Dickey C, Ziegner U, Agadjanyan M G, Srikantan V, Refaeli Y, Prabhu A, Sato A, Williams W V, Weiner D B, Ugen K E
Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, Tampa, Florida, USA.
DNA Cell Biol. 2000 Apr;19(4):243-52. doi: 10.1089/104454900314519.
The human immunodeficiency virus (HIV)-1 envelope glycoprotein is synthesized as a precursor (gp160) and subsequently cleaved to generate the external gp120 and transmembrane gp41 glycoproteins. Both gp120 and gp41 have been demonstrated to mediate critical functions of HIV, including viral attachment and fusion with the cell membrane. The antigenic variability of the HIV-1 envelope glycoprotein has presented a significant problem in the design of appropriate and successful vaccines and offers one explanation for the ability of HIV to evade immune surveillance. Therefore, the development and characterization of functional antibodies against conserved regions of the envelope glycoprotein is needed. Because of this need, we generated a panel of murine monoclonal antibodies (MuMabs) against the HIV-1 envelope glycoprotein. To accomplish this, we immunized Balb/C mice with a recombinant glycoprotein 160 (gp160) that was synthesized in a baculovirus expression system. From the growth-positive hybridomas, three MuMabs were generated that demonstrated significant reactivity with recombinant gp120 but failed to show reactivity against HIV-1 gp41, as determined by enzyme-linked immunosorbent assay (ELISA). Using vaccinia constructs that synthesize variant truncated subunits of gp160, we were able to map reactivity of all three of the Mabs (ID6, AC4, and AD3) to the first 204 residues of gp120 (i.e., the N terminus of gp120) via Western blot analysis. Elucidation of the epitopes for these Mabs may have important implications for inhibition of infection by HIV-1. Our initial attempts to map these Mabs with linear epitopes have not elucidated a specific antigenic determinant; however, several physical characteristics have been determined that suggest a continuous surface epitope. Although these antibodies failed to neutralize cell-free or cell-associated infection by HIV-1, they did mediate significant antibody-dependent cellular cytotoxicity (ADCC) activity, indicating potential therapeutic utility. In summary, these data suggest the identification of a potentially novel site in the first 200 aa of gp120 that mediates ADCC.
人类免疫缺陷病毒1型（HIV-1）包膜糖蛋白以前体形式（gp160）合成，随后被切割产生外膜gp120和跨膜gp41糖蛋白。gp120和gp41均已被证明介导HIV的关键功能，包括病毒附着以及与细胞膜融合。HIV-1包膜糖蛋白的抗原变异性给设计合适且成功的疫苗带来了重大问题，这也为HIV逃避免疫监视的能力提供了一种解释。因此，需要开发并鉴定针对包膜糖蛋白保守区域的功能性抗体。基于这一需求，我们制备了一组针对HIV-1包膜糖蛋白的鼠单克隆抗体（MuMabs）。为此，我们用在杆状病毒表达系统中合成的重组糖蛋白160（gp160）免疫Balb/C小鼠。从生长阳性杂交瘤中产生了三种MuMabs，通过酶联免疫吸附测定（ELISA）确定，它们与重组gp120表现出显著反应性，但对HIV-1 gp41无反应性。使用合成gp160变异截短亚基的痘苗构建体，我们能够通过蛋白质印迹分析将所有三种单克隆抗体（ID6、AC4和AD3）的反应性定位到gp120的前204个残基（即gp120的N端）。阐明这些单克隆抗体的表位可能对抑制HIV-1感染具有重要意义。我们最初用线性表位定位这些单克隆抗体的尝试尚未阐明特定的抗原决定簇；然而，已经确定了一些物理特征，提示存在连续的表面表位。尽管这些抗体未能中和HIV-1的游离病毒或细胞相关感染，但它们确实介导了显著的抗体依赖性细胞毒性（ADCC）活性，表明具有潜在的治疗效用。总之，这些数据表明在gp120的前200个氨基酸中鉴定出了一个潜在的新位点，该位点介导ADCC。
