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: 8329172
• 负责人: MIROSLAW K GORNY
• 金额: $ 32.28万
• 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 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8329172
• 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. PUBLIC HEALTH RELEVANCE: The goal of the proposed research is to test in non-human primates, used as preclinical models, the possibility to induce cross-neutralizing antibodies against HIV-1. The monkeys will be immunized with rationally designed antigen stimulating the B cells to produce antibodies encoded only by selected immunoglobulin genes. The antibodies encoded by these genes are pre-adapted to the V3 antigen on the virus envelope that leads to rapid maturation of antibody response and efficient HIV-1 neutralization compared to other control vaccine immunogen.

描述（由申请人提供）：可以保护健康个体免受HIV感染的广泛跨中和抗体的诱导仍然是疫苗开发的主要挑战。这些中和ABS在天然HIV-1感染过程中观察到，在感染后2至3年出现，这引发了一个问题，即在相对较短的免疫时期是否可以诱导它们。我们提出了一种新方法来（a）采用免疫原（MIMOTOPE融合蛋白），该免疫原（MIMOTOPE融合蛋白）靶向选定的免疫球蛋白（IG）基因编码的ABS上的ABS上的ABS上，这些ABS是抗V3中和ABS的前体，并（B）（B）监测Pseudizations Pseudizations Pseudizations pseudizations psseudizations psseudizations的亲和力成熟和开发。朝向这些目标，我们基于VH5-51模拟率开发了一种合理设计的免疫原，该免疫原模拟了由人类跨中和抗V3单克隆ABS（MABS）所识别的高度保守的V3表位（MABS）通过VH5-51和VL Lambda基因配对编码的。 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.在第一个目的中，我们将从源自用两种免疫原子的恒河猕猴衍生而来的抗原特异性单B细胞产生单克隆抗V3 ABS，以特异性地引起由VH5-51或其他非VH5-51基因编码的抗V3 ABS。两组包括三个由三只恒河猴组成，将与GP160 DNA Prime免疫，并与VH5-51 MIMOTOPE-CTB融合蛋白或GP160 DNA Prime结合使用，并具有对照非VH5-51 MIMOTOPE-CTB。在免疫预免疫，在免疫时以及腹膜后1、3、6、12和18个月时，将抽取每只动物的血液标本。每组中，将选择来自一组动物的纵向PBMC标本，用于从IgG+单个B细胞中生产使用生物素化V3-FC融合蛋白的抗V3 mAb。来自V3特异性B细胞的IG变量基因将被放大 通过RT-PCR，将克隆到表达载体中，并且在质粒共转染后将由293T细胞产生全长IgG mAb。在第二个目标中，我们将通过测量突变速率，相对亲和力（50％最大结合）和中和活性（IC50）（IC50）来监测由用VH5-51和非VH5-51和非VH5-51免疫原免疫产生的抗V3 mAb的成熟。在VH5-51-和非VH5-51衍生的抗V3 mAB之间，将比较亲和力和中和活动的变化的特征。结果表明，靶向特定Ig基因编码的V3 B细胞受体的可行性将为靶向其他具有不同特异性中和ABS的Ig基因的机会。为了进行疫苗的发育，基于Ig基因靶向模拟物的免疫原可用于与GP120合并增强，以激发针对中和中和表位的免疫反应。 公共卫生相关性：拟议的研究的目的是在非人类灵长类动物（用作临床前模型）中测试，这种模型的可能性是诱导针对HIV-1的跨中和抗体。猴子将通过理性设计的抗原刺激B细胞进行免疫，以产生仅由选定的免疫球蛋白基因编码的抗体。这些基因编码的抗体与其他对照疫苗的免疫原相比，预先适应了病毒包膜上的V3抗原，从而导致抗体反应快速成熟和有效的HIV-1中和。