SIVmac239感染中国猕猴艾滋病潜伏模型的创建及潜伏激活剂对体内病毒库的作用和机制研究

AIDS remains incurable owing to the presence of HIV-1 reservoir. The resting CD4+ T cells horbouring intergated HIV-1 proviruses are considered as the main celluar poor of HIV latency. To attenuate and purge viral reservoir, a “kick and kill” strategy is proposed to reawake the latent proviruses through latency reversing agents (LRAs) and subsequently kill the reactivated viruses or their host cells by highly active antiretroviral therapy (HAART) or host immune system, which is a focus in HIV-1/AIDS therapy. At present, compounds are beening extensively screened in vitro in some cell models and a large number of LRAs with mechanistical distince were identified. But the further development of LRAs are greatly limited due to the lack of a suitable animal model for evaluation of LRAs. In our previous study, we have constructed a nonhuman primate HIV/AIDS models by infected Chinese rhesus macaque with a SIVmac239 strain, which is the most widely used animal model to resemble HIV-1 infection. In the present study, through long term HAART treatment, a persistent viral reservoir will be pregnant in SIVmac239/ Chinese rhesus macaque model, this viral reservoir should be mainly consisted of resting CD4+ T cells. Firstly, the resting CD4+ T cells in SIV infected Chinese rhesus macaques will be seperated and enriched, and different LRAs or their combinations will be added into the cultures of resting CD4+ T cells to disrupt the latent viruses. Secondly, LRAs which can rewake virus replication will be selected to explore their effects and mechanisms, such as the upregulation of nucleosomal histone acetylation, inhibition of BRD4 and reactivation of NF-κB. Finally, a group of LRAs or their combination with low toxicity and high activity will be selected and administrated into the macaques. The reactivation of latent viruses and attenuation of viral reservoir will be measured, and the influences of restriction factors, CD8+ T cell and neutralizing antibody upon viral reservoir will be analyzed in vivo. This research will contribute to further explore therapeutic strategies of LRAs for eradication HIV-1 reservoir, and help to establish a potential nonhuman primate animal model for preclinical evaluation of LRAs.

HIV-1病毒库的存在是艾滋病不能治愈的根本原因。静息性CD4+ T细胞是HIV-1主要潜伏细胞。通过潜伏激活剂诱导潜伏病毒活化，再杀灭活化的病毒和细胞，即“激活并杀灭”策略，是艾滋病治疗领域的研究热点和重点。目前体外筛选已获得多种潜伏激活剂，但缺乏适合的动物模型极大地限制了其研发。本研究拟在前期构建的SIVmac239病毒感染中国猕猴艾滋病模型基础上：通过长期抗病毒治疗，创建SIV感染中国猕猴的潜伏模型；分离静息性CD4+ T细胞，与不同机制的潜伏激活剂及其组合共孵育，检测潜伏病毒的激活情况；鉴定潜伏细胞核小体的组蛋白乙酰化、BRD4的抑制和NF-κB的激活作用；优化出一个低毒高效的激活剂配伍刺激猴体，研究其对体内病毒库的激活、变异和衰减的作用；分析限制因子、CD8+ T细胞及中和抗体在控制病毒库中的作用机制。为潜伏激活剂的研发和治疗策略的制定，以及动物模型评估体系的建立奠定科学依据。

艾滋病不能治愈的根本原因是HIV-1病毒库的存在。由于缺乏适合的艾滋病潜伏动物模型，难以进行HIV-1病毒库和潜伏病毒机制的研究。静息性CD4+ T细胞是HIV-1主要潜伏细胞。通过潜伏激活剂诱导潜伏病毒活化，再杀灭活化的病毒和细胞，即“激活并杀灭”策略，是艾滋病治疗领域的研究热点和重点。目前只能在体外系统中评估潜伏激活剂的活性，极大地制约了潜伏激活剂的研发。在本研究中，首先，我们建立了SIV感染北平顶猴潜伏模型，但使用各种潜伏激活剂组合未能激活体内病毒。随后，我们创建了HIV感染北平顶猴潜伏模型，并发现一种潜伏激活剂组合JQ1和Prostratin可激活体内潜伏病毒，但不降低潜伏库。然后，我们建立新型HIV-1持续性复制的北平顶猴动物模型并进行了潜伏机制的研究：用基因工程的方法构建了以我国流行的HIV-1毒株为骨架的1株新型HIV-1病毒株(HIV-1NL4-R3A)和1株替换SIV-vif 的HSIV病毒株(stHIV-1sv)，用它们分别感染4只北平顶猴，发现这两种病毒在感染早期，形成较高水平复制；在感染的慢性期，形成了持续性感染。并进行了基因组和转录组测序研究病毒潜伏机制。因此，本研究创建了HIV-1潜伏动物模型，为潜伏激活剂评估体系的建立奠定基础。在研究过程中，我们培养博士研究生4名，硕士研究生1名。发表论文11篇；其中SCI论文10篇。其中，HIV-1感染北平顶猴潜伏模型的创建被科技日报、中国科学报、中国科学院网、科学网、人民网、新浪网、搜狐网、凤凰网等学术和大众网站广泛报道，取得良好的社会效益。