Ditzel H J, Binley J M, Moore J P, Sodroski J, Sullivan N, Sawyer L S, Hendry R M, Yang W P, Barbas C F, Burton D R
Department of Immunology, Scripps Research Institute, La Jolla, CA 92037.
J Immunol. 1995 Jan 15;154(2):893-906.
As part of the goal of assembling a mixture of neutralizing human mAbs for possible prophylaxis and therapy of HIV-1 disease, we describe a strategy by which neutralizing human Abs to a weakly immunogenic epitope can be accessed. From a phage display library derived from an asymptomatic HIV-1 seropositive donor, a panel of recombinant Fabs against the CD4 binding site (CD4bs) of gp120 was retrieved by affinity selection using recombinant gp120 (strain LAI). Two Fabs corresponding to the dominant clones were used to mask the CD4bs epitope(s) before repeating the selection procedure. Four Fabs were then retrieved that had novel heavy chain sequences. Three recognized a novel epitope distinct from that recognized by conventional CD4bs Abs and were defined by the following criteria: 1) second V region (V2 region) dependence indicated by sensitivity to amino acid changes in the V2 loop and by competition with murine anti-V2 mAbs; 2) CD4bs dependence indicated by sensitivity to amino acid changes usually associated with CD4 binding and by inhibition of Fab binding to gp120 by soluble CD4; this dependence seemed to arise via conformational changes rather than by direct binding, as CD4bs Abs enhanced binding of two of the novel Fabs and, in a reversal of the competition format, the novel Fabs did not inhibit soluble CD4 binding to gp120; and 3) equivalent binding to glycosylated and deglycosylated gp120 and significant, although much reduced, binding to denatured gp120 in contrast with CD4bs Abs, which do not bind to deglycosylated or denatured gp120. One of the novel Fabs efficiently neutralized the MN and LAI strains of HIV-1. These results indicate the presence of a novel neutralizing conformational epitope on gp120 sensitive to the V2 loop and the CD4bs and further highlight the conformational flexibility of gp120. The strategy of masking highly immunogenic epitopes with Abs to rescue a broader range of specific Abs from combinatorial libraries should be widely applicable.
作为组装用于HIV-1疾病预防和治疗的中和性人源单克隆抗体混合物目标的一部分，我们描述了一种可获取针对弱免疫原性表位的中和性人源抗体的策略。从一名无症状HIV-1血清阳性供体来源的噬菌体展示文库中，通过使用重组gp120（LAI株）进行亲和选择，获得了一组针对gp120 CD4结合位点（CD4bs）的重组Fab片段。在重复选择过程之前，使用对应于优势克隆的两个Fab片段来掩盖CD4bs表位。然后获得了四个具有新重链序列的Fab片段。其中三个识别出一个不同于传统CD4bs抗体所识别表位的新表位，并由以下标准定义：1）对V2环中氨基酸变化的敏感性以及与鼠抗V2单克隆抗体的竞争所表明的对第二V区（V2区）的依赖性；2）对通常与CD4结合相关的氨基酸变化的敏感性以及可溶性CD4对Fab与gp120结合的抑制所表明的对CD4bs的依赖性；这种依赖性似乎是通过构象变化而非直接结合产生的，因为CD4bs抗体增强了两个新Fab片段的结合，并且在竞争形式的逆转中，新Fab片段不抑制可溶性CD4与gp120的结合；3）与糖基化和去糖基化gp120的等效结合以及与变性gp120的显著结合（尽管大幅降低），与不与去糖基化或变性gp120结合的CD4bs抗体形成对比。其中一个新Fab片段有效地中和了HIV-1的MN和LAI株。这些结果表明在gp120上存在一个对V2环和CD4bs敏感的新的中和性构象表位，并进一步突出了gp120的构象灵活性。用抗体掩盖高免疫原性表位以从组合文库中拯救更广泛特异性抗体的策略应具有广泛的适用性。
