Generation and characterization of a human monoclonal antibody that neutralizes diverse HIV-1 isolates in vitro.
Generation and characterization of a human monoclonal antibody that neutralizes diverse HIV-1 isolates in vitro.
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可在体外中和多种 HIV-1 分离株的人单克隆抗体的生成和表征。
DOI：
10.1097/00002030-199201000-00002
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发表时间：
1992
期刊：
影响因子：
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通讯作者：
Hersh,EM
中科院分区：
文献类型：
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作者：
Lake,DF;Kawamura,T;Tomiyama,T;RobinsonJr,WE;Matsumoto,Y;Masuho,Y;Hersh,EM
目的本研究的目的是开发和表征中和 HIV-1 的人单克隆抗体 (HuMAb)。设计基于先前涉及中和其他包膜病毒的 HuMAb 生成的研究，我们认为生成中和 HIV-1 的 HuMAb 是可行的。方法通过将 HIV 阳性患者的脾 B 细胞与小鼠骨髓瘤细胞系融合来生成 HuMAb。使用流式细胞术测定 HuMAb 对 HIV 感染和未感染细胞的表面反应性。采用放射免疫沉淀法来阐明 HuMAb 识别的抗原。使用细胞存活测定来确定 HuMAb 在补体存在或不存在的情况下中和不同 HIV-1 分离株的能力。开发了 gp120-CD4 抑制酶联免疫吸附测定 (ELISA)，以便启动研究以确定 HuMAb 的中和机制。结果产生了一种抗 HIV HuMAb，它可以中和两种不含补体的 HIV-1 分离株（IIIB 和 MN），并在补体存在的情况下中和一种不同分离株 (RF) 和一种临床分离株。通过流式细胞分析发现，这种被命名为 S1-1 的 HuMAb 可以与 HIV-1 感染的细胞表面发生反应，但不会与未感染的细胞发生反应。放射免疫沉淀分析表明，S1-1 与天然 HIV gp120 结合，但不与二硫苏糖醇 (DTT) 处理的 gp120 结合。此外，在蛋白质印迹分析中，HuMAb S1-1 不与变性的 HIV 抗原结合。 HuMAb S1-1 在 ELISA 中有效抑制 gp 120 与可溶性 CD4 的结合。结论这些结果表明，S1-1 识别的表位在不同的 HIV-1 分离株中是构象和保守的，并且可能代表 gp120 上 CD4 结合域内或附近的一个未表征的 HIV 中和域。 HuMAb S1-1 可能在疫苗开发或被动免疫治疗中发挥作用。
ObjectiveThe purpose of this study was to develop and characterize human monoclonal antibodies (HuMAb) that neutralize HIV-1.DesignBased upon previous studies involving the generation of HuMAb that neutralize other enveloped viruses, we thought it feasible to generate HuMAb that might neutralize HIV-1.MethodsA HuMAb was generated by fusing splenic B-cells from an HIV-positive patient with a mouse myeloma cell line. Flow cytometry was used to determine surface reactivity of the HuMAb on HIV-infected and non-infected cells. Radioimmuno-precipitation was employed to elucidate the antigen recognized by the HuMAb. A cell survival assay was used to determine the ability of the HuMAb to neutralize divergent isolates of HIV-1 in the presence or absence of complement. A gp120-CD4 inhibition enzyme-linked immunosorbent assay (ELISA) was developed in order to initiate studies to determine the mechanism of neutralization by the HuMAb.ResultsAn anti-HIV HuMAb was generated that neutralized two HIV-1 isolates (IIIB and MN) without complement and which neutralized one divergent isolate (RF) and one clinical isolate in the presence of complement. This HuMAb, designated S1–1, was found, by flow cytometric analysis, to react with the surface of HIV-1-infected but not with uninfected cells. Radioimmunoprecipitation analysis demonstrated that S1–1 binds to native HIV gp120, but not dithiothreitol (DTT)-treated gp120. In addition, HuMAb S1–1 did not bind to denatured HIV antigens in Western blot analysis. HuMAb S1–1 effectively inhibited the binding of gp 120 to soluble CD4 in ELISA.ConclusionsThese results suggest that the epitope recognized by S1–1 is con-formational and conserved among diverse HIV-1 isolates and may represent an uncharacterized HIV neutralizing domain within or close to the CD4 binding domain on gp120. HuMAb S1–1 might have a role to play in vaccine development or passive immunotherapy.