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Analysis of the interaction between chemokine receptor and V3 region of HIV-gp120 by neutralizing antibodies

中和抗体分析趋化因子受体与HIV-gp120 V3区的相互作用

基本信息

批准号：
10670418
负责人：
MATSUSHITA Shuzo
金额：
$ 2.11万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670418/
关键词：
neutralizing antibody anti-anti-idiotype HIV chemokine receptor

项目摘要

The V3 region of an external envelope protein gp120 of HIV plays a crucial role in its chemokine receptor usage as a coreceptor. We have succeed in inducing an anti-idiotypic antibodies in rabbits by immunizing them with three monoclonal antibodies (mAbs) to V3 region. We titrated the anti-idiotype antibodies by an assay measuring the inhibitory activity in the binding of the biotinylated mAb to its epitope peptide. Anti-idiotype activity was most prominent in the combination of certain mAb and its anti-mAb anti-idiotype. However, cross-reactivity of anti-idiotypic antibody to the other mAb was observed to some extent. Anti-idiotype IgG was purified by immunoaffinity chromatography and the purity of anti-idiotype was more than 90%. We then analyzed the effect of these mAbs and anti-idiotypes in the interaction of chemokine and chemokine receptor (CCR5, CXCR4). We used 125I labeled RANTES, MIPα, and SDF1. However, no inhibitory activity of the chemokine binding to CCR5 transfected CEM cells (CEM/CCR5) was observed for these mAbs and their anti-idiotypes. We attempted to induce anti-anti-idiotype to induce neutralizing antibodies in mice. Although we immunized mice in the same titer of purified anti-idiotypes strung neutralizing activity was observed for the anti-anti-RC25 as compared to the other two. We have been trying to get an anti-idiotypic mAbs. However we have not succeeded to establish clone(s) that have activity comparable to the rabbit anti-idiotypic antiserum. Because high activity anti-idiotypic mAb is essential for further elucidating the utility of the anti-idiotype in vaccine development and immunotherapy we are continuing an effort to get such mAb producing cells.

HIV外膜蛋白gp 120的V3区在其作为辅助受体的趋化因子受体使用中起着至关重要的作用。我们用三种抗V3区单克隆抗体免疫家兔，成功地诱导出抗独特型抗体。我们通过测定生物素化mAb与其表位肽结合的抑制活性来滴定抗独特型抗体。抗独特型活性在某些mAb及其抗mAb抗独特型组合中最突出。然而，抗独特型抗体与另一种mAb在一定程度上观察到交叉反应性。用免疫亲和层析法纯化抗独特型IgG，纯度大于90%。然后我们分析了这些单克隆抗体和抗独特型在趋化因子和趋化因子受体（CCR 5，CXCR 4）相互作用中的作用。我们使用125 I标记的RANTES、MIPα和SDF 1。然而，对于这些mAb及其抗独特型，未观察到趋化因子与CCR 5转染的CEM细胞（CEM/CCR 5）结合的抑制活性。我们尝试诱导抗-抗-独特型以在小鼠中诱导中和抗体。尽管我们以相同滴度的纯化抗独特型抗体免疫小鼠，但与其他两种抗体相比，观察到抗-抗-RC 25的中和活性。我们一直在尝试获得抗独特型单克隆抗体。然而，我们还没有成功地建立具有与兔抗独特型抗血清相当的活性的克隆。因为高活性抗独特型mAb对于进一步阐明抗独特型在疫苗开发和免疫治疗中的效用是必不可少的，所以我们正在继续努力获得这样的mAb产生细胞。