Immunogenicity of G145R mutant HBsAg "a" determinant with synthetic peptides
Objective To study the change of antigenicity and immunogenicity of G145R mutant HBsAg. Methods Two peptides were synthesized which were specific to HBsAg "a" determinant. P1 wt represents wild type and P2 145R represents G145R mutant. The conformation structure was analyzed with 2 ME denature test. Then the antigenicity and immunogenicity of G145R mutant HBsAg were studied with ELISA, competitive inhibition test and Western blot. Results SDS PAGE results revealed that in the absence of reducing agents, both peptides had a heterogeneous mixture of multiple forms with molecular masses ranging from 4 kD to 30 kD and the main bands were at 5 kD and 10 kD, whereas in denatured condition, it showed one band of 4 kD. In a direct ELISA, peptide 1(P1 wt) showed a high degree of cross reactivity with polyclonal anti HBsAg antisera, the highest titer of antisera could reach as high as 1/32 000, whereas the antigenicity of peptide 2(P2 145R) was significantly reduced, that means when the titer of antisera was 1/8000, the result became negative. And 6 weeks after being immunized with these peptides, mice produced a high level of serum antibody against respective peptides such as P1 wt and P2 145R, but the anti peptide antibodies bound to the corresponding native peptide with an apparent affinity higher than the others. Conclusion All of the cysteine sulfhydryl groups may spontaneously form disulfide bonds to yield a conformational, disulfide dependent "a" determinant of HBsAg. And the antigenicity and immunogenicty changed significantly after amino acid mutate at amino acid 145. This result suggest that it is necessary to reevaluate the effect of present hepatitis B vaccine, set up G145R mutant HBV specific diagnostic methods and to develop new vaccines.