Nanoparticle HIV Protein Vaccines for Cellular Responses

描述（由申请人提供）：目前，没有有效的艾滋病毒感染疫苗。HIV疫苗可潜在地用于预防感染或治疗性地控制暴露后HIV复制的水平。 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，以引发对重组达特（1-72）和Gag p24蛋白的增强的Th 1、细胞毒性T淋巴细胞（CTL）和体液免疫应答。所开发的基于纳米颗粒的HIV-1达特（1-72）和Gag p24疫苗的免疫原性将通过以下方式进一步增强：1）树突状细胞靶向配体甘露五糖与纳米颗粒的连接，和2）用CpG包被纳米颗粒以靶向toll样受体-9（TLR-9）。 将优化的纳米颗粒疫苗与基于表达抗原和佐剂化蛋白质的牛痘的异源初免加强策略进行比较。 重组HIV达特（1-72）和Gag p24蛋白以及His标记蛋白将由约翰霍普金斯的Nath博士实验室合成并纯化至均一，并检测其生物活性。在肯塔基州大学的Mumper博士的实验室中，蛋白质将被涂覆在阴离子纳米颗粒上（1型）或附着在表面含有少量可接近镍的纳米颗粒上（2型）。这两种类型的固体纳米颗粒（<100 nm）将由新型水包油微乳液前体制成。Mumper博士的实验室将表征纳米颗粒，在小鼠和人类树突状细胞中进行体外摄取和活化研究，并进行动物实验以确定基于纳米颗粒的制剂的体液免疫反应，并进行免疫组织化学分析。伍德沃德博士在肯塔基州大学的实验室将负责ELISPOT、CTL、多探针核糖核酸酶、四聚体、细胞增殖测定以及共聚焦显微镜和流式细胞术实验。 博士Nath的实验室将进行研究，证明来自免疫小鼠的血清可以抑制Tat诱导的LTR反式激活，并将评估达特和Gag p24抗血清对HIV复制的影响。此外，纳特博士的实验室将生产表达抗原的牛痘。

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.