Mechanisms of in vivo protection from HIV infection

描述(由申请人提供)：我们建议开发和评估两种治疗方法，作为“概念验证”，旨在增强艾滋病毒感染者的免疫反应。首先，我们将评估通过转导HIV-1感染者的CD34+造血干细胞(HSC)或CD8+T淋巴细胞与慢病毒载体的鸡尾酒混合物来增强HIV-1感染者的HIV-1特异性免疫的有效性。慢病毒载体编码多种HIV-1特异性TCR，限制在接受者的I类HLA，将在自体移植后诱导分化为有效的HIV-1特异性CTL。具体地说，我们将研究是否可以通过将编码来自强大的HIV特异性CTL的TCR的α链和β链的基因转移到CD34+HSC或CD8+T淋巴细胞来产生功能性的HIV特异性CD8+CTL。为此，我们将鉴定人类HIV特异性CTL的体外和体内功能，从最有效的HIV特异性CD8+CTL克隆中克隆出TCRα和β链基因，并将其插入到新型慢病毒载体中。然后，我们将确定这些载体的能力，将克隆的TCR基因导入人造血祖细胞或CD8+淋巴细胞，并对其进行编程，使其分化为HIV特异性CD8+CTL，显示出在体外和体内识别和消除HIV感染细胞的能力。其次，我们将研究最近的发现与HIV免疫的相关性，即阻断PD-1/PD-L1抑制通路可以恢复“耗尽”的LCMV特异性CTL的功能活性。我们建议通过密切相关和协调的目标来实现这一目标：1)评估编码来自强大的人类HIV-1特异性CTL的TCRα链和β链的慢病毒载体在转导人类CD8+淋巴细胞后产生功能性人类HIV特异性CTL的能力；2)确定表达HIV特异性TCRα链和β链的慢病毒载体是否能够编程人CD34+HSC分化为功能强大的HIV特异性CTL；以及3)研究Fc-PD1或LG-PD1融合蛋白处理PD1/PD-L1抑制通路是否会增加HIV特异性CTL的体外和体内功能。这些方法通过重定向免疫反应以产生识别免疫保护性表位的CTL，并通过阻断PD-1的激活(由激活的CTL表达的抑制分子)来增强HIV特异性CTL的功能，为提高HIV特异性免疫提供了新的方式。

DESCRIPTION (provided by applicant): We propose to develop and evaluate as a "proof-of-concept" two therapeutic approaches designed to augment the immune response of HIV-infected individuals. First, we will evaluate the effectiveness of augmenting HIV-1-specific immunity in HIV-1-infected individuals by transducing their CD34+ hematopoietic stem cells (HSC) or CD8+ T lymphocytes with a cocktail of lentiviral vectors encoding various HIV-1-specific TCRs restricted to the recipient's Class I HLA that will induce differentiation into effective HIV-1-specific CTLs after autologous transplantation. Specifically we will examine whether functional HIV-specific CD8+ CTLs can be generated by delivering genes encoding the alpha and beta chain of TCRs derived from potent HIV-specific CTLs into CD34+ HSC or CD8+ T lymphocytes. To this end we will characterize the in vitro and in vivo function of human HIV-specific CTLs, clone out the TCR alpha and beta chain cDNA from the most potent HIV-specific CD8+ CTL clones and insert them into novel lentiviral vectors. We will then determine the capacity of these vectors to introduce the cloned TCR genes into human hematopoietic precursor cells or CD8+ lymphocytes and program them to differentiate into HIV-specific CD8+ CTLs that display the in vitro and in vivo capacity to recognize and eliminate HIV-infected cells. Second, we will examine the relevance to HIV immunity of the recent finding that blocking the PD-1/PD-L1 inhibitory pathway restores functional activity to "exhausted" LCMV-specific CTLs. We propose to achieve this goal via closely related and coordinated aims: 1 )To evaluate the capacity of lentiviral vectors encoding TCR alpha and beta chains derived from potent human HIV-1-specific CTLs to generate functional human HIV-specific CTL after transduction of human CD8+ lymphocytes, 2) To determine whether lentiviral vectors expressing HIV- specific TCR alpha and beta chains can program human CD34+ HSC to differentiate into functional HIV- specific CTLs, and 3) To investigate whether blockade of the PD1/PD-L1 inhibitory pathway by treatment with Fc-PD1 or lg-PD1 fusion proteins increases the in vitro and in vivo function of HIV-specific CTLs. These approaches provide new modalities for increasing HIV-specific immunity by redirecting the immune response to produce CTLs that recognize immunoprotective epitopes and by blocking activation of PD-1, an inhibitory molecule expressed by activated CTL, enhances the function of HIV-specific CTLs.