Yeast expression and DNA immunization of hepatitis B virus S gene with second-loop deletion of alpha determinant region.
OBJECTIVE
Immune escape mutations of HBV often occur in the dominant epitope, the second-loop of the a determinant of hepatitis B surface antigen (HBsAg). To let the hosts respond to the subdominant epitopes in HBsAg may be an effective way to decrease the prevalence of immune escape mutants. For this reason, a man-made clone of HBV S gene with the second-loop deletion was constructed. Its antigenicity was evaluated by yeast expression analysis and DNA immunization in mice.


METHODS
HBV S gene with deleted second-loop, amino acids from 139 to 145, was generated using splicing by overlap extension. HBV deleted S gene was then cloned into the yeast expression vector pPIC9 and the mammalian expression vector pcDNA3 to generate pHB-SDY and pHB-SD, respectively. The complete S gene was cloned into the same vectors as controls. The deleted recombinant HBsAg expressed in yeasts was detected using Abbott IMx HBsAg test kits, enzyme-linked immunoadsorbent assay (ELISA) and immune dot blotting to evaluate its antigenicity in vitro. The anti-HBs responses to DNA immunization in BALB/c mice were detected using Abbott IMx AUSAB test kits to evaluate the antigenicity of that recombinant protein in vivo.


RESULTS
Both deleted and complete HBsAg were successfully expressed in yeasts. They were intracellular expressions. The deleted HBsAg could not be detected by ELISA, in which the monoclonal anti-HBs against the alpha determinant was used, but could be detected by Abbott IMx and immune dot blotting, in which multiple monoclonal anti-HBs and polyclonal anti-HBs were used, respectively. The activity of the deleted HBsAg detected by Abbott IMx was much lower than that of complete HBsAg (the ratio of sample value/cut off value, 106+/-26.7 vs 1 814.4+/-776.3, P<0.01, t = 5.02). The anti-HBs response of pHB-SD to DNA immunization was lower than that of complete HBV S gene vector pHB (the positive rate 2/10 vs 6/10, 4.56+/-3.52 mIU/mL vs 27.60+/-17.3 mIU/mL, P = 0.02, t = 2.7).


CONCLUSIONS
HBsAg with deleted second-loop of the alpha determinant still has antigenicity, and can also raise weak anti-HBs response in mice to DNA immunization, suggesting that it is possible to develop a subdominant vaccine for preventing infections of immune escape mutants of HBV.
Hu
Hui
H
Department of Infectious Diseases, the Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510630, Guangdong Province, China.
Peng
Xiao-Mou
XM
Huang
Yang-Su
YS
Gu
Lin
L
Xie
Qi-Feng
QF
Gao
Zhi-Liang
ZL
D000818:Animals; D000939:Epitopes; D005260:Female; D015966:Gene Expression Regulation, Fungal; D006509:Hepatitis B / Q000235:genetics* / Q000276:immunology / Q000821:virology; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D007114:Immunization; D051379:Mice; D012150:Polymorphism, Restriction Fragment Length; D011994:Recombinant Proteins / Q000235:genetics* / Q000276:immunology; D017384:Sequence Deletion; D019444:Vaccines, DNA; D015003:Yeasts / Q000235:genetics*
D000939:Epitopes; D006514:Hepatitis B Surface Antigens; D011994:Recombinant Proteins; D019444:Vaccines, DNA