乙型肝炎治疗性疫苗的研究
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摘要
乙型肝炎是一种严重危险人类健康的全球性疾病,疫苗是预防和治疗乙肝的重要手段。本研究构建了表达HBV(中国流行株)表面抗原的DNA疫苗,并在表达载体中引入了人特异的CpG免疫刺激序列,摸索了一套适合于人用DNA疫苗中试生产的工艺路线。经过两步纯化,有效地去除菌液中的蛋白质和杂质核酸,降低了内毒素含量,使得到的DNA疫苗的质量符合有关标准。从体液免疫、细胞免疫全面地考察了乙肝DNA疫苗的免疫原性,发现DNA疫苗能在免疫小鼠体内表达目的抗原,能诱导出抗原特异性的体液反应和细胞免疫,诱导的抗体的IgG亚型疫IgG_(2(?))为主。进一步考察了乙肝DNA疫苗对HBV转基因小鼠的治疗作用。DNA疫苗免疫后,转基因小鼠血清中HBsAg水平均有不同程度地下降,部分小鼠HBsAg完全转阴;DNA疫苗能诱导出特异性的抗体反应,说明DNA疫苗能打破转基因小鼠对乙肝病毒的免疫耐受。
鉴于DNA疫苗的免疫原性较低,本课题分别通过改善疫苗的免疫方案、改善疫苗的剂型,来提高乙肝DNA疫苗的免疫效果。采用DNA疫苗、蛋白质疫苗联合免疫的策略,从不同免疫组合、不同免疫次序、不同免疫次数的8种免疫方案中筛选得到的一种最佳联合方案(2次DNA疫苗初免和1次蛋白质疫苗加强免疫的组合),能明显地增强体液免疫和细胞免疫。本课题采用PLGA微球制剂的两种剂型分别包裹或吸附乙肝DNA疫苗,系统地考察了疫苗新剂型的免疫效果,并探讨了其增效的分子机制。结果表明两种微球给药系统明显地增强DNA疫苗的免疫原性,PLGA包裹的乙肝DNA口服疫苗缓慢释放质粒DNA,不仅诱导了系统的抗原特异性免疫,还能诱导高效的消化道粘膜免疫反应。增强的免疫原性与微球制剂的缓释、靶向给药的特点有关。研究发现乙肝DNA疫苗PLGA微球制剂延长目的基因的转录和表达,还能靶向性向抗原提呈细胞递送DNA疫苗。
HBx是一种多功能蛋白,为病毒基因组转录所必需。HBx与许多共刺激因子、转录因子相互作用,调控靶基因的转录,从而影响HBV感染的细胞的功能。由于HBx在乙肝以及乙肝引起的肝硬化、肝癌的肝组织上较HBV其他亚基有更高的表达率,所以本研究将其作为靶抗原,联合应用超基序法、延展基序法、量化基序法等生物信息学的表位预测方案,并结合生物学功能实验加以验证,筛选到3个来源于HBx、并能与HLA-A*0201分子高亲和性结合的抗原九肽VLHKRTLGL(92-100),
Current antigen-based vaccines have been widely used by inducing effective humoral immunity. The major shortcoming of these vaccines is the disability to induce cellular immunity, which is essential for the prophylaxis and treatment of the disease. DNA vaccine has been well-documented to elicit durable humoral and cell-mediated immunity including CTL and cytokines in rodents and primates. It is a good candidate for immunization of non-responders of recombinant HBsAg vaccines and for therapeutic vaccination.The envelope gene of HBV was amplified by PCR from HBV genome (adr subtype of Chinese epidemic strain) and cloned into plasmid vector. Three copies of CpG motif, immunostimulatory sequence were also constructed into pVAX1. Optimal conditions for purification of supercoiled plasmid DNA in a large scale of semi-works production were identified. Several procedures involving alkaline lysis, ion exchange chromatography and gel chromatography were performed to separate plasmid isoforms from each other and from other host cell contaminants, including RNA, genomic DNA, protein, and endotoxins. Finally, the purified supercoiled plasmid DNA was suitable for use in DNA vaccine applications, according to Points to Consider on Plasmid DNA Vaccine set by FDA.The immunogecity of HBV DNA vaccine were tested in both normal mice and HBV-Tg mice. HBsAg was detected in serum and bilateral tibialis anterior muscle of normal mice post-immunization. HBsAg-specific humoral and cellular immune response was efficiently elicited. The therapeutic value of DNA vaccine was demonstrated in HBV-Tg mice injected with HBV DNA vaccine, resulting in the clearance of HBsAg from 2 out of 5 HBV-Tg mice and the HBsAg level decreased in the other mice. Anti-HBs was detected in all HBV-Tg mice at 4 weeks after vaccination. The results indicated that DNA-based vaccine was capable to break the immune tolerance of HBV-Tg and would be a promising therapeutic choice for the treatment of HBV carriers.However, some phase 1 clinical trials of HBV DNA vaccine and other DNA vaccines have demonstrated that the magnitude of immune responses induced in human is generally lower than that in small animals, and the amount of DNA required for effective immunization is much larger. Therefore, the potency of DNA vaccines must be increased to enable this technology for successful human application.Several reports have indicated that combinatorial regimens with DNA and protein vaccines can
elicit both strong immune responses, to circumvent the limits of each vaccine. Surprisingly little was known on HBV vaccine. Here, we investigated the immunization effects of several regimens involving different components, different orders and different times of vaccination in BALB/c mice. The regimen involving twice priming pVAX(S) encoding HBsAg and once rHBsAg boosting, induced strong and homogenous antibody responses. The regimen induced significant stronger responses of IL-12 and IFN-y in splenocytes and elicited stronger CD8+ CTL responses than that elicited by immunization with rHBsAg or pVAX(S) alone. The efficacy of immunoprotection against the challenge of HBsAg-expressing tumor was also examined. A greater immunopretectional efficacy was found in group of mice immunized with rHBsAg after twice pVAX(S) priming. Our regimen may thus provide a strategy for developing an improved immunization against HBV and many other pathogens.Plasmid DNA encoding HBV envelope antigen was encapsulated into or absorbed onto PLGA and generated highly uniform microparticles. The immunogenicity of PLGA-DNA microparticles was demonstrated by significantly enhanced humoral and cellular immune responses in immunized mice, including higher levels of antibody, IFN^y and CTL activity, compared with that elicited by naked ' plasmid DNA. A greater efficacy of immunoprotection against the challenge of transplanted HBsAg-expressing tumor cells was also shown in the group of mice immunized with PLGA-DNA microparticles. Oral administration of PLGA-DNA microparticles induced a long-lasting and stable intestinal IgA and cellular immune responses in GALT of immunized mice. The feature of controlled release of PLGA-DNA microparticles was demonstrated in vitro and in vivo. Flow cytometry and confocal laser scanning microscopy were used to localize and quantitatively evaluate APCs involved in HBsAg expression. Much higher percentage of CDllc+ APCs involved in HBsAg expressing at 24 h and day 14 postinoculation, as well as prolonged transcription of plasmid DNA detected by RT-PCR, was demonstrated in the draining lymph nodes from mice immunized with PLGA-DNA microparticles, compared with that in mice vaccinated by naked DNA. The detection of controlled release in vitro and in vivo of PLGA-DNA microparticles provided direct evidence that the formulation not only directed the delivery of plasmid DNA to APCs, but also prolonged the existence of plasmid DNA expressing target antigen. The significant association between the feature of controlled release of microparticles and its augmented immunogenicity revealed some mechanism of immunopotentiation. The results may facilitate the development of a new type of protective HBV vaccine in humans.HBx is a multifunctional protein, participating in viral pathogenesis as a transactivator of viral
鉴于DNA疫苗的免疫原性较低,本课题分别通过改善疫苗的免疫方案、改善疫苗的剂型,来提高乙肝DNA疫苗的免疫效果。采用DNA疫苗、蛋白质疫苗联合免疫的策略,从不同免疫组合、不同免疫次序、不同免疫次数的8种免疫方案中筛选得到的一种最佳联合方案(2次DNA疫苗初免和1次蛋白质疫苗加强免疫的组合),能明显地增强体液免疫和细胞免疫。本课题采用PLGA微球制剂的两种剂型分别包裹或吸附乙肝DNA疫苗,系统地考察了疫苗新剂型的免疫效果,并探讨了其增效的分子机制。结果表明两种微球给药系统明显地增强DNA疫苗的免疫原性,PLGA包裹的乙肝DNA口服疫苗缓慢释放质粒DNA,不仅诱导了系统的抗原特异性免疫,还能诱导高效的消化道粘膜免疫反应。增强的免疫原性与微球制剂的缓释、靶向给药的特点有关。研究发现乙肝DNA疫苗PLGA微球制剂延长目的基因的转录和表达,还能靶向性向抗原提呈细胞递送DNA疫苗。
HBx是一种多功能蛋白,为病毒基因组转录所必需。HBx与许多共刺激因子、转录因子相互作用,调控靶基因的转录,从而影响HBV感染的细胞的功能。由于HBx在乙肝以及乙肝引起的肝硬化、肝癌的肝组织上较HBV其他亚基有更高的表达率,所以本研究将其作为靶抗原,联合应用超基序法、延展基序法、量化基序法等生物信息学的表位预测方案,并结合生物学功能实验加以验证,筛选到3个来源于HBx、并能与HLA-A*0201分子高亲和性结合的抗原九肽VLHKRTLGL(92-100),
Current antigen-based vaccines have been widely used by inducing effective humoral immunity. The major shortcoming of these vaccines is the disability to induce cellular immunity, which is essential for the prophylaxis and treatment of the disease. DNA vaccine has been well-documented to elicit durable humoral and cell-mediated immunity including CTL and cytokines in rodents and primates. It is a good candidate for immunization of non-responders of recombinant HBsAg vaccines and for therapeutic vaccination.The envelope gene of HBV was amplified by PCR from HBV genome (adr subtype of Chinese epidemic strain) and cloned into plasmid vector. Three copies of CpG motif, immunostimulatory sequence were also constructed into pVAX1. Optimal conditions for purification of supercoiled plasmid DNA in a large scale of semi-works production were identified. Several procedures involving alkaline lysis, ion exchange chromatography and gel chromatography were performed to separate plasmid isoforms from each other and from other host cell contaminants, including RNA, genomic DNA, protein, and endotoxins. Finally, the purified supercoiled plasmid DNA was suitable for use in DNA vaccine applications, according to Points to Consider on Plasmid DNA Vaccine set by FDA.The immunogecity of HBV DNA vaccine were tested in both normal mice and HBV-Tg mice. HBsAg was detected in serum and bilateral tibialis anterior muscle of normal mice post-immunization. HBsAg-specific humoral and cellular immune response was efficiently elicited. The therapeutic value of DNA vaccine was demonstrated in HBV-Tg mice injected with HBV DNA vaccine, resulting in the clearance of HBsAg from 2 out of 5 HBV-Tg mice and the HBsAg level decreased in the other mice. Anti-HBs was detected in all HBV-Tg mice at 4 weeks after vaccination. The results indicated that DNA-based vaccine was capable to break the immune tolerance of HBV-Tg and would be a promising therapeutic choice for the treatment of HBV carriers.However, some phase 1 clinical trials of HBV DNA vaccine and other DNA vaccines have demonstrated that the magnitude of immune responses induced in human is generally lower than that in small animals, and the amount of DNA required for effective immunization is much larger. Therefore, the potency of DNA vaccines must be increased to enable this technology for successful human application.Several reports have indicated that combinatorial regimens with DNA and protein vaccines can
elicit both strong immune responses, to circumvent the limits of each vaccine. Surprisingly little was known on HBV vaccine. Here, we investigated the immunization effects of several regimens involving different components, different orders and different times of vaccination in BALB/c mice. The regimen involving twice priming pVAX(S) encoding HBsAg and once rHBsAg boosting, induced strong and homogenous antibody responses. The regimen induced significant stronger responses of IL-12 and IFN-y in splenocytes and elicited stronger CD8+ CTL responses than that elicited by immunization with rHBsAg or pVAX(S) alone. The efficacy of immunoprotection against the challenge of HBsAg-expressing tumor was also examined. A greater immunopretectional efficacy was found in group of mice immunized with rHBsAg after twice pVAX(S) priming. Our regimen may thus provide a strategy for developing an improved immunization against HBV and many other pathogens.Plasmid DNA encoding HBV envelope antigen was encapsulated into or absorbed onto PLGA and generated highly uniform microparticles. The immunogenicity of PLGA-DNA microparticles was demonstrated by significantly enhanced humoral and cellular immune responses in immunized mice, including higher levels of antibody, IFN^y and CTL activity, compared with that elicited by naked ' plasmid DNA. A greater efficacy of immunoprotection against the challenge of transplanted HBsAg-expressing tumor cells was also shown in the group of mice immunized with PLGA-DNA microparticles. Oral administration of PLGA-DNA microparticles induced a long-lasting and stable intestinal IgA and cellular immune responses in GALT of immunized mice. The feature of controlled release of PLGA-DNA microparticles was demonstrated in vitro and in vivo. Flow cytometry and confocal laser scanning microscopy were used to localize and quantitatively evaluate APCs involved in HBsAg expression. Much higher percentage of CDllc+ APCs involved in HBsAg expressing at 24 h and day 14 postinoculation, as well as prolonged transcription of plasmid DNA detected by RT-PCR, was demonstrated in the draining lymph nodes from mice immunized with PLGA-DNA microparticles, compared with that in mice vaccinated by naked DNA. The detection of controlled release in vitro and in vivo of PLGA-DNA microparticles provided direct evidence that the formulation not only directed the delivery of plasmid DNA to APCs, but also prolonged the existence of plasmid DNA expressing target antigen. The significant association between the feature of controlled release of microparticles and its augmented immunogenicity revealed some mechanism of immunopotentiation. The results may facilitate the development of a new type of protective HBV vaccine in humans.HBx is a multifunctional protein, participating in viral pathogenesis as a transactivator of viral
引文
1. Perspectives on therapy of hepatitis B. Journal of Hepatology 2003; 39: S220-S223
2.李用国 陈敏 张大志等.自体树突状细胞疫苗治疗慢性乙型肝炎的临床研究.中华肝脏病杂志.2003;11(4):206-9.
3. Akbar SM, Horiike N, Onji M, et al. Dendritic cells and chronic hepatitis virus carriers. Intervirology. 2001; 44(4): 199-208. 5.
4. Geissler M, Tokushige K, Chante CC, Zurawski VR Jr, Wands JR. Cytokine and hepatitis B virus DNA co-immunizations enhance cellular and humoral responses to the middle but not to the large hepatitis B virus surface antigen in mice. Hepatology 1998; 28: 202-210.
5. Wai CT, Lok AS. Treatment of hepatitis B. J Gastroenterol. 2002; 37(10): 771-8
6. Oka Y, Akbar SM, Horiike N, Joko K, Onji M. Mechanism and therapeutic potential of DNA-based immunization against the envelope proteins of hepatitis B virus in normal and transgenic mice. Immunology 2001; 103: 90-97.
7. Le Guerhier F, Thermet A, Guerret S, Chevallier M, Jamard C, Gibbs CS, et al. Antiviral effect of adefovir in combination with a DNA vaccine in the duck hepatitis B virus infection model. J Hepatol. 2003; 38: 328-334.
8. Davis HL, Brazolot Millan CL, Mancini M, McCluskie MJ, Hadchouel M, Comanita L, et al. DNA vaccine for hepatitis B: evidence for immunogenicity in chimpanzees and comparison with other vaccines. Proc Natl Acad Sci U S A. 1996; 93: 7213-7218.
9. Rottinghaus ST, Poland GA, Jacobson RM, Barr LJ, Roy MJ. Hepatitis B DNA vaccine induces protective antibody responses in human non-responders to conventional vaccination. Vaccine. 2003; 21(31): 4604-8.
10. Zhou FJ, Hu ZL, Dai JX, Chen RW, Shi K, Lin Y, et al. Protection of tree shrews by pVAX-PS DNA vaccine against HBV infection. DNA Cell Biol. 2003, 22: 475-478.
11. Sandberg LM, Bjurling A, Busson P, et al. Thiophilic interaction chromatography for supercoiled plasmid DNA purification. J Biotechnol. 2004; 109(1-2): 193-9.
12. Lemmens R, Olsson U, Nyhammar T, et al. Supercoiled plasmid DNA: selective purification
2.李用国 陈敏 张大志等.自体树突状细胞疫苗治疗慢性乙型肝炎的临床研究.中华肝脏病杂志.2003;11(4):206-9.
3. Akbar SM, Horiike N, Onji M, et al. Dendritic cells and chronic hepatitis virus carriers. Intervirology. 2001; 44(4): 199-208. 5.
4. Geissler M, Tokushige K, Chante CC, Zurawski VR Jr, Wands JR. Cytokine and hepatitis B virus DNA co-immunizations enhance cellular and humoral responses to the middle but not to the large hepatitis B virus surface antigen in mice. Hepatology 1998; 28: 202-210.
5. Wai CT, Lok AS. Treatment of hepatitis B. J Gastroenterol. 2002; 37(10): 771-8
6. Oka Y, Akbar SM, Horiike N, Joko K, Onji M. Mechanism and therapeutic potential of DNA-based immunization against the envelope proteins of hepatitis B virus in normal and transgenic mice. Immunology 2001; 103: 90-97.
7. Le Guerhier F, Thermet A, Guerret S, Chevallier M, Jamard C, Gibbs CS, et al. Antiviral effect of adefovir in combination with a DNA vaccine in the duck hepatitis B virus infection model. J Hepatol. 2003; 38: 328-334.
8. Davis HL, Brazolot Millan CL, Mancini M, McCluskie MJ, Hadchouel M, Comanita L, et al. DNA vaccine for hepatitis B: evidence for immunogenicity in chimpanzees and comparison with other vaccines. Proc Natl Acad Sci U S A. 1996; 93: 7213-7218.
9. Rottinghaus ST, Poland GA, Jacobson RM, Barr LJ, Roy MJ. Hepatitis B DNA vaccine induces protective antibody responses in human non-responders to conventional vaccination. Vaccine. 2003; 21(31): 4604-8.
10. Zhou FJ, Hu ZL, Dai JX, Chen RW, Shi K, Lin Y, et al. Protection of tree shrews by pVAX-PS DNA vaccine against HBV infection. DNA Cell Biol. 2003, 22: 475-478.
11. Sandberg LM, Bjurling A, Busson P, et al. Thiophilic interaction chromatography for supercoiled plasmid DNA purification. J Biotechnol. 2004; 109(1-2): 193-9.
12. Lemmens R, Olsson U, Nyhammar T, et al. Supercoiled plasmid DNA: selective purification