Molecular Basis for Broad Neuraminidase Immunity: Conserved Epitopes in Seasonal and Pandemic H1N1 as Well as H5N1 Influenza Viruses

Reduction of plaque size by cross-reactive group B MAbs. MDCK cells growing in six-well plates were inoculated with cloned, wt BR/07, attenuated VN/04 (ΔVN/04), or reassortant H6N1_CA/09 virus and overlaid with agar supplemented with 1 μg/ml (the top three rows) or 10 μg/ml (the bottom row) of MAbs. Shown are images of plaques formed by these viruses in the presence of strain-specific group A MAb 3A2 and cross-reactive group B MAbs 1H5 and 3H10. The virus control shows plaques formed in the absence of MAb.

Effect of single amino acid mutations on MAb binding to N1. Binding of group A MAb 3A2 (A), group A MAb 4G2 (B), and group C MAb 1C7 (C) to mutant N1s was examined by cell-based ELISAs using HEK293 cells transfected with a panel of plasmids carrying each of 14 different single amino acid mutations. The signal generated with each MAb (1 μg/ml) was normalized to that of mouse hyperimmune serum and was expressed as relative binding.

Location of 12 critical residues in N1 epitopes on an N1 dimer (Protein Data Bank code 3TI6). The image in the top panel was generated with PyMol software (Delano Scientific). Residues recognized by strain-specific group A MAbs and cross-reactive group B and E MAbs are highlighted in magenta, green, and yellow, respectively; residue 309 recognized by group F MAb 4C4 is at the bottom of the NA region and is shown in blue. Note that residues 339, 341, and 397 are shared by epitopes recognized by two or three groups of MAbs (Table 4 and Fig. 3). The bottom panel shows an alignment of the 12 residues in N1 viruses described in the present study.

Prophylactic efficacy of cross-reactive group B MAbs in mice against H1N1 and H5N1 challenge. (A to I) DBA/2 mice (9/group) and (J to L) BALB/c mice (15/group) were treated with group A MAb 3A2 and group B MAb 1H5 or 3H10 (15 mg/kg) intraperitoneally 12 h before virus challenge. Mice treated with PBS served as a control. DBA/2 mice were challenged intranasally with 10 MLD₅₀ of seasonal H1N1 BR/07 (A to C), 09pdm H1N1 CA/09-X179A (D to F), or attenuated H5N1 ΔVN/04 virus (G to I). BALB/c mice were challenged with 20 MLD₅₀ of wt VN/04 virus. Mortality (A, D, G, and J) and body weight (B, E, H, and K) were monitored daily for 14 days, and on day 3 postchallenge, animals (4/group for DBA/2 mice and 5/group for BALB/c mice) were euthanized, and lung viral titers (C, F, I, and L) determined in MDCK cells.

Group B MAbs protect mice against H1N1 but not H3N2 virus challenge. DBA/2 mice (5/group) were treated with group B MAb 3H10 (15 mg/kg) intraperitoneally 12 h before challenge with 10 MLD₅₀ of 09pdm H1N1 CA/09-X179A or H3N2 A/Hong Kong/1/1968-X31 (HK/68-X31) virus. Mice treated with PBS and challenged with HK/68-X31 served as a control. Mortality (A) and body weight (B) were monitored daily for 14 days.