William G. H. Abbott

Serum hepatitis B surface antigen and hepatitis B e antigen titers: disease phase influences correlation with viral load and intrahepatic hepatitis B virus markers.

Although threshold levels for hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) titers have recently been proposed to guide therapy for chronic hepatitis B (CHB), their relationship to circulating hepatitis B virus (HBV) DNA and intrahepatic HBV replicative intermediates, and the significance of emerging viral variants, remains unclear. We therefore tested the hypothesis that HBsAg and HBeAg titers may vary independently of viral replication in vivo. In all, 149 treatment‐naïve CHB patients were recruited (HBeAg‐positive, n = 71; HBeAg‐negative, n = 78). Quantification of HBeAg and HBsAg was performed by enzyme immunoassay. Virological characterization included serum HBV DNA load, HBV genotype, basal core promoter (BCP)/precore (PC) sequence, and, in a subset (n = 44), measurement of intrahepatic covalently closed circular DNA (cccDNA) and total HBV DNA, as well as quantitative immunohistochemical (IHC) staining for HBsAg. In HBeAg‐positive CHB, HBsAg was positively correlated with serum HBV DNA and intrahepatic cccDNA and total HBV DNA (r = 0.69, 0.71, 0.76, P < 0.01). HBeAg correlated with serum HBV DNA (r = 0.60, P < 0.0001), although emerging BCP/PC variants reduced HBeAg titer independent of viral replication. In HBeAg‐negative CHB, HBsAg correlated poorly with serum HBV DNA (r = 0.28, P = 0.01) and did not correlate with intrahepatic cccDNA nor total HBV DNA. Quantitative IHC for hepatocyte HBsAg confirmed a relationship with viral replication only in HBeAg‐positive patients. Conclusion: The correlation between quantitative HBsAg titer and serum and intrahepatic markers of HBV replication differs between patients with HBeAg‐positive and HBeAg‐negative CHB. HBeAg titers may fall independent of viral replication as HBeAg‐defective variants emerge prior to HBeAg seroconversion. These findings provide new insights into viral pathogenesis and have practical implications for the use of quantitative serology as a clinical biomarker. (HEPATOLOGY 2010)

High serum IL-21 levels after 12 weeks of antiviral therapy predict HBeAg seroconversion in chronic hepatitis B

BACKGROUND & AIMS Interleukin-21 (IL-21) stimulates T cell and B cell responses and plays a role in control of chronic viral infections. The role of IL-21 in chronic hepatitis B virus (HBV) infection is not understood. METHODS Serum IL-21 levels were measured by enzyme immunoassay in 75 HBeAg-positive chronic hepatitis B (CHB) patients undergoing telbivudine treatment. The findings were validated in 103 patients from a separate clinical trial of telbivudine. A complete response to telbivudine was defined as having both HBeAg seroconversion and serum HBV-DNA level <300 copies/ml by treatment week 52. The proportions of T-cells producing IL-21 and/or expressing programmed death 1 (PD-1) in peripheral blood mononuclear cells were assessed longitudinally during treatment by intracellular cytokine staining and flow cytometry. RESULTS Median serum IL-21 levels at treatment week 12 were significantly higher in patients who did achieve vs. patients who did not achieve a complete response in both the initial (128.4 vs. 69.2 pg/ml, p=0.003) and the validation (142.2 vs. 89.9 pg/ml, p=0.004) trials. Serum levels of IL-21 (p=0.005) or HBV-DNA (p=0.003) levels at treatment week 12 independently predicted HBeAg seroconversion in the first year of treatment. The decrease in PD-1 expression on CD4(+) and CD8(+) T cells during the first 12 weeks on telbivudine treatment was not correlated with changes in IL-21 concentrations. CONCLUSIONS Serum IL-21 levels may be a biomarker for HBeAg seroconversion, and may contribute to individualization of antiviral therapy in HBeAg-positive CHB. IL-21 may also have a role in immunotherapy for CHB.

Circulating chemokine (C‐X‐C Motif) receptor 5+CD4+ T cells benefit hepatitis B e antigen seroconversion through IL‐21 in patients with chronic hepatitis B virus infection

Given the clinical significance of hepatitis B e antigen (HBeAg) seroconversion in chronic hepatitis B virus (HBV) infection, it is critical to elucidate the mechanisms regulating this process. In the present study, we found that the frequency of circulating chemokine (C‐X‐C motif) receptor 5 (CXCR5)+CD4+ T cells was higher in patients who had achieved HBeAg seroconversion in both cross‐sectional (P < 0.001) and longitudinal (P = 0.009) studies. These cells were able to produce a significantly higher level of intracellular interleukin 21 (IL‐21) after stimulation with HBV peptides in patients with telbivudine‐induced HBeAg seroconversion (P = 0.007). Furthermore, sorted CXCR5+CD4+ T cells from HBeAg seroconverters boosted a higher frequency of antibody against hepatitis B e antigen (anti‐HBe)‐secreting B cells in coculture assay (P = 0.011). Of note, the increase in frequency of anti‐HBe‐secreting B cells was abrogated by soluble recombinant IL‐21 receptor‐Fc chimera (P = 0.027), whereas exogenous recombinant IL‐21 enhanced this effect (P = 0.043). Additionally, circulating CXCR5+CD4+ T cells shared similar phenotypic markers, and were positively correlated in frequency with, splenic follicular T helper cells. Conclusion: Circulating CXCR5+CD4+ T cells, by producing IL‐21, may have a significant role in facilitating HBeAg seroconversion in patients with chronic HBV infection. (Hepatology 2013;58:1277–1286)

Interleukin-21 is upregulated in hepatitis B-related acute-on-chronic liver failure and associated with severity of liver disease.

Summary.  The immune mechanism(s) that lead to hepatitis B‐related acute‐on‐chronic liver failure (HB‐ACLF) are poorly understood. Interleukin‐21 is a newly discovered cytokine that is involved in autoimmune and inflammatory diseases. Its potential role in HB‐ACLF remains unknown. The serum levels of 12 immune cytokines measured by cytometric bead arrays and the frequency of IL‐21‐secreting CD4+ T cells in peripheral blood mononuclear cells (PBMC) measured by intracellular cytokine staining were compared in moderate chronic hepatitis B (M‐CHB, n = 20), severe chronic hepatitis B (S‐CHB, n = 20), HB‐ACLF (n = 39) and healthy controls (n = 10). PBMC from M‐CHB patients or healthy subjects were stimulated with rhIL‐21 in vitro, and cytokines in supernatants were measured by FlowCytomix. The frequencies of IL‐21‐secreting CD4+ T cells were higher in HB‐ACLF (both P < 0.001) and S‐CHB (P = 0.002 and 0.001) as compared to M‐CHB patients and controls. Serum IL‐21 levels were highest (P < 0.001) in HB‐ACLF and positively associated with high MELD score (P = 0.001) and mortality (P = 0.038). Recovery from HB‐ACLF was associated with reduced serum IL‐21 levels (P = 0.003) and lower CD4+IL‐21+ T‐cell frequency (P = 0.006). The secretions of IL‐1β (P < 0.001), IL‐6 (P < 0.001), IL‐10 (P < 0.001), IFN‐γ (P = 0.001) and TNF‐α (P = 0.042) from PBMC were significantly increased with rhIL‐21 stimulation. In summary, IL‐21 has a causal role in the development of severe liver inflammation, which is upregulated in HB‐ACLF and associated with severity of liver disease.

Restored Circulating Invariant NKT Cells Are Associated with Viral Control in Patients with Chronic Hepatitis B

Invariant NKT (iNKT) cells are involved in the pathogenesis of various infectious diseases. However, their role in hepatitis B virus (HBV) infection is not fully understood, especially in human species. In this study, 35 chronic hepatitis B (CHB) patients, 25 inactive carriers (IC) and 36 healthy controls (HC) were enrolled and the proportions of circulating iNKT cells in fresh isolated peripheral blood mononuclear cells (PBMC) were detected by flow cytometry. A longitudinal analysis was also conducted in 19 CHB patients who received antiviral therapy with telbivudine. Thereafter, the immune functions of iNKT cells were evaluated by cytokine secretion and a two-chamber technique. The median frequency of circulating iNKT cells in CHB patients (0.13%) was lower than that in HC (0.24%, P = 0.01) and IC (0.19%, P = 0.02), and increased significantly during antiviral therapy with telbivudine (P = 0.0176). The expressions of CC chemokine receptor 5 (CCR5) and CCR6 were dramatically higher on iNKT cells (82.83%±9.87%, 67.67%±16.83% respectively) than on conventional T cells (30.5%±5.65%, 14.02%±5.92%, both P<0.001) in CHB patients. Furthermore, iNKT cells could migrate toward the CC chemokine ligand 5. Patients with a high ratio (≥1.0) of CD4−/CD4+ iNKT cells at baseline had a higher rate (58.33%) of HBeAg seroconversion than those with a low ratio (<1.0, 0%, P = 0.0174). In conclusion, there is a low frequency of peripheral iNKT cells in CHB patients, which increases to normal levels with viral control. The ratio of CD4−/CD4+ iNKT cells at baseline may be a useful predictor for HBeAg seroconversion in CHB patients on telbivudine therapy.

Associations between HLA Class I Alleles and Escape Mutations in the Hepatitis B Virus Core Gene in New Zealand-Resident Tongans

ABSTRACT The full repertoire of hepatitis B virus (HBV) peptides that bind to the common HLA class I molecules found in areas with a high prevalence of chronic HBV infection has not been determined. This information may be useful for designing immunotherapies for chronic hepatitis B. We identified amino acid residues under positive selection pressure in the HBV core gene by phylogenetic analysis of cloned DNA sequences obtained from HBV DNA extracted from the sera of Tongan subjects with inactive, HBeAg-negative chronic HBV infections. The repertoires of positively selected sites in groups of subjects who were homozygous for either HLA-B*4001 (n = 10) or HLA-B*5602 (n = 7) were compared. We identified 13 amino acid sites under positive selection pressure. A significant association between an HLA class I allele and the presence of nonsynonymous mutations was found at five of these sites. HLA-B*4001 was associated with mutations at E77 (P = 0.05) and E113 (P = 0.002), and HLA-B*5602 was associated with mutations at S21 (P = 0.02). In addition, amino acid mutations at V13 (P = 0.03) and E14 (P = 0.01) were more common in the seven subjects with an HLA-A*02 allele. In summary, we have developed an assay that can identify associations between HLA class I alleles and HBV core gene amino acids that mutate in response to selection pressure. This is consistent with published evidence that CD8+ T cells have a role in suppressing viral replication in inactive, HBeAg-negative chronic HBV infection. This assay may be useful for identifying the clinically significant HBV peptides that bind to common HLA class I molecules.

Polymorphisms of CARD15/NOD2 and CD14 genes in New Zealand Crohn's disease patients.

Polymorphisms in the CARD15/NOD2 gene, which encodes a cytosolic protein involved in bacterial recognition, are associated with development of Crohns disease (CD). Other potential susceptibility genes such as CD14 may compound the risk of developing CD. We examined the frequency of the three major CARD15 risk alleles (3020insC/L1007fsinsC, G908R and R702W), and a functional polymorphism (–159C/T) in the promoter of the CD14 gene in 185 CD patients in New Zealand and 187 ethnically matched controls. The frequencies of the 3020insC (8.1 vs 0.8%, P < 0.0001), G908R (3.5 vs 2.4%, P = 0.37) and R702W (7.3 vs 5.1%, P = 0.21) alleles in CD patients and controls, respectively, were similar to those described in Australia, and the ancestral countries of Scotland, Ireland and the UK. Only the 3020insC polymorphism was found to be a significant risk factor for CD in our New Zealand cohort (odds ratio = 10.91 [95% confidence intervals 3.30–36.08]; P < 0.0001 for heterozygotes), but not a single patient was homozygous for the 3020insC polymorphism. The T allele (51 vs 50%, P = 0.77) and TT genotype (26 vs 24%, P = 0.84) frequencies of the –159C/T CD14 gene promoter polymorphism did not significantly differ between CD patients and controls. In summary, our findings provide evidence that the CARD15 3020insC risk allele influences disease susceptibility in a small proportion (<17%) of New Zealand CD patients, whereas there was no evidence that the CD14 –159C/T polymorphism is associated with CD.

Hepatitis B virus genotype B with G1896A and A1762T/G1764A mutations is associated with hepatitis B related acute-on-chronic liver failure.

The existence of statistical associations between hepatitis B‐related acute‐on‐chronic liver failure and both hepatitis B virus (HBV) genotype and mutations in the basal core promoter (BCP) and precore (PC) regions needs to be confirmed. A total of 322 patients with a chronic HBV infection, including 77 with hepatitis B‐related acute‐on‐chronic liver failure, 109 with hepatocellular carcinoma (HCC) and 136 with chronic hepatitis B (CHB) were enrolled. The HBV genotype and the presence of mutations in the BCP/PC regions were determined by direct sequencing, and the frequencies were compared in the three patient groups. Overall, 198/322 (61.5%) were infected with genotype B and 124/322 (38.5%) with genotype C. Genotype B was significantly more frequent in patients with acute‐on‐chronic liver failure than CHB (92.2% vs. 60.3%, P < 0.001). As a contrast, genotype C was more common in patients with HCC than CHB (58.7% vs. 39.7%, P = 0.003). In genotype B patients, the A1762T/G1764A, A1846T, and G1896A mutations were significantly more prevalent in patients with acute‐on‐chronic liver failure than CHB (50.7% vs. 28.0%, P = 0.004; 59.2% vs. 34.1%, P = 0.002; 69.0% vs. 41.5%, P = 0.001, respectively). In multivariate analysis, the risk factors for acute‐on‐chronic liver failure were genotype B, A1762T/G1764A, and G1896A. In conclusion, CHB patients with genotype B, G1896A, and A1762T/G1764A had a higher tendency to develop liver failure than patients with genotype C. Therefore, HBV genotyping and detecting G1896A and A1762T/G1764A mutations might have important clinical implications as predictive risk factors for hepatitis B‐related acute‐on‐chronic liver failure. J. Med. Virol. 83:1544–1550, 2011.

Cellular immune responses in patients with hepatitis B surface antigen seroclearance induced by antiviral therapy.

BackgroundThe mechanisms by which chronic hepatitis B is completely resolved through antiviral therapy are unknown, and the contribution of acquired T cell immunity to hepatitis B surface antigen (HBsAg) seroclearance has not been investigated. Therefore, we measured the T-cell responses to core and envelope antigens in patients with HBsAg seroclearance.MethodsFourteen subjects with HBsAg seroclearance following antiviral treatment for chronic hepatitis B, 7 HBeAg-positive immunotolerant HBV carriers and 9 HBeAg-negative inactive HBsAg carriers were recruited. HBV-specific T-cell responses to recombinant HBV core (rHBcAg) and envelope (rHBsAg) proteins and pools of core and envelope peptides were measured using an ELISPOT assay detecting interferon-gamma and intracellular cytokine staining (ICS) assays detecting interferon-gamma or interleukin 2.ResultsInterferon-gamma ELISPOT assays showed a low frequency of weak responses to the rHBsAg and S peptide pool in the HBsAg seroclearance group, and the response frequency to the rHBcAg and the C peptide pool was higher than to the rHBsAg (P < 0.001) and S peptide pool (P = 0.001) respectively. A higher response frequency to C than S peptide pools was confirmed in the interferon-gamma ICS assays for both CD4+ (P = 0.033) and CD8+ (P = 0.040) T cells in the HBsAg seroclearance group. The responses to C and S antigens in the inactive carriers were similar.ConclusionsThere was a low frequency of CD4+ and CD8+ T cell immune responses to envelope antigens in Chinese subjects with HBsAg seroclearance following antiviral therapy. It is unlikely that these immune responses are responsible for HBsAg seroclearance in these subjects.

Complementarity-Determining Region 3 Size Spectratypes of T Cell Receptor β Chains in CD8+ T Cells following Antiviral Treatment of Chronic Hepatitis B

ABSTRACT An increased CD8+ T cell response to hepatitis B virus (HBV) peptides occurs between 12 and 24 weeks after starting antiviral therapy for chronic hepatitis B. It is not known whether these cells have antiviral function. The aim of this study was to determine whether clonal expansions of CD8+ T cells at these time points predict the virological response to therapy. Peripheral blood CD8+ T cells were obtained from 20 patients treated with lamivudine or telbivudine for chronic hepatitis B at baseline, 12 weeks, and 24 weeks. The CDR3 spectratype of each T cell receptor (TCR) β chain variable region (Vβ) gene family was analyzed, and the changes in the numbers of Vβ families with clonal expansions were compared in subjects with (n = 12) and without (n = 8) a virological response (52 week HBV DNA < 300 copies/ml). The number of CD8+ TCR Vβ families with clonal expansions at 12 weeks relative to baseline (median [10th to 90th percentile], +2.5 [0 to +7] versus +1 [0 to +2], P = 0.03) and at 24 weeks relative to 12 weeks (+1 [0 to +2] versus −1 [−3 to +4], P = 0.006) was higher in subjects with a virological response versus subjects without a virological response, as were interleukin-2 (IL-2) but not IL-21 mRNA levels in peripheral blood mononuclear cells. The duration of new expansions at 12 weeks was higher (P < 0.0001) in responders. Increased numbers of CD8+ T cell expansions after antiviral therapy are associated with a virological response to treatment. These CD8+ T cells are a potential target for a therapeutic vaccine for chronic hepatitis B.