavy chains contain a terminal lysine). As is known in the art, the C-terminal lysine may also be trimmed off during antibody production, resulting in an antibody having a heavy chain lacking the C-terminal lysine. Alternatively, nucleic acids that do not contain the C-terminal lysine can be used to produce antibody heavy chains.

[0061] The term "antigen" refers to a molecule capable of being recognized by an antibody. Examples of antigens include polypeptides, peptides, lipids, polysaccharides, and nucleic acids that contain antigenic determinants such as those recognized by immune cells.

[0062] "Agonist" refers to a substance that promotes (e.g., induces, causes, enhances, or increases) the biological activity or effect of another molecule. The term agonist includes substances (such as antibodies) that bind to a molecule and promote the activity of that molecule.

[0063] "Antagonist" refers to a substance that blocks, blocks, inhibits, neutralizes, or reduces the biological activity or effect of another molecule, such as a receptor. The term antagonist includes substances (such as antibodies) that bind to a molecule and prevent or reduce the activity of that molecule.

[0064] The term "binding affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by commonly used methods known in the art. Low affinity antibodies generally bind antigens slowly and tend to dissociate quickly, while high affinity antibodies generally bind antigens faster and tend to remain bound longer. In particular, the term "binding affinity" is intended to refer to the dissociation rate of a particular antigen-antibody interaction. K D is the association rate, or "on rate (k on )" or "k a " for "off speed (k off )" or "k d is the ratio of the dissociation rates, also known as K D is k off / k on (or k d / k a ) and expressed as molar concentration (M). Therefore, K D The smaller the K, the stronger the binding affinity. Therefore, a K of 1 μM D has a K of 1 nM D It shows weaker binding affinity compared to the K D The K value can be determined using methods well established in the art. D One exemplary method for determining K is by using surface plasmon resonance (SPR), typically using a biosensor system such as a BIACORE system. BIACORE kinetic analysis involves analyzing the binding and dissociation of antigens from a chip with immobilized molecules (e.g., molecules containing epitope-binding domains) on their surface. DAnother method for determining e This is by using biolayer interferometry using a system (ForteBio). Alternatively or additionally, the KinExA (Kinetic Exclusion Assay) assay available from Sapidyne Instruments (Boise, ID) can be used.

[0065] A "bispecific antibody" refers to a molecule that has binding specificities for at least two different epitopes. In some embodiments, a bispecific antibody can simultaneously bind to two different antigens. In other embodiments, the two different epitopes can be present on the same antigen.

[0066] "Chimeric antibody" refers to an antibody whose variable region sequences are derived from one species and whose constant region sequences are derived from another species, such as an antibody whose variable region sequences are derived from a mouse antibody and whose constant region sequences are derived from a human antibody.

[0067] The term "compete" as used herein with respect to antibodies means that a first antibody binds to an epitope in a manner sufficiently similar to that of a second antibody, such that the resultant binding of the second antibody to its cognate epitope is detectably reduced in the presence of the first antibody compared to binding of the second antibody in the absence of the first antibody. Another possibility may arise, but not necessarily, in which the binding of the first antibody to its epitope is also detectably reduced in the presence of the second antibody. That is, a first antibody may inhibit the binding of a second antibody to its epitope without the second antibody inhibiting binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits binding of the other antibody to its cognate epitope or ligand to the same, greater, or lesser extent, the antibodies are said to "cross-compete" with each other for binding of their respective epitopes. Both competing and cross-competing antibodies are encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), one of skill in the art will understand, based on the teachings provided herein, that such competing or cross-competing antibodies are encompassed and may be useful in the methods disclosed herein.

[0068] The term "conservative substitution" refers to the substitution of an amino acid with a chemically similar amino acid. Conservative amino acid substitutions that provide functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0069] The "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination. The IgG heavy chain constant region contains three sequential immunoglobulin domains (CH1, CH2, and CH3), with a hinge region between the CH1 and CH2 domains. The IgG light chain constant region contains a single immunoglobulin domain (CL).

[0070] The term "degenerate variant" of a reference polynucleotide refers to a polynucleotide that differs in nucleotide sequence from the reference polynucleotide but encodes the same polypeptide sequence as that encoded by the reference polynucleotide.There are 20 naturally occurring amino acids, most of which are specified by more than one codon.For example, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine.Therefore, at any position where arginine is specified in a protein coding sequence, the codon can be changed to any of the corresponding codons listed without changing the encoded protein.Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide.

[0071] The term "effective amount" refers to the amount of an agent that is sufficient to produce a desired response. For example, this may be the amount necessary to inhibit viral replication or to measurably alter the outward symptoms of a viral infection.

[0072] "Effector cells" refer to leukocytes that express one or more FcRs and perform effector function. In certain embodiments, effector cells express at least FcgRIII and perform ADCC effector function. Examples of leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, macrophages, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, for example, from blood.

[0073] The term "epitope" (or "antigenic determinant" or "antigenic site") refers to the region of an antigen to which an antibody, B-cell receptor, or T-cell receptor binds or responds. Epitopes can be formed from contiguous or non-contiguous amino acids juxtaposed by secondary, tertiary, or quaternary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by higher order folding are typically lost on treatment with denaturing solvents.

[0074] The term "F0 polypeptide" (F0) when used in reference to the hMPV F protein refers to the precursor polypeptide of the hMPV F protein, which is composed of a signal polypeptide sequence, an F1 polypeptide sequence, and an F2 polypeptide sequence. With rare exceptions, the F0 polypeptides of known hMPV strains consist of 539 amino acids.

[0075] The term "F0 polypeptide" (F0) when used in reference to the PIV1 F protein refers to the precursor polypeptide of the PIV1 F protein, which is composed of a signal polypeptide sequence, an F1 polypeptide sequence, and an F2 polypeptide sequence. Examples of F0 polypeptides of known PIV1 strains are provided in Table 4 and consist of 555 amino acids.

[0076] The term "F0 polypeptide" (F0) when used in reference to the PIV3 F protein refers to the precursor polypeptide of the PIV3 F protein, which is composed of a signal polypeptide sequence, an F1 polypeptide sequence, and an F2 polypeptide sequence. Examples of F0 polypeptides of known PIV3 strains are provided in Table 6 and consist of 539 amino acids.

[0077] The term "F1 polypeptide" (F1) when used in reference to the hMPV F protein refers to the polypeptide chain of the mature hMPV F protein. Native F1 comprises approximately residues 103-539 of the hMPV F0 precursor and is composed, from the N- to C-terminus, of an extracellular region (approximately residues 103-489), a transmembrane domain (approximately residues 490-514), and a cytoplasmic domain (also referred to as the intracellular domain) (approximately residues 515-539). As used herein, the term encompasses both native F1 polypeptides and F1 polypeptides containing modifications (e.g., amino acid substitutions, insertions, or deletions) from the native sequence, such as modifications designed to stabilize the F variant or to enhance the immunogenicity of the F variant.

[0078] The term "F1 polypeptide" (F1) when used in reference to the PIV1 F protein refers to the polypeptide chain of the mature PIV1 F protein. Native F1 comprises approximately residues 113-555 of the PIV1 F precursor and is composed, from the N- to C-terminus, of an extracellular region (approximately residues 103-496), a transmembrane domain (approximately residues 497-517), and a cytoplasmic domain (also referred to as the intracellular domain) (approximately residues 518-555). As used herein, the term encompasses both native F1 polypeptides and F1 polypeptides containing modifications (e.g., amino acid substitutions, insertions, or deletions) from the native sequence, such as modifications designed to stabilize the F variant or to enhance the immunogenicity of the F variant.

[0079] The term "F1 polypeptide" (F1) when used in reference to PIV3 proteins refers to the polypeptide chain of the mature PIV3 F protein. Native F1 comprises approximately residues 103-539 of the PIV3 F0 precursor and is composed, from the N- to C-terminus, of an extracellular region (approximately residues 103-493), a transmembrane domain (approximately residues 494-514), and a cytoplasmic domain (also referred to as the intracellular domain) (approximately residues 515-539). As used herein, the term encompasses both native F1 polypeptides and F1 polypeptides containing modifications (e.g., amino acid substitutions, insertions, or deletions) from the native sequence, such as modifications designed to stabilize the F variant or to enhance the immunogenicity of the F variant.

[0080] The term "F2 polypeptide" (F2) when used in reference to the hMPV F protein refers to the polypeptide chain of the mature hMPV F protein. Native F2 comprises approximately residues 19-102 of the hMPV F0 precursor. As used herein, the term encompasses both native F2 polypeptides and F2 polypeptides containing modifications (e.g., amino acid substitutions, insertions, or deletions) from the native sequence, such as modifications designed to stabilize the F variant or enhance the immunogenicity of the F variant. In the native hMPV F protein, the F2 polypeptide is linked to the F1 polypeptide by two disulfide bonds to form an F2-F1 heterodimer.

[0081] The term "F2 polypeptide" (F2) when used in reference to a PIV1 protein refers to the polypeptide chain of the mature PIV1 F protein. Native F2 comprises approximately residues 22-112 of the PIV1 F precursor. As used herein, the term encompasses both native F2 polypeptides and F2 polypeptides containing modifications (e.g., amino acid substitutions, insertions, or deletions) from the native sequence, such as modifications designed to stabilize the F variant or enhance the immunogenicity of the F variant. In native PIV1 F protein, the F2 polypeptide is linked to the F1 polypeptide by two disulfide bonds to form an F2-F1 heterodimer.

[0082] The term "F2 polypeptide" (F2) when used in reference to the PIV3 F protein refers to the polypeptide chain of the mature PIV3 F protein. Native F2 comprises approximately residues 19-109 of the PIV3 F0 precursor. As used herein, the term encompasses both native F2 polypeptides and F2 polypeptides containing modifications (e.g., amino acid substitutions, insertions, or deletions) from the native sequence, such as modifications designed to stabilize the F variant or enhance the immunogenicity of the F variant. In native PIV3 F protein, the F2 polypeptide is linked to the F1 polypeptide by two disulfide bonds to form an F2-F1 heterodimer.

[0083] "Fc domain" refers to the portion of an immunoglobulin (Ig) molecule that correlates with the crystallizable fragment obtained by papain digestion of the Ig molecule. As used herein, the term refers to the two-chain constant region of an antibody, each chain excluding the first constant region immunoglobulin domain. Within the Fc domain, there are two "Fc chains" (e.g., "first Fc chain" and "second Fc chain"). "Fc chain" generally refers to the C-terminal portion of an antibody heavy chain. Thus, the Fc chain refers to the last two constant region immunoglobulin domains (CH2 and CH3) of IgA, IgD, and IgG heavy chains, and the last three constant region immunoglobulin domains of IgE and IgM heavy chains, and optionally the flexible hinge N-terminal to these domains.

[0084] Although the boundaries of the Fc chain can vary, the human IgG heavy chain Fc chain is usually defined to include residues C226 or P230 at its carboxyl terminus, numbering according to the EU index of Edelman et al., Proc. Natl. Acad. Sci. USA 1969;63(1):78-85, and as described in Kabat et al., 1991. Typically, an Fc chain includes amino acid residues from about 236 to about 447 of the human IgG1 heavy chain constant region. "Fc chain" can refer to this polypeptide in isolation or in the context of a larger molecule (e.g., an antibody heavy chain or an Fc fusion protein).

[0085] A "functional" Fc domain refers to an Fc domain that possesses at least one effector function of a native-sequence Fc domain. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation, etc. Such effector functions generally require that the Fc domain be combined with a binding domain (e.g., an antibody variable region), and can be assessed using a variety of assays known in the art for evaluating such antibody effector functions.

[0086] A "native sequence" Fc chain refers to an Fc chain comprising an amino acid sequence identical to that of an Fc chain found in nature. A "variant" Fc chain comprises an amino acid sequence that differs from that of a native sequence Fc chain by virtue of at least one amino acid modification.

[0087] "Fc receptor" (FcR) refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR binds IgG antibodies (gamma receptors) and includes receptors of the FcgRI, FcgRII, and FcgRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcgRII receptors include FcgRIIA ("activating receptor") and FcgRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcgRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcgRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 1997;15:203-234). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 1991;9:457-92; Capel et al., Immunomethods 1994;4:25-34; and de Haas et al., J. Lab. Clin. Med. 1995;126:330-41. Other FcRs, including those identified in the future, are encompassed herein by the term "Fc receptor." The term "Fc receptor" also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 1976;117:587, and Kim et al., J. Immunol. 1994;24:249) and in regulating immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 1997;18(12):592-598; Ghetie et al., Nature Biotechnology 1997;15(7):637-640; Hinton et al., J. Biol. Chem. 2004;279(8):6213-6216; WO2004 / 92219).

[0088] The term "foldon" or "foldon domain" refers to an amino acid sequence capable of forming a trimer. One example of such a foldon domain is the peptide sequence derived from bacteriophage T4 fibritin, which has the sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 7).

[0089] The term "mammal" refers to any animal species of the class Mammalia. Examples of mammals include humans; non-human primates such as monkeys; laboratory animals such as rats, mice, and guinea pigs; domestic animals such as cats, dogs, rabbits, cows, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, and elephants.

[0090] The term "glycoprotein" refers to a protein containing oligosaccharide chains (glycans) covalently attached to polypeptide side chains. Carbohydrates are attached to proteins during translation or in a post-translational modification known as glycosylation. The term "glycosylation site" refers to an amino acid sequence on the surface of a polypeptide, such as a protein, that can accommodate the attachment of a glycan. An N-linked glycosylation site is a triplet sequence, N-X(S / T), where N is asparagine, X is any residue except proline, and (S / T) is a serine or threonine residue. A glycan is a polysaccharide or oligosaccharide. Glycan can also be used to refer to the carbohydrate moiety of a glycoconjugate such as a glycoprotein, glycolipid, or proteoglycan.

[0091] A "monoclonal antibody" (mAb) refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies of interest for use in accordance with the present invention may be produced by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be produced by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. In another example, the monoclonal antibodies can be isolated from phage libraries, such as those produced using the techniques described in McCafferty et al., 1990, Nature 348:552-554.

[0092] A "monospecific antibody" refers to an antibody that contains one or more antigen-binding sites per molecule, such that each and every binding site of the antibody specifically recognizes the same epitope on the antigen. Thus, when a monospecific antibody has more than one antigen-binding site, the binding sites compete with each other for binding to a single antigen molecule.

[0093] The term "hMPV-2 mAb" refers to an hMPV AF protein pre-fusion specific antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:360 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:361.

[0094] Half-maximal effective concentration (EC 50 The term "EC" refers to the concentration of a therapeutic agent that produces a response halfway between baseline and maximum after a specified exposure time. The therapeutic agent can produce inhibition or stimulation. 50The value is commonly used and is used herein as a measure of efficacy.

[0095] The term "host cells" refers to cells in which a vector can be propagated and its DNA or RNA expressed. The cells can be prokaryotic or eukaryotic.

[0096] "Human antibody" refers to an antibody that has an amino acid sequence corresponding to that of an antibody produced by humans, or that is produced using any technique for producing a fully human antibody. For example, a fully human antibody can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins, or by library (e.g., phage, yeast, or ribosome) display techniques to prepare a fully human antibody. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0097] A "humanized" antibody refers to a non-human (e.g., murine) antibody that is a chimeric antibody containing minimal sequence derived from non-human immunoglobulin. Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's CDR are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit that has the desired specificity, affinity, and capacity. A humanized antibody may contain residues that are not found in the recipient antibody or in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. The terms "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that, when compared and aligned for maximum correspondence, are the same or have a specified percentage of identical amino acid residues or nucleotides. Methods for aligning sequences for comparison are well known in the art. Once aligned, the number of matches is determined by counting the number of positions where identical nucleotides or amino acid residues occur in both sequences. Percent sequence identity is determined by dividing the number of matches by the length of the sequence specified in the specified sequence, or by the stated length (such as 100 consecutive nucleotides or amino acid residues from the sequence specified in the specified sequence), and then multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166÷1554*100=75.0).

[0098] Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443, 1970, by the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley and Sons, New York, Supplement to 104, 2013).

[0099] The term "immunogenicity" refers to the ability of a substance, with or without the presence of an adjuvant, to provoke, induce, stimulate, or induce an immune response in an animal against a particular antigen.

[0100] The term "immune response" refers to any detectable response of one or more cells of the immune system of a host mammal to a stimulus (e.g., an immunogen), including, but not limited to, innate immune responses (e.g., activation of the Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells, such as antigen-specific T cells, and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells, such as the production of antibodies and secretion of antibodies into plasma, lymph, and / or tissue fluids). Examples of immune responses include altered (e.g., increased) Toll-like receptor activation, lymphokine (e.g., cytokine (e.g., Th1, Th2, or Th17-type cytokine) or chemokine) expression or secretion, macrophage activation, dendritic cell activation, T cell (e.g., CD4+ or CD8+ T cell) activation, NK cell activation, B cell activation (e.g., antibody production and / or secretion), binding of an immunogen (e.g., an antigen (e.g., an immunogenic polypeptide)) to an MHC molecule, induction of a cytotoxic T lymphocyte ("CTL") response, induction of a B cell response (e.g., antibody production), and expansion (e.g., growth of cell populations) of cells of the immune system (e.g., T cells and B cells), and increased antigen processing and presentation by antigen-presenting cells. The term "immune response" also encompasses any detectable response to a particular substance (such as an antigen or immunogen) by one or more components of a vertebrate's immune system in vitro. The term "immunogen" refers to a compound, composition, or substance that is immunogenic as defined herein below.

[0101] The term "immunogenic composition" refers to a composition that includes an immunogen.

[0102] The term "MPE8" refers to the antibody described in Corti et al. [Corti, D., Bianchi, S., Vanzetta, F., Minola, A., Perez, L., Agatic, G., Lanzavecchia, A. Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature, 501(7467), 439-443(2013)], which has a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 358 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 359.

[0103] The terms "mutant" of a wild-type hMPV F protein, "mutant" of an hMPV F protein, "hMPV F protein mutant," or "modified hMPV F protein" refer to a polypeptide that exhibits mutations introduced relative to the wild-type F protein and is immunogenic relative to the wild-type F protein.

[0104] The terms "mutant" of a wild-type PIV1 F protein, "mutant" of a PIV1 F protein, "PIV1 F protein mutant," or "modified PIV1 F protein" refer to a polypeptide that exhibits mutations introduced relative to the wild-type F protein and is immunogenic relative to the wild-type F protein.

[0105] The terms "mutant" of a wild-type PIV3 F protein, "mutant" of a PIV3 F protein, "PIV3 F protein mutant," or "modified PIV3 F protein" refer to a polypeptide that exhibits mutations introduced relative to the wild-type F protein and is immunogenic relative to the wild-type F protein.

[0106] The term "mutation" refers to the deletion, addition, or substitution of an amino acid residue in the amino acid sequence of a protein or polypeptide compared to the amino acid sequence of a reference protein or polypeptide. Throughout this specification and claims, an amino acid substitution at one specific position in a protein sequence is referred to using the notation "(amino acid residue in wild-type protein) (amino acid position) (amino acid residue in engineered protein)." For example, the notation Y75A refers to the substitution of a tyrosine (Y) residue at position 75 of the amino acid sequence of the reference protein with an alanine (A) residue (in a mutant of the reference protein). When there is a variation in the amino acid residue at the same position between different wild-type sequences, the amino acid code preceding the position number, such as "75A," may be omitted in the notation.

[0107] The term "native" or "wild-type" protein, sequence, or polypeptide refers to a protein, sequence, or polypeptide that occurs in nature and has not been artificially modified by selective mutation.

[0108] The term "pharmaceutically acceptable carrier" refers to a material or composition that, when combined with an active ingredient, is compatible with the active ingredient and does not cause any adverse or otherwise undesirable reactions when administered to a subject, particularly a mammal. Examples of pharmaceutically acceptable carriers include solvents, surfactants, suspending agents, buffers, lubricants, emulsifiers, absorbents, dispersion media, coatings, and stabilizers.

[0109] The term "PIA174 mAb" refers to a PIV3 F protein prefusion-specific antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 364 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 365. The amino acid sequence of SEQ ID NO: 364 comprises the heavy chain variable domain and constant domain of PIA174 mAb, and the amino acid sequence of SEQ ID NO: 365 comprises the light chain variable domain and constant domain of PIA174 mAb. The heavy chain variable domain of PIA174 mAb has the amino acid sequence of SEQ ID NO: 553. The light chain variable domain of PIA174 mAb has the amino acid sequence of SEQ ID NO: 554.

[0110] The term "PIV1-8 mAb" (also referred to as hPIV1-8 mAb) refers to a PIV1 F protein prefusion-specific antibody having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 362 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 363.

[0111] The term "pre-fusion specific antibody" refers to an antibody that specifically binds to the F glycoprotein in the pre-fusion conformation but not to the F protein in the post-fusion conformation. Exemplary pre-fusion specific antibodies include MPE8, hMPV-2, and PIV1-8 antibodies.

[0112] The term "prime-boost vaccination" refers to an immunotherapy regimen that involves administering a first immunogenic composition (primer vaccine) to a subject, followed by a second immunogenic composition (booster vaccine) to induce an immune response. The primer vaccine and the booster vaccine typically contain the same immunogen and are presented in the same or similar format. However, they can also be presented in different formats, for example, one in the form of a vector and the other in the form of a naked DNA plasmid. Those skilled in the art will understand the appropriate time interval between the administration of the primer vaccine and the booster vaccine. Furthermore, the primer vaccine, the booster vaccine, or both the primer vaccine and the booster vaccine additionally contain an adjuvant.

[0113] The term "pre-fusion conformation" refers to the structural conformation adopted by the F protein or mutant to which pre-fusion-specific antibodies, such as, for example, MPE8 mAb against hMPV A, hMPV-2 mAb against hMPV B, PIV1-8 mAb against PIV1, and PIA174 mAb against PIV3, can specifically bind.

[0114] The term "post-fusion conformation" refers to the structural conformation adopted by the F protein to which MPE8 mAb, hMPV-2 mAb, or PIV1-8 do not specifically bind. Native F proteins adopt a post-fusion conformation after fusion of the viral envelope with the host cell membrane. F proteins can also adopt a post-fusion conformation outside the context of a fusion event, under stress conditions such as heat and hypotonicity, when extracted from membranes, when expressed as an ectodomain, or during storage.

[0115] The term "soluble protein" refers to a protein that can dissolve and remain dissolved in an aqueous solution. The solubility of a protein can vary depending on the concentration of the protein in the water-based liquid, the buffering state of the liquid, the concentrations of other solutes in the liquid, e.g., salt and protein concentrations, and the temperature of the liquid.

[0116] The term "specifically binds" in the context of antibody binding to a given target molecule refers to the antibody binding to the target molecule with a higher affinity than its binding to other test substances. For example, an antibody that specifically binds to hMPV F protein in the pre-fusion conformation is an antibody that binds to hMPV F protein in the pre-fusion conformation with a higher affinity than it binds to hMPV F protein in the post-fusion conformation.

[0117] The term "therapeutically effective amount" refers to that amount of an agent that is sufficient to prevent, treat (including prevent), reduce, and / or ameliorate the symptoms and / or underlying causes of a disorder.

[0118] The term "vaccine" refers to a pharmaceutical composition containing an immunogen that can induce a prophylactic or therapeutic immune response in a subject. Typically, a vaccine induces an antigen-specific immune response against an antigen of a pathogen, e.g., a viral pathogen.

[0119] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions, which contribute to the formation of the antigen-binding site of the antibody. When a variant of a subject variable region is desired, particularly one with an amino acid residue substitution outside the CDR region (e.g., in the framework region), appropriate amino acid substitutions, preferably conservative amino acid substitutions, can be identified by comparing the subject variable region with the variable regions of other antibodies containing CDR1 and CDR2 sequences in the same standard class as the subject variable region (Chothia and Lesk, J Mol Biol 196(4):901-917, 1987).

[0120] In certain embodiments, the delineation of CDRs and the identification of the residues comprising the antibody binding site are achieved by elucidating the structure of an antibody or the structure of an antibody-ligand complex. In certain embodiments, this can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various analytical methods can be employed to identify or estimate CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, the contact definition, the extended definition, and the conformational definition.

[0121] The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDR regions. See, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8. The Chothia definition is similar to the Kabat definition, but takes into account the location of certain structural loop regions. See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83. The extended definition is a combination of the Kabat and Chothia definitions. The AbM definition uses a unified suite of computer programs created by the Oxford Molecular Group to model antibody structure. See, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of antibodies from primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. The contact definition is based on an analysis of available complex crystal structures. See, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another approach, referred to herein as "conformational definition" of CDRs, CDR positions can be identified as residues that contribute enthalpicly to antigen binding. See, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166.Still other CDR boundary definitions may not strictly follow one of the above methods, but still be considered to overlap with at least a portion of Kabat CDRs, although they may be shortened or lengthened in light of predictions or experimental findings that certain residues or groups of residues do not significantly affect antigen binding.As used herein, CDR may refer to CDRs defined by any method known in the art, including a combination of methods.The method used herein may utilize CDRs defined according to any of these methods.For any given embodiment that contains more than one CDR, the CDR may be defined according to any one or more of Kabat, Chothia, extended, AbM, contact, or conformational definitions.Unless otherwise stated, the CDRs disclosed herein are defined according to Kabat.

[0122] The term "vector" refers to a nucleic acid molecule capable of transporting or transferring a foreign nucleic acid molecule. The term encompasses both expression vectors and transcription vectors. The term "expression vector" refers to a vector capable of expressing an insert in a target cell and generally contains control sequences, such as enhancer, promoter, and terminator sequences, that drive the expression of the insert. The term "transcription vector" refers to a vector that can be transcribed but not translated. Transcription vectors are used to amplify their inserts. The foreign nucleic acid molecule is referred to as an "insert" or "transgene." A vector generally consists of an insert and a larger sequence that serves as the backbone of the vector. Based on the structure or origin of the vector, the main types of vectors include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus (Ad) vectors, and artificial chromosomes.

[0123] B. hMPV mutants The present disclosure relates to hMPV F protein variants, immunogenic compositions comprising hMPV F protein variants, methods for producing hMPV F protein variants, compositions comprising hMPV F protein variants, and nucleic acids encoding hMPV F protein variants.

[0124] 1. Exemplary Embodiment (E) of the Present Invention Exemplary embodiments (E) of the invention provided herein include the following: E1. A mutant of a wild-type hMPV F protein, the mutant comprising an F1 polypeptide and an F2 polypeptide, the mutant comprising at least one amino acid mutation compared to the amino acid sequence of the wild-type hMPV F protein, the amino acid mutation being: (1) at least one engineered disulfide bond mutation; (2) at least one cavity-filling mutation; (3) at least one proline substitution mutation; (4) at least one glycine replacement mutation; (5) a combination of at least one engineered disulfide mutation and at least one cavity-filling mutation; (6) a combination of at least one engineered disulfide mutation and at least one proline substitution mutation; (7) a combination of at least one engineered disulfide mutation and at least one glycine replacement mutation; (8) a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one proline substitution mutation; (9) a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one glycine-replacement mutation; (10) a combination of at least one engineered disulfide mutation, at least one proline substitution mutation, and at least one glycine replacement mutation; and (11) A combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, at least one proline substitution mutation, and at least one glycine replacement mutation. A mutant of the wild-type hMPV F protein selected from the group consisting of: E2. The variant of E1, comprising an engineered disulfide mutation selected from the group consisting of G366C and D454C, T411C and Q434C, I137C and A159C, A140C and S149C, L141C and A159C, L141C and A161C, E146C and T160C, V148C and L158C, and T150C and R156C. E3. The variant of E1 or E2, wherein the engineered disulfide mutation is selected from the group consisting of T411C and Q434C, A140C and S149C, L141C and A161C, E146C and T160C, and T150C and R156C. E4. The variant described in E2, wherein the engineered disulfide mutations are G366C and D454C. E5. A variant as described in E2, in which the engineered disulfide mutations are T411C and Q434C. E6. The variant described in E2, wherein the engineered disulfide mutations are I137C and A159C. E7. The variant described in E2, wherein the engineered disulfide mutations are A140C and S149C. E8. The variant described in E2, wherein the engineered disulfide mutations are L141C and A159C. E9. The variant described in E2, wherein the engineered disulfide mutations are L141C and A161C. E10. The mutant described in E2, in which the engineered disulfide mutations are E146C and T160C. E11. The mutant described in E2, in which the engineered disulfide mutations are V148C and L158C. E12. The mutant described in E2, wherein the engineered disulfide mutations are T150C and R156C. E13. The variant as described in E1, comprising two engineered disulfide mutations selected from T411C and Q434C, A140C and S149C, L141C and A161C, and E146C and T160C. E14. The variant as described in E13, wherein the two engineered disulfide mutations are selected from T411C and Q434C, and A140C and S149C. E15. The variant as described in E13, wherein the two engineered disulfide mutations are selected from T411C and Q434C, and L141C and A161C. E16. The mutant of any one of E1 to E15, comprising a cavity-filling mutation. E17. The mutant of E16, wherein the cavity-filling mutation is selected from T49I, S149T, A159V, S291I, T365I, and L473F. E18. The mutant of E17, wherein the cavity-filling mutation is selected from T49I, S149T, and T365I. E19. The mutant described in E17, wherein the cavity-filling mutation is T49I. E20. The mutant described in E17, wherein the cavity-filling mutation is S149T. E21. The mutant described in E17, wherein the cavity-filling mutation is A159V. E22. The mutant described in E17, wherein the cavity-filling mutation is S291I. E23. The mutant described in E17, wherein the cavity-filling mutation is T365I. E24. The mutant described in E17, wherein the cavity-filling mutation is L473F. E25. The variant as described in E16, comprising two cavity-filling mutations selected from T49I, S149T, A159V, S291I, T365I, and L473F. E26. The variant of E25, comprising two cavity-filling mutations selected from T49I, S149T, and T365I. E27. The variant described in E26, wherein the cavity-filling mutations are T49I and S149T. E28. The mutant as described in E26, wherein the cavity-filling mutations are T49I and T365I. E29. The variant described in E26, wherein the cavity-filling mutations are S149T and T365I. E30. The variant described in E16, comprising the cavity-filling mutations T49I, S149T, and T365I. E31. The variant of any one of E1 to E30, comprising a proline substitution mutation. E32. The variant of E31, wherein the proline substitution mutation is selected from the group consisting of L66P, ​​L110P, S132P, N145P, L187P, V449P, and A459P. E33. The variant of E32, wherein the proline substitution mutation is L66P. E34. The variant of E32, wherein the proline substitution mutation is L110P. E35. The variant of E32, wherein the proline substitution mutation is S132P. E36. The variant of E32, wherein the proline substitution mutation is N145P. E37. The variant of E32, wherein the proline substitution mutation is L187P. E38. The variant of E32, wherein the proline substitution mutation is V449P. E39. The variant of E32, wherein the proline substitution mutation is A459P. E40. The variant of any one of E1 to E39, comprising a glycine replacement mutation. E41. The variant of E40, wherein the glycine replacement mutation is selected from the group consisting of G106A, G121A, and G239A. E42. The variant of any one of E2 to E41, comprising mutations Q100R and S101R. E43. (1) A140C and S149C, (2) A140C, S149C, T411C and Q434C; (3) A140C, S149C, T411C, Q434C and A459P; (4) A140C, S149C, T411C, Q434C, and T365I; (5) A140C, S149C, T411C, Q434C, and G239A; (6) A140C, S149C, T411C, Q434C, A459P, G239A, T49I, and T365I; (7) T411C, Q434C, L141C and A161C; (8) T411C, Q434C, L141C, A161C and A459P; (9) T411C, Q434C, L141C, A161C and T49I; (10) T411C, Q434C, L141C, A161C and T365I; (11) T411C, Q434C, L141C, A161C, and G239A; (12) T411C, Q434C, L141C, A161C and S149T; (13) T411C, Q434C, L141C, A161C, A459P, G239A, T49I, S149T, and T365I, and (14) T411C, Q434C, E146C, T160C, A459P, G239A, T49I, S149T, and T365I The variant described in E1, comprising a mutation selected from the group consisting of: E44. (1) T150C, R156C and A459P; (2) T150C, R156C and T49I; (3) T150C, R156C, T49I and A459P; (4) A140C, S149C, T411C, and Q434C; (5) L141C, A161C, T411C, and 434C; (6) A140C, S149C, T411C, Q434C, and A459P; (7) A140C, S149C, G239A, T411C, and Q434C; (8) L141C, A161C, T411C, Q434C and A459P; (9) L141C, A161C, G239A, T411C, and Q434C; (10) T49I, T150C, R156C, G239A, and A459P; (11) A140C, S149C, G239A, T411C, Q434C, and A459P; (12) T49I, A140C, S149C, G239A, T411C, Q434C, and A459P; (13) T49I, A140C, S149C, G239A, T365I, T411C, Q434C, and A459P; (14) L141C, A161C, G239A, T411C, Q434C, and A459P; (15) T49I, L141C, A161C, G239A, T411C, Q434C, and A459P; (16) L141C, A161C, S149T, G239A, T411C, Q434C, and A459P; (17) T49I, L141C, A161C, S149T, G239A, T411C, Q434C, and A459P; (18) T49I, L141C, A161C, S149T, G239A, T365I, T411C, Q434C, and A459P; (19) T49I, S149T and A459P; (20) A140C, S149C and A459P; (21) T49I, A140C and S149C; (22) T49I, A140C, S149C and A459P; (23) T49I, L141C, A161C, T411C, and Q434C; (24) T49I, L141C, A161C, T411C, Q434C, and A459P; (25) L141C, A161C and S149T; (26) L141C, A161C, S149T, and A459P; (27) T49I, L141C, A161C and S149T, and (28) T49I, L141C, A161C, S149T, and A459P The variant described in E1, comprising a mutation selected from the group consisting of: E45. (1) L66P; (2) L187P; (4) A140C, S149C and L187P; (5) T49I; (6) T365I; and (7) T49I and T365I The variant described in E1, comprising a mutation selected from the group consisting of: E46. (1) L187P, Q100R, and S101R; and (2) A140C, S149C, L187P, Q100R, and S101R The variant described in E1, comprising a mutation selected from the group consisting of: E47. (a) containing cysteines (C) at positions 140 (140C) and 149 (149C); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 30, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 29; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 30, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 29; or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (b) containing cysteines (C) at positions 411, 434, 140, and 149; (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 40, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 39; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 40, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 39. or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (c) cysteines (C) at positions 411, 434, 140, and 149 and proline at position 459; (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 76, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 75; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 76, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 75. or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (d) containing cysteines (C) at positions 411, 434, 140, and 149 and an alanine at position 239; (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 80, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 79; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 80, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 79. or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (e) cysteine ​​(C) at positions 411, 434, 140, and 149, proline at position 459, alanine at position 239, and isoleucine at positions 49 and 365; (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 94, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 93; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 94, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 93; comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of The mutant described in E1. E48. The variant of any one of E1 to E47, wherein the F1 polypeptide lacks the entire cytoplasmic domain. E49. The variant of any one of E1 to E48, wherein the F1 polypeptide lacks the cytoplasmic domain and part or all of the transmembrane domain, preferably the F1 polypeptide lacks the cytoplasmic domain and the transmembrane domain. E50. The variant of any one of E1 to E47, wherein the F1 polypeptide comprises an ectodomain, a transmembrane domain, and a cytoplasmic domain, and in a preferred embodiment, the variant comprises a full-length F1 polypeptide and a full-length F2 polypeptide. E51. The variant of any one of E1 to E49, wherein the variant is linked to a trimerization domain, preferably wherein the trimerization domain is a GCN4 leucine zipper or a phage T4 fibritin foldon. E52. The variant of E51, wherein the trimerization domain is a phage T4 fibritin foldon. E53. The variant of E52, wherein the trimerization domain is the phage T4 fibritin foldon of SEQ ID NO: 7. E54. The variant of any one of E50 to E53, wherein the trimerization domain is linked to the C-terminus of the F1 polypeptide. E55. The variant of E54, wherein the trimerization domain is linked to the C-terminus of the F1 polypeptide via a linker. E56. The variant of any one of E50 to E55, wherein the trimerization domain is linked to the C-terminus of the F1 polypeptide via a linker selected from the group consisting of GG, GS, GGGS, or SAIG. E57. The variant of E56, wherein the linker is GGGS. E58. The variant of any one of E1 to E57, in a trimeric form. E59. The variant of any one of E1 to E58, which is in a pre-fusion conformation. E60. A variant of any one of E1 to E59, which is in a pre-fusion conformation and specifically binds to an antibody specific for the hMPV F ectodomain in the pre-fusion conformation but not the hMPV F ectodomain in the post-fusion conformation (such as MPE8 mAb against an hMPV A mutant or hMPV-2 mAb against an hMPV B mutant). E61. The variant of any one of E1 to E60, which is in the prefusion conformation and specifically binds to MPE8 mAb or hMPV-2 mAb, preferably as measured by the ELISA disclosed in the Examples. E62. The variant of any one of E1 to E61, having increased stability compared to the corresponding wild-type hMPV F protein, wherein the stability is measured by binding of the variant to the antibody MPE8 (for an hMPV A variant) or hMPV-2 (for an hMPV B variant). E63. The variant of any one of E1 to E62, wherein the wild-type hMPV F protein is SEQ ID NO:1. E64. The variant of any one of E1 to E62, wherein the wild-type hMPV F protein is SEQ ID NO:2. E65. The variant of any one of E1 to E62, wherein the wild-type hMPV F protein is SEQ ID NO: 3. E66. The variant of any one of E1 to E62, wherein the wild-type hMPV F protein is SEQ ID NO:4. E67. The variant of any one of E1 to E62, wherein the wild-type hMPV F protein is SEQ ID NO:5 or SEQ ID NO:6. E68. The variant of any one of E1 to E62, wherein the wild-type hMPV F protein is SEQ ID NO: 99. E69. The variant of any one of E1 to E62, wherein the wild-type hMPV is subtype A. E70. The variant of any one of E1 to E62, wherein the wild-type hMPV is subtype B. E71. The variant of any one of E1 to E62, wherein the amino acid positions correspond to the reference amino acid sequence of SEQ ID NO: 1. E72. The variant of any one of E1 to E62, wherein the amino acid positions correspond to the reference amino acid sequence of SEQ ID NO:4. E73. A nucleic acid comprising at least one coding sequence encoding at least one mutant of a wild-type hMPV F protein, or an immunogenic fragment or variant thereof, as described in any one of embodiments E1 to E72, preferably E50, wherein the nucleic acid comprises at least one heterologous untranslated region (UTR). E74. The nucleic acid of any one of the preceding embodiments, wherein the at least one heterologous untranslated region is selected from at least one heterologous 5'-UTR and / or at least one heterologous 3'-UTR. E75. At least one heterologous 3'-UTR is CΨCGAGCΨGGΨ ACΨGCAΨGCA CGCAAΨGCΨA GCΨGCCCCΨΨ ΨCCCGΨCCΨG GGΨACCCCGA GΨCΨCCCCCG ACCΨCGGGΨC CCAGGΨAΨGC ΨCCCACCΨCC ACCΨGCCCCA CΨCACCACCΨ CΨGCΨAGΨΨC CAGACACCΨC CCAAGCACGC AGCAAΨGCAG CΨCAAAACGC ΨΨAGCCΨAGC CACACCCCCA CGGGAAACAG CAGΨGAΨΨAA CCΨΨΨAGCAA ΨAAACGAAAG ΨΨΨAACΨAAG CΨAΨACΨAAC CCCAGGGΨΨG 10. The nucleic acid of any one of the preceding embodiments, comprising or consisting of a nucleic acid sequence having at least, and at most exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to GΨCAAΨΨΨCG ΨGCCAGCCAC ACCCΨGGAGC ΨAGC, or a percentage identity between any two thereof. E76. The nucleic acid of any one of the preceding embodiments, wherein at least one heterologous 5'-UTR comprises or consists of a nucleic acid sequence having at least, and at most, exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to GAAΨAAAC ΨAGΨAΨΨCΨΨ CΨGGΨCCCCA CAGACΨCAGA GAGAACCCGC CACC, or a percentage identity between any two thereof. E77. The nucleic acid of any one of the preceding embodiments, comprising at least one poly(A) sequence, preferably comprising 30 to 200 adenosine nucleotides, and / or at least one poly(C) sequence, preferably comprising 10 to 40 cytosine nucleotides. E78. The nucleic acid of any one of the preceding embodiments, which is DNA or RNA. E79. The nucleic acid of any one of the preceding embodiments, which is a coding RNA. E80. The nucleic acid of E79, wherein the coding RNA is mRNA, self-replicating RNA, circular RNA, or replicon RNA. E81. The nucleic acid of any one of the preceding embodiments, wherein the nucleic acid, preferably the coding RNA, is mRNA. E82. The nucleic acid of E81, wherein the mRNA is not a replicon RNA or a self-replicating RNA. E83. The nucleic acid of any one of the preceding embodiments E80 to E82, wherein the mRNA comprises at least one poly(A) sequence comprising 30 to 200 adenosine nucleotides, and the 3' terminal nucleotide is an adenosine. E84. The nucleic acid of any one of the preceding embodiments E78 to E83, wherein the RNA, preferably the coding RNA, comprises a 5'-cap structure, preferably an m7G, cap0, cap1, cap2, modified cap0, or modified cap1 structure, preferably a 5'-cap1 structure. E85. The nucleic acid of any one of the preceding embodiments E78 to E84, wherein the RNA is codon-optimized. E86. The nucleic acid of any one of the preceding embodiments E78 to E85, wherein the RNA comprises chemically modified nucleotides. E87. The nucleic acid of any one of the preceding embodiments E78 to E86, wherein the RNA comprises a 1-methylpseudouridine substitution, preferably all uridines of the RNA are replaced by 1-methylpseudouridine. E88. The nucleic acid of any one of the preceding embodiments E78 to E87, wherein the RNA is purified RNA, preferably RNA purified by RP-HPLC and / or TFF. E89. The nucleic acid of any one of the preceding embodiments E78 to E88, wherein the RNA comprises the nucleic acid sequence of any of SEQ ID NO:391, SEQ ID NO:393, SEQ ID NO:395; SEQ ID NO:397, SEQ ID NO:399, SEQ ID NO:512, SEQ ID NO:514, SEQ ID NO:516, SEQ ID NO:518, SEQ ID NO:520, SEQ ID NO:522, SEQ ID NO:524 and SEQ ID NO:526. E90. A composition comprising at least one nucleic acid of any one of the preceding embodiments E73 to E89. E91. A composition comprising at least one nucleic acid of any one of the preceding embodiments E73 to E89, comprising at least one pharmaceutically acceptable carrier. E92. A composition comprising at least one nucleic acid according to any one of the preceding embodiments E73 to E89, which is a multivalent composition comprising a plurality or more than at least one of the nucleic acids according to any one of E73 to E89. E93. A composition comprising at least one nucleic acid of any one of the preceding embodiments E73 to E89, wherein the composition comprises RNA having an RNA integrity of 70% or greater. E94. A composition comprising at least one nucleic acid of any one of the preceding embodiments E73 to E89, wherein the composition comprises RNA with a degree of capping of 70% or more, preferably at least 70%, 80%, or 90% of mRNA species contain Cap 1 structures. E95. A composition comprising at least one nucleic acid of any one of the preceding embodiments E73 to E89, wherein the at least one nucleic acid is complexed or associated, or at least partially complexed or partially associated, with one or more cationic or polycationic compounds, preferably cationic or polycationic polymers, cationic or polycationic polysaccharides, cationic or polycationic lipids, cationic or polycationic proteins, cationic or polycationic peptides, or any combination thereof. E96. A composition comprising at least one nucleic acid of any one of the preceding embodiments E73 to E89, wherein the at least one nucleic acid is complexed or associated with one or more lipids or lipid-based carriers, thereby forming a liposome, lipid nanoparticle (LNP), lipoplex, and / or nanoliposome, preferably encapsulating the at least one nucleic acid. E97. A composition comprising at least one nucleic acid of any one of the preceding embodiments E73 to E89, wherein the at least one nucleic acid is complexed with one or more lipids, thereby forming a lipid nanoparticle. E98.LNP has formula III-3:

[0125] [ka] The composition of any one of the preceding embodiments E95 to E96, comprising a cationic lipid according to E99.LNP has formula (IVa):

[0126] [ka] The composition of any one of the preceding embodiments E96 to E98, comprising a PEG lipid of The composition of embodiment E99, wherein E100.n has an average value ranging from 30 to 60, preferably n has an average value of about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and most preferably n has an average value of 49 or 45. E101.LNP has formula (IVa):

[0127] [ka] The composition of any one of the preceding embodiments E96 to E100, comprising a PEG lipid of formula (I), where n is an integer selected such that the average molecular weight of the PEG lipid is about 2500 g / mol. E102. The composition of any one of the preceding embodiments E96 to E101, wherein the LNP comprises one or more neutral lipids, and / or one or more steroids or steroid analogues. E103. The composition of any one of the preceding embodiments E96 to E101, wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and preferably the molar ratio of cationic lipid to DSPC is in the range of about 2:1 to about 8:1. E104. The composition of any one of the preceding embodiments E96 to E103, wherein the steroid is cholesterol, and preferably the molar ratio of cationic lipid to cholesterol is in the range of about 2:1 to about 1:1. E105. The composition of any one of the preceding embodiments E96 to E104, wherein the LNP comprises: (i) at least one cationic lipid, preferably a lipid of formula (III), more preferably lipid III-3; (ii) at least one neutral lipid, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) at least one steroid or steroid analog, preferably cholesterol; and (iv) at least one polymer-conjugated lipid, preferably a PEG lipid derived from formula (IVa, having n=49), wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG lipid. E106. The composition of any one of the preceding embodiments E96 to E105, wherein the LNP comprises: (i) at least one cationic lipid, preferably a lipid of formula (III), more preferably lipid III-3; (ii) at least one neutral lipid, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) at least one steroid or steroid analog, preferably cholesterol; and (iv) at least one polymer-conjugated lipid, preferably a PEG lipid from formula (IVa, having n=45), wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG lipid. E107. The composition of any one of the preceding embodiments E105 to E106, wherein (i)-(iv) are in a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7, or more preferably 47.4:10:40.9:1.7. E108. The composition of any one of the preceding embodiments E96 to E107, wherein the nucleic acid is RNA and the composition comprises less than about 20% free (uncomplexed or unencapsulated) RNA, preferably less than about 15% free RNA, more preferably less than about 10% free RNA. E109. The composition of any one of the preceding embodiments E96 to E108, wherein the wt / wt ratio of lipid to nucleic acid is from about 10:1 to about 60:1, preferably from about 20:1 to about 30:1, for example about 25:1. E110. The composition of any one of the preceding embodiments E96 to E109, wherein the n / p ratio of the LNPs encapsulating the nucleic acid is in the range of about 1 to about 10, preferably in the range of about 5 to about 7, and more preferably about 6. E111. The composition of any one of the preceding embodiments E96 to E110 having a polydispersity index (PDI) value of less than about 0.4, preferably less than about 0.3, more preferably less than about 0.2, and most preferably less than about 0.1. E112. The composition of any one of the preceding embodiments E96 to E111, wherein the LNPs have a Z-average size in the range of about 60 nm to about 120 nm, preferably less than about 120 nm, more preferably less than about 100 nm, and most preferably less than about 80 nm. E113. The composition of any one of the preceding embodiments E96 to E112, wherein the LNPs comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of LNPs having a particle size greater than about 500 nm. E114. The composition of any one of the preceding embodiments E96 to E113, wherein the LNPs comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% LNPs having a particle size smaller than about 20 nm. E115. The composition of any one of the preceding embodiments E96 to E114, wherein the LNP comprises (i) at least one cationic lipid; (ii) at least one neutral lipid; (iii) at least one steroid or steroid analog; and (iv) at least one PEG-lipid, wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid. E116. The composition of any one of the preceding embodiments E96 to E115, wherein the LNP comprises (i) at least one cationic lipid according to formula III-3; (ii) DSPC; (iii) cholesterol; and (iv) a PEG-lipid according to formula IVa, wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid. E117. The composition of any one of the preceding embodiments E90 to E116, which is a freeze-dried composition. E118. An immunogenic composition comprising a variant according to any one of E1 to E72, a nucleic acid according to any one of E73 to E89, or a composition according to any one of E90 to E117. E119. The immunogenic composition of E118, comprising a variant described in any one of E1 to E72, a nucleic acid described in any one of E73 to E89, or a composition described in any one of E90 to E117, wherein the wild-type hMPV F protein is subtype A, and a variant described in any one of E1 to E72, a nucleic acid described in any one of E73 to E89, or a composition described in any one of E90 to E117, wherein the wild-type hMPV F protein is subtype B. E120. The immunogenic composition of any one of embodiments E118 to E119, further comprising a PIV1 antigen selected from the group consisting of a variant of the wild-type PIV1 F protein, and a nucleic acid encoding a variant of the wild-type PIV1 F protein. E121. The immunogenic composition of embodiment E120, wherein the PIV1 antigen is a variant of the wild-type PIV1 F protein. E122. The immunogenic composition of embodiment E121, wherein the PIV1 antigen is a variant of a wild-type PIV1 F protein from the present disclosure, preferably from any of E1 to E56 of section C of the present disclosure. E123. The immunogenic composition of embodiment E121, wherein the PIV1 antigen comprises a nucleic acid encoding a variant of the wild-type PIV1 F protein. E124. The immunogenic composition of embodiment E123, wherein the PIV1 antigen comprises a nucleic acid encoding a variant of the wild-type PIV1 F protein from the present disclosure, preferably a nucleic acid from any of E57 to E73 of section C of the present disclosure. E125. The immunogenic composition of any one of embodiments E118 to E124, further comprising a PIV3 antigen selected from the group consisting of a variant of the wild-type PIV3 F protein, and a nucleic acid encoding a variant of the wild-type PIV3 F protein. E126. The immunogenic composition of embodiment E125, wherein the PIV3 antigen is a variant of the wild-type PIV3 F protein. E127. The immunogenic composition of embodiment E125, wherein the PIV3 antigen is a variant of a wild-type PIV3 F protein from the present disclosure, preferably from any of E1 to E52 of section D of the present disclosure. E128. The immunogenic composition of embodiment E125, wherein the PIV3 antigen is a variant of the wild-type PIV3 F protein disclosed in WO2018081289 or WO22207839. E129. The immunogenic composition of embodiment E125, wherein the PIV3 antigen comprises a nucleic acid encoding a variant of the wild-type PIV3 F protein. E130. The immunogenic composition of embodiment E125, wherein the PIV3 antigen comprises a nucleic acid encoding a variant of a wild-type PIV3 F protein from the present disclosure, preferably a nucleic acid from any of E53 to E69 of section D of the present disclosure. E131. The immunogenic composition of embodiment E125, wherein the PIV3 antigen comprises a nucleic acid encoding a variant of the wild-type PIV3 F protein disclosed in WO2018081289 or WO2022207839. E132. The immunogenic composition of any one of E118 to E131, further comprising a RSV antigen selected from the group consisting of a subtype A wild-type RSV F protein variant and a nucleic acid encoding a subtype A wild-type RSV F protein variant. E133. The immunogenic composition of embodiment E132, wherein the RSV antigen is a variant of a subtype A wild-type RSV F protein. E134. The immunogenic composition of embodiment E132, wherein the RSV antigen is a nucleic acid encoding a variant of a subtype A wild-type RSV F protein. E135. The immunogenic composition of any one of embodiments E132 to E134, wherein the variant of the subtype A wild-type RSV F protein is disclosed in one of WO2009 / 079796, WO2010 / 149745, WO2011 / 008974, WO2014 / 160463, WO2014 / 174018, WO2014 / 202570, WO2015 / 013551, WO2015 / 177312, WO2017 / 005848, WO2017 / 174564, WO2017 / 005844, WO2017 / 109629, WO2022 / 002894, and WO2018 / 109220. E136. The immunogenic composition of any one of embodiments E118 to E135, further comprising a RSV antigen selected from the group consisting of a subtype B wild-type RSV F protein variant and a nucleic acid encoding a subtype B wild-type RSV F protein variant. E137. The immunogenic composition of embodiment E136, wherein the RSV antigen is a variant of the wild-type RSV F protein of subtype B. E138. The immunogenic composition of embodiment E136, wherein the RSV antigen comprises a nucleic acid encoding a variant of a wild-type RSV F protein of subtype B. E139. The immunogenic composition of embodiment E137 or E138, wherein the variant of the subtype B wild-type RSV F protein is disclosed in one of WO2009 / 079796, WO2010 / 149745, WO2011 / 008974, WO2014 / 160463, WO2014 / 174018, WO2014 / 202570, WO2015 / 013551, WO2015 / 177312, WO2017 / 005848, WO2017 / 174564, WO2017 / 005844, WO2017 / 109629, WO2022 / 002894, and WO2018 / 109220.

[0128] 2. hMPV F protein mutants In some aspects, the present invention provides mutants of wild-type hMPV F proteins, which exhibit introduced mutations in their amino acid sequences compared to the amino acid sequence of the corresponding wild-type hMPV F protein and are immunogenic against the wild-type hMPV F protein in a pre-fusion conformation or against viruses containing the wild-type F protein. In certain embodiments, the hMPV F mutants possess certain beneficial characteristics compared to the corresponding wild-type F protein, such as increased immunogenic properties or improved stability of the mutant's pre-fusion conformation or the mutant's pre-fusion trimer conformation. In still other embodiments, the present disclosure provides hMPV F mutants that exhibit one or more introduced mutations described herein and bind to a pre-fusion-specific antibody selected from MPE8 mAb (against an hMPV A mutant) or hMPV-2 mAb (against an hMPV B mutant).

[0129] The amino acid mutations introduced in the hMPV F protein mutant include amino acid substitutions, deletions, or additions. In some embodiments, the only mutations in the amino acid sequence of the mutant are amino acid substitutions compared to the wild-type hMPV F protein.

[0130] The amino acid sequences of numerous native hMPV F proteins from various hMPV subtypes, as well as the nucleic acid sequences encoding such proteins, are known in the art. For example, the sequences of several subtype A and B hMPV F0 precursor proteins are set forth in SEQ ID NOS: 1-6 and 99.

[0131] The native hMPV F protein exhibits significant sequence conservation across hMPV subtypes. For example, the hMPV subtype A and B consensus sequences share approximately 94% sequence identity across the F precursor molecule. Nearly all identified hMPV F precursor sequences are 539 amino acids in length, with slight differences in length. The sequence identity across various native hMPV F proteins is known in the art (see, for example, Yang et al., Virology Journal 2009, 6:138).

[0132] Given the substantial conservation of hMPV F protein sequences, one skilled in the art can easily compare amino acid positions among various native hMPV F protein sequences to identify corresponding hMPV F protein amino acid positions among various hMPV strains and subtypes. For example, the protease cleavage site is located at the same amino acid position across nearly all identified native hMPV F precursor proteins. Thus, the conservation of native hMPV F protein sequences across strains and subtypes allows for the use of a reference hMPV F protein sequence for comparison of amino acids at specific positions within the hMPV F protein. For purposes of this disclosure (unless the context indicates otherwise), hMPV F protein amino acid positions are provided with reference to the sequence of the F precursor polypeptide set forth in SEQ ID NO: 1 (corresponding to the amino acid sequence of the full-length native F precursor polypeptide of the hMPV A2b strain; GenBank identifiers ACJ53569.1 (amino acid) and EU857558.1 (nucleotide)).

[0133] For purposes of this disclosure (unless the context indicates otherwise), hMPV AF protein amino acid positions are given with reference to the sequence of the F0 precursor polypeptide set forth in SEQ ID NO: 1 (amino acid sequence of the full-length native F precursor polypeptide of hMPV A2b strain; corresponding to Genbank identifiers ACJ53569.1 (amino acids) and EU857558.1 (nucleotides)).

[0134] For purposes of this disclosure (unless the context indicates otherwise), hMPV BF protein amino acid positions are given with reference to the sequence of the F0 precursor polypeptide set forth in SEQ ID NO: 4 (the amino acid sequence of the full-length consensus F precursor polypeptide of hMPV B strain).

[0135] The consensus sequence for hMPV B was obtained as follows: the entire genome sequence for hMPV B was downloaded from the NCBI GenBank database as a GenBank file. The fusion protein gene sequences were filtered by sequence length to include only the complete coding DNA sequence. The translated fusion protein sequences were then analyzed from the GenBank file and saved as a FASTA file. Multiple sequence alignment of the collected sequences was performed using Muscle v5. A position-specific score matrix (PSSM) was created to summarize the alignment information. For each column in the alignment, the number of each amino acid character was counted and summed. The consensus sequence at each position was calculated as the most commonly occurring amino acid type in the PSSM table. The final consensus sequence was then extracted and saved as a FASTA file.

[0136] However, it should be noted, and one of skill in the art will understand, that different hMPV F0 sequences may have different numbering systems, e.g., if there are additional amino acid residues added or removed compared to SEQ ID NO: 1. Thus, when specific amino acid residues are referred to by their numbers, it should be understood that the description is not limited to only the amino acid at that exact numbered position when counting from the beginning of a given amino acid sequence, but rather that the equivalent / corresponding amino acid residue in any and all hMPV F sequences is intended, even if that residue is not at the same exact numbered position, e.g., the hMPV sequence is shorter or longer than SEQ ID NO: 1 or has insertions or deletions compared to SEQ ID NO: 1.

[0137] 2-1. Structure of hMPV F protein mutants The hMPV F protein variants provided by the present disclosure comprise an F1 polypeptide and an F2 polypeptide. In some embodiments, the variants further comprise a trimerization domain. In some embodiments, either the F1 polypeptide or the F2 polypeptide comprises at least one introduced modification (e.g., an amino acid substitution), as described in detail herein below. In some other embodiments, each of the F1 polypeptide and the F2 polypeptide comprises at least one introduced modification (e.g., an amino acid substitution), as described in detail herein below.

[0138] 2-1(a). F1 and F2 polypeptides of hMPV F mutants In some embodiments, the variant is in the mature form of the hMPV F protein, which comprises two separate polypeptide chains, ie, an F1 polypeptide and an F2 polypeptide.

[0139] The F1 polypeptide chain of a variant can be the same length as the full-length F1 polypeptide of the corresponding wild-type hMPV F protein; however, it can also have a deletion, such as a deletion of 1 to up to 36 amino acid residues from the C-terminus of the full-length F1 polypeptide. The full-length F1 polypeptide of an hMPV F variant corresponds to amino acids 103 to 539 of the native hMPV F precursor and includes (N- to C-terminus) the extracellular region (residues 103-489), the transmembrane domain (residues 490-514), and the cytoplasmic domain (residues 515-539). It should be noted that amino acid residues 490 and beyond in the native F1 polypeptide sequence are optional in the F1 polypeptides of the hMPV F variants provided herein and therefore may be absent from the variant F1 polypeptides.

[0140] In some embodiments, the F1 polypeptide of the hMPV F mutant lacks the entire cytoplasmic domain. In other embodiments, the F1 polypeptide lacks the cytoplasmic domain and part or all of the transmembrane domain. In some specific embodiments, the mutant comprises an F1 polypeptide in which amino acid residues 490-539 are absent. Typically, for mutants linked to a trimerization domain, such as a foldon, amino acids 490-539 may be absent. Thus, in some specific embodiments, amino acid residues 490-539 are absent from the mutant F1 polypeptide. In yet other specific embodiments, the F1 polypeptide of the hMPV F mutant comprises or consists of amino acid residues 103-489 of a native F0 polypeptide sequence, such as any of the F0 precursor sequences set forth in SEQ ID NOS: 1-6 and 99.

[0141] On the other hand, the F1 polypeptide of an hMPV F mutant may include a C-terminal linkage to a trimerization domain, such as a foldon. Many of the sequences of the hMPV F mutants disclosed herein include PreScission cleavage sites and Strep Tag II sequences that are not essential for the function of the hMPV F protein, such as inducing an immune response. Those of skill in the art will recognize such sequences and understand that, where appropriate, these sequences are not included in the disclosed hMPV F mutants.

[0142] In the hMPV F mutants provided by the present disclosure, the F2 polypeptide chain can be the same length as the full-length F2 polypeptide of the corresponding wild-type hMPV F protein; it can also have a deletion, such as a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues from the N-terminus or C-terminus of the F2 polypeptide.

[0143] Variants of the F0 form (i.e., a single-chain polypeptide comprising an F2 polypeptide joined to an F1 polypeptide) or the F1-F2 heterodimeric form can form protomers. Variants can also be in the form of trimers comprising three identical protomers. Furthermore, variants can be glycosylated proteins (i.e., glycoproteins) or non-glycosylated proteins. Variants of the F0 form can contain or lack a signal peptide sequence.

[0144] The F1 and F2 polypeptides of the hMPV F protein variants into which one or more mutations have been introduced can be derived from any wild-type hMPV F protein known in the art or discovered in the future, including, without limitation, the F protein amino acid sequences of hMPV subtype A and subtype B strains, or any other subtype. In some embodiments, the hMPV F variants comprise F1 and / or F2 polypeptides from an hMPV A virus, e.g., F1 and / or F2 polypeptides from a known hMPV F0 precursor protein, such as those set forth in any one of SEQ ID NOS: 1-3, into which one or more mutations have been introduced. In some other embodiments, the hMPV F variants comprise F1 and / or F2 polypeptides from an hMPV B virus, e.g., F1 and / or F2 polypeptides from a known hMPV F0 precursor protein, such as those set forth in any one of SEQ ID NOS: 4-6 or 99, into which one or more mutations have been introduced.

[0145] In some embodiments, the hMPV F protein variant comprises an F1 polypeptide, an F2 polypeptide, and one or more introduced amino acid mutations described herein below, wherein the F1 polypeptide comprises 350 contiguous amino acids and is at least 90, 95, 98, or 99 percent identical to amino acids 103 to 489 of any of SEQ ID NOs: 1 to 3, and the F2 polypeptide comprises 70 contiguous amino acids and is at least 90, 95, 98, or 99 percent identical to amino acids 21 to 102 of any of SEQ ID NOs: 1 to 3, and the hMPV F protein variant is stabilized in a pre-fusion trimer conformation both as a monomer and as a trimer.

[0146] In some embodiments, the hMPV F protein variant comprises an F1 polypeptide, an F2 polypeptide, and one or more introduced amino acid mutations described herein below, wherein the F1 polypeptide comprises 350 contiguous amino acids and is at least 90, 95, 98, or 99 percent identical to amino acids 103-489 of any of SEQ ID NOs: 4-6 or 99, and the F2 polypeptide comprises 70 contiguous amino acids and is at least 90, 95, 98, or 99 percent identical to amino acids 21-102 of any of SEQ ID NOs: 4-6 or 99, and the hMPV F protein variant is stabilized in a pre-fusion trimer conformation both as a monomer and as a trimer.

[0147] 2-1(b) Trimerization domain In some embodiments, the hMPV F mutants provided by the present disclosure are linked to a trimerization domain, which in some embodiments promotes the formation of trimers of three F1 / F2 heterodimers.

[0148] Some exogenous trimerization domains that promote the formation of stable trimers of soluble proteins are known in the art.Non-limiting examples of such trimerization domains that can be linked to the variants provided by the present disclosure include: (1) GCN4 leucine zipper (Harbury et al., 1993 Science 262:1401-1407); (2) trimerization motif from pulmonary surfactant protein (Hoppe et al., 1994 FEBS Lett 344:191-195); (3) collagen (McAlinden et al., 2003 Biol Chem 278:42200-42207); and (4) phage T4 fibritin foldon (Miroshnikov et al., 1998 Protein Eng 11:329-414).

[0149] Typically, the trimerization domain is located C-terminal to the F1 polypeptide. It may be directly attached to the F1 polypeptide chain. Optionally, the multimerization domain is connected to the F1 polypeptide via an amino acid linker, such as a linker with the sequence GG, GS, GGGS, or SAIG. The linker may also be a longer linker (e.g., containing a repeat sequence GG). A preferred linker is GGGS. Many conformationally neutral linkers are known in the art and can be used in the variants provided by the present disclosure. In some embodiments, the F1 variant containing a foldon domain comprises a protease cleavage site for removing the foldon domain from the F1 polypeptide, such as a thrombin site between the F1 polypeptide and the foldon domain.

[0150] In some embodiments, the foldon domain is linked to the F mutant at the C-terminus of the F1 polypeptide. In a specific embodiment, the foldon domain is a T4 fibritin foldon domain, such as the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 7).

[0151] 2-2. Mutations introduced into hMPV F protein mutants The hMPV F mutants provided by the present disclosure include an F1 polypeptide and an F2 polypeptide, wherein either (1) the F1 polypeptide or (2) the F2 polypeptide, or (3) both the F1 polypeptide and the F2 polypeptide, contain one or more introduced amino acid mutations compared to the amino acid sequence of the corresponding native F protein. The introduction of such amino acid mutations in hMPV F mutants confers beneficial properties to the mutants, such as enhanced immunogenicity, improved stability, improved expression or formation, or improved stability of a particular desired physical form or conformation of the mutant. Such introduced amino acid mutations are referred to as "engineered disulfide bond mutations," "cavity-filling mutations," "proline substitution mutations," or "glycine replacement mutations," and are described in detail herein below. hMPV F mutants containing any additional mutations are also encompassed by the present invention, so long as the immunogenic properties of the mutants are not substantially adversely affected by the additional mutations.

[0152] 2-2(a) Engineered disulfide bond mutations In some embodiments, the hMPV F mutants provided by the present disclosure contain one or more engineered disulfide bond mutations. The term "engineered disulfide bond mutation" refers to the mutation of a pair of amino acid residues in the wild-type hMPV F protein to a pair of cysteine ​​residues. The pair of introduced cysteine ​​residues allows for the formation of a disulfide bond between the introduced cysteine ​​residues, and the disulfide bond serves to stabilize the conformation or oligomeric state of the protein, such as the pre-fusion conformation. To stabilize the pre-fusion conformation of the mutant, the pair of residues mutated to cysteines should be in close proximity in the pre-fusion conformation but far apart in the post-fusion conformation. Preferably, the distance between the pair of residues (e.g., beta carbons) is less than 8 Å in the pre-fusion conformation but greater than 20 Å in the post-fusion conformation.

[0153] In some embodiments, the hMPV F protein mutant contains only one engineered disulfide mutation (a "single engineered disulfide mutation"). In other embodiments, the hMPV F protein mutant contains at least two engineered disulfide mutations, with each pair of cysteine ​​residues of the engineered disulfide mutations appropriately positioned when the hMPV F protein mutant is in the prefusion conformation (a "double engineered disulfide mutation").

[0154] In some specific embodiments, the present disclosure provides hMPV F mutants comprising at least one engineered disulfide bond mutation, wherein the mutants comprise the same introduced mutations as in any of the exemplary mutants provided in Tables 9, 13-16, 19, and 23-28. The exemplary hMPV F mutants provided in Tables 9, 13-16, 19, and 23-28 are based on the same native F sequence of hMPV A strain TN / 95 / 3-54 (SEQ ID NO: 128) or the consensus F sequence of hMPV B strain (SEQ ID NO: 129), depending on whether the mutant is an hMPV A or hMPV B F protein mutant. The same introduced mutations in each of the mutants can be made to the native F polypeptide sequence of any other hMPV subtype or strain to arrive at different hMPV F mutants, such as the native F polypeptide sequences set forth in any of SEQ ID NOs: 1-6 and 99 or from any other hMPV A or B strain. hMPV F mutants based on the native F0 polypeptide sequence of any other hMPV subtype or strain and containing any engineered disulfide mutations are also within the scope of the present invention. In certain embodiments, the hMPV F protein mutant comprises at least one engineered disulfide mutation selected from the group consisting of G366C and D454C, T411C and Q434C, I137C and A159C, A140C and S149C, L141C and A159C, L141C and A161C, E146C and T160C, V148C and L158C, and T150C and R156C, 366C and 454C, 411C and 434C, 137C and 159C, 140C and 149C, 141C and 159C, 141C and 161C, 146C and 160C, 148C and 158C, and 150C and 156C.

[0155] 2-2(b) Cavity-filling mutation In other embodiments, the present disclosure provides hMPV F mutants containing one or more cavity-filling mutations. The term "cavity-filling mutation" refers to the replacement of an amino acid residue in the wild-type hMPV F protein with an amino acid predicted to fill the internal cavity of the mature hMPV F protein. In one application, such cavity-filling mutations contribute to stabilizing the pre-fusion conformation of the hMPV F protein mutant. For example, the amino acids to be replaced for cavity-filling mutations typically include small aliphatic amino acids (e.g., Gly, Ala, and Val) or small polar amino acids (e.g., Ser and Thr). They may also include amino acids that are buried in the pre-fusion conformation but exposed to the solvent in the post-fusion conformation. The replacement amino acids may include aliphatic amino acids (Val, Ile, Leu, and Met) that are larger in size than the amino acid being replaced, aromatic amino acids (His, Phe, Tyr, and Trp), or polar amino acids (Thr).

[0156] In some specific embodiments, the hMPV F protein mutant is (1) amino acid substitutions at positions 49, 291, or 365 with I, V, L, M, F, Y, or H; (2) substitution of the amino acid at position 149 with T, V, or I; (3) substitution of the amino acid at position 159 with V, I, or L; (4) substitution of amino acid at position 473 with F or W The mutations comprise one or more cavity-filling mutations selected from the group consisting of:

[0157] In some specific embodiments, the disclosure provides hMPV F mutants comprising one or more cavity-filling mutations, wherein the mutants comprise cavity-filling mutations in any of the mutants provided in Tables 12, 15, 16, 22, 25, 26, 27, and 29. The hMPV F mutants provided in Tables 12, 15, 16, 22, 25, 26, 27, and 29 are based on the same native F sequence of hMPV A strain TN / 95 / 3-54 (SEQ ID NO: 128) or the consensus F sequence of hMPV B strain (SEQ ID NO: 129), depending on whether the mutant is an hMPV A or hMPV B F protein mutant. The same introduced mutations in each of the mutants can be made to the native F polypeptide sequence of any other hMPV subtype or strain to arrive at different hMPV F mutants, such as the native F polypeptide sequences set forth in any of SEQ ID NOs: 1-6 and 99 or from any other hMPV A or B strain. hMPV F mutants based on the native F0 polypeptide sequence of any other hMPV subtype or strain and containing one or more cavity-filling mutations are also within the scope of the present invention. In certain embodiments, the hMPV F protein mutants provided by the present disclosure contain at least one cavity-filling mutation selected from the group consisting of T49I, S149T, or T365I.

[0158] 2-2(c) Proline substitution mutation In yet other embodiments, the present disclosure provides hMPV F protein variants comprising one or more proline substitution mutations, the term "proline substitution mutation" referring to the substitution of an amino acid with a proline that prevents structural refolding that occurs during the transition from the pre-fusion to the post-fusion conformation.

[0159] In some specific embodiments, the hMPV F protein mutant comprises at least one proline substitution mutation selected from the group consisting of 66P, 110P, 132P, 145P, 187P, 449P, and 459P, such as L66P, ​​L110P, S132P, N145P, L187P, V449P, and A459P. In some specific embodiments, the present disclosure provides hMPV F mutants comprising one or more proline substitution mutations, wherein the mutant comprises a proline substitution mutation in any of the mutants provided in Tables 10, 13, 16, 20, 23, and 27-29. The hMPV F mutants provided in Tables 10, 13, 16, 20, 23, and 27-29 are based on the same native F sequence of hMPV A strain TN / 95 / 3-54 (SEQ ID NO: 128) or the consensus F sequence of hMPV B strain (SEQ ID NO: 129), depending on whether the mutant is an hMPV A or hMPV B F protein mutant. The same introduced mutations in each of the mutants can be performed on the native F polypeptide sequence of any other hMPV subtype or strain to arrive at different hMPV F mutants, such as the native F polypeptide sequences set forth in any of SEQ ID NOs: 1-6 and 99 or from any other hMPV A or B strain. hMPV F mutants based on the native F polypeptide sequence of any other hMPV subtype or strain and containing one or more proline substitution mutations are also within the scope of the present invention. In certain embodiments, the hMPV F protein mutant contains the mutation A459P. In certain embodiments, the hMPV F protein mutant comprises the mutation L66P or L187P.

[0160] 2-2(d) Glycine replacement mutation In yet other embodiments, the disclosure provides hMPV F protein variants comprising one or more glycine replacement mutations, the term "glycine replacement mutation" referring to the replacement of glycine in the center of the α-helix with another amino acid, preferably an amino acid without a Cβ substitution, such as Ala, Leu, or Met, to improve protein stability.

[0161] In some specific embodiments, the hMPV F protein mutant comprises at least one glycine replacement mutation selected from the group consisting of G106A, G121A, and G239A. In some specific embodiments, the present disclosure provides hMPV F mutants comprising one or more glycine replacement mutations, wherein the mutant comprises a glycine replacement mutation in any of the mutants provided in Tables 11, 14, 16, 21, 24, 26, and 28. The hMPV F mutants provided in Tables 11, 14, 16, 21, 24, 26, and 28 are based on the same native F sequence of hMPV A strain TN / 95 / 3-54 (SEQ ID NO: 128) or the consensus F sequence of hMPV B strain (SEQ ID NO: 129), depending on whether the mutant is an hMPV A or hMPV B F protein mutant. The same introduced mutations in each of the variants can be made to the native F0 polypeptide sequence of any other hMPV subtype or strain to arrive at different hMPV F variants, such as the native F0 polypeptide sequence set forth in any of SEQ ID NOS: 1-6 and 99, or from any other hMPV A or B strain. hMPV F variants based on the native F0 polypeptide sequence of any other hMPV subtype or strain and containing one or more glycine replacement mutations are also within the scope of the present invention. In certain embodiments, the hMPV F protein variant contains the mutation G239A.

[0162] 2-2(e) Engineered combinations of disulfide bond mutations, cavity-filling mutations, proline substitution mutations, and glycine replacement mutations In another aspect, the present disclosure provides hMPV F protein variants that include a combination of two or more different types of mutations selected from engineered disulfide bond mutations, cavity-filling mutations, proline substitution mutations, and glycine replacement mutations, each of which is described above.

[0163] In some embodiments, the mutant comprises at least one engineered disulfide bond mutation and at least one cavity-filling mutation. In some specific embodiments, the hMPV F mutant comprises a combination of mutations set forth in Tables 15, 16, 25, 26, and 27.

[0164] In some further embodiments, the hMPV F protein mutant comprises at least one engineered disulfide mutation and at least one proline substitution mutation, hi some specific embodiments, the hMPV F mutant comprises a combination of mutations set forth in Tables 13, 16, 23, and 26-28.

[0165] In some further embodiments, the hMPV F protein mutant comprises at least one engineered disulfide mutation and at least one glycine replacement mutation, hi some specific embodiments, the hMPV F mutant comprises a combination of mutations set forth in Tables 14, 16, 24, 26, and 28.

[0166] In some further embodiments, the hMPV F protein mutant comprises at least one proline substitution mutation and at least one cavity-filling mutation. In some specific embodiments, the hMPV F mutant comprises a combination of mutations set forth in Table 29.

[0167] In some further embodiments, the hMPV F protein mutant comprises at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one proline substitution mutation, hi some specific embodiments, the hMPV F mutant comprises a combination of mutations set forth in Tables 16, 26, and 27.

[0168] In some further embodiments, the hMPV F protein mutant comprises at least one engineered disulfide mutation, at least one proline substitution mutation, and at least one glycine replacement mutation, hi some specific embodiments, the hMPV F mutant comprises a combination of mutations set forth in Tables 16, 27, and 28.

[0169] In some further embodiments, the hMPV F protein mutant comprises at least one engineered disulfide mutation, at least one cavity-filling mutation, at least one proline substitution mutation, and at least one glycine replacement mutation, hi some specific embodiments, the hMPV F mutant comprises a combination of mutations set forth in Tables 16 and 26.

[0170] In certain embodiments, the hMPV F protein mutant comprises the mutations A140C, S149C, and L187P.

[0171] In certain embodiments, the hMPV F protein mutant comprises any of the mutations or combinations of mutations disclosed above in combination with Q100R and S101R.

[0172] In certain embodiments, the hMPV F protein mutant is, for example, E26C and G439C; N46C and L158C, T49C and A161C, L50C and V162C, E51C and R163C; E51C and K166C; V104C and N457C, L110C and N322C, A113C and D336C, A116C and A338C, A140C and A147C, S291C and S443C; S293C and S443C; S293C and S444C; S355C and V442C; T365C and V463C, S22C and H435 C; G53C and K166C; G53C and V169C; E305C and N457C; S291C and L302C, V47C and A159C; T127C and N153C, G121C and I / F258C, F48C and T160C, and / or T365C and Q455C, L219K, V2311, S376T, G366S, S194Q, K166E, T49E, L187F, L473F, S347Q, H435E, H435D or H435N, G106W, A107F, T160 M, L158W, I128F, A190M, V118F, V118M, Q426W, L165F, V191I, T160V, S149V, I137L, S149I, V169I, N46V, T49I, V / I122L, S192L, T317L, V162 F, V162W, L105I, L105F, L105W, L134I, A117M, S347M, S347K, S347Q, V47M, G261M, I268M, S470Y, V231I, A374V, I217V, S355F, A86P, A107P, A 113P, T114P, V148P, S443P, D461P, L130P, L141P, K142P, E146P, L151P, N153P, V162P, A / D185P, D186P, L187P, K188P, N342P, A344P, L66N, L73E, N145E, Q195K, E453Q, L66D, K188R, H368R, D461E, T49E, V262D, etc., including any of the mutations or combinations of mutations disclosed above in combination with any mutation disclosed in WO2022076669.

[0173] In certain other specific embodiments, the invention provides an hMPV F mutant, wherein the mutant comprises a cysteine ​​(C) at position 140 (140C) and at position 149 (149C), (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 30, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 29; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 30, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 29; The present invention provides an hMPV F mutant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0174] In certain other specific embodiments, the invention provides an hMPV F mutant, wherein the mutant comprises a cysteine ​​(C) at positions 411, 434, 140, and 149, (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 40, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 39; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 40, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 39. The present invention provides an hMPV F mutant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0175] In certain other specific embodiments, the invention provides an hMPV F mutant, wherein the mutant comprises cysteines (C) at positions 411, 434, 140, and 149 and a proline at position 459, (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 76, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 75; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 76, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 75. The present invention provides an hMPV F mutant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0176] In certain other specific embodiments, the invention provides an hMPV F mutant, wherein the mutant comprises cysteines (C) at positions 411, 434, 140, and 149 and an alanine at position 239, (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 80, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 79; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 80, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 79; The present invention provides an hMPV F mutant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0177] In certain other specific embodiments, the invention provides an hMPV F mutant, wherein the mutant comprises cysteine ​​(C) at positions 411, 434, 140, and 149, proline at position 459, alanine at position 239, and isoleucine at positions 49 and 365, (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 94, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 93; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 94, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 93; The present invention provides an hMPV F mutant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0178] The hMPV F protein variants provided by the present disclosure can be prepared by routine methods known in the art, such as by expression in a recombinant host system using an appropriate vector. Suitable recombinant host cells include, for example, insect cells, mammalian cells, avian cells, bacteria, and yeast cells. Examples of suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (clonal isolates derived from the parent Trichoplusia ni BTI-TN-5B1-4 cell line (Invitrogen)). Examples of suitable mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi293 cells, typically transformed with sheared adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Suitable avian cells include, for example, chicken embryonic stem cells (e.g., EBx.RTM. cells), chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts (e.g., ELL-O), and duck cells. Suitable insect cell expression systems, such as baculovirus vector systems, are known to those skilled in the art and are described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin, No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego, Calif. Avian cell expression systems are also known to those skilled in the art and are described, for example, in U.S. Patent Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668. Similarly, bacterial and mammalian cell expression systems are known in the art and are described, for example, in Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths, London.

[0179] Many suitable vectors for expressing recombinant proteins in insect or mammalian cells are well known in the art and have been used conventionally.Suitable vectors may contain several components, including, but not limited to, one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as transcriptional control elements (e.g., promoters, enhancers, terminators), and / or one or more translation signals; and a signal or leader sequence for targeting the secretory pathway in a selected host cell (e.g., of mammalian origin, or from a heterologous mammalian or non-mammalian species).For example, for expression in insect cells, a suitable baculovirus expression vector, such as pFastBac (Invitrogen), is used to produce recombinant baculovirus particles.The baculovirus particles are amplified and used to infect insect cells to express recombinant proteins.For expression in mammalian cells, a vector that drives the expression of the construct in the desired mammalian host cell (e.g., Chinese hamster ovary cells) is used.

[0180] hMPV F protein mutant polypeptides can be purified using any suitable method. For example, methods for purifying hMPV F protein mutant polypeptides by immunoaffinity chromatography are known in the art. Ruiz-Arguello et al., J. Gen. Virol., 85:3677-3687 (2004). Suitable methods for purifying desired proteins, including precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelating, and size exclusion, are well known in the art. Two or more of these or other suitable methods can be used to create a suitable purification scheme. If desired, hMPV F protein mutant polypeptides can contain a "tag" that facilitates purification, such as an epitope tag, a strep II tag, or a histidine (HIS) tag. Such tagged polypeptides can be conveniently purified, for example, from conditioned medium by chelating or affinity chromatography.

[0181] Table 1 below provides representative sequences from hMPV A and B F0 polypeptides.

[0182] [Table 1-1]

[0183] [Table 1-2]

[0184] [Table 1-3]

[0185] Table 2 provides the amino acid sequences of the F1 polypeptide lacking the transmembrane and intracellular domains, and the F2 polypeptide of the mutant hMPV083 variant (based on the F protein sequence from the TN / 95 / 3-54 strain), to illustrate how a particular set of mutations can be applied to any hMPV A wild-type F protein.

[0186] [Table 2-1]

[0187] [Table 2-2]

[0188] Table 3 provides the amino acid sequences of the F1 polypeptide, lacking the transmembrane and intracellular domains, and the F2 polypeptide of the mutant hMPV083 variant (based on the hMPV B consensus sequence), to illustrate how a particular set of mutations can be applied to any hMPV B wild-type F protein.

[0189] [Table 3-1]

[0190] [Table 3-2]

[0191] 3. Nucleic acids encoding hMPV F protein mutants In another aspect, the present invention provides nucleic acid molecules encoding the hMPV F protein mutants described hereinabove. These nucleic acid molecules include DNA, cDNA, and RNA sequences. Nucleic acid molecules encoding only the F2 polypeptide or only the F1 polypeptide of an hMPV F mutant are also encompassed by the present invention. The nucleic acid molecules can be incorporated into a vector, such as an expression vector.

[0192] In some embodiments, the nucleic acid molecule encodes a precursor F0 polypeptide that, when expressed in a suitable cell, is processed into the disclosed hMPV F mutants. In some embodiments, the nucleic acid molecule encodes a precursor F0 polypeptide that, when expressed in a suitable cell, is processed into the disclosed hMPV F mutants, the precursor F0 polypeptide comprising, from N- to C-terminus, a signal peptide, an F2 polypeptide, and an F1 polypeptide. In some embodiments, the signal peptide comprises the amino acid sequence set forth as positions 1-18 of any one of SEQ ID NOs: 1-6 and 99, wherein the amino acid positions correspond to the reference amino acid sequence of SEQ ID NO: 1.

[0193] In a preferred embodiment, the nucleic acid is RNA, more preferably mRNA. In a preferred embodiment, the mRNA, when expressed in an appropriate cell, encodes a precursor F0 polypeptide that is processed into a full-length hMPV F protein variant disclosed herein (i.e., comprising one or more mutations, a full-length F1 polypeptide, and a full-length F2 polypeptide). The full-length F1 polypeptide of the hMPV F variant corresponds to amino acids 103 to 539 of the native hMPV F0 precursor and includes (from the N- to C-terminus) the extracellular region (residues 103 to 489), the transmembrane domain (residues 490 to 514), and the cytoplasmic domain (residues 515 to 539). In a preferred embodiment, the nucleic acid is mRNA comprising chemically modified nucleotides. In a preferred embodiment, the nucleic acid is mRNA comprising chemically modified nucleotides, preferably 1-methylpseudouridine. Preferably, all uridines in the RNA are replaced by 1-methylpseudouridine.

[0194] In some embodiments, the nucleic acid molecule is (1) variants containing at least one engineered disulfide bond mutation; (2) mutants containing at least one cavity-filling mutation; (3) mutants containing at least one proline substitution mutation; (4) mutants containing at least one glycine replacement mutation; (5) variants containing a combination of at least one engineered disulfide mutation and at least one cavity-filling mutation; (6) variants containing a combination of at least one engineered disulfide mutation and at least one proline substitution mutation; (7) variants containing a combination of at least one engineered disulfide mutation and at least one glycine replacement mutation; (8) variants containing a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one proline substitution mutation; (9) variants containing a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one glycine replacement mutation; (10) A variant comprising a combination of at least one engineered disulfide mutation, at least one proline substitution mutation, and at least one glycine replacement mutation; and (11) A mutant comprising a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, at least one proline substitution mutation, and at least one glycine replacement mutation. The compound encodes a variant selected from the group consisting of:

[0195] In some specific embodiments, the present disclosure provides: (1) a mutant containing a combination of substitutions 140C and 149C; (2) a variant containing a combination of substitutions 140C, 149C, 411C, and 434C; (3) a variant containing a combination of substitutions 140C, 149C, 411C, 434C, and 459P; (4) variants containing combinations of substitutions 140C, 149C, 411C, 434C, and 365I; (5) a mutant containing a combination of substitutions 140C, 149C, 411C, 434C, and G239A; (6) mutants containing combinations of substitutions 140C, 149C, 411C, 434C, 459P, G239A, 49I, and 365I; (7) a variant containing a combination of substitutions 411C, 434C, 141C, and 161C; (8) a variant containing a combination of substitutions 411C, 434C, 141C, 161C, and 459P; (9) variants containing combinations of substitutions 411C, 434C, 141C, 161C, and 49I; (10) variants containing a combination of substitutions 411C, 434C, 141C, 161C, and 365I; (11) a mutant containing a combination of substitutions 411C, 434C, 141C, 161C, and G239A; (12) a variant containing a combination of substitutions 411C, 434C, 141C, 161C, and 149T; (13) A variant containing a combination of substitutions 411C, 434C, 141C, 161C, 459P, G239A, 49I, 149T, and 365I; and (14) A variant containing a combination of substitutions 411C, 434C, 141C, 161C, and 365I; and (15) Substitutions 411C, 434C, 146C, 160C, 459P, G239A, 49I, 149T, and 365I Nucleic acid molecules encoding variants selected from the group consisting of:

[0196] In some specific embodiments, the present disclosure provides a nucleic acid molecule, preferably an mRNA, more preferably an mRNA in which all uridines are replaced by 1-methylpseudouridine, said nucleic acid, when expressed in a suitable cell, (1) A140C and S149C, (2) A140C, S149C, T411C and Q434C, (3) A140C, S149C, T411C, Q434C and A459P, (4) A140C, S149C, T411C, Q434C and T365I, (5) A140C, S149C, T411C, Q434C and G239A, (6) A140C, S149C, T411C, Q434C, A459P, G239A, T49I and T365I, (7) T411C, Q434C, L141C and A161C, (8) T411C, Q434C, L141C, A161C and A459P, (9) T411C, Q434C, L141C, A161C and T49I, (10) T411C, Q434C, L141C, A161C and T365I, (11) T411C, Q434C, L141C, A161C and G239A, (12) T411C, Q434C, L141C, A161C and S149T, (13) T411C, Q434C, L141C, A161C, A459P, G239A, T49I, S149T, and T365I, and (14) T411C, Q434C, E146C, T160C, A459P, G239A, T49I, S149T, and T365I The present invention provides a nucleic acid molecule encoding a precursor F0 polypeptide that is processed into a full-length hMPV F protein mutant disclosed herein, comprising a mutation selected from the group consisting of:

[0197] In some specific embodiments, the present disclosure provides a nucleic acid molecule, preferably an mRNA, more preferably an mRNA in which all uridines are replaced by 1-methylpseudouridine, said nucleic acid, when expressed in a suitable cell, (1) T150C, R156C and A459P; (2) T150C, R156C and T49I; (3) T150C, R156C, T49I and A459P; (4) A140C, S149C, T411C, and Q434C; (5) L141C, A161C, T411C, and 434C; (6) A140C, S149C, T411C, Q434C, and A459P; (7) A140C, S149C, G239A, T411C, and Q434C; (8) L141C, A161C, T411C, Q434C and A459P; (9) L141C, A161C, G239A, T411C, and Q434C; (10) T49I, T150C, R156C, G239A, and A459P; (11) A140C, S149C, G239A, T411C, Q434C, and A459P; (12) T49I, A140C, S149C, G239A, T411C, Q434C, and A459P; (13) T49I, A140C, S149C, G239A, T365I, T411C, Q434C, and A459P; (14) L141C, A161C, G239A, T411C, Q434C, and A459P; (15) T49I, L141C, A161C, G239A, T411C, Q434C, and A459P; (16) L141C, A161C, S149T, G239A, T411C, Q434C, and A459P; (17) T49I, L141C, A161C, S149T, G239A, T411C, Q434C, and A459P; (18) T49I, L141C, A161C, S149T, G239A, T365I, T411C, Q434C, and A459P; (19) T49I, S149T and A459P; (20) A140C, S149C and A459P; (21) T49I, A140C and S149C; (22) T49I, A140C, S149C and A459P; (23) T49I, L141C, A161C, T411C, and Q434C; (24) T49I, L141C, A161C, T411C, Q434C, and A459P; (25) L141C, A161C and S149T; (26) L141C, A161C, S149T, and A459P; (27) T49I, L141C, A161C and S149T, and (28) T49I, L141C, A161C, S149T, and A459P The present invention provides a nucleic acid molecule encoding a precursor F0 polypeptide that is processed into the disclosed full-length hMPV F protein mutant, comprising a mutation selected from the group consisting of:

[0198] In some specific embodiments, the present disclosure provides a nucleic acid molecule, preferably an mRNA, more preferably an mRNA in which all uridines are replaced by 1-methylpseudouridine, said nucleic acid, when expressed in a suitable cell, (1) L66P; (2) L187P; (4) A140C, S149C and L187P; (5) T49I; (6) T365I; and (7) T49I and T365I The present invention provides a nucleic acid molecule encoding a precursor F0 polypeptide that is processed into a full-length hMPV F protein mutant disclosed herein, comprising a mutation selected from the group consisting of:

[0199] In some specific embodiments, the present disclosure provides a nucleic acid molecule, preferably an mRNA, more preferably an mRNA in which all uridines are replaced by 1-methylpseudouridine, said nucleic acid, when expressed in a suitable cell, (1) L187P, Q100R, and S101R; and (2) A140C, S149C, L187P, Q100R, and S101R The present invention provides a nucleic acid molecule encoding a precursor F0 polypeptide that is processed into a full-length hMPV F protein mutant disclosed herein, comprising a mutation selected from the group consisting of:

[0200] C. PIV1 mutants The present disclosure relates to PIV1 F protein variants, immunogenic compositions comprising the PIV1 F protein variants, methods for producing the PIV1 F protein variants, compositions comprising the PIV1 F protein variants, and nucleic acids encoding the PIV1 F protein variants.

[0201] 1. Exemplary Embodiment (E) of the Present Invention E1. A variant of a wild-type PIV1 F protein, the variant comprising an F1 polypeptide and an F2 polypeptide, the variant comprising at least one amino acid mutation compared to the amino acid sequence of the wild-type PIV1 F protein, the amino acid mutation being: (1) at least one engineered disulfide bond mutation; (2) at least one cavity-filling mutation; (3) at least one proline substitution mutation; (4) at least one glycine replacement mutation; (5) cleavage site mutation; (6) a combination of at least one engineered disulfide mutation and at least one cavity-filling mutation; (7) a combination of at least one engineered disulfide mutation and at least one proline substitution mutation; (8) a combination of at least one engineered disulfide mutation and at least one glycine replacement mutation; (9) a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one proline substitution mutation; (10) a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one glycine-replacement mutation; (11) a combination of at least one engineered disulfide mutation, at least one proline replacement mutation, and at least one glycine replacement mutation; (12) a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, at least one proline substitution mutation, and at least one glycine replacement mutation; (13) a combination of a cleavage site mutation and at least one engineered disulfide mutation; (14) a combination of a cleavage site mutation and at least one cavity-filling mutation; (15) a combination of a cleavage site mutation and at least one proline substitution mutation; (16) a combination of a cleavage site mutation and at least one glycine replacement mutation; (17) a combination of a cleavage site mutation and at least one engineered disulfide mutation and at least one cavity-filling mutation; (18) a combination of a cleavage site mutation and at least one engineered disulfide mutation and at least one proline substitution mutation; (19) a combination of a cleavage site mutation and at least one engineered disulfide mutation and at least one glycine replacement mutation; (20) a combination of a cleavage site mutation, at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one proline substitution mutation; (21) a combination of a cleavage site mutation, at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one glycine-replacement mutation; (22) a combination of a cleavage site mutation, at least one engineered disulfide mutation, at least one proline replacement mutation, and at least one glycine replacement mutation; (23) a combination of a cleavage site mutation, at least one engineered disulfide mutation, at least one cavity-filling mutation, at least one proline substitution mutation, and at least one glycine replacement mutation; (24) a combination of a cleavage site mutation, at least one cavity-filling mutation, and at least one proline substitution mutation; (25) a combination of a cleavage site mutation, at least one cavity-filling mutation, and at least one glycine replacement mutation; (26) a combination of a cleavage site mutation, at least one proline substitution mutation, and at least one glycine replacement mutation; (27) a combination of at least one cavity-filling mutation and at least one proline substitution mutation; (28) a combination of at least one cavity-filling mutation and at least one glycine-replacement mutation; (29) A combination of at least one proline substitution mutation and at least one glycine replacement mutation: (30) A combination of at least one cavity-filling mutation, at least one proline substitution mutation, and at least one glycine replacement mutation. A mutant of the wild-type PIV1 F protein selected from the group consisting of: E2. A variant as described in E1, comprising an engineered disulfide mutation. E3. The variant of E1 or E2, wherein the engineered disulfide mutation is Q92C-G134C. E4. The variant of any one of E1 to E3, comprising a cavity-filling mutation. E5. The variant of any one of E1 to E4, wherein the cavity-filling mutation is selected from T198A, Q92A, Q92L, A466L, A466V, A466I, S473V, S473L, S473I, S473A, A480L, and A480V. E6. The variant described in E5, wherein the cavity-filling mutation is T198A. E7. The variant described in E5, wherein the cavity-filling mutation is Q92A. E8. The variant described in E5, wherein the cavity-filling mutation is Q92L. E9. The mutant described in E5, wherein the cavity-filling mutation is A466L. E10. The mutant described in E5, wherein the cavity-filling mutation is A466V. E11. The mutant described in E5, wherein the cavity-filling mutation is S473V. E12. The mutant described in E5, wherein the cavity-filling mutation is S473L. E13. The mutant described in E5, wherein the cavity-filling mutation is S473I. E14. The mutant described in E5, wherein the cavity-filling mutation is S473A. E15. The mutant described in E5, wherein the cavity-filling mutation is A480L. E16. The mutant described in E5, wherein the cavity-filling mutation is A480V. E17. The variant of any one of E1 to E4, comprising two or three cavity-filling mutations selected from T198A, Q92A, Q92L, A466L, A466V, A466I, S473V, S473L, S473I, S473A, A480L, and A480V. E18. The mutant described in E17, wherein the cavity-filling mutations are A466L and S473L. E19. The variant described in E17, wherein the cavity-filling mutations are A466I and S473I. E20. The mutant of any one of E18 or E19, further comprising a cavity-filling mutation A480L or A480V. E21. The variant of any one of E1 to E20, comprising a proline substitution mutation. E22. The variant of E21, wherein the proline substitution mutation is A128P. E23. The variant of any one of E1 to E22, comprising a glycine replacement mutation. E24. The variant of E23, wherein the glycine replacement mutation is G134A or G134L. E25. The variant according to E24, wherein the glycine replacement mutation is G134A. E26. The variant of any one of E1 to E25, comprising a cleavage site mutation. E27. The variant according to E26, wherein the cleavage site mutations are F113G and F114S. E28. (1) Q92C-G134C; (2) A466L; (3) A466V; (4) S473V; (5) S473L; (6) A480L; (7) A466L and S473A; (8) A466L and S473L; (9) T198A; (10) G134A; (11) A128P; (12) F113G, F114S, Q92C-G134C, A466L, S473L and A480L; (13) Q92C-G134C, A466L, S473L and A480L; (14) Q92C-G134C, A466L and S473L; (15) F113G, F114S, Q92C-G134C, A466V, S473V and A480V; (16) Q92C-G134C, A466V, S473V and A480V; (17) Q92C-G134C, A466V and S473V; (18) F113G, F114S, A466L, S473L, A480L, and G134A; (19) A466L, S473L, A480L and G134A; (20) A466L, S473L and G134A; (21) F113G, F114S, A466L, S473L, A480L, Q92A, and G134A; (22) F113G, F114S, A466L, S473L, and G134A; (23) A466L, S473L, A480L, Q92A, G134A; (24) A466L, S473L, Q92A, G134A; (25) F113G, F114S, Q92L, G134A; (26) A466L, S473L, A480L, Q92L, and G134A; (27) A466L, S473L, Q92L, and G134A; (28) F113G, F114S, A466L, S473L, A480L, Q92A, and G134L; (29) A466L, S473L, A480L, Q92A, and G134L; (30) F113G, F114S, Q92C-G134C, A466I, S473I, and A480L; (31) F113G, F114S, Q92C-G134C, A466I, and S473I; and (32) A466I, S473I, A480L, Q92L, and G134A The variant described in E1, comprising a mutation selected from the group consisting of: E29. A variant described in E1, comprising the mutations A466L, S473L, A480L, and G134A. E30. A variant as described in E1, comprising the mutations F113G, F114S, A466L, S473L, and G134A. E31. The variant described in E1, comprising the mutations F113G, F114S, A466L, S473L, A480L, and G134A. E32. The variant described in E1, comprising the mutations F113G, F114S, Q92C, G134C, A466L, S473L, and A480L. E33. (a) leucine at positions 466, 473, and 480 (466L, 473L, and 480L) and alanine at position 134 (134A); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 255, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 254; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 255, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 254. or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (b) glycine (G) at position 113 (113G), serine at position 114 (114S), leucine at positions 466 and 473 (466L and 473L), and alanine at position 134 (134A); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 291, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 290; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 291, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 290. comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (c) glycine (G) at position 113 (113G), serine at position 114 (114S), leucine at positions 466, 473, and 480 (466L, 473L, and 480L), and alanine at position 134 (134A); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 277, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 276; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 277, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 276. or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (d) glycine (G) at position 113 (113G), serine at position 114 (114S), leucine at positions 466, 473, and 480 (466L, 473L, and 480L), and cysteine ​​at positions 92 and 134 (92C and 134C); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 273, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 272; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 273, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 272; comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of The mutant described in E1. E34. The variant of any one of E1 to E33, wherein the F1 polypeptide lacks the entire cytoplasmic domain. E35. The variant of any one of E1 to E34, wherein the F1 polypeptide lacks the cytoplasmic domain and part or all of the transmembrane domain, preferably the F1 polypeptide lacks the cytoplasmic domain and the transmembrane domain, preferably the F1 polypeptide comprises or consists of amino acid residues 113 to 477, preferably the F1 polypeptide comprises or consists of amino acid residues 113 to 480. E36. The variant of any one of E1 to E33, wherein the F1 polypeptide comprises an ectodomain, a transmembrane domain, and a cytoplasmic domain, and in a preferred embodiment, the variant comprises a full-length F1 polypeptide and a full-length F2 polypeptide. E37. The variant of any one of E1 to E36, wherein said variant is linked to a trimerization domain. E38. The variant of E37, wherein the trimerization domain is a GCN4 leucine zipper or a phage T4 fibritin foldon. E39. The variant of E38, wherein the trimerization domain is a phage T4 fibritin foldon. E40. The variant of E39, wherein the trimerization domain is the phage T4 fibritin foldon of SEQ ID NO:7. E41. The variant of any one of E37 to E40, wherein the trimerization domain is linked to the C-terminus of the F1 polypeptide. E42. The variant of any one of E37 to E40, wherein the trimerization domain is linked to the C-terminus of the F1 polypeptide via a linker. E43. The variant of E42, wherein the trimerization domain is linked to the C-terminus of the F1 polypeptide via a linker selected from the group consisting of GG, GS, GGGS, or SAIG. E44. The variant of E43, wherein the linker is GGGS. E45. The variant of any one of E1 to E44, in a trimeric form. E46. The variant of any one of E1 to E45, wherein the variant is in a pre-fusion conformation. E47. A variant of any one of E1 to E45, which is in a pre-fusion conformation and specifically binds to an antibody (such as PIV1-8 mAb) specific for the PIV1 F ectodomain in the pre-fusion conformation but not the PIV1 F ectodomain in the post-fusion conformation. E48. The variant of any one of E1 to E45, which is in the prefusion conformation and specifically binds to PIV1-8 mAb, preferably as measured by the ELISA disclosed in the Examples. E49. The variant of any one of E1 to E45, having increased stability compared to the corresponding wild-type PIV1 F protein, wherein the stability is measured by binding of the variant to the antibody PIV1-8 mAb. E50. The variant of any one of E1 to E49, wherein the wild-type PIV1 F protein is SEQ ID NO: 206. E51. The variant of any one of E1 to E49, wherein the wild-type PIV1 F protein is SEQ ID NO: 207. E52. The variant of any one of E1 to E49, wherein the wild-type PIV1 F protein is SEQ ID NO: 208. E53. The variant of any one of E1 to E49, wherein the wild-type PIV1 F protein is SEQ ID NO: 209. E54. The variant of any one of E1 to E49, wherein the wild-type PIV1 F protein is SEQ ID NO: 210. E55. The variant of any one of E1 to E49, wherein the wild-type PIV1 F protein is SEQ ID NO: 211. E56. The variant of any one of E1 to E49, wherein the amino acid positions correspond to the reference amino acid sequence of SEQ ID NO: 206. E57. A nucleic acid comprising at least one coding sequence encoding at least one mutant of the wild-type PIV1 F protein, or an immunogenic fragment or variant thereof, of any one of embodiments E1 to E56, preferably E36, wherein the nucleic acid comprises at least one heterologous untranslated region (UTR). E58. The nucleic acid of any one of the preceding embodiments, wherein the at least one heterologous untranslated region is selected from at least one heterologous 5'-UTR and / or at least one heterologous 3'-UTR. E59. At least one heterologous 3'-UTR is CΨCGAGCΨGGΨ ACΨGCAΨGCA CGCAAΨGCΨA GCΨGCCCCΨΨ ΨCCCGΨCCΨG GGΨACCCCGA GΨCΨCCCCCG ACCΨCGGGΨC CCAGGΨAΨGC ΨCCCACCΨCC ACCΨGCCCCA CΨCACCACCΨ CΨGCΨAGΨΨC CAGACACCΨC CCAAGCACGC AGCAAΨGCAG CΨCAAAACGC ΨΨAGCCΨAGC CACACCCCCA CGGGAAACAG CAGΨGAΨΨAA CCΨΨΨAGCAA ΨAAACGAAAG ΨΨΨAACΨAAG CΨAΨACΨAAC CCCAGGGΨΨG 10. The nucleic acid of any one of the preceding embodiments, comprising or consisting of a nucleic acid sequence having at least, and at most exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to GΨCAAΨΨΨCG ΨGCCAGCCAC ACCCΨGGAGC ΨAGC, or a percentage identity between any two thereof. E60. The nucleic acid of any one of the preceding embodiments, wherein at least one heterologous 5'-UTR comprises or consists of a nucleic acid sequence having at least, and at most, exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to GAAΨAAAC ΨAGΨAΨΨCΨΨ CΨGGΨCCCCA CAGACΨCAGA GAGAACCCGC CACC, or a percentage identity between any two thereof. E61. The nucleic acid of any one of the preceding embodiments, comprising at least one poly(A) sequence, preferably comprising 30 to 200 adenosine nucleotides, and / or at least one poly(C) sequence, preferably comprising 10 to 40 cytosine nucleotides. E62. The nucleic acid of any one of the preceding embodiments, which is DNA or RNA. E63. The nucleic acid of any one of the preceding embodiments, which is a coding RNA. E64. The nucleic acid of E63, wherein the coding RNA is mRNA, self-replicating RNA, circular RNA, or replicon RNA. E65. The nucleic acid of any one of the preceding embodiments, wherein the nucleic acid, preferably the coding RNA, is mRNA. E66. The nucleic acid of E63, wherein the mRNA is not a replicon RNA or a self-replicating RNA. E67. The nucleic acid of any one of the preceding embodiments E63 to E66, wherein the mRNA comprises at least one poly(A) sequence comprising 30 to 200 adenosine nucleotides, and the 3' terminal nucleotide is an adenosine. E68. The nucleic acid of any one of the preceding embodiments E62 to E67, wherein the RNA, preferably the coding RNA, comprises a 5'-cap structure, preferably an m7G, cap0, cap1, cap2, modified cap0, or modified cap1 structure, preferably a 5'-cap1 structure. E69. The nucleic acid of any one of the preceding embodiments E62 to E68, wherein the RNA is codon-optimized. E70. The nucleic acid of any one of the preceding embodiments E62 to E69, wherein the RNA comprises chemically modified nucleotides. E71. The nucleic acid of any one of the preceding embodiments E62 to E70, wherein the RNA comprises 1-methylpseudouridine substitutions, preferably all uridines of the RNA are replaced by 1-methylpseudouridine. E72. The nucleic acid of any one of the preceding embodiments E62 to E71, wherein the RNA is purified RNA, preferably RNA purified by RP-HPLC and / or TFF. E73. The nucleic acid of any one of the preceding embodiments E62 to E72, wherein the RNA comprises the nucleic acid sequence of any of SEQ ID NO: 403, SEQ ID NO: 405, SEQ ID NO: 407, and SEQ ID NO: 409. E74. A composition comprising at least one nucleic acid of any one of the preceding embodiments E57 to E73. E75. A composition comprising at least one nucleic acid of any one of the preceding embodiments E57 to E73, comprising at least one pharmaceutically acceptable carrier. E76. A composition comprising at least one nucleic acid according to any one of the preceding embodiments E57 to E73, which is a multivalent composition comprising a plurality or more than at least one of the nucleic acids according to any one of E57 to E73. E77. A composition comprising at least one nucleic acid of any one of the preceding embodiments E57 to E73, wherein the composition comprises RNA having an RNA integrity of 70% or greater. E78. A composition comprising at least one nucleic acid according to any one of the preceding embodiments E57 to E73, wherein the composition comprises RNA with a degree of capping of 70% or more, preferably at least 70%, 80%, or 90% of mRNA species contain Cap 1 structures. E79. A composition comprising at least one nucleic acid of any one of the preceding embodiments E57 to E73, wherein the at least one nucleic acid is complexed or associated, or at least partially complexed or partially associated, with one or more cationic or polycationic compounds, preferably cationic or polycationic polymers, cationic or polycationic polysaccharides, cationic or polycationic lipids, cationic or polycationic proteins, cationic or polycationic peptides, or any combination thereof. E80. A composition comprising at least one nucleic acid of any one of the preceding embodiments E57 to E73, wherein the at least one nucleic acid is complexed or associated with one or more lipids or lipid-based carriers, thereby forming a liposome, lipid nanoparticle (LNP), lipoplex, and / or nanoliposome, preferably encapsulating the at least one nucleic acid. E81. A composition comprising at least one nucleic acid of any one of the preceding embodiments E57 to E73, wherein the at least one nucleic acid is complexed with one or more lipids, thereby forming a lipid nanoparticle. E82.LNP has formula III-3:

[0202] [ka] The composition of any one of the preceding embodiments E80 to E81, comprising a cationic lipid according to E83.LNP has formula (IVa):

[0203] [ka] The composition of any one of the preceding embodiments E80 to E82, comprising a PEG lipid of E84. The composition of embodiment E83, wherein n has an average value ranging from 30 to 60, preferably n has an average value of about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and most preferably n has an average value of 49 or 45. E85.LNP has formula (IVa):

[0204] [ka] The composition of any one of the preceding embodiments E80 to E84, comprising a PEG lipid of formula (I), where n is an integer selected such that the average molecular weight of the PEG lipid is about 2500 g / mol. E86. The composition of any one of the preceding embodiments E80 to E85, wherein the LNP comprises one or more neutral lipids, and / or one or more steroids or steroid analogues. E87. The composition of any one of the preceding embodiments E80 to E86, wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and preferably the molar ratio of cationic lipid to DSPC is in the range of about 2:1 to about 8:1. E88. The composition of any one of the preceding embodiments E80 to E87, wherein the steroid is cholesterol, and preferably the molar ratio of cationic lipid to cholesterol is in the range of about 2:1 to about 1:1. E89. The composition of any one of the preceding embodiments E80 to E88, wherein the LNP comprises: (i) at least one cationic lipid, preferably a lipid of formula (III), more preferably lipid III-3; (ii) at least one neutral lipid, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) at least one steroid or steroid analog, preferably cholesterol; and (iv) at least one polymer-conjugated lipid, preferably a PEG lipid derived from formula (IVa, having n=49), wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG lipid. E90. The composition of any one of the preceding embodiments E80 to E89, wherein the LNP comprises: (i) at least one cationic lipid, preferably a lipid of formula (III), more preferably lipid III-3; (ii) at least one neutral lipid, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) at least one steroid or steroid analog, preferably cholesterol; and (iv) at least one polymer-conjugated lipid, preferably a PEG lipid from formula (IVa, having n=45), wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG lipid. E91. The composition of any one of the preceding embodiments E80 to E90, wherein (i)-(iv) are in a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7, or more preferably 47.4:10:40.9:1.7. E92. The composition of any one of the preceding embodiments E80 to E91, wherein the nucleic acid is RNA and the composition comprises less than about 20% free (uncomplexed or unencapsulated) RNA, preferably less than about 15% free RNA, more preferably less than about 10% free RNA. E93. The composition of any one of the preceding embodiments E80 to E92, wherein the wt / wt ratio of lipid to nucleic acid is from about 10:1 to about 60:1, preferably from about 20:1 to about 30:1, such as about 25:1. E94. The composition of any one of the preceding embodiments E80 to E93, wherein the n / p ratio of the LNPs encapsulating the nucleic acid is in the range of about 1 to about 10, preferably in the range of about 5 to about 7, and more preferably about 6. E95. The composition of any one of the preceding embodiments E80 to E94, having a polydispersity index (PDI) value of less than about 0.4, preferably less than about 0.3, more preferably less than about 0.2, and most preferably less than about 0.1. E96. The composition of any one of the preceding embodiments E80 to E95, wherein the LNPs have a Z-average size in the range of about 60 nm to about 120 nm, preferably less than about 120 nm, more preferably less than about 100 nm, and most preferably less than about 80 nm. E97. The composition of any one of the preceding embodiments E80 to E96, wherein the LNPs comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of LNPs having a particle size greater than about 500 nm. E98. The composition of any one of the preceding embodiments E80 to E97, wherein the LNPs comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% LNPs having a particle size smaller than about 20 nm. E99. The composition of any one of the preceding embodiments E80 to E98, wherein the LNP comprises (i) at least one cationic lipid; (ii) at least one neutral lipid; (iii) at least one steroid or steroid analog; and (iv) at least one PEG-lipid, wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid. E100. The composition of any one of the preceding embodiments E80 to E99, wherein the LNP comprises: (i) at least one cationic lipid according to formula III-3; (ii) DSPC; (iii) cholesterol; and (iv) a PEG-lipid according to formula IVa, wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid. E101. The composition of any one of the preceding embodiments E74 to E100, which is a lyophilized composition. E102. An immunogenic composition comprising a variant according to any one of E1 to E56, a nucleic acid according to any one of E57 to E73, or a composition according to any one of E74 to E101. E103. The method further comprises: providing an hMPV A antigen selected from the group consisting of a mutant of a wild-type hMPV AF protein and a nucleic acid encoding the mutant of a wild-type hMPV AF protein; in one embodiment, the hMPV A antigen is selected from a mutant of a wild-type hMPV AF protein and a nucleic acid encoding the mutant of a wild-type hMPV AF protein disclosed in any of WO16103238, WO20234300, WO21222639, WO22076669, WO22214678, WO23102373, WO23110618, WO23217988, and WO23102388; in one embodiment, the hMPV A antigen is selected from a mutant of a wild-type hMPV AF protein and a nucleic acid encoding the mutant of a wild-type hMPV AF protein disclosed in Battles et al., Nature communication 8:1528 (2017). The immunogenic composition of embodiment E102, which is a nucleic acid encoding a variant of the AF protein or a variant of the wild-type hMPV AF protein comprising the mutations of mutant 115-BV. E104. The immunogenic composition of embodiment E103, wherein the hMPV A antigen is a mutant of the wild-type hMPV AF protein. E105. The immunogenic composition of embodiment E103, wherein the hMPV A antigen is a variant of a wild-type hMPV AF protein from the present disclosure, preferably from any of E1 to E72 of section B of the present disclosure. E106. The immunogenic composition of embodiment E103, wherein the hMPV A antigen comprises a nucleic acid encoding a variant of the wild-type hMPV AF protein. E107. The immunogenic composition of embodiment E103, wherein the hMPV A antigen comprises a nucleic acid encoding a variant of a wild-type hMPV AF protein from the present disclosure, preferably a nucleic acid from any of E73 to E89 of section B of the present disclosure. E108. The method further comprises: providing an hMPV B antigen selected from the group consisting of a mutant of a wild-type hMPV BF protein and a nucleic acid encoding the mutant of a wild-type hMPV BF protein; in one embodiment, the hMPV B antigen is selected from a mutant of a wild-type hMPV BF protein and a nucleic acid encoding the mutant of a wild-type hMPV BF protein disclosed in any of WO16103238, WO20234300, WO21222639, WO22076669, WO22214678, WO23102373, WO23110618, WO23217988, and WO23102388; in one embodiment, the hMPV B antigen is a wild-type hMPV B antigen comprising the mutations of mutant 115-BV disclosed in Battles et al., Nature communication 8:1528 (2017). The immunogenic composition of any one of embodiments E102 to E107, which is a nucleic acid encoding a variant of the BF protein or a variant of the wild-type hMPV BF protein comprising the mutations of mutant 115-BV. E109. The immunogenic composition of embodiment E108, wherein the hMPV B antigen is a mutant of the wild-type hMPV BF protein. E110. The immunogenic composition of embodiment E108, wherein the hMPV B antigen is a variant of a wild-type hMPV BF protein from the present disclosure, preferably from any of E1 to E72 of section B of the present disclosure. E111. The immunogenic composition of embodiment E108, wherein the hMPV B antigen comprises a nucleic acid encoding a variant of the wild-type hMPV BF protein. E112. The immunogenic composition of embodiment E108, wherein the hMPV B antigen comprises a nucleic acid encoding a variant of a wild-type hMPV BF protein from the present disclosure, preferably a nucleic acid from any of E73 to E89 of section B of the present disclosure. E113. The immunogenic composition of any one of embodiments E102 to E112, further comprising a PIV3 antigen selected from the group consisting of a variant of the wild-type PIV3 F protein, and a nucleic acid encoding a variant of the wild-type PIV3 F protein. E114. The immunogenic composition of embodiment E113, wherein the PIV3 antigen is a variant of the wild-type PIV3 F protein. E115. The immunogenic composition of embodiment E113, wherein the PIV3 antigen is a variant of a wild-type PIV3 F protein from the present disclosure, preferably from any of E1 to E52 of section D of the present disclosure. E116. The immunogenic composition of embodiment E113, wherein the PIV3 antigen is a variant of the wild-type PIV3 F protein disclosed in WO2018081289 or WO22207839. E117. The immunogenic composition of embodiment E113, wherein the PIV3 antigen comprises a nucleic acid encoding a variant of the wild-type PIV3 F protein. The immunogenic composition of embodiment E113, wherein the PIV3 antigen comprises a nucleic acid encoding a variant of a wild-type PIV3 F protein from the present disclosure, preferably a nucleic acid from any of E53 to E69 of section D of the present disclosure. E119. The immunogenic composition of embodiment E113, wherein the PIV3 antigen comprises a nucleic acid encoding a variant of the wild-type PIV3 F protein disclosed in WO2018081289 or WO2022207839. E120. The immunogenic composition of any one of E102 to E119, further comprising a RSV antigen selected from the group consisting of a subtype A wild-type RSV F protein variant and a nucleic acid encoding a subtype A wild-type RSV F protein variant. E121. The immunogenic composition of embodiment E120, wherein the RSV antigen is a variant of the wild-type RSV F protein of subtype A. E122. The immunogenic composition of embodiment E120, wherein the RSV antigen is a nucleic acid encoding a variant of a subtype A wild-type RSV F protein. E123. The immunogenic composition of embodiment E120, wherein the variant of the subtype A wild-type RSV F protein is disclosed in one of WO2009 / 079796, WO2010 / 149745, WO2011 / 008974, WO2014 / 160463, WO2014 / 174018, WO2014 / 202570, WO2015 / 013551, WO2015 / 177312, WO2017 / 005848, WO2017 / 174564, WO2017 / 005844, WO2017 / 109629, WO2022 / 002894, and WO2018 / 109220. E124. The immunogenic composition of any one of embodiments E102 to E123, further comprising a RSV antigen selected from the group consisting of a subtype B wild-type RSV F protein variant and a nucleic acid encoding a subtype B wild-type RSV F protein variant. E125. The immunogenic composition of embodiment E124, wherein the RSV antigen is a variant of a wild-type RSV F protein of subtype B. E126. The immunogenic composition of embodiment E124, wherein the RSV antigen is a nucleic acid encoding a variant of a wild-type RSV F protein of subtype B. E127. The immunogenic composition of embodiment E124, wherein the variant of the subtype B wild-type RSV F protein is disclosed in one of WO2009 / 079796, WO2010 / 149745, WO2011 / 008974, WO2014 / 160463, WO2014 / 174018, WO2014 / 202570, WO2015 / 013551, WO2015 / 177312, WO2017 / 005848, WO2017 / 174564, WO2017 / 005844, WO2017 / 109629, WO2022 / 002894, and WO2018 / 109220.

[0205] 2. PIV1 F protein mutants In some aspects, the present invention provides mutants of wild-type PIV1 F proteins, where the mutants exhibit introduced mutations in their amino acid sequences compared to the corresponding wild-type PIV1 F protein amino acid sequence and are immunogenic against the wild-type PIV1 F protein in its pre-fusion conformation or against viruses containing the wild-type F protein. In certain embodiments, the PIV1 F mutants possess certain beneficial characteristics, such as increased immunogenic properties or improved stability of the mutant's pre-fusion conformation or the mutant's pre-fusion trimer conformation, compared to the corresponding wild-type F protein. In still other embodiments, the present disclosure provides PIV1 F mutants that exhibit one or more introduced mutations described herein and that bind to a pre-fusion-specific antibody selected from PIV1-8 mAb.

[0206] The amino acid mutations introduced in the PIV1 F protein variant include amino acid substitutions, deletions, or additions. In some embodiments, the only mutations in the amino acid sequence of the variant are amino acid substitutions compared to the wild-type PIV1 F protein.

[0207] The amino acid sequences of numerous native PIV1 F proteins from various strains, as well as the nucleic acid sequences encoding such proteins, are known in the art. For example, the sequences of several PIV1 F0 precursor proteins are set forth in SEQ ID NOs: 206-210.

[0208] The native PIV1 F protein exhibits remarkable sequence conservation across different lineages.

[0209] Given the substantial conservation of the PIV1 F protein sequence, those skilled in the art can easily compare amino acid positions between various native PIV1 F protein sequences to identify corresponding PIV1 F protein amino acid positions between various PIV1 strains. For example, the protease cleavage site is located at the same amino acid position across nearly all identified native PIV1 F precursor proteins. Thus, the conservation of native PIV1 F protein sequences across strains and subtypes allows the use of a reference PIV1 F sequence for comparison of amino acids at specific positions within the PIV1 F protein. For purposes of this disclosure (unless the context indicates otherwise), PIV1 F protein amino acid positions are provided with reference to the sequence of the F precursor polypeptide set forth in SEQ ID NO: 206 (corresponding to the amino acid sequence of the full-length native F precursor polypeptide of the PIV1 strain; GenBank identifier AFP49460.1 (amino acids)).

[0210] The consensus sequence for PIV1 (corresponding to SEQ ID NO: 206) was obtained as follows: The entire genome sequence for PIV1 was downloaded from the NCBI GenBank database as a GenBank file. The fusion protein gene sequences were filtered by sequence length to include only complete coding DNA sequence features. The translated fusion protein sequences were then analyzed from the GenBank file and saved as FASTA files. Muscle v5 was used to perform multiple sequence alignment of the collected sequences. A position-specific score matrix (PSSM) was created to summarize the alignment information. For each column in the alignment, the number of each amino acid character was counted and summed. The consensus sequence at each position was calculated as the most commonly occurring amino acid type in the PSSM table. The final consensus sequence was then extracted and saved as a FASTA file.

[0211] However, it should be noted, and one of skill in the art would understand, that different PIV1 F0 sequences may have different numbering systems if there are additional amino acid residues added or removed compared to, for example, SEQ ID NO: 206. Thus, when specific amino acid residues are referred to by their numbers, it should be understood that the description is not limited to only the amino acid at that exact numbered position when counting from the beginning of a given amino acid sequence, but rather that the equivalent / corresponding amino acid residue in any and all PIV1 F sequences is intended, even if that residue is not at the same exact numbered position, for example, if the PIV1 sequence is shorter or longer than SEQ ID NO: 206 or has an insertion or deletion compared to SEQ ID NO: 206.

[0212] 2-1. Structure of PIV1 F protein mutants The PIV1 F protein variants provided by the present disclosure include an F1 polypeptide and an F2 polypeptide. In some embodiments, the variants further include a trimerization domain. In some embodiments, either the F1 polypeptide or the F2 polypeptide comprises at least one introduced modification (e.g., amino acid substitution) as described in detail herein below. In some other embodiments, each of the F1 polypeptide and the F2 polypeptide comprises at least one introduced modification (e.g., amino acid substitution) as described in detail herein below.

[0213] 2-1(a). F1 and F2 polypeptides of PIV1 F mutants In some embodiments, the variant is in the mature form of the PIV1 F protein, which comprises two separate polypeptide chains, ie, an F1 polypeptide and an F2 polypeptide.

[0214] The F1 polypeptide chain of a variant can be the same length as the full-length F1 polypeptide of the corresponding wild-type PIV1 F protein; however, it can also have a deletion, such as a deletion of 1 to up to 36 amino acid residues from the C-terminus of the full-length F1 polypeptide. The full-length F1 polypeptide of a PIV1 F variant corresponds to amino acids 113 to 555 of the native PIV1 F precursor and includes (from N- to C-terminus) the extracellular region (residues 113-496), the transmembrane domain (residues 497-517), and the cytoplasmic domain (residues 518-555). It should be noted that amino acid residues 477 and beyond in the native F1 polypeptide sequence are optional in the F1 polypeptides of the PIV1 F variants provided herein and therefore may be absent from the variant F1 polypeptides.

[0215] In some embodiments, the F1 polypeptide of a PIV1 F variant lacks the entire cytoplasmic domain. In other embodiments, the F1 polypeptide lacks the cytoplasmic domain and part or all of the transmembrane domain. In some specific embodiments, the variant comprises an F1 polypeptide in which amino acid residues 477-555 are absent. Typically, for variants linked to a trimerization domain, such as a foldon, amino acids 477-555 may be absent. Thus, in some specific embodiments, amino acid residues 477-555 are absent from the variant F1 polypeptide. In yet other specific embodiments, the F1 polypeptide of a PIV1 F variant comprises or consists of amino acid residues 103-477 of a native F0 polypeptide sequence, such as any of the F0 precursor sequences set forth in SEQ ID NOs: 206-210.

[0216] In some embodiments, the PIV1 F protein variant comprises a mutation at position 480. In such cases, the F1 polypeptide of the PIV1 F variant comprises or consists of amino acid residues 103 to 480 of the native F0 polypeptide sequence.

[0217] On the other hand, the F1 polypeptide of a PIV1 F variant can include a C-terminal linkage to a trimerization domain such as a foldon. Many of the sequences of the PIV1 F variants disclosed herein contain PreScission cleavage sites and Strep Tag II sequences that are not essential for the function of the PIV1 F protein, such as inducing an immune response. Those of skill in the art will recognize such sequences and understand that, where appropriate, these sequences are not included in the disclosed PIV1 F variants.

[0218] In the PIV1 F mutants provided by the present disclosure, the F2 polypeptide chain can be the same length as the full-length F2 polypeptide of the corresponding wild-type PIV1 F protein; it can also have a deletion, such as a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues from the N-terminus or C-terminus of the F2 polypeptide.

[0219] Variants of the F0 form (i.e., a single-chain polypeptide comprising an F2 polypeptide joined to an F1 polypeptide) or the F1-F2 heterodimeric form can form protomers. Variants can also be in the form of trimers comprising three identical protomers. Furthermore, variants can be glycosylated proteins (i.e., glycoproteins) or non-glycosylated proteins. Variants of the F0 form can contain or lack a signal peptide sequence.

[0220] The F1 and F2 polypeptides of the PIV1 F protein variants having one or more mutations can be derived from any wild-type PIV1 F protein known in the art or discovered in the future. In some embodiments, the PIV1 F variants include F1 and / or F2 polypeptides derived from a known PIV1 F0 precursor protein, such as those set forth in any one of SEQ ID NOS: 206-210, having one or more mutations introduced therein.

[0221] In some embodiments, the PIV1 F protein variant comprises an F1 polypeptide, an F2 polypeptide, and one or more introduced amino acid mutations described herein below, wherein the F1 polypeptide comprises 350 contiguous amino acids and is at least 90, 95, 98, or 99 percent identical to amino acids 113-477 or 113-480 of any of the sequences set forth in SEQ ID NOs: 206-210, and the F2 polypeptide comprises 70 contiguous amino acids and is at least 90, 95, 98, or 99 percent identical to amino acids 22-112 of any of the sequences set forth in SEQ ID NOs: 206-210, and the PIV1 F protein variant is stabilized in a pre-fusion trimer conformation both as a monomer and as a trimer.

[0222] 2-1(b) Trimerization domain In some embodiments, the PIV1 F variants provided by the present disclosure are linked to a trimerization domain, which in some embodiments promotes the formation of trimers of three F1 / F2 heterodimers.

[0223] Some exogenous trimerization domains that promote the formation of stable trimers of soluble proteins are known in the art.Non-limiting examples of such trimerization domains that can be linked to the variants provided by the present disclosure include: (1) GCN4 leucine zipper (Harbury et al., 1993 Science 262:1401-1407); (2) trimerization motif from pulmonary surfactant protein (Hoppe et al., 1994 FEBS Lett 344:191-195); (3) collagen (McAlinden et al., 2003 Biol Chem 278:42200-42207); and (4) phage T4 fibritin foldon (Miroshnikov et al., 1998 Protein Eng 11:329-414).

[0224] Typically, the trimerization domain is located C-terminal to the F1 polypeptide. It may be directly attached to the F1 polypeptide chain. Optionally, the multimerization domain is connected to the F1 polypeptide via an amino acid linker, such as a linker with the sequence GG, GS, GGGS, or SAIG. The linker may also be a longer linker (e.g., containing a repeat sequence GG). A preferred linker is GGGS. Many conformationally neutral linkers are known in the art and can be used in the variants provided by the present disclosure. In some embodiments, the F1 variant containing a foldon domain comprises a protease cleavage site for removing the foldon domain from the F1 polypeptide, such as a thrombin site between the F1 polypeptide and the foldon domain.

[0225] In some embodiments, the foldon domain is linked to the F mutant at the C-terminus of the F1 polypeptide. In a specific embodiment, the foldon domain is a T4 fibritin foldon domain, such as the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 7).

[0226] 2-2.Introduced mutations in PIV1 F protein mutants The PIV1 F variants provided by the present disclosure include an F1 polypeptide and an F2 polypeptide, wherein either (1) the F1 polypeptide or (2) the F2 polypeptide, or (3) both the F1 polypeptide and the F2 polypeptide, contain one or more introduced amino acid mutations compared to the amino acid sequence of the corresponding native F protein. Introduction of such amino acid mutations in a PIV1 F variant confers beneficial properties to the variant, such as enhanced immunogenicity, improved stability, improved expression or formulation, or improved stability of a particular desired physical form or conformation of the variant. Such introduced amino acid mutations are referred to as "engineered disulfide bond mutations," "cavity-filling mutations," "proline substitution mutations," "cleavage site mutations," or "glycine replacement mutations."

[0227] The nature and purpose of the "engineered disulfide bond mutations," "cavity-filling mutations," "proline substitution mutations," and "glycine replacement mutations" have already been disclosed above in connection with hMPV protein mutants.

[0228] A "cleavage site mutation" prevents cleavage of the PIV1 F protein variant between amino acids 113 and 114. In such a case, the F1 and F2 polypeptides form a single polypeptide rather than two separate polypeptides linked by a disulfide bond. Examples of cleavage site mutations are F113G and F114S. PIV1 F protein variants containing any additional mutations are also encompassed by the present invention, as long as the immunogenic properties of the variant are not substantially adversely affected by the additional mutations.

[0229] 2-2(a) Engineered disulfide bond mutations In some embodiments, the PIV1 protein F mutants provided by the present disclosure contain one or more engineered disulfide bond mutations. The term "engineered disulfide bond mutation" refers to the mutation of a pair of amino acid residues in the wild-type PIV1 F protein to a pair of cysteine ​​residues. The introduced pair of cysteine ​​residues allows for the formation of a disulfide bond between the introduced cysteine ​​residues, and the disulfide bond serves to stabilize the conformation or oligomeric state of the protein, such as the pre-fusion conformation. To stabilize the pre-fusion conformation of the mutant, the residue pair for mutation to cysteine ​​should be in close proximity in the pre-fusion conformation but far apart in the post-fusion conformation. Preferably, the distance between the residue pair (e.g., beta carbons) is less than 8 Å in the pre-fusion conformation but greater than 20 Å in the post-fusion conformation.

[0230] In some embodiments, the PIV1 F protein variant contains only one engineered disulfide mutation (a "single engineered disulfide mutation"). In other embodiments, the PIV1 F protein variant contains at least two engineered disulfide mutations, with each pair of cysteine ​​residues of the engineered disulfide mutations appropriately positioned when the PIV1 F protein variant is in the prefusion conformation (a "double engineered disulfide mutation").

[0231] In some specific embodiments, the present disclosure provides PIV1 F variants comprising at least one engineered disulfide bond mutation, wherein the variants comprise the same introduced mutation as any of the exemplary variants provided in Tables 32, 36, and 37. The exemplary PIV1 F variants provided in Tables 32, 36, and 37 are based on the same F0 sequence of PIV1 of SEQ ID NO: 211. The same introduced mutations in each of the variants can be performed on the native F0 polypeptide sequence of any other PIV1 subtype or strain to arrive at a different PIV1 F variant, such as the native F0 polypeptide sequence set forth in any of SEQ ID NOs: 206-210 or from any other PIV1 strain. PIV1 F variants based on the native F0 polypeptide sequence of any other PIV1 subtype or strain and comprising any of the engineered disulfide mutations are also within the scope of the present invention. In certain embodiments, the PIV1 F protein variants comprise at least one engineered disulfide mutation, such as 92C and 134C, preferably Q92C-G134C.

[0232] 2-2(b) Cavity-filling mutation In other embodiments, the present disclosure provides PIV1 F mutants containing one or more cavity-filling mutations. The term "cavity-filling mutation" refers to the replacement of an amino acid residue in the wild-type PIV1 F protein with an amino acid predicted to fill the internal cavity of the mature PIV1 F protein. In one application, such cavity-filling mutations contribute to stabilizing the pre-fusion conformation of the PIV1 F protein mutant. For example, the amino acids to be replaced for cavity-filling mutations typically include small aliphatic amino acids (e.g., Gly, Ala, and Val) or small polar amino acids (e.g., Ser and Thr). They may also include amino acids that are buried in the pre-fusion conformation but exposed to the solvent in the post-fusion conformation. The replacement amino acids may include aliphatic amino acids (Val, Ile, Leu, and Met), aromatic amino acids (His, Phe, Tyr, and Trp), or polar amino acids (Thr) that are larger in size than the amino acids being replaced.

[0233] In some specific embodiments, the PIV1 F protein variant comprises one or more cavity-filling mutations at positions 198, 92, 466, 473, and 480, preferably at positions 466, 473, and 480.

[0234] In some specific embodiments, the disclosure provides PIV1 F variants comprising one or more cavity-filling mutations, wherein the variants comprise the cavity-filling mutations in any of the variants provided in Tables 34 and 37. The PIV1 F variants provided in Tables 34 and 37 are based on the same native F0 sequence of PIV1 of SEQ ID NO: 211. The same introduced mutations in each of the variants can be made to the native F0 polypeptide sequence of any other PIV1 subtype or strain to arrive at a different PIV1 F variant, such as the native F0 polypeptide sequence set forth in any of SEQ ID NOs: 206-210 or from any other PIV1 strain. PIV1 F variants based on the native F0 polypeptide sequence of any other PIV1 subtype or strain and comprising one or more cavity-filling mutations are also within the scope of the invention. In certain embodiments, the PIV1 F protein variant comprises at least one cavity-filling mutation selected from the group consisting of T198A, Q92A, Q92L, A466L, A466V, A466I, S473V, S473L, S473I, S473A, A480L, and A480V.

[0235] In certain embodiments, the PIV1 F protein variants provided by the present disclosure comprise at least one cavity-filling mutation selected from the group consisting of A466L, S473L, A480L.

[0236] 2-2(c) Proline substitution mutation In yet other embodiments, the present disclosure provides PIV1 F protein variants comprising one or more proline substitution mutations. The term "proline substitution mutation" refers to the substitution of an amino acid with a proline that prevents structural refolding during the transition from the pre-fusion to the post-fusion conformation.

[0237] In some specific embodiments, the PIV1 F protein variant comprises the proline mutation A128P. In some specific embodiments, the present disclosure provides PIV1 F variants comprising one or more proline substitution mutations as provided in Table 33. The PIV1 F variants provided in Table 33 are based on the native F0 sequence of PIV1 of SEQ ID NO: 211. The same introduced mutations in the variants can be performed on the native F0 polypeptide sequence of any other PIV1 subtype or strain to arrive at a different PIV1 F variant, such as the native F0 polypeptide sequence set forth in any of SEQ ID NOs: 206-210 or from any other PIV1 strain. PIV1 F variants based on the native F0 polypeptide sequence of any other PIV1 subtype or strain and comprising one or more promine substitution mutations are also within the scope of the present invention. In some specific embodiments, the PIV1 F protein variant comprises the mutation A128P.

[0238] 2-2(d) Glycine replacement mutation In still other embodiments, the disclosure provides PIV1 F protein variants comprising one or more glycine replacement mutations. The term "glycine replacement mutation" refers to the replacement of glycine with another amino acid in the center of the α-helix, preferably an amino acid without a Cβ substitution, such as Ala, Leu, or Met, to improve protein stability. In some specific embodiments, the PIV1 F protein variant comprises at least one glycine replacement mutation at position 134. In some specific embodiments, the disclosure provides PIV1 F variants comprising one or more glycine replacement mutations, wherein the variants comprise a glycine replacement mutation in a variant provided in Table 35.

[0239] The PIV1 F variants provided in Table 35 are based on the native F0 sequence of PIV1 of SEQ ID NO: 211. The same introduced mutations in each of the variants can be performed on the native F0 polypeptide sequence of any other PIV1 subtype or strain to arrive at different PIV1 F variants, such as the native F0 polypeptide sequence set forth in any of SEQ ID NOs: 206-210 or from any other PIV1 strain. PIV1 F variants based on the native F0 polypeptide sequence of any other PIV1 subtype or strain and containing one or more glycine replacement mutations are also within the scope of the present invention. In certain embodiments, the PIV1 F protein variant contains the mutation G134A or G134L, preferably G134A.

[0240] 2-2(e) Cleavage site mutation The "cleavage site mutation" was introduced to prevent cleavage of the PIV1 F protein mutant between amino acids 112 and 113. However, when recombinantly expressed in CHO cells, the PIV1 F protein mutant disclosed herein appeared to be inefficiently cleaved between amino acids 112 and 113, even in the absence of any cleavage site mutation. As a result, the F1 and F2 polypeptides form a single polypeptide rather than two separate polypeptides linked by a disulfide bond. Unexpectedly, the "cleavage site mutation," while not preventing cleavage that would not occur in the expression system used, provided some unexpected benefits in terms of the thermostability of the produced polypeptide.

[0241] In some specific embodiments, the disclosure provides PIV1 F mutants comprising one or more cleavage site mutations, wherein the mutants comprise cleavage site mutations in the mutants provided in Table 37.

[0242] The PIV1 F variants provided in Table 37 are based on the native F0 sequence of PIV1 of SEQ ID NO: 211. The same introduced mutations in each of the variants can be made to the native F0 polypeptide sequence of any other PIV1 subtype or strain to arrive at a different PIV1 F variant, such as the native F0 polypeptide sequence set forth in any of SEQ ID NOs: 206-210 or from any other PIV1 strain. PIV1 F variants based on the native F0 polypeptide sequence of any other PIV1 subtype or strain and containing one or more cleavage site mutations are also within the scope of the present invention. In certain embodiments, the PIV1 F protein variant contains the mutations F113G and F114S.

[0243] In certain other specific embodiments, the invention provides a PIV1 F mutant, wherein the mutant comprises leucine at positions 466, 473, and 480 (466L, 473L, and 480L) and an alanine at position 134 (134A), (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 255, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 254; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 255, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 254. The present invention provides a PIV1 F variant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0244] In certain other specific embodiments, the invention provides a PIV1 F mutant, wherein the mutant comprises a glycine (G) at position 113 (113G), a serine at position 114 (114S), a leucine at positions 466 and 473 (466L and 473L), and an alanine at position 134 (134A), (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 291, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 290; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 291, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 290. The present invention provides a PIV1 F variant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0245] In certain other specific embodiments, the invention provides a PIV1 F mutant, wherein the mutant comprises a glycine (G) at position 113 (113G), a serine at position 114 (114S), a leucine at positions 466, 473, and 480 (466L, 473L, and 480L), and an alanine at position 134 (134A), (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 277, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 276; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 277, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 276; The present invention provides a PIV1 F variant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0246] In certain other specific embodiments, the invention provides a PIV1 F mutant, wherein the mutant comprises a glycine (G) at position 113 (113G), a serine at position 114 (114S), leucines at positions 466, 473, and 480 (466L, 473L, and 480L), and cysteines at positions 92 and 134 (92C and 134C), (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 273, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 272; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 273, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 272; The present invention provides a PIV1 F variant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0247] The PIV1 F protein variants provided by the present disclosure can be prepared by routine methods known in the art, such as by expression in a recombinant host system using an appropriate vector. Suitable recombinant host cells include, for example, insect cells, mammalian cells, avian cells, bacteria, and yeast cells. Examples of suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (clonal isolates derived from the parent Trichoplusia ni BTI-TN-5B1-4 cell line (Invitrogen)). Examples of suitable mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi293 cells, typically transformed with sheared adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Suitable avian cells include, for example, chicken embryonic stem cells (e.g., EBx.RTM. cells), chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts (e.g., ELL-O), and duck cells. Suitable insect cell expression systems, such as baculovirus vector systems, are known to those skilled in the art and are described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin, No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego, Calif. Avian cell expression systems are also known to those skilled in the art and are described, for example, in U.S. Patent Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668. Similarly, bacterial and mammalian cell expression systems are known in the art and are described, for example, in Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths, London.

[0248] Many suitable vectors for expressing recombinant proteins in insect or mammalian cells are well known in the art and have been used conventionally.Suitable vectors may contain several components, including, but not limited to, one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as transcriptional control elements (e.g., promoters, enhancers, terminators), and / or one or more translation signals; and a signal or leader sequence for targeting the secretory pathway in a selected host cell (e.g., of mammalian origin, or from a heterologous mammalian or non-mammalian species).For example, for expression in insect cells, a suitable baculovirus expression vector, such as pFastBac (Invitrogen), is used to produce recombinant baculovirus particles.The baculovirus particles are amplified and used to infect insect cells to express recombinant proteins.For expression in mammalian cells, a vector that drives the expression of the construct in the desired mammalian host cell (e.g., Chinese hamster ovary cells) is used.

[0249] PIV1 F protein variant polypeptides can be purified using any suitable method. For example, methods for purifying PIV1 F protein variant polypeptides by immunoaffinity chromatography are known in the art. Ruiz-Arguello et al., J. Gen. Virol., 85:3677-3687 (2004). Suitable methods for purifying desired proteins, including precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelate, and size exclusion, are well known in the art. Two or more of these or other suitable methods can be used to create a suitable purification scheme. If desired, PIV1 F protein variant polypeptides can contain a "tag" that facilitates purification, such as an epitope tag, a strep II tag, or a histidine (HIS) tag. Such tagged polypeptides can be conveniently purified, for example, from conditioned medium by chelate or affinity chromatography.

[0250] Table 4 below provides representative sequences from the PIV1 F0 polypeptide.

[0251] [Table 4-1]

[0252] [Table 4-2]

[0253] Table 5 provides the amino acid sequences of the F1 polypeptide lacking the transmembrane and intracellular domains, and the F2 polypeptide of the mutant PIV1054 variant (based on the F protein sequence from the HPIV1 / WI / 629-D00712 / 2009 strain), to illustrate how a particular set of mutations applies to any PIV1 wild-type F protein.

[0254] [Table 5-1]

[0255] [Table 5-2]

[0256] 3. Nucleic acids encoding PIV1 F protein variants In another aspect, the present invention provides nucleic acid molecules encoding the PIV1 F protein variants described hereinabove. These nucleic acid molecules include DNA, cDNA, and RNA sequences. Nucleic acid molecules encoding only the F2 polypeptide or only the F1 polypeptide of a PIV1 F protein variant are also encompassed by the present invention. The nucleic acid molecules can be incorporated into a vector, such as an expression vector.

[0257] In some embodiments, the nucleic acid molecule encodes a precursor F0 polypeptide that, when expressed in an appropriate cell, is processed into the disclosed PIV1 F protein variants. In some embodiments, the nucleic acid molecule encodes a precursor F0 polypeptide that, when expressed in an appropriate cell, is processed into the disclosed PIV1 F protein variants, the precursor F0 polypeptide comprising, from N- to C-terminus, a signal peptide, an F2 polypeptide, and an F1 polypeptide. In some embodiments, the signal peptide comprises the amino acid sequence set forth as positions 1-21 of any one of SEQ ID NOs:206-210, wherein the amino acid positions correspond to the reference amino acid sequence of SEQ ID NO:206.

[0258] In a preferred embodiment, the nucleic acid is RNA, more preferably mRNA. In a preferred embodiment, the mRNA, when expressed in an appropriate cell, encodes a precursor F0 polypeptide that is processed into a full-length PIV1 F protein variant disclosed herein (i.e., including one or more mutations, a full-length F1 polypeptide, and a full-length F2 polypeptide). The full-length F1 polypeptide of the PIV1 F variant corresponds to amino acids 113 to 555 of the native PIV1 F0 precursor and includes (from the N- to C-terminus) the extracellular region (residues 113 to 496), the transmembrane domain (residues 497 to 517), and the cytoplasmic domain (residues 518 to 555). In a preferred embodiment, the nucleic acid is mRNA containing chemically modified nucleotides. In a preferred embodiment, the nucleic acid is mRNA containing chemically modified nucleotides, preferably 1-methylpseudouridine. Preferably, all uridines in the RNA are replaced by 1-methylpseudouridine.

[0259] In some embodiments, the nucleic acid molecule is (1) variants containing at least one engineered disulfide bond mutation; (2) mutants containing at least one cavity-filling mutation; (3) mutants containing at least one proline substitution mutation; (4) mutants containing at least one glycine replacement mutation; (5) mutants containing at least one cleavage site mutation; (6) variants containing a combination of at least one engineered disulfide mutation and at least one cavity-filling mutation; (7) variants containing a combination of at least one engineered disulfide mutation and at least one proline substitution mutation; (8) variants containing a combination of at least one engineered disulfide mutation and at least one glycine replacement mutation; (9) a variant containing a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one proline substitution mutation; (10) a variant containing a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one glycine replacement mutation; (11) a variant comprising a combination of at least one engineered disulfide mutation, at least one proline replacement mutation, and at least one glycine replacement mutation; (12) a variant containing a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, at least one proline substitution mutation, and at least one glycine replacement mutation; (13) mutants containing a combination of a cleavage site mutation and at least one engineered disulfide mutation; (14) mutants containing a combination of a cleavage site mutation and at least one cavity-filling mutation; (15) mutants containing a combination of a cleavage site mutation and at least one proline substitution mutation; (16) A mutant containing a combination of a cleavage site mutation and at least one glycine replacement mutation; (17) A mutant containing a combination of a cleavage site mutation, at least one engineered disulfide mutation, and at least one cavity-filling mutation; (18) A mutant containing a combination of a cleavage site mutation, at least one engineered disulfide mutation, and at least one proline substitution mutation; (19) A mutant containing a combination of a cleavage site mutation, at least one engineered disulfide mutation, and at least one glycine replacement mutation; (20) A mutant containing a cleavage site mutation and a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one proline substitution mutation; (21) A mutant containing a cleavage site mutation and a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one glycine replacement mutation; (22) A mutant containing a cleavage site mutation and a combination of at least one engineered disulfide mutation, at least one proline substitution mutation, and at least one glycine replacement mutation; (23) A mutant containing a cleavage site mutation and a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, at least one proline substitution mutation, and at least one glycine replacement mutation; (24) mutants containing a combination of a cleavage site mutation, at least one cavity-filling mutation, and at least one proline substitution mutation; (25) mutants containing a combination of a cleavage site mutation, at least one cavity-filling mutation, and at least one glycine replacement mutation; (26) a mutant containing a combination of a cleavage site mutation, at least one proline substitution mutation, and at least one glycine replacement mutation; (27) a combination of at least one cavity-filling mutation and at least one proline substitution mutation; (28) a combination of at least one cavity-filling mutation and at least one glycine-replacement mutation; (29) a combination of at least one proline replacement mutation and at least one glycine replacement mutation; (30) A combination of at least one cavity-filling mutation, at least one proline substitution mutation, and at least one glycine replacement mutation. The PIV1 F protein variant encodes a PIV1 F protein variant selected from the group consisting of:

[0260] In some specific embodiments, the present disclosure provides: (1) Q92C-G134C; (2) A466L; (3) A466V; (4) S473V; (5) S473L; (6) A480L; (7) A466L and S473A; (8) A466L and S473L; (9) T198A; (10) G134A; (11) A128P; (12) F113G, F114S, Q92C-G134C, A466L, S473L and A480L; (13) Q92C-G134C, A466L, S473L and A480L; (14) Q92C-G134C, A466L and S473L; (15) F113G, F114S, Q92C-G134C, A466V, S473V and A480V; (16) Q92C-G134C, A466V, S473V and A480V; (17) Q92C-G134C, A466V and S473V; (18) F113G, F114S, A466L, S473L, A480L, and G134A; (19) A466L, S473L, A480L and G134A; (20) A466L, S473L and G134A; (21) F113G, F114S, A466L, S473L, A480L, Q92A, and G134A; (22) F113G, F114S, A466L, S473L, and G134A; (23) A466L, S473L, A480L, Q92A, G134A; (24) A466L, S473L, Q92A, G134A; (25) F113G, F114S, Q92L, G134A; (26) A466L, S473L, A480L, Q92L, and G134A; (27) A466L, S473L, Q92L, and G134A; (28) F113G, F114S, A466L, S473L, A480L, Q92A, and G134L; (29) A466L, S473L, A480L, Q92A, and G134L; (30) F113G, F114S, Q92C-G134C, A466I, S473I, and A480L; (31) F113G, F114S, Q92C-G134C, A466, and S473I; and (32) A466I, S473I, A480L, Q92L, and G134A Nucleic acid molecules encoding the variants are provided, which include a mutation selected from the group consisting of:

[0261] In some specific embodiments, the present disclosure provides a nucleic acid molecule, preferably an mRNA, more preferably an mRNA in which all uridines are replaced by 1-methylpseudouridine, said nucleic acid, when expressed in a suitable cell, (1) Q92C-G134C; (2) A466L; (3) A466V; (4) S473V; (5) S473L; (6) A480L; (7) A466L and S473A; (8) A466L and S473L; (9) T198A; (10) G134A; (11) A128P; (12) F113G, F114S, Q92C-G134C, A466L, S473L and A480L; (13) Q92C-G134C, A466L, S473L and A480L; (14) Q92C-G134C, A466L and S473L; (15) F113G, F114S, Q92C-G134C, A466V, S473V and A480V; (16) Q92C-G134C, A466V, S473V and A480V; (17) Q92C-G134C, A466V and S473V; (18) F113G, F114S, A466L, S473L, A480L, and G134A; (19) A466L, S473L, A480L and G134A; (20) A466L, S473L and G134A; (21) F113G, F114S, A466L, S473L, A480L, Q92A, and G134A; (22) F113G, F114S, A466L, S473L, and G134A; (23) A466L, S473L, A480L, Q92A, G134A; (24) A466L, S473L, Q92A, G134A; (25) F113G, F114S, Q92L, G134A; (26) A466L, S473L, A480L, Q92L, and G134A; (27) A466L, S473L, Q92L, and G134A; (28) F113G, F114S, A466L, S473L, A480L, Q92A, and G134L; (29) A466L, S473L, A480L, Q92A, and G134L; (30) F113G, F114S, Q92C-G134C, A466I, S473I, and A480L; (31) F113G, F114S, Q92C-G134C, A466I, and S473I; and (32) A466I, S473I, A480L, Q92L, and G134A The present invention provides a nucleic acid molecule encoding a precursor F0 polypeptide that is processed into a full-length PIV1 F protein variant disclosed herein, comprising a mutation selected from the group consisting of:

[0262] In some specific embodiments, the present disclosure provides a nucleic acid molecule, preferably an mRNA, more preferably an mRNA in which all uridines are replaced by 1-methylpseudouridines, which, when expressed in a suitable cell, encodes a precursor F0 polypeptide that is processed into a full-length PIV1 F protein variant disclosed herein, including the mutations F113G, F114S, Q92C, G134C, A466L, S473L, and A480L.

[0263] D.PIV3 The present disclosure relates to PIV3 F protein variants, immunogenic compositions comprising the PIV3 F protein variants, methods for producing the PIV3 F protein variants, compositions comprising the PIV3 F protein variants, and nucleic acids encoding the PIV3 F protein variants.

[0264] 1. Exemplary embodiment (E) of the present invention E1. A variant of a wild-type PIV3 F protein, the variant comprising an F1 polypeptide and an F2 polypeptide, the variant comprising at least one amino acid mutation compared to the amino acid sequence of the wild-type PIV3 F protein, the amino acid mutation being: (1) at least one engineered disulfide bond mutation; (2) at least one cavity-filling mutation; (3) at least one proline substitution mutation; (4) at least one glycine replacement mutation; (5) electrostatic mutation; (6) a combination of at least one engineered disulfide mutation and at least one cavity-filling mutation; (7) a combination of at least one engineered disulfide mutation and at least one proline substitution mutation; (8) a combination of at least one engineered disulfide mutation and at least one glycine replacement mutation; (9) a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one proline substitution mutation; (10) a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one glycine-replacement mutation; (11) a combination of at least one engineered disulfide mutation, at least one proline replacement mutation, and at least one glycine replacement mutation; (12) a combination of at least one engineered disulfide mutation, at least one cavity-filling mutation, at least one proline substitution mutation, and at least one glycine replacement mutation; (13) a combination of electrostatic mutations and at least one engineered disulfide mutation; (14) a combination of electrostatic mutations and at least one cavity-filling mutation; (15) a combination of electrostatic mutations and at least one proline substitution mutation; (16) a combination of an electrostatic mutation and at least one glycine replacement mutation; (17) a combination of electrostatic mutations and at least one engineered disulfide mutation and at least one cavity-filling mutation; (18) a combination of electrostatic mutations and at least one engineered disulfide mutation and at least one proline substitution mutation; (19) a combination of electrostatic mutations and at least one engineered disulfide mutation and at least one glycine replacement mutation; (20) a combination of electrostatic mutations and at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one proline substitution mutation; (21) a combination of electrostatic mutations and at least one engineered disulfide mutation, at least one cavity-filling mutation, and at least one glycine-replacement mutation; (22) a combination of electrostatic mutations and at least one engineered disulfide mutation, at least one proline replacement mutation, and at least one glycine replacement mutation; (23) a combination of electrostatic mutations and at least one engineered disulfide mutation, at least one cavity-filling mutation, at least one proline substitution mutation, and at least one glycine replacement mutation; (24) a combination of electrostatic mutations, at least one cavity-filling mutation, and at least one proline substitution mutation; (25) a combination of an electrostatic mutation, at least one cavity-filling mutation, and at least one glycine replacement mutation; (26) a combination of an electrostatic mutation, at least one proline replacement mutation, and at least one glycine replacement mutation; (27) Cleavage site mutations, and (28) Cleavage site mutations in combination with any of the mutations or combinations of mutations listed in items (1) to (26) above. A mutant of the wild-type PIV3 F protein selected from the group consisting of: E2. The variant of E1, comprising an engineered disulfide mutation selected from the group consisting of V175C-A202C, S160C-V170C, E209C-L234C, E209C-S233C, G85C-E209C, and Q162C-L168C. E3. The variant of E2, wherein the engineered disulfide mutation is V175C-A202C or Q162C-L168C. E4. The variant described in E2, wherein the engineered disulfide mutation is S160C-V170C. E5. The variant described in E2, wherein the engineered disulfide mutation is E209C-L234C. E6. The variant according to E2, comprising two engineered disulfide mutations selected from the group consisting of V175C-A202C, S160C-V170C, E209C-L234C, and Q162C-L168C, preferably S160C-V170C and E209C-L234C. E7. The variant of any one of E1 to E6, comprising a cavity-filling mutation. E8. The variant of E7, wherein the cavity-filling mutation is selected from the group consisting of T277V, S470A, S470L, S477A, ​​A463L, I474F, and I474Y. E9. The variant of E7, wherein the cavity-filling mutation is selected from the group consisting of S470A, I474F, S477A, ​​and A463L. E10. The mutant described in E8, wherein the cavity-filling mutation is S470A or S470L. E11. The mutant described in E8, wherein the cavity-filling mutation is S477A. E12. The mutant described in E8, wherein the cavity-filling mutation is A463L. E13. The mutant described in E4, wherein the cavity-filling mutation is I474Y or I474F. E14. The variant of any one of E1 to E13, comprising two or three cavity-filling mutations selected from S470A, S470L, S477A, ​​A463L, I474F, and I474Y. E15. The mutant described in E14, wherein the cavity-filling mutations are S470A and S477A. E16. The mutant as described in E14, wherein the cavity-filling mutation is A463L and I474F, A463L and S470L, or A463L and I474F. E17. The variant of any one of E1 to E16, comprising a proline substitution mutation. E18. The mutant of E17, wherein the proline substitution mutation is S164P or G219P. E19. The variant of any one of E1 to E18, comprising a cleavage site mutation. E20. The variant described in E19, wherein the cleavage site mutations include F110G and F111S. E21. The variant of any one of E1 to E20, comprising a glycine replacement mutation. E22. The variant of E21, wherein the glycine replacement mutation is G196A or G230A. E23. The variant described in E21, wherein the glycine replacement mutation is G196A. E24. The variant of E21, wherein the glycine replacement mutation is G230A. E25. The variant of any one of E1 to E24, comprising an electrostatic mutation. E26. The mutant of E25, wherein the electrostatic mutation is E182L or D455S. E27. (1) V175C and A202C; (2) S160C and V170C; (3) S164P; (4) G196A; (5) G219P; (6) G230A; (7) E182L; (8) S470A; (9) S477A; (10) S470A and S477A; (11) D455S; (12) A463L; (13) Q162C, L168C, S470A and S477A; (14) S160C, V170C, S470A and S477A; (15) G230A, S470A, and S477A; (16) A463L, S470A and S477A; (17) E209C and L234C; (18) A463L and S470L; (19) S160C, V170C, E209C, L234C, A463L and S470L; (20) S160C, V170C, E209C, L234C, A463L and I474F; (21) S160C, V170C, E209C, L234C, A463L, S470L, F110G, F111S; (22) S160C, V170C, A463L and S470L; (23) Q162C, L168C, G230A, A463V, and I474Y; (24) Q162C, L168C, G230A, S470A, and S477A; (25) Q162C, L168C, G230A, and A463L; (26) Q162C, L168C, G230A, A463L, S470A, and S477A; (27) S160C, V170C, G230A, A463V and I474Y; (28) S160C, V170C, G230A, S470A and S477A; (29) S160C, V170C, G230A and A463L; (30) S160C, V170C, G230A, A463L, S470A and S477A; (31) S160C, V170C and A463L; (32) E209C and S233C; (33) G85C and E209C; (34) T277V; (35) A463L and I474F; (36) A463I, S470I (37) S160C, V170C, E209C, S233C, A463L, and S470L; (38) S160C, V170C, E209C, S233C, A463L and I474F; (39) S160C, V170C, G85C, E209C, A463L and S470L; (40) S160C, V170C, G85C, E209C, A463L and I474F; (41) S160C, V170C, E209C, L234C, T277V, A463L and S470L; (42) S160C, V170C, E209C, L234C, T277V, A463L and I474F; (43) S160C, V170C, E209C, S233C, T277V, A463L and S470L; (44) S160C, V170C, E209C, S233C, T277V, A463L and I474F; (45) S160C, V170C, G85C, E209C, T277V, A463L and I474F; (46) S160C, V170C, E209C, L234C, D455S, A463L and S470L; (47) S160C, V170C, E209C, S233C, D455S, A463L, and S470L; (48) S160C, V170C, G85C, E209C, D455S, A463L and S470L; (49) S160C, V170C, E209C, L234C, T277V, D455S, A463L and S470L; (50) S160C, V170C, E209C, S233C, T277V, D455S, A463L and S470L; (51) S160C, V170C, G85C, E209C, T277V, D455S, A463L and S470L; (52) S160C, V170C and S470L; (53) R106G, T107S, E108A, R109S, S160C, V170C, E209C, L234C, A463L, and S470L; (54) R106G, T107S, E108A, R109S, S160C, V170C, E209C, S233C, A463L and S470L; (55) R106G, T107S, E108A, R109S, S160C, V170C, G85C, E209C, A463L and S470L; (56) F110G, F111S, S160C, V170C, E209C, L234C, A463L, and S470L; (57) F110G, F111S, S160C, V170C, E209C, S233C, A463L, and S470L; (58) F110G, F111S, S160C, V170C, A463L, and S470L; (59) F110G, F111S, S160C, V170C, and S470L; (60) S160C, V170C, A463L and S477L; (61) S160C, V170C, E209C, L234C, A463L and S470L; and (62) S160C, V170C, and S470L The variant described in E1, comprising a mutation selected from the group consisting of: E28. (1) G230A, S470A and S477A; (2) S160C, V170C, G230A and A463L; (3) S160C, V170C, S470A and S477A; (4) S160C, V170C, G230A, S470A and S477A; (5) S160C, V170C, G230A, A463L, S470A and S477A (6) S160C, V170C, E209C, L234C, A463L and S470L; (7) S160C, V170C, E209C, L234C, A463L and I474F; (8) S160C, V170C, E209C, L234C, A463L, S470L, F110G, F111S; and (9) S160C, V170C, A463L, and S470L, and (10) E209C and L234C The variant described in E1, comprising a mutation selected from the group consisting of: E29. The variant of E1, wherein the variant comprises a mutation selected from S160C, V170C, A463L, and S470L, and wherein the F1 polypeptide comprises or consists of amino acid residues 110-484. E30. The variant of any one of E1 to E29, further comprising a mutation selected from the amino acid substitutions R106G, T107S, E108A, and R109S. E31. (a) containing alanines at positions 230, 470, and 477 (230A, 470A, and 477A); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 329, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 328; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 329, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 328. or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (b) cysteine ​​at positions 160 (160C) and 170 (170C), leucine at position 463 (463L), and alanine at position 230 (230A); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 353, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 352; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 353, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 352. or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (c) cysteines at positions 160 (160C) and 170 (170C) and alanines at positions 470 (470A) and 477 (477A); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 339, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 338; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 339, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 338. or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (d) cysteines at positions 160 (160C) and 170 (170C) and alanines at positions 230 (230A), 470 (470A), and 477 (477A); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 351, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 350; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 351, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 350. or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (e) cysteine ​​at positions 160 (160C) and 170 (170C), leucine at position 463 (463L), and alanine at positions 230 (230A), 470 (470A), and 477 (477A); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 355, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 354; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 355, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 354. comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of The mutant described in E1. E32. (a) cysteines at positions 160 (160C), 170 (170C), 209 (209C), and 234 (234C) and leucines at positions 463 (463L) and 470 (470L); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 438, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 437; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 438, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 437; or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (b) cysteine ​​at positions 160 (160C), 170 (170C), 209 (209C), and 234 (234C), leucine at position 463 (463L), and phenylalanine at position 474 (474F); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 440, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 439; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 440, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 439. or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (c) cysteine ​​at positions 160 (160C), 170 (170C), 209 (209C), and 234 (234C), leucine at positions 463 (463L) and 470 (470L), glycine at position 110 (110G), and serine at position 111 (111S); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 482, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 481; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 482, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 481; or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (d) cysteines at positions 160 (160C) and 170 (170C) and leucines at positions 463 (463L) and 470 (470L); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 494, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 493; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 494, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 493. comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of The mutant described in E1. the E33.F1 polypeptide lacks the entire cytoplasmic domain, or The F1 polypeptide lacks the cytoplasmic domain and part or all of the transmembrane domain. The mutant according to any one of E1 to E32. E34. The variant of any one of E1 to E30, wherein the F1 polypeptide lacks the cytoplasmic and transmembrane domains. E35. The variant of any one of E1 to E33, wherein the F1 polypeptide comprises or consists of amino acid residues 110 to 481. E36. The variant of any one of E1 to E33, wherein the F1 polypeptide comprises or consists of amino acid residues 110 to 484. E37. The variant of any one of E1 to E31, wherein the F1 polypeptide comprises an ectodomain, a transmembrane domain, and a cytoplasmic domain, and in a preferred embodiment, the variant comprises a full-length F1 polypeptide and a full-length F2 polypeptide. E38. The variant of any one of E1 to E37, wherein said variant is linked to a trimerization domain. E39. The variant of E38, wherein the trimerization domain is a GCN4 leucine zipper or a phage T4 fibritin foldon. E40. The variant of E39, wherein the trimerization domain is a phage T4 fibritin foldon. E41. The variant of E40, wherein the trimerization domain is the phage T4 fibritin foldon of SEQ ID NO:7. E42. The variant of any one of E38 to E41, wherein the trimerization domain is linked to the C-terminus of the F1 polypeptide. E43. The variant of any one of E38 to E42, wherein the trimerization domain is linked to the C-terminus of the F1 polypeptide via a linker. E44. The variant of E43, wherein the trimerization domain is linked to the C-terminus of the F1 polypeptide via a linker selected from the group consisting of GG, GS, GGGS, or SAIG. E45. The variant as described in E44, wherein the linker is GGGS. E46. The variant of any one of E1 to E45, in a trimeric form. E47. The variant of any one of E1 to E46, wherein the variant is in a pre-fusion conformation. E48. The variant of any one of E1 to E46, which is in a pre-fusion conformation and specifically binds to an antibody (such as PIA174 mAb) specific for the PIV3 F ectodomain in the pre-fusion conformation but not the PIV3 F ectodomain in the post-fusion conformation. E49. The variant of any one of E1 to E47, wherein the variant is in the pre-fusion conformation and specifically binds to PIA174 mAb, preferably as measured by ELISA as disclosed in the Examples. E50. The variant of any one of E1 to E46, having increased stability compared to a corresponding wild-type PIV3 F protein, wherein the stability is measured by binding of the variant to the antibody PIA174 mAb. E51. The variant of any one of E1 to E46, wherein the wild-type PIV3 F protein is SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, or SEQ ID NO: 304, preferably wherein the wild-type PIV3 F protein is SEQ ID NO: 300. E52. The variant of any one of E1 to E51, wherein the amino acid positions correspond to the reference amino acid sequence of SEQ ID NO: 300. E53. A nucleic acid comprising at least one coding sequence encoding at least one mutant of a wild-type PIV3 F protein, or an immunogenic fragment or variant thereof, as described in any one of embodiments E1 to E52, preferably E37, wherein the nucleic acid comprises at least one heterologous untranslated region (UTR). E54. The nucleic acid of E53, wherein the at least one heterologous untranslated region is selected from at least one heterologous 5'-UTR and / or at least one heterologous 3'-UTR. E55. At least one heterologous 3'-UTR is CΨCGAGCΨGGΨ ACΨGCAΨGCA CGCAAΨGCΨA GCΨGCCCCΨΨ ΨCCCGΨCCΨG GGΨACCCCGA GΨCΨCCCCCG ACCΨCGGGΨC CCAGGΨAΨGC ΨCCCACCΨCC ACCΨGCCCCA CΨCACCACCΨ CΨGCΨAGΨΨC CAGACACCΨC CCAAGCACGC AGCAAΨGCAG CΨCAAAACGC ΨΨAGCCΨAGC CACACCCCCA CGGGAAACAG CAGΨGAΨΨAA CCΨΨΨAGCAA ΨAAACGAAAG ΨΨΨAACΨAAG CΨAΨACΨAAC CCCAGGGΨΨG 10. The nucleic acid of any one of the preceding embodiments, comprising or consisting of a nucleic acid sequence having at least, and at most exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to GΨCAAΨΨΨCG ΨGCCAGCCAC ACCCΨGGAGC ΨAGC, or a percentage identity between any two thereof. E56. The nucleic acid of any one of the preceding embodiments, wherein at least one heterologous 5'-UTR comprises or consists of a nucleic acid sequence having at least, and at most, exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to GAAΨAAAC ΨAGΨAΨΨCΨΨ CΨGGΨCCCCA CAGACΨCAGA GAGAACCCGC CACC, or a percentage identity between any two thereof. E57. The nucleic acid of any one of the preceding embodiments, comprising at least one poly(A) sequence, preferably comprising 30 to 200 adenosine nucleotides, and / or at least one poly(C) sequence, preferably comprising 10 to 40 cytosine nucleotides. E58. The nucleic acid of any one of the preceding embodiments, which is DNA or RNA. E59. The nucleic acid of any one of the preceding embodiments, which is a coding RNA. E60. The nucleic acid of E59, wherein the coding RNA is mRNA, self-replicating RNA, circular RNA, or replicon RNA. E61. The nucleic acid of any one of the preceding embodiments, wherein the nucleic acid, preferably the coding RNA, is mRNA. E62. The nucleic acid of E1, wherein the mRNA is not a replicon RNA or a self-replicating RNA. E63. The nucleic acid of any one of the preceding embodiments E59 to E62, wherein the mRNA comprises at least one poly(A) sequence comprising 30 to 200 adenosine nucleotides, and the 3' terminal nucleotide is an adenosine. E64. The nucleic acid of any one of the preceding embodiments E58 to E63, wherein the RNA, preferably the coding RNA, comprises a 5'-cap structure, preferably an m7G, cap0, cap1, cap2, modified cap0, or modified cap1 structure, preferably a 5'-cap1 structure. E65. The nucleic acid of any one of the preceding embodiments E58 to E64, wherein the RNA is codon-optimized. E66. The nucleic acid of any one of the preceding embodiments E58 to E65, wherein the RNA comprises chemically modified nucleotides. E67. The nucleic acid of any one of the preceding embodiments E58 to E66, wherein the RNA comprises 1-methylpseudouridine substitutions, preferably all uridines of the RNA are replaced by 1-methylpseudouridine. E68. The nucleic acid of any one of the preceding embodiments E58 to E67, wherein the RNA is purified RNA, preferably RNA purified by RP-HPLC and / or TFF. E69. The nucleic acid of any one of the preceding embodiments E58 to E68, wherein the RNA comprises the nucleic acid sequence of any of SEQ ID NO:411, SEQ ID NO:413, SEQ ID NO:415, SEQ ID NO:417 and SEQ ID NO:419, SEQ ID NO:504, SEQ ID NO:506, SEQ ID NO:508 and SEQ ID NO:510, SEQ ID NO:295, SEQ ID NO:297 and SEQ ID NO:299. E70. A composition comprising at least one nucleic acid of any one of the preceding embodiments E53 to E69. E71. A composition comprising at least one nucleic acid of any one of the preceding embodiments E53 to E69, comprising at least one pharmaceutically acceptable carrier. E72. A composition comprising at least one nucleic acid according to any one of the preceding embodiments E53 to E69, which is a multivalent composition comprising a plurality or more than at least one of the nucleic acids according to any one of E53 to E69. E73. A composition comprising at least one nucleic acid of any one of the preceding embodiments E53 to E69, wherein the composition comprises RNA having RNA integrity of 70% or greater. E74. A composition comprising at least one nucleic acid of any one of the preceding embodiments E53 to E69, wherein the composition comprises RNA with a degree of capping of 70% or more, preferably at least 70%, 80%, or 90% of mRNA species contain Cap 1 structures. E75. A composition comprising at least one nucleic acid of any one of the preceding embodiments E53 to E69, wherein the at least one nucleic acid is complexed or associated, or at least partially complexed or partially associated, with one or more cationic or polycationic compounds, preferably cationic or polycationic polymers, cationic or polycationic polysaccharides, cationic or polycationic lipids, cationic or polycationic proteins, cationic or polycationic peptides, or any combination thereof. E76. A composition comprising at least one nucleic acid of any one of the preceding embodiments E53 to E69, wherein the at least one nucleic acid is complexed or associated with one or more lipids or lipid-based carriers, thereby forming a liposome, lipid nanoparticle (LNP), lipoplex, and / or nanoliposome, preferably encapsulating the at least one nucleic acid. E77. A composition comprising at least one nucleic acid of any one of the preceding embodiments E53 to E69, wherein the at least one nucleic acid is complexed with one or more lipids, thereby forming a lipid nanoparticle. E78.LNP has formula III-3:

[0265] [ka] The composition of any one of the preceding embodiments E76 to E77, comprising a cationic lipid according to E79. E80.LNP has formula (IVa):

[0266] [ka] The composition of any one of the preceding embodiments E76 to E78, comprising a PEG lipid of E81. The composition of embodiment E79, wherein n has an average value ranging from 30 to 60, preferably n has an average value of about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and most preferably n has an average value of 49 or 45. E82.LNP has formula (IVa):

[0267] [ka] The composition of any one of the preceding embodiments E76 to E80, comprising a PEG lipid of formula (I), where n is an integer selected such that the average molecular weight of the PEG lipid is about 2500 g / mol. E83. The composition of any one of the preceding embodiments E76 to E81, wherein the LNP comprises one or more neutral lipids, and / or one or more steroids or steroid analogues. E84. The composition of any one of the preceding embodiments E76 to E82, wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and preferably the molar ratio of cationic lipid to DSPC is in the range of about 2:1 to about 8:1. E85. The composition of any one of the preceding embodiments E76 to E83, wherein the steroid is cholesterol, and preferably the molar ratio of cationic lipid to cholesterol is in the range of about 2:1 to about 1:1. E86. The composition of any one of the preceding embodiments E76 to E84, wherein the LNP comprises: (i) at least one cationic lipid, preferably a lipid of formula (III), more preferably lipid III-3; (ii) at least one neutral lipid, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) at least one steroid or steroid analog, preferably cholesterol; and (iv) at least one polymer-conjugated lipid, preferably a PEG lipid derived from formula (IVa, having n=49), wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG lipid. E87. The composition of any one of the preceding embodiments E76 to E85, wherein the LNP comprises: (i) at least one cationic lipid, preferably a lipid of formula (III), more preferably lipid III-3; (ii) at least one neutral lipid, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) at least one steroid or steroid analog, preferably cholesterol; and (iv) at least one polymer-conjugated lipid, preferably a PEG lipid from formula (IVa, having n=45), wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG lipid. E88. The composition of any one of the preceding embodiments E76 to E86, wherein (i)-(iv) are in a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7, or more preferably 47.4:10:40.9:1.7. E89. The composition of any one of the preceding embodiments E76 to E87, wherein the nucleic acid is RNA and the composition comprises less than about 20% free (uncomplexed or unencapsulated) RNA, preferably less than about 15% free RNA, more preferably less than about 10% free RNA. E90. The composition of any one of the preceding embodiments E76 to E88, wherein the wt / wt ratio of lipid to nucleic acid is from about 10:1 to about 60:1, preferably from about 20:1 to about 30:1, such as about 25:1. E91. The composition of any one of the preceding embodiments E76 to E89, wherein the n / p ratio of the LNP encapsulating the nucleic acid is in the range of about 1 to about 10, preferably in the range of about 5 to about 7, and more preferably about 6. E92. The composition of any one of the preceding embodiments E76 to E90 having a polydispersity index (PDI) value of less than about 0.4, preferably less than about 0.3, more preferably less than about 0.2, and most preferably less than about 0.1. E93. The composition of any one of the preceding embodiments E76 to E91, wherein the LNPs have a Z-average size in the range of about 60 nm to about 120 nm, preferably less than about 120 nm, more preferably less than about 100 nm, and most preferably less than about 80 nm. E94. The composition of any one of the preceding embodiments E76 to E92, wherein the LNPs comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of LNPs having a particle size greater than about 500 nm. E95. The composition of any one of the preceding embodiments E76 to E93, wherein the LNPs comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% LNPs having a particle size smaller than about 20 nm. E96. The composition of any one of the preceding embodiments E76 to E94, wherein the LNP comprises (i) at least one cationic lipid; (ii) at least one neutral lipid; (iii) at least one steroid or steroid analog; and (iv) at least one PEG-lipid, wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid. E97. The composition of any one of the preceding embodiments E76 to E95, wherein the LNP comprises: (i) at least one cationic lipid according to formula III-3; (ii) DSPC; (iii) cholesterol; and (iv) a PEG-lipid according to formula IVa, wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid. E98. The composition of any one of the preceding embodiments E70 to E96, which is a lyophilized composition. E99. An immunogenic composition comprising a variant according to any one of E1 to E52, a nucleic acid according to any one of E53 to E69, or a composition according to any one of E70 to E97. E100. The method further comprises: providing an hMPV A antigen selected from the group consisting of a mutant of a wild-type hMPV AF protein and a nucleic acid encoding the mutant of a wild-type hMPV AF protein; in one embodiment, the hMPV A antigen is selected from a mutant of a wild-type hMPV AF protein and a nucleic acid encoding the mutant of a wild-type hMPV AF protein disclosed in any of WO16103238, WO20234300, WO21222639, WO22076669, WO22214678, WO23102373, WO23110618, WO23217988, and WO23102388; in one embodiment, the hMPV A antigen is selected from a mutant of a wild-type hMPV AF protein and a nucleic acid encoding the mutant of a wild-type hMPV AF protein disclosed in Battles et al., Nature communication 8:1528 (2017). The immunogenic composition of E98, which is a nucleic acid encoding a variant of the AF protein or a variant of the wild-type hMPV AF protein that includes the mutations of mutant 115-BV. E101. The immunogenic composition of embodiment E99, wherein the hMPV A antigen is a mutant of the wild-type hMPV AF protein. E102. The immunogenic composition of embodiment E99, wherein the hMPV A antigen is a variant of a wild-type hMPV AF protein from the present disclosure, preferably from any of E1 to E72 of section B of the present disclosure. E103. The immunogenic composition of embodiment E99, wherein the hMPV A antigen comprises a nucleic acid encoding a variant of the wild-type hMPV AF protein. E104. The immunogenic composition of embodiment E99, wherein the hMPV A antigen comprises a nucleic acid encoding a variant of the wild-type hMPV AF protein from the present disclosure, preferably a nucleic acid from any of E73 to E89 of section B of the present disclosure. E105. The method further comprises: providing an hMPV B antigen selected from the group consisting of a mutant of a wild-type hMPV BF protein and a nucleic acid encoding the mutant of a wild-type hMPV BF protein; in one embodiment, the hMPV B antigen is selected from a mutant of a wild-type hMPV BF protein and a nucleic acid encoding the mutant of a wild-type hMPV BF protein disclosed in any of WO16103238, WO20234300, WO21222639, WO22076669, WO22214678, WO23102373, WO23110618, WO23217988, and WO23102388; in one embodiment, the hMPV B antigen is a wild-type hMPV B antigen comprising the mutations of mutant 115-BV disclosed in Battles et al., Nature communication 8:1528 (2017). The immunogenic composition of any one of embodiments E98 to E103, which is a nucleic acid encoding a variant of the BF protein or a variant of the wild-type hMPV BF protein comprising the mutations of mutant 115-BV. E106. The immunogenic composition of embodiment E104, wherein the hMPV B antigen is a mutant of the wild-type hMPV BF protein. E107. The immunogenic composition of embodiment E104, wherein the hMPV B antigen is a variant of a wild-type hMPV BF protein from the present disclosure, preferably from any of E1 to E72 of section B of the present disclosure. E108. The immunogenic composition of embodiment E104, wherein the hMPV B antigen comprises a nucleic acid encoding a variant of the wild-type hMPV BF protein. E109. The immunogenic composition of embodiment E104, wherein the hMPV B antigen comprises a nucleic acid encoding a variant of a wild-type hMPV BF protein from the present disclosure, preferably a nucleic acid from any of E73 to E89 of section B of the present disclosure. E109. The immunogenic composition of any one of embodiments E98 to E108, further comprising a PIV1 antigen selected from the group consisting of a variant of the wild-type PIV1 F protein, and a nucleic acid encoding a variant of the wild-type PIV1 F protein. E110. The immunogenic composition of embodiment E109, wherein the PIV1 antigen is a variant of the wild-type PIV1 F protein. E111. The immunogenic composition of embodiment E109, wherein the PIV1 antigen is a variant of a wild-type PIV1 F protein from the present disclosure, preferably from any of E1 to E56 of section C of the present disclosure. E112. The immunogenic composition of embodiment E109, wherein the PIV1 antigen comprises a nucleic acid encoding a variant of the wild-type PIV1 F protein. E113. The immunogenic composition of embodiment E109, wherein the PIV1 antigen comprises a nucleic acid encoding a variant of a wild-type PIV1 F protein from the present disclosure, preferably from any of E57 to E73 of section C of the present disclosure. E114. The immunogenic composition of embodiment E109, wherein the PIV1 antigen comprises a nucleic acid encoding a variant of the wild-type PIV1 F protein disclosed in WO2018081289 or WO2022207839. E115. The immunogenic composition of any one of E98 to E114, further comprising a RSV antigen selected from the group consisting of a subtype A wild-type RSV F protein variant and a nucleic acid encoding a subtype A wild-type RSV F protein variant. E116. The immunogenic composition of embodiment E115, wherein the RSV antigen is a variant of a subtype A wild-type RSV F protein. E117. The immunogenic composition of embodiment E115, wherein the RSV antigen is a nucleic acid encoding a variant of a subtype A wild-type RSV F protein. E118. The immunogenic composition of embodiment E115, wherein the variant of the subtype A wild-type RSV F protein is disclosed in one of WO2009 / 079796, WO2010 / 149745, WO2011 / 008974, WO2014 / 160463, WO2014 / 174018, WO2014 / 202570, WO2015 / 013551, WO2015 / 177312, WO2017 / 005848, WO2017 / 174564, WO2017 / 005844, WO2017 / 109629, WO2022 / 002894, and WO2018 / 109220. E119. The immunogenic composition of any one of embodiments E98 to E118, further comprising a RSV antigen selected from the group consisting of a subtype B wild-type RSV F protein variant and a nucleic acid encoding a subtype B wild-type RSV F protein variant. E120. The immunogenic composition of embodiment E119, wherein the RSV antigen is a variant of the wild-type RSV F protein of subtype B. E121. The immunogenic composition of embodiment E119, wherein the RSV antigen is a nucleic acid encoding a variant of a wild-type RSV F protein of subtype B. E122. The immunogenic composition of embodiment E119, wherein the variant of the subtype B wild-type RSV F protein is disclosed in one of WO2009 / 079796, WO2010 / 149745, WO2011 / 008974, WO2014 / 160463, WO2014 / 174018, WO2014 / 202570, WO2015 / 013551, WO2015 / 177312, WO2017 / 005848, WO2017 / 174564, WO2017 / 005844, WO2017 / 109629, WO2022 / 002894, and WO2018 / 109220.

[0268] 2. PIV3 F protein variants In some aspects, the present invention provides mutants of wild-type PIV3 F proteins, which exhibit introduced mutations in their amino acid sequences compared to the amino acid sequence of the corresponding wild-type PIV3 F protein and are immunogenic against the wild-type PIV3 F protein in a pre-fusion conformation or against viruses containing the wild-type F protein. In certain embodiments, the PIV3 F mutants possess certain beneficial characteristics, such as increased immunogenic properties or improved stability of the mutant pre-fusion conformation or the mutant pre-fusion trimer conformation, compared to the corresponding wild-type F protein. In still other embodiments, the present disclosure provides PIV3 F mutants that exhibit one or more introduced mutations described herein and that bind to a pre-fusion-specific antibody selected from PIA174 mAb.

[0269] The amino acid mutations introduced in the PIV3 F protein variant include amino acid substitutions, deletions, or additions. In some embodiments, the only mutations in the amino acid sequence of the variant are amino acid substitutions compared to the wild-type PIV3 F protein.

[0270] The amino acid sequences of numerous native PIV3 F proteins from various strains, as well as the nucleic acid sequences encoding such proteins, are known in the art. For example, the sequences of several PIV3 F0 precursor proteins are set forth in SEQ ID NOs: 300-304.

[0271] The native PIV3 F protein exhibits remarkable sequence conservation across different lineages.

[0272] Given the substantial conservation of PIV3 F protein sequences, those skilled in the art can easily compare amino acid positions between various native PIV3 F protein sequences to identify corresponding PIV3 F protein amino acid positions between various PIV3 strains. For example, the protease cleavage site is located at the same amino acid position across nearly all identified native PIV3 F precursor proteins. Thus, the conservation of native PIV3 F protein sequences across strains and subtypes allows the use of a reference PIV3 F sequence for comparison of amino acids at specific positions within the PIV3 F protein. For purposes of this disclosure (unless the context indicates otherwise), PIV3 F protein amino acid positions are provided with reference to the sequence of the F precursor polypeptide set forth in SEQ ID NO: 300 (corresponding to the amino acid sequence of the full-length native F precursor polypeptide of PIV3 strain HPIV3 / MEX / 2545 / 2006; GenBank identifier AGT75285.1 (amino acids)).

[0273] The consensus sequence for PIV3 (corresponding to SEQ ID NO: 300) was obtained as follows: The entire genome sequence for PIV3 was downloaded from the NCBI GenBank database as a GenBank file. The fusion protein gene sequences were filtered by sequence length to include only the complete coding DNA sequence. The translated fusion protein sequences were then analyzed from the GenBank file and saved as a FASTA file. Muscle v5 was used to perform multiple sequence alignment of the collected sequences. A position-specific score matrix (PSSM) was created to summarize the alignment information. For each column in the alignment, the number of each amino acid character was counted and summed. The consensus sequence at each position was calculated as the most commonly occurring amino acid type in the PSSM table. The final consensus sequence was then extracted and saved as a FASTA file.

[0274] However, it should be noted, and one of skill in the art would understand, that different PIV3 F0 sequences may have different numbering systems if, for example, there are additional amino acid residues added or removed compared to SEQ ID NO: 300. Thus, when specific amino acid residues are referred to by their numbers, it should be understood that the description is not limited to only the amino acid at that exact numbered position when counting from the beginning of a given amino acid sequence, but rather that the equivalent / corresponding amino acid residue in any and all PIV3 F sequences is intended, even if that residue is not at the same exact numbered position, for example, if the PIV3 sequence is shorter or longer than SEQ ID NO: 300 or has an insertion or deletion compared to SEQ ID NO: 300.

[0275] 2-1. Structure of PIV3 F protein mutants The PIV3 F protein variants provided by the present disclosure include an F1 polypeptide and an F2 polypeptide. In some embodiments, the variants further include a trimerization domain. In some embodiments, either the F1 polypeptide or the F2 polypeptide comprises at least one introduced modification (e.g., amino acid substitution) as described in detail herein below. In some other embodiments, each of the F1 polypeptide and the F2 polypeptide comprises at least one introduced modification (e.g., amino acid substitution) as described in detail herein below.

[0276] 2-1(a). F1 and F2 polypeptides of PIV3 F mutants The mature form of the PIV3 F protein comprises two separate polypeptide chains, i.e., an F1 polypeptide and an F2 polypeptide, linked by a disulfide bond. In some embodiments, the variants of the present disclosure are not cleaved, and the F2 polypeptide and the F1 polypeptide form a single polypeptide. The expression system (CHO cells) used to produce the variants may not contain a protease that cleaves the PIV3 F protein in its native environment and therefore will exhibit limited cleavage.

[0277] The F1 polypeptide chain of a variant can be the same length as the full-length F1 polypeptide of the corresponding wild-type PIV3 F protein; however, it can also have a deletion, such as a deletion of 1 to up to 36 amino acid residues from the C-terminus of the full-length F1 polypeptide. The full-length F1 polypeptide of a PIV3 F variant corresponds to amino acids 103 to 539 of the native PIV3 F precursor and includes (from N- to C-terminus) the extracellular region (residues 103 to 493), the transmembrane domain (residues 494 to 514), and the cytoplasmic domain (residues 515 to 539). It should be noted that amino acid residues 481 and beyond in the native F1 polypeptide sequence are optional in the F1 polypeptides of the PIV3 F variants provided herein and therefore may be absent from the variant F1 polypeptides.

[0278] In some embodiments, the F1 polypeptide of a PIV3 F variant lacks the entire cytoplasmic domain. In other embodiments, the F1 polypeptide lacks the cytoplasmic domain and part or all of the transmembrane domain. In some specific embodiments, the variant comprises an F1 polypeptide in which amino acid residues 482-539 are absent. In some specific embodiments, the variant comprises an F1 polypeptide in which amino acid residues 485-539 are absent. Typically, for variants linked to a trimerization domain, such as a foldon, amino acids 482-539 may be absent. Thus, in some specific embodiments, amino acid residues 482-539 are absent from the variant F1 polypeptide. In yet other specific embodiments, the F1 polypeptide of a PIV3 F variant comprises or consists of amino acid residues 110-481 of a native F0 polypeptide sequence, such as any of the F0 precursor sequences set forth in SEQ ID NOs: 300-304.

[0279] On the other hand, the F1 polypeptide of a PIV3 F variant can include a C-terminal linkage to a trimerization domain such as a foldon. Many of the sequences of the PIV3 F variants disclosed herein contain PreScission cleavage sites and Strep Tag II sequences that are not essential for the function of the PIV3 F protein, such as inducing an immune response. Those skilled in the art will recognize such sequences and understand that, where appropriate, these sequences are not included in the disclosed PIV3 F variants.

[0280] In the PIV3 F variants provided by the present disclosure, the F2 polypeptide chain can be the same length as the full-length F2 polypeptide of the corresponding wild-type PIV3 F protein; it can also have a deletion, such as a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues from the N-terminus or C-terminus of the F2 polypeptide.

[0281] Variants of the F0 form (i.e., a single-chain polypeptide comprising an F2 polypeptide joined to an F1 polypeptide) or the F1-F2 heterodimeric form can form protomers. Variants can also be in the form of trimers comprising three identical protomers. Furthermore, variants can be glycosylated proteins (i.e., glycoproteins) or non-glycosylated proteins. Variants of the F0 form can contain or lack a signal peptide sequence.

[0282] The F1 and F2 polypeptides of the PIV3 F protein variants having one or more mutations introduced therein can be derived from any wild-type PIV3 F protein known in the art or discovered in the future. In some embodiments, the PIV3 F variants comprise F1 and / or F2 polypeptides derived from a known PIV3 F0 precursor protein from a PIV3 virus, such as those set forth in any one of SEQ ID NOS: 300-304, having one or more mutations introduced therein.

[0283] In some embodiments, the PIV3 F protein variant comprises an F1 polypeptide, an F2 polypeptide, and one or more introduced amino acid mutations described herein below, wherein the F1 polypeptide comprises 350 contiguous amino acids and is at least 90, 95, 98, or 99 percent identical to amino acids 110-481 of any of the sequences set forth in SEQ ID NOs: 300-304, the F2 polypeptide comprises 70 contiguous amino acids and is at least 90, 95, 98, or 99 percent identical to amino acids 22-112 of any of the sequences set forth in SEQ ID NOs: 300-304, and the PIV3 F protein variant is stabilized in a pre-fusion trimer conformation both as a monomer and as a trimer.

[0284] 2-1(b) Trimerization domain In some embodiments, the PIV3 F variants provided by the present disclosure are linked to a trimerization domain, which in some embodiments promotes the formation of trimers of three F1 / F2 heterodimers.

[0285] Some exogenous trimerization domains that promote the formation of stable trimers of soluble proteins are known in the art.Non-limiting examples of such trimerization domains that can be linked to the variants provided by the present disclosure include: (1) GCN4 leucine zipper (Harbury et al., 1993 Science 262:1401-1407); (2) trimerization motif from pulmonary surfactant protein (Hoppe et al., 1994 FEBS Lett 344:191-195); (3) collagen (McAlinden et al., 2003 Biol Chem 278:42200-42207); and (4) phage T4 fibritin foldon (Miroshnikov et al., 1998 Protein Eng 11:329-414).

[0286] Typically, the trimerization domain is located C-terminal to the F1 polypeptide. It may be directly attached to the F1 polypeptide chain. Optionally, the multimerization domain is connected to the F1 polypeptide via an amino acid linker, such as a linker with the sequence GG, GS, GGGS, or SAIG. The linker may also be a longer linker (e.g., containing a repeat sequence GG). A preferred linker is GGGS. Many conformationally neutral linkers are known in the art and can be used in the variants provided by the present disclosure. In some embodiments, the F1 variant containing a foldon domain comprises a protease cleavage site for removing the foldon domain from the F1 polypeptide, such as a thrombin site between the F1 polypeptide and the foldon domain.

[0287] In some embodiments, the foldon domain is linked to the F mutant at the C-terminus of the F1 polypeptide. In a specific embodiment, the foldon domain is a T4 fibritin foldon domain, such as the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 7).

[0288] 2-2.Introduced mutations in PIV3 F protein mutants The PIV3 F variants provided by the present disclosure include an F1 polypeptide and an F2 polypeptide, wherein either (1) the F1 polypeptide or (2) the F2 polypeptide, or (3) both the F1 polypeptide and the F2 polypeptide, contain one or more introduced amino acid mutations compared to the amino acid sequence of the corresponding native F protein. The introduction of such amino acid mutations in the PIV3 F variants confers beneficial properties to the variant, such as enhanced immunogenicity, improved stability, improved expression or formulation, or improved stability of a particular desired physical form or conformation of the variant. Such introduced amino acid mutations are referred to as "engineered disulfide bond mutations," "cavity-filling mutations," "proline substitution mutations," "cleavage site mutations," "glycine replacement mutations," or "electrostatic mutations."

[0289] The nature and purpose of the "engineered disulfide bond mutations," "cavity-filling mutations," "proline substitution mutations," and "glycine replacement mutations" have already been disclosed above in connection with hMPV protein mutants.

[0290] 2-2(a) Engineered disulfide bond mutations In some embodiments, the PIV3 protein F mutants provided by the present disclosure contain one or more engineered disulfide bond mutations. The term "engineered disulfide bond mutation" refers to the mutation of a pair of amino acid residues in the wild-type PIV3 F protein to a pair of cysteine ​​residues. The introduced pair of cysteine ​​residues allows for the formation of a disulfide bond between the introduced cysteine ​​residues, and the disulfide bond serves to stabilize the conformation or oligomeric state of the protein, such as the pre-fusion conformation. To stabilize the pre-fusion conformation of the mutant, the residue pair for mutation to cysteine ​​should be in close proximity in the pre-fusion conformation but far apart in the post-fusion conformation. Preferably, the distance between the residue pair (e.g., beta carbons) is less than 8 Å in the pre-fusion conformation but greater than 20 Å in the post-fusion conformation.

[0291] In some embodiments, the PIV3 F protein variant contains only one engineered disulfide mutation (a "single engineered disulfide mutation"). In other embodiments, the PIV3 F protein variant contains at least two engineered disulfide mutations, with each pair of cysteine ​​residues of the engineered disulfide mutations appropriately positioned when the PIV3 F protein variant is in the prefusion conformation (a "double engineered disulfide mutation").

[0292] In some specific embodiments, the present disclosure provides PIV3 F mutants comprising at least one engineered disulfide bond mutation, wherein the mutants comprise the same introduced mutations as in any of the exemplary mutants provided in Tables 42 and 47.

[0293] The exemplary PIV3 F variants provided in Tables 42 and 47 are based on the same F0 sequence of PIV3 of SEQ ID NO: 305. The same introduced mutations in each of the variants can be made to the native F0 polypeptide sequence of any other PIV3 subtype or strain to arrive at a different PIV3 F variant, such as the native F0 polypeptide sequence set forth in any of SEQ ID NOs: 300-304 or from any other PIV3 strain. PIV3 F variants based on the native F0 polypeptide sequence of any other PIV3 subtype or strain and including any of the engineered disulfide mutations are also within the scope of the invention. In certain embodiments, the PIV3 F protein variant comprises at least one engineered disulfide mutation such as 175C-202C, 160C-170C, 209C-234C, 209C-233C, 85C-209C, and 162C-168C, preferably V175C-A202C, S160C-V170C, E209C-L234C, E209C-S233C, G85C-E209C, and Q162C-L168C. ​​In certain embodiments, the PIV3 F protein variant comprises at least one engineered disulfide mutation such as 160C-170C, preferably S160C-V170C.

[0294] 2-2(b) Cavity-filling mutation In other embodiments, the present disclosure provides PIV3 F mutants containing one or more cavity-filling mutations. The term "cavity-filling mutation" refers to the replacement of an amino acid residue in a wild-type PIV3 F protein with an amino acid predicted to fill the internal cavity of the mature PIV3 F protein. In one application, such cavity-filling mutations contribute to stabilizing the pre-fusion conformation of the PIV3 F protein mutant. For example, the amino acids to be replaced for cavity-filling mutations typically include small aliphatic amino acids (e.g., Gly, Ala, and Val) or small polar amino acids (e.g., Ser and Thr). They may also include amino acids that are buried in the pre-fusion conformation but exposed to the solvent in the post-fusion conformation. The replacement amino acids may include aliphatic amino acids (Val, Ile, Leu, and Met), aromatic amino acids (His, Phe, Tyr, and Trp), or polar amino acids (Thr) that are larger in size than the amino acids being replaced.

[0295] In some specific embodiments, the PIV3 F protein variant comprises one or more cavity-filling mutations at positions 463, 470, 474, and 477.

[0296] In some specific embodiments, the present disclosure provides PIV3 F variants comprising one or more cavity-filling mutations, wherein the variants comprise a cavity-filling mutation in any of the variants provided in Tables 44 and 47. The PIV3 F variants provided in Tables 44 and 47 are based on the same native F sequence of PIV3 of SEQ ID NO: 305. The same introduced mutations in each of the variants can be performed on the native F polypeptide sequence of any other PIV3 subtype or strain to arrive at a different PIV3 F variant, such as a native F polypeptide sequence set forth in any of SEQ ID NOs: 300-304 or from any other PIV3 strain. PIV3 F variants based on the native F polypeptide sequence of any other PIV3 subtype or strain and comprising one or more cavity-filling mutations are also within the scope of the present invention. In some specific embodiments, the PIV3 F protein variant comprises at least one cavity-filling mutation selected from the group consisting of S470A, S470L, S477A, ​​A463L, I474F, and I474Y.

[0297] 2-2(c) Proline substitution mutation In yet other embodiments, the present disclosure provides PIV3 F protein variants comprising one or more proline substitution mutations. The term "proline substitution mutation" refers to the substitution of an amino acid with a proline that prevents structural refolding during the transition from the pre-fusion to the post-fusion conformation.

[0298] In some specific embodiments, the PIV3 F protein variants include the proline mutations S164P, G219P, or S164P and G219P. In some specific embodiments, the present disclosure provides PIV3 F variants including one or more proline substitution mutations provided in Tables 43 and 44. The PIV3 F variants provided in Tables 43 and 44 are based on the native F0 sequence of PIV3 of SEQ ID NO: 305. The same introduced mutations in the variants can be performed on the native F0 polypeptide sequence of any other PIV3 subtype or strain to arrive at different PIV3 F variants, such as the native F0 polypeptide sequence set forth in any of SEQ ID NOs: 300-304 or from any other PIV3 strain. PIV3 F variants based on the native F0 polypeptide sequence of any other PIV3 subtype or strain and including one or more proline substitution mutations are also within the scope of the present invention. In some specific embodiments, the PIV3 F protein variants include the mutation A128P.

[0299] 2-2(d) Glycine replacement mutation In still other embodiments, the disclosure provides PIV3 F protein variants comprising one or more glycine replacement mutations. The term "glycine replacement mutation" refers to the replacement of glycine with another amino acid in the center of the α-helix, preferably an amino acid without a Cβ substitution, such as Ala, Leu, or Met, to improve protein stability. In some specific embodiments, the disclosure provides PIV3 F variants comprising one or more glycine replacement mutations, wherein the variants comprise the glycine replacement mutations in the variants provided in Table 44. The PIV3 F variants provided in Table 44 are based on the native F0 sequence of PIV3 of SEQ ID NO: 305. The same introduced mutations in each of the variants can be performed on the native F0 polypeptide sequence of any other PIV3 subtype or strain to arrive at different PIV3 F variants, such as the native F0 polypeptide sequence set forth in any of SEQ ID NOs: 300-304 or from any other PIV3 strain. PIV3 F variants based on the native F0 polypeptide sequence of any other PIV3 subtype or strain and comprising one or more glycine replacement mutations are also within the scope of the invention. In certain embodiments, the PIV3 F protein mutant comprises the mutations G196A, G230A, or G196A and G230A.

[0300] 2-2(e) Electrostatic Mutation In yet other embodiments, the disclosure provides PIV3 F protein variants that include one or more electrostatic mutations.

[0301] The term "electrostatic mutation" refers to an amino acid mutation introduced into a wild-type PIV3 F protein that reduces ionic repulsion or increases ionic attraction between residues in the protein that are close to each other in the folded structure.

[0302] In some specific embodiments, the present disclosure provides PIV3 F variants comprising one or more electrostatic mutations, where the variants comprise the electrostatic mutations in the variants provided in Tables 44 and 45. The PIV3 F variants provided in Tables 44 and 45 are based on the native F0 sequence of PIV3 of SEQ ID NO: 305. The same introduced mutations in each of the variants can be performed on the native F0 polypeptide sequence of any other PIV3 subtype or strain to arrive at a different PIV3 F variant, such as the native F0 polypeptide sequence set forth in any of SEQ ID NOs: 300-304 or from any other PIV3 strain. PIV3 F variants based on the native F0 polypeptide sequence of any other PIV3 subtype or strain and comprising one or more electrostatic mutations are also within the scope of the present invention. In some specific embodiments, the PIV3 F protein variant comprises the mutations E182L, D455S, or E182L and D455S.

[0303] 2-2(f) Cleavage site mutation The "cleavage site mutation" was introduced to prevent cleavage of the PIV3 F protein variant between amino acids 109 and 110. However, when recombinantly expressed in CHO cells, the PIV3 F protein variant disclosed herein appeared to be inefficiently cleaved between amino acids 109 and 110, even in the absence of any cleavage site mutation. As a result, the F1 and F2 polypeptides form a single polypeptide rather than two separate polypeptides linked by a disulfide bond. Unexpectedly, the "cleavage site mutation," while not preventing cleavage that would not occur in the expression system used, provided some unexpected benefits in terms of the thermostability of the produced polypeptide.

[0304] In some specific embodiments, the present disclosure provides PIV3 F mutants comprising one or more cleavage site mutations, wherein the mutants comprise cleavage site mutations in the mutants provided in Table 46.

[0305] The PIV3 F variants provided in Table 46 are based on the native F0 sequence of PIV3 of SEQ ID NO: 305. The same introduced mutations in each of the variants can be made to the native F0 polypeptide sequence of any other PIV3 subtype or strain to arrive at a different PIV3 F variant, such as the native F0 polypeptide sequence set forth in any of SEQ ID NOs: 300-304 or from any other PIV3 strain. PIV3 F variants based on the native F0 polypeptide sequence of any other PIV3 subtype or strain and containing any one or more cleavage site mutations are also within the scope of the invention.

[0306] In some embodiments, the cleavage site mutations comprise the following substitutions: R106G, T107S, E108A, and R109S.

[0307] In some embodiments, the cleavage site mutation comprises the following substitutions F110G and F111S.

[0308] PIV3 F protein variants containing any additional mutations are also encompassed by the present invention, so long as the immunogenic properties of the variant are not substantially adversely affected by the additional mutations.

[0309] In certain other specific embodiments, the invention provides a PIV3 F mutant, wherein the mutant comprises alanines at positions 230, 470, and 477 (230A, 470A, and 477A), (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 329, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 328; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 329, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 328. The present invention provides a PIV3 F variant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0310] In certain other specific embodiments, the invention provides a PIV3 F mutant, wherein the mutant comprises cysteines at positions 160 (160C) and 170 (170C), a leucine at position 463 (463L), and an alanine at position 230 (230A), (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 353, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 352; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 353, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 352. The present invention provides a PIV3 F variant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0311] In certain other specific embodiments, the invention provides a PIV3 F mutant, wherein the mutant comprises cysteines at positions 160 (160C) and 170 (170C) and alanines at positions 470 (470A) and 477 (477A), (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 339, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 338; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 339, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 338. The present invention provides a PIV3 F variant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0312] In certain other specific embodiments, the invention provides a PIV3 F mutant, wherein the mutant comprises cysteines at positions 160 (160C) and 170 (170C) and alanines at positions 230 (230A), 470 (470A), and 477 (477A), (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 351, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 350; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 351, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 350. The present invention provides a PIV3 F variant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0313] In certain other specific embodiments, the invention provides a PIV3 F mutant, wherein the mutant comprises cysteines at positions 160 (160C) and 170 (170C), a leucine at position 463 (463L), and alanines at positions 230 (230A), 470 (470A), and 477 (477A), (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 355, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 354; (2) an F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 355, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 354. The present invention provides a PIV3 F variant comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of:

[0314] The PIV3 F protein variants provided by the present disclosure can be prepared by routine methods known in the art, such as by expression in a recombinant host system using an appropriate vector. Suitable recombinant host cells include, for example, insect cells, mammalian cells, avian cells, bacteria, and yeast cells. Examples of suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (clonal isolates derived from the parent Trichoplusia ni BTI-TN-5B1-4 cell line (Invitrogen)). Examples of suitable mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi293 cells, typically transformed with sheared adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Suitable avian cells include, for example, chicken embryonic stem cells (e.g., EBx.RTM. cells), chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts (e.g., ELL-O), and duck cells. Suitable insect cell expression systems, such as baculovirus vector systems, are known to those skilled in the art and are described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin, No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego, Calif. Avian cell expression systems are also known to those skilled in the art and are described, for example, in U.S. Patent Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668. Similarly, bacterial and mammalian cell expression systems are known in the art and are described, for example, in Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths, London.

[0315] Many suitable vectors for expressing recombinant proteins in insect or mammalian cells are well known in the art and have been used conventionally.Suitable vectors may contain several components, including, but not limited to, one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as transcriptional control elements (e.g., promoters, enhancers, terminators), and / or one or more translation signals; and a signal or leader sequence for targeting the secretory pathway in a selected host cell (e.g., of mammalian origin, or from a heterologous mammalian or non-mammalian species).For example, for expression in insect cells, a suitable baculovirus expression vector, such as pFastBac (Invitrogen), is used to produce recombinant baculovirus particles.The baculovirus particles are amplified and used to infect insect cells to express recombinant proteins.For expression in mammalian cells, a vector that dr...

Claims

1. A mutant of a wild-type PIV1 F protein, the mutant comprising an F1 polypeptide and an F2 polypeptide, the mutant comprising at least one amino acid mutation compared to the amino acid sequence of the wild-type PIV1 F protein, the amino acid mutation being (1) at least one engineered disulfide bond mutation; (2) at least one cavity-filling mutation; (3) at least one proline substitution mutation; (4) at least one glycine replacement mutation; and (5) Cleavage site mutation A mutant of the wild-type PIV1 F protein selected from the group consisting of:

2. The mutant of claim 1 , comprising an engineered disulfide mutation.

3. The mutant of claim 1 or 2, wherein the engineered disulfide mutation is Q92C-G134C.

4. A mutant according to any one of claims 1 to 3, comprising a cavity-filling mutation.

5. 5. The mutant of any one of claims 1 to 4, wherein the cavity-filling mutation is selected from T198A, Q92A, Q92L, A466L, A466V, A466I, S473V, S473L, S473I, S473A, A480L, and A480V.

6. The mutant of claim 5 , wherein the cavity-filling mutation is A466L.

7. The mutant of claim 5 , wherein the cavity-filling mutation is S473L.

8. The mutant of claim 5 , wherein the cavity-filling mutation is A480L.

9. 5. The variant of any one of claims 1 to 4, comprising two or three cavity-filling mutations selected from T198A, Q92A, Q92L, A466L, A466V, A466I, S473V, S473L, S473I, S473A, A480L, and A480V.

10. The mutant of claim 9, wherein the cavity-filling mutations are A466L and S473L.

11. 11. The mutant of claim 9 or 10, further comprising a cavity-filling mutation A480L or A480V.

12. 12. The variant of any one of claims 1 to 11, comprising a proline substitution mutation.

13. The mutant of claim 12, wherein the proline substitution mutation is A128P.

14. 14. The mutant of any one of claims 1 to 13, comprising a glycine replacement mutation.

15. The mutant of claim 14, wherein the glycine replacement mutation is G134A or G134L.

16. 16. The mutant of claim 15, wherein the glycine replacement mutation is G134A.

17. 17. A mutant according to any one of claims 1 to 16, comprising a cleavage site mutation.

18. 18. The mutant of claim 17, wherein the cleavage site mutations are F113G and F114S.

19. (1) Q92C-G134C; (2) A466L; (3) A466V; (4) S473V; (5) S473L; (6) A480L; (7) A466L and S473A; (8) A466L and S473L; (9) T198A; (10) G134A; (11) A128P; (12) F113G, F114S, Q92C-G134C, A466L, S473L and A480L; (13) Q92C-G134C, A466L, S473L and A480L; (14) Q92C-G134C, A466L and S473L; (15) F113G, F114S, Q92C-G134C, A466V, S473V and A480V; (16) Q92C-G134C, A466V, S473V and A480V; (17) Q92C-G134C, A466V and S473V; (18) F113G, F114S, A466L, S473L, A480L and G134A; (19) A466L, S473L, A480L and G134A; (20) A466L, S473L and G134A; (21) F113G, F114S, A466L, S473L, A480L, Q92A, and G134A; (22) F113G, F114S, A466L, S473L and G134A; (23) A466L, S473L, A480L, Q92A, G134A; (24) A466L, S473L, Q92A, G134A; (25) F113G, F114S, Q92L, G134A; (26) A466L, S473L, A480L, Q92L, and G134A; (27) A466L, S473L, Q92L, and G134A; (28) F113G, F114S, A466L, S473L, A480L, Q92A, and G134L; (29) A466L, S473L, A480L, Q92A, and G134L; (30) F113G, F114S, Q92C-G134C, A466I, S473I, and A480L; (31) F113G, F114S, Q92C-G134C, A466I, and S473I; and (32) A466I, S473I, A480L, Q92L, and G134A 2. The mutant of claim 1, comprising a mutation selected from the group consisting of:

20. 2. The mutant of claim 1, comprising the mutations A466L, S473L, A480L, and G134A.

21. 2. The mutant of claim 1, comprising the mutations F113G, F114S, A466L, S473L, and G134A.

22. 2. The mutant of claim 1, comprising the mutations F113G, F114S, A466L, S473L, A480L, and G134A.

23. 2. The mutant of claim 1, comprising the mutations F113G, F114S, Q92C, G134C, A466L, S473L, and A480L.

24. comprising leucine at positions 466, 473, and 480 (466L, 473L, and 480L) and alanine at position 134 (134A); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 255, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 254; (2) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 255, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

254. comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: The mutant of claim 1.

25. comprising glycine (G) at position 113 (113G), serine at position 114 (114S), leucine at positions 466 and 473 (466L and 473L), and alanine at position 134 (134A); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 291, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 290; (2) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 291, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

290. comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: The mutant of claim 1.

26. comprising glycine (G) at position 113 (113G), serine at position 114 (114S), leucine at positions 466, 473, and 480 (466L, 473L, and 480L), and alanine at position 134 (134A); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 277, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 276; (2) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 277, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

276. comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: The mutant of claim 1.

27. comprising glycine (G) at position 113 (113G), serine at position 114 (114S), leucine at positions 466, 473, and 480 (466L, 473L, and 480L), and cysteine ​​at positions 92 and 134 (92C and 134C); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 273, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 272; (2) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 273, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

272. comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: The mutant of claim 1.

28. 28. The variant of any one of claims 1 to 27, wherein the F1 polypeptide lacks the cytoplasmic and transmembrane domains.

29. 28. The variant of any one of claims 1 to 27, wherein the F1 polypeptide lacks the cytoplasmic domain and a portion of the transmembrane domain.

30. 28. The variant of any one of claims 1 to 27, wherein the F1 polypeptide comprises or consists of amino acid residues 113 to 477, or comprises or consists of amino acid residues 113 to 480.

31. 28. The variant of any one of claims 1 to 27, wherein the F1 polypeptide comprises an ectodomain, a transmembrane domain, and a cytoplasmic domain.

32. 32. A variant according to any one of claims 1 to 31, which is linked to a trimerization domain.

33. 33. The variant of claim 32, wherein the trimerization domain is phage T4 fibritinfoldon.

34. 34. The mutant of claim 33, wherein the trimerization domain is the phage T4 fibritinfoldon of SEQ ID NO:

7.

35. 35. The variant of any one of claims 32 to 34, wherein the trimerization domain is linked to the C-terminus of the F1 polypeptide.

36. 36. The variant of any one of claims 32 to 35, wherein the trimerization domain is linked to the C-terminus of the F1 polypeptide via a linker.

37. 37. The variant of claim 36, wherein the linker is GGGS.

38. 38. A mutant according to any one of claims 1 to 37 in the form of a trimer.

39. 39. The mutant of any one of claims 1 to 38, in a pre-fusion conformation.

40. 40. The variant of any one of claims 1 to 39, wherein the variant is in a pre-fusion conformation and specifically binds to an antibody (such as a PIV1-8 mAb) that is specific for the PIV1 F ectodomain in the pre-fusion conformation but not the PIV1 F ectodomain in the post-fusion conformation.

41. The mutant of any one of claims 1 to 40, wherein the wild-type PIV1 is SEQ ID NO:

206.

42. 42. The variant of any one of claims 1 to 41, wherein the amino acid positions correspond to the reference amino acid sequence of SEQ ID NO:

206.

43. A nucleic acid comprising at least one coding sequence encoding at least one mutant of a wild-type PIV1 F protein described in any one of claims 1 to 42, or an immunogenic fragment or immunogenic variant thereof, the nucleic acid comprising at least one heterologous untranslated region (UTR).

44. 44. The nucleic acid of claim 43, wherein the at least one heterologous untranslated region is selected from at least one heterologous 5'-UTR and / or at least one heterologous 3'-UTR.

45. The at least one heterologous 3'-UTR is CΨCGAGCΨGGΨ ACΨGCAΨGCA CGCAAΨGCΨA GCΨGCCCΨΨ ΨCCCGΨCCΨG GGΨACCCCGA GΨCΨCCCCCG ACCΨCGGGΨC CCAGGΨAΨGC ΨCCCACCΨCC ACCΨGCCCCA CΨCACCACCΨ CΨGCΨAGΨΨC CAGACCΨC CCAAGCACGC AGCAAΨGCAG CΨCAAAACGC ΨΨAGCCΨAGC CACACCCCCA 45. The nucleic acid of claim 43 or 44, comprising or consisting of a nucleic acid sequence having at least, and at most exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to: CGGGAAACAG CAGGAAACAAA CCCCCCCCAGCAA AAACGAAAC AAAC AAAC AAAC CCCCCCAC AAAC GGAGCCAC AAAC GGAGCCAC AAAC

46. 46. ​​The nucleic acid of any one of claims 43 to 45, wherein at least one heterologous 5'-UTR comprises or consists of a nucleic acid sequence having at least, and at most exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to GAAAC ΨAGΨAΨΨCΨΨ CΨGGΨCCCCA CAGACΨCAGA GAGAACCCGC CACC, or a percentage identity between any two thereof.

47. 47. The nucleic acid according to any one of claims 43 to 46, comprising at least one poly(A) sequence, preferably comprising 30 to 200 adenosine nucleotides, and / or at least one poly(C) sequence, preferably comprising 10 to 40 cytosine nucleotides.

48. 48. A nucleic acid according to any one of claims 43 to 47, which is DNA or RNA.

49. 49. The nucleic acid of claim 48, which is a coding RNA.

50. 50. The nucleic acid of claim 49, wherein the coding RNA is mRNA, self-replicating RNA, circular RNA, or replicon RNA.

51. 51. The nucleic acid of claim 50, wherein the nucleic acid, preferably the coding RNA, is mRNA.

52. 52. The nucleic acid of claim 51, wherein the mRNA is not a replicon RNA or a self-replicating RNA.

53. 53. The nucleic acid of any one of claims 50 to 52, wherein the mRNA comprises at least one poly(A) sequence comprising 30 to 200 adenosine nucleotides, the 3' terminal nucleotide being adenosine.

54. 54. The nucleic acid of any one of claims 48 to 53, wherein the RNA, preferably the coding RNA, comprises a 5'-cap structure, preferably m7G, cap 0, cap 1, cap 2, modified cap 0, or modified cap 1 structure, preferably a 5'-cap 1 structure.

55. 55. The nucleic acid of any one of claims 48 to 54, wherein the RNA is codon-optimized.

56. 56. The nucleic acid of any one of claims 48 to 55, wherein the RNA comprises chemically modified nucleotides.

57. 57. The nucleic acid of any one of claims 49 to 56, wherein the RNA comprises 1-methylpseudouridine substitutions.

58. 58. The nucleic acid of claim 57, wherein all uridines in the RNA are replaced by 1-methylpseudouridine.

59. 59. The nucleic acid of any one of claims 49 to 58, wherein the RNA is purified RNA, preferably RNA purified by RP-HPLC and / or TFF.

60. 60. The nucleic acid of any one of claims 48 to 59, wherein the RNA comprises the nucleic acid sequence of any of SEQ ID NO:403, SEQ ID NO:405, SEQ ID NO:407, and SEQ ID NO:

409.

61. 61. A composition comprising at least one nucleic acid according to any one of claims 43 to 60.

62. 61. A composition comprising at least one nucleic acid according to any one of claims 43 to 60, comprising at least one pharmaceutically acceptable carrier.

63. 61. A composition comprising at least one nucleic acid of any one of claims 43 to 60, which is a multivalent composition comprising a plurality or more than at least one of the nucleic acids of any one of claims 43 to 60.

64. 61. A composition comprising at least one nucleic acid of any one of claims 43 to 60, wherein the composition comprises RNA with 70% or greater RNA integrity.

65. 61. A composition comprising at least one nucleic acid described in any one of claims 43 to 60, wherein the composition comprises RNA having a degree of capping of 70% or more, preferably at least 70%, 80%, or 90% of mRNA species contain Cap 1 structures.

66. 61. A composition comprising at least one nucleic acid according to any one of claims 43 to 60, wherein the at least one nucleic acid is complexed or associated, or at least partially complexed or partially associated with one or more cationic or polycationic compounds, preferably cationic or polycationic polymers, cationic or polycationic polysaccharides, cationic or polycationic lipids, cationic or polycationic proteins, cationic or polycationic peptides, or any combination thereof.

67. 61. A composition comprising at least one nucleic acid according to any one of claims 43 to 60, wherein the at least one nucleic acid is complexed or associated with one or more lipids or lipid-based carriers, thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes, preferably encapsulating the at least one nucleic acid.

68. 61. A composition comprising at least one nucleic acid according to any one of claims 43 to 60, wherein the at least one nucleic acid is complexed with one or more lipids, thereby forming a lipid nanoparticle.

69. LNP has the formula III-3: 【Chemistry 1】 69. The composition of any one of claims 67 or 68, comprising a cationic lipid according to

70. LNP has the formula (IVa): 【Chemistry 2】 70. The composition of any one of claims 67 to 69, comprising a PEG lipid of

71. 71. The composition of claim 70, wherein n has an average value ranging from 30 to 60, preferably n has an average value of about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and most preferably n has an average value of 49 or 45.

72. LNP has the formula (IVa): 【Transformation 3】 72. The composition of any one of claims 67 to 71, comprising a PEG-lipid of formula: wherein n is an integer selected such that the average molecular weight of the PEG-lipid is about 2500 g / mol.

73. 73. The composition of any one of claims 67 to 72, wherein the LNP comprises one or more neutral lipids and / or one or more steroids or steroid analogs.

74. 74. The composition of claim 73, wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and preferably the molar ratio of cationic lipid to DSPC is in the range of about 2:1 to about 8:

1.

75. 75. The composition of claim 73 or 74, wherein the steroid is cholesterol, and preferably the molar ratio of cationic lipid to cholesterol is in the range of about 2:1 to about 1:

1.

76. 76. The composition of any one of claims 67 to 75, wherein the LNPs comprise: (i) at least one cationic lipid, preferably a lipid of formula (III), more preferably lipid III-3; (ii) at least one neutral lipid, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) at least one steroid or steroid analog, preferably cholesterol; and (iv) at least one polymer-conjugated lipid, preferably a PEG lipid derived from formula (IVa, having n=49), wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG lipid.

77. 77. The composition of any one of claims 67 to 76, wherein the LNPs comprise: (i) at least one cationic lipid, preferably a lipid of formula (III), more preferably lipid III-3; (ii) at least one neutral lipid, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) at least one steroid or steroid analog, preferably cholesterol; and (iv) at least one polymer-conjugated lipid, preferably a PEG lipid derived from formula (IVa, having n=45), wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG lipid.

78. 78. The composition of claim 76 or 77, wherein (i) to (iv) are in a molar ratio of about 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7, or more preferably 47.4:10:40.9:1.

7.

79. 79. The composition of any one of claims 67 to 78, wherein the nucleic acid is RNA and the composition comprises less than about 20% free (uncomplexed or unencapsulated) RNA, preferably less than about 15% free RNA, more preferably less than about 10% free RNA.

80. 80. The composition of any one of claims 67 to 79, wherein the wt / wt ratio of lipid to nucleic acid is from about 10:1 to about 60:1, preferably from about 20:1 to about 30:1, for example about 25:

1.

81. 81. The composition of any one of claims 67 to 80, wherein the n / p ratio of the LNPs encapsulating the nucleic acid is in the range of about 1 to about 10, preferably in the range of about 5 to about 7, and more preferably about 6.

82. 82. The composition of any one of claims 67 to 81, having a polydispersity index (PDI) value of less than about 0.4, preferably less than about 0.3, more preferably less than about 0.2, and most preferably less than about 0.

1.

83. 83. The composition of any one of claims 67 to 82, wherein the LNPs have a Z-average size in the range of about 60 nm to about 120 nm, preferably less than about 120 nm, more preferably less than about 100 nm, and most preferably less than about 80 nm.

84. 84. The composition of any one of claims 67 to 83, wherein the LNPs comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of LNPs having a particle size greater than about 500 nm.

85. 85. The composition of any one of claims 67 to 84, wherein the LNPs comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of LNPs having a particle size smaller than about 20 nm.

86. 86. The composition of any one of claims 67 to 85, wherein the LNP comprises: (i) at least one cationic lipid; (ii) at least one neutral lipid; (iii) at least one steroid or steroid analog; and (iv) at least one PEG lipid, wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG lipid.

87. 87. The composition of any one of claims 67 to 86, wherein the LNP comprises: (i) at least one cationic lipid according to Formula III-3; (ii) DSPC; (iii) cholesterol; and (iv) a PEG lipid according to Formula IVa, wherein (i)-(iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG lipid.

88. 88. The composition of any one of claims 67 to 87, which is a freeze-dried composition.

89. 89. An immunogenic composition comprising a variant according to any one of claims 1 to 42, or a nucleic acid according to any one of claims 43 to 60, or a composition according to any one of claims 61 to 88.

90. 90. The immunogenic composition of claim 89, further comprising an hMPV A antigen selected from the group consisting of a variant of a wild-type hMPV A F protein and a nucleic acid encoding a variant of a wild-type hMPV A F protein.

91. 91. The immunogenic composition of claim 90, wherein the hMPV A antigen is a variant of the wild-type hMPV A F protein.

92. 92. The immunogenic composition of claim 91, wherein the hMPV A antigen is a variant of a wild-type hMPV A F protein from this disclosure, preferably from any of E1 to E72 of Section B of this disclosure.

93. 91. The immunogenic composition of claim 90, wherein the hMPV A antigen comprises a nucleic acid encoding a variant of a wild-type hMPV A F protein.

94. 94. The immunogenic composition of claim 93, wherein the hMPV A antigen comprises a nucleic acid encoding a variant of a wild-type hMPV A F protein from the present disclosure, preferably from any of E73 to E89 of section B of the present disclosure.

95. 95. The immunogenic composition of any one of claims 89 to 94, further comprising an hMPV B antigen selected from the group consisting of a mutant of a wild-type hMPV B F protein and a nucleic acid encoding a mutant of a wild-type hMPV B F protein.

96. 96. The immunogenic composition of claim 95, wherein the hMPV B antigen is a variant of the wild-type hMPV B F protein.

97. 97. The immunogenic composition of claim 96, wherein the hMPV B antigen is a variant of a wild-type hMPV B F protein from this disclosure, preferably from any of E1 to E72 of section B of this disclosure.

98. 96. The immunogenic composition of claim 95, wherein the hMPV B antigen comprises a nucleic acid encoding a variant of the wild-type hMPV B F protein.

99. 98. The immunogenic composition of claim 97, wherein the hMPV B antigen comprises a nucleic acid encoding a variant of a wild-type hMPV B F protein from the present disclosure, preferably a nucleic acid from any of E73 to E89 of section B of the present disclosure.

100. 100. The immunogenic composition of any one of claims 89-99, further comprising a PIV3 antigen selected from the group consisting of a variant of a wild-type PIV3 F protein and a nucleic acid encoding a variant of a wild-type PIV3 F protein.

101. The immunogenic composition of claim 100, wherein the PIV3 antigen is a mutant of the wild-type PIV3 F protein.

102. 102. The immunogenic composition of claim 101, wherein the PIV3 antigen is a variant of a wild-type PIV3 F protein from the present disclosure, preferably from any of E1 to E52 of section D of the present disclosure.

103. The immunogenic composition of claim 101, wherein the PIV3 antigen is a mutant of the wild-type PIV3 F protein disclosed in WO2018081289 or WO2022207839.

104. The immunogenic composition of claim 100, wherein the PIV3 antigen comprises a nucleic acid encoding a variant of the wild-type PIV3 F protein.

105. 105. The immunogenic composition of claim 104, wherein the PIV3 antigen comprises a nucleic acid encoding a variant of a wild-type PIV3 F protein from the present disclosure, preferably a nucleic acid from any of E53 to E69 of section D of the present disclosure.

106. The immunogenic composition of claim 104, wherein the PIV3 antigen comprises a nucleic acid encoding a variant of the wild-type PIV3 F protein disclosed in WO2018081289 or WO2022207839.

107. 107. The immunogenic composition of any one of claims 89 to 106, further comprising an RSV antigen selected from the group consisting of a subtype A wild-type RSV F protein variant and a nucleic acid encoding a subtype A wild-type RSV F protein variant.

108. 108. The immunogenic composition of claim 107, wherein the RSV antigen is a variant of a wild-type RSV F protein of subtype A.

109. 108. The immunogenic composition of claim 107, wherein the RSV antigen is a nucleic acid encoding a variant of a subtype A wild-type RSV F protein.

110. The immunogenic composition of claim 108, wherein the variant of a wild-type RSV F protein of subtype A is disclosed in one of WO2009 / 079796, WO2010 / 149745, WO2011 / 008974, WO2014 / 160463, WO2014 / 174018, WO2014 / 202570, WO2015 / 013551, WO2015 / 177312, WO2017 / 005848, WO2017 / 174564, WO2017 / 005844, WO2017 / 109629, WO2022 / 002894, and WO2018 / 109220.

111. The immunogenic composition of any one of claims 89 to 110, further comprising an RSV antigen selected from the group consisting of a subtype B wild-type RSV F protein variant and a nucleic acid encoding a subtype B wild-type RSV F protein variant.

112. 112. The immunogenic composition of claim 111, wherein the RSV antigen is a variant of a wild-type RSV F protein of subtype B.

113. 112. The immunogenic composition of claim 111, wherein the RSV antigen is a nucleic acid encoding a variant of a subtype B wild-type RSV F protein.

114. The immunogenic composition of claim 111, wherein the variant of a wild-type RSV F protein of subtype B is disclosed in one of WO2009 / 079796, WO2010 / 149745, WO2011 / 008974, WO2014 / 160463, WO2014 / 174018, WO2014 / 202570, WO2015 / 013551, WO2015 / 177312, WO2017 / 005848, WO2017 / 174564, WO2017 / 005844, WO2017 / 109629, WO2022 / 002894, and WO2018 / 109220.

115. A mutant of the wild-type hMPV F protein as defined in any of embodiments E1 to E72 disclosed in Section B above.

116. A nucleic acid comprising at least one coding sequence encoding at least one variant of a wild-type hMPV F protein, wherein the nucleic acid is as defined in any of embodiments E73 to E89 disclosed in Section B above.

117. A composition as defined in any of embodiments E90 to E117 disclosed in Section B above.

118. An immunogenic composition as defined in any of embodiments E118 to E139 disclosed in section B above.

119. A mutant of the wild-type PIV3 F protein as defined in any of embodiments E1 to E52 disclosed in section D above.

120. 120. The mutant of claim 119, comprising the mutations E209C and L234C.

121. 121. The mutant of claim 120, comprising the mutations E209C, L234C, S160C, and V170C.

122. (1) G230A, S470A and S477A; (2) S160C, V170C, G230A and A463L; (3) S160C, V170C, S470A and S477A; (4) S160C, V170C, G230A, S470A and S477A; (5) S160C, V170C, G230A, A463L, S470A and S477A (6) S160C, V170C, E209C, L234C, A463L and S470L; (7) S160C, V170C, E209C, L234C, A463L and I474F; (8) S160C, V170C, E209C, L234C, A463L, S470L, F110G, F111S; (9) S160C, V170C, A463L and S470L, and (10) E209C, L234C 120. The variant of claim 119, comprising a mutation selected from the group consisting of:

123. (a) comprising cysteines at positions 160 (160C), 170 (170C), 209 (209C), and 234 (234C) and leucines at positions 463 (463L) and 470 (470L); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 438, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 437; (2) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 438, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

437. or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (b) containing cysteine ​​at positions 160 (160C), 170 (170C), 209 (209C), and 234 (234C), leucine at position 463 (463L), and phenylalanine at position 474 (474F); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 440, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 439; (2) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 440, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

439. or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (c) cysteine ​​at positions 160 (160C), 170 (170C), 209 (209C), and 234 (234C), leucine at positions 463 (463L) and 470 (470L), glycine at position 110 (110G), and serine at position 111 (111S); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 482, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 481; (2) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 482, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

481. or comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: (d) cysteines at positions 160 (160C) and 170 (170C) and leucines at positions 463 (463L) and 470 (470L); (1) an F2 polypeptide comprising the amino acid sequence of SEQ ID NO: 494, and an F1 polypeptide comprising the amino acid sequence of SEQ ID NO: 493; (2) An F2 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 494, and an F1 polypeptide comprising an amino acid sequence at least 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

493. comprising an F1 polypeptide and an F2 polypeptide selected from the group consisting of: The mutant of claim 119.

124. A nucleic acid comprising at least one coding sequence encoding at least one variant of a wild-type PIV3 F protein, wherein the nucleic acid is as defined in any of embodiments E53 to E69 disclosed in section D above.

125. 125. The nucleic acid of claim 124, which is an mRNA and comprises any of the nucleic acid sequences of SEQ ID NO:411, SEQ ID NO:413, SEQ ID NO:415, SEQ ID NO:417, and SEQ ID NO:419, SEQ ID NO:504, SEQ ID NO:506, SEQ ID NO:508, and SEQ ID NO:510, SEQ ID NO:295, SEQ ID NO:297, and SEQ ID NO:

299.

126. A composition defined in any of embodiments E70 to E97 disclosed in Section D above.

127. The immunogenic composition defined in any of embodiments E99 to E122 disclosed in section D above.

128. An isolated antibody that binds to human metapneumovirus (hMPV), comprising a heavy chain variable region (hMPV-VH) and a light chain variable region (hMPV-VL) comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO:360, and the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO:

361.

129. 1. An isolated antibody that binds to hMPV, comprising a heavy chain variable region (hMPV-VH) and a light chain variable region (hMPV-VL), comprising a CDR-H1 sequence according to SEQ ID NO:523 or 524; a CDR-H2 sequence according to SEQ ID NO:525 or 526; a CDR-H3 sequence according to SEQ ID NO:527 or 528, and comprising a CDR-L1 sequence according to SEQ ID NO:529; a CDR-L2 sequence according to SEQ ID NO:530; and a CDR-L3 sequence according to SEQ ID NO:

531.

130. 130. The antibody of claim 128 or 129, comprising the hMPV-VH sequence of SEQ ID NO: 360 and the hMPV-VL sequence of SEQ ID NO:

361.

131. An isolated antibody that binds to parainfluenza virus type 1 (PIV1), comprising a heavy chain variable region (PIV1-VH) and a light chain variable region (PIV1-VL) comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO:362, and the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO:

363.

132. 1. An isolated antibody that binds to PIV1, comprising a heavy chain variable region (PIV1-VH) and a light chain variable region (PIV1-VL), the heavy chain variable region (PIV1-VH) comprising a CDR-H1 sequence according to SEQ ID NO: 534 or 535; a CDR-H2 sequence according to SEQ ID NO: 536 or 537; a CDR-H3 sequence according to SEQ ID NO: 538 or 539; and the light chain variable region (PIV1-VL) comprising a CDR-L1 sequence according to SEQ ID NO: 540; a CDR-L2 sequence according to SEQ ID NO: 541; and a CDR-L3 sequence according to SEQ ID NO:

542.

133. 134. The antibody of any of claims 132 or 133, comprising the PIV1-VH sequence of SEQ ID NO: 362 and the PIV1-VL sequence of SEQ ID NO: 363.

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