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发表于 2019-10-29 20:24 |只看该作者 |倒序浏览 |打印
Editorial         Open Access




   
   Fine‐Tuning TLR‐7‐Based Therapy for Functional HBV Cure                                 Antonio Bertoletti M.D.            
            Nina Le Bert Ph.D.            
         
      
   
         First published: 01 October 2019
      https://doi.org/10.1002/hep4.1420
   
                  Supported by the National Medical Research Council (MOH‐STaR17nov‐0001 and NMRC/TCR/014‐NUHS/2015).
      
               Potential conflict of interest: Dr. Bertoletti consults for, advises, and received grants from Gilead and Spring Bank.
      
               See Article on Page 1296
      
   
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                     Abstract                     This is an invited editorial on the editorial on the original manuscript HEP4‐19‐0099.R1 “Liver‐targeted TLR7 agonist combined with Entecavir promotes a functional cure in the woodchuck model of chronic HBV.”            
                              
                                 
         
   
                  Abbreviations                              anti‐HBs                           antibody to hepatitis B surface antigen                           CHB                           chronic hepatitis B                           HBV                           hepatitis B virus                           IFN                           interferon                           IL                           interleukin                           NK                           natural killer                           TLR                           toll‐like receptor                           WHeAg                           woodchuck hepatitis e antigen                           WHV                           woodchuck hepatitis virus                  
                              Although nucleos(t)ide analogue therapy is efficient in reducing hepatitis B virus (HBV) replication and liver inflammation in patients with chronic hepatitis B (CHB), the therapy rarely achieves functional cure of HBV, a virological situation characterized by negativity of HBV virological parameters and antibody to hepatitis B surface antigen (anti‐HBs) seroconversion. This is why new therapies that not only inhibit viral replication but stimulate the defective antiviral immunity of CHB patients are being developed.
                  Toll‐like receptor (TLR) (TLR‐7, TLR‐8, TLR‐9) and retinoic acid–inducible gene 1 (RIG‐I) agonists are the major representatives of such class of immune therapy. They are designed to activate host antiviral immunity in a non–antigen‐specific way. Their rationale resides in the fact that even though HBV replication is not activating innate immunity in hepatocytes, it is responsive to the antiviral effect of interferons (IFNs) and other cytokines. Thus, activating type I IFN responses directly in hepatocytes (by RIG‐I agonists) or intrahepatic immune cells (by TLR‐7, TLR‐8, TLR‐9 agonists) should inhibit HBV replication. Furthermore, the localized activation of IFN type I responses may trigger a cascade of immunological events (e.g., release of cytokines, alteration of antigen presentation) that lead to natural killer (NK) cell activation and possibly recovery of HBV‐specific T cells and B cells (Fig. 1).1
                                                Figure 1                      Open in figure viewerPowerPoint
                  
                  TLR‐7 agonist's immunological mechanisms. TLR‐7 agonist (as APR002) acts by targeting intrahepatic plasmacytoid dendritic cells (PDCs) and B cells. TLR‐7 agonist triggers type I IFN production in PDC. Type I IFNs have a direct antiviral effect on HBV‐infected hepatocytes but also activate intrahepatic NK or T cells. TLR‐7 agonist on B cells can induce their functional maturation to plasma cells (both in the liver and peripheral blood).
                                                      In this issue of Hepatology Communications, Korolowizc et al. published a study describing the safety and therapeutic efficacy of a new TLR‐7‐activating compound (APR002) used alone or in association with entecavir in the woodchuck model of woodchuck hepatitis virus (WHV) infection. Therapy with TLR‐7 agonists has already been reported in chimpanzees,2 woodchucks,3 and humans.4, 5 The difference with this new compound (APR002) in comparison with others (i.e., GS‐9620) is the fact that APR002 has been designed to have preferentially intrahepatic delivery. By minimizing systemic exposure, the authors argued that it should reduce the poor tolerability observed in animal models treated previously with TLR‐7 agonist. The hepato‐selective design of APR002 is principally mediated by active uptake by the organic‐anion‐transporting polypeptide 1B1/3 transporters, highly expressed on the sinusoidal membrane of hepatocytes. Korolowizc et al. first showed in mice that APR002 is indeed preferentially localized in the liver with a serum to liver ratio of approximately 30, an undeniable improvement in comparison to the ratio of approximately 6 for GS‐9620. They also reported in mice that APR002 induces lower release of proinflammatory cytokines (interleukin [IL]‐6 and tumor necrosis factor α) in comparison to GS‐9620 and then demonstrated APR002’s ability to activate IFN‐α genes in the liver of normal uninfected woodchucks. APR002’s therapeutic efficacy was then tested: Four comparable cohorts of woodchucks chronically infected with WHV (5 woodchucks per group) were treated either with entecavir alone, APR002 monotherapy, or a complex combination of entecavir and different doses of APR002 (see table 1 of the article for posology).         
                  Treatment with APR002 was well tolerated. The weekly oral dosing of APR002 for 12 weeks did not induce systemic immunotoxicity or changes in clinical chemistry and hematological parameters. Increases of liver enzymes were observed primarily but not exclusively in the APR002‐treated animals and reversed in all animals at the end of the study. However, elevated liver enzymes were also observed in a single animal treated with APR002, only it was found dead. The mortality was attributed to preexisting kidney disfunction.
                  As expected, APR002 treatment (alone or in combination) induced a clear activation of innate immunity in the liver and blood of infected animals (tested by measuring the activation of IFN‐stimulated genes) and clear reduction of WHV parameters (WHV DNA and WH surface antigen [WHsAg] and WH e antigen [WHeAg]) that was less pronounced than in the animals treated with entecavir only. Remarkably, only in the animals treated with combination therapy (groups 3 and 4) did the authors observe a sustained negativity of WHV antigen and seroconversion to anti‐HBs (3 animals), which occurred approximately 8 to 10 weeks after therapy.
                  This small but very well conducted study provides important new information that might help fine‐tune the treatment with these immunotherapeutic agents. First, the preferential hepato‐selectivity and the absence of signs of systemic toxicity and alteration of hematological parameters, linked with an efficient activation of intrahepatic innate immunity, suggest that APR002 might have a better safety profile than GS‐9620. Perhaps future clinical trials with APR002 in CHB patients will be designed using a drug posology that is similar to what was used in animals that seroconverted. It is important to remember that the lack of therapeutic efficacy observed in the human trials of GS‐9620 could have been principally caused by the low dose used to minimize possible side effects (a dose approximately 35 to 100 times lower than the maximum ones used in woodchucks and chimpanzees was administered to patients4, 5).         
                  However, it appears inevitable that immunotherapies, designed to trigger intrahepatic immunity, activate liver inflammation. Despite the lack of systemic toxicity, APR002 treatment induced in many animals a transient alteration of transaminase, and this clinical perturbation should perhaps be considered a direct sign of drug activity and not an undesirable side effect. Nevertheless, this remains a problem in the design of human trials because it is difficult to fully predict the severity of hepatic flares after activation of intrahepatic innate immunity. Their severity is likely multifactorial, related to environmental, clinical, and genetic factors. A very recent publication on a case of fulminant hepatic failure in a child infected with hepatitis A virus has identified a deletion in IL‐18‐binding protein (IL‐18BP) as the cause of excessive activation of NK cells with uncontrolled killing of hepatocytes.6 If this case illustrates the importance of genetic factors in the development of severe hepatic failure after intrahepatic innate immune activation, it also shows that IL‐18BP might be the perfect antidote to control hepatic flares caused by innate immune activation, and therefore a possible new alley for future experimental trials on immunotherapy for CHB.         
                  One other interesting observation of the Korolowizc et al. study is the fact that the virological and immunological features of the APR002‐treated woodchucks that achieved the equivalence of functional HBV cure (anti‐WHs+) indicate that the therapeutic efficacy of APR002 is linked with immunological modulation (particularly on B cells) rather than increased antiviral potency. The analysis of virological markers (WHV DNA, WHsAg, and WHeAg) in animals treated with entecavir alone or with the APR002 and entecavir combination showed that the inhibition of WHV virological markers was more marked in the entecavir‐alone group. The authors suggested that a low level of hepatic metabolism induced by APR002 reduced entecavir’s antiviral potency, because the drug needs to be transformed into its active form. Regardless of the causes of such virological findings, the fact is that the anti‐WHs seroconversion detected in animals treated with combination therapy was therefore not directly related to an increased level of viral inhibition (and type I IFN gene activation in the liver).
                  Therefore, even though a threshold of viral inhibition appears indispensable for subsequent seroconversion (not a single animal treated with APR002 monotherapy developed anti‐WHs antibodies), the success of the therapy in these animals is likely dependent on the ability of TLR‐7 agonists to induce functional maturation of antiviral B‐cell responses.7
                  The study of humoral immunity in patients with CHB has been neglected for a long time, but the development of new methods has recently permitted the study of HBV‐specific B cells at a single‐cell level8, 9 and led to the demonstration that hepatitis B surface antigen–specific B cells in CHB patients are not fully deleted but rather present functional maturation defects.         
                  It appears that the correct use of immunotherapies (such as TLR‐7 agonist) targeting B‐cell maturation cannot be separated from more precise understanding of the requirement of HBs‐specific B‐cell restoration in the treated patients. The recent analysis of T‐cell and NK‐cell immunological parameters in GS‐9620‐treated CHB patients5 has shown that, despite the lack of therapeutic efficacy, the treatment induced a boost of HBV‐specific T‐cell responses. These data open the question of why not a single CHB patient experienced anti‐HBs seroconversion despite a partial recovery of T‐cell immunity. The features of HBV‐specific B cells were not analyzed, but the lack of patients experiencing anti‐HBs seroconversion clearly argues that a functional reconstitution of the B‐cell compartment did not occur.         
                  As we discussed previously, the therapeutic discrepancy between TLR‐7 agonist efficacy in animal models and patients may be explained by the different quantity and frequency of drug administration tested.
                  However, in our opinion, one other variable that should be considered is that while TLR‐7 agonist therapy has been tested in classic adult anti‐HBe+ CHB patients, the features of the animal models (WHeAg+, barely elevated or normality of alanine aminotransferase infected for 1‐2 years and not for decades) treated with TLR‐7 agonists appear to be more similar to the HBeAg+ chronic infection (or immunotolerant subjects). We do not know whether HBV‐specific B cells might be less or differently altered, as we have seen for T cells,10 in different phases of CHB infection.         
                  In conclusion, this carefully designed study not only reports the development of a new and potentially safer compound but stimulates immunological analysis that can help fine‐tune the treatment with TLR‐7 agonists and achieve functional HBV cure in selected CHB patients.
               

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发表于 2019-10-29 20:29 |只看该作者

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发表于 2019-10-29 20:30 |只看该作者
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基于TLR-7的微调功能性HBV疗法
安东尼奥·贝托莱蒂(医学博士)
Nina Le Bert博士
首次发布:2019年10月1日
https://doi.org/10.1002/hep4.1420
由国家医学研究理事会(MOH-STaR17nov-0001和NMRC / TCR / 014-NUHS / 2015)支持。
潜在的利益冲突:Bertoletti博士为Gilead和Spring Bank提供咨询,建议并获得了赠款。
请参阅第1296页的文章
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这是原始手稿HEP4-10-19999的社论的受邀社论。“ R1靶向肝的TLR7激动剂与恩替卡韦联合可促进慢性HBV土拨鼠模型的功能性治愈。”
图片

缩略语

抗HBs
乙肝表面抗原抗体
CHB
慢性乙型肝炎
乙肝病毒
乙肝病毒
干扰素
干扰素
白介素
白介素
NK
天生杀手
TLR
Toll样受体
抗原
土拨鼠戊型肝炎抗原
超高压
土拨鼠肝炎病毒

尽管核苷类似物疗法可有效减少慢性乙型肝炎(CHB)患者的乙型肝炎病毒(HBV)复制和肝脏炎症,但该疗法很少能实现HBV的功能性治愈,这是一种以HBV病毒学阴性为特征的病毒学情况乙型肝炎表面抗原(anti-HBs)血清转化的参数和抗体。这就是为什么正在开发不仅抑制病毒复制而且刺激CHB患者的抗病毒免疫缺陷的新疗法的原因。

Toll样受体(TLR)(TLR-7,TLR-8,TLR-9)和视黄酸诱导基因1(RIG-1)激动剂是这类免疫疗法的主要代表。它们旨在以非抗原特异性的方式激活宿主的基本原理。直接在肝细胞(通过RIG-I激动剂)或肝内免疫细胞(通过TLR-7,TLR-8,TLR-9激动剂)中产生的IFN应答应抑制HBV复制。此外,I型IFN反应的局部激活可能触发一系列免疫事件(例如,细胞因子释放,抗原呈递改变),从而导致自然杀伤(NK)细胞激活,并可能恢复HBV特异性T细胞和B细胞(图1).1
图片
图1
在图形查看器中打开PowerPoint
TLR-7激动剂(作为APR002)的作用是靶向肝内浆细胞样树突状细胞(PDC)和B细胞。 TLR-7激动剂触发PDC中的I型干扰素产生。 I型干扰素对HBV感染的肝细胞有直接的抗病毒作用,但也会激活肝内NK或T细胞。 B细胞上的TLR-7激动剂可以诱导其功能成熟到浆细胞(在肝脏和外周血中)。

在本期《肝病通讯》中,Korolowizc等人。发表了一项研究,描述了在土拨鼠肝炎病毒(WHV)的土拨鼠模型中单独使用或与恩替卡韦联用的新型TLR-7活化化合物(APR002)的安全性和治疗功效,已有关于用TLR-7激动剂进行治疗的报道。黑猩猩,2土拨鼠,3和人类。4、5与这种新化合物(APR002)相比,该新化合物(GS-9620)的不同之处在于,APR002通过最大程度地减少全身性暴露,认为应该减少在先前使用TLR-7激动剂的动物模型中观察到的耐受性差。 APR002的肝选择性设计主要是由有机阴离子转运多肽1B1 / 3转运蛋白的主动摄取介导的,该转运蛋白在肝细胞的正弦膜上高度表达。 Korolowizc等。首次在小鼠中发现APR002 ind Eed优先定位在肝脏中,血清与肝脏的比率约为30,与GS-9620的比率约为6相比,这是不可否认的改善。他们还在小鼠中报告说,与GS-9620相比,APR002诱导的促炎细胞因子(白介素[IL] _6和肿瘤坏死因子α)的释放更低,然后498 APR002激活正常未感染的土拨鼠肝脏中IFN-α基因的能力。然后测试了APR002的治疗功效:单独用恩替卡韦,APR002单一疗法或恩替卡韦和不同剂量的APR002的复杂组合治疗四个可同组的慢性感染WHV的土拨鼠(每组5只土拨鼠)(参见本文表1) (用于本体论)。

APR002的治疗耐受性良好。每周口服APR002 12周,不会引起全身免疫毒性或临床化学和血液学参数的变化。在研究结束时,主要但并非仅在APR002处理的动物中观察到肝酶的增加,并且在所有动物中均观察到了逆转。但是,在一只用APR002治疗的动物中也观察到肝酶升高,只有它被发现死亡。死亡率归因于先前存在的肾功能不全。

如预期的那样,APR002处理(单独或联合使用)可在被感染动物的肝脏和血液中明显激活先天免疫(通过测量IFN刺激基因的激活进行测试),并明显降低WHV参数(WHV DNA和WH表面)抗原[WHsAg]和WH e抗原[WHeAg])比仅用恩替卡韦治疗的动物更不明显。值得注意的是,只有在接受联合疗法治疗的动物(第3组和第4组)中,观察到WHV抗原持续呈阴性,并且血清转化为抗HBs的动物(3只动物)在治疗后约8至10周发生。

这项规模很小但进行得很好的研究提供了重要的新信息,这些信息可能有助于微调这些免疫治疗剂的治疗方法。首先,优先肝选择性,缺乏全身毒性和血液学参数改变以及肝内先天免疫的有效激活,提示APR002的安全性可能比GS-9620好。也许将来将在APR002上对CHB患者进行临床试验时,将使用与血清转化动物相似的药物疗法来进行设计。重要的是要记住,在GS-9620的人体试验中观察到的缺乏治疗功效可能主要是由于用于将可能的副作用减至最小的低剂量所致(该剂量比最大剂量低约35至100倍)。病人接受了土拨鼠和黑猩猩4、5)。

但是,似乎不可避免的是,旨在触发肝内免疫的免疫疗法会激活肝脏炎症。尽管缺乏全身毒性,但APR002治疗在许多动物中诱导了转氨酶的瞬时改变,这种临床扰动应被视为药物活性的直接标志,而不是不良的副作用。然而,这在人体试验设计中仍然是一个问题,因为在激活肝内先天免疫后很难完全预测肝耀斑的严重程度。它们的严重性可能是多因素的,与环境,临床和遗传因素有关。最近的一篇有关感染甲型肝炎病毒的儿童暴发性肝衰竭病例的出版物已确定IL-18结合蛋白(IL-18BP)的缺失是导致NK细胞过度活化而肝细胞杀伤失控的原因。 6如果该病例说明遗传因素在肝内固有免疫激活后严重肝衰竭发展中的重要性,则还表明IL-18BP可能是控制由固有免疫激活引起的肝耀斑的完美解毒剂,因此可能是新的未来进行CHB免疫疗法实验性试验的小巷。

对Korolowizc等人的另一有趣观察。研究是事实,经过APR002处理的土拨鼠的病毒学和免疫学特征达到了功能性HBV治愈(抗WHs +)的水平,表明APR002的治疗功效与免疫调节有关(尤其是对B细胞),而不是增加抗病毒效力。对单独使用恩替卡韦或APR002和恩替卡韦联合治疗的动物进行病毒学标记(WHV DNA,WHsAg和WHeAg)分析表明,在单独使用恩替卡韦的组中,对WHV病毒学标记的抑制作用更为明显。作者认为,APR002引起的肝脏代谢水平低会降低恩替卡韦的抗病毒效力,因为该药物需要转化为活性形式。不管造成这种病毒学发现的原因是什么,事实是,因此在用联合疗法治疗的动物中检测到的抗WHs血清转化与病毒抑制水平的提高(和肝脏中I型IFN基因的激活水平)没有直接关系。

因此,尽管病毒抑制的阈值似乎对于随后的血清转化是必不可少的(不是一只用APR002单药治疗的动物开发出抗WHs抗体),但在这些动物中治疗的成功可能仍取决于TLR-7激动剂的抗病毒能力。诱导抗病毒B细胞反应的功能成熟7

CHB患者的体液免疫研究长期以来一直被忽略,但是新方法的发展最近允许在单细胞水平上研究HBV特异性B细胞[8,9],并证明了乙型肝炎CHB患者中的表面抗原特异性B细胞并未完全缺失,但存在功能成熟缺陷。
似乎不能将针对B细胞成熟的免疫疗法(例如TLR-7激动剂)的正确使用与对所治疗患者的HBs特异性B细胞修复的需求的更精确的了解区分开来。最近对经GS-9620治疗的CHB患者中T细胞和NK细胞免疫学参数的分析5显示,尽管缺乏治疗功效,该治疗仍可引起HBV特异性T细胞应答的增强。这些数据提出了一个问题,即为什么尽管T细胞免疫力部分恢复,但没有一名CHB患者经历抗HBs血清转化。并未分析HBV特异性B细胞的特征,但是缺乏抗HBs血清转化患者,这清楚地表明,并未发生B细胞区室的功能重建。

正如我们之前所讨论的,在动物模型和患者中TLR-7激动剂疗效之间的治疗差异可能由所测试药物的不同数量和频率来解释。

但是,我们认为,应该考虑的另一个变量是,虽然已经在典型的成人抗HBe + CHB患者中测试了TLR-7激动剂治疗,但动物模型的特征(WHeAg +,感染丙氨酸氨基转移酶的程度很少或正常) TLR-7激动剂治疗1至2年而不是数十年)似乎与HBeAg +慢性感染(或免疫耐受受试者)更相似。我们不知道HBV特异性B细胞是否会减少或发生不同的变化,正如我们在CHB感染的不同阶段中的T细胞[10]所见。

总之,这项精心设计的研究不仅报告了一种新的,可能更安全的化合物的开发,而且还刺激了免疫学分析,可以帮助微调TLR-7激动剂的治疗方法,并在部分CHB患者中实现功能性HBV治愈。

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发表于 2019-10-29 20:45 |只看该作者
本帖最后由 newchinabok 于 2019-10-29 20:47 编辑

双免疫疗法是出路。比如治疗性疫苗+tlr—7,…等等
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