The mechanism of antigenic drift in influenza virus: sequence changes in the haemagglutinin of variants selected with monoclonal hybridoma antibodies

Abstract

Antigenic variants of the A/PR8 (H0N1) and A/Hong Kong/68 (H3N2) strains of influenza virus were isolated after a single passage of these viruses in the presence of monoclonal hybridoma antibodies to the haemagglutinin. Hyperimmune rabbit antisera reacted (in haemagglutination-inhibition tests) to high titre with both wild-type and variant viruses, but the monoclonal antibodies, which reacted with the wild-type virus to titres of the order of 1/10 5 did not react at all (or to very low titre) with the variants that they selected. This suggests that the changes occurring in the monoclonal variants are restricted to a single antigenic site out of many on the haemagglutinin molecule. Amino acid analysis of the soluble tryptic peptides from the haemagglutinin ‘spikes’ of wild-type and variant viruses suggest that the dramatic loss in the ability of the variants to bind the monoclonal antibody used in their selection is associated with a single change in the amino acid sequence of the large haemagglutinin polypeptide, HA 1 . For PR8 virus, eight out of ten variants selected with one monoclonal antibody showed the same sequence change of serine to leucine in the HA 1 polypeptide. The change in the other two variants was not determined. No sequence data on PR8 haemagglutinin are available, so the experiments were continued with a Hong Kong (H3N2) strain where much of the sequence of HA 1 and HA 2 is known. Three different monoclonal hybridoma antibodies to A/Mem/1/71 (H3N2) haemagglutinin were used to select a total of ten variants of this virus. Variants selected with one monoclonal antibody were not recognized by the other two monoclonal antibodies as being different from wild-type virus, suggesting that the three antibodies bound to different sites on the surface of the haemagglutinin molecules. Each of the variants occurred with a frequency of about 1 in 105 in the wild-type virus. One group of our variants selected with H14/A2 monoclonal antibody showed the same antigenic properties and the same sequence change (asparagine to lysine) in the N-terminal half of HA 1 . Of three variants selected with H14/A20, two showed a different change at a locus also in the N-terminal region of HA 1 (a proline was replaced by serine in one variant and by leucine in the other). Of the other three variants (selected with H14/A21 monoclonal antibody) one showed a change in HAX of serine to tyrosine. This change occurred in residue number 37 of cyanogen bromide fragment 2 (CN2). In the other two variants the change in HAX has not been determined, but in these a tryptic peptide comprising residues 49-56 of CN2 was missing. The tryptic peptides of the HA 1 polypeptide, showing changes in the variants selected with monoclonal antibodies, were also found to undergo sequence changes in naturally occurring Hong Kong variants isolated from man. In each case, however, the sequence changes in the monoclonal variants were different from those in the field strains. No changes were found in the HA 2 polypeptide from any of the variants.