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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 mutate the membrane protein of a human immunodeficiency virus type 1 (HIV-1) CRF_07BC subtype to expose conserved neutralizing epitopes and thereby enhance their neutralizing activity. Methods Mutated clones were obtained by PCR site-directed mutagenesis. The pseudoviruses were used to evaluate the virus infectivity and the effect of transformation. Results The FE, FE-2G12, FE-S365A, FE-22L, FE-N197D, FE-N197Q and FE-N301Q mutants were obtained and compared to their preimproved strain S939 in terms of infectivity: FE- 45%, while the FE-N301Q decreased about 90%, N197Q lost the ability to infect other mutants without significant changes in the neutralization activity: FE 2F5 sensitivity increased by 11 times; FE-N197D monoclonal antibody Anti-2F5 was 63-fold more sensitive, 3-fold more resistant to MAb 4E10, 17-fold more MAb b12, and more than twice as sensitive to 5 positive plasmids; FE-N301Q vs. The sensitivity of anti-2F5 increased 119-fold, the sensitivities to MAb 4E10 and b12 were enhanced, and the neutralization sensitivity to 6 positive plasma increased more than 2 folds. FE-S365A and FE-F22L had little effect on virus neutralization activity. Conclusion The modification of key sites of HIV-1 membrane protein can significantly enhance the neutralization sensitivity of the virus. The immunogenicity of the modified membrane antigen needs further evaluation.