反转录下游机制减少但不是乙肝表面抗原HBV DNA血清水平在慢

StephenW

Research
Mechanisms downstream of reverse transcription reduce serum levels of HBV DNA but not of HBsAg in chronic hepatitis B virus infection

    Simon B. Larsson Email author, Sebastian Malmström, Charles Hannoun, Gunnar Norkrans and Magnus Lindh

Virology Journal201512:213

DOI: 10.1186/s12985-015-0447-5

©  Larsson et al. 2015

Received: 30 October 2015

Accepted: 3 December 2015

Published: 9 December 2015
Abstract
Background

Hepatitis B virus (HBV) DNA in serum of chronically infected patients declines by 3–4 log10 units at loss of HBe antigen (HBeAg) from serum. The mechanisms behind this decline, and the much smaller decline of surface antigen (HBsAg) levels, are still not well known. The aim of this study was to get a better understanding of this process by analysing both serum and intrahepatic markers of HBV replication.
Methods

Levels of HBV DNA and HBsAg in serum, and covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA) and S-RNA and total intrahepatic HBV DNA (ihDNA) in liver biopsies from 84 chronically infected patients (16 positive and 68 negative for HBeAg) were analysed.
Results

Lower HBV DNA levels within HBeAg-positive stage reflected lower levels of cccDNA and pgRNA with strong correlation. In HBeAg-negative patients, ihDNA levels were greater and HBV DNA levels in serum lower than expected from pgRNA levels. A lower HBV DNA/HBsAg ratio corresponded with lower pgRNA/cccDNA (p < 0.01) and higher S-RNA/cccDNA (p < 0.0001) ratios, suggesting that in HBeAg-negative patients transcription of pgRNA, but not of S-RNA, becomes suppressed.
Conclusions

The marked reduction of HBV DNA in serum after loss of HBeAg appears to be due to combined reduction of cccDNA, pgRNA and yet unidentified mechanisms downstream of reverse transcription. Such mechanisms include faster clearance of circulating virus or blocked secretion of virions, the latter supported by the observed relative increase of ihDNA in HBeAg-negative patients. The smaller reduction of S-RNA than of pgRNA partly explains why HBsAg remain high in the HBeAg-negative stage, supporting the possibility of HBsAg synthesis from integrated HBV DNA.
Keywords
HBV DNA HBsAg cccDNA Replication pgRNA

Background

Despite the availability of an effective vaccine, chronic hepatitis B virus (HBV) infection remains an important cause of liver cirrhosis and hepatocellular carcinoma, in particular in East Asia and Sub-Saharan Africa [1]. In clinical diagnostics, HBV DNA levels in serum are the most important marker for the assessment of prognosis and treatment outcome during chronic HBV infection. In early stage of chronic infection HBV DNA usually persists at high levels, above 7–8 log10 IU/mL, for many years, often decades. Later in life the HBV DNA levels decline when the immune response evolves to more efficiently suppress viral replication. The greatest reduction of HBV DNA is observed at loss of hepatitis B e antigen (HBeAg) from the blood, a critical step that typically represents a transition from high to low replicative stage, usually also conferring clinical improvement [2]. Mechanisms that explain this pronounced reduction of viremia are not fully clarified. Part of the reduction of HBV DNA in serum is a result of reduced intrahepatic levels of covalently closed circular DNA (cccDNA), and of the pregenomic RNA (pgRNA), a transcript from cccDNA that is reverse transcribed to minus strand HBV DNA during formation of new viral particles [3]. The cccDNA minichromosome is also template for transcripts that are translated into hepatitis B surface antigens (HBsAg) embedded in the envelope of both virions and so-called subviral particles (SVP), of which the latter are produced in great excess [4].

In diagnostics, detection of HBsAg has been used as the basic test for identification of HBV infection for decades. Recently, quantification of HBsAg has come into focus as a complement to HBV DNA levels for monitoring both the natural course of infection and the response to treatment [5]. Levels of HBsAg, which mainly represent the levels of SVP [4], are more stable over time than HBV DNA, with a much smaller decline during the course of infection. This difference is in particular striking during loss of HBeAg when HBV DNA declines by 3–4 log10 or even more, whereas the HBsAg levels often are reduced by only 1 log10 [6–8]. As a consequence, the ratio between these two serum markers (HBV DNA/HBsAg) decreases when HBV DNA reaches very low or even undetectable levels while HBsAg remains at significant levels [9]. Previous studies indeed indicate that reduced transcription of pgRNA from cccDNA may contribute to the decline in HBV DNA [10]. It is however not well known to what extent such effects are specific for pgRNA, i.e. are not also reducing S-RNA. In the present study we explored the relations between HBV markers in serum and liver biopsies from both HBeAg-positive and HBeAg-negative patients, searching for explanations to the strong reduction of HBV DNA in serum and the shift in HBV DNA/HBsAg ratio that occur in parallel with loss of HBeAg. The contribution of different steps of replication to the reduced serum levels of HBV DNA and HBsAg is discussed.

Discussion

This study of intrahepatic HBV RNA and HBV DNA and their correlation with each other and with levels of HBV DNA and HBsAg in serum may help to understand how HBV replication is suppressed during the course of infection and how low serum levels of HBV DNA and HBsAg are achieved. Part of our results agree well with published studies [11–13], but the observations of an increased S-RNA/pgRNA ratio after loss of HBeAg, and of the much greater decline of serum HBV DNA than of intrahepatic HBV DNA have to our knowledge not been described before.

Within HBeAg-positive patients, lower serum levels of HBV DNA essentially reflected lower levels of the cccDNA template in combination with reduced amounts of pgRNA per cccDNA. Our results, with a 2 log10 lower cccDNA level in HBeAg-negative patients are identical with the observations by Werle-Lapostelle et al. [11], whereas other studies have reported smaller reduction (≈1 log10) after loss of HBeAg [12, 13]. The reduced levels of cccDNA after loss of HBeAg is probably mainly a result of eradication of infected hepatocytes by cytotoxic T cells [14] in combination with lower cccDNA content in each infected cell.

Concern has been raised regarding the specificity of cccDNA PCR assay, i.e. whether this assay amplifies only cccDNA. When this method was introduced by Köck and Schlicht they showed that the degree of cross-reactivity, i.e. amplification by cccDNA primers of other forms of HBV DNA, was low, corresponding to a 3 log10 lower sensitivity [15]. We found the same degree of cross-reactivity when we repeated their experiments using real-time PCR (results not shown). When analysing liver biopsies, we found that cccDNA levels were on average 1.90 log10 lower than ihDNA, and not 3 log10 lower as one would expect if the cccDNA findings were due to cross-reactivity. This indicates that a low specificity of the assay should have limited impact on our cccDNA results.

In addition to lower cccDNA levels, we found in HBeAg-negative patients a lower pgRNA/cccDNA ratio. This mechanism of reduced virus production has been proposed by others, and might be mediated by different mechanisms such as accelerated intracellular clearance of pgRNA-containing capsids induced by IFN-alfa/-beta [16], or by IFN-alfa mediated suppression of HBV replication by epigenetic control of cccDNA function and transcription [17]. Among our patients, the reduction of the pgRNA/cccDNA ratio was quite moderate (0.57 log10), whereas Volz et al. and Laras et al. found larger reductions (0.88 and 1.46 log10) [12, 13].

Within HBeAg-positive stage, pgRNA and serum HBV DNA correlated strongly with a slope of ≈ 1, meaning that 1 log10 decline of pgRNA results in 1 log10 decline of HBV DNA in serum. In HBeAg-negative patients, the relations between cccDNA, replicative intermediates (pgRNA and ihDNA) and levels of HBV DNA in serum were more complex.

Firstly, levels of pgRNA showed poorer correlation with cccDNA, suggesting that suppression acting on this step varies considerably between patients.

Secondly, in patients with lower cccDNA there were relatively higher levels of ihDNA, as indicated by both the slope of the ihDNA/pgRNA regression line and the higher ihDNA/pgRNA ratio in HBeAg-negative patients. The explanation to this relative increase of ihDNA among HBeAg-negative patients is uncertain. It might be due to production of non-productive replicative intermediates, or retention of viral particles that are not secreted into the blood. The latter possibility would agree with a study describing that viral particles can be blocked by anti-HBs antibodies internalized in liver cells [18]. Another study did however not report differences in virion release between HBeAg-positive and negative patients [19], and our finding and possible explanations need to be further explored. One also has to consider the possibility that integrated HBV DNA contributes to ihDNA, because such integrations would be detected by our real-time PCR assays (if they include the target region). In early stage their contribution would probably be minimal, but when the production of relaxed circular DNA in virions declines in HBeAg-negative patients the amount of integrated HBV DNA might be high enough to influence the measured ihDNA level.

Thirdly, in several HBeAg-negative patients the HBV DNA level in serum was reduced much more than what would be expected by the reduction of ihDNA, suggesting that factors acting downstream of reverse transcription may have great impact on HBV DNA in serum. Such factors might, as mentioned above, be reduced release of virions from infected cells, or shorter half-life of viral particles in serum. The possibility that enhanced clearance of free virions from the blood contributes to the pronounced reduction of HBV DNA levels in serum after loss of HBeAg has not been much studied or discussed. Mathematical calculations based on HBV kinetics under antiviral therapy [20] suggested that the half-life of free virions is ~25 h in HBeAg-positive and ~13 h in HBeAg-negative patients [21]. Another study reported similar half-lives in HBeAg-positive patients (17 h), but extremely short half-life in HBeAg-negative patients (0.6 s) [19], data that subsequently were questioned [22]. Despite the discrepancies it seems likely that stronger immunologic responses may lead to faster clearance of virions, but how much this influences serum levels of HBV DNA remains to be elucidated.

Whereas HBV DNA levels were more than 4.0 log10 lower in HBeAg-negative patients, levels of HBsAg were on average only 1.1 log10 lower. Accordingly, the HBV DNA/HBsAg ratio was 3.3 log10 lower in HBeAg-negative patients, as observed also in previous studies [13, 23, 24]. To some extent this was explained by a reduced ratio (by 2.0 log10) between the corresponding transcripts (pgRNA/S-RNA) in HBeAg-negative patients. This reduction of pgRNA/S-RNA is greater than the 1.2 log10 difference reported by Volz et al. [13], and was explained by both reduced pgRNA/cccDNA (as mentioned above) and an increased S-RNA/cccDNA ratio by 1.0 log10. This finding suggests that the smaller reduction of HBsAg after loss of HBeAg than expected from the cccDNA decline might be due to an enhanced transcription of S-RNA from cccDNA, or possibly transcription of S-RNA from integrated sequences of HBV DNA that contain the complete S region, including the promoter. Such transcription occurs in the Alexander hepatoma cell line [25], but whether this type of integrations occur during natural infection remains to be demonstrated.

The ratio between HBsAg/S-RNA was unchanged after loss of HBeAg, indicating that post-transcriptional processing of S-RNA and turnover of subviral particles remain unchanged after loss of HBeAg.

StephenW

研究
反转录下游机制减少但不是乙肝表面抗原HBV DNA血清水平在慢性乙型肝炎病毒感染

    西蒙·拉尔森电子邮件的作者，塞巴斯蒂安·马姆斯特罗姆，查尔斯Hannoun，贡纳尔Norkrans和马格努斯·林德

病毒学Journal201512：213

DOI：10.1186 / s12985-015-0447-5

©Larsson等人。 2015年

收稿日期：2015年10月30日

接受2015 12月3日

发布时间：2015年12月9日
抽象
背景

乙型肝炎病毒（HBV）在血清慢性感染病人的DNA的下降了3-4日志10单位在HBe的从血清抗原（HBeAg）的损失。这背后下降，表面抗原（HBsAg）水平小得多下降的机制，仍然是不为人所熟知。这项研究的目的是通过分析两种血清HBV复制的肝内标志，以获得更好的理解这个过程。
方法

HBV DNA与HBsAg血清水平，和共价从84慢性感染病人（16积极和68负的闭合环状DNA（cccDNA的），前基因组RNA（pgRNA）和S-RNA和肝内总HBV DNA（ihDNA）肝活检HBeAg的）进行了分析。
结果

在HBeAg阳性阶段较低的HBV DNA水平反映的cccDNA及pgRNA水平较低具有很强的相关性。在HBeAg阴性患者，ihDNA水平在降低血清从pgRNA水平比预期的更大，HBV DNA水平。较低的HBV DNA / HBsAg的比例对应着较低的pgRNA / cccDNA的（P <0.01），更高的S-RNA / cccDNA的（P <0.0001）的比例，这表明在pgRNA HBeAg阴性患者中的转录，但不是S-RNA的，成为抑制。
结论

HBV DNA的血清HBeAg消失后显着下降似乎是由于结合减少的cccDNA，pgRNA的和反转录的下游尚未鉴定的机制。这些机制包括流行病毒的清除速度更快或病毒颗粒的分泌受阻，后者由ihDNA在HBeAg阴性患者中观察到的相对增加的支持。较小减少S-RNA比pgRNA的部分解释了为什么乙肝表面抗原维持高位，在HBeAg阴性阶段，从整合的HBV DNA的HBsAg配套合成的可能性。
关键词
HBV DNA的HBsAg cccDNA的复制pgRNA

背景

尽管有效疫苗的有效性，慢性乙型肝炎病毒（HBV）感染仍肝硬化和肝癌的一个重要原因，特别是在东亚和撒哈拉以南非洲地区[1]。在临床诊断，血清HBV DNA水平对预后和治疗结果在慢性乙肝病毒感染的评估中最重要的标志。在慢性感染HBV DNA的早期阶段通常持续在高水平，上述7-8日志10 IU /毫升，多年，常常几十年。后来在生活中的HBV DNA水平下降，当免疫反应的发展能够更有效地抑制病毒的复制。 HBV DNA的下降幅度最大，在从失血乙肝e抗原（HBeAg）的观察，通常代表从高分到低分复制阶段的过渡，平时也赋予的临床改善的一个关键步骤[2]。机制，解释这种明显减少病毒血症都没有完全阐明。 HBV DNA的血清中减少的部分是共价闭合环状DNA（cccDNA的）的减小的肝内的水平的结果，和所述前基因组RNA（pgRNA），从cccDNA的转录物被逆转录成负链HBV DNA的形成过程中的新的病毒颗粒[3]。所述cccDNA的微染色体也是模板转录物被翻译成B型肝炎表面抗原（HBsAg的）嵌入在两个病毒体和所谓的亚病毒颗粒（SVP）的包络线，其中后者都产大过量[4]。

在诊断，检测的HBsAg已经用作基本试验用于鉴定HBV感染的几十年。近来，乙肝表面抗原的量化已成为焦点，以补充HBV DNA水平监测感染的两个自然病程和对治疗的反应[5]。 HBsAg的，这主要是指SVP [4]的水平的水平，是随着时间的推移比HBV DNA更稳定，与感染的过程中，更小的下降。这种差异是特别引人注目HBeAg消失期间时HBV DNA的下降由3-4日志10或甚至更多，而HBsAg水平往往只由1日志10 [6-8]减小。其结果是，（HBV DNA / HBsAg的）这两种血清标记物之间的比率时HBV DNA的到达非常低，甚至检测不到的水平降低，而HBsAg的保持在显著水平[9]。以前的研究确实表明pgRNA从cccDNA的降低的转录可能有助于在HBV DNA的减少[10]。然而，不公知到什么程度这种效果是特异性的pgRNA，即是不是还减少的S-RNA。在本研究中，我们探讨了血清和肝活检的HBeAg阳性和HBeAg阴性患者乙肝病毒标志物之间的关系，寻找解释的强还原HBV DNA血清和所发生的转变，HBV DNA /乙肝表面抗原比值在HBeAg消失平行。不同的步骤复制到HBV DNA和HBsAg的降低血清水平的贡献进行了讨论。

讨论

肝内HBV RNA和HBV DNA和它们相互之间的和与HBV DNA和乙肝表面抗原的血清水平可能有助于相关这项研究，以了解如何HBV复制的感染过程中抑制，并且有多低HBV DNA和乙肝表面抗原的血清水平是实现。我们的研究结果部分吻合较好，已发表的研究[11-13]，但HBeAg消失后，增加的S-RNA / pgRNA比例的意见，和血清HBV DNA比对肝内HBV DNA的更大下降对我们的知识以前尚未描述。

在HBeAg阳性患者，HBV DNA的降低血清水平基本上反映的cccDNA模板在每cccDNA的pgRNA量的减少相结合较低的水平。我们的研究结果，在HBeAg阴性患者2日志10 cccDNA的较低水平是相同的意见通过Werle-Lapostelle等。 [11]，而其他研究报道较小减少（≈1日志10）HBeAg消失后[12，13]。的cccDNA的水平降低HBeAg消失后是可能主要是消除感染的肝细胞的通过中，在每个感染细胞低级cccDNA的内容结合细胞毒性T细胞[14]的结果。

关注已经提出关于cccDNA的PCR检测的特异性，即该法是否仅放大cccDNA的。当该方法是由KOCK和SCHLICHT引入它们表明，交叉反应性，即扩增的其他形式的HBV DNA的引物的cccDNA的程度，是低的，对应于3日志10较低的灵敏度[15]。我们发现交叉反应的程度相同，当我们使用实时PCR（结果未示出）重复他们的实验。在分析肝活检，我们发现，cccDNA的水平上比ihDNA平均1.90日志10下，而不是3日志10下作为一个希望，如果cccDNA的结果是由于交叉反应。这表明该测定的一个低特异性应该对我们的cccDNA结果的影响有限。

除了降低cccDNA的水平，我们HBeAg阴性患者中发现更低的pgRNA / cccDNA的比例。减少病毒产生的这种机制已经提出了其他人，可能是由不同的机制，例如通过诱导IFN-阿尔法/ -beta [16]，或由干扰素介导干扰素α的HBV抑制含pgRNA衣壳加速细胞内清除率来介导通过cccDNA的功能和转录[17]的表观遗传调控复制。在我们的患者中，pgRNA / cccDNA的比率的减少是相当适中（0.57日志10），而VOLZ等。和LARAS等。发现较大的减少（0.88和1.46日志10）[12，13]。

在HBeAg阳性阶段，pgRNA和血清HBV DNA相关性强烈的≈1的斜率，这意味着1日志10下降pgRNA导致血清1日志10下降HBV DNA的。在HBeAg阴性患者，cccDNA的，复制中间体（pgRNA和ihDNA）之间和HBV DNA血清水平之间的关系则较为复杂。

首先，pgRNA的水平与cccDNA的相关性较差，表明抑制作用在这一步患者之间差别很大。

其次，在患者下cccDNA的有相对较高的水平ihDNA的，由ihDNA / pgRNA回归线的两个斜坡和HBeAg阴性患者更高ihDNA / pgRNA比表示。该解释ihDNA之间HBeAg阴性患者这相对增加是不确定的。这可能是由于生产的非生产性复制中间体，或保留病毒颗粒的未分泌到血液中。后者可能会同意一项研究描述了病毒颗粒可阻断内化于肝细胞[18]抗-HBs抗体。另一项研究也不过没有报告在HBeAg阳性和阴性的患者[19]之间的病毒体释放的差异，我们的发现和可能的解释需要进一步探讨。我们还必须考虑整合的HBV DNA有助于ihDNA，因为这样的集成会由我们的实时PCR测定来检测（如果它们包括的对象区域）的可能性。在早期阶段，他们的贡献很可能是最小的，但是当松弛环状DNA的生产病毒粒子下跌HBeAg阴性患者的综合性HBV DNA量可能会高到足以影响测量ihDNA水平。

第三，在一些HBeAg阴性患者血清中HBV DNA水平下降比将由ihDNA的减少预期多得多，这表明因素作用逆转录下游可能对HBV DNA的影响很大血清。这样的因素可能，如上所述，从感染的细胞，或在血清中病毒颗粒的短半衰期减少病毒颗粒的释放。该增强的游离的病毒颗粒的间隙从血液的可能性有助于明显降低血清HBV DNA水平的后HBeAg消失并没有受到太多的研究或讨论。基于在抗病毒治疗乙肝病毒动力学[20]数学计算表明，游离病毒粒子的半衰期是~~ 13小时在HBeAg阴性患者HBeAg阳性25小时和[21]。另一项研究报告了类似的半衰期在HBeAg阳性患者（17小时），但非常短的半衰期在HBeAg阴性患者（0.6秒）[19]，数据随后遭到质疑[22]。尽管差异这很可能是更强的免疫反应可能导致病毒粒子的通关速度更快，但是这多少影响HBV DNA血清水平仍有待澄清。

而HBV DNA水平均超过4.0日志10低于HBeAg阴性患者，乙肝表面抗原水平平均只有1.1 LOG10低。因此，HBV DNA / HBsAg的比例在HBeAg阴性患者3.3日志10时，观测到的也是在以往的研究[13，23，24]。在一定程度上，这是由相应的成绩单（pgRNA / S-RNA）在HBeAg阴性患者之间的下降比例（2.0日志10）解释。该还原pgRNA / S-RAN的是大于1.2日志10差报道VOLZ等。 [13]，并通过这两个减压pgRNA / cccDNA的解释（如上所述）和增加的S-RAN / cccDNA的比率由1.0日志10。这一发现表明，HBeAg消失后的小减少的HBsAg来自cccDNA的下降比预期的可能是由于S-RAN从cccDNA的，或者可能从HBV DNA的整合序列包含完整的转录S-RAN的增强转录S区，包括启动子。这种转录发生在亚历山大肝癌细胞系[25]，但自然感染过程中是否会出现这种类型的整合还有待证明。

的HBsAg / S-RAN之间的比率是HBeAg消失后保持不变，这表明S-RAN的转录后加工和周转亚病毒颗粒的HBeAg消失后保持不变。

StephenW

一个非常有意义的论文，推荐。全篇论文：
http://virologyj.biomedcentral.c ... 6/s12985-015-0447-5