RNAi manipulations of DC to enhance HIV immunogenicity

描述（由申请人提供）：迄今为止，没有有效的艾滋病毒感染疫苗。尽管最近基于同源重组病毒初免/加强的主要疫苗试验失败，但后来在猴模型中进行的一项研究表明，采用更合适的异源初免/加强方案，有可能引发强烈的T细胞应答，保护免受强毒病毒攻击。因此，要使疫苗有效，它应该能够引起强烈和广泛的T细胞反应。此外，通过在临床前模型中进行HIV攻击来预先测试新疫苗方法的相关性，将大大有助于防止疫苗在人体临床试验中失败的痛苦。由于树突状细胞对于诱导T细胞免疫应答是至关重要的，我们假设用靶向树突状细胞的HIV蛋白免疫，其中选择的负性免疫调节分子如SOCS-1、PD-L1、L2和IL-10已被RNA干扰抑制，这将引发有效的多功能CD 8 + T细胞应答。我们的假设是基于我们的初步结果，其中通过靶向siRNA递送至DC来沉默SOCS-1足以在体外引发对几种HIV gag表位（包括亚优势表位）的强烈初级T细胞反应。此外，我们最近已经显示了使用最新版本的人源化小鼠模型来测试siRNA介导的干预在HIV感染中的功效的可行性，因此能够测试人DC靶向方法是否在体内有效。在本提案的具体目标1中，我们将开发用于将HIV抗原和免疫调节siRNA试剂靶向递送至人DC的方法和试剂。这些将包括经修饰以结合siRNA以及递送HIV抗原的DC靶向肽、与HIV蛋白融合并经进一步修饰以结合siRNA的两种DC靶向抗体以及允许以mRNA形式靶向递送siRNA和HIV抗原的脂质体制剂。在目的2中，我们将评估通过这些方法中的任一种共递送HIV免疫原与不同的免疫调节siRNA（单独和组合）是否能够在体外诱导广泛和多功能的初级HIV特异性CD 8 T细胞应答。在目标3中，我们将验证体外发现以及测试我们的方法在HLA-A2和HLA-B27转基因的人源化BLT小鼠模型中实际赋予体内HIV攻击保护的功效。该项目将开发用于HIV抗原递送和RNAi操作的DC靶向方法，作为一种新的HIV疫苗策略，以诱导有效的病毒特异性T细胞免疫。如果成功，这些研究将为基于重组病毒载体的疫苗提供替代方案。此外，在临床前动物模型中的体内研究将为保护的免疫相关性提供新的见解。

DESCRIPTION (provided by applicant): To date there is no effective vaccine for HIV infection. Although a major vaccine trial based on homologous recombinant viral prime/boost failed recently, a study undertaken later, in a monkey model, suggests that with a more appropriate heterologous prime/boost regimen, it is possible to elicit a strong T cell response that protects against virulent viral challenge. Thus, for a vaccine to be effective, it should be able to evoke a strong and broad-based T cell response. Moreover, pretesting the relevance of novel vaccine approaches by HIV challenge in preclinical models would greatly help prevent the agony of vaccine failures in human clinical trials. As Dendritic cells are critical for induction of T cell immune responses, we hypothesize that immunization with HIV proteins targeted to dendritic cells in which select negative immunomodulatory molecules such as SOCS-1, PD-L1, L2 and IL-10 have been suppressed by RNA interference will elicit a potent polyfunctional CD8+ T cell response. Our hypothesis is based on our preliminary results in which silencing of SOCS-1 via targeted siRNA delivery to DC was enough to elicit a robust primary T cell response in vitro to several HIV gag epitopes, including subdominant ones. Moreover, we have recently shown the feasibility of using the latest versions of humanized mouse models to test the efficacy of siRNA mediated interventions in HIV infection and are thus are in a position to test whether the human DC- targeted methods are effective in vivo. In Specific Aim 1 of this proposal we will develop methods and reagents for targeted delivery of HIV-antigens and immunomodulatory siRNA reagents to human DCs. These will include a DC targeting peptide modified to bind siRNA as well as to deliver HIV antigens, two DC-targeting antibody fused to HIV proteins and further modified to bind siRNAs and a liposomal formulation that allows targeted delivery of siRNA and HIV antigen in mRNA form. In Aim 2, we will evaluate whether co-delivery of HIV immunogen with the different immunomodulatory siRNA (singly and in combination) by any of these methods is able to induce a broad and polyfunctional primary HIV-specific CD8 T cell response in vitro. In Aim 3, we will validate the in vitro findings as well as test the efficacy of our methods to actually confer protection from in vivo HIV challenge in the humanized BLT mouse model transgenic for HLA-A2 and HLA-B27. This project will develop DC-targeted approaches for HIV antigen delivery and RNAi manipulations as a novel HIV vaccine strategy to induce potent virus-specific T cell immunity. If successful, the studies would provide an alternative to the recombinant viral vector-based vaccines. In addition, the in vivo studies in a preclinical animal model will provide novel insights into the immune correlates of protection.