Nanoengineered HIV-1 Vaccines Based on Tat

描述（由申请方提供）：人类免疫缺陷病毒（HIV）的达特蛋白是一种在感染早期产生的蛋白质，是病毒有效复制所必需的。达特在HIV亚型之间的变异很小。达特以相对高的水平从细胞中释放，并且可以在HIV感染个体的血清中检测到。 细胞内达特被主要组织相容性复合物（MHC）I类有效加工以呈递给细胞毒性T淋巴细胞（CTL）。几个研究小组已经提出了这样的想法，即达特将成为一种合乎逻辑的预防性疫苗候选物，并且需要中和抗体和CTL应答。我们的研究的总体目标是开发基于纳米颗粒的HIV-1达特疫苗，以引发增强的Th 1，CTL和体液免疫反应的纳米颗粒，无论是佐剂蛋白或“裸”质粒DNA单独。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疫苗可以从新型微乳液前体工程化，以在小鼠局部应用或皮下注射后将重组达特（r达特）或表达达特的质粒DNA（pDNA-达特）递送至树突状细胞（DC）。稳定的阴离子纳米颗粒（1型）或阳离子纳米颗粒（2型）将使用廉价的、可重复的和可扩展的方法从新型微乳液前体工程化，并分别用于通过rTat和pDNA-Tat进行涂覆。两个具体目标如下：具体目标#1：制备纳米工程HIV-1疫苗。i）证明阴离子纳米颗粒可以用rTat包被，且阳离子纳米颗粒可以用pDNA-Tat包被，ii）证明树突细胞特异性配体甘露聚糖可以包被在两种类型纳米颗粒上，以及iii）根据颗粒尺寸、表面电荷、稳定性和人树突细胞中的细胞摄取和/或转染来表征基于纳米颗粒的疫苗。具体目标#2：在小鼠中测试纳米工程HIV-1疫苗。ii）在局部和皮下给药后，用这些甘露聚糖包被的纳米颗粒疫苗证明Th 1、CTL和体液免疫应答增强，超过含ALU佐剂的rTat或“裸”质粒DNA，和iii）证明使用rTat和pDNA-Tat的基于纳米颗粒的HIV-1疫苗的异源初免-加强方案上级单独使用rTat或pDNA-Tat免疫的同源方案。该提议涉及含有r达特或pDNA-达特的树突状细胞靶向纳米颗粒的新颖和创新的疫苗发现和开发概念，以及达特（1-72）产生保护性Th 1、CTL和体液应答的用途。 适当的基于纳米颗粒的HIV-1达特疫苗的开发也将有益于可能掺入多种HIV基因如达特、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.