The prevalence of hepatitis B virus vaccine escape mutants has increased as a consequence of the introduction of global vaccination programs. Furthermore and as a consequence of the organization of the genome of hepatitis B virus (HBV) into overlapping reading frames, the selection of polymerase mutants during long-term lamivudine therapy can select viruses with changes in the overlapping S gene coding for the hepatitis B small antigen (HBsAg). We have investigated the role of lamivudine in selecting HBV mutants with antigenically altered HBsAg protein using pooled human vaccine sera in enzyme immunosorbent assays and radioimmunoassays. HBsAg proteins containing the vaccine escape mutations G145R and D144E/G145R demonstrated markedly reduced binding to anti-HBs antibody. HBsAg mutants including E164D, W196S, I195M, M198I, and E164D/I195M (corresponding to the polymerase protein changes of V519L, M550I, L526M/M550V V553I, and V519L/L526M/M550V) selected during lamivudine treatment also demonstrated reduced binding to anti-HBs antibody. These findings raise the possibility of lamivudine-resistant mutants arising that possess antigenically distinct HBsAg proteins.
The introduction of lamivudine (LMV) for the treatment of chronic hepatitis B infection has been an important advance in the management of this disease. However, the long-term efficacy of LMV may become limited by the emergence of antiviral-resistant hepatitis B virus (HBV) mutants. The two most common LMV-resistant mutants produce changes in the viral polymerase protein (rt) of rtM204I and rtL180M/M204V (previously rtM550I and rtL526M/M550V). A number of studies have demonstrated that these HBV mutants appear to be replication impaired, both in vitro and in vivo. The detection and selection of compensatory mutations in the polymerase protein that restore the replication phenotype of these HBV mutants have been poorly described to date. The effects of mutations in the fingers subdomain of the viral polymerase protein arising as a consequence of vaccine and hepatitis B immune globulin (HBIg) selected changes in the overlapping envelope gene (S), and a determinant of the hepatitis Bs antigen (HBsAg) were analyzed in vitro. The LMV-resistant HBV mutants rtM204I and rtL180M/M204V produced substantially weaker HBV DNA replicative intermediate signals by Southern blot analysis and less total intracellular HBV DNA by real-time PCR compared to wild-type virus. The viral polymerase protein of these mutants produced little detectable radiolabeled HBV DNA in an endogenous polymerase assay. In contrast, the HBV a determinant HBIg/vaccine escape mutants sP120T, sT123N, sG145R, and sD144E/G145R (that produce rtT128N, Q130P, rtW153Q, and rtG153E respectively) yielded as much virus as wild-type HBV while the sM133L (rtY141S) mutant was replication impaired. Two of these mutants, rtT128N and rtW153Q, when introduced into a replication-competent HBV vector containing the rtL180M/M204V polymerase mutation restored the replication phenotype of this LMV-resistant mutant. These viruses produced levels of intracellular HBV DNA as determined by Southern blot and real-time PCR that were comparable to those of wild-type HBV, indicating that the changes in the fingers subdomain were able to compensate for the reduced replication of the LMV-resistant mutations. Since these viruses carry mutations in the a determinant of HBsAg that may potentially decrease the ability of anti-HBs antibody to neutralize these viruses, these HBV mutants also have the potential to behave as vaccine escape mutants.
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