Neutralization escape mutants define a dominant immunogenic neutralization site on hepatitis A virus

Abstract

Hepatitis A virus is an hepatotrophic human picornavirus which demonstrates little antigenic variability. To topologically map immunogenic sites on hepatitis A virus which elicit neutralizing antibodies, eight neutralizing monoclonal antibodies were evaluated in competition immunoassays employing radiolabeled monoclonal antibodies and HM-175 virus. Whereas two antibodies (K3-4C8 and K3-2F2) bound to intimately overlapping epitopes, the epitope bound by a third antibody (B5-B3) was distinctly different as evidenced by a lack of competition between antibodies for binding to the virus. The other five antibodies variably blocked the binding of both K3-4C8-K3-2F2 and B5-B3, suggesting that these epitopes are closely spaced and perhaps part of a single neutralization immunogenic site. Several combinations of monoclonal antibodies blocked the binding of polyclonal human convalescent antibody by greater than 96%, indicating that the neutralization epitopes bound by these antibodies are immunodominant in humans. Spontaneously arising HM-175 mutants were selected for resistance to monoclonal antibody-mediated neutralization. Fourteen clonally isolated mutants demonstrated substantial resistance to multiple monoclonal antibodies, including K3-4C8-K3-2F2 and B5-B3. In addition, 13 mutants demonstrated a 10-fold or greater reduction in neutraliztion mediated by polyclonal human antibody. Neutralization resistance was associated with reduced antibody binding. These results suggest that hepatitis A virus may differ from poliovirus in possessing a single, dominant neutralization immunogenic site and therefore may be a better candidate for synthetic peptide or antiidiotype vaccine development.

Direct detection of hepatitis C virus (HCV) RNA from whole blood, and comparison with HCV RNA in plasma and peripheral blood mononuclear cells

“…PCR products and controls were electrophoresed on 0.8% agarose gels, visualized by ethidium bromide staining, and transferred to nitrocellulose [Sambrook et al, 19891. 32P-dCTP labeling of nested HCV PCR product was performed by random priming a s previously described [Stapleton and Lemon, 19871, and used for hybridization with the Southern transfer [Stapleton and Lemon, 1987;Naides et al, 19931. Specificity was confirmed by including PGEM-3Zf-5'UTR and HCV antibody negative PCR products on the agarose gel.…”

A detailed comparative analysis on the overall codon usage patterns in Hepatitis A virus

“…HAV has been shown to possess a single conserved immunogenic neutralization site, and isolates from different parts of the world belong to a single serotype (Stapleton and Lemon, 1987;Hollinger and Emerson, 2001). Nevertheless, the study of the HAV evolution in cell culture revealed the presence of some antigenic variants in the mutant spectra that were generated even in the absence of immune selection (Sanchez et al, 2003).…”

Recombinant proteins VP1 and VP3 of hepatitis A virus prime for neutralizing response

“…Epitope mapping using peptide antigens was little help in identifying neutralizing antigenic sites because only one of the peptides was reported to induce HAV-neutralizing antibodies [Emini et al, 1985;Wheeler et al, 1986;Gauss-Muller and Deinhardt, 19881. Two immunodominant antigenic sites within VP1 and VP3 were identified by determination of the amino acid sequence of neutralization mutants [Stapleton and Lemon, 1987;Ping et al, 19881. Both sites (residue 70 of VP3, Asp, and residue 102 of VP1, Ser) are parts of knob or loop structures predicted by the three-dimensional model and align with antigenic sites in polio and rhino virus [Luo et al, 19881. In our study, different parts of VP1 and VP3 were expressed and the antigenicity and immunogenicity of the resulting polypeptides were tested, to determine the most probable antigenic sites on these viral proteins.…”

“…Figure 1 depicts the overlapping HAV expression plasmids A to F and their mapping positions along the HAV genome. All plasmids contain one of two amino acids (Asp 70 in VP3 or Ser 102 in VP1) which have been described as immunodominant by analysis of neutralization escape mutants [Stapleton and Lemon, 1987;Ping et al, 19881. P-galactosidase-HAV fusion proteins were expressed after induction with IPTG, separated on SDS-PAGE, and detected by Coomassie blue staining (Table I and Fig.…”