Mumps virus and measles virus immunogens and their uses

Abstract

Immunogen embodiments are provided that include a recombinant mumps (MuV) F ectodomain trimer stabilized in a prefusion conformation or a recombinant measles (MeV) F ectodomain trimer stabilized in a prefusion conformation. Also provided are immunogen embodiments that include a chimeric protein comprising the recombinant MuV or MeV F ectodomain trimer and one or more MuV HN or MeV H ectodomains. Nucleic acids encoding the immunogens and methods for their production are also disclosed. Methods for inducing an immune response in a subject by administering an immunogen of the present disclosure to the subject are also provided. In some embodiments, the immune response treats or inhibits MuV and / or MeV infection in the subject. TIFF2023506170000066.tif98128

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the earlier filing date of U.S. Provisional Application No. 62 / 946,902, filed December 11, 2019, which is incorporated herein by reference in its entirety.

[0002] Field This disclosure relates to polypeptides, polynucleotides, compositions, and methods of their use for eliciting and detecting immune responses to mumps virus (MuV) and measles virus (MeV). [Background technology]

[0003] background MeV and MuV are highly contagious paramyxoviruses that can be transmitted through respiratory droplets from an infected person or through direct contact with an infected person. The resulting disease can lead to severe complications or death in children. Existing vaccines for MeV and MuV are live-attenuated viruses that are administered as two subcutaneous doses, one at age 1 year and one as early as 1 month later. Two doses of the measles, mumps, and rubella combined vaccine are 97% effective against measles and 88% effective against mumps. A single dose of the measles, mumps, and rubella combined vaccine is 93% effective against measles and 78% effective against mumps.

[0004] Despite the effectiveness of currently licensed vaccines against MeV and MuV, the incidence of both has increased in recent years due to declining vaccination rates caused by vaccine hesitancy and the circulation of divergent strains that offer limited protection from the licensed MMR vaccine.

[0005] In the case of MuV, recent studies have shown that immunity wanes significantly after the second dose of MMR vaccine, typically administered during childhood. Additionally, currently circulating MuV strains have undergone genotypic shifts away from the Jeryl-Lynn strain in the standard mumps vaccine. In response to recent recurrent outbreaks of MuV disease in the United States and Europe, the Advisory Committee on Immunization Practices has recommended a third dose of MMR vaccination to boost protection. However, pre-existing immunity neutralizes the third dose of MMR vaccination, limiting its effectiveness. Summary of the Invention

[0006] overview Disclosed herein are recombinant MuV F ectodomain trimers and recombinant MeV F ectodomain trimers, which comprise promoters containing one or more modifications (such as amino acid substitutions) to stabilize the pre-fusion conformation. Further provided are embodiments of recombinant MuV F ectodomain trimers and recombinant MeV F ectodomain trimers linked to a MuV HN ectodomain or a MeV H ectodomain. Chimeric proteins are provided that include a combination of a MuV F ectodomain trimer and a MeV H ectodomain, or a combination of a MeV F ectodomain trimer and a MuV HN ectodomain. Embodiments of such proteins have been demonstrated to generate excellent immune responses in animal models and can be used, for example, to induce or boost immune responses against MeV and / or MuV in subjects.

[0007] In some embodiments, the immunogen comprises a recombinant MuV F ectodomain trimer stabilized in a pre-fusion conformation by one or more amino acid substitutions in the trimer promoter, including cysteine ​​substitutions that form a non-native disulfide bond to stabilize the MuV F ectodomain trimer in the pre-fusion conformation. In some embodiments, the recombinant MuV F ectodomain trimer is stabilized in the pre-fusion conformation by a non-native disulfide bond between cysteine ​​substitutions at MuV F positions 206 and 223 in the trimer promoter. In some embodiments, the trimer promoter further comprises a mutation to remove the F1 / F2 furin cleavage site in the MuV F ectodomain. In some embodiments, the recombinant MuV F ectodomain trimer promoter is fused at its C-terminus to a trimerization domain, such as the GCN4 trimerization domain. In a further embodiment, the recombinant MuV F ectodomain trimer protomer is linked to a heterologous protein, such as the MuV HN ectodomain or the MeV H ectodomain.

[0008] In some embodiments, the immunogen comprises a recombinant MeV F ectodomain trimer stabilized in a pre-fusion conformation by one or more amino acid substitutions in the trimer promoter, including cysteine ​​substitutions that form a non-native disulfide bond to stabilize the MeV F ectodomain trimer in the pre-fusion conformation. In some embodiments, the recombinant MeV F ectodomain trimer is stabilized in the pre-fusion conformation by a non-native disulfide bond between cysteine ​​substitutions at MeV F positions 165 and 171 in the trimer promoter. In some embodiments, the trimer promoter further comprises a mutation to remove the F1 / F2 furin cleavage site in the MeV F ectodomain. In some embodiments, the recombinant MeV F ectodomain trimer promoter is fused at its C-terminus to a trimerization domain, such as the GCN4 trimerization domain. In a further embodiment, the recombinant MeV F ectodomain trimer protomer is linked to a heterologous protein, such as the MuV HN ectodomain or the MeV H ectodomain.

[0009] In some embodiments, an immunogen is provided that comprises a trimer of fusion proteins, each fusion protein comprising, from N- to C-terminus, a trimerization domain and one or more MuV HN ectodomains or MeV H ectodomains.

[0010] In some embodiments, the immunogen comprises a dimer of MeV H ectodomain heads.

[0011] Nucleic acid molecules encoding the proteins of the present disclosure are also provided, as are vectors containing the nucleic acid molecules and methods for their production.

[0012] Immunogenic compositions comprising the immunogens of the present disclosure suitable for administration to a subject are also provided, which may be contained in unit dosage form. The immunogen may also contain a carrier to facilitate presentation to the immune system.

[0013] Methods for inducing an immune response in a subject are disclosed, as are methods for inhibiting or preventing MuV or MeV infection in a subject by administering to the subject an effective amount of an immunogen, nucleic acid molecule, or vector of the present disclosure.

[0014] [The present invention 1001] 1. An immunogen comprising a recombinant mumps virus (MuV) F ectodomain trimer stabilized in a pre-fusion conformation by one or more amino acid substitutions in the trimer's promoter, wherein the amino acid substitutions include cysteine ​​substitutions that form non-native disulfide bonds to stabilize the MuV F ectodomain trimer in the pre-fusion conformation. [The present invention 1002] 1001. The immunogen of the present invention, wherein the cysteine ​​substitutions are located in one or more of MuV F positions 86 and 215, positions 155 and 161, positions 165 and 231, positions 206 and 223, positions 209 and 214, and positions 221 and 255. [The present invention 1003] 1001 or 1002, wherein the recombinant MuV F ectodomain trimer is stabilized in the pre-fusion conformation by a non-native disulfide bond between the cysteine ​​substitutions at MuV F positions 206 and 223 in the protomer of the trimer. [The present invention 1004] The cysteine ​​substitution is MuV F positions 86 and 215 have N86C and A215C substitutions; MuV F positions 155 and 161 have K155C and L161C substitutions; MuV F positions 165 and 231 have V165C and M231C substitutions; MuV F positions 206 and 223 have V206C and A223C substitutions; MuV F positions 209 and 214 have P209C and P214C substitutions; MuV F positions 221 and 255 are I221C and M255C substitutions; Any of the immunogens of the present invention. [The present invention 1005] The recombinant MuV F ectodomain trimer protomer comprises or consists of MuV F positions 20 to 100. 2 and a MuV F protein comprising or consisting of positions 104-469, 104-476, or 104-483. 1 Any of the immunogens of the present invention, comprising an ectodomain. [The present invention 1006] Any of the aforementioned immunogens of the invention, wherein the trimer protomer further comprises a mutation to remove the F1 / F2 furin cleavage site of the MuV F ectodomain and optionally the first residue of the fusion peptide of the F1 ectodomain. [The present invention 1007] 1006. An immunogen of the present invention, wherein the mutation to remove the F1 / F2 furin cleavage site and the first residue of the fusion peptide comprises a deletion of MuV F positions 101-103 and, accordingly, positions 100 and 104 fused with a peptide linker. [The present invention 1008] The immunogen of the present invention 1007, wherein the peptide linker is a Gly-Gly-Gly linker. [The present invention 1009] Any of the immunogens of the invention described above, wherein the positioning of the amino acid substitutions is according to the reference MuV F protein sequence shown as SEQ ID NO:1. [The present invention 1010] The MuV F ectodomain trimer protomer is Residues 20 to 483 of any one of SEQ ID NOs: 3 to 8; Residues 20-476 of any one of SEQ ID NOs: 11-16, 26, or 51; or Residues 20 to 469 of any one of SEQ ID NOs: 19 to 24 comprising an amino acid sequence at least 90% identical to the protomer contains the one or more amino acid substitutions that stabilize the MuV F ectodomain trimer in the pre-fusion conformation. Any of the immunogens of the present invention. [The present invention 1011] The MuV F ectodomain trimer protomer is Residues 20 to 483 of any one of SEQ ID NOs: 3 to 8; Residues 20-476 of any one of SEQ ID NOs: 11-16, 26, or 51; or Residues 20 to 469 of any one of SEQ ID NOs: 19 to 24 1010. An immunogen of the present invention, comprising or consisting of the amino acid sequence shown below. [The present invention 1012] Any of the immunogens of the present invention, wherein the recombinant MuV F ectodomain trimer protomer is fused to a trimerization domain at its C-terminus. [The present invention 1013] 1012. The immunogen of the present invention, wherein the trimerization domain comprises a GCN4 trimerization domain, a T4 fibritin trimerization domain, or both. [The present invention 1014] the GCN4 trimerization domain TIFF0007801221000001.tif4128 and comprising the amino acid sequence shown as the T4 fibritin trimerization domain TIFF0007801221000002.tif4128 and comprising the amino acid sequence shown as A trimerization domain comprising both the GCN4 trimerization domain and the T4 fibritin trimerization domain TIFF0007801221000003.tif4156 1013. An immunogen of the present invention, comprising the amino acid sequence shown as: [The present invention 1015] a MuV F ectodomain trimer protomer fused to the trimerization domain, Residues 20 to 513 of any one of SEQ ID NOs: 3 to 8; Residues 20-506 of any one of SEQ ID NOs: 11-16, 26, or 51; or Residues 20 to 499 of any one of SEQ ID NOs: 19 to 24 comprising an amino acid sequence at least 90% identical to the protomer contains the one or more amino acid substitutions that stabilize the MuV F ectodomain trimer in the pre-fusion conformation. An immunogen according to any one of 1012 to 1014 of the present invention. [The present invention 1016] the MuV F ectodomain trimer protomer fused to the trimerization domain comprises: Residues 20 to 513 of any one of SEQ ID NOs: 3 to 8; Residues 20-506 of any one of SEQ ID NOs: 11-16, 26, or 51; or Residues 20 to 499 of any one of SEQ ID NOs: 19 to 24 An immunogen of the present invention, comprising or consisting of the amino acid sequence shown below. [The present invention 1017] Any of the aforementioned immunogens of the present invention, wherein the recombinant MuV F ectodomain trimer protomer is linked to a heterologous protein. [The present invention 1018] The immunogen of the present invention 1017, wherein the heterologous protein is the head of the ectodomain or the stalk and head of the ectodomain of MeV H protein or MuV HN protein. [The present invention 1019] An immunogen of the present invention 1018, wherein the head of the ectodomain or the stalk and head of the ectodomain of the MeV H protein or the MuV HN protein is fused to a trimerization domain at its C-terminus, and the trimerization domain is fused to a promoter of the recombinant MuV F ectodomain trimer at its C-terminus. [The present invention 1020] The immunogen of the present invention, wherein the MuV F ectodomain trimer promoter linked to the trimerization domain and the MeV H protein ectodomain or the MuV HN protein ectodomain comprises the amino acid sequence set forth as residues 20 to 966 of SEQ ID NO:27, residues 21 to 981 of SEQ ID NO:28, or residues 20 to 1006 of SEQ ID NO:29. [The present invention 1021] 1. An immunogen comprising a recombinant measles virus (MeV) F ectodomain trimer stabilized in a pre-fusion conformation by one or more amino acid substitutions in the protomers of the trimer, wherein the amino acid substitutions are: cysteine ​​substitutions at one or more of MeV F positions 48 and 284, positions 90 and 225, positions 141 and 270, positions 165 and 171, positions 173 and 245, positions 175 and 241, positions 212 and 236, positions 216 and 233, positions 219 and 224, positions 99 and 117, positions 100 and 117, positions 101 and 117, positions 102 and 117, and positions 103 and 117 that form non-native disulfide bonds; a phenylalanine substitution at MeV F position 175, and Proline substitution at MeV F position 194 An immunogen comprising one or more of: [The present invention 1022] 1021. The immunogen of the present invention, wherein the recombinant MeV F ectodomain trimer is stabilized in the pre-fusion conformation by a non-native disulfide bond between the cysteine ​​substitutions at MeV F positions 165 and 171 in the protomer of the trimer. [The present invention 1023] the cysteine ​​substitutions at MeV F positions 48 and 284 are R48C and A284C substitutions; the cysteine ​​substitutions at MeV F positions 90 and 225 are A90C and I225C substitutions; the cysteine ​​substitutions at MeV F positions 141 and 270 are M141C and T270C substitutions; the cysteine ​​substitutions at MeV F positions 165 and 171 are R165C and M171C substitutions; the cysteine ​​substitutions at MeV F positions 173 and 245 are L173C and V245C substitutions; the cysteine ​​substitutions at MeV F positions 175 and 241 are V175C and D241C substitutions; the cysteine ​​substitutions at MeV F positions 212 and 236 are E212C and Y236C substitutions; the cysteine ​​substitutions at MeV F positions 216 and 233 are L216C and A233C substitutions; the cysteine ​​substitutions at MeV F positions 219 and 224 are P219C and P224C substitutions; the cysteine ​​substitutions at MeV F positions 99 and 117 are R99C-V117C substitutions; the cysteine ​​substitutions at MeV F positions 100 and 117 are P100C-V117C substitutions; the cysteine ​​substitutions at MeV F positions 101 and 117 are V101C-V117C substitutions; the cysteine ​​substitutions at MeV F positions 102 and 117 are Q102C-V117C substitutions; the cysteine ​​substitutions at MeV F positions 103 and 117 are S103C-V117C substitutions; the phenylalanine substitution at MeV F position 175 is a V175F substitution; the proline substitution at MeV F position 194 is an S194P substitution; The immunogen of the present invention 1021 or 1022. [The present invention 1024] The recombinant MeV F ectodomain trimer protomer comprises or consists of MeV F positions 24 to 110. 2 protein and F comprising or consisting of MeV F positions 114-486 1 An immunogen according to any one of claims 1021 to 1023, comprising an ectodomain. [The present invention 1025] The immunogen of any of claims 1021 to 1024, wherein the trimer protomer further comprises a mutation to remove the F1 / F2 furin cleavage site of the MeV F ectodomain and optionally the first residue of the fusion peptide of the F1 ectodomain. [The present invention 1026] 1025. An immunogen of the present invention, wherein the mutation to remove the F1 / F2 furin cleavage site and the first residue of the fusion peptide comprises a deletion of MeV F positions 111-113 and positions 110 and 114 fused with a peptide linker. [The present invention 1027] The immunogen of the present invention 1026, wherein the peptide linker is a Gly-Gly-Gly linker. [The present invention 1028] The immunogen of any of claims 1021 to 1027, wherein the positioning of the amino acid substitutions is according to the reference MeV F protein shown as SEQ ID NO:36. [The present invention 1029] the MeV F ectodomain trimer protomer comprises an amino acid sequence at least 90% identical to residues 21-483 of any one of SEQ ID NOs: 37-43, 53-55, 62-68, or 72-80, or residues 21-490 of any one of SEQ ID NOs: 70-72; the protomer comprises the one or more amino acid substitutions that stabilize the MeV F ectodomain trimer in the pre-fusion conformation. An immunogen according to any one of claims 1021 to 1028 of the present invention. [The present invention 1030] 1029. The immunogen of the present invention, wherein the MeV F ectodomain trimer protomer comprises or consists of an amino acid sequence set forth as residues 21 to 483 of any one of SEQ ID NOs: 37 to 43, 53 to 55, 62 to 68, or 72 to 80, or residues 21 to 490 of any one of SEQ ID NOs: 70 to 72. [The present invention 1031] The immunogen of any of claims 1021 to 1030, wherein the protomer of the recombinant MeV F ectodomain trimer is fused to a trimerization domain at the C-terminus. [The present invention 1032] 1031. The immunogen of the present invention, wherein the trimerization domain comprises a GCN4 trimerization domain, a T4 fibritin trimerization domain, or both. [The present invention 1033] the GCN4 trimerization domain TIFF0007801221000004.tif4128 and comprising the amino acid sequence shown as the T4 fibritin trimerization domain TIFF0007801221000005.tif4128 and comprising the amino acid sequence shown as A trimerization domain comprising both the GCN4 trimerization domain and the T4 fibritin trimerization domain TIFF0007801221000006.tif4156 An immunogen of the present invention 1032, comprising the amino acid sequence shown as: [The present invention 1034] the MeV F ectodomain trimer protomer fused to the trimerization domain comprises an amino acid sequence at least 90% identical to residues 21-513 of any one of SEQ ID NOs: 37-43, 53-55, 62-68, or 72-80, or residues 21-520 of any one of SEQ ID NOs: 70-72; the protomer comprises the one or more amino acid substitutions that stabilize the MeV F ectodomain trimer in the pre-fusion conformation. An immunogen according to any one of claims 1031 to 1033 of the present invention. [This invention 1035] The immunogen of the present invention 1034, wherein the MeV F ectodomain trimer protomer fused to the trimerization domain comprises or consists of the amino acid sequence set forth as residues 21 to 513 of any one of SEQ ID NOs: 37 to 43, 53 to 55, 62 to 68, or 72 to 80, or residues 21 to 520 of any one of SEQ ID NOs: 70 to 72. [The present invention 1036] The immunogen of any of claims 1021 to 1035, wherein the recombinant MeV F ectodomain trimer protomer is linked to a heterologous protein. [This invention 1037] 1036. The immunogen of the present invention, wherein said heterologous protein is the head of the ectodomain or the stalk and head of the ectodomain of MeV H protein or MuV HN protein. [The present invention 1038] An immunogen of the present invention 1037, wherein the head of the ectodomain or the stalk and head of the ectodomain of the MeV H protein or the MuV HN protein is fused to a trimerization domain at its C-terminus, and the trimerization domain is fused to a promoter of a recombinant MuV F ectodomain trimer at its C-terminus. [This invention 1039] 1038. The immunogen of the present invention, wherein the trimerization domain and the MeV F ectodomain trimer promoter linked to the ectodomain of the MeV H protein or the MuV HN protein comprise the amino acid sequence set forth as residues 21 to 959 of SEQ ID NO:56, or residues 21 to 973 of SEQ ID NO:57, or residues 21 to 988 of SEQ ID NO:81. [The present invention 1040] Any of the immunogens of the present invention, wherein the recombinant MeV F ectodomain trimer or the protomer of the recombinant MuV F ectodomain trimer further comprises one or more additional amino acid substitutions. [This invention 1041] a trimer of fusion proteins, each fusion protein comprising or consisting of, from N- to C-terminus, a trimerization domain, an optional peptide linker, and one or more copies of the MuV HN ectodomain head or the MeV H ectodomain head; or a trimer of fusion proteins, each fusion protein comprising or consisting of, from N-terminus to C-terminus, one or more copies of a MuV HN ectodomain head or a MeV H ectodomain head, an optional peptide linker, a trimerization domain, an optional peptide linker, and one or more copies of a MuV HN ectodomain head or a MeV H ectodomain head; or A trimer of fusion proteins, each fusion protein comprising or consisting of, from N-terminus to C-terminus, one or more copies of a MuV HN ectodomain head or stalk and head, or a MeV H ectodomain head or stalk and head, an optional peptide linker, a trimerization domain, an optional peptide linker, and one or more copies of a MuV HN ectodomain head or stalk and head, or a MeV H ectodomain head or stalk and head. 1. An isolated immunogen comprising: [The present invention 1042] the head of the MuV HN ectodomain comprises or consists of the amino acid sequence set forth as residues 59-510 of SEQ ID NO:58, or a sequence at least 90% identical to residues 59-510 of SEQ ID NO:58; the stalk and head of the MuV HN ectodomain comprise residues 22-550 of SEQ ID NO:90, residues 22-543 of SEQ ID NO:91, residues 22-541 of SEQ ID NO:92, or residues 22-549 of SEQ ID NO:93, or a sequence at least 90% identical to residues 22-550 of SEQ ID NO:90, residues 22-543 of SEQ ID NO:91, residues 22-541 of SEQ ID NO:92, or residues 22-549 of SEQ ID NO:93; the head of the MeV H ectodomain comprises or consists of the amino acid sequence set forth as residues 59-496 of SEQ ID NO:59, or a sequence at least 90% identical to residues 59-496 of SEQ ID NO:59; or the stalk and head of the MeV H ectodomain comprise residues 22-580 of SEQ ID NO:86, residues 22-577 of SEQ ID NO:87, residues 22-579 of SEQ ID NO:88, or residues 22-572 of SEQ ID NO:89, or a sequence at least 90% identical to residues 22-580 of SEQ ID NO:86, residues 22-577 of SEQ ID NO:87, residues 22-579 of SEQ ID NO:88, or residues 22-572 of SEQ ID NO:89; The immunogen of the present invention 1041. [This invention 1043] the GCN4 trimerization domain TIFF0007801221000007.tif4128 and comprising the amino acid sequence shown as the T4 fibritin trimerization domain TIFF0007801221000008.tif4128 and comprising the amino acid sequence shown as A trimerization domain comprising both the GCN4 trimerization domain and the T4 fibritin trimerization domain TIFF0007801221000009.tif4156 The immunogen of the present invention 1041 or 1042, comprising the amino acid sequence shown below. [This invention 1044] 1043. The immunogen of any of claims 1041 to 1043, wherein the fusion protein in the trimer comprises or consists of residues 24 to 510 of SEQ ID NO:58, residues 24 to 496 of SEQ ID NO:59, residues 22 to 950 of SEQ ID NO:82, residues 25 to 985 of SEQ ID NO:83, residues 22 to 948 of SEQ ID NO:84, or residues 22 to 981 of SEQ ID NO:85, or a sequence at least 90% identical to any one of residues 24 to 510 of SEQ ID NO:58, residues 24 to 496 of SEQ ID NO:59, residues 22 to 950 of SEQ ID NO:82, residues 25 to 985 of SEQ ID NO:83, residues 22 to 948 of SEQ ID NO:84, or residues 22 to 981 of SEQ ID NO:85. [This invention 1045] MeV H ectodomain head dimer, MeV H ectodomain stalk and head dimers, a dimer of the MuV HN ectodomain head, or Stalk and head dimers of the MuV HN ectodomain 1. An isolated immunogen comprising: [The present invention 1046] the head of the MeV H ectodomain comprises or consists of the amino acid sequence set forth as residues 22-459 of SEQ ID NO:60, or a sequence at least 90% identical to residues 22-459 of SEQ ID NO:60; the stalk and head of the MeV H ectodomain comprise residues 22-580 of SEQ ID NO:86, residues 22-577 of SEQ ID NO:87, residues 22-579 of SEQ ID NO:88, or residues 22-572 of SEQ ID NO:89, or a sequence at least 90% identical to residues 22-580 of SEQ ID NO:86, residues 22-577 of SEQ ID NO:87, residues 22-579 of SEQ ID NO:88, or residues 22-572 of SEQ ID NO:89; the head of the MuV H ectodomain comprises or consists of the amino acid sequence set forth as SEQ ID NO:30, or a sequence at least 90% identical to SEQ ID NO:30; or the stalk and head of the MuV HN ectodomain comprise or consist of residues 22-550 of SEQ ID NO:90, residues 22-543 of SEQ ID NO:91, residues 22-541 of SEQ ID NO:92, or residues 22-549 of SEQ ID NO:93, or a sequence at least 90% identical to residues 22-550 of SEQ ID NO:90, residues 22-543 of SEQ ID NO:91, residues 22-541 of SEQ ID NO:92, or residues 22-549 of SEQ ID NO:93; The immunogen of the present invention 1045. [This invention 1047] Any of the preceding immunogens of the present invention conjugated to a heterologous carrier. [This invention 1048] Any of the immunogens of the present invention that are soluble. [This invention 1049] Any of the immunogens of the present invention, wherein the recombinant MeV F ectodomain trimer protomer, the recombinant MuV F ectodomain trimer protomer, the fusion protein trimer, or the MeV H ectodomain head dimer is fused to a transmembrane domain via a peptide linker or directly to the transmembrane domain. [The present invention 1050] The recombinant MeV F ectodomain trimer or the recombinant MuV F ectodomain trimer protomer is a full-length F 1 The immunogen of the present invention 1049, comprising a protein. [This invention 1051] A virus-like particle comprising any of the immunogens of the present invention. [This invention 1052] A self-assembling protein nanoparticle comprising any of the immunogens of the present invention. [This invention 1053] A nucleic acid molecule encoding any of the immunogens of the present invention. [This invention 1054] The nucleic acid molecule of the present invention 1053, operably linked to a promoter. [This invention 1055] A vector comprising a nucleic acid molecule of the present invention. [This invention 1056] The vector of the present invention 1055, which is an RNA vector. [This invention 1057] 1. A method for producing an immunogen, comprising: A step of expressing any one of the nucleic acid molecules of the present inventions 1055 to 1056 in a host cell; and purifying the immunogen A method comprising: [This invention 1058] An immunogen produced by the method of the present invention. [This invention 1059] An immunogenic composition comprising the immunogen, nucleic acid molecule, vector, or virus-like particle of any of the present inventions 1001 to 1056 and 1058, and a pharmaceutically acceptable carrier. [The present invention 1060] A method for inducing an immune response to MuV F, MeV F, MuV NH, and / or MeV H in a subject, comprising administering to the subject an effective amount of an immunogenic composition of the present invention 1059 to induce the immune response. [The present invention 1061] The method of claim 1060, wherein said immune response inhibits MuV and / or MeV infection in said subject. [The present invention 1062] The method of claim 1060 or claim 1061, wherein the subject is an adult who has previously been immunized with a live attenuated vaccine against MeV and / or MuV, and wherein said administration boosts the immune response against said MuV and / or MeV. The above and other features and advantages of the present disclosure will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0015] [Figure 1A] Figures 1A-1E. Structure-based design of disulfide-stabilized prefusion mumps F trimers. (Figure 1A) Structure-based design of prefusion mumps F glycoprotein trimers by systematic screening of disulfides and GCN4 attachment sites. Both the yield and the percentage of prefusion versus postfusion F conformations as determined by negative-stain EM are shown. [Figure 1B] (Figure 1B) By combining V206C / A223C with 476-GCN4, a uniform pre-fusion F trimer was observed in high yield (top panel). Post-fusion is shown in the bottom panel. [Figure 1C] (Figure 1C) S200 gel filtration analysis shows the monodispersity of glycosylated and deglycosylated prefusion F (PreF), HN, and prefusion F-HN. [Figure 1D] (Figure 1D) Crystal structure of the pre-fusion mumps F trimer (SEQ ID NO:11) at 2.16 Å resolution. The protomer is shown, and residues that undergo a conformational change of >5 Å to transition to the post-fusion conformation are shown in black. The GCN4 trimerization (TD) motif tethered to F residue 476 is shown in dotted lines. The enlarged view highlights mutated residues that stabilize the trimeric pre-fusion F structure. [Figure 1E] (Figure 1E) A single protomer of the prefusion mumps trimer showing the D1-3 subregion and the location of the six N-linked glycans per protomer. [Figure 2-1]Figures 2A-2H. Structural comparison of the mumps and PIV5 prefusion F proteins and analysis of genotypic variation in mumps F and mumps HN. (Figure 2A) Structural superposition of the mumps prefusion F trimer with the closely related paramyxovirus PIV5 prefusion F trimer, showing similar overall topology (RMSD = 1.68 Å) and 49% sequence identity. (Figure 2B) The apical loops of mumps preF assemble into a "closed cap" assembly, whereas in PIV5, these loops are spread apart (top panel). Residues that stabilize the apical end of mumps preF are T178, T179, and N181 (bottom panel). (Fig. 2C) The mumps F glycoprotein is glycosylated with six glycans per protomer, for a total of 18 glycans per trimer, of which six glycans are in conformationally flexible regions (black) and 12 glycans are in conformationally less flexible regions, providing substantial glycan shielding to the trimer. [Figure 2-2] (Fig. 2D) Phylogenetic analysis of fusion glycoproteins from mumps genotypes A to J. (Fig. 2E) Phylogenetic analysis of hemagglutinin-neuraminidase glycoproteins from mumps genotypes A to J. [Figure 2-3] (Figure 2F and Figure 2G) Sequence alignment of the fusion glycoproteins from the Jeryl Lynn vaccine strain (genotype A, SEQ ID NO:98) and mumps genotypes C (SEQ ID NO:102), D (SEQ ID NO:103), F (SEQ ID NO:104), G (SEQ ID NO:105), and H (SEQ ID NO:106). [Figure 2-4] See description of Figure 2-3. [Figure 2-5] (Figure 2H) Structural mapping of prefusion F (left panel) and HN (right panel) mumps genotype variants. A-J genotype variants (top panel) and genotype G vs. A (Jeryl Lynn (JL)) (bottom panel) are shown, indicating residue differences and glycan differences. [Figure 3A]Figure 3A and Figure 3B. Design and immunization scheme for mumps F and preF-HN immunogen mutants. (Figure 3A) CB6F1 / J mice were immunized three times at 10 μg / dose with poly(I:C) adjuvant at weeks 0, 3, and 10, and blood samples were collected at weeks 2, 5, 12, and 16. [Figure 3B] (Figure 3B) Negative staining characterization of mumps F and preF-HN proteins confirming the conformational and molecular identity of the immunogens, and mumps neutralizing titers for postfusion F (left), prefusion F (center), and prefusion F-HN (right) immunogens elicited after one, two, or three immunizations (indicated by arrows) in mice, as well as PRN titers for genotype G virus, Jeryl Lynn genotype A virus, and genotype H virus. [Figure 4-1] Figures 4A-4E. Design of the prefusion mumps F-HN immunogen and analysis of sera for specificity and durability of neutralizing titers against three different mumps genotypes. (Figure 4A) Negative staining characterization of the mumps preF-HN fusion protein. The positions of the preF head, GCN4 trimerization domain, and three HN heads are indicated. (Figure 4B) Octet Bioinferometry binding titers of immunized mice using the mumps prefusion F probe and (Figure 4C) the HN probe. [Figure 4-2] (Figure 4D) Persistence analysis of mice immunized with three doses of 10 μg of prefusion F protein with poly(I:C) followed by monthly serum sampling and PRNT analysis against genotype G, Jeryl Lynn, and genotype H viruses over a 6-month period. [Figure 4-3] (Figure 4E) Persistence analysis of mice immunized with three doses of 10 μg of prefusion F-HN protein with poly(I:C) followed by monthly serum sampling and PRNT analysis against genotype G virus, Jeryl Lynn virus, and genotype H virus over a 6-month period. [Figure 5A] Figures 5A-5C. Structure-based design of prefusion measles F. (Figure 5A) Structure-based design of prefusion-stabilizing measles F glycoprotein mutations that stabilize the prefusion conformation. [Figure 5B] (Figure 5B) Negative stain EM analysis of measles F mutants containing the non-native disulfide bonds and C-terminal GCN4 trimerization domain indicated. [Figure 5C] (Figure 5C) Immunogenicity of pre-fusion (MeV F R165C / M171C-486-GCN4, SEQ ID NO:38) and post-fusion (native MeV F sequence with -486-GCN4) measles F glycoprotein. [Figure 6A] Figures 6A-6C. Design of mumps pre-F-measles H chimeric immunogens. (Figure 6A) Negative staining characterization of measles H dimer (top left), measles H trimer (top center), mumps HN trimer (top right), and mumps pre-F with measles H (MuV F 206C / 223C-476+GCN4 / Fd+MeV-H, SEQ ID NO:28) (bottom). [Figure 6B] (FIG. 6B) Measles plaque reduction neutralization titer assay (PRNT). [Figure 6C] (FIG. 6C) Mumps (genotype G) PRNT assay.

[0016] Sequence Listing The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each sequence is shown, but it is understood that any reference to the displayed strand includes the complementary strand. The sequence listing has been submitted as an ASCII text file entitled "Sequence.txt" (approximately 456 kb), created on December 11, 2020, which is incorporated herein by reference.

[0017] Structural coordinates The atomic coordinates of the crystal structure of the MuV F ectodomain trimer stabilized in the prefusion conformation are set forth in Table 1 of U.S. Provisional Application No. 62 / 946,902, filed December 11, 2019, which is incorporated herein by reference in its entirety and submitted as an ASCII text file entitled "Table_1.txt" (approximately 555 KB), created December 9, 2019. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description I. Terminology Overview Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes X (Jones & Bartlett Publishers, 2009) and Meyers et al., eds., The Encyclopedia of Cell Biology and Molecular Medicine (16 volumes, Wiley-VCH, 2008) and other similar references. As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless the context dictates otherwise. For example, the term "an antigen" encompasses single or multiple antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." Furthermore, any and all base size or amino acid size and any molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are understood to be approximate and provided for illustrative purposes, unless otherwise indicated. Suitable methods and materials are specifically described below, but many methods and materials similar or equivalent to those described herein can be used. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are merely illustrative and not intended to be limiting. The following explanations of terms are provided to facilitate review of various embodiments.

[0019] Adjuvant: A medium used to enhance antigenicity. In some embodiments, adjuvants include suspensions of minerals (alum, aluminum hydroxide, or phosphate) to which antigens are adsorbed, or water-in-oil emulsions, for example, in which an antigen solution is emulsified in mineral oil (Freund's incomplete adjuvant), optionally including killed mycobacteria to further enhance antigenicity (Freund's complete adjuvant) (to inhibit antigen degradation and / or induce macrophage influx). In some embodiments, the adjuvant used in the immunogenic compositions of the present disclosure is a combination of lecithin and carbomer homopolymer (e.g., ADJUPLEX™ adjuvant available from Advanced BioAdjuvants, LLC; see also Wegmann, Clin Vaccine Immunol, 22(9):1004-1012, 2015). Additional adjuvants for use in the immunogenic compositions of the present disclosure include QS21 purified plant extract, Matrix M, AS01, MF59, and ALFQ adjuvants. Immunostimulatory oligonucleotides (such as those containing CpG motifs) can also be used as adjuvants. Adjuvants also include biological molecules ("biological adjuvants") such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, immune stimulating complex (ISCOM) matrix, and Toll-like receptor (TLR) agonists, such as TLR-9 agonists, poly I:C, or poly ICLC. (See, e.g., Singh, ed., Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007).

[0020] Administration: The introduction of a composition into a subject by a selected route. Administration can be local or systemic. For example, if the selected route is intranasal, the composition (e.g., a recombinant MuV F ectodomain trimer or a composition comprising a recombinant MeV F ectodomain trimer of the present disclosure) is administered by introducing the composition into the nasal passages of a subject. Exemplary routes of administration include, but are not limited to, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (e.g., topical), intranasal, vaginal, and inhalation routes.

[0021] Amino acid substitution: The replacement of an amino acid in a polypeptide with one or more different amino acids. In the context of protein sequences, an amino acid substitution is also called a mutation.

[0022] Antibody: An immunoglobulin, antigen-binding fragment, or derivative thereof that specifically binds and recognizes an analyte (antigen), such as MuV or MeV F protein, an antigenic fragment thereof, or an antigen dimer or multimer. The term "antibody" is used in the broadest sense herein and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins, as well as variants and fragments thereof that retain binding affinity for the antigen. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel, eds., Antibody Engineering, Vols. 1-2, 2002). nd Ed., Springer Press, 2010).

[0023] Carrier: An immunogenic molecule to which an antigen can be linked. When linked to a carrier, the antigen can become more immunogenic. The carrier is chosen to increase the immunogenicity of the antigen and / or to elicit antibodies to the carrier that are diagnostically, analytically, and / or therapeutically useful. Useful carriers include polymeric carriers, which can be natural (e.g., proteins from bacteria or viruses), semi-synthetic, or synthetic, containing one or more functional groups to which a reactant moiety can be attached.

[0024] Conservative variant: A "conservative" amino acid substitution is one that does not substantially affect or reduce the function of a protein, for example, the ability of the protein to induce an immune response when administered to a subject. The term conservative variation also encompasses the use of a substituted amino acid in place of an unsubstituted parent amino acid. Furthermore, individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small number of amino acids (e.g., less than 5%, in some embodiments less than 1%) in an encoded sequence are conservative variations if the modifications result in the replacement of amino acids with chemically similar amino acids.

[0025] The following six groups are examples of amino acids that are considered conservative substitutions for one another: 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).

[0026] Non-conservative substitutions reduce the activity or function of the recombinant MuV or MeV F ectodomain trimer, such as its ability to induce an immune response when administered to a subject. For example, if an amino acid residue is essential for the function of the protein, even an otherwise conservative substitution may destroy its activity. Thus, conservative substitutions do not alter the basic function of the protein of interest.

[0027] Control: Reference standard. In some embodiments, the control is a negative control sample obtained from a healthy patient. In another embodiment, the control is a positive control sample obtained from a patient diagnosed with MuV or MeV infection. In yet another embodiment, the control is a historical control or a standard reference value or standard reference value range (e.g., a previously tested control sample, such as a group of MuV or MeV patients with known prognosis or outcome, or a group of samples representing baseline or normal values).

[0028] The difference between the test sample and the control may be an increase or a decrease. The difference may be a qualitative difference or a quantitative difference, for example, a statistically significant difference. In some examples, the difference is an increase or decrease of at least about 5%, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or more than 500% compared to the control.

[0029] Degenerate variant: In the context of this disclosure, the term "degenerate variant" refers to a polynucleotide encoding a polypeptide that contains a sequence that is degenerate as a result of the genetic code. There are 20 naturally occurring amino acids, most of which are specified by more than one codon. Thus, all degenerate nucleotide sequences that encode a peptide are included, as long as the amino acid sequence of the peptide encoded by the nucleotide sequence remains unchanged.

[0030] Effective amount: An amount of an agent, such as an immunogen, sufficient to elicit a desired response, such as an immune response, in a subject. It is understood that obtaining a protective immune response to an antigen of interest may require multiple administrations of an immunogen of the present disclosure, and / or administration of an immunogen of the present disclosure as a "prime" in a prime-boost protocol (where the boost immunogen can be different from the prime immunogen). Thus, an effective amount of an immunogen of the present disclosure can be an amount of the immunogen sufficient to elicit a priming immune response in a subject, who can then be boosted with the same or a different immunogen to elicit a protective immune response.

[0031] In one example, the desired response is to inhibit, reduce, or prevent MuV infection. MuV infection need not be completely eliminated, reduced, or prevented for the method to be effective. For example, administration of an effective amount of an agent can reduce MuV infection (e.g., as measured by infection of cells with MuV or by the number or percentage of subjects infected with MuV) by a desired amount (elimination or prevention of detectable MuV infection) compared to an appropriate control, e.g., by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%.

[0032] In one example, the desired response is to inhibit, reduce, or prevent MeV infection. MeV infection need not be completely eliminated, reduced, or prevented for the method to be effective. For example, administration of an effective amount of an agent can reduce MeV infection (e.g., as measured by infection of cells with MeV or by the number or percentage of subjects infected with MeV) by a desired amount (elimination or prevention of detectable MeV infection) compared to a suitable control, such as by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%.

[0033] In one example, the desired response is to inhibit, reduce, or prevent both MuV and MeV infection. MuV and MeV infection need not be completely eliminated, reduced, or prevented for the method to be effective. For example, administration of an effective amount of an agent can reduce MuV and MeV infection (e.g., as measured by infection of cells with MuV and / or MeV, or by the number or percentage of subjects infected with MuV and / or MeV) by a desired amount (elimination or prevention of detectable MuV and / or MeV infection) compared to a suitable control, e.g., by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%.

[0034] Expression: The transcription or translation of a nucleic acid sequence. For example, a gene is expressed if its DNA is transcribed into RNA or an RNA fragment, which in some instances is processed into mRNA. A gene can also be expressed if its mRNA is translated into an amino acid sequence, such as a protein or protein fragment. In one particular example, a heterologous gene is expressed if it is transcribed into RNA. In another example, a heterologous gene is expressed if its RNA is translated into an amino acid sequence. The term "expression" is used herein to refer to either transcription or translation. Regulation of expression can include control of transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or activation, inactivation, compartmentalization, or degradation of specific protein molecules after they are produced.

[0035] Expression control sequence: A nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence operably linked to it. An expression control sequence is operably linked to a nucleic acid sequence if the expression control sequence controls and regulates the transcription and, if appropriate, translation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (ATG) in front of a protein-encoding gene, splicing signals for introns, maintaining the correct reading frame of the gene to allow proper translation of mRNA, and stop codons. The term "control sequence" is intended to include, at a minimum, components whose presence can affect expression, and can also include additional components whose presence is beneficial, such as leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

[0036] A promoter is a minimal sequence sufficient to direct transcription. It also includes promoter elements sufficient to render promoter-dependent gene expression controllable, such as cell type-specific, tissue-specific, or inducible by external signals or agents; such elements can be located in the 5' or 3' region of the gene. Both constitutive and inducible promoters are included (see, e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in a bacterial system, inducible promoters such as pL, plac, ptrp, and ptac (ptrp-lac hybrid promoter) of bacteriophage lambda can be used. In one embodiment, when cloning in a mammalian cell system, promoters derived from the genome of a mammalian cell (e.g., metallothionein promoter) or from a mammalian virus (e.g., retroviral long terminal repeat; adenovirus late promoter; vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used to provide for transcription of nucleic acid sequences.

[0037] Expression vector: A vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked plasmids or plasmids contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating a recombinant polynucleotide.

[0038] GCN4 trimerization domain: A trimerization domain from the GCN4 protein, comprising a leucine zipper amino acid sequence that naturally forms a trimer structure. Embodiments of the GCN4 trimerization domain are described, for example, in Harbury et al. (1993 Science 262:1401-1407). In some examples, the amino acid sequence of a recombinant protein of the present disclosure can include a GCN4 trimerization domain so that the recombinant protein trimerizes. Non-limiting examples of GCN4 trimerization domain sequences for use in embodiments of the present disclosure include: Given as TIFF0007801221000010.tif4128.

[0039] Heterologous: Derived from different genetic sources.

[0040] Host cell: A cell in which a vector can be propagated and its nucleic acid expressed. The cell can be prokaryotic or eukaryotic. The term also encompasses any progeny of the subject host cell. It is understood that all progeny may not be identical to the parent cell since there may be mutations that occur during replication. However, when the term "host cell" is used, such progeny are included.

[0041] Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, resulting in the production of specific antibodies.

[0042] Immunogen: A compound, composition, or substance (e.g., a recombinant MuV or MeV F ectodomain trimer) that can elicit an immune response in an animal. This includes compositions that are injected or absorbed into an animal. Administration of the immunogen to a subject can lead to protective immunity against the pathogen of interest.

[0043] Immunogenic composition: A composition comprising an immunogen of the present disclosure that, when administered to a subject, induces a measurable CTL response against MuV or MeV or induces a measurable B cell response (e.g., antibody production) against MuV or MeV. This also refers to isolated nucleic acid molecules and vectors encoding a recombinant MuV or MeV F ectodomain trimer protomer of the present disclosure that can be used to express the protomer (and thus elicit an immune response against the recombinant MuV or MeV F ectodomain trimer). For in vivo use, the immunogenic composition will typically comprise a nucleic acid molecule encoding a recombinant MuV or MeV F ectodomain trimer or a recombinant MuV or MeV F ectodomain trimer protomer in a pharmaceutically acceptable carrier, and may also include other agents, such as an adjuvant.

[0044] Inhibition or treatment of disease: Inhibition of the full development of a disease or condition in a subject at risk for a disease, such as MuV infection or MeV infection. "Treatment" refers to a therapeutic intervention that ameliorates signs or symptoms of a disease or pathological condition after it has begun to develop. The term "ameliorate," with respect to a disease or pathological condition, refers to any observable beneficial effect of the treatment. Inhibition of disease can include preventing the disease or reducing the risk of the disease, for example, preventing or reducing the risk of viral infection. A beneficial effect can be evidenced, for example, by delaying the onset of clinical symptoms of the disease in a susceptible subject, reducing the severity of some or all clinical symptoms of the disease, slowing disease progression, reducing viral load, improving the overall health or well-being of the subject, or other parameters specific to the particular disease. "Prophylactic" treatment is treatment administered to a subject who does not exhibit signs of the disease or who exhibits only early signs, with the aim of reducing the risk of developing the pathology.

[0045] Isolated: An "isolated" biological component is substantially separated or purified from other biological components, such as other biological components that naturally accompany the component, e.g., other chromosomal and extrachromosomal DNA, RNA, and proteins. "Isolated" proteins, peptides, nucleic acids, and viruses include those purified by standard purification methods. "Isolated" does not require absolute purity and can include proteins, peptides, nucleic acids, or viral molecules that are at least 50% isolated, e.g., at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

[0046] Linker and linked: A bifunctional molecule that can be used to link two molecules into one continuous molecule. Non-limiting examples of peptide linkers include glycine-serine peptide linkers. References to "linking" a first polypeptide to a second polypeptide, or to two polypeptides "linked" to each other, or to a first polypeptide having a "link" to a second polypeptide, refer to a covalent linkage by a peptide bond (e.g., via a peptide linker) such that the first and second polypeptides form a continuous polypeptide chain, unless the context indicates otherwise. When a peptide linker is involved, the covalent linkage between the first and second polypeptides can be to the N-terminus and C-terminus of the peptide linker. Typically, such linkage is achieved by genetically engineering DNA encoding a first polypeptide linked to a second polypeptide by a peptide linker using molecular biology techniques.

[0047] Native protein, sequence, or disulfide bond: A polypeptide, sequence, or disulfide bond that has not been modified, such as by selective mutation, e.g., selective mutation to focus the antigenicity of an antigen on a target epitope or to introduce disulfide bonds into a protein that are not found in the native protein. A native protein or sequence is also referred to as a wild-type protein or sequence. A non-native disulfide bond is a disulfide bond that is not present in the native protein, e.g., a disulfide bond formed in a protein by introducing one or more cysteine ​​residues into the protein by genetic engineering.

[0048] Measles: An infectious disease caused by the measles virus. Symptoms usually develop 10–12 days after exposure to an infected person and last for 7–10 days. Early symptoms typically include fever, cough, runny nose, and bloodshot eyes. Two or three days after the onset of symptoms, small white spots called Koplik spots may form inside the mouth. A red, flat rash, usually beginning on the face and then spreading to the rest of the body, typically begins 3–5 days after the onset of symptoms. Common complications include diarrhea, middle ear infections, and pneumonia, which occur in part due to measles-induced immune suppression. Less common complications include seizures, blindness, or brain inflammation.

[0049] Measles virus: A non-segmented, negative-strand RNA virus of the Morbillivirus genus in the Paramyxoviridae family that causes measles disease. The measles virus genomic RNA contains six linked transcription units encoding open reading frames for eight proteins: nucleoprotein (N), phosphoprotein (P), C protein, V protein, matrix (M) protein, fusion (F) protein, hemagglutinin (H) protein, and large (L) protein. There are at least seven known genotypes of MeV currently circulating worldwide, designated genotypes A, B, C, D, F, G, and H.

[0050] MeV fusion (F) protein: The MeV envelope glycoprotein that facilitates fusion of the viral and cellular membranes. In nature, the F protein from MeV is first synthesized as a single polypeptide precursor, approximately 550 amino acids long, designated F0. F0 contains an N-terminal signal peptide that directs localization to the endoplasmic reticulum, where it is proteolytically cleaved. The remaining F0 residues oligomerize to form a trimer, which is then proteolytically processed by cellular proteases to generate two disulfide-linked fragments, F1 and F2. In the case of MeV F, the cleavage site is located approximately between residues 113 and 114. The smaller of these fragments, F2, is derived from the N-terminal portion of the F0 precursor (approximately residues 24–113). The larger of these fragments, F1, contains the C-terminal portion of the F0 precursor (approximately residues 114-550), including the extracellular / luminal region (approximately residues 110-486) ​​and the transmembrane and cytosolic regions (approximately residues 487-550). The extracellular portion of the MeV F protein is the MeV F ectodomain, which includes the F2 protein and the F1 ectodomain.

[0051] The MeV F protein exhibits significant sequence conservation among MeV strains. This conservation allows one of skill in the art to easily compare amino acid positions of different MeV F proteins. The numbering of MeV F amino acids follows SEQ ID NO:36 (NCBI Reference Sequence P35973.1, incorporated herein by reference), unless the context indicates otherwise: This is done by referencing TIFF0007801221000011.tif26160.

[0052] Three MeV F protomers oligomerize to form the mature F protein, which adopts a metastable pre-fusion conformation. Upon contact with the target cell membrane, this conformation is triggered to undergo a conformational change to a post-fusion conformation. This conformational change exposes a hydrophobic sequence located at the N-terminus of the F1 ectodomain, called the fusion peptide, which associates with the host cell membrane and promotes fusion of the viral or infected cell membrane with the target cell membrane.

[0053] A MeV F ectodomain trimer "stabilized in a pre-fusion conformation" contains one or more amino acid substitutions, deletions, or insertions that, compared to the corresponding native MeV F sequence, increase the retention of the pre-fusion conformation relative to a MeV F ectodomain trimer formed from the corresponding native MeV F sequence. "Stabilization" of the pre-fusion conformation can be, for example, energetic stabilization (e.g., reducing the energy of the pre-fusion conformation relative to the post-fusion open conformation) and / or kinetic stabilization (e.g., reducing the rate of transition from the pre-fusion conformation to the post-fusion conformation). Additionally, stabilization of the MeV F ectodomain trimer in the pre-fusion conformation can include increased resistance to denaturation compared to the corresponding native MeV F sequence. Methods for determining whether a MeV F ectodomain trimer is in a pre-fusion conformation are provided herein, including, but not limited to, negative stain electron microscopy and antibody binding assays using pre-fusion conformation-specific antibodies. The term "pre-F," with respect to the MeV F protein, refers to a molecule that is a trimeric class I fusion protein stabilized in the pre-fusion conformation by one or more amino acid substitutions.

[0054] MeV F prefusion-specific antibody: An antibody that specifically binds to the MeV F protein in the prefusion conformation but not the MeV F protein in the postfusion conformation.

[0055] MeV hemagglutinin (H) protein: MeV envelope glycoprotein, a type II membrane protein that facilitates attachment of MeV to the host cell membrane. The full-length H protein has an N-terminal cytoplasmic tail and transmembrane domains (CT and TM, approximately amino acids 1-58), as well as an ectodomain (approximately amino acids 59-617) that includes a stalk region (approximately amino acids 59-179) and a head region (approximately amino acids 180-617). An exemplary MeV H protein sequence is herein referred to as SEQ ID NO:49 (NCBI Reference Sequence AAA56644.1, incorporated herein by reference): Provided as TIFF0007801221000012.tif29160.

[0056] As used herein, MeV H residue positions are made with reference to the sequence shown as SEQ ID NO:49.

[0057] Mumps: An infectious disease caused by the mumps virus. Mumps is characterized by inflammation of the salivary glands, typically the parotid gland. Serious complications of mumps virus infection can occur, including meningitis, encephalitis, pancreatitis, oophoritis (in women), orchitis (in men), and hearing loss.

[0058] Mumps virus (MuV): A non-segmented, negative-strand RNA virus of the Paramyxovirinae subfamily, Paramyxovirinae, genus Rubulavirus that causes mumps disease. The mumps virus genomic RNA contains seven tandemly linked transcription units encoding open reading frames for the nucleoprotein (N), phosphoprotein (P), V protein, I protein, matrix (M) protein, fusion (F) protein, small hydrophobic (SH) protein, hemagglutinin-neuraminidase (HN) protein, and large (L) protein. A schematic diagram of the mumps virus genome is shown in Figure 6. The P gene (also referred to as the "V / P / I gene") generates three mRNA transcripts corresponding to the V, P, and I proteins through RNA editing by the insertion of guanine nucleotides. Specifically, faithful transcription of the P gene yields the V protein; insertion of two guanine nucleotides yields the mRNA encoding the P protein; insertion of four guanine residues yields the mRNA encoding the I protein. The SH gene is the most variable gene among different MuV genotypes and is therefore commonly used as the basis for genotyping. There are 12 known MuV genotypes currently circulating worldwide, designated genotypes A, B, C, D, F, G, H, I, J, K, L, and N. Current MuV vaccines are based on genotype A (Jeryl Lynn), genotype B (Urabe-AM9), or undetermined genotype (Leningrad-Zagreb) viruses.

[0059] MuV fusion (F) protein: The MuV envelope glycoprotein that facilitates fusion of the viral and cellular membranes. In nature, the F protein from MuV is first synthesized as a single polypeptide precursor, approximately 538 amino acids long, designated F0. F0 contains an N-terminal signal peptide that directs localization to the endoplasmic reticulum, where it is proteolytically cleaved. The remaining F0 residues oligomerize to form a trimer, which is then proteolytically processed by cellular proteases to generate two disulfide-linked fragments, F1 and F2. In the case of MuV F, the cleavage site is located approximately between residues 103 and 104. The smaller of these fragments, F2, is derived from the N-terminal portion of the F0 precursor (approximately residues 20–103). The larger of these fragments, F1, contains the C-terminal portion of the F0 precursor (approximately residues 104-538), including the extracellular / luminal region (approximately residues 110-483) and the transmembrane and cytosolic regions (approximately residues 484-538). The extracellular portion of the MuV F protein is the MuV F ectodomain, which includes the F2 protein and the F1 ectodomain.

[0060] MuV F proteins exhibit significant sequence conservation among MuV strains. This conservation allows one of skill in the art to easily compare amino acid positions of different MuV F proteins. MuV F amino acid numbering follows SEQ ID NO:1 (NCBI Reference Sequence P09458.1, incorporated herein by reference), unless the context indicates otherwise: This is done by referencing TIFF0007801221000013.tif25160.

[0061] Three MuV F protomers oligomerize to form the mature F protein, which adopts a metastable prefusion conformation. Upon contact with the target cell membrane, this conformation is triggered to undergo a conformational change to a postfusion conformation. This conformational change exposes a hydrophobic sequence located at the N-terminus of the F1 ectodomain, called the fusion peptide, which associates with the host cell membrane and promotes fusion of the viral or infected cell membrane with the target cell membrane.

[0062] A MuV F ectodomain trimer "stabilized in the pre-fusion conformation" contains one or more amino acid substitutions, deletions, or insertions that, compared to the corresponding native MuV F sequence, increase the retention of the pre-fusion conformation relative to a MuV F ectodomain trimer formed from the corresponding native MuV F sequence. "Stabilization" of the pre-fusion conformation can be, for example, energetic stabilization (e.g., reducing the energy of the pre-fusion conformation relative to the post-fusion open conformation) and / or kinetic stabilization (e.g., reducing the rate of transition from the pre-fusion conformation to the post-fusion conformation). Additionally, stabilization of the MuV F ectodomain trimer in the pre-fusion conformation can include increased resistance to denaturation compared to the corresponding native MuV F sequence. Methods for determining whether a MuV F ectodomain trimer is in the pre-fusion conformation are provided herein, including, but not limited to, negative stain electron microscopy and antibody binding assays using pre-fusion conformation-specific antibodies. The term "pre-F," with respect to the MuV F protein, refers to a molecule that is a trimeric class I fusion protein stabilized in the pre-fusion conformation by one or more amino acid substitutions.

[0063] MuV hemagglutinin-neuraminidase (HN) protein: A MuV envelope glycoprotein, a type II membrane protein that facilitates attachment of MuV to the host cell membrane. The full-length MuV HN protein has an N-terminal cytoplasmic tail and transmembrane domains (CT and TM, approximately amino acids 1-53), as well as an ectodomain (approximately amino acids 54-582) that includes a stalk region (approximately amino acids 54-130) and a head region (approximately amino acids 131-582). An exemplary MuV HN protein sequence is herein set forth as SEQ ID NO:50 (NCBI Reference Sequence AQT03695.1, incorporated herein by reference): Provided as TIFF0007801221000014.tif30160.

[0064] As used herein, the location of MuV HN residues is made with reference to the sequence shown as SEQ ID NO:50.

[0065] MuV F prefusion-specific antibody: An antibody that specifically binds to the MuV F protein in the prefusion conformation but not the MuV F protein in the postfusion conformation.

[0066] Nucleic acid molecule: A polymeric form of nucleotide, which may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms, as well as mixed polymers of the above. Nucleotide refers to ribonucleotides, deoxynucleotides, or modified forms of either type of nucleotide. As used herein, the terms "nucleic acid molecule" are synonymous with "nucleic acid" and "polynucleotide." Nucleic acid molecules are typically at least 10 bases in length, unless otherwise specified. The term encompasses single-stranded and double-stranded forms of DNA. Polynucleotides may contain either or both natural and modified nucleotides linked to each other by natural and / or non-natural nucleotide linkages. "cDNA" refers to DNA complementary to or identical to mRNA, in single-stranded or double-stranded form. "Encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template in biological processes for the synthesis of other polymers and macromolecules having either a predetermined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a predetermined amino acid sequence and the biological properties that result therefrom.

[0067] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence if the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0068] Pharmaceutically acceptable carriers: Useful pharmaceutically acceptable carriers are conventional. Remington's Pharmaceutical Sciences by E.W. Martin (Mack Publishing Co., Easton, PA, 19th ed., 1995) describes compositions and formulations suitable for pharmaceutical delivery of the immunogens of this disclosure.

[0069] Generally, the nature of the carrier will depend on the particular mode of administration being used. For example, parenteral formulations usually comprise injectable fluids containing pharmaceutically and physiologically acceptable fluids as a vehicle, such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, or the like. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the administered pharmaceutical composition (e.g., immunogenic composition) can also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate. In certain embodiments suitable for administration to a subject, the carrier is sterile and / or can be suspended or otherwise contained in a unit dosage form containing one or more metered doses of the composition appropriate for inducing the desired immune response. A drug for its intended use in treatment can also be included. The unit dosage form may be, for example, in a sealed vial containing sterile contents, may be lyophilized for later dissolution and administration, or may be in a solid or controlled release formulation.

[0070] Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). "Polypeptide" applies to amino acid polymers, including natural and unnatural amino acid polymers, and those in which one or more amino acid residues are unnatural amino acids, e.g., artificial chemical mimetics of the corresponding natural amino acids. "Residue" refers to an amino acid or amino acid mimetic incorporated into the polypeptide by an amide bond or amide bond mimetic. Polypeptides have an amino terminus (N-terminus) and a carboxy terminus (C-terminus). "Polypeptide" is used interchangeably with peptide or protein and is used herein to refer to a polymer of amino acid residues.

[0071] Prime-boost vaccination: Immunotherapy involving administering a first immunogenic composition (primer vaccine) to a subject to induce an immune response, followed by administering a second immunogenic composition (booster vaccine). The primer vaccine and / or booster vaccine contain a vector (such as a viral vector, RNA, or DNA vector) expressing an antigen to which an immune response is directed. The booster vaccine is administered to a subject after the primer vaccine, and examples of appropriate time intervals and time frames between the administration of the primer vaccine and the booster vaccine are disclosed herein. In some embodiments, the primer vaccine, the booster vaccine, or both the primer vaccine and the booster vaccine further contain an adjuvant. In one non-limiting example, the primer vaccine is a DNA-based vaccine (or other vaccine based on gene delivery), and the booster vaccine is a vaccine based on protein subunits or protein nanoparticles.

[0072] Protein nanoparticles: Self-assembling, multi-subunit, protein-based polyhedral structures, each composed of a protein or polypeptide (e.g., a glycosylated polypeptide) and optionally one or more of the following features: nucleic acids, prosthetic groups, organic compounds, and inorganic compounds. Non-limiting examples of protein nanoparticles include ferritin nanoparticles (see, e.g., Zhang, Y. Int. J. Mol. Sci., 12:5406-5421, 2011, incorporated herein by reference), encapsulin nanoparticles (see, e.g., Sutter et al., Nature Struct. and Mol. Biol., 15:939-947, 2008, incorporated herein by reference), sulfur oxygenase / reductase (SOR) nanoparticles (see, e.g., Urich et al., Science, 311:996-1000, 2006, incorporated herein by reference), lumazine synthase nanoparticles (see, e.g., Zhang et al., J. Mol. Biol., 306:1099-1114, 2001), or pyruvate dehydrogenase nanoparticles (see, e.g., Izard et al., PNAS 96:1240-1245, 1999). Ferritin, encapsulin, SOR, lumazine synthase, and pyruvate dehydrogenase are monomeric proteins that self-assemble into globular protein complexes, which optionally consist of 24, 60, 24, 60, and 60 protein subunits, respectively. In some examples, ferritin, encapsulin, SOR, lumazine synthase, or pyruvate dehydrogenase monomers are linked to a recombinant MuV or MeV F ectodomain, which self-assemble into protein nanoparticles that display the recombinant MuV or MeV F ectodomain trimer on their surface, which can be administered to a subject to stimulate an immune response to the antigen.

[0073] Recombinant: A recombinant nucleic acid molecule is one that has a sequence that is not naturally occurring, e.g., a sequence that contains one or more nucleic acid substitutions, deletions, or insertions, and / or a sequence that is made by the artificial combination of two naturally separated segments of sequence, which can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

[0074] A recombinant virus is one that contains a genome that includes a recombinant nucleic acid molecule.

[0075] A recombinant protein is one that has a sequence that is not naturally occurring or that is created by the artificial combination of two otherwise separated sequence segments. In some embodiments, a recombinant protein is encoded by a heterologous (e.g., recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell, or introduced into the genome of a recombinant virus.

[0076] Sequence identity: The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, also known as sequence identity. Sequence identity is often measured by a percentage identity; the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide will have a relatively high degree of sequence identity when aligned using standard methods.

[0077] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al., Computer Appls. In the Biosciences 8,155-65, 1992; and Pearson et al. al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990 provides a detailed discussion of sequence alignment methods and homology calculations.

[0078] Polypeptide variants are typically characterized by retaining at least about 75% sequence identity, e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, when counted in a full-length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequence will exhibit increased percentage identities, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, when assessed by this method. When sequences being compared for sequence identity are shorter than the entire sequence, homologs and variants typically share at least 80% sequence identity over a short window of 10-20 amino acids, and may share at least 85%, or at least 90%, or 95% sequence identity, depending on their respective similarity to the reference sequence. Methods for determining sequence identity over such short windows are available on the Internet at the NCBI website.

[0079] As used herein, when "at least 90% identical" is used, it (or similar expressions) refers to "at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity" to a specified reference sequence.

[0080] Signal peptide: A short amino acid sequence (e.g., approximately 18-25 amino acids in length) that targets a newly synthesized secretory or membrane protein to and translocates it through a membrane (e.g., the endoplasmic reticulum membrane). A signal peptide is typically located at the N-terminus of a polypeptide and is removed by a signal peptidase after the polypeptide crosses the membrane. Signal peptide sequences typically contain three common structural features: an N-terminal polar basic region (n-region), a hydrophobic core, and a hydrophilic c-region. An exemplary signal peptide sequence is shown as MKAFSVTCLSFAVFSSSIC (residues 1-19 of SEQ ID NO:2).

[0081] Specific binding: With respect to the formation of an antibody:antigen-protein complex or protein:protein complex, this refers to a binding reaction that determines the presence of a target protein, peptide, or polysaccharide (e.g., a glycoprotein) in the presence of a heterogeneous population of proteins and other biologics. Thus, under specified conditions, a particular antibody or protein preferentially binds to a specific target protein, peptide, or polysaccharide (e.g., an antigen present on the surface of a pathogen, e.g., an antigenic site at the membrane-distal apical end of the pre-fusion conformation of the MuV F or MeV F ectodomain trimer) and does not bind in significant amounts to other proteins or polysaccharides present in the sample or subject, or alternative conformations of the same protein (e.g., the post-fusion conformation of the MuV or MeV F protein). Specific binding can be determined by methods known in the art. The first protein or antibody is selected based on the K D is 10 -6 Less than molar, e.g., 10 -7 Less than 10 molar -8 Less than 10 molar -9 Less than molar, or even 10 -10 When present in submolar concentrations, it specifically binds to the target protein.

[0082] Soluble protein: A protein that can dissolve and remain dissolved in an aqueous liquid at room temperature. The solubility of a protein can vary depending on the concentration of the protein in the aqueous liquid, the buffer conditions of the liquid, the concentration of other solutes in the liquid, such as salt concentration and protein concentration, and the temperature of the liquid. In some embodiments, a soluble protein is one that dissolves to a concentration of at least 0.5 mg / ml in phosphate buffered saline (pH 7.4) at room temperature and remains dissolved for at least 48 hours.

[0083] Subject: A living multicellular vertebrate organism, a category that includes humans and non-human mammals. In one example, the subject is a human. In a particular example, the subject is a neonate / newborn. In a further example, a subject in need of inhibition of MuV or MeV infection is selected. For example, the subject may be uninfected and at risk for MuV or MeV infection, or infected and in need of treatment.

[0084] T4 fibritin trimerization domain: The T4 fibritin trimerization domain, also known as the "foldon" domain, comprises an amino acid sequence that naturally forms a trimer structure. In some examples, the amino acid sequence of a recombinant protein of the present disclosure can include a T4 fibritin trimerization domain so that the antigen forms a trimer. In one example, the T4 fibritin trimerization domain is TIFF0007801221000015.tif4128. Some embodiments include a T4 fibritin trimerization domain that can be cleaved from the purified protein, such as by incorporating a thrombin cleavage site next to the T4 fibritin trimerization domain that can be used for cleavage.

[0085] Transmembrane domain: An amino acid sequence that is inserted into a lipid bilayer, for example, the lipid bilayer of a cell or a virus or virus-like particle. A transmembrane domain can be used to anchor an antigen to a membrane. In some examples, the transmembrane domain is a MuV F transmembrane domain. In another example, the transmembrane domain is a MeV F transmembrane domain.

[0086] Under conditions sufficient for: A phrase used to describe any environment that allows for a desired activity.

[0087] Vaccine: A preparation of immunogenic material capable of stimulating an immune response administered to prevent, ameliorate, or treat infectious or other types of disease. The immunogenic material may include attenuated or killed microorganisms (such as bacteria or viruses), or antigenic proteins, peptides, or DNA derived therefrom. Vaccines may include an immunogen of the present disclosure (such as a recombinant MuV or MeV F ectodomain trimer or a nucleic acid molecule encoding same), a virus, a cell, or one or more cellular components. Vaccines can elicit both prophylactic (preventative or protective) and therapeutic responses. Methods of administration vary depending on the vaccine but may include inoculation, ingestion, inhalation, or other forms of administration. Vaccines may be administered with an adjuvant to boost the immune response. In one specific, non-limiting example, a vaccine prevents and / or reduces the severity of symptoms associated with MuV infection and / or reduces viral load compared to a control. In another specific, non-limiting example, the vaccine prevents and / or reduces the severity of symptoms associated with MeV infection and / or reduces viral load compared to a control.

[0088] Vector: An entity containing a DNA or RNA molecule operably linked to a coding sequence of an antigen of interest and carrying a promoter capable of expressing the coding sequence. Non-limiting examples include naked or packaged (lipid and / or protein) DNA, naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication-incompetent, or a virus or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. A recombinant DNA vector is a vector containing recombinant DNA. A vector can contain nucleic acid sequences, such as an origin of replication, that enable it to replicate in a host cell. A vector can also contain one or more selectable marker genes and other genetic elements known in the art. A viral vector is a recombinant nucleic acid vector that contains at least some nucleic acid sequences derived from one or more viruses.

[0089] Virus-like particle (VLP): A non-replicating viral shell derived from any of several viruses. VLPs are generally composed of one or more viral proteins, including, but not limited to, proteins referred to as capsid, coat, shell, surface, and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of proteins in an appropriate expression system. Methods for producing specific VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as electron microscopy, biophysical characterization, etc. Furthermore, VLPs can be isolated by known techniques, such as density gradient centrifugation, and identified by characteristic density banding. See, e.g., Baker et al. (1991) Biophys. J. 60:1445-1456 and Hagensee et al. (1994) J. Virol. 68:4503-4505; Vincente, J Invertebr Pathol., 2011; Schneider-Ohrum and Ross, Curr. Top. Microbiol. Immunol., 354:53073, 2012).

[0090] II. Immunogens A. Recombinant MuV F ectodomain trimer Disclosed herein are recombinant MuV F ectodomain trimers that have been modified from their native form (e.g., by introducing one or more amino acid substitutions) to stabilize them in the pre-fusion conformation. As described in the Examples, embodiments of the disclosed MuV F ectodomain trimers were selected through multiple rounds of structure-based design for optimized solubility, stability, expression, and immunogenicity. Recombinant MuV F ectodomain trimers are useful for inducing immune responses against MuV in vertebrates (such as humans). Exemplary embodiments have been shown to generate superior immune responses in animal models compared to corresponding MuV F ectodomain trimers that are not stabilized in the pre-fusion conformation.

[0091] In some embodiments, the immunogen comprises a recombinant MuV F ectodomain trimer comprising a protomer containing one or more amino acid substitutions or deletions that stabilize the MuV F ectodomain trimer in the pre-fusion conformation.

[0092] In some embodiments, the immunogen comprises a recombinant MuV F ectodomain trimer stabilized in a pre-fusion conformation by one or more amino acid substitutions in the trimer's promoter, including cysteine ​​substitutions that form non-native disulfide bonds to stabilize the MuV F ectodomain trimer in the pre-fusion conformation. The non-native disulfide bonds are not found in the native MuV F protein and are introduced by protein engineering (e.g., by including one or more substituted cysteine ​​residues that form non-native disulfide bonds). For example, in some embodiments, any of the recombinant MuV F proteins of the present disclosure can be stabilized in a pre-fusion conformation by any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-native disulfide bonds.

[0093] Disulfide bond-forming cysteine ​​residues can be introduced into the native MuV F sequence by one or more amino acid substitutions. For example, in some embodiments, a single amino acid substitution introduces a cysteine ​​that forms a disulfide bond with a cysteine ​​residue present in the native MuV F sequence. Alternatively, two cysteine ​​residues can be introduced into the native MuV F sequence to form a disulfide bond. The location of the non-native disulfide bond cysteine(s) can be determined by one of skill in the art using the disclosed structure of the MuV F ectodomain trimer in the prefusion conformation.

[0094] The amino acid positions of these cysteines are typically within a sufficiently close distance for disulfide bond formation in the pre-fusion conformation of the MuV F protein trimer. Methods for determining whether two residues are within a sufficiently close distance for disulfide bond formation using three-dimensional structural data (e.g., that provided in Table 1) are known (see, e.g., Peterson et al., Protein engineering, 12:535-548, 1999 and Dombkowski, Bioinformatics, 19:1852-1853, 3002 (disclosing DISULFIDE BY DESIGN™); each of these references is incorporated herein by reference). Residues can be selected manually based on the three-dimensional structure of the MuV F trimer in the pre-fusion conformation provided herein, or software such as DISULFIDEBYDESIGN™ can be used. Without being bound by theory, the ideal distances for disulfide bond formation are generally believed to be approximately 5.6 Å for the Cα-Cα distance, approximately 2.02 Å for the Sγ-Sγ distance, and approximately 3.5-4.25 Å for the Cβ-Cβ distance (using optimal rotomers). Those skilled in the art will appreciate that variations from these distances are encompassed when selecting residues in the three-dimensional structure that can be used in place of cysteines to introduce disulfide bonds. For example, in some embodiments, the selected residues have a Cα-Cα distance of less than 7.0 Å and / or a Cβ-Cβ distance of less than 4.7 Å. In some embodiments, the selected residues have a Cα-Cα distance of 2.0-8.0 Å and / or a Cβ-Cβ distance of 2.0-6.0 Å.

[0095] In some embodiments, the recombinant MuV F ectodomain trimer protomers contain cysteine ​​substitutions at MuV F positions 86 and 215 (e.g., N86C and A215C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0096] In some embodiments, the protomers of the recombinant MuV F ectodomain trimer contain cysteine ​​substitutions at MuV F positions 155 and 161 (e.g., K155C and L161C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0097] In some embodiments, the protomers of the recombinant MuV F ectodomain trimer contain cysteine ​​substitutions at MuV F positions 165 and 231 (e.g., V165C and M231C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0098] In some embodiments, the protomers of the recombinant MuV F ectodomain trimer contain cysteine ​​substitutions at MuV F positions 206 and 223 (e.g., V206C and A223C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0099] In some embodiments, the recombinant MuV F ectodomain trimer protomers contain cysteine ​​substitutions at MuV F positions 209 and 214 (e.g., P209C and P214C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0100] In some embodiments, the recombinant MuV F ectodomain trimer protomers contain cysteine ​​substitutions at MuV F positions 221 and 255 (e.g., I221C and M255C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0101] Any of the above recombinant MuV F proteins can further include modifications to eliminate the protease cleavage site between the F1 and F2 polypeptides to generate a "single-chain" recombinant F protein. For example, any of the above recombinant MuV proteins can include a deletion of MuV F positions 101-103 and fusion of positions 100 and 104 with a peptide linker. This modification removes the F2 / F1 furin cleavage site and also removes the first residue of the fusion peptide (which is hydrophobic). Any suitable peptide linker that fuses the F2 and F1 ectodomains and allows folding of the F ectodomain into its pre-fusion conformation can be used. In some embodiments, the peptide linker is a glycine, serine, or glycine-serine peptide linker. In some embodiments, the peptide linker is a Gly-Gly-Gly linker.

[0102] In one non-limiting example, a recombinant MuV F ectodomain trimer is provided that includes a promoter having V206C and A223C substitutions to form a non-native disulfide bond and a deletion of MuV F positions 101-103 with positions 100 and 104 fused with a Gly-Gly-Gly peptide linker.

[0103] In some embodiments, the recombinant MuV F ectodomain protomer can include one or more additional amino acid substitutions to enhance stabilization of the pre-fusion conformation or for other purposes, such as to increase solubility or reduce an unwanted immune response.

[0104] The non-native disulfide bonds listed above stabilize the membrane-distal portion of the MuV F ectodomain in the pre-fusion conformation. Any of these mutations can be combined with modifications to the membrane-proximal portion (e.g., stem) of the MuV F ectodomain, for example, to enhance ectodomain trimerization.

[0105] In some embodiments, the N-terminal position of the recombinant F2 polypeptide in the protomer can be one of MuV F positions 20-30 (e.g., position 20), and the C-terminal position of the F1 ectodomain can be a position from the stem region of the ectodomain, e.g., one of MuV F positions 469-483 (e.g., position 476).

[0106] In one non-limiting example, a recombinant MuV F ectodomain trimer is provided, comprising a protomer comprising MuV positions 20-476 with V206C and A223C substitutions to form a non-native disulfide bond and a deletion of MuV F positions 101-103, with positions 100 and 104 fused with a Gly-Gly-Gly peptide linker.

[0107] Non-limiting examples of MuV F ectodomain trimer protomers containing amino acid substitutions for stabilization in the pre-fusion conformation are provided herein. In some embodiments, the MuV F ectodomain trimer protomers contain an amino acid sequence that is at least 90% identical to residues 20-483 of any one of SEQ ID NOs:3-8, residues 20-476 of any one of SEQ ID NOs:11-16, 26, or 51, or residues 20-469 of any one of SEQ ID NOs:19-24, wherein the protomers contain one or more amino acid substitutions that stabilize the MuV F ectodomain trimer in the pre-fusion conformation. In some embodiments, a MuV F ectodomain trimer protomer comprises residues 20-483 of any one of SEQ ID NOs:3-8, residues 20-476 of any one of SEQ ID NOs:11-16, 26, or 51, or residues 20-469 of any one of SEQ ID NOs:19-4.

[0108] In some embodiments, the recombinant MuV F ectodomain trimer is a soluble protein complex for use, for example, as a recombinant subunit vaccine. In some such embodiments, the promoters of the recombinant MuV F ectodomain trimer can each comprise a C-terminal linkage to a trimerization domain, such as a GCN4 trimerization domain, a T4 fibritin trimer trimer, or both. The trimerization domain promotes trimerization and stabilization of the membrane-proximal aspect of the recombinant MuV F ectodomain trimer. For example, the C-terminal residue of the promoter of the recombinant MuV F ectodomain trimer (e.g., a residue in the stem region of the trimer) can be directly linked to the trimerization domain or indirectly linked to the trimerization domain via a peptide linker. Exemplary linkers include glycine and glycine-serine linkers. Non-limiting examples of exogenous multimerization domains that promote stable trimers of soluble recombinant proteins include the GCN4 leucine zipper, the T4 fibritin trimerization domain, and trimerization motifs derived from pulmonary surfactant proteins (Hoppe et al. 1994 FEBS Lett 344:191-195) or collagen (McAlinden et al. 2003 J Biol Chem 278:42200-42207), all of which can be linked to the C-terminus of recombinant MuV F ectodomain promoters to promote trimerization, as long as the recombinant MuV F ectodomain trimer maintains its prefusion conformation. In some examples, the protomers of the recombinant MuV F ectodomain trimer can be linked to a MuV trimerization domain, e.g., each protomer in the trimer can include a C-terminal linkage to a GCN4 trimerization domain, e.g., a linkage to any one of MuV F positions 469-483, e.g., MuV F position 469, MuV F position 476, or MuV F position 483. In a specific example, the GCN4 trimerization domain has the amino acid sequence In a specific example, the T4 fibritin trimerization domain comprises or consists of the amino acid sequence TIFF0007801221000016.tif4128. TIFF0007801221000017.tif4128. In a specific example, the GCN4 trimerization domain fused to the fibritin trimerization domain comprises or consists of the amino acid sequence It contains or consists of the same amino acid sequence as TIFF0007801221000018.tif10129.

[0109] In one non-limiting example, a recombinant MuV F ectodomain trimer is provided, comprising a protomer comprising MuV positions 20-476 with V206C and A223C substitutions to form a non-native disulfide bond, a deletion of MuV F positions 101-103 with positions 100 and 104 fused with a Gly-Gly-Gly peptide linker, and a GCN4 trimerization domain linked to the C-terminus of the protomer ectodomain.

[0110] Non-limiting examples of MuV F ectodomain trimer protomers comprising amino acid substitutions for stabilization in the pre-fusion conformation and a C-terminal linkage to a trimerization domain are provided herein. In some embodiments, the MuV F ectodomain trimer protomers comprise an amino acid sequence at least 90% identical to residues 20-513 of any one of SEQ ID NOs:3-8, residues 20-506 of any one of SEQ ID NOs:11-16, 26, or 51, or residues 20-499 of any one of SEQ ID NOs:19-24, wherein the protomers comprise one or more amino acid substitutions that stabilize the MuV F ectodomain trimer in the pre-fusion conformation. In some embodiments, a promoter of the MuV F ectodomain trimer comprises residues 20-513 of any one of SEQ ID NOs:3-8, residues 20-506 of any one of SEQ ID NOs:11-16, 26, or 51, or residues 20-499 of any one of SEQ ID NOs:19-24.

[0111] In some embodiments, the recombinant MuV F ectodomain trimer can be a membrane-anchored protein complex, e.g., for use in attenuated virus vaccines or virus-like particle vaccines. Membrane anchoring can be achieved, e.g., by C-terminal linkage of the recombinant MuV F ectodomain trimer protomer to the transmembrane domain and, optionally, the cytoplasmic tail, e.g., to the transmembrane domain and cytoplasmic tail of MuV F. In some embodiments, one or more peptide linkers (e.g., gly-ser linkers, e.g., a 10-amino acid glycine-serine peptide linker) can be used to link the recombinant MuV F ectodomain trimer protomer to the transmembrane domain. Non-limiting examples of transmembrane domains for use in embodiments of the present disclosure include: Non-limiting examples of transmembrane domains and cytoplasmic tails for use in embodiments of the present disclosure include: MuV F transmembrane domain and cytoplasmic tail, such as TIFF0007801221000020.tif4137.

[0112] Native MuV F proteins from different MuV strains are known, along with nucleic acid sequences and methods encoding such proteins, which can be modified using the instructions provided herein to generate recombinant MuV F ectodomain trimers.

[0113] The recombinant MuV F ectodomain trimer can be derivatized or linked to another molecule (e.g., another peptide or protein). Generally, the recombinant MuV F ectodomain is derivatized so that binding of the recombinant MuV F protein trimer to a neutralizing antibody is not adversely affected by the derivatization or labeling. For example, the recombinant MuV F ectodomain can be functionally linked (by chemical coupling, genetic fusion, noncovalent association, etc.) to one or more other molecular entities, such as a carrier protein, an antibody, a heterologous protein, or a detection tag.

[0114] In some embodiments, the recombinant MuV F ectodomain trimer comprises one or more MuV HN ectodomains, e.g. It is fused to the ectodomain head of the MuV HN sequence shown as TIFF0007801221000021.tif33160.

[0115] In some embodiments, the recombinant MuV F ectodomain trimer is fused to one or more MuV HN ectodomains, e.g., the ectodomain stalk and ectodomain head of any one of MuV HN positions 54-130 through MuV HN position 582 (e.g., MuV HN positions 54-63 through MuV HN position 582, e.g., positions 54-582, 61-582, 63-582, or 55-582). In some embodiments, the recombinant MuV F ectodomain trimer is fused to one or more MuV HN ectodomains, e.g., residues 22-550 of SEQ ID NO:90, residues 22-543 of SEQ ID NO:91, residues 22-541 of SEQ ID NO:92, or residues 22-549 of SEQ ID NO:93.

[0116] For example, each of the recombinant MuV F ectodomain trimer protomers is fused to a MuV HN ectodomain. Fusion can be direct or via a peptide linker. In some embodiments, the MuV HN ectodomain can be fused directly or indirectly via a peptide linker to the C-terminus of the MuV F ectodomain trimer protomers. In some such embodiments, the MuV HN ectodomain can be fused directly or indirectly via a peptide linker to the C-terminus of a trimerization domain (e.g., a GCN4 or T4 fibritin trimer domain) fused to the C-terminus of the MuV F ectodomain trimer protomers. In some such embodiments, the MuV F ectodomain trimer promoter linked to the trimerization domain and the MuV HN ectodomain comprises the amino acid sequence set forth as residues 20-966 of SEQ ID NO:27, or an amino acid sequence at least 90% identical to residues 20-966 of SEQ ID NO:27.

[0117] In some embodiments, the recombinant MuV F ectodomain trimer comprises one or more MeV H ectodomains, e.g. It is fused to the ectodomain head of the H sequence shown as TIFF0007801221000022.tif56160.

[0118] In some embodiments, the recombinant MuV F ectodomain trimers are fused to one or more MeV H ectodomains, e.g., the ectodomain stalk and ectodomain head of any one of MeV H positions 59-179 through MeV H position 617 (e.g., any one of MeV H positions 59-67 through MeV H position 617, e.g., positions 59-617, 62-617, 60-617, or 67-617). In some embodiments, the recombinant MuV F ectodomain trimers are fused to one or more MeV H ectodomains, e.g., the ectodomain stalk and ectodomain head of the sequence set forth as residues 22-580 of SEQ ID NO:86, residues 22-577 of SEQ ID NO:87, residues 22-579 of SEQ ID NO:88, or residues 22-572 of SEQ ID NO:89.

[0119] For example, each of the recombinant MuV F ectodomain trimer protomers stabilized in a prefusion conformation is fused to a MeV H ectodomain. Fusion can be direct or via a peptide linker. In some embodiments, the MeV H ectodomain can be fused directly or indirectly via a peptide linker to the C-terminus of the prefusion MuV F ectodomain trimer protomers. In some such embodiments, the MeV H ectodomain can be fused directly or indirectly via a peptide linker to the C-terminus of a trimerization domain (e.g., a GCN4 or T4 fibritin trimer domain) fused to the C-terminus of the MuV F ectodomain trimer protomers. In some such embodiments, the MuV F ectodomain trimer promoter linked to the trimerization domain and the MeV H ectodomain comprises the amino acid sequence set forth as residues 21-981 of SEQ ID NO:28 or residues 20-1006 of SEQ ID NO:29, or comprises an amino acid sequence at least 90% identical to residues 21-981 of SEQ ID NO:28 or residues 20-1006 of SEQ ID NO:29.

[0120] Non-limiting examples of sequences containing the MuV F ectodomain with amino acid substitutions for stabilization in the pre-fusion conformation are listed below. TIFF0007801221000023.tif232160TIFF0007801221000024.tif232160TIFF0007801221000025.tif200160

[0121] The above sequences contain an N-terminal signal peptide, a MuV F ectodomain, and a GCN4 trimerization domain. It will be understood that alternative trimerization domains, such as the T4 fibritin trimerization domain, can be used. Additionally, many of the above sequences contain a GGG linker to remove the native furin cleavage site separating the F1 and F2 subunits. Alternative glycine linkers, such as GSG, GGS, or SGG, can also be used. Additionally, a native furin cleavage site can be included in any of the sequences in place of the GGG linker. It will be understood that the N-terminal signal peptide is removed during intracellular processing and is not present in the purified protein. Additionally, the MuV F ectodomain of any of the above sequences can be included in a full-length MuV F protein to provide a membrane-anchored pre-fusion MuV F protein, for example, for mRNA immunization.

[0122] Non-limiting examples of sequences containing the MuV F ectodomain with amino acid substitutions for stabilization in the pre-fusion conformation linked to the MuV HN ectodomain or the MeV H ectodomain are listed below. TIFF0007801221000026.tif167160

[0123] The above sequences contain an N-terminal signal peptide and MuV F ectodomain in combination with various other elements, such as a GCN4 trimerization domain, a T4 fibritin trimerization domain, a peptide cleavage site (e.g., thrombin), a HIS tag, a Strep tag, and various linker residues between segments. Purified forms of these proteins typically lack the N-terminal signal peptide and C-terminal residues that are removed by peptide cleavage.

[0124] B. Recombinant MeV F ectodomain trimer Disclosed herein are recombinant MeV F ectodomain trimers that have been modified from their native form (e.g., by introducing one or more amino acid substitutions) to stabilize them in the pre-fusion conformation. As described in the Examples, embodiments of the disclosed MeV F ectodomain trimers were selected through multiple rounds of structure-based design for optimized solubility, stability, expression, and immunogenicity. Recombinant MeV F ectodomain trimers are useful for inducing immune responses against MeV in vertebrates (such as humans). Exemplary embodiments are shown to generate superior immune responses in animal models compared to corresponding MeV F ectodomain trimers that are not stabilized in the pre-fusion conformation.

[0125] In some embodiments, the immunogen comprises a recombinant MeV F ectodomain trimer comprising a protomer containing one or more amino acid substitutions or deletions that stabilize the MeV F ectodomain trimer in the pre-fusion conformation.

[0126] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 48 and 284 (e.g., R48C and A284C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0127] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 90 and 225 (e.g., A90C and I225C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0128] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 141 and 270 (e.g., M141C and T270C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0129] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 165 and 171 (e.g., R165C and M171C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0130] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 173 and 245 (e.g., L173C and V245C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0131] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 175 and 241 (e.g., V175C and D241C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0132] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 212 and 236 (e.g., E212C and Y236C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0133] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 216 and 233 (e.g., L216C and A233C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0134] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 219 and 224 (e.g., P219C and P224C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0135] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 99 and 117 (e.g., R99C and V117C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0136] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 100 and 117 (e.g., P100C and V117C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0137] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 101 and 117 (e.g., V101C and V117C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0138] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 102 and 117 (e.g., Q102C and V117C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0139] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 103 and 117 (e.g., S103C and V117C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0140] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 165 and 171 (e.g., R165C and M171C substitutions) and positions 141 and 270 (e.g., M141C and T270C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0141] In some embodiments, the promoters of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 165 and 171 (e.g., R165C and M171C substitutions) and positions 212 and 236 (e.g., E212C and Y236C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0142] In some embodiments, the protomers of the recombinant MeV F ectodomain trimer contain cysteine ​​substitutions at MeV F positions 165 and 171 (e.g., R165C and M171C substitutions) and positions 48 and 284 (e.g., R48C and A284C substitutions) that form non-native intraprotomer disulfide bonds for stabilization in the prefusion conformation.

[0143] In some embodiments, the recombinant MeV F ectodomain trimer protomer contains a phenylalanine at MeV F position 175 (e.g., a V175F substitution) for stabilization in the pre-fusion conformation. This phenylalanine substitution can be combined with any of the disclosed cysteine ​​substitutions or a proline substitution at MeV F position 194 to stabilize the recombinant MeV F ectodomain trimer in the pre-fusion conformation.

[0144] In some embodiments, the recombinant MeV F ectodomain trimer promoter contains a proline substitution (e.g., an S194P substitution) at MeV F position 194 for stabilization in the pre-fusion conformation. This proline substitution can be combined with any of the cysteine ​​substitutions disclosed herein for stabilizing the recombinant MeV F ectodomain trimer in the pre-fusion conformation.

[0145] Any of the above recombinant MeV F proteins can further include modifications to eliminate the protease cleavage site between the F1 and F2 polypeptides to generate a "single-chain" recombinant F protein. For example, any of the above recombinant MeV F proteins can include a deletion of MeV F positions 111-113 and positions 110 and 114 fused with a peptide linker. This modification removes the F2 / F1 furin cleavage site and also removes the first residue of the fusion peptide (which is hydrophobic). Any suitable peptide linker that fuses the F2 and F1 ectodomains and allows folding of the F ectodomain into a pre-fusion conformation can be used. In some embodiments, the peptide linker is a glycine, serine, or glycine-serine peptide linker. In some embodiments, the peptide linker is a Gly-Gly-Gly linker.

[0146] In one non-limiting example, a recombinant MeV F ectodomain trimer is provided that includes a promoter having R165C and M171C substitutions to form non-native disulfide bonds and a deletion of MeV F positions 111-113 with positions 110 and 114 fused with a Gly-Gly-Gly peptide linker.

[0147] In some embodiments, the recombinant MeV F ectodomain protomer can include one or more additional amino acid substitutions to enhance stabilization of the pre-fusion conformation or for other purposes, such as to increase solubility or reduce unwanted immune responses.

[0148] The non-native disulfide bonds listed above stabilize the membrane-distal portion of the MeV F ectodomain in the pre-fusion conformation. Any of these mutations can be combined with modifications of the membrane-proximal portion (e.g., stem) of the MeV F ectodomain, for example, to enhance ectodomain trimerization.

[0149] In some embodiments, the N-terminal position of the recombinant F2 polypeptide in the protomer can be one of MeV F positions 24-34 (e.g., position 24), and the C-terminal position of the F1 ectodomain can be a position from the stem region of the ectodomain, e.g., one of MeV F positions 472-486 (e.g., position 486).

[0150] In one non-limiting example, a recombinant MeV F ectodomain trimer is provided, comprising a protomer comprising MeV positions 24-486 with R165C and M171C substitutions to form non-native disulfide bonds and a deletion of MeV F positions 111-113 with positions 110 and 114 fused with a Gly-Gly-Gly peptide linker.

[0151] Non-limiting examples of MeV F ectodomain trimer protomers containing amino acid substitutions for stabilization in the pre-fusion conformation are provided herein. In some embodiments, the MeV F ectodomain trimer protomers contain an amino acid sequence at least 90% identical to residues 21-483 of any one of SEQ ID NOs: 37-43 or 53-55, wherein the protomers contain one or more amino acid substitutions that stabilize the MeV F ectodomain trimer in the pre-fusion conformation. In some embodiments, the MeV F ectodomain trimer protomers contain residues 21-483 of any one of SEQ ID NOs: 37-43 or 53-55.

[0152] In some embodiments, the recombinant MeV F ectodomain trimer is a soluble protein complex for use, for example, as a recombinant subunit vaccine. In some such embodiments, the protomers of the recombinant MeV F ectodomain trimer can each comprise a C-terminal linkage to a trimerization domain, such as a GCN4 trimerization domain or a T4 fibritin trimer domain, or both. The trimerization domain promotes trimerization and stabilization of the membrane-proximal aspect of the recombinant MeV F ectodomain trimer. For example, the C-terminal residue of the recombinant MeV F ectodomain trimer protomer (e.g., a residue in the stem region of the trimer) can be directly linked to the trimerization domain or indirectly linked to the trimerization domain via a peptide linker. Exemplary linkers include glycine and glycine-serine linkers. Non-limiting examples of exogenous multimerization domains that promote stable trimers of soluble recombinant proteins include the GCN4 leucine zipper, the T4 fibritin trimerization domain, and trimerization motifs derived from pulmonary surfactant proteins (Hoppe et al. 1994 FEBS Lett 344:191-195) or collagen (McAlinden et al. 2003 J Biol Chem 278:42200-42207), all of which can be linked to the C-terminus of recombinant MeV F ectodomain promoters to promote trimerization, as long as the recombinant MeV F ectodomain trimer maintains its pre-fusion conformation. In some examples, the protomers of the recombinant MeV F ectodomain trimer can be linked to a MeV trimerization domain, e.g., each protomer in the trimer can include a C-terminal linkage to a GCN4 trimerization domain, e.g., a linkage to any one of MeV F positions 472-486, e.g., MeV F position 486. In a specific example, the GCN4 trimerization domain has the amino acid sequence In a specific example, the T4 fibritin trimerization domain comprises or consists of the amino acid sequence TIFF0007801221000027.tif4128. TIFF0007801221000028.tif4128. In a specific example, the GCN4 trimerization domain fused to the fibritin trimerization domain comprises or consists of the amino acid sequence It contains or consists of the same amino acid sequence as TIFF0007801221000029.tif4156.

[0153] In one non-limiting example, a recombinant MeV F ectodomain trimer is provided, comprising a protomer comprising MeV positions 24-486 with R165C and M171C substitutions to form a non-native disulfide bond, a deletion of MeV F positions 111-113 with positions 110 and 114 fused with a Gly-Gly-Gly peptide linker, and a GCN4 trimerization domain linked to the C-terminus of the protomer ectodomain.

[0154] Non-limiting examples of MeV F ectodomain trimer protomers comprising amino acid substitutions for stabilization in the pre-fusion conformation and a C-terminal linkage to a trimerization domain are provided herein. In some embodiments, the MeV F ectodomain trimer protomers comprise an amino acid sequence at least 90% identical to residues 21-513 of any one of SEQ ID NOs: 37-43 or 53-55, wherein the protomers comprise one or more amino acid substitutions that stabilize the MeV F ectodomain trimer in the pre-fusion conformation. In some embodiments, the MeV F ectodomain trimer protomers comprise residues 21-513 of any one of SEQ ID NOs: 37-43 or 53-55.

[0155] In some embodiments, the recombinant MeV F ectodomain trimer can be a membrane-anchored protein complex, e.g., for use in an attenuated virus vaccine or a virus-like particle vaccine. Membrane anchoring can be achieved, e.g., by C-terminal linkage of the recombinant MeV F ectodomain trimer protomer to the transmembrane domain and optionally the cytoplasmic tail, e.g., to the MeV F transmembrane domain and cytoplasmic tail. In some embodiments, one or more peptide linkers (e.g., gly-ser linkers, e.g., a 10 amino acid glycine-serine peptide linker) can be used to link the recombinant MeV F ectodomain trimer protomer to the transmembrane domain. Non-limiting examples of transmembrane domains for use in embodiments of the present disclosure include: Non-limiting examples of transmembrane domains for use in embodiments of the present disclosure include: Examples include the MeV F transmembrane domain, such as TIFF0007801221000031.tif4143.

[0156] Native MeV F proteins from different MeV strains are known, along with nucleic acid sequences and methods encoding such proteins, which can be modified using the instructions provided herein to generate recombinant MeV F ectodomain trimers.

[0157] The recombinant MeV F ectodomain trimer can be derivatized or linked to another molecule (e.g., another peptide or protein). Generally, the recombinant MeV F ectodomain is derivatized so that binding of the recombinant MeV F protein to a cross-neutralizing antibody to the trimer is not adversely affected by the derivatization or labeling. For example, the recombinant MeV F ectodomain can be functionally linked (by chemical coupling, genetic fusion, non-covalent association, etc.) to one or more other molecular entities, such as a carrier protein, an antibody, a heterologous protein, or a detection tag.

[0158] In some embodiments, the recombinant MeV F ectodomain trimer comprises one or more MuV HN ectodomains, e.g. It is fused to the ectodomain of the MuV HN sequence shown as TIFF0007801221000032.tif27160.

[0159] For example, each of the recombinant MeV F ectodomain trimer protomers is fused to a MuV HN ectodomain. Fusion can be direct or via a peptide linker. In some embodiments, the MuV HN ectodomain can be fused directly or indirectly via a peptide linker to the C-terminus of the MeV F ectodomain trimer protomers. In some such embodiments, the MuV HN ectodomain can be fused directly or indirectly via a peptide linker to the C-terminus of a trimerization domain (e.g., a GCN4 or T4 fibritin trimer domain) fused to the C-terminus of the MeV F ectodomain trimer protomers. In some such embodiments, the MeV F ectodomain trimer protomers linked to the trimerization domain and MuV HN ectodomain comprise the amino acid sequence set forth as residues 20-966 of SEQ ID NO:27, or an amino acid sequence at least 90% identical to residues 20-966 of SEQ ID NO:27.

[0160] In some embodiments, the recombinant MeV F ectodomain trimer comprises one or more MeV H ectodomains, e.g. It is fused to the ectodomain of the H sequence shown as TIFF0007801221000033.tif56160.

[0161] For example, each of the recombinant MeV F ectodomain trimer protomers is fused to an MeV H ectodomain. Fusion can be direct or via a peptide linker. In some embodiments, the MeV H ectodomain can be fused directly or indirectly via a peptide linker to the C-terminus of the MeV F ectodomain trimer protomers. In some such embodiments, the MeV H ectodomain can be fused directly or indirectly via a peptide linker to the C-terminus of a trimerization domain (e.g., a GCN4 or T4 fibritin trimer domain) fused to the C-terminus of the MeV F ectodomain trimer protomers. In some such embodiments, the MeV F ectodomain trimer protomers linked to the trimerization domain and MeV H ectodomain comprise the amino acid sequence set forth as residues 21-959 of SEQ ID NO:56 or positions 21-973 of SEQ ID NO:57, or an amino acid sequence at least 90% identical thereto.

[0162] Non-limiting examples of sequences containing the MeV F ectodomain with amino acid substitutions for stabilization in the pre-fusion conformation are listed below. TIFF0007801221000034.tif49160TIFF0007801221000035.tif232160TIFF00078012210 00036.tif232160TIFF0007801221000037.tif228160TIFF0007801221000038.tif232160

[0163] The above sequences contain an N-terminal signal peptide, a MeV F ectodomain, and a GCN4 trimerization domain. It will be understood that alternative trimerization domains, such as the T4 fibritin trimerization domain, can be used. Additionally, many of the above sequences contain a GGG linker to remove the native furin cleavage site separating the F1 and F2 subunits. Alternative glycine linkers, such as GSG, GGS, or SGG, can also be used. Additionally, a native furin cleavage site can be included in any of the sequences in place of the GGG linker. It will be understood that the N-terminal signal peptide is removed during intracellular processing and is not present in the purified protein. Additionally, the MeV F ectodomain of any of the above sequences can be included in a full-length MeV F protein to provide a membrane-anchored pre-fusion MeV F protein, for example, for mRNA immunization.

[0164] Non-limiting examples of sequences containing the MeV F ectodomain with amino acid substitutions for stabilization in the pre-fusion conformation linked to the MuV HN ectodomain or the MeV H ectodomain are listed below. TIFF0007801221000039.tif165160

[0165] The above sequences comprise an N-terminal signal peptide, MeV F ectodomain, trimerization domain (GCN4 and / or T4 fibritin), and MeV H ectodomain head region or MuV HN ectodomain head region. The MeV F ectodomain sequence includes a GGG linker to remove the native furin cleavage site separating the F1 and F2 subunits. Alternative glycine linkers, such as GSG, GGS, or SGG, can also be used. Additionally, either sequence can include a native furin cleavage site instead of the GGG linker. It will be understood that the N-terminal signal peptide is removed during intracellular processing and will not be present in the purified protein.

[0166] C. MuV HN and MuV H multimers In some embodiments, immunogens are provided that comprise multimers of the MuV HN ectodomain and / or MeV H ectodomain. The H ectodomain or HN ectodomain can comprise the ectodomain head, or can comprise the stalk and head of the ectodomain.

[0167] In some embodiments, the immunogen comprises a trimer of fusion proteins, each fusion protein comprising one or more MuV HN ectodomains or MeV H ectodomains and a trimerization domain (e.g., a GCN4 trimerization domain, a T4 fibritin trimerization domain, or a GCN4 trimerization domain fused to a T4 fibritin trimerization domain).

[0168] In some embodiments, the fusion protein comprises, from N- to C-terminus, a trimerization domain (e.g., a GCN4 trimerization domain, a T4 fibritin trimerization domain, or a GCN4 trimerization domain fused to a T4 fibritin trimerization domain) and one or more (e.g., one, two, or three) MuV HN ectodomains or MeV H ectodomains. The trimerization domains interact to form trimers. In some embodiments, a fragment of an ectodomain is included, e.g., the head region of the MuV HN ectodomain or the head region of the MeV H ectodomain can be fused to the trimerization domain, optionally via a peptide linker. In some embodiments, the fusion protein in the trimer comprises or consists of the amino acid sequence set forth as residues 24-510 of SEQ ID NO:58 or residues 24-496 of SEQ ID NO:59, or a sequence at least 90% identical to any one of residues 25-510 of SEQ ID NO:58 or residues 25-496 of SEQ ID NO:59.

[0169] In some embodiments, the fusion protein comprises, from N- to C-terminus, one or more (e.g., one, two, or three) MuV HN ectodomains or Mev H ectodomains, a trimerization domain (e.g., a GCN4 trimerization domain, a T4 fibritin trimerization domain, or a GCN4 trimerization domain fused to a T4 fibritin trimerization domain), and one or more (e.g., one, two, or three) MuV HN ectodomains or Mev H ectodomains. The trimerization domains interact to form trimers. In some embodiments, a fragment of an ectodomain is included, e.g., the head region of the MuV HN ectodomain or the head region of the MeV H ectodomain can be fused to the trimerization domain, optionally via a peptide linker. In some embodiments, the fusion protein in the trimer comprises or consists of an amino acid sequence set forth as residues 22-950 of SEQ ID NO:82, residues 25-985 of SEQ ID NO:83, residues 22-948 of SEQ ID NO:84, or residues 22-981 of SEQ ID NO:85, or a sequence at least 90% identical to any one of residues 22-950 of SEQ ID NO:82, residues 25-985 of SEQ ID NO:83, residues 22-948 of SEQ ID NO:84, or residues 22-981 of SEQ ID NO:85.

[0170] In some embodiments, the multimer is a dimer of the MeV H ectodomain head region. The MeV H ectodomain head region can be expressed in mammalian cells and spontaneously form dimers in physiological solution. The dimer can then be purified and used as an immunogen. In some embodiments, the subunits of the dimer comprise or consist of the amino acid sequence set forth as residues 22-459 of SEQ ID NO:60, or a sequence at least 90% identical to residues 22-459 of SEQ ID NO:60.

[0171] In some embodiments, the multimer is a dimer of the stalk and head regions of the MeV H ectodomain. The stalk and head regions of the MeV H ectodomain can be expressed in mammalian cells and spontaneously form dimers in physiological solution. The dimer can then be purified and used as an immunogen. In some embodiments, the subunits of the dimer comprise or consist of the amino acid sequence from any one of MeV H positions 59-197 to MeV H position 617 (e.g., from any one of MeV H positions 59-67 to MeV H position 617, e.g., positions 59-617, 62-617, 60-617, or 67-617). In some embodiments, a subunit of the dimer comprises or consists of an amino acid sequence set forth as residues 22-580 of SEQ ID NO:86, residues 22-577 of SEQ ID NO:87, residues 22-579 of SEQ ID NO:88, or residues 22-572 of SEQ ID NO:89, or a sequence at least 90% identical to residues 22-580 of SEQ ID NO:86, residues 22-577 of SEQ ID NO:87, residues 22-579 of SEQ ID NO:88, or residues 22-572 of SEQ ID NO:89.

[0172] In some embodiments, the multimer is a dimer of MuV HN ectodomain head regions. In some embodiments, the subunits of the dimer comprise or consist of the amino acid sequence set forth as SEQ ID NO:30, or a sequence at least 90% identical to SEQ ID NO:30.

[0173] In some embodiments, the multimer is a dimer of the stalk and head regions of the MuV H ectodomain. In some embodiments, the subunits of the dimer comprise or consist of the amino acid sequence from any one of MuV H positions 54-130 to MuV HN position 582 (e.g., from any one of MuV H positions 54-63 to MuV HN position 582, e.g., positions 54-582, 61-582, 63-582, or 55-582). In some embodiments, the subunits of the dimer comprise or consist of the amino acid sequence set forth as residues 22-550 of SEQ ID NO:90, residues 22-543 of SEQ ID NO:91, residues 22-541 of SEQ ID NO:92, or residues 22-549 of SEQ ID NO:93, or a sequence at least 90% identical to residues 22-550 of SEQ ID NO:90, residues 22-543 of SEQ ID NO:91, residues 22-541 of SEQ ID NO:92, or residues 22-549 of SEQ ID NO:93.

[0174] D. Additional Explanation Protomers in a recombinant MuV or MeV F ectodomain trimer can include modifications of the native MuV F or MeV F sequence, such as amino acid substitutions, deletions, or insertions, glycosylation, and / or covalent linkage to unrelated proteins (e.g., protein tags), in addition to those described above, so long as the recombinant MuV or MeV F ectodomain trimer remains stabilized in the pre-fusion conformation and retains immunogenicity. Furthermore, in embodiments comprising heterologous MuV HN ectodomains or MeV H ectodomains, or multimers of MuV HN ectodomains or MeV H ectodomains, the HN or H ectodomains can include modifications of the native HN or H sequence, such as amino acid substitutions, deletions, or insertions, glycosylation, and / or covalent linkage to unrelated proteins (e.g., protein tags), so long as the HN or H ectodomain retains immunogenicity. These variations in the sequence can be natural mutations or can be engineered using genetic engineering techniques known to those of skill in the art. Examples of such techniques can be found, for example, in 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 & Sons, New York, supplement 104, 2013), both of which are incorporated herein by reference in their entireties.

[0175] In some embodiments, the protomers in the recombinant MuV F ectodomain trimer comprise one or more amino acid substitutions compared to the corresponding native MuV F sequence. For example, in some embodiments, the F2 polypeptide, the F1 ectodomain, or both, can comprise up to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the native MuV F sequence.

[0176] In some embodiments, the protomers in the recombinant MeV F ectodomain trimer comprise one or more amino acid substitutions compared to the corresponding native MeV F sequence. For example, in some embodiments, the F2 polypeptide, the F1 ectodomain, or both, can comprise up to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the native MeV F sequence.

[0177] In some embodiments, the MuV HN ectodomain comprises one or more amino acid substitutions compared to the corresponding native MuV HN ectodomain sequence, e.g., in some embodiments, the MuV HN ectodomain comprises up to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the native MuV HN ectodomain sequence.

[0178] In some embodiments, the MeV H ectodomain comprises one or more amino acid substitutions compared to the corresponding native MeV H ectodomain sequence, e.g., in some embodiments, the MeV H ectodomain comprises up to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) amino acid substitutions (e.g., conservative amino acid substitutions) compared to the native MeV H ectodomain sequence.

[0179] The simplest modifications involve the substitution of one or more amino acids with amino acids having similar biochemical properties, e.g., conservative amino acid substitutions. Such substitutions are likely to have minimal effect on the activity of the resulting protein.

[0180] In some embodiments, the recombinant MeV F ectodomain trimer or the protomers in the MuV F ectodomain trimer can be joined at one end to other unrelated sequences (e.g., non-MuV F or MeV F protein sequences, non-viral envelope, or non-viral protein sequences).

[0181] In some embodiments, the recombinant MuV F ectodomain trimer or MeV F ectodomain trimer, or fusions of these protomers with heterologous proteins, such as MuV HN ectodomain or MeV H ectodomain, are soluble in aqueous solution. In some embodiments, the recombinant MuV F ectodomain trimer, MeV F ectodomain trimer, or corresponding fusion with a heterologous protein, e.g., the MuV HN ectodomain or the MeV H ectodomain, dissolves in an aqueous solution (e.g., phosphate buffered saline (pH 7.4) or 350 mM NaCl (pH 7.0)) at room temperature (e.g., 20-22 degrees Celsius) to a concentration of at least 0.5 mg / ml (e.g., at least 1.0 mg / ml, 1.5 mg / ml, 2.0 mg / ml, 3.0 mg / ml, 4.0 mg / ml, or at least 5.0 mg / ml) and remains dissolved for at least 12 hours (e.g., at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 1 month, or longer). In one embodiment, the phosphate buffered saline solution contains NaCl (137 mM), KCl (2.7 mM), NaHPO (10 mM), KHPO (1.8 mM) at pH 7.4. In some embodiments, the phosphate buffered saline solution further contains CaCl (1 mM) and MgCl (0.5 mM). Those skilled in the art are familiar with methods for determining whether a protein remains dissolved over time. For example, standard methods can be used to test the concentration of a protein dissolved in an aqueous solution over time.

[0182] In some embodiments, the immunogen is provided as a homogeneous population of soluble trimers that are substantially in the pre-fusion conformation, with limited or no MuV F ectodomain trimers and / or MeV F ectodomain trimers in the post-fusion conformation. The conformation of the MeV F ectodomain trimers or MuV F ectodomain trimers can be detected, for example, by negative staining electron microscopy and / or specific binding with a pre-fusion or post-fusion specific antibody. In some embodiments, at least about 95% of the recombinant MuV F ectodomain trimers or MeV F ectodomain trimers in the homogeneous population (e.g., at least about 95%, 96%, 97%, 98%, 99%, or 99.9% of the MuV or MeV F proteins) are stabilized in the pre-fusion conformation.

[0183] In some embodiments, the recombinant MuV F ectodomain trimer or MeV F ectodomain trimer retains specific binding to a prefusion-specific antibody after incubation in phosphate buffered saline at 50° C. for 1 hour. In some embodiments, the recombinant MuV F ectodomain trimer or MeV F ectodomain trimer retains specific binding to a prefusion-specific antibody after incubation in phosphate buffered saline at 4° C. for 6 months.

[0184] In certain embodiments, the immunogens provided herein may be further modified to contain additional nonprotein moieties that are known in the art and readily available. Moieties suitable for derivatizing immunogens include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and when two or more polymers are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular properties or functions of the immunogen to be improved or modified, whether the immunogen derivative will be used therapeutically under certain conditions, etc.

[0185] Some of the sequences comprising the recombinant MuV F ectodomain trimer or MeV F ectodomain trimer or MuV HN ectodomain or MeV H ectodomain provided herein contain sequences for protease cleavage sites (e.g., thrombin sites), protein tags (e.g., His tags, Strep tag II, Avi tags, etc.), and signal peptides, and such sequences can be removed from isolated immunogens comprising the recombinant MuV F ectodomain trimer or MeV F ectodomain trimer or MuV HN ectodomain or MeV H ectodomain for therapeutic use.

[0186] E. Protein Nanoparticles In some embodiments, protein nanoparticles are provided that comprise one or more of the recombinant MuV F ectodomain trimers or recombinant MeV F ectodomain trimers of the present disclosure, or MuV HN or MeV H multimers, or chimeras thereof.

[0187] In some embodiments, the protein nanoparticles comprise MeV F ectodomain trimers or MuV F ectodomain trimers displayed on the two-component self-assembling nanoparticle platform described in Marcandalli et al., "Induction of potent neutralizing antibody responses by a designed protein nanoparticle vaccine for respiratory syncytial virus," Cell, 176(6):1420-1431, 2019, which is incorporated herein by reference.

[0188] Non-limiting examples of nanoparticles include ferritin nanoparticles, encapsulin nanoparticles, sulfur oxidase / reductase (SOR) nanoparticles, and lumazine synthase nanoparticles, which are composed of assemblies of monomer subunits containing ferritin protein, encapsulin protein, SOR protein, and lumazine synthase, respectively. To construct such protein nanoparticles, a protomer of a recombinant MuV F ectodomain trimer or a recombinant MeV F ectodomain trimer, or a subunit of a MuV HN or MeV H multimer, is linked to a subunit of the protein nanoparticle (e.g., ferritin protein, encapsulin protein, SOR protein, or lumazine synthase protein) and expressed in cells under appropriate conditions. The fusion protein self-assembles into nanoparticles and can be purified.

[0189] In some embodiments, ferritin nanoparticles can be constructed by linking the recombinant MuV F ectodomain trimer or recombinant MeV F ectodomain trimer protomers, or subunits of MuV HN or MeV H multimers, of the present disclosure, to ferritin subunits. Ferritin nanoparticles and their use for immunization (e.g., immunization against influenza antigens) have been described in the art (see, e.g., Kanekiyo et al., Nature, 499:102-106, 2013, incorporated herein by reference in its entirety). Spherical ferritin nanoparticles are made of monomeric subunits, which are polypeptides with molecular weights of approximately 17-20 kDa. These monomeric subunit proteins self-assemble into spherical ferritin proteins after production. Thus, spherical ferritin contains 24 monomeric subunit proteins and has a capsid-like structure with 432 symmetry. Methods for constructing ferritin nanoparticles are further described herein (see, e.g., Zhang, Int. J. Mol. Sci., 12:5406-5421, 2011, which is incorporated by reference in its entirety). An example of one amino acid sequence of such a monomeric ferritin subunit is: Represented by TIFF0007801221000040.tif11158.

[0190] In specific examples, the ferritin polypeptide is E. coli ferritin, Helicobacter pylori ferritin, human light chain ferritin, bullfrog ferritin, or a hybrid thereof, such as E. coli-human hybrid ferritin, E. coli-bullfrog hybrid ferritin, or human-bullfrog hybrid ferritin. Exemplary amino acid sequences of ferritin polypeptides and nucleic acid sequences encoding ferritin polypeptides for use in making ferritin nanoparticles comprising recombinant MuV or MeV F ectodomain trimers can be found in GENBANK®, e.g., under Accession Nos. ZP_03085328, ZP_06990637, EJB64322.1, AAA35832, NP_000137 AAA49532, AAA49525, AAA49524, and AAA49523, each of which sequences available as of April 10, 2015, are specifically incorporated herein by reference in their entirety. In some embodiments, a recombinant MuV or MeV F ectodomain trimer protomer can be linked to a ferritin subunit comprising an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 97%) identical to the amino acid sequence set forth as SEQ ID NO:45.

[0191] In some embodiments, lumazine synthase nanoparticles can be constructed by linking the recombinant MuV F ectodomain trimer or recombinant MuV or MeV F ectodomain trimer protomers, or subunits of MuV HN or MeV H multimers of the present disclosure to lumazine synthase subunits. Spherical lumazine synthase nanoparticles are made of monomeric subunits, and an example sequence of one such lumazine synthase subunit is: It is provided as an amino acid sequence shown as TIFF0007801221000041.tif11158.

[0192] In some embodiments, a protomer of a recombinant MuV F ectodomain trimer or a recombinant MeV F ectodomain trimer, or a subunit of a MuV HN or MeV H multimer of the present disclosure can be linked to a lumazine synthase subunit comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 97%) identical to the amino acid sequence set forth as SEQ ID NO:46.

[0193] In some embodiments, encapsulin nanoparticles can be constructed by linking the recombinant MuV F ectodomain trimer or recombinant MeV F ectodomain trimer protomers of the present disclosure, or subunits of MuV HN or MeV H multimers, to encapsulin nanoparticle subunits. Spherical encapsulin nanoparticles are made of monomeric subunits, and an example of one sequence of such an encapsulin nanoparticle subunit is: It is provided as an amino acid sequence shown as TIFF0007801221000042.tif15158.

[0194] In some embodiments, a protomer of a recombinant MuV F ectodomain trimer or a recombinant MeV F ectodomain trimer, or a subunit of a MuV HN or MeV H multimer of the present disclosure can be linked to an encapsulin subunit comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 97%) identical to the amino acid sequence set forth as SEQ ID NO:47.

[0195] Encapsulin proteins, also known as linocin-like proteins, are a conserved family of bacterial proteins that form large protein assemblies that serve as minimal compartments for packaging enzymes. Encapsulin assemblies are made of monomeric subunits, which are polypeptides with molecular weights of approximately 30 kDa. After production, these monomeric subunits self-assemble into spherical encapsulin assemblies containing 60, or in some cases 180, monomeric subunits. Methods for constructing encapsulin nanoparticles have been further described (see, e.g., Sutter et al., Nature Struct. and Mol. Biol., 15:939-947, 2008, incorporated herein by reference in its entirety). In specific examples, the encapsulin polypeptide is a bacterial encapsulin, such as an encapsulin from Thermotoga maritime or Pyrococcus furiosus or Rhodococcus erythropolis or Myxococcus xanthus.

[0196] In some embodiments, recombinant MuV F ectodomain trimer or recombinant MeV F ectodomain trimer protomers, or subunits of MuV HN or MeV H multimers of the present disclosure can be linked to sulfur oxidase / reductase (SOR) subunits to construct recombinant SOR nanoparticles. It may contain the amino acid sequence shown as TIFF0007801221000043.tif15158.

[0197] In some embodiments, a protomer of a recombinant MuV F ectodomain trimer or a recombinant MeV F ectodomain trimer, or a subunit of a MuV HN or MeV H multimer of the present disclosure can be linked to a SOR subunit comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 97%) identical to the amino acid sequence set forth as SEQ ID NO:48.

[0198] The SOR protein is a microbial protein (e.g., derived from the thermoacidophilic archaeon Acidianus ambivalens) that forms a protein assembly of 24 subunits. Methods for constructing SOR nanoparticles are described in Urich et al., Science, 311:996-1000, 2006, which is incorporated herein by reference in its entirety. An example of the amino acid sequence of the SOR protein for use in making SOR nanoparticles is provided in Urich et al., Science, 311:996-1000, 2006, which is incorporated herein by reference in its entirety.

[0199] For production purposes, the recombinant MuV F ectodomain or MeV F ectodomain, or the subunit of a MuV HN or MeV H multimer linked to the nanoparticle subunit can include an N-terminal signal peptide that is cleaved during intracellular processing. For example, the recombinant MuV F ectodomain protomer or MeV F ectodomain protomer linked to the protein nanoparticle subunit can include a signal peptide at its N-terminus, including, for example, the native MuV or MeV F signal peptide.

[0200] Protein nanoparticles can be expressed in suitable cells (e.g., HEK293 Freestyle cells), and the fusion protein can be secreted from the cells in a self-assembled form. The nanoparticles can be purified using known techniques, for example, by several different chromatographic methods, such as Mono Q (anion exchange) followed by size exclusion (SUPEROSE® 6) chromatography.

[0201] The fusion protein need not contain the entire sequence of a monomeric subunit polypeptide of a ferritin, encapsulin, SOR, or lumazine synthase protein: any portion or region of the monomeric subunit polypeptide can be used, so long as the portion contains an amino acid sequence that directs the self-assembly of the monomeric subunits into a globular protein.

[0202] II. Polynucleotides and Expression Polynucleotides encoding any of the immunogens of the present disclosure are also provided. For example, polynucleotides encoding a MuV F ectodomain trimer protomer stabilized in a prefusion conformation, a MeV F ectodomain trimer protomer stabilized in a prefusion conformation, a chimera of one of these protomers linked to a MuV HN or MeV H ectodomain, or a subunit of a self-assembling protein nanoparticle containing a recombinant MuV or MeV F ectodomain. These polynucleotides include DNA, cDNA, and RNA sequences, including vectors containing DNA, cDNA, and RNA sequences, such as DNA or RNA vectors used for immunization. The genetic code can be used to construct functionally equivalent nucleic acids, such as nucleic acids that differ in sequence but encode the same protein sequence, or nucleic acids that encode conjugates or fusion proteins containing the nucleic acid sequence.

[0203] An exemplary nucleic acid sequence encoding the full-length MeV F protein is SEQ ID NO:94: Provided as TIFF0007801221000044.tif78159.

[0204] An exemplary nucleic acid sequence encoding the full-length MuV F protein is SEQ ID NO:95: Provided as TIFF0007801221000045.tif78159.

[0205] An exemplary nucleic acid sequence encoding the full-length MeV H protein is SEQ ID NO:96: Provided as TIFF0007801221000046.tif89159.

[0206] An exemplary nucleic acid sequence encoding the full-length MuV HN protein is SEQ ID NO:97: Provided as TIFF0007801221000047.tif82159.

[0207] These exemplary nucleic acid sequences (or corresponding RNA sequences) can be modified to encode any of the immunogens provided herein.

[0208] In some embodiments, the nucleic acid molecule encodes a MuV or MeV F ectodomain trimer protomer or MeV F ectodomain trimer promoter that, when expressed in an appropriate cell, is processed into an F ectodomain trimer protomer or MuV HN or MeV H multimer subunit that can self-assemble into the corresponding trimers or multimers, or a chimera of such a protomer with a MuV HN or MeV H ectodomain, or a precursor of a MuV HN or MeV H multimer subunit. For example, the nucleic acid molecule can encode a MuV or MeV F ectodomain trimer protomer or MeV F ectodomain trimer promoter that includes an N-terminal signal sequence for entry into the cellular secretory system that is proteolytically cleaved during processing of the recombinant F ectodomain in the cell.

[0209] In some embodiments, the nucleic acid molecule encodes an F0 polypeptide that, when expressed in a suitable cell, is processed by a MuV or MeV F ectodomain trimer protomer comprising an F2 polypeptide linked to an F1 ectodomain, or a MeV F ectodomain trimer promoter, wherein the recombinant F2-F1 ectodomain protomer comprises any of the prefusion stabilizing modifications described herein and can optionally be linked to a trimerization domain, such as a GCN4 trimerization domain and / or a T4 fibritin trimerization domain.

[0210] In some embodiments, the nucleic acid molecule, when expressed in a suitable cell, encodes a MuV or MeV F ectodomain trimer protomer comprising an F2 polypeptide linked to an F1 polypeptide comprising an F1 transmembrane and cytosolic tail, or a full-length F0 polypeptide processed by a MuV or MeV F ectodomain trimer promoter, wherein the recombinant F2-F1 ectodomain protomer comprises any of the pre-fusion stabilizing modifications described herein.

[0211] Exemplary nucleic acid can be prepared by cloning technique.The example of suitable cloning technique and sequencing technique and the procedure manual sufficient to guide those skilled in the art in many cloning operations are known (for example, see Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, supplement 104, 2013).

[0212] Nucleic acids can also be prepared by amplification techniques, including polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), and self-sustained sequence replication systems (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methods are well known to those skilled in the art.

[0213] Polynucleotides encoding protomers of MuV or MeV F ectodomain trimers or subunits of MuV HN or MeV H multimers can include recombinant DNA incorporated into vectors (e.g., expression vectors), autonomously replicating plasmids or viruses, or incorporated into the genomic DNA of prokaryotes or eukaryotes, or recombinant DNA present as a separate molecule (e.g., cDNA) independent of other sequences. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term encompasses single-stranded and double-stranded forms of DNA.

[0214] A polynucleotide sequence encoding a MuV or MeV F ectodomain trimer protomer, or a MeV F ectodomain trimer promoter, or a chimera of such a protomer with a MuV HN or MeV H ectodomain, or a subunit of a MuV HN or MeV H multimer can be operably linked to an expression control sequence. An expression control sequence operably linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequence. Expression control sequences include, but are not limited to, an appropriate promoter, enhancer, transcription terminator, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signals for introns, maintenance of the correct reading frame of the gene to allow proper translation of mRNA, and stop codons.

[0215] DNA sequences encoding MuV or MeV F ectodomain trimer protomers, MeV F ectodomain trimer promoters, chimeras of such protomers with MuV HN or MeV H ectodomains, or subunits of MuV HN or MeV H multimers can be expressed in vitro by DNA transfer into appropriate host cells. The cells can be prokaryotic or eukaryotic. This term also encompasses any progeny of the subject host cell. It is understood that not all progeny will be identical to the parent cell, as mutations may occur during replication. Methods for stable transfer, which means that foreign DNA is continuously maintained in the host, are known in the art.

[0216] Hosts can include microbial, yeast, insect, and mammalian organisms. Methods for expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Non-limiting examples of suitable host cells include bacteria, archaea, insects, fungi (e.g., yeast), plants, and animal cells (e.g., mammalian cells, e.g., human cells). Useful exemplary cells include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells, Neurospora, and immortalized myeloid and lymphoid mammalian cell lines. Techniques for growing mammalian cells in culture are well known (see, e.g., Helgason and Miller, eds., 2012, Basic Cell Culture Protocols (Methods in Molecular Biology), 4th ed., Humana Press). Examples of commonly used mammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK, and COS cell lines, although cell lines such as cells engineered for higher expression levels, desired glycosylation patterns, or other characteristics may also be used. In some embodiments, host cells include HEK293 cells or derivatives thereof, such as GnTI cells. - / - cells (ATCC® No. CRL-3022), or HEK-293F cells.

[0217] Transformation of host cells with recombinant DNA can be carried out by conventional techniques. In some embodiments where the host is a prokaryotic host (such as, but not limited to, E. coli), competent cells capable of taking up DNA can be prepared from cells harvested after exponential growth phase and then treated with CaCl. Alternatively, MgCl or RbCl can be used. Transformation can also be carried out after forming a protoplast of the host cell, if desired, or by electroporation.

[0218] If the host is a eukaryotic organism, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical procedures such as microinjection, electroporation, insertion of a liposome-encapsulated plasmid, or viral vectors can be used. Eukaryotic cells can also be co-transformed with a polynucleotide sequence encoding the antigen of the present disclosure and a second foreign DNA molecule encoding a selectable phenotype, such as a herpes simplex thymidine kinase gene. Another method is to transiently infect or transform eukaryotic cells with a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papillomavirus, to express the protein (see, for example, Viral Expression Vectors, Springer Press, edited by Muzyczka, 2011). Suitable expression systems, such as plasmids and vectors, are useful for producing proteins in cells, including higher eukaryotic cells such as COS, CHO, HeLa, and myeloma cell lines.

[0219] In one non-limiting example, the immunogens of the present disclosure are expressed using the pVRC8400 vector (described in Barouch et al., J. Virol., 79, 8828-8834, 2005, incorporated herein by reference).

[0220] Nucleic acids encoding the immunogens of the present disclosure can be modified without reducing their biological activity. Such modifications can be made to facilitate cloning, expression, or incorporation of targeting molecules into fusion proteins. Exemplary modifications include stop codons, methionine added to the amino terminus to provide an initiation site, additional amino acids at either end to create restriction sites at convenient locations, or additional amino acids (e.g., polyHis) to aid in purification steps.

[0221] In some embodiments, a promoter of the MuV or MeV F ectodomain trimer, or a promoter of the MeV F ectodomain trimer, or a chimera of such a promoter with the MuV HN or MeV H ectodomain, or a subunit of the MuV HN or MeV H multimer, can be expressed in cells under conditions in which the promoter self-assembles into a trimer that is secreted from the cell into the cell culture medium, as described, for example, for the RSV F protein (see, e.g., PCT Publication Nos. WO2014160463, McLellan et al., Science, 340: 1113-1117, 2013, McLellan et al., Science, 342: 592-598, 2013, each of which is incorporated herein by reference in its entirety). In such embodiments, the promoter contains a leader sequence (signal peptide) that directs the protein into the secretion system, and the signal peptide is cleaved, and the promoter forms a trimer and is then secreted into the cell culture medium. The medium can be centrifuged and the recombinant MuV or MeV F ectodomain trimer or recombinant MuV or MeV F ectodomain trimer or chimeras thereof with MuV HN or MeV H ectodomain purified from the supernatant.

[0222] III. Viral Vectors The nucleic acid molecule encoding the immunogen of the present disclosure can be included in a viral vector, for example, for expressing the immunogen in host cells or for the immunization of the control disclosed herein.In some embodiments, the viral vector is administered to a subject as part of a prime-boost vaccination.Typically, such a viral vector comprises a nucleic acid molecule encoding an immunogen that contains a transmembrane domain.In some embodiments, the viral vector is included in a vaccine, such as a primer vaccine or a booster vaccine, for use in a prime-boost vaccination.

[0223] In some instances, a viral vector can be replication-competent, e.g., the viral vector can have a mutation in the viral genome (e.g., an insertion of a nucleic acid encoding a protomer) that attenuates but does not completely prevent viral replication in a host cell.

[0224] In some embodiments, viral vectors can be delivered via the respiratory tract. For example, hPIV vectors, such as bovine parainfluenza virus (BPIV) vectors (e.g., BPIV1, BPIV2, or BPIV3 vectors) or human hPIV vectors (e.g., hPIV3 vectors), metapneumovirus (MPV) vectors, Sendai virus vectors, Newcastle disease virus (NCDV) (vectors), mumps virus vectors, measles virus vectors, or other paramyxoviruses or pneumoviruses, are used to express antigens of the present disclosure.

[0225] Other viral vectors can be used to express the disclosed antigens, such as polyoma or SV40 (Madzak et al., 1992, J. Gen. Virol., 73:1533-1536), adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol., 158:39-6; Berliner et al., 1988, BioTechniques, 6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992, Proc. Natl. Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239, Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256), vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol., 158:91-123, On et al., 1990, Gene, 89:279-282), herpes viruses including HSV, EBV, and CMV (Margolskee, 1992, Curr. Top. Microbiol. Immunol., 158:67-90, Johnson et al., 1992, J. Virol., 66:295-2965, Fink et al. al., 1992, Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol., 1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199), Sindbis virus (H. Herweijer et al., 1995, Human Gene Therapy 6:1161-1167; U.S. Patent Nos. 5,091,309 and 5,2217,879), alphavirus (S. Schlesinger, 1993, Trends Biotechnol.11:18-22, I. Frolov et al., 1996, Proc. Natl. Acad. Sci. USA 93:11371-11377), and birds (Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754, Petropouplos et al. al., 1992, J. Virol., 66: 3391-3397), mouse (Miller, 1992, Curr. Top. Microbiol. Immunol., 158: 1-24, Miller et al., 1985, Mol. Cell Biol., 5: 431-437, Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737, Mann et al. Retroviruses of human origin (Page et al., 1990, J. Virol., 64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739) can also be used. Baculovirus (Autographa californica multinuclear polyhedrosis virus; AcMNPV) vectors are also known in the art and are available from commercial sources (e.g., PharMingen (San Diego, CA), Protein Sciences Corp. (Meriden, CT), Stratagene (La Jolla, CA)).

[0226] IV. Virus-like particles In some embodiments, virus-like particles (VLPs) containing the immunogens of the present disclosure are provided. Typically, such VLPs contain an immunogen containing a transmembrane domain, such as a recombinant MuV F ectodomain trimer with a promoter containing the MuV F transmembrane domain and cytosolic tail, or a recombinant MeV F ectodomain trimer with a promoter containing the MeV F transmembrane domain and cytosolic tail. VLPs lack viral components necessary for viral replication and are therefore significantly attenuated, replication-incompetent viruses. However, VLPs can display polypeptides similar to those expressed on infectious viral particles (e.g., recombinant MuV or MeV F ectodomain trimers) and, when administered to a subject, can induce an immune response against MuV or MeV. Exemplary virus-like particles and methods for their production, as well as viruses known to form VLPs, include human papillomavirus, HIV (Kang et al., Biol. Chem. 380:353-64 (1999)), Semliki Forest virus (Notka et al., Biol. Chem. 380:341-52 (1999)), human polyomavirus (Goldmann et al., J. Virol. 73:4465-9 (1999)), rotavirus (Jiang et al., Vaccine 17:1005-13 (1999)), parvovirus (Casal, Biotechnology and Applied Biochemistry, Vol. 29, Part 2, pp. 141-150 (1999)), canine parvovirus (Hurtado et al., J. Virol. 17:1005-13 (1999)), and avian parvovirus (Hurtado et al., J. Virol. 17:1005-13 (1999)). Viral proteins from several viruses, including hepatitis E virus (Li et al., J. Virol. 70:5422-9 (1996)), hepatitis E virus (Li et al., J. Virol. 71:7207-13 (1997)), and Newcastle disease virus. The formation of such VLPs can be detected by any suitable technique. Examples of suitable techniques for detecting VLPs in culture media include, for example, electron microscopy, dynamic light scattering (DLS), selective chromatographic separation (e.g., ion exchange, hydrophobic interaction, and / or size exclusion chromatographic separation of VLPs), and density gradient centrifugation.

[0227] V. Immunogenic compositions Immunogenic compositions comprising an immunogen of the present disclosure (e.g., a recombinant MuV F ectodomain trimer, a recombinant MeV F ectodomain trimer, or a corresponding fusion with the MuV HN ectodomain or MeV H ectodomain, or a MuV HN or MeV H multimer) and a pharmaceutically acceptable carrier are also provided. Such compositions can be administered to a subject by various modes of administration, such as intramuscular, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, or parenteral routes. In some embodiments, pharmaceutical compositions comprising one or more of the immunogens of the present disclosure are immunogenic compositions. Actual methods for preparing administrable compositions are described in more detail in publications such as Remington's Pharmaceutical Sciences (19th ed., Mack Publishing Company, Easton, PA, 1995).

[0228] Thus, the immunogens described herein can be formulated using pharmaceutically acceptable carriers to help preserve biological activity and also promote increased stability during storage within acceptable temperature ranges. Potential carriers include, but are not limited to, physiologically balanced culture media, phosphate-buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), various types of humectants, cryoprotective additives or stabilizers, such as proteins, peptides, or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., monosodium glutamate), or other protective agents. The resulting aqueous solution can be packaged for immediate use or lyophilized. Lyophilized preparations are mixed with sterile solutions prior to administration for single or multiple doses.

[0229] Formulated compositions, particularly liquid compositions, may contain effective concentrations (usually ≦1% w / v) of bacteriostatic agents, such as, but not limited to, benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben, to prevent or minimize degradation during storage. Because bacteriostatic agents may be contraindicated for some patients, the lyophilized formulations can be reconstituted in solutions containing or lacking such ingredients.

[0230] The immunogenic compositions of the present disclosure can contain, as a pharmaceutically acceptable vehicle, substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

[0231] The immunogenic composition can optionally contain an adjuvant to enhance the host's immune response. Adjuvants such as aluminum hydroxide (e.g., ALHYDROGEL® available from Brenntag Biosector, Copenhagen, Denmark, and Amphogel® (Wyeth Laboratories, Madison, New Jersey)), Freund's adjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Indiana) and IL-12 (Genetics Institute, Cambridge, Massachusetts), TLR agonists (e.g., TLR-9 agonists, such as cytidine-phospho-guanosine oligodeoxynucleotide (CpG-ODN) 1018), and many other suitable adjuvants known in the art can be included in the composition. Suitable adjuvants include, for example, Toll-like receptor agonists, alum, AlPO4, alhydrogel, lipid A and its derivatives or variants, oil emulsions, saponins, neutral liposomes, liposomes containing vaccines and cytokines, nonionic block copolymers, and chemokines. Nonionic block polymers containing polyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Indiana), and IL-12 (Genetics Institute, Cambridge, Massachusetts) may also be used as adjuvants (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants are advantageous in that they non-specifically stimulate the immune system, thus helping to enhance the immune response to pharmaceutical products.

[0232] In some examples, the adjuvant formulation is an inorganic salt, such as a calcium salt or an aluminum (alum) salt, such as calcium phosphate, aluminum phosphate, or aluminum hydroxide. In some embodiments, the immunogens of the disclosure comprise one or more phosphoserine modifications and are used with an alum adjuvant. In some embodiments, the adjuvant comprises an oil and water emulsion, such as an oil-in-water emulsion (e.g., MF59 (Novartis) or AS03 (GlaxoSmithKline)). One example of an oil-in-water emulsion comprises a metabolizable oil, such as squalene, a tocol, e.g., a tocopherol such as alpha-tocopherol, and a surfactant, such as sorbitan trioleate (Span 85) or polyoxyethylenesorbitan monooleate (Tween 80), in an aqueous carrier.

[0233] In some instances, it may be desirable to combine the immunogens of the present disclosure with other pharmaceutical products (e.g., vaccines) that induce protective responses against other agents. For example, compositions comprising the recombinant MuV F ectodomain trimers, recombinant MeV F ectodomain trimers, or corresponding fusions with the MuV HN ectodomain or MeV H ectodomain described herein can be administered simultaneously (typically separately) or sequentially with other vaccines recommended by the Advisory Committee on Immunization Practices (ACIP; cdc.gov / vaccines / acip / index.html) for target age groups (e.g., infants approximately 1-6 months of age). Thus, the immunogens of the present disclosure described herein can be administered simultaneously or sequentially with vaccines against, for example, hepatitis B (HepB), diphtheria, tetanus, and pertussis (DTaP), streptococcus (PCV), Haemophilus influenzae type b (Hib), polio, influenza, and rotavirus.

[0234] In some embodiments, the composition can be provided as a sterile composition. The immunogenic composition typically contains an effective amount of the immunogen of the present disclosure and can be prepared by conventional techniques. Typically, the amount of immunogen in each dose of the immunogenic composition is selected to induce an immune response without significant adverse side effects. In some embodiments, the composition can be provided in a unit dosage form for use in inducing an immune response in a subject, for example, for use in inhibiting MuV and / or MeV infection in a subject. The unit dosage form contains a preselected single dosage amount suitable for administration to a subject, or a segmented or measured multiple of two or more preselected unit dosage amounts, and / or a metering mechanism for administering the unit dose or multiples thereof.

[0235] VI. Methods of Inducing an Immune Response To induce an immune response against MuV and / or MeV in a subject, an immunogen of the present disclosure (e.g., a recombinant MuV F ectodomain trimer, a recombinant MeV F ectodomain trimer, or a corresponding fusion with the MuV HN ectodomain or MeV H ectodomain, a MuV HN or MeV H multimer, a nucleic acid molecule (e.g., an RNA molecule) encoding an immunogen of the present disclosure, or a protein nanoparticle or virus-like particle comprising the immunogen) can be administered to the subject. In certain examples, the subject is a human. The immune response can be a protective immune response, e.g., a response that inhibits subsequent infection by MuV and / or MeV. Induction of an immune response can also be used to treat or inhibit MuV and / or MeV infection and diseases associated therewith.

[0236] Subjects who have or are at risk of developing a MuV or MeV infection due to exposure or potential exposure to MuV or MeV can be selected for treatment. After administration of the immunogen of the present disclosure, subjects can be monitored for MuV and / or MeV infection and / or symptoms associated therewith.

[0237] Typical subjects intended for treatment with the therapeutic agents and methods of the present disclosure include humans. In some embodiments, the subject is a human subject who is seronegative for MuV and / or MeV-specific antibodies. In some embodiments, the subject is a human subject who is seropositive for MuV and / or MeV-specific antibodies, and the immunogen is administered to boost the subject's immune response to MeV and / or MuV. To identify subjects for treatment with the methods of the present disclosure, accepted screening methods are used to determine risk factors associated with the target or suspected disease or condition, or to determine the status of an existing disease or condition in the subject. These screening methods include, for example, conventional workups to determine environmental factors, familial factors, occupational factors, and other similar risk factors that may be associated with the target or suspected disease or condition, as well as diagnostic methods, such as various ELISAs and other immunoassays for detecting and / or characterizing MuV and / or MeV infection. These and other conventional methods enable clinicians to select patients in need of treatment with the methods and immunogenic compositions of the present disclosure. In accordance with these methods and principles, compositions can be administered as a stand-alone prevention or treatment program, or as a follow-up, adjunctive, or coordinate treatment to other treatments, in accordance with the teachings herein or other conventional methods.

[0238] The purpose of administration of the immunogens of the present disclosure can be prophylactic or therapeutic. When provided prophylactically, the immunogens can be provided prior to any symptoms, e.g., prior to infection. Prophylactic administration serves to prevent or ameliorate any subsequent infection. In some embodiments, the methods can involve selecting a subject at risk for MuV and / or MeV infection and administering a therapeutically effective amount of an immunogen of the present disclosure to the subject. The immunogen can be provided prior to anticipated exposure to MuV and / or MeV, such that the anticipated severity, duration, or extent of infection and / or associated disease symptoms is attenuated after exposure or suspected exposure to the virus, or after the onset of infection. Populations that may benefit from prophylactic use of the immunogens of the present disclosure (e.g., as a booster immunization) include children (e.g., 5 years old) at the time of school entry and adolescents (e.g., 15-18 years old) at the time of entering high school or college or joining the military. Transplant recipients may also need to be revaccinated, or immunocompromised children, such as HIV+, including pregnant women, would benefit from a protein vaccine rather than a live attenuated virus, which may be unsafe.

[0239] When provided therapeutically, the immunogens of the present disclosure are provided at or after the onset of symptoms of MuV and / or MeV infection, or after diagnosis of MuV and / or MeV infection. Treating MuV by inhibiting MuV replication or infection can include delaying and / or reducing signs or symptoms of MuV infection in a subject. Treating MeV by inhibiting MeV replication or infection can include delaying and / or reducing signs or symptoms of MeV infection in a subject. In some examples, treatment using the methods disclosed herein extends the survival of a subject.

[0240] In some embodiments, administration of the immunogens of the present disclosure to a subject can induce the production of an immune response that is protective against and reduces disease symptoms when the subject is subsequently infected or reinfected with wild-type MuV and / or MeV. Naturally circulating viruses may still be capable of causing infection, but vaccination may result in a reduced likelihood of severe or life-threatening symptoms, and subsequent infection with wild-type viruses may result in enhanced resistance. Following vaccination, there are detectable levels of host-produced serum and host-produced secretory antibodies capable of neutralizing homologous (same subgroup) wild-type viruses in vitro and in vivo. In many cases, host antibodies will also neutralize wild-type viruses of different subgroups that are not the vaccine subgroup.

[0241] The immunogens and immunogenic compositions thereof described herein are provided to a subject, preferably a human, in an amount effective to induce or enhance an immune response to MuV and / or MeV in the subject. The actual dosage of the immunogens of the present disclosure will vary depending on factors such as the indication and the subject's specific condition (e.g., the subject's age, size, health, severity of symptoms, susceptibility factors, etc.), the time and route of administration, other drugs or treatments administered concomitantly, and the specific pharmacology of the composition to elicit the desired activity or biological response in the subject. Dosage regimens can be adjusted to obtain an optimal prophylactic or therapeutic response.

[0242] Immunogenic compositions comprising one or more of the immunogens of the present disclosure can be used in coordinate (or prime-boost) vaccination protocols or combinatorial formulations. In certain embodiments, novel combinatorial immunogenic compositions and coordinate immunization protocols use separate immunogens or formulations, each of which aims to induce an antiviral immune response, such as an immune response against the MuV F protein and / or the MeV F protein. Separate immunogenic compositions that induce antiviral immune responses can be combined as a multivalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (as monovalent immunogenic compositions) in a coordinate (or prime-boost) immunization protocol.

[0243] Several boosts can be administered, and each boost can be a different immunogen of the present disclosure. In some examples, a boost can be the same immunogen as another boost or prime. The prime and boost can be administered as a single dose or as multiple doses, e.g., two, three, four, five, six, or more doses to a subject over the course of several days, weeks, or months. Multiple boosts can also be given, e.g., one to five (e.g., one, two, three, four, or five boosts) or more. Different dosages can be used in a series of sequential immunizations, e.g., a relatively higher dose for the initial immunization, followed by a relatively lower dose for the boost.

[0244] In some embodiments, the boost can be administered about 2, about 3-8, or about 4 weeks after the prime, or approximately several months after the prime. In some embodiments, the boost can be administered about 5, about 6, about 7, about 8, about 10, about 12, about 18, or about 24 months after the prime, or thereabouts. Periodic additional boosts can also be used at appropriate times to strengthen the subject's "immunological memory." The appropriateness of selected vaccination parameters, such as formulation, dose, and regimen, can be determined by collecting aliquots of serum from the subject and assaying the antibody titer during the immunization program. In addition, the subject's clinical status can be monitored for the desired effect, such as inhibition of MuV and / or MeV infection or amelioration of disease status (e.g., reduction in viral load). If such monitoring indicates suboptimal vaccination, the subject can be boosted with additional doses of the immunogenic composition, and vaccination parameters can be modified to anticipate an enhanced immune response.

[0245] In some embodiments, the prime-boost method can include a DNA primer and protein boost vaccination protocol for the subject. The method can include two or more administrations of the nucleic acid molecule or protein.

[0246] For protein therapeutics, typically each human dose will contain 1 to 1000 μg of protein, for example about 1 μg to about 100 μg, for example about 1 μg to about 50 μg, for example about 1 μg, about 2 μg, about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 40 μg or about 50 μg.

[0247] The amount utilized in the immunogenic composition is selected based on the subject population (e.g., infants or the elderly). Optimal amounts for a particular composition can be ascertained by standard testing involving observation of antibody titers and other responses in subjects. It is understood that an effective amount of an immunogen of the present disclosure, e.g., a recombinant MuV or MeV F ectodomain trimer or a recombinant MeV F ectodomain trimer or chimera thereof with MuV HN or MeV H ectodomain, a viral vector, or a nucleic acid molecule, in an immunogenic composition can include an amount that is not effective in eliciting an immune response in a single administration, but is effective when administered multiple times, e.g., in a prime-boost administration protocol.

[0248] Upon administration of the immunogens of the present disclosure, the subject's immune system typically responds to the immunogenic composition by producing antibodies specific to the viral proteins, and such a response indicates that an immunologically effective amount has been delivered to the subject.

[0249] For each particular subject, the specific dosing regimen can be evaluated and adjusted over time according to the individual needs and the judgment of the professional administering or supervising the administration of the immunogenic composition. The dosage and frequency of administration will depend on the situation. For example, in adults or those primed by previous MuV and / or MeV infection or immunization, a single dose may be a sufficient booster. In naive subjects, in some instances, at least two doses, e.g., at least three doses, will be administered. In some embodiments, an annual boost is given, for example, in conjunction with an annual influenza vaccination.

[0250] In some embodiments, the subject's antibody response will be determined in connection with evaluating an effective dosage / immunization protocol. In most cases, it will be sufficient to evaluate the antibody titer in serum or plasma obtained from the subject. Decisions regarding whether to administer a booster vaccination and / or change the amount of therapeutic agent administered to an individual can be based, at least in part, on the antibody titer level. The antibody titer level can be based, for example, on an immune binding assay that measures the concentration of antibodies in serum that bind to antigens, including MuV F protein and / or MeV F protein.

[0251] Determination of effective dosages is typically guided by an administration protocol that significantly reduces the occurrence or severity of symptoms or pathology of the targeted disease in a subject, or that induces a desired response (e.g., a neutralizing immune response) in a subject, based on animal model testing and subsequent human clinical trials. Suitable models in this regard include, for example, mice, rats, pigs, cats, ferrets, non-human primates, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models (e.g., immunological and histopathological assays). Using such models, routine calculations and adjustments are all that is required to determine appropriate concentrations and doses for administering an effective amount of a composition (e.g., an amount effective to induce a desired immune response or alleviate one or more symptoms of a targeted disease). In an alternative embodiment, an effective amount or dose of a composition may simply inhibit or enhance one or more selected biological activities correlated with a disease or pathology identified herein for therapeutic or diagnostic purposes.

[0252] Administration of an immunogenic composition that elicits an immune response to reduce or prevent infection can eliminate, but not necessarily completely eliminate, such infection, so long as the infection is measurably reduced. For example, administration of an effective amount of the agent can reduce MuV or MeV infection (e.g., as measured by infection of cells by MuV or MeV or by the number or percentage of subjects infected with MuV or MeV) by a desired amount, such as at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (also known as sterilizing immunity, elimination or prevention of detectable MuV or MeV infection compared to an appropriate control).

[0253] In some embodiments, administering an effective amount of one or more immunogens of the present invention to a subject induces a neutralizing immune response in the subject. To assess neutralizing activity, serum can be collected from the subject at an appropriate time point after immunization, frozen, and stored for neutralization testing. Methods for assaying neutralizing activity include, but are not limited to, plaque reduction neutralizing titer (PRNT) assay, microneutralization assay, flow cytometry-based assay, and single-cycle infection assay. In some embodiments, serum neutralizing activity can be assayed using a panel of MuV and / or MeV pseudoviruses.

[0254] One approach to nucleic acid administration is direct immunization with plasmid DNA, e.g., mammalian expression plasmids. Immunization with nucleic acid constructs is well known in the art and is taught, for example, in U.S. Pat. No. 5,643,578 (which describes a method of immunizing vertebrates by introducing DNA encoding a desired antigen to elicit a cell-mediated or humoral response) and U.S. Pat. Nos. 5,593,972 and 5,817,637 (which describe operably linking a nucleic acid sequence encoding an antigen to a regulatory sequence enabling expression). U.S. Pat. No. 5,880,103 describes several methods for delivering nucleic acids encoding immunogenic peptides or other antigens to organisms. These methods include liposomal delivery of nucleic acids (or synthetic peptides themselves) and immunostimulatory constructs, or ISCOMs, which are negatively charged cage-like structures 30-40 nm in size that spontaneously form when cholesterol and Quil A (saponin) are mixed. The use of ISCOMs™ as antigen delivery vehicles has produced protective immunity in a variety of experimental models of infection, including toxoplasmosis and Epstein-Barr virus-induced tumors (Mowat and Donachie, Immunol. Today 12:383, 1991). Doses of as little as 1 μg of antigen encapsulated in ISCOMs™ have been found to generate class I-mediated CTL responses (Takahashi et al., Nature 344:873, 1990).

[0255] In some embodiments, plasmid DNA vaccines are used to express the immunogens of the present disclosure in subjects.For example, to induce immune responses against the F protein of MuV or MeV, nucleic acid molecules encoding the immunogens of the present disclosure can be administered to subjects.In some embodiments, nucleic acid molecules can be included in the plasmid vectors for DNA immunization, such as pVRC8400 vector (described in Barouch et al., J.Virol, 79, 8828-8834, 2005, which is incorporated herein by reference).

[0256] In another approach to using nucleic acids for immunization, the immunogens of the present disclosure can be expressed by attenuated viral hosts (e.g., attenuated MuV or MeV vectors) or by attenuated viral or bacterial vectors. Recombinant vaccinia virus, adeno-associated virus (AAV), herpesvirus, retrovirus, cytomegalovirus, paramyxovirus, pneumovirus, or other viral vectors can be used to express peptides or proteins and thereby elicit CTL responses. For example, vaccinia vectors and methods useful in immunization protocols are described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette-Guerin) is another vector for expressing peptides (see Stover, Nature 351:456-460, 1991).

[0257] In another example, an immunogen of the present disclosure can be administered to a subject using RNA immunization, e.g., a lipid-encapsulated mRNA immunization platform (see, e.g., Roth et al., "A Modified mRNA Vaccine Targeting Immunodominant NS Epitopes Protects Against Dengue Virus Infection in HLA Class I Transgenic Mice," Fort Immunol., June 21, 2019, Vol. 10, Article 1424; Jagger et al., J Infect Dis, "Protective Efficacy of Nucleic Acid Vaccines Against Transmission of Zika Virus During Pregnancy in Mice," jiz338, July 1, 2019; Feldman et al., "mRNA Vaccines Against H10N8 and H7N9 Influenza Viruses of Pandemic Potential Are Immunogenic and Well Tolerated in Healthy Adults in Phase 1 Randomized Clinical Trials," Vaccine, 37 (25), 3326-3334, 2019, and Hasset et al., “Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines,” Mol Ther Nucleic Acids, 15:1-11, 2019.

[0258] In one embodiment, nucleic acids encoding the MuV F or MeV F ectodomain trimer protomers of the present disclosure are directly introduced into cells. For example, the nucleic acids can be loaded onto gold microspheres by standard methods and then introduced into the skin using a device such as Bio-Rad's HELIOS™ gene gun. The nucleic acids can be "naked" and consist of a plasmid under the control of a strong promoter. Typically, DNA is injected intramuscularly, but can also be injected directly into other sites. The dosage for injection is usually around 0.5 μg / kg to about 50 mg / kg, typically about 0.005 mg / kg to about 5 mg / kg (see, e.g., U.S. Patent No. 5,589,466).

[0259] In another embodiment, mRNA-based immunization protocols can be used to directly deliver the nucleic acid encoding the immunogen of the present disclosure into cells.In some embodiments, mRNA-based nucleic acid-based vaccines can be a powerful alternative to the above-mentioned approaches.mRNA vaccines eliminate the safety concerns about DNA integration into host genome and can be directly translated in the cytoplasm of host cells.Furthermore, the simple cell-free in vitro synthesis of RNA avoids the manufacturing problems associated with viral vectors. Two exemplary forms of RNA-based vaccination that can be used to deliver nucleic acids encoding immunogens of the present disclosure include conventional, non-amplified mRNA immunization (see, e.g., Petsch et al., "Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection," Nature biotechnology, 30(12):1210-6, 2012), and self-replicating mRNA immunization (see, e.g., Geall et al., "Nonviral delivery of self-amplifying RNA vaccines," PNAS, 109(36):14604-14609, 2012; Magini et al., "Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge," PLoS One, 11(8):e0161193, 2016; and Brito et al., "Self-amplifying mRNA vaccines," Adv. Genet.,89:179-233,2015).

[0260] In some embodiments, lipid nanoparticles containing mRNA encoding an immunogen of the present disclosure are used in methods for eliciting an immune response, such as those described in WO2017070626, US2019 / 0192646, and in Jackson et al., "An mRNA vaccine against SARS-CoV2 - preliminary report," N. Engl. J. Med., 383(20):1920-1931, 2020, for the mRNA-1273 vaccine, each of which is incorporated herein by reference. As described in WO2017070626, the mRNA encoding the immunogen can be formulated into lipid nanoparticles using 50 mol% ionizable lipid, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% (PEG2000DMG). Furthermore, the mRNA encoding the immunogen can be a modified mRNA with 1-methylpseudouridine instead of uridine and a 7mG(5')ppp(5')N1mpNp cap (enzymatic) in addition to the 5'UTR, 3'UTR and polyA tail. [Example]

[0261] Examples are provided below to illustrate particular features of certain embodiments, but the claims should not be limited to those exemplified features.

[0262] Example 1 MuV F protein stabilized in the prefusion conformation and its fusion with the MuV HN ectodomain This example illustrates an embodiment of a MuV F ectodomain trimer stabilized in a pre-fusion conformation by one or more amino acid substitutions. MuV F ectodomain trimers linked to a MuV HN ectodomain are also provided. Pre-fusion stabilized MuV F ectodomain trimers and corresponding fusions with the MuV HN ectodomain are useful, for example, for inducing a neutralizing immune response to MuV in a subject.

[0263] Until the introduction of mumps-containing vaccines in the late 1960s, mumps caused a wide range of morbidity characterized by fever, parotitis, and, less commonly, orchitis, meningitis, encephalitis, and hearing loss. The combined measles, mumps, and rubella (MMR) vaccine dramatically reduced the incidence of mumps worldwide. Although two doses of the MMR vaccine are approximately 88% effective in preventing mumps, since 2006, the number of mumps cases has increased again worldwide among highly vaccinated populations, with >30,000 affected individuals in the United States. Contributing factors include waning immunity, ineffective antibody responses, and antigenic differences between the Jeryl Lynn strain used in the MMR vaccine and circulating wild-type strains. Genotype G has become the predominant mumps genotype characterizing recent outbreaks in the United States and Europe.

[0264] As described herein, structure-based design was used to engineer a MuV F glycoprotein stabilized in a prefusion conformation. The crystal structure of the mumps fusion glycoprotein at 2.16 Å resolution reveals the basis for prefusion conformation stabilization. Potent cross-mumps genotype plaque reduction neutralization titers (PRNTs) were elicited in mice from chimeric fusion glycoproteins of the mumps prefusion-stabilized F glycoprotein or the prefusion-stabilized mumps F trimer linked to genotype G mumps hemagglutinin-neuraminidase (HN). The prefusion F-HN mumps chimera was able to elicit the highest PRNTs against genotype A, G, and H mumps viruses, 100-fold higher than reported human protective titers. Additionally, monoclonal antibodies against mumps prefusion F and HN were isolated from immunized mice and were capable of neutralizing genotype G mumps virus with a range of potencies. Structural and binding analyses of these prefusion F-specific antibodies revealed binding to four discontinuous neutralizing antigenic sites. The engineered immunogens are potential mumps vaccines, either as novel vaccines or booster vaccines.

[0265] result Disulfide bonds and membrane-proximal coiled-coil stabilization robustly stabilize the soluble prefusion mumps F trimer and enable its production.

[0266] The MuV F ectodomain linked to the C-terminal GCN4 trimerization domain forms trimers that spontaneously transition to the pre-fusion conformation when produced in cells. Unstabilized recombinant MuV F-GCN4 is so unstable that 100% of the molecules transition to the post-fusion conformation at the time of evaluation (EM). Protein expression is also substantially reduced without stabilization. Therefore, using structure-based vaccine design, we identified mutations that stabilize the MuV F ectodomain in the pre-fusion conformation and eliminated the F1 / F2 cleavage site, thereby producing a "single-chain" MuV F protein with increased expression.

[0267] Using the crystal structure of the simian prefusion parainfluenza virus 5 (PIV5) F glycoprotein (PDB IDs 4GIP, 4WSG) (Welch, BD et al. Proc Natl Acad Sci USA 109, 16672-16677, 2012), we constructed a homology model of the prefusion mumps F protein, consisting of three intertwined monomers that form a quaternary assembly of the DI, DII, DIII, and HRB domains.

[0268] To "lock" the MuV F ectodomain in the prefusion conformation, we used a multiple stabilization strategy, including the introduction of disulfide bonds and proline substitutions. The selection of residues in MuV F to be mutated to cysteines was based on homology design from the PIV5 prefusion F structure (PDB 4WSG) and on residues predicted to undergo conformational changes during the transition from the prefusion to the postfusion conformation. The C beta atoms of residue pairs were confirmed to be within 5 Å and in an orientation that would allow disulfide bond formation. A total of approximately 60 different mutants were designed, expressed, purified, and assessed for expression levels and prefusion conformation by negative-stain EM.

[0269] Mutations were introduced into the MuV F ectodomain (based on C-terminal truncations at MuV F positions 469, 476, or 483) and linked to the C-terminal GCN4 trimerization domain, and the resulting mutants were screened as described above. The ectodomain also contained mutations to remove the F1 / F2 furin cleavage site. Prefusion-stabilizing mutations evaluated included cysteine ​​substitutions at one or more of MuV F positions 86 and 215, 155 and 161, 163 and 235, 165 and 231, 206 and 223, 209 and 214, and 221 and 255, which form non-native disulfide bonds, as well as a proline substitution at MuV F position 184. The relevant sequences are shown below. Figure 1A summarizes the initial successful prefusion-stabilizing mutations.

[0270] Expression and purification of the single-chain prefusion stabilized MuV F protein showed a substantial increase in expression levels compared to unmodified MuV F.

[0271] As illustrated in Figure 1A and Figure 1B, negative stain EM can be used to distinguish MuV F ectodomain trimers in the pre-fusion conformation from those in the post-fusion conformation.

[0272] By creating a matrix of disulfide bond positions and C-terminal coiled-coil-GCN4 attachment positions, we assessed protein expression levels and the proportion of proteins adopting pre- or post-fusion trimer conformations using negative-stain EM (Fig. 1A). Five combinations of disulfide bond positions and GCN4 attachment positions were observed to result in 100% pre-fusion trimers, and one such combination (V206C-A223C and 476-GCN4) resulted in high protein expression yields of approximately 4.8 mg / L from Expi293 cells. The negative-stain EM 2D average of this design (Fig. 1B, top panel) contrasted with the post-fusion mumps F glycoprotein trimer (Fig. 1B, bottom panel), consistent with previous observations of parainfluenza virus F protein conformations.

[0273] The crystal structure of the prefusion mumps F glycoprotein trimer at 2.16 Å resolution reveals the stabilizing disulfide design and the location of polymorphic residues. Size-exclusion chromatography of the prefusion stabilized mumps F protein trimer (MuV F V206C-A223C-GGG-476-GCN4, SEQ ID NO:11) revealed a homogeneous peak (Figure 1C) that was deglycosylated and could be crystallized when expressed in the presence of kifunensine. The three-dimensional coordinates of the crystal are provided here as Table 1. The X-ray structure at 2.16 Å resolution (Figures 1D and 1E) showed an overall architecture similar to that of the prefusion PIV5 and other paramyxovirus prefusion F trimers (Stewart-Jones et al., Proc Natl Acad Sci USA 115, 12265-12270, 2018; Welch, B.D. et al., Proc Natl Acad Sci USA 109, 16672-16677, 2012; Xu, K. et al., PLoS Pathog 11(12)e1005322, 2015). However, the mumps prefusion F trimer adopts a "closed lid" conformation composed of the loop N177-S184 at the apical end of the trimer, formed by interactions between side chains T178, Q179, and N181 (Figures 2A and 2B). The remaining N-acetylglucosamine moieties from six glycans at positions N73, N182, N352, N427, N433, and N457 were visible in the electron density, and a glycosylation model was constructed (Figure 2C). The V206C-A223C disulfide was clearly defined by the electron density, with a Cα-Cα atom distance of 4.8 Å, whereas in the homologous postfusion PIV3 F trimer structure [PDB ID 1ZTM], the Cα-Cα atoms are located 5.8 Å apart. The DI-DIII domain has a volume of approximately 20,000 Å, similar to that observed in the PIV5, PIV3, and nipah prefusion F structures. 3 The protomers surround a large aqueous cavity measuring 6,500 Å. 2(Stewart-Jones et al., Proc Natl Acad Sci USA 115, 12265-12270, 2018; Welch, B.D. et al., Proc Natl Acad Sci USA 109, 16672-16677, 2012; Xu, K. et al., PLoS Pathog 11(12):e1005322, 2015). Residues that are thought to undergo conformational changes between the pre-fusion and post-fusion conformations (based on the PIV5 pre-F structure [PDB ID 4WSG] and PIV3 post-F [PDB ID 1ZTM]) are shown in Figure 1E and correspond to MuV F residues 92–253.

[0274] Although sequence identity between mumps F and HN is relatively high among genotypes (Figures 2A–2C), we mapped polymorphic variation in the structures of prefusion mumps F and HN (Figure 2D). Mapping prefusion F variation across all genotypes, particularly between genotype G and Jeryl Lynn (genotype A), revealed that the majority of the protein surface exposed for antibody recognition was conserved, while numerous variable amino acids were located within the aqueous cavity in the core of the prefusion trimer (Figure 2D, left). In contrast, the mumps HN dimer structure revealed that most polymorphic amino acids were solvent-exposed, including the glycan variation at position N464 between Jeryl Lynn HN and genotype G HN (Figure 2D, right). The predominance of solvent-exposed polymorphic residues on HN compared with prefusion F suggests that HN rather than prefusion F may explain the resistance of genotype G to Jeryl Lynn vaccine-induced humoral immunity.

[0275] Cross-strain efficacy of stabilizing mutations. To demonstrate that prefusion stabilizing mutations are effective against F across MuV strains, these mutations were tested on F from several different MuV strains. MuV F V206C-A223C-GGG-476-GCN4 (SEQ ID NO:11) is based on genotype C MuV F. Introduction of these prefusion stabilizing mutations into the genotype A (Jeryl Lynn) MuV F protein (MuV-JL F 206C-A223C-GGG-476-GCN4 (SEQ ID NO:26)) and the genotype G MuV F protein (MuV-IL17 F 206C-A223C-GGG-476-GCN4 (SEQ ID NO:51)) similarly conferred prefusion stabilization. Furthermore, introduction of these prefusion stabilizing mutations into F proteins from the following MuV strains also conferred prefusion stabilization as measured by negative stain EM and / or prefusion-specific antibody binding: Canada (Urabe), Albany (genotype A), Hoshino (genotype B), India (genotype C), Netherlands (genotype D), China (genotype F), NethL11 (genotype G), NY14 (genotype G), IA14 (genotype G), MA16 (genotype G), LA17 (genotype G), IL17 (genotype G), Virginia (genotype H), Taiwan (genotype J), ​​Taiwan (genotype K), Netherlands (genotype L), MG15 (genotype A).

[0276] F-HN Chimera. To increase the immunogenic footprint of the MuV F ectodomain trimer, the MuV HN ectodomain was genetically fused to the C-terminus of the trimerization domain of each protomer in the trimer. The format is diagrammed in Figure 4A. The corresponding sequences are shown below. Negative-stain EM shows that the F ectodomain maintains its prefusion conformation, with three HN ectodomains (one linked to each F protomer) positioned C-terminal to the trimerization domain. This design yielded approximately 0.3 mg / L from Expi293 cells, was monodisperse by size-exclusion chromatography, and showed the expected assembly by negative-stain EM (Figures 1C and 4A).

[0277] Prefusion-stabilized mumps F and prefusion-stabilized F-HN chimeric trimers induce high-titer neutralizing antibodies in mice. The ability of prefusion-stabilized mumps F to induce neutralizing antibodies was evaluated in comparison with other mumps immunogens. Groups of 10 CB6F1 / J mice were immunized at weeks 0, 3, and 10 with a dose of 10 μg of mumps glycoprotein combined with 10 μg of polyinosinic-polycytidylic acid (poly I:C) adjuvant, and the ability of sera to prevent mumps virus infection of HEp-2 cells was measured (Figure 3A).

[0278] The immunogens evaluated were the MuV F ectodomain trimer in the postfusion conformation (native ectodomain with -476-GCN4), the MuV F ectodomain trimer in the prefusion conformation (MuV F V206C-A223C-GGG-476-GCN4, SEQ ID NO:11), the MuV HN ectodomain monomer, and the MuV F ectodomain trimer in the prefusion conformation in which the trimer protomer is fused to the MuV HN ectodomain (MuV F 206C-223C-GGG-476+GCN4+MuV HN_G (SEQ ID NO:27)).

[0279] When mice were immunized with prefusion mumps F-containing immunogens (preF or preF-HN), a specific response to preF was detected in the serum, with lower levels of binding in postfusion-immunized mice (Fig. 4B). Recombinant HN monomer bound only to HN-immunized mouse sera, whereas monomeric HN-immunized mice showed little binding, whereas sera from preF-HN-immunized mice showed substantial levels of HN binding, suggesting that the multivalency of HN drives robust humoral responses (Fig. 4C).

[0280] To analyze the induction of neutralizing antibody titers by three immunizations with either post-fusion F, pre-fusion F, or pre-fusion F-HN, PRNTs were analyzed 2 weeks after each immunization (Figure 3B). A stepwise increase in neutralizing titers was observed after each immunization, but the third immunization showed a smaller increase than the second immunization. Neutralizing antibodies were also observed with post-fusion immunizations, but the pre-fusion F immunogen showed 3.5- and 2.5-fold higher neutralization than post-fusion F after the second and third immunizations, respectively (geometric mean infectious dose (ID ) against genotype G mumps virus). 60 The preF-HN chimeric mutant elicited neutralizing titers 12-fold higher than postfusion F and 5-fold higher than prefusion F after the third immunization, and showed ID values ​​against genotype G viruses. 60 The value was 3930.

[0281] Next, PRNT against Jeryl Lynn and genotype H viruses was assessed to characterize the cross-neutralizing antibodies elicited from the recombinant immunogens. For postfusion F and prefusion F, higher levels of neutralization were observed for Jeryl Lynn virus than for genotype G virus, whereas equivalent PRNT was observed for the prefusion F-HN chimera. At week 16, sera from both the preF and preF-HN groups showed robust PRNT against genotype H virus, indicating that these recombinant immunogens can elicit antibodies capable of cross-neutralizing numerous mumps genotypes. The persistence of PRNT against these three mumps viruses was monitored for an additional 6 months, and despite a decline in titers, the ID was still high after 3 months. 60 A plateau was formed, and the geometric mean PRNT of preF-HN was found to be approximately 640, 800, and 1700 for genotype G, Jeryl Lynn, and genotype H viruses, respectively (Fig. 4D). 60 The PRNT plateaus were approximately 100, 660, and 3153 (Fig. 4E ). Overall, prefusion-stabilized F mumps linked to HN gave higher neutralizing titers than F alone and represents a design strategy for stoichiometrically combining both viral surface antigens in a single immunogen.

[0282] Consideration Human immunity against mumps after MMR vaccination is typically characterized by a PRNT of around 220 for Jeryl Lynn and around 40 for genotype G (Rasheed et al., Proc Natl Acad Sci USA 116(38):19071-19076, 2019). The results provided herein for the immunogens of the present disclosure appear to confer increased efficacy as measured by PNRT, assuming mouse model data correlates with responses in humans.

[0283] Because mumps outbreaks have occurred among recipients of two doses of the vaccine, improvements to the current mumps vaccine are needed to reduce disease incidence and the burden on public health resources. The Advisory Committee on Immunization Practices (ACIP) recommends a third dose of a mumps-containing vaccine for individuals at risk of contracting mumps in outbreak situations. A third dose of live-attenuated MMR resulted in a transient increase in neutralizing titers that persisted for approximately 12 months (Fiebelkorn AP et al. Open Forum Infect Dis 1(3):ofu094, 2014). Additionally, after the third dose of MMR, the risk of developing mumps infection was reduced by 78% compared with individuals who received two doses of MMR (Cardemil C. et al., Effectiveness of a Third Dose of MMR Vaccine for Mumps Outbreak Control. N Engl J Med. 377(10):947-956, 2017). The recombinant protein vaccine candidate described in this example offers an alternative vaccine modality to administration of MMR in mumps outbreak situations, offering increased durability and efficacy.

[0284] A prefusion F-HN chimera containing two key neutralization targets on the mumps virion can elicit potent cross-genotype neutralization responses, including those against the predominant outbreak-causing mumps genotype G and two other genotypes, A and H, representing a universal vaccine candidate against mumps strains worldwide.

[0285] array: TIFF0007801221000048.tif164160TIFF0007801221000049.tif232160TIFF0007801221 000050.tif232160TIFF0007801221000051.tif234160TIFF0007801221000052.tif86160

[0286] The above sequences include an N-terminal signal peptide, a MuV F ectodomain, a GCN4 trimerization domain, an optional MuV HN ectodomain, a thrombin cleavage site, a HIS tag and a Strep tag, and various linker residues between the segments.

[0287] Example 2 MeV F protein stabilized in a prefusion conformation and its fusions with the MeV H ectodomain or the MuV HN ectodomain This example illustrates an embodiment of a MeV F ectodomain trimer stabilized in a pre-fusion conformation by one or more amino acid substitutions. MeV F ectodomain trimers linked to a MeV H ectodomain are further provided. Pre-fusion stabilized MeV F ectodomain trimers and corresponding fusions with the MeV H ectodomain are useful, for example, for inducing a neutralizing immune response to MeV in a subject.

[0288] The MeV F ectodomain linked to the C-terminal GCN4 trimerization domain forms trimers that spontaneously transition to the prefusion conformation when produced in cells. Unstabilized recombinant MeV F-GCN4 is so unstable that 100% of the molecules transition to the postfusion conformation at the time of evaluation (EM). Protein expression levels are also substantially reduced without stabilization.

[0289] Therefore, we used structure-based vaccine design to identify mutations that stabilize the MeV F ectodomain in the prefusion conformation (based on the prefusion PIV5 F structure PDB ID 4WSG and the MeV F structure PDB ID 5YXW) and eliminate the F1 / F2 cleavage site, thereby producing a "single-chain" MeV F protein with increased expression. A multiplex stabilization strategy, including the introduction of disulfide bonds and proline substitutions, was used to "lock" the MeV F ectodomain in the prefusion conformation. A total of approximately 40 different mutants were designed, expressed, purified, and assessed for expression levels and prefusion conformation by negative-stain EM.

[0290] Mutations were introduced into the MeV F ectodomain (based on a C-terminal truncation at MeV F position 486) and linked to the C-terminal GCN4 trimerization domain, and the resulting mutants were screened as described above. The ectodomain also contained mutations to remove the F1 / F2 furin cleavage site. Prefusion-stabilizing mutations evaluated included cysteine ​​substitutions at one or more of MeV F positions 48 and 284, 90 and 225, 141 and 270, 165 and 171, 173 and 245, 175 and 241, 212 and 236, 216 and 233, and 219 and 224, which form non-native disulfide bonds, as well as a proline substitution at MeV F position 194. The relevant sequences are shown below.

[0291] Expression and purification of the single-chain prefusion stabilized MeV F protein showed a substantial increase in expression levels compared to unmodified MeV F.

[0292] As illustrated in Figure 5, negative EM can be used to distinguish MeV F ectodomain trimers in the pre-fusion conformation from those in the post-fusion conformation. Furthermore, MeV F R165C-M171C-486-GCN4 (SEQ ID NO:38) demonstrated an excellent combination of pre-fusion stabilization and protein expression, and was purified as a monodisperse protein by S200 gel filtration. To further confirm the pre-fusion conformation, this and other constructs were analyzed by electron microscopy.

[0293] Immunization assays were performed using MeV F ectodomain trimers in the postfusion conformation and MeV F ectodomain trimers in the prefusion conformation (MeV F R165C-M171C-486-GCN4 (SEQ ID NO:38)). The immunization protocol followed that shown in Figure 3A. Groups of 10 CB6F1 / J mice were immunized with 10 μg of protein per dose in PolyIC adjuvant at weeks 0 and 3, and the neutralizing titers of week 5 sera from immunized mice were assessed. Immune sera were evaluated in an MeV neutralization assay (Figure 5C). Sera from animals immunized with MeV F R165C-M171C-486-GCN4 (SEQ ID NO:38) were found to neutralize MeV 200-fold more than sera from animals immunized with postfusion MeV F, exceeding the protective threshold.

[0294] array: TIFF0007801221000053.tif83160TIFF0007801221000054.tif235160TIFF00078012210 00055.tif229160TIFF0007801221000056.tif232160TIFF0007801221000057.tif131160

[0295] The above sequence includes the N-terminal signal peptide, the MeV F ectodomain, and the GCN4 trimerization domain, as well as various linker residues between the segments.

[0296] In addition, chimeric constructs containing the MeV F ectodomain with amino acid substitutions for stabilization in the prefusion conformation linked to the MuV HN ectodomain or the MeV H ectodomain were designed as follows. TIFF0007801221000058.tif162160

[0297] The above sequences include the N-terminal signal peptide, the MeV F ectodomain, and the GCN4 trimerization domain, optionally the T4 fibritin trimerization domain, the MeV H ectodomain, and various linker residues between the segments.

[0298] Example 3 Multimeric MuV HN, multimeric MeV H, and MuV pre-F-MeV H chimeras This example describes one embodiment of a recombinant MuV pre-F ectodomain trimer linked to the MeV H ectodomain to provide a chimeric immunogen that elicits cross-neutralizing immune responses against MeV and MuV. Additionally, multimeric MuV HN and multimeric MeV H are also described.

[0299] To increase the immunogenic footprint of the MuV F ectodomain trimer, the MeV H ectodomain was genetically fused to the C-terminus of the trimerization domain of each promoter in the trimer. The construct evaluated included a MuV F ectodomain containing V206C-A223C, mutations to remove the F1 / F2 furin cleavage site, a trimerization domain fused to position 476 of the ectodomain, and a MeV H ectodomain linked to the C-terminus of the trimerization domain. In this embodiment, the trimerization domain contained both the GCN4 trimerization domain and the T4 fibritin trimerization domain in tandem, although either of these domains can also be used alone. The format is illustrated in Figure 6A. The corresponding sequences are shown below. TIFF0007801221000059.tif52160

[0300] Negative-stain EM of purified MuV F 206C-223C-476+GCN4 / Fd+MeV-H (SEQ ID NO:28) shows that the F ectodomain maintains its prefusion conformation and that three H ectodomains (one linked to each F protomer) are aligned C-terminal to the trimerization domain (Figure 6A). Negative-stain EM shows that this construct assembles in a conformation similar to that of MuV F-MuV HN described in Example 1.

[0301] Additional immunogens were constructed containing multimers of the MeV H ectodomain head region or the MuV HN ectodomain head region.

[0302] Trimeric MuV HN ectodomain head regions and trimeric MeV H ectodomain head regions were constructed by linking the N-terminus of the head regions to the T4 fibritin trimerization domain. The sequences are listed below. TIFF0007801221000060.tif65160

[0303] Dimeric MeV H was constructed by expressing the MeV H ectodomain head region in mammalian cells and purifying the resulting protein complex. The MeV H head dimerizes in physiological solution. The sequence of the MeV H head region is listed below. TIFF0007801221000061.tif28160

[0304] Dimeric MeV H containing the stalk and head regions can be constructed by expressing the stalk and head regions of the MeV H ectodomain in mammalian cells and purifying the resulting protein complex. The MeV H stalk and head dimerize in physiological solutions. Exemplary sequences of the MeV H stalk and head regions are listed below. TIFF0007801221000062.tif146160

[0305] MuV HN, including the stalk and head regions, can be constructed by expressing the stalk and head regions of the MuV HN ectodomain in mammalian cells and purifying the resulting protein. Exemplary sequences of the stalk and head regions of MuV HN are listed below. TIFF0007801221000063.tif131160

[0306] Additionally, chimeric trimeric MuV HN ectodomain head regions and trimeric MeV H ectodomain head regions were constructed by linking these molecules to the N- and C-termini of the T4 fibritin trimerization domain and / or the GCN4 trimerization domain. The sequences are listed below. TIFF0007801221000064.tif221160

[0307] MeV H ectodomain head dimer (SEQ ID NO:60), MeV H ectodomain head trimer (SEQ ID NO:59), and MuV HN ectodomain head trimer (SEQ ID NO:58) were designed, expressed, purified, and characterized by negative-stain EM (see Figure 6A).

[0308] Mice were immunized with the purified constructs, and sera were evaluated for MeV and MuV neutralization by PRNT. The immunogens evaluated were MeV H dimer or trimer (SEQ ID NO: 59 or 60), prefusion MuV F ectodomain trimer (MuV F V206C-A223C-GGG-476-GCN4, SEQ ID NO: 11), MuV pre-F-MeV H chimera (MuV F 206C-223C-476+GCN4 / Fd+MeV_H_3INB-tHS (SEQ ID NO: 28)), and MuV HN trimer (SEQ ID NO: 58).

[0309] For MeV neutralization (Fig. 6B ), MeV H dimers had an average PRNT ID of 66,000, MeV H trimers had an average of 30,000, and the trimerized H on the C-terminus of prefusion MuV F had an average of 35,000. 60 These results were quite surprising, given the much lower neutralizing titers observed with immunization with the prefusion MeV F trimer (PRNT of 873; see Figure 5C). Human PRNT titers after two doses of MMR vaccination average approximately 650. The extremely high MeV PRNT titers with this MeV H design were unexpected, as we initially expected that the prefusion F ectodomain trimer would confer a better immune response (similar to other paramyxoviruses, such as RSV). Unexpectedly, the multimeric MeV H immunogen exhibited extremely good immunogenicity, up to 75-fold more potent than the prefusion-stabilized MeV F ectodomain trimer and approximately 100-fold higher than the human response after MMR vaccination.

[0310] For MuV neutralization (Fig. 6C), each of the immunogens evaluated elicited an immune response above the protective threshold, with the soluble trimerized MuV HN eliciting an extremely potent immune response. This was quite surprising, given the negligible response elicited by the soluble MuV HN monomer (Fig. 4C). Surprisingly, there was a 13.4-fold increase in neutralization potency between the MuV HN ectodomain monomer and the trimeric MuV HN ectodomain.

[0311] Protein production, analysis, and immunizations were performed as described above.

[0312] It will be apparent that modifications or variations can be made to the precise details of the methods or compositions described without departing from the spirit of the described embodiments, and we claim all such variations and modifications that come within the scope and spirit of the following claims.

Claims

1. An immunogen comprising a recombinant mumps virus (MuV) F ectodomain trimer stabilized in a pre-fusion conformation by one or more amino acid substitutions in the protomers of the trimer; the amino acid substitutions include cysteine ​​substitutions that form non-native disulfide bonds to stabilize the MuV F ectodomain trimer in the pre-fusion conformation; an immunogen, wherein the cysteine ​​substitutions are located at one or more of MuV F positions 86 and 215, positions 155 and 161, positions 165 and 231, positions 206 and 223, positions 209 and 214, and positions 221 and 255;

2. 2. The immunogen of claim 1, wherein the cysteine ​​substitutions are located at one or more of MuV F positions 86 and 215, positions 165 and 231, positions 206 and 223, and positions 209 and 214.

3. 3. The immunogen of claim 1 or claim 2, wherein the cysteine ​​substitutions are located at MuV F positions 206 and 223.

4. The cysteine ​​substitution is MuV F positions 86 and 215 have N86C and A215C substitutions; MuV F positions 155 and 161 have K155C and L161C substitutions; MuV F positions 165 and 231 have V165C and M231C substitutions; MuV F positions 206 and 223 have V206C and A223C substitutions; MuV F positions 209 and 214 have P209C and P214C substitutions; MuV F positions 221 and 255 are I221C and M255C substitutions; The immunogen according to any one of claims 1 to 3.

5. The recombinant MuV F ectodomain trimer protomer comprises or consists of MuV F positions 20-100. 2 and a MuV F protein comprising or consisting of positions 104-469, 104-476, or 104-483. 1 The immunogen of any one of claims 1 to 4, comprising an ectodomain.

6. 6. The immunogen of any one of claims 1 to 5, wherein the trimeric protomer further comprises mutations to remove the F1 / F2 furin cleavage site of the MuV F ectodomain and the first residue of the fusion peptide of the F1 ectodomain.

7. 7. The immunogen of claim 6, wherein the mutation to remove the F1 / F2 furin cleavage site and the first residue of the fusion peptide comprises a deletion of MuV F positions 101-103 and, consequently, positions 100 and 104 fused with a peptide linker.

8. The immunogen of claim 7, wherein the peptide linker is a Gly-Gly-Gly linker.

9. 9. The immunogen of any one of claims 1 to 8, wherein the positioning of the amino acid substitutions is according to the reference MuV F protein sequence shown as SEQ ID NO:

1.

10. The MuV F ectodomain trimer protomer is Residues 20-483 of any one of SEQ ID NOs:3-8; Residues 20 to 476 of any one of SEQ ID NOs: 11-16, 26, or 51; or Residues 20-469 of any one of SEQ ID NOs: 19-24 comprising an amino acid sequence at least 90% identical to the protomer contains the one or more amino acid substitutions that stabilize the MuV F ectodomain trimer in the pre-fusion conformation. The immunogen according to any one of claims 1 to 9.

11. The MuV F ectodomain trimer protomer is Residues 20-483 of any one of SEQ ID NOs:3-8; Residues 20 to 476 of any one of SEQ ID NOs: 11-16, 26, or 51; or Residues 20-469 of any one of SEQ ID NOs: 19-24 11. The immunogen of claim 10, comprising or consisting of the amino acid sequence shown below.

12. The immunogen of any one of claims 1 to 11, wherein the recombinant MuV F ectodomain trimer protomer is fused to a trimerization domain at the C-terminus.

13. 13. The immunogen of claim 12, wherein the trimerization domain comprises a GCN4 trimerization domain, a T4 fibritin trimerization domain, or both.

14. the GCN4 trimerization domain IEDKIEEILSKIYHIENEIARIKKLIGEAP (SEQ ID NO: 33) and comprising the amino acid sequence shown as the T4 fibritin trimerization domain GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 34) and comprising the amino acid sequence shown as A trimerization domain comprising both the GCN4 trimerization domain and the T4 fibritin trimerization domain IEDKIEEILSKIYHIENEIARIKKLIGEAPGSGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 35) 14. The immunogen of claim 13, comprising the amino acid sequence shown as:

15. a MuV F ectodomain trimer protomer fused to the trimerization domain, Residues 20 to 513 of any one of SEQ ID NOs: 3 to 8; Residues 20 to 506 of any one of SEQ ID NOs: 11-16, 26, or 51; or Residues 20-499 of any one of SEQ ID NOs:19-24 comprising an amino acid sequence at least 90% identical to the protomer contains the one or more amino acid substitutions that stabilize the MuV F ectodomain trimer in the pre-fusion conformation. The immunogen according to any one of claims 12 to 14.

16. the MuV F ectodomain trimer protomer fused to the trimerization domain comprises: Residues 20 to 513 of any one of SEQ ID NOs: 3 to 8; Residues 20 to 506 of any one of SEQ ID NOs: 11-16, 26, or 51; or Residues 20-499 of any one of SEQ ID NOs:19-24 16. The immunogen of claim 15, comprising or consisting of the amino acid sequence shown below:

17. The immunogen of any one of claims 1 to 16, wherein the recombinant MuV F ectodomain trimer protomer is linked to a heterologous protein.

18. 18. The immunogen of claim 17, wherein the heterologous protein is the head of the ectodomain or the stalk and head of the ectodomain of the MeV H protein or the MuV HN protein.

19. The immunogen of claim 18, wherein the MeV H protein or the MuV HN protein ectodomain head or the ectodomain stalk and head is fused at its C-terminus to a trimerization domain, and the trimerization domain is fused at its C-terminus to the recombinant MuV F ectodomain trimer promoter.

20. 20. The immunogen of claim 19, wherein the MuV F ectodomain trimer promoter linked to the trimerization domain and the MeV H protein ectodomain or the MuV HN protein ectodomain comprises the amino acid sequence set forth as residues 20 to 966 of SEQ ID NO:27, residues 21 to 981 of SEQ ID NO:28, or residues 20 to 1006 of SEQ ID NO:

29.

21. 1. An immunogen, comprising a recombinant measles virus (MeV) F ectodomain trimer stabilized in a pre-fusion conformation by one or more amino acid substitutions in the protomers of the trimer, wherein the amino acid substitutions are: cysteine ​​substitutions at one or more of MeV F positions 48 and 284, positions 141 and 270, positions 165 and 171, and positions 212 and 236 that form non-native disulfide bonds; An immunogen comprising one or more of:

22. 22. The immunogen of claim 21, wherein the recombinant MeV F ectodomain trimer is stabilized in the prefusion conformation by a non-native disulfide bond between the cysteine ​​substitutions at MeV F positions 165 and 171 in the protomer of the trimer.

23. the cysteine ​​substitutions at MeV F positions 48 and 284 are R48C and A284C substitutions; the cysteine ​​substitutions at MeV F positions 141 and 270 are M141C and T270C substitutions; the cysteine ​​substitutions at MeV F positions 165 and 171 are R165C and M171C substitutions; the cysteine ​​substitutions at MeV F positions 212 and 236 are E212C and Y236C substitutions; 23. The immunogen of claim 21 or claim 22.

24. The recombinant MeV F ectodomain trimer protomer comprises or consists of MeV F positions 24-110. 2 protein and F comprising or consisting of MeV F positions 114-486. 1 24. The immunogen of any one of claims 21 to 23, comprising an ectodomain.

25. 25. The immunogen of any one of claims 21 to 24, wherein the trimeric protomer further comprises mutations to remove the F1 / F2 furin cleavage site of the MeV F ectodomain and the first residue of the fusion peptide of the F1 ectodomain.

26. 26. The immunogen of claim 25, wherein the mutation to remove the F1 / F2 furin cleavage site and the first residue of the fusion peptide comprises a deletion of MeV F positions 111-113 and positions 110 and 114 fused with a peptide linker.

27. 27. The immunogen of claim 26, wherein the peptide linker is a Gly-Gly-Gly linker.

28. 28. The immunogen of any one of claims 21 to 27, wherein the positioning of the amino acid substitutions is according to the reference MeV F protein shown as SEQ ID NO:

36.

29. the MeV F ectodomain trimer protomer comprises an amino acid sequence at least 90% identical to residues 21-483 of any one of SEQ ID NOs: 38, 53-55, 65, 68, 72-77, or 80, or residues 21-490 of any one of SEQ ID NOs: 71 or 72; the protomer comprises the one or more amino acid substitutions that stabilize the MeV F ectodomain trimer in the pre-fusion conformation. The immunogen of any one of claims 21 to 28.

30. 30. The immunogen of claim 29, wherein the MeV F ectodomain trimer protomer comprises or consists of the amino acid sequence set forth as residues 21 to 483 of any one of SEQ ID NOs: 38, 53-55, 65, 68, 72-77, or 80, or residues 21 to 490 of any one of SEQ ID NOs: 71 or 72.

31. The immunogen of any one of claims 21 to 30, wherein the recombinant MeV F ectodomain trimer protomer is fused to a trimerization domain at the C-terminus.

32. 32. The immunogen of claim 31, wherein the trimerization domain comprises a GCN4 trimerization domain, a T4 fibritin trimerization domain, or both.

33. the GCN4 trimerization domain IEDKIEEILSKIYHIENEIARIKKLIGEAP (SEQ ID NO: 33) and comprising the amino acid sequence shown as the T4 fibritin trimerization domain GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 34) and comprising the amino acid sequence shown as A trimerization domain comprising both the GCN4 trimerization domain and the T4 fibritin trimerization domain IEDKIEEILSKIYHIENEIARIKKLIGEAPGSGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 35) 33. The immunogen of claim 32, comprising the amino acid sequence shown as:

34. the MeV F ectodomain trimer protomer fused to the trimerization domain comprises an amino acid sequence at least 90% identical to residues 21-513 of any one of SEQ ID NOs: 38, 53-55, 65, 68, 72-77, or 80, or residues 21-520 of any one of SEQ ID NOs: 71 or 72; the protomer comprises the one or more amino acid substitutions that stabilize the MeV F ectodomain trimer in the pre-fusion conformation. The immunogen of any one of claims 31 to 33.

35. 35. The immunogen of claim 34, wherein the MeV F ectodomain trimer protomer fused to the trimerization domain comprises or consists of the amino acid sequence set forth as residues 21 to 513 of any one of SEQ ID NOs: 38, 53-55, 65, 68, 72-77, or 80, or residues 21 to 520 of any one of SEQ ID NOs: 71 or 72.

36. The immunogen of any one of claims 21 to 35, wherein the recombinant MeV F ectodomain trimer protomer is linked to a heterologous protein.

37. 37. The immunogen of claim 36, wherein the heterologous protein is the head of the ectodomain or the stalk and head of the ectodomain of the MeV H protein or the MuV HN protein.

38. 38. The immunogen of claim 37, wherein the MeV H protein or the MuV HN protein ectodomain head or the ectodomain stalk and head is fused at its C-terminus to a trimerization domain, and the trimerization domain is fused at its C-terminus to a recombinant MuV F ectodomain trimer promoter.

39. 39. The immunogen of claim 38, wherein the MeV F ectodomain trimer protomer linked to the trimerization domain and the ectodomain of the MeV H protein or the MuV HN protein comprises the amino acid sequence set forth as residues 21 to 959 of SEQ ID NO:56, residues 21 to 973 of SEQ ID NO:57, or residues 21 to 988 of SEQ ID NO:

81.

40. The immunogen of any one of claims 1 to 39, wherein the recombinant MeV F ectodomain trimer or the protomer of the recombinant MuV F ectodomain trimer further comprises one or more additional amino acid substitutions.

41. An isolated immunogen comprising a trimer of fusion proteins, each fusion protein comprising, from N-terminus to C-terminus: a trimerization domain and one or more copies of a MuV HN ectodomain head or a MeV H ectodomain head, or one or more copies of a MuV HN ectodomain head or a MeV H ectodomain head, a trimerization domain, and one or more copies of a MuV HN ectodomain head or a MeV H ectodomain head, or one or more copies of the MuV HN ectodomain head or stalk and head, or the MeV H ectodomain head or stalk and head, a trimerization domain, and one or more copies of the MuV HN ectodomain head or stalk and head, or the MeV H ectodomain head or stalk and head comprising or consisting of Isolated immunogens.

42. the head of the MuV HN ectodomain comprises or consists of the amino acid sequence set forth as residues 59-510 of SEQ ID NO:58, or a sequence at least 90% identical to residues 59-510 of SEQ ID NO:58; the stalk and head of the MuV HN ectodomain comprise residues 22-550 of SEQ ID NO:90, residues 22-543 of SEQ ID NO:91, residues 22-541 of SEQ ID NO:92, or residues 22-549 of SEQ ID NO:93, or a sequence at least 90% identical to residues 22-550 of SEQ ID NO:90, residues 22-543 of SEQ ID NO:91, residues 22-541 of SEQ ID NO:92, or residues 22-549 of SEQ ID NO:93; the head of the MeV H ectodomain comprises or consists of the amino acid sequence set forth as residues 59-496 of SEQ ID NO:59, or a sequence at least 90% identical to residues 59-496 of SEQ ID NO:59; or the stalk and head of the MeV H ectodomain comprise residues 22-580 of SEQ ID NO:86, residues 22-577 of SEQ ID NO:87, residues 22-579 of SEQ ID NO:88, or residues 22-572 of SEQ ID NO:89, or a sequence at least 90% identical to residues 22-580 of SEQ ID NO:86, residues 22-577 of SEQ ID NO:87, residues 22-579 of SEQ ID NO:88, or residues 22-572 of SEQ ID NO:89; 42. The immunogen of claim 41.

43. the trimerization domain IEDKIEEILSKIYHIENEIARIKKLIGEAP (SEQ ID NO: 33) or comprising the amino acid sequence shown as the trimerization domain GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 34) or comprising the amino acid sequence shown as the trimerization domain IEDKIEEILSKIYHIENEIARIKKLIGEAPGSGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 35) 43. The immunogen of claim 41 or claim 42, comprising the amino acid sequence shown as:

44. 44. The immunogen of any one of claims 41 to 43, wherein the fusion protein in the trimer comprises or consists of residues 24 to 510 of SEQ ID NO:58, residues 24 to 496 of SEQ ID NO:59, residues 22 to 950 of SEQ ID NO:82, residues 25 to 985 of SEQ ID NO:83, residues 22 to 948 of SEQ ID NO:84, or residues 22 to 981 of SEQ ID NO:85, or a sequence at least 90% identical to any one of residues 24 to 510 of SEQ ID NO:58, residues 24 to 496 of SEQ ID NO:59, residues 22 to 950 of SEQ ID NO:82, residues 25 to 985 of SEQ ID NO:83, residues 22 to 948 of SEQ ID NO:84, or residues 22 to 981 of SEQ ID NO:

85.

45. 45. The immunogen of any one of claims 1 to 44, wherein the recombinant MeV F ectodomain trimer protomer, the recombinant MuV F ectodomain trimer protomer, or the fusion protein trimer is fused to a transmembrane domain by a peptide linker or directly to the transmembrane domain.

46. The recombinant MeV F ectodomain trimer or the recombinant MuV F ectodomain trimer protomer is a full-length F 1 46. ​​The immunogen of claim 45, comprising a protein.

47. 47. A virus-like particle comprising the immunogen of any one of claims 1 to 46.

48. 47. A self-assembling protein nanoparticle comprising the immunogen of any one of claims 1 to 46.

49. A nucleic acid molecule encoding the immunogen of any one of claims 1 to 46.

50. 50. The nucleic acid molecule of claim 49, operably linked to a promoter.

51. 51. A vector comprising the nucleic acid molecule of claim 50.

52. 52. The vector of claim 51, which is an RNA vector.

53. 1. An in vitro method for producing an immunogen, comprising: Expressing the nucleic acid molecule of any one of claims 49 to 50 in a host cell; and purifying the immunogen A method comprising:

54. 54. An immunogen produced by the method of claim 53.

55. 55. An immunogenic composition comprising the immunogen, nucleic acid molecule, vector, or virus-like particle of any one of claims 1 to 52 and claim 54, and a pharmaceutically acceptable carrier.

56. 56. The immunogenic composition of claim 55, for inducing an immune response against MuV F, MeV F, MuV HN, and / or MeV H in a subject.

57. 57. The immunogenic composition of claim 56, wherein the immune response inhibits MuV and / or MeV infection in the subject.

58. The immunogenic composition of claim 56 or claim 57, wherein the subject is an adult who has previously been immunized with a live attenuated vaccine against MeV and / or MuV, and administration to the subject boosts the immune response against the MuV and / or MeV.

59. the cysteine ​​substitutions are located at one or more of MuV F positions 86 and 215, positions 165 and 231, positions 206 and 223, and positions 209 and 214; and The recombinant MuV F ectodomain trimer protomer comprises or consists of MuV F positions 104-476, or 104-483. 1 Contains the ectodomain The immunogen according to any one of claims 5 to 20.

60. 42. The immunogen of claim 41, further comprising a peptide linker at the C-terminus, the N-terminus, or both the C-terminus and the N-terminus of the trimerization domain.

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