Combination Anti-PD-1 and Anti-retroviral Therapy Provides Therapeutic Benefit Against SIV

Therapeutic strategies that enhance anti-viral immunity and reduce the viral reservoir are critical to achieving durable remission of HIV. The co-inhibitory receptor programmed death-1 (PD-1) regulates CD8+ T cell dysfunction during chronic HIV and SIV infections 1-4. In addition, PD-1+ CD4+ T cells constitute a significant fraction of the HIV/SIV viral reservoir5-7. We previously demonstrated that in vivo blockade of PD-1 during chronic SIV infection improves the function of anti-viral CD8+ T cells and B cells 4,8. However, the immunological effects of PD-1 blockade during anti-retroviral therapy (ART) and the potential to destabilize the persistent HIV/SIV reservoir have not been studied. Here, we show that administration of anti-PD-1 antibody (PD-1 Ab) 10 days prior to initiation of ART rapidly enhanced anti-viral CD8+ T cell function and diminished interferon stimulated genes (ISGs) that resulted in markedly improved viral suppression in plasma and better Th17 cell reconstitution in the rectal mucosa following ART initiation. In addition, PD-1 blockade during suppressive ART resulted in transient increases in plasma viremia, induction of gene signatures associated with effector CD8+ T cell function and ISGs, and lower levels of cell associated replication-competent virus suggesting destabilization of the viral reservoir. Following ART interruption, PD-1 Ab treated animals showed markedly higher expansion of proliferating CXCR5+ Perforin+ Granzyme B+ effector CD8+ T cells and lower regulatory T cells that resulted in delayed viral rebound and better control of viremia. PD-1 blockade administered only during suppressive ART however was only partially effective. Our results indicate an optimal regimen of PD-1 blockade administered in combination with ART, that augments anti-viral CD8+ T cell function and reduces the viral reservoir leading to improved control of viral rebound after ART interruption. These results have important implications for developing novel therapeutic interventions to achieve durable remission of HIV. Overall design: To determine the effects of PD-1 blockade in combination with ART and its potential to enhance anti-viral immunity and reduce the viral reservoirs, we performed PD-1 blockade in two phases: I) prior to initiation of ART and II) during suppressive ART. In phase I of the study, we administered 5 doses of PD-1 Ab (3 mg/kg/dose) intravenously on days 0, 3, 7, 10, and 14 to 10 SIVmac251-infected rhesus macaques (RMs) between 24-30 weeks after infection (Fig. 1A). As a control, we treated 10 SIVmac251-infected RMs with saline. We initiated ART in all animals at day 10 after the initiation of PD-1 blockade. To determine the effects of PD-1 blockade on a global scale, we performed RNASeq to compare gene signatures in the blood of 10 PD-1 Ab treated and 5 saline treated animals at day 0 and day 10 of PD-1 blockade. To determine the additive biological effects of PD-1 blockade on anti-viral immunity and the persistent viral reservoirs when administered during suppressive ART (phase II), the 10 RMs given PD-1 Ab during phase I were again treated with PD-1 Ab (double treated) at 26-30 weeks following ART initiation. Three monthly infusions of PD-1 Ab were administered at 10mg/kg/dose (Fig. 3A). To test the influence of PD-1 blockade administered only during suppressive ART, we split the 10 RMs from the saline group into two groups and gave either PD-1 Ab (single treated group; n=5) or saline (saline control group; n=5). RNASeq was only performed on the double treated and single treated groups, not the saline control group. We again looked to compare gene signatures in the blood of PD-1 Ab treated RMs from day 0 of first PD-1 Ab infusion under ART and 7 days after.

能够增强抗病毒免疫并缩减病毒储存库的治疗策略，对于实现HIV的持久缓解至关重要。共抑制受体程序性死亡蛋白-1（programmed death-1, PD-1）在慢性HIV与猴免疫缺陷病毒（simian immunodeficiency virus, SIV）感染期间，调控CD8+ T细胞功能衰竭[1-4]。此外，PD-1+ CD4+ T细胞在HIV/SIV病毒储存库中占比可观[5-7]。我们此前已证实，在慢性SIV感染期间体内阻断PD-1，可改善抗病毒CD8+ T细胞与B细胞的功能[4,8]。然而，抗反转录病毒治疗（antiretroviral therapy, ART）期间PD-1阻断的免疫学效应，以及其是否可能破坏持续性HIV/SIV病毒储存库的稳定性，尚未得到研究。本研究显示，在启动ART前10天给予抗PD-1抗体（PD-1 Ab），可快速增强抗病毒CD8+ T细胞功能，并降低干扰素刺激基因（interferon stimulated genes, ISGs）的表达，进而显著提升血浆病毒抑制效果，并在ART启动后更好地实现直肠黏膜的Th17细胞重建。此外，在抑制性ART期间实施PD-1阻断，会导致血浆病毒血症一过性升高，诱导与效应性CD8+ T细胞功能及ISGs相关的基因特征表达，同时降低细胞相关复制型病毒的水平，提示病毒储存库发生了不稳定化。在ART中断后，接受PD-1抗体治疗的动物，其增殖性CXCR5+ 穿孔素+ 颗粒酶B+ 效应性CD8+ T细胞的扩增能力显著更强，调节性T细胞水平更低，这使得病毒反弹延迟，并实现了对病毒血症的更佳控制。不过，仅在抑制性ART期间实施PD-1阻断的效果仅为部分有效。我们的研究结果表明，PD-1阻断与ART联合应用的最优方案，可增强抗病毒CD8+ T细胞功能并缩减病毒储存库，进而在ART中断后改善对病毒反弹的控制。上述结果为开发实现HIV持久缓解的新型治疗干预手段提供了重要参考。总体实验设计：为明确PD-1阻断联合ART的效应，以及其增强抗病毒免疫与缩减病毒储存库的潜力，我们分两个阶段实施PD-1阻断：I）ART启动前阶段；II）抑制性ART期间阶段。在本研究的第一阶段，我们于感染后24~30周，对10只感染SIVmac251的恒河猴（rhesus macaques, RMs）在第0、3、7、10、14天静脉给予5剂抗PD-1抗体（3 mg/kg/剂）（图1A）。对照组则对10只感染SIVmac251的恒河猴给予生理盐水处理。我们于PD-1阻断启动后第10天，为所有动物启动ART。为从全局层面明确PD-1阻断的效应，我们通过RNA测序（RNASeq），对比了PD-1抗体治疗组10只动物与生理盐水处理组5只动物在PD-1阻断第0天与第10天的血液基因特征。为明确在抑制性ART期间实施PD-1阻断时，其对抗病毒免疫与持续性病毒储存库的附加生物学效应（第二阶段），我们对第一阶段接受PD-1抗体治疗的10只恒河猴，在ART启动后26~30周再次给予PD-1抗体治疗（双重治疗组），共给予3次每月一次的抗PD-1抗体输注，剂量为10mg/kg/剂（图3A）。为测试仅在抑制性ART期间实施PD-1阻断的影响，我们将生理盐水组的10只恒河猴分为两组，分别给予抗PD-1抗体（单一治疗组；n=5）或生理盐水（生理盐水对照组；n=5）。RNA测序仅在双重治疗组与单一治疗组中开展，未纳入生理盐水对照组。我们同样对比了ART条件下首次PD-1抗体输注第0天与7天后恒河猴血液的基因特征。