RNAi manipulations of DC to enhance HIV immunogenicity

描述(申请人提供)：到目前为止，还没有有效的艾滋病毒感染疫苗。尽管最近一项基于同源重组病毒Prime/Boost的主要疫苗试验失败了，但后来在猴子模型上进行的一项研究表明，使用更合适的异源Prime/Boost方案，有可能引发强大的T细胞反应，以保护其免受毒力病毒的挑战。因此，对于一种有效的疫苗来说，它应该能够激发强大和广泛的T细胞反应。此外，在临床前模型中通过HIV挑战来预先测试新疫苗方法的相关性，将极大地帮助防止在人类临床试验中疫苗失败的痛苦。由于树突状细胞在诱导T细胞免疫应答中起关键作用，我们假设以HIV蛋白为靶点的树突状细胞免疫，其中选择的免疫调节分子如SOCS-1、PD-L1、L2和IL-10已被RNA干扰抑制，将诱导强大的多功能CD8 T细胞应答。我们的假设是基于我们的初步结果，即通过靶向向DC传递siRNA来沉默SoCS-1足以在体外诱导出对几个HIV Gag表位的强大的初级T细胞反应，包括亚优势表位。此外，我们最近已经证明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感染中的有效性的可行性，从而能够测试人类DC靶向方法是否在体内有效。在这项提议的具体目标1中，我们将开发定向递送HIV抗原和免疫调节siRNA试剂到人树突状细胞的方法和试剂。其中包括一种可与siRNA结合并传递HIV抗原的DC靶向多肽，两种与HIV蛋白融合并进一步修饰以结合siRNA的DC靶向抗体，以及一种允许以mRNA形式靶向传递siRNA和HIV抗原的脂质体配方。在目标2中，我们将评估通过这些方法中的任何一种方法将HIV免疫原与不同免疫调节的siRNA(单独或联合)共同传递是否能够在体外诱导广泛而多功能的原代HIV特异性CD8 T细胞反应。在目标3中，我们将验证体外研究结果，并测试我们的方法在人源化转人类白细胞抗原A2和人类白细胞抗原B27的BLT小鼠模型中抵御体内HIV攻击的有效性。该项目将开发针对DC的HIV抗原传递和RNAi操作方法，作为一种新的HIV疫苗策略，以诱导强大的病毒特异性T细胞免疫。如果成功，这些研究将提供重组病毒载体疫苗的替代方案。此外，临床前动物模型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.