Analysis of surface antigen amino acid variations in occult hepatitis B among Indonesian blood donors

Abstract

Hepatitis B virus (HBV) infection presents an example of complex host-pathogen interactions in disease progression. One of hepatitis B outcomes is occult hepatitis B infection (OBI), with its characteristically low HBV DNA titer and serologically undetectable hepatitis B surface antigen (HBsAg) - a diagnostic marker of hepatitis B. OBI poses problems in modern medicine, particularly in blood transfusion and tissue/organ transplantation. With similar transmissibility and pathogenicity as wild type HBV, occult HBV presents potential threat to the community, particularly in hepatitis B endemic areas. Indonesia as a region with moderate-to-high hepatitis B endemicity, also has a multitude of host genetic diversity. This presents an interesting field for investigation of host-pathogen interaction, which may influence the evolution of HBV in specific host background. Previous study found that from a total of 7,913 samples of regular blood donors from various regions of Indonesia, OBI was observed at approximately 10.18% prevalence rate. Research on OBI epidemiology and its molecular background will broaden our understanding of the reciprocal effects between host immunity response and pathogen evolution. The seronegativity of HBsAg in OBI may be caused by several factors, including variations of the HBsAg antigenic determinant. Screening of potentially significant HBsAg variations was performed in this study, utilizing Jameson-Wolf antigenic index calculation and protein tertiary conformation prediction to pick out amino acid variations that showed altered HBsAg antigenicity. Out of 27 novel HBsAg variations, in silico analysis highlighted 12 substitution patterns with notable antigenicity changes - most of which related to alterations of structurally important amino acids. Molecular cloning analysis confirmed the presence of 4 such mutations (sF134S, sC147Y, sS154L, and sW163R), with the existence of quasispecies population in majority of the isolates, indicating long-term chronic infection and/or co-infection of more than one HBV isolate. Other than the direct effect on HBsAg antigenicity, nucleotide changes also affect the translation of HBV polymerase gene, potentially changing the replication ability of the variant HBV. Immunoassay studies to determine the sensitivity of commercial diagnostic assays in detecting variant HBsAg were further investigated in this study. Short synthetic peptides representing the main epitopes of HBsAg - the 'a' determinant region -were tested against three HBsAg diagnostic kits commonly used in Indonesia. However, results of this immunoassay analysis should be further confirmed with development of more suitable assay method. Overall, this study underlined the significance of novel HBsAg variants in OBI and their molecular characteristics. It emphasized the importance of understanding OBI - a potential public health hazard - particularly in hepatitis B endemic regions such as Indonesia. Applications of the results of this study may lead to the development of methods to select for biologically important genomic data, employable in the improvement of hepatitis B management, including diagnostic assay formulation, therapy management, and active inhibition of viral transmission. Following this study, further analysis should be performed to confirm the biological significance of these novel HBsAg variations in both pathogen and host point of views, such as potential effect of HBV variations in altering viral replication capacity and modulating activation of host immune response.