多载体艾滋病疫苗序贯和重复免疫动物的实验研究

发布时间：2018-03-03 02:18

本文选题：人免疫缺陷病毒　切入点：艾滋病　出处：《中国疾病预防控制中心》2017年博士论文　论文类型：学位论文

【摘要】：由人免疫缺陷病毒(HIV)感染导致的获得性免疫缺陷综合征(AIDS)已在全球范围内流行了超过30年,迄今尚未研制出安全有效的HIV疫苗。本课题组在前期的研究中,提出了使用多载体H1V疫苗进行序贯和重复免疫的策略。动物实验的结果证明,该策略能够诱导持续时间较长、反应强度较高的HIV特异性免疫应答。基于这一免疫策略,本研究中继续开展了多载体HIV/SIV疫苗序贯和重复免疫的相关工作。我们首先在小鼠中进行了表达SIVmac239-gag/env基因的三载体疫苗联合免疫试验,以评价疫苗免疫原性,为后续的恒河猴实验提供依据。其后,我们应用前期构建的四种载体 SIVmac239Gag 疫苗,包括 DNA 疫苗、rAd5-SIVgag、rMVA-SIVgag 和 rAd5F35-SIVgag在SIV感染恒河猴模型中对多载体疫苗预防或治疗SIV感染的有效性进行了评价。15只中国恒河猴被分为预防性免疫组、治疗性免疫组和对照组,分别以上述四种载体疫苗免疫,并以500TCID50与疫苗同源的SIVmac239通过静脉途径进行攻击。实验结果显示,通过四种载体序贯和重复免疫,能够在部分受试动物中诱导强度较高、抗原表位识别范围较广以及维持有效应答时间较长的SIV Gag特异性Elispot反应。有效的免疫反应强度与SIV感染后病毒载量变化呈负相关性,对被感染动物的免疫功能具有保护作用,具有改善疾病进展和结局的能力。预防性免疫组中80%(4/5)的受试动物受疫苗诱导产生了有效的特异性免疫反应。在受到SIV感染后,虽未能阻断感染,但病毒载量峰值显著低于对照组,提示疫苗对降低急性期病毒载量起到了保护作用。其中,40%(2/5)的受试动物病毒载量下降了2 log10copies/mL以上。治疗性免疫组对疫苗的反应显示出较大的个体差异,40%(2/5)的受试动物受诱导产生了有效强度的免疫应答,随之病毒载量下降了约2 log10 copies/mL,下降水平优于对照组,显示多载体疫苗有能力修复或重建被SIV感染动物受损的免疫功能。在先前多载体HIV疫苗序贯免疫恒河猴研究工作的基础上,我们分别使用Ad5载体疫苗和MVA载体疫苗,在恒河猴中进行了多载体疫苗重复免疫策略的评价。结果显示,受试动物接受rAd5-HIVgag疫苗重复免疫后,在疫苗的诱导作用下,高水平的HIV gag特异性细胞免疫应答被重新激活,并且高强度的特异性Elispot反应分别持续了 52w和69w,免疫应答的宽度亦得到了一定程度的增强。多载体HIV疫苗重复免疫的策略具有重新诱导并维持较长时间的高水平特异性免疫应答的能力。Ad5-HIVgag是本课题组实施的多载体联合免疫治疗HIV感染者/AIDS病人临床研究中继DNA疫苗和rMVA疫苗后的第三个疫苗。在临床研究实施之前,我们建立了基于HIV感染者PBMCs在体外对Ad5-HIVgag的免疫原性进行评价的检测方法。分别使用健康人和AIDS病人PBMCs对检测模型中Ad5-HIVgag的使用剂量、刺激时间进行了探索和优化。根据优化的检测条件,我们采集了 24份HIV感染者的PBMCs,对Ad5-HIVgag的免疫原性进行了评价。结果表明,Ad5-HIVgag能够在体外有效的激活HIVgag特异性的细胞免疫应答,并且,病人是否曾接受过药物治疗对Ad5-HIVgag的激活能力没有影响。此外,由于我国近年来HIV-1优势流行株发生了显著的变化,CRF_01 AE和CRF_07/08BC亚型逐渐成为了主要的流行毒株,我们构建了表达HIV-1 AE/BC亚型gag基因的治疗性DNA疫苗和重组MVA疫苗。以从HIV-1感染者中分离的病毒基因组为模板,分别对AE/BC亚型gag基因进行表达优化设计。使用经优化的AEgagi和BCgagi基因分别构建了 DNA疫苗(pVR-HIVAEgagi/pVR-HIVBCgagi)和重组MVA 疫苗(rMVA-HIVAEgagi/rMVA-HIVBCgagi)。初步鉴定结果显示,重组MVA能够在允许细胞中携带外源基因进行稳定的传代和复制;同时,DNA疫苗和rMVA疫苗均能够在细胞内高效表达HIV-1 Gag抗原。我们将继续对以上疫苗的免疫效果进行评价,并联合同样基于优化AE/BC gagi基因构建的重组Ad5疫苗开展多载体HIV疫苗序贯和重复免疫的相关研究。
[Abstract]:By the human immunodeficiency virus (HIV) infection causes acquired immunodeficiency syndrome (AIDS) has been popular worldwide for more than 30 years, has yet to develop safe and effective HIV vaccines. This group in the previous study, proposed the use of multi carrier H1V vaccine and immune strategy of repeated sequential animal. The experiment results show that this strategy can induce longer duration, HIV specific immune response in high intensity. This immunization strategy based on this study, continue to carry out related work of multi carrier HIV/SIV vaccine and sequential repeated immunization. We first conducted three test vector vaccine combined with immune gene expression of SIVmac239-gag/env in mice to evaluate the immunogenicity of the vaccine, and provide the basis for the subsequent Ganges RIver monkeys. Subsequently, four kinds of SIVmac239Gag vaccine vector we constructed before application, including DNA vaccine, RAd5-SIVgag, rMVA-SIVgag and rAd5F35-SIVgag in SIV infected monkey model of Ganges RIver multi carrier vaccine for prevention or treatment of SIV infection effectiveness was evaluated.15 only China Ganges RIver monkey were divided into preventive immunization group, immunity treatment group and control group, respectively, the above four kinds of vector vaccine immunity, and are attacked by 500TCID50 and vaccine homologous SIVmac239 intravenously. The experimental results show that the four kinds of carrier sequential and repeated immunization, can induce high strength in some animal subjects, SIV Gag antigen specific Elispot response a wide range of recognition and maintain effective response time longer. There was a negative correlation between changes in viral load and immune response SIV infection effectively, has a protective effect on the immune function of infected animal, has the ability to improve disease progression and outcomes. Preventive immunization group (4/5) by 80% The vaccine induced specific immune response in animal test. Effective by SIV after infection, although failed to block the infection, but peak viral load was significantly lower than the control group, suggesting that the vaccine protection effect on reducing acute viral load. Among them, 40% (2/5) of the tested animal viral load decreased by more than 2 log10copies/mL. The treatment group of immune response to the vaccine showed large individual differences, 40% (2/5) was induced by the immune response of the effective strength of animal test, then viral load decreased by about 2 log10 copies/mL level than the control group, showing multi carrier vaccine have the ability to repair or reconstruction of the damaged immune function of animal SIV infection. Based on previous sequential vector HIV vaccine immunization in Ganges RIver monkey research work, we use Ad5 vector vaccine and MVA vaccine respectively, the carrier in Ganges RIver monkey disease Evaluation of seedling immunization strategies. The results showed that animal subjects underwent repeated immunization with rAd5-HIVgag vaccine after induction in vaccine, HIV gag specific cellular immune response level is reactivated, and the specificity of the Elispot high strength for 52w and 69w respectively, the width of the immune response also has been enhanced to a certain extent. The multi carrier HIV vaccine immunization strategy repeated with a high level of specific immune response to induce and maintain a longer period of time the ability of.Ad5-HIVgag to clinical researchers with /AIDS relay DNA vaccine and rMVA vaccine after the third vaccine vector combined with immunotherapy of HIV infection. The implementation of the research group before conducting clinical research, we established HIV method for detection of PBMCs infection in vitro immunogenicity of Ad5-HIVgag. Based on the evaluation of healthy individuals and patients with AIDS were used to test PBMCs The dose measurement model Ad5-HIVgag, stimulation time were explored and optimized. According to the optimization of detection conditions, we collected 24 HIV infected PBMCs, the immunogenicity of Ad5-HIVgag was evaluated. The results show that Ad5-HIVgag can activate HIVgag in cellular immune responses, specific and effective in vitro and patients have you had the ability to activate the drugs have no effect on Ad5-HIVgag. In addition, in recent years in China because of the advantages of HIV-1 strains has undergone significant changes, CRF_01 AE and CRF_07/08BC subtype has gradually become the main epidemic strains, we constructed the HIV-1 expression of AE/BC subtype gag gene therapeutic DNA vaccine and recombinant MVA vaccine to isolated from HIV-1 infected the virus genome as template, respectively for AE/BC subtype gag gene expression by AEgagi and optimization design. The optimization of the BCgagi gene were used. The DNA vaccine (pVR-HIVAEgagi/pVR-HIVBCgagi) and recombinant MVA vaccine (rMVA-HIVAEgagi/rMVA-HIVBCgagi). Preliminary identification results showed that recombinant MVA can allow cells carrying exogenous gene and replication were stable; at the same time, DNA vaccine and rMVA vaccine can be effective expression of HIV-1 antigen in Gag cells. We will continue on the above immune effect of vaccine for evaluation, and also combined with related research optimization construction of AE/BC gagi gene recombinant Ad5 vaccine to carry out multi carrier HIV vaccine based on sequential and repeated immunization.

【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】：2017
【分类号】：R392

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