Cross-& Intra-Clade Conserved V3 Neutralization Targets

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• 批准号: 6495921
• 负责人: SAMUEL C KAYMAN
• 金额: $ 23.34万
• 依托单位: PUBLIC HEALTH RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-15 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6495921
• 关键词: AIDS education /prevention; affinity chromatography; antiAIDS agent; antiviral antibody; chemical structure function; chimeric proteins; enzyme linked immunosorbent assay; epitope mapping; genetic strain; genetically modified animals; host organism interaction; human immunodeficiency virus 1; human tissue; immunologic substance development /preparation; intermolecular interaction; laboratory mouse; microorganism immunology; neutralizing antibody; vaccine development; virus classification; virus infection mechanism; virus protein

DESCRIPTION: (Provided by Applicant) A protective vaccine against HIV-1 is widely believed to require, in part, induction of a potent neutralizing humoral response, but current vaccine candidates do not approach this goal, nor are the epitopes that might mediate such neutralization known. Although the sequence diversity in the V3 loop is generally believed to make this domain unsuitable as a vaccine target, our recent results suggest that there are conserved epitopes within V3 that mediate potent cross-neutralization of primary viruses within and across clades. These results were obtained using a novel V3 antigen, produced in mammalian cells as a fusion protein, that is properly glycosylated and at least ten times more reactive with HIV-1+ human patient sera than are synthetic peptides. Fusion proteins expressing clade B and clade A V3 domains have been used to isolate polyclonal V3-reactive antibody fractions from human sera from patients infected with clade B or clade A viruses and to screen new V3-reactive human monoclonal antibodies (mabs) with a number of different patterns of cross-neutralization activity against primary isolates. This proposal seeks to further characterize such polyclonal and monoclonal V3-reactive antibodies in order to better define the epitopes that mediate cross-neutralization. Polyclonal V3-reactive neutralizing antibodies will be analyzed by determining the ability of various antigens to adsorb out the neutralizing activity. These antigens will include synthetic peptides, de-glycosylated V3 fusion proteins, fusion proteins expressing linearized V3 domains, and fusion proteins expressing V3 domains with alanine substitutions at different locations. V3-reactive mabs will be characterized by ELISA against these antigens, against soluble gp120, and against Env complexes on the viral surface. We will attempt to exploit our finding that neutralization sensitivity to V3-directed antibodies is regulated by the Vl/V2 domain to generate chimeric Envs suitable for extending studies on the breadth of cross-neutralization mediated by polyclonal V3-reactive antibody fractions in which the concentration of relevant antibodies may be low. We will also isolate and characterize new V3-reactive mabs using transgenic mice that express fully human lgGs (XenoMouse, Abgenix). We will also determine whether isolated V3 domains expressed on fusion proteins and carrying a synthetic TH epitope are able to induce high titer neutralizing antibodies against primary viruses. These studies will increase our understanding of conserved neutralizing epitopes in V3, and may lead directly to a vaccine candidate for further development.

描述：（由申请人提供）针对HIV-1的保护疫苗是 人们普遍认为需要部分诱导有效的中和体液 回应，但目前的候选疫苗没有实现此目标，也不是 可能介导这种中和已知的表位。虽然序列 通常认为V3环中的多样性使该领域不合适 作为疫苗目标，我们最近的结果表明有保守 V3中的表位介导了原代病毒的有效跨境化 内部和整个进化枝。这些结果是使用新型V3抗原获得的 在哺乳动物细胞中作为融合蛋白产生，已适当糖基化 至少用HIV-1+人类患者血清反应性的十倍 合成肽。表达进化枝B和进化枝A V3结构域的融合蛋白 已用于分离人类的多克隆V3反应抗体级分 感染了进化枝B或进化枝A病毒的患者的血清并筛查新的 V3反应性人类单克隆抗体（mAb）具有多种不同的 针对原代分离株的跨中性化活性的模式。这 提案旨在进一步表征这种多克隆和单克隆 V3反应性抗体，以更好地定义介导的表位 交叉中和化。多克隆V3反应性中和抗体将是 通过确定各种抗原吸附的能力来分析 中和活性。这些抗原将包括合成肽， 去糖基化的V3融合蛋白，表达线性化V3的融合蛋白 域和融合蛋白表达具有丙氨酸取代的V3结构域 在不同的位置。 V3反应性mab将以ELISA为特征 这些抗原，针对可溶性GP120，并针对病毒上的Env复合物。 表面。我们将尝试利用中和敏感性的发现 v3定向抗体受VL/V2结构域调节以生成嵌合 ENV适用于扩展有关跨中和的广度研究 由多克隆V3反应抗体介导的，其中 相关抗体的浓度可能很低。我们还将隔离和 使用完全表达充分表达的转基因小鼠表征新的V3反应性mAb 人类LGGS（Xenomouse，abgenix）。我们还将确定孤立的V3是否 在融合蛋白上表达并携带合成表位的域是 能够诱导对原发病毒的抗体中和抗体的高滴度。 这些研究将增加我们对保守中和的理解 V3中的表位，可能直接导致候选疫苗 发展。