TRANSGENIC MICE HAVING A HUMAN MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) PHENOTYPE, EXPERIMENTAL USES AND APPLICATIONS

Abstract

The present invention relates to transgenic mice and isolated transgenic mouse cells, the mice and mouse cells comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA class I transgene, and a functional HLA class II transgene. In embodiments, the transgenic mouse or mouse cells are deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene. In embodiments, the transgenic mouse or mouse cell has the genotype HLA-A2⁺HLA-DR1+β2m°IAβ°. The invention also relates to methods of using a transgenic mouse of the invention.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is based on and claims the benefit of U.S. Provisional Application No. 60/490,945, filed Jul. 30, 2003 (Attorney Docket No. 03495.6093), the entire disclosure of which is relied upon and incorporated by reference herein for all purposes.

BACKGROUND OF THE INVENTION

[0002] Many vaccines are currently being developed for human cancer immunotherapy and for treatment of infectious diseases, such as malaria, AIDS, hepatitis C virus, and SARS. Given the rapidity with which new emerging pathogens can appear, it is important to improve animal models that could be used to evaluate vaccination strategies and the protective capacity of different epitopes quickly and reliably. Furthermore, in vivo studies are already required to assess crucial variables of vaccine behavior that are not easily evaluated or impossible to measure in vitro, such as vaccine immunogenicity, vaccine formulation, route of administration, tissue distribution, and involvement of primary and secondary lymphoid organs. Because of their simplicity and flexibility, small animals, such as mice represent an attractive alternative to more cumbersome and expensive model systems, such as nonhuman primates, at least for initial vaccine development studies.

[0003] The moderate efficacy observed in several clinical trials of vaccines, which were found to be protective in wild-type animal studies (McMichael, A. J. & Henke, T. Nat Med 9, 874-880 (2003)), may be partly explained by the different influence that human and animal MHC have on the outcome of the immune response, since animal MHC and human HLA molecules do not present the same optimal epitopes (Rotzschke, O. et al. Nature 348, 252-254 (1990)). Thus, despite some limitations, transgenic mice expressing human HLA should represent a useful Improvement over wild-type mice as a preclinical model for testing vaccine candidates, evaluating the potential risk that the vaccines could induce autoimmune disorders, and devising better therapeutic strategies based on the human restriction element.

Cytotoxic T Cells

[0004] Cytotoxic T cells (CTL) play a crucial role in the eradication of infectious diseases and in some cases, cancer (P. Aichele, H. Hengartner, R. M. Zinkernagel and M. Schulz, J Exp Med 171 (1990), p. 1815; L. BenMohamed, H. Gras-Masse, A. Tartar, P. Daubersies, K Brahimi, M. Bossus, A. Thomas and P. Druhile, Eur J Immunol 27 (1997), p. 1242; D. J. Diamond, J. York, J. Sun, C. L. Wright and S. J. Forman, Blood 90 (1997), p. 1751). Recombinant protein vaccines do not reliably induce CTL responses (Habeshaw J A, Dalgleish A G, Bountiff L, Newell A L, Wilks, D, Walker L C, Manca F. 1990 November; 11(11): 418-25; Miller S B, Tse H, Rosenspire A J, King S R. Virology. 1992 December; 191 (2):9 73-7). The use of otherwise immunogenic vaccines consisting of attenuated pathogens in humans is hampered, in several important diseases, by overriding safety concerns. In the last few years, epitope-based approaches have been proposed as a possible strategy to develop novel prophylactic and immunotherapeutic vaccines (Melief C J, Offringa R, Toes R E, Kast W M. Curr Opin Immunol. 1996 October, 8(5):651-7; Chesnut R W, Design testing of peptide based cytotoxic T-cell mediated immunotherapeutic to treat inflction disease, cancer, in Powell, M F, Newman, M J (eds.): Vaccine Design: The Subunit, Adjuvant Approach, Plenum Press, New-York 1995, 847). This approach offers several advantages, including selection of naturally processed epitopes, which forces the immune system to focus on highly conserved and immunodominant epitopes of a pathogen (R. G. van der Most, A. Sette, C. Oseroff, J. Alexander, K. Murali-Krishna, L. L. Lau, S, Southwood, J. Sidney, R. W. Chesnut, M. Matioubian and R. Ahmed, J Immunol 157 (1996), p. 5543) and induction of multiepitopic responses to prevent escape by mutation such observed in HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. It also allows the elimination of suppressive T cell determinants, which might preferably elicit a TH2 response, in conditions where a TH1 responses is desirable, or vice-versa (Pfeiffer C, Murray J, Madri J, Bottomly K. Immunol Rev. 1991 October; 123:65-84; P Chaturvedi, Q Yu, S Southwood, A Sette, and B Singh Int Immunol 1996 8: 745-755). It finally provides the possibility to get rid of autoimmune T cell determinants in antigens, which might induce undesirable autoimmune diseases. Protective antiviral or anti-tumoral immunity using CTL epitope-peptides has been achieved in several experimental models (D. J. Diamond, J. York, J. Sun, C. L. Wright and S. J. Forman, Blood 90 1997, p. 1751; J. E. J. Blaney, E. Nobusawa, M. A. Brehm, R. H. Bonneau, L. M. Mylin, T. M. Fu, Y. Kawaoka and S. S. Tevethia, J Virol 72 (1998), p. 9567).

[0005] CTL epitope definition based on the usage of human lymphocytes might be misleading due to environmental and genetic heterogeneity that lead to incomplete results, and due to technical difficulties in isolating CTL clones. HLA class I or class II transgenic mice described to date have proved to be a valuable tool to overcome these limitations as illustrated by the identification with such animal models of novel CTL and T helper epitopes (Hill A V. Annu Rev Immunol. 1998;16:593-617; Carmon L, El-Shaml K M, Paz A., Pascolo S, Tzehoval E, Tirosb B, Koren R, Feldman M, Fridkin M, Lemonnier F A, Eisenbach L. Int J Cancer, 2000 Feb. 1; 85(3):391-7). These mice have also been used to demonstrate: i) good correlation between peptide HLA binding affinity and immunogenicity (Lustgarten J, Theobald M, Labadie C, LaFace D, Peterson P, Disis M L, Cheaver M A, Sherman L A. Hum Immunol. 1997 February; 52(2):109-18; Bakker A B, van der Burg S H, Huijbens R J, DRijfhout J W, Melief C J, Adema G J, Figdor C G. Int J Cancer. 1997 Jan. 27; 70(3):302-9), ii) significant overlap between the murine and human CTL system at the level of antigen processing (same epitopes generated), and iii) comparable mobilization against most antigens of the CTL repertoires in HLA transgenic mice and humans (Wentworth, P. A., A. Vifiello, J. Sidney, E. Keogh, P, W. Chesnut, H. Grey, A. Sette. 1996. Eur. J. Immunol. 26:97; Alexander, J., C. Oserof, J. Sidney, P. Wentworth, E. Keogh, G. Hermanson, F. V. Chisari R. T, Kubo, H. M, Grey, A, Sette, 1997. J. Immunol. 159:4753).

[0006] To date, synthetic peptide-based CTL epitope vaccines have been developed as immunotherapeutics against a number of human diseases [18-20]. However, only moderate efficacy was observed in several clinical trials (21). This may be partly explained by the failure of these vaccines to induce sufficiently strong CTL responses. Indeed, recent reports suggest the need for CD4+ T-cell help to obtain maximum CTL response (A. J. Zajac, K. Murali-Krishna, J. N. Biattman and R. Ahmed, Curr Opin Immunol 10 (1998), p. 444; Firat H, Garcia-Pons F, Tourdot S, Pascolo S, Scardino A, Garcia Z, Michel M L, Jack R W, Jung O, Kosmatopoulos K, Mateo L, Suhrbier A, Lemonnier F A, Langlade-Demoyen P Eur J Immunol 29, 3112, 1999).

[0007] CTL are critical components of protective immunity against viral infections, but the requirements for in vivo priming of CTL are not completely understood. It is now accepted that Th cells are usually essential for CTL priming with synthetic peptides. With respect to synthetic CTL epitopic peptides, several studies point to a mandatory need for Th lymphocyte stimulation to induce optimal CTL responses (C. Fayolle, E. Deriaud and C. Leclerc, J Immunol 147 (1991), p, 4069; C. Widmann, P. Romero, J. L. Maryanski, G. Corradin and D. Valmori, J Immunol Meth 155 (1992), p. 95; M. Shirai, C. D. Pendkton, J. Ahlers, T. Takeshita, M. Newman and J. A. Berzofsky, J Immunol 152 (1994), p. 549; J. P. Sauet, H. Gras-Masse, J. G. Guillet and E. Gomard, Int Immunol 8 (1996). p. 457). Several of these studies showed that activation of a CD8+ T cell requires simultaneous interaction of a CD4+ T helper cell and a CD8+ T cell with the same antigen-presenting cell presenting their cognate epitopes (Ridge J P, DI Rosa F, Matzinger P. Nature. 1998 Jun. 4; 3 93 (6684):474-8). The relevance of this three-cell interaction for priming of CTLs is confirmed by studies with viral epitopes, and animal models, since in vivo induction of CTLs was most efficient when CTL and Th epitopes were physically linked rather than administered as an unlinked mixture (Shirai M, Pendleton C D, Ahlers J, Takeshita T, Newman M, Berzohky J A. J Immunol. 1994 Jan. 15; 152(2): 549-56; Oseroff C, Sette A, Wentworth P, Celis E, Maewal A, Dahlberg C, Fikes J, Kubo R T, Chesnut R W, Grey B X Alexander J. Vaccine. 1998 May; 16(8): 823-33). The capacity of CTL and Th antigenic peptides to efficiently induce CTL responses has been demonstrated both in experimental models (C. Fayolle, E. Deriaud and C. Leclerc, J Immunol 147 (1991), p, 4069; C. Widmann, P. Romero, J. L. Maryanski, G. Corradin and D. Vaimori, J Immunol Meth 155 (1992), p. 95) and in humans (A. Vitiello, G. Ishioka, H. M. Grey, R. Rose, P. Famess, R. LaFond, L. Yuan, F. V. Chisari, J. Furze and R. Bartholomeuz, J Clin Invest 95 (1995), p. 341; B. Livingston, C. Crimi, H. Grey, G. Ishioka, F. V. Chisari, J. Fikes, H. M. Grey, R. Chesnut and A. Sette, J Immunol 159 (1997), p. 1383). Moreover, a potent Th response plays an important role not only for optimal induction of CTL responses, but also for maintenance of CTL memory (E A. Walter, P. D. Greenberg, M. J. Gilbert, R. J. Finch, K-S. Watanabe, E. D. Thomas and S. R. Riddell, N Engl J Med 333 (1995), p. 1038; Riddell S R, Greenberg P D, In Thomas E D, Blume K G, Forman S J (eds): Hematopoietic Cell Transplantation, 2nd edn. Malden, M A: Blackwell Science Inc., 1999). Finally, it has long been documented that CD4+ T "helper" cells are crucial in coordinating cellular and humoral immune responses against exogenous antigens.

[0008] Recently, a transgenic (Tg) mouse that expresses both HLA-A*0201 class I and HLA-DR1 class II molecules was established (BenMohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond D J, Hum, Immunol. 2000 August; 61(8):764-79). The authors reported that both HLA-A*0201 and HLA-DR1 transgenes are functional in vivo, that both MHC class-I and class-II molecules were utilized as restriction elements, and that the product of the HLA-DR1 transgene enhances the HLA-A*0201-restricted antigen-specific CTL responses (BenMohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond DJ, Hum, Immunol. 2000 August; 61(8):76479).

[0009] It is noteworthy that these HLA-A*0201/DR1 Tg mice expressed their own MHC H-2 class I and class II molecules. Because HLA class I transgenic mice expressing endogenous mouse MHC class I genes preferentially and often exclusively develop H-2 restricted cm response (C Barra, H Goumier, Z Garcia, P N Marche, E Jouvin-Marche, P Briand, P Fillipi, and F A Lemonnier J Immunol 1993 150: 3681-3689; Epstein H, Hardy F., May J S, Johnson M. H., Holmes N. Eur J Immunol. 1989 September; 19(9):1575-83; Le A X; E J Bernhard, M J Holterman, S Strub, P Parham, E Lacy, and V H Engelhard J Immunol 1989 142: 13 66-1371; Vitiello A, Marchesini D, Furze J, Sherman L A, Chesnut R W., J Exp Med. 1991 Apr. 1; 173(4):100715), and HLA class II transgenic mice expressing endogenous mouse MHC class II genes fail to induce reliable HLA class II restricted antigen-specific responses (Nishimura Y, Iwanaga T, Inamitsu T, Yanagawa Y, Yasunami M, Kimura A, Hirokawa K, Sasazuki T., J Immunol 1990 Jul. 1; 145(0:353-60), these HLA-A*0201/DR1 Tg mice are of limited utility to assess human-specific responses to antigen.

[0010] However, in HLA class I transgenic H-2'class I knock-out mice, or HLA class II transgenic H-2 class II knock-out mice, only HLA-restricted CTL immune responses occur (Pascolo S, Bervas N, Ure J M, Smith A G, Lemonnier F A, Peramau, B., J Exp Med. 1997 Jun. 16; 185(12). 2043-51; Madsen L, Labrecque N, Engberg J, Dierich A, Svejgaard A, Benoist C, Mathis D, Fugger L., Proc Natl Acad Sci USA-1999 Aug. 31; 96(18):10338-43). In fact, HLA-A2.1-transgenic H-2 class I-knock-out (KO) mice exhibit the ability to mount enhanced HLA-A2.1-restricted responses as compared to HLA A2.1-transgenic mice that still express the endogenous murine H-2 class I molecules (Pascolo, S. et al. J Exp Med 185, 2043-2051 (1997); Ureta-Vidal, A., Firat, H., Peramau, B. & Lemonnier, F. A. J Immunol 1163, 2555-2560 (1999); Firat, H. et al., Int Immunol 14, 925-934 (2002); Rohrlich, P. S. et al., Int Immunol 15, 765-772 (2003)). The inventors have made similar observations with HLA-DR1-transgenic mice, depending on whether or not they are deficient in H-2 class II molecules (A. Pajot, unpublished results). Furthermore, in the absence of competition from murine MHC molecules, the HLA-A2.1-transgenic H-2-class I-KO or HLA-DP1-transgenic H-2-class II-KO mice generate only HLA-restricted immune responses (Pascolo, S. et al. J Exp Med 185, 2043-2051 (1997)) (A. Pajot, unpublished results), facilitating the monitoring of HLA-restricted CD8⁺ and CD4⁺ T cell responses. However, protective immune responses against pathogens, which often require collaboration between T helper and cytotoxic CD8⁺ T cells, cannot be studied in the single HLA class I- or HLA class II-transgenic mice, which do not allow the simultaneous assessment of HLA class I and II human responses in the same mouse.

[0011] Accordingly, there exists a need in the art for a convenient animal model system to test the immunogenicity of human vaccine candidates comprising constructs containing human CTL epitopes and, in some cases, with the inclusion of high potency CD4+ Th (helper T lymphocyte) epitopes to sustain antiviral and antitumoral CD8+T-cell activity (A. J. Zajac, K. Murall-Krishna, J. N. Blattman and R. Ahmed, Curr Opin Immunol 10(1998), p. 444; Firat H, Garcia-Pons F, Tourdot S, Pascolo S; Scardino A, Garcia Z, Michel M L, Jack R W, Jung O, Kosmatopoulos K, Mateo L, Suhrbier A, Lemonnier F A, Langlade-Dernoyen P, Eur J Immunol 29, 3112, 1999). There is also a need for a system that allows the simultaneous assessment of the mutual coordination between a CTL response, a TH response (in particular s TH₁ or TH₂ response), and, optionally, a humoral response.

SUMMARY OF THE INVENTION

[0012] The inventors have met this need and more by providing mice transgenic for both HLA-A2.1 and HLA-DR1 molecules, in a background that Is deficient for both H-2 class I and class II molecules. Specifically, the invention provides mice comprising (1) mutated H-2 class I and class II molecules; and (2) expressing HLA class I transgenic molecules, or HLA class II transgenic molecules, or HLA class I transgenic molecules and HLA class II transgenic molecules. These mice provide a model useful in the development and optimization of vaccine constructs with maximum in vivo immunogenicity for human use. Specifically, such mice enable a complete analysis of the three components of the immune adaptive response (antibody, helper and cytolytic) in a single animal, as well as an evaluation of the protection specifically conferred by vaccination against an antigenic challenge.

[0013] Mice of the invention, which comprise a knock-out for both H-2 class I and class II molecules, and express HLA class I transgenic molecules and HLA class II transgenic molecules represent a completely humanized experimental mouse that can be used to simultaneously detect the presence of antigen-specific antibodies, an antigen-specific HLA-DRI restricted T cell response, and an antigen-specific HLA-A2 restricted T cell response. These mice will be useful to study how mutual coordination operates between a CTL response, a TH response (in particular a TH₁ or TH₂ response), and, optionally, a humoral response. These mice represent an optimized tool for basic and applied vaccinology studies.

[0014] A first embodiment of the invention provides a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, and a functional HLA class I or class II transgene.

[0015] A second embodiment of the invention provides a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA class I transgene, and a functional HLA class II transgene.

[0016] In some embodiments, the HLA class I transgene is an HLA-A2 transgene and the HLA class II transgene is an HLA-DR1 transgene. In other embodiments, the HLA-A2 transgene comprises the HLA-A2 sequence provided in the sequence listing and the HLA-DR1 transgene comprises the HLA-DR1 sequence provided in the sequence listing.

[0017] A further embodiment of the invention provides a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene. In an embodiment, the mouse has the genotype HLA-A2⁺HLA-DR1+β2m°IAβ°. In some embodiments the HLA-A2 transgene comprises the HLA-A2 sequence provided in the sequence listing and the HLA-DR1 transgene comprises the HLA-DR1 sequence provided in the sequence listing.

[0018] Another embodiment of the invention provides a method of simultaneously identifying the presence of one or more epitopes in a candidate antigen or group of antigens, where the one or more epitopes elicits a specific humoral response, a TH HLA-DR1 restricted response, and/or a CTRL HLA-A2 restricted response. The method comprises administering the candidate antigen or group of candidate antigens to a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA-A2 transgene, and a functional FILA-DR1 transgene, or a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene, and has the genotype HLA-A2⁺FILA-DR1⁺β2m°IAβ°; assaying for a specific humoral response in the mouse to the antigen; assaying for a TH HLA-DR1 restricted response in the mouse to the antigen; and assaying for a CTRL HLA-A2 restricted response in the mouse to the antigen. Observation of a specific humoral response in the mouse to the antigen identifies an epitope that elicits a humoral response in the antigen. Observation of a TH HLA-DR1 restricted response in the mouse to the antigen identifies an epitope that elicits a TH HLA-DR1 restricted response in the antigen. Observation of a CTRL HLA-A2 restricted response in the mouse to the antigen identifies an epitope which elicits a CTRL HLA-A2 restricted response in the antigen.

[0019] In some embodiments, the method includes assaying for a Th1-specific response in the mouse to the antigen and assaying for a Th2-specific response in the mouse to the antigen. In this case, observation of a Th1-specific response in the mouse to the antigen identifies an epitope that elicits a Th1-specific response in the mouse to the antigen, and observation of a Th2-specific response in the mouse to the antigen identifies an epitope that elicits a Th2-specific response in the mouse to the antigen.

[0020] This invention also provides a method of identifying the presence of an HLA DR1-restricted T helper epitope in a candidate antigen or group of candidate antigens, the method comprising administering the candidate antigen or group of candidate antigens to a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA-A2 transgene, and a functional HLA-DR1 transgene, or a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene, and has the genotype HLA-A2⁺HLA-DR1⁺β2m°IAβ°; and assaying for a TH HLA-DR1 restricted T helper epitope response in the mouse to the antigen. Observation of a TH HLA-DR1 restricted T helper epitope response in the mouse to the antigen identifies an epitope that elicits a TH HLA-DR1 restricted T helper epitope response in the antigen.

[0021] In addition, this invention provides an isolated antigen comprising an HLA DR1-restricted T helper epitope identified by the method of the preceding paragraph. In some embodiments, the isolated antigen further includes an epitope that elicits a humoral response and/or an epitope that elicits a CTRL HLA-A2 restricted response. In some embodiments, the antigen comprising an HLA DR1-restricted T helper epitope comprises a polypeptide. In other embodiments, the antigen comprising an HLA DR1-restricted T helper epitope comprises a polynucleotide. In further embodiments, the antigen comprising an HLA DR1-restricted T helper epitope comprises DNA, RNA, or DNA and RNA.

[0022] Further, this invention provides a method of identifying the presence of an HLA-A2-restricted T cytotoxic (CTL) epitope in a candidate antigen or group of candidate antigens, the method comprising administering the candidate antigen or group of candidate antigens to a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA-A2 transgene, and a functional HLA-DR1 transgene, or a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene, and has the genotype HLA-A2⁺HLA-DR1⁺β2m°IAβ°; and assaying for an HLA-A2-restricted T cytotoxic (CTL) response in the mouse to the antigen or group of antigens. Observation of an HLA-A2-restricted T cytotoxic (CTL) response in the mouse to the antigen or group of antigens identifies an epitope that elicits a an HLA-A2-restricted T-cytotoxic (CTL) response in the antigen or group of antigens.

[0023] This invention provides an isolated antigen comprising an HLA-A2-restricted T cytotoxic (CTL) epitope identified by the method of the preceding paragraph. In some embodiments, the antigen further comprises an epitope that elicits a humoral response and/or an epitope that elicits a TH HLA-DR1 restricted T helper epitope response. In some embodiments, the antigen comprising an HLA-A2-restricted T cytotoxic (CTL) epitope comprises a polypeptide. In other embodiments, the antigen comprising an HLA-A2-restricted T cytotoxic (CTL) epitope comprises a polynucleotide. In further embodiments, the antigen comprising an HLA-A2-restricted T cytotoxic (CTL) epitope comprises, DNA, RNA, or DNA and RNA.

[0024] This invention also provides a method of comparing the efficiency of the T-helper cell response induced by two or more vaccines. This method comprises administering a first candidate vaccine to a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA-A2 transgene, and a functional HLA-DR1 transgene, or a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene, and has the genotype HLA-A2⁺HLA-DR1⁺β2m°IAβ°, and measuring the T-helper cell response induced in the mouse by the first candidate vaccine; administering a second candidate vaccine to a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA-A2 transgene, and a functional HLA-DR1 transgene, or a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene, and has the genotype HLA-A2⁺HLA-DR1⁺β2m°IAβ°, and measuring the T-helper cell response induced in the mouse by the second candidate vaccine; administering each additional candidate vaccine to be compared to a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA-A2 transgene, and a functional HLA-DR1 transgene, or a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional FILA class I transgene and a functional HLA class II transgene, and has the genotype HLA-A2⁺HLA-DR1⁺β2m°IAβ°, and measuring the T-helper cell response induced in the mouse by the additional candidate vaccine, and determining the efficiency of each candidate vaccine to induce a T-helper cell response by comparing the T-helper cell responses to each of the vaccines to be compared with each other. In some embodiments the T-helper cell response is an HLA-DR1 restricted response.

[0025] In addition, this invention provides a method of comparing the efficiency of T cytotoxic cell responses induced by two or more vaccines. The method includes administering a first candidate vaccine to a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA-A2 transgene, and a functional HLA-DR1 transgene, or a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene, and has the genotype HLA-A2⁺HLA-DR1⁺β2m°IAβ°, and measuring the T cytotoxic cell response induced in the mouse by the first candidate vaccine; administering a second candidate vaccine to a mouse of a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA-A2 transgene, and a functional HLA-DR1 transgene, or a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene, and has the genotype HLA-A2⁺HLA-DR1⁺β2m°IAβ°, and measuring the T cytotoxic cell response induced in the mouse by the second candidate vaccine; administering each additional candidate vaccine to be compared to a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA-A2 transgene, and a functional HLA-DR1 transgene, or a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene, and has the genotype HLA-A2⁺HLA-DR1⁺β2m°IAβ°, and measuring the T cytotoxic cell response induced in the mouse by the additional candidate vaccine; and determining the efficiency of each candidate vaccine to induce a T cytotoxic cell response by comparing the T cytotoxic cell responses to each of the vaccines to be compared with each other. In some embodiments the T cytotoxic cell response is an HLA-A2 restricted response.

[0026] Further, this invention provides a method of simultaneously comparing the efficiency of T-helper cell response and T cytotoxic cell response induced by two or more vaccines. The method comprises administering a first candidate vaccine to a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA-A2 transgene, and a functional HLA-DR1 transgene, or a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene, and has the genotype HLA-A2⁺HLA-DR1⁺β2m°IAβ°, and measuring the T-helper cell response and T cytotoxic cell response induced in the mouse by the first candidate vaccine; administering a second candidate vaccine to a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA-A2 transgene, and a functional HLA-DR1 transgene, or a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class 1 transgene and a functional HLA class II transgene, and has the genotype HLA-A2⁺HLA-DR1⁺β2m°IAβ°, and measuring the T-helper cell response and T cytotoxic cell response induced in the mouse by the second candidate vaccine; administering each additional candidate vaccine to be compared to a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene; a functional HLA-A2 transgene, and a functional HLA-DR1 transgene, or a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene, and has the genotype HLA-A2⁺HLA-DR1⁺β2m°IAβ°, and measuring the T-helper cell response and T cytotoxic cell response induced in the mouse by each additional candidate vaccine; and determining the efficiency of each candidate vaccine to induce a T-helper cell response and T cytotoxic cell response by comparing the T-helper cell response and T cytotoxic cell response to each of the vaccines to be compared with each other. In some embodiments the T-helper cell response is an HLA-DR1 restricted response, and the T cytotoxic cell response is an HLA-A2 restricted response.

[0027] This invention also provides a method of simultaneously determining the humoral response, the T-helper cell response, and the T cytotoxic cell response of a mouse following its immunization with an antigen or a vaccine comprising one or more antigens. The method comprises administering the antigen or the vaccine comprising one or more antigens to a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA-A2 transgene, and a functional HLA-DR1 transgene, or a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene, and has the genotype HLA-A2⁺HLA-DR1⁺β2m°IAβ°, and assaying for a specific humoral response in the mouse to the antigen or vaccine comprising one or more antigens, assaying for a T-helper cell response in the mouse to the antigen or vaccine comprising one or more antigens, and assaying for a T cytotoxic cell response in the mouse to the antigen or vaccine comprising one or more antigens. In some embodiments the T-helper cell response is a TH HLA-DR1 restricted response. In some embodiments the T cytotoxic cell response is a CTRL HLA-A2 restricted response.

[0028] This invention also provides a method of optimizing two or more candidate vaccine compositions for administration to a human, based on preselected criteria. The method includes simultaneously determining the humoral response, the T-helper cell response, and the T cytotoxic cell response of a mouse following its immunization with the two or more candidate vaccine compositions, using a method comprising administering the antigen or the vaccine comprising one or more antigens to a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA-A2 transgene, and a functional HLA-DR1 transgene, or a transgenic mouse deficient or both H2 class Land_class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional. HLA class II transgene, and has the genotype HLA-A2⁺-HLA-DR1⁺β2m°IAβ°, assaying for a specific humoral response in the mouse to the antigen or vaccine comprising one or more antigens, assaying for a T-helper cell response in the mouse to the antigen or vaccine comprising one or more antigens; assaying for a T cytotoxic cell response in the mouse to the antigen or vaccine comprising one or more antigens, and selecting an optimized vaccine by applying preselected criteria to the results. In some embodiments, the two or more vaccine candidates differ only in the ratio of antigen to adjuvant present in the vaccine. In some embodiments, the two or more vaccine candidates differ only in the type of adjuvant present in the vaccine.

[0029] In another aspect, the invention provides a method of determining whether a vaccine poses a risk of induction of an autoimmune disease when administered to a human. The method comprises administering the vaccine to a transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA-A2 transgene, and a functional HLA-DR1 transgene, or a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene, and has the genotype HLA-A2⁺HLA-DR1⁺β2m°IAβ°, and assaying for an autoimmune response in the mouse, where observation of an autoimmune response in the mouse indicates that the vaccine poses a risk of induction of an autoimmune disease when administered to a human.

[0030] This invention also provides an isolated transgenic mouse cell comprising a disrupted H2 class I gene, a disrupted H2 class II gene, and a functional HLA class I or class II transgene.

[0031] In addition, the invention provides an isolated transgenic mouse cell comprising a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA class I transgene, and a functional HLA class II transgene.

[0032] In some embodiments, the HLA class I transgene is an HLA-A2 transgene and the HLA class II transgene is an HLA-DR1 transgene. In other embodiments, the HLA-A2 transgene comprises the HLA-A2 sequence provided in the sequence listing and the HLA-DR1 transgene comprises the HLA-DR1 sequence provided in the sequence listing.

[0033] Further, this invention provides an isolated transgenic mouse cell deficient for both H2 class I and class II molecules, wherein the transgenic mouse cell comprises a functional HLA class I transgene and a functional HLA class II transgene. In some embodiments, the transgenic mouse cell has the genotype HLA-A2⁺HLA-DR1⁺β2m°IAβ°. In other embodiments, the HLA-A2 transgene comprises the HLA-A2 sequence provided in the sequence listing and the HLA-DR1 transgene comprises the HLA-DR1 sequence provided in the sequence listing.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The invention will be more fully described with reference to the drawings in which:

[0035] FIG. 1 shows a flow cytometric analysis of the cell-surface expression of the indicated transgenic molecules. (a) Splenocytes from HLA-DR1-transgenic H-2 class II-KO (DR1⁺ CII^-, left panel), HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-KO (A2⁺ DR1⁺ CI^- CII^-, central panel), and HLA-A2.1 transgenic H-2 class I-KO (A2⁺ CI^-, right panel) mice were stained with either FITC-labeled W6/32 (anti-HLA-ABC, in abcissas) or biotinylated 28-8-6S (anti-H-2K^b/D^b, in ordinates) m.Ab, the latter revealed With PE-labeled anti-mouse IgG. (b) B220+ splenic B lymphocytes from the same strains of mice, were stained with FITC-labeled L243 (anti-HLA-DR1, upper panels) and PE-labeled AF6-120.1 (anti-H-2 IAβ^b, lower panels) m.Ab.

[0036] FIG. 2 shows CD8⁺ and CD4⁺ splenic T cell numbers and BV segment usage (based on an immunoscope analysis) in mice of the indicated genotypes. (a) Splenocytes from HLA-DR1-transgenic H-2 class II-KO (DR1⁺ CII^-, left panel), HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-KO (A2⁺ DR1⁺ CI^-CII^-, central panel), and HLA-A2.1-transgenic H-2 class I-KO (A2⁺ CI^-, right panel) mice were stained with PE-labeled CT-CD4 (anti-mouse CD4, in ordinates) and FITC-labeled 53-6.7 (anti-mouse CD8, in abcissas) m.Ab. Numbers correspond to percentages of CD4⁺ (upper left square) or CD8⁺ (lower right square) T cells in total splenocytes. (b and c) Immunoscope RT-PCR analysis of purified splenic CD8⁺ (b) and CD4⁺ (c) T cells for BV segment family (1-20) usage using forward BV family (1-20) specific and reverse BC primers. A typical profile for a BV segment family productively rearranged includes a series of peaks with a Gaussian-like distribution differing in length by 3 nucleotides. The Figure illustrates the results obtained with a HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-KO representative mouse.

[0037] FIG. 3 shows HBs-specific antibody, cytolytic and proliferative responses. HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-KO mice were or not immunized by intramuscular injection of HBsAg-encoding plasmid-DNA and then individually tested. (a) Humoral (upper panel), cytolytic (middle panel) and proliferative (lower panel) responses and specificity controls of a representative HBsAg-DNA-immunized mouse. The antibody (IgG) titer against HBsAg particles containing both middle and small HBV envelope proteins and against the preS2_109-134 peptide were determined in an ELISA assay. Cytolytic activity at different effector/target (E/T) ratios was assessed using RMAS-HHD target cells pulsed with either relevant (HBsAg_348-357, HLA-A2.1-restricted .diamond-solid.) or control (HBsAg_371-378, H-2 K^b-restricted Δ, and MAGE-3_271-279, HLA-A2.1-restricted quadrature) peptide. Proliferative responses were detected using either relevant (HBsAg_180-195, HLA-DR1-restricted) or control (HBsAg_126-138, H-2 IA^b-restricted and HIV 1 Gag_263-278. HLA-DR1-restricted) peptide. (b) Similar evaluation of the antibody (IgG, upper panel), cytolytic (middle panel) and proliferative (lower panel) responses of 6 (1-6) HBsAg-DNA-immunized mice as compared to mean responses of 6 naive mice (0). Cytolytic activity at a 30/1 E/T ratio was assessed on RMAS-HHD target cells pulsed with either HBsAg_346-357, immunodominant (filled bars) or HBsAg_335-343, subdominant (grey bars) peptide.

[0038] FIG. 4 shows results of protection assays. HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-/KO mice were or not immunized twice with plasmid DNA encoding HBsAg. Fifteen days after the last immunization, they were challenged intraperitoneally with 10⁷ PFU of rVV expressing either the HBsAg or the HBx protein. Four days later, animals were tested individually for viral titers in ovaries. The results (rVV PFU/ovary in log 10) are given for the HBsAg-DNA-immunized mice challenged with rVV-HBsAg (I, n=10), naive mice challenged with rVV-HBsAg (N, n=6) , HBsAg-immune mice challenged with rVV-HBx (Ix, n=6) and naive mice challenged with rVV-HBx (Nx, n=6).

[0039] FIG. 5 shows the AC anti-Pre S2 response in HLA-A2+DR1+CI-CII-mice following a pcmv S2/S immunization.

[0040] FIG. 6 shows the T CD4 proliferative response to HLA-DR1 restricted epitopes following immunization of HLA-A2+DR1+CI-CII-mice with pcmv S2-S.

[0041] FIG. 7 shows the T CD8 cytotoxic response to the HLA-A2 restricted HBS (348-357) peptide following an immunization of HLA-A2+DR1+CI-CII-mice with pcmv S2/S.

SEQUENCES

[0042] SEQ ID NO:1 contains the following subparts: Nucleotides 1-1205 comprise the HLA-A2 promoter; nucleotides 1206-1265 the HLA-A2 leader sequence; nucleotides 1266-1565 the human β2 microgobulin cDNA; nucleotides 1566-1610 a (Gly4Ser)₃ linker, nucleotides 1611-2440 a segment containing exon 2 and part of intron 3 of HLA-A2; and nucleotides 2441-4547 a segment containing part of intron 3, exons 4 to 8, and part of the 3' non-coding region of the H₂D^b gene.

[0043] SEQ ID NO:2 is the nucleotide sequence of the DRA*0101 gene. Nucleotides 1-15279 are the promoter located 5' to the HLA-DR alpha gene, nucleotides 15280-15425 are exon 1, nucleotides 15344-15346 are the ATG start codon, nucleotides 17838-18083 are exon 2, nucleotides 18575-18866 are exon 3, nucleotides 19146-19311 are exon 4, and nucleotides 20008-20340 are exon 5.

[0044] SEQ ID NO:3 is the nucleotide sequence of the DRB1*010101 gene. Nucleotides 7391-7552 are exon 1, nucleotides 7453-7455 are the ATG start codon, nucleotides 15809-16079 are exon 2, nucleotides 19536-19817 are exon 3, nucleotides 20515-20624 are exon 4, nucleotides 21097-21121 are exon 5, and nucleotides 21750-22085 are exon 6.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0046] This invention provides mice comprising (1) mutated H-2 class I and class II molecules; and (2) expressing HLA class I transgenic molecules, or HLA class II transgenic molecules, or HLA class I transgenic molecules and HLA class II transgenic molecules. Mice of the invention, which comprise a knock-out for both H-2 class I and class II molecules, and express HLA class I transgenic molecules and HLA class II transgenic molecules represent a completely humanized experimental mouse that can be used to simultaneously detect the presence of antigen-specific antibodies, an antigen-specific HLA-DRI restricted T cell response, and an antigen-specific HLA-A2 restricted T cell response. These mice are useful to study how mutual coordination operates between a CTL response, a TH response (in particular a TH₁ or TH₂ response), and, optionally, a humoral response. These mice represent an optimized tool for basic and applied vaccinology studies.

[0047] The invention provides transgenic mouse comprising a disrupted H2 class I gene, a disrupted H2 class II gene, and a functional HLA class I or class II transgene. In some embodiments, the transgenic mouse comprises a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA class I transgene, and a functional HLA class II transgene. Such a mouse can be said to be a completely humanized experimental mouse, because it can be used to simultaneously detect the presence of antigen-specific antibodies, an antigen-specific HLA-DRI restricted T cell response, and an antigen-specific HLA-A2 restricted T cell response.

[0048] As shown, in part, in the Examples provided herein, and as is generally clear to one of skill in the art from the disclosure, HLA-A2.1-/HLA-DR9-transgenic H-2 class I-/class II-KO mice have the capacity to develop HBsAg-specific antibody, CD4⁺ helper and CD8⁺ cytolytic T cell responses following DNA immunization. These responses, observed in every single mouse tested, were directed at the same immunodominant epitopes as human responses and conferred to the immunized animals specific protection against a HBsAg recombinant vaccinia virus.

[0049] T helper cells are essential for full maturation of antibody responses (Katz, D. H. & Benacerraf, B., Adv Immunol 15, 1-94 (1972)) CTL priming against many epitopes (von Boehmer, H. & Haas, W., J Exp Med 150, 1134-1142 (1979); Keene, J. A. & Forman, J., J Exp Med 155, 768-782 (1982)) and CTL long-term maintenance (Matioubian, M., Concepcion, R. J. & Ahmed, R., J Virol 68, 8056-8063 (1994)). Both antibodies (Lefrancois, L., J Virol 51, 208-214 (1984)) and CTL (Zinkernagel, R. M. & Welsh, R. M., J Immunol 117, 1495-1502 (1976)) are critical components of protective immunity against viral infections. Potent HBsAg-specific antibody and CTL responses were in fact observed in HLA-A2.1-/HLA-DR1-double transgenic, H-2 class I-/class II-KO mice, but not in HLA-A2.1-single transgenic, H-2 class I-/class II-KO mice. Thus, HBsAg-specific CD4⁺ T cell help is essential for generating efficient HBsAg-specific CTL and antibody responses. These results are consistent with studies on HBsAg-immunized mice (Milich, D. R., Semin Liver Dis 11, 93-112(1991)) and HBsAg-vaccinated humans (Cells, E., Kung, P. C. & Chang, T. W., J Immunol 132, 1511-1516 (1984)), which suggest that production of an anti-HBs antibody response is dependent on CD4⁺ T cells.

[0050] Transgenic mice expressing both HLA-A2.1 class I and HLA-DR1 class II molecules have already been derived (BenMohamed, L. et al. Hum Immunol 61, 764-779 (2000)). The authors reported that both the HLA-A2.1 and HLA-DR1 molecules are functional restriction elements in vivo and that the product of the HLA-DR1 transgene enhances the HLA-A2.1-restricted antigen-specific CTL responses. However, the human relevance of the immune responses in these mice is dwarfed by the fact that they still expressed their own H-2 class I and class II molecules, which are usually preferentially and often exclusively used as restricting elements in response to antigens (Ureta-Vidal, A., Firat, H., Peramau, B. & Lemonnier, F. A., J Immunol 163, 2555-2560 (1999); Rohrlich, P. S. et al., Int Immunol 15, 765-772 (2003)) (A. Pajot, unpublished results). The invention described herein overcomes this limitation by providing HLA-A2.1-/HLA-DR1-transgenic, H-2 class I-/class II-KO mice.

[0051] In some embodiments the HLA-A2.1-/HL4-DR1-transgenic, H-2 class I-/class II-KO mice express, in a βm-KO context, a HLA-A2.1 monochain in which the human β2m is covalently linked by a peptidic arm to the HLA-A2.1 heavy chain. They further lack cell surface expression of conventional H-2 IA and IE class II molecules as a result of the inactivation of the H-2 IAβ^b gene, since H-2 IEσ is a pseudogene in the H-2^b haplotype. The results provided herein demonstrate that such mice are deprived of cell surface expression of H-2 class I and class II molecules. However, it was reported in one case that a free class I heavy chain, in particular H-2 D^b, may exist on the surface of a β2m-KO mouse, and could induce an alloreactivity response. Even if this is so, because such mice are empty of peptide, they should not interfere in antigen-specific immune response (Bix, M. & Raulet, D., J Exp Med 176, 829-834 (1992)). This is supported by the report of Allen et al (Allen, H., Fraser, J., Flyer, D., Calvin, S. & Flavell, R., Proc Natl Acad Sci USA 83, 7447-7451 (1986)), in which they confirmed that H-2 D^b is expressed at the cell surface even when there is no β2m present within the cell, but that such D^b antigen is recognized by neither D^b-allospecific or D^b-restricted cytotoxic T lymphocytes. Furthermore, D^b antigens are not recognized by most monoclonal antibodies of the native D^b.

[0052] Nonetheless, in HLA-DRα single transgenic mice, it was reported that unconventional HLA-DRα/H-2 IEβ^b hybrid complexes may be expressed to some extent on the cell surface, at least in the absence of the HLA-DRβ chain (Lawrance, S. K. et al., Cell 58, 583-594 (1989)). In spite of this observation, these unconventional molecules were not detected serologically on cell surfaces in HLA-A2.1-/HLA-DR1-transgenic, H-2 class I-/class II-KO mice, even with mAb (17-3-3S), which is known to react with such hybrid molecules (Ozato, K., Mayer, N. & Sachs, D. H., J Immunol 124, 533-540 (1980)) (FIG. 1a and data not shown). In addition, the results obtained on studying HBsAg-specific and HIV 1-Gag-specific T cell responses of these mice were all indicative of exclusive usage of the HLA-A2.1 and HLA-DR1 molecules as restricting elements. This argues that the unconventional HLA-DRα/H-2 IEβ^b were likely unstable compared to conventional HLA-DRα/HLA-DRβ molecules and that they may exist only in the absence of the HLA-DRβ chain. Mouse strains in which the entire (H-2 IAβ^b, IAα^b, IEβ^b) H-2 class II region has been deleted (Madsen, L. et al., Proc Natl Acad Sci USA 96, 10338-10343 (1999)), as well as the H-2 D^b gene, are being analyzed to completely exclude this possibility. Preliminary analysis of splenocytes obtained from the first animals revealed a CD4⁺ T cell pool restoration similar to that observed in HLA-DR1-transgenic H-2 class II-KO (Iaβ^b) mice, suggesting that the HLA-DR1-restricted CD4⁺ T cell responses of these new mice should be equivalent to those of the HLA-A2.1-/HLA-DR1-transgenic, H-2 class I-/class II-KO mice.

[0053] The peripheral CD8⁺ T lymphocytes of HLA-A2.1-/HLA-DR1-transgenic, H-2 class I-/class II-KO mice, compared to parental HLA-A2.1-transgenic H-2 class I-KO mice, are quantitatively and qualitatively similar with full diversification, at least in terms of BV segment usage, of the TCR repertoire. Partial restoration compared to wild-type animals, especially of the CD8⁺ T cell pool, has been a constant observation in single HLA-transgenic mice expressing a chimeric (α3 domain of mouse origin) HLA-A2.1 molecule (Pascolo, S. et at., J Exp Med 185, 2043-2051 (1997)). Regardless of the α3 domain substitution, the interaction remains suboptimal between mouse CD8 and HLA-A2.1 molecules, since co-crystal analysis has documented that human CD8 also contacts the HLA-A2.1 heavy chain α2 domain (Gao, G. F. et al., Nature 387, 630-634 (1997)). Suboptimal cooperation might also occur in the endoplasmic reticulum where many molecules (TAP, tapasine, ERp 57) assist MHC class I molecule biosynthesis. However, at this stage, the only documented functional difference between these mice and human endoplasmic reticulum molecules, namely the efficient transport by human but not mouse TAP of COON-terminus positively charged cytosolic peptides (Momburg, F., Neefjes, J. J. & Hammerling, G. J., Curr Opin Immunol 6, 32-37 (1994)), is not relevant for HLA-A2.1 molecules which bind peptides with a hydrophobic C-terminus, since these peptides are transported efficiently by mouse and human TAP. Even though the number of CD8⁺ T lymphocytes is lower in both single HLA-A2.1-transgenic, H-2 class I-KO mice and in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-KO mice, they respond efficiently against HBsAg and, importantly, the latter mice develop antibody, helper and cytolytic cell responses similar to humans.

[0054] One of the difficulties hampering the design of T-epitope-based vaccines targeting T lymphocytes is HLA class I/class II molecule polymorphism. HLA-A2.1 and HLA-DR1-molecules are expressed by a significant proportion of individuals in human populations (30 to 50% for HLA-A2.1, 6 to 18% for HLA-DR1). Even though the functional clustering of HLA class I molecules in superfamilies is based on significant redundancy of the presented sets of peptides³⁴, individual analysis of the responses elicited by each HLA class I isotypic or allelic variant remains desirable to identify the optimal epitopes they present. This is particularly important to devise a new reagent, such as tetramer (HLA-class I or HLA-class II) to monitor the immune response. For the same reason, it would be helpful to obtain strains of mice co-expressing HLA-A2.1 with other HLA class II molecules, even if the binding of peptides to HLA class II molecules is less restrictive than to class I molecules. Based on the disclosure herein, additional HLA class I-/class II-transgenic, H-2 class I-/class II-KO mice can be constructed for these and other purposes.

[0055] Whereas HLA-transgenic H-2-KO mice enable a detailed analysis and optimization of the immunogenicity of antigenic peptides with excellent transposability to humans (Rohrlich, P. S. et al., Int Immunol 15, 765-772 (2003); Loirat, D., Lemonnier, F. A. & Michel, M. L., J Immunol 165, 4748-4755 (2000); Scardino, A. et al., Eur J Immunol 31, 3261-3270 (2001)) this is less evident for vaccine adjuvant-formulation studies. This could be due to differences between the two species in the various effectors that are mobilized early in response to an antigenic challenge. Increasing fundamental knowledge of innate immunity might, in the future, lead to a more complete humanization of the mouse immune system.

[0056] In conclusion, the disclosure herein describes an optimized, humanized transgenic mouse model, whose H-2 class I (mouse β2m) and class II (H-2 IAβ^b) genes have been deleted and replaced with equivalent human genes HHD (HLA-A*0201), HLA-DRA*0101 and HLA-DRB1*0101. Cellular immunity in the HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-KO mice is completely restricted by the human HLA molecules, with a complete absence of immune responses restricted by the murine MHC molecules. The absence of competition between murine MHC and human (transgenic) HLA immune responses allows for use of these mice to characterize epitopes in human vaccines that require collaboration between HLA-restricted CD4⁺ T helper and HLA-restricted CD8⁺ T cytolytic cells.

[0057] "HLA" is the human MHC complex, and "H-2" the mouse MHC complex. The human complex comprises three class I α-chain genes, HLA-A, HLA-B, and HLA-C, and three pairs of MHC class II α- and β-chain genes, HLA-DR, -DP, and -DQ. In many haplotypes, the HLA-DR cluster contains an extra β-chain gene whose product can pair with the DRα chain, and so the three sets of genes give rise to four types of MHC class II molecules. In the mouse, the three class I a-chain genes are H-2-L, H-2-D, and H-2-K. The mouse MHC class II genes are H-2-A and H-2-E.

[0058] It is known in the art that genetic diversity exists between the HLA genes of different individuals as a result of both polymorphic HLA antigens and distinct HLA alleles. Accordingly, embodiments of the invention disclosed herein may substitute one polymorphic HLA antigen for another or one HLA allele for another. Examples of HLA polymorphisms and alleles can be found, for example, at http://www.anthonynolan.org.uk/HIG/data.html and http://www.eblac.uk/imgt/hla, and in Genetic diversity of HLA: Functional and Medical Implication, Dominique Charon (Ed.), EDK Medical and Scientific International Publisher, and The HLA FactsBook, Steven G. E. Marsh, Peter Parham and Linda Barber, AP Academic Press, 2000.

[0059] A "disrupted" gene is one that has been mutated using homologous recombination or other approaches known in the art. A disrupted gene can be either a hypomorphic allele of the gene or a null allele of the gene. One of skill in the art will recognize that the type of allele to be used can be selected for any particular context. In many embodiments of the invention, a null allele is preferred.

[0060] "Homologous recombination" is a general approach for targeting mutations to a preselected, desired gene sequence of a cell in order to produce a transgenic animal (Mansour, S. L. et al., Nature 336:348-352 (1988); Capecchi, M. R., Trends Genet. 5:70-76 (1989); Capecchi, M. R., Science 24:1288-1292 (1989); Capecchl, M. R. et al., In: Current Communications in Molecular Biology, Capecchi, M. R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), pp. 45-52; Frohman, M. A. et al., Cell 56:145-147 (1989)).

[0061] It is now be feasible to deliberately alter any gene in a mouse (Capecchi, M. R., Trends Genet. 5:70-76 (1989); Frohman, M. A. et al., Cell 56:145-147 (1989)). Gene targeting involves the use of standard recombinant DNA techniques to introduce a desired mutation into a cloned DNA sequence of a chosen locus. That mutation is then transferred through homologous recombination to the genome of a pluripotent, embryo-derived stem (ES) cell. The altered stem cells are microinjected into mouse blastocysts and are incorporated into the developing mouse embryo to ultimately develop into chimeric animals. In some cases, germ line cells of the chimeric animals will be derived from the genetically altered ES cells, and the mutant genotypes can be transmitted through breeding.

[0062] Gene targeting has been used to produce chimeric and transgenic mice in which an nptll gene has been inserted into the β₂-microglobulin locus (Koller, B. H. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:8932-8935 (1989); Zijlstra, M. et al., Nature 342:435-438 (1989); Zijlstra, M. et al., Nature 344:742-746 (1989); DeChiaba et al., Nature 345:78-80 (1990)). Similar experiments have enabled the production of chimeric and transgenic animals having a c-abl gene which has been disrupted by the insertion of an nptll gene (Schwartzberg, P. L. et al., Science 246:799-803 (1989)). The technique has been used to produce chimeric mice in which the en-2 gene has been disrupted by the insertion of an nptll gene (Joyner, A. L. et al., Nature 338:153-155 (1989)).

[0063] In order to utilize the "gene targeting" method, the gene of interest must have been previously cloned, and the intron-exon boundaries determined. The method results in the insertion of a marker gene (e.g., an nptll gene) into a translated region of a particular gene of interest. Thus, use of the gene targeting method results in the gross destruction of the gene of interest.

[0064] Significantly, the use of gene targeting to alter a gene of a cell results in the formation of a gross alteration in the sequence of that gene. The efficiency of gene targeting depends upon a number of variables, and is different from construct to construct.

[0065] The chimeric or transgenic animal cells of the present invention are prepared by introducing one or more DNA molecules into a cell, which may be a precursor pluripotent cell, such as an ES cell, or equivalent (Robertson, E. J., In: Current Communications in Molecular Biology, Capecchi, M. R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), pp. 39-44). The term "precursor" is intended to denote only that the pluripotent cell is a precursor to the desired ("transfected") pluripotent cell, which is prepared in accordance with the teachings of the present invention. The pluripotent (precursor or transfected) cell can be cultured in vivo in a manner known in the art (Evans, M. J. et al., Nature 292:154-156 (1981)) to form a chimeric or transgenic animal.

[0066] Any ES cell can be used in accordance with the present invention. It is, however, preferred to use primary isolates of ES cells. Such isolates can be obtained directly from embryos, such as the CCE cell line disclosed by Robertson, E. J., In: Current Communications in Molecular Biology, Capecchi, M. R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), pp. 39-44), or from the clonal isolation of ES cells from the CCE cell line (Schwartzberg, P. A. et al., Science 246:799-803 (1989), which reference is incorporated herein by reference). Such clonal isolation can be accomplished according to the method of E. J. Robertson (In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, (E. J. Robertson, Ed.), IRL Press, Oxford, 1987), which reference and method are incorporated herein by reference. The purpose of such clonal propagation is to obtain ES cells, which have a greater efficiency for differentiating into an animal. Clonally selected ES cells are approximately 10-fold more effective in producing transgenic animals than the progenitor cell line CCE. For the purposes of the recombination methods of the present invention, clonal selection provides no advantage.

[0067] An example of ES cell lines, which have been clonally derived from embryos, are the ES cell lines, AB1 (hprt⁺) or AB2.1 (hprt^-). The ES cells are preferably cultured on stromal cells (such as STO cells (especially SNC4 STO cells) and/or primary embryonic fibroblast cells) as described by E. J. Robertson (in: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, (E. J. Robertson, Ed., IRL Press, Oxford, 1987, pp 71-112), which reference is incorporated herein by reference. Methods for the production and analysis of chimeric mice are disclosed by Bradley, A. (In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, (E. J. Robertson, Ed.), IRL Press, Oxford, 1987, pp 113-151), which reference is incorporated herein by reference. The stromal (and/or fibroblast) cells serve to eliminate the clonal overgrowth of abnormal ES cells. Most preferably, the cells are cultured in the presence of leukocyte inhibitory factor ("lif") (Gough, N. M. et al., Reprod. Fertil. Dev. 1:281-288 (1989); Yamamori, Y. et al., Science 246:1412-1416 (1989), both of which references are incorporated herein by reference). Since the gene encoding lif has been cloned (Gough, N. M. et al., Reprod. Fertil. Dev. 1:281-288 (1989)), it is especially preferred to transform stromal cells with this gene, by means known in the art, and to then culture the ES cells on transformed stromal cells that secrete lif into the culture medium.

[0068] As used herein, the term "transgene" refers to a nucleic acid sequence, which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can be operably linked to one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid. Exemplary transgenes of the present invention encode, for instance an H-2 polypeptide. Other exemplary transgenes are directed to disrupting one or more HLA genes by homologous recombination with genomic sequences of an HLA gene.

[0069] A "functional transgene" is one that produces an mRNA transcript, which in turn produces a properly processed protein in at least one cell of the mouse comprising the transgene. One of skill will realize that the diverse set of known transcriptional regulatory elements and sequences directing posttranscriptional processing provide a library of options from which to direct the expression of a transgene is a host mouse. In many embodiments of the invention, expression of an HLA transgene under the control of an H-2 gene regulatory element may be preferred.

[0070] In some embodiments, the HLA class I transgene is an HLA-A2 transgene and the HLA class II transgene is an HLA-DR1 transgene. An example of an HLA-A2 transgene is one that comprises the HLA-A2 sequence provided in the sequence listing. An example of an HLA-DR1 transgene is one that comprises the HLA-DR1 sequence provided in the sequence listing.

[0071] In an embodiment, the invention provides a transgenic mouse deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene. In some embodiments, the mouse has the genotype HLA-A2⁺HLA-DR1⁺β2m°IAβ°. In other embodiments the HLA-A2 transgene comprises the HLA-A2 sequence provided in the sequence listing and the HLA-DR1 transgene comprises the HLA-DR1 sequence provided in the sequence listing.

[0072] The invention also provides isolated transgenic mouse cells. In some cases the cell comprises a disrupted H2 class I gene, a disrupted H2 class II gene, and a functional HLA class I or class II transgene. In others, the cell comprises a disrupted H2 class I gene, a disrupted H2 class II gene, a functional HLA class I transgene, and a functional HLA class II transgene. The HLA class I transgene can be an HLA-A2 transgene and the HLA class II transgene can be an HLA-DR1 transgene. In some the HLA-A2 transgene comprises the HLA-A2 sequence provided in the sequence listing and the HLA-DR1 transgene comprises the HLA-DR1 sequence provided in the sequence listing.

[0073] In an embodiment, the invention provides an isolated transgenic mouse cell deficient for both H2 class I and class II molecules, wherein the transgenic mouse comprises a functional HLA class I transgene and a functional HLA class II transgene. The isolated transgenic mouse cells can have the genotype HLA₇A2⁺HLA-DR1⁺β2m°IAβ°. The HLA-A2 transgene can comprise the HLA-A2 sequence provided in the sequence listing and the HLA-DR1 transgene can comprise the HLA-DR1 sequence provided in the sequence listing.

[0074] The isolated transgenic mouse cells of the invention can have the genotype of any mouse of the invention. However, the set of genotypes of the isolated transgenic mouse cells of the invention, and the set of genotypes of the mice of the invention are not necessarily entirely overlapping.

[0075] The isolated mouse cells of the invention can be obtained from a mouse or mouse embryo. In one embodiment, the mouse or mouse embryo has the same genotype as the cell to be obtained. In another embodiment, the mouse or mouse embryo has a different genotype than the cell to be obtained. After the cell is obtained from the mouse or mouse embryo, a gene of the cell can be disrupted by, for example, homologous recombination. Additionally, a functional transgene can be introduced into the genome of the cell by, for example, transfection. One of skill in the art will recognize that any suitable method known in the art can be applied to modify the genome of the cell to thereby obtain an isolated mouse cell having the desired genotype.

[0076] An additional object of the invention is an isolated transgenic mouse cell deficient for both H2 class I and class II molecules, wherein the transgenic mouse cell comprises a functional HLA class I transgene and a functional HLA class II transgene. In some embodiments, the transgenic mouse cell has the genotype HLA-A2⁺HLA-DR1⁺β2m°IA°. In other embodiments, the HLA-A2 transgene comprises the HLA-A2 sequence provided in the sequence listing and the HLA-DR1 transgene comprises the HLA-DR1 sequence provided in the sequence listing.

[0077] T cells play a central role in many aspects of acquired immunity, carrying out a variety of regulatory and defensive functions. When some T cells encounter an infected or cancerous cell, they recognize it as foreign and respond by acting as killer cells, killing the host's own cells as part of the cell-mediated immune response. Other T cells, designated helper T cells, respond to perceived foreign antigens by stimulating B cells to produce antibodies, or by suppressing certain aspects of a humoral or cellular immune response.

[0078] T helper cells (Th) orchestrate much of the immune response via the production of cytokines. Although generally identifiable as bearing the CD4 cell surface Marker, these cells are functionally divided into Th1 or Th2 subpopulations according to the profile of cytokines they produce and their effect on other cells of the immune system.

[0079] The Th1 cells detect invading pathogens or cancerous host cells through a recognition system referred to as the T cell antigen receptor. Termed cellular immunity, Th1-related processes generally involve the activation of non-B cells and are frequently characterized by the production of IFN-γ. Nevertheless, although the Th1 system is primarily independent from the production of humoral antibodies, Th1 cytokines do promote immunoglobulin class switching to the IgG₂a isotype.

[0080] Upon detection of a foreign antigen, most mature Th1 cells direct the release of IL-2, IL-3, IFN-γ, TNF-β, GM-CSF, high levels of TNF-α, MIP-1α, MIP-1β, and RANTES. These cytokines promote delayed-type hypersensitivity and general cell-mediated immunity. IL-2, for instance, is a T cell growth factor that promotes the production of a clone of additional T cells sensitive to the particular antigen that was initially detected. The sensitized T cells attach to and attack cells or pathogens containing the antigen.

[0081] In contrast, mature Th2 cells tend to promote the secretion of IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, 1L-13, GM-CSF, and low levels of TNF-α. In addition, the Th2 response promotes humoral immunity by activating B cells, stimulating antibody production and secretion, and inducing class switching to IgA, IgG₁ and IgE isotypes.

[0082] As used herein, an "antigen" comprises: 1) at least one HTL epitope, or 2) at least one CTL epitope or, 3) at least one B cell epitope, or 4) at least one HTL epitope and at least one CTL epitope, or 5) at least one HTL epitope and at least one B cell epitope, or 6) at least one CTL epitope and at least one B cell epitope, or 7) at least one HTL epitope and at least one CTL epitope and at least one B cell epitope. A "candidate antigen" is a molecule that is under investigation to determine whether it functions as an antigen.

[0083] A "humoral immune response" is antibody-mediated specific immunity.

[0084] An "epitope" is a site on an antigen that is recognized by the immune system. An antibody epitope is a site on an antigen recognized by an antibody. A T-cell epitope is a site on an antigen that binds to an MHC molecule. A TH epitope is one that binds to an MHC class II molecule. A cm epitope is one that binds to an MHC class I molecule.

[0085] The antigen can comprise a polypeptide sequence or a polynucleotide sequence, which can comprise RNA, DNA, or both. In one embodiment, the antigen comprises at least one polynucleotide sequence operationally encoding one or more antigenic polypeptides. Used in this context, the word "comprises" intends that at least one antigenic polypeptide is provided by the transcription and/or translation apparatus of a host cell acting upon an exogenous polynucleotide that encodes at least one antigenic polypeptide, as described, for example in U.S. Pat. Nos. 6,194,389 and 6,214,808.

[0086] Antigens of the invention can be any antigenic molecule. Antigenic molecules include: proteins, lipoproteins, and glycoproteins, including viral, bacterial, parasitic, animal, and fungal proteins such as albumins, tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacterial outer membrane proteins (including meningococcal outer membrane protein), RSV-F protein, malarial derived peptide, B-lactoglobulin B, aprotinin, ovalbumin, lysozyme, and tumor associated antigens such as carcinoembryonic antigen (CEA), CA 15-3, CA 125, CA 19-9, prostrate specific antigen (PSA), and the TAA complexes of U.S. Pat. No. 5,478,556, which is incorporated herein by reference in its entirety; carbohydrates, including naturally-occurring and synthetic polysaccharides and other polymers such as ficoll, dextran, carboxymethyl cellulose, agarose, polyacrylamide and other acrylic resins, poly (lactide-co-glycolide), polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, polyvinyipryrolidine, Group B Steptococcal and Pneumococcal capsular polysaccharides (including type III), Pseudomonas aeruginosa mucoexopolysaccharide, and capsular polysaccharides (including fisher type I), and Haemophilus influenzae polysaccharides (including PRP); haptens, and other moieties comprising low molecular weight molecules, such as TNP, saccharides, oligosaccharides, polysaccharides, peptides, toxins, drugs, chemicals, and allergens; and haptens and antigens derived from bacteria, rickettsiae, fungi, viruses, parasites, including Diphtheria, Pertussis, Tetanus, H. influenzae, S. pneumoniae, E. Coli, Klebsiella, S. aureus, S. epidermidis, N. meningiditis, Polio, Mumps, measles, rubella; Respiratory Syncytial Virus, Rabies, Ebola; Anthrax; Listeria, Hepatitis A, B, C, Human Immunodeficiency Virus I and II, Herpes simplex types 1 and 2, CMV, EBV, Varicella Zoster, Malaria, Tuberculosis, Candida albicans, and other candida, Pneumocystis carinii, Mycoplasma, Influenzae virus A and B, Adenovirus, Group A streptococcus, Group B streptococcus, Pseudomonas aeryinosa, Rhinovirus, Leishmania, Parainfluenzae, types 1, 2 and 3, Coronaviruses, Salmonella, Shigella, Rotavirus, Toxoplasma, Enterovirusses, and Chlamydia trachomatis and pneumoniae.

[0087] As used herein, a pharmaceutical composition or vaccine comprises at least one immunological composition, which can be dissolved, suspended, or otherwise associated with a pharmaceutically acceptable carrier or vehicle. Any pharmaceutically acceptable carrier can be employed for administration of the composition. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, 18th Edition (A. Gennaro, ed., 1990) Mack Pub., Easton, Pa., which is incorporated herein by reference in its entirety. Carriers can be sterile liquids, such as water, polyethylene glycol, dimethyl sulfoxide (DMSO), oils, including petroleum oil, animal oil, vegetable oil, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Carriers can be in the form of mists, sprays, powders, waxes, creams, suppositories, implants, salves, ointments, patches, poultices, films, or cosmetic preparations.

[0088] Proper formulation of the pharmaceutical composition or vaccine is dependent on the route of administration chosen. For example, with intravenous administration by bolus injection or continuous infusion, the compositions are preferably water soluble, and saline is a preferred carrier. For transcutaneous, intranasal, oral, gastric, intravaginal, intrarectal, or other transmucosal administration, penetrants appropriate to the barrier to be permeated can be included in the formulation and are known in the art. For oral administration, the active ingredient can be combined with carriers suitable for inclusion into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like. Time-sensitive delivery systems are also applicable for the administration of the compositions of the invention. Representative systems include polymer base systems, such as poly(lactide-glycoside), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid and polyanhydrides. These and like polymers can be formulated into microcapsules according to methods known in the art, for example, as taught in U.S. Pat. No. 5,075,109, which is incorporated herein by reference in its entirety. Alternative delivery systems appropriate for the administration of the disclosed immunostimulatory compounds of the invention include those disclosed in U.S. Pat. Nos. 6,194,389, 6,024,983 5,817,637, 6,228,621, 5,804,212, 5,709,879, 5,703,055, 5,643,605, 5,643,574, 5,580,563, 5,239,660, 5,204,253, 4,748,043, 4,667,014, 4,452,775, 3,854,480, and 3,832,252 (each of which is incorporated herein by reference in its entirety).

[0089] Aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable or aerosol solutions. For administration by aerosol, as by pressurized spray or nebulizer, suitable propellants can be added as understood by those familiar with the art. The immunological composition can also be formulated with solubilizing agents; emulsifiers; stabilizers; dispersants; flavorants; adjuvants; carriers; topical anesthetics, such as lidocaine, xylocaine, and the like; antibiotics; and known or suspected anti-viral, anti-fungal, anti-parasitic, or anti-tumor compounds.

[0090] An "adjuvant" is a composition that promotes or enhances an immune response to a target antigen. One of skill in the art can select an appropriate adjuvant for use in practicing the present invention in view of the disclosure herein.

[0091] The present invention encompasses methods of treating a patient in need of immune stimulation by administering a composition comprising one or more antigens of the invention. As used herein, treatment encompasses corrective, restorative, ameliorative, and preventive methods relating to any disease, condition, abnormality, or symptom. Treatment further encompasses the elicitation or suppression of an immune response in an experimental animal or ex vivo.

[0092] Thus, treatment comprises administering an immunostimulatory amount of any of the immunostimulatory compositions of the invention by any method familiar to those of ordinary skill in the art, commonly including oral and intranasal routes, and intravenous, intramuscular, and subcutaneous injections, but also encompassing, intraperitoneal, intracorporeal, intra-articular, intraventricular, intrathecal, topical, tonsillar, mucosal, transdermal, intravaginal administration and by gavage.

[0093] As is recognized by the skilled practitioner, choosing an appropriate administration method may contribute to the efficacy of a treatment, and local administration may be preferred for some applications. Acceptable routes of local administration include subcutaneous, intradermal, intraperitoneal, intravitreal, inhalation or lavage, oral, intranasal, and directed injection into a predetermined tissue, organ, joint, tumor, or cell mass. For example, mucosal application or injection into mucosal lymph nodes or Peyer's patches may promote a humoral immune response with substantial IgA class switching. Alternatively, targeted injection into a lesion, focus, or affected body site may be applicable for the treatment of solid tumors, localized infections, or other situs requiring immune stimulation.

[0094] Alternatively, cells of the immune system (e.g., T cells, B cells, NK cells, or oligodendrocytes) can be removed from a host and treated in vitro. The treated cells can be further cultured or reintroduced to a patient (or to a heterologous host) to provide immune stimulation to the patient or host. For example, bone marrow cells can be aspirated from a patient and treated with an HDR to stimulate global or specific immunity. High-dose radiation, or comparable treatments, can then be used to destroy. the remaining immune cells in the patient. Upon re-implantation, the autologous stimulated cells will restore normal immune function in the patient. Alternatively, NK and/or T cells isolated from a patient suffering from cancer may be exposed in vitro to one or more antigens specific to the patient's cancer. Upon re-implantation into the patient, the antigen-stimulated cells will deploy a vigorous cellular immune response against the cancerous cells.

[0095] An immunostimulatory (efficacious) amount refers to that amount of vaccine that is able to stimulate an immune response in a patient, which is sufficient to prevent, ameliorate, or otherwise treat a pathogenic challenge, allergy, or immunologic abnormality or condition. An immunostimulatory amount is that amount, which provides a measurable increase in a humoral or cellular immune response to at least one epitope of the antigen as compared to the response obtained if the antigen is administered to the patient without prior treatment with the vaccine: Thus, for example, an immunostimulatory amount refers to that amount of an antigen-containing composition that is able to promote the production of antibodies directed against an antigenic epitope of interest or stimulate a detectable protective effect against a pathogenic or allergenic challenge or to promote a protective CTL response against an antigenic epitope of interest.

[0096] Treatment with an immunostimulatory amount of an antigen-containing composition of the invention comprises effecting any directly, indirectly, or statistically observable or measurable increase or other desired change in the immune response in a host, specifically including an ex vivo tissue culture host, comprising at least one cell of the immune system or cell line derived therefrom. Host cells can be derived from human or animal peripheral blood, lymph nodes or the like. Preferred tissue culture hosts include freshly isolated T cells, B cells, macrophages, oligodendrocytes, NK cells, and monocytes, each of which can be isolated or purified using standard techniques. Observable or measurable responses include, B or T cell proliferation or activation; increased antibody secretion; isotype switching; increased cytokine release, particularly the increased release of one or more of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, GM-CSF, IFN-γ, TNF-α, TNF-β, GM-CSF, MIP-1α, MIP-1β, or RANTES; increased antibody titer or avidity against a specific antigen; reduced morbidity or mortality rates associated with a pathogenic infection; promoting, inducing, maintaining, or reinforcing viral latency; suppressing or otherwise ameliorating the growth, metastasis, or effects of malignant and non-malignant tumors; and providing prophylactic protection from a disease or the effects of a disease.

[0097] Where the suppression of an immunological response is desired, for example, in the treatment of autoimmune disease or allergy, an effective amount also encompasses that amount sufficient to effect a measurable or observable decrease in a response associated with the condition or pathology to be treated.

[0098] The amount of an antigen-containing composition to be administered and the frequency of administration can be determined empirically and will take into consideration the age and size of the patient being treated, and the condition or disease to be addressed. An appropriate dose is within the range of 0.01 μg to 100 μg per inoculum, but higher and lower amounts may also be indicated. Secondary booster immunizations can be given at intervals ranging from one week to many months later.

[0099] The following examples demonstrate certain embodiments of the invention. One of ordinary skill in the art will recognize, the numerous modifications and variations that may be performed without altering the spirit or scope of the present invention. Such modifications and variations are believed to be encompassed within the scope of the invention. The examples do not in any way limit the invention.

EXAMPLES

[0100] The following experimental techniques and reagents were used to demonstrate certain nonlimiting embodiments of the invention.

[0101] Transgenic Mice

[0102] The HLA-DR1-transgenic H-2 class II-KO (IA β^b°) mice were obtained at the Institut Pasteur of Lille by crossing HLA-DR1-transgenic mice (Altmann, D. M. et al., J Exp Med 181, 867-875 (1995)) with H-2 class II-KO (IA β^b°) mice (Rohrlich, P. S. et al., Int Immunol 15, 765-772 (2003)). The HLA-A2.1-transgenic mice, expressing a chimeric monochain (HHD molecule: α1-α2 domains of HLA-A2.1, α3 to cytoplasmic domains of H-2 D^b, linked at its N-terminus to the C terminus of human β2m by a 15 amino-acid peptide linker) were created (Pascolo, S. et al., J Exp Med 185, 2043-2051 (1997)). HLA-A2.1 (HHD)-transgenic H-2 class I-KO and HLA-DR1-transgenic H-2 class II-KO (IA β^b°) mice were intercrossed and progenies screened until HLA-A2.1⁺/-/HLA-DR1⁺/- double transgenic H-2-class I (β2m⁰)-/class II (IAβ⁰)-KO animals were obtained and used for the experiments described herein. HLA-A2.1⁺/- single transgenic H-2-class I (β2m⁰)-/class II (IAβ⁰)-KO mice were used as controls in the protection assays. Mice were bred in the animal facilities at the Institut Pasteur, Paris; all protocols were reviewed by the Institut Pasteur competent authority for compliance with the French and European regulations on Animal Welfare and with Public Health Service recommendations.

[0103] Genotyping

[0104] The HLA-DRB1*0101, HLA-DRA*0101 and HLA-A*0201 transgenes were detected by PCR. Tall-DNA was extracted after overnight incubation at 56° C. in 100 mM NaCl, 50 mM Tris-HCl pH 7.2, 100 mM EDTA, 1% SDS and 0.5 mg/ml proteinase K, followed by the addition of 250 μl of saturated NaCl solution and isopropanol precipitation. The samples were washed (3×) in 70% ethanol and resuspended in 150 μl of 10 mM Tris-HCl, 1 mM EDTA pH 8. PCR conditions were: 1.5 mM MgCl₂, 1.25 U of Taq Polymerase, buffer supplied by the manufacturer (InVitrogen, Carlsbad, Calif.), 1 cycle (7 min, 94° C.), 40 cycles (30 sec, 94° C.; 30 sec, 60° C.; 1 min, 72° C.), 1 cycle (4 min, 72° C.), using as forward and reverse primers, for HHD: 5'CAT TGA GAC AGA GCG CTT GGC ACA GAA GCA G 3' and 5'GGA TGA CGT GAG TM ACC TGA ATC TTT GGA GTA CGC 3', for HLA-DRB1*0101: 5' TTC TIC MC GGG ACG GAG CGG GTG 3' and 5' CTG CAC TGT GAA GCT CTC ACC MC 3', and for HLA-DRA*0101: 5' CTC CM GCC CTC TCC CAG AG 3' and 5' ATG TGC CTT ACA GAG GCC CC 3'.

[0105] FACS Analysis

[0106] Cytofluorimetry studies were performed on red-blood cell-depleted, Lympholyte M-purified (Tebu-bio, Le Perray en Yvelines, France) splenocytes using FITC-conjugated W6/32 (anti-HLA-ABC, Sigma, St Louis, Mo.) and biotinilated anti-28-8-68 (anti-H-2 K^b/D^b, BD Biosciences, San Diego, Calif.) m.Ab. CD4⁺ and CD8⁺ T lymphocytes were stained using PE-labeled CT-CD4 anti-mouse CD4 (CALTAG, South San Francisco, Calif.) and FITC-labeled 53-6.7 anti-mouse CD8 m.Ab (BD Biosciences). Analysis of MHC class II molecule expression was performed on B220⁺ B lymphocytes positively selected on MS columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Following saturation of Fc receptors with 2.4G2 m.Ab, expression of HLA-DR1 and H-2 IA^b was analyzed using FITC-labeled L243 (anti-HLA-DR) and PE-labeled AF6-120.1 (anti-H-2 IAβ^b) m.Ab (BD Biosciences). Paraformaldehyde fixed cells were analyzed with a FACSCalibur (Becton Dickinson, Bedford, Mass.).

[0107] Immunoscope Analyses

[0108] CD4⁺ and CD8⁺ T cells from naive mice were positively selected on Auto-Macs (Miltenyl Biotec), RNA prepared using RNA Easy Kit (Qiagen, Hilden, Germany) and used for cDNA synthesis. The cDNA was PCR-amplified using forward primers specific for each BV segment family and a reverse primer shared by the two BC segments. PCR-products were subjected to a run-off-elongation with internal BC FAM-tagged primer. The run-off products were loaded on a 6% acrylamide/8 M urea gel for separation (7 h, 35 W) with a 373A DNA sequencer (Perkin Elmer Applied Biosystem, Foster City, Calif.). Data were analyzed using immunoscope software (Pannetier, C. et al., Proc Natl Acad Sci USA 90, 4319-4323 (1993)).

[0109] Peptides

[0110] The HLA-A2 binding peptides HBsAg_348-357GLSPTVWLSV and HBsAg_335-343 WLSLLVPFV, the H-2 K^b binding peptide HBsAg_371-378 ILSPFLPL, the HLA-DR1 binding peptide HBsAg_180-195 QAGFFLLTRILTIPQS, the H-2 IA^b binding peptide HBsAg_126-138RGLYFPAGGSSSG and the preS2 peptide HBsAg_109-134 MQWNSTTFHQTLQDPRVRGLYFPAGG were synthesized by Neosystem (Strasbourg, France) and dissolved in PBS-10% DMSO at a concentration of 1 mg/ml. The numbering of the amino acid sequence of peptides starts from the first methionine of the HBV ayw subtype preS1 domain.

[0111] Immunization with DNA Encoding the S2-S Proteins of HBV

[0112] The pCMV-S2.S plasmid vector (Michel, M. L. et al., Proc Natl Acad Sci USA 92, 5307-5311 (1995)) coding for the preS2 and the S HBV surface antigens expressed under the control of the human CMV immediate early gene promotor was purified on Plasmid Giga Kit columns under endotoxin free conditions (Qiagen). Anesthesized mice were injected (50 μg each side) into regenerating tibialis anterior muscles, as previously described (Davis, H. L., Michel, M. L. & Whalen, R. G., Hum Mol Genet 2, 1847-1851 (1993)).

T Cell Proliferation Assay

[0113] Twelve days after the last immunization, red-blood cell-depleted, Ficoll-purified splenocytes (5.10⁸ cells/ 25 cm² culture flask (Techno Plastic Products (TPP), Trasadingen, Switzerland)) were co-cultured with peptide-pulsed-(20 μg/ml), γ-irradiated (180 Gy) LPS-blasts (5.10⁶ cells/ culture flask) in RPMI medium supplemented with 10% FCS, 10 mM HEPES, 1 mM sodium pyruvate, 5×10^-5 M 2-mercaptoethanol, 100 I.U/ml penicillin and 100 μg streptomycin, as described (Loirat, D., Lemonnier , F. A. & Michel, M. L., J Immunol 165, 4748-4755 (2000)). On day 7, for proliferation assays, cells were plated (5×10⁵ cells/well of flat bottomed 96 well microplates, (TPP)) with peptide-pulsed irradiated LPS-Blasts (2×10⁵ cells/well) for 72 h in complete RPMI medium supplemented with 3% FCS. Cells were pulsed for the final 16 h with 1 μCi of (³H)-thymidine per well before being harvested on filtermates with a TOMTEC collector (Perkin Elmer Applied Biosystem), and incorporated radioactivity was measured on a micro-β counter (Perkin Elmer Applied Biosystem). Results are given as stimulation index (SI)=cpm with specific peptide/cpm with irrelevant peptide.

[0114] Measurement of CTL Activity

[0115] Cytotoxicity assays were performed on the same immune splenocyte populations as the proliferation assays. Responder cells (5.10⁶ cells/25 cm² culture flask, TPP) and stimulating peptide-pulsed (20 μg/ml), γ-irradiated (180 Gy) LPS-blasts (5.10⁶ cells/ culture flask) were co-cultured for 7 days in the same supplemented RPMI medium as for proliferation assays. Cytolytic activity was tested in a standard 4 h ⁵¹Cr assay against RMA-S HHD target cells pulsed with 10 μg/ml of the experimental or control peptides. Specific lysis, in %, was calculated in duplicates, according to: [experimental-spontaneous release]/[maximal-spontaneous release]×100, subtracting the non-specific lysis observed with the control peptide.

[0116] Measurement of In Vivo Antibody Production

[0117] At various times before and after DNA injection, blood was collected from mice by retrobulbar puncture with heparinized glass pipettes, and sera recovered by centrifugation were assayed for anti-HBs and anti-preS2 by specific ELISA. Purified recombinant particles containing HBV small S protein (1 ug/ml) or preS2 (120-145) synthetic peptide (1 ug/ml) were used as the solid phase. After blocking with PBST (PBS containing 0.1% Tween 20) supplemented with 10% FCS, serial dilutions were added. After extensive washing, the bound antibodies were detected with anti mouse Ig (total IgG) labeled with horseradish peroxidase (Amersham, Little Chalfont, UK). Antibody titers were determined by the serial end-point dilution method. Mouse sera were tested individually, and titers were the mean of at least three determinations. Serum dilutions below 1/100 were considered negative.

[0118] Antibody Titration

[0119] Sera from immunized mice were individually assay_ed by ELISA (Michel, M. L. et al., Proc Natl Acad Sci USA 92, 5307-5311 (1995)) on either purified HBV middle and small proteins or preS2 synthetic HBS_109-134. peptide, After blocking with PBS 1× supplemented with 0.1% Tween 20, 10%. FCS and washings (×3), bound antibodies were detected with horseradish peroxidase-labeled anti-mouse IgG (Amersham, Little Chalfont, UK). Antibody titers (means of at least 3 determinations) were determined by the serial end-point dilution method. Titers below 1/100 were considered negative.

[0120] Vaccinia Challenge and Plaque Assay

[0121] DNA-injected mice were challenged intraperitoneally 12 days post fast injection with 10⁷PFU of recombinant vaccinia virus (Western Reserve strain) expressing either the HbsAg (Smith, G. L., Mackett, M. & Moss, B., Nature 302, 490-495 (1983)) or the HBx protein (Schek, N., Bartenschlager, R., Kuhn, C. & Schaller, H., Oncogene 6, 1735-1744. (1991)) kindly provided, respectively, by Dr B. Moss and Dr H. Schaller. Four days later, ovaries were assayed for rVV titers by plaque assay on BHK 21 cells (Buffer, R. M. & Wallace, G. D., Lab Anim Sci 35, 473-476 (1985).

Example 1

Cell Surface Expression of MHC Molecules

[0122] Cell surface expression of the HLA-A2.1, H-2 K^b/D^b, HLA-DR1, and H-2 IA^b molecules was evaluated on splenocytes by flow cytometry. As illustrated in FIG. 1a, a similar level of HLA-A2.1 expression was observed in HLA-A2.1-/HLA-DR1-transgenic, H-2 class I-/class II-KO mice and HLA-A2.1-transgenic, H-2 class I-KO mice, while HLA-A2.1 was absent and H-2 K^b/D^b expressed exclusively in HLA-DR1-transgenic, H-2 class II-KO mice. Cell surface expression of HLA-DR1 and H-2 IA^b was measured on B220⁺-enriched B cells. As shown in FIG. 1b, a similar level of HLA-DR1 expression was observed in HLA-A2.1-/HL4-DR1-transgenic, H-2 class I-/class II-KO mice and HLA-DR1-transgenic, H-2 class II-KO mice, whereas no expression was detected in HLA-A2.1-transgenic, H-2 class I-KO mice. Cell surface expression of the transgenic molecules (especially HLA-DR1) was, however, lower than the expression of endogenous H-2 class I and class II molecules.

Example 2

Peripheral CD4⁺ and CD8⁺ T Cells

[0123] CD4⁺ and CD8⁺ splenic T cell numbers were determined by immunostaining and flow cytometry analysis as illustrated in FIG. 2a.

[0124] CD4⁺ T cells represented 13-14% of the splenocyte population in both HLA-A2.1-/HLA-DRI-transgenic, H-2 class I-/class II-KO mice and HLA-DR1-transgenic, H-2 class II-KO mice. In contrast, only 2-3% of the cells were CD4⁺ in H-2 class II-KO mice (data not shown), in agreement with the initial report on mice lacking MHC class II molecules (Cosgrove, D. et al., Cell 66, 1051-1066 (1991)). As expected, expression of transgenic HLA-A2.1 molecules led to an increase in the size of the peripheral CD8⁺ T cell population, which reached 2-3% of the total splenocytes in both HLA A2.1/HLA-DR1-transgenic, H-2 class I-/class II-KO mice and HL4A2.1-transgenic, H-2 class I-KO mice, compared to 0.6-1% in the β2 microglobulin (β2m)-KO MHC class I-deficient mice (Pascolo, S. et al., J Exp Med 185, 2043-2051 (1997)).

[0125] The results presented in Examples 1 and 2 show that: [0126] (1) In the HLA-A2⁺HLA-DR1⁺ β2m°IAβ° mouse, the expression of HLA-A2 molecules, the absence of expression of H2-K^b molecules, the number of CD8⁺ peripheral T-lymphocytes, and the diversity of the CD8⁺ T repertoire are generally comparable to the HLA-A2⁺ β2m° mouse; [0127] (2) In the HLA-A2⁺HLA-DR1⁺ β2m°IAβ° mouse, the expression of HLA-DR1 molecules, the absence of expression of H2-IA^b molecules, the number of CD4⁺T-Iymphocytes, and the diversity of the CD4⁺ repertoire are generally comparable to the HLA-DR1⁺IAβ mouse; and [0128] (3) The HLA-A2⁺HLA-DR1⁺β2m°IAβ° mouse has all the characteristic advantages found in HLA-A2⁺ β2m° mice, and the HLA-DR1⁺IA β° mice.

Example 3

TCR BV Segment Usage

[0129] As the presence of a single MHC class I and single MHC class II molecule could diminish the size and diversity of the TCR repertoire, the expression of the various BV families and the CDR3 length diversity was studied as previously described (Cochet, M. et al., Eur J Immunol 22, 2639-2647 (1992)) by the RT-PCR-based immunoscope technique, on purified splenic CD4⁺ or CD8⁺ T cells. Peaks of significant magnitude with a Gaussian-like distribution were observed for most BV families (15 out of the 20 analyzed) in both CD8⁺ (FIG. 2b) and CD4⁺ (FIG. 2c) populations of T cells. Such profiles observed on peripheral T lymphocytes are typical of functionally rearranged BV segments with a 3 nucleotide length variation of the CDR3 subregions from one peak to the next (Cochet, M. et al., Eur J Immunol 22, 2639-2647 (1992)).

[0130] Absence of expansion (or profoundly altered profile) as observed for BV 5.3 and 17 were expected since these two BV segments are pseudogenes in C57BU6 mice (Wade, T., Bill, J., Marrack, P. C., Palmer, E. & Kappler, J. W., J Immunol 141, 2165-2167 (1988)); Chou, H. S. et al., Proc Natl Acad Sci USA 84, 1992-1996 (1987). However, the altered profiles observed for BV 5.1, 5.2 and 11 segments were due to a small subpopulation of corresponding BV-expressing T cells (they represent lower than 5% in C57BU6 mice, and around 2% in HLA-DR1-transgenic class II-KO mice) (data not shown). Other than these instances, both CD4⁺ and CD8⁺ T cells in HLA-A2.1-/HLA-DR1-transgenic, H-2 class I-/class II-KO mice display, respectively, a pattern of TCR BV chain usage and CDR3 diversity, which is similar to that of non-transgenic C57BL/6 mice.

Example 4

Functional Characterization

[0131] HLA-A2⁺ HLA-DR1⁺ β2m°IAβ° mice immunized with Ag HBs (hepatitis B envelope protein) were analyzed. FIG. 5 shows the specific humoral response, as indicated by the production of HBs S2 antibodies. FIG. 6 shows the specific DR1-restricted CD4⁺ T proliferation response of HBS_348-357. And FIG. 7 shows the specific HLA-A2-restricted CD8⁺ cytolytic T response of the HBs_348-357 or HBs_335-343.

[0132] These results show that the HLA-A2⁺HLA-DR1⁺ β2m°IAβ° mouse allows for simultaneous analysis of the specific humoral response, of the Ag-specific HLA-DR1 -restricted response of CD4⁺ T helper cells, and of the cytolitic response of Ag-specific HLA-A2-restricted CD8⁺ T cells in an immunized individual.

[0133] Additional data obtained from these mice is provided in the following Tables 1-3.

TABLE-US-00001 TABLE 1 Proliferative responses of T CD4+ against HBV virus envelope HLA-DR1 epitopes from HLA-A2 + DR1 + H-2 CI-CII-transgenic mice injected with pcmv S2-S Respon- der/ Stimu- tested lation position Amino Acid sequence mice index 109-134 MQWNSTTFHQTLQDPRVRGLYFPAGG (12/12) 3-4 200-214 TSLNFLGGTTVCLGQ (6/12) 3-4 16/31 QAGFFLLTRILTIPQS (12/12) 3-6 337/357 SLLVPFVQWFVGLSPTVWLSV (5/12) 4-5

TABLE-US-00002 TABLE 2 Cytolytic response to HLA-A2 + DR1 + H-2 CI-CII-transgenic mice injected with pcmv S2-S Amino Acid Responder/ Maximal position sequence tested mice lysis 348-357 GLSPTVWLS (12/12) 20-70% 335-343 WLSLLVPVF (4/12) 30%

TABLE-US-00003 TABLE 3 Anti-PreS2 Antibody response anti of HLA-A2 + DR1 + H-2 CI-II transgenic mice injected with pcmv S2-S Responder/ position Amino Acid sequence tested mice preS2 MQWNSTTFHQTLQDPRVRGLYFPAGG (9/12)

Example 5

Immune Response to HBsAg-DNA-Vaccine

[0134] To evaluate the immunological potential of HLA-A2.1-/HLA-DR1-transgenic, H-2 class I-/class II-KO mice, and to compare their humoral, CD4⁺ and CD8⁺ T cell responses to those of humans, mice were immunized with an HBsAg-DNA plasmid. This plasmid encodes two hepatitis B virus envelope proteins (preS2/S middle and S/small) that self-assemble in particles carrying hepatitis B surface antigen. The currently used vaccine against hepatitis B comprises these two proteins.

[0135] As illustrated in FIG. 3a for a representative mouse, HBsAg-specific antibodies were first detected at day 12 after Injection of the HBsAg-DNA-vaccine (FIG. 3a, upper panel), and the titer of these antibodies increased up to day 24 (12 days after the second DNA immunization, data non shown). This early antibody response was specific for the preS2-B cell epitope (HBs_109-434) carried by the middle HBV envelope protein and for HBsAg particles, in agreement with a similar response reported in HBsAg-DNA-Immunized mice (Michel, M. L. et al., Proc Natl Aced Sci USA 92, 5307-5311 (1995)) and in HBsAg vaccinated humane (Moulia-Pelat, J. P. et al., Vaccine 12, 499-502 (1994)).

[0136] The CD8⁺ CTL response to HBsAg was examined to determine whether the CD8⁺ T cells in the periphery of the HLA-A2.1-/HLA-DRI-transgenic, H-2 class I-/class II-KO mouse were functionally restricted by the transgenic human class I molecules. In HBV-infected HLA-A2.1⁺ humans, the immunodominant HLA-A2.1-restricted HBsAg-specific CTL response is directed at the HBsAg_348-357 (Maini, M. K. et al., Gastroenterology 117, 1386-1396 (1999)) and at the HBsAg_335-343 (Nayersina, R. et al., J Immunol 150, 4659-4671 (1993)) peptide (i.e., a multi-epitopic response is observed). In C57BL/6 mice, the H-2 K^b-restricted HBsAg-specific CTL response is directed at the HBsAg_371-378 peptide (Schirmbeck, R., Wild, J. & Reimann, J., Eur J Immunol 128, 4149-4161 (1998)). To evaluate whether the humanized mouse may respond as humans, splenic T cells were restimulated for 7 days, as described herein, with either relevant (HBsAg_348-357, HLA-A2.1-restricted), or control (HBsAg_371-378, H-2 K^b-restricted; MAGE-3_271-279, HLA-A2.1-restricted) peptide. FIG. 3a (middle panel) shows that HBsAg-DNA-immunization elicited a strong HBsAg₃₄-357-specific CTL response, but no response to either HBsAg_371-378 or the MAGE-3_271-279 peptide.

[0137] To determine whether the CD4' T cells in the periphery of this HLA-A2.1-/HLA-DR1-transgenic, H-2 class I-/class II-KO mouse may be functionally restricted by the transgenic human class II molecules, the CD4⁺ T cell response to the HBsAg protein was examined. In HBsAg-vaccinated or HBV-Infected HLA-DR1⁺ humans, an immunodominant HLA-DR1-restricted HBsAg-specific CD4⁺ T cell response is directed at the HBsAg_180-195 peptide (Mm, W. P. et al., Hum Immunol 46, 93-99 (1996)). In C57BL6 mice, the H-2 IA^b-restricted HBsAg-specific CD4⁺ T cell response is directed at the HBsAg_126-138 peptide (Milich, D. R., Semin Liver Dis 11, 93-112(1991)). To compare the humanized mouse with humans and wild-type mice, splenic T cells were restimulated in vitro with either relevant (HBsAg_180-195, HLA-DR1-restricted) or control (HBsAg_126-138, H-2 IA^b-restricted; HIV 1 Gag_263-278, HLA-DR1-restricted) peptides. FIG. 3a (lower panel) shows a strong proliferative response directed against the HLA-DR1-restricted HBsAg_180-195 peptide, while the H-2 IA.sup.quadrature-restricted peptide was not efficient at stimulating a response, as expected. Similarly, no response was induced by the HIV 1 Gag_263-278 peptide. Moreover, an additional in vitro recall with the HBsAg_180-195 peptide increased several-fold the specific proliferative index (data not shown).

[0138] Having documented in a first HBsAg-DNA-Immunized HLA A2.1/HLA-DR1-transgenic H-2 class I-/class II-KO mouse the development and the specificity of the HBsAg-specific antibody, proliferative and cytolytic T cell responses, 6 additional HBsAg-DNA-Immunized and 6 naive control HLA A2.1-/HLA-DR1-transgenic H-2 class I-/class II-KO mice were also tested individually for the same three responses. As illustrated in FIG. 3b, the three reponses were simultaneously documented in the 6 immunized animals tested and not in control naive mice. Interestingly, 2 immunized mice were able to develop CTL responses against both HBsAg_348-357 and HBsAg_335-343 HLA-A2.1 restricted peptides (FIG. 3b, middle panel).

Example 6

Protection Assays

[0139] The above examples document the induction of HBsAg-specific humoral, CD4⁺ and CD8⁺ T cell responses in HLA-A2.1-/HLA-DR1-transgenic, H-2 class I-/class II-KO mice, and show that they are directed at the same immunodominant epitopes as those of naturally-infected or HBsAg-vaccinated humans. This example tested whether these responses conferred protection to vaccinated animals. Since mice are not permissive to HBV, a HBsAg-recombinant vaccinia virus (rVV-HBsAg) was used for these experiments. Mice were immunized twice intramuscularly with 100 μg of HBsAg-DNA. Twelve days after the last immunization, mice were challenged intraperitoneally with 10⁷ PFU of rVV-HBsAg. Four days later, virus titers were determined according to published methods and recorded as rVV PFU/ovary (Buller, R. M. & Wallace, G. D., Lab Anim Sci 35, 473-476 (1985)).

[0140] The results are illustrated in FIG. 4. Naive animals that had not been immunized with HBsAg-DNA showed evidence of rVV-HBsAg replication after challenge. In contrast, the virus titers in mice immunized with HBsAg-DNA were more than 4 orders of magnitude lower. These results strongly suggest that vaccination with HBsAg-DNA induced protective HBsAg-specific immune responses that controlled the infection with rVV-HBsAg.

[0141] The specificity of the protection conferred by HBsAg-DNA-vaccination was documented by challenging HBsAg-DNA-immunized mice with another HBx-recombinant VV (encoding hepatitis Bx protein). No reduction of rVV-HBx replication was observed in HBsAg-DNA-immunized mice compared to unimmunized controls.

Example 7

HLA-DR1-Restricted CD4⁺ T Cells Are Critical for Antibody and CTL Responses and Protection Against Viral Infection

[0142] To evaluate whether HLA-DR1-restricted T helper lymphocytes contribute to antibody and CTL responses in the humanized mice, the immune response and the efficiency of viral infection were compared in single (HLA-A2.1) and double (HLA-A2.1/HLA-DR1) transgenic, H-2 class I-/class II-KO mice. As shown in Table 4, a potent HBsAg_348-357-specific CTL response was observed in HLA-A2.1-/HLA-DR1-double transgenic, H-2 class I/class II-KO mice, but not in HLA-A2.1-single transgenic H-2 class I-/class II-KO mice. Furthermore, anti-HBs antibodies could not be detected in HBsAg-DNA-vaccinated HLA A2.1-single transgenic H-2 class I-/class II-KO mice. As a consequence, HBsAg-DNA-immunized HLA-A2.1-single transgenic H-2 class I-/class II-KO mice were not protected against rVV-HBsAg infection.

TABLE-US-00004 TABLE 4 Table 4 Antibody, cytolytic, and proliferative responses of HBsAg-DNA-immunized mice, and protection against rVV-HBsAg-challenge Specific Lysis Proliferation rVV-HBsAg (%) (SI) Antibody PFU/ovary Mice 348-357 335-343 179-194 Titer (log10) A 1 0 0 1 0 2.5 10⁸ 2 0 0 1 0 2.5 10⁸ 3 0 0 1 0 10⁸ 4 0 0 1 0 2.5 10⁸ 5 0 0 1 0 10⁸ 6 0 0 1 0 1.5 10⁸ B 1 30 15 4.7 2000 10⁴ 2 14 0 3.9 3000 3 10³ 3 30 11 4 7500 4 10³ 4 5 0 2.5 6500 7.5 10³ 5 50 30 6.3 13000 7.5 10² 6 40 18 4 16000 5 10³ 7 6 7 2.9 1500 2 10⁴ 8 5 5 3 2500 1.5 10⁴ 9 24 36 4.5 3000 <10² 10 23 14 5 15000 5 10³ C 1 0 0 1 0 10⁸ 2 0 0 1 0 2 10⁸ 3 0 0 1 0 1.5 10⁸ 4 0 0 1 0 10⁸ 5 0 0 1 0 2.5 10⁸ 6 0 0 1 0 10⁸

Naive HLA A2.1-/HLA-DR1-double transgenic H-2 class I-/class II-KO mice (A 1-6), HBsAg-DNA-immunized HLA A2.1/HLA-DR1-double transgenic H-2 class I-/class II-KO mice (B 1-10) and HBsAg-DNA-immunized HLA-A2.1-single transgenic H-2 class I-/class II-KO mice (C 1-6) were challenged intraperitoneally with 10⁷ PFU of rVV-HBsAg. Four days later, PFU per ovary, cytolytic and proliferative splenic T cell responses and serum antibody titers were assessed individually using either HBsAg_348-357, (immunodominant) or HBsAg_335-343 (subdominant), HLA-A2.1-restricted peptides-loaded RMAS-HHD target cells (E/T ratio 30/1) for cytolytic assays, HBsAg_179-194 HLA-DR1-restricted peptide for proliferation assays and preS2_109-134 peptide for the determination of antibody (IgG) titers.

[0143] The entire contents of all references, patents and published patent applications cited throughout this application are herein incorporated by reference In their entirety.

Sequence CWU 1

2014547DNAArtificial SequenceDescription of Artificial Sequence Synthetic construct 1gaattcttag gtttaaatac attgttttat ggattttaat acatccatct acagagccta 60gcagggtgtc cttggcagtt gtcttttaat acctcatgtg ggtctgccta aaaactaatt 120ttttatgtta atcaggttta aaaaatacta agtgttccta taaaatatac acaacactta 180gaagtggata cttcctaaaa acaggcagtg catgagcact agtgaggggc attgtgagtg 240cattgaacag ttgcaacttt gaggtgaata aagcctgtaa tcgcttctgg ttgcaacata 300taggaacaca gtcgctactt tgtattgagg agatgtcctg gactcacaca gaaactcaga 360gctatggaat gatggtaaat ttaaaatact acaaccagga gtcacagata cattgtctgg 420gaaactgcaa cttagtagct ttgtgagtcc tgttgtaagg cttttggaca catttataca 480tcaaggggct aaagtcacat tttttaccta ttagattcct gatcattcag gggttaccaa 540gattctgcta cccactgtag ttaataaaca aagagcaaat tggtctctat tctgtctcat 600gcactcaggc gcaactcttc ccgattaaaa acaaaaacaa caacaacaaa aatctacacc 660tccattccca gagcaagctt actctctggc accaaactcc atgggatgat ttttcttcta 720gaagagtcca ggtggacagg taaggagtgg gagtcaggga gtccagttca gggacagaga 780ttacgggata aaaagtgaaa ggagagggac ggggcccatg ccgagggttt ctcccttgtt 840tctcagacag ctcttgggcc aagactcagg gagacattga gacagagcgc ttggcacaga 900agcagagggg tcagggcgaa gtcccagggc cccaggcgtg gctctcaggg tctcaggccc 960cgaaggcggt gtatggattg gggagtccca gccttgggga ttccccaact ccgcagtttc 1020ttttctccct ctcccaacct atgtagggtc cttcttcctg gatactcacg acgcggaccc 1080agttctcact cccattgggt gtcgggtttc cagagaagcc aatcagtgtc gtcgcggtcg 1140cggttctaaa gtccgcacgc acccaccggg actcagattc tccccagacg ccgaggatgg 1200ccgtcatggc gccccgaacc ctcgtcctgc tactctcggg ggctctggcc ctgacccaga 1260cctgggcgat ccagcgtact ccaaagattc aggtttactc acgtcatcca gcagagaatg 1320gaaagtcaaa tttcctgaat tgctatgtgt ctgggtttca tccatccgac attgaagttg 1380acttactgaa gaatggagag agaattgaaa aagtggagca ttcagacttg tctttcagca 1440aggactggtc tttctatctc ttgtactaca ctgaattcac ccccactgaa aaagatgagt 1500atgcctgccg tgtgaaccat gtgactttgt cacagcccaa gatagttaag tgggatcgag 1560acatgggagg tggcggatcc ggcggaggcg gctcgggtgg cggcggctct ggatctcact 1620ccatgaggta tttcttcaca tccgtgtccc ggcccggccg cggggagccc cgcttcatcg 1680cagtgggcta cgtggacgac acgcagttcg tgcggttcga cagcgacgcc gcgagccaga 1740ggatggagcc gcgggcgccg tggatagagc aggagggtcc ggagtattgg gacggggaga 1800cacggaaagt gaaggcccac tcacagactc accgagtgga cctggggacc ctgcgcggct 1860actacaacca gagcgaggcc ggtgagtgac cccggcccgg ggcgcaggtc acgacctctc 1920atcccccacg gacgggccag gtcgcccaca gtctccgggt ccgagatccg ccccgaagcc 1980gcgggacccc gagacccttg ccccgggaga ggcccaggcg cctttacccg gtttcatttt 2040cagtttaggc caaaaatccc cccaggttgg tcggggcggg gcggggctcg ggggaccggg 2100ctgaccgcgg ggtccgggcc aggttctcac accgtccaga ggatgtatgg ctgcgacgtg 2160gggtcggact ggcgcttcct ccgcgggtac caccagtacg cctacgacgg caaggattac 2220atcgccctga aagaggacct gcgctcttgg accgcggcgg acatggcagc tcagaccacc 2280aagcacaagt gggaggcggc ccatgtggcg gagcagttga gagcctacct ggagggcacg 2340tgcgtggagt ggctccgcag atacctggag aacgggaagg agacgctgca gcgcacgggt 2400accaggggcc acggggcgcc tccctgatcg cctgtagatc ctgtgtgaca tacctgtacc 2460ttgtcctcca gagtcagggg ctgggagtca ttttctctgg ctacagactt tgtgatggct 2520gttcactcgg actgacagtt aacgttggtc agcaagatga ccacaatggt tgagtctcag 2580tggtgggacc cttccagtag catatgcccc taattttgat atgaactcaa acagatatta 2640aattacttat tttccattcc ctattccatt ctgtgactat ctctctcatg ctattgaaca 2700tcacataagg atggccatgt tcacccactg gctcatgtgg attccctctt agcttctttg 2760tcccaaaaga aaatgtgcag tcctgtgctg aggggaccag ctctgctttt ggtcactagt 2820gcaatgacag tgtagtgtca aatagacaca tagttcactc tcatcattga tttaactgag 2880tcttgtgtag atttcagttt gtcttgttaa ttgtggaatt tcttaaatct tccacacaga 2940ttccccaaag gcacatgtga cccatcaccc cagatctaaa ggtgaagtca ccctgaggtg 3000ctgggccctg ggcttctacc ctgctgacat caccctgacc tggcagttga atggggagga 3060gctgacccag gacatggagc ttgtggagac caggcctgca ggggatggaa ccttccagaa 3120gtgggcatct gtggtggtgc ctcttgggaa ggagcagaat tacacatgcc gtgtgtacca 3180tgaggggctg cctgagcccc tcaccctgag atggggtaag gagggtgtgg gtgcagagct 3240ggggtcaggg aaagctggag ccctctgcag accctgagct ggtcagggat gagagctggg 3300gtcataaccc tcaccttcat ttcctgtacc tgtccttccc agagcctcct ccgtccactg 3360actcttacat ggtgatcgtt gctgttctgg gtgtccttgg agctatggcc atcattggag 3420ctgtggtggc ttttgtgatg aagagaagga gaaacacagg taagaaaggg cagggtctga 3480gttttctctc agcctccttt agaagtgtgc tctgctcatt aatggggaac acagccacac 3540cccacattgc tactgtctct aactgggtct gctgtcagtt ctgggaattt ccagtgtcaa 3600gatcttcctt gaactctcac agcttttctt ttcacaggtg gaaaaggagg ggactatgct 3660ctggctccag gttagtgtgg ggacaggatc gtctggggga cattggagtg aagttggaga 3720tgatgggagc tctgggaatc cataatagct cctccagaga aatcttctag gggcctgagt 3780tgtgccatga agtgaataca ttcatgtaca tatgcatata catttgtttt gttttaccct 3840aggctcccag agctctgaaa tgtctctccg agattgtaaa ggtgacactc tagggtctga 3900ttggggaggg gcaatgtgga catgattggg tttcagggac tcccagaatc tcctgagagt 3960gagtggtggg ttgctggaat gttgtcttca cagtgatggt tcatgactct cattctctag 4020cgtgaagaca gctgcctgga ctgtactgag tgacagacga tgtgttcagg tctctcctgt 4080gacatccaga gccctcagtt ctctttacac aacattgtct gatgttccct gtgagcttgg 4140gttcagtgtg aagaactgtg gagcccagcc tgccctgcac accaggaccc tatccctgca 4200ctgccctgtg ttcccttcca tagccaacct tgctgctcca gccaaacact gggggacatc 4260tgcatcctgt aagctccatg ctaccctgag ctgcagctcc tcacttccac actgagaata 4320ataatttgaa tgtgggtggc tggagagatg gctcagcgct gactgctctt ccaaaggtcc 4380tgagttcaaa tcccagcaac cacatggtgg ctcacaacca tctgtaatgg gatctaacac 4440cctcttctgc agtgtctgaa gacagctaca gtgtacttac atataataat aaataagtct 4500ttaaaaaata atttgaaagt gacccttgat tgttaacatc ttgatct 4547229133DNAHomo sapiens 2aaaaaattaa gtatataaag tttaaaaagt tagagtaagc taaggttaat tattgtagaa 60aaacattttt cataaattta atgttgtctt agttacagta tttataaagt ctacagtaat 120gtatagtaat gccttaggcc ctcgcattca ctcaccactc actcactgac tcatcagggc 180aacttccagt cctgcaagct ccattcatgg taagtgtcct agaaagatct accatttaaa 240aatctttcat atggtatttt caccacacct tttgtatgtt tagatacata aacagttagc 300attgtgttac aattaccaat agtattcaat acagtcacat gctgtacagg tttgtcgcct 360aggggtaata gggtgtacca tatagcctaa atgtatagta ggctataaca tctagtttgc 420gtaaggacac tctgtgatgt tcacacaaag atgaaatcac ctaatgacac atttcttaga 480gcttgtccct ttagctaagt gatgcatgac ttcagttttg ccccatttct agagcatagt 540cctcaatgac tttcaatgaa aaacccgata gctttcatct tctcaatcct gaagagctga 600aggagattta ggctgaactt aaagaaattt tcagcttagc tcattagtct tctactccat 660acatcttcaa catttaacaa gtgttttgaa aaagacacct acaaagtgct tgaagtcatc 720aactctcaaa tcttgtcatt gcagcaccac gtcaaatgac aaaacacttg ctattttctt 780agtccactgg aggagcctat tgtcagaggc caaacctgga ttattagctc caaacaagca 840ctcagatcag taagtgtcct caggtgataa gtggttgttg ctacttggca tcaattcacc 900agttcttctg aaacttacgt ctgttttgtt ttagggccct tatcaatggt aggtctttgt 960ttcctcaaca ccactggaca gtgaaagatt ttgcactgcc tttcagaagt tgacacttta 1020gttttttgtt ttaccttcta ccgtagcatc agaagttaac caacgtgttt tgaagaaacc 1080agagtgtttg agatgcctca gttttctagt tacatcacac tggccccata attgctgctg 1140atttctttct tacagcagaa aactgtagga aaattgtagc agaaaacttt tctacagcag 1200aaaacggtag cagaaaaatg gcactaaaac gcagcgtaca cttgcaaaca gcaaatgcta 1260ccaagagaaa cagtgatgtc caaacgtcag cttacatttg catggttctt ctttggaatt 1320tttattcatc tagtcctatt tactttctta gctaaacaat gctttttaaa aatatacctt 1380taaaatttta tcctattttt gtagttgttg ccagtgggac aatttgtcct actgtgaccc 1440taatgcatct tatactgtgg tggaaaaaag aataagattt taaattgtgc tttctgaaaa 1500actggatata gaaacagaca atggccagac catatataaa aataggcctg gctgggcacg 1560gtggctcacg cctgtaatcc cagcactttg ggaggccaag gcggatggat catgaggtca 1620agagatcgag accatcctgg ccaacatggt gaaacccctg cctctactaa aaatacaaat 1680ttagctgggc atggtggcgc gcagctgtag tcccagctac tcgggaggct gaggcaggaa 1740aatcacttga gcccaggagg tggaggttgc agtgagctga gatcgtgcca ctgcactcca 1800gcctggcgac agagcaagac tccatctaag aaaaaaaaaa aaaaaataga cctttgaccc 1860acagcctaca gcagcctgcc tggggaacca attcccttat cttcaataaa caatccagca 1920aggtagtctg cttaagtccg acttgcagga agtcagattg ctgtctctag taacaatcca 1980ggaggctaaa taataacttt tataacaatt gttttaaaat ggccaggact tgattaataa 2040ctgacagttc ccccaatatt tgtgcctgct tccaacttag gaccaaccag ggaaagctaa 2100atatgcatcc tacccaatta cataggatac tccacttcta gttacccctt aagcattccc 2160catgccaaca gcctccaatc aggtcctttt taaccactat aaagtttcct acttctttgc 2220ctgtctttga gtctctgcca aaatgcaaaa gatggtggct gactcctttg ttatagcaat 2280ttgtgaataa tttttgctct tttcatttgg ttgatcttca tgtattttca cattattaag 2340ctttatataa attaaaatcc aagaggctaa catttaatta atgacattta agatcttcta 2400tatcggataa tgctatacat tatattaggt ttaatatttc tattaaatat agatttagta 2460aattactaaa aatgctaaaa attcatcaaa tatatatgta agtacaaata aggaaaatgc 2520aaagagagat attagaaagg ggtaatatat tcaggaataa atattcaaga tattttaagt 2580tggagatatt gtctgttggt actaaatcaa tttccccctg ttttgtgctt ttttccatat 2640cacttggggt tgaagcctgg acaccacttc ttccagagtc cctttcttag gaaggcactc 2700acttgcgatt agaaggcagt ggaaaattgc tgtcattctg cttctgacag caagtagcag 2760cagctgccag gagtgtgggt ttgtttagtg ctgcagggcc aatagtagct tcctgcagtt 2820cctgaccttt ggaagcacaa ttttgctttt tctgtcctta caaaactttt gcaatgcact 2880tcactgtatt acatctctct gggcttaaaa taccttgagt gtgttttttc ccccttgtaa 2940atctgggcta gactgaataa tcttgtaagt atgtaaatat aagcaactat tttaaaataa 3000cctgggtttt taaatgtaat acagatgctc ttcaacttat gatggggtta actcccaata 3060aatccagtgt aaattgaaaa tattgtgagt tgaaagtgta gagtataagt tgttcacctt 3120catgatcatg tggctgaggc tgcctggcat tgtgaaagag tatcttactg agtatcgctg 3180gtctggaata agatcaaaat ttaaagtatg gtttatacag aatggatatt gcttttacac 3240cattgaaaag tcaaaaattc ctaagtcaaa ccatcttaag tcaggtgtgt ctgtagtttt 3300aaaaaaatta caaataaaga atatccagtg ttgttgggag tgcagagaag atttacaagg 3360taaacattga tttgtttaaa gtttgagaga aaaaattaga taatatgctt tatgattttt 3420aaatgttaat ttcaaagtaa ttatacattc acaggagttg atgaaaatag tacagagagg 3480tcccttgtac ccttcaccca gtttccccca atggttacat catacataac tatagcacaa 3540tatcgaaaca aggaaatcga cactgataca atgtatttgc agttttctac tttatcacat 3600gtgtagattc atgtaaccac cactgtgatc aaaatacaga actatattcc atcaccacaa 3660agatcttcct catgccactc gccctcctta agagtcacac cattccccca cccccaccat 3720ccctacactg tgccaaccac taatttgatt ttcatctgta taattttatc atttagaaaa 3780tgttatataa atggaattat actatatgtg accttccgag actggcattt tgtactcaga 3840ataatgccct tgggatctgt attaggtgct ccagagcggt tgtactaaca ggatatgtat 3900atatagaaag atatttcttt taaagaattt gctcacatga ttgtggaagc ttactgagtc 3960caaattctga tggaagaggc cagcagtgga ggagactggg acagagttgc agtttgagcc 4020caaaggtagt ctgctgtgga accaggaaga gccaggattg cagatggagt ctgaggcaat 4080ctgttggaga gttccctctt atgctagtca ggcattcaac tgattaaatg aggggaaccc 4140agttatggag ggcaatgtac tttacttaaa atctactgac ttaaatatgt aactctcacc 4200ccaaaactgc cagattatgt gaaattccat gtcctctact tggctccatt gacactcaga 4260tggagtagat taaacaacag acatttactg aaagtcctca cttaacatca tcaataggtt 4320cttagaagct gtgactttaa gcaaaatgac atataataaa actaatttga ccataggcta 4380attcagcgat ccccaacatt tttggcacca gggactggtt ttgtggaaga aaattttgcc 4440atggatgggg gttggggact agcggtggca gggagtggga tggcacaacc tagatccctc 4500gcatgggcag tccacaatac agttcacaaa ggtttgcact cctgtgagaa tccaatgcct 4560ctgccgatct gacagcaggc cattagtggt ctgtggccca ggggttggga acccctgggc 4620taattgatgc gaacaagatt taagttccta tggcttattt ctggtcacaa acacatcacc 4680aaactcctaa ataaagactc agaacacttc taatattaaa cattaaaata aatgggaact 4740atatatacat ttaaggtagg tttataataa caagtaagat aattaattat ccagtttttg 4800gtgaattagt gagtgatggt ggtcacagtg gtggtgggtt acattaagga acaaatgttt 4860gtaaaatgaa aatggtaagg agcacctcct gccaccacac agctcaaacg caaagaagaa 4920caaatacgtt gaactcactg agtacttttg taccccattg tttactattg tacagttgta 4980tgaatatcat gtactttaca aatttttatt ttagaaacat ttctattcat tcgcttattc 5040attttccaac ctgcttattc cagttcaagg tcatggatga ctggagccta tcccggcagc 5100tcaaggacaa gagaggaacc aaccttgtat aggatgccat cccatccatt gtgggatgca 5160gacacacaca cacatacaca cacacacaca cacacacaca aagtcactct gctgggacaa 5220tttagactca ccaattaacc taacatgcat gtctttggga tgtgggataa aactcaaata 5280cacaaagaaa acccatgcgg acgtggggag aacacacaaa ctcctcatgg ccagtggccc 5340tggccaggaa cctatttatt ttctcaccaa cattgtaaca aaacgttgaa caaaacaatg 5400ctataggagg accctctgtg tttctcacag tcctggaggc tgggaagtcc aagatcaaga 5460tgctgacagg ttcaattcct ggtgaactta gaactgaagg ctctctggca ggggtgcctt 5520gtggctgcag gctgggtata gaaactcagg ctccccacta ggcctccact tacagaatcc 5580tgactgggag ggagagggtc tcatcagcgc tcccacatgg cctctactga caccaggaag 5640ggagaagtgc ctccttacac ctggacagtg gtgaaagtcc cagctttcta cttggcctcc 5700tctgacaaca ccttggcaaa gtgggtgagg agtgcttcct tgcaacaggg caggtggaag 5760tccaggctct tcacatgggc ttcactaaca ccacagtgtg gaggtggctg attactgata 5820ggcaggggca aaagtcctag gtccccagtt ggcttcctct gacataagcc tgatgggtct 5880aggtagtgtc tcattatcgc caggcaatgg gataagacaa agctcctcac tcagtgtttg 5940ctgactgagg cgggatggaa gcccctgatt tttctgtatt tgactggagt agtgcggtta 6000ctgtcagtta tctgcctggt aggctgctct ttcttgttcc cttggataga gaaacatgct 6060ttccttagga tatttttgtc tgtgactact gatgtttcct gttttccagt ttctccagca 6120ctcattcctg gatatattag gcagaaagaa gacctatgaa actcaccact ctgtcattcc 6180ccaatcccat ggtctgaggc caacctgctt ctcctctcca tcattcaagg gctttttatg 6240tctgtctgta gctgtactta gcaggaagaa taggaagaat tgtacctact tcatcttgtc 6300ttagaaccag aaatctctca ccatattttt taaaatatgt ttttgtcata tattaaaata 6360ttatacatct atccttagat ccttaaataa acatataatc tatccttaga gttaagttaa 6420tttggtaaca aaaataaaac aagactaaaa ctattaattg tgttaaagcc ataaaaaata 6480tgcaaatttt tgcccaaaat atgggaaatg tgcgtgtgtg tgtgtgtatc tcctatgtat 6540acacataaaa aaagacataa aatgaaaatt gctgatgtat caatacccgg gggcagggag 6600tattctcagg tttaactaag tactcatatt caagttttta ccataggcca cacctggctc 6660tcagattcac ttagaaggat attagacagg agtcaaagta tgccaaagtg ctgaatcagg 6720tctttttctt cagtgggaga agttcttgaa acagttcata atttattcca ggtgctagtt 6780tcatcctctg cccccatccc ccaagtgaca actcaggtac aaggagctga atttacacct 6840gtggaagttg tgtccaccgt agcttagaat cctcatgtca tctacgagct agtacctctt 6900ataacaaacc catgggcaca gcttccagag tccccgtaaa gggcatgctc agttacaagg 6960gtcactgcat ttggaaatac ccaaactatg ggtccccgtc atttgttacg gttcatgaaa 7020tattcttccc agtaaagata caaaatgcca accagaagcc atttgtgcca taagcaatgt 7080tgtctaaaaa tccagctgac attcttcctc catcaggttt ccagaaaaca gctagaaaat 7140tagcctaaga ttaaatacat catggagaag tagaaagggt gttataaagc atttatccac 7200aagattcaaa atgaaataca gttaattttg tccgttttaa gacattattt caaccttcaa 7260attatttaaa agaagtacat cctatatttt gtgtgcttat tcaaaaaagg catggtaata 7320cttataaaaa gactttaaat atttttataa gttttaaata ttttataagt aattttataa 7380atgaaattac aaaccattta agtgacctaa ttaaatcaaa cacactttga gtatgcacac 7440aagaaaaaaa ttagttgaag catcctgact taagaaatcc ttgatctttc ataaggtgtc 7500tgaatactca atgtcaaaaa cacttatgaa gaattaaaca ctgttgacca caagagggaa 7560acctagtccc agttatacta taaattagaa aatcaaggga aaaatatgtg tcctgagaac 7620ttttgaaata gtcacatata aacatagtat acaagaaaaa accaaccgtc atccctaccc 7680aaggatatgt ttgtggtatg agtggtttta gtgttttgag tggactggtt cttggactcc 7740acatattatt ggctacagag atagagactt gatttagaaa atcacagttg ccactttcta 7800agtaagccct tgaccaaaag actagatttc tttaaaccca gttttctcag gtaaaatgga 7860aatacaacta ttatctaata aatataagta agctttagtg tcatagtcat agcagtagta 7920ttttcaattg gtaaaaagaa actggacccc aaaaaagaat ttcagtgaaa gcagtaacag 7980tcttctggca tatttctcac ctttctttct accttaaagg ttcaaagttc ctaagtaatc 8040tcagaaacct aaaatagttt attctctatc ctcactattg gtttttaaaa aacattttgc 8100agcatggacc actgctcatg tacagatgct ctccaactta acaatagggt tatgtcccaa 8160taaacccatt ataacttgaa aatatcttaa gctgaaaatg catttaatac accaataaac 8220ccatcataaa gttgaacaat cataagccaa attataagtc agagaccatc tgtattagct 8280taagtcttgg aatggtttat tttttagatg ccatttagcc acttatattc tcttctattt 8340tattgtgaga actaattccc ctcttacatt ctgtgcttga cccatgctat acttagtgtg 8400aacaagagcc accttcttct catgacttct atttttttgt gaaaatttcc ttcactcatt 8460cacgacattt ggatttgaaa tcttacctac ttaagtactt taaaaaatca ttttctacca 8520tctttcttat caggagcctc tagtgattcc ttctccacac ttctaacttc tcatcttcac 8580actccttgtc ttcctaactt cactacagta agtgttttac atgtttagaa ctcagctcct 8640ttactatgat tgctaaccat gtaccttaaa taaaccgtct tctagttttt tgtttcttac 8700tctcaattat accttttaga aaagaattaa gagtagaaaa agactgctac atagacattc 8760ttatgatctt cagaaatgag cacagatcat gcttaatgaa aaaagatttc caaataatgc 8820tgcatatgtc cagagaaaag gtggcagaaa tgactgtcgt ttgggggcac tattgtctgg 8880acatggccag ttctcagaac tccagtccct aaattccctt ctaactaaag gaaaagcctc 8940ttaagggtct tatagaaatc ctgccacttt cacctgaaag aataatcttc agttatgtgg 9000cacatggcca agagtaaaag tctttagtca cttggaagca gacagacact gtaatgctaa 9060ataattggac ataacatgga acttactgag gcctcaaata tcaattttac tttgggaaaa 9120agagcagcaa ctttaaaagt gattgaaagt aactcaagtt tattccttaa cagagtgatg 9180cttaatctaa caaaaaacat gttatatgca cactcttctc cattaccttg taagaaaact 9240ggactaggaa acacagctga aatggccagt tctgcctcca tttcctaaac cgtgttataa 9300ttatgtctat gtgaccagta acagacaatg accatgattt atactttttc atatgtttgt 9360tgttttgttt tcaatgtttg tggtctttcc tcagtatcag ctaagaggcc attaacacag 9420atatctattt atggacatgc gagactgttg ttcacctctt ttgcagaatt cataaagaaa 9480tgatggggaa aacacatcaa agatagagtg gataaagcaa atgtgccaca tatacaccat 9540ggaatactat gcagccatga aaaagaatga gttcatgtcc tttgcaggga catggatgaa 9600gctggaaacc atcattctca gcaaaataac acaggaacag aaaaccaaac actgaatttt 9660ctcactcata agtgggagtt gaacaatgag aacacatgga cacaggggcc tgttgggggg 9720gtggggggca aggggaggag agcattagga caaataccta gtgcttgagg agcttaaaac 9780ctagatgacg ggttgatggg agcagaaaac caccacggca catgtatacc tatgtaacaa 9840acctgcatgt tctgcacatg tatcccagaa cttaaagtag aataaaataa ataagtaaat 9900aaggaatgat gggacaaaca agtttctgtt attgtctctc tactgaccaa agggtggtca 9960gagagtatag gatgaagcag atttgtgata tccttgaata gatctgctct ttactatgaa 10020ttctatcatc tactcccagc gtatgtggga aagggaccaa cttacttgcc tggaatttag 10080tgaaattgtt ttctaggggg accaagagtt tcctctactt gatatgaagt tgggtggttg 10140aagatgatag gattggcttc tgcttccatc agaatcctaa agggcagggt atatggacta 10200gttggtattg gatcttggaa actgtgatgc attgggaatg gtcacactcc cagagtttgt 10260ggacacaaag aatgttttag tgttccctac acaccagaca cgggccatga aggaatctga 10320agagcctacc aaaccttgca caagagaaaa gctttacttg gaacatcatc caggctcaga 10380gaacacaaat atttcatttc cagtaagacg

tttctggtct ttttctcttc ctccccttcc 10440ctgaacctac cctagatgag ctatggcctc aaagtgccag tagaacgtaa gaaggaagga 10500gaaccacact cattcctgcc ttcaacaatt tacacaggga tagaaagaga tttatattaa 10560atcaagttgg gactttcaat tattatatag taccaaacaa tctaattgct gaactaagat 10620atacttgtgc aatttaaggg aattgtagaa tagcatatta attagaatca agaaaataat 10680tcatgaagta tgctataatt cctacccaag cgcaggggaa tagcatctct aatgaaattc 10740tctaaagagg caagagcagg cacaatgagt ttttgtttga ttaaagattc catttagtgc 10800ttatccaacc tagcaattac atttgtatgc ttcagatgtt tttaaaaaaa taaacaaaag 10860aaagtacctt aaataaagaa taggatcaaa tagtatttaa acaattgagt aaattaaaaa 10920attatatgaa ttagattgat tgaaattgat actttcctaa ttctcctcct tcaacacaca 10980gacacacaca cacacacaca cacacacaca cacgtatgca tacaaacaca tctgaattct 11040ataaaatcat tctgaccttg atgagattcc atagtttact catgcaacag aacataatgt 11100ctaaatgaag tttctggtct ctgttttaca tggatgattg agtaaaatca ttccctattc 11160ctggaagaat agctaagaaa ggattcacag gtgaggacat gcgttttttc agaagatgag 11220aacaaagatg agaagatgag agcaacagaa tgtcctatat cctaatgctc tgtgctgact 11280tcggagtggc caatatgata gagatggaag gaactctgaa aacaaattgc cagaatttct 11340aaggaacagg agatgttgag tgagtgaatc aagccatgga ctggctgtat gggggcagct 11400attagagaca actaccctta gacttctttg gtgattggtc aagctaatct tttccttcag 11460agtctctcaa ttataagact tagcttgtgc catttagaac agacaagaac acagagaatt 11520atagaacaat ctgactacag gttcttaagt tatagcaatg aaacttgtag ttggccggca 11580ggaaaatatt ctgagatgtg gattcaaagt ttctaagtgt gcacacgtac acacacacac 11640ccctacctgc atgcgttttc taatttacaa agactactca agtaaagagg ggtaatttca 11700caccccagga ggtctgtata aagataactc tggtctttaa agcatcgggt ttcaggtaga 11760ggtgaagaga gaatgaatca aactcaaact gccatcctcc caggttaaag atgagtccag 11820tcattgtgga gccctctatt aacacaggac atgctaggaa ggcccattaa cccactgccc 11880tagcacattt gttaacgtcc tagtgcattt gttgatatca acagttcaca gtttttattc 11940tgatagggat ctattccagc agaccagctt ctgtgacctc tcaggatgcg aaaaagtaac 12000acaagaaaag cttcttatgt agtgaattga gaaggaaata cctagatcaa tattccctca 12060gcacctctgg taggaagtcc ttagtaggag aaaaacacca tgaagaccct tagtgcagaa 12120ggaaaagggg gtagggggtg gtggaaggga agctaaaaga aggggctgga ggttctcaga 12180attcaaacca cacaaacaaa tgaagtattg aggtcccaga cttgatctgg gcccagtgtg 12240aaagccctaa cttatttctc cagaagaata tgtcctctgg ttttagactt ggcactgtgg 12300ggagaaccag agtgatctat ggtggatata cacacaaaca tagacacaca tatttgcatt 12360tagtaatttt tgtaaaattt ccatttgctt ctctgatcct gtctgtatct ttgggaatag 12420atgtaagaat attacatctc tcaggcttgc tctgccccag gtttctgaac gtggaataca 12480tttctccagg gaaactcagt attatgagat ttgggaggtg gaagttaggc cacagccatc 12540tcagggacag gtttcacaga catgagtttt ggcagcagcc ttgtgttcta aagacattta 12600ctcctagggg ctctagagga tctgcaacat cagcagaggc ttcctgtggg ttcctgatct 12660tttaaaatta gggttctgca gtgacttctg ctcctccaga ccccctaaca gttttaaggg 12720ctaattccct gtaatatatt cagttctgct tagactgatt acagggattc ctatttcttg 12780actgaattct catggctata gtggctcgtc accatttgac atcaccaaga agtcctcatt 12840caggtgcctt tggaaattcc ctcaaacaca caggaaatta gagtttgaaa gaaaacggag 12900aaccatgagc actgtccaaa taggaacttc tctcctatca cagagaaagg gaactgaaag 12960tcatttctca agtctcccaa atttagtaat ctcacaagaa gaaccaatca gtgttctagg 13020actaaacagt gtcataagtt gctgagcaac aacttggatt gaagatgcta ttataatata 13080tgaaatgtct ttgaatttac catgtttttc tcaagcacca tttaagaaca aggcattatg 13140gcagccagca aagggcagac atagaaaatt atacatggtt ttgcctctaa aagaggagat 13200gacaagctta aatcatagga tcagactctt agcacagact gataccatag gctctcatct 13260ggcccattct cctgactctt tacctttcag gaaaggtatt cctgaaaaat tgcaggagag 13320accatgctgt aggtctcttt ctagcgatct aggagttaat gccacagtgt gttcaaagcc 13380ctttgatgcg atcagataat cagtaatgta tggaatattt gtgttcataa cttgtgagaa 13440cggctgcatg gcaggacaag accccagcac aacagtatgg aaaatccacc ctaagcagac 13500atgtcatgac tgatgttgaa caatggactc accagccagg cacggtggct catgcctgta 13560atcccagcac tttgggaggc agaagcaggc agatcacgag gtcaggagat caaaaccatc 13620ctggttaaca tggtgcaacc ccgtctctac tgaaaataca aaaaaaaaaa aaaaaaaatt 13680ggccgggcat ggtggcgggt gcctgtagtc ctagctactc gggaggctga ggcaggagaa 13740tggcgtgaac ccaggaggca gagctttcag tgagccgaga tcgtgccact gcactccagc 13800ctgggcgaca gagcaagact tccgtctcga aaacaaaaca acaacaaaaa aaacaatgga 13860ttcaccatcc gatgggctcc ctcactgcca ggtcactctt catggaagta tttgtattcc 13920agtcctttct gtggaaagaa cttaacattc tccttttcat aacactgtat cttcagaaac 13980aagagagtcg aagtctccta attttcagga gtgtctatgt tgaacatcaa aatatattct 14040ttagagcaga tctttaataa tcatatgaca agagaaaaac tttcataatc ttatgacatg 14100agggaaggaa tattaaagcc gttctgtggg ttattatctc taacgttccc aatagaatag 14160gctttgccag ctgggtgcgg tggctcatgc ctgtaatccc agcactttga gaggccaagg 14220cgggcaaatc acgaggtcag gagtctgaga ccagcctgac caacatggtg aaaccccgtc 14280tctactaaaa atacaaaaat tagccgggca tggtggtggg cgcctgtaat cccagctact 14340caggaggctg aggcaggaga atcgcttgaa cccgggaggc ggagattaca atgagctgag 14400atcacgccac caactccagc ttgggcgaca gagcaagact ctgtctaaaa aaaaaaaaaa 14460aaaaaaaaaa aaaaaaaaaa aaaaaaaaat aggctttgcc cattatactc tctcatattc 14520attgacctga atcctcaaat gaggtgtgtc cattagtcaa ctccaatctc ttgtcatata 14580taagatggta gagatgagaa gaaggtagct cctttacagc ccactatttc cactaactac 14640tacctgtgtt tcaagataca gcctttcatc cttctccagt gttgagagtg ttgaacctca 14700gagtttctcc tcttattttc tctaaatgag atacaatgcc agccatccca agctcttggc 14760ctgagttgtt catcttgaag tctaggactc caagaagcat gaaagagctt ctttagtgaa 14820gctatgtcct cagtactgcc aaaattcaga caatctccat ggcctgacaa tttaccttct 14880atttgggtaa tttattgtcc cttacgcaaa ctctccagct gtcatggcac agacatatga 14940tctgtattta gctctcactt taggtgtttc cattgattct attctcacta atgtgcttca 15000ggtatatccc tgtctagaac tcagattggg gttaaagagt ctgtccgtca ttgaccaaca 15060gtcttaaata cttgatttgt tgtcgttgtt gtcctgtttg tttaagaact ttacttcttt 15120atccaatgaa cggagtatct tgtgtcctgg accctttgca agaacccttc ccctagcaac 15180agatgcgtca tctcaaaata tttttctgat tggccaaaga gtaattgatt tgcattttaa 15240tggtcagact ctattacacc ccacattctc ttttctttta ttcttgtctg ttctgcctca 15300ctcccgagct ctactgactc ccaacagagc gcccaagaag aaaatggcca taagtggagt 15360ccctgtgcta ggatttttca tcatagctgt gctgatgagc gctcaggaat catgggctat 15420caaaggtagg tgctgaggga atgaaatctg ggacgataga ctacgaagca ttggagaaaa 15480gacctatgga catttggaag ataatgtgtg gagtgaaaga atagtgtgac aggtattatg 15540tggtctcgac agaaagtata acaaattgtg gtttggtgga gttcttccct caccacaaac 15600tgaagtaagt caaatttggt ttagaggatc aaaactgagt tgtgtattga tgaatagcaa 15660ggtcctgcta caagccaaac tgggggtggg ggtgggggtg ggggaggaag aatattttct 15720ggcaagcatt aacaagttat atttctgggc tttaattatt ctttctggaa aattagtaaa 15780attaaaaact aaaaaccaca catagttttg ctagaattaa atgaaaaaaa aagttattag 15840ccctgttctt atctgaatac atgatacagt agttattttt tggagtgtaa atcctgtcgg 15900tatatattga gcacatatat tgtgttgaag attactagaa ggaaaagtca tcaaaaagca 15960acaatttacc ccaggaaaag gggagggaag gcatgctgat atgagttgcc tcatgggaca 16020gtgatagcca ttccctgcct tcccatctcc atggtacagc agatcttata tcatgttaac 16080ttagtaatat ttccaagaga gtagaaaaat gagtaaggaa atggggaatc tgatattatt 16140ctctctcatc tccagagcaa cattggtgct gttgtaaaga tgtactgtag aaaagtattc 16200ttcacccagc atgaccccca cagaaggtgt caggtagact tgaaataagc aaagtaataa 16260cccagctccc atacccatag tggcaattgt agatttctat tgccccaaaa gagccataca 16320tagggatact tacctagaaa gacagaggct cttcccttgg tttgtgaaga ggcagctagt 16380atatttgtgt gtgtttgcat agatgcaaac ggtaaataaa ttcctaggtt tatcaataca 16440cagtcaaaca ttaaagtctc tcatcttggc tgggcacggt ggctcacgcc tgtaatccca 16500gcactttggg aggccgaggc aggcggatca cgaggtcaag agatcgagac cgtcctgggc 16560aacatggtga aaccccgtct ctactaaaaa tacaaaaaat tagctgggta tggtggcaca 16620cgcctgtagt cccagctact cgggaggctg aggcaggagg attgcttgag cccaggaggc 16680ggaggttgca gtgagctgag atggtgccac tgcactccag cctggcgata gagcaagact 16740ccgtctcaaa caaccaaacc aaaacaaaac aaaatatctc accttatctt tgaagactaa 16800ggaaaaaaaa atctcccact catcgataca ctccacagag gcagcatact ctccaagtgt 16860agctttctct tttcatgttc attattccct tggtgttggt tattctcaat gtcaatcata 16920acagaacatc ttccataata acagtcccaa tttaaggagc attaagataa aaggtggaat 16980tgccaaggtc aatccagacg agaaccttct catagaggta accaccgtgt gggtttggat 17040gctgggaagc agggggacta tgacgctaca aggtctcagt cttaattttt ggagtatttc 17100agtccccagg tatattttcc atagatttgg cccttaaata aaaagaagct tctgactcta 17160aaatgtaaac agtgcttgtt acagtcttgt tgatatatta agaaattact caccttatct 17220catttaatct taaaaacaaa cccctgacag gatcaaaacc acagcagggc tacataatag 17280gaaaactata cataaatagg tagaataatc tgctcaggat cactaggtaa gttgctgaat 17340aagaattcaa gatgtttttg atcccagagt ttaaaaccca acctttcaaa cagcgtttct 17400ttcttcttag agtacaatgt tctgagaaag agatcctctg gaattctggc ctaagtgtat 17460ttaatgcccg ggtaaagaaa gtgagagaac atttctcttt aggggctgct gctggatttc 17520taaaaagaaa ataatttctc agctagtaac atggagccaa acaacagctt cacaagactc 17580tgggttcttt agccctcatc tccttcaatc caccctcttt ataaccagtc cttcttgttt 17640ttcccctccc agctttgttc agcagcatgc ccttcaccca gaccttgtct tgtcactcat 17700ccctactcgc catcattctt tcattcctct tggcccaatc tctctccacc acttcctgcc 17760tacatgtatg taggttattc atttccctct cttgattccc cccacccaac tctctttctc 17820cacttctcgc ctttcagaag aacatgtgat catccaggcc gagttctatc tgaatcctga 17880ccaatcaggc gagtttatgt ttgactttga tggtgatgag attttccatg tggatatggc 17940aaagaaggag acggtctggc ggcttgaaga atttggacga tttgccagct ttgaggctca 18000aggtgcattg gccaacatag ctgtggacaa agccaacctg gaaatcatga caaagcgctc 18060caactatact ccgatcacca atggtacctc cctctctgct gcactcctgg acatgggaat 18120ccatagtttg aaagtagttg cttcagctct ttgtgttaga ttattgtaac tgattttccc 18180tccaagggcc taaccttgcc attaacaagc cccaaattct catgccagag gtctgagaac 18240tttatgggtt tgatcctatc ttgttgtgct caagtcttgt ctctgtcatc catggtctcc 18300tacaaagtca ttgccctaag ttcatgctgg gggagccaga agggaagtcc ttggatatct 18360tatacctcaa tattggctca atttcttggg gagggggtgc tgtcagagat tgttatctga 18420ggatgtgaca tagatttctc agggcacaat ttcaactact ttttcagctt tagggttttt 18480agatacgttt gtaccacaat tgagcatggg agggagaggg gtgagcctaa gcagtgatgg 18540ctgatttctg tcatgtctgt catgtgtccc ccagtacctc cagaggtaac tgtgctcacg 18600aacagccctg tggaactgag agagcccaac gtcctcatct gtttcataga caagttcacc 18660ccaccagtgg tcaatgtcac gtggcttcga aatggaaaac ctgtcaccac aggagtgtca 18720gagacagtct tcctgcccag ggaagaccac cttttccgca agttccacta tctccccttc 18780ctgccctcaa ctgaggacgt ttacgactgc agggtggagc actggggctt ggatgagcct 18840cttctcaagc actggggtat ggaccaacac tcaatctcct ttatttcaag gtttcctcct 18900atgatgcttg tgtgaaactt ggtgttctaa ctgtttcata atatctgcta caattaatat 18960aactgtcttc tcctactatc cagcttccgc ctttttttaa tctgtaattc tctcaataca 19020tcattctgtc ttcctcttct ttaatctatg aataactttt ctctttatta agaaccctac 19080atttgattct gagtgttact tcttcccaca ctcattacca tgtactctgc cttatttccc 19140cccagagttt gatgctccaa gccctctccc agagactaca gagaacgtgg tgtgtgccct 19200gggcctgact gtgggtctgg tgggcatcat tattgggacc atcttcatca tcaagggatt 19260gcgcaaaagc aatgcagcag aacgcagggg gcctctgtaa ggcacatgga ggtgagttag 19320gtgtggtcag aggaagacat atatggagat atctgaggga ggaaaacagg gtggggaaag 19380gaaatgtaat gcatttaaga gacaaggtag gaacagatgt ggctcttgat ttctctttgc 19440tagaacgaat cagacattgg tatcatctgg tatcccaaag cttcagggtc tgtcatccct 19500ttctatagac gggcaccttg atcacggctc cagtcttaga aatcatctcc agtacctaaa 19560accattgttt cacattagaa tactgagtct agggatctag aaaatactga gtctagggat 19620ctagaaaaat aagcctcaag atttgggcac atcctagctt gtatttcctg gggcaggtca 19680tcagttcaga agcatttcca gatcctggct cctttcaggt tagggtcaat tcattgcatg 19740aaatgggaat ctcttagagg ccaatgcctg cttttgcttc tttagtctca aatgtagtat 19800gagaaactct aaaaaaaggt aaagcatggt tgcttattat gttcagttgg agagtagggt 19860atacagttag ttcatgttgg aaaggttaga tgaacattga aagaattttg caaagtcaaa 19920ggattaagag agaagaggaa ggaatctgaa gcaaggagct caaaactgat cttaaactcc 19980ttggtaacta tgtgtgtctt gctataggtg atggtgtttc ttagagagaa gatcactgaa 20040gaaacttctg ctttaatggc tttacaaagc tggcaatatt acaatccttg acctcagtga 20100aagcagtcat cttcagcatt ttccagccct atagccaccc caagagtggt tatgcctcct 20160cgattgctcc atactctaac atctagctgg cttccctgtc tattgccttt tcctgtatct 20220attttcctct atttcctatc attttattat caccatgcaa tgcctctgga ataaaacata 20280caggagtctg tctctgctat ggaatgcccc atggggcatc tcttgtgtac ttattgttta 20340aggtttcctc aaactgtgat ttttctgaac acaataaact attttgaaga tcttgggtgg 20400aatttttggt gtttaagcca gttctttggg tggcggtggg gggtggggag tcggtcctgg 20460ggaatatatg tgatcctttc ccggtaaaat atctgaatgt tgaatttatc ttataaattc 20520tagaattcat cagacatatc ccggttcatt tgggcttggt ctcattttgt gcatctgcag 20580gcaaccctct tgttgtggtc tagtcctcat caggaaaacc taaagtgggg ttggtttgtt 20640gggagatctc tactgagcaa tgatataact ctatcttcag tagagtgaat ctgaaacccc 20700aaggtatgga tctcagaatg catgggatgg aggggagcag atggggttag agtggggaga 20760aggaagacag aagaatccat aaacattgca ggatttacat atcaacatcg ttcattccag 20820atttaatgag caaagagatt ggacactgaa gactggcctt acccattctg ttagacatag 20880tctcagatgc ctattttatt accgagagag tagtctgact gattcttgaa accaccttat 20940atttgaagat gtgtctttga gtggaaaagc tgagtgaaat ttggggttgg ggagaaagat 21000atgacattaa gatgagagga aggaatattt gaaacacgat gaactgttgc tcatttgtct 21060ataaaactat gacttgatat ttatctctaa aatagtttct agaacctgcc ataaaccact 21120aagataaact attcatgata gtgtggtaga ctgcaaataa atgctgttga aatgagttag 21180gcttgggttt catcttggct gtatcattta ctagctatgt tttcactggt atcttactta 21240acttagcctc acattactca tgaaaatact ggtgttaatt tttactacat tgaattaata 21300tcagaattaa aaggaaaacg caagcaaagt aattagatac atgcttagtg ataataaaat 21360attgcaaaaa attatacatt ctgttgtttt tctcaaaatt tctatagagt gatgataaaa 21420atctaagaga agctaaacaa aacaaggata aaccaaagca tcatgacctt ctaagcctta 21480ctaataaata agaagtttct cggctgggca cggtggctca cgcctgtaat ccagcacttt 21540gggaggccga ggtgggcgga tcacaaggtc aggaaatcaa gaccatcctg gccaacatgg 21600tgaaacccca tctctactaa aaatacaaaa attagccagg cgtggtgata ggcgcctgta 21660atcccagcta ctctggaggt tgaggcagga gaatctcttg aatccgggag gcagaggttg 21720cagtgagccg agatcgcacc actgcgctcc tgcctggcaa cagactgaga ctccgtctca 21780aaaaaaaaaa aaaaaaaaaa aaaaagtttc tctactgttg gttcagagaa tcaaagcaga 21840atcttgagac tactgacggt agaataggta tgaatgtctt tcttacatga ctacaaactt 21900tattataaaa taaatagctt aacacagaga atacactaaa acttagacaa gcatggatta 21960agaaagcaaa aagtaaaccc atatactacc atgtaagaaa accatttttg gccaggcgtg 22020gtggctcacg cctgtaatcc cagcactttg ggaggccgag gcgggcggat cacgaggtca 22080ggagatcgag accatcctgg ctaacatggt gaaaccccgt ctctactaaa aaaaaaaaaa 22140aaaattagcc gggtgtggtg gcgggtgcct gtagtcccag ctactcgaga agttgaggca 22200ggaaaatggc gtgaacccaa gaggcagagc ttgcagtaag ccgagatcgc accactgcac 22260tccagcctgg gcgacagagc gagactccat ctcaaaaaaa agaaaaaaaa aaaaaaaaaa 22320aaaggaaaac cattttaata gacttttatt tttagagctg ttttaagcta acagaaaaat 22380tgcagaaatt gtatacagag ctcccccacc cccagtttct acaatgctta acatcctgta 22440ttaatgtggt acacttgtta caattgatga accaatacta ataattatta ttaactaaaa 22500ttcatagtta tacgagggtt cactctgtat tacacagtta tatgggttct gacaaataca 22560taatatcata tatccaccat tacaggatta aacaaaatag cttcactgat ctaaaaatga 22620cccaggctcc atctactcat ccttccttcc tccctctgaa ccattggcat tctctgagct 22680atttactagt gttttgcctt tttcagaatg tcacatactt gtaatcatac agcatagagc 22740tttttcagat gagattcttt tgcttagcca tatgcataca ggtttcctgc gtatattgtc 22800atagcttgat agcttatttt tctttaatgt taaataatac tccattgtat aaatgtacta 22860tggtttattt acccattaat ctattgaagg acatcttggt tgcttctaat ttttggcaat 22920tatgaataaa gctgctataa acatccatga acagatgttt gtgcagacac aagttttcca 22980ctttggataa atacatagaa gggcagttgc tggatcatat ggtaagagta tgtttagctt 23040tgtaagaaac aactagaata tcttccaaaa tggctgtatc attttgcatt cctaccagca 23100acgaatgaga gtccctgttg ttctatatcc ttgccagcat ttggtattct ggggtttggg 23160atttcagcaa gaaagccatt ttaatatttt tttattttaa aataattata gattcagggg 23220aaattgcaaa gacagtatag agacattctg catacgcctt cacccagttt ctccaaatgt 23280ttatatttta agtaattata gcacagtagc aaaaccaaga aaataccttg atacaatgtg 23340tatgtatagt tttatgcatg tcttaccaca tttgtagatt catgtaacca ccaccacaat 23400caagcacaga gctattccat atcacagaga tcttcatcat gcttcccttt atagccaaat 23460tccccccaca caatcacctt aacaacttaa aaccactaat ttctttgcta ttaatctcta 23520gaatagtgtc attttgaaaa tactagttaa atggaatcat gcagtatgtg actggtgttt 23580ttcacttagc ataataccca tgagatccat ccaagctgct gcatatatca acaatctttt 23640tttttttatt gctaagtagt attccatggt ctaaatgcag cacagtttgc ttaactattt 23700gcctattgaa ggacattttg gctgtttcta gtttggggtc actataaata aggctgtttt 23760gaacatgtgt ttaaggtttt tctatgagca tgagttcatg agttttcatt tctctggtat 23820aaatgtctgg gatataattc atgggcatat ggaaatatat gtttagtttt tcaagaaact 23880gccaaactta gccaagtatg atggcttata cctgtaatcc cagcactttg ggaggccaag 23940gaggaaggat aaattgaggc caggaatttg aggccagccc cagcgtctac actttttttt 24000ttttttgaga cagagtctcg ctctgttgcc agactggagt gccatgatgc gatctcggct 24060cactgcaacc tccgcctccc aggttcaagc aattcttctg cctcagcctc tcgagtagct 24120gagactacag gtgcacacca ccacgcccaa ttaatttttg tatttttagt agagacaggg 24180tttcaccatg ttggccagga tggtcttgac ctcatgacct cgtgatccgc ttgccttggc 24240ctcccaaagt gctgagatta caggcatgag ccaccgtgcc cggccaaatg ttttgttttg 24300tttttgtttt ttgttttttt gtcaggtgga tgaggtggca tgcccctata gtcacagcta 24360cttgggaggc tgaggtggga ggattgcttg agcccaggaa ttcgaggctg cagtgagcca 24420ctgcacttca gcctatctga cagagcaaga tcctgtctcc aaaaggaagg aagggaggga 24480agaagcaagg aaggaaggaa ggaaggaagg aaggaaggaa ggaaggagaa aaaagaaggt 24540agggagggag gaaggaaggg agggagggag gaaaaaagaa agaagaaagg aagttaaaaa 24600gaagggaggg agggaggaag gaagaaagga aagatggaag aaaggaagga agggagggag 24660gagaaagaga aagaaaaaga aggaaggaag aagggaagga gggagggaag ggaggaaggg 24720agggagggtg aaaggaagga aagaaggaag gaaggagaaa gaaaaggaag agagaaagag 24780aaagaaaaaa gaaagaagaa agaaagaaga aagaaagaga gagaaggaaa ggaaagaagg 24840aaggaaagga aagaaagaaa aagaaaaagg aaggaaggaa agaaggaagg aagaaagaaa 24900aagaaagaaa gaaggaagga aagaaagaaa gaaagagaaa gaaagaaacc gataaactat 24960tctctaattg ctttgtggga gtatggccac tttcatcata ttgatttttc cttttttttt 25020tttttttttt ttttttgcga tagagtctgg ctctgtcgcc caggctggag tgcaatggcg 25080tgatttcggc tcactgaaac ctctgcctcc tgggttcagg tgattctcct gcctcagcct 25140ccctagtagc tgggattaca ggtgcacacc atcacgcctg gataattttt ttgtattttt 25200actagagatg gggtttcacc atgttggcca ggttggtctc aaattcctga cctcaggtga 25260ttcgcctgcc ttggcctccg gaagtgctag gattacagat gtgagccacc gcgcccagac 25320aatattgatt cttccttttc catgaacatg atattttttt ccatttattt gtgtcatctc 25380tgagttcttt gagcagtggt ttgtagtttt ccttgtagag atctttctcc tccctagtta 25440gctgtattcc taggtatttc gtgtgtgtgt

ggcaatcgtg aatgggatta cgttcctgat 25500ttggctctca gcttgactgt tgtggtgtat aggaatgtta gtaatttttc cacattaatt 25560ttgaatgcca agacttcgct gaagttgtta attagcttaa agagcttttg ggctgagact 25620atggggtttt cttgatatag gatcatgcca tctgcaaata ggcatagttc aatttcctct 25680cttcctgttt ggatgccttt aattcttttt cttgcgtgtt gccctggcca agacttccaa 25740tactatgttg gataggagta gtgagagagg gtatccttgt cttgcgctgg ttttcaaggg 25800gaatgcttct agctttttcc catttagtat ggtattagct gtggggttgt cacagaaggc 25860tcttattatt ttaagttatg ttcacttact actcagttta ttaagagttt ttaaatgaag 25920ggatattgaa ttttatcaaa aaccattcct gcatctattg agctaatcat gtggcttctg 25980tctttagtac tgcttatgta atgaatcaaa tttattgatt tgcatatgtt gaactaacct 26040tgcatcacca agataaagca tacttgatca ttgtagatta gctttttaat gtactgctgg 26100attcagtttg ccagtatttt gtggaggatt tttgcataaa tcttcatcaa taatatttgc 26160ctgaagtttt cttttgtgtg tgtgtctgcc aggttttggt gctgatcctg atgatgctgg 26220cctcatagaa tgagttagag aggtatccct cttcctcaat tttttggact aattataaca 26280ggaatggtac cagctcttct ttgtacatca ggcagaattc agctgtgaat tattctagtc 26340ctaggggttt tttttgtttg gtagtctact tattactgat ttaatttctg agatcattat 26400cagtctgttc agggattgaa tttcttcctg gttctgtctt gggagggtgt acgtgtccag 26460aaatttatca atttcttcta gttttcctag tttatgtgca tagaggtgtt tttaatattc 26520tctgatggtt atttgtgttt ctgtggggtc agtggtaata tccccattgt aatttctgag 26580tgtgattatt tgaatcttct ctcttttctt ctttattagt ctaactagag gtcttttttt 26640tttattaatt tttttttagg aaaccaattc ctggactcat tgatcttttg agtgttgttt 26700ttttttctgt ctcaatctcc tttagttcag ctctgatttt ggttatttct tgtcttctgc 26760tagccttgat attggtttgt acctggttga ccagttcttt tagttgtgat gttaggttgt 26820taaattgagg tctttctttt tcatgtgggc atttgatgca taaatttccc acttaacact 26880gccttagctg tgtcccagag attctggtat gttgtatcgt tgttctcatc agttttaaag 26940aacttctcaa tttcttcctt aatttcatta tttacacaaa agtcattcag gagcaggcgg 27000ttcaacttcc atgtaattgt agggttttga atgaatttct tagtcttaat ttctaatttg 27060attgcactgt tgtctgaaag attgtttttt atgatttcag ttcttgtgca tttgctgagg 27120agtatttgac ttccgattat gtgatcaatt ttagagtaca tgccatgtgg tgatgagaag 27180aatgtgtata ctgttgtttt ggtgtggata attctataga tgtctatcag gtccatttga 27240ttcagtgctg agttcaagtc ctgaatatct ttgttaattt tttgtctcga tgatctgtct 27300aatattatca gtgagttgtt aacatctcca agtattattg tgttggagtc taagtctctt 27360tgaaggtccc taagaacttg ctttatgaat ctgggtgttc ctgtgttggg tgctgatctg 27420gtttggctgt gttcccattc aaatctcacc ttgaattgta gctcccacaa ttctcacatg 27480ccacgggagg cacctggtgg gaggtaattg aatcatgggt gcgggtcttt cccatgctat 27540tctcatcata gtgaataagt ctcatgagat ctgatagttt tataaagagg agtttccctg 27600cacaagttct cttgtcttgt ctgccaccat gtgagatgtg attttcacct tccatcatga 27660ttgtgaggca tccctagcca tgtggaactg tcagtcaatt aaatttcttt cttttgtaaa 27720ttgcccagtc tcaggtacat ctttgtcagc agcataacag actaatagag gagagtggag 27780cactgctgaa aagatatctg aaaatgtgga agtgactttg gaactgggta acaggcagag 27840gttgaaacag tttggagggc tcagaagaag ataggaaaat gtgggaaatt ttggaacttc 27900ctagagactt gttgaatgcc tttgcccaaa atgctgatgg tgatgtggac aataatgtcc 27960aggctaaggt agtctcagat ggaaatgagg aacttgttgg gaactggagc aaaggtgact 28020cattatgctt tagcaaagag actggtggca ttttgtccct gtcctagaga cttgtggaac 28080tttgaacttg agagagatga tttagggtat ctggcagaag atatttctaa gcagcaaagc 28140attcaagagg ttacttgcgt gctgttaaag ccattcagtt ttataaggga agcagagcat 28200aaatgtttgg aaaatttgca gcctgacaat gcaatagaaa agaaaatcca attttctgag 28260gataaattca agctggctgc agaaatttca tgggtaacga ggagctgaat gttaattatt 28320aagacaatgg ggaaaatgtc tccaaggcat gtcagaggtg tttttttttt ttttccagag 28380tctcgctctg tcgcccaggc tggagtgcag tggtatgatc tcagctcact gcaagctctg 28440cctgccaggt tcatgccatt ctcctgcctc agccttccaa gtagctggga ctacaggcat 28500ccgccaccac acctggctaa ttttttgtat ttttagtaga gacggggttt caccatgtta 28560gccaggatag tctcgatctc ctgacctcat gatccaccca cctcggcctc ccaaagtgct 28620gggattacag gtgtgagcca ccatgcctgg ccatgtcaga ggtcttgatg gcagccctgc 28680ccatcacagg cctggaggcc taggaggaaa gaatggtttc ttgggctggg cccagtgtcc 28740ccgtgctgta tgcggtcttt ggacttggtg ccctgtgtct cagccgctcc agctgtgact 28800aaaaggggcc aacatagagc tcaggccacg acttcagagg atgcaagccc caagccttgg 28860cagcttccat gtggtgttga gcctacgtgt acacagaagt caagagttga ggtttgggaa 28920cctccaccta gatttcagag gatgtatgga aatgcctgga tgtccaggca gaagtttgct 28980gcctgggcag ggcactcatg tggaacctct gctagggcag tgcagaaagg aaatgtggag 29040tgggcaccct cacacagagt tctcaatggg gcagtgccta gtggagtttt gaaaagagga 29100acaccatcct ccagactcca gagtgatgga tcc 29133322485DNAHomo sapiens 3cttgttaact actattgtat tctctgcttc tatgagcttg agtattttag ttacctcata 60taagaggaat tctgcaatat ttgtcttcct gtaactggct tatttcactc agcataagcg 120aaactaaaaa gcttttgcat agcaaagaaa acaataaaca gaatgaaaag ataacctgca 180gaatggaaga aaatatttgc aaaccatata tttgataagg ggttaatttc aaaaatgtat 240aaggaactca tacaactcaa gagcaaaaca accaaccaaa caaacaagct gattaaaaaa 300tgggcaaagg acttgaacag atatgtcttg gaaaaagatg ttatagacta aatgtttgtg 360ttccctccta atcatatgtt aaatcctaag ccccaatatt ataggattag aaggtgggcc 420ctttggaagg aattaggtct agagtctatc tagataaaca ctccaattaa ttgcccatat 480gggaacacaa attggtttct gatggtgtaa agttattaag gaaaaataat aacaataaat 540ggataaacag gatcaatttc ttcttaccat ggagggagga tatgtatcct caacagaaga 600ctgtgccatt taaaggccat attagtattc atcagagttg ataggaccct ttcctgtaag 660atttcagcaa tgtcatccat cgaggaactt tgggtggagc cagtctttgc accatggcac 720tatctaggtc atggctgtaa gtctacaagt gaaaaaacca tggtcaatat taatgatatt 780gcaattagta tcagcaatac cactgaagga aacgttgaca gttaaaataa tttaggaggc 840tgggcacagt ggctcacgcc tgtaatccca gcactttggg aggccaaggt caagagatag 900agaccatcct agccaacatg gtgaaacccc atctccacta aaaatacaaa aattagctgg 960gcatggtcgt gtgcacctgt agtcccagct actcgggagg ctgaggcagg agaatagcct 1020gaacccagga ggcagaggtt gcagttagtc aagatcacgg cactgcactc cagcctggca 1080acagagccag actctgtctc aaaaaaaaaa aaaagaaaaa aagaaaaaaa aaagaattta 1140gtgtttaaat cctgttctgg agagaaaata aacccatgcc tatgtatttt gcctcagtgg 1200cagaaccagt tgcttttaga actgattctt gttatgcttt tttgtttttc atagctttag 1260aacttgtaat agcaataaag attcctcact tctggaaaaa ctcaaaaatt tctaataatc 1320cttatattat cttctgctgt ggttattata aagtattttc ctctggttaa taatatagct 1380atggcttaga ttatttttta ctagctggta ccttttttct tattctaaca gttgactaga 1440catctcttta aggtcaaatc caaaattgtg aactatggac actgacaggg ttttgtttgc 1500acccaaatca tctttagcat ctctagatgg atacttaata gtaccaagtt ttacaccttt 1560atcttgctgg tgtaacccct tgaatgacca catgaccaac acagggatgc tagcaatagt 1620tagtgtggtt cttatgcttc atttttctca gaattgctgt atgatactga gagttgcttt 1680gtttgacagg cacacagggt aggaagagat gtcatgacaa aaggattagc taattctttt 1740gcacacacat aagttgattt tatttaggta aaatgttgtt aacataaata agtactagta 1800agtttacttt ttcaagataa taaattgtct gcaaggatga gctattagaa aaataactgg 1860taaaagattt tattatttat tttaaataaa ataagcaaca aaccaggcac acacttcaca 1920ggacagattg caaagaggtt ggtacagctc aacaggttca tcaatgtctt cactgtccac 1980actgcagtca ctggggagtg ggctagtgga gcaccaaaat gaaattttga aaaataacct 2040ggcctatttt ggactggata gatagttgga tacttaccac agcacaatta acataagagc 2100tagaaagata aatcaaaaga aaattctttg tacttacaat attagtagaa aggcctatgg 2160gaattctagt tgttttctct atatgcaatg gcaagcattc tgttataaaa cagctaagtt 2220gagccaaaga atgacatttg gaagttaaag aagtcctgtc aactgaccta gatagtgtca 2280tggtgtaatg attggcgcta ccaaagtttc ttaatggagg acattgctga gattttactg 2340gaaagggtcc tatcaagtgt gatgaatact aataaggcct ctaaatggca cagttttctg 2400ttcaggatac atatcttcca tgctgagaag aaactggttc tgtttatcca tttattgtta 2460ttgtcatttt ttctttagat ataaggatta gtaatctgct ttttacttta aattccagat 2520agcagtggtt gctttctgaa aactcctgag aacattttgt gactgcttgg acaagtgaca 2580gacattctat gataaccagg gcttgctgat agccaaaaaa gcctattttt ccaaaatgtg 2640ttgcaagctt tcagccttac tgcaagtgag ccccattctg tgacaatcgg taggactcaa 2700gacattctgt aatcctacca atgtcaacct gcctgttgtt cagcaaaagt ctgtgcttct 2760gattttacct gtttagtcct gagaatgctg ggatttattt cctgtaaaac caagcctatg 2820gtttcattca tcaagaccag agaccatgca acttccgtac ggaaaaccac tttcttccag 2880aaaccaattt aattgccatt tcctcaggaa ataagctctc gtatttgaca cctctcacac 2940acagcagagt tgagaggatt cttctcttgt tttcagtgca aaagttcagc ctagagatca 3000cctgtacgat tgatggtttg gtgacagttg ctacattgtt tagcctgaac tatgcttata 3060ttgaatattg aaggaaagtg tgagatatat aatttaaaga agcatatctg ttatagttta 3120agtgtgtcgc ctttaaaatt caggtaggac tcctcactga tgaatgggat taaggccctt 3180gtaaaaaacg cttcacacag tttttagcct cttgtctttc caccttctac catgtgagaa 3240cacagtattc atcccctctg taggatgcaa ggtgccatct tggaagtgga gagcatgccc 3300tttgccagac attgaacctg ctggtgcctt aatcttggac ttctcagcct ccatgtattc 3360tttataaatt acccagtcta ccgtatttta tcacagcaca aataaactaa gacaacatct 3420ttgttactca taaatgtata aaacatacag aatttagttc attgccattg gccatttttt 3480aaatataaat ttgtatgatt tatgaatagc gtgtcaaatt tatataaaca aattttgaaa 3540atttctcttt aactatattt tagtacattc ttgatgtaga attacttatt tttgcctctg 3600catttatcct gtaataataa ggtgaacctt agcttccttt cctagattac cacaccacaa 3660ttagttaatt agtaaattac gatttcctat tatcaaatga aatgtgatat tctcctggtt 3720gcaattgcac aattgtcaat agtactgaat tatcactgta gtgtaggaac acagtttgtt 3780ctcaaatcca gggacttcta cccaacctct ccaagaaatc ttcaactttc cacattaaca 3840gaaatatatt tttccaaagt aaacgagaca ctatttttat tctattctga aatgatcaaa 3900cttgcactac ttcgtgctga gaaattagaa atgatgattg agagtaataa accggagtta 3960ggaatgttga ggctgttgtt atctttaata agaaggagat ttgtgcagga gctatgggtg 4020tccttatgta acataaacgc aaactgtgtc atttccagag gagagtaacc atgatgatga 4080ggggaatctt ttgaaggaac tagcattgct acacagtatc taccccaaag tctataaaat 4140gttgccattc actaaaagaa gtagtcttac tgatttgcac agccatgaat taaagggata 4200aaaataattt tagtataagg gacataattc tctttagaaa ttgaatgtga ggtagtataa 4260tacaacagta gagcctgagg ggtttggaat cacatatata ataccttggt tcaatagagt 4320tgacagaaaa actctgcttt aaaataatta atattttatg tgaagagtgt tcaatccctc 4380attcctggct cccattatga tctcctcatt tgtttgaggc tatggccctt tactattcca 4440cttctcttgt tttatcataa agggagatat aagaagactt tgctggccgg atgcactggc 4500tcatgcctgt aatcccagca ctttgggagg ccgagttaca attctgagaa ttgctacaat 4560tctgagtaca aagcaaaatg ctcaaaaatt gctgaagaaa ttttagtcat ttttattgca 4620gcatggtgag tatcccaacc ctagaaacac taaggcacac aaggaaggag tgtgtatcag 4680aactgtggta gggtgtgaat taatgcagaa ctttatctgt atagttgtac tttgaagtcc 4740attctgaatc ttagatgcta catttatata aatataaagc ataataagta tctaaatgta 4800gaattatatg tttaaaatta tatgattaca ttaactgatg taattcatag attttcccta 4860gggttctgtt tcctgaacat tctgtaacgt attagttagc aaagtctttt ttttttttga 4920aactgagtct cactctatcg accagactgg agtgcagtgg catgctcccg actcactgca 4980acctctgcct cctggaatca agcaattctc gtgcttcagc ctcctgagta gctgggatta 5040caggcatgca ccaccacacc cagctgattt ttgtattttt tctgttagta gagacaaggt 5100ttcaccatgt gggccaggct ggtttcaaac tcctgacctc aagtggtcca cccacctcgg 5160cctcccaaag tgctgggatt acaggcatga cccactgtgt cctgccagca aagtcttctt 5220atatattccc ttatgataaa acaagagaag tggaacagta aagggccata gcctcaatca 5280aatgaggaaa tcctaatggg aaccaggaat gagggattga acactcttca cataaaatat 5340taattatttt aaaactattg ggggaaattc agccagatat caggcaaaat tcacccccga 5400tatttcacgt agtttctttt ctatattccc taagtgtcgg ccggtctgag aaataaaggg 5460acagagtacc aaagagagaa attttaaagc tgggtgtccc caggagacgt cacatgttgg 5520caggttctgt gatgccccac aagccacaaa accagcaagt ttttattagt gattttcaaa 5580aggggagggg gagtgtatga atagggtgtg ggtcacagag atcacatgct tcacaaggta 5640atagaatatc acaaggcaaa tggaggcagg gcgagatcac aggaccacag gaccggggcg 5700aaattaaaat tgctaatgaa gtttcgggca ccattgtcat tgataacatc ttatcaggag 5760acagggtttg agagcagaca accggtctga tcaaaaattt attaggcggg aatttcctca 5820tcctaataag cctgggagcg ctatgggaga ctggggttta tttcatccct aagcttgacc 5880acagaagacg gccaccccct gaagcagcca tttcagaggc ctaacctcag ggaagtattc 5940tctttctcag ggatgttcct tgctgagaaa aagaattcag cgatatttct cccatttgct 6000tttgaaagaa gagaaatatg gctctgttcc acgtggctca ccagtggtca gagtttaagg 6060ttatctctct tgttccctga acattgctgt tatcctgttc ttttttcaag gtgcccagat 6120ttcatattgt tcaaacacac atgctctaca aacaatttgt gcagttaaca caatcatcac 6180agggtcctga ggtgacatac atcctcctca gattacaaag atgacaggat taagagatta 6240aagtaaagac agggatagga aatcacaagg gtattgattg gggaagtgaa gtgtccatga 6300aatcttcaca atttatgttg agagattgca gtaaagacag gtgtaagaaa ttataaaagt 6360attaatttgg ggaactaata aacgtccatg aaatcttcac aatctatgtt cttctgccat 6420ggcttcaggc ggtccctcca ttcggggtcc ctgacttccc gcaacaaaaa cagagttgtt 6480ctgtcaacag ctgactttga gtccttgatc ggtctctcag acccctgaat acttggattg 6540cacagttgac cttatcacat tgttagggta agtgcataca aaggcaactt cagaccctcc 6600attgcacata ggtggcccct gcaagccgct tgcctgtgtg tgttctggag ctgccactaa 6660acttggggac agcatcagga gatacacttg aaaaaaccct ttttactcag attaaattat 6720taacaaactt tccatttcct ttaacttact aaagaatctc tacctgtaaa taggtacaga 6780ttaaactcgc tagtcaacag ctatcattct gtcatatcaa cagatactcg tggctgctgc 6840tccttgaggc atccacagaa tcacagcatt ttccagtatt gaaagacctg aaagatcacg 6900gtgccttcat tttaactgtg agacatgaag taattttccc aagtctacaa cagtaagata 6960tggtgcaata aggaccagat taaaagtctc ctgatttgca accatgttcc ctccatctcc 7020tttactccta agcacactca cacactcact cctgcaaaca attctcttgt caagtgggaa 7080atgaatgctc ttacaaggct caaatttgtg aacacatcac tgaccagcac agagctggct 7140aacaataggg acacaattaa ggtgttttac acgcaactgg ttcaaacctt tcaagtacta 7200aattaaaaca atcctttaaa gaaggaaatt gtttcagaaa aggaccttca tacagcatct 7260ctgaccagcg actgatgatg ctattgtact cagatgctga ttcgttctcc aacactagat 7320tacccaatcc acgagcaagg aaatcagtaa cttcttccct ataatttgga atgtgggtgg 7380agaggggtca tagttctccc tgagtgagac tcacctgctc ctctggcccc tggtcctgtc 7440ctgttctcca gcatggtgtg tctgaagttc cctggaggct cctgcatggc agctctgaca 7500gtgacactga tggtgctgag ctccccactg gctttggctg gggacacccg acgtaagtgc 7560acattgtggg tgctgaccta ctatggggtg gggaaaaaag ggagttgtgt taacattgtg 7620cccaggccat gtcccttaag aaagtgtgac attttcttca gggattgccc atctttatca 7680tatggatccc aaattatttc caccacaaat ggaacttggc tacttgccct attcatgaga 7740ctgtgtaaag ggcctttgta caggccatgt tttactttaa atctctacca ataaaacctt 7800tgcatcacat gtcctcaggg tctttagagg atttagaaat aaggatgcta aaataaattc 7860ctcatacagc acttcccttt atcatgttga cttatgtcag acgaaacaag gttttgtttt 7920gaaaattttg tgggagtcaa aggaattcaa agggtctctc ctagacgatc ctgtgttgtc 7980ctccacagga cctgtggtgt tggcccctct tcctcatatg tgaggatgta cccagtggcc 8040tccccattgt ttcctttctt ttttttctga actccagtgt ttataaagcc tgtatccctg 8100tagcatatgt aggttctctg acagaagtta tacttagtgc tctttctttc ttatggggaa 8160aaatccctgg atctgaaact gacatcttta gtacttggag tcaccctaca ggtaaagacc 8220atttatgagg tattcattgg tgcctcctct tgatcggtct ctcagacccc tgaatacttg 8280gatactcctc aagaacttaa ggcatcctct gaaaaactgg cccagattag tgcttattat 8340taatctttta taacctttct atacttgttt ctcctgcatg ctctaactag acatgacaga 8400agagattcaa ctaacatagg ataaattata tgaaattcta tttttgtaag tcaaaaatag 8460tcaaatacca gaaaattaat aatgttcaaa ctatatactc tgtgtggggt taccgagacg 8520acgtggacat tgttcacatc taatagggct gaaagtcaat gaagaagtcc tggaaactcc 8580ttgtcttact ggggtcttgt cctaaatttc ataggttcac ccatcatgcc ctcagctttc 8640cttaattagc catgtctgct tatctctacc tccagtttct ctctattttt ccccagctat 8700gttgtcatca tttccagaaa tctctaaaac ttgcaaagat ccttagcact atgagatcca 8760ttgaaagaga taattttttt ctttttgaga cagggcttgg ttctgtcacc caggctgtag 8820tgcagtggtg tgatctaggc tcactgcaac ctctgcttcc cacgctcaag tgatcctccc 8880tcctcagcct ccagagtagc ggagactaca ggcaggcaaa catgtgcagc taattttcat 8940gattttgtta gagatgagat tttgccatgt tgcccaggct gttcttaaac tcctggactc 9000aagcaatcct cctgccttag cctcccaata tgctaggatt atagatgtga gccattgtgc 9060ccaggcaaaa agagatgaac cttaatttaa aaatttcctt tttcttaaat cactgtttct 9120ctatctgtga attcttcttc caactagaag gaggagaaag aagaagtttg cctgtatttc 9180tcaccaggag gaggagtcta gtgtgatatc aaaatgaaag agtgctggag cttgatcccc 9240ttcttgcttt ccaggatccc tgcagtgatc agttcccaca ccctggttta ttcatgtaaa 9300gcacacttat ttttttcagc agctactctt tactgggctc cattctaagt tcaaatcatt 9360ctatttgagt aagatagaga gggtcccgac tctcatggaa gttacacaag agtagaggag 9420acagacacta acccaataag catttaacaa agaagaaaat gttagagaga catagtgcac 9480tgaagaaaag acatcaggtt tgtgaaaaag agagacatgg attcacttac tttggttcat 9540atgcttaggc agctataact gagaaagtga cattcagctg agacaacaaa ataaatagac 9600agtcgtgaag atctaaagga cgaaagttcc agggagaatg aatggggggg aagctctggt 9660gtgggaaatt atgtggaagg acagaaagaa ggctagaggg actgaactat agcaagcaag 9720gaaatggaga ggcagaagat gaggtaggac acagagagga agtcaggagc ctcatcatat 9780tagactctga tggccatggt aaaaaaattg aattttattt tatttttatt tattttttga 9840gacggagatt tgttcttgtt gcccaggctg gagtgcaatg gcgcgatctc gactcactgc 9900aacctctgcc tcctgggttc aagtgattct cctgcctcag cttcccaagt agctgggatt 9960acaggtgcct gcgaccatac tcggcttatt tttttgtatt tttagtagag acagggtatc 10020accatgttgg ccaggctggt ctcaaactcc tgacctcaga taatctgcct ggcttcccaa 10080agtgctgaga ttacaggcgt gagccaccat gcccaacctg aattttattt gaatagatat 10140gagaagctac tgtatggtta caaggacagt caatttatat tcgatttttt ttttttgaga 10200cagagtcttg ctctgttgcc caggctagat tgcagtggta caatctcagc tcactgcaac 10260ctctgcctcc tgggttccag caattctcct gcctcagcct cccaagtagc tgagaccaca 10320ggtacatgcc actacacctg gctaattttt tgtattttta gtagagatgg ggtttcaccg 10380tgttagccag gatggtcttg atctcctgac ctcgtgatcc actcccctcg gcctcccaaa 10440gtgctgggat tacaggtgtg agccaccacg cccggcctat attcaattat taaaattaat 10500tctagctact ctgtggggat tggattgttg ggtttcacaa gtggtcagga agactattta 10560ggatcacagc agggaattct ccagggaaaa caggcttgtg gcttcataga gtgcattagt 10620gataaagaca gtgaaaacga caaagtggac agactaggca tgtatttttg cttagcttgt 10680taatggatta ctctaaaggg ggtagaaaaa tcaagcttat tcctaaggat tttgttttga 10740caaataagtg gatggtggtg tttattgaga taggaaaaac tgtgggagga aatgatttga 10800agtgggtggt tggaaataaa agttttgttt aaatttgaga tgatttattg acatttatgt 10860ggagcaatcc gaaggtcaat ggcatttaag agactcatgg tgaggtgagg ccagggcttc 10920aggtatttat gttggcggca tcagtacgtg taatgtgtta aattccaggg agtggaagag 10980gatacatagg gagatggatt gtgtggagaa aaaagaagag ggtacaggcc agcaaagggg 11040gctgagacag agcccaggga tgctggagaa aacccaagag aacataatgg gtgtaagtca 11100tggaaaatag attattttca aggagaaggg agaggtcaat tgtggtgagt accactaaga 11160ggagggggaa gtgagaacgt gacagagaag caagtgctgg gtttgctgga gttgatattt 11220gcagtcaatg gagtatccag ggaggaaact ggattggacc atttgaagag caagtagaag 11280tgaggacgag gttaagggtg actattttaa gtagagagct tcagggaagg actgtgctct 11340gggttcaggg

agcctgctgg atctaaagga aaagggctga agaggctgaa gagaaggagg 11400aggacctgtg aaccagagat actgagttat tattagcaag gaaatactag agggtccctg 11460tgtgcagtgc tgactgctca tgcaaaaggt cacacagaca atatttcaca cagccagtat 11520ttattagtga catagaatat gccagttatt actctaggtc atgagaatag agtgataaat 11580aaaatgaatc tggtcgccat cggtatatgc catgtaacat tttgcagtga ctgtgtacca 11640ggcctatgaa tttcagtatg caatttcaat aacgatcctg ttgtatctgt ggtgtttaaa 11700aacatataca tctctggaat ctaaaattga gaggatataa gtaaaaccca gtattagaaa 11760tttagtgctg gaaatcagac tgcagtttaa atctgagcat atagaaagtc cctttcttct 11820atgtcagcag atgccttttg tgtgaggttt aggtatacta cattattaga cataaaccag 11880tgattctgcc ctatgttttc agaatgacaa ttctttatga aactaataga agaacagaag 11940acaattgcaa aatcatgatg aagatgctag tggctttaga accaaggaat acaaaaaata 12000atgtgagctg cagttatagg gattataaaa gttaaaatgg gaatgcattt gagtgtttat 12060tatgtgatca gtgctaataa gagtcatcat ttaattttac acttaacaat aatcctgtga 12120ggattaagct attattaaat gcatttgata gattacaaaa aggcttaccg ttggtaaaaa 12180ttgacccaag gggaagaggt cacattttta ttcagatttt ctgattctag agtttgagag 12240tctgtccatc attagtgagt agtgacaata ctgtgtctaa attatcgaca gaatttctga 12300tattcatatg tactatgttg tttcttagag tgtgggcaga gattcagggc tgctagttcc 12360aatgtatagg agaaactttc attcattgtg catttatcat tttaaaagtt ctaggctggg 12420tgcggtggct catgcctgta atcccagcac tttgggaggc caaggcgggc agatcacgag 12480gtcaggagat gaagaccatc ctggctaaca tggtgaaacc tcgtctctac taaaaataca 12540aaaaattagc tgggcgtggt ggtgtgcacc tgtagtccca gctacttggg aggctgaggc 12600aggagaatgg catgaacctg ggaggcggag cttgcagtga gctgagatcg tgccactgca 12660ctccagctcc accctgggca aaagagcgaa actccgtctc aaaaaaaaaa aaaagttcta 12720tatgtctgtc atggcatatg ttgaagaaca caaggaagta ttaaatcact ccttctgagg 12780tttgtctagc aagttgggct aggattgcca aataaaatac aggtttctag ttaaatctga 12840atttcagata cacaactata atttactgaa aatccaaatg taacttggca tcctctgatt 12900ttatttgcca aatctgtcaa ccctacatga gacacatgag catggattac ggtgttaccc 12960atggaagcca cagccacagt gacagcgact tcacacatgt ttatttttta actttctctc 13020tgtaaagaaa gtgcttagat aatttaggga taaaaagata gacattgttt gatccaggat 13080gcactcctct ctgccatcgt ttctaaaggg caaagagaga tttccacagg tcttactcac 13140agtctgactc acagtctggg gacctgctca tgctttgaaa ctgtctgtat gagaatgtca 13200ttttcttggt ttctcccttt ctgaggggac ttgactacaa aactgagagt tctacctctg 13260gccaaggctg gaaatttgat gcctgctagt attgttggga atgggagact gaaataaatg 13320agttagttgg ggcattaaac aggaataaaa tagctgtggt tgtgattcat tactacaatt 13380agtggactag tggcagagaa attaagaaag aagatgatgt gagagataaa ttatatgatt 13440tggtaaggca agggaatcag taaatcttgg ttctgaacaa gttcattttc tggaaagata 13500gcactgtact gggaccagaa ttctacaaaa catccgtttt atgtaagacc aagattttca 13560acaaatattt ttcaatgcag ttctcagctg ctccataact aatagtgact tattcaacac 13620agatattttc agatggttca cacccatgtt tcttacccag ggacagttca ccacccctcc 13680ccttccctcc catcactctt gaggaacatg tggcaatgtt agaataattt ttggttgtca 13740caacaggggt ttcttctgat atttaatgag cagaagccag ggacactgct agagaaccca 13800caatgttcag aatagactcc atcaccaacc aagatttatc tcgtccaaaa tgtcaatagt 13860gctgaggctg gaagcattgg ttcacactgt gctctttctg aaaaatgtag actcgctttt 13920tttttttttt tttttgagat ggggtcttgc tctgtcgccc agactggagt gcagtggctc 13980catctcagct cactacaacc tctgcctccc aggttcaagc gattctcctg tctcagcctc 14040cccagtagct gggattacag gtgcaccctg ccatgcccgg ctaatttttt gtattttagt 14100agagatgggg tttcaccatg ttgcccaggc tggtctcgaa ctcctgagct caggcaatcc 14160acccgctttg gtctcccaaa gtgctaggat tacacgcatg agccaccgcg cccggcctag 14220actcacatct tttatacact tactgcccaa ttcagttctt tatggtttat ttttgcttgt 14280ttcattataa aaaactagac agttgcataa attcaaccac ttacttgttg aatccattta 14340gtcaatgcaa gctcaacatt ttcatattta ttttttgcct tatgcaatat tgttcaacat 14400tttcataagt tgttggtcag cactatctct attaactttc aacagtttgc ccttctaagt 14460cacaaatagt gatgctgctg caattatttt tcactaacat gcctcagatt tctgtagtga 14520ttctacattt gatattattc acaatgtaaa atgcttctat ttattcattt cacttttacc 14580cacaggatta tttttaagtt atttttgtca ttttcacact tcaaccaaac ataaagacaa 14640aaacatcaaa aatatgtaca tagtgttata cataggtgta tatttacaca catatatgca 14700catatgttta tatgtattga aactacagaa gcacatgtca ccaataagag ctctgagaca 14760cctttgacca cttaccctta tcagatgaga tttgccaaat gagttttggg aacaaatttc 14820ttttaactga atttctgagc tttgtggatt tagaaatgca actgaaagtt tgtggacatt 14880tacgaggatc atagttttat tctccttaaa actcttcaat actttcccat tgtctttagt 14940aaatccaaaa tcctaacacc actcacgagg cttttcaaca cctggcttct tgtgatttct 15000ccaatctaac cttttaccct ccttcccctc agcctctctg ctttagtgaa ctttgttcta 15060gttttttgaa gttcatcatc aattcaagct tttgtacatg ggatttccta aacctgaaat 15120gtgcctccgg ttttgtccaa acagacacac aggctccact ctgccccctg gctcacacct 15180gcttaacttg ttaagtcaca tctgtaactg tcactcttct ctggcaccct aaaggaattg 15240agatcatcct attattctct gttctagaac tccacacttc tgaaatttct cattcctgtc 15300taagctcttg tgtgtttggt ttttggccat cactttcact gctcttaaag ctcccccagc 15360ggagtggaga ggtctgtttt cccgtgtttg gattcctaga ggcagcgcag gcctggcaca 15420aggtcatcac taaggaagtg ttcacaggat gaaagcggtg cgtgctgttt aaggaaaggg 15480taaagccttt aaatggtaaa gggttgagag aaggagcaaa gtgcctttgg ggtggaggct 15540cccaggagga ggcggcgcgg gctgcggtgc tggacggatc ctcctccagc tcctgcctgg 15600aggtctccag aacaggctgg aggcagggag ggggtcccaa aagccttggg atcagaggta 15660gtttttccac ctggtccccc agacccccgt ccgcctcaga aagacagagg atgagcccct 15720gggctgcgtg ttgtcggggt tgcgggtggg gccagatagt gtcttccccg gaggccgctt 15780ctgtaaccgg atcgttcttg tccccccagc acgtttcttg gagcaggtta aacatgagtg 15840tcatttcttc aacgggacgg agcgggtgcg gttcctggac agatacttct atcaccaaga 15900ggagtacgtg cgcttcgaca gcgacgtggg ggagtaccgg gcggtgacgg agctggggcg 15960gcctgatgcc gagtactgga acagccagaa ggacctcctg gagcagaagc gggccgcggt 16020ggacacctac tgcagacaca actacggggt tggtgagagc ttcacagtgc agcggcgagg 16080tgagcgcggc gcggggcggg gcctgagtcc ctgtgagcgg agaatctgag tgtgtgtgtg 16140tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgagagag agagagagag agagagagag 16200agagagcgcc atctgtgagc atttagaatc ctctctatcc tgagcaagga gttctgcggg 16260cacaggtgtg tgtgtagagt gtggatttgt ccgtgtctgt gaggctgttg tgggagggga 16320ggcaggaggg ggctgcttct tattcttgga gacttctgtg gggaggtgac aagggaggtg 16380ggtgctgggg gctggagaga gaggcgacct tgattgtctc gggtccttag agatgcaagg 16440aagggaaatg tatggggtgt gtggttgggg tgaaggttta ggggaggaga gctgaggggt 16500aaggaaggtt tgggataatg tgaagaggcc agtttcagac tgtccctggc acacaccctt 16560catgtaatct ctgaaataaa agtgtgtgct gtttgtttgt aaaagcatta gattaacttc 16620taggggaatt gagtagacct ctgaggcacc tctgaagctt ctttaggtat aaatttcttg 16680ctagtttttt gttttcttag tgttatattt ttacatagtt gaaatgactg tgaaactaac 16740tttttgaatt aaagtttgaa aacactgtta ctattttatt ataatgctaa taatttcata 16800gttacttttt aaatatataa tagttgtgac acaaattacc tcactttctt tgtttttttt 16860tttcttacac tttaagtttt agggtacatg tgcacaacgt gcaggtttgt tacatatgta 16920tacatgtgcc atgttggtgt gctgcaccca ttaactcgtc atttaacatt aggtatatct 16980cctaatgcta tccctcccca cccccccacc ccacaacagg ccccagtgtg tgatgttccc 17040cttcctgtgt ccatgtgttc tcactgttca attcccacct atgagtgaga acatgcggtg 17100ttcggttttt tgtccttgcc atagtttgct gagaatgatg gtttccagct tcatccatgt 17160ccctacaaag gacatgaact cattcttttt tgtggctgca tagtattcca tagtgtatat 17220gtgccacatt ttcttaatcc agtctatcat tgttggacat ttgggttggt tccaagtctt 17280tgctattgtg aatagtgccg caataaacat acatgtgcat atgtctttat agcagcatga 17340tttataatcc ttgggttata tacccagtaa tgggatggct gggtcaaatg gtatttctag 17400ttctagatcc ctgaggaatc gccacactga cttccacaat ggttgaacta gtttagagtc 17460ccaccaacag ggtaaaagtg ttcctatttc tccacatcct ctccagcacc tgttgcttcc 17520tgacttttta atgatcgcca ttctaactgg tgtgagatgg tatctcattg tggttttgat 17580ttgcaattct ctgatggcca gtgatgatga gcattttttc atgtgtcttt tggctgcata 17640aatgtcttct tttgagaagt gtctgttcat gtcctttgcc cactttttga tggggtattt 17700tgtttttttc ttgtaaattt gtttgagttc attgcagatt ctggatatta gccctttgtc 17760atatgagtag attgcaaaaa ttttctccca ttctgtaggt tgcctcttca ctctgatggt 17820agtttctttt gctgtgcaga agctctttag tttaattaga tcccatttgt ccattttggc 17880ttttgttgcc attgcttttg gtgttttaga catgaagttc ttgcccatgc ctatgtcctg 17940aatggtattg cctaggtttt cttctagggt ttttatggtt tcaggtctaa catttaagtc 18000tttaatccat cttgaattaa tttttgtata agcaaattac gtcactttcc ccattgatga 18060cctttattat gacattcacc aatagttgaa aatgtatgtt tctggttaat ttttgattta 18120tatttttttg atttgtaatt attttgaatt attttgacct atttattggc cagttgtaat 18180tactgctctg ctctacgaat tacctgttgt atttggtagg taatggacaa tgatctattg 18240tctcttatct ttagggctta gtatttttct cagtgacttt gtgggtttgt tgtactgtaa 18300gattattaac actttattga tatttgattc agtattttct ccagtttgtg gtatgtatat 18360tttgaaaatt cttttccatg ttaagaattt gaacattttt atttaataaa atatattgca 18420aaatgttaat taatgattca caaactagct caagtctacc attttgtggt attgatgtct 18480ccaggtttct ccttccttct taaaaaaaaa tgtatttatt gagagtatgc tagtgtcagg 18540gatttcccta ggcataagca ctccaagtaa tgagtcccag acactgcctt gatccaaatg 18600tcattctgga aagaaaaatc attttacagt gataagccta ataatagtta tacttgtttt 18660gcctgggaga tgcattgatc agctaaatgt aaatataaga actttcaaaa ctaaaatgac 18720gttccttaat ctttctctct gctttaggaa tcatgctttc ttaggaactt aaagatttgg 18780agaatcattt ctgtctgtcc caccttccca ggagcataac catttctgtg gtgttctaag 18840gtgtgagtgc atggcagtag tattcctaaa aatccatatt cagtttcctc atgtgcccta 18900ctccgtccct ttctctatcc acattgcttt aaatcatatt tttctctcaa ggtgtacaag 18960gatgataaat aggtgccaag tggagaaccc aagtgtgacg agccctctca cagtagaatg 19020gagtgagaag ctttctgacc tcataaattg aaggctatcg taattcattc ttttatatat 19080tttacttgca ttaatcctca tataacctca agaggtaaat taatataatt atcctccatt 19140attggagaga aagttgagac acaaaagaat caaaaactct tccaggatca accagtaaaa 19200ggcagacctt ggatttgaac caggcaacct ggctcagaag tcagttttaa ttaccacact 19260ctgtactttc aaagatttgt aaacgctttg acaatgcatg tcaatttcaa gctatgaaga 19320gccaaacata atttttcaca atatctctca aatctaatgg gtccccacta taaagattaa 19380attccaggct gatgacactg tgaggccaca tggccagctg tgctggaggc ctgctcaagg 19440ccagagccta ggtttacaga gaagcagaca aaaagctaaa caaggagact tactctgtct 19500gcatgactta ttccctctac cttgttttct cctagtctat cctgaggtga ctgtgtatcc 19560tgcaaagacc cagcccctgc agcaccacaa cctcctggtc tgctctgtga atggtttcta 19620tccaggcagc attgaagtca ggtggttccg gaacggccag gaagagaaga ctggggtggt 19680gtccacaggc ctgatccaga atggagactg gaccttccag accctggtga tgctggaaac 19740agttcctcgg agtggagagg tttacacctg ccaagtggag cacccaagcc tgacgagccc 19800tctcacagtg gaatggagtg agcagctttc tgacttcata aatttctcac ccaccaagac 19860gcgaacttta ctaatccctg agtatcaggc ttctcctatc ccacatccta ttttcatttg 19920ctccacgttc tcatctccat cagcacaggt cactgggggg tagccctgta atactttcta 19980gaaacacctg taccccctgg ggaagcagtc atgcctgcca ggcaggagag gctgtccctc 20040ttttgaacct ccccatgatg tcacaagtcg gggtcacctg ctgtctgtgg gctccaggcc 20100ctgcctctgg gtctgagact gagtttctgg tactgttgct ctgagtcgtt tgttgtaatc 20160tgagaagagg agaagtatag ggaccttcct gacatgaggg gagtccaatc tcagctccgc 20220cttttattag atctgtcact ctaggcaact acttaacctc attgggtctc aggctttctg 20280ttcatcagat gttgaagtcc tgtcttacat caaggctgta atatttgaat gagtttgatg 20340actgaacctt gtaactgttc agtgtgattt gaaaaccttt ctcaagaaat ggtcagttat 20400tttagttctt gcagagcagc cttctttctc attttcaaag ctctgaatct caaggtgtca 20460attaaagagg ttccatttgg gataaaaatc actaaacctg gcttcctctc tcaggagcac 20520ggtctgaatc tgcacagagc aagatgctga gtggagtcgg gggcttcgtg ctgggcctgc 20580tcttccttgg ggccgggctg ttcatctact tcaggaatca gaaaggtgag gagcctttgg 20640tagctggctg tctccatacg cttttctgga ggaggaacta tggctttgct gaagttggtt 20700ctcagcatat gaatggccct ggataaagcc tctctactcc caaatgacct ccaatgttct 20760gcaaatccag aaatcatcag tgcatggttg ctatgtcaaa gcataatagc ttgtggccta 20820cagagataac agaaagatta acaggtatag gtgctttggt tgagatcgtg gagcaaatta 20880aggaagagca actaaagcta atacaattac actggatcct gtgacagaca cttcacactt 20940catgggtcac atggtctgtt tctgctcctc tctgccctgg ctggtgtggg ttgtggtgtc 21000agagaactct caggtgggag atctggagct gggacattgt gttggaggac agatttgctt 21060ccatatcctt taagtgtata tcttctcttt ttcctaggac actctggact tcagccaaca 21120ggtaatacct tttcatcctc tttaagaaac agatttggag gccaggcgca gtggctcacg 21180cctgtaatcc cagcactttg ggaggccgag gcgggcgaat catgaggtca ggagttcgag 21240accagcctga ccaacgtggt gaaaccccgt ctctactaaa aatacaaaaa aaaatcagtc 21300gggcgtggtg gtgtgcgcct gtaatcccag ctactcagga ggccaaggca ggagaatcgc 21360tggaacccag gaggcagagg ttgcagtgag ccgagattgg gccactgcac tccagcctag 21420gtgacagagt gagaccccat ctcaaaaaaa caaaaaaaag aaagaaagaa acagatttcc 21480tttccctaga atgatggtag aggtaataag gcatgagaca gaagtaatag caaagacatt 21540ggatccaaat ttctgatcag gcaatttaca ccagaactcc tcctctccac ttagaaaagg 21600cctgtgctct gcaggagtat tgactcatgg agacttcaga acttgttttt cttcttcctg 21660cagtgctctc atctgagtcc ttgaaagagg gcaaaataaa ctgttagtag agccaggtct 21720gaaaacaaca ctttcttgcg tctctgcagg attcctgagc tgaagtgaag atgaccacat 21780tcaaggaaga accttctgcc ccagctttgc aggatgaaac acttccccgc ttggctctca 21840ttcttccaca agagagacct ttctccggac ctggttgcta ctggttcagc agctctgcag 21900aaaatgtcct cccttgtggc tgcctcagct cgtacctttg gcctgaagtc ccagcattaa 21960tggcagcccc tcatcttcca agttttgtgc tcccctttac ctaatgcttc ctgcctccca 22020tgcatctgta ctcctgctgt gccacaaaca cattacatta ttaaatgttt ctcaaacatg 22080gagttaaaaa tcgtctggtc atttggcccc aaggacaaaa aataaaaaga aaagaaaaag 22140tgaagattat ttcccgatag aataatggtt ttcatggata tgtcataagt atgtgagata 22200gtgcatatgt taaataggtt gatttagaca ttttacacta caggcatata tcaaaacttc 22260atgctgtatg acataaatgc acaattttta cttgtcaatt taaaaagtaa acctaacgtt 22320taaaaaggtg atgcataaaa actgagaaca gactataaga actgaaacaa acttggcaaa 22380catgagatga taaaccagct agcaagtcaa tcagaactct ttctcaaccc cgtctacaat 22440attgtgtgtc tataactgta aattagtata tagtttttca ttcca 22485415PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 4Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 15531DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 5cattgagaca gagcgcctgg cacagaagca g 31636DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 6ggatgacgtg agtaaacctg aatctttgga gtacgc 36724DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 7ttcttcaacg ggacggagcg ggtg 24824DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 8ctgcactgtg aagctctcac caac 24920DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 9ctccaagccc tctcccagag 201020DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 10atgtgcctta cagaggcccc 201110PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 11Gly Leu Ser Pro Thr Val Trp Leu Ser Val1 5 10129PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 12Trp Leu Ser Leu Leu Val Pro Phe Val1 5138PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 13Ile Leu Ser Pro Phe Leu Pro Leu1 51416PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 14Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser1 5 10 151513PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 15Arg Gly Leu Tyr Phe Pro Ala Gly Gly Ser Ser Ser Gly1 5 101626PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 16Met Gln Trp Asn Ser Thr Thr Phe His Gln Thr Leu Gln Asp Pro Arg1 5 10 15Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly 20 251715PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 17Thr Ser Leu Asn Phe Leu Gly Gly Thr Thr Val Cys Leu Gly Gln1 5 10 151821PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 18Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr1 5 10 15Val Trp Leu Ser Val 20199PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 19Gly Leu Ser Pro Thr Val Trp Leu Ser1 5209PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 20Trp Leu Ser Leu Leu Val Pro Val Phe1 5

Claims

1. A transgenic mouse comprising: a) a disrupted H2 class I gene; b) a disrupted H2 class \I gene; and c) a functional HLA class I or class II transgene.

2.-7. (canceled)

8. A method of simultaneously identifying the presence of one or more epitopes in a candidate antigen or group of antigens, wherein the epitope elicits a specific humoral response, a TH HLA-DR1 restricted response, and/or a CTRL HLA-A2 restricted response, the method comprising: a) administering the candidate antigen or group of candidate antigens to the mouse of claim 3 or claim 6; b) assaying for a specific humoral response in the mouse to the antigen; c) assaying for a TH HLA-DR1 restricted response in the mouse to the antigen; and d) assaying for a GTRL HLA-A2 restricted response in the mouse to the antigen; wherein, observation of a specific humoral response in the mouse to the antigen identifies an epitope which elicits a humoral response in the antigen; observation of a TH HLA-DR1 restricted response in the mouse to the antigen identifies an epitope which elicits a TH HLA-DR1 restricted response in the antigen; and observation of a GTRL HLA-A2 restricted response In the mouse to the antigen identifies an epitope which elicits a GTRL HLA-A2 restricted response in the antigen.

9. The method of claim 8, further comprising assaying for a Th1-specific response in the mouse to the antigen and assaying for a Th2-specific response in the mouse to the antigen; wherein observation of a Th1-specific response In the mouse to the antigen identifies an epitope which elicits a Th1-specific response In the mouse to the antigen; and observation of a Th2-specific response in the mouse to the antigen identifies an epitope which elicits a Th2-specific response in the mouse to the antigen.

10. A method of identifying the presence of an HLA DR1-restricted T helper epitope in a candidate antigen or group of candidate antigens, the method comprising: a) administering the candidate antigen or group of candidate antigens to the mouse of claim 3 or claim 6; and b) assaying for a TH HLA-DR1 restricted T helper epitope response in the mouse to the antigen; wherein, observation of a TH HLA-DR1 restricted T helper epitope response in the mouse to the antigen identifies an epitope which elicits a TH HLA-DR1 restricted T helper epitope response In the antigen.

11. An isolated antigen comprising an HLA DR1-restricted T helper epitope identified by the method of claim 10.

12. The isolated antigen of claim 11, wherein the antigen further comprises an epitope which elicits a humoral response and/or an epitope which elicits a CTRL HLA-A2 restricted response.

13. The isolated antigen of claim ii, wherein the antigen comprising an HLA DR1-restricted T helper epitope comprises a polypeptide.

14. The isolated antigen of claim 11, wherein the antigen comprising an HLA DR1-restricted T helper epitope comprises a polynucleotide.

15. The isolated antigen of claim 14, wherein the antigen comprising an HLA DR1-restricted. T helper epitope comprises, DNA, RNA, or DNA and RNA.

16. A method of Identifying the presence of an HLA-A2-restricted T cytotoxio (CTL) epitope in a candidate antigen or group of candidate antigens, the method comprising: a) administering the candidate antigen or group or candidate antigens to the mouse of claim 3 or claim 6; and b) assaying for an HLA-A2-restricted T cytotoxic (CIL) response in the mouse to the antigen or group of antigens; wherein, observation of an HLA-A2-restricted T cytotoxic (CTL) response in the mouse to the antigen or group of antigens identifies an epitope which elicits a an HLA-A2 restricted T cytotoxic (eTL) response in the antigen group of antigens.

17. An isolated antigen comprising an HLA-A2-restricted T cytotoxic (CTL) epitope identified by the method of claim 16.

18. The isolated antigen of claim 17, wherein the antigen further comprises an epitope which elicits a humoral response and/or an epitope which elicits a TH HLA-DR1 restricted T helper epitope response.

19. The isolated antigen of claim 17, wherein the antigen comprising an HLA-A2-restricted cytotoxic (CTL) epitope comprises a polypeptide.

20. The isolated antigen of claim 17, wherein the antigen comprising an HLA A2-restricted T cytotoxic (CTL) epitope comprises a polynucleotide.

21. The isolated antigen of claim 20, wherein the antigen comprising an HLA-A2-restricted T cytotoxic (eTI) epitope comprises, DNA, RNA, or DNA and RNA.

22. A method of comparing the efficiency of T-helper cell response induced by two or more vaccines, the method comprising: a) administering a first candidate vaccine to a mouse of claim 3 or claim 6 and measuring the T-helper cell response Induced in the mouse by the first candidate vaccine; b) administering a second candidate vaccine to a mouse of claim 3 or claim 6 and measuring the T-helper cell response induced in the mouse by the second candidate vaccine; c) administering each additional candidate vaccine to be compared to a mouse of claim 3 or claim 6 and measuring the T-helper cell response induced in the mouse by each additional candidate vaccine to be compared; and d) determining the efficiency of each candidate vaccine to Induce a T-helper cell response by comparing the T-helper cell responses to each of the vaccines to be compared with each other.

23. The method of claim 22, wherein the T-helper cell response is an HLA DR1 restricted response.

24. A method of comparing the efficiency of T cytotoxic cell response induced by two or more vaccines, the method comprising: a) administering a first candidate vaccine to a mouse of claim 3 or claim 6 and measuring the T cytotoxic cell response induced In the mouse by the first candidate vaccine; b) administering a second Candidate vaccine to a mouse of claim 3 or claim 6 and measuring the T cytotoxic cell response induced in the mouse by second candidate vaccine; c) administering each additional candidate vaccine to be compared to a mouse of claim 3 or claim 6 and measuring the T cytotoxic cell response induced in the mouse by each additional candidate vaccine to be compared; and d) determining the efficiency of each candidate vaccine to induce a T cytotoxic cell response by comparing the T cytotoxic cell responses to each of the vaccines to be compared with each other.

25. The method of claim 24, wherein the T cytotoxic cell response is an HLA-A2 restricted response.

26. A method of simultaneously comparing the efficiency of T-helper cell response and T cytotoxic cell response induced by two or more vaccines, the method comprising: a) administering a first candidate vaccine to a mouse of claim 3 or claim 6 and measuring the T-helper cell response and T cytotoxic cell response induced in the mouse by the first candidate vaccine; b) administering a second candidate vaccine to a mouse of claim 3 or claim 6 and measuring the T-helper cell response and T cytotoxic cell response induced in the mouse by the second candidate vaccine; c) administering each additional candidate vaccine to be compared to a mouse of claim 3 or claim 6 and measuring the T-helper cell response and T cytotoxic cell response induced in the mouse by each additional candidate vaccine to be compared; and d) determining the efficiency of each candidate vaccine to induce a T-helper cell response and T cytotoxic cell response by comparing the T-helper cell response and T cytotoxic cell response to each of the vaccines to be compared with each other.

27. The method of claim 26, wherein the T-helper cell response is an HLADR1 restricted response, and wherein the T cytotoxic cell response is an HLA-A2 restricted response.

28. A method of simultaneously determining the humoral response, the T-helper cell response, and the T cytotoxic cell response of a mouse following its immunization with an antigen or a vaccine comprising one or more antigens, the method comprising: a) administering the antigen or the vaccine comprising one or more antigens to a mouse of claim 3 or claim 6; b) assaying for a specific humoral response In the mouse to the antigen or vaccine comprising one or more antigens; c) assaying for a T-helper cell response in the mouse to the antigen or vaccine comprising one or more antigens; and d) assaying for a T cytotoxic cell response in the mouse to the antigen or vaccine comprising one or more antigens.

29. The method of claim 28, wherein the T-helper cell response is a TH HLADR1 restricted response.

30. The method of claim 28, wherein the T cytotoxic cell response is a CTRL HLA-A2 restricted response.

31. A method of optimizing two or more candidate vaccine compositions for administration to a human, based on preselected criteria, the method comprising: simultaneously determining the humoral response, the T-helper cell response, and the T cytotoxic cell response of a mouse following its immunization with the two or more candidate vaccine compositions, according to claim 28; and selecting an optimized vaccine by applying preselected criteria to the results.

32. The method according to claim 31, wherein the two or more candidate vaccines differ only in the ratio of antigen to adjuvant present in the vaccine.

33. The method according to claim 31, wherein the two, or more candidate vaccines differ only in the type of adjuvant present in the vaccine.

34. A method of determining whether a vaccine poses a risk of induction of an `autoimmune disease when administered to a human`, the method comprising: a) administering the vaccine to a mouse of claim 3 or claim 6; and b) assaying for an autoimmune response In the mouse; wherein observation of an autoimmune response in the mouse indicates that the vaccine poses a risk of induction of an autoimmune disease when administered to a human.

35. An isolated transgenic mouse cell comprising: a) a disrupted H2 class I gene; b) a disrupted H2 class II gene; and c) a functional HLA Class I or class II transgene.

36.-41. (canceled)

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