Localization of epitopes recognized by monoclonal antibodies that neutralized the H3N2 influenza viruses in man

Abstract

Through extensive isolation of neutralizing mAbs against H3N2 influenza viruses representing the in vivo repertoire in a human donor, we examined the relationships between antigenic drift of influenza virus and protective antibodies generated in an infected individual. The majority of mAbs isolated from a donor born in 1960 were divided into three major groups with distinct strain specificity: 1968-1973, 1977-1993 and 1997-2003. In the present study, we developed a new method that allowed us to comprehensively determine the location of epitopes recognized by many mAbs. Original haemagglutinins (HAs) of several strains and chimaeric variants, in which one of the seven sites (A, B1, B2, C1, C2, D or E) was replaced by some other strain-derived sequence, were artificially expressed on the cell surface. The binding activity of mAbs to the HAs was examined by flow cytometry. By using this method, we determined the location of epitopes recognized by 98 different mAbs. Clones that neutralize the 1968-1973 strains bind to site B2/D, A or A/B1. While sites C, E and B were recognized by clones that neutralized the 1977-1993 strains, the majority of these clones bind to site C. Clones that neutralize the 1997-2003 strains bind to site B, A/B1, A/B2 or E/C2.

Fig. 1.

Amino acid sequences of the seven antigenic sites in template and chimaeric HAs constructed in this study. (a) HAs of five vaccine strains, Aichi/68 (Aic68), Yamanashi/77 (Yam77), Fukuoka/85 (Fuk85), Sydney/97 (Syd97), Wyoming/2003 (Wyo03), were used as starting materials. The deduced amino acid sequences showed discrepancies with the reported data (Okada et al., 2010) at six residues, which are highlighted in grey. Since we used these plasmids for the expression of HA the actual sequence determined is indicated in this figure. The name of the chimaeric HA and the amino acids that replaced the original ones are indicated under the template HA. Bars indicate the same amino acids as those of Aichi/68. (b) Location of the amino acids replaced in the chimaeric HA is indicated in the 3D structure of HA by using the examples of the Sydney/97 strain. The amino acids that formed a receptor-binding site were marked in grey. The amino acids replaced in the chimaera were marked as follows: site A, purple (121A), orange (131A) or red (142A); B1, blue; B2, light blue; C1, green; C2, light green; D, pink; E, yellow.

Fig. 2.

Attachment of reticulocytes to the cells expressing wild-type (wt) or chimaeric HAs of Aichi/68 influenza strain. 293T cells were transfected with plasmid containing wt or chimaeric HA genes of the Aichi/68 stain and incubated with guinea-pig red blood cells. As a control, plasmid without the HA gene was used.

Fig. 3.

FCM analyses of wt and chimaeric HAs of five influenza strains. F49 is a mouse mAb that recognizes an epitope commonly present on HA of H3 subtype influenza viruses (Hay et al., 2001). The Fab-PP form of Ab was used for F033-161 (a), F032-072 (b) and F008-007 (c).

Fig. 4.

Summary of FCM analyses of 98 mAbs that bound to HA and showed neutralizing activity. Plus (+) indicates that the Ab reacted with HA expressed on the cell surface. Minus (−) indicates that the Ab did not react with the HA expressed on the cell surface at all. Blanks indicate that the analysis was not performed. Recognized site: the location of the epitope recognized by respective clones. (a) The first set of clones, (b) the second set and (c) the third set.

Fig. 5.

Comparison of the sites assigned by the EMAC method with those determined by the isolation of escape mutants. (a) Amino acid sequences of Wyoming/03 HA, those replaced in the chimaeric HA and those in the escape mutants are indicated. Bars indicate the same amino acid as those of Wyo/03. (b) Locations of the epitopes identified by the EMAC method and by the isolation of escape mutants are indicated in the 3D structure of HA. The illustrations were constructed by using the structure of H3 HA [Protein Data Bank (PDB) accession code 1HA0] according to molecular graphics viewer RasMol 2.7.5. Amino acids indicated in various colours correspond to those used in (a).

Fig. 6.

The antigenic site of HA recognized by 98 mAbs that showed binding and neutralizing activity against H3 influenza viruses. Illustrations of the 3D model were constructed in the same way as described in Fig. 5(b). The amino acids that formed a receptor-binding site are marked in grey. When loss of Ab binding was observed in some chimaeric HA, the amino acids replaced in the chimaera were marked in the same way as in Fig. 1(b).