Hepatitis B Virus Vaccines

Abstract

This document provides methods and materials for producing immune responses against hepatitis B viruses. For example, polypeptides, nucleic acid molecules encoding such polypeptides, virus-like particles containing such polypeptides, vaccine preparations containing one or more polypeptides provided herein, vaccine preparations containing one or more nucleic acid molecules provided herein, vaccine preparations containing one or more virus-like particles provided herein, and methods for inducing immune responses against hepatitis B viruses within mammals (e.g., humans) are provided.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 14/207,135, filed Mar. 12, 2014, which claims the benefit of U.S. Provisional Application Ser. No. 61/785,838, filed Mar. 14, 2013. The disclosure of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.

BACKGROUND

[0002] 1. Technical Field

[0003] This document relates to methods and materials for producing immune responses against hepatitis B viruses. For example, this document provides vaccines (e.g., nucleic acid vaccines, virus-like particle vaccines, and polypeptide vaccines) capable of being administered to mammals (e.g., humans) under conditions that induce production of immune responses against hepatitis B viruses.

[0004] 2. Background Information

[0005] Hepatitis B virus (HBV) is a hepadnavirus that causes an inflammatory illness of the liver. About a third of the world population is infected at one point in their lives, and about 350 million people are chronic carriers. The virus can be transmitted by exposure to infectious blood or body fluids. The acute illness causes liver inflammation, vomiting, jaundice, and, in rare cases, death. Chronic hepatitis B infections may cause cirrhosis and liver cancer.

SUMMARY

[0006] This document provides methods and materials for producing immune responses against hepatitis B viruses. For example, this document provides vaccines (e.g., nucleic acid vaccines, virus-like particle vaccines, and polypeptide vaccines) capable of being administered to mammals (e.g., humans) under conditions that induce production of immune responses against hepatitis B viruses as well as methods for producing immune responses against hepatitis B viruses within a mammal (e.g., a human).

[0007] As described herein, polypeptides having the amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 (or a sequence at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identical to such as sequence) can be produced and formulated into a vaccine preparation having the ability to produce an immune response against hepatitis B viruses when administered to a mammal (e.g., a human). Such vaccine preparations can be administered to a mammal prior to the mammal being exposed to hepatitis B viruses. In such cases, the administered vaccine preparation can provide increased protection against hepatitis B virus infection. In some cases, such vaccine preparations can be administered to a mammal after the mammal is infected with hepatitis B virus (e.g., an acutely hepatitis B virus infected or chronically hepatitis B virus infected mammal). In such cases, administration of the vaccine preparation can be used to treat the hepatitis B virus infection. For example, administration of a vaccine preparation provided herein to a mammal infected with hepatitis B virus can result in a reduction in hepatitis B viral load, a reduction in the severity of the symptoms of the hepatitis B virus infection, a reduction in the degree of liver cirrhosis, a reduction in the incidence of hepatocellular cancer, or a clearance of the hepatitis B virus from the liver.

[0008] In some cases, a polypeptide provided herein (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58, or an amino acid sequence that is at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identical to the amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58) can be formulated into virus-like particles that can be used as vaccine preparations having the ability to produce an immune response against hepatitis B viruses when administered to a mammal (e.g., a human). The vaccine preparations containing such virus-like particles can be administered to a mammal prior to the mammal being exposed to hepatitis B viruses. In such cases, the administered vaccine preparation containing virus-like particles can provide increased protection against hepatitis B virus infection. In some cases, a vaccine preparation containing a virus-like particle provided herein can be administered to a mammal after the mammal is infected with hepatitis B virus (e.g., an acutely hepatitis B virus infected or chronically hepatitis B virus infected mammal). In such cases, administration of the vaccine preparation containing a virus-like particle can be used to treat the hepatitis B virus infection. For example, administration of a vaccine preparation containing a virus-like particle provided herein to a mammal infected with hepatitis B virus can result in a reduction in hepatitis B viral load, a reduction in the severity of the symptoms of the hepatitis B virus infection, a reduction in the degree of liver cirrhosis, a reduction in the incidence of hepatocellular cancer, or a clearance of the hepatitis B virus from the liver.

[0009] As also described herein, nucleic acid molecules encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 (or a sequence at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identical to such as sequence) can be obtained and formulated into a nucleic acid vaccine preparation having the ability to express the encoded polypeptide and produce an immune response against hepatitis B viruses when administered to a mammal (e.g., a human). Such nucleic acid vaccine preparations can be administered to a mammal prior to the mammal being exposed to hepatitis B viruses. In such cases, the administered nucleic acid vaccine preparation can provide increased protection against hepatitis B virus infection. In some cases, such nucleic acid vaccine preparations can be administered to a mammal after the mammal is infected with hepatitis B virus (e.g., an acutely hepatitis B virus infected or chronically hepatitis B virus infected mammal). In such cases, administration of the nucleic acid vaccine preparation can be used to treat the hepatitis B virus infection. For example, administration of a nucleic acid vaccine preparation provided herein to a mammal infected with hepatitis B virus can result in a reduction in hepatitis B viral load, a reduction in the severity of the symptoms of the hepatitis B virus infection, a reduction in the degree of liver cirrhosis, a reduction in the incidence of hepatocellular cancer, or a clearance of the hepatitis B virus from the liver.

[0010] Having the ability to use the vaccine preparations provided herein produce immune responses against hepatitis B viruses can allow clinicians to provide their patients with increased protection against hepatitis B virus infections. In addition, the vaccine preparations provided herein can allow clinicians to treat patients previously infected with hepatitis B virus.

[0011] In general, one aspect of this document features a polypeptide comprising an amino acid sequence that is at least 97 percent identical (e.g., at least 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:10.

[0012] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 96 percent identical (e.g., at least 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:12.

[0013] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 96 percent identical (e.g., at least 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:14.

[0014] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 95 percent identical (e.g., at least 96, 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:16.

[0015] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 97 percent identical (e.g., at least 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:18.

[0016] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 93 percent identical (e.g., at least 94, 95, 96, 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:20.

[0017] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 97 percent identical (e.g., at least 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:22.

[0018] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 89 percent identical (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:24.

[0019] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 89 percent identical (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:26.

[0020] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 95 percent identical (e.g., at least 94, 95, 96, 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:28.

[0021] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 94 percent identical (e.g., at least 94, 95, 96, 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:30.

[0022] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 96 percent identical (e.g., at least 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:32.

[0023] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 96 percent identical (e.g., at least 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:34.

[0024] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 96 percent identical (e.g., at least 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:36.

[0025] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 93 percent identical (e.g., at least 94, 95, 96, 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:38.

[0026] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 97 percent identical (e.g., at least 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:40.

[0027] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 93 percent identical (e.g., at least 94, 95, 96, 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:42.

[0028] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 93 percent identical (e.g., at least 94, 95, 96, 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:44.

[0029] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 95 percent identical (e.g., at least 96, 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:46.

[0030] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 96 percent identical (e.g., at least 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:48.

[0031] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 93 percent identical (e.g., at least 94, 95, 96, 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:50.

[0032] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 93 percent identical (e.g., at least 94, 95, 96, 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:52.

[0033] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 92 percent identical (e.g., at least 93, 94, 95, 96, 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:54.

[0034] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 86 percent identical (e.g., at least 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:56.

[0035] In another aspect, this document features a polypeptide comprising an amino acid sequence that is at least 89 percent identical (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identical) to the amino acid sequence set forth in SEQ ID NO:58.

[0036] Any one of the polypeptides described in the above 25 paragraphs can comprise the ability to induce an immune response against a hepatitis B virus when the polypeptide is administered to a mammal. In some cases, any one of the polypeptides described in the above 25 paragraphs can be a fusion polypeptide comprising a second amino acid sequence. The second amino acid sequence can encode a tag selected from the group consisting of GST, FLAG, GFP, and c-myc. The second amino acid sequence can encode a cytokine selected from the group consisting of GM-CSF, IL-2, and IL-12. The polypeptide can be substantially pure. The polypeptide can be an isolated polypeptide.

[0037] In another aspect, this document features a virus-like particle comprising one or more polypeptides selected from the group consisting of the polypeptides described in the above said 25 paragraphs. The virus-like particle can comprise one polypeptide selected from the group. The virus-like particle can comprise two, three, four, or five different polypeptides selected from the group. The virus-like particle can comprise the ability to induce an immune response against a hepatitis B virus when the particle is administered to a mammal.

[0038] In another aspect, this document features a vaccine preparation comprising one or more polypeptides selected from the group consisting of the polypeptides described in the above said 25 paragraphs. The vaccine preparation can comprise one polypeptide selected from the group. The vaccine preparation can comprise two, three, four, or five different polypeptides selected from the group. The vaccine preparation can comprise the ability to induce an immune response against a hepatitis B virus when the vaccine preparation is administered to a mammal. The vaccine preparation can comprise an adjuvant. The adjuvant can be selected from the group consisting of aluminum-based compounds, Montanide ISA 51, Montanide ISA 720, and CpG oligodeoxynucleotides. The adjuvant can comprise alum or Al.sub.2O.sub.3.

[0039] In another aspect, this document features a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide selected from the group consisting of the polypeptides described in the above said 25 paragraphs. The nucleic acid molecule can comprise a promoter sequence operably linked to the nucleic acid sequence. The nucleic acid molecule can be a vector. The vector can be a plasmid. The vector can be a viral vector. The viral vector can be selected from the group consisting of adenoviral vectors, adeno-associated virus vectors, and vaccinia viral vectors.

[0040] In another aspect, this document features a vaccine preparation comprising one or more nucleic acid molecules. The nucleic acid molecule comprises a nucleic acid sequence encoding a polypeptide selected from the group consisting of the polypeptides described in the above said 25 paragraphs. The nucleic acid molecule can comprise a promoter sequence operably linked to the nucleic acid sequence. The nucleic acid molecule can be a vector. The vector can be a plasmid. The vector can be a viral vector. The viral vector can be selected from the group consisting of adenoviral vectors, adeno-associated virus vectors, and vaccinia viral vectors. The vaccine preparation can comprise a nucleic acid molecule comprising a nucleic acid sequence encoding one polypeptide selected from the group. The vaccine preparation can comprise one or more nucleic acid molecules comprising a nucleic acid sequence encoding two, three, four, or five different polypeptides selected from the group. The vaccine preparation can comprise the ability to induce an immune response against a hepatitis B virus when the vaccine preparation is administered to a mammal and the polypeptide is expressed within the mammal. The vaccine preparation can comprise an adjuvant. The adjuvant can be selected from the group consisting of aluminum-based compounds, Montanide ISA 51, Montanide ISA 720, and CpG oligodeoxynucleotides. The adjuvant can comprise alum or Al.sub.2O.sub.3.

[0041] In another aspect, this document features a method for inducing an immune response against a hepatitis B virus within a mammal. The method comprises administering a vaccine preparation to the mammal. The vaccine preparation comprises (a) one or more polypeptides selected from the group consisting of the polypeptides described in the above said 25 paragraphs, (b) one or more virus-like particles comprising one or more polypeptides selected from the group consisting of the polypeptides described in the above said 25 paragraphs, or (c) one or more nucleic acid molecules comprising a nucleic acid sequence encoding a polypeptide selected from the group consisting of the polypeptides described in the above said 25 paragraphs. The mammal can be a human. The mammal can be a human not previously infected with a hepatitis B virus. The mammal can be a human acutely infected with a hepatitis B virus. The mammal can be a human chronically infected with a hepatitis B virus. The immune response can comprise the production of anti-hepatitis B virus antibodies. The method can comprise administering a second vaccine preparation to the mammal after the step of administering the vaccine preparation to the mammal, wherein the second vaccine preparation comprises an hepatitis B virus antigen. The method can comprise administering a second vaccine preparation to the mammal after the step of administering the vaccine preparation to the mammal, wherein the second vaccine preparation comprises (a) one or more polypeptides selected from the group consisting of the polypeptides described in the above said 25 paragraphs, (b) one or more virus-like particles comprising one or more polypeptides selected from the group consisting of the polypeptides described in the above said 25 paragraphs, or (c) one or more nucleic acid molecules comprising a nucleic acid sequence encoding a polypeptide selected from the group consisting of the polypeptides described in the above said 25 paragraphs.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0043] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 is a schematic of the strategy used in designing the strain-specific oligonucleotide primers used to keep the `a` epitope loop constrained during the DNA shuffling reaction. Thirty nucleotides of the HBV (strain ayw) `a` loop sequence were held constant and flanked by fifteen nucleotides on each side derived from the related HBs genes of chimpanzee/gibbon, woodchuck, and woolly monkey. A restriction endonuclease BsrB I site sequence (CCGCTC) was introduced in the sequence encoding the HBV "a" loop.

[0045] FIG. 2 shows the positive (+) strain oligonucleotide sequences used to hold the HBV "a` loop sequence constant during DNA shuffling. A restriction endonuclease BsrB I site sequence (CCGCTC) was introduced, and a BspM I site sequence (ACCTGCN4) was knocked out by the two conservative changes shown in lower case (t and c). These altered restriction sites were engineered into the oligonucleotides to enable a rapid evaluation of oligo incorporation by restriction analysis. The conserved 30 nucleotides from the AYW `a` epitope loop are shown above the dashed line and underlined in the sequences below the dashed line. All primers are positive sense as indicated by (+). Primers shown: chibbon (+) primer (chimpanzee/gibbon); WM (+) primer (woolly monkey); WD (+) primer (woodchuck).

[0046] FIG. 3 shows representative ELISA data from primary screening of a first shuffled library are shown. Anti-Hepatitis B antibody levels present in the sera of mice injected with clones from the first shuffled library were measured by ELISA and expressed as milli-International Units per mL. A reference level typically used is the highest antibody level induced in mice by a positive-control DNA vaccine (measured as mIU/mL using a commercial anti-HBsAg detection kit). The striped bar indicates antibody levels induced by a wild-type clone. The black bars indicate antibody levels above reference value induced by shuffled clones.

[0047] FIG. 4 contains a listing of the nucleic acid sequence and amino acid sequence for one of the human parental clone used in the initial shuffling.

[0048] FIG. 5 contains a listing of the nucleic acid sequence and amino acid sequence for one of the woolly monkey parental clone used in the initial shuffling.

[0049] FIG. 6 contains a listing of the nucleic acid sequence and amino acid sequence for one of the human/woodchuck parental clone used in the initial shuffling.

[0050] FIG. 7 contains a listing of the nucleic acid sequence and amino acid sequence for one of the composite chimpanzee-gibbon parental clone used in the initial shuffling.

[0051] FIG. 8 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6101 obtained from the initial shuffling.

[0052] FIG. 9 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6102 obtained from the initial shuffling.

[0053] FIG. 10 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6103 obtained from the initial shuffling.

[0054] FIG. 11 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6104 obtained from the initial shuffling.

[0055] FIG. 12 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6105 obtained from the initial shuffling.

[0056] FIG. 13 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6106 obtained from the initial shuffling.

[0057] FIG. 14 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6107 obtained from the initial shuffling.

[0058] FIG. 15 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6108 obtained from the initial shuffling.

[0059] FIG. 16 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6109 obtained from the initial shuffling.

[0060] FIG. 17 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6111 obtained from the initial shuffling.

[0061] FIG. 18 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6201 obtained from a second shuffling.

[0062] FIG. 19 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6202 obtained from a second shuffling.

[0063] FIG. 20 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6203 obtained from a second shuffling.

[0064] FIG. 21 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6204 obtained from a second shuffling.

[0065] FIG. 22 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6205 obtained from a second shuffling.

[0066] FIG. 23 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6206 obtained from a second shuffling.

[0067] FIG. 24 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6207 obtained from a second shuffling.

[0068] FIG. 25 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6208 obtained from a second shuffling.

[0069] FIG. 26 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6209 obtained from a second shuffling.

[0070] FIG. 27 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6210 obtained from a second shuffling.

[0071] FIG. 28 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6211 obtained from a second shuffling.

[0072] FIG. 29 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6212 obtained from a second shuffling.

[0073] FIG. 30 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6213 obtained from a second shuffling.

[0074] FIG. 31 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6214 obtained from a second shuffling.

[0075] FIG. 32 contains a listing of the nucleic acid sequence and amino acid sequence for clone 6215 obtained from a second shuffling.

[0076] FIG. 33 is a nucleic acid sequence alignment of the four parental clones encoding hepatitis surface antigen proteins obtained from HBV, woolly monkey hepatitis virus (WMHV), human/woodchuck hepatitis virus (HWHV, and chimpanzee-gibbon hepatitis virus (CGHV), and the indicated clones.

[0077] FIG. 34 is an amino acid sequence alignment of the hepatitis surface antigen proteins encoded by the four parental clones from HBV, woolly monkey hepatitis virus (WMHV), human/woodchuck hepatitis virus (HWHV), and chimpanzee-gibbon hepatitis virus (CGHV), and the indicated clones.

[0078] FIG. 35 contains a wild-type hepatitis B virus sequence with a summary of the differences observed in clones 6101-6109, 6111, and 6201-6215.

[0079] FIG. 36 contains a diagram of a vector used to express HBsAg clones.

[0080] FIG. 37. Geometric Mean Titers (GMT) of anti-HBsAg obtained with first round clones. Geometric Mean Titers .+-.SEM of anti-HBsAg were measured by ELISA in sera obtained by immunizing test groups of mice each with one of ten improved clones selected as inducing the highest expression levels in the initial screening procedure. The clone names are shown on the X axis. HBV is a plasmid used as a control that wild-type hepatitis B envelope gene-containing plasmid used as a control.

[0081] FIG. 38. Geometric Mean Titers of anti-HBsAg obtained with second round clones. A second round library was obtained by shuffling the ten improved clones selected from the first round library. One of ten second round library clones selected based on their induction of anti-HBsAg levels, or the wild-type human hepatitis B envelope gene (HBV), was used to immunize each group of mice. Geometric Mean Titers of anti-HBsAg in the sera from the immunized mice were measured by ELISA. The clone names are shown on the X axis; each bar represents one group of mice. The Y-axis shows the geometric mean titer .+-.SEM of anti-HBsAg for each group in International units.

[0082] FIG. 39 is a bar graph plotting the GMT (mIU/mL) for the indicated clones when administered to 30 six-week old C57BL/6 mice.

[0083] FIG. 40 is a bar graph plotting the GMT (mIU/mL) for the indicated clones when administered to mice alone (DNA prime; gray) or when administered to mice prior to the mice receiving a boost with wild-type protein (Protein Boost; black). The bars labeled "Engerix" are from groups of mice that received one protein injection (gray bar) or the initial priming protein injection followed by the boosting injection (black bar).

[0084] FIG. 41 is a bar graph plotting the GMT (mIU/mL) for the indicated clones when administered to outbred mice.

[0085] FIG. 42 is a bar graph plotting the GMT (mIU/mL) for the indicated clones when administered to non-responder mice.

DETAILED DESCRIPTION

[0086] This document provides methods and materials for producing immune responses against hepatitis B viruses. For example, this document provides polypeptides, nucleic acid molecules encoding such polypeptides, virus-like particles containing such polypeptides, vaccine preparations containing one or more polypeptides provided herein, vaccine preparations containing one or more nucleic acid molecules provided herein, vaccine preparations containing one or more virus-like particles provided herein, and methods for inducing immune responses against hepatitis B viruses within mammals (e.g., humans).

[0087] As described herein, a polypeptide provided herein can be designed to include the amino acid sequence as set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 (or an amino acid sequence that is at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identical to the amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58). Such polypeptides can be used to induce an immune response against hepatitis B viruses within a mammal. In some cases, a polypeptide provided herein can have the amino acid sequence as set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 and can have the identity to SEQ ID NO:2, 4, 6, and 8 as indicated in Table 1.

TABLE-US-00001 TABLE 1 Percentages of amino acid residues of the indicated clones that are identical to those of the indicated parental clones. Clone SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID No. NO:.sup.a NO: 2.sup.b NO: 4.sup.c NO: 6.sup.d NO: 8.sup.e 6101 10 87.6 96.9 59.7 78.5 6102 12 95.6 88.1 60.6 82.9 6103 14 95.6 88.1 62.8 80.3 6104 16 95.1 92.0 60.2 78.5 6105 18 96.5 88.1 60.6 82.9 6106 20 92.9 90.7 62.8 78.5 6107 22 96.9 87.6 61.5 80.3 6108 24 88.1 84.1 65.6 79.4 6109 26 88.1 84.1 65.6 79.4 6111 28 94.7 92.5 61.9 77.6 6201 30 93.4 91.2 60.2 79.4 6202 32 95.6 87.2 61.1 83.3 6203 34 95.1 92.0 60.2 78.5 6204 36 95.1 92.0 60.2 78.5 6205 38 92.5 91.2 60.6 79.4 6206 40 96.0 89.4 61.5 82.0 6207 42 92.5 90.3 62.4 78.1 6208 44 92.9 86.3 62.8 81.6 6209 46 94.2 87.6 61.1 84.2 6210 48 95.1 92.5 61.1 78.5 6211 50 92.5 91.2 60.6 79.4 6212 52 92.0 91.6 60.2 78.9 6213 54 91.6 91.2 61.1 82.9 6214 56 84.5 85.0 65.6 72.4 6215 58 88.9 87.6 67.0 77.6 .sup.aprovided SEQ ID NO: is for the amino acid sequence of the indicated clone number. .sup.bSEQ ID NO: 2 is the amino acid sequence for the human parental clone. .sup.cSEQ ID NO: 4 is the amino acid sequence for the woolly monkey parental clone. .sup.dSEQ ID NO: 6 is the amino acid sequence for the human/woodchuck parental clone. .sup.eSEQ ID NO: 8 is the amino acid sequence for the chimpanzee/gibbon parental clone.

[0088] In some cases, a polypeptide provided herein can be an isolated polypeptide. The term "isolated" as used herein with reference to polypeptides refers to (a) a polypeptide that is not associated with proteins that such polypeptide is normally associated with in nature or (b) a polypeptide that does not occur or exist in nature.

[0089] In some cases, a polypeptide provided herein can be a substantially pure polypeptide. The term "substantially pure" as used herein with reference to polypeptides refers to a polypeptide preparation that is substantially free of other polypeptides, lipids, carbohydrates, and nucleic acid. In some cases, a substantially pure polypeptide can be a polypeptide that is at least 60 percent pure or is a chemically synthesized polypeptide. A substantially pure polypeptide can be at least about 60, 65, 70, 75, 80, 85, 90, 95, or 99 percent pure. Typically, a substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel.

[0090] In some cases, a polypeptide provided herein can include the amino acid sequence as set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 except that the amino acid sequence includes a variation (e.g., a substitution, addition, or deletion) at one or more positions (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, 19, 20, or more positions). Such variant sequences, e.g., those having one or more amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58, can be prepared and modified as described herein. For example, a polypeptide provided herein can include the amino acid sequence as set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 except that the amino acid sequence includes a conservative or non-conservative amino acid substitution at one or more positions (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, 13, 14, 15, 16, 17, 18, 19, 20, or more positions). In some cases, an amino acid substitution can be made by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at a particular site, or (c) the bulk of the side chain. For example, naturally occurring residues can be divided into groups based on side-chain properties: (1) hydrophobic amino acids (methionine, alanine, valine, leucine, and isoleucine); (2) neutral hydrophilic amino acids (cysteine, serine, and threonine); (3) acidic amino acids (aspartic acid and glutamic acid); (4) basic amino acids (asparagine, glutamine, histidine, lysine, and arginine); (5) amino acids that influence chain orientation (glycine and proline); and (6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Substitutions made within these groups can be considered conservative substitutions. Non-limiting examples of substitutions include, without limitation, substitution of valine for alanine, lysine for arginine, glutamine for asparagine, glutamic acid for aspartic acid, serine for cysteine, asparagine for glutamine, aspartic acid for glutamic acid, proline for glycine, arginine for histidine, leucine for isoleucine, isoleucine for leucine, arginine for lysine, leucine for methionine, leucine for phenylalanine, glycine for proline, threonine for serine, serine for threonine, tyrosine for tryptophan, phenylalanine for tyrosine, and/or leucine for valine.

[0091] Further examples of conservative substitutions that can be made at any position within the amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 include, without limitation, those set forth in Table 2.

TABLE-US-00002 TABLE 2 Examples of conservative amino acid substitutions. Original Residue Exemplary substitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln, His, Lys, Arg Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln, Lys, Arg Ile Leu, Val, Met, Ala, Phe Leu Ile, Val, Met, Ala, Phe Lys Arg, Gln, Asn Met Leu, Phe, Ile Phe Leu, Val, Ile, Ala Pro Gly Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe, Thr, Ser Val Ile, Leu, Met, Phe, Ala

[0092] In some case, an amino acid sequence used to make a polypeptide provided herein can include one or more non-conservative substitutions. Non-conservative substitutions typically entail exchanging a member of one of the classes described above for a member of another class.

[0093] In some cases, a polypeptide provided herein can have an amino acid sequence with at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a reference sequence (e.g., SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58). Percent sequence identity is calculated by determining the number of matched positions in aligned amino acid sequences (target amino acid sequence aligned to an identified amino acid sequence), dividing the number of matched positions by the number of amino acids of the identified amino acid sequence (e.g., SEQ ID NO:10), and multiplying by 100. A matched position refers to a position in which identical amino acids occur at the same position in aligned amino acid sequences. Percent sequence identity also can be determined for nucleic acid sequences.

[0094] Percent sequence identity is determined by comparing a target amino acid sequence to the identified amino acid sequence (e.g., SEQ ID NO:10) using the ClustalW alignment tool provided in the Geneious software platform (version 6.05, Biomatters Ltd, Auckland, New Zealand).

[0095] Any appropriate method can be used to obtain a polypeptide provided herein. For example, common polypeptide purification techniques such as affinity chromatography and HPLC as well as polypeptide synthesis techniques can be used. In addition, any appropriate material can be used as a source to obtain a polypeptide provided herein. For example, cultured cells engineered to over-express a particular polypeptide provided herein (e.g., a cell line designed to include a nucleic acid molecule provided herein) can be used to produce a polypeptide provided herein. Such cells can be prokaryotic cells (e.g., bacterial cells such as E. coli, Bacillus subtilis, or Pseudomonas cells) or eukaryotic cells (e.g., yeast cells such as Saccharomyces cerevisiae, Hansenula polymorpha or Pichia pastoris cells, insect cells such as Drosophila melanogaster (e.g., Schneider 2 or Schneider 3 cells), Spodoptera frugiperda, or Trichoplusia ni cells, or mammalian cells such as CHO, HEK 293, MRC, or PER-C6 cells). In some cases, a polypeptide provided herein can be designed to contain an amino acid sequence that allows the polypeptide to be captured onto an affinity matrix. For example, a tag such as c-myc, hemagglutinin, polyhistidine, Flag.TM. tag (Kodak), Strep-Tag, V5, or VSV-G can be used to aid polypeptide purification. Such tags can be inserted anywhere alone a polypeptide including at either the carboxyl or amino termini.

[0096] In some cases, a polypeptide provided herein can be a fusion polypeptide. Such a fusion polypeptide can include one or more additional amino acid sequences in addition to the amino acid sequence as set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 (or an amino acid sequence that is at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identical to the amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58). The additional amino acid sequences can be the amino acid sequence of a tag as described above, a marker such as GFP or Luciferase, an enzyme such as alkaline phosphatase or GST, or a cytokine such as GM-CSF, IL-2, or IL-12, or a chemokine such as IP-10, MCP-3, or RANTES.

[0097] This document also provides nucleic acid molecules that encode a polypeptide provided herein. The term "nucleic acid" as used herein encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

[0098] The term "isolated" as used herein with reference to nucleic acid refers to a naturally-occurring nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally-occurring genome of the organism or virus from which it is derived. For example, an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.

[0099] The term "isolated" as used herein with reference to nucleic acid also includes any non-naturally-occurring nucleic acid since non-naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally occurring genome. For example, non-naturally-occurring nucleic acid such as an engineered nucleic acid is considered to be isolated nucleic acid. Engineered nucleic acid can be made using common molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally-occurring nucleic acid can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote. In addition, a non-naturally-occurring nucleic acid can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence.

[0100] It will be apparent to those of skill in the art that a nucleic acid existing among hundreds to millions of other nucleic acid molecules within, for example, cDNA or genomic libraries, or gel slices containing a genomic DNA restriction digest is not to be considered an isolated nucleic acid.

[0101] A nucleic acid molecule provided herein (e.g., an isolated nucleic acid molecule) can encode any of the polypeptides provided herein. For example, a nucleic acid molecule provided herein can encode a polypeptide having the amino acid sequence as set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 (or an amino acid sequence that is at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identical to the amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58). Examples of such nucleic acid molecules include, without limitation, nucleic acid molecules that have the nucleic acid sequence set forth in SEQ ID NO:9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57. In some cases, nucleic acid molecule provided herein can be codon-optimized to express the encoded polypeptide in cells of a particular species (e.g., codon-optimized for expression in bacterial cells, yeast cells, insect cells, fungal cells, algal cells, mammalian cells, or human cells). For example, a nucleic acid molecule provided herein encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:10 can include a codon-optimized version of the nucleic acid sequence set for in SEQ ID NO:9.

[0102] In some cases, a nucleic acid molecule provided herein can be a vector. A vector can be is a replicon, such as a plasmid, phage, virus, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. An "expression vector" is a vector that includes one or more expression control sequences. An "expression control sequence" is a sequence (e.g., a DNA sequence) that controls or regulates the transcription and/or translation of another sequence (e.g., another DNA sequence).

[0103] In expression vectors, a nucleic acid molecule provided herein (e.g., a nucleic acid encoding a polypeptide provided herein) can be operably linked to one or more expression control sequences. As used herein, "operably linked" means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 to 500 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, the translation initiation site of the translational reading frame of the polypeptide can be positioned between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is "operably linked" and "under the control" of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the polypeptide encoded by the coding sequence.

[0104] Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Invitrogen/Life Technologies (Carlsbad, Calif.).

[0105] An expression vector can include a tag sequence designed to facilitate subsequent manipulation of the expressed nucleic acid sequence (e.g., purification or localization). Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag.TM. tag (Kodak, New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus.

[0106] A nucleic acid molecule provided herein can be obtained using any appropriate method including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, PCR can be used to obtain a nucleic acid containing a nucleic acid sequence sharing similarity to a nucleic acid molecule provided herein, and common mutagenesis techniques can be used to introduce desired nucleotide additions, deletions, substitutions, or combinations thereof into the obtained nucleic acid. PCR refers to a procedure or technique in which target nucleic acid is amplified in a manner similar to that described in U.S. Pat. No. 4,683,195, and subsequent modifications of the procedure described therein. Generally, sequence information from the ends of the region of interest or beyond are used to design oligonucleotide primers that are identical or similar in sequence to opposite strands of a potential template to be amplified. Using PCR, a nucleic acid sequence can be amplified from RNA or DNA. For example, a nucleic acid sequence can be isolated by PCR amplification from total cellular RNA, total genomic DNA, and cDNA as well as from bacteriophage sequences, plasmid sequences, viral sequences, and the like. When using RNA as a source of template, reverse transcriptase can be used to synthesize complimentary DNA strands.

[0107] This document also provides virus-like particles that include one or more of the polypeptides provided herein. For example, a virus-like particle provided herein can be designed to include a polypeptide having the amino acid sequence as set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 (or an amino acid sequence that is at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identical to the amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58). In some cases, a single virus-like particle can include more than one different polypeptide provided herein. For example, a single virus-like particle provided herein can include both a polypeptide having the amino acid sequence as set forth in SEQ ID NO:16 and a polypeptide having the amino acid sequence as set forth in SEQ ID NO:24. Other combinations of polypeptides that can be used to make virus-like particles having a collection of different polypeptides include, without limitation, those set forth in Table 3.

TABLE-US-00003 TABLE 3 Combinations of polypeptides for virus-like particles (VLPs). Sequence identifiers of polypeptides VLP ID No. to be included in the VLP 7101 14, 16, 18 7102 22, 24, 28 7103 14, 16, 18, 22, 24 7104 36, 38 7105 32, 52, 58 7106 32, 36, 38, 52, 58 7107 14, 16, 36, 38 7108 22, 24, 28, 32, 52, 58 7109 16, 18, 24, 32, 36, 38, 52, 58

[0108] Any appropriate method can be used to make virus-like particles provided herein. For example, a virus-like particle provided herein can be made using a method described elsewhere. See, e.g., U.S. Pat. No. 4,803,164, which describes a method to produce HBs-containing VLPs in yeast cells; U.S. Pat. No. 6,551,820, which describes a method to produce HBs-containing VLPs in transgenic plants; and European Patent Application No. EP0241021 A2, which describes a method to produce HBs-containing VLPs in Chinese Hamster Ovary cells or normal liver cells.

[0109] This document also provides vaccine preparations for inducing an immune response within a mammal against a hepatitis B virus. In some cases, a vaccine preparation provided herein can include one or more of the polypeptides provided herein. For example, a vaccine preparation provided herein can be designed to include a polypeptide having the amino acid sequence as set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 (or an amino acid sequence that is at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identical to the amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58). In some cases, a single vaccine preparation can include more than one different polypeptide provided herein. For example, a single vaccine preparation provided herein can include both a polypeptide having the amino acid sequence as set forth in SEQ ID NO:16 and a polypeptide having the amino acid sequence as set forth in SEQ ID NO:24. Other combinations of polypeptides that can be used to make a vaccine preparation having a collection of different polypeptides include, without limitation, those set forth in Table 4.

TABLE-US-00004 TABLE 4 Combinations of polypeptides for vaccine preparations. Vaccine Sequence identifiers of polypeptides to ID No. be included in the vaccine preparation 8101 14, 16, 18 8102 22, 24, 28 8103 14, 16, 18, 22, 24 8104 36, 38 8105 32, 52, 58 8106 32, 36, 38, 52, 58 8107 14, 16, 36, 38 8108 22, 24, 28, 32, 52, 58 8109 16, 18, 24, 32, 36, 38, 52, 58

[0110] In some cases, a vaccine preparation provided herein can include one or more of the nucleic acid molecules provided herein. For example, a vaccine preparation provided herein can be designed to include a nucleic acid vector having a promoter operably linked to a nucleic acid sequence that encodes a polypeptide having the amino acid sequence as set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 (or an amino acid sequence that is at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identical to the amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58). In some cases, a single vaccine preparation can include more than one different nucleic acid molecule provided herein. For example, a single vaccine preparation provided herein can include both a nucleic acid vector designed to express a polypeptide having the amino acid sequence as set forth in SEQ ID NO:16 and a nucleic acid vector designed to express a polypeptide having the amino acid sequence as set forth in SEQ ID NO:24. Other combinations of vectors that can be used to make a vaccine preparation having a collection of different vectors include, without limitation, those set forth in Table 5. In some cases, a single nucleic acid vector can encode two or more polypeptides provided herein. For example, a single nucleic acid vector can be designed to encode a combination of polypeptides as set forth in Table 4.

TABLE-US-00005 TABLE 5 Combinations of vectors for vaccine preparations. Sequence identifiers of polypeptides Vaccine to be encoded by individual vectors ID No. included in the vaccine preparation 9101 14, 16, 18 9102 22, 24, 28 9103 14, 16, 18, 22, 24 9104 36, 38 9105 32, 52, 58 9106 32, 36, 38, 52, 58 9107 14, 16, 36, 38 9108 22, 24, 28, 32, 52, 58 9109 16, 18, 24, 32, 36, 38, 52, 58

[0111] In some cases, a vaccine preparation provided herein can include one or more of the virus-like particles provided herein. For example, a vaccine preparation provided herein can be designed to include a virus-like particle that includes a polypeptide having the amino acid sequence as set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58 (or an amino acid sequence that is at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent identical to the amino acid sequence set forth in SEQ ID NO:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 58). In some cases, a single vaccine preparation can include more than one different virus-like particles provided herein. For example, a single vaccine preparation provided herein can include both a virus-like particle that includes a polypeptide having the amino acid sequence as set forth in SEQ ID NO:16 and a virus-like particle that includes a polypeptide having the amino acid sequence as set forth in SEQ ID NO:24. Other combinations of virus-like particles that can be used to make a vaccine preparation having a collection of different virus-like particles include, without limitation, those set forth in Table 6.

TABLE-US-00006 TABLE 6 Combinations of VLPs for vaccine preparations. Vaccine Specific VLPs to be included ID No. in the vaccine preparation 10101 VLP ID#7101, VLP ID#7102 10102 VLP ID#7104, VLP ID#7105 10103 VLP ID#7101, VLP ID#7104 10104 VLP ID#7102, VLP ID#7105 10105 VLP ID#7101, VLP ID#7102, VLP ID#7104, VLP ID#7105 10106 VLP ID#7103, VLP ID#7106 10107 VLP ID#7101, VLP ID#7104, VLP ID#7105

[0112] Any appropriate method can be used to formulate a vaccine preparation provided herein (e.g., a polypeptide vaccine, nucleic acid vaccine, or VLP vaccine provided herein). For example, a polypeptide vaccine provided herein, a nucleic acid vaccine provided herein, or a VLP vaccine provided herein can be formulated to include one or more adjuvants. An adjuvant can be an immunological compound that can enhance an immune response against a particular antigen such as a polypeptide provided herein. Suitable adjuvants include, without limitation, alum as well as other aluminum-based compounds (e.g., Al.sub.2O.sub.3) that can be obtained from various commercial suppliers. For example, REHYDRAGEL.RTM. adjuvants can be obtained from Reheis Inc. (Berkeley Heights, N.J.). REHYDRAGEL.RTM. adjuvants are based on crystalline aluminum oxyhydroxide, and are hydrated gels containing crystalline particles with a large surface area (about 525 m.sup.2/g). Their Al.sub.2O.sub.3 content typically ranges from about 2 percent to about 10 percent. Rehydragel LG, for example, has an Al.sub.2O.sub.3 content of about 6 percent, and flows readily upon slight agitation. Rehydragel LG also has a protein binding capacity of 1.58 (i.e., 1.58 mg of bovine serum albumin bound per 1 mg of Al.sub.2O.sub.3), a sodium content of 0.02 percent, a chloride content of 0.28 percent, undetectable sulphate, an arsenic level less than 3 ppm, a heavy metal content less than 15 ppm, a pH of 6.5, and a viscosity of 1090 cp. Rehydragel LG can be combined with a polypeptide solution (e.g., a polypeptide in PBS) to yield Al(OH).sub.3. In some cases, ALHYDROGEL.TM., an aluminum hydroxy gel adjuvant (Alhydrogel 1.3%, Alhydrogel 2.0%, or Alhydrogel "85") obtained from Brenntag Stinnes Logistics, can be used.

[0113] In some cases, Montanide ISA 51 can be included in a vaccine preparation provided herein. MN51 (MONTANIDE.RTM. Incomplete SEPPIC Adjuvant (ISA) 51) as well as MN720 are available from Seppic (Paris, France). MN51 contains mannide oleate (MONTANIDE.RTM. 80, also known as anhydro mannitol octadecenoate) in mineral oil solution (Drakeol 6 VR). MONTANIDE.RTM. 80 is a limpid liquid with a maximum acid value of 1, a saponification value of 164-172, a hydroxyl value of 89-100, an iodine value of 67-75, a maximum peroxide value of 2, a heavy metal value less than 20 ppm, a maximum water content of 0.35%, a maximum color value of 9, and a viscosity at 25.degree. C. of about 300 mPas. MONTANIDE.RTM. associated with oil (e.g., mineral oil, vegetable oil, squalane, squalene, or esters) is known as MONTANIDE.RTM. ISA. Drakeol 6 VR is a pharmaceutical grade mineral oil. Drakeol 6 VR contains no unsaturated or aromatic hydrocarbons, and has an A.P.I. gravity of 36.2-36.8, a specific gravity at 25.degree. C. of 0.834-0.838, a viscosity at 100.degree. F. of 59-61 SSU or 10.0-10.6 centistokes, a refractive index at 25.degree. C. of 1.458-1.463, a better than minimum acid test, is negative for fluorescence at 360 nm, is negative for visible suspended matter, has an ASTM pour test value of 0-15.degree. F., has a minimum ASTM flash point of 295.degree. F., and complies with all RN requirements for light mineral oil and ultraviolet absorption. MN51 contains about 8 to 12 percent anhydro mannitol octadecenoate and about 88 to 92 percent mineral oil.

[0114] Other immunostimulatory components that can be used include, without limitation, plant extracts derived from the Soap bark tree (Quillaja species) containing members of a family of plant-based compounds called saponins.

[0115] Other adjuvants that can be included in a vaccine preparation provided herein include, without limitation, immuno-stimulating complexes (ISCOMs) that can contain such components as cholesterol and saponins. Examples include, without limitation, ISCOMATRIX.TM. and MATRIX-M.TM.. ISCOM matrices can be prepared and conjugated to Cu.sup.2+. Adjuvants such as FCA, FIA, MN51, MN720, and Al(OH).sub.3 are commercially available from companies such as Seppic, Difco Laboratories (Detroit, Mich.), and Superfos Biosector A/S (Vedbeak, Demark).

[0116] Other immunostimulatory components include, without limitation, muramyldipeptide (e.g., N-acetylmuramyl-L-alanyl-D-isoglutamine; MDP), monophosphoryl-lipid A (MPL), formyl-methionine containing tripeptides such as N-formyl-Met-Leu-Phe, or a bacterial lipopolysaccharide. Such compounds are commercially available from Sigma Chemical Co. (St. Louis, Mo.) and RIBI ImmunoChem Research, Inc. (Hamilton, Mont.), for example. In some cases, an adjuvant can be Complete Freund's Adjuvant or Incomplete Freund's Adjuvant.

[0117] In some cases, a nucleic acid vaccine preparation provided herein can be formulated to lack an adjuvant. For example, a nucleic acid vaccine preparation provided herein can be designed to include a nucleic acid molecule that encodes a polypeptide provided herein without including any adjuvant.

[0118] In some cases, a polypeptide vaccine provided herein, a nucleic acid vaccine provided herein, or a VLP vaccine provided herein can be formulated to include other components such as cytokines, chemokines, monoclonal antibodies, or co-stimulatory molecules such as B7.

[0119] This document also provides methods for preparing a vaccine preparation provided herein. Such methods can involve suspending an amount of a polypeptide provided herein, a VLP provided herein, or a nucleic acid vector provided herein in a suitable amount of a physiological buffer (e.g., PBS). The polypeptides, VLPs, or nucleic acid vectors then can optionally be combined with a suitable amount of an adjuvant/immunostimulatory compound. The combining step can be achieved by any appropriate method, including, for example, stirring, shaking, vortexing, or passing back and forth through a needle attached to a syringe.

[0120] A vaccine preparation provided herein can be prepared in batch, such that enough unit doses are obtained for multiple injections (e.g., injections into multiple mammals or multiple injections into the same mammal). A "unit dose" of a vaccine preparation provided herein refers to the amount of a vaccine preparation administered to a mammal at one time. A unit dose of a vaccine preparation provided herein can contain an amount of polypeptides, VLPs, or nucleic acid molecules effective to induce an immune response against a hepatitis B virus. For example, a unit dose of a vaccine preparation provided herein can contain between about 0.1 .mu.g and about 1 g (e.g., 1 .mu.g, 10 .mu.g, 15 .mu.g, 25 .mu.g, 30 .mu.g, 50 .mu.g, 100 .mu.g, 250 .mu.g, 280 .mu.g, 300 .mu.g, 500 .mu.g, 750 .mu.g, 1 mg, 10 mg, 15 mg, 25 mg, 30 mg, 50 mg, 100 mg, 250 mg, 280 mg, 300 mg, 500 mg, 750 mg, or more) of polypeptides, VLPs, or nucleic acid molecules. In the case of vaccine preparations containing viral vectors, a unit dose of a vaccine preparation can have a titer between about 10.sup.3 to 10.sup.10 (e.g., 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, or 10.sup.10) viral particles or plaque forming units.

[0121] This document also provides methods for inducing an immune response within a mammal (e.g., a human) against a hepatitis B virus. For example, one or more polypeptide vaccines provided herein, one or more nucleic acid vaccines provided herein, one or more VLP vaccine provided herein, or combinations thereof can be administered to a mammal (e.g., a human) to induce an immune response against hepatitis B viruses. In some cases, an immune response against hepatitis B viruses can be induced by administering one or more nucleic acid vaccines provided herein followed by a vaccine that includes a hepatitis B virus antigen or followed by one or more polypeptide vaccines provided herein. For example, Vaccine ID No. 9101 can be administered to a human, and 1 to 30 days later, Vaccine ID No. 8105 can be administered to that human. In some cases, Vaccine ID No. 9101 can be administered followed 20 to 60 days later by Vaccine ID No. 7101. In some cases, Vaccine ID No. 9109 can be administered followed 30 to 120 days later by Vaccine ID No. 7106.

[0122] In some cases, a vaccine preparation provided herein can be delivered as a prophylactic vaccine to increase a mammal's resistance to a hepatitis B virus infection. For example, one or more polypeptide vaccines provided herein, one or more nucleic acid vaccines provided herein, one or more VLP vaccine provided herein, or combinations thereof can be administered to a human who has not been infected with a hepatitis B virus.

[0123] In some cases, a vaccine preparation provided herein can be used to treat a mammal after the mammal is infected with hepatitis B virus (e.g., an acutely hepatitis B virus infected or chronically hepatitis B virus infected mammal). For example, one or more polypeptide vaccines provided herein, one or more nucleic acid vaccines provided herein, one or more VLP vaccine provided herein, or combinations thereof can be administered to a human acutely or chronically infected with hepatitis B virus. Administration of a vaccine preparation provided herein to a mammal infected with hepatitis B virus can result in a reduction in hepatitis B viral load, a reduction in the severity of the symptoms of the hepatitis B virus infection, a reduction in the degree of liver cirrhosis, a reduction in the incidence of hepatocellular cancer, or a clearance of the hepatitis B virus from the liver.

[0124] This document also provides methods for priming a mammal (e.g., a human) to receive a vaccine containing a hepatitis B virus antigen. For example, one or more polypeptide vaccines provided herein, one or more nucleic acid vaccines provided herein, one or more viral vaccines provided herein, one or more VLP vaccine provided herein, or combinations thereof can be administered to a mammal (e.g., a human) to prime that mammal for generating an enhanced immune response against hepatitis B viruses. Once primed, the mammal can be treated with a vaccine containing a hepatitis B virus antigen (e.g., Engerix) or a vaccine preparation provided herein.

[0125] A vaccine preparation provided herein can be administered using any appropriate method. For example, the administration can be, for example, topical (e.g., transdermal or intranasal), pulmonary (e.g., by inhalation or insufflation of powders or aerosols), oral, or parenteral (e.g., by intradermal, subcutaneous, intramuscular, or intraperitoneal injection, or by intravenous drip). Administration can be rapid (e.g., by injection) or can occur over a period of time (e.g., by slow infusion or administration of slow release formulations). In some cases, a nucleic acid vaccine can be delivered intramuscularly followed 1-90 days (e.g., between 1 and 90, between 1 and 80, between 1 and 70, between 1 and 60, between 1 and 50, between 5 and 90, between 10 and 90, between 20 and 90, between 5 and 75, between 10 and 75, between 10 and 50, between 20 and 50, between 25 and 50, or between 30 and 60 days) later by a VLP vaccine containing adjuvant.

[0126] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1

Producing Shuffled Hepatitis B Surface Antigen

[0127] The parental DNA sequences encoding the preS2 and S regions of the hepatitis envelope protein were derived from the human, chimpanzee, gibbon, woolly monkey, and woodchuck hepadnaviruses and were obtained as follows.

[0128] The preS2+S envelope sequence of the human HBV corresponding to that of the ayw subtype (GenBank.RTM. Accession No. J02203; GI No. 329640) was amplified by PCR from a plasmid vector called pCAG-M-Kan. The nucleic acid sequence for this obtained DNA is set forth in SEQ ID NO:1 and encodes a hepatitis B surface antigen polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0129] The woolly monkey envelope (preS2+S) sequence (GenBank.RTM. Accession No. AF046996; GI No. 3150070) was synthesized by oligonucleotide gene assembly (Stemmer et al., Gene, 164(1):49-53 (1995)). The nucleic acid sequence for this obtained DNA is set forth in SEQ ID NO:3 and encodes a hepatitis B surface antigen polypeptide having the amino acid sequence set forth in SEQ ID NO:4.

[0130] A hybrid envelope gene was designed by combining the preS2 sequence of the human HBV (adw2 subtype; GenBank.RTM. Accession No. X02763; GI No. 59418) and the S sequence of the woodchuck hepatitis virus (strain WHV8; GenBank.RTM. Accession No. J04514; GI No. 336146). The gene was synthesized by oligonucleotide gene assembly. The nucleic acid sequence for this obtained DNA is set forth in SEQ ID NO:5 and encodes a hepatitis B surface antigen polypeptide having the amino acid sequence set forth in SEQ ID NO:6.

[0131] The amino acid sequence differences between the gibbon (GenBank.RTM. Accession No. U46935; GI No. 1814218) and the chimpanzee hepatitis (GenBank.RTM. Accession No. D00220; GI No. 163838595) preS2+S envelope proteins were minimal. A single composite parent gene was therefore synthesized by oligonucleotide gene assembly that contained the sum of all the amino acid changes in the chimpanzee and gibbon sequences relative to the corresponding human sequence. During the course of the synthesis of this composite chimp-gibbon sequence, a mutation occurred which led to the introduction of isoleucine at amino acid number 197 in place of the methionine found in the wild-type chimpanzee and gibbon sequences. The nucleic acid sequence for this obtained DNA is set forth in SEQ ID NO:7 and encodes a hepatitis B surface antigen polypeptide having the amino acid sequence set forth in SEQ ID NO:8.

[0132] The workflow for the hepatitis B surface antigen (HBsAg) library was as follows. The original library was generated using DNAse treatment of the four parental sequences described above (SEQ ID NOs:1, 3, 5, and 7). The shuffling of four parental sequences was achieved by assembling DNase I-digested gene fragments. Basically, each parental gene fragment was made from plasmids in PCR reactions using recombinant Thermus thermophilus with two vector primers (Pre-for-1 (+):GCA GCT CCT TGC TCC TAA CAG (SEQ ID NO:64); and Pre-rev-2(-):GTA TCA CGA GGC CCT TTC GTC (SEQ ID NO:65)). The PCR was performed at 30 cycles of 1 minute at 95.degree. C., 1 minute at 60.degree. C. and 2 minutes at 72.degree. C. The amplified products were purified in gel electrophoresis and digested by DNase I into fragments ranging from 25 bp to 100 bp, which were further purified in gel. These fragments and the three "a"-epitope constrained primers were mixed and assembled with Taq/Pfu (9:1) polymerase in two rounds of 25 cycle PCR reactions (30 seconds at 96.degree. C., 30 seconds at 40.degree. C. and 30 seconds at 72.degree. C.). In the assembly reactions, the percentage of "a" sequence incorporation was controlled by adjusting the ratio of the constrained "a" oligos to DNase I-digested gene fragments based on their molecular weights (FIG. 1). Assembled HBs recombinants were rescued in 30 cycles of PCR by Taq polymerase (Qiagen) with two nested primers (V1521+: GCT GAC AGA CTA ACA GAC TG (SEQ ID NO:66); and V2453 (-): AAC AGA TGG CTG GCA ACT AG (SEQ ID NO:67); 30 seconds at 95.degree. C., 30 seconds at 55.degree. C., and 1 minute at 72.degree. C.). Subsequently, the shuffled HBs fragments were digested with EcoR I/Asp718, purified in gel, and cloned into EcoR I/Asp718-cut pMKan vector using a standard cloning procedure.

[0133] The HBs "a" loop is a major neutralizing epitope and induces protective neutralizing antibodies against hepatitis B virus infection. To preserve this sequence, "a" epitope-constrained libraries were generated. In the assembly reactions, the individual "a" epitope-constrained primer was mixed with the DNase I-digested parental HBs fragments at three concentrations, 0.5:1, 1:1, and 2:1. The incorporation of `a` epitope primers in shuffling reactions were identified by BsrB I restriction enzyme digestion of the assembled HBs fragments. The rate was further evaluated with BsrB I-digested plasmid DNA samples. The ratio of 0.5:1 exhibited about 30% incorporation, and both 1:1 and 2:1 ratios exhibited 60-65% incorporation. Sequencing analyses of plasmid clones indicated that the three constrained `a` epitopes were equally incorporated.

`a` Loop-Constrained Primers

[0134] The thirty nucleotides of the human `a` loop 1 sequence were held constant and flanked by fifteen nucleotides on each side derived from the related HBs genes of Chimpanzee/Gibbon (chibbon), Woolly Monkey (WM), or Woodchuck (WD) (FIG. 2). The primers were designed for human HBs `a` constrained epitopes in the chimpanzee/gibbon hybrid (SEQ ID NO:7), Woolly monkey (SEQ ID NO:3), and human/woodchuck hybrid (SEQ ID NO:5).

Screening

[0135] Clones from the library were screened for protein expression using an immunofluorescence assay (IFA) for expression. Briefly, DNA prepared by BioRobot 9600 was transfected to Cos-7 cells (ATCC #CRL-1651) using SuperFect.TM. transfection reagent in 96-well format as described by the manufacturer (Qiagen). At 48-hour post-transfection, the cells were fixed with paraformaldehyde and stained with goat anti-HBs antibody (Dako #B0560), and then FITC-conjugated rabbit anti-goat immunoglobulins (Dako #PO449). The FITC-positive cells were visualized under fluorescent microscopy. Clones that produced positive IFA signals were then screened in mice by direct intramuscular injection of the plasmid DNA, injecting one shuffled clone in a single mouse. Antibody responses were measured using validated clinical kits (either the Monolisa anti-HBs 3.0 ELISA from Sanofi Diagnostics or the AUSAB EIA ELISA from Abbott Laboratories) and by standard ELISA assays using commercial preparations of the HBsAg obtained from (Seradyn recombinant HBsAg #ABH0705) or the wild-type ayw or adw2 preS2 peptides (custom synthesized by Seradyn) (FIG. 3).

[0136] The most immunogenic clones were further analyzed in larger groups of mice (FIG. 37). Briefly, purified DNA (Aldevron LLC, Fargo, N. Dak.) was diluted to a final concentration of 100 .mu.g/mL in sterile PBS. For each test group, 10 six-week old C57BL/6 mice were injected intramuscularly with 50 .mu.L DNA solution in the tibialis anterior muscle of each leg muscle (10 .mu.g DNA total per mouse). Sera were analyzed 4 weeks after treatment for the level of anti-HBsAg as measured by the Sanofi Monolisa anti-HBS kit (expressed above as the GMT.+-.SEM for each group in mIU per mL). Titers were calculated for mice that received clones 6101, 6102, 6103, 6104, 6105, 6106, 6107, 6108, 6109, or 6111, or the wild-type human hepatitis B envelope gene (HBV). Mice injected with clones 6103, 6104, 6105, 6107, or 6111 from the first round library developed titers that were significantly higher than those in mice that were injected with the wild-type hepatitis B envelope gene containing plasmid control (HBV) (FIG. 37).

[0137] Based on these results, ten clones were selected (clones 6101, 6102, 6103, 6104, 6105, 6106, 6107, 6108, 6109, and 6111) and then shuffled to generate a second round library. This library was screened as before (IFA and mouse immunization), and immunogenic clones were selected and analyzed further in larger groups of animals (FIG. 39). Briefly, purified DNA (Aldevron LLC, Fargo, N. Dak.) was diluted to a final concentration of 100 .mu.g/mL in sterile PBS. For each test group, 10 six-week old C57BL/6 mice were anesthetized and injected intramuscularly with 50 .mu.L DNA solution in the tibialis anterior muscle of each leg muscle (10 .mu.g DNA total per mouse). Sera was analyzed 4 weeks after treatment for the level of anti-hepatitis B antibody as measured by the Sanofi Monolisa anti-HBS kit (expressed above as the GMT.+-.SEM for each group in International Units). Titers were calculated for mice that received clones 6201, 6202, 6204, 6205, 6208, 6209, 6210, 6212, 6213, or 6215, or the wild-type human hepatitis B envelope gene (HBV). Mice injected with clones 6201, 6202, 6204, 6205, 6208, 6209, 6210, 6212, 6213, or 6215 developed titers that were significantly higher than those in mice that were injected with the wild-type hepatitis B envelope gene containing plasmid control (HBV) (FIG. 38).

[0138] The nucleic acid and amino acid sequences of the original four parents (FIGS. 4-7), the ten clones selected from the first round of shuffling (FIGS. 8-17), and the fifteen clones selected from the second round of shuffling (FIGS. 18-32) are presented in FIGS. 4-32. An alignment of the parental and shuffled DNA and protein sequences were obtained using the Clustal W algorithm within the AlignX component of Vector NTI ver 6.0 and are shown in FIGS. 33 and 34, respectively. A summary of the amino acid changes for the clones shown in FIGS. 8-32 is shown in FIG. 35, along with an indication of the various regions of the protein. The nucleotide sequence and component locations of the vector used in this work are shown in FIG. 36.

[0139] All but two of the clones (clones 6101 and 6111) were obtained from a shuffled library in which the main antigenic determinant (the so-called "a-loop") of the human HBV envelope protein was held constant during the shuffling reaction. This was accomplished by including specific oligonucleotides in the shuffling reaction to generate clones in which this sequence was held constant. Several obtained clones did not retain this conserved a-loop in its entirety due to a spontaneous mutation not found in any of the parents used in the shuffling reaction (proline at amino acid number 183 in the place of alanine in the wild-type sequence).

[0140] To obtain additional reliable data, an experiment was conducted in which three first round clones (6104, 6105, and 6108) and five second round clones (6202, 6204, 6205, 6212, and 6215) were used to immunize groups of 30 mice per clone. In this experiment, four plasmids were used that express each of the parental envelope sequences used in the initial shuffling reaction (designated HBV, WM, CH, and WD in FIG. 39). Briefly, purified DNA (Aldevron LLC, Fargo, N. Dak.) was diluted to a final concentration of 100 .mu.g/mL in sterile PBS. For each test group, 30 six-week old C57BL/6 mice were anesthetized and injected intramuscularly with 50 .mu.L DNA solution in the tibialis anterior muscle of each leg muscle (10 .mu.g DNA total per mouse). Sera were analyzed four weeks after treatment for the level of anti-hepatitis B antibody as measured by the Abbott AUSZYME ELISA (expressed above as the GMT.+-.SEM for each group in International Units). Titers were calculated for mice that received DNA clones from the first round library (6104, 6105, and 6108), the second round library (6202, 6204, 6205, 6212, and 6215), and the parents from the original shuffling reaction (wild-type human hepatitis B envelope gene (HBV), woolly monkey hepatitis envelope gene (WM), chimpanzee/gibbon hepatitis envelope gene (CH), and woodchuck hepatitis envelope (WD)). Another group of mice were injected with 1 .mu.g of the commercially available hepatitis B recombinant protein vaccine (Engerix-B, SmithKline Beecham), which was derived from a wild-type HBV gene sequence.

[0141] Mice injected with clones 6104, 6105, 6108, 6202, 6204, 6205, 6212, and 6215 developed titers that were significantly higher than mice that were injected with the original parental clones including the wild-type Hepatitis B envelope gene containing plasmid control (HBV) (FIG. 39). The commercial protein vaccine containing purified protein from the wild-type human Hepatitis B envelope gene adjuvanted with aluminum elicited a superior titer compared to DNA injection of the gene by itself (compare "Engerix" to "HBV"). The titer obtained with the commercial vaccine (Engerix) was surpassed by clones 6104, 6105, 6202, 6204, 6205, 6212, and 6215.

[0142] Clones 6104, 6105, 6108, 6202, 6204, 6205, 6212, and 6215 were further analyzed for their ability to prime the immune system to respond to wild-type hepatitis B surface antigen. Briefly, purified DNA (Aldevron LLC, Fargo, N. Dak.) was diluted to a final concentration of 100 .mu.g/mL in sterile PBS. For each test group, 20 six-week old C57BL/6 mice were anesthetized and injected intramuscularly with 50 .mu.L DNA solution in the tibialis anterior muscle of each leg muscle (10 .mu.g DNA total per mouse). Three weeks after the initial DNA immunization, 10 mice in each group were injected intraperitoneally with 1 .mu.g of the commercially available hepatitis B recombinant protein vaccine (Engerix-B, SmithKline Beecham) diluted four fold in endotoxin free PBS (5 .mu.g/mL final). Two weeks after the protein injection sera from all mice was analyzed for the level of anti-hepatitis B antibody as measured by the Abbott AUSZYME ELISA (expressed above as the GMT for each group in International Units). Titers were calculated for mice that received DNA clones 6104, 6105, 6108, 6202, 6204, 6205, 6212, or 6215, one of the four parental clones, or the backbone DNA plasmid containing an irrelevant gene sequence.

[0143] Clones 6202, 6204, 6205, 6212, and 6215 were superior to the wild-type hepatitis B envelope protein gene for priming the immune system to respond to the hepatitis B protein (FIG. 40). This improved priming was exemplified by the significantly larger memory responses observed after the protein injection in mice that received the shuffled clones as compared to mice that received the wild-type clone (compare black bars of 6104, 6105, 6108, 6202, 6204, 6205, 6212, and 6215 with black bar of HBV in FIG. 40). The relative titers between groups that only received the DNA prime (grey bars) were very similar to the relationships seen in previous experiments (see FIG. 37), but the post boost titers did not maintain this relationship. This change of profile indicated that the high memory response was not solely due to a simple boosting of the higher titer produced by primary injection but rather may indicate that there are aspects of the shuffled genes that enable them to enhance the immune system's ability to respond to a second exposure (compare grey and black bars of 6202 to those of 6205).

[0144] In another experiment, clones 6102, 6103, 6104, 6105, 6108, 6205, 6212, and 6213 were tested in a population of outbred mice. Briefly, purified DNA (Aldevron LLC, Fargo, N. Dak.) was diluted to a final concentration of 1000 .mu.g/mL in sterile PBS. For each test group, 10 six-week old outbred Swiss Webster mice were anesthetized and injected intramuscularly with 50 .mu.L DNA solution in the tibialis anterior muscle of each leg muscle (100 .mu.g DNA total per mouse). Sera were analyzed four weeks after treatment for the level of anti-hepatitis B antibody as measured by Abbott AUSZYME ELISA. Titers were calculated for groups of ten mice each that received clones 6102, 6103, 6104, 6105, 6108, 6205, 6212, or 6213, or for two groups of ten mice each that received the wild-type human hepatitis B envelope gene (HBV). Groups of mice injected with clones 6103, 6105, 6108, 6205, 6212, and 6213 exhibited a higher percentage of seroconverting mice and/or higher anti-HBsAg titers than groups of mice that were injected with the wild-type hepatitis B envelope gene containing plasmid control (HBV) (FIG. 41). This population of outbred mice more accurately reflects the heterogeneity seen in natural populations. The ability of shuffled clones to outperform the wild-type clone in this environment suggests that the shuffled clones may outperform the wild-type sequences in other outbred populations such as the human population.

[0145] In another experiment, clones 6104 and 6105 were tested in a population of non-responder mice. Briefly, purified DNA (Aldevron LLC, Fargo, N. Dak.) was diluted to a final concentration of 1000 .mu.g/mL in sterile PBS. For each test group, 10 six-week old B10M mice were anesthetized and injected intramuscularly with 50 .mu.L DNA solution in the tibialis anterior muscle of each leg muscle (100 .mu.g DNA total per mouse). Sera were collected by retro-orbital methods four weeks after treatment, and the mice were then given a second dose of DNA. Three weeks after the second treatment, sera was collected. The level of anti-hepatitis B antibody as measured by the Abbott AUSZYME ELISA (expressed as the GMT.+-.SEM for each group in International Units) was determined for both the sera after a single treatment (grey bars in FIG. 42) and the sera after two doses (black bars in FIG. 42). Titers were calculated for mice that received DNA clones 6104 and 6105 and wild-type human hepatitis B envelope gene (HBV) (FIG. 42).

[0146] Shuffled clones 6104 and 6105 were able to stimulate strong immune responses in a mouse strain that typically does not respond to hepatitis B antigens. These results were particularly interesting because the B10.M non-responder strain of mice may be similar to a subset of chronically infected humans who also do not respond to HBsAg. The ability of the shuffled clones to overcome this lack of response demonstrates that the shuffled clones provided herein can be used as a therapeutic against chronic hepatitis B.

Other Embodiments

[0147] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Sequence CWU 1

1

691846DNAArtificial Sequencesynthetic nucleic acid 1atgcagtgga attccacaac cttccaccaa actctgcaag atcccagagt gagaggcctg 60tatttccctg ctggtggctc cagttcagga acagtaaacc ctgttctgac tactgcctct 120cccttatcgt caatcttctc gaggattggg gaccctgcgc tgaacatgga gaacatcaca 180tcaggattcc taggacccct tctcgtgtta caggcggggt ttttcttgtt gacaagaatc 240ctcacaatac cgcagagtct agactcgtgg tggacttctc tcaattttct agggggaact 300accgtgtgtc ttggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcaagg tatgttgccc 480gtttgtcctc taattccagg atcctcaaca accagcacgg gaccatgccg gacctgcatg 540actactgctc aaggaacctc tatgtatccc tcctgttgct gtaccaaacc ttcggacgga 600aattgcacct gtattcccat cccatcatcc tgggctttcg gaaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgtaggg 720ctttccccca ctgtttggct ttcagttata tggatgatgt ggtattgggg gccaagtctg 780tacagcatct tgagtccctt tttaccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 8462281PRTArtificial Sequencesynthetic polypeptide 2Met Gln Trp Asn Ser Thr Thr Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Val Leu Thr Thr Ala Ser Pro Leu Ser Ser Ile Phe Ser Arg 35 40 45 Ile Gly Asp Pro Ala Leu Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile65 70 75 80 Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Thr Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Arg Thr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly225 230 235 240 Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Met Met Trp Tyr Trp 245 250 255 Gly Pro Ser Leu Tyr Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 3852DNAArtificial Sequencesynthetic nucleic acid 3atgcagtgga attccacttc cttccagagt tatcttcaga atccaaaggt cagaggcctc 60tactttcctg ctggtggctc aacttcaagc attgtcaacc ctgttccgac cactgcctcc 120accacatcgt caagcttctc gacgactggg gtccctgtca gcaccatgga catcacttca 180tcaggattcc taggacccct tctcgcatta caggcggtgt ttttcttgtt gacaaaaatc 240ctcacaatgc cacagagtct agactcgttg tggacttctc tcaattttct agggggaaca 300ccagcgtgtc ctggcctaaa ttcgcagtcc ccaacctcca gtcactcacc aacctgctgt 360ccaccgactt gtcctgggta tcgctggatg tgtttgcggc gttctatcat cttcctcttc 420atcctgcttc tatgcctcat cttcttgttg gttcttctgg actaccaagg tatgttgccc 480gtgtgtcctc ttctaccaac agttacagga acaacaacaa caacgggacc ctgcaggacc 540tgcacgccaa ttgttccagg catctcttcg tatccctcat gttgctgtac caaacctacg 600gacggaaact gcacttgtat tcccatcccc tcatcatggg ctttcgcaaa gttcctatgg 660gactgggcct tagcccgttt ctcctggctc aattcacttc tgccatttgt tcagtggttc 720gcagggcttt cccccactgt atggctttta gttatatgga tgatgtggtt ctgggggcca 780agtctgttca gcatcttgag tcccttcttg cctctgttac cacttttctt ttggctttgg 840gtatacattt aa 8524283PRTArtificial Sequencesynthetic polypeptide 4Met Gln Trp Asn Ser Thr Ser Phe Gln Ser Tyr Leu Gln Asn Pro Lys1 5 10 15 Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly Ser Thr Ser Ser Ile Val 20 25 30 Asn Pro Val Pro Thr Thr Ala Ser Thr Thr Ser Ser Ser Phe Ser Thr 35 40 45 Thr Gly Val Pro Val Ser Thr Met Asp Ile Thr Ser Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Ala Leu Gln Ala Val Phe Phe Leu Leu Thr Lys Ile65 70 75 80 Leu Thr Met Pro Gln Ser Leu Asp Ser Leu Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Pro Ala Cys Pro Gly Leu Asn Ser Gln Ser Pro Thr 100 105 110 Ser Ser His Ser Pro Thr Cys Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Ser Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Leu Pro Thr Val Thr Gly Thr Thr Thr Thr Thr Gly 165 170 175 Pro Cys Arg Thr Cys Thr Pro Ile Val Pro Gly Ile Ser Ser Tyr Pro 180 185 190 Ser Cys Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys Ile Pro 195 200 205 Ile Pro Ser Ser Trp Ala Phe Ala Lys Phe Leu Trp Asp Trp Ala Leu 210 215 220 Ala Arg Phe Ser Trp Leu Asn Ser Leu Leu Pro Phe Val Gln Trp Phe225 230 235 240 Ala Gly Leu Ser Pro Thr Val Trp Leu Leu Val Ile Trp Met Met Trp 245 250 255 Phe Trp Gly Pro Ser Leu Phe Ser Ile Leu Ser Pro Phe Leu Pro Leu 260 265 270 Leu Pro Leu Phe Phe Trp Leu Trp Val Tyr Ile 275 280 5834DNAArtificial Sequencesynthetic nucleic acid 5atgcagtgga attccactgc cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tattgcctct 120cacatctcgt caatctccgc gaggactggg gaccctgtga cgaacatgtc accatcaagt 180ctcctaggac tcctcgcagg attacaggtg gtgtatttct tgtggacaaa aatcctaaca 240atagctcaga atctagattg gtggtggact tctctcagtt ttccaggggg cataccagag 300tgcactggcc aaaattcgca gttccaaact tgcaaacact tgccaacctc ctgtccacca 360acttgcaatg gctttcgttg gatgtatctg cggcgtttta tcatatacct attagtcctg 420ctgctgtgcc tcatcttctt gttggttctc ctggactgga aaggtttaat acctgtctgt 480cctcttcaac ccacaacaga aacaacagtc aattgcagac aatgcacaat ctctgcacaa 540gacatgtata ctcctcctta ctgttgttgt ttaaaaccta cggcaggaaa ttgcacttgt 600tggcccatcc cttcatcatg ggctttagga aattacctat gggagtgggc cttagcccgt 660ttctcttggc tcaatttact agtgcccttg cttcaatggt taggaggaat ttccctcatt 720gcgtggtttt tgcttatatg gatgatttgg ttttgggggc ccgcacttct gagcatctta 780ccgccattta ttcccatatt tgttctgttt ttcttgattt gggtatacat ttaa 8346277PRTArtificial Sequencesynthetic polypeptide 6Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Thr Gly Asp Pro Val Thr Asn Met Ser Pro Ser Ser Leu Leu Gly Leu 50 55 60 Leu Ala Gly Leu Gln Val Val Tyr Phe Leu Trp Thr Lys Ile Leu Thr65 70 75 80 Ile Ala Gln Asn Leu Asp Trp Trp Trp Thr Ser Leu Ser Phe Pro Gly 85 90 95 Gly Ile Pro Glu Cys Thr Gly Gln Asn Ser Gln Phe Gln Thr Cys Lys 100 105 110 His Leu Pro Thr Ser Cys Pro Pro Thr Cys Asn Gly Phe Arg Trp Met 115 120 125 Tyr Leu Arg Arg Phe Ile Ile Tyr Leu Leu Val Leu Leu Leu Cys Leu 130 135 140 Ile Phe Leu Leu Val Leu Leu Asp Trp Lys Gly Leu Ile Pro Val Cys145 150 155 160 Pro Leu Gln Pro Thr Thr Glu Thr Thr Val Asn Cys Arg Gln Cys Thr 165 170 175 Ile Ser Ala Gln Asp Met Tyr Thr Pro Pro Tyr Cys Cys Cys Leu Lys 180 185 190 Pro Thr Ala Gly Asn Cys Thr Cys Trp Pro Ile Pro Ser Ser Trp Ala 195 200 205 Leu Gly Asn Tyr Leu Trp Glu Trp Ala Leu Ala Arg Phe Ser Trp Leu 210 215 220 Asn Leu Leu Val Pro Leu Leu Gln Trp Leu Gly Gly Ile Ser Leu Ile225 230 235 240 Ala Trp Phe Leu Leu Ile Trp Met Ile Trp Phe Trp Gly Pro Ala Leu 245 250 255 Leu Ser Ile Leu Pro Pro Phe Ile Pro Ile Phe Val Leu Phe Phe Leu 260 265 270 Ile Trp Val Tyr Ile 275 7846DNAArtificial Sequencesynthetic nucleic acid 7atgcagtgga attctacagt attccaccaa gctctgcaag atcccagagt acggggccta 60tactttcctg ttggtggctc cagttcagga acattgaacc ctgttccgaa tactgcctct 120cacatctcgt cagtcttctc gacgactggg gaccctgcac cgaacatgga gaacatcaca 180tcaggattcc taggacccct gctcgtgtta caggcggggt ttttcttgtt gacaaaaatc 240ctcacaatac cacagagtct agactcgtgg tggacttctc tcaattttct agggggagca 300cccgtgtgtc ctggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaattt gtcctggcta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcaagg tatgttgccc 480gtttgtcctc tacttccagg atcatcgacc accagcacgg gaccatgcaa aacctgcacg 540atccctgctc aaggaacctc tttgattccc tcatgttgtt gtacaaaacc ttcggacgga 600aattgcactt gtattcccat cccatcgtct tgggctttcg caaaattcct atgggagtgg 660gcctcagtcc gtttctcctg gctcagttta ctagctccat ttgttcagtg gttcgcaggg 720ctttccccca ctgcttggct tttagctata tggatcatct ggtattgggg gccaaatctg 780tacaacatct tgaatccatt tataccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 8468281PRTArtificial Sequencesynthetic polypeptide 8Met Gln Trp Asn Ser Thr Val Phe His Gln Ala Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Phe Pro Val Gly Gly Ser Ser Ser Gly Thr Leu 20 25 30 Asn Pro Val Pro Asn Thr Ala Ser His Ile Ser Ser Val Phe Ser Thr 35 40 45 Thr Gly Asp Pro Ala Pro Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Lys Ile65 70 75 80 Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Ala Pro Val Cys Pro Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Leu Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Lys Thr Cys Thr Ile Pro Ala Gln Gly Thr Ser Leu Ile Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Ala Lys Phe Leu Trp Glu Trp Ala Ser Val Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Ala Pro Phe Val Gln Trp Phe Ala Gly225 230 235 240 Leu Ser Pro Thr Ala Trp Leu Leu Ala Ile Trp Ile Ile Trp Tyr Trp 245 250 255 Gly Pro Asn Leu Tyr Asn Ile Leu Asn Pro Phe Ile Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 9864DNAArtificial Sequencesynthetic nucleic acid 9atgcagtgga attctacagt attccaccaa actctgcaag atcccagagt ggggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgttctgac tactgcctct 120cccttatcgt caatcttctc gaggattggg gaccctgcgc tgaacatgga gaacatcaca 180tcaggattcc taggacccct tctcgtgtta caggcggggt ttttcttgat gacaaaaatc 240ctcacaatgc cgcagagtct agactcgtgg tggacttctc tcaattttct agggagagca 300cccgtgtgtc ctggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaattt gtcctggcta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc tacttccagg atcatcgacc accagcacgg gaccatgcaa aacctgcacg 540atccctgctc aaggaacctc tttgattccc tcatgttgtt gtacaaaacc ttcggacgga 600aattgcactt gtattcccat cccatcgtct tgggctttcg caaaattcct atgggagtgg 660gcctcagtcc gtttctcctg gctcagttta ctagctccat ttgttcagcg gttcgcaggg 720ctttccccca ctgcttggct tttagctata tggatcatct ggtattgggg gccaaatctg 780tacaacatct tgagtccctt cttgccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaaatgc tagaggtacc ctga 86410281PRTArtificial Sequencesynthetic polypeptide 10Met Gln Trp Asn Ser Thr Val Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Gly Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Val Leu Thr Thr Ala Ser Pro Leu Ser Ser Ile Phe Ser Arg 35 40 45 Ile Gly Asp Pro Ala Leu Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Met Thr Lys Ile65 70 75 80 Leu Thr Met Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Arg Ala Pro Val Cys Pro Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Leu Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Lys Thr Cys Thr Ile Pro Ala Gln Gly Thr Ser Leu Ile Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Ala Lys Phe Leu Trp Glu Trp Ala Ser Val Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Ala Pro Phe Val Gln Arg Phe Ala Gly225 230 235 240 Leu Ser Pro Thr Ala Trp Leu Leu Ala Ile Trp Ile Ile Trp Tyr Trp 245 250 255 Gly Pro Asn Leu Tyr Asn Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 11846DNAArtificial Sequencesynthetic nucleic acid 11atgcagtgga attccacaac cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tactgcctct 120cacatctcgt cagtcttctc gacgactggg gaccctgcac cgaacatgga gaacatcaca 180tcaggattcc taggacccct gctcgtgtta caggcggggt ttttcttgtt gacaaaaatc 240ctcacaatgc cacagagtct agactcgttg tggacttctc tcaattttct agggggaaca 300ccagcgtgtc ttggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc taattccagg atcctcaaca accagcacgg gaccttgcag gacctgtatg 540actaccgctc aaggaacctc tatgtatccc tcatgttgtt gtacaaaacc ttcggacgga 600aattgcacct gtattcccat cccatcatcc tgggctttcg gaaaattcct atgggactgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagcg gttcgcaggg 720ctttctccca ctgcttggct ttcagttata tggatgatgt ggtattgggg accaagtctg 780tacagcatct tgagtccctt tttaccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84612281PRTArtificial Sequencesynthetic polypeptide 12Met Gln Trp Asn Ser Thr Thr Phe His Gln Thr Leu Gln Asp Pro Arg1 5

10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Thr Ala Ser His Ile Ser Ser Val Phe Ser Thr 35 40 45 Thr Gly Asp Pro Ala Pro Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Lys Ile65 70 75 80 Leu Thr Met Pro Gln Ser Leu Asp Ser Leu Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Pro Ala Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Arg Thr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Asp Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Arg Phe Ala Gly225 230 235 240 Leu Ser Pro Thr Ala Trp Leu Ser Val Ile Trp Met Met Trp Tyr Trp 245 250 255 Gly Pro Ser Leu Tyr Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 13846DNAArtificial Sequencesynthetic nucleic acid 13atgcagtgga attccactgc cttccaccaa actctgcagg atcccagagt caggggtctg 60tatctccctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tattgcctct 120cacatctcgt caatctccgc gaggactggg gaccctgcac cgaacatgga gaacatcaca 180tcaggattcc taggacccct gctcgtgtta caggcggtgt ttttcttgtt gacaagaatc 240ctcacaatac cgcagagtct agactcgtgg tggacttctc tcaattttct agggggaact 300accgtgtgtc ctggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat atacctatta 420gtcctgctgc tgtgcctcat cttcttgttg gttcttctgg actatcaagg tatgttgccc 480gtttgtcctc taattccagg atcctcaaca accagcacgg gaccatgcaa aacctgtatg 540actaccgctc aaggaacctc tatgtatccc tcatgttgtt gtacaaaacc ttcggacgga 600aattgcactt gtattcccat cccatcatcc tgggctttcg gaaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagcg gttcgcaggg 720ctttccccca ctgtttggct ttcagttata tggatgattt ggttttgggg gccaagtctg 780tacagcatct tgagtccctt tttaccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84614281PRTArtificial Sequencesynthetic polypeptide 14Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Thr Gly Asp Pro Ala Pro Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Val Phe Phe Leu Leu Thr Arg Ile65 70 75 80 Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Thr Val Cys Pro Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Tyr Leu Leu Val Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Lys Thr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Arg Phe Ala Gly225 230 235 240 Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Met Ile Trp Phe Trp 245 250 255 Gly Pro Ser Leu Tyr Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 15846DNAArtificial Sequencesynthetic nucleic acid 15atgcagtgga attctacaac cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tattgcctct 120cacatctcgt caatctccgc gaggactggg gaccctgcgc tgaacatgga gaacatcaca 180tcaggattcc taggacccct tctcgtgtta caggcggggt ttttcttgtt gacaagaatc 240ctcacaatac cacagagtct agactcgtgg tggacttctc tcaattttct agggggaact 300accgtgtgtc ttggcctaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgcttc tatgcctcat cttcttgttg gttcttctgg actatcaagg tatgttgccc 480gtttgtcccc taattccagg atcctcaaca accagcacgg gaccatgcaa aacctgtatg 540actacccctc aaggaacctc tatgtatccc tcatgttgct gtaccaaacc ttcggacgga 600aattgcacct gtattcccat cccatcatcc tgggctttcg gaaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgtaggg 720ctttccccca ctgcttggct tttggctata tggatcatct ggtattgggg gccaaatctg 780tacaacatct tgaatccctt tttaccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84616281PRTArtificial Sequencesynthetic polypeptide 16Met Gln Trp Asn Ser Thr Thr Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Thr Gly Asp Pro Ala Leu Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile65 70 75 80 Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Thr Val Cys Leu Gly Leu Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Lys Thr Cys Met Thr Thr Pro Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly225 230 235 240 Leu Ser Pro Thr Ala Trp Leu Leu Ala Ile Trp Ile Ile Trp Tyr Trp 245 250 255 Gly Pro Asn Leu Tyr Asn Ile Leu Asn Pro Phe Leu Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 17846DNAArtificial Sequencesynthetic nucleic acid 17atgcagtgga attccactgc cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagctcagga acagtaaacc ctgctccgaa tattgcctct 120cacatctcgt caatctccgc gaggattggg gaccctgcgc tgaacatgga gaacatcaca 180tcaggattcc taggacccct tctcgcatta caggcggtgt ttttcttgtt gacaaaaatc 240ctcacaatgc cacagagtct agactcgtgg tggacttctc tcaattttct agggggaact 300accgtgtgtc ttggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc taattccagg atcctcaaca accagcacgg gaccatgcaa aacctgtatg 540actaccgctc aaggaacctc tatgtatccc tcatgttgtt gtacaaaacc ttcggacgga 600aattgcacct gtattcccat cccatcatcc tgggctttcg caaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgtaggg 720ctttccccca ctgtttggct ttcagttata tggatgatgt ggtattgggg gccaagtctg 780ttcagcatct tgagtccctt cttgcctctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84618281PRTArtificial Sequencesynthetic polypeptide 18Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Ile Gly Asp Pro Ala Leu Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Ala Leu Gln Ala Val Phe Phe Leu Leu Thr Lys Ile65 70 75 80 Leu Thr Met Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Thr Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Lys Thr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Ala Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly225 230 235 240 Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Met Met Trp Tyr Trp 245 250 255 Gly Pro Ser Leu Phe Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 19846DNAArtificial Sequencesynthetic nucleic acid 19atgcagtgga attccacaac cttccaccaa actctgcaag atcccagagt gagaggcctg 60tatttccctg ctggtggctc cagttcagga acagtaaacc ctgttctgac tactgcctct 120cccttatcgt caatcttctc gaggattggg gaccctgcgc tgaacatgga gaacatcaca 180tcaggattcc taggacccct tctcgtgtta caggcggggt ttttcttgtt gacaagaatc 240ctcacaatac cgcagagtct agactcgttg tggacttctc tcagttttcc agggggcata 300ccagagtgca ctggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc tacttccagg atcctcaaca accagcacgg gaccctgcag gacctgtatg 540actaccgctc aaggaacctc tatgtatccc tcatgttgct gtaccaaacc ttcggacgga 600aattgcacct gtattcccat cccatcatcc tgggctttcg caaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgtaggg 720ctttccccca ctgtttggct ttcagttata tggatcatct ggtattgggg gccaaatctg 780tacaacatct tgaatccatt tataccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84620281PRTArtificial Sequencesynthetic polypeptide 20Met Gln Trp Asn Ser Thr Thr Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Val Leu Thr Thr Ala Ser Pro Leu Ser Ser Ile Phe Ser Arg 35 40 45 Ile Gly Asp Pro Ala Leu Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile65 70 75 80 Leu Thr Ile Pro Gln Ser Leu Asp Ser Leu Trp Thr Ser Leu Ser Phe 85 90 95 Pro Gly Gly Ile Pro Glu Cys Thr Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Leu Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Arg Thr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Ala Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly225 230 235 240 Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Ile Ile Trp Tyr Trp 245 250 255 Gly Pro Asn Leu Tyr Asn Ile Leu Asn Pro Phe Ile Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 21846DNAArtificial Sequencesynthetic nucleic acid 21atgcagtgga attccactgc cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tattgcctct 120cccttatcgt caatcttctc gacgactggg gaccctgcac cgaacatgga gaacatcaca 180tcaggattcc taggacccct tctcgtgtta caggcggggt ttttcttgtt gacaagaatc 240ctcacaatac cgcagagtct agactcgtgg tggacttctc tcaattttct agggggagca 300cccgtgtgtc ttggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctgggta tcgctggatg cgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc taattccagg atcctcaaca accggcacgg gaccctgcag gacctgtatg 540actactgctc aaggaacctc tatgtatccc tcatgttgtt gtacaaaacc ttcggacgga 600aattgcactt gtattcccat cccatcatcc tgggctttcg gaaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgtaggg 720ctttcccccg ctgtatggct ttcagttata tggatgatgt ggtattgggg gccaagtctg 780tacagcatct tgagtccctt tttaccgctg ttaccaattt tcttttggct ttgggtatac 840atttaa 84622281PRTArtificial Sequencesynthetic polypeptide 22Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser Pro Leu Ser Ser Ile Phe Ser Thr 35 40 45 Thr Gly Asp Pro Ala Pro Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile65 70 75 80 Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Ala Pro Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Arg Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155

160 Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Gly Thr Gly Pro Cys 165 170 175 Arg Thr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly225 230 235 240 Leu Ser Pro Ala Val Trp Leu Ser Val Ile Trp Met Met Trp Tyr Trp 245 250 255 Gly Pro Ser Leu Tyr Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro 260 265 270 Ile Phe Phe Trp Leu Trp Val Tyr Ile 275 280 23840DNAArtificial Sequencesynthetic nucleic acid 23atgcagtgga attctactgc cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tattgcctct 120cacatctcgt caatctccgc gaggactggg gaccctgtga cgaacatgtc accatcaagt 180ctcctaggac tcctcgcagg attacaggtg gtgtatttct tgtggacaaa aatcctaaca 240ataccgcaga gtctagactc gtggtggact tctctcaatt ttctaggggg agcacccgtg 300tgtcctggcc aaaattcgca gtccccaacc tccaatcact caccaacctc ttgtcctcca 360atttgtcctg gttatcgctg gatgtgtctg cggcgtttta tcatcttcct cttcatcctg 420cttctatgcc tcatcttctt gttggttctt ctggactatc aaggtatgtt gcccgtttgt 480cctctaattc caggatcatc gaccaccagc acgggaccat gcaaaacctg tatgactacc 540cctcaaggaa cctctatgta tccctcatgt tgttgtacca aaccttcgga cggaaattgc 600acctgtattc ccatcccatc atcctgggct ttcggaaaat tcctatggga gtgggcctca 660gtccgtttct cctggctcag tttactagct ccatttgttc agcggttcgt agggctttcc 720cccactgttt ggctttcagt tatatggatg atgtggttct gggggccaag tctgttcagc 780atcttgagtc ccttcttgcc tctgttacca cttttctttt ggctttgggt atacatttaa 84024279PRTArtificial Sequencesynthetic polypeptide 24Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Thr Gly Asp Pro Val Thr Asn Met Ser Pro Ser Ser Leu Leu Gly Leu 50 55 60 Leu Ala Gly Leu Gln Val Val Tyr Phe Leu Trp Thr Lys Ile Leu Thr65 70 75 80 Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly 85 90 95 Gly Ala Pro Val Cys Pro Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn 100 105 110 His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met 115 120 125 Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu 130 135 140 Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys145 150 155 160 Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr 165 170 175 Cys Met Thr Thr Pro Gln Gly Thr Ser Met Tyr Pro Ser Cys Cys Cys 180 185 190 Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser 195 200 205 Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Val Arg Phe Ser 210 215 220 Trp Leu Ser Leu Leu Ala Pro Phe Val Gln Arg Phe Val Gly Leu Ser225 230 235 240 Pro Thr Val Trp Leu Ser Val Ile Trp Met Met Trp Phe Trp Gly Pro 245 250 255 Ser Leu Phe Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro Leu Phe 260 265 270 Phe Trp Leu Trp Val Tyr Ile 275 25840DNAArtificial Sequencesynthetic nucleic acid 25atgcagtgga attctactgc cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tattgcctct 120cacatctcgt caatctccgc gaggactggg gaccctgtga cgaacatgtc accatcaagt 180ctcctaggac tcctcgcagg attacaggtg gtgtatttct tgtggacaaa aatcctaaca 240ataccgcaga gtctagactc gtggtggact tctctcaatt ttctaggggg agcacccgtg 300tgtcctggcc aaaattcgca gtccccaacc tccaatcact caccaacctc ttgtcctcca 360atttgtcctg gttatcgctg gatgtgtctg cggcgtttta tcatcttcct cttcatcctg 420cttctatgcc tcatcttctt gttggttctt ctggactatc aaggtatgtt gcccgtttgt 480cctctaattc caggatcatc gaccaccagc acgggaccat gcaaaacctg tatgactacc 540cctcaaggaa cctctatgta tccctcatgt tgttgtacca aaccttcgga cggaaattgc 600acctgtattc ccatcccatc atcctgggct ttcggaaaat tcctatggga gtgggcctca 660gtccgtttct cctggctcag tttactagct ccatttgttc agcggttcgt agggctttcc 720cccactgttt ggctttcagt tatatggatg atgtggttct gggggccaag tctgttcagc 780atcttgagtc ccttcttgcc tctgttacca cttttctttt ggctttgggt atacatttaa 84026279PRTArtificial Sequencesynthetic polypeptide 26Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Thr Gly Asp Pro Val Thr Asn Met Ser Pro Ser Ser Leu Leu Gly Leu 50 55 60 Leu Ala Gly Leu Gln Val Val Tyr Phe Leu Trp Thr Lys Ile Leu Thr65 70 75 80 Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly 85 90 95 Gly Ala Pro Val Cys Pro Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn 100 105 110 His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met 115 120 125 Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu 130 135 140 Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys145 150 155 160 Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr 165 170 175 Cys Met Thr Thr Pro Gln Gly Thr Ser Met Tyr Pro Ser Cys Cys Cys 180 185 190 Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser 195 200 205 Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Val Arg Phe Ser 210 215 220 Trp Leu Ser Leu Leu Ala Pro Phe Val Gln Arg Phe Val Gly Leu Ser225 230 235 240 Pro Thr Val Trp Leu Ser Val Ile Trp Met Met Trp Phe Trp Gly Pro 245 250 255 Ser Leu Phe Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro Leu Phe 260 265 270 Phe Trp Leu Trp Val Tyr Ile 275 27846DNAArtificial Sequencesynthetic nucleic acid 27atgcagtgga attccactgc cttccaccaa actctgcaag atcccagagt acggggccta 60tactttcctg ttggtggctc cagttcagga acagtaaacc ctgctccgaa tattgcctct 120cccttatcgt caatcttctc gaggattggg gaccctgcgc tgaacatgga gaacatcaca 180tcaggattcc taggacccct gctcgtgtta caggcggggt ttttcttgtt gacaaaagtc 240ctcacaatac cgcagagcct agactcgtgg tggacttctc tcaattttct agggggaact 300accgtgtgtc ttggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc taattccagg atcctcaaca accagcacgg gaccctgcag gacctgtatg 540actaccgctc aaggaacctc tatgtatccc tcatgttgtt gtacaaaacc ttcggacgga 600aattgcactt gtattcccat cccatcatcc tgggctttcg gaaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgtaggg 720ctttccccca ctgcttggct tttagctata tggatcatct ggtattgggg gccaaatctg 780tacaacatct tgaatccatt tataccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84628281PRTArtificial Sequencesynthetic polypeptide 28Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Phe Pro Val Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser Pro Leu Ser Ser Ile Phe Ser Arg 35 40 45 Ile Gly Asp Pro Ala Leu Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Lys Val65 70 75 80 Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Thr Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Arg Thr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly225 230 235 240 Leu Ser Pro Thr Ala Trp Leu Leu Ala Ile Trp Ile Ile Trp Tyr Trp 245 250 255 Gly Pro Asn Leu Tyr Asn Ile Leu Asn Pro Phe Ile Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 29846DNAArtificial Sequencesynthetic nucleic acid 29atgcagtgga attccactgc cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagctcagga acagtaaacc ctgctccgaa tattgcctct 120cacatctcgt caatctccgc gaggattggg gaccctgcgc tgaacatggg gaacatcaca 180tcaggattcc taggacccct tctcgtgtta caggcggggt ttttcttgat gacaaaaatc 240ctcacaatgc cgcagagtct agactcgtgg tggacttctc tcaattttct agggggaact 300accgtgcgtc ctggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc tacttccagg gtcctcaaca accagcacgg gaccctgcag gacctgtatg 540actaccgctc aaggaacctc tatgtatccc tcatgttgct gtaccaaacc ttcggacgga 600aattgcacct gtattcccat cccatcatcc tgggctttcg caaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgtaggg 720ctttccccca ctgtttggct ttcagttata tggatcatct ggtattgggg gccaaatctg 780tacaacatct tgaatccatt tataccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84630281PRTArtificial Sequencesynthetic polypeptide 30Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Ile Gly Asp Pro Ala Leu Asn Met Gly Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Met Thr Lys Ile65 70 75 80 Leu Thr Met Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Thr Val Arg Pro Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Leu Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Arg Thr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Ala Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly225 230 235 240 Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Ile Ile Trp Tyr Trp 245 250 255 Gly Pro Asn Leu Tyr Asn Ile Leu Asn Pro Phe Ile Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 31846DNAArtificial Sequencesynthetic nucleic acid 31atgcagtgga attccactgc cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagctcagga acagtaaacc ctgctccgaa tattgcctct 120cacatctcgt caatctccgc gaggattagg gaccctgcgc tgaacatgga gaacatcgca 180tcaggattcc taggacccct tctcgcatta caggcggtgt ttttcttgtt gacaaaaatc 240ctcacaatgc cacagagtct agactcgtgg tggacttctc tcaattttct agggggaact 300accgtgtgtc ttggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc taattccagg atcctcaaca accagcacgg gaccatgcaa aacctgtatg 540actaccgctc aaggaacctc tatgtatccc tcatgttgtt gtacaaaacc ttcggacgga 600aattgcacct gtattcccat cccatcatcc tgggctttcg caaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgtaggg 720ctttccccca ctgtttggct ttcagttata tggatgatgt ggtattgggg accaagtctg 780tacagcatct tgagtccctt cttgcctctg ttaccacttt tcttttggct ttgggtatac 840atttaa 84632281PRTArtificial Sequencesynthetic polypeptide 32Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Ile Arg Asp Pro Ala Leu Asn Met Glu Asn Ile Ala Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Ala Leu Gln Ala Val Phe Phe Leu Leu Thr Lys Ile65 70 75 80 Leu Thr Met Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Thr Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Lys Thr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Ala Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly225 230 235 240 Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Met Met Trp Tyr Trp 245 250 255 Gly Pro Ser Leu Tyr Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro 260 265 270 Leu Phe Phe Trp Leu Trp Val Tyr Ile 275 280 33846DNAArtificial Sequencesynthetic nucleic acid 33atgcagtgga attctacaac cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tattgcctct 120cacatctcgt caatctccgc gaggactggg gaccctgcgc tgaacatgga gaacatcaca 180tcaggattcc taggacccct tctcgtgtta caggcggggt ttttcttgtt gacaagaatc 240ctcacaatac cacagagtct

agactcgtgg tggacttctc tcaattttct agggggagca 300cccgtgtgtc ctggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccgattt gtcctggcta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc tacttccagg atcctcaaca accagcacgg gaccatgccg gacctgcatg 540actaccgctc aaggaacctc tatgtatccc tcatgttgct gtaccaaacc ttcggacgga 600agctgcactt gtattcccat cccatcatcc tgggctttcg caaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgtaggg 720ctttcccccg ctgtatggct ttcagttata tggatgattt ggttttgggg gccaagtctg 780tacagcatct tgagtccctt tttaccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84634281PRTArtificial Sequencesynthetic polypeptide 34Met Gln Trp Asn Ser Thr Thr Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Thr Gly Asp Pro Ala Leu Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile65 70 75 80 Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Thr Val Cys Leu Gly Leu Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Lys Thr Cys Met Thr Thr Pro Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly225 230 235 240 Leu Ser Pro Thr Ala Trp Leu Leu Ala Ile Trp Ile Ile Trp Tyr Trp 245 250 255 Gly Pro Asn Leu Tyr Asn Ile Leu Asn Pro Phe Leu Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 35846DNAArtificial Sequencesynthetic nucleic acid 35atgcagtgga attctacaac cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tattgcctct 120cacatctcgt caatctccgc gaggactggg gaccctgcgc tgaacatgga gaacatcaca 180tcaggattcc taggacccct tctcgtgtta caggcggggt ttttcttgtt gacaagaatc 240ctcacaatac cacagagtct agactcgtgg tggacttctc tcaattttct agggggaact 300accgtgtgtc ttggcctaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgcttc tatgcctcat cttcttgttg gttcttctgg actatcaagg tatgttgccc 480gtttgtcccc taattccagg atcctcaaca accagcacgg gaccatgcaa aacctgtatg 540actacccctc aaggaacctc tatgtatccc tcatgttgct gtaccaaacc ttcggacgga 600aattgcacct gtattcccat cccatcatcc tgggctttcg gaaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgtaggg 720ctttccccca ctgcttggct tttggctata tggatcatct ggtattgggg gccaaatctg 780tacaacatct tgaatccctt tttaccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84636281PRTArtificial Sequencesynthetic polypeptide 36Met Gln Trp Asn Ser Thr Thr Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Thr Gly Asp Pro Ala Leu Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile65 70 75 80 Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Thr Val Cys Leu Gly Leu Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Lys Thr Cys Met Thr Thr Pro Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly225 230 235 240 Leu Ser Pro Thr Ala Trp Leu Leu Ala Ile Trp Ile Ile Trp Tyr Trp 245 250 255 Gly Pro Asn Leu Tyr Asn Ile Leu Asn Pro Phe Leu Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 37846DNAArtificial Sequencesynthetic nucleic acid 37atgcagtgga attccacaac cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tactgcctct 120cacatctcgt cagtcttctc gacgactggg gaccctgcac cgaacatgga gaacatcaca 180tcaggattcc taggacccct gctcgtgtta caggcggggt ttttcttgtt gacaaaaatc 240ctcacaatgc cacagagtct agactcgttg tggacttctc tcaattttct agggggaaca 300ccagcgtgtc ttggccaaaa ttcgcagtcc ccaaccccca atcactcacc aacctcttgc 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgcttc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc taattccagg atcctcaaca accagcacgg gaccatgcaa aacctgtatg 540actacccctc aaggaacctc tatgtatccc tcatgttgct gtaccaaacc ttcggacgga 600aattgcacct gtattcccat cccatcatcc tgggctttcg gaaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgtaggg 720ctttccccca ctgcttggct tttggctata tggatcatct ggtattgggg gccaaatctg 780tacaacatct tgaatccctt tttaccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84638281PRTArtificial Sequencesynthetic polypeptide 38Met Gln Trp Asn Ser Thr Thr Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Thr Ala Ser His Ile Ser Ser Val Phe Ser Thr 35 40 45 Thr Gly Asp Pro Ala Pro Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Lys Ile65 70 75 80 Leu Thr Met Pro Gln Ser Leu Asp Ser Leu Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Pro Ala Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Pro Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Lys Thr Cys Met Thr Thr Pro Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly225 230 235 240 Leu Ser Pro Thr Ala Trp Leu Leu Ala Ile Trp Ile Ile Trp Tyr Trp 245 250 255 Gly Pro Asn Leu Tyr Asn Ile Leu Asn Pro Phe Leu Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 39846DNAArtificial Sequencesynthetic nucleic acid 39atgcagtgga attccactgc cttccaccaa actctgcagg atcccagagt caggggtctg 60tatctccctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tattgcctct 120cacatctcgt caatctccgc gaggactggg gaccctgcac cgaacatgga gaacatcaca 180tcaggattcc taggacccct gctcgtgtta caggcggtgt ttttcttgtt gacaagaatc 240ctcacaatac cgcagagtct agactcgtgg tggacttctc tcaattttct agggggaaca 300cccgtgtgtc ctggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgcttc tatgcctcat cttcttgttg gttcttctgg actatcaagg tatgttgccc 480gtttgtcccc taattccagg atcctcaaca accagcacgg gaccatgcaa aacctgtatg 540actacccctc aaggaacctc tatgtatccc tcatgttgct gtaccaaacc ttcggacgga 600aattgcacct gtattcccat cccatcatcc tgggctttcg gaaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagcg gttcgcaggg 720ctttccccca ctgtttggct ttcagttata tggatgattt ggttttgggg gccaagtctg 780tacagcatct tgagtccctt tttaccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84640281PRTArtificial Sequencesynthetic polypeptide 40Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Thr Gly Asp Pro Ala Pro Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Val Phe Phe Leu Leu Thr Arg Ile65 70 75 80 Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Pro Val Cys Pro Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Lys Thr Cys Met Thr Thr Pro Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Arg Phe Ala Gly225 230 235 240 Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Met Ile Trp Phe Trp 245 250 255 Gly Pro Ser Leu Tyr Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 41846DNAArtificial Sequencesynthetic nucleic acid 41atgcagtgga attccacaac cttccaccaa actctgcaag atcccagagt gagaggcctg 60tatttccctg ctggtggctc cagttcagga acagtaaacc ctgttctgac tactgcctct 120cccgtatcgt caatcttctc gaggattggg gaccctgcgc tgaacatgga gaacatcaca 180tcaggattcc taggacccct tctcgtgtta caggcggggt ttttcttgtt gacaagaatc 240ctcacaatac cgcagagtct agactcgttg tggacttctc tcagttttcc aaggggcata 300ccagagtgca ctggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc tacttccagg atcctcaaca accagcacgg gaccctgcag gacctgtatg 540actaccgctc aaggaacctc tatgtatccc tcatgttgct gtaccaaacc ttcggacgga 600aattgcacct gtattcccat cccatcatcc tgggctttcg caaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgtaggg 720ctttccccca ctgtttggct ttcagttata tggatcatct ggtattgggg gccaaatctg 780tacaacatct tgaatccatt tataccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84642281PRTArtificial Sequencesynthetic polypeptide 42Met Gln Trp Asn Ser Thr Thr Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Val Leu Thr Thr Ala Ser Pro Val Ser Ser Ile Phe Ser Arg 35 40 45 Ile Gly Asp Pro Ala Leu Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile65 70 75 80 Leu Thr Ile Pro Gln Ser Leu Asp Ser Leu Trp Thr Ser Leu Ser Phe 85 90 95 Pro Arg Gly Ile Pro Glu Cys Thr Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Leu Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Arg Thr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Ala Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly225 230 235 240 Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Ile Ile Trp Tyr Trp 245 250 255 Gly Pro Asn Leu Tyr Asn Ile Leu Asn Pro Phe Ile Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 43846DNAArtificial Sequencesynthetic nucleic acid 43atgcagtgga attccacaac cttccaccaa actctgcaag atcccagagt gagaggcctg 60tatttccctg ctggtggctc cagttcagga acagtaaacc ctgttctgac tactgcctct 120cccttatcgt caatcttctc gaggattggg gaccctgcgc tgaacatgga gaacatcaca 180tcaggattcc taggacccct tctcgtgtta caggcggggt ttttcttgtt gacaagaatc 240ctcacaatac cgcagagtct agactcgttg tggacctctc tcagttttcc agggggcata 300ccagagtgca ctggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc tacttccagg atcctcaaca accagcacgg gaccctgcag gacctgtatg 540actaccgctc aaggaacctc tatgtatccc tcatgttgct gtaccaaacc ttcggacgga 600aattgcacct gtattcccat cccatcatcc tgggctttcg gaaaattcct atgggagtgg 660gcctcagtcc gtttctcctg gctcagttta ctagctccat ttgttcagcg gttcgtaggg 720ctttccccca ctgtttggct ttcagttata tggatgatgt ggttctgggg gccaagtctg 780ttcagcatct tgagtccctt cttgcctctg ttaccacttt tcttttggct ttgggtatac 840atttaa 84644281PRTArtificial Sequencesynthetic polypeptide 44Met Gln Trp Asn Ser Thr Thr Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Val Leu Thr Thr Ala Ser Pro Leu Ser Ser Ile Phe Ser Arg 35

40 45 Ile Gly Asp Pro Ala Leu Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile65 70 75 80 Leu Thr Ile Pro Gln Ser Leu Asp Ser Leu Trp Thr Ser Leu Ser Phe 85 90 95 Pro Gly Gly Ile Pro Glu Cys Thr Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Leu Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Arg Thr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Val Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Ala Pro Phe Val Gln Arg Phe Val Gly225 230 235 240 Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Met Met Trp Phe Trp 245 250 255 Gly Pro Ser Leu Phe Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro 260 265 270 Leu Phe Phe Trp Leu Trp Val Tyr Ile 275 280 45846DNAArtificial Sequencesynthetic nucleic acid 45atgcagtgga attccactgc cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagctcagga acagtaaacc ctgctccgaa tattgcctct 120cacatctcgt caatctccgc gaggattggg gaccctgcgc tgaacatgga gaacatcaca 180tcaggattcc taggacccct tctcgcatta caggcggtgt ttttcttgtt gacaaaaatc 240ctcacaatgc cacagagtct agactcgtgg tggacttctc tcaattttct agggggaact 300accgtgtgtc ttggcctaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tgtgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc tacttccagg atcctcaaca accagcacgg gaccatgccg gacctgcatg 540actaccgctc aaggaacctc tatgtatccc tcatgttgct gtaccaaacc ttcggacgga 600agctgcactt gtattcccat cccctcatcc tgggctttcg gaaaattcct atgggagtgg 660gcctcagtcc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgcaggg 720ctttccccca ctgtatggct tttagttata tggatgatgt ggttctgggg gccaagtctg 780ttcagcatct tgagtccctt cttgcctctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84646281PRTArtificial Sequencesynthetic polypeptide 46Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Ile Gly Asp Pro Ala Leu Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Ala Leu Gln Ala Val Phe Phe Leu Leu Thr Lys Ile65 70 75 80 Leu Thr Met Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Thr Val Cys Leu Gly Leu Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Leu Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Arg Thr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Ser Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Val Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Ala Gly225 230 235 240 Leu Ser Pro Thr Val Trp Leu Leu Val Ile Trp Met Met Trp Phe Trp 245 250 255 Gly Pro Ser Leu Phe Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 47846DNAArtificial Sequencesynthetic nucleic acid 47atgcagtgga attccactgc cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tattgcctct 120cccttatcgt caatcttctc gacgactggg gaccctgcgc tgaacatgga gaacatcaca 180tcaggattcc taggacccct tctcgtgtta caggcggggt ttttcttgtt gacaagaatc 240ctcacaatac cgcagagtct agactcgtgg tggacttctc tcaattttct agggggagca 300cccgtgtgtc ttggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc taattccagg atcctcaaca accagcacgg gaccctgcag gacctgtatg 540actgccgctc aaggaacctc tatgtatccc tcatgttgct gtaccaaacc ttcggacgga 600aattgcacct gtattcccat cccatcatcc tgggctttcg caaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgtaggg 720ctttccccca ctgtttggct ttcagttata tggatcatct ggtattgggg gccaaatctg 780tacaacatct tgaatccatt tataccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84648281PRTArtificial Sequencesynthetic polypeptide 48Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser Pro Leu Ser Ser Ile Phe Ser Thr 35 40 45 Thr Gly Asp Pro Ala Leu Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile65 70 75 80 Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Ala Pro Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Arg Thr Cys Met Thr Ala Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Ala Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly225 230 235 240 Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Ile Ile Trp Tyr Trp 245 250 255 Gly Pro Asn Leu Tyr Asn Ile Leu Asn Pro Phe Ile Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 49846DNAArtificial Sequencesynthetic nucleic acid 49atgcagtgga attccacaac cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tactgcctct 120cacatctcgt cagtcttctc gacgactggg gaccctgcac cgaacatgga gaacatcaca 180tcaggattcc taggacccct gctcgtgtta caggcggggt ttttcttgtt gacaaaaatc 240ctcacaatgc cacagagtct agactcgttg tggacttctc tcaattttct agggggaaca 300ccagcgtgtc ttggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtca tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc taattccagg atcctcaaca accagcacgg gaccatgcaa aacctgtatg 540actacccctc aaggaacctc tatgtatccc tcatgttgct gtaccaaacc ttcggacgga 600aattgcacct gtattcccat cccatcatcc tgggctttcg gaaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgtaggg 720ctttccccca ctgcttggct tttggctata tggatcatct ggtattgggg gccaaatctg 780tacaacatct tgaatccctt tttaccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84650281PRTArtificial Sequencesynthetic polypeptide 50Met Gln Trp Asn Ser Thr Thr Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Thr Ala Ser His Ile Ser Ser Val Phe Ser Thr 35 40 45 Thr Gly Asp Pro Ala Pro Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Lys Ile65 70 75 80 Leu Thr Met Pro Gln Ser Leu Asp Ser Leu Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Pro Ala Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly His Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Lys Thr Cys Met Thr Thr Pro Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly225 230 235 240 Leu Ser Pro Thr Ala Trp Leu Leu Ala Ile Trp Ile Ile Trp Tyr Trp 245 250 255 Gly Pro Asn Leu Tyr Asn Ile Leu Asn Pro Phe Leu Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 51846DNAArtificial Sequencesynthetic nucleic acid 51atgcagtgga attccacaac cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tactgcctct 120cacatctcgt cagtcttctc gacgactggg gaccctgcac cgaacatgga gaacatcaca 180tcaggattcc taggacccct gctcgtgtta caggcggggt ttttcttgtt gacaaaaatc 240ctcacaatgc cacagagtct agactcgttg tggacttctc tcaattttct agggggaaca 300ccagcgtgtc ttggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtca tcgctggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc taattccagg atcctcaaca accagcacgg gaccatgcaa aacctgtatg 540actacccctc aaggaacctc tatgtatccc tcatgttgct gtaccaaacc ttcggacgga 600aattgcacct gtattcccat cccatcatcc tgggctttcg gaaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagtg gttcgtaggg 720ctttccccca ctgcttggct tttggctata tggatcatct ggtattgggg gccaaatctg 780tacaacatct tgaatccctt tttaccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84652281PRTArtificial Sequencesynthetic polypeptide 52Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Ile Gly Asp Pro Ala Leu Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Ala Leu Gln Ala Val Phe Phe Leu Leu Thr Lys Ile65 70 75 80 Leu Thr Met Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Thr Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Lys Thr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys 180 185 190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Ala Pro Phe Val Gln Arg Phe Ala Gly225 230 235 240 Leu Ser Pro Thr Ala Trp Leu Leu Ala Ile Trp Ile Ile Trp Tyr Trp 245 250 255 Gly Pro Asn Leu Tyr Asn Ile Met Asn Pro Phe Leu Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 53846DNAArtificial Sequencesynthetic nucleic acid 53atgcagtgga attccactgc cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tactgcctct 120cacatctcgt cagtcttctc gacgactggg gaccctgcac cgaacatgga gaacatcaca 180tcaggattcc taggacccct gctcgtgtta caggcggggt ttttcttgtt gacaaaaatc 240ctcacaatgc cacagagtct agactcgttg tggacttctc tcaattttct agggggaaca 300ccagcgtgtc ttggccaaaa ttcgcagtcc ccaacctcca atcactcacc aacctcttgt 360cctccaactt gtcctggtta tcactggatg tgtctgcggc gttttatcat cttcctcttc 420atcctgctgc tatgcctcat cttcttgttg gttcttctgg actatcgagg tatgttgccc 480gtttgtcctc tacttccagg atcatcgacc accagcacgg gaccatgcaa aacctgcacg 540atccctgctc aaggaacctc tttgattccc tcatgttgtt gtacaaaacc ttcggacgga 600aattgcactt gtattcccat cccatcgtct tgggctttcg caaaattcct atgggagtgg 660gcctcagccc gtttctcctg gctcagttta ctagtgccat ttgttcagcg gttcgcaggg 720ctttccccca ctgtttggct ttcagttata tggatgattt ggttttgggg gccaagtctg 780tacagcatct tgagtccctt tttaccgctg ttaccaattt tcttttgtct ttgggtatac 840atttaa 84654281PRTArtificial Sequencesynthetic polypeptide 54Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Thr Ala Ser His Ile Ser Ser Val Phe Ser Thr 35 40 45 Thr Gly Asp Pro Ala Pro Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Lys Ile65 70 75 80 Leu Thr Met Pro Gln Ser Leu Asp Ser Leu Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Pro Ala Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr His 115 120 125 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 130 135 140 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro145 150 155 160 Val Cys Pro Leu Leu Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys 165 170 175 Lys Thr Cys Thr Ile Pro Ala Gln Gly Thr Ser Leu Ile Pro Ser Cys 180 185

190 Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 195 200 205 Ser Ser Trp Ala Phe Ala Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg 210 215 220 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Arg Phe Ala Gly225 230 235 240 Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Met Ile Trp Phe Trp 245 250 255 Gly Pro Ser Leu Tyr Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro 260 265 270 Ile Phe Phe Cys Leu Trp Val Tyr Ile 275 280 55840DNAArtificial Sequencesynthetic nucleic acid 55atgcagtgga attctactgc cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tattgcctct 120cacatctcgt caatctccgc gaggactggg gaccctgtga cgaacatgtc accatcaagt 180ctcctaggac tcctagcagg attacaggtg gtgtatttct tgtggacaaa aatcctaaca 240ataccgcaga gtctagactc gtggtggact tctctcaatt ttctaggggg agcacccgtg 300tgtcctggcc aaaattcgca gcccccaacc cccaatcact caccaacctc ttgtccttca 360acttgtcctg ggtatcgctg gatgcgtctg tggcgtttta tcatcttcct cttcatcctg 420ctgctatgcc tcatcttctt gttggttctt ctggactatc gaggtgtgtt gcccgtttgt 480cctccaattc caggatcctc aacaaccagc acgggaccat gcaaaacctg tatgactacc 540gctcaaggaa cctctatgta tccctcatgt tgttgtacaa aaccttcgga cggaaattgc 600acctgtattc ccatcccatc atcctgggct ttcggaaaat tcctatggga gtgggcctca 660gcccgtttct cctggctcag tttactagtg ccatttgttc agtggttcgt agggctttcc 720cccactgctt ggcttttggc tacatggatc atctggtatt gggggccaaa tctgtacaac 780atcttgaatc cctttttacc gctgttacca attttctttt gtctttgggt atacatttaa 84056279PRTArtificial Sequencesynthetic polypeptide 56Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Thr Gly Asp Pro Val Thr Asn Met Ser Pro Ser Ser Leu Leu Gly Leu 50 55 60 Leu Ala Gly Leu Gln Val Val Tyr Phe Leu Trp Thr Lys Ile Leu Thr65 70 75 80 Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly 85 90 95 Gly Ala Pro Val Cys Pro Gly Gln Asn Ser Gln Pro Pro Thr Pro Asn 100 105 110 His Ser Pro Thr Ser Cys Pro Ser Thr Cys Pro Gly Tyr Arg Trp Met 115 120 125 Arg Leu Trp Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu 130 135 140 Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Val Leu Pro Val Cys145 150 155 160 Pro Pro Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr 165 170 175 Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys Cys Cys 180 185 190 Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser 195 200 205 Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg Phe Ser 210 215 220 Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser225 230 235 240 Pro Thr Ala Trp Leu Leu Ala Thr Trp Ile Ile Trp Tyr Trp Gly Pro 245 250 255 Asn Leu Tyr Asn Ile Leu Asn Pro Phe Leu Pro Leu Leu Pro Ile Phe 260 265 270 Phe Cys Leu Trp Val Tyr Ile 275 57840DNAArtificial Sequencesynthetic nucleic acid 57atgcagtgga attctactgc cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tattgcctct 120cacatctcgt caatctccgc gaggactggg gaccctgtga cgaacatgtc accatcaagt 180ctcctaggac tcctcgcagg attacaggtg gtgtatttct tgtggacaaa aatcctaaca 240ataccgcaga gtctagactc gtggtggact tctctcaatt ttctaggggg agcacccgtg 300tgtcctggcc aaaattcgca gtccccaacc tccaatcact caccaacctc ttgtcctcca 360acttgtcctg gttatcgctg gatgtgtctg cggcgtttta tcatcttcct cttcatcctg 420ctgctatgcc tcatcttctt gttggttctt ctggactatc gaggtatgtt gcccgtttgt 480cctctacttc caggatcctc aacaaccagc acgggaccct gcaggacctg tatgactacc 540gctcaaggaa cctctatgta tccctcatgt tgctgtacca aaccttcgga cggaaattgc 600acctgtattc ccatcccatc atcctgggct ttcgcaaaat tcctatggga gtgggcctca 660gcccgtttct cctggctcag tttactagtg ccatttgttc agtggttcgt agggctttcc 720cccactgttt ggctttcagt tatatggatc atctggtatt gggggccaaa tctgtacaac 780atcttgagtc catttatacc gctgttacca attttctttt gtctttgggt atacatttaa 84058279PRTArtificial Sequencesynthetic polypeptide 58Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Thr Gly Asp Pro Val Thr Asn Met Ser Pro Ser Ser Leu Leu Gly Leu 50 55 60 Leu Ala Gly Leu Gln Val Val Tyr Phe Leu Trp Thr Lys Ile Leu Thr65 70 75 80 Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly 85 90 95 Gly Ala Pro Val Cys Pro Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn 100 105 110 His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg Trp Met 115 120 125 Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu 130 135 140 Ile Phe Leu Leu Val Leu Leu Asp Tyr Arg Gly Met Leu Pro Val Cys145 150 155 160 Pro Leu Leu Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys Arg Thr 165 170 175 Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys Cys Cys 180 185 190 Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser 195 200 205 Trp Ala Phe Ala Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg Phe Ser 210 215 220 Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser225 230 235 240 Pro Thr Val Trp Leu Ser Val Ile Trp Ile Ile Trp Tyr Trp Gly Pro 245 250 255 Asn Leu Tyr Asn Ile Leu Ser Pro Phe Ile Pro Leu Leu Pro Ile Phe 260 265 270 Phe Cys Leu Trp Val Tyr Ile 275 59280PRThepatitis B 59Met Gln Trp Asn Ser Thr Thr Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Val Leu Thr Thr Ala Ser Pro Leu Ser Ser Ile Phe Ser Arg 35 40 45 Ile Gly Asp Pro Ala Leu Asn Met Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile65 70 75 80 Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly Gly Thr Thr Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr 100 105 110 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg 115 120 125 Trp Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys 130 135 140 Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro Val145 150 155 160 Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys Arg 165 170 175 Thr Cys Met Thr Thr Ala Gln Gly Thr Ser Met Tyr Pro Ser Cys Cys 180 185 190 Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser 195 200 205 Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Ala Arg Phe 210 215 220 Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly Leu225 230 235 240 Ser Pro Thr Val Trp Leu Ser Val Ile Trp Met Met Trp Tyr Trp Gly 245 250 255 Pro Ser Leu Tyr Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile 260 265 270 Phe Phe Cys Leu Trp Val Tyr Ile 275 280 6030DNAhepatitis B 60tatgactacc gctcaaggaa cctctatgta 306159DNAhepatitis B 61accatgcaaa cctgtatgac taccgctcaa ggaacctcta tgtatccctc atgttgttg 596260DNAhepatitis B 62accctgcagg acctgtatga ctaccgctca aggaacctct atgtatccct catgttgctg 606360DNAhepatitis B 63caattgcaga caatgtatga ctaccgctca aggaacctct atgtatcctt actgttgttg 606421DNAArtificial Sequencevector 64gcagctcctt gctcctaaca g 216521DNAArtificial Sequencevector 65gtatcacgag gccctttcgt c 216620DNAArtificial Sequencevector 66gctgacagac taacagactg 206720DNAArtificial Sequencevector 67aacagatggc tggcaactag 2068837DNAArtificial Sequencesynthetic nucleic acid 68atgcagtgga attctactgc cttccaccaa actctgcagg atcccagagt caggggtctg 60tatcttcctg ctggtggctc cagttcagga acagtaaacc ctgctccgaa tattgcctct 120cacatctcgt caatctccgc gaggactggg gaccctgtga cgaacatgtc accatcaagt 180ctcctaggac tcctcgcagg attacaggtg gtgtatttct tgtggacaaa aatcctaaca 240ataccgcaga gtctagactc gtggtggact tctctcaatt ttctaggggg agcacccgtg 300tgtcctggcc aaaattcgca gtccccaacc tccaatcact caccaacctc ttgtcctcca 360atttgtcctg gttatcgctg gatgtgtctg cggcgtttta tcatcttcct cttcatcctg 420cttctatgcc tcatcttctt gttggttctt ctggactatc aaggtatgtt gcccgtttgt 480cctctaattc caggatcatc gaccaccagc acgggaccat gcaaaacctg tatgactacc 540cctcaaggaa cctctatgta tccctcatgt tgttgtacca aaccttcgga cggaaattgc 600acctgtattc ccatcccatc atcctgggct ttcggaaaat tcctatggga gtgggcctca 660gtccgtttct cctggctcag tttactagct ccatttgttc agcggttcgt agggctttcc 720cccactgttt ggctttcagt tatatggatg atgtggttct gggggccaag tctgttcagc 780atcttgagtc ccttcttgcc tctgttacca cttttctttt ggctttgggt atacatt 83769279PRTArtificial Sequencesynthetic polypeptide 69Met Gln Trp Asn Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15 Val Arg Gly Leu Tyr Leu Pro Ala Gly Gly Ser Ser Ser Gly Thr Val 20 25 30 Asn Pro Ala Pro Asn Ile Ala Ser His Ile Ser Ser Ile Ser Ala Arg 35 40 45 Thr Gly Asp Pro Val Thr Asn Met Ser Pro Ser Ser Leu Leu Gly Leu 50 55 60 Leu Ala Gly Leu Gln Val Val Tyr Phe Leu Trp Thr Lys Ile Leu Thr65 70 75 80 Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly 85 90 95 Gly Ala Pro Val Cys Pro Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn 100 105 110 His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met 115 120 125 Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu 130 135 140 Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys145 150 155 160 Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr 165 170 175 Cys Met Thr Thr Pro Gln Gly Thr Ser Met Tyr Pro Ser Cys Cys Cys 180 185 190 Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser 195 200 205 Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp Ala Ser Val Arg Phe Ser 210 215 220 Trp Leu Ser Leu Leu Ala Pro Phe Val Gln Arg Phe Val Gly Leu Ser225 230 235 240 Pro Thr Val Trp Leu Ser Val Ile Trp Met Met Trp Phe Trp Gly Pro 245 250 255 Ser Leu Phe Ser Ile Leu Ser Pro Phe Leu Pro Leu Leu Pro Leu Phe 260 265 270 Phe Trp Leu Trp Val Tyr Ile 275

Claims

1-5. (canceled)

6. A nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence, wherein said amino acid sequence is at least 97 percent identical to the amino acid sequence set forth in SEQ ID NO:18.

7. The nucleic acid molecule of claim 6, wherein said amino acid sequence is at least 98 percent identical to the amino acid sequence set forth in SEQ ID NO:18.

8. The nucleic acid molecule of claim 6, wherein said amino acid sequence is at least 99 percent identical to the amino acid sequence set forth in SEQ ID NO:18.

9. A nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence, wherein said amino acid sequence is at least 96 percent identical to the amino acid sequence set forth in SEQ ID NO:14.

10. The nucleic acid molecule of claim 9, wherein said amino acid sequence is at least 97 percent identical to the amino acid sequence set forth in SEQ ID NO:14.

11. The nucleic acid molecule of claim 9, wherein said amino acid sequence is at least 98 percent identical to the amino acid sequence set forth in SEQ ID NO:14.

12. The nucleic acid molecule of claim 9, wherein said amino acid sequence is at least 99 percent identical to the amino acid sequence set forth in SEQ ID NO:14.

13. A nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence, wherein said amino acid sequence is at least 97 percent identical to the amino acid sequence set forth in SEQ ID NO:22.

14. The nucleic acid molecule of claim 13, wherein said amino acid sequence is at least 98 percent identical to the amino acid sequence set forth in SEQ ID NO:22.

15. The nucleic acid molecule of claim 13, wherein said amino acid sequence is at least 99 percent identical to the amino acid sequence set forth in SEQ ID NO:22.

16. A nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence, wherein said amino acid sequence is at least 95 percent identical to the amino acid sequence set forth in SEQ ID NO:28.

17. The nucleic acid molecule of claim 16, wherein said amino acid sequence is at least 96 percent identical to the amino acid sequence set forth in SEQ ID NO:28.

18. The nucleic acid molecule of claim 16, wherein said amino acid sequence is at least 97 percent identical to the amino acid sequence set forth in SEQ ID NO:28.

19. The nucleic acid molecule of claim 16, wherein said amino acid sequence is at least 98 percent identical to the amino acid sequence set forth in SEQ ID NO:28.

20. The nucleic acid molecule of claim 16, wherein said amino acid sequence is at least 99 percent identical to the amino acid sequence set forth in SEQ ID NO:28.

21. A nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence, wherein said amino acid sequence is at least 96 percent identical to the amino acid sequence set forth in SEQ ID NO:12.

22. The nucleic acid molecule of claim 21, wherein said amino acid sequence is at least 97 percent identical to the amino acid sequence set forth in SEQ ID NO:12.

23. The nucleic acid molecule of claim 21, wherein said amino acid sequence is at least 98 percent identical to the amino acid sequence set forth in SEQ ID NO:12.

24. The nucleic acid molecule of claim 21, wherein said amino acid sequence is at least 99 percent identical to the amino acid sequence set forth in SEQ ID NO:12.