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Abstract
BackgroundSelective 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.MethodsTo 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.ResultsWestern-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.ConclusionsDrug-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.
Highlights
The implementation of the Hepatitis B Immunization program in China resulted in a decrease in the incidence of Hepatitis B virus (HBV) infections, from approximately 10% to 7% in the general population [1]
These results indicated that 293 T cells transfected with either wild-type or Hepatitis B surface antigen (HBsAg) mutants had very comparable levels of HBsAg production (Figure 1)
Negligible decline in the antigenicity of sT118M-rtM204I or sT118M-rtM204V mutant Of the five commercial HBsAg Enzyme-linked immunosorbent assay (ELISA) kits used in this study, four kits (LZ, WT, GBT, and BioNeovan Limited Company (BN)) recognized the sT118M immune-escaped mutant and recombinant sT118M-rtM204I mutant, yielding similar titration curves, and indicating that rtM204I may contribute marginally to the antigenicity of sT118M HBsAg
Summary
The implementation of the Hepatitis B Immunization program in China resulted in a decrease in the incidence of HBV infections, from approximately 10% to 7% in the general population [1]. Selection pressure from the specific immune response, whether from the passive application of hyperimmune globulin (HBIG) prophylaxis [12] or from active HBsAg vaccination [13,14], could drive the emergence of HBsAg mutant viruses. An amino acid change from glycine to arginine at position 145 (sG145R) for the immunodominant determinant of HBsAg, which alone can be responsible for vaccine escape, is most commonly reported and has been well documented [7,15]. Selective pressure from either the immune response or the use of nucleoside analogs in antiviral therapy could be driving the emergence of HBV mutants. 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
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