Study On The Relationship Between Hepatitis B Virus Mutations And Acute-on-chronic Liver Failure

Hepatitis B virus (HBV) is responsible for chronic infection. There are approximately 300 million chronic HBV carriers worldwide and about 120 million HBV carriers in China. HBV infection can lead to a series of spectrums of liver diseases including asymptomatic carrier, chronic hepatitis B (CHB), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) or acute-on-chronic liver failure (ACLF). According to the Asian Pacific Association for the Study of the Liver including acute hepatic insult, ACLF was characterized by jaundice and coagulopathy, complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease. Similarly, in China, the diagnosis of ACLF was based on recent development of jaundice [total bilirubin (TB)≥171.0μmol/l] or rapidly rising levels of TB (≥17.1μmol/L/day) and plasma prothrombin activity (PTA)≤40%. These were accompanied by the development of complications such as hepatic encephalopathy (≥grade 2), abrupt and obvious increase in ascites or spontaneous bacterial peritonitis, or hepatorenal syndrome. In China, HBV-related ACLF cases account for more than 80% of ACLF patients. ACLF has been as a result of the high incidence of chronic HBV infection and was reported to have a high mortality rate (60-80%) in the absence of liver transplantation.The pathogenesis of ACLF remains unclear. Both viral and immune factors may play a role. HBV mutations in the basal core promoter (BCP) and precore (PC) have attracted special attention. Previous studies have clarified that a higher prevalence of the BCP double mutations and PC mutation were in patients with acute liver failure. Because the BCP mutations may enhance HBV replication in vitro and the PC mutation abrogates translation of HBeAg, which is considered a tolerogen buffering any immune attack on the infected hepatocytes.HBV DNA contains four open reading frames. One of them is the C gene, which encodes a core peptide (HBcAg). HBcAg has been postulated to be an immunological target of cytotoxic T lymphocytes (CTL). Studies on endogenously processed viral peptides demonstrated that a peptide could be recognized by CTL and is the principal motive power which can activate specific cell immune response. When the mutate site just located in the epitope sequence, it will be possible to change the immunological characteristics and then have an impact on the host responsibility to HBV. Investigation of changes in such regions may help to predict the occurrence of ACLF.PurposeIn this study, the nucleotide sequences of the precore and core region of HBV were analyzed to investigate the relationship of HBV mutations with ACLF. Moreover, we looked into the association between ACLF and the epitopes in HBcAg, who have prototype and variant.MethodsThirty-nine patients with ACLF and 38 with severe hepatitis B (SHB) were involved in our study. SHB was defined as ALT≥600 IU/1 associated with TB≥3.0 mg/dl and PTA≤50%. Forty four chronic hepatitis B (CHB) patients with ALT level within the range of 80-400 IU/1 and TB≤1.0 mg/dl were enrolled as control. The patients with ACLF and SHB were followed up for average 10.6 months (ranged for 3-17m).Sequences of PreC/C region, HBV genotype was determined. The line epitopes, including Th epitope and CTL epitope, were analyzed by epitope prediction WEB SYFPEITHI and BIMAS. The 3D data of B cell epitopes were presented from the X-ray structure of the HBcAg by Chimera software. The pentamer staining was performed to analyze epitope-specific CTL. Results1) There were significant differences in TB, serum albumin (ALB) and PTA between patients with ACLF and SHB. Creatinine (CR) and white blood cell (WBC) were significantly high in ACLF compared with SHB. Of the 39 patients with ACLF,18 had a fatal outcome and 21 survived more than 3 months after onset of liver failure. Lower PTA and higher WBC were detected as risk factors, while a high CR was only probable as a risk factor for mortality.2) The distribution of genotype B was statistically higher in SHB Patients than that in CHB patients (72.2% vs.50%, P=0.044). And genotype B was found more often in ACLF compared with in CHB, although there was no statistically significant difference (P=0.069).3) The prevalence of BCP (A1762T/G1764A) mutations were found significantly more often in ACLF than in CHB (56.7% vs.32.4%, P=0.046). The rate of PC (G1896A) mutation was higher in ACLF compared with CHB (50% vs.15.9%, P=0.003). Moreover, compared with the distribution of HBV BCP/PC mutation patterns in investigated groups, the patterns of BCP-/PC+and BCP+/PC+ occurred more frequently in ACLF patients than that in CHB patients.4) The HBeAg negative rate increased in a stepwise manner in patients with BCP+/PC-(40%), BCP-/PC-(53.3%), BCP-/PC+(76.5%) and BCP+/PC+ (83.3%) viruses. And the rate of HBeAg negativity was not lowest in patients with BCP-/PC-virus. Furthermore, the rate of HBeAg negativity was compared among CHB, SHB and ACLF. The rate of HBeAg negativity was significantly high in ACLF patients..5) The prevalence of T1846 was found significantly high in ACLF than in SHB [80.6%(25/31) vs.16.7%(5/30), P<0.001] or than in CHB [80.6%(25/31) vs.11.1% (4/36),P<0.001].6) In ACLF patients, the sites with amino acid variability of more than 10% in PreC/C protein included aa5, aa60, aa97, aa83, aa27, aa98 and aa35. And there were statistically significant difference in above amino acids variability compared with ACLF and CHB. Th epitopes and CTL epitopes, were analyzed by epitope prediction WEB SYFPEITHI and BIMAS. The affinity of CTL18-27 to HLA-A*0201 was enhanced when I varied into V at the site of aa27. From the 3D structure of HBc tetramers, the observed major B epitopes (HBcAg76-87 and HBcAg130-135) appeared predominantly on the outer surface of the HBc protein. The one of most variable positions aa135 within the B epitope HBcAg130-135 was the most externally exposed.7) Amino acid mutation in aa5 always accompanied with aa60; and the index of Pearson correlation was 0.632 (P<0.001). aa5 P-T accompanied with aa60 L-V and aa5 T-P with aa60 V-L in followed-up patients. Shown in the 3D structure of HBc tetramers, these two sites closely contacted within the four-helix bundle. Moreover, the affinity of epitope with variant in aa5 to HLA-A*0201 was enhanced, while the affinity of epitope with mutant in aa60 was decreased by epitope prediction WEB.8) The prevalence of V27 was found significantly more often in ACLF than in CHB [10/39 (25.6%) vs.3/44 (6.8%); P=0.019], although there was no statistically significant difference between SHB and CHB [8/38 (21.1%) vs.3/44 (6.8%); P=0.059].9) Ten patients primarily infected with pure V27 or the mixture of 127 and V27 and 19 primarily infected with pure 127 were followed up for at least 3 months in SHB and ACLF groups. In two cases (2/29), the sequence of the HBcAg18-27 encoding region could not be amplified. The remaining 27 patients were including 9 with pure V27 or the mixture of 127 and V27 and 19 with pure 127. These 9 patients with pure V27 or the mixture of 127 and V27 were classified into two groups according to HLA-A2 type. Among 4 HLA-A2 positive cases, V27 was not detected and it turned into 127. On contrary, V27 could still be detected among 5 HLA-A2 negative cases. Amino acid at position 27 was unchanged in 18 cases originally infected with 127, in whom the longest period of follow-up was 17 months. We used clones sequence to verify the virus change.10) The low frequency of epitope-specific CD8+T cells by pentamer staining ex vivo was unavailable in our study, thus the residue PBMCs were stimulated with I27/V27 peptide mixture, and then the pentamer staining was performed. The results of the pentamer staining showed V27 specific CD8 T cells were detected at high level, while the 127 specific CD8 T cells were found to be much less common.11) Mean HBV DNA level was significantly lower in ACLF than in CHB [(5.75±1.73 vs.9.16±1.72) log10 copies/ml; P<0.001], and a significant difference was also found between SHB and CHB [(5.55±1.61 vs.9.16±1.72) log10 copies/ml; P< 0.001]. HBV DNA level was lower in patients with V27 than in ones with 127 in ACLF and SHB, although there was no statistically significant difference [(5.14±1.31 vs.5.80±1.74) log10 copies/ml; P=0.093].Conclusions1) In this study, we found the prevalence of genotype B was 72% and 74% in ACLF and SHB, respectively, which was much higher in CHB. And BCP and PC mutations were more often in ACLF than in CHB. These suggested HBV genotype B and BCP/PC mutations were significantly associated with ACLF.2) The HBeAg negative rate increased in a stepwise manner in patients with BCP+/PC-, BCP-/PC-, BCP-/PC+ and BCP+/PC+ viruses. The loss of HBeAg caused by BCP+/PC+ and BCP-/PC+ was related with the pathogenesy of ACLF.3) Amino acid mutation in aa5 accompanied with aa60. These two sites closely contacted within the four-helix bundle in the 3D structure of HBc tetramers. The affinity of epitope with variant(T) in aa5 to HLA-A*0201 was enhanced by epitope prediction WEB. The above data suggested aa5T was associated with ACLF.4) In HBcAg, the epitope drift or up regulation of immunodominance at multiply sites play a role of the development of ACLF.5) HBcAg18-27 epitope with V27 may be associated with the development of ACLF and CTL response produced by the epitope involves the process of controlling the virus replication.