Induction of HIV Neutralizing Antibodies by Targeting Macaque B Cell Receptors

描述（由申请方提供）：诱导可保护健康个体免受HIV感染的广泛交叉中和抗体（Abs）仍然是疫苗开发的主要挑战。这些中和抗体在自然HIV-1感染的过程中观察到，在感染后2至3年出现，这提出了它们是否可以在相对较短的免疫期内诱导的问题。我们提出了一种新的方法，以（a）采用靶向幼稚B细胞上的选择的免疫球蛋白（IG）基因编码的Ab的免疫原（模拟表位融合蛋白），所述抗体是抗V3中和Ab的前体，和（B）监测亲和力成熟和假病毒交叉中和效力的发展。为了实现这些目标，我们已经开发了一种基于VH 5 -51模拟表位的合理设计的免疫原，该模拟表位模拟由VH 5 -51和VL λ基因的配对编码的人交叉中和抗V3单克隆抗体（mAb）识别的高度保守的V3表位。我们假设VH 5 -51模拟表位可以靶向猕猴B细胞受体（由VH 5 -51和VL λ基因编码），与对照模拟表位（非VH 5 -51）相比，它将诱导具有显著增强的亲和力成熟的Ab，对照模拟表位（非VH 5 -51）将诱导由不同IG基因编码的抗V3 Ab。在第一个目标中，我们将从来自恒河猴的抗原特异性单个B细胞产生单克隆抗V3抗体，所述抗原特异性单个B细胞用两种免疫原免疫以特异性地引发由VH 5 -51或由其他非VH 5 -51基因编码的抗V3抗体。将用gp 160 DNA引发剂与VH 5 - 51模拟表位-CTB融合蛋白的组合或gp 160 DNA引发剂与对照非VH 5 -51模拟表位-CTB的组合免疫两组，每组包括三只恒河猴。将在免疫前、免疫期间和末次免疫后1、3、6、12和18个月采集每只动物的血样。将选择每组中1只动物的纵向PBMC标本，用于从使用生物素化V3-Fc融合蛋白选择的IgG+单个B细胞中生产抗V3 mAb。将扩增来自V3特异性B细胞的IG可变基因 通过RT-PCR，克隆到表达载体中，并且在质粒共转染后，将从293 T细胞产生全长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（IC 50）来监测抗V3 mAb的成熟，所述抗V3 mAb由用VH 5 -51和非VH 5 -51免疫原免疫的猕猴顺序产生。将在VH 5 -51衍生的抗V3 mAb和非VH 5 -51衍生的抗V3 mAb之间比较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提供靶向编码具有不同特异性的中和Ab的其它IG基因的机会。对于疫苗开发，基于靶向IG基因的模拟表位的免疫原可用于与gp 120的组合加强，以激发针对特定包膜中和表位的免疫应答。 公共卫生相关性：拟议研究的目标是在非人灵长类动物中测试，用作临床前模型，诱导抗HIV-1交叉中和抗体的可能性。将用合理设计的抗原免疫猴子，刺激B细胞产生仅由选定的免疫球蛋白基因编码的抗体。由这些基因编码的抗体预先适应病毒包膜上的V3抗原，与其他对照疫苗免疫原相比，这导致抗体应答的快速成熟和有效的HIV-1中和。

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.