Construction and expression of hepatitis B surface antigen escape variants within the "a" determinant by site directed mutagenesis.
BACKGROUND
The antibody response to hepatitis B surface antigen (HBsAg) controls hepatitis B virus infection. The "a" determinant of HBsAg is the most important target for protective antibody response, diagnosis and immunoprophylaxis. Mutations in this area may induce immune escape mutants and affect the performance of HBsAg assays.


OBJECTIVE
To construct clinically relevant recombinant mutant forms of HBsAg and assessment of their reactivity with anti-HBs monoclonal antibodies (MAbs).


METHODS
Wild type (wt) and mutant (mt) HBsAg genes were constructed by site directed mutagenesis and SEOing PCR. The amplified genes were inserted into pCMV6-neo plasmid and transfected in CHO cell line. The expression of wt- and mtHBsAg was assessed by commercial ELISA assays and stable cells were established and cloned by limiting dilution. The recombinant mutants were further characterized using a panel of anti-HBs monoclonal antibodies (MAbs) and the pattern of their reactivity was assessed by ELISA.


RESULTS
Ten HBsAg mutants having single mutation within the "a" determinant including P120E, T123N, Q129H, M133L, K141E, P142S, D144A, G145R, N146S and C147S together with a wt form were successfully constructed and expressed in CHO cells. Reactivity of anti-HBs MAbs with mtHBsAgs displayed different patterns. The effect of mutations on antibody binding differed depending on the amino acid involved and its location within the ''a'' determinant. Mutation at amino acids 123 and 145 resulted in either complete loss or significant reduction of binding to all anti-HBs MAbs.


CONCLUSIONS
Our panel of mtHBsAgs is a valuable tool for assessment of the antibody response to HBV escape mutants and may have substantial implications in HBV immunological diagnostics.
Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, e-mail: forough_golsaz@yahoo.com.
Forough
F
Golsaz Shirazi
Mohammad Mehdi
MM
Amiri
Hamed
H
Mohammadi
Ali Ahmad
AA
Bayat
Azam
A
Roohi
Jalal
J
Khoshnoodi
Amir Hassan
AH
Zarnani
Mahmood
M
Jeddi-Tehrani
Gholam Ali
GA
Kardar
Fazel
F
Shokri
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D016466:CHO Cells; D003412:Cricetulus; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D006509:Hepatitis B / Q000276:immunology*; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000276:immunology*; D006515:Hepatitis B virus / Q000276:immunology*; D006801:Humans; D057131:Immune Evasion / Q000276:immunology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D000914:Antibodies, Viral; D000939:Epitopes; D006514:Hepatitis B Surface Antigens; D011994:Recombinant Proteins