Discrimination of hepatitis B virus (HBV) subtypes using monoclonal antibodies to the PreS1 and PreS2 domains of the viral envelope

We report the production and characterization of murine anti-PreS2 and anti-PreS1 monoclonal antibodies (mAb) and demonstrate their utility in discriminating hepatitis B virus (HBV) subtypes. On the basis of Western blotting and reciprocal competition binding to HBV virions, at least five distinct epitopes have been identified in the PreS domain: two within the PreSi region and three within the PreS2 region. All PreS2 mAb bind M protein (gp33 and gp36) but only one group binds strongly to M and L proteins (p39 and gp42). This group determinant was mapped to peptide residues 120–145. The second group bound to an endoglycosidase F-sensitive epitope which is defined by a mannose-rich glycan at ASN 123 in the PreS2 region. The third group was mapped to peptide residues 150–174 and was reactive with the M envelope proteins but not L or S proteins on Western blots. All PreS1 mAb bind L protein but not M protein on Western blots. Using these mAb, HBV subtype assays were developed allowing evaluation of the Paris 1975 HBsAg subtype panel members along with other HBsAg-positive specimens. All Paris subtype members (except ayw 2 and ayw 3) could be easily distinguished by differential PreS2 mAb reactivity. The Paris subtypes, adw 2, adw 4 and adr, could be classified as distinct groups by PreS2 and PreS1 mAb binding. Specimens from Hong Kong and the United States classified as adw 2 in the S region fell into two groups based on PreS2 mAb binding: one having reactivity similar to Paris adw 2 subtype and the other having identical reactivity to Paris ayw 1 subtype. Furthermore, some specimens classified as adr in the S region gave similar reactivity to the Paris ayr subtype in the PreS2 and PreS1 regions. One complicating factor in this approach toward subtyping was the discovery that some HBsAg positive sera may contain factors which block PreS epitopes. Grouping of HBV subtypes by PreSi, PreS2, and S mAb reactivity may allow better correlation with groupings based on HBV DNA sequence homology.