乙型肝炎的新病毒生物标志物：我们能够改变实践吗？

StephenW

New Viral Biomarkers for Hepatitis B: Are We Able to Change Practice?


Christoph Höner zu Siederdissen; Benjamin Maasoumy; Markus Cornberg
J Viral Hepat. 2018;25(11):1226-1235.  

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Hepatitis B Surface Antigen (HBsAg)

The HBsAg is part of the viral envelope but also exists in large excess as noninfectious subviral particles. Measurement of HBsAg level has been widely adapted in clinical practice and clinical trials.[10] It is formed by a small protein (SHBs), a middle protein (MHBs) and a large protein (LHBs).[11] The MHBs and the LHBs account for up to one–third of the viral envelope proteins and are equally distributed, whereas the SHBs accounts for the rest. HBsAg serves several functions as part of the infectious virion as well as noninfectious particles. By binding of SHBs and, more importantly, LHBs (contains pre–S1) to the heparan sulphate proteoglycan (HSPG) and the sodium taurocholate cotransporting polypeptide, these proteins are required for transmission of the virion.[12]

As noninfectious subviral particle, HBsAg may inhibit the host immune system.[13] Importantly, HBsAg levels allow for important conclusions about the state of the viral infection and the interplay with the host. Levels of HBsAg reflect the transcriptional activity of cccDNA predominantly in HBeAg–positive infection and to a lesser degree in HBeAg–negative patients.[14,15] In general, lower HBsAg levels are associated with better immune control.[16,17] However, as HBsAg can also be produced from integrated HBV DNA, HBsAg levels can be independent from transcriptional activity of cccDNA.[18,19]

Standardized assays are available for quantification and are commercially available, that is, the Architect HBsAg assay (Abbott Diagnostics, Abbott Park, IL, USA), the Elecsys HBsAg II quant assay (Roche Diagnostics, Indianapolis, IN, USA) and the DiaSorin Liaison XL (DiaSorin, Saluggia, Italy). All assays correlate strongly and can be compared independently.[20–23] However, the assays do not discriminate between the three different HBs proteins.

The role of the different HBsAg proteins in virions and subviral particles and their reflection of the disease state as well as their roles as potential biomarkers have gained attention in recent years. Nevertheless, to date, no commercial test to detect the different types of HBsAg proteins is available and measuring of these proteins is based on in–house quantitative enzyme–linked immunosorbent assays or Western blot analysis.[24,2

StephenW

乙型肝炎的新病毒生物标志物：我们能够改变实践吗？


ChristophHönerzuSiederdissen;本杰明马苏米; Markus Cornberg
J病毒肝病。 2018; 25（11）：1226至1235年。

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乙型肝炎表面抗原（HBsAg）

HBsAg是病毒包膜的一部分，但也作为非感染性亚病毒颗粒大量过量存在。 HBsAg水平的测量已在临床实践和临床试验中得到广泛应用。[10]它由小蛋白质（SHBs），中间蛋白质（MHBs）和大蛋白质（LHBs）组成。[11] MHB和LHB占病毒包膜蛋白的三分之一并且分布均匀，而SHB占其余部分。 HBsAg作为感染性病毒粒子的一部分以及非感染性粒子具有多种功能。通过结合SHB，更重要的是，LHBs（含有pre-S1）与硫酸乙酰肝素蛋白多糖（HSPG）和牛磺胆酸钠协同转运多肽，这些蛋白质是病毒体传播所必需的[12]。

作为非感染性亚病毒颗粒，HBsAg可能抑制宿主免疫系统。[13]重要的是，HBsAg水平可以得出关于病毒感染状态和与宿主相互作用的重要结论。 HBsAg的水平反映了cccDNA的转录活性，主要是HBeAg阳性感染，HBeAg阴性患者的转录活性较低[14,15]一般来说，较低的HBsAg水平与更好的免疫控制有关[16,17]。由于HBsAg也可以从整合的HBV DNA中产生，HBsAg水平可以独立于cccDNA的转录活性。[18,19]

标准化测定可用于定量并且可商购，即Architect HBsAg测定（Abbott Diagnostics，Abbott Park，IL，USA），Elecsys HBsAg II定量测定（Roche Diagnostics，Indianapolis，IN，USA）和DiaSorin Liaison。 XL（DiaSorin，Saluggia，意大利）。所有分析都强烈相关，可以独立比较。[20-23]然而，这些分析不区分三种不同的HBs蛋白。

近年来，不同HBsAg蛋白在病毒体和亚病毒颗粒中的作用及其对疾病状态的反映以及它们作为潜在生物标志物的作用已引起关注。然而，到目前为止，还没有检测不同类型的HBsAg蛋白的商业测试，这些蛋白质的测量是基于内部定量酶联免疫吸附测定或Western印迹分析。[24,25]

StephenW

Hepatitis B Core–related Antigen (HBcrAg)

Hepatitis B core antigen, hepatitis e antigen (HBeAg) and a small core–related protein (p22cr) share a 149 long amino acid sequence. This sequence is detected by HBcrAg assays.[28,29] HBcrAg correlates well with HBV DNA. Thus, it may be used as a marker of viral replication.[28,30,31] There is a weaker correlation between HBcrAg and HBsAg.[28] Furthermore, it has been shown that HBcrAg levels correlate well with the amount of cccDNA in both HBeAg–positive and HBeAg–negative patients.[32–34] Since 2014, a fully automated HBcrAg assay is commercially available (Lumipulse G, Fujirebio).
乙型肝炎核心相关抗原（HBcrAg）

乙型肝炎核心抗原，肝炎e抗原（HBeAg）和小核心相关蛋白（p22cr）共有149个长氨基酸序列。 该序列通过HBcrAg检测进行检测[28,29] HBcrAg与HBV DNA有很好的相关性。 因此，它可以用作病毒复制的标志物。[28,30,31] HBcrAg和HBsAg之间的相关性较弱[28]。 此外，已显示HBcrAg水平与HBeAg阳性和HBeAg阴性患者中的cccDNA数量相关[32-34]。自2014年以来，全自动HBcrAg测定可商购获得（Lumipulse G，Fujirebio）

StephenW

Hepatitis B Virus RNA (HBV RNA)

The blueprint of HBV replication is the cccDNA. HBV RNA (mRNA transcripts) is the template for protein synthesis and is synthesized from cccDNA. Thus, HBV RNA is a marker for viral replication and pregenomic RNA can be detected in the serum of HBV–infected patients.[7]

Importantly, no commercial assay is available, but in–house assays are used to detect HBV RNA. It correlates with HBV DNA in untreated patients.[35]
It is important to note that HBV RNA is influenced by many variables, such as genotype and presence of mutations.[36] Still, similar assumptions can be made about other diagnostic markers. Knowing about these factors may become an advantage in future studies and make HBV RNA more reliable.

乙型肝炎病毒RNA（HBV RNA）

HBV复制的蓝图是cccDNA。 HBV RNA（mRNA转录物）是蛋白质合成的模板，由cccDNA合成。 因此，HBV RNA是病毒复制的标志物，可以在HBV感染患者的血清中检测到前基因组RNA [7]。

重要的是，没有商业化验可用，但内部测定用于检测HBV RNA。 它与未经治疗的患者的HBV DNA相关。[35]

值得注意的是，HBV RNA受许多变量的影响，如基因型和突变的存在。[36] 仍然，可以对其他诊断标记进行类似的假设。 了解这些因素可能成为未来研究的一个优势，使HBV RNA更可靠。

StephenW

HBeAg–positive Infection and HBeAg–positive Hepatitis

HBeAg–positive infection is characterized by high viral replication, but no signs of inflammation. It is commonly seen in the first decade(s) of infection. The major question during this phase is to distinguish between patients, who have infection from patients with active hepatitis. The latter require immediate treatment.

Viral markers of HBeAg–positive infection are positivity for HBeAg and high levels of HBsAg, HBV DNA and HBcrAg.[28,37] HBsAg values above 4.9 log IU/mL are highly suggestive for HBeAg–positive infection in European and Asian patients.[15,17,38] Not unexpectedly, high levels of HBcrAg ≥7 log IU/mL also help to identify HBeAg–positive infection.[28,37] Recently, the assumption that HBeAg–positive infection is immunologically inert and has no consequences for the patient has been challenged.[39] During this phase, HBV DNA integration occurs to a significant degree and also clonal hepatocyte expansion. Additionally, it has been recognized that ALT is a poor surrogate marker for ongoing hepatitis. Thus, it may be plausible that within the next few years patient separation between HBeAg–positive infection and HBeAg–positive hepatitis will be done based on one or, more likely, a combination of several new markers.

The transition from HBeAg–positive infection to HBeAg–positive hepatitis usually occurs during the second–third decade, as a result of the host's immune response against HBV. Patients can undergo anti–HBe seroconversion and may develop HBeAg–negative infection, with a more favourable long–term outcome. Without antiviral therapy, spontaneous conversion is expected to occur at an annual rate of 5%–10%.[40,41] On the contrary, if patients fail to achieve anti–HBe seroconversion and have ongoing (immune mediated) hepatitis, they are at an increased risk of developing liver cirrhosis and liver–related adverse events.

In clinical practice, prediction of spontaneous anti–HBe seroconversion would be helpful; however, to date no predictor to reliably forecast anti–HBe seroconversion exists.[38] A small study in 43 patients examined HBcrAg levels, which seem to be predictive for future HBeAg seroconversion.[42] In comparison with patients, who do not undergo anti–HBe seroconversion, HBcrAg levels were lower and declined more noticeably in patients with anti–HBe seroconversion. HBcrAg levels of 4.9 log IU/mL had a positive predictive value (PPV) of 73.9% and a negative predictive value (NPV) of 96.7% for HBeAg seroconversion. A cut–off of 2 log IU/mL for the decline in HBcrAg levels over 28 weeks had a PPV of 76.2% and a NPV of 93.8%.[42]

If no spontaneous anti–HBe seroconversion occurs, the ongoing immune response may lead to liver damage. The ongoing inflammation might be detected by an increased ALT, but also lower HBsAg levels may raise suspicion. In fact, lower HBsAg levels in HBeAg–positive hepatitis have been identified to be associated with a higher risk for advanced fibrosis.[43,44]

A particular challenge is the detection of ongoing low–level inflammation in the liver, which often is not detected by ALT testing. A recent study has assessed the value of quantitative anti–HBc as a marker for biopsy–proven inflammation.[45] Even more importantly, the study included a subset of patients with low ALT. The AUROC for significant (>G2) liver inflammation in patients with ALT <64 U/L for quantitative anti–HBc was 0.888. The cut–off of 4.01 log IU/mL had 89.5%/75.5% sensitivity/specificity and 72.3%/90.9% PPV/NPV. Thus, anti–HBc may be a useful marker to distinguish infection from hepatitis.[42]

HBeAg阳性感染和HBeAg阳性肝炎

HBeAg阳性感染的特征在于高病毒复制，但没有炎症迹象。它常见于感染的第一个十年。这一阶段的主要问题是区分患有活动性肝炎患者感染的患者。后者需要立即治疗。

HBeAg阳性感染的病毒标志物对HBeAg阳性，HBsAg，HBV DNA和HBcrAg水平高[28,37] HBsAg值高于4.9 log IU / mL，高度提示欧洲和亚洲患者的HBeAg阳性感染[ 15,17,38]出乎意料的是，高水平的HBcrAg≥7logIU/ mL也有助于鉴别HBeAg阳性感染。[28,37]最近，HBeAg阳性感染具有免疫惰性且无后果的假设。病人受到了挑战。[39]在该阶段期间，HBV DNA整合在很大程度上发生并且还发生克隆肝细胞扩增。此外，已经认识到ALT是正在进行的肝炎的不良替代标志物。因此，可能有理由认为，在未来几年内，HBeAg阳性感染和HBeAg阳性肝炎之间的患者分离将基于一种或更可能的几种新标志物的组合来完成。

从HBeAg阳性感染到HBeAg阳性肝炎的转变通常发生在第二个三十年期间，这是由于宿主对HBV的免疫反应。患者可以接受抗HBe血清转换，并可能出现HBeAg阴性感染，并有更有利的长期预后。如果没有抗病毒治疗，预计自发转换率将以每年5％-10％的速度发生。[40,41]相反，如果患者未能实现抗HBe血清学转换并且持续（免疫介导的）肝炎，他们是发生肝硬化和肝脏相关不良事件的风险增加。

在临床实践中，预测自发性抗HBe血清转换将是有帮助的;然而，迄今为止还没有可靠预测抗HBe血清学转换的预测因子。[38]一项针对43名患者的小型研究检测了HBcrAg水平，这似乎可预测未来的HBeAg血清学转换。[42]与未接受抗HBe血清学转换的患者相比，抗HBe血清学转换患者的HBcrAg水平较低且下降更明显。 HBcrAg水平为4.9 log IU / mL，阳性预测值（PPV）为73.9％，HBeAg血清转换阴性预测值（NPV）为96.7％。对于28周内HBcrAg水平下降的截止值为2 log IU / mL，PPV为76.2％，NPV为93.8％。[42]

如果没有发生自发的抗HBe血清转换，正在进行的免疫反应可能导致肝损伤。 ALT升高可能会检测到持续的炎症，但HBsAg水平降低也可能引起怀疑。事实上，已确定HBeAg阳性肝炎中较低的HBsAg水平与晚期纤维化的高风险有关。[43,44]

特别的挑战是检测肝脏中持续的低水平炎症，这通常不会通过ALT测试检测到。最近的一项研究评估了定量抗-HBc作为活检证实炎症的标志物的价值。[45]更重要的是，该研究包括一部分ALT低的患者。 ALT <64 U / L定量抗-HBc患者的AUROC显着（> G2）肝脏炎症为0.888。 4.01log IU / mL的截止值具有89.5％/ 75.5％的灵敏度/特异性和72.3％/ 90.9％PPV / NPV。因此，抗HBc可能是区分感染与肝炎的有用标志物。[42]

StephenW

HBeAg–negative Infection and HBeAg–negative Hepatitis

Patients with successful anti–HBe seroconversion and immune control of the HBV infection have HBeAg–negative infection. This phase is characterized by persistently low levels of HBV DNA (<2000 IU/mL) and no signs of ongoing hepatitis without antiviral treatment. In general, these patients have a low risk for disease progression and the development of HCC (Table 1).

These data are based on findings of the REVEAL study, which showed cumulative incidence rates of HCC after 12 years from 3.57% to >12% for patients with HBV DNA >2000 IU/mL and >20 000 IU/mL, respectively.[46]

Importantly, mutations in the precore and basal core promoter region may mimic HBeAg–negative status by escaping the anti–HBe antibody response. Additionally, the natural course is variable. Thus, patients may experience ongoing intrahepatic inflammation, but mimicking HBeAg–negative infection. Consequently, in clinical practice, differentiation of HBeAg–negative infection from HBeAg–negative hepatitis is important.

Quantitative HBsAg levels help to distinguish between the different phases. HBsAg level <1000 U/mL with HBV DNA <2000 IU/mL has a 87.9% PPV and 96.7% NPV for HBeAg–negative infection (in genotype D).[47] Even lower values <100 IU/mL for HBsAg increase the specificity for HBeAg–negative infection. However, such low levels of HBsAg occur rarely in clinical practice and are therefore of limited value. More importantly, repeated measurements help to reliably detect HBeAg–negative infection.[48]

As mentioned, the phases of HBV infection are not linear and reactivation may be possible or fluctuations of HBV DNA and ALT may mimic HBeAg–negative infection. In HBeAg–negative patients, levels of HBsAg allow to assess the risk of developing HBeAg–negative hepatitis from HBeAg–negative infection. In one study, the annual rate of conversion from infection to hepatitis was 2.0%; however, in patients with HBsAg <1000 IU/mL, the rate of annual reactivation was reduced to 1.1%.[49] Thus, low HBsAg levels in addition to ALT and HBV DNA can identify patients with HBeAg–negative infection and low risk for adverse events.

HBsAg may also be useful to predict HBsAg loss (functional cure) in patients with HBeAg–negative infection. Several studies have shown that HBsAg level <100 IU/mL are predictive for subsequent functional cure.[48,50,51]

However, several factors can influence HBsAg levels; higher levels are expected for genotypes A and D, whereas lower levels are seen in genotypes B and C.[17] Mutations in the HBV genome may additionally reduce the production and the levels of HBsAg.[52]

As already described, HBsAg consists of LHBs, MHBs and SHBs. These can be measured and show distinct ratios in specific phases of infection.[25] Among the three proteins, LHBs is significantly different to a very high degree, with values of 1.9 ± 0.5 log ng/mL in patients with HBeAg–negative infection vs 2.5 ± 0.5 log ng/mL in HBeAg–negative hepatitis. The AUROC for HBeAg–negative infection for LHBs was 0.89, whereas the AUROC for HBsAg in general was 0.62 in this study. Thus, measurement of LHBs could become a potentially better discriminator for HBeAg infection vs HBeAg–negative hepatitis. However, it must be noted that measurement is currently based on in–house assays and the present data are based on 90 patients.[25]

HBcrAg has also been suggested as an additional marker in this setting. One recent study in 202 HBeAg–negative patients suggested that HBcrAg may be even more accurate compared with HBsAg to identify true inactive carriers with a single measurement regardless of the genotype. The combination of HBcrAg ≤3 log U/mL plus HBV DNA ≤2000 IU/mL showed a PPV and diagnostic accuracy >85%.[53]

Also, quantification of anti–HBc has been evaluated as a marker. Significantly higher levels can be found in HBeAg–negative hepatitis in comparison with HBeAg–negative infection.[54] The AUROC was 0.947. The cut–off of 12 489 IU/mL had 81.8% sensitivity, 94.4% specificity, 76.1% PPV and 96.4% NPV. Anti–HBc has also been evaluated as a marker to assess liver inflammation in HBeAg–negative patients, although performance is poor in comparison with HBeAg–positive patients.[45] The AUROC for biopsy–proven inflammation <G2 was 0.628. The sensitivity/specificity was 57.7%/69.6% and the PPV/NPV is 46.9%/78.0% for a cut–off of 4.23 log IU/mL.

HBeAg阴性感染和HBeAg阴性肝炎

具有成功的抗HBe血清转换和HBV感染免疫控制的患者具有HBeAg阴性感染。该阶段的特征是持续低水平的HBV DNA（<2000 IU / mL），并且没有抗病毒治疗的情况下没有正在进行的肝炎的迹象。通常，这些患者的疾病进展风险和HCC的发展风险较低（表1）。

这些数据基于REVEAL研究的结果，该研究显示，HBV DNA> 2000 IU / mL和> 20 000 IU / mL的患者在12年后的HCC累积发病率从3.57％增加到> 12％[46]。 ]

重要的是，前核心和基础核心启动子区域的突变可以通过逃避抗-HBe抗体反应来模拟HBeAg阴性状态。此外，自然过程是可变的。因此，患者可能经历持续的肝内炎症，但模仿HBeAg阴性感染。因此，在临床实践中，HBeAg阴性感染与HBeAg阴性肝炎的分化是重要的。

定量HBsAg水平有助于区分不同阶段。 HBs DNA水平<1000 U / mL，HBV DNA <2000 IU / mL，对HBeAg阴性感染（基因型D）的PPV为87.9％，NPV为96.7％。[47] HBsAg甚至更低的值<100 IU / mL会增加HBeAg阴性感染的特异性。然而，这种低水平的HBsAg在临床实践中很少发生，因此价值有限。更重要的是，重复测量有助于可靠地检测HBeAg阴性感染。[48]

如上所述，HBV感染的阶段不是线性的，可能是再激活或HBV DNA和ALT的波动可能模拟HBeAg阴性感染。在HBeAg阴性患者中，HBsAg水平可以评估HBeAg阴性感染引​​起HBeAg阴性肝炎的风险。在一项研究中，从感染到肝炎的年转换率为2.0％;然而，在HBsAg <1000 IU / mL的患者中，年复活率降至1.1％。[49]因此，除ALT和HBV DNA外，低HBsAg水平可以识别HBeAg阴性感染患者和不良事件风险低。

HBsAg也可用于预测HBeAg阴性感染患者的HBsAg消失（功能性治愈）。一些研究表明，HBsAg水平<100 IU / mL可预测随后的功能性治愈[48,50,51]。

但是，有几个因素会影响HBsAg水平;预计基因型A和D的水平较高，而基因型B和C的水平较低[17]。 HBV基因组中的突变可能会进一步降低HBsAg的产生和水平。[52]

如上所述，HBsAg由LHB，MHB和SHB组成。这些可以测量并在感染的特定阶段显示不同的比例。[25]在这三种蛋白质中，LHBs在很大程度上有显着差异，HBeAg阴性感染患者的HB值为1.9±0.5 log ng / mL，HBeAg阴性肝炎患者的值为2.5±0.5 log ng / mL。 LURB的HBeAg阴性感染的AUROC为0.89，而本研究中HBsAg的AUROC一般为0.62。因此，LHBs的测量可能成为HBeAg感染与HBeAg阴性肝炎相比更好的鉴别者。但是，必须指出的是，目前的测量基于内部检测，目前的数据基于90名患者。[25]

HBcrAg也被建议作为此设置中的附加标记。 202例HBeAg阴性患者最近的一项研究表明，与HBsAg相比，HBcrAg可能更准确，无论基因型如何，只需一次测量即可识别真正的非活动性携带者。 HBcrAg≤3logU/ mL加HBVDNA≤2000IU/ mL的组合显示PPV，诊断准确度> 85％。[53]

此外，已经评估了抗-HBc的定量作为标记。与HBeAg阴性感染相比，HBeAg阴性肝炎的水平显着升高[54]。 AUROC是0.947。截止值为12 489 IU / mL，敏感性为81.8％，特异性为94.4％，PPV为76.1％，NPV为96.4％。抗HBc也被评估为评估HBeAg阴性患者肝脏炎症的标志物，尽管与HBeAg阳性患者相比表现较差[45]。活检证实的炎症<G2的AUROC为0.628。截止值为4.23 log IU / mL时，灵敏度/特异度为57.7％/ 69.6％，PPV / NPV为46.9％/ 78.0％。