Shotton C, Arnold C, Sattentau Q, Sodroski J, McKeating J A
Institute of Cancer Research, Hybridoma Unit, Sutton, Surrey, London.
J Virol. 1995 Jan;69(1):222-30. doi: 10.1128/JVI.69.1.222-230.1995.
We have identified six monoclonal antibodies (MAbs) mapping to both linear and conformation-dependent epitopes within the V2 region of the human immunodeficiency virus type 1 clone HXB10. Three of the MAbs (12b, 66c, and 66a) were able to neutralize the molecular clones HXB10 and HXB2, with titers in the range of 9.5 to 20.0 micrograms/ml. MAbs mapping to the crown of the V2 loop (12b, 60b, and 74) bound poorly to cell surface-expressed oligomeric gp120, suggesting an explanation for the poor or negligible neutralizing activity of MAbs to this region. In contrast, MAbs 12b and 60b demonstrated good reactivity with recombinant gp120 in an enzyme-linked immunosorbent assay format, suggesting differential epitope exposure between the recombinant and native forms of gp120. Cross-competition analysis of these MAbs and additional V1V2 MAbs for gp120 binding enabled us to assign the MAbs to six groups (A to F). Selection of neutralization escape mutants with MAbs 10/76b and 11/68b, belonging to nonoverlapping competition groups, identified amino acid changes at residues 165 (I to T) and 185 (D to N), respectively. Interestingly, these escape variants remained sensitive to neutralization by the nonselecting V2 MAbs. All MAbs demonstrated good recognition of IIIB viral gp120 yet failed to neutralize nonclonal stocks of IIIB. In addition, MAbs 12b and 62c bound MN and RF viral gp120, respectively, yet failed to neutralize the respective isolates. Cloning and expression of a library of gp120 and V1V2 fragments from IIIB-, MN-, and RF-infected H9 cultures identified a number of polymorphic sites, resulting in antigenic variation and subsequent loss of V2 MAb recognition. In contrast, the V3 region from the clones of the same isolates showed no amino acid changes, suggesting that the V2 region is polymorphic in long-term-passaged laboratory isolates and may account for the reduced antibody recognition observed.
我们已经鉴定出六种单克隆抗体（MAb），它们可定位到人免疫缺陷病毒1型克隆HXB10的V2区域内的线性表位和构象依赖性表位。其中三种单克隆抗体（12b、66c和66a）能够中和分子克隆HXB10和HXB2，效价范围为9.5至20.0微克/毫升。定位到V2环冠部的单克隆抗体（12b、60b和74）与细胞表面表达的寡聚gp120结合较差，这为单克隆抗体对该区域的中和活性较差或可忽略提供了解释。相比之下，单克隆抗体12b和60b在酶联免疫吸附测定中与重组gp120表现出良好的反应性，这表明重组形式和天然形式的gp120之间表位暴露存在差异。对这些单克隆抗体和其他V1V2单克隆抗体与gp120结合进行交叉竞争分析，使我们能够将单克隆抗体分为六组（A至F）。用属于非重叠竞争组的单克隆抗体10/76b和11/68b筛选中和逃逸突变体，分别鉴定出第165位残基（I到T）和第185位残基（D到N）的氨基酸变化。有趣的是，这些逃逸变体对未用于筛选的V2单克隆抗体的中和作用仍敏感。所有单克隆抗体对IIIB病毒gp120均表现出良好的识别，但未能中和IIIB的非克隆毒株。此外，单克隆抗体12b和62c分别与MN和RF病毒gp120结合，但未能中和相应的分离株。对来自感染IIIB、MN和RF的H9培养物的gp120和V1V2片段文库进行克隆和表达，鉴定出许多多态性位点，导致抗原变异以及随后V2单克隆抗体识别的丧失。相比之下，相同分离株克隆的V3区域未显示氨基酸变化，这表明V2区域在长期传代的实验室分离株中具有多态性，可能是观察到抗体识别降低的原因。
