Decreased antigenicity profiles of immune-escaped and drug-resistant hepatitis B surface antigen (HBsAg) double mutants.
BACKGROUND
Selective pressure from either the immune response or the use of nucleoside analogs in antiviral therapy could be driving the emergence of HBV mutants. Because of the overlap of the open reading frame (ORF) S for the HBsAg and ORF P for viral polymerase, rtM204I and rtM204V mutations in the polymerase would produce sI195M and sW196S in the HBsAg. The combined effects of immune-escaped mutations (sT118M, sG145K, sG145R) and drug-resistant mutations (rtM204I, rtM204V) on the antigenicity profiles of HBsAg has not been widely explored.


METHODS
To determine the combined effects of immune-escaped and drug-resistant mutants on the antigenicity profiles of HBsAg, recombinant plasmids encoding HBsAg double mutants were constructed using site-directed mutagenesis. The supernatant from each plasmid transfection was analyzed for HBsAg in the western-blotting and five of the most commonly used commercial ELISA kits in China.


RESULTS
Western-blotting assay showed the successful expression of each HBsAg mutant. All five ELISA kits manifested similar avidity, which were demonstrated by the slope of the curves, for the sT118M mutant, and sT118M-rtM204I (sT118M-sI195M) and sT118M-rtM204V (sT118M-sW196S) double mutants, suggesting that drug-resistant YMDD mutants caused negligible losses in the antigenicity of immune-escaped sT118M HBsAg. In contrast, the presence of the rtM204I (sI195M) mutation, but not rtM204V (sW196S) in combination with the sG145K mutation significantly reduced the avidity of sG145K HBsAg. The rtM204I (sI195M) mutation also decreased the antigenicity profiles for sG145R HBsAg.


CONCLUSIONS
Drug-resistant mutations rtM204I (sI195M) and rtM204V (sW196S) caused significant reduction in antigenicity for the immune-escaped HBsAg mutants sG145K and sG145R, which may hamper HBV diagnosis and disease control from HBV blood-transfusion transmissions in China. The development of ELISA kits with a greater sensitivity for drug-resistant and immune-escaped HBsAg warrants further consideration.
Department of Infectious Disease, the First Affiliated Hospital of Nanjing Medical University, Nanjing, China. caixubing@sina.com.
Mingshun
M
Zhang
Guohong
G
Ge
Yonglin
Y
Yang
Xubing
X
Cai
Qiang
Q
Fu
Jie
J
Cai
Zuhu
Z
Huang
D015153:Blotting, Western; D002681:China; D024882:Drug Resistance, Viral; D004797:Enzyme-Linked Immunosorbent Assay; D006509:Hepatitis B / Q000175:diagnosis* / Q000821:virology; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000276:immunology*; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics / Q000276:immunology*; D006801:Humans; D057131:Immune Evasion; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000276:immunology; D020125:Mutation, Missense
D006514:Hepatitis B Surface Antigens; D050505:Mutant Proteins