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

描述(由申请人提供)：尽管做出了巨大的科学努力，但开发有效的艾滋病毒疫苗仍然是一个巨大的挑战。迄今为止的努力在很大程度上忽略了病毒在表面糖基化方面的一个关键脆弱性。通过这项申请，我们建议迈出第一步，利用病毒在糖基化方面的脆弱性，这是一种不同于目前和早期尝试识别单抗和识别和诱导广泛中和多克隆血清的方法。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。病毒防御该蛋白的一部分是编码大量的糖基化位点，以阻止抗体识别Env表面的区域。Env保守结构域中的糖基化位点可能会因它们的存在而不同，在某些情况下，40%的分离株会缺失。我们推测，可以防止感染的抗体可以靶向于缺少碳水化合物结合部位的Env表面结构。我们将碳水化合物和表面结构分成六个锥体-碳水化合物-封闭的中和表位。在对大量传播病毒的调查中，我们发现70%的病毒至少缺少两个保守的碳水化合物，这表明这些病毒将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锥体开发免疫原，这些免疫原可用于产生抗体，这些抗体与全长环境中的每个锥体特异结合。考虑到这六个锥体属于Env的表面区，这些区域相对保守，但在大多数分离物中至少有1-2个锥体暴露的可能性，我们的策略在组合使用时有可能产生广泛的中和效应。我们将首先设计显示单个锥体的可溶性蛋白质支架(目标1)，方法是识别显示与给定锥体相似的表面补片的低分子蛋白质支架，并重新设计支架以显示锥体。然后我们将筛选多克隆人血清，以识别与特定锥体结合的自然产生的抗体。这将为维持保守区域糖基化位点高水平流行的选择压力提供证据(目标2)。然后，我们将根据特定锥体上是否存在特定的碳水化合物附着位点来测试对锥体模拟物起反应的血清中和假型病毒的能力(目标2)。然后，我们将免疫兔子，以提高多克隆抗体的锥体模拟，然后测试它们的血清中和特性(目标3)。通过使用伪型病毒(目标4)测试更广泛的Env蛋白组中的血清来确定抗体活性的广度，将向我们提供潜在的广泛中和抗体。我们将研究糖基化位点的存在与其对病毒适合性的影响之间的关系(目标5)。

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).