Adenoviral vector encoding hepatitis B virus antigen fused to herpes virus glycoprotein D and methods of use thereof

Abstract

This article provides non-natural variants of the hepatitis B virus (HBV) Core protein, the N-terminal domain of HBV polymerase, and the C-terminal domain of HBV polymerase, as well as immunogenic fragments thereof. It also provides fusion proteins comprising HBV variants fused to a sequence of herpes simplex virus (HSV) glycoprotein (gD), and methods for using such fusion proteins.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 958,809, filed January 9, 2020; U.S. Provisional Application No. 62 / 958,827, filed January 9, 2020; U.S. Provisional Application No. 62 / 967,242, filed January 29, 2020; U.S. Provisional Application No. 62 / 967,104, filed January 29, 2020; U.S. Provisional Application No. 63 / 064,506, filed August 12, 2020; U.S. Provisional Application No. 63 / 064,571, filed August 12, 2020; and U.S. Provisional Application No. 63 / 112,202, filed November 11, 2020; and U.S. Provisional Application No. 63 / 112,219, filed November 11, 2020, the disclosure of each of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence list, which was electronically submitted in ASCII format and incorporated herein by reference in its entirety. The ASCII copy was created on January 7, 2021, and is named 111876_000035_SL.txt, with a size of 151,446 bytes. Technical Field

[0005] This article discloses non-natural variants of the hepatitis B virus (HBV) Core protein, the N-terminal domain of HBV polymerase, and the C-terminal domain of HBV polymerase, as well as their immunogenic fragments and fusion proteins containing them. Background Technology

[0006] The World Health Organization estimates that in 2015, 257 million people were chronically infected with hepatitis B (defined as HBsAg positive), and hepatitis B caused an estimated 887,000 deaths, mostly due to cirrhosis and hepatocellular carcinoma (i.e., primary liver cancer). Assuming that women of reproductive age comprise 25.3% of the world's population (UN data), chronically infected adults could include 65 million women of reproductive age who could potentially transmit HBV to their infants (WHO 2017 Global Hepatitis Report, available at apps_who_int / iris / bitstream / handle / 10665 / 255016 / 9789241565455-eng.pdf;jsessionid=D78616700ED7322D4109CA4541FB94EA?sequence=1). The overall incidence rate in 2016 was 1.0 case per 100,000 people (Centers for Disease Control and Prevention. Viral Hepatitis Surveillance—United States, 2017. Atlanta: US Department of Health and Human Services, Centers for Disease Control and Prevention; 2019, available at www_cdc_gov / hepatitis / statistics / 2017surveillance / index.htm). In 2017 alone, a total of 3,407 cases of acute hepatitis B were reported to the Centers for Disease Control and Prevention (CDC).

[0007] Despite the availability of preventative HBV vaccines, the burden of chronic HBV infection remains a significant unresolved global medical problem due to inadequate treatment options and persistent new infections in much of the developing world. Invention Overview

[0009] This article provides the hepatitis B virus (HBV) Core protein containing the amino acid sequence of SEQ ID NO:6 or an immunogenic fragment thereof.

[0010] It also provides an N-terminal domain of HBV polymerase containing the amino acid sequence of SEQ ID NO:8 or an immunogenic fragment thereof.

[0011] The C-terminal domain of HBV polymerase containing the amino acid sequence of SEQ ID NO:10 or its immunogenic fragment is also disclosed.

[0012] It also provides a fusion protein comprising: an N-terminal herpes simplex virus (HSV) glycoprotein (gD) sequence or a variant thereof; a disclosed HBV Core protein, an N-terminal domain of HBV polymerase, a C-terminal domain of HBV polymerase or an immunogenic fragment thereof; and a C-terminal HSV gD sequence or a variant thereof.

[0013] This article also provides a fusion protein comprising: an N-terminal herpes simplex virus (HSV) glycoprotein (gD) sequence or a variant thereof; a combination of a disclosed HBV Core protein, an N-terminal domain of HBV polymerase, a C-terminal domain of HBV polymerase, and / or an immunogenic fragment thereof; and a C-terminal HSV gD sequence or a variant thereof.

[0014] This article discloses nucleic acid molecules encoding disclosed proteins or fusion proteins, vectors containing said nucleic acid molecules, and vaccines containing disclosed vectors.

[0015] This article also provides a method for inducing an immune response to HBV in subjects, which involves providing subjects with an effective amount of any publicly available fusion protein, nucleic acid molecule, vector, or vaccine to induce an immune response to HBV. Brief description of the attached diagram

[0017] The explanation and the following detailed description will become more clear when read in conjunction with the accompanying drawings. To illustrate the disclosed proteins, vaccines, and methods, exemplary embodiments of the proteins, vaccines, and methods are shown in the drawings; however, the proteins, vaccines, and methods are not limited to the specific embodiments disclosed. In the drawings:

[0018] Figure 1 The frequency of core amino acids for epitope optimization is shown. Amino acid residues are displayed on the X-axis; the percentage of sequence similarity across all analyzed genomes is displayed on the Y-axis.

[0019] Figure 2A , Figure 2B , Figure 2C , Figure 2D , Figure 2E and Figure 2F This study demonstrated the frequency of vaccine insert-specific T cells in C57Bl / 6 mice after intramuscular (im) injection of a specified dose of the following substance: a replication-defective adenovirus vector (AdC6-gDCore) containing a genetically fused epitope-optimized Core sequence (SEQ ID NO:15) of chimpanzee serotype 6 (AdC6). Figure 2A and Figure 2D); AdC6 (AdC6-gDPolC) containing a genetically optimized polymerase C-terminal domain sequence (SEQ ID NO:19) fused to gD. Figure 2B and Figure 2E ); and AdC6 (AdC6-gDPolN) containing a polymerase N-terminal domain sequence (SEQ ID NO:17) optimized by an epitope fused into gD. Figure 2C and Figure 2F Blood was collected from mice 14 days post-injection, and T cell frequencies for various HBV inserts were analyzed by intracellular cytokine staining (ICS) against interferon (IFN)γ after cell stimulation with overlapping peptides representing HBV sequences. Control cells were cultured without the peptides. Coordinate plots show the results for individual mice, with the median represented by a line. Figure 2A-2C Insertion-specific CD8 was demonstrated + T cell frequency; Figure 2D-2F Displaying insert-specific CD4 + T cell frequency.

[0020] Figure 3A , Figure 3B and Figure 3C This study illustrates the T cell frequencies of different mouse strains (A: C57Bl / 6 mice; B: BALB / c mice; C: HLA-A2 transgenic (tg) mice) in response to peptide pools representing the HBV sequences shown. Results were obtained using spleen cells harvested 4 weeks post-immunization and analyzed by an ICS assay targeting IFN-γ. Peptides are arranged in a matrix so that identifying two pools identifies one peptide. Coordinate plots show responses to different pools; responses to pools containing all peptides are shown on the right. Background frequencies obtained without peptides are subtracted. The bottom of each plot lists the pools considered to elicit a response and the peptides identified in response to different pools. CD8 counts of BALB / c mice are shown. + T cells and CD4 + T cell response; showed CD8 in HLA-A2 tg mice + T cell response; the mouse carries human MHC class I molecules but mouse MHC class II molecules. Activated CD44 +On the cellular level, T cells are gated. Each sequentially numbered "peptide" consists of 15 amino acids starting from amino acids 1, 6, 11, etc., of the Core, PolN, or PolC sequence. Thus, for example, peptide 1 of Core corresponds to amino acids 1-15 of SEQ ID NO:6 (i.e., the epitope-optimized Core amino acid sequence), peptide 2 of Core corresponds to amino acids 6-20 of SEQ ID NO:6, peptide 3 of Core corresponds to amino acids 11-25 of SEQ ID NO:6, and so on. Similarly, peptide 1 of PolN corresponds to amino acids 1-15 of SEQ ID NO:8 (i.e., the epitope-optimized PolN amino acid sequence), peptide 2 of PolN corresponds to amino acids 6-20 of SEQ ID NO:8, peptide 3 of PolN corresponds to amino acids 11-25 of SEQ ID NO:8, and so on. Similarly, peptide 1 of PolC corresponds to amino acids 1-15 of amino group SEQ ID NO:10 (i.e., the epitope-optimized amino acid sequence of PolC), peptide 2 of PolC corresponds to amino acids 6-20 of SEQ ID NO:10, peptide 3 of PolC corresponds to amino acids 11-25 of SEQ ID NO:10, and so on.

[0021] Figure 4A , Figure 4B and Figure 4C The graph shows the IFN-γ response in C57Bl / 6 mice immunized with different doses of the vectors shown, after boosting with AdC6-gDCore (A), AdC6-gDPolC (B), and AdC6-gDPolN (C). The left graph shows the response tested in blood 2 weeks after priming with the AdC6 vector. After 8 weeks, mice were boosted with the same dose of the AdC7 vector expressing the same insert. The right graph shows the response in blood 2 weeks after boosting.

[0022] Figure 5A , Figure 5B and Figure 5CThis study illustrates the T cell frequencies of different mouse strains (A: C57Bl / 6 mice; B: BALB / c mice; C: HLA-A2tg mice) in response to peptide pools representing the HBV sequences shown. Mice were primed with an AdC6 vector expressing any of three inserts (i.e., Core, PolC, or PolN) and boosted 8 weeks later with an AdC7 vector expressing the same insert. Results were obtained using spleen cells harvested 4 weeks post-immunization and analyzed by an ICS assay targeting IFN-γ. Peptides are arranged in a matrix so that identifying two pools identifies one peptide. Coordinate plots show responses to different pools; responses to pools containing all peptides are shown on the right. Background frequencies obtained without peptides are subtracted. The bottom of each plot lists the pools considered to elicit a response and the peptides identified in response to different pools. CD8 values ​​of BALB / c mice are shown. + T cells and CD4 + T cell response; showed CD8 in HLA-A2 tg mice + T cell response; the mice carried human MHC class I molecules but mouse MHC class II molecules. In activated CD44... + On the cellular level, T cells are gated. Each sequentially numbered "peptide" consists of 15 amino acids starting from amino acids 1, 6, 11, etc., of the Core, PolN, or PolC sequence. Thus, for example, peptide 1 of Core corresponds to amino acids 1-15 of SEQ ID NO:6 (i.e., the epitope-optimized Core amino acid sequence), peptide 2 of Core corresponds to amino acids 6-20 of SEQ ID NO:6, peptide 3 of Core corresponds to amino acids 11-25 of SEQ ID NO:6, and so on. Similarly, peptide 1 of PolN corresponds to amino acids 1-15 of SEQ ID NO:8 (i.e., the epitope-optimized PolN amino acid sequence), peptide 2 of PolN corresponds to amino acids 6-20 of SEQ ID NO:8, peptide 3 of PolN corresponds to amino acids 11-25 of SEQ ID NO:8, and so on. Similarly, peptide 1 of PolC corresponds to amino acids 1-15 of amino group SEQ ID NO:10 (i.e., the epitope-optimized amino acid sequence of PolC), peptide 2 of PolC corresponds to amino acids 6-20 of SEQ ID NO:10, peptide 3 of PolC corresponds to amino acids 11-25 of SEQ ID NO:10, and so on.

[0023] Figure 6 This study demonstrated the effect of vaccination on the HBV genome copy number in serum following AAV-1.3 HBV challenge. (Using 1x10⁻¹² HBV) 10 1x10 11 Or 1.5x10 11A group of three mice was challenged with an adeno-associated virus 8 (AAV8)-1.3HBV vector containing one viral genome (vg), and then inoculated with AdC6-gDPolN eight weeks later. Viral titers were measured eight weeks post-vaccination and compared with pre-vaccination titers. Viral changes relative to baseline are shown for each treatment group.

[0024] Figure 7A , Figure 7B , Figure 7C , Figure 7D and Figure 7E An exemplary HBV epitope shifting experiment is described. Figure 7A Mice were immunized with the AdC6-gDPolN vaccine. Four weeks later, spleen cells were tested for intracellular cytokine staining in response to the IFN-γ pool representing the PolN sequence. T cell markers were stained on stimulated T cells. Figure 7B - Used from 1x10 10 Spleen cells from mice challenged with 1000 Vg of AAV8-1.3-HBV were obtained using the same assay. Four weeks later, the mice were vaccinated, and T-cell responses were tested from the spleen ten weeks later. Figure 7C -Using from 1.5x10 11 Spleen cells from mice challenged with 1000 Vg of AAV8-1.3-HBV were obtained using the same assay. Mice were vaccinated 4 weeks later, and T-cell responses were tested from the spleen 10 weeks later. Figure 7A , 7B And 7C shows relative to all CD44 + CD8 + T cells produce IFN-γ-CD44 + CD8 + The frequency of T cells was subtracted from the background response obtained by incubating spleen cells without a peptide pool. Figure 7D –Peptide pool. Figure 7E – Individual peptide sequences. Figure 7E SEQ ID NO:55-68 and 189-233 are disclosed in the order of their appearance.

[0025] Figure 8A , Figure 8B and Figure 8C Data from the same experiment described in Figure 7 above are shown. Based on the response to the peptide pools, which individual peptides (pools and peptides shown in Figure 7) were identified as positive. The coordinate plot shows the response to all peptides. Each peptide was present in both pools, thus obtaining two frequency values ​​for each peptide; only the lower data points are shown in this plot.

[0026] Figure 9A , Figure 9B and Figure 9C The results of an exemplary immunogenicity study were presented in C57Bl / 6 mice (n=5 per group), which were injected with different doses of the exemplary AdC6-gDCore, AdC6-gDPolN, or AdC6-gDPolC vectors, and boosted two months after the first injection with an AdC7 vector containing the same insert (i.e., AdC7-gDCore, AdC7-gDPolN, or AdC7-gDPolC vector). Figure 9A Explain the immunogenicity of the antigen. Figure 9B This indicates the duration of the reaction, and Figure 9C This explains the prime-boost response.

[0027] Figure 10 This indicates that after primary immunization with AdC6-gDPolN and booster immunization with AdC7-gDPolN, the CD8+ of the PolN epitope was observed in BALB / c, C57B1 / 6, and HLA-A2 transgenic mice. + T-cell peptide recognition. CD8... + T-cell peptide recognition is calculated as the fraction of positive peptides identified two weeks after primary or booster vaccination, divided by the total number of 8 overlapping peptides from PolN (out of a total of 59 peptides).

[0028] Figure 11A and Figure 11B This demonstrates that in the livers of C57B1 / 6 mice injected with the indicated vector, vaccine-induced HBV-specific CD8+ was observed. + T cell response. *p values ​​were 0.01–0.05; ***p values ​​were 0.0001–0.001; measured using one-way ANOVA.

[0029] Figure 12A , 12B , Figure 12C , 12D , Figure 12E and Figure 12F This image illustrates hematoxylin and eosin staining of liver samples from C57Bl / 6 mice injected with the vector shown. 20x magnification. Arrows indicate areas of lymphocyte infiltration.

[0030] Figure 13A and Figure 13B This indicates that CD8+ levels in the livers of C57B1 / 6 mice injected with the indicated vector were elevated. + Vaccine-induced biomarkers for T cell activation / depletion. **p values ​​were 0.001–0.01; ***p values ​​were 0.0001–0.001; by one-way ANOVA.

[0031] Figure 14A and Figure 14B This study describes the HBV virus kinetics in C57Bl / 6 mice injected with the exemplary AdC6-gDPolN vector. Median HBV DNA VL / ml at week 4 is provided as 7.3 log. 10 cps / mL. n=7; one mouse was excluded due to lack of data.

[0032] Figure 15A and Figure 15B This demonstrates the effect of AAV-induced HBV on CD8 in C57B1 / 6 mice. + The effect of T cell response was observed in the C57Bl / 6 mice, which were first injected with 10... 10 Or 10 11 Av-1.3HBV per vg, then 10 after 4 weeks. 10 An example of an enhanced AdC6-gDPolN vector for vp. Figure 15B In the diagram, each small block represents an individual epitope, and its size indicates the proportion of the total epitopes; only responses >0.1% are included. Pullout represents epitopes identified only in mice infected with AAV8-1.3HBV.

[0033] Figure 16 This demonstrates that individual C57Bl / 6 mice produce IFN-γ on CD8. + The frequency of T cells in the mouse was 10 IV-administered. 10 One gram of AAV8-1.3HBV vector was administered, and 5 x 10^6 doses were inoculated 4 weeks later. 9 The mice were given the AdC6-gDPolN vector of VP and boosted with the same dose of AdC7-gDPolN vaccine 2 months later. Control mice received only the vaccine. (Immature) Mice were used as an additional control.

[0034] Figure 17A and Figure 17B Note: A) CD8 in liver lymph infiltrates from individual mice + The percentage of T cells; and B) PolN-tetramer in the same infiltration. + CD8 + T cell frequency. C57Bl / 6 mice were injected intravenously with 10... 10 Or 10 11 One gram of AAV8-1.3HBV vector was administered, and 5 x 10^6 doses were inoculated 4 weeks later. 9 The mice were given the AdC6-gDPolN vector and boosted with the same dose of AdC7-gDPolN vaccine after 2 months. Control mice received only the vaccine. Naïve mice served as an additional control.

[0035] Figure 18A , Figure 18B, Figure 18C , Figure 18D , Figure 18E and Figure 18F This describes the infiltrative tetramer. + CD8 + T cells and immature (i.e., tetramers) - CD44 - CD8 + Phenotypic results for T cells compared to T cells, analyzed using mean fluorescence intensity (MFI) of the indicated markers. Lines marked with an asterisk indicate significant differences after multiple t-tests. (*) p ≤ 0.05–0.01, (**) p ≤ 0.01–0.001, (***) p ≤ 0.001–0.0001, (****) p ≤ 0.0001.

[0036] Figure 19A , Figure 19B , Figure 19C , Figure 19D , Figure 19E and Figure 19F This indicates that Tet is positive for the indicated marker. + Or childish CD8 + The percentage of T cells. The lines marked with an asterisk above indicate significant differences after multiple t-tests. (*) p ≤ 0.05-0.01, (**) p ≤ 0.01-0.001, (***) p ≤ 0.001-0.0001, (****) p ≤ 0.0001.

[0037] Figure 20A , Figure 20B , Figure 20C , Figure 20D , Figure 20E and Figure 20F This explains CD8 + T cell responses to individual peptides spanning the PolN sequence. Total pool – response to a mixture of all PolN peptides; Naïve – response of naïve mice to a mixture of all PolN peptides. Figure 20A and Figure 20D The CD8 count of mice that received only the AdC6-gDPolN vaccine was shown. + T cell response. Figure 20B and Figure 20E The results showed that 10 doses were injected 4 weeks prior to AdC6-gDPolN administration. 10 CD8 of mice with 1.3 HBV AAV8-1.3 HBV + T cell response. Figure 20C and Figure 20F The results showed that 10 doses were injected 4 weeks prior to AdC6-gDPolN administration. 11 CD8 of mice with 1.3 HBV AAV8-1.3 HBV +T cell response. Figure 20A , 20B And 20C can be used Figure 7D The peptide pool shown and its utilization Figure 7E The breadth of the immune response for each epitope is calculated from the identified peptide sequences.

[0038] Figure 21A and Figure 21B This indicates the presence of PolN-specific CD8 in the spleen or liver of mice. + T cells. Figure 21A The left figure shows whether or not 5×10 was subsequently accepted. 10 CD8 in the spleen of AAV8-1.3HBV mice injected with the AdC6-gDPolN vaccine. + T cell response. Figure 21A The middle image shows CD8 in mouse liver. + T cell frequency was measured in mice that received different doses of AAV8-1.3HBV in a primary immunization booster regimen before receiving the vaccine. Figure 21A The right figure shows PolN-specific CD8 from the same experiment. + T cells or immature CD8 + Tox-1 expression level in T cells. Figure 21B This explains IFN-γ + CD8 + Cell percentage.

[0039] Figure 22A and Figure 22B This indicates that A) CD8 levels in the blood of mice injected with the indicated AdC6 vector were high. + T cell frequency; and B) tetramer + CD8 + T cell frequency.

[0040] Figure 23 This indicates that CD8 levels in the blood of mice injected with the indicated AdC7 vector were elevated. + T cell frequency.

[0041] Figure 24A , Figure 24B , Figure 24C , Figure 24D , Figure 24E and Figure 24F This describes the CD8+ levels of the gDHBV2 and gDHBV3 inserts in the blood of mice injected with the indicated AdC7 vector (“post-primary”) and then boosted with the corresponding AdC6 vector (“post-boost”). + ( Figures 24A-24C ) and CD4 + ( Figures 24D-24FT cell frequency. The coordinate graph shows the frequency of T cells producing IFN-γ, the frequency of T cells producing TNF-α, and the sum of the frequencies of T cells producing any cytokine.

[0042] Figure 25A and Figure 25B This indicates the HBV DNA viral titer in C57Bl / 6 mice, which were treated with 1x10⁻⁶ mice. 9 Av8-1.3HBV was attacked in one vg and 1x10 vg was inoculated 4 weeks later. 10 AdC6-gDPolN (“gDPolN”), AdC6-gDHBV2 (“gDHBV2”), AdC6-gDHBV3 (“gDHBV3”), or AdC6-HBV2 without gD (“HBV2”) were used as controls; AAV-infected, unvaccinated animals (“naïve”) and non-AAV-infected, unvaccinated animals (data not shown) were used as controls. Figure 25A This describes the viral titers in each group at weeks 4 and 8 after the AAV attack; Figure 25B The results for each mouse at weeks 4 and 8 after AAV challenge were presented.

[0043] Figure 26A , Figure 26B , Figure 26C and Figure 26D This study demonstrated that parental CD8+ producing IFN-γ and / or TNF-α was observed at 2 and 8 weeks post-primary immunization and 2 and 4 weeks post-boost immunization (as average) using the illustrated construct. + T cells ( Figure 26A CD44 + CD8 + T cells ( Figure 26B CD4 + T cells ( Figure 26C ) or CD44 + CD4 + T cells ( Figure 26D The percentage of ).

[0044] Figure 27A , Figure 27B and Figure 27C CD8 at multiple time points + T cells: 4 weeks post-primary immunization with the indicated constructs (PolN = gDPolN; HBV2 = gDHBV2; HBV3 = gDHBV3) Figure 27A ); Strengthened in the following two weeks ( Figure 27B ); and 4 weeks after intensification ( Figure 27C The chart shows the production of IFN-γ as assessed by ICS. + CD8 + Overall frequency of T cells.

[0045] Figure 28A , Figure 28B and Figure 28C This indicates the levels of CD4+ producing cytokines as assessed by ICS at multiple time points. + T cells: 4 weeks post-primary immunization with the indicated constructs (PolN = gDPolN; HBV2 = gDHBV2; HBV3 = gDHBV3) Figure 28A ); Strengthened in the following two weeks ( Figure 28B ); and 4 weeks after intensification ( Figure 28C Based on results from juvenile mice, the dashed line represents the cutoff value for a positive response.

[0046] Figure 29A and Figure 29B This indicates that 4 weeks after initial immunization with the shown constructs (PolN = gDPolN; HBV2 = gDHBV2), on CD8... + T cells ( Figure 29A ) or CD44 + CD8 + T cells ( Figure 29B The results of gated tetramer staining were obtained.

[0047] Figure 30A , Figure 30B , Figure 30C , Figure 30D , Figure 30E and Figure 30F This explains the tetramer + CD8 + The T cell phenotype is shown as the average fluorescence intensity of the dye conjugated to the antibody shown: Figure 30A -An anti-PD1 antibody conjugated with BV605; Figure 30B -An anti-LAG3 antibody conjugated with BV650; Figure 30C - Anti-TIM3 antibody conjugated with Pe-Cy7-A; 30D- anti-CTLA4 antibody conjugated with PE-A; Figure 30E -An anti-EOMES antibody conjugated with AF488; and Figure 30F -An anti-T-bet antibody conjugated with BV786.

[0048] Figure 31 This indicates that the primary immunization dose is 5 x 10. 10 vp AdC7-gDHBV2, followed by 5x10 doses two months later. 10 CD8 after vpAdC6-gDHBV2 + T cell response. The numbers on the X-axis correspond to the SEQ ID NO provided in this article.

[0049] Figure 32This indicates that the primary immunization dose is 5 x 10. 9 vp AdC7-gDHBV2, followed by 5x10 doses two months later. 9 CD8 after vp AdC6-gDHBV2 + T cell response. The numbers on the X-axis correspond to the SEQ ID NO provided in this article.

[0050] Figure 33 The primary immunization dose was shown as 5x10. 10 vp AdC7-gDHBV3, followed by 5x10 doses two months later. 10 Immunogenicity after vpAdC6-gDHBV3. The numbers on the X-axis correspond to the SEQ ID NO provided herein.

[0051] Figure 34 The immunogenicity of the AdC6-gDHBV2 and AdC7-gDHBV2 vaccines corresponding to SEQ ID NO (X-axis) provided herein was demonstrated. The Core, PolC, and PolN regions in both HBV2 constructs were immunogenic.

[0052] Figure 35 The immunogenicity of the AdC6-gDHBV3 and AdC7-gDHBV3 vaccines corresponding to SEQ ID NO (X-axis) provided herein was demonstrated. The Core, PolC, and PolN regions in both HBV3 constructs were immunogenic. Detailed Implementation

[0053] The disclosed proteins, vaccines, and methods can be more readily understood by referring to the following detailed description, taken in conjunction with the accompanying drawings, which form part of this disclosure. It should be understood that the disclosed proteins, vaccines, and methods are not limited to the specific proteins, vaccines, and methods described and / or shown herein, and the terminology used herein is for the purpose of illustrating particular embodiments only by way of example and is not intended to limit the claimed proteins, vaccines, and methods.

[0054] Unless otherwise expressly stated, any description of possible mechanisms or modes of action or reasons for improvement is intended to be illustrative only, and the disclosed proteins, vaccines, and methods are not limited by the correctness or incorrectness of any such proposed mechanisms or modes of action or reasons for improvement.

[0055] Throughout this disclosure, the description relates to proteins and methods of using said proteins. Where this disclosure describes or claims features or embodiments related to proteins, such features or embodiments also apply to methods of using said proteins. Similarly, where this disclosure describes or claims features or embodiments related to methods of using proteins, such features or embodiments also apply to the proteins themselves.

[0056] In the case of numerical ranges enumerated or established herein, the range includes its endpoints and all individual integers and decimals within that range, and also includes each of the narrower ranges formed by all the various possible combinations of these endpoints and internal integers and decimals, to form a subgroup of groups of larger values ​​within said range, to the same extent as each of these narrower ranges is explicitly enumerated. Where a numerical range is described herein as being greater than the stated value, the range remains finite and its upper limit is bounded by a value operable in the context of the invention described herein. Where a numerical range is described herein as being less than the stated value, the range remains bounded by a non-zero value at its lower limit. It is not intended to limit the scope of the invention to the specific values ​​enumerated when the range is defined. All ranges are inclusive and composable.

[0057] When a value is expressed as an approximation using the antecedent “approximately,” it should be understood that the specific value forms another implementation. References to a specific numerical value must include at least that specific value, unless the context explicitly states otherwise.

[0058] It should be understood that certain features of the disclosed proteins, vaccines, and methods described herein in the context of individual embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the disclosed proteins, vaccines, and methods described herein in the context of individual embodiments for brevity may also be provided individually or in any sub-combination.

[0059] As used in this article, the singular forms “a” and “the” include the plural.

[0060] Various terms related to the described aspects are used throughout the specification and claims. Unless otherwise stated, these terms shall be given their ordinary meaning in the art. Other specifically defined terms shall be interpreted in a manner consistent with the definitions provided herein.

[0061] As used herein, “the immunogenic fragment” refers to a portion of the disclosed HBV Core (Core), HBV polymerase N-terminal domain (PolN), or HBV polymerase C-terminal domain (PolC) that can elicit an immune response in a subject.

[0062] As used herein, “provided to a subject” and similar terms refer to a procedure for delivering a fusion protein, nucleic acid molecule, vector, or vaccine to a subject, thereby bringing the subject’s target cells, tissues, or body parts into contact with the fusion protein, nucleic acid molecule, vector, or vaccine. “Provided to a subject” includes parenteral and non-parenteral administration routes.

[0063] The term "biosimilar" in the context of an approved reference product / biologic (i.e., a reference-listed drug) refers to a biological product that, despite minor differences in clinically inactive ingredients, is highly similar to a reference product and, based on data from: (a) analytical studies demonstrating high similarity between the biological product and the reference product despite minor differences in clinically inactive ingredients; (b) animal studies (including toxicity assessments); and / or (c) one or more clinical studies (including assessments of immunogenicity and pharmacokinetics or pharmacodynamics) sufficient to demonstrate safety, purity, and efficacy under one or more appropriate conditions of use that are for which the reference product is licensed and intended for use, and which the biosimilar is seeking license for. A biosimilar can be an interchangeable product that can be used to replace a pharmacy-approved reference product without the intervention of a prescribing healthcare professional. To meet additional criteria for “interchangeability,” the biosimilar is expected to produce the same clinical outcomes as the reference product in any given patient, and the risk of reduced safety or efficacy from alternating or switching between the use of the biosimilar and the reference product if the biosimilar is administered to an individual more than once is no greater than the risk of using the reference product without such alternation or switching. The biosimilar uses the same mechanism of action for the recommended applicable disease conditions, provided that these mechanisms are known to the reference product. One or more applicable disease conditions specified, recommended, or suggested in the labeling of the biosimilar have previously been approved for use with the reference product. The biosimilar has the same route of administration, dosage form, and / or strength as the reference product, and is manufactured, processed, packaged, or stored in a facility that meets standards designed to ensure that the biosimilar continues to be safe, pure, and effective. Compared to the reference product, the biosimilar may include minor modifications to the amino acid sequence, such as N- or C-terminal truncation that is not expected to alter the properties of the biosimilar. Biosimilars of the disclosed proteins and fusion proteins are included within the scope of this disclosure.

[0064] As used herein, the term "subject" is intended to refer to any animal, particularly a mammal. Although this document illustrates the induction of an immune response in mice, the disclosed methods can be used to treat any type of mammal. Therefore, this method is applicable to both human and non-human animals, although it is preferred for mice and humans, and most preferably for humans.

[0065] The term “comprising” is intended to include instances covered by the terms “substantially composed of” and “composed of”; similarly, the term “substantially composed of” is intended to include instances covered by the term “composed of”.

[0066] This article uses the following abbreviations: Hepatitis B virus (HBV); adenovirus (Ad); herpes simplex virus (HSV); glycoprotein (gD); and viral genome (vg).

[0067] This document provides non-naturally occurring variants of the hepatitis B virus (HBV) Core protein. The disclosed HBV Core protein may comprise the amino acid sequence of SEQ ID NO:6 or an immunogenic fragment thereof. Exemplary immunogenic fragments of SEQ ID NO:6 include SEQ ID NO:20-54 provided in Table 3 below. In some embodiments, the immunogenic fragment of the HBV Core protein comprises the amino acid sequence of SEQ ID NO:180. In some embodiments, the immunogenic fragment of the HBV Core protein comprises the amino acid sequence of SEQ ID NO:183.

[0068] Also provided are nucleic acid molecules encoding the HBV Core protein or an immunogenic fragment thereof. The nucleic acid molecule may encode the HBV Core protein comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:7. The nucleic acid molecule may encode the Core fragment provided in Table 3. In some embodiments, the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:180. In some embodiments, the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:183.

[0069] Vectors containing nucleic acid molecules encoding the HBV Core protein or its immunogenic fragments are also provided. Suitable vectors include viral vectors, such as lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus replicons, herpesvirus vectors, poxvirus vectors, and rhabdovirus vectors. In some embodiments, the viral vector is an adenovirus vector. The adenovirus vector may be a chimpanzee-derived adenovirus vector. In some aspects, the vector is the AdC68 vector, which is described in Farina SF, Gao GP, Xiang ZQ, Rux JJ, Burnett RM, Alvira MR, Marsh J, Ertl HC, Wilson JM. “Replication-defective vector based on a chimpanzee adenovirus.” J Virol. 2001 Dec; 75(23):11603-13. In some respects, the vector is the AdC7 vector, described in Reyes-Sandoval A, Fitzgerald JC, Grant R, Roy S, Xiang ZQ, Li Y, Gao GP, Wilson JM, Ertl HC. “Human immunodeficiency virus type 1-specific immune responses in primates upon sequential immunization with adenoviral vaccine carriers of human and simian serotypes” J Virol. 2004 Jul; 78(14):7392-9. In some respects, the vector is the AdC6 vector, described in Pinto AR, Fitzgerald JC, Giles-Davis W, Gao GP, Wilson JM, Ertl HC. “Induction of CD8 + T cells to an HIV-1antigen through a prime boost regimen with heterologous E1-deleted adenoviral vaccine carriers” J Immunol. 2003Dec 15;171(12):6774-9.

[0070] In some embodiments, the vector comprises a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO:7. In some embodiments, the vector is an AdC6 vector containing a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO:7. In some embodiments, the vector is an AdC7 vector containing a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO:7.

[0071] In some embodiments, the vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:180. In some aspects, the vector is an AdC6 vector. In some aspects, the vector is an AdC7 vector. In some embodiments, the vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:183. In some aspects, the vector is an AdC6 vector. In some aspects, the vector is an AdC7 vector.

[0072] The vaccine also discloses a vector comprising a nucleic acid molecule encoding the HBV Core protein or an immunogenic fragment thereof. In some embodiments, the vaccine comprises a vector containing a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:7. In some embodiments, the vaccine comprises an AdC6 vector containing a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:7. In some embodiments, the vaccine comprises an AdC7 vector containing a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:7. In some embodiments, the vaccine comprises an AdC6 vector containing a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:180. In some embodiments, the vaccine comprises an AdC7 vector containing a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:180. In some embodiments, the vaccine comprises an AdC6 vector containing a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:183. In some embodiments, the vaccine comprises an AdC7 vector containing a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:183.

[0073] Vaccines may also contain pharmaceutically acceptable carriers or pharmaceutically acceptable excipients. As used herein, “pharmaceutically acceptable carriers” or “pharmaceutically acceptable excipients” include any substance that, when combined with the disclosed fusion protein, nucleic acid, or carrier, allows the fusion protein, nucleic acid, or carrier to retain its biological activity and is unresponsive to the immune system of the subject. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions, such as oil / water emulsions, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate-buffered saline or physiological saline (0.9%). Compositions containing such carriers are formulated using well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0074] This document also discloses non-naturally occurring variants of the N-terminal domain (PolN) and C-terminal domain (PolC) of HBV polymerase. The disclosed N-terminal domain of HBV polymerase may comprise the amino acid sequence of SEQ ID NO:8 or an immunogenic fragment thereof. Exemplary immunogenic fragments of SEQ ID NO:8 include SEQ ID NO:55-113 provided in Table 4 below. In some embodiments, the immunogenic fragment of HBV PolN comprises the amino acid sequence of SEQ ID NO:178. In some embodiments, the immunogenic fragment of HBV PolN comprises the amino acid sequence of SEQ ID NO:181. The disclosed C-terminal domain of HBV polymerase may comprise the amino acid sequence of SEQ ID NO:10 or an immunogenic fragment thereof. Exemplary immunogenic fragments of SEQ ID NO:10 include SEQ ID NO:114-172 provided in Table 5 below. In some embodiments, the immunogenic fragment of HBV PolC comprises the amino acid sequence of SEQ ID NO:179. In some implementations, the immunogenic fragment of HBV PolC contains the amino acid sequence of SEQ ID NO:182.

[0075] Also provided are nucleic acid molecules encoding the N-terminal domain of HBV polymerase or an immunogenic fragment thereof, or the C-terminal domain of HBV polymerase or an immunogenic fragment thereof. The nucleic acid molecule may encode the N-terminal domain of HBV polymerase comprising the amino acid sequence of SEQ ID NO:8. In some embodiments, the nucleic acid molecule encoding the N-terminal domain of HBV polymerase comprises the nucleotide sequence of SEQ ID NO:9. The nucleic acid molecule may encode the N-terminal domain fragment of HBV polymerase provided in Table 4. The nucleic acid molecule may encode the C-terminal domain of HBV polymerase comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the nucleic acid molecule encoding the C-terminal domain of HBV polymerase comprises the nucleotide sequence of SEQ ID NO:11. The nucleic acid molecule may encode the C-terminal domain fragment of HBV polymerase provided in Table 5. In some embodiments, the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:178. In some embodiments, the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:181. In some embodiments, the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:179. In some implementations, the nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:182.

[0076] Vectors comprising nucleic acid molecules encoding the N-terminal domain or an immunogenic fragment thereof or the C-terminal domain or an immunogenic fragment thereof of HBV polymerase are also provided. Suitable vectors include those described above. In some embodiments, the vector comprises a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO:9. In some embodiments, the vector comprises a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO:11. In some aspects, the vector is an adenovirus vector. Suitable adenovirus vectors include, for example, AdC6 or AdC7 vectors. In some embodiments, the vector is an AdC6 vector containing a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO:9. In some embodiments, the vector is an AdC7 vector containing a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO:9. In some embodiments, the vector is an AdC6 vector containing a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO:11. In some embodiments, the vector is an AdC7 vector containing a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO:11. In some embodiments, the vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:178. In some aspects, the vector is an AdC6 vector. In some aspects, the vector is an AdC7 vector. In some embodiments, the vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:181. In some aspects, the vector is an AdC6 vector. In some aspects, the vector is an AdC7 vector. In some embodiments, the vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:179. In some aspects, the vector is an AdC6 vector. In some aspects, the vector is an AdC7 vector. In some embodiments, the vector comprises a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:182. In some aspects, the vector is an AdC6 vector. In some aspects, the vector is an AdC7 vector.

[0077] The vaccine also discloses a vector comprising a nucleic acid molecule encoding an N-terminal domain of HBV polymerase or an immunogenic fragment thereof, or a C-terminal domain of HBV polymerase or an immunogenic fragment thereof. In some embodiments, the vaccine comprises a vector containing a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:9. The vaccine may comprise an AdC6 vector containing a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:9. The vaccine may comprise an AdC7 vector containing a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:9. In some embodiments, the vaccine comprises a vector containing a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:11. The vaccine may comprise an AdC6 vector containing a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:11. The vaccine may comprise an AdC7 vector containing a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:11. The vaccine may also comprise a pharmaceutically acceptable vector or a pharmaceutically acceptable excipient as described above. In some embodiments, the vaccine comprises a vector containing a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:178. In some aspects, the vector is an AdC6 vector. In some aspects, the vector is an AdC7 vector. In some embodiments, the vaccine comprises a vector containing a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:181. In some aspects, the vector is an AdC6 vector. In some aspects, the vector is an AdC7 vector. In some embodiments, the vaccine comprises a vector containing a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:179. In some aspects, the vector is an AdC6 vector. In some aspects, the vector is an AdC7 vector. In some embodiments, the vaccine comprises a vector containing a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:182. In some aspects, the vector is an AdC6 vector. In some aspects, the vector is an AdC7 vector.

[0078] This document also provides fusion proteins comprising a combination of the disclosed HBV Core protein or an immunogenic fragment thereof, an N-terminal domain of HBV polymerase or an immunogenic fragment thereof, and / or a C-terminal domain of HBV polymerase or an immunogenic fragment thereof. For example, the fusion protein may comprise:

[0079] (1) HBV Core protein containing the amino acid sequence of SEQ ID NO:6 or its immunogenic fragment and HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO:8 or its immunogenic fragment;

[0080] (2) One or more immunogenic fragments of the HBV Core protein containing the amino acid sequence of SEQ ID NO:6 and one or more immunogenic fragments of the N-terminal domain of the HBV polymerase containing the amino acid sequence of SEQ ID NO:8. For example, one or more of SEQ ID NO:20-54 (immunogenic fragments of SEQ ID NO:6) provided in Table 3 and one or more of SEQ ID NO:55-113 (immunogenic fragments of SEQ ID NO:8) provided in Table 4;

[0081] (3) HBV Core protein containing the amino acid sequence of SEQ ID NO:6 or its immunogenic fragment and HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO:10 or its immunogenic fragment.

[0082] (4) One or more immunogenic fragments of the HBV Core protein containing the amino acid sequence of SEQ ID NO:6 and one or more immunogenic fragments of the C-terminal domain of the HBV polymerase containing the amino acid sequence of SEQ ID NO:10. For example, one or more of SEQ ID NO:20-54 (immunogenic fragments of SEQ ID NO:6) provided in Table 3 and one or more of SEQ ID NO:114-172 (immunogenic fragments of SEQ ID NO:10) provided in Table 5;

[0083] (5) An HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO:8 or its immunogenic fragment thereof and an HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO:10 or its immunogenic fragment thereof.

[0084] (6) One or more immunogenic fragments comprising the N-terminal domain of HBV polymerase containing the amino acid sequence of SEQ ID NO:8 and one or more immunogenic fragments comprising the C-terminal domain of HBV polymerase containing the amino acid sequence of SEQ ID NO:10. For example, one or more of SEQ ID NO:55-113 (immunogenic fragments of SEQ ID NO:8) provided in Table 4 and one or more of SEQ ID NO:114-172 (immunogenic fragments of SEQ ID NO:10) provided in Table 5;

[0085] (7) HBV Core protein containing the amino acid sequence of SEQ ID NO:6 or its immunogenic fragment, HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO:8 or its immunogenic fragment, and HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO:10 or its immunogenic fragment.

[0086] (8) One or more immunogenic fragments of HBV Core protein containing the amino acid sequence of SEQ ID NO:6, one or more immunogenic fragments of HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO:8, and one or more immunogenic fragments of HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO:10. For example, one or more of SEQ ID NO:20-54 (immunogenic fragments of SEQ ID NO:6) provided in Table 3, one or more of SEQ ID NO:55-113 (immunogenic fragments of SEQ ID NO:8) provided in Table 4, and one or more of SEQ ID NO:114-172 (immunogenic fragments of SEQ ID NO:10) provided in Table 5;

[0087] (9) An HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO:178 or an immunogenic fragment thereof, an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO:179 or an immunogenic fragment thereof, and an HBV Core protein comprising the amino acid sequence of SEQ ID NO:180 or an immunogenic fragment thereof; or

[0088] (10) An HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO:181 or an immunogenic fragment thereof, an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO:182 or an immunogenic fragment thereof, and an HBV Core protein comprising the amino acid sequence of SEQ ID NO:183 or an immunogenic fragment thereof.

[0089] The fusion protein may comprise an N-terminal domain of HBV polymerase containing the amino acid sequence of SEQ ID NO:178 or an immunogenic fragment thereof, a C-terminal domain of HBV polymerase containing the amino acid sequence of SEQ ID NO:179 or an immunogenic fragment thereof, and an HBV Core protein containing the amino acid sequence of SEQ ID NO:180 or an immunogenic fragment thereof. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:174.

[0090] The fusion protein may comprise an N-terminal domain of HBV polymerase containing the amino acid sequence of SEQ ID NO:181 or an immunogenic fragment thereof, a C-terminal domain of HBV polymerase containing the amino acid sequence of SEQ ID NO:182 or an immunogenic fragment thereof, and an HBV Core protein containing the amino acid sequence of SEQ ID NO:183 or an immunogenic fragment thereof. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:175.

[0091] This article also provides fusion proteins comprising the sequence of herpes simplex virus (HSV) glycoprotein (gD) and the disclosed HBV Core protein, HBV polymerase N-terminal domain, HBV polymerase C-terminal domain, or various combinations thereof.

[0092] HSV gD is the receptor-binding glycoprotein of HSV. The extracellular domain of gD comprises two structurally and functionally distinct regions: an N-terminus, which includes a signal sequence and a receptor binding site; and a C-terminus, which includes a pro-fusion domain and a transmembrane domain. gD interacts with herpesvirus entry mediator (HVEM) receptors and connecton receptors. The interaction of gD with the receptors leads to downregulation of HVEM receptors that bind to BTLA or CD160, immunosuppressive molecules expressed on T cells. In some embodiments, the disclosed fusion protein, comprising gD and the disclosed HBV Core protein, the N-terminal domain of HBV polymerase, the C-terminal domain of HBV polymerase (referred to as “gDCore”, “gDPolN”, or “gDPolC”, respectively), or combinations thereof, is expected to enhance the subject’s immune response to HBV to a greater extent compared to HBV Core and / or polymerase antigen alone (i.e., without gD).

[0093] Suitable HSV gD proteins for the disclosed fusion proteins include wild-type or mutant gDs that retain the following capabilities: 1) enhancing CD8 + T cell stimulation of antigen response; and / or 2) disruption of HVEM-BTLA pathway activity.

[0094] The fusion protein may comprise: the HBV Core protein disclosed herein or an immunogenic fragment thereof, the N-terminal domain of HBV polymerase or an immunogenic fragment thereof, the C-terminal domain of HBV polymerase or an immunogenic fragment thereof, or any combination thereof, an N-terminal HSV gD protein sequence, and a C-terminal HSV gD protein sequence. The HBV Core protein, the N-terminal domain of HBV polymerase, and the C-terminal domain of HBV polymerase may be those provided in Table 9 or the immunogenic fragments provided in Tables 3-5. The HBV Core protein, the N-terminal domain of HBV polymerase, the C-terminal domain of HBV polymerase, or an immunogenic fragment thereof may be inserted between the N-terminal HSV gD protein sequence and the C-terminal HSV gD protein sequence. In some aspects, the N-terminal HSV gD protein sequence comprises the amino acid sequence of SEQ ID NO: 12, and the C-terminal HSV gD protein sequence comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the N-terminal HSV gD protein sequence comprises amino acid residues 26-269 of SEQ ID NO: 12.

[0095] Fusion proteins may include:

[0096] N-terminal HSV gD sequence or a variant thereof;

[0097] HBV Core protein, comprising the amino acid sequence of SEQ ID NO:6 or an immunogenic fragment thereof; and

[0098] C-terminal HSV gD sequence or its variants.

[0099] The immunogenic fragment of the HBV Core protein may include any one of SEQ ID NO:20-54, 180, or 183.

[0100] Fusion proteins may include:

[0101] N-terminal HSV gD sequence or a variant thereof;

[0102] HBV Core protein, comprising the amino acid sequence of SEQ ID NO:180 or SEQ ID NO:183; and

[0103] C-terminal HSV gD sequence or its variants.

[0104] Fusion proteins may include:

[0105] N-terminal HSV gD sequence or a variant thereof;

[0106] The N-terminal domain of HBV polymerase, comprising the amino acid sequence of SEQ ID NO:8 or an immunogenic fragment thereof; and

[0107] C-terminal HSV gD protein sequence or its variants.

[0108] The immunogenic fragment of the N-terminal domain of HBV polymerase may include any one of SEQ ID NO:55-113, 178 or 181.

[0109] Fusion proteins may include:

[0110] N-terminal HSV gD sequence or a variant thereof;

[0111] The N-terminal domain of HBV polymerase, comprising the amino acid sequence of SEQ ID NO:178 or SEQ ID NO:181; and

[0112] C-terminal HSV gD protein sequence or its variants.

[0113] Fusion proteins may include:

[0114] N-terminal HSV gD sequence or a variant thereof;

[0115] The C-terminal domain of HBV polymerase, comprising the amino acid sequence of SEQ ID NO:10 or an immunogenic fragment thereof; and

[0116] C-terminal HSV gD protein sequence or its variants.

[0117] The immunogenic fragment of the C-terminal domain of HBV polymerase may include any one of SEQ ID NO:114-172, 179 or 182.

[0118] Fusion proteins may include:

[0119] N-terminal HSV gD sequence or a variant thereof;

[0120] The C-terminal domain of HBV polymerase, comprising the amino acid sequence of SEQ ID NO:179 or SEQ ID NO:182; and

[0121] C-terminal HSV gD protein sequence or its variants.

[0122] Fusion proteins may include:

[0123] N-terminal HSV gD sequence or a variant thereof;

[0124] HBV sequence, which includes:

[0125] (1) HBV Core protein containing the amino acid sequence of SEQ ID NO:6 or its immunogenic fragment and HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO:8 or its immunogenic fragment;

[0126] (2) One or more immunogenic fragments of the HBV Core protein containing the amino acid sequence of SEQ ID NO:6 and one or more immunogenic fragments of the N-terminal domain of the HBV polymerase containing the amino acid sequence of SEQ ID NO:8. For example, one or more of SEQ ID NO:20-54 (immunogenic fragments of SEQ ID NO:6) provided in Table 3 and one or more of SEQ ID NO:55-113 (immunogenic fragments of SEQ ID NO:8) provided in Table 4;

[0127] (3) HBV Core protein containing the amino acid sequence of SEQ ID NO:6 or its immunogenic fragment and HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO:10 or its immunogenic fragment.

[0128] (4) One or more immunogenic fragments of the HBV Core protein containing the amino acid sequence of SEQ ID NO:6 and one or more immunogenic fragments of the C-terminal domain of the HBV polymerase containing the amino acid sequence of SEQ ID NO:10. For example, one or more of SEQ ID NO:20-54 (immunogenic fragments of SEQ ID NO:6) provided in Table 3 and one or more of SEQ ID NO:114-172 (immunogenic fragments of SEQ ID NO:10) provided in Table 5;

[0129] (5) An HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO:8 or its immunogenic fragment thereof and an HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO:10 or its immunogenic fragment thereof.

[0130] (6) One or more immunogenic fragments comprising the N-terminal domain of HBV polymerase containing the amino acid sequence of SEQ ID NO:8 and one or more immunogenic fragments comprising the C-terminal domain of HBV polymerase containing the amino acid sequence of SEQ ID NO:10. For example, one or more of SEQ ID NO:55-113 (immunogenic fragments of SEQ ID NO:8) provided in Table 4 and one or more of SEQ ID NO:114-172 (immunogenic fragments of SEQ ID NO:10) provided in Table 5;

[0131] (7) An HBV Core protein comprising the amino acid sequence of SEQ ID NO:6 or an immunogenic fragment thereof, an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO:8 or an immunogenic fragment thereof, and an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO:10 or an immunogenic fragment thereof; or

[0132] (8) One or more immunogenic fragments of HBV Core protein containing the amino acid sequence of SEQ ID NO:6, one or more immunogenic fragments of HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO:8, and one or more immunogenic fragments of HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO:10. For example, one or more of SEQ ID NO:20-54 (immunogenic fragments of SEQ ID NO:6) provided in Table 3, one or more of SEQ ID NO:55-113 (immunogenic fragments of SEQ ID NO:8) provided in Table 4, and one or more of SEQ ID NO:114-172 (immunogenic fragments of SEQ ID NO:10) provided in Table 5;

[0133] (9) An HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO:178 or an immunogenic fragment thereof, an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO:179 or an immunogenic fragment thereof, and an HBV Core protein comprising the amino acid sequence of SEQ ID NO:180 or an immunogenic fragment thereof; or

[0134] (10) An HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO:181 or an immunogenic fragment thereof, an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO:182 or an immunogenic fragment thereof, and an HBV Core protein comprising the amino acid sequence of SEQ ID NO:183 or an immunogenic fragment thereof; and

[0135] C-terminal HSV gD protein sequence or its variants.

[0136] In some embodiments, the N-terminal HSV gD sequence may contain at least amino acids 1-269 of the HSV gD. For example, the N-terminal HSV gD sequence may contain the amino acid sequence of SEQ ID NO:12. In some embodiments, the N-terminal HSV gD sequence contains amino acid residues 26-269 of SEQ ID NO:12.

[0137] In some embodiments, the C-terminal HSV gD sequence contains the transmembrane domain of the HSV gD. For example, the C-terminal HSV gD sequence may contain the amino acid sequence of SEQ ID NO:13.

[0138] The fusion protein may contain the amino acid sequence of SEQ ID NO:14 (corresponding to gDCore) or its immunogenic fragment. The fusion protein may contain the amino acid sequence of SEQ ID NO:16 (corresponding to gDPolN) or its immunogenic fragment. The fusion protein may contain the amino acid sequence of SEQ ID NO:18 (corresponding to gDPolC) or its immunogenic fragment. In some embodiments, the amino acid sequence of any one of SEQ ID NO:14, 16, or 18 or its immunogenic fragment does not contain a 25-amino acid signal peptide at the N-terminus.

[0139] The fusion protein may contain the amino acid sequence of SEQ ID NO:185 (gDHBV2). The fusion protein may contain the amino acid sequence of SEQ ID NO:187 (gDHBV3).

[0140] Nucleic acid molecules encoding any publicly disclosed fusion protein are also provided. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:15 (corresponding to gDCore). In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:17 (corresponding to gDPolN). In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:19 (corresponding to gDPolC).

[0141] Nucleic acid molecules may contain the nucleotide sequence of SEQ ID NO:184 (gDHBV2). Nucleic acid molecules may contain the nucleotide sequence of SEQ ID NO:186 (gDHBV3).

[0142] Vectors containing nucleic acid molecules encoding fusion proteins are also disclosed. Suitable vectors include those described above, including, for example, adenoviral vectors. In some embodiments, the adenoviral vector is an AdC6 vector. In some embodiments, the adenoviral vector is an AdC7 vector. The vector may contain the nucleotide sequence of SEQ ID NO:184 (gDHBV2). In some aspects, the vector is an AdC6 vector containing the nucleotide sequence of SEQ ID NO:184 (gDHBV2). In some aspects, the vector is an AdC7 vector containing the nucleotide sequence of SEQ ID NO:184 (gDHBV2). The vector may contain the nucleotide sequence of SEQ ID NO:186 (gDHBV3). In some aspects, the vector is an AdC6 vector containing the nucleotide sequence of SEQ ID NO:186 (gDHBV3). In some aspects, the vector is an AdC7 vector containing the nucleotide sequence of SEQ ID NO:186 (gDHBV3).

[0143] Vaccines containing any of the disclosed vectors are also provided. Vaccines may also contain pharmaceutically acceptable vectors or excipients as disclosed above. Vaccines may contain a vector containing the nucleotide sequence of SEQ ID NO:184 (gDHBV2). In some aspects, vaccines contain an AdC6 vector containing the nucleotide sequence of SEQ ID NO:184 (gDHBV2). In some aspects, vaccines contain an AdC7 vector containing the nucleotide sequence of SEQ ID NO:184 (gDHBV2). Vaccines may contain a vector containing the nucleotide sequence of SEQ ID NO:186 (gDHBV3). In some aspects, vaccines contain an AdC6 vector containing the nucleotide sequence of SEQ ID NO:186 (gDHBV3). In some aspects, vaccines contain an AdC7 vector containing the nucleotide sequence of SEQ ID NO:186 (gDHBV3).

[0144] This document provides a method for inducing an immune response to HBV in a subject, the method comprising providing the subject with an effective amount of any publicly disclosed fusion protein, any publicly disclosed nucleic acid molecule, any publicly disclosed vector, or any publicly disclosed vaccine, thereby inducing an immune response to HBV. In some embodiments, the method comprises providing the subject with an effective amount of any publicly disclosed fusion protein, thereby inducing an immune response to HBV. In some embodiments, the method comprises providing the subject with an effective amount of any publicly disclosed nucleic acid molecule, thereby inducing an immune response to HBV. In some embodiments, the method comprises providing the subject with an effective amount of any publicly disclosed vector, thereby inducing an immune response to HBV. In some embodiments, the method comprises providing the subject with an effective amount of any publicly disclosed vaccine, thereby inducing an immune response to HBV.

[0145] This method may include administering to a subject an effective amount of a vaccine comprising an AdC6 vector, wherein the AdC6 vector comprises a fusion protein containing an amino acid sequence of any one of SEQ ID NO:14, 16, or 18, or an immunogenic fragment thereof. In some embodiments, the method further includes, after administering the vaccine comprising the AdC6 vector, administering to the subject a vaccine comprising an AdC7 vector, wherein the AdC7 vector comprises a fusion protein containing an amino acid sequence of any one of SEQ ID NO:14, 16, or 18, or an immunogenic fragment thereof. Such a prime-boost method may include:

[0146] - A vaccine comprising an AdC6 vector is administered to the subject, the AdC6 vector comprising a fusion protein containing the amino acid sequence of SEQ ID NO:14 or an immunogenic fragment thereof, and subsequently, a vaccine comprising an AdC7 vector is administered to the subject, the AdC7 vector comprising a fusion protein containing the amino acid sequence of SEQ ID NO:14 or an immunogenic fragment thereof. In some embodiments, the amino acid sequence of SEQ ID NO:14 or its immunogenic fragment thereof does not contain a signal peptide consisting of 25 N-terminal amino acids;

[0147] - A vaccine comprising an AdC6 vector is administered to the subject, the AdC6 vector comprising a fusion protein containing the amino acid sequence of SEQ ID NO:16 or an immunogenic fragment thereof, and subsequently, a vaccine comprising an AdC7 vector is administered to the subject, the AdC7 vector comprising a fusion protein containing the amino acid sequence of SEQ ID NO:16 or an immunogenic fragment thereof. In some embodiments, the amino acid sequence of SEQ ID NO:16 or the immunogenic fragment thereof does not contain a signal peptide of the N-terminal 25 amino acids; or

[0148] - A vaccine comprising an AdC6 vector containing a fusion protein containing the amino acid sequence of SEQ ID NO:18 or an immunogenic fragment thereof is administered to the subject, and subsequently, a vaccine comprising an AdC7 vector containing a fusion protein containing the amino acid sequence of SEQ ID NO:18 or an immunogenic fragment thereof is administered to the subject. In some embodiments, the amino acid sequence of SEQ ID NO:18 or the immunogenic fragment thereof does not contain a signal peptide consisting of the N-terminal 25 amino acids.

[0149] This method may include administering to a subject an effective amount of a vaccine comprising an AdC7 vector, wherein the AdC7 vector comprises a fusion protein containing an amino acid sequence of any one of SEQ ID NO:14, 16, or 18, or an immunogenic fragment thereof. In some embodiments, the method further includes, after administering the vaccine comprising the AdC7 vector, administering to the subject a vaccine comprising an AdC6 vector, wherein the AdC6 vector comprises a fusion protein containing an amino acid sequence of any one of SEQ ID NO:14, 16, or 18, or an immunogenic fragment thereof. Such prime-boost methods may include:

[0150] - A vaccine comprising an AdC7 vector is administered to the subject, the AdC7 vector comprising a fusion protein containing the amino acid sequence of SEQ ID NO:14 or an immunogenic fragment thereof, and subsequently, a vaccine comprising an AdC6 vector is administered to the subject, the AdC6 vector comprising a fusion protein containing the amino acid sequence of SEQ ID NO:14 or an immunogenic fragment thereof. In some embodiments, the amino acid sequence of SEQ ID NO:14 or the immunogenic fragment thereof does not contain a signal peptide of the N-terminal 25 amino acids;

[0151] - A vaccine comprising an AdC7 vector is administered to the subject, the AdC7 vector comprising a fusion protein containing the amino acid sequence of SEQ ID NO:16 or an immunogenic fragment thereof, and subsequently, a vaccine comprising an AdC6 vector is administered to the subject, the AdC6 vector comprising a fusion protein containing the amino acid sequence of SEQ ID NO:16 or an immunogenic fragment thereof. In some embodiments, the amino acid sequence of SEQ ID NO:16 or the immunogenic fragment thereof does not contain a signal peptide of the N-terminal 25 amino acids; or

[0152] - A vaccine comprising an AdC7 vector containing a fusion protein containing the amino acid sequence of SEQ ID NO:18 or an immunogenic fragment thereof is administered to the subject, followed by a vaccine comprising an AdC6 vector containing a fusion protein containing the amino acid sequence of SEQ ID NO:18 or an immunogenic fragment thereof. In some embodiments, the amino acid sequence of SEQ ID NO:18 or its immunogenic fragment thereof does not contain a signal peptide consisting of the N-terminal 25 amino acids.

[0153] This method may include administering to a subject an effective amount of a vaccine comprising an AdC6 vector, wherein the AdC6 vector comprises a fusion protein containing the amino acid sequence of SEQ ID NO:185 or 187 or an immunogenic fragment thereof. In some embodiments, the method further includes, after administering the vaccine comprising the AdC6 vector, administering to the subject a vaccine comprising an AdC7 vector, wherein the AdC7 vector comprises a fusion protein containing the amino acid sequence of SEQ ID NO:185 or 187 or an immunogenic fragment thereof. Such prime-boost methods may include:

[0154] - The subject is provided with a vaccine comprising an AdC6 vector containing a fusion protein containing the amino acid sequence of SEQ ID NO:185 or an immunogenic fragment thereof, and subsequently, the subject is provided with a vaccine comprising an AdC7 vector containing a fusion protein containing the amino acid sequence of SEQ ID NO:185 or an immunogenic fragment thereof. In some embodiments, the amino acid sequence of SEQ ID NO:185 or an immunogenic fragment thereof does not contain a signal peptide consisting of 25 N-terminal amino acids; or

[0155] - A vaccine comprising an AdC6 vector containing a fusion protein having the amino acid sequence of SEQ ID NO:187 or an immunogenic fragment thereof is administered to the subject, and subsequently, a vaccine comprising an AdC7 vector containing a fusion protein having the amino acid sequence of SEQ ID NO:187 or an immunogenic fragment thereof is administered to the subject. In some embodiments, the amino acid sequence of SEQ ID NO:187 or an immunogenic fragment thereof does not contain a signal peptide consisting of the N-terminal 25 amino acids.

[0156] This method may include administering to a subject an effective amount of a vaccine comprising an AdC7 vector, wherein the AdC7 vector comprises a fusion protein containing the amino acid sequence of SEQ ID NO:185 or 187 or an immunogenic fragment thereof. In some embodiments, the method further includes, after administering the vaccine comprising the AdC7 vector, administering to the subject a vaccine comprising an AdC6 vector, wherein the AdC6 vector comprises a fusion protein containing the amino acid sequence of SEQ ID NO:185 or 187 or an immunogenic fragment thereof. Such prime-boost methods may include:

[0157] - A vaccine comprising an AdC7 vector is administered to the subject, the AdC7 vector comprising a fusion protein containing the amino acid sequence of SEQ ID NO:185 or an immunogenic fragment thereof, and subsequently, a vaccine comprising an AdC6 vector is administered to the subject, the AdC6 vector comprising a fusion protein containing the amino acid sequence of SEQ ID NO:185 or an immunogenic fragment thereof. In some embodiments, the amino acid sequence of SEQ ID NO:185 or an immunogenic fragment thereof does not contain a signal peptide of the N-terminal 25 amino acids; or

[0158] - A vaccine comprising an AdC7 vector containing a fusion protein having the amino acid sequence of SEQ ID NO:187 or an immunogenic fragment thereof is administered to the subject, and subsequently, a vaccine comprising an AdC6 vector containing a fusion protein having the amino acid sequence of SEQ ID NO:187 or an immunogenic fragment thereof is administered to the subject. In some embodiments, the amino acid sequence of SEQ ID NO:187 or an immunogenic fragment thereof does not contain a signal peptide consisting of the N-terminal 25 amino acids.

[0159] The immune response induced by the disclosed method includes, but is not limited to, T-cell responses, B-cell responses, or both (i.e., cellular and / or humoral immune responses). The immune response can be a primary or secondary immune response. Compared to HBVCore or polymerase antigen alone (i.e., without gD), the disclosed method can induce a greater degree of immune response against HBV in the subject.

[0160] The disclosed methods can be used for therapeutic treatment and preventative or preventative measures, and can reduce the severity and / or frequency of symptoms, eliminate symptoms and / or the underlying cause of symptoms, reduce the frequency or likelihood of symptoms and / or their underlying cause, and improve or remedy damage caused directly or indirectly by HBV. Treatment also includes prolonging survival compared to the expected survival of untreated subjects. Subjects to be treated include those with HBV, those susceptible to HBV, or those seeking HBV prevention.

[0161] The amount of the disclosed fusion protein, nucleic acid molecule, vector, or vaccine required to induce an immune response to HBV (e.g., the “effective amount”) can vary depending on factors such as the subject’s disease state, age, sex, and weight, as well as the ability of the fusion protein, nucleic acid molecule, vector, or vaccine to elicit the desired response in the subject. Exemplary indicators of the effective amount include, for example, improved health in the subject and reduction, elimination, or prevention of HBV symptoms.

[0162] It also provides the use of any disclosed fusion protein, nucleic acid molecule, vector, or vaccine in the preparation of a medicament for inducing an immune response against HBV in subjects.

[0163] It also provides disclosed fusion proteins, nucleic acid molecules, vectors, or vaccines for inducing an immune response to HBV in subjects.

[0164] Example

[0165] To further describe some of the embodiments disclosed herein, the following examples are provided. These examples are intended to illustrate, and not limit, the disclosed embodiments.

[0166] Epitope-optimized Core sequence generation

[0167] Based on phylogenetic clustering, hepatitis B virus (HBV) can be classified into several genotypes. To aid in the development of antigen inserts for multi-genotype HBV vaccines for patients with chronic infection, preliminary bioinformatics assessments were performed on the genes encoding HBV Core and HBV polymerase in genotypes A, B, C, and D.

[0168] Core amino acid sequences of four major HBV clades were downloaded from the Hepatitis B Virus Database (HBVdb) (release version 45.0; last updated August 2, 2018) as ClustalW sequences for alignment. The amino acid sequences represent thousands of HBV genomes input by European users, as summarized in the table below.

[0169] Table 1. Number of unique Core genomes analyzed

[0170]

[0171] First, use Los Alamos National Laboratory (Los Alamos National Laboratory) www.hiv.lanl.gov / content / sequence / ENTROPY / entropy The Shannon Entropy tool, developed by the director, identifies a “shared” core sequence for each genotype. This tool calculates the variation and frequency at each amino acid position. These calculations are repeated for each genotype, generating four “shared” core sequences, one for each analyzed genotype (SEQ ID NO: 1-4):

[0172] Common sequence for genotype A (SEQ ID NO:1)

[0173] MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLeDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC-

[0174] Common sequence for genotype B (SEQ ID NO:2)

[0175]

[0176] Genotype C common sequence (SEQ ID NO:3)

[0177]

[0178] Common sequence for genotype D (SEQ ID NO:4)

[0179]

[0180] (Bold residues with underlined text represent amino acids with a frequency of less than 90%).

[0181] The previously mentioned "shared" Core sequences were combined to generate epitope-optimized Core sequences. Conserved amino acids were identified at each amino acid residue of the Core protein from each genotype (A, B, C, and D), and frequencies and variations within a given genotype genomic sample were determined. To select amino acids at variant sites, each variant was tested across multiple HLA types using an epitope prediction algorithm, and the most immunogenic sequence was selected. Specifically:

[0182] (1) Each residue was kept identical across the four genotypes. Genomic weighted frequencies were also calculated to understand variability, and spacers were added where applicable to align sequence differences.

[0183] (2) Dissimilar residues in the four genotypes were identified, and the amino acid differences were documented (see Table 2). The initial core sequence (SEQ ID NO:5) is provided below, with the residues that are distinct in the four genotypes labeled X1–X. 11 Residues with a frequency below 90% are displayed in bold underlined font:

[0184]

[0185] Table 2. Different residues in the four genotypes

[0186]

[0187] (3) To determine the final amino acids at these positions, an epitope prediction algorithm was used to select appropriate amino acids. For amino acids exhibiting variability among genotypes, amino acids present in all three genotypes were selected, or the most immunogenic amino acids were selected using MHC class I epitope prediction software. This approach maximizes potential immunogenicity across the largest number of HLA types. The epitope-optimized core sequences for all genotypes and within genotypes are shown below (SEQ ID NO:6):

[0188] DIDPYKEFGATVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC

[0189] The mean variation at each site across all genomes was calculated, weighted by the number of clade-specific genomes analyzed, and higher and larger conserved regions and residues were shown. Figure 1 .

[0190] Epitope-optimized polymerase sequence generation

[0191] As discussed above regarding the Core sequence, epitope-optimized polymerase sequences were generated from the four major HBV clades. Because the polymerase is quite long, two fragments were generated—an N-terminal fragment (from which highly variable regions between genotypes were removed) and a C-terminal fragment. Both fragments are approximately 300 amino acids in length. The epitope-optimized polymerase amino acid sequences are shown below and in Table 9:

[0192] Epitope-optimized N-terminal amino acid sequence of HBV polymerase (SEQ ID NO:8):

[0193] PLSYQHFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFPKIHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWK AGILYKRETTRSASFCGSPYSWEQELQHGSCWWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGITRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHIPLHPAAMP

[0194] Epitope-optimized C-terminal amino acid sequence of HBV polymerase (SEQ ID NO:10):

[0195] HLLVGSSGLSRYVARLSSNSRIINHQHGTMQNLHDSCSRNLYVSLLLLYKTFGRKLHLYSHPIILKTKRWGYSLNFMGYVIGSWGSLPQDHIIQKIKECFRKLPVNRPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQSKQAFTFS PTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAMGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKILGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDPSRGRLGLSRPLLRLPFRPTTGRTSLYAVSPSV

[0196] Generation of AdC6 and AdC7 vectors with optimized core and polymerase sequences for expressing epitopes

[0197] Genes encoding epitope-optimized core or polymerase amino acid sequences were cloned into transfer vectors containing the herpes simplex virus (HSV) glycoprotein D (gD) sequence controlled by the CMV promoter. The genes were then cloned into replication-defective adenovirus vectors with E1 deletions and E3 ORF3, 4, 5, 6, and 7 deletions (as described in PCT / US2017 / 043315) to generate the following vectors:

[0198] • AdC6 (AdC6-gDCore) containing an epitope-optimized Core sequence fused to gD;

[0199] • AdC6 (AdC6-gDPolN) contains an epitope-optimized N-terminal polymerase sequence fused to gD;

[0200] • AdC6 (AdC6-gDPolC) contains an epitope-optimized C-terminal polymerase sequence fused to gD;

[0201] • AdC7 (AdC7-gDCore) containing an epitope-optimized Core sequence fused to gD;

[0202] • AdC7 (AdC7-gDPolN) containing an epitope-optimized N-terminal polymerase sequence fused to gD; and

[0203] • AdC7 (AdC7-gDPolC) contains an epitope-optimized C-terminal sequence of polymerase fused to gD.

[0204] Correct clones were identified by restriction enzyme digestion, and cloning sites were sequenced. Vectors were rescued and amplified in HEK 293 cells, purified by cesium chloride (CsCl) gradient centrifugation, and vector concentration (vp) was determined spectrophotometrically. After amplification in HEK 293 cells in a series of dilutions, the vectors were titrated against infectious units, followed by RNA isolation and reverse transcription, and hexon-specific nested PCR. Genetic integrity of the vectors was determined by restriction enzyme digestion followed by gel electrophoresis of the purified viral DNA. Protein expression was determined by Western blotting using a gD-specific antibody. Genetic stability was determined by serial passage of the vectors in HEK 293 cells (12–15) followed by restriction enzyme digestion and gel electrophoresis of the purified viral DNA.

[0205] Testing the immunogenicity of the vaccine in mice

[0206] C57Bl / 6, BALB / c, and HLA-A2 tg mice (n=5 per group) were injected with different concentrations of each of the above-mentioned vectors. Naïve mice served as controls. Blood samples were collected from the mice at different time points after injection, and insert-specific CD8+ staining was performed using intracellular cytokine staining (ICS) against IFN-γ. + and CD4 + T cell frequency. Two months after the first injection, mice were boosted with AdC6 immunization using a heterologous vector (AdC7) expressing the same insert. HBV-specific T cell frequency was tested again. Results after primitivism are shown in... Figures 2A-2F as well as Figure 3A (C57Bl / 6 mice) Figure 3B (BALB / c mice) and Figure 3C In (HLA-A2 mice). Enhanced results are shown in... Figures 4A-4C In 5A-5B.

[0207] C57Bl / 6 mice exhibited highly robust CD8 response to epitope-optimized N-terminal polymerase sequences. + T cell responses showed lower responses to epitope-optimized polymerase C-terminal sequences and epitope-optimized Core sequences, while CD4 showed higher responses to epitope-optimized Core sequences and epitope-optimized polymerase C-terminal sequences. + The response was good. Figures 2A-2F Epitope mapping in C57Bl / 6 mice showed a higher and broader response to PolN than to PolC. Figure 3A Within PolN, CD8 + T cells recognized a total of 14 peptides, but within PolC, only two adjacent peptides were recognized, which likely reflect an epitope. CD4 + T cells did not respond to PolN or PolC. This pattern was primarily observed in BALB / c mice, where CD8... + T cells responded most strongly to PolN, recognizing 12 peptides, followed by PolC, which recognized 4 peptides. Figure 3B The response to Core was low, but surprisingly broad, identifying 10 peptides (). Figure 3B ). BALB / c CD4 + T cells responded best to Core, recognizing 15 peptides, while recognition of PolC (4 peptides) or PolN (2 peptides) was lower. CD8 was also tested in HLA-A2 tg mice. + T cell response, in which PolN again triggered the highest response involving 12 peptides ( Figure 3C The response to PolC was lower but broader (16 peptides), while only one peptide from Core was detected. Figure 3C The sequences of the peptides tested in the primiparity assays are provided in Tables 3 (Core peptides), 4 (PolN peptides), and 5 (PolC peptides). Tables 6–8 provide the peptide composition from the peptide pools of the primiparity assays. Overall, these data indicate that the inserts elicited detectable T-cell responses, which in most cases targeted multiple epitopes within each sequence.

[0208] Table 3. Epitope-optimized Core peptides

[0209]

[0210]

[0211] B / c=BALB / c; Bl / 6=C57Bl / 6; HLA=HLA-A2

[0212] Table 4. Epitope-optimized PolN peptides

[0213]

[0214]

[0215] B / c=BALB / c; Bl / 6=C57Bl / 6; HLA=HLA-A2

[0216] Table 5. Epitope-optimized PolC peptides

[0217]

[0218]

[0219] B / c=BALB / c; Bl / 6=C57Bl / 6; HLA=HLA-A2

[0220] Table 6. Core Pools with Tablet Optimization

[0221] Core Matrix A B C D E F G 1 2 3 4 5 6 H 7 8 9 10 11 12 I 13 14 15 16 17 18 J 19 20 21 22 23 24 K 25 26 27 28 29 30 L 31 32 33 34 35

[0222] Table 7. Epitope-optimized PolN pool

[0223] Pol N matrix A B C D E F G H I 1 2 3 4 5 6 7 8 J 9 10 11 12 13 14 15 16 K 17 18 19 20 21 22 23 24 L 25 26 27 28 29 30 31 32 M 33 34 35 36 37 38 39 40 N 41 42 43 44 45 46 47 48 O 49 50 51 52 53 54 55 56 P 57 58 59

[0224] Table 8. Epitope-optimized PoLC pool

[0225]

[0226]

[0227] In C57Bl / 6, BALB / c, and HLA-A2 tg mice, the increased response after boosting was primarily observed at the insert and the dose of the carrier that induced a suboptimal response after primary immunization, i.e., for a dose of 1x102. 9 Core of the vector dose test for vp ( Figures 4A-4C Although booster immunization failed to increase the response to PolN or PolC when the carrier was injected at high doses, the booster still broadened the T-cell response. Figures 5A-5C ).

[0228] Immunogenicity summary

[0229] The above results indicate that:

[0230] • Vaccines are immunogenic: effective against CD8+. + For T cells, PolN > PolC > Core; for CD4 cells... +In terms of T cell response, Core > PolC > PolN;

[0231] Heterogeneous vaccine delivery vectors can enhance the immune response;

[0232] • Broad immune response; and

[0233] • The breadth of T cell responses increases after enhancement.

[0234] Effects of vaccination on HBV titer challenge with low-dose AAV-1.3HBV

[0235] Use 1x10 10 1x10 11 Or 1.5x10 11 Three mice were challenged with one dose of AAV-1.3HBV and then vaccinated with AdC6-gDPolN eight weeks later. Viral titers were tested eight weeks post-vaccination and compared with pre-vaccination titers. Figure 6 The changes in virus count for each treatment group relative to the baseline are shown.

[0236] Epitope shift

[0237] During chronic HBV infection, CD8 antigens are targeted. + T cells become exhausted. Compared to secondary dominant epitopes, CD8+ targeting dominant epitopes... + The progression of T cell exhaustion is faster and more pronounced. The fundamental reason is that exhaustion is driven by overwhelming antigen-driven stimulation via T cell receptors. Dominant epitopes are presented at higher levels on MHC class I antigens expressed by antigen-presenting cells compared to secondary dominant epitopes with lower affinity for their restricting elements. Typical vaccine approaches primarily induce an immune response against dominant epitopes. Therapeutic vaccines should take into account the loss of T cells targeting dominant epitopes during chronic viral infection and should be designed to facilitate the expansion of CD8+ targeting secondary dominant epitopes. + T cells with secondary dominant epitopes are more likely to resist disease-driven exhaustion, thus translating into better disease control.

[0238] Epitope profiles were determined in juvenile mice immunized with an adenovirus vector containing a nucleic acid sequence encoding the N-terminal domain (PolN) of HBV polymerase fused to herpes simplex virus glycoprotein D (“AdC6-gDPolN”, where the amino acid sequence of gDPolN is SEQ ID NO:16). The responses of mice not pretreated with the AAV8-1.3HBV vector were compared with those obtained in mice infected with an AAV8 vector expressing the 1.3HBV genome prior to inoculation with AdC6-gDPolN. The AAV8-1.3HBV vector induced high titers of HBV in serum, which may drive CD8... + T cell depletion.

[0239] In the first series of experiments, peptide pooling matrices were used to identify epitopes in mice inoculated with the AdC6-gDPolN vector but not challenged with the AAV-1.3HBV vector. Numerous epitopes eliciting potent responses (e.g., greater than 1% of CD8+ producing IFN-γ) were identified in these naïve mice. + CD44 + T cells, Figure 7A and Figure 8A The region is ). In the second experiment, 1x10 10 Mice were challenged with the AAV-1.3HBV vector containing one viral genome (vg), and 4 weeks later they were inoculated with the AdC6 vector [which expresses the same HBV polymerase sequence (gDPolN) as the unchallenged mice in the initial experiment]. Ten weeks later, HBV PolN-specific CD8+ was measured in spleen cells from mice that had been challenged before inoculation using a peptide pooling matrix. + T-cell epitope profile. Figure 7B and Figure 8B Using more stringent conditions, by using 1.5x10 11 Mice were challenged with a vg dose of AAV8-1.3HBV vector, and the experiment was repeated. Mice were vaccinated again after 4 weeks, and CD8 was tested approximately 10 weeks post-vaccination. + T cell response to peptide pool matrix. Figure 7C and Figure 8C In both experiments, a shift in the epitope profile was observed in AAV8-1.3HBV-infected mice compared to results obtained in unvaccinated mice, which had 10 [units of something] per milliliter of serum at the time of vaccination. 7 -10 9The effect was more pronounced in mice challenged with high doses of the AAV8-1.3HBV vector, with a high viral load of 1000 mg / vg. A reduced response was observed in both experiments. Furthermore, particularly in mice challenged with high doses of AAV8-1.3HBV, results showed a significant reduction in the CD8 response, targeting numerous epitopes (which exhibited an immune advantage in vaccinated, uninfected mice). + The loss of T cells (e.g., in the region represented by peptides 50–59, Fig. 8), the subdominant epitopes (e.g., those in the region represented by peptides 2–8), and the better retention of novel epitopes, such as in the region represented by peptides 10–29. These data confirm a shift from recognizing dominant epitopes to recognizing subdominant epitopes.

[0240] Based on these data, Sheng Chen developed a new N-terminal domain insert for HBV polymerase (HBV PolN v2) (SEQ ID NO:173):

[0241] HFRKLLLLDEEAGPLEEELPRLADEGLNRRRVAEDLNLGNLPEWQTPSFPKIHLQEDIVDRCKQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIKPYYPEHAVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGSCWLQFRNSKPCSEYCLTHLVNLLEDWGPCDEHGEHHIRIPRTPARVT

[0242] This insert primarily induces CD8 + The T cell response to secondary dominant epitopes remains intact in mice with high HBV viral load.

[0243] Immunogenicity and efficacy of gDCore, gDPolN and gDPolC vaccines

[0244] The immunogenicity and efficacy of the AdC6-gDCore, AdC6-gDPolN, AdC6-gDPolC, AdC7-gDCore, AdC7-gDPolN, and AdC7-gDPolC vaccines in the AAV8-HBV mouse model were analyzed.

[0245] Methods - Immunogenicity

[0246] C57Bl / 6 mice (n=5 per group) were injected with different doses of the following substances: AdC6-gDCore (corresponding to the gDCore nucleic acid sequence of SEQ ID NO:15), AdC6-gDPolN (corresponding to the gDPolN nucleic acid sequence of SEQ ID NO:17), or AdC6-gDPolC (corresponding to the gDPolC nucleic acid sequence of SEQ ID NO:19). Two months after the first injection, mice immunized with AdC6 vectors were boosted with AdC7 vectors containing the same inserts (e.g., AdC7-gDCore, AdC7-gDPolN, or AdC7-gDPolC). Blood was collected from mice at 14 and 56 days post-injection, and T cell frequencies against various HBV inserts were analyzed by intracellular cytokine staining (ICS) with interferon (IFN)-γ after cell stimulation with overlapping peptides representing HBV sequences. Control cells were cultured in the absence of peptides. CD8 was tested by staining with MHC I tetramers. + Frequency and phenotype of T cells targeting an immunodominant epitope within PolN. Epitope mapping via spleen cells (CD8+ as measured by ICS of IFN-γ). + T cells) perform CD8 + The breadth and specificity of T cell responses to individual peptides within the target sequence.

[0247] To assess CD8 in the liver + T cells were administered to C57Bl / 6 mice (n=8 per group) via tail vein at a dose of 1 x 10⁻⁶. 10 AAV8-1.3HBV with 1 x 10 viral genomes (vg) 11 Administer AAV8-1.3HBV intravenously, or without administration, after 4 weeks, 5x10 9 A single IM injection of AdC6-gDPolN containing one viral particle (vp) was administered. Eight weeks after the IM injection, mice were sacrificed, livers were removed, lymphocytes were isolated and stained with T-cell markers and tetramers that recognize T-cell receptors targeting the immunodominant epitopes present in the PolN sequence.

[0248] In separate experiments, three groups of C57Bl / 6 mice (n=4 per group) received 5 x 10⁻⁶ doses at week 4. 9 A single IM injection of AdC6-gDPolN of vp (-), or 4 weeks later, with or without 5x10 9 In the case of a single IM injection of AdC6-gDPolN in vp, 1x10 is administered via tail vein. 11Mice were intravenously administered AAV8-1.3HBV, containing one viral genome (vg). Approximately two months after AAV8-1.3HBV administration, mice were sacrificed, and livers were harvested. Liver sections were prepared from each of the three groups, stained with hematoxylin and eosin, and lymphocyte infiltration was assessed. In the same experiment, cells were stained with antibodies against T-bet specific tetramers and fluorescently labeled (clone 4B10, BV785), or antibodies against PD-1 (clone 29F.1A12, BF605), TIM-3 (clone RMT3-23, Pe / Cy7), CTLA-4 (clone UC10-4B9, PE), or LAG-3 (clone C9B7W, BV650). Cells were analyzed by flow cytometry and their activity on CD44. + Gating was performed on CD8 tetramer-positive cells, and then gating was performed on the biomarker. The percentage of biomarker-positive cells was identified from the histogram compared to naive T cells.

[0249] Methods - Efficacy

[0250] AAV8-1.3HBV Vector Study—To evaluate the effect of AdC6-gDPolN on chronic HBV exposure, 1x10⁻¹⁰ HBV vectors were used. 10 One vg of AAV8-1.3HBV was administered intravenously to C57Bl / 6 mice (n=8 per group) via the tail vein, and after 4 weeks, the mice were administered a single intravenous injection of 5 x 10 HBV. 9 Immunization with AdC6-gDPolN was performed on vp. HBV DNA viral titers were assessed by qPCR; changes in pre- and post-vaccination levels relative to baseline were reported (log). 10 (Copies / mL). Viral genome copy number was assessed at 4, 6, 8, 10, and 12 weeks post-AAV8 challenge. Viral kinetics over time were assessed by PCR, and log10 changes per mL of HBV copies were evaluated. The number of mice exhibiting 1, 2, or 3 log reductions at different time points post-treatment was assessed.

[0251] Over time, chronic HBV exposure affects CD8 + The impact of AAV8-1.3HBV on vaccine-induced liver CD8 antigen recognition was evaluated. + The effect on T cells. Four weeks after vaccination, the effect was measured after a single IM injection of 5x10 [units of technology / method / etc.]. 9 Epitope profile in spleen cells of naïve mice immunized with AdC6-gDPolN. Mice, which were immunized with 1x10 10 and 1.5x10 11 Av8-1.3HBV attack of 1 vg followed by vaccination with 5x10 vg 4 weeks later. 9vp's AdC6-gDPolN, 10 weeks post-vaccination (14 weeks post-AAV injection), showed CD8+ in spleen cells. + T-cell epitope profiling was performed. Epitope profiles were compared between AAV-naïve and AAV-treated vaccinated animals. PolN-specific CD8+ from the liver was analyzed. + T cell differentiation markers.

[0252] result

[0253] Immunogenicity - Vaccination induced CD8 + T cells exhibit robust and sustained responses to PolN (relative to all circulating CD8). + The median frequency of T cells was 6.0% and the median frequency of responses to PolC and Core was 1.0% and 0.4%, respectively. Figure 9A and Figure 9B The intensification performed in week 8 increased the response in all regions, with significant changes observed for the Core (p = 0.007). Figure 9C ). Figures 9A-9C Showing all CD8 values ​​relative to each mouse + %CD8% of T cells + T cells, median, are represented by the line. The booster further enhanced vaccine-induced CD8 cell counts. + Extensive epitope recognition by T cells (27% to 34%); Figure 10 ).

[0254] Twelve weeks after AdC6-gDPolN vaccination, vaccine-treated mice infected with AAV8-1.3HBV showed liver CD8 abnormalities. + Preferential increase of infiltration ( Figures 11A-11B and Figure 12A-12F ), vaccine-induced HBV-specific CD8 + The presence of T cells is reduced ( Figure 11A and Figure 11B ) and T-bet levels were slightly reduced (suggesting loss of effector function) Figures 13A-13B ). Figure 11A The percentage of CD8 in all recovered lymphocytes from each liver is shown. + T cells. Figure 11B It showed a tetramer-positive CD8 + The percentage of cells, identified from a histogram compared to naive T cells. However, no clear pattern of cellular markers indicative of T cell differentiation into an exhausted phenotype was observed between vaccinated AAV1.3HBV-infected and uninfected mice. Figures 13A-13B ).

[0255] effect- Following a single IM injection of the AdC6-gDPolN vector, serum HBV DNA levels in AAV8-1.3HBV-infected mice showed a multiple logarithmic decrease, which persisted throughout the 8 weeks post-vaccination (Figure 14). The median decrease in serum HBV DNA viral load at 4 and 8 weeks post-vaccination was 0.86 and 2.69 log, respectively. 10 cps / mL ( Figure 14A At week 8, all animals had >1 log [value missing] relative to baseline. 10 The decrease in cps / mL was observed in 6 / 7 (86%) of the animals with >2 log 10 The decrease in cps / mL was observed in 2 / 7 (29%) of the animals with >3 log 10 The decrease in cps / mL ( Figure 14B ).

[0256] Following a single injection of the AdC6-gDPolN vector, when comparing AAV-HBV-infected mice and juvenile mice, different CD8 responses to the PolN peptide were observed in spleen cells. + T-cell recognition patterns. Figure 15A and Figure 15B This illustrates the results from the experiment, in which mice were first injected with AAV-1.3HBV, and then 10 mg / L was administered 4 weeks later. 10 The adC6-gDPolN vector of vp was boosted, and spleen cells were harvested 8 weeks post-immunization. After brief in vitro stimulation with various peptides spanning the PolN sequence, the cells were tested via ICS targeting IFN-γ. Background frequencies obtained without the peptides were subtracted. Figure 15A The peptide recognition profiles of mice that received only the AdC6-gDPolN vaccine are shown, followed by those mice that were first injected with the indicated dose of the AAV8-1.3HBV vector. Figure 15B The pie chart in the image shows the results for all CD44. + CD8 + The corresponding response of peptides at the 0.1% threshold of cells (data corresponding to) Figure 15A (Those in the image). Each small patch / color represents the frequency of response to individual peptides, and the size indicates the total proportion; only responses greater than 0.1% are included. Pull-out indicators are epitopes recognized only in mice infected with AAV8-1.3HBV. Pretreatment with AAV was found to reduce both the number of epitopes recognized after a single IM priming and the number of CD8 cells that generate IFN-γ. + The total number of T cells relative to CD8 + The magnitude of the immune response in the T cell pool. AAV pretreatment shifts T cell recognition to a new epitope, which accounts for approximately [percentage missing] of detectable CD8 [cells missing]. +One-third of the T cell response. Functional HBV-specific CD8. + The percentage of T cell responses was highest in juvenile mice (4.4%). Figure 15B However, it decreased in the presence of low and high doses of AAV8-1.3HBV (by 2.0% and 0.6%, respectively). Figure 15B Animals uninfected with AAV8-1.3HBV showed strong CD8 response to many epitopes. + T cell responses, which are reduced and transformed in AAV-HBV-infected animals, include T cell recognition of novel epitopes.

[0257] discuss

[0258] A checkpoint inhibitor targeting early CD8 was generated using gD as the gene encoding. + A T-cell activated HBV therapeutic vaccine, and its efficacy has been demonstrated:

[0259] • Inducing CD8 + Effective and durable T cell response to key HBV antigens (Figure 9);

[0260] • Stimulates a very broad CD8+ T cell response ( Figure 10 ), including secondary dominant epitope identification (Figure 15); and

[0261] • Achieving sustained multi-log reduction in HBV DNA viral load in an AAV mouse model (Figure 14), with preferential reduction of functional CD8+ viral load. + T cells are transported to the liver (Figures 11 and 12).

[0262] In publicly available AAV studies, AAV-induced HBV infection led to CD8+ infection after AdC6-gDPolN inoculation. + T cells lose recognition of the dominant epitope of PolN (Figure 15). Without being bound by theory, it is believed that this loss is caused by gD-induced CD8... + The breadth of T cells and their ability to recognize secondary dominant epitopes lead to a sustained immune response and multiple logarithmic suppression of HBV.

[0263] Immunogenicity of AdC6 / 7-gDPolN in blood and liver of AAV-induced HBV-infected animals after vaccination

[0264] The following studies were conducted to evaluate CD8 levels in the blood, spleen, and liver of animals in the presence of pre-existing AAV-induced HBV infection. + T cell response to the AdC6-gDPolN vaccine.

[0265] Experiment #1 - AAV8-1.3HBV infection of CD8 in mice +T-cell response: Reaction kinetics in the blood

[0266] Objective – To evaluate the effect of sustained HBV antigen titers on CD8. + The effect of T cell response to gDPolN antigen expressed in the AdC6 vector.

[0267] method -Administer 10 mg IV injections to C57Bl / 6 mice 10 The AAV8-1.3HBV vector was used. Four weeks later, they were inoculated with 5 x 10-1 9 The vaccine was administered via the AdC6-gDPolN vector. Control mice received only the AdC6-gDPolN vector. Naïve mice served as an additional control. Two months later, mice were boosted with the same dose of the AdC7-gDPolN vaccine. Blood was collected at different time points after the initial and booster immunizations, and PBMCs were tested for IFN-γ-producing CD8+ T cells.

[0268] result -like Figure 16 As shown, mice exhibited strong PolN-specific CD8 uptake two weeks after vaccination. + T-cell responses gradually declined by week 8, then increased again after a booster. The booster-mediated CD8+ T-cell response was more stable than that after primary immunization. At most time points, mice injected with the AAV8-1.3HBV vector showed lower test responses than the uninjected control group.

[0269] Experiment #2 - AAV8-1.3HBV infection of CD8 in mice + T cell response: Response in the liver

[0270] Purpose –Evaluate CD8 in the livers of vaccinated mice infected with AAV8-1.3HBV. + T cell responses, including markers indicating T cell exhaustion.

[0271] method -Administer 10 mg IV injections to C57Bl / 6 mice 10 Or 10 11 The AAV8-1.3HBV vector was used to inoculate vg. After 4 weeks, they were inoculated with 5 x 10⁻⁶ cells / mL. 9 The vp-based AdC6-gDPolN vector. Control mice received only the AdC6-gDPolN vector. Naïve mice served as an additional control. Two months later, mice were boosted with the same dose of the AdC7-gDPolN vaccine.

[0272] To obtain hepatic lymphocytes, liver fragments were cut into small pieces and treated in L15 with 2 mg / ml collagenase P, 1 mg / ml DNase I (both from Roche, Basel Switzerland), and 2% FBS (Tissue Culture Biologicals, Tulare, CA) for 1 hour with stirring. The liver fragments were homogenized, filtered through a 70 μm filter, purified by Percoll gradient centrifugation, and washed with DMEM supplemented with 10% FBS. Lymphocytes were stained in the dark at +4°C for 30 min with the following staining agents: Violet Live / Dead Dye (Thermo Fisher Scientific), anti-CD8-APC (clone 53-6.7, BioLegend), anti-CD44-Alexa Flour 700 (clone IM7, BioLegend), and anti-EOMES-Alexa. Fluor488 (clone Dan11mag, eBioscience), anti-PD1-BV605 (clone 29F.1A12, BioLegend), anti-LAG3-BV650 (clone C9B7W, BioLegend), anti-T-bet-BV786 (clone 4B10, BioLegend), anti-CTLA-4-PE-A (clone UC10-4B9, BioLegend), anti-TIM-3-Pe-Cy7-A (clone RMT3-23, BioLegend), and APC-tagged MHC class I tetramers corresponding to amino acids 396-404 of HBV polymerase, namely FAVPNLQSL (SEQ ID NO:188) (peptide 55), were analyzed (NIHtetramer Facility, Emory University, Atlanta GA). Cells were washed and analyzed using BD FACS Celesta (BD Biosciences, San Jose, CA) and DiVa software. Post-acquisition analysis was performed using FlowJo(TreeStar,Ashland,OR).

[0273] result -Analysis of CD8 in lymphocyte liver infiltrates + The frequency of T cells. Compared to juvenile mice, vaccinated mice showed higher levels of CD8+ in liver infiltrates from lymphocytes. + The frequency of T cells increased, and a further increase was observed in mice that had been injected with the AAV8-1.3HBV vector prior to vaccination. Figure 17AIn mice injected with AAV-1.3HBV, PolN-specific CD8 was identified by staining with tetramers that specifically target epitopes present in the PolN insert. + The frequency of T cells decreased ( Figure 17B ).

[0274] By measuring the average fluorescence intensity of the dye conjugated with a given antibody ( Figures 18A-18F ), and by evaluating CD8 that are positive for the indicated markers. + Percentage of T cells ( Figures 19A-19F To evaluate the infiltrating tetramer + CD8 + T cells and immature CD8 + T cells (i.e., tetramers) - CD44 - CD8 + Phenotype compared to T cells.

[0275] Compared with the vaccine-only group, mice injected with AAV8-1.3HBV before vaccination had significantly higher liver CD8 counts. + On T cells, many CD8 cells are controlled. + T-betting of T cell function was reduced. No increase in exhaustion markers was observed in the AAV8-1.3HBV pretreatment group, suggesting that PolN-specific CD8+ is less effective in the presence of HBV. + The loss of T cells is unlikely to be due to classical CD8. + T cell exhaustion caused by ( Figures 18A-18F , Figures 19A-19F ).

[0276] Experiment #3 - PolN-specific CD8 in HBV-infected mice + Breadth of T cell response

[0277] Purpose -Assess whether the presence of HBV affects CD8. + The breadth of T cell response to PolN expressed in gD by the AdC vaccine.

[0278] method - Administer 10 via IV injection to mice 10 Or 10 11 The mice were administered the AAV8-1.3HBV vector and boosted with the corresponding AdC7 vector after 2 months. Control mice received only the AdC6-gDPolN vector. Mice were euthanized after 10 weeks, and pooled spleen cells were tested for peptide pooling in non-AAV-infected animal studies. Results are provided in Figures 20A-20C middle.

[0279] In the second experiment, mice were injected intravenously with 10 10 Or 10 11 The AAV8-1.3HBV vector was used to inoculate vg. After 4 weeks, they were inoculated with 5 x 10⁻⁶ cells / mL. 10 The vp-based AdC6-gDPolN vector. Control mice received only the AdC6-gDPolN vector. Naïve mice served as an additional control. After 6 weeks, CD8+ IFN-γ was generated in response to the production of individual peptides across the PolN sequence. + T cells were analyzed in spleen cells. Results are provided in […]. Figures 20D-20F middle.

[0280] result - The presence of HBV, especially high titers, such as in the injection of 10 11 After receiving a dose of VG AAV8-HBV1.3, not only did it reduce CD8 + The overall response of T cells to the PolN sequence presented by the AdC6-gDPolN vaccine also leads to a shift in the epitope recognition spectrum.

[0281] Experiment #4 – Liver PolN-specific CD8 in AAV8-1.3HBV-infected mice + T cell function

[0282] Purpose –Assess liver-invasive PolN-specific CD8 + Do T cells retain function in mice infected with AAV8-1.3HBV?

[0283] method - In the first experiment, C57BL / 6 mice were intravenously injected with 3x10 11 One group received 1.3 HBV AAV8. Eight weeks later, one group received 5 x 10 doses. 10 The vp-based AdC6-gDPolN vector. Another group was unvaccinated. Mice were euthanized after 4.5 months, and CD8+ receptors targeting the PolN peptide pool were used to produce IFN-γ. + The frequency of T cells was measured in spleen cells.

[0284] In the second experiment, mice were injected with graded concentrations of AAV8-1.3HBV (1x10⁻¹⁰). 10 4x10 10 Or 1x10 11 Four weeks later, all mice were vaccinated with 5x10 10The mice were given the AdC6-gDPolN vector. Two months later, they were boosted with the same dose of the AdC7-gDPolN vector. Two months later, the mice were euthanized, and lymphocytes were isolated from the liver. CD8 cells targeting the IFN-γ production pool in response to the PolN peptide pool were then analyzed. + T-cell testing was used to assess lymphocytes. Cells were also stained with an antibody against Tox, a transcription factor that increases in exhausted T cells.

[0285] result -As shown in Figure 21, vaccine-induced CD8 + T cells remained functional in mice injected with the AAV8-1.3HBV vector.

[0286] Experiment #5 - Effects of vaccination on liver histology in mice infected with AAV8-1.3HBV

[0287] Purpose - Evaluate whether AdC6 / 7-gDPolN vaccination in AAV.8-1.3HBV-inoculated mice leads to persistent liver injury.

[0288] method - Administer 10 via IV injection to mice 10 One vg of AAV8-1.3 HPV. One month later, they were vaccinated with 5 x 10 9 The mice were treated with the AdC6-gDPolN vector. Two months later, they were boosted with the same dose of the AdC7-gDPolN vector. The mice were euthanized approximately two months later. Liver sections were collected and fixed in 10% formaldehyde. Sections (approximately 3 μm thick) were prepared and stained with hematoxylin and eosin (H&E). They were examined under an optical microscope at 20x magnification.

[0289] result - In 33 slices from mice that received both the AAV vector and the vaccine, one showed a small lymphocyte infiltration located at the edge of the liver slice.

[0290] like Figure 21B As shown, in HLA-A2-tg mice, following a single dose of gDPolN vaccination, compared to mice vaccinated only, mice receiving AAV produced IFN-γ-producing liver CD8+. + The frequency of T cells is reduced.

[0291] in conclusion

[0292] • In mice infected with AAV8-1.3HBV, CD8 + T cells respond less to PolN. Nevertheless, they are still detectable.

[0293] • In animals pretreated with AAV8-1.3HBV, no increase was observed in exhaustion markers, suggesting that PolN-specific CD8 was not observed in the presence of HBV. + The loss of T cells is unlikely to be due to classical CD8. + Caused by T cell depletion.

[0294] AAV-induced HBV infection leads to CD8 + The epitope recognition spectrum of T cell responses to PolN undergoes a shift.

[0295] • Vaccine-induced CD8 + T cells remained functional in mice previously infected with the AAV8-1.3HBV vector.

[0296] • The vaccine used in the primary immunization booster regimen did not cause significant liver damage in HBV-positive mice.

[0297] Generation of HBV PolN-PolC-Core builders

[0298] Two multi-antigen inserts (second-generation PolN-PolC-Core and third-generation PolN-PolC-Core) were generated. The sequences of these inserts are shown below:

[0299] Second-generation HBV vaccine insert (“HBV2”) (Pol N (italic)-Pol C (underline)-Core) (SEQ ID NO:174)

[0300]

[0301] Third-generation HBV vaccine insert (“HBV3”) (Pol N (italic)-Pol C (underline)-Core) (SEQ ID NO:175)

[0302]

[0303]

[0304] The second-generation HBV (“HBV2”) insert includes immunodominant PolN epitopes identified in mice that were not infected with the AAV8-1.3HBV vector before vaccination. In mouse models of chronic HBV infection induced by pre-administration of the AAV8-1.3HBV vector (as defined in “Epitope Shifts” above), numerous epitopes were lost. The third-generation HBV (“HBV3”) insert selected contiguous regions of PolN that were preferentially recognized by mice with high HBV loads (see above). Regions of Core and PolC were selected for both constructs using the following protocol: regions exhibiting the highest immune response in either the primary (Fig. 3) or booster (Fig. 5) regions in C57Bl / 6, BALBc, and HLA-A2 tg mice, with the aim of selecting large, contiguous regions rather than unique epitopes, and inserting spacer sequences between them.

[0305] Genetic integrity and stability of second- and third-generation HBV inserts (HBV2 and HBV3)

[0306] Protein blot - The ability of purified recombinant viral vector formulations (AdC6-gDHBV2, AdC6-gDHBV3, AdC7-gDHBV2, and AdC7-gDHBV3) to induce transgenic product expression in vitro was evaluated. For this purpose, Western blot analysis was performed to assess gD protein expression in cell lysates after cell cultures were infected with the target vectors. HEK293 adherent cell monolayers were infected with known amounts of the purified vectors and harvested 48 hours post-infection. Cells were resuspended in lysis and extraction buffer containing protease inhibitors and lysed by sonication. Total protein extracts were denatured using dithiothreitol as a redox agent and subjected to electrophoresis on a 12% Bis-Tris polyacrylamide gel (PAGE). After protein separation by SDS-PAGE, samples were transferred to activated polyvinylidene fluoride membranes via wet electrophoresis. The membrane was immunostained for 1 hour at room temperature with gD primary antibody (clone PA1-30233, Invitrogen, Carlsbad, CA) diluted 1:1000 in saline to detect gD protein. The membrane was washed with 1X TBS-T and then incubated for 1 hour at room temperature with HRP-conjugated goat anti-rabbit secondary IgG (ab6721, Abcam, Cambridge UK). A luminol-based chemiluminescent substrate was then added. The stained membrane was exposed to autoradiographic film, and signal emission was assessed after processing in an automated film developer. After recording gD protein expression in lysates of infected HEK293 cells, the membrane was peeled off, and the presence of β-actin in the total protein extract sample was re-probeed. This staining step was used to assess the consistency of the PAGE loading procedure, thus better supporting the semi-quantitative analysis of gD protein expression stimulated in vitro by recombinant viral vectors.

[0307] stability To ensure the genetic integrity of the viral constructs, the genetic stability of each recombinant viral vector lot was assessed through successive viral passages in HEK293 adherent cell cultures. The recombinant viral pools generated from each transfection were cultured for a total of 12 passages under standard growth conditions. In the final passage, the viral pools were expanded, and the crude harvest was purified using a cesium chloride gradient. After vector purification, viral DNA was isolated using the QIAGEN DNeasy Blood & Tissue Kit and evaluated by restriction enzyme digestion with AseI and BglII, two restriction enzymes that cleave the DNA template with different construct-specific predefined banding patterns. After digestion, samples were subjected to electrophoresis on a 1% agarose gel containing ethidium bromide to visualize the digested bands, and the results were recorded using a digital gel imaging system. Viral formulations exhibiting the same banding pattern as those in earlier passages were considered to have maintained the initial molecular clonal structure and were therefore considered stable at the end of 12 viral passages.

[0308] result - The banding pattern of the viral vector DNA remained stable after 12 passages, compared to 5 passages, indicating that the vector genome was stable (data not shown).

[0309] Immunogenicity of second- and third-generation HBV inserts (HBV2 and HBV3) expressed in AdC6 or AdC7 vectors

[0310] Purpose -Evaluate CD8 + T cell response to HBV2 and HBV3 inserts expressed in the AdC6 or AdC7 vector.

[0311] method - Inject 5x10 mmol / L of C57Bl / 6 mice 9 Or 5x10 10 The HBV2 or AdC6-gDHBV3 vector was used. Mice injected with the same dose of the AdC6-gDPolN vector served as positive controls; juvenile mice served as negative controls. Blood was collected from mice 14 days later, and CD8+ IFN-γ was generated in response to the peptide pool corresponding to the HBV insert. + T cell frequency was measured in PBMCs. Four weeks later (six weeks after vaccination), blood was collected again from mice and tested for PolN-specific tetramers. AdC6-gDHBV3-immunized mice were excluded because this insert lacked the epitope corresponding to the tetramer.

[0312] C57Bl / 6 mice were injected with 5x10 9Or 5x10 10 vp's AdC7-gDHBV2 or 5x10 10 The adC7-gDHBV3 vector of vp. Naïve mice were used as a negative control. Blood was collected from mice 14 days later, and CD8+ IFN-γ was generated in response to the peptide pool corresponding to the HBV insert. + T cell frequency assay PBMC.

[0313] AdC7 primary immunization / AdC6 enhanced immunogenicity

[0314] Approximately 4 weeks later, blood was collected from mice, and CD8 inhibitors were used to target the production of IFN-γ and / or TNF-α in response to the intercalation peptides. + T cells were used to retest PBMCs via ICS. Two months after priming, mice were boosted with a heterologous vector expressing the same insert at the same dose. PBMCs were tested by ICS two weeks later, and CD8+ levels were compared before and after booster. + and CD4 + T cell response. Following primary immunization, the AdC7-gDHBV2 vector induces the production of IFN-γ and / or TNF-α by CD8+ cells. + The robust frequency of T cells increased after AdC6-gDHBV2 enhancement, which was also observed after low carrier doses and for CD8 cells producing IFN-γ. + T cells were particularly affected. The AdC7-gDHBV3 vector had poor immunogenicity, but after enhancement with AdC6-gDHBV3, CD8 cells showed significant improvement. + T-cell responses become positive. Similarly, CD4+ responses increase after primary immunization. + T cell responses were negligible but increased after enhancement. CD4 responses to HBV2 or HBV3 inserts showed no significant difference.

[0315] in conclusion

[0316] Both the AdC6-gDHBV2 and AdC7-gDHBV2 vectors are highly immunogenic. Figure 22A , Figure 22B and Figure 23 Furthermore, the response increased after enhancement with a heterologous AdC vector expressing the same insert (Fig. 24).

[0317] The AdC7-gDHBV2 and AdC7-gDHBV3 vectors exhibited critical immunogenicity consistent with their design because they lacked epitopes corresponding to the tetramers used. Figure 22A and Figure 23 ).

[0318] • Enhance CD8 with AdC7-gDHBV2 using AdC6-gDHBV2 + T cell response.

[0319] Comparison of HBV DNA viral titers in mice infected with AdC6-gDPolN, AdC6-gDHBV2, AdC6-gDHBV3, or AdC6-HBV2 AAV

[0320] method

[0321] Use 1x10 9 Five groups of C57Bl / 6 mice were challenged with AAV8-1.3HBV at a dose of 1 g each, and 4 weeks later, they were vaccinated with 1 x 10 g of AAV8-1.3HBV. 10 The viral loads included AdC6-gDPolN (n=10), AdC6-gDHBV2 (n=10), AdC6-gDHBV3 (n=10), or AdC6-HBV2 without gD (n=10); AAV-infected, unvaccinated animals (“naïve”) (n=10) and non-AAV-infected, unvaccinated animals (n=2–5) as controls. Viral titers were tested 4 weeks after AAV injection (before vaccination) and compared with levels 4 weeks after vaccination (8 weeks after AAV injection).

[0322] result

[0323] At week 8, the median HBV viral titer increased by 0.98 log in naïve mice. 10 The cps / mL remained unchanged in mice inoculated with AdC6-HBV2, but decreased by -0.04, -1.09, and -2.13 log in animals inoculated with AdC6-gDHBV3, AdC6-gDPolN, and AdC6-gDHBV2, respectively. 10 cps / mL ( Figure 25A Results for each mouse showed... Figure 25B In China, all animals vaccinated with AdC6-gDPolN and AdC6-gDHBV2 showed more than 1 and 2 log, respectively. 10 A decrease in copies / mL; conversely, no 1 log [value] was observed in nausea, animals vaccinated with AdC6-HBV2, or animals vaccinated with AdC6-gDHBV3 at week 8. 10 A decrease in copies / mL or a greater decrease.

[0324] Immunogenicity studies of gDHBV2 and gDHBV3

[0325] The CD8+ activity of fragments of HBV Core and polymerase contained in gDHBV2 or gDHBV3 was evaluated after a single primordial immunization or a booster immunization with a heterologous vector containing the same insert. + Induction and extent of T cell responses.

[0326] Experiment 1

[0327] Purpose: IFN-γ was evaluated in C57Bl / 6 mice following primary and booster vaccination with gD-HBV2 and gD-HBV3 expressed by heterologous chimpanzee adenovirus vectors (AdC6 and AdC7). + CD8 + T cell response.

[0328] method Four groups (n=5 per group) of C57Bl / 6 mice were immunized by intramuscular injection as follows: (a) 5 x 10 10 vp's AdC7-gDHBV2, after two months, is 5x10 10 vp's AdC6-gDHBV2; (b) 5x10 9 vp's AdC7-gDHBV2, after two months, is 5x10 9 vp's AdC6-gDHBV2; (c) 5x10 10 vp's AdC7-gDHBV3, after two months, is 5x10 10 vp AdC6-gDHBV3; or (d) no vaccine. IFN-γ levels in blood were assessed by ICS at 2 and 6 weeks after initial immunization, before booster, and 2 and 4 weeks after booster. + CD8 + T cell response.

[0329] result: At all time points during testing, each vaccine construct was found to induce IFN-γ. + CD8 + T cells. Figure 26 shows parental CD8 cells that produce IFN-γ and / or TNF-α. + T cells ( Figure 26A CD44 + CD8 + T cells ( Figure 26B CD4 + T cells ( Figure 26C ) or CD44 + CD4 + T cells ( Figure 26D The percentage of ) is shown. Immune responses of individual mouse PBMCs, as assessed by ICS, are shown as mean values ​​at 2 and 8 weeks after primary immunization and at 2 and 4 weeks after booster immunization.

[0330] Experiment 2

[0331] PurposeIn C57Bl / 6 mice, heterologous chimpanzee adenovirus vectors (AdC6 and AdC7) were used to compare IFN-γ levels after primary and booster vaccination with different doses of gD-HBV2 and gD-HBV3 versus gD-PolN. + CD8 + T cell response.

[0332] method Immunize the C57Bl / 6 mouse group (n=5 mice / group) as follows:

[0333] gDPolN group

[0334] (a) 5x10 9 vp's AdC6-gDPolN, 5x10 after three months 9 vp's AdC7-gDPolN; and

[0335] (b)5x10 10 vp's AdC6-gDPolN, 5x10 after three months 10 vp's AdC7-gDPolN

[0336] gDHBV2 group

[0337] (c)5x10 9 vp's AdC6-gDHBV2, 5x10 after three months 9 vp's AdC7-gDHBV2 and;

[0338] (d)5x10 10 vp's AdC6-gDHBV2, 5x10 after three months 10 vp's AdC7-gDHBV2

[0339] gDHBV3 group

[0340] (e)5x10 9 vp's AdC6-gDHBV3, 5x10 after three months 9 vp's AdC7-gDHBV3 and;

[0341] (f)5x10 10 vp's AdC6-gDHBV3, 5x10 after three months 10 vp's AdC7-gDHBV3

[0342] No treatment was used as a control.

[0343] For all treatment groups, immunogenic CD8 +T-cell responses were elicited by ICS targeting IFN-γ at 2 and 6 weeks post-primary immunization, before booster, and 2 and 6 weeks post-booster. + Blood samples were evaluated. Immunogenicity was also assessed at week 4 post-primary immunization using tetramer staining with APC-labeled MHC class I tetramer (NIH tetramer Facility, Emory University, Atlanta GA), which corresponds to amino acids 396-404 of HBV polymerase, namely FAVPNLQSL (peptide 55). HBV3 does not contain the FAVPNLQSL peptide.

[0344] result: At all time points, each tested vaccine was found to induce IFN-γ. + CD8 + T cells. Results obtained using the gDHBV2 vaccine were similar to those obtained using the gDPolN vaccine; the gDHBV3 vaccine showed lower immunogenicity. Following tetramer staining, specific CD8+ was observed between the two vaccines. + T cell frequencies were comparable; many activation markers tended to be tetramerized in the gDHBV2 immune group. + CD8 + Higher expression on T cells. Figure 27 shows CD8 at multiple time points. + T cells: 4 weeks after primary immunization ( Figure 27A ); Strengthened in the following two weeks ( Figure 27B ); and 4 weeks after intensification ( Figure 27C The coordinate plot shows the production of IFN-γ as evaluated by ICS. + CD8 + Overall frequency of T cells.

[0345] Figure 28 shows the cytokine-producing CD4 counts as assessed by ICS at multiple time points. + T cells: 4 weeks after primary immunization ( Figure 28A ); Strengthened in the following two weeks ( Figure 28B ); and 4 weeks after intensification ( Figure 28C Based on results from juvenile mice, the dashed line represents the cutoff value for a positive response.

[0346] Figure 29 shows the CD8 levels 4 weeks after initial immunization. + T cells ( Figure 29A ) or CD44 + CD8 + T cells ( Figure 29B The results of gated tetramer staining.

[0347] Figure 30 shows the tetramer + CD8 +The T cell phenotype is shown as the average fluorescence intensity of the dye conjugated to the antibody shown: Figure 30A Anti-PD1 antibody conjugated with BV605; Figure 30B Anti-LAG3 antibody conjugated with BV650; Figure 30C Anti-TIM3 antibody conjugated with Pe-Cy7-A; Figure 30D Anti-CTLA4 antibody conjugated with PE-A; Figure 30E Anti-EOMES antibodies conjugated with AF488; and Figure 30F Anti-T-bet antibody conjugated with BV786.

[0348] Experiment 3

[0349] The breadth of response was assessed using aggregated spleen cells from vaccinated C57BL / 6 mice, targeting individual peptides present in the HBV vaccine insert via ICS testing of the aggregated spleen cells.

[0350] method: Four groups (n=5 per group) of C57Bl / 6 mice were immunized by intramuscular injection as follows: (a) 5 x 10 10 vp's AdC7-gDHBV2, 5x10 after two months 10 vp's AdC6-gDHBV2; (b) 5x10 9 vp's AdC7-gDHBV2, 5x10 after two months 9 vp's AdC6-gDHBV2; (c) 5x10 10 vp's AdC7-gDHBV3, 5x10 after two months 10 vp AdC6-gDHBV3; or (3) no vaccine. Animals were sacrificed eight weeks after booster and pooled spleen cells were assessed by ICS for IFN-γ of individual HBV2 or HBV3 peptides. + CD8 + T-cell response (the cutoff for a positive response was set at 0.1%).

[0351] result: Regardless of dose, the primary immunization booster regimen with gDHBV2 vaccine induced responses to several epitopes in the Core and polymerase. Figure 31 Displayed 5x10 10 The VP AdC7-gDHBV2 primary vaccination is followed by 5 x 10 doses two months later. 10 CD8 after AdC6-gDHBV2 of vp + T cell response. The numbers on the X-axis correspond to the SEQ ID NO provided in this article. Figure 32 Display using 5x10 9The VP AdC7-gDHBV2 primary vaccination is followed by 5 x 10 doses two months later. 9 CD8 after AdC6-gDHBV2 of vp + T cell response. The numbers on the X-axis correspond to the SEQ ID NO provided in this article. Figure 33 Displayed using 5x10 10 The VP AdC7-gDHBV3 primary vaccination is followed by 5 x 10 doses two months later. 10 Immunogenicity of vp after AdC6-gDHBV3. The numbers on the X-axis correspond to the SEQ ID NO provided herein.

[0352] Experiment 4

[0353] The breadth of response was assessed using aggregated spleen cells from vaccinated BALB / c mice, and aggregated spleen cells were tested by ICS for each peptide present in the HBV vaccine insert.

[0354] method: Five groups (n=5 per group) of BALB / c mice were immunized by intramuscular injection as follows: (a) 5 x 10 10 vp's AdC6-gDHBV2; (b) 5x10 10 vp's AdC6-gDHBV3; (c) 5x10 10 vp's AdC7-gDHBV2; (d) 5x10 10 vp AdC7-gDHBV3; or (e) no vaccine. Animals were sacrificed 12 weeks after vaccination, spleens were collected, and the pooled spleen cells were assessed by ICS for IFN-γ of individual HBV2 or HBV3 peptides. + CD8 + T-cell response (the cutoff for a positive response was set at 0.1%).

[0355] result At week 12, each vaccine construct was found to be immunogenic in multiple regions of the vaccine delivery core and polymerase gene. Figure 34 The immunogenicity of the AdC6-gDHBV2 and AdC7-gDHBV2 vaccines corresponding to SEQ ID NO (X-axis) provided herein is shown. The Core, PolC, and PolN regions in both HBV2 constructs are immunogenic. Figure 35 The immunogenicity of the AdC6-gDHBV3 and AdC7-gDHBV3 vaccines corresponding to SEQ ID NO (X-axis) provided herein is shown. The Core, PolC, and PolN regions in both HBV3 constructs are immunogenic.

[0356] Experiment 5

[0357] method : Using 1x10 9 Five groups of C57Bl / 6 mice were attacked with AAV8-1.3HBV (vg) and vaccinated with 1 x 10⁻⁶ HBV at 4 weeks later (“primitive vaccination”). 10 The mice were injected with AdC6-gDPolN (n=10), AdC6-gDHBV2 (n=10), AdC6-gDHBV3 (n=10), or AdC6-HBV2 without gD (n=10); unvaccinated animals infected with AAV (n=10) and unvaccinated animals not infected with AAV (n=2-5) served as controls. Blood samples were collected from mice at different time points after injection, and insert-specific CD8 was determined by intracellular cytokine staining (ICS) against IFN-γ. + and CD4 + T cell frequency. PCR was performed at 2, 6, and 8 weeks post-primary vaccination, and T cell counts were performed 4 weeks post-primary vaccination.

[0358] Eight weeks after the primary vaccination, mice were boosted with the AdC7 vector (“booster vaccination”), which contained the same antigen insert used in the primary vaccination, and CD8 levels in blood and serum were tested at different time points post-vaccination as previously described. + / CD4 + T cells. PCR was performed at 2, 6, and 10 weeks after booster vaccination, and T cell assays were performed at 4 and 12 weeks after booster vaccination.

[0359] Those skilled in the art will understand that many changes and modifications can be made to the preferred embodiments of the invention, and these changes and modifications can be made without departing from the spirit of the invention. Therefore, the appended claims are intended to cover all such equivalent variations that fall within the true spirit and scope of the invention.

[0360] The disclosure of each patent, patent application, and publication cited or described in this document is incorporated herein in its entirety by reference.

[0361] Table 9. Sequence

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373] Implementation Plan

[0374] The following list of implementation schemes is intended to supplement, rather than replace or supersede, the previous description.

[0375] Implementation Scheme 1. Hepatitis B virus (HBV) Core protein, comprising the amino acid sequence of SEQ ID NO:6 or an immunogenic fragment thereof.

[0376] Implementation Scheme 2. The HBV Core protein as described in Implementation Scheme 1, wherein the immunogenic fragment comprises any one of SEQ ID NO: 20-54.

[0377] Implementation Scheme 3. Hepatitis B virus (HBV) Core protein, comprising the amino acid sequence of SEQ ID NO:180 or an immunogenic fragment thereof, or the amino acid sequence of SEQ ID NO:183 or an immunogenic fragment thereof.

[0378] Implementation Scheme 4. Nucleic acid molecules encoding the HBV Core protein as described in any one of Implementation Schemes 1-3.

[0379] Implementation Scheme 5. The nucleic acid molecule as described in Implementation Scheme 4, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:7.

[0380] Implementation Scheme 6. A vector comprising the nucleic acid molecule described in Implementation Scheme 4 or 5.

[0381] Implementation Scheme 7. The vector as described in Implementation Scheme 6, wherein the vector is an adenovirus vector.

[0382] Implementation Scheme 8. The vector as described in Implementation Scheme 7, wherein the adenovirus vector is an AdC6 vector or an AdC7 vector.

[0383] Implementation Plan 9. A vaccine comprising any one of the vectors described in Implementation Plans 6-8.

[0384] Implementation Scheme 10. HBV polymerase N-terminal domain, comprising the amino acid sequence of SEQ ID NO:8 or an immunogenic fragment thereof.

[0385] Implementation Scheme 11. The N-terminal domain of HBV polymerase as described in Implementation Scheme 10, wherein the immunogenic fragment comprises any one of SEQ ID NO: 55-113.

[0386] Implementation Scheme 12. HBV polymerase N-terminal domain, comprising the amino acid sequence of SEQ ID NO:178 or an immunogenic fragment thereof, or the amino acid sequence of SEQ ID NO:181 or an immunogenic fragment thereof.

[0387] Implementation Scheme 13. HBV polymerase C-terminal domain, comprising the amino acid sequence of SEQ ID NO:10 or an immunogenic fragment thereof.

[0388] Implementation Scheme 14. The HBV polymerase C-terminal domain as described in Implementation Scheme 13, wherein the immunogenic fragment comprises any one of SEQ ID NO: 114-172.

[0389] Implementation Scheme 15. HBV polymerase C-terminal domain, comprising the amino acid sequence of SEQ ID NO:179 or an immunogenic fragment thereof, or the amino acid sequence of SEQ ID NO:182 or an immunogenic fragment thereof.

[0390] Implementation Scheme 16. Nucleic acid molecule encoding the HBV polymerase described in any one of Implementation Schemes 10-15.

[0391] Implementation Scheme 17. The nucleic acid molecule as described in Implementation Scheme 16, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:9.

[0392] Implementation Scheme 18. The nucleic acid molecule as described in Implementation Scheme 16, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:11.

[0393] Example 19. A vector comprising any one of the nucleic acid molecules described in embodiments 16-18.

[0394] Implementation Scheme 20. The vector as described in Implementation Scheme 19, wherein the vector is an adenovirus vector.

[0395] Implementation Scheme 21. The vector as described in Implementation Scheme 20, wherein the adenovirus vector is an AdC6 vector or an AdC7 vector.

[0396] Implementation Scheme 22. A vaccine comprising any one of the vectors described in Implementation Schemes 19-21.

[0397] Implementation scheme 23. A fusion protein comprising one or more of the following:

[0398] The HBV Core protein containing the amino acid sequence of SEQ ID NO:6 or its immunogenic fragment, the N-terminal domain of HBV polymerase containing the amino acid sequence of SEQ ID NO:8 or its immunogenic fragment, and the C-terminal domain of HBV polymerase containing the amino acid sequence of SEQ ID NO:10 or its immunogenic fragment.

[0399] Implementation Scheme 24. The fusion protein as described in Implementation Scheme 23, comprising:

[0400] (1) HBV Core protein containing the amino acid sequence of SEQ ID NO:6 or its immunogenic fragment and HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO:8 or its immunogenic fragment;

[0401] (2) One or more of SEQ ID NO:20-54 (immunogenic fragments of SEQ ID NO:6) and one or more of SEQ ID NO:55-113 (immunogenic fragments of SEQ ID NO:8);

[0402] (3) HBV Core protein containing the amino acid sequence of SEQ ID NO:6 or its immunogenic fragment and HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO:10 or its immunogenic fragment.

[0403] (4) One or more of SEQ ID NO:20-54 (immunogenic fragments of SEQ ID NO:6) and one or more of SEQ ID NO:114-172 (immunogenic fragments of SEQ ID NO:10);

[0404] (5) An HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO:8 or its immunogenic fragment thereof and an HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO:10 or its immunogenic fragment thereof.

[0405] (6) One or more of SEQ ID NO:55-113 (immunogenic fragments of SEQ ID NO:8) and one or more of SEQ ID NO:114-172 (immunogenic fragments of SEQ ID NO:10);

[0406] (7) An HBV Core protein comprising the amino acid sequence of SEQ ID NO:6 or its immunogenic fragment, an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO:8 or its immunogenic fragment, and an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO:10 or its immunogenic fragment; or

[0407] (8) One or more of SEQ ID NO:20-54 (immunogenic fragments of SEQ ID NO:6), one or more of SEQ ID NO:55-113 (immunogenic fragments of SEQ ID NO:8), and one or more of SEQ ID NO:114-172 (immunogenic fragments of SEQ ID NO:10).

[0408] Implementation Scheme 25. Fusion protein, comprising:

[0409] The HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO:178 or its immunogenic fragment, the HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO:179 or its immunogenic fragment, and the HBV Core protein containing the amino acid sequence of SEQ ID NO:180 or its immunogenic fragment.

[0410] Implementation Scheme 26. The fusion protein as described in Implementation Scheme 25, comprising the amino acid sequence of SEQ ID NO:174.

[0411] Example 27. Fusion protein, comprising:

[0412] The HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO:181 or its immunogenic fragment, the HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO:182 or its immunogenic fragment, and the HBV Core protein containing the amino acid sequence of SEQ ID NO:183 or its immunogenic fragment.

[0413] Implementation Scheme 28. The fusion protein as described in Implementation Scheme 27, comprising the amino acid sequence of SEQ ID NO:175.

[0414] Implementation Scheme 29. Fusion protein, comprising:

[0415] N-terminal herpes simplex virus (HSV) glycoprotein (gD) sequence or a variant thereof;

[0416] HBV Core protein, comprising the amino acid sequence of SEQ ID NO:6 or an immunogenic fragment thereof; and

[0417] C-terminal HSV gD sequence or its variants.

[0418] Implementation Scheme 30. The fusion protein as described in Implementation Scheme 29, wherein the immunogenic fragment comprises any one of SEQ ID NO: 20-54.

[0419] Implementation scheme 31. Fusion protein, comprising:

[0420] N-terminal HSV gD sequence or a variant thereof;

[0421] The N-terminal domain of HBV polymerase, comprising the amino acid sequence of SEQ ID NO:8 or an immunogenic fragment thereof; and

[0422] C-terminal HSV gD protein sequence or its variants.

[0423] Implementation Scheme 32. The fusion protein as described in Implementation Scheme 31, wherein the immunogenic fragment comprises any one of SEQ ID NO: 55-113.

[0424] Implementation scheme 33. Fusion protein, comprising:

[0425] N-terminal HSV gD sequence or a variant thereof;

[0426] The C-terminal domain of HBV polymerase, comprising the amino acid sequence of SEQ ID NO:10 or an immunogenic fragment thereof; and

[0427] C-terminal HSV gD protein sequence or its variants.

[0428] Implementation Scheme 34. The fusion protein as described in Implementation Scheme 33, wherein the immunogenic fragment comprises any one of SEQ ID NO: 114-172.

[0429] Implementation scheme 35. Fusion protein, comprising:

[0430] N-terminal HSV gD sequence or a variant thereof;

[0431] HBV sequence, which includes:

[0432] (1) HBV Core protein containing the amino acid sequence of SEQ ID NO:6 or its immunogenic fragment and HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO:8 or its immunogenic fragment;

[0433] (2) One or more of SEQ ID NO:20-54 (immunogenic fragments of SEQ ID NO:6) and one or more of SEQ ID NO:55-113 (immunogenic fragments of SEQ ID NO:8);

[0434] (3) HBV Core protein containing the amino acid sequence of SEQ ID NO:6 or its immunogenic fragment and HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO:10 or its immunogenic fragment.

[0435] (4) One or more of SEQ ID NO:20-54 (immunogenic fragments of SEQ ID NO:6) and one or more of SEQ ID NO:114-172 (immunogenic fragments of SEQ ID NO:10);

[0436] (5) An HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO:8 or its immunogenic fragment thereof and an HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO:10 or its immunogenic fragment thereof.

[0437] (6) One or more of SEQ ID NO:55-113 (immunogenic fragments of SEQ ID NO:8) and one or more of SEQ ID NO:114-172 (immunogenic fragments of SEQ ID NO:10);

[0438] (7) An HBV Core protein comprising the amino acid sequence of SEQ ID NO:6 or its immunogenic fragment, an HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO:8 or its immunogenic fragment, and an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO:10 or its immunogenic fragment; or

[0439] (8) One or more of SEQ ID NO:20-54 (immunogenic fragments of SEQ ID NO:6), one or more of SEQ ID NO:55-113 (immunogenic fragments of SEQ ID NO:8), and one or more of SEQ ID NO:114-172 (immunogenic fragments of SEQ ID NO:10), and

[0440] C-terminal HSV gD protein sequence or its variants.

[0441] Implementation scheme 36. Fusion protein, comprising:

[0442] N-terminal HSV gD sequence or a variant thereof;

[0443] HBV Core protein comprising the amino acid sequence of SEQ ID NO:180 or an immunogenic fragment thereof, or the amino acid sequence of SEQ ID NO:183 or an immunogenic fragment thereof; and

[0444] C-terminal HSV gD sequence or its variants.

[0445] Implementation scheme 37. Fusion protein, comprising:

[0446] N-terminal HSV gD sequence or a variant thereof;

[0447] The N-terminal domain of HBV polymerase, comprising the amino acid sequence of SEQ ID NO:178 or an immunogenic fragment thereof, or the amino acid sequence of SEQ ID NO:181 or an immunogenic fragment thereof; and

[0448] C-terminal HSV gD protein sequence or its variants.

[0449] Implementation scheme 38. Fusion protein, comprising:

[0450] N-terminal HSV gD sequence or a variant thereof;

[0451] The C-terminal domain of HBV polymerase, comprising the amino acid sequence of SEQ ID NO:179 or an immunogenic fragment thereof, or the amino acid sequence of SEQ ID NO:182 or an immunogenic fragment thereof; and

[0452] C-terminal HSV gD protein sequence or its variants.

[0453] Implementation scheme 39. Fusion protein, comprising:

[0454] N-terminal HSV gD sequence or a variant thereof;

[0455] HBV sequence, which includes:

[0456] (1) An HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO:178 or its immunogenic fragment thereof, an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO:179 or its immunogenic fragment thereof, and an HBV Core protein comprising the amino acid sequence of SEQ ID NO:180 or its immunogenic fragment thereof; or

[0457] (2) An HBV polymerase N-terminal domain comprising the amino acid sequence of SEQ ID NO:181 or an immunogenic fragment thereof, an HBV polymerase C-terminal domain comprising the amino acid sequence of SEQ ID NO:182 or an immunogenic fragment thereof, and an HBV Core protein comprising the amino acid sequence of SEQ ID NO:183 or an immunogenic fragment thereof; and

[0458] C-terminal HSV gD protein sequence or its variants.

[0459] Implementation Scheme 40. The fusion protein as described in Implementation Scheme 39, wherein the HBV sequence comprises an HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO:178 or an immunogenic fragment thereof, an HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO:179 or an immunogenic fragment thereof, and an HBV Core protein containing the amino acid sequence of SEQ ID NO:180 or an immunogenic fragment thereof.

[0460] Implementation Scheme 41: The fusion protein as described in Implementation Scheme 40, wherein the HBV sequence comprises the amino acid sequence of SEQ ID NO:174.

[0461] Implementation Scheme 42. The fusion protein as described in Implementation Scheme 39, wherein the HBV sequence comprises an HBV polymerase N-terminal domain containing the amino acid sequence of SEQ ID NO:181 or an immunogenic fragment thereof, an HBV polymerase C-terminal domain containing the amino acid sequence of SEQ ID NO:182 or an immunogenic fragment thereof, and an HBV Core protein containing the amino acid sequence of SEQ ID NO:183 or an immunogenic fragment thereof.

[0462] Implementation Scheme 43. The fusion protein as described in Implementation Scheme 42, wherein the HBV sequence comprises the amino acid sequence of SEQ ID NO:175.

[0463] Implementation Scheme 44. The fusion protein as described in any one of Implementation Schemes 29-43, wherein the N-terminal HSV gD sequence comprises the amino acid sequence of SEQ ID NO:12.

[0464] Implementation Scheme 45. The fusion protein of any one of Implementation Schemes 29-43, wherein the N-terminal HSV gD sequence comprises amino acid residues 26-269 of SEQ ID NO:12.

[0465] Implementation Scheme 46. The fusion protein of any one of Implementation Schemes 29-45, wherein the C-terminal HSV gD sequence comprises the transmembrane domain of HSV gD.

[0466] Implementation Scheme 47. The fusion protein of any one of Implementation Schemes 29-46, wherein the C-terminal HSV gD sequence comprises the amino acid sequence of SEQ ID NO:13.

[0467] Implementation Scheme 48. The fusion protein of any one of Implementation Schemes 29-47, wherein the fusion protein comprises the amino acid sequence of any one of the following: SEQ ID NO:14 or an immunogenic fragment thereof, SEQ ID NO:16 or an immunogenic fragment thereof, or SEQ ID NO:18 or an immunogenic fragment thereof.

[0468] Implementation Scheme 49. The fusion protein as described in any one of Implementation Schemes 39-47, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:185.

[0469] Implementation Scheme 50. The fusion protein as described in any one of Implementation Schemes 39-47, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:187.

[0470] Implementation Scheme 51. Nucleic acid molecules encoding the fusion protein described in any one of Implementation Schemes 23-50.

[0471] Implementation Scheme 52. The nucleic acid molecule as described in Implementation Scheme 51, wherein the nucleic acid molecule comprises a nucleotide sequence of any one of SEQ ID NO: 15, 17 or 19.

[0472] Implementation Scheme 53. The nucleic acid molecule as described in Implementation Scheme 51, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:176.

[0473] Implementation Scheme 54. The nucleic acid molecule as described in Implementation Scheme 51, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:177.

[0474] Implementation Scheme 55. The nucleic acid molecule as described in Implementation Scheme 51, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:184.

[0475] Implementation Scheme 56. The nucleic acid molecule as described in Implementation Scheme 51, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:186.

[0476] Implementation Scheme 57. A vector comprising any one of the nucleic acid molecules described in Implementation Schemes 51-56.

[0477] Implementation scheme 58. The vector as described in implementation scheme 57, wherein the vector is an adenovirus vector.

[0478] Implementation Scheme 59. The vector as described in Implementation Scheme 58, wherein the adenovirus vector is an AdC6 vector or an AdC7 vector.

[0479] Implementation Plan 60. A vaccine comprising the vector described in any one of Implementation Plans 57-59.

[0480] Implementation Scheme 61. A method for inducing an immune response to HBV in a subject, the method comprising providing the subject with an effective amount of any one of the fusion protein of Implementation Scheme 23-50, any one of the nucleic acid molecules of Implementation Scheme 51-56, any one of the vectors of Implementation Scheme 57-59, or the vaccine of Implementation Scheme 60, thereby inducing an immune response to HBV.

[0481] Implementation Scheme 62. The method of Implementation Scheme 61, wherein the vaccine comprises an AdC6 vector, the AdC6 vector comprises a fusion protein, the fusion protein comprising an amino acid sequence of any one of SEQ ID NO: 14, 16 or 18 or an immunogenic fragment thereof.

[0482] Implementation Scheme 63. The method of Implementation Scheme 62 further includes, after providing the vaccine comprising the AdC6 vector, providing the subject with a vaccine comprising the AdC7 vector, the AdC7 vector comprising a fusion protein comprising an amino acid sequence of any one of SEQ ID NO: 14, 16 or 18 or an immunogenic fragment thereof.

[0483] Implementation Scheme 64. The method of Implementation Scheme 61, wherein the vaccine comprises an AdC7 vector, the AdC7 vector comprises a fusion protein, the fusion protein comprising an amino acid sequence of any one of SEQ ID NO: 14, 16 or 18 or an immunogenic fragment thereof.

[0484] Implementation Scheme 65. The method of Implementation Scheme 64 further comprises, after providing the vaccine comprising the AdC7 vector, providing the subject with a vaccine comprising the AdC6 vector, the AdC6 vector comprising a fusion protein comprising an amino acid sequence of any one of SEQ ID NO: 14, 16 or 18 or an immunogenic fragment thereof.

[0485] Implementation Scheme 66. The method of Implementation Scheme 61, wherein the vaccine comprises an AdC6 vector, the AdC6 vector comprises a fusion protein, the fusion protein comprising the amino acid sequence of SEQ ID NO:185.

[0486] Implementation Scheme 67. The method of Implementation Scheme 66 further includes, after providing the vaccine comprising the AdC6 vector, providing the subject with a vaccine comprising the AdC7 vector, the AdC7 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO:185.

[0487] Implementation Scheme 68. The method of Implementation Scheme 61, wherein the vaccine comprises an AdC7 vector, the AdC7 vector comprises a fusion protein, the fusion protein comprising the amino acid sequence of SEQ ID NO:185.

[0488] Implementation Scheme 69. The method of Implementation Scheme 68 further includes, after providing the vaccine comprising the AdC7 vector, providing the subject with a vaccine comprising the AdC6 vector, the AdC6 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO:185.

[0489] Implementation Scheme 70. The method of Implementation Scheme 61, wherein the vaccine comprises an AdC6 vector, the AdC6 vector comprises a fusion protein, the fusion protein comprising the amino acid sequence of SEQ ID NO:187.

[0490] Implementation Scheme 71. The method of Implementation Scheme 70 further includes, after providing the vaccine comprising the AdC6 vector, providing the subject with a vaccine comprising the AdC7 vector, the AdC7 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO:187.

[0491] Implementation Scheme 72. The method of Implementation Scheme 61, wherein the vaccine comprises an AdC7 vector, the AdC7 vector comprises a fusion protein, the fusion protein comprising the amino acid sequence of SEQ ID NO:187.

[0492] Implementation Scheme 73. The method of Implementation Scheme 72 further includes, after providing the vaccine comprising the AdC7 vector, providing the subject with a vaccine comprising the AdC6 vector, the AdC6 vector comprising a fusion protein comprising the amino acid sequence of SEQ ID NO:187.

[0493] Implementation Scheme 74. The method of any one of Implementation Schemes 61-73, wherein the amino acid sequence or immunogenic fragment of any one of SEQ ID NO: 14, 16, 18, 185 or 187 does not contain a signal peptide of 25 amino acids at the N-terminus. sequence list <110> Virus Therapy Limited Liability Company Wistar Institute <120> Adenovirus vector encoding hepatitis B virus antigen fused with herpesvirus glycoprotein D and its usage <130> 111876.000035 <140> <141> <150> 63 / 112,219 <151> 2020-11-11 <150> 63 / 112,202 <151> 2020-11-11 <150> 63 / 064,571 <151> 2020-08-12 <150> 63 / 064,506 <151> 2020-08-12 <150> 62 / 967,242 <151> 2020-01-29 <150> 62 / 967,104 <151> 2020-01-29 <150> 62 / 958,827 <151> 2020-01-09 <150> 62 / 958,809 <151> 2020-01-09 <160> 233 <170> PatentIn version 3.5 <210> 1 <211> 185 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 1 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val Arg Arg Arg Asp Arg Gly Arg Ser Pro Arg Arg 145 150 155 160 Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg 165 170 175 Arg Ser Gln Ser Arg Glu Ser Gln Cys 180 185 <210> 2 <211> 183 <212> PRT <213> artificial sequence <220> <221> source <223> / Remarks = "Description of artificial sequence: synthetic polypeptide" <400> 2 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser Arg Glu Ser Gln Cys 180 <210> 3 <211> 182 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 3 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Ser Gln 165 170 175 Ser Arg Glu Ser Gln Cys 180 <210> 4 <211> 183 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 4 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Gly Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Phe Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser Arg Glu Ser Gln Cys 180 <210> 5 <211> 184 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <220> <221> variants <222> (12)..(12) <223> / replaces "S" <220> <221> variants <222> (27)..(27) <223> / replaces "I" <220> <221> variants <222> (66)..(66) <223> / replaces "N" <220> <221> variants <222> (73)..(73) <223> / Replaces "S" or "G" <220> <221> variants <222> (82)..(82) <223> / replaces "E" <220> <221> variants <222> (86)..(86) <223> / replaces "S" <220> <221> variants <222> (90)..(90) <223> / replaces "V" <220> <221> variants <222> (96)..(96) <223> / replaces "F" <220> <221> variants <222> (115)..(115) <223> / replaces "I" <220> <221> variants <222> (152) (153) <223> / replaces=" " <220> <221> SITE <222> (1)..(184) <223> / Note: The variant residues given in the sequence are not biased relative to those in the variant location annotation. / <400> 5 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Asp Leu Leu Asp Thr 20 25 30 Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser 35 40 45 Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu 50 55 60 Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Pro Ala Ser 65 70 75 80 Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile 85 90 95 Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu 100 105 110 Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro 115 120 125 Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu 130 135 140 Thr Thr Val Val Arg Arg Arg Asp Arg Gly Arg Ser Pro Arg Arg Arg 145 150 155 160 Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg 165 170 175 Ser Gln Ser Arg Glu Ser Gln Cys 180 <210> 6 <211> 184 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 6 Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser 1 5 10 15 Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp Thr 20 25 30 Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser 35 40 45 Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu 50 55 60 Met Thr Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala Ser 65 70 75 80 Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys Ile 85 90 95 Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu 100 105 110 Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro 115 120 125 Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu 130 135 140 Thr Thr Val Val Arg Arg Arg Asp Arg Gly Arg Ser Pro Arg Arg Arg 145 150 155 160 Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg 165 170 175 Ser Gln Ser Arg Glu Ser Gln Cys 180 <210> 7 <211> 552 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic polynucleotide" <400> 7 gacatcgacc cctacaagga gttcggcgcc accgtggagc tgctgagctt cctgcccagc 60 gacttcttcc ccagcatcag ggacctgctg gacaccgcca gcgccctgta cagggaggcc 120 ctggagagcc ccgagcactg cagcccccac cacaccgccc tgaggcaggc catcctgtgc 180 tggggcgagc tgatgaccct ggccacctgg gtgggcagca acctggagga ccccgccagc 240 agggagctgg tggtgagcta cgtgaacgtg aacatgggcc tgaagatcag gcagctgctg 300 tggttccaca tcagctgcct gaccttcggc agggagaccg tgatcgagta cctggtgagc 360 ttcggcgtgt ggatcaggac cccccccgcc tacaggcccc ccaacgcccc catcctgagc 420 accctgcccg agaccaccgt ggtgaggagg agggacaggg gcaggagccc caggaggagg 480 acccccagcc ccaggaggag gaggagccag agccccagga ggaggaggag ccagagcagg 540 gagagccagt gc 552 <210> 8 <211> 305 <212> PRT <213> Artificial Sequence <220> <221> Source <223> / Remark="Description of artificial sequence: Synthetic polypeptide" <400> 8 Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp Glu Glu 1 5 10 15 Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gly Leu 20 25 30 Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Val Ser 35 40 45 Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Ser Ser 50 55 60 Thr Val Pro Val Phe Asn Pro Glu Trp Gln Thr Pro Ser Phe Pro Lys 65 70 75 80 Ile His Leu Gln Glu Asp Ile Val Asp Arg Cys Lys Gln Phe Val Gly 85 90 95 Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pro Ala 100 105 110 Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gly Ile 115 120 125 Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr Phe Gln Thr Arg 130 135 140 His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Arg Glu 145 150 155 160 Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu Gln 165 170 175 Glu Leu Gln His Gly Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys 180 185 190 Pro Cys Ser Glu Tyr Cys Leu Thr His Leu Val Asn Leu Leu Glu Asp 195 200 205 Trp Gly Pro Cys Asp Glu His Gly Glu His His Ile Arg Ile Pro Arg 210 215 220 Thr Pro Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro 225 230 235 240 His Asn Thr Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser 245 250 255 Arg Gly Ile Thr Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu 260 265 270 Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu 275 280 285 Asp Val Ser Ala Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met 290 295 300 Pro 305 <210> 9 <211> 915 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic polynucleotide" <400> 9 cccctgagct accagcactt caggaagctg ctgctgctgg accagaggc cggccccctg gaggaggagc tgcccaggct ggccgacgag ggcctgaaca ggagggtggc cgaggacctg 120 aacctgggca acctgaacgt gagcatcccc tggacccaca aggtgggcaa cttcaccggc ctgtacagca gcaccgtgcc cgtgttcaac cccgagtggc agaccccag cttccccag 240 atccacctgc aggaggacat cgtggacagg tgcaagcagt tcgtgggccc cctgaccgtg 300 aacgagaaga ggaggctgaa gctgatcatg cccgccaggt tctaccccaa cgtgaccaag tacctgcccc tggacaaggg catcaagccc tactacccg agcacgccgt gaaccactac ttccagacca ggcactacct gcacaccctg tggaaggccg gcatcctgta caagagggag 480 accaccagga gcgccagctt ctgcggcagc ccctacagct gggagcagga gctgcagcac 540 ggcagctgct ggtggctgca gttcaggac agcaagccct gcagcgagta ctgcctgacc 600 cacctggtga acctgctgga ggactggggc ccctgcgacg agcacggcga gcaccacatc 660 aggatcccca ggacccccgc cagggtgacc ggcggcgtgt tcctggtgga caagaacccc 720 cacaacaccg ccgagagcag gctggtggtg gacttcagcc agttcagcag gggcatcacc 780 agggtgagct ggcccaagtt cgccgtgccc aacctgcaga gcctgaccaa cctgctgagc 840 agcaacctga gctggctgag cctggacgtg agcgccgcct tctaccacat ccccctgcac 900 cccgccgcca tgccc 915 <210> 10 <211> 303 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 10 His Leu Leu Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu 1 5 10 15 Ser Ser Asn Ser Arg Ile Ile Asn His Gln His Gly Thr Met Gln Asn 20 25 30 Leu His Asp Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu 35 40 45 Tyr Lys Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile 50 55 60 Leu Lys Thr Lys Arg Trp Gly Tyr Ser Leu Asn Phe Met Gly Tyr Val 65 70 75 80 Ile Gly Ser Trp Gly Ser Leu Pro Gln Asp His Ile Ile Gln Lys Ile 85 90 95 Lys Glu Cys Phe Arg Lys Leu Pro Val Asn Arg Pro Ile Asp Trp Lys 100 105 110 Val Cys Gln Arg Ile Val Gly Leu Leu Gly Phe Ala Ala Pro Phe Thr 115 120 125 Gln Cys Gly Tyr Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln Ser 130 135 140 Lys Gln Ala Phe Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Ser Lys 145 150 155 160 Gln Tyr Leu Asn Leu Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys 165 170 175 Gln Val Phe Ala Asp Ala Thr Pro Thr Gly Trp Gly Leu Ala Met Gly 180 185 190 His Gln Arg Met Arg Gly Thr Phe Val Ala Pro Leu Pro Ile His Thr 195 200 205 Ala Glu Leu Leu Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Lys 210 215 220 Ile Leu Gly Thr Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser 225 230 235 240 Phe Pro Trp Leu Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr 245 250 255 Ser Phe Val Tyr Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro Ser 260 265 270 Arg Gly Arg Leu Gly Leu Ser Arg Pro Leu Leu Arg Leu Pro Phe Arg 275 280 285 Pro Thr Thr Gly Arg Thr Ser Leu Tyr Ala Val Ser Pro Ser Val 290 295 300 <210> 11 <211> 909 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic polynucleotide" <400> 11 cacctgctgg tgggcagcag cggcctgagc aggtacgtgg ccaggctgag cagcaacagc 60 aggatcatca accaccagca cggcaccatg cagaacctgc acgacagctg cagcaggaac 120 ctgtacgtga gcctgctgct gctgtacaag accttcggca ggaagctgca cctgtacagc 180 caccccatca tcctgaagac caagaggtgg ggctacagcc tgaacttcat gggctacgtg 240 atcggcagct ggggcagcct gccccaggac cacatcatcc agaagatcaa ggagtgcttc 300 aggaagctgc ccgtgaacag gcccatcgac tggaaggtgt gccagaggat cgtgggcctg 360 ctgggcttcg ccgccccctt cacccagtgc ggctaccccg ccctgatgcc cctgtacgcc 420 tgcatccaga gcaagcaggc cttcaccttc agccccacct acaaggcctt cctgagcaag 480 cagtacctga acctgtaccc cgtggccagg cagaggcccg gcctgtgcca ggtgttcgcc 540 gacgccaccc ccaccggctg gggcctggcc atgggccacc agaggatgag gggcaccttc 600 gtggcccccc tgcccatcca caccgccgag ctgctggccg cctgcttcgc caggagcagg 660 agcggcgcca agatcctggg caccgacaac agcgtggtgc tgagcaggaa gtacaccagc 720 ttcccctggc tgctgggctg cgccgccaac tggatcctga ggggcaccag cttcgtgtac 780 gtgcccagcg ccctgaaccc cgccgacgac cccagcaggg gcaggctggg cctgagcagg 840 cccctgctga ggctgccctt caggcccacc accggcagga ccagcctgta cgccgtgagc 900 cccagcgtg 909 <210> 12 <211> 269 <212> PRT <213> Artificial Sequence <220> <221> Source <223> / Remarks="Description of artificial sequence: Synthetic polypeptide" <400> 12 Met Gly Gly Ala Ala Ala Arg Leu Gly Ala Val Ile Leu Phe Val Val 1 5 10 15 Ile Val Gly Leu His Gly Val Arg Gly Lys Tyr Ala Leu Ala Asp Ala 20 25 30 Ser Leu Lys Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp Leu Pro 35 40 45 Val Leu Asp Gln Leu Thr Asp Pro Pro Gly Val Arg Arg Val Tyr His 50 55 60 Ile Gln Ala Gly Leu Pro Asp Pro Phe Gln Pro Pro Ser Leu Pro Ile 65 70 75 80 Thr Val Tyr Tyr Ala Val Leu Glu Arg Ala Cys Arg Ser Val Leu Leu 85 90 95 Asn Ala Pro Ser Glu Ala Pro Gln Ile Val Arg Gly Ala Ser Glu Asp 100 105 110 Val Arg Lys Gln Pro Tyr Asn Leu Thr Ile Ala Trp Phe Arg Met Gly 115 120 125 Gly Asn Cys Ala Ile Pro Ile Thr Val Met Glu Tyr Thr Glu Cys Ser 130 135 140 Tyr Asn Lys Ser Leu Gly Ala Cys Pro Ile Arg Thr Gln Pro Arg Trp 145 150 155 160 Asn Tyr Tyr Asp Ser Phe Ser Ala Val Ser Glu Asp Asn Leu Gly Phe 165 170 175 Leu Met His Ala Pro Ala Phe Glu Thr Ala Gly Thr Tyr Leu Arg Leu 180 185 190 Val Lys Ile Asn Asp Trp Thr Glu Ile Thr Gln Phe Ile Leu Glu His 195 200 205 Arg Ala Lys Gly Ser Cys Lys Tyr Ala Leu Pro Leu Arg Ile Pro Pro 210 215 220 Ser Ala Cys Leu Ser Pro Gln Ala Tyr Gln Gln Gly Val Thr Val Asp 225 230 235 240 Ser Ile Gly Met Leu Pro Arg Phe Ile Pro Glu Asn Gln Arg Thr Val 245 250 255 Ala Val Tyr Ser Leu Lys Ile Ala Gly Trp His Gly Pro 260 265 <210> 13 <211> 127 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 13 Gly Pro Lys Ala Pro Tyr Thr Ser Thr Leu Leu Pro Pro Glu Leu Ser 1 5 10 15 Glu Thr Pro Asn Ala Thr Gln Pro Glu Leu Ala Pro Glu Asp Pro Glu 20 25 30 Asp Ser Ala Leu Leu Glu Asp Pro Val Gly Thr Val Ala Pro Gln Ile 35 40 45 Pro Pro Asn Trp His Ile Pro Ser Ile Gln Asp Ala Ala Thr Pro Tyr 50 55 60 His Pro Pro Ala Thr Pro Asn Asn Met Gly Leu Ile Ala Gly Ala Val 65 70 75 80 Gly Gly Ser Leu Leu Ala Ala Leu Val Ile Cys Gly Ile Val Tyr Trp 85 90 95 Met His Arg Arg Thr Arg Lys Ala Pro Lys Arg Ile Arg Leu Pro His 100 105 110 Ile Arg Glu Asp Asp Gln Pro Ser Ser His Gln Pro Leu Phe Tyr 115 120 125 <210> 14 <211> 580 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 14 Met Gly Gly Ala Ala Ala Arg Leu Gly Ala Val Ile Leu Phe Val Val 1 5 10 15 Ile Val Gly Leu His Gly Val Arg Gly Lys Tyr Ala Leu Ala Asp Ala 20 25 30 Ser Leu Lys Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp Leu Pro 35 40 45 Val Leu Asp Gln Leu Thr Asp Pro Pro Gly Val Arg Arg Val Tyr His 50 55 60 Ile Gln Ala Gly Leu Pro Asp Pro Phe Gln Pro Pro Ser Leu Pro Ile 65 70 75 80 Thr Val Tyr Tyr Ala Val Leu Glu Arg Ala Cys Arg Ser Val Leu Leu 85 90 95 Asn Ala Pro Ser Glu Ala Pro Gln Ile Val Arg Gly Ala Ser Glu Asp 100 105 110 Val Arg Lys Gln Pro Tyr Asn Leu Thr Ile Ala Trp Phe Arg Met Gly 115 120 125 Gly Asn Cys Ala Ile Pro Ile Thr Val Met Glu Tyr Thr Glu Cys Ser 130 135 140 Tyr Asn Lys Ser Leu Gly Ala Cys Pro Ile Arg Thr Gln Pro Arg Trp 145 150 155 160 Asn Tyr Tyr Asp Ser Phe Ser Ala Val Ser Glu Asp Asn Leu Gly Phe 165 170 175 Leu Met His Ala Pro Ala Phe Glu Thr Ala Gly Thr Tyr Leu Arg Leu 180 185 190 Val Lys Ile Asn Asp Trp Thr Glu Ile Thr Gln Phe Ile Leu Glu His 195 200 205 Arg Ala Lys Gly Ser Cys Lys Tyr Ala Leu Pro Leu Arg Ile Pro Pro 210 215 220 Ser Ala Cys Leu Ser Pro Gln Ala Tyr Gln Gln Gly Val Thr Val Asp 225 230 235 240 Ser Ile Gly Met Leu Pro Arg Phe Ile Pro Glu Asn Gln Arg Thr Val 245 250 255 Ala Val Tyr Ser Leu Lys Ile Ala Gly Trp His Gly Pro Asp Ile Asp 260 265 270 Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu Pro 275 280 285 Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp Thr Ala Ser Ala 290 295 300 Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His His 305 310 315 320 Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr Leu 325 330 335 Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala Ser Arg Glu Leu 340 345 350 Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys Ile Arg Gln Leu 355 360 365 Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Ile 370 375 380 Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr 385 390 395 400 Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val 405 410 415 Val Arg Arg Arg Asp Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser 420 425 430 Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser 435 440 445 Arg Glu Ser Gln Cys Gly Pro Lys Ala Pro Tyr Thr Ser Thr Leu Leu 450 455 460 Pro Pro Glu Leu Ser Glu Thr Pro Asn Ala Thr Gln Pro Glu Leu Ala 465 470 475 480 Pro Glu Asp Pro Glu Asp Ser Ala Leu Leu Glu Asp Pro Val Gly Thr 485 490 495 Val Ala Pro Gln Ile Pro Pro Asn Trp His Ile Pro Ser Ile Gln Asp 500 505 510 Ala Ala Thr Pro Tyr His Pro Pro Ala Thr Pro Asn Asn Met Gly Leu 515 520 525 Ile Ala Gly Ala Val Gly Gly Ser Leu Leu Ala Ala Leu Val Ile Cys 530 535 540 Gly Ile Val Tyr Trp Met His Arg Arg Thr Arg Lys Ala Pro Lys Arg 545 550 555 560 Ile Arg Leu Pro His Ile Arg Glu Asp Asp Gln Pro Ser Ser His Gln 565 570 575 Pro Leu Phe Tyr 580 <210> 15 <211> 1743 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic polynucleotide" <400> 15 atgggggggg ctgccgccag gttgggggcc gtgattttgt ttgtcgtcat agtgggcctc 60 catggggtcc gcggcaaata tgccttggcg gatgcctctc tcaagatggc cgaccccaat 120 cgctttcgcg gcaaagacct tccggtcctg gaccagctga ccgaccctcc gggggtccgg 180 cgcgtgtacc acatccaggc gggcctaccg gacccgttcc agccccccag cctcccgatc 240 acggtttact acgccgtgtt ggagcgcgcc tgccgcagcg tgctcctaaa cgcaccgtcg 300 gaggcccccc agattgtccg cggggcctcc gaagacgtcc ggaaacaacc ctacaacctg 360 accatcgctt ggtttcggat gggaggcaac tgtgctatcc ccatcacggt catggagtac 420 accgaatgct cctacaacaa gtctctgggg gcctgtccca tccgaacgca gccccgctgg 480 aactactatg acagcttcag cgccgtcagc gaggataacc tggggttcct gatgcacgcc 540 cccgcgtttg agaccgccgg cacgtacctg cggctcgtga agataaacga ctggacggag 600 attacacagt ttatcctgga gcaccgagcc aagggctcct gtaagtacgc cctcccgctg 660 cgcatccccc cgtcagcctg cctctccccc caggcctacc agcagggggt gacggtggac 720 agcatcggga tgctgccccg cttcatcccc gagaaccagc gcaccgtcgc cgtatacagc 780 ttgaagatcg ccgggtggca cgggcccgac atcgacccct acaaggagtt cggcgccacc 840 gtggagctgc tgagcttcct gcccagcgac ttcttcccca gcatcaggga cctgctggac 900 accgccagcg ccctgtacag ggaggccctg gagagccccg agcactgcag cccccaccac 960 accgccctga ggcaggccat cctgtgctgg ggcgagctga tgaccctggc cacctgggtg 1020 ggcagcaacc tggaggaccc cgccagcagg gagctggtgg tgagctacgt gaacgtgaac 1080 atgggcctga agatcaggca gctgctgtgg ttccacatca gctgcctgac cttcggcagg 1140 gagaccgtga tcgagtacct ggtgagcttc ggcgtgtgga tcaggacccc ccccgcctac 1200 aggcccccca acgcccccat cctgagcacc ctgcccgaga ccaccgtggt gaggaggagg 1260 gacaggggca ggagccccag gaggaggacc cccagcccca ggaggaggag gagccagagc 1320 cccaggagga ggaggagcca gagcagggag agccagtgcg ggcccaaggc cccatacacg 1380 agcaccctgc tgcccccgga gctgtccgag acccccaacg ccacgcagcc agaactcgcc 1440 ccggaagacc ccgaggattc ggccctcttg gaggaccccg tggggacggt ggcgccgcaa 1500 atcccaccaa actggcacat cccgtcgatc caggacgccg cgacgcctta ccatcccccg 1560 gccaccccga acaacatggg cctgatcgcc ggcgcggtgg gcggcagtct cctggcagcc 1620 ctggtcattt gcggaattgt gtactggatg caccgccgca ctcggaaagc cccaaagcgc 1680 atacgcctcc cccacatccg ggaagacgac cagccgtcct cgcaccagcc cttgttttac 1740 tag 1743 <210> 16 <211> 701 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 16 Met Gly Gly Ala Ala Ala Arg Leu Gly Ala Val Ile Leu Phe Val Val 1 5 10 15 Ile Val Gly Leu His Gly Val Arg Gly Lys Tyr Ala Leu Ala Asp Ala 20 25 30 Ser Leu Lys Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp Leu Pro 35 40 45 Val Leu Asp Gln Leu Thr Asp Pro Pro Gly Val Arg Arg Val Tyr His 50 55 60 Ile Gln Ala Gly Leu Pro Asp Pro Phe Gln Pro Pro Ser Leu Pro Ile 65 70 75 80 Thr Val Tyr Tyr Ala Val Leu Glu Arg Ala Cys Arg Ser Val Leu Leu 85 90 95 Asn Ala Pro Ser Glu Ala Pro Gln Ile Val Arg Gly Ala Ser Glu Asp 100 105 110 Val Arg Lys Gln Pro Tyr Asn Leu Thr Ile Ala Trp Phe Arg Met Gly 115 120 125 Gly Asn Cys Ala Ile Pro Ile Thr Val Met Glu Tyr Thr Glu Cys Ser 130 135 140 Tyr Asn Lys Ser Leu Gly Ala Cys Pro Ile Arg Thr Gln Pro Arg Trp 145 150 155 160 Asn Tyr Tyr Asp Ser Phe Ser Ala Val Ser Glu Asp Asn Leu Gly Phe 165 170 175 Leu Met His Ala Pro Ala Phe Glu Thr Ala Gly Thr Tyr Leu Arg Leu 180 185 190 Val Lys Ile Asn Asp Trp Thr Glu Ile Thr Gln Phe Ile Leu Glu His 195 200 205 Arg Ala Lys Gly Ser Cys Lys Tyr Ala Leu Pro Leu Arg Ile Pro Pro 210 215 220 Ser Ala Cys Leu Ser Pro Gln Ala Tyr Gln Gln Gly Val Thr Val Asp 225 230 235 240 Ser Ile Gly Met Leu Pro Arg Phe Ile Pro Glu Asn Gln Arg Thr Val 245 250 255 Ala Val Tyr Ser Leu Lys Ile Ala Gly Trp His Gly Pro Pro Leu Ser 260 265 270 Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp Glu Glu Ala Gly Pro 275 280 285 Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gly Leu Asn Arg Arg 290 295 300 Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Val Ser Ile Pro Trp 305 310 315 320 Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Ser Ser Thr Val Pro 325 330 335 Val Phe Asn Pro Glu Trp Gln Thr Pro Ser Phe Pro Lys Ile His Leu 340 345 350 Gln Glu Asp Ile Val Asp Arg Cys Lys Gln Phe Val Gly Pro Leu Thr 355 360 365 Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pro Ala Arg Phe Tyr 370 375 380 Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gly Ile Lys Pro Tyr 385 390 395 400 Tyr Pro Glu His Ala Val Asn His Tyr Phe Gln Thr Arg His Tyr Leu 405 410 415 His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Arg Glu Thr Thr Arg 420 425 430 Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu Gln Glu Leu Gln 435 440 445 His Gly Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser 450 455 460 Glu Tyr Cys Leu Thr His Leu Val Asn Leu Leu Glu Asp Trp Gly Pro 465 470 475 480 Cys Asp Glu His Gly Glu His His Ile Arg Ile Pro Arg Thr Pro Ala 485 490 495 Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr 500 505 510 Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Ile 515 520 525 Thr Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu 530 535 540 Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser 545 550 555 560 Ala Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro Gly Pro 565 570 575 Lys Ala Pro Tyr Thr Ser Thr Leu Leu Pro Pro Glu Leu Ser Glu Thr 580 585 590 Pro Asn Ala Thr Gln Pro Glu Leu Ala Pro Glu Asp Pro Glu Asp Ser 595 600 605 Ala Leu Leu Glu Asp Pro Val Gly Thr Val Ala Pro Gln Ile Pro Pro 610 615 620 Asn Trp His Ile Pro Ser Ile Gln Asp Ala Ala Thr Pro Tyr His Pro 625 630 635 640 Pro Ala Thr Pro Asn Asn Met Gly Leu Ile Ala Gly Ala Val Gly Gly 645 650 655 Ser Leu Leu Ala Ala Leu Val Ile Cys Gly Ile Val Tyr Trp Met His 660 665 670 Arg Arg Thr Arg Lys Ala Pro Lys Arg Ile Arg Leu Pro His Ile Arg 675 680 685 Glu Asp Asp Gln Pro Ser Ser His Gln Pro Leu Phe Tyr 690 695 700 <210> 17 <211> 2106 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic polynucleotide" <400> 17 atgggggggg ctgccgccag gttgggggcc gtgattttgt ttgtcgtcat agtgggcctc 60 catggggtcc gcggcaaata tgccttggcg gatgcctctc tcaagatggc cgaccccaat 120 cgctttcgcg gcaaagacct tccggtcctg gaccagctga ccgaccctcc gggggtccgg 180 cgcgtgtacc acatccaggc gggcctaccg gacccgttcc agccccccag cctcccgatc 240 acggtttact acgccgtgtt ggagcgcgcc tgccgcagcg tgctcctaaa cgcaccgtcg 300 gaggcccccc agattgtccg cggggcctcc gaagacgtcc ggaaacaacc ctacaacctg 360 accatcgctt ggtttcggat gggaggcaac tgtgctatcc ccatcacggt catggagtac 420 accgaatgct cctacaacaa gtctctgggg gcctgtccca tccgaacgca gccccgctgg 480 aactactatg acagcttcag cgccgtcagc gaggataacc tggggttcct gatgcacgcc 540 cccgcgtttg agaccgccgg cacgtacctg cggctcgtga agataaacga ctggacggag 600 attacacagt ttatcctgga gcaccgagcc aagggctcct gtaagtacgc cctcccgctg 660 cgcatccccc cgtcagcctg cctctccccc caggcctacc agcagggggt gacggtggac 720 agcatcggga tgctgccccg cttcatcccc gagaaccagc gcaccgtcgc cgtatacagc ttgaagatcg ccgggtggca cggggcccccc ctgagctacc agcacttcag gaagctgctg 840 ctgctggacg aggaggccgg ccccctggag gaggagctgc ccaggctggc cgacgagggc 900 ctgaacagga gggtggccga ggacctgaac ctgggcaacc tgaacgtgag catcccctgg acccacaagg tgggcaactt caccggcctg tacagcagca ccgtgcccgt gttcaacccc gagtggcaga cccccagctt ccccaagatc cacctgcagg aggacatcgt ggacaggtgc 1080 aagcagttcg tgggtcccct gaccgtgaac gagaagagga ggctgaagct gatcatgccc gccaggttct accccaacgt gccaagtac ctgcccctgg acaagggcat caagccctac taccccgagc accccgtga ccactacttc cagaccaggc actacctgca caccctgtgg aaggccggca tcctgtacaa gagggagacc accaggagcg cggcttctg cggcagcccc tacagctggg agcaggagct gcagcacggc agctgctggt ggctgcagtt caggaacagc aagccctgca gcgagtactg cctgacccac ctggtgaacc tgctggagga ctggggtccc 1440 tgcgacgagc acggcgagca ccacatcagg atccccagga cccccgccag ggtgaccggc 1500 ggcgtgttcc tggtggacaa gaacccccac aacaccgccg agagcaggct ggtggtggac 1560 ttcagccagt tcagcagggg catcaccagg gtgagctggc ccaagttcgc cgtgcccaac 1620 ctgcagagcc tgaccaacct gctgagcagc aacctgagct ggctgagcct ggacgtgagc 1680 gccgccttct accacatccc cctgcacccc gccgccatgc ccgggcccaa ggccccatac 1740 acgagcaccc tgctgccccc ggagctgtcc gagaccccca acgccacgca gccagaactc 1800 gccccggaag accccgagga ttcggccctc ttggaggacc ccgtggggac ggtggcgccg 1860 caaatcccac caaactggca catcccgtcg atccaggacg ccgcgacgcc ttaccatccc 1920 ccggccaccc cgaacaacat gggcctgatc gccggcgcgg tgggcggcag tctcctggca 1980 gccctggtca tttgcggaat tgtgtactgg atgcaccgcc gcactcggaa agccccaaag 2040 cgcatacgcc tcccccacat ccgggaagac gaccagccgt cctcgcacca gcccttgttt 2100 tactag 2106 <210> 18 <211> 699 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 18 Met Gly Gly Ala Ala Ala Arg Leu Gly Ala Val Ile Leu Phe Val Val 1 5 10 15 Ile Val Gly Leu His Gly Val Arg Gly Lys Tyr Ala Leu Ala Asp Ala 20 25 30 Ser Leu Lys Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp Leu Pro 35 40 45 Val Leu Asp Gln Leu Thr Asp Pro Pro Gly Val Arg Arg Val Tyr His 50 55 60 Ile Gln Ala Gly Leu Pro Asp Pro Phe Gln Pro Pro Ser Leu Pro Ile 65 70 75 80 Thr Val Tyr Tyr Ala Val Leu Glu Arg Ala Cys Arg Ser Val Leu Leu 85 90 95 Asn Ala Pro Ser Glu Ala Pro Gln Ile Val Arg Gly Ala Ser Glu Asp 100 105 110 Val Arg Lys Gln Pro Tyr Asn Leu Thr Ile Ala Trp Phe Arg Met Gly 115 120 125 Gly Asn Cys Ala Ile Pro Ile Thr Val Met Glu Tyr Thr Glu Cys Ser 130 135 140 Tyr Asn Lys Ser Leu Gly Ala Cys Pro Ile Arg Thr Gln Pro Arg Trp 145 150 155 160 Asn Tyr Tyr Asp Ser Phe Ser Ala Val Ser Glu Asp Asn Leu Gly Phe 165 170 175 Leu Met His Ala Pro Ala Phe Glu Thr Ala Gly Thr Tyr Leu Arg Leu 180 185 190 Val Lys Ile Asn Asp Trp Thr Glu Ile Thr Gln Phe Ile Leu Glu His 195 200 205 Arg Ala Lys Gly Ser Cys Lys Tyr Ala Leu Pro Leu Arg Ile Pro Pro 210 215 220 Ser Ala Cys Leu Ser Pro Gln Ala Tyr Gln Gln Gly Val Thr Val Asp 225 230 235 240 Ser Ile Gly Met Leu Pro Arg Phe Ile Pro Glu Asn Gln Arg Thr Val 245 250 255 Ala Val Tyr Ser Leu Lys Ile Ala Gly Trp His Gly Pro His Leu Leu 260 265 270 Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Ser Ser Asn 275 280 285 Ser Arg Ile Ile Asn His Gln His Gly Thr Met Gln Asn Leu His Asp 290 295 300 Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu Tyr Lys Thr 305 310 315 320 Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Lys Thr 325 330 335 Lys Arg Trp Gly Tyr Ser Leu Asn Phe Met Gly Tyr Val Ile Gly Ser 340 345 350 Trp Gly Ser Leu Pro Gln Asp His Ile Ile Gln Lys Ile Lys Glu Cys 355 360 365 Phe Arg Lys Leu Pro Val Asn Arg Pro Ile Asp Trp Lys Val Cys Gln 370 375 380 Arg Ile Val Gly Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly 385 390 395 400 Tyr Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala 405 410 415 Phe Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Ser Lys Gln Tyr Leu 420 425 430 Asn Leu Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys Gln Val Phe 435 440 445 Ala Asp Ala Thr Pro Thr Gly Trp Gly Leu Ala Met Gly His Gln Arg 450 455 460 Met Arg Gly Thr Phe Val Ala Pro Leu Pro Ile His Thr Ala Glu Leu 465 470 475 480 Leu Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Lys Ile Leu Gly 485 490 495 Thr Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp 500 505 510 Leu Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val 515 520 525 Tyr Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg 530 535 540 Leu Gly Leu Ser Arg Pro Leu Leu Arg Leu Pro Phe Arg Pro Thr Thr 545 550 555 560 Gly Arg Thr Ser Leu Tyr Ala Val Ser Pro Ser Val Gly Pro Lys Ala 565 570 575 Pro Tyr Thr Ser Thr Leu Leu Pro Pro Glu Leu Ser Glu Thr Pro Asn 580 585 590 Ala Thr Gln Pro Glu Leu Ala Pro Glu Asp Pro Glu Asp Ser Ala Leu 595 600 605 Leu Glu Asp Pro Val Gly Thr Val Ala Pro Gln Ile Pro Pro Asn Trp 610 615 620 His Ile Pro Ser Ile Gln Asp Ala Ala Thr Pro Tyr His Pro Pro Ala 625 630 635 640 Thr Pro Asn Asn Met Gly Leu Ile Ala Gly Ala Val Gly Gly Ser Leu 645 650 655 Leu Ala Ala Leu Val Ile Cys Gly Ile Val Tyr Trp Met His Arg Arg 660 665 670 Thr Arg Lys Ala Pro Lys Arg Ile Arg Leu Pro His Ile Arg Glu Asp 675 680 685 Asp Gln Pro Ser Ser His Gln Pro Leu Phe Tyr 690 695 <210> 19 <211> 2100 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic polynucleotide" <400> 19 atgggggggg ctgccgccag gttgggggcc gtgattttgt ttgtcgtcat agtgggcctc 60 catggggtcc gcggcaaata tgccttggcg gatgcctctc tcaagatggc cgaccccaat 120 cgctttcgcg gcaaagacct tccggtcctg gaccagctga ccgaccctcc gggggtccgg 180 cgcgtgtacc acatccaggc gggcctaccg gacccgttcc agccccccag cctcccgatc 240 acggtttact acgccgtgtt ggagcgcgcc tgccgcagcg tgctcctaaa cgcaccgtcg 300 gaggcccccc agattgtccg cggggcctcc gaagacgtcc ggaaacaacc ctacaacctg 360 accatcgctt ggtttcggat gggaggcaac tgtgctatcc ccatcacggt catggagtac 420 accgaatgct cctacaacaa gtctctgggg gcctgtccca tccgaacgca gccccgctgg 480 aactactatg acagcttcag cgccgtcagc gaggataacc tggggttcct gatgcacgcc 540 cccgcgtttg agaccgccgg cacgtacctg cggctcgtga agataaacga ctggacggag 600 attacacagt ttatcctgga gcaccgagcc aagggctcct gtaagtacgc cctcccgctg 660 cgcatccccc cgtcagcctg cctctccccc caggcctacc agcagggggt gacggtggac 720 agcatcggga tgctgccccg cttcatcccc gagaaccagc gcaccgtcgc cgtatacagc 780 ttgaagatcg ccgggtggca cgggccccac ctgctggtgg gcagcagcgg cctgagcagg 840 tacgtggcca ggctgagcag caacagcagg atcatcaacc accagcacgg caccatgcag 900 aacctgcacg acagctgcag caggaacctg tacgtgagcc tgctgctgct gtacaagacc 960 ttcggcagga agctgcacct gtacagccac cccatcatcc tgaagaccaa gaggtggggc 1020 tacagcctga acttcatggg ctacgtgatc ggcagctggg gcagcctgcc ccaggaccac 1080 atcatccaga agatcaagga gtgcttcagg aagctgcccg tgaacaggcc catcgactgg 1140 aaggtgtgcc agaggatcgt gggcctgctg ggcttcgccg cccccttcac ccagtgcggc 1200 taccccgccc tgatgcccct gtacgcctgc atccagagca agcaggcctt caccttcagc 1260 cccacctaca aggccttcct gagcaagcag tacctgaacc tgtaccccgt ggccaggcag 1320 aggcccggcc tgtgccaggt gttcgccgac gccaccccca ccggctgggg cctggccatg 1380 ggccaccaga ggatgagggg caccttcgtg gcccccctgc ccatccacac cgccgagctg 1440 ctggccgcct gcttgccag gagcaggagc ggcgccaaga tcctgggcac cgacaacagc 1500 gtggtgctga gcaggaagta caccagcttc ccctggctgc tgggctgcgc cgccaactgg 1560 atcctgaggg gcaccagctt cgtgtacgtg cccagcgccc tgaaccccgc cgacgacccc 1620 agcaggggca ggctgggcct gagcaggccc ctgctgaggc tgcccttcag gcccaccacc 1680 ggcaggacca gcctgtacgc cgtgagcccc agcgtggggc ccaaggcccc atacacgagc 1740 accctgctgc ccccggagct gtccgagacc cccaacgcca cgcagccaga actcgccccg 1800 gaagaccccg aggattcggc cctcttggag gaccccgtgg ggacggtggc gccgcaaatc 1860 ccaccaaact ggcacatccc gtcgatccag gacgccgcga cgccttacca tcccccggcc 1920 accccgaaca acatgggcct gatcgccggc gcggtgggcg gcagtctcct ggcagccctg 1980 gtcatttgcg gaattgtgta ctggatgcac cgccgcactc ggaaagcccc aaagcgcata 2040 cgcctccccc acatccggga agacgaccag ccgtcctcgc accagccctt gttttactag 2100 <210> 20 <211> 15 <212> PRT <213> Artificial Sequence <220> <221> Source <223> / Remark="Description of artificial sequence: Synthetic peptide" <400> 20 Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 <210> 21 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> twenty one Lys Glu Phe Gly Ala Thr Val Glu Leu Leu Ser Phe Leu Pro Ser 1 5 10 15 <210> twenty two <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> twenty two Thr Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser 1 5 10 15 <210> twenty three <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> twenty three Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu 1 5 10 15 <210> twenty four <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> twenty four Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp Thr Ala Ser Ala 1 5 10 15 <210> 25 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 25 Ile Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala 1 5 10 15 <210> 26 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 26 Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu 1 5 10 15 <210> 27 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 27 Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys Ser Pro His 1 5 10 15 <210> 28 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 28 Leu Glu Ser Pro Glu His Cys Ser Pro His His Thr Ala Leu Arg 1 5 10 15 <210> 29 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 29 His Cys Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys 1 5 10 15 <210> 30 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 30 His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met 1 5 10 15 <210> 31 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 31 Gln Ala Ile Leu Cys Trp Gly Glu Leu Met Thr Leu Ala Thr Trp 1 5 10 15 <210> 32 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 32 Trp Gly Glu Leu Met Thr Leu Ala Thr Trp Val Gly Ser Asn Leu 1 5 10 15 <210> 33 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 33 Thr Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala Ser 1 5 10 15 <210> 34 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 34 Val Gly Ser Asn Leu Glu Asp Pro Ala Ser Arg Glu Leu Val Val 1 5 10 15 <210> 35 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 35 Glu Asp Pro Ala Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val 1 5 10 15 <210> 36 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 36 Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys 1 5 10 15 <210> 37 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 37 Ser Tyr Val Asn Val Asn Met Gly Leu Lys Ile Arg Gln Leu Leu 1 5 10 15 <210> 38 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 38 Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser 1 5 10 15 <210> 39 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 39 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly 1 5 10 15 <210> 40 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 40 Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Ile 1 5 10 15 <210> 41 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 41 Cys Leu Thr Phe Gly Arg Glu Thr Val Ile Glu Tyr Leu Val Ser 1 5 10 15 <210> 42 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 42 Arg Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile 1 5 10 15 <210> 43 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 43 Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 1 5 10 15 <210> 44 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 44 Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn 1 5 10 15 <210> 45 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 45 Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser 1 5 10 15 <210> 46 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 46 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr 1 5 10 15 <210> 47 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 47 Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg 1 5 10 15 <210> 48 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 48 Thr Leu Pro Glu Thr Thr Val Val Arg Arg Arg Asp Arg Gly Arg 1 5 10 15 <210> 49 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 49 Thr Val Val Arg Arg Arg Asp Arg Gly Arg Ser Pro Arg Arg Arg 1 5 10 15 <210> 50 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 50 Arg Asp Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg 1 5 10 15 <210> 51 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 51 Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln 1 5 10 15 <210> 52 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 52 Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg 1 5 10 15 <210> 53 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 53 Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Arg Ser Gln Ser Arg 1 5 10 15 <210> 54 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 54 Ser Pro Arg Arg Arg Arg Arg Ser Gln Ser Arg Glu Ser Gln Cys 1 5 10 15 <210> 55 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 55 Pro Leu Ser Tyr Gln His Phe Arg Lys Leu Leu Leu Leu Asp Glu 1 5 10 15 <210> 56 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 56 His Phe Arg Lys Leu Leu Leu Leu Asp Glu Glu Ala Gly Pro Leu 1 5 10 15 <210> 57 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 57 Leu Leu Leu Asp Glu Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro 1 5 10 15 <210> 58 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 58 Glu Ala Gly Pro Leu Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu 1 5 10 15 <210> 59 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 59 Glu Glu Glu Leu Pro Arg Leu Ala Asp Glu Gly Leu Asn Arg Arg 1 5 10 15 <210> 60 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 60 Arg Leu Ala Asp Glu Gly Leu Asn Arg Arg Val Ala Glu Asp Leu 1 5 10 15 <210> 61 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 61 Gly Leu Asn Arg Arg Val Ala Glu Asp Leu Asn Leu Gly Asn Leu 1 5 10 15 <210> 62 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 62 Val Ala Glu Asp Leu Asn Leu Gly Asn Leu Asn Val Ser Ile Pro 1 5 10 15 <210> 63 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 63 Asn Leu Gly Asn Leu Asn Val Ser Ile Pro Trp Thr His Lys Val 1 5 10 15 <210> 64 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 64 Asn Val Ser Ile Pro Trp Thr His Lys Val Gly Asn Phe Thr Gly 1 5 10 15 <210> 65 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 65 Trp Thr His Lys Val Gly Asn Phe Thr Gly Leu Tyr Ser Ser Thr 1 5 10 15 <210> 66 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 66 Gly Asn Phe Thr Gly Leu Tyr Ser Ser Thr Val Pro Val Phe Asn 1 5 10 15 <210> 67 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 67 Leu Tyr Ser Ser Thr Val Pro Val Phe Asn Pro Glu Trp Gln Thr 1 5 10 15 <210> 68 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 68 Val Pro Val Phe Asn Pro Glu Trp Gln Thr Pro Ser Phe Pro Lys 1 5 10 15 <210> 69 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 69 Pro Glu Trp Gln Thr Pro Ser Phe Pro Lys Ile His Lys Leu Gln Glu 1 5 10 15 <210> 70 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 70 Pro Ser Phe Pro Lys Ile His Lys Leu Gln Glu Asp Ile Val Asp Arg 1 5 10 15 <210> 71 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 71 Ile His Lys Leu Gln Glu Asp Ile Val Asp Arg Cys Lys Gln Phe Val 1 5 10 15 <210> 72 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 72 Glu Asp Ile Val Asp Arg Cys Lys Gln Phe Val Gly Pro Leu Thr Val 1 5 10 15 <210> 73 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 73 Arg Cys Lys Gln Phe Val Gly Pro Leu Thr Val Asn Glu Lys Arg Arg 1 5 10 15 <210> 74 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 74 Val Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met 1 5 10 15 <210> 75 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 75 Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pro Ala Arg Phe Tyr 1 5 10 15 <210> 76 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 76 Arg Leu Lys Leu Ile Met Pro Ala Arg Phe Tyr Pro Asn Val Thr Lys 1 5 10 15 <210> 77 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 77 Met Pro Ala Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp 1 5 10 15 <210> 78 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 78 Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gly Ile Lys Pro 1 5 10 15 <210> 79 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 79 Lys Tyr Leu Pro Leu Asp Lys Gly Ile Lys Pro Tyr Tyr Pro Glu His 1 5 10 15 <210> 80 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 80 Asp Lys Gly Ile Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr 1 5 10 15 <210> 81 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 81 Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr Phe Gln Thr Arg His 1 5 10 15 <210> 82 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 82 His Ala Val Asn His Tyr Phe Gln Thr Arg His Tyr Leu His Thr Leu 1 5 10 15 <210> 83 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 83 Tyr Phe Gln Thr Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile 1 5 10 15 <210> 84 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 84 His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Arg Glu 1 5 10 15 <210> 85 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 85 Leu Trp Lys Ala Gly Ile Leu Tyr Lys Arg Glu Thr Thr Arg Ser Ala 1 5 10 15 <210> 86 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 86 Ile Leu Tyr Lys Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser 1 5 10 15 <210> 87 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 87 Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu 1 5 10 15 <210> 88 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 88 Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu Gln Glu Leu Gln His 1 5 10 15 <210> 89 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 89 Ser Pro Tyr Ser Trp Glu Gln Glu Leu Gln His Gly Ser Cys Trp Trp 1 5 10 15 <210> 90 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 90 Glu Gln Glu Leu Gln His Gly Ser Cys Trp Trp Leu Gln Phe Arg Asn 1 5 10 15 <210> 91 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 91 His Gly Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser 1 5 10 15 <210> 92 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 92 Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser Glu Tyr Cys Leu Thr 1 5 10 15 <210> 93 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 93 Asn Ser Lys Pro Cys Ser Glu Tyr Cys Leu Thr His Leu Val Asn Leu 1 5 10 15 <210> 94 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 94 Ser Glu Tyr Cys Leu Thr His Leu Val Asn Leu Leu Glu Asp Trp Gly 1 5 10 15 <210> 95 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 95 Thr His Leu Val Asn Leu Leu Glu Asp Trp Gly Pro Cys Asp Glu His 1 5 10 15 <210> 96 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 96 Leu Leu Glu Asp Trp Gly Pro Cys Asp Glu His Gly Glu His His Ile 1 5 10 15 <210> 97 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 97 Gly Pro Cys Asp Glu His Gly Glu His His Ile Arg Ile Pro Arg Thr 1 5 10 15 <210> 98 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 98 His Gly Glu His His Ile Arg Ile Pro Arg Thr Pro Ala Arg Val Thr 1 5 10 15 <210> 99 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 99 Ile Arg Ile Pro Arg Thr Pro Ala Arg Val Thr Gly Gly Val Phe Leu 1 5 10 15 <210> 100 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 100 Thr Pro Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro 1 5 10 15 <210> 101 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 101 Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Ala Glu 1 5 10 15 <210> 102 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 102 Leu Val Asp Lys Asn Pro His Asn Thr Ala Glu Ser Arg Leu Val Val 1 5 10 15 <210> 103 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 103 Pro His Asn Thr Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe 1 5 10 15 <210> 104 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 104 Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Ile Thr 1 5 10 15 <210> 105 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 105 Val Asp Phe Ser Gln Phe Ser Arg Gly Ile Thr Arg Val Ser Trp Pro 1 5 10 15 <210> 106 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 106 Phe Ser Arg Gly Ile Thr Arg Val Ser Trp Pro Lys Phe Ala Val Pro 1 5 10 15 <210> 107 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 107 Thr Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu 1 5 10 15 <210> 108 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 108 Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser 1 5 10 15 <210> 109 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 109 Pro Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp 1 5 10 15 <210> 110 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 110 Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val 1 5 10 15 <210> 111 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 111 Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr 1 5 10 15 <210> 112 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 112 Trp Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr His Ile Pro Leu His 1 5 10 15 <210> 113 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 113 Val Ser Ala Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro 1 5 10 15 <210> 114 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 114 His Leu Leu Val Gly Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg 1 5 10 15 <210> 115 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 115 Ser Ser Gly Leu Ser Arg Tyr Val Ala Arg Leu Ser Ser Asn Ser Arg 1 5 10 15 <210> 116 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 116 Arg Tyr Val Ala Arg Leu Ser Ser Asn Ser Arg Ile Ile Asn His Gln 1 5 10 15 <210> 117 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 117 Leu Ser Ser Asn Ser Arg Ile Ile Asn His Gln His Gly Thr Met Gln 1 5 10 15 <210> 118 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 118 Arg Ile Ile Asn His Gln His Gly Thr Met Gln Asn Leu His Asp Ser 1 5 10 15 <210> 119 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 119 Gln His Gly Thr Met Gln Asn Leu His Asp Ser Cys Ser Arg Asn Leu 1 5 10 15 <210> 120 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 120 Gln Asn Leu His Asp Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu 1 5 10 15 <210> 121 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 121 Ser Cys Ser Arg Asn Leu Tyr Val Ser Leu Leu Leu Leu Tyr Lys Thr 1 5 10 15 <210> 122 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 122 Leu Tyr Val Ser Leu Leu Leu Leu Tyr Lys Thr Phe Gly Arg Lys Leu 1 5 10 15 <210> 123 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 123 Leu Leu Leu Tyr Lys Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His 1 5 10 15 <210> 124 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 124 Thr Phe Gly Arg Lys Leu His Leu Tyr Ser His Pro Ile Ile Leu Lys 1 5 10 15 <210> 125 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 125 Leu His Leu Tyr Ser His Pro Ile Ile Leu Lys Thr Lys Arg Trp Gly 1 5 10 15 <210> 126 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 126 His Pro Ile Ile Leu Lys Thr Lys Arg Trp Gly Tyr Ser Leu Asn Phe 1 5 10 15 <210> 127 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 127 Lys Thr Lys Arg Trp Gly Tyr Ser Leu Asn Phe Met Gly Tyr Val Ile 1 5 10 15 <210> 128 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 128 Gly Tyr Ser Leu Asn Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Ser 1 5 10 15 <210> 129 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 129 Phe Met Gly Tyr Val Ile Gly Ser Trp Gly Ser Leu Pro Gln Asp His 1 5 10 15 <210> 130 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 130 Ile Gly Ser Trp Gly Ser Leu Pro Gln Asp His Ile Ile Gln Lys Ile 1 5 10 15 <210> 131 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 131 Ser Leu Pro Gln Asp His Ile Ile Gln Lys Ile Lys Glu Cys Phe Arg 1 5 10 15 <210> 132 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 132 His Ile Ile Gln Lys Ile Lys Glu Cys Phe Arg Lys Leu Pro Val Asn 1 5 10 15 <210> 133 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 133 Ile Lys Glu Cys Phe Arg Lys Leu Pro Val Asn Arg Pro Ile Asp Trp 1 5 10 15 <210> 134 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 134 Arg Lys Leu Pro Val Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg 1 5 10 15 <210> 135 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 135 Asn Arg Pro Ile Asp Trp Lys Val Cys Gln Arg Ile Val Gly Leu Leu 1 5 10 15 <210> 136 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 136 Trp Lys Val Cys Gln Arg Ile Val Gly Leu Leu Gly Phe Ala Ala Pro 1 5 10 15 <210> 137 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 137 Arg Ile Val Gly Leu Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly 1 5 10 15 <210> 138 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 138 Leu Gly Phe Ala Ala Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met 1 5 10 15 <210> 139 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 139 Pro Phe Thr Gln Cys Gly Tyr Pro Ala Leu Met Pro Leu Tyr Ala Cys 1 5 10 15 <210> 140 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 140 Gly Tyr Pro Ala Leu Met Pro Leu Tyr Ala Cys Ile Gln Ser Lys Gln 1 5 10 15 <210> 141 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 141 Met Pro Leu Tyr Ala Cys Ile Gln Ser Lys Gln Ala Phe Thr Phe Ser 1 5 10 15 <210> 142 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 142 Cys Ile Gln Ser Lys Gln Ala Phe Thr Phe Ser Pro Thr Tyr Lys Ala 1 5 10 15 <210> 143 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 143 Gln Ala Phe Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Ser Lys Gln 1 5 10 15 <210> 144 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 144 Ser Pro Thr Tyr Lys Ala Phe Leu Ser Lys Gln Tyr Leu Asn Leu Tyr 1 5 10 15 <210> 145 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 145 Ala Phe Leu Ser Lys Gln Tyr Leu Asn Leu Tyr Pro Val Ala Arg Gln 1 5 10 15 <210> 146 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 146 Gln Tyr Leu Asn Leu Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys 1 5 10 15 <210> 147 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 147 Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp 1 5 10 15 <210> 148 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 148 Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Thr Gly 1 5 10 15 <210> 149 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 149 Cys Gln Val Phe Ala Asp Ala Thr Pro Thr Gly Trp Gly Leu Ala Met 1 5 10 15 <210> 150 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 150 Asp Ala Thr Pro Thr Gly Trp Gly Leu Ala Met Gly His Gln Arg Met 1 5 10 15 <210> 151 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 151 Gly Trp Gly Leu Ala Met Gly His Gln Arg Met Arg Gly Thr Phe Val 1 5 10 15 <210> 152 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 152 Met Gly His Gln Arg Met Arg Gly Thr Phe Val Ala Pro Leu Pro Ile 1 5 10 15 <210> 153 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 153 Met Arg Gly Thr Phe Val Ala Pro Leu Pro Ile His Thr Ala Glu Leu 1 5 10 15 <210> 154 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 154 Val Ala Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe 1 5 10 15 <210> 155 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 155 Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Ala Arg Ser Arg Ser 1 5 10 15 <210> 156 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 156 Leu Leu Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Lys Ile Leu 1 5 10 15 <210> 157 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 157 Phe Ala Arg Ser Arg Ser Gly Ala Lys Ile Leu Gly Thr Asp Asn Ser 1 5 10 15 <210> 158 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 158 Ser Gly Ala Lys Ile Leu Gly Thr Asp Asn Ser Val Val Leu Ser Arg 1 5 10 15 <210> 159 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 159 Leu Gly Thr Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Phe 1 5 10 15 <210> 160 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 160 Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly 1 5 10 15 <210> 161 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 161 Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Ala Asn Trp 1 5 10 15 <210> 162 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 162 Phe Pro Trp Leu Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr 1 5 10 15 <210> 163 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 163 Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val 1 5 10 15 <210> 164 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 164 Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser Ala Leu Asn 1 5 10 15 <210> 165 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 165 Thr Ser Phe Val Tyr Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro 1 5 10 15 <210> 166 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 166 Val Pro Ser Ala Leu Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu 1 5 10 15 <210> 167 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 167 Asn Pro Ala Asp Asp Pro Ser Arg Gly Arg Leu Gly Leu Ser Arg Pro 1 5 10 15 <210> 168 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 168 Pro Ser Arg Gly Arg Leu Gly Leu Ser Arg Pro Leu Leu Arg Leu Pro 1 5 10 15 <210> 169 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 169 Leu Gly Leu Ser Arg Pro Leu Leu Arg Leu Pro Phe Arg Pro Thr Thr 1 5 10 15 <210> 170 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 170 Pro Leu Leu Arg Leu Pro Phe Arg Pro Thr Thr Gly Arg Thr Ser Leu 1 5 10 15 <210> 171 <211> 16 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 171 Pro Phe Arg Pro Thr Thr Gly Arg Thr Ser Leu Tyr Ala Val Ser Pro 1 5 10 15 <210> 172 <211> 13 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 172 Thr Gly Arg Thr Ser Leu Tyr Ala Val Ser Pro Ser Val 1 5 10 <210> 173 <211> 200 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 173 His Phe Arg Lys Leu Leu Leu Leu Asp Glu Glu Ala Gly Pro Leu Glu 1 5 10 15 Glu Glu Leu Pro Arg Leu Ala Asp Glu Gly Leu Asn Arg Arg Val Ala 20 25 30 Glu Asp Leu Asn Leu Gly Asn Leu Pro Glu Trp Gln Thr Pro Ser Phe 35 40 45 Pro Lys Ile His Leu Gln Glu Asp Ile Val Asp Arg Cys Lys Gln Phe 50 55 60 Val Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met 65 70 75 80 Pro Ala Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys 85 90 95 Gly Ile Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr Phe Gln 100 105 110 Thr Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys 115 120 125 Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp 130 135 140 Glu Gln Glu Leu Gln His Gly Ser Cys Trp Trp Leu Gln Phe Arg Asn 145 150 155 160 Ser Lys Pro Cys Ser Glu Tyr Cys Leu Thr His Leu Val Asn Leu Leu 165 170 175 Glu Asp Trp Gly Pro Cys Asp Glu His Gly Glu His His Ile Arg Ile 180 185 190 Pro Arg Thr Pro Ala Arg Val Thr 195 200 <210> 174 <211> 380 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 174 Tyr Leu Pro Leu Asp Lys Gly Ile Lys Pro Tyr Tyr Pro Glu His Ala 1 5 10 15 Val Asn His Tyr Phe Gln Thr Arg His Tyr Leu His Thr Leu Trp Lys 20 25 30 Ala Gly Ile Leu Tyr Lys Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys 35 40 45 Gly Ser Pro Tyr Ser Trp Glu Gln Glu Leu Gln His Gly Ser Cys Trp 50 55 60 Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser Glu Tyr Cys Leu Thr 65 70 75 80 His Leu Val Asn Leu Leu Glu Asp Trp Gly Pro Cys Asp Glu His Gly 85 90 95 Glu His His Ile Arg Ile Pro Arg Thr Pro Ala Arg Val Thr Gly Gly 100 105 110 Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Ala Glu Ser Arg Leu 115 120 125 Val Val Asp Phe Ser Gln Phe Ser Arg Gly Ile Thr Arg Val Ser Trp 130 135 140 Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser 145 150 155 160 Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Gln Ala Phe Thr Phe Ser 165 170 175 Pro Thr Tyr Lys Ala Phe Leu Ser Lys Gln Tyr Leu Asn Leu Tyr Pro 180 185 190 Val Ala Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Thr 195 200 205 Pro Thr Gly Trp Gly Leu Ala Met Gly His Gln Arg Met Arg Gly Thr 210 215 220 Phe Val Ala Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys 225 230 235 240 Phe Ala Arg Ser Arg Ser Gly Ala Lys Ile Leu Gly Thr Asp Asn Ser 245 250 255 Val Val Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys 260 265 270 Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser 275 280 285 Ala Leu Asn Pro Ala Asp Asp Val Gly Ser Asn Leu Glu Asp Pro Ala 290 295 300 Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys 305 310 315 320 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 325 330 335 Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 340 345 350 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 355 360 365 Glu Thr Thr Val Val Arg Arg Arg Asp Arg Gly Arg 370 375 380 <210> 175 <211> 410 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 175 His Phe Arg Lys Leu Leu Leu Leu Asp Glu Glu Ala Gly Pro Leu Glu 1 5 10 15 Glu Glu Leu Pro Arg Leu Ala Asp Glu Gly Leu Asn Arg Arg Val Ala 20 25 30 Glu Asp Leu Asn Leu Gly Asn Leu Pro Glu Trp Gln Thr Pro Ser Phe 35 40 45 Pro Lys Ile His Leu Gln Glu Asp Ile Val Asp Arg Cys Lys Gln Phe 50 55 60 Val Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met 65 70 75 80 Pro Ala Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys 85 90 95 Gly Ile Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr Phe Gln 100 105 110 Thr Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys 115 120 125 Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp 130 135 140 Glu Gln Glu Leu Gln His Gly Ser Cys Trp Trp Leu Gln Phe Arg Asn 145 150 155 160 Ser Lys Pro Cys Ser Glu Tyr Cys Leu Thr His Leu Val Asn Leu Leu 165 170 175 Glu Asp Trp Gly Pro Cys Asp Glu His Gly Glu His His Ile Arg Ile 180 185 190 Pro Arg Thr Pro Ala Arg Val Thr Gln Ala Phe Thr Phe Ser Pro Thr 195 200 205 Tyr Lys Ala Phe Leu Ser Lys Gln Tyr Leu Asn Leu Tyr Pro Val Ala 210 215 220 Arg Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Thr 225 230 235 240 Gly Trp Gly Leu Ala Met Gly His Gln Arg Met Arg Gly Thr Phe Val 245 250 255 Ala Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Ala 260 265 270 Arg Ser Arg Ser Gly Ala Lys Ile Leu Gly Thr Asp Asn Ser Val Val 275 280 285 Leu Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Ala 290 295 300 Asn Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser Ala Leu 305 310 315 320 Asn Pro Ala Asp Asp Val Gly Ser Asn Leu Glu Asp Pro Ala Ser Arg 325 330 335 Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys Ile Arg 340 345 350 Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr 355 360 365 Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro 370 375 380 Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr 385 390 395 400 Thr Val Val Arg Arg Arg Asp Arg Gly Arg 405 410 <210> 176 <211> 1140 <212> DNA <213> Artificial sequence <220> <221> Source <223> / Remark = "Description of artificial sequence: synthetic polynucleotide" <400> 176 tatctgccgc tggataaagg cattaaaccg tattatccgg aacatgcggt gaaccattat 60 tttcagaccc gccattatct gcataccctg tggaaagcgg gcattctgta taaacgcgaa 120 accacccgca gcgcgagctt ttgcggcagc ccgtatagct gggaacagga actgcagcat 180 ggcagctgct ggtggctgca gtttcgcaac agcaaaccgt gcagcgaata ttgcctgacc 240 catctggtga acctgctgga agattgggga ccgtgcgatg aacatggcga acatcatatt 300 cgcattccgc gcaccccggc gcgcgtgacc ggcggcgtgt ttctggtgga taaaaacccg 360 cataacaccg cggaaagccg cctggtggtg gattttagcc agtttagccg cggcattacc 420 cgcgtgagct ggccgaaatt tgcggtgccg aacctgcaga gcctgaccaa cctgctgagc 480 agcaacctga gctggctgag cctggatgtg caggcgttta cctttagccc gacctataaa 540 gcgtttctga gcaaacagta tctgaacctg tatccggtgg cgcgccagcg cccgggcctg 600 tgccaggtgt ttgcggatgc gaccccgacc ggctggggcc tggcgatggg ccatcagcgc 660 atgcgcggca cctttgtggc gccgctgccg attcataccg cggaactgct ggcggcgtgc 720 tttgcgcgca gccgcagcgg cgcgaaaatt ctgggcaccg ataacagcgt ggtgctgagc 780 cgcaaatata ccagctttcc gtggctgctg ggctgcgcgg cgaactggat tctgcgcggc 840 accagctttg tgtatgtgcc gagcgcgctg aacccggcgg atgatgtggg cagcaacctg 900 gaagatccgg cgagccgcga actggtggtg agctatgtga acgtgaacat gggcctgaaa 960 attcgccagc tgctgtggtt tcatattagc tgcctgacct ttggccgcga aaccgtgatt 1020 gaatatctgg tgagctttgg cgtgtggatt cgcaccccgc cggcgtatcg cccgccgaac 1080 gcgccgattc tgagcaccct gccggaaacc accgtggtgc gccgccgcga tcggggccgc 1140 <210> 177 <211> 1230 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / Remark="Description of artificial sequence: Synthetic polynucleotide" <400> 177 cattttcgca aactgctgct gctggatgaa gaagcgggac cgctggaaga agaactgccg 60 cgcctggcgg atgaaggcct gaaccgccgc gtggcggaag atctgaacct gggcaacctg 120 ccggaatggc agaccccgag ctttccgaaa attcatctgc aggaagatat tgtggatcgc 180 tgcaaacagt ttgtgggacc gctgaccgtg aacgaaaaac gccgcctgaa actgattatg 240 ccggcgcgct tttatccgaa cgtgaccaaa tatctgccgc tggataaagg cattaaaccg 300 tattatccgg aacatgcggt gaaccattat tttcagaccc gccattatct gcataccctg 360 tggaaagcgg gcattctgta taaacgcgaa accacccgca gcgcgagctt ttgcggcagc 420 ccgtatagct gggaacagga actgcagcat ggcagctgct ggtggctgca gtttcgcaac 480 agcaaaccgt gcagcgaata ttgcctgacc catctggtga acctgctgga agattgggga 540 ccgtgcgatg aacatggcga acatcatatt cgcattccgc gcaccccggc gcgcgtgacc 600 caggcgttta cctttagccc gacctataaa gcgtttctga gcaaacagta tctgaacctg 660 tatccggtgg cgcgccagcg cccgggcctg tgccaggtgt ttgcggatgc gaccccgacc 720 ggctggggcc tggcgatggg ccatcagcgc atgcgcggca cctttgtggc gccgctgccg 780 attcataccg cggaactgct ggcggcgtgc tttgcgcgca gccgcagcgg cgcgaaaatt 840 ctgggcaccg ataacagcgt ggtgctgagc cgcaaatata ccagctttcc gtggctgctg 900 ggctgcgcgg cgaactggat tctgcgcggc accagctttg tgtatgtgcc gagcgcgctg 960 aacccggcgg atgatgtggg cagcaacctg gaagatccgg cgagccgcga actggtggtg 1020 agctatgtga acgtgaacat gggcctgaaa attcgccagc tgctgtggtt tcatattagc 1080 tgcctgacct ttggccgcga aaccgtgatt gaatatctgg tgagctttgg cgtgtggatt 1140 cgcaccccgc cggcgtatcg cccgccgaac gcgccgattc tgagcaccct gccggaaacc 1200 accgtggtgc gccgccgaga tcgaggccgc 1230 <210> 178 <211> 170 <212> PRT <213> Artificial Sequence <220> <221> Source <223> / Remarks="Description of artificial sequence: Synthetic polypeptide" <400> 178 Tyr Leu Pro Leu Asp Lys Gly Ile Lys Pro Tyr Tyr Pro Glu His Ala 1 5 10 15 Val Asn His Tyr Phe Gln Thr Arg His Tyr Leu His Thr Leu Trp Lys 20 25 30 Ala Gly Ile Leu Tyr Lys Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys 35 40 45 Gly Ser Pro Tyr Ser Trp Glu Gln Glu Leu Gln His Gly Ser Cys Trp 50 55 60 Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser Glu Tyr Cys Leu Thr 65 70 75 80 His Leu Val Asn Leu Leu Glu Asp Trp Gly Pro Cys Asp Glu His Gly 85 90 95 Glu His His Ile Arg Ile Pro Arg Thr Pro Ala Arg Val Thr Gly Gly 100 105 110 Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Ala Glu Ser Arg Leu 115 120 125 Val Val Asp Phe Ser Gln Phe Ser Arg Gly Ile Thr Arg Val Ser Trp 130 135 140 Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser 145 150 155 160 Ser Asn Leu Ser Trp Leu Ser Leu Asp Val 165 170 <210> 179 <211> 125 <212> PRT <213> Synthetic Sequence <220> <221> Source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 179 Gln Ala Phe Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Ser Lys Gln 1 5 10 15 Tyr Leu Asn Leu Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys Gln 20 25 30 Val Phe Ala Asp Ala Thr Pro Thr Gly Trp Gly Leu Ala Met Gly His 35 40 45 Gln Arg Met Arg Gly Thr Phe Val Ala Pro Leu Pro Ile His Thr Ala 50 55 60 Glu Leu Leu Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Lys Ile 65 70 75 80 Leu Gly Thr Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Phe 85 90 95 Pro Trp Leu Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser 100 105 110 Phe Val Tyr Val Pro Ser Ala Leu Asn Pro Ala Asp Asp 115 120 125 <210> 180 <211> 85 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 180 Val Gly Ser Asn Leu Glu Asp Pro Ala Ser Arg Glu Leu Val Val Ser 1 5 10 15 Tyr Val Asn Val Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe 20 25 30 His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Ile Glu Tyr Leu 35 40 45 Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro 50 55 60 Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg 65 70 75 80 Arg Asp Arg Gly Arg 85 <210> 181 <211> 200 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 181 His Phe Arg Lys Leu Leu Leu Leu Asp Glu Glu Ala Gly Pro Leu Glu 1 5 10 15 Glu Glu Leu Pro Arg Leu Ala Asp Glu Gly Leu Asn Arg Arg Val Ala 20 25 30 Glu Asp Leu Asn Leu Gly Asn Leu Pro Glu Trp Gln Thr Pro Ser Phe 35 40 45 Pro Lys Ile His Leu Gln Glu Asp Ile Val Asp Arg Cys Lys Gln Phe 50 55 60 Val Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met 65 70 75 80 Pro Ala Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys 85 90 95 Gly Ile Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr Phe Gln 100 105 110 Thr Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys 115 120 125 Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp 130 135 140 Glu Gln Glu Leu Gln His Gly Ser Cys Trp Trp Leu Gln Phe Arg Asn 145 150 155 160 Ser Lys Pro Cys Ser Glu Tyr Cys Leu Thr His Leu Val Asn Leu Leu 165 170 175 Glu Asp Trp Gly Pro Cys Asp Glu His Gly Glu His His Ile Arg Ile 180 185 190 Pro Arg Thr Pro Ala Arg Val Thr 195 200 <210> 182 <211> 125 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 182 Gln Ala Phe Thr Phe Ser Pro Thr Tyr Lys Ala Phe Leu Ser Lys Gln 1 5 10 15 Tyr Leu Asn Leu Tyr Pro Val Ala Arg Gln Arg Pro Gly Leu Cys Gln 20 25 30 Val Phe Ala Asp Ala Thr Pro Thr Gly Trp Gly Leu Ala Met Gly His 35 40 45 Gln Arg Met Arg Gly Thr Phe Val Ala Pro Leu Pro Ile His Thr Ala 50 55 60 Glu Leu Leu Ala Ala Cys Phe Ala Arg Ser Arg Ser Gly Ala Lys Ile 65 70 75 80 Leu Gly Thr Asp Asn Ser Val Val Leu Ser Arg Lys Tyr Thr Ser Phe 85 90 95 Pro Trp Leu Leu Gly Cys Ala Ala Asn Trp Ile Leu Arg Gly Thr Ser 100 105 110 Phe Val Tyr Val Pro Ser Ala Leu Asn Pro Ala Asp Asp 115 120 125 <210> 183 <211> 85 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 183 Val Gly Ser Asn Leu Glu Asp Pro Ala Ser Arg Glu Leu Val Val Ser 1 5 10 15 Tyr Val Asn Val Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe 20 25 30 His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Ile Glu Tyr Leu 35 40 45 Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro 50 55 60 Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg 65 70 75 80 Arg Asp Arg Gly Arg 85 <210> 184 <211> 2331 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: Synthetic polynucleotide" <400> 184 atgggggggg ctgccgccag gttgggggcc gtgattttgt ttgtcgtcat agtgggcctc 60 catggggtcc gcggcaaata tgccttggcg gatgcctctc tcaagatggc cgaccccaat 120 cgctttcgcg gcaaagacct tccggtcctg gaccagctga ccgaccctcc gggggtccgg 180 cgcgtgtacc acatccaggc gggcctaccg gacccgttcc agccccccag cctcccgatc 240 acggtttact acgccgtgtt ggagcgcgcc tgccgcagcg tgctcctaaa cgcaccgtcg 300 gaggcccccc agattgtccg cggggcctcc gaagacgtcc ggaaacaacc ctacaacctg 360 accatcgctt ggtttcggat gggaggcaac tgtgctatcc ccatcacggt catggagtac 420 accgaatgct cctacaacaa gtctctgggg gcctgtccca tccgaacgca gccccgctgg 480 aactactatg acagcttcag cgccgtcagc gaggataacc tggggttcct gatgcacgcc 540 cccgcgtttg agaccgccgg cacgtacctg cggctcgtga agataaacga ctggacggag 600 attacacagt ttatcctgga gcaccgagcc aagggctcct gtaagtacgc cctcccgctg 660 cgcatccccc cgtcagcctg cctctccccc caggcctacc agcagggggt gacggtggac 720 agcatcggga tgctgccccg cttcatcccc gagaaccagc gcaccgtcgc cgtatacagc 780 ttgaagatcg ccgggtggca cgggccctat ctgccgctgg ataaaggcat taaaccgtat 840 tatccggaac atgcggtgaa ccattatttt cagacccgcc attatctgca taccctgtgg 900 aaagcgggca ttctgtataa acgcgaaacc acccgcagcg cgagcttttg cggcagcccg 960 tatagctgggg aacaggaact gcagcatggc agctctctggt ggctgcagtt tcgcaacagc 1020 aaaccgtgca gcgaatattg cctgacccat ctggtgaacc tgctggaaga ttggggaccg 1080 tgcgatgaac atggcgaaca tcatattcgc attccgcgca ccccggcgcg cgtgaccggc 1140 ggcgtgtttc tggtggataa aaacccgcat aacaccgcgg aaagccgcct ggtggtggat 1200 tttagccagt ttagccgcgg cattacccgc gtgagctggc cgaaatttgc ggtgccgaac 1260 ctgcagagcc tgaccaacct gctgagcagc aacctgagct ggctgagcct ggatgtgcag 1320 gcgtttacct ttagcccgac ctataaagcg tttctgagca aacagtatct gaacctgtat 1380 ccggtggcgc gccagcgccc gggcctgtgc caggtgtttg cggatgcgac cccgaccggc 1440 tggggcctgg cgatgggcca tcagcgcatg cgcggcacct ttgtggcgcc gctgccgatt 1500 cataccgcgg aactgctggc ggcgtgcttt gcgcgcagcc gcagcggcgc gaaaattctg 1560 ggcaccgata acagcgtggt gctgagccgc aatatacca gctttccgtg gctgctgggc 1620 tgcgcggcga actggattct gcgcggcacc agctttgtgt atgtgccgag cgcgctgaac 1680 ccggcggatg atgtgggcag caacctggaa gatccggcga gccgcgaact ggtggtgagc 1740 tatgtgaacg tgaacatggg cctgaaaatt cgccagctgc tgtggtttca tattagctgc 1800 ctgaccttg gccgcgaaac cgtgattgaa tatctggtga gctttggcgt gtggattcgc 1860 accccgccgg cgtatcgccc gccgaacgcg ccgattctga gcaccctgcc ggaaaccacc 1920 gtggtgcgcc gccgcgatcg gggccgcggg cccaaggccc catacacgag caccctgctg 1980 ccccggagc tgtccgagac ccccaacgcc acgcagccag aactcgcccc ggaagacccc 2040 gaggattcgg ccctcttgga ggaccccgtg gggacggtgg cgccgcaaat cccaccaaac 2100 tggcacatcc cgtcgatcca ggacgccgcg acgccttacc atcccccggc caccccgaac 2160 aacatgggcc tgatcgccgg cgcggtgggc ggcagtctcc tggcagccct ggtcatttgc 2220 ggaattgtgt actggatgca ccgccgcact cggaaagccc caaagcgcat acgcctcccc 2280 cacatccggg aagacgacca gccgtcctcg caccagccct tgttttacta g 2331 <210> 185 <211> 776 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 185 Met Gly Gly Ala Ala Ala Arg Leu Gly Ala Val Ile Leu Phe Val Val 1 5 10 15 Ile Val Gly Leu His Gly Val Arg Gly Lys Tyr Ala Leu Ala Asp Ala 20 25 30 Ser Leu Lys Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp Leu Pro 35 40 45 Val Leu Asp Gln Leu Thr Asp Pro Pro Gly Val Arg Arg Val Tyr His 50 55 60 Ile Gln Ala Gly Leu Pro Asp Pro Phe Gln Pro Pro Ser Leu Pro Ile 65 70 75 80 Thr Val Tyr Tyr Ala Val Leu Glu Arg Ala Cys Arg Ser Val Leu Leu 85 90 95 Asn Ala Pro Ser Glu Ala Pro Gln Ile Val Arg Gly Ala Ser Glu Asp 100 105 110 Val Arg Lys Gln Pro Tyr Asn Leu Thr Ile Ala Trp Phe Arg Met Gly 115 120 125 Gly Asn Cys Ala Ile Pro Ile Thr Val Met Glu Tyr Thr Glu Cys Ser 130 135 140 Tyr Asn Lys Ser Leu Gly Ala Cys Pro Ile Arg Thr Gln Pro Arg Trp 145 150 155 160 Asn Tyr Tyr Asp Ser Phe Ser Ala Val Ser Glu Asp Asn Leu Gly Phe 165 170 175 Leu Met His Ala Pro Ala Phe Glu Thr Ala Gly Thr Tyr Leu Arg Leu 180 185 190 Val Lys Ile Asn Asp Trp Thr Glu Ile Thr Gln Phe Ile Leu Glu His 195 200 205 Arg Ala Lys Gly Ser Cys Lys Tyr Ala Leu Pro Leu Arg Ile Pro Pro 210 215 220 Ser Ala Cys Leu Ser Pro Gln Ala Tyr Gln Gln Gly Val Thr Val Asp 225 230 235 240 Ser Ile Gly Met Leu Pro Arg Phe Ile Pro Glu Asn Gln Arg Thr Val 245 250 255 Ala Val Tyr Ser Leu Lys Ile Ala Gly Trp His Gly Pro Tyr Leu Pro 260 265 270 Leu Asp Lys Gly Ile Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His 275 280 285 Tyr Phe Gln Thr Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile 290 295 300 Leu Tyr Lys Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro 305 310 315 320 Tyr Ser Trp Glu Gln Glu Leu Gln His Gly Ser Cys Trp Trp Leu Gln 325 330 335 Phe Arg Asn Ser Lys Pro Cys Ser Glu Tyr Cys Leu Thr His Leu Val 340 345 350 Asn Leu Leu Glu Asp Trp Gly Pro Cys Asp Glu His Gly Glu His His 355 360 365 Ile Arg Ile Pro Arg Thr Pro Ala Arg Val Thr Gly Gly Val Phe Leu 370 375 380 Val Asp Lys Asn Pro His Asn Thr Ala Glu Ser Arg Leu Val Val Asp 385 390 395 400 Phe Ser Gln Phe Ser Arg Gly Ile Thr Arg Val Ser Trp Pro Lys Phe 405 410 415 Ala Val Pro Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu 420 425 430 Ser Trp Leu Ser Leu Asp Val Gln Ala Phe Thr Phe Ser Pro Thr Tyr 435 440 445 Lys Ala Phe Leu Ser Lys Gln Tyr Leu Asn Leu Tyr Pro Val Ala Arg 450 455 460 Gln Arg Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Thr Gly 465 470 475 480 Trp Gly Leu Ala Met Gly His Gln Arg Met Arg Gly Thr Phe Val Ala 485 490 495 Pro Leu Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Ala Arg 500 505 510 Ser Arg Ser Gly Ala Lys Ile Leu Gly Thr Asp Asn Ser Val Val Leu 515 520 525 Ser Arg Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Ala Asn 530 535 540 Trp Ile Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser Ala Leu Asn 545 550 555 560 Pro Ala Asp Asp Val Gly Ser Asn Leu Glu Asp Pro Ala Ser Arg Glu 565 570 575 Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys Ile Arg Gln 580 585 590 Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val 595 600 605 Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala 610 615 620 Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr 625 630 635 640 Val Val Arg Arg Arg Asp Arg Gly Arg Gly Pro Lys Ala Pro Tyr Thr 645 650 655 Ser Thr Leu Leu Pro Pro Glu Leu Ser Glu Thr Pro Asn Ala Thr Gln 660 665 670 Pro Glu Leu Ala Pro Glu Asp Pro Glu Asp Ser Ala Leu Leu Glu Asp 675 680 685 Pro Val Gly Thr Val Ala Pro Gln Ile Pro Pro Asn Trp His Ile Pro 690 695 700 Ser Ile Gln Asp Ala Ala Thr Pro Tyr His Pro Pro Ala Thr Pro Asn 705 710 715 720 Asn Met Gly Leu Ile Ala Gly Ala Val Gly Gly Ser Leu Leu Ala Ala 725 730 735 Leu Val Ile Cys Gly Ile Val Tyr Trp Met His Arg Arg Thr Arg Lys 740 745 750 Ala Pro Lys Arg Ile Arg Leu Pro His Ile Arg Glu Asp Asp Gln Pro 755 760 765 Ser Ser His Gln Pro Leu Phe Tyr 770 775 <210> 186 <211> 2421 <212> DNA <213> Artificial sequence <220> <221> Source <223> / Remark="Description of artificial sequence: synthetic polynucleotide" <400> 186 atgggggggg ctgccgccag gttgggggcc gtgattttgt ttgtcgtcat agtgggcctc 60 catggggtcc gcggcaaata tgccttggcg gatgcctctc tcaagatggc cgaccccaat 120 cgctttcgcg gcaaagacct tccggtcctg gaccagctga ccgaccctcc gggggtccgg 180 cgcgtgtacc acatccaggc gggcctaccg gacccgttcc agccccccag cctcccgatc 240 acggtttact acgccgtgtt ggagcgcgcc tgccgcagcg tgctcctaaa cgcaccgtcg 300 gaggcccccc agattgtccg cggggcctcc gaagacgtcc ggaaacaacc ctacaacctg 360 accatcgctt ggtttcggat gggaggcaac tgtgctatcc ccatcacggt catggagtac 420 accgaatgct cctacaacaa gtctctgggg gcctgtccca tccgaacgca gccccgctgg 480 aactactatg acagcttcag cgccgtcagc gaggataacc tggggttcct gatgcacgcc 540 cccgcgtttg agaccgccgg cacgtacctg cggctcgtga agataaacga ctggacggag 600 attacacagt ttatcctgga gcaccgagcc aagggctcct gtaagtacgc cctcccgctg 660 cgcatccccc cgtcagcctg cctctccccc caggcctacc agcagggggt gacggtggac 720 agcatcggga tgctgccccg cttcatcccc gagaaccagc gcaccgtcgc cgtatacagc 780 ttgaagatcg ccgggtggca cgggccccat tttcgcaaac tgctgctgct ggatgaagaa 840 gcgggaccgc tggaagaaga actgccgcgc ctggcggatg aaggcctgaa ccgccgcgtg 900 gcggagaatc tgaacctggg caacctgccg gaatggcaga ccccgagctt tccgaaaatt 960 catctgcagg aagatattgt ggatcgctgc aaacagtttg tgggaccgct gaccgtgaac 1020 gaaaaacgcc gcctgaaact gattatgccg gcgcgctttt atccgaacgt gaccaaatat 1080 ctgccgctgg ataaaggcat taaaccgtat tatccggaac atgcggtgaa ccattatttt 1140 cagacccgcc attatctgca taccctgtgg aaagcgggca ttctgtataa acgcgaaacc 1200 acccgcagcg cgagcttttg cggcagcccg tatagctgggg aacaggaact gcagcatggc 1260 agctgctggt ggctgcagtt tcgcaacagc aaaccgtgca gcgaatattg cctgacccat 1320 ctggtgaacc tgctggaaga ttggggaccg tgcgatgaac atggcgaaca tcatattcgc 1380 attccgcgca ccccggcgcg cgtgacccag gcgtttacct ttagcccgac ctataaagcg 1440 tttctgagca aacagtatct gaacctgtat ccggtggcgc gccagcgccc gggcctgtgc 1500 caggtgtttg cggatgcgac cccgaccggc tggggcctgg cgatgggcca tcagcgcatg 1560 cgcggcacct ttgtggcgcc gctgccgatt cataccgcgg aactgctggc ggcgtgcttt 1620 gcgcgcagcc gcagcggcgc gaaaattctg ggcaccgata acagcgtggt gctgagccgc 1680 aaatatacca gctttccgtg gctgctgggc tgcgcggcga actggattct gcgcggcacc 1740 agctttgtgt atgtgccgag cgcgctgaac ccggcggatg atgtgggcag caacctggaa 1800 gatccggcga gccgcgaact ggtggtgagc tatgtgaacg tgaacatggg cctgaaaatt 1860 cgccagctgc tgtggtttca tattagctgc ctgacctttg gccgcgaaac cgtgattgaa 1920 tatctggtga gctttggcgt gtggattcgc accccgccgg cgtatcgccc gccgaacgcg 1980 ccgattctga gcaccctgcc ggaaaccacc gtggtgcgcc gccgagatcg aggccgcggg 2040 cccaaggccc catacacgag caccctgctg cccccggagc tgtccgagac ccccaacgcc 2100 acgcagccag aactcgcccc ggaagacccc gaggattcgg ccctcttgga ggaccccgtg 2160 gggacggtgg cgccgcaaat cccaccaaac tggcacatcc cgtcgatcca ggacgccgcg 2220 acgccttacc atcccccggc caccccgaac aacatgggcc tgatcgccgg cgcggtgggc 2280 ggcagtctcc tggcagccct ggtcatttgc ggaattgtgt actggatgca ccgccgcact 2340 cggaaagcccc caaagcgcat acgcctcccc cacatccggg aagacgacca gccgtcctcg 2400 caccagccct tgttttacta g 2421 <210> 187 <211> 806 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic polypeptide" <400> 187 Met Gly Gly Ala Ala Ala Arg Leu Gly Ala Val Ile Leu Phe Val Val 1 5 10 15 Ile Val Gly Leu His Gly Val Arg Gly Lys Tyr Ala Leu Ala Asp Ala 20 25 30 Ser Leu Lys Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp Leu Pro 35 40 45 Val Leu Asp Gln Leu Thr Asp Pro Pro Gly Val Arg Arg Val Tyr His 50 55 60 Ile Gln Ala Gly Leu Pro Asp Pro Phe Gln Pro Pro Ser Leu Pro Ile 65 70 75 80 Thr Val Tyr Tyr Ala Val Leu Glu Arg Ala Cys Arg Ser Val Leu Leu 85 90 95 Asn Ala Pro Ser Glu Ala Pro Gln Ile Val Arg Gly Ala Ser Glu Asp 100 105 110 Val Arg Lys Gln Pro Tyr Asn Leu Thr Ile Ala Trp Phe Arg Met Gly 115 120 125 Gly Asn Cys Ala Ile Pro Ile Thr Val Met Glu Tyr Thr Glu Cys Ser 130 135 140 Tyr Asn Lys Ser Leu Gly Ala Cys Pro Ile Arg Thr Gln Pro Arg Trp 145 150 155 160 Asn Tyr Tyr Asp Ser Phe Ser Ala Val Ser Glu Asp Asn Leu Gly Phe 165 170 175 Leu Met His Ala Pro Ala Phe Glu Thr Ala Gly Thr Tyr Leu Arg Leu 180 185 190 Val Lys Ile Asn Asp Trp Thr Glu Ile Thr Gln Phe Ile Leu Glu His 195 200 205 Arg Ala Lys Gly Ser Cys Lys Tyr Ala Leu Pro Leu Arg Ile Pro Pro 210 215 220 Ser Ala Cys Leu Ser Pro Gln Ala Tyr Gln Gln Gly Val Thr Val Asp 225 230 235 240 Ser Ile Gly Met Leu Pro Arg Phe Ile Pro Glu Asn Gln Arg Thr Val 245 250 255 Ala Val Tyr Ser Leu Lys Ile Ala Gly Trp His Gly Pro His Phe Arg 260 265 270 Lys Leu Leu Leu Leu Asp Glu Glu Ala Gly Pro Leu Glu Glu Glu Leu 275 280 285 Pro Arg Leu Ala Asp Glu Gly Leu Asn Arg Arg Val Ala Glu Asp Leu 290 295 300 Asn Leu Gly Asn Leu Pro Glu Trp Gln Thr Pro Ser Phe Pro Lys Ile 305 310 315 320 His Leu Gln Glu Asp Ile Val Asp Arg Cys Lys Gln Phe Val Gly Pro 325 330 335 Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pro Ala Arg 340 345 350 Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gly Ile Lys 355 360 365 Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr Phe Gln Thr Arg His 370 375 380 Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Arg Glu Thr 385 390 395 400 Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu Gln Glu 405 410 415 Leu Gln His Gly Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro 420 425 430 Cys Ser Glu Tyr Cys Leu Thr His Leu Val Asn Leu Leu Glu Asp Trp 435 440 445 Gly Pro Cys Asp Glu His Gly Glu His His Ile Arg Ile Pro Arg Thr 450 455 460 Pro Ala Arg Val Thr Gln Ala Phe Thr Phe Ser Pro Thr Tyr Lys Ala 465 470 475 480 Phe Leu Ser Lys Gln Tyr Leu Asn Leu Tyr Pro Val Ala Arg Gln Arg 485 490 495 Pro Gly Leu Cys Gln Val Phe Ala Asp Ala Thr Pro Thr Gly Trp Gly 500 505 510 Leu Ala Met Gly His Gln Arg Met Arg Gly Thr Phe Val Ala Pro Leu 515 520 525 Pro Ile His Thr Ala Glu Leu Leu Ala Ala Cys Phe Ala Arg Ser Arg 530 535 540 Ser Gly Ala Lys Ile Leu Gly Thr Asp Asn Ser Val Val Leu Ser Arg 545 550 555 560 Lys Tyr Thr Ser Phe Pro Trp Leu Leu Gly Cys Ala Ala Asn Trp Ile 565 570 575 Leu Arg Gly Thr Ser Phe Val Tyr Val Pro Ser Ala Leu Asn Pro Ala 580 585 590 Asp Asp Val Gly Ser Asn Leu Glu Asp Pro Ala Ser Arg Glu Leu Val 595 600 605 Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys Ile Arg Gln Leu Leu 610 615 620 Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Ile Glu 625 630 635 640 Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg 645 650 655 Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr Thr Val Val 660 665 670 Arg Arg Arg Asp Arg Gly Arg Gly Pro Lys Ala Pro Tyr Thr Ser Thr 675 680 685 Leu Leu Pro Pro Glu Leu Ser Glu Thr Pro Asn Ala Thr Gln Pro Glu 690 695 700 Leu Ala Pro Glu Asp Pro Glu Asp Ser Ala Leu Leu Glu Asp Pro Val 705 710 715 720 Gly Thr Val Ala Pro Gln Ile Pro Pro Asn Trp His Ile Pro Ser Ile 725 730 735 Gln Asp Ala Ala Thr Pro Tyr His Pro Pro Ala Thr Pro Asn Asn Met 740 745 750 Gly Leu Ile Ala Gly Ala Val Gly Gly Ser Leu Leu Ala Ala Leu Val 755 760 765 Ile Cys Gly Ile Val Tyr Trp Met His Arg Arg Thr Arg Lys Ala Pro 770 775 780 Lys Arg Ile Arg Leu Pro His Ile Arg Glu Asp Asp Gln Pro Ser Ser 785 790 795 800 His Gln Pro Leu Phe Tyr 805 <210> 188 <211> 9 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 188 Phe Ala Val Pro Asn Leu Gln Ser Leu 1 5 <210> 189 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 189 Pro Glu Trp Gln Thr Pro Ser Phe Pro Lys Ile His Leu Gln Glu 1 5 10 15 <210> 190 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 190 Pro Ser Phe Pro Lys Ile His Leu Gln Glu Asp Ile Val Asp Arg 1 5 10 15 <210> 191 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 191 Ile His Leu Gln Glu Asp Ile Val Asp Arg Cys Lys Gln Phe Val 1 5 10 15 <210> 192 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 192 Asp Ile Val Asp Arg Cys Lys Gln Phe Val Gly Pro Leu Thr Val 1 5 10 15 <210> 193 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 193 Cys Lys Gln Phe Val Gly Pro Leu Thr Val Asn Glu Lys Arg Arg 1 5 10 15 <210> 194 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 194 Gly Pro Leu Thr Val Asn Glu Lys Arg Arg Leu Lys Leu Ile Met 1 5 10 15 <210> 195 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 195 Asn Glu Lys Arg Arg Leu Lys Leu Ile Met Pro Ala Arg Phe Tyr 1 5 10 15 <210> 196 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 196 Leu Lys Leu Ile Met Pro Ala Arg Phe Tyr Pro Asn Val Thr Lys 1 5 10 15 <210> 197 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 197 Pro Ala Arg Phe Tyr Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp 1 5 10 15 <210> 198 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 198 Pro Asn Val Thr Lys Tyr Leu Pro Leu Asp Lys Gly Ile Lys Pro 1 5 10 15 <210> 199 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 199 Tyr Leu Pro Leu Asp Lys Gly Ile Lys Pro Tyr Tyr Pro Glu His 1 5 10 15 <210> 200 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 200 Lys Gly Ile Lys Pro Tyr Tyr Pro Glu His Ala Val Asn His Tyr 1 5 10 15 <210> 201 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 201 Tyr Tyr Pro Glu His Ala Val Asn His Tyr Phe Gln Thr Arg His 1 5 10 15 <210> 202 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 202 Ala Val Asn His Tyr Phe Gln Thr Arg His Tyr Leu His Thr Leu 1 5 10 15 <210> 203 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 203 Phe Gln Thr Arg His Tyr Leu His Thr Leu Trp Lys Ala Gly Ile 1 5 10 15 <210> 204 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 204 Tyr Leu His Thr Leu Trp Lys Ala Gly Ile Leu Tyr Lys Arg Glu 1 5 10 15 <210> 205 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 205 Trp Lys Ala Gly Ile Leu Tyr Lys Arg Glu Thr Thr Arg Ser Ala 1 5 10 15 <210> 206 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 206 Leu Tyr Lys Arg Glu Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser 1 5 10 15 <210> 207 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 207 Thr Thr Arg Ser Ala Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu 1 5 10 15 <210> 208 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 208 Ser Phe Cys Gly Ser Pro Tyr Ser Trp Glu Gln Glu Leu Gln His 1 5 10 15 <210> 209 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 209 Pro Tyr Ser Trp Glu Gln Glu Leu Gln His Gly Ser Cys Trp Trp 1 5 10 15 <210> 210 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 210 Gln Glu Leu Gln His Gly Ser Cys Trp Trp Leu Gln Phe Arg Asn 1 5 10 15 <210> 211 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 211 Gly Ser Cys Trp Trp Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser 1 5 10 15 <210> 212 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 212 Leu Gln Phe Arg Asn Ser Lys Pro Cys Ser Glu Tyr Cys Leu Thr 1 5 10 15 <210> 213 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 213 Ser Lys Pro Cys Ser Glu Tyr Cys Leu Thr His Leu Val Asn Leu 1 5 10 15 <210> 214 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 214 Glu Tyr Cys Leu Thr His Leu Val Asn Leu Leu Glu Asp Trp Gly 1 5 10 15 <210> 215 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 215 His Leu Val Asn Leu Leu Glu Asp Trp Gly Pro Cys Asp Glu His 1 5 10 15 <210> 216 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 216 Leu Glu Asp Trp Gly Pro Cys Asp Glu His Gly Glu His His Ile 1 5 10 15 <210> 217 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 217 Pro Cys Asp Glu His Gly Glu His His Ile Arg Ile Pro Arg Thr 1 5 10 15 <210> 218 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 218 Gly Glu His His Ile Arg Ile Pro Arg Thr Pro Ala Arg Val Thr 1 5 10 15 <210> 219 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 219 Arg Ile Pro Arg Thr Pro Ala Arg Val Thr Gly Gly Val Phe Leu 1 5 10 15 <210> 220 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 220 Pro Ala Arg Val Thr Gly Gly Val Phe Leu Val Asp Lys Asn Pro 1 5 10 15 <210> 221 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 221 Gly Gly Val Phe Leu Val Asp Lys Asn Pro His Asn Thr Ala Glu 1 5 10 15 <210> 222 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 222 Val Asp Lys Asn Pro His Asn Thr Ala Glu Ser Arg Leu Val Val 1 5 10 15 <210> 223 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 223 His Asn Thr Ala Glu Ser Arg Leu Val Val Asp Phe Ser Gln Phe 1 5 10 15 <210> 224 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 224 Ser Arg Leu Val Val Asp Phe Ser Gln Phe Ser Arg Gly Ile Thr 1 5 10 15 <210> 225 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 225 Asp Phe Ser Gln Phe Ser Arg Gly Ile Thr Arg Val Ser Trp Pro 1 5 10 15 <210> 226 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 226 Ser Arg Gly Ile Thr Arg Val Ser Trp Pro Lys Phe Ala Val Pro 1 5 10 15 <210> 227 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 227 Arg Val Ser Trp Pro Lys Phe Ala Val Pro Asn Leu Gln Ser Leu 1 5 10 15 <210> 228 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 228 Lys Phe Ala Val Pro Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser 1 5 10 15 <210> 229 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 229 Asn Leu Gln Ser Leu Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp 1 5 10 15 <210> 230 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 230 Thr Asn Leu Leu Ser Ser Asn Leu Ser Trp Leu Ser Leu Asp Val 1 5 10 15 <210> 231 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 231 Ser Asn Leu Ser Trp Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr 1 5 10 15 <210> 232 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 232 Leu Ser Leu Asp Val Ser Ala Ala Phe Tyr His Ile Pro Leu His 1 5 10 15 <210> 233 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthetic peptide" <400> 233 Ser Ala Ala Phe Tyr His Ile Pro Leu His Pro Ala Ala Met Pro 1 5 10 15

Claims

1. A nucleic acid molecule comprising: The nucleotide sequence encoding the N-terminal domain of the HBV polymerase shown in SEQ ID NO: 178; The nucleotide sequence encoding the C-terminal domain of the HBV polymerase shown in SEQ ID NO: 179; and The nucleotide sequence encodes the HBV Core protein represented by the amino acid sequence of SEQ ID NO:

180.

2. The nucleic acid molecule of claim 1, which encodes the fusion protein represented by the amino acid sequence of SEQ ID NO:

174.

3. The nucleic acid molecule of claim 1 or 2 further comprises a nucleotide sequence encoding the following: N-terminal HSV gD sequence; C-terminal HSV gD sequence; or Both.

4. The nucleic acid molecule as described in claim 3, comprising: The nucleotide sequence encoding the N-terminal HSV gD sequence; A nucleotide sequence encoding an HBV fusion protein, the HBV fusion protein comprising: The amino acid sequence of SEQ ID NO: 178 shows the N-terminal domain of HBV polymerase. The amino acid sequence of SEQ ID NO: 179 shows the C-terminal domain of HBV polymerase, and The amino acid sequence of SEQ ID NO: 180 represents the HBV Core protein; and The nucleotide sequence encoding the C-terminal HSV gD sequence.

5. The nucleic acid molecule of claim 4, wherein the nucleotide sequence encodes the HBV fusion protein represented by the amino acid sequence of SEQ ID NO:

174.

6. The nucleic acid molecule of claim 5, wherein the nucleotide sequence encodes the N-terminal HSV gD sequence shown in the amino acid sequence of SEQ ID NO:

12.

7. The nucleic acid molecule of claim 5, wherein the nucleotide sequence encodes the N-terminal HSV gD sequence of amino acid residues 26-269 of SEQ ID NO:

12.

8. The nucleic acid molecule of any one of claims 5-7, wherein the nucleotide sequence encodes a C-terminal HSV gD sequence comprising a transmembrane domain of HSV gD.

9. The nucleic acid molecule of claim 8, wherein the nucleotide sequence encodes the C-terminal HSV gD sequence shown in the amino acid sequence of SEQ ID NO:

13.

10. The nucleic acid molecule according to any one of claims 4-9, comprising: Nucleotide sequence encoding the N-terminal HSV gD sequence shown in amino acid residues 26-269 of SEQ ID NO: 12; A nucleotide sequence encoding an HBV fusion protein, the HBV fusion protein comprising: The amino acid sequence of SEQ ID NO: 178 shows the N-terminal domain of HBV polymerase. The amino acid sequence of SEQ ID NO: 179 shows the C-terminal domain of HBV polymerase, and The amino acid sequence of SEQ ID NO: 180 represents the HBV Core protein; and The nucleotide sequence, which encodes the C-terminal HSV gD sequence shown in SEQ ID NO:

13.

11. The nucleic acid molecule of claim 10, wherein the nucleotide sequence encodes the N-terminal HSV gD sequence shown in the amino acid sequence of SEQ ID NO:

12.

12. The nucleic acid molecule of claim 10, wherein the nucleotide sequence encodes the HBV fusion protein represented by the amino acid sequence of SEQ ID NO:

174.

13. The nucleic acid molecule of any one of claims 10-12, wherein the nucleotide sequence encodes the fusion protein represented by the amino acid sequence of SEQ ID NO:

185.

14. The nucleic acid molecule of claim 12, wherein the nucleic acid molecule is the nucleotide sequence shown in SEQ ID NO:

176.

15. The nucleic acid molecule of claim 13, wherein the nucleic acid molecule is the nucleotide sequence shown in SEQ ID NO:

184.

16. A fusion protein comprising: The amino acid sequence of SEQ ID NO: 178 shows the N-terminal domain of HBV polymerase, the amino acid sequence of SEQ ID NO: 179 shows the C-terminal domain of HBV polymerase, and the amino acid sequence of SEQ ID NO: 180 shows the HBV Core protein.

17. The fusion protein of claim 16, wherein the amino acid sequence is as shown in SEQ ID NO:

174.

18. The fusion protein of claim 16 or 17 further comprises an N-terminal HSV gD sequence, a C-terminal HSV gD protein sequence, or both.

19. The fusion protein of claim 18, comprising: N-terminal HSV gD sequence, The HBV sequence comprising the N-terminal domain of HBV polymerase shown in SEQ ID NO: 178, the C-terminal domain of HBV polymerase shown in SEQ ID NO: 179, and the HBV Core protein shown in SEQ ID NO: 180; and C-terminal HSV gD protein sequence.

20. The fusion protein of claim 19, wherein the HBV sequence is the amino acid sequence shown in SEQ ID NO:

174.

21. The fusion protein according to any one of claims 18-20, wherein the N-terminal HSV gD sequence is the amino acid sequence shown in SEQ ID NO:

12.

22. The fusion protein according to any one of claims 18-20, wherein the N-terminal HSV gD sequence is as shown in amino acid residues 26-269 of SEQ ID NO:

12.

23. The fusion protein according to any one of claims 18-22, wherein the C-terminal HSV gD sequence is represented by the transmembrane domain of the HSV gD.

24. The fusion protein according to any one of claims 18-23, wherein the C-terminal HSV gD sequence is the amino acid sequence shown in SEQ ID NO:

13.

25. The fusion protein according to any one of claims 18-24, wherein the fusion protein has the amino acid sequence shown in SEQ ID NO:

185.

26. A virus comprising any one of claims 1-15.

27. The virus of claim 26, wherein the virus is an adenovirus.

28. The virus of claim 27, wherein the adenovirus is AdC6 or AdC7.

29. A vaccine comprising the virus according to any one of claims 26-28.

30. Use of the nucleic acid molecule of any one of claims 1-15, the fusion protein of any one of claims 16-25, the virus of any one of claims 26-28, or the vaccine of claim 29 in the preparation of a medicament for inducing an immune response against HBV in a subject.

31. The use as claimed in claim 30, wherein the vaccine comprises an AdC6 vector, the AdC6 vector comprising a nucleic acid molecule encoding a fusion protein represented by the amino acid sequence of SEQ ID NO:

185.

32. The use as claimed in claim 31, wherein after providing the vaccine comprising the AdC6 vector, the subject is further administered a vaccine comprising the AdC7 vector, the AdC7 vector comprising a nucleic acid molecule encoding a fusion protein represented by the amino acid sequence of SEQ ID NO:

185.

33. The use as claimed in claim 30, wherein the vaccine comprises an AdC7 vector, the AdC7 vector comprising a nucleic acid molecule encoding a fusion protein represented by the amino acid sequence of SEQ ID NO:

185.

34. The use as claimed in claim 33, wherein after providing the vaccine comprising the AdC7 vector, the subject is further administered a vaccine comprising the AdC6 vector, the AdC6 vector comprising a nucleic acid molecule encoding a fusion protein represented by the amino acid sequence of SEQ ID NO:

185.

35. The use as described in any one of claims 30-34, wherein the amino acid sequence of SEQ ID NO: 185 does not contain a signal peptide with 25 amino acids at the N-terminus.

36. A nucleic acid molecule comprising a nucleotide sequence that encodes the N-terminal domain of HBV polymerase represented by the amino acid sequence of SEQ ID NO:

178.

37. The nucleic acid molecule according to claim 36, wherein the nucleotide sequence of nucleic acid 1-510 of SEQ ID NO: 176 is shown.

38. An N-terminal domain of an HBV polymerase, the amino acid sequence of which is shown in SEQ ID NO:

178.

39. A nucleic acid molecule comprising a nucleotide sequence that encodes the C-terminal domain of HBV polymerase represented by the amino acid sequence of SEQ ID NO:

179.

40. The nucleic acid molecule according to claim 39, wherein the nucleotide sequence of nucleic acid 511-885 of SEQ ID NO: 176 is shown.

41. A C-terminal domain of an HBV polymerase, the amino acid sequence of which is shown in SEQ ID NO:

179.

42. A nucleic acid molecule comprising a nucleotide sequence that encodes the HBV Core protein represented by the amino acid sequence of SEQ ID NO:

180.

43. The nucleic acid molecule according to claim 42, wherein the nucleotide sequence of nucleic acid 886-1140 of SEQ ID NO: 176 is shown.

44. An HBV Core protein, the amino acid sequence of which is shown in SEQ ID NO:

180.

45. A vector comprising the nucleic acid molecule of any one of claims 36, 37, 39, 40, 42 or 43.

46. ​​The vector according to claim 45, wherein the vector is an adenovirus vector.

47. The vector according to claim 46, wherein the adenovirus vector is an AdC6 vector or an AdC7 vector.

48. A vaccine comprising the vector of any one of claims 45-47.

Images