Role of Viral Reservoirs in the Pathogenesis of HIV Disease

十多年前，我们证明了静息的CD4+T细胞隔间中潜伏的病毒储存库在几乎所有接受临床有效的抗逆转录病毒疗法的HIV感染者中持续存在。此外，我们还证明，在长期接受抗逆转录病毒治疗的慢性感染者中，艾滋病毒持续以低水平复制。因此，持久的病毒库已成为在接受抗逆转录病毒疗法的感染者中根除艾滋病毒的主要障碍。近年来，主要的研究工作致力于更好地了解持续艾滋病毒感染的发病机制，并制定旨在根除接受抗逆转录病毒治疗的感染者的治疗策略。在过去的一年里，我们的研究集中在：1)检测ART对精英控制者中HIV宿主的影响；2)开发针对持续感染的CD4+T细胞的基于免疫的治疗策略。 首先，我们研究了抗逆转录病毒治疗对精英控制员中持续HIV宿主的影响。虽然ART可以将绝大多数HIV感染者的血浆病毒血症抑制到检测到的限度以下，但仅靠治疗无法根除病毒，部分原因是病毒储存在外周血液、淋巴组织和其他隔离部位。因此，在缺乏抗逆转录病毒治疗的情况下，人们越来越有兴趣制定治疗策略，以消除持久的艾滋病毒宿主和/或增强宿主对病毒的免疫力，以控制病毒复制。在这方面，已经表明，在没有ART(控制者)的情况下，一小部分艾滋病毒感染者自发地控制血浆病毒血症。此外，这类患者的一部分(精英控制员)能够在不需要抗逆转录病毒治疗的情况下，在数年至数十年内保持相对正常的CD4+T细胞计数和无法检测到的血浆病毒血症水平。尽管以前的研究表明，精英控制员可能会发生正在进行的病毒复制，但在这一患者群体中，携带复制能力病毒的感染的CD4+T细胞的确切频率和/或ART对免疫学和病毒学参数的影响尚未完全描述。因此，我们进行了一项研究，以确定ART对持续HIV储存库动态和精英控制者的各种免疫学参数的影响。我们证明了短疗程(9个月)的抗逆转录病毒疗法显著降低了精英控制者的传染性艾滋病毒负担，提供了令人信服的证据，表明在这些无菌个体中会发生感染/残留病毒复制。在停止抗逆转录病毒疗法后，精英控制者的CD4+T细胞室中的传染性艾滋病毒负荷反弹到其原始基线水平，这表明即使在病毒复制水平非常低的情况下，也存在病毒学设定点，而且宿主免疫可以有效地控制血浆病毒血症，而无需完全消除受感染的CD4+T细胞。我们还证明，精英控制员的HIV特异性CD8+T细胞水平在ART开始后逐渐下降，进一步支持了病毒复制水平较低的概念。我们的数据提供了令人信服的证据，表明正在进行的艾滋病毒复制发生在精英控制者身上。考虑到这些人有效地控制了病毒复制，并且在没有ART的情况下疾病没有进展，我们的研究对在面临ART停止的情况下在非控制者中实现持续的病毒学缓解而不完全抑制或消除病毒复制的可行性有影响。 其次，我们研究了使用表达嵌合抗原受体(CAR)的自体CD8+T细胞来消除HIV感染的CD4+T细胞的可行性。最近的研究表明，慢性淋巴细胞白血病患者在输注表达CD19结合CD3-Zeta B细胞抗原的自体CD8+T细胞和共刺激分子后，可以达到完全缓解。受这些研究的启发，我们调查了表达针对HIV env的CAR的自体CD8+T细胞是否可以从接受ART的HIV感染者中清除持续感染的CD4+T细胞。对三种汽车结构进行了评估。所有构建物包括用于识别感染靶标上的HIV Env的CD4，用于细胞毒活性的CD3-Zeta，以及下列共刺激分子之一：mock、4-1BB或CD28。将表达上述CAR蛋白的自体CD8+T细胞与接受抗逆转录病毒治疗的HIV感染者的预刺激的CD4+T细胞共培养，并用p24-EL ISA监测病毒复制水平。我们的初步数据表明，当表达包含CD28的CAR的CD8+T细胞与受感染的靶细胞共同培养时，可以达到最高水平的HIV抑制。我们的数据还表明，表达上述CAR结构的CD8+T细胞与表达HIV Env的细胞株孵育后，与表达CAR的模型或4-1BB细胞相比，产生更高水平的细胞内细胞因子和趋化因子(如干扰素-g、肿瘤坏死因子-α、MIP-1)。综上所述，我们的初步数据表明，通过表达识别HIV env的CAR的自体CD8+T细胞，可以在体外消除HIV感染者体内持续感染的CD4+T细胞。由于CD8+细胞毒性T细胞不需要病毒抗原特异性(除了识别HIV Env)来杀伤，这种效应细胞可能在不诱导病毒逃逸突变的情况下消除受感染的靶细胞。此外，考虑到在癌症患者中观察到的转导CAR的CD8+T细胞的寿命(几年到几十年)，这些细胞可能会在很长一段时间内限制病毒在体内的复制。

Over a decade ago, we demonstrated that the latent viral reservoir in the resting CD4+ T cell compartment persists in virtually all HIV-infected individuals receiving clinically effective ART. In addition, we demonstrated that HIV continually replicates at low levels in chronically infected individuals who are consistently aviremic during prolonged periods of receiving ART. Consequently, the persistent viral reservoir has become a major impediment to the eradication of HIV in infected individuals receiving ART. In recent years, major research efforts have been dedicated to a better understanding of the pathogenesis of persistent HIV infection and to the development of therapeutic strategies aimed at eradicating virus in infected individuals receiving ART. During the past year, we have focused our research on: 1) examining the effect of ART on HIV reservoirs in elite controllers and 2) developing immune-based therapeutic strategies that target persistently infected CD4+ T cells. First, we investigated the effect of ART on the persistent HIV reservoirs in elite controllers. Although ART suppresses plasma viremia to below the limit of detection in the vast majority of HIV-infected individuals, the virus cannot be eradicated by therapy alone due in part to the persistence of viral reservoirs in the peripheral blood, lymphoid tissues, and other sequestered sites. Consequently, there is a growing interest in developing therapeutic strategies to eliminate persistent HIV reservoirs and/or to enhance host immunity against the virus in order to control viral replication in the absence of ART. In this regard, it has been shown that a small proportion of HIV-infected individuals spontaneously control plasma viremia in the absence of ART (controllers). Moreover, a subset of such individuals (elite controllers) are capable of maintaining relatively normal CD4+ T cell counts and undetectable levels of plasma viremia for years to decades without the need for ART. Although previous studies have suggested that ongoing viral replication may occur in elite controllers, precise frequencies of infected CD4+ T cells carrying replication-competent virus and/or the impact of ART on immunologic and virologic parameters have not been fully delineated in this patient population. Therefore, we conducted a study in order to determine the effect of ART on the dynamics of persistent HIV reservoirs and various immunologic parameters in elite controllers. We demonstrated that a short course (9 months) of ART dramatically decreased the infectious HIV burden in elite controllers, providing compelling evidence that onging/residual viral replication occurs in such aviremic individuals. The infectious HIV burden in the CD4+ T cell compartment of the elite controllers rebounded back to their original baseline levels upon cessation of ART, an indication that a virologic set-point exists even at this very low level of viral replication, and that host immunity can efficiently control plasma viremia without completely eliminating the infected CD4+ T cells. We also demonstrated that the level of HIV-specific CD8+ T cells in elite controllers gradually declined upon initiation of ART, further supporting the concept that low levels of viral replication occur in these infected individuals. Our data provide compelling evidence that ongoing HIV replication occurs in elite controllers. Given that these individuals effectively control viral replication and lack disease progression in the absence of ART, our study has implications for the feasibility of attaining sustained virologic remission in non-controllers without complete suppression or elimination of viral replication in the face of discontinuation of ART. Second, we investigated the feasibility of eliminating HIV-infected CD4+ T cells using autologous CD8+ T cells that express a chimeric antigen receptor (CAR). Recent studies have demonstrated that complete remission can be achieved in patients with chronic lymphoid leukemia upon infusion of autologous CD8+ T cells expressing CAR with specificity for the B cell antigen CD19 coupled with CD3-zeta, and costimulatory molecules. Inspired by these studies, we investigated whether autologous CD8+ T cells expressing CAR specific for HIV Env could eliminate persistently infected CD4+ T cells from HIV-infected individuals receiving ART. Three CAR constructs were evaluated. All constructs included CD4 for recognition of HIV Env on infected targets, CD3-zeta for cytotoxic activity, and one of the following constimulatory molecules: mock, 4-1BB, or CD28. Autologous CD8+ T cells expressing the above CAR proteins were cocultured with pre-stimulated CD4+ T cells from HIV-infected individuals receiving ART and the level of viral replication was monitored by p24 ELISA over time. Our preliminary data suggest that the highest level of HIV suppression can be achieved when CD8+ T cells expressing the CAR that contains CD28 are cocultured with infected target cells. Our data also show that CD8+ T cells expressing the above CAR construct produce higher levels of intracellular cytokines and chemokines (such as IFN-g, TNF-a, MIP-1) compared to those expressing CAR with mock or 4-1BB upon incubation with a cell line expressing HIV Env. Taken together, our preliminary data suggest that the elimination of persistently infected CD4+ T cells from HIV-infected individuals ex vivo can be achieved with autologous CD8+ T cells that express a CAR that recognizes HIV Env. Because CD8+ cytotoxic T cells do not require viral antigen specificity (besides recognition of HIV Env) for killing, such effector cells could potentially eliminate infected target cells without inducing viral escape mutants. In addition, considering the longevity of CAR-transduced CD8+ T cells observed in cancer patients (years to decades), these cells could restrict viral replication in vivo for extended periods of time.