Germline-Specific Immunogens for the Induction of Neutralizing Antibodies to HIV-

描述（申请人提供）：目前迫切需要疫苗来减缓HIV/AIDS流行病的传播，能够诱导中和抗体的免疫原仍然是疫苗研究的重要目标。近年来，越来越多的人单克隆抗体（mAb）已从显示出非常广泛和有效的中和反应的个体中分离出来。再加上过去的工作，这些结果支持了人类免疫系统可以产生罕见但有效的病毒保护性抗体的想法。 许多工作，包括我们自己的工作，都集中在识别HIV-1包膜糖蛋白（Env）与广泛中和抗体结合的形式上。与优化Env与这些高度亲和力成熟抗体结合的方法相反，我们和其他人现在提出鉴定可以与广泛中和抗体的种系形式结合的免疫原，作为更有效的免疫方法。 虽然抗体-抗原相互作用的三维结构已经为抗原设计提供了信息，但相应的种系抗体序列很少显示出与HIV-1 Env的任何结合。因此，结构信息是不容易获得的合理设计的生殖系免疫原。因此，我们建议采用定向分子进化的方法来识别生殖系特异性免疫原的问题。这种免疫原可以刺激携带成熟广泛中和抗体的种系前体的未成熟B细胞。因为任何单个的种系序列仅代表所有重排的免疫球蛋白基因的百分之几，所以用种系特异性免疫原激活特异性B细胞可以增加诱导适当抗体的可能性。还可以设想使用一系列免疫原，其沿着特定路径刺激部分亲和力成熟的抗体沿着形成成熟的中和抗体。 最近表征的VRC 01 mAb非常适合这种方法，特别是因为与大多数其他广泛中和mAb相比，CDRH 3结构域对于中和活性的重要性较低。这最小化了预测生殖系VRC 01前体结构的限制之一。我们将创建许多VRC 01样mAb，这些mAb逐渐回复到种系序列，并将其用作试剂来筛选通过体外同源DNA重组创建的Env变体文库。回复突变体可以以逐步递归的方式使用，由此结合最小回复的mAb的Env变体可以用作亲本以产生额外变体的文库，用于以更高度回复的形式进行筛选。我们将评估这些种系特异性Env免疫原与骨髓瘤细胞结合的能力，这些骨髓瘤细胞在细胞表面表达VRC 01样种系抗体，这种相互作用类似于B细胞活化的第一步之一。 该提案代表了鉴定HIV-1疫苗候选免疫原的创新方法。它对已经鉴定出保护性（或广泛保护性）mAb的其他病原体具有普遍适用性（例如，RSV或流感病毒），但这些抗体特异性的诱导已被证明是有问题的。 与公共卫生的关系：艾滋病毒/艾滋病流行病继续造成死亡和新的感染，约有3 300万人患有这种疾病。可以减少感染的疫苗是预防措施的重要组成部分。这项研究计划旨在创造能够防止艾滋病毒感染细胞的候选疫苗。

DESCRIPTION (provided by applicant): Vaccines are urgently needed to slow the spread of the HIV/AIDS epidemic, and immunogens that can induce neutralizing antibodies remain an important goal of vaccine research. An increasing number of human monoclonal antibodies (mAbs) have been isolated in recent years from individuals who show remarkably broad and potent neutralizing responses. Coupled with past work, these results support the idea that the human immune system can generate rare but potent protective antibodies to the virus. Much work, including our own, has focused on identifying forms of the HIV-1 envelope glycoprotein (Env) that bind to broadly neutralizing antibodies. In contrast to approaches that optimize Env for binding to these highly affinity-matured antibodies, we and others now propose to identify immunogens that can bind to the germline form of the broadly neutralizing antibodies as a more productive approach to immunization. While three-dimensional structures of antibody-antigen interactions have informed antigen design, the corresponding germline antibody sequences rarely show any binding to the HIV-1 Env. As a result, structural information is not readily available for rational design of germline immunogens. We propose therefore to employ a directed molecular evolution approach to the problem of identifying germline-specific immunogens. Such immunogens could stimulate immature B cells that carry the germline precursor of a mature broadly neutralizing antibody. Because any individual germline sequence represents only a few percent of all rearranged immunoglobulin genes, activating specific B cells with germline-specific immunogens could increase the likelihood of inducing an appropriate antibody. One can also envision using a series of immunogens that stimulate partially affinity-matured antibodies along a particular path to form a mature neutralizing antibody. The recently characterized VRC01 mAb is well suited to this approach, notably because the CDRH3 domain is less important for the neutralization activity compared to most of the other broadly neutralizing mAbs. This minimizes one of the constraints in predicting the structure of the germline VRC01 precursor. We will create a number of VRC01-like mAbs that are increasing reverted to the germline sequence and use these as reagents to screen libraries of Env variants created by in vitro homologous DNA recombination. The revertants can be used in a stepwise recursive fashion, whereby Env variants that bind minimally reverted mAbs can be used as parents to create libraries of additional variants for screening with more highly reverted forms. We will evaluate the ability of these germline-specific Env immunogens to bind to myeloma cells that express VRC01-like germline antibodies on the cell surface, an interaction that resembles one of the first steps of B-cell activation. This Proposal represents an innovative approach to the identification of candidate immunogens for HIV-1 vaccines. It has general applicability to other pathogens for which protective (or broadly protective) mAbs have been identified (e.g., RSV or influenza) but induction of those antibody specificities has proven problematic. PUBLIC HEALTH RELEVANCE: The HIV/AIDS epidemic continues to cause death and new infections with some 33 million people living with the disease. A vaccine that can reduce infection is an essential component of preventative measures. This research proposal is designed to create vaccine candidates that can prevent the HIV virus from infecting cells.