Induction of HIV Neutralizing Antibodies by Targeting Macaque B Cell Receptors

描述（由申请人提供）：诱导能够保护健康个体免受艾滋病毒感染的广泛交叉中和抗体（Abs）仍然是疫苗开发的主要挑战。在自然感染HIV-1的过程中观察到这些中和抗体，在感染后2至3年出现，这提出了一个问题，即它们是否可以在相对较短的免疫期间诱导产生。我们提出了一种新的方法：(a)利用免疫原（mimotope-fusion protein）靶向免疫球蛋白（Ig）基因编码的抗体，这些抗体是抗v3中和抗体的前体；(B)监测假病毒的亲和成熟和交叉中和能力的发展。为了实现这些目标，我们开发了一种基于VH5-51模位的合理设计的免疫原，该免疫原模拟了由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模组可以靶向猕猴B细胞受体（由VH5-51和VL lambda基因编码），与对照模组（非VH5-51）相比，它可以诱导具有明显增强亲和力成熟的抗体，从而诱导由不同Ig基因编码的抗v3抗体。在第一个目标中，我们将从接种了两种免疫原的恒河猴的抗原特异性单B细胞中产生单克隆抗v3抗体，以特异性地引发由VH5-51或其他非VH5-51基因编码的抗v3抗体。两组，每组包括3只恒河猴，将用gp160 DNA引物与VH5-51 mimotope-CTB融合蛋白或gp160 DNA引物与对照非VH5-51 mimotope-CTB结合进行免疫。将在免疫前、免疫期间以及最后一次免疫后1、3、6、12和18个月抽取每只动物的血液标本。每组选择一只动物的纵向PBMC标本，用生物素化V3-Fc融合蛋白选择IgG+单个B细胞，生产抗v3单克隆抗体。来自v3特异性B细胞的Ig可变基因将被扩增

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.