【作者】 李学仁；

【导师】 阮力； 谭文杰；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2008， 博士

【摘要】 自从1981年发现艾滋病以来,艾滋病已在全世界范围流行,成为严重危害人类健康的传染病。目前,宣传教育、行为干预以及抗病毒药物治疗等措施在防控艾滋病中发挥了重要作用。然而,有效的疫苗将在艾滋病的防控中发挥关键性作用。由于HIV具有高度变异、攻击免疫系统,造成持续感染和诱发免疫病理损伤的特点,使该疫苗的研制遭遇了前所未有的困难。使用能诱发机体产生针对HIV主要亚型病毒起保护性免疫反应的抗原做疫苗靶抗原是HIV疫苗研发的首要策略,确定这类抗原是HIV疫苗研究关键而艰巨的工作。二十多年来,这方面的研究尚无明显突破。使用HIV主要中和抗原Env和主要细胞免疫抗原Gag等为疫苗的临床实验均告失败,更说明研究确定HIV疫苗靶抗原的关键性和艰巨性。与近些年来使用一种或多种完整的或修饰过的Env为HIV疫苗重要靶抗原的研究策略不同,本研究尝试了使用Env中具有一定广谱中和活性的HIV非主要中和抗原表位或称在自然感染过程中不能发挥明显保护作用的弱中和抗原表位做靶抗原的HIV疫苗研究新策略。鉴于这些抗原表位的免疫原性均较弱,本研究提出:若使用这类抗原作疫苗,一定要设法诱发出针对这类抗原的强免疫反应,否则不能有效识别自然感染过程中这类弱保护性抗原,也就不能有效抵抗这类病毒感染。即:诱发针对这类抗原的强免疫反应,可能是研制成功这类病毒疫苗的关键所在。这一设想是否正确,需要实验证明。如何来证实这个设想,就成了本研究要解决的中心问题。本研究的思路是:为了增强HIV-1交叉中和表位的免疫原性,将HIV-1三个具有一定广谱中和活性的线性抗原表位ELDKWA(简称2F5)、NWFDIT(简称4E10)和GPGRAFY(简称447-52D)的编码基因分别融合到能高效表达外源抗原表位的HBV S基因3’末端,选用佐剂效应不同的载体分别表达这类抗原,通过这类抗原单独或者几种不同类型疫苗的联合免疫,在小鼠模型中造成针对这类抗原强度不同的免疫状态,对其诱生的结合抗体和中和抗体水平进行评价。主要结果如下:第一、构建了分别表达HBV S抗原与HIV交叉中和表位的四种融合基因的天坛株重组痘苗病毒疫苗,分别命名为RVJ1175S-2F5、RVJ1175S-4E10、RVJ1175S-44752D和RVJ1175S-串联表位(前述三种表位的串联体),PCR和测序结果表明,这四种融合基因序列正确重组到痘苗病毒TK区,免疫荧光检测结果显示,HBV S抗原均可在Hela细胞中很好的表达。HBsAg的ELISA检测表明这四种融合蛋白有效表达并分泌到细胞培养上清液中。第二、使用上述四种重组痘苗病毒感染的细胞培养上清液经分离纯化制备了四种相应的蛋白亚单位疫苗PS-2F5、PS-4E10、PS-447-52D和PS-串联表位,SDS-PAGE显示四种纯化后的融合蛋白均含分子量为23kD和27kD两种典型HBsAg条带,Western blot证明这两个条带均能与HBsAg抗体反应,并分别能与三种表位相应的HIV-1单抗2F5、4E10和447-52D反应。第三、构建了包含HBV S抗原与HIV交叉中和表位四种融合基因的复制型DNA疫苗,限制性内切酶酶切和序列测定确定了这四个质粒的正确性,ELISA确定了pSCA中插入S-2F5、S-4E10、S-447-52D和S-串联表位基因均能在Hela中有效表达,并能分泌到细胞上清。第四、建立了HIV假病毒中和试验技术。用HIV慢病毒载体系统的双质粒(包装质粒pSG3deltaEnv、表达HIV B、C亚型不同株包膜蛋白的质粒pcDNA3.1-Env)共同转染293FT细胞,获得分别针对B、C亚型Env的五种假病毒颗粒,分别命名为SVPB6(B亚型)、SVPB12(B亚型)、SVPB13(B亚型)、SVPB8(B亚型)、SVPC5(C亚型)。中和试验结果显示,这五种HIV假病毒能分别被交叉中和单抗2F5、4E10和447-52D所中和。第五、对重组痘苗病毒、亚单位疫苗和DNA疫苗单独免疫或几种不同类型的疫苗联合免疫的免疫学特性进行了比较研究。小鼠免疫结果显示,这四种重组痘苗病毒疫苗和蛋白亚单位疫苗均能诱发较高水平的HBsAg抗体和相应HIV-1交叉中和表位的结合抗体,蛋白亚单位疫苗诱生的这两类抗体均明显高于对应的重组痘苗病毒疫苗。中和抗体检测结果表明,单独免疫组中只有三针PS-串联表位蛋白亚单位疫苗免疫血清表现出较弱的特异和交叉中和活性,疫苗联合免疫组中DNA初免三针蛋白加强一针、痘苗病毒初免两针蛋白加强一针与蛋白单独免疫三针产生的抗体水平相当,联合免疫未进一步增强交叉中和抗体水平。上述结果表明,HIV中和表位与HBV S抗原融合蛋白多次免疫可以诱发产生一定程度的HIV交叉中和抗体,而不同类型疫苗联合免疫未能进一步增强HIV交叉中和抗体反应。这些结果提示,使用相同融合蛋白多次加强免疫更多增强的是融合抗原中载体蛋白的免疫反应,而不能有效增强融合抗原中HIV这种弱中和表位的免疫反应;同时提示,使用不同载体蛋白构建的含HIV弱中和表位的融合抗原开展进一步提高HIV交叉中和抗原免疫反应的研究可能是有价值的。本研究对HIV广谱中和抗体疫苗进行了有意义的探索,为深入进行这类疫苗的研究提供了有价值的实验资料。更多还原

【Abstract】 Since the discovery of AIDS in 1981, the global spread of HIV has reached pandemic proportions, representing a global developmental and public health threat. Although the propaganda and education, behavior intervention and antiretroviral drugs have led to a remarkable progress in prevention and control of AIDS, the development of a safe, globally effective and affordable HIV vaccine will offer the best hope for the future control of the pandemic. Because of the unique aspects of the virus itself, i.e. high variation, attacking immune system, resulting in persistence infection and inducing immunopathogenesis damage, HIV vaccine research is facing with unprecedented difficulties and challenges. It’s the prime approach to develop the cross-protection HIV-1 vaccine, using the antigen inducing different subtype HIV protective immune response, as the main target of vaccine. Finding the antigen is a key and hard job in HIV vaccine study field. In recent 20 years, substantive breakthroughs haven’t been achieved on the study. HIV clinical experiment’s failure using major neutralizing antigen Env or major cellular immunization antigen Gag illustrated that finding HIV vaccine target antigen was very key and difficult.Different from the study strategy using one or more intact or modified Env as target antigen of HIV vaccine in recent years, our study tried to use broadly neutralizing activity subdominant antigens or internal conserved antigens in the process of natural infection, as the main target of vaccine. As the immunogenicity of these antigen epitopes is very weak, we bring forward such point: It can’t be considered as the vaccine antigen, which can’t induce strong immune response. The antigen must induce strong immune response, which is the base of successful this virus vaccine. It must be proved whether the assumption is right or not. Central to the strategy is how to prove this assume.The strategy of our investigation is: To enhance immunogenicity of HIV-1 cross neutralizing epitopes, three HIV-1 cross neutralizing epitopes(ELDKWA, NWFDIT, GPGRAFY) or the three-epitope concatemer were fused to 3’ end of HBV S gene by PCR cloning technology, respectively. Different immune vectors will be selected to express these antigens separately. Different intensity immune response to these antigens in the mouse model will be explored, using different combinations of antigens and immune vectors, such as single antigen, single antigen in different vectors (prime-boost). Combination antibodies and neutralizing antibodies were evaluated. The major results were summarized as follow:Firstly, Four vaccinia virus (Tiantan strain) recombinants expressing separately the four fusion genes were constructed, named as RVJ1175S-2F5(ELDKWA), RVJ1175S-4E10(NWFDIT), RVJ1175S-44752D(GPGRAFY) and RVJ1175S-multiepitopes (the three-epitope concatemer) respectively. It was confirmed by PCR and sequencing that the fusion genes were inserted into the TK locus of vaccinia virus Tiantan strain correctly. The fusion proteins were expressed efficiently and secreted into supernatant of the infected cells, which was demonstrated by HBsAg ELISA test. Immunofluorescence proved the well expression of the four fusion protein in rVV-infected Hela cells.Secondly, From the supernatants of CEF cells infected by these vaccinia recombinants, four subunit vaccines (PS-2F5, PS-4E10, PS-447-52D and PS- multiepitopes) were prepared after purification. Two typical HBsAg bands of 23kD and 27kD were detected in all the purified samples by SDS-PAGE. These two electrophoresis bands were reacted well with HBsAb and corresponded HIV-1 monoclonal antibodies 2F5,4E10 and 447-52D.Thirdly, Four DNA plasmids expressing separately HIV-1 crossing neutralizing epitopes and HBV S fusion genes were constructed. It was confirmed by restriction digest and sequencing that the fusion genes were correct. ELISA proved the well expression of the four fusion proteins and secreted into supernatant of the transient transfected Hela cells.Fourthly, Construction of HIV-1 pseudotyped virus neutralizing test platformThe lentiviral vector system used in this study consists of the backbone plasmid (pSG3deltaEnv), and the envelop plasmid encoding the HIV-1 subtype B or C Env protein (pcDNA3.1-Env). These plasmids were cotransfected in 293FT cells. Five HIV pseudotyped viruses were successfully packaged, named as SVPB6 (subtype B) , SVPB12 (subtype B) , SVPB13 (subtype B) , SVPB8 (subtype B) , SVPC5 (subtype C) separately. Neutralizing test results showed the five pseudotyped viruses could be neutralized by monoclonal antibodies 2F5, 4E10 and 447-52D.Fifthly, BALB/c mice were immunized with subunit vaccine or recombinant vaccinia vaccine or DNA vaccine separately or combined immunization with different type vaccines. High levels of HBsAb and anti-HIV-1 cross neutralizing epitope combination antibodies in peripheral blood of vaccinia virus and subunit vaccine immunized mice were tested by ELIS A, and all the antibody levels induced by subunit vaccines were higher than that induced by correlated vaccinia recombinants in mice. Neutralizing test results showed that mice serum immunized with three needles protein subunit vaccine (PS-multiepitopes) have weak special and crossing neutralizing activity. The neutralizing antibody levels of mice immunized by three needles DNA vaccine followed by one needle protein (prime-boost) or two needles vaccinia virus vaccine followed by one needle protein (prime-boost) or three needles protein were equivalent. Combined immunization can’t enhance crossing neutralizing antibodies level.These above results showed that immunization with HIV crossing neutralizing epitopes fused with HBV S protein many times may induce HIV crossing neutralizing antibodies to a certain extent, but combined immunization with different type vaccines can’t enhance HIV crossing neutralizing antibody response. These results hinted that immunization with the same fusion protein more times enhance the immune response of carrier protein only, can’t enhance the immune response of HIV weak neutralizing epitope. At the same time, the study on immunization with HIV weak neutralizing epitope and different carriers’ fusion protein boosting HIV crossing neutralizing epitope immune response may be valuable. This work undertakes effective exploration on HIV broad-spectrum neutralizing antibody vaccine, also provides valuable experiment data for future study on the virus vaccine.更多还原