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。这些中和抗体是在自然感染HIV-1的过程中观察到的，出现在感染后2到3年，提出了在相对较短的免疫期间是否可以诱导它们的问题。我们提出了一种新的方法来(A)使用一种免疫原(模拟表位融合蛋白)来靶向作为抗V3中和抗体前体的幼稚B细胞上的选择免疫球蛋白(Ig)基因编码的抗体，以及(B)监测伪病毒的亲和力成熟和交叉中和能力的发展。为了达到这些目标，我们开发了一种基于VH5-51模拟表位的合理设计的免疫原，它模拟了由VH5-51和VL lambda基因配对编码的人交叉中和抗V3单抗(MAbs)识别的高度保守的V3表位。我们假设VH5-51模拟表位可以靶向恒河猴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，它将诱导亲和力显著增强的抗体成熟，从而诱导由不同Ig基因编码的抗V3抗体。在第一个目标中，我们将从两种免疫原免疫的恒河猴单个B细胞中产生抗V3的单抗，以特异性地诱导由VH5-51或其他非VH5-51基因编码的抗V3抗体。两组各包括三只恒河猴，分别用gp160 DNA PRIME和VH5-51模拟表位-CTB融合蛋白或gp160 DNA PRIME和对照非VH5-51模拟表位-CTB免疫。每只动物的血液样本将在免疫前、免疫期间以及最后一次免疫后1、3、6、12和18个月抽取。每组一只动物的纵向PBMC样本将被选择用于从使用生物素化的V3-Fc融合蛋白选择的免疫球蛋白+单个B细胞中产生抗V3单抗。将从V3特异性B细胞中扩增Ig可变区基因 通过RT-PCR，将其克隆到表达载体上，共转染293T细胞，可获得全长的抗体。在第二个目标中，我们将通过检测变异率、相对亲和力(50%最大结合)和中和活性(IC50)来监测经VH5-51免疫和非VH5-51免疫的猕猴依次产生的抗V3单抗的成熟度。我们将比较VH5-51和非VH5-51衍生的抗V3单抗亲和力和中和活性的变化。这些结果证明了靶向特定Ig基因编码的V3B细胞受体的可行性，为靶向其他编码不同特异性中和抗体的Ig基因提供了机会。在疫苗开发中，基于Ig基因靶向模拟表位的免疫原可用于与gp120联合加强免疫，以增强针对特定包膜中和表位的免疫反应。

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.