Quantitation of large, middle and small hepatitis B surface proteins in HBeAg-positive patients treated with peginterferon alfa-2a.

BACKGROUND & AIMS: Hepatitis B virus (HBV) contains three viral surface proteins, large, middle and small hepatitis B surface protein (LHBs, MHBs, SHBs). Proportions of LHBs and MHBs are lower in patients with inactive vs active chronic infection. Interferon alfa may convert hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) to an inactive carrier state, but prediction of sustained response is unsatisfactory. The aim of this study was to test the hypothesis that quantification of MHBs and LHBs may allow for a better prognosis of therapeutic response than total hepatitis B surface antigen (HBsAg) concentration. METHODS: Hepatitis B surface proteins were measured before and during peginterferon alfa-2a therapy in serum from 127 Asian patients with HBeAg-positive CHB. Sustained response was defined as HBeAg seroconversion 24 weeks post-treatment. RESULTS: Mean total HBs levels were significantly lower in responders vs nonresponders at all time points (P < .05) and decreased steadily during the initial 24 weeks treatment (by 1.16 vs 0.86 ng/mL in responders/nonresponders respectively) with unchanged relative proportions. Genotype B had a two-fold higher proportion of LHBs than genotype C (13% vs 6%). HBV DNA, HBeAg, HBsAg and HBs protein levels predicted response equally well but not optimally (area under the receiver operating characteristic curve values >0.70). CONCLUSIONS: Hepatitis B surface protein levels differ by HBV genotype. However, quantification of HBs proteins has no advantage over the already established HBsAg assays to predict response to peginterferon alfa-2a therapy in HBeAg-positive patients.

Hepatitis B virus-specific T cell responses after stopping nucleos(t)ide analogue therapy in HBeAg-negative chronic hepatitis B.

BACKGROUND & AIMS: Treatment with nucleos(t)ide analogues (NA) leads to hepatitis B virus (HBV) DNA suppression in most patients with chronic hepatitis B (CHB), but HBV surface antigen (HBsAg) loss rates are low. Upon NA discontinuation, HBV DNA can return rapidly with ensuing alanine aminotransferase flares and induction of cytokines. Several studies reported higher HBsAg loss rates after stopping therapy, but at present it is unclear if cell-mediated immune responses are altered after treatment discontinuation. The aim of this study was to characterise T cell responses during the early phase of virological relapse, following discontinuation of NA therapy in HBeAg-negative patients. METHODS: A total of 15 HBeAg-negative patients with CHB on long-term NA treatment were included in a prospective study and subjected to structured NA discontinuation. T cell responses were studied at the end of NA therapy and 4, 8 and 12 weeks thereafter. RESULTS: The T cell phenotype of patients with CHB on long-term NA therapy was markedly different compared to healthy individuals, but was only slightly altered after discontinuation of therapy. T cells from patients with HBsAg loss expressed low levels of KLRG1 and PD-1 at all time-points and high levels of Ki-67 and CD38 at week 12 after treatment cessation. In vitro peptide stimulated HBV-specific T cell responses were increased in several patients after NA cessation. Blocking of PD-L1 further enhanced HBV-specific T cell responses, especially after discontinuation of therapy. CONCLUSION: Relapse of active HBV replication after stopping therapy may trigger an immunological environment that enhances the responsiveness of HBV-specific T cells in vitro. Together with other immune interventions, this approach might be of interest for the development of novel therapeutic options to induce HBsAg loss in CHB. LAY SUMMARY: Relapse of hepatitis B virus replication after discontinuation of nucleos(t)ide analogue therapy in certain patients with chronic hepatitis B may alter the phenotype of T cells and enhance the responsiveness of hepatitis B virus-specific T cells to in vitro peptide stimulation. Blocking PD-L1 can further augment these hepatitis B virus-specific T cell responses. Interestingly, T cells of patients that subsequently achieve hepatitis B surface antigen loss are less exhausted at all time-points after stopping treatment and display a higher proliferative capacity 12-weeks after treatment discontinuation. These findings contribute to the understanding of the immunological events that occur during discontinuation of nucleos(t)ide analogue therapy.

Increased NK Cell Function After Cessation of Long-Term Nucleos(t)ide Analogue Treatment in Chronic Hepatitis B Is Associated With Liver Damage and HBsAg Loss.

Background: Treatment with nucleos(t)ide analogues (NA) suppresses hepatitis B virus (HBV) DNA but rarely leads to functional cure of chronic hepatitis B (CHB). Following NA cessation, some hepatitis B e antigen (HBeAg)-negative CHB patients experience hepatitis B s antigen (HBsAg) loss. Cellular immune responses, including natural killer (NK) cell responses, explaining virological events following NA treatment cessation remain elusive. Methods: In a single-center prospective trial, 15 HBeAg-negative CHB patients on long-term NA treatment underwent structured NA cessation and were studied longitudinally. The NK cell compartment was assessed using high-dimensional flow cytometry and correlated with the clinical course. Results: Unsupervised stochastic neighbor embedding analysis revealed NA-treated CHB patients to have a significantly affected NK cell compartment compared to controls. Cessation of NA treatment resulted in minor phenotypic alterations, but it significantly augmented NK cell natural cytotoxicity responses in the CHB patients. This increased NK cell functionality correlated with alanine aminotransferase flares in the patients and was particularly enhanced in patients experiencing HBsAg seroclearance at long-term follow-up. Conclusions: Increased NK cell function is associated with active hepatitis and HBsAg seroclearance following structured NA cessation. This adds to our knowledge of the immunological events that develop following cessation of NA treatment in CHB.

Hepatitis E virus ORF 1 induces proliferative and functional T-cell responses in patients with ongoing and resolved hepatitis E.

BACKGROUND AND AIMS: Hepatitis E virus (HEV) is a major cause of acute viral hepatitis with >3 million symptomatic cases per year accounting for 70 000 HEV-related deaths. HEV-specific T-cell responses have been investigated against structural proteins expressed by open reading frames (ORF) 2 and 3. T-cell responses against non-structural HEV proteins encoded by ORF1 are hardly studied. The aim of this study was to determine HEV ORF1-specific T-cell responses in comparison to ORF2/3 in patients exposed to HEV. METHODS: HEV-specific CD4+ and CD8+ T-cell responses against HEV genotype 3 were investigated in patients with acute and chronic hepatitis E as well as in HEV seropositive and seronegative individuals. HEV-specific T-cell responses were determined by proliferation and intracellular cytokine assay upon stimulation of PBMCs with HEV-specific overlapping peptide pools spanning the entire HEV genome. HEV-antigen was measured using an anti-HEV antigen-specific ELISA. RESULTS: Broad HEV ORF1-specific T-cell responses were detected in patients with acute, resolved and chronic hepatitis E without distinct dominant regions. The magnitude and frequency in recognition of ORF1-specific T-cell responses were similar compared to responses against HEV ORF2/3. Longitudinal studies of HEV-specific T-cell responses displayed similar behaviour against structural and non-structural proteins. HEV-antigen levels were inversely correlated with HEV-specific T-cell responses. CONCLUSIONS: HEV-specific T-cell responses are detectable against the entire HEV genome including the non-structural proteins. HEV-specific T-cell responses are associated with control of HEV infection. These findings have implications for the design of HEV vaccines.

Viral and Host Responses After Stopping Long-term Nucleos(t)ide Analogue Therapy in HBeAg-Negative Chronic Hepatitis B.

This prospective study investigated viral and host markers after stopping long-term therapy with nucleos(t)ide analogues in noncirrhotic patients with hepatitis B e antigen-negative chronic hepatitis B. After stopping therapy, 13 of 15 patients experienced a virological relapse. Rebound of hepatitis B virus DNA and hepatitis B core-related antigen was associated with induction of plasma tumor necrosis factor, interleukin (IL) 10 , IL-12p70, CXCL10 and subsequent decline in hepatitis B surface antigen (HBsAg), with 20% HBsAg loss after long-term follow-up. The peak levels of hepatitis B virus DNA and hepatitis B core-related antigen after cessation of therapy were positively correlated with the level of HBsAg decline at week 48. Thus, stopping or interrupting NA treatment should be further investigated as a strategy to accelerate HBsAg loss.