Induction of HIV Neutralizing Antibodies by Targeting Macaque B Cell Receptors

通过靶向猕猴 B 细胞受体诱导 HIV 中和抗体

• 批准号: 8462899
• 负责人: MIROSLAW K GORNY
• 金额: $ 16.86万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8462899
• 关键词: 3-Dimensional; Affinity; Animals; Antibodies; Antibody Formation; Antigens; Avidity; B-Lymphocytes; Binding; Binding Sites; Blood specimen; Cell Separation; Cells; Chimeric Proteins; Clinical Trials; Complementary DNA; Cyclic Peptides; DNA; Development; Epitopes; Flow Cytometry; Gene Targeting; Genes; Goals; HIV; HIV Envelope Protein gp120; HIV Infections; HIV vaccine; HIV-1; Human; Immune response; Immunization; Immunogenetics; Immunoglobulin G; Immunoglobulin Genes; Immunoglobulins; Individual; Infection; Inhibitory Concentration 50; Length; Macaca; Macaca mulatta; Measures; Mediating; Monitor; Monkeys; Monoclonal Antibodies; Mutation; Peptides; Peripheral Blood Mononuclear Cell; Plasmids; Pre-Clinical Model; Process; Production; Proteins; Receptors, Antigen, B-Cell; Regimen; Relative (related person); Research; Reverse Transcriptase Polymerase Chain Reaction; Serum; Shapes; Somatic Mutation; Specificity; Specimen; Statistical Methods; Testing; Time; Transfection; Vaccines; antigen binding; base; design; env Gene Products; expression vector; gp160; immunoglobulin receptor; innovation; neutralizing antibody; neutralizing monoclonal antibodies; nonhuman primate; novel strategies; receptor; vaccine development; virus envelope

DESCRIPTION (provided by applicant): The induction of broadly cross-neutralizing antibodies (Abs) that can protect healthy individuals against HIV infection remains a major challenge for vaccine development. These neutralizing Abs are observed in the course of natural HIV-1 infection, appearing 2 to 3 years post infection raising the question of whether or not they can be induced during a relatively short period of immunization. We propose a new approach to (a) employ an immunogen (mimotope-fusion protein) which targets selected immunoglobulin (Ig) gene-encoded Abs on naive B cells that are the precursors of anti-V3 neutralizing Abs and (b) monitor for an affinity maturation and development of cross-neutralization potency of pseudoviruses. Toward these goals, we have developed a rationally designed immunogen based on VH5-51 mimotope that mimics the highly conserved V3 epitopes recognized by human cross-neutralizing anti-V3 monoclonal Abs (mAbs) encoded by a pairing of the VH5-51 and VL lambda genes. We hypothesize that a VH5-51 mimotope can be targeted to macaque B cell receptors (encoded by the VH5-51 and VL lambda genes), where it will induce Abs with significantly enhanced affinity maturation compared to the control mimotope (non-VH5-51), which will induce anti-V3 Abs encoded by different Ig genes. In the first aim, we will generate monoclonal anti-V3 Abs from antigen-specific single B cells derived from rhesus macaques immunized with two immunogens to specifically elicit anti-V3 Abs encoded by the VH5-51 or by other non-VH5-51 genes. Two groups, each comprising three rhesus monkeys, will be immunized with gp160 DNA prime in combination with a VH5-51 mimotope-CTB fusion protein or gp160 DNA prime with control non-VH5-51 mimotope-CTB. Blood specimens from each animal will be drawn at pre-immunization, during immunization and at 1, 3, 6, 12 and 18 months post-last immunization. The longitudinal PBMC specimens from one animal in each group will be chosen for production of anti-V3 mAbs from IgG+ single B cells selected using the biotinylated V3-Fc fusion protein. The Ig variable genes from V3-specific B cells will be amplified by RT-PCR, cloned into expression vectors, and full-length IgG mAbs will be produced from 293T cells upon plasmid co-transfection. In the second aim, we will monitor the maturation of anti-V3 mAbs, sequentially produced from macaques immunized with the VH5-51 and non-VH5-51 immunogens by measuring mutation rates, relative affinity (50% maximal binding) and neutralizing activity (IC50). The profile of changes in the affinity and neutralizing activities wil be compared between VH5-51- and non-VH5-51-derived anti-V3 mAbs. Results that demonstrate the feasibility of targeting the particular Ig gene-encoded V3 B cell receptor would provide opportunities to target other Ig genes encoding neutralizing Abs with different specificities. For vaccine development, the immunogen based on a Ig gene-targeted mimotope can be used for combined boosting with gp120 to spike the immune response against particular envelope neutralizing epitope.

描述（由申请人提供）：诱导可保护健康个体免受 HIV 感染的广泛交叉中和抗体 (Ab) 仍然是疫苗开发的主要挑战。这些中和抗体是在自然 HIV-1 感染过程中观察到的，在感染后 2 至 3 年出现，这就提出了它们是否可以在相对较短的免疫时间内诱导的问题。我们提出了一种新方法：(a) 采用免疫原（模拟表位融合蛋白），其靶向初始 B 细胞上选定的免疫球蛋白 (Ig) 基因编码的抗体，这些抗体是抗 V3 中和抗体的前体；(b) 监测假病毒的亲和力成熟和交叉中和效力的发展。为了实现这些目标，我们开发了一种基于 VH5-51 模拟表位的合理设计的免疫原，该免疫原模仿由 VH5-51 和 VL lambda 基因配对编码的人类交叉中和抗 V3 单克隆抗体 (mAb) 识别的高度保守的 V3 表位。我们假设 VH5-51 模拟表位可以靶向猕猴 B 细胞受体（由 VH5-51 和 VL lambda 基因编码），与对照模拟表位（非 VH5-51）相比，它将诱导亲和力成熟显着增强的抗体，从而诱导由不同 Ig 基因编码的抗 V3 抗体。第一个目标是，我们将从用两种免疫原免疫的恒河猴的抗原特异性单个 B 细胞中产生单克隆抗 V3 抗体，以特异性引发由 VH5-51 或其他非 VH5-51 基因编码的抗 V3 抗体。将用与VH5-51模拟表位-CTB融合蛋白组合的gp160 DNA引发剂或与对照非VH5-51模拟表位-CTB组合的gp160 DNA引发剂免疫两组，每组包含三只恒河猴。将在免疫前、免疫期间以及最后一次免疫后1、3、6、12和18个月时从每只动物抽取血液样本。将选择每组中一只动物的纵向 PBMC 标本，用于从使用生物素化的 V3-Fc 融合蛋白选择的 IgG+ 单 B 细胞生产抗 V3 mAb。来自 V3 特异性 B 细胞的 Ig 可变基因将被扩增 通过 RT-PCR，克隆到表达载体中，并在质粒共转染后从 293T 细胞中产生全长 IgG mAb。在第二个目标中，我们将通过测量突变率、相对亲和力（50% 最大结合）和中和活性（IC50）来监测抗 V3 mAb 的成熟，这些 mAb 是由用 VH5-51 和非 VH5-51 免疫原免疫的猕猴依次产生的。将比较VH5-51和非VH5-51衍生的抗V3 mAb之间的亲和力和中和活性的变化概况。证明靶向特定 Ig 基因编码的 V3 B 细胞受体的可行性的结果将为靶向编码具有不同特异性的中和抗体的其他 Ig 基因提供机会。对于疫苗开发，基于 Ig 基因靶向模拟表位的免疫原可与 gp120 联合加强，以激发针对特定包膜中和表位的免疫反应。