HIV-1超感染者体内广谱中和抗体与病毒共进化的研究

By far, there is neither ideal vaccine for HIV-1 prevention nor practical way for HIV cure. Broadly neutralizing antibodies (bnAbs) are important targets for both vaccine and immunotherapy in HIV infection. Although dozens of bnAbs have been identified from natural HIV-1 infected donors in the world, unfortunately, bnAbs still cannot be elicited in the human body through active immunization by far. Both our group and the foreign researchers found that HIV-1 infected persons often developed broadly neutralizing activities after super infected with another heterogeneous HIV-1. Moreover, some bnAbs have been identified from super infected cases. Because heterologous antigens are provided in HIV-1 super infected cases, the mechanism of bnAb development among super infection maybe different from single strain infected cases, but the detail process still need further study. In this study, an expanded screening will be done on patients from mem who have sex with men acute HIV infection cohort previously established by our group. Next generation sequencing and single-genome amplification will be used to analyze the trajectory of viral evolution among super infected cases since the initial HIV infection. In vitro pseudovirus neutralization assay will be used to identify elite neutralizer and the dynamics of heterologous neutralization activities among the super infected cases. Pseudotyped viruses based on autologous transmitted founder virus and representative mutant/recombinant strains among super infected cases will be used to analyze the initiation and development of autologous neutralizing activities. The potential temporal link between the viral evolution and neutralizing activities will be explored. The epitope mapping of bnAbs and their evolution processes among super infected cases will be first explored with yeast antigen display library, then verified with env clones containing known epitope-specific knockout mutations, peptide binding and epitope-specific antigens blocking assays. The trajectories of targeting epitope will be compared with the sites of autologous viral mutations. Through exploring the viral features that determine the key points of bnAbs initiation and maturation, this study will provide basis on precisely designing sequential immune strategy with multiple HIV envelope immunogens aiming at eliciting bnAb against HIV-1 in human body.

HIV感染尚无理想疫苗，亦无可行根治方法，需终身用药。广谱中和抗体（bnAb）是疫苗及免疫治疗的重要目标，全球已筛选到数十种bnAb，但迄今仍无法通过主动免疫诱导人体产生bnAb。本团队及国外研究均发现人发生HIV超感染后，常获得广谱中和能力，多个已知bnAb来自超感染者。由于异源抗原序贯刺激，超感染者bnAb产生机理可能与单毒株感染者不同，具体过程尚未解明。本研究拟从急性感染队列中扩大筛选超感染者，以下一代测序、单基因组扩增分析超感染者体内env基因进化轨迹；以假病毒中和实验筛选超感染者中的精英中和者，揭示其自体及异体中和活性发展历程及与病毒进化的时序关系；以酵母表面展示抗原片段文库、突变假病毒中和、多肽及表位特异性抗原封闭试验解析超感染者bnAb识别表位及演变过程；通过病毒与bnAb共进化研究，揭示决定bnAb发生发展的关键变异病毒特征，为精准设计序贯免疫策略诱导bnAb提供科学依据。

全球HIV疫苗研发历时30余年，从HIV感染者体内鉴定到上百株广谱中和抗体（bnAb），但仍无法通过主动免疫方式诱导产生bnAb，提示仍需深入解析bnAb的自然产生过程，为疫苗研发提供思路。2个月前，美国学者在Science上撰文报道采用基于生发中心设计种系靶向诱导免疫原成功诱导出bnAb前体反应，进一步支持了本研究解析病毒与抗体共进化轨迹的重要性及必要性。.本项目首次发现，在初始感染后1-2年发生超感染与产生广谱中和能力相关，提示超感染可作为解析序贯免疫疫苗效果的天然模型。从HIV-1急性感染队列筛选到3例超感染精英中和者，病毒env基因进化轨迹各不相同：①超感染病毒与初始感染病毒重组，②两株病毒较长时间各自进化，③从初期的多样性病毒准种到后期病毒趋同进化，提示在宿主不同免疫选择压力下，病毒向逃逸抗体的选择压力的方向进化。 .本项目对2例有性伴关系的超感染者进行对比发现:其中1例精英中和者中和宽度达到75%，并持续保持广谱中和能力，其中和抗体主要由超感染毒株及其子代毒株驱动产生，初始感染病毒作用很小；而其性伴在感染早期，初始感染病毒家族具有较强的诱导中和应答的能力，但感染一年后，病毒诱导中和抗体的能力减弱，该患者最终并未发展出广谱中和能力。本项目利用已知单克隆广谱中和抗体中和假病毒的方法，初步筛选到影响中和抗体应答的病毒区域，再用突变病毒技术，解析了中和抗体靶向区域及逃逸自体中和应答的关键表位。精英中和者持续在病毒CD4bs区累积点突变驱动了中和抗体逐步发展成熟，最终产生广谱中和能力。而其性伴先后通过V2、V3及CD4bs不同区域的变异驱动抗体应答反应，但异体中和活性并未逐步发展。本研究结果提示在同一表位连续变异病毒的持续刺激，可能是驱动广谱中和抗体的必要条件，上述发现为序贯免疫策略的抗原设计奠定了实验基础。.本项目首次发现超感染者不同时期可产生多种重组病毒，且均可在人群中传播，为解释重组结构相似但不完全相同的HIV新重组株起源，及应对新型重组株传播提供了科学依据。