Peptide Vaccines to Elicit HIV-1-Neutralizing Antibodies

描述：(申请人提供)三株广谱中和单抗 抗体(MAb)B12、2F5和2G12已从猪B细胞中克隆出来 持续感染艾滋病毒-1的人。这些单抗针对的是包膜蛋白 HIV-1和最近单独使用它们的被动免疫研究，以及在 结合在一起，已经显示出对猕猴抵抗IV-和 新城疫病毒致病毒株的黏膜攻击剂量。这些结果表明， 广谱中和抗体可以产生对病毒的灭菌保护。 我们设计艾滋病疫苗的目的是诱导出与 主动免疫B12、2F5和2G12。我们正在开发能结合的多肽 与这些单抗紧密而具体地联系在一起，并计划将它们用于一部小说 针对这些相同抗体特异性产生的免疫策略 在幼稚的动物身上。首先，我们将用包膜蛋白启动动物以诱导 针对整体的多克隆反应中的少量靶向抗体 蛋白。下一步，将使用单抗特异性多肽来提高产量。 只有目标抗体。强大的T细胞表位将在 Env蛋白底物和多肽增强以维持B细胞反应。单抗b12， 2F5，可能还有2G12，都有非常长的H3超变量环路 老鼠“不能产生抗体。因此，我们也必须使用能够产生这种抗体的动物。 我们用B12单抗在这种方法上取得了最大的进展。这种多肽， B2.1，与B12单抗结合紧密，并与其特异性结合。中的B2.1的结构 与B12 Fab的复合体已经被阐明，并正在被用于进一步的工程 B2.1多肽。我们发表的研究清楚地表明，B12的亲和力 重组B2.1融合成更大的蛋白质比其更强 合成肽类似物。因此，我们建议免疫兔子XenoMouseTM 动物，最终是猕猴，噬菌体携带Env蛋白，其次是 增强与噬菌体展示B2.1肽。将对血清抗体效价进行检测 B12样反应性的存在，并了解分子和 这些反应的细胞基础，含有抗B2.1抗体和抗gp120的B细胞 用流式细胞仪分离ABS，用RT-PCR方法克隆其表达的V基因 在单个细胞上。将对具有所需反应性的克隆抗体进行测试 HIV-1-中和。化验应该足够灵敏，可以检测到低浓度 类B12抗体及其B细胞水平。通过这种方式，高滴度的应答可能 从最初较低的基础上建造。我们已经开发出单抗2F5的多肽导联 和2G12，并正在为新发现的单抗4E10和Z13这样做；这些 也会受到同样的考验。

DESCRIPTION: (Provided by Applicant) Three broadly-neutralizing monoclonal antibodies (MAbs), b12, 2F5 and 2G12, have been cloned from the B cells of people with on-going HIV-1 infections. These MAbs target envelope proteins of HIV-1 and recent passive-immunization studies using them alone, and in combination, have shown protection of macaques against IV- and mucosal-challenge doses of pathogenic SHIV strains. These results indicate that broadly-neutralizing Abs can produce sterilizing protection against the virus. Our aim in designing an AIDS vaccine is to induce the same Ab specificities as b12, 2F5 and 2G12 by active immunization. We are developing peptides that bind tightly and specifically to these MAbs, and plan to use them in a novel immunization strategy to target the production of these same Ab specificities in naive animals. First, we will prime animals with envelope proteins to elicit small amounts of the targeted Abs in the polyclonal response against the whole protein. Next, the MAb-specific peptides will be used to boost the production of only the targeted Abs. Strong T-cell epitopes will be conserved between the Env protein primes and peptide boosts to maintain B-cell responses. MAbs b12, 2F5, and probably 2G12, have very long H3 hypervariable loops that "normal mice" cannot produce. Thus, we also must use animals that can produce such Abs. We have made the most progress in this approach with the b12 MAb. The peptide, B2.1, binds tightly and specifically to the b12 MAb. The structure of B2.1 in complex with b12 Fab has been elucidated, and is being used to further engineer the B2.1 peptide. Our published studies clearly show that the affinity of b12 is stronger for recombinant B2.1 fused to a larger protein than for its synthetic-peptide analog. Thus, we propose to immunize rabbits, XenoMouseTM animals, and eventually macaques, with phage bearing Env proteins, followed by boosts with phage displaying B2.1 peptide. Serum Ab titers will be tested for the presence of b12-like reactivity, and to understand the molecular and cellular bases of these responses, B-cells bearing anti-B2.1 Abs and anti-gp120 Abs will be isolated by FACS, and their expressed V-genes cloned using RT-PCR on single cells. Cloned Abs having the desired reactivities will be tested for HIV-1-neutralization. The assays should be sensitive enough to detect low levels of b12-like Abs and their B cells. In this way, high-titer responses may be built from initially low ones. We have developed peptide leads for Mabs 2F5 and 2G12, and are doing so for the newly-discovered Mabs 4E10 and Z13; these will be similarly tested.