An analysis of the properties of monoclonal antibodies directed to epitopes on influenza virus hemagglutinin

Abstract

Monoclonal antibodies (MAbs) specific for the hemagglutinin (HA) of the H3 subtype of influenza A virus were grouped according to their inability to bind to particular MAb-selected neutralization escape mutants of the virus having an amino acid substitution in one of the five postulated antigenic sites on the molecule. Additional residues critical to the binding of the MAbs were deduced from their patterns of reactivity with a panel of field strains and receptor mutants of the H3 subtype. The relationship of these residues to the actual epitopes recognized by the MAbs was inferred from their location on the three-dimensional structure of the HA molecule. In this way it was generally possible to identify a number of residues that are critical to the integrity of the epitope recognized by each of the MAbs examined. It was found that: (1) Several of these epitopes appear to be discontinuous and some may depend on residues contributed by more than one monomer. For example, residue 205, in the interface between monomers of the HA, was found to affect the integrity of the epitopes for several MAbs, possibly by stabilizing the conformation of residues around the receptor-binding pocket and/or in site B on the adjacent monomer. The activity of these particular MAbs was greatly decreased if the virus was exposed to pH 5. (2) All the MAbs tested neutralized viral infectivity and inhibited hemagglutination, although the single MAb directed to site C, which is the most distant from the receptor-binding site, was the least efficient. (3) Hemagglutination inhibition, and particularly neutralization tests, were more discriminating than ELISA in discerning subtle differences between the corresponding epitopes recognized by MAbs on different field strains. (4) Efficiency of neutralization of infectivity did not correlate consistently with hemagglutination inhibiting efficiency; MAbs postulated to bind to epitopes close to the receptor-binding pocket were very efficient at inhibiting hemagglutination, whereas neutralization efficiency tended to be more influenced by the affinity of binding of the MAb. (5) A MAb binding to any particular epitope could affect the binding of a second MAb directed to an epitope within the same or even a different antigenic site. The observed effect was most commonly inhibition of binding, which was not always reciprocal; enhancement of binding was also observed with certain combinations of MAbs. The relative affinity of the MAbs, in addition to steric constraints, were shown to be important factors in the ability to compete for interaction with HA.

Sequential Seasonal H1N1 Influenza Virus Infections Protect Ferrets against Novel 2009 H1N1 Influenza Virus

“…Binding of antibodies found in different sera might be cooperative. Cooperativity of binding between different anti-HA antibodies has been observed (36,37). In addition, the effects of bound antibodies on hemagglutination, and on protection in vivo, might be synergistic.…”

Neutralization escape mutants of type A influenza virus are readily selected by antisera from mice immunized with whole virus: a possible mechanism for antigenic drift

“…The finding of an amino acid substitution in site A distinguishes our mutants from adsorptive non-antigenic mutants derived by others using mixtures of MAbs (Yewdell et al, 1986) and confirms our mutants as neutralizing antibody escape mutants. The minor changes in the Group II escape mutants are reminiscent of conformational rearrangements which are thought to accompany an amino acid substitution at another site (Brown et al, 1990), and have been noted by others (Laver et al, 1981). The ability of a Group I escape mutant to react with its selecting antiserum was reduced with respect to wt virus R. Lambkin and others from 50 to 75%, consistent with the escape mutation conferring upon them a selective advantage in vivo.…”

High and low efficiency neutralization epitopes on the haemagglutinin of type A influenza virus.

“…Values obtained by equilibrium filtration with polyclonal anti-influenza virus antibody averaged 10 −* M (Fazekas de St Groth & Webster, 1963), while data obtained by radioimmunoassay with MAbs gave values up to 10 −"! M (Frankel & Gerhard, 1979) and for 12 different MAbs averaged 6i10 −* M (Brown et al, 1990). Values for neutralizing IgG antibodies specific to viruses other than influenza, and assayed by ELISA with virions or virus proteins ranged from 1n2i10 −"!…”