恶性疟疾多表位DNA疫苗M.RCAg-1在不同免疫方案中的免疫原性及其免疫机制的研究
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摘要
疟疾是目前世界上对人类危害最严重的蚊媒寄生原虫病。全世界每年有5亿人感染疟原虫，约三百万人死于恶性疟疾，其中80％是五岁以下的儿童和孕妇。恶性疟原虫及蚊媒抗药性的不断增加和蔓延，使得疟疾的防治成为世界性难题之一。疟原虫生活周期复杂，抗原具有阶段特异性、虫株特异性和高度变异性等特点，长期以来形成研制抗疟原虫疫苗的技术瓶颈。因此，针对恶性疟原虫不同生活时期的免疫反应特点，选取诱导产生多种免疫反应类型的不同抗原构建多表位疫苗已经成为研究抗疟疫苗的热点。
本课题组在前期工作中借鉴了分子育种技术(即DNA改组)的随机重组原理，建立了表位改组技术并构建了多表位基因库，用库免疫血清筛选出高抗原性的阳性克隆，经实验证实其中VR312(后改名为M．RCAg-1)基因克隆及其编码蛋白不仅能在小鼠模型中诱导交叉免疫保护，而且能在家兔模型中诱导抑制抗恶性疟原虫生长的抗体。
由于蛋白质疫苗成本昂贵，不适宜在非洲等落后地区推广使用；单一DNA疫苗的免疫原性较低，难以达到预期的免疫反应强度和令人满意的免疫保护效果。因此本研究试图采用不同的免疫策略以观察对DNA疫苗免疫原性的影响，并分析其免疫反应机制，重点观察了基因枪免疫中不同粒径金颗粒对免疫佐剂效应的影响。初步证实将恶性疟疾多表位DNA疫苗M．RCAg-1D通过基因枪皮内免疫小鼠，能够获得高滴度特异性抗体水平，诱生明显的CD4~+T细胞反应，而且在用P．yoelii攻击时能明显保护小鼠延长其存活时间。提示基因枪皮内免疫能够显著提高基因疫苗的免疫原性，这可能与其提高外源基因的转染及表达有关。与此同时，应用活体电穿孔免疫小鼠，也能够获得较高滴度特异性抗体水平和诱生明显的CD4~+T细胞反应，但是不能对P．yoelii产生交叉保护。而脂质体混合基因疫苗M．RCAg-1DDNA后肌肉注射所诱导产生的免疫原性尽管显著高于裸DNA肌肉注射和裸DNA皮下注射，但是诱导产生的抗体不能对P．yoelii产生交叉免疫保护。基因枪免疫时诱导产生的抗体水平受到金粉粒径的影响，其中1.0μm金颗粒作为质粒DNA载体进行基因免疫时，在小鼠体内诱导产生的特异性抗体水平最高。以上工作对DNA疫苗的应用和改进均提供了有价值的实验依据。
Human malaria infections caused by Plasmodium falciparum claim up to 3 million lives annually (especially children and pregnant women). In view of its high morbidity and motality rates and the progression of drug resistence that threatens treatment efficacy, populations living in epidemic areas are in great need of novel effective antimalaria control measures, such as an antimalaria vaccine. The unique obstacles to develop antimalarial vaccines have been Plasmodium' s complex life cycles, antigenic stage-specificity, diversity and variation for a long time. Thus, the ultimate malaria vaccine will require the delivery of multiple antigens from different stages of the complex malaria life cycle. Delivery of combinations of malaria antigens can evoke enhanced immune responses and protect to a greater extent than can a single antigen alone, as well as overcome genetic restrictions in different Plasmodium strains.
In the previous work, we have constructed polyepitope libraries with epitope shuffling technology, screened positive clones with high antigenicity by dot blot and obtained a positive clone named M. RCAg-1 which could induce cross-protection in mice and inhibitory antibodies against Plasmodium falicparum in rabbits.
Protein vaccine is not suitable for generalization in poor countries owing to its high-cost, and a single DNA vaccine is difficult to induce satisfactory protection and obtain diversity of immune response after immunization for its low immunogenicity.
So this study focuses on the immunogenicity and protection of M. RCAg-1D vaccines in different immunization methods, and the adjuvant effect of different gold beads in the gene gun vaccination.
The results are shown in the following. The multiepitope DNA vaccine against Plasmodium falciparum M. RCAg-1D induced significant CD4~+ T cell responses and high titers of specific antibodies in immunized mice by gene gun, which could effectively protect immunized mice from deaths when challenged by P. yoelii. This suggests that gene gun immunization by intradermal could improve the immunogenicity of DNA vaccine significantly, which might be related to up-regulation of the transfection, expression of the exogenous gene and release of the M. RCAg-1 protein. By electroporation in vivo M. RCAg-1D gene vaccine could induce high titers of specific antibodies but with low avidity in mice which could not effectively protect immunized mice from deaths when challenged by P. yoelii. M. RCAg-1D DNA vaccine encapsued by liposome could induce significantly higher titers of specific antibodies than naked DNA vaccinated intramusculary or subcutaneous, although there are not enough protective antibodies in immunized mice by these three methods. This might be related with the same presentation pattern of gene vaccine by in vivo electroporation or by liposome capsued DNA vaccination as those by naked DNA vaccinated intramusculary and a single improvement of the transfection efficiency. Results of comparision of different gold beads as adjuvant tell us that using different gold beads can induced different levels of antibody in gene gun immunization and when using 1.0μm as the plasmid DNA vector in gene gun vaccination, M. RCAg-1D DNA vaccine could induce the highest titer of specific antibody in immunized mice, this maybe related to the effection of different gold beads to release, transfection pattern of the exogenous genes.
本课题组在前期工作中借鉴了分子育种技术(即DNA改组)的随机重组原理，建立了表位改组技术并构建了多表位基因库，用库免疫血清筛选出高抗原性的阳性克隆，经实验证实其中VR312(后改名为M．RCAg-1)基因克隆及其编码蛋白不仅能在小鼠模型中诱导交叉免疫保护，而且能在家兔模型中诱导抑制抗恶性疟原虫生长的抗体。
由于蛋白质疫苗成本昂贵，不适宜在非洲等落后地区推广使用；单一DNA疫苗的免疫原性较低，难以达到预期的免疫反应强度和令人满意的免疫保护效果。因此本研究试图采用不同的免疫策略以观察对DNA疫苗免疫原性的影响，并分析其免疫反应机制，重点观察了基因枪免疫中不同粒径金颗粒对免疫佐剂效应的影响。初步证实将恶性疟疾多表位DNA疫苗M．RCAg-1D通过基因枪皮内免疫小鼠，能够获得高滴度特异性抗体水平，诱生明显的CD4~+T细胞反应，而且在用P．yoelii攻击时能明显保护小鼠延长其存活时间。提示基因枪皮内免疫能够显著提高基因疫苗的免疫原性，这可能与其提高外源基因的转染及表达有关。与此同时，应用活体电穿孔免疫小鼠，也能够获得较高滴度特异性抗体水平和诱生明显的CD4~+T细胞反应，但是不能对P．yoelii产生交叉保护。而脂质体混合基因疫苗M．RCAg-1DDNA后肌肉注射所诱导产生的免疫原性尽管显著高于裸DNA肌肉注射和裸DNA皮下注射，但是诱导产生的抗体不能对P．yoelii产生交叉免疫保护。基因枪免疫时诱导产生的抗体水平受到金粉粒径的影响，其中1.0μm金颗粒作为质粒DNA载体进行基因免疫时，在小鼠体内诱导产生的特异性抗体水平最高。以上工作对DNA疫苗的应用和改进均提供了有价值的实验依据。
Human malaria infections caused by Plasmodium falciparum claim up to 3 million lives annually (especially children and pregnant women). In view of its high morbidity and motality rates and the progression of drug resistence that threatens treatment efficacy, populations living in epidemic areas are in great need of novel effective antimalaria control measures, such as an antimalaria vaccine. The unique obstacles to develop antimalarial vaccines have been Plasmodium' s complex life cycles, antigenic stage-specificity, diversity and variation for a long time. Thus, the ultimate malaria vaccine will require the delivery of multiple antigens from different stages of the complex malaria life cycle. Delivery of combinations of malaria antigens can evoke enhanced immune responses and protect to a greater extent than can a single antigen alone, as well as overcome genetic restrictions in different Plasmodium strains.
In the previous work, we have constructed polyepitope libraries with epitope shuffling technology, screened positive clones with high antigenicity by dot blot and obtained a positive clone named M. RCAg-1 which could induce cross-protection in mice and inhibitory antibodies against Plasmodium falicparum in rabbits.
Protein vaccine is not suitable for generalization in poor countries owing to its high-cost, and a single DNA vaccine is difficult to induce satisfactory protection and obtain diversity of immune response after immunization for its low immunogenicity.
So this study focuses on the immunogenicity and protection of M. RCAg-1D vaccines in different immunization methods, and the adjuvant effect of different gold beads in the gene gun vaccination.
The results are shown in the following. The multiepitope DNA vaccine against Plasmodium falciparum M. RCAg-1D induced significant CD4~+ T cell responses and high titers of specific antibodies in immunized mice by gene gun, which could effectively protect immunized mice from deaths when challenged by P. yoelii. This suggests that gene gun immunization by intradermal could improve the immunogenicity of DNA vaccine significantly, which might be related to up-regulation of the transfection, expression of the exogenous gene and release of the M. RCAg-1 protein. By electroporation in vivo M. RCAg-1D gene vaccine could induce high titers of specific antibodies but with low avidity in mice which could not effectively protect immunized mice from deaths when challenged by P. yoelii. M. RCAg-1D DNA vaccine encapsued by liposome could induce significantly higher titers of specific antibodies than naked DNA vaccinated intramusculary or subcutaneous, although there are not enough protective antibodies in immunized mice by these three methods. This might be related with the same presentation pattern of gene vaccine by in vivo electroporation or by liposome capsued DNA vaccination as those by naked DNA vaccinated intramusculary and a single improvement of the transfection efficiency. Results of comparision of different gold beads as adjuvant tell us that using different gold beads can induced different levels of antibody in gene gun immunization and when using 1.0μm as the plasmid DNA vector in gene gun vaccination, M. RCAg-1D DNA vaccine could induce the highest titer of specific antibody in immunized mice, this maybe related to the effection of different gold beads to release, transfection pattern of the exogenous genes.
引文
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9. Green, S., et al., Liposomal vaccines. Adv Exp Med Biol, 1995. 383: p. 83-92.
10. Burghaus, P.A., et al., Immunization of Aotus nancymai with recombinant C terminus of Plasmodium falciparum merozoite surface protein 1 in liposomes and alum adjuvant does not induce protection against a challenge infection. Infect Immun, 1996. 64(9): p. 3614-9.
11. Heppner, D.G., et al., Safety, immunogenicity, and efficacy of Plasmodium falciparum repeatless circumsporozoite protein vaccine encapsulated in liposomes. J Infect Dis, 1996. 174(2): p. 361-6.
12. Hui, G.S. and C.N. Hashimoto, Pathways for potentiation of immunogenicity during adjuvant-assisted immunizations with Plasmodium falciparum major merozoite surface protein 1. Infect Immun, 1998. 66(11): p. 5329-36.
13. Richards, R.L., et al., Liposomes containing lipid A serve as an adjuvant for induction of antibody and cytotoxic T-cell responses against RTS,S malaria antigen. Infect Immun, 1998. 66(6): p. 2859-65.
14. Tanaka, M., et al., Inhibition of RL male 1 tumor growth in BALB/c mice by introduction of the RLakt gene coding for antigen recognized by cytotoxic T-lymphocytes and the GM-CSF gene by in vivo electroporation. Cancer Sci, 2004. 95(2): p. 154-9.
15. Kalat, M., et al., In vivo plasmid electroporation induces tumor antigen-specific CD8+ T-cell responses and delays tumor growth in a syngeneic mouse melanoma model. Cancer Res, 2002. 62(19): p. 5489-94.
16. Widera, G, et al., Increased DNA vaccine delivery and immunogenicity by electroporation in vivo. J Immunol, 2000. 164(9): p. 4635-40.
17. Quaglino, E., et al., Electroporated DNA vaccine clears away multifocal mammary carcinomas in her-2/neu transgenic mice. Cancer Res, 2004. 64(8): p. 2858-64.
18. Dayball, K., et al., Electroporation enables plasmid vaccines to elicit CD8+ T cell responses in the absence of CD4+ T cells. J Immunol, 2003. 171(7): p. 3379-84.
19. Miyachi, K., et al., Arg-gingipain A DNA Vaccine Prevents Alveolar Bone Loss in Mice. J Dent Res, 2007. 86(5): p. 446-50.
20. Maestre, L., et al., Generation of a New Monoclonal Antibody Against MALT1 by Genetic Immunization. Hybridoma (Larchmt), 2007. 26(2): p. 86-91.
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22. Pertmer, T.M., et al., Gene gun-based nucleic acid immunization: elicitation of humoral and cytotoxic T lymphocyte responses following epidermal delivery of nanogram quantities of DNA. Vaccine, 1995. 13(15): p. 1427-30.
23. Weiss, R., et al., Genetic vaccination against malaria infection by intradermal and epidermal injections of a plasmid containing the gene encoding the Plasmodium berghei circumsporozoite protein. Infect Immun, 2000. 68(10): p. 5914-9.
24. Belperron, A.A., et al., Immune responses induced by gene gun or intramuscular injection of DNA vaccines that express immunogenic regions of the serine repeat antigen from Plasmodium falciparum. Infect Immun, 1999. 67(10): p. 5163-9.
25. Rainczuk, A., et al., A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent Plasmodium chabaudi adami DS malaria. Infect Immun, 2004. 72(10): p. 5565-73.
26. Lobo, C.A., R. Dhar, and N. Kumar, Immunization of mice with DNA-based Pfs25 elicits potent malaria transmission-blocking antibodies. Infect Immun, 1999. 67(4): p. 1688-93.
27. Tongren, J.E., et al., Epitope-specific regulation of immunoglobulin class switching in mice immunized with malarial merozoite surface proteins. Infect Immun, 2005. 73(12): p. 8119-29.
28. Dunachie, S.J., et al., A DNA prime-modified vaccinia virus ankara boost vaccine encoding thrombospondin-related adhesion protein but not circumsporozoite protein partially protects healthy malaria-naive adults against Plasmodium falciparum sporozoite challenge. Infect Immun, 2006. 74(10): p. 5933-42.
29. Walther, M., et al., Safety, immunogenicity, and efficacy of prime-boost immunization with recombinant poxvirus FP9 and modified vaccinia virus Ankara encoding the full-length Plasmodium falciparum circumsporozoite protein. Infect Immun, 2006. 74(5): p. 2706-16.
30. Wang, R., et al., Boosting of DNA vaccine-elicited gamma interferon responses in humans by exposure to malaria parasites. Infect Immun, 2005. 73(5): p. 2863-72.
31. Good, M.F., Towards a blood-stage vaccine for malaria: are we following all the leads? Nat Rev Immunol, 2001. 1(2): p. 117-25.
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2. Waters, A., Malaria: new vaccines for old? Cell, 2006. 124(4): p. 689-93.
3. Good, M.F., et al., The immunological challenge to developing a vaccine to the blood stages of malaria parasites. Immunol Rev, 2004. 201: p. 254-67.
4. Cai, Q.L., et al., Immunogenicity of polyepitope libraries assembled by epitope shuffling: an approach to the development of chimeric gene vaccination against malaria. Vaccine, 2004. 23(2): p. 267-77.
5. Lai, W.C. and M. Bennett, DNA vaccines. Crit Rev Immunol, 1998. 18(5): p. 449-84.
6. Stevenson, F.K., DNA vaccines against cancer: from genes to therapy. Ann Oncol, 1999. 10(12): p. 1413-8.
7. McCluskie, M. J., et al., Route and method of delivery of DNA vaccine influence immune responses in mice and non-human primates. Mol Med, 1999. 5(5): p. 287-300.
8. Krishnan, L., et al., Archaeosome vaccine adjuvants induce strong humoral, cell-mediated, and memory responses: comparison to conventional liposomes and alum. Infect Immun, 2000. 68(1): p. 54-63.
9. Green, S., et al., Liposomal vaccines. Adv Exp Med Biol, 1995. 383: p. 83-92.
10. Burghaus, P.A., et al., Immunization of Aotus nancymai with recombinant C terminus of Plasmodium falciparum merozoite surface protein 1 in liposomes and alum adjuvant does not induce protection against a challenge infection. Infect Immun, 1996. 64(9): p. 3614-9.
11. Heppner, D.G., et al., Safety, immunogenicity, and efficacy of Plasmodium falciparum repeatless circumsporozoite protein vaccine encapsulated in liposomes. J Infect Dis, 1996. 174(2): p. 361-6.
12. Hui, G.S. and C.N. Hashimoto, Pathways for potentiation of immunogenicity during adjuvant-assisted immunizations with Plasmodium falciparum major merozoite surface protein 1. Infect Immun, 1998. 66(11): p. 5329-36.
13. Richards, R.L., et al., Liposomes containing lipid A serve as an adjuvant for induction of antibody and cytotoxic T-cell responses against RTS,S malaria antigen. Infect Immun, 1998. 66(6): p. 2859-65.
14. Tanaka, M., et al., Inhibition of RL male 1 tumor growth in BALB/c mice by introduction of the RLakt gene coding for antigen recognized by cytotoxic T-lymphocytes and the GM-CSF gene by in vivo electroporation. Cancer Sci, 2004. 95(2): p. 154-9.
15. Kalat, M., et al., In vivo plasmid electroporation induces tumor antigen-specific CD8+ T-cell responses and delays tumor growth in a syngeneic mouse melanoma model. Cancer Res, 2002. 62(19): p. 5489-94.
16. Widera, G, et al., Increased DNA vaccine delivery and immunogenicity by electroporation in vivo. J Immunol, 2000. 164(9): p. 4635-40.
17. Quaglino, E., et al., Electroporated DNA vaccine clears away multifocal mammary carcinomas in her-2/neu transgenic mice. Cancer Res, 2004. 64(8): p. 2858-64.
18. Dayball, K., et al., Electroporation enables plasmid vaccines to elicit CD8+ T cell responses in the absence of CD4+ T cells. J Immunol, 2003. 171(7): p. 3379-84.
19. Miyachi, K., et al., Arg-gingipain A DNA Vaccine Prevents Alveolar Bone Loss in Mice. J Dent Res, 2007. 86(5): p. 446-50.
20. Maestre, L., et al., Generation of a New Monoclonal Antibody Against MALT1 by Genetic Immunization. Hybridoma (Larchmt), 2007. 26(2): p. 86-91.
21. O'Brien, J.A. and S.C. Lummis, Biolistic transfection of neuronal cultures using a hand-held gene gun. Nat Protoc, 2006. 1(2): p. 977-81.
22. Pertmer, T.M., et al., Gene gun-based nucleic acid immunization: elicitation of humoral and cytotoxic T lymphocyte responses following epidermal delivery of nanogram quantities of DNA. Vaccine, 1995. 13(15): p. 1427-30.
23. Weiss, R., et al., Genetic vaccination against malaria infection by intradermal and epidermal injections of a plasmid containing the gene encoding the Plasmodium berghei circumsporozoite protein. Infect Immun, 2000. 68(10): p. 5914-9.
24. Belperron, A.A., et al., Immune responses induced by gene gun or intramuscular injection of DNA vaccines that express immunogenic regions of the serine repeat antigen from Plasmodium falciparum. Infect Immun, 1999. 67(10): p. 5163-9.
25. Rainczuk, A., et al., A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent Plasmodium chabaudi adami DS malaria. Infect Immun, 2004. 72(10): p. 5565-73.
26. Lobo, C.A., R. Dhar, and N. Kumar, Immunization of mice with DNA-based Pfs25 elicits potent malaria transmission-blocking antibodies. Infect Immun, 1999. 67(4): p. 1688-93.
27. Tongren, J.E., et al., Epitope-specific regulation of immunoglobulin class switching in mice immunized with malarial merozoite surface proteins. Infect Immun, 2005. 73(12): p. 8119-29.
28. Dunachie, S.J., et al., A DNA prime-modified vaccinia virus ankara boost vaccine encoding thrombospondin-related adhesion protein but not circumsporozoite protein partially protects healthy malaria-naive adults against Plasmodium falciparum sporozoite challenge. Infect Immun, 2006. 74(10): p. 5933-42.
29. Walther, M., et al., Safety, immunogenicity, and efficacy of prime-boost immunization with recombinant poxvirus FP9 and modified vaccinia virus Ankara encoding the full-length Plasmodium falciparum circumsporozoite protein. Infect Immun, 2006. 74(5): p. 2706-16.
30. Wang, R., et al., Boosting of DNA vaccine-elicited gamma interferon responses in humans by exposure to malaria parasites. Infect Immun, 2005. 73(5): p. 2863-72.
31. Good, M.F., Towards a blood-stage vaccine for malaria: are we following all the leads? Nat Rev Immunol, 2001. 1(2): p. 117-25.
32. Medi, B.M. and J. Singh, Skin targeted DNA vaccine delivery using electroporation in rabbits II. Safety. Int J Pharm, 2006. 308(1-2): p. 61-8.
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