血清HBV RNA和HBcrAg已被证明能反映肝内cccDNA转录活性，能更好地对慢性乙型肝炎（CHB）患者的疾病状态进行分类以及监测患者的功能性治愈情况。然而，在大规模的真实世界队列中对这两种标志物联合应用的研究数据仍然有限。在今年的美国肝病研究学会年会（AASLD2021）上，法国里昂大学Fabien Zoulim教授团队的一项题为“CROSS-SECTIONAL STUDY OF SERUM HBV RNA AND HBCRAG IN A REAL-LIFE PROSPECTIVE COHORT OF 1500 CHRONIC HEPATITIS B PATIENTS FOLLOWED IN FRANCE AND ITALY”的研究公布（LP28）^[1]。会期《国际肝病》特别采访了Fabien Zoulim教授，并邀其针对该研究的相关话题作进一步分享。  

 

 

《国际肝病》：血清HBV RNA和HBcrAg作为慢乙肝新型生物标志物，二者有何特点？其作用机制是什么？

 

Zoulim教授：首先，我们正在尝试开发新的非侵入性生物标记物，用患者的血清就可以检测，但能够反映肝脏中的病毒储存库，主要是肝内cccDNA的水平。由于HBsAg既可以来自cccDNA，也可以来自整合的HBV DNA，因此定量检测HBsAg不能充分反映cccDNA库的情况。所以如果我们要寻找根除感染和消除cccDNA的新疗法或新策略，那么定量HBsAg可能不是最好的标志物。我们需要能真正反映cccDNA的生物标志物。

 

第一种是血清中的HBV RNA。确切的说，是血清中的前基因组RNA，它主要来自cccDNA而非整合的基因。有几项研究表明，前基因组RNA与肝内cccDNA的转录活性之间存在相关性。已经有很多新的手段可用来检测前基因组RNA，从首个诞生于实验室的方法，到现在由很多诊断试剂公司开发的方法。我们看到了很多非常有趣的研究，本次AASLD年会上也有多项关于前基因组RNA的研究公布。

 

第二种是血清中的HBcrAg，通过酶联免疫吸附法（ELISA）来检测。它反映了核心蛋白和核心相关前蛋白以及HBsAg的表达，因为它主要由cccDNA表达，不由整合基因表达，因此也被认为能反映cccDNA的情况。几项研究表明，血清中的HBcrAg与cccDNA转录RNA的能力之间存在相关性。这是一个非常有趣的检测，因为通过非常简单的ELISA方法就能反映有转录活性的cccDNA库。

 

目前已经有多项临床试验开展，研究这些新的生物标志物。

 

Hepatology Digest: What are the characteristics of serum HBV RNA and HBcrAg as new biomarkers of chronic hepatitis B? What is the mechanism of action?

 

Dr Zoulim: First of all, what we are trying to develop is novel biomarkers that are non-invasive, so they can be tested in the serum of patients, and that reflect the reservoir of the virus in the liver, and mainly trying to reflect the cccDNA in the liver. The first interesting biomarker is viral RNA in serum. We need these biomarkers because we are seeing that quantitative HBsAg is not sufficient to predict the pool of cccDNA, because HBsAg can come either from cccDNA or from integrated HBV DNA. So if we are looking at new treatments or new strategies aimed at curing infection and eliminating cccDNA, then quantitative HBsAg may not be the best marker because it can come from integrated forms. So we need to have biomarkers that really reflect cccDNA. The first one is viral RNA in serum. When we say viral RNA, we really need to mention that it should be the pregenomic RNA in serum, because pregenomic RNA comes mainly from cccDNA and not from integration. This biomarker is very interesting. There are several studies that have shown the correlation between pregenomic RNA and the transcriptional ability of cccDNA in the liver. Now there are novel tests to measure pregenomic RNA. We are seeing the first of many laboratory-developed tests, and now there are even more tests being developed by diagnostic companies, such as Abbott and Roche Diagnostics. We have seen very interesting studies on that, and maybe we can comment later on those results. These biomarkers and the assays are now in development. We have seen many studies at AASLD that have used pregenomic RNA as a marker. The second one is the HBcrAg or core-related antigen in serum. This time, it is an ELISA, because we are measuring the protein, not the RNA. Those tests for RNA are PCR-based assays, but here it is an ELISA for core-related antigen. It reflects the expression of core protein and proteins that are related to core as well as HBsAg, and it is also thought to be reflective of cccDNA because it is mainly expressed by cccDNA and not by integrated forms. There are several studies that have shown a correlation between the HBcrAg in serum and the cccDNA’s ability to transcribe viral RNA. So it is a very interesting test, because it is an easy ELISA test that reflects the reservoir of transcriptionally active cccDNA. Many studies are being performed to show that. The assay is developed by Fujirebio from Japan. It is also being developed here by diagnostic companies. So we are getting these different biomarkers and there are clinical studies ongoing. We can talk about them in the next question.

 

《国际肝病》：结合该研究，请您概述一下在不同的慢乙肝治疗人群中，HBV RNA和HBcrAg预测价值是怎样的？

 

Zoulim教授：我们目前用来治疗慢乙肝的核苷（酸）类似物（NUCs）有一个很大的问题，那就是我们能否在某一时刻停药而不需要终生治疗。停止NUCs治疗后，一种情况是病毒复制和炎症复发，之后伴随HBsAg清除，HBsAg消失率增加。但也有另一种情况是，病毒复制和炎症复发可能重新引发慢性肝病。因此，在停药前预测患者是有HBsAg消失（这将是非常有利的）的机会还是有疾病活动、复发的风险，非常重要。

 

但直到最近也没有合适的预测因子。在今年的AASLD上，有几项研究探索了上述问题。迄今为止，最简单的生物标志物是定量HBsAg。不同研究表明，HBsAg越低，HBsAg消失的可能性越大。但其预测价值不是很强。

 

若是加上停药时的前基因组RNA和HBcrAg这两个标志物，可能具有很高的预测价值。来自世界不同地区（中国、日本、西方国家）的多项研究表明，将这三种标志物结合起来可以有更好的预测价值。如果患者HBsAg较低（<100 IU/mL）、无法检测到前基因组RNA和HBcrAg较低，那么HBsAg消失的概率可能更高。不过，由于这些研究一般都是回顾性的，因此，我们需要进行前瞻性研究来进一步分析。

 

利用这三种生物标志物，找到各自的最佳阈值，并建立一个预测算法——我们现在似乎正在向这个方向发展，我想这非常重要。对于新的治疗方法，新作用模式药物的临床试验也非常有趣，但我们需要学习。在AASLD会议期间，我们看到了关于衣壳组装调节剂和siRNA以及二者联合应用的研究，还有使用血清HBcrAg和前基因组RNA作为终点的研究。因此，对于这些新的药物和新的生物标志物，我们还处于学习阶段，现在说如何将这些生物标志物用于新药治疗还为时过早，但我相信它们对监测新药的抗病毒疗效会非常有帮助。

 

Hepatology Digest: In conjunction with this study, could you please outline the predictive value of HBV RNA and HBcrAg in the treatment of different populations with chronic hepatitis B?

 

Dr Zoulim: Since these biomarkers are very important, they have been used in clinical studies. Regarding nucleoside analog therapies, the drugs that we currently have, one of the big questions is whether we can stop NUCs at some point and not do lifelong therapy. In that setting, the question is whether stopping nucleoside analog therapy will allow relapse of viral replication and inflammation, which would be followed by the clearance of HBsAg and an increase in the rate of HBsAg loss. On the other hand, the other scenario is the relapse of viral replication and inflammation, which may just restart the chronic liver disease. So there are two main scenarios. It is important to predict before stopping treatment of the chance or the risk for the patient to either lose HBsAg (which would be very favorable), or to have an active relapse of the disease. Up until very recently, the field was very confused. There were no predictors. There have been several presentations at AASLD looking at the risk of relapse or the chance of achieving HBsAg loss. Again, it is not very easy to have a simple take-home message. The easiest biomarker so far was quantitative HBsAg, because HBV DNA is undetectable by definition, as patients are already on NUCs. It was suggested by different studies again at AASLD that the lower the HBsAg is, the higher the likelihood of losing HBsAg. But the predictive value is not very strong. That is a problem. So now, adding pregenomic RNA and core-related antigen at the time of stopping NUCs, may be of high value. There are several studies from different parts of the world (China, Japan, Western countries) suggesting that combining the three markers could add better predictive value. If patients have low HBsAg (<100), undetectable pregenomic RNA, and low HBcrAg, then the chances of losing HBsAg may be higher. But we will need to address that in a prospective manner, because for most of the studies, the analysis of the biomarkers was done retrospectively. It seems that we are going in that direction - to use these three biomarkers, and finding the best threshold for the different biomarkers together, and building an algorithm to predict. I think that will be very important. For novel therapies, it is also very interesting in clinical trials with novel modes of action, but we need to learn. We have seen very interesting studies with capsid assembly modulators and siRNAs during AASLD, as well as combinations of capsid assembly modulators and siRNAs. We have seen studies of endpoints using HBcrAg and pregenomic RNA in serum, and I think we are learning, together with the new drugs but also with the new biomarkers. It is still a bit early to say how to use these biomarkers with the new drugs, because we have two new things - biomarkers and drugs. But the studies are really interesting, and I am sure it will be very helpful to have these novel biomarkers for monitoring the efficacy of the novel antiviral treatments.

 

《国际肝病》：在对乙肝病毒进行检测的过程中，cccDNA非常关键。那么HBV RNA和HBcrAg分别与cccDNA的相关性如何？未来是否有必要在乙肝五项检查指标的基础上加上这两项指标？

 

Zoulim教授：如前所述，不同的学者独立证明了前基因组RNA和HBcrAg是能很好反映cccDNA转录活性的生物标志物。相比分析cccDNA所需的肝活检，它们是非侵入性的。这一定很好，也很重要。

 

现在的问题是我们将如何使用这些生物标志物？它们能否成为更好的疾病分期工具——区分免疫耐受、慢性肝炎、非活动携带者等？有这种可能，但我们需要做更多的研究。NIH小组最近发表了一些研究，但我们需要在这些研究的基础上，进一步分析这些生物标志物能否更好地描述患者特征和诊断患者。

 

它们是否有预测价值？在HBsAg检测不到之前，预测HBsAg的消失、从而预测功能性治愈？这一点非常重要。我们需要在HBsAg消失的患者中开展前瞻性研究，以确定前基因组RNA和HBcrAg是否比定量HBsAg表现更好，是否有更大的预测价值，可用于临床。

 

第三个应用，我想是用于新药的监测，并且是针对所有不同的药物。根据药物的作用方式，生物标志物的演变可能有所不同。我们需要在新抗病毒药物的临床开发过程中学习如何使用生物标志物，以了解它们如何帮助监测患者。我觉得目前进展很顺利，在一两年内，我们将更好地了解如何使用这些生物标志物。在AASLD会议期间，我们看到了很多在研新药，如衣壳组装调节剂、siRNA、免疫调节剂和检查点抑制剂——中国报告了一项针对PD-L1抗体的临床试验。这些都是很有希望的。我们需要将这些药物结合起来治愈感染，而生物标志物对于协助这些药物的开发，以及以后在常规临床环境中慢性乙型肝炎的管理将是极其重要的。

 

Hepatology Digest: In the process of detecting hepatitis B virus, cccDNA is very critical. So how do HBV RNA and HBcrAg correlate with cccDNA respectively? Is it necessary to add these two biomarkers in the management of hepatitis B in the future?

 

Dr Zoulim: As I said at the beginning in my response to the first question, it was nicely shown by different investigators independently that pregenomic RNA and HBcrAg are really good biomarkers of cccDNA transcriptional activity. They are very interesting. They are non-invasive, compared to the liver biopsy that is required for cccDNA analysis. That is very good and very important. Now the question will be how are we going to use these biomarkers? Will it be as tools to better classify patients into the different phases of disease - from immune-tolerant to chronic hepatitis to inactive carriers, and so on? That will be one possibility, and we need to do more studies. There were some studies published recently by the NIH group, but we need to build on those studies to see whether these biomarkers can add value for better characterization of patients, and better diagnoses. Will it be a predictive value - a prediction of loss of HBsAg, therefore a prediction of functional cure, before HBsAg is undetectable? That would be extremely important, and we need to have prospective studies where patients lose HBsAg to determine whether pregenomic RNA and HBcrAg perform even better than quantitative HBsAg. We need these prospective studies to see whether these biomarkers will have a strong predictive value that can be used in the clinic. The third application, I think, will be for the monitoring of the new drugs. But then we will need to learn how to use these biomarkers across all the different drugs. Depending on the mode of action of the drugs, the evolution of the biomarker may be different. We need to learn with the clinical development of the new antivirals how to use the biomarkers to see how they may help with the monitoring of patients. I think it is going very well, and we are learning. I think within one or two years, we will know much better how to use these biomarkers. At AASLD, we have seen new drugs in development, like capsid assembly modulators, siRNA, and immune modulators and checkpoint inhibitors. Yesterday, we saw a PD-L1 antibody in a clinical trial from China. All these are very promising. We need to combine all these drugs together to cure the infection, and the use of biomarkers will be extremely important to assist in the development of these drugs, and later on in the future, to manage patients with chronic hepatitis B in the routine clinical setting.

 

参考来源：

 

[1]Fabien Zoulim, Barbara Testoni, Carrie-Lynn Newsom, et al. CROSS-SECTIONAL STUDY OF SERUM HBV RNA AND HBCRAG IN A REAL-LIFE PROSPECTIVE COHORT OF 1500 CHRONIC HEPATITIS B PATIENTS FOLLOWED IN FRANCE AND ITALY. AASLD2021, LP28.