Novel adjuvants of activating RIG-I and inhibiting immune inhibitors for HIV vacc

描述(申请人提供)：尽管进行了密集的努力，但接种疫苗或自然感染诱导的免疫反应未能有效预防和控制艾滋病毒感染。因此，探索替代免疫途径和新型佐剂，以诱导优于自然免疫的保护性免疫应答，对抗HIV感染具有重要意义。树突状细胞(DC)在免疫反应的激活和维持中起关键作用，受刺激信号和抑制信号的调节。最近，我们发现泛素修饰酶A20是RIG-I、TLR和TNFR信号的负调控因子，在限制APC的免疫刺激活性和对自身抗原的自身反应中发挥关键作用。我们证明，沉默A20显著增强了TLR激动剂和DC疫苗诱导T细胞和抗体反应的刺激效力。在这项研究中，我们的目标是通过抑制A20和刺激促炎信号级联反应来探索和开发新的和有效的HIV疫苗佐剂。本研究的中心假设是，激活RIG-I并抑制A20的5‘-三磷酸A20 siRNA(3P-siA20)可用作一种新型疫苗佐剂，以增强抗HIV细胞和体液对更高水平的应答，这是目前所描述的疫苗接种方法无法达到的，并可能能够克服HIV的免疫逃避和抑制。R21期探索性研究的具体目的是：1.体外检测5‘-PPP-siA20是否具有激活RIG-I和抑制RIG-I、TLR和TNFR信号转导的关键负调控因子A20的双重功能。2.检测双功能siA20是否能有效刺激DC诱导更强的全身和粘膜HIV特异性CTL和Th应答。在完成R21研究的里程碑之后，我们将继续进行R33阶段的研究(3-5年)，以实现以下目标：1.测试3P-siA20是否能有效地刺激DC更有效地诱导小鼠抗HIV env的全身和粘膜抗体应答；2.研究3P-siA20作为佐剂体内免疫和HIV env突变体是否能更有效地诱导小鼠和兔的记忆T细胞和中和抗HIV抗体应答。这项研究是首次尝试开发和测试刺激RIG-I和抑制促炎信号的关键负性调节因子的双重功能分子作为一种新型的HIV疫苗体内佐剂。这种新型佐剂可以克服APC的生物抑制屏障，从而诱导针对HIV上弱免疫原性、保护性表位的免疫反应。 公共卫生相关性：在这项拟议的研究中，我们的目标是开发一种新型的体内佐剂，能够激活RIG-I信号并抑制艾滋病毒疫苗接种中促炎症信号的关键抑制物，以诱导更有效的保护性细胞和体液免疫反应。R21期研究旨在证明5‘三磷酸(3P)A20-siRNA(SiA20)将具有激活RIG-I和抑制树突状细胞中TLR、TNFR和RIG-I信号的关键负调控因子A20的独特双重功能。我们将进一步证明，启动3P-siA20介导的信号转导和延长和增强的促炎信号将有效地刺激DC在小鼠体内诱导更强的HIV特异性T细胞应答。在完成R21研究的里程碑之后，我们将继续R33阶段的研究，以确定3P-siA20是否有效地刺激DC诱导更强的HIV特异性抗体反应。我们将进一步测试3P-siA20作为佐剂和HIV Env在体内免疫是否更有效地诱导小鼠和兔的记忆T细胞和针对HIV的中和抗体反应。这项研究是首次尝试开发和测试刺激RIG-I和抑制促炎信号的关键负性调节因子的双重功能分子作为一种新型的HIV疫苗体内佐剂。

DESCRIPTION (provided by applicant): Despite intensive efforts, the immune responses induced by vaccination or natural infection fail to effectively prevent and control HIV infection. Thus, it is important to explore alternative immunization approaches and novel adjuvants to induce protective immune response that is superior to the natural immunity against HIV infection. Dendritic cells (DCs) play a critical role in the activation and maintenance of immune responses, and they are regulated by stimulatory as well as inhibitory signaling. Recently, we found that the ubiquitin-modifying enzyme A20, a negative regulator of RIG-I, TLR and TNFR signaling, play critical roles in limiting the immunostimulatory potency of APCs and the autoreactive response against self-antigens. We demonstrated that silencing of A20 drastically enhanced the stimulatory potency of TLR agonists and DC vaccines to induce both T cell and antibody responses. In this study we aim to explore and develop novel and potent adjuvants for HIV vaccines by inhibiting A20 and stimulating proinflammatory signaling cascades. The central hypothesis of this study is that 5'- triphosphate A20 siRNA (3P-siA20) that activates RIG-I and inhibits A20 can be used as a novel vaccine adjuvant to enhance anti-HIV cellular and humoral responses to higher levels that cannot be achieved by currently described vaccination approaches and may be capable of overcoming HIV's immune evasion and suppression. The specific aims for the exploratory R21 phase study are: 1. To in vitro test whether 5'-PPP-siA20 has a unique dual function of activating RIG-I and inhibiting the key negative regulator A20 of RIG-I, TLR, and TNFR signaling in DCs. 2. To test whether the bifunctional siA20 can potently stimulate DCs to induce stronger systemic and mucosal HIV-specific CTL and Th responses in mice. After completing the milestones of R21 study, we will proceed to the R33 phase study (years 3-5) to accomplish the following aims: 1. To test whether 3P-siA20 can potently stimulate DCs to more efficiently induce systemic and mucosal antibody responses against HIV Env in mice; and 2. To investigate whether in vivo immunization of 3P-siA20 as an adjuvant and HIV Env mutants with enhanced exposure of protective epitopes more efficiently induces memory T cells and neutralizing antibody responses against HIV in mice and rabbits. This study represents the first attempt of developing and testing a dual function molecule of stimulating RIG-I and inhibiting the key negative regulator of proinflammatory signaling as a novel in vivo adjuvant for HIV vaccination. This novel adjuvant could overcome the biological inhibitory barrier in APCs to allow the induction of immune responses against the weakly immunogenic, protective epitopes on HIV. PUBLIC HEALTH RELEVANCE: In this proposed study, we aim to develop a novel in vivo adjuvant capable of activating RIG-I signaling and inhibiting the key inhibitor of proinflammatory signaling for HIV vaccination to induce more potent protective cellular and humoral immune responses. The R21 phase study is intend to prove the concept that 5' triphosphate (3P) A20-siRNA (siA20) will have a unique dual function of activating RIG-I and inhibiting the key negative regulator A20 of TLR, TNFR, and RIG-I signaling in DCs. We will further prove the concept that the initiation of 3P-siA20- mediated signaling and the prolonged and enhanced proinflammatory signaling will potently stimulate DCs to induce stronger HIV-specific T cell responses in mice. After completing the milestones of R21 study, we will proceed to the R33 phase study to determine whether 3P- siA20 will potently stimulate DCs in inducing stronger HIV-specific antibody responses. We will further test whether in vivo immunization of 3P-siA20 as an adjuvant and HIV Env more efficiently induces memory T cells and neutralizing antibody responses against HIV in mice and rabbits. This study represents the first attempt of developing and testing a dual function molecule of stimulating RIG-I and inhibiting the key negative regulator of proinflammatory signaling as a novel in vivo adjuvant for HIV vaccination.