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

描述（由申请人提供）：该多项目HIVRAD计划申请的总体目标是引发针对主要HIV-1包膜（Env）糖蛋白的CD 4结合位点（CD 4 bs）的中和抗体。本P01计划建议书包括三个主要项目，以及两个支持主要项目活动的核心。以下是本计划不同项目/核心中提出的主要活动的摘要。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-1 R5 Envs的变异如何影响嗜性、中和和疫苗开发（项目1）。HIV-1 R5包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（mac-tropism）的变化对与Env相关的其他生物学特性（包括中和敏感性）的影响。目标3：研究CD 4 bs抗原性的变化如何影响一级Env蛋白的中和敏感性和免疫原性（项目2）。将通过mAb探测几组原代Env的CD 4 bs抗原性，每种Env均具有其独特的生物学特征。我们将使用DMA初免-蛋白加强免疫方法检查高CD 4 bs抗原性和对CD 4 bs mAb介导的中和的高敏感性是否会导致关键代表性Env的高免疫原性。目标4：研究受体结合位点的修饰作为HIV-1疫苗设计的方法（项目3）。我们将测试由特异性聚糖修饰引起的变化是否会导致受体结合位点中保守表位的稳定性或可及性增加，以及这些保守位点的更大可及性是否会增强其作为免疫原的功能，以引发交叉反应性NAb应答。目标5：为Env结构分析、新抗原结构和抗原抗体相互作用研究（核心A）等重大项目提供支持。 项目1：HIV-1 R5 Envs的变异：嗜性、中和和疫苗的后果 （查塔姆，P） 项目1描述（由申请方提供）：HIV-1 R5病毒的表型差异很大，包括嗜巨噬细胞性和对中和抗体的敏感性。因此，高嗜性包膜（env）利用低量的CD 4和/或CCR 5进行感染，并且与需要高CD 4水平并且无效地感染巨噬细胞的非嗜性R5 env形成对比。高嗜性envs使用低CD 4/CCR 5的能力表明，这些变异体可能在表达低受体水平的其他CD 4+细胞类型中赋予更广泛的嗜性，并可能在病毒传播过程中具有优势。我们最近的数据，使用中和单克隆抗体（mAb）和进入抑制剂，强烈表明，高度mac-tropic R5 envs携带更多暴露的CD 4结合位点（CD 4 bs），可能是脆弱的中和抗体（NAbs）诱导的疫苗。我们建议在以下四个目标中广泛研究不同R5 envs的向性和中和敏感性：目标1。HIV-1 R5巨噬细胞嗜性对不同CD 4+细胞群感染的影响我们将研究是否高度mac-tropic R5信封赋予更广泛的嗜性之间的CD 4 + T细胞群体。目标2.进一步分析巨噬细胞感染的包膜决定因素以及在进化枝B和非进化枝B包膜中使用低CD 4。我们将分析进化枝B和非进化枝B env，以确定向性的决定因素，并进一步研究CD 4结合环的作用。目标3：研究巨噬细胞嗜性中R5 env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、中和单克隆抗体和疫苗诱导的中和抗体的敏感性。目标4。模拟CD 4 bs的细菌产生的非糖基化蛋白质构建体的开发和表征。我们已经设计并生产了CD 4 bs模拟物，其在可以在细菌中产生的非糖基化肽中携带CD 4 bs的关键元件。这些CD 4 bs模拟物结合CD 4和b12。将研究其结构和诱导中和抗体的能力。我们希望提供重要的见解（1）R5 env的嗜性和中和敏感性的变化，（2）env氨基酸和结构决定因素参与不同的嗜性和中和敏感性和（3）这种变化在设计新的env疫苗的影响。

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.