HIV-1流行株囊膜假病毒系统的构建及广谱中和抗体中和效价的测定

Construction of HIV-1 Env-pseudotyped Virus System and Evaluation of Broadly Neutralizing Antibody Titers

人类免疫缺陷病毒主要感染人CD4+T细胞与单核-巨噬细胞,造成高致命性的免疫系统衰竭。近年来,抗HIV-1的广谱中和抗体被用于HIV-1感染的阻断和临床免疫治疗效果显著。然而,针对HIV-1抗体中和活性评价假病毒谱系仍有待补充,本研究旨在构建一种易于量化,生物安全性高的HIV-1流行毒株假病毒谱系,用于定量HIV-1中和抗体抗病毒活力。研究从10种亚型中选取了24株代表性HIV-1国内外流行毒株,构建囊膜表达质粒并与囊膜缺失骨架质粒pNL4-3-copGFP-ΔENV共转染制备慢病毒。通过定量p24抗原和测定TCID50分别获得病毒物理和活性滴度。以本实验室表达、纯化的HIV广谱中和抗体VRC01、3BNC117和N6经多梯度稀释后,分别与200 TCID50的假病毒孵育后,感染敏感指示细胞TZM-bl。随后通过测定荧光素酶活性定量假病毒的感染水平并计算出抗体的中和活性。结果表明:三种广谱中和抗体均能中和24种假病毒并计算获得了IC50。本研究成功建立了24种假病毒,可为HIV的抗体筛选功能验证和疫苗免疫效果提供中和活性评价平台。

HIV-1 primarily infects CD4~+T cells,monocyte and macrophages,leading to a damaged immune system.Recently,a broad spectrum of neutralizing HIV-1 antibodies has been used to block HIV-1 entry and to offer clinical immunotherapy.However,many novel pseudoviruses for evaluating the neutralizing antibodies or vaccine against HIV-1 are currently desired.In this study,a panel of HIV-1 pseudoviruses including 24 viral strains derived from China and other countries,covering the majority of contemporaneous subtypes and circulating recombinant forms were prepared by co-transfecting 293T cells with the backbone plasmid(pNL4-3-CopGFP-ΔENV)and envelope expression plasmid.Pseudovirus stocks were characterized for p24 antigen concentration by antigen enzyme-linked immunosorbent assay and for infectious titer on TZM-bl and C8166cells,respectively.HIV-1 broadly neutralizing antibodies VRC01,3BNC117 and N6 which were expressed and purified in our laboratory were serially-diluted and incubated with pseudovirus of 200 TCID_(50)to infect HIV-1reporter cell line TZM-bl.Neutralization antibody activity was measured using a TZM-bl cell based assay employing pseudoviruses and a luciferase reporter gene readout.These results indicated that a panel of 24pseudoviruses prepared in our lab could be neutralized by all 3 broadly neutralizing antibodies,and half-maximal inhibitory concentrations(IC50 values)for each antibody-pseudovirus pair were calculated.Altogether,these pseudoviruses have been successfully prepared and will be applied in drug screening,neutralizing antibody activity detection,and the evaluation of vaccine effects.