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重大挑战。迄今为止的努力在很大程度上忽略了病毒在其表面糖基化方面的关键脆弱性。通过这种应用，我们建议采取第一步，利用病毒的脆弱性，其糖基化，这是一种方法，这是从目前和早期的尝试，以确定单克隆抗体，并确定和引起广泛的中和多克隆血清不同。病毒与宿主免疫系统进行持续的战斗，包括体液抗体反应。中和抗体的唯一靶标是病毒Env蛋白。病毒防御这种蛋白质的一部分是编码大量的糖基化位点，以封闭Env表面区域，使其不被抗体识别。Env保守结构域中的糖基化位点在其存在下可以变化，在某些情况下在40%的分离株中缺失。我们假设可以防止感染的抗体可以靶向Env的表面结构，其中碳水化合物附着位点缺失。我们将碳水化合物和表面结构分为六个锥形-碳水化合物封闭的中和表位。在对大量传播病毒的调查中，我们已经证明，这些病毒中有70%缺失至少两种保守碳水化合物，这表明这些病毒对针对六种CONE的中和组合方法敏感。因此，我们假设有可能为每个CONE开发免疫原，其可用于产生在全长Env的背景下特异性结合每个CONE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具有广泛的中和作用。我们将首先设计可溶性蛋白质支架，显示个别锥（目标1），通过识别低分子量蛋白质支架，显示表面补丁，这是类似于给定的锥和重新设计的支架，以显示锥。然后，我们将筛选多克隆人血清，以确定天然存在的抗体结合到特定的锥。这将为保持保守区域中糖基化位点的高水平流行的选择压力提供证据（目的2）。然后，我们将测试与CONE模拟物反应的血清基于特定CONE处是否存在特定碳水化合物附着位点来中和假型病毒的能力（目的2）。然后，我们将免疫兔子以产生针对CONE模拟物的多克隆抗体，然后测试它们的血清的中和特性（目的3）。通过使用假型病毒在更广泛的Env蛋白组中测试血清来确定抗体活性的宽度（目的4）将告知我们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应性的影响之间的关系（目的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).