Immunogens of mumps virus and measles virus and their use

Abstract

The present invention provides polypeptides, polynucleotides, and the like for inducing and detecting immune responses to mumps virus (MuV) and measles virus (MeV). [Solution] An embodiment of an immunogen is provided comprising a MuV F ectodomain trimer or a MeV F ectodomain trimer stabilized in a pre-fusion conformation. Also provided is an embodiment of an immunogen comprising a chimeric protein comprising the recombinant MuV or MeV F ectodomain trimer and one or more MuV HN or MeV H ectodomains. Also disclosed are nucleic acids encoding the immunogen and methods for producing them. Methods for inducing an immune response in a subject by administering the immunogen of this disclosure are also provided. In some embodiments, the immune response treats or inhibits MuV infection and / or MeV infection in the subject.

Description

[Technical Field]

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

[0002] field This disclosure relates to polypeptides, polynucleotides, compositions, and methods of using them for inducing 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 infected individuals or through direct contact with infected individuals. The resulting illness can lead to serious complications or death in children. Existing vaccines for MeV and MuV are attenuated live viruses and are administered as two subcutaneous doses at 1 year of age and as early as 1 month later. Two doses of the measles, mumps, and rubella combined vaccine are effective in 97% of cases against measles and 88% against mumps. A single dose of the measles, mumps, and rubella combined vaccine is effective in 93% of cases against measles and 78% against mumps.

[0004] Despite the effectiveness of currently approved vaccines against MeV and MuV, the incidence of both diseases has been increasing in recent years. Factors contributing to this include declining vaccination rates due to vaccination hesitation and the circulation of divergent strains that offer only limited protection against approved MMR vaccines.

[0005] Recent studies have shown that in the case of MuV, immunity significantly diminishes after the second MMR vaccine, typically administered in childhood. In addition, MuV strains currently in circulation are exhibiting genotypic shifts, moving away from the Jeryl-Lynn strain found in standard mumps vaccines. In response to recent MuV outbreaks in the United States and Europe, the Advisory Committee on Immunization Practices has recommended a third MMR vaccination to boost protection. However, existing immunity may neutralize the effectiveness of a third MMR vaccination, limiting its efficacy. [Overview of the project]

[0006] overview This specification discloses recombinant MuV F ectodomain trimers and recombinant MeV F ectodomain trimers comprising protomers containing one or more modifications (such as amino acid substitutions) to stabilize the pre-fusion conformation. Embodiments of recombinant MuV F ectodomain trimers and recombinant MeV F ectodomain trimers linked to MuV HN ectodomains or MeV H ectodomains are further provided. Chimeric proteins are provided, comprising combinations of MuV F ectodomain trimers and MeV H ectodomains, or combinations of MeV F ectodomain trimers and MuV HN ectodomains. Embodiments of such proteins have been demonstrated to produce superior immune responses in animal models and can be used, for example, to induce or boost an immune response to MeV and / or MuV in a subject.

[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 protomer, wherein the amino acid substitutions include cysteine ​​substitutions that form a non-native disulfide bond for stabilizing the MuV F ectodomain trimer in a pre-fusion conformation. In some embodiments, the recombinant MuV F ectodomain trimer is stabilized in a pre-fusion conformation by a non-native disulfide bond between cysteine ​​substitutions at MuV F positions 206 and 223 in the trimer protomer. In some embodiments, the trimer protomer further comprises mutations for removing the F1 / F2 furin cleavage site of the MuV F ectodomain. In some embodiments, the recombinant MuV F ectodomain protomer is fused at the C-terminus to a trimerizing domain such as the GCN4 trimerizing domain. In a further embodiment, the recombinant MuV F ectodomain trimer protomer is ligated 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 protomer, wherein the amino acid substitutions include cysteine ​​substitutions that form a non-native disulfide bond for stabilizing the MeV F ectodomain trimer in a pre-fusion conformation. In some embodiments, the recombinant MeV F ectodomain trimer is stabilized in a pre-fusion conformation by a non-native disulfide bond between cysteine ​​substitutions at MeV F positions 165 and 171 in the trimer protomer. In some embodiments, the trimer protomer further comprises a mutation for removing the F1 / F2 furin cleavage site of the MeV F ectodomain. In some embodiments, the recombinant MeV F ectodomain protomer is fused at the C-terminus to a trimerizing domain such as a GCN4 trimerizing domain. In a further embodiment, the recombinant MeV F ectodomain trimer protomer is ligated to a heterologous protein such as a MuV HN ectodomain or a MeV H ectodomain.

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

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

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

[0012] Immunogenic compositions containing the immunogen of this disclosure, suitable for administration to a target, are also provided, which may be contained in a 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 an effective amount of the immunogen, nucleic acid molecule, or vector of this disclosure to the subject.

[0014] The aforementioned and other features and advantages of this disclosure will become even more apparent from the detailed description of several embodiments set forth below with reference to the accompanying drawings. [Brief explanation of the drawing]

[0015] [Figure 1A] Figures 1A-1E. Structure-based design of disulfide-stabilized pre-fusion mumps F trimer. (Figure 1A) Structure-based design of pre-fusion mumps F glycoprotein trimer by systematic screening of disulfide and GCN4 attachment sites. Both yield and the percentage of pre-fusion conformation to post-fusion conformation determined by negative staining EM are shown. [Figure 1B] (Figure 1B) The combination of V206C / A223C and 476-GCN4 resulted in a high yield of uniform pre-fusion F trimers (upper figure). The post-fusion product is shown in the lower figure. [Figure 1C] (Figure 1C) S200 gel filtration analysis shows the monodispersity of pre-glycosylation and deglycosylation fusion F (PreF), HN, and pre-fusion 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 cause a >5 Å conformational change to transition to the post-fusion conformation are shown in black. The GCN4 trimerization (TD) motif linked to F residue 476 is shown by a dotted line. In the magnified view, mutant residues that stabilize the pre-fusion F structure are highlighted. [Figure 1E](FIG. 1E) Single protomer of pre-fusion mumps trimer showing D1-3 subregions and the location of six N-linked glycans per protomer. [Figure 2-1] FIGS. 2A-2H. Structural comparison of pre-fusion F of mumps and PIV5, and analysis of genotypic variations of mumps F and mumps HN. (FIG. 2A) Superposition of the structures of pre-fusion F trimer of mumps and pre-fusion F trimer of the related paramyxovirus PIV5. Although showing a similar overall topology (RMSD = 1.68 Å), the sequence identity is 49%. (FIG. 2B) The apical loops of mumps preF associate in a “closed cap” assembly, while in PIV5 these loops are spread out (upper figure), and the residues stabilizing the mumps preF apex are T178, T179, and N181 (lower figure). (FIG. 2C) The mumps F glycoprotein is glycosylated with six glycans per protomer and a total of 18 glycans per trimer, of which six glycans are in conformationally mobile regions (black) and 12 glycans are in regions with less conformational variability, providing substantial glycan shielding to the trimer. [Figure 2-2] (FIG. 2D) Phylogenetic analysis of fusion glycoproteins from mumps genotypes A-J. (FIG. 2E) Phylogenetic analysis of hemagglutinin-neuraminidase glycoproteins from mumps genotypes A-J. [Figure 2-3] (FIGS. 2F and 2G) Sequence alignment of 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 the description of FIGS. 2-3. [Figure 2-5](Fig. 2H) Structural mapping of mumps genotype mutations of F (left panel) and HN (right panel) before fusion. A-J genotype mutations (upper panel) and genotype G to A (Jeryl Lynn (JL)) (lower panel) are shown, indicating residue differences and glycan differences. [Figure 3A] Figs. 3A and 3B. Design and immunization scheme for mumps F and preF-HN immunogens. (Fig. 3A) In CB6F1 / J mice, immunization was performed three times at 0, 3, and 10 weeks at 10 μg per time using poly(I:C) adjuvant, and blood samples were collected at 2, 5, 12, and 16 weeks. [Figure 3B] (Fig. 3B) Negative staining characterization of mumps F and preF-HN proteins confirming conformational and molecular identity of the immunogens, and mumps neutralization titers for post-fusion F (left), pre-fusion F (center) and pre-fusion F-HN (right) immunogens induced after 1, 2 or 3 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] Figs. 4A-4E. Design of pre-fusion mumps F-HN immunogen, serum analysis of specificity and persistence of neutralization titers for three different mumps genotype viruses. (Fig. 4A) Negative staining characterization of mumps preF-HN fusion protein. Positions of preF head, GCN4 trimerization domain, and three HN heads are shown. (Fig. 4B) Binding titers of immunized mice using mumps pre-fusion F probe and (Fig. 4C) HN probe by Octet Bioinferometry. [Figure 4-2] (Fig. 4D) Persistence analysis of mice immunized by administering 10 μg of pre-fusion F protein three times together with poly(I:C), with monthly serum sample collection and PRNT analysis against genotype G virus, Jeryl Lynn virus and genotype H virus over 6 months. [Figure 4-3] (Figure 4E) Sustained-release analysis of mice immunized with 10 μg of pre-fusion F-HN protein in three doses with poly(I:C), and serum samples were collected monthly for 6 months, along with PRNT analysis for genotype G virus, Jeryl Lynn virus, and genotype H virus. [Figure 5A] Figures 5A-5C. Structural design of pre-fusion measles F. (Figure 5A) Structural design of pre-fusion-stabilized measles F glycoprotein mutations that stabilize the pre-fusion conformation. [Figure 5B] (Figure 5B) Negative staining EM analysis of the measles F mutant, including the non-native disulfide bond and the C-terminal GCN4 trimerization domain shown. [Figure 5C] (Figure 5C) Immunogenicity of measles F glycoprotein before fusion (MeV F R165C / M171C-486-GCN4, SEQ ID NO:38) and after fusion (native MeV F sequence with -486-GCN4). [Figure 6A] Figures 6A-6C. Design of the mumps pre-F-measles H chimeric immunogen. (Figure 6A) Negative staining characterization of measles H dimer (upper left), measles H trimer (upper center), mumps HN trimer (upper right), and mumps pre-F with measles H (MuV F 206C / 223C-476+GCN4 / Fd+MeV-H, SEQ ID NO:28) (bottom). [Figure 6B] (Figure 6B) Measles plaque reduction neutralization assay (PRNT). [Figure 6C] (Figure 6C) Mumps (genotype G) PRNT assay.

[0016] Sequence List The nucleic acid and amino acid sequences listed in the attached sequence listing are indicated using standard letter abbreviations for nucleotide bases and three-letter symbols for amino acids, as specified in 37C.FR1.822. Only one strand of each sequence is shown, but references to the shown strand are understood to include the complementary strand. The sequence listing was submitted as an ASCII text file named "Sequence.txt" (approximately 456kb), created on December 11, 2020, and is incorporated herein by reference.

[0017] Structural coordinates The atomic coordinates of the crystal structure of the MuV F ectodomain trimer stabilized in the pre-fusion conformation are presented in Table 1 of U.S. Provisional Application No. 62 / 946,902, filed on 11 December 2019, which is incorporated herein by reference in its entirety and has also been submitted as an ASCII text file named "Table_1.txt" (approximately 555KB), created on 9 December 2019. [Modes for carrying out the invention]

[0018] Detailed explanation I. Glossary of Terms Unless otherwise noted, technical terms are used according to conventional usage. Definitions of general terms in molecular biology can be found in Benjamin Lewin, Genes X (Jones & Bartlett Publishers, 2009) and Meyers et al., 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 singular and plural, unless the context indicates otherwise. For example, the term “an antigen” encompasses one or more antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” Furthermore, any base size or amino acid size and any molecular weight or molecular mass values ​​given to nucleic acids or polypeptides are approximate and provided for illustrative purposes only, unless otherwise indicated. Appropriate methods and materials are described below in detail, but many similar or equivalent methods and materials can be used. In case of any inconsistency, this specification shall prevail, including the definition of terms. Furthermore, the materials, methods, and examples are illustrative and not intended to be limiting. A definition of terms is provided below to facilitate consideration of various embodiments.

[0019] Adjuvant: A medium used to enhance antigenicity. In some embodiments, the adjuvant includes a suspension of an inorganic substance (alum, aluminum hydroxide, or phosphate) on which the antigen is adsorbed, or a water-in-oil emulsion in which, for example, the antigen solution is emulsified in mineral oil (Freund's incomplete adjuvant), and possibly includes dead mycobacteria to further enhance antigenicity (Freund's complete adjuvant) (which inhibits the degradation of the antigen and / or induces macrophage influx). In some embodiments, the adjuvant used in the immunogenic compositions of this disclosure is a combination of lecithin and a carbomer homopolymer (see also ADJUPLEX® adjuvant, e.g., available from Advanced BioAdjuvants, LLC; Wegmann, Clin Vaccine Immunol, 22(9):1004-1012, 2015). Further adjuvants for use in the immunogenic compositions of this disclosure include QS21 purified plant extract, Matrix M, AS01, MF59, and ALFQ adjuvants. Immunostimulating oligonucleotides (such as those containing CpG motifs) can also be used as adjuvants. Adjuvants also include biological molecules such as co-stimulatory molecules ("biological adjuvants"). 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, immunostimulating complex (ISCOM) matrices, and Toll-like receptor (TLR) agonists, such as TLR-9 agonists, poly-I:C, or poly-ICLC. (See, for example, Singh (ed.), Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007).

[0020] Administration: Introduction of the composition to the subject via a selected route. Administration may be topical or systemic. For example, if the selected route is intranasal, the composition (e.g., a composition comprising recombinant MuV F ectodomain trimer or recombinant MeV F ectodomain trimer as disclosed herein) is administered by introducing the composition into the nasal passages of the 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 one or more different amino acids in a polypeptide. In relation to protein sequences, amino acid substitutions are also called mutations.

[0022] Antibody: An immunoglobulin, antigen-binding fragment, or derivative thereof that specifically binds to and recognizes an analyte (antigen), such as the MuV or MeV F protein, its antigenic fragment, or a dimer or multimer of the antigen. The term “antibody” is used most broadly 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, as long as they exhibit the desired antigen-binding activity. Non-limited examples of antibodies include, for example, intact immunoglobulins, as well as their variants and fragments that retain binding affinity to 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 the entire antibody, or those newly synthesized using recombinant DNA methods (e.g., Kontermann and Dubel, Antibody Engineering, Vols. 1-2, 2 nd (See Ed., Springer Press, 2010).

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

[0024] Conservative Variants: A “conservative” amino acid substitution is one that does not substantially alter or diminish the function of a protein, such as its ability to induce an immune response when administered to a target. The term conservative mutation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Furthermore, individual substitutions, deletions, or additions that modify, add, or delete a single amino acid or a small fraction of amino acids (e.g., less than 5%, and in some embodiments less than 1%) in the encoded sequence are also considered conservative mutations if their modification results in the substitution of an amino acid with a chemically similar amino acid.

[0025] The following six groups are examples of amino acids that are considered to be conserved substitutions with each other: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0026] Non-conservative substitutions reduce the activity or function of recombinant MuV or MeV F ectodomain trimers, such as their ability to induce an immune response when administered to a target. For example, if a certain amino acid residue is essential to the function of the protein, even an otherwise conservative substitution can disrupt its activity. Thus, conservative substitutions do not alter the fundamental 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 standard reference value or standard reference range (e.g., a previously tested control sample, e.g., a group of MuV or MeV patients with known prognosis or outcome, or a sample group 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, such as a statistically significant difference. In some cases, 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 Variants: For the purposes of this disclosure, “degenerate variant” means a polynucleotide encoding a polypeptide that contains a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by two or more codons. Therefore, a degenerate nucleotide sequence encoding a peptide includes all such sequences, insofar as the amino acid sequence of the peptide encoded by that nucleotide sequence remains unchanged.

[0030] Effective dose: The amount of an active substance, such as an immunogen, sufficient to induce a desired response, such as an immune response, in a subject. It is understood that obtaining a protective immune response against an antigen of interest may require multiple administrations of the immunogen of this disclosure, and / or administration of the immunogen of this disclosure as a "prime" in a prime-boost protocol (in which case the boost immunogen may differ from the prime immunogen). Therefore, the effective dose of the immunogen of this disclosure may be the amount of immunogen sufficient to induce a priming immune response in a subject, and then a protective immune response can be induced by boosting the subject with the same or a different immunogen.

[0031] In one example, the desired response is to inhibit, reduce, or prevent MuV infection. For this method to be effective, it is not necessary for MuV infection to be completely eliminated, reduced, or prevented. For example, administration of an effective amount of the active ingredient can reduce MuV infection (measured, for example, by infection of cells with MuV, or by the number or percentage of subjects infected with MuV) by a desired amount compared to a suitable control, for example, 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% (elimination or prevention of detectable MuV infection).

[0032] In one example, the desired response is to inhibit, reduce, or prevent MeV infection. For this method to be effective, it is not necessary for MeV infection to be completely eliminated, reduced, or prevented. For example, administration of an effective amount of the active ingredient can reduce MeV infection (measured, for example, by MeV infection of cells or by the number or percentage of subjects infected with MeV) by a desired amount compared to a suitable control, for example, 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% (elimination or prevention of detectable MeV infection).

[0033] In one example, the desired response is to inhibit, reduce, or prevent both MuV and MeV infections. For this method to be effective, it is not necessary for MuV and MeV infections to be completely eliminated, reduced, or prevented. For example, administration of an effective dose of the active ingredient can reduce MuV and MeV infections (measured, for example, 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 compared to a suitable control, for example, 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% (elimination or prevention of detectable MuV and / or MeV infections).

[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 cases is processed into mRNA. A gene may also be expressed if its mRNA is translated into an amino acid sequence, such as a protein or a protein fragment. In certain specific cases, 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 may 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 their production.

[0035] Regulatory Sequence: A nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence to which it is functionally linked. A regulatory sequence is functionally linked to a nucleic acid sequence if it controls and regulates the transcription and, where appropriate, the translation of the nucleic acid sequence. Therefore, regulatory sequences can include appropriate promoters, enhancers, transcriptional terminators, start codons (ATGs) before protein-coding genes, splicing signals for introns, maintenance of the correct reading frame of the gene to enable proper translation of mRNA, and stop codons. The term "regulatory sequence" includes at least a component whose presence can affect expression, and may also include additional components whose presence is beneficial, such as leader sequences and fusion partner sequences. Regulatory sequences can include promoters.

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

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

[0038] GCN4 trimerizing domain: A trimerizing domain from the GCN4 protein containing a leucine zipper amino acid sequence that spontaneously forms a trimer structure. Embodiments of the GCN4 trimerizing domain are described, for example, in Harbury et al. (1993 Science 262:1401-1407). In some examples, the amino acid sequence of the recombinant protein of this disclosure may include the GCN4 trimerizing domain so that the recombinant protein trimers. Non-limiting examples of GCN4 trimerizing domain sequences for use in embodiments of this disclosure are: It is given in TIFF2026062907000002.tif4128.

[0039] Different species: Derived from different genetic sources.

[0040] Host cell: A cell within which a vector can grow and express its nucleic acid. The cell can be prokaryotic or eukaryotic. This term also encompasses any offspring of the target host cell. It is understood that not all offspring are identical to the parent cell due to possible mutations during replication. However, when the term "host cell" is used, such offspring are included.

[0041] Immune response: The response of cells of the immune system, such as B cells, T cells, or monocytes, to a stimulus. In one embodiment, the response is specific to a particular antigen ("antigen-specific response"). In one embodiment, the immune response is a T cell response, e.g., 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., recombinant MuV or MeV F ectodomain trimer) that can induce an immune response in an animal. This includes compositions that are injected or absorbed by the animal. Administration of an immunogen to a subject may lead to protective immunity against the pathogen of interest.

[0043] Immunogenic composition: A composition comprising the immunogen of the present disclosure that, when administered to a subject, induces a measurable CTL response to MuV or MeV, or a measurable B-cell response (e.g., antibody production) to MuV or MeV. This also refers to isolated nucleic acid molecules and vectors encoding the recombinant MuV or MeV F ectodomain trimer protomer of the present disclosure, which can be used to express the protomer (and therefore to induce an immune response to the recombinant MuV or MeV F ectodomain trimer). When used in vivo, the immunogenic composition would typically contain the recombinant MuV or MeV F ectodomain trimer or a nucleic acid molecule encoding the recombinant MuV or MeV F ectodomain trimer protomer in a pharmaceutically acceptable carrier, and may also contain other active agents such as adjuvants.

[0044] Disease inhibition or treatment: Inhibition of the complete onset of a disease or condition in a person at risk of a disease, such as MuV or MeV infection. "Treatment" refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has begun to develop. The term "improve" with respect to a disease or pathological condition refers to any observable, beneficial effect of the treatment. Disease inhibition can include preventing a disease or reducing the risk of a disease, such as preventing a viral infection or reducing the risk of a viral infection. Beneficial effects can be demonstrated, for example, by delaying the onset of clinical symptoms of a disease in a susceptible person, 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 person, or by other parameters specific to that particular disease. "Prophylactic" treatment is a treatment performed on a person who does not show signs of a disease or shows only early signs, with the aim of reducing the risk of the pathology developing.

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

[0046] Linkers and linked molecules: Bifunctional molecules that can be used to link two molecules together into a single continuous molecule. A non-limiting example of a peptide linker is the glycine-serine peptide linker. When we say that a first polypeptide "links" a second polypeptide, or that two polypeptides are "linked" to each other, or that the first polypeptide has a "linking" to the second polypeptide, we mean a covalent linking via peptide bonds (e.g., via a peptide linker) such that the first polypeptide and the second polypeptide form a continuous polypeptide chain, unless the context indicates otherwise. When a peptide linker is involved, the covalent linking of the first polypeptide and the second polypeptide can be linking to the N-terminus and C-terminus of the peptide linker. Typically, such linking is achieved by using molecular biology techniques to genetically engineer the DNA encoding the first polypeptide linked to the second polypeptide by a peptide linker.

[0047] Native protein, sequence, or disulfide bond: A polypeptide, sequence, or disulfide bond that has not been modified by selective mutation or other means. For example, a selective mutation used to concentrate the antigenicity of an antigen on a target epitope, or to introduce a disulfide bond into a protein that is not found in the native protein. The native protein or native sequence is also called the wild-type protein or wild-type sequence. A non-native disulfide bond is a disulfide bond that is not present in the native protein, for example, a disulfide bond that is formed in a protein by introducing one or more cysteine ​​residues into the protein through genetic engineering.

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

[0049] Measles virus: A non-segmented minus-strand RNA virus belonging to the genus Morbillivirus of the family Paramyxoviridae, which causes measles disease. The measles virus genomic RNA contains six linked transcription units that encode 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, which are called genotypes A, B, C, D, F, G, and H.

[0050] MeV fusion (F) protein: The envelope glycoprotein of MeV that facilitates the fusion of the viral membrane and the cell membrane. In nature, the F protein from MeV is first synthesized as a single polypeptide precursor called F0, which is approximately 550 amino acids long. 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 proteolytically processed by cellular proteases to produce 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), which includes the extracellular / luminal region (approximately residues 110–486), as well as the transmembrane and cytosolic regions (approximately residues 487–550). The extracellular portion of the MeV F protein is the MeV F ectodomain, which contains the F2 protein and the F1 ectodomain.

[0051] The MeV F protein exhibits remarkable sequence conservation within the MeV strain. Because of this conservation, those skilled in the art can easily compare the 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 otherwise indicated by the context. This is done by referring to TIFF2026062907000003.tif26160.

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

[0053] A MeV F ectodomain trimer “stabilized in a pre-fusion conformation” includes one or more amino acid substitutions, deletions, or insertions that increase the retention rate of the pre-fusion conformation compared to a MeV F ectodomain trimer formed from the corresponding native MeV F sequence, compared to 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 compared 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). In addition, stabilization of the MeV F ectodomain trimer in a pre-fusion conformation may include increased resistance to denaturation compared to the corresponding native MeV F sequence. Methods for determining whether a MeV F ectodomain trimer is present in a pre-fusion conformation are provided herein and include, but are not limited to, negative staining electron microscopy and antibody-binding assays using pre-fusion conformation-specific antibodies. With respect to MeV F proteins, the term "pre-F" refers to a molecule that is a trimer class I fusion protein stabilized in its pre-fusion conformation by one or more amino acid substitutions.

[0054] MeV F pre-fusion specific antibody: An antibody that specifically binds to the MeV F protein in its pre-fusion conformation but does not specifically bind to the MeV F protein in its post-fusion conformation.

[0055] MeV hemagglutinin (H) protein: A type II membrane protein, a MeV envelope glycoprotein that facilitates the 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) containing a stalk region (approximately amino acids 59-179) and a head region (approximately amino acids 180-617). An exemplary MeV H protein sequence is shown herein as SEQ ID NO: 49 (NCBI reference sequence AAA56644.1 incorporated herein by reference): It will be provided as TIFF2026062907000004.tif29160.

[0056] The positioning of MeV H residues used herein is performed by referring to the sequence indicated 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 glands. 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 minus-strand RNA virus belonging to the genus Rubulavirus, subfamily Paramyxovirinae, family Paramyxoviridae, that causes mumps disease. The mumps virus genome RNA contains seven sequentially linked transcription units that encode 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 called the "V / P / I gene") yields three mRNA transcripts corresponding to the V, P, and I proteins through RNA editing by guanine nucleotide insertion. Specifically, faithful transcription of the P gene produces the V protein, insertion of two guanine nucleotides produces mRNA encoding the P protein, and insertion of four guanine residues produces 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 currently 12 known MuV genotypes circulating worldwide, which are called genotypes A, B, C, D, F, G, H, I, J, K, L, and N. Modern MuV vaccines are based on viruses of genotype A (Jeryl Lynn), genotype B (Urabe-AM9), or undetermined genotype (Leningrad-Zagreb).

[0059] MuV fusion (F) protein: The envelope glycoprotein of MuV that facilitates the fusion of the viral membrane and the cell membrane. In nature, the F protein from MuV is first synthesized as a single polypeptide precursor called F0, approximately 538 amino acids long. 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 proteolytically processed by cellular proteases to produce 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), which includes the extracellular / luminal region (approximately residues 110–483), as well as the transmembrane and cytosolic regions (approximately residues 484–538). The extracellular portion of the MuV F protein is the MuV F ectodomain, which contains the F2 protein and the F1 ectodomain.

[0060] The MuV F protein exhibits remarkable sequence conservation within the MuV strain. Because of this conservation, those skilled in the art can easily compare the amino acid positions of different MuV F proteins. The numbering of MuV F amino acids follows SEQ ID NO:1 (NCBI reference sequence P09458.1, incorporated herein by reference), unless otherwise indicated by the context. This will be done by referring to TIFF2026062907000005.tif25160.

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

[0062] A MuV F ectodomain trimer “stabilized in a pre-fusion conformation” includes one or more amino acid substitutions, deletions, or insertions that increase the retention rate of the pre-fusion conformation compared 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 compared 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). In addition, stabilization of the MuV F ectodomain trimer in a pre-fusion conformation may include increased resistance to denaturation compared to the corresponding native MuV F sequence. Methods for determining whether a MuV F ectodomain trimer is present in a pre-fusion conformation are provided herein and include, but are not limited to, negative staining electron microscopy and antibody-binding assays using pre-fusion conformation-specific antibodies. With respect to MuV F proteins, the term "pre-F" refers to a molecule that is a trimer class I fusion protein stabilized in its pre-fusion conformation by one or more amino acid substitutions.

[0063] MuV hemagglutinin-neuraminidase (HN) protein: A type II membrane protein, the MuV envelope glycoprotein, which facilitates the 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) containing a stalk region (approximately amino acids 54-130) and a head region (approximately amino acids 131-582). An exemplary MuV HN protein sequence is shown herein as SEQ ID NO: 50 (NCBI reference sequence AQT03695.1 incorporated herein by reference): It will be provided as TIFF2026062907000006.tif30160.

[0064] The MuV HN residues used herein are located by referring to the sequence indicated as SEQ ID NO:50.

[0065] MuV F pre-fusion specific antibody: An antibody that specifically binds to the MuV F protein in its pre-fusion conformation but does not specifically bind to the MuV F protein in its post-fusion conformation.

[0066] Nucleic acid molecules: Polymerized nucleotides, which may include both the sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic types, as well as mixed polymers of the above. A nucleotide refers to a ribonucleotide, a deoxynucleotide, or a modified form of either type of nucleotide. As used herein, the term “nucleic acid molecule” is synonymous with “nucleic acid” and “polynucleotide.” Unless otherwise specified, a nucleic acid molecule is typically at least 10 nucleotides long. This term encompasses single-stranded and double-stranded DNA. Polynucleotides may include one or both of native and modified nucleotides linked together by native and / or non-native nucleotide linkages. “cDNA” refers to DNA that is complementary to or identical to mRNA, and is single-stranded or double-stranded. "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, that it serves as a template for synthesizing other polymers and macromolecules that possess either a given nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a given amino acid sequence, along with the biological properties it imposes, in biological processes.

[0067] Functionally linked: If a first nucleic acid sequence is positioned in a functional relationship with a second nucleic acid sequence, then the first nucleic acid sequence is functionally linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, then the promoter is functionally linked to that coding sequence. In general, functionally linked nucleic acid sequences are contiguous and, when two protein-coding regions need to be joined, they are in the same reading frame.

[0068] Pharmacokinetically acceptable carriers: Useful pharmaceutically acceptable carriers remain the same as before. EW Martin's Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pennsylvania, 19th edition, 1995) describes compositions and formulations suitable for the pharmaceutically acceptable delivery of the immunogens of this disclosure.

[0069] Generally, the properties of the carrier will depend on the specific administration method used. For example, parenteral formulations typically contain an injectable fluid with a pharmaceutically and physiologically acceptable fluid as the medium, such as water, saline, equilibrium salt solutions, dextrose aqueous solution, or glycerol. In the case of solid compositions (e.g., powders, pills, tablets, or capsules), conventional non-toxic solid carriers include, for example, pharmaceutically-grade mannitol, lactose, starch, or magnesium stearate. The administered pharmaceutical composition (such as an immunogenic composition) may also contain trace amounts of non-toxic adjuncts, such as sodium acetate or sorbitan monolaurate, in addition to a biologically neutral carrier, such as wetting or emulsifying agents, preservatives, and pH buffers. In specific embodiments suitable for administration to a subject, the carrier may be sterile and / or contained in a unit dosage form containing one or more measured doses of the composition suitable for inducing a desired immune response, suspended or otherwise included. The drug for use for treatment purposes may also be present. The unit dosage form may be, for example, in a sealed vial containing sterile contents, or it may be lyophilized for later dissolution and administration, or it may be in a solid or controlled-release form.

[0070] Polypeptide: Any chain of amino acids, regardless of length or post-translational modifications (e.g., glycosylation or phosphorylation). “Polypeptide” applies to natural and non-natural amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are non-natural amino acids, e.g., artificial chemical mimetics of the corresponding natural amino acids. “Residue” means 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 carboxyl terminus (C-terminus). “Polypeptide” is used interchangeably with peptide or protein, and in this specification, it is used to refer to a polymer of amino acid residues.

[0071] Primer-boost vaccination: An immunotherapy comprising administering a first immunogenic composition (primer vaccine) to a subject to induce an immune response, followed by the administration of a second immunogenic composition (booster vaccine). The primer vaccine and / or booster vaccine comprises a vector (such as a viral vector, RNA, or DNA vector) expressing an antigen that is the target of the immune response. The booster vaccine is administered to the subject after the primer vaccine, and appropriate time intervals and examples of such time frames between the administration of the primer vaccine and the administration of 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 comprises 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, polyhedral structures based on proteins. Each subunit consists of a protein or polypeptide (e.g., 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, incorporated herein by reference). Examples include (see 96:1240-1245, 1999). Ferritin, encapsulin, SOR, lumazine synthase, and pyruvate dehydrogenase are monomeric proteins that self-assemble to form a globular protein complex, which may consist of 24, 60, 24, 60, and 60 protein subunits, respectively. In some cases, ferritin, encapsulin, SOR, lumazine synthase, or pyruvate dehydrogenase monomers are linked to recombinant MuV or MeV F ectodomains, and they self-assemble to form protein nanoparticles that present recombinant MuV or MeV F ectodomain trimers on their surface, which can be administered to targets to stimulate an immune response to an antigen.

[0073] Recombinant: Recombinant nucleic acid molecules have sequences that are not native, such as sequences containing substitutions, deletions, or insertions of one or more nucleic acids, and / or sequences created by the artificial combination of two sequence segments that would normally be separated. This artificial combination can be achieved by chemical synthesis or, more generally, by the artificial manipulation of isolated nucleic acid segments, for example, by genetic engineering techniques.

[0074] Recombinant viruses contain a genome that includes recombinant nucleic acid molecules.

[0075] Recombinant proteins are those that have non-natural sequences or sequences created by artificially combining two sequence segments that would normally be separated. In some embodiments, recombinant proteins are encoded by heterologous (e.g., recombinant) nucleic acids that have been introduced into host cells such as bacterial or eukaryotic cells, or into the genome of a recombinant virus.

[0076] Sequence Identity: The similarity between amino acid sequences is expressed by sequence similarity, also known as sequence identity. Sequence identity is often measured by an identity percentage, with a higher percentage indicating greater similarity between the two sequences. Homologs, orthologues, or variants of polypeptides will exhibit 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. This is described in al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Bio. 215:403-10, 1990, provides a detailed discussion of sequence alignment methods and homology calculations.

[0078] Polypeptide variants typically retain at least approximately 75% sequence identity, for example, at least approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, when counted in full-length alignment with the amino acid sequence of interest. Proteins with greater similarity to the reference sequence will show increased identity percentages when evaluated by this method, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When the sequence being compared for sequence identity is shorter than the entire sequence, homologs and variants typically have at least 80% sequence identity in a short window of 10–20 amino acids, and may have 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 in such short windows are available on the NCBI website.

[0079] In this specification, when we say “at least 90% identity” (or a similar expression), we mean “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” with respect to a given reference array.

[0080] Signal peptides are short amino acid sequences (e.g., approximately 18–25 amino acids long) that direct newly synthesized secretory or membrane proteins toward and across the membrane (e.g., the endoplasmic reticulum membrane). Signal peptides are typically located at the N-terminus of a polypeptide and are removed by signal peptidases after the polypeptide has crossed 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 refers to a binding reaction that determines the presence of a target protein, peptide, or polysaccharide (e.g., glycoprotein) in the presence of a heterogeneous population of proteins and other biologics, in relation to the formation of antibody:antigen-protein complexes or protein:protein complexes. Therefore, 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., the antigenic site at the distal apex of the membrane 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 or alternative conformations of the same protein (e.g., the post-fusion conformation of the MuV or MeV F protein) present in the sample or subject. Specific binding can be determined by methods known in the art. The first protein or antibody is the K of its interaction. D 10 -6 Less than molar concentration, for example, 10 -7 Less than molar concentration, 10 -8 Less than molar concentration, 10 -9 Less than molar concentration, or even less than 10 -10 When the concentration is below molar level, it binds specifically to the target protein.

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

[0083] Subjects: Living multicellular vertebrates, this category includes humans and non-human mammals. In one example, the subject is human. In a specific example, the subject is a neonatal / newborn. In yet another example, subjects requiring inhibition of MuV or MeV infection are selected. For example, subjects are uninfected and at risk of MuV or MeV infection, or are infected and require treatment.

[0084] T4 fibrintin trimerizing domain: Also known as the "foldon" domain, the T4 fibrintin trimerizing domain contains an amino acid sequence that naturally forms a trimer structure. In some cases, the T4 fibrintin trimerizing domain can be included in the amino acid sequence of the recombinant protein of this disclosure so that the antigen forms a trimer. In one example, the T4 fibrintin trimerizing domain is: The amino acid sequence is represented as TIFF2026062907000007.tif4128. Some embodiments include a T4 fibrintin trimer domain that can be cleaved from the purified protein, for example, by incorporating a thrombin cleavage site adjacent to the T4 fibrintin trimer domain that can be used for cleavage.

[0085] Transmembrane domain: An amino acid sequence inserted into a lipid bilayer, such as the lipid bilayer of a cell, virus, or virus-like particle. Transmembrane domains can be used to immobilize antigens to the membrane. In some examples, the transmembrane domain is the MuV F transmembrane domain. In other examples, the transmembrane domain is the MeV F transmembrane domain.

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

[0087] Vaccine: A preparation of immunogenic material that can stimulate an immune response, administered for the prevention, improvement, or treatment of an infectious disease or other type of disease. Immunogenic material may include attenuated or dead microorganisms (such as bacteria or viruses), or antigenic proteins, peptides, or DNA derived therefrom. A vaccine may include the immunogen of this disclosure (such as recombinant MuV or MeV F ectodomain trimers or nucleic acid molecules encoding them), viruses, cells, or one or more cellular components. A vaccine may induce both a prophylactic (preventive or protective) response and a therapeutic response. The method of administration varies depending on the vaccine and may include inoculation, ingestion, inhalation, or other forms of administration. A vaccine may be administered with an adjuvant to boost the immune response. In one non-limiting specific example, a vaccine may prevent and / or reduce the severity of symptoms associated with MuV infection, and / or reduce the viral load, compared to a control. In another non-specific example, the vaccine prevents and / or reduces the severity of symptoms associated with MeV infection, and / or reduces the viral load, compared to a control.

[0088] A vector is an entity containing a DNA or RNA molecule that is functionally linked to the coding sequence of an antigen of interest and holds a promoter capable of expressing the coding sequence. Non-limiting examples include naked or packaged (lipid and / or protein) DNA, naked or packaged RNA, subcomponents of viruses or bacteria or other microorganisms that may be non-replicable, and viruses or bacteria or other microorganisms that may be replicable. A vector is sometimes called a construct. A recombinant DNA vector is a vector that contains recombinant DNA. A vector may contain nucleic acid sequences, such as an origin of replication, that enable it to replicate in a host cell. A vector may 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 several nucleic acid sequences derived from one or more viruses.

[0089] Virus-like particles (VLPs): Non-replicating viral shells derived from one of several viruses. VLPs generally consist of one or more viral proteins, such as, but not limited to, capsid, coat, shell, surface, and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can be spontaneously formed during recombinant expression of proteins in a suitable expression system. Methods for producing specific VLPs are known in the art. The presence of VLPs after recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as electron microscopy and biophysical characterization. Furthermore, VLPs can be isolated using known techniques, such as density gradient centrifugation, and identified by characteristic density band formation. See, for example, Baker et al. (1991) Biophys. J. 60:1445-1456 and Hagensee et al. (1994) J. Virol. 68:4503-4505, Vincente, J Invertebr Pathol., 2011, and Schneider-Ohrum and Ross, Curr. Top. Microbiol. Immunol., 354:53073, 2012).

[0090] II. Immunogens A. Recombinant MuV F ectodomain trimer Recombinant MuV F ectodomain trimers modified from the native form (e.g., by introducing one or more amino acid substitutions) to stabilize the pre-fusion conformation are disclosed herein. As described in the examples, embodiments of the MuV F ectodomain trimers of this disclosure were selected by multiple rounds of structure-based design to obtain optimized solubility, stability, expression, and immunogenicity. Recombinant MuV F ectodomain trimers are useful for inducing an immune response to MuV in vertebrates (such as humans). Exemplary embodiments are shown to produce a superior immune response in animal models compared to the corresponding MuV F ectodomain trimer that is 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 a 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 protomer, the amino acid substitutions comprising cysteine ​​substitutions that form a non-native disulfide bond for stabilizing the MuV F ectodomain trimer in a pre-fusion conformation. The non-native disulfide bond is not found in the native MuV F protein and is introduced by protein engineering (e.g., by including one or more substituted cysteine ​​residues that form a non-native disulfide bond). For example, in some embodiments, any recombinant MuV F protein of the present disclosure may 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] Cysteine ​​residues that form disulfide bonds 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 cysteine(s) of the unnatural disulfide bond can be determined by those skilled in the art using the structure of the MuV F ectodomain trimer in the pre-fusion conformation of this disclosure.

[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., those 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®); these references are incorporated herein by reference, respectively). Residues can be manually selected 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 adhering strictly to theory, the ideal distances for disulfide bond formation are generally considered to be around 5.6 Å for the Cα-Cα distance, 2.02 Å for the Sγ-Sγ distance, and 3.5–4.25 Å for the Cβ-Cβ distance (using the optimal rotomer). It will be understood by those skilled in the art that variations from these distances are involved when selecting residues in a three-dimensional structure that can be used in place of cysteine ​​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 protomer includes cysteine ​​substitutions (e.g., N86C and A215C substitutions) at MuV F positions 86 and 215, which form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0096] In some embodiments, the recombinant MuV F ectodomain trimer protomer includes cysteine ​​substitutions (e.g., K155C and L161C substitutions) at MuV F positions 155 and 161, which form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0097] In some embodiments, the recombinant MuV F ectodomain trimer protomer includes cysteine ​​substitutions at MuV F positions 165 and 231 (e.g., V165C and M231C substitutions) that form intra-protomer disulfide bonds in the non-natural protomer for stabilization in the pre-fusion conformation.

[0098] In some embodiments, the recombinant MuV F ectodomain trimer protomer includes cysteine ​​substitutions at MuV F positions 206 and 223 (e.g., V206C and A223C substitutions) that form intra-protomer disulfide bonds in the non-natural protomer for stabilization in the pre-fusion conformation.

[0099] In some embodiments, the recombinant MuV F ectodomain trimer protomer includes cysteine ​​substitutions (e.g., P209C and P214C substitutions) at MuV F positions 209 and 214, which form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0100] In some embodiments, the recombinant MuV F ectodomain trimer protomer includes cysteine ​​substitutions (e.g., I221C and M255C substitutions) at MuV F positions 221 and 255, which form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0101] Any of the recombinant MuV F proteins described above may further include modifications to eliminate the protease cleavage site between the F1 and F2 polypeptides to produce a "single-chain" recombinant F protein. For example, any of the recombinant MuV proteins described above may include deletions at MuV F positions 101-103 and positions 100 and 104 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 can be used to fuse the F2 and F1 ectodomains and enable the folding of the F ectodomain into the pre-fusion conformation. 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 a non-limiting example, recombinant MuV F ectodomain trimers are provided, including protomers having V206C and A223C substitutions to form non-natural disulfide bonds, as well as deletions of MuV F positions 101-103 and consequently positions 100 and 104 fused with a Gly-Gly-Gly peptide linker.

[0103] In some embodiments, the recombinant MuV F ectodomain protomer may contain one or more additional amino acid substitutions to enhance the stabilization of the pre-fusion conformation or for other purposes, such as increasing solubility or reducing unwanted immune responses.

[0104] The non-native disulfide bonds listed above stabilize the distal membrane portion of the MuV F ectodomain in the pre-fusion conformation. All of these mutations can be combined with modifications to the proximal membrane portion (e.g., the 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 one of the positions from the stem region of the ectodomain, for example, one of MuV F positions 469-483 (e.g., position 476).

[0106] In a non-limiting example, recombinant MuV F ectodomain trimers are provided, including protomers containing MuV positions 20-476, having V206C and A223C substitutions to form unnatural disulfide bonds, as well as deletions of MuV F positions 101-103 and consequently positions 100 and 104 fused with a Gly-Gly-Gly peptide linker.

[0107] Non-limiting examples of protomers of MuV F ectodomain trimers, including amino acid substitutions for stabilization in the pre-fusion conformation, are provided herein. In some embodiments, a protomer of a MuV F ectodomain trimer comprises an amino acid sequence that is at least 90% identical to one residue 20-483 of any SEQ ID NO:3-8, one residue 20-476 of any SEQ ID NO:11-16, 26, or 51, or one residue 20-469 of any SEQ ID NO:19-24, wherein the protomer comprises one or more amino acid substitutions that stabilize the MuV F ectodomain trimer in the pre-fusion conformation. In some embodiments, the protomer of the MuV F ectodomain trimer contains one residue 20-483 of any SEQ ID NO:3-8, one residue 20-476 of any SEQ ID NO:11-16, 26, or 51, or one residue 20-469 of any SEQ ID NO:19-4.

[0108] In some embodiments, recombinant MuV F ectodomain trimers are soluble protein complexes for use, for example, as recombinant subunit vaccines. In some such embodiments, each protomer of the recombinant MuV F ectodomain trimer may contain a C-terminal linkage to a trimerizing domain, such as a GCN4 trimerizing domain or a T4 fibrintin trimerizing domain or both. The trimerizing domain facilitates trimerization and stabilization of the membrane proximal morphology of the recombinant MuV F ectodomain trimer. For example, the C-terminal residue of a recombinant MuV F ectodomain trimer protomer (e.g., residues in the stem region of the trimer) can be linked directly to the trimerizing domain or indirectly to the trimerizing domain via a peptide linker. Examples of linkers include glycine and glycine-serine linkers. Non-limiting examples of exogenous multimerizing domains that promote stable trimerization of soluble recombinant proteins include the GCN4 leucine zipper, the T4 fibrintin trimerizing domain, and trimerizing motifs derived from lung surfactant protein (Hoppe et al. 1994 FEBS Lett 344:191-195) or collagen (McAlinden et al. 2003 J Biol Chem 278:42200-42207). All of these can be ligated to the C-terminus of a recombinant MuV F ectodomain protomer to promote trimerization, provided that the recombinant MuV F ectodomain trimer maintains its pre-fusion conformation. In some cases, a recombinant MuV F ectodomain trimer protomer can be linked to the MuV trimerizing domain, for example, each protomer in the trimer may include a C-terminal linkage to the GCN4 trimerizing domain, for example, to one of MuV F positions 469-483, for example, MuV F position 469, MuV F position 476, or MuV F position 483. In a specific example, the GCN4 trimerizing domain is linked to the amino acid sequence TIFF2026062907000008.tif4128 contains or consists of the same amino acid sequence. A specific example is the T4 fibrin trimerization domain containing the amino acid sequence TIFF2026062907000009.tif4128 is included or consists of the same amino acid sequence. In a specific example, the GCN4 trimerization domain fused to the fibrintin trimerization domain is the amino acid sequence. Contains or consists of the same amino acid sequence as TIFF2026062907000010.tif10129.

[0109] In a non-limiting example, a recombinant MuV F ectodomain trimer is provided, comprising a protomer containing MuV positions 20-476, having V206C and A223C substitutions to form unnatural disulfide bonds, deletion of MuV F positions 101-103 and consequently positions 100 and 104 fused with a Gly-Gly-Gly peptide linker, and a GCN4 trimerizing domain linked to the C-terminus of the protomer's ectodomain.

[0110] Non-limiting examples of protomers of MuV F ectodomain trimers, including amino acid substitutions for stabilization in the pre-fusion conformation and C-terminal linking to the trimerizing domain, are provided herein. In some embodiments, a protomer of MuV F ectodomain trimer comprises an amino acid sequence that is at least 90% identical to one residue 20-513 of any SEQ ID NO:3-8, one residue 20-506 of any SEQ ID NO:11-16, 26, or 51, or one residue 20-499 of any SEQ ID NO:19-24, wherein the protomer comprises one or more amino acid substitutions that stabilize the MuV F ectodomain trimer in the pre-fusion conformation. In some embodiments, the protomer of the MuV F ectodomain trimer contains one residue 20-513 of any of SEQ ID NO:3-8, one residue 20-506 of any of SEQ ID NO:11-16, 26, or 51, or one residue 20-499 of any of SEQ ID NO:19-24.

[0111] In some embodiments, the recombinant MuV F ectodomain trimer can be a membrane-immobilized protein complex for use, for example, in attenuated viral vaccines or virus-like particle vaccines. Membrane immobilization can be achieved by C-terminal linking of the recombinant MuV F ectodomain trimer protomer to, for example, the transmembrane domain and cytoplasmic tail of MuV F. In some embodiments, one or more peptide linkers (e.g., gly-ser linkers, e.g., 10-amino acid glycine-serine peptide linkers) 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 this disclosure include: Examples include MuV F transmembrane domains such as TIFF2026062907000011.tif4128. Non-limiting examples of transmembrane domains and cytoplasmic tails for use in aspects of this disclosure include: Examples include MuV F transmembrane domains and cytoplasmic tails such as TIFF2026062907000012.tif4137.

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

[0113] Recombinant MuV F ectodomain trimers can be derivatized or linked to other molecules (e.g., other peptides or proteins). Generally, recombinant MuV F ectodomains are derivatized so that their binding to neutralizing antibodies against recombinant MuV F protein trimers is not adversely affected by derivatization or labeling. For example, recombinant MuV F ectodomains can be functionally linked (by chemical coupling, gene fusion, non-covalent association, etc.) to one or more other molecular entities, such as carrier proteins, antibodies, heterologous proteins, or detection tags.

[0114] In some embodiments, the recombinant MuV F ectodomain trimer comprises one or more MuV HN ectodomains, for example It is fused to the ectodomain head of the MuV HN sequence, which is represented as TIFF2026062907000013.tif33160.

[0115] In some embodiments, a recombinant MuV F ectodomain trimer is fused to one or more MuV HN ectodomains with an ectodomain stalk and ectodomain head of any one of the sequences from MuV HN positions 54-130 to MuV HN position 582 (e.g., from MuV HN positions 54-63 to MuV HN position 582, e.g., positions 54-582, 61-582, 63-582, or 55-582). In some embodiments, a recombinant MuV F ectodomain trimer is fused to one or more MuV HN ectodomains with an ectodomain stalk and ectodomain head of a sequence represented, for example, 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 protomer of a recombinant MuV F ectodomain trimer is fused to a MuV HN ectodomain. The 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 a MuV F ectodomain trimer protomer. In some such embodiments, the MuV HN ectodomain can be fused directly or indirectly via a peptide linker to the C-terminus of a trimerizing domain (e.g., a GCN4 or T4 fibrintin trimerizing domain) fused to the C-terminus of a MuV F ectodomain trimer protomer. In some such embodiments, the protomer of the MuV F ectodomain trimer linked to the trimerizing domain and the MuV HN ectodomain contains an amino acid sequence represented as residues 20-966 of SEQ ID NO:27, or an amino acid sequence that is 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, for example It is fused to the ectodomain head of the H sequence, which is represented as TIFF2026062907000014.tif56160.

[0118] In some embodiments, a recombinant MuV F ectodomain trimer is fused to one or more MeV H ectodomains with an ectodomain stalk and ectodomain head of any one of the sequences from MeV H positions 59-179 to MeV H position 617 (e.g., any one from 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 recombinant MuV F ectodomain trimer is fused to one or more MeV H ectodomains with an ectodomain stalk and ectodomain head of a sequence represented, for example, 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 the pre-fusion conformation is fused to a MeV H ectodomain. The 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 pre-fusion MuV F ectodomain trimer protomer. In some such embodiments, the MeV H ectodomain can be fused directly or indirectly via a peptide linker to the C-terminus of the trimerizing domain (e.g., GCN4 or T4 fibrintin trimerizing domain) fused to the C-terminus of the MuV F ectodomain trimer protomer. In some such embodiments, the protomer of the MuV F ectodomain trimer linked to the trimerizing domain and the MeV H ectodomain contains an amino acid sequence represented as residues 21-981 of SEQ ID NO:28 or residues 20-1006 of SEQ ID NO:29, or contains an amino acid sequence that is at least 90% identical to residues 21-981 of SEQ ID NO:28 or residues 20-1006 of SEQ ID NO:29.

[0120] A non-restrictive example of a sequence containing a MuV F ectodomain with amino acid substitutions for stabilization in the pre-fusion conformation is shown below. TIFF2026062907000015.tif232160TIFF2026062907000016.tif232160TIFF2026062907000017.tif200160

[0121] The sequences described above include an N-terminal signal peptide, a MuV F ectodomain, and a GCN4 trimerization domain. It will be understood that alternative trimerization domains, such as a T4 fibrintin trimerization domain, can be used. In addition, many of the sequences described above include a GGG linker to remove the native furin cleavage site that separates the F1 and F2 subunits. Alternative glycine linkers such as GSG, GGS, or SGG can also be used. Furthermore, in any of the sequences, the native furin cleavage site can be included instead 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. In addition, by including the MuV F ectodomain of any of the sequences described above in a full-length MuV F protein, a membrane-immobilized pre-fusion MuV F protein can also be provided, for example, for mRNA immunotherapy.

[0122] The following are non-limiting examples of sequences containing a MuV F ectodomain with amino acid substitutions for stabilization in the pre-fusion conformation, linked to a MuV HN ectodomain or a MeV H ectodomain. TIFF2026062907000018.tif167160

[0123] The sequences described above contain the N-terminal signal peptide and MuV F ectodomain in combination with various other elements, such as the GCN4 trimer domain, T4 fibrintin trimer domain, peptide cleavage sites (e.g., thrombin), HIS tag, Strep tag, and various linker residues between segments. These proteins in purified form typically lack the N-terminal signal peptide and C-terminal residues that are removed by peptide cleavage.

[0124] B. Recombinant MeV F ectodomain trimer Recombinant MeV F ectodomain trimers modified from the native form (e.g., by introducing one or more amino acid substitutions) to stabilize the pre-fusion conformation are disclosed herein. As described in the examples, embodiments of the MeV F ectodomain trimers of this disclosure were selected by multiple rounds of structure-based design to obtain optimized solubility, stability, expression, and immunogenicity. Recombinant MeV F ectodomain trimers are useful for inducing an immune response to MeV in vertebrates (such as humans). Exemplary embodiments are shown to produce a superior immune response 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 a pre-fusion conformation.

[0126] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 48 and 284 of the MeV F (e.g., R48C and A284C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0127] In some embodiments, the recombinant MeV F ectodomain trimer protomer includes cysteine ​​substitutions at positions 90 and 225 of MeV F (e.g., A90C and I225C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0128] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 141 and 270 of MeV F (e.g., M141C and T270C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0129] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 165 and 171 of the MeV F (e.g., R165C and M171C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0130] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 173 and 245 of MeV F (e.g., L173C and V245C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0131] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 175 and 241 of MeV F (e.g., V175C and D241C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0132] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 212 and 236 of MeV F (e.g., E212C and Y236C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0133] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 216 and 233 of MeV F (e.g., L216C and A233C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0134] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 219 and 224 of the MeV F (e.g., P219C and P224C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0135] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 99 and 117 of MeV F (e.g., R99C and V117C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0136] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 100 and 117 of the MeV F (e.g., P100C and V117C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0137] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 101 and 117 of MeV F (e.g., V101C and V117C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0138] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 102 and 117 of MeV F (e.g., Q102C and V117C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0139] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 103 and 117 of MeV F (e.g., S103C and V117C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0140] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 165 and 171 (e.g., R165C and M171C substitutions) and positions 141 and 270 (e.g., M141C and T270C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0141] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 165 and 171 (e.g., R165C and M171C substitutions) and positions 212 and 236 (e.g., E212C and Y236C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

[0142] In some embodiments, the recombinant MeV F ectodomain trimer protomers include cysteine ​​substitutions at positions 165 and 171 (e.g., R165C and M171C substitutions) and positions 48 and 284 (e.g., R48C and A284C substitutions) that form intra-protomer disulfide bonds for stabilization in the pre-fusion conformation.

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

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

[0145] Any of the recombinant MeV F proteins described above may further include modifications to eliminate the protease cleavage site between the F1 and F2 polypeptides to produce a "single-chain" recombinant F protein. For example, any of the recombinant MeV F proteins may include deletions at MeV F positions 111-113 and fusion at positions 110 and 114 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 can be used to fuse the F2 and F1 ectodomains and enable the folding of the F ectodomain into the pre-fusion conformation. 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 a non-limiting example, recombinant MeV F ectodomain trimers are provided, including protomers having R165C and M171C substitutions to form unnatural disulfide bonds, as well as deletions of MeV F positions 111-113 and consequently positions 110 and 114 fused with a Gly-Gly-Gly peptide linker.

[0147] In some embodiments, recombinant MeV F ectodomain protomers may include one or more additional amino acid substitutions to enhance pre-fusion conformation stabilization or for other purposes, such as increasing solubility or reducing unwanted immune responses.

[0148] The non-native disulfide bonds listed above stabilize the distal membrane portion of the MeV F ectodomain in the pre-fusion conformation. All of these mutations can be combined with modifications to the proximal membrane portion (e.g., the 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, one of MeV F positions 472-486 (e.g., position 486).

[0150] In a non-limiting example, recombinant MeV F ectodomain trimers are provided, including protomers containing MeV positions 24-486, having R165C and M171C substitutions to form unnatural disulfide bonds, as well as deletions of MeV F positions 111-113 and consequently positions 110 and 114 fused with a Gly-Gly-Gly peptide linker.

[0151] Non-limiting examples of MeV F ectodomain trimer protomers including amino acid substitutions for stabilization in the pre-fusion conformation are provided herein. In some embodiments, a MeV F ectodomain trimer protomer comprises an amino acid sequence that is at least 90% identical to residues 21-483 of either SEQ ID NO: 37-43 or 53-55, wherein the protomer comprises one or more amino acid substitutions that stabilize the MeV F ectodomain trimer in the pre-fusion conformation. In some embodiments, a MeV F ectodomain trimer protomer comprises residues 21-483 of either SEQ ID NO: 37-43 or 53-55.

[0152] In some embodiments, recombinant MeV F ectodomain trimers are soluble protein complexes for use, for example, as recombinant subunit vaccines. In some such embodiments, each protomer of the recombinant MeV F ectodomain trimer may contain a C-terminal linkage to a trimerizing domain, such as a GCN4 trimerizing domain or a T4 fibrintin trimerizing domain or both. The trimerizing domain facilitates trimerization and stabilization of the membrane proximal morphology of the recombinant MeV F ectodomain trimer. For example, the C-terminal residue of a recombinant MeV F ectodomain trimer protomer (e.g., a residue in the stem region of the trimer) can be linked directly to the trimerizing domain or indirectly to the trimerizing domain via a peptide linker. Examples of linkers include glycine and glycine-serine linkers. Non-limiting examples of exogenous multimerizing domains that promote stable trimerization of soluble recombinant proteins include the GCN4 leucine zipper, the T4 fibrintin trimerizing domain, and trimerizing motifs derived from lung surfactant protein (Hoppe et al. 1994 FEBS Lett 344:191-195) or collagen (McAlinden et al. 2003 J Biol Chem 278:42200-42207). All of these can be ligated to the C-terminus of a recombinant MeV F ectodomain protomer to promote trimerization, provided that the recombinant MeV F ectodomain trimer maintains its pre-fusion conformation. In some cases, a protomer of a recombinant MeV F ectodomain trimer can be linked to the MeV trimerizing domain, for example, each protomer in the trimer may include a C-terminal linkage to the GCN4 trimerizing domain, for example, to one of the MeV F positions 472-486, for example, to MeV F position 486. In a specific example, the GCN4 trimerizing domain is linked to the amino acid sequence Contains TIFF2026062907000019.tif4128 or consists of the same amino acid sequence. A specific example is the T4 fibrin trimerization domain, which is part of the amino acid sequence. TIFF2026062907000020.tif4128 is included or consists of the same amino acid sequence. In a specific example, the GCN4 trimerization domain fused to the fibrintin trimerization domain is the amino acid sequence. Contains TIFF2026062907000021.tif4156 or consists of the same amino acid sequence.

[0153] In a non-limiting example, a recombinant MeV F ectodomain trimer is provided, comprising a protomer containing MeV positions 24-486, having R165C and M171C substitutions for forming unnatural disulfide bonds, deletion of MeV F positions 111-113 and consequently fusion of positions 110 and 114 with a Gly-Gly-Gly peptide linker, and a GCN4 trimerizing domain linked to the C-terminus of the protomer's ectodomain.

[0154] Non-limiting examples of MeV F ectodomain trimer protomers, including amino acid substitutions for stabilization in the pre-fusion conformation and C-terminal linking to the trimerizing domain, are provided herein. In some embodiments, the MeV F ectodomain trimer protomer comprises an amino acid sequence that is at least 90% identical to residues 21-513 of either SEQ ID NO: 37-43 or 53-55, wherein the protomer comprises 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 protomer comprises residues 21-513 of either SEQ ID NO: 37-43 or 53-55.

[0155] In some embodiments, the recombinant MeV F ectodomain trimer can be a membrane-immobilized protein complex for use, for example, in attenuated viral vaccines or virus-like particle vaccines. Membrane immobilization can be achieved, for example, by C-terminal linking of the recombinant MeV F ectodomain trimer protomer to the transmembrane domain and optionally to the cytoplasmic tail, for example, the MeV F transmembrane domain and the cytoplasmic tail. In some embodiments, one or more peptide linkers (e.g., gly-ser linkers, e.g., 10-amino acid glycine-serine peptide linkers) 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 this disclosure include: Examples of MeV F transmembrane domains include TIFF2026062907000022.tif4128. Non-limiting examples of transmembrane domains for use in the embodiments of this disclosure include: Examples include MeV F transmembrane domains such as TIFF2026062907000023.tif4143.

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

[0157] Recombinant MeV F ectodomain trimers can be derivatized or linked to other molecules (e.g., other peptides or proteins). Generally, recombinant MeV F ectodomains are derivatized so that their binding to cross-neutralizing antibodies against recombinant MeV F protein trimers is not adversely affected by derivatization or labeling. For example, recombinant MeV F ectodomains can be functionally linked (by chemical coupling, gene fusion, non-covalent association, etc.) to one or more other molecular entities, such as carrier proteins, antibodies, heterologous proteins, or detection tags.

[0158] In some embodiments, the recombinant MeV F ectodomain trimer comprises one or more MuV HN ectodomains, for example It is fused to the ectodomain of the MuV HN sequence, which is represented as TIFF2026062907000024.tif27160.

[0159] For example, each protomer of a recombinant MeV F ectodomain trimer is fused to a MuV HN ectodomain. The 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 protomer. In some such embodiments, the MuV HN ectodomain can be fused directly or indirectly via a peptide linker to the C-terminus of the trimerizing domain (e.g., a GCN4 or T4 fibrin trimerizing domain) fused to the C-terminus of the MeV F ectodomain trimer protomer. In some such embodiments, the MeV F ectodomain trimer protomer linked to the trimerizing domain and the MuV HN ectodomain contains an amino acid sequence represented 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, for example It is fused to the ectodomain of the H sequence, which is represented as TIFF2026062907000025.tif56160.

[0161] For example, each protomer of a recombinant MeV F ectodomain trimer is fused to a MeV H ectodomain. The 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 protomer. In some such embodiments, the MeV H ectodomain can be fused directly or indirectly via a peptide linker to the C-terminus of the trimerizing domain (e.g., a GCN4 or T4 fibrin trimerizing domain) fused to the C-terminus of the MeV F ectodomain trimer protomer. In some such embodiments, the MeV F ectodomain trimer protomer linked to the trimerizing domain and the MeV H ectodomain contains an amino acid sequence indicated 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] A non-restrictive example of a sequence containing a MeV F ectodomain with amino acid substitutions for stabilization in the pre-fusion conformation is shown below. TIFF2026062907000026.tif49160TIFF2026062907000027.tif232160TIFF20260629070 00028.tif232160TIFF2026062907000029.tif228160TIFF2026062907000030.tif232160

[0163] The sequences described above include an N-terminal signal peptide, a MeV F ectodomain, and a GCN4 trimerization domain. It will be understood that alternative trimerization domains, such as a T4 fibrintin trimerization domain, can be used. In addition, many of the sequences described above include a GGG linker to remove the native furin cleavage site that separates the F1 and F2 subunits. Alternative glycine linkers such as GSG, GGS, or SGG can also be used. Furthermore, in any of the sequences, the native furin cleavage site can be included instead 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. In addition, by including the MeV F ectodomain of any of the sequences described above in a full-length MeV F protein, a membrane-immobilized pre-fusion MeV F protein can be provided, for example, for mRNA immunotherapy.

[0164] The following are non-limiting examples of sequences containing a MeV F ectodomain with amino acid substitutions for stabilization in the pre-fusion conformation, linked to a MuV HN ectodomain or a MeV H ectodomain. TIFF2026062907000031.tif165160

[0165] The above sequences include an N-terminal signal peptide, a MeV F ectodomain, a trimerization domain (GCN4 and / or T4 fibrintin), and a MeV H ectodomain head region or a MuV HN ectodomain head region. The MeV F ectodomain sequence contains 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 may also be used. In addition, in any of the sequences, the native furin cleavage site may be included instead 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.

[0166] C. MuV HN and MuV H polymers In some embodiments, an immunogen is provided comprising a multimer of MuV HN ectodomains and / or MeV H ectodomains. The H ectodomain or HN ectodomain may include an ectodomain head or may include a 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 trimerizing domain (e.g., a GCN4 trimerizing domain, a T4 fibrintin trimerizing domain, or a GCN4 trimerizing domain fused to a T4 fibrintin trimerizing domain).

[0168] In some embodiments, the fusion protein includes, from the N-terminus to the C-terminus, a trimerizing domain (e.g., a GCN4 trimerizing domain, a T4 fibrintin trimerizing domain, or a GCN4 trimerizing domain fused to a T4 fibrintin trimerizing domain) and one or more (e.g., one, two, or three) MuV HN ectodomains or MeV H ectodomains. The trimerizing domains interact to form a trimer. In some embodiments, fragments of ectodomains are included, and, for example, the head region of a MuV HN ectodomain or the head region of a MeV H ectodomain can be fused to the trimerizing domain, optionally by a peptide linker. In some embodiments, the fusion protein in the trimer contains, or consists of, an amino acid sequence represented as residues 24-510 of SEQ ID NO:58 or residues 24-496 of SEQ ID NO:59, or a sequence that is at least 90% identical to one of either 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-terminus to C-terminus, one or more (e.g., one, two, or three) MuV HN ectodomains or Mev H ectodomains, a trimerizing domain (e.g., a GCN4 trimerizing domain, a T4 fibrintin trimerizing domain, or a GCN4 trimerizing domain fused to a T4 fibrintin trimerizing domain), and one or more (e.g., one, two, or three) MuV HN ectodomains or Mev H ectodomains. The trimerizing domains interact to form a trimer. In some embodiments, the ectodomain fragments are included, and, for example, the head region of a MuV HN ectodomain or the head region of a MeV H ectodomain can be fused to the trimerizing domain, optionally by a peptide linker. In some embodiments, the fusion protein in the trimer contains, or consists of, an amino acid sequence represented 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 that is at least 90% identical to one of the following: 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 dimerize in physiological solution. This dimer can then be purified and used as an immunogen. In some embodiments, the dimer's subunits contain or consist of an amino acid sequence represented as residues 22-459 of SEQ ID NO:60 or a sequence that is 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. These dimers can then be purified and used as immunogens. In some embodiments, the dimer subunits contain, or consist of, an 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, the dimer subunit contains, or consists of, an amino acid sequence represented 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 that is 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 polymer is a dimer of the MuV HN ectodomain head region. In some embodiments, the subunit of the dimer contains or consists of an amino acid sequence indicated as SEQ ID NO:30 or a sequence that is at least 90% identical to SEQ ID NO:30.

[0173] In some embodiments, the polymer is a dimer of the stalk and head regions of the MuV H ectodomain. In some embodiments, the dimer subunit contains or consists of an 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 dimer subunit contains, or consists of, an amino acid sequence represented 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 that is 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 recombinant MuV or MeV F ectodomain trimers may, in addition to those described above, include modifications to the native MuV F or MeV F sequence, such as amino acid substitution, deletion or insertion, glycosylation, and / or covalent linkage to unrelated proteins (e.g., protein tags), provided that the recombinant MuV or MeV F ectodomain trimer remains stable in its 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 may include modifications to the native HN or H sequence, such as amino acid substitution, deletion or insertion, glycosylation, and / or covalent linkage to unrelated proteins (e.g., protein tags), provided that the HN or H ectodomain retains immunogenicity. These mutations in the sequence may be native mutations or may be engineered using genetic engineering techniques known to those skilled in the art. Examples of such techniques can be found, for example, in 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), both of which are incorporated herein by reference in their entirety.

[0175] In some embodiments, the protomer in the recombinant MuV F ectodomain trimer contains 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 may contain 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., conserved amino acid substitutions) compared to the native MuV F sequence.

[0176] In some embodiments, the protomer in the recombinant MeV F ectodomain trimer contains 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 may contain 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., conserved amino acid substitutions) compared to the native MeV F sequence.

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

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

[0179] The simplest modifications involve the substitution of one or more amino acids with similar biochemical properties, such as conserved amino acid substitutions. Such substitutions are likely to have only a minimal effect on the activity of the resulting protein.

[0180] In some embodiments, a protomer in a recombinant MeV F ectodomain trimer or MuV F ectodomain trimer can be conjugated at one end to another unrelated sequence (e.g., a non-MuV F or MeV F protein sequence, a non-viral envelope, or a non-viral protein sequence).

[0181] In some embodiments, recombinant MuV F ectodomain trimers or MeV F ectodomain trimers, or fusions of these protomers with heterologous proteins, such as MuV HN ectodomains or MeV H ectodomains, are soluble in aqueous solution. In some embodiments, recombinant MuV F ectodomain trimers, MeV F ectodomain trimers, or corresponding fusions with heterologous proteins, such as MuV HN ectodomain or MeV H ectodomain, are dissolved 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 remain 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 is pH 7.4 and contains NaCl (137 mM), KCl (2.7 mM), Na2HPO4 (10 mM), and KH2PO4 (1.8 mM). In some embodiments, the phosphate-buffered saline further contains CaCl2 (1 mM) and MgCl2 (0.5 mM). Those skilled in the art are familiar with methods for determining whether a protein remains dissolved over time. For example, the concentration of a protein dissolved in an aqueous solution can be tested over time using standard methods.

[0182] In some embodiments, the immunogen is provided as a homogeneous population of soluble trimers, substantially in the pre-fusion conformation, with limited or absent post-fusion conformations of MuV F ectodomain trimers and / or MeV F ectodomain trimers. The conformation of MeV F ectodomain trimers or MuV F ectodomain trimers can be detected, for example, by negative staining electron microscopy and / or by specific binding with pre-fusion or post-fusion specific antibodies. In some embodiments, at least about 95% (e.g., at least about 95%, 96%, 97%, 98%, 99%, or 99.9% of MuV or MeV F proteins) of the recombinant MuV F ectodomain trimers or MeV F ectodomain trimers in the homogeneous population are stabilized in the pre-fusion conformation.

[0183] In some embodiments, recombinant MuV F ectodomain trimers or MeV F ectodomain trimers retain specific binding to pre-fusion specific antibodies after incubation in phosphate-buffered saline at 50°C for 1 hour. In some embodiments, recombinant MuV F ectodomain trimers or MeV F ectodomain trimers retain specific binding to pre-fusion specific antibodies 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 include additional non-protein moieties that are known and readily available in the art. Suitable moieties for derivatization of 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 homopolymers, prolypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may offer manufacturing advantages due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if two or more polymers are attached, they may be the same molecule or different molecules. In general, the number and / or type of polymers used in derivatization can be determined based on considerations such as, for example, the specific properties or functions of the immunogen to be improved or modified, and whether the immunogen derivative is intended to be used therapeutically under certain conditions.

[0185] Some of the sequences provided herein that include recombinant MuV F ectodomain trimers or MeV F ectodomain trimers or MuV HN ectodomains or MeV H ectodomains include sequences of 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 containing recombinant MuV F ectodomain trimers or MeV F ectodomain trimers or MuV HN ectodomains or MeV H ectodomains 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, 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 a two-component self-assembling nanoparticle platform described by 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] In addition, non-limiting examples of nanoparticles include ferritin nanoparticles, encapsulin nanoparticles, sulfur oxidase / reductase (SOR) nanoparticles, and lumazine synthase nanoparticles, which are composed of aggregates of monomeric subunits containing ferritin protein, encapsulin protein, SOR protein, and lumazine synthase, respectively. To construct such protein nanoparticles, recombinant MuV F ectodomain trimers or recombinant MeV F ectodomain trimer protomers, or subunits of MuV HN or MeV H multimers, are ligated to a protein nanoparticle subunit (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 that can be purified.

[0189] In some embodiments, ferritin nanoparticles can be constructed by linking a recombinant MuV F ectodomain trimer or a recombinant MeV F ectodomain trimer protomer, or a subunit of a MuV HN or MeV H macromer, to a ferritin subunit. Ferritin nanoparticles and their use for immunotherapy (e.g., for immunotherapy against influenza antigens) have been disclosed in the Art (see, for example, Kanekiyo et al., Nature, 499:102-106, 2013, which is incorporated herein by reference in its entirety). Spherical ferritin nanoparticles are composed of monomeric subunits, which are polypeptides with a molecular weight of approximately 17-20 kDa. These monomeric subunit proteins self-assemble after production to form a spherical ferritin protein. Thus, spherical ferritin contains 24 monomeric subunit proteins and has a capsid-like structure with 432 symmetries. Methods for constructing ferritin nanoparticles are described further herein (see, for example, Zhang, Int. J. Mol. Sci., 12:5406-5421, 2011, which is incorporated herein in its entirety by reference). An example of a single amino acid sequence of such a monomeric ferritin subunit is: It is represented as TIFF2026062907000032.tif11158.

[0190] In specific examples, ferritin polypeptides include E. coli ferritin, Helicobacter pylori ferritin, human light chain ferritin, bullfrog ferritin, or hybrids 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 the production of ferritin nanoparticles containing recombinant MuV or MeV F ectodomain trimers can be found in GENBANK®, e.g., accession numbers ZP_03085328, ZP_06990637, EJB64322.1, AAA35832, NP_000137 AAA49532, AAA49525, AAA49524 and AAA49523, and these sequences available as of April 10, 2015, are 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 containing 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 indicated as SEQ ID NO:45.

[0191] In some embodiments, lumazine synthase nanoparticles can be constructed by linking a recombinant MuV F ectodomain trimer or a recombinant MuV or MeV F ectodomain trimer protomer, or a subunit of a MuV HN or MeV H polymer, to a lumazine synthase subunit. The spherical lumazine synthase nanoparticles are composed of monomeric subunits, and an example of one sequence of such lumazine synthase subunits is: The amino acid sequence is provided as TIFF2026062907000033.tif11158.

[0192] In some embodiments, the recombinant MuV F ectodomain trimer or recombinant MeV F ectodomain trimer protomer or subunit of the MuV HN or MeV H macromer of the present disclosure can be linked to a lumazine synthase subunit having 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 indicated as SEQ ID NO:46.

[0193] In some embodiments, encapsulin nanoparticles can be constructed by linking a recombinant MuV F ectodomain trimer or a recombinant MeV F ectodomain trimer protomer, or a subunit of a MuV HN or MeV H polymer, to an encapsulin nanoparticle subunit. The spherical encapsulin nanoparticles are composed of monomeric subunits, and an example of one sequence of such encapsulin nanoparticle subunits is: The amino acid sequence is provided as TIFF2026062907000034.tif15158.

[0194] In some embodiments, a recombinant MuV F ectodomain trimer or a recombinant MeV F ectodomain trimer protomer, or a subunit of a MuV HN or MeV H macromer of the present disclosure, can be linked to an encapsrin subunit having 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 indicated as SEQ ID NO:47.

[0195] Encapsrin proteins, also known as linocin-like proteins, are a conserved family of bacterial proteins that form large protein assemblies that function as minimal compartments for packaging enzymes. Encapsrin assemblies are composed of monomeric subunits, which are polypeptides with a molecular weight of approximately 30 kDa. These monomeric subunits self-assemble after production to form spherical encapsrin assemblies containing 60, or possibly 180, monomeric subunits. Methods for constructing encapsrin nanoparticles are further described (see, for example, Sutter et al., Nature Struct. and Mol. Biol., 15:939-947, 2008, which is incorporated herein by reference in its entirety). In specific examples, encapsrin polypeptides are bacterial encapsrins, such as those of Thermotoga maritime, Pyrococcus furiosus, Rhodococcus erythropolis, or Myxococcus xanthus.

[0196] In some embodiments, recombinant SOR nanoparticles can be constructed by linking a recombinant MuV F ectodomain trimer or a recombinant MeV F ectodomain trimer protomer, or a subunit of a MuV HN or MeV H polymer, to a sulfur oxidase / reductase (SOR) subunit. In some embodiments, the SOR subunit is It may contain the amino acid sequence shown as TIFF2026062907000035.tif15158.

[0197] In some embodiments, a recombinant MuV F ectodomain trimer or a recombinant MeV F ectodomain trimer protomer, or a subunit of a MuV HN or MeV H macromer of the present disclosure, can be linked to a SOR subunit having 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 indicated as SEQ ID NO:48.

[0198] The SOR protein is a microbial protein that forms a 24-subunit protein assembly (e.g., derived from the thermophilic acidophilic archaeon Acidianus ambivalens). A method for constructing SOR nanoparticles is 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 the construction of SOR nanoparticles is shown 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 recombinant MeV F ectodomain, or the MuV HN or MeV H polymer subunits linked to the nanoparticle subunits, may contain an N-terminal signal peptide that is cleaved during intracellular processing. For example, a recombinant MuV F ectodomain protomer or recombinant MeV F ectodomain protomer linked to a protein nanoparticle subunit may contain a signal peptide, such as a native MuV or MeV F signal peptide, at its N-terminus.

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

[0201] The fusion protein does not need to contain the full-length sequence of a monomeric subunit polypeptide of ferritin, encapsrin, SOR, or rumazine synthase protein. A portion or region of the monomeric subunit polypeptide can be used, as long as that portion contains the amino acid sequence that directs the monomeric subunit to self-assemble into a spherical protein.

[0202] II. Polynucleotides and Expression Polynucleotides encoding any of the immunogens of this disclosure are also provided. For example, a protomer of a MuV F ectodomain trimer stabilized in a pre-fusion conformation, a protomer of a MeV F ectodomain trimer stabilized in a pre-fusion conformation, a chimera of one of these protomers linked to a MuV HN or MeV H ectodomain, or a polynucleotide encoding 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 a variety of functionally equivalent nucleic acids, such as nucleic acids with different sequences but encoding the same protein sequence, or nucleic acids encoding a conjugate or fusion protein containing that nucleic acid sequence.

[0203] The exemplary nucleic acid sequence encoding the full-length MeV F protein is SEQ ID NO:94: It will be provided as TIFF2026062907000036.tif78159.

[0204] The exemplary nucleic acid sequence encoding the full-length MuV F protein is SEQ ID NO:95: It will be provided as TIFF2026062907000037.tif78159.

[0205] The exemplary nucleic acid sequence encoding the full-length MeV H protein is SEQ ID NO:96: It will be provided as TIFF2026062907000038.tif89159.

[0206] The exemplary nucleic acid sequence encoding the full-length MuV HN protein is SEQ ID NO:97: It will be provided as TIFF2026062907000039.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, nucleic acid molecules encode MuV or MeV F ectodomain trimer protomers or MeV F ectodomain trimer promoters, or chimeras of such protomers with MuV HN or MeV H ectodomains, or precursors of MuV HN or MeV H multimer subunits, which, when expressed in suitable cells, can self-assemble into corresponding trimers or multimers. For example, a nucleic acid molecule may encode a MuV or MeV F ectodomain trimer protomer or MeV F ectodomain trimer promoter, which includes an N-terminal signal sequence for entry into the cell's secretory system, which is proteolytically cleaved during the processing of 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 into a MuV or MeV F ectodomain trimer protomer containing an F2 polypeptide linked to an F1 ectodomain, or into a promoter of a MeV F ectodomain trimer, wherein the recombinant F2-F1 ectodomain protomer comprises any of the pre-fusion stabilization modifications described herein and may optionally be linked to a trimer domain such as a GCN4 trimer domain and / or a T4 fibrintin trimer domain.

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

[0211] Exemplary nucleic acids can be prepared by cloning techniques. Examples of suitable cloning and sequencing techniques, as well as sufficient procedures to guide those skilled in the art to many cloning operations, are publicly known (see, for example, 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, up to 2013)).

[0212] Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), and self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cell 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 may include recombinant DNA that is incorporated into a vector (e.g., an expression vector), incorporated into an autonomously replicating plasmid or virus, or incorporated into the genomic DNA of a prokaryotic or eukaryote, or recombinant DNA that exists as a separate molecule independent of other sequences (e.g., cDNA). Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. This term encompasses single-stranded and double-stranded DNA.

[0214] Protomers of MuV or MeV F ectodomain trimers, promoters of MeV F ectodomain trimers, or chimeras of such protomers with MuV HN or MeV H ectodomains, or polynucleotide sequences encoding subunits of MuV HN or MeV H multimers can be functionally ligated to regulatory sequences. A regulatory sequence functionally ligated to a coding sequence is ligated so that expression of the coding sequence is achieved under conditions compatible with that regulatory sequence. Regulatory sequences include, but are not limited to, suitable promoters, enhancers, transcription terminators, pre-protein coding gene start codons (i.e., ATGs), splicing signals for introns, maintenance of the correct reading frame of the gene to enable 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 suitable host cells. These cells may be prokaryotic or eukaryotic. The term also encompasses any offspring of the target host cell. It is understood that not all offspring will be identical to the parent cell due to the possibility of mutations occurring during replication. Stable transfer methods, meaning that the foreign DNA is continuously maintained within the host, are known in the art.

[0216] Suitable hosts include microorganisms, yeasts, insects, and mammals. Methods for expressing DNA sequences containing eukaryotic or viral sequences in prokaryotes are well known in the art. Non-limiting examples of suitable host cells include bacteria, archaea, insects, fungi (e.g., yeast), plant, and animal cells (e.g., mammalian cells, 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 vesicles. 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 edition, Humana Press). Commonly used mammalian host cell lines include VERO and HeLa cells, CHO cells, and WI38, BHK, and COS cell lines, but cell lines such as those designed to obtain higher expression levels, desired glycosylation patterns, or other characteristics may also be used. In some embodiments, the host cell may be HEK293 cells or their derivatives, e.g., GnTI - / - This includes cells (ATCC® number CRL-3022) or HEK-293F cells.

[0217] Transformation of host cells with recombinant DNA can be carried out using conventional techniques. In some embodiments where the host is a prokaryotic host (but not limited to, such as E. coli), competent cells with DNA uptake ability can be prepared from cells harvested after the exponential growth phase and treated with CaCl2. Alternatively, MgCl2 or RbCl can be used. Transformation can be carried out after protoplast formation in the host cells, if desired, or by electroporation.

[0218] When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical procedures such as microinjection, electroporation, insertion of liposome-encapsulated plasmids, or viral vectors can be used. Eukaryotic cells can also be co-transformed with a polynucleotide sequence encoding the antigen of this disclosure and a second exogenous DNA molecule encoding a selectable phenotype, such as a herpesthymidine kinase gene. Another method is to transiently infect or transform eukaryotic cells with eukaryotic viral vectors, e.g., Simian virus 40 (SV40) or bovine papillomavirus, to express the protein (see, e.g., Viral Expression Vectors, Springer Press, Muzyczka, ed., 2011). Suitable expression systems, such as plasmids and vectors, that are useful for producing the protein in cells, including higher eukaryotic cells such as COS, CHO, HeLa, and myeloma cell lines.

[0219] In a non-limiting example, the immunogens of this 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 immunogens of this disclosure can be modified without diminishing 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 placed at either end to create a restriction site at a favorable location, or additional amino acids (e.g., polyHis) to assist in purification processes.

[0221] In some embodiments, protomers of MuV or MeV F ectodomain trimers, promoters of MeV F ectodomain trimers, chimeras of such protomers with MuV HN or MeV H ectodomains, or nucleic acids encoding subunits of MuV HN or MeV H multimers can be expressed in cells under conditions in which the protomer self-assembles into a trimer secreted from the cell into the cell medium, as described, for example, for the RSV F protein (see, e.g., PCT publication number WO2014160463, McLellan et al., Science, 340:1113-1117, 2013, McLellan et al., Science, 342:592-598, 2013; these publications are incorporated herein by reference in their entirety). In such embodiments, the protomer contains a leader sequence (signal peptide) that leads the protein into the secretory system, the signal peptide is cleaved, the protomer forms a trimer, and then secretes into the cell medium. The culture medium can be centrifuged to purify recombinant MuV or MeV F ectodomain trimers, or chimeras of these with MuV HN or MeV H ectodomains, from the supernatant.

[0222] III. Viral Vectors The nucleic acid molecules encoding immunogens of this disclosure may be included in a viral vector, for example, for the expression of the immunogen in host cells or for the immunization of a 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 containing a transmembrane domain. In some embodiments, the viral vector is included in a vaccine, such as a primer vaccine or booster vaccine, for use in a prime-boost vaccination.

[0223] In some cases, viral vectors can possess replication ability. For example, viral vectors may have mutations in their viral genome that attenuate but do not completely block viral replication in host cells (e.g., insertions of nucleic acids encoding a protocol).

[0224] In some embodiments, viral vectors can be delivered via the respiratory tract. For example, hPIV vectors, e.g., 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 can be used to express the antigens of this disclosure.

[0225] The expression of the disclosed antigens can be achieved using other viral vectors, such as polyomas, namely SV40 (Madzak et al., 1992, J. Gen. Virol., 73:1533-1536), adenoviruses (Berkner, 1992, Cur. Top. Microbiol. Immunol., 158:39-6, Berliner et al., 1988, Bio Techniques, 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), herpesviruses including HSV, EBV and CMV (Margolskee, 1992, Curr. Top. Microbiol. Immunol., 158:67-90, Johnson et al., 1992, J. Virol., 66:29522965, 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, Sindbisvirus (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., 1985, J. Virol., 54:401-407, Buchschalcher et al., 1992, J. Virol., 66:2731-2739) are also available. 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, California), Protein Sciences Corp. (Meriden, Connecticut), Stratagene (La Jolla, California)).

[0226] IV. Virus-like particles In some embodiments, virus-like particles (VLPs) comprising the immunogens of the present disclosure are provided. Typically, such VLPs comprise immunogens containing transmembrane domains, such as recombinant MuV F ectodomain trimers comprising a protomer containing the transmembrane domain and cytosol tail of MuV F, or recombinant MeV F ectodomain trimers comprising a protomer containing the transmembrane domain and cytosol tail of MeV F. VLPs are significantly attenuated, non-replicating viruses because they lack the viral components necessary for viral replication. However, VLPs can display polypeptides similar to those expressed on infectious virus particles (e.g., recombinant MuV or MeV F ectodomain trimers) and, when administered to a subject, can induce an immune response to MuV or MeV. Exemplary virus-like particles and methods for their production, as well as viruses known to form VLPs, such as human papillomavirus, HIV (Kang et al., Biol. Chem. 380:353-64 (1999)), Semlik 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)), and canine parvovirus (Hurtado et al.). Viral proteins from several viruses, including hepatitis E virus (Li et al., J. Virol. 70:5422-9 (1996)) 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 are also provided, comprising the immunogens of this disclosure (e.g., recombinant MuV F ectodomain trimers, recombinant MeV F ectodomain trimers, or corresponding fusions with MuV HN ectodomains or MeV H ectodomains, or MuV HN or MeV H macromers) and a pharmaceutically acceptable carrier. Such compositions can be administered to a subject by various modes of administration, e.g., intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, or parenteral routes. In some embodiments, a pharmaceutical composition comprising one or more of the immunogens of this disclosure is an immunogenic composition. Practical methods for preparing administerable compositions are described in more detail in publications such as Remingtons Pharmaceutical Sciences (19th edition, Mack Publishing Company, Easton, Pennsylvania, 1995).

[0228] Therefore, the immunogens described herein can be formulated using pharmaceutically acceptable carriers to help maintain biological activity and to promote increased stability during storage within an acceptable temperature range. Potential carriers include, but are not limited to, physiological equilibrium culture media, phosphate-buffered saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions), various types of wetting agents, 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 may be packaged for immediate use or lyophilized. The lyophilized preparation is mixed with a sterile solution prior to administration for single or multiple doses.

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

[0230] The immunogenic compositions of this disclosure may contain, as pharmaceutically acceptable media, substances necessary to approximate physiological conditions, such as pH adjusters and buffers, tonicity adjusters, and wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

[0231] The immunogenic composition may optionally include adjuvants to enhance the host 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 adjuvants, MPL® (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Indiana), and IL-12 (Genetics Institute, Cambridge, Massachusetts), TLR agonists (e.g., TLR-9 agonists, e.g., cytidine-phospho-guanosine oligodeoxynucleotide (CpG-ODN) 1018), and many other suitable adjuvants well known in the art may be included in the composition. Suitable adjuvants include, for example, Toll-like receptor agonists, alum, AlPO4, alpha-hydrogels, 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 copolymer, 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 because they stimulate the immune system nonspecifically, thereby helping to enhance the immune response to pharmaceutical products.

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

[0233] In some cases, it may be desirable to combine the immunogens of this disclosure with other pharmaceutical products (e.g., vaccines) that induce a protective response to other agents. For example, compositions comprising the recombinant MuV F ectodomain trimer, recombinant MeV F ectodomain trimer, or corresponding fusions with MuV HN ectodomain or MeV H ectodomain described herein may be administered concurrently (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 this disclosure described herein may be administered concurrently 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 compositions may be provided as sterile compositions. The immunogenic compositions typically contain an effective amount of the immunogen of this disclosure and can be prepared by conventional techniques. Typically, the amount of immunogen in each dose of the immunogenic composition is selected as an amount that induces an immune response without significant adverse side effects. In some embodiments, the compositions may be provided in unit dosage forms 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 forms include a suitable pre-selected single dose for administration to a subject, or a divided or measured multiple dose of two or more pre-selected unit doses, and / or a measuring mechanism for administering a unit dose or multiple doses thereof.

[0235] VI. Methods for inducing an immune response To induce an immune response to MuV and / or MeV in a subject, the immunogens of this disclosure (e.g., recombinant MuV F ectodomain trimers, recombinant MeV F ectodomain trimers, or corresponding fusions with MuV HN ectodomain or MeV H ectodomain, MuV HN or MeV H multimers, nucleic acid molecules encoding the immunogens of this disclosure (e.g., RNA molecules), or protein nanoparticles or virus-like particles containing the immunogens) may be administered to the subject. In certain specific cases, the subject is human. The immune response may be a protective immune response, such as a response that inhibits subsequent infection by MuV and / or MeV. Induction of an immune response may also be used to treat or inhibit MuV and / or MeV infection and related diseases.

[0236] Subjects who have or are at risk of developing MuV or MeV infection due to or potential exposure to MuV or MeV may be selected for treatment. After administration of the immunogens of this disclosure, subjects may be monitored for MuV and / or MeV infection, or related symptoms, or both.

[0237] Typical subjects for which treatment by the therapeutic agents and methods of this disclosure are intended include humans. In some embodiments, the subjects are human subjects who are seronegative for MuV and / or MeV-specific antibodies. In some embodiments, the subjects are human subjects who are seronegative for MuV and / or MeV-specific antibodies, and the immunogen is administered to boost the immune response to MeV and / or MuV in the subjects. To identify subjects for treatment by the methods of this disclosure, accepted screening methods are used to determine risk factors associated with the target or suspected disease or condition, or to determine the pre-existing disease or condition status in the subjects. These screening methods include, for example, conventional precision tests to determine environmental, family, occupational, 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 allow clinicians to select patients who require treatment with the methods and immunogenic compositions of this disclosure. In accordance with these methods and principles, the compositions may be administered as an independent preventive or treatment program, or as a continuation, adjunct, or coordinate treatment to other treatments, in accordance with the teachings herein or other conventional methods.

[0238] The purpose of administering the immunogens of this disclosure may be prevention or treatment. When administered prophylactically, the immunogens may be administered prior to any symptoms, for example, prior to infection. Prophylactic administration may help prevent or improve any subsequent infection. In some embodiments, the method may involve the steps of selecting subjects at risk of developing MuV and / or MeV infection and administering a therapeutically effective dose of the immunogens of this disclosure to those subjects. The immunogens may be administered prior to anticipated exposure to MuV and / or MeV to reduce the expected severity, duration, or degree of infection and / or associated disease symptoms after exposure to the virus or suspected exposure, or after the onset of infection. Populations that may benefit from the prophylactic use of the immunogens of this disclosure (e.g., as a booster immunization treatment) include children at school entry (e.g., age 5) and young adults at high school or college entry or military enlistment (e.g., ages 15-18). Transplant recipients may also need revaccination, or immunocompromised children, such as those who are HIV-positive, would benefit from protein vaccines rather than potentially unsafe live attenuated viruses, including pregnant women.

[0239] Where provided therapeutically, the immunogens of this disclosure are provided at or after the onset of symptoms of MuV and / or MeV infection, or after a diagnosis of MuV and / or MeV infection. Treatment of MuV by inhibiting MuV replication or infection may include delaying and / or reducing the signs or symptoms of MuV infection in a subject. Treatment of MeV by inhibiting MeV replication or infection may include delaying and / or reducing the signs or symptoms of MeV infection in a subject. In some cases, treatment using the methods disclosed herein extends the survival time of a subject.

[0240] In some embodiments, administration of the immunogens of this disclosure to a subject may induce the production of an immune response that is protective against and reduces disease symptoms when the subject subsequently becomes infected or reinfected with wild-type MuV and / or MeV. While naturally circulating viruses may still have the ability to cause infection, it is conceivable that vaccination reduces the likelihood of severe or life-threatening symptoms, and that subsequent infection with wild-type viruses may lead to enhanced resistance. After vaccination, detectable levels of host-produced serum and host-produced secreted antibodies are present that have the ability to neutralize 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 part of the vaccine subgroup.

[0241] The immunogens and immunogenic compositions described herein are provided to a subject, preferably a human, in an amount effective in inducing or enhancing an immune response to MuV and / or MeV. The actual dosage of the immunogens of this disclosure will vary depending on factors such as the indication and subject-specific conditions (e.g., age, size, health status, severity of symptoms, susceptibility factors of the subject), the time and route of administration, other drugs or treatments administered concurrently, and the pharmacology specific to the composition for inducing the desired activity or biological response in the subject. The dosage regimen can be adjusted to obtain an optimal prophylactic or therapeutic response.

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

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

[0244] In some embodiments, the booster may be administered approximately 2, 3-8, or 4 weeks after the prime, or approximately several months after the prime. In some embodiments, the booster may be administered approximately 5, 6, 7, 8, 10, 12, 18, or 24 months after the prime, or around that time. Periodic additional boosters may also be used at appropriate times to enhance the subject's "immunological memory." The appropriateness of selected vaccination parameters, such as formulation, dosage, and regimen, can be determined by taking aliquots of serum from the subject during the course of the immunization program and assaying antibody titers. In addition, the clinical status of the subject can be monitored for desired effects, such as inhibition of MuV and / or MeV infection or improvement of disease status (e.g., reduction of viral load). If such monitoring indicates that vaccination is below optimal, the subject may be given a booster dose of an additional immunogenic composition, and the vaccination parameters may be modified so that an enhancement of the immune response is expected.

[0245] In some embodiments, the prime-boost method may include a DNA primer and protein-boosting vaccination protocol for the target. The method may include two or more doses of nucleic acid molecules or proteins.

[0246] In the case of protein-based therapeutics, typically each human dose would 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 used in an immunogenic composition is selected based on the target population (e.g., infants or older adults). The optimal amount for a particular composition can be confirmed by standardized testing involving observation of antibody titers and other responses in the target population. It is understood that an effective amount of the immunogen of this disclosure in an immunogenic composition, such as recombinant MuV or MeV F ectodomain trimers or chimeras with recombinant MeV F ectodomain trimers or their MuV HN or MeV H ectodomains, viral vectors, or nucleic acid molecules, may be insufficient to induce an immune response with a single dose, but may be effective with multiple doses, for example, in a prime-boost dosing protocol.

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

[0249] For each specific target group, the specific drug regimen may be evaluated and adjusted over time in accordance with individual needs and the judgment of the professional administering or managing 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 cases, at least two doses, e.g., at least three doses, will be given. In some embodiments, an annual boost may be given, for example, in conjunction with an annual influenza vaccination.

[0250] In some embodiments, the antibody response of the subject will be determined in relation to the evaluation of an effective dosage / immunotherapy protocol. In most cases, it will suffice to evaluate the antibody titer in serum or plasma obtained from the subject. Decisions regarding whether to perform a booster inoculation and / or to change the amount of therapeutic agent administered to the individual can be based, at least in part, on antibody titer levels. Antibody titer levels can be based on immunobinding assays that measure the concentration of antibodies in serum that bind to antigens, such as MuV F protein and / or MeV F protein.

[0251] The determination of an effective dosage is typically guided by a dosage protocol that significantly reduces the onset or severity of symptoms or pathologies of the targeted disease in the subject, or by a dosage protocol that induces a desired response (e.g., a neutralizing immune response) in the subject, based on animal model studies 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, the effective dosage can be determined using in vitro models (e.g., immunological and histopathological assays). Using such models, the appropriate concentration and dose for administering an effective amount of composition (e.g., an amount effective to induce a desired immune response or alleviate one or more symptoms of the targeted disease) can be determined by standard calculations and adjustments alone. In an alternative embodiment, the effective amount or effective dose of composition may simply inhibit or enhance one or more selected biological activities correlated with the diseases or pathologies described herein, for therapeutic or diagnostic purposes.

[0252] Administration of immunogen compositions that induce an immune response to reduce or prevent infections can eliminate such infections, but not necessarily completely, as long as the infection is reduced to a measurable degree. For example, administration of an effective amount of the active ingredient can reduce MuV or MeV infection (measured, for example, by infection of cells with MuV or MeV, or by the number or percentage of objects infected with MuV or MeV) by a desired amount, for example, at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (also known as sterilizing immunity, which, in comparison to a suitable control, eliminates or prevents detectable MuV or MeV infection).

[0253] In some embodiments, administration of 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 after immunization, frozen, and stored for neutralization testing. Methods for assaying neutralizing activity include, but are not limited to, plaque reduction titer (PRNT) assays, microneutralization assays, flow cytometry-based assays, and single-cycle infection assays. In some embodiments, serum neutralizing activity can be assayed using a panel of MuV and / or MeV pseudoviruses.

[0254] One approach for nucleic acid administration is direct immunization with plasmid DNA, such as mammalian expression plasmids. Immunotherapy with nucleic acid constructs is well known in the art and is taught, for example, in U.S. Patent No. 5,643,578 (describes a method for immunizing vertebrates by introducing DNA encoding a desired antigen to induce a cell-mediated or humoral response) and U.S. Patents No. 5,593,972 and 5,817,637 (describes the functional ligation of an antigen-encoding nucleic acid sequence to a regulatory sequence that enables expression). U.S. Patent No. 5,880,103 describes several methods for delivering immunogenic peptides or nucleic acids encoding other antigens to organisms. These methods include liposome delivery of nucleic acids (or synthetic peptides themselves) and immunostimulatory constructs, i.e., ISCOM®, which are negatively charged cage-like structures 30-40 nm in size that are spontaneously formed when cholesterol and Quil A® (saponin) are mixed. By using ISCOM® as an antigen delivery medium, protective immunity has been generated in experimental models of various infections, including toxoplasmosis and Epstein-Barr virus-induced tumors (Mowat and Donachie, Immunol. Today 12:383, 1991). Even a dose of as little as 1 μg of antigen encapsulated in ISCOM® has been found to induce a class I-mediated CTL response (Takahashi et al., Nature 344:873, 1990).

[0255] In some embodiments, plasmid DNA vaccines are used to express the immunogen of the Disclosure in a subject. For example, a nucleic acid molecule encoding the immunogen of the Disclosure may be administered to a subject to induce an immune response to the F protein of MuV or MeV. In some embodiments, the nucleic acid molecule may be included on a plasmid vector for DNA immunization, such as the pVRC8400 vector (described in Barouch et al., J. Virol, 79, 8828-8834, 2005, incorporated herein by reference).

[0256] Another approach to using nucleic acids in immunotherapy involves expressing the immunogens of this disclosure using attenuated viral hosts (e.g., attenuated MuV or MeV vectors) or attenuated viral or bacterial vectors. Recombinant vaccinia viruses, adeno-associated viruses (AAVs), herpesviruses, retroviruses, cytomegaloviruses, paramyxoviruses, pneumoviruses, or other viral vectors can be used to express peptides or proteins and thereby induce a CTL response. For example, useful vaccinia vectors and methods in immunotherapy 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, RNA immunization can be used to target the immunogen of this disclosure, for example, in a lipid-encapsulated mRNA immunization platform (e.g., Roth et al., "A Modified mRNA Vaccine Targeting Immunodominant NS Epitopes Protects Against Dengue Virus Infection in HLA Class I Transgenic Mice", Frot 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, Jul 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). See (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 aspect, a nucleic acid encoding a protomer of a MuV F or MeV F ectodomain trimer of the present disclosure is directly introduced into a cell. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad's HELIOS™ gene gun. The nucleic acid can consist of a "naked" plasmid under the control of a strong promoter. Typically, DNA is injected intramuscularly, but it can also be injected directly into other sites. Dosages for injection are usually on the order of about 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, an mRNA-based immunization protocol can be used to directly deliver the nucleic acid encoding the immunogen of this disclosure into cells. In some embodiments, mRNA-based nucleic acid vaccines can be a powerful alternative to the approaches described above. mRNA vaccines eliminate safety concerns regarding DNA integration into the host genome and can be directly translated in the cytoplasm of host cells. Furthermore, simple cell-free in vitro synthesis of RNA avoids manufacturing problems associated with viral vectors. Two exemplary forms of RNA-based vaccines that can be used to deliver the nucleic acids encoding the immunogens of this disclosure are conventional non-amplifying 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) is one example.

[0260] In some embodiments, lipid nanoparticles comprising mRNA encoding an immunogen of the present disclosure are used in methods of inducing an immune response, such as those described in WO2017070626, US2019 / 0192646, and the method described for the mRNA-1273 vaccine in Jackson et al., "An mRNA vaccine against SARS-CoV2 - preliminary report", N. Engl. J. Med., 383(20):1920 - 1931, 2020, which are hereby incorporated by reference herein. 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). Further, the mRNA encoding the immunogen can be a modified mRNA having 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 certain features of certain embodiments, but the claims should not be limited to these illustrated features.

[0262] Example 1 MuV F protein stabilized in a pre-fusion conformation and its fusion with the MuV HN ectodomain In this example, embodiments of a MuV F ectodomain trimer stabilized in a pre-fusion conformation by one or more amino acid substitutions are illustrated. A MuV F ectodomain trimer linked to the MuV HN ectodomain is further provided. The pre-fusion stabilized MuV F ectodomain trimer and the corresponding fusion with the MuV HN ectodomain are useful, for example, in inducing a neutralizing immune response against MuV in a subject.

[0263] Until the introduction of the mumps-containing vaccine in the late 1960s, mumps caused a wide range of pathological conditions characterized by fever, parotitis, and, less commonly, orchitis, meningitis, encephalitis, and hearing loss. The measles, mumps, and rubella (MMR) combined vaccine dramatically reduced the incidence of mumps worldwide. While two doses of the MMR vaccine are approximately 88% effective in preventing mumps, since 2006, the number of mumps cases has been increasing again worldwide, even in highly vaccinated populations, with the number of affected individuals in the United States being >30,000. Factors contributing to this increase include waning immunity, weaker antibody responses, and antigenic differences between the Jeryl Lynn strain used in the MMR vaccine and the circulating wild-type strain. The dominant mumps genotype characterizing recent outbreaks in the United States and Europe is genotype G.

[0264] As described herein, we engineered a pre-fusion conformation-stabilized MuV F glycoprotein using structure-based design. The crystal structure of the mumps fusion glycoprotein at 2.16 Å resolution clarifies the basis for pre-fusion conformation stabilization. A potent cross-reactive mumps genotype plaque reduction neutralization titer (PRNT) was induced in mice from either the mumps pre-fusion stabilized F glycoprotein or a chimeric fusion glycoprotein of the pre-fusion stabilized mumps F trimer linked to genotype G mumps hemagglutinin neuraminidase (HN). The pre-fusion F-HN mumps chimer was able to induce the highest PRNT against genotypes A, G, and H mumps viruses, 100 times higher than the reported human protective titer. In addition, monoclonal antibodies against pre-fusion F and HN genotypes of mumps were isolated from immunized mice, and they possessed the ability to neutralize genotype G mumps virus with a certain range of potency. Structural and binding analysis of these pre-fusion F-specific antibodies revealed binding to four discontinuous neutralizing antigen sites. The engineered immunogens are candidate vaccines for mumps, either as novel vaccines or as booster vaccines.

[0265] result Disulfide bond and membrane-proximal coiled-coil stabilization robustly stabilize the soluble pre-fusion mumps F trimer, enabling its production.

[0266] The MuV F ectodomain linked to the C-terminal GCN4 trimerizing domain forms a trimer that spontaneously transitions to the pre-fusion conformation when produced intracellularly. Unstabilized recombinant MuV F-GCN4 is highly unstable, and at the time of evaluation (EM), 100% of the molecule has transitioned to the post-fusion conformation. Protein expression levels are also substantially reduced without stabilization. Therefore, using a structure-based vaccine design, we identified mutations that stabilize the MuV F ectodomain in the pre-fusion conformation and produced a "single-chain" MuV F protein with increased expression levels by eliminating the F1 / F2 cleavage site.

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

[0268] To "lock" the MuV F ectodomain in the pre-fusion conformation, a multiple stabilization strategy was employed, including the introduction of disulfide bonds and proline substitutions. The selection of residues in MuV F to be mutated to cysteine ​​was based on homology design from the PIV5 pre-fusion F structure (PDB 4WSG) and on residues that were predicted to undergo conformational changes during the transition from the pre-fusion to the post-fusion conformation. The C beta atoms of the residue pairs were confirmed to be within 5 angstroms and in an orientation that would allow for the formation of disulfide bonds. In total, approximately 60 different mutants were designed, expressed, purified, and their expression levels were evaluated, and their pre-fusion conformations were assessed by negative staining EM.

[0269] Mutations were introduced into the MuV F ectodomain (based on C-terminal cleavage 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 included mutations to remove the F1 / F2 furin cleavage site. Prefusion stabilization mutations evaluated included cysteine ​​substitutions at one or more of the following 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 proline substitutions at MuV F position 184. The relevant sequences are shown below. Figure 1A summarizes the first successful prefusion stabilization mutation.

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

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

[0272] By creating matrices of disulfide substitutions and C-terminal coiled-coil-GCN4 attachment sites, protein expression levels and the ratio of proteins adopting pre-fusion trimer conformations or post-fusion trimer conformations were evaluated using negative-stained EM (Figure 1A). Five combinations of disulfide bond sites and GCN4 attachment sites resulted in 100% pre-fusion trimer formation, and one such combination was observed to yield high-yield protein expression of approximately 4.8 mg / L from Expi293 cells (V206C-A223C and 476-GCN4). The negative-stained EM 2D mean of this design (Figure 1B, top) contrasted with the post-fusion mumps F glycoprotein trimer (Figure 1B, bottom), consistent with previous observations regarding the parainfluenza virus F protein conformation.

[0273] The crystal structure of the prefusion mumps F glycoprotein trimer at 2.16 Å resolution reveals the designed stabilizing disulfides 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) showed a homogeneous peak (Figure 1C) that could be deglycosylated and crystallized when expressed in the presence of kifunensine. The 3D 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 prefusion PIV5 and other paramyxovirus prefusion F trimers (Stewart-Jones et al., Proc Natl Acad Sci U S A 115, 12265-12270, 2018, Welch, B.D. et al. Proc Natl Acad Sci U S A 109, 16672-16677, 2012, Xu K. et al. PLoS Pathog 11(12)e1005322, 2015), which has a "closed lid" structure composed of the loop N177~S184 at the trimer apex of the mumps prefusion F trimer, formed by interactions between the side chains T178, Q179 and N181 (Figures 2A and 2B). The remaining N-acetylglucosamine moieties from the six glycans at positions N73, N182, N352, N427, N433 and N457 could be seen in the electron density, and a glycosylation model was constructed (Figure 2C). The V206C-A223C disulfide was clearly defined by the electron density, and the Cα-Cα atomic distance was 4.8 Å, while in the homologous postfusion PIV3 F trimer structure [PDB ID 1ZTM], the Cα-Cα atoms were located 5.8 Å apart. The DI~DIII domains surround a large aqueous cavity with a volume of approximately 20,000 Å 3 as measured, and the protomer is 6,500 Å 2These residues covered the interface (Stewart-Jones et al., Proc Natl Acad Sci USA 115,12265-12270,2018; Welch, BD et al. Proc Natl Acad Sci USA 109,16672-16677,2012; Xu K. et al. PLoS Pathog 11(12):e1005322,2015). Figure 1E shows residues that are thought to cause conformational changes between the pre-fusion and post-fusion conformations (based on PIV5 pre-F structure [PDB ID 4WSG] and PIV3 post-F [PDB ID 1ZTM]), which correspond to MuV F residues 92-253.

[0274] While sequence identity between mumps F and HN is relatively high between genotypes (Figures 2A-2C), we mapped polymorphic variations in the pre-fusion structure of mumps F and HN (Figure 2D). Mapping of pre-fusion F mutations for 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 aqueous cavities in the core of the pre-fusion trimer (Figure 2D, left). In contrast, the mumps HN dimer structure revealed that most polymorphic amino acids, including the glycan mutation at position N464 between Jeryl Lynn HN and genotype G HN, were exposed to the solvent (Figure 2D, right). The fact that polymorphic residues exposed to the solvent are more prevalent on HN than on pre-fusion F suggests that the resistance of genotype G to Jeryl Lynn vaccine-induced humoral immunity can be explained more by HN than by pre-fusion F.

[0275] Cross-strain efficacy of stabilizing mutations. To demonstrate that pre-fusion stabilizing mutations are effective against F cells across MuV strains, these mutations were tested in F cells from several different MuV strains. MuV F V206C-A223C-GGG-476-GCN4 (SEQ ID NO:11) is based on genotype C MuV F. Introducing 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 resulted in prefusion stabilization. Furthermore, introducing these prefusion-stabilizing mutations into the F protein from the following MuV strains also resulted in prefusion stabilization, as measured by negative staining 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 of the trimer. The format is illustrated in Figure 4A. The corresponding sequences are shown below. Negative staining EM shows that the F ectodomain maintains its pre-fusion conformation, and the three HN ectodomains (one linked to each F protomer) are positioned towards the C-terminus of the trimerization domain. This design yielded approximately 0.3 mg / L from Expi293 cells, was monodisperse on size exclusion chromatography, and showed the expected assembly on negative staining EM (Figure 1C and Figure 4A).

[0277] Pre-fusion-stabilized mumps F and pre-fusion-stabilized F-HN chimeric trimers induce high-titer neutralizing antibodies in mice. The neutralizing antibody-inducing ability of pre-fusion-stabilized mumps F was evaluated compared to that of multiple mumps immunogens. Ten CB6F1 / J mice were immunized at weeks 0, 3, and 10 with 10 μg of mumps glycoprotein combined with 10 μg of polyinosine-polycytidic acid (Poly-I:C) adjuvant, and the serum's ability to prevent mumps virus infection of HEp-2 cells was measured (Figure 3A).

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

[0279] When mice were immunized with the pre-fusion mumps F-containing immunogen (preF or preF-HN), specific responses to preF were detected in the serum, and the level of binding to post-fusion immunized mice was lower than that (Figure 4B). The recombinant HN monomer bound only to the serum of HN-immunized mice, while the serum from preF-HN-immunized mice showed sufficient HN binding levels, while monomeric HN-immunized mice showed little binding, suggesting that the multivalency of HN drives a robust humoral response (Figure 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, the PRNT two weeks after each immunization was analyzed (Figure 3B). A stepwise increase in neutralizing titers was observed after each immunization, but the increase shown by the third immunization was even smaller than that after the second immunization. Neutralizing antibodies were also observed in post-fusion immunizations, but the pre-fusion F immunogen showed 3.5-fold and 2.5-fold higher neutralization than post-fusion F, respectively, after the second and third immunizations (geometric mean infectious dose (ID 60 values) were 322 and 789, respectively, against genotype G mumps virus). The preF-HN chimeric mutant induced a neutralizing titer 12-fold higher than post-fusion F and 5-fold higher than pre-fusion F after the third immunization, and the ID 60 value against genotype G virus was 3930.

[0281] Next, to characterize the cross-neutralizing antibodies induced from recombinant immunogens, PRNT against Jeryl Lynn and genotype H virus was evaluated. In the case of post-fusion F and pre-fusion F, higher levels of neutralization against Jeryl Lynn virus were observed compared to against genotype G virus, while equivalent PRNT was observed in the case of pre-fusion F-HN chimeras. At week 16, serum from both the pre-F and pre-F-HN groups showed robust PRNT against genotype H virus. This indicates that these recombinant immunogens can induce antibodies capable of cross-neutralizing numerous mumps genotypes. Further monitoring of the persistence of PRNT against these three mumps viruses for another 6 months revealed a decrease in titers, but ID remained after 3 months. 60 A plateau was formed, and the geometric mean PRNTs of preF-HN were found to be approximately 640, 800, and 1700 for genotype G, Jeryl Lynn, and genotype H viruses, respectively (Figure 4D). ID formed by preF immunogen for genotype G, Jeryl Lynn, and genotype H viruses 60 The PRNT plateaus were approximately 100, 660, and 3153 (Figure 4E). Overall, pre-fusion-stabilized F parotitis linked to HN yielded higher neutralization titers than F alone, representing a design strategy for stoichiometrically combining both viral surface antigens in a single immunogen.

[0282] Consideration Human immunity to mumps after MMR vaccination is typically characterized by approximately 220 PRNTs for Jeryl Lynn and approximately 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 this disclosure appear to give an increase in efficacy as measured by PNRT, assuming that the mouse model data correlate with the human response.

[0283] Given the recent mumps outbreaks among those who received 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 the mumps-containing vaccine for individuals at risk of contracting mumps during outbreaks. A third dose of attenuated live MMR resulted in a temporary increase in neutralizing titer that lasted approximately 12 months (Fiebelkorn AP et al. Open Forum Infect Dis 1(3):ofu094, 2014). In addition, a third dose of MMR reduced the risk of mumps infection by 78% compared to 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 embodiment provides an alternative vaccine modality to MMR administration in the context of a mumps outbreak, offering increased persistence and efficacy.

[0284] A pre-fusion F-HN chimera containing two key neutralizing targets on the mumps virion can induce a potent cross-genotypic neutralizing response, including a neutralizing response to the outbreak-causing dominant mumps genotype G and two other genotypes A and H, making it a universal vaccine candidate against mumps strains worldwide.

[0285] array: TIFF2026062907000040.tif164160TIFF2026062907000041.tif232160TIFF2026062907 000042.tif232160TIFF2026062907000043.tif234160TIFF2026062907000044.tif86160

[0286] The above sequence includes the N-terminal signal peptide, MuV F ectodomain, GCN4 trimer domain, optionally MuV HN ectodomain, thrombin cleavage site, HIS tag and Strep tag, as well as various linker residues between segments.

[0287] Example 2 A fusion of the MeV F protein stabilized in its pre-fusion conformation with its MeV H ectodomain or 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. A MeV F ectodomain trimer linked to a MeV H ectodomain is further provided. The pre-fusion stabilized MeV F ectodomain trimer and the corresponding fusion 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 trimerizing domain forms a trimer that spontaneously transitions to the pre-fusion conformation when produced intracellularly. Unstabilized recombinant MeV F-GCN4 is highly unstable, and at the time of evaluation (EM), 100% of the molecule has transitioned to the post-fusion conformation. Protein expression levels are also substantially reduced without stabilization.

[0289] Therefore, using structure-based vaccine design, we identified mutations that stabilize the MeV F ectodomain in the pre-fusion conformation (based on pre-fusion PIV5 F structure PDB ID 4WSG and MeV F structure PDB ID 5YXW), and produced increased-expression "single-chain" MeV F proteins by eliminating the F1 / F2 cleavage site. Multiple stabilization strategies, including the introduction of disulfide bonds and proline substitutions, were used to "lock" the MeV F ectodomain in the pre-fusion conformation. In total, approximately 40 different mutants were designed, expressed, purified, and evaluated for expression levels, and their pre-fusion conformation was assessed by negative-stained EM.

[0290] Mutations were introduced into the MeV F ectodomain (based on C-terminal cleavage 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 included mutations to remove the F1 / F2 furin cleavage site. Pre-fusion stabilization mutations evaluated included cysteine ​​substitutions at one or more of the following 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 proline substitutions at MeV F position 194. The relevant sequences are shown below.

[0291] Expression and purification of the single-chain pre-fusion 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 between the MeV F ectodomain trimer in the pre-fusion conformation and that in the post-fusion conformation. Furthermore, MeV F R165C-M171C-486-GCN4 (SEQ ID NO:38) showed 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 construct and other constructs were analyzed by electron microscopy.

[0293] Immunotherapy assays were performed using the post-fusion conformation MeV F ectodomain trimer and the pre-fusion conformation MeV F ectodomain trimer (MeV F R165C-M171C-486-GCN4 (SEQ ID NO:38)). The immunotherapy protocol shown in Figure 3A was followed. Ten CB6F1 / J mice were immunized with 10 μg of protein in a poly-IC adjuvant at weeks 0 and 3, and the neutralization titer of serum from the immunized mice at week 5 was evaluated. When the immune serum was evaluated using the MeV neutralization assay (Figure 5C), it was found that the immune serum from animals immunized with MeV F R165C-M171C-486-GCN4 (SEQ ID NO:38) neutralized MeV 200 times more effectively than the serum from animals immunized with post-fusion MeV F, exceeding the defense threshold.

[0294] array: TIFF2026062907000045.tif83160TIFF2026062907000046.tif235160TIFF20260629070 00047.tif229160TIFF2026062907000048.tif232160TIFF2026062907000049.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 segments.

[0296] In addition, we designed a chimeric construct having a MeV F ectodomain with amino acid substitutions for stabilization in the pre-fusion conformation, linked to either a MuV HN ectodomain or a MeV H ectodomain, as follows. TIFF2026062907000050.tif162160

[0297] The above sequence includes the N-terminal signal peptide, the MeV F ectodomain, and the GCN4 trimer domain, optionally the T4 fibrintin trimer domain, the MeV H ectodomain, and various linker residues between segments.

[0298] Example 3 MuV HN multimer, MeV H multimer, and MuV pre-F-MeV H chimera This example describes one embodiment of a recombinant MuV pre-F ectodomain trimer linked to a MeV H ectodomain for providing a chimeric immunogen that induces a cross-neutralizing immune response against MeV and MuV. In addition, the multimer MuV HN and the multimer 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 protomer of the trimer. The construct evaluated included a MuV F ectodomain containing V206C-A223C, a mutation to remove the F1 / F2 furin cleavage site, a trimerization domain fused at position 476 of the ectodomain, and a MeV H ectodomain ligated to the C-terminus of the trimerization domain. In this embodiment, the trimerization domain contained both the GCN4 trimerization domain and the T4 fibrintin trimerization domain in series, but either of these domains can also be used alone. The format is illustrated in Figure 6A. The corresponding sequences are shown below. TIFF2026062907000051.tif52160

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

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

[0302] By ligating the N-terminus of the head region to the T4 fibrintin trimerization domain, we constructed the trimer MuV HN ectodomain head region and the trimer MeV H ectodomain head region. The sequences are shown below. TIFF2026062907000052.tif65160

[0303] Dimerized 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 shown below. TIFF2026062907000053.tif28160

[0304] The dimerized MeV H, containing both 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 stalk and head of MeV H dimerize in physiological solution. Exemplary sequences of the stalk and head regions of MeV H are shown below. TIFF2026062907000054.tif146160

[0305] MuV HN, which includes 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 shown below. TIFF2026062907000055.tif131160

[0306] In addition, chimeric trimer MuV HN ectodomain head regions and trimer MeV H ectodomain head regions were constructed by ligating these molecules to the N-terminus and C-terminus of the T4 fibrintin trimerization domain and / or GCN4 trimerization domain. The sequences are listed below. TIFF2026062907000056.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 staining EM (see Figure 6A).

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

[0309] Regarding MeV neutralization (Figure 6B), the average PRNT ID for MeV H dimers is 66,000, for MeV H trimers it is 30,000, and for trimerized H at the C-terminus of pre-fusion MuV F it is 35,000. 60 This induced [results]. These results were quite surprising, given the much lower neutralization titers observed with immunization using the pre-fusion MeV F trimer (PRNT of 873, see Figure 5C). The average human PRNT titer after two MMR vaccinations is approximately 650. The extremely high MeV PRNT titers with this MeV H design were unexpected, as we initially thought that the pre-fusion F ectodomain trimer would give a better immune response (similar to other paramyxoviruses such as RSV). Surprisingly, the multimer MeV H immunogen showed excellent immunogenicity, being up to 75 times more potent than the pre-fusion stabilized MeV F ectodomain trimer and approximately 100 times higher than the human response after MMR vaccination.

[0310] Regarding MuV neutralization (Figure 6C), each of the immunogens evaluated induced an immune response exceeding the defense threshold, and soluble trimerized MuV HN induced an extremely potent immune response. This is highly surprising, considering that the response induced by the soluble MuV HN monomer is very weak (Figure 4C). Surprisingly, there was a 13.4-fold increase in neutralizing potency between the MuV HN ectodomain monomer and the trimerized MuV HN ectodomain.

[0311] Protein production, analysis, and immunization were carried out as described above.

[0312] It will be apparent that the exact details of the described methods or compositions may be modified or altered without departing from the gist of the described embodiments. The inventors claim all such modifications and alterations that are included in the scope and gist of the claims below.

[0313] Sequence information SEQUENCE LISTING <110> The United States of America, as Represented by the Secretary, Department of Health and Human Services <120> MUMPS AND MEASLES VIRUS IMMUNOGENS AND THEIR USE <150> US 62 / 946,902 <151> 2019-12-11 <160> 106 <170> PatentIn version 3.5 <210> 1 <211> 538 <212> PRT <213> Mumps virus <400> 1 Met Lys Ala Phe Ser Val Thr Cys Leu Gly Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asn Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Lys Arg Phe Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Ile Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Tyr Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Ile Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Val Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Thr Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Val Ser Val Asn 465 470 475 480 Ser Lys Ile Gly Ala Ile Ile Val Ala Ala Leu Val Leu Ser Ile Leu 485 490 495 Ser Ile Ile Ile Ser Leu Leu Phe Cys Cys Trp Ala Tyr Ile Ala Thr 500 505 510 Lys Glu Ile Arg Arg Ile Asn Phe Lys Thr Asn His Ile Asn Thr Ile 515 520 525 Serum Serum Serum Val Asp Asp Leu Ile Arg Tyr 530 535 <210> 2 <211> 513 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 2 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Cys Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Cys Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Val Ser Val Asn 465 470 475 480 Ser Lys Ile Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr 485 490 495 His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Ala 500 505 510 Pro <210> 3 <211> 513 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 3 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Cys Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Cys Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Val Ser Val Asn 465 470 475 480 Ser Lys Ile Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr 485 490 495 His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Ala 500 505 510 Pro <210> 4 <211> 513 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 4 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Cys Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Cys Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Val Ser Val Asn 465 470 475 480 Ser Lys Ile Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr 485 490 495 His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Ala 500 505 510 Pro <210> 5 <211> 513 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 5 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Cys Gln Leu Thr Asn Cys Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Val Ser Val Asn 465 470 475 480 Ser Lys Ile Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr 485 490 495 His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Ala 500 505 510 Pro <210> 6 <211> 513 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 6 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Cys Glu Gly Thr Gln Gln 145 150 155 160 Cys Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Val Ser Val Asn 465 470 475 480 Ser Lys Ile Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr 485 490 495 His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Ala 500 505 510 Pro <210> 7 <211> 513 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 7 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Cys Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Cys Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Val Ser Val Asn 465 470 475 480 Ser Lys Ile Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr 485 490 495 His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Ala 500 505 510 Pro <210> 8 <211> 513 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 8 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Cys Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Cys Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Val Ser Val Asn 465 470 475 480 Ser Lys Ile Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr 485 490 495 His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Ala 500 505 510 Pro <210> 9 <211> 513 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 9 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Pro Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Val Ser Val Asn 465 470 475 480 Ser Lys Ile Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr 485 490 495 His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Ala 500 505 510 Pro <210> 10 <211> 506 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 10 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Cys Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Cys Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Ile Glu Asp Lys 465 470 475 480 Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala 485 490 495 Arg Ile Lys Lys Leu Ile Gly Glu Ala Pro 500 505 <210> 11 <211> 506 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 11 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Cys Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Cys Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Ile Glu Asp Lys 465 470 475 480 Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala 485 490 495 Arg Ile Lys Lys Leu Ile Gly Glu Ala Pro 500 505 <210> 12 <211> 506 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 12 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Cys Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Cys Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Ile Glu Asp Lys 465 470 475 480 Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala 485 490 495 Arg Ile Lys Lys Leu Ile Gly Glu Ala Pro 500 505 <210> 13 <211> 506 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 13 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Cys Gln Leu Thr Asn Cys Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Ile Glu Asp Lys 465 470 475 480 Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala 485 490 495 Arg Ile Lys Lys Leu Ile Gly Glu Ala Pro 500 505 <210> 14 <211> 506 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 14 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Cys Glu Gly Thr Gln Gln 145 150 155 160 Cys Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Ile Glu Asp Lys 465 470 475 480 Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala 485 490 495 Arg Ile Lys Lys Leu Ile Gly Glu Ala Pro 500 505 <210> 15 <211> 506 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 15 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Cys Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Cys Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Ile Glu Asp Lys 465 470 475 480 Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala 485 490 495 Arg Ile Lys Lys Leu Ile Gly Glu Ala Pro 500 505 <210> 16 <211> 506 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 16 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Cys Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Cys Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Ile Glu Asp Lys 465 470 475 480 Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala 485 490 495 Arg Ile Lys Lys Leu Ile Gly Glu Ala Pro 500 505 <210> 17 <211> 506 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 17 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Pro Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Ile Glu Asp Lys 465 470 475 480 Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala 485 490 495 Arg Ile Lys Lys Leu Ile Gly Glu Ala Pro 500 505 <210> 18 <211> 499 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 18 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Cys Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Cys Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys 465 470 475 480 Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly 485 490 495 Glu Ala Pro <210> 19 <211> 499 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 19 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Cys Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Cys Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys 465 470 475 480 Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly 485 490 495 Glu Ala Pro <210> 20 <211> 499 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 20 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Cys Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Cys Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys 465 470 475 480 Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly 485 490 495 Glu Ala Pro <210> 21 <211> 499 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 21 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Cys Gln Leu Thr Asn Cys Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys 465 470 475 480 Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly 485 490 495 Glu Ala Pro <210> 22 <211> 499 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 22 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Cys Glu Gly Thr Gln Gln 145 150 155 160 Cys Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys 465 470 475 480 Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly 485 490 495 Glu Ala Pro <210> 23 <211> 499 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 23 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Cys Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Cys Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys 465 470 475 480 Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly 485 490 495 Glu Ala Pro <210> 24 <211> 499 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 24 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Cys Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Cys Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys 465 470 475 480 Tyr His Glu, Asn Glu, Arg, Lys, Leu, and Gly 485,490,495 Glu Wing Pro <210> 25 <211> 499 <212> PRT <213> Artificial Sequence <220> <223> Recombinant F protein <400> 25 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Pro Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Val Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Ala Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys 465 470 475 480 Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly 485 490 495 Glu Ala Pro <210> 26 <211> 506 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 26 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Val Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asn Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Cys Phe Gln 195 200 205 Pro Gln Leu Ile Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Cys Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Val Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Val Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Arg Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Ser Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Thr Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ser Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Ile Glu Asp Lys 465 470 475 480 Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala 485 490 495 Arg Ile Lys Lys Leu Ile Gly Glu Ala Pro 500 505 <210> 27 <211> 966 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 27 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Cys Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Cys Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Ile Glu Asp Lys 465 470 475 480 Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala 485 490 495 Arg Ile Lys Lys Leu Ile Gly Glu Ala Pro Gly Ser Gly Gly Gly Gly 500 505 510 Gly Gly Asn Ile Pro Leu Val Asn Asp Leu Arg Phe Ile Asn Gly Ile 515 520 525 Asn Lys Phe Ile Ile Glu Asp Tyr Ala Thr His Asp Phe Ser Ile Gly 530 535 540 His Pro Leu Asn Met Pro Ser Phe Ile Pro Thr Ala Thr Ser Pro Asn 545 550 555 560 Gly Cys Thr Arg Ile Pro Ser Phe Ser Leu Gly Lys Thr His Trp Cys 565 570 575 Tyr Thr His Asn Val Ile Asn Ala Asn Cys Lys Asp His Thr Ser Ser 580 585 590 Asn Gln Tyr Val Ser Met Gly Ile Leu Val Gln Thr Ala Ser Gly Tyr 595 600 605 Pro Met Phe Lys Thr Leu Lys Ile Gln Tyr Leu Ser Asp Gly Leu Asn 610 615 620 Arg Lys Ser Cys Ser Ile Ala Thr Val Pro Asp Gly Cys Ala Met Tyr 625 630 635 640 Cys Tyr Val Ser Thr Gln Leu Glu Thr Asp Asp Tyr Ala Gly Ser Ser 645 650 655 Pro Pro Thr Gln Lys Leu Thr Leu Leu Phe Tyr Asn Asp Thr Val Thr 660 665 670 Glu Arg Thr Ile Ser Pro Ser Gly Leu Glu Gly Asn Trp Ala Thr Leu 675 680 685 Val Pro Gly Val Gly Ser Gly Ile Tyr Phe Glu Asn Lys Leu Ile Phe 690 695 700 Pro Ala Tyr Gly Gly Val Leu Pro Asn Ser Thr Leu Gly Val Lys Ser 705 710 715 720 Ala Arg Glu Phe Phe Arg Pro Val Asn Pro Tyr Asn Pro Cys Ser Gly 725 730 735 Pro Gln Gln Asp Leu Asp Gln Arg Ala Leu Arg Ser Tyr Phe Pro Ser 740 745 750 Tyr Phe Ser Asn Arg Arg Ile Gln Ser Ala Phe Leu Val Cys Ala Trp 755 760 765 Asn Gln Ile Leu Val Thr Asn Cys Glu Leu Val Val Pro Ser Ser Asn 770 775 780 Gln Thr Met Met Gly Ala Glu Gly Arg Val Leu Leu Ile Asn Asn Arg 785 790 795 800 Leu Leu Tyr Tyr Gln Arg Ser Thr Ser Trp Trp Pro Tyr Glu Leu Leu 805 810 815 Tyr Glu Ile Ser Phe Thr Phe Thr Asn Ser Gly Pro Ser Ser Val Asn 820 825 830 Met Ser Trp Ile Pro Ile Tyr Ser Phe Thr Arg Pro Gly Ser Gly Asn 835 840 845 Cys Ser Gly Glu Asn Val Cys Pro Thr Ala Cys Val Ser Gly Val Tyr 850 855 860 Leu Asp Pro Trp Pro Leu Thr Pro Tyr Ser His Gln Ser Gly Ile Asn 865 870 875 880 Arg Asn Phe Tyr Phe Thr Gly Ala Leu Leu Asn Ser Ser Thr Thr Arg 885 890 895 Val Asn Pro Thr Leu Tyr Val Ser Ala Leu Asn Asn Leu Lys Val Leu 900 905 910 Ala Pro Tyr Gly Thr Gln Gly Leu Phe Ala Ser Tyr Thr Thr Thr Thr 915 920 925 Cys Phe Gln Asp Thr Gly Asp Ala Ser Val Tyr Cys Val Tyr Ile Met 930 935 940 Glu Leu Ala Ser Asn Ile Val Gly Glu Phe Gln Ile Leu Pro Val Leu 945 950 955 960 Thr Arg Leu Thr Ile Thr 965 <210> 28 <211> 981 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 28 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Cys Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Cys Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Ile Glu Asp Lys 465 470 475 480 Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala 485 490 495 Arg Ile Lys Lys Leu Ile Gly Glu Ala Pro Gly Ser Gly Tyr Ile Pro 500 505 510 Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp 515 520 525 Val Leu Leu Ser Thr Phe Leu Gly Ser Gly Gly Gly Gly Gly Gly Phe 530 535 540 Leu Ala Val Ser Lys Gly Asn Cys Ser Gly Pro Thr Thr Ile Arg Gly 545 550 555 560 Gln Phe Ser Asn Met Ser Leu Ser Leu Leu Asp Leu Tyr Leu Gly Arg 565 570 575 Gly Tyr Asn Val Ser Ser Ile Val Thr Met Thr Ser Gln Gly Met Tyr 580 585 590 Gly Gly Thr Tyr Leu Val Glu Lys Pro Asn Leu Ser Ser Lys Arg Ser 595 600 605 Glu Leu Ser Gln Leu Ser Met Tyr Arg Val Phe Glu Val Gly Val Ile 610 615 620 Arg Asn Pro Gly Leu Gly Ala Pro Val Phe His Met Thr Asn Tyr Leu 625 630 635 640 Glu Gln Pro Val Ser Asn Asp Leu Ser Asn Cys Met Val Ala Leu Gly 645 650 655 Glu Leu Lys Leu Ala Ala Leu Cys His Gly Glu Asp Ser Ile Thr Ile 660 665 670 Pro Tyr Gln Gly Ser Gly Lys Gly Val Ser Phe Gln Leu Val Lys Leu 675 680 685 Gly Val Trp Lys Ser Pro Ala Asp Met Gln Ser Trp Val Pro Leu Ser 690 695 700 Thr Asp Asp Pro Val Ile Asp Arg Leu Tyr Leu Ser Ser His Arg Gly 705 710 715 720 Val Ile Ala Asp Asn Gln Ala Lys Trp Ala Val Pro Thr Thr Arg Thr 725 730 735 Asp Asp Lys Leu Arg Met Glu Thr Cys Phe Gln Gln Ala Cys Lys Gly 740 745 750 Lys Ile Gln Thr Leu Cys Glu Asn Pro Glu Trp Ala Pro Leu Lys Asp 755 760 765 Asn Arg Ile Pro Ser Tyr Gly Val Leu Ser Val Asp Leu Ser Leu Thr 770 775 780 Val Glu Leu Lys Ile Lys Ile Ala Ser Gly Phe Gly Pro Leu Ile Thr 785 790 795 800 His Gly Ser Gly Met Asp Leu Tyr Lys Ser Asn His Asn Asn Val Tyr 805 810 815 Trp Leu Thr Ile Pro Pro Met Lys Asn Leu Ala Leu Gly Val Ile Asn 820 825 830 Thr Leu Glu Trp Ile Pro Arg Phe Lys Val Ser Pro Tyr Leu Phe Thr 835 840 845 Val Pro Ile Lys Glu Ala Gly Glu Asp Cys His Ala Pro Thr Tyr Leu 850 855 860 Pro Ala Glu Val Asp Gly Asp Val Lys Leu Ser Ser Asn Leu Val Ile 865 870 875 880 Leu Pro Gly Gln Asp Leu Gln Tyr Val Leu Ala Thr Tyr Asp Thr Ser 885 890 895 Arg Val Glu His Ala Val Val Tyr Tyr Val Tyr Ser Pro Gly Arg Ser 900 905 910 Phe Ser Tyr Phe Tyr Pro Phe Arg Leu Pro Ile Lys Gly Val Pro Ile 915 920 925 Glu Leu Gln Val Glu Cys Phe Thr Trp Asp Gln Lys Leu Trp Cys Arg 930 935 940 His Phe Cys Val Leu Ala Asp Ser Glu Ser Gly Gly His Ile Thr His 945 950 955 960 Ser Gly Met Val Gly Met Gly Val Ser Cys Thr Val Thr Arg Glu Asp 965 970 975 Gly Thr Asn Arg Arg 980 <210> 29 <211> 1006 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 29 Met Lys Ala Phe Ser Val Thr Cys Leu Ser Phe Ala Val Phe Ser Ser 1 5 10 15 Ser Ile Cys Val Asn Ile Asn Ile Leu Gln Gln Ile Gly Tyr Ile Lys 20 25 30 Gln Gln Val Arg Gln Leu Ser Tyr Tyr Ser Gln Ser Ser Ser Ser Tyr 35 40 45 Ile Val Val Lys Leu Leu Pro Asn Ile Gln Pro Thr Asp Asp Ser Cys 50 55 60 Glu Phe Lys Ser Val Thr Gln Tyr Asn Lys Thr Leu Ser Asn Leu Leu 65 70 75 80 Leu Pro Ile Ala Glu Asn Ile Asn Asn Ile Ala Ser Pro Ser Pro Gly 85 90 95 Ser Arg Arg His Gly Gly Gly Ala Gly Ile Ala Ile Gly Ile Ala Ala 100 105 110 Leu Gly Val Ala Thr Ala Ala Gln Val Thr Ala Ala Val Ser Leu Val 115 120 125 Gln Ala Gln Thr Asn Ala Arg Ala Ile Ala Ala Met Lys Asn Ser Ile 130 135 140 Gln Ala Thr Asn Arg Ala Val Phe Glu Val Lys Glu Gly Thr Gln Gln 145 150 155 160 Leu Ala Ile Ala Val Gln Ala Ile Gln Asp His Ile Asn Thr Ile Met 165 170 175 Asn Thr Gln Leu Asn Asn Met Ser Cys Gln Ile Leu Asp Asn Gln Leu 180 185 190 Ala Thr Ser Leu Gly Leu Tyr Leu Thr Glu Leu Thr Thr Cys Phe Gln 195 200 205 Pro Gln Leu Thr Asn Pro Ala Leu Ser Pro Ile Ser Ile Gln Cys Leu 210 215 220 Arg Ser Leu Leu Gly Ser Met Thr Pro Ala Val Val Gln Ala Thr Leu 225 230 235 240 Ser Thr Ser Ile Ser Ala Ala Glu Ile Leu Ser Ala Gly Leu Met Glu 245 250 255 Gly Gln Ile Ile Ser Val Leu Leu Asp Glu Met Gln Met Ile Val Lys 260 265 270 Ile Asn Ile Pro Thr Ile Val Thr Gln Ser Asn Ala Leu Val Ile Asp 275 280 285 Phe Tyr Ser Ile Ser Ser Phe Ile Asn Asn Gln Glu Ser Ile Ile Gln 290 295 300 Leu Pro Asp Arg Ile Leu Glu Ile Gly Asn Glu Gln Trp Ser Tyr Pro 305 310 315 320 Ala Lys Asn Cys Lys Leu Thr Arg His His Ile Phe Cys Gln Tyr Asn 325 330 335 Glu Ala Glu Arg Leu Ser Leu Glu Ser Lys Leu Cys Leu Ala Gly Asn 340 345 350 Ile Ser Ala Cys Val Phe Ser Pro Ile Ala Gly Ser Tyr Met Arg Arg 355 360 365 Phe Val Ala Leu Asp Gly Thr Ile Val Ala Asn Cys Arg Ser Leu Thr 370 375 380 Cys Leu Cys Lys Ser Pro Ser Tyr Pro Ile Tyr Gln Pro Asp His His 385 390 395 400 Ala Val Thr Thr Ile Asp Leu Thr Ala Cys Gln Thr Leu Ser Leu Asp 405 410 415 Gly Leu Asp Phe Ser Ile Val Ser Leu Ser Asn Ile Thr Tyr Ala Glu 420 425 430 Asn Leu Thr Ile Ser Leu Ser Gln Thr Ile Asn Thr Gln Pro Ile Asp 435 440 445 Ile Ser Thr Glu Leu Ser Lys Val Asn Ala Ser Leu Gln Asn Ala Val 450 455 460 Lys Tyr Ile Lys Glu Ser Asn His Gln Leu Gln Ser Ile Glu Asp Lys 465 470 475 480 Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala 485 490 495 Arg Ile Lys Lys Leu Ile Gly Glu Ala Pro Gly Ser Gly Tyr Ile Pro 500 505 510 Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp 515 520 525 Val Leu Leu Ser Thr Phe Leu Gly Ser Gly Gly Gly Gly Gly Gly Ala 530 535 540 Asp Val Ala Ala Glu Glu Leu Met Asn Ala Leu Val Asn Ser Thr Leu 545 550 555 560 Leu Glu Ala Arg Ala Thr Asn Gln Phe Leu Ala Val Ser Lys Gly Asn 565 570 575 Cys Ser Gly Pro Thr Thr Ile Arg Gly Gln Phe Ser Asn Met Ser Leu 580 585 590 Ser Leu Leu Asp Leu Tyr Leu Ser Arg Gly Tyr Asn Val Ser Ser Ile 595 600 605 Val Thr Met Thr Ser Gln Gly Met Tyr Gly Gly Thr Tyr Leu Val Gly 610 615 620 Lys Pro Asn Leu Ser Ser Lys Gly Ser Glu Leu Ser Gln Leu Ser Met 625 630 635 640 His Arg Val Phe Glu Val Gly Val Ile Arg Asn Pro Gly Leu Gly Ala 645 650 655 Pro Val Phe His Met Thr Asn Tyr Phe Glu Gln Pro Val Ser Asn Asp 660 665 670 Phe Ser Asn Cys Met Val Ala Leu Gly Glu Leu Lys Phe Ala Ala Leu 675 680 685 Cys His Arg Glu Asp Ser Ile Thr Ile Pro Tyr Gln Gly Ser Gly Lys 690 695 700 Gly Val Ser Phe Gln Leu Val Lys Leu Gly Val Trp Lys Ser Pro Thr 705 710 715 720 Asp Met Arg Ser Trp Val Pro Leu Ser Thr Asp Asp Pro Val Ile Asp 725 730 735 Arg Leu Tyr Leu Ser Ser His Arg Gly Val Ile Ala Asp Asn Gln Ala 740 745 750 Lys Trp Ala Val Pro Thr Thr Arg Thr Asp Asp Lys Leu Arg Met Glu 755 760 765 Thr Cys Phe Gln Gln Ala Cys Lys Gly Lys Asn Gln Ala Leu Cys Glu 770 775 780 Asn Pro Glu Trp Ala Pro Leu Lys Asp Asn Arg Ile Pro Ser Tyr Gly 785 790 795 800 Val Leu Ser Val Asn Leu Ser Leu Thr Val Glu Leu Lys Ile Lys Ile 805 810 815 Ala Ser Gly Phe Gly Pro Leu Ile Thr His Gly Ser Gly Met Asp Leu 820 825 830 Tyr Lys Thr Asn His Asn Asn Val Tyr Trp Leu Thr Ile Pro Pro Met 835 840 845 Lys Asn Leu Ala Leu Gly Val Ile Asn Thr Leu Glu Trp Ile Pro Arg 850 855 860 Phe Lys Val Ser Pro Asn Leu Phe Thr Val Pro Ile Lys Glu Ala Gly 865 870 875 880 Glu Asp Cys His Ala Pro Thr Tyr Leu Pro Ala Glu Val Asp Gly Asp 885 890 895 Val Lys Leu Ser Ser Asn Leu Val Ile Leu Pro Gly Gln Asp Leu Gln 900 905 910 Tyr Val Leu Ala Thr Tyr Asp Thr Ser Arg Val Glu His Ala Val Val 915 920 925 Tyr Tyr Val Tyr Ser Pro Ser Arg Ser Phe Ser Tyr Phe Tyr Pro Phe 930 935 940 Arg Leu Pro Ile Lys Gly Val Pro Ile Glu Leu Gln Val Glu Cys Phe 945 950 955 960 Thr Trp Asp Lys Lys Leu Trp Cys Arg His Phe Cys Val Leu Ala Asp 965 970 975 Ser Glu Ser Gly Gly His Ile Thr His Ser Gly Met Val Gly Met Gly 980 985 990 Val Ser Cys Thr Val Thr Arg Glu Asp Gly Thr Asn Arg Arg 995 1000 1005 <210> 30 <211> 452 <212> PRT <213> Mumps virus <400> 30 Asn Ile Pro Leu Val Asn Asp Leu Arg Phe Ile Asn Gly Ile Asn Lys 1 5 10 15 Phe Ile Ile Glu Asp Tyr Ala Thr His Asp Phe Ser Ile Gly His Pro 20 25 30 Leu Asn Met Pro Ser Phe Ile Pro Thr Ala Thr Ser Pro Asn Gly Cys 35 40 45 Thr Arg Ile Pro Ser Phe Ser Leu Gly Lys Thr His Trp Cys Tyr Thr 50 55 60 His Asn Val Ile Asn Ala Asn Cys Lys Asp His Thr Ser Ser Asn Gln 65 70 75 80 Tyr Val Ser Met Gly Ile Leu Val Gln Thr Ala Ser Gly Tyr Pro Met 85 90 95 Phe Lys Thr Leu Lys Ile Gln Tyr Leu Ser Asp Gly Leu Asn Arg Lys 100 105 110 Ser Cys Ser Ile Ala Thr Val Pro Asp Gly Cys Ala Met Tyr Cys Tyr 115 120 125 Val Ser Thr Gln Leu Glu Thr Asp Asp Tyr Ala Gly Ser Ser Pro Pro 130 135 140 Thr Gln Lys Leu Thr Leu Leu Phe Tyr Asn Asp Thr Val Thr Glu Arg 145 150 155 160 Thr Ile Ser Pro Ser Gly Leu Glu Gly Asn Trp Ala Thr Leu Val Pro 165 170 175 Gly Val Gly Ser Gly Ile Tyr Phe Glu Asn Lys Leu Ile Phe Pro Ala 180 185 190 Tyr Gly Gly Val Leu Pro Asn Ser Thr Leu Gly Val Lys Ser Ala Arg 195 200 205 Glu Phe Phe Arg Pro Val Asn Pro Tyr Asn Pro Cys Ser Gly Pro Gln 210 215 220 Gln Asp Leu Asp Gln Arg Ala Leu Arg Ser Tyr Phe Pro Ser Tyr Phe 225 230 235 240 Ser Asn Arg Arg Ile Gln Ser Ala Phe Leu Val Cys Ala Trp Asn Gln 245 250 255 Ile Leu Val Thr Asn Cys Glu Leu Val Val Pro Ser Ser Asn Gln Thr 260 265 270 Met Met Gly Ala Glu Gly Arg Val Leu Leu Ile Asn Asn Arg Leu Leu 275 280 285 Tyr Tyr Gln Arg Ser Thr Ser Trp Trp Pro Tyr Glu Leu Leu Tyr Glu 290 295 300 Ile Ser Phe Thr Phe Thr Asn Ser Gly Pro Ser Ser Val Asn Met Ser 305 310 315 320 Trp Ile Pro Ile Tyr Ser Phe Thr Arg Pro Gly Ser Gly Asn Cys Ser 325 330 335 Gly Glu Asn Val Cys Pro Thr Ala Cys Val Ser Gly Val Tyr Leu Asp 340 345 350 Pro Trp Pro Leu Thr Pro Tyr Ser His Gln Ser Gly Ile Asn Arg Asn 355 360 365 Phe Tyr Phe Thr Gly Ala Leu Leu Asn Ser Ser Thr Thr Arg Val Asn 370 375 380 Pro Thr Leu Tyr Val Ser Ala Leu Asn Asn Leu Lys Val Leu Ala Pro 385 390 395 400 Tyr Gly Thr Gln Gly Leu Phe Ala Ser Tyr Thr Thr Thr Thr Cys Phe 405 410 415 Gln Asp Thr Gly Asp Ala Ser Val Tyr Cys Val Tyr Ile Met Glu Leu 420 425 430 Ala Ser Asn Ile Val Gly Glu Phe Gln Ile Leu Pro Val Leu Thr Arg 435 440 445 Leu Thr Ile Thr 450 <210> 31 <211> 438 <212> PRT <213> Measles virus <400> 31 Phe Leu Ala Val Ser Lys Gly Asn Cys Ser Gly Pro Thr Thr Ile Arg 1 5 10 15 Gly Gln Phe Ser Asn Met Ser Leu Ser Leu Leu Asp Leu Tyr Leu Gly 20 25 30 Arg Gly Tyr Asn Val Ser Ser Ile Val Thr Met Thr Ser Gln Gly Met 35 40 45 Tyr Gly Gly Thr Tyr Leu Val Glu Lys Pro Asn Leu Ser Ser Lys Arg 50 55 60 Ser Glu Leu Ser Gln Leu Ser Met Tyr Arg Val Phe Glu Val Gly Val 65 70 75 80 Ile Arg Asn Pro Gly Leu Gly Ala Pro Val Phe His Met Thr Asn Tyr 85 90 95 Leu Glu Gln Pro Val Ser Asn Asp Leu Ser Asn Cys Met Val Ala Leu 100 105 110 Gly Glu Leu Lys Leu Ala Ala Leu Cys His Gly Glu Asp Ser Ile Thr 115 120 125 Ile Pro Tyr Gln Gly Ser Gly Lys Gly Val Ser Phe Gln Leu Val Lys 130 135 140 Leu Gly Val Trp Lys Ser Pro Ala Asp Met Gln Ser Trp Val Pro Leu 145 150 155 160 Ser Thr Asp Asp Pro Val Ile Asp Arg Leu Tyr Leu Ser Ser His Arg 165 170 175 Gly Val Ile Ala Asp Asn Gln Ala Lys Trp Ala Val Pro Thr Thr Arg 180 185 190 Thr Asp Asp Lys Leu Arg Met Glu Thr Cys Phe Gln Gln Ala Cys Lys 195 200 205 Gly Lys Ile Gln Thr Leu Cys Glu Asn Pro Glu Trp Ala Pro Leu Lys 210 215 220 Asp Asn Arg Ile Pro Ser Tyr Gly Val Leu Ser Val Asp Leu Ser Leu 225 230 235 240 Thr Val Glu Leu Lys Ile Lys Ile Ala Ser Gly Phe Gly Pro Leu Ile 245 250 255 Thr His Gly Ser Gly Met Asp Leu Tyr Lys Ser Asn His Asn Asn Val 260 265 270 Tyr Trp Leu Thr Ile Pro Pro Met Lys Asn Leu Ala Leu Gly Val Ile 275 280 285 Asn Thr Leu Glu Trp Ile Pro Arg Phe Lys Val Ser Pro Tyr Leu Phe 290 295 300 Thr Val Pro Ile Lys Glu Ala Gly Glu Asp Cys His Ala Pro Thr Tyr 305 310 315 320 Leu Pro Ala Glu Val Asp Gly Asp Val Lys Leu Ser Ser Asn Leu Val 325 330 335 Ile Leu Pro Gly Gln Asp Leu Gln Tyr Val Leu Ala Thr Tyr Asp Thr 340 345 350 Ser Arg Val Glu His Ala Val Val Tyr Tyr Val Tyr Ser Pro Gly Arg 355 360 365 Ser Phe Ser Tyr Phe Tyr Pro Phe Arg Leu Pro Ile Lys Gly Val Pro 370 375 380 Ile Glu Leu Gln Val Glu Cys Phe Thr Trp Asp Gln Lys Leu Trp Cys 385 390 395 400 Arg His Phe Cys Val Leu Ala Asp Ser Glu Ser Gly Gly His Ile Thr 405 410 415 His Ser Gly Met Val Gly Met Gly Val Ser Cys Thr Val Thr Arg Glu 420 425 430 Asp Gly Thr Asn Arg Arg 435 <210> 32 <211> 463 <212> PRT <213> Measles virus <400> 32 Ala Asp Val Ala Ala Glu Glu Leu Met Asn Ala Leu Val Asn Ser Thr 1 5 10 15 Leu Leu Glu Ala Arg Ala Thr Asn Gln Phe Leu Ala Val Ser Lys Gly 20 25 30 Asn Cys Ser Gly Pro Thr Thr Ile Arg Gly Gln Phe Ser Asn Met Ser 35 40 45 Leu Ser Leu Leu Asp Leu Tyr Leu Ser Arg Gly Tyr Asn Val Ser Ser 50 55 60 Ile Val Thr Met Thr Ser Gln Gly Met Tyr Gly Gly Thr Tyr Leu Val 65 70 75 80 Gly Lys Pro Asn Leu Ser Ser Lys Gly Ser Glu Leu Ser Gln Leu Ser 85 90 95 Met His Arg Val Phe Glu Val Gly Val Ile Arg Asn Pro Gly Leu Gly 100 105 110 Ala Pro Val Phe His Met Thr Asn Tyr Phe Glu Gln Pro Val Ser Asn 115 120 125 Asp Phe Ser Asn Cys Met Val Ala Leu Gly Glu Leu Lys Phe Ala Ala 130 135 140 Leu Cys His Arg Glu Asp Ser Ile Thr Ile Pro Tyr Gln Gly Ser Gly 145 150 155 160 Lys Gly Val Ser Phe Gln Leu Val Lys Leu Gly Val Trp Lys Ser Pro 165 170 175 Thr Asp Met Arg Ser Trp Val Pro Leu Ser Thr Asp Asp Pro Val Ile 180 185 190 Asp Arg Leu Tyr Leu Ser Ser His Arg Gly Val Ile Ala Asp Asn Gln 195 200 205 Ala Lys Trp Ala Val Pro Thr Thr Arg Thr Asp Asp Lys Leu Arg Met 210 215 220 Glu Thr Cys Phe Gln Gln Ala Cys Lys Gly Lys Asn Gln Ala Leu Cys 225 230 235 240 Glu Asn Pro Glu Trp Ala Pro Leu Lys Asp Asn Arg Ile Pro Ser Tyr 245 250 255 Gly Val Leu Ser Val Asn Leu Ser Leu Thr Val Glu Leu Lys Ile Lys 260 265 270 Ile Ala Ser Gly Phe Gly Pro Leu Ile Thr His Gly Ser Gly Met Asp 275 280 285 Leu Tyr Lys Thr Asn His Asn Asn Val Tyr Trp Leu Thr Ile Pro Pro 290 295 300 Met Lys Asn Leu Ala Leu Gly Val Ile Asn Thr Leu Glu Trp Ile Pro 305 310 315 320 Arg Phe Lys Val Ser Pro Asn Leu Phe Thr Val Pro Ile Lys Glu Ala 325 330 335 Gly Glu Asp Cys His Ala Pro Thr Tyr Leu Pro Ala Glu Val Asp Gly 340 345 350 Asp Val Lys Leu Ser Ser Asn Leu Val Ile Leu Pro Gly Gln Asp Leu 355 360 365 Gln Tyr Val Leu Ala Thr Tyr Asp Thr Ser Arg Val Glu His Ala Val 370 375 380 Val Tyr Tyr Val Tyr Ser Pro Ser Arg Ser Phe Ser Tyr Phe Tyr Pro 385 390 395 400 Phe Arg Leu Pro Ile Lys Gly Val Pro Ile Glu Leu Gln Val Glu Cys 405 410 415 Phe Thr Trp Asp Lys Lys Leu Trp Cys Arg His Phe Cys Val Leu Ala 420 425 430 Asp Ser Glu Ser Gly Gly His Ile Thr His Ser Gly Met Val Gly Met 435 440 445 Gly Val Ser Cys Thr Val Thr Arg Glu Asp Gly Thr Asn Arg Arg 450 455 460 <210> 33 <211> 30 <212> PRT <213> Artificial sequence <220> <223> GCN4 trimerization domain <400> 33 Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu 1 5 10 15 Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Ala Pro 20 25 30 <210> 34 <211> 27 <212> PRT <213> Artificial sequence <220> <223> T4 Fibritin Trimerization domain <400> 34 Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys 1 5 10 15 Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 20 25 <210> 35 <211> 59 <212> PRT <213> Artificial sequence <220> <223> GCN4 / T4 Fibritin trimerization domain <400> 35 Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu 1 5 10 15 Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu Ala Pro Gly Ser 20 25 30 Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys 35 40 45 Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 50 55 <210> 36 <211> 550 <212> PRT <213> Measles virus <400> 36 Met Gly Leu Lys Val Asn Val Ser Ala Ile Phe Met Ala Val Leu Leu 1 5 10 15 Thr Leu Gln Thr Pro Thr Gly Gln Ile His Trp Gly Asn Leu Ser Lys 20 25 30 Ile Gly Val Val Gly Ile Gly Ser Ala Ser Tyr Lys Val Met Thr Arg 35 40 45 Ser Ser His Gln Ser Leu Val Ile Lys Leu Met Pro Asn Ile Thr Leu 50 55 60 Leu Asn Asn Cys Thr Arg Val Glu Ile Ala Glu Tyr Arg Arg Leu Leu 65 70 75 80 Arg Thr Val Leu Glu Pro Ile Arg Asp Ala Leu Asn Ala Met Thr Gln 85 90 95 Asn Ile Arg Pro Val Gln Ser Val Ala Ser Ser Arg Arg His Lys Arg 100 105 110 Phe Ala Gly Val Val Leu Ala Gly Ala Ala Leu Gly Val Ala Thr Ala 115 120 125 Ala Gln Ile Thr Ala Gly Ile Ala Leu His Gln Ser Met Leu Asn Ser 130 135 140 Gln Ala Ile Asp Asn Leu Arg Ala Ser Leu Glu Thr Thr Asn Gln Ala 145 150 155 160 Ile Glu Ala Ile Arg Gln Ala Gly Gln Glu Met Ile Leu Ala Val Gln 165 170 175 Gly Val Gln Asp Tyr Ile Asn Asn Glu Leu Ile Pro Ser Met Asn Gln 180 185 190 Leu Ser Cys Asp Leu Ile Gly Gln Lys Leu Gly Leu Lys Leu Leu Arg 195 200 205 Tyr Tyr Thr Glu Ile Leu Ser Leu Phe Gly Pro Ser Leu Arg Asp Pro 210 215 220 Ile Ser Ala Glu Ile Ser Ile Gln Ala Leu Ser Tyr Ala Leu Gly Gly 225 230 235 240 Asp Ile Asn Lys Val Leu Glu Lys Leu Gly Tyr Ser Gly Gly Asp Leu 245 250 255 Leu Gly Ile Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile Thr His Val 260 265 270 Asp Thr Glu Ser Tyr Leu Ile Val Leu Ser Ile Ala Tyr Pro Thr Leu 275 280 285 Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val Ser Tyr 290 295 300 Asn Ile Gly Ser Gln Glu Trp Tyr Thr Thr Val Pro Lys Tyr Val Ala 305 310 315 320 Thr Gln Gly Tyr Leu Ile Ser Asn Phe Asp Glu Ser Ser Cys Thr Phe 325 330 335 Met Pro Glu Gly Thr Val Cys Ser Gln Asn Ala Leu Tyr Pro Met Ser 340 345 350 Pro Leu Leu Gln Glu Cys Leu Arg Gly Ser Thr Lys Ser Cys Ala Arg 355 360 365 Thr Leu Val Ser Gly Ser Phe Gly Asn Arg Phe Ile Leu Ser Gln Gly 370 375 380 Asn Leu Ile Ala Asn Cys Ala Ser Ile Leu Cys Lys Cys Tyr Thr Thr 385 390 395 400 Gly Thr Ile Ile Asn Gln Asp Pro Asp Lys Ile Leu Thr Tyr Ile Ala 405 410 415 Ala Asp His Cys Pro Val Val Glu Val Asn Gly Val Thr Ile Gln Val 420 425 430 Gly Ser Arg Arg Tyr Pro Asp Ala Val Tyr Leu His Arg Ile Asp Leu 435 440 445 Gly Pro Pro Ile Leu Leu Glu Arg Leu Asp Val Gly Thr Asn Leu Gly 450 455 460 Asn Ala Ile Ala Lys Leu Glu Asp Ala Lys Glu Leu Leu Glu Ser Ser 465 470 475 480 Asp Gln Ile Leu Arg Ser Met Lys Gly Leu Ser Ser Thr Cys Ile Val 485 490 495 Tyr Ile Leu Ile Ala Val Cys Leu Gly Gly Leu Ile Gly Ile Pro Ala 500 505 510 Leu Ile Cys Cys Cys Arg Gly Arg Cys Asn Lys Lys Gly Glu Gln Val 515 520 525 Gly Met Ser Arg Pro Gly Leu Lys Pro Asp Leu Thr Gly Thr Ser Lys 530 535 540 Ser Tyr Val Arg Ser Leu 545 550 <210> 37 <211> 513 <212> PRT <213> Artificial sequence <220> <223> Recombinant F protein <400> 37 Met Tyr Ser Met Gln Leu Ala Ser Cys Val Thr Leu Thr Leu Val Leu 1 5 10 15 Leu Val Asn Ser Gln Ile His Trp Gly Asn Leu Ser Lys Ile Gly Val 20 25 30 Val Gly Ile Gly Ser Ala Ser Tyr Lys Val Met Thr Arg Ser Ser His 35 40 45 Gln Ser Leu Val Ile Lys Leu Met Pro Asn Ile Thr Leu Leu Asn Asn 50 55 60 Cys Thr Arg Val Glu Ile Ala Glu Tyr Arg Arg Leu Leu Arg Thr Val 65 70 75 80 Leu Glu Pro Ile Arg Asp Cys Leu Asn Ala Val Thr Gln Asn Ile Arg 85 90 95 Pro Val Gln Ser Val Ala Ser Ser Arg Arg His Gly Gly Gly Ala Gly 100 105 110 Val Val Leu Ala Gly Ala Ala Leu Gly Val Ala Thr Ala Ala Gln Ile 115 120 125 Thr Ala Gly Ile Ala Leu His Gln Ser Met Leu Asn Ser Gln Ala Ile 130 135 140 Asp Asn Leu Arg Ala Ser Leu Glu Thr Thr Asn Gln Ala Ile Glu Ala 145 150 155 160 Ile Arg Gln Ala Gly Gln Glu Met Ile Leu Ala Val Gln Gly Val Gln 165 170 175 Asp Tyr Ile Asn Asn Glu Leu Ile Pro Ser Met Asn Gln Leu Ser Cys 180 185 190 Asp Leu Ile Gly Gln Lys Leu Gly Leu Lys Leu Leu Arg Tyr Tyr Thr 195 200 205 Glu Ile Leu Ser Leu Phe Gly Pro Ser Leu Arg Asp Pro Cys Ser Ala 210 215 220 Glu Ile Ser Ile Gln Ala Leu Ser Tyr Ala Leu Gly Gly Asp Ile Asn 225 230 235 240 Lys Val Leu Glu Lys Leu Gly Tyr Ser Gly Gly Asp Leu Leu Gly Ile 245 250 255 Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile Thr His Val Asp Thr Glu 260 265 270 Ser Tyr Phe Ile Val Leu Ser Ile Ala Tyr Pro Thr Leu Ser Glu Ile 275 280 285 Lys Gly Val Ile Val His Arg Leu Glu Gly Val Ser Tyr Asn Ile Gly 290 295 300 Ser Gln Glu Trp Tyr Thr Thr Val Pro Lys Tyr Val Ala Thr Gln Gly 305 310 315 320 Tyr Leu Ile Ser Asn Phe Asp Glu Ser Ser Cys Thr Phe Met Pro Glu 325 330 335 Gly Th...

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 trimer protomer, wherein the amino acid substitution includes a cysteine ​​substitution that forms a non-natural disulfide bond for stabilizing the MuV F ectodomain trimer in the pre-fusion conformation.

2. The immunogen according to claim 1, wherein the cysteine ​​substitution is located at one or more of the following MuV F positions: 86 and 215, 155 and 161, 165 and 231, 206 and 223, 209 and 214, and 221 and 255.

3. The immunogen according to claim 1 or 2, 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.

4. The cysteine ​​substitution described above is MuV positions 86 and 215 are replaced by N86C and A215C. MuV positions 155 and 161 are replaced by K155C and L161C. MuV positions 165 and 231 are replaced by V165C and M231C. MuV positions 206 and 223 are V206C and A223C substitutions. At MuV positions 209 and 214, P209C and P214C substitutions are performed. At MuV positions 221 and 255, I221C and M255C are substituted. The immunogen according to any one of the above claims.

5. The aforementioned recombinant MuV F ectodomain trimer protomer contains or comprises F at positions 20 to 100. 2 Proteins and F2 containing or consisting of MuV F2 positions 104-469, 104-476, or 104-483. 1 The immunogen according to any one of the claims, comprising an ectodomain.

6. The immunogen according to any one of the claims, wherein the protomer of the trimer further comprises mutations for removing the F1 / F2 furin cleavage site of the MuV F ectodomain and optionally the first residue of the fusion peptide of the F1 ectodomain.

7. The immunogen according to claim 6, wherein the mutation for removing the F1 / F2 furin cleavage site and the first residue of the fusion peptide includes deletion of MuV F positions 101-103 and consequently positions 100 and 104 fused by the peptide linker.

8. The immunogen according to claim 7, wherein the peptide linker is a glycy-glycy-glycy linker.

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

1.

10. The protomer of the aforementioned MuV F ectodomain trimer is SEQ ID NO: 20–483 of any one residue from 3–8, SEQ ID NO: One residue from 20 to 476 of any of 11-16, 26, or 51, or SEQ ID NO: Residues 20-469 of any one of the 19-24 residues It contains at least 90% identical amino acid sequences, The protomer comprises 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 the above claims.

11. The protomer of the aforementioned MuV F ectodomain trimer is SEQ ID NO: 20–483 of any one residue from 3–8, SEQ ID NO: One residue from 20 to 476 of any of 11-16, 26, or 51, or SEQ ID NO: Residues 20-469 of any one of the 19-24 residues The immunogen according to claim 10, comprising or consisting of the amino acid sequence shown as.

12. The immunogen according to any one of the claims, wherein the protomer of the recombinant MuV F ectodomain trimer is fused to the trimerizing domain at its C-terminus.

13. The immunogen according to claim 12, wherein the trimerizing domain comprises a GCN4 trimerizing domain, a T4 fibrintin trimerizing domain, or both.

14. The aforementioned GCN4 trimerizing domain It includes the amino acid sequence shown as, The T4 fibrin trimerizing domain It includes the amino acid sequence shown as, The trimerization domain, which includes both the GCN4 trimerization domain and the T4 fibrintin trimerization domain, The immunogen according to claim 13, comprising an amino acid sequence shown as.

15. The protomer of the MuV F ectodomain trimer fused to the aforementioned trimer domain is SEQ ID NO: Residues 20-513 of any one of 3-8, SEQ ID NO: one residue from 20 to 506 of 11-16, 26, or 51, or SEQ ID NO: Residues 20-499 of any one of the 19-24 residues It contains at least 90% identical amino acid sequences, The protomer comprises 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 protomer of the MuV F ectodomain trimer fused to the trimer domain is SEQ ID NO: Residues 20-513 of any one of 3-8, SEQ ID NO: one residue from 20 to 506 of 11-16, 26, or 51, or SEQ ID NO: Residues 20-499 of any one of the 19-24 residues The immunogen according to claim 15, comprising or consisting of the amino acid sequence shown as.

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

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

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

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

29.

21. 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 trimer protomer, wherein the amino acid substitution is Cysteine ​​substitutions at one or more of the following positions of MeV F forming unnatural disulfide bonds: 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, 219 and 224, 99 and 117, 100 and 117, 101 and 117, 102 and 117, and 103 and 117. Phenylalanine substitution at position 175 of MeV F, and Proline substitution at position 194 of MeV F An immunogen containing one or more of the following.

22. The immunogen according to claim 21, 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 positions 165 and 171 of MeV F 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 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 ​​substitution at MeV F positions 99 and 117 is R99C-V117C substitution. The cysteine ​​substitution at MeV F positions 100 and 117 is P100C-V117C substitution. The cysteine ​​substitution at MeV F positions 101 and 117 is a V101C-V117C substitution. The cysteine ​​substitution at MeV F positions 102 and 117 is a Q102C-V117C substitution. The cysteine ​​substitution at MeV F positions 103 and 117 is S103C-V117C substitution. The phenylalanine substitution at position 175 of MeV is a V175F substitution. The proline substitution at position 194 of MeV F is an S194P substitution. The immunogen according to claim 21 or claim 22.

24. The aforementioned recombinant MeV F ectodomain trimer protomer contains or comprises MeV F positions 24-110. 2 Proteins and MeV F positions 114–486 or consisting of them. 1 The immunogen according to any one of claims 21 to 23, comprising an ectodomain.

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

26. The immunogen according to claim 25, wherein the mutation for removing the F1 / F2 furin cleavage site and the first residue of the fusion peptide includes deletions at MeV F positions 111-113 and positions 110 and 114 fused by the peptide linker.

27. The immunogen according to claim 26, wherein the peptide linker is a glycy-glycy-glycy linker.

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

36.

29. The protomer of the MeV F ectodomain trimer contains an amino acid sequence that is at least 90% identical to residues 21-483 of any one of SEQ ID NO:37-43, 53-55, 62-68, or 72-80, or residues 21-490 of any one of SEQ ID NO:70-72. The protomer comprises one or more amino acid substitutions that stabilize the MeV F ectodomain trimer in the pre-fusion conformation. The immunogen according to any one of claims 21 to 28.

30. The immunogen according to claim 29, wherein the protomer of the MeV F ectodomain trimer comprises or consists of an amino acid sequence represented as residues 21-483 of any one of SEQ ID NO: 37-43, 53-55, 62-68, or 72-80, or residues 21-490 of any one of SEQ ID NO: 70-72.

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

32. The immunogen according to claim 31, wherein the trimerizing domain comprises a GCN4 trimerizing domain, a T4 fibrintin trimerizing domain, or both.

33. The aforementioned GCN4 trimerizing domain It includes the amino acid sequence shown as, The T4 fibrin trimerizing domain It includes the amino acid sequence shown as, The trimerization domain, which includes both the GCN4 trimerization domain and the T4 fibrintin trimerization domain, The immunogen according to claim 32, comprising the amino acid sequence shown as.

34. The protomer of the MeV F ectodomain trimer fused to the trimerizing domain contains an amino acid sequence that is at least 90% identical to residues 21-513 of any one of SEQ ID NO:37-43, 53-55, 62-68, or 72-80, or residues 21-520 of any one of SEQ ID NO:70-72. The protomer comprises one or more amino acid substitutions that stabilize the MeV F ectodomain trimer in the pre-fusion conformation. The immunogen according to any one of claims 31 to 33.

35. The immunogen according to claim 34, wherein the protomer of the MeV F ectodomain trimer fused to the trimerizing domain includes or consists of an amino acid sequence represented as one residue 21-513 of SEQ ID NO: 37-43, 53-55, 62-68, or 72-80, or one residue 21-520 of SEQ ID NO: 70-72.

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

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

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

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

81.

40. The immunogen according to any one of the claims, 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. A trimer of fusion proteins, wherein each fusion protein contains, or consists of, a trimer, a trimer, a trimer, a trimer, a trimer, and, from the N-terminus to the C-terminus, a trimer, or, A trimer of a fusion protein, wherein each fusion protein contains, from the N-terminus to the C-terminus, one or more copies of a MuV HN ectodomain head or a MeV H ectodomain head, an optional peptide linker, a trimer, an optional peptide linker, and one or more copies of a MuV HN ectodomain head or a MeV H ectodomain head, or is a trimer composed of these, A trimer of a fusion protein, wherein each fusion protein contains, from the N-terminus to the C-terminus, one or more copies of a head or stalk and head of a MuV HN ectodomain, or a head or stalk and head of a MeV H ectodomain, an optional peptide linker, a trimer, and one or more copies of a head or stalk and head of a MuV HN ectodomain, or a head or stalk and head of a MeV H ectodomain, or a trimer composed of these. Isolated immunogens containing [specific substances].

42. The head of the MuV HN ectodomain contains, or comprises, the amino acid sequence shown as residues 59-510 of SEQ ID NO:58, or a sequence that is at least 90% identical to residues 59-510 of SEQ ID NO:

58. The stalk and head of the MuV HN ectodomain contain at least 90% identical sequences 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, or 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 contains, or consists of, the amino acid sequence shown as residues 59-496 of SEQ ID NO:59, or a sequence that is at least 90% identical to residues 59-496 of SEQ ID NO:59, or The stalk and head of the MeV H ectodomain contain at least 90% identical sequences 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 according to claim 41.

43. The aforementioned GCN4 trimerizing domain It includes the amino acid sequence shown as, The T4 fibrin trimerizing domain It includes the amino acid sequence shown as, The trimerization domain, which includes both the GCN4 trimerization domain and the T4 fibrintin trimerization domain, The immunogen according to claim 41 or claim 42, comprising an amino acid sequence shown as.

44. The immunogen according to any one of claims 41 to 43, wherein the fusion protein in the trimer contains, or consists of, a sequence that is at least 90% identical to any one of the following: residues 24-510 of SEQ ID NO: 58, residues 24-496 of SEQ ID NO: 59, 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 residues 24-510 of SEQ ID NO: 58, residues 24-496 of SEQ ID NO: 59, 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.

45. MeV H ectodomain head dimer, MeV H ectodomain stalk and head dimer, MuV HN ectodomain head dimer, or MuV HN ectodomain stalk and head dimer Isolated immunogens containing [specific substances].

46. The head of the MeV H ectodomain contains, or comprises, the amino acid sequence shown as residues 22-459 of SEQ ID NO:60, or a sequence that is at least 90% identical to residues 22-459 of SEQ ID NO:

60. The stalk and head of the MeV H ectodomain contain at least 90% identical sequences 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, or 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 contains, or consists of, the amino acid sequence indicated as SEQ ID NO:30 or a sequence that is at least 90% identical to SEQ ID NO:30, or The stalk and head of the MuV HN ectodomain contain, or consist of, at least 90% identical sequences 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, or 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 according to claim 45.

47. The immunogen according to any one of the claims, conjugated to a heterogeneous carrier.

48. The immunogen according to any one of the above claims, which is soluble.

49. The immunogen according to any one of the claims, wherein the protomer of the recombinant MeV F ectodomain trimer, the protomer of the recombinant MuV F ectodomain trimer, the trimer of the fusion protein, or the dimer of the head of the MeV H ectodomain is fused to a transmembrane domain by a peptide linker, or is directly fused to the transmembrane domain.

50. The recombinant MeV F ectodomain trimer or the protomer of the recombinant MuV F ectodomain trimer is full-length F 1 The immunogen according to claim 49, comprising a protein.

51. A virus-like particle comprising the immunogen described in any one of the preceding claims.

52. Self-assembling protein nanoparticles comprising the immunogen according to any one of the above claims.

53. A nucleic acid molecule encoding the immunogen according to any one of the above claims.

54. A nucleic acid molecule according to claim 53, functionally linked to a promoter.

55. A vector comprising the nucleic acid molecule described in claim 54.

56. The vector according to claim 55, which is an RNA vector.

57. A method for producing immunogens, A step of expressing a nucleic acid molecule according to any one of claims 55 to 56 in a host cell, and Steps to purify the immunogen Methods that include...

58. An immunogen produced by the method described in claim 57.

59. An immunogenic composition comprising an immunogen, nucleic acid molecule, vector, or virus-like particle according to any one of claims 1 to 56 and claim 58, and a pharmaceutically acceptable carrier.

60. A method for inducing an immune response to MuV F, MeV F, MuV NH, and / or MeV H in a subject, comprising the step of administering to the subject an effective amount of the immunogenic composition according to claim 59 for inducing the immune response.

61. The method according to claim 60, wherein the immune response inhibits MuV and / or MeV infection in the subject.

62. The method according to claim 60 or 61, wherein the subject is an adult that has previously received immunization with a live attenuated vaccine against MeV and / or MuV, and the administration boosts the immune response against the MuV and / or MeV.

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