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

描述（由申请人提供）：疫苗可以减缓HIV/AIDS流行病的传播，鉴定诱导中和抗体的免疫原仍然是HIV疫苗研究的重要目标。越来越多的广泛中和的人单克隆抗体（mAb）已被分离出感染的个人谁显示出显着的广泛和有效的中和反应。再加上过去的研究，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观点。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，我们现在提出鉴定可以与广泛中和抗体的种系形式结合的Env变体，作为更有效的免疫方法。虽然抗体-抗原相互作用的结构研究为抗原设计提供了信息，但相应的种系抗体序列很少显示与HIV-1 Env的任何结合。因此，详细的信息是不容易获得的合理设计的种系结合的Env。因此，我们建议使用定向分子进化的方法来确定生殖系特异性免疫原。这种免疫原可以更有效地刺激幼稚B细胞。广泛中和的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的mAb相比，CDRH 3结构域对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。我们将基于猕猴种系序列创建许多VRC 01样mAb，这些序列逐渐回复为种系序列，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。我们将用这些种系特异性变体免疫猕猴，并使用大规模并行DNA测序技术分析这些基于Env的免疫原刺激其所选择的特定种系亲和力成熟的能力。基于初始免疫研究的结果，我们将鉴定出额外的免疫原，其沿着特定路径刺激部分亲和力成熟的抗体沿着以形成成熟的中和抗体。在10个月的时间内，将使用种系特异性和额外的免疫原对猕猴进行免疫。对抗体基因突变模式的分析将表明是否有可能沿着特定途径指导抗体成熟。即使我们没有成功地诱导VRC 01样活性，这项研究的结果将导致对免疫原的初始反应以及它在多大程度上可以被操纵和控制的更深入的理解。这一建议代表了一种创新的方法来确定候选免疫原的HIV-1疫苗。它对已经鉴定出保护性（或广泛保护性）mAb的其他病原体（例如RSV或流感）具有普遍适用性，但这些特异性的诱导已被证明是有问题的。

DESCRIPTION (provided by applicant): Vaccines can slow the spread of the HIV/AIDS epidemic, and the identification of immunogens that induce neutralizing antibodies remains an important goal of HIV vaccine research. An increasing number of broadly neutralizing human monoclonal antibodies (mAbs) have been isolated from infected individuals who show remarkably broad and potent neutralizing responses. Coupled with past studies, 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 now propose to identify Env variants that can bind to the germline form of the broadly neutralizing antibodies as a more productive approach to immunization. While structural studies 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, detailed information is not readily available for the rational design of germline-binding Env. We propose therefore to use a directed molecular evolution approach to identify germline-specific immunogens. Such immunogens could more effectively stimulate naive B cells. The broadly neutralizing 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 based on macaque germline sequences 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. We will immunize macaques with these germline-specific variants and analyze, using massively parallel DNA sequencing technologies, the ability of these Env-based immunogens to stimulate affinity maturation of the specific germline for which they were selected. Based on the results of an initial immunization study, we will identify additional immunogens that stimulate partially affinity-matured antibodies along a particular path to form a mature neutralizing antibody. The germline-specific and the additional immunogens will be used to immunize macaques over a 10-month period. Analysis of the pattern of mutation in the antibody genes will indicate whether it is possible to direct antibody maturation along a specific pathway. Even if we do not succeed in eliciting VRC01-like activity, the results of this study will lead to a greater understanding of the initial response tothe immunogen and to what extent can it be manipulated and controlled. 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 where induction of those specificities has proven problematic.