自1981年HIV被发现以来，尽管人们经过了40多年的不懈努力，但至今仍未找到彻底治愈艾滋病的方法。目前，临床治疗主要依靠抗逆转录病毒疗法（Antiretroviral therapy，ART），这种疗法可有效抑制HIV复制，将患者血浆病毒载量控制在检测线以下，显著降低AIDS相关疾病的发病率和死亡率以及 HIV 传播的风险。然而，由于HIV病毒储存库的存在，ART无法彻底清除患者体内的病毒，一旦停药病毒将迅速反弹，患者需终身服药。由此造成的患者治疗依从性差、药物不良反应和病毒耐药性是亟待解决的问题，新型抗逆转录病毒药物的研发刻不容缓。

HIV感染靶细胞是一个多步骤、多环节、多因素的复杂过程。其中，病毒进入是感染的关键初始环节，针对这一环节设计的进入抑制剂能够充当体内的“守门员”，兼具治疗和预防双重作用，因而成为人们的研究热点。根据作用靶点的不同，进入抑制剂可分为受体拮抗剂，辅助受体拮抗剂和膜融合抑制剂。研究表明，单一用药虽可抑制病毒的复制，但容易导致耐药性进而引发病毒反弹，因此，开发同时靶向不同位点的双/多功能抑制剂具有重要意义。

截至目前，全球仅报道9例艾滋病患者被成功治愈，除日内瓦患者外，8例患者都是通过移植CCR5/Δ32造血干细胞而实现，这一进展表明CCR5是艾滋病治疗的关键靶标。基于此，本研究聚焦于HIV进入过程的CCR5辅助受体以及病毒融合蛋白gp41，联合靶向CCR5的单克隆抗体PRO140和靶向gp41的短肽融合抑制剂2P23，将2P23通过柔性连接子（GGGGS）n分别偶联到PRO140原型抗体轻链和/或重链的N端或C端，通过融合蛋白形式表达，获得多个不同构型的双功能进入抑制剂。2P23偶联在PRO140抗体轻链N端的融合蛋白2P23-PRO140L和2P23偶联在PRO140抗体轻链和重链C端的融合蛋白PRO140HL-2P23显示出最好的抗病毒活性。

接下来，本研究对双功能抑制剂2P23-PRO140L和PRO140HL-2P23进行了深入表征。首先验证了双功能抑制剂通过CCR5结合至细胞膜上并发挥抗病毒作用。进一步对双功能抑制剂抗病毒活性进行了系统评价，发现其不仅能够有效抑制CXCR4嗜性HIV-1毒株，还对多种不同亚型HIV-1、HIV-2以及SIV代表性毒株以及HIV-1包膜蛋白介导的细胞-细胞融合均表现出强效广谱的抑制活性，显著优于单体分子2P23和PRO140。此外，双功能抑制剂对2P23耐药株仍能保持高效抑制，提示其具有更高的耐药屏障。体外细胞毒性和稳定性实验结果显示两个双功能抑制剂均具有极低的细胞毒性和良好的体外稳定性。重要的是，两个双功能抑制剂在大鼠体内均具有强大的抗病毒活性和体内稳定性。

综上，本研究联合靶向辅助受体CCR5的单克隆抗体PRO140和靶向病毒融合蛋白gp41的短肽2P23，成功设计并筛选出高效广谱的双功能抑制剂2P23-PRO140L和PRO140HL-2P23，为临床治疗提供了潜在候选药物，并为多靶点抗病毒药物开发提供了新策略。

学位论文: 06-18 13:00:14 Since the discovery of HIV in 1981, despite over 40 years of persistent efforts, a definitive cure for AIDS remains elusive. Current clinical treatment primarily relies on antiretroviral therapy (ART), which effectively suppresses HIV replication, maintains plasma viral load below detection limits, and significantly reduces AIDS-related morbidity and mortality as well as HIV transmission risk. However, due to the persistence of viral reservoirs, ART cannot completely eradicate the virus, leading to rapid viral rebound upon treatment discontinuation and necessitating lifelong medication. Consequently, challenges including poor patient adherence, drug-related adverse effects, and viral drug resistance remain critical issues that demand urgent solutions, highlighting the pressing need for novel antiretroviral drug development. HIV infection of target cells is a complex, multi-step process involving multiple factors. Viral entry represents the critical initial step, and entry inhibitors targeting this stage can function as molecular "gatekeepers" with both therapeutic and prophylactic potential, making them a key research focus. Based on their distinct targets, entry inhibitors can be classified into receptor antagonists, coreceptor antagonists, and fusion inhibitors. Studies have shown that while monotherapy can suppress viral replication, it often leads to drug resistance and subsequent viral rebound. Therefore, developing dual/multifunctional inhibitors targeting different viral entry sites is of paramount importance. To date, only nine cases of HIV cure have been reported worldwide. With the exception of the Geneva patient, eight cases were achieved through CCR5Δ32 hematopoietic stem cell transplantation, demonstrating CCR5 as a critical therapeutic target for AIDS. Building on this, our study focuses on the HIV entry coreceptor CCR5 and the viral fusion protein gp41. We engineered bifunctional entry inhibitors by conjugating the CCR5-targeting monoclonal antibody PRO140 with the gp41-targeting peptide fusion inhibitor 2P23 through flexible (GGGGS)n linkers at the N- or C-termini of PRO140's light and/or heavy chains, generating multiple fusion protein constructs with distinct configurations. Among these, 2P23-PRO140L (with 2P23 conjugated to the N-terminus of PRO140's light chain) and PRO140HL-2P23 (with 2P23 conjugated to the C-termini of both heavy and light chains) demonstrated optimal antiviral activity. We subsequently conducted comprehensive characterization of the bifunctional inhibitors 2P23-PRO140L and PRO140HL-2P23. Initial validation confirmed their CCR5-mediated membrane binding and antiviral function. Systematic evaluation revealed that these inhibitors not only effectively suppressed CXCR4-tropic HIV-1 strains but also exhibited potent and broad-spectrum inhibitory activity against diverse HIV-1 subtypes, HIV-2, representative SIV strains, and HIV-1 Env-mediated cell-cell fusion, significantly outperforming the monomeric molecules 2P23 and PRO140. Moreover, the bifunctional inhibitors maintained high efficacy against 2P23-resistant strains, suggesting an enhanced genetic barrier to resistance. In vitro cytotoxicity and stability assays demonstrated minimal cellular toxicity and excellent stability for both inhibitors. Importantly, both compounds showed robust in vivo antiviral activity and stability in rat models. In summary, by strategically combining the CCR5-targeting monoclonal antibody PRO140 with the gp41-targeting peptide 2P23, we successfully designed and identified two highly potent and broad-spectrum bifunctional inhibitors, 2P23-PRO140L and PRO140HL-2P23. These candidates hold significant promise for clinical application and provide a novel strategic framework for developing multi-target antiviral therapeutics 学位论文: 06-18 13:02:13 S2022035001-欧梦蝶