Engineered Envelope Glycoprotein Trimers for HIV-1 Vaccine Immunogens

本申请要求对授予R21 AI 087472“工程信封”进行行政补充 用于HIV-1疫苗免疫原的糖蛋白三聚体”，这是2009年授予的一笔赠款，现在是第二笔免费赠款。 扩展名.由于飓风桑迪期间的停电，从用 两个修饰的GP 140蛋白质不可挽回地丢失。虽然抗Env抗体滴度的初步测量 在兔血清中的结果表明，该项目有希望的结果，我们无法完成我们的分析， 评估血清中和活性的程度和广度。补充资金将使我们能够 重复我们的兔子免疫实验。这些免疫原性研究将为新R 01提供平台 研究，旨在设计和开发HIV-1 Env免疫原，其在一种免疫系统中呈现关键的四级表位。 能够在人体内引发广泛中和的HIV-1抗体的稳定状态。 实验1：制备和表征生物化学稳定的化合物的均质制剂 HIV-1 gp 140三聚体。 实验2：在家兔中进行免疫原性研究，以确定稳定的天然gp 140 三聚体可以改善中和抗体反应。 我们的总体目标是了解特定的gp 41-gp 41相互作用如何调节三聚体和稳定性。 融合前Env复合物，并应用这一知识设计天然Env-三聚体模拟物， 中和表位的免疫原性。为此，我们将表达和纯化均一性修饰的HIV-1 JR-FL gp 140三聚体，并评估其在兔体内的免疫原性。这些相互关联的实验旨在 在兔子模型中生成概念验证信息，这些信息可用于解决我们的总体问题， 天然Env三聚体中特异性协同亚基间相互作用导致其固有不稳定性的假设 并且可以被工程化以增强免疫原性。在不增加费用的情况下扩大支助将大大 克服桑迪造成的材料损失，我们迫切需要这些材料，以实现中心目标 家长补助金

This application requests an Administrative Supplement to Grant R21 AI087472 “Engineered envelope glycoprotein trimers for HIV-1 vaccine immunogens”, a grant awarded in 2009, and now in a second no-cost extension. As a result of power outages during Hurricane Sandy, sera collected from rabbits immunized with two modified gp140 proteins were irretrievably lost. While preliminary measurements of anti-Env antibody titers in the rabbit serum suggest promising outcomes for this project, we are unable to complete our analysis to assess the extent and breadth of the serum neutralizing activity. The supplemental funds will enable us to repeat our rabbit immunization experiments. These immunogenicity studies will provide a platform for new R01 research, intended to design and develop HIV-1 Env immunogens that present key quaternary epitopes in a stable state capable of eliciting broadly neutralizing HIV-1 antibodies in humans. Experiment 1: To produce and characterize homogeneous preparations of biochemically stabilized HIV-1 gp140 trimers. Experiment 2: To conduct immunogenicity studies in rabbits to determine whether stable native gp140 trimers can improve neutralizing antibody responses. Our overall goal is to understand how specific gp41-gp41 interactions modulate the trimerization and stability of the prefusion Env complex and to apply this knowledge to design native Env-trimer mimics with enhanced immunogenicity of neutralizing epitopes. To do this, we will express and purify to homogenicity modified HIV-1 JR-FL gp140 trimers, and evaluate their immunogenicity in rabbits. These interlinked experiments are intended to generate proof-of-concept information in the rabbit model that can be used to address our overarching hypothesis that specific cooperative inter-subunit interactions in native Env trimer lead to its inherent instability and can be engineered to enhance immunogenicity. The with-cost extension of support would significantly overcome losses of materials incurred by Sandy that we need desperately in order to fulfill the central objective of the parent grant.