Induction of neutralizing antibodies targeting CD4 binding region of HIV-1 Env

描述(由申请人提供)：这个多项目HIVRAD计划应用的总体目标是诱导针对主要HIV-1包膜(Env)糖蛋白的CD4结合位点(CD4bs)的中和抗体。这份P01计划提案由三个重大项目组成，外加两个支持重大项目活动的核心。以下是该计划不同项目/核心中提议的主要活动的摘要。目标1：组织和管理一个高度互动和富有成效的研究团队(核心B)。目标2：了解HIV-1R5 env基因变异如何影响趋向性、中和性和疫苗开发(项目1)。HIV-1R5囊膜感染巨噬细胞的能力差异很大。我们建议研究巨噬细胞嗜性的变化对与Env相关的其他生物学特性的影响，包括中和敏感性。目标3：研究CD4b抗原性的变化如何影响原代Env蛋白的中和敏感性和免疫原性(项目2)。单抗将检测几组初级包膜病毒的CD4b抗原性，每组包膜病毒都有自己独特的生物学特性。我们将使用DMA Prime-Protein Boost免疫方法研究高CD4b抗原性和对CD4bs单抗介导的中和高敏感性是否会导致关键代表病毒的高免疫原性。目标4：研究受体结合位点的修饰作为HIV-1疫苗设计的一种方法(项目3)。我们将测试特定的多糖修饰引起的变化是否会导致受体结合部位保守表位的稳定性或可及性增加，以及这些保守表位的可及性是否会增强它们作为免疫原的功能，以引发交叉反应的NAB反应。目标5：为环境病毒结构分析和研究新抗原的结构以及抗原-抗体相互作用的主要项目提供支助(核心A)。 项目1：HIV-1R5环境变异：趋向性、中和性和疫苗的后果 (查普汉姆，P) 项目1描述(由申请人提供)：HIV-1R5病毒在表型上有很大的差异，包括巨噬细胞嗜性和对中和抗体的敏感性。因此，高度嗜Mac包膜(Env)利用低数量的CD4和/或CCR5进行感染，与需要高CD4水平和低效感染巨噬细胞的非Mac嗜性R5包膜形成对比。高度嗜Mac的env使用低的CD4/CCR5的能力表明，这种变体可能在其他表达低受体水平的CD4细胞类型中提供更广泛的趋向性，并在病毒传播过程中具有优势。我们最近使用中和单抗(MAbs)和进入抑制剂的数据有力地表明，高度嗜Mac的R5env携带更多暴露的CD4结合位点(CD4bs)，可能容易受到疫苗诱导的中和抗体(NAb)的影响。我们建议在以下四个方面广泛研究不同的R5env的趋向性和中和敏感性：目的1.HIV-1R5巨噬细胞趋向性在不同CD4细胞群感染中的作用。我们将检查高度嗜Mac的R5包膜是否在CD4T细胞群体中赋予更广泛的取向。目的2.进一步分析巨噬细胞感染的包膜决定因素，以及在B分支和非B分支包膜中使用低CD4的情况。我们将分析B分支和非B分支环境，以确定趋向性的决定因素，并进一步研究CD4结合环的作用。目的3.研究巨噬细胞趋化中R5env变异对中和抗体敏感性的影响。我们将研究R5 env变异如何影响对HIV-1人血清、中和单抗和疫苗诱导的中和抗体的敏感性。目的4.细菌产生的、非糖基化的模拟CD4b的蛋白质结构的开发和特性。我们设计并生产了CD4bs模拟物，这种模拟物携带CD4bs的关键成分，是一种可以在细菌中产生的非糖基化多肽。这些CD4bs模拟结合CD4和B12。将研究它们的结构和诱导中和抗体的能力。我们期望对以下方面提供重要的见解：(1)R5env的趋向性和中和敏感性的变化，(2)与不同的趋向性和中和敏感性有关的env氨基酸和结构决定因素，以及(3)这种变化对新型env疫苗设计的影响。

DESCRIPTION (provided by applicant): The overall objective of this multi-project HIVRAD program application is to elicit neutralizing antibodies that target the CD4 binding site (CD4bs) of primary HIV-1 envelope (Env) glycoproteins. This P01 program proposal consists of three major projects, plus two cores to support the activities of major projects. The following is a summary of major activities as proposed in different Projects/Cores of this program. Goal 1: To organize and manage a highly interactive and productive research team (Core B). Goal 2: To understand how the variation in HIV-1 R5 Envs affect tropism, neutralization and vaccine development (Project 1). HIV-1 R5 envelopes vary extensively in their capacity to infect macrophages. We propose to investigate the impact of variation in macrophage tropism (mac-tropism) on other biological properties associated with Env including neutralization sensitivity. Goal 3: To study how the variation of antigenicity of CD4bs will affect the neutralization sensitivity and immunogenicity of primary Env proteins (Project 2). The CD4bs antigenicity of several panels of primary Envs, each with their own unique biological features, will be probed by mAbs. We will examine whether high CD4bs antigenicity and high sensitivity to CD4bs mAb mediated neutralization will lead to high immunogenicity for key representative Env using the DMA prime-protein boost immunization approach. Goal 4: To study the modification of receptor binding site as an approach to HIV-1 vaccine design (Project 3). We will test whether changes resulting from specific glycan modifications will lead to increased stability or accessibility of conserved epitopes in the receptor binding site and whether greater accessibility of these conserved sites will enhance their function as immunogen to elicit cross-reactive NAb responses. Goal 5: To provide support to major projects on structure analysis of Env and to study the structure of novel antigens, and antigen-antibody interactions (Core A). PROJECT 1: Variation in HIV-1 R5 Envs: Consequences for tropism, neutralization and vaccines (Chapham, P) PROJECT 1 DESCRIPTION (provided by applicant): HIV-1 R5 viruses vary considerably in phenotypes, including macrophage-tropism and sensitivity to neutralizing antibodies. Thus, highly mac-tropic envelopes (envs) exploit low amounts of CD4 and/or CCR5 for infection and contrast with non-mac-tropic R5 envs that require high CD4 levels and infect macrophages inefficiently. The capacity of highly mac-tropic envs to use low CD4/CCR5 suggests that such variants may confer a broader tropism among other CD4+ cell types that express low receptor levels and may have an advantage during virus transmission. Our recent data, using neutralizing monoclonal antibodies (mAbs) and entry inhibitors, strongly suggests that highly mac-tropic R5 envs carry a more exposed CD4 binding site (CD4bs) and may be vulnerable to neutralizing antibodies (NAbs) induced by vaccines. We propose to extensively investigate tropism and neutralization sensitivity of diverse R5 envs in the following four aims: Aim 1. The effect of HIV-1 R5 macrophage-tropism on infection of different CD4+ cell populations. We will examine whether highly mac-tropic R5 envelopes confer a broader tropism among CD4+ T-cell populations. Aim 2. Further analysis of the envelope determinants for macrophage infection and use of low CD4 in clade B and non-clade B envelopes. We will analyze clade B and non-clade B envs to identify determinants of tropism and to further examine the role of the CD4 binding loop. Aim 3. Investigation of the impact of R5 env variation in macrophage-tropism on sensitivity to neutralizing antibodies. We will investigate how R5 env variation impacts on sensitivity to HIV-1 + human sera, neutralizing mabs and vaccine induced neutralizing antibodies. Aim 4. Development and characterization of bacterially-produced, unglycosylated protein constructs that mimic the CD4bs. We have designed and produced CD4bs mimics that carry the critical elements of the CD4bs in an unglycosylated peptide that can be produced in bacteria. These CD4bs mimics bind CD4 and b12. Their structure and capacity to induce neutralizing antibodies will be investigated. We expect to provide important insights into (1) the variation of R5 envs for tropism and neutralization sensitivity, (2) the env amino acids and structural determinants involved in varying tropism and neutralization sensitivity and (3) the impact of this variation in the design of novel env-based vaccines.