Peptide Vaccines to Elicit HIV-1-Neutralizing Antibodies

描述：（由申请人提供）三种广泛中和的单克隆抗体 已经从以下小鼠的B细胞克隆了抗体（MAb）b12、2F 5和2G 12， HIV-1感染者。这些单克隆抗体靶向的包膜蛋白 HIV-1和最近单独使用它们的被动免疫研究， 组合，已经显示出保护猕猴免受IV-和 粘膜攻击剂量的致病性SHIV毒株。这些结果表明 广泛中和的抗体可以产生针对病毒的杀菌保护。 我们设计艾滋病疫苗的目的是诱导与 b12、2F 5和2G 12主动免疫。我们正在开发一种肽， 紧密和具体到这些单克隆抗体，并计划在小说中使用它们 针对这些相同Ab特异性产生的免疫策略 在天真的动物身上。首先，我们将用包膜蛋白来引导动物， 在针对整个抗体的多克隆反应中， 蛋白下一步，MAb特异性肽将用于促进生产 只针对目标腹肌强T细胞表位将在免疫球蛋白之间保守。 Env蛋白引发和肽加强以维持B细胞应答。单克隆抗体b12， 2F 5，可能还有2G 12，有很长的H3高变环， 老鼠不能生产。因此，我们也必须使用能够产生这种抗体的动物。 我们在b12单克隆抗体方面取得了最大的进展。肽， B2.1与b12 MAb紧密特异性结合。B2.1的结构 已经阐明了与b12 Fab的复合物，并正在用于进一步工程化 B2.1肽。我们发表的研究清楚地表明，b12的亲和力 对于与较大蛋白融合的重组B2.1， 合成肽类似物。因此，我们建议免疫兔子，XenoMouseTM 动物，最终是猕猴，与携带Env蛋白的噬菌体，然后是 用展示B2.1肽的噬菌体增强。将检测血清Ab滴度， B12样反应的存在，并了解分子和 这些反应的细胞基础，携带抗B2.1抗体和抗gp 120的B细胞 将通过FACS分离Ab，并使用RT-PCR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检测， HIV-1中和。检测试剂盒应足够灵敏， b12样抗体及其B细胞的水平。通过这种方式，高滴度反应可以 从最初的低层开始建造。我们已经开发了单克隆抗体2F 5的肽先导化合物 和2G 12，并正在为新发现的Mab 4 E10和Z13这样做;这些 也将接受类似的测试。

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.