Novel HIV 1 Envelope immunogens derived from broadly neutralizing plasmas

描述(由申请人提供)：我们研究的总体目标是设计能够诱导广泛反应性中和抗体(NAB)的新型HIV包膜免疫原。尽管通过接种疫苗来激发广谱中和抗体反应的尝试没有成功，但在一些慢性感染HIV+的受试者中发现了异常有效的广中和抗体反应。我们建议测试从一个特征良好的HIV+受试者分离的包膜序列中提取的新免疫原，该受试者具有针对CD4受体结合位点(CD4-BS)的异常广泛的中和活性。我们假设，来自HIV-1感染者的HIV-1包膜免疫原，其血浆对CD4-BS具有强大的、广泛的交叉中和活性，将诱导能够广泛交叉中和的NAB。这项提议分为两个不同的阶段。在第一阶段，我们提出如下建议：首先，构建和鉴定来自广泛中和血浆的自体Env序列的可溶三聚体gp140 Env蛋白。其次，我们将使用这些三聚体gp140 env作为免疫原，并监测它们在动物体内诱导广泛中和抗体反应的能力。我们将仔细分析疫苗接种引起的NAB反应，以确定引发的NAB反应的质量和表位靶点。 在第二阶段，我们将从接种疫苗后产生广泛中和抗体的动物中分离出新的单克隆中和抗体。我们将利用噬菌体展示文库和抗原特异性单个B细胞克隆从免疫动物的脾和骨髓组织中分离抗体。新的抗体和抗体将被分离出来，并广泛用于结合和中和活性的表征。有希望的纤维蛋白将被制成完整的免疫球蛋白G分子，并广泛地表征交叉中和潜力和结合表位。这项研究的新的抗HIV中和抗体将为研究界提供有价值的新工具，并有助于在HIV-1包膜尖峰上定义新的中和表位。 公共卫生相关性：这项提案将测试新的HIV-1候选疫苗，这些候选疫苗来自HIV-1+受试者的传播病毒，这些受试者产生了广泛的中和抗体，以应对自然感染。我们将用HIV-1包膜蛋白免疫兔子，监测它们是否产生中和抗体，并在那些产生广泛中和抗体反应的动物中分离出新的中和抗体。该项目将评估在自然产生广泛中和抗体的受试者中存在的HIV-1外膜序列是否可以通过接种疫苗在其他受试者中引发相同类型的免疫反应。

DESCRIPTION (provided by applicant): The overall aim of our studies is to design novel HIV Envelope immunogens capable of eliciting broadly reactive neutralizing antibodies (NAbs). Although attempts at eliciting broadly neutralizing antibody responses by vaccination have been unsuccessful, exceptionally potent broadly neutralizing antibody responses have been detected in some chronically infected HIV+ subjects. We propose to test new immunogens that are derived from Envelope sequences that were isolated from a well-characterized HIV+ subject whose exceptional broadly neutralizing activity targeted the CD4 receptor binding site (CD4-BS). We hypothesize that HIV-1 Envelope immunogens derived from HIV-1 infected subjects whose plasma exhibits potent, broad cross-neutralizing activity to the CD4-BS will elicit NAbs that are capable of broad cross-neutralization. This proposal is separated into two distinct phases. In the first phase, we propose the following: First, construct and characterize soluble trimeric gp140 Env proteins derived from autologous Env sequences isolated from well-characterized broadly neutralizing plasmas. Second, we will use these trimeric gp140 Envs as immunogens and monitor their ability to elicit broadly neutralizing antibody responses in animals. We will carefully dissect the NAb responses elicited by vaccination to determine both the quality and the epitope targets of the elicited NAb responses. In the second phase, we will isolate new monoclonal neutralizing antibodies from animals that develop broadly neutralizing antibodies in response to vaccinations. We will utilize both phage display libraries and antigen-specific single B cell cloning to isolate antibodies from spleen and bone marrow tissue of immunized animals. Novel antibodies and Fabs will be isolated and extensively characterized for binding and neutralizing activity. Promising Fabs will be made into full immunoglobulin G molecules and extensively characterized for cross-neutralizing potential and binding epitope. Novel anti-HIV neutralizing antibodies from this study will provide valuable new tools to the research community, and help to define new neutralization epitopes on the HIV-1 Envelope spike. PUBLIC HEALTH RELEVANCE: This proposal will test new HIV-1 vaccine candidates derived from the circulating viruses of HIV-1+ subjects who developed broadly neutralizing antibodies in response to natural infection. We will immunize rabbits with HIV-1 Envelope proteins, monitor them for the development of neutralizing antibodies, and isolate new neutralizing antibodies in those animals that develop broadly neutralizing antibody responses. This project will evaluate the possibility that HIV-1 Envelope sequences that are present in subjects who naturally developed broadly neutralizing antibodies can be used to elicit the same type of immune response in others by vaccination.