Potent Phage T4 Derived V2 Immunogens as HIV Vaccines

描述（由申请人提供）：该提案的主要目标是开发一种新的实验范式，以设计预防性HIV-1疫苗。HIV-1包膜的V1/V2结构域是疫苗开发的重要靶标，原因有几个。V2与粘膜T细胞的<$4 <$7整联蛋白的相互作用可能在HIV-1病毒的有效捕获中起关键作用。 在性传播过程中最初接触的部位。V1/V2结构域也是调节HIV-1中和敏感性的关键因素，并且由迄今报道的一些最有效的广泛中和抗体识别的表位组成。在这里，我们假设通过接种V1/V2免疫原诱导抗体，干扰，特别是V2 <$4 <$7相互作用，赋予对HIV-1获得的保护。 由于V2的构象是高度可变的，V2结构域将通过将其移植到来自噬菌体T4的复合折叠的9 kDa小外壳蛋白（Soc）中而受到限制。为了选择功能上重要的V2构象，将构建由创始者/急性病毒序列、不同糖基化模式和随机突变组成的V2结构域变体文库，并在CHO细胞中表达为分泌蛋白。将开发高通量测定，一种用于选择V4、V7结合构象，另一种用于选择V2构象抗体结合变体。 通过将纯化的Soc-V2蛋白与Soc-负Hoc-负噬菌体一起孵育，将生物活性Soc-V2构象变体排列在噬菌体T4纳米颗粒上，每个颗粒高达870个拷贝。Hoc融合的靶向配体如Dec 205 mAb也将组装在相同的衣壳上，每个衣壳多达155个拷贝，以将T4-V2纳米颗粒靶向抗原呈递树突细胞。 T4-V2纳米颗粒的免疫原性将通过肌内途径以及使用热不稳定肠毒素作为佐剂的经皮（皮肤）途径在小鼠中进行评价。将对V2-<$4 <$7干扰抗体、病毒中和抗体和传播阻断抗体的免疫应答进行定量。然后将在兔模型中检测从这些试验中向下选择的最佳V2变体。 将开发一种新的HIV-1传播检测方法，该方法将精确定量暴露于CD 4+、CD 4 + 7+和CCR 5 + T细胞几分钟后穿过宿主膜的病毒基因组数量。基于实时PCR，该检测将是非常敏感，快速，高通量。使用该试验，将选择可诱导抗体的V2变体，这些抗体通过干扰初始病毒-宿主相互作用来阻断病毒进入。这些可能作为预防性HIV-1疫苗候选物，用于在非人灵长类动物和临床试验中的进一步研究。 公共卫生相关性：艾滋病毒在暴露部位的传播机制以及如何开发能够预防艾滋病毒的疫苗仍然是开发预防性艾滋病毒疫苗的关键目标。该提案旨在分析HIV包膜组件之一，V1/V2结构域，该结构域参与病毒与宿主之间的初始相互作用。利用噬菌体T4展示、分子遗传学、高通量筛选和新的HIV传播测定的组合，可以诱导传播阻断抗体的V1/V2纳米颗粒免疫原将被选择作为潜在的HIV预防性疫苗。

DESCRIPTION (provided by applicant): A major goal of this proposal is to develop a novel experimental paradigm to engineer preventative HIV-1 vaccines. The V1/V2 domain of HIV-1 envelope is an important target for vaccine development for several reasons. V2 interaction with the ¿4¿7 integrin of mucosal T cells may play a key role in the efficient capture of HIV-1 virus at the site of initial exposure during sexual transmission. The V1/V2 domain is also a key player in regulating neutralization sensitivity of HIV-1, and consists of epitopes recognized by some of the most potent broadly neutralizing antibodies reported to date. Here, we hypothesize that induction of antibodies by vaccination with V1/V2 immunogens that interferes, specifically with the V2 ¿4¿7 interactions, confers protection against HIV-1 acquisition. Since the conformation of V2 is highly variable, the V2 domain will be constrained by transplanting it into the compactly folded 9 kDa small outer capsid protein (Soc) from bacteriophage T4. To select for the functionally important V2 conformations, libraries of V2 domain variants consisting of founder/acute virus sequences, different glycosylation patterns, and random mutations will be constructed and expressed in CHO cells as secretory proteins. High throughput assays will be developed, one to select ¿4¿7 binding conformations and another to select V2 conformational antibody binding variants. The biologically active Soc-V2 conformational variants will be arrayed on phage T4 nanoparticles by incubating the purified Soc-V2 proteins with Soc-minus Hoc-minus phage, up to 870 copies per particle. A Hoc- fused targeting ligand such as Dec205 mAb will also be assembled on the same capsid, up to 155 copies per capsid, to target the T4-V2 nanoparticles to the antigen-presenting dendritic cells. The immunogenicity of T4-V2 nanoparticles will be evaluated in mice by intramuscular route as well as transcutaneous (skin) route using heat labile enterotoxin as an adjuvant. The immune responses will be quantified for V2- ¿4¿7 interfering antibodies, virus neutralizing antibodies and transmission-blocking antibodies. The best V2 variants down-selected from these assays will then be tested in the rabbit model. A novel HIV-1 transmission assay will be developed, which will precisely quantify the number of virus genomes that cross the host membrane after a few minutes of exposure to CD4+, ¿4¿7+ and CCR5+ T cells. Based on real-time PCR, this assay would be extremely sensitive, rapid, and high throughput. Using this assay, V2 variants that can induce antibodies which block virus entry by interfering with the initial virus-host interactions will be selected. These might potentially serve as preventative HIV-1 vaccine candidates for further studies in nonhuman primates and clinical trials. PUBLIC HEALTH RELEVANCE: The mechanism of HIV transmission at the site of exposure and how to develop vaccines that can prevent it remained as key goals in the development of preventative HIV vaccines. This proposal aims to analyze one of the HIV envelope components, the V1/V2 domain, which is involved in the initial interactions between the virus and the host. Using a combination of bacteriophage T4 display, molecular genetics, high throughput screening, and a new HIV transmission assay, V1/V2 nanoparticle immunogens that can induce transmission blocking antibodies will be selected as potential HIV preventative vaccines.