Impact of core protein naturally selected mutants associated with HBeAg-negative status in HBV biosynthesis.

Hepatitis B e antigen (HBeAg) loss represents a late stage of chronic hepatitis B virus (HBV) infection associated with a drastic decrease in HBV-DNA, a lower risk of disease progression, and the occurrence of several mutations in the preCore/core region. However, the underlying mechanisms supporting the downregulation of viral replication have yet to be elucidated. In the present study, the analysis of the frequency of subgenotype D1 core protein (HBc) mutations associated with HBeAg status revealed a higher mutation rate in HBeAg-negative sequences compared to HBeAg-positive ones. Particularly, 22 amino acids exhibited a higher frequency of mutation in HBeAg-negative sequences, while the remaining residues showed a high degree of conservation. Subsequently, the assessment of HBc mutants derived from HBeAg-negative patients in viral structure and replicative capacity revealed that HBc mutations have the ability to modulate the subcellular localization of the protein (either when the protein was expressed alone or in the context of viral replication), capsid assembly, and, depending on specific mutation patterns, alter covalently closed circular DNA (cccDNA) recycling and up- or downregulate viral replication. In conclusion, HBc mutations associated with HBeAg-negative status impact on various stages of the HBV life cycle modulating viral replication during the HBeAg-negative stage of infection.

Different mutation rates in the basal core promoter and precore regions between hepatitis B virus subgenotype F1b clusters.

Hepatitis B virus (HBV) is the main etiological agent of hepatocellular carcinoma (HCC) worldwide. It has been classified into nine genotypes and several subgenotypes, with uneven global distribution. There is growing evidence that the viral genotype influences the course and outcome of chronic hepatitis B infection. Two evolutionarily different clusters of the subgenotype F1b, called basal and cosmopolitan, have been described. The two clusters have constrained geographical distribution, with the particular feature that the basal cluster is present in regions of high HCC incidence, while the Cosmopolitan cluster is found in regions of low HCC incidence. The BCP/pC region was sequenced in 68 cases chronically infected with the F1b subgenotype to determine if there was a differential pattern of pathogenic-associated mutations between both clusters. Twenty-two of the 68 cases belonged to the subgenotype F1b basal cluster and 46 to the cosmopolitan cluster. Among the HBeAg-negative patients the A1762T/G1764A and G1896A mutations were more frequently found in the basal samples (85.7 and 92.9%) compared to the cosmopolitan ones (50 and 18.2%). Interestingly, no HBeAg loss-associated mutations were observed in 7.1 and 36.4% of the basal and cosmopolitan cases, respectively. The different rate of mutations associated with a more severe course of chronic hepatitis in the basal cluster would support the difference in the HCC incidence rate in the geographical regions where the basal cluster is restricted.

Intrahepatic immune infiltrate in chronic hepatitis B and chronic hepatitis C: Similar but not the same.

In chronic hepatitis B (CHB) and C (CHC) infections, the composition of the immune cell microenvironment at the site of infection is poorly understood. Thus, our aim was to characterize and compare liver infiltrates to identify shared and exclusive hepatic immune components. Immunohistochemistry was performed on 26 CHB and 42 CHC liver biopsies to determine Th (CD4+), Th1 (T-bet+), Th17 (IL-17A+), Treg (Foxp3+) and CTL (CD8+) cells frequency in portal/periportal and intralobular areas and relate them to liver damage. CHB and CHC cases shared a portal/periportal CD4+ lymphocyte predominance and a lobular CD8+ lymphocyte majority. However, CHC exhibited a concomitant lobular T-bet+ cell dominance while in CHB FoxP3+ cells prevail. CHC disclosed higher frequencies of P/P FoxP3+, IL-17A+ and T-bet+ cells and intralobular CD4+, IL-17A+ and T-bet+ lymphocytes. HBeAg+ chronic hepatitis and CHC cell frequencies were similar except for lobular T-bet+ that remained higher among CHC cases. Comparison among cases with less severe liver disease revealed lower lymphocyte frequencies in CHB samples, while no differences were observed between patients with more severe stages. Interestingly, in CHB portal/periportal CD4+ and lobular CD4+, CD8+ and IL-17A+ cells were associated with severe hepatitis. Even when all studied populations were identified in both infections preferential lymphocyte frequencies and prevalence at different areas along with their association with liver damage highlighted that CHB and CHC immune responses are not the same.

Chronic Hepatitis C Pathogenesis: Immune Response in the Liver Microenvironment and Peripheral Compartment.

Chronic hepatitis C (CHC) pathogenic mechanisms as well as the participation of the immune response in the generation of liver damage are still a topic of interest. Here, we evaluated immune cell populations and cytokines in the liver and peripheral blood (PB) to elucidate their role in CHC pathogenesis. B, CTL, Th, Treg, Th1, Th17, and NK cell localization and frequency were evaluated on liver biopsies by immunohistochemistry, while frequency, differentiation, and functional status on PB were evaluated by flow cytometry. TNF-α, IL-23, IFN-γ, IL-1ß, IL-6, IL-8, IL-17A, IL-21, IL-10, and TGF-ß expression levels were quantified in fresh liver biopsy by RT-qPCR and in plasma by CBA/ELISA. Liver CTL and Th1 at the lobular area inversely correlated with viral load (r = -0.469, p =0.003 and r = -0.384, p = 0.040). Treg correlated with CTL and Th1 at the lobular area (r = 0.784, p < 0.0001; r = 0.436, p = 0.013). Th17 correlated with hepatic IL-8 (r = 0.52, p < 0.05), and both were higher in advanced fibrosis cases (Th17 p = 0.0312, IL-8 p = 0.009). Hepatic cytokines were higher in severe hepatitis cases (IL-1ß p = 0.026, IL-23 p = 0.031, IL-8 p = 0.002, TGF-ß, p= 0.037). Peripheral NK (p = 0.008) and NK dim (p = 0.018) were diminished, while NK bright (p = 0.025) was elevated in patients vs. donors. Naïve Th (p = 0.011) and CTL (p = 0.0007) were decreased, while activated Th (p = 0.0007) and CTL (p = 0.0003) were increased. IFN-γ production and degranulation activity in NK and CTL were normal. Peripheral cytokines showed an altered profile vs. donors, particularly elevated IL-6 (p = 0.008) and TGF-ß (p = 0.041). Total hepatic CTLs favored damage. Treg could not prevent fibrogenesis triggered by Th17 and IL-8. Peripheral T-lymphocyte differentiation stage shift, elevated cytokine levels and NK-cell count decrease would contribute to global disease.

Viral hepatitis update: Progress and perspectives.

Viral hepatitis, secondary to infection with hepatitis A, B, C, D, and E viruses, are a major public health problem and an important cause of morbidity and mortality. Despite the huge medical advances achieved in recent years, there are still points of conflict concerning the pathogenesis, immune response, development of new and more effective vaccines, therapies, and treatment. This review focuses on the most important research topics that deal with issues that are currently being solved, those that remain to be solved, and future research directions. For hepatitis A virus we will address epidemiology, molecular surveillance, new susceptible populations as well as environmental and food detections. In the case of hepatitis B virus, we will discuss host factors related to disease, diagnosis, therapy, and vaccine improvement. On hepatitis C virus, we will focus on pathogenesis, immune response, direct action antivirals treatment in the context of solid organ transplantation, issues related to hepatocellular carcinoma development, direct action antivirals resistance due to selection of resistance-associated variants, and vaccination. Regarding hepatitis D virus, we describe diagnostic methodology, pathogenesis, and therapy. Finally, for hepatitis E virus, we will address epidemiology (including new emerging species), diagnosis, clinical aspects, treatment, the development of a vaccine, and environmental surveillance.

Hepatic lymphocytes involved in the pathogenesis of pediatric and adult non-alcoholic fatty liver disease.

The immune response is critical in NAFLD pathogenesis, but the liver infiltrate's composition and the role of each T cell population is still up for debate. To characterize liver pathogenesis in pediatric and adult cases, frequency and localization of immune cell populations [Cytotoxic T Lymphocytes (CD8+), T helper Lymphocytes (CD4+), Regulatory T lymphocytes (Foxp3+) and Th17 (IL-17A+)] were evaluated. In portal/periportal (P/P) tracts, both age groups displayed a similar proportion of CD8+ and CD4+ lymphocytes. However, comparable Foxp3+ and IL-17A+ cell frequencies were observed in pediatric cases, meanwhile, in adults Foxp3+ was higher than IL-17A+ cells. Interestingly, IL-17A+ lymphocytes seemed to be nearly exclusive of P/P area in both age groups. In intralobular areas, both pediatric and adult cases showed CD8+ lymphocytes predominance with lower frequencies of CD4+ lymphocytes followed by Foxp3+ . Severe inflammation was associated with higher intralobular Foxp3+ lymphocytes (p = 0.026) in children, and lower P/P Foxp3+ and higher IL-17A+ lymphocytes in adults. All cases with fibrosis ≥ 2 displayed P/P low Foxp3+ and high IL-17A+ lymphocyte counts. Pediatric cases with worse steatosis showed high P/P CD4+ (p = 0.023) and intralobular CD8+ (p = 0.027) and CD4+ cells (p = 0.012). In NAFLD cases, the lymphocyte liver infiltrate composition differs between histological areas. Treg and Th17 balance seems to condition damage progression, denoting their important role in pathogenesis.

Chronic hepatitis B: The interplay between intrahepatic lymphocyte population and viral antigens in relation to liver damage.

In Chronic hepatitis B (CHB) infection, virus and immune response interplay is thought to be responsible for pathogenesis. Yet, the impact of each immune cell population and viral protein expression in liver damage is still unknown. Our aim was to study the interplay between intrahepatic immune response and viral activity in relation to CHB liver damage. Immunostaining was performed in 29 liver biopsies from untreated CHB patients to characterize liver infiltrate [Th (CD4+), CTL (CD8+), Treg (FoxP3+), Th17 (IL-17A+) and Th1 (T-bet+)] and viral antigen expression (HBsAg and HBcAg). Inflammatory activity and fibrosis were assessed using the HAI and METAVIR scoring system. All studied populations were identified in the portal-periportal (P-P) areas with a CD4+ lymphocyte predominance, while only CD8+ and FoxP3+ cells were observed in the intralobular area. Both P-P CD4+ and intralobular CD8+ cell frequencies were increased among severe hepatitis cases. Concerning HBsAg and HBcAg expression, a mutually exclusive pattern was observed. HBcAg was mainly detected among HBeAg-positive patients and was associated with hepatitis severity and higher frequency of P-P FoxP3+, intralobular CD8+ and FoxP3+ cells. HBsAg was identified among HBeAg-negative cases with less severe hepatitis grade and lower frequency of P-P CD4+ and intralobular FoxP3+ lymphocytes. In conclusion, the HBV antigen profile expression seen during CHB infection may be reflecting different stages of viral replication which impacts the host immune response and liver damage process. While HBcAg might be an inducer of a regulatory microenvironment, the intralobular CTL population seemed to have a key role in hepatitis severity.