Edinger A L, Ahuja M, Sung T, Baxter K C, Haggarty B, Doms R W, Hoxie J A
Department of Pathology and Laboratory Medicine, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Virol. 2000 Sep;74(17):7922-35. doi: 10.1128/jvi.74.17.7922-7935.2000.
In an attempt to generate broadly cross-reactive, neutralizing monoclonal antibodies (MAbs) to simian immunodeficiency virus (SIV), we compared two immunization protocols using different preparations of oligomeric SIV envelope (Env) glycoproteins. In the first protocol, mice were immunized with soluble gp140 (sgp140) from CP-MAC, a laboratory-adapted variant of SIVmacBK28. Hybridomas were screened by enzyme-linked immunosorbent assay, and a panel of 65 MAbs that recognized epitopes throughout the Env protein was generated. In general, these MAbs detected Env by Western blotting, were at least weakly positive in fluorescence-activated cell sorting (FACS) analysis of Env-expressing cells, and preferentially recognized monomeric Env protein. A subset of these antibodies directed toward the V1/V2 loop, the V3 loop, or nonlinear epitopes were capable of neutralizing CP-MAC, a closely related isolate (SIVmac1A11), and/or two more divergent strains (SIVsmDeltaB670 CL3 and SIVsm543-3E). In the second protocol, mice were immunized with unfixed CP-MAC-infected cells and MAbs were screened for the ability to inhibit cell-cell fusion. In contrast to MAbs generated against sgp140, the seven MAbs produced using this protocol did not react with Env by Western blotting and were strongly positive by FACS analysis, and several reacted preferentially with oligomeric Env. All seven MAbs potently neutralized SIVmac1A11, and several neutralized SIVsmDeltaB670 CL3 and/or SIVsm543-3E. MAbs that inhibited gp120 binding to CD4, CCR5, or both were identified in both groups. MAbs to the V3 loop and one MAb reactive with the V1/V2 loop interfered with CCR5 binding, indicating that these regions of Env play similar roles for SIV and human immunodeficiency virus. Remarkably, several of the MAbs generated against infected cells blocked CCR5 binding in a V3-independent manner, suggesting that they may recognize a region analogous to the conserved coreceptor binding site in gp120. Finally, all neutralizing MAbs blocked infection through the alternate coreceptor STRL33 much more efficiently than infection through CCR5, a finding that has important implications for SIV neutralization assays using CCR5-negative human T-cell lines.
为了产生对猴免疫缺陷病毒（SIV）具有广泛交叉反应性的中和单克隆抗体（MAb），我们比较了两种使用不同制备的寡聚SIV包膜（Env）糖蛋白的免疫方案。在第一个方案中，用来自CP-MAC（SIVmacBK28的实验室适应变体）的可溶性gp140（sgp140）免疫小鼠。通过酶联免疫吸附测定筛选杂交瘤，并产生了一组65种识别整个Env蛋白表位的MAb。一般来说，这些MAb通过蛋白质印迹法检测Env，在表达Env的细胞的荧光激活细胞分选（FACS）分析中至少呈弱阳性，并且优先识别单体Env蛋白。这些针对V1/V2环、V3环或非线性表位的抗体亚组能够中和CP-MAC、一种密切相关的分离株（SIVmac1A11）和/或另外两种差异更大的毒株（SIVsmDeltaB670 CL3和SIVsm543-3E）。在第二个方案中，用未固定的CP-MAC感染细胞免疫小鼠，并筛选MAb抑制细胞间融合的能力。与针对sgp140产生的MAb不同，使用该方案产生的七种MAb通过蛋白质印迹法不与Env反应，而通过FACS分析呈强阳性，并且有几种优先与寡聚Env反应。所有七种MAb都能有效中和SIVmac1A11，并且有几种能中和SIVsmDeltaB670 CL3和/或SIVsm543-3E。在两组中都鉴定出了抑制gp120与CD4、CCR5或两者结合的MAb。针对V3环的MAb和一种与V1/V2环反应的MAb干扰了CCR5结合，表明Env的这些区域对SIV和人类免疫缺陷病毒起类似作用。值得注意的是，针对感染细胞产生的几种MAb以不依赖V3的方式阻断CCR5结合，表明它们可能识别与gp120中保守的共受体结合位点类似的区域。最后，所有中和MAb阻断通过替代共受体STRL33的感染比通过CCR5的感染更有效，这一发现对使用CCR5阴性人T细胞系的SIV中和试验具有重要意义。
