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

描述（由申请人提供）：尽管付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项重大挑战。迄今为止的努力在很大程度上忽视了病毒在其表面糖基化方面的一个关键弱点。有了这个应用，我们建议在利用病毒的糖基化脆弱性方面迈出第一步，这是一种不同于当前和早期尝试鉴定单克隆抗体和鉴定并引发广泛中和的多克隆血清的方法。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Env蛋白。病毒防御这种蛋白的一部分是编码大量的糖基化位点，以阻止Env表面的区域被抗体识别。Env保守结构域的糖基化位点的存在可能有所不同，在某些情况下，40%的分离株中缺失糖基化位点。我们假设可以防止感染获得的抗体可以靶向Env表面结构中缺少碳水化合物附着位点的地方。我们将碳水化合物和表面结构分为六个锥体-碳水化合物闭塞中和表位。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这种保守碳水化合物，这表明这些病毒将容易受到针对六种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细胞开发免疫原，这些免疫原可用于产生抗体，在全长Env环境中特异性结合每个锥细胞。考虑到这6个锥体属于相对保守的Env表面区域，但在大多数分离株中至少有1-2个锥体暴露的可能性，我们的策略在组合使用时具有广泛中和效果的潜力。我们将首先设计可显示单个锥体的可溶性蛋白支架（目标1），通过识别低分子量蛋白质支架，显示与给定锥体相似的表面斑块，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，以鉴定与特定锥体结合的天然抗体。这将为在保守区域维持糖基化位点高水平流行的选择压力提供证据（目的2）。然后，我们将根据特定的CONE上是否存在特定的碳水化合物附着位点，测试对CONE模拟物有反应的血清中和假型病毒的能力（目标2）。然后，我们将免疫兔子以培养针对CONE模拟物的多克隆抗体，然后测试其血清的中和特性（目的3）。通过使用假型病毒在更广泛的Env蛋白中检测血清（Aim 4）来确定抗体活性的广度，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及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).