Hepatitis B Virus Variants and Cytokine Patterns in Acute Liver Failure and Transplant-Free Survival

Abstract

Background & aims: Only 25% of hepatitis B-related acute liver failure (HBV-ALF) patients survive without liver transplantation (transplant-free survival, TFS). There is limited study of immunological and virological profiles in these patients. We analysed the association between hepatitis B viremia and cytokine patterns on TFS of HBV-ALF patients.

Methods: We identified 48 acute and 20 history of HBV infection ALF patients from the US ALF Study Group registry (> 3400 patients). The inclusion criteria were age > 18 years, diagnosis of HBV-ALF with hepatic encephalopathy and INR ≥ 1.5. Data were collected from ICU admission through day 21. Serum collected at admission and at days 3-5 were used for cytokine quantification by Luminex and novel HBV biomarkers, genotypes and variants.

Results: In 48 acute (50% F, median age 40 years) and 20 history/reactivation (40% F, median age 53 years) HBV-ALF patients, there were 26 (54%) and 5 (25%) TFS, respectively. Detectable HBV DNA by clinical PCR assay (median 3.39 log₁₀IU/mL, aOR 5.308; 95% CI: 1.217-23.155, p = 0.026) and qAHBc levels (median 4.5 log₁₀IU/mL, aOR 4.466, 95% CI: 0.968-20.608, p = 0.050) were associated with TFS in acute HBV-ALF patients. HBV variants associated with anti-viral immune escape were more frequently detected in acute HBV-ALF TFS patients compared to non-TFS (p < 0.05). TFS with acute HBV-ALF had higher angiogenic factors (PDGF-AA, p = 0.008; PDGF-BB, p = 0.0006; VEGF-A, p = 0.014) and lower pro-inflammatory cytokine levels (IL-1α, p = 0.031; IL-2, p = 0.014; IL-6, p = 0.039). Significant differences in HBV viremia were not observed in history/reactivation of HBV-ALF patients.

Conclusions: Acute HBV-ALF patients with TFS were often viremic with immune escape variants, increased angiogenic factors and decreased pro-inflammatory cytokines.

Trial registration: ClinicalTrials.gov identifier: NCT00518440.

Keywords: acute liver failure; cytokines; hepatitis B; hepatitis B virus quasispecies; transplant‐free survival.

FIGURE 1

(A) From baseline (day 0) to days 3–5 follow‐up, angiogenic factors were upregulated in de novo acute HBV‐ALF transplant‐free survivors (TFS). Whereas (B) acute HBV‐ALF patients that received liver transplantation (LT) and/or died at day 21 post‐admission had higher levels of pro‐inflammatory cytokines and chemokines at day 3–5 follow‐up compared to baseline. (C) IL‐10 (anti‐inflammatory cytokine) levels increased from baseline to days 3–5 follow‐up in both TFS and LT/died acute HBV‐ALF patient cohorts. (D and E) In HBV‐ALF patients with history and/or reactivation of HBV, PDGF‐BB angiogenic factor and pro‐inflammatory cytokines and chemokines were elevated from baseline to day 3–5 follow‐up in both transplant‐free survivors and LT/died patint cohorts at day 21 post‐admission. Mean with standard error of the mean bars are plotted. Wilcoxon signed‐ranked test was performed for paired comparisons. Mann Whitney U‐test was performed for comparisons between unpaired groups. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001.

FIGURE 2

Next‐generation sequencing was performed to analyse HBV surface and core gene variants in de novo acute hepatitis B ALF patients. We found increased frequency of single nucleotide polymorphisms associated with (A) host anti‐viral immune escape in transplant‐free survivors (TFS) compared to those that received liver transplantation (LT) and/or died at baseline (day 0) and days 3–5 follow‐up. More specifically, we observed HBV D144A/E variant frequency to decrease at days 3–5 follow‐up in TFS. There were no significant differences observed in HBV variants associated with (B) anti‐viral therapy resistance (overlapping HBV polymerase region) and (C) increased risk of cirrhosis and hepatocellular carcinoma development. Mean with standard error of the mean bars are plotted. Wilcoxon signed‐ranked test was performed for paired comparisons at different time points. Mann Whitney U‐test for performed for comparisons between unpaired groups *p < 0.05. **p < 0.01.

FIGURE 3

Increased frequency of single nucleotide polymorphisms associated with (B) anti‐viral therapy resistance (overlapping HBV polymerase and surface gene regions) and (C) increased risk of cirrhosis and hepatocellular carcinoma development (HBV core gene) were observed in history or reactivation of HBV‐ALF patients with who received liver transplantation (LT) and/or died compared to transplant‐free survivors (TFS) (Next‐generation sequencing). There were no significant differences observed in HBV variants associated with (A) host anti‐viral immune escape (HBV surface gene). Mean with standard error of the mean bars are plotted. Wilcoxon signed‐ranked test and Mann Whitney U‐test were performed for comparisons between paired and unpaired groups, respectively. *p < 0.05.