JP2006528000A - Transgenic mice with human major histocompatibility complex (MHC) phenotype, methods for their use and application - Google Patents

Abstract

The present invention relates to transgenic mice and cells of isolated transgenic mice, wherein the mice and mouse cells comprise a disrupted H-2 class I gene, a disrupted H-2 class II gene, It includes a functional HLA class I transgene and a functional HLA class II transgene. In an embodiment, the transgenic mouse or transgenic mouse cells lack both H-2 class I and class II molecules and have a functional HLA class I and a functional HLA class II transgene. Contains. In an embodiment, the transgenic mouse or cells of the transgenic mouse have a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ °. The invention also relates to a method using the transgenic mouse according to the invention.
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Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is based on and is based on the benefit of US Provisional Application No. 60/490945 (Attorney No. 03495.6093) filed July 30, 2003. The entire disclosure of this provisional application is the basis of this application for all purposes and is incorporated by reference in its entirety.

  Currently, many vaccines have been developed for immunotherapy of human cancer and for the treatment of infectious diseases such as malaria, AIDS, hepatitis C virus and severe acute respiratory syndrome (SARS). Yes. Given the speed with which emerging pathogens emerge, it is important to improve animal models that can be used to make a quick and reliable assessment of the vaccine strategy and the protective ability of various epitopes. Furthermore, in vivo studies cannot be easily assessed or measured in vitro, such as vaccine immunogenicity, vaccine formulation, route of administration, tissue distribution, and involvement of primary and secondary lymphoid organs. There is already a need to assess the significant variation in vaccine behavior that cannot be done. Because of its simplicity and flexibility, small animals such as mice are attractive, at least for early vaccine development studies, to replace more cumbersome and more costly model systems like non-human primates. Alternative.

  It has been shown that vaccines that have been found to be protective in wild-type animal studies have shown moderate efficacy in several clinical trials (McMichael, AJ & Hanke, T. Nat Med). 9, 874-880 (2003)), since animal MHC molecules and human HLA molecules do not have the same optimal epitope, the effects of human and animal MHC on the results of immune responses are different. This can be explained in part (Rotzschke, O. et al. “Nature” 348, 252-254 (1990)). Thus, despite some limitations, transgenic mice expressing human HLA have been tested for vaccine candidates, assessed the potential risk that the vaccine will induce autoimmune disease, and based on human binding factors. It is a useful improvement over wild-type mice as a preclinical model for developing good treatment methods.

Cytotoxic T cells Cytotoxic T cells (CTL) play an important role in the eradication of infectious diseases and in some cases cancer (P. Aichele, H. Hengartner, RM Zinkneragel 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. Druh. Eur J Immunol 27 (1997), p. 1242; DJ Diamond, J York, J Sun, CL Wright and SJ Forman, Blood 90 (1997), p. .1751). Recombinant protein vaccines do not reliably elicit CTL responses (Habeshave JA, Dalleish AG, Boundiff L, Newell AL, Wilks D, Walker LC, Manca F. November 1990; 11 (11): 418-25; Miller SB, Tse H, Rosenspire AJ, King SR, “Virology” Dec. 1992; 191 (2): 973-7). Other immunogenic vaccines composed of attenuated pathogens cannot be used in humans due to the most important safety concerns in some major diseases. In recent years, an epitope-based approach has been proposed as a promising method for developing new vaccines for prevention and immunotherapy (Melief CJ, Offringa R, Toes RE, Kast WM. “Curr Opin Immunol.” October 1996, 8 (5): 651-7; Chesnut RW, “Design testing of peptide based biotoxic T-cell medium immunotherapeutic tot, (Ed.) “Vaccine Design: The Subunit, Adjuvant Approach”, Pleum Press, New-York, 1995, 847). This approach has several advantages, including the selection of naturally processed epitopes that focus the immune system on epitopes of highly conserved and immunologically dominant pathogens. (RG van der Most, A. Sette, C. Oseroff, J. Alexander, K. Murali-Krishna, L. L. Lau, S, Southwood, J. Sidney, R. W.) Chesnut, M. Matoubian and R. Ahmed, “J Immunol” 157 (1996), p. 5543), and as seen in HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) infections Multiple epitoes that prevent escape by There are included to induce reactions involved. The approach also makes it possible to eliminate the determinants of suppressor T cells, which can favorably generate a TH2 reaction under conditions where a TH1 reaction is desired, and vice versa. (Pfeiffer C, Murray J, Madri J, Bottomory K. “Immunol Rev.” 1991 Oct; 123: 65-84; P Chaturvedi, Q Yu, S Southwood, A Sette, and B Singh, “Int 19”, Int 19 ” 8: 745-755). Finally, the approach offers the possibility of removing autoimmune T cell determinants in the antigen, which can induce unwanted autoimmune diseases. Protective antiviral immunity or protective antitumor immunity using CTL epitope peptides has been successful in several experimental models (DJ Diamond, J York, J Sun, C.L. Wright and SJ Forman, Blood 90, 1997, p.1751; JEJ Blaney, E. Nobusawa, MA Brehm, RH Bonneau, LM Mylin, T M. Fu, Y. Kawaoka and SS Tevecia, “J Virol” 72 (1998), p. 9567).

  The determination of CTL epitopes based on the use of human lymphocytes is error-prone due to environmental and genetic diversity leading to incomplete results and technical difficulties in isolating CTL clones. May occur. The HLA class I or class II transgenic mice described to date have demonstrated these limitations, as demonstrated by the identification of new CTL and helper T cell epitopes in such animal models. Proven to be an effective tool to overcome (Hill AV. Annu Rev Immunol. 1998; 16: 593-617; Carmon L, El-Shami KM, Paz A, Pascolo S, Tzehoval E, Tirosb B, Koren R, Feldman M, Fridkin M, Lemonier FA, Eisenbach L. “Int J Cancer”, February 1, 2000; 85 (3): 391-7). These mice also showed i) good correlation between peptide HLA binding affinity and immunogenicity (Lustgarten J, Theobald M, Labadie C, LaFace D, Peterson P, Diss ML, Cheaver MA, Sherman LA "Hum Immunol." February 1997; 52 (2): 109-18; Baker Ak, van der Burg SH, Huijbens RJ, DRijfhout JW, Melief CJ, Adema GJ, Fig. CG. Jan 27; 70 (3): 302-9), ii) significant overlap between the mouse and human CTL lines at the level of antigen processing (production of identical epitopes), And iii) comparable mobility of the CTL repertoire in HLA transgenic mice and humans to most antigens (Wentworth, PA, A. Vifiello, J. Sidney, E. Keoh, PW Chesnut, H. Grey, A. Sette. 1996. "Eur. J. Immunol." 26:97; Alexander, J., C. Oserof, J. Sidney, P. Wentworth, E. Keohgh, G. Hermanson, F.V. Chisari, RT Kubo, HM Grey, A. Sette, 1997. “J. Immunol.” 159: 4753).

To date, synthetic peptide-based CTL epitope vaccines have been developed as immunotherapy for many human diseases [18-20]. However, only moderate efficacy has been observed in some clinical trials (21). This can be explained in part by the inability of these vaccines to elicit a sufficiently strong CTL response. Indeed, recent reports suggest that CD4 ⁺ T cell assistance is required to obtain maximal CTL responses (AJ Zajac, K. Murali-Krishna, J. Blatman. and R. Ahmed, “Curr Opin Immunol” 10 (1998), p. 444; Firat H, Garcia-Pons F, Tourdo S, Pascolo S, Scardono A, Garcia Z, Michel O, J Mateo L, Suhrbier A, Lemonier FA, Langade-Dernoyen P “Eur J Immunol”, 291212, 1999).

Although CTLs are an important component of protective immunity against viral infections, the need for CTL priming in vivo is not well understood. It is now recognized that Th cells are usually essential for CTL priming with synthetic peptides. With respect to synthetic CTL epitope peptides, several studies have pointed out that Th lymphocyte stimulation is essential to elicit an optimal CTL response (C. Fayole, E. Deiraud 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; Shirai, C. P. Pendkton, J. Ahlers, T. Takeshita, M. Newman and JA Berzovsky, J Immunol 152 (1994), p. 549; Masse JG Guillet and E. Gomard, “Int Immunol” 8 (1996), p.457). Some of these studies, CD4 ⁺ helper T cells and CD8 ⁺ T cells, may interact simultaneously with one antigen-presenting cells with their cognate epitopes, required for the activation of CD8 ⁺ T cells (Ridge JP, Di Rosa F, Matzinger P. Nature, June 4, 1998; 393 (6684): 474-8).
The interaction of these three cells in CTL priming is more physically observed in studies using viral epitopes and animal models than when administered as a mixture of unbound CTL and Th cell epitopes. It was validated because in vivo induction of CTL was most effective when bound (Shirai M, Pendleton CD, Ahlers J, Takeshita T, Newman M, Berzoky JA. “J Immunol” 1994. January 15; 152 (2): 549-56; Oseroff C, Sette A, Wentworth P, Celis E, Maewal A, Dahlberg C, Fikes J, Kubo RT, Chesnut RW, Grey BX Alex nder "J.Vaccine" May 1998; 16 (8): 823-33). The ability of CTL antigenic peptides and Th cell antigenic peptides to effectively induce CTL responses has been shown by experimental models (C. Fayole, E. Deiraud 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), human (A. Vitiello, G. Ishioka, H.); M. Grey, R. Rose, P. Famess, R. LaFondo, L. Yuan, FV Chisari, J. Furze and R. Bartholomeuz, “J Clin Invest” 95 (1995), p. 341; .Li ingston, C. Crimi, H. Grey, G. Ishioka, FV Chisari, J. Fikes, HM Grey, R. Chesnut and A. Sette, “J Immunol” 159 (1997), p. ). In addition, a strong Th response plays an important role not only in optimal induction of CTL responses, but also in maintaining CTL memory (EA Walter, PD Greenberg, MJ Gilbert). , RJ Finch, KS Watanabe, ED Thomas and SR Riddell, “N Engl J Med” 333 (1995), p. 1038; Thomas ED, Blume KG, Forman SJ (edited) ) “Hematopoietic Cell Transplantation” 2nd edition, Malden, MA: Blackwell Science Inc., 1999, Riddell SR, Greenberg PD). Finally, it has long been demonstrated that CD4 ⁺ “helper” T cells are important in coordinating cellular and humoral immune responses against foreign antigens.

Recently, transgenic (Tg) mice have been established that express both HLA-A ^* 0201 class I and HLA-DR1 class II molecules (BenMohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus). J, Diamond DJ, “Hum, Immunol” August 2000; 61 (8): 764-79). The authors indicate that both HLA-A ^* 0201 and HLA-DR1 transgenes are functional in vivo, that both MHC class I and class II molecules are used as constraining factors, and HLA- It has been reported that the product of the DR1 transgene promotes an HLA-A ^* 0201-restricted antigen-specific CTL reaction (BenMohamed L, Krishnan R, Longmate J, Age C, Low L, Primus J, Diamond) DJ, “Hum, Immunol” August 2000; 61 (8): 764-79).

It is noteworthy that these HLA-A ^* 0201 / DR1 Tg mice expressed their own MHC, H-2 class I and class II molecules. HLA class I transgenic mice expressing endogenous mouse MHC class I genes preferentially and often exclusively show H-2 restricted CTL responses (C Barra, H Gournier, Z Garcia, PN Marche , E Jovin-Marche, P Briand, P Filippi, and FA Lemonnier “J Immunol” 1993, 150: 3681-3689; Epstein H, Hardy F, May JS, Johnson MH, Holm N, 1989 Moon; 19 (9): 1575-83; Le AX; EJ Bernhard, MJ Holtman, S Strub, P Parham, E Lacy, and VH Engelhard, J munol ”1989, 142: 1366-1371; Vitiello A, Marchesini D, Furze J, Sherman LA, Chesnut RW“ J Exp Med ”Apr. 1, 1991; 173 (4): 100715), endogenous mouse MHC class HLA class II transgenic mice expressing the II gene cannot elicit a reliable HLA class II-restricted antigen-specific response (Nishimura Y, Iwanaga T, Inamitsu T, Yanagawa Y, Yasunami M , Kimura a, Hirokawa K, Sasazuki T "J Immunol" July 1, 1990; 145 (1) 353-60), Tg between these HLA-a ^* 0201 / DR1 Scan, to evaluate the specific response of the human to the antigen has a limited availability.

However, only HLA-restricted CTL immune responses occur in H-2 class I knockout mice transfected with HLA class I or H-2 class II knockout mice transfected with HLA class II (Pascolo). S, Bervas N, Ure JM, Smith AG, Lemonier FA, Pernarau, B "J Exp Med" June 16, 1997; 185 (12): 2043-51; Madsen L, Labreque N, Enberg J, Diger J Svejgaard A, Benoist C, Mathis D, Fugger L, Proc Natl Acad Sci USA, August 31, 1999; 96 (18): 10338-43). Indeed, H-2 class I knockout (KO) mice that have been transfected with HLA-A2.1 are HLA-A2.1 transgenic mice that remain expressing endogenous mouse H-2 class I molecules. (Pascolo, S. et al. “J Ex Med” 185, 2043-2051 (1997); Ureta-Vidal, A., Firat, H., Perarnau, B. & Lemonier, FA “J Immunol” 163, 2555-2560 (1999); Firat, H. et al., “Int Immunol” 14, 925-934 (2002); S. et al., “Int Immunol” 15, 765-772 ( 003)). The present inventors have made similar observations in HLA-DR1 transgenic mice depending on whether they lack H-2 class II molecules (A. Pajot, unpublished results). In addition, when there is no competition with mouse MHC molecules, H-2 class I-KO mice transfected with HLA-A2.1 or H-2 class II-KO mice transfected with HLA-DR1 are: Generates only HLA-restricted immune responses (Pascolo, S. et al. “J Exp Med” 185, 2043-2051 (1997)) (A. Pajot, unpublished results), HLA-restricted CD8 ⁺ T cells Monitoring of responses and CD4 ⁺ T cell responses is facilitated. However, protective immune responses against pathogens often require coordination between helper T cells and cytotoxic CD8 ⁺ T cells, and simultaneously assess responses in HLA class I and class II humans in one mouse This is not possible with HLA class I only transgenic mice or HLA class II only transgenic mice.

Thus, in the art, potent CD4 ⁺ Th (helper T lymphocytes) consisting of structures containing human CTL epitopes and in some cases to maintain antiviral and antitumor CD8 ⁺ T cell activity. There is a need for a suitable animal model system to test the immunogenicity of human vaccine candidates, consisting of epitope-containing structures (AJ Zajac, K. Murali-Krishna, J. N. Blatman and R). Ahmed, “Curr Opin Immunol” 10 (1998), p. 444; , Su rbier A, Lemonnier FA, Langlade-Dernoyen P "Eur J Immunol" 29,3112,1999). There is also a need for a system that allows the simultaneous coordination between CTL responses, TH responses (especially TH ₁ responses or TH ₂ responses) and possibly humoral immune responses to be assessed simultaneously.
US Pat. No. 6,194,389 US Pat. No. 6,214,808 BenMohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond DJ “Hum, Immunol”. August 2000; 61 (8): 764-79) A. J. et al. Zajac, K .; Murali-Krishna, J. et al. N. Blattman and R.M. Ahmed “Curr Opin Immunol” 10 (1998), p. 444 Firat H, Garcia-Pons F, Tourdot S, Pascolo S, Scardino A, Garcia Z, Michel ML, Jack RW, Jung O, Kosmatopoulos K, Mateo L, Suhrber 29, 3112, 1999

  The inventors have provided mice that have been transfected with both HLA-A2.1 and HLA-DR1 molecules in a genetic background that lacks both H-2 class I and class II molecules. Satisfying this need and meeting further needs. Specifically, the present invention provides a mouse, which comprises (1) a mutated H-2 class I molecule and a class II molecule, and (2) a transgenic HLA class I molecule, Alternatively, a gene-introduced HLA class II molecule, or both a gene-introduced HLA class I molecule and a gene-introduced HLA class II molecule are expressed. These mice provide a useful model in developing and optimizing vaccine structures with maximum immunogenicity in vivo for use in humans. Specifically, such mice specifically allow for complete analysis of the three components of the immune adaptive response (antibodies, helper T cells and cytolysis) in animal individuals, as well as by vaccination against antigen challenge. It is possible to evaluate the protection given.

The mouse of the present invention knocks out both H-2 class I molecule and class II molecule, expresses the introduced HLA class I molecule and the introduced HLA class II molecule, and is completely humanized. An experimental mouse, which simultaneously detects the presence or absence of an antigen-specific antibody, an antigen-specific HLA-DR1-restricted T cell reaction, and an antigen-specific HLA-A2-restricted T cell reaction Can be used. These mice are useful for studying how mutual coordination occurs between CTL responses, TH responses (especially TH ₁ or TH ₂ responses), and in some cases humoral immune responses. These mice will be the best tools for basic and applied vaccine research.

  A first embodiment of the invention is a transgenic comprising a disrupted H-2 class I gene, a disrupted H-2 class II gene, and a functional HLA class I or class II transgene. A mouse is provided.

  A second embodiment of the invention comprises a disrupted H-2 class I gene, a disrupted H-2 class II gene, a functional HLA class I transgene, and a functional HLA class II transgene. The present invention provides a transgenic mouse.

  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 another embodiment, the HLA-A2 transgene comprises a sequence of HLA-A2 listed in the sequence list, and the HLA-DR1 transgene comprises a sequence of HLA-DR1 listed in the sequence list.

A further embodiment of the invention provides a transgenic mouse lacking both H-2 class I and class II molecules, wherein the transgenic mouse is a functional HLA class I transgene and Contains a functional HLA class II transgene. In one embodiment, the mouse has a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ °. In some embodiments, the HLA-A2 transgene includes a sequence of HLA-A2 listed in the sequence list, and the HLA-DR1 transgene includes a sequence of HLA-DR1 listed in the sequence list.

Another embodiment of the invention provides a method for simultaneously identifying the presence or absence of one or more epitopes in one candidate antigen or group of candidate antigens, wherein in one embodiment, one or more Epitopes cause a specific humoral immune response, a TH HLA-DR1 restricted response, and / or a CTL HLA-A2 restricted response. The method may be applied to a transgenic mouse comprising a disrupted H-2 class I gene, a disrupted H-2 class II gene, a functional HLA-A2 transgene, and a functional HLA-DR1 transgene, or An H-2 class I molecule and class comprising a functional HLA class I transgene and a functional HLA class II transgene and having a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° Administering a candidate antigen or group of candidate antigens to a transgenic mouse lacking both of the II molecules, analyzing a specific humoral immune response to the antigen in the mouse, HLA-DR1 restriction of TH to the antigen in the mouse Analyzing sexual response and HLA-A2 restriction of CTL to antigen in mice It includes analyzing sexual reactions. The specific humoral immune response to the antigen is seen in the mouse, confirming the epitope that causes the humoral immune response to the antigen. The presence of a TH HLA-DR1-restricted response to the antigen in the mouse confirms an epitope that causes a TH HLA-DR1-restricted response in the antigen. The presence of an CTL HLA-A2 restricted response to the antigen in the mouse confirms the epitope that causes the CTL HLA-A2 restricted response to the antigen.

  In some embodiments, the method includes analyzing a Th1-specific response to the antigen in the mouse and analyzing a Th2-specific response to the antigen in the mouse. In this case, an epitope that causes a Th1-specific response to the antigen in the mouse is confirmed by a Th1-specific reaction to the antigen in the mouse, and a Th2-specific reaction to the antigen is observed in the mouse, whereby the antigen in the mouse is confirmed. Epitopes that produce a Th2-specific response to are identified.

The present invention also provides a method for identifying the presence or absence of an HLA-DR1-restricted helper T cell epitope in one candidate antigen or group of candidate antigens, which method comprises a disrupted H-2 class I gene. In transgenic mice containing a disrupted H-2 class II gene, a functional HLA-A2 transgene and a functional HLA-DR1 transgene, or functional with a functional HLA class I transgene Transgenic mice lacking both H-2 class I and class II molecules, which contain an HLA class II transgene and have the genotype HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ °, Administration of a candidate antigen or group of candidate antigens, and in mice, the HLA-D of TH against the antigen Analysis of R1 restricted helper T cell epitope responses. In mice, the TH HLA-DR1-restricted helper T cell epitope response to the antigen is seen, confirming the epitope that causes the TH HLA-DR1-restricted helper T cell epitope response in the antigen.

  Furthermore, the present invention provides an isolated antigen comprising the epitope of HLA-DR1 restricted helper T cells identified by the method of the previous paragraph. In some embodiments, the isolated antigen further comprises an epitope that produces a humoral immune response and / or an epitope that produces a CTL HLA-A2 restricted response. In another embodiment, the antigen comprising an epitope of HLA-DR1 restricted helper T cells comprises a polypeptide. In another embodiment, the antigen comprising an epitope of HLA-DR1 restricted helper T cells comprises a polynucleotide. In a further embodiment, the antigen comprising an epitope of HLA-DR1 restricted helper T cells comprises DNA, RNA, or both DNA and RNA.

Furthermore, the present invention also provides a method for identifying the presence or absence of HLA-A2 restricted cytotoxic T cell (CTL) epitopes in one candidate antigen or group of candidate antigens, the method comprising disrupted H A transgenic mouse containing a C-2 class I gene, a disrupted H-2 class II gene, a functional HLA-A2 transgene and a functional HLA-DR1 transgene, or a functional HLA class I Lacking both H-2 class I and class II molecules, which contain a transgene and a functional HLA class II transgene and have the genotype HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° Administering a candidate antigen or group of candidate antigens to a transgenic mouse and against the antigen or group of antigens in the mouse Analyzing the HLA-A2 restricted cytotoxic T cell (CTL) response. Epitope that causes HLA-A2-restricted cytotoxic T cell (CTL) reaction in antigen or antigen group when HLA-A2-restricted cytotoxic T cell (CTL) reaction to antigen or antigen group is observed in mouse Is confirmed.

  The present invention provides an isolated antigen comprising an HLA-A2 restricted cytotoxic T cell (CTL) epitope identified by the method of the previous paragraph. In some embodiments, the antigen further comprises an epitope that produces a humoral immune response and / or an epitope that produces an HLA-DR1 restricted helper T cell epitope response of TH. In another embodiment, the antigen comprising an HLA-A2 restricted cytotoxic T cell (CTL) epitope comprises a polypeptide. In other embodiments, the antigen comprising an HLA-A2 restricted cytotoxic T cell (CTL) epitope comprises a polynucleotide. In further embodiments, the antigen comprising an HLA-A2 restricted cytotoxic T cell (CTL) epitope comprises DNA, RNA, or both DNA and RNA.

The present invention also provides a method for comparing the effectiveness of helper T cell responses elicited by two or more vaccines. This method can be applied to transgenic mice comprising a disrupted H-2 class I gene, a disrupted H-2 class II gene, a functional HLA-A2 transgene and a functional HLA-DR1 transgene, or Both H-2 class I and class II molecules comprising a functional HLA class I and a functional HLA class II transgene and having the genotype HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° A first candidate vaccine is administered to a transgenic mouse that lacks the protein, and the response of helper T cells induced in the mouse by the first candidate vaccine is determined, the disrupted H-2 class I gene is disrupted H-2 class II gene, functional HLA-A2 transgene and functional HLA- Transgenic mice containing a DR1 transgene or a functional HLA class I and a functional HLA class II transgene and having a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° A second candidate vaccine is administered to a transgenic mouse lacking both H-2 class I and class II molecules and the response of helper T cells induced in the mouse by the second candidate vaccine is measured A transgenic mouse comprising a disrupted H-2 class I gene, a disrupted H-2 class II gene, a functional HLA-A2 transgene and a functional HLA-DR1 transgene or function HLA class I transgene and functional HLA class II tiger Additional candidate vaccines to be compared to transgenic mice lacking both H-2 class I and class II molecules, which contain the gene and the genotype HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° And measuring the helper T cell responses elicited in mice by additional candidate vaccines, and comparing the helper T cell responses to each vaccine to be compared with each other to determine the helper T cell responses. Determining the effectiveness of each candidate vaccine to induce. In some embodiments, the helper T cell response is an HLA-DR1 restricted response.

Furthermore, the present invention provides a method for comparing the effectiveness of cytotoxic T cell responses elicited by two or more vaccines. The method can be applied to transgenic mice comprising a disrupted H-2 class I gene, a disrupted H-2 class II gene, a functional HLA-A2 transgene and a functional HLA-DR1 transgene, or H-2 class I molecules and class II comprising a functional HLA class I transgene and a functional HLA class II transgene and having a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° Administering a first candidate vaccine to a transgenic mouse lacking both molecules and measuring the response of cytotoxic T cells induced in the mouse by the first candidate vaccine, disrupted H-2 class I gene, disrupted H-2 class II gene, functional HLA-A2 transgene and functional HLA -A transgenic mouse containing a DR1 transgene or a functional HLA class I and a functional HLA class II transgene with a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° A transgenic mouse lacking both H-2 class I and class II molecules is administered a second candidate vaccine and the cytotoxic T cell response elicited in the mouse by the second candidate vaccine A transgenic mouse comprising a disrupted H-2 class I gene, a disrupted H-2 class II gene, a functional HLA-A2 transgene and a functional HLA-DR1 transgene, or A functional HLA class I transgene and a functional HLA class II transgene Further candidates to be compared to transgenic mice lacking both H-2 class I and class II molecules that contain sgene and have the genotype HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° Administering each of the vaccines, measuring the cytotoxic T cell responses elicited in mice by additional candidate vaccines, and comparing the cytotoxic T cell responses for each vaccine to be compared to each other Determining the effectiveness of each candidate vaccine to elicit a T cell response. In some embodiments, the cytotoxic T cell response is an HLA-A2 restricted response.

Furthermore, the present invention also provides a method for simultaneously comparing the effectiveness of helper T cell responses and cytotoxic T cell responses elicited by two or more vaccines. The method can be applied to transgenic mice comprising a disrupted H-2 class I gene, a disrupted H-2 class II gene, a functional HLA-A2 transgene and a functional HLA-DR1 transgene, or H-2 class I molecules and class II comprising a functional HLA class I transgene and a functional HLA class II transgene and having a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° Administering a first candidate vaccine to a transgenic mouse lacking both molecules and measuring a helper T cell response and cytotoxic T cell response elicited in the mouse by the first candidate vaccine, destruction H-2 class I gene, disrupted H-2 class II gene, functional HLA-A2 transgene Or a functional HLA-DR1 transgene or a functional HLA class I and a functional HLA class II transgene, HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ Transgenic mice having a genotype of ° and lacking both H-2 class I and class II molecules were administered a second candidate vaccine and induced in mice by the second candidate vaccine Measuring helper and cytotoxic T cell responses, disrupted H-2 class I genes, disrupted H-2 class II genes, functional HLA-A2 transgenes and functional Transgenic mice containing HLA-DR1 transgene or functional HLA class I Lacking both H-2 class I and class II molecules, which contain a transgene and a functional HLA class II transgene and have the genotype HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° Transgenic mice are administered each of the additional candidate vaccines to be compared, and the helper T cell response and cytotoxic T cell response elicited in the mouse by each of the additional candidate vaccines are measured and compared Comparing the response of helper T cells and cytotoxic T cells to each vaccine to determine the effectiveness of each candidate vaccine to elicit the response of helper T cells and cytotoxic T cells Contains. In some embodiments, the helper T cell response is an HLA-DR1 restricted response and the cytotoxic T cell response is an HLA-A2 restricted response.

The present invention also relates to mouse humoral immune response, helper T cell reaction, and cytotoxic T cell according to immunization with one antigen or immunization with a vaccine having one or more antigens. It also proposes a method for simultaneously judging the reactions of The method can be applied to transgenic mice comprising a disrupted H-2 class I gene, a disrupted H-2 class II gene, a functional HLA-A2 transgene and a functional HLA-DR1 transgene, or H-2 class I molecules and class II comprising a functional HLA class I transgene and a functional HLA class II transgene and having a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° Specific antigens in mice for administering antigens or vaccines containing one or more antigens to transgenic mice lacking both molecules of Analysis of sex immune responses, antigens, or vaccines containing one or more antigens Analyzing the response of helper T cells in the mouse and analyzing the response of cytotoxic T cells in mice to an antigen, or a vaccine comprising one or more antigens. In some embodiments, the helper T cell response is an HLA-DR1 restricted response of TH. In some embodiments, the cytotoxic T cell response is a CTL HLA-A2 restricted response.

The present invention also provides a method for optimizing the composition of two or more candidate vaccines for administration to humans based on pre-selected criteria. The method comprises simultaneously determining a humoral immune response, helper T cell response and cytotoxic T cell response in a mouse according to immunization with a composition of two or more candidate vaccines; The method used here is for transgenic mice comprising a disrupted H-2 class I gene, a disrupted H-2 class II gene, a functional HLA-A2 transgene and a functional HLA-DR1 transgene. Or an H-2 class I molecule comprising a functional HLA class I transgene and a functional HLA class II transgene and having a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ °; Administer antigens or vaccines containing one or more antigens to transgenic mice lacking both class II molecules Analyzing a specific humoral immune response in mice against an antigen, or a vaccine comprising one or more antigens, helper T in mice against a vaccine comprising an antigen or one or more antigens Analyzing cellular responses, analyzing the response of cytotoxic T cells in mice to an antigen or vaccine containing one or more antigens, and applying preselected criteria to the results Selecting an optimized vaccine. 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.

In another aspect, the present invention provides a method for determining whether a vaccine is at risk of inducing an autoimmune disease when administered to a human. The method can be applied to a transgenic mouse comprising a disrupted H-2 class I gene, a disrupted H-2 class II gene, a functional HLA-A2 transgene and a functional HLA-DR1 transgene, or Both H-2 class I and class II molecules comprising a functional HLA class I and a functional HLA class II transgene and having a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° Administering a vaccine to a transgenic mouse lacking and analyzing an autoimmune response in the mouse, wherein an autoimmune response is seen in the mouse, wherein the vaccine is administered to a human This indicates that there is a risk of inducing an autoimmune disease.

  The present invention also relates to an isolated transgenic comprising a disrupted H-2 class I gene, a disrupted H-2 class II gene, and a functional HLA class I or class II transgene. It also provides mouse cells.

  Further, the present invention has been isolated comprising a disrupted H-2 class I gene, a disrupted H-2 class II gene, a functional HLA class I transgene and a functional HLA class II transgene. It also provides cells for transgenic mice.

  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 another embodiment, the HLA-A2 transgene comprises a sequence of HLA-A2 listed in the sequence list, and the HLA-DR1 transgene comprises a sequence of HLA-DR1 listed in the sequence list.

Furthermore, the present invention provides isolated transgenic mouse cells lacking both H-2 class I and class II molecules, wherein the transgenic mouse cells are functional. A typical HLA class I transgene and a functional HLA class II transgene. In some embodiments, the cells of the transgenic mouse have a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ °. In other embodiments, the HLA-A2 transgene comprises a sequence of HLA-A2 listed in the sequence list, and the HLA-DR1 transgene comprises a sequence of HLA-DR1 listed in the sequence list.

  The invention will be more fully described with reference to the following drawings.

FIG. 1 shows a flow cytometric analysis of cell surface expression of the indicated transgenic molecule. (A) Spleen cells derived from H-2 class II-KO mice transfected with HLA-DR1 (DR1 ⁺ CII ⁻ , left panel), H-2 class transfected with HLA-A2.1 / HLA-DR1 Splenocytes derived from I / class II-KO mice (A2 ⁺ DR1 ⁺ CI ⁻ CII ⁻ , middle panel) and splenocytes derived from H-2 class I-KO mice transfected with HLA-A2.1 (A2 ⁺ CI ⁻ , right panel) was FITC labeled W6 / 32 (anti-HLA-ABC, abscissa) or biotinylated 28-8-6S (anti-H-2K ^b / D ^b , ordinate) m. Stained with either Ab, the latter visualized with PE-labeled anti-mouse IgG. (B) B220 ⁺ splenic B lymphocytes derived from the same mouse strain were isolated from FITC-labeled L243 (anti-HLA-DR1, upper panel) and PE-labeled AF6-120.1 (anti-H-2IAβ ^b , lower panel) m. Stained with Ab.

FIG. 2 shows the number of CD8 ⁺ and CD4 ⁺ splenic T cells and the use of BV segments (based on immunoscope analysis) in mice of the indicated genotype. (A) Spleen cells derived from H-2 class II-KO mice transfected with HLA-DR1 (DR1 ⁺ CII ⁻ , left panel), H-2 class transfected with HLA-A2.1 / HLA-DR1 Splenocytes derived from I / class II-KO mice (A2 ⁺ DR1 ⁺ CI ⁻ CII ⁻ , middle panel) and splenocytes derived from H-2 class I-KO mice transfected with HLA-A2.1 (A2 ⁺ CI ^-, right panel) the, PE-labeled CT-CD4 (anti-mouse CD4, ordinate) and FITC-labeled 53-6.7 (anti-mouse CD8, abscissa) m. Stained with Ab. The numbers correspond to the percentage of CD4 ⁺ T cells (upper left square) or CD8 ⁺ T cells (lower right square) in the total splenocytes. (B and c) Purified splenic CD8 ⁺ T cells (b) for use of BV segment family (1-20) with forward and BC reverse primers specific for BV family (1-20) (b ) And immunoscope RT-PCR analysis of CD4 ⁺ T cells (c). A typical graph feature of a productively rearranged BV segment family has a series of peaks that differ in length by three nucleotides in a distribution such as a Gaussian distribution. This figure shows the results obtained with a representative mouse of H-2 class I / class II-KO into which HLA-A2.1 / HLA-DR1 was introduced.

FIG. 3 shows HBs-specific antibody reaction, cell lysis reaction and proliferation reaction. H-2 class I / class II-KO mice transfected with HLA-A2.1 / HLA-DR1 were immunized by intramuscular injection of plasmid DNA encoding HBsAg, and not immunized and then individually Tested. (A) Control of humoral immune response (upper panel), cytolytic response (middle panel) and proliferation response (lower panel) and specificity of representative mice immunized with HBsAg-DNA. The antibody (IgG) titer against HBsAg particles containing both the middle and small HBV envelope proteins and the antibody titer against the preS2 _109-134 peptide were determined by ELISA assay. _Cytolytic activity at various effector / target (E / T) ratios is related to related peptide (HBsAg _348-357 , HLA-A2.1 restricted ◆) or control peptide (HBsAg _371-378 , H-2K ^b restricted). Δ, and MAGE-3 _271-279 , HLA-A2.1 restricted □) were evaluated using RMAS-HHD target cells pulsed. Proliferative response uses _either related peptide (HBsAg _180-195 , HLA-DR1 restricted) or control peptide (HBsAg _126-138 , H-2IA ^b restricted and HIV1 _{Gag 263-278} , HLA-DR1 restricted) Detected. (B) Similar evaluation of antibody response (IgG, upper panel), cytolytic response (middle panel) and proliferation response (lower panel) of 6 mice (1-6) immunized with HBsAg-DNA, Compared to the average response of one untreated mouse (0). _Cytolytic activity at an E / T ratio of 30/1 was _{measured using the immunodominant} peptide HBsAg _348-357 (filled bar) or the subdominant peptide HBsAg _335-343 (gray bar). RMAS-HHD target cells pulsed with either of these were evaluated.

FIG. 4 shows the result of the defense analysis. H-2 class I / class II-KO mice transfected with HLA-A2.1 / HLA-DR1 were prepared with and without immunization twice with plasmid DNA encoding HBsAg. . Fifteen days after the last immunization, the mice were challenged intraperitoneally with 10 ⁷ PFU of rVV expressing either HBsAg or HBx protein. Four days later, mice were individually tested for virus titer in the ovaries. Results (rVV PFU / ovary in base 10 logarithm) were immunized with HBsAg-DNA challenged with rVV-HBsAg (I, n = 10), untreated mice challenged with rVV-HBsAg (N, n = 6), shown for mice immunized with HBsAg challenged with rVV-HBx (Ix, n = 6) and untreated mice challenged with rVV-HBx (Nx, n = 6) Yes.

FIG. 5 shows the anti-PreS2 response of AC in HLA-A2 ⁺ DR1 ⁺ CI ⁻ CII ⁻ mice following immunization with pcmvS2 / S.

FIG. 6 shows the proliferative response of CD4 ⁺ T cells to HLA-DR1 restricted epitopes following immunization of HLA-A2 ⁺ DR1 ⁺ CI ⁻ CII ⁻ mice with pcmvS2-S.

FIG. 7 shows the cytotoxic response of CD8 ⁺ T cells to HLA-A2 restricted HBS (348-357) peptide according to immunization of HLA-A2 ⁺ DR1 ⁺ CI ⁻ CII ⁻ mice with pcmvS2 / S. Show.

The sequence SEQ ID NO: 1 contains the lower part, nucleotides 1-1205, the lower part, contains the HLA-A2 promoter, nucleotides 1206-1265 contain the HLA-A2 leader sequence, nucleotides 1266-1565 Contains human β2 microglobulin cDNA, nucleotides 1566-1610 contain a (Gly4Ser) ₃ linker, nucleotides 1611-2440 contain a segment containing exon 2 and part of intron 3 of HLA-A2, and nucleotides 2441-4547 is part of intron 3, exon 4 8, and include a segment containing the 3 'portion of the non-coding region of the H ₂ D ^b gene.

SEQ ID NO: 2 is the nucleotide sequence of the DRA ^* 0101 gene. Nucleotides 1-15279 is the promoter located in the 5 'to HLA-DRalpha gene, nucleotides 15280-15425 are exon 1, nucleotides 15344-15346 are the ATG start codon, and nucleotides 1738-18083 are exon 2 , Nucleotides 18575-18866 are exon 3, nucleotides 19146-19311 are exon 4, and nucleotides 20008-20340 are exon 5.

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

  The practice of the present invention, unless otherwise indicated, includes cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are included in the art. Conventional techniques shall be used. Such techniques are explained fully in previous studies. For example, Sambrook, Fritsch and Maniatis ed. “Molecular Cloning A Laboratory Manual” 2nd edition (Cold Spring Harbor Laboratory Press: 1989); “DNA Cloning” Volume I and Volume II (D. N, 1988); “Oligonucleotide Synthesis” (MJ Gait, 1984); “Mullis et al. U.S. Pat. No. 4,683,195; “Nucleic Acid Hybridization” (BD Hames & SJ Higgins, 1984); “Transscript And Translation” (BD Hames & S. J. Higgs; 198). Culture Of Animal Cells (R.I. Freshney, Alan R.Liss, Inc., 1987); “Immobilized Cells And Enzymes” (IRL Press, 1986); Perbal "A Practical Guide To Molecular Cloning" (1984); "Methods In ENZYMOLOGY" (J. Abelson and M. Simon Editor-in-Chief, Academic Press, Inc., Volume 15, Volume 15) (Edited by Wu et al.) And Volume 185 “Gene Expression Technology” (Edited by D. Goeddel); “Gene Transfer Vectors for Mammalian Cells” (Edited by J. H. Miller and MP P. Calos, 1987, Calis. Laboratory); “Immunochemical Me "Thods In Cell And Molecular Biology" (Mayer and Walker, Academic Press, London, 1987); "Handbook Of Experimental Immunology", Volumes I to C. W. D.M. And see "Manipulating the Mouse Embryo" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

The present invention provides a mouse, which comprises (1) a mutated H-2 class I molecule and a class II molecule, and (2) a transgenic HLA class I molecule or gene It expresses the introduced HLA class II molecule, or both the introduced HLA class I molecule and the introduced HLA class II molecule. The mouse according to the present invention knocks out both H-2 class I and class II molecules, expresses the transfected HLA class I molecule and the introduced HLA class II molecule, and is fully humanized An experimental mouse, which simultaneously detects the presence or absence of an antigen-specific antibody, an antigen-specific HLA-DR1-restricted T cell reaction, and an antigen-specific HLA-A2-restricted T cell reaction Can be used for To study how these mice interact with each other between CTL responses, TH responses (especially TH ₁ or TH ₂ responses), and possibly humoral immune responses Useful. These mice will be the best tools for basic and applied vaccine research.

  The present invention provides a transgenic mouse comprising a disrupted H-2 class I gene, a disrupted H-2 class II gene, and a functional HLA class I or class II transgene. is there. In some embodiments, the transgenic mouse has a disrupted H-2 class I gene, a disrupted H-2 class II gene, a functional HLA class I transgene, and a functional HLA class II transgene. Contains. Such a mouse can be said to be a fully humanized laboratory mouse, because it is antigen specific antibody, antigen specific HLA-DR1 restricted T cell response and antigen specific. This is because it can be used to simultaneously detect the presence or absence of a typical HLA-A2 restricted T cell reaction.

Generating HLA-A2.1 / HLA-DR1 as partly shown in the examples given in the present invention and as will be generally apparent to those skilled in the art from the disclosure. H-2 class I / class II-KO mice have the ability to exhibit antibody responses specific for HBsAg, CD4 ⁺ helper T cell responses and cytolytic CD8 ⁺ T cell responses following immunization with DNA ing. These responses were seen in all mice tested, but occurred against the same immunodominant epitope as the human response, and the immunized mice exhibited specific protection against HBsAg recombinant vaccinia virus. Indicated.

Helper T cells are found to be fully matured in the antibody response (Katz, DH & Bencerraf, B., “Adv Immunol” 15, 1-94 (1972)), CTL priming against many epitopes (von Boehmer, H. et al. & Haas, W., "J Exp Med" 150, 1134-1142 (1979); Keene, JA & Forman, J., "J Exp Med" 155, 768-782 (1982)), and the long term of CTL It is indispensable for maintenance (Matlaubian, M., Conception, RJ & Ahmed, R., J Virol 68, 8056-8063 (1994)). Antibodies (Lefrancois, L., “J Virol” 51, 208-214 (1984)) and CTL (Zinkernagel, RM & Welsh, RM, “J Immunol” 117, 1495-1502 (1976)) Both are important components of protective immunity against viral infection. Indeed, antibody responses and CTL responses specific for strong HBsAg are seen in H-2 class I / class II-KO mice transgenic for both HLA-A2.1 and HLA-DR1, whereas HLA- It is not observed in H-2 class I / class II-KO mice in which only A2.1 is introduced. Therefore, the help of CD4 ⁺ T cells specific for HBsAg is essential to generate an effective HBsAg-specific CTL and antibody response. These results show that mice immunized with HBsAg (Millich, DR, “Semi Liver Dis” 11, 93-112 (1991)) and humans vaccinated with HBsAg (Celis, E., Kung, P C. & Chang, TW, “J Immunol” 132, 1511-1516 (1984)), which indicates that the development of anti-HBs antibody responses in CD4 ⁺ T cells It suggests that it depends.

  Transgenic mice expressing both HLA-A2.1 class I and HLA-DR1 class II molecules have already been obtained (BenMohamed, L. et al. “Hum Immunol” 61, 764-779 (2000)). . The authors found that both HLA-A2.1 and HLA-DR1 molecules are functionally restricted elements in vivo, and that the product of the HLA-DR1 transgene is a HLA-A2.1 restricted antigen. It has been reported to enhance specific CTL responses. However, the relevance of the immune response in these mice to humans is related to their own H-2 class I molecules and class II, which mice are usually used preferentially and often exclusively as a constraining element in response to antigens. It is underestimated because it still expresses the molecule (Ureta-Vidal, A., Firat, H., Perarnau, B. & Lemonier, FA, J Immunol 163, 2555-2560 (1990) Rohrrich, PS et al., “Int Immunol” 15, 765-772 (2003) (A. Pajott, unpublished results). The present invention described herein overcomes this limitation by providing H-2 class I / class II-KO mice transgenic for HLA-A2.1 / HLA-DR1.

In some embodiments, an H-2 class I / class II-KO mouse transgenic for HLA-A2.1 / HLA-DR1 in the background of β2m-KO is a single strand of HLA-A2.1. Expressed and in the single chain, human β2m is covalently linked to the heavy chain of HLA-A2.1 by peptide arms. The mice also lack normal cell surface expression of H-2 class II IA and IE molecules as a result of inactivation of the H-2IAβ ^b gene, because H-2 IEα is H -2 because it is a pseudo gene of ^b haplotype. The results listed here indicate that such mice have lost cell surface expression of H-2 class I and class II molecules. However, in some cases, the heavy chain of the class I unbound, particularly H-2D ^b present on the cell surface of .beta.2m-KO mice, it has been pointed out that there is a potential to induce alloreactive . Even if this is correct, since such mice do not have peptides, they do not participate in antigen-specific immune responses (Bix, M. & Raulet, D., “J Exp Med” 176, 829-834 (1992)). This is also described in the report of Allen et al (Allen, H., Fraser, J., Flyer, D., Calvin, S. & Flavel, R., "Proc Natl Acad Sci USA" 83, 7447-7451. (1986)), Allen et al. Show that H-2D ^b is expressed on the cell surface even in the absence of β2m in the cell, but such D ^b antigens are D ^b allospecific or D ^b Confirmed not to be recognized by any of the restricted cytotoxic T lymphocytes. Furthermore, D ^b antigens are not recognized by most of the monoclonal antibodies of natural D ^b.

Nevertheless, in mice only HLA-DR.alpha. Introduced gene, in the absence of at least HLA-DR beta chain, that special HLA-DRα / H-2IEβ ^b hybrid composite to some extent may be expressed on the cell surface (Lawrence, SK et al., “Cell” 58, 583-594 (1989)). Despite this observation, these special molecules are also serologically expressed by HLA-A2.1 by mAb (17-3-3S), which is known to react with such mixed components. / HLA-DR1 transgenic H-2 class I / class II-KO mice were not detected on the cell surface (Ozato, K., Mayer, N. & Sachs, DH, “J Immunol” 124, 533-540 (1980)) (FIG. 1a and data not shown). Furthermore, all of the results obtained by studying the T cell response specific for HBsAg and the 1-Gag of HIV in these mice all have HLA-A2.1 molecules and HLA as binding elements. -Shown that only DR1 molecules were used. Therefore, special HLA-DRα / H-2IEβ ^b hybrid body, it appears to be less stable than a normal HLA-DRα / HLA-DRβ molecule and,該混adult, HLA-DR beta It can be said that it can exist only when there is no chain. Mouse strains (Madsen, L. et al., “Proc Natl Acad Sci USA” 96, 10338-10343 (1999) in which all H-2 class II regions (H-2IAβ ^b , IAα ^b , IEβ ^b ) have been deleted. )), As well as mouse strains in which the H-2D ^b gene has been deleted are being analyzed to completely eliminate this possibility. In a preliminary analysis of splenocytes obtained from the first mouse, recovery of the CD4 ⁺ T cell pool similar to that seen in H-2 class II-KO mice (Iaβ ^b °) transfected with HLA-DR1 was found. HLA-DR1-restricted CD4 ⁺ T cell responses in these new mice were seen, and HLA-DR1 restriction in H-2 class I / class II-KO mice transfected with HLA-A2.1 / HLA-DR1 This suggests that it is equivalent to a sex CD4 ⁺ T cell response.

Peripheral CD8 ⁺ T lymphocytes of H-2 class I / class II-KO mice transfected with HLA-A2.1 / HLA-DR1 are H-2 class I transgenic for parental HLA-A2.1. -Compared to KO mice, it is quantitatively and qualitatively similar, at least in terms of BV segment usage, and the TCR repertoire is fully diversified. Compared to wild-type mice, in particular, partial recovery of the CD8 ⁺ T cell pool is consistently observed in mice transfected with one HLA that expresses chimeric (mouse-derived α3 domain) HLA-A2.1 molecules. (Pascolo, S. et al., “J Exp Med” 185, 2043-2051 (1997)). Co-crystal analysis demonstrated that human CD8 also has contact with the α2 domain of the HLA-A2.1 heavy chain, so mouse CD8 and HLA-A2.1 are independent of α3 domain substitution. Interactions between molecules remain inadequate (Gao, GF et al., “Nature” 387, 630-634 (1997)). Inadequate coordination can also occur in the endoplasmic reticulum, where many molecules (TAP, tapasin, ERp57) assist in the biosynthesis of MHC class I molecules. However, at this stage, the only demonstrated functional difference between these mouse and human endoplasmic reticulum molecules, ie, human TAP, but not mouse TAP, has a positively charged cytoplasm. Effective transport of the COOH termini of peptides (Momburg, F., Neefjes, JJ & Hammerling, GJ, Curr Opin Immunol 6, 32-37 (1994)) This is not a problem for HLA-A2.1 molecules that bind peptides with termini, because these peptides are effectively transported by mouse and human TAP. In both H-2 class I-KO mice transfected with only HLA-A2.1 and H-2 class I / class II-KO mice transfected with HLA-A2.1 / HLA-DR1, CD8 ⁺ T Even with a small number of lymphocytes, the lymphocytes effectively respond to HBsAg, and importantly, the latter mice have antibody responses similar to humans, helper T cell responses and cytolytic responses. Indicates.

One of the difficulties that hinders the design of vaccines based on T cell epitopes that target T lymphocytes is the polymorphism of HLA class I / class II molecules. HLA-A2.1 and HLA-DR1 molecules are expressed in a significant proportion of individuals in the human population (from 30% for HLA-A2.1 to 6% for HLA-DR1 18%). The functional clustering of HLA class I molecules into superfamily is based on a significant remainder of the presented set of peptides ³⁴ , but each of the HLA class I isotype variants or allelic polymorphisms Individual analyzes of the reactions generated by the need still be made to identify the optimal epitopes they present. This is particularly important for developing new reagents such as tetramers (HLA class I or HLA class II) for monitoring immune responses. For similar reasons, one obtains a mouse strain that co-expresses HLA-A2.1 with other HLA class II molecules, even though the binding of the peptide to HLA class II molecules is less restrictive than the binding to class I molecules. It will be beneficial. Based on the disclosure herein, additional H-2 class I / class II-KO mice transfected with HLA class I / class II can be configured for these or other purposes.

  H-2-KO mice transgenic for HLA enable detailed analysis and optimization of the immunogenicity of antigen peptides that have the potential to be replaced by humans (Rohrrich, P. S. et al., "Int Immunol" 15, 765-772 (2003); Loirat, D., Lemonnier, FA & Michel, ML, "J Immunol" 165, 4748-4755 (2000). ); Scardino, A. et al., “Eur J Immunol” 31, 3261-3270 (2001)), which is less obvious for vaccine adjuvant formulation studies. This may be due to differences in the various effectors that are recruited early in the response to antigenic attack between the two species. Increasing basic knowledge about innate immunity may lead to a more complete humanization of the mouse immune system in the future.

Finally, the disclosure herein describes a model of an optimized and humanized transgenic mouse, the mouse H-2 class I gene (mouse β2m) and class II gene (H-2IAβ ^b ). Are deleted and replaced with the equivalent human genes HHD (HLA-A ^* 0201), HLA-DRA ^* 0101 and HLA-DRB1 ^* 0101. Cellular immunity in H-2 class I / class II-KO mice transfected with HLA-A2.1 / HLA-DR1 is completely restricted by human HLA molecules and immune responses restricted by mouse MHC molecules There is no. Due to the lack of competition between the murine MHC immune response and the human (transgenic) HLA immune response, these mice were treated with HLA-restricted CD4 ⁺ helper T cells and HLA-restricted CD8 ⁺ cytolysis in human vaccines. It can be used to identify epitopes that require coordination between sex T cells.

  “HLA” is the human MHC complex and “H-2” is the mouse MHC complex. The human complex is three class I α chain genes, HLA-A, HLA-B and HLA-C, and three pairs of MHC class II α chain gene and β chain gene, HLA-DR, -DP And -DQ. In many haplotypes, the HLA-DR cluster contains an extra β chain gene, and the product of the gene can be paired with the DR α chain, so that the three sets of genes consist of four types of MHC classes. II molecule will be produced. In mice, the three class I α 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.

  It is known in the art that genetic diversity exists between the HLA genes of various individuals as a result of both polymorphic HLA antigens and different HLA alleles. Thus, embodiments of the invention disclosed herein can replace one polymorphic HLA antigen with another, or one HLA allele with another. Examples of HLA polymorphisms and alleles are described, for example, at http: // www. anthonynolan. org. uk / HIG / data. html and http: // www. ebi. ac. uk / imgt / hla, edited by Dominique Charon, “Genetic diversity of HLA: Functional and Medical Implication”, EDK Medical and Scientific International Publisher, G. E. Marsh, Peter Parham and Linda Barber, “The HLA FactsBook”, AP Academic Press, 2000.

  A “disrupted” gene is a gene that has been mutated using homologous recombination or other approaches known in the art. The disrupted gene can be either a hypomorphic allele of the gene or a null allele of the gene. One skilled in the art will appreciate that the type of allele used can be selected for any particular background. In many embodiments of the invention, invalid alleles are selected.

  “Homologous recombination” is a general approach for making transgenic animals by mutating a preselected, desired gene sequence in a cell (Mansour, SL et al. , Nature 336: 348-352 (1988); Capecchi, MR, Trends Genet. 5: 70-76 (1989); Capechi, MR, Science 244: 1288-1292 ( 1989); Capecchi, MR et al., Capecchi, edited by MR, Current Communications in Molecular Biology, Cold Spring Harbor Press, Cold Spring Harbor, NY (198). .), Pp.45-52; Frohman, M.A.et al, "Cell" 56: 145-147 (1989)).

  Thus, any gene in the mouse can be arbitrarily modified (Capecchi, MR, “Trends Genet.” 5: 70-76 (1989); Frohman, MA et al. "Cell" 56: 145-147 (1989)). Gene targeting uses standard recombinant DNA techniques to introduce desired mutations into the cloned DNA sequence at a selected locus. The mutation is then introduced into the genome of pluripotent embryonic stem cells (ES cells) through homologous recombination. The modified stem cells are microinjected into a mouse blastocyst, integrated into a developing mouse embryo, and finally grown into a chimeric mouse. In some cases, germline cells of chimeric mice can be derived from genetically modified ES cells and the mutated genotype can be transmitted through reproduction.

  Gene targeting has been used to generate chimeric and transgenic mice in which the nptII gene has been inserted into the β2 microglobulin locus (Koller, BH 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 allowed the generation of chimeric and transgenic mice that have the c-abl gene disrupted by the insertion of the nptII gene (Schwartzberg, PL et al., Science. 246: 799-803 (1989)). The technique has been used to create chimeric mice in which the en-2 gene is disrupted by the insertion of the nptII gene (Joyner, AL et al., Nature 338: 153-155 (1989). )).

  In order to use the “gene targeting” method, the gene of interest must be cloned before that, and the intron-exon boundary must be determined. By this method, a marker gene (for example, nptII gene) is inserted into a translated region of a specific gene of interest. Therefore, when gene targeting is used, the gene of interest is significantly destroyed.

  Significantly, the use of gene targeting to modify a cell's genes results in considerable modification in the gene sequence. The effectiveness of gene targeting depends on the number of variable regions and varies from structure to structure.

  The cells of the chimeric or transgenic mice of the invention are prepared by introducing one or more DNA molecules, which can be pluripotent progenitor cells such as ES cells or equivalents, into the cells. (Capecchi, M. R., "Current Communications in Molecular Biology", Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), Robertson, E.J., 44.) The term “progenitor” means only that the pluripotent cell is a precursor to the (transfected) pluripotent cell of interest, said pluripotent cell being prepared according to the teachings of the present invention. The Pluripotent cells (precursor or transfected) can be obtained by methods known in the art (Evans, MJ et al., “Nature”) to generate chimeric or transgenic mice. 292: 154-156 (1981)).

  Any ES cell can be used according to the present invention. However, it is preferred to use primary isolates of ES cells. Such isolates are described in Capecchi, M .; R. Ed. “Current Communications in Molecular Biology”, Cold Spring Harbor Press, Cold Spring Harbor, N .; Y. (1989) Inventory, Robertson, E .; J. et al. pp. Clone isolation of ES cells directly from embryos, such as the CCE cell line disclosed by 39-44 or from CCE cell lines (Schwartzberg, PA et al., Science 246: 799-803). (1989), which is incorporated herein by reference). Such clonal isolation is described in E. coli. J. et al. Robertson's method (edited by EJ Robertson, “Teratocarcinomas and Embryonic Stem Cells: A Practical Approach”, described in IRL Press, Oxford, 1987). It constitutes a part of the invention. The purpose of such clonal expansion is to obtain ES cells that have great effectiveness for differentiation into mice. Clone-selected ES cells are approximately 10 times more effective in producing transgenic mice than the primary cell line CCE. There is no advantage of clonal selection for the recombinant method of the present invention.

Examples of ES cell lines obtained by clonal isolation from embryos are ES cell lines, AB1 (hprt ⁺ ) or AB2.1 (hprt ⁻ ). ES cells are J. et al. Cell as described by Robertson (E. J. Robertson, “Teratocarcinomas and Embryonic Stem Cells: A Practical Approach”, IRL Press, Oxford, 1987, pp. 71-112). (Especially SNC4 STO cells) and / or primary embryonic fibroblasts, etc., which are part of the present invention by reference. Methods for the generation and analysis of chimeric mice are described in Bradley, A. et al. (E. J. Robertson, “Teratocarcinomas and Embryonic Stem Cells: A Practical Approach”, IRL Press, Oxford, 1987, pp. 113-151), which is incorporated herein by reference. Part of it. Stromal cells (and / or fibroblasts) help to eliminate excessive clonal expansion of abnormal ES cells. Most preferably, the cells are leukocyte inhibitory factor (“lif”) (Gough, NM et al., Reprod. Fertil. Dev. 1: 281-288 (1989); Yamamori, Y. et al. , "Science" 246: 1412-1416 (1989), both of which are incorporated by reference in their entirety). Since the gene encoding life has been cloned (Gough, NM et al., “Reprod. Fertil. Dev.” 1: 281-288 (1989)), the stromal cells carrying this gene are It is particularly preferred that the cells are transformed by methods known in the art and that the ES cells are cultured on transformed stromal cells that secrete the lif into the medium.

  As used herein, the term “transgene” refers to a nucleic acid sequence that is partially or wholly heterogeneous, ie, that is not present in the transgenic animal or cell into which it is introduced, or Homologous to the endogenous gene of the transgenic animal or cell to be introduced, but designed to be inserted into the genome of the animal in such a way as to alter the genome of the cell into which it is inserted, or Refers to an inserted nucleic acid sequence (eg, the nucleic acid sequence is inserted at a location different from that of the native gene, otherwise the insertion results in a knockout). A transgene can be operably linked to one or more transcriptional control sequences and any other nucleic acid that may be required for optimal expression of the selected nucleic acid, such as an intron. it can. A typical transgene of the present invention encodes, for example, an H-2 polypeptide. Other typical transgenes disrupt one or more HLA genes by homologous recombination with the genomic sequence of the HLA gene.

  A “functional transgene” is one that produces an mRNA transcript, which produces an appropriately processed protein in at least one cell of the mouse that contains the transgene. Those skilled in the art will understand that there are a range of options with various combinations of known transcriptional control elements and sequences that direct post-transcriptional processing, from which the host mouse directs the expression of the transgene. It is. In many embodiments of the invention, it is preferred that the HLA transgene is expressed under the control of the H-2 gene regulatory element.

  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 includes an HLA-A2 sequence listed in a sequence list. An example of an HLA-DR1 transgene includes an HLA-DR1 sequence listed in a sequence list.

In one embodiment, the invention provides a transgenic mouse that lacks both H-2 class I and class II molecules, in which the transgenic mouse is a functional HLA class I transgene. And a functional HLA class II transgene. In some embodiments, the mouse has a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ °. In another embodiment, the HLA-A2 transgene comprises a sequence of HLA-A2 listed in the sequence list and the HLA-DR1 transgene comprises a sequence of HLA-DR1 listed in the sequence list.

  The present invention also provides isolated transgenic mouse cells. In some cases, the cell contains a disrupted H-2 class I gene, a disrupted H-2 class II gene, and a functional HLA class I or class II transgene. In other cases, the cell comprises a disrupted H-2 class I gene, a disrupted H-2 class II gene, a functional HLA class I transgene, and a functional HLA class II transgene. Yes. The HLA class I transgene may be an HLA-A2 transgene and the HLA class II transgene may be an HLA-DR1 transgene. In some cases, the HLA-A2 transgene includes the sequence of HLA-A2 listed in the sequence list, and the HLA-DR1 transgene includes the sequence of HLA-DR1 listed in the sequence list.

In one embodiment, the invention provides isolated transgenic mouse cells lacking both H-2 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 cells of the isolated transgenic mouse can have a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ °. The HLA-A2 transgene can include the sequence of HLA-A2 listed in the sequence list, and the HLA-DR1 transgene can include the sequence of HLA-DR1 listed in the sequence list.

  The cells of the isolated transgenic mouse of the present invention can have any mouse genotype of the present invention. However, the set of genotypes of cells of the isolated transgenic mouse of the present invention and the set of genotypes of the mouse of the present invention do not necessarily overlap completely.

  Isolated mouse cells of the present 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 cells are obtained from a mouse or mouse embryo, the cellular genes can be disrupted, eg, by homologous recombination. Furthermore, functional genes can be introduced into the genome of the cell, for example by transfection. A person skilled in the art can apply any suitable method known in the art to modify the genome of a cell, so that an isolated mouse cell having the desired genotype can be obtained. It is understood that it is obtained.

Another object of the invention is an isolated transgenic mouse cell lacking both H-2 class I and class II molecules, in which the transgenic mouse cell is functional. It contains an HLA class I transgene and a functional HLA class II transgene. In some embodiments, the cells of the transgenic mouse have a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ °. In other embodiments, the HLA-A2 transgene comprises a sequence of HLA-A2 listed in the sequence list, and the HLA-DR1 transgene comprises a sequence of HLA-DR1 listed in the sequence list.

  T cells play a central role in many aspects of acquired immunity and perform a variety of regulatory and protective functions. When some T cells encounter infected or cancerous cells, they recognize them as xenogeneic and respond by acting as killer cells, as part of the cellular immune response. Kill. Other T cells, called helper T cells, are recognized by stimulating B cells to produce antibodies or by suppressing certain aspects of humoral or cellular immune responses. Reacts to different antigens.

  Helper T cells (Th) organize many immune responses through the production of cytokines. Although generally identified as having CD4 cell surface markers, these cells functionally depend on the cytokine profile they produce and their effects on other cells of the immune system , Th1 or Th2.

Th1 cells detect a host cell that is a pathogen or cancer that is about to enter through a recognition system called a T cell antigen receptor. A process involving Th1, called cellular immunity, generally involves activation of cells other than B cells and is often characterized by the production of IFN-γ. Nevertheless, Th1 cytokines promote immunoglobulin class switching to the IgG _2a isotype, even though the Th1 system is independent of humoral antibody production in the first place.

  Upon detection of the heterologous antigen, most mature Th1 cells become IL-2, IL-3, IFN-γ, TNF-β, GM-CSF, high concentrations of TNF-α, high concentrations of MIP-1α, Command the release of high concentrations of MIP-1β and RANTES. These cytokines promote delayed-type hypersensitivity and general cellular immunity. For example, IL-2 is a T cell growth factor that promotes the generation of additional T cell clones that are sensitive to the specific antigen first detected. Sensitized T cells attach and attack antigen-containing cells or pathogens.

In contrast, mature Th2 cells are expressed in IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF, and low concentrations of TNF-α. Tend to promote the secretion of. Moreover, Th2 response, to activate the B cells, it stimulates the production and secretion of antibodies, and to promote humoral immunity by inducing IgA, the class switching to IgG ₁ and IgE isotypes.

  As used herein, an “antigen” is 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 under study to determine whether it functions as an antigen.

  A “humoral immune response” is specific immunity mediated by antibodies.

  An “epitope” is a site on an antigen that is recognized by the immune system. An antibody epitope is a site on an antigen that is recognized by an antibody. A T cell epitope is a site on an antigen that is bound to an MHC molecule. A TH epitope is a site on an antigen that is bound to an MHC class II molecule. A CTL epitope is a site on an antigen that is bound to an MHC class I molecule.

  An antigen can comprise a polypeptide or polynucleotide sequence, which can consist of RNA, DNA, or both. In one embodiment, the antigen comprises at least one polynucleotide sequence that functionally encodes one or more antigenic polypeptides. As used in this context, the term “comprising” means a transcriptional apparatus of a host cell in which at least one antigenic polypeptide acts on an exogenous polynucleotide encoding at least one antigenic polypeptide and / or Or by a translation device, which is described, for example, in US Pat. No. 6,194,389 and US Pat. No. 6,214,808.

  The antigen of the present invention may be any antigenic molecule. Antigenic molecules include proteins, lipoproteins, and glycoproteins, including albumin, tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacterial outer membrane proteins (including meningococcal outer membrane proteins) , RSV-F protein, malaria-derived peptide, B-lactoglobulin B, aprotinin, ovalbumin, lysozyme, and other viral, bacterial, parasitic, animal and fungal proteins, and carcinoembryonic antigen ( CEA), CA15-3, CA125, CA19-9, prostate specific antigen (PSA), and tumor associated antigens such as the TAA complex of US Pat. No. 5,478,556, the specification of which is incorporated by reference The whole forms part of the present invention. Antigenic molecules also include carbohydrates, including natural and synthetic polysaccharides, and other polymers, including ficoll, dextran, carboxymethylcellulose, agarose, polyacrylamide and other acrylic resins, lactic acid Glycolic acid copolymer, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, polyvinylpyrrolidine, group B streptococcal capsular polysaccharide and pneumococcal capsular polysaccharide (including type III), Pseudomonas aeruginosa cells Outer mucopolysaccharides and capsular polysaccharides (including Fisher type I), as well as hemophilus influenza polysaccharides (including PRP). Antigenic molecules also include haptens and other components including low molecular weight molecules such as TNPs, sugars, oligosaccharides, polysaccharides, peptides, toxins, drugs, chemicals and allergens. And antigenic molecules also include haptens and antigens derived from bacteria, rickettsia, fungi, viruses, parasites, including diphtheria, pertussis, tetanus, hemophilus influenza virus, pneumococci, E. coli , Klebsiella, Staphylococcus aureus, Staphylococcus epidermidis, Neisseria meningitidis, Poliovirus, Mumps virus, Measles virus, Rubella virus, Respiratory syncytial virus, Rabies virus, Ebola virus, Anthrax, Listeria monocytogenes, Hepatitis A Virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus I and II, herpes simplex virus type 1 and 2, CMV, EBV, varicella-zoster virus, plasmodium, tuberculosis, Candida albicans, other Candida Fungus, Pneumocystis carini, Mycoplasm , A and B influenza viruses, adenovirus, group A streptococci, group B streptococci, Pseudomonas aeruginosa, rhinovirus, leishmania, parainfluenza virus type 1, type 2 and type 3, coronavirus, salmonella, dysentery Fungi, rotavirus, toxoplasma, enterovirus, and Chlamydia trachomatis and Chlamydia pneumoniae are included.

  As used in the present invention, a pharmaceutical composition or vaccine comprises at least one immune composition that is dissolved in a pharmaceutically acceptable carrier or excipient, It can become cloudy or bind. Any pharmaceutically acceptable carrier can be used for administration of the composition. Suitable pharmaceutical carriers are A. Gennaro, “Remington's Pharmaceutical Sciences”, 18th edition, 1990, Mack Pub. Easton, Pa. Which is incorporated by reference in its entirety to form part of the present invention. The carrier can be a sterilized liquid such as water, polyethylene glycol, dimethyl sulfoxide (DMSO), and oils including petroleum, animal oils, vegetable oils, peanut oil, soybean oil, mineral oil, sesame oil and the like. The carrier can be in the form of a mist, spray, powder, wax, cream, suppository, implant, ointment, salve, patch, poultice, film, or cosmetic.

  The appropriate formulation of a pharmaceutical composition or vaccine will depend on the route of administration chosen. For example, for intravenous administration by rapid intravenous infusion or continuous infusion, the composition is preferably water-soluble and saline is the preferred carrier. For transdermal, nasal, oral, intragastric, vaginal, rectal, or other transmucosal administration, penetrants appropriate to the barrier to be permeated can be included in the formulation, It is known in the art. For oral administration, the active ingredient can be combined with carriers suitable for inclusion in tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like. Time-dependent delivery systems are also applicable for administration of the compositions of the present invention. Typical systems include systems based on polymers such as lactic acid glycolic acid copolymers, copolymerized oxalates, polycaprolactones, polyester amides, polyorthoesters, polyhydroxybutyric acid and polyanhydrides. Although these polymers and similar polymers can be formulated into microcapsules according to methods known in the art, such methods are taught, for example, in US Pat. No. 5,075,109, which is referred to Thus, the whole constitutes a part of the present invention. Alternative delivery systems suitable for administration of the disclosed immunostimulatory compounds of the present invention are US 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, 4,804,043, Nos. 4,667,014, 4,452,775, 3,854,480, and 3,832,252, each of which is incorporated by reference in its entirety. Includes what is being done.

  Dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable or spray solutions. For spray administration, a suitable propellant can be added, as is understood by those skilled in the art, as is the case with a pressurized spray or nebulizer. Immunological compositions are also envisioned as solubilizers, emulsifiers, stabilizers, dispersants, fragrances, adjuvants, carriers, local anesthetics such as lidocaine or xylocaine, antibodies, and known or possible. Antiviral, antibacterial, antiparasitic, or antitumor compounds can also be formulated.

  An “adjuvant” is a composition that promotes or enhances an immune response to a target antigen. One skilled in the art can select an appropriate adjuvant for use in practicing the present invention in light of the disclosure herein.

  The present invention includes a method of treating a patient in need of immunostimulation by administering a composition comprising one or more of the antigens of the present invention. As used in the present invention, treatment includes correction, recovery, amelioration and prevention methods associated with any disease, illness, abnormality or condition. Furthermore, treatment includes generating or suppressing an immune response in the laboratory animal or ex vivo.

  Accordingly, treatment includes administering an immunostimulatory amount of any immunostimulatory composition of the present invention by any method familiar to one of ordinary skill in the art, which generally includes the oral route. , Including transdermal route, intravenous injection, intramuscular injection and subcutaneous injection. In addition, intraperitoneal administration, intracorporeal administration, intraarticular administration, intraventricular administration, intrathecal administration, topical administration, tonsil administration, mucosal administration Including transdermal, vaginal and gavage.

  As recognized by skilled practitioners, choosing an appropriate method of administration may contribute to the effectiveness of the treatment, and local administration may be preferred for certain applications. There is. Acceptable routes of topical administration include subcutaneous, intradermal, intraperitoneal, intravitreal, inhalation or irrigation, oral, nasal, and infusion directed to a given tissue, organ, joint, tumor, or cell population Is included. For example, applying or injecting the mucosa into the lymph nodes or Peyer's patches of the mucosa may promote a humoral immune response with a significant class switch to IgA. Alternatively, infusion targeting a lesion site, lesion, or infected body site can be applied to the treatment of solid tumors, local infections, or other sites that require immunostimulation.

  In addition, immune system cells (eg, T cells, B cells, NK cells, or oligodendrocytes) can be collected from the host and processed in vitro. The treated cells can be further cultured and reintroduced into the patient (or into 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 HDR to stimulate global or specific immunity. High dose radiation treatment or equivalent treatment can then be used to destroy immune cells remaining in the patient. Upon retransplantation, the activated autologous cells will restore normal immune function in the patient. Alternatively, NK cells and / or T cells isolated from a patient affected with cancer can be exposed in vitro to one or more antigens specific for the patient's cancer. When re-implanted into a patient, antigen-stimulated cells develop an active cellular immune response against cancer cells.

  An immunostimulatory (effective) amount refers to the amount of vaccine that can stimulate an immune response in a patient, preventing and ameliorating pathogenic attacks, allergies, or immune abnormalities or immunological diseases, Or an amount sufficient to treat. The immunostimulatory amount measures the humoral or cellular immune response to at least one epitope of the antigen compared to the response obtained when the antigen is administered to a patient who has not received prior treatment with the vaccine The amount is as high as possible. Thus, for example, an immunostimulatory amount promotes the production of antibodies to the antigenic epitope of interest, or activates a detectable protective effect against pathogenic or allergic attacks, or is of interest Refers to the amount of an antigen-containing composition that can promote a protective CTL response to an antigenic epitope.

  Treatment with an immunostimulatory amount of an antigen-containing composition of the invention is directly, indirectly, or statistically observed in an immune response in a host comprising at least one immune system cell or cell line derived therefrom. Inducing any increase or other desired change that is possible or measurable, particularly including hosts that have been cultured tissue ex vivo. Host cells can be derived from human or animal peripheral blood, lymph nodes, and the like. Ex vivo tissue culture hosts preferably include freshly isolated T cells, B cells, macrophages, oligodendrocytes, NK cells, and monocytes, each of which is isolated or purified using standard techniques. can do. Observable or measurable responses include B or T cell proliferation or activation, increased antibody secretion, isotype switching, increased cytokine release, particularly one or more 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- Increased release of CSF, MIP-1α, MIP-1β or RANTES, increased antibody titer or affinity for specific antigens, decreased morbidity or mortality associated with pathogenic infection, promoted, induced, maintained or viral latency Includes strengthening, growth of malignant and non-malignant tumors, metastasis, suppression or mitigation of effects, and prophylactic protection from disease or disease effects.

  In cases where suppression of the immune response is desired, for example in the treatment of autoimmune diseases or allergies, an effective amount is also sufficient to measurably or observably reduce the response associated with the disease or lesion to be treated. Amount included.

  The amount of antigen-containing composition to be administered and the frequency of administration can be determined empirically and will take into account the age and size of the patient being treated, the disease or condition being treated. Appropriate volumes fall within the range of 0.01 μg to 100 μg per inoculum, although higher or lower amounts may be indicated. Immunization with secondary boosts can occur after an interval of one week to several months.

  The following examples illustrate some embodiments of the present invention. Those skilled in the art will recognize numerous modifications and variations that can be made without changing the spirit or scope of the invention. These modifications and variations are considered to be within the scope of the present invention. The examples are not intended to limit the invention in any way.

  The following experimental techniques and reagents were used to illustrate some non-limiting embodiments of the present invention.

  Transgenic mouse

H-2 class II-KO mice transfected with HLA-DR1 (IAβ ^b °) were tested at the Pasteur Institute of Lille (Institut Pasteur of Lille) and mice transfected with HLA-DR1 (Altmann, DM) Et al., “J Exp Med” 181, 867-875 (1995)) and H-2 class II-KO mice (IAβ ^b °) (Rohrrich, PS et al., “Int Immunol” 15, 765-772 (2003)). Chimera single chain (HHD molecule: α1-α2 domain of HLA-A2.1, α-2 to H-2D ^{b from} the α3 to intracellular domain at its N-terminus by a 15 amino acid peptide linker at the C-terminus of human β2m Mice in which HLA-A2.1 was transfected (Pascolo, S. et al., “J Exp Med” 185, 2043-2051 (1997)). An H-2 class I-KO mouse into which HLA-A2.1 (HHD) has been introduced and an H-2 class II-KO mouse into which HLA-DR1 has been introduced (IAβ ^b °) are cross-bred, Selection was performed until H-2 class I (β2m ⁰ ) / class II (IAβ ⁰ ) -KO mice into which HLA-A2.1 ^+/− / HLA-DR1 ^+/− were introduced were obtained. Used for the experiments described. H-2 class I (β2m ⁰ ) / class II (IAβ ⁰ ) -KO mice transfected with only HLA-A2.1 ^+/− were used as controls in the protection analysis. Mice will be housed in the animal facility of the Pasteur Institute in Paris (Institut Pasteur, Paris) and all procedures will be passed to ensure compliance with French and European regulations for animal protection and the recommendations of the Public Health Service. Under investigation by the competent authority of the Tool Institute.

  Genotyping

HLA-DRB1 ^* 0101, HLA-DRA ^* 0101 and HLA-A ^* 0201 transgenes were detected by PCR. Incubate overnight at 56 ° C. in 100 mM NaCl, 50 mM Tris-HCI pH 100, 100 mM EDTA, 1% SDS and 0.5 mg / ml proteinase K, then 250 μl saturated NaCl After adding a solution and performing isopropanol precipitation, the tail part of DNA was extracted. The specimens were washed in 70% ethanol (x3) and resuspended in 150 μl EDTA, 10 mM Tris-HCI, 1 mM pH 8. PCR conditions are as follows. 1.5 mM MgCl ₂ , 1.25 U Taq polymerase, buffer provided by the manufacturer (InVitrogen, Carlsbad, Calif.), 1 cycle (7 minutes, 94 ° C.), 40 cycles ( 30 seconds, 94 ° C .; 30 seconds, 60 ° C .; 1 minute, 72 ° C.), 1 cycle (4 minutes, 72 ° C.), 5′CAT TGA GAC AGA GCG CTT for HHD as forward primer and reverse primer GGC ACA GAA GCA G3 ′ and 5 ′ GGA TGA CGT GAG TAA ACC TGA ATC TTT GGA GTA CGC 3 ′ are used for HLA-DRB1 ^* 0101 and 5 ′ TTC TTC AAC GGG 3 AGG GGG CGG CAC TGT GAA GC 'Used for HLA-DRA ^* 0101 is, 5'CTC CAA GCC CTC TCC CAG AG3 ' CTC ACC AAC 3 with and 5'ATG TGC CTT ACA GAG GCC CC 3 '.

  FACS analysis

Fluorescence quantification of cells was performed on W6 / 32 (anti-HLA-ABC, Sigma, St. Louis, MO) and biotinylated anti 28-8-6S (anti ^{H-2K b / D b,} BD Biosciences , Inc., San Diego, CA) m. Performed with Ab. CD4 ⁺ and CD8 ⁺ T lymphocytes were isolated from PE-labeled CT-CD4 anti-mouse CD4 (CALTAG, South San Francisco, Calif.) And FITC-labeled 53-6.7 anti-mouse CD8-m. Staining was performed using 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). 2.4G2m. After the Fc receptor is saturated with ab, HLA-DR1 and the expression of H-2IA ^b, FITC-labeled L243 (anti-HLA-DR) and PE-labeled AF6-120.1 (anti H-2IAβ ^b) m. Analysis was performed using Ab (BD Biosciences). Cells fixed with paraformaldehyde were analyzed on a FACSCalibur (Becton Dickinson, Bedford, Mass.).

  Immunoscope analysis

CD4 ⁺ and CD8 ⁺ T cells derived from untreated mice were positive-selected with Auto-Macs (Miltenyi Biotec), RNA was prepared using RNA Easy Kit (Qiagen, Hilden, Germany), cDNA Used for synthesis. The cDNA was PCR amplified using a forward primer specific for each BV segment family and a reverse primer common to the two BC segments. The PCR product was extended in run-off using an internal BC primer tagged with FAM. The run-off products were loaded on a 6% acrylamide / 8M urea gel for separation (7h, 35W) on 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)).

  peptide

HLA-A2 binding peptides HBsAg _348-357 GLSPTVWLSV and HBsAg _335-343 WLSLLVPFV, H-2K ^b binding peptide HBsAg _371-378 ILSPFLPL, HLA-DR1 binding peptides HBsAg _180-195 QAGFFLLTRILTIPQS, the H-2ia ^b binding peptide HBsAg _126-138 RGLYFPAGGSSG, and the preS2 peptide HBsAg _109-134 MQWNSTTFHQTLQDPRVRLYFPAGG 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 the peptide starts with the first methionine of the preS1 region of the ayw subtype of HBV.

  Immunization with DNA encoding S2-S protein of HBV

  PCMV-S2, which encodes the surface antigens preS2 and S of HBV expressed under the control of the human CMV immediate early gene promoter. S plasmid vector (Michel, ML et al., “Proc Natl Acad Sci USA” 92, 5307-5311 (1995)) was purified on a column of Plasmid Giga Kit (Qiagen) under conditions of endotoxin free. did. Regeneration of anesthetized mice as described in the prior literature (Davis, HL, Michel, ML & Whalen, RG, “Hum Mol Genet” 2, 1847-1851 (1993)). Injection into the anterior tibial muscle (50 μg on each side).

T cell proliferation analysis Twelve days after the last immunization, as described in the literature (Loirat, D., Lemonier, FA & Michel, ML, J Immunol, 165, 4748-4755 (2000 )), Ficoll-purified splenocytes (5 × 10 ⁶ cells / 25 cm ² culture flask (Techno Plastic Products (TPP), Trasadingen, Switzerland)) from which red blood cells have been removed, pulsed with peptide (20 μg / ml), LPS immature cells (5 × 10 ⁶ cells / culture flask) that were γ-radiated (180 Gy), 10% FCS, 10 mM HEPES, 1 mM sodium pyruvate, 5 × 10 ⁻⁵ M 2- Mercaptoethanol, 100I × U / ml penicillin and 100 μg strep In RPMI medium mycin was added, they were co-cultured. On day 7, for proliferation analysis, cells were cultured in RPMI complete medium supplemented with LPS immunized cells (2 × 10 ⁵ cells / well) pulsed with peptide and irradiated for 72 hours with 3% FCS. Planar culture was performed (5 × 10 ⁵ cells / well in a flat-bottom 96-well microplate (TPP)). Cells were pulsed with 1 μCi of ( ³ H) -thymidine per well for 16 hours prior to harvesting with filtermates (Perkin Elmer Applied Biosystem) using a TOMTEC collector, and the incorporated radioactivity was measured with a microβ counter (Perkin Elmer Applied). (Biosystem). Results were expressed as activation index (SI) = cpm with specific peptide / cpm with unrelated peptide.

  Measurement of CTL activity

As a proliferation analysis, a cytotoxicity analysis was performed on the same group of immune spleen cells. Responder cells (5 × 10 ⁶ cells / 25 cm ² culture flask, TPP) and pulsed peptide (20 μg / ml) for stimulation and emitted γ rays (180 Gy) LPS immature antigen cells (5 × 10 ⁶ cells / culture flask) were co-cultured in the same supplemented RPMI medium for 7 days. Cytolytic activity was tested in standard 4h ⁵¹ Cr assay on RMA-S-HHD target cells pulsed with 10 μg / ml experimental peptide or control peptide. The specific lysis expressed in% was calculated in two steps, which was calculated as [experimental spontaneous release] / [maximum spontaneous release] × 100 minus the nonspecific lysis seen with the control peptide. It is a thing.

  Measuring antibody production in vivo

  At various times before and after DNA injection, blood was collected from mice using a heparinized glass pipette by retrobulbar puncture and serum collected by centrifugation was collected for anti-HBs and anti-preS2 by specific ELISA. analyzed. Purified recombinant particles containing HBV smallS protein (1 μg / ml) or preS2 (120-145) synthetic peptide (1 μg / ml) were used as the solid phase. Serial dilutions were performed after blocking with PBST (PBS containing 0.1% Tween 20) with 10% FCS. After extensive washing, the bound antibody was detected with anti-mouse Ig (total IgG) labeled with horseradish peroxidase (Amersham, Little Chalfont, UK). Antibody titer was measured by serial end point dilution method. Mouse sera were tested individually and the titer was the average of at least three measurements. When serum was diluted to less than 1/100, it was considered negative.

  Antibody titer measurement

Sera from immunized mice were individually purified by ELISA (Michel, ML et al., “Proc Natl Acad Sci USA” 92, 5307-5311 (1995)) and the HBV middle protein and small. Either protein or preS2 synthetic HBs _109-134 peptide was analyzed. After blocking with 1 × PBS with 0.1% Tween 20 and 10% FCS and washing (× 3), the bound antibody was anti-mouse IgG labeled with horseradish peroxidase (Amersham, Little Chalfont, UK) ). Antibody titers (average of at least 3 measurements) were measured by serial end point dilution. Titers less than 1/100 were considered negative.

  Vaccine attack and plaque assay

Mice injected with DNA were either HbsAg protein (Smith, GL, Macckett, M. & Moss, B., Nature 302, 490-495 (1983)) or HBx protein (Schek, N., Bartenschlager, R , Kuhn, C. & Schaller, H., Oncogene 6, 1735-1744 (1991)), 10 ⁷ PFU of recombinant vaccinia virus (Western Reserve strain), last injection The vaccinia virus expressing HBsAg protein and the vaccinia virus expressing HBx protein were attacked intraperitoneally 12 days later. Dr. Moss and H.C. Courtesy of Dr. Schaller. Four days later, ovaries were analyzed for rVV titers by plaque assay on BHK21 cells (Buller, RM & Wallace, GD, Lab Anim Sci 35, 473-476 (1985)).

Cell surface expression HLA-A2.1 MHC ^{molecule, H-2K b / D b} , the cell surface expression of HLA-DR1 and H-2ia ^b molecules, by flow cytometry, was evaluated in spleen cells. As shown in FIG. 1a, similar levels of HLA-A2.1 expression were observed in H-2 class I / class II-KO mice transfected with HLA-A2.1 / HLA-DR1, and HLA HLA-A2.1 was not observed in H-2 class II-KO mice transfected with HLA-DR1, whereas -A2.1 was transfected in H-2 class I-KO mice only H-2K ^b / D ^b was expressed. The HLA-DR1 and H-2ia ^b of cell surface expression was measured in rich B cells B220 ^+. As shown in FIG. 1b, similar levels of HLA-DR1 expression were observed in H-2 class I / class II-KO mice transfected with HLA-A2.1 / HLA-DR1, and HLA-DR1. However, no expression was detected in H-2 class I-KO mice transfected with HLA-A2.1. However, cell surface expression of the transduced molecules (especially HLA-DR1) was lower than that of endogenous H-2 class I and class II molecules.

The number of peripheral CD4 ⁺ T cells and CD8 ⁺ T cells CD4 ⁺ and CD8 ⁺ splenic T cells were determined by immunostaining and flow cytometric analysis as shown in FIG. 2a.

CD4 ⁺ T cells are found in both H-2 class I / class II-KO mice transfected with HLA-A2.1 / HLA-DR1 and H-2 class II-KO mice transfected with HLA-DR1. , 13% to 14% of the splenocyte group. In contrast, in H-2 class II-KO mice, only 2% to 3% of the cells are CD4 ⁺ (data not shown), and initial reports for mice lacking MHC class II molecules (Cosgrove, D. et al., “Cell” 66, 1051-1066 (1991)). As expected, the expression of the transgenic HLA-A2.1 molecule increased the proportion of peripheral CD8 ⁺ T cell populations and was 0 in mice lacking MHC class I that KO for β2 microglobulin (β2m). (Pascolo, S. et al., “J Exp Med” 185, 2043-2051 (1997)), HLA-A2.1 / HLA-DR1 gene-transfected H -2 class I / class II-KO mice and H-2 class I-KO mice transfected with HLA-A2.1 reached 2% to 3% of total splenocytes.

The results shown in Example 1 and Example 2 are:
(1) In HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° mice, expression of HLA-A2 molecule, non-expression of H-2- ^Kb molecule, number of CD8 ⁺ peripheral T lymphocytes, and CD8 ⁺ T repertoire That the diversity of HLA-A2 ⁺ β2m ° mice is comparable
(2) In the ^{HLA-A2 + HLA-DR1 +} β2m ° IAβ ° mice, the expression of HLA-DR1 molecules, non-expression of H-2-IA ^b molecules, CD4 ⁺ T lymphocyte numbers and CD4 ⁺ repertoire diversity Gender is roughly comparable to HLA-DR1 ⁺ IAβ ° mice, and
(3) HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° mice have all of the characteristic advantages seen in HLA-A2 ⁺ β2m ° mice and HLA-DR1 ⁺ IAβ ° mice,
Is shown.

Use of TCR BV Segments The presence of one MHC class I molecule and one MHC class II molecule can reduce the number and diversity of TCR repertoires, as described previously (Cochet, M Et al., “Eur J Immunol” 22, 2639-2647 (1992)), various BV family expression and CDR3 length diversity were purified by RT-PCR based immunoscope technology. Cellular CD4 ⁺ or CD8 ⁺ T cells were studied. A large peak with a distribution similar to the Gaussian distribution was seen in both the CD8 ⁺ group of CD cells (FIG. 2b) and the CD4 ⁺ group (FIG. 2c) for most BV families (15 out of 20 analyzed). . The characteristics of such a graph seen in peripheral T lymphocytes are typical for functionally rearranged BV segments, from the first peak to the next, the 3 nucleotide length of the CDR3 subregion. With changes (Cochet, M. et al., Eur J Immunol 22, 2639-2647 (1992)).

It was expected that there was no expansion (or greatly altered graph features) as seen in BV5.3 and 17, which is that these two BV segments were sham in C57BL / 6 mice. Because it is a gene (Wade, T., Bill, J., Marack, PC, Palmer, E. & Kappler, JW, "J Immunol" 141, 2165-2167 (1988); S. et al., "Proc Natl Acad Sci USA" 84, 1992-1996 (1987)). However, the graph changes seen for the segments of BV5.1, 5.2 and 11 are attributed to the small BV-expressing T cell subgroup (the T cell subgroup is in C57BL / 6 mice). Less than 5%, approximately 2% in H-2 class II-KO mice transgenic for HLA-DR1) (data not shown). Other than these cases, both CD4 ⁺ and CD8 ⁺ T cells in H-2 class I / class II-KO mice transgenic for HLA-A2.1 / HLA-DR1, respectively, were nontransgenic C57BL / 6 shows a pattern of TCR BV chain usage and CDR3 diversity similar to that of 6 mice.

Functional analysis HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° mice immunized with AgHBs (hepatitis B virus envelope protein) were analyzed. FIG. 5 shows the specific humoral immune response shown by the production of HBsS2 antibody. FIG. 6 shows the specific DR1-restricted CD4 ⁺ T cell proliferative response to HBs _348-357 . FIG. 7 shows the response of specific HLA-A2 restricted CD8 ⁺ cytolytic T cells to HBs _348-357 or HBs _335-343 .

These results show that in individuals immunized with HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ ° mice, a specific humoral immune response, an Ag-specific HLA-DR1 restricted response of CD4 ⁺ T helper cells, and , Indicating that the cytolytic reaction of Ag-specific HLA-A2 restricted CD8 ⁺ T cells can be analyzed simultaneously.

  Additional data obtained from these mice is shown in Tables 1 to 3 below.

Immune response to HBsAg-DNA vaccines To assess the immunological potential of H-2 class I / class II-KO mice transgenic for HLA-A2.1 / HLA-DR1 and their humoral immune response In order to compare the CD4 ⁺ and CD8 ⁺ T cell responses with those of humans, mice were immunized with the HBsAg-DNA plasmid. This plasmid encodes two hepatitis B virus envelope proteins (middle, preS2 / S and small, S), which are self-assembled with particles with hepatitis B surface antigen. The vaccine currently used against hepatitis B contains these two proteins.

As shown in FIG. 3a for representative mice, HBsAg-specific antibodies were first detected 12 days after injection of the HBsAg-DNA vaccine (FIG. 3a, upper panel) and the titers of these antibodies Increased until day 24 (data not shown 12 days after the second DNA immunization). The early response of this antibody is specific to the preS2-B cell epitope (HBs _109-134 ) and HBsAg particles of the middle HBV envelope protein and immunized with HBsAg-DNA (Michel, ML. Et al., “Proc Natl Acad Sci USA” 92, 5307-5311 (1995)) and humans vaccinated with HBsAg (Moulia-Pelat, JP et al., “Vaccine” 12, 499-). 502 (1994)).

CD8 ⁺ CTL responses to HBsAg were tested, and CD8 ⁺ T cells in the periphery of H-2 class I / class II-KO mice transfected with HLA-A2.1 / HLA-DR1 were transfected with human class I It was determined whether it was functionally constrained by the molecule. In HLA-A2.1 ⁺ humans infected with HBV, an immunologically dominant HLA-A2.1-restricted HBsAg-specific CTL response is _expressed by the HBsAg _348-357 peptide (Maini, MK et al., “ Gastroenterology ”117, 1386-1396 (1999)) and HBsAg _335-343 peptide (Nayersina, R. et al.,“ J Immunol ”150, 4659-4671 (1993)) (ie, multiple epitopes) Reaction was seen). In C57BL / 6 mice, H-2K ^b -restricted HBsAg specific CTL response, HBsAg _371-378 peptide (Schirmbeck, R., Wild, J . & Reimann, J., "Eur J Immunol" 28,4149-4161 ( 1998)). To evaluate whether humanized mice can respond like humans, spleen T cells were _{treated with} related peptides (HBsAg _348-357 , HLA-A2.1 for 7 days as described herein. restricted) or control peptide (HBsAg _371-378, H-2K ^b -restricted, MAGE-3 _271-279, and re-activated with either HLA-A2.1-restricted). FIG. 3a (middle panel) produces a HBsAg _348-357 specific CTL response strongly immunized with HBsAg-DNA but no response to either HBsAg _371-378 peptide or MAGE-3 _271-279 peptide. It shows that.

Whether CD4 ⁺ T cells in the periphery of H-2 class I / class II-KO mice transfected with this HLA-A2.1 / HLA-DR1 are functionally restricted by the transfected human class II molecule To determine if, the CD4 ⁺ T cell response to HBsAg protein was tested. In HLA-DR1 ⁺ humans vaccinated with HBsAg or infected with HBV, an immunologically dominant HLA-DR1 restricted HBsAg-specific CD4 ⁺ T cell response was _{observed in} the HBsAg _180-195 peptide (Mm, W. P. et al., “Hum Immunol” 46, 93-99 (1996)). In C57BL / 6 mice, H-2IA ^b -restricted HBsAg-specific CD4 ⁺ T cell response, HBsAg _126-138 peptide (Milich, D.R., "Semin Liver Dis" 11,93-112 (1991)) in Against. To compare humanized mice with human and wild type mice, splenic T cells were _{treated with} related peptides (HBsAg _180-195 , HLA-DR1 restricted) or control peptides (HBsAg _126-138 , H-2IA ^b restricted). , HIV1Gag _263-278 , HLA-DR1 restricted). FIG. 3a (bottom panel) shows a strong proliferative response to the HLA-DR1-restricted HBsAg _180-195 peptide, as expected, while the H-2IA ^b- restricted peptide was not effective in stimulating the reaction. . Similarly, no response was elicited by the HIV1Gag _263-278 peptide. Furthermore, when further reproduced in vitro by the HBsAg _180-195 peptide, the specific proliferation index increased several fold (data not shown).

First, HBsAg-specific antibody response, T cell proliferation response and cells in H-2 class I / class II- mice immunized with HBsAg-DNA and transfected with HLA-A2.1 / HLA-DR1 Although the generation and specificity of the lytic reaction was demonstrated, for the same three reactions, another 6 mice immunized with HBsAg-DNA and 6 untreated HLA-A2.1 / HLA controls H-2 class I / class II-KO mice transfected with -DR1 were also tested individually. As shown in FIG. 3b, the three responses were demonstrated simultaneously in the 6 immunized mice tested, but not in the control untreated mice. Interestingly, the two immunized mice were capable of showing CTL responses to both HBsAg _348-357 and HBsAg _335-343 HLA-A2.1 restricted peptides (FIG. 3b, middle panel).

Protection analysis The above examples show that HBsAg-specific humoral immunity, CD4 ⁺ and CD8 ⁺ T cells in H-2 class I / class II-KO mice transfected with HLA-A2.1 / HLA-DR1 It demonstrates the induction of a response and shows that the response occurs against an immunologically dominant epitope that is identical to that of a naturally infected or HBsAg vaccinated human. In this example, it was tested whether these responses show protection in vaccinated mice. Since mice are not tolerant to HBV, vaccinia virus transgenic for HBsAg (rVV-HBsAg) was used in these experiments. Mice were immunized intramuscularly twice with 100 μg HBsAg-DNA. Twelve days after the last immunization, mice were challenged intraperitoneally with 10 ⁷ PFU of rVV-HBsAg. After 4 days, virus titers were measured according to known methods and recorded in rVV PFU / ovary (Buller, RM & Wallace, GD, Lab Anim Sci, 35, 473-476 (1985)). ).

  The results are shown in FIG. Untreated mice that were not immunized with HBsAg-DNA showed that rVV-HBsAg was replicated after challenge. In contrast, virus titers in mice immunized with HBsAg-DNA were 4 or more orders of magnitude smaller. These results strongly suggest that vaccination with HBsAg-DNA elicits a protective HBsAg-specific immune response that controls infection with rVV-HBsAg.

  The specificity of protection conferred by vaccination with HBsAg-DNA is demonstrated by attacking mice immunized with HBsAg-DNA with other HBx transgenic VVs (encoding the hepatitis B x protein) It was done. There was no reduction in rVV-HBx replication in mice immunized with HBsAg-DNA compared to non-immunized controls.

HLA-DR1-restricted CD4 ⁺ T cells are important for antibodies and CTL responses and protection against viral infection.
In order to evaluate whether HLA-DR1-restricted helper T lymphocytes contribute to the antibody response and CTL response in humanized mice, one gene (HLA-A2.1) is used for the immune response and virus infection efficiency. Comparison was made in H-2 class I / class II-KO mice that were introduced and double-transfected (HLA-A2.1 / HLA-DR1). As shown in Table 4, a strong HBsAg _348-357 specific CTL response was _observed in H-2 class I / class II-KO mice in which HLA-A2.1 / HLA-DR1 was double-transfected. Although it was observed, it was not observed in H-2 class I / class II-KO mice into which only HLA-A2.1 was introduced. Furthermore, anti-HBs antibodies could not be detected in H-2 class I / class II-KO mice in which HBsAg-DNA was vaccinated and only HLA-A2.1 was introduced. Consequently, H-2 class I / class II-KO mice immunized with HBsAg-DNA and transfected with only HLA-A2.1 were not protected against infection with rVV-HBsAg.

  The entire contents of all references, patent documents and published patent applications cited throughout this application are hereby incorporated by reference in their entirety.

Figure 5 shows a flow cytometric analysis of cell surface expression of the displayed transgenic molecules. Figure 5 shows a flow cytometric analysis of cell surface expression of the displayed transgenic molecules. Shows the number of CD8 ⁺ and CD4 ⁺ splenic T cells and the use of BV segments (based on immunoscope analysis) in mice of the indicated genotype. Shows the number of CD8 ⁺ and CD4 ⁺ splenic T cells and the use of BV segments (based on immunoscope analysis) in mice of the indicated genotype. Shows the number of CD8 ⁺ and CD4 ⁺ splenic T cells and the use of BV segments (based on immunoscope analysis) in mice of the indicated genotype. HBs specific antibody response, cell lysis reaction and proliferation response are shown. HBs specific antibody response, cell lysis reaction and proliferation response are shown. The result of the defense test is shown. FIG. 5 shows AC anti-PreS2 response in HLA-A2 ⁺ DR1 ⁺ CI ⁻ CII ⁻ mice following immunization with pcmvS2 / S. FIG. 6 shows the proliferative response of CD4 ⁺ T cells to HLA-DR1 restricted epitopes following immunization of HLA-A2 ⁺ DR1 ⁺ CI ⁻ CII ⁻ mice with pcmvS2-S. FIG. 9 shows the cytotoxic response of CD8 ⁺ T cells to HLA-A2 restricted HBS (348-357) peptide according to immunization of HLA-A2 ⁺ DR1 ⁺ CI ⁻ CII ⁻ mice with pcmvS2 / S.

Claims (41)

a) a disrupted H-2 class I gene,
b) a disrupted H-2 class II gene, and c) a functional HLA class I or class II transgene,
A transgenic mouse comprising a) a disrupted H-2 class I gene,
b) a disrupted H-2 class II gene,
c) a functional HLA class I transgene, and
d) a functional HLA class II transgene;
A transgenic mouse comprising   The transgenic mouse according to claim 2, wherein the HLA class I transgene is an HLA-A2 transgene and the HLA class II transgene is an HLA-DR1 transgene.   4. The transgenic of claim 3, wherein the HLA-A2 transgene comprises a sequence of HLA-A2 listed in the sequence list and the HLA-DR1 transgene comprises a sequence of HLA-DR1 listed in the sequence list. mouse.   A transgenic mouse lacking both an H-2 class I molecule and an H-2 class II molecule comprising a functional HLA class I transgene and a functional HLA class II transgene. The transgenic mouse according to claim 5, which has a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ °.   7. The transgenic of claim 6, wherein the HLA-A2 transgene comprises a sequence of HLA-A2 listed in the sequence list and the HLA-DR1 transgene comprises a sequence of HLA-DR1 listed in the sequence list. mouse. Simultaneous identification of the presence or absence of one or more epitopes in a candidate antigen or group of candidate antigens that produce a specific humoral immune response, a TH HLA-DR1 restricted response, and / or a CTL HLA-A2 restricted response And the method comprises:
a) administering the candidate antigen or group of candidate antigens to the mouse of claim 3 or claim 6;
b) analyzing the specific humoral immune response to the antigen in the mouse;
c) analyzing the HLA-DR1 restricted response of TH to the antigen in the mouse; and
d) analyzing the HLA-A2 restricted response of CTL to the antigen in the mouse,
In the analysis,
-The presence of a specific humoral immune response to the antigen in the mouse confirms the epitope that causes the humoral immune response in the antigen;
-The presence of a TH HLA-DR1 restricted response to the antigen in the mouse confirms the epitope that produces a TH HLA-DR1 restricted response in the antigen;
-A method in which an epitope causing an HLA-A2-restricted response of CTL in an antigen is confirmed by observing an HLA-A2-restricted response of CTL to an antigen in a mouse. Further comprising analyzing a Th1 specific response to the antigen in the mouse and analyzing a Th2 specific response to the antigen in the mouse, wherein
The presence of a Th1 specific response to the antigen in the mouse confirms the epitope that produces a Th1 specific response to the antigen in the mouse;
9. The method of claim 8, wherein the presence of a Th2-specific response to the antigen in the mouse confirms an epitope that produces a Th2-specific response to the antigen in the mouse. A method for identifying the presence or absence of an HLA-DR1 restricted helper T cell 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) analyzing the HLA-DR1-restricted helper T cell epitope response of TH to the antigen in the mouse,
In the analysis, a method in which an HLA-DR1-restricted helper T cell epitope response of TH to an antigen is observed in a mouse, thereby confirming an epitope that causes a HLA-DR1-restricted helper T cell epitope response of TH in the antigen.   11. An isolated antigen comprising an HLA-DR1 restricted helper T cell epitope identified by the method of claim 10.   12. The isolated antigen of claim 11, wherein the antigen further comprises an epitope that produces a humoral immune response and / or an epitope that produces a CTL HLA-A2 restricted response.   12. The isolated antigen of claim 11, wherein the antigen comprising an HLA-DR1 restricted helper T cell epitope comprises a polypeptide.   12. The isolated antigen of claim 11, wherein the antigen comprising an HLA-DR1 restricted helper T cell epitope comprises a polynucleotide.   15. The isolated antigen of claim 14, wherein the antigen comprising an HLA-DR1 restricted helper T cell epitope comprises DNA, RNA, or both DNA and RNA. A method for identifying the presence or absence of an HLA-A2 restricted cytotoxic T cell (CTL) 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) analyzing an HLA-A2 restricted cytotoxic T cell (CTL) response to an antigen or group of antigens in a mouse,
In this analysis, an HLA-A2 restricted cytotoxic T cell (CTL) response to the antigen or antigen group is observed in the mouse, whereby an HLA-A2 restricted cytotoxic T cell (CTL) reaction in the antigen or antigen group is observed. A method in which the epitope giving rise to is confirmed.   An isolated antigen comprising an HLA-A2 restricted cytotoxic T cell (CTL) epitope identified by the method of claim 16.   18. The isolated antigen of claim 17, further comprising an epitope that produces a humoral immune response and / or an epitope that produces an HLA-DR1-restricted helper T cell epitope response of TH.   18. The isolated antigen of claim 17, wherein the antigen comprising an HLA-A2 restricted cytotoxic T cell (CTL) epitope comprises a polypeptide.   18. The isolated antigen of claim 17, wherein the antigen comprising an HLA-A2 restricted cytotoxic T cell (CTL) epitope comprises a polynucleotide.   21. The isolated antigen of claim 20, wherein the antigen comprising an HLA-A2 restricted cytotoxic T cell (CTL) epitope comprises DNA, RNA, or both DNA and RNA. A method for comparing the efficacy of helper T cell responses elicited by two or more vaccines, comprising:
a) administering a first candidate vaccine to the mouse of claim 3 or claim 6 and measuring a helper T cell response elicited in the mouse by the first candidate vaccine;
b) administering a second candidate vaccine to the mouse of claim 3 or claim 6 and measuring a helper T cell response elicited in the mouse by the second candidate vaccine;
c) administering each of the additional candidate vaccines to be compared to the mouse of claim 3 or claim 6 and measuring the helper T cell response elicited in the mouse by each additional candidate vaccine to be compared; and
d) determining the effectiveness of each candidate vaccine that elicits a helper T cell response by comparing the helper T cell responses for each candidate vaccine to be compared with each other.   23. The method of claim 22, wherein the helper T cell response is an HLA-DR1 restricted response. A method for comparing the efficacy of a cytotoxic T cell response elicited by two or more vaccines, the method comprising:
a) administering a first candidate vaccine to the mouse of claim 3 or claim 6 and measuring the cytotoxic T cell response elicited in the mouse by the first candidate vaccine;
b) administering a second candidate vaccine to the mouse of claim 3 or claim 6 and measuring the cytotoxic T cell response elicited in the mouse by the second candidate vaccine;
c) administering each of the additional candidate vaccines to be compared to the mouse of claim 3 or claim 6 and measuring the cytotoxic T cell response elicited in the mouse by each of the additional candidate vaccines to be compared; and ,
d) determining the effectiveness of each candidate vaccine to elicit a cytotoxic T cell response by comparing the cytotoxic T cell responses against each vaccine to be compared to each other.   25. The method of claim 24, wherein the cytotoxic T cell response is an HLA-A2 restricted response. A method for simultaneously comparing the effectiveness of a helper T cell response and a cytotoxic T cell response elicited by two or more vaccines, comprising:
a) administering a first candidate vaccine to the mouse of claim 3 or claim 6 and measuring the helper T cell response and cytotoxic T cell response elicited in the mouse by the first candidate vaccine;
b) administering a second candidate vaccine to the mouse of claim 3 or claim 6 and measuring the helper T cell response and cytotoxic T cell response elicited in the mouse by the second candidate vaccine;
c) administering each of the additional candidate vaccines to be compared to the mice of claim 3 or claim 6 and determining the helper and cytotoxic T cell responses elicited in the mice by each of the additional candidate vaccines to be compared. Measuring and
d) determining the effectiveness of each candidate vaccine to elicit a helper T cell response and cytotoxic T cell response by comparing the helper T cell response and cytotoxic T cell response to each vaccine to be compared; Including methods.   27. The method of claim 26, wherein the helper T cell response is an HLA-DR1 restricted response and the cytotoxic T cell response is an HLA-A2 restricted response. A method of simultaneously determining a humoral immune response, a helper T cell response and a cytotoxic T cell response in a mouse after immunization with an antigen or a vaccine containing one or more antigens, the method comprising:
a) administering an antigen or a vaccine comprising one or more antigens to the mouse of claim 3 or claim 6;
b) analyzing a specific humoral immune response in mice against an antigen or a vaccine comprising one or more antigens;
c) analyzing a helper T cell response in mice against an antigen or a vaccine comprising one or more antigens; and
d) A method comprising analyzing a cytotoxic T cell response in a mouse against an antigen or a vaccine comprising one or more antigens.   29. The method of claim 28, wherein the helper T cell response is an HLA-DR1 restricted response of TH.   29. The method of claim 28, wherein the cytotoxic T cell response is a CTL HLA-A2 restricted response. A method of optimizing a composition of two or more candidate vaccines for administration to a human based on preselected criteria, the method comprising:
-Simultaneously determining the humoral immune response, helper T cell response and cytotoxic T cell response in mice after immunization with the composition of two or more candidate vaccines according to claim 28; and
-A method comprising selecting an optimized vaccine by applying preselected criteria to the results.   32. The method of claim 31, wherein the two or more candidate vaccines differ only in the ratio of antigen to adjuvant present in the vaccine.   32. The method of claim 31, wherein the two or more candidate vaccines differ only in the type of adjuvant present in the vaccine. A method for determining whether a vaccine has a risk of inducing an autoimmune disease when administered to a human, the method comprising:
a) administering the vaccine to the mouse of claim 3 or claim 6; and
b) analyzing an autoimmune response in a mouse, wherein
A method wherein the presence of an autoimmune response in a mouse indicates that there is a risk of inducing an autoimmune disease when the vaccine is administered to a human. a) a disrupted H-2 class I gene,
b) a disrupted H-2 class II gene, and
c) a functional HLA class I or class II transgene;
An isolated transgenic mouse cell comprising a) a disrupted H-2 class I gene,
b) a disrupted H-2 class II gene,
c) a functional HLA class I transgene, and
d) a functional HLA class II transgene;
An isolated transgenic mouse cell comprising   37. The transgenic mouse cell of claim 36, wherein the HLA class I transgene is an HLA-A2 transgene and the HLA class II transgene is an HLA-DR1 transgene.   38. The transgenic of claim 37, wherein the HLA-A2 transgene comprises a sequence of HLA-A2 listed in the sequence list and the HLA-DR1 transgene comprises a sequence of HLA-DR1 listed in the sequence list. Mouse cells.   An isolated transgenic mouse cell that lacks both an H-2 class I molecule and an H-2 class II molecule and contains a functional HLA class I transgene and a functional HLA class II transgene. 40. The transgenic mouse cell of claim 39, which has a genotype of HLA-A2 ⁺ HLA-DR1 ⁺ β2m ° IAβ °. 41. The transgenic of claim 40, wherein the HLA-A2 transgene comprises a sequence of HLA-A2 listed in a sequence list and the HLA-DR1 transgene comprises a sequence of HLA-DR1 listed in a sequence list. Mouse cells.

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