A new HBsAg screening assay designed for sensitive detection of HBsAg subtypes and variants.

The design of a new HBsAg screening assay, the Hepanostika HBsAg Ultra is based on the use of monoclonal antibodies raised against native wild-type HBsAg and reactive with HBsAg in which the common 'a'-determinant is modified by site-directed mutagenesis of four of the cysteine moieties. The design was checked using the same cysteine variants and samples from patients known to be infected with HBsAg variants. The results found were compared with other state-of-the-art commercial screening assays. The design of the Hepanostika HBsAg Ultra enabled detection of all variant HBsAg-positive samples in contrast to the other commercial assays. An additional 980 samples were tested to assess the specificity and sensitivity of the Hepanostika HBsAg Ultra. Screening of presumed negative serum and plasma samples resulted in a specificity of 100%. This makes the Hepanostika HBsAg Ultra the first screening assay with a design able to detect HBsAg variants with high sensitivity and specificity.

Anti-HBs after hepatitis B immunization with plasma-derived and recombinant DNA-derived vaccines: binding to mutant HBsAg.

The G145R mutant of the small S-protein is a major escape mutant of hepatitis B virus observed in natural infection, after immunization and HBIG therapy. In a previous study we found that plasma-derived and recombinant DNA-derived vaccine HBsAg reacted differently with monoclonal antibodies sensitive for the G145R change. In the present study we investigated the binding of polyclonal anti-HBs obtained after immunization with plasma vaccine and recombinant DNA vaccine to synthetic peptides (adw(2), adr) and rHBsAg (HepG2) (ayw(3); wild type and a 145R mutant). Anti-HBs binding to synthetic peptids (25-mers, 7aa overlap) from the "a"-loop was significantly reduced by the G145R substitution and by changing the amino acid sequence from adw(2) into adr. With mutant G145R rHBsAg the inhibitory activity of vaccine anti-HBs was decreased compared to rHBsAg wild type. In general only minor differences were observed between plasma vaccine and recombinant DNA vaccine related antibody responses. However, the individual heterogeneity in epitope specific reactivity with its possible consequences for protection (against escape mutants) is not reflected in an anti-HBs titer by standard anti-HBs assays. The presented differentiation in anti-HBs response after immunization may deliver new tools for evaluation of future vaccines.

Characterization of the reactivity pattern of murine monoclonal antibodies against wild-type hepatitis B surface antigen to G145R and other naturally occurring "a" loop escape mutations.

The hepatitis B surface antigen (HBsAg) "a" domain harbors major B-cell epitopes. Viruses with mutations in this region emerge after vaccination or during hepatitis B immune globulin (HBIg) prophylaxis. A strain with G145R replacement has been almost invariably isolated as a major escape mutant. We investigated mutant antigen-antibody interactions with direct binding assays. G145R and 16 other naturally occurring recombinant HBsAg mutants were expressed in mammalian Cos-1 cells. The reactivity of a panel of 28 murine anti-hepatitis B surface antigen (anti-HBs) monoclonal antibodies to mutant antigens was measured with enzyme immunoassay and expressed as percentage compared with the wild-type (wt) HBsAg signal for each antibody. All point-mutated proteins displayed diffuse intracellular immunofluorescent labeling corresponding to a secretory pathway. Monoclonal antibodies (mAbs) were classified according to different binding patterns. The effect of mutations on antibody binding differs depending on the amino acid involved and on the location within the "a" loop. As expected, most antibodies had absent or negligible binding (<40%), notably with residue 145 replacements. However, we identified antibodies that reacted with conformational epitopes but nevertheless had adequate reactivity (>40%) with all mutant antigens, including G145R. The effect of G145R was more pronounced than that of G145A. A subgroup of antibodies had substantially increased recognition (>120%) of antigens with mutations in the first loop. We demonstrated that antibodies can be selected or combined that react with all mutants investigated, including G145R. These data offer perspectives for improving anti-HBs-based protection against hepatitis B.

Localization of a unique hepatitis B virus epitope sheds new light on the structure of hepatitis B virus surface antigen.

In a search for monoclonal antibodies (MAbs) that can bind hepatitis B virus surface antigen (HBsAg) with amino acid substitutions in the immune dominant 'a' region (escape mutants) we investigated the epitope recognition site of the human MAb 4-7B. Pepscan analysis and experiments with alanine substitution as well as substitutions known from nature pointed to residues 178-186 in the small S protein with the amino acid sequence PFVQWFVGL (key amino acids in bold) as the minimal epitope. Single amino acid substitutions at positions 122(R/K)(d/y), 134(Y/F), 145(G/R), 148(T/A) and 160(K/R)(w/r), representing 'a' region variants in recombinant HBsAg COS-I cells, did not influence binding of MAb 4-7B. Synthetic peptides (residues 175-189) including the 4-7B epitope sequence were able to evoke an anti-HBs response in rabbits. According to established polypeptide models, the 4-7B epitope region is located in the lipid layer of 20 nm HBsAg particles. The present results, however, suggest that residues 178-186 are exposed on the surface of the 20 nm particle. This may change our view of the structure of HBsAg.