增强核酸疫苗免疫效果的策略及其作用机制的研究
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摘要
核酸疫苗(DNA疫苗)是指将含有编码抗原基因的真核表达载体直接接种体内,在体内表达相应抗原刺激机体产生针对该抗原的免疫应答,产生保护性免疫。核酸疫苗由于其独特的双免疫激活作用而受到越来越多的关注,有一些核酸疫苗已经进而Ⅱ期、Ⅲ期临床研究,然而,核酸疫苗的免疫效果却始终不甚理想,在动物实验中,对于猴子、猩猩等大动物,疫苗用量过大,为此,探索增强核酸疫苗免疫效果及降低疫苗用量的策略研究是十分必要的。
剂型改造是提高DNA疫苗免疫效果的方法之一。本课题中探讨了一种新型的DNA疫苗给药系统,用聚乳酸/羟基乙酸共聚物(PLGA)包裹DNA疫苗形成微球疫苗后免疫小鼠,观察免疫后小鼠体液免疫、细胞免疫及粘膜免疫的效果。实验中,以乙肝治疗性DNA疫苗和多表位口蹄疫DNA疫苗为候选疫苗,应用PLGA包裹形成微球疫苗,首先在体外实验中验证了疫苗微球对抗原递呈细胞的靶向作用。小鼠巨噬细胞系Raw264.7细胞被用于体外吞噬实验,模拟抗原递呈细胞对疫苗抗原的摄取过程。通过姬姆萨染色,证实了微球疫苗的可吞噬性;荧光标记的疫苗微球的应用,可以更准确的动态观察微球的吞噬过程,在吞噬的2小时、4小时、8小时后分别收集细胞荧光显微镜观察,可以看到随着时间延长,细胞内点状荧光逐渐融合,荧光强度逐渐增加,进一步证实微球的可吞噬性。吞噬后的微球能否成功释放疫苗,疫苗抗原能否正确表达?后续实验中,在体内、体外两个层面上证实了抗原成份在抗原递呈细胞中的正确表达。当应用PLGA包裹的乙肝DNA疫苗口服免疫小鼠后,可全面的激发机体的细胞免疫和体液免疫,还可在局部引流淋巴结中观察到疫苗抗原的表达,观察到体现粘膜免疫效果的IgA的分泌,充分证实了口服乙肝DNA疫苗良好的免疫效果,获得了有应用前景的口服治疗性乙肝DNA疫苗。在多表位口蹄疫DNA疫苗的研究中发现,PLGA包裹可以降低疫苗的免疫剂量、减少疫苗的免疫次数,使新型口蹄疫DNA疫苗更廉价、使用更方便。
膜联蛋白B1基因是本课题组以免疫筛选法从猪囊尾蚴cDNA文库中得到的一个新基因,其基因产物是猪带绦虫囊虫病的诊断用抗原,也是很好的保护性抗原,以annexin B1为抗原基础构建的囊虫DNA疫苗,在猪的保护性实验中可保护仔猪免于虫卵冲击后的感染或提高减虫率。对于一个高免疫原性的疫苗抗原,其结构的解析、生物学活性的认识是安全、有效的利用疫苗抗原的基础,为了找到能
Nucleic acid vaccine(DNA vaccine) is a kind of DNA vector which can carry exogenous gene encoding immunogenic antigen. Generally, the plasmids or some viruses can be used as DNA vector. When the plasmid carrying target gene is inoculated into the body, the transfected muscular cells or antigen-presenting cells(APC) can express the immunogenic antigen in cytoplasm and present the antigen to the Th cells to induce immune response, which can protect the body from infection. More and more attentions are paid to the usage of DNA vaccine for its special immunogenicity that can induce strong cellular and humoral immune responses. Furthermore, some phase Ⅰ-Ⅱ clinical trials of DNA vaccines are ongoing recently. However, the potency of DNA vaccines in humans is disappointing. In addition, a large amount of plasmid DNA was required to induce immune response on the larger animals, such as rhesus macaques, chimpanzee, and so on. Therefore, it is urgent to develop new strategies in order to increase the efficacy of DNA vaccine with decreased amount of plasmid DNA.Novel formulation is one of the strategies to enhance the immunopotency of DNA vaccines. A new vaccine delivery system was selected and two kind of DNA vaccines were used as candidates in our experiments. At first, the plasmid DNA encoding HBsAg or FMD multiple epitope antigen (SG) was encapsulated by PLGA in order to form vaccine microparticles. It was confirmed that microparticles could be phagocytosed by APC. The phagocytosis process was simulated by the Raw 264.7 cells in vitro. After the cells have been incubated with particles for one hour, Giemsa agent was used to dye the cells. Many microparticles with no dye could be found in the cytoplasm. For the further showing of the phagocytotic process, the fluorescent dye was added to the plasmid solution in order to prepare fluorescent microparticles, which were added to cover slides seeded with Raw264.7 cells. Phagocytosis was observed directly and photographed using a digital camera linked to a fluorescence microscope. The fluorescent dots became confluent and larger with the prolongation
of incubation time. These two phagocytosis experiments confirmed that the plasmid DNA encapsulated by PLGA could be taked up by APC directly. Whether the vaccine DNA could be released suitably and whether the target antigen could be expressed in the cytoplasm were the key points to the subsequent immune responses. The expression of target antigen was verified in vitro and in vivo. It was also found that the capsulation of PLGA could prolong the persistence of antigen in local tissue or draining lymph nodes. By this mechanism, DNA vaccine's immunopotency was enhanced. Moreover, the single oral administration HBV DNA vaccine encapsulated by PLGA could induce not only the cellular and humoral immune response, but also the mucosal immunity. In the research of FMD multiple epitope DNA vaccine, the expression of multiple epitope antigen(SG) was verified by immunocytochemistry. The results add support to the conclusion that the PLGA capsulation helps the vaccine target APC and prolong the persistence of DNA in vivo and in vitro, which facilitates the antigen presentation to Thl cells in draining lymph nodes. The encapsulated FMD DNA vaccine produced a broad spectrum of immune response. Meanwhile, the new formulation elicited the desired immune response with less amount of plasmid DNA and less injection times compared with naked plasmid DNA. So this kind of immune method against FMDV is more convenient, safer and cheaper.Annexin Bl gene was found by immune screening from cDNA library of Cysticercus cellulosae. The annexin Bl protein can be used as an antigen to diagnose Cysticercus cellulosae disease, and is a protective antigen. The DNA vaccine encoding annexin Bl could protect the pigs against Taenia solium cysticercosis. As far as a vaccine antigen with high immunogenicity was concerned, analyzing the structure and learning the bioactivity are the basis of using vaccine antigen safely. According to the data from the homology modeling three-dimensional structure of annexin Bl, we constructed three domain-deleted mutants in order to study the bioactivity of annexin Bl and membrane binding properties of its domains. The phosphatidylserine(PS) binding properties of wild annexin Bl and three domain-deleted mutants were confirmed in vivo and in vitro. PS exposure at the
cellular surface is one of the earliest detectable molecular events in apoptosis and the main event in blood coagulation initiation. So the PS binding bioactivities of annexin Bl made it be used as anticoagulant protein to facilitate the migration of Cysticercus cellulosae in human body. The physiological and pathological roles of annexin Bl were elucidated. In reality, annexin Bl can be developed to be anticoagulant and imaging agent for apoptotic cells.The illumination of PS binding property of annexin Bl contributed to the understanding of annexin Bl possessing the adjuvant function. It was suggested that annexin Bl binding to the PS molecular on the surface of early apoptotic APC could protect the transfected APC from the phagocytosis which depended the PS as the apoptotic singal. It was assumed that the annexin Bl played adjuvant role by prolonging the life span of tranfected APCs and the expression of antigen in local tissue and draining lymph nodes.On the whole, this study developed a series of experiments surrounding the topic how to enhance the immunopotency of DNA vaccines. A novel delivery system, PLGA microparticle system, was confirmed to be effective on improving the immunogenicity of encapsulated DNA vaccines. The PS binding property of annexin Bl was observed on active platelets, apoptotic cells and PS lipid beads. The mechanism of annexin Bl as a novel adjuvant could be explained by blocking the PS on surface of early apoptotic APC.
剂型改造是提高DNA疫苗免疫效果的方法之一。本课题中探讨了一种新型的DNA疫苗给药系统,用聚乳酸/羟基乙酸共聚物(PLGA)包裹DNA疫苗形成微球疫苗后免疫小鼠,观察免疫后小鼠体液免疫、细胞免疫及粘膜免疫的效果。实验中,以乙肝治疗性DNA疫苗和多表位口蹄疫DNA疫苗为候选疫苗,应用PLGA包裹形成微球疫苗,首先在体外实验中验证了疫苗微球对抗原递呈细胞的靶向作用。小鼠巨噬细胞系Raw264.7细胞被用于体外吞噬实验,模拟抗原递呈细胞对疫苗抗原的摄取过程。通过姬姆萨染色,证实了微球疫苗的可吞噬性;荧光标记的疫苗微球的应用,可以更准确的动态观察微球的吞噬过程,在吞噬的2小时、4小时、8小时后分别收集细胞荧光显微镜观察,可以看到随着时间延长,细胞内点状荧光逐渐融合,荧光强度逐渐增加,进一步证实微球的可吞噬性。吞噬后的微球能否成功释放疫苗,疫苗抗原能否正确表达?后续实验中,在体内、体外两个层面上证实了抗原成份在抗原递呈细胞中的正确表达。当应用PLGA包裹的乙肝DNA疫苗口服免疫小鼠后,可全面的激发机体的细胞免疫和体液免疫,还可在局部引流淋巴结中观察到疫苗抗原的表达,观察到体现粘膜免疫效果的IgA的分泌,充分证实了口服乙肝DNA疫苗良好的免疫效果,获得了有应用前景的口服治疗性乙肝DNA疫苗。在多表位口蹄疫DNA疫苗的研究中发现,PLGA包裹可以降低疫苗的免疫剂量、减少疫苗的免疫次数,使新型口蹄疫DNA疫苗更廉价、使用更方便。
膜联蛋白B1基因是本课题组以免疫筛选法从猪囊尾蚴cDNA文库中得到的一个新基因,其基因产物是猪带绦虫囊虫病的诊断用抗原,也是很好的保护性抗原,以annexin B1为抗原基础构建的囊虫DNA疫苗,在猪的保护性实验中可保护仔猪免于虫卵冲击后的感染或提高减虫率。对于一个高免疫原性的疫苗抗原,其结构的解析、生物学活性的认识是安全、有效的利用疫苗抗原的基础,为了找到能
Nucleic acid vaccine(DNA vaccine) is a kind of DNA vector which can carry exogenous gene encoding immunogenic antigen. Generally, the plasmids or some viruses can be used as DNA vector. When the plasmid carrying target gene is inoculated into the body, the transfected muscular cells or antigen-presenting cells(APC) can express the immunogenic antigen in cytoplasm and present the antigen to the Th cells to induce immune response, which can protect the body from infection. More and more attentions are paid to the usage of DNA vaccine for its special immunogenicity that can induce strong cellular and humoral immune responses. Furthermore, some phase Ⅰ-Ⅱ clinical trials of DNA vaccines are ongoing recently. However, the potency of DNA vaccines in humans is disappointing. In addition, a large amount of plasmid DNA was required to induce immune response on the larger animals, such as rhesus macaques, chimpanzee, and so on. Therefore, it is urgent to develop new strategies in order to increase the efficacy of DNA vaccine with decreased amount of plasmid DNA.Novel formulation is one of the strategies to enhance the immunopotency of DNA vaccines. A new vaccine delivery system was selected and two kind of DNA vaccines were used as candidates in our experiments. At first, the plasmid DNA encoding HBsAg or FMD multiple epitope antigen (SG) was encapsulated by PLGA in order to form vaccine microparticles. It was confirmed that microparticles could be phagocytosed by APC. The phagocytosis process was simulated by the Raw 264.7 cells in vitro. After the cells have been incubated with particles for one hour, Giemsa agent was used to dye the cells. Many microparticles with no dye could be found in the cytoplasm. For the further showing of the phagocytotic process, the fluorescent dye was added to the plasmid solution in order to prepare fluorescent microparticles, which were added to cover slides seeded with Raw264.7 cells. Phagocytosis was observed directly and photographed using a digital camera linked to a fluorescence microscope. The fluorescent dots became confluent and larger with the prolongation
of incubation time. These two phagocytosis experiments confirmed that the plasmid DNA encapsulated by PLGA could be taked up by APC directly. Whether the vaccine DNA could be released suitably and whether the target antigen could be expressed in the cytoplasm were the key points to the subsequent immune responses. The expression of target antigen was verified in vitro and in vivo. It was also found that the capsulation of PLGA could prolong the persistence of antigen in local tissue or draining lymph nodes. By this mechanism, DNA vaccine's immunopotency was enhanced. Moreover, the single oral administration HBV DNA vaccine encapsulated by PLGA could induce not only the cellular and humoral immune response, but also the mucosal immunity. In the research of FMD multiple epitope DNA vaccine, the expression of multiple epitope antigen(SG) was verified by immunocytochemistry. The results add support to the conclusion that the PLGA capsulation helps the vaccine target APC and prolong the persistence of DNA in vivo and in vitro, which facilitates the antigen presentation to Thl cells in draining lymph nodes. The encapsulated FMD DNA vaccine produced a broad spectrum of immune response. Meanwhile, the new formulation elicited the desired immune response with less amount of plasmid DNA and less injection times compared with naked plasmid DNA. So this kind of immune method against FMDV is more convenient, safer and cheaper.Annexin Bl gene was found by immune screening from cDNA library of Cysticercus cellulosae. The annexin Bl protein can be used as an antigen to diagnose Cysticercus cellulosae disease, and is a protective antigen. The DNA vaccine encoding annexin Bl could protect the pigs against Taenia solium cysticercosis. As far as a vaccine antigen with high immunogenicity was concerned, analyzing the structure and learning the bioactivity are the basis of using vaccine antigen safely. According to the data from the homology modeling three-dimensional structure of annexin Bl, we constructed three domain-deleted mutants in order to study the bioactivity of annexin Bl and membrane binding properties of its domains. The phosphatidylserine(PS) binding properties of wild annexin Bl and three domain-deleted mutants were confirmed in vivo and in vitro. PS exposure at the
cellular surface is one of the earliest detectable molecular events in apoptosis and the main event in blood coagulation initiation. So the PS binding bioactivities of annexin Bl made it be used as anticoagulant protein to facilitate the migration of Cysticercus cellulosae in human body. The physiological and pathological roles of annexin Bl were elucidated. In reality, annexin Bl can be developed to be anticoagulant and imaging agent for apoptotic cells.The illumination of PS binding property of annexin Bl contributed to the understanding of annexin Bl possessing the adjuvant function. It was suggested that annexin Bl binding to the PS molecular on the surface of early apoptotic APC could protect the transfected APC from the phagocytosis which depended the PS as the apoptotic singal. It was assumed that the annexin Bl played adjuvant role by prolonging the life span of tranfected APCs and the expression of antigen in local tissue and draining lymph nodes.On the whole, this study developed a series of experiments surrounding the topic how to enhance the immunopotency of DNA vaccines. A novel delivery system, PLGA microparticle system, was confirmed to be effective on improving the immunogenicity of encapsulated DNA vaccines. The PS binding property of annexin Bl was observed on active platelets, apoptotic cells and PS lipid beads. The mechanism of annexin Bl as a novel adjuvant could be explained by blocking the PS on surface of early apoptotic APC.
引文
1.孙树汉。《核酸疫苗》 第二军医大学出版社, 2000年。
2. Chinsangaram J, Beard C, Mason PW, Zellner MK, Ward G, Grubman MJ. Antibody response in mice inoculated with DNA expressing foot-and-mouth disease virus capsid proteins. J Virol. 1998,72(5):4454-7.
3. Donnelly JJ, Wahren B, Liu MA. DNA vaccines: progress and challenges. J Immunol. 2005, 175(2):633-9.
4. Girard MP, Osmanov SK, Kieny MP. A review of vaccine research and development: The human immunodeficiency virus (HIV). Vaccine. 2006, 24(19):4062-81.
5. Guevara-Patino JA, Engelhorn ME, Turk MJ, Liu C, Duan F, Rizzuto G, Cohen AD, Merghoub T, Wolchok JD, Houghton AN. Optimization of a self antigen for presentation of multiple epitopes in cancer immunity J Clin Invest. 2006,116(5):1382-90.
6. A.P.D. Souzal, L. Hautl, A. Reyes-Sandova12 and A.R. Pintol. Recombinant viruses as vaccines against viral diseases Recombinant viruses as vaccines. Brazilian Journal of Medical and Biological Research (2005) 38:509-522
7. Someya K, Ami Y, Nakasone T, Izumi Y, Matsuo K, Horibata S, Xin KQ, Yamamoto H, Okuda K, Yamamoto N, Honda M. Induction of positive cellular and humoral immune responses by a prime-boost vaccine encoded with simian immunodeficiency virus gag/pol. J Immunol. 2006, 176(3):1784-95.
8. Peng S, Kim TW, Lee JH, Yang M, He L, Hung CF, Wu TC. Vaccination with dendritic cells transfected with BAK and BAX siRNA enhances antigen-specific immune responses by prolonging dendritic cell life. Hum Gene Ther. 2005;16(5):584-93
9. C.M. Stach, X. Turnay, R.E. Voll, P.M. Kern, W. Kolowos, T.D.Beyer, J.R. Kalden, M. Herrmann, Treatment with annexin V increases immunogenicity of apoptotic human T-cells in Balb/c mice, Cell Death Differ. 7 (2000) 911-915.
10. Samantha Jilek, Hans P. Merkle, Elke Walter. DNA-loaded biodegradable microparticles as vaccine delivery systems and their interaction with dendritic ceils. Advanced Drug Delivery Reviews 57 (2005) 377-390
11. Michele A. Kutzler and David B. Weiner. Developing DNA vaccines that call to dendritic cells. J. Clin. Invest. 114:1241 1244
2. Chinsangaram J, Beard C, Mason PW, Zellner MK, Ward G, Grubman MJ. Antibody response in mice inoculated with DNA expressing foot-and-mouth disease virus capsid proteins. J Virol. 1998,72(5):4454-7.
3. Donnelly JJ, Wahren B, Liu MA. DNA vaccines: progress and challenges. J Immunol. 2005, 175(2):633-9.
4. Girard MP, Osmanov SK, Kieny MP. A review of vaccine research and development: The human immunodeficiency virus (HIV). Vaccine. 2006, 24(19):4062-81.
5. Guevara-Patino JA, Engelhorn ME, Turk MJ, Liu C, Duan F, Rizzuto G, Cohen AD, Merghoub T, Wolchok JD, Houghton AN. Optimization of a self antigen for presentation of multiple epitopes in cancer immunity J Clin Invest. 2006,116(5):1382-90.
6. A.P.D. Souzal, L. Hautl, A. Reyes-Sandova12 and A.R. Pintol. Recombinant viruses as vaccines against viral diseases Recombinant viruses as vaccines. Brazilian Journal of Medical and Biological Research (2005) 38:509-522
7. Someya K, Ami Y, Nakasone T, Izumi Y, Matsuo K, Horibata S, Xin KQ, Yamamoto H, Okuda K, Yamamoto N, Honda M. Induction of positive cellular and humoral immune responses by a prime-boost vaccine encoded with simian immunodeficiency virus gag/pol. J Immunol. 2006, 176(3):1784-95.
8. Peng S, Kim TW, Lee JH, Yang M, He L, Hung CF, Wu TC. Vaccination with dendritic cells transfected with BAK and BAX siRNA enhances antigen-specific immune responses by prolonging dendritic cell life. Hum Gene Ther. 2005;16(5):584-93
9. C.M. Stach, X. Turnay, R.E. Voll, P.M. Kern, W. Kolowos, T.D.Beyer, J.R. Kalden, M. Herrmann, Treatment with annexin V increases immunogenicity of apoptotic human T-cells in Balb/c mice, Cell Death Differ. 7 (2000) 911-915.
10. Samantha Jilek, Hans P. Merkle, Elke Walter. DNA-loaded biodegradable microparticles as vaccine delivery systems and their interaction with dendritic ceils. Advanced Drug Delivery Reviews 57 (2005) 377-390
11. Michele A. Kutzler and David B. Weiner. Developing DNA vaccines that call to dendritic cells. J. Clin. Invest. 114:1241 1244