Insight into highly conserved H1 subtype-specific epitopes in influenza virus hemagglutinin

Abstract

Influenza viruses continuously undergo antigenic changes with gradual accumulation of mutations in hemagglutinin (HA) that is a major determinant in subtype specificity. The identification of conserved epitopes within specific HA subtypes gives an important clue for developing new vaccines and diagnostics. We produced and characterized nine monoclonal antibodies that showed significant neutralizing activities against H1 subtype influenza viruses, and determined the complex structure of HA derived from a 2009 pandemic virus A/Korea/01/2009 (KR01) and the Fab fragment from H1-specific monoclonal antibody GC0587. The overall structure of the complex was essentially identical to the previously determined KR01 HA-Fab0757 complex structure. Both Fab0587 and Fab0757 recognize readily accessible head regions of HA, revealing broadly shared and conserved antigenic determinants among H1 subtypes. The β-strands constituted by Ser110-Glu115 and Lys169-Lys170 form H1 epitopes with distinct conformations from those of H1 and H3 HA sites. In particular, Glu112, Glu115, Lys169, and Lys171 that are highly conserved among H1 subtype HAs have close contacts with HCDR3 and LCDR3. The differences between Fab0587 and Fab0757 complexes reside mainly in HCDR3 and LCDR3, providing distinct antigenic determinants specific for 1918 pdm influenza strain. Our results demonstrate a potential key neutralizing epitope important for H1 subtype specificity in influenza virus.

Figure 1. Binding affinities of monoclonal antibodies against H1 HAs.

ELISA analysis results of monoclonal antibodies against KR01 and CU44 HAs that were coated on the 96-well plates to which 0 to 10 µg/ml of antibodies were added. Absorbance was measured after addition of TMB solution to each well at 490 nm.

Figure 2. Overview of the structures of KR01 HA and its complex with Fab0587.

(A) Structure of KR01 HA-Fab0587 complex. HA are colored in orange and light brown, H-chains are blue and green, and L-chains are light blue and lime. (B) Superposition of KR01 HA in Fab0587 complex and head domain of KR01 HA structures, and (C) superposition of stem region between Fab0587 bound KR01 HA and Free KR01 HA. Gray color represents free HA, and orange color represents HA in complex.

Figure 3. Sequence alignments and antigenic sites.

(A) Sequence alignments of variable region of GC0587 and GC0757 (upper panel) and H1 HAs (lower panel). Residues in CDRs are in blue open boxes and residues that interact with HA are highlighted in blue filled boxes. Epitopes in H1 HAs are highlighted in pink, and more conserved residues are highlighted in red. Potential glycosylation sites are highlighted in green. (B) Surface representations of KR01 HAs (gray) and Fab fragments (dark blue and light blue for H-chain and L-chain, respectively). Antigenic sites are colored in red for highly conserved residues and pink for moderately conserved residues. Amino acid residues involved in the interactions between KR01 HA and Fab0587 are colored in blue and slate. Insets are surface charge representations with contours from −10 (red) to+10 (blue) kT through 0 (white). (C) Detailed interactions of HA with Fab0587. HA and Fab are colored in orange and blue, respectively. Residues that contribute to the interactions are represented as stick models. LCDR1 and LCDR3 are colored in purple and green, respectively (upper panel), and HCDR2 and HCDR3 are colored in magenta and cyan, respectively (lower panel). (D) Residues found at other structurally characterized antibody complexes are colored in yellow, those at both Fab0587 and other antibody complexes are in orange, and those against GC0587 are in red. Classical antigenic sites are colored in light pink (Ca, Cb, Sa, and Sb) and those at both Fab0587 antigenic sites are in pink.

Figure 4. Comparison of Fab binding sites.

Residues involved in interactions between HA and Fab0587 are represented as a ball-and-stick model and hydrophilic interactions as dotted line. Residues of HA in complex with Fab0587 are colored in orange and H-chains of Fab0587 and Fab0757 are in dark and light blue, respectively. (A) Comparison of LCDR3, (B) HCDR3. (C) Superposition of KR01 HA bound to Fab0587 (magenta), KR01 HA bound to Fab0757 (dark gray), free KR01 HA (yellow), 1918 pdm HA (green), Fab0587 (purple), and Fab0757 (light teal). Surface representations are based on the KR01HA-Fab0587 complex structure.