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病毒性肝炎以乙型肝炎和丙型肝炎最为常见。肝炎、肝硬化和肝癌，被称作肝癌发展的“三步曲”疾病的发生发展伴随着不同的作用机制，对应的也就有不同的治疗手段。当前乙肝丙肝等的研究热点是什么？丙肝导致肝癌的作用机制如何？面对乙肝新药如何选择？……围绕这些问题，在今年的美国肝病研究学会年会（AASLD2021）期间，《国际肝病》特别邀请到本次大会主席、美国马萨诸塞州总医院肝病和肝脏中心主任、美国哈佛大学医学院医学教授Raymond Chung博士为我们一一解答。

 

《国际肝病》：目前慢乙肝的治疗还不能达到彻底治愈，理想的治疗终点是功能性治愈。您觉得当前重点该关注乙肝的哪些方面？面对众多的乙肝新药，该如何选择？

 

Raymond Chung博士：这些都是很好的问题。正如已经指出的，实现慢乙肝的彻底治愈是一个非常大的挑战。因为乙型肝炎病毒是一种高度潜伏的病毒，在感染细胞的细胞核中有一种非常稳定的形式——封闭的共价环状DNA（cccDNA）——一旦建立，就很难消除。

 

乙肝新的治疗方法

 

对于慢性乙型肝炎患者，我们希望通过核苷（酸）类似物（NUC）治疗阻断HBV DNA聚合酶而抑制病毒复制。新的治疗选择给了我们希望，将能够提高功能性治愈率，即慢性感染患者的HBsAg消失。而单独使用NUC治疗，难以达到这样的终点。

 

此外还有针对病毒生命周期其他步骤的抑制剂其中最突出的是衣壳组装调节剂，其具有抗病毒作用，而它的抗病毒作用实际上是对NUC治疗作用的叠加。这些衣壳组装调节剂也可能在生命周期的其他步骤中起作用，包括细胞核内HBV cccDNA的补充。因此，这类疗法很令人欣喜。

 

还有另一类疗法通过靶向从细胞核中释放出来的HBV核酸来阻止病毒蛋白质的产生。例如，HBV基因组转录的RNA在细胞质中被翻译成病毒蛋白，这种所谓的前基因组RNA（pgRNA）可以成为RNAi（干扰RNA）或寡核苷酸反义分子的治疗靶点，这些分子可以再次导致这些mRNAs的降解，并阻止产物的翻译。这类疗法的特点在于它实际上可以降低包括HBsAg在内的蛋白质水平，以及在病毒生命周期里病毒粒子的产生过程中负责病毒复制或是病毒复制必不可少的关键蛋白质的水平。

 

另一类疗法是免疫治疗。这些方法针对免疫应答，或者说使感染期间低下的免疫应答增强，包括治疗性疫苗、抗体、以及针对PD-1等在慢性感染期间失调的耗竭分子。我们的假设是，通过恢复这些免疫应答，可使T细胞识别病毒感染的细胞，从而提高功能性治愈率

 

我认为，针对慢性感染期间免疫反应减弱的治疗，可能会对我们获得功能性治愈产生一定的影响。之所以这么说，是因为当我们谈论功能性治愈时，最好的例子实际上是在自然急性感染期间实现乙型肝炎的自发清除。在经历急性感染的具有成熟免疫应答的成年患者中，清除和功能性治愈是常态，而不是例外。这些个体最终HBsAg消失，并以抗HBs的形式产生强烈的中和抗体反应。在这些患者身上可以清楚地看到，他们对乙型肝炎有着很好且广泛的T细胞反应。我们在慢性感染者中做的尝试是希望能重新激活这些反应，从根本上说，是为慢性感染者实现在急性感染时看到的病毒清除或治疗反应铺平道路。

 

那么，我们能模拟急性感染的临床路径吗？我对能够调整免疫反应途径的方法非常感兴趣，这些方法可能与我前面描述的其他治疗方法结合使用，这些治疗方法在特定的病毒生命周期步骤中发挥作用，包括RNAi途径，它本身负责HBsAg的产生。例如，将RNAi与免疫疗法联合治疗的特点在于HBsAg分泌实际上可能会破坏宿主的免疫反应。除了我们提到的免疫治疗策略外，抑制HBsAg或许能在一定程度上恢复免疫应答。因此，就乙肝免疫应答的恢复而言，联合疗法可能会带来更令人印象深刻的结果。

 

Hepatology Digest: Cure of hepatitis B is still a mission impossible at present. The ideal endpoint of treatment for now is socalled “functional cure”. In your opinion, which aspects of hepatitis B should be the focus at present? How should we make choice among the new hepatitis B drugs?

 

Dr Chung: These are all very good questions. As you have already pointed out, chronic hepatitis B is a real challenge to think about the notion of cure, in a large measure, because it is a highly latent virus with a very stable form in the nucleus of the infected cell - the covalently closed circular or cccDNA - that is extremely difficult to eliminate once established. In persons with chronic hepatitis B, our goals, at least to this point, have really been more modest. These include suppression with nucleoside analog treatments that block the HBV DNA polymerase, which of course is the engine of HBV nucleic acid replication. There are new classes of therapy as you implied that I think give us hope that we will be able to enhance the rates of functional cure, namely the loss of HBsAg in chronically infected patients, which is a highly elusive endpoint on NUC therapy alone. These other classes of therapy include inhibitors of other life cycle steps, the most prominent of which would be the assembly of the virus aided and abetted by a protein known as the HBV capsid protein. There are a number of inhibitors of these capsids, called capsid assembly modulators, which have an antiviral effect, and whose antiviral effect is actually additive to the effects of nucleoside analog therapy. It is quite possible that these capsid assembly modulators may also work on other steps in the life cycle, including replenishment of the HBV cccDNA in the nucleus. So, there is excitement around that class, and another class of therapies that block viral protein production by specifically targeting HBV nucleic acids as they emerge from the nucleus. For instance, there is an HBV transcribed RNA that emerges from the nucleus to be translated into viral proteins, and this so-called pregenomic RNA (pgRNA) can be the target of therapies known as either RNAi (interfering RNAs) or oligonucleotide antisense molecules that can again bring about the degradation of these mRNAs and prevention of the translation of the products. The attractiveness of this class of therapies is that this can actually knock down levels of proteins including HBsAg as well as other key proteins that are responsible or necessary for viral replication in the viral life cycle in virion production. Then another class of therapy is immunotherapy. These, of course, are therapies directed against the immune responses or enhancing immune responses that have become much more feeble during infection. These approaches range from therapeutic vaccines, to antibodies, to exhaustion molecules such as PD-1, that would potentially be unregulated during chronic infection. The premise here is that by restoring these responses, we may be able to enhance functional cure rates by allowing T-cells to recognize chronically infected cells that they don’t recognize during chronic infection. I think the promise really lies in the fact that therapies that are targeted against that immune response that is weakened during chronic infection, are likely to have a disproportionate impact on our likelihood of obtaining functional cure. The reason I say that is because when we talk about achieving a functional cure, the best example of that is actually the spontaneous clearance of hepatitis B during natural acute infection. In persons with mature immune responses (namely adults) who experience acute infection, clearance and functional cure is the rule, rather than the exception. These individuals eventually lose HBsAg and develop strong neutralizing antibody responses in the form of anti-HBs. What is clear in those patients is that they have excellent and broad T-cell responses against hepatitis B. What we are looking to try to do in chronic infection is really to reinvigorate those responses, and essentially pave the way for a clearing or curative response that might be seen in acute resolving infection in persons who are chronically infected. So can we emulate that roadmap that we see in acute infection? I am excited about approaches that would tweak that immune response pathway, potentially in conjunction with some of the other classes of therapy that I described earlier that work on specific viral life cycle steps, including the RNAi pathway, which is responsible for HBsAg production itself. The attractiveness of including RNAi with, for instance, immunotherapy, is the notion that HBsAg secretion (which we see in excess in chronic infection) may actually contribute to the subversion of those host immune responses. Knocking down HBsAg may have the net effect of liberating some of those immune responses, in addition to the immune therapy strategies we talked about. So, it is possible that combination approaches may result in even more impressive responses in terms of recovery of immune response against hepatitis B.

 

《国际肝病》：您是国际公认的丙型肝炎病毒学、发病机制和肝癌发生研究领域的领导者。对于一些获得SVR的丙肝患者继发肝癌您是怎么看的？丙肝导致肝癌的作用机制又是怎样的？

 

Raymond Chung博士：这是一个非常好的问题。随着对丙型肝炎的治愈性直接作用抗病毒疗法的引入，我们无疑取得了革命性的进展。看到慢性丙型肝炎患者的治愈率接近98%~100%，这是非常好的结果。然而，对于那些纤维化程度更高（即晚期纤维化和肝硬化）的患者来说，丙肝治疗使他们中的绝大多数人获得了持续应答，这些患者进展为肝细胞癌的风险有所降低，但并未消除。其中一个重要的因素是，对于晚期纤维化患者，我们还没有完全完成对这些患者的管理，他们仍会面临肝癌发生的风险。这一风险经治疗管理降低了70%~75%，但仍然存在HCC进展的残余风险。这些患者需要进行进一步的HCC检查。

 

作用机制

 

当我们认识到存在发生HCC的持续风险机制后，必须指出HCV与HBV的不同。HBV是另一种已知可导致肝癌的病毒，但它们的机制不同，因为HBV是一种将遗传物质整合到宿主体内的DNA病毒。因此，在乙型肝炎感染中发生插入突变事件的可能性非常大。相比之下，HCV不发生整合或潜伏。所以这种治愈实际上是可以实现的，因为这种病毒必须继续复制才能生存。这就是为什么我们能够在如此多的慢性丙型肝炎感染病例中实现治愈的原因。那么在持续风险方面会发生什么呢？

 

众所周知，HCV影响多种信号通路，包括表皮生长因子信号通路和其他与生长相关的通路。另外包括Hippo信号通路和LCA（溶血磷脂酸）通路。

 

在丙型肝炎感染的情况下，所有这些通路都可能紊乱。这些生长通路可能反过来增加肝癌发生的风险。人们期望这些通路在感染治愈后会恢复正常，但实际上，在已经治愈的丙型肝炎患者肝脏中的mRNA转录研究已经表明，尽管病毒被清除，这些通路可能仍然处于开启状态或仅部分受到抑制。因此，这就提出了一个问题，即在病毒消失后，肝脏或肝脏环境中的哪些因素仍然存在，以解释这些持续性的改变。

 

研究表明，可能存在许多持续性改变，包括持续的氧化应激，这反过来又会导致肝癌的发生，以及所谓的表观遗传变化——这些表观遗传变化与其说是DNA序列本身的变化，还不如说是DNA周围的染色质的变化，而染色质的变化会导致遗传物质的转录。如我前面所述，这些表观遗传变化可能允许某些生长相关基因的更多转录。

 

因此，这些表观遗传变化在慢性丙型肝炎感染后——甚至有早期研究表明在丙型肝炎感染清除后，可能会更加持久。所以我们认为有一些不同于乙肝的机制在发挥作用，这些机制可能仍然存在。所以我认为，了解这些患者中哪些患者将面临持续性肝癌风险是非常重要的。基于这些似乎持续存在的一些改变，开发预测工具将非常重要。

 

Hepatology Digest: You are among the internationally acknowledged leaders in the field of HCV virology, pathogenesis and liver cancer. What do you think about the development of liver cancer in some HCV patients who have achieved SVR? What is the mechanism of HCV related liver cancer?

 

Dr Chung: This is a really excellent question. We certainly have made revolutionary advances with the introduction of curative direct-acting antiviral therapy for hepatitis C. It has really been an extraordinary marvel to see the cure rates approaching 98-100% in our patients with chronic hepatitis C. And yet, in those patients with more established fibrosis (i.e. advanced fibrosis and cirrhosis) who have achieved sustained responses (and that is the great majority of those patients), there is a reduction, but not an elimination of the risk of those patients for progression to hepatocellular carcinoma. An important practical element of this is that in patients who do have advanced fibrosis, we are not completely done with managing these patients, because they still have residual liver disease that puts them at risk for HCC. Now that risk goes down by about 70~75%, but still there is a remnant risk for HCC progression. Those patients need to be surveyed for HCC moving forward. You get at the mechanisms of that persistent risk, and when we think about HCC, we have to state that it is different from hepatitis B virus, another well-known virus that causes and contributes to liver cancer,  but it’s mechanisms are going to be different, because HBV is a DNA virus that integrates genetic material into the host. So there is the very real possibility of an insertional mutagenesis event occurring in hepatitis B infection. In contrast, in hepatitis C, there is no integration or latency of this virus. So that cure is in fact achievable, because this virus must continue to replicate for its viability. That is the reason we are able to achieve cure in so many instances of chronic hepatitis C infection. So what goes on there in terms of persistent risk? Well, it is known that HCV affects multiple signaling pathways, including the epidermal growth factor signaling pathway and other growth related pathways. The Hippo signaling pathway is another example, and the LCA (lysophosphatidic acid) pathway. All are potentially deranged in the context of hepatitis C infection. Those growth pathways may then in turn contribute to the risk of hepatocarcinogenesis. Interestingly, one would expect those pathways to calm down after cure of infection, but in reality, transcriptional studies of mRNAs in the livers of patients who have been cured of hepatitis C have shown that these pathways may remain switched on or only partially suppressed despite clearance of the virus. So this raises questions as to what in the hepatic or liver environment persists now that the virus is gone to explain these persistent alterations. Studies have demonstrated perhaps a number of persistent alterations, including the persistence of oxidative stress, which in turn can contribute to hepatocarcinogenesis, as well as so-called epigenetic changes - changes not so much in the DNA sequences themselves, but in the chromatin that surrounds the DNA that lead to its access to transcription of the genetic material. These epigenetic alterations may be permissive for more transcription of certain growth related genes as I described earlier. So these epigenetic changes may actually be more persistent after chronic hepatitis C infection, and even after clearance of hepatitis C infection as early studies have demonstrated. We think there are probably some other mechanisms at work that differ from those we have historically attributed to hepatitis B, for instance, and that these may remain. So I think it is important for us to understand which among those patients are going to be at risk for persistent hepatocarcinogenesis risk moving forward. These prognostic tools are going to be very important and interesting to develop based on some of these alterations that appear to persist.

 

《国际肝病》：乙型肝炎病毒和丙型肝炎病毒等是肝癌发生的主要病因。您能分享一下目前在这些领域内研究的热点有哪些？您还进行了哪些方面的研究？麻省总医院在这些方面又有哪些新的研究成果呈现？

 

Raymond Chung博士：我认为对于乙型肝炎病毒来说，我们确实是在试图理解恢复那些本来已经耗尽的免疫反应是多么重要。对于T细胞来说尤其如此，T细胞确实是清除病毒感染的肝细胞的主要引擎，但也有其他类型的细胞，包括先天免疫细胞和适应性免疫细胞，已被证明参与了病毒控制和发病机制。因此，我认为我们真的要尝试了解慢性乙型肝炎感染对这些细胞类型的影响，以及随着时间的推移这些细胞功能应答的耗竭。当然，也要了解如何通过合适和正确的手段来逆转这些应答。我认为这个问题的答案从恢复T细胞和其他免疫应答角度上对描述如何逆转慢性感染将有很大的帮助。这将是乙型肝炎领域研究中令人感兴趣的工作方向。

 

当然，还有其他关于肝癌发生的研究，以及我在回答前一个问题时提到的那些研究。这将是非常重要的。这些可以通过基于细胞的模型、乙型肝炎感染的动物模型，甚至所谓的类器官模型来实现，这些模型在体外更能代表多个细胞在三维空间相互作用的例子，从而更准确地复制体内情况。我认为这些模型在告诉我们肝癌发生的机制、免疫反应和免疫反应的重塑以及对功能性治愈，甚至是生物治疗的贡献方面将是最有用的。

 

从肝癌发生、纤维化形成和疾病进展的其他研究角度来看，我们对寻找化学预防方法非常感兴趣。正如我前面所说，众所周知，晚期肝病患者仍然存在持续的风险，不仅是肝癌的发生，而且还有持续纤维化和疾病进展的风险。我们不能对所有患者进行移植治疗。移植并不是一种足够广泛的资源，而且费力、费用高。

 

因此，对于那些患有晚期肝病的患者，我们需要讨论一些策略，以防止他们出现严重的肝病并发症。这在乙型肝炎中尤其如此，在其他病因相关肝病中也是如此，比如非酒精性脂肪肝就是一个很好的例子。基于我之前提到的这些基因特征的持续性，以及疾病进展和肝癌风险的持续存在，我们正在研究可能的化学预防化合物的作用，这些化合物可能会阻断我之前描述的那些可能在患者中持续存在的途径，即使是那些治愈了的丙型肝炎患者。

 

他汀类药物或阿司匹林，甚至其他更为特异的通路抑制剂，是否有助于防止疾病进展为肝硬化和肝癌？这是我们一直在进行的一个积极的研究领域。我们也非常有兴趣研究乙肝与其他辅助因素的相互作用，例如脂肪肝疾病，以及HIV合并感染，这两种因素都会加速HBV相关疾病的进程，反之亦然。

 

我们已经对可能导致疾病进展的新途径——不仅是乙肝，也包括艾滋病和脂肪肝——进行了研究，正在探索。我们在这方面也有一些发现，例如缺氧诱导因子等因素的发现。我们正在探索其中的许多相互作用，并对阐明新途径很有兴趣，这些新途径可以特异性地靶向预防这些重要形式肝病的进一步进展。

 

Hepatology Digest: HBV and HCV infection are the main causes of liver cancer. Could you please share the current hot-spots in the research of these fields??Are you interested in other aspects? Could you please show us some new findings from Massachusetts General Hospital?

 

Dr Chung: Thank you for the question. I think for hepatitis B virus, it is really trying to understand how important it is to try to restore those immune responses that have otherwise become exhausted. This is particularly true for T-cells, which are really the main engine of clearance of virally-infected hepatocytes, but there are other cell types, both innate and adaptive cells, that have been shown to be involved in both viral control and pathogenesis. So I think it is really trying to understand what chronic hepatitis B infection does to those cell types and their disarming of those functional responses over time, as well, of course, understanding how reversible those responses are with proper and appropriate manipulations. I think the answer to that question is really going to go a long way towards describing how reversible chronic infection is in terms of restoring those T-cell and other immune responses. That is going to be the exciting line of work in hepatitis B, and of course, other studies of hepatocarcinogenesis, as well as those I have alluded to in my answer to the previous question. That is going to be very important. These can be done using cell-based models, animal models of hepatitis B infection, and even so-called organoid models, which in vitro are really more representative of examples of multiple cells in three-dimensions with one another to more accurately replicate the in vivo situation. I think these models are really going to be most informative in terms of telling us about mechanisms of hepatocarcinogenesis, as well as the immune response and the reshaping of that immune response and the contribution to functional cure, and even biological cure. From the vantage point of other research on hepatocarcinogenesis, fibrogenesis and disease progression in general, we have been very interested in looking at chemopreventive approaches. It is well-known, as I said earlier, that there is still persistent risk for patients with advanced liver disease, not only for hepatocarcinogenesis, but also for continued fibrosis and disease progression. We can’t transplant all patients. Transplantation is just not a widespread enough resource. It is also labor and capital intensive. So for those with advanced liver disease, we need to talk about strategies that would keep them from developing the serious complications of that liver disease, even if we can’t cure the underlying driver of that disease. That is particularly true in hepatitis B, and also true in other causes, like non-alcoholic fatty liver disease as a good example. Based on the persistence of these gene signatures that I referred to earlier, and the persistence of risk for disease progression and liver cancer, we are examining the role of possible chemopreventive compounds that might work to block those pathways that I described earlier that may persist in patients, even in those patients cured of hepatitis C for example. Is there a role for agents such as statins or aspirin, or even other more specific pathway inhibitors, to help prevent disease progression to the feared clinical complications of cirrhosis and liver cancer? This is an active area of investigation that we have been pursuing. We have also been very interested in investigating the interaction of hepatitis B as an example with other cofactors such as fatty liver disease, as well as HIV coinfection, both of which accelerate the course of HBV-related disease, and vice versa. We have investigated and are exploring new pathways that may contribute to disease progression for not just HBV disease, but also HIV and fatty liver disease. We are quite excited about some of our findings in that regard in terms of the contribution of factors such as hypoxia-inducible factor, just as one example. We are exploring many of these interactions, and are excited about the idea of elucidating new pathways that can be specifically targeted to prevent further progression of these important forms of liver disease.