Development Structure and Function of Broadly Neutralizing anti-HIV Antibodies

描述（由申请人提供）： 该P01计划项目申请旨在深入了解抗体（Abs）保护免受HIV感染的机制，以促进改进的Abs和有效免疫原的设计。开发控制感染的有效疫苗或递送的Ab将需要理解Ab与抗原和介导效应子功能的Ab受体的相互作用。使用在人源化小鼠中响应于HIV感染而出现什么Env突变的知识，允许对哪些特征促进Ab逃避进行结构/生物信息学分析，这是设计对病毒逃避的常见途径不敏感的广泛中和抗体（bNAb）所需的信息。这种知识将允许优化bNAb被动递送（通过注射和基因治疗“反向疫苗接种”）的广度/效力，并且是疫苗的有效免疫原设计所需的，因此我们的项目与传统和“反向”疫苗策略相关，以对抗HIV。为了实现这些目标并建立Ab介导保护的基本原则，我们将联合收割机结合Nussenzweig、Ravetch和Bjorkman实验室在HIV bNAb和HIV感染的人源化小鼠模型表征、抗体效应子功能评价和改进以及Ab-HIV和Ab-受体相互作用的结构生物学方面的专业知识。我们的建议包括三个独立的，但相互关联和相互依赖的合作项目，具有以下目的：（1）测试设计的bNAb在人源化小鼠模型的HIV感染，序列抗性HIV株，评估bNAb的能力，以控制建立在人源化小鼠中的HIV感染，并评估新的免疫原在小鼠模型中;（2）在体外和体内研究Fc效应子功能对HIV bNAb的贡献，包括在新的体内研究中。 HIV进入的小鼠模型和人源化小鼠中基于AAV的反向免疫模型;（3）通过解析与HIV Env蛋白和Fc受体复合的设计和天然bNAb的晶体结构来确定广泛/有效中和和改善的效应子功能的结构相关性;设计和测试用于引发bNAb的免疫原。这些项目将得到一个行政核心和三个科学核心的支持，包括一个细胞/生化自动化核心，用于进行自动化体外HIV中和和平板结合试验，一个蛋白表达核心，用于表达和纯化功能和结构研究所需的重组蛋白，以及一个动物服务核心，用于生成/维持体内实验所需的小鼠。 相关性：艾滋病毒/艾滋病仍然是一种全球流行病，迫切需要疫苗和/或新的治疗方法。我们的项目目标是发现抗HIV抗体可以预防或治疗感染的机制（通过Fab介导的中和和Fc介导的效应子功能）以及HIV如何通过突变逃逸，这是改善天然bNAb作为治疗剂和设计免疫原以引发bNAb所需的关键知识。 项目1 -人类艾滋病毒抗体 项目负责人（PL）：Nussenzweig，Michel 描述（由申请人提供）：绝大多数HIV感染者会产生病毒抗体。在大多数情况下，抗体仅针对自体毒株，但一些个体对广泛的不同病毒分离株产生中和血清学应答。这些反应是令人感兴趣的，因为将具有广泛中和活性的单克隆抗体被动转移至人源化小鼠或猴可预防感染。根据这些观察，有人提出，一种能产生广泛中和抗体的疫苗将对艾滋病毒具有保护作用。然而，人们对广泛中和反应的性质知之甚少。迄今为止，仅对少数患者进行了研究，选择这些患者中的大多数是因为其血清学活性集中在病毒包膜刺突的CD 4结合位点。该提案的长期目标是表征新的广泛中和抗体在体内的功能，并了解HIV-1如何在体内对这些抗体产生耐药性。为了实现这些目标，我们提出了三个具体目标。首先，我们将开发一种体内试验来评估广泛抗体防止病毒进入小鼠体内的能力。目前使用TZM-bl细胞在体外测定HIV中和。新的小鼠模型将用于检查不同单克隆抗体的相对功效以及先天效应器机制对阻断体内HIV进入的贡献。第二，我们将确定在HIV感染的人源化小鼠中对广泛中和抗体产生抗性的基础。我们将使用基于结构的合理设计方法的信息，以迭代方式增强抗体的广度和效力。这部分提案的最终目标是确定目前可用的大量抗体中的哪一种可能对被动疫苗和免疫原设计方法最有用。最后，我们将评估潜在的免疫原设计使用结构和其他数据在敲入小鼠含有人类生殖系抗体前体基因。总之，这些实验应该有助于为未来的临床研究提供信息，在这些研究中，中和抗体可能被考虑用于被动治疗或预防研究，以及它们的靶点中哪一个对免疫策略最有用。 相关性：虽然目前还没有艾滋病毒疫苗，但少数感染者会产生抗体，可以预防感染。拟议的研究旨在了解这些抗体，长期目标是能够重新引发它们作为疫苗的组成部分，用于未感染的个体。

DESCRIPTION (provided by applicant): This P01 Program Project application seeks to develop insights into mechanisms by which antibodies (Abs) protect against HIV infection to facilitate design of improved Abs and effective immunogens. Development of effective vaccines or delivered Abs to control infection will require understanding of Ab interactions with antigen and with Ab receptors that mediate effector functions. Using knowledge of what Env mutations arise in response to HIV infection in humanized mice allows structural/bioinformatic analyses of which features promote Ab evasion, required information for designing broadly neutralizing antibodies (bNAbs) that are insensitive to common routes of viral evasion. This knowledge will allow optimization of the breadth/potency of bNAbs for passive delivery (both by injection and gene therapy "reverse vaccination") and is required for effective immunogen design for vaccines, thus our project is relevant to both traditional and "reverse" vaccine strategies to combat HIV. To accomplish these goals and to establish basic principles underlying Ab-mediated protection, we will combine the expertise of the Nussenzweig, Ravetch, and Bjorkman laboratories in characterization of HIV bNAbs and humanized mouse models of HIV infection, antibody effector function evaluation and improvement, and the structural biology of Ab-HIV and Ab-receptor interactions. Our proposal comprises three separate, but inter-related and inter-dependent collaborative projects, with the following aims: (1) Test designed bNAbs in a humanized mouse model of HIV infection, sequence resistant HIV strains, evaluate bNAbs for ability to control established HIV infection in humanized mice, and evaluate novel immunogens in a mouse model; (2) Investigate the contributions of Fc effector function to HIV bNAbs in vitro and in vivo, including in a new in vivo mouse model for HIV entry and an AAV-based reverse immunization model in humanized mice; (3) Determine structural correlates of broad/potent neutralization and improved effector functions by solving crystal structures of designed and natural bNAbs complexed with HIV Env proteins and Fc receptors; design and test immunogens for eliciting bNAbs. These projects will be supported by an administrative core and three scientific cores comprising a cell/biochemical automation core to perform automated in vitro HIV neutralization and plate-binding assays, a protein expression core to express and purify recombinant proteins required for functional and structural studies, and an animal services core to generate/maintain mice required for in vivo experiments. RELEVANCE: HIV/AIDS remains a global epidemic with an urgent need for a vaccine and/or new therapies. Our project goals are to discover the mechanisms by which anti-HIV antibodies can prevent or treat infection (through Fab-mediated neutralization and Fc-mediated effector functions) and how HIV can escape through mutation, critical knowledge required for improving natural bNAbs as therapeutics and designing immunogens to elicit bNAbs. Project 1 - Human Antibodies to HIV Project Leader (PL): Nussenzweig, Michel DESCRIPTION (provided by applicant): The vast majority of HIV infected individuals develop antibodies to the virus. In most cases the antibodies only target the autologous strain, but some individuals develop neutralizing serologic responses to a broad range of different viral isolates. These responses are of interest because passive transfer of monoclonal antibodies with broad neutralizing activity to humanized mice or monkeys prevents infection. On the basis of these observations it has been proposed that a vaccine that elicits broadly neutralizing antibodies would be protective against HIV. However, little is known about the nature of the broadly neutralizing response. Only a small number of patients have been studied to date, and the majority of these patients are selected because their serologic activity focuses on the CD4 binding site of the viral envelope spike. The long-term goals of this proposal are to characterize new broadly neutralizing antibodies in terms of their functions in vivo and to understand how HIV-1 develops resistance to these antibodies in vivo. To accomplish these goals we propose three specific aims. First, we will develop an in vivo assay to assess the ability of broadly antibodies to prevent viral entry in mice. HIV neutralization is currently assayed in vitro using TZM-bl cells. The new mouse model will be used to examine the relative efficacy of different monoclonal antibodies and the contribution of innate effector mechanisms to blocking HIV entry in vivo. Second, we will define the basis for development of resistance to broadly neutralizing antibodies in HIV infected humanized mice. We will use the information for structure based rational design approaches to iteratively enhance antibody breadth and potency. The ultimate goal of this part of the proposal is to determine which of the large group of currently available antibodies might be most useful for passive vaccine and immunogen design approaches. Finally, we will evaluate potential immunogens designed using structural and other data in knock-in mice containing human germline antibody precursor genes. Taken together, these experiments should help inform future clinical studies in which neutralizing antibodies might be considered for use in passive therapy or prevention studies and which of their targets would be most useful for immunization strategies. RELEVANCE: Although there is still no vaccine for HIV, a small number of infected individuals develop antibodies that can prevent the infection. The proposed research aims to develop an understanding of these antibodies with the long term goal of being able to elicit them de novo as a component of a vaccine to be used in un-infected individuals.