HIV-1膜蛋白V5区多态性对CD4bs特异性中和抗体轻链进化的影响及其作用机制

Our previous study found that the heavy chain of the CD4bs-directed neutralizing antibody is fully evolved but its light chain development is lagging in the HIV-1 infected individual. Structural analysis suggests that the V5 region of HIV-1 Env hinders the evolution of light chain, but its mechanism is still uncertain. We plan to select HIV-1 infected individuals whose plasma present different CD4bs-directed neutralization activities, and observe the association between the V5 polymorphism and the evolutional level of the light chain of the CD4bs-directed neutralizing antibody. Next, we will evaluate the binding affinity between the HIV-1 Env carrying the characteristic V5 sequence and the CD4bs-directed neutralizing antibody. Combined with the structural analysis of antigen-antibody complex, we will explore the mechanism of the V5 region affecting the evolution of the light chain of the CD4bs-directed neutralizing antibody. The implementation of this project not only reveal the novel mechanism of HIV-1 escaping humoral immunity, but also get the neutralizing antibodies with independent intellectual property rights, and provide a theoretical basis for designing immunogens that induce broadly neutralizing antibodies.

前期研究发现HIV-1感染者体内的CD4结合位点（CD4bs）特异性中和抗体存在重链充分进化而轻链发育滞后的现象，导致其中和能力有限。结构生物学分析提示HIV-1膜蛋白V5区阻碍了轻链进化，但其作用机制尚不明确。本研究拟选取血浆具有不同CD4bs特异性中和活性的HIV-1感染者为研究对象，观察不同个体内HIV-1膜蛋白V5区序列多态性和CD4bs特异性中和抗体轻链进化水平的差异，分析二者之间的关联。随后评价携带特征V5序列的膜蛋白与CD4bs特异性中和抗体的亲和力，结合抗原-抗体复合物结构分析，探讨膜蛋白V5区影响CD4bs特异性中和抗体轻链进化的机制。该项目的实施不仅可以揭示HIV-1逃逸体液免疫的新机制，还可以获得具有我国自主知识产权的中和抗体产品，并为设计可诱导广谱中和抗体的免疫原提供理论依据。

利用CD4bs（CD4结合位点， CD4 binding site）分子探针RSC3和敲除CD4bs的分子探针ΔRSC3筛选了114份HIV-1 BC亚型病毒感染者血浆和106份HIV-1 B亚型感染者的血浆，获得了一批血浆内含有针对CD4bs抗体的样本。通过单细胞分选和抗体基因克隆从感染者CBJC508体内获得了7个膜蛋白特异性抗体，5个识别gp41, 2个为gp120结合抗体；从CBJC263感染者体内分离出一个VRC01类抗体263A9，其中和能力较弱，研究提示263A9的轻链存在着进化上的缺陷，阻碍了该抗体的中和能力。将263A9轻链的CDR1和CDR3替换为VRC01轻链的相应区域可以显著提高嵌合抗体的中和宽度。对CBJC263感染者抗体库进行了深度测序，发现263A9抗体的轻链家族发生了偏轨进化，同时对感染者体内的HIV膜区开展SGA分析和抗原-抗体相互作用分析发现V5区的N460/N463糖基化位点可能干扰CD4bs抗体轻链的进化。从精英中和者CBJC451和CBJC363体内鉴定出了两个可以介导ADCC杀伤效应的CD4bs抗体CBJC451-B4和CBJC363-B4。构建了一批来自于精英中和者CBJC261、CBJC451和广谱中和者CBJC263体内不同时间点的假病毒，并对CBJC261和CBJC451的抗体库进行了无偏移测序和基本分析。发现HIV-1 Rev提前出现的终止密码子对CRF07_BC病毒的复制无影响，而削弱了B亚型病毒的复制能力。本研究成果为中和抗体评价和疫苗免疫原研发提供了理论支持。