RESUMO
OBJECTIVE: A convenient, reproducible biomarker of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) transcriptional activity is lacking. We measured circulating HBV RNA (cirB-RNA) in untreated and nucleos(t)ide analogues (NUC) treated chronic hepatitis B (CHB) patients to define its correlation with intrahepatic viral markers and HBV core-related antigen (HBcrAg). DESIGN: Paired liver biopsy and serum samples were collected from 122 untreated and 30 NUC-treated CHB patients. We measured cirB-RNA, HBV DNA, hepatitis B surface antigen (HBsAg), HBcrAg and alanine aminotransferase levels. cirB-RNA was quantified using an investigational HBV RNA assay for use on the cobas 6800 system. The test detects a region spanning the HBV canonical polyadenylation site. cccDNA and 3.5 kb RNA in liver tissue were assessed by quantitative PCR and droplet digital PCR. RESULTS: cirB-RNA was detectable in 100% of HBeAg(+) chronic hepatitis (CH), 57% and 14% of HBeAg(-) CH and chronic infection untreated patients and 47% of NUC-treated patients. cirB-RNA undetectability was associated with lower intrahepatic cccDNA transcriptional activity, as well as serum HBcrAg, but no significant differences in HBsAg, in both untreated and treated patients. In untreated HBeAg(-) patients, cirB-RNA correlated with intrahepatic 3.5 kb RNA and cccDNA transcriptional activity, serum HBV DNA and HBcrAg, but not with HBsAg or total cccDNA levels. Combined undetectability of both cirB-RNA and HBcrAg detection in untreated HBeAg(-) patients identified a subgroup with the lowest levels of intrahepatic transcriptionally active cccDNA. CONCLUSION: Our results support the usefulness of quantification of circulating HBV RNA expressed from cccDNA as an indicator of intrahepatic active viral reservoir in both untreated and NUC-treated CHB patients. TRIAL REGISTRATION NUMBER: NCT02602847.
Assuntos
Hepatite B Crônica , Humanos , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/tratamento farmacológico , Vírus da Hepatite B/genética , Antígenos de Superfície da Hepatite B , Antígenos E da Hepatite B , DNA Circular , DNA Viral , Antivirais/uso terapêutico , Fígado/patologia , Antígenos do Núcleo do Vírus da Hepatite B , RNA , BiomarcadoresRESUMO
BACKGROUND & AIMS: It has been proposed that serum hepatitis B core-related antigen (HBcrAg) reflects intrahepatic covalently closed circular (ccc)DNA levels. However, the correlation of HBcrAg with serum and intrahepatic viral markers and liver histology has not been comprehensively investigated in a large sample. We aimed to determine if HBcrAg could be a useful therapeutic marker in patients with chronic hepatitis B. METHODS: HBcrAg was measured by chemiluminescent enzyme immunoassay in 130 (36 hepatitis B e antigen [HBeAg]+ and 94 HBeAg-) biopsy proven, untreated, patients with chronic hepatitis B. HBcrAg levels were correlated with: a) serum hepatitis B virus (HBV)-DNA, quantitative hepatitis B surface antigen and alanine aminotransferase levels; b) intrahepatic total (t)HBV-DNA, cccDNA, pregenomic (pg)RNA and cccDNA transcriptional activity (defined as pgRNA/cccDNA ratio); c) fibrosis and necroinflammatory activity scores. RESULTS: HBcrAg levels were significantly higher in HBeAg+ vs. HBeAg- patients and correlated with serum HBV-DNA, intrahepatic tHBV-DNA, pgRNA and cccDNA levels, and transcriptional activity. Patients who were negative for HBcrAg (<3 LogU/ml) had less liver cccDNA and lower cccDNA activity than the HBcrAg+ group. Principal component analysis coupled with unsupervised clustering identified that in a subgroup of HBeAg- patients, higher HBcrAg levels were associated with higher serum HBV-DNA, intrahepatic tHBV-DNA, pgRNA, cccDNA transcriptional activity and with higher fibrosis and necroinflammatory activity scores. CONCLUSIONS: Our results indicate that HBcrAg is a surrogate marker of both intrahepatic cccDNA and its transcriptional activity. HBcrAg could be useful in the evaluation of new antiviral therapies aiming at a functional cure of HBV infection either by directly or indirectly targeting the intrahepatic cccDNA pool. LAY SUMMARY: Hepatitis B virus causes a chronic infection which develops into severe liver disease and liver cancer. The viral covalently closed circular DNA (cccDNA) is responsible for the persistence of the infection in hepatocytes. To better manage patient treatment and follow-up, and to develop new antiviral treatments directly targeting the intrahepatic pool of cccDNA, serum surrogate markers reflecting the viral activity in the liver are urgently needed. In this work, we demonstrate that quantification of hepatitis B core-related antigen in serum correlates with cccDNA amount and activity and could be used to monitor disease progression.
Assuntos
DNA Circular/genética , DNA Viral/genética , Antígenos do Núcleo do Vírus da Hepatite B/sangue , Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Hepatite B Crônica/imunologia , Ativação Transcricional , Adolescente , Adulto , Idoso , Antivirais/uso terapêutico , Biomarcadores/sangue , Estudos de Coortes , Progressão da Doença , Feminino , Genótipo , Antígenos E da Hepatite B/sangue , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Humanos , Fígado/patologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Hepatitis B virus (HBV) infection remains a major public health concern worldwide with 240 million individuals chronically infected and at risk of developing cirrhosis and hepatocellular carcinoma. Current treatments rarely cure chronic hepatitis B infection, highlighting the need for new anti-HBV drugs. Nucleic acid polymers (NAPs) are phosphorothioated oligonucleotides that have demonstrated a great potential to inhibit infection with several viruses. In chronically infected human patients, NAPs administration lead to a decline of blood HBsAg and HBV DNA and to HBsAg seroconversion, the expected signs of functional cure. NAPs have also been shown to prevent infection of duck hepatocytes with the Avihepadnavirus duck hepatitis B virus (DHBV) and to exert an antiviral activity against established DHBV infection in vitro and in vivo. In this study, we investigated the specific anti-HBV antiviral activity of NAPs in the HepaRG human hepatoma cell line and primary cultures of human hepatocytes. NAPs with different chemical features (phosphorothioation, 2'O-methyl ribose, 5-methylcytidine) were assessed for antiviral activity when provided at the time of HBV inoculation or post-inoculation. NAPs dose-dependently inhibited HBV entry in a phosphorothioation-dependent, sequence-independent and size-dependent manner. This inhibition of HBV entry by NAPs was impaired by 2'O-methyl ribose modification. NAP treatment after viral inoculation did not elicit any antiviral activity.
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Antivirais/farmacologia , Vírus da Hepatite B/fisiologia , Ácidos Nucleicos/química , Polímeros/farmacologia , Internalização do Vírus/efeitos dos fármacos , Antivirais/química , Antivirais/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citidina/análogos & derivados , Citidina/química , DNA Viral/sangue , Antígenos de Superfície da Hepatite B/sangue , Antígenos E da Hepatite B/sangue , Vírus da Hepatite B/genética , Hepatócitos/citologia , Hepatócitos/virologia , Humanos , Imunoensaio , Fosfatos/química , Polímeros/química , Polímeros/toxicidade , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND & AIMS: Hepatitis B virus (HBV) persistence and the pathobiology of chronic HBV (CHB) infections result from the interplay between viral replication and host immune responses. We aimed to comprehensively analyse the expression of intrahepatic host genes as well as serum and liver HBV markers in a large cohort of untreated CHB patients. METHODS: One-hundred and five CHB patients untreated at the time of liver biopsy (34 HBeAg[+] and 71 HBeAg[-]) were analysed for the intrahepatic expression profile of 67 genes belonging to multiple innate immunity pathways. Results were correlated to serological (quantification of HBsAg [qHBsAg] and HBV DNA) and intrahepatic viral markers (total HBV DNA, pre-genomic RNA and covalently closed circular HBV DNA). RESULTS: Intrahepatic gene expression profiling revealed a strong downregulation of antiviral effectors, interferon stimulated genes, Toll-like and pathogen recognition receptor pathways in CHB patients as compared to non-infected controls, which was not directly correlated to HBV replication. A subset of genes [CXCL10, GBP1, IFITM1, IFNB1, IL10, IL6, ISG15, TLR3, SOCS1, SOCS3] was more repressed in HBeAg(-) respect to HBeAg(+) patients (median of serum HBV DNA 7.9×103vs. 7.9×107IU/ml, respectively). Notably, HBeAg(-) patients with lower qHBsAg (<5×103IU/ml) showed a relief of repression of genes belonging to multiple pathways. CONCLUSIONS: Our results show a strong impairment of innate immune responses in the liver of CHB patients. The association of low levels of qHBsAg with gene repression, if confirmed, might prove useful for the identification of patients who would most benefit from immune-modulators and/or HBsAg targeting agents as strategies to restore immune responsiveness. LAY SUMMARY: Chronic hepatitis B virus (HBV) infections represent a major public health problem worldwide. Over 200 million people are chronically infected and at risk of developing chronic hepatitis, liver cirrhosis and cancer. Our work aimed to understand the molecular consequences of chronic hepatitis B in the infected liver. It was conducted in a large cohort of untreated chronically infected HBV patients and analysed the expression of immunity and liver disease-related genes in the liver, with respect to markers of viral replication and persistence. Our results indicate that chronic HBV infection has a suppressive effect on immune responses, which was more pronounced with high levels of hepatitis B virus surface antigen (HBsAg). These data provide novel insight into the mechanisms of HBV persistence in the liver and suggest that approaches aimed at reducing HBsAg levels, may restore immune responsiveness against the virus.
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Hepatite B Crônica/imunologia , Fígado/imunologia , Adulto , Regulação para Baixo , Feminino , Antígenos de Superfície da Hepatite B/análise , Antígenos E da Hepatite B/análise , Hepatite B Crônica/patologia , Humanos , Imunidade Inata , Masculino , Pessoa de Meia-Idade , TranscriptomaRESUMO
UNLABELLED: The monoclonal antibody (mAb) D32.10 recognizes a discontinuous epitope encompassing three regions E1 (amino acids 297-306), E2A (amino acids 480-494), and E2B (amino acids 613-621) juxtaposed on the surface of serum-derived hepatitis C virus (HCV) particles (HCVsp). The mAb D32.10 inhibits efficiently and specifically the binding of HCVsp to human hepatocytes. Therefore, we investigated the clinical relevance of anti-E1E2A,B response in the serum of patients infected with HCV. To this end, an enzyme-linked immunosorbent assay (ELISA) using synthetic E1-, E2A-, and E2B-derived peptides was used. The ELISA was validated in terms of sensitivity, specificity, and test efficiency. The detection of the anti-E1E2 D32.10 epitope-binding antibodies during natural HCV infection in more than 300 HCV-positive sera demonstrated significantly (P < 0.001) higher prevalence of these antibodies: (1) in patients who spontaneously cured HCV infection (46 of 52, 88.5%) showing high titers (70% ≥ 1/1000) compared to never-treated patients with chronic hepatitis C (7 of 50, 14%) who actively replicated the virus, and (2) in complete responders (20 of 52, 38.5%) who cleared virus following treatment and achieved a sustained viral response compared to nonresponders (4 of 40, 10%). Serum anti-E1E2 antibodies were monitored before, during, and after the current standard-of-care therapy (pegylated interferon plus ribavirin) in responder and nonresponder patients. Optimal cutoff values were assessed by receiver operating characteristic curve analysis. One month prior to therapy initiation, the threshold of 1131 (optical density × 1000) gave 100% and 86% positive and negative predictive values, respectively, for achieving or not achieving a sustained viral response. CONCLUSION: The anti-E1E2 D32.10 epitope-binding antibodies are associated with control of HCV infection and may represent a new relevant prognostic marker in serum. This unique D32.10 mAb may also have immunotherapeutic potential.
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Anticorpos Anti-Idiotípicos/uso terapêutico , Antivirais/uso terapêutico , Hepatite C/tratamento farmacológico , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Epitopos/imunologia , Epitopos/uso terapêutico , Seguimentos , Hepacivirus/imunologia , Hepatite C/sangue , Hepatite C/imunologia , Humanos , Imunoglobulinas/sangue , Estudos Longitudinais , Peptídeos/imunologia , Reprodutibilidade dos Testes , Estudos Soroepidemiológicos , Resultado do TratamentoRESUMO
The genomic diversity of HCV embraces 6 genoptypes and at least 52 subtypes with clinical and epidemiological correlations. There is a paucity of studiens assessing HCV genotypes and biomolecular epidemiology in Brazil. We studied genotypes distribution and epidemiological aspects in 232 HCV carriers, 133 ( 57,9 per cent) males and 99 (42,1 per cent) females, followed in the liver disease referral unit in Salvador, BA, northeastern Brazil. All of them were anti-HCV positive by 3rd generation ELISA assay, and HCV infection showed that 93 (40 per cent) had past blood transfusion, 14 (6 per cent) intravenous drug use, 19 (8 per cent) inhalation of cocaine, 28 (12 per cent) tattooing, 15 (7 per cent) were health care workers, 5 (2 per cent) had reused disposable syringes, 5 (2 per cent) had multiple risk factors and in 53 (23 per cent) no risk factor was determined.Genotypes 1a was observed in 75 (32 per cent), 1b in 72 (31 per cent), 3a in 61 (26 per cent), 2b in 14 (6 per cent): 5 (2.5 per cent) had mixed genotypes and 5 (2.5 per cent) were undetermined. Patients with genotype 1 had a higher mean age (P<0.05) and no particular risk factors were associated with a specific genotype. Genotype 1 largely predominates in northeast Brazil followed by genotype 3 which, in this population, does not seem to be related to intravenous drug abuse, in contrast to some European studies. Although 80 porcentage of the Salvador population comprise African-Brazilians, no Africa genotype was identified, which way mean that HCV was introduced into this region via European immigration. This study demonstrated some peculiarities of HCV epidemiology in Brazil and strongly suggests that HCV introduction to this region was probably related to European immigration.