Nanoengineered HIV-1 Vaccines Based on Tat

描述（由申请人提供）：人类免疫缺陷病毒（HIV）的Tat蛋白是在感染早期产生的蛋白质，是病毒有效复制所必需的。这表明艾滋病毒亚型之间的差异很小。Tat从细胞中以相对较高的水平释放，可以在HIV感染者的血清中检测到。细胞内Tat被主要组织相容性复合体（MHC） I类有效处理，并提呈给细胞毒性T淋巴细胞（CTL）。一些研究小组提出了这样一种想法，即Tat将成为一种合乎逻辑的预防性候选疫苗，并且同时需要中和抗体和CTL反应。我们研究的总体目标是开发基于纳米颗粒的HIV-1 Tat疫苗，通过纳米颗粒在佐剂蛋白或“裸”质粒DNA上单独引发增强的Th1、CTL和体液免疫反应。这项为期24个月的研究计划的具体目标是证明两种不同类型的基于纳米颗粒的HIV-1疫苗可以由新型微乳前体制成，在局部应用或皮下注射小鼠后，将重组Tat （rTat）或表达Tat的质粒DNA （pDNA-Tat）递送到树突状细胞（dc）。稳定的阴离子纳米颗粒（1型）或阳离子纳米颗粒（2型）将采用一种廉价、可复制和可扩展的工艺从新型微乳液前体中设计出来，分别用于rTat和pDNA-Tat的涂层。两个特定目标如下：特定目标#1：制备纳米工程HIV-I疫苗。i)证明阴离子纳米颗粒可以包被rTat，阳离子纳米颗粒可以包被pDNA-Tat， ii)证明树突细胞特异性配体甘露聚糖可以包被两种类型的纳米颗粒，以及iii)在颗粒大小、表面电荷、稳定性以及人类树突细胞的细胞摄取和/或转染方面表征纳米颗粒基疫苗。具体目标2：在小鼠体内测试纳米工程HIV-1疫苗。i)证明纳米颗粒在体内靶向dc，并在体外测试原型纳米颗粒的佐剂性，ii)在局部和皮下给药后，这些甘露聚糖包被的纳米颗粒疫苗在铝佐剂rTat或“裸”质粒DNA上增强了Th1、CTL和体液免疫反应。iii)证明使用rTat和pDNA-Tat的基于纳米颗粒的HIV-1疫苗的异源启动-增强方案优于单独使用rTat或pDNA-Tat免疫的同源方案。该建议涉及树突状细胞靶向纳米颗粒含有rTat或pDNA-Tat的新型和创新的疫苗发现和开发概念，以及使用Tat（1-72）产生保护性Th1， CTL和体液反应。开发一种合适的基于纳米颗粒的HIV-1 Tat疫苗也将有利于整合多种HIV基因（如Tat、Env、Gag、Pol和Rev）的策略。

DESCRIPTION (provided by applicant): The Tat protein of the human immunodeficiency virus (HIV) is a protein produced early in infection and is required for efficient replication of the virus. Tat shows very little variation between HIV subtypes. Tat is released from cells at relatively high levels and can be detected in the serum of HIV infected individuals. Intracellular Tat is efficiently processed by major histocompatibility complex (MHC) class I for presentation to cytotoxic T lymphocytes (CTL). Several groups have advanced the idea that Tat would make a logical prophylactic vaccine candidate and that both neutralizing antibody and CTL responses are needed. The overall goal of our research is to develop nanoparticle-based HIV-1 Tat vaccines to elicit enhanced Th1, CTL, and humoral immune responses with nanoparticles over either adjuvanted protein or 'naked' plasmid DNA alone. The specific goal of this 24 month Research Plan is to demonstrate that two different types of nanoparticle-based HIV-1 vaccines can be engineered from novel microemulsion precursors to deliver either recombinant Tat (rTat) or plasmid DNA expressing Tat (pDNA-Tat) to dendritic cells (DCs) after either topical application or subcutaneous injection in mice. Stable anionic nanoparticles (type 1) or cationic nanoparticles (type 2) will be engineered from novel microemulsion precursors using an inexpensive, reproducible, and scalable process and used for coating by rTat and pDNA-Tat, respectively. The two Specific Aims are as follows: Specific Aim #1: Preparation of Nanoengineered HIV-I Vaccines. i) demonstrate that anionic nanoparticles can be coated with rTat, and that cationic nanoparticles can be coated with pDNA-Tat, ii) demonstrate that a dendritic cell-specific ligand, mannan, can be coated on both types of nanoparticles, and iii) characterize the nanoparticle-based vaccines in terms of particle size, surface charge, stability, and cell uptake and/or transfection in human dendritic cells. Specific Aim #2: Testing of Nanoengineered HIV-1 Vaccines in Mice. i) demonstrate nanoparticle targeting to DCs in-vivo and test adjuvanticity of prototype nanoparticles in-vitro, ii) demonstrate enhanced Th1, CTL, and humoral immune responses with these mannan-coated nanoparticle vaccines over either ALUM-adjuvanted rTat or 'naked' plasmid DNA after both topical and subcutaneous administration, and iii) demonstrate that a heterologous prime-boost regimen of nanoparticle-based HIV-1 vaccines using rTat and pDNA-Tat is superior to a homologous regimen using either rTat or pDNA-Tat immunization alone. This proposal relates to novel and innovative vaccine discovery and development concepts of dendritic cell-targeted nanoparticles containing either rTat or pDNA-Tat, and the use of Tat (1-72) to generate protective Th1, CTL, and humoral responses. The development of an appropriate nanoparticle-based HIV-1 Tat vaccine will also benefit strategies that may incorporate multiple HIV genes such as Tat, Env, Gag, Pol, and Rev.