Vaccine compositions, methods, and uses thereof

Abstract

The present invention provides immunogenic compositions comprising a secreted fusion protein, wherein the secreted fusion protein comprises a soluble influenza or rabies virus antigen linked by in-frame fusion to a collagen C-terminal portion capable of self-trimerization to form a disulfide-linked trimeric fusion protein. The present invention also provides uses of the immunogenic compositions for generating an immune response against influenza or rabies infection and for use in vaccine compositions. The present invention also provides methods of producing recombinant peptides and proteins, methods of prevention, treatment, and / or diagnosis, and related kits.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority and benefit from International Patent Application No. PCT / CN2020 / 095296, filed on June 10, 2020, and International Patent Application No. PCT / CN2021 / 087074, filed on April 13, 2021, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0003] Sequence list submitted as an ASCII text file

[0004] The following content, submitted in ASCII text file format, is incorporated herein by reference in its entirety: Computer-readable form of sequence listings (CRF) (filename: 165762000342SEQLIST.TXT, record date: June 9, 2021, size: 99.5KB). Technical Field

[0005] This disclosure relates in some aspects to immunogenic compositions comprising recombinant peptides and proteins, said recombinant peptides and proteins comprising viral antigens and immunogens, such as influenza HA protein peptides for treating and / or preventing influenza infection, and rabies virus glycoprotein (G) peptides for treating and / or preventing rabies virus infection. Background Technology

[0006] RNA viruses, such as influenza and rabies viruses, are major contributors to morbidity and mortality worldwide. Various immunization strategies against viral pathogens such as influenza and rabies viruses include egg-based inactivated vaccines and inactivated recombinant subunit vaccines. Improvements in strategies are needed to enhance vaccination efficacy, for example, by increasing the speed of vaccine preparation. This document provides compositions, methods, uses, and articles that meet these and other needs. Summary of the Invention

[0007] In some embodiments, this document discloses a method for preventing rabies virus infection in mammals, the method comprising immunizing mammals with an effective amount of a recombinant subunit vaccine comprising a soluble rabies virus surface antigen, the soluble rabies virus surface antigen being linked to a C-terminal portion of collagen via in-frame fusion to form a disulfide-linked trimer fusion protein. In some embodiments, the rabies virus is the CTN-1 strain. In some embodiments, the rabies virus is the PM strain. In any of the above embodiments, the rabies virus surface antigen may comprise a G protein or a fragment thereof or an epitope. In any of the above embodiments, the rabies virus surface antigen may comprise a peptide or a fragment thereof or an epitope binding to the nerve growth factor receptor NGFR (p75), the neural cell adhesion molecule NCAM, and / or the nicotinic acetylcholine receptor nAchR. In any of the above embodiments, the fusion protein may comprise the sequence shown in SEQ ID NO:3. In any of the above embodiments, the fusion protein may comprise the sequence shown in SEQ ID NO:4. In any of the above embodiments, the fusion protein may comprise the sequence shown in SEQ ID NO:5. In any of the above embodiments, the fusion protein may comprise the sequence shown in SEQ ID NO:6. In any of the above embodiments, the fusion protein may comprise a first sequence shown in any of SEQ ID NO:10-15 linked to a second sequence shown in any of SEQ ID NO:16-31, wherein the C-terminus of the first sequence is directly or indirectly linked to the N-terminus of the second sequence.

[0008] In any of the above embodiments, the recombinant subunit vaccine can be administered by intramuscular injection. In any of the above embodiments, the recombinant subunit vaccine can be administered by intranasal spray. In any of the above embodiments, the recombinant subunit vaccine can be administered as a single dose or as a series of doses spaced at intervals of weeks or months. In any of the above embodiments, the recombinant subunit vaccine can be administered without adjuvant, with an adjuvant, or with more than one adjuvant.

[0009] In some embodiments, this document discloses a method for detecting antibodies against rabies virus from mammalian serum, the method comprising contacting the serum with a soluble rabies virus surface antigen, the soluble rabies virus surface antigen being linked to a C-terminal portion of collagen via in-frame fusion to form a disulfide-linked trimer fusion protein. In some embodiments, the soluble rabies virus surface antigen is a G protein or peptide.

[0010] In some embodiments, this document discloses a method for using a recombinant subunit vaccine comprising a soluble surface antigen from rabies virus, the soluble rabies virus surface antigen being linked to a C-terminal portion of collagen via in-frame fusion to form a disulfide-linked trimer fusion protein. The method includes: immunizing a mammal, purifying the resulting neutralizing antibody, and treating a patient infected with the rabies virus via passive immunization using the neutralizing antibody. In some embodiments, the neutralizing antibody comprises a polyclonal antibody. In some embodiments, the neutralizing antibody is a monoclonal antibody.

[0011] In one aspect, this article provides a protein comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising an influenza virus hemagglutinin (HA) protein peptide or a fragment or epitope linked to a C-terminal propeptide of collagen, wherein the C-terminal propeptide of said recombinant polypeptide forms an interpeptide disulfide bond.

[0012] In some embodiments, this document discloses a recombinant subunit vaccine comprising an extracellular domain (e.g., lacking transmembrane and cytoplasmic domains) of an influenza HA protein or a fragment thereof, said extracellular domain being fused within a collagen C propeptide box capable of forming disulfide bonds to form a homotrimeric form. The resulting recombinant subunit vaccine, such as the HA trimer, can be expressed and purified from transfected cells and is expected to exhibit a native-like conformation in trimer form. This addresses the misfolding problem frequently encountered when viral antigens are expressed as recombinant peptides or proteins in soluble forms lacking transmembrane and / or cytoplasmic domains. Such misfolded viral antigens do not accurately retain the native viral antigen conformation and often fail to elicit neutralizing antibodies.

[0013] In some of any embodiments, the influenza virus is an influenza A virus or an influenza B virus, optionally wherein the influenza A virus belongs to the H1, H3, or H5 subtype, such as H1N1 or H3N2. In some of any embodiments, the epitope is a linear epitope or a conformational epitope.

[0014] In some embodiments, the HA protein peptide comprises an HA1 subunit peptide, an HA2 subunit peptide, or any combination thereof, wherein the protein comprises three recombinant polypeptides. In some embodiments, the HA protein peptide comprises a signal peptide, a stalk peptide, a vestigial esterase (VE) peptide, a receptor-binding domain (RBD) peptide, a fusion peptide (FP), a helical A peptide, a cyclic B peptide, a helical C peptide, a helical D peptide, a proximal membrane region (MPR) peptide, or any combination thereof. In some embodiments, the HA protein peptide comprises an HA1 subunit or an HA2 subunit of an HA protein. In some embodiments, the HA protein peptide comprises both an HA1 subunit and an HA2 subunit of an HA protein, optionally wherein the HA1 subunit and the HA2 subunit are linked by a disulfide bond or an artificially introduced linker. In some embodiments, the HA protein peptide does not contain a transmembrane (TM) domain peptide and / or a cytoplasmic (CP) domain peptide.

[0015] In some of any embodiments, the HA protein peptide includes a protease cleavage site, wherein the protease is optionally furin, a transmembrane serine protease such as TMPRSS2, trypsin, factor Xa, or cathepsin L. In some of any embodiments, the HA protein peptide does not include a protease cleavage site, wherein the protease is optionally furin, a transmembrane serine protease such as TMPRSS2, trypsin, factor Xa, or cathepsin L.

[0016] In some of any embodiments, the HA protein peptide is soluble or does not bind directly to a lipid bilayer (e.g., a membrane or viral envelope). In some of any embodiments, the HA protein peptide may be the same or different among the recombinant polypeptides of the protein. In some of any embodiments, the HA protein peptide is fused directly to a C-terminal propeptide or linked to a C-terminal propeptide via a linker such as a linker comprising a glycine-XY repeat sequence, wherein X and Y are independently any amino acid, optionally proline or hydroxyproline.

[0017] In some of any embodiments, the provided protein is soluble. In some of any embodiments, the protein does not bind directly to a lipid bilayer (e.g., a membrane or viral envelope). In some of any embodiments, the protein is capable of binding to cell surface attachment factors or receptors of a subject, optionally wherein the subject is a mammal, such as a primate, for example, a human.

[0018] In some of any embodiments, the C-terminal propeptide belongs to human collagen. In some of any embodiments, the C-terminal propeptide comprises a C-terminal polypeptide or fragment thereof of proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI), or proα3(XI). In some of any embodiments, the C-terminal propeptide may be the same or different among the recombinant polypeptides.

[0019] In some embodiments, the C-terminal propeptide comprises SEQ ID NO:16 or an amino acid sequence having at least 90% identity with it, capable of forming interpeptide disulfide bonds and trimerizing the recombinant polypeptide. In some embodiments, the C-terminal propeptide comprises SEQ ID NO:22 or an amino acid sequence having at least 90% identity with it, capable of forming interpeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0020] In any of the above embodiments, the C-terminal propeptide may comprise an amino acid sequence containing a glycine-XY repeating sequence linked to the N-terminus of any of SEQ ID NO:16-31, wherein X and Y are independently any amino acid, optionally proline or hydroxyproline, or an amino acid sequence having at least 90% identity with it and capable of forming interpeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0021] This document provides an immunogen, such as an immunogen comprising any of the provided proteins. This document also provides a protein nanoparticle, such as a protein nanoparticle comprising any of the provided proteins directly or indirectly linked to the nanoparticle. This document further provides a virus-like particle (VLP), such as a VLP comprising any of the provided proteins.

[0022] In some embodiments, the isolated nucleic acid is operatively linked to a promoter. In some embodiments, the isolated nucleic acid is operatively linked to a promoter. In some embodiments, the isolated nucleic acid is a DNA molecule.

[0023] In some embodiments, the isolated nucleic acid is an RNA molecule. Optionally, the mRNA molecule is, for example, nucleoside-modified mRNA, non-amplified mRNA, self-amplified mRNA, or trans-amplified mRNA.

[0024] This document provides a vector, such as a vector comprising any of the provided nucleic acids. In some embodiments, the vector is a viral vector.

[0025] This document also provides viruses, pseudoviruses, or cells comprising any of the vectors provided herein. Optionally, said virus or cell has a recombinant genome.

[0026] This article provides an immunogenic composition comprising any of the provided protein, immunogen, protein nanoparticles, VLP, isolated nucleic acid, vector, virus, pseudovirus or cell, and pharmaceutically acceptable carrier.

[0027] This document provides a vaccine comprising any of the provided immunogenic compositions. Optionally, in the presence of an adjuvant, the vaccine is optionally a subunit vaccine. In some embodiments, the vaccine is a prophylactic and / or therapeutic vaccine.

[0028] This document also provides a method for producing a protein, the method comprising expressing any one of a provided isolated nucleic acid or vector in a host cell to produce any one of the provided proteins; and purifying the protein. Proteins produced by this method are provided herein.

[0029] This article provides a method for generating an immune response in a subject to HA protein and / or G protein peptide or fragments or epitopes of influenza virus and / or rabies virus, the method comprising administering to the subject an effective amount of any of the provided protein, immunogen, protein nanoparticles, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine to generate the immune response.

[0030] In some embodiments, the method is used to treat or prevent influenza virus and / or rabies virus infection. In some embodiments, an immune response is generated that inhibits or reduces the replication of influenza virus and / or rabies virus in the subject. In some embodiments, the immune response includes a cell-mediated response and / or a humoral response, optionally including the generation of one or more neutralizing antibodies, such as polyclonal or monoclonal antibodies. In some embodiments, the immune response targets the HA protein peptide or fragments or epitopes of influenza virus and / or rabies virus, but not the C-terminal propeptide.

[0031] In some embodiments, the administration does not lead to antibody-dependent enhancement (ADE) due to prior exposure of the subject to one or more influenza viruses and / or rabies viruses. In some embodiments, the administration does not lead to antibody-dependent enhancement (ADE) when the subject is subsequently exposed to one or more influenza viruses and / or rabies viruses.

[0032] In some implementations, the method further includes an initial exemption step and / or a strengthening step.

[0033] In some of any embodiments, the administration step is performed via local, transdermal, subcutaneous, intradermal, oral, intranasal (e.g., nasal spray), intratracheal, sublingual, buccal, rectal, vaginal, inhalation, intravenous (e.g., intravenous injection), intra-arterial, intramuscular (e.g., intramuscular injection), intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intra-articular, periarticular, local, or transepidermal administration. In some of any embodiments, the effective amount is administered as a single dose or as a series of doses spaced at one or more intervals. In some of any embodiments, the effective amount is administered without adjuvant. In some of any embodiments, the effective amount is administered with an adjuvant.

[0034] This document provides a method comprising administering to a subject an effective amount of any of the provided proteins to generate neutralizing antibodies or neutralizing antiserum against influenza virus and / or rabies virus. In some embodiments, the subject is a mammal. Optionally, a human or a non-human primate.

[0035] In some embodiments, the method further includes isolating the neutralizing antibody or neutralizing antiserum from the subject. In some embodiments, the method further includes administering an effective amount of the isolated neutralizing antibody or neutralizing antiserum to a human subject via passive immunization to prevent or treat influenza virus and / or rabies virus infection. In some embodiments, the neutralizing antiserum comprises a polyclonal antibody against HA protein and / or G protein peptide or fragments or epitopes thereof, optionally wherein the neutralizing antibody contains no or substantially no antibody against collagen C-terminal propeptide. In some embodiments, the neutralizing antibody comprises a monoclonal antibody against HA protein peptide or fragments or epitopes thereof, optionally wherein the neutralizing antibody contains no or substantially no antibody against collagen C-terminal propeptide.

[0036] In some of any embodiments, the provided protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine is used to induce an immune response against influenza and / or rabies virus in a subject, and / or to treat or prevent influenza virus and / or rabies virus infection.

[0037] This document provides for the use of any of the provided proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines for inducing an immune response against influenza and / or rabies virus in subjects and / or for the treatment or prevention of influenza and / or rabies virus infection.

[0038] This document provides the use of any of the provided proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines for the manufacture of pharmaceuticals or prophylactic agents for inducing an immune response against influenza and / or rabies virus in a subject and / or for the treatment or prevention of influenza and / or rabies virus infection.

[0039] This article also provides a method for analyzing a sample, the method comprising: contacting the sample with any of the provided proteins, and detecting the binding between the protein and an analyte capable of specifically binding to an HA protein or G protein peptide or fragment or epitope thereof for influenza and / or rabies virus.

[0040] In some of any embodiments, the analyte is an antibody, receptor, or cell that recognizes the HA protein peptide or a fragment or epitope thereof. In some of any embodiments, the binding indicates the presence of the analyte in the sample and / or the presence of influenza and / or rabies virus infection in the subject from whom the sample was derived.

[0041] This document also provides a kit comprising any of the provided proteins and a substrate, liner, or vial for containing or immobilizing the protein, optionally wherein the kit is an ELISA or flow assay kit. Attached Figure Description

[0042] Figure 1 The expression levels of an exemplary fusion protein containing HA are shown. Figure 1 A schematic diagram depicts the HA protein (top) and an exemplary fusion protein construct containing HA (bottom). SP, signal peptide; TM, transmembrane domain; CT, cytoplasmic domain. Figure 1 B depicts the cell density and cell viability from day 3 to day 9 during the fed-batch process. Figure 1 C. 10% SDS-PAGE analysis of exemplary fusion protein expression containing HA in fed-batch serum-free cell cultures in shake flasks. Exemplary fusion protein expression in 10 μL of cell-free conditioned medium from day 3 to day 9 was analyzed under non-reducing conditions, followed by Coomassie blue staining. Arrows indicate HA trimers.

[0043] Figure 2 The purification and structural characterization of an exemplary fusion protein containing HA are shown. Figure 2A depicts the SDS-PAGE and Western blot analyses of the purified exemplary fusion protein under both reducing and non-reducing conditions. 2 μg of purified protein was analyzed by 10% SDS-PAGE and stained with Coomassie blue. 0.2 μg of purified protein was analyzed by Western blot using CR6261, an anti-tag monoclonal antibody, and a polyclonal anti-exemplary fusion protein antibody, respectively. Analysis was performed by SEC-HPLC and OD... 280 The purity assessment of the exemplary fusion protein is shown in the figure. Figure 2 In section B, the prime peak area of ​​the exemplary fusion protein is 95%. Figure 2 C depicts the representative structure of the exemplary fusion protein containing HA under a negative staining electron microscope (EM). Hemagglutination activity analysis of the exemplary fusion protein containing HA and control live H1N1 virus is shown in [the figure]. Figure 2 In step D, serially diluted purified exemplary fusion protein (starting concentration 1 mg / mL) and virus were mixed with washed chicken RBCs, and hemagglutination activity was read after 30 minutes at room temperature. Figure 2 E depicts the kinetic parameters of binding of an exemplary fusion protein to bNAb CR6261, evaluated by biolayer interferometry. The CR6261 antibody was first captured on a protein A (Pro A) sensor, and the binding was measured and plotted in real time by applying the sensor at gradient concentrations (2.5 μg / mL–20 μg / mL) of the exemplary fusion protein. Deglycosylation of the exemplary fusion protein containing HA and PNG enzyme F is shown in [the figure]. Figure 2 In F, the lanes represent molecular weight markers, exemplary fusion proteins, and exemplary fusion proteins treated with PNG enzyme F, respectively.

[0044] Figure 3 An in vivo immune response to an exemplary fusion protein containing HA was described. Figure 3 A schematic diagram of the vaccination regimen is shown. BALB / c mice (n = 6 mice / group) were vaccinated twice on days 0 and 21 with an SAS-adjuvanted exemplary HA-containing fusion protein, a quadrivalent inactivated vaccine (QIV), or phosphate-buffered saline (PBS). Three weeks after the last vaccination, the mice were challenged with autologous influenza virus. Antibody titers after vaccination are shown in [Figure / Diagram / Illustration]. Figure 3 In B, mice were vaccinated twice, on days 0 and 21, with either 1.5 μg of the exemplary fusion protein or 1.5 μg of QIV, and blood samples were collected on days 14 and 35. HA-specific IgG titers were determined using an ELISA assay. Serum (immunized with PBS) served as a negative control. Serum was collected 14 days after the last vaccination. Figure 3As shown in Figure C, the HI titer against autologous H1N1 virus in the antiserum of mice vaccinated with the exemplary fusion protein or QIV was determined, with immature serum (immunized with PBS) serving as a negative control. Figure 3 D depicts the micro-neutralizing (MN) titers in the antiserum of mice vaccinated with the exemplary fusion protein or QIV 14 days after the last H1N1 vaccination, with naive serum (immunized with PBS) serving as a negative control. Competition of the antiserum for bnAbCR6261 is shown in... Figure 3 In E, the binding of antiserum from mice immunized with the exemplary fusion protein or QIV to recombinant HA was tested at 100 ng / mL CR6261 14 days after the last immunization. Immature serum (immunized with PBS) served as a negative control. The dashed line indicates the detection limit. Statistical analysis was performed using a two-tailed Student's t-test; **p < 0.01, ***p < 0.001.

[0045] Figure 4 This study describes the provision of immune protection against irritation by a deadly influenza virus in mice. BALB / c mice (n = 6 mice / group) were vaccinated twice, on days 0 and 21, with either a SAS-adjuvanted exemplary HA-containing fusion protein or QIV. Mice with PBS (pre-vaccinated saline) and virus-free healthy mice served as negative and healthy controls, respectively. Three weeks after the last vaccination, mice were irritated with autologous H1N1 virus, and weight loss was monitored. Figure 4 A) Changes in body temperature Figure 4 B) Survival rate Figure 4 C) and signs of infection in lung morphology ( Figure 4 D).

[0046] Figure 5 An analysis of passive immunization of mice with serum IgG was described. BALB / c mice (n = 6 mice / group) were passively immunized (intraperitoneally) with serum IgG, purified from antiserum collected on day 42 following immunization with an exemplary fusion protein containing HA or QIV, 24 hours before stimulation with autologous H1N1 influenza virus. Weight loss was monitored. Figure 5 A) and survival rate Figure 5 B), and identified signs of infection in the lung morphology by H&E staining. Figure 5 C).

[0047] Figure 6The figure above shows the relative positions and amino acid numbers of antigenic sites (i, ii, ii, iv, and a) within the extracellular domain of rabies G. The numbering relates to the mature glycoprotein (after removal of the 19-mer signal peptide). The location of disulfide bridges is indicated by comparison with G of vesicular stomatitis virus (vSv) (solid line) or by prediction (dashed line). Figure 6 The figure below illustrates an exemplary G trimer fusion protein construct. The 458aa of rabies G includes a 19-meric signal peptide, which is fused with a 311aa trimer tag sequence.

[0048] Figure 7 The expression of G-trimers from the CTN-1 strain and the PM strain in mammalian cells is shown. Fusion protein expression was analyzed under non-reducing conditions (-ME, -β-mercaptoethanol) and reducing conditions (+ME, +β-mercaptoethanol). G-trimer formation was shown under non-reducing conditions, while under reducing conditions, the trimer dissociated into monomers of the expected molecular weight.

[0049] Figure 8 The kinetic parameters of binding of the exemplary CTN-1 strain G trimer fusion protein to NGFR-Fc, evaluated by biolayer interferometry, were depicted. NGFR-Fc was first captured on a protein A (Pro A) sensor, and the binding curves were measured and plotted in real time by applying the sensor to a gradient concentration of CTN-1 strain G trimer.

[0050] Figure 9 The effects of one, two, and three doses of CTN-1 strain G trimer alone, CTN-1 strain G trimer with adjuvant 1, CTN-1 strain G trimer with adjuvant 2, and combinations of CTN-1 strain G trimer with adjuvants 1 and 2 on neurotrophic protein receptor (p75) in immunized mice were demonstrated. NTR Detection of competitive titers. Figure 9 The figure above shows the results of increasing the antigen dose (1 μg, 3 μg, and 10 μg) on ​​day 14 after three doses on days 0, 3, and 7. Figure 9 The following figures show the results in animals that received one, two, and three doses of the vaccine. A commercially available rabies vaccine, HDCV, containing inactivated virus, was used as a control group. Individual animals are represented by dots in each figure. IC50 is shown. 50 The geometric mean titer (GMT) of the value.

[0051] Figure 10 The left figure shows that specific IgG against the CTN-1 strain G protein was detected in immunized mice after one dose of CTN-1G trimer in combination with adjuvants 1 and 2, one dose of CTN-1G trimer in combination with adjuvant 3, and one or two doses of HDCV. Figure 10The right figure shows the p75 levels in immunized mice following one dose of CTN-1 strain G trimer with adjuvants 1 and 2, one dose of CTN-1 strain G trimer with adjuvant 3, and one or two doses of HDCV. NTR Detection of competing titers. Detailed Implementation

[0052] In some embodiments, compositions and methods of using recombinant soluble surface antigens from RNA viruses in the form of covalently linked trimers are disclosed. In some embodiments, the resulting fusion protein is secreted as a disulfide-linked homotrimer, which is structurally more stable while retaining the conformation of the natural-like trimer viral antigen, and therefore can be used as a more effective vaccine against these dangerous pathogens.

[0053] In some embodiments, this document discloses a method of using a viral antigen trimer as a vaccine or as part of a multivalent vaccine, with or without an adjuvant or with more than one adjuvant, optionally administered via intramuscular injection or intranasal administration to prevent viral infection.

[0054] In some embodiments, this document discloses a method for using viral antigen trimers as diagnostic antigens for viral infection by detecting antibodies (e.g., IgM or IgG) that recognize viral antigens, such as neutralizing antibodies.

[0055] In some embodiments, this document discloses methods for using viral antigen trimers as antigens to generate polyclonal or monoclonal antibodies that can be used for passive immunization, such as neutralizing mAbs for treating influenza and / or rabies virus infections.

[0056] In some embodiments, this document discloses viral antigen trimers as part of a vaccine or a multivalent vaccine, wherein the vaccine comprises multiple trimeric subunit vaccines, the multiple trimeric subunit vaccines comprising a viral antigen of the same viral protein or a viral antigen comprising two or more different proteins of one or more viruses or one or more strains of the same virus.

[0057] In some embodiments, a monovalent vaccine is disclosed herein, the monovalent vaccine comprising the viral antigen trimer disclosed herein. In some embodiments, a bivalent vaccine is disclosed herein, the bivalent vaccine comprising the viral antigen trimer disclosed herein. In some embodiments, a trivalent vaccine is disclosed herein, the trivalent vaccine comprising the viral antigen trimer disclosed herein. In some embodiments, a quadrivalent vaccine is disclosed herein, the quadrivalent vaccine comprising the viral antigen trimer disclosed herein.

[0058] In some embodiments, a monovalent vaccine is disclosed herein, comprising the influenza HA trimer disclosed herein. In some embodiments, a bivalent vaccine is disclosed herein, comprising the influenza HA trimer disclosed herein. In some embodiments, a bivalent vaccine is disclosed herein, comprising at least one influenza HA trimer comprising a first HA protein antigen and at least one influenza HA trimer comprising a second HA protein antigen. In some embodiments, the first and second HA protein antigens are derived from the same HA protein of one or more viral species or strains / subtypes, or from two or more different HA proteins of one or more viral species or one or more strains / subtypes of the same viral species. In some embodiments, a trivalent vaccine is disclosed herein, comprising the influenza HA trimer disclosed herein. In some embodiments, a trivalent vaccine is disclosed herein, comprising at least one influenza HA trimer comprising a first HA protein antigen, at least one influenza HA trimer comprising a second HA protein antigen, and at least one influenza HA trimer comprising a third HA protein antigen. In some embodiments, the first, second, and third HA protein antigens originate from the same HA protein of one or more viral species or strains / subtypes, or from two, three, or more different HA proteins of one or more viral species or one or more strains / subtypes of the same viral species. In some embodiments, a quadrivalent vaccine is disclosed herein comprising the HA trimer disclosed herein. In some embodiments, a quadrivalent vaccine is disclosed herein comprising at least one influenza HA trimer containing a first HA protein antigen, at least one influenza HA trimer containing a second HA protein antigen, at least one influenza HA trimer containing a third HA protein antigen, and at least one influenza HA trimer containing a fourth HA protein antigen. In some embodiments, the first, second, third, and fourth HA protein antigens originate from the same HA protein of one or more viral species or strains / subtypes, or from two, three, four, or more different HA proteins of one or more viral species or one or more strains / subtypes of the same viral species.

[0059] In some embodiments, a monovalent vaccine is disclosed herein, comprising the rabies G trimer disclosed herein. In some embodiments, a bivalent vaccine is disclosed herein, comprising the rabies G trimer disclosed herein. In some embodiments, a bivalent vaccine is disclosed herein, comprising at least one rabies G trimer comprising a first G protein antigen and at least one rabies G trimer comprising a second G protein antigen. In some embodiments, the first and second G protein antigens are derived from the same G protein of one or more viral species or strains / subtypes, or from two or more different G proteins of one or more viral species or one or more strains / subtypes of the same viral species. In some embodiments, a trivalent vaccine is disclosed herein, comprising the rabies G trimer disclosed herein. In some embodiments, a trivalent vaccine is disclosed herein, comprising at least one rabies G trimer comprising a first G protein antigen, at least one rabies G trimer comprising a second G protein antigen, and at least one rabies G trimer comprising a third G protein antigen. In some embodiments, the first, second, and third G protein antigens originate from the same G protein of one or more viral species or strains / subtypes, or from two, three, or more different G proteins of one or more viral species or one or more strains / subtypes of the same viral species. In some embodiments, a quadrivalent vaccine is disclosed herein, comprising the rabies G trimer disclosed herein. In some embodiments, a quadrivalent vaccine is disclosed herein, comprising at least one rabies G trimer containing a first G protein antigen, at least one rabies G trimer containing a second G protein antigen, at least one rabies G trimer containing a third G protein antigen, and at least one rabies G trimer containing a fourth G protein antigen. In some embodiments, the first, second, third, and fourth G protein antigens originate from the same G protein of one or more viral species or strains / subtypes, or from two, three, four, or more different G proteins of one or more viral species or one or more strains / subtypes of the same viral species.

[0060] I. Viral antigens and immunogens

[0061] This document provides a protein comprising a plurality of recombinant polypeptides, each recombinant polypeptide containing a viral antigen. In some embodiments, the polypeptides are further linked to a C-terminal propeptide of collagen. In some embodiments, the C-terminal propeptide of the recombinant polypeptide forms an interpeptide disulfide bond.

[0062] Viral genomes can contain either RNA or DNA. RNA viruses can have single-molecule or segmental genomes with positive or negative polarity. Some RNA viruses have double-stranded genomes. Typically, eukaryotic host cells lack replication mechanisms for negative-stranded or double-stranded RNA genomes. Therefore, except for retroviridae viruses, RNA viruses encode and / or transport their own RNA-dependent RNA polymerases to catalyze the synthesis of new genomic RNA and mRNA to produce viral proteins and progeny. Thus, negative-sense, deproteinized RNA molecules lacking the associated RNA-dependent RNA polymerase are not infectious. Conversely, positive RNA is generally considered infectious because typical eukaryotic cellular mechanisms are sufficient for viral replication and protein production.

[0063] The viral RNA genome must be packaged inside the viral particle for the virus to spread. Some viral RNA capsids are encapsulated or sealed by the lipid membrane of the infected host cell, while others have a viral protein coat but no lipid bilayer. Viral proteins are generally classified into structural proteins and non-structural proteins. Generally, non-structural proteins are involved in genome replication, transcriptional regulation, and packaging. Structural proteins typically perform three main functions, including: (1) binding the genomic RNA (i.e., the nucleocapsid proteins of influenza A virus), (2) maintaining the relationship between the packaged RNA and other proteins (i.e., matrix proteins), and (3) constructing the outermost viral outer layer (i.e., surface proteins such as HA and NA). Assembly into viral particles ensures the efficient transmission of the viral RNA genome to another host within the same species or across species.

[0064]

[0065]

[0066] 1. Influenza hemagglutinin

[0067] Influenza viruses belong to the Orthomyxoviridae family and can be further classified into three subtypes: influenza A, B, and C (Subbarao, The Lancet 390:697-708, 2017). Seasonal epidemics can be caused by any of influenza A, B, or C, but influenza C is rarely diagnosed. Pandemic influenza specifically refers to influenza A strains because influenza A is characterized by a large number of animal hosts, thus infecting both animals (such as pigs and chickens) and humans.

[0068] The lack of large animal hosts for influenza B viruses is key to the emergence of pandemic influenza A strains. However, during interpandemic periods, the cumulative impact of annual epidemics exceeds that of pandemics. Although the morbidity and mortality rates attributable to influenza B are lower than those of influenza A H3N2 virus, they are higher than those of influenza A H1N1 virus (Thompson et al., JAMA(11):1330,2004).

[0069] The evolutionary characteristic of influenza B viruses is the co-circulation of lineages with different antigens and genes over a long period. Currently, two lineages are distinguished, represented by the prototype virus B / Victoria / 2 / 87 (Victoria lineage) and B / Yamagata / 16 / 88 (Yamagata lineage) (Ahmed et al., Applied Microbiology, 2006). B / Yamagata was the dominant lineage until the isolation of the B / Victoria lineage virus in the 1980s. Since then, variants of both influenza B lineages have co-circulated globally during recent flu seasons.

[0070] Both influenza A and B viruses contain two major surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA). HA and NA proteins are used to differentiate influenza A virus subtypes. To date, 18 HA and 11 NA subtypes of influenza A viruses (H1 to H18 and N1 to N11) have been isolated (Monto, Emerging Infectious Diseases 12:55-60, 2006).

[0071] The influenza genome is segmented, with eight gene segments encoding at least 11 proteins. The HA protein mediates virus / receptor interactions via sialic acid and facilitates viral entry into the host cell solute. The NA protein is an enzyme that functions as a budding virion release agent on the host cell surface by cleaving salivary oligosaccharide residues. Antibodies targeting the viral HA, NA, and matrix 2 (M2) proteins have been observed following natural infection and vaccination.

[0072] Seasonal epidemics of influenza A (e.g., H1N1, H1N1pdm, H3N2) and influenza B (e.g., B / Yamagata and B / Victoria) cause 3 to 5 million infections and 250,000 to 500,000 deaths worldwide. In the United States, more than 200,000 hospitalizations and 30,000 to 50,000 deaths are attributed annually to seasonal influenza infections (Zhu et al., Int J Mol Sci (18), 2017). High-risk groups such as the elderly, infants, children under 5 years of age, pregnant women, and those with chronic diseases are more susceptible to infection and severe illness (Nolan et al., JAMA 303:37-46, 2016).

[0073] Pandemics of influenza have occurred multiple times in human history. The 1918 H1N1 "Spanish flu" outbreak was the deadliest in modern history, killing approximately 50 million people worldwide (Johnson NP et al., Bull Hist Med 76:105-115, 2002). Recent pandemics include the 2009 H1N1 "swine flu" (Peiris et al., J Clin Virol 45:169-173, 2009).

[0074] Protective immunity following vaccination is primarily mediated by antibodies against HA. These antibodies mostly target receptor binding sites located on the globular head of HA and function to inhibit the interaction with host cell receptors, thereby blocking viral attachment and entry (Smith et al., PNAS(103)16936-16941, 2006).

[0075] As of 2007, all commercially available flu vaccines were produced from embryo-containing eggs. Egg-based vaccine production has limitations, including distribution time. Traditional flu vaccine manufacturing can take four to eight months or more, severely hindering pandemic preparedness. Other limitations include egg allergies in a small segment of the population, and potential problems with the capacity to saturate egg supplies or deplete them due to an outbreak of avian influenza.

[0076] Influenza A, B, and C viruses are capable of antigenic drift, where antigenic sites accumulate mutations and deviate from wild-type sequences. Due to their numerous animal hosts, influenza A is also capable of antigenic shift, where new genomic segments from different influenza A viruses are packaged into budding virions. Antigenic shift is fundamental to the potential for influenza A pandemics because the new genetic combinations result in a state of initial immunity in the population. Alterations to key antigenic sites through drift and / or shift necessitate seasonal evaluation and modification of influenza vaccines. Viruses expected to dominate the upcoming flu season are selected for inclusion in the vaccine. However, virus predictions can be inaccurate, leading to vaccine “mismatches” and significantly reduced overall efficacy. While it is possible to predict the pandemic probability of emerging influenza viruses, pandemic influenza vaccine production protocols are largely reactive because reagent preparation will depend on the exact antigenic characteristics of the virus.

[0077] Therefore, there is a need to design an influenza vaccine that can provide protection against multiple influenza strains for several years, or even throughout an individual's lifespan, as is the standard practice for some other viral pathogens, such as polio. In some respects, a universal vaccine is one that provides protection against multiple strains of the same virus, such as multiple influenza strains. Developing an effective universal influenza vaccine would reduce the cost and labor of formulating seasonal vaccines and allow for more robust pandemic preparedness.

[0078] Since the HA protein of the transmissible strain is available shortly after viral isolation, recombinant vaccines based on the extracellular HA domain have been investigated. Neutralizing antibodies against influenza virus have been reported to be induced by the variable globular head (HA1 region) and HA2 region of hemagglutinin (Wiley et al., 1981, Nature 29:373-78; Gocnik et al., 2008, J Gen Virol 89:958-67; Pica et al., 2012, PNAS 109:2573-78).

[0079] Systems for expressing HA in cell lines such as insect and mammalian cells are under development and / or in clinical trials. In 2007, the European Union approved Novartis' Optaflu vaccine, produced using mammalian cell lines (Extance et al., Nat Rev Drug Discov 10:246, 2006). In 2013, Protein Sciences' recombinant HA vaccine (Flublok), manufactured in insect cells, was also approved in the United States (Yang et al., Drugs 73:1357-1366, 2013).

[0080] Given the limitations of traditional influenza vaccines, recombinant technology is a potential alternative for influenza vaccine design. Several previous studies have demonstrated that recombinant HA vaccines purified from baculovirus expression systems are safe and effective against H1N1 and H3N2 influenza viruses (Lakey et al., J Infect Dis(174):838-841, 1996; Powers et al., J Infect Dis(175):342-351, 1997). FluBlok, the first recombinant HA subunit vaccine produced from the insect cell line Sf9, was recently approved by the U.S. Food and Drug Administration (FDA) for human use (Traynor, Am J Health Syst Pharm(70):382, 2013). However, these recombinant HA antigens are expressed in insect cells as membrane proteins along with transmembrane domains, requiring detergent dissolution followed by multiple purification steps. Although the cell culture production cycle of insect cells is relatively short, the extremely low cell viability (40-50%), relatively low antigen expression levels (up to 20 mg / L), and the need for detergent lysis of cells and dissolution of recombinant HA (Wang et al., Vaccine (24) 216-2185, 2006) pose significant challenges to vaccine production and are the reason for the lengthy delay in NDA approval of Flublok. Furthermore, the recombinant HA protein expressed in insect cells has a lower degree of glycosylation compared to the secreted His-labeled HA antigen produced by CHO cells, resulting in nearly 10-fold lower virus-neutralizing activity (Lin et al., PLoS One (8), 2013; Corper et al., Science (303) 1866-1870, 2004). These results suggest that HA antigens produced from insect cells may exhibit a different conformation compared to natural viral antigens and could explain why Flublok requires a dose three times higher than that of egg-based vaccines.

[0081] There is a need for alternative influenza vaccine manufacturing platforms. In some respects, the methods provided allow for the safe production of subunit vaccines using a simple and robust manufacturing process. In other respects, the methods provided allow for subunit vaccines in which the HA subunit resembles the natural HA trimer conformation from the virus, thus enabling robust immune responses that target protective conformational epitopes in the HA.

[0082] This document provides influenza virus antigens and immunogens. In some embodiments, the recombinant polypeptide is a viral antigen. In some embodiments, the viral antigen is an influenza virus hemagglutinin (HA) protein peptide or a fragment or epitope thereof.

[0083] There are 18 subtypes of influenza A, defined by their hemagglutinin (“HA”) proteins. These 18 HAs, H1-H18, can be divided into two groups. Group 1 consists of subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18, while Group 2 includes subtypes H3, H4, H7, H10, H14, and H15. For these reasons, there is an urgent need for a vaccine that induces broadly neutralizing antibodies against all influenza A virus subtypes and their annual variants. Furthermore, broadly neutralizing heterologous subtype antibodies could be administered as a drug for the prevention or treatment of influenza A infection.

[0084] In some embodiments, the viral antigen is an influenza A virus hemagglutinin (HA) protein peptide or a fragment or epitope thereof. In some embodiments, the influenza A virus belongs to the H1, H3, or H5 subtype, such as H1N1 or H3N2.

[0085] Like influenza A virus, influenza B virus infects cells by binding to sialic acid residues on the surface of target cells. After endocytosis, the influenza virus fuses its membrane with the endosome membrane and releases the genome transcriptase complex into the cytoplasm. Both receptor binding and membrane fusion are mediated by the HA glycoprotein. The HA of both influenza A and B viruses comprises two structurally distinct regions: a globular head region containing the receptor-binding site responsible for viral attachment to target cells and involved in the hemagglutination activity of HA; and a stem region containing the fusion peptide necessary for membrane fusion between the viral envelope and the endosome membrane. The HA protein is a trimer, where each monomer consists of two disulfide-linked glycopeptides, HA1 and HA2, generated during the proteolytic cleavage of the precursor (HA0) during infection. Cleavage is essential for viral infectivity because it is required to initiate membrane fusion of HA, allowing for conformational changes. Activation of the initiating molecule occurs in the endosome at low pH levels between pH 5 and pH 6 and requires significant structural changes in HA.

[0086] HA is synthesized as the homotrimeric precursor polypeptide HA0. Each monomer can be independently cleaved post-translational to form two polypeptides linked by a single disulfide bond, HA1 and HA2. The larger N-terminal fragment (HA1, 320-330 amino acids) forms the distal globular domain, containing the receptor-binding site and most of the determinants for recognition by viral neutralizing antibodies. The HA1 polypeptide of HA is responsible for viral attachment to the cell surface. The receptor-binding domain (HA-RBD) forms the distal head of the molecule and inserts into the HA1 subunit. During viral entry, the HA-RBD binds to sialic acid-containing receptors on the host cell surface, subsequently internalizing the virus via endocytosis. The smaller C-terminal portion (HA2, approximately 180 amino acids) forms a stem structure that anchors the globular domain to the cell or viral membrane. The HA2 polypeptide mediates viral-cell membrane fusion in endosomes, allowing the release of the ribonucleoprotein complex into the cytoplasm.

[0087] Structurally and functionally, HA-RBD is a member of the lectin superfamily, and the specificity of the binding pocket determines the host range of influenza viruses. For example, HA proteins from human viruses typically prefer α(2,6) sialic acid, while HA proteins from avian viruses prefer α(2,3) sialic acid. Triggered by the low pH environment of the endosome, the HA protein undergoes an irreversible conformational change, during which the intact HA-RBD dissociates from the stalk of the trimer.

[0088] In some embodiments, the HA protein peptide comprises an HA1 subunit peptide, an HA2 subunit peptide, or any combination thereof, wherein the protein comprises three recombinant polypeptides. In some embodiments, the HA protein peptide comprises a signal peptide, a stalk peptide, a degenerate esterase (VE) peptide, a receptor-binding domain (RBD) peptide, a fusion peptide (FP), a helical A peptide, a cyclic B peptide, a helical C peptide, a helical D peptide, a proximal membrane region (MPR) peptide, or any combination thereof. In some embodiments, the HA protein peptide comprises an HA1 subunit or an HA2 subunit of an HA protein. In some embodiments, the HA protein peptide comprises both an HA1 subunit and an HA2 subunit of an HA protein, optionally wherein the HA1 subunit and the HA2 subunit are linked by a disulfide bond or an artificially introduced linker. In some embodiments, the HA protein peptide does not contain a transmembrane (TM) domain peptide and / or a cytoplasmic (CP) domain peptide. In some embodiments, the HA protein peptide comprises a protease cleavage site, wherein the protease is optionally furin, a transmembrane serine protease such as TMPRSS2, trypsin, factor Xa, or cathepsin L. In some embodiments, the HA protein peptide does not contain a protease cleavage site, wherein the protease is optionally furin, a transmembrane serine protease such as TMPRSS2, trypsin, factor Xa, or cathepsin L. In some embodiments, the HA protein peptide is soluble or does not bind directly to a lipid bilayer (e.g., a membrane or viral envelope). In some embodiments, the HA protein peptide is the same or different among the recombinant polypeptides of the protein.

[0089] In some embodiments, the HA protein peptide in each recombinant polypeptide is in a pre-fusion conformation or a post-fusion conformation.

[0090] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:7. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:7, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions.

[0091] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:8. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:8, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions.

[0092] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:9. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:9, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions.

[0093] In some embodiments, the viral antigen or immunogen is generated from a codon-optimized nucleic acid sequence. In other embodiments, the viral antigen or immunogen is generated from a non-codon-optimized nucleic acid sequence.

[0094] In some embodiments, the viral antigen or immunogen referred to herein may include a recombinant polypeptide or fusion polypeptide comprising said viral antigen or immunogen. The term viral antigen or immunogen may be used to refer to a protein comprising a recombinant receptor containing an influenza virus antigen or immunogen. In some cases, the influenza virus antigen or immunogen is an influenza protein peptide as provided herein.

[0095] 2. Rabies G protein

[0096] Rabies virus (RVV) is a non-segmental negative-sense RNA virus belonging to the Rhabdoviridae family. The RVV virion consists of two main structural components: a nucleocapsid or ribonucleoprotein (RNP) and an envelope surrounding the RNP core in the form of a bilayer membrane. The infectious component of all Rhabdoviruses is the RNP core, which comprises an RNA genome encased in the nucleocapsid (N) protein and two minor proteins: RNA-dependent RNA polymerase (L) and a phosphoprotein (P). The membrane surrounding the RNP core consists of two proteins: a transmembrane glycoprotein (G) and a matrix (M) protein located at an internal site on the membrane. The G protein, also known as the spike protein, is responsible for cell attachment and membrane fusion in RVV and is also a major target of the host's immune system. The region of amino acids 330 to 340 of the G protein (called antigenic site III) has been identified as responsible for viral virulence, particularly the Arg residue at position 333. All RVV strains share this virulence-determining antigenic site III. With few exceptions, rabies always causes fatal neurological disorders in humans and animals and remains a serious global public health problem.

[0097] In some embodiments, the G protein is 62-67 kDa and is a type I glycoprotein of 505 amino acids. In some embodiments, the G protein forms protrusions covering the outer surface of the virion envelope. Studies have shown that the G protein can induce virus-neutralizing antibodies. The G protein has at least five neutralizing epitopes, where epitope II is a discontinuous spatial epitope containing 34-42 amino acid residues and 198-200 amino acid residues, and epitope III is a linear epitope containing 330-338 amino acid residues. Approximately 97% of reporter antibodies recognize epitopes II and III. The rabies virus neutralizing antibody CR4098 binds to epitope III. Antibodies recognize epitopes I and IV less frequently. The rabies virus neutralizing antibody CR57 recognizes linear epitope I at positions 218-240, with its core binding domain being KLCGVL at positions 226-231. Epitope IV contains residues 251 and 264. Another epitope is microepitope a, separated from epitope III by three amino acid residues that do not overlap with epitope III; it consists of only two amino acid residues, 342-343. Numbering applies to mature glycoproteins (after removal of the 19-mer signal peptide), such as... Figure 6 As shown in the image above.

[0098] In some embodiments, the rabies G antigen or immunogen is or comprises the 1-439 amino acid sequence of SEQ ID NO: 10 or 13 (a G protein sequence without a signal peptide). In some embodiments, the rabies G antigen or immunogen is or comprises the 1-458 amino acid sequence of SEQ ID NO: 11 or 14 (a G protein sequence with a signal peptide).

[0099] In some embodiments, the rabies G antigen or immunogen is or comprises an amino acid sequence between any one of residues 34, 42, 198, 200, 226, 231, 251, 264, 330, 338, 342, 343, and 439 of SEQ ID NO: 10 or 13. In some embodiments, the rabies G antigen or immunogen comprises one or more antigenic sites (e.g., antigenic sites I, II, III, or IV) of SEQ ID NO: 10, 11, 13, or 14.

[0100] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:10. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:10, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions.

[0101] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:11. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:11, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions.

[0102] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:12. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:12, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions.

[0103] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:13. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:13, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions.

[0104] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:14. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:14, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions.

[0105] In some embodiments, the viral antigen or immunogen comprises the sequence shown in SEQ ID NO:15. In some embodiments, the viral antigen or immunogen comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:15, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions.

[0106] In some embodiments, the rabies G protein peptide may include any G protein sequence known in the art, such as those disclosed in U.S. Patent No. 10,722,571, which is incorporated herein by reference in its entirety for all purposes.

[0107] In some embodiments, the viral antigen or immunogen is generated from a codon-optimized nucleic acid sequence. In other embodiments, the viral antigen or immunogen is generated from a non-codon-optimized nucleic acid sequence.

[0108] In some embodiments, the viral antigen or immunogen mentioned herein may include a recombinant polypeptide or fusion polypeptide comprising said viral antigen or immunogen. The term viral antigen or immunogen may be used to refer to a protein comprising a recombinant receptor containing a rabies virus antigen or immunogen. In some cases, the rabies virus antigen or immunogen is a rabies protein peptide as provided herein.

[0109] In some embodiments, the viral antigen or immunogen is generated from a codon-optimized nucleic acid sequence. In other embodiments, the viral antigen or immunogen is generated from a non-codon-optimized nucleic acid sequence.

[0110] In some embodiments, the viral antigen or immunogen mentioned herein may include a recombinant polypeptide or fusion polypeptide comprising said viral antigen or immunogen. The term viral antigen or immunogen may be used to refer to a protein comprising a recombinant receptor containing a rabies virus antigen or immunogen. In some cases, the rabies virus antigen or immunogen is a rabies protein peptide as provided herein.

[0111] II. Recombinant peptides and proteins

[0112] In some embodiments, compositions and methods of using recombinant soluble surface antigens from RNA viruses in the form of covalently linked trimers are disclosed. In some embodiments, the resulting fusion protein is secreted as a disulfide-linked homotrimer, which is structurally more stable while retaining the conformation of the natural-like trimer viral antigen, and therefore can be used as a more effective vaccine against these dangerous pathogens.

[0113] The influenza virus antigens and immunogens provided herein, such as the influenza HA protein peptide (see Section I), are envisioned to be combined, e.g., linked, with other proteins or peptides to form recombinant polypeptides, including fusion peptides. In some embodiments, individual recombinant polypeptides (e.g., monomers) provided herein associate to form multimers, such as trimers, of the recombinant polypeptide. In some embodiments, association of individual recombinant polypeptide monomers occurs through covalent interactions. In some embodiments, association of individual recombinant polypeptide monomers occurs through non-covalent interactions. In some embodiments, the interactions (e.g., covalent or non-covalent) are influenced by the protein or peptide to which the influenza virus antigen or immunogen (e.g., the influenza HA protein peptide) is linked. In some embodiments, for example when the influenza virus antigen or immunogen is the influenza HA protein peptide as described herein, the protein or peptide to which it will be linked can be selected to preserve the native homotrimeric structure of the glycoprotein. This may be beneficial in evoking a strong and effective immunogenic response to the influenza HA protein peptide. For example, preserving and / or maintaining the native conformation of the influenza virus antigen or immunogen (e.g., the influenza HA protein peptide) can improve or allow access to antigenic sites capable of generating an immune response. In some cases, recombinant polypeptides containing the influenza HA protein peptides described herein (see, for example, Section I) are alternatively referred to herein as recombinant influenza HA antigen, recombinant influenza HA immunogen, or recombinant influenza HA protein.

[0114] It is also envisioned that, in some cases, the recombinant polypeptide or its polymerized recombinant polypeptide may aggregate or can aggregate to form a protein comprising multiple influenza virus antigens and / or immunogenic recombinant polypeptides. The formation of such proteins may facilitate a strong and effective immunogenic response to influenza virus antigens and / or immunogens. For example, the formation of a protein comprising multiple recombinant polypeptides, and thus multiple influenza virus antigens (e.g., influenza HA protein peptides), can preserve the tertiary and / or quaternary structures of the viral antigens, thereby allowing for enhanced immune responses against native structures. In some cases, aggregation can confer structural stability to the influenza virus antigens or immunogens, thereby allowing access to potential antigenic sites that can promote an immune response.

[0115] The rabies virus antigen and immunogen provided herein, such as the rabies G protein peptide (see Section I), are envisioned to be combined, e.g., linked, with other proteins or peptides to form recombinant polypeptides, including fusion peptides. In some embodiments, individual recombinant polypeptides (e.g., monomers) provided herein associate to form multimers, such as trimers, of the recombinant polypeptide. In some embodiments, association of individual recombinant polypeptide monomers occurs through covalent interactions. In some embodiments, association of individual recombinant polypeptide monomers occurs through non-covalent interactions. In some embodiments, the interactions (e.g., covalent or non-covalent) are influenced by the protein or peptide to which the rabies virus antigen or immunogen (e.g., the rabies G protein peptide) is linked. In some embodiments, for example when the rabies virus antigen or immunogen is the rabies G protein peptide as described herein, the protein or peptide to which it will be linked can be selected to preserve the native homotrimeric structure of the glycoprotein. This may be beneficial in evoking a strong and effective immunogenic response to the rabies G protein peptide. For example, retaining and / or maintaining the native conformation of rabies virus antigens or immunogens (e.g., rabies G protein peptides) can improve or allow access to antigenic sites capable of generating an immune response. In some cases, recombinant polypeptides comprising the rabies G protein peptides described herein (e.g., see Section I) are alternatively referred to herein as recombinant rabies G antigen, recombinant rabies G immunogen, or recombinant rabies G protein.

[0116] It is also envisioned that, in some cases, the recombinant polypeptides or polymerized recombinant polypeptides thereof may aggregate or aggregate to form proteins comprising multiple rabies virus antigens and / or immunogenic recombinant polypeptides. The formation of such proteins may facilitate a strong and effective immunogenic response to rabies virus antigens and / or immunogens. For example, the formation of proteins comprising multiple recombinant polypeptides, and thus multiple rabies virus antigens (e.g., rabies G protein peptides), may preserve the tertiary and / or quaternary structures of the viral antigens, thereby allowing for enhanced immune responses against native structures. In some cases, aggregation may confer structural stability to the rabies virus antigens or immunogens, thereby allowing access to potential antigenic sites that can promote an immune response.

[0117] 1. Fusion peptides and recombinant peptides

[0118] In some embodiments, the influenza virus antigen or immunogen may be C-terminus-linked (C-terminal linking) to a trimerizing domain to promote monomer trimerization. In some embodiments, trimerization stabilizes the proximal membrane state of the influenza virus antigen or immunogen in a trimer conformation. In some embodiments, trimerization stabilizes the proximal membrane state of the influenza virus antigen or immunogen (e.g., influenza HA protein peptide) in a trimer conformation.

[0119] Non-limiting examples of exogenous polymerizing domains that promote stable trimers of soluble recombinant proteins include: GCN4 leucine zippers (Harbury et al., 1993 Science 262:1401-1407), trimerizing motifs from pulmonary surfactant proteins (Hoppe et al., 1994 FEBS Lett 344:191-195), collagen (McAlinden et al., 2003 J Biol Chem 278:42200-42207), and phage T4 minor fibrin folds (Miroshnikov et al., 1998 Protein Eng 11:329-414), any of which can be linked to the recombinant influenza virus antigens or immunogens described herein (e.g., by linking to the C-terminus of the HA domain) to promote the trimerization of the recombinant viral antigens or immunogens. See also U.S. Patent Nos. 7,268,116, 7,666,837, 7,691,815, 10,618,949, 10,906,944 and 10,960,070, and US2020 / 0009244, which are incorporated herein by reference in their entirety for all purposes.

[0120] In some embodiments, one or more peptide linkers (such as gly-serine linkers, for example, a 10-amino acid glycine-serine peptide linker) may be used to link the recombinant viral antigen or immunogen to the transmembrane domain. The trimer may include any stabilizing mutation (or combination thereof) described herein, provided that the recombinant viral antigen or immunogen trimer retains the desired properties (e.g., pre-fusion conformation).

[0121] For therapeutic feasibility, the desired trimerized protein moiety for biopharmaceutical design should meet the following criteria. Ideally, it should be a part of a naturally secreted protein, such as immunoglobulin Fc, abundant in circulation (non-toxic), derived from humans (lacking immunogenicity), relatively stable (long half-life), and capable of efficient self-trimerization (enhanced by interchain covalent disulfide bonds), thus ensuring the stable structure of the trimerized influenza virus antigen or immunogen.

[0122] Collagen belongs to the fibrous protein family and is a major component of the extracellular matrix. It is the most abundant protein in mammals, accounting for nearly 25% of total body protein. Collagen plays a major structural role in the formation of bones, tendons, skin, cornea, cartilage, blood vessels, and teeth. The fibrous types of collagen I, II, III, IV, V, and XI are synthesized into larger trimer precursors called procollagen, which consists of a central, uninterrupted triple helix domain composed of hundreds of "GXY" repeat sequences (or glycine repeat sequences) flanked by a non-collagenous domain (NC), an N-propeptide, and a C-propeptide. The C-terminal and N-terminal extensions are proteased after procollagen secretion, an event that triggers the assembly of mature proteins into collagen fibrils, thereby forming the insoluble cellular matrix. BMP-1 is a protease that recognizes specific peptide sequences in procollagen near the junction between the glycine repeat sequence and the collagen C-propeptide and is responsible for removing the propeptide. The concentration of type I collagen desquamated trimer propeptide C in normal adult human serum ranges from 50 to 300 ng / mL, with much higher levels in children, indicating active bone formation. In individuals with familial hypertrophy of serum type I collagen C propeptide, these levels can be as high as 1–6 μg / mL without significant abnormalities, suggesting that C propeptide is non-toxic. Structural studies of collagen trimer propeptide C have revealed a trefoil structure in which all three subunits aggregate in a linker region near their N-terminus, connecting to the remainder of the procollagen molecule. The geometry of this fused protein, extending in one direction, resembles that of an Fc dimer.

[0123] Types I, IV, V, and XI collagen primarily assemble into heterotrimeric forms consisting of two α-1 chains and one α-2 chain (for types I, IV, and V) or three highly homologous chains on different sequences (for type XI). Types II and III collagen are homotrimers of the α-1 chain. Even type I collagen, the most abundant form of collagen, forms stable α(I) homotrimers and exists at variable levels in different tissues. Most of these collagen C-propeptide chains can self-assemble into homotrimers when overexpressed alone in cells. Although the N-propeptide domain is synthesized first, molecular assembly into trimeric collagen begins with the mutual registration and association of the C-propeptides. The C-propeptide complex is thought to be stabilized by the formation of interchain disulfide bonds, but the necessity of disulfide bond formation for suitable chain registration is unclear. A repeating glycine triple helix then propagates from the associated C-terminus to the N-terminus in a zipper-like manner. This understanding has led to the creation of non-natural collagen matrices by exchanging C-propeptides from different collagen chains using recombinant DNA technology. Non-collagenous proteins such as cytokines and growth factors have also been fused to the N-terminus of procollagen or mature collagen to form new collagen matrices, a move designed to allow for the slow release of non-collagenous proteins from the cellular matrix. However, in both cases, the C-propeptide needs to be cleaved before the recombinant collagen fibrils assemble into the insoluble cellular matrix.

[0124] While other protein trimerizing domains, such as those from yeast GCN4, bacteriophage T4 minor fibrin, and Escherichia coli aspartate transcarbamate, have previously been described as allowing the trimerization of heterologous proteins, these trimerizing proteins are not native human proteins, nor are they naturally secreted proteins. Therefore, any trimerizing fusion protein must be produced intracellularly, which not only can lead to misfolding of naturally secreted proteins (such as soluble receptors) but also makes it difficult to purify such fusion proteins from thousands of other intracellular proteins. Furthermore, a fatal drawback of using such non-human protein trimerizing domains (e.g., from yeast, bacteriophages, and bacteria) for trimerizing biopharmaceutical design is their putative immunogenicity in humans, rendering these fusion proteins ineffective shortly after injection.

[0125] Therefore, the use of collagen in recombinant peptides, as described herein, has many advantages, including: (1) collagen is the most abundant protein secreted in mammals, accounting for nearly 25% of total protein in the body; (2) the main form of collagen naturally exists as a trimeric helix, with its globular C-propeptide responsible for initiating trimerization; (3) collagen trimeric C-propeptide released by the hydrolysis of mature collagen has been found to be naturally present in mammalian blood at submicrogram / mL levels and is known to be non-toxic to the body; (4) the linear triple helix region of collagen can be included as a linker, with the predicted spacing between each residue being [missing information]. Alternatively, it can be excluded from the fusion protein portion, thus allowing precise adjustment of the distance between the protein to be trimerized and the collagen C propeptide for optimal bioactivity; (5) the BMP1 recognition site of the C propeptide cleaved from the procollagen can be mutated or deleted to prevent disruption of the trimer fusion protein; (6) the C propeptide domain trimers itself via disulfide bonds, providing a universal affinity tag that can be used to purify any secreted fusion protein produced. In some embodiments, the collagen C propeptide linked to influenza virus antigens and immunogens (e.g., influenza HA protein peptides) enables recombination to produce soluble, covalently linked homotrimeric fusion proteins.

[0126] In some embodiments, the influenza virus antigen or immunogen is linked to a C-terminal propeptide of collagen to form a recombinant polypeptide. In some embodiments, the C-terminal propeptide of the recombinant polypeptide forms an interpeptide disulfide bond. In some embodiments, the recombinant protein forms a trimer. In some embodiments, the influenza virus antigen or immunogen is an influenza HA protein peptide as described in Section I.

[0127] In some embodiments, the C-terminal propeptide belongs to human collagen. In some embodiments, the C-terminal propeptide comprises a C-terminal polypeptide or fragment thereof of proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI), or proα3(XI). In some embodiments, the C-terminal propeptide is or comprises a C-terminal polypeptide of proα1(I).

[0128] In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO:16. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:16. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO:17. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:17. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO:18. In some embodiments, the C-terminal propeptide presents an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:18. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO:19. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO: 19. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO: 20. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO: 20. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO: 21. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO: 21.

[0129] In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO:22. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:22. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO:23. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:23. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO:24. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:24. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO:25. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:25. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO:26. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:26. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO:27. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:27.

[0130] In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO:28. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:28. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO:29. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:29. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO:30. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:30. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence shown in SEQ ID NO:31. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:31.

[0131] In some embodiments, the C-terminal propeptide is or contains an amino acid sequence of a collagen trimerization domain (e.g., the C-terminal propeptide of human α1(I) collagen), wherein the aspartic acid (D) at the BMP-1 site is replaced with asparagine (N), for example, wherein RA D Mutation to RA N In some embodiments, the C-terminal propeptide is or contains an amino acid sequence comprising a collagen trimerization domain (e.g., the C-terminal propeptide of human α1(I) collagen), wherein alanine (A) at the BMP-1 site is substituted with asparagine (N), for example, wherein R A D mutation to R N D. In some embodiments, the C-terminal propeptide herein may contain a mutated BMP-1 site, such as RSAN replacing DDAN. In some embodiments, the C-terminal propeptide herein may contain a BMP-1 site, such as a sequence containing a RAD (e.g., RADDAN) sequence instead of a RAN (e.g., RANDAN) or RND (e.g., RNDDAN) sequence, which may be used in the fusion peptide disclosed herein.

[0132] In some embodiments, the C-terminal propeptide is or comprises an amino acid sequence as a fragment of any one of SEQ ID NO:16-31.

[0133] In some embodiments, the C-terminal propeptide may comprise a sequence containing a glycine-XY repeating sequence, wherein X and Y are independently any amino acid, or have at least 85%, 90%, 92%, 95%, or 97% identity with it, and are amino acid sequences capable of forming interpeptide disulfide bonds and trimerizing the recombinant polypeptide. In some embodiments, X and Y are independently proline or hydroxyproline.

[0134] In some cases where an influenza HA peptide (e.g., an influenza virus antigen or immunogen, see Section I) is linked to a C-terminal propeptide to form a recombinant polypeptide, the recombinant polypeptide forms a trimer, thereby producing a homotrimer of the influenza HA protein peptide. In some embodiments, the trimerized recombinant polypeptide contains a cane-shaped, rod-shaped HA protein peptide trimer. In some embodiments, the influenza HA protein peptide of the trimerized recombinant polypeptide is in a pre-fusion conformation. In some embodiments, the influenza HA protein peptide of the trimerized recombinant polypeptide is in a post-fusion conformation. In some embodiments, the conformational state allows access to different antigenic sites on the HA protein peptide. In some embodiments, the antigenic site is an epitope, such as a linear epitope or a conformational epitope. One advantage of having the trimerized recombinant polypeptide is that it can enhance the immune response against a variety of potentially different antigenic sites.

[0135] In some embodiments, the trimerized recombinant polypeptide includes individual recombinant polypeptides containing the same viral antigen or immunogen. In some embodiments, the trimerized recombinant polypeptide includes individual recombinant polypeptides, each containing a viral antigen or immunogen different from the others. In some embodiments, the trimerized recombinant polypeptide includes individual recombinant polypeptides, wherein one of the individual recombinant polypeptides contains a viral antigen or immunogen different from the others. In some embodiments, the trimerized recombinant polypeptide includes individual recombinant polypeptides, wherein two of the individual recombinant polypeptides contain the same viral antigen or immunogen, and the viral antigen or immunogen is different from the viral antigen or immunogen contained in the remaining recombinant polypeptides.

[0136] In some embodiments, the recombinant polypeptide comprises any influenza virus antigen or immunogen described in Section I. In some embodiments, the recombinant polypeptide comprises any influenza virus antigen or immunogen described in Section I, which is linked to a collagen C-terminal propeptide as described herein.

[0137] In some embodiments, the recombinant polypeptide or the fusion protein comprises a first sequence shown in any one of SEQ ID NO:7-15, which is linked to a second sequence shown in any one of SEQ ID NO:16-31, wherein the C-terminus of the first sequence is directly linked to the N-terminus of the second sequence.

[0138] In some embodiments, the recombinant polypeptide or the fusion protein comprises a first sequence shown in any one of SEQ ID NO: 7-15 linked to a second sequence shown in any one of SEQ ID NO: 16-31, wherein the C-terminus of the first sequence is indirectly linked to the N-terminus of the second sequence, for example, via a linker. In some embodiments, the linker comprises a sequence containing a glycine-XY repeat sequence.

[0139] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:1. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:1, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions.

[0140] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:2. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:2, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions.

[0141] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:3. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:3, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions.

[0142] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:4. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:4, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions.

[0143] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:5. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:5, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions.

[0144] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO:6. In some embodiments, the recombinant polypeptide is or comprises an amino acid sequence having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:6, including sequences comprising substitutions, deletions, and / or insertions at one or more amino acid positions.

[0145] As noted above, in some embodiments, the recombinant peptides provided herein not only associate to form trimers, but can also aggregate or be aggregated to produce proteins comprising multiple recombinant peptides. In some embodiments, the formed proteins have a macroscopic structure. In some cases, the macroscopic structure can confer structural stability to the influenza virus antigen or immunogenic recombinant peptides, thereby allowing access to potential antigenic sites that can promote an immune response.

[0146] In some embodiments, the trimerized recombinant peptides aggregate to form a protein containing a plurality of trimerized recombinant peptides. In some embodiments, the plurality of trimerized recombinant peptides form a protein with a macroscopic structure.

[0147] In some embodiments, this document provides a complex comprising any suitable combination of recombinant polypeptides selected from SEQ ID NO:1-2 or fragments, variants, or mutants thereof. In some embodiments, this document provides a complex comprising a trimer of a recombinant polypeptide selected from SEQ ID NO:1-2 or fragments, variants, or mutants thereof, wherein the recombinant polypeptide trimers to form the trimer via interpeptide disulfide bonds.

[0148] In some embodiments, this document provides a complex comprising any suitable combination of recombinant polypeptides selected from SEQ ID NO:3-6, or fragments, variants, or mutants thereof. In some embodiments, this document provides a complex comprising a trimer of a recombinant polypeptide selected from SEQ ID NO:3-6, or fragments, variants, or mutants thereof, wherein the recombinant polypeptide trimers via interpeptide disulfide bonds to form the trimer.

[0149] In some embodiments, the protein comprising multiple recombinant peptides described herein is an immunogen. In some embodiments, the protein comprising multiple recombinant peptides described herein is contained in nanoparticles. For example, in some embodiments, the protein is directly linked to nanoparticles, such as protein nanoparticles. In some embodiments, the protein is indirectly linked to nanoparticles. In some embodiments, the protein comprising multiple recombinant peptides described herein is contained in virus-like particles (VLPs).

[0150] 2. Polynucleotides and carriers

[0151] Also provided are polynucleotides (nucleic acid molecules) encoding the influenza antigens or immunogens and recombinant peptides provided herein, as well as vectors for genetically engineered cells to express such influenza antigens or immunogens and recombinant peptides.

[0152] In some embodiments, a polynucleotide encoding the recombinant polypeptide provided herein is provided. In some aspects, the polynucleotide contains a single nucleic acid sequence, such as a nucleic acid sequence encoding a recombinant polypeptide. In other cases, the polynucleotide contains a first nucleic acid sequence encoding a recombinant polypeptide comprising a specific influenza virus antigen or immunogen and a second nucleic acid sequence encoding a recombinant polypeptide comprising a different influenza virus antigen or immunogen.

[0153] In some embodiments, the polynucleotide encoding the recombinant polypeptide contains at least one promoter operatively linked to control the expression of the recombinant polypeptide. In some embodiments, the polynucleotide contains two, three, or more promoters operatively linked to control the expression of the recombinant polypeptide.

[0154] In some embodiments, for example, when the polynucleotide contains two or more nucleic acid coding sequences, such as sequences encoding recombinant polypeptides containing different influenza virus antigens or immunogens, at least one promoter is operatively linked to control the expression of the two or more nucleic acid sequences. In some embodiments, the polynucleotide contains two, three, or more promoters operatively linked to control the expression of the recombinant polypeptide.

[0155] In some embodiments, the expression of the recombinant polypeptide is inducible or conditional. Therefore, in some aspects, the polynucleotide encoding the recombinant polypeptide contains a conditional promoter, enhancer, or trans-activator. In some such aspects, the conditional promoter, enhancer, or trans-activator is an inducible promoter, enhancer, or trans-activator, or an inhibitory promoter, enhancer, or trans-activator. For example, in some embodiments, inducible or conditional promoters can be used to confine the expression of the recombinant polypeptide to a specific microenvironment. In some embodiments, expression driven by an inducible or conditional promoter is regulated by exposure to exogenous factors such as heat, radiation, or drugs.

[0156] In cases where a polynucleotide contains more than one nucleic acid sequence encoding a recombinant polypeptide, the polynucleotide may also include a nucleic acid sequence encoding a peptide between one or more nucleic acid sequences. In some cases, the peptide encoded by the nucleic acid located between the nucleic acid sequences separates the translation products of the nucleic acid sequences during or after translation. In some embodiments, the peptide contains an internal ribosome entry site (IRES), a self-cleaving peptide, or a peptide that causes ribosome jumping, such as the T2A peptide.

[0157] In some embodiments, a polynucleotide encoding a recombinant polypeptide is introduced into a composition containing cultured cells (e.g., host cells), such as through retroviral transduction, transfection, or transformation. In some embodiments, this may allow expression (e.g., production) of the recombinant polypeptide. In some embodiments, the expressed recombinant polypeptide is purified.

[0158] In some embodiments, the polynucleotides (nucleic acid molecules) provided herein encode influenza virus antigens or immunogens as described herein. In some embodiments, the polynucleotides (nucleic acid molecules) provided herein encode recombinant polypeptides containing influenza virus antigens or immunogens, such as the influenza F peptide protein described herein.

[0159] Vectors or constructs containing nucleic acid molecules as described herein are also provided. In some embodiments, the vector or construct contains one or more promoters operatively linked to a nucleic acid molecule encoding a recombinant polypeptide to drive its expression. In some embodiments, the promoters are operatively linked to one or more nucleic acid molecules, such as nucleic acid molecules encoding recombinant polypeptides containing different influenza virus antigens or immunogens.

[0160] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the retroviral vector is a gamma retroviral vector.

[0161] In some embodiments, the vector or construct includes a single promoter that drives the expression of one or more nucleic acid molecules of a polynucleotide. In some embodiments, such a promoter may be polycistronic (bicistronic or tricistronic, see, for example, U.S. Patent No. 6,060,273). For example, in some embodiments, the transcription unit may be engineered to be a bicistronic unit containing an IRES (internal ribosome entry site), thereby allowing co-expression of gene products (e.g., encoding different recombinant polypeptides) via signals from a single promoter. In some embodiments, the vectors provided herein are bicistronic, thereby allowing the vector to contain and express two nucleic acid sequences. In some embodiments, the vectors provided herein are tricistronic, thereby allowing the vector to contain and express three nucleic acid sequences.

[0162] In some embodiments, a single promoter directs RNA expression containing two or three genes (e.g., encoding a chimeric signaling receptor and a recombinant receptor) within a single open reading frame (ORF), separated from each other by sequences encoding self-cleaving peptides (e.g., 2A sequences) or protease recognition sites (e.g., furin proteases). The ORF thus encodes a single polypeptide, which is processed into an individual protein during or after translation (in the case of 2A). In some cases, the peptide, such as T2A, may cause ribosome skipping (ribosome jumping) of the C-terminal peptide bond of a 2A element, resulting in the 2A sequence terminus separating from the next downstream peptide (see, for example, de Felipe. Genetic Vaccines and Ther. 2:13 (2004); and de Felipe et al., Traffic 5:616-626 (2004)). Many 2A elements are known in the art. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein include, but are not limited to, the 2A sequences from foot-and-mouth disease virus (F2A), equine rhinitis virus (E2A), those from the swine tussock moth virus (Thosea asigna) (T2A), and those from porcine swine tussock virus-1 (P2A) as described in U.S. Patent Publication No. 20070116690.

[0163] In some embodiments, the vector is contained within a virus. In some embodiments, the virus is a pseudovirus. In some embodiments, the virus is a virus-like particle. In some embodiments, the vector is contained within a cell. In some embodiments, the virus or cell containing the vector contains a recombinant genome.

[0164] In some embodiments, the polynucleotide is operatively linked to a promoter. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA, such as an mRNA molecule, such as nucleoside-modified mRNA, non-amplified mRNA, self-amplified mRNA, or trans-amplified mRNA.

[0165] III. Immunogenic compositions and formulations

[0166] In some embodiments, this document provides an immunogenic composition comprising a recombinant polypeptide trimer, the recombinant polypeptide trimer comprising sequences selected from SEQ ID NO:1-2 or fragments thereof, variants or mutants, or combinations of any two or more of the trimers. In some embodiments, a unit dose of the immunogenic composition may comprise about 10 μg to about 100 μg of the HA antigen, preferably about 25 μg to about 75 μg of the HA antigen, preferably about 40 μg to about 60 μg of the HA antigen, or about 50 μg of the HA antigen. In some embodiments, the dose contains 3 μg of the HA antigen. In other embodiments, the dose contains 9 μg of the HA antigen. In other embodiments, the dose contains 30 μg of the HA antigen.

[0167] In some embodiments, this document provides an immunogenic composition comprising a recombinant polypeptide trimer, the recombinant polypeptide trimer comprising sequences selected from SEQ ID NO:3-6 or fragments thereof, variants or mutants, or combinations of any two or more of the trimers. In some embodiments, a unit dose of the immunogenic composition may comprise about 10 μg to about 100 μg of the rabies G antigen, preferably about 25 μg to about 75 μg of the rabies G antigen, preferably about 40 μg to about 60 μg of the rabies G antigen, or about 50 μg of the rabies G antigen. In some embodiments, the dose contains 3 μg of the rabies G antigen. In other embodiments, the dose contains 9 μg of the rabies G antigen. In other embodiments, the dose contains 30 μg of the rabies G antigen.

[0168] In some cases, it may be necessary to combine the disclosed immunogen with other drugs (e.g., vaccines) that induce a protective response against other factors. For example, a composition including the recombinant influenza HA or rabies G antigen (e.g., trimer or protein) described herein may be administered simultaneously (usually separately) or sequentially with other vaccines recommended by the Advisory Committee on Immunization Practices (ACIP; cdc.gov / vaccines / acip / index.html) for the target age group (e.g., infants approximately 1 to 6 months old), such as influenza vaccines, rabies vaccines, or varicella-zoster vaccines. Therefore, the disclosed immunogen including the recombinant influenza HA or rabies G antigen described herein may be administered simultaneously or sequentially with vaccines, for example, those against hepatitis B (HepB), diphtheria, tetanus and pertussis (DTaP), pneumococcus (PCV), Haemophilus influenzae type b (Hib), poliomyelitis, rotavirus, influenza, and rabies.

[0169] Multivalent or combination vaccines provide protection against multiple pathogens. In some cases, multivalent vaccines can provide protection against multiple strains or strains of the same pathogen. For example, the combination vaccine Tdap provides protection against strains of tetanus, pertussis, and diphtheria. Multivalent vaccines are ideal for minimizing the number of immunizations required to provide protection against multiple pathogens or strains, reducing administration costs, and increasing coverage. This can be particularly useful, for example, when vaccinating infants or children.

[0170] In some embodiments, for example, the vaccine comprising the immunogenic composition described herein is a multivalent vaccine. In some embodiments, the antigenic material used in the multivalent vaccine composition incorporated herein is derived from various types of viruses or combinations thereof. The antigen used in the multivalent vaccine composition incorporated herein may be derived from a single influenza or rabies virus strain or multiple strains, for example, two to five strains, to provide a broader range of protection. In one embodiment, the antigen used in the multivalent vaccine composition incorporated herein is derived from multiple influenza or rabies viruses. Other available antigens include live viruses, attenuated viruses, and inactivated viruses, such as inactivated poliovirus (Jiang et al., J. Biol. Stand., (1986) 14:103-9), attenuated hepatitis A virus strains (Bradley et al., J. Med. Virol., (1984) 14:373-86), attenuated measles virus (James et al., N. Engl. J. Med., (1995) 332:1262-6), and pertussis virus epitopes (e.g., ACEL-IMUNErM acellular DTP, Wyeth-Lederle vaccine, and pediatric medications).

[0171] In some respects, the vaccines described herein are universal vaccines. In some implementations, a universal vaccine is one that provides protection against multiple strains of the same virus, such as multiple strains of influenza or rabies virus. Developing an effective universal influenza or rabies vaccine would reduce the cost and labor required for, for example, the formulation of seasonal vaccines and allow for more robust pandemic preparedness.

[0172] In some respects, universal vaccines are vaccines that contain multiple epitopes derived from different viral strains. In other respects, universal vaccines contain a single epitope that is conserved across different viral strains. For example, universal vaccines may be based on relatively conserved domains of the influenza HA protein or the rabies G protein.

[0173] Immunogenic compositions comprising the disclosed immunogen (e.g., the disclosed recombinant influenza HA or rabies G trimer, or a nucleic acid molecule encoding a protopolymer of the disclosed recombinant influenza HA or rabies G trimer) and a pharmaceutically acceptable carrier are also provided. In some embodiments, the immunogenic composition comprises a trimerized recombinant polypeptide provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a protein containing a plurality of trimerized recombinant polypeptides provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises protein nanoparticles provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a VLP provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises isolated nucleic acids provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a vector provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a virus provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises the pseudoviruses provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises the cells provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition as described herein is a vaccine. In some embodiments, the vaccine is a prophylactic vaccine. In some embodiments, the vaccine is a therapeutic vaccine. In some embodiments, the vaccine is both a prophylactic and a therapeutic vaccine. Such pharmaceutical compositions can be administered to subjects via a variety of routes of administration known to those skilled in the art, such as intramuscular, intradermal, subcutaneous, intravenous, intraarticular, intra-articular, intraperitoneal, intranasal, sublingual, tonsillar, oropharyngeal, or other parenteral and mucosal routes. In several embodiments, a pharmaceutical composition comprising one or more of the disclosed immunogens is an immunogenic composition. Practical methods for preparing dosable compositions will be known or apparent to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Sciences, 19th edition, Mack Publishing Company, Easton, Pa., 1995.

[0174] Therefore, immunogens, such as recombinant influenza HA antigen or rabies G antigen, such as the trimers and proteins described herein, can be formulated with pharmaceutically acceptable carriers to help preserve biological activity while also contributing to increased stability during storage within an acceptable temperature range. Possible carriers include, but are not limited to, physiologically balanced 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 protectants. The resulting aqueous solution can be used as is or lyophilized. Lyophilized formulations are combined with sterile solutions prior to administration for single or multiple doses.

[0175] Formulated compositions, especially liquid formulations, may contain antibacterial agents to prevent or minimize degradation during storage, including but not limited to effective concentrations (typically 1% w / v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben. Some patients may be contraindicated with antibacterial agents; therefore, lyophilized formulations can be reconstituted in solutions with or without such ingredients.

[0176] The immunogenic compositions disclosed herein may contain pharmaceutically acceptable mediators to approximate physiological conditions, such as pH adjusters and buffers, osmotic pressure modifiers, wetting agents, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. The immunogenic compositions may optionally include adjuvants to enhance the host's immune response. Suitable adjuvants include, for example, Toll-like receptor agonists, aluminum agents, AlPO4, aluminum 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 polypropylene (POP), such as POE-POP-POE block copolymers, MPL, etc., are also suitable. TM(3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.) as well as many other suitable adjuvants well known in the art can be used as adjuvants (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). The advantage of these adjuvants is that they help to stimulate the immune system in a non-specific manner, thereby enhancing the immune response to the drug. In some embodiments, the immunogenic compositions of this disclosure may include more than one adjuvant or be administered with more than one adjuvant. In some embodiments, the immunogenic compositions of this disclosure may include two adjuvants or be administered with two adjuvants. In some embodiments, the immunogenic compositions of this disclosure may include multiple adjuvants or be administered with multiple adjuvants. For example, in some cases, vaccines, such as those comprising the immunogenic compositions provided herein, may include multiple adjuvants or be administered in combination with multiple adjuvants.

[0177] Examples of suitable adjuvants for vaccine compositions include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, and MPL. TM And IL-12. In some embodiments, the vaccine compositions or nanoparticle immunogens disclosed herein (e.g., influenza or rabies vaccine compositions) can be formulated as controlled-release or timed-release formulations. This can be achieved in compositions containing sustained-release polymers or via microencapsulation delivery systems or bioadhesive gels. Various pharmaceutical compositions can be prepared according to standard procedures well known in the art.

[0178] In some embodiments, the immunogenic compositions of the present invention may contain an adjuvant formulation comprising a metabolizable oil (e.g., squalene) and α-tocopherol in the form of an oil-in-water emulsion, and polyoxyethylene sorbitan monooleate (Tween-80). In some embodiments, the adjuvant formulation may comprise about 2% to about 10% squalene, about 2% to about 10% α-tocopherol (e.g., D-α-tocopherol), and about 0.3% to about 3% polyoxyethylene sorbitan monooleate. In some embodiments, the adjuvant formulation may comprise about 5% squalene, about 5% tocopherol, and about 0.4% polyoxyethylene sorbitan monooleate. In some embodiments, the immunogenic compositions of the present disclosure may contain 3-O-deacylated monophosphoryl lipid A (3D-MPL) and an adjuvant in the form of an oil-in-water emulsion, the adjuvant comprising a metabolizable oil, α-tocopherol, and polyoxyethylene sorbitan monooleate. In some embodiments, the immunogenic compositions of this disclosure may contain QS21 (Quillajasaponaria Molina extract: fraction 21), 3D-MPL, and an oil-in-water emulsion, wherein the oil-in-water emulsion comprises metabolizable oils, α-tocopherol, and polyoxyethylene sorbitan monooleate. In some embodiments, the immunogenic compositions of this disclosure may contain QS21, 3D-MPL, and an oil-in-water emulsion, wherein the oil-in-water emulsion has the following composition: metabolizable oils such as squalene, α-tocopherol, and Tween-80. In some embodiments, the immunogenic compositions of this disclosure may contain an adjuvant in the form of a liposome composition.

[0179] In some embodiments, the immunogenic compositions of this disclosure may contain an adjuvant formulation comprising a metabolizable oil (e.g., squalene), polyoxyethylene sorbitan monooleate (Tween-80), and Span 85. In some embodiments, the adjuvant formulation may comprise about 5% (w / v) squalene, about 0.5% (w / v) polyoxyethylene sorbitan monooleate, and about 0.5% (w / v) Span 85.

[0180] In some embodiments, the immunogenic compositions of this disclosure may contain, for example, an adjuvant formulation in the form of a nanoparticle composition, said adjuvant formulation comprising saponins, cholesterol, and phospholipids. In some embodiments, the immunogenic compositions of this disclosure may contain a mixture of separately purified saponin fractions, subsequently formulated with cholesterol and phospholipids.

[0181] In some embodiments, the immunogenic composition of this disclosure may contain a compound selected from MF59. TM Matrix-A TM Matrix-C TM Matrix-M TM Adjuvants to AS01, AS02, AS03 and AS04.

[0182] In some embodiments, the immunogenic compositions of this disclosure may contain a Toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide of 8 to 35 nucleotides in length containing an unmethylated cytidine-phosphate-guanosine (also known as CpG or cytosine-phosphate-guanosine) motif, the presence of the influenza or rabies antigen (e.g., HA or G protein) and the oligonucleotide in the immunogenic composition being sufficient to effectively stimulate an immune response to the influenza or rabies antigen in a desired mammalian subject, such as a human subject. TLR9 (CD289) recognizes the unmethylated cytidine-phosphate-guanosine (CpG) motif found in microbial DNA, which can be mimicked using synthetic CpG-containing oligodeoxynucleotides (CpG-ODN). CpG-ODN is known to enhance antibody production and stimulate T helper 1 (Th1) cell responses (Coffman et al., Immunity, 33:492-503, 2010). Optimal oligonucleotide TLR9 agonists typically contain a palindromic sequence following the general formula: 5'-purine-purine-CG-pyrimidine-pyrimidine-3', or 5'-purine-purine-CG-pyrimidine-pyrimidine-CG-3'. U.S. Patent No. 6,589,940, which is incorporated herein by reference in its entirety. In some embodiments, the CpG oligonucleotide is linear. In other embodiments, the CpG oligonucleotide is cyclic or includes a hairpin ring. The CpG oligonucleotide can be single-stranded or double-stranded. In some embodiments, the CpG oligonucleotide may contain modifications. Modifications include, but are not limited to, modifications of 3'OH or 5'OH groups, modifications of nucleotide bases, modifications of sugar components, and modifications of phosphate groups. Modified bases may be included in the palindromic sequence of the CpG oligonucleotide, provided that the modified bases maintain the same specificity to their natural complement via Watson-Crick base pairing (e.g., the palindromic portion remains self-complementary). In some embodiments, the CpG oligonucleotide contains atypical bases. In some embodiments, the CpG oligonucleotide contains modified nucleosides. In some embodiments, the modified nucleosides are selected from 2'-deoxy-7-deazonoguanosine, 2'-deoxy-6-thioguanosine, arabinosylguanosine, 2'-deoxy-2'-substituted arabinosylguanosine, and 2'-O-substituted arabinosylguanosine. The CpG oligonucleotide may contain phosphate group modifications. For example, in addition to phosphodiester bonds, phosphate modifications include, but are not limited to, methyl phosphonate, thiophosphate, phosphoramide (bridged or non-bridged), triphosphate, and dithiophosphate, and may be used in any combination. Other non-phosphate ester bonds may also be used. In some embodiments, the oligonucleotide contains only a thiophosphate backbone. In some embodiments, the oligonucleotide contains only a phosphodiester backbone.In some embodiments, the oligonucleotide comprises a combination of phosphate ester bonds in its phosphate backbone, such as a combination of phosphodiester bonds and thiophosphate bonds. Oligonucleotides with a thiophosphate backbone may be more immunogenic than oligonucleotides with a phosphodiester backbone and appear to be more resistant to degradation after injection into the host (Braun et al., J Immunol, 141:2084-2089, 1988; and Latimer et al., Mol Immunol, 32:1057-1064, 1995). The CpG oligonucleotides of this disclosure comprise at least one, two, or three internucleotide thiophosphate bonds. In some embodiments, when multiple CpG oligonucleotide molecules are present in a pharmaceutical composition comprising at least one excipient, both stereoisomers of the thiophosphate bond are present in the multiple CpG oligonucleotide molecules. In some embodiments, all internucleotide bonds of the CpG oligonucleotide are thiophosphate bonds, or in other words, the CpG oligonucleotide has a thiophosphate backbone.

[0183] Any suitable CpG oligodeoxynucleotide (ODN) or combination thereof can be used as an adjuvant in this disclosure. For example, K-type ODNs (also known as B-type) encode multiple CpG motifs on a phosphate thioester backbone. K-type ODNs can be based on the following sequences: Compared to natural phosphodiester nucleotides, the use of thiophosphate nucleotides enhances resistance to nuclease digestion, resulting in a substantially longer in vivo half-life. K-type ODNs induce pDC differentiation and TNF-α production, and trigger B cell proliferation and IgM secretion. D-type ODNs (also known as A-type) are constructed from a mixed phosphodiester / thiophosphate backbone, containing a single CpG motif flanked by palindromic sequences, and poly-G tails (a structural motif that facilitates multiplying) at the 3′ and 5′ ends. D-type ODNs can be based on the following sequence: GGTGCAT CG ATGCAGGGGGG. Type D ODN induces pDC maturation and IFN-α secretion, but has no effect on B cells. Type C ODN is similar to Type K ODN in that it is composed entirely of phosphate-thioester nucleotides, but is similar to Type D ODN in that it contains a palindromic CpG motif. Type C ODN can be based on the following sequence: These types of ODNs stimulate B cells to secrete IL-6 and stimulate pDCs to produce IFN-α. P-type ODNs contain two palindromic sequences, enabling them to form a more highly ordered structure. P-type ODNs can be based on the following sequences: P-type ODN activates B cells and pDCs and induces substantially higher IFN-α production compared to C-type ODN. In this section, bold letters in the ODN sequence indicate self-complementary palindromic sequences, and CpG motifs are underlined.

[0184] Exemplary CpG ODNs, such as CpG 7909 (5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′) and CpG 1018 (5′-TGACTGTGAACGTTCGAGATGA-3′), are known and disclosed in U.S. Patent Nos. 7,255,868, 7,491,706, 7,479,285, 7,745,598, 7,785,610, 8,003,115, 8,133,874, 8,114,418, 8,222,398, 8,333,980, 8,597,665, 8,669,237, 9,028,845, and 10,052,378, application publication US 2020 / 0002704, and Bode et al., “CpG as a Vaccine Adjuvant”. In Expert Rev Vaccines (2011), 10(4):499-511, all of these references are incorporated herein by reference in their entirety for all purposes.

[0185] One or more adjuvants may be used in combination and may include, but are not limited to, aluminum hydroxide (aluminum salt), oil-in-water emulsions, water-in-oil emulsions, liposomes, and microparticles, such as poly(lactide-co-glycolic acid) microparticles (Shah et al., Methods Mol, 1494:1-14, 2017). In some embodiments, the immunogenic composition further comprises an aluminum salt adjuvant that adsorbs influenza or rabies antigens. In some embodiments, the aluminum salt adjuvant comprises one or more of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate. In some embodiments, the aluminum salt adjuvant comprises one or both of aluminum hydroxide and aluminum phosphate. In some embodiments, the aluminum salt adjuvant comprises aluminum hydroxide. In some embodiments, the unit dose of the immunogenic composition comprises about 0.25 to about 0.50 mg Al. 3+ Or approximately 0.35 mg Al 3+In some embodiments, the immunogenic composition further comprises other adjuvants. Other suitable adjuvants include, but are not limited to, water-coated squalene emulsions (e.g., MF59 or AS03), TLR3 agonists (e.g., polyIC or polyICLC), TLR4 agonists (e.g., bacterial lipopolysaccharide derivatives such as monophospholipid A (MPL) and / or saponins such as Quil A or QS-21, as in AS01 or AS02), TLR5 agonists (bacterial flagellin), and TLR7, TLR8, and / or TLR9 agonists (imidazoquinoline derivatives such as imiquimod and resiquimod) (Coffman et al., Immunity, 33:492-503, 2010). In some embodiments, other adjuvants comprise MPL and aluminum (e.g., AS04). For veterinary use and for antibody production in nonhuman animals, mitogenic components of Freund's adjuvants (complete and incomplete) may be used.

[0186] In some embodiments, the immunogenic composition comprises pharmaceutically acceptable excipients, including, for example, solvents, extenders, buffers, osmotic modifiers, and preservatives (Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the immunogenic composition may comprise excipients that function as one or more of the solvent, extender, buffer, and osmotic modifier functions (e.g., sodium chloride in saline can simultaneously act as an aqueous medium and an osmotic modifier).

[0187] In some embodiments, the immunogenic composition comprises an aqueous mediator as a solvent. Suitable mediators include, for example, sterile water, saline solution, phosphate-buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0188] The immunogenic composition may contain a buffer. The buffer controls pH to inhibit degradation of the active agent during processing, storage, and optional remodeling. Suitable buffers include, for example, salts, including acetates, citrates, phosphates, or sulfates. Other suitable buffers include, for example, amino acids such as arginine, glycine, histidine, and lysine. The buffer may also contain hydrochloric acid or sodium hydroxide. In some embodiments, the buffer maintains the pH of the composition in the range of 6 to 9. In some embodiments, the pH is greater than (lower limit) 6, 7, or 8. In some embodiments, the pH is less than (upper limit) 9, 8, or 7. That is, the pH is in the range of about 6 to 9, where the lower limit is less than the upper limit.

[0189] The immunogenic composition may contain an osmotic modifier. Suitable osmotic modifiers include, for example, dextran, glycerol, sodium chloride, glycerol, and mannitol.

[0190] The immunogenic composition may contain a expander. Expanders are particularly useful when the pharmaceutical composition is lyophilized prior to administration. In some embodiments, the expander is a protectant that helps stabilize and prevent degradation of the active agent during freeze-drying or spray drying and / or storage. Suitable expanders are sugars (monosaccharides, disaccharides, and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, and raffinose.

[0191] The immunogenic composition may contain preservatives. Suitable preservatives include, for example, antioxidants and antimicrobial agents. However, in a preferred embodiment, the immunogenic composition is prepared under sterile conditions and in a single-use container, therefore it is not necessary to include preservatives.

[0192] In some embodiments, the composition may be provided as a sterile composition. The pharmaceutical composition typically contains an effective amount of the disclosed immunogen and can be prepared by conventional methods. Generally, the amount of immunogen in each dose of the immunogenic composition is selected to induce an immune response without significant adverse side effects. In some embodiments, the composition may be provided in unit dosage forms for inducing an immune response in a subject. The unit dosage form contains a suitable single preselected dose to administer the drug to a subject, or multiple doses of two or more preselected unit doses, appropriately labeled or measured, and / or a dosing mechanism for administering the unit dose or multiple unit doses. In other embodiments, the composition further includes an adjuvant.

[0193] IV. Methods for inducing immune responses

[0194] In some embodiments, this document discloses a method of using a viral antigen trimer as a vaccine or as part of a multivalent vaccine, with or without an adjuvant or with more than one adjuvant, optionally administered via intramuscular injection or intranasal administration to prevent viral infection.

[0195] In some embodiments, this document discloses methods for preventing pandemic avian influenza or swine influenza by intramuscular or intranasal administration of a viral antigen trimer as a vaccine or as part of a multivalent vaccine, with or without an adjuvant or with more than one adjuvant.

[0196] In some embodiments, this document discloses a method for using viral antigen trimers as diagnostic antigens for viral infection by detecting antibodies (e.g., IgM or IgG) that recognize viral antigens, such as neutralizing antibodies.

[0197] In some embodiments, this document discloses methods for using viral antigen trimers as antigens to generate polyclonal or monoclonal antibodies that can be used for passive immunization, such as neutralizing mAbs for treating influenza infections.

[0198] In some embodiments, this document discloses viral antigen trimers as part of a vaccine or a multivalent vaccine, wherein the vaccine comprises multiple trimeric subunit vaccines, the multiple trimeric subunit vaccines comprising a viral antigen of the same viral protein or a viral antigen comprising two or more different proteins of one or more viruses or one or more strains of the same virus.

[0199] This document provides recombinant polypeptides comprising influenza virus hemagglutinin (HA) protein peptides or fragments or epitopes thereof. In some embodiments, the recombinant polypeptide is linked to a C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an interpeptide disulfide bond. Engineered virus-like particles (VLPs) comprising the provided polypeptides can be used in vaccination methods and in the preparation of the provided immunogenic compositions.

[0200] In some embodiments, a monovalent vaccine is disclosed herein, the monovalent vaccine comprising the viral antigen trimer disclosed herein. In some embodiments, a bivalent vaccine is disclosed herein, the bivalent vaccine comprising the viral antigen trimer disclosed herein. In some embodiments, a trivalent vaccine is disclosed herein, the trivalent vaccine comprising the viral antigen trimer disclosed herein. In some embodiments, a quadrivalent vaccine is disclosed herein, the quadrivalent vaccine comprising the viral antigen trimer disclosed herein.

[0201] In some embodiments, a monovalent vaccine is disclosed herein, comprising the HA trimer disclosed herein. In some embodiments, a bivalent vaccine is disclosed herein, comprising the HA trimer disclosed herein. In some embodiments, a bivalent vaccine is disclosed herein, comprising at least one HA trimer comprising a first HA protein antigen and at least one HA trimer comprising a second HA protein antigen. In some embodiments, the first and second HA protein antigens are derived from the same HA protein of one or more viral species or strains / subtypes, or from two or more different HA proteins of one or more viral species or one or more strains / subtypes of the same viral species. In some embodiments, a trivalent vaccine is disclosed herein, comprising the HA trimer disclosed herein. In some embodiments, a trivalent vaccine is disclosed herein, comprising at least one HA trimer comprising a first HA protein antigen, at least one HA trimer comprising a second HA protein antigen, and at least one HA trimer comprising a third HA protein antigen. In some embodiments, the first, second, and third HA protein antigens originate from the same HA protein of one or more viral species or strains / subtypes, or from two, three, or more different HA proteins of one or more viral species or one or more strains / subtypes of the same viral species. In some embodiments, a quadrivalent vaccine is disclosed herein comprising the HA trimer disclosed herein. In some embodiments, a quadrivalent vaccine is disclosed herein comprising at least one HA trimer containing a first HA protein antigen, at least one HA trimer containing a second HA protein antigen, at least one HA trimer containing a third HA protein antigen, and at least one HA trimer containing a fourth HA protein antigen. In some embodiments, the first, second, third, and fourth HA protein antigens originate from the same HA protein of one or more viral species or strains / subtypes, or from two, three, four, or more different HA proteins of one or more viral species or one or more strains / subtypes of the same viral species. In any of the above embodiments, the HA protein antigen may be derived from influenza A virus or influenza B virus, optionally wherein the influenza A virus belongs to the H1, H3 or H5 subtype, such as H1N1 or H3N2, or any combination of influenza virus subtypes / strains.

[0202] Several universal vaccine formulations are currently under investigation. In some respects, universal vaccines contain multiple epitopes derived from different viral strains. In other respects, universal vaccines contain a single epitope conserved across different viral strains. For example, universal vaccines could be based on relatively conserved domains of the influenza HA protein, such as the HA stem conserved region, which may be entirely derived from HA2 but may contain some residues from the N-terminus and C-terminus of HA1.

[0203] T-cell vaccines based on highly conserved CD4 epitopes have been evaluated in a Phase II irritation study, demonstrating positive protective responses against various influenza strains, including pandemic strains. A recombinant multiepitope vaccine called Multimeric-001, incorporating conserved epitopes from nine different influenza proteins on B cells, CD4 T cells, and CD8 T cells, is also being tested in trials. Fusion protein vaccines consisting of nucleoproteins (NPs) in gold nanoparticles and adjuvant-linked B-cell epitopes M2e and M2e peptides combined with CpG are also under development.

[0204] Multivalent or combination vaccines provide protection against multiple pathogens. In some cases, multivalent vaccines can provide protection against multiple strains of the same pathogen, such as the quadrivalent inactivated influenza vaccine. In others, multivalent vaccines provide protection against multiple pathogens, such as the Tdap combination vaccine, which provides protection against strains of tetanus, pertussis, and diphtheria. Multivalent vaccines are ideal for minimizing the number of immunizations required to provide protection against multiple pathogens or strains, reducing administration costs, and increasing coverage. This can be particularly useful, for example, when vaccinating infants or children.

[0205] The disclosed immunogen (e.g., recombinant influenza HA or rabies G trimer, nucleic acid molecules (e.g., RNA molecules) or vectors encoding protomers of the disclosed recombinant influenza HA or rabies G trimer, or protein nanoparticles or virus-like particles containing the disclosed recombinant influenza HA or rabies G trimer) can be administered to a subject to induce an immune response against the corresponding influenza HA or rabies G trimer. In one particular embodiment, the subject is a human. The immune response can be a protective immune response, such as a response that inhibits subsequent infection with the corresponding influenza or rabies virus. Inducing an immune response can also be used to treat or suppress infections and diseases associated with the corresponding influenza or rabies virus.

[0206] In some embodiments, this document provides a method for inducing an immune response to influenza surface antigen in a subject, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from SEQ ID NO:1-2. In some embodiments, this document provides a method for inducing an immune response to influenza surface antigen in a subject, wherein the surface antigen comprises an HA protein or an antigenic fragment thereof, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from SEQ ID NO:1-2. In some embodiments, this document provides a method for inducing an immune response to influenza surface antigen in a subject, wherein the surface antigen comprises a sequence selected from SEQ ID NO:7-9, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from SEQ ID NO:1-2. In some embodiments, this document provides a method for inducing an immune response to influenza surface antigen in a subject, wherein the surface antigen comprises an influenza HA protein or an antigenic fragment thereof, optionally, the surface antigen comprises any one or more sequences of SEQ ID NO:7-9 or antigenic fragments thereof, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide containing a sequence shown in any one of SEQ ID NO:1-2.

[0207] In some embodiments, this document provides a method for generating an immune response to an influenza surface antigen in a subject, wherein the surface antigen comprises an HA protein or an antigenic fragment thereof, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide containing a sequence selected from SEQ ID NO:1-2, or a combination of two or more complexes.

[0208] In some implementations, subjects who have been or may be infected with an influenza virus corresponding to the HA protein in the immunogen (e.g., due to exposure to or potential exposure to an influenza virus) may be selected for treatment. Following administration of the disclosed immunogen, the subject may be monitored for infection status or influenza-related symptoms, or both.

[0209] In some embodiments, this document provides a method for inducing an immune response to rabies surface antigen in a subject, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from SEQ ID NO:3-6. In some embodiments, this document provides a method for inducing an immune response to rabies surface antigen in a subject, wherein the surface antigen comprises a G protein or an antigenic fragment thereof, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from SEQ ID NO:3-6. In some embodiments, this document provides a method for inducing an immune response to rabies surface antigen in a subject, wherein the surface antigen comprises a sequence selected from SEQ ID NO:10-15, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from SEQ ID NO:3-6. In some embodiments, this document provides a method for generating an immune response to a rabies surface antigen in a subject, wherein the surface antigen comprises a rabies G protein or an antigenic fragment thereof, optionally, the surface antigen comprises a sequence of any one or more of SEQ ID NO:10-15 or an antigenic fragment thereof, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide containing a sequence shown in any one of SEQ ID NO:3-6.

[0210] In some embodiments, this document provides a method for generating an immune response to a rabies surface antigen in a subject, wherein the surface antigen comprises a G protein or an antigenic fragment thereof, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide containing a sequence selected from SEQ ID NO:3-6, or a combination of two or more such complexes.

[0211] In some implementations, subjects who are already or may be infected with rabies virus corresponding to the G protein in the immunogen (e.g., due to exposure or potential exposure to rabies virus) may be selected for treatment. Following administration of the disclosed immunogen, the subject may be monitored for infection status or rabies-related symptoms, or both.

[0212] Typical subjects intended for treatment with the therapeutic agents and methods of this disclosure include humans, as well as non-human primates and other animals. To identify subjects for prevention or treatment according to the methods of this disclosure, recognized screening methods are employed to determine risk factors associated with the target or suspected disease or condition, or to determine the subject's existing disease or condition status. These screening methods include, for example, routine examinations to identify environmental, family, occupational, and other such risk factors that may be associated with the target or suspected disease or condition, and diagnostic methods for detecting and / or characterizing influenza or rabies virus infection, such as various ELISA and other immunoassay methods. These and other conventional methods allow clinicians to select patients requiring treatment using the methods and pharmaceutical compositions of this disclosure. Based on these methods and principles, the compositions may be administered according to the teachings herein or other conventional methods, as a standalone preventative or therapeutic regimen, or as a follow-up, adjunctive, or complementary treatment to other therapies.

[0213] The disclosed immunogen can be administered for prophylactic or therapeutic purposes. When administered prophylactically, the disclosed therapeutic agent is administered before any symptoms appear, such as before infection. Prophylactic administration of the disclosed therapeutic agent is for the prevention or improvement of any secondary infection. When administered therapeutically, the disclosed therapeutic agent is administered at or after the onset of symptoms of illness or infection, such as after the appearance of symptoms of infection with influenza virus corresponding to the HA protein in the immunogen, or after a diagnosis of influenza virus infection. The therapeutic agent can therefore be administered before anticipated exposure to the influenza virus in order to reduce the anticipated severity, duration, or extent of the symptoms of infection and / or related illness after exposure to or suspected exposure to the virus or after the actual onset of infection.

[0214] The immunogen and immunogenic compositions described herein are provided to the subject in an amount that effectively induces or enhances an immune response in a subject (preferably a human) to the influenza virus HA protein in the immunogen. The actual dose of the disclosed immunogen will vary depending on numerous factors, such as the subject's disease indications and specific condition (e.g., age, body size, health status, symptom severity, susceptibility factors, etc.), the time and route of administration, any concurrent medications or treatments, and the specific pharmacology of the composition used to elicit the desired active or biological response in the subject. Dosing regimens may be adjusted to provide optimal prophylactic or therapeutic responses.

[0215] Immunogenic compositions comprising one or more of the disclosed immunogens may be used in coordinated (or priming-boost) vaccination regimens or combination formulations. In some embodiments, novel combination immunogenic compositions and coordinated immunization regimens employ individual immunogens or formulations, each targeting the initiation of an antiviral immune response, such as an immune response to the influenza virus HA protein. Individual immunogenic compositions that induce an antiviral immune response may be combined in a multivalent immunogenic composition administered to a subject in a single immunization step, or they may be administered separately (in a monovalent immunogenic composition) in a coordinated (or priming-boost) immunization regimen.

[0216] Several booster immunizations may be administered, and each booster may use a different, disclosed immunogen. In some instances, a booster may use the same immunogen as another booster or primary immunization. The primary and booster immunizations may be administered as single or multiple doses, for example, two, three, four, five, six, or more doses may be given to the subject over days, weeks, or months. Multiple boosters may also be administered, for example, one to five times (e.g., 1, 2, 3, 4, or 5 boosters) or more. Different doses may be used in a series of sequential immunizations. For example, a relatively large dose may be used in the first immunization, followed by a relatively smaller dose in the boosters.

[0217] In some embodiments, the booster may be administered approximately 2 weeks, 3 to 8 weeks, or 4 weeks after primary immunization, or approximately several months after primary immunization. In some embodiments, the booster may be administered approximately 5, 6, 7, 8, 10, 12, 18, or 24 months after primary immunization, or more or less time after primary immunization. Regular additional boosters may also be used at appropriate times to enhance the subject's "immune memory." The adequacy of the selected vaccination parameters, such as formulation, dosage, regimen, etc., can be determined by obtaining aliquots of serum from the subject during the immunization program and measuring antibody titers. Furthermore, the subject's clinical condition can be monitored to identify desired effects, such as prevention of infection or improvement in disease status (e.g., reduction in viral load). If such monitoring indicates that vaccination is suboptimal, the subject may be boosted with an additional dose of immunogenic composition, and vaccination parameters may be modified in a manner intended to enhance the immune response.

[0218] In some embodiments, the primary-boost method may include providing the subject with a DNA primary immunization and a protein booster vaccination regimen. The method may include two or more administrations of a nucleic acid molecule or protein.

[0219] For protein therapeutic agents, each human dose typically contains 1-1000 μg of protein, such as about 1 μg to about 100 μg, for example about 1 μg to about 50 μg, such as 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.

[0220] The amount used in an immunogenic composition is selected based on the subject population (e.g., infants or the elderly). The optimal dosage of a particular composition can be determined through standard studies, including observation of antibody titers and other responses in the subjects. It is understood that the therapeutically effective amount of the disclosed immunogen in the immunogenic composition, such as the disclosed recombinant influenza virus HA trimer, viral vector, or nucleic acid molecule, can include amounts that are ineffective in evoking an immune response with a single dose but effective with multiple doses, for example, in an initiation-boost dosing regimen.

[0221] Upon administration of the immunogen disclosed herein, the immune system of a subject typically responds to the immunogenic composition by producing antibodies specific to the influenza virus HA trimer included in the immunogen. Such a response indicates that an immunogenic dose has been delivered to the subject.

[0222] In some implementations, the antibody response of a subject is determined in the context of evaluating an effective dose / immunization regimen. In most cases, assessing the antibody titer in the serum or plasma obtained from the subject is sufficient. Decisions regarding whether to administer a booster dose and / or change the amount of therapeutic agent administered to an individual may be based at least in part on antibody titer levels. Antibody titer levels may be based, for example, on an immune binding assay that measures the concentration of antibodies in serum that bind to antigens, including, for example, recombinant influenza virus HA trimer included in the immunogen.

[0223] The method is effective only if the complete elimination, reduction, or prevention of influenza or rabies virus infection is not required. For example, compared to influenza or rabies virus infection in the absence of an immunogen, inducing an immune response against influenza or rabies virus with one or more of the disclosed immunogens can reduce or suppress influenza or rabies virus infection by the amount required, such as at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable infected cells). In other embodiments, viral replication can be reduced or suppressed by the disclosed method. The method is effective only if the complete elimination of influenza or rabies virus replication is not required. For example, compared to influenza or rabies virus replication in the absence of an immunogen, an immune response induced by one or more disclosed immunogens can reduce the replication of the corresponding influenza or rabies virus by the required amount, such as at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (eliminating or preventing detectable influenza or rabies virus replication).

[0224] In some embodiments, the disclosed immunogen is administered to the subject concurrently with the administration of an adjuvant. In other embodiments, the disclosed immunogen is administered to the subject after the administration of the adjuvant and for a period of time sufficient to induce an immune response.

[0225] One method of administering nucleic acids is direct immunization with plasmid DNA, such as mammalian expression plasmids. Immunization via nucleic acid constructs is well known in the art and is taught, for example, in U.S. Patent No. 5,643,578 (describing a method of immunizing vertebrates by inducing a cell-mediated or humoral response by introducing DNA encoding a desired antigen) and U.S. Patent Nos. 5,593,972 and 5,817,637 (describing the operative linking of a nucleic acid sequence encoding an antigen to a regulatory sequence that enables its expression). U.S. Patent No. 5,880,103 describes several methods of delivering nucleic acids encoding immunogenic peptides or other antigens to an organism. These methods include liposome delivery of nucleic acids (or the synthetic peptide itself) and immunostimulatory constructs or ISCOMS. TM That is, in a mixture of cholesterol and Quil A TM (Saponin) spontaneously forms a negatively charged cage-like structure of 30-40 nm in size. ISCOMS has been used in various infection experimental models, including toxoplasmosis and Epstein-Barr virus-induced tumors. TM As an antigen delivery medium, it generated protective immunity (Mowat and Donachie, Immunol. Today 12:383, 1991). Encapsulation in ISCOMS at levels as low as 1 μg has been found to produce this effect.TM The antigen dose produced a class I mediated CTL response (Takahashi et al., Nature 344:873, 1990).

[0226] In some embodiments, a plasmid DNA vaccine is used to express the disclosed immunogen in a subject. For example, a nucleic acid molecule encoding the disclosed immunogen can be administered to the subject to induce an immune response to an influenza virus HA protein included in the immunogen. In some embodiments, the nucleic acid molecule can 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, which is incorporated herein by reference).

[0227] In another method of immunization using nucleic acids, the disclosed recombinant influenza virus HA or recombinant influenza virus HA trimer can be expressed via an attenuated viral host or vector or bacterial vector. In another embodiment, a viral vector-based immunization protocol can be used to directly deliver nucleic acids encoding the disclosed recombinant influenza virus HA or influenza virus HA trimer into cells. Many virus-based systems, such as retroviruses and adenoviruses, have been described for gene transfer purposes. Recombinant vaccinia virus, adeno-associated virus (AAV), herpesvirus, retrovirus, cytomegalovirus, or other viral vectors can be used to express the peptide or protein, thereby evoking a CTL response. For example, U.S. Patent No. 4,722,848 describes vaccinia vectors and methods that can be used in immunization protocols. BCG (Bacillus Calmette-Guérin) provides another vector for expressing peptides (see Stover, Nature 351:456-460, 1991).

[0228] In one implementation, nucleic acids encoding the disclosed recombinant influenza virus HA or influenza virus HA trimer are directly introduced into cells. For example, the nucleic acids can be loaded onto gold microspheres using standard methods and delivered via HELIOS, such as Bio-Rad. TM Devices such as gene guns are introduced into the skin. Nucleic acids can be "naked," consisting of plasmids controlled by strong promoters. Typically, DNA is injected into muscle, but it can also be injected directly into other sites. Injection doses are typically from about 0.5 μg / kg to about 50 mg / kg, and usually from about 0.005 mg / kg to about 5 mg / kg (see, for example, U.S. Patent No. 5,589,466).

[0229] For example, nucleic acids can be loaded onto gold microspheres using standard methods and then processed via HELIOS, such as those from Bio-Rad. TMDevices such as gene guns are introduced into the skin. Nucleic acids can be "naked," consisting of plasmids controlled by strong promoters. Typically, DNA is injected into muscle, but it can also be injected directly into other sites. Injection doses are typically from about 0.5 μg / kg to about 50 mg / kg, and usually from about 0.005 mg / kg to about 5 mg / kg (see, for example, U.S. Patent No. 5,589,466).

[0230] In another embodiment, mRNA-based immunization protocols can be used to directly deliver nucleic acids encoding the disclosed recombinant influenza virus HA or influenza virus HA trimer into cells. In some embodiments, mRNA-based nucleic acid-based vaccines can provide an effective alternative to the aforementioned methods. mRNA vaccines eliminate safety concerns regarding DNA integration into the host genome and can be translated directly in the host cytoplasm. Furthermore, the simple cell-free in vitro synthesis of RNA avoids the manufacturing complexities associated with viral vectors. Two exemplary forms of RNA-based vaccination that can be used to deliver nucleic acids encoding the disclosed recombinant influenza virus HA or influenza virus HA trimer include conventional non-amplified mRNA immunization (see, for example, 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-amplifying mRNA immunization (see, for example, 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 Antitigens Confer Protection against Homologous and Heterotyped Viral Infections”). Heterosubtypic ViralChallenge), PLoS One, 11(8):e0161193, 2016; and Brito et al., “Self-amplifying mRNA vaccines”, Adv Genet., 89:179-233, 2015. In some embodiments, the isolated nucleic acid is an RNA molecule. In some embodiments, the nucleic acid is an mRNA molecule, such as nucleoside-modified mRNA, non-amplified mRNA, self-amplifying mRNA, or trans-amplified mRNA.

[0231] In some implementations, nucleic acids encoding the disclosed recombinant influenza virus HA or influenza virus HA trimer are directly introduced into cells. For example, the nucleic acid or protein may be contained within virus-like particles (VLPs). Virus-like particles (VLPs) are multiprotein structures that mimic the organization and structure of a standard natural virus but lack the viral genome. Several studies have demonstrated that recombinant influenza proteins can self-assemble into VLPs in cell cultures using mammalian plastids or baculovirus vectors. For example, Neumann et al. (PNAS(16)9345-9350, 2000) developed a mammalian plastid-based system that generates infectious influenza-like virus particles entirely from transfected cDNA.

[0232] In some embodiments, administration of a therapeutically effective dose of one or more of the disclosed immunogens to a subject induces a neutralizing immune response in the subject. To assess neutralizing activity, serum may be collected from the subject at appropriate time points after immunization, frozen, and stored for neutralization assays. Methods for determining neutralizing activity are known to those skilled in the art and are further described herein, including but not limited to plaque reduction neutralization (PRNT) assays, micro-neutralization assays, flow cytometry-based assays, and single-cycle infection assays.

[0233] In some embodiments, administration of a therapeutically effective dose of one or more of the disclosed immunogens to a subject induces a neutralizing immune response in the subject. To assess neutralizing activity, serum may be collected from the subject at appropriate time points after immunization, frozen, and stored for neutralization testing. Methods for determining neutralizing activity are known to those skilled in the art and are further described herein, including but not limited to plaque reduction neutralization (PRNT) assays, micro-neutralization assays, flow cytometry-based assays, and single-cycle infection assays. In some embodiments, a panel of influenza or rabies virus pseudoviruses may be used to determine serum neutralizing activity.

[0234] The information disclosed in this article regarding influenza virus HA peptides and nucleic acids generally applies to rabies G peptides and nucleic acids, for example, for inducing immune responses using rabies G peptides and / or nucleic acids.

[0235] In some embodiments, a neutralizing immune response induced by the immunogens disclosed herein generates neutralizing antibodies against RNA viruses such as influenza virus or rabies virus. In some embodiments, the neutralizing antibodies of this document bind to cellular receptors of RNA viruses such as influenza virus or rabies virus, or components thereof. In some embodiments, the viral receptor is an orthomyxovirus receptor or co-receptor, preferably a pneumovirus receptor or co-receptor, more preferably an influenza virus receptor or co-receptor. In some embodiments, the viral receptor is a rhabdovirus receptor or co-receptor, preferably a rabies virus receptor or co-receptor. In some embodiments, the neutralizing antibodies of this document regulate, reduce, antagonize, mitigate, block, inhibit, eliminate, and / or interfere with the activity or binding of at least one RNA virus such as influenza virus or rabies virus, or the activity or binding of an RNA virus receptor such as influenza virus or rabies virus receptor, such as influenza virus or rabies virus release, influenza virus or rabies virus receptor signaling, membrane influenza virus or rabies virus cleavage, influenza virus or rabies virus activity, influenza virus or rabies virus production and / or synthesis, in vitro, in situ, and / or in vivo. In some embodiments, the immunogens disclosed herein induce neutralizing antibodies against RNA viruses such as influenza virus or rabies virus, said neutralizing antibodies regulating, reducing, antagonizing, mitigating, blocking, inhibiting, eliminating and / or interfering with the binding of RNA viruses to receptors or co-receptors such as nerve growth factor receptor NGFR (p75), neural cell adhesion molecule NCAM, nicotinic acetylcholine receptor nAchR and / or sialic acid (SA, N-acetylneuraminic acid) of cell surface glycoproteins and glycolipids.

[0236] V. Products or reagent kits

[0237] Articles or kits containing the provided recombinant peptides, proteins, and immunogenic compositions are also provided. The articles may include a container and a label or packaging insert on or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, test tubes, IV infusion bags, etc. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container has a sterile inlet. Exemplary containers include intravenous infusion bags and vials, including those with stoppers that can be punctured by an injection needle. The articles or kits may also include packaging inserts indicating that the composition can be used to treat a specific condition, such as the condition described herein (e.g., influenza infection). Alternatively, or additionally, the articles or kits may also include another or the same container containing a pharmaceutically acceptable buffer. It may also include other materials such as other buffers, diluents, filters, needles, and / or syringes.

[0238] The label or packaging insert may indicate that the composition is intended for the treatment of an individual with influenza infection. The label or packaging insert on or accompanying the container may provide guidance regarding formulation reconfiguration and / or use. The label or packaging insert may also indicate that the formulation is intended for or designed for use with the infusion bag via subcutaneous, intravenous, or other modes of administration for the treatment or prevention of an individual with influenza infection.

[0239] In some embodiments, the container contains the composition alone or in combination with another composition that can effectively treat, prevent, and / or diagnose the condition. The article or kit may include (a) a first container containing a composition (i.e., a first drug), wherein the composition comprises the immunogenic composition or its protein or recombinant polypeptide; and (b) a second container containing a composition (i.e., a second drug), wherein the composition comprises other agents, such as adjuvants or other therapeutic agents. The article or kit may also include instructions on the label or packaging insert regarding the treatment of the subject with the second drug in an effective amount.

[0240] the term

[0241] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some instances, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein is not to be construed as indicating a material difference from the commonly understood meaning in the art.

[0242] The terms "peptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Peptides (including the provided receptors and other peptides, such as linkers or peptides) may include amino acid residues, including native and / or non-native amino acid residues. The term also includes post-expression modifications of the peptide, such as glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, peptides may contain modifications relating to their original or native sequences, provided that the protein retains the desired activity. These modifications can be intentional, such as through site-directed mutagenesis, or accidental, such as through mutations in the host producing the protein or errors due to PCR amplification.

[0243] As used herein, "subject" is a mammal, such as a human or other animal, typically a human. In some embodiments, the subject (e.g., a patient) who is administered one or more agents, cells, cell populations, or compositions is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject can be male or female and can be of any suitable age, including infants, adolescents, teenagers, adults, and the elderly. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[0244] As used herein, “treatment” (and its grammatical variations) means the complete or partial improvement or relief of a disease or ailment or condition, or its associated symptoms, adverse reactions or outcomes, or phenotype. The desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or relief of the disease state, and relief or improvement of prognosis. The term does not imply a complete cure of the disease or the complete elimination of any symptoms, or effectiveness against all symptoms or consequences.

[0245] As used herein, “delaying disease progression” means delaying, hindering, slowing, stabilizing, inhibiting, and / or postponing the development of a disease (such as cancer). The length of this delay may vary depending on the individual’s medical history and / or the individual being treated. In some implementations, sufficient or significant delay can actually encompass prevention, as the individual will not develop the disease. For example, the development of advanced cancer, such as metastasis, can be delayed.

[0246] As used herein, “prevention” includes providing prevention of the onset or recurrence of disease in subjects who may be susceptible to the disease but have not yet been diagnosed with it. In some embodiments, the provided cells and compositions are used to delay the development of the disease or slow its progression.

[0247] As used herein, "inhibition" function or activity refers to a reduction in function or activity when compared to a condition that is otherwise identical except for the target condition or parameter, or to another condition. For example, cells that inhibit tumor growth reduce the tumor growth rate compared to the tumor growth rate in the absence of said cells.

[0248] In the context of drug administration, the “effective amount” of a pharmaceutical agent, such as a drug formulation, cell, or composition, refers to the amount of dose / volume and time period necessary to effectively achieve the desired outcome, such as a therapeutic or preventative outcome.

[0249] A "therapeutic effective amount" of a pharmaceutical agent, such as a drug formulation, cell, or composition, refers to the amount of dose and time necessary to effectively achieve a desired therapeutic outcome, such as treating a disease, ailment, or condition, and / or a pharmacokinetic or pharmacodynamic effect of the treatment. Therapeutic effective amounts can vary depending on many factors, such as the subject's disease state, age, sex, and weight, as well as the cell population administered. In some embodiments, the provided method includes administering the cells and / or composition at an effective amount (e.g., a therapeutic effective amount).

[0250] "Prophylactic effective dose" refers to the amount of dose and time period necessary to effectively achieve the desired preventive outcome. It is usually, but not always, necessary because the preventive dose is administered to the subject before or in the early stages of disease, and therefore the prophylactic effective dose is less than the therapeutic effective dose. In cases of low cancer burden, the prophylactic effective dose may be higher than the therapeutic effective dose in some aspects.

[0251] As used herein, the term "about" refers to the typical range of error for various values ​​that is readily known to those skilled in the art. References to "about" a value or parameter herein include (and describe) implementations for that value or parameter itself.

[0252] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural referents. For example, “a” or “an” means “at least one (kind)” or “one (kind) or more (kinds).”

[0253] Throughout this disclosure, all aspects of the claimed subject matter are presented in scope. It should be understood that this scope-based description is for convenience and brevity only and should not be considered a rigid limitation on the scope of the claimed subject matter. Therefore, the scope description should be considered as having explicitly disclosed all possible sub-scopes and individual values ​​within those scopes. For example, in the case of providing a range of values, it should be understood that each intermediate value between the upper and lower limits of the range, as well as any other stated or intermediate values ​​within that range, is covered within the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also covered within the claimed subject matter, subject only to any explicit exclusions within those ranges. When the scope includes one or more limits, the scope excluding any one or both of those included limits is also included within the claimed subject matter. This applies to any scope width.

[0254] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It can be a solution, suspension, liquid, powder, paste, aqueous solution, non-aqueous solution, or any combination thereof.

[0255] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors incorporated into the genome of a host cell that has already been introduced with them. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0256] The term "influenza virus subtype" in relation to influenza A virus refers to a variant of influenza A virus characterized by various combinations of hemagglutinin (H) and neuraminidase (N) viral surface proteins. Influenza A virus subtypes can be referred to by their number of H, such as "influenza virus containing H1 or H3 subtype HA" or "H1 influenza virus" or "H3 influenza virus," or by a combination of the number of H and N, such as "influenza virus subtype H3N2" or "H3N2." The term "influenza virus subtype" explicitly includes all individual influenza virus "strains" within each subtype, which are usually caused by mutations and exhibit different pathogenic characteristics. Such strains may also be referred to as various "isolated strains" of the virus subtype. Therefore, as used herein, the terms "strain" and "isolated strain" are used interchangeably.

[0257] The term "influenza hemagglutinin," also known as "influenza HA," is a trimeric glycoprotein found on the surface of influenza viruses. It mediates viral attachment (via HA1 binding to α-2,3-sialic acid and α-2,6-sialic acid) and entry (via conformational changes) into host cells. HA comprises two structures: a globular head domain containing receptor-binding sites (affected by high-frequency antigenic mutations) and a stem region (relatively conserved across different influenza virus strains). Influenza HA is synthesized as a precursor (HA0), which undergoes proteolytic processing to produce two subunits (HA1 and HA2), which associate to form the stem / globular head structure. Viral HA is the most variable antigen on the virus (the 18 subtypes can be divided into two groups), but the stem (HA2) is highly conserved across these groups.

[0258] As used in this article, the term "influenza infection" is also known as "influenza," referring to a severe acute respiratory illness caused by the influenza virus. The term encompasses respiratory infection and symptoms, including high fever, headache, body aches, fatigue and weakness, and in some cases, extreme fatigue, nasal congestion, sneezing, sore throat, chest discomfort, cough, shortness of breath, bronchitis, pneumonia, and, in severe cases, death.

[0259] Exemplary Implementation

[0260] Implementation Method 1. A protein comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising an influenza virus hemagglutinin (HA) or a rabies G protein peptide or a fragment or epitope thereof linked to a C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an interpeptide disulfide bond.

[0261] Implementation Method 2. The protein according to Implementation Method 1, wherein the influenza virus is an influenza A virus or an influenza B virus, optionally wherein the influenza A virus belongs to the H1, H3 or H5 subtype, such as H1N1 or H3N2.

[0262] Implementation 3. The protein according to Implementation 1 or 2, wherein the epitope is a linear epitope or a conformational epitope.

[0263] Embodiment 4. The protein according to any one of Embodiments 1 to 3, wherein the HA protein peptide comprises HA1 subunit peptide, HA2 subunit peptide or any combination thereof, wherein the protein comprises three recombinant polypeptides.

[0264] Embodiment 5. The protein according to any one of Embodiments 1 to 4, wherein the HA protein peptide comprises a signal peptide, a stalk peptide, a degenerate esterase (VE) peptide, a receptor-binding domain (RBD) peptide, a fusion peptide (FP), a helical A peptide, a cyclic B peptide, a helical C peptide, a helical D peptide, a proximal membrane region (MPR) peptide, or any combination thereof.

[0265] Embodiment 6. The protein according to any one of Embodiments 1 to 5, wherein the HA protein peptide comprises the HA1 subunit or HA2 subunit of the HA protein.

[0266] Embodiment 7. The protein according to any one of Embodiments 1 to 6, wherein the HA protein peptide comprises an HA1 subunit and an HA2 subunit of an HA protein, optionally wherein the HA1 subunit and the HA2 subunit are linked by a disulfide bond or an artificially introduced linker.

[0267] Embodiment 8. The protein according to any one of Embodiments 1 to 7, wherein the HA protein peptide does not contain transmembrane (TM) domain peptides and / or cytoplasmic (CP) domain peptides.

[0268] Embodiment 9. The protein according to any one of Embodiments 1 to 8, wherein the HA protein peptide comprises a protease cleavage site, wherein the protease is optionally furin, a transmembrane serine protease such as TMPRSS2, trypsin, factor Xa, or cathepsin L.

[0269] Example 10. The protein according to any one of Examples 1 to 8, wherein the HA protein peptide does not contain a protease cleavage site, wherein the protease is optionally furin, a transmembrane serine protease such as TMPRSS2, trypsin, factor Xa, or cathepsin L.

[0270] Embodiment 11. The protein according to any one of Embodiments 1 to 10, wherein the HA or G protein peptide is soluble or does not bind directly to a lipid bilayer, such as a membrane or viral envelope.

[0271] Embodiment 12. The protein according to any one of Embodiments 1 to 11, wherein the HA or G protein peptides are the same or different among the recombinant polypeptides of the protein.

[0272] Embodiment 13. The protein according to any one of Embodiments 1 to 12, wherein the HA or G protein peptide is directly fused to the C-terminal propeptide, or linked to the C-terminal propeptide via a linker such as a linker comprising a glycine-XY repeating sequence, wherein X and Y are independently any amino acid, optionally proline or hydroxyproline.

[0273] Embodiment 14. The protein according to any one of Embodiments 1 to 13, wherein the protein is soluble.

[0274] Embodiment 15. The protein according to any one of Embodiments 1 to 14, wherein the protein does not bind directly to a lipid bilayer, such as a membrane or viral envelope.

[0275] Embodiment 16. A protein according to any one of Embodiments 1 to 15, wherein the protein is capable of binding to a cell surface attachment factor or receptor of a subject, optionally wherein the subject is a mammal, such as a primate, for example a human.

[0276] Embodiment 17. The protein according to any one of Embodiments 1 to 16, wherein the C-terminal propeptide is a human collagen.

[0277] Embodiment 18. The protein according to any one of Embodiments 1 to 17, wherein the C-terminal propeptide comprises a C-terminal polypeptide or fragment thereof of proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI) or proα3(XI).

[0278] Embodiment 19. The protein according to any one of Embodiments 1 to 18, wherein the C-terminal propeptide is the same or different among the recombinant polypeptides.

[0279] Example 20. The protein according to any one of Examples 1 to 19, wherein the C-terminal propeptide comprises any one of SEQ ID NO: 16-31 or has at least 90% identity with it, and is capable of forming interpeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0280] Example 21. The protein according to any one of Examples 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO:16 or has at least 90% identity with it, an amino acid sequence capable of forming interpeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0281] Example 22. The protein according to any one of Examples 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO:17 or has at least 90% identity with it, an amino acid sequence capable of forming interpeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0282] Example 23. The protein according to any one of Examples 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO:18 or has at least 90% identity with it, an amino acid sequence capable of forming interpeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0283] Example 24. The protein according to any one of Examples 1 to 20, wherein the C-terminal propeptide comprises an amino acid sequence of SEQ ID NO:19 or having at least 90% identity with it, capable of forming interpeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0284] Example 25. The protein according to any one of Examples 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO:20 or has at least 90% identity with it, an amino acid sequence capable of forming interpeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0285] Example 26. The protein according to any one of Examples 1 to 20, wherein the C-terminal propeptide comprises or has at least 90% identity with SEQ ID NO:21, an amino acid sequence capable of forming interpeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0286] Example 27. The protein according to any one of Examples 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO:22 or has at least 90% identity with it, an amino acid sequence capable of forming interpeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0287] Example 28. The protein according to any one of Examples 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO:23 or has at least 90% identity with it, an amino acid sequence capable of forming interpeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0288] Example 29. The protein according to any one of Examples 1 to 20, wherein the C-terminal propeptide comprises SEQ ID NO:24 or has at least 90% identity with it, an amino acid sequence capable of forming interpeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0289] Embodiment 30. The protein according to any one of Embodiments 1 to 29, wherein the C-terminal propeptide comprises a sequence containing a glycine-XY repeating sequence linked to the N-terminus of any one of SEQ ID NO: 16-31, wherein X and Y are independently any amino acid, optionally proline or hydroxyproline, or an amino acid sequence having at least 90% identity with them, capable of forming interpeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0290] Embodiment 31. The protein according to any one of Embodiments 1 to 30, wherein the HA protein peptide in each recombinant polypeptide is in a pre-fusion conformation or a post-fusion conformation.

[0291] Embodiment 32. The protein according to any one of Embodiments 1 to 31, wherein the HA protein peptide in each recombinant polypeptide comprises any one of SEQ ID NO: 7-9 or an amino acid sequence having at least 80% identity with it, and the G protein peptide in each recombinant polypeptide comprises any one of SEQ ID NO: 10-15 or an amino acid sequence having at least 80% identity with it.

[0292] Embodiment 33. The protein according to any one of Embodiments 1 to 32, wherein the recombinant polypeptide comprises any one of SEQ ID NO: 1-6 or has at least 80% identity with the amino acid sequence thereon.

[0293] Embodiment 34. An immunogen comprising a protein according to any one of Embodiments 1 to 33.

[0294] Embodiment 35. A protein nanoparticle comprising a protein according to any one of Embodiments 1 to 33, which is directly or indirectly connected to the nanoparticle.

[0295] Embodiment 36. A virus-like particle (VLP) comprising a protein according to any one of Embodiments 1 to 33.

[0296] Embodiment 37. An isolated nucleic acid, said isolated nucleic acid encoding one, two, three or more recombinant polypeptides of a protein according to any one of Embodiments 1 to 33.

[0297] Implementation Method 38. The isolated nucleic acid according to Implementation Method 37, wherein the polypeptide encoding the HA protein peptide is fused within a polypeptide frame to the polypeptide encoding the C-terminal propeptide of collagen.

[0298] Implementation 39. The isolated nucleic acid according to Implementation 37 or 38, wherein the isolated nucleic acid is operatively linked to a promoter.

[0299] Implementation Method 40. The isolated nucleic acid according to any one of Implementation Methods 37 to 39, wherein the isolated nucleic acid is a DNA molecule.

[0300] Implementation Method 41. The isolated nucleic acid according to any one of Implementation Methods 37 to 39, wherein the isolated nucleic acid is an RNA molecule, optionally an mRNA molecule such as nucleoside-modified mRNA, non-amplified mRNA, self-amplified mRNA, or trans-amplified mRNA.

[0301] Embodiment 42. A vector comprising isolated nucleic acid according to any one of Embodiments 37 to 41.

[0302] Implementation Method 43. The vector according to Implementation Method 42, wherein the vector is a viral vector.

[0303] Embodiment 44. A virus, pseudovirus, or cell comprising the vector according to Embodiment 42 or 43, optionally wherein the virus or cell has a recombinant genome.

[0304] Embodiment 45. An immunogenic composition comprising a protein, immunogen, protein nanoparticles, VLP, isolated nucleic acid, vector, virus, pseudovirus or cell, and pharmaceutically acceptable carrier according to any one of Embodiments 1 to 44.

[0305] Embodiment 46. A vaccine comprising the immunogenic composition according to Embodiment 45 and an optional adjuvant, wherein the vaccine is optionally a subunit vaccine, and / or wherein the vaccine is optionally a prophylactic and / or therapeutic vaccine.

[0306] Implementation method 47. The vaccine according to implementation method 46, wherein the vaccine comprises a variety of different adjuvants.

[0307] Embodiment 48. A method for producing a protein, the method comprising: expressing an isolated nucleic acid or vector according to any one of Embodiments 37 to 43 in a host cell to produce a protein according to any one of Embodiments 1 to 33; and purifying the protein.

[0308] Implementation 49. Protein produced according to the method described in Implementation 48.

[0309] Implementation 50. A method for generating an immune response in a subject to an HA protein peptide of an influenza virus or a fragment or epitope thereof, the method comprising administering to the subject an effective amount of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine according to any one of Implementations 1 to 47 and 49 to generate the immune response.

[0310] Implementation Method 51. The method according to Implementation Method 50, the method being used to treat or prevent influenza virus infection.

[0311] Implementation 52. The method according to implementation 50 or 51, wherein the immune response is generated by inhibiting or reducing the replication of influenza virus in the subject.

[0312] Embodiment 53. The method according to any one of Embodiments 50 to 52, wherein the immune response includes a cell-mediated response and / or a humoral response, optionally including the production of one or more neutralizing antibodies, such as polyclonal antibodies or monoclonal antibodies.

[0313] Implementation 54. The method according to any one of Implementations 50 to 53, wherein the immune response is directed against the HA protein peptide of the influenza virus or a fragment or epitope thereof, but not against the C-terminal propeptide.

[0314] Implementation 55. The method according to any one of Implementations 50 to 54, wherein the administration does not cause antibody-dependent enhancement (ADE) due to prior exposure of the subject to one or more influenza viruses.

[0315] Implementation 56. The method according to any one of Implementations 50 to 55, wherein the administration does not cause antibody-dependent enhancement (ADE) when the subject is subsequently exposed to one or more influenza viruses.

[0316] Implementation 57. The method according to any one of Implementations 50 to 56, the method further comprising an initial exemption step and / or an enhancement step.

[0317] Implementation Method 58. The method according to any one of Implementation Methods 50 to 57, wherein the administration step is performed by local, transdermal, subcutaneous, intradermal, oral, intranasal (e.g., intranasal spray), intratracheal, sublingual, buccal, rectal, vaginal, inhalation, intravenous (e.g., intravenous injection), intra-arterial, intramuscular (e.g., intramuscular injection), intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intra-articular, periarticular, local, or transepidermal administration.

[0318] Embodiment 59. The method according to any one of Embodiments 50 to 58, wherein the effective amount is administered as a single dose or as a series of doses spaced apart by one or more intervals.

[0319] Embodiment 60. The method according to any one of Embodiments 50 to 59, wherein the effective amount is administered without adjuvant.

[0320] Embodiment 61. The method according to any one of Embodiments 50 to 59, wherein the effective amount is administered together with an adjuvant.

[0321] Implementation Method 62. A method comprising administering to a subject an effective amount of a protein according to any one of Implementation Methods 1 to 33 to generate neutralizing antibodies or neutralizing antiserum against an influenza virus in the subject.

[0322] Implementation 63. The method according to implementation 62, wherein the subject is a mammal, optionally a human or a non-human primate.

[0323] Implementation 64. The method according to implementation 62 or 63, the method further comprising isolating the neutralizing antibody or neutralizing antiserum from the subject.

[0324] Implementation 65. The method according to Implementation 64, the method further comprising administering an effective amount of the isolated neutralizing antibody or neutralizing antiserum to a human subject via passive immunization to prevent or treat influenza virus infection.

[0325] Embodiment 66. The method according to any one of Embodiments 62 to 65, wherein the neutralizing antibody or neutralizing antiserum comprises a polyclonal antibody against the HA protein peptide or a fragment or epitope thereof, optionally wherein the neutralizing antibody or neutralizing antiserum contains no or substantially no antibody against the C-terminal propeptide of the collagen.

[0326] Embodiment 67. The method according to any one of Embodiments 62 to 65, wherein the neutralizing antibody comprises a monoclonal antibody against the HA protein peptide or a fragment or epitope thereof, optionally wherein the neutralizing antibody contains no or substantially no antibody against the C-terminal propeptide of the collagen.

[0327] Implementation Method 68. A protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine according to any one of Implementation Methods 1 to 47 and 49, used to induce an immune response against influenza virus in a subject and / or for the treatment or prevention of influenza virus infection.

[0328] Implementation 69. The use of any of the proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions or vaccines according to any one of Implementations 1 to 47 and 49, for inducing an immune response against an influenza virus in a subject, and / or for treating or preventing influenza virus infection.

[0329] Implementation 70. The use of any of the proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions or vaccines according to any one of Implementations 1 to 47 and 49 for the manufacture of a medicament or prophylactic agent for inducing an immune response against an influenza virus in a subject and / or for the treatment or prevention of influenza virus infection.

[0330] Embodiment 71. A method for analyzing a sample, the method comprising: contacting the sample with a protein according to any one of Embodiments 1 to 33, and detecting the binding between the protein and an analyte capable of specifically binding the HA protein peptide or a fragment or epitope thereof to the influenza virus.

[0331] Implementation 72. The method according to Implementation 71, wherein the analyte is an antibody, receptor, or cell that recognizes the HA protein peptide or a fragment or epitope thereof.

[0332] Implementation 73. The method according to Implementation 71 or 72, wherein the combination indicates the presence of the analyte in the sample and / or the subject from whom the sample was derived is infected with the influenza virus.

[0333] Embodiment 74. A kit comprising a protein according to any one of Embodiments 1 to 33 and a substrate, liner or vial for containing or immobilizing the protein, optionally wherein the kit is an ELISA or flow assay kit.

[0334] Example

[0335] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0336] Example 1: Generation of an exemplary influenza HA fusion protein

[0337] In-frame fusion of human α1(I) collagen C propeptide (trimeric tag) with the extracellular domain of H1 hemagglutinin (HA) resulted in high-level production of disulfide-linked soluble HA trimers in serum-free CHO cells. Following two-step purification, the resulting HA trimers not only folded appropriately into compact native homotrimers, as observed under negative-stain EM microscopy, but also retained high affinity for the broadly neutralizing antibody CR6261.

[0338] To generate an exemplary fusion protein containing HA, cDNA encoding amino acid residues 1-518 of the HA extracellular domain of the A / California / 07 / 2009-pdm(H1N1) virus (EpiFluDatabase accession number EPI516535) was synthesized using mouse preferred codon genes from GenScript, Inc. The cDNA was cloned into the Hind III and Bgl II sites of the pTRIMER expression vector (GenHunter, Inc.) to allow in-frame HA fusion (Liu et al., 2017, SciRep 7:8953).

[0339] The pTRIMER expression vector containing the HA extracellular domain coding sequence was transfected into the GH-CHO (dhfr- / -) cell line (GenHunter, USA) using FUGENE 6 (Roche, Mannheim, Germany) and grown in IMDM medium containing 10% FBS. After stepwise gene amplification with increasing MTX (Sigma) concentrations (0.0–0.5 μM), clones producing the highest exemplary fusion protein titer were adapted to SFM-4-CHO (Hyclone, Logan, UT, USA) serum-free medium. Exemplary fusion proteins were produced in 1 L shake flasks using a fed-batch process, with CellBoost 2 supplement (Hyclone) added every other day starting on day 3 until harvest on day 9. Cell density and viability, as well as the exemplary fusion protein titer, were monitored daily.

[0340] The cDNA template corresponding to residues 1-518 of HA in the A / California / 07 / 2009-pdm(H1N1) virus was cloned into the pTRIMER expression vector, allowing HA to fuse within the trimer tag frame. Figure 1 A). The transmembrane region and cytoplasmic tail of HA are excluded to facilitate the secretion of the antigenic extracellular domain into the cell culture medium. Following stepwise gene amplification with increasing methotrexate (MTX) concentration, CHO cells transfected with high-level expression clones of the exemplary fusion protein vector were screened and adapted to serum-free medium. The exemplary fusion protein was produced in serum-free medium using a fed-batch process over 9 days, achieving a cell density of over 7 million cells / mL and a cell viability exceeding 90%, after which the cells were harvested. Figure 1 B). An exemplary fusion protein titer is close to 200 mg / L, with very little contaminating cellular protein. Figure 1 C). Therefore, the soluble HA in trimer form was successfully expressed.

[0341] The exemplary fusion protein was purified from cell-free medium by centrifugation at 3000g for 20 min, followed by elution with a 5 mL blue agarose gel column (GE Healthcare, Logan, UT, USA) along a salt gradient (0.1–0.5 M NaCl). The fraction corresponding to the exemplary fusion protein was further processed by gel filtration using a Superdex 200 (GE Healthcare), with buffer changes according to the manufacturer's instructions, followed by ultrafiltration to concentrate in PBS for bioassays. The purity of the HA trimer was determined by reducing and non-reducing SDS-PAGE and SEC-HPLC (Sepax Zenix-C SEC 300).

[0342] Western blot analysis was performed on purified exemplary fusion proteins (0.2 μg) under reducing (+β-mercaptoethanol) or non-reducing (-β-mercaptoethanol) conditions using 10% SDS-PAGE with antibodies as described below, followed by goat anti-human IgG-HRP (Southern Biotech, Birmingham, AL, USA) or goat anti-mouse IgG-HRP (Southern Biotech, Birmingham, AL, USA). The reacting proteins were visualized using an ECL kit according to the manufacturer's protocol. The primary antibodies used for visualization were anti-HA CR6261 (ACRO Biosystems), anti-tag 12B11D11 (Clover Biopharmaceutical Co., Ltd., Chengdu, China), and anti-HA polyclonal mouse antiserum. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo-Fisher Scientific).

[0343] via SDS-PAGE ( Figure 2 A) and size exclusion high performance liquid chromatography (SEC-HPLC) Figure 2 B) The purity of the exemplary fusion protein was confirmed after two-step purification. Western blotting using HA and tag-specific antibodies confirmed the structural features and integrity of the exemplary fusion protein. Figure 2 A) The exemplary fusion protein exists essentially as a disulfide-linked homotrimer under non-reducing conditions.

[0344] Samples were prepared using a continuous carbon mesh method, where the mesh was made of nitrocellulose loaded with 400-mesh copper. 5 μL of sample (approximately 20 μg / mL protein) was applied to the cleaned mesh, blotted dry with filter paper, and immediately stained with 1% (w / w) uranyl formate. Images were recorded at 120,000x magnification on a 4096×4096 CCD (charge-coupled device) detector (FEI Eagle) with a Tecnai F20 electron microscope (FEI) operating at an accelerating voltage of 120 kV.

[0345] Negative staining electron microscopy (EM) confirmed that the exemplary fusion protein formed a compact homotrimer with a two-headed structure, consisting of a rod-shaped HA trimer at one end and a collagen C propeptide at the other. Figure 2 C). Unlike influenza viruses, which can induce hemagglutination due to the numerous HA trimer spikes on their surface that can bind to multiple red blood cells, the exemplary fusion protein does not induce hemagglutination, as predicted based on the monovalent properties of the structure confirmed by EM ( Figure 2 D).

[0346] The ability of HA to agglutinate red blood cells can be determined by a chicken red blood cell agglutination assay. Whole blood is thoroughly mixed with PBS, then centrifuged at 1500×g for 8 minutes at room temperature, and the supernatant is discarded. This process is then repeated three times. After absolute washing, 50 μL of 1% (v / v) chicken RBC suspension in PBS is added to 50 μL of purified HA trimer protein or influenza virus serially diluted in PBS in a U-bottom 96-well plate. After incubation at room temperature for 30 minutes, blood cell agglutination is recorded.

[0347] The affinity of bNAb CR6261 for binding to the exemplary fusion protein was assessed using biolayer interference (Octet) measurements (ForteBio). CR6261 (7.5 μg / mL) was immobilized on a protein A (ProA) biosensor (Pall). Real-time binding curves were measured by applying the sensor to a two-fold dilution series of the analyte in PBS. The concentration of the exemplary fusion protein ranged from 20 to 2.5 μg / mL. Kinetic parameters (Kp) were analyzed using Octet software version 9.0 (Pall). 缔合 and K 解离 ) and affinity. Using a 1:1 binding model of bNAb with an exemplary fusion protein, steady-state analysis was employed to determine the dissociation constant (Ka). D ).

[0348] Figure 2 The results shown in E indicate that the exemplary fusion protein formed an extremely tight complex with CR6261, with apparent K D Value <1.0E-12M. These results indicate that the exemplary fusion protein reproduces the bNAb epitope, thus accurately mimicking the conformation of natural HA.

[0349] An exemplary fusion protein purified was digested with peptide-N-glycosidase F (PNG enzyme F) to digest N-linked oligosaccharides, and the digestion products were analyzed on SDS-PAGE. Figure 2 The results shown in F indicate that the recombinant protein is extensively glycosylated by N-linked oligosaccharides, as evidenced by the visible changes in molecular weight. Glycosylation is known to be crucial for the biological function of HA, and the trimerized soluble HA produced in CHO cells is appropriately glycosylated.

[0350] Example 2: Functional characterization of an exemplary influenza HA fusion protein vaccine

[0351] Mice (BALB / c, female, 6-8 weeks old, n=6 / group) were vaccinated intramuscularly (im) in the hind leg on day 0, given a booster on day 21, and administered 1000×TCID10 on day 42. 50 A / California / 07 / 2009-pdm(H1N1) virus irritation ( Figure 3 A) Control animals were vaccinated using PBS. Each animal was vaccinated twice with either 1.5 μg of an exemplary fusion protein containing HA or 1.5 μg of the 2014-2015 quadrivalent inactivated influenza vaccine (QIV). All immunogens were mixed with the adjuvant preparation SigmaAdjuvant System (Sigma) at a 1:1 ratio. Blood was collected 14 days after each immunization, and serum was separated. Following viral challenge, animals were monitored daily for body temperature, weight loss, and decreased activity.

[0352] To measure the total antibody titer induced by the exemplary fusion protein, 96-well plates (Corning) were coated with 1 μg / mL of the exemplary fusion protein (100 μL / well), blocked with 1 mg / mL BSA (Roche), and then incubated with serially diluted antiserum. After three deep washes with PBST (PBS containing 0.05% Tween-20), the plates were incubated with goat anti-mouse IgG-HRP (Southern Biotech, Birmingham, AL, USA). The plates were washed three times with PBST, and the signal was visualized using TMB substrate (Thermo Scientific). The colorimetric reaction was terminated after 10 minutes by adding 2M HCl. Optical density (OD) was measured at 450 nm. The antibody titer of a given serum sample was defined as the reciprocal of the highest dilution, where its OD signal was up to twice that of the negative control. All immunized mice elicited a robust immune response with high serum HA-specific IgG antibodies, and the exemplary fusion protein titer was higher than that of the QIV group ( Figure 3 B) indicates that the exemplary fusion protein elicits excellent immunogenicity.

[0353] Serum samples were treated overnight at 37°C with receptor-destroying enzyme (RDE) (Sigma) to remove nonspecific agglutination inhibitors. Serum samples were serially diluted in U-bottom 96-well microtiter plates and then mixed with 25 μL (8 HAU / 50 μL) of virus for 30 minutes. A 1% suspension of chicken erythrocytes (RBCs) was then added. RBCs were allowed to settle at room temperature for 30 minutes, and the hemagglutinin inhibition (HI) titer was determined by the reciprocal of the final serum dilution that completely inhibited RBC hemagglutination. A negative titer was defined as 1:16. HI titers were tested to obtain viral neutralizing efficacy, which correlated well with total antibody levels against HA. Figure 3 C). The exemplary fusion protein vaccine group achieved an HI titer of 2048, which was sufficient to protect the animals from viral irritation; in contrast, all HA titers in the dummy vaccine mice were below the detection limit. Figure 3 C).

[0354] Serum neutralizing antibodies were determined using the micro-neutralization (MN) assay with the A / California / 07 / 2009-pdm (H1N1) virus. Martin-Darby canine kidney (MDCK) cells were seeded at 15,000 cells / well in 96-well plates. Two serially diluted RDE-treated sera were prepared in assay medium, mixed with the virus at 37°C for 1 hour, and then added to the MDCK cells to a final concentration of 100 × TCID⁻¹. 50 One virus / well. Cytopathic effect (CPE) was measured after 20 hours of incubation. Neutralizing titers of various antisera were measured using a micro-neutralization (MN) assay based on MDCK cells; serum from vaccinated mice showed robust neutralization against homologous influenza virus. Figure 3 D).

[0355] The plate was coated overnight at 4°C with 1 μg / mL of the exemplary fusion protein. After blocking with 1 mg / mL BSA and washing three times with PBST, the plate was incubated at room temperature for 1 hour with serially diluted mouse immune serum containing 100 ng / mL CR6261. After washing with PBST, 1:20000 diluted goat anti-human IgG-HRP (Southern Biotech, Birmingham, AL, USA) was added. After washing with PBST, TMB (Thermo Scientific) was added for signal visualization. The percentage of competition was calculated as follows: competition% = (AP) / A × 100), where A is the maximum OD signal of CR6261 binding to the exemplary fusion protein in the absence of serum, and P is the OD signal of CR6261 binding to the exemplary fusion protein in the presence of a given dilution of serum (Bommakanti et al., 2012, J Virol 86:13434-44). IC50 of a given serum sample 50The titer is defined as the reciprocal of the dilution at which the sample exhibits 50% competition.

[0356] Compared to the QIV vaccination group, the exemplary fusion protein-induced antiserum showed a higher level of competition for CR6261bNAb, consistent with the improved biophysical / biochemical properties of the immunogen. As a control, serum from mice simulating vaccination failed to compete with CR6261. The competition assay supports the presence of CR6261-like bNAb after immunization with the exemplary fusion protein. Figure 3 E).

[0357] These results demonstrate that the exemplary fusion protein containing the HA immunogen elicits high levels of HA antibodies, exhibits high immunogenicity, and shows promise as an influenza vaccine.

[0358] Following immunization, an in vivo mouse model was challenged with live virus. Lung tissue collected from the mice was fixed in 10% formalin and then embedded in paraffin. Sections (5 μm) were prepared and stained with hematoxylin and eosin (H&E). All tissue staining images were captured using an upright microscope (BX53, Olympus, Japan).

[0359] Such as changes in animal weight ( Figure 4 A) Survival rate Figure 4 B) Changes in body temperature Figure 4 C) and lung morphology ( Figure 4 As shown in D), the exemplary fusion protein containing HA provides high protection against the homologous H1N1 influenza virus. Mice that mimicked the vaccine died shortly after viral irritation, exhibiting a histopathological pattern of acute pneumonia and bronchopneumonia or bronchitis with abundant alveolar cavities filled with neutrophils, macrophages, and hyaline membranes; in contrast, no such histological abnormalities were observed in control mice or mice vaccinated with the exemplary fusion protein or QIV. Figure 4 D). The exemplary fusion protein provides complete protection against lethal irritation from homologous viral infection in mice and demonstrates a level of protective efficacy similar to that of a quadrivalent inactivated influenza vaccine.

[0360] Mice were vaccinated twice with the exemplary fusion protein or QIV vaccine, and serum was collected 42 days later. Serum IgG from each group of mice was purified by protein G column according to the manufacturer's instructions. Serum IgG transfer assays were performed to test whether HA-specific IgG induced by the exemplary fusion protein provided protection against lethal H1N1 virus exposure. Naïve mice received 1 mg / 200 μL of serum IgG or PBS (simulated group) from mice vaccinated with the exemplary fusion protein or QIV vaccine 24 hours prior to exposure to homologous H1N1 influenza virus. Mice in the control group were not exposed to the virus. Although mice in the simulated group died from infection, mice receiving serum IgG from mice vaccinated with the exemplary fusion protein or QIV vaccine were completely protected from infection, as evidenced by changes in animal weight (…). Figure 5 A) Survival rate Figure 5 B) and lung histopathology ( Figure 5 As shown in C), the efficacy of the exemplary fusion protein as an effective vaccine has been confirmed.

[0361] In summary, the exemplary fusion protein vaccine reproduces the epitopes of the natural HA antigen both in vitro and in vivo, and the trimer tag technology can provide a new platform for the rapid and safe production of recombinant subunit vaccines against influenza viruses.

[0362] A soluble exemplary fusion protein containing the A / California / 07 / 2009-pdm(H1N1) virus was produced in a CHO cell expression system (Liu et al., Sci Rep(7)8953, 2017). The exemplary fusion protein was secreted in its native form into serum-free cell culture medium with cell viability exceeding 90%, and was subsequently harvested, resulting in antigen titers and initial purity that were nearly 10 times higher than those of HA vaccines produced using insect cells (Wang et al., Vaccine(24)2176, 2006).

[0363] For downstream processes, the exemplary fusion protein is purified directly from cell-free culture medium to near homogeneity using only a two-step chromatographic method, without the need for detergent dissolution. Therefore, in some respects, the overall CMC process for the exemplary fusion protein is simpler and more scalable than producing HA antigens from insect cells. Once the influenza virus sequence is obtained, a soluble HA-encoding cDNA sequence can be rapidly synthesized. Subcloning this cDNA into the expression vector provided herein (as described in Example 1), followed by transfection, allows for the establishment of transfected cell lines within 4 weeks. Thus, a recombinant exemplary fusion protein vaccine can be produced within 100 days, potentially enabling timely response to any emerging pandemic.

[0364] Physical and chemical analyses of the highly purified exemplary fusion protein containing HA confirmed that the fusion protein not only existed as a disulfide-linked trimer easily identifiable by both non-reducing and reducing SDS-PAGE, but was also extensively glycosylated, with diols comprising approximately 10% of the total mass of the exemplary fusion protein. EM analysis showed that the exemplary fusion protein existed primarily in a compact two-headed structure, with a rod-shaped HA trimer at one end and a disulfide-linked trimer collagen C propeptide at the other end, consistent with the native structures of the two previously reported peptides (Sriwilalijaroen, Proc Jpn AcadSer B Phys Biol Sci (88) 226-249, 2012; Bouhrhis et al., Nat Struct Mol iol (19) 1031-1036, 2012).

[0365] In contrast to HA vaccines produced from insect cells that exist in a rosette form within heterogeneous oligomers and involve hemagglutination activity (Buckland et al., Vaccine(32)5496-5502, 2014), the exemplary fusion protein, as a single subunit vaccine, is structurally more homogeneous and therefore, as predicted, lacks hemagglutination activity. ForteBio Octet molecular interaction analysis revealed that the exemplary fusion protein binds to the K+ of bNAb CR6261. D Value <1.0E-12M, compared to recombinant HA produced from insect cells showing a binding strength 2-3 orders of magnitude weaker, K D The value is approximately 3.8E-9M. These comprehensive structural studies strongly support that the exemplary fusion protein reproduces the native HA trimer on the viral surface and retains bNAb epitopes like those in CR6261.

[0366] The efficacy of the exemplary fusion protein was investigated in a mouse model. Immunogenic efficacy was first measured at the humoral response level. All immunized mice elicited robust immune responses with high serum HA-specific antibodies, and the titers of the exemplary fusion protein group were higher than those of the commercially available QIV vaccine group, indicating that the exemplary fusion protein elicited superior immunogenicity compared to the conventional vaccine.

[0367] HI and MN assays are key parameters for evaluating influenza vaccine efficacy. The exemplary fusion protein induced high titers of HI and MN antibodies after immunization. Antiserum from mice immunized with the exemplary fusion protein showed a higher degree of competition for CR6261 compared to the QIV vaccination group, consistent with the improved biophysical / biochemical properties of the novel trimeric subunit immunogen. The competition assay indicated the presence of CR6261-like bNAb after immunization with the exemplary fusion protein. Vaccine efficacy was also quantified by measuring prophylaxis against morbidity and mortality in vivo after live virus infection. Following mouse immunization, the exemplary fusion protein demonstrated complete protection against irritation by autologous H1N1 virus. To further confirm the efficacy of the exemplary fusion protein as an effective vaccine, a passive transfer assay of serum IgG was performed. The results were consistent with the aforementioned irritation assays, indicating that HA-specific antibodies purified solely from vaccinated animals can provide complete protection against viral infection.

[0368] In summary, the exemplary fusion proteins containing HA maintained a conformation that accurately reproduced the native HA antigenic epitope both in vitro and in vivo.

[0369] Example 3: Generation of an exemplary rabies G fusion protein

[0370] The fusion of human α1(I) collagen C propeptide (trimeric tag) with the rabies G extracellular domain resulted in high levels of disulfide-linked soluble G trimers in serum-free CHO cells. Following two-step purification, the resulting G trimers not only folded appropriately to form trimers but also retained high affinity for the rabies G receptor, the nerve growth factor receptor NGFR (p75).

[0371] To generate an exemplary fusion protein containing the rabies G extracellular domain, cDNA encoding amino acid residues 1-458 (including the signal peptide) of the rabies CTN-1 or PM strain G protein was synthesized using mouse preferred codon genes from GenScript, Inc. The cDNA was cloned into the pTRIMER expression vector (GenHunter, Inc.) to allow in-frame fusion of the G extracellular domain and the trimer tag sequence. Figure 6 The transmembrane region and cytoplasmic tail of G are excluded to facilitate the secretion of the antigenic extracellular domain into the cell culture medium. The pTRIMER expression vector containing the coding sequence for the G extracellular domain was transfected into the GH-CHO(dhfr- / -) cell line (GenHunter Biosciences, USA). Figure 7Soluble G in trimer form was successfully expressed on SDS-PAGE under both non-reducing (-β-mercaptoethanol) and reducing (+β-mercaptoethanol) conditions. These results demonstrate that disulfide-linked soluble G trimers are correctly formed, and that when the disulfide bonds between polypeptide chains are broken under reducing conditions, the trimer decomposes into G trimer-tagged fusion peptide monomers of the desired molecular weight.

[0372] The affinity of the G trimer for binding to the receptor p75 was assessed using octet measurements (ForteBio). NGFR-Fc was immobilized on a protein A (ProA) biosensor (Pall). Real-time binding curves were measured by applying the sensor to a series of analyte dilutions in PBS. Kinetic parameters (K0) were analyzed. 缔合 and K 解离 ) and affinity (K D ). Figure 8 The results shown indicate that the CTN-1G trimer forms a tight complex with its receptor p75. These results demonstrate that the exemplary fusion protein mimics the native G protein conformation in trimer form.

[0373] Example 4: Functional characterization of an exemplary rabies G fusion protein vaccine

[0374] Mice were vaccinated with either the CTN-1 strain G-trimer antigen alone, the CTN-1 strain G-trimer with adjuvant 1, the CTN-1 strain G-trimer with adjuvant 2, a combination of the CTN-1 strain G-trimer with adjuvant 1 and adjuvant 2, or the CTN-1 strain G-trimer with adjuvant 3. Adjuvants 1 through 3 belong to three different classes of adjuvants, including aluminum hydroxide-based adjuvants, oligodeoxynucleotide-based adjuvants, and metabolizable oil-based adjuvants (e.g., squalene). Control animals were vaccinated with PBS-mimicked vaccines or with a commercially available inactivated rabies virus (HDCV) vaccine. Animals were vaccinated on day 0 / day 3 / day 7 (three doses), day 0 / day 3 (two doses), or day 0 (one dose), and blood was collected and serum separated 14 days after each immunization. The neurotrophin receptor (p75) was analyzed in mice immunized after one, two, and three doses of vaccination. NTR Competitive titers. Figure 9 The figure above shows the results of increasing the antigen dose (1 μg, 3 μg, and 10 μg) on ​​day 14 after three doses. Figure 9 The figure below shows the results for animals receiving one, two, and three doses. Because HDCV was administered three times, therefore... Figure 9The results in the figure below indicate that similar p75 competitive antibody titers can be achieved using only a single dose of CTN-1 strain G trimer with adjuvant 1 and / or adjuvant 2. Higher p75 competitive antibody titers can be achieved using only two doses of CTN-1 strain G trimer with adjuvant 1 or CTN-1 strain G trimer with adjuvant 1 and adjuvant 2 compared to three doses of HDCV. Another adjuvant, adjuvant 3, can also be used to induce a stronger neutralizing immune response than HDCV, such as... Figure 10 As shown.

[0375] In summary, the exemplary fusion protein containing a soluble G protein peptide mimics the conformation of the natural G protein in its trimer form. Furthermore, the soluble G trimer can induce a level of neutralizing immune response comparable to that of commercially available HDCV vaccines, even without any adjuvants, and the immune response can be further enhanced using a variety of adjuvants (alone or in combination).

[0376] This invention is not intended to be limited in scope to the specific disclosed embodiments, which are provided for the purpose of illustrating various aspects of the invention, for example. Various modifications to the compositions and methods will be apparent from the description and teachings herein. These changes may be made without departing from the true scope and spirit of this disclosure and are intended to fall within its scope.

[0377] sequence

[0378]

[0379]

[0380]

[0381] sequence list <110> Sichuan Clover Biopharmaceutical Co., Ltd. <120> Vaccine Compositions, Methods and Uses <130> 16576–20003.42 <140> Not yet allocated <141> Submitted at the same time <150> PCT / CN2021 / 087074 <151> 2021-04-13 <150> PCT / CN2020 / 095296 <151> 2020-06-10 <160> 31 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 812 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 1 Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr Val 1 5 10 15 Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn Leu 20 25 30 Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val Ala 35 40 45 Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly Asn 50 55 60 Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile Val 65 70 75 80 Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe Ile 85 90 95 Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe Glu 100 105 110 Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp Ser 115 120 125 Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser Phe 130 135 140 Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro Lys 145 150 155 160 Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val Leu 165 170 175 Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu Tyr 180 185 190 Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser Lys 195 200 205 Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln Glu 210 215 220 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys Ile 225 230 235 240 Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe Ala 245 250 255 Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro Val 260 265 270 His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr 275 280 285 Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys Pro 290 295 300 Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn 305 310 315 320 Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 325 330 335 Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His 340 345 350 His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr 355 360 365 Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu 370 375 380 Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu 385 390 395 400 Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu 405 410 415 Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 420 425 430 Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys 435 440 445 Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys 450 455 460 Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys 465 470 475 480 Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn 485 490 495 Arg Glu Glu Ile Asp Arg Ser Asn Gly Leu Pro Gly Pro Ile Gly Pro 500 505 510 Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pro 515 520 525 Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe 530 535 540 Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala His Asp Gly 545 550 555 560 Gly Arg Tyr Tyr Arg Ala Asn Asp Ala Asn Val Val Arg Asp Arg Asp 565 570 575 Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn 580 585 590 Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg 595 600 605 Asp Leu Lys Met Cys His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile 610 615 620 Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn 625 630 635 640 Met Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala 645 650 655 Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val 660 665 670 Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly 675 680 685 Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg 690 695 700 Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn 705 710 715 720 Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu 725 730 735 Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser 740 745 750 Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser His Thr Gly 755 760 765 Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys Thr Ser Arg 770 775 780 Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala Pro Asp Gln 785 790 795 800 Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu 805 810 <210> 2 <211> 829 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 2 Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn 1 5 10 15 Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val 50 55 60 Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly 65 70 75 80 Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp 130 135 140 Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser 145 150 155 160 Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro 165 170 175 Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val 180 185 190 Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu 195 200 205 Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser 210 215 220 Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln 225 230 235 240 Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys 245 250 255 Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe 260 265 270 Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro 275 280 285 Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn 290 295 300 Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys 305 310 315 320 Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg 325 330 335 Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly 340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr 355 360 365 His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser 370 375 380 Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile 385 390 395 400 Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His 405 410 415 Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe 420 425 430 Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn 435 440 445 Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu 450 455 460 Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly 465 470 475 480 Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val 485 490 495 Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu 500 505 510 Asn Arg Glu Glu Ile Asp Arg Ser Asn Gly Leu Pro Gly Pro Ile Gly 515 520 525 Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val Gly Pro 530 535 540 Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly 545 550 555 560 Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala His Asp 565 570 575 Gly Gly Arg Tyr Tyr Arg Ala Asn Asp Ala Asn Val Val Arg Asp Arg 580 585 590 Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu 595 600 605 Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys 610 615 620 Arg Asp Leu Lys Met Cys His Ser Asp Trp Lys Ser Gly Glu Tyr Trp 625 630 635 640 Ile Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val Phe Cys 645 650 655 Asn Met Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser Val 660 665 670 Ala Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg His 675 680 685 Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly 690 695 700 Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr Phe Leu 705 710 715 720 Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His Cys Lys 725 730 735 Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala 740 745 750 Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn 755 760 765 Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser His Thr 770 775 780 Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys Thr Ser 785 790 795 800 Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala Pro Asp 805 810 815 Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu 820 825 <210> 3 <211> 750 <212> PRT <213> Synthetic sequence <220> <223> Synthetic construct <400> 3 Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro 1 5 10 15 Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp 20 25 30 Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val 35 40 45 Gly Tyr Ile Ser Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val 50 55 60 Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr 65 70 75 80 Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ser 85 90 95 Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu 100 105 110 His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val Lys Thr Thr 115 120 125 Lys Glu Ser Val Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro 130 135 140 Tyr Asp Lys Ser Leu His Ser Arg Val Phe Pro Arg Gly Lys Cys Ser 145 150 155 160 Gly Ile Thr Val Ser Ser Ala Tyr Cys Ser Thr Asn His Asp Tyr Thr 165 170 175 Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Thr Ser Cys Asp Ile Phe 180 185 190 Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Lys Thr Cys Gly 195 200 205 Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys 210 215 220 Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp 225 230 235 240 Val Ala Ile Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Gln 245 250 255 Leu Val Asn Leu His Asp Phe His Ser Asp Glu Ile Glu His Leu Val 260 265 270 Val Glu Glu Leu Val Lys Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu 275 280 285 Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu 290 295 300 Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys 305 310 315 320 Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn 325 330 335 Glu Ile Ile Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His 340 345 350 Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp 355 360 365 Gly His Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His 370 375 380 Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Met His Pro Leu Ala 385 390 395 400 Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Val Glu Asp Phe Val 405 410 415 Glu Val His Leu Pro Asp Val His Lys Gln Val Ser Gly Val Asp Leu 420 425 430 Gly Leu Pro Asn Trp Gly Lys Arg Ser Asn Gly Leu Pro Gly Pro Ile 435 440 445 Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val Gly 450 455 460 Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala 465 470 475 480 Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala His 485 490 495 Asp Gly Gly Arg Tyr Tyr Arg Ala Asn Asp Ala Asn Val Val Arg Asp 500 505 510 Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser Gln Gln Ile 515 520 525 Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr 530 535 540 Cys Arg Asp Leu Lys Met Cys His Ser Asp Trp Lys Ser Gly Glu Tyr 545 550 555 560 Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val Phe 565 570 575 Cys Asn Met Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser 580 585 590 Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg 595 600 605 His Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr 610 615 620 Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr Phe 625 630 635 640 Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His Cys 645 650 655 Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys Lys 660 665 670 Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly 675 680 685 Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser His 690 695 700 Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys Thr 705 710 715 720 Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala Pro 725 730 735 Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu 740 745 750 <210> 4 <211> 769 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 4 Met Ile Pro Gln Ala Leu Leu Phe Val Pro Leu Leu Val Phe Pro Leu 1 5 10 15 Cys Phe Gly Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro 20 25 30 Trp Ser Pro Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val 35 40 45 Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu 50 55 60 Leu Lys Val Gly Tyr Ile Ser Ala Ile Lys Val Asn Gly Phe Thr Cys 65 70 75 80 Thr Gly Val Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr 85 90 95 Val Thr Thr Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala 100 105 110 Cys Arg Ser Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu 115 120 125 Glu Ser Leu His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val 130 135 140 Lys Thr Thr Lys Glu Ser Val Val Ile Ile Ser Pro Ser Val Ala Asp 145 150 155 160 Leu Asp Pro Tyr Asp Lys Ser Leu His Ser Arg Val Phe Pro Arg Gly 165 170 175 Lys Cys Ser Gly Ile Thr Val Ser Ser Ala Tyr Cys Ser Thr Asn His 180 185 190 Asp Tyr Thr Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Thr Ser Cys 195 200 205 Asp Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Lys 210 215 220 Thr Cys Gly Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly 225 230 235 240 Ala Cys Lys Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp 245 250 255 Gly Thr Trp Val Ala Ile Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro 260 265 270 Pro Asp Gln Leu Val Asn Leu His Asp Phe His Ser Asp Glu Ile Glu 275 280 285 His Leu Val Val Glu Glu Leu Val Lys Lys Arg Glu Glu Cys Leu Asp 290 295 300 Ala Leu Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu 305 310 315 320 Ser His Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile 325 330 335 Phe Asn Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg 340 345 350 Thr Trp Asn Glu Ile Ile Pro Ser Lys Gly Cys Leu Arg Val Gly Gly 355 360 365 Arg Cys His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu 370 375 380 Gly Pro Asp Gly His Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu 385 390 395 400 Gln Gln His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Met His 405 410 415 Pro Leu Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Val Glu 420 425 430 Asp Phe Val Glu Val His Leu Pro Asp Val His Lys Gln Val Ser Gly 435 440 445 Val Asp Leu Gly Leu Pro Asn Trp Gly Lys Arg Ser Asn Gly Leu Pro 450 455 460 Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly 465 470 475 480 Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro 485 490 495 Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu 500 505 510 Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala Asn Asp Ala Asn Val 515 520 525 Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser 530 535 540 Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro 545 550 555 560 Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His Ser Asp Trp Lys Ser 565 570 575 Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile 580 585 590 Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr 595 600 605 Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys 610 615 620 Asp Lys Arg His Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln 625 630 635 640 Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln 645 650 655 Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr 660 665 670 Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn 675 680 685 Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg 690 695 700 Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys 705 710 715 720 Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr 725 730 735 Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val 740 745 750 Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe 755 760 765 Leu <210> 5 <211> 750 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 5 Lys Phe Pro Ile Tyr Thr Ile Pro Asp Glu Leu Gly Pro Trp Ser Pro 1 5 10 15 Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp 20 25 30 Glu Gly Cys Thr Asn Leu Ser Glu Phe Ser Tyr Met Glu Leu Lys Val 35 40 45 Gly Tyr Ile Ser Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val 50 55 60 Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr 65 70 75 80 Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala 85 90 95 Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu 100 105 110 His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val Arg Thr Thr 115 120 125 Lys Glu Ser Leu Ile Ile Ile Ser Pro Ser Val Thr Asp Leu Asp Pro 130 135 140 Tyr Asp Lys Ser Leu His Ser Arg Gly Phe Pro Gly Gly Lys Cys Ser 145 150 155 160 Gly Ile Thr Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr 165 170 175 Ile Trp Met Pro Glu Asn Pro Gly Pro Arg Thr Pro Cys Asp Ile Phe 180 185 190 Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Asn Lys Thr Cys Gly 195 200 205 Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Arg 210 215 220 Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp 225 230 235 240 Val Ala Met Gln Thr Ser Asp Glu Thr Lys Trp Cys Pro Pro Asp Gln 245 250 255 Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu His Leu Val 260 265 270 Val Glu Glu Leu Val Lys Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu 275 280 285 Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu 290 295 300 Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys 305 310 315 320 Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn 325 330 335 Glu Ile Ile Pro Ser Lys Gly Cys Leu Lys Val Gly Gly Arg Cys His 340 345 350 Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp 355 360 365 Gly His Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His 370 375 380 Met Glu Leu Leu Lys Ser Ser Val Ile Pro Leu Met His Pro Leu Ala 385 390 395 400 Asp Pro Ser Thr Val Phe Lys Glu Gly Asp Glu Ala Glu Asp Phe Val 405 410 415 Glu Val His Leu Pro Asp Val Tyr Lys Gln Ile Ser Gly Val Asp Leu 420 425 430 Gly Leu Pro Asn Trp Gly Lys Arg Ser Asn Gly Leu Pro Gly Pro Ile 435 440 445 Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val Gly 450 455 460 Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala 465 470 475 480 Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala His 485 490 495 Asp Gly Gly Arg Tyr Tyr Arg Ala Asn Asp Ala Asn Val Val Arg Asp 500 505 510 Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser Gln Gln Ile 515 520 525 Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr 530 535 540 Cys Arg Asp Leu Lys Met Cys His Ser Asp Trp Lys Ser Gly Glu Tyr 545 550 555 560 Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val Phe 565 570 575 Cys Asn Met Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser 580 585 590 Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg 595 600 605 His Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr 610 615 620 Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr Phe 625 630 635 640 Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His Cys 645 650 655 Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys Lys 660 665 670 Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly 675 680 685 Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser His 690 695 700 Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys Thr 705 710 715 720 Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala Pro 725 730 735 Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu 740 745 750 <210> 6 <211> 769 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 6 Met Val Pro Gln Val Leu Leu Phe Val Pro Leu Leu Gly Phe Ser Leu 1 5 10 15 Cys Phe Gly Lys Phe Pro Ile Tyr Thr Ile Pro Asp Glu Leu Gly Pro 20 25 30 Trp Ser Pro Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val 35 40 45 Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Glu Phe Ser Tyr Met Glu 50 55 60 Leu Lys Val Gly Tyr Ile Ser Ala Ile Lys Val Asn Gly Phe Thr Cys 65 70 75 80 Thr Gly Val Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr 85 90 95 Val Thr Thr Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala 100 105 110 Cys Arg Ala Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu 115 120 125 Glu Ser Leu His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val 130 135 140 Arg Thr Thr Lys Glu Ser Leu Ile Ile Ile Ser Pro Ser Val Thr Asp 145 150 155 160 Leu Asp Pro Tyr Asp Lys Ser Leu His Ser Arg Gly Phe Pro Gly Gly 165 170 175 Lys Cys Ser Gly Ile Thr Val Ser Ser Thr Tyr Cys Ser Thr Asn His 180 185 190 Asp Tyr Thr Ile Trp Met Pro Glu Asn Pro Gly Pro Arg Thr Pro Cys 195 200 205 Asp Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Asn Lys 210 215 220 Thr Cys Gly Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly 225 230 235 240 Ala Cys Arg Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp 245 250 255 Gly Thr Trp Val Ala Met Gln Thr Ser Asp Glu Thr Lys Trp Cys Pro 260 265 270 Pro Asp Gln Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu 275 280 285 His Leu Val Val Glu Glu Leu Val Lys Lys Arg Glu Glu Cys Leu Asp 290 295 300 Ala Leu Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu 305 310 315 320 Ser His Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile 325 330 335 Phe Asn Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg 340 345 350 Thr Trp Asn Glu Ile Ile Pro Ser Lys Gly Cys Leu Lys Val Gly Gly 355 360 365 Arg Cys His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu 370 375 380 Gly Pro Asp Gly His Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu 385 390 395 400 Gln Gln His Met Glu Leu Leu Lys Ser Ser Val Ile Pro Leu Met His 405 410 415 Pro Leu Ala Asp Pro Ser Thr Val Phe Lys Glu Gly Asp Glu Ala Glu 420 425 430 Asp Phe Val Glu Val His Leu Pro Asp Val Tyr Lys Gln Ile Ser Gly 435 440 445 Val Asp Leu Gly Leu Pro Asn Trp Gly Lys Arg Ser Asn Gly Leu Pro 450 455 460 Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly 465 470 475 480 Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro 485 490 495 Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu 500 505 510 Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala Asn Asp Ala Asn Val 515 520 525 Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser 530 535 540 Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro 545 550 555 560 Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His Ser Asp Trp Lys Ser 565 570 575 Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile 580 585 590 Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr 595 600 605 Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys 610 615 620 Asp Lys Arg His Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln 625 630 635 640 Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln 645 650 655 Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr 660 665 670 Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn 675 680 685 Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg 690 695 700 Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys 705 710 715 720 Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr 725 730 735 Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val 740 745 750 Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe 755 760 765 Leu <210> 7 <211> 501 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 7 Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr Val 1 5 10 15 Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn Leu 20 25 30 Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val Ala 35 40 45 Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly Asn 50 55 60 Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile Val 65 70 75 80 Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe Ile 85 90 95 Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe Glu 100 105 110 Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp Ser 115 120 125 Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser Phe 130 135 140 Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro Lys 145 150 155 160 Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val Leu 165 170 175 Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu Tyr 180 185 190 Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser Lys 195 200 205 Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln Glu 210 215 220 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys Ile 225 230 235 240 Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe Ala 245 250 255 Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro Val 260 265 270 His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr 275 280 285 Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys Pro 290 295 300 Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn 305 310 315 320 Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 325 330 335 Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His 340 345 350 His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr 355 360 365 Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu 370 375 380 Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu 385 390 395 400 Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu 405 410 415 Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 420 425 430 Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys 435 440 445 Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys 450 455 460 Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys 465 470 475 480 Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn 485 490 495 Arg Glu Glu Ile Asp 500 <210> 8 <211> 518 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 8 Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn 1 5 10 15 Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val 50 55 60 Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly 65 70 75 80 Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp 130 135 140 Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser 145 150 155 160 Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro 165 170 175 Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val 180 185 190 Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu 195 200 205 Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser 210 215 220 Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln 225 230 235 240 Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys 245 250 255 Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe 260 265 270 Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro 275 280 285 Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn 290 295 300 Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys 305 310 315 320 Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg 325 330 335 Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly 340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr 355 360 365 His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser 370 375 380 Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile 385 390 395 400 Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His 405 410 415 Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe 420 425 430 Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn 435 440 445 Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu 450 455 460 Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly 465 470 475 480 Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val 485 490 495 Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu 500 505 510 Asn Arg Glu Glu Ile Asp 515 <210> 9 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 9 Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn 1 5 10 15 Ala <210> 10 <211> 439 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 10 Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro Trp Ser Pro 1 5 10 15 Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp 20 25 30 Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val 35 40 45 Gly Tyr Ile Ser Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val 50 55 60 Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr 65 70 75 80 Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ser 85 90 95 Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu 100 105 110 His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val Lys Thr Thr 115 120 125 Lys Glu Ser Val Val Ile Ile Ser Pro Ser Val Ala Asp Leu Asp Pro 130 135 140 Tyr Asp Lys Ser Leu His Ser Arg Val Phe Pro Arg Gly Lys Cys Ser 145 150 155 160 Gly Ile Thr Val Ser Ser Ala Tyr Cys Ser Thr Asn His Asp Tyr Thr 165 170 175 Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Thr Ser Cys Asp Ile Phe 180 185 190 Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Lys Thr Cys Gly 195 200 205 Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Lys 210 215 220 Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp 225 230 235 240 Val Ala Ile Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro Pro Asp Gln 245 250 255 Leu Val Asn Leu His Asp Phe His Ser Asp Glu Ile Glu His Leu Val 260 265 270 Val Glu Glu Leu Val Lys Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu 275 280 285 Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu 290 295 300 Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys 305 310 315 320 Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn 325 330 335 Glu Ile Ile Pro Ser Lys Gly Cys Leu Arg Val Gly Gly Arg Cys His 340 345 350 Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp 355 360 365 Gly His Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His 370 375 380 Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Met His Pro Leu Ala 385 390 395 400 Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Val Glu Asp Phe Val 405 410 415 Glu Val His Leu Pro Asp Val His Lys Gln Val Ser Gly Val Asp Leu 420 425 430 Gly Leu Pro Asn Trp Gly Lys 435 <210> 11 <211> 458 <212> PRT <213> artificial sequence <220> <223> composite structure <400> 11 Met Ile Pro Gln Ala Leu Leu Phe Val Pro Leu Leu Val Phe Pro Leu 1 5 10 15 Cys Phe Gly Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro 20 25 30 Trp Ser Pro Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val 35 40 45 Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu 50 55 60 Leu Lys Val Gly Tyr Ile Ser Ala Ile Lys Val Asn Gly Phe Thr Cys 65 70 75 80 Thr Gly Val Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr 85 90 95 Val Thr Thr Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala 100 105 110 Cys Arg Ser Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu 115 120 125 Glu Ser Leu His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val 130 135 140 Lys Thr Thr Lys Glu Ser Val Val Ile Ile Ser Pro Ser Val Ala Asp 145 150 155 160 Leu Asp Pro Tyr Asp Lys Ser Leu His Ser Arg Val Phe Pro Arg Gly 165 170 175 Lys Cys Ser Gly Ile Thr Val Ser Ser Ala Tyr Cys Ser Thr Asn His 180 185 190 Asp Tyr Thr Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Thr Ser Cys 195 200 205 Asp Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Lys 210 215 220 Thr Cys Gly Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly 225 230 235 240 Ala Cys Lys Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp 245 250 255 Gly Thr Trp Val Ala Ile Gln Thr Ser Asn Glu Thr Lys Trp Cys Pro 260 265 270 Pro Asp Gln Leu Val Asn Leu His Asp Phe His Ser Asp Glu Ile Glu 275 280 285 His Leu Val Val Glu Glu Leu Val Lys Lys Arg Glu Glu Cys Leu Asp 290 295 300 Ala Leu Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu 305 310 315 320 Ser His Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile 325 330 335 Phe Asn Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg 340 345 350 Thr Trp Asn Glu Ile Ile Pro Ser Lys Gly Cys Leu Arg Val Gly Gly 355 360 365 Arg Cys His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu 370 375 380 Gly Pro Asp Gly His Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu 385 390 395 400 Gln Gln His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Met His 405 410 415 Pro Leu Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Val Glu 420 425 430 Asp Phe Val Glu Val His Leu Pro Asp Val His Lys Gln Val Ser Gly 435 440 445 Val Asp Leu Gly Leu Pro Asn Trp Gly Lys 450 455 <210> 12 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 12 Met Ile Pro Gln Ala Leu Leu Phe Val Pro Leu Leu Val Phe Pro Leu 1 5 10 15 Cys Phe Gly <210> 13 <211> 439 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 13 Lys Phe Pro Ile Tyr Thr Ile Pro Asp Glu Leu Gly Pro Trp Ser Pro 1 5 10 15 Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val Val Glu Asp 20 25 30 Glu Gly Cys Thr Asn Leu Ser Glu Phe Ser Tyr Met Glu Leu Lys Val 35 40 45 Gly Tyr Ile Ser Ala Ile Lys Val Asn Gly Phe Thr Cys Thr Gly Val 50 55 60 Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr Val Thr Thr 65 70 75 80 Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala Cys Arg Ala 85 90 95 Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu Glu Ser Leu 100 105 110 His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val Arg Thr Thr 115 120 125 Lys Glu Ser Leu Ile Ile Ile Ser Pro Ser Val Thr Asp Leu Asp Pro 130 135 140 Tyr Asp Lys Ser Leu His Ser Arg Gly Phe Pro Gly Gly Lys Cys Ser 145 150 155 160 Gly Ile Thr Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr 165 170 175 Ile Trp Met Pro Glu Asn Pro Gly Pro Arg Thr Pro Cys Asp Ile Phe 180 185 190 Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Asn Lys Thr Cys Gly 195 200 205 Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly Ala Cys Arg 210 215 220 Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp Gly Thr Trp 225 230 235 240 Val Ala Met Gln Thr Ser Asp Glu Thr Lys Trp Cys Pro Pro Asp Gln 245 250 255 Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu His Leu Val 260 265 270 Val Glu Glu Leu Val Lys Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu 275 280 285 Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His Leu 290 295 300 Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile Phe Asn Lys 305 310 315 320 Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg Thr Trp Asn 325 330 335 Glu Ile Ile Pro Ser Lys Gly Cys Leu Lys Val Gly Gly Arg Cys His 340 345 350 Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu Gly Pro Asp 355 360 365 Gly His Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His 370 375 380 Met Glu Leu Leu Lys Ser Ser Val Ile Pro Leu Met His Pro Leu Ala 385 390 395 400 Asp Pro Ser Thr Val Phe Lys Glu Gly Asp Glu Ala Glu Asp Phe Val 405 410 415 Glu Val His Leu Pro Asp Val Tyr Lys Gln Ile Ser Gly Val Asp Leu 420 425 430 Gly Leu Pro Asn Trp Gly Lys 435 <210> 14 <211> 458 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 14 Met Val Pro Gln Val Leu Leu Phe Val Pro Leu Leu Gly Phe Ser Leu 1 5 10 15 Cys Phe Gly Lys Phe Pro Ile Tyr Thr Ile Pro Asp Glu Leu Gly Pro 20 25 30 Trp Ser Pro Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val 35 40 45 Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Glu Phe Ser Tyr Met Glu 50 55 60 Leu Lys Val Gly Tyr Ile Ser Ala Ile Lys Val Asn Gly Phe Thr Cys 65 70 75 80 Thr Gly Val Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr 85 90 95 Val Thr Thr Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala 100 105 110 Cys Arg Ala Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu 115 120 125 Glu Ser Leu His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val 130 135 140 Arg Thr Thr Lys Glu Ser Leu Ile Ile Ile Ser Pro Ser Val Thr Asp 145 150 155 160 Leu Asp Pro Tyr Asp Lys Ser Leu His Ser Arg Gly Phe Pro Gly Gly 165 170 175 Lys Cys Ser Gly Ile Thr Val Ser Ser Thr Tyr Cys Ser Thr Asn His 180 185 190 Asp Tyr Thr Ile Trp Met Pro Glu Asn Pro Gly Pro Arg Thr Pro Cys 195 200 205 Asp Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Asn Lys 210 215 220 Thr Cys Gly Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys Gly 225 230 235 240 Ala Cys Arg Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met Asp 245 250 255 Gly Thr Trp Val Ala Met Gln Thr Ser Asp Glu Thr Lys Trp Cys Pro 260 265 270 Pro Asp Gln Leu Val Asn Leu His Asp Phe Arg Ser Asp Glu Ile Glu 275 280 285 His Leu Val Val Glu Glu Leu Val Lys Lys Arg Glu Glu Cys Leu Asp 290 295 300 Ala Leu Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg Leu 305 310 315 320 Ser His Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile 325 330 335 Phe Asn Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val Arg 340 345 350 Thr Trp Asn Glu Ile Ile Pro Ser Lys Gly Cys Leu Lys Val Gly Gly 355 360 365 Arg Cys His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile Leu 370 375 380 Gly Pro Asp Gly His Val Leu Ile Pro Glu Met Gln Ser Ser Leu Leu 385 390 395 400 Gln Gln His Met Glu Leu Leu Lys Ser Ser Val Ile Pro Leu Met His 405 410 415 Pro Leu Ala Asp Pro Ser Thr Val Phe Lys Glu Gly Asp Glu Ala Glu 420 425 430 Asp Phe Val Glu Val His Leu Pro Asp Val Tyr Lys Gln Ile Ser Gly 435 440 445 Val Asp Leu Gly Leu Pro Asn Trp Gly Lys 450 455 <210> 15 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic constructs <400> 15 Met Val Pro Gln Val Leu Leu Phe Val Pro Leu Leu Gly Phe Ser Leu 1 5 10 15 Cys Phe Gly <210> 16 <211> 244 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 16 Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys 1 5 10 15 Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg 20 25 30 Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His Ser Asp 35 40 45 Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu 50 55 60 Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys Val 65 70 75 80 Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys 85 90 95 Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met Thr Asp 100 105 110 Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp Val 115 120 125 Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala Ser Gln 130 135 140 Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Gln 145 150 155 160 Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile 165 170 175 Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Val 180 185 190 Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu 195 200 205 Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val Ala Pro 210 215 220 Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro 225 230 235 240 Val Cys Phe Leu <210> 17 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 17 Arg Ser Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr 1 5 10 15 Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly 20 25 30 Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His 35 40 45 Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys 50 55 60 Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr 65 70 75 80 Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile 85 90 95 Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met 100 105 110 Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala 115 120 125 Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala 130 135 140 Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp 145 150 155 160 Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn 165 170 175 Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val 180 185 190 Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val 195 200 205 Ile Glu Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val 210 215 220 Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val 225 230 235 240 Gly Pro Val Cys Phe Leu 245 <210> 18 <211> 309 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 18 Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr 1 5 10 15 Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly 20 25 30 Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln 35 40 45 Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala Asn 50 55 60 Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr Leu 65 70 75 80 Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser 85 90 95 Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His Ser 100 105 110 Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn 115 120 125 Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys 130 135 140 Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser 145 150 155 160 Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met Thr 165 170 175 Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp 180 185 190 Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala Ser 195 200 205 Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp Gln 210 215 220 Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu 225 230 235 240 Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr 245 250 255 Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile 260 265 270 Glu Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val Ala 275 280 285 Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val Gly 290 295 300 Pro Val Cys Phe Leu 305 <210> 19 <211> 309 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 19 Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr 1 5 10 15 Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly 20 25 30 Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln 35 40 45 Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Asn Asp 50 55 60 Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr Leu 65 70 75 80 Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser 85 90 95 Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His Ser 100 105 110 Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn 115 120 125 Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys 130 135 140 Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser 145 150 155 160 Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met Thr 165 170 175 Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp 180 185 190 Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala Ser 195 200 205 Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp Gln 210 215 220 Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu 225 230 235 240 Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr 245 250 255 Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile 260 265 270 Glu Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val Ala 275 280 285 Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val Gly 290 295 300 Pro Val Cys Phe Leu 305 <210> 20 <211> 311 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic Construct <400> 20 Arg Ser Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly 1 5 10 15 Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro 20 25 30 Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu 35 40 45 Pro Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg 50 55 60 Ala Asn Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr 65 70 75 80 Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu 85 90 95 Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys 100 105 110 His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly 115 120 125 Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu 130 135 140 Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr 145 150 155 160 Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser 165 170 175 Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro 180 185 190 Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu 195 200 205 Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met 210 215 220 Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser 225 230 235 240 Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser 245 250 255 Val Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr 260 265 270 Val Ile Glu Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp 275 280 285 Val Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp 290 295 300 Val Gly Pro Val Cys Phe Leu 305 310 <210> 21 <211> 311 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 21 Gly Ser Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly 1 5 10 15 Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro 20 25 30 Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu 35 40 45 Pro Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg 50 55 60 Ala Asn Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr 65 70 75 80 Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu 85 90 95 Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys 100 105 110 His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly 115 120 125 Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu 130 135 140 Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr 145 150 155 160 Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser 165 170 175 Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro 180 185 190 Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu 195 200 205 Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met 210 215 220 Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser 225 230 235 240 Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser 245 250 255 Val Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr 260 265 270 Val Ile Glu Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp 275 280 285 Val Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp 290 295 300 Val Gly Pro Val Cys Phe Leu 305 310 <210> 22 <211> 244 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 22 Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys 1 5 10 15 Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg 20 25 30 Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His Ser Asp 35 40 45 Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu 50 55 60 Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys Val 65 70 75 80 Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys 85 90 95 Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met Thr Asp 100 105 110 Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp Val 115 120 125 Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala Ser Gln 130 135 140 Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Gln 145 150 155 160 Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Lys Gly Ser Asn Glu Ile 165 170 175 Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Val 180 185 190 Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu 195 200 205 Tyr Lys Thr Thr Lys Ser Ser Arg Leu Pro Ile Ile Asp Val Ala Pro 210 215 220 Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro 225 230 235 240 Val Cys Phe Leu <210> 23 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 23 Arg Ser Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr 1 5 10 15 Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly 20 25 30 Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His 35 40 45 Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys 50 55 60 Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr 65 70 75 80 Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile 85 90 95 Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met 100 105 110 Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala 115 120 125 Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala 130 135 140 Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp 145 150 155 160 Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Lys Gly Ser Asn 165 170 175 Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val 180 185 190 Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val 195 200 205 Ile Glu Tyr Lys Thr Thr Lys Ser Ser Arg Leu Pro Ile Ile Asp Val 210 215 220 Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val 225 230 235 240 Gly Pro Val Cys Phe Leu 245 <210> 24 <211> 309 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 24 Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr 1 5 10 15 Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly 20 25 30 Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln 35 40 45 Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala Asn 50 55 60 Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr Leu 65 70 75 80 Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser 85 90 95 Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His Ser 100 105 110 Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn 115 120 125 Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys 130 135 140 Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser 145 150 155 160 Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met Thr 165 170 175 Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp 180 185 190 Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala Ser 195 200 205 Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp Gln 210 215 220 Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Lys Gly Ser Asn Glu 225 230 235 240 Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr 245 250 255 Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile 260 265 270 Glu Tyr Lys Thr Thr Lys Ser Ser Arg Leu Pro Ile Ile Asp Val Ala 275 280 285 Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val Gly 290 295 300 Pro Val Cys Phe Leu 305 <210> 25 <211> 309 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 25 Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr 1 5 10 15 Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly 20 25 30 Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln 35 40 45 Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Asn Asp 50 55 60 Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr Leu 65 70 75 80 Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser 85 90 95 Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His Ser 100 105 110 Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn 115 120 125 Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys 130 135 140 Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser 145 150 155 160 Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met Thr 165 170 175 Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp 180 185 190 Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala Ser 195 200 205 Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp Gln 210 215 220 Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Lys Gly Ser Asn Glu 225 230 235 240 Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr 245 250 255 Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile 260 265 270 Glu Tyr Lys Thr Thr Lys Ser Ser Arg Leu Pro Ile Ile Asp Val Ala 275 280 285 Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val Gly 290 295 300 Pro Val Cys Phe Leu 305 <210> 26 <211> 311 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 26 Arg Ser Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly 1 5 10 15 Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro 20 25 30 Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu 35 40 45 Pro Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg 50 55 60 Ala Asn Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr 65 70 75 80 Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu 85 90 95 Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys 100 105 110 His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly 115 120 125 Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu 130 135 140 Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr 145 150 155 160 Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser 165 170 175 Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro 180 185 190 Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu 195 200 205 Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met 210 215 220 Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Lys Gly Ser 225 230 235 240 Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser 245 250 255 Val Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr 260 265 270 Val Ile Glu Tyr Lys Thr Thr Lys Ser Ser Arg Leu Pro Ile Ile Asp 275 280 285 Val Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp 290 295 300 Val Gly Pro Val Cys Phe Leu 305 310 <210> 27 <211> 311 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 27 Gly Ser Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly 1 5 10 15 Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro 20 25 30 Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu 35 40 45 Pro Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg 50 55 60 Ala Asn Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr 65 70 75 80 Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu 85 90 95 Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys 100 105 110 His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly 115 120 125 Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu 130 135 140 Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr 145 150 155 160 Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser 165 170 175 Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro 180 185 190 Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu 195 200 205 Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met 210 215 220 Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Lys Gly Ser 225 230 235 240 Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser 245 250 255 Val Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr 260 265 270 Val Ile Glu Tyr Lys Thr Thr Lys Ser Ser Arg Leu Pro Ile Ile Asp 275 280 285 Val Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp 290 295 300 Val Gly Pro Val Cys Phe Leu 305 310 <210> 28 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 28 Asp Glu Ile Met Thr Ser Leu Lys Ser Val Asn Gly Gln Ile Glu Ser 1 5 10 15 Leu Ile Ser Pro Asp Gly Ser Arg Lys Asn Pro Ala Arg Asn Cys Arg 20 25 30 Asp Leu Lys Phe Cys His Pro Glu Leu Lys Ser Gly Glu Tyr Trp Val 35 40 45 Asp Pro Asn Gln Gly Cys Lys Leu Asp Ala Ile Lys Val Phe Cys Asn 50 55 60 Met Glu Thr Gly Glu Thr Cys Ile Ser Ala Asn Pro Leu Asn Val Pro 65 70 75 80 Arg Lys His Trp Trp Thr Asp Ser Ser Ala Glu Lys Lys His Val Trp 85 90 95 Phe Gly Glu Ser Met Asp Gly Gly Phe Gln Phe Ser Tyr Gly Asn Pro 100 105 110 Glu Leu Pro Glu Asp Val Leu Asp Val Gln Leu Ala Phe Leu Arg Leu 115 120 125 Leu Ser Ser Arg Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser 130 135 140 Ile Ala Tyr Met Asp Gln Ala Ser Gly Asn Val Lys Lys Ala Leu Lys 145 150 155 160 Leu Met Gly Ser Asn Glu Gly Glu Phe Lys Ala Glu Gly Asn Ser Lys 165 170 175 Phe Thr Tyr Thr Val Leu Glu Asp Gly Cys Thr Lys His Thr Gly Glu 180 185 190 Trp Ser Lys Thr Val Phe Glu Tyr Arg Thr Arg Lys Ala Val Arg Leu 195 200 205 Pro Ile Val Asp Ile Ala Pro Tyr Asp Ile Gly Gly Pro Asp Gln Glu 210 215 220 Phe Gly Val Asp Val Gly Pro Val Cys Phe 225 230 <210> 29 <211> 244 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 29 Glu Pro Met Asp Phe Lys Ile Asn Thr Asp Glu Ile Met Thr Ser Leu 1 5 10 15 Lys Ser Val Asn Gly Gln Ile Glu Ser Leu Ile Ser Pro Asp Gly Ser 20 25 30 Arg Lys Asn Pro Ala Arg Asn Cys Arg Asp Leu Lys Phe Cys His Pro 35 40 45 Glu Leu Lys Ser Gly Glu Tyr Trp Val Asp Pro Asn Gln Gly Cys Lys 50 55 60 Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys 65 70 75 80 Ile Ser Ala Asn Pro Leu Asn Val Pro Arg Lys His Trp Trp Thr Asp 85 90 95 Ser Ser Ala Glu Lys Lys His Val Trp Phe Gly Glu Ser Met Asp Gly 100 105 110 Gly Phe Gln Phe Ser Tyr Gly Asn Pro Glu Leu Pro Glu Asp Val Leu 115 120 125 Asp Val Gln Leu Ala Phe Leu Arg Leu Leu Ser Ser Arg Ala Ser Gln 130 135 140 Asn Ile Thr Tyr His Cys Lys Asn Ser Ile Ala Tyr Met Asp Gln Ala 145 150 155 160 Ser Gly Asn Val Lys Lys Ala Leu Lys Leu Met Gly Ser Asn Glu Gly 165 170 175 Glu Phe Lys Ala Glu Gly Asn Ser Lys Phe Thr Tyr Thr Val Leu Glu 180 185 190 Asp Gly Cys Thr Lys His Thr Gly Glu Trp Ser Lys Thr Val Phe Glu 195 200 205 Tyr Arg Thr Arg Lys Ala Val Arg Leu Pro Ile Val Asp Ile Ala Pro 210 215 220 Tyr Asp Ile Gly Gly Pro Asp Gln Glu Phe Gly Val Asp Val Gly Pro 225 230 235 240 Val Cys Phe Leu <210> 30 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 30 Ser Glu Pro Met Asp Phe Lys Ile Asn Thr Asp Glu Ile Met Thr Ser 1 5 10 15 Leu Lys Ser Val Asn Gly Gln Ile Glu Ser Leu Ile Ser Pro Asp Gly 20 25 30 Ser Arg Lys Asn Pro Ala Arg Asn Cys Arg Asp Leu Lys Phe Cys His 35 40 45 Pro Glu Leu Lys Ser Gly Glu Tyr Trp Val Asp Pro Asn Gln Gly Cys 50 55 60 Lys Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr 65 70 75 80 Cys Ile Ser Ala Asn Pro Leu Asn Val Pro Arg Lys His Trp Trp Thr 85 90 95 Asp Ser Ser Ala Glu Lys Lys His Val Trp Phe Gly Glu Ser Met Asp 100 105 110 Gly Gly Phe Gln Phe Ser Tyr Gly Asn Pro Glu Leu Pro Glu Asp Val 115 120 125 Leu Asp Val Gln Leu Ala Phe Leu Arg Leu Leu Ser Ser Arg Ala Ser 130 135 140 Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Ile Ala Tyr Met Asp Gln 145 150 155 160 Ala Ser Gly Asn Val Lys Lys Ala Leu Lys Leu Met Gly Ser Asn Glu 165 170 175 Gly Glu Phe Lys Ala Glu Gly Asn Ser Lys Phe Thr Tyr Thr Val Leu 180 185 190 Glu Asp Gly Cys Thr Lys His Thr Gly Glu Trp Ser Lys Thr Val Phe 195 200 205 Glu Tyr Arg Thr Arg Lys Ala Val Arg Leu Pro Ile Val Asp Ile Ala 210 215 220 Pro Tyr Asp Ile Gly Gly Pro Asp Gln Glu Phe Gly Val Asp Val Gly 225 230 235 240 Pro Val Cys Phe Leu 245 <210> 31 <211> 246 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 31 Arg Ser Glu Pro Met Asp Phe Lys Ile Asn Thr Asp Glu Ile Met Thr 1 5 10 15 Ser Leu Lys Ser Val Asn Gly Gln Ile Glu Ser Leu Ile Ser Pro Asp 20 25 30 Gly Ser Arg Lys Asn Pro Ala Arg Asn Cys Arg Asp Leu Lys Phe Cys 35 40 45 His Pro Glu Leu Lys Ser Gly Glu Tyr Trp Val Asp Pro Asn Gln Gly 50 55 60 Cys Lys Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu 65 70 75 80 Thr Cys Ile Ser Ala Asn Pro Leu Asn Val Pro Arg Lys His Trp Trp 85 90 95 Thr Asp Ser Ser Ala Glu Lys Lys His Val Trp Phe Gly Glu Ser Met 100 105 110 Asp Gly Gly Phe Gln Phe Ser Tyr Gly Asn Pro Glu Leu Pro Glu Asp 115 120 125 Val Leu Asp Val Gln Leu Ala Phe Leu Arg Leu Leu Ser Ser Arg Ala 130 135 140 Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Ile Ala Tyr Met Asp 145 150 155 160 Gln Ala Ser Gly Asn Val Lys Lys Ala Leu Lys Leu Met Gly Ser Asn 165 170 175 Glu Gly Glu Phe Lys Ala Glu Gly Asn Ser Lys Phe Thr Tyr Thr Val 180 185 190 Leu Glu Asp Gly Cys Thr Lys His Thr Gly Glu Trp Ser Lys Thr Val 195 200 205 Phe Glu Tyr Arg Thr Arg Lys Ala Val Arg Leu Pro Ile Val Asp Ile 210 215 220 Ala Pro Tyr Asp Ile Gly Gly Pro Asp Gln Glu Phe Gly Val Asp Val 225 230 235 240 Gly Pro Val Cys Phe Leu 245

Claims

1. A trimeric fusion protein, which is a trimeric fusion protein formed by the in-frame fusion of soluble influenza virus surface antigen and the C-terminal portion of collagen linked by disulfide bonds, wherein the sequence of the fusion protein is shown in SEQ ID NO:1 or SEQ ID NO:

2.

2. Use of the trimeric fusion protein according to claim 1 in the preparation of a recombinant subunit vaccine for the prevention of H1N1 influenza virus infection in mammals.

3. The use according to claim 2, wherein the recombinant subunit vaccine is suitable for administration by intramuscular injection.

4. The use according to claim 2, wherein the recombinant subunit vaccine is suitable for administration by intranasal spray.

5. The use according to any one of claims 2 to 4, wherein the recombinant subunit vaccine is suitable for administration in a single dose or in a series of doses spaced apart at intervals of weeks or months.

6. The use according to any one of claims 2 to 4, wherein the recombinant subunit vaccine is adjuvant-free.

7. The use according to any one of claims 2 to 4, wherein the recombinant subunit vaccine comprises an adjuvant.

8. The use according to any one of claims 2 to 4, wherein the recombinant subunit vaccine comprises more than one adjuvant.

9. Use of the trimer fusion protein of claim 1 in the preparation of a reagent used in a method for detecting antibodies against influenza A virus H1N1 from mammalian serum, the method comprising the step of contacting the serum with the trimer fusion protein.

10. The use of the trimeric fusion protein according to claim 1 in the preparation of a recombinant subunit vaccine, wherein the recombinant subunit vaccine is used to prepare neutralizing antibodies in patients infected with influenza A virus H1N1 via passive immunization, wherein the preparation comprises: Neutralizing antibodies produced by purification in immune mammals.

11. The use according to claim 10, wherein the neutralizing antibody comprises a polyclonal antibody.

12. The use according to claim 10, wherein the neutralizing antibody is a monoclonal antibody.

13. Use of the trimeric fusion protein according to claim 1 in the preparation of a recombinant subunit vaccine for the prevention of H1N1 influenza virus infection in poultry or pigs.

14. The use according to claim 13, wherein the recombinant subunit vaccine is suitable for administration by intramuscular injection.

15. The use according to claim 13, wherein the recombinant subunit vaccine is suitable for administration by intranasal spray.

16. The use according to any one of claims 13 to 15, wherein the recombinant subunit vaccine is suitable for administration in a single dose or in a series of doses spaced apart at intervals of weeks or months.

17. The use according to any one of claims 13 to 15, wherein the recombinant subunit vaccine is adjuvant-free.

18. The use according to any one of claims 13 to 15, wherein the recombinant subunit vaccine comprises an adjuvant.

19. The use according to any one of claims 13 to 15, wherein the recombinant subunit vaccine comprises more than one adjuvant.

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