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

描述（由申请人提供）：尽管经过了大量的努力，但由于疫苗接种或自然感染引起的免疫反应并不能有效地预防和控制HIV感染。因此，探索替代免疫方法和新型佐剂来诱导优于HIV感染天然免疫的保护性免疫反应是很重要的。树突状细胞（dc）在免疫应答的激活和维持中起着至关重要的作用，它们受到刺激和抑制信号的调节。最近，我们发现泛素修饰酶A20是RIG-I、TLR和TNFR信号的负调节因子，在限制apc的免疫刺激效力和对自身抗原的自身反应性反应中起关键作用。我们证明A20的沉默极大地增强了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）可以作为一种新的疫苗佐剂，增强抗HIV细胞和体液反应到更高水平，这是目前描述的疫苗接种方法无法实现的，并且可能能够克服HIV的免疫逃避和抑制。探索性R21期研究的具体目标是：1。体外测试5'-PPP-siA20是否具有独特的双重功能，即激活RIG-I并抑制dc中RIG-I、TLR和TNFR信号通路的关键负调控因子A20。2. 测试双功能siA20是否能有效刺激dc诱导小鼠更强的全身和粘膜hiv特异性CTL和Th反应。在完成R21研究的里程碑后，我们将进入R33期研究（3-5年），以实现以下目标：测试3P-siA20是否能有效刺激dc，更有效地诱导小鼠对HIV Env的全身和粘膜抗体反应；和2。研究在小鼠和家兔体内免疫作为佐剂的3P-siA20和增强保护性表位暴露的HIV Env突变体是否更有效地诱导记忆T细胞和中和针对HIV的抗体反应。这项研究首次尝试开发和测试一种双重功能分子，可以刺激rig - 1并抑制促炎信号的关键负调节因子，作为HIV疫苗接种的新型体内佐剂。这种新型佐剂可以克服apc中的生物抑制屏障，从而诱导针对HIV上弱免疫原性保护性表位的免疫应答。

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.