Identification of a Hepatitis B Virus S Gene Mutant in Lamivudine-Treated Patients Experiencing HBsAg

SeroclearanceCHAO-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

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 analogue2. 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

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

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

Question:

Whether the clearance of HBsAg after long-term lamivudine therapy is truly due to 1. eradication of the virus2. 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.

Amino Acid Substitutions in Patients With HBsAg Seroclearance After Lamivudine

Therapy

RNA Northern blot

Lanes 1, 6: RNA of Huh-7 cells + pRc/CMVLanes 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/CMVLanes 5, 10: DNA of pCMV-S

Characterization of mutant HBsAg

Western blot of Huh-7 cells

Detection of secreted HBsAg

Antibodies from:MO1,2: recombinant HBsAg derived from yeast MO3: culture fluid of human culture cells expressing HBsAgMO4: purified native HBsAg

Discussion:

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

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.

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.