1. Identification of a Hepatitis B Virus S Gene Mutant in Lamivudine-Treated Patients Experiencing HBsAg Seroclearance CHAO-WEI HSU, CHAU-TING YEH, MING-LING CHANG, and YUN-FAN LIAW Liver Research Unit, Chang Gung Memorial Hospital, Taipei, Taiwan GASTROENTEROLOGY 2007;132:543–550 Speaker: 彭佳琇

2. Chronic HBV infection Severe sequelae (cirrhosis and hepatocellular carcinoma) It has been estimated that there are currently 350 million patients chronically infected with hepatitis B virus (HBV) worldwide. Lamivudine 1. Is a nucleoside analogue 2. Potent inhibitory effect (the RNA- and DNA-dependent DNA polymerase activities of HBV polymerase) 3. It is able to suppress viral replication in the majority of patients Introduction

3. Prolonged usage of lamivudine is therefore required, whereby mergence of resistance mutants casts a challenge for hepatologists. The lamivudine- resistant mutants were discovered to have characteristic amino acid substitutions over the YMDD motif of the HBV polymerase. YMDD locus （ tyr-met-asp-asp ） methionine → valine （ YVDD ） methionine → isoleucine （ YIDD ） Introduction

4. Because the HBV polymerase gene overlaps with the surface gene, mutations of HBV polymerase sometimes alter the amino acid codons of the surface gene. Substitutions of the amino acid residues in the surface gene have been reported to alter the antigenicity of surface antigen (HBsAg). Introduction

5. Question: Whether the clearance of HBsAg after long-term lamivudine therapy is truly due to 1. eradication of the virus 2. merely because of the detection failure of HBsAg In this report, they have analyzed the surface gene sequences in patients who experienced seroclearance of HBsAg after lamivudine therapy.

7. Amino Acid Substitutions in Patients With HBsAg Seroclearance After Lamivudine Therapy

8. RNA Northern blot Lanes 1, 6: RNA of Huh-7 cells + pRc/CMV Lanes 2, 7: RNA of Huh-7 cells + pCMV-Smt (mutant surface antigen) Lanes 3, 8: RNA of Huh-7 cells + pCMV-S (wild-type surface antigen) Lanes 4, 9: DNA of pRc/CMV Lanes 5, 10: DNA of pCMV-S Characterization of mutant HBsAg

9. Western blot of Huh-7 cells

11. Detection of secreted HBsAg Antibodies from: MO1,2: recombinant HBsAg derived from yeast MO3: culture fluid of human culture cells expressing HBsAg MO4: purified native HBsAg

12. Discussion: A polymerase mutation (rtM204I) occurred in patient 9, which was likely responsible for the higher HBV-DNA level in this patient.

13. Altered Antigenicity in sP120A The “ a ” determinant amino acids 124 – 147 has been designated to a hydrophilic region at, a region located between the transmembrane domain II and III of surface protein. 120 was upstream of the “ a ” determinant, several studies indicated that mutations in this upstream region altered the antigenicity of HBsAg  affecting the correct folding or topology of the “ a ” determinant, resulting in detection failure.

14. Conclusion: 1.The sP120A mutant was not directly selected by lamivudine as a drug-resistant mutant 2.Seroclearance of HBsAg during lamivudine therapy may not indicate viral clearance. 3.Specifically, it may be caused by a point mutation in the S gene, which results in detection failure. 4.In such patients, further verification and follow-up using a sensitive HBV-DNA test are advised.

15. Lamivudine A nucleoside analogue with a potent inhibitory effect on both the RNA- and DNA dependent DNA polymerase activities of HBV polymerase, is able to suppress viral replication in the majority of patients. Relapse of hepatitis occurs shortly after withdrawal of the drug, even after hepatitis B e antigen (HBeAg) seroconversion. The lamivudine-resistant mutants were discovered to have characteristic amino acid substitutions over the YMDD motif of the HBV polymerase