Systemic cytokine and viral antigen-specific responses in hepatitis D virus RNA positive versus HDV RNA negative patients.

Background: Hepatitis B virus (HBV)/Hepatitis D Virus (HDV) co-infection increases the risk of severe liver disease compared to HBV mono-infection. Adaptive immune responses to HDV are weakly detectable, and the involvement of innate immunity in the progression of HDV-related liver fibrosis is suggested. We hypothesize that an overall innate immune activation in HBV/HDV co-infection plays a role in liver disease progression and also impacts virus specific T cell response. Methods: Sixteen HBV/HDV-co-infected-patients (median age 42y/7F/6 Asian/4 White/6 Black/15 HBeAg-) and 8 HBV monoinfected-patients (median age 39y/4F/4 Asian/3 Black/1 White/HBeAg-) with median follow-up of 5 years were enrolled. Liver fibrosis was assessed by liver stiffness measurement (LSM, FibroScan®). Proliferation of CD3 + CD4+ T cells in response to viral antigens using CFSE assays and cytokine secreting monocytes was analyzed by flow cytometry. Results: Of 16 HBV/HDV, 11 were HDV-RNA+ (HBV-DNA 0-1,040 IU/mL), 5/11 Interferon (IFN) + Nucleos/tide Analog (NA), 3/11 NA monotherapy, median ALT 77 U/L at the time of sample collection, median LSM of 9.8. In 5 HDV RNA-, median HBV DNA 65 IU/mL, 4/5 prior IFN and/or NA, ALT 31 U/L, and median LSM 8.5 kPa. In 8 HBV controls, median HBV-DNA, ALT, LSM was 69 IU/mL, 33 U/L,5 kPa, respectively. PBMC stimulation with HBV core antigen (HBcAg) and HDV antigen (HDAg) showed weaker CD3 + CD4 + T-cell proliferation in HDV-RNA+ vs. HDV RNA- and HBV-mono-infected patients (p < 0.05). In HDV-RNA+ patients, a correlation between ALT and TNF-α (r = 0.76, p = 0.008), higher IL-10 levels and increased proportion of CD14 + TNF-α+ cells were found. Conclusion: In summary, during HBV/HDV coinfection, HDV RNA+ patients had weaker HBV and HDV specific responses, associated with increased TNF-α + monocytes irrespective of IFN treatment.

Reduced immune responses to hepatitis B primary vaccination in obese individuals with nonalcoholic fatty liver disease (NAFLD).

Obesity and cirrhosis are associated with poor hepatitis B virus (HBV) vaccine responses, but vaccine efficacy has not been assessed in nonalcoholic fatty liver disease (NAFLD). Sixty-eight HBV-naïve adults with NAFLD were enrolled through the Canadian HBV network and completed three-dose HBV or HBV/HAV vaccine (Engerix-B®, or Twinrix®, GlaxoSmithKline). Anti-HBs titers were measured at 1-3 months post third dose. In 31/68 subjects enrolled at the coordinating-site, T-cell proliferation and follicular T-helper cells (pTFH) were assessed using PBMC. Immune response was also studied in NAFLD mice. NAFLD patients were stratified as low-risk-obesity, BMI < 35 (N = 40) vs. medium-high-risk obesity, BMI > 35 (N = 28). Anti-HBs titers were lower in medium/high-risk obesity, 385 IU/L ± 79 vs. low-risk obesity class, 642 IU/L ± 68.2, p = 0.02. High-risk obesity cases, N = 14 showed lower vaccine-specific-CD3+ CD4+ T-cell response compared to low-risk obesity patients, N = 17, p = 0.02. Low vaccine responders showed dysfunctional pTFH. NAFLD mice showed lower anti-HBs levels and T-cell response vs. controls. In conclusion, we report here that obese individuals with NAFLD exhibit decreased HBV vaccine-specific immune responses.

Differences in HBV Replication, APOBEC3 Family Expression, and Inflammatory Cytokine Levels Between Wild-Type HBV and Pre-core (G1896A) or Basal Core Promoter (A1762T/G1764A) Mutants.

BACKGROUND: Chronic hepatitis B virus (HBV) infection is the leading cause of hepatocellular carcinoma (HCC) world-wide. HBV variants, particularly the G1896A pre-core (PC) and A1762T/G1764A basal core promoter (BCP) mutations, are established risk factors for cirrhosis and HCC, but the molecular biological basis is unclear. We hypothesized that these variants result in differential HBV replication, APOBEC3 family expression, and cytokine/chemokine expression. METHODS: HepG2 cells were transfected with monomeric full-length containing wild-type, PC, or BCP HBV. Cells and supernatant were collected to analyze viral infection markers (i.e., HBsAg, HBeAg, HBV DNA, and RNA). Cellular APOBEC3 expression and activity was assessed by quantitative real-time (qRT)-PCR, immunoblot, differential DNA denaturation PCR, and sequencing. Cytokine/chemokines in the supernatant and in serum from 11 CHB carriers (4 non-cirrhotic; 7 cirrhotic and/or HCC) with predominantly wild-type, PC, or BCP variants were evaluated by Luminex. RESULTS: HBeAg expression was reduced in PC and BCP variants, and higher supernatant HBV DNA and HBV RNA levels were found with A1762T/G1764A vs. G1896A mutant (p < 0.05). Increased APOBEC3G protein levels in wild-type vs. mutant were not associated with HBV covalently closed circular DNA G-to-A hypermutations. Differences in cytokine/chemokine expression in culture supernatants, especially IL-13 were observed amongst the variants analyzed. Noticeable increases of numerous cytokines/chemokines, including IL-4 and IL-8, were observed in ex vivo serum collected from CHB carriers with PC mutant. CONCLUSION: HBV sequence variation leads to differences in HBV protein production (HBeAg) and viral replication in addition to altered host innate antiviral restriction factor (APOBEC3) and cytokine/chemokine expression.

Oncogenic HBV variants and integration are present in hepatic and lymphoid cells derived from chronic HBV patients.

The hepatitis B virus (HBV) is a major cause of hepatocellular carcinoma (HCC), partly driven by viral integration and specific oncogenic HBV variants. However, the biological significance of HBV genomes within lymphoid cells (i.e., peripheral blood mononuclear cells, PBMCs) is unclear. Here, we collected available plasma, PBMC, liver, and tumor from 52 chronic HBV (CHB) carriers: 32 with HCC, 19 without HCC, and one with dendritic cell sarcoma, DCS. Using highly sensitive sequencing techniques, next generation sequencing, and AluPCR, we demonstrate that viral genomes (i.e., HBV DNA, RNA, and cccDNA), oncogenic variants, and HBV-host integration are often found in all sample types collected from 52 patients (including lymphoid cells and a DCS tumor). Viral integration was recurrently identified (n = 90 such hits) in genes associated with oncogenic consequences in lymphoid and liver cells. Further, HBV genomes increased in PBMCs derived from 7 additional (treated or untreated) CHB carriers after extracellular mitogen stimulation. Our study shows novel HBV molecular data and replication not only liver, but also within 63.8% of lymphoid cells analysed (including a representative lymphoid cell malignancy), that was enhanced in ex vivo stimulated PBMC.

Hepatitis B and Pregnancy: Virologic and Immunologic Characteristics.

The hepatitis B virus (HBV) is an important human pathogen. Unvaccinated infants infected through mother-to-child transmission (MTCT) are at >95% risk of developing serum hepatitis B surface antigen-positive chronic hepatitis B (CHB). Despite complete passive-active HBV immunoprophylaxis, approximately 10% of infants born to mothers who are highly viremic develop CHB, and thus maternal treatment with nucleos(t)ide analogs (tenofovir disoproxil fumarate, lamivudine, or telbivudine) is recommended in the third trimester of pregnancy to reduce MTCT risk. Viral rebound usually occurs after stopping treatment and, in the context of maternal immunologic reconstitution postpartum, can also precipitate host immune-mediated hepatic (biochemical) flares. In this article, we review the epidemiology of HBV MTCT, discuss management and potential mechanisms of HBV vertical transmission, and highlight recent studies on virologic and immunologic aspects of hepatitis B in pregnancy and postpartum.

Presence of Precore (C)/C Promoter Mutants in Peripheral Blood Mononuclear Cells of Chronic Hepatitis B (CHB) Carriers During Pregnancy Does Not Correlate with Increased Risk of Liver Disease in 4 Years of Follow-Up.

PURPOSE: HBV precore (PC) and basal core promoter (BCP) mutants are associated with liver disease severity, yet have been suggested to protect against HBV vertical transmission. HBV within peripheral blood mononuclear cells (PBMC) has been reported in association with intrauterine HBV infection. We analyzed HBV replication status in PBMC and PC/BCP mutants in PBMC from pregnant chronic hepatitis B (CHB) patients. METHODS: Pregnant CHB carriers were assessed for HBeAg, HBV-DNA, ALT in second-third trimester and liver stiffness measurement (LSM) postpartum. HBV-DNA, HBV-cccDNA, and HBV-mRNA were tested in PBMC by in-house PCR. BCP/PC variants were determined by Sanger sequencing and analyzed using MEGA7. RESULTS: In 37 CHB pregnant carriers, median age 32 years, 53% Asian, median ALT 19 versus 26 U/L, median HBV-DNA 2.6 versus 8.1 logIU/mL (untreated vs. treated), eight HBeAg+, with genotype 10%A, 29%B, 21%C, 10%D, 19%E, eight received tenofovir in pregnancy to reduce vertical transmission risk. HBV-DNA was detected in ~ 55% (25/45) PBMC, and PC/BCP mutations were found in 36% (9/25) and 4% (1/25), respectively. All infants received HBV immunoprophylaxis and tested HBV surface antigen negative at 9-12 months of age. During a median 4 years (IQR 3-5), follow-up all mothers showed normal LSM, with no significant change in ALT, HBeAg status, or HBV-DNA levels compared to baseline in untreated CHB carriers. CONCLUSION: In this multiethnic cohort of pregnant CHB carriers, HBV replicative intermediates and PC/BCP mutants were found in significant proportion of PBMC, but were not associated with increased risk of HBV immunoprophylaxis failure or liver disease severity over long-term follow-up.

Analysis of serum hepatitis B virus RNA levels in a multiethnic cohort of pregnant chronic hepatitis B carriers.

BACKGROUND: Hepatitis B virus (HBV) flares have been reported due to alterations in the immune system during pregnancy. Recent studies in non-pregnant chronic hepatitis B (CHB) carriers have indicated that serum HBV RNA is a novel viral marker to assess treatment response and risk of disease flares. OBJECTIVES: To analyze serum HBV RNA levels in association with established HBV markers in pregnant and/or post-partum CHB carriers. STUDY DESIGN: In this prospective cohort study, serum and plasma were collected from 46 pregnant and/or post-partum CHB patients. Clinical data included demographics, hepatitis B e antigen (HBeAg) status (Abbott), quantitative hepatitis B s antigen (qHBsAg) levels (Abbott), HBV DNA (Abbott, sensitivity 10 IU/mL), alanine aminotransferase (ALT), liver stiffness measurement (LSM, post-partum), and treatment regime. Serum HBV total RNA and pre-genomic (pg)RNA were quantified using in-house assays, and HBV genotype was determined by direct population sequencing of HBV surface gene. Parametric and non-parametric statistical methods were used for analysis. RESULTS: In this study, we found that serum HBV total RNA levels correlated with the HBeAg status, HBV DNA, qHBsAg, ALT, and LSM while serum HBV pgRNA levels did not (p < 0.05, N = 46). Additionally, HBV total RNA & pgRNA levels increased, HBV DNA levels decreased, and qHBsAg levels remained unchanged throughout tenofovir disoproxil fumarate (TDF) treatment (N = 2). CONCLUSIONS: The associations between serum HBV total RNA with other validated markers indicates it may be a complementary HBV marker to monitor liver disease and HBV replication during pregnancy.

Hepatitis B virus lymphotropism: emerging details and challenges.

The hepatitis B virus (HBV) is predominantly a hepatotropic virus but also infects cells of the lymphatic system. HBV genomes (DNA, messenger (m)RNA, covalently closed circular (ccc) DNA) and proteins have been found in extrahepatic sites such as peripheral blood mononuclear cells (PBMC), lymph nodes, spleen, bone marrow and cerebrospinal fluid. HBV entry into hepatocytes occurs by binding of the HBV preS1 surface protein to its specific receptor, the bile acid transporter, sodium taurocholate co-transporting polypeptide (NTCP). Although the mechanism of HBV entry into lymphatic cells is unknown, the pre S1 encoded surface protein is thought to be involved. Extrahepatic HBV infection has been studied in both chronic HBV (CHB) and in occult HBV infection (OBI). Studies have shown that HBV genomes are present in different PBMC subsets from chronically infected carriers. Unique HBV variants have been found in PBMC compared to plasma or liver in both nucleos(t)ide analogue (NA) treated and untreated CHB carriers, suggesting replication and compartment specific evolution of HBV. In HBV coinfection, HBV genomes were found in PBMC from hepatitis C virus (HCV), human immunodeficiency virus (HIV) and hepatitis delta virus (HDV) co-infected individuals. Moreover, during pregnancy, the trans placental passage of HBV infected PBMC from highly viremic mothers to infants is one of the postulated means of vertical transmission of HBV. Taken together, HBV infection in extrahepatic sites (i.e., PBMC) is implicated in multiple facets of HBV pathogenesis such as persistence, viral evolution and vertical transmission.

Chronic hepatitis B carriers with acute on chronic liver failure show increased HBV surface gene mutations, including immune escape variants.

BACKGROUND: The pathogenesis of acute-on-chronic liver failure (ACLF) in chronic hepatitis B (CHB) is not well understood. The aim of this study was to investigate whether there is an association between HBV polymerase (P)/overlapping surface (S) gene and basal core promoter (BCP)/precore (PC) variants and development of ACLF in CHB. METHODS: Two CHB patient cohorts were compared: (i) ACLF (N = 12) (11/12 M, median age 52 yrs., 5/9 genotype C, 6/12 HBeAg+), (ii) 27 treatment native CHB carriers (15/27 M, median age 44 yrs., 9 genotype B, 10 genotype C, 1 genotype A, 5 genotype D, 2 genotype E). Clonal sequencing of PCR-amplified HBV P/S and BCP/PC gene fragments was done and HBV diversity, frequency of immune escape (IE) and drug resistance (DR) mutations and mutations in BCP/PC gene (G1896A and A1762T/G1764A), were compared between each group. RESULTS: Our data showed the incidence of IE and clusters of mutations in the HBV S region was significantly greater in ACLF patients vs. treatment naïve CHB patients (p < 0.05). Additionally, a significantly higher frequency of G1896A and A1762T/G1764A mutations were found in HBeAg negative than in ACLF patients (p < 0.0001). CONCLUSION: In our study, ACLF was not associated with a specific genomic mutation. However, higher frequency of IE mutations along with various mutations clustering in the HBV S region could contribute to or be an outcome of ACLF in CHB infection. (words 226).

Peripartum cytokine flares in a multiethnic cohort of chronic hepatitis B carriers does not correlate with hepatitis B virus suppression or increased risk of liver disease.

PROBLEM: In chronic hepatitis B (CHB) carriers, alanine transaminase (ALT) flares are common in the peripartum period. There are limited data on immunological changes of pregnancy in CHB. We hypothesize that in pregnant CHB carriers, the Th1/Th2 cytokine ratio is altered resulting in changes in biochemical/virological and liver fibrosis markers. STUDY METHODS: Serum from 38 pregnant/post-partum CHB carriers (median age 32 years, 53% Asian, 8 HBeAg+ ) was tested for HBV DNA, quantitative HBV surface antigen, ALT and liver fibrosis by transient elastography (TE). Serum cytokines were analyzed using a Luminex assay. RESULTS: Untreated CHB cases had mild ALT flares post-partum, but showed normal TE, and no change in viral markers despite increased Th1 cytokines compared to healthy controls (P<.05). CONCLUSION: CHB carriers show increased Th1 cytokines in the peripartum period albeit with no effect on viral load or liver disease suggesting an aborted antiviral immune response.