中国HIV-1临床分离株感染性假病毒的构建及其中和特征分析

Infectious Pseudovirus Construction and Neutralization Susceptibility Analysis of Clinical Isolates of HIV-1 in China

本研究从中国人类免疫缺陷病毒1型(Human immunodeficiency virus-1, HIV-1)感染者血浆样本中分离和构建HIV-1 Envelope(Env)感染性假病毒,并分析其对单克隆广谱中和抗体的敏感性。采用逆转录和巢式PCR方法从感染者样本中扩增HIV-1 env基因,利用同源重组方法将env基因克隆至真核表达载体pVAX1中,包装HIV-1Env假病毒后使用TZM-bl/假病毒中和实验方法检测单克隆广谱中和抗体的中和能力,基于已有抗体的结构数据分析病毒逃逸中和抗体的关键突变位点。本研究从两个HIV-1感染者donor A和donor B中分别获得了8株和3株携带CRF07_BC亚型env基因的感染性假病毒;序列分析显示两组假病毒Env氨基酸同源性仅为83.2%~85.2%,存在一定的差异;11株假病毒对于VRC01和10E8中和抗体依旧敏感,而对于35O22和VRC34.01耐受;donor B来源的假病毒存在N133D和R170E突变而可能影响了PG16抗体的中和能力,donor A来源的假病毒可能由于携带了S291F和E336K突变而逃逸了VRC-PG05抗体,donorA来源的假病毒还可能通过G324E突变进一步影响PGT121中和抗体的活性。本研究成功获得了11株中国HIV-1 CRF07_BC亚型的Env假病毒,评估了临床分离株病毒对已有中和抗体的敏感性,揭示了病毒逃逸中和抗体的关键突变位点,为中国HIV-1感染者应用单克隆中和抗体治疗提供了选择依据,同时也丰富了HIV-1假病毒与中和抗体领域的研究。

We aimed to isolate and construct infectious human immunodeficiency virus(HIV)-1 Envelope(Env)pseudoviruses from HIV-1-infected Chinese donors and evaluate their neutralization susceptibilities.The HIV-1 env gene was amplified by reverse transcription and nested-polymerase chain reaction(PCR),and then cloned into the eukaryotic expression vector pVAX1 by homologous recombination.The neutralization susceptibility of HIV-1 pseudovirus was measured by the TZM-bl/pseudovirus assay.The key mutation site mediating virus escape was analyzed based on available structural information on broadly neutralizing antibodies.Eight and three HIV-1 pseudoviruses carrying the CRF07_BC env gene were obtained from two Chinese individuals(donorA and donorB).Sequencing analysis showed that the amino-acid homology between pseudoviruses from donorA and donorB was 83.2%to 85.2%.These 11 pseudoviruses could be neutralized by VRC01 and 10E8,but escaped neutralization by 35O22 and VRC34.01.N133D and R170E mutations might have impacted the neutralizing activity of PG16 against pseudoviruses derived from donor B.S291F and E336K mutations might have led to donorA pseudoviruses escaping from VRC-PG05.The G324E mutation might have further decreased the neutralizing activity of PGT121 against donorA pseudoviruses.In this study,we identified 11 infectious pseudoviruses carrying the HIV-1 CRF07_BC env gene from two Chinese HIV-1-infected donors and evaluated their neutralization susceptibilities for seven monoclonal broadly neutralizing antibodies.These findings revealed the key mutations mediating evasion of the HIV-1 antibody,provided a reference and guideline for Chinese HIV-1-infected donors receiving monoclonal antibodies,and enriched the research of HIV-1 pseudoviruses and neutralizing antibodies.