Investigation On The Molecular Basis Of Immune Escape Mutations Within The Surface Antigen Of Hepatitis B Virus

The hepatitis B virus (HBV) caused severe liver disease, including acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. More than350million people worldwide infected with hepatitis B virus. The surface antigen of hepatitis B virus is an important diagnostic marker, and its amino acid sequence contains a highly hydrophilic domain, locating in the region of amino acid99-169, which termed as the major hydrophilic region (MHR). The "a" determinant (amino acids124-147) within the MHR serves as the most important epitopes of HbsAg, stimulating B cells to produce antibodies, and involving in surface antigen diagnosis. The binding of the antibody after successful vaccination to the "a" determinant provides immune protection.Escape mutations in the major hydrophilic region (MHR) of hepatitis B surface antigen (HBsAg) are reported widely worldwide; these mutations lead to diagnostic problems, emergence of vaccine-escape mutants, and hepatitis B immunoglobulin (HBIG) therapy failure. However, the prevalence of these mutations in different genotypes remains to be studied systematically. In the current study,11,221non-redundant hepatitis B virus(HBV) sequences of8genotypes(from A to H), obtained from the National Center for Biotechnology Information(NCBI),were analyzed to determine the prevalence of HBsAg escape mutations that were previously described. Eight important mutations associated with diagnostic failure, P120T, T126S, Q129H, G130N, S143L, D144A and G145A/R, were prevalent in one or more genotypes, with the frequency of no less than1%. With regard to escape variants that evade vaccine or immunoglobulin therapy, mutations were located mainly at positions120,126,129,130,133,134,137,140,143,144and145. The majority of such mutations showed genotypic heterogeneity, indicating the different distribution of the escape mutations. Most of the escape mutations clustered in the "a"determiniant,indicating that this region was more likely to be affected by immune selection or antiviral therapy than other regions. Understanding the prevalence and heterogeneity of escape mutations could provide useful guidance for the improvement of diagnostic assays,design of new vaccines, and prevention of failure of HBIG therapy.In addition, this study investigated the distribution of the immune escape mutatons in the surface antigen across the natural history of hepatitis B virus. A total of206patients were divided into four periods according to the natural history of hepatitis B virus, which were immune tolerance (IT)(N=62) phase, immune clearance phase (IC)(N=60), the inactive phase (IN)(N=40) and HBeAg-negative hepatitis (ENH)(N=44) phase. First we compared the frequency of mutations within the Th epitopes among the four groups in the natural history. The difference was significantly different (p<0.05) when comparing the mutations locating in the regions17-31,37-51,67-81, while there was no statistical differnce among other regions such as139-146,165-179,176-190(p>0.05). We next compared the frequences of mutations among the surface antigen between two groups, and we discovered the difference was the same as the the four groups when comparing the HBeAg-positive samples, which were divided into IT and IC groups. While there was no difference between the IN and the ENH group (p>0.05). In addition, significant difference was also discovered between the two groups before and after seroconversion (IC and IN)(p<0.05).When analyzing the frequency of mutations locating in CTL epitopes among the four groups in the natural history, the significant difference was found locating in regions14-22and41-49(p<0.05). There was no significant difference among the four groups in regions95-109,150-158,190-197and196-206(p>0.05). Further analysis also showed differences was found between HBeAg-positive samples(p<0.05), while no difference in HBeAg negative samples(p>0.05). This result indicated that mutations within the immune epitopes accumulated in the IC phase across the the natural history, indicating a potential role in evading the immune system attacks. There was no significant increase in the frequency of mutations in immune epitopes within surface antigen across other stages. Additionally, in this study, immune escape mutations in B cell epitopes have been found across the the four periods in the natural history of hepatitis B virus, namely Q101K, P120S, T126S, Q129H, M133L, M133I, and the M133T. The frequency of such escape mutations were almost all the same as what we predicted in the Chapter Two,confirming the results of our analysis.