摘要
由于DNA疫苗在诱导机体特异性细胞免疫方面具有独特优势,已作为针对慢性乙型肝炎具有潜力的免疫治疗手段被广泛研究。如何加强DNA疫苗的效果是推动DNA疫苗发展的关键。其中一个研究方向就是通过选择佐剂和调整免疫程序加强DNA疫苗的保护效率。
异源性载体prime/boost免疫策略是利用不同性质的载体表达同一个保护抗原,一般是DNA疫苗初次免疫(prime),然后用病毒载体加强(boost)。DNA疫苗抗原纯度高,持续表达低剂量的抗原,可多次重复免疫以及诱导高效价记忆性细胞毒性T淋巴细胞(CTL)反应,是理想的用于初次免疫的疫苗;重组病毒能高效表达目的抗原,模拟病毒感染诱导机体免疫的天然过程,能诱发多种细胞因子,而且有利于高亲和性抗体和T细胞受体的选择,可以有效地产生Th1型和CTL反应。
白细胞介素18(IL-18)最早被称为IFN-γ诱导因子γ,它不仅能促进T细胞和NK细胞产生IFN-γ、IL-2和GM-CSF等细胞因子,并能增强Th1细胞和NK细胞表达Fas配体,从而介导细胞毒作用。
本课题在构建含HBV不同抗原基因的重组MVA假病毒颗粒的基础上,采取DNA prime/VLP boost的免疫程序,辅以细胞因子IL-18作为佐剂,评价其治疗HBV慢性感染的可能价值。
首先将表达乙型肝炎病毒PreS2-S、C、PreC/C等抗原基因,以及分别突变2个和4个核苷酸的PreC/C基因分别克隆入与痘苗病毒Ankara株(MVA)基因组同源的穿梭载体pSC11中,构建并筛选重组质粒,将所得转染MVA病毒经过9次传代培养,筛选分别携带这些目的基因的重组MVA病毒,通过PCR扩增证实重组病毒分别正确携带目的基因,并且在传代过程中成功形成单克隆,间接免疫荧光鉴定重组病毒可以被所表达抗原的特异性抗体识别,证实重组病毒正确表达目的抗原并具有良好的抗原性。
大量制备DNA疫苗、重组病毒以及鼠白细胞介素-18(mIL-18)质粒,分别采用DNA疫苗单独免疫、rMVA单独免疫、DNA prime/rMVA boost、DNA prime/rMVAboost+mIL-18的免疫程序免疫C57BL/6小鼠,采用ELISA、ELISPOT方法评价体液免疫和细胞免疫的效果。结果显示,表达PreS2-S、C两种抗原的各组疫苗在抗体产生的时间和滴度方面均没有统计学差异,用ELISPOT检测IFN-γ的产生可知,所有免疫组均可引起针对特异性抗原的细胞免疫,rMVA单独免疫组强于DNA疫苗单独免疫组,弱于DNA prime/rMVA boost和DNA prime/rMVAboost+mIL-18组,DNA prime/rMVA boost和DNA prime/rMVA boost+mIL-18之间没有显著差别。
研究表明,携带HBV保护性抗原基因的重组MVA病毒可以诱导针对抗原的特异性细胞免疫和体液免疫,将其与表达相同抗原的DNA疫苗联合,采用DNAprime/rMVA boost的免疫方案,可以诱导比两者单独应用更强的细胞免疫反应,实验结果将为治疗型HBV疫苗的应用研究提供科学依据。
DNA vaccine,with a predominant advantage in inducing specific cellular immunity in vivo,DNA vaccine has been widely determined as a potential immunotherapy method for treating CHB.How to enhance the efficiency should be particularly focused on DNA vaccine discovery.One possible way is to adopt suitable immunoadjuvants as well as to optimize immunization strategies.
The immunization strategy of the heterologous vector(prime/boost)is achieved by the co-expression of the same protective antigen with different vectors,i.e.prime immunization by a DNA vaccine(prime)and boost immunization by a viral vector (boost).As an ideal prime immunization vaccine,the DNA vaccine has the advantages of high purity,continuous expression of antigens with low dose, repeatable immunization and inducement of memory CTL with high titer.Similarly, recombinant virus can not only highly express the target antigen,simulate the inducement process of virus infection on body immunity,and induce various cytokines,but also be beneficial to the interaction of high-affinity antibodies with T cell receptors,and the generation of Th1 reaction and CTL reaction.
Interleukin-18(IL-18),also called interferon-gamma inducing factor(IGIF), can not only promote T cells and NK cells to generate cytokines,such as IFN-γ,IL-2 and GM-CSF,but also strengthen the expression on Fas ligand in Th1 cells and NK cells in order to mediate cytotoxicity and promote the development of Th1 cells.
In this paper,the genes expressing PreS2-S、C、PreC/C and two mutanted PreC/C of HBV adr subtype were inserted into a shuttle vector pSC11,which is homologous with modified vaccinia virus Ankara(MVA),to construct and screen recombinant plasmid.The recombinant plasmid was then transfected into MVA virus. Recombinant MVA viruses respectively carrying the genes were screened after subculture of nine generations.Besides,monoclonal virus without wild virus was obtained during the subculture process.Indirect immunofluorescent analysis was used to identify the recombinant virus.The results showed that the recombinant virus could be recognized by the specific antibody against the antigen expressed by the recombinant virus.It was thus proved that the recombinant virus could express target antigen correctly and had good antigenicity.
The DNA vaccine,the recombinant virus and mouse IL-18(mIL-18)were prepared on large scale.C57BL/6 mice were immunized by different immunization processes,including single DNA vaccine immunization,single rMVA immunization, DNA prime/rMVA boost and DNA prime/rMVA boost + mIL-18.The effects of humoral immunity and cellular immunity were evaluated by ELISA and ELISPOT. The results showed that there was no statistic difference among the vaccine groups capable of expressing two antigens,in the respect of antibody generation time and titer.The ELISPOT detection of IFN-γshowed that all immunization groups could induce cellular immunity against the specific antigen.The effect of the single rMVA immunization group was better than that of the single DNA vaccine immunization group,while weaker than that of the DNA prime/rMVA boost group and the DNA prime/rMVA boost + mIL-18 group.There was no significant difference between the DNA prime/rMVA boost group and the DNA prime/rMVA boost + mIL-18 group.
The recombinant MVA virus induced specific cellular and humoral immune response toward HBV antigen.With the strategy of DNA prime/rMVA boost, stronger cellular immune were induced than DNA vaccine or rMVA alone,which provides basic evidence for the application of gene therapy for HBV.
引文
1.Mancini-Bourgine M,Fontaine H,Brechot C,et al.Immunogenicity of a hepatitis B DNA vaccine administered to chronic HBV carriers.Vaccine,2006,24(21):4482-4489.
2.Davis HL,Michel ML,Whalen RG.DNA-based immunization induces continuous secretion of hepatitis B surface antigen and high level of circulating antibody.Hum Mol Genet.1993,2(11):1847-1851.
3.Panther E,Spangenberg HC,Neumann-Haefelin C,et al.The role of the vires specific T-cell response in acute and chronic HBV and HCV infection.J Gastroenterol.2004,42(1):39-46.
4.Milich D,Liang YJ.Exploring the biological basis of hepatitis B e antigen in hepatitis B virus infection.Hepatology.2003,38(5):1075-1086.
5.Gurunathan S,Klinman DM,Seder RA.DNA vaccine:immunology,application,and optimization.Ann Rev Immunol.2000,18:927-974.
6.Beckebaum S,Cicinnati VR,Gerken G.DNA-based immunotherapy:89 potential for treatment of chronic viral hepatitis? Med Virol.2002,12:297-319
7.Ramsburg E,Rose NF,Preston A,et al.Highly effective control of an AIDS virus challenge in macaques by using vesicular stomatitis virus and modified vaccinia virus Ankara vaccine vectors in a single-boost protocol.J Virol.2004,78(8):3930-3940.
8.Murray K,Bruce SA,Hinnen A,et al.Hepatitis B virus antigens made in microbiaocells immunize against viral infection.EMBO J,1984,3(3):645-650.
9.Iwarson S,Tabor E,Thomas HC,et al.Protection against hepatitis B virus infection by immunization with hepatitis B core antigen.Gastroenterology.1985,88(3):763-767.
10.赫兢,辛绍杰,侯俊,等.增强子Ⅰ对乙型肝炎病毒DNA疫苗免疫应答的影响.解放军医学.2006,02,132-134.
11.张敏,辛绍杰,赫兢,等.乙型肝炎病毒e抗原前C区基因变异的血清型和基因型分布特点研究.实用预防医学.2007,04,338-341.
12.Lee YS,Yoon SJ,Kwon tk,et al.Immune response induced by immunization with hepatitis B vires core DNA isolated from chronic active hepatitis patients.Immunology Letters.2001,78:13-20.
13.Riedl P,Buschle M,Reimann J,et al.The immunopathogenesis of viral hepatitis.Rom J Intern Med.2004,42(1):59-67.
14.Webster GJ,Bertoletti A.Quantity and quality of virus-specific CD8 cell response:relevance to the design of a therapeutic vaccine for chronic HBV infection .Mol Immunol .2001:467-473.
15. Isogawa M ,Kakimi K, Kamamoto H ,et al.Differential dynamics of the peripheral and intrahepatic cytotoxic T lymphocyte response to hepatitis B surface antigen. Virology.2005 ,15,333(2):293-300.
16. Sette AD ,Oseroff C ,Sidney J ,et al .Overcoming T cell tolerance to the hepatitis B virus surface antigen in hepatitis B virus-transgenic mice.J Immunol , 2001,166:1389-1397.
1.Mayr A,Hochstein-Mintzel V,Stickl H.Passage history,properties,and applicability of the attenuated vaccinia virus strain MVA.Infection.1975,3:6-14.
2.Greiner S,Humrich JY,Thuman P,et al.The highly attenuated vaccinia virus strain modified virus Ankara induces apoptosis in melanoma cells and allows bystander dendritic cells to generate a potent anti-tumoral immunity.Clin Exp Immunol.2006,146(2):344-353.
3.Moorthy VS,Pinder M,Reece WH,et al.Safety and immunogenicity of DNA/modified vaccinia virus ankara malaria vaccination in African adults.J Infect Dis.2003,15,188(8):1239-1244.
4.Moroziewicz D,Kaufman HL.Gene therapy with poxvirus vectors.Curr Opin Mol Ther.2005,7(4):317-25.
5.Harrer E,Bauerle M,Ferstl B,et al.Therapeutic vaccination of HIV-1-infected patients on HAART with a recombinant HIV-1 nef-exPressing MVA: safety, immunogenicity and influence on viral load during treatment interruption. Antivir Ther. 2005,10(2): 285-300.
6. Hebben M, Brants J, Birck C.High level protein expression in mammalian cells using a safe viral vector: Modified vaccinia virus Ankara. Protein Expr Purif. 2007,56(2):269-278c
7. Wang ZD,Rosa CL, Maas R,et al.Recombinant modified vaccinia virus Ankara expressing a soluble form of glycoprotein B causes durable Immunity and neutralizing antibodies against multiple strains of human cytomegalovirus, J Virol. 2004, 78(8): 3965-3976.
8. Staib C, Drexler I, Sutter G.Construction and isolation of recombinant MVA. Methods Mol Biol .2004,269:77-100.
9. Cosma A, Nagaraj R, Buhler S, et al. Therapeutic vaccination with MVA-HIV-1 nef elicits Nef-specific T-helper cell responses in chronically HIV-1 infected individuals. Vaccine 2003, 22:21-29.
10. Hodge JW, Poole DJ, Aarts WM,et al.Modified vaccinia virus Ankara recombinants are as potential vaccinia recombinants in diversified prime and boost vaccine regimens to elicit therapeutic antitumor responses.Cancer Res .2003, 63:7942-7949.
1.Chow YH,Chiang BL,Lee YL,et al.Development of Th1 and Th2 populations and the nature of immune response to hepatitis B virus DNA vaccine can be modulated by codelivery of various cytokine genes.J Immunol.1998,160(3):1320-1329.
2.Dunachie SJ,Hill AV.Prime-boost strategies for malaria vaccine development.J Experimental Biology.2003,206:3771-3779.
3.Gherardi MM,Ramirez JC,Esteban M.Towards a new generation of vaccines:the cytokine IL-12 as an adjuvant to enhance cellular immune responses to pathogens during prime booster vaccination regimens.Histol Histopathol.2001,16(2):655-667.
4.Hanke T,Samuel RV,Blanchard TJ,et al.,Effective induction of simian immunodeficiency virus-specific cytotoxic T lymphocytes in macaques by using a multiepitope gene and DNA prime-modified vaccinia virus Ankara boost vaccination regimen.J Virol.1999,73:7524-7532.
5.Heath WR,Carbone FR.Cross-Presentation in viral immunity and selftolerance.Nat Rev Immunology.2001,1:126-134.
6.Kim JJ,Yang JS,Manson KH,et al.Modulation of antigen specific cellular immune response to DNA vaccine in rhesus macaques through the use of IL-2,IFN gamma,or IL-4 gene adjuvant.Vaccine.2001;19(1719):2496-2505
7.Tsai SL,Sheen IS,Chien RN,et al.Activation of Th1 immunity is a common immune mechanism for the successful treatment of hepatitis B and C:tetramer assay and therapeutic implication.J Biomed Sci.2003,10:120-135.
8. Yim HJ, Lok ASF. Natural history of chronic hepatitis B virus infection: What we knew in 1981 and what we know in 2005. Hepatology. 2006, 43(2):S173-S181.
9. Moore AC ,Hill AV .Progress in DNA-based heterologouse prime-boost immuniazation strategies for malaria. Immunological Reviews.2004; 199: 126-143.
10. Stambas J ,Brown SA. long lived multi-isotype anti-HIV antibody response fllowing a prime-boost immuniazation strategy.Vaccine.2005,23:2452-2462.
11. Vases A ,Egeruan B I ,Hill AV .Phase I evaluation of 3 highly immunogenic prime-boost regimens ,including a 12-month reboosting vaccination ,for malaria vaccination in Gambian men J Infect.Dis.2004,189:2213-2219.
12. Sun Y Jglesias E ,a systematic comparison of methods to measure HIV-1 specific CD8 T cells ,J Immuno Methods .2003,272:23-34.
13. Shata MT, Pfahler W, Brotman B et al, Attempted therapeutic immunization in a chimpanzee chronic HBV carrier with a high viral load. J Med Primatol. 2006,35(3):165-171.
14. Rochlitz C, Figlin R, Squiban P, et al. Phase I immunotherapy with a modifed vaccinia virus (MVA) expressing human MUC1 as antigen-specific immunotherapy in patients with MUC1-positive advanced cancer. J Gene Med. 2003, 5:690-699.
15. Moorthy VS, Pinder M, Reece WH, et al.Safety and immunogenicity of DNA/modi?ed vaccinia virus Ankara malaria vaccination in African adults. J Infect Dis .2003,188:1239-1244.
1.World Health Organization.Hepatitis B.World Health Organization Fact Sheet 204 dex.(Revised October 2000).WHO Web site.http://www.who.int/mediacentre/factsheets/fs204/en/in html.
2、卫生部公布全国人群乙型肝炎血清流行病学调查结果我国乙型肝炎免疫预防工作取得显著成绩.http://www.cpmama.org.cn/newshtml/10/20080430114936.asp
3、中华医学会肝病学分会,中华医学会感染病学分会.慢性乙型肝炎防治指南.中华肝脏病杂志,2005,12:881-91.
4.Yao GB,Cui ZY,Wang BN,et al.An extended two-year trial of lamivudine in Chinese patients with chronic hepatitis B.Chin Med J.2002,11 5:1814-18.
5 Lai CL, Ratziu V, Yuen MF, Poynard T. Viral hepatitis B. Lancet. 2003, 362:2089-2094.
6 Chisari FV. Hepatitis B virus immunopathogenesis. Annu Rev Immunol. 1995,13:29-60.
7 Bertoni R, Sidney J, Fowler P, et al, Human histocompatibility leucocyte antigen-binding supermotifs Predict broadly cross-reactive cytotoxic T lymphocyte responses in patients with acute hepatitis. J Clin Invest. 1997,100:503 13.
8 Guidotti LG, Ishikawa T, Hobbs hV, et al. Chisari FV: Intracellular inactivation of the hepatitis B virus by cytotoxic Tlymphocytes. Immunity. 1996,4:25-36.
9 Maini MK, Boni C, Lee CK, et al.The role of virus-specific CD8(~+) cells in liver damage and viral control during persistent hepatitis B virus infection. J Exp Mcd .2000,191:1269-1280.
10 Webster GJ, Reignat S, Maini MK, et al. Incubation phase of acute hepatitis B in man: Dynamic of cellular immune mechanisms. Hepatology. 2000,32:1117-1124.
11 Rehermann B, Lau D, Hoofnagle JH, et al. Cytotoxic T lymphocyte responsiveness after resolution of chronic hepatitis B virus infection. J Clin Invest .1996,97:1655-65.
12 Webster GJ, Reignat S, Brown D , et al. Longitudinal analysis of CD8~+ T cells specific for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B: implications for immunotherapy. J Virol. 2004, 78(11):5707-5719.
13 Boni C, Penna A, Ogg GS, et al. Lamivudine treatment can overcome cytotoxic T-cell hypores-ponsiveness in chronic hepatitis B: new perspectives for immune therapy. Hepatology .2001,33:963-71.
14、Boni C,Penna A,Bertoletti A,et al.Transient restoration of anti-viral T cell responses induced by lamivudine therapy in chronic hepatitis B.J Hepatol.2003,39:595-605.
15、Shouval D,Ilan Y.Transplantation of hepatitis B immune lymphocytes as means for adoptive transfer of immunity to hepatitis B virus.J Hepatol.1995,23:98-101.
16、Lau GK,Suri D,Liang R,et al.Resolution of chronic hepatitis B and anti-HBs seroconversion in humans by adoptive transfer of immunity to hepatitis B core antigen.Gastroenterology.2002,122:614-24.
17、Pol S,Driss F,Michel M-L,et al.Specific vaccine therapy in chronic hepatitis B infection.Lancet.1994,344:342.
18、Pol S,Nalpas B,Driss F,et al.Efficacy and limitations of a specific immunotherapy in chronic hepatitis B.J Hepatol.2001,34:917-21.
19、Jung MC,Gruner N,Zachoval R,et al.Immunological monitoring during therapeutic vaccination as a Prerequisite for the design of new effective therapies:induction of a vaccine-specific CD4+ T-cell proliferative response in chronic hepatitis B carriers.Vaccine.2002,20:3598-3612.
20、Dahmen A,Herzog-Hauff S,Bocher WO,et al.Clinical and immunological efficacy of intradermal vaccine plus lamivudine with or without interleukin-2in patients with chronic hepatitis B.J Med Virol.2002,66:452-460.
21、Xu DZ,Huang KL,Zhao K,et al.Vaccination with recombination with recombinant HBsAg-HIBC complex in healthy adults.Vaccine.2005,23:2658-2664.
22、Vitiello A,Ishioka G,Grey HM,et al.Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection.I.Induction of a primary cytotoxic T lymphocyte response in humans.J Clin Invest.1995,95:341-9.
23、管孝鞠,吴玉章,贾政才,等.乙型肝炎治疗性多肽免疫刺激复合物的制备及鉴定.第三军医大学学报.2000,10:927-930.
24 Rabinovich N R,McInnes P,Klein D L.Vaccine technologies: View to the future.Science,1994,265:1401-1404.
25 Oka Y, Akbar SM, Horiike N ,et al.Mechanism and therapeutic potential of DNA-based immunization against the envelope proteins of hepatitis B virus in normal and transgenic mice. Immunology. 2001 ,103(1):90-97.
26 张延龄, 张晖主编. 疫苗学.北京: 科学出版社, 2004,301-302.
27 Wolff JA ,Mallon RW, Williams P ,et al. Direct gene transfer into mouse muscle in vivo. Science. 1990,23,247:1465-1468.
28 Loirat D, Mancini-Bourgine M, Abastado JP,et al. HBsAg/HLA- A2 transgenic mice: a model for T cell tolerance to hepatitis B surface antigen in chronic hepatitis B virus infection. Int Immunol .2003,15: 1125-1136.
29 Roy MJ, Wu MS, Barr LJ, et al. Induction of antigen-specific CD8~+ T cells, T helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis B virus DNA vaccine. Vaccine 2000,19:764-778.
30 Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis.Annu Rev Immunol. 1995,13:29-60.
31 Tacket CO,Roy MJ,Widera G,et al.Phase I safety and immune response studies of a DNA vaccine encoding hepatitis B surface antigen delivered by a gene delivery device.Vaccine. 1999,17(22):2826-2829.
32 Roy MJ, Wu MS,Barr LJ,et al.Induction of antigen-specific CD8~+T cells,T helper cells,and protective levels of antibody in human by particle-mediated administration of a hepatitis B virus DNA vaccine. Vaccine. 2000, 19(7-8):764-778.
33 Mancini-Bourgine M, Fontaine H, Scott-Algara D. Induction or expansion of T-cell responses by a hepatitis B DNA vaccine administered to chronic HBV carriers.Hepatology. 2004,40(4):874-82.
34 Chen M, Sallberg M, Hughes J,et al. Immune tolerance split between hepatitis B virus precore and core proteins.J Virol. 2005,79(5):3016-3027.
35 Milich D, Liang T J. Exploring the biological basis of hepatitis B e antigen in hepatitis B virus infection. Hepatology. 2003,38(5):1075-1086.
36 Kuhober A, Pudollek HP, Reifenberg K, et al. DNA immunization induces antibody and cytotoxic T cell responses to hepatitis B core antigen in H-2b miceJ Immunol. 1996,156:3687-3695.
37 Kuhober A,Wild J, Pudollek HP,et al. DNA vaccination with plasmids encoding the intracellular (HBcAg) or secreted (HBeAg) form of the core protein of hepatitis B virus primes T cell responses to two overlapping Kb- and Kd-restricted epitopes. Inter Immunol.1997,9(8):1203-1212.
38 Huang ZH, Zhuang H, Guo RH, et al.Humoral and cellular immunogenecity of nucleic acid vaccine of hepatitis B core antigen in rhesus monkeys. Chin J Infect Dis.2001,19(3):140-143.
39 Estcourt MJ, Brooks A ,Brooks A, et al., Prime-boost immunization generates a high frequency, high-avidity CD8(~+) cytotoxic T lymphocyte population. Int. Immunol. 2002; 14 (1) :31-37.
40 Andrade BP, Gazzinelli RT, Del V M, et al. Protective immunization against murine cytomegalovirus infection using adenoviruses and poxviruses expressing hepatitis B virus chimeras. Int Microbiol. 2007,10(4):261 -269.
41 Pancholi P, Lee DH, Liu Q, et al. DNA prime/canarypox boost-based immunotherapy of chronic hepatitis B virus infection in a chimpanzee. Hepatology .2001,33:448-454.
42 Kennedy R, Poland GA. T-Cell epitope discovery for variola and vaccinia viruses, Rev. Med. Virol. 2007,17: 93-113.
43 Moroziewicz D,Kaufman HL. Gene therapy with poxvirus vectors. Curr Opin Mol Ther. 2005 ,7(4):317-325.
44 Harrer E, Bauerle M, Ferstl B,et al.Therapeutic vaccination of HIV-1 -infected patients on HAART with a recombinant HIV-1 nef-exPressing MVA: safety, immunogenicity and influence on viral load during treatment interruption. Antivir Ther. 2005,10(2):285-300.
45 McShane H, Behboudi S, Goonetilleke N ,et al, Protective immunity against Mycobacterium tuberculosis induced by dendritic cells pulsed with both CD8~+- and CD4~+-T-cell epitopes from antigen 85A. Infect Immun .2002, 70:1623-1626.
46 Thermet A, Rollier C, Zoulim F,et al. Progress in DNA vaccine for prophylaxis and therapy of hepatitis B.Vaccine.2003,21:659-662.
47 Guerhier FL, Thermet A, Guerret S, et al. Antiviral effect of adefovir in combination with a DNA vaccine in the duck hepatitis B virus infection model.Journal of Hepatology. 2003,38:328-334.
48 Shata MT, Pfahler W, Brotman B, et al. Attempted therapeutic immunization in a chimpanzee chronic HBV carrier with a high viral load. J Med Primatol. 2006,35(3):165-171.
49 Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future trends. Immunol Cell Biol. 2004 , 82(5):488-496.
50 Scheerlinck J-P Y. Genetic adjuvants for DNA vaccines,Vaccine . 2001,19:2647-2656.
51 Horton HM, Dorigo O, Hernandez P, IL-2 Plasmid Therapy of Murine Ovarian Carcinoma Inhibits the Growth of Tumor Ascites and Alters Its Cytokine Profile, The Journal of Immunology. 1999, 163: 6378-6385.
52 Rao JB, Chamberlain RS, Bronte V ,et al.IL-12 is an effective adjuvant to recombinant vaccinia virus-based tumor vaccines: enhancement by simultaneous B7-1 expression,!. Immunol. 1996,156: 3357 - 3365.
53 Trembleau S, Penna G, Gregori G, et al.IL-12 administration accelerates autoimmune diabetes in both wild-type and IFN-γ -deficient nonobese diabetic mice, revealing pathogenic and protective effects of IL-12-Induced IFN-γ . J Immuno. 2003,170: 5491-5501.
54 Constantinescu CS, Wysocka M, et al. Antibodies against IL-12 prevent superantigen-induced and spontaneous relapses of experimental autoimmune encephalomyelitis. J Immunol. 1998, 161: 5097 - 5104.
55 Chen JZ, Zhu HH, Liu KZ, et al,Enhancing cellular immune response to HBV M DNA vaccine in mice by codelivery of interleukin-18 recombinant. J Zhejiang Univ Sci. 2004,5(4):467-71.
56 Xin KQ, Hamajima K, Sasaki S, etal.IL-15 expressing plasmid enhances cell mediated immunity induced by an HIV-1 DNA vaccine. Vaccine, 1999, 17 : 858 - 866.
57 Hohlfeld R, Engel AGThe immunobiology of muscle.Immunol Today. 1994,15:269-74.