Development and evaluation of a novel SOCS1-silenced HIV vaccine

描述(由申请人提供)：旨在通过修改人类免疫缺陷病毒(HIV)抗原和使用各种递送系统和佐剂来刺激免疫反应的努力，迄今未能生产出预防初级感染的艾滋病毒疫苗，这突显了继续开发替代和有效的候选疫苗的必要性。其中许多方法的一个共同特点是使用阳性免疫刺激来增强免疫反应。然而，在疫苗设计的背景下，较少考虑的是用于缓和免疫反应的抑制信号。抗原提呈细胞，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着关键作用，因此DC是刺激免疫信号的疫苗的关键靶点。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(SOCS1)蛋白通过限制Janus激活的激酶信号转导和转录激活因子以及Toll样受体信号通路而发挥抗原提呈衰减器的功能。抑制树突状细胞中的SOCS1信号可以诱导更高水平的Th1和Th2极化细胞因子，从而增强记忆、抗原特异性T和B细胞反应以及激活的自然杀伤细胞。在此，我们假设基于腺病毒(Ad)的HIV疫苗的免疫原性将通过抑制SOCS1信号通路而增强。为了验证这一假设，该项目的具体目标是(1)通过Ad介导的SOCS1小干扰(Si)RNA抑制DC SOCS1信号转导，(2)评估SOCS1沉默对DC功能的影响(S)，以及(3)通过测量HIV抗原特异性免疫反应的疫苗接种效率来确定SOCS1沉默对DC功能的影响。为了实现目标1，我们将首先生成表达SOCS1 siRNA的Ad载体。然后，利用定量RT-PCR和Western blotting方法，我们将显示感染表达siRNA的Ad的DC产生显著降低的SOCS1mRNA和蛋白水平。在第二个目标中，我们将确定SOCS1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SOCS1瞬时沉默的DC动物对艾滋病毒疫苗的免疫反应。这一目标将通过测量艾滋病毒特异性体液和细胞介导的反应以及疫苗接种方案缓解体内病毒挑战的能力来实现。SOCS1对信号转导通路的负调控对于细胞因子刺激的适当反应至关重要，然而，通过瞬时打破平衡，该项目代表了HIV疫苗设计的一个潜在的新方向。通过抑制抗原呈递的抑制物，如SOCS1，该项目代表了一种普遍适用的替代战略，以提高预防性和治疗性艾滋病毒疫苗的效力。公共卫生相关性：目前有4000多万人感染艾滋病毒，考虑到在可预见的未来缺乏预防性疫苗，这一数字预计将呈指数级增长，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影响因素来显著提高艾滋病毒疫苗的效力。这种改进的相关性在于疫苗的开发和潜在的全球影响，该疫苗提供了预防艾滋病毒感染的保护。

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.