Nanoparticle HIV Protein Vaccines for Cellular Responses

描述(申请人提供)：目前，尚无有效的艾滋病毒感染疫苗。艾滋病毒疫苗有可能用于预防感染或在治疗上控制暴露后的艾滋病毒复制水平。 这项为期5年的研究计划的总体目标是开发基于纳米颗粒的HIV-1疫苗，以诱导增强的Th1、细胞毒性T淋巴细胞(CTL)和对重组TAT(1-72)和Gag p24蛋白的体液免疫反应。所开发的基于纳米颗粒的HIV-1TAT(1-72)和Gag p24疫苗的免疫原性将进一步增强，方法是：1)将树突状细胞靶向配体甘露五糖附着到纳米颗粒上，2)将CpG包裹在纳米颗粒上，以靶向Toll样受体-9(TLR-9)。 优化的纳米颗粒疫苗将与基于表达抗原和佐剂蛋白的牛痘疫苗的异种主要增强策略进行比较。 重组HIV Tat(1-72)和Gag p24蛋白以及His标记的蛋白将由约翰·霍普金斯大学的Nath博士的实验室合成和纯化至均一，并进行生物活性测试。在肯塔基大学芒珀博士的实验室里，这些蛋白质将被覆盖在阴离子纳米颗粒上(类型1)，或者附着在由表面少量可接触到的镍制成的纳米颗粒上(类型2)。这两种类型的固体纳米颗粒(&lt；100 nm)都将由新型水包油微乳液前体制成。芒珀博士的实验室将对纳米颗粒进行表征，在小鼠和人类树突状细胞中进行体外摄取和激活研究，并进行动物实验，以确定纳米颗粒配方的体液免疫反应，并进行免疫组织化学分析。伍德沃德博士在肯塔基大学的实验室将负责ELISPOT、CTL、多探针核糖核酸酶、四聚体、细胞增殖分析以及共聚焦显微镜和流式细胞术实验。 纳特博士的实验室将进行研究，证明免疫小鼠的血清可以抑制TAT诱导的LTR反式激活，并将评估TAT和Gag p24抗血清对HIV复制的影响。此外，纳特博士的实验室还将生产表达抗原的牛痘疫苗。

DESCRIPTION (provided by applicant): Currently, there is no effective vaccine for HIV infection. HIV vaccines can potentially be used for prevention of infection or therapeutically to control the level of HIV replication post-exposure. The overall goal of this 5-year research proposal is to develop nanoparticle-based HIV-1 vaccines to elicit enhanced Th1, cytotoxic T lymphocyte (CTL), and humoral immune responses to recombinant Tat (1-72) and Gag p24 proteins. The immunogenicity of the developed nanoparticle-based HIV-1 Tat (1-72) and Gag p24 vaccine will further be enhanced by, 1) the attachment of a dendritic cell targeting ligand, mannopentaose, to the nanoparticles, and 2) coating the nanoparticles with CpG to target the toll-like receptor-9 (TLR-9). Optimized nanoparticles vaccines will be compared to a heterologous prime boost strategy based on vaccinia expressing antigen and adjuvanted protein. Recombinant HIV Tat (1-72) and Gag p24 proteins and his-tagged proteins will be synthesized and purified to homogeneity by Dr. Nath's laboratory at Johns Hopkins and tested for bioactivity. In Dr. Mumper's laboratory at the University of Kentucky, the proteins will be either coated on anionic nanoparticles (Type 1) or attached to nanoparticles made with a small amount of accessible nickel at the surface (Type 2). Both types of solid nanoparticles (<100 nm) will be made from novel oil-in-water microemulsion precursors. Dr. Mumper's lab will characterize the nanoparticles, perform in-vitro uptake and activation studies in mouse and human dendritic cells, and perform animal experiments to determine the humoral immune responses of the nanoparticle-based formulations, and perform immunohistochemical analyses. Dr. Woodward's laboratory at the University of Kentucky will be responsible for ELISPOT, CTL, multi-probe ribonuclease, tetramer, cell proliferation assays, as well as confocal microscopy and flow cytometry experiments. Dr. Nath's laboratory will perform studies demonstrating that sera from immunized mice can suppress Tat-induced LTR-transactivation, and will assess the effect of Tat and Gag p24 antisera on HIV replication. In addition, Dr. Nath's lab will produce vaccinia expressing antigen.