Novel HIV 1 Envelope immunogens derived from broadly neutralizing plasmas

摘要 我们研究的总体目标是设计新型HIV包膜免疫原 能够引发广泛反应性中和抗体（NAb）。虽然尝试 通过疫苗接种引发广泛中和抗体应答的研究 不成功的、特别有效的广泛中和抗体应答已经被 在一些慢性感染的HIV+受试者中检测到。我们建议测试新的 免疫原来源于分离自孔的包膜序列， 特征性HIV+受试者，其异常的广泛中和活性针对 CD 4受体结合位点（CD 4-BS）。我们假设HIV-1包膜 来自HIV-1感染受试者的免疫原， 对CD 4-BS的广泛交叉中和活性将引发能够 广泛的交叉中和。 这项建议分为两个不同的阶段。在第一阶段，我们 首先，构建并鉴定可溶性三聚体gp 140 Env 来自分离自充分表征的细胞的自体Env序列的蛋白 广泛中和等离子体。其次，我们将使用这些三聚体gp 140 Env作为 免疫原和监测他们的能力，引发广泛中和抗体反应， 动物我们将仔细分析疫苗接种引起的NAb反应， 确定引发的NAb应答的质量和表位靶标。 在第二阶段，我们将分离新的单克隆中和抗体， 接种疫苗后产生广泛中和抗体的动物。我们 将利用噬菌体展示文库和抗原特异性单B细胞克隆， 从免疫动物脾和骨髓组织分离抗体。 小说 抗体和Fab将被分离并广泛表征结合， 中和活性 有希望的Fab将被制成完整的免疫球蛋白G 分子，并广泛表征交叉中和潜力和结合 表位这项研究中的新型抗HIV中和抗体将提供有价值的新的 工具的研究界，并帮助确定新的中和表位上的 HIV-1包膜峰值。

ABSTRACT 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.