Highlander S L, Sutherland S L, Gage P J, Johnson D C, Levine M, Glorioso J C
Department of Microbiology, University of Michigan Medical School, Ann Arbor 48109.
J Virol. 1987 Nov;61(11):3356-64. doi: 10.1128/JVI.61.11.3356-3364.1987.
Nine monoclonal antibodies specific for glycoprotein D (gD) of herpes simplex virus type 1 were selected for their ability to neutralize virus in the presence of complement. Four of these antibodies exhibited significant neutralization titers in the absence of complement, suggesting that their epitope specificities are localized to site(s) which contribute to the role of gD in virus infectivity. Each of these antibodies was shown to effectively neutralize virus after virion adsorption to cell surfaces, indicating that neutralization did not involve inhibition of virus attachment. Although some of the monoclonal antibodies partially inhibited adsorption of radiolabeled virions, this effect was only observed at concentrations much higher than that required to neutralize virus and did not correlate with complement-independent virus-neutralizing activity. All of the monoclonal antibodies slowed the rate at which virus entered cells, further suggesting that antibody binding of gD inhibits virus penetration. Experiments were carried out to determine the number of different epitopes recognized by the panel of monoclonal antibodies and to identify epitopes involved in complement-independent virus neutralization. Monoclonal antibody-resistant (mar) mutants were selected by escape from neutralization with individual gD-specific monoclonal antibodies. The reactivity patterns of the mutants and antibodies were then used to construct an operational antigenic map for gD. This analysis identified a minimum of six epitopes on gD that could be grouped into four antigenic sites. Antibodies recognizing four distinct epitopes contained in three antigenic sites were found to neutralize virus in a complement-independent fashion. Moreover, mar mutations in these sites did not affect the processing of gD, rate of virus penetration, or the ability of the virus to replicate at high temperature (39 degrees C). Taken together, these results (i) confirm that gD is a major target antigen for neutralizing antibody, (ii) indicate that the mechanism of neutralization can involve inhibition of virus penetration of the cell surface membrane, and (iii) strongly suggest that gD plays a direct role in the virus entry process.
选择了9种针对单纯疱疹病毒1型糖蛋白D（gD）的单克隆抗体，因其在补体存在下具有中和病毒的能力。其中4种抗体在无补体时表现出显著的中和效价，这表明它们的表位特异性定位于有助于gD在病毒感染性中发挥作用的位点。这些抗体中的每一种在病毒粒子吸附到细胞表面后都能有效中和病毒，表明中和作用不涉及抑制病毒附着。虽然一些单克隆抗体部分抑制了放射性标记病毒粒子的吸附，但这种效应仅在远高于中和病毒所需浓度时才观察到，并且与不依赖补体的病毒中和活性无关。所有单克隆抗体都减缓了病毒进入细胞的速度，进一步表明gD的抗体结合抑制了病毒穿透。进行实验以确定该组单克隆抗体识别的不同表位数量，并鉴定与不依赖补体的病毒中和相关的表位。通过用单个gD特异性单克隆抗体进行中和逃逸筛选出单克隆抗体抗性（mar）突变体。然后利用突变体和抗体的反应模式构建gD的操作抗原图谱。该分析确定了gD上至少6个表位，可分为4个抗原位点。发现识别3个抗原位点中包含的4个不同表位的抗体以不依赖补体的方式中和病毒。此外，这些位点的mar突变不影响gD的加工、病毒穿透速率或病毒在高温（39℃）下的复制能力。综上所述，这些结果（i）证实gD是中和抗体的主要靶抗原，（ii）表明中和机制可能涉及抑制病毒穿透细胞表面膜，（iii）强烈提示gD在病毒进入过程中起直接作用。
