1例慢性HIV-1感染者体内CD4结合位点特异性记忆B细胞分选及抗体表达研究

Expression of CD4bs-directed antibody from a chronic HIV-1 infected individual using single B cell sorting

目的通过抗原特异性记忆B细胞(Memory B)分选、抗体基因克隆表达和鉴定方法,从艾滋病病毒1型(HIV-1)感染者中获得HIV-1中和抗体,并进一步探讨获得抗体的特征。方法通过检测血浆中和抗体活性和表位筛选,挑选出1例含有CD4结合位点(CD4bs)特异性抗体的慢性HIV-1感染者。采用一对含有/缺失CD4-bs的探针RSC3/ΔRSC3进行Memory B分选,获得抗体可变区基因后进行抗体的表达纯化,并检验所得抗体的结合能力和中和能力。结果从9×106外周血单核淋巴细胞(PBMCs)中分离得6个特异性Memory B。通过反转录聚合酶链反应(RT-PCR)分别扩增出3对重链和轻链可变区基因,配对得到3个抗体,其中有一个抗体具有HIV-1CD4bs结合能力。中和实验表明,这个抗体能中和毒株SF162[50%抑制浓度(IC50=0.89μg/mL]。抗体可变区基因家系分析表明,该抗体属于IGHV1-18家系,重链可变区(VH)自体突变率为12%,低于广谱中和抗体VRC01VH的自体突变率(32%)。结论建立的单克隆特异性Memory B分选方法,可以获得有中和能力的抗体。该平台有希望获得具有我国自主知识产权的广谱中和抗体,并为开发抗体药物和设计新型免疫原提供技术支持。

Objective To obtain HIV-1neutralizing antibody from HIV-1infected patients though antigen-specific memory B cell sorting,gene cloning,antibody expression and identification,and explore the characteristics of obtained antibody.Methods We chose a chronic HIV-1B-infected individual whose plasma presented a CD4bs-specific antibody activity by measuring nAb responses and CD4 binding site（CD4bs response）.We sorted CD4bs-specific memory B cells using probes RSC3/ΔRSC3,and expressed immunoglobulin（IgG）from sorted single cells,and determined their binding capabilitiec to CD4 bs and neutralization activities.Results We got six RSC3-specific memory B cells from 9×106 PBMCs.Pairings of heavy and light chains of three single cells were acquired by RT-PCR and three antibodies were expressed subsequently.Only one antibody presented binding activity to CD4 bs and neutralized SF162 pseudovirus efficiently（IC50=0.89μg/ml）.The VH of this antibody derived from IGHV1-18 had a somatic mutation rate of 12%,lower than VRC01（32%）,indicating its limited neutralizing capability compared with VRC01.Conclusion Our results demonstrated that we could get neutralizing antibody through single B cell sorting platform,and hopefully acquire the broadly neutralizing antibody with China intellectual property rights by this platform,which provides solid basis for developing antibody-related drugs and designing novel vaccine immunogens.