Immunogenicity of the newly identified V3 crown vulnerable site

为了能够诱导针对定义的HIV-1包膜（Env）刺突易损位点的抗体（Ab）应答， 通过广泛中和抗体（bnAbs）将可能导致保护性疫苗。许多HIV bnAb已经被 已经发现了它们的表位，并且它们的表位已经被仔细地绘制在Env的表面上，并且这些表位已经被 它被分为几个很好的类别。然而，持续诱导bnAb 针对任何目前已明确特征的脆弱地点的应对措施仍然极为困难， 这些bnAb及其相关易受攻击位点的不寻常特征，例如需要长时间的 CDR H3穿透Env加标物的聚糖屏蔽。因此，确定其他易受攻击的网站， 更有利的合理靶向特征可能有助于提高我们诱导bnAb的能力 应答我们最近通过定位bnAb M4008_N1的表位鉴定了一个新的脆弱位点， 表征其抗体-抗原相互作用。M4008_N1是一种bnAb，可以中和约40%的 测试的原代分离株及其谱系类别转换为IgG和伊加。我们通过冷冻电镜发现， 在融合前，M4008_N1的表位以gp 120的第三可变环（V3）的冠区为中心 Env三聚体。M4008_N1表位的独特特征及其与V3冠的相互作用模式 提出了通过V3初免和异源加强诱导M4008_N1样Ab应答策略， 稳定的Env三聚体。正如我们先前所证明的，V3冠状抗体应答相对较低。 直接被V3免疫原诱导。因此，我们假设，由于V3冠发夹是一个 共同结构和M4008_N1可以结合V3环本身，抗体通过β- 片层相互作用，如M4008_N1，可由V3免疫原诱导，且这种M4008_N1样 然后可以用V3稳定的Env三聚体进行后续免疫来加强抗体应答， 其具有bnAb M4008_N1的整个表位。已知SOSIP免疫原的早期版本 诱导非中和性V3抗体，但一些V3稳定版本似乎能够清除非中和性V3抗体， 中和V3抗体应答。因此，这些V3稳定的SOSIP三聚体可用于制备聚体。 - 异源加强以诱导M4008_N1样抗体应答。我们将在兔子模型中测试我们的假设 使用一组精心选择的在异源初免/加强策略中是最佳的，并且我们有两个目的。 (1)最佳免疫原组合的选择，（2）：兔免疫和鉴定 抗体反应。该项目的预期成果是对 针对新发现的V3冠易感位点的免疫原性，为未来 广泛的免疫原性研究，包括潜在的非人灵长类动物研究。

To be able to induce antibody (Ab) responses against the HIV-1 envelope (Env) spike vulnerable sites defined by broadly neutralizing antibodies (bnAbs) will likely lead to a protective vaccine. Many HIV bnAbs have been discovered and their epitopes have been carefully mapped on the surface of Env, and these epitopes have been categorized into a number of well characterized classes. However, a consistently induction of bnAb responses targeting any of those currently well characterized vulnerable sites is still extremely difficult because of the unusual features of these bnAbs and their associated vulnerable sites, such as the requirement of a long CDR H3 to penetrate the glycan shield of the Env spike. Thus, identification of additional vulnerable sites with more favorable characteristics for rational targeting may help to increase our capability of induction of bnAb responses. We have identified recently a new vulnerable site by mapping the epitope of bnAb M4008_N1 and characterizing its antibody-antigen interaction. M4008_N1 is a bnAb that can neutralize about 40% of the primary isolates tested and its lineage class-switched to both IgG and IgA. We showed that by cryo-EM the epitope of M4008_N1 is centered at the crown region of the third variable loop (V3) of gp120 in the prefusion Env trimer. The unique characteristics of the M4008_N1 epitope and its mode of interaction with the V3 crown suggest a strategy to induce M4008_N1-like Ab responses by a V3 priming and heterologous boosting with stabilized Env trimer. As we have demonstrated previously, the V3 crown Ab responses are relatively straightforward to elicit by V3 immunogens. We therefore hypothesize that, since the V3 crown -hairpin is a common structure and M4008_N1 can bind V3 loop itself, antibodies that engage the N-terminal strand by a β- sheet interaction, like that of M4008_N1, can be induced by V3 immunogens, and such M4008_N1-like antibody responses can then be boosted with a subsequent immunization with a V3-stabilized Env trimer, which harbors the whole epitope of bnAb M4008_N1. Early versions of the SOSIP immunogens were known to induce non-neutralizing V3 antibodies, but some V3-stabilized versions seemed to be able to purge non- neutralizing V3 antibody responses. These V3-stabilized SOSIP trimers can therefore be used for the heterologous boost to induce M4008_N1-like antibody responses. We will test our hypothesis in a rabbit model using a set of carefully selected to be optimal in the heterologous prime/boost strategy, and we have two Aims. (1) Selection of the best immunogen combination, (2): Rabbit immunization and characterization of the antibody responses. The expected outcome of this project is a proof of principle demonstration for the immunogenicity targeting the newly identified V3 crown vulnerable site and to lay a foundation for future extensive immunogenicity studies including potentially non-human primate studies.