RSV vaccine compositions, methods, and uses thereof

Abstract

The present invention provides an immunogenic composition comprising recombinant peptides and proteins, the recombinant peptides and proteins comprising respiratory syncytial virus (RSV) viral antigens and immunogens, such as RSV F protein peptides. The immunogenic composition comprises a secreted fusion protein comprising a soluble RSV viral antigen, which is linked to the collagen C-terminal portion by in-frame fusion to form a disulfide-linked trimeric fusion protein. The immunogenic composition can be used to generate an immune response, such as for treating or preventing RSV infection. The immunogenic composition can be used in vaccine compositions, such as as part of a preventive and / or therapeutic vaccine. Also provided herein are methods for producing the recombinant peptides and proteins, prevention, treatment and / or diagnostic methods, and related kits.

Description

RSV vaccine compositions, methods, and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of International Patent Application No. PCT / CN2020 / 095295 filed on June 10, 2020, and International Patent Application No. PCT / CN2021 / 087045 filed on April 13, 2021, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0003] Sequence listings submitted as ASCII text files

[0004] The following content submitted in the form of an ASCII text file is incorporated herein by reference in its entirety: Computer Readable Form (CRF) of the Sequence Listing (file name: 165762000242SEQLIST.TXT, recording date: June 9, 2021, size: 229KB). Technical Field

[0005] The present disclosure relates in some aspects to an immunogenic composition comprising recombinant peptides and proteins comprising respiratory syncytial virus (RSV) viral antigens and immunogens, such as RSV F protein peptides, for use in treating and / or preventing RSV infection. Background Art

[0006] Respiratory syncytial virus (RSV) causes respiratory tract infections in adults and children and is a major cause of lower respiratory tract infections and hospitalizations in infancy and childhood. Although RSV infection rates are high, treatment options, including prophylactics, therapeutics, and vaccines, are limited or unavailable. Improved methods are needed to treat RSV. Provided herein are compositions, methods, uses, and products that meet these and other needs. Summary of the Invention

[0007] In one aspect, the present invention provides a protein comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising a respiratory syncytial virus (RSV) F protein peptide or a fragment or epitope thereof connected to a collagen C-terminal propeptide, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond. In some embodiments, the RSV belongs to subtype A or subtype B. In some embodiments, the epitope is a linear epitope or a conformational epitope.

[0008] In some embodiments, disclosed herein are recombinant subunit vaccines comprising an extracellular domain (e.g., without a transmembrane domain and a cytoplasmic domain) of an RSV F protein or a fragment thereof, the extracellular domain being fused in-frame to a collagen C propeptide capable of forming a disulfide-linked homotrimer. The resulting recombinant subunit vaccine, such as an F trimer, can be expressed and purified from transfected cells and is expected to be in a natural-like conformation in the form of a trimer. This solves the misfolding problem often encountered when viral antigens are expressed as recombinant peptides or proteins in a soluble form without a transmembrane domain and / or a cytoplasmic domain. Such misfolded viral antigens cannot accurately retain the conformation of the native viral antigen and often fail to elicit neutralizing antibodies.

[0009] In some embodiments, the F protein peptide comprises an F1 subunit peptide, an F2 subunit peptide, or any combination thereof, and the protein comprises three recombinant polypeptides. In some embodiments, the F protein peptide comprises a signal peptide, a heptad repeat C (HRC) peptide, a pep27 peptide, a fusion peptide (FP), a heptad repeat A (HRA) peptide, a domain I peptide, a domain II peptide, or a heptad repeat B (HRB) peptide, or any combination thereof. In some embodiments, the F protein peptide comprises the F1 subunit of the F protein but does not comprise the F2 subunit, or vice versa. In some embodiments, the F protein peptide comprises the F1 subunit and the F2 subunit of the F protein, optionally in the absence of pep27, and optionally wherein the F1 subunit and the F2 subunit are connected by a disulfide bond or an artificially introduced linker. In some embodiments, the F protein peptide does not comprise a transmembrane (TM) domain peptide and / or a cytoplasmic (CP) domain peptide. In some embodiments, the protein F peptide comprises a protease cleavage site, wherein the protease is optionally furin, trypsin, factor Xa, thrombin, or cathepsin L. In some embodiments, the protein F peptide does not comprise a protease cleavage site, wherein the protease is optionally furin, trypsin, factor Xa, thrombin, or cathepsin L.

[0010] In some embodiments, the F protein peptide is soluble or does not directly bind to a lipid bilayer, such as a membrane or viral envelope. In some embodiments, the F protein peptide is the same or different among the recombinant polypeptides of the protein. In some embodiments, the F protein peptide is fused directly to the C-terminal propeptide or is linked to the 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.

[0011] In some embodiments, the protein is soluble or does not directly bind to a lipid bilayer, such as a membrane or viral envelope. In some embodiments, the protein is capable of forming a rosette-like oligomer comprising an F protein peptide trimer. In some embodiments, the protein is capable of binding to a cell surface attachment factor or receptor in a subject, optionally wherein the subject is a mammal, such as a primate, e.g., a human.

[0012] In some embodiments, the C-terminal propeptide belongs to human collagen. In some embodiments, the C-terminal propeptide comprises the C-terminal polypeptide of proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI) or proα3(XI) or a fragment thereof. In some embodiments, the C-terminal propeptides are the same or different between the recombinant polypeptides. In some embodiments, the C-terminal propeptide comprises any one of SEQ ID NO:48-63 or an amino acid sequence having at least 90% identity thereto, capable of forming an inter-polypeptide disulfide bond and allowing the recombinant polypeptide to trimerize.

[0013] In some embodiments, the F protein peptide in each recombinant polypeptide is in a pre-fusion conformation or a post-fusion conformation, optionally wherein the protein comprises a rosette-like oligomer comprising a crutch-shaped rod-shaped F protein peptide trimer. In any of the above embodiments, the F protein peptide in each recombinant polypeptide can comprise any one of SEQ ID NOs: 17-47 or an amino acid sequence having at least 80% identity thereto.

[0014] In any of the above embodiments, the recombinant polypeptide may comprise any one of SEQ ID NOs: 1-16, or an amino acid sequence at least 80% identical thereto. In any of the above embodiments, the recombinant polypeptide may comprise any one of SEQ ID NOs: 17-47, or an amino acid sequence at least 80% identical thereto, linked directly or indirectly to any one of SEQ ID NOs: 48-63, or an amino acid sequence at least 90% identical thereto.

[0015] Also provided herein is an immunogen comprising a protein as provided herein. Provided herein is a protein nanoparticle comprising a protein as provided herein directly or indirectly attached to a nanoparticle. Provided herein is a virus-like particle (VLP) comprising a protein as provided herein.

[0016] This paper also provides a kind of isolated nucleic acid, the protein 1,2,3 or more recombinant polypeptides that described isolated nucleic acid encoding this paper provides.In some embodiments, the polypeptide of coding F protein peptide and the polypeptide frame of coding collagen protein C-terminal propeptide merge.In some embodiments, the isolated nucleic acid that this paper provides is operably connected to promotor.

[0017] In some embodiments, the isolated nucleic acid provided herein is a DNA molecule. In some embodiments, the isolated nucleic acid provided herein is an RNA molecule, optionally an mRNA molecule, such as a nucleoside-modified mRNA, a non-amplified mRNA, a self-amplified mRNA, or a trans-amplified mRNA.

[0018] Also provided herein is a vector comprising the isolated nucleic acid provided herein.In some embodiments, the vector is a viral vector.

[0019] In some aspects, a virus, pseudovirus or cell comprising a vector as provided herein is provided herein, optionally wherein the virus or cell has a recombinant genome. In some aspects, an immunogenic composition is provided herein, comprising a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus or cell as provided herein, and a pharmaceutically acceptable carrier.

[0020] Also provided herein is a vaccine comprising the immunogenic composition provided herein and an optional adjuvant, wherein the vaccine is optionally a subunit vaccine. In some embodiments, the vaccine is a prophylactic and / or therapeutic vaccine.

[0021] In some aspects, the present invention provides a method for producing a protein, comprising: expressing the isolated nucleic acid or vector provided herein in a host cell to produce the protein provided herein; and purifying the protein. The present invention provides a protein produced by the method provided herein.

[0022] Provided herein is a method for generating an immune response to the F protein peptide of RSV or its fragment or epitope in a subject, 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 as provided herein to generate the immune response. In some embodiments, the method provided herein is used to treat or prevent RSV infection. In some embodiments, generating an immune response inhibits or reduces the replication of RSV in the subject. In some embodiments, the immune response comprises a cell-mediated response and / or a humoral response, optionally comprising generating one or more neutralizing antibodies, such as polyclonal antibodies or monoclonal antibodies. In some embodiments, the immune response is directed against the F protein peptide of RSV or its fragment or epitope, but not against the C-terminal propeptide. In some embodiments, administration to the subject will not result in antibody-dependent enhancement (ADE) due to exposure to one or more RSVs before the subject. In some embodiments, when the subject is subsequently exposed to one or more RSVs, the administration will not result in antibody-dependent enhancement (ADE). In some embodiments, the method further comprises a primary immunization step and / or a booster step. In some embodiments, the administering step is performed topically, transdermally, subcutaneously, intradermally, orally, intranasally (e.g., intranasal spray), intratracheally, sublingually, buccally, rectally, vaginally, by inhalation, intravenously (e.g., intravenous injection), intraarterially, intramuscularly (e.g., intramuscular injection), intracardially, intraosseously, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, topically, or epidermally. In some embodiments, the effective amount is administered as a single dose or in a series of doses separated by one or more intervals. In some embodiments, the effective amount is administered without an adjuvant. In some embodiments, the effective amount is administered with an adjuvant.

[0023] This article provides a method, which includes administering an effective amount of the protein provided herein to a subject to produce neutralizing antibodies or neutralizing antiserum for RSV in the subject. In some embodiments, the subject is a mammal, optionally a human or non-human primate. In some embodiments, the method also includes isolating the neutralizing antibody or neutralizing antiserum from the subject. In some embodiments, the method also includes administering an effective amount of isolated neutralizing antibody or neutralizing antiserum to a human subject by passive immunization to prevent or treat RSV infection. In some embodiments, neutralizing antibodies or neutralizing antiserum for RSV comprise polyclonal antibodies to RSV F protein peptides or fragments or epitopes thereof, optionally wherein the neutralizing antibodies or neutralizing antiserum do not contain or are substantially free of antibodies to collagen C-terminal propeptide. In some embodiments, the neutralizing antibodies comprise monoclonal antibodies to RSV F protein peptides or fragments or epitopes thereof, optionally wherein the neutralizing antibodies do not contain or are substantially free of antibodies to collagen C-terminal propeptide.

[0024] In some aspects, the proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions, or vaccines provided herein are used to induce an immune response to RSV in a subject and / or to treat or prevent RSV infection.

[0025] In some respects, the purposes of protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, carrier, virus, pseudovirus, cell, immunogenic composition or vaccine that this paper provides are provided herein, for inducing the immunne response to RSV and / or for treating or preventing RSV infection in a subject. In some respects, the purposes of protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, carrier, virus, pseudovirus, cell, immunogenic composition or vaccine that this paper provides are provided herein, for preparing medicine or prophylactic agent, and described medicine or prophylactic agent are used for inducing the immunne response to RSV virus and / or for treating or preventing RSV infection in a subject.

[0026] This paper also provides the method for analyzing sample, described method comprises: sample is contacted with the protein that this paper provides, and detects described protein and can specifically bind to the F protein peptide of RSV or its fragment or the combination between the analyte of epi-position.In some embodiments, described analyte is the antibody, receptor or cell that identifies F protein peptide or its fragment or epi-position.In some embodiments, described combination shows that there is RSV infection in the experimenter in described analyte and / or described sample source in described sample.

[0027] Provided herein is a kit comprising a protein provided herein and a substrate, pad or vial containing or immobilizing the protein, optionally wherein the kit is an ELISA or lateral flow assay kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figures 1A-1C show the expression level and purification of exemplary fusion peptides comprising RSV F protein peptides. Figure 1A shows a schematic diagram of an exemplary fusion peptide comprising an extracellular F domain fused to a trimerization peptide. Figure 1B shows an 8% SDS-PAGE analysis of the expression of an exemplary fusion peptide in serum-free fed-batch cell culture. 10 mL of cell-free conditioned medium from day 1 to day 12 was separated under non-reducing conditions and then stained with Coomassie blue. Figure 1C shows the purity assessment of the exemplary fusion peptide by SEC-HPLC, based on OD 280 The main peak area of ​​the exemplary protein was detected to be 94.6%.

[0029] Figures 2A-2C show the characterization of exemplary purified fusion peptides comprising RSV F protein peptides. Figure 2A shows SDS-PAGE and Western blot analysis of exemplary fusion peptides comprising RSV F protein peptides under non-reducing and reducing conditions. 2 μg of purified protein was loaded to be stained with Coomassie blue by 8% SDS-PAGE, and 0.1 μg of purified protein was loaded to be Western blotted using antibodies specific to F and collagen propeptide, respectively. Figure 2B shows a negative staining electron micrograph of an exemplary fusion peptide protein comprising RSV F protein peptides, which exhibits a cane-shaped molecule that is in the form of an individual or mostly a rosette-like oligomer. Examples of individual and rosette-like molecules are shown below, with their structural diagrams attached. Figure 2C shows a binding study conducted by biolayer interferometry of palivizumab and an exemplary fusion peptide comprising RSV F protein peptides. First, 5 μg / mL palivizumab was fixed to a protein A sensor, and then the sensor was immersed in exemplary fusion peptides of varying concentrations to measure binding kinetics. The resulting curve was fitted to a 1:1 binding model by subtracting the buffer reference value to obtain K 缔合 and K 解离 The values ​​are shown in the table below. The resulting K values ​​for palivizumab and an exemplary fusion peptide comprising a RSV F protein peptide D Less than 1 picomolar.

[0030] Figures 3A-3E show that immunization with an exemplary fusion peptide comprising RSV F protein peptide protects against RSV infection. Figure 3A shows a schematic diagram of the experimental method: mice were immunized on day 0 and day 21, and then challenged intranasally (in) with RSV after collecting serum on day 49. Figure 3B shows serum anti-F IgG ELISA titers for purified exemplary fusion peptides comprising RSV F protein peptide. Figure 3C is a virus microneutralization assay, showing serum neutralizing antibody titers that provide 50% inhibition for CPE formation infected with RSV. Figure 3D shows the RSV titers obtained in the lungs of immunized mice by plaque assay 5 days after challenge. The values ​​represent the number of plaques per gram of lung tissue. Figure 3E shows the competitive IgG titers for palivizumab that provide 50% inhibition for the binding of palivizumab to heat-inactivated RSV (HI-RSV) particles, as measured by serum sample dilutions. Values ​​are expressed as log2 and mean ± SEM.

[0031] Figure 4 shows that vaccination with an exemplary fusion peptide comprising an RSV F protein peptide protects against vaccine-induced disease enhancement. Lung tissue collected 5 days after challenge was fixed in 10% neutral buffered formalin, embedded in paraffin, sliced ​​at 5 μm, stained with H&E, and photographed at 200x magnification. DETAILED DESCRIPTION

[0032] In some embodiments, compositions and methods of use of 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 native-like trimeric viral antigen and can therefore be used as a more effective vaccine against these dangerous pathogens.

[0033] In some embodiments, disclosed herein are methods of preventing viral infection using viral antigen trimers as a vaccine or as part of a multivalent vaccine, with or without an adjuvant or with more than one adjuvant, optionally by intramuscular injection or intranasal administration.

[0034] In some embodiments, disclosed herein is a method for diagnosing viral infection using viral antigen trimers as antigens by detecting antibodies, such as IgM or IgG, such as neutralizing antibodies, that recognize the viral antigens.

[0035] In some embodiments, disclosed herein are 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 RSV infection in infants.

[0036] In some embodiments, disclosed herein is a viral antigen trimer as a vaccine or as part of a multivalent vaccine, wherein the vaccine comprises multiple trimeric subunit vaccines, wherein the multiple trimeric subunit vaccines comprise viral antigens of the same viral protein or viral antigens of two or more different proteins of one or more viruses or one or more strains of the same virus.

[0037] In some embodiments, disclosed herein is a monovalent vaccine comprising a viral antigen trimer disclosed herein. In some embodiments, disclosed herein is a bivalent vaccine comprising a viral antigen trimer disclosed herein. In some embodiments, disclosed herein is a trivalent vaccine comprising a viral antigen trimer disclosed herein. In some embodiments, disclosed herein is a tetravalent vaccine comprising a viral antigen trimer disclosed herein.

[0038] In some embodiments, disclosed herein is a monovalent vaccine comprising an F trimer disclosed herein. In some embodiments, disclosed herein is a bivalent vaccine comprising an F trimer disclosed herein. In some embodiments, disclosed herein is a bivalent vaccine comprising at least one F trimer comprising a first F protein antigen and at least one F trimer comprising a second F protein antigen. In some embodiments, the first and second F protein antigens are derived from the same F protein from one or more viral species, strains, or subtypes, or from two or more different F proteins from one or more viral species, or from one or more strains or subtypes of the same viral species. In some embodiments, disclosed herein is a trivalent vaccine comprising an F trimer disclosed herein. In some embodiments, disclosed herein is a trivalent vaccine comprising at least one F trimer comprising a first F protein antigen, at least one F trimer comprising a second F protein antigen, and at least one F trimer comprising a third F protein antigen. In some embodiments, the first, second, and third F protein antigens are derived from the same F protein from one or more viral species, strains, or subtypes, or from two, three, or more different F proteins from one or more viral species, or from one or more strains or subtypes of the same viral species. In some embodiments, disclosed herein is a tetravalent vaccine comprising an F trimer disclosed herein. In some embodiments, disclosed herein is a tetravalent vaccine comprising at least one F trimer comprising a first F protein antigen, at least one F trimer comprising a second F protein antigen, at least one F trimer comprising a third F protein antigen, and at least one F trimer comprising a fourth F protein antigen. In some embodiments, the first, second, third, and fourth F protein antigens are derived from the same F protein of one or more viral species or strains / subtypes, or from two, three, four, or more different F proteins of one or more viral species or one or more strains / subtypes of the same viral species.

[0039] Provided herein are immunogenic compositions, methods, and uses of fusion peptides and proteins comprising RSV viral antigens or immunogens for, e.g., prophylactic or therapeutic treatment of RSV infection. Respiratory syncytial virus (RSV) is considered a leading cause of acute lower respiratory tract infections (ALRTI) in infants and young children, killing 7,000 to 20,000 more children worldwide each year. RSV infection is the second most common cause of death in infants in developing countries. In addition, RSV can cause severe disease in the elderly and immunocompromised populations. Despite the disease burden caused by RSV, there is currently no approved vaccine. The effective prophylactic humanized mAb palivizumab It should only be used as a passive immunization measure for infants at high risk of RSV infection.

[0040] Despite decades of research, RSV vaccine development has been unsuccessful for a variety of reasons. For example, production, stability, and efficacy issues of RSV vaccine candidates have been difficult to overcome. In particular, safety is a major concern due to the recognition that formalin-inactivated RSV (FI-RSV) vaccines mediate vaccine-induced disease enhancement (VED).

[0041] The protein that comprises RSV viral antigen and immunogen provided herein, comprises recombinant polypeptide and fusion protein, can be used for effectively and safely treating (for example therapeutically, prophylactically) RSV and infect.For example, the protein therapy RSV that comprises RSV viral antigen and immunogen provided herein infects, without considering VED and / or antibody-dependent enhancement (ADE).In addition, the protein that comprises RSV viral antigen and immunogen provided herein is easy to produce, and shows stability under high stress conditions such as high temperature, extreme pH and hyperosmotic pressure and hypoosmotic pressure.Therefore, the protein that this paper provided and immunogenic composition circumvent and meet the production, stability, safety and effectiveness problem that hinder RSV vaccine development.

[0042] In some aspects, the RSV viral antigens and immunogens provided herein comprise RSV glycoprotein (F), also referred to herein as RSV F protein peptide or peptide. RSV F protein peptide is a homotrimeric type I transmembrane protein that mediates membrane and viral penetration into host cells. RSV F protein peptide is synthesized as F0 proprotein precursor, which is converted into disulfide-linked F1 and F2 mature forms after being cleaved by furin at two sites. RSV F protein peptide is highly conserved between RSV A and B strains. Neutralizing antibodies such as palivizumab target the antigenic site of F, and provide protection for respiratory diseases caused by RSV infection.

[0043] In some embodiments, the protein comprising RSV viral antigens or immunogens, such as RSV F protein peptides, can produce an immune response, such as an immune response to RSV F peptide protein. In some embodiments, the immune response suppresses or reduces the duplication of RSV in a subject, such as a patient. In some embodiments, the immune response comprises producing one or more neutralizing antibodies, such as polyclonal and / or monoclonal antibodies. In some embodiments, the neutralizing antibodies suppress or reduce the duplication of RSV in a subject, such as a patient. In some embodiments, the subject is administered protein (such as in the form of an immunogenic composition) and will not cause antibody-dependent enhancement (ADE) due to prior exposure to RSV. In some respects, the protein comprising RSV viral antigens and immunogens, such as RSV F protein peptides, is used as a vaccine.

[0044] In some embodiments, RSV viral antigen and immunogen, for example RSV F protein peptides, are connected to protein or peptide to form fusion rotein or recombinant polypeptide.In some embodiments, the protein to which RSV viral antigen or immunogen are connected or peptide can be associated with protein or peptide, such as protein or peptide of fusion rotein or recombinant polypeptide, for example covalently or non-covalently connected.Therefore, in some cases, the protein to which RSV viral antigen or immunogen are connected or peptide are multimerization domains.

[0045] In some embodiments, RSV viral antigens and immunogens, such as RSV F protein peptides, are connected to collagen propeptides, such as collagen propeptide C-terminus, to form fusion peptides or recombinant polypeptides. Therefore, in some embodiments, the protein provided herein comprises a recombinant polypeptide containing RSV viral antigens and immunogens, such as RSV F protein peptides or fragments or epitopes thereof, connected to collagen C-terminal propeptides. In some embodiments, the collagen propeptides are derived from human α1 collagen C propeptides and are capable of self-trimerization.

[0046] In some embodiments, RSV viral antigens and immunogens, such as RSV F protein peptides are connected to collagen propeptide, such as collagen propeptide C-terminal, contribute to the ability of protein to produce an immune response. For example, the production of recombinant protein can retain the tertiary and quaternary structures of RSV F protein peptides, which may be important to the stability of the natural conformation of RSV F protein peptides, and then to the accessibility of antigenic sites on the surface of the protein (such as neutralizing antibodies) that can induce an immune response. In addition, RSV F protein peptides are connected to proteins or peptides that can self-trimerize, allowing recombinant protein to assemble, thereby simulating the natural homotrimeric structure of the RSV F protein peptides on the viral envelope.

[0047] In some embodiments, RSV F protein peptide is connected to collagen C-terminal propeptide to produce self-trimerized recombinant polypeptide.In some embodiments, protein provided herein comprises propeptide of multiple self-trimerized RSV F protein peptide and collagen recombinant polypeptide, optionally wherein said multiple recombinant protein forms the structure of for example rosette (referring to for example Fig. 2 B).In some embodiments, the trimeric characteristic of recombinant protein contributes to the stability of protein.In some embodiments, the macrostructure (for example rosette) of multiple self-trimerization recombinant proteins contributes to the stability of protein.In some embodiments, the trimeric characteristic of recombinant protein and the macrostructure (for example rosette) of multiple self-trimerization recombinant proteins contribute to the stability of protein.In some embodiments, the trimeric characteristic of recombinant protein contributes to the ability of protein to produce immune response.In some embodiments, the macrostructure (for example rosette) of multiple self-trimerization recombinant proteins contributes to the ability of protein to produce immune response.In some embodiments, the trimeric characteristic of recombinant protein and the macrostructure (for example rosette) of multiple self-trimerization recombinant proteins contribute to the ability of protein to produce immune response.

[0048] Also provided herein are immunogenic compositions comprising the proteins provided herein, methods of producing the proteins provided herein, methods of treating subjects with the proteins and compositions provided herein, and kits.

[0049] All publications, including patent documents, scientific papers, and databases, mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. To the extent that definitions set forth herein are contrary to or inconsistent with definitions set forth in patents, applications, published applications, and other publications incorporated by reference herein, the definitions set forth herein take precedence over those incorporated by reference herein.

[0050] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0051] I. Viral Antigens and Immunogens

[0052] Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infection in infants and young children and represents a major burden of disease in the elderly. Despite being characterized half a century ago, there is currently no vaccine against RSV, and development efforts have been hampered by vaccine-mediated disease enhancement in children administered formalin-inactivated RSV in the 1960s. Challenges in antigen production, purity, stability, and potency of RSV vaccine candidates have also been obstacles to development.

[0053] In some embodiments, the protein provided herein comprises RSV viral antigens and / or immunogens. In some embodiments, RSV viral antigens and / or immunogens can promote or stimulate cell-mediated response and / or humoral response. In some embodiments, response (such as cell-mediated response or humoral response) includes producing antibodies, such as neutralizing antibodies. In some embodiments, neutralizing antibodies (NAb) directed against viral antigens and / or immunogens provide adaptive immune defense for RSV exposure by blocking the infection of susceptible cells. In some embodiments, the efficacy of vaccines for several viruses is owing to and / or is relevant to the ability of their induction of NAb. In some embodiments, RSV viral antigens or immunogens are RSV F protein peptides disclosed herein.

[0054] The RSV F protein peptide is an envelope glycoprotein of respiratory syncytial virus (RSV). The RSV F protein peptide is translated into a single precursor polypeptide (designated F0). The RSV F protein mediates viral entry into cells and cell-cell fusion, is the target of neutralizing antibodies, and is highly conserved between RSV A and B strains. F0 can be cleaved by cellular furin at Arg109 and Arg136 into three fragments. The shorter F2 polypeptide is covalently linked to the longer F1 polypeptide via two disulfide bonds at the N-terminus. The latter has an 18-amino acid fusion domain at the N-terminus and a hydrophobic transmembrane region near the C-terminus; the central 27-amino acid fragment is released. The neutralizing monoclonal antibodies palivizumab and motavizumab bind to RSV F antigenic site II (Asn258-Val278) and have been shown to protect against RSV disease in the lower and upper respiratory tracts in high-risk and full-term infants. The structures of the RSV F epitope polypeptides that bind these neutralizing antibodies are larger than linear peptides, with palivizumab binding to RSV F with nanomolar affinity and motavizumab binding to RSV F with picomolar affinity. Modeling predicts that full binding of palivizumab and motavizumab requires amino acids from one or two RSV F protomers, respectively. Therefore, preserving RSV F tertiary and quaternary structures may be very important in developing RSV F vaccines that retain the native conformation of this important neutralizing region.

[0055] In some embodiments, the F0 precursor polypeptide is 574 amino acids in length, as shown in SEQ ID NO:31.

[0056]

[0057] In some embodiments, the F0 precursor polypeptide is 574 amino acids in length, as shown in SEQ ID NO:32.

[0058]

[0059] In some embodiments, the RSV F protein peptide herein comprises a proline or alanine at residue 102. In some embodiments, the RSV F protein peptide herein comprises a replacement, deletion, and / or insertion at residue 102 and / or near residue 102 of SEQ ID NO:31 or 32. In some embodiments, the RSV F protein peptide herein comprises a glutamic acid or alanine at residue 218. In some embodiments, the RSV F protein peptide herein comprises a replacement, deletion, and / or insertion at residue 218 and / or near residue 218 of SEQ ID NO:31 or 32. In some embodiments, the RSV F protein peptide herein comprises a valine or isoleucine at residue 379. In some embodiments, the RSV F protein peptide herein comprises a replacement, deletion, and / or insertion at residue 379 and / or near residue 31 of SEQ ID NO:31 or 32. In some embodiments, the RSV F protein peptide herein comprises a valine or methionine at residue 447. In some embodiments, the RSV F protein peptides herein comprise substitutions, deletions, and / or insertions at and / or near residue 447 of SEQ ID NO:31 or 32. In some embodiments, the RSV F protein peptides herein comprise substitutions, deletions, and / or insertions at and / or near any one or more of the proline or alanine at residue 102, the glutamic acid or alanine at residue 218, the valine or isoleucine at residue 379, and the valine or methionine at residue 447. In some embodiments, the RSV F protein peptides herein comprise substitutions, deletions, and / or insertions at and / or near any one or more of residues 102, 218, 379, and 447 of SEQ ID NO:31 or 32. In some embodiments, the RSV F protein peptides herein comprise substitutions, deletions, and / or insertions at and / or near any one or more of the other residues of SEQ ID NO:31 or 32.

[0060] In some embodiments, the RSV F protein peptides herein comprise substitutions, deletions, and / or insertions at and / or near residues 106, 107, 108, and / or 109 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein comprise glutamine or asparagine at residues 108 and / or 109. In some embodiments, the RSV F protein peptides herein comprise glutamine at residues 108 and 109. In some embodiments, the RSV F protein peptides herein comprise asparagine at residues 108 and 109. In some embodiments, the RSV F protein peptides herein comprise substitutions, deletions, and / or insertions at and / or near residues 131, 132, 133, 134, 135, and / or 136 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein comprise glycine, arginine, glutamine, or asparagine at and / or near residues 131, 132, 133, 134, 135, and / or 136 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein comprise glutamine at residues 131, 132, 133, 134, 135, and / or 136 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein comprise glutamine at residues 133, 135, and 136 of SEQ ID NO: 31 or 32.

[0061] In some embodiments, the RSV F protein peptides herein comprise substitutions, deletions, and / or insertions at and / or near residues 109, 136, 161, and / or 215 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein comprise an alanine or proline at any one or more of residues 109, 136, 161, and / or 215 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein comprise an alanine at residue 109 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein comprise an alanine at residue 136 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein comprise an alanine at residues 109 and 136 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein comprise a proline at residue 161 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein comprise a proline at residue 215 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein comprise a proline at residues 161 and 215 of SEQ ID NO: 31 or 32. In some embodiments, the RSV F protein peptides herein comprise alanine at residues 109 and 136 and proline at residues 161 and 215 of SEQ ID NO: 31 or 32.

[0062] In some embodiments, the RSV F protein peptide herein comprises a replacement, deletion, and / or insertion of any one or more of and / or near residues 131-154 of SEQ ID NO:31 or 32. In some embodiments, the RSV F protein peptide herein comprises a deletion of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more of residues 131-154 of SEQ ID NO:31 or 32. In some embodiments, the RSV F protein peptide herein comprises a deletion of any one or more of residues 137-154 of SEQ ID NO:31 or 32. In some embodiments, the RSV F protein peptide herein comprises a deletion of any one or more of residues 137-146 of SEQ ID NO:31 or 32. In some embodiments, the RSV F protein peptides herein comprise glutamine at residues 133, 135, and 136 of SEQ ID NO: 31 or 32 and a deletion of residues 137-146.

[0063] In some embodiments, the RSV F protein peptide herein comprises amino acids 1-25 of the F0 precursor, which is the signal peptide MELLILKANAITTILTAVTFCFASG (SEQ ID NO: 33). In some embodiments, the precursor polypeptide F0 forms a precursor trimer. In some embodiments, the RSV F protein peptide herein is hydrolyzed and cleaved by one or more cellular proteases, for example, at a conserved furin consensus cleavage site, to produce Pep 27 polypeptides (also referred to as p27), F1 polypeptides, and F2 polypeptides. In some embodiments, the Pep 27 polypeptides (e.g., amino acids 110-136 of the F0 precursor) are excised and, in some aspects, do not become part of a mature RSV F trimer. In some embodiments, the F2 polypeptide (alternatively referred to herein as "F2" or "F2 subunit peptide") comprises amino acid residues 26-109 of the F0 precursor. In some embodiments, the F1 polypeptide (alternatively referred to herein as "F1" or "F1 subunit peptide") comprises amino acid residues 137-574 of the F0 precursor and can include an extracellular region (e.g., residues 137-524), a transmembrane domain (e.g., residues 525-550), and a cytoplasmic domain (e.g., residues 551-574).

[0064] In some embodiments, the RSV F protein peptide herein comprises F1 and F2 polypeptides, which are connected by a disulfide bond to form a heterodimer, referred to as RSV F "protomer". In some embodiments, the RSV F protein peptide herein comprises three protomers that form an RSV F trimer, so it is a homotrimer of the three protomers. In some embodiments, the RSV F protein peptide herein is a mature RSV F trimer. In some embodiments, the RSV F protein peptide herein is membrane-bound. In some embodiments, the RSV F protein peptide herein is not membrane-bound. In some embodiments, the RSV F protein peptide herein is soluble and lacks a transmembrane region and a cytoplasmic region or a fragment thereof. For example, conversion to a soluble form can be achieved by truncating the RSV F protein at amino acid 513 (by removing the amino acid starting at 514), 514, 515, 516, 517, 518, 519, 520, 521, 522, 523 or 524. In nature, ripe RSV F trimer mediation virus and cell membrane fusion.Mature RSV F tripolymer fusion pre-conformation (this paper can be referred to as " pre-F " or before fusion) highly unstable (metastable) of RSV F trimer.But, once RSV virus and cell membrane dock after, RSV F protein trimer just carries out a series of conformational changes and transitions to highly stable fusion after (" post-F ") conformation.

[0065] In some embodiments, the RSV viral antigen or immunogen comprises a signal peptide (SP) (e.g., amino acids 1-22 of SEQ ID NO: 31 or 32) or a fragment and / or mutant sequence thereof, a heptad repeat C (HRC) (e.g., F2, which can be amino acids 23-109 of SEQ ID NO: 31 or 32) or a fragment and / or mutant sequence thereof, a furin cleavage site (FCS) (e.g., at the junction between amino acids 109 / 110 of SEQ ID NO: 31 or 32) or a fragment and / or mutant sequence thereof, a 27-mer fragment (pep27) (e.g., amino acids 110-136 of SEQ ID NO: 31 or 32) or a fragment and / or mutant sequence thereof, a putative fusion peptide (FP) (e.g., amino acids 137-155 of SEQ ID NO: 31 or 32) or a fragment and / or mutant sequence thereof, a heptad repeat A (HRA) (e.g., amino acids 156-214 of SEQ ID NO: 31 or 32) or a fragment and / or mutant sequence thereof, domains I and II (e.g., SEQ ID NO: 31 or 32) or fragments and / or mutant sequences thereof, heptad repeat sequence B (HRB) (e.g., amino acids 477-524 of SEQ ID NO: 31 or 32) or fragments and / or mutant sequences thereof, transmembrane (TM) domain (e.g., amino acids 525-550 of SEQ ID NO: 31 or 32) or fragments and / or mutant sequences thereof, and / or any suitable combination of cytoplasmic (CP) domain (e.g., amino acids 551-574 of SEQ ID NO: 31 or 32) or fragments and / or mutant sequences thereof.

[0066] In some embodiments, the RSV viral antigen or immunogen are the RSV F protein peptides of RSV A hypotype. In some embodiments, the RSV viral antigen or immunogen are the RSV F protein peptides of RSV A2 hypotype. In some embodiments, the RSV viral antigen or immunogen are the RSV F protein peptides of RSV B hypotype. In some cases, the RSV F protein peptides are conservative between RSV hypotypes.

[0067] In some cases, the RSV viral antigen or immunogen is a fragment of the RSV F protein peptide. In some embodiments, the RSV viral antigen or immunogen is an epitope of the RSV F protein peptide. In some embodiments, the epitope is a linear epitope. In some embodiments, the epitope is a conformational epitope. In some embodiments, the epitope is a neutralizing epitope site, such as site I, II or IV. In some embodiments, all neutralizing epitopes of the RSV F protein peptide or its fragments exist as RSV viral antigens or immunogens.

[0068] In some cases, such as when the RSV viral antigen or immunogen is a fragment of an RSV F protein peptide, only a single subunit of the RSV F protein peptide is present.

[0069] In some embodiments, the RSV viral antigen or immunogen is or comprises an F1 subunit peptide. In some embodiments, the F1 subunit peptide is or comprises the amino acid sequence of 137-574 of wild-type F protein. In some embodiments, the RSV viral antigen or immunogen is or comprises an F2 subunit peptide. In some embodiments, the RSV viral antigen or immunogen comprises an RSV F protein peptide containing a signal peptide, a heptad repeat C (HRC) peptide, a pep27 peptide, a fusion peptide (FP), a heptad repeat A (HRA) peptide, a domain I peptide, a domain II peptide or a heptad repeat B (HRB) peptide or any combination thereof. In some embodiments, the RSV viral antigen or immunogen comprises an RSV F protein peptide containing a signal peptide. In some embodiments, the RSV viral antigen or immunogen comprises an RSV F protein peptide containing a pep27 peptide. In some embodiments, the RSV viral antigen or immunogen comprises an RSV F protein peptide containing a fusion peptide (FP) (also known as a fusion domain (FD)). In some embodiments, the RSV viral antigen or immunogen comprises an RSV F protein peptide comprising a signal peptide, a pep27 peptide, and a fusion peptide (FP).

[0070] In some embodiments, the RSV viral antigens or immunogens comprise the RSV F protein peptides containing the F1 subunit and the F2 subunit of the F protein. In some embodiments, the RSV viral antigens or immunogens comprise the RSV F protein peptides containing the F1 subunit and the F2 subunit of the F protein. In some embodiments, the RSV viral antigens or immunogens comprise the RSV F protein peptides containing the F1 subunit and the F2 subunit of the F protein and not containing the pep 27 peptide. In some embodiments, the RSV viral antigens or immunogens comprise the RSV F protein peptides containing the F1 subunit, the F2 subunit, and the pep 27 peptide of the F protein. In some embodiments, the RSV viral antigens or immunogens comprise the RSV F protein peptides containing the F1 subunit, the F2 subunit, the pep 27 peptide and the FP of the F protein.

[0071] In some cases, such as when the viral antigen or immunogen comprises both the F1 subunit peptide and the F2 subunit peptide of the RSV F protein peptide, the F1 and F2 subunits are linked. In some embodiments, the F1 and F2 subunits are linked by a disulfide bond. In some embodiments, the F1 and F2 subunits are linked by an artificially introduced linker. In some embodiments, the F1 and F2 subunits are linked by a pep27 peptide. For example, in some embodiments, the orientation from the N-terminus to the C-terminus is or comprises F2-pep27-F1. In some embodiments, the orientation from the N-terminus to the C-terminus is or comprises F2-pep27-FP-F1 (F2-pep27-FD-F1). In some embodiments, FP is considered a structural feature of the F1 subunit peptide.

[0072] In some cases, RSV viral antigen or immunogen are RSV F protein peptides that do not contain transmembrane (TM) domain peptides.In some cases, described RSV F albumen does not contain cytoplasm (CP) domain peptides.In some cases, described RSV F albumen does not contain TM domain peptides or CP domain peptides.

[0073] In some embodiments, the RSV viral antigen or immunogen comprises an RSV F protein peptide that contains a protease cleavage site. In some embodiments, the protease cleavage site is specific to the cutting of the protease furin. In some embodiments, the protease cleavage site is specific to the cutting of the protease trypsin. In some embodiments, the protease cleavage site is specific to the cutting of protease factor Xa. In some embodiments, the protease cleavage site is specific to the cutting of the protease cathepsin L.

[0074] In some cases, the RSV viral antigens or immunogens comprise RSV F protein peptides that do not contain a protease cleavage site. In some cases, the RSV viral antigens or immunogens comprise RSV F protein peptides that do not contain a protease cleavage site specific for the cutting of the protease furin. In some cases, the RSV viral antigens or immunogens comprise RSV F protein peptides that do not contain a protease cleavage site specific for the cutting of the protease trypsin. In some cases, the RSV viral antigens or immunogens comprise RSV F protein peptides that do not contain a protease cleavage site specific for the cutting of the protease factor Xa. In some cases, the RSV viral antigens or immunogens comprise RSV F protein peptides that do not contain a protease cleavage site specific for the cutting of the protease cathepsin L.

[0075] In some embodiments, the RSV viral antigen or immunogen comprise soluble RSV F protein peptides. In some embodiments, the soluble RSV F protein peptides lack TM domain peptides and CP domain peptides. In some embodiments, the soluble RSV F protein peptides are not attached to a lipid bilayer, such as a film or a viral envelope.

[0076] In some embodiments, the RSV F protein peptide is produced by a codon-optimized nucleic acid sequence. In some embodiments, the RSV F protein peptide is produced by a non-codon-optimized nucleic acid sequence.

[0077] In some embodiments, the RSV F protein peptide can comprise any F protein sequence known in the art, such as those disclosed in US Patent No. 10,017,543, which is herein incorporated by reference in its entirety for all purposes.

[0078] In some embodiments, the RSV viral antigen or immunogen is or comprises an RSV F protein peptide having an amino acid sequence of 1-520 of SEQ ID NO: 31 or 32. In some embodiments, the RSV viral antigen or immunogen is or comprises an RSV F protein peptide having an amino acid sequence of 26-520 of SEQ ID NO: 31 or 32.

[0079] In some embodiments, the RSV viral antigen or immunogen are or comprise the sequence of F2, the sequence of pep27 and the sequence of F1 (such as F2-pep27-F1).In some embodiments, the RSV viral antigen or immunogen comprise fusogenic peptide and expose and have fusion rear conformation.In some embodiments, the RSV viral antigen or immunogen comprise furin cleavage site sudden change.In some embodiments, the RSV viral antigen or immunogen comprise furin site I sudden change (for example R109A) and / or furin site II sudden change (for example R136A), in these examples some in, the RSV viral antigen or immunogen have fusion rear conformation, and in other examples, the RSV viral antigen or immunogen have fusion front conformation.In some embodiments, the RSV viral antigen or immunogen comprise furin site I sudden change and furin site II sudden change (for example R109A / R136A), in these examples some in, the RSV viral antigen or immunogen comprise full length F0, do not have fusogenic peptide and expose and have fusion rear conformation. In some embodiments, the RSV viral antigen or immunogen include one or more mutations, and the mutation prevents the formation of long spirals and / or stable α4-α5 hinge loops. In some embodiments, the RSV viral antigen or immunogen include one or more mutations, and the mutation retains conformation before fusion. In some embodiments, the RSV viral antigen or immunogen include one or more mutations, and the mutation improves expression. In some embodiments, the replacement of position 161,182 and 215 (for example, with proline) causes higher expression levels, and E161P and S215P also increase protein stability. In some embodiments, the RSV viral antigen or immunogen include E161P and / or S215P and have conformation before fusion. In some embodiments, the RSV viral antigen or immunogen include R109A, R136A, E161P and / or S215P and have conformation before fusion.

[0080] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 17. 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 to the sequence of SEQ ID NO: 17, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0081] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 18. 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 to the sequence of SEQ ID NO: 18, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0082] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 19. 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 to the sequence of SEQ ID NO: 19, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0083] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 20. 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 to the sequence of SEQ ID NO: 20, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0084] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 21. 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 to the sequence of SEQ ID NO: 21, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0085] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 22. 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 to the sequence of SEQ ID NO: 22, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0086] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 23. 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 to the sequence of SEQ ID NO: 23, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0087] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 24. 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 to the sequence of SEQ ID NO: 24, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0088] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 25. 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 to the sequence of SEQ ID NO: 25, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0089] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 26. 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 to the sequence of SEQ ID NO: 26, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0090] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 27. 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 to the sequence of SEQ ID NO: 27, 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 set forth in SEQ ID NO: 28. 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 to the sequence of SEQ ID NO: 28, 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 set forth in SEQ ID NO: 29. 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 to the sequence of SEQ ID NO: 29, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0093] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 30. 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 to the sequence of SEQ ID NO: 30, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0094] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 31. 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 to the sequence of SEQ ID NO: 31, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0095] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 32. 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 to the sequence of SEQ ID NO: 32, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0096] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 33. 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 to the sequence of SEQ ID NO: 33, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0097] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 34. 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 to the sequence of SEQ ID NO: 34, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0098] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 35. 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 to the sequence of SEQ ID NO: 35, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0099] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 36. 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 to the sequence of SEQ ID NO: 36, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0100] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 37. 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 to the sequence of SEQ ID NO: 37, 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 set forth in SEQ ID NO: 38. 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 to the sequence of SEQ ID NO: 38, 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 set forth in SEQ ID NO: 39. 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 to the sequence of SEQ ID NO: 39, 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 set forth in SEQ ID NO: 40. 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 to the sequence of SEQ ID NO: 40, 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 set forth in SEQ ID NO: 41. 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 to the sequence of SEQ ID NO: 41, 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 set forth in SEQ ID NO: 42. 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 to the sequence of SEQ ID NO: 42, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0106] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 43. 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 to the sequence of SEQ ID NO: 43, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0107] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 44. 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 to the sequence of SEQ ID NO: 44, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0108] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 45. 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 to the sequence of SEQ ID NO: 45, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0109] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 46. 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 to the sequence of SEQ ID NO: 46, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0110] In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 47. 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 to the sequence of SEQ ID NO: 47, including sequences comprising substitutions, deletions and / or insertions at one or more amino acid positions.

[0111] In some embodiments, the viral antigens or immunogens herein may comprise RSV glycoprotein (G) or a fragment, variant or mutant thereof; RSV small hydrophobic protein (SH) or a fragment, variant or mutant thereof; RSV fusion protein (F) or a fragment, variant or mutant thereof; RSV matrix protein (M) or a fragment, variant or mutant thereof; RSV nucleoprotein (N) or a fragment, variant or mutant thereof; RSV phosphoprotein (P) or a fragment, variant or mutant thereof; RSV "large" protein (L) or a fragment, variant or mutant thereof; M2-1 protein or a fragment, variant or mutant thereof; RSV M2-2 protein or a fragment, variant or mutant thereof; RSV NS-1 protein or a fragment, variant or mutant thereof; or RSV Ns-2 protein or a fragment, variant or mutant thereof; or any combination thereof.

[0112] In some embodiments, the viral antigen or immunogen is produced by a codon-optimized nucleic acid sequence. In some embodiments, the viral antigen or immunogen is produced by a non-codon-optimized nucleic acid sequence.

[0113] In some embodiments, RSV viral antigen or immunogen mentioned herein can comprise recombinant polypeptide or the fusion polypeptide that comprises described viral antigen or immunogen.Term viral antigen or immunogen can be used for referring to and comprise RSV viral antigen or immunogenic protein.In some cases, described RSV viral antigen or immunogen are RSV protein peptides as provided herein.

[0114] II. Recombinant Peptides and Proteins

[0115] It is envisioned that RSV viral antigens and immunogens provided herein, for example RSV F protein peptides (referring to Section 1) can be combined with other proteins or peptides, for example, connected to form recombinant polypeptides, including fusogenic peptides. In some embodiments, individual recombinant polypeptides (for example monomers) provided herein associate to form a multimer, for example a trimer, of a recombinant polypeptide. In some embodiments, the association of individual recombinant polypeptide monomers occurs by covalent interactions. In some embodiments, the association of individual recombinant polypeptide monomers occurs by non-covalent interactions. In some embodiments, the protein or peptide to which the interaction (for example covalent or non-covalent) is connected by RSV viral antigens or immunogens (for example RSV F protein peptides) affects. In some embodiments, for example, when RSV viral antigens or immunogens are RSV F protein peptides as described herein, the protein or peptide to which it will be connected can be selected to retain the natural homotrimeric structure of glycoprotein. This may be conducive to stimulating the strong and effective immunogenic response to RSV F protein peptides. For example, retaining and / or keeping the natural conformation of RSV viral antigens or immunogen (such as RSV F protein peptides) can improve or allow approaching the antigenic site that can produce an immune response.In some cases, the recombinant polypeptide comprising RSV F protein peptides as herein described (such as referring to Section 1) is alternatively referred to as recombinant RSV F antigen, recombinant RSV F immunogen or recombinant RSV F protein in this article.

[0116] In some cases, it is also envisioned that the recombinant polypeptide or its polymerized recombinant polypeptide gather or can gather to form a protein that comprises multiple RSV viral antigens and / or immunogen recombinant polypeptide.The formation of this type of protein may help RSV viral antigens and / or immunogen produce strong and effective immunogenicity response.For example, the formation of the protein that comprises multiple recombinant polypeptides and therefore the formation of multiple RSV viral antigens (such as RSV F protein peptides) can retain tertiary and / or quaternary structure of viral antigen, thereby allow strengthening the immunne response for native structure.In some cases, gathering can give RSV viral antigen or immunogen structural stability, and then can approach the potential antigenic site that can promote immunne response.

[0117] 1. Fusion peptides and recombinant peptides

[0118] In some embodiments, RSV viral antigen or immunogen can be connected (C-terminus is connected) to trimerization domain at its C-terminus, to promote monomer trimerization.In some embodiments, trimerization has stabilized the membrane proximal situation of RSV viral antigen or immunogen (such as RSV F protein peptide) in trimeric conformation.

[0119] Non-limiting examples of exogenous multimerization domains that promote stable trimers of soluble recombinant proteins include: GCN4 leucine zipper (Harbury et al., 1993 Science 262: 1401-1407), a trimerization motif from lung surfactant protein (Hoppe et al., 1994 FEBS Lett 344: 191-195), collagen (McAlinden et al., 2003 J Biol Chem 278: 42200-42207), and bacteriophage T4 minor fibrillar protein fold (Miroshnikov et al., 1998 Protein Eng 11: 329-414), any of which can be linked to a recombinant RSV viral antigen or immunogen described herein (e.g., by linking to the C-terminus of the RSV F peptide) to promote trimerization of the recombinant viral antigen or immunogen. 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 US 2020 / 0009244, which are incorporated herein by reference in their entirety for all purposes.

[0120] In some embodiments, one or more peptide connectors (such as gly-ser connectors, e.g., 10 amino acid glycine-serine peptide connectors) can be used to connect the recombinant viral antigen or immunogen to the multimerization domain. The trimer can include any stabilizing mutations (or combinations thereof) described herein, as long as the recombinant viral antigen or immunogen trimer retains the desired properties (e.g., pre-fusion conformation).

[0121] To be therapeutically viable, the desired trimerization protein portion for biopharmaceutical design should meet the following criteria. Ideally, it should be a portion of a naturally secreted protein, such as immunoglobulin Fc, also abundant in the circulation (non-toxic), of human origin (lack of immunogenicity), relatively stable (long half-life), and able to efficiently trimerize itself (enhanced by interchain covalent disulfide bonds), so that the trimeric RSV viral antigen or immunogen structure is stable.

[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, comprising nearly 25% of the total protein in the body. Collagen plays a major structural role in the formation of bones, tendons, skin, cornea, cartilage, blood vessels, and teeth. The fibrillar types of collagen I, II, III, IV, V, and XI are synthesized as large, trimeric precursors called procollagens, consisting of a central, uninterrupted triple-helical domain composed of hundreds of "GXY" repeats (or glycine repeats) flanked by a non-collagenous domain (NC), an N-propeptide, and a C-propeptide. Both the C-terminal and N-terminal extensions undergo proteolytic processing after procollagen secretion, triggering the assembly of the mature protein into collagen fibrils, thereby forming an insoluble cellular matrix. BMP-1 is a protease that recognizes a specific peptide sequence in procollagen near the junction between the glycine repeats and the collagen C-proper domain and is responsible for removing the propeptide. The shed trimeric C-propeptide of type I collagen is found in normal adult human serum at concentrations ranging from 50 to 300 ng / mL, with levels much higher in children, indicating active bone formation. In individuals with familial high serum type I collagen C-propeptide concentrations, levels can be as high as 1-6 μg / mL without apparent abnormalities, suggesting that the C-propeptide is non-toxic. Structural studies of the collagen trimeric C-propeptide have revealed a trilobal structure in which all three subunits come together in a linker region near their N-termini, connecting to the rest of the procollagen molecule. The geometry of this protein to be fused, with one direction extending outward, resembles that of an Fc dimer.

[0123] Type I, IV, V, and XI collagens primarily assemble into heterotrimeric forms composed of two α-1 chains and one α-2 chain (for types I, IV, and V) or three distinct, highly sequenced chains (for type XI). Type II and III collagens are homotrimers of α-1 chains. Type I collagen, the most abundant collagen form, also forms stable α(I) homotrimers, present 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 mutually aligned association of the C propeptides. The C propeptide complex is believed to be stabilized by the formation of interchain disulfide bonds, but the necessity of disulfide bond formation for proper chain alignment is unclear. The glycine triple helix repeats and then propagates in a zipper-like manner from the associated C-terminus to the N-terminus. This understanding has led to the creation of non-natural collagen matrices by exchanging the C-propeptides of different collagen chains using recombinant DNA technology. Non-collagenous proteins, such as cytokines and growth factors, have also been fused to the N-termini of procollagen or mature collagen to form new collagen matrices, a move intended to allow for the slow release of non-collagenous proteins from the cell matrix. However, in both cases, the C-propeptide must be cleaved before the recombinant collagen fibrils can assemble into an insoluble cell matrix.

[0124] Although other protein trimerization domains have been described previously, such as those from yeast GCN4, the minor fibrin of bacteriophage T4, and the aspartate transcarbamylase of Escherichia coli, which allow the trimerization of heterologous proteins, none of these trimerizing proteins are native human proteins, nor are they naturally secreted proteins. Therefore, any trimeric fusion protein must be produced intracellularly, which not only may cause naturally secreted proteins (such as soluble receptors) to fold incorrectly, but also makes it difficult to purify the fusion protein from thousands of other intracellular proteins. Furthermore, a fatal drawback of using such non-human protein trimerization domains (e.g., from yeast, phage, and bacteria) for trimeric biopharmaceutical design is their presumed immunogenicity in humans, rendering such fusion proteins ineffective shortly after they are injected into humans.

[0125] Therefore, the use of collagen in recombinant polypeptides as described herein has many advantages, including: (1) collagen is the most abundant protein secreted by mammals, comprising nearly 25% of the total protein in the body; (2) the major form of collagen exists naturally as a trimeric helix, with its globular C propeptide responsible for initiating trimerization; (3) the collagen trimeric C propeptide, released by hydrolysis of mature collagen, is found naturally in mammalian blood at submicrogram / ml levels and is known to be non-toxic to the body; (4) the linear triple helical region of collagen can be included as a linker, with a predicted spacing of each residue of Alternatively, it may be excluded from the fusion protein portion, thereby enabling precise adjustment of the distance between the protein to be trimerized and the collagen C propeptide for optimal biological activity; (5) the BMP1 recognition site that cleaves the C propeptide from the procollagen may be mutated or deleted to prevent disruption of the trimeric fusion protein; (6) the C propeptide domain trimerizes itself via disulfide bonds, which provides a universal affinity tag that can be used to purify any secreted fusion protein produced. In some embodiments, the collagen C propeptide to which RSV viral antigens and immunogens (e.g., RSV F protein peptides) are attached enables the recombinant production of a soluble, covalently linked homotrimeric fusion protein.

[0126] In some embodiments, RSV viral antigen or immunogen are connected to collagen C-terminal propeptide to form recombinant polypeptide.In some embodiments, the C-terminal propeptide of described recombinant polypeptide has formed interpolypeptide disulfide bond.In some embodiments, described recombinant protein has formed trimer.In some embodiments, described RSV viral antigen or immunogen are RSV F protein peptides as described in Section 1.

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

[0128] In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO:48. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:48. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO:49. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:49. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO:50. In some embodiments, the C-terminal propeptide exhibits an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:50. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO:51. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 51. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 52. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 53.

[0129] In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO:54. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:54. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO:55. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:55. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO:56. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:56. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO:57. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 57. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 58. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 58. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO: 59. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to the sequence of SEQ ID NO: 59.

[0130] In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO:60. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:60. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO:61. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:61. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO:62. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity with the sequence of SEQ ID NO:62. In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence set forth in SEQ ID NO:63. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95% or 97% sequence identity to the sequence of SEQ ID NO:63.

[0131] In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence of a collagen trimerization domain (e.g., the C-propeptide of human α1(I) collagen), wherein the aspartic acid (D) in the BMP-1 site is replaced with asparagine (N), such as wherein RA D Mutation into RA N In some embodiments, the C-terminal propeptide is or comprises the amino acid sequence of a collagen trimerization domain (e.g., the C-propeptide of human α1(I) collagen), wherein the alanine (A) in the BMP-1 site is replaced with asparagine (N), e.g., wherein R A D mutates to R N D. In some embodiments, the C-terminal propeptide herein may comprise a mutated BMP-1 site, such as RSAN instead of DDAN. In some embodiments, the C-terminal propeptide herein may comprise a BMP-1 site, such as a sequence comprising 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 polypeptides disclosed herein.

[0132] In some embodiments, the C-terminal propeptide is or comprises an amino acid sequence that is a fragment of any one of SEQ ID NOs: 48-63.

[0133] In some embodiments, the C-terminal propeptide may comprise a sequence comprising a glycine-XY repeating sequence, wherein X and Y are independently any amino acid, or an amino acid sequence having at least 85%, 90%, 92%, 95%, or 97% identity thereto, capable of forming an interpolypeptide disulfide bond and trimerizing the recombinant polypeptide. In some embodiments, X and Y are independently proline or hydroxyproline.

[0134] In some cases where RSV F peptide protein (such as RSV viral antigen or immunogen, for example, referring to Section 1) is connected to C-terminal propeptide to form recombinant polypeptide, the recombinant polypeptide has formed a trimer, thereby produces the homotrimer of RSV F protein peptide. In some embodiments, the trimerized recombinant polypeptide contains the F protein peptide trimer of crutch-shaped rod. In some embodiments, the RSV F protein peptide of the trimerized recombinant polypeptide is in the conformation before fusion. In some embodiments, the RSV F protein peptide of the trimerized recombinant polypeptide is in the conformation after fusion. In some embodiments, the conformational state allows to approach the different antigenic sites on the F protein peptide. In some embodiments, the antigenic site is an epitope, such as a linear epitope or a conformational epitope. An advantage with the trimerized recombinant polypeptide is that the immune response for various potential different antigenic sites can be strengthened.

[0135] In some embodiments, the trimerized recombinant polypeptide comprises individual recombinant polypeptides comprising the same viral antigen or immunogen. In some embodiments, the trimerized recombinant polypeptide comprises individual recombinant polypeptides each comprising a viral antigen or immunogen different from the other recombinant polypeptides. In some embodiments, the trimerized recombinant polypeptide comprises individual recombinant polypeptides wherein one of the individual recombinant polypeptides comprises a viral antigen or immunogen different from the other recombinant polypeptides. In some embodiments, the trimerized recombinant polypeptide comprises individual recombinant polypeptides wherein two of the individual recombinant polypeptides comprise the same viral antigen or immunogen, and the viral antigen or immunogen is different from the viral antigen or immunogen comprised by the remaining recombinant polypeptides.

[0136] In some embodiments, the recombinant polypeptide comprises any RSV viral antigen or immunogen described in Section 1. In some embodiments, the recombinant polypeptide comprises any RSV viral antigen or immunogen described in Section 1, as described herein, 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 NOs: 17-47 connected to a second sequence shown in any one of SEQ ID NOs: 48-63, wherein the C-terminus of the first sequence is directly connected to the N-terminus of the second sequence.

[0138] In some embodiments, the recombinant polypeptide or fusion protein comprises a first sequence as set forth in any one of SEQ ID NOs: 17-47 linked to a second sequence as set forth in any one of SEQ ID NOs: 48-63, 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 comprising 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 the sequence shown in SEQ ID NO: 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (relative to SEQ ID NO: 1) In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 106, 114, 116, 122, 129, 130, 147, 151, 161, 170, 189, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 251, 252, 253, 261, 262, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 310, 311, 312, 324, 335, 336, 337, 340, 359, 361, 362, 363, 364, 375

[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 the sequence shown in SEQ ID NO: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the sequence of SEQ ID NO: 2, including at least one or more amino acid positions such as 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (relative to SEQ ID NO: 2). In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 106, 114, 116, 122, 129, 130, 131, or 132) or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 2 comprising any one, two, three, four, five or more mutations selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V, or any combination thereof.

[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 the sequence shown in SEQ ID NO: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the sequence of SEQ ID NO: 3, including at least one or more amino acid positions such as 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (relative to SEQ ID NO: 3). In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 106, 114, 116, 122, 129, 130, 131, or 132) or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 3 comprising any one, two, three, four, five or more mutations selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V, or any combination thereof.

[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 the sequence shown in SEQ ID NO: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the sequence of SEQ ID NO: 4, including at least one or more amino acid positions such as 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (relative to SEQ ID NO: 4). In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 106, 114, 116, 122, 129, 130, 131, or 132) or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 4 comprising any one, two, three, four, five or more mutations selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V, or any combination thereof.

[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 the sequence shown in SEQ ID NO: 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, including at one or more amino acid positions such as 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379 or 447 (relative to SEQ ID NO:5) In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 106, 114, 116, 122, 129, 130, 131, or 132) or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 5 comprising any one, two, three, four, five or more mutations selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V, or any combination thereof.

[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 the sequence shown in SEQ ID NO: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the sequence of SEQ ID NO: 6, including at least one or more amino acid positions such as 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (relative to SEQ ID NO: 6). In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 106, 114, 116, 122, 129, 130, 131, or 132) or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 6 comprising any one, two, three, four, five or more mutations selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V, or any combination thereof.

[0145] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 7. In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to the sequence of NO:7, including at least one or more amino acid positions such as 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (relative to SEQ ID NO:7). In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 106, 114, 116, 122, 129, 130, 131, or 132) or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 7 comprising any one, two, three, four, five or more mutations selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V, or any combination thereof.

[0146] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 8. In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 8 has 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, including at one or more amino acid positions such as 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379 or 447 (relative to SEQ ID NO: In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 106, 114, 116, 122, 129, 130, 131, or 132) or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 8 comprising any one, two, three, four, five or more mutations selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V, or any combination thereof.

[0147] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 9. In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 98, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (relative to SEQ ID NO:9) In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 106, 114, 116, 122, 129, 130, 131, or 132) or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 9 comprising any one, two, three, four, five or more mutations selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V, or any combination thereof.

[0148] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 10. In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: The sequence of NO: 10 has 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, including at one or more amino acid positions such as 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379 or 447 (relative to SEQ ID NO: In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 100, 101, 102, 103, 104, 105, 106, 107, 108, 112, 119, 120, 130, 131, 132, 140, 158, 160, 161, 109, 112, 121, 133, 144, 159, 161, 162, 163, 164, 171, 175, 180, 181, 192, 202, 203, 204, 205, 206, 207, 208, 209, 210, 21

[0149] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 11. In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: The sequence of NO: 11 has 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, including at one or more amino acid positions such as 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379 or 447 (relative to SEQ ID NO: In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 106, 110, 111a, 111b, 111c ...

[0150] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 12. In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity, including at least one or more amino acid positions such as 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (relative to SEQ ID NO: In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 106, 114, 116, 129, 131, or 132) or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 12 comprising any one, two, three, four, five or more mutations selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V, or any combination thereof.

[0151] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 13. In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (relative to SEQ ID NO: 13) In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 106, 114, 116, 122, 129, 130, 131, or 132) or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 13 comprising any one, two, three, four, five or more mutations selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V, or any combination thereof.

[0152] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 14. In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: The sequence of NO: 14 has 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, including at one or more amino acid positions such as 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379 or 447 (relative to SEQ ID NO: In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 106, 114, 116, 117, 118, 120, 131, or 132) or any combination thereof. In some embodiments, the recombinant polypeptide is or comprises a variant of SEQ ID NO: 14 comprising any one, two, three, four, five or more mutations selected from P102A, R109A, R136A, E161P, E218A, S215P, I379A and M447V, or any combination thereof.

[0153] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 15. In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity, including at least one or more amino acid positions such as 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379, or 447 (relative to SEQ ID NO: In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 106, 114, 116, 122, 129, 130, 141, 153, 169, 171, 172, 173, 180, 181, 192, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 251, 252, 253, 254, 261, 262, 263, 271, 272, 273, 274, 275, 280, 286, 287, 288, 290, 309, 310, 311, 322, 312, 323, 313, 324, 335, 290, 314, 336, 290, 315, 325, 337, 290, 316, 338, 291, 317, 326, 292, 339, 293, 339, 294, 339

[0154] In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: 16. In some embodiments, the recombinant polypeptide is or comprises the sequence shown in SEQ ID NO: The sequence of NO:16 has 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, including at one or more amino acid positions such as 102, 106, 107, 108, 109, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 161, 215, 218, 379 or 447 (relative to SEQ ID NO: In some embodiments, the recombinant polypeptide comprises a sequence comprising substitutions, deletions and / or insertions at amino acid positions (i.e., amino acid positions 106, 114, 116, 122, 129, 130, 147, 151, 169, 170, 182, 184, 190, 200, 206, 208, 210, 211, 212, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 251, 261, 271, 272, 273, 274, 289, 290, 300, 310, 311, 322, 28

[0155] As pointed out above, in some embodiments, recombinant polypeptide provided herein not only associates to form a trimer, but also can assemble or be assembled to produce a protein that comprises a plurality of recombinant polypeptides.In some embodiments, formed protein has macrostructure.In some cases, macrostructure can give RSV viral antigen or immunogen recombinant polypeptide structural stability, and then can approach the potential antigenic site that can promote immunne response.

[0156] In some embodiments, the trimerized recombinant polypeptides aggregate to form a protein comprising multiple trimerized recombinant polypeptides. In some embodiments, the multiple trimerized recombinant polypeptides form a protein having a macrostructure. In some embodiments, the protein comprises a rosette-like oligomer comprising a crutch-shaped rod-shaped F protein peptide trimer.

[0157] In some embodiments, provided herein is a complex comprising any suitable combination of a recombinant polypeptide selected from SEQ ID NOs: 1-16 or fragments, variants, or mutants thereof. In some embodiments, provided herein is a complex comprising a trimer of a recombinant polypeptide selected from SEQ ID NOs: 1-16 or fragments, variants, or mutants thereof, wherein the recombinant polypeptides are trimerized via inter-polypeptide disulfide bonds to form the trimer.

[0158] In some embodiments, the proteins comprising multiple recombinant polypeptides described herein are immunogens. In some embodiments, the proteins comprising multiple recombinant polypeptides described herein are contained in nanoparticles. For example, in some embodiments, the proteins are directly attached to nanoparticles, such as protein nanoparticles. In some embodiments, the proteins are indirectly attached to nanoparticles. In some embodiments, the proteins comprising multiple recombinant polypeptides described herein are contained in virus-like particles (VLPs).

[0159] 2. Polynucleotides and Vectors

[0160] Also provided are polynucleotides (nucleic acid molecules) encoding the RSV antigens or immunogens and recombinant polypeptides provided herein, as well as vectors for genetically engineering cells to express such RSV antigens or immunogens and recombinant polypeptides.

[0161] In some embodiments, the polynucleotides of the recombinant polypeptide that coding this paper provides are provided.Aspect some, described polynucleotides contain single nucleotide sequence, as the nucleotide sequence of encoding recombinant polypeptide.In other cases, described polynucleotides contain the first nucleotide sequence that coding comprises specific RSV viral antigen or immunogenic recombinant polypeptide and coding comprises different RSV viral antigen or immunogenic second nucleotide sequence of recombinant polypeptide.

[0162] In some embodiments, the polynucleotide encoding the recombinant polypeptide contains at least one promoter operably linked to control the expression of the recombinant polypeptide. In some embodiments, the polynucleotide contains two, three or more promoters operably linked to control the expression of the recombinant polypeptide.

[0163] In some embodiments, for example, when the polynucleotide contains two or more nucleic acid encoding sequences, such as encoding a sequence of a recombinant polypeptide comprising different RSV viral antigens or immunogens, at least one promoter is operably connected to control the expression of the two or more nucleic acid sequences. In some embodiments, the polynucleotide contains two, three or more promoters, and the promoter is operably connected to control the expression of the recombinant polypeptide.

[0164] 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 transactivator. In some such aspects, the conditional promoter, enhancer or transactivator is an inducible promoter, enhancer or transactivator, or an inhibitory promoter, enhancer or transactivator. For example, in some embodiments, an inducible or conditional promoter 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.

[0165] In the case where the 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 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 skipping, such as a T2A peptide.

[0166] In some embodiments, the polynucleotide encoding the recombinant polypeptide is introduced into a composition containing cultured cells (e.g., host cells), such as by retroviral transduction, transfection, or transformation. In some embodiments, this can allow expression (e.g., production) of the recombinant polypeptide. In some embodiments, the expressed recombinant polypeptide is purified.

[0167] In some embodiments, provided herein are polynucleotides (nucleic acid molecules) encoding RSV viral antigens or immunogens as described herein. In some embodiments, provided herein are polynucleotides (nucleic acid molecules) encoding recombinant polypeptides comprising RSV viral antigens or immunogens, such as RSV F peptide proteins as described herein.

[0168] Also provided are vectors or constructs containing nucleic acid molecules as described herein. In some embodiments, the vector or construct contain one or more promoters, and the promoter is operably connected to the nucleic acid molecules encoding recombinant polypeptides to drive their expression. In some embodiments, the promoter is operably connected to one or more than one nucleic acid molecules, such as nucleic acid molecules encoding recombinant polypeptides containing different RSV viral antigens or immunogens.

[0169] 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 gammaretroviral vector.

[0170] In some embodiments, described vector or construct include single promoter, and described promoter drives the expression of one or more nucleic acid molecules of polynucleotide.In some embodiments, this type of promoter can be polycistronic (bicistronic or tricistronic, referring to, for example, U.S. Patent number 6,060,273).For example, in some embodiments, transcription unit can be engineered to contain the bicistronic unit of IRES (internal ribosome entry site), thus allows by the signal coexpression gene product (for example, encoding different recombinant polypeptides) from single promoter.In some embodiments, provided herein is a bicistronic carrier, thereby allows described carrier to contain and express two nucleotide sequences.In some embodiments, provided herein is a tricistronic carrier, thereby allows described carrier to contain and express three nucleotide sequences.

[0171] In some embodiments, a single promoter guides RNA expression, and the RNA contains two or three genes (e.g., encoding a chimeric signaling receptor and encoding a recombinant receptor) in a single open reading frame (ORF), and the genes are separated from each other by sequences encoding self-cleaving peptides (e.g., 2A sequences) or protease recognition sites (e.g., furin). The ORF therefore encodes a single polypeptide, which is processed into individual proteins during translation (in the case of 2A) or after translation. In some cases, the peptide, such as T2A, may cause the ribosome to skip (ribosome skipping) the peptide bond at the C-terminus of the synthetic 2A element, resulting in the 2A sequence end being separated from the next downstream peptide (see, e.g., 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, 2A sequences from foot-and-mouth disease virus (F2A), equine rhinitis virus (E2A), Thosea asigna virus (T2A), and porcine teschovirus-1 (P2A) as described in U.S. Patent Publication No. 20070116690.

[0172] In some embodiments, the vector is contained in 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 in a cell. In some embodiments, the virus or cell containing the vector contains a recombinant genome.

[0173] III. Immunogenic Compositions and Formulations

[0174] In some embodiments, there is provided herein an immunogenic composition, the immunogenic composition comprising a trimer of a recombinant polypeptide or any two or more combinations of the trimer, the recombinant polypeptide comprising a sequence selected from SEQ ID NO: 1-16. In some embodiments, the unit dose of the immunogenic composition may include RSV F antigens of about 10 μg to about 100 μg, preferably RSV F antigens of about 25 μg to about 75 μg, preferably RSV F antigens of about 40 μg to about 60 μg or RSV F antigens of about 50 μg. In some embodiments, the dosage contains RSV F antigens of 3 μg. In other embodiments, the dosage contains RSV F antigens of 9 μg. In other embodiments, the dosage contains RSV F antigens of 30 μg.

[0175] In some cases, it may be necessary to combine the disclosed immunogen with other medicines (e.g., vaccines) that induce the protective response to other factors. For example, compositions comprising recombinant RSV F antigens as described herein (e.g., trimers or proteins) can be administered simultaneously (usually separately) or sequentially with other vaccines such as influenza vaccines or varicella zoster vaccines recommended by the U.S. Advisory Committee on Immunization Practices (ACIP; cdc.gov / vaccines / acip / index.html) for target age groups (e.g., approximately 1 to 6 month old infants). Therefore, disclosed immunogens comprising recombinant RSV F antigens as described herein can be administered simultaneously or sequentially with vaccines such as for hepatitis B (HepB), diphtheria, tetanus, and pertussis (DTaP), pneumococcus (PCV), Haemophilus influenzae type b (Hib), polio, influenza, and rotavirus.

[0176] 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. In some cases, multivalent vaccines provide protection against multiple pathogens, such as the combination vaccine Tdap, 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 or strains of pathogens, reducing administration costs, and increasing coverage. This can be particularly useful, for example, when vaccinating infants or children.

[0177] In some embodiments, for example, vaccines comprising immunogenic compositions as described herein are multivalent vaccines. In some embodiments, the antigenic material for incorporation into the multivalent vaccine compositions of the present invention is derived from type A or type B RSV or a combination thereof. The antigens for incorporation into the multivalent vaccine compositions of the present invention can be derived from one RSV or multiple strains, for example, two to five strains, in order to provide a wider range of protection. In one embodiment, the antigens for incorporation into the multivalent vaccine compositions of the present invention are derived from multiple strains of RSV viruses. Other useful antigens include live, attenuated, and inactivated viruses, such as inactivated poliovirus (Jiang et al., J. Biol. Stand., (1986) 14: 103-9), attenuated hepatitis A virus (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 cell-free DTP, Wyeth-Lederle vaccine, and children's medicine).

[0178] In some aspects, the vaccine provided herein is a universal vaccine. In some embodiments, a universal vaccine is a vaccine that provides protection against multiple strains of the same virus, such as multiple RSV strains. Developing an effective universal RSV vaccine would reduce the cost and labor of, for example, seasonal vaccine preparations, and allow for more robust pandemic preparedness.

[0179] In some respects, universal vaccine is the vaccine that comprises a plurality of epi-positions that derive from different virus strains.In some respects, universal vaccine is included in the single epi-position that is conservative between different virus strains.For example, universal vaccine can be based on the relatively conservative structural domain of RSV F albumen.

[0180] Also provided are immunogenic compositions comprising a disclosed immunogen (e.g., a disclosed recombinant RSV F antigen or a nucleic acid molecule encoding a protomer of a disclosed recombinant RSV F antigen) and a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a trimerized recombinant polypeptide as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a protein containing multiple trimerized recombinant polypeptides as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a protein nanoparticle as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a VLP as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises an isolated nucleic acid as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a carrier as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a virus as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a pseudovirus as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises a cell as provided herein and an optional pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition, such as the immunogenic composition 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 a prophylactic vaccine and a therapeutic vaccine. Such pharmaceutical compositions can be administered to a subject by various modes of administration known to those of ordinary skill, such as intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, intranasal, sublingual, tonsil, oropharyngeal, or other parenteral and mucosal routes. In several embodiments, a pharmaceutical composition comprising one or more disclosed immunogens is an immunogenic composition. Actual methods for preparing administrable 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.

[0181] The present invention relates to the preparation of the present invention and the invention relates to immunogens, for example recombinant RSV F antigens, for example tripolymers as herein described, proteins, can be prepared together with a pharmaceutically acceptable carrier, to help retain biological activity, and also to contribute to increase the stability during storage in an acceptable temperature range. Possible carriers include but are not limited to physiological equilibrium culture medium, phosphate buffered saline solution, water, emulsions (for example oil / water or water / oil emulsions), various types of wetting agents, cryoprotectant additives or stabilizers such as protein, peptide or hydrolyzate (for example albumin, gelatin), sugar (for example sucrose, lactose, sorbitol), amino acids (for example sodium glutamate) or other protective agents. The gained aqueous solution can be used by former state or freeze-dried packaging. Freeze-dried preparations are combined with sterile solution before administration for single or multiple administration.

[0182] Formulated compositions, especially liquid formulations, may contain bacteriostatic agents to prevent or minimize degradation during storage, including but not limited to benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and / or propylparaben at effective concentrations (typically 1% w / v). Some patients may be averse to bacteriostatic agents; therefore, lyophilized formulations can be reconstituted in solutions with or without such ingredients.

[0183] The immunogenic compositions of the present invention may contain pharmaceutically acceptable vehicle substances as needed to approach physiological conditions, such as pH regulators and buffers, osmotic pressure regulators, wetting agents, and the like, such as 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 polyoxypropylene (POP), such as POE-POP-POE block copolymers, MPL 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 stimulate the immune system in a non-specific manner, thereby enhancing the immune response to the drug. In some embodiments, the immunogenic compositions of the present disclosure can include more than one adjuvant or be administered with more than one adjuvant. In some embodiments, the immunogenic compositions of the present disclosure can include two adjuvants or be administered with two adjuvants. In some embodiments, the immunogenic compositions of the present disclosure can include multiple adjuvants or be administered with multiple adjuvants. For example, in some cases, a vaccine, such as one comprising the immunogenic compositions provided herein, can include multiple adjuvants or be administered in combination with multiple adjuvants.

[0184] For vaccine compositions, examples of suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, MPL TM and IL-12. In some embodiments, the vaccine compositions or nanoparticle immunogens disclosed herein (e.g., RSV vaccine compositions) can be formulated as controlled-release or timed-release formulations. This can be achieved in compositions containing sustained-release polymers or by microencapsulation delivery systems or bioadhesive gels. Various pharmaceutical compositions can be prepared according to standard procedures well known in the art.

[0185] In some embodiments, the immunogenic compositions of the present invention may contain an adjuvant formulation comprising a metabolizable oil (e.g., squalene) and alpha tocopherol, and polyoxyethylene sorbitan monooleate (Tween-80) in the form of an oil-in-water emulsion. In some embodiments, the adjuvant formulation may comprise about 2% to about 10% squalene, about 2 to about 10% alpha tocopherol (e.g., D-alpha 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 containing a metabolizable oil, alpha tocopherol, and polyoxyethylene sorbitan monooleate. In some embodiments, the immunogenic compositions of the present disclosure may contain QS21 (Quillaja saponaria Molina extract: fraction 21), 3D-MPL, and an oil-in-water emulsion, wherein the oil-in-water emulsion comprises a metabolizable oil, alpha-tocopherol, and polyoxyethylene sorbitan monooleate. In some embodiments, the immunogenic compositions of the present disclosure may contain QS21, 3D-MPL, and an oil-in-water emulsion, wherein the oil-in-water emulsion has the following composition: a metabolizable oil such as squalene, alpha-tocopherol, and Tween-80. In some embodiments, the immunogenic compositions of the present disclosure may contain an adjuvant in the form of a liposomal composition.

[0186] In some embodiments, the immunogenic compositions of the present 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.

[0187] In some embodiments, the immunogenic compositions of the present disclosure may contain an adjuvant formulation, for example in the form of a nanoparticle composition, comprising Quillaja saponins, cholesterol, and a phospholipid. In some embodiments, the immunogenic compositions of the present disclosure may contain a mixture of separately purified Quillaja fractions that are subsequently formulated with cholesterol and a phospholipid.

[0188] In some embodiments, the immunogenic composition of the present disclosure may contain a TM Matrix-A TM Matrix-C TM 、Matrix-M TM , AS01, AS02, AS03 and AS04 adjuvants.

[0189] In some embodiments, the immunogenic composition of the present invention may contain a toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide having a length of 8 to 35 nucleotides and comprising an unmethylated cytidine-phosphate-guanosine (also referred to as CpG or cytosine-phosphate-guanosine) motif, and the RSV antigen and the oligonucleotide are present in the immunogenic composition in an amount that can effectively stimulate an immune response of a mammalian subject in need thereof, such as a human subject, to the RSV antigen. TLR9 (CD289) recognizes unmethylated cytidine-phosphate-guanosine (CpG) motifs found in microbial DNA, which can be simulated 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). The best oligonucleotide TLR9 agonists generally contain a palindromic sequence that follows the following 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 annular or includes a hairpin loop. The CpG oligonucleotide can be single-stranded or double-stranded. In some embodiments, the CpG oligonucleotide can 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. The modified base can be included in the palindromic sequence of the CpG oligonucleotide, as long as the modified base maintains the same specificity to its natural complement by Watson-Crick base pairing (e.g., the palindromic portion is still self-complementary). In some embodiments, the CpG oligonucleotide comprises an atypical base. In some embodiments, the CpG oligonucleotide comprises a modified nucleoside. In some embodiments, the modified nucleoside is selected from 2'-deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2' substituted-arabinoguanosine and 2'-O-substituted-arabinoguanosine. The CpG oligonucleotide can contain the modification of a phosphate group. For example, in addition to a phosphodiester bond, phosphate modification also includes but is not limited to methylphosphonate, phosphorothioate, phosphoramide (bridged or non-bridged), phosphotriester and dithiophosphate, and can be used in any combination. Other non-phosphate bonds can also be used. In some embodiments, the oligonucleotide only comprises a thiophosphate backbone. In some embodiments, the oligonucleotide only comprises a phosphodiester backbone. In some embodiments, the oligonucleotide comprises a combination of phosphate bonds in the phosphate backbone, such as a combination of phosphodiester bonds and thiophosphate bonds.Oligonucleotides with thiophosphate backbones may be more immunogenic than oligonucleotides with phosphodiester backbones 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 the present disclosure include 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, two stereoisomers of thiophosphate bonds are present in multiple CpG oligonucleotide molecules. In some embodiments, all internucleotide bonds of the CpG oligonucleotides are thiophosphate bonds, or in other words, the CpG oligonucleotides have a thiophosphate backbone.

[0190] Any suitable CpG oligodeoxynucleotide (ODN) or combination thereof can be used as adjuvant in the present disclosure. For example, K-type ODN (also referred to as B-type) encodes multiple CpG motifs on a phosphorothioate backbone. K-type ODN can be based on the following sequence: Compared to natural phosphodiester nucleotides, the use of phosphorothioate 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 induce B cell proliferation and IgM secretion. D-type ODNs (also known as A-types) are constructed from a mixed phosphodiester / phosphorothioate backbone, contain a single CpG motif flanked by palindromic sequences, and have polyG tails (a structural motif that facilitates concatemer formation) at the 3' and 5' ends. D-type ODNs can be based on the following sequence: GGTGCAT CG ATGCAGGGGGG. Type D ODN triggers pDC maturation and IFN-α secretion but has no effect on B cells. Type C ODN is similar to type K in that it is composed entirely of phosphorothioate nucleotides but is similar to type D in that it contains a palindromic CpG motif. Type C ODN can be based on the following sequence: This type of ODN stimulates B cells to secrete IL-6 and pDCs to produce IFN-α. P-type ODNs contain two palindromic sequences, which enable 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 paragraph, bold letters in ODN sequences indicate self-complementary palindromic sequences, and CpG motifs are underlined.

[0191] 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 vaccine adjuvants.” DNA" (CpG DNA as a vaccine adjuvant), Expert Rev Vaccines (2011), 10(4): 499-511, all of which are incorporated herein by reference in their entirety for all purposes.

[0192] One or more adjuvants can be used in combination and may include, but are not limited to, aluminum hydroxide (aluminum salts), oil-in-water emulsions, water-in-oil emulsions, liposomes, and microparticles, such as poly(lactide-co-glycolide) microparticles (Shah et al., Methods Mol, 1494: 1-14, 2017). In some embodiments, the immunogenic composition further comprises an aluminum salt adjuvant that adsorbs RSV 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 about 0.35 mg Al 3+. In some embodiments, the immunogenic composition further comprises other adjuvants. Other suitable adjuvants include, but are not limited to, squalene in water emulsion (e.g., MF59 or AS03), TLR3 agonists (e.g., poly IC or poly ICLC), TLR4 agonists (e.g., bacterial lipopolysaccharide derivatives such as monophosphoryl lipid A (MPL) and / or saponins such as Quil A or QS-21, such 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 include MPL and an aluminum agent (e.g., AS04). For veterinary use and for producing antibodies in non-human animals, the mitogenic component of Freund's adjuvant (complete and incomplete) can be used.

[0193] In some embodiments, the immunogenic composition comprises a pharmaceutically acceptable excipient, including, for example, a solvent, a bulking agent, a buffer, a tonicity regulator, and a preservative (Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments, the immunogenic composition may comprise an excipient that functions as one or more of a solvent, a bulking agent, a buffer, and a tonicity regulator (e.g., sodium chloride in saline may serve as both an aqueous vehicle and a tonicity regulator).

[0194] In some embodiments, the immunogenic composition comprises an aqueous vehicle as a solvent. Suitable vehicles include, for example, sterile water, saline solution, phosphate-buffered saline, and Ringer's solution. In some embodiments, the composition is isotonic.

[0195] The immunogenic composition may include a buffer. The buffer controls the pH to suppress degradation of the active agent during processing, storage, and optional reconstitution. 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 include hydrochloric acid or sodium hydroxide. In some embodiments, the buffer maintains the pH of the composition within 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 within the range of approximately 6 to 9, wherein the lower limit is less than the upper limit.

[0196] The immunogenic composition may comprise a tonicity adjusting agent. Suitable tonicity adjusting agents include, for example, dextrose, glycerol, sodium chloride, glycerol and mannitol.

[0197] The immunogenic composition may include an extender. When the pharmaceutical composition is lyophilized before administration, an extender is particularly useful. In some embodiments, the extender is a protective agent that helps to stabilize and prevent the active agent from degrading during freezing or spray drying and / or storage. Suitable extenders are sugars (monosaccharides, disaccharides, and polysaccharides), such as sucrose, lactose, trehalose, mannitol, sorbitol, glucose, and raffinose.

[0198] The immunogenic composition may contain a preservative. Suitable preservatives include, for example, antioxidants and antimicrobials. However, in a preferred embodiment, the immunogenic composition is prepared under sterile conditions and in a single-use container and therefore does not need to include a preservative.

[0199] In some embodiments, the composition can be provided as a sterile composition. The pharmaceutical composition generally contains an effective amount of the disclosed immunogen and can be prepared by conventional methods. Generally, the amount of the immunogen in each dose of the immunogenic composition is selected to be an amount that induces an immune response without significant adverse side effects. In some embodiments, the composition can be provided in a unit dosage form for inducing an immune response in a subject. The unit dosage form contains a suitable single preselected dose to supply the drug to the subject, or two or more preselected unit doses of suitable markings or measurements, and / or a metering mechanism for administering a unit dose or multiple unit doses. In other embodiments, the composition further comprises an adjuvant.

[0200] IV. Methods of Inducing an Immune Response

[0201] In some embodiments, provided herein is a method for generating an immune response to an RSV 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 NOs: 1-16. In some embodiments, provided herein is a method for generating an immune response to an RSV surface antigen in a subject, wherein the surface antigen comprises an F 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 NOs: 1-16. In some embodiments, provided herein is a method for generating an immune response to an RSV surface antigen in a subject, wherein the surface antigen comprises a sequence selected from SEQ ID NOs: 17-47, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from SEQ ID NOs: 1-16. In some embodiments, the present invention provides a method for generating an immune response to an RSV surface antigen in a subject, wherein the surface antigen comprises the F protein of RSV or an antigenic fragment thereof, optionally, the surface antigen comprises a sequence of any one or more of SEQ ID NOs: 17-47 or an antigenic fragment thereof, and the method comprises administering to the subject an effective amount of a complex comprising a recombinant polypeptide comprising a sequence shown in any one of SEQ ID NOs: 1-16.

[0202] In some embodiments, provided herein is a method for generating an immune response to an RSV surface antigen in a subject, wherein the surface antigen comprises an F protein or an antigenic fragment thereof, the method comprising administering to the subject an effective amount of a complex comprising a recombinant polypeptide comprising a sequence selected from SEQ ID NOs: 1-16, or a combination of any two or more complexes.

[0203] Disclosed immunogens (e.g., recombinant RSV F antigens, such as trimers, proteins, nucleic acid molecules (e.g., RNA molecules) or vectors encoding the protomers of disclosed recombinant RSV F antigens as described herein, or protein nanoparticles or virus-like particles comprising disclosed recombinant RSV F antigens) can be administered to a subject to induce an immune response to the corresponding RSV F antigen in the subject. In a specific instance, the subject is a human. The immune response can be a protective immune response, such as one that suppresses the response of subsequent infection with the corresponding RSV. Inducing an immune response can also be used to treat or suppress infections and diseases relevant to the corresponding RSV.

[0204] In some embodiments, subjects who have or are at risk of RSV infection (e.g., due to exposure or potential exposure to RSV) can be selected for treatment. Following administration of the disclosed immunogens, the subject can be monitored for infection or symptoms associated with RSV, or both.

[0205] Typical subjects that are intended to be treated with the therapeutic agents and methods of the present invention include humans, as well as non-human primates and other animals. In order to identify a subject for prevention or treatment according to the methods of the present invention, generally recognized screening methods are used to determine the risk factors relevant to the target or suspected disease or illness, or to determine the situation of the subject's existing disease or illness. These screening methods include, for example, routine inspections to determine the environment, family, occupation and other such risk factors that may be relevant to the target or suspected disease or illness, and diagnostic methods for detecting and / or characterizing RSV infection, such as various ELISAs and other immunoassays. These and other conventional methods allow clinicians to use the methods and pharmaceutical compositions of the present invention to select patients who need treatment. According to these methods and principles, compositions can be administered according to the teachings of this paper or other conventional methods, as an independent prevention or treatment regimen, or as a follow-up, auxiliary or coordinated treatment regimen for other treatments.

[0206] In some embodiments, the present invention provides the therapeutic agent of the present invention.Disclosed immunogen, for example RSV F antigen, for example tripolymer, protein administration can be used for preventive or therapeutic purpose.When providing prophylactically, disclosed therapeutic agent is before any symptom, for example, provides before infection.The prophylactic administration of disclosed therapeutic agent is used to prevent or improve any secondary infection.When providing therapeutically, disclosed therapeutic agent is when disease or infection symptom outbreak or afterwards, for example, after the RSV infection symptom corresponding to RSV F antigen occurs, or provides after being diagnosed as RSV and infect.Therefore described therapeutic agent can provide before expection is exposed to RSV, so that after exposure or suspected exposure to described virus or after infecting actual beginning, weaken the severity, duration or degree of the estimate of infection and / or related disease symptom.

[0207] The immunogens and immunogenic compositions described herein are provided to the subject in an amount that effectively induces or enhances an immune response of the subject (preferably a human) to the RSV F antigen. The actual dosage of the disclosed immunogen will vary according to many factors, such as the subject's disease indications and specific state (e.g., the subject's age, size, health status, symptom level, predisposition factors, etc.), the time and route of administration, other drugs or treatments administered concurrently, and the specific pharmacology of the composition for eliciting the desired activity or biological response in the subject. The dosage regimen can be adjusted to provide optimal preventive or therapeutic response.

[0208] Immunogenic compositions comprising one or more disclosed immunogens can be used to coordinate (or initially immunize-boost) vaccination regimens or combined formulations. In certain embodiments, new combined immunogenic compositions and coordinated vaccination regimens employ separate immunogens or formulations, each directed against eliciting an antiviral immune response, such as an immune response to RSV F antigen. Separate immunogenic compositions that elicit an antiviral immune response can be combined in a multivalent immunogenic composition administered to a subject in a single vaccination step, or they can be administered separately (in a monovalent immunogenic composition) in a coordinated (or initially immunize-boost) vaccination regimen.

[0209] Can carry out several reinforcements, and each reinforcement can be different disclosed immunogens.In some instances, reinforcement can be the immunogen identical with another reinforcement or initial immunization.Described initial immunization and reinforcement can be administered as single dose or multiple doses, for example, can be administered two doses, three doses, four doses, five doses, six doses or more to the subject in a few days, weeks or months.Can also carry out multiple reinforcements, for example 1 to 5 times (for example 1,2,3,4 or 5 reinforcements) or more times.Different doses can be used in a series of sequential immunizations.For example, a relatively large dose in the first immunization, then a relatively small dose is used in reinforcement.

[0210] In some embodiments, the strengthening can be at about 2 weeks, about 3 to 8 weeks or about 4 weeks after the initial exemption, or at about several months administration after the initial exemption. In some embodiments, the strengthening can be at about 5, about 6, about 7, about 8, about 10, about 12, about 18, about 24 months after the initial exemption or at more or less time administration after the initial exemption. It is also possible to use regular additional reinforcement at the time point that is suitable, to enhance " immune memory " of the experimenter. The adequacy of the vaccination parameters such as preparation, dosage, scheme etc. selected can be determined by obtaining aliquots of serum from the experimenter and measuring antibody titer in the immunization program process. In addition, the clinical condition of the experimenter can be monitored to find required effect, such as the prevention of infection or the improvement (such as reduction of viral load) of morbid state. If this type of monitoring shows that vaccination is suboptimal, then the experimenter can be strengthened with extra immunogenic composition dosage, and the vaccination parameters can be modified in the mode of expected enhanced immune response.

[0211] In certain embodiments, the prime-boost method may comprise providing a subject with a DNA prime and protein boost vaccination regimen.The method may comprise two or more administrations of the nucleic acid molecule or protein.

[0212] For protein therapeutics, typically, each human dose comprises 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.

[0213] The amount utilized in the immunogenic composition is selected based on a subject population (such as an infant or the elderly). By standard studies, including observing the antibody titer and other responses of the subject, the optimal dose of a particular composition can be determined. It is understood that the disclosed immunogen in the immunogenic composition, such as the disclosed recombinant RSV F antigen (such as a trimer, protein), viral vector or nucleic acid molecule, can include a single dose of administration that is ineffective in eliciting an immune response but is effective after administering multiple doses, such as in a primary immunity-boosting regimen.

[0214] After administration of an immunogen disclosed herein, the subject's immune system typically responds to the immunogenic composition by producing antibodies specific for the RSV F protein peptides included in the immunogen. Such a response indicates that an immunologically effective dose has been delivered to the subject.

[0215] In some embodiments, the antibody response of experimenter is determined under the background of assessment effective dose / immunization scheme.In most cases, it is enough to assess the antibody titer in the serum or plasma obtained from the experimenter.About whether to administer booster vaccination and / or change the decision of the amount of the therapeutic agent administered to an individual may be at least partially based on antibody titer levels.Antibody titer levels can be based on, for example, immune binding assays, and the immune binding assays measure the antibody concentration combined with antigen (comprising, for example, recombinant RSV F antigen, for example, tripolymer, protein) in serum.

[0216] There is no need to completely eliminate or reduce or prevent RSV infection for the method to be considered effective. For example, compared with RSV infection when there is no immunogen, the immune response to RSV caused by one or more disclosed immunogens can reduce or suppress the required amount of RSV infection, 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 infected cells). In other embodiments, RSV replication can be reduced or suppressed by the disclosed method. There is no need to completely eliminate RSV replication for the method to be considered effective. For example, compared with RSV replication when there is no immunogen, the immune response caused by one or more disclosed immunogens can reduce the required amount of corresponding RSV replication, 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 RSV replication).

[0217] In some embodiments, the disclosed immunogens are administered to a subject concurrently with administration of an adjuvant. In other embodiments, the disclosed immunogens are administered to a subject after administration of an adjuvant and within an amount of time sufficient to induce an immune response.

[0218] A method for administering nucleic acid is to use plasmid DNA, such as direct immunization with mammalian expression plasmids. Immunization by nucleic acid constructs is well known in the art and is taught in, for example, U.S. Patent No. 5,643,578 (describing the method for initiating cell-mediated response or humoral response by introducing DNA encoding the desired antigen to immunize vertebrates) and U.S. Patent No. 5,593,972 and 5,817,637 (describing the nucleic acid sequence operably connected to encode antigens and the regulatory sequence enabling their expression). U.S. Patent No. 5,880,103 describes several methods for delivering nucleic acids encoding immunogenic peptides or other antigens to organisms. Described method includes liposome delivery of nucleic acid (or synthetic peptide itself) and immunostimulatory constructs or ISCOMS. TM , that is, in a mixture of cholesterol and Quil A TM ISCOMS is a 30-40 nm negatively charged cage-like structure that spontaneously forms after the addition of saponin. ISCOMS has been used in various infection models, including toxoplasmosis and Epstein-Barr virus-induced tumors. TM As an antigen delivery vehicle, it has produced protective immunity (Mowat and Donachie, Immunol. Today 12:383, 1991). It has been found that as little as 1 μg of the antigen encapsulated in ISCOMS TMAntigen doses in the range of 1:1 to 2 generated class I-mediated CTL responses (Takahashi et al., Nature 344:873, 1990).

[0219] In some embodiments, a plasmid DNA vaccine is used to express the disclosed immunogens in a subject. For example, a nucleic acid molecule encoding the disclosed immunogen can be administered to a subject to induce an immune response to the RSV F antigen. In some embodiments, the nucleic acid molecule can be included in 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).

[0220] In another method for immunization using nucleic acid, the disclosed recombinant RSV F antigen, such as a trimer or protein, can be expressed by an attenuated viral host or vector or a bacterial vector. Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalovirus or other viral vectors can be used to express the peptide or protein, thereby inducing CTL responses. For example, U.S. Patent No. 4,722,848 describes vaccinia virus vectors and methods that can be used for immunization protocols. BCG (BCG) provides another vector for expressing peptides (see Stover, Nature 351:456-460, 1991).

[0221] In one embodiment, nucleic acids encoding the disclosed recombinant RSV F antigens are introduced directly into cells. For example, nucleic acids can be loaded onto gold microspheres by standard methods and expressed by, for example, Bio-Rad's HELIOS TM The nucleic acid can be introduced into the skin using a device such as a gene gun. The nucleic acid can be "naked," consisting of a plasmid under the control of a strong promoter. Typically, DNA is injected into muscle, but it can also be injected directly into other sites. The injection dose is typically about 0.5 μg / kg to about 50 mg / kg, typically about 0.005 mg / kg to about 5 mg / kg (see, for example, U.S. Patent No. 5,589,466).

[0222] For example, nucleic acids can be loaded onto gold microspheres by standard methods and analyzed by a HELIOS microscope, such as that from Bio-Rad. TM The nucleic acid can be introduced into the skin using a device such as a gene gun. The nucleic acid can be "naked," consisting of a plasmid under the control of a strong promoter. Typically, DNA is injected into muscle, but it can also be injected directly into other sites. The injection dose is typically about 0.5 μg / kg to about 50 mg / kg, typically about 0.005 mg / kg to about 5 mg / kg (see, for example, U.S. Patent No. 5,589,466).

[0223] In another embodiment, an mRNA-based immunization protocol can be used to deliver nucleic acids encoding the disclosed recombinant RSV F antigens directly 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 about DNA integration into the host genome and can be translated directly in the host cytoplasm. In addition, the simple cell-free in vitro synthesis of RNA avoids the manufacturing complexity associated with viral vectors. Two exemplary formats of RNA-based vaccination that can be used to deliver nucleic acids encoding the disclosed recombinant RSV F antigens include conventional non-amplified mRNA vaccination (see, e.g., Petsch et al., “Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection,” Nature biotechnology, 30(12):1210-6, 2012) and self-amplifying mRNA vaccination (see, e.g., Geall et al., “Nonviral delivery of self-amplifying RNA vaccines,” PNAS, 109(36):14604-14609, 2012; Magini et al., “Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenges.” Challenge), PLoS One, 11(8):e0161193, 2016; and Brito et al., “Self-amplifying mRNA vaccines”, Adv Genet., 89:179-233, 2015).

[0224] In some embodiments, one or more disclosed immunogens of a subject's administration of a therapeutically effective dose are induced in the subject to induce a neutralizing immune response. In order to assess neutralizing activity, after subject immunization, serum can be collected from the subject at an appropriate time point, frozen and stored to carry out a neutralization test. The method for measuring neutralizing activity is known to those of ordinary skill in the art and is further described herein, including but not limited to plaque reduction neutralization (PRNT) assay, microneutralization assay, flow cytometry-based assay, single-cycle infection assay. In some embodiments, a group of RSV pseudoviruses can be used to measure serum neutralization activity.

[0225] In some embodiments, one or more disclosed immunogens of a subject's administration of a therapeutically effective dose are induced in the subject to induce a neutralizing immune response. In order to assess neutralizing activity, after subject immunization, serum can be collected from the subject at an appropriate time point, frozen and stored to carry out a neutralization test. The method for measuring neutralizing activity is known to those of ordinary skill in the art and is further described herein, including but not limited to plaque reduction neutralization (PRNT) assay, microneutralization assay, flow cytometry-based assay, single-cycle infection assay. In some embodiments, a group of RSV pseudoviruses can be used to measure serum neutralization activity.

[0226] In some embodiments, neutralizing antibodies against RSV are produced by the neutralizing immune response induced by the immunogen disclosed herein. In some embodiments, the neutralizing antibodies herein bind to the cell receptors or co-receptors of RSV or its components. Nucleolin is the entry co-receptor of RSV, and also mediates the cell entry of influenza virus, parainfluenza virus, some enteroviruses and the bacteria that cause tularemia. The combination of RSV-F glycoprotein and insulin-like growth factor 1 receptor (IGF1R) before fusion may also trigger the activation of protein kinase C ζ (PKCζ), thereby recruiting nucleolin to the cytoplasmic membrane from the nucleus to bind to the RSV-F on the virion. In some embodiments, the viral receptor or co-receptor is a paramyxovirus receptor or co-receptor, preferably a pneumonia virus receptor or co-receptor, more preferably a human RSV receptor or co-receptor. For example, CCR1, CCR2, CCR3, CCR4, CCR5 and / or CCR8 receptors may be involved in human RSV infection. RhoA is another example of a host cell RSV receptor or co-receptor. In some embodiments, the neutralizing antibodies herein regulate, reduce, antagonize, alleviate, block, suppress, eliminate and / or interfere with at least one RSV activity or combination or RSV receptor activity or combination in vitro, in situ and / or in vivo, such as RSV release, RSV receptor signaling, membrane RSV cutting, RSV activity, RSV production and / or synthesis. In some embodiments, immunogens disclosed herein induce neutralizing antibodies for RSV, which regulate, reduce, antagonize, alleviate, block, suppress, eliminate and / or interfere with the combination of RSV and RSV receptors or auxiliary receptors such as nucleolin, IGF1R, CCR1, CCR2, CCR3, CCR4, CCR5, CCR8 and / or RhoA.

[0227] V. Products or Kits

[0228] Also provided are articles or kits containing the provided recombinant polypeptides, proteins, and immunogenic compositions. The articles can include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, test tubes, IV bags, etc. The container can be formed from a variety of materials such as glass or plastic. In some embodiments, the container has a sterile access port. Exemplary containers include intravenous bags, vials, including those with stoppers that can be pierced by an injection needle. The articles or kits can also include a package insert indicating that the composition can be used to treat a specific illness, such as an illness as described herein (e.g., RSV infection). Alternatively, or additionally, the articles or kits can also include another or the same container containing a pharmaceutically acceptable buffer. It can also include other materials, such as other buffers, diluents, filters, needles, and / or syringes.

[0229] The label or package insert can indicate that the composition is used to treat individual RSV infection. The label or package insert accompanying the container can indicate guidance about preparation reconstruction and / or use. The label or package insert can also indicate that the preparation can be used for or is intended to be used for subcutaneous, intravenous or other modes of administration, to treat or prevent individual RSV infection.

[0230] In some embodiments, the container contains the composition itself or in combination with another composition that is effective for treating, preventing and / or diagnosing the condition. The article of manufacture or kit may include (a) a first container containing a composition (i.e., a first medicament), wherein the composition includes the immunogenic composition or a protein or recombinant polypeptide thereof; and (b) a second container containing a composition (i.e., a second medicament), wherein the composition includes other agents, such as adjuvants or other therapeutic agents, and the article of manufacture or kit further includes instructions on the label or package insert for treating the subject with the second medicament in an effective amount.

[0231] the term

[0232] Unless otherwise defined, all special 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 belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference from what is generally understood in the art.

[0233] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and do not impose a minimum length restriction. Polypeptides (including the provided receptors and other polypeptides, such as linkers or peptides) can include amino acid residues, including natural and / or non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, a polypeptide may contain modifications with respect to the native or native sequence, so long as the protein retains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or accidental, such as through mutations in the host that produces the protein or errors due to PCR amplification.

[0234] As used herein, a "subject" is a mammal, such as a human or other animal, typically a human. In some embodiments, the subject (e.g., patient) to whom one or more agents, cells, cell populations, or compositions are administered 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, teenagers, adolescents, adults, and elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[0235] As used herein, "treating" (and grammatical variations thereof) refers to the complete or partial improvement or alleviation of a disease or illness or condition, or symptoms, adverse reactions or consequences, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. The term does not mean to completely cure the disease or completely eliminate any symptoms or to be effective for all symptoms or consequences.

[0236] As used herein, "delaying disease progression" means delaying, hindering, slowing, retarding, stabilizing, inhibiting, and / or postponing the development of a disease (e.g., cancer). The length of this delay may vary depending on the history of the disease and / or the individual being treated. In some embodiments, a sufficient or significant delay may actually encompass prevention, in that the individual will not develop the disease. For example, the development of advanced cancers, such as metastases, may be delayed.

[0237] As used herein, "prevention" includes providing protection against the occurrence or recurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease. In some embodiments, provided cells and compositions are used to delay the development of a disease or slow the progression of a disease.

[0238] As used herein, "inhibiting" a function or activity means reducing the function or activity when compared to otherwise identical conditions except for the conditions or parameters of interest, or when compared to another condition. For example, a cell that inhibits tumor growth reduces the tumor growth rate compared to the tumor growth rate in the absence of the cell.

[0239] In the context of administration, an "effective amount" of an agent, such as a pharmaceutical preparation, cell, or composition, refers to an amount effective, at dosages / amounts, and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.

[0240] A "therapeutically effective amount" of an agent, such as a pharmaceutical formulation, cell, or composition, refers to the amount of dosage and time period necessary to effectively achieve a desired therapeutic outcome, such as the pharmacokinetic or pharmacodynamic effect of treating a disease, illness, or condition and / or a therapeutic effect. A therapeutically effective amount can vary depending on factors such as the disease state, age, sex, and weight of the subject, as well as the cell population being administered. In some embodiments, provided methods comprise administering the cells and / or composition in an effective amount (e.g., a therapeutically effective amount).

[0241] A "prophylactically effective amount" refers to an amount effective at the dosage and for the period of time necessary to achieve the desired prophylactic result. Typically, but not necessarily, a prophylactic effective amount is less than a therapeutically effective amount because a prophylactic dose is used in subjects before or at an early stage of disease. In cases where the tumor burden is low, the prophylactically effective amount in some aspects may be higher than the therapeutically effective amount.

[0242] As used herein, the term "about" refers to the typical error range for each value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments for that value or parameter itself.

[0243] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more."

[0244] Throughout this disclosure, various aspects of the claimed subject matter are presented in the form of ranges. It should be understood that the description in the form of ranges is merely for convenience and brevity and should not be considered as a rigid limitation on the scope of the claimed subject matter. Therefore, the description of a range should be considered as having clearly disclosed all possible sub-ranges and individual numerical values ​​within the range. For example, where a range of values ​​is provided, it should be understood that each intermediate value between the upper and lower limits of the range and any other described or intermediate values ​​within the described range are encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges can be independently included in a smaller range and are also encompassed within the claimed subject matter, subject only to any explicitly excluded limitations within the described range. When the described range includes one or more limits, the scope excluding any one or two of those included limits is also encompassed within the claimed subject matter. This applies to any range width.

[0245] 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.

[0246] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0247] Exemplary embodiments

[0248] Embodiment 1. A protein comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising a respiratory syncytial virus (RSV) F protein peptide or a fragment or epitope thereof linked to a collagen C-terminal propeptide, wherein the C-terminal propeptide of the recombinant polypeptide forms an inter-polypeptide disulfide bond.

[0249] Embodiment 2. The protein of embodiment 1, wherein the RSV belongs to subtype A or subtype B.

[0250] Embodiment 3. The protein of embodiment 1 or 2, wherein the epitope is a linear epitope or a conformational epitope.

[0251] Embodiment 4. The protein of any one of embodiments 1 to 3, wherein the F protein peptide comprises an F1 subunit peptide, an F2 subunit peptide, or any combination thereof, and the protein comprises three recombinant polypeptides.

[0252] Embodiment 5. A protein according to any one of embodiments 1 to 4, wherein the F protein peptide comprises a signal peptide, a heptad repeat C (HRC) peptide, a pep27 peptide, a fusion peptide (FP), a heptad repeat A (HRA) peptide, a domain I peptide, a domain II peptide or a heptad repeat B (HRB) peptide or any combination thereof.

[0253] Embodiment 6. The protein of any one of embodiments 1 to 5, wherein the F protein peptide comprises the F1 subunit but not the F2 subunit of the F protein, or vice versa.

[0254] Embodiment 7. A protein according to any one of embodiments 1 to 6, wherein the F protein peptide comprises the F1 subunit and the F2 subunit of the F protein, optionally in the absence of pep27, optionally wherein the F1 subunit and the F2 subunit are connected by a disulfide bond or an artificially introduced linker.

[0255] Embodiment 8. The protein of any one of embodiments 1 to 7, wherein the F protein peptide does not comprise a transmembrane (TM) domain peptide and / or a cytoplasmic (CP) domain peptide.

[0256] Embodiment 9. The protein of any one of embodiments 1 to 8, wherein the F protein peptide comprises a protease cleavage site, wherein the protease is optionally furin, trypsin, Factor Xa, or cathepsin L.

[0257] Embodiment 10. The protein of any one of embodiments 1 to 8, wherein the F protein peptide does not comprise a protease cleavage site, wherein the protease is optionally furin, trypsin, Factor Xa, or cathepsin L.

[0258] Embodiment 11. The protein of any one of embodiments 1 to 10, wherein the F protein peptide is soluble or does not directly bind to a lipid bilayer, such as a membrane or viral envelope.

[0259] Embodiment 12. The protein of any one of embodiments 1 to 11, wherein the F protein peptides are the same or different among recombinant polypeptides of the protein.

[0260] Embodiment 13. The protein of any one of embodiments 1 to 12, wherein the F protein peptide is fused directly to the C-terminal propeptide or is linked to the 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.

[0261] Embodiment 14. The protein of any one of embodiments 1 to 13, which is soluble or not directly bound to a lipid bilayer, such as a membrane or viral envelope.

[0262] Embodiment 15. The protein of any one of embodiments 1 to 14, wherein the protein is capable of forming rosette-like oligomers comprising F protein peptide trimers.

[0263] Embodiment 16. The protein of any one of embodiments 1 to 15, wherein the protein is capable of binding to a cell surface attachment factor or receptor in a subject, optionally wherein the subject is a mammal, such as a primate, eg, a human.

[0264] Embodiment 17. The protein of any one of embodiments 1 to 16, wherein the C-terminal propeptide is of human collagen.

[0265] Embodiment 18. A protein according to any one of embodiments 1 to 17, wherein the C-terminal propeptide comprises the C-terminal polypeptide of proα1(I), proα1(II), proα1(III), proα1(V), proα1(XI), proα2(I), proα2(V), proα2(XI) or proα3(XI) or a fragment thereof.

[0266] Embodiment 19. The protein of any one of embodiments 1 to 18, wherein the C-terminal propeptide is the same or different among the recombinant polypeptides.

[0267] Embodiment 20. A protein according to any one of embodiments 1 to 19, wherein the C-terminal propeptide comprises SEQ ID NO: 48 or an amino acid sequence that is at least 90% identical thereto and is capable of forming an inter-polypeptide disulfide bond and trimerizing the recombinant polypeptide.

[0268] Embodiment 21. A protein according to any one of embodiments 1 to 19, wherein the C-terminal propeptide comprises SEQ ID NO: 49 or an amino acid sequence that is at least 90% identical thereto and is capable of forming an inter-polypeptide disulfide bond and trimerizing the recombinant polypeptide.

[0269] Embodiment 22. A protein according to any one of embodiments 1 to 19, wherein the C-terminal propeptide comprises SEQ ID NO: 50 or an amino acid sequence that is at least 90% identical thereto and is capable of forming an inter-polypeptide disulfide bond and trimerizing the recombinant polypeptide.

[0270] Embodiment 23. A protein according to any one of embodiments 1 to 19, wherein the C-terminal propeptide comprises SEQ ID NO: 51 or an amino acid sequence that is at least 90% identical thereto and is capable of forming an inter-polypeptide disulfide bond and trimerizing the recombinant polypeptide.

[0271] Embodiment 24. A protein according to any one of embodiments 1 to 19, wherein the C-terminal propeptide comprises SEQ ID NO: 52 or an amino acid sequence that is at least 90% identical thereto and is capable of forming an inter-polypeptide disulfide bond and trimerizing the recombinant polypeptide.

[0272] Embodiment 25. A protein according to any one of embodiments 1 to 19, wherein the C-terminal propeptide comprises SEQ ID NO: 53 or an amino acid sequence that is at least 90% identical thereto and is capable of forming an inter-polypeptide disulfide bond and trimerizing the recombinant polypeptide.

[0273] Embodiment 26. A protein according to any one of embodiments 1 to 19, wherein the C-terminal propeptide comprises SEQ ID NO: 54 or an amino acid sequence that is at least 90% identical thereto and is capable of forming an inter-polypeptide disulfide bond and trimerizing the recombinant polypeptide.

[0274] Embodiment 27. A protein according to any one of embodiments 1 to 19, wherein the C-terminal propeptide comprises any one of SEQ ID NOs: 55-59 or an amino acid sequence that is at least 90% identical thereto and is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0275] Embodiment 28. A protein according to any one of embodiments 1 to 19, wherein the C-terminal propeptide comprises SEQ ID NO: 60 or an amino acid sequence that is at least 90% identical thereto and is capable of forming an inter-polypeptide disulfide bond and trimerizing the recombinant polypeptide.

[0276] Embodiment 29. A protein according to any one of embodiments 1 to 19, wherein the C-terminal propeptide comprises any one of SEQ ID NOs: 61-63 or an amino acid sequence that is at least 90% identical thereto and is capable of forming inter-polypeptide disulfide bonds and trimerizing the recombinant polypeptide.

[0277] Embodiment 30. The protein of any one of embodiments 1 to 29, wherein the C-terminal propeptide comprises an amino acid sequence comprising a glycine-XY repeat sequence linked to the N-terminus of any one of SEQ ID NOs: 48-63, wherein X and Y are independently any amino acid, optionally proline or hydroxyproline, or an amino acid sequence having at least 90% identity thereto, capable of forming an interpolypeptide disulfide bond and allowing trimerization of the recombinant polypeptide.

[0278] Embodiment 31. A protein according to any one of embodiments 1 to 30, wherein the F protein peptide in each recombinant polypeptide is in a pre-fusion conformation or a post-fusion conformation, optionally wherein the protein comprises a rosette-like oligomer comprising a crutch-shaped rod-shaped F protein peptide trimer.

[0279] Embodiment 32. The protein of any one of embodiments 1 to 31, wherein the F protein peptide in each recombinant polypeptide comprises any one of SEQ ID NOs: 17-47 or an amino acid sequence at least 80% identical thereto.

[0280] Embodiment 33. The protein of any one of embodiments 1 to 31, wherein the recombinant polypeptide comprises any one of SEQ ID NOs: 1-16 or an amino acid sequence at least 80% identical thereto.

[0281] Embodiment 34. An immunogen comprising the protein of any one of embodiments 1 to 33.

[0282] Embodiment 35. A protein nanoparticle comprising the protein according to any one of embodiments 1 to 33 directly or indirectly attached to the nanoparticle.

[0283] Embodiment 36. A virus-like particle (VLP) comprising the protein of any one of embodiments 1 to 33.

[0284] Embodiment 37. An isolated nucleic acid encoding one, two, three or more recombinant polypeptides of the protein according to any one of embodiments 1 to 33.

[0285] Embodiment 38. The isolated nucleic acid of embodiment 37, wherein the polypeptide encoding the F protein peptide is fused in frame with the polypeptide encoding the collagen C-terminal propeptide.

[0286] Embodiment 39. The isolated nucleic acid of embodiment 37 or 38, wherein the isolated nucleic acid is operably linked to a promoter.

[0287] Embodiment 40. The isolated nucleic acid of any one of embodiments 37 to 39, wherein the isolated nucleic acid is a DNA molecule.

[0288] Embodiment 41. The isolated nucleic acid of any one of embodiments 37 to 39, which is an RNA molecule, optionally an mRNA molecule such as a nucleoside-modified mRNA, a non-amplified mRNA, a self-amplified mRNA, or a trans-amplified mRNA.

[0289] Embodiment 42. A vector comprising the isolated nucleic acid according to any one of embodiments 37 to 41.

[0290] Embodiment 43. The vector of embodiment 42, wherein the vector is a viral vector.

[0291] Embodiment 44. A virus, pseudovirus or cell comprising the vector of embodiment 42 or 43, optionally wherein the virus or cell has a recombinant genome.

[0292] Embodiment 45. An immunogenic composition comprising the protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus or cell of any one of embodiments 1 to 44, and a pharmaceutically acceptable carrier.

[0293] Embodiment 46. A vaccine comprising the immunogenic composition of embodiment 45 and optionally an adjuvant, wherein the vaccine is optionally a subunit vaccine, and / or optionally wherein the vaccine is a prophylactic and / or therapeutic vaccine.

[0294] Embodiment 47. The vaccine of embodiment 46, wherein the vaccine comprises a plurality of different adjuvants.

[0295] Embodiment 48. A method of producing a protein, the method comprising: expressing the isolated nucleic acid or vector of any one of embodiments 37 to 43 in a host cell to produce the protein of any one of embodiments 1 to 33; and purifying the protein.

[0296] Embodiment 49. A protein produced according to the method of embodiment 48.

[0297] Embodiment 50. A method for generating an immune response to an F protein peptide of RSV or a fragment or epitope thereof in a subject, 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 embodiments 1 to 47 and 49 to generate the immune response.

[0298] Embodiment 51. The method of embodiment 50, for treating or preventing RSV infection.

[0299] Embodiment 52. The method of embodiment 50 or 51, wherein generating the immune response inhibits or reduces RSV replication in the subject.

[0300] Embodiment 53. A method according to any one of embodiments 50 to 52, wherein the immune response comprises a cell-mediated response and / or a humoral response, optionally comprising the production of one or more neutralizing antibodies, such as polyclonal antibodies or monoclonal antibodies.

[0301] Embodiment 54. The method of any one of embodiments 50 to 53, wherein the immune response is directed against a peptide of the RSV F protein or a fragment or epitope thereof, but not against the C-terminal propeptide.

[0302] Embodiment 55. The method of any one of embodiments 50 to 54, wherein the administering does not result in antibody-dependent enhancement (ADE) due to the subject's prior exposure to one or more RSVs.

[0303] Embodiment 56. The method of any one of embodiments 50 to 55, wherein the administering does not result in antibody-dependent enhancement (ADE) upon subsequent exposure of the subject to one or more RSVs.

[0304] Embodiment 57. The method according to any one of embodiments 50 to 56, further comprising a priming step and / or a boosting step.

[0305] Embodiment 58. The method of any one of embodiments 50 to 57, wherein the administering step is performed topically, transdermally, subcutaneously, intradermally, orally, intranasally (e.g., intranasal spray), intratracheally, sublingually, buccally, rectally, vaginally, by inhalation, intravenously (e.g., intravenous injection), intraarterially, intramuscularly (e.g., intramuscular injection), intracardially, intraosseously, intraperitoneally, transmucosally, intravitreally, subretinally, intraarticularly, periarticularly, topically, or transdermally.

[0306] Embodiment 59. The method of any one of embodiments 50 to 58, wherein the effective amount is administered as a single dose or a series of doses separated by one or more intervals.

[0307] Embodiment 60. The method of any one of embodiments 50 to 59, wherein the effective amount is administered without an adjuvant.

[0308] Embodiment 61. The method of any one of embodiments 50 to 59, wherein the effective amount is administered with an adjuvant.

[0309] Embodiment 62. A method comprising administering to a subject an effective amount of a protein according to any one of embodiments 1 to 33 to produce neutralizing antibodies or neutralizing antiserum against RSV in the subject.

[0310] Embodiment 63. The method of embodiment 62, wherein the subject is a mammal, optionally a human or a non-human primate.

[0311] Embodiment 64. The method of embodiment 62 or 63, further comprising isolating the neutralizing antibody or neutralizing antiserum from the subject.

[0312] Embodiment 65. The method of embodiment 64, further comprising administering an effective amount of the isolated neutralizing antibody or neutralizing antiserum to a human subject by passive immunization to prevent or treat RSV infection.

[0313] Embodiment 66. A method according to any one of embodiments 62 to 65, wherein the neutralizing antibodies or neutralizing antiserum against the RSV comprises polyclonal antibodies against the RSV F protein peptide or a fragment or epitope thereof, optionally wherein the neutralizing antibodies or neutralizing antiserum does not contain or is substantially free of antibodies against the collagen C-terminal propeptide.

[0314] Embodiment 67. The method of any one of embodiments 62 to 65, wherein the neutralizing antibodies comprise monoclonal antibodies to the RSV F protein peptide or fragment or epitope thereof, optionally wherein the neutralizing antibodies are free or substantially free of antibodies to the collagen C-terminal propeptide.

[0315] Embodiment 68. A protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine according to any one of embodiments 1 to 47 and 49 for inducing an immune response to RSV in a subject, and / or for treating or preventing RSV infection.

[0316] Embodiment 69. Use of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine according to any one of embodiments 1 to 47 and 49 for inducing an immune response to RSV in a subject, and / or for treating or preventing RSV infection.

[0317] Embodiment 70. Use of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine according to any one of embodiments 1 to 47 and 49 for the manufacture of a medicament or prophylactic agent for inducing an immune response to RSV in a subject and / or for treating or preventing RSV infection.

[0318] 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 that can specifically bind to the F protein peptide or a fragment or epitope thereof of the RSV.

[0319] Embodiment 72. The method of embodiment 71, wherein the analyte is an antibody, receptor, or cell that recognizes the F protein peptide or fragment or epitope thereof.

[0320] Embodiment 73. The method of embodiment 71 or 72, wherein said binding indicates the presence of said analyte in said sample and / or that the subject from which said sample was derived is infected with said RSV.

[0321] Embodiment 74. A kit comprising a protein according to any one of embodiments 1 to 33 and a substrate, liner or vial containing or immobilizing the protein, optionally wherein the kit is an ELISA or lateral flow assay kit.

[0322] Example

[0323] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0324] Example 1: Production of recombinant polypeptides containing RSV F protein peptides

[0325] A secreted form of a recombinant polypeptide containing RSV F protein peptides was generated as a vaccine candidate.

[0326] The RSV F glycoprotein construct was derived from the RSV A2 strain (accession number AAC55970). The sequence encoding residues 1 to 520 of the F protein peptide was codon-optimized, synthesized, and subcloned into the Hind III and Bgl II sites of a mammalian expression vector encoding human α1 collagen C propeptide. Figure 1A shows a schematic diagram of an exemplary recombinant polypeptide.

[0327] The recombinant plasmid was transfected into GH-CHO (dfhr-) cells, and hypoxanthine thymidine (HT) (Invitrogen) was selected, and the gene was gradually amplified with MTX (Sigma) concentration increase to express the fusion protein at high titer under serum-free culture using CD007-4 TM1 culture medium (Jianshun Biosciences). The exemplary recombinant polypeptide was initially purified using salt gradient elution by affinity binding with Endo180, and then further purified on a Superdex 200 gel filtration column (GE Healthcare). The purity of the exemplary recombinant polypeptide comprising the RSV F peptide was determined according to the manufacturer's instructions (Sepax Technologies).

[0328] In a serum-free fed-batch culture process, it was found that the production titer of disulfide-linked fusion peptides (e.g., trimers) was as high as about 0.15 g / L ( FIG. 1B ). Conditioned medium containing the trimerized recombinant polypeptide was first purified by affinity binding to the Fc-tagged collagen receptor uPARAP / Endo180 (a member of the mannose receptor family) pre-captured on a protein A chromatography column (Thomas et al., (2005) J. Biol. Chem. 280, 22596-22605), followed by gel filtration chromatography. SEC-HPLC analysis showed that the purity of the exemplary recombinant polypeptide trimer was about 95% ( FIG. 1C ).

[0329] The exemplary trimerized recombinant polypeptide (0.1 μg) of the purification was separated under non-reducing or reducing conditions on 8% SDS-PAGE and transferred to a PVDF membrane. After blocking with PBS containing 5% skim milk, the membrane was incubated with mouse anti-RSV F monoclonal antibody (Millipore) or palivizumab (AbbVie) and rabbit anti-type I procollagen C propeptide (CICP) polyclonal antibody (Millipore). 2 μg of purified exemplary recombinant polypeptide trimer was loaded for Coomassie blue staining. FIG2A shows that the fusion peptide is expressed as a covalently linked trimeric protein.

[0330] The structural identity and integrity of the purified recombinant polypeptide were further confirmed by Western blot analysis using antibodies specific for F and the trimerization peptide ( FIG. 2A ).

[0331] The purified recombinant polypeptide trimers were analyzed by negative staining electron microscopy. The purified recombinant polypeptide was diluted to 50 μg / mL and applied to a carbon-coated 400 CU grid that had been glow-discharged at 12 mA for 20 seconds. The grid was negatively stained with 1% (w / v) uranyl formate for 20 seconds. Samples were collected using a FEI Tecnai spirit electron microscope operated at 120 KeV, and micrographs were taken at 180,000 times magnification. Figure 2B shows the recombinant polypeptide trimers aggregated in the protein, with the macrostructure mainly in the form of rosette-like oligomers, which is similar to the observations for the full-length F protein (Calder et al., (2000) Virology 271, 122-131; Smith et al., (2012) PloS One 7, e50852). The molecules in the rosette are cane-shaped rods with their wider ends protruding outward from the center, consistent with the reported post-fusion conformation of F (Swanson et al., (2011) Prot. Natl. Acad. Sci. USA 108, 9619-9624).

[0332] Affinity binding of the monoclonal antibody palivizumab to an exemplary recombinant polypeptide trimer was measured using biolayer interferometry (BLI) on a fortebio OCTET QKe system (Pall). 5 μg / mL palivizumab was directly immobilized on a protein A sensor, then equilibrated in PBS and placed into wells containing a 2-fold dilution of the fusion peptide (starting at 20 μg / mL). Dissociation was performed by immersion in PBS, and data were processed into a 1:1 binding model by subtracting a buffer reference value using data analysis software.

[0333] The binding affinity of palivizumab to the purified exemplary recombinant polypeptides exhibited a K of less than 1 picomolar D ( FIG. 2C ), indicating that antigenic site II is exposed on the exemplary fusion polypeptide.

[0334] Example 2: Functional Characterization of Recombinant Polypeptides Containing RSV F Protein Peptides

[0335] To evaluate the immunogenicity and protective efficacy of the exemplary recombinant polypeptides produced as described in Example 1, randomized BALB / c mice were immunized intramuscularly twice on days 0 and 21 with one of three doses (1, 6, and 30 μg) of the exemplary fusion polypeptide with or without aluminum adsorption (Imject aluminum adjuvant (Thermo Scientific)). Another group immunized with PBS served as a control group. Serum was collected on day 49 and then treated with 1×10 6 Animals were challenged intranasally with 10 pfu RSV A2 strain (Figure 3A). Animals were observed daily and euthanized on day 54 to collect lung tissue.

[0336] Serum was evaluated with enzyme-linked immunosorbent assay (ELISA). In brief, 96-well plates were coated overnight with the exemplary fusion peptide (in PBS) of 2 μg / mL purification at 4°C and blocked with 1mg / mL BSA. The plate was washed with PBST and subsequently incubated at room temperature for 2 hours with a serial 2-fold dilution (1:64 to 1:262,144) of serum. The antibody bound was detected at room temperature for 1 hour by HRP-conjugated goat anti-mouse IgG (SouthernBiotech). Enzymatic reaction was carried out with TMB (Thermo) and the absorbance at 450nm was recorded by adding 2M HCl to stop the enzymatic reaction. The PBS immune inoculation mouse serum using the same dilution was used as the negative control group, and the antibody titer was defined as the serum dilution that caused the ratio of OD RSV F trimer to OD PBS to be 2.0.

[0337] Serum analysis showed that all groups immunized with the exemplary recombinant polypeptides had high levels of RSV F protein peptide-specific antibodies in a dose-dependent and adjuvant-dependent manner ( FIG. 3B ). The neutralizing activity of anti-F antibodies was measured by microneutralization assay.

[0338] RSV microneutralization assays were performed using HeLa cells and RSV A2 strain. Serum was heat-inactivated at 56°C for 30 minutes and serially diluted in serum-free DMEM (50 μL / well) in a 96-well cell culture plate. An equal volume of virus (1,000 pfu / mL, prepared in serum-free DMEM) was added to the plate, and the serum / virus mixture was incubated at 37°C for 1 hour. Approximately 5 × 10 4100 μL of DMEM supplemented with 10% FBS was added to each HeLa cell and incubated at 37°C until the positive control (virus only) well showed 100% CPE. The plate was washed with PBST and fixed with PBS containing 80% pre-cooled acetone for 10 minutes. 100 ng / mL Palivizumab was added to the wells, blocked with 1 mg / mL BSA for 1 hour, and incubated at room temperature for 2 hours. After three washes, HRP-conjugated goat anti-human IgG (SouthernBiotech) was added, the enzymatic reaction was performed, and the OD at 450 nm was recorded. The dilution that caused 50% inhibition of CPE formation was determined as the neutralizing antibody titer.

[0339] Microneutralization assays showed that the anti-F antibodies induced by the exemplary recombinant polypeptides had potent RSV neutralizing activity. At the same dose of the exemplary recombinant polypeptides, co-injection with an aluminum adjuvant induced higher neutralizing antibody titers. These results were consistent with those of the anti-F antibodies (Figure 3C).

[0340] The protective efficacy of the exemplary recombinant polypeptides was evaluated by measuring viral replication in the lungs on day 5 after viral challenge. Five days after intranasal RSV challenge, mice were sacrificed, and the harvested left lungs were weighed and homogenized in 1 mL of serum-free DMEM. The homogenates were clarified by centrifugation at 1,000 × g for 10 minutes at 4°C, and the virus in the lung samples was titrated by plaque assay as described above.

[0341] The results showed that all mice immunized with the exemplary recombinant polypeptides were fully protected from RSV replication and no virus was detected, while the PBS-immunized control group showed high levels of viral load in the lungs ( FIG. 3D ).

[0342] Because antigenic site II is exposed on the exemplary recombinant polypeptides (see Example 1 and FIG. 2C ), a palivizumab competitive ELISA was performed to determine whether the exemplary recombinant polypeptides induced antibodies directed against this site.

[0343] Use coated 5×10 6Palivizumab competitive ELISA was performed on 96-well ELISA plates containing 50 pfu / mL heat-inactivated RSV (HI-RSV, in 50 mM carbonate-bicarbonate buffer, pH 9.2) and incubated overnight at 4°C. The uncoated surface was blocked with 1 mg / mL BSA. Two-fold dilutions of the serum mixture (1:32 to 1:4,096) were added to the wells together with 100 ng / mL palivizumab and incubated at room temperature for 2 hours. Bound palivizumab was detected using HRP-conjugated goat anti-human IgG (SouthernBiotech) and TMB substrate. The wells containing PBS-immunized mouse serum represent non-competitive positive controls, and the percentage of inhibition is calculated as ((OD PBS-OD RSV F trimer) / OD PBS)×100%. Competitive binding titers are expressed as the dilution that causes 50% inhibition.

[0344] In the presence or absence of aluminum adjuvant, inhibition of palivizumab binding to heat-inactivated RSV (HI-RSV) particles was observed in all serum samples collected from mice immunized with RSV F trimers (Fig. 3E). These results demonstrate that the neutralizing antibodies induced by exemplary fusion peptide immunization can effectively prevent RSV replication in the lungs of irritated mice at least by targeting antigenic site II of the virus.

[0345] Because FI-RSV vaccination unexpectedly enhances disease severity (Kim et al., (1969) Am. J, Epidemiol. 89, 422-434; Chin et al., (1969) Am. J. Epidemiol. 89, 449-463), safety monitoring is a top priority for RSV vaccine candidate development (Murata, Y. (2009) Clin. Lab. Med. 29, 725-739). Histological examination of lung tissue obtained from immunized mice 5 days after challenge was performed to assess the safety of the exemplary recombinant polypeptides.

[0346] The collected right lung tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 μm, and stained with H&E for histopathological evaluation. Photos were taken at 200× magnification under a Nikon microscope.

[0347] H&E staining shows that the control group of PBS immunization shows a certain degree of alveolitis, bronchiolitis and perivascular inflammation, and is accompanied by obvious inflammatory cell infiltration (Fig. 4).By contrast, no matter there is or do not exist aluminum agent adjuvant, limited immune cell infiltration and no obvious pathological change (Fig. 4) occur in the animal of exemplary recombinant polypeptide immunization at any dosage.This result supports that exemplary recombinant polypeptide immunization can protect against vaccine-mediated enhanced disease after RSV infection.

[0348] The present invention is not intended to be limited in scope to the specific disclosed embodiments, which are provided for purposes of illustration, for example, of illustrating various aspects of the invention. Various modifications of the compositions and methods will be apparent from the description and teachings herein. Such variations may be implemented without departing from the true scope and spirit of the present disclosure and are intended to be within the scope of the present disclosure.

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[0369] Sequence Listing <110> Sichuan Clover Biopharmaceutical Co., Ltd. <120> RSV vaccine compositions, methods, and uses thereof <130> 16576-20002.42 <140> Not yet allocated <141> Submit together <150> PCT / CN2021 / 087045 <151> 2021-04-13 <150> PCT / CN2020 / 095295 <151> 2020-06-10 <160> 63 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 806 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 1Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser1 5 10 15Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile20 25 30Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp35 40 45Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala50 55 60Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn65 70 75 80Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn85 90 95Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Arg Phe100 105 110Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala115 120 125Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser130 135 140Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn GlyVal145 150 155 160SerVal Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys165 170 175Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile180 185 190Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile195 200 205Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr210 215 220Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro225 230 235 240Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val245 250 255Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu260 265 270Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys275 280 285Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly290 295 300Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn305 310 315 320Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln325 330 335Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser340 345 350Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys355 360 365Lys Ile Met Thr Ser Lys Thr AspVal Ser Ser Ser Val Ile Thr Ser370 375 380Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser385 390 395 400Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr405 410 415Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr420 425 430Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro435 440 445Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp450 455 460Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe465 470 475 480Ile Arg Lys Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys Arg485 490 495Ser Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg500 505 510Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro515 520 525Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro530 535 540Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala545 550 555 560Asn Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr565 570 575Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser ProGlu Gly580 585 590Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His595 600 605Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys610 615 620Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr625 630 635 640Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile645 650 655Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met660 665 670Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala675 680 685Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala690 695 700Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp705 710 715 720Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn725 730 735Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val740 745 750Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val755 760 765Ile Glu Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val770 775 780Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val785 790 795 800Gly ProVal Cys Phe Leu805<210> 2<211> 831<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 2Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr1 5 10 15Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe20 25 30Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu35 40 45Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile50 55 60Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys65 70 75 80Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu85 90 95Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro100 105 110Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr115 120 125Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val130 135 140Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu145 150Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln LeuLeu Pro Ile Val Asn195 200 205Lys Gln Ser Cys Ser Ile Ser Asn Ile Ala Thr Val Ile Glu Phe Gln210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu245 250 255Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys260 265 270Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe340 345 350Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val370 375 380Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys405 410415Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile420 425 430Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp435 440 445Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly450 455 460Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro465 470 475 480Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn485 490 495Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu500 505 510Leu His Asn Val Asn Ala Gly Lys Arg Ser Asn Gly Leu Pro Gly Pro515 520 525Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val530 535 540Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser545 550 555 560Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala565 570 575His Asp Gly Gly Arg Tyr Tyr Arg Ala Asn Asp Ala Asn Val Val Arg580 585 590Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser Gln Gln595 600 605Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg610 615 620Thr Cys Arg Asp Leu Lys MetCys His Ser Asp Trp Lys Ser Gly Glu 625 630 635 640 Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val 645 650 655 Phe Cys Asn Met Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro 660 665 670 Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys 675 680 685 Arg His Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln Phe Glu 690 695 700 Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr 705 710 715 720 Phe Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His 725 730 735 Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys 740 745 750 Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu 755 760 765 Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser 770 775 780 His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys 785 790 795 800 Thr Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala 805 810 815 Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu 820 825 830 <210> 3 <211> 806 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400>3Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser1 5 10 15Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile20 25 30Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp35 40 45Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala50 55 60Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn65 70 75 80Arg Ala Arg Ala Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn85 90 95Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Arg Phe100 105 110Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala115 120 125Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser130 135 140Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val145 150 155 160Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys165 170 175Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile180 185 190Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile195 200 205Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr ThrPro Val Ser Thr210 215 220Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro225 230 235 240Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val245 250 255Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu260 265 270Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys275 280 285Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly290 295 300Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn305 310 315 320Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln325 330 335Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser340 345 350Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys355 360 365Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser370 375 380Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser385 390 395 400Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr405 410 415Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr420 425430Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro435 440 445Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp450 455 460Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe465 470 475 480Ile Arg Lys Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys Arg485 490 495Ser Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg500 505 510Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro515 520 525Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro530 535 540Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala545 550 555 560Asn Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr565 570 575Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly580 585 590Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His595 600 605Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys610 615 620Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr625 630 635 640Cys Val Tyr Pro Thr GlnPro Ser Val Ala Gln Lys Asn Trp Tyr Ile 645 650 655 Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met 660 665 670 Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala 675 680 685 Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala 690 695 700 Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp 705 710 715 720 Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn 725 730 735 Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val 740 745 750 Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val 755 760 765 Ile Glu Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val 770 775 780 Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val 785 790 795 800 Gly Pro Val Cys Phe Leu 805 <210> 4 <211> 831 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 4 Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr 1 5 10 15 Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly TyrLeu Ser Ala Leu35 40 45Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile50 55 60Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys65 70 75 80Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu85 90 95Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Ala Glu Leu Pro100 105 110Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr115 120 125Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val130 135 140Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu145 150 155 160Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys165 170 175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn195 200 205Lys Gln Ser Cys Ser Ile Ser Asn Ile Ala Thr Val Ile Glu Phe Gln210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu245 250 255Leu Leu SerLeu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys260 265 270Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe340 345 350Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val370 375 380Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys405 410 415Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile420 425 430Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp435 440 445Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly450 455 460Lys Ser Leu Tyr Val Lys Gly Glu Pro IleIle Asn Phe Tyr Asp Pro465 470 475 480Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn485 490 495Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu500 505 510Leu His Asn Val Asn Ala Gly Lys Arg Ser Asn Gly Leu Pro Gly Pro515 520 525Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val530 535 540Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser545 550 555 560Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala565 570 575His Asp Gly Gly Arg Tyr Tyr Arg Ala Asn Asp Ala Asn Val Val Arg580 585 590Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser Gln Gln595 600 605Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg610 615 620Thr Cys Arg Asp Leu Lys Met Cys His Ser Asp Trp Lys Ser Gly Glu625 630 635 640Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val645 650 655Phe Cys Asn Met Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro660 665 670Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys675680 685Arg His Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln Phe Glu690 695 700Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr705 710 715 720Phe Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His725 730 735Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys740 745 750Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu755 760 765Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser770 775 780His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys785 790 795 800Thr Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala805 810 815Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu820 825 830<210> 5<211> 806<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 5Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser1 5 10 15Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile20 25 30Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp35 40 45Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys TyrLys Asn Ala50 55 60Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn65 70 75 80Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn85 90 95Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Ala Phe100 105 110Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala115 120 125Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser130 135 140Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val145 150 155 160Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys165 170 175Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile180 185 190Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile195 200 205Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr210 215 220Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro225 230 235 240Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val245 250 255Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu260 265 270Ala Tyr ValVal Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys275 280 285Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly290 295 300Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn305 310 315 320Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln325 330 335Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser340 345 350Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys355 360 365Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser370 375 380Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser385 390 395 400Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr405 410 415Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr420 425 430Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro435 440 445Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp450 455 460Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe465 470 475 480Ile Arg Lys Ser Asp Glu Leu Leu HisAsn Val Asn Ala Gly Lys Arg485 490 495Ser Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg500 505 510Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro515 520 525Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro530 535 540Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala545 550 555 560Asn Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr565 570 575Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly580 585 590Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His595 600 605Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys610 615 620Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr625 630 635 640Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile645 650 655Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met660 665 670Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala675 680 685Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala690695 700Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp705 710 715 720Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn725 730 735Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val740 745 750Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val755 760 765Ile Glu Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val770 775 780Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val785 790 795 800Gly Pro Val Cys Phe Leu805<210> 6<211> 831<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 6Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr1 5 10 15Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe20 25 30Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu35 40 45Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile50 55 60Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys65 70 75 80Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu85 90 95Met Gln Ser Thr Pro Ala ThrAsn Asn Arg Ala Arg Arg Glu Leu Pro100 105 110Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr115 120 125Leu Ser Lys Lys Arg Lys Arg Ala Phe Leu Gly Phe Leu Leu Gly Val130 135 140Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu145 150 155 160Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys165 170 175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn195 200 205Lys Gln Ser Cys Ser Ile Ser Asn Ile Ala Thr Val Ile Glu Phe Gln210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu245 250 255Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys260 265 270Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His ThrSer Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe340 345 350Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val370 375 380Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys405 410 415Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile420 425 430Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp435 440 445Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly450 455 460Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro465 470 475 480Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn485 490 495Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu500 505 510Leu His Asn Val Asn Ala Gly Lys Arg Ser Asn Gly Leu Pro Gly Pro515 520 525Ile GlyPro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val530 535 540Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser545 550 555 560Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala565 570 575His Asp Gly Gly Arg Tyr Tyr Arg Ala Asn Asp Ala Asn Val Val Arg580 585 590Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser Gln Gln595 600 605Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg610 615 620Thr Cys Arg Asp Leu Lys Met Cys His Ser Asp Trp Lys Ser Gly Glu625 630 635 640Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val645 650 655Phe Cys Asn Met Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro660 665 670Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys675 680 685Arg His Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln Phe Glu690 695 700Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr705 710 715 720Phe Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His725 730 735Cys Lys Asn Ser Val Ala Tyr MetAsp Gln Gln Thr Gly Asn Leu Lys740 745 750Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu755 760 765Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser770 775 780His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys785 790 795 800Thr Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala805 810 815Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu820 825 830<210> 7<211> 806<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 7Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser1 5 10 15Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile20 25 30Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp35 40 45Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala50 55 60Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn65 70 75 80Arg Ala Arg Ala Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn85 90 95Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Ala Phe100 105 110Leu Gly Phe Leu Leu GlyVal Gly Ser Ala Ile Ala Ser Gly Val Ala115 120 125Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser130 135 140Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val145 150 155 160Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys165 170 175Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile180 185 190Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile195 200 205Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr210 215 220Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro225 230 235 240Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val245 250 255Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu260 265 270Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys275 280 285Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly290 295 300Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn305 310 315 320Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu ThrCys Lys Val Gln325 330 335Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser340 345 350Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys355 360 365Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser370 375 380Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser385 390 395 400Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr405 410 415Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr420 425 430Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro435 440 445Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp450 455 460Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe465 470 475 480Ile Arg Lys Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys Arg485 490 495Ser Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg500 505 510Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro515 520 525Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro530 535 540GlnPro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala545 550 555 560Asn Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr565 570 575Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly580 585 590Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His595 600 605Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys610 615 620Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr625 630 635 640Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile645 650 655Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met660 665 670Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala675 680 685Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala690 695 700Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp705 710 715 720Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn725 730 735Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val740 745 750Thr Val Asp Gly Cys Thr SerHis Thr Gly Ala Trp Gly Lys Thr Val755 760 765Ile Glu Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val770 775 780Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val785 790 795 800Gly Pro Val Cys Phe Leu805<210> 8<211> 831<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 8Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr1 5 10 15Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe20 25 30Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu35 40 45Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile50 55 60Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys65 70 75 80Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu85 9150 155 160Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys165 170 175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn195 200 205Lys Gln Ser Cys Ser Ile Ser Asn Ile Ala Thr Val Ile Glu Phe Gln210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu245 250 255Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys260 265 270Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe340 345 350Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp355 360 365Thr Met Asn Ser LeuThr Leu Pro Ser Glu Val Asn Leu Cys Asn Val370 375 380Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys405 410 415Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile420 425 430Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp435 440 445Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly450 455 460Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro465 470 475 480Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn485 490 495Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu500 505 510Leu His Asn Val Asn Ala Gly Lys Arg Ser Asn Gly Leu Pro Gly Pro515 520 525Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val530 535 540Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser545 550 555 560Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala565 570 575His Asp Gly Gly Arg Tyr Tyr Arg Ala Asn AspAla Asn Val Val Arg580 585 590Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser Gln Gln595 600 605Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg610 615 620Thr Cys Arg Asp Leu Lys Met Cys His Ser Asp Trp Lys Ser Gly Glu625 630 635 640Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val645 650 655Phe Cys Asn Met Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro660 665 670Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys675 680 685Arg His Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln Phe Glu690 695 700Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr705 710 715 720Phe Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His725 730 735Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys740 745 750Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu755 760 765Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser770 775 780His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys785 790 795800Thr Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala805 810 815Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu820 825 830<210> 9<211> 806<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 9Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser1 5 10 15Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile20 25 30Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp35 40 45Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala50 55 60Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn65 70 75 80Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn8Lys165 170 175Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Pro Asn Ile180 185 190Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile195 200 205Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr210 215 220Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro225 230 235 240Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val245 250 255Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu260 265 270Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys275 280 285Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly290 295 300Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn305 310 315 320Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln325 330 335Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser340 345 350Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys355 360 365Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser370 375 380Leu Gly Ala IleVal Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser385 390 395 400Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr405 410 415Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr420 425 430Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro435 440 445Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp450 455 460Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe465 470 475 480Ile Arg Lys Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys Arg485 490 495Ser Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg500 505 510Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro515 520 525Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro530 535 540Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala545 550 555 560Asn Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr565 570 575Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly580 585 590Ser Arg Lys Asn Pro Ala Arg Thr Cys ArgAsp Leu Lys Met Cys His595 600 605Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys610 615 620Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr625 630 635 640Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile645 650 655Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met660 665 670Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala675 680 685Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala690 695 700Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp705 710 715 720Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn725 730 735Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val740 745 750Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val755 760 765Ile Glu Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val770 775 780Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val785 790 795 800Gly Pro Val Cys Phe Leu805<210> 10<211> 831<212> PRT<213>Artificial Sequence <220><223> Synthetic construct <400> 10Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr1 5 10 15Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe20 25 30Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu35 40 45Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile50 55 60Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys65 70 75 80Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu85 90 95Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro100 105 110Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr115 120 125Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val130 135 140Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu145 150 155 160Pro Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys165 170 175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn195 200 205Lys Gln Ser Cys SerIle Pro Asn Ile Ala Thr Val Ile Glu Phe Gln210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu245 250 255Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys260 265 270Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe340 345 350Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val370 375 380Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys405 410 415Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn LysAsn Arg Gly Ile Ile420 425 430Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp435 440 445Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly450 455 460Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro465 470 475 480Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn485 490 495Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu500 505 510Leu His Asn Val Asn Ala Gly Lys Arg Ser Asn Gly Leu Pro Gly Pro515 520 525Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val530 535 540Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser545 550 555 560Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala565 570 575His Asp Gly Gly Arg Tyr Tyr Arg Ala Asn Asp Ala Asn Val Val Arg580 585 590Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser Gln Gln595 600 605Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg610 615 620Thr Cys Arg Asp Leu Lys Met Cys His Ser Asp Trp Lys Ser Gly Glu625 630 635640 Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val645 650 655Phe Cys Asn Met Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro660 665 670Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys675 680 685Arg His Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln Phe Glu690 695 700Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr705 710 715 720Phe Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His725 730 735Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys740 745 750Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu755 760 765Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser770 775 780His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys785 790 795 800Thr Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala805 810 815Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu820 825 830<210> 11<211> 806<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 11Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser ThrCys Ser Ala Val Ser1 5 10 15Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile20 25 30Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp35 40 45Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala50 55 60Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn65 70 75 80Arg Ala Arg Ala Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn85 90 95Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Ala Phe100 105 110Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala115 120 125Val Ser Lys Val Leu His Leu Pro Gly Glu Val Asn Lys Ile Lys Ser130 135 140Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val145 150 155 160Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys165 170 175Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Pro Asn Ile180 185 190Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile195 200 205Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr210 215 220Tyr Met Leu ThrAsn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro225 230 235 240Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val245 250 255Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu260 265 270Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys275 280 285Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly290 295 300Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn305 310 315 320Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln325 330 335Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser340 345 350Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys355 360 365Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser370 375 380Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser385 390 395 400Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr405 410 415Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr420 425 430Tyr Val Asn Lys Gln Glu Gly Lys Ser LeuTyr Val Lys Gly Glu Pro435 440 445Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp450 455 460Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe465 470 475 480Ile Arg Lys Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys Arg485 490 495Ser Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg500 505 510Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro515 520 525Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro530 535 540Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala545 550 555 560Asn Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr565 570 575Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly580 585 590Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His595 600 605Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys610 615 620Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr625 630 635 640Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile645650 655Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met660 665 670Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala675 680 685Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala690 695 700Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp705 710 715 720Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn725 730 735Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val740 745 750Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val755 760 765Ile Glu Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val770 775 780Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val785 790 795 800Gly Pro Val Cys Phe Leu805<210> 12<211> 831<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 12Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr1 5 10 15Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe20 25 30Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu35 4Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile50 55 60Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys65 70 75 80Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu85 90 95Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Ala Glu Leu Pro100 105 110Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr115 120 125Leu Ser Lys Lys Arg Lys Arg Ala Phe Leu Gly Phe Leu Leu Gly Val130 135 140Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu145 150 155 160Pro Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys165 170 175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn195 200 205Lys Gln Ser Cys Ser Ile Pro Asn Ile Ala Thr Val Ile Glu Phe Gln210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu245 250 255Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp GlnLys Lys260 265 270Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe340 345 350Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val370 375 380Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys405 410 415Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile420 425 430Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp435 440 445Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly450 455 460Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro465 470 475 480Leu ValPhe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn485 490 495Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu500 505 510Leu His Asn Val Asn Ala Gly Lys Arg Ser Asn Gly Leu Pro Gly Pro515 520 525Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val530 535 540Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser545 550 555 560Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala565 570 575His Asp Gly Gly Arg Tyr Tyr Arg Ala Asn Asp Ala Asn Val Val Arg580 585 590Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser Gln Gln595 600 605Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg610 615 620Thr Cys Arg Asp Leu Lys Met Cys His Ser Asp Trp Lys Ser Gly Glu625 630 635 640Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val645 650 655Phe Cys Asn Met Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro660 665 670Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys675 680 685Arg His Val Trp Phe Gly Glu Ser MetThr Asp Gly Phe Gln Phe Glu690 695 700Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr705 710 715 720Phe Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His725 730 735Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys740 745 750Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu755 760 765Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser770 775 780His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys785 790 795 800Thr Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala805 810 815Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu820 825 830<210> 13<211> 741<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 13Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser1 5 10 15Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile20 25 30Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp35 40 45Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala50 55 60Val Thr Glu Leu GlnLeu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn65 70 75 80Arg Ala Arg Ala Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn85 90 95Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Ala Phe100 105 110Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala115 120 125Val Ser Lys Val Leu His Leu Pro Gly Glu Val Asn Lys Ile Lys Ser130 135 140Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val145 150 155 160Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys165 170 175Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Pro Asn Ile180 185 190Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile195 200 205Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr210 215 220Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro225 230 235 240Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val245 250 255Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu260 265 270Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile AspThr Pro Cys275 280 285Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly290 295 300Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn305 310 315 320Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln325 330 335Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser340 345 350Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys355 360 365Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser370 375 380Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser385 390 395 400Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr405 410 415Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr420 425 430Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro435 440 445Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp450 455 460Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe465 470 475 480Ile Arg Lys Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys Arg485 490 495SerAla Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr Leu500 505 510Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser515 520 525Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His Ser530 535 540Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn545 550 555 560Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys565 570 575Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser580 585 590Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met Thr595 600 605Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp610 615 620Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala Ser625 630 635 640Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp Gln645 650 655Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu660 665 670Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr675 680 685Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile690 695 700Glu Tyr Lys Thr Thr Lys Thr SerArg Leu Pro Ile Ile Asp Val Ala 705 710 715 720 Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val Gly 725 730 735 Pro Val Cys Phe Leu 740 <210> 14 <211> 766 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 14 Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr 1 5 10 15 Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Ala Glu Leu Pro 100 105 110 Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr 115 1170 175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn195 200 205Lys Gln Ser Cys Ser Ile Pro Asn Ile Ala Thr Val Ile Glu Phe Gln210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu245 250 255Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys260 265 270Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe340 345 350Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val370 375 380Asp Ile Phe Asn Pro LysTyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys405 410 415Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile420 425 430Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp435 440 445Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly450 455 460Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro465 470 475 480Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn485 490 495Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu500 505 510Leu His Asn Val Asn Ala Gly Lys Arg Ser Ala Asn Val Val Arg Asp515 520 525Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser Gln Gln Ile530 535 540Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr545 550 555 560Cys Arg Asp Leu Lys Met Cys His Ser Asp Trp Lys Ser Gly Glu Tyr565 570 575Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val Phe580 585 590Cys Asn Met Glu Thr Gly Glu Thr Cys Val Tyr ProThr Gln Pro Ser595 600 605Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg610 615 620His Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr625 630 635 640Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr Phe645 650 655Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His Cys660 665 670Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys Lys675 680 685Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly690 695 700Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser His705 710 715 720Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys Thr725 730 735Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala Pro740 745 750Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu755 760 765<210> 15<211> 806<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 15Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser1 5 10 15Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile20 25 30Thr Ile Glu Leu Ser Asn Ile LysGlu Asn Lys Cys Asn Gly Thr Asp35 40 45Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala50 55 60Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Pro Thr Asn Asn65 70 75 80Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn85 90 95Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Arg Phe100 105 110Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala115 120 125Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser130 135 140Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val145 150 155 160Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys165 170 175Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile180 185 190Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile195 200 205Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr210 215 220Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro225 230 235 240Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val245 250255Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu260 265 270Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys275 280 285Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly290 295 300Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn305 310 315 320Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln325 330 335Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser340 345 350Glu Ile Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys355 360 365Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser370 375 380Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser385 390 395 400Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr405 410 415Val Ser Asn Lys Gly Met Asp Thr Val Ser Val Gly Asn Thr Leu Tyr420 425 430Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro435 440 445Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp450 455 460Ala Ser Ile Ser Gln Val AsnGlu Lys Ile Asn Gln Ser Leu Ala Phe465 470 475 480Ile Arg Lys Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys Arg485 490 495Ser Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg500 505 510Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro515 520 525Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro530 535 540Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala545 550 555 560Asn Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr565 570 575Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly580 585 590Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His595 600 605Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys610 615 620Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr625 630 635 640Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile645 650 655Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met660 665 670Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly SerAsp Pro Ala675 680 685Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala690 695 700Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp705 710 715 720Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn725 730 735Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val740 745 750Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val755 760 765Ile Glu Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val770 775 780Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val785 790 795 800Gly Pro Val Cys Phe Leu805<210> 16<211> 831<212> PRT<213> Artificial Sequence<220><223> Synthetic Construct<400> 16Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr1 5 10 15Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe20 25 30Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu35 40 45Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile50 55 60Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys65 70 75 80Gln GluLeu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu85 90 95Met Gln Ser Thr Pro Pro Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro100 105 110Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr115 120 125Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val130 135 140Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu145 150 155 160Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys165 170 175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn195 200 205Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu245 250 255Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys260 265 270Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu AlaTyr Val Val Gln Leu Pro290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe340 345 350Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Ile Asn Leu Cys Asn Val370 375 380Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys405 410 415Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile420 425 430Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Met Asp435 440 445Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly450 455 460Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro465 470 475 480Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn485 490 495Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu500505 510Leu His Asn Val Asn Ala Gly Lys Arg Ser Asn Gly Leu Pro Gly Pro515 520 525Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val530 535 540Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser545 550 555 560Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala565 570 575His Asp Gly Gly Arg Tyr Tyr Arg Ala Asn Asp Ala Asn Val Val Arg580 585 590Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser Gln Gln595 600 605Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg610 615 620Thr Cys Arg Asp Leu Lys Met Cys His Ser Asp Trp Lys Ser Gly Glu625 630 635 640Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val645 650 655Phe Cys Asn Met Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro660 665 670Ser Val Ala Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys675 680 685Arg His Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln Phe Glu690 695 700Tyr Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr705 710 715 720Phe Leu Arg Leu MetSer Thr Glu Ala Ser Gln Asn Ile Thr Tyr His725 730 735Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys740 745 750Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu755 760 765Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser770 775 780His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys785 790 795800Thr Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala805 8—10 815Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu820 825 830<210> 17<211> 495<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 17Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser1 5 10 15Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile20 25 30Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp35 40 45Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala50 55 60Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn65 70 75 80Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn85 90 95Ala Lys Lys It should be noted that there seems to be an incomplete or unclear part in the original text around "8—10" which might need further clarification for a more accurate translation. Also, the "800" seems to be in an odd position in the original sequence presentation.Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Arg Phe100 105 110Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala115 120 125Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser130 135 140Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val145 150 155 160Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys165 170 175Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile180 185 190Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile195 200 205Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr210 215 220Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro225 230 235 240Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val245 250 255Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu260 265 270Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys275 280 285Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly290 295 300Ser Asn Ile Cys Leu Thr Arg Thr Asp ArgGly Trp Tyr Cys Asp Asn305 310 315 320Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln325 330 335Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser340 345 350Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys355 360 365Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser370 375 380Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser385 390 395 400Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr405 410 415Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr420 425 430Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro435 440 445Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp450 455 460Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe465 470 475 480Ile Arg Lys Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys485 490 495<210> 18<211> 520<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 18Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr1 5 10 15Ala Val ThrPhe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe20 25 30Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu35 40 45Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile50 55 60Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys65 70 75 80Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu85 90 95Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro100 105 110Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr115 120 125Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val130 135 140Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu145 150 155 160Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys165 170 175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn195 200 205Lys Gln Ser Cys Ser Ile Ser Asn Ile Ala Thr Val Ile Glu Phe Gln210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe SerVal Asn225 230 235 240Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu245 250 255Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys260 265 270Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe340 345 350Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val370 375 380Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys405 410 415Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile420 425 430Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp435 440 445Thr ValSer Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly450 455 460Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro465 470 475 480Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn485 490 495Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu500 505 510Leu His Asn Val Asn Ala Gly Lys515 520<210> 19<211> 495<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 19Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser1 5 10 15Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile20 25 30Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp35 40 45Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala50 55 60Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn65 70 75 80Arg Ala Arg Ala Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn85 90 95Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Arg Phe100 105 110Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala115 120 125Val Ser Lys Val Leu His Leu GluGly Glu Val Asn Lys Ile Lys Ser130 135 140Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val145 150 155 160Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys165 170 175Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile180 185 190Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile195 200 205Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr210 215 220Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro225 230 235 240Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val245 250 255Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu260 265 270Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys275 280 285Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly290 295 300Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn305 310 315 320Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln325 330 335Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr LeuPro Ser340 345 350Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys355 360 365Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser370 375 380Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser385 390 395 400Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr405 410 415Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr420 425 430Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro435 440 445Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp450 455 460Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe465 470 475 480Ile Arg Lys Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys485 490 495<210> 20<211> 520<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 20Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr1 5 10 15Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe20 25 30Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu35 40 4Thr Ile Glu Leu Ser Asn Ile50 55 60Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys65 70 75 80Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu85 90 95Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Ala Glu Leu Pro100 105 110Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr115 120 125Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val130 135 140Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu145 150 155 160Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys165 170 175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn195 200 205Lys Gln Ser Cys Ser Ile Ser Asn Ile Ala Thr Val Ile Glu Phe Gln210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu245 250 255Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys260 265270Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe340 345 350Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val370 375 380Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys405 410 415Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile420 425 430Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp435 440 445Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly450 455 460Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro465 470 475 480Leu Val Phe Pro Ser AspGlu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Ala Gly Lys 515 520 <210> 21 <211> 806 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 21 Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser 1 5 10 15 Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile 20 25 30 Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp 35 40 45 Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala 50 55 60 Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn 65 70 75 80 Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn 85 90 95 Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Ala Phe 100 105 110 Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala 115 120 125 Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser 130 135 140 Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val 145 150 155 160 Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys AsnTyr Ile Asp Lys165 170 175Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile180 185 190Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile195 200 205Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr210 215 220Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro225 230 235 240Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val245 250 255Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu260 265 270Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys275 280 285Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly290 295 300Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn305 310 315 320Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln325 330 335Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser340 345 350Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys355 360 365Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser370 375 380LeuGly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser385 390 395 400Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr405 410 415Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr420 425 430Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro435 440 445Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp450 455 460Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe465 470 475 480Ile Arg Lys Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys Arg485 490 495Ser Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg500 505 510Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro515 520 525Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro530 535 540Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala545 550 555 560Asn Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr565 570 575Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly580 585 590Ser Arg Lys Asn Pro Ala ArgThr Cys Arg Asp Leu Lys Met Cys His595 600 605Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys610 615 620Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr625 630 635 640Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile645 650 655Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser Met660 665 670Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala675 680 685Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr Glu Ala690 695 700Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr Met Asp705 710 715 720Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn725 730 735Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val740 745 750Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val755 760 765Ile Glu Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val770 775 780Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val785 790 795 800Gly Pro Val Cys Phe Leu805<210> 22<211> 520<212>PRT<213> Artificial Sequence<220><223> Synthetic Construct<400> 22Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr1 5 10 15Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe20 25 30Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu35 40 45Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile50 55 60Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys65 70 75 80Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu85 90 95Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro100 105 110Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr115 120 125Leu Ser Lys Lys Arg Lys Arg Ala Phe Leu Gly Phe Leu Leu Gly Val130 135 140Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu145 150 155 160Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys165 170 175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn195 200 205Lys Gln SerCys Ser Ile Ser Asn Ile Ala Thr Val Ile Glu Phe Gln210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu245 250 255Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys260 265 270Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe340 345 350Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val370 375 380Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys405 410 415Tyr Gly Lys Thr Lys Cys Thr Ala SerAsn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly 450 455 460 Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro 465 470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu 500 505 510 Leu His Asn Val Asn Ala Gly Lys 515 520 <210> 23 <211> 495 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 23 Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser 1 5 10 15 Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile 20 25 30 Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp 35 40 45 Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala 50 55 60 Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn 65 70 75 80 Arg Ala Arg Ala Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn 85 90 95 Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg AlaPhe100 105 110Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala115 120 125Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser130 135 140Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val145 150 155 160Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys165 170 175Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile180 185 190Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile195 200 205Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr210 215 220Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro225 230 235 240Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val245 250 255Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu260 265 270Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys275 280 285Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly290 295 300Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn305 310 315 320Ala Gly SerVal Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln325 330 335Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser340 345 350Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys355 360 365Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser370 375 380Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser385 390 395 400Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr405 410 415Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr420 425 430Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro435 440 445Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp450 455 460Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe465 470 475 480Ile Arg Lys Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys485 490 495<210> 24<211> 520<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 24Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr1 5 10 15Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu GluPhe20 25 30Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu35 40 45Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile50 55 60Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys65 70 75 80Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu85 90 95Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Ala Glu Leu Pro100 105 110Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr115 120 125Leu Ser Lys Lys Arg Lys Arg Ala Phe Leu Gly Phe Leu Leu Gly Val130 135 140Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu145 150 155 160Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys165 170 175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn195 200 205Lys Gln Ser Cys Ser Ile Ser Asn Ile Ala Thr Val Ile Glu Phe Gln210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro ValSer Thr Tyr Met Leu Thr Asn Ser Glu245 250 255Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys260 265 270Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe340 345 350Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val370 375 380Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys405 410 415Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile420 425 430Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp435 440 445Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys GlnGlu Gly450 455 460Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro465 470 475 480Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn485 490 495Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu500 505 510Leu His Asn Val Asn Ala Gly Lys515 520<210> 25<211> 495<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 25Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser1 5 10 15Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile20 25 30Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp35 40 45Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala50 55 60Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn65 70 75 80Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn85 90 95Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Arg Phe100 105 110Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala115 120 125Val Ser Lys Val Leu His Leu Pro Gly Glu Val Asn Lys Ile Lys Ser1Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val145 150 155 160Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys165 170 175Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Pro Asn Ile180 185 190Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile195 200 205Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr210 215 220Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro225 230 235 240Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val245 250 255Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu260 265 270Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys275 280 285Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly290 295 300Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn305 310 315 320Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln325 330 335Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser340 345 350Glu Val Asn Leu Cys Asn ValAsp Ile Phe Asn Pro Lys Tyr Asp Cys355 360 365Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser370 375 380Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser385 390 395 400Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr405 410 415Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr420 425 430Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro435 440 445Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp450 455 460Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe465 470 475 480Ile Arg Lys Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys485 490 495<210> 26<211> 520<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 26Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr1 5 10 15Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe20 25 30Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu35 40 45Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile50 55 60Lys Glu AsnLys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys65 70 75 80Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu85 90 95Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro100 105 110Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr115 120 125Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val130 135 140Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu145 150 155 160Pro Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys165 170 175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn195 200 205Lys Gln Ser Cys Ser Ile Pro Asn Ile Ala Thr Val Ile Glu Phe Gln210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu245 250 255Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys260 265 270Leu Met Ser Asn Asn Val Gln Ile Val Arg GlnGln Ser Tyr Ser Ile275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe340 345 350Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val370 375 380Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys405 410 415Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile420 425 430Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp435 440 445Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly450 455 460Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro465 470 475 480Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn485 490495Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu500 505 510Leu His Asn Val Asn Ala Gly Lys515 520<210> 27<211> 495<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 27Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser1 5 10 15Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile20 25 30Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp35 40 45Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala50 55 60Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn65 70 75 80Arg Ala Arg Ala Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn85 90 95Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Ala Phe100 105 110Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala115 120 125Val Ser Lys Val Leu His Leu Pro Gly Glu Val Asn Lys Ile Lys Ser130 135 140Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val145 150 155 160Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys165 170 175Gln Leu Leu Pro IleVal Asn Lys Gln Ser Cys Ser Ile Pro Asn Ile180 185 190Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile195 200 205Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr210 215 220Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro225 230 235 240Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val245 250 255Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu260 265 270Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys275 280 285Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly290 295 300Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn305 310 315 320Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln325 330 335Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser340 345 350Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys355 360 365Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser370 375 380Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr LysCys Thr Ala Ser 385 390 395 400 Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr 405 410 415 Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr 420 425 430 Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro 435 440 445 Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp 450 455 460 Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe 465 470 475 480 Ile Arg Lys Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys 485 490 495 <210> 28 <211> 520 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 28 Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr 1 5 10 15 Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Ser Thr Pro Ala Thr Asn AsnArg Ala Arg Ala Glu Leu Pro100 105 110Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr115 120 125Leu Ser Lys Lys Arg Lys Arg Ala Phe Leu Gly Phe Leu Leu Gly Val130 135 140Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu145 150 155 160Pro Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys165 170 175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn195 200 205Lys Gln Ser Cys Ser Ile Pro Asn Ile Ala Thr Val Ile Glu Phe Gln210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu245 250 255Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys260 265 270Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe340 345 350Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val370 375 380Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys405 410 415Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile420 425 430Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp435 440 445Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly450 455 460Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro465 470 475 480Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn485 490 495Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu500 505 510Leu His Asn Val Asn Ala Gly Lys515 520<210> 29<211> 495<212> PRT<213> Artificial Sequence<220><223>Synthetic construct <400> 29 Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser1 5 10 15Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile20 25 30Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp35 40 45Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala50 55 60Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Pro Thr Asn Asn65 70 75 80Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn85 90 95Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Arg Phe100 105 110Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala115 120 125Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser130 135 140Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val145 150 155 160Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys165 170 175Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile180 185 190Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile195 200 205Thr Arg Glu Phe Ser Val Asn Ala GlyVal Thr Thr Pro Val Ser Thr210 215 220Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro225 230 235 240Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val245 250 255Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu260 265 270Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys275 280 285Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly290 295 300Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn305 310 315 320Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln325 330 335Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser340 345 350Glu Ile Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys355 360 365Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser370 375 380Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser385 390 395 400Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr405 410 415Val Ser Asn Lys Gly Met Asp Thr Val Ser Val Gly Asn Thr LeuTyr420 425 430Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro435 440 445Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp450 455 460Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe465 470 475 480Ile Arg Lys Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys485 490 495<210> 30<211> 520<212> PRT<213> Artificial Sequence<220><223> Synthetic construct<400> 30Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr1 5 10 15Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe20 25 30Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu35 40 45Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile50 55 60Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys65 70 75 80Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu85 90 95Met Gln Ser Thr Pro Pro Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro100 105 110Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr115 120 125Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe LeuLeu Gly Val130 135 140Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu145 150 155 160Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys165 170 175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn195 200 205Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu245 250 255Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys260 265 270Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile275 280 285Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro290 295 300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe340 345 350ProGln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp355 360 365Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Ile Asn Leu Cys Asn Val370 375 380Asp Ile Phe Asn Pro Lys Tyr Asp Cys L...

Claims

1. A recombinant subunit vaccine for preventing respiratory syncytial virus (RSV) infection, comprising a disulfide-linked trimeric fusion protein formed by in-frame fusion of a soluble RSV viral F protein and the C-terminal portion of collagen, wherein the fusion protein consists of the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:

2.

2. Use of the recombinant subunit vaccine according to claim 1 in the preparation of a medicament for preventing respiratory syncytial virus (RSV) infection in mammals.

3. The use according to claim 2, wherein the mammal comprises a human.

4. The method according to claim 2 or 3, wherein the recombinant subunit vaccine is administered by intramuscular injection.

5. The use according to any one of claims 2 to 4, wherein the recombinant subunit vaccine is administered as a single dose or as a series of doses separated by intervals of weeks or months.

6. The use according to any one of claims 2 to 5, wherein the recombinant subunit vaccine is administered without an adjuvant.

7. The use according to any one of claims 2 to 5, wherein the recombinant subunit vaccine is administered together with an adjuvant.

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