Molecular basis for broad neuraminidase immunity: conserved epitopes in seasonal and pandemic H1N1 as well as H5N1 influenza viruses

Abstract

Influenza A viruses, including H1N1 and H5N1 subtypes, pose a serious threat to public health. Neuraminidase (NA)-related immunity contributes to protection against influenza virus infection. Antibodies to the N1 subtype provide protection against homologous and heterologous H1N1 as well as H5N1 virus challenge. Since neither the strain-specific nor conserved epitopes of N1 have been identified, we generated a panel of mouse monoclonal antibodies (MAbs) that exhibit different reactivity spectra with H1N1 and H5N1 viruses and used these MAbs to map N1 antigenic domains. We identified 12 amino acids essential for MAb binding to the NA of a recent seasonal H1N1 virus, A/Brisbane/59/2007. Of these, residues 248, 249, 250, 341, and 343 are recognized by strain-specific group A MAbs, while residues 273, 338, and 339 are within conserved epitope(s), which allows cross-reactive group B MAbs to bind the NAs of seasonal H1N1 and the 1918 and 2009 pandemic (09pdm) H1N1 as well as H5N1 viruses. A single dose of group B MAbs administered prophylactically fully protected mice against lethal challenge with seasonal and 09pdm H1N1 viruses and resulted in significant protection against the highly pathogenic wild-type H5N1 virus. Another three N1 residues (at positions 396, 397, and 456) are essential for binding of cross-reactive group E MAbs, which differ from group B MAbs in that they do not bind 09pdm H1N1 viruses. The identification of conserved N1 epitopes reveals the molecular basis for NA-mediated immunity between H1N1 and H5N1 viruses and demonstrates the potential for developing broadly protective NA-specific antibody treatments for influenza.

Fig 1

Reactivity of cross-reactive group B MAbs to N1 and other NA subtypes in IFAs. MDCK cells were infected with BR/07 (human H1N1) (A), CA/09 (human H1N1) (B), A/Chongqing/150/2007 (human H1N1) (C), A/Guangdong/51/2008 (human H1N1) (D), A/duck/Eastern China/1/2008 (avian H6N1) (E), A/duck/Eastern China/103/03 (Y103, avian H1N1) (F), A/duck/Eastern China/233/03 (Y233, avian H3N1) (G), A/turkey/Ontario/6188/68 (avian H8N4) (F), and other viruses as listed in Table 1. IFAs were performed with all group B MAbs, and the same reactivity patterns were observed. Shown are images generated with group B MAb 1H5. The remaining viruses tested (listed in Table 1) were negative for staining with group B MAbs, and therefore images are not shown.

Fig 2

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.

Fig 3

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.

Fig 4

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.

Fig 5

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.

Fig 6

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.

Fig 7

Dose-dependent prophylactic efficacy of group B MAbs against BR/07 (H1N1). DBA/2 mice (9/group) were treated with group B MAb 3H10 (15, 5, 2, 0.5, and 0.1 mg/kg) intraperitoneally 12 h before challenge with 10 MLD₅₀ of BR/07. Mice treated with the 09pdm H1N1 NA-specific MAb CD6 (Table 3) at 15 mg/kg served as a control. Mortality (A) and weight (B) were monitored daily for 14 days. (C) lung viral titers were measured for 4 mice per group on days 3 and 6 postchallenge.