Wang et al. Virol J (2021) 18:4 https://doi.org/10.1186/s12985-020-01471-2 RESEARCH Open Access Serum HBV RNA correlated with intrahepatic cccDNA more strongly than other HBV markers during peg-interferon treatment Xiaomei Wang1, Xiumei Chi1, Ruihong Wu1, Hongqin Xu1, Xiuzhu Gao1, Lei Yu2, Longgen Liu3, Mingxiang Zhang4, Youwen Tan5, Junqi Niu1 and Qinglong Jin1* Abstract Background: Serum hepatitis B virus RNA (HBV RNA) has been reported to be a surrogate marker of intrahepatic cccDNA during nucleos(t)ide analogs therapy. However, in HBeAg-positive patients treated with peg-interferon (peg- IFN), whether HBV RNA is superior to other HBV markers in refecting cccDNA profle is still unclear. Methods: Serum HBV RNA, HBcrAg, HBV DNA, and HBsAg were longitudinally assessed among 30 HBeAg-positive patients during 48-week peg-IFN treatment. Besides, intrahepatic cccDNA was detected at baseline and week 48 respectively. Then, the individual correlations between HBV RNA, HBcrAg, HBV DNA, HBsAg, and cccDNA were statisti- cally analyzed. Results: HBV RNA levels decreased more rapidly in patients with HBeAg seroconversion than those without HBeAg seroconversion. Among all patients, cccDNA correlated better with HBV RNA than with HBcrAg, HBV DNA, and HBsAg at baseline. After 48 weeks peg-IFN treatment, cccDNA still correlated more strongly with HBV RNA than other HBV markers. Further analysis indicated that in patients with HBeAg seroconversion cccDNA strongly correlated with HBV RNA and HBcrAg, whereas not correlate with HBV DNA and HBsAg. While in patients without HBeAg seroconversion, cccDNA highly correlated with HBV RNA and HBV DNA, moderately correlated with HBcrAg, and not correlated with HBsAg. Conclusion: Compared to HBcrAg, HBV DNA, and HBsAg, serum HBV RNA correlated more strongly with intrahepatic cccDNA levels before and after 48-week peg-IFN treatment. The level of serum HBV RNA may be a superior surrogate marker in refecting the intrahepatic cccDNA profle in HBeAg-positive patients during peg-IFN treatment. Trial registration ClinicalTrials, NCT03546530. Registered 1 January 2015. https ://clini caltr ials.gov/ct2/resul ts?cond &term NCT03 54653 0. = = Keywords: Hepatitis B virus, HBV RNA, HBcrAg, HBV DNA, cccDNA, Interferon Background risk of terminal liver diseases, such as cirrhosis, hepatic Hepatitis B virus (HBV) infection is a major global health decompensation, and hepatocellular carcinoma, which problem. Chronic HBV infection greatly increases the contribute to more than 780,000 deaths every year world- wide [1, 2]. Currently, the goal for the treatment of HBV is to suppress viral replication to the lowest possible *Correspondence: [email protected] level thus halting disease progression. Nowadays, the 1 Department of Hepatology, The First Hospital of Jilin University, 71 approved agents for the treatment of chronic HBV infec- Xinmin Street, Changchun 130021, Jilin Province, China Full list of author information is available at the end of the article tions mainly belong to two classes: pegylated interferon © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wang et al. Virol J (2021) 18:4 Page 2 of 10 (peg-IFN) and nucleotide/nucleoside analogs (NAs) [3]. Aim of the study Among these agents, peg-IFN, which has the efects of In the present study, our aim was to fnd a better sur- suppressing HBV replication and immunomodulation, is rogate marker of cccDNA in HBeAg-positive patients still widely used in the clinic as the frst-line therapeutic with peg-IFN treatment through evaluating the corre- option for patients with HBeAg-positive chronic hepati- lations of HBV RNA, HBcrAg, HBV DNA, and HBsAg tis B (CHB) [4]. with cccDNA. At the same time, the dynamic changes of Although antiviral agents can efectively reduce the serum HBV RNA and HBV DNA were also investigated. serum HBV DNA level in CHB patients, complete elimi- nation of HBV is difcult due to the existence of intrahe- Materials and methods patic covalently closed circular DNA (cccDNA) [5]. HBV Study participants cccDNA, which serves as the template for the transcrip- A total of 30 HBeAg-positive, noncirrhotic CHB patients tion of a 3.5-kb pregenomic RNA, can produce progeny were recruited from a multicenter, randomized, con- viral DNA and proteins even in the absence of detect- trolled clinical trial between Mar. 2015 and Dec. 2017 able HBV DNA or HBsAg in the blood [6–8]. Low lev- (ClinicalTrials.gov Identifer: NCT03546530). All els of intrahepatic cccDNA predict a sustained virologic recruited patients completed the full course of 48 weeks response after cessation of antiviral treatment. Tere- treatment, also had paired liver biopsy at baseline fore, quantitation of intrahepatic cccDNA is suggested to and week 48 and serial serum during treatment. Te be a valuable marker in evaluating the cure of CHB and patients were treated with peg-IFN (Kawin Technol- assessing treatment endpoints. However, the invasive ogy, China) at a dosage of 1.5 μg/kg body weight once nature of liver biopsy is the major obstacle for quantita- weekly for 48 weeks. Briefy, the inclusion criteria were tion of cccDNA, which greatly limits the use of cccDNA as follows: 18 to 60 years old; positive HBsAg for at least as a marker in real-world clinical practice. Terefore, 6 months; HBeAg-positive and anti-HBe negative; HBV 5 fnding non-invasive surrogate markers of intrahepatic DNA ≥ 10 IU/mL; ALT ≥ 2 and < 10 × the upper limit cccDNA is clinically meaningful. of normal; no treatment history. Te criteria for exclu- Several traditional serum markers, such as quantitative sion were: positivity for antibodies against HCV, HDV, HBsAg and HBV DNA have been proposed to refect the or HIV; other infammatory diseases such as rheuma- intrahepatic cccDNA profle [9, 10]. However, the corre- toid arthritis, diabetes, or autoimmune hepatitis; hyper- lations between cccDNA and those traditional markers tension; kidney disease; or a recent history of infectious are too weak to apply them in clinical practice [11]. disease. Recently, serum HBV RNA, which is transcribed from Te research protocol was approved by the Ethics cccDNA, has been reported as a potential intrahepatic Committee of the First Hospital of Jilin University and cccDNA surrogate marker in CHB patients, as well as other participating institutions, according to the Helsinki during NAs therapy [12–14]. Serum HBV RNA was also Declaration of 1975. Written informed consent forms identifed as a good predictor of HBeAg seroconversion were obtained from all patients. in HBeAg-positive patients during therapy with peg-IFN [4]. In addition, a mouse model experiment revealed that Samples HBV RNA correlated positively with cccDNA before Peripheral serum samples obtained from all patients at treatment, whereas no correlation between them after baseline, week 4, week 12, week 24, week 36, and week 48 6 weeks of peg-IFN therapy [12]. during treatment were stored at − 80 °C for virological HBV core-related antigen (HBcrAg) is another new analyses. Paired liver biopsies were collected at baseline potential marker of HBV infection, which consists of and week 48 and were snap frozen in liquid nitrogen until three species of related proteins, including hepatitis cccDNA analyses. B core antigen, hepatitis B e antigen, and a truncated 22KDa precore protein [15]. Serum HBcrAg was also Standard laboratory assessments considered to be correlated with cccDNA activity and a Serum HBV DNA was quantifed by quantitative poly- good biomarker in predicting HBeAg seroconversion in merase chain reaction (qPCR) using a Roche COBAS patients treated with peg-IFN [16, 17]. AmpliPrep/COBAS TaqMan system (Roche Diagnos- Despite the fndings above, it remains unclear whether tics, Mannheim, Germany) with a lower detection limit HBV RNA or HBcrAg could ofer better predictive per- of 20 IU/mL. HBV genotypes were determined by real- formance compared with traditional serum viral markers time PCR with a commercial kit (Shanghai ZJ Bio-Tech, such as HBV DNA or HBsAg in refecting the intrahe- China). HBsAg, HBeAg, anti-HBs, anti-HBe, and anti- patic cccDNA among HBeAg-positive patients during HBc were quantitated by chemiluminescence micropar- peg-IFN treatment. ticle immunoassays using the Architect i2000SR platform Wang et al. Virol J (2021) 18:4 Page 3 of 10 and Abbott Architect reagents (Abbott Laboratories, between the patients with HBeAg seroconversion and Chicago, IL) according to manufactures’ instruction. Lab- without HBeAg seroconversion were performed using oratory assessments were performed at baseline, week 4, the chi-squared test for categorical data and the Mann– week 12, week 24, week 36, and week 48. Whitney U-test for continuous data. Serum HBV RNA, HBcrAg, HBV DNA, HBsAg, and intrahepatic cccDNA were expressed in the logarithm. Diferences in mean Serum HBV RNA quantifcation log-transformed values were calculated using Student’s Serum HBV RNA was quantitatively measured in the t-test or one-way ANOVA where it is appropriate.