诱导HIV-1膜蛋白保护性抗体免疫策略的研究

发布时间：2018-04-28 22:34

本文选题：HIV-1 + 膜蛋白　；参考：《中国疾病预防控制中心》2016年博士论文

【摘要】：自发现艾滋病和其病原体人类免疫缺陷病毒(HIV)30多年来,HIV已经感染和杀死超过7000万人,是威胁人类健康最严重的传染病。尽管因抗HIV药物取得重大突破,艾滋病己从死亡率最高的“超级癌症”演化为可控制的慢性病,但因无法治愈而需长期服药,感染者终身携带病毒并可能感染他人。每年全球仍有数百万人被HIV感染,我国每年发现的HIV感染和艾滋病人也从十年前的几万人增加到现在的十余万人。国际共识是人类需要有效的疫苗才能最终控制艾滋病。HIV-1膜蛋白是HIV疫苗研究的重要免疫原,因为它是以广谱中和抗体为主的保护性抗体的的唯一靶位。由于HIV高度变异,其在全球流行过程中产生出众多的亚型、簇和流行株。如何诱导广谱中和抗体反应一直是HIV疫苗研究的一个重点也是最大的难点。许多研究显示,在自然感染中仅有少数人可产生抗HIV广谱中和抗体。在疫苗研究中,天然HIV-1膜蛋白和常规的免疫策略也不能产生广谱中和抗体和其他具有免疫保护性的抗体反应。因此,本论文研究针对HIV疫苗研究的这一难点,沿着免疫原改造和优化免疫策略的两条紧密联系的技术路线,展开课题的研究,力求获得能提高HIV膜蛋白保护性抗体反应和优化的免疫策略。本研究以中国主要流行的B'/C重组亚型的CN54株作为疫苗研究的出发毒株,在第一条技术路线中对两个基于马传染性贫血病毒弱毒疫苗的突变体gp140免疫原2M和5M与CN54 gp140免疫原通过DNA初免-蛋白加强诱导的抗体免疫反应进行了系统的比较,然后纯化不同结构的CN54gp140蛋白,比较不同结构的gp140蛋白免疫豚鼠诱导的抗体免疫反应,随后选择三种亚型的HIV-1膜蛋白和HIV-2的膜蛋白研究交叉免疫诱导的抗体免疫反应。为了比较两个突变体gp140 2M和CN54 gp140免疫原诱导产生的抗体免疫反应情况,三种gp140蛋白在真核表达系统得到表达并纯化,通过DNA初免-蛋白加强的方式免疫小鼠和豚鼠,最后一次免疫后三周采集血清检测诱导产生的抗体免疫反应。检测结果显示相比于野生型gp140免疫组,gp140 2M免疫组诱导更高水平的膜蛋白特异性IgG、IgG1和IgG3结合抗体反应,gp140 5M免疫组诱导更高水平的膜蛋白特异性IgG2a、IgG3和IgA结合抗体反应。进一步分析显示gp140 2M免疫组诱导Th1型免疫反应,而gp140和gp140 5M免疫组诱导Th0型免疫反应。在诱导膜蛋白特异性IgG2b结合抗体和IgM结合抗体上,三组没有显著差异。与未突变gp140以及gp140 5M相比,gp140 2M能诱导产生更好的IgG3,IgA等保护性结合抗体,以及更高水平的针对tier 1假病毒的中和抗体反应。提示gp140 2M可能是更好的候选疫苗。为了研究不同结构的gp140蛋白的免疫原性,我们表达纯化了多聚体gp140、三聚体gp140和单体gp140蛋白,并免疫豚鼠检测其抗体免疫反应情况。免疫结果显示,三聚体gp140蛋白可以诱导产生比多聚体和单体gp140蛋白更高水平的结合抗体和中和抗体反应。提示三聚体gp140蛋白可能是更好的免疫原。在第二条技术路线中,为了提高免疫原诱导产生的具有交叉免疫反应的中和抗体反应,选择了三种不同HIV-1亚型的膜蛋白和HIV-2膜蛋白用于评价交叉免疫产生的抗体免疫反应。结果显示相比于BC亚型单独免疫组,不同亚型混合免疫组可以提高中和抗体的广谱性和中和强度,对于B亚型和AE亚型的膜蛋白特异性结合抗体反应和V1V2特异性结合抗体反应也显著提高。HIV-1和HIV-2免疫原的交叉免疫也能提高中和抗体的广谱性和中和强度,对于B亚型和AE亚型的膜蛋白特异性结合抗体反应和AE亚型的V1V2特异性结合抗体反应也显著提高。交叉免疫可以提高保护性结合抗体和中和抗体反应,不同的免疫顺序会影响诱导产生的抗体免疫反应。为了提高保护性抗体免疫反应,我们从本实验室已经分离到广谱中和抗体DRVIA7的HIV-1 B亚型病毒感染者DRVI01不同时间点扩增相应的膜蛋白抗原,克隆表达后免疫豚鼠研究其免疫原性,然后采取不同的免疫程序以求提高免疫原诱导的保护性抗体反应。结果显示不同免疫程序可以提高针对易中和的tier 1的HIV-1假病毒的中和抗体反应,但对于难中和的tier 2的HIV-1假病毒的中和抗体反应影响不大。这些结果显示广谱中和患者体内可以分离出具有一定中和活性的膜蛋白抗原,不同的免疫顺序会影响诱导产生的抗体免疫反应。
[Abstract]:Since the discovery of AIDS and its pathogen, human immunodeficiency virus (HIV) for more than 30 years, HIV has infected and killed more than 70 million people and is the most serious infectious disease that threatens human health. Despite a major breakthrough in anti HIV drugs, AIDS has evolved from the highest "supercancer" to a controlled chronic disease, but it is not cured. The infected people carry the virus for a long time and can infect others for a long time. Millions of people worldwide are still infected with HIV every year. The HIV infection and aids people found in our country have increased from tens of thousands of people ten years ago to more than 10, 000 people. The international consensus is that human need effective vaccine to control AIDS.HIV-1 membrane protein finally. It is the important immunogen of HIV vaccine research, because it is the only target of protective antibody with broad spectrum and neutralization antibody. Because of the high variation of HIV, it produces numerous subtypes, clusters and epidemic strains in the global epidemic process. How to induce the broad-spectrum neutralization antibody reaction has been a key and the most difficult point in the study of the HIV vaccine. Many studies have shown that only a few people can produce anti HIV broad-spectrum neutralizing antibodies in natural infections. In vaccine studies, natural HIV-1 membrane proteins and conventional immunization strategies do not produce broad-spectrum neutralizing antibodies and other immune protective antibodies. Therefore, this paper aims at the difficulty of the study of HIV vaccines. The two closely related technical routes of immunization and optimization of immunization were carried out in order to obtain the immunization strategy which could improve the protective antibody response and optimization of HIV membrane protein. This study took the CN54 strain of B'/C recombinant in China as the starting strain of the vaccine research, and two in the first technical route. A systematic comparison of the immune response of the mutant gp140 immunogen 2M and 5M based on the vaccine of the equine infectious anemia virus (5M) and CN54 gp140 immunogen was systematically compared with the Immunogenicity Induced by the DNA primer protein immunogen, and then the CN54gp140 protein of different structures was purified to compare the immune response induced by the different structural gp140 protein immunized guinea pigs. Then select three subtypes of HIV-1 membrane protein and HIV-2 membrane protein to study the antibody immunoreaction induced by cross immunization. In order to compare the antibody immunoreaction induced by the two mutant gp140 2M and CN54 gp140 immunogen, three gp140 proteins were expressed and purified in eukaryotic expression system, and through the strengthening of DNA first immune protein. Immune mice and guinea pigs were immunized three weeks after the last immunization. The results showed that compared to the wild type gp140 immune group, the gp140 2M immune group induced a higher level of membrane protein specific IgG, IgG1 and IgG3 combined with antibody reaction, and the gp140 5M immune group induced a higher level of membrane protein specific I. GG2a, IgG3 and IgA combined with antibody reaction. Further analysis showed that gp140 2M immune group induced Th1 type immune response, while gp140 and gp140 5M immunization group induced Th0 type immune response. There was no significant difference between the three groups in inducing membrane specific IgG2b binding antibody and IgM binding antibody. IgG3, IgA and other protective binding antibodies, as well as a higher level of neutralizing antibody response to the tier 1 pseudo virus, suggest that gp140 2M may be a better candidate vaccine. In order to study the immunogenicity of gp140 proteins of different structures, we purified the gp140, three polymer gp140 and mono gp140 protein, and immunized guinea pigs. The antibody immune response was measured. The immune results showed that the trimer gp140 protein could induce a higher binding antibody and neutralizing antibody reaction than the polymer and the monomer gp140 protein. It suggests that the trimer gp140 protein may be a better immunogen. In the second technical route, the interaction between the immunogen and the immunogen is improved. Three different HIV-1 subtypes of membrane proteins and HIV-2 membrane proteins were selected to evaluate the antibody immunoreaction produced by cross immunization. The results showed that the different subtypes of mixed immune groups could improve the broad-spectrum and neutralization intensity of neutralizing antibodies compared to the BC subtype individual immune group, and for the B subtype and the AE subtype. The membrane protein specific binding antibody reaction and V1V2 specific binding antibody reaction also significantly increase the cross immunization of.HIV-1 and HIV-2 immunogens and improve the broad-spectrum and neutralization strength of neutralizing antibodies. The specific binding antibody reaction of the B subtype and AE subtype and the V1V2 specific binding antibody reaction of the AE subtype also significantly increased. Cross immunization improves the protective binding antibody and neutralizing antibody response, and the different immune sequence affects the induced antibody immunoreaction. In order to improve the protective antibody immune response, we have isolated the HIV-1 B subtype infection of the broad-spectrum neutralizing antibody DRVIA7 from our laboratory and amplified the corresponding DRVI01 at different time points. Membrane protein antigen, after cloning and expression, immunized guinea pigs to study its immunogenicity, and then adopted different immunization programs to improve immunogen induced protective antibody response. The results showed that different immunization programs could improve the neutralizing antibody response to the HIV-1 pseudo virus of the easily neutralized tier 1, but for the difficult neutralizing HIV-1 pseudo virus of the tier 2 The neutralization antibody reaction is not affected. These results show that the membrane protein antigen with certain neutralization activity can be isolated in the broad spectrum and in the patient's body, and the different immune order may affect the induced antibody immune response.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R392

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