HIV-1膜蛋白抗原表位突变对感染性以及中和特性的影响

Epitope mutagenesis of HIV-1 envelope glycoprotein and its impact on infectivity and neutralization

目的对1株人类免疫缺陷病毒1型(HIV-1)CRF_07BC亚型的膜蛋白进行突变,以暴露保守的中和表位,从而增强其中和活性。方法通过PCR定点突变获得突变克隆。以假病毒系统评价病毒感染力和改造效果。结果获得了FE、FE-2G12、FE-S365A、FE-22L、FE-N197D、FE-N197Q和FE-N301Q突变体,与其改造前的病毒株S939比较,在感染能力方面:FE-S365A增强约45%,而FE-N301Q下降约90%,N197Q失去了感染能力,其他突变体无明显变化;在中和活性方面:FE对2F5敏感性提高11倍以上;FE-N197D对单克隆抗体(单抗)2F5敏感性增强了63倍,对单抗4E10增强了3倍,对单抗b12增强了17倍,而且对5份阳性血浆的中和敏感性增强了2倍以上;FE-N301Q对单抗2F5的敏感性增强了119倍,对单抗4E10和b12的敏感性有一定增强,而且对6份阳性血浆的中和敏感性增强了2倍以上。FE-S365A和FE-F22L对病毒中和活性基本无影响。结论对HIV-1膜蛋白关键位点的改造能够显著增强病毒的中和敏感性。对改造后膜抗原的免疫原性需要进一步评价。

Objective To enhance the immunogenicity of HIV-1 envelope（Env） glycoprotein by exposing the conserved neutralizing epitopes through mutagenesis analysis.Methods HIV-1 Env mutants were prepared by site-directed mutagenesis and the pseudoviruses were produced to compare the viral infectivity and its ability of antibody-mediated neutralization to the wild-type Env.Results Seven mutants including FE,FE-2G12,FE-S365A,FE-F22L,FE-N197D,FE-N197Q and FE-N301Q were constructed from a parental strain S939 and among which,FE-S365A showed approximately 45% increase while FE-N301Q approximately 90% reduction in viral infectivity.There were no detectable infectivity of mutant strain N197Q and no significant changes in viral infectivity were found in other mutants.In regard to the susceptibility of neutralization to different antibodies,mutant FE demonstrated an over 11-fold increase to antibody 2F5;FE-N197D showed 63-fold,3-fold,17-fold and 2-fold increase to antibodies 2F5,4E10,b12 and 5 positive plasma,respectively;FE-N301Q was detected 119-fold and 2-fold increase to antibody 2F5 and six positive plasma,respectively;FE-S365A and FE-F22L didn′t show significant changes in neutralization.Conclusions The mutagenesis of key sites of the HIV-1 Env glycoprotein may significantly increase the susceptibility of neutralization to different monoclonal antibodies and HIV-1 positive plasma.