Antiviral resistance mutations potentiate hepatitis B virus immune evasion through disruption of its surface antigen a determinant

Background: The hepatitis B virus (HBV) pol gene overlaps the S gene encoding surface antigen (HBsAg). It has been reported previously that drug-induced changes in HBsAg alter its binding to sera from humans immunized against HBV. We investigate here the changes to specific epitopes in the a determinant (the major target of neutralizing antibody) caused by a number of drug-resistant mutations. Methods: Recombinant HBsAgs, produced by transfection of Chinese hamster ovary cells with S gene plasmids into which lamivudine, adefovir and entecavir resistance and common antibody-escape mutations had been introduced, were probed with monoclonal antibodies to epitopes in the first and second loops of the a determinant. Results: The mutations rtF166L/sF158Y (lamivudine-associated, compensatory) and rtI169T/sF161L (entecavir-associated, primary) acting alone, and the mutations rtV173L/sE164D (lamivudine-associated, compensatory) and rtSilent/sD144E (antibody escape-associated) each when combined with rtM204V/sI195M (lamivudine-associated, primary) led to decreases in antibody reactivity to epitopes in the first or second loop, or in both loops. The rtM204V/ sI195M+rtV173L/sE164D mutations yielded an epitope-antibody profile similar to the rtR153Q/sG145R vaccine escape mutant. The rtM204V/sI195M mutation combined with the rtF166L/sF158Y or rtR153Q/sG145R mutation restored reactivity to second-loop epitopes previously abrogated by single mutations. Conclusions: Mutations associated with resistance to nucleos(t)ide analogue therapy, singly or in combination with each other or antibody escape-associated mutations, alter HBsAg immunoreactivity through concomitant amino acid substitutions at codons within and downstream of the a determinant. The findings have implications for understanding the native structure of HBsAg, optimizing treatment of chronic hepatitis B and evaluating the success of immunization programmes.