男男同性恋者HIV-1新型重组株形成与宿主HLA限制性CTL应答及中和抗体反应关系的研究

As the increasing complexity of the HIV spread in the world, recombinants continue to exert a growing impact on the global HIV epidemic. The issue of HIV-1 recombination is one of the bottlenecks of the diagnosis, treatment and prevention of AIDS. A few of studies have showed the close relationships between the vital recombination and host immune responses selection through the viral phylogenetic analysis, mathematical model and case reports. The dynamics between the host immune responses and viral escape mutation created the preconditions for the emergence of viral recombination. However, the research on the immune mechanisms of HIV recombination is not enough. Whether a new recombinant can emerge or spread and the recombination pattern is related with the epidemic strains as well as the specific human populations. The immune mechanisms of the HIV new recombinants in China is not clear, which greatly confined the effectiveness of prevent and control efforts. Now in China, Men who have sex with men population is the fastest growing group for HIV spreading. Our team has found many genotypes of HIV co-epidemic and several strains spread quickly in various regions of China. In Addition, two new circulation recombinant forms, identified by our team in Guangdong, Hunan and Liaoning provinces recently, add to the mounting evidence showing that the MSM population has got the necessary condition for the new HIV recombinants emerging and spreading. In this study, the relationship between the emergence of new HIV-1 recombinants in MSM and host adaptive immune responses are studied on both population level and individual level. On population level, HIV infected MSM were selected from previous identified areas with high new HIV-1 recombinants incidence, among which, new recombinants were screened though small gene fragments amplification and analysis, and then confirmed in the near full-length genome sequences through single genome amplification assay. HLA-I alleles were detected with PCR-SSCP, the HLA-I restricted cytotoxic T-lymphocyte (CTL) epitopes were identified through comparing with previous identified optimal CTL epitopes and prediction with bioinformatics tools. The relationships between the epitopes distribution and breakpoints in HIV genome were analyzed. In addition, the difference on the neutralizing antibody responses to the wild type viruses and recombinants were detected and compared through pseudovirus neutralization assay. On individual level, previous identified acute HIV-1 dual infection cases were selected, who had been proved to develop recombinants in the course of HIV disease progression. Longitudinal samples were used to study the relationships between HIV-1 recombination development and the dynamic of HIV specific CTL responses and neutralizing antibody responses, to disclose the immunity mechanisms and to provide basis for diagnosis, treatment and vaccine studies responding to the new HIV recombinants.

HIV重组是困扰艾滋病的诊断、治疗、预防研究的瓶颈问题之一。目前HIV重组研究多围绕监测新重组株，揭示其来源和进化关系，对重组形成机理研究尚不充分。本研究拟在前期男男同性恋人群(MSM)中鉴定两株新型重组株的基础上，从群体和个体水平上解析宿主获得性免疫应答在HIV-1新型重组株形成中的作用。一方面有针对性地选择新型重组株高发地区的MSM人群，以基因序列分析法筛选并鉴定新型重组株、明确其发生频率及重组断点，PCR-SSCP和表位预测法研究人群水平HLA限制性细胞毒性T细胞应答表位分布与重组断点的关系，假病毒法测定感染人群对野生株和重组株的中和抗体反应的差异；另一方面，选择已知在病程中自然发生重组的急性多重感染者，动态研究病毒重组的发生、发展与HLA限制性CTL应答及中和性抗体反应的变化关系，揭示重组的免疫学成因，从而为我国HIV感染诊断、治疗乃至疫苗研究提出有效应对策略提供依据。

HIV通过重组引起基因水平的巨大改变，影响药物敏感性及获得性免疫应答反应，是困扰艾滋病的诊断、治疗、预防研究的瓶颈问题之一。目前HIV重组研究多围绕监测新重组株，揭示其来源和进化关系，对重组形成机理研究尚不充分。本研究对全国9城市MSM高危人群进行了大规模横断面研究，明确了各地MSM人群中CRF55_01B、CRF59_01B及新重组型HIV-1毒株的发生频率，确定了重组断点的位置及分布规律；新建基于NGS技术的HIV超感染筛选方法，通过急性感染者前瞻性队列研究，揭示了我国MSM人群HIV超感染的发生率为15.6%，明确了当前我国MSM人群中HIV新重组株不断出现具有广泛的人群基础；对HIV急性感染队列的MSM进行了HLA-I等位基因分型，明确了目标人群HLA-I等位基因的分布，揭示了当前MSM人群的主要HIV流行株在常见保护性HLA限制的CTL应答作用下，表位发生普遍发生适应性进化，对常见保护性CTL应答反应不敏感；对HIV超感染者进行了病毒时序性进化研究，揭示了HIV发生重组与HLA限制性细胞毒性T细胞（CTL）应答表位之间的联系，发现重组断点常常位于T细胞表位上或表位侧区；对不同基因型HIV急性感染者进行了中和抗体研究，发现多重感染者比单毒株感染者中和抗体中和能力好；对HIV急性感染者进行了个体内病毒进化与中和抗体应答发生发展关系的研究，发现针对V4区的抗体引起病毒持续通过V4区变异，逃逸自体中和抗体应答反应，但并无助于产生bNAb。本研究从群体和个体水平上解析宿主获得性免疫应答在HIV-1新型重组株形成中的作用及与重组断点之间的关系，探索了重组的免疫学成因，从而为我国HIV感染诊断、治疗乃至疫苗研究提出有效应对策略提供依据。