Diverse antigenic site targeting of influenza hemagglutinin in the murine antibody recall response to A(H1N1)pdm09 virus

Abstract

Here we define the epitopes on HA that are targeted by a group of 9 recombinant monoclonal antibodies (rmAbs) isolated from memory B cells of mice, immunized by infection with A(H1N1)pdm09 virus followed by a seasonal TIV boost. These rmAbs were all reactive against the HA1 region of HA, but display 7 distinct binding footprints, targeting each of the 4 known antigenic sites. Although the rmAbs were not broadly cross-reactive, a group showed subtype-specific cross-reactivity with the HA of A/South Carolina/1/18. Screening these rmAbs with a panel of human A(H1N1)pdm09 virus isolates indicated that naturally-occurring changes in HA could reduce rmAb binding, HI activity, and/or virus neutralization activity by rmAb, without showing changes in recognition by polyclonal antiserum. In some instances, virus neutralization was lost while both ELISA binding and HI activity were retained, demonstrating a discordance between the two serological assays traditionally used to detect antigenic drift.

Keywords: A/H1N1/pdm09; Antigenic drift; Antigenic site; Epitope mapping; Influenza; Monoclonal antibody; Virus neutralization.

Fig. 1. Epitope mapping using a recHA point mutation panel

A panel of 20 respective single point mutations in recHA was constructed. (A) Sequence changes are shown; (B) Their locations are indicated on the 3D structure of HA.

Fig. 2. Epitope map of rmAb as determined by Biolayer Interferometry (BLI)

(A) rmAb binding affinity to each mutant HA and the percent response compared to wild-type CA/07 HA was determined. A greater than 50% reduction in binding activity was the cutoff for significance. (B) Epitopes whose mutations lead to a significant reduction in rmAb binding are indicated on the 3D structure of CA/07 HA.

Fig. 2. Epitope map of rmAb as determined by Biolayer Interferometry (BLI)

(A) rmAb binding affinity to each mutant HA and the percent response compared to wild-type CA/07 HA was determined. A greater than 50% reduction in binding activity was the cutoff for significance. (B) Epitopes whose mutations lead to a significant reduction in rmAb binding are indicated on the 3D structure of CA/07 HA.