Compositions of influenza hemagglutinins with heterologous epitopes and / or altered mature cleavage sites

Abstract

Disclosed are modified forms of hemagglutinin (HA) proteins, including those with modified immunodominant regions and with modified mature cleavage sites, and viruses and virus-like particles containing the same.

Description

[0001] This application is a divisional application of Chinese patent application No. 2017800188563 (Application date: February 2, 2017, Invention name: Compositions of influenza hemagglutinins with heterologous epitopes and / or altered mature cleavage sites).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 290,894, filed on February 3, 2016, entitled “Compositions of Influenza Hemagglutinin with Heterologous Epitopes and / or Altered Maturation Cleavage Sites and Methods of Use Thereof,” the contents of which are incorporated by reference in their entirety.

[0004] Incorporation by Reference into the Sequence Listing

[0005] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file named 758252000140SeqList.TXT, created on January 30, 2017, and having a size of 173,061 bytes. The information in the electronic sequence listing is incorporated by reference in its entirety. Technical Field

[0006] The present invention belongs to the field of immunology and virology. More specifically, the present invention relates to compositions of recombinant influenza hemagglutinin (HA) proteins having altered and heterologous epitopes and / or altered mature cleavage sites. 1 Background Art

[0007] Influenza, commonly known as "flu", is a contagious respiratory disease caused by infection with influenza viruses that are transmitted to humans or animals. Influenza outbreaks occur during the cold months of each year (commonly known as "flu season"). Although most influenza infections have mild symptoms, these annual epidemics cause 3-5 million cases of serious illness and up to 500,000 deaths worldwide (World Wide Web, who.int / mediacentre / factsheets / fs211 / en / , World Health Organization: Influenza (Seasonal)). Every century, there are several influenza epidemics when influenza viruses infect a large part of the population and cause significant morbidity and mortality worldwide. The Spanish flu pandemic of 1918, which killed 3% to 5% of the world's population, was the deadliest epidemic in history.

[0008] Current strategies for eliciting stalk-domain-reactive antibodies

[0009] The elicitation of broadly neutralizing antibodies (bNAbs) against all influenza viruses is the ultimate goal of influenza vaccine design. Most isolated bNAbs recognize conserved epitopes in the HA stem domain. However, antibodies elicited by natural influenza virus infection or current influenza vaccines primarily recognize antigenic sites surrounding the receptor binding site in the HA globular head domain due to the immunodominance of these antigenic sites. Due to the high variability of these antigenic sites among influenza virus strains, these antibodies are generally strain-specific. Therefore, current influenza vaccines do not produce universal immunity to influenza viruses.

[0010] Hemagglutinin (HA) is an integral membrane glycoprotein. It is the most abundant protein in the influenza virus lipid bilayer envelope. An estimated 500 HA molecules are present on the surface of the virion. HA is the only protein required for viral attachment and penetration into host cells. It binds to host cell receptors and enables fusion between the virion lipid envelope and the host cell membrane. Through its ability to bind to host cell receptors, HA determines the host range of an influenza virus strain.

[0011] HA is synthesized in the endoplasmic reticulum as a single-chain precursor named HA0 (Stevens, J., et al., Science (2006) 312: 404-410). The HA0 precursor has a signal peptide at its N-terminus and a membrane anchor sequence at its C-terminus. During the process of HA being transported and anchored to the host cell membrane, the N-terminal signal peptide is removed. HA is assembled into trimers of identical subunits in the endoplasmic reticulum and then exported to the cell surface via the Golgi network. HA0 is cleaved by a specific host trypsin-like protease at the C-terminus of the mature cleavage site (MCS) and converted into a mature form consisting of two disulfide-linked polypeptides HA1 and HA2. HA1 is the larger N-terminal part of HA0, while HA2 is the smaller C-terminal part of HA0. HA2 has a transmembrane region with a short C-terminal intracellular tail and anchors HA to the membrane. The total molecular weight of each mature HA is about 70 kDa, of which HA1 is about 45 kDa and HA2 is about 25 kDa. The atomic structure of the extracellular portion of HA from many influenza virus strains has been determined. The structural model of mature HA is shown in Figure 1 All HAs, whether HA0 or mature forms, have the same overall trimeric structure with a globular head on a stalk. Each HA monomer is anchored to the membrane by a single C-terminal transmembrane region with a short hydrophilic cytoplasmic tail.

[0012] The globular head domain is composed entirely of HA1 and contains immunodominant epitopes. The stem domain is composed primarily of HA2 and contains conserved regions of subdominant immunogenicity. The globular head domain has an eight-stranded β-sheet structure at its core, along with surface loops and helices. The membrane-proximal stem domain is composed of a left-handed supercoil of a triple coiled-coil structure of residues from both HA1 and HA2. Each HA monomer has multiple glycosylation sites, the molecular weight of the total carbohydrate of the multiple glycosylation sites being approximately 13 kDa or 19% of the total HA molecular weight. Most glycosylation is located in the stem domain near the membrane surface. HA is also modified by palmitoylation of cysteine ​​residues on the cytoplasmic tail (Veit, M., et al., J. Virol (1991) 65: 2491-2500).

[0013] To circumvent the immunodominance of the globular head domain, immunogens have been designed based on the HA stem domain without the globular head domain (headless HA). These attempts have been largely unsuccessful due to the difficulty in producing such molecules with appropriate tertiary structure (Krammer, F. and Palese, P. Curr Opin Virol (2013) 3: 521-530; Eckert, DM, and Kay, MS, PNAS (2010) 107: 13563-13564). HA2 itself has been expressed in E. coli in a soluble form that folds into its most stable post-fusion low pH-induced conformation (Chen, J., et al., PNAS (1995) 92: 12205-12209). By incorporating designed mutations to destabilize the low pH conformation of HA2, another HA2 construct (HA6) based on H3 HA was expressed in E. coli and refolded into the desired neutral pH pre-fusion conformation (Bommakanti, G., et al., PNAS (2010) 107: 13701-13706). HA6 is highly immunogenic in mice and protects mice from infection with homologous influenza A virus. However, sera from HA6-immunized mice were unable to neutralize the virus in vitro, which may be due to the limitation of the assay that only detects the virus neutralizing activity of antibodies that recognize the globular head domain. Another "headless" HA stem domain construct has been described that has HA2 and a region of HA1 in the stem domain (Steel, J., et al., mBio (2010) 1: e00018-10). This immunogen protected mice from homologous influenza challenge and elicited antisera that cross-reacted with heterologous HA within the same panel. As with HA6, the antisera did not show neutralizing activity in vitro. These designs are based on protein minimization by eliminating immunodominant regions.

[0014] Recently, stable trimeric H1 HA stem-purified immunogens (headless mini-HA) were generated by structure-based rational design of construct libraries and iterative bNAb selection (Impagliazzo, A., et al., Science (2015) 349:1301-1306; Yassing, H., M., et al., Nat Med (2015) 21:1065-1070; patent application WO 2014 / 191435_A1). These stem-purified mini-HA immunogens elicited the desired antibodies against the HA stem domain. Mice and ferrets immunized with these mini-HA immunogens were protected from lethal challenge with a highly pathogenic H5 virus. In addition, the mini-HA immunogens elicited H5 neutralizing antibodies in cynomolgus macaques. These mini-HA immunogens were selected for their binding to specific bNAbs. The tedious iterative bNAb selection process needs to be repeated for different types of bNAbs. Although the conformation of the bNAb epitope is preserved in these mini-HA immunogens, the overall structure differs from the stalk domain of native HA. Due to these structural changes, these headless mini-HA immunogens are less likely to assemble into influenza viruses or influenza virus-like particles (VLPs). In addition, these headless mini-HA immunogens do not have receptor binding sites for host cell binding.

[0015] Chimeric HA has been designed to direct host immune responses to the stem domain. Most neutralizing antibodies induced by pandemic H1N1 infection broadly cross-react with epitopes in the HA stem and globular head domains of multiple influenza strains (Wrammert, J., et al., J. Exp Med (2011) 208: 181-193). Immunization with HA derived from an H5N1 influenza strain that does not circulate in humans (Group 1 HA) greatly increased HA stem-specific responses to Group 1 circulating seasonal strains (Ellebedy, AH, et al., PNAS (2014) 111: 13133-13138; Nachbagauer, R., et al., J Virol (2014) 88: 13260-13268; Whittle, JRR, et al., J. Virol (2014) 88: 4047-4057). After each pandemic influenza strain emerged in 1957, 1968, and 2009, the existing seasonal virus strain was replaced in the human population by the new pandemic strain.

[0016] It is hypothesized that exposure to novel pandemic influenza virus strains with different globular head domains results in affinity-matured memory responses to conserved epitopes in the stem domain (Palese, P. and Wang, TT, mBio (2011) 2: e00150-11). In support of this hypothesis, engineered recombinant chimeric HAs with H6, H9, or H5 globular head domains and an H1 stem domain generated high titers of stem-specific neutralizing antibodies (Pica, N., et al., PNAS (2012) 109: 2573-2578; Krammer, F., et al., J. Virol (2013) 87: 6542-6550). In these cases, the globular head domain of the H1 HA was replaced with the globular head domain of H6, H9, or H5 from the same group 1 HA native to most of the human population. These substitutions were made by replacing the H1 HA sequence between cysteine ​​52 and cysteine ​​277 of HA1 with the corresponding sequences of H6, H9, or H5. Cysteine ​​52 and cysteine ​​277 form a disulfide bond in the hinge region between the globular head domain and the stem domain. Similar chimeric HAs were prepared using the H3 stem domain to develop vaccines for Group 2 influenza strains (Krammer, F., et al., J. Virol (2014) 88:2340-2343; Margine, I., et al., J. Virol (2013) 87:10435-10446). Immunization of animals with these chimeric HAs induced stem-specific antibodies with broad neutralizing activity against each group of viruses. Humans have pre-existing immunity to circulating H1 (Group 1), H3 (Group 2), and influenza B strains. Vaccination with chimeric HAs increased antibody levels against HAs of circulating strains and the stem domain common to chimeric HAs. The novel globular head domain on the human-specific chimeric HA induced only a primary response. Subsequent boosting with a second chimeric HA with the same stalk domain but a different head domain further increased stalk-specific antibody levels. The results suggest that altering host exposure to immunodominant epitopes in the globular head domain can increase broadly protective immune responses against immunosubdominant epitopes in the stalk domain.

[0017] The six amino acid loop of antigenic site B of A / WSN / 33 (H1N1) hemagglutinin can be replaced by residues from the homologous antigenic site B of HA from A / Japan / 57 (H2N2) and A / Hong Kong / 8 / 68 (H3N2) (Li, S., et al., J. Virol (1992) 66: 399-404). These replacements do not interfere with the receptor binding function of HA. Recombinant influenza viruses with these chimeric HAs were replicated in MDCK (Beagle kidney epithelial) cell cultures. Viruses with chimeric HAs of A / WSN / 33 (H1N1) and A / Hong Kong / 8 / 68 (H3N2) induced antibodies against both A / WSN / 33 (H1N1) and A / Hong Kong / 8 / 68 (H3N2). These results indicate that the immunodominant antigenic site of HA can be replaced by the homologous corresponding immunodominant antigenic site of other HAs from different strains. These replacements alter the antigenic specificity of the resulting chimeric HA.

[0018] Thus, in the preceding paragraph, an immunodominant epitope in one influenza strain is replaced by a homologous immunodominant epitope from another strain.

[0019] Another strategy is to suppress immunodominant epitopes and refocus the host immune response on the stalk domain. Immunodominant antigenic sites in the globular head domain can be shielded by introducing additional glycosylation sites for hyperglycosylation (Eggink, D., et al., J. Virol (2014) 88: 699-704, Patent application US 2014 / 0004149_A1). Hyperglycosylation in the globular head domain does not alter the binding affinity of stalk-reactive antibodies. Immunization of mice with hyperglycosylated HA induced high titers of stalk-reactive antibodies and prevented morbidity and mortality when challenged with different seasonal viruses. Patent application US 2013 / 0315929_A1 discloses another approach to suppress immunodominant epitopes in the globular head domain by replacing some residues of those epitopes with other amino acids that are unlikely to be part of the epitope. The results suggest that shielding immunodominant epitopes in the globular head domain can increase broadly protective immune responses against immunosubdominant epitopes in the stalk domain.

[0020] HA as a carrier for presenting foreign epitopes

[0021] Influenza HA has been used as a carrier for the epitope of the V3 loop of the HIV-1 envelope protein. The insertion of an immunodominant epitope peptide of 12 to 22 residues in length from the HIV-1 envelope protein gp120 to the HA immunodominant antigenic site (site A or site B) produces a chimeric HA with a single HIV-1 epitope in the globular head domain. The residues of site A and site B are not removed. The immunodominant HIV-1 epitope is inserted into the site. Chimeric HA induces an immune response to the HIV-1 V3 loop epitope in animals (Kalyan, NK, et al., Vaccine (1994) 12: 753-760; U.S. Patent No. 5,591,823_A; Li, S., et al., J. Virol (1993) 67: 6659-6666). The immunogenicity of the inserted epitope appears to be enhanced by HA, because very low doses of chimeric HA protein are sufficient to induce antibodies specific for the inserted epitope. These results indicate that immunodominant exogenous epitopes from proteins other than HA can be inserted into the immunodominant antigenic site of HA. These chimeric HA molecules with inserted exogenous immunodominant epitopes can induce an immune response to the inserted exogenous immunodominant epitopes. Summary of the Invention

[0022] The present invention relates to modified forms of influenza hemagglutinin proteins, to vaccines, virus-like particles and viruses containing the same, and to recombinant methods and materials for their production. Typically, these modifications involve replacing the immunodominant region of the globular head domain of the HA protein with an alternative epitope to produce antibodies and / or modifying the mature cleavage site (MCS) in the stem domain of the HA protein. HA0 between the MCS and the fusion peptide is cut to produce a free N-terminus of the HA2 necessary for cell entry. By altering the MCS to prevent it from being cleaved by animal proteases, viruses whose MCS has been cut allow infection of host cells, wherein the virus can reproduce, but its offspring are not infectious. Therefore, the presence of any epitope, including those occupying the immunodominant site, is amplified, and the offspring virus will not further infect the host.

[0023] In one aspect, the present invention relates to an influenza vaccine comprising a modified HA or a virus or virus-like particle containing the same, wherein an immunodominant region of the HA protein contains a conserved alternative epitope of the same influenza strain or another influenza strain inserted into the region. This provides a more successful immunogenic form of inserted alternative epitopes that are immunosubdominant in their native position. Alternative epitopes are those that are not derived from the globular head domain of other HAs. These alternative epitopes are typically derived from the HA stem domain or from non-HA influenza proteins, such as M2.

[0024] In another aspect, the present invention relates to modified influenza viruses (which can also be used as vaccines) that have been modified to contain MCSs that are not cleaved by animal proteases. As described above, this allows the virus to be amplified without generating its infectious form. This virus can be further modified by replacing one or more immunodominant regions with alternative or heterologous epitopes, which can be influenza epitopes or exogenous epitopes, including, for example, epitopes unique to other viruses, bacteria, or tumor-associated antigens.

[0025] In other aspects, the present invention relates to recombinant materials and methods for preparing proteins, viruses or virus-like particles of modified HA proteins, as well as methods for generating antibodies using these proteins, virus-like particles or modified viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The X-ray crystal structure of the extracellular portion of the mature HA of A / California / 07 / 2009 (a swine-derived influenza A virus H1N1 strain) is shown (PDB: 3LZG). This structure was used to guide the design of the constructs of the present invention. The peptide sequence of this HA was used to prepare the parent wild-type (WT) H1 HA construct. Figure 1 A shows the HA trimer, with one HA monomer in the ribbon diagram and the other two in the backbone diagram. The orientation relative to the membrane, the position of the globular head domain, and the stalk domain are shown. HA1 is shown as a light shaded ribbon, and HA2 is shown as a dark shaded ribbon. Figure 1 B shows a single HA monomer with the locations of HA1 and HA2 and the N- and C-termini of HA1 and HA2. The fusion peptide is circled.

[0027] Figure 2 The approximate location of the immunodominant antigenic site of the H3 HA mapped to the H1 HA monomer of A / California / 07 / 2009 is shown. Figure 2 A shows the Figure 1 Ribbon diagram of . H3 HA immunodominant antigenic sites A, B, C, D, and E are shown in darker shading. Figure 2 B is above the globular head domain at the distal end of the membrane Figure 2 Top view of A.

[0028] Figure 3 The approximate location of the immunodominant antigenic site of the H1 HA mapped to the H1 HA monomer of A / California / 07 / 2009 is shown. Figure 3 A shows the Figure 1 The H1 HA immunodominant antigenic sites Ca1, Ca2, Cb, Sa, and Sb are shown in darker shading. Figure 3 B is above the globular head domain at the distal end of the membrane Figure 3Top view of A. Many of these immunodominant antigenic sites surround the host cell receptor binding site indicated by the arrows.

[0029] Figure 4 Schematic design of the H1 HA construct is shown. Figure 4 A is a schematic diagram of the nucleic acid features of the construct. The locations of restriction sites used for subcloning are marked. Figure 4 B is Figure 4 Schematic diagram of the protein profile of the constructs showing the relative proportions of the nucleic acids shown in A. GP67ss represents the N-terminal GP67 secretion signal (signal peptide). HA1 represents HA HA1. MCS represents the HA mature cleavage site, which is part of HA1. HA2 represents HA HA2. TEV represents the TEV cleavage site. Foldon represents the foldon sequence. His represents the C-terminal 10-histidine tag (10xHis tag).

[0030] Figure 5 The position of the heterologous epitope in the HA globular head domain and the position of the changed mature cleavage site are shown. The parent construct peptide sequence (WT) is compared with the HA sequences of H3 (A / Aichi / 2 / 1968H3N2) and H1 (A / Puerto Rico / 8 / 1934H1N1). The amino acid position of each sequence is marked on the right based on the HA0 numbering, wherein the first residue after removing the signal sequence is used as position 1. The mature cleavage site (MCS) is indicated by arrows and vertical lines. The immunodominant antigenic sites A, B, C, D and E of H3 HA are marked above the H3 sequence. The immunodominant antigenic sites Ca1, Ca2, Cb, Sa and Sb of H1 HA are marked below the H1 sequence. The underlined sequence in WT is replaced by the heterologous epitope shown below the sequence. The changed mature cleavage site is shown below the mature cleavage site (MCS). The naturally occurring subdominant antigenic sites of broadly neutralizing antibodies (bNAbs), FI6, 1C9, and CR8020 are marked by lines above the sequences, respectively.

[0031] Figure 6 A model of an HA monomer is shown, wherein the composite CR8020 epitope is located at the immunodominant antigenic site of the globular head domain. Figure 1 The ribbon diagram shown. The immunodominant antigenic site replaced by the composite CR8020 epitope is shown in dark shading. The original native CR8020 epitope in the stem domain is also shown in dark shading. Figure 6 A shows the model of CR8020Sa4. Figure 6 B shows the model of CR8020Ca.

[0032] Figure 7The expression data of the HA constructs described in the present invention are shown. Cell culture medium of SF9 cells infected with recombinant baculovirus of the indicated constructs was harvested and captured by Ni-NTA resin (Ni pull-down). The Ni-NTA resin was washed with 1xPBS to remove unbound proteins. Stained SDS-PAGE ( Figure 7 A. Figure 7 B. Figure 7 C and Figure 7 D) or anti-His Western blot ( Figure 7 E) Analysis of proteins bound to Ni-NTA resin. The arrow indicates the full-length HAO.

[0033] Figure 8 The purified HA constructs used in the present invention are shown. Cell culture medium of SF9 cells infected with recombinant baculovirus containing the constructs shown was harvested and captured by Ni-NTA resin (Ni pull-down). The Ni-NTA resin was washed with 1xPBS. Bound proteins were eluted with imidazole and purified by The arrow indicates the full-length HAO. DETAILED DESCRIPTION

[0034] Because the present invention relates to modifications of the HA protein, further description of this protein and its function, as well as the proteins provided above, may be helpful.

[0035] Protease cleavage of HA0 is a prerequisite for influenza A virus infectivity. The distribution of these proteases in the host is one of the determinants of tissue tropism and, therefore, pathogenicity. Several trypsin-like proteases have been identified in the respiratory tract and lungs that cleave most HAs with a single-base mature cleavage site (Kido, H., et al., J. Biol Chem (1992) 267: 13573-13579; Peitsch, C., et al., J. Virol (2014) 88: 282-291; Zhimov, O., P., et al., J. Virol (2002) 76: 8682-8689). These proteases are serine proteases. One of them is the trypsin-like protease Clara, which was first isolated from rat bronchiolar epithelial Clara cells. The narrow tissue distribution of these proteases that cleave HA0 limits influenza virus infection to the respiratory tract and lungs of mammals.

[0036] The proteases involved in HA maturation have not been fully characterized. Like trypsin, these proteases cleave the C-terminal peptide bond to a basic residue, such as arginine (R) or lysine (K). The maturation cleavage site (MCS) is located at the C-terminus of HA1, and cleavage at the C-terminus of the MCS is essential for infectivity.

[0037] The polybasic sequences of the mature cleavage sites of H5 and H7 subtypes result in susceptibility to cleavage by a wider range of cellular proteases, such as furin and subtilisin, which is associated with the broader tissue tropism and higher pathogenicity of these viruses in mammals ( A., et al., EMBO J (1992) 11: 2407-2414; Maines, TR, et al., J. Virol (2005) 79: 11788-11800). Many highly pathogenic avian influenza (HPAI) subtypes are H5 and H7. During HPAI outbreaks in humans, reported case fatality rates are higher than those for epidemic and seasonal influenza viruses (Morens, DM, et al., Nature (2012) 486: 335-340). These polybasic sequences in MCS also lead to viral replication in multiple organs of avian species, resulting in high mortality in these species.

[0038] The HA of seasonal influenza viruses and non-pathogenic avian influenza viruses is cleaved extracellularly by specific proteases that restrict their tissue tropism in the respiratory tract and lungs. On the other hand, the HA of highly pathogenic viruses is cleaved intracellularly by ubiquitous proteases. These highly pathogenic viruses undergo multiple cycles of replication in various tissues to cause systemic infection (Steinhauer, DA, Virus. Virol (1999) 258: 1-20; Taubenberger, JK, PNAS (1998) 95: 9713-9715). The 1918 pandemic human influenza virus strain also utilizes a wide range of cellular proteases and uses its neuraminidase (NA) to recruit plasminogen for HA cleavage (Goto, H. and Kawaoka, Y. PNAS (1998) 95: 10224-10228; Chaipan, C., et al., J. Virol (2009) 83: 3200-3211).

[0039] Cleavage of HAO produces HA2, which has a new free N-terminus necessary for fusion of the virion with the host cell. The cleaved form is the mature form of HA, which has all the functions of receptor binding and membrane fusion. HAO has the ability to bind to receptors but does not mediate membrane fusion. Both the precursor and mature forms of HA are present on the surface of the virion. Viruses containing only HAO have no fusion activity and cannot cause infection.

[0040] The N-terminal twelve residues of HA2 are called "fusion peptides" (Skehel, et al., Biochem Soc Trans (2001) 29: 623-626). The fusion peptide has a hydrophobic sequence. In HAO, the MCS and the fusion peptide form a surface loop. After cleavage, the newly generated N-terminal fusion peptide is inserted into the HA trimer interface. After binding to the host cell receptor, the attached influenza virus particles are internalized by the host cell into the endosome. The fusion potential of mature HA is activated at endosomal pH (depending on the specific influenza virus strain, between pH 5 and 6). Extensive changes in the structure of HA at low pH lead to the extrusion of the fusion peptide into the cell membrane. Insertion of the fusion peptide into the host endosomal membrane results in fusion of the surrounding host endosomal membrane with the viral membrane containing the C-terminal membrane anchor region of HA. Fusion releases the viral RNA segment into the cytoplasm of the host cell to enter the host cell nucleus where viral replication occurs.

[0041] HA determines the host range of the influenza virus strain. HA binds to terminal sialic acids on host cell surface glycoproteins and glycolipids. HA from human influenza strains binds almost exclusively to sialic acid oligosaccharides terminated by SAα2,6Gal, whereas HA from avian and equine influenza strains binds to SAα2,3Gal. Humans have predominantly SAα2,6Gal sialic acid oligosaccharides, whereas avian species have predominantly SAα2,3Gal sialic acid oligosaccharides. Figure 3 As schematically shown in FIG, the host receptor binding site on HA is located in the globular head domain composed solely of HA1. The receptor specificity of influenza virus HA has been well characterized. The amino acid residues associated with the recognition of SAα2,6Gal or SAα2,3Gal have been mapped. It has been shown that single amino acid residue substitutions in the receptor binding pocket alter receptor binding specificity (Rogers, GN, et al., Nature (1983) 304: 76-78). Different isolates of the 1918 pandemic influenza virus have different receptor binding specificities due to single amino acid substitutions in the receptor binding site of HA (Glaser, L., et al., J. Virol (2005) 79: 11533-11536). Strain A / South Carolina / 1 / 18 HA preferentially binds to the human cell receptor SAα2,6Gal, while strain A / New York / 1 / 18 HA binds to both the human cell receptor SAα2,6Gal and the avian cell receptor SAα2,3Gal, revealing the dynamic nature of host adaptation to influenza viruses.

[0042] Although a single mutation in the receptor binding site of HA changes the receptor binding specificity from avian to human, efficient transmission of H5 avian influenza strains in humans requires several other changes in HA outside the receptor binding site and changes in other influenza proteins (Imai, M., et al., Nature (2012) 486: 420-428; Herfst, S., et al., Science (2012) 336: 1534-1541; Chen, LM., et al., Virol (2012) 422: 105-113; Russell, CA, et al., Science (2012) 336: 1541-1547). Together with other changes, only four amino acid substitutions in H5 HA were sufficient to enable the mutant avian H5 virus to spread through droplets in ferrets. In addition to understanding cross-species influenza virus transmission, these studies also established laboratory procedures to study the transmission of non-mammalian influenza viruses in mammals, which can be used to evaluate universal influenza vaccine candidates.

[0043] Delineation of Antigenic Sites of Anti-Influenza Antibodies and HA

[0044] As the most abundant protein in the influenza virus lipid bilayer envelope, HA is the major antigen of the influenza virus and has a primary epitope for neutralizing antibodies. Most human anti-influenza antibodies are directed against HA and NA. Sixty percent (60%) of influenza-reactive antibodies elicited by vaccination react to HA (Wrammert, J., et al., Nature (2008) 453: 667-671). Most HA-reactive antibodies recognize antigenic sites surrounding the receptor binding site in the globular head domain. Some of these antibodies are virus-neutralizing antibodies. These neutralizing antibodies (NAbs) generally interfere with HA binding to host cells and exhibit HA inhibitory (HI) activity. Due to the high variability of these antigenic sites, these neutralizing antibodies are generally strain-specific and therefore lack the desired broad neutralizing activity (Wang, TT and Palese, P., Nat Struct Mol Biol (2009) 16: 233-234).

[0045] The widespread infection of influenza viruses is the result of the ability of the virus to change its antigenic properties. Changes in the antigenic properties of influenza viruses are the result of low-fidelity replication of the viral genome by the error-prone viral RNA-dependent RNA polymerase complex. High mutation rates lead to antigenic drift, a gradual change in the antigenic properties of HA. Antigenic drift occurs in all types of influenza viruses. Sequence analysis of the HA gene shows that although silent nucleic acid sequence changes are found throughout the HA gene, most of the changes in the amino acid sequence are located in HA1 (Palese, P. and Young, JF, Science (1982) 215: 1468-1474). HA2 is more conservative than HA1.

[0046] When two different strains of virus co-infect a host simultaneously, the segmented nature of the viral genome also leads to the reassortment of viral genome segments. New viral strains are formed from this reassortment using different viral surface proteins, which leads to antigenic shift, i.e., a sudden change in the antigenic properties of HA. Antigenic shift only occurs in influenza A viruses. Antigenic drift and antigenic shift allow influenza viruses to escape neutralization by existing antibodies.

[0047] H3 influenza

[0048] The first detailed structure of influenza HA, determined 35 years ago, elucidated the antibody binding site of HA and provided a molecular explanation for antigenic drift and antigenic shift (Wilson, IA, et al., Nature (1981) 289:366-373; Wiley, DC, et al., Nature (1981) 289:373-378; Wiley, DC and Skehel, JJ, Ann Rev Biochem (1987) 6:365-394).

[0049] Comparison of HA sequences from antigenically distinct viruses identified immunodominant antigenic sites within the globular head domain of H3 HA (group 2 subtype HA). The description of the immunodominant antigenic sites of H3 HA is confirmed by other references (Both, GW, et al., J. Virol (1983) 48: 52-60). The approximate locations of these antigenic sites within the HA structure are schematically shown in FIG. Figure 2 The sequence locations of these antigenic sites are as follows Figure 5 Site A is located in a surface loop from residues 133 to 148 of the HA of the A / Aichi / 2 / 1968 H3N2 strain ( Figure 5(numbered H3 in [1]). This loop, called the 140-loop, extends from the globular head domain and is located at the lower edge of the receptor binding pocket. Site B is located at the top of the globular head domain and comprises a surface α-helix of residues 187 to 196 and an adjacent surface loop of residues 155 to 160 along the upper edge of the receptor binding pocket. Site C surrounds the disulfide bond between Cys52 and Cys277. The intersection of the loop centered on Cys52 (residues 46 to 55) and the loop centered on Cys277 (residues 271 to 280) forms a bulge at the hinge between the globular head domain and the stalk domain. Site D is located in the interface region between the HA monomer subunits of the HA trimer. It is centered on two β-strands, residues 200 to 214, in the eight-stranded β-sheet structure in the core of the globular head domain. Residues at the corners of other β-strands can also be part of site D. Site D is primarily buried in the HA trimer interface. It is currently unclear how site D functions as an antigenic site. Site E is located between sites A and C on the side of the globular head domain and is composed of residues 62 to 63, a surface loop of residues 78 to 83, and a beta strand of residues 119 to 122 on the edge of the eight-stranded beta sheet. These residues in site E together form a continuous surface on the side of the globular head domain. Comparison of amino acid substitutions and antigenic properties of influenza viruses that appeared between 1968 and 2003 showed that substitutions associated with major antigenic changes were only located in sites A and B (Smith, DJ, et al., Science (2004) 305: 371-376; Koel, BF, et al., Science (2013) 342: 976-979). Substitutions in sites C, D, and E appear to cause minor antigenic changes. The results indicate that most strain-specific neutralizing antibodies bind to sites A and B outside the receptor binding site of the globular head domain.

[0050] H1 Flu

[0051] Genetic analysis of the antigenically distinct group 1 H1 HA of virus strain A / PR / 8 / 34 identified distinct antigenic sites within the globular head domain, designated as the Ca1, Ca2, Cb, Sa, and Sb sites (Caton, AJ, et al., Cell (1982) 31: 417-427; Gerhard, W., et al., Nature (1981) 290: 713-717). The approximate locations of these antigenic sites within the HA structure are schematically shown in FIG. Figure 3 The sequence locations of these antigenic sites are as follows Figure 5 The Ca1 site is located at the corner of the β strand of the eight-stranded β sheet structure. One of the corners from residues 165 to 169 (in Figure 5The Ca1 site (numbered H1 in the figure) is surface exposed. Residue 207 of the Ca1 site is located at the turn connecting the two β-strands corresponding to site D of H3 HA. The Ca1 site of H1 HA generally corresponds to site D of H3 HA. The Ca2 site is also composed of two segments, which are separate in the primary structure but together in the tertiary structure. One segment of Ca2 is located in a surface loop of residues 136 to 141 at the corresponding position of site A of H3 HA. The other segment of Ca2 is composed of residues 220 to 221 on a long surface loop. In the HA trimer, the Ca2 site is located on the opposite side of Ca1 in the globular head domain of the HA monomer, but is adjacent to the Cal site of another HA monomer. In the trimeric structure, the Cal site of one HA monomer forms a continuous surface with the Ca2 site of another HA monomer. The Cb site is a linear epitope of residues 70 to 75 that forms a surface loop adjacent to the eight-stranded β-sheet structure. It corresponds to site E of H3 HA. Sites Sa and Sb can be considered subsites corresponding to site B of H3 HA. Site Sa, residues 154 to 163, overlaps with a loop corresponding to the site B loop of H3 HA. Another segment of the Sa site is a nearby turn from residues 124 to 125. Site Sb corresponds to the site B α-helix of H3 HA and has a majority of α-helical residues 188 to 194.

[0052] Broadly neutralizing antibodies and conserved epitopes

[0053] Natural influenza virus infection or vaccination with a trivalent inactivated influenza vaccine (TIV) also elicits low levels of antibodies against conserved HA epitopes, which are primarily located in the membrane proximal stem domain (Ellebedy, AH, et al., PNAS (2014) 111: 13133-13138). These antibodies recognize epitopes conserved in many influenza virus strains. Those antibodies that provide protection against several strains are called broadly neutralizing antibodies (bNAbs). These bNAbs generally do not have HA inhibitory activity and do not prevent HA from binding to host cell surface receptors. The first cross-strain antibody, C179, was characterized 20 years ago (Okuno, Y, et al., J. Virol (1993) 67: 2552-2558). It recognizes conformational epitopes at residues 318 to 322 (TGLRN) of HA1 and residues 47 to 58 (GITNKVNSVIEK) of HA2, which are conserved in H1 and H2 strains. C179 inhibits the fusion activity of HA, resulting in virus neutralization.

[0054] Many bNAbs have been detected or isolated from patients infected with influenza virus (Ekiert, DC, and Wilson, IA, Curr Opin Virol (2012) 2:134-141). bNAbs have been shown to neutralize group 1, group 2, or both group 1 and group 2 influenza A viruses. Numerous epitopes recognized by bNAbs have been identified and characterized. These epitopes are conformational epitopes of linear segments of the HA sequence or of multiple linear segments of the HA sequence. Many epitopes recognized by bNAbs are located in the less variable stalk domain of the HA protein. These epitopes include, but are not limited to, peptide sequences surrounding the fusion peptide and the MCS.

[0055] Many bNAbs and their epitopes have been structurally characterized. For example, bNAb FI6 recognizes residues of the MCS and fusion peptide in HA0 (Corti, D., et al., Science (2011) 333:850-856). Peptide mapping of HA identified two peptides as the FI6 epitope, namely RKKRGLFGAIAGFIE, which consists of the MCS and most of the fusion peptide, and KESTQKAIDGVTNKVNS, which consists of the coiled-coil peptide in HA2. The proposed mechanism of FI6 neutralization is to inhibit membrane fusion and prevent HA maturation by blocking protease access to the MCS of HA0. Another bNAb, F10, recognizes a conformational epitope surrounding the fusion peptide in the mature form of HA (Su, J., et al., Nat Struct Mol Biol (2009) 16:265-273). F10 may inhibit all group 1 influenza A viruses by preventing membrane fusion.

[0056] Monoclonal bNAb 1C9 inhibits cell fusion in vitro (US Patent 8,540,995; Prabhu, N., et al., J. Virol (2009) 83:2553-2562). 1C9 recognizes a linear epitope of GLFGAIAGF, the N-terminus of the fusion peptide of H5 HA2 of HPAI H5N1 (Immune Epitope Database URL: iedb.org / assaydetails.php?assayId=1599077). 1C9 has been shown to protect mice from infection with the highly pathogenic avian influenza (HPAI) H5N1 virus. Monoclonal bNAbs have also been isolated from memory B cells (Hu, W., et al., Virol (2013) 435:320-328). Some of these monoclonal antibodies recognize a linear epitope of FIGGWTGMVDGWYGYHH from HA2 of the 2009 pandemic H1N1 influenza virus. This epitope is C-terminal to the 1C9 epitope of the fusion peptide. The 14-residue sequence of the HA fusion peptide is highly conserved among influenza A and B viruses.

[0057] The conserved nature of HA fusion peptides has been explored to develop universal influenza vaccines. Based on the highly conserved sequence of HA0 of influenza B virus (including the MCS of HA2 and the last 9 amino acid residues of HA1 of the fusion peptide on both sides of the break bond), peptide conjugate vaccines elicited protective immune responses against lethal challenge with virus strains of antigenically different lineages of influenza B virus (Bianchi, E., et al., J. Virol (2005) 79: 7380-7388).

[0058] The monoclonal bNAb CR6261 recognizes a highly conserved region in the HA2 helix A and HA1 residues in the stem domain (Ekiert, DC, et al., Science (2009) 324:246-251). CR6261 neutralizes group 1 influenza viruses by preventing HA from converting to a post-fusion conformation. CR6261 belongs to a group of viruses that use the same V H bNAb to the heavy chain of 1-69 germline antibody. H 1-69 monoclonal antibody CR8020 neutralizes group 2 influenza viruses. CR8020 is in close proximity to the membrane (approximately 15 to ) binds to HA at the base of the stem domain, similar to those anti-HIV antibodies that recognize the membrane proximal external region (MPER) from the HIV gp41 subunit (Ekiert, DC, et al., Science (2011) 333:843-850). The two main components of the CR8020 epitope consist of the C-terminal portion of the fusion peptide (HA2 residues 15 to 19, EGMID of H3) and the outermost strand of the 5-stranded β-sheet near the base of the stem domain (HA2 residues 30 to 36, EGTGQAA of H3). These two components are separated by 10 residues in the primary structure of H3. Most bNAbs that bind to the HA stem domain neutralize either group 1 (H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16) or group 2 (H3, H4, H7, H10, H14, and H15) of influenza A virus HA. These antibodies do not inhibit the binding of HA to host cells, but can prevent the fusion of viral membranes and host cell membranes.

[0059] The HA receptor binding site is a pocket at the top of the globular head domain (Wilson, IA, et al., Nature (1981) 289: 366-373; Wiley, DC and Skehel, JJ, Ann Rev Biochem (1987) 56: 365-394). The pocket is formed by amino acid residues that are highly conserved among many influenza strains. The edges of the pocket are formed by immunodominant antigenic sites (such as site A and site B in H3 described above). Cloning of human monoclonal antibodies (mAbs) from healthy human subjects identified bNAbs that recognize conserved residues in close proximity to the receptor binding site in the globular head domain of HA from H1, H2, and H3 strains (Krause, JC, et al., J. Virol (2011) 85: 10905-10908; Krause, JC, et al., J. Virol (2012) 86: 6334-6340). Structural studies have shown that at least some of these bNAbs mimic the interaction of sialic acid with the receptor binding pocket (Whittle, JRR, et al., PNAS (2011) 108: 14216-14221; Ekiert, DC, et al., Nature (2012) 489: 526-532).

[0060] Stem-reactive antibodies are rare in natural infections and are even rarer in current seasonal influenza vaccines. Only a subset of these antibodies are neutralizing. Due to the conserved nature of the stem domain, most neutralizing antibodies are bNAbs. These rare broadly neutralizing stem-reactive antibodies can now be routinely identified by cloning from antibody libraries prepared from human subjects (Kashyap, A.K., et al., PNAS (2008) 105: 5986-5991; Wrammert, J., et al., Nature (2008) 453: 667-671). The rare occurrence of these stem-reactive antibodies has led to the hypothesis that these epitopes in the stem domain are immunodominant, while epitopes in the globular head domain are immunodominant to which most antibodies are directed (Krammer, F. and Palese, P., Nature Rev Drug Disc (2015) 14: 167-182). Repeated exposure to circulating seasonal influenza viruses or immunization with seasonal influenza vaccines results in the production of antibodies against immunodominant antigenic sites in the globular head domain. It has been suggested that the presence of immunodominant epitopes in the globular head domain may bias secondary responses away from the stem domain (Russell, CJ, N Engl J Med (2011) 365: 1541-1542). Individuals who have been infected with current strains or vaccinated against them may have a more difficult time achieving a universal response than those who are naturally immune.

[0061] M2 protein and its epitope

[0062] M2 is a single-pass transmembrane protein that forms a homotetrameric proton channel in the viral envelope (Lamb. RA, et al., Cell (1985) 40:627-633; Pielak, RM and Chou, JJ, Biochim Biophys Acta. (2011) 1808:522-529). Compared to HA in the viral envelope, M2 is present at much lower abundance (the M2:HA ratio is 1:10 to 1:100). The M2 proton channel function is important for regulating the pH inside the virus to release viral proteins into the host cell cytosol and regulating the pH of the Golgi lumen to transport HA to the cell surface. The influenza A virus M2 (AM2) protein has 97 residues, including an extracellular N-terminal domain (residues 1 to 23) called M2e, a transmembrane (TM) domain (residues 24 to 46), and an intracellular C-terminal domain (residues 47 to 97). The TM domains from four M2 molecules form a four-helix bundle that functions as a pH-sensitive proton channel to regulate pH across the viral membrane during cell entry and across the trans-Golgi membrane of infected cells during viral assembly and egress. The large cytoplasmic domain is essential for stable tetramer formation and plays a role in viral assembly through its association with the M1 protein of the virion inner shell. Aside from the HXXXW sequence motif in the TM domain that is essential for proton channel function, the M2 proteins of influenza A, B, and C viruses share little sequence homology. However, the 10 N-terminal extracellular residues of AM2 are conserved among all influenza A viruses.

[0063] Influenza A virus M2 (AM2) is the target of the antiviral drugs amantadine and rimantadine, which block the AM2 proton channel. Rimantadine, a drug that stabilizes the closed state of the proton channel, binds to a lipid-facing pocket near the C-terminus of the channel domain. Amantadine is an antiviral drug with activity against influenza A viruses, but not against influenza B viruses. Due to widespread drug resistance caused by mutations in the AM2 channel domain, the use of these channel blockers has been discontinued. Many of these mutations result in slightly attenuated viruses that are less infective than wild-type (WT) virus. These resistant mutants can revert to WT in the absence of drug selection pressure.

[0064] The M2 protein is an integral membrane protein expressed in large quantities on the surface of host cells infected with influenza virus (Lamb, RA, et al., Cell (1985) 40: 627-633). M2 has been shown to be a cell surface antigen of cytotoxic T lymphocytes (CTLs) that respond to influenza virus. Infection with influenza A virus elicits only low titers of anti-M2 antibodies (Feng, J., et al., Virol J. (2006) 3: 102-115). The high structural conservation of M2e may be due in part to a poor M2e-specific antibody response, resulting in no pressure changes. Anti-M2 antibody responses are more robust in individuals with pre-existing antibodies to the M2 protein (Zhong, W., et al., J. Infect Dis (2014) 209: 986-994). Anti-M2 antibodies induced by infection with the 2009 pandemic influenza A (H1N1) virus cross-react with the M2 protein of seasonal influenza A virus. Treatment of mice with anti-M2e antibodies significantly delayed disease progression and led to the isolation of M2e escape mutants, suggesting the potential of using M2e as a vaccine against influenza A virus infection (Zharikova, D., et al., J. Virol (2005) 79:6644-6654).

[0065] A DNA vaccine based on the fusion of M2e with HA has been described (Park, KS, et al., Vaccine (2011) 29:5481-5487). This fusion protein contains a human M2e peptide and an avian M2e peptide, located at the N-terminus of the HA protein via a 20-residue linker. Expression of the encoded M2e-HA fusion protein was confirmed. Mice immunized with the M2e-HA fusion DNA vaccine showed enhanced T cell responses to M2e and complete protection against lethal challenge with a heterologous avian influenza virus.

[0066] A recombinant fusion protein containing four tandem repeats of M2e genetically fused to the C-terminus of the Mycobacterium tuberculosis HSP70 (mHSP70) protein has been shown to protect against multiple influenza virus strains in mice (Ebrahimi, SM, et al., Virology (2012) 430:63-72). The M2e peptide SLLTEVETPIRNEWGCRCNDSSD has been conjugated to a cationic liposome delivery vehicle, along with peptides from other influenza proteins and BM2, a homolog of M2e from influenza B virus, as a vaccine (patent application US 2010 / 086584_A1). This vaccine elicits an immune response against M2e in mice. Mice vaccinated with the M2e peptide were protected from lethal influenza virus challenge. An inactivated influenza vaccine supplemented with M2e VLPs conferred enhanced and long-lasting cross-protection against antigenically distinct influenza A viruses in mice (Song, JM, et al., PNAS (2011) 108:757-761).

[0067] Modification of immunodominant regions

[0068] The present invention includes genetically modified recombinant influenza hemagglutinin (HA) genes and proteins produced by replacing the immunodominant region of the globular head domain of HA with alternative epitopes or inserting alternative epitopes therein to direct the host immune response to these epitopes. Some of these epitopes are recognized as HA by bNAbs. The present invention also relates to modified HA proteins and genes with modified MCSs and combinations of these modifications.

[0069] In one embodiment, the modified recombinant HA has the immunodominant antigenic site of the globular head domain replaced by the extracellular domain of the M2 protein (M2e), or M2e is inserted into the immunodominant antigenic site.

[0070] Guided by the three-dimensional structure of the HA protein, a specific surface peptide or peptides in the immunodominant region can be replaced with a heterologous peptide or peptides from another region of the same HA, or a heterologous peptide or peptides from HA of another subtype or strain of influenza virus. These heterologous peptides can be inserted into the immunodominant region. In addition, a specific surface peptide or peptides of HA can be replaced with a heterologous peptide from another protein unrelated to HA, or such a peptide can be inserted therein. These heterologous peptides can be natural proteins or artificially designed. Currently known antibodies may not recognize them.

[0071] The immunodominant regions of HA are located on the surface of the globular head domain of HA. These surface regions to be modified are surface-exposed helices, beta strands, or loops. These surface regions are preferably immunodominant antigenic sites and epitopes, or portions of such antigenic sites and epitopes, or immediately adjacent to such antigenic sites and epitopes.

[0072] In some embodiments, the immunodominant region of the HA globular head domain is replaced with a peptide representing an immunologically subdominant epitope of HA (recognized as HA by bNAbs). In other embodiments, the immunodominant region of the HA globular head domain is replaced with a peptide representing an immunologically subdominant epitope that is recognized by antibodies that neutralize influenza viruses. These immunologically subdominant epitopes are selected from the HA stem domain, or include HA fusion peptides or HA mature cleavage sites.

[0073] The recombinant modified HA proteins of the present invention are expressed in cell culture and secreted into the cell culture medium at levels similar to those of recombinant wild-type HA. These designs are applicable to any influenza HA of all influenza A, B, and C virus strains, including but not limited to influenza viruses that infect humans, as well as influenza viruses that infect other mammalian and avian species. In one embodiment, these modified HA proteins can be used as immunogens to prepare vaccines against influenza virus infection in humans or animals.

[0074] These immunodominant regions can be altered through site-directed mutagenesis. Solvent-exposed residues in the antigenic site are identified by the three-dimensional structure of the HA protein. One or more solvent-exposed residues in the antigenic site are altered through site-directed mutagenesis or replaced with peptides containing specific variations of these residues. The new site has the same secondary structure as the original site.

[0075] Exemplary HA with antigenic sites replaced by heterologous epitopes

[0076] For example, based on Figure 3 The HA structure is shown. Several positions in HA were selected to illustrate the feasibility of replacing immunodominant antigenic sites in the globular head domain by heterologous peptides from the stalk domain of HA.

[0077] The peptide KTSS of residues 119 to 122 (in Figure 5 The WT (numbered in Figure 1) is a surface-exposed helical structure near residues 124 to 125 of the Sa site. The Sa site is located on the side of the globular head domain of the HA trimer and is away from the receptor binding site. Modification at this site is unlikely to alter the receptor binding site and therefore unlikely to alter host receptor binding by the modified HA. The four-residue peptide KTSS is replaced by the bNAb epitope of the stem domain. HAGAKS at residues 137-142 at the Ca2 site corresponding to site A of H3 HA is the primary immunodominant site. KKGNSYPKLSKS at residues 153 to 164 is a surface loop at the Sa site corresponding to site B of H3 HA. In some constructs, the N-terminal portion of this peptide is replaced at KKGNS at residues 153 to 157. TSADQQSLYQNA at residues 184 to 195 of the Sb site forms a helix at the top of the HA globular head domain.

[0078] The surface loop of the Sa site and the helix of the Sb site are adjacent to each other in the globular head domain of HA and correspond to site B of H3. The Sa site and Sb site can jointly accommodate a conformational epitope with a loop and helix structure. However, because these sites are close to the receptor binding site, replacement of these peptides may disrupt the receptor binding site. EIAIRPKVRDQE at residues 213 to 224 of the Ca2 site is a loop near the interface between two HA monomers. This loop contacts the β-strand of the adjacent HA monomer corresponding to site D of H3 HA. A portion of the loop is surface-exposed. The Ca2 site is located within this peptide. In some constructs, this peptide was partially replaced.

[0079] In addition, the heterologous peptide can be placed in Figure 5 Any other antigenic sites as indicated and Figures 1 to 3 In or near the surface loops or helices shown. In addition, a heterologous peptide can be inserted into any of these positions without any deletion of antigenic site residues.

[0080] The bNAb CR8020 epitope is located in the stem domain. Figure 5 and Figure 6 As shown, the CR8020 epitope has two main components, which are composed of the outermost beta strand of the 5-stranded beta sheet near the base of the stem domain (HA2 residues 30 to 36, H3 HA's EGTGQAA, SEQ ID NO: 26) and the C-terminal portion of the fusion peptide (HA2 residues 15 to 19, H3 HA's EGMID, SEQ ID NO: 25). These two components are separated by 10 amino acid residues in the primary structure. The composite CR8020 epitope EGMIDYEGTGQAA (SEQ ID NO: 27) was designed, in which the two epitope components are connected by a tyrosine (Y). This composite CR8020 epitope is used as a heterologous peptide to replace the immunodominant site.

[0081] Another bNAb 1C9 epitope is a fusion peptide in the N-terminus of HA2 present in the stalk domain. The modified 1C9 epitope peptide GIFGAIAGFIEG (SEQ ID NO: 36) was designed to be displayed at an immunodominant site. This modified 1C9 peptide, designated I1C9, has an isoleucine (I) substitution for leucine (L) at position 2 of the H5 fusion peptide recognized by bNAb 1C9. This I1C9 fusion peptide is present in H1 HA and HA of some H6 and H9 viruses. This I1C9 peptide is placed in a different immunodominant site. Multiple constructs have been prepared using I1C9 to alter the position of the antigenic site.

[0082] The bNAb FI6 epitope is a conformational epitope containing two peptides that form a continuous surface on the tertiary structure of the HA precursor HA0 but are separated in the primary structure ( Figure 5 ). One FI6 epitope peptide, RKKRGLFGAIAGFIE, is the mature cleavage site and fusion peptide of HA0. Another FI6 peptide, KESTQKAIDGVTNKVNS, has a coiled-coil structure in HA2. Constructs have been prepared in which these two FI6 epitope peptides are placed in the globular head domain. The FI6 epitope peptide RKKRGLFGAIAGFIE is placed in the Sa site at residues 153 to 164, and the coiled-coil FI6 epitope peptide KESTQKAIDGVTNKVNS is placed in the Sb site at residues 184 to 195. In another construct, the FI6 epitope peptide RKKRGLFGAIAGFIE is placed in the Sa site without a second FI6 epitope peptide.

[0083] Many bNAb epitopes have been characterized. As described above, any of them can be placed at or near an immunodominant antigenic site or surface loop in the globular head domain.

[0084] In other embodiments, the heterologous peptide is a peptide of the extracellular domain of the M2 protein (M2e peptide). The M2e peptide is conserved and less variable among influenza A viruses. Although the M2e peptides from influenza A and influenza B viruses are different, the M2e peptide is also conserved and less variable among influenza B viruses.

[0085] In some embodiments, the heterologous peptide is an artificially designed peptide that has been specifically altered at an immunodominant antigenic site through site-directed mutagenesis. In some embodiments, the artificial peptide combines the desired features of both the immunodominant antigenic site and the heterologous peptide. In some embodiments, the artificial peptide has residues that interact with bNAbs and residues from the original immunodominant antigenic site to maintain the three-dimensional structure of the HA globular head domain. These artificial peptides are rationally designed based on the three-dimensional structure of HA or by screening randomly generated peptide libraries.

[0086] Constructs have been prepared to place the M2e peptide SLLTEVETPTRNGWECKCSDS in either the Ca2 site or the Sb site for expression using either a baculovirus expression system or a mammalian expression system. The M2e peptide can be further optimized based on consensus sequences from different influenza strains.

[0087] HA with a modified mature cleavage site

[0088] The present invention includes the following embodiments, in which the protease susceptibility of the HA mature cleavage site (MCS) is changed so that the resulting HA is sensitive to different types of proteases and resistant to those trypsin-like proteases that cut the natural HA mature cleavage sites of all strains. The changed MCS is designed to be recognized by a specific protease that does not exist in the known natural host of influenza virus. This makes the resulting HA resistant to maturation in all natural hosts of influenza virus. In the presence of this specific protease that recognizes the changed mature cleavage site, the HA of these gains is cut into mature forms containing HA1 and HA2. The recombinant influenza virus prepared by the resulting HA in the presence of this specific protease forms mature HA and is infective to the natural influenza host. However, due to the lack of appropriate protease in the natural host, the viral offspring replicated in the infected natural host do not have infectivity.

[0089] H1 HA molecules typically have a single basic residue in the MCS. This single basic residue is typically an arginine (R) residue at the N-terminus of the cleavage bond. Adding multiple basic residues, such as arginine (R) or lysine (K), to the MCS of H1 HA increases the infectivity of recombinant influenza viruses containing modified HA. Replacing the native H1 MCS with an H5 MCS containing polybasic residues increases the infectivity of recombinant influenza viruses containing modified HA (Kong, Wp., et al., PNAS (2006) 103: 15987-15991). These polybasic residues are more easily cleaved by many intracellular trypsin-like proteases. In some embodiments, the H1 HA MCS is modified by replacing it with an H5 HA MCS or a polybasic residue. The H1 HA MCS is also modified by inserting a polybasic residue between the last residue of HA1 (arginine) and the first residue of HA2 (glycine).

[0090] Further disclosed are genetically modified recombinant influenza HA genes and proteins having altered MCS sequences. The native MCS is replaced with a sequence from an MCS of another HA. The alteration includes replacing the native MCS of the HA with another MCS from a different HA, which is known to make the recombinant influenza virus more infective by making the resulting HA more susceptible to maturation and cleavage by an unidentified host protease. In a preferred embodiment, the native MCS of the H1 HA is replaced with the MCS of the H5 HA, or with polybasic residues of arginine (R) and lysine (K).

[0091] Several mammalian proteases such as factor Xa and enterokinase have their respective cleavage recognition sites that are completely located at the N-terminal side of the break bond. Their cleavage recognition sites can also be used to replace the natural HA mature cleavage site. As with natural HA, the C-terminal side of the break bond of these protease cleavage sites is glycine (G). The thrombin cleavage site also has glycine (G) on the C-terminal side of the break bond. After thrombin cleavage, a new glycine (G) N-terminus is produced. All of these proteases are present in many natural influenza hosts. However, these proteases are usually absent or present in trace amounts in the cell culture medium or cell line used to produce recombinant HA.

[0092] The TEV protease of tobacco etch virus (TEV) recognizes the cleavage site of ENLYFQG and cuts between glutamic acid (Q) and glycine (G). The free N-terminus produced by TEV protease cleavage is glycine (G), which is the same as the N-terminal residue of HA2 after HA maturation. Unlike the MCS of natural HA, the TEV cleavage site does not have basic residues. In some embodiments, the entire HA MCS is replaced by the TEV cleavage site, or some residues of the MCS are replaced by the TEV cleavage site, or only the arginine (R) N-terminus of the broken bond is replaced, for example, by the TEV cleavage site. HA constructs with MCS replaced by the TEV cleavage site are expressed using a baculovirus expression system or a mammalian expression system, and constructs with the same expression level as wild-type HA have been identified.

[0093] In the presence of TEV protease that cuts the modified MCS, the modified HA undergoes maturation and is converted into HA1 and HA2 with the natural N-terminus of the fusion peptide. The mature modified recombinant HA has the ability to bind to the corresponding host cell receptor and undergo membrane fusion. In the absence of TEV protease, the newly synthesized modified HA is still an uncut HA0 precursor. The lack of a free N-terminus of the fusion peptide prevents the HA from undergoing membrane fusion. The progeny HA protein produced in the infected host cell is still HA0 and is not processed into a mature functional form due to the lack of TEV protease in the host. The newly prepared virus with HA0 does not have membrane fusion ability and is therefore non-infectious.

[0094] Combination of antigenic site and mature cleavage site

[0095] The changes in the antigenic sites in the globular domain of HA and the changes in the MCS in the stem domain of HA can be any combination. A specific heterologous peptide can be introduced into the antigenic site in the globular head domain of the HA protein with different altered mature cleavage sites. In addition, two or more heterologous peptides can be introduced into a single HA protein at different antigenic sites. In one embodiment, the genetically modified recombinant influenza HA has an M2e peptide that replaces the antigenic site in the globular head domain of HA and the TEV protease site as the MCS. In another embodiment, the genetically modified recombinant influenza H1 HA has an M2e peptide that replaces the antigenic site in the globular head domain of HA and the H5 MCS (instead of the H1 natural MCS).

[0096] Production method

[0097] In order to produce modified HA, the protein is secreted into the cell culture medium in a soluble form by the cell, and the protein is not an integral membrane protein or is not attached to the cell membrane or cell surface. In some embodiments, the natural HA signal sequence is used for secretion in insect cells and mammalian cells. In other embodiments, the natural HA signal sequence is replaced by an insect cell signal sequence secreted in insect cells or a mammalian signal sequence secreted in mammalian cells. The protease cleavage site is placed at the C-terminus of the HA extracellular domain rather than the HA transmembrane domain and the intracellular domain, and then a "foldon" sequence derived from phage T4 fibritin is placed to stabilize the HA trimer, and a C-terminal His tag is placed for purification. The recombinant HA protein is made into a full-length, uncut precursor HA0 with a removed signal sequence, or a mature form containing HA1 and HA2 subunits with a wild-type fusion peptide at the N-terminus of HA2. In a preferred embodiment, the finally purified genetically modified HA forms a trimer as wild-type HA.

[0098] The gene of the designed recombinant influenza HA is prepared by de novo gene synthesis. Gene synthesis technology has been perfected and reviewed (Kosuri, S. and Church, GM, Nat Methods (2014) 11: 499-507). Genes longer than 10 kb are usually prepared by commercial providers. Gene synthesis provides the ability to modify specific HA sequences according to the needs of the expression host or specific genetic engineering and the opportunity for codon optimization. Restriction sites are designed at specific positions of the synthesized HA gene to facilitate the exchange of HA fragments between different constructs. A comprehensive scheme is used to incorporate a synthetic HA gene with a signal sequence and a protease site, a foldon sequence, and a C-terminal His tag into the baculovirus genome so that the expression of the modified HA protein is guided by the baculovirus polyhedrin promoter. In other embodiments, the same set of HA genes are cloned into a mammalian expression vector for expression in mammalian cells.

[0099] In some embodiments, baculovirus vectors are used to codon-optimize the HA construct for expression in insect cells. In other embodiments, the HA construct is codon-optimized for expression in mammalian cells, including but not limited to CHO cells (Chinese hamster ovary cells) and HEK 293 cells (human embryonic kidney 293 cells). Codon optimization of any organism is generally performed as follows: commercial software such as the Lasergene software package from DNASTAR (3801 Regent Street, Madison, WI 53705 USA), online web servers such as OPTIMIZER (on the World Wide Web, genomes.urv.es / OPTIMIZER / ), or by using its proprietary algorithm by a gene synthesis service provider. Codon optimization takes into account the elimination of G / C content, cryptic splice sites and RNA destabilization sequence elements, and the avoidance of stable RNA secondary structure. Codons are also manually adjusted according to the codon usage database (on the World Wide Web, kazusa.or.jp / codon / ). Due to codon degeneracy, the gene sequence can be changed without changing the encoded amino acid sequence. Restriction sites are introduced at specific positions by changing the codons without altering the amino acid sequence. Using any of these methods, the gene sequence is typically generated by back-translating the protein sequence using a computer algorithm or manually, with or without codon optimization for a specific organism.

[0100] Other embodiments include methods for preparing recombinant non-infectious influenza viruses using established cell culture methods from genetically modified HA genes with a TEV protease recognition site changed from the MCS. Reverse genetics systems have been developed that allow influenza viruses to be produced from RNA segments or plasmids with cloned cDNAs with or without the use of helper viruses (Luytjes, W., et al., Cell (1989) 59: 1107-1113; Neumann, G., et al., PNAS (1999) 96: 9345-9350; Fodor, E., et al., J. Virol (1999) 73: 9679-9682; de Wit, E., et al., J. Gen Virol (2007) 88: 1281-1287). Infectious influenza viruses are prepared by transiently transfecting mammalian cells with influenza RNA segments or plasmids with cDNAs for influenza RNAs. These viruses isolated from cell culture medium are used to infect embryonated chicken eggs to produce live infectious influenza viruses for vaccine production. Some influenza RNA segments are replaced by exogenous genes. Recombinant influenza A viruses are prepared using influenza C virus HEF protein replacing the HA protein (Gao, Q., et al., J. Virol (2008) 82: 6419-6426). In these embodiments, cDNA or RNA of the modified HA is co-transfected into mammalian cells with cDNAs of seven other influenza RNAs or seven other RNA segments prepared by standard molecular techniques and gene synthesis. Multiple identical or different HA proteins can be packaged into a single virion using established methods or similar methods (Uraki, R., et al., J. Virol (2013) 87: 7874-7881).

[0101] The method for preparing recombinant infectious influenza virus by genetically modified HA gene uses the established cell culture method as described above. In these embodiments, the genetically modified HA gene having H5 HA MCS or polybasic sequence at MCS is co-transfected into mammalian cells with cDNA of other 7 influenza RNAs or other 7 RNA segments prepared by the standard reverse genetics system as described above. These modified mature cleavage sites increase the efficiency of HA maturation in influenza natural hosts or cell cultures (Kong, Wp., et al., PNAS (2006) 103: 15987-15991). Mature functional HA protein has the ability to bind to host cells and fuse with host cell membranes.

[0102] Other embodiments include methods for preparing recombinant influenza virus-like particles (VLPs) from genetically modified HA genes using established cell culture methods in mammalian cells, insect cells, and plant cells (Chen, BJ, et al., J. Virol (2007) 81:7111-7123; Smith, GE, et al., Vaccine (2013) 31:4305-4313; D'Aoust, MA, et al., Plant Biotech (2010) 8:607-619).

[0103] Other embodiments include methods of preparing DNA vaccines from genetically modified HA genes using established methods (Jiang, Y., et al., Antiviral Res (2007) 75:234-241; Alexander, J., et al., Vaccine (2010) 28:664-672; Rao, SS, et al., PLoS ONE (2010) 5:e9812).

[0104] The expression results disclosed herein demonstrate that the MCS of H1 HA can be modified without affecting the expression of the resulting HA. Constructs with either the H5 HAMCS or a polybasic MCS have been shown to achieve expression levels equivalent to wild-type H1 HA. Furthermore, the MCS of HA has been replaced with a TEV protease cleavage site that lacks any basic residues. By varying the position of the TEV protease cleavage site, constructs with the TEV cleavage site as the MCS have been prepared that achieve expression levels equivalent to wild-type H1 HA.

[0105] The expression results further demonstrated that many antigenic sites in the globular head domain of HA1 can be replaced with heterologous peptides from the stem domain of the same HA or a different HA. Some constructs expressed at levels comparable to wild-type HA, while others expressed less. The nature of the heterologous peptide and its location within the globular head domain significantly affected the expression level of each resulting HA construct. More constructs can be prepared and tested in the same manner for expression to identify the best expressing construct.

[0106] In addition, recombinant HA was prepared by replacing certain immunodominant antigenic sites of the HA globular head domain with the M2e epitope from the influenza M2 protein. It is conceivable that any epitope from other influenza proteins can be used as a replacement peptide to replace the immunodominant antigenic sites in the HA globular head domain. In addition, the heterologous replacement peptide can be derived from another protein unrelated to the influenza virus. Specific antigenic sites in the HA globular head domain can be selected for the replacement peptide to maintain as many of the native structural features of the replacement peptide as possible to present the replacement peptide to the host immune system.

[0107] Baculovirus expression system

[0108] Since its introduction about 30 years ago (U.S. Patent No. 4,745,051; Summers, MD and Smith, GE, (1987) A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures.Texas Agricultural Experiment Station Bulletin No.1555 .), the baculovirus expression vector system (BEVS) has been used to express many different types of human and viral proteins, including intracellular proteins, membrane proteins, and secreted proteins. BEVS has been used to produce virus-like particles (VLPs). BEVS is a eukaryotic expression system using insect cells as hosts that provides post-translational modifications of proteins similar to those of mammalian cells (Jarvis, DL, "Baculovirus Expression Vectors," ed. The Baculoviruses In (Miller, LK, ed. (1997) pp. 389-420, Plenum Press, New York). BEVS based on Autographa californica nuclear polyhedrosis virus (AcNPV) have been developed. Many commercial kits are available for the production of recombinant baculovirus. BEVS has been successfully used to produce recombinant vaccines sold in the United States. Two FDA-approved vaccines have been produced using BEVS: Cervarix. TM - A bivalent recombinant human papillomavirus (types 16 and 18) vaccine in the form of non-infectious virus-like particles (VLPs) against cervical cancer (patent application WO 2010 / 012780A1); and (US Patents 5,762,939 A and 5,858,368 A) - A trivalent influenza vaccine against influenza made from membrane-bound hemagglutinin precursor (HAO).

[0109] To express a target protein using BEVS, the gene encoding the target protein is first subcloned into a transfer vector, which is an E. coli plasmid containing baculovirus sequences flanking the baculovirus polyhedrin gene. Polyhedrin is an abundant viral protein in wild-type baculovirus that is not required for baculovirus propagation in cell culture. The transfer vector is propagated in E. coli and isolated using standard molecular biology techniques (Sambrook, J., et al., (1989)). Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor). A transfer vector containing the gene of interest is recombined with baculovirus genomic DNA to produce a recombinant baculovirus genome in Escherichia coli or insect cells. The recombinant baculovirus genome directs the production of recombinant baculovirus and the expression of the target protein. The original method relies on homologous recombination between identical sequences flanking the polyhedrin gene in the transfer vector and in the viral genomic DNA in the insect cells. The isolated transfer vector DNA is co-transfected with the baculovirus genomic DNA into the insect cells. The recombinant baculovirus is selected for the absence of polyhedrin. Current commercial kits, such as BacPAK from Clontech, are available. TM Baculovirus expression system and BacMagic from Millipore TM Systems, using linearized baculovirus genomic DNA, which does not produce live virus without recombination. These kits allow for the production of recombinant baculovirus with high efficiency and minimal contamination with non-recombinant virus. Gateway recombination reactions (BaculoDirect TM The baculovirus expression system (Life Technologies) allows for in vitro recombination of viral genomes. Another method for producing recombinant viruses is through site-specific transposition in E. coli (Bac-to-Bac system, Life Technologies). The pFastBac-based transfer vector contains a transposon that allows the production of bacmids (large plasmids containing the baculovirus genome) in E. coli (U.S. Patent No. 5,348,886). Recombination of the target gene with the bacmid can be easily confirmed by polymerase chain reaction (PCR) before transfection of the bacmid into insect cells.

[0110] Recombinant baculovirus is propagated in insect cell culture. A small amount of virus is used to infect insect cells. After a few days, the conditioned medium containing the amplified virus is harvested as a viral stock. This amplification process is typically repeated several times to produce large quantities of viral stock. The viral stock is typically stored refrigerated in the dark for months or even years. Supplementing the viral stock with 5%-10% fetal bovine serum (FBS) has been used to preserve the viral stock and is believed to extend its shelf life. The viral stock is sometimes frozen for long-term storage at -70°C. Viruses are also amplified and stored in the form of baculovirus-infected insect cells (BIIC) (Wasilko, DJ, et al., Prot Exp Purif (2009) 65:122-132). Infected insect cells are harvested as BIIC stock before lysis and frozen following standard cell freezing procedures. The BIIC stock is stored in liquid nitrogen or at ultra-low temperatures of -65°C to -85°C for extended periods of time and used as a viral stock to infect insect cells for protein expression. Frozen BIIC stocks offer longer storage times than liquid viral stocks.

[0111] Commonly used insect cell lines for BEVS are SF9 and SF21 cells from the fall armyworm (Spodoptera frugiperda) and Hi5 or Trichoplusia ni cells from the cabbage looper (Trichoplusia ni). Other insect cell lines from species of the silkworm (Bombyx mori), the honeycomb moth (Galleria mellonella), and the gypsy moth (Lymantria dispar) have also been used. SF9, SF21, and Hi5 (or Trichoplusia ni) cells have been adapted for suspension culture in serum-free medium. These cell lines and culture media are available from a number of commercial sources. Cell cultures of these cell lines are typically maintained in small volumes of 1 or 2 liters in shake flasks in a shaking incubator at a temperature range of 22°C to 28°C in an unsupplemented ambient atmosphere at room temperature. The cell cultures are scaled up in stirred tank bioreactors or disposable bioreactors. Conditions for large-scale insect cell culture in bioreactors have been well established (WAVE Bioreactor System - Cell Culture Procedures. GE Healthcare). Supplemental oxygen is also commonly used in large-scale insect cell culture to increase cell density.

[0112] A range of conditions for expressing a specific target protein are tested to optimize the expression of the target protein using different cell lines by varying the virus-to-cell ratio and the time of harvest after infection. SF9, SF21, and Hi5 (or Trichoplusia ni) cells are the most common cell lines used for BEVS protein expression. The target protein can be expressed better in one cell line than in other cell lines. Some reports show that Hi5 (or Trichoplusia ni) cells express certain secreted proteins more. The virus-to-cell ratio, commonly referred to as the multiplicity of infection (MOI), is tested to determine the optimal infection conditions for target protein expression. Culture samples are collected at different time points after infection. Cells and conditioned medium are separated by centrifugation or filtration. Protein expression levels are determined by standard methods. The cell density, cell viability, and cell size of the culture are monitored, which provide information on cell growth and culture conditions. The glucose level, dissolved oxygen, and pH of the culture are sometimes monitored for nutrient consumption in the culture medium. Conditions that result in optimal target protein expression are selected for large-scale production of the protein.

[0113] While determination of viral stock titer and MOI are widely used to determine infection conditions for viral amplification and protein expression using BEVS, the TIPS method described above provides a faster method for determining optimal infection conditions for viral amplification and protein expression. Insect cells increase in size after infection with recombinant baculovirus. Cell division also ceases following infection. Recombinant baculovirus causes cell lysis approximately 48 hours after infection. Cell division is monitored by counting cells as a function of the culture's cell density. Cell lysis is monitored as a function of cell viability by counting live and dead cells in the culture. Viable cell size is measured as cell diameter. Cell density, cell viability, and viable cell size are commonly measured by many cell counting instrument models. Cell density and cell viability can also be measured manually using a hemocytometer. Together, cell density, cell viability, and viable cell size provide information on infection kinetics. The TIPS method uses infection kinetics to determine the optimal conditions for expressing a specific target protein using a specific viral stock. It eliminates the time-consuming measurement of viral titer for MOI calculation. The TIPS method is often used to achieve consistent viral amplification and protein expression.

[0114] In addition to insect cells, living insects infected with recombinant baculovirus have been used to express many secretory and membrane proteins. Mature HA has been expressed in larvae of tobacco budworm (Heliothis virescens) (Kuroda, K., et al., J. Virol (1989) 63: 1677-1685).

[0115] Expression systems based on other types of baculoviruses, such as Bombyx mori nuclear polyhedrosis virus (BmNPV), have also been developed. BmNPV may have better biosafety than AcNPV because BmNPV has a narrower host range and does not grow as a pest in the field. Baculovirus expression systems based on BmNPV have been used to express functional proteins in cell culture and in silkworm (Bombyx mori) larvae (Maeda, S., et al., Nature (1985) 315: 592-594).

[0116] BEVS are commonly used to produce protein complexes and VLPs. Two or more genes are cloned into a single vector to express multiple proteins. Two or more viral stocks, each directing the expression of a single protein, are used to co-infect insect cells to produce protein complexes or VLPs. Influenza VLPs have been produced using the baculovirus expression system (Bright, RA, et al., PLoS ONE (2008) 3:e1501).

[0117] HA transfer vector for baculovirus expression

[0118] Genes to be expressed using BEVS are typically under the control of the polyhedrin promoter, a strong late promoter of baculovirus that leads to protein expression prior to baculovirus-induced insect cell death. Other early or late promoters are also used for protein expression. In order for the recombinant target protein to be secreted into the cell culture medium by insect cells, a DNA segment encoding a signal peptide is engineered at the 5' end of the frame of the gene of interest. Two commonly used signal peptides for insect cells are the honey bee melittin signal sequence or the AcNPV envelope surface glycoprotein GP67 signal sequence. The Signal Sequence Database (on the World Wide Web, signalpeptide.de / index.php) lists many other possible signal peptides for consideration. After secretion, the signal peptide is removed by cellular proteases that process the signal peptide. Although influenza virus is not an insect virus, the HA signal peptide has been used as a secretion signal peptide for secretion or membrane insertion of recombinant proteins in BEVS. The HA signal peptide of MKTIIALSYIFCLVFA is commonly used to express recombinant transmembrane G protein-coupled receptors (GPCRs) in insect cells (Rosenbaum, DM, et al., Science (2007) 318: 1266-1273; Zou, Y., et al., PLoS One (2012) 7: e46039). HA has been expressed in insect cells using its native signal peptide. In this particular case, overall expression was lower than when using the insect cell signal peptide, but the expressed HA was a mature form containing HA1 and HA2 (U.S. Patent No. 5,858,368A). HA signal peptides are not conserved among HAs. For example, the aforementioned HA signal peptide differs from the H1 HA signal peptide of SEQ ID NO: 7. Empirical selection of the HA signal peptide can improve HA expression in insect cells.

[0119] Commercial vectors can be used to prepare recombinant baculoviruses by homologous recombination using the Bac-to-Bac system or the Gateway system (Life Technologies). Features of these transfer vectors, such as promoter and signal sequences, can be customized using standard molecular biology techniques. Entire transfer vectors can now be synthesized completely by gene synthesis. Gene synthesis allows the design of transfer vectors with specific sequences and features.

[0120] The method of expressing influenza HA using BEVS has been perfected on a laboratory scale (Stevens, J., et al., Science (2004) 303: 1866-1870). For example, the transfer vector contains HA from the 1918 influenza virus under the control of the polyhedrin promoter. The 1918 influenza virus HA expression construct has a GP67 signal peptide at the N-terminus instead of the HA signal peptide for secretion. A thrombin cleavage site is introduced into the extracellular domain instead of the C-terminus of the transmembrane domain, and then a "foldon" sequence derived from bacteriophage T4 fibritin is placed to stabilize the HA trimer, and a C-terminal His tag is placed to facilitate purification. The foldon sequence and His-tag can be removed by thrombin cleavage. Similar designs of HA expression transfer vectors have been used to express different HAs from many influenza strains (Stevens, J., et al., Science (2006) 312:404-410; Xu, R., et al., Science (2010) 328:357-360; Whittle, JRR, et al., PNAS (2011) 108:14216-14221).

[0121] In addition to foldon, other trimerization domains can be used to stabilize the trimer of recombinant HA. For example, the thermostable HIV-1 glycoprotein 41 (gp41) trimerization domain or the first 16 of the 31 residues of the GCN4 leucine zipper sequence have been used to facilitate trimerization of truncated HA constructs (Impagliazzo, A., et al., Science (2015) 349: 1301-1306; Yassing, HM, et al., Nat Med (2015) 21: 1065-1070).

[0122] As disclosed in U.S. Patents 5,762,939 A, 5,858,368 A and 6,245,532 B1, BEVS has been used on a commercial scale to produce recombinant HA precursors (HA0) with transmembrane and intracellular domains from influenza A and B viruses. These recombinant HA proteins are FDA-approved for use as influenza vaccines. Ingredients in. The HA0 in the assay was prepared by replacing the HA signal peptide with the baculovirus chitinase signal peptide (referred to as the 61K signal peptide). The recombinant HA binds to the peripheral membranes of insect cells. The recombinant HA is extracted from the membranes using detergents and further purified.

[0123] Mammalian expression

[0124] Chinese hamster ovary (CHO) cells and human embryonic kidney 293 (HEK293) cells are commonly used mammalian cell hosts for transient transfection gene expression of recombinant proteins. Both CHO cells and HEK293 cells have suspension cell lines and adherent cell lines. These cells are usually cultured in culture medium with or without FBS. When cells are cultured in the absence of FBS, chemically defined culture medium is usually used. All suspension and adherent cell lines can be used to express HA or other secreted proteins.

[0125] Transient transfection is a well-established method for introducing DNA into cells for protein expression. Many commercial transfection reagents are available. Similar to the expression optimization process for baculovirus expression, culture medium samples are collected regularly after transfection to analyze protein expression using standard methods. HA protein is easily detected by affinity capture of the His tag in the HA construct or by Western blotting using a number of commercially available anti-HA antibodies. Protein expression typically begins approximately 48 hours after transfection and can increase within several days after transfection. Supplements are sometimes added after transfection to increase target protein expression. Other mammalian cell lines, such as COS-1, a fibroblast-like cell line derived from African green monkey kidney tissue, have also been used for HA expression. Influenza VLPs are produced by co-transfection of multiple plasmids containing cDNAs for proteins encoded by all 10 influenza viruses (Mena, I., et al., J. Virol (1996) 70: 5016-5024; Chen, BJ, et al., J. Virol (2007) 81: 7111-7123).

[0126] Another method for delivering genes to mammalian cells uses a recombinant baculovirus called BacMam to transfer baculovirus genes into mammalian cells (Boyce, FM and Bucher, NL, PNAS (1996) 93: 2348-2352; Dukkipatia. A., et al., Protein Expr Purif (2008) 62: 160-170). Baculovirus is modified by incorporating a mammalian expression cassette into a baculovirus expression vector for transgene expression in mammalian cells. Recombinant BacMam baculovirus is produced by standard baculovirus production methods. A stock of amplified recombinant BacMam baculovirus is added to CHO or HEK293 cultures to deliver the mammalian expression cassette to CHO or HEK293 cells for protein expression. The BacMam platform can easily transduce large numbers of mammalian cells.

[0127] Gene expression vectors transfected into mammalian cells can be integrated into the cell chromosomes to establish stable cell lines for expressing the target protein. Stable CHO cell lines are the most common hosts for producing therapeutic biological agents such as monoclonal antibodies. This technology is very suitable for large-scale production of therapeutic proteins and vaccines. In order to establish stable cell lines, cells transfected with the expression vector are selected based on the selection marker on the expression vector. Typically, multiple copies of the target gene are integrated into the genome of the stable cell line.

[0128] HA vectors for mammalian expression

[0129] The HA expression construct for mammalian expression has the same design and amino acid sequence as baculovirus expression. Codons have been optimized for CHO cell expression or HEK293 cell expression. Baculovirus codons are also suitable for mammalian cells, and vice versa. The HA signal peptide has been used as a signal peptide to secrete or membrane insert recombinant proteins in mammalian expression systems. Other commonly used mammalian signal peptides include human IL-2 signal peptide, tissue plasminogen activator (tPA) signal peptide and many other signal peptides found in the signal sequence database (on the world wide web, signalpeptide.de / index.php). Many mammalian expression vectors are commercially available. Each vector usually has an enhancer-promoter for high-level expression, a polyadenylation signal and transcription termination sequence for mRNA stability, an SV40 starting point for episomal replication, an antibiotic resistance gene and a pUC starting point (for selection and maintenance in Escherichia coli). Common promoters for mammalian expression include the CMV (cytomegalovirus) promoter, the hEF1-HTLV promoter, a composite promoter comprising the elongation factor-1α (EF-1α) core promoter and the R segment of the long terminal repeat of human T-cell leukemia virus (HTLV) type 1 and part of the U5 sequence (R-U5′), or other promoters found in MPromDb (Mammalian Promoter Database, on the World Wide Web, mpromdb.wistar.upenn.edu / ) or the Eukaryotic Promoter Database (EPD, on the World Wide Web, epd.vital-it.ch / ). Transfer vectors typically have another selection marker for generating stable cell lines.

[0130] The following examples are offered to illustrate, but not to limit, the present invention.

[0131] Example 1

[0132] Construction of transfer vector for H1 HA production by recombinant baculovirus

[0133] In this example, a parent plasmid that can be modified to prepare the modified HA protein of the present invention is described. The basic outline of this plasmid is shown in Figure 4 In which the insert in the standard plasmid for expression in baculovirus is bracketed by the restriction sites of this insert, and there are restriction sites that allow replacement of the MCS of the HA protein and replacement of the immunodominant region in the globular head domain of HA. The encoded protein is fused to an affinity tag of 10 histidines (10xHis tag) for purification, which can be removed after purification by the performance of the inserted protease cleavage site. This vector is named H1WT.

[0134] A / California / 07 / 2009, the swine-derived influenza A virus H1N1 strain recommended by the WHO for 2009 influenza vaccine production (as described on the World Wide Web at who.int / csr / resources / publications / swineflu / vaccine_recommendations / en / ), its HA sequence (SEQ ID NO: 1) was used as the parent sequence for incorporation of heterologous peptides. The accession number for this HA sequence is UniProt: C3W5X2. All constructs have Figure 4 The same design as schematically shown in FIG. The N-terminal HA signal peptide (SEQ ID NO: 7) was replaced by the GP67 signal peptide MVSAIVLYVLLAAAAHSAFA (SEQ ID NO: 2). Based on the HA construct first described by Stevens, J., et al., Science (2006) 312: 404-410, the C-terminal transmembrane (TM) domain with the ILAIYSTVASSLVLVVSLGAISFWMCS sequence and the intracellular domain with the NGSLQCRICI sequence of HA were replaced by the TEV cleavage site (SEQ ID NO: 4), and then replaced by foldon (SEQ ID NO: 5) and 10xHis tag (SEQ ID NO: 6), i.e., GAENLYFQGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHHHHHH (SEQ ID NO: 3). The foldon stabilizes the recombinant HA, while the 10xHis tag facilitates purification of the HA from the cell culture medium. The foldon and 10xHis tags can be removed by TEV protease which recognizes the TEV cleavage site located between the HA sequence and the foldon sequence.The gene was codon optimized for baculovirus expression and synthesized by standard methods (GENEWIZ, South Plainfield, NJ 07080, USA).

[0135] like Figure 4As schematically shown in the figure, unique restriction sites spaced apart: ClaI site, NsiI site and BamHI site were introduced into the HA sequence to facilitate sequence changes and exchanges. The resulting construct GP67-H1WT-TEV-foldon-10His (denoted as "H1WT" (SEQ ID NO: 9)) was subcloned into the pFastBac plasmid between the NcoI site and the HindIII site. The sequence between the ClaI and NsiI sites encodes the most immunodominant antigenic sites of the globular head domain. The sequence between the NsiI and BamHI sites encodes the MCS. The expression of the H1WT construct is controlled by the baculovirus polyhedrin promoter. The resulting plasmid is a transfer vector for expressing the recombinant wild-type HA of A / California / 07 / 2009. The H1WT construct is the parent construct from which the genetically modified HA construct is prepared.

[0136] To prepare genetically modified HA with a heterologous peptide, the wild-type fragment of H1WT was replaced with the aforementioned restriction sites using a synthetic DNA fragment encoding a specific variation of the HA. To modify the MCS, the NsiI-BamHI fragment of H1WT was replaced with a DNA fragment encoding the altered mature cleavage site. To alter the antigenic site in the HA1 globular head domain, the ClaI-NsiI fragment of H1WT was replaced with a DNA fragment encoding the altered antigenic site of the heterologous peptide. By simply exchanging restriction fragments from different plasmids, constructs with different combinations of mature cleavage sites and antigenic sites were prepared. The resulting constructs were confirmed by DNA sequencing.

[0137] Example 2

[0138] Design and construction of H1 HA constructs with modified mature cleavage sites

[0139] In this example, the segment of H1WT between the Nsil and BamH1 restriction sites of the MCS was replaced by an alternative sequence that modified the MCS. In several cases, a TEV cleavage site was included in the modified MCS.

[0140] The modified mature cleavage site was introduced into the transfer vector of H1 HA described in Example 1. The constructs with the modified mature cleavage site are listed in Table 1.

[0141] Table 1 List of H1 HA constructs with altered mature cleavage sites

[0142]

[0143] a Construct names are based on the mature cleavage site. H1 indicates H1 HA. MCS indicates the mature cleavage site.

[0144] b The constructs are described by their sequence characteristics. GP67ss represents the GP67 signal sequence. TEV-foldon-10His represents the sequence of the TEV cleavage site, foldon sequence, and 10xHis tag at the C-terminus of the construct. Figure 4 shown.

[0145] c H1WT is the wild-type H1 HA construct with the native MCS.

[0146] The positions of these modifications are shown in Figure 5 .

[0147] Construct H1H5cs (SEQ ID NO: 11) has the MCS of wild-type H1 HA (SEQ ID NO: 9) replaced by the H5 MCS (SEQ ID NO: 12). Construct H1R5cs (SEQ ID NO: 14) has the MCS of wild-type H1 replaced by five arginines. Construct H1IR5cs (SEQ ID NO: 16) has four arginines inserted between the HA1 and HA2 sequences while retaining all native HA1 residues. An Nsil-BamHI fragment with the changes was synthesized and subcloned into H1WT between the Nsil and BamHI sites to replace the wild-type fragment.

[0148] To test whether the H1 HA MCS could be modified to contain a TEV cleavage site that did not contain any basic residues, several constructs were prepared by varying the number of residues in the H1 HA MCS. Construct H1TEV1 (SEQ ID NO: 18) had the 7-residue PSIQSRG at the H1 HA MCS replaced by the 7-residue TEV cleavage site of ENLYFQG (SEQ ID NO: 4). Construct H1TEV2 (SEQ ID NO: 20) had the 8-residue IPSIQSRG of the H1 HA MCS replaced by SPENLYFQG containing the TEV cleavage site. Construct H1TEV3 (SEQ ID NO: 22) had the last 3 residues (QSR) of the H1 HA MCS (SEQ ID NO: 8) replaced by the TEV cleavage site.

[0149] Example 3

[0150] Design and construction of H1 HA constructs harboring heterologous epitopes in the globular head domain

[0151] In this example, a basic plasmid and a plasmid with a modified MCS were modified between the ClaI and NsiI sites by alternative nucleotide sequences in which various immunodominant sites were replaced with heterologous epitopes.

[0152] Heterologous epitopes were introduced into the constructs described in Examples 1 and 2. The heterologous epitopes were inserted around or replaced with the immunodominant antigenic sites of the HA globular head domain. The constructs with modified antigenic sites are listed in Table 2.

[0153] Table 2 List of H1 HA constructs with modified immunodominant antigenic sites in the globular head domain

[0154]

[0155]

[0156]

[0157] The positions of these modifications are shown in Figure 5 .

[0158] The 13 amino acid residue composite CR8020 epitope peptide replaced the 12 residue EIAIRPKVRDQE surface loop surrounding the antigenic site Ca2. A ClaI-NsiI fragment with the composite CR8020 epitope peptide replacement was synthesized and subcloned between the ClaI and NsiI sites of H1H5cs to generate the HA construct designated H1H5cs-CR8020Ca (SEQ ID NO: 24). To generate H1TEV2-CR8020Ca (SEQ ID NO: 29), the ClaI-NsiI fragment of H1H5cs-CR8020Ca was isolated and subcloned between the ClaI and NsiI sites of H1TEV2.

[0159] The construct H1TEV2-CR8020Sa3 (SEQ ID NO: 31) has a composite CR8020 epitope peptide replacing residues KKGNS at one of the Sa sites, which corresponds to the site B loop of the H3 HA at the top of the globular head domain. A ClaI-Nsil fragment with the CR8020 modification was synthesized and subcloned between the ClaI and Nsil sites of H1TEV2 to generate the HA construct denoted H1TEV2-CR8020Sa3.

[0160] The construct H1TEV2-CR8020Sa4 (SEQ ID NO: 33) contains a composite CR8020 epitope peptide that replaces the helical structure of residue KTSS near one of the Sa sites. This position is located on the side of the globular head domain of the HA trimer and distal to the receptor binding site. This modification is unlikely to alter receptor binding. A ClaI-NsiI fragment with the CR8020 modification was synthesized and subcloned between the ClaI and NsiI sites of H1TEV2 to generate the HA construct denoted H1TEV2-CR8020Sa4.

[0161] The HA monomer models of constructs CR8020Sa4 and CR8020Ca are shown in Figure 6 Each HA monomer has a composite CR8020 epitope peptide in the globular head domain and a native CR8020 epitope in the stalk domain.

[0162] Several HA constructs were prepared with the I1C9 epitope peptide of GIFGAIAGFIEG (SEQ ID NO: 36). One construct, H1TEV2-I1C9Ca1 (SEQ ID NO: 35), had the I1C9 peptide replacing RPKVRDQE at residues 217 to 224 around the Ca2 site. Another construct, H1H5cs-I1C9Ca (SEQ ID NO: 46), had the I1C9 peptide replacing the longer peptide EIAIRPKVRDQE at residues 213 to 224 of Ca2. Other constructs had the I1C9 peptide replacing the Ca2 site HAGAKS, Sa site KKGNS, and Sa site KTSS, corresponding to site A of H3 HA, respectively. Each ClaI-NsiI fragment with a change was synthesized and subcloned between the ClaI and NsiI sites of H1TEV2 to generate HA constructs designated H1TEV2-I1C9Ca1 (SEQ ID NO: 35), H1TEV2-I1C9Ca2 (SEQ ID NO: 38), H1TEV2-I1C9Sa3 (SEQ ID NO: 40), and H1TEV2-I1C9Sa4 (SEQ ID NO: 42), respectively. A ClaI-NsiI fragment with a change in I1C9Ca was synthesized and subcloned between the ClaI and NsiI sites of H1H5cs to generate the HA construct H1H5cs-I1C9Ca. The I1C9 peptide was also used in the same manner to replace residues TSADQQSLYQNA at the Sb position of the H1H5cs construct, such as H1H5cs-I1C9Sb (SEQ ID NO: 44). The Sb site corresponds to the site B helix of H3 HA.

[0163] The ClaI-NsiI fragment was gene synthesized using the FI6 epitope peptide RKKRGLFGAIAGFIE (SEQ ID NO: 49) at the Sa site and the coiled-coil FI6 epitope peptide KESTQKAIDGVTNKVNS (SEQ ID NO: 50) at the Sb site. The ClaI-NsiI fragment with the FI6 substitution was subcloned between the ClaI and NsiI sites of H1H5c and H1WT, generating constructs H1H5cs-FI6Sab (SEQ ID NO: 48) and H1WT-FI6Sab (SEQ ID NO: 52), respectively. Another ClaI-NsiI fragment was gene synthesized using the FI6 epitope peptide RKKRGLFGAIAGFIE (SEQ ID NO: 49) at the Sa site. This fragment replaced the ClaI-NsiI fragment of H1TEV1 to generate construct H1TEV1-FI6Sa (SEQ ID NO: 54).

[0164] Example 4

[0165] Design and construction of an H1 HA construct harboring an M2e peptide at the antigenic site of the globular head domain

[0166] In this example, the nucleotide sequence of the ClaI-NsiI fragment was replaced by a nucleotide sequence in which a portion of the Ca2 site or the Sb site was replaced by a nucleotide sequence encoding the M2e peptide. The constructs having the M2e peptide are listed in Table 2. The positions of these modifications are shown in FIG. Figure 5 .

[0167] Gene synthesis generated ClaI-NsiI fragments (SEQ ID NO: 57) with the M2e peptide replacing EIAIRPKVRDQE at the Ca2 site or replacing the helix of residues TSADQQSLYQNA at the Sb site. HA constructs with the M2e peptide were first prepared in H1H5cs, where the ClaI-NsiI fragment of H1H5cs was replaced with a single ClaI-NsiI fragment containing the M2e peptide, resulting in constructs H1H5cs-M2eCa (SEQ ID NO: 56) and H1H5cs-M2eSb (SEQ ID NO: 59), respectively. To transfer the ClaI-Nsil fragment of H1H5cs-M2eCa to the H1TEV2 construct, the ClaI-Nsil fragment of H1H5cs-M2eCa was isolated and subcloned between the ClaI and Nsil sites of H1TEV2, resulting in the construct named H1TEV2-M2eCa (SEQ ID NO: 61).

[0168] Example 5

[0169] Production of recombinant baculovirus using the Bac-to-Bac baculovirus expression system

[0170] This example describes techniques for preparing insect cells containing the expression systems contained in the constructs prepared in Examples 1-4.

[0171] Recombinant baculovirus was generated from the pFastBac-based transfer vector of the construct (Bac-to-Bac Baculovirus Expression System, Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Briefly, the transfer vector was transformed into E. coli DH10Bac chemically competent cells. White colonies harboring the recombinant bacmid were selected on plates containing Blue-gal. Bacmid DNA was isolated by standard alkaline lysis miniprep methods according to the manufacturer's instructions. Recombinant bacmids were identified by PCR using the M13 forward (-40) primer (GTTTTCCCAGTCACGAC) and the M13 reverse primer (CAGGAAACAGCTATGAC). The recombinant bacmid produced a PCR product of approximately 4 kb.

[0172] use Reagent, other commercially available transfection reagents, or polyethyleneimine (PEI) were used to transfect SF9 insect cells attached to 12-well plates with the recombinant bacmid. 4-7 days after transfection or when the virus infection caused the SF9 cells to enlarge, the cell culture medium was harvested as the P0 virus stock. Each P0 virus stock was used to infect 2 x 10 6 50ml SF9 cells with 10 cells / ml were added to amplify the virus. A Cedex cell counter (Roche Diagnostics Corporation, Indianapolis, IN, USA) was used to monitor the culture. The harvest time was determined by the average viable cell size and cell viability. When the average viable cell size was 4-7 μm larger than that of uninfected cells and the activity range was 50%-70% within 4-5 days after infection, the cells were removed by centrifugation, the cell culture supernatant was collected and stored as P1 virus stock solution in the dark at 4°C. Sometimes the virus stock solution was filtered through a 0.2 μm sterile filter to keep it sterile.

[0173] To further amplify the viral stock, 250 μl of P1 viral stock was used to infect 500 ml of a 2x10 6SF9 cells were grown at 100 cells / ml. The culture was monitored using a Cedex cell counter. When the average viable cell size was 4-7 μm larger than that of uninfected cells and the viability ranged from 50% to 70% within 4-5 days after infection, the cells were removed by centrifugation, and the cell culture supernatant was collected and stored as P2 virus stock at 4°C in the dark. The virus stock was sometimes filtered through a 0.2 μm sterile filter to maintain sterility.

[0174] Example 6

[0175] Expression of recombinant HA protein.

[0176] This example demonstrates expression in insect cells of the various constructs described in Examples 1 to 4. Depending on the specific construct, different levels of target protein expression were obtained.

[0177] To detect the expression of recombinant HA, the cell culture medium of SF9 cells infected with a recombinant baculovirus stock (usually a P1 or P2 virus stock as described in the examples) was collected and incubated with Ni-NTA resin (Qiagen, Germantown, MD USA) to perform affinity capture via the 10xHis tag of the recombinant HA. After washing the Ni-NTA resin with phosphate-buffered saline (1xPBS), the Ni-NTA resin was boiled with gel loading buffer in the presence or absence of a reducing agent for analysis by SDS-PAGE or Western blotting using an anti-His antibody. A protein band of approximately 63 kDa on stained gels or anti-His Western blots indicated the expression of secreted recombinant HA full-length protein.

[0178] like Figure 7 and Figure 8 As shown, expression of the H1 HA protein of the parental construct H1 WT was detected as expected. HA proteins with altered mature cleavage sites with multiple base residues: H1H5cs, H1R5cs, and H1IR5cs were expressed at the same level as H1WT. H1TEV1, in which the entire H1 HA mature cleavage site was replaced with the TEV cleavage site, was expressed much less than H1WT. By retaining more native residues in the mature cleavage site, H1TEV2 and H1TEV3 expressed well compared to H1WT ( Figure 7 and Figure 8 Not all substitutions produce the same level of expression. By adjusting the position of the TEV cleavage site in the construct, expression levels of wild-type HA were achieved.

[0179] Figure 7 and Figure 8It was also shown in the literature that placing the same immunodominant bNAb epitope at different immunodominant antigenic sites resulted in different expression levels. Constructs of composite CR8020 epitope peptides at the Sa site (H1TEV2-CR8020Sa4) or the Ca2 site (H1H5cs-CR8020Ca) expressed at the same level as wild-type HA, while the H1TEV2-CR8020Sa3 construct expressed poorly. Therefore, the exact location of heterologous epitope placement may be important for the expression of the resulting HA. The nature of the immunodominant bNAb epitope may also affect the expression level of the resulting HA protein.

[0180] Individual placement of the I1C9 epitope at several antigenic sites also revealed different expression levels of the resulting HA proteins ( Figure 7 and Figure 8 Construct H1H5cs-I1C9Ca showed higher expression than construct H1TEV2-I1C9Ca1. The peptide sequences in the antigenic sites replaced by the I1C9 epitope peptide differed slightly between these two constructs. Similar to constructs with TEV cleavage sites such as the MCS, the position of the substitution affected expression levels.

[0181] Constructs with the M2e peptide at the Ca2 site, H1H5cs-M2eCa and H1TEV2-M2eCa, gave the same expression as H1WT regardless of the mature cleavage site ( Figure 7 and Figure 8 However, the construct H1H5cs-M2eSb, which has the M2e peptide at the Sb site, expressed poorly. The Sb site exhibits a helical structure, which may be required for proper folding of HA.

[0182] The successful expression of numerous H1 HA constructs demonstrates that the immunodominant antigenic site of the HA globular head domain can be replaced with an immunodominant bNAb epitope from the stalk domain or with an M2e peptide from another protein unrelated to HA. These results reveal the plasticity of the globular head domain and demonstrate the feasibility of enabling functional HA to present heterologous epitopes within its globular head domain. In summary, Examples 1-6 illustrate methods for constructing HA with heterologous epitopes and / or altered MCSs and selecting modified HAs with good expression.

[0183] Example 7

[0184] Purification of recombinant HA protein

[0185] This example demonstrates the purification of the various constructs described in Examples 1-4.

[0186] In shake flasks, infect 50 ml to 1 L of baculovirus stock at a density of 2 x 10 cells with 1-4 ml of P1 or P2 virus stock to 1 L of cells.6 SF9 cells were grown at 100 cells / ml. The cultures were monitored using a Cedex cell counter. The harvest time was determined by the mean viable cell size and cell viability. When the mean viable cell size was 4-7 μm larger than that of uninfected cells and the viability was about 80% within 2 to 3 days after infection, the cells were removed by centrifugation and the conditioned cell culture medium was collected and stored at 4°C. The conditioned cell culture medium was incubated with Ni-NTA resin by shaking on a rotating grill at 4°C for 2-4 hours. The Ni-NTA resin was collected by centrifugation and washed with 1xPBS. The washed Ni-NTA resin was loaded into a gravity flow column and further washed with 1xPBS supplemented with 50 mM imidazole. The Ni-NTA resin was then eluted with 400 mM imidazole in 1xPBS by gravity flow. The eluted HA protein buffer was exchanged with 1xPBS and concentrated by ultrafiltration. By Figure 8 SDS-PAGE analysis of purified HA protein is shown.

[0187] appendix

[0188] Description of influenza viruses, types, and hosts

[0189] Influenza viruses are composed of three genera in the Orthomyxoviridae family of viruses: influenza A, B, and C. Each genus contains only one virus: influenza A, B, and C. Influenza A, B, and C are also referred to as influenza A, influenza B, and influenza C, respectively. Influenza viruses are enveloped, negative-sense, single-stranded RNA viruses. The viral genome is encoded in a single RNA segment. Influenza A and B viruses each have eight RNA segments encoding ten proteins. Influenza C viruses each have seven RNA segments encoding nine proteins. The ten characteristic proteins of influenza A are PB2, PB1, and PA polymerases, hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix proteins M1 and M2, and nonstructural proteins NS1 and NS2 (Webster, RG, et al., Microbiol Rev (1992) 56:152-179). Influenza C virus has a hemagglutinin-esterase-fusion (HEF) protein that combines the functions of HA and NA (Herrler, G., et al., J. Gen Virol (1988) 69:839-846). The classification of influenza A, B, and C viruses is based on antigenic differences in their NP and matrix proteins.

[0190] Influenza A viruses are small particles with a diameter of 80-120 nm. They consist of a host-derived lipid bilayer envelope studded with the virally encoded membrane proteins HA, NA, and M2, an inner shell made of the M1 matrix protein, and a central nucleocapsid containing the viral genome of the individual RNA segments (Webster, RG, et al., Microbiol Rev (1992) 56:152-179). The RNA segments are loosely encapsidated by multiple NP molecules. A complex of three viral polymerase proteins (PB1, PB2, and PA) is located at the end of the nucleocapsid. All RNA segments are required for the production of infectious viral particles.

[0191] RNA segment 1, the slowest migrating RNA species by gel electrophoresis, encodes the PB2 RNA polymerase. RNA segment 2 encodes the PB1 RNA polymerase, as well as two other transcripts for the PB1-N40 and PB1-F2 proteins, using different reading frames from the same RNA sequence. The PB1-N40 and PB1-F2 proteins induce apoptosis in host cells. RNA segment 3 encodes the PA RNA polymerase, which forms an RNA-dependent RNA polymerase complex with PB1 and PB2. RNA segment 4 encodes HA, the major surface antigen of the influenza virion. Each virion has approximately 500 HA molecules, which appear as evenly distributed protrusions on the virion surface (Ruigrok, RWH, et al., J. Gen Virol (1984) 65:799-902; Murti, KG and Webster, RG, Virol (1986) 149:36-43). HA determines the host range due to its binding to host receptors. RNA segment 5 encodes the NP nucleoprotein. NP encapsidates the viral RNA and is transported to the host cell nucleus. NP is synthesized in large quantities in infected cells and is the second most abundant protein in influenza virus virions. It is the main target of the host's cytotoxic T cell immune response. RNA segment 6 encodes neuraminidase (NA), which is the second major surface antigen of influenza virus virions. NA is an enzyme that cleaves terminal sialic acid from glycoproteins or glycolipids to release the virus particle from host cell receptors. Its function is essential for viral propagation. Each virion has approximately 100 NA molecules on its surface. NA forms tetramers and is located in discrete patches on the virion envelope (Murti, KG and Webster, RG, Virol (1986) 149: 36-43). RNA segment 7 is bicistronic and encodes both matrix proteins M1 and M2. M1 forms a shell around the virion nucleocapsid below the virion envelope and is the most abundant protein in influenza virus virions. M2 is composed of different splicing forms of the same transcript of M1. M2 is an integral membrane protein and serves as a proton channel to control the pH of the Golgi network during the production of influenza virus in host cells. M2 can be partially replaced by the alternative splice variant M42. Approximately 3,000 matrix protein molecules are required to produce one virion. RNA segment 8 encodes two nonstructural proteins, NS1 and NS2, required for viral replication. NS2 is derived from a different reading frame than NS1. Both proteins are abundant in infected cells but are not incorporated into progeny virions.

[0192] Among the three genera of influenza viruses, influenza A virus is the most deadly human pathogen and causes the most severe disease in humans. Influenza A virus is classified into subtypes based on the antigenic properties of its surface glycoproteins HA and NA. A total of 18 HA subtypes have been identified, named H1 to H18, and 10 NA subtypes, named N1 to N10 (Tong, S., et al., PLoS Pathogens (2013) 9: e1003657). The common nomenclature of influenza A subtypes is derived from different combinations of HA subtypes and NA subtypes, such as H1N1, H3N2 and H7N9 (BullWorld Health Organ (1980) 58: 585-591). Waterfowl are the natural hosts of subtypes H1 to H16 and N1 to N9. Subtypes H17N10 and H18N10 have recently been found in American fruit bats. In nature, when a host is co-infected with two subtypes of influenza A virus, the segmented genome of influenza A virus allows for the reassortment of RNA segments. RNA segments from two different subtypes can be packaged into a single virion to produce a new subtype. Of the many subtypes of influenza A virus, only H1N1, H2N2, and H3N2 have developed the ability to spread efficiently among humans. These three subtypes are the so-called seasonal influenza viruses that predominate. Therefore, the human population has an inherent immune response to many influenza A subtypes. Occasionally, other subtypes of influenza A virus jump species and infect humans, causing epidemic outbreaks with high mortality rates.

[0193] Based on their phylogenetic relationships, influenza A HA subtypes cluster into two distinct groups. Group 1 includes 10 of the 16 subtypes: H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16. Group 2 accounts for the remaining six subtypes: H3, H4, H7, H10, H14, and H15.

[0194] Influenza B viruses are antigenically distinct from influenza A viruses. They co-circulate with influenza A viruses and cause human epidemics. Influenza B viruses have stably adapted to humans in the absence of a known animal reservoir. Unlike the vast genetic variation in HA of influenza A viruses, only one HA serotype has been reported for influenza B viruses, although three lineages are defined by the phylogenetic relationships of the HA genes. Influenza B viruses do not appear to recombine with influenza A viruses, as there have been no reports of reassortment of RNA segments between influenza A and influenza B viruses.

[0195] Both influenza A and B viruses use terminal sialic acid on the surface of host cells as cellular receptors. The HA of both virus types has the same structural features and binds to the sialic acid receptor to enter the host cell.

[0196] Subtypes of influenza A and influenza B viruses are further classified into strains. There are many different strains of influenza A and influenza B viruses. Each flu season is dominated by several strains of influenza A and influenza B viruses, which are often genetically different from the strains of the previous flu season. Viruses of a strain isolated in different geographic locations or at different times during the flu season (called isolates) often have genetic variations.

[0197] Infection with influenza C virus is usually asymptomatic or causes a mild illness that primarily affects children or young adults. Influenza C virus is associated with sporadic cases and minor local outbreaks. Compared to influenza A and B viruses, influenza C virus has a much smaller disease burden. A large proportion of the population shows seroconversion, indicating that influenza C virus is widely circulated in the population. Influenza C virus has also been isolated from animals. The hemagglutinin-esterase-fusion (HEF) protein of influenza C virus combines the functions of HA and NA of influenza A and B viruses. Unlike the HA of influenza A and B viruses, the HEF of influenza C virus uses the terminal 9-O-acetyl-N-acetylneuraminic acid (9-O-Ac-NeuAc) as a cellular receptor (Herrler, G., et al., EMBO J. (1985) 4: 1503-1506).

[0198] Description of the viral infection process and life cycle

[0199] Influenza virus is spread from person to person through droplets or pollutants (any object or material that can carry infectious organisms). The virus infects the epithelial cells of the respiratory tract. After binding to the cell surface receptors, the attached virions are endocytosed by the host cell to endosomes, where low pH triggers a conformational change in HA, causing its hydrophobic fusion peptide to insert into the vacuole membrane of the host cell, and triggering the fusion of the virus and the vacuole membrane. Fusion releases the contents of the virions into the cytoplasm of infected cells. The viral nucleocapsid migrates into the host cell nucleus, and their associated polymerase complexes begin the primary transcription of mRNA to translate viral proteins. Simultaneously, the translation of host mRNA is blocked. Newly synthesized viral RNA is wrapped, and viral structural proteins are synthesized and transported to the host cell surface, where they are integrated into the host cell membrane. Influenza viruses germinate from the apical surface of polarized epithelial cells, such as bronchial epithelial cells, and thus are generally pneumophilic (Roth, MG, et al., PNAS (1979) 76:6430-6434; Nayak, DP, et al., Virus Res (2009) 143:147-161). Transmission of influenza viruses between pigs and humans has been demonstrated. After infection, the immune system produces antibodies against the influenza virus. This is the body's primary source of protection.

[0200] In summary, this application includes but is not limited to the following:

[0201] 1. A modified hemagglutinin (HA) protein of influenza A or B, wherein an immunodominant region of the HA protein contains a conserved alternative influenza epitope by insertion into the region or by replacing the region with the conserved alternative influenza epitope.

[0202] 2. The modified HA protein of claim 1, wherein the modified HA protein is contained in a live or attenuated virus or virus-like particle (VLP).

[0203] 3. The modified HA protein of item 2, which is contained in a live virus, and the modified HA protein further comprises a modified mature cleavage site (MCS) that is not cleaved by animal proteases.

[0204] 4. The modified HA protein of claim 3, wherein the modified MCS has been cleaved by a suitable protease.

[0205] 5. The modified HA protein of any one of items 1-4, wherein the alternative epitope of influenza is an epitope from the stem domain of the influenza HA protein or an epitope of the M2 influenza protein.

[0206] 6. The modified HA protein of any one of items 1-4, wherein more than one immunodominant region of the HA protein has been modified to contain alternative influenza epitopes.

[0207] 7. The modified HA protein of claim 6, wherein at least some of the alternative influenza epitopes contained in each of the more than one immunodominant regions are different from each other.

[0208] 8. An influenza vaccine comprising the modified HA protein according to any one of items 1 to 4.

[0209] 9. A modified influenza virus, wherein the virus comprises an influenza HA protein that has been modified to contain an MCS that is not cleaved by an animal protease.

[0210] 10. The modified influenza virus of claim 9, wherein the MCS has been cleaved by a suitable protease.

[0211] 11. The modified influenza virus of claim 9, wherein the HA protein further contains a heterologous immunogenic epitope in at least one immunodominant region.

[0212] 12. The modified influenza virus of claim 11, wherein the heterologous immunogenic epitope is an epitope of a tumor-associated antigen or an epitope of a virus other than influenza virus or a surrogate influenza epitope.

[0213] 13. The modified influenza virus of claim 11, wherein more than one immunodominant region of the HA protein has been modified to contain heterologous epitopes.

[0214] 14. The modified influenza virus of claim 13, wherein at least some of the heterologous epitopes contained in each of the more than one immunodominant regions are different from each other.

[0215] 15. A modified HA protein, which is modified to contain an MCS that is not cleaved by animal proteases and further contains an inserted heterologous immunogenic epitope, or an MCS that replaces at least one immunodominant region.

[0216] 16. The modified HA protein of claim 15, wherein the MCS has been cleaved by a suitable protease, and / or wherein the heterologous immunogenic epitope is an epitope of a tumor-associated antigen or an epitope of a virus other than influenza virus or a surrogate influenza epitope.

[0217] 17. A method for producing anti-influenza antibodies, the method comprising administering the influenza vaccine of item 8 to a non-human subject.

[0218] 18. The influenza vaccine of claim 8 for use in a method of protecting a subject from influenza, the method comprising administering the vaccine to the subject.

[0219] 19. A method of producing antibodies against a desired antigen, the method comprising administering to a subject the modified influenza virus of any one of items 9 to 14.

[0220] 20. A nucleic acid encoding at least a significant portion of an influenza HA protein, wherein a nucleotide sequence encoding an alternative epitope of the influenza protein is inserted into or replaces a nucleotide sequence encoding an immunodominant region of the HA protein.

[0221] 21. The nucleic acid of item 20, wherein the nucleotide sequence encoding MCS is modified to encode an MCS that is not cleaved by animal proteases.

[0222] 22. The nucleic acid of claim 20, wherein more than one region encoding the immunodominant region of the HA protein has been modified to contain a nucleotide sequence encoding an alternative influenza epitope.

[0223] 23. The nucleic acid of claim 21, wherein more than one region encoding the immunodominant region of the HA protein has been modified to contain a nucleotide sequence encoding an alternative influenza epitope.

[0224] 24. A nucleic acid encoding at least a significant portion of an influenza HA protein, wherein the nucleotide sequence encoding the MCS is modified to encode an MCS that is not cleaved by an animal protease.

[0225] 25. The nucleic acid of claim 24, wherein a nucleotide sequence encoding a heterologous epitope is inserted into or replaces a nucleotide sequence encoding an immunodominant region of the HA protein.

[0226] 26. The nucleic acid of claim 25, wherein more than one region encoding the immunodominant region of the HA protein has been modified to contain a nucleotide sequence encoding a heterologous epitope.

[0227] 27. A nucleic acid expressed in a recombinant host cell encoding one or more nucleotide sequences as described in any one of items 20-26.

[0228] 28. A recombinant host cell containing the nucleic acid of item 27.

[0229] 29. A method for preparing the modified HA protein of item 1, the method comprising culturing a recombinant host cell comprising a nucleic acid encoding at least a significant portion of an influenza HA protein, wherein a nucleotide sequence encoding an alternative epitope of the influenza protein is inserted into or replaces a nucleotide sequence encoding an immunodominant region of the HA protein under conditions in which the nucleic acid is expressed and the modified HA protein is recovered.

[0230] 30. A method for preparing the modified HA protein of item 3, the method comprising culturing a recombinant host cell comprising a nucleic acid encoding at least a valid portion of an influenza HA protein, wherein, under conditions where the nucleic acid is expressed and the modified HA protein is recovered, a nucleotide sequence encoding an alternative epitope of the influenza protein is inserted into or replaces a nucleotide sequence encoding an immunodominant region of the HA protein, and wherein the nucleotide sequence encoding the MCS is modified to encode an MCS that is not cleaved by an animal protease.

[0231] 31. A method for preparing the modified influenza virus of item 9, the method comprising culturing a recombinant host cell comprising a nucleic acid encoding at least a valid portion of an influenza HA protein, wherein, under conditions where the nucleic acid is expressed and the modified influenza virus is recovered, the nucleotide sequence encoding the MCS is modified to encode an MCS that is not cleaved by an animal protease.

[0232] 32. A method for preparing the modified influenza virus of item 11, the method comprising culturing a recombinant host cell comprising a nucleic acid encoding at least a valid portion of an influenza HA protein, wherein, under conditions where the nucleic acid is expressed and the modified influenza virus is recovered, the nucleotide sequence encoding the MCS is modified to encode an MCS that is not cleaved by an animal protease, and wherein at least one nucleotide sequence encoding the immunodominant region of the HA protein has been modified to contain a nucleotide sequence encoding a heterologous epitope.

[0233] 33. A method of producing anti-influenza antibodies, the method comprising administering the nucleic acid of any one of items 20-27 to a non-human subject.

[0234] 34. The nucleic acid of any one of items 20 to 27 for use in a method of protecting a subject from influenza, the method comprising administering the nucleic acid to the subject.

[0235] 35. A method of producing antibodies against a desired antigen, the method comprising administering to a subject the nucleic acid of any one of items 20-27.

[0236] Sequence Listing

[0237] SEQ ID NO: 1: Peptide sequence: Peptide sequence of HA of A / California / 07 / 2009, a swine-derived influenza A virus H1N1 strain (UniProt: C3W5X2)

[0238]

[0239] SEQ ID NO: 2: Peptide sequence of GP67 secretion signal (GP67ss)

[0240]

[0241] SEQ ID NO: 3: Peptide sequence of TEV cleavage site, foldon and 10xHis tag

[0242]

[0243] SEQ ID NO: 4: Peptide sequence of TEV cleavage site

[0244]

[0245] SEQ ID NO: 5: Peptide sequence of foldon

[0246]

[0247] SEQ ID NO: 6: 10xHis-tagged peptide sequence

[0248]

[0249] SEQ ID NO: 7: Peptide sequence of the signal peptide of HA of A / California / 07 / 2009 (UniProt: C3W5X2)

[0250]

[0251] SEQ ID NO: 8: Peptide sequence of H1 HA mature cleavage site (MCS)

[0252]

[0253] SEQ ID NO: 9: Peptide sequence of GP67ss-H1WT-TEV-foldon-10His (H1WT)

[0254]

[0255]

[0256] SEQ ID NO: 10: Nucleotide sequence of GP67ss-H1WT-TEV-foldon-10His (H1WT)

[0257]

[0258] SEQ ID NO: 11: Peptide sequence of GP67ss-H1H5cs-TEV-foldon-10His (H1H5cs)

[0259]

[0260]

[0261] SEQ ID NO: 12: Peptide sequence of the mature cleavage site of H5

[0262]

[0263] SEQ ID NO: 13: Nucleotide sequence of GP67ss-H1H5cs-TEV-foldon-10His (H1H5cs)

[0264]

[0265] SEQ ID NO: 14: Peptide sequence of GP67ss-H1R5cs-TEV-foldon-10His (H1R5cs)

[0266]

[0267]

[0268] SEQ ID NO: 15: Nucleotide sequence of GP67ss-H1R5cs-TEV-foldon-10His (H1R5cs)

[0269]

[0270] SEQ ID NO: 16: Peptide sequence of GP67ss-H1IR5cs-TEV-foldon-10His (H1IR5cs)

[0271]

[0272]

[0273] SEQ ID NO: 17: Nucleotide sequence of GP67ss-H1IR5cs-TEV-foldon-10His (H1IR5cs)

[0274]

[0275] SEQ ID NO: 18: Peptide sequence of GP67ss-H1TEV1-TEV-foldon-10His (H1TEV1)

[0276]

[0277]

[0278] SEQ ID NO: 19: Nucleotide sequence of GP67ss-H1TEV1-TEV-foldon-10His (H1TEV1)

[0279]

[0280] SEQ ID NO: 20: Peptide sequence of GP67ss-H1TEV2-TEV-foldon-10His (H1TEV2)

[0281]

[0282]

[0283] SEQ ID NO: 21: Nucleotide sequence of GP67ss-H1TEV2-TEV-foldon-10His (H1TEV2)

[0284]

[0285] SEQ ID NO: 22: Peptide sequence of GP67ss-H1TEV3-TEV-foldon-10His (H1TEV3)

[0286]

[0287]

[0288] SEQ ID NO: 23: Nucleotide sequence of GP67ss-H1TEV3-TEV-foldon-10His (H1TEV3)

[0289]

[0290] SEQ ID NO: 24: Peptide sequence of GP67ss-H1H5cs-CR8020Ca-TEV-foldon-10His (H1H5cs-CR8020Ca)

[0291]

[0292]

[0293] SEQ ID NO: 25: Peptide sequence of the CR8020 epitope of HA2 residues 15-19

[0294]

[0295] SEQ ID NO: 26: Peptide sequence of the CR8020 epitope of HA2 residues 30-36

[0296]

[0297] SEQ ID NO: 27: Peptide sequence of composite C8020 epitope

[0298]

[0299] SEQ ID NO: 28: Nucleotide sequence of GP67ss-H1H5cs-CR8020Ca-TEV-foldon-10His (H1H5cs-CR8020Ca)

[0300]

[0301]

[0302] SEQ ID NO: 29: Peptide sequence of GP67ss-H1TEV2-CR8020Ca-TEV-foldon-10His (H1TEV2-CR8020Ca)

[0303]

[0304] SEQ ID NO: 30: Nucleotide sequence of GP67ss-H1TEV2-CR8020Ca-TEV-foldon-10His (H1TEV2-CR8020Ca)

[0305]

[0306]

[0307] SEQ ID NO: 31: Peptide sequence of GP67ss-H1TEV2-CR8020Sa3-TEV-foldon-10His (H1TEV2-CR8020Sa3)

[0308]

[0309] SEQ ID NO: 32: Nucleotide sequence of GP67ss-H1TEV2-CR8020Sa3-TEV-foldon-10His (H1TEV2-CR8020Sa3)

[0310]

[0311]

[0312] SEQ ID NO: 33: Peptide sequence of GP67ss-H1TEV2-CR8020Sa4-TEV-foldon-10His (H1TEV2-CR8020Sa4)

[0313]

[0314] SEQ ID NO: 34: Nucleotide sequence of GP67ss-H1TEV2-CR8020Sa4-TEV-foldon-10His (H1TEV2-CR8020Sa4)

[0315]

[0316]

[0317] SEQ ID NO: 35: Peptide sequence of GP67ss-H1TEV2-I1C9Ca1-TEV-foldon-10His (H1TEV2-I1C9Ca1)

[0318]

[0319] SEQ ID NO: 36: Peptide sequence of I1C9

[0320]

[0321] SEQ ID NO: 37: Nucleotide sequence of GP67ss-H1TEV2-I1C9Ca1-TEV-foldon-10His (H1TEV2-I1C9Ca1)

[0322]

[0323]

[0324] SEQ ID NO: 38: Peptide sequence of GP67ss-H1TEV2-I1C9Ca2-TEV-foldon-10His (H1TEV2-I1C9Ca2)

[0325]

[0326] SEQ ID NO: 39: Nucleotide sequence of GP67ss-H1TEV2-I1C9Ca2-TEV-foldon-10His (H1TEV2-I1C9Ca2)

[0327]

[0328]

[0329] SEQ ID NO: 40: Peptide sequence of GP67ss-H1TEV2-I1C9Sa3-TEV-foldon-10His (H1TEV2-I1C9Sa3)

[0330]

[0331] SEQ ID NO: 41: Nucleotide sequence of GP67ss-H1TEV2-I1C9Sa3-TEV-foldon-10His (H1TEV2-I1C9Sa3)

[0332]

[0333]

[0334] SEQ ID NO: 42: Peptide sequence of GP67ss-H1TEV2-I1C9Sa4-TEV-foldon-10His (H1TEV2-I1C9Sa4)

[0335]

[0336] SEQ ID NO: 43: Nucleotide sequence of GP67ss-H1TEV2-I1C9Sa4-TEV-foldon-10His (H1TEV2-I1C9Sa4)

[0337]

[0338]

[0339] SEQ ID NO: 44: Peptide sequence of GP67ss-H1H5cs-I1C9Sb-TEV-foldon-10His (H1H5cs-I1C9Sb)

[0340]

[0341] SEQ ID NO: 45: Nucleotide sequence of GP67ss-H1H5cs-I1C9Sb-TEV-foldon-10His (H1H5cs-I1C9Sb)

[0342]

[0343]

[0344] SEQ ID NO: 46: Peptide sequence of GP67ss-H1H5cs-I1C9Ca-TEV-foldon-10His (H1H5cs-I1C9Ca)

[0345]

[0346] SEQ ID NO: 47: Nucleotide sequence of GP67ss-H1H5cs-I1C9Ca-TEV-foldon-10His (H1H5cs-I1C9Ca)

[0347]

[0348]

[0349] SEQ ID NO: 48: Peptide sequence of GP67ss-H1H5cs-FI6Sab-TEV-foldon-10His (H1H5cs-FI6Sab)

[0350]

[0351] SEQ ID NO: 49: Peptide sequence of the mature cleavage site of the FI6 epitope

[0352]

[0353] SEQ ID NO: 50: Peptide sequence of coiled-coil peptide of FI6 epitope

[0354]

[0355] SEQ ID NO: 51: Nucleotide sequence of GP67ss-H1H5cs-FI6Sab-TEV-foldon-10His (H1H5cs-FI6Sab)

[0356]

[0357]

[0358] SEQ ID NO: 52: Peptide sequence of GP67ss-H1WT-FI6Sab-TEV-foldon-10His (H1WT-FI6Sab)

[0359]

[0360] SEQ ID NO: 53: Nucleotide sequence of GP67ss-H1WT-FI6Sab-TEV-foldon-10His (H1WT-FI6Sab)

[0361]

[0362] SEQ ID NO: 54: Peptide sequence of GP67ss-H1TEV1-FI6Sa-TEV-foldon-10His (H1TEV1-FI6Sa)

[0363]

[0364] SEQ ID NO: 55: Nucleotide sequence of GP67ss-H1TEV1-FI6Sa-TEV-foldon-10His (H1TEV1-FI6Sa)

[0365]

[0366] SEQ ID NO: 56: Peptide sequence of GP67ss-H1H5cs-M2eCa2-TEV-foldon-10His (H1H5cs-M2eCa)

[0367]

[0368] SEQ ID NO: 57: Peptide sequence of M2e peptide

[0369]

[0370] SEQ ID NO: 58: Nucleotide sequence of GP67ss-H1H5cs-M2eCa2-TEV-foldon-10His (H1H5cs-M2eCa)

[0371]

[0372] SEQ ID NO: 59: Peptide sequence of GP67ss-H1H5cs-M2eSb-TEV-foldon-10His (H1H5cs-M2eSb)

[0373]

[0374]

[0375] SEQ ID NO: 60: Nucleotide sequence of GP67ss-H1H5cs-M2eSb-TEV-foldon-10His (H1H5cs-M2eSb)

[0376]

[0377] SEQ ID NO: 61: Peptide sequence of GP67ss-H1TEV2-M2eCa2-TEV-foldon-10His (H1TEV2-M2eCa)

[0378]

[0379]

[0380] SEQ ID NO: 62: Nucleotide sequence of GP67ss-H1TEV2-M2eCa2-TEV-foldon-10His (H1TEV2-M2eCa)

[0381] Sequence Listing <110> CG Exploration Co., Ltd. <120> Compositions of influenza hemagglutinins with heterologous epitopes and / or altered mature cleavage sites and methods of use thereof <130> 758252000140 <140> Not yet transferred <141> At the same time with this <150> US 62 / 290,894 <151> 2016-02-03 <160> 62 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 566 <212> PRT <213> Influenza A virus <400> 1 Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn 1 5 10 15 Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val 50 55 60 Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly 65 70 75 80 Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp 130 135 140 Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser 145 150 155 160 Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro 165 170 175 Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val 180 185 190 Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu 195 200 205 Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser 210 215 220 Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln 225 230 235 240 Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys 245 250 255 Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe 260 265 270 Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro 275 280 285 Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn 290 295 300 Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys 305 310 315 320 Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg 325 330 335 Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly 340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr 355 360 365 His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser 370 375 380 Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile 385 390 395 400 Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His 405 410 415 Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe 420 425 430 Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn 435 440 445 Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu 450 455 460 Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly 465 470 475 480 Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val 485 490 495 Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu 500 505 510 Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr 515 520 525 Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Val 530 5�5 540 Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545 550 555 560 Gln Cys Arg Ile Cys Ile 565 <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 2 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala 20 <210> 3 <211> 49 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 3 Gly Ala Glu Asn Leu Tyr Phe Gln Gly Gly Ser Gly Tyr Ile Pro Glu 1 5 10 15 Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val 20 25 30 Leu Leu Ser Thr Phe Leu Gly His His His His His His His 35 40 45 His <210> 4 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 4 Glu Asn Leu Tyr Phe Gln Gly 1 5 <210> 5 <211> 29 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 5 Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val 1 5 10 15 Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 20 25 <210> 6 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 6 His His His His His His His His 1 5 10 <210> 7 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 7 Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn 1 5 10 15 Ala <210> 8 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 8 Ile Pro Ser Ile Gln Ser Arg 1 5 <210> 9 <211> 581 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 9 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln 195 200 205 Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser 210 215 220 Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val 225 230 235 240 Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro 245 250 255 Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg 260 265 270 Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser 275 280 285 Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly 290 295 300 Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile 305 310 315 320 Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr 325 330 335 Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala 340 345 350 Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp 355 360 365 Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp 370 375 380 Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn 385 390 395 400 Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu 405 410 415 Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp 420 425 430 Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu 435 440 445 Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn 450 455 460 Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile 465 470 475 480 Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met 485 490 495 Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu 500 505 510 Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr 515 520 525 Arg Ile Tyr Gln Gly Ala Glu Asn Leu Tyr Phe Gln Gly Gly Ser Gly 530 535 540 Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp 545 550 555 560 Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His His His His His 565 570 575 His His His His His 580 <210> 10 <211> 1757 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 10 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acaatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactctg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caaatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgacttcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtgaa gaagggcaac agctacccta 540 agctgtctaa gagctacatc aacgacaagg gcaaagaagt gctggtgctg tggggaatcc 600 accaccctag cacaagcgcc gatcagcaga gcctgtacca gaatgccgat gcctatgtgt 660 ttgtgggcag cagcagatac agcaaaaagt tcaagcctga aattgccatt agacccaaag 720 tgagagatca ggaaggcaga atgaattact actggaccct ggtggaacct ggcgataaga 780 tcacatttga ggccaccgga aatctggtgg tgcctagata tgcatttgct atggagagaa 840 atgctggctc tggcatcatt atctctgata cccctgtgca cgactgtaat accacctgtc 900 agacacctaa gggcgccatt ataccagcc tgcccttcca gatattcac cctatcacca 960 tcggcaagtg tcctaagtat gtgaagagca ccaagctgag actggctacc ggtctgagaa 1020 atatccctag catccagagc agaggcctgt ttggagccat cgccggcttt attgaggg 1080 gatggaccgg aatggtggat ggctggtacg gctatcacca ccagaatgag cagggatccg 1140 gatatgccgc cgatctgaag tctacacaga acgccatcga cgagatcaca aaaaggtga 1200 acagcgtgat cgagaagatg aacacccagt ttacagctgt gggcaggg ttcaaccacc 1260 tggagagag atcgagaac ctgacaga aagtggacga cggcttccctg gatatttgga 1320 cctacaatgc cgagctgctc gtgctcctgg agatgagag aaccctggac taccacgaca 1380 gcaatgtgaa gaacctgtac gagaaggtga gaagccagct gagaacaat gccaaggaga 1440 tcggcaacgg ctgctttgag ttctaccaca agtgtgacaa cacctgtatg gagtcttgtga 1500 agaacggcac ctacgactac cctaagtata gcgaggaggc caagctgaat aggaggaga 1560 tcgacggcgt gaactggaa agcacagaa tctatcaggg cgctgaaac ctgtattttc 1620 agggcggttc tggttacatc ccggaagctc cgcgtgacgg tcaggcttac gttcgtaaag 1680 acggtgaatg ggttctgctg tctaccttcc tgggtcacca tcatcaccac catcaccatc 1740 atcactgata aaagctt 1757 <210> 11 <211> 584 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 11 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln 195 200 205 Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser 210 215 220 Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val 225 230 235 240 Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro 245 250 255 Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg 260 265 270 Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser 275 280 285 Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly 290 295 300 Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile 305 310 315 320 Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr 325 330 335 Gly Leu Arg Asn Ser Pro Gln Arg Glu Arg Arg Lys Lys Arg Gly Leu 340 345 350 Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val 355 360 365 Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr 370 375 380 Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn 385 390 395 400 Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val 405 410 415 Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys 420 425 430 Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu 435 440 445 Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn 450 455 460 Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn Ala 465 470 475 480 Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn 485 490 495 Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 500 505 510 Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu 515 520 525 Glu Ser Thr Arg Ile Tyr Gln Gly Ala Glu Asn Leu Tyr Phe Gln Gly 530 535 540 Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val 545 550 555 560 Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His His 565 570 575 His His His His His His His His 580 <210> 12 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 12 Gln Arg Glu Arg Arg Lys Lys Arg 1 5 <210> 13 <211> 1766 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 13 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acaatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactctg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caaatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgacttcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtgaa gaagggcaac agctacccta 540 agctgtctaa gagctacatc aacgacaagg gcaaagaagt gctggtgctg tggggaatcc 600 accaccctag cacaagcgcc gatcagcaga gcctgtacca gaatgccgat gcctatgtgt 660 ttgtgggcag cagcagatac agcaaaaagt tcaagcctga aattgccatt agacccaaag 720 tgagagatca ggaaggcaga atgaattact actggaccct ggtggaacct ggcgataaga 780 tcacatttga ggccaccgga aatctggtgg tgcctagata tgcatttgct atggagagaa 840 atgctggctc tggcatcatt atctctgata cccctgtgca cgactgtaat accacctgtc 900 agacacctaa gggcgccatt aataccagcc tgcccttcca gaatattcac cctatcacca 960 tcggcaagtg tcctaagtat gtgaagagca ccaagctgag actggctacc ggtctgagaa 1020 atagccctca gagggagaga cgcaagaaga gaggcctgtt tggagccatc gccggcttta 1080 ttgagggagg atggaccgga atggtggatg gctggtacgg ctatcaccac cagaatgagc 1140 agggatccgg atatgccgcc gatctgaagt ctacacagaa cgccatcgac gagatcacaa 1200 acaaggtgaa cagcgtgatc gagaagatga acacccagtt tacagctgtg ggcaaggagt 1260 tcaaccacct ggagaagaga atcgagaacc tgaacaagaa agtggacgac ggcttcctgg 1320 atatttggac ctacaatgcc gagctgctcg tgctcctgga gaatgagaga accctggact 1380 accacgacag caatgtgaag aacctgtacg agaaggtgag aagccagctg aagaacaatg 1440 ccaaggagat cggcaacggc tgctttgagt tctaccacaa gtgtgacaac acctgtatgg 1500 agtctgtgaa gaacggcacc tacgactacc ctaagtatag cgaggaggcc aagctgaata 1560 gagaggagat cgacggcgtg aaactggaaa gcacaagaat ctatcagggc gctgaaaacc 1620 tgtattttca gggcggttct ggttacatcc cggaagctcc gcgtgacggt caggcttacg 1680 ttcgtaaaga cggtgaatgg gttctgctgt ctaccttcct gggtcaccat catcaccacc 1740 atcaccatca tcactgataa aagctt 1766 <210> 14 <211> 581 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 14 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln 195 200 205 Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser 210 215 220 Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val 225 230 235 240 Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro 245 250 255 Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg 260 265 270 Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser 275 280 285 Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly 290 295 300 Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile 305 310 315 320 Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr 325 330 335 Gly Leu Arg Asn Ile Pro Arg Arg Arg Arg Arg Gly Leu Phe Gly Ala 340 345 350 Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp 355 360 365 Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp 370 375 380 Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn 385 390 395 400 Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu 405 410 415 Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp 420 425 430 Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu 435 440 445 Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn 450 455 460 Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile 465 470 475 480 Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met 485 490 495 Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu 500 505 510 Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr 515 520 525 Arg Ile Tyr Gln Gly Ala Glu Asn Leu Tyr Phe Gln Gly Gly Ser Gly 530 535 540 Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp 545 550 555 560 Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His His His His His 565 570 575 His His His His His 580 <210> 15 <211> 1757 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 15 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgactcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtga gaagggcaac agctacccta 540 agctgtctaa gagctacatc aacgacaagg gcaagaagt gctggtgctg tgggaatcc 600 accaccctag cacaagcgcc gatcagcaga gcctgtacca gatgccgat gcctatgtgt 660 ttgtgggcag cagcagatac agcaaagt tcaagcctga aattgccatt agacccaaag 720 tgagagatca ggaggcaga atgattact actgaccct ggtgaacct ggcgataaga 780 tcacatttga ggccaccgga aatctggtgg tgcctagata tgcatttgct atggagagaa 840 atgctggctc tggcatcatt atctctgata cccctgtgca cgactgtaat accacctgtc 900 agacacctaa gggcgcatt aataccagcc tgcccttcca gaatattcac cctatcacca 960 tcggcaagtg tcctaagtat gtgaagagca ccaagctgag actggctacc ggtctgagaa 1020 atatccctag gagacgcaga agaggcctgt ttggagccat cgccggcttt attgagggag 1080 gatggaccgg aatggtggat ggctggtacg gctatcacca ccagaatgag cagggatccg 1140 gatatgccgc cgatctgaag tctacacaga acgccatcga cgagatcaca aacaaggtga 1200 acagcgtgat cgagaagatg aacacccagt ttacagctgt gggcaaggag ttcaaccacc 1260 tggagaag aatcgagaac ctgaacaaga aagtggacga cggcttcctg gatatttgga 1320 cctacaatgc cgagctgctc gtgctcctgg agaatgagag aaccctggac taccacgaca 1380 gcaatgtgaa gaacctgtac gagaaggtga gaagccagct gaagaacaat gccaaggaga 1440 tcggcaacgg ctgctttgag ttctaccaca agtgtgacaa cacctgtatg gagtctgtga 1500 agaacggcac ctacgactac cctaagtata gcgaggaggc caagctgaat agagaggaga 1560 tcgacggcgt gaaactggaa agcacaagaa tctatcaggg cgctgaaaac ctgtattttc 1620 agggcggttc tggttacatc ccggaagctc cgcgtgacgg tcaggcttac gttcgtaaag 1680 acggtgaatg ggttctgctg tctaccttcc tgggtcacca tcatcaccac catcaccatc 1740 atcactgata aaagctt 1757 <210> 16 <211> 585 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 16 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln 195 200 205 Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser 210 215 220 Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val 225 230 235 240 Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro 245 250 255 Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg 260 265 270 Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser 275 280 285 Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly 290 295 300 Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile 305 310 315 320 Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr 325 330 335 Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Arg Arg Arg Arg Arg Gly 340 345 350 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 355 360 365 Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 370 375 380 Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr 385 390 395 400 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala 405 410 415 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn 420 425 430 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 435 440 445 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 450 455 460 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn 465 470 475 480 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp 485 490 495 Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys 500 505 510 Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys 515 520 525 Leu Glu Ser Thr Arg Ile Tyr Gln Gly Ala Glu Asn Leu Tyr Phe Gln 530 535 540 Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr 545 550 555 560 Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His 565 570 575 His His His His His His His His His 580 585 <210> 17 <211> 1769 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 17 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acaatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactctg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caaatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgacttcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg gagccaagtc cttttacaag aacctgatct ggctggtgaa gaagggcaac agctacccta agctgtctaa gagctacatc aacgacaagg gcaaagaagt gctggtgctg tggggaatcc accaccctag cacaagcgcc gatcagcaga gcctgtacca gatgccgat gcctatgtgt 660 ttgtgggcag cagcagatac agcaaaagt tcaagcctga aattgccatt agcaccaaag tgagagatca ggaaggcaga atgaattact actggaccct ggtggaacct ggcgataaga tcacatttga ggccaccgga aatctggtgg tgcctagata tgcatttgct atggagaga 840 atgctggctc tggcatcatt atctctgata cccctgtgca cgactgtaat accacctgtc agacacctaa gggcgccatt aataccagcc tgcccttcca gaatattcac cctatcacca tcggcaagtg tcctaagtat gtgaagagca ccaagctgag actggctacc ggtctgagaa atatccctag catccagagc aggagacgca gaagaggcct gtttggagcc atcgccggct ttattgaggg aggatggacc ggaatggtgg atggctggta cggctatcac caccagaatg agcagggatc cggatatgcc gccgatctga agtctacaca gaacgccatc gacgagatca 1200 caaacaaggt gaacagcgtg atcgagaaga tgaacaccca gtttacagct gtgggcaagg 1260 agttcaacca cctggagaag agaatcgaga acctgaacaa gaaagtggac gacggcttcc 1320 tggatatttg gacctacaat gccgagctgc tcgtgctcct ggagaatgag agaaccctgg 1380 actaccacga cagcaatgtg aagaacctgt acgagaaggt gagaagccag ctgaagaaca 1440 atgccaagga gatcggcaac ggctgctttg agttctacca caagtgtgac aacacctgta 1500 tggagtctgt gaagaacggc acctacgact accctaagta tagcgaggag gccaagctga 1560 atagagagga gatcgacggc gtgaaactgg aaagcacaag aatctatcag ggcgctgaaa 1620 acctgtattt tcagggcggt tctggttaca tcccggaagc tccgcgtgac ggtcaggctt 1680 acgttcgtaa agacggtgaa tgggttctgc tgtctacctt cctgggtcac catcatcacc 1740 accatcacca tcatcactga taaaagctt 1769 <210> 18 <211> 581 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic <400> 18 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln 195 200 205 Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser 210 215 220 Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val 225 230 235 240 Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro 245 250 255 Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg 260 265 270 Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser 275 280 285 Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly 290 295 300 Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile 305 310 315 320 Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr 325 330 335 Gly Leu Arg Asn Ile Glu Asn Leu Tyr Phe Gln Gly Leu Phe Gly Ala 340 345 350 Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp 355 360 365 Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp 370 375 380 Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn 385 390 395 400 Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu 405 410 415 Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp 420 425 430 Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu 435 440 445 Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn 450 455 460 Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile 465 470 475 480 Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met 485 490 495 Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu 500 505 510 Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr 515 520 525 Arg Ile Tyr Gln Gly Ala Glu Asn Leu Tyr Phe Gln Gly Gly Ser Gly 530 535 540 Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp 545 550 555 560 Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His His His His His 565 570 575 His His His His His 580 <210> 19 <211> 1757 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 19 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgactcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtga gaagggcaac agctacccta 540 agctgtctaa gagctacatc aacgacaagg gcaagaagt gctggtgctg tgggaatcc 600 accaccctag cacaagcgcc gatcagcaga gcctgtacca gatgccgat gcctatgtgt 660 ttgtgggcag cagcagatac agcaaagt tcaagcctga aattgccatt agacccaaag 720 tgagagatca ggaggcaga atgattact actgaccct ggtgaacct ggcgataaga 780 tcacatttga ggccaccgga aatctggtgg tgcctagata tgcatttgct atggagagaa 840 atgctggctc tggcatcatt atctctgata cccctgtgca cgactgtaat accacctgtc 900 agacacctaa gggcgcatt aataccagcc tgcccttcca gaatattcac cctatcacca 960 tcggcaagtg tcctaagtat gtgaagagca ccaagctgag actggctacc ggtctgagaa 1020 atatcgaaaa cctgttattt caaggcctgt ttggagccat cgccggcttt attgagggag 1080 gatggaccgg aatggtggat ggctggtacg gctatcacca ccagaatgag cagggatccg 1140 gatatgccgc cgatctgaag tctacacaga acgccatcga cgagatcaca aacaaggtga 1200 acagcgtgat cgagaagatg aacacccagt ttacagctgt gggcaaggag ttcaaccacc 1260 tggagaag aatcgagaac ctgaacaaga aagtggacga cggcttcctg gatatttgga 1320 cctacaatgc cgagctgctc gtgctcctgg agaatgagag aaccctggac taccacgaca 1380 gcaatgtgaa gaacctgtac gagaaggtga gaagccagct gaagaacaat gccaaggaga 1440 tcggcaacgg ctgctttgag ttctaccaca agtgtgacaa cacctgtatg gagtctgtga 1500 agaacggcac ctacgactac cctaagtata gcgaggaggc caagctgaat agagaggaga 1560 tcgacggcgt gaaactggaa agcacaagaa tctatcaggg cgctgaaaac ctgtattttc 1620 agggcggttc tggttacatc ccggaagctc cgcgtgacgg tcaggcttac gttcgtaaag 1680 acggtgaatg ggttctgctg tctaccttcc tgggtcacca tcatcaccac catcaccatc 1740 atcactgata aaagctt 1757 <210> 20 <211> 582 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 20 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln 195 200 205 Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser 210 215 220 Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val 225 230 235 240 Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro 245 250 255 Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg 260 265 270 Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser 275 280 285 Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly 290 295 300 Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile 305 310 315 320 Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr 325 330 335 Gly Leu Arg Asn Ser Pro Glu Asn Leu Tyr Phe Gln Gly Leu Phe Gly 340 345 350 Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly 355 360 365 Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala 370 375 380 Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val 385 390 395 400 Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys 405 410 415 Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val 420 425 430 Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val 435 440 445 Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys 450 455 460 Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu 465 470 475 480 Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys 485 490 495 Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu 500 505 510 Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser 515 520 525 Thr Arg Ile Tyr Gln Gly Ala Glu Asn Leu Tyr Phe Gln Gly Gly Ser 530 535 540 Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys 545 550 555 560 Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His His His His 565 570 575 His His His His His His 580 <210> 21 <211> 1760 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 21 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acaatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactctg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caaatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgacttcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtgaa gaagggcaac agctacccta 540 agctgtctaa gagctacatc aacgacaagg gcaaagaagt gctggtgctg tggggaatcc 600 accaccctag cacaagcgcc gatcagcaga gcctgtacca gaatgccgat gcctatgtgt 660 ttgtgggcag cagcagatac agcaaaaagt tcaagcctga aattgccatt agacccaaag 720 tgagagatca ggaaggcaga atgaattact actggaccct ggtggaacct ggcgataaga 780 tcacatttga ggccaccgga aatctggtgg tgcctagata tgcatttgct atggagagaa 840 atgctggctc tggcatcatt atctctgata cccctgtgca cgactgtaat accacctgtc 900 agacacctaa gggcgccatt aataccagcc tgcccttcca gaatattcac cctatcacca 960 tcggcaagtg tcctaagtat gtgaagagca ccaagctgag actggctacc ggtctgagaa 1020 atagccctga aaacctgtat tttcaaggcc tgtttggagc catcgccggc tttattgagg 1080 gaggatggac cggaatggtg gatggctggt acggctatca ccaccagaat gagcagggat 1140 ccggatatgc cgccgatctg aagtctacac agaacgccat cgacgagatc acaaacaagg 1200 tgaacagcgt gatcgagaag atgaacaccc agtttacagc tgtgggcaag gagttcaacc 1260 acctggagaa gagaatcgag aacctgaaca agaaagtgga cgacggcttc ctggatattt 1320 ggacctacaa tgccgagctg ctcgtgctcc tggagaatga gagaaccctg gactaccacg 1380 acagcaatgt gaagaacctg tacgagaagg tgagaagcca gctgaagaac aatgccaagg 1440 agatcggcaa cggctgcttt gagttctacc acaagtgtga caacacctgt atggagtctg 1500 tgaagaacgg cacctacgac taccctaagt atagcgagga ggccaagctg aatagagagg 1560 agatcgacgg cgtgaaactg gaaagcacaa gaatctatca gggcgctgaa aacctgtatt 1620 ttcagggcgg ttctggttac atcccggaag ctccgcgtga cggtcaggct tacgttcgta 1680 aagacggtga atgggttctg ctgtctacct tcctgggtca ccatcatcac caccatcacc 1740 atcatcactg ataaaagctt 1760 <210> 22 <211> 584 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 22 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln 195 200 205 Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser 210 215 220 Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val 225 230 235 240 Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro 245 250 255 Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg 260 265 270 Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser 275 280 285 Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly 290 295 300 Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile 305 310 315 320 Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr 325 330 335 Gly Leu Arg Asn Ile Pro Ser Ile Glu Asn Leu Tyr Phe Gln Gly Leu 340 345 350 Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val 355 360 365 Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr 370 375 380 Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn 385 390 395 400 Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val 405 410 415 Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys 420 425 430 Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu 435 440 445 Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn 450 455 460 Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn Ala 465 470 475 480 Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn 485 490 495 Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 500 505 510 Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu 515 520 525 Glu Ser Thr Arg Ile Tyr Gln Gly Ala Glu Asn Leu Tyr Phe Gln Gly 530 535 540 Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val 545 550 555 560 Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His His 565 570 575 His His His His His His His His 580 <210> 23 <211> 1766 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 23 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgactcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtga gaagggcaac agctacccta 540 agctgtctaa gagctacatc aacgacaagg gcaagaagt gctggtgctg tgggaatcc 600 accaccctag cacaagcgcc gatcagcaga gcctgtacca gatgccgat gcctatgtgt 660 ttgtgggcag cagcagatac agcaaagt tcaagcctga aattgccatt agacccaaag 720 tgagagatca ggaggcaga atgattact actgaccct ggtgaacct ggcgataaga 780 tcacatttga ggccaccgga aatctggtgg tgcctagata tgcatttgct atggagagaa 840 atgctggctc tggcatcatt atctctgata cccctgtgca cgactgtaat accacctgtc 900 agacacctaa gggcgccatt aataccagcc tgcccttcca gaatattcac cctatcacca 960 tcggcaagtg tcctaagtat gtgaagagca ccaagctgag actggctacc ggtctgagaa 1020 atatccctag catcgaaaac ctgtattttc aaggcctgtt tggagccatc gccggcttta 1080 ttgagggagg atggaccgga atggtggatg gctggtacgg ctatcaccac cagaatgagc 1140 agggatccgg atatgccgcc gatctgaagt ctacacagaa cgccatcgac gagatcacaa 1200 acaaggtgaa cagcgtgatc gagaagatga acacccagtt tacagctgtg ggcaaggagt 1260 tcaaccacct ggagaagaga atcgagaacc tgaacaagaa agtggacgac ggcttcctgg 1320 atatttggac ctacaatgcc gagctgctcg tgctcctgga gaatgagaga accctggact 1380 accacgacag caatgtgaag aacctgtacg agaaggtgag aagccagctg aagaacaatg 1440 ccaaggagat cggcaacggc tgctttgagt tctaccacaa gtgtgacaac acctgtatgg 1500 agtctgtgaa gaacggcacc tacgactacc ctaagtatag cgaggaggcc aagctgaata 1560 gagaggagat cgacggcgtg aaactggaaa gcacaagaat ctatcagggc gctgaaaacc 1620 tgtattttca gggcggttct ggttacatcc cggaagctcc gcgtgacggt caggcttacg 1680 ttcgtaaaga cggtgaatgg gttctgctgt ctaccttcct gggtcaccat catcaccacc 1740 atcaccatca tcactgataa aagctt 1766 <210> 24 <211> 585 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 24 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln 195 200 205 Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser 210 215 220 Arg Tyr Ser Lys Lys Phe Lys Pro Glu Gly Met Ile Asp Tyr Glu Gly 225 230 235 240 Thr Gly Gln Ala Ala Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu 245 250 255 Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro 260 265 270 Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile 275 280 285 Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys 290 295 300 Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr 305 310 315 320 Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala 325 330 335 Thr Gly Leu Arg Asn Ser Pro Gln Arg Glu Arg Arg Lys Lys Arg Gly 340 345 350 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 355 360 365 Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 370 375 380 Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr 385 390 395 400 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala 405 410 415 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn 420 425 430 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 435 440 445 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 450 455 460 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn 465 470 475 480 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp 485 490 495 Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys 500 505 510 Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys 515 520 525 Leu Glu Ser Thr Arg Ile Tyr Gln Gly Ala Glu Asn Leu Tyr Phe Gln 530 535 540 Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr 545 550 555 560 Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His 565 570 575 His His His His His His His 580 585 <210> 25 <211> 5 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 25 Glu Gly Met Ile Asp 1 5 <210> 26 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 26 Glu Gly Thr Gly Gln Ala Ala 1 5 <210> 27 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 27 Glu Gly Met Ile Asp Tyr Glu Gly Thr Gly Gln Ala Ala 1 5 10 <210> 28 <211> 1770 <212> DNA <213> Artificial sequence <220> <223> Synthesized <400> 28 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acaatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactctg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caaatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgacttcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtgaa gaagggcaac agctacccta 540 agctgtctaa gagctacatc aacgacaagg gcaaagaagt gctggtgctg tggggaatcc 600 accaccctag cacaagcgcc gatcagcaga gcctgtacca gaatgccgat gcctatgtgt 660 ttgtgggcag cagcagatac agcaaagt tcaagcctga agcatgat gattacgaag 720 gcacaggcca ggcagccggc agaatgaatt actactggac cctggtggaa cctggcgata 780 agatcacatt tgaggccacc ggaaatctgg tggtgcctag atatgcattt gctatggaga 840 gaaatgctgg ctctggcatc attatctctg atacccctgt gcacgactgt ataccacct 900 gtcagacacc taagggcgcc attack gcctgccctt ccagaatatt cacctatca 960 ccatcggcaa gtgtcctaag tatgtgaaga gcaccaagct gagactggct accggtctga 1020 gaaatagccc tcagagggag agacgcaga aggaggcct gtttggaggcc atcgccggct 1080 ttattgagggg aggatggacc ggaatggtgg atggctgta cggctatcac caccagaatg 1140 agcagggatc cggatatgcc gccgatctga agtctacaca gaacgccac gacgagatca 1200 haaaaaggt gaacagcgtg atcgagaga tgaacacca gtttacagct gtggggcaagg 1260 agttcaacca cctggagaag agaatcgaga acctgaaca gaagtggac gacggcttcc 1320 tggatatttg gacctacaat gccgagctgc tcgtgctcct ggagaatgag agaaccctgg 1380 actaccacga cagcaatgtg aagaacctgt acgagaaggt gagaagccag ctgaagaaca 1440 atgccaagga gatcggcaac ggctgctttg agttctacca caagtgtgac aacacctgta 1500 tggagtctgt gaagaacggc acctacgact accctaagta tagcgaggag gccaagctga 1560 atagagagga gatcgacggc gtgaaactgg aaagcacaag aatctatcag ggcgctgaaa 1620 acctgtattt tcagggcggt tctggttaca tcccggaagc tccgcgtgac ggtcaggctt 1680 acgttcgtaa agacggtgaa tgggttctgc tgtctacctt cctgggtcac catcatcacc 1740 accatcacca tcatcactga taaaaagctt 1770 <210> 29 <211> 583 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 29 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln 195 200 205 Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser 210 215 220 Arg Tyr Ser Lys Lys Phe Lys Pro Glu Gly Met Ile Asp Tyr Glu Gly 225 230 235 240 Thr Gly Gln Ala Ala Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu 245 250 255 Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro 260 265 270 Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile 275 280 285 Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys 290 295 300 Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr 305 310 315 320 Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala 325 330 335 Thr Gly Leu Arg Asn Ser Pro Glu Asn Leu Tyr Phe Gln Gly Leu Phe 340 345 350 Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp 355 360 365 Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala 370 375 380 Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys 385 390 395 400 Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly 405 410 415 Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys 420 425 430 Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu 435 440 445 Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val 450 455 460 Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys 465 470 475 480 Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr 485 490 495 Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser 500 505 510 Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu 515 520 525 Ser Thr Arg Ile Tyr Gln Gly Ala Glu Asn Leu Tyr Phe Gln Gly Gly 530 535 540 Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg 545 550 555 560 Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His His His 565 570 575 His His His His His His His 580 <210> 30 <211> 1763 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 30 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acaatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactctg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caaatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgacttcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtgaa gaagggcaac agctacccta 540 agctgtctaa gagctacatc aacgacaagg gcaaagaagt gctggtgctg tggggaatcc 600 accaccctag cacaagcgcc gatcagcaga gcctgtacca gaatgccgat gcctatgtgt 660 ttgtgggcag cagcagatac agcaaaaagt tcaagcctga aggcatgatt gattacgaag 720 gcacaggcca ggcagccggc agaatgaatt actactggac cctggtggaa cctggcgata 780 agatcacatt tgaggccacc ggaaatctgg tggtgcctag atatgcattt gctatggaga 840 gaaatgctgg ctctggcatc attatctctg atacccctgt gcacgactgt aataccacct 900 gtcagacacc taagggcgcc attaatacca gcctgccctt ccagaatatt caccctatca 960 ccatcggcaa gtgtcctaag tatgtgaaga gcaccaagct gagactggct accggtctga 1020 gaaatagccc tgaaaacctg tattttcaag gcctgtttgg agccatcgcc ggctttattg agggaggatg gaccggaatg gtggatggct ggtacggcta tcaccaccag aatgagcagg 1140 gatccggata tgccgccgat ctgaagtcta cacagaacgc catcgacgag atcacaaaca aggtgaacag cgtgatcgag aagatgaaca cccagtttac agctgtgggc aaggagttca accacctgga gaagagaatc gagaacctga acaagaaagt ggacgacggc ttcctggata tttggaccta caatgccgag ctgctcgtgc tcctggaga tgagagacc ctggactacc acgacagcaa tgtgaagaac ctgtacgaga aggtgagaag ccagctgaag aacaatgcca aggagatcgg caacggctgc tttgagttct accacaagtg tgacaacacc tgtatggagt ctgtgaagaa cggcacctac gactacccta agtatagcga ggaggccaag ctgaatagag aggagatcga cggcgtgaaa ctggaaagca caagatcta tcagggcgct gaaaaacctgt attttcaggg cggttctggt tacatcccgg aagctccgcg tgacggtcag gcttacgttc 1680. gtaaagacgg tgaatgggtt ctgctgtcta ccttcctggg tcaccatcat caccaccatc 1740. accatcatca ctgataaaag ctt 1763 <210> 31 <211> 590 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 31 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Glu Gly Met Ile 165 170 175 Asp Tyr Glu Gly Thr Gly Gln Ala Ala Tyr Pro Lys Leu Ser Lys Ser 180 185 190 Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His 195 200 205 His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp 210 215 220 Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser Lys Lys Phe Lys Pro 225 230 235 240 Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn 245 250 255 Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala 260 265 270 Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg Asn 275 280 285 Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn 290 295 300 Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro Phe 305 310 315 320 Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys 325 330 335 Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn Ser Pro Glu Asn 340 345 350 Leu Tyr Phe Gln Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly 355 360 365 Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn 370 375 380 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala 385 390 395 400 Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn 405 410 415 Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg 420 425 430 Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp 435 440 445 Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu 450 455 460 Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser 465 470 475 480 Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe 485 490 495 Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr 500 505 510 Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu 515 520 525 Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr Gln Gly Ala Glu 530 535 540 Asn Leu Tyr Phe Gln Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg 545 550 555 560 Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser 565 570 575 Thr Phe Leu Gly His His His His His His His His His His 580 585 590 <210> 32 <211> 1785 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 32 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acaatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactctg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caaatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgacttcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtgga aggcatgatt gattacgaag 540 gcacaggcca ggcagcctac cctaagctgt ctaagagcta catcaacgac aagggcaaag 600 aagtgctggt gctgtgggga atccaccacc ctagcacaag cgccgatcag cagagcctgt 660 accagaatgc cgatgcctat gtgttttgtgg gcagcagcag atacagcaaa aagttcaagc 720 ctgaaattgc cattagacccc aaagtgagag atcaggagg caggaatgaat tactactgga 780 ccctggtgga acctggcgat aagatcacat ttgaggccac cggaatctg gtggtgccta 840 gatatgcatt tgctatggag agaatgctg gctctggcat cattatctct gatacccctg 900 tgcacgactg taataccacc tgtcagacac ctaagggcgc cattaatacc agcctgccct 960 tccagaatat tcacctatc accatcggca agtgtcctaa gtatgtgaag agcaccaagc 1020 tgagactggc taccggtctg agaataagcc ctgaaaacct gtattttcaa ggcctgtttg 1080 gagccatcgc cggctttatt gagggaggt ggaccggaat ggtggatggc tggtacggct 1140 atcaccacca gatgagcag ggatccggat atgccgccga tctgaagtct accagaacg 1200 ccatcgacga gatcacaac aaggtgaaca gcgtgatcga gagatgaac acccagttta 1260 cagctgtggg caggagttc aaccacctgg agagagaat cgagaacctg aacaagaag 1320 tggacgacgg cttcctggat atttggacct acatgccga gctgctcgtg ctcctggaga 1380 atgagagaac cctggactac cacgacagca atgtgaagaa cctgtacgag aaggtgagaa 1440 gccagctgaa gaacaatgcc aaggagatcg gcaacggctg ctttgagttc taccacaagt 1500 gtgacaacac ctgtatggag tctgtgaaga acggcaccta cgactaccct aagtatagcg 1560 aggaggccaa gctgaataga gaggagatcg acggcgtgaa actggaaagc acaagaatct 1620 atcagggcgc tgaaaacctg tattttcagg gcggttctgg ttacatcccg gaagctccgc 1680 gtgacggtca ggcttacgtt cgtaaagacg gtgaatgggt tctgctgtct accttcctgg 1740 gtcaccatca tcaccaccat caccatcatc actgataaaa agctt 1785 <210> 33 <211> 591 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 33 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Glu Gly Met Ile Asp Tyr 130 135 140 Glu Gly Thr Gly Gln Ala Ala Trp Pro Asn His Asp Ser Asn Lys Gly 145 150 155 160 Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser Phe Tyr Lys Asn 165 170 175 Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro Lys Leu Ser Lys 180 185 190 Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile 195 200 205 His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala 210 215 220 Asp Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser Lys Lys Phe Lys 225 230 235 240 Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met 245 250 255 Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu 260 265 270 Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg 275 280 285 Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys 290 295 300 Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro 305 310 315 320 Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val 325 330 335 Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn Ser Pro Glu 340 345 350 Asn Leu Tyr Phe Gln Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 355 360 365 Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Gln 370 375 380 Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn 385 390 395 400 Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met 405 410 415 Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu Glu Lys 420 425 430 Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile 435 440 445 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr 450 455 460 Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Arg 465 470 475 480 Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu 485 490 495 Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys Asn Gly 500 505 510 Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu 515 520 525 Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr Gln Gly Ala 530 535 540 Glu Asn Leu Tyr Phe Gln Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro 545 550 555 560 Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu 565 570 575 Ser Thr Phe Leu Gly His His His His His His His His His His 580 585 590 <210> 34 <211> 1787 <212> DNA<​​​​​​​​​​​​​​​​​​tggaaacccc tagcagcgac aatggcacct gttaccctgg cgacttcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctgaag 420 gcatgattga ttacgaaggc acaggccagg cagcctggcc taatcacgat tctaataagg 480 gagtgacagc cgcctgtcct catgccggag ccaagtcctt ttacaagaac ctgatctggc 540 tggtgaagaa gggcaacagc taccctaagc tgtctaagag ctacatcaac gacaagggca 600 aagaagtgct ggtgctgtgg ggaatccacc accctagcac aagcgccgat cagcagagcc 660 tgtaccagaa tgccgatgcc tatgtgtttg tgggcagcag cagatacagc aaaaagttca 720 agcctgaaat tgccattaga cccaaagtga gagatcagga aggcagaatg aattactact 780 ggaccctggt ggaacctggc gataagatca catttgaggc caccggaaat ctggtggtgc 840 ctagatatgc atttgctatg gagaaatg ctggctctgg catcattatc tctgataccc 900 ctgtgcacga ctgtaatacc acctgtcaga cacctaaggg cgccattaat accagcctgc 960 ccttccagaa tattcaccct atcaccatcg gcaagtgtcc taagtatgtg aagagcacca 1020 agctgagact ggctaccggt ctgagaata gccctgaaaa cctgtatttt caaggcctgt ttggagccat cgccggcttt attgagggag gatggaccgg aatggtggat ggctggtacg 1140 gctatcacca ccagaatgag cagggatccg gatatgccgc cgatctgaag tctacacaga acgccatcga cgagatcaca aacaaggtga acagcgtgat cgagagatg aacacccagt ttacagctgt gggcaaggag ttcaaccacc tggagaagag aatcgagaac ctgaacaaga aagtggacga cggcttcctg gatatttgga cctacaatgc cgagctgctc gtgctcctgg aatgagag aaccctggac taccacgaca gcaatgtgaa gaacctgtac gagaaggtga gaagccagct gaagacaat gccaaggaga tcggcaacgg ctgctttgag ttctaccaca agtgtgacaa cacctgtatg gagtctgtga agaacggcac ctacgactac cctaagtata gcgaggaggc rich rich tcgacggcgt rich rich tctatcaggg cgctgaaaac ctgtattttc agggcggttc tggttacatc ccggaagctc 1680. cgcgtgacgg tcaggcttac gttcgtaaag acggtgaatg ggttctgctg tctaccttcc tgggtcacca tcatcaccac catcaccatc atcactgata aaagctt 1787 <210> 35 <211> 586 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 35 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln 195 200 205 Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser 210 215 220 Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Gly Ile Phe Gly 225 230 235 240 Ala Ile Ala Gly Phe Ile Glu Gly Gly Arg Met Asn Tyr Tyr Trp Thr 245 250 255 Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu 260 265 270 Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly 275 280 285 Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln 290 295 300 Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His 305 310 315 320 Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu 325 330 335 Arg Leu Ala Thr Gly Leu Arg Asn Ser Pro Glu Asn Leu Tyr Phe Gln 340 345 350 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly 355 360 365 Met Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser 370 375 380 Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile 385 390 395 400 Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr 405 410 415 Ala Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu 420 425 430 Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala 435 440 445 Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp 450 455 460 Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn 465 470 475 480 Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys 485 490 495 Asp Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro 500 505 510 Lys Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val 515 520 525 Lys Leu Glu Ser Thr Arg Ile Tyr Gln Gly Ala Glu Asn Leu Tyr Phe 530 535 540 Gln Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala 545 550 555 560 Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly 565 570 575 His His His His His His His His His His 580 585 <210> 36 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 36 Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly 1 5 10 <210> 37 <211> 1772 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 37 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acaatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactctg tgaatctgct ggaggataag cacaatggca 180 <223> Synthetic agctgtgtaa gctgagagga gttgcccctc tgcacctggg caaatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgacttcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtgaa gaagggcaac agctacccta 540 agctgtctaa gagctacatc aacgacaagg gcaaagaagt gctggtgctg tggggaatcc 600 accaccctag cacaagcgcc gatcagcaga gcctgtacca gaatgccgat gcctatgtgt 660 ttgtgggcag cagcagatac agcaaaaagt tcaagcctga aattgccatt ggcattttcg 720 gcgctatcgc cggcttcatt gagggaggca gaatgaatta ctactggacc ctggtggaac 780 ctggcgataa gatcacattt gaggccaccg gaaatctggt ggtgcctaga tatgcatttg 840 ctatggagag aaatgctggc tctggcatca ttatctctga tacccctgtg cacgactgta 900 ataccacctg tcagacacct aagggcgcca ttaataccag cctgcccttc cagaatattc 960 accctatcac catcggcaag tgtcctaagt atgtgaagag caccaagctg agactggcta 1020 ccggtctgag aaatagccct gaaaacctgt attttcaagg cctgtttgga gccatcgccg 1080 gctttattga gggaggatgg accggaatgg tggatggctg gtacggctat caccaccaga 1140 atgagcaggg atccggatat gccgccgatc tgaagtctac acagaacgcc atcgacgaga 1200 tcacaaacaa ggtgaacagc gtgatcgaga agatgaacac ccagtttaca gctgtgggca 1260 aggagttcaa ccacctggag aagagaatcg agaacctgaa caagaaagtg gacgacggct 1320 tcctggatat ttggacctac aatgccgagc tgctcgtgct cctggagaat gagagaaccc 1380 tggactacca cgacagcaat gtgaagaacc tgtacgagaa ggtgagaagc cagctgaaga 1440 acaatgccaa ggagatcggc aacggctgct ttgagttcta ccacaagtgt gacaacacct 1500 gtatggagtc tgtgaagaac ggcacctacg actaccctaa gtatagcgag gaggccaagc 1560 tgaatagaga ggagatcgac ggcgtgaaac tggaaagcac aagaatctat cagggcgctg 1620 aaaacctgta ttttcagggc ggttctggtt acatcccgga agctccgcgt gacggtcagg 1680 cttacgttcg taaagacggt gaatgggttc tgctgtctac cttcctgggt caccatcatc 1740 accaccatca ccatcatcac tgataaaagc tt 1772 <210> 38 <211> 588 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 38 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro Gly Ile Phe 145 150 155 160 Gly Ala Ile Ala Gly Phe Ile Glu Gly Phe Tyr Lys Asn Leu Ile Trp 165 170 175 Leu Val Lys Lys Gly Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile 180 185 190 Asn Asp Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro 195 200 205 Ser Thr Ser Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr 210 215 220 Val Phe Val Gly Ser Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile 225 230 235 240 Ala Ile Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr 245 250 255 Trp Thr Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly 260 265 270 Asn Leu Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly 275 280 285 Ser Gly Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr 290 295 300 Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn 305 310 315 320 Ile His Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr 325 330 335 Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn Ser Pro Glu Asn Leu Tyr 340 345 350 Phe Gln Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp 355 360 365 Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln 370 375 380 Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp 385 390 395 400 Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln 405 410 415 Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu 420 425 430 Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr 435 440 445 Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr 450 455 460 His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu 465 470 475 480 Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His 485 490 495 Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp 500 505 510 Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp 515 520 525 Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr Gln Gly Ala Glu Asn Leu 530 535 540 Tyr Phe Gln Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly 545 550 555 560 Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe 565 570 575 Leu Gly His His His His His His His His His His 580 585 <210> 39 <211> 1778 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 39 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgactcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctggcattt 480 tcggcgctat cgccggcttc attgagggat tttacagaa cctgatctgg ctggtgaaga 540 agggcacag ctaccctaag ctgtctaaga gctacatcaa cgacaagggc aagaagtgc 600 tggtgctgtg gggaatccac cacctagca caagcgccga tcagcagagc ctgtaccaga 660 atgccgatgc ctatgtgttt gtggggcagca gcagatacag caaaagttc aagcctgaaa 720 ttgccattag acccaagtg agagatcagg aaggcagaat gatttac tggaccctgg 780 tggaacctgg cgataagatc acatttgagg ccaccggaaa tctggtggtg cctagatatg 840 catttgctat ggagagaaat gctggctctg gcatcattat ctctgatacc cctgtgcacg 900 actgtaatac cacctgtcag acacctaagg gcgccattaa taccagcctg cccttccaga 960 atattcaccc tatcaccatc ggcaagtgtc ctaagtatgt gaagagcacc aagctgagac 1020 tggctaccgg tctgagaaat agccctgaaa acctgtattt tcaaggcctg tttggagcca 1080 tcgccggctt tattgaggga ggatggaccg gaatggtgga tggctggtac ggctatcacc 1140 accagaatga gcagggatcc ggatatgccg ccgatctgaa gtctacacag aacgccatcg 1200 acgagatcac aaacaaggtg aacagcgtga tcgagaagat gaacacccag tttacagctg 1260 tgggcaagga gttcaaccac ctggagaaga gaatcgagaa cctgaacaag aaagtggacg 1320 acggcttcct ggatatttgg acctacaatg ccgagctgct cgtgctcctg gagaatgaga 1380 gaaccctgga ctaccacgac agcaatgtga agaacctgta cgagaaggtg agaagccagc 1440 tgaagaacaa tgccaaggag atcggcaacg gctgctttga gttctaccac aagtgtgaca 1500 acacctgtat ggagtctgtg aagaacggca cctacgacta ccctaagtat agcgaggagg 1560 ccaagctgaa tagagaggag atcgacggcg tgaaactgga aagcacaaga atctatcagg 1620 gcgctgaaaa cctgtatttt cagggcggtt ctggttacat cccggaagct ccgcgtgacg 1680 gtcaggctta cgttcgtaaa gacggtgaat gggttctgct gtctaccttc ctgggtcacc 1740 atcatcacca ccatcaccat catcactgat aaaagctt 1778 <210> 40 <211> 589 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 40 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Gly Ile Phe Gly 165 170 175 Ala Ile Ala Gly Phe Ile Glu Gly Tyr Pro Lys Leu Ser Lys Ser Tyr 180 185 190 Ile Asn Asp Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His 195 200 205 Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp Ala 210 215 220 Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu 225 230 235 240 Ile Ala Ile Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn Tyr 245 250 255 Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr 260 265 270 Gly Asn Leu Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala 275 280 285 Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr 290 295 300 Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln 305 310 315 320 Asn Ile His Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser 325 330 335 Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn Ser Pro Glu Asn Leu 340 345 350 Tyr Phe Gln Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly 355 360 365 Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu 370 375 380 Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile 385 390 395 400 Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr 405 410 415 Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile 420 425 430 Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr 435 440 445 Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp 450 455 460 Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln 465 470 475 480 Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr 485 490 495 His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr 500 505 510 Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile 515 520 525 Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr Gln Gly Ala Glu Asn 530 535 540 Leu Tyr Phe Gln Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp 545 550 555 560 Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr 565 570 575 Phe Leu Gly His His His His His His His His His His 580 585 <210> 41 <211> 1781 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 41 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acaatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactctg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caaatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgacttcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtggg cattttcggc gctatcgccg 540 gcttcattga gggataccct aagctgtcta agagctacat caacgacaag ggcaaagaag 600 tgctggtgct gtggggaatc caccacccta gcacaagcgc cgatcagcag agcctgtacc 660 agaatgccga tgcctatgtg tttgtgggca gcagcagata cagcaaaaag ttcaagcctg 720 aaattgccat tagacccaaa gtgagagatc aggaaggcag aatgaattac tactggaccc 780 tggtggaacc tggcgataag atcacatttg aggccaccgg aaatctggtg gtgcctagat 840 atgcatttgc tatggagaga aatgctggct ctggcatcat tatctctgat acccctgtgc 900 acgactgtaa taccacctgt cagacaccta agggcgccat taataccagc ctgcccttcc 960 agaatattca ccctatcacc atcggcaagt gtcctaagta tgtgaagagc accaagctga 1020 gactggctac cggtctgaga aatagccctg aaaacctgta ttttcaaggc ctgtttggag 1080 ccatcgccgg ctttattgag ggagatgga ccggaatggt ggatggctgg tacggctatc 1140 accaccagaa tgagcaggga tccggatatg ccgccgatct gaagtctaca caaacgcca 1200 tcgacgagat cacaacaag gtgaacagcg tgatcgagaa gatgaacacc cagtttacag 1260 ctgtgggcaa ggagttcaac cacctggaga agaatcga gaacctgaac aagaaagtgg 1320 acgacggctt cctggatatt tggacctaca atgccgagct gctcgtgctc ctggagaatg 1380 agagaaccct ggactaccac gacagcaatg tgaagaacct gtacgagaag gtgagaagcc 1440 1500 1560 aggccaagct gatagagag gagatcgacg gcgtgaaact ggaaagcaca agaatctatc 1620 agggcgctga aaacctgtat tttcagggcg gttctggtta catcccggaa gctccgcgtg 1680 acggtcaggc ttacgttcgt aaagacggtg aatgggttct gctgtctacc ttcctgggtc 1740 accatcatca ccaccatcac catcatcact gataaaagct t 1781 <210> 42 <211> 590 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 42 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 ... Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Gly Ile Phe Gly Ala Ile 130 135 140 Ala Gly Phe Ile Glu Gly Trp Pro Asn His Asp Ser Asn Lys Gly Val 145 150 155 160 Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser Phe Tyr Lys Asn Leu 165 170 175 Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro Lys Leu Ser Lys Ser 180 185 190 Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His 195 200 205 His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp 210 215 220 Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser Lys Lys Phe Lys Pro 225 230 235 240 Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn 245 250 255 Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala 260 265 270 Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg Asn 275 280 285 Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn 290 295 300 Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro Phe 305 310 315 320 Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys 325 330 335 Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn Ser Pro Glu Asn 340 345 350 Leu Tyr Phe Gln Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly 355 360 365 Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn 370 375 380 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala 385 390 395 400 Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn 405 410 415 Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg 420 425 430 Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp 435 440 445 Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu 450 455 460 Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser 465 470 475 480 Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe 485 490 495 Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr 500 505 510 Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu 515 520 525 Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr Gln Gly Ala Glu 530 535 540 Asn Leu Tyr Phe Gln Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg 545 550 555 560 Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser 565 570 575 Thr Phe Leu Gly His His His His His His His His His His 580 585 590 <210> 43 <211> 1784 <212> DNA <213> Synthetic sequence <220> <223> Synthetic <400> 43 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acaatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactctg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caaatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgacttcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctggca 420 tttcggcgc tatcgccggc ttcattgagg gatggcctaa tcacgattct aataagggag 480 tgacagccgc ctgtcctcat gccggagcca agtcctttta caagaacctg atctggctgg 540 tgaagaaggg caacagctac cctaagctgt ctaagagcta catcaacgac aagggcaaag 600 aagtgctggt gctgtgggga atccaccacc ctagcacaag cgccgatcag cagagcctgt 660 accagaatgc cgatgcctat gtgtttgtgg gcagcagcag atacagcaaa aagttcaagc 720 ctgaaattgc cattagacccc aaagtgagag atcaggagg caggaatgaat tactactgga 780 ccctggtgga acctggcgat aagatcacat ttgaggccac cggaatctg gtggtgccta 840 gatatgcatt tgctatggag agaatgctg gctctggcat cattatctct gatacccctg 900 tgcacgactg taataccacc tgtcagacac ctaagggcgc cattaatacc agcctgccct 960 tccagaatat tcacctatc accatcggca agtgtcctaa gtatgtgaag agcaccaagc 1020 tgagactggc taccggtctg agaataagcc ctgaaaacct gtattttcaa ggcctgtttg 1080 gagccatcgc cggctttatt gagggaggt ggaccggaat ggtggatggc tggtacggct 1140 atcaccacca gatgagcag ggatccggat atgccgccga tctgaagtct accagaacg 1200 ccatcgacga gatcacaac aaggtgaaca gcgtgatcga gagatgaac acccagttta 1260 cagctgtggg caggagttc aaccacctgg agagagaat cgagaacctg aacaagaag 1320 tggacgacgg cttcctggat atttggacct acatgccga gctgctcgtg ctcctggaga 1380 atgagagaac cctggactac cacgacagca atgtgagaa cctgtacgag aaggtgagaa 1440 gccagctgaa gaacaatgcc aaggagatcg gcaacggctg ctttgagttc taccacaagt 1500 gtgacaacac ctgtatggag tctgtgaaga acggcaccta cgactaccct aagtatagcg 1560 aggaggccaa gctgaataga gaggagatcg acggcgtgaa actggaaagc acaagaatct 1620 atcagggcgc tgaaaacctg tattttcagg gcggttctgg ttacatcccg gaagctccgc 1680 gtgacggtca ggcttacgtt cgtaaagacg gtgaatgggt tctgctgtct accttcctgg 1740 gtcaccatca tcaccaccat caccatcatc actgataaaa gctt 1784 <210> 44 <211> 584 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 44 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Ser Gly Ile Phe Gly Ala 195 200 205 Ile Ala Gly Phe Ile Glu Gly Asp Ala Tyr Val Phe Val Gly Ser Ser 210 215 220 Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val 225 230 235 240 Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro 245 250 255 Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg 260 265 270 Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser 275 280 285 Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly 290 295 300 Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile 305 310 315 320 Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr 325 330 335 Gly Leu Arg Asn Ser Pro Gln Arg Glu Arg Arg Lys Lys Arg Gly Leu 340 345 350 Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val 355 360 365 Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr 370 375 380 Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn 385 390 395 400 Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val 405 410 415 Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys 420 425 430 Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu 435 440 445 Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn 450 455 460 Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn Ala 465 470 475 480 Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn 485 490 495 Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 500 505 510 Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu 515 520 525 Glu Ser Thr Arg Ile Tyr Gln Gly Ala Glu Asn Leu Tyr Phe Gln Gly 530 535 540 Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val 545 550 555 560 Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His His 565 570 575 His His His His His His His His 580 <210> 45 <211> 1766 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 45 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acaatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactctg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caaatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgacttcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtgaa gaagggcaac agctacccta 540 agctgtctaa gagctacatc aacgacaagg gcaaagaagt gctggtgctg tggggaatcc 600 accaccctag cggcattttc ggcgctatcg ccggcttcat tgagggagat gcctatgtgt 660 ttgtgggcag cagcagatac agcaaaaagt tcaagcctga aattgccatt agacccaaag 720 tgagagatca ggaaggcaga atgaattact actggaccct ggtggaacct ggcgataaga 780 tcacatttga ggccaccgga aatctggtgg tgcctagata tgcatttgct atggagagaa 840 atgctggctc tggcatcatt atctctgata cccctgtgca cgactgtaat accacctgtc 900 agacacctaa gggcgccatt aataccagcc tgcccttcca gaatattcac cctatcacca 960 tcggcaagtg tcctaagtat gtgaagagca ccaagctgag actggctacc ggtctgagaa 1020 atagccctca gagggagaga cgcaagaaga gaggcctgtt tggagccatc gccggcttta 1080 ttgagggagg atggaccgga atggtggatg gctggtacgg ctatcaccac cagaatgagc 1140 agggatccgg atatgccgcc gatctgaagt ctacacagaa cgccatcgac gagatcacaa 1200 acaaggtgaa cagcgtgatc gagaagatga acacccagtt tacagctgtg ggcaaggagt 1260 tcaaccacct ggagaagaga atcgagaacc tgaacaagaa agtggacgac ggcttcctgg 1320 atatttggac ctacaatgcc gagctgctcg tgctcctgga gaatgagaga accctggact 1380 accacgacag caatgtgaag aacctgtacg agaaggtgag aagccagctg aagaacaatg 1440 ccaaggagat cggcaacggc tgctttgagt tctaccacaa gtgtgacaac acctgtatgg 1500 agtctgtgaa gaacggcacc tacgactacc ctaagtatag cgaggaggcc aagctgaata 1560 gagaggagat cgacggcgtg aaactggaaa gcacaagaat ctatcagggc gctgaaaacc 1620 tgtattttca gggcggttct ggttacatcc cggaagctcc gcgtgacggt caggcttacg 1680 ttcgtaaaga cggtgaatgg gttctgctgt ctaccttcct gggtcaccat catcaccacc 1740 atcaccatca tcactgataa aagctt 1766 <210> 46 <211> 584 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 46 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln 195 200 205 Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser 210 215 220 Arg Tyr Ser Lys Lys Phe Lys Pro Gly Ile Phe Gly Ala Ile Ala Gly 225 230 235 240 Phe Ile Glu Gly Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro 245 250 255 Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg 260 265 270 Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser 275 280 285 Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly 290 295 300 Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile 305 310 315 320 Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr 325 330 335 Gly Leu Arg Asn Ser Pro Gln Arg Glu Arg Arg Lys Lys Arg Gly Leu 340 345 350 Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val 355 360 365 Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr 370 375 380 Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn 385 390 395 400 Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val 405 410 415 Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys 420 425 430 Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu 435 440 445 Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn 450 455 460 Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn Ala 465 470 475 480 Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn 485 490 495 Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr 500 505 510 Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu 515 520 525 Glu Ser Thr Arg Ile Tyr Gln Gly Ala Glu Asn Leu Tyr Phe Gln Gly 530 535 540 Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val 545 550 555 560 Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His His 565 570 575 His His His His His His His His 580 <210> 47 <211> 1766 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 47 ccatggtaag cgctattgtt ttatatgtgc ttttgcggc ggcggcgcat tctgccttg 60 cggatacact gtgtattggc taccacgcca acatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgactcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtga gaagggcaac agctacccta 540 agctgtctaa gagctacacc aacgacaagg gcaagaagt gctggtgctg tgggaatcc 600 accaccctag cacaagcgcc gatcagcaga gcctgtacca gatgccgat gcctatgtgt 660 ttgtgggcag cagcagatac agcaaagt tcaagcctgg cattttcggc gctatcgccg 720 gcttcattga gggaggcaga atgaattact actggaccct ggtggaacct ggcgataaga 780 tcacatttga ggccaccgga aatctggtgg tgcctagata tgcatttgct atggagagaa 840 atgctggctc tggcatcatt atctctgata cccctgtgca cgactgtaat accacctgtc 900 agacacctaa gggcgccatt aataccagcc tgcccttcca gaatattcac cctatcacca 960 tcggcaagtg tcctaagtat gtgaagagca ccaagctgag actggctacc ggtctgagaa 1020 atagccctca gagggagaga cgcaagaaga gaggcctgtt tggagccatc gccggcttta 1080 ttgagggagg atggaccgga atggtggatg gctggtacgg ctatcaccac cagaatgagc 1140 agggatccgg atatgccgcc gatctgaagt ctacacagaa cgccatcgac gagatcacaa 1200 acaaggtgaa cagcgtgatc gagaagatga acacccagtt tacagctgtg ggcaaggagt 1260 tcaaccacct ggagaagaga atcgagaacc tgaacaagaa agtggacgac ggcttcctgg 1320 atatttggac ctacaatgcc gagctgctcg tgctcctgga gaatgagaga accctggact 1380 accacgacag caatgtgaag aacctgtacg agaaggtgag aagccagctg aagaacaatg 1440 ccaaggagat cggcaacggc tgctttgagt tctaccacaa gtgtgacaac acctgtatgg 1500 ccaaggagat cggcaacggc tgctttgagt tctaccacaa gtgtgacaac acctgtatgg 1500 agtctgtgaa gaacggcacc tacgactacc ctaagtatag cgaggaggcc aagctgaata 1560 agtctgtgaa gaacggcacc tacgactacc ctaagtatag cgaggaggcc aagctgaata 1560 gagaggagat cgacggcgtg aaactggaaa gcacaagaat ctatcagggc gctgaaaacc 1620 gagaggagat cgacggcgtg aaactggaaa gcacaagaat ctatcagggc gctgaaaacc 1620 tgtattttca gggcggttct ggttacatcc cggaagctcc gcgtgacggt caggcttacg 1680 tgtattttca gggcggttct ggttacatcc cggaagctcc gcgtgacggt caggcttacg 1680 ttcgtaaaga cggtgaatgg gttctgctgt ctaccttcct gggtcaccat catcaccacc 1740 ttcgtaaaga cggtgaatgg gttctgctgt ctaccttcct gggtcaccat catcaccacc 1740 atcaccatca tcactgataa aagctt 17-66 atcaccatca tcactgataa aagctt 1766 <210> 48<210> 48 <211> 592<211> 592 <212> PRT<212> PRT <213> 人工序列<213> Artificial sequence <220><220><o003199> <223> 合成的<223> Synthetic <400> 48 <400> 48 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 5--5 60 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Arg Lys Lys Arg 165 170 175 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Tyr Ile Asn Asp Lys 180 185 190 Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Lys Glu 195 200 205 Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Asp 210 215 220 Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser Lys Lys Phe Lys Pro 225 230 235 240 Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn 245 250 255 Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala 260 265 270 Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg Asn 275 280 285 Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn 290 295 300 Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro Phe 305 310 315 320 Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys 325 330 335 Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln Arg 340 345 350 Glu Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile 355 360 365 Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His His 370 375 380 Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln 385 390 395 400 Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys 405 410 415 Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu Glu 420 425 430 Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp 435 440 445 Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg 450 455 460 Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val 465 470 475 480 Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe 485 490 495 Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys Asn 500 505 510 Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn Arg 515 520 525 Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr Gln Gly 530 535 540 Ala Glu Asn Leu Tyr Phe Gln Gly Gly Ser Gly Tyr Ile Pro Glu Ala 545 550 555 560 Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu 565 570 575 Leu Ser Thr Phe Leu Gly His His His His His His His 580 585 590 <210> 49 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 49 Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 1 5 10 15 <210> 50 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 50 Lys Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn 1 5 10 15 Ser <210> 51 <211> 1790 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 51 ccatggtaag cgctattgtt ttatatgtgc ttttgcggc ggcggcgcat tctgccttg 60 cggatacact gtgtattggc taccacgcca acatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgactcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtgag aaagagaga ggcctgtttg 540 gagccatcgc cggctttatt gagtacatca acgacaaggg caagaagtg ctggtgctgt 600 ggggaatcca ccacctagc aaggagtcta cacagaaggc cattgatggc gttacaata 660 aggtcaattc tgatgcctat gtgtttgtgg gcagcagcag atacagcaaa aagttcaagc 720 ctgaaattgc cattagacccc aaagtgagag atcaggagg caggaatgaat tactactgga 780 ccctggtgga acctggcgat aagatcacat ttgaggccac cggaatctg gtggtgccta 840 gatatgcatt tgctatggag agaatgctg gctctggcat cattatctct gatacccctg 900 tgcacgactg taataccacc tgtcagacac ctaagggcgc cattaatacc agcctgccct 960 tccagaatat tcacctatc accatcggca agtgtcctaa gtatgtgaag agcaccaagc 1020 tgagactggc taccggtctg agaatagcc ctcagaggga gagacgcaag aagagaggcc 1080 tgtttggagc catcgccggc tttattgagg gaggatggac cggaatggtg gatggctggt 1140 acggctatca ccaccagaat gagcagggat ccggatatgc cgccgatctg aagtctacac 1200 agaacgccat cgacgagatc aaaaaagg tgaacagcgt gatcgagaag atgacaccc 1260 agtttacagc tgtgggcaag gagttcacc acctggagaa gagaatcgag aacctgaaca 1320 agaaagtgga cgacggcttc ctggatattt ggacctacaa tgccgagctg ctcgtgctcc 1380 tggagaatga gagaaccctg gactaccacg acagcaatgt gagaacctg tacgagaagg 1440 tgagaagcca gctgaagaac aatgccaagg agatcggcaa cggctgcttt gagttctacc 1500 acaagtgtga caacacctgt atggagtctg tgaagaacgg cacctacgac taccctaagt 1560 atagcgagga ggccaagctg aatagagagg agatcgacgg cgtgaaactg gaaagcacaa 1620 gaatctatca gggcgctgaa aacctgtatt ttcagggcgg ttctggttac atcccggaag 1680 ctccgcgtga cggtcaggct tacgttcgta aagacggtga atgggttctg ctgtctacct 1740 tcctgggtca ccatcatcac caccatcacc atcatcactg ataaaagctt 1790 <210> 52 <211> 589 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 52 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Arg Lys Lys Arg 165 170 175 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Tyr Ile Asn Asp Lys 180 185 190 Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Lys Glu 195 200 205 Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Asp 210 215 220 Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser Lys Lys Phe Lys Pro 225 230 235 240 Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn 245 250 255 Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala 260 265 270 Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg Asn 275 280 285 Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn 290 295 300 Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro Phe 305 310 315 320 Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys 325 330 335 Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn Ile Pro Ser Ile 340 345 350 Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly 355 360 365 Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu 370 375 380 Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile 385 390 395 400 Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr 405 410 415 Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile 420 425 430 Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr 435 440 445 Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp 450 455 460 Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln 465 470 475 480 Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr 485 490 495 His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr 500 505 510 Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile 515 520 525 Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr Gln Gly Ala Glu Asn 530 535 540 Leu Tyr Phe Gln Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp 545 550 555 560 Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr 565 570 575 Phe Leu Gly His His His His His His His His His His 580 585 <210> 53 <211> 1781 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 53 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acaatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactctg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caaatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgactcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtgag aaagagaga ggcctgtttg 540 gagccatcgc cggctttatt gagtacatca acgacaaggg caagaagtg ctggtgctgt 600 ggggaatcca ccacctagc aaggagtcta cacagaaggc cattgatggc gttacaata 660 aggtcaattc tgatgcctat gtgtttgtgg gcagcagcag atacagcaaa aagttcaagc 720 ctgaaattgc cattagacccc aaagtgagag atcaggagg caggaatgaat tactactgga 780 ccctggtgga acctggcgat aagatcacat ttgaggccac cggaatctg gtggtgccta 840 gatatgcatt tgctatggag agaatgctg gctctggcat cattatctct gatacccctg 900 tgcacgactg taataccacc tgtcagacac ctaagggcgc cattaatacc agcctgccct 960 tccagaatat tcacctatc accatcggca agtgtcctaa gtatgtgaag agcaccaagc 1020 tgagactggc taccggtctg agaaatatcc ctagcatcca gagcagaggc ctgtttggag 1080 ccatcgccgg ctttattgag ggagatgga ccggaatggt ggatggctgg tacggctatc 1140 accaccagaa tgagcaggga tccggatatg ccgccgatct gaagtctaca caaacgcca 1200 tcgacgagat cacaacaag gtgaacagcg tgatcgagaa gatgaacacc cagtttacag 1260 ctgtgggcaa ggagttcaac cacctggaga agaatcga gaacctgaac aagaaagtgg 1320 acgacggctt cctggatatt tggacctaca atgccgagct gctcgtgctc ctggagaatg 1380 agagaaccct ggactaccac gacagcaatg tgaagaacct gtacgagaag gtgagaagcc 1440 1500 1560 aggccaagct gatagagag gagatcgacg gcgtgaaact ggaaagcaca agaatctatc 1620 agggcgctga aaacctgtat tttcagggcg gttctggtta catcccggaa gctccgcgtg 1680 acggtcaggc ttacgttcgt aaagacggtg aatgggttct gctgtctacc ttcctgggtc 1740 accatcatca ccaccatcac catcatcact gataaaagct t 1781 <210> 54 <211> 585 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 54 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Arg Lys Lys Arg 165 170 175 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Tyr Ile Asn Asp 180 185 190 Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr 195 200 205 Ser Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe 210 215 220 Val Gly Ser Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile 225 230 235 240 Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr 245 250 255 Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu 260 265 270 Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly 275 280 285 Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln 290 295 300 Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His 305 310 315 320 Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu 325 330 335 Arg Leu Ala Thr Gly Leu Arg Asn Ile Glu Asn Leu Tyr Phe Gln Gly 340 345 350 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 355 360 365 Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 370 375 380 Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr 385 390 395 400 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala 405 410 415 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn 420 425 430 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 435 440 445 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 450 455 460 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn 465 470 475 480 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp 485 490 495 Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys 500 505 510 Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys 515 520 525 Leu Glu Ser Thr Arg Ile Tyr Gln Gly Ala Glu Asn Leu Tyr Phe Gln 530 535 540 Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr 545 550 555 560 Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His 565 570 575 His His His His His His His His His 580 585 <210> 55 <211> 1769 <212> DNA <213> Artificial sequence <220> <223> Synthesized <400> 55 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acaatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactctg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caaatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgacttcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtgag aaagaagaga ggcctgttcg 540 gcgctatcgc cggcttcatt gagggataca tcaacgacaa gggcaaagaa gtgctggtgc 600 tgtggggaat ccaccaccct agcacaagcg ccgatcagca gagcctgtac cagaatgccg 660 atgcctatgt gtttgtgggc agcagcagat acagcaaaa gttcaagcct gaaattgcca 720 ttagacccaa agtgagagat caggaggca gatgaatta ctactggacc ctggtggaac 780 ctggcgataa gatcacattt gaggccaccg gaaatctggt ggtgcctaga tatgcattg 840 ctatggagag aaatgctggc tctggcatca ttatctctga taccctgtg cacgactgta 900 ataccacctg tcagacacct aagggcgcca ttaataccag cctgccctc cagaatattc 960 accctatcac catcggcaag tgtcctaagt atgtgaagg caccaagctg agactggcta 1020 ccggtctgag aaatatcgaa aacctgtatt ttcaggcct gttggagcc atcgccggct 1080 ttattgagggg aggatggacc ggaatggtgg atggctgta cggctatcac caccagaatg 1140 agcagggatc cggatatgcc gccgatctga agtctacaca gaacgccac gacgagatca 1200 haaaaaggt gaacagcgtg atcgagaga tgaacacca gtttacagct gtggggcaagg 1260 agttcaacca cctggagaag agaatcgaga acctgaaca gaagtggac gacggcttcc 1320 tggatatttg gacctacaat gccgagctgc tcgtgctcct ggagaatgag agaaccctgg 1380 actaccacga cagcaatgtg aagaacctgt acgagaaggt gagaagccag ctgaagaaca 1440 atgccaagga gatcggcaac ggctgctttg agttctacca caagtgtgac aacacctgta 1500 tggagtctgt gaagaacggc acctacgact accctaagta tagcgaggag gccaagctga 1560 atagagagga gatcgacggc gtgaaactgg aaagcacaag aatctatcag ggcgctgaaa 1620 acctgtattt tcagggcggt tctggttaca tcccggaagc tccgcgtgac ggtcaggctt 1680 acgttcgtaa agacggtgaa tgggttctgc tgtctacctt cctgggtcac catcatcacc 1740 accatcacca tcatcactga taaaagctt 1769 <210> 56 <211> 593 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 56 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln 195 200 205 Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser 210 215 220 Arg Tyr Ser Lys Lys Phe Lys Pro Ser Leu Leu Thr Glu Val Glu Thr 225 230 235 240 Pro Thr Arg Asn Gly Trp Glu Cys Lys Cys Ser Asp Ser Gly Arg Met 245 250 255 Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu 260 265 270 Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg 275 280 285 Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys 290 295 300 Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro 305 310 315 320 Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val 325 330 335 Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln 340 345 350 Arg Glu Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 355 360 365 Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His 370 375 380 His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr 385 390 395 400 Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu 405 410 415 Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu 420 425 430 Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu 435 440 445 Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 450 455 460 Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys 465 470 475 480 Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys 485 490 495 Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys 500 505 510 Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn 515 520 525 Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr Gln 530 535 540 Gly Ala Glu Asn Leu Tyr Phe Gln Gly Gly Ser Gly Tyr Ile Pro Glu 545 550 555 560 Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val 565 570 575 Leu Leu Ser Thr Phe Leu Gly His His His His His His His His 580 585 590 His <210> 57 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 57 Ser Leu Leu Thr Glu Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Cys 1 5 1​​​​​​​​​​​​​​​​​​​​​cggatacact gtgtattggc taccacgcca acatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgactcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtga gaagggcaac agctacccta 540 agctgtctaa gagctacacc aacgacaagg gcaagaagt gctggtgctg tgggaatcc 600 accaccctag cacaagcgcc gatcagcaga gcctgtacca gatgccgat gcctatgtgt 660 ttgtgggcag cagcagatac agcaaagt tcaagccttc cctgctgacc gaggtggaga 720 cccccaccag gaacggctgg gagtgcaagt gctccgactc cggcagaatg attack 780 ggaccctggt ggaacctggc gataagatca catttgaggc caccggaaat ctggtggtgc 840 ctagatatgc atttgctatg gagaaatg ctggctctgg catcattatc tctgataccc 900 ctgtgcacga ctgtaatacc acctgtcaga cacctaaggg cgccattaat accagcctgc 960 ccttccagaa tattcaccct atcaccatcg gcaagtgtcc taagtatgtg aagagcacca 1020 agctgagact ggctaccggt ctgagaaata gccctcagag ggagagacgc aagaagagag 1080 gcctgtttgg agccatcgcc ggctttattg agggaggatg gaccggaatg gtggatggct 1140 ggtacggcta tcaccaccag aatgagcagg gatccggata tgccgccgat ctgaagtcta 1200 cacagaacgc catcgacgag atcacaaaca aggtgaacag cgtgatcgag aagatgaaca 1260 cccagtttac agctgtgggc aaggagttca accacctgga gaagagaatc gagaacctga 1320 acaagaaagt ggacgacggc ttcctggata tttggaccta caatgccgag ctgctcgtgc 1380 tcctggagaa tgagagaacc ctggactacc acgacagcaa tgtgaagaac ctgtacgaga 1440 aggtgagaag ccagctgaag aacaatgcca aggagatcgg caacggctgc tttgagttct 1500 accacaagtg tgacaacacc tgtatggagt ctgtgaagaa cggcacctac gactacccta 1560 agtatagcga ggaggccaag ctgaatagag aggagatcga cggcgtgaaa ctggaaagca 1620 caagaatcta tcagggcgct gaaaacctgt attttcaggg cggttctggt tacatcccgg 1680 aagctccgcg tgacggtcag gcttacgttc gtaaagacgg tgaatgggtt ctgctgtcta 1740 ccttcctggg tcaccatcat caccaccatc accatcatca ctgataaaag ctt 1793 <210> 59 <211> 593 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 59 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30 Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Ser Ser Leu Leu Thr Glu 195 200 205 Val Glu Thr Pro Thr Arg Asn Gly Trp Glu Cys Lys Cys Ser Asp Ser 210 215 220 Asp Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser Lys Lys Phe Lys 225 230 235 240 Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met 245 250 255 Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu 260 265 270 Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg 275 280 285 Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys 290 295 300 Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro 305 310 315 320 Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val 325 330 335 Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln 340 345 350 Arg Glu Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 355 360 365 Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His 370 375 380 His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr 385 390 395 400 Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu 405 410 415 Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu 420 425 430 Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu 435 440 445 Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 450 455 460 Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys 465 470 475 480 Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys 485 490 495 Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys 500 505 510 Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn 515 520 525 Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr Gln 530 535 540 Gly Ala Glu Asn Leu Tyr Phe Gln Gly Gly Ser Gly Tyr Ile Pro Glu �45 550 555 560 Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val 565 570 575 Leu Leu Ser Thr Phe Leu Gly His His His His His His His His His 580 585 590 His <210> 60 <211> 1793 <212> DNA <213> Artificial sequence <220> <↵23> Synthetic <400> 60 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 ‍cggatacact gtgtattggc taccacgcca acaatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactctg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caaatgtaat attgccggct 240<‍ [[ID=3‍8]]ggattctggg aaatcctgaa tgtgaaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgacttcatc gattacgagg 360 [[ID=‍41]] It should be noted that there seems to be some encoding or formatting issues in the original text which might cause some inaccuracies in the translation. For example, the "↵" character in line 26 might be an encoding error. The above translation tries to make sense of the text as much as possible while following the rules.agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtgaa gaagggcaac agctacccta 540 agctgtctaa gagctacatc aacgacaagg gcaaagaagt gctggtgctg tggggaatcc 600 accaccctag ctccctgctg accgaggtgg agacccccac caggaacggc tgggagtgca 660 agtgctccga ctccgatgcc tatgtgtttg tgggcagcag cagatacagc aaaaagttca 720 agcctgaaat tgccattaga cccaaagtga gagatcagga aggcagaatg aattactact 780 ggaccctggt ggaacctggc gataagatca catttgaggc caccggaaat ctggtggtgc 840 ctagatatgc atttgctatg gagaaatg ctggctctgg catcattatc tctgataccc 900 ctgtgcacga ctgtaatacc acctgtcaga cacctaaggg cgccattaat accagcctgc 960 ccttccagaa tattcaccct atcaccatcg gcaagtgtcc taagtatgtg aagagcacca 1020 agctgagact ggctaccggt ctgagaaata gccctcagag ggagagacgc aagaagagag 1080 gcctgtttgg agccatcgcc ggctttattg aggaggatg gaccggaatg gtggatggct 1140 ggtacggcta tcaccaccag aatgagcagg gatccggata tgccgccgat ctgaagtcta cacagaacgc catcgacgag atcacaaaca aggtgaacag cgtgatcgag aagatgaaca cccagtttac agctgtgggc aaggagttca accacctgga gaagagaatc gagaacctga acaagaaagt ggacgacggc ttcctggata tttggaccta caatgccgag ctgctcgtgc tcctggagaa tgagagaacc ctggactacc acgacagcaa tgtgaagaac ctgtacgaga aggtgagag ccagctgag aacaatgcca aggagatcgg caacggctgc tttgagttct accacaagtg tgacaacacc tgtatggagt ctgtgaagaa cggcacctac gactacccta agtatagcga ggaggccaag ctgaatagag agtagatcga cggcgtgaaa ctggaagca caagaatcta tcagggcgct gaaaacctgt attttcaggg cggttctggt tacatcccgg 1680. aagctccgcg tgacggtcag gcttacgttc gtaaagacgg tgaatgggtt ctgctgtcta 1740. ccttcctggg tcaccatcat caccaccatc accatcatca ctgataaaag ctt <210> 61 <211> 591 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 61 Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His 1 5 10 15 Ser Ala Phe Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser 20 25 30<00XX3827>Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His \n35 40 45 Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu 50 55 60 Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp 65 70 75 80 Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp 85 90 95 Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro 100 105 110 Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val 115 120 125 Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 130 135 140 Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly 145 150 155 160 Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn 165 170 175 Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu 180 185 190 Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln 195 200 205 Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser 210 215 220 Arg Tyr Ser Lys Lys Phe Lys Pro Ser Leu Leu Thr Glu Val Glu Thr 225 230 235 240 Pro Thr Arg Asn Gly Trp Glu Cys Lys Cys Ser Asp Ser Gly Arg Met 245 250 255 Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu 260 265 270 Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg 275 280 285 Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys 290 295 300 Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro 305 310 315 320 Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val 325 330 335 Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn Ser Pro Glu 340 345 350 Asn Leu Tyr Phe Gln Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu 355 360 365 Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Gln 370 375 380 Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn 385 390 395 400 Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met 405 410 415 Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His Leu Glu Lys 420 425 430 Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu Asp Ile 435 440 445 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr 450 455 460 Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Arg 465 470 475 480 Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu 485 490 495 Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys Asn Gly 500 505 510 Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu 515 520 525 Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr Gln Gly Ala 530 535 540 Glu Asn Leu Tyr Phe Gln Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro 545 550 555 560 Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu 565 570 575[[ID=三十一]] Ser Thr Phe Leu Gly His His His His His His His His His 580 585 590 <210> 62 <211> 1787 <212> DNA [[ID=四十二]]<213> Artificial Sequence <220> <223> Synthetic <400> 62 ccatggtaag cgctattgtt ttatatgtgc ttttggcggc ggcggcgcat tctgcctttg 60 cggatacact gtgtattggc taccacgcca acaatagcac cgataccgtg gatacagtgc 120 tggagaagaa tgtgaccgtg acccactctg tgaatctgct ggaggataag cacaatggca 180 agctgtgtaa gctgagagga gttgcccctc tgcacctggg caaatgtaat attgccggct 240 ggattctggg aaatcctgaa tgtgaaagcc tgtctacagc cagcagctgg tcttatatcg 300 tggaaacccc tagcagcgac aatggcacct gttaccctgg cgacttcatc gattacgagg 360 agctgagaga acagctgtct agcgtgtcca gcttcgagag attcgagatc ttccctaaga 420 caagcagctg gcctaatcac gattctaata agggagtgac agccgcctgt cctcatgccg 480 gagccaagtc cttttacaag aacctgatct ggctggtgaa gaagggcaac agctacccta 540 agctgtctaa gagctacatc aacgacaagg gcaaagaagt gctggtgctg tggggaatcc 600 accaccctag cacaagcgcc gatcagcaga gcctgtacca gaatgccgat gcctatgtgt 660 ttgtgggcag cagcagatac agcaaaaagt tcaagccttc cctgctgacc gaggtggaga 720 cccccaccag gaacggctgg gagtgcaagt gctccgactc cggcagaatg aattactact 780 ggaccctggt ggaacctggc gataagatca catttgaggc caccggaaat ctggtggtgc 840 ctagatatgc atttgctatg gagaaatg ctggctctgg catcattatc tctgataccc 900 ctgtgcacga ctgtaatacc acctgtcaga cacctaaggg cgccattaat accagcctgc 960 ccttccagaa tattcaccct atcaccatcg gcaagtgtcc taagtatgtg aagagcacca 1020 agctgagact ggctaccggt ctgagaaata gccctgaaaa cctgtatttt caaggcctgt 1080 ttggagccat cgccggcttt attgagggag gatggaccgg aatggtggat ggctggtacg 1140 gctatcacca ccagaatgag cagggatccg gatatgccgc cgatctgaag tctacacaga 1200 acgccatcga cgagatcaca aacaaggtga acagcgtgat cgagaagatg aacccagt 1260 ttacagctgt gggcaaggag ttcaaccacc tggagaagag aatcgagaac ctgaacaaga 1320 aagtggacga cggcttcctg gatatttgga cctacaatgc cgagctgctc gtgctcctgg 1380 aatgagag aaccctggac taccacgaca gcaatgtgaa gaacctgtac gagaaggtga gaagccagct gaagacaat gccaaggaga tcggcaacgg ctgctttgag ttctaccaca agtgtgacaa cacctgtatg gagtctgtga agaacggcac ctacgactac cctaagtata gcgaggaggc rich rich tcgacggcgt rich rich tctatcaggg cgctgaaaac ctgtattttc agggcggttc tggttacatc ccggaagctc 1680. cgcgtgacgg tcaggcttac gttcgtaaag acggtgaatg ggttctgctg tctaccttcc tgggtcacca tcatcaccac catcaccatc atcactgata aaagctt

Claims

1. A modified influenza virus, wherein the virus comprises an influenza HA protein, wherein the influenza HA protein comprises (i) a globular head domain, wherein at least one immunodominant region in the globular head domain contains an HA stem epitope, wherein the stem epitope comprises at least one stem peptide of a homologous influenza virus HA stem domain, and (ii) a modified mature cleavage site, wherein the influenza HA protein comprises a sequence as shown in any one of SEQ ID NO: 24, 29, 31, 33, 35, 38, 40, 42, 44, 46, 48, 52, 54, 56, 59 or 61.

2. The modified influenza virus according to claim 1, wherein the modified mature cleavage site is a mature cleavage site that has been modified to be cleavable by tobacco etch virus protease.

3. The modified influenza virus according to claim 1, wherein the modified mature cleavage site has been cleaved by a suitable protease.

4. The modified influenza virus of claim 2, wherein the modified mature cleavage site has been cleaved by a tobacco etch virus protease.

5. The modified influenza virus of claim 1, wherein the HA stem epitope is recognized by a broadly neutralizing antibody against influenza virus.

6. The modified influenza virus of claim 1, wherein more than one immunodominant region of the HA protein has been modified to contain HA stem epitopes.

7. The modified influenza virus of claim 6, wherein at least some of the HA stem epitopes contained in each of the more than one immunodominant regions are different from each other.

8. An immunogenic composition comprising the modified influenza virus according to claim 1.

9. An immunogenic composition comprising the modified influenza virus according to claim 3.

10. An immunogenic composition comprising the modified influenza virus according to claim 5.

11. Use of the immunogenic composition according to any one of claims 8 to 10 in the preparation of a medicament for generating anti-influenza antibodies in a subject.

12. Use of the immunogenic composition according to any one of claims 8 to 10 in the preparation of a medicament for use in the prophylactic treatment of a patient against influenza.

13. Use of the modified influenza virus according to claim 1 in the preparation of a medicament for use in the prophylactic treatment of a subject against influenza.

14. A nucleic acid encoding an influenza HA protein, wherein the influenza HA protein comprises (i) a globular head domain, wherein at least one immunodominant region in the globular head domain contains an HA stem epitope, wherein the stem epitope comprises at least one stem peptide of a homologous influenza virus HA stem domain, and (ii) a modified mature cleavage site, wherein the influenza HA protein comprises a sequence as set forth in any one of SEQ ID NO: 24, 29, 31, 33, 35, 38, 40, 42, 44, 46, 48, 52, 54, 56, 59 or 61.

15. The nucleic acid of claim 14, wherein the modified mature cleavage site is modified to encode a mature cleavage site cleavable by a tobacco etch virus protease.

16. A recombinant host cell that produces a modified virus or virus-like particle comprising the HA protein encoded by the nucleic acid according to claim 14.

17. A method for preparing a virus or virus-like particle comprising a modified HA protein, the method comprising culturing the recombinant host cell according to claim 16 under conditions where the nucleic acid is expressed, and recovering the virus or virus-like particle comprising the HA protein.

18. A method for producing a modified influenza HA protein, the method comprising: (i) culturing cells in a culture medium, wherein the cells contain an expression vector, wherein the expression vector contains the nucleic acid sequence according to claim 14 or 15; and (ii) isolating the modified influenza HA protein from the cells and / or the culture medium.

19. The method of claim 18, wherein the expression vector is a recombinant baculovirus vector, and wherein the cell is an insect cell.

20. The method of claim 18, wherein the HA stem epitope has been inserted into or has replaced at least one immunodominant region in the globular head domain of the HA protein, wherein the HA stem epitope is recognized by broadly neutralizing antibodies against influenza virus.

21. The method of claim 20, wherein more than one immunodominant region of the HA protein has been modified to contain HA stem epitopes.

22. The method of claim 21, wherein at least some of the HA stem epitopes contained in each of the more than one immunodominant regions are different from each other.

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