A Prospective Cohort Study of Novel Markers of Hepatitis B Virus Replication in Human Immunodeficiency Virus Coinfection.

BACKGROUND & AIMS: The contribution of the novel biomarkers, hepatitis B virus (HBV) RNA and HBV core-related antigen (HBcrAg), to characterization of HBV-human immunodeficiency virus (HIV) coinfection is unclear. We evaluated the longitudinal dynamics of HBV RNA and HBcrAg and their association with classical HBV serum biomarkers and liver histology and viral staining. METHODS: HBV-HIV co-infected adults from 8 North American centers entered a National Institutes of Health-funded prospective cohort study. Demographic, clinical, serological, and virological data were collected at entry and every 24 to 48 weeks for up to 192 weeks. Participants with HBV RNA and HBcrAg measured ≥2 times (N = 95) were evaluated; 56 had paired liver biopsies obtained at study entry and end of follow-up. RESULTS: Participants had a median age of 50 years; 97% were on combination anti-viral therapy. In hepatitis B e antigen (HBeAg)+ participants, there were significant declines in HBV RNA and HBcrAg over 192 weeks that tracked with declines in HBeAg, hepatitis B surface antigen, HBV DNA, and hepatitis B core antigen (HBcAg) hepatocyte staining grade (all P < .05). In HBeAg- participants, there were not significant declines in HBV RNA (P = .49) and HBcrAg (P = .63), despite modest reductions in hepatitis B surface antigen (P < .01) and HBV DNA (P = .03). HBV serum biomarkers were not significantly related to change in hepatic activity index, Ishak fibrosis score, or hepatocyte HBcAg loss (all P > .05). CONCLUSIONS: In HBV-HIV coinfected adults on suppressive dually active antiviral therapy, the use of novel HBV markers reveals continued improvement in suppression of HBV transcription and translation over time. The lack of further improvement in HBV serum biomarkers among HBeAg- patients suggests limits to the benefit of combination anti-viral therapy and provide rationale for additional agents with distinct mechanisms of action.

Ultrasensitive Assay for Hepatitis B Core-Related Antigen Predicts Hepatocellular Carcinoma Incidences During Entecavir.

Serum hepatitis B core-related antigen (HBcrAg) and surface antigen (HBsAg) are surrogate markers of intrahepatic covalently closed circular DNA. The measurement range of the current HBcrAg assay is relatively narrow. Thus, we examined the potential of HBcrAg and HBsAg measured by ultrasensitive assays for predicting hepatocellular carcinoma (HCC) development in patients with chronic hepatitis B treated with entecavir (ETV). We conducted a retrospective cohort study of 180 patients who received ETV for >1 year. All patients had hepatitis B e-antigen negativity at baseline. Serum HBcrAg and HBsAg levels at baseline and year 1 were measured in all patients by ultrasensitive assays using immunoassay for total antigen including complex by pretreatment (iTACT) technology. During the median follow-up of 11.0 years, 22 patients developed HCC (11.8/1,000 person-years). Baseline HBsAg levels were not associated with HCC development during ETV treatment. However, high HBcrAg levels at baseline and at year 1 were significantly associated with HCC development (log-rank test; P < 0.001). In 110 patients (61.1%) with ≥4.0 log U/mL at baseline (high HBcrAg cohort), HBcrAg declined to ≤2.9 log U/mL at year 1 in 25 patients (22.7%). The adjusted hazard ratio for HCC incidence was significantly lower in patients with HBcrAg ≤2.9 log U/mL at year 1 than in those in the high HBcrAg cohort. Conclusion: Measurement of HBcrAg by ultrasensitive assay has better potential for predicting HCC during antiviral treatment than the current HBcrAg assay.

Seroprevalencia de infecciones hemotransmisibles en donantes de sangre / Seroprevalence of transmissible infections in blood donors

Introducción. La seguridad transfusional es el objetivo primordial de los bancos de sangre, sin embargo, conlleva un alto riesgo de eventos adversos como son las infecciones transmisibles por transfusión (ITT). El conocimiento de la prevalencia de estas infecciones fue de particular interés en esta investigación, donde se determinó su frecuencia, coinfección y relación con el tipo de donantes admitidos. Metodología. Estudio observacional retrospectivo de 2017 y 2018, en el que se incluyeron todos los registros de donantes de sangre que contenían datos demográficos y resultados de los marcadores obligatorios en el país (Ecuador), tanto de pruebas serológicas como moleculares. Se obtuvo el permiso del custodio de la información y del subcomité de bioética de investigaciones en seres humanos. Para el análisis de los datos se utilizó estadística descriptiva e inferencial. Resultados. Se determinó una prevalencia del 3,18 % de resultados reactivos para una o más ITT, el rango de edad más prevalente fue de 29 a 40 años, el 89,8 % fueron donantes compensatorios, y de ellos el 90 % fueron reactivos para una o más ITT. El marcador serológico más prevalente fue el anti-core del virus de la hepatitis B (anti-HBc), seguido por el de sífilis y los anticuerpos contra el virus de la hepatitis C (VHC). La coinfección más prevalente fue con sífilis y hepatitis B. Se encontró una diferencia estadísticamente significativa entre los resultados obtenidos en las pruebas serológicas y las moleculares (x2=26,9; p=0,000). Conclusión. Las ITT en los bancos de sangre son un riesgo latente, por lo que es necesario conocer las variaciones epidemiológicas que existen en cada población. El conocimiento de la prevalencia de las ITT en donantes de sangre permite establecer nuevas estrategias de selección del donante, que garanticen la mejor seguridad posible en las transfusiones, además debe verificarse siempre la metodología utilizada y hacer monitoreo permanente del sistema de calidad establecido

Introduction. Transfusion safety is the primary objective of blood banks, however one of the adverse reactions to blood transfusion are the transfusion transmissible infections (TTIs). Knowledge of the prevalence of these infections was of particular interest in this study where we determined their frequency, co-infection and relationship with the type of donors admitted. Methodology. Retrospective observational study during 2017 and 2018, in which all blood donor records containing demographic data and results of the country's (Ecuador) mandatory serological markers of both serological and molecular tests were included. Permission was obtained from the data custodian and the Human Research Bioethics Subcommittee. Descriptive and inferential statistics were used for data analysis. Results. A prevalence of 3,18% of reactive results to one or more TTIs was determined, the most prevalent age range was 29 to 40 years, 89.8% were compensatory donors and 90% of them were reactive to one or more TTIs. The anti- core serological marker of the hepatitis B virus (anti-HBc) was the most prevalent, followed by syphilis and hepatitis C antibodies. Syphilis and hepatitis B were identified as the most prevalent coinfection. The correlation between the results obtained in the serological and molecular tests was determined to be different and statistically significant (x2=26.9; p=0.000). Conclusion. TTIs in blood banks are a latent risk, so it is necessary to know the epidemiological variations that exist in every population. Knowledge of the prevalence of TTIs in blood donors facilitates new donor selection strategies that guarantee the best possible safety in transfusions. In addition, the methodology used must always be verified and the established quality system must be permanently monitored

Analysis of HBsAg Immunocomplexes and cccDNA Activity During and Persisting After NAP-Based Therapy.

Therapy with nucleic acid polymers (NAPs), tenofovir disoproxil fumarate (TDF), and pegylated interferon (pegIFN) achieve high rates of HBsAg loss/seroconversion and functional cure in chronic hepatitis B virus (HBV) infection. The role of hepatitis B surface antigen (HBsAg) seroconversion and inactivation of covalently closed circular DNA (cccDNA) in establishing functional cure were examined. Archived serum from the REP 401 study was analyzed using the Abbott ARCHITECT HBsAg NEXT assay (Chicago, IL), Abbott research use-only assays for HBsAg immune complexes (HBsAg ICs), circulating HBV RNA, and the Fujirebio assay for hepatitis B core-related antigen (HBcrAg; Malvern, PA). HBsAg became < 0.005 IU/mL in 23 participants during NAP exposure, which persisted in all participants with functional cure. HBsAg IC declined during lead-in TDF monotherapy and correlated with minor declines in HBsAg. Following the addition of NAPs and pegIFN, minor HBsAg IC increases (n = 13) or flares (n = 2) during therapy were not correlated with HBsAg decline, hepatitis B surface antibody (anti-HBs) titers, or alanine aminotransferase. HBsAg IC universally declined during follow-up in participants with virologic control or functional cure. Universal declines in HBV RNA and HBcrAg during TDF monotherapy continued with NAP + pegIFN regardless of therapeutic outcome. At the end of therapy, HBV RNA was undetectable in only 5 of 14 participants with functional cure but became undetectable after removal of therapy in all participants with functional cure. Undetectable HBV RNA at the end of therapy in 5 participants was followed by relapse to virologic control or viral rebound. Conclusion: Anti-HBs-independent mechanisms contribute to HBsAg clearance during NAP therapy. Inactivation of cccDNA does not predict functional cure following NAP-based therapy; however, functional cure is accompanied by persistent inactivation of cccDNA. Persistent HBsAg loss with functional cure may also reflect reduction/clearance of integrated HBV DNA. Clinicaltrials.org number NCT02565719.

Quantitative serum HBV markers in predicting phases of natural history of chronic HBV infection.

BACKGROUND: Clinical importance of commercially available quantitative HBV markers has not been fully investigated. OBJECTIVE: To choice and to evaluate clinically valuable HBV markers for predicting phases of natural history with chronic HBV infection. METHODS: 472 naïve patients with chronic HBV infection were enrolled, in which 21 and 220 were confirmed as HBeAg-positive inactive and active hepatitis (EPIH and EPAH), respectively, and 106 and 125 were confirmed as HBeAg-negative inactive and active hepatitis (ENIH and ENAH), respectively. HBsAg, HBcrAg and anti- HBc were measured using chemiluminescent immunoassay, and HBV DNA was measured using PCR-fluorescence probing assay. RESULTS: There were all statistical differences in medians of HBsAg, anti-HBc, HBcrAg and HBV DNA between EPIH and EPAH and between ENIH and ENAH (all P < 0.01). According to binary logistic stepwise regressions, HBsAg and anti-HBc were preferred variables for predicting EPAH, and HBcrAg and HBV DNA were preferred variables for predicting ENAH. Based on normalization for coefficients of preferred variables entering regression equations, a handy model of MEPAH for predicting EPAH and of MENAH for predicting ENAH was constructed, respectively. Area under receiver operating characteristic curves of MEPAH and MENAH for predicting EPAH and ENAH were 0.882 and 0.931, respectively. With standard of MEPAH ≤ 5.997 and MENAH > 10.535, sensitivity or specificity of which for predicting EPAH and ENAH were about 81.0 % and 87.0 %, respectively. CONCLUSION: HBsAg and anti-HBc for predicting EPAH and HBcrAg and HBV DNA for predicting ENAH are dependable markers; MEPAH for predicting EPAH and MENAH for predicting ENAH have very good performance.

Novel Biomarkers of Hepatitis B Virus and Their Use in Chronic Hepatitis B Patient Management.

Even though an approved vaccine for hepatitis B virus (HBV) is available and widely used, over 257 million individuals worldwide are living with chronic hepatitis B (CHB) who require monitoring of treatment response, viral activity, and disease progression to reduce their risk of HBV-related liver disease. There is currently a lack of predictive markers to guide clinical management and to allow treatment cessation with reduced risk of viral reactivation. Novel HBV biomarkers are in development in an effort to improve the management of people living with CHB, to predict disease outcomes of CHB, and further understand the natural history of HBV. This review focuses on novel HBV biomarkers and their use in the clinical setting, including the description of and methodology for quantification of serum HBV RNA, hepatitis B core-related antigen (HBcrAg), quantitative hepatitis B surface antigen (qHBsAg), including ultrasensitive HBsAg detection, quantitative anti-hepatitis B core antigen (qAHBc), and detection of HBV nucleic acid-related antigen (HBV-NRAg). The utility of these biomarkers in treatment-naïve and treated CHB patients in several clinical situations is further discussed. Novel HBV biomarkers have been observed to provide critical clinical information and show promise for improving patient management and our understanding of the natural history of HBV.

Comparison of HBV RNA and Hepatitis B Core Related Antigen With Conventional HBV Markers Among Untreated Adults With Chronic Hepatitis B in North America.

BACKGROUND AND AIMS: The clinical utility of two biomarkers, hepatitis B virus (HBV) RNA and hepatitis B core-related antigen (HBcrAg), as compared to conventional markers of HBV replication and disease activity, is unclear. APPROACH AND RESULTS: Untreated participants in the North American Hepatitis B Research Network Adult Cohort Study were categorized by chronic hepatitis B (CHB) phases based on HBsAg and HBeAg status and HBV DNA and alanine aminotransferase (ALT) levels. HBV RNA and HBcrAg were measured (Abbott HBV pgRNA Research Assay and Fujirebio Lumipulse Immunoassay, respectively), and cross-sectional associations with conventional CHB markers were tested. Among 1,409 participants across all CHB phases, median HBV DNA was 3.8 log10 IU/mL and ALT was 34 U/L. HBV RNA was quantifiable in 99% of HBeAg+ and 58% of HBeAg- participants; HBcrAg was quantifiable in 20% of HBeAg+ (above linear range in the other 80%) and 51% of HBeAg- participants. Both markers differed across CHB phases (P < 0.001), with higher levels in the HBeAg+ and HBeAg- immune active phases. HBV RNA and HBcrAg correlated moderately strongly with HBV DNA in both HBeAg+ and HBeAg- phases (HBV RNA: e+ ρ = 0.84; e- ρ = 0.78; HBcrAg: e+ ρ = 0.66; e- ρ = 0.56; P for all, <0.001), but with HBsAg levels among HBeAg+ phases only (HBV RNA: e+ ρ = 0.71; P < 0.001; e- ρ = 0.18; P = 0.56; HBcrAg: e+ ρ = 0.51; P < 0.001; e- ρ = 0.27; P < 0.001). Associations of higher HBV RNA and HBcrAg levels with higher ALT, APRI, and Fibrosis-4 levels were consistent in HBeAg- , but not HBeAg+ , phases. CONCLUSIONS: Despite clear relationships between HBV RNA and HBcrAg levels and CHB phases, these markers have limited additional value in differentiating CHB phases because of their strong association with HBV DNA and, to a lesser extent, with clinical disease indicators.

Novel viral markers and the prediction of off-treatment relapse in chronic hepatitis B patients: A systematic review.

BACKGROUND AND AIM: Hepatitis B core-related antigen (HBcrAg) and hepatitis B virus RNA (HBV RNA) are novel markers that reflect intrahepatic cccDNA and could be useful in the prediction of relapse after nucleos(t)ide analogues (NA) discontinuation. The aim of the study is to perform a systematic review on this issue. METHODS: Medline/Pubmed database was searched using text terms related to HBcrAg, RNA, NAs, discontinuation, and relapse. Included studies were those that enrolled adult patients who had been on NAs for more than 6 months with available information on end-of-treatment (EOT) HBcrAg and/or HBV RNA and relapse rates. RESULTS: Sixteen studies were included. Virological and clinical relapse rates ranged from 11% to 100% and 11% to 73%, respectively. Low or undetectable EOT HBcrAg levels were associated with low off-treatment relapse rates in most studies with area under the receiver operating characteristic curve (AUROC) of 0.69-0.70 for predicting virological relapse (VR) and 0.61-0.77 for predicting clinical relapse (CR). Undetectable EOT HBV RNA was associated with a lower risk of off-treatment relapse with AUROC of 0.65-0.76 for predicting VR and 0.66-0.73 for predicting CR. Combined EOT HBcrAg and HBV RNA performed better in predicting off-treatment relapse than either test alone with AUROC of 0.816-0.846 for predicting CR. None of the patients with double-negative HBV RNA and HBcrAg developed CR. CONCLUSION: Combining HBcrAg with HBV RNA or HBsAg improved the discriminating abilities in the prediction of off-treatment relapse of each test. Patients with double-negative HBcrAg and HBV RNA at EOT had low risks of relapse and could be considered for NA discontinuation.

Occult and active hepatitis B virus detection in donated blood in São Paulo, Brazil.

BACKGROUND: The present study determined the HBV antigen, antibody, and DNA status in blood donations deemed to be HBV positive. Individuals with an occult HBV infection (OBI), defined as being positive for HBV DNA but negative for HBV surface antigen (HBsAg), as well as those with active infection (HBsAg-positive), were identified and characterized. STUDY DESIGN AND METHODS: From a total pool if 198,363 blood donations, we evaluated in a cross-sectional study, 1106 samples that were positive in screening tests for antibody to HBV core antigen (HBcAb), HBsAg, and/or HBV DNA by nucleic acid testing (NAT-HBV). The presence of genetic variants in the HBV pol/S gene in individuals with an active HBV infection was also determined. RESULTS: OBIs were detected in six of 976 samples (0.6%) that were positive only for HBcAb. The rate of HBV active infection was 0.024% (48/198,363) and there was a predominance of HBV sub-genotype A1 (62.2%, 28/45), followed by D3 (17.8%, 8/45). Mutations in the S gene were found in 57.8% (26/45) and immune escape mutations in 37.8% (17/45) of active HBV-infected donors. Among them, T123N, G145A, and D144G high-impact immune escape mutations were identified. CONCLUSION: Highly sensitive molecular tests improve the capacity to detect OBIs. When NAT is performed in pooled samples, HBcAb test has value in the detection of donors with OBI and improves transfusion safety. Mutations in the S gene are frequent in HBsAg-positive blood, including those associated with diagnostic failure and vaccine escape mutations.

Hepatitis C virus core antigen as alternative diagnostic algorithm for active hepatitis C virus infection among haemodialysis population: Cost implications.

AIMS: In Malaysia, majority anti-HCV positive haemodialysis patients do not undergo hepatitis C confirmation due to the high cost of HCV RNA. HCV Core Antigen might be a cost-effective diagnostic test to identify HD patients who have active HCV infection eligible for Direct Acting Anti-viral therapy. METHODS: A cross-sectional study was conducted to assess the correlation between HCV Ag and HCV RNA and the cost implications of different diagnostic algorithms to diagnose active HCV infection using Anti-HCV, HCV Ag, and HCV RNA. Pre-dialysis blood was tested for both HCV Ag and HCV RNA. HCV Ag was tested with Abbott ARCHITECT HCV Ag test. RESULTS: Two-hundred twenty-seven haemodialysis patients were recruited from 20 centres with mean age of 57.68 ± 12.48 years, and male constitutes 56.8% (129) of the study population. HCV Ag correlated well with HCV RNA (Spearman test coefficient 0.943, p < .001) with sensitivity of 93.9%, specificity 99.3%, and the accuracy was 97.36%. Cost analysis indicated that a sequential test involving Anti-HCV antibody as initial screening, followed by HCV Ag on Anti-HCV positive and HCV RNA on HCV Ag negative cases translated to a modest cost-saving algorithm compared to standard diagnostic algorithm. CONCLUSION: HCV Ag correlated well with HCV RNA and can potentially be fused in an alternative diagnostic algorithm to generate cost savings methods to diagnose active HCV infection among haemodialysis patients. This alternative algorithm is especially relevant in low to middle-income countries such as Malaysia to optimize the use of the healthcare resource and gains in clinical outcomes.

Characterization and Application of Precore/Core-Related Antigens in Animal Models of Hepatitis B Virus Infection.

BACKGROUND AND AIMS: The hepatitis B core-related antigen (HBcrAg), a composite antigen of precore/core gene including classical hepatitis B core protein (HBc) and HBeAg and, additionally, the precore-related antigen PreC, retaining the N-terminal signal peptide, has emerged as a surrogate marker to monitor the intrahepatic HBV covalently closed circular DNA (cccDNA) and to define meaningful treatment endpoints. APPROACH AND RESULTS: Here, we found that the woodchuck hepatitis virus (WHV) precore/core gene products (i.e., WHV core-related antigen [WHcrAg]) include the WHV core protein and WHV e antigen (WHeAg) as well as the WHV PreC protein (WPreC) in infected woodchucks. Unlike in HBV infection, WHeAg and WPreC proteins were N-glycosylated, and no significant amounts of WHV empty virions were detected in WHV-infected woodchuck serum. WHeAg was the predominant form of WHcrAg, and a positive correlation was found between the serum WHeAg and intrahepatic cccDNA. Both WHeAg and WPreC antigens displayed heterogeneous proteolytic processing at their C-termini, resulting in multiple species. Analysis of the kinetics of each component of the precore/core-related antigen, along with serum viral DNA and surface antigens, in HBV-infected chimpanzees and WHV-infected woodchucks revealed multiple distinct phases of viral decline during natural resolution and in response to antiviral treatments. A positive correlation was found between HBc and intrahepatic cccDNA but not between HBeAg or HBcrAg and cccDNA in HBV-infected chimpanzees, suggesting that HBc can be a better marker for intrahepatic cccDNA. CONCLUSIONS: In conclusion, careful monitoring of each component of HBcrAg along with other classical markers will help understand intrahepatic viral activities to elucidate natural resolution mechanisms as well as guide antiviral development.

Pilot Study Using Machine Learning to Identify Immune Profiles for the Prediction of Early Virological Relapse After Stopping Nucleos(t)ide Analogues in HBeAg-Negative CHB.

Treatment with nucleos(t)ide analogues (NAs) may be stopped after 1-3 years of hepatitis B virus DNA suppression in hepatitis B e antigen (HBeAg)-negative patients according to Asian Pacific Association for the Study of Liver and European Association for the Study of Liver guidelines. However, virological relapse (VR) occurs in most patients. We aimed to analyze soluble immune markers (SIMs) and use machine learning to identify SIM combinations as predictor for early VR after NA discontinuation. A validation cohort was used to verify the predictive power of the SIM combination. In a post hoc analysis of a prospective, multicenter therapeutic vaccination trial (ABX-203, NCT02249988), hepatitis B surface antigen, hepatitis B core antigen, and 47 SIMs were repeatedly determined before NA was stopped. Forty-three HBeAg-negative patients were included. To detect the highest predictive constellation of host and viral markers, a supervised machine learning approach was used. Data were validated in a different cohort of 49 patients treated with entecavir. VR (hepatitis B virus DNA ≥ 2,000 IU/mL) occurred in 27 patients. The predictive value for VR of single SIMs at the time of NA stop was best for interleukin (IL)-2, IL-17, and regulated on activation, normal T cell expressed and secreted (RANTES/CCL5) with a maximum area under the curve of 0.65. Hepatitis B core antigen had a higher predictive power than hepatitis B surface antigen but lower than the SIMs. A supervised machine-learning algorithm allowed a remarkable improvement of early relapse prediction in patients treated with entecavir. The combination of IL-2, monokine induced by interferon γ (MIG)/chemokine (C-C motif) ligand 9 (CCL9), RANTES/CCL5, stem cell factor (SCF), and TNF-related apoptosis-inducing ligand (TRAIL) was reliable in predicting VR (0.89; 95% confidence interval: 0.5-1.0) and showed viable results in the validation cohort (0.63; 0.1-0.99). Host immune markers such as SIMs appear to be underestimated in guiding treatment cessation in HBeAg-negative patients. Machine learning can help find predictive SIM patterns that allow a precise identification of patients particularly suitable for NA cessation.

Serum HBV RNA correlated with intrahepatic cccDNA more strongly than other HBV markers during peg-interferon treatment.

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 reflecting cccDNA profile 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 statistically 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 reflecting the intrahepatic cccDNA profile in HBeAg-positive patients during peg-IFN treatment. Trial registration ClinicalTrials, NCT03546530. Registered 1 January 2015. https://clinicaltrials.gov/ct2/results?cond=&term=NCT03546530 .

HBV RNA Profiles in Patients With Chronic Hepatitis B Under Different Disease Phases and Antiviral Therapy.

BACKGROUND AND AIMS: Large-scale comprehensive studies on HBV RNA in chronic hepatitis B are lacking. We aimed to study the HBV RNA profile and its correlation with other viral markers in patients with chronic hepatitis B who are treatment-naïve and patients receiving nucleos(t)ide analogues (NA). APPROACH AND RESULTS: Biomarkers, including HBV RNA and hepatitis B core-related antigen (HBcrAg), were measured in 388 patients. Of these, 246 were treatment-naïve and were categorized into HBeAg-positive chronic infection (n = 41), HBeAg-positive chronic hepatitis (n = 81), HBeAg-negative chronic infection (n = 39), HBeAg-negative chronic hepatitis (n = 66), and HBsAg seroclearance (n = 19). These biomarkers were also measured in 142 patients who were NA-treated receiving tenofovir or entecavir at baseline, week 48, and week 96. The pattern of serum HBV RNA levels mirrored HBV DNA (1-2 logs higher than HBV RNA) and HBcrAg in patients who were treatment-naïve. HBV RNA correlated best with HBcrAg (r = 0.84) and to a lesser extent with HBV DNA (r = 0.737) (both P < 0.001). In patients with HBsAg seroclearance, 15.8% and 15.8% had detectable serum HBV RNA and HBcrAg, respectively. NA treatment reduced serum HBV RNA by 1.46 logs and 1.77 logs at weeks 48 and 96, respectively. At week 96 of NA therapy, only 19.1% patients who were tenofovir-treated and 25.7% patients who were entecavir-treated had unquantifiable HBV RNA (P > 0.05). In patients who were treated and had undetectable HBV DNA, 77.5% and 30% had quantifiable HBV RNA and HBcrAg, respectively. CONCLUSIONS: HBV RNA showed distinct and corresponding profiles in patients with HBV in different disease phases. HBV RNA and HBcrAg could be used to monitor residual transcriptional activities in patients with HBsAg seroclearance. NA led to reduction of serum HBV RNA. Monitoring of viral activities can still be achieved in patients with undetectable HBV DNA by serum HBV RNA.

Distribution and factors associated with serum HBV pregenomic RNA levels in Chinese chronic hepatitis B patients.

Correlations between serum hepatitus B virus (HBV) pregenomic RNA (pgRNA), hepatitus B surface antigen (HBsAg), and hepatitus B core-related antigen (HBcrAg) levels, and influencing factors of serum HBV pgRNA levels in Chinese chronic hepatitis B (CHB) patients are rarely reported. This was a retrospective cohort study consisting of 204 outpatients with CHB. Serum levels of HBV pgRNA, HBsAg, and HBcrAg were quantitative measured in frozen blood samples. The linear regression and multivariate logistic regression analysis were performed to determine associated factors of serum HBV pgRNA levels. In this cohort, the median serum HBV pgRNA level was 4.12 log10 copies/ml and 33.33% (68/204) of them had serum HBV pgRNA under low limit of detection (LLD) (<500 copies/ml); and the percentage of patients with serum HBV pgRNA under LLD in hepatitis B e antigen (HBeAg)-positive patients was significantly lower than that in HBeAg-negative patients (15.75% [23/46] vs. 77.59% [45/58], p < .001). Overall, serum HBV pgRNA strongly correlated with HBcrAg (r = 0.760, p < .001), and moderately correlated with HBV DNA (r = 0.663, p < .001) and HBsAg (r = 0.670, p < .001). As compared with HBsAg and HBV DNA, only HBcrAg showed stable correlation with serum HBV pgRNA both in HBeAg-positive and HBeAg-negative patients. Serum HBV pgRNA level differed between HBeAg-positive and HBeAg-negative patients; and it had better and more stable correlation with serum HBcrAg than serum HBV DNA and HBsAg, irrespective of HBeAg status.

Identification of the association between HBcAg-specific T cell and viral control in chronic HBV infection using a cultured ELISPOT assay.

Hepatitis B virus (HBV)-specific T cells play a critical role in determining the outcome of HBV infection. However, T cell response induced by predominant Ag in chronic infection is hardly detectable owing to the lack of a suitable assay. We herein established an optimized method to enumerate HBV-specific T cells and assessed the association between HBV surface Ag (HBsAg) and HBV DNA. Sixty chronic HBV infection patients were enrolled. HBV-specific T cells were expanded by using overlapping peptide pools covering the entire sequence of HBV genotypes B and C. IFN-γ-producing HBV-specific T cells were detected by a cultured enzyme-linked immunospot (ELISPOT) assay, ex vivo ELISPOT assay, or flow cytometry staining. The association between HBV-specific T cells and serum levels of HBsAg and HBV DNA were analyzed. Cultured ELISPOT assay had a higher sensitivity than ex vivo ELISPOT in the detection of HBV-specific T cells. Moreover, consistent results were acquired by flow cytometry analysis and cultured ELISPOT assay, but the latter required only a limited number of cells for detection. Interestingly, HBV core peptide pool induced a robust HBV-specific T cell response in patients with lower levels of HBV DNA and HBsAg. Specifically, the frequency of HBV core Ag-specific IFN-γ+ spot-forming cells was inversely correlated with serum levels of HBV DNA and HBsAg. An optimized cultured ELISPOT assay reveals the association between HBV core Ag-induced T cell response and HBV control; this method may favor the investigation of HBV-specific T cell in chronic HBV infection.

Rapid Turnover of Hepatitis B Virus Covalently Closed Circular DNA Indicated by Monitoring Emergence and Reversion of Signature-Mutation in Treated Chronic Hepatitis B Patients.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a pivotal role in the establishment and persistence of HBV infection. Understanding the turnover time of preexisting cccDNA pools would be helpful in designing strategies to clear HBV by fully blocking the de novo generation of cccDNA. APPROACH AND RESULTS: In this study, we retrospectively monitored the emergence and reversion of the rtM204I/V mutant, a signature lamivudine resistance (LAMR ) mutation serving as a biomarker of cccDNA turnover in liver biopsies and longitudinal serum samples from two clinical trials. Methodologies were optimized to differentially isolate and sequence HBV virion DNA, cccDNA, and HBV RNA from clinical samples. A strong correlation was observed between LAMR composition of cccDNA with that of serum and intrahepatic HBV RNA in paired liver and serum samples (r = 0.96 and 0.90, respectively), suggesting that serum HBV RNA can serve as a surrogate marker of cccDNA genetic composition when liver biopsies are unavailable. LAMR mutations emerged and increased from undetectable to 40%-90% within 16-28 weeks in serum HBV RNA from telbivudine-treated patients experiencing virological breakthrough. Similarly, in lamivudine-resistant patients who switched to interferon therapy, serum HBV-RNA population bearing 100% LAMR mutations fully reversed back to wild type within 24-48 weeks. CONCLUSIONS: The genetic composition dynamics of serum HBV RNA and biopsy cccDNA in treated HBV patients indicates that cccDNA turnover occurs relatively rapidly (several months), offering a possibility of HBV cure with finite therapy through completely blocking cccDNA replenishment.

Characterization of Hepatitis B Precore/Core-Related Antigens.

Current therapies rarely cure chronic hepatitis B virus (HBV) infection due to the persistence of the viral episome, the covalently closed circular DNA (cccDNA), in hepatocytes. The hepatitis B virus core-related antigen (HBcrAg), a mixture of the viral precore/core gene products, has emerged as one potential marker to monitor the levels and activities of intrahepatic cccDNA. In this study, a comprehensive characterization of precore/core gene products revealed that HBcrAg components included the classical hepatitis B virus core antigen (HBc) and e antigen (HBeAg) and, additionally, the precore-related antigen, PreC, retaining the N-terminal signal peptide. Both HBeAg and PreC antigens displayed heterogeneous proteolytic processing at their C termini resulting in multiple species, which varied with viral genotypes. HBeAg was the predominant form of HBcrAg in HBeAg-positive patients. Positive correlations were found between HBcrAg and PreC, between HBcrAg and HBeAg, and between PreC and HBeAg but not between HBcrAg and HBc. Serum HBeAg and PreC shared similar buoyant density and size distributions, and both displayed density and size heterogeneity. HBc, but not HBeAg or PreC antigen, was found as the main component of capsids in DNA-containing or empty virions. Neither HBeAg nor PreC protein was able to form capsids in cells or in vitro under physiological conditions. In conclusion, our study provides important new quantitative information on levels of each component of precore/core gene products as well as their biochemical and biophysical characteristics, implying that each component may have distinct functions and applications in reflecting intrahepatic viral activities.IMPORTANCE Chronic hepatitis B virus (HBV) infection afflicts approximately 257 million people, who are at high risk of progressing to chronic liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. Current therapies rarely achieve cure of HBV infection due to the persistence of the HBV episome, the covalently closed circular DNA (cccDNA), in the nuclei of infected hepatocytes. Peripheral markers of cccDNA levels and transcriptional activities are urgently required to guide antiviral therapy and drug development. Serum hepatitis B core-related antigen (HBcrAg) is one such emerging peripheral marker. We have characterized the components of HBcrAg in HBV-infected patients as well as in cell cultures. Our results provide important new quantitative information on levels of each HBcrAg component, as well as their biochemical and biophysical characteristics. Our findings suggest that each HBcrAg component may have distinct functions and applications in reflecting intrahepatic viral activities.

Quantitative HBcrAg and HBcAb versus HBsAg and HBV DNA in predicting liver fibrosis levels of chronic hepatitis B patients / HBcrAg y HBcAb cuantitativos versus HBsAg y ADN del VHB en la predicción de los niveles de fibrosis hepática de los pacientes con hepatitis B crónica

OBJECTIVE: To evaluate the performance of the quantitative markers of hepatitis B core-related antigen (HBcrAg) and anti-hepatitis B core antigen antibodies HbcAb versus hepatitis B surface antigen (HBsAg) and hepatitis B virus DNA (HBV DNA) in predicting liver fibrosis levels in chronic hepatitis B patients. METHODS: Two hundred and fifty hepatitis B e antigen (HBeAg)-positive and 245 HBeAg-negative patients were enrolled. With reference to the Scheuer standard, stage 2 or higher and stage 4 liver disease were defined as significant fibrosis and cirrhosis, respectively. A receiver operating characteristic (ROC) curve was used to evaluate the performance of the HBV markers investigated. RESULTS: The areas under the ROC curves (AUCs) of HBcrAg in predicting significant fibrosis and cirrhosis in HBeAg-positive patients (0.577 and 0.700) were both close to those of HBsAg (0.617 and 0.762) (both P> 0.05). In HBeAg-negative patients (0.797 and 0.837), they were both significantly greater than those of HBV DNA (0.723 and 0.738) (P=0.0090 and P=0.0079). The AUCs of HBcAb in predicting significant fibrosis and cirrhosis in HBeAg-positive patients (0.640 and 0.665) were both close to those of HBsAg. In HBeAg-negative patients (0.570 and 0.621), they were both significantly less than those of HBcrAg (P <0.0001 and P=0.0001). Specificity in predicting significant fibrosis and sensitivity in predicting cirrhosis in HBeAg-positive patients, using a single cut-off of HBsAg ≤5,000 IU/ml, were 76.5% and 72.7%, respectively. In HBeAg-negative patients, using a single cut-off of HBcrAg>80kU/ml, they were 85.9% and 81.3%, respectively. CONCLUSIONS: HBsAg has good performance in predicting liver fibrosis levels in HBeAg-positive and HBeAg-negative patients, and HBcrAg has very good performance in predicting liver fibrosis levels in HBeAg-negative patients

OBJETIVO: Evaluar el rendimiento de los marcadores cuantitativos del antígeno central de la hepatitis B (HBcrAg) y los anticuerpos contra el antígeno central de la hepatitis B (HBcAb) frente al antígeno de superficie de la hepatitis B (HBsAg) y el ADN del virus de la hepatitis B (ADN del VHB) en la predicción de los niveles de fibrosis hepática de los pacientes con hepatitis B crónica. MÉTODOS: Se inscribieron 250 pacientes con HBsAg positivo y 245 pacientes con HBeAg negativo. Con referencia al estándar de Scheuer, la etapa patológica hepática 2 o superior y la etapa 4 se definieron como fibrosis y cirrosis significativas, respectivamente. Se utilizó la curva característica de funcionamiento del receptor (ROC) para evaluar el rendimiento de los marcadores del VHB investigados. RESULTADOS: Las áreas bajo la curva ROC (AUC) del HBcrAg en la predicción de la fibrosis y cirrosis significativa de los pacientes positivos para el HBeAg (0,577 y 0,700) fueron ambas cercanas a las del HBsAg (0,617 y 0,762) (ambas p > 0,05); de los pacientes negativos para el HBeAg (0,797 y 0,837) fueron ambas significativamente mayores que las del ADN del VHB (0,723 y 0,738) (p = 0,0090 y p = 0,0079); las AUC del HBcAb en la predicción de la fibrosis y cirrosis significativa de los pacientes positivos para el HBeAg (0,640 y 0,665) fueron ambas cercanas a las del HBsAg; de los pacientes negativos para el HBeAg (0,570 y 0,621) fueron ambas significativamente menores que las del HBcrAg (p < 0,0001 y p = 0,0001). La especificidad en la predicción de la fibrosis significativa y la sensibilidad en la predicción de la cirrosis de los pacientes positivos para el HBeAg, utilizando un solo corte de HBsAg ≤ 5.000 UI/mL fueron 76,5 y 72,7%, respectivamente; de los pacientes negativos para el HBeAg utilizando un solo corte de HBcrAg > 80 kU/mL fueron 85,9 y 81,3%, respectivamente. CONCLUSIONES: El HBsAg tiene un buen rendimiento en la predicción de los niveles de fibrosis hepática de los pacientes HBeAg positivos y negativos, mientras que HBcrAg tiene un muy buen rendimiento en la predicción de los niveles de fibrosis de los pacientes HBaAg negativos

Quantitative HBcrAg and HBcAb versus HBsAg and HBV DNA in predicting liver fibrosis levels of chronic hepatitis B patients.

OBJECTIVE: To evaluate the performance of the quantitative markers of hepatitis B core-related antigen (HBcrAg) and anti-hepatitis B core antigen antibodies HbcAb versus hepatitis B surface antigen (HBsAg) and hepatitis B virus DNA (HBV DNA) in predicting liver fibrosis levels in chronic hepatitis B patients. METHODS: Two hundred and fifty hepatitis B e antigen (HBeAg)-positive and 245 HBeAg-negative patients were enrolled. With reference to the Scheuer standard, stage 2 or higher and stage 4 liver disease were defined as significant fibrosis and cirrhosis, respectively. A receiver operating characteristic (ROC) curve was used to evaluate the performance of the HBV markers investigated. RESULTS: The areas under the ROC curves (AUCs) of HBcrAg in predicting significant fibrosis and cirrhosis in HBeAg-positive patients (0.577 and 0.700) were both close to those of HBsAg (0.617 and 0.762) (both P> 0.05). In HBeAg-negative patients (0.797 and 0.837), they were both significantly greater than those of HBV DNA (0.723 and 0.738) (P=0.0090 and P=0.0079). The AUCs of HBcAb in predicting significant fibrosis and cirrhosis in HBeAg-positive patients (0.640 and 0.665) were both close to those of HBsAg. In HBeAg-negative patients (0.570 and 0.621), they were both significantly less than those of HBcrAg (P <0.0001 and P=0.0001). Specificity in predicting significant fibrosis and sensitivity in predicting cirrhosis in HBeAg-positive patients, using a single cut-off of HBsAg ≤5,000 IU/ml, were 76.5% and 72.7%, respectively. In HBeAg-negative patients, using a single cut-off of HBcrAg>80kU/ml, they were 85.9% and 81.3%, respectively. CONCLUSIONS: HBsAg has good performance in predicting liver fibrosis levels in HBeAg-positive and HBeAg-negative patients, and HBcrAg has very good performance in predicting liver fibrosis levels in HBeAg-negative patients.