Impact of “a” determinant mutations on detection of hepatitis B surface antigen (HBsAg) in HBV strains from Chinese patients with occult hepatitis B

Abstract

This study was designed to detect mutations that occur within the "a" determinant in the S gene of the hepatitis B virus (HBV) in patients with occult hepatitis B (OHB), and to analyze the influence of these mutations on expression and reactivity of the hepatitis B surface antigen (HBsAg). Twenty-three certified OHB samples were compared to 32 HBsAg positive samples from patients with chronic hepatitis B. The median HBV DNA levels in the OHB group were significantly lower than those in the control group (P < 0.0001). Mutations within the "a" determinant were analyzed by gene amplification and sequencing. This revealed mixed infections in which clones within a sample displayed either different mutations or mutations in association with clones that exhibited wild type amino acid patterns. Sequencing analysis also showed a significant difference between the proportions of amino acid mutations observed in the OHB and control groups. Seven recombinant S (rS) proteins with corresponding OHB mutations and three wild type alleles were expressed and purified in the Pichia pastoris expression system to preserve conformational attributes, and their reactivity analyzed using six commercial HBsAg assays. The OHB sera were HBsAg nonreactive while the rS proteins with corresponding OHB mutations were universally reactive. Thus, we postulate that the reduced binding affinity between mutated HBsAg and its antibody may not be as important in defining OHB as is the effect of specific mutations in the preS/S region of the genome that affect the synthesis and secretion of the S protein and/or the virion.

Molecular and Serological Characterization of Hepatitis B Virus (HBV)-Positive Samples with Very Low or Undetectable Levels of HBV Surface Antigen

“…In the present study, there was no significant difference between HBsAgNx-negative and HBsAgNx-positive NAT yield or OBI groups in the frequency of samples with substitutions or in the average number of substitutions per sample for the PreS1, PreS2, or S regions (Table 1). In contrast, there were significantly more S substitutions in the OBI samples than in the comparison HSLD group in which HBsAg was readily detectable (Tables 1 and 6), a finding similar to other reports on the increased frequency of MHR substitutions in OBI [16,[22][23][24][25]. HBsAg substitutions, particularly in the 'a' determinant within the MHR, have been associated with the lack of detection by affecting antigen binding to antibodies used in HBsAg immunoassays [16].…”

“…Thus, the observed HBsAgNx-negative results were likely due to very low levels of virus rather than assay failure. A similar conclusion was reported in a study comparing the HBsAg detection of six native samples carrying ten 'a' determinant mutations with recombinant proteins derived from the native samples [25]. Although the native samples were HBsAg-negative by sensitive immunoassays, all ten of the recombinant proteins were detected.…”

Occult hepatitis B infection and hepatocellular carcinoma: Epidemiology, virology, hepatocarcinogenesis and clinical significance

“…Studies have shown that some patients with OBI harbour a higher proportion of mutations in the preS/S region than patients with CHB, which may result in a reduced antigenicity for HBsAg detection or impairment in HBsAg production or secretion. 7,12,[36][37][38][39][40][41] The mutations in pre-S1 may also alter the B and T epitopes that affect immune recognition. 7 However, HBV DNA isolated from individuals with OBI is fully replication competent in vitro, 42 and the virus can also be transmitted via blood transfusion or organ transplantation.…”

Pro-oncogenic, intra host viral quasispecies in Diffuse large B cell lymphoma patients with occult Hepatitis B Virus infection

“…The ‘a’ determinant within the major hydrophilic loop of the S protein contains viral epitopes which are not only targets for neutralizing antibody responses in natural infection and vaccine recipients but also the region detected in most detection assays 34,35 . Hence amino acid substitutions in this region often result in immune escapes mutants, vaccine failures and also evade detection, technically resulting in OBI 17,34,36 . We found mutant T528C (Met125Thr) within this region in all ten of our patients analyzed which may explain HBsAg negativity accounting for OBI.…”