Potent Phage T4 Derived V2 Immunogens as HIV Vaccines

有效的噬菌体 T4 衍生 V2 免疫原作为 HIV 疫苗

• 批准号: 8494569
• 负责人: Venigalla B. Rao
• 金额: $ 36.65万
• 依托单位: CATHOLIC UNIVERSITY OF AMERICA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8494569
• 关键词: Acute; Adjuvant; Affinity; Antibodies; Antigens; Bacteriophage T4; Bacteriophages; Binding; Biological Assay; Biological Process; Blocking Antibodies; CCR5 gene; Capsid; Capsid Proteins; Cells; Chinese Hamster Ovary Cell; Clinical Trials; Dendritic Cells; Development; Engineering; Enterotoxins; Epitopes; Exposure to; Genome; Goals; HIV; HIV vaccine; HIV-1; Heating; Immune response; In Vitro; Incubated; Integrins; Intramuscular; Length; Libraries; Ligands; Membrane; Modeling; Molecular Conformation; Molecular Genetics; Monoclonal Antibodies; Mus; Mutation; Oryctolagus cuniculus; Pattern; Phase; Play; Protein Binding; Proteins; Reporting; Route; Sexual Transmission; Site; Skin; Specificity; Surface; System; T-Lymphocyte; Testing; Time; Transplantation; Vaccination; Vaccines; Variant; Virus; base; fitness; glycosylation; high throughput screening; immunogenicity; nanoparticle; neutralizing antibody; nonhuman primate; novel; particle; prevent; prophylactic; scaffold; secretory protein; transmission process; vaccine candidate; vaccine delivery; vaccine development; virus host interaction

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.

描述（由申请人提供）：该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 HIV-1 疫苗。出于多种原因，HIV-1 包膜的 V1/V2 结构域是 V2 疫苗开发的重要目标。与 ¿ 的互动粘膜T细胞的4¿7整合素可能在高效捕获HIV-1病毒中发挥关键作用 V1/V2 结构域也是调节 HIV-1 中和敏感性的关键因素，由迄今为止报道的一些最有效的广泛中和抗体所识别的表位组成。通过接种 V1/V2 免疫原（特别是 V2 干扰）来诱导抗体 ¿ 4¿7 相互作用，提供针对 HIV-1 获取的保护，因为 V2 的构象高度可变，因此将通过将其移植到来自噬菌体 T4 的紧凑折叠的 9 kDa 小外衣壳蛋白 (Soc) 中来限制 V2 结构域。对于功能上重要的 V2 构象，将构建由创始人/急性病毒变体、不同糖基化模式和随机突变组成的 V2 结构域文库，并在 CHO 细胞中作为分泌表达将开发高通量检测方法，供选择 ¿ 4¿7 结合构象和另一个选择 V2 构象抗体结合变体 通过将纯化的 Soc-V2 蛋白与 Soc-minus Hoc-minus 噬菌体（最多 870）一起孵育，将具有生物活性的 Soc-V2 构象变体排列在噬菌体 T4 纳米颗粒上。每个颗粒的拷贝数，例如 Dec205 mAb 等 Hoc 融合靶向配体也将组装在同一衣壳上，最多 155 个拷贝每个衣壳，将 T4-V2 纳米颗粒靶向呈递抗原的树突状细胞。将使用热不稳定肠毒素作为免疫佐剂，通过肌内途径和经皮（皮肤）途径评估 T4-V2 纳米颗粒的免疫原性。响应将被量化为 V2- ¿然后将从这些测定中筛选出来的最佳 V2 变异体在兔子模型中进行测试，从而精确量化 4¿7 干扰抗体、病毒中和抗体和传播阻断抗体。暴露于 CD4+ 几分钟后穿过宿主膜的病毒基因组数量，¿基于实时 PCR 的 4¿7+ 和 CCR5+ T 细胞，该检测极其灵敏、快速且高通量。使用该检测，V2 变体可诱导抗体通过干扰初始病毒来阻止病毒进入。这些可能作为预防性 HIV-1 疫苗候选物，用于非人类灵长类动物的进一步研究和临床试验。