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

描述（由申请人提供）：长期目标是引发广泛中和抗体，但通过定义灵长类动物中env特异性，疫苗引发的B细胞反应主要针对CD4结合位点来推进这一目标。利用这些信息，通过迭代设计，我们将改进针对这个和其他保守的中和决定因素的响应。项目1名为免疫原设计和选择，将生成基于HIV包膜糖蛋白的候选疫苗免疫原，以便在HIVRAD中进行分析。免疫原将通过基于结构的设计和选择得到，并将在其他程序组件中分析对它们的反应。项目2，B细胞分析和Ig遗传学，将检查记忆B细胞库的多样性，通过可溶性三聚体在短时间和长时间间隔内，通过新设计的三聚体小分子，以及骨髓中长寿的浆细胞，来检测方案对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，还将分析接种env三聚体后的env特异性记忆IgM室，以寻找过渡到IgG室的缺陷。项目四，生物信息学与

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.