High Resolution Analysis of Env-directed B Cells to Accelerate Vaccine Design

描述（由申请方提供）：长期目标是引发广泛中和抗体，但通过定义主要针对CD 4结合位点的灵长类动物中Env特异性疫苗引发的B细胞应答来推进这一目标。利用这些信息，通过迭代设计，我们将改善针对这个和其他保守的中和决定簇的反应。项目1，题为免疫原设计和选择，将产生艾滋病毒包膜糖蛋白为基础的候选疫苗免疫原进行分析，在艾滋病毒研究和开发。免疫原将通过基于结构的设计以及选择来获得，并且对它们的反应将在其他程序组件中进行分析。项目2，B细胞分析和免疫球蛋白遗传学，将检查记忆B细胞库的多样性，针对由可溶性三聚体以短和长的间隔、由新设计的三聚体免疫原以及骨髓中的长寿命浆细胞引起的Env的选定区域，以检查方案对V基因使用和体细胞超突变的影响。项目3，记忆B细胞分离和抗体表征，将使用Env特异性FACS分离B细胞，并将表征分离的Env特异性抗体的表位特异性，以帮助免疫原重新设计，还将分析Env三聚体接种后的Env特异性记忆IgM区室，以寻找向IgG区室过渡的缺陷。项目4，生物信息学和 结构，将使用从NHP免疫原性实验获得的B细胞转录物的深度测序的生物信息学分析，并表征免疫应答中产生的抗原特异性抗体群体。第二个重点将是使用x射线晶体学来确定与gp120复合的高兴趣抗体的原子水平结构。这些工作将得到行政核心A的支持，行政核心A将制定一项管理计划，以确保HIVRAD和NHP核心B之间的明确沟通，NHP核心B将饲养动物，使用项目1的免疫原进行免疫原性实验，并为项目2、3和4的B细胞反应分析提供样本。 相关性：本HIVRAD和本概述的总体目标是引发针对人类病原体HIV-1的广泛中和抗体。这种抗体的成功诱导将是朝着产生广泛有效的HIV-1疫苗的目标迈出的一大步，并将对改善人类公共卫生产生重大影响。 ******************************************************************************************************************** 项目1：免疫原设计和选择 项目负责人（PL）：Richard Wyatt，PHD 描述（由申请方提供）：项目1，标题为免疫原设计和选择，在生成本HIVRAD中待分析的基于HIV包膜糖蛋白的候选疫苗免疫原方面发挥主要作用。在目标1中，我们将基于三种不同的策略设计新型三聚体免疫原。最初，我们将利用我们的合作小组对从用HIV Env三聚体免疫原免疫的非人灵长类动物（NHP）中分离的CD 4结合位点定向（CD 4bs）中和抗体（nAbs）进行的最新绘图数据。我们比较了NHP CD4bs nAbs与已知的、更广泛的nAbs，这些nAbs是通过丙氨酸扫描诱变HIV-1 gp120从HIV-1感染的人类受试者中分离的。使用丙氨酸扫描信息，我们在NHP CD4bs Mab和人bNab的结构定义的足迹之间产生了不同的mAb足迹模式。有了这些信息，我们将设计稳定的三聚体，优先呈现中和表位， 激活与感染相关的VH基因引发bNab，并通过接种引发中和抗体应答。在第二种方法中，我们将评估环缺失的构象稳定的三聚体在兔中更快地诱导CD4b定向的能力，以在NHP中这样做。将评估改良版本引发CD4bs单克隆抗体或与全长三聚体协同引发CD4bs导向的B细胞应答的能力。在目标2中，我们将显示最佳抗原性的半胱氨酸稳定的三聚体缀合到脂质体上，以确定它们是否会增加对Env的抗体应答的持久性以及更好地引发中和抗体。最后，在目标3中，我们通过定向进化选择新的三聚体免疫原作为Env疫苗候选物。作为合理Env设计的补充方法，将评估通过Michael Zwick博士建立的定向进化和选择标准稳定的Env三聚体在NHP中引发B细胞应答的能力。此外，通过Zwick实验室建立的病毒定向进化，选择HIV-1 Env三聚体用于被CD4bs bNabs的NHP种系等同物（例如VH1 - 02或VH4 - 59）识别。 相关性：项目1的主要目标是设计新的HIV Env免疫原，这是HIVRAD的总体目标所必需的任务，即引发针对人类病原体HIV-1的广泛中和抗体。这种抗体的成功诱导将是朝着产生广泛有效的HIV-1疫苗的目标迈出的一大步，并将对改善人类公共卫生产生重大影响。

DESCRIPTION (provided by applicant): The long term goal is to elicit broadly neutralizing antibodies, but to forward this objective by defining the Env-specific, vaccine-elicited B cell responses in primates primarily directed at the CD4 binding site. Using this information, through iterative design, we will improve responses directed at this and other conserved neutralizing determinants. Project 1, entitled Immunogen Design and Selection, will generate the HIV envelope glycoprotein-based candidate vaccine immunogens to be analyzed in the HIVRAD. The immunogens will be derived by structure-based design, as well as by selection, and responses to them will be analyzed in the other program components. Project 2, B cell Analysis and Ig Genetics, will examine the diversity of the memory B cell repertoire against selected regions of Env elicited by soluble trimers at both short and longer intervals, by newly designed trimeric imijnunogens, as well as long-lived plasma cells in the bone marrow to examine the impact of regimen on V gene usage and somatic hypermutation. Project 3, Memory B Cell Isolation and Antibody Characterization, will isolate B cells using Env-specific FACS and will characterize the isolated Env-specific antibodies for their epitope specificity to aid immunogen redesign and will also analyze the Env-specific memory IgM compartment following Env-trimer inoculation to look for defects in transition to the IgG compartment. Project 4, Bioinformatics and Structure, will use bioinformatics analysis of deep sequencing of B cell transcripts, obtained from the NHP immunogenicity experiments and to characterize antigen-specific antibody populations that arise in response to immunization. The second focus will be to use x-ray crystallography to determine atomic-level structures of high-interest antibodies in complex with gp120. These efforts will be supported by the Administrative Core A, which will institute a management plan to ensure clear communications within the HIVRAD and the NHP Core B which will house the animals, perform immunogenicity experiments with immunogens from Project 1 and provide samples for the analysis of B cell responses for Projects 2, 3 and 4. RELEVANCE: The overall goal of this HIVRAD, and this Overview, is to elicit broadly neutralizing antibodies to the human pathogen HIV-1. The successful elicitation of such antibodies would be a large step forward toward the goal of generating a broadly effective HIV-1 vaccine and would have a substantial impact on improving human public health. ******************************************************************************************************************** Project 1: Immunogen Design and Selection Project Leader (PL): Richard Wyatt, PHD DESCRIPTION (provided by applicant): Project 1, entitled Immunogen Design and Selection, plays the major role of generating the HIV envelope glycoprotein-based candidate vaccine immunogens to be analyzed in this HIVRAD. In Aim 1, we will design novel trimeric immunogens based upon three distinct strategies. Initially, we will utilize recent mapping data performed in our collaborative group on CD4 binding-site directed (CD4bs) neutralizing antibodies (nAbs) isolated from non-human primates (NHPs) immunized with HIV Env trimeric immungens. We compared the NHP CD4bs nAbs to the known, more broadly nAbs isolated from HIV-1-infected human subjects by alanine scanning mutagenesis ofthe HIV-1 gp120. Using the alanine scan information, we generated divergent mAb footprint patterns between the NHP CD4bs Mabs and the structurally defined footprint of the human bNabs. Armed with this information, we will design stabilized trimers that preferentially present neutralizing epitopes to activate VH genes associated with the infection elicited bNabs and to elicit neutralizing antibody responses by vaccination. In the 2nd approach, we will assess the ability of loop-deleted, conformationally stabilized trimers, which more rapidly elicit CD4bs-directed in rabbits, to do so in NHPs. Improved versions will be assessed for their capacity to elicit CD4bs Mabs or to synergize with full-length trimers to elicit CD4bsdirected B cell responses. In Aim 2, we conjugate cysteine stabilized trimers displaying an optimal antigenic profile to liposomses to the determine if they will increase the durability ofthe antibody response to Env as well as better elicit neutralizing antibodies. Finally, in Aim 3 we select novel trimeric immunogens by directed evolution as Env vaccine candidates. Performed as a complementary approach to rational Env design, Env trimers, stabilized by directed evolution and selection criteria established by Dr. Michael Zwick, will be assessed for their ability to elicit B cell responses in NHPs. In addition, HIV-1 Env trimers selected for recognition by NHP germline equivalents of CD4bs bNabs (e.g. VH1-02 or VH4-59) by directed evolution of virus as established in the Zwick laboratory. RELEVANCE: The major objective of Project 1 is to design novel HIV Env immunogens, an undertaking which is essential for the overall goal of the HIVRAD to elicit broadly neutralizing antibodies to the human pathogen HlV-1. The successful elicitation of such antibodies would be a large step forward toward the goal of generating a broadly effective HIV-1 vaccine and would have a substantial impact on improving human public health.