Vaccine- and hepatitis B immune globulin-induced escape mutations of hepatitis B virus surface antigen.

Hepatitis B virus surface antigen (HBsAg) vaccination has been shown to be effective in preventing hepatitis B virus (HBV) infection. The protection is based on the induction of anti-HBs antibodies against a major cluster of antigenic epitopes of HBsAg, defined as the 'a' determinant region of small HBsAg. Prophylaxis of recurrent HBV infection in patients who have undergone liver transplantation for hepatitis B-related end-stage liver disease is achieved by the administration of hepatitis B immune globulins (HBIg) derived from HBsAg-vaccinated subjects. The anti-HBs-mediated immune pressure on HBV, however, seems to go along with the emergence and/or selection of immune escape HBV mutants that enable viral persistence in spite of adequate antibody titers. These HBsAg escape mutants harbor single or double point mutations that may significantly alter the immunological characteristics of HBsAg. Most escape mutations that influence HBsAg recognition by anti-HBs antibodies are located in the second 'a' determinant loop. Notably, HBsAg with an arginine replacement for glycine at amino acid 145 is considered the quintessential immune escape mutant because it has been isolated consistently in clinical samples of HBIg-treated individuals and vaccinated infants of chronically infected mothers. Direct binding studies with monoclonal antibodies demonstrated a more dramatic impact of this mutation on anti-HBs antibody recognition, compared with other point mutations in this antigenic domain. The clinical and epidemiological significance of these emerging HBsAg mutants will be a matter of research for years to come, especially as data available so far document that these mutants are viable and infectious strains. Strategies for vaccination programs and posttransplantation prophylaxis of recurrent hepatitis need to be developed that may prevent immune escape mutant HBV from spreading and to prevent these strains from becoming dominant during the next decennia.

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.

Anti-HBs characteristics after hepatitis B immunisation with plasma-derived and recombinant DNA-derived vaccines.

Hepatitis B surface antigen derived from chronic hepatitis B carriers has been replaced almost completely by recombinant DNA-derived HBsAg for use as hepatitis B vaccine. Similarly, recombinant DNA-derived HBsAg is replacing plasma-derived HBsAg in standard anti-HBs assays. We analysed the influence of a change from plasma-derived HBsAg to recombinant DNA-derived HBsAg on antigen presentation in immunoassays and the characteristics of the anti-HBs antibodies after immunisation. Antigens and/or antibodies were subjected to three types of experiments: (a) binding of 'a'-loop specific monoclonal (anti-S) antibody conjugates to immobilised vaccine-HBsAg; (b) binding of post-vaccination anti-HBs to immobilised (vaccine-)HBsAg and (c) inhibition of HBsAg binding to immobilised monoclonal anti-HBs after pre-incubation with post-vaccination antibodies. Our results show that, in both antigen presentation and anti-HBs binding properties, yeast recombinant HBsAg and related antibodies could be clearly distinguished from plasma-derived HBsAg and related antibodies. Divergent results were also obtained in the inhibition assay with recombinant DNA-derived HBsAg but not with serum HBsAg from the vaccine HBsAg subtype. It is concluded that both antigen presentation in vaccines and in anti-HBs assays can markedly influence the quantitation anti-HBs response. It is suggested that a challenge with an heterologous hepatitis B virus may encounter reduced efficacy of vaccine antibodies.

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.

Epitope mapping and serodiagnosis using Ata- and (Lys)7 peptides.

The general applicability of the new peptide immobilization strategy in which the peptide of interest is N-terminally extended with an acetyl-thio-acetyl group or (poly)-Lys extension during synthesis, has been demonstrated in epitope-mapping experiments and serodiagnosis. Ala-scanning experiments and minimal epitope determination showed that the antigenicity of Ata-extended peptides derived from the human chorionic gonadotropin (hCG) and hepatitis B virus (HBV) amino acid sequence, was superior to the free parent peptides. Further, it could be shown that the choice of the epitope-mapping procedure (peptide in solution or immobilized on a solid support) may lead to a different perception of which residues constitute the epitope. In addition, a time-consuming conjugation process could be circumvented since the ELISA reactivity of BSA-conjugates was comparable to that of Ata-extended peptides. In the serodiagnosis using sera from various HIV-positive individuals, the lysyl-peptide showed a signal/noise ratio 10 times higher than the parent peptide, indicating that sensitivity increased as a result of this N-terminal lysyl tail. In all experiments we observed that antibody detection could be performed at roughly 10 times lower amounts of peptide when N-terminally linked to an Ata-group or lysyl-extension compared to the free parent peptide or the BSA-conjugated equivalent.

The effect of copper on the embryonic development and hatching of Sepia officinalis L.

The influence of copper on embryonic development and hatching of Sepia offinalis was investigated. Copper exerts a profound effect on both hatching stage and time-to-hatching. At high copper concentrations (50-200 ppb Cu2+), the embryos hatch earlier than the controls but have a lower survival potential. No external morphological malformations were found. Whereas copper does not accumulate in the embryo or in the vitellus, it is absorbed by the envelope and/or the chorion.