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: 8685883
• 负责人: Venigalla B. Rao
• 金额: $ 48.54万
• 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/8685883
• 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（由应用程序提供）：该提案的一个主要目标是开发一种新型的实验范式，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目标，原因有几个。 V2与粘膜T细胞的4€7整联蛋白的相互作用在有效捕获HIV-1病毒的关键作用 性传播期间初次接触的地点。 V1/V2结构域也是控制HIV-1神经敏感性的关键参与者，由迄今为止报告的一些最有效的广泛中和抗体所识别的表位组成。在这里，我们假设通过使用干扰V2/V2免疫原的V1/V2免疫原子（特别是V2€4€7相互作用）来诱导抗体，从而赋予了对HIV-1采集的保护。由于V2的构象高度可变，因此V2结构域将通过将其移植到细菌噬菌体T4中的9 kDa小型外皮蛋白（SOC）中来限制。为了选择功能上重要的V2构象，V2结构域变体的库由创始人/急性病毒序列，不同的糖基化模式和随机突变组成，将在CHO细胞中构造并表达为秘密蛋白质。将开发高吞吐量测定，一种选择€4€7结合会议，另一个选择V2宪法抗体结合变体。通过将纯化的SOC-V2蛋白与Soc-Minus Hoc-Minus噬菌体孵育，每个粒子的纯净SOC-V2蛋白将孵育纯化的SOC-V2蛋白，将在噬菌体T4纳米颗粒上批准生物活性SOC-V2会议变体。诸如DEC205 MAB之类的HOC粘合靶向配体也将在同一帽质上组装，最多可达155份人均拷贝，以将T4-V2纳米颗粒靶向抗原呈现的树突状细胞。 T4-V2纳米颗粒的免疫原性将通过肌内途径以及使用热标记肠毒素作为可调的小鼠评估。免疫呼应将用于V2-€4€4的干​​扰抗体，中和抗体和传输阻断抗体。从这些测定法下选择的最佳V2变体将在兔模型中测试。将开发出一种新型的HIV-1传输测定法，该测定将准确地量化几分钟接触CD4+，4¿7+和CCR5+ T细胞的病毒基因组数量。基于实时PCR，该测定法将非常敏感，快速且高吞吐量。使用此测定，将选择可以诱导抗体来诱导抗体来阻断病毒进入的V2变体，从而干扰初始病毒 - 宿主相互作用。这些可能有可能作为预防性HIV-1疫苗候选者，以进一步研究非人类隐私和临床试验。