Development and evaluation of a novel SOCS1-silenced HIV vaccine

描述（由申请人提供）：旨在通过修饰人类免疫缺陷病毒（HIV）抗原和使用各种递送系统和佐剂来刺激免疫应答的努力迄今为止未能生产出保护免受原发性感染的HIV疫苗，这突出了继续开发替代和有效候选疫苗的必要性。许多这些方法的共同特征是使用阳性免疫刺激来增强免疫应答。然而，在疫苗设计的背景下，很少考虑用于调节免疫应答的抑制信号。抗原呈递细胞如树突状细胞（DC）在建立和维持针对HIV感染的免疫应答中起关键作用;因此DC是刺激免疫信号传导的疫苗的关键靶标。我们的建议是基于这样的发现，即细胞因子信号转导抑制因子1（SOCS 1）蛋白通过限制Janus激活的激酶信号转导子和转录激活子以及Toll样受体信号转导通路而作为抗原呈递衰减子发挥功能。在DC中抑制SOCS 1信号传导显示诱导更高水平的Th 1和Th 2极化细胞因子，导致记忆、抗原特异性T细胞和B细胞应答和活化的自然杀伤细胞的增强。在此，我们假设腺病毒（Ad）为基础的HIV疫苗的免疫原性将通过抑制SOCS 1信号通路增强。为了验证这一假设，该项目的具体目的是（1）通过Ad介导的SOCS 1小干扰（si）RNA的递送抑制DC SOCS 1信号传导，（2）评估SOCS 1沉默对DC功能的影响，以及（3）通过测量HIV抗原特异性免疫应答的疫苗接种效率来确定SOCS 1沉默在Ad靶向DC中的影响。为了实现目标1，我们将首先产生表达SOCS 1 siRNA的Ad载体。然后，使用定量RT-PCR和Western印迹方法，我们将显示用表达siRNA的Ad感染的DC产生显著降低水平的SOCS 1 mRNA和蛋白。在第二个目标中，我们将确定SOCS 1沉默对DC功能的影响。这将通过DC表型标志物和细胞因子表达谱的流式细胞术分析来完成。该项目的第三阶段旨在定量评估具有短暂SOCS 1沉默DC的动物对HIV疫苗的免疫应答。这一目标将通过测量HIV特异性体液和细胞介导的应答以及疫苗接种方案减轻体内病毒攻击的能力来实现。SOCS 1对信号转导通路的负调节对于细胞因子刺激的适当反应至关重要，然而通过短暂地打破平衡，该项目代表了HIV疫苗设计的潜在新方向。通过“抑制抗原呈递的抑制剂”，如SOCS 1，该项目代表了用于增强预防性和治疗性HIV疫苗效力的普遍适用的替代策略。公共卫生相关性：目前有4 000多万人感染艾滋病毒，考虑到在可预见的将来缺乏预防性疫苗，预计这一数字将成倍上升，特别是在世界不发达地区。该项目的目标是评估是否可以通过调节免疫反应的关键效应器来显着提高艾滋病毒疫苗的效力。这种改进的意义在于研制出一种能够保护人们不受艾滋病毒感染的疫苗，并可能产生全球影响。

DESCRIPTION (provided by applicant): Efforts aimed at stimulating immune responses by modifying human immunodeficiency virus (HIV) antigens and using various delivery systems and adjuvants have so far failed to produce a HIV vaccine that protects against primary infections, highlighting the need for the continued development of alternative and effective vaccine candidates. A common feature of many of these approaches is the use of positive immunostimulation to enhance the immune response. Less often considered in the context of vaccine design however are the inhibitory signals that serve to moderate the immune response. Antigen-presenting cells, such as dendritic cells (DC), play a critical role in the establishment and maintenance of the immune responses against HIV infection; therefore DCs are key targets for vaccines that stimulate immune signaling. Our proposal is based on findings that the suppressor of cytokine signaling 1 (SOCS1) protein functions as an antigen-presentation attenuator by restricting the Janus-activated kinase-signal transducers and activators of transcription and Toll-like receptor-signaling pathways. Inhibition of SOCS1 signaling in DCs was shown to induce higher levels of both Th1- and Th2-polarizing cytokines, resulting in the enhancement of memory, antigen-specific T- and B- cell responses and activated natural killer cells. Herein we hypothesize that the immunogenicity of an adenovirus (Ad)-based HIV vaccine will be enhanced by suppressing the SOCS1 signaling pathway. To test this hypothesis, the Specific Aims of the project are to (1) inhibit DC SOCS1 signaling with Ad-mediated delivery of SOCS1 small interfering (si) RNA, (2) evaluate the effect(s) of SOCS1-silencing on DC function, and (3) determine the effect of SOCS1-silencing in Ad-targeted DCs by measuring the vaccination efficiency of HIV antigen-specific immunes responses. To accomplish Aim 1 we will first generate Ad vectors that express SOCS1 siRNA. Then, using quantitative RT-PCR and Western blotting methods, we will show that DCs infected with the siRNA-expressing Ad produce significantly reduced levels of SOCS1 mRNA and protein. In the second aim we will determine the effects of SOCS1-silencing on DC function. This will be done by flow cytometric analyses of DC phenotypic markers and cytokines expression profiles. The third phase of the project is designed to quantitatively assess the immune responses to an HIV vaccine in animals with transiently SOCS1-silenced DCs. This aim will be accomplished by measuring HIV-specific humoral and cellular-mediated responses and the ability of the vaccination protocol to mitigate an in vivo viral challenge. The negative regulation of signal transduction pathways exerted by SOCS1 is vitally important for an appropriate response to cytokine stimulation, however by transiently tipping the balance this project represents a potentially novel direction in the design of HIV vaccines. By "inhibiting the inhibitors" of antigen-presentation, such a SOCS1, this project represents a generally applicable and alternative strategy for enhancing the potency of prophylactic and therapeutic HIV vaccines. PUBLIC HEALTH RELEVANCE: Over 40 million people are currently infected with HIV and, considering the lack of a prophylactic vaccine in the foreseeable future, this number is expected to rise exponentially particularly in the underdeveloped regions of the world. The goal of this project is to evaluate whether the efficacy of an HIV vaccine can be significantly improved by modulating key effectors of the immune response. The relevance of such an improvement lies in the development and potential global impact of a vaccine that affords protection from HIV infection.