Figure 1.
Five antigenic sites A–E are mapped on the HA1 surface of H3N2 influenza viruses. (A). Antigenic site A (red color) and antigenic site B (blue color) are localized on the top of HA around the receptor binding pocket. (B). The “190 helix” and “B loop” create antigenic site B. The “A loop” is a part of antigenic site A. Figure 1A was made from PDB ID 2VIR [24] using PyMol (Schrödinger, LLC). Figure 1B was made from an Oklahoma/309 HA structural model made by SWISS-MODEL.
Table 1.
Mutations made in antigenic site A and B of A/Oklahoma/309/2006 HA.
Figure 2.
Expression of wild type and mutant HAs in the baculovirus system.
1. Wild type 309 HA 2. Mutant HL156-157KS 3. Mutant KFK158-160GST 4. NDQI189-192QEQT 5. A196V 6. N133D 7. TSSS135-138GSNA 8. K140I. Supernatant (25 µl) from a 25 cm2 flask of Sf9 cells infected with the recombinant baculoviruses expressing wild type and mutant HA was loaded on a 12% SDS-polyacrylamide gel. HA was visualized by immunoblotting assay with anti-HA tag (YPYDVPDYA) polyclonal antiserum.
Figure 3.
Quantitation of expressed HA by immunoblotting assay using anti-HA-tag antibody.
Different concentrations of standard protein GST-HA tag were visualized in the same membrane as HAs. 1–2, 309 HA (10 µl, 7 µl); 3–4, mutant HA HL156-157KS (10 µl, 7 µl); 5, mock infected; 6–9. GST-HA tag 4 ng, 6 ng, 8 ng, 10 ng, respectively. The bands were scanned and quantitated using ImageQuant software (Molecular Dynamics). Amounts of HA were determined from a standard curve of GST-HA tag fusion protein. Standard curves were built for each sample of HA.
Table 2.
Binding of human monoclonal Abs 1_C02 and E05 to 309 HA and mutants.
Table 3.
Dissociation constants (Kd) for binding of human monoclonal Abs 1_C02 and E05 to H3N2 viruses.
Figure 4.
Analysis of human plasma samples vaccinated in season 2006–07 (H3 component A/Wisconsin/67/05) and season 2008–09 (H3 component A/Uruguay/716/07).
A, B. Overall affinity (Kd ± St. dev.) of antibodies in human plasma against wt 309 HA and mutants. Overall affinity labeled as dissociation constant (Kd) of binding of antibodies in plasma from subjects to wild type and mutants in antigenic site A or B; higher Kd is lower affinity. Kds were measured as described in Materials and Methods and the units are µl plasma in the standard assay. Results are plotted as mean Kd ± standard deviation over 3 experiments. C, D. Relative Kd of antibodies in human plasma against mutants relative to binding of wild type A/OK/309/06 HA. Kds of binding of plasma antibodies to mutants HA are calculated by normalizing Kd of wild type 309 HA to 1.0.
Table 4.
Decreased binding to site A and B mutants by human plasma after vaccination.
Figure 5.
Overall dissociation constants (Kd) of antibodies in human plasma after vaccination.
Kd of antibodies in season 2006–2007 (A) and season 2008–2009 (B). Plasma samples were tested against H3 vaccine viruses A/Wisconsin/67/05 (2006–2007 vaccine) and A/Uruguay/716/07 (2008–2009) and against escape mutant viruses EM 1_C02, EM E05. The median Kd is represented by a horizontal bar, and the p values are from the Student's T test.
Table 5.
Differences in HA1 sequence between Wisconsin/05 and Uruguay/07 PR8 reassortant vaccine strains.