Potent Phage T4 Derived V2 Immunogens as HIV Vaccines

描述(由申请人提供)：这项提案的一个主要目标是开发一种新的实验范例来设计预防性艾滋病毒-1疫苗。HIV-1包膜的V1/V2结构域是疫苗开发的一个重要目标，原因有几个。V2与粘膜T细胞整合素的相互作用可能在有效捕获HIV-1病毒的过程中发挥关键作用 在性传播过程中最初暴露的地方。V1/V2结构域也是调节HIV-1中和敏感性的关键因素，它由一些迄今报道的最有效的广谱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的相互作用，提供对HIV-1感染的保护。由于V2的构象高度可变，将其移植到T4噬菌体紧密折叠的9 kDa小外壳蛋白(SOC)中将限制V2结构域的表达。为了筛选出具有重要功能的V2构象，我们将构建V2结构域变异体文库，该文库包括方正病毒/急性病毒序列、不同的糖基化模式和随机突变，并在CHO细胞中以分泌蛋白的形式表达。将开发高通量分析，一种用于选择结合构象，另一种用于选择V2构象抗体结合变异体。生物活性的Soc-V2构象变体将通过与Soc-阴性噬菌体孵育纯化的Soc-V2蛋白而排列在噬菌体T4纳米颗粒上，每个颗粒最多可达870个拷贝。一个特定融合的靶向配体，如Dec205单抗，也将组装在同一衣壳上，每个衣壳最多155个拷贝，以将T4-V2纳米颗粒靶向抗原递呈的树突状细胞。T4-V2纳米粒子的免疫原性将通过肌肉注射和经皮(皮肤)途径进行评估，以不耐热的肠毒素作为佐剂。将对V2-4干扰抗体、病毒中和抗体和传播阻断抗体的免疫反应进行量化。从这些检测中挑选出最好的V2变种，然后在兔模型中进行测试。将开发一种新的HIV-1传播分析方法，该方法将精确地量化接触CD4、4和CCR5 T细胞几分钟后穿过宿主膜的病毒基因组的数量。基于实时荧光定量聚合酶链式反应，该方法具有非常灵敏、快速、高通量的特点。使用这种检测方法，可以通过干扰最初的病毒-宿主相互作用来诱导抗体阻止病毒进入的V2变种将被挑选出来。这些可能作为预防性HIV-1疫苗候选，用于在非人类灵长类动物上的进一步研究和临床试验。 公共卫生相关性：艾滋病毒在接触地点的传播机制以及如何开发能够预防它的疫苗仍然是开发预防性艾滋病毒疫苗的关键目标。这项建议旨在分析HIV包膜成分之一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、高通量筛选和一种新的HIV传播试验相结合的方法，可以诱导传播阻断抗体的V1/V2纳米颗粒免疫原将被选为潜在的HIV预防疫苗。

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.