Immunogen Design to Target Carbohydrate-Occluded Epitopes on the HIV envelope

针对 HIV 包膜上碳水化合物封闭表位的免疫原设计

• 批准号: 8681357
• 负责人: NIKOLAY DOKHOLYAN
• 金额: $ 50.32万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8681357
• 关键词: Accounting; Animals; Antibodies; Antibody Formation; Antigens; Binding; Biological Assay; Carbohydrates; Clinical; Development; Engineering; Enzyme-Linked Immunosorbent Assay; Epitopes; Growth; HIV; HIV Antibodies; HIV vaccine; HIV-1; Human; Immune system; Immunization; Individual; Infection; Length; Measures; Methodology; Molecular Weight; Monoclonal Antibodies; Mutation; Oryctolagus cuniculus; Prevalence; Property; Protein Engineering; Proteins; Regimen; Relative (related person); Research; Scaffolding Protein; Serum; Site; Structure; Surface; Surveys; Testing; Vaccination; Vaccines; Viral; Virus; base; combinatorial; cost; design; env Gene Products; fitness; glycosylation; immunogenic; mimetics; neutralizing antibody; neutralizing monoclonal antibodies; novel; polyclonal antibody; pressure; prevent; scaffold; screening; stability testing; vaccine development

DESCRIPTION (provided by applicant): Despite enormous scientific efforts, development of an effective vaccine for HIV remains a significant challenge. Efforts to date have largely overlooked a key vulnerability of the virus with respect to its surface glycosylation. With this application we propose to take the first step in exploiting the vulnerability of the virus with respect to its glycosylation, which is an approach that is distinct from the current and earlier attempts to identify monoclonal antibodies and to identify and elicit broadly neutralizing polyclonal sera. The virus carries on a continuous battle with the host immune system, including the humoral antibody response. The sole target of neutralizing antibodies is the viral Env protein. One part of the viruses' defense of this protein is encoding a large number of glycosylation sites to occlude regions of Env surface from recognition by antibodies. The sites for glycosylation in the conserved domains of Env can vary in their presence, in some cases missing in 40% of isolates. We hypothesize that antibodies that can prevent the acquisition of infection can be targeted to surface structures of Env where a carbohydrate attachment site is missing. We have grouped the carbohydrates and surface structures into six CONEs - Carbohydrate-Occluded Neutralization Epitopes. In a survey of a large number of transmitted viruses we have documented that 70% of these viruses are missing at least two of these conserved carbohydrates, which indicates that these viruses will be susceptible to a combinatorial approach of neutralization directed at the six CONEs. Thus, we hypothesize that it is possible is to develop immunogens for each CONE that can be used to generate antibodies that bind specifically to each of the CONEs in the context full-length Env. Given that these six CONEs belong to surface regions of Env that are relatively conserved but with the possibility of exposure of at least 1-2 CONEs in most isolates, our strategy has the potential when used combinatorially to have a broad neutralization effect. We will first design soluble protein scaffolds that display the individual CONEs (Aim 1) by identifying low molecular weight protein scaffolds that display a surface patch that is similar to the given CONE and redesigning the scaffolds to display the CONEs. We will then screen polyclonal human sera to identify naturally occurring antibodies that bind to specific CONEs. which will provide evidence for the selective pressure that maintains the high level of prevalence of the glycosylation sites in the conserved regions (Aim 2). We will then test sera that react to the CONE mimetics for their ability to neutralize pseudotyped viruses based on the presence or absence of specific carbohydrate attachment sites at the specific CONE (Aim 2). We will then immunize rabbits to raise polyclonal antibodies to CONE mimetics and then test their sera for neutralization properties (Aim 3). Determining the breadth of antibody activity by testing the sera in a broader panel of Env proteins using pseudotyped viruses (Aim 4) will inform us of potential broadly neutralizing antibody. We will examine the relationship between the presence of a glycosylation site and its impact on viral fitness (Aim 5).

描述（由申请人提供）：尽管付出了巨大的科学努力，开发有效的艾滋病毒疫苗仍然是一个重大挑战。迄今为止的努力在很大程度上忽视了病毒在表面糖基化方面的一个关键脆弱性。通过这一应用，我们建议迈出第一步，利用病毒在糖基化方面的脆弱性，这是一种不同于当前和早期识别单克隆抗体以及识别和引发广泛中和多克隆血清的尝试的方法。该病毒与宿主免疫系统进行持续的战斗，包括体液抗体反应。中和抗体的唯一目标是病毒包膜蛋白。病毒对该蛋白的防御之一是编码大量糖基化位点，以阻止 Env 表面区域被抗体识别。 Env 保守结构域中的糖基化位点的存在情况可能有所不同，在某些情况下，40% 的分离株缺失。我们假设可以防止获得感染的抗体可以靶向缺少碳水化合物附着位点的 Env 表面结构。我们将碳水化合物和表面结构分为六个锥体 - 碳水化合物封闭的中和表位。在对大量传播病毒的调查中，我们记录了 70% 的病毒至少缺少两种保守的碳水化合物，这表明这些病毒容易受到针对六个锥体的组合中和方法的影响。因此，我们假设有可能为每个 CONE 开发免疫原，该免疫原可用于生成与全长 Env 上下文中的每个 CONE 特异性结合的抗体。鉴于这 6 个 CONE 属于相对保守的 Env 表面区域，但在大多数分离株中可能暴露至少 1-2 个 CONE，因此我们的策略在组合使用时有可能产生广泛的中和效果。我们将首先设计显示单个 CONE 的可溶性蛋白质支架（目标 1），方法是识别显示与给定 CONE 相似的表面斑块的低分子量蛋白质支架，并重新设计支架以显示 CONE。然后，我们将筛选多克隆人血清，以鉴定与特定 CONE 结合的天然抗体。这将为维持保守区域糖基化位点的高流行水平的选择压力提供证据（目标 2）。然后，我们将根据特定 CONE 上是否存在特定碳水化合物附着位点，测试对 CONE 模拟物起反应的血清中和假型病毒的能力（目标 2）。然后，我们将对兔子进行免疫接种，以产生针对 CONE 模拟物的多克隆抗体，然后测试其血清的中和特性（目标 3）。通过使用假型病毒测试更广泛的 Env 蛋白组中的血清来确定抗体活性的广度（目标 4）将告诉我们潜在的广泛中和抗体。我们将研究糖基化位点的存在及其对病毒适应性的影响之间的关系（目标 5）。