Selection of a Multiple Drug-Resistant Hepatitis B Virus Strain in a Liver-Transplanted Patient

Background & Aims: Sequential anti-hepatitis B virus (HBV) therapy may lead to the selection of complex mutants. We analyzed the genetic and phenotypic evolution of the viral quasispecies of a patient who received successively lamivudine, add-on adefovir+lamivudine, followed by lamivudine+adefovir+hepatitis B immunoglobulins (HBIg) after orthotopic liver transplantation. Methods: For genotypic analysis, a 1310-bp region of the polymerase gene was amplified, cloned, and sequenced. Huh-7 cells were transfected to compare the replication fitness of HBV mutants and their susceptibility to drugs. Results: At baseline, all HBV genomes carried a wild-type (wt) RT gene but 22% harbored the sP120S and 55% the sC107stop mutations within the surface (S) gene associated with vaccine escape. Following viral breakthrough to lamivudine monotherapy, a complex mixture of lamivudine-resistant HBV strains prevailed. Interestingly, among these mutants emerged a population harboring only the rtL180M+A181V mutations, conferring lamivudine-resistance in vitro. After addition of adefovir to the ongoing treatment, viral load dropped, and the patient underwent an orthotopic liver transplantation and received HBIg. As viral load rose again, a single viral population was progressively selected, harboring the rtV173L+L180M+A181V+N236T and sP120S mutations. In vitro, this last mutant showed a level of replication reduced by only 30% compared to wt HBV and a strong resistance to both lamivudine (>1000-fold) and adefovir (>10-fold). It remained sensitive to tenofovir both in vitro and in vivo. Conclusions: We report the selection of a complex HBV mutant that escaped the antiviral pressure of lamivudine, adefovir, and HBIg, and provide insight on the process of selection via genotypic and phenotypic analysis.