Figure 1.
MAbs react with H3 hemagglutinin by western blot.
(A) MAb 12D1 binds the A/Pan/2007/1999 hemagglutinin within the HA2 subunit. mAbs 7A7 and 39A4 do not react with hemagglutinin under reducing conditions. (B) MAbs 7A7, 12D1 and 39A4 react with the A/HK/1/1968 hemagglutinin under non-reducing conditions. MAbs 7A7 (lane 1) and 39A4 (lane 3) bind HA trimer complexes. mAb 12D1 (lane 2) binds HA trimer complexes and HA0.
Table 1.
Pattern of reactivity of anti-H3 mAbs.
Figure 2.
Reactivity of anti-H3 mAbs by ELISA.
(A) mAbs react with purified A/HK/1968 (H3) virus. (B) mAbs react with purified A/Alabama/1981 (H3) virus. mAb XY102 is specific for the hemagglutinin of A/HK/1968 virus.
Table 2.
Reactivity of mAbs at 5ug/ml by immunofluorescence against MDCK cells infected with a panel of randomly chosen viruses.
Figure 3.
Anti-H3 mabs in microneutralization assay.
Neutralization of virus expressing the HA from either (A) A/Hong Kong/1/1968 virus or (B) A/Panama/2007/1999 virus. mAb XY102 is specific for A/HK/1968 virus. Purified mouse IgG was used for the negative control.
Figure 4.
Activity of anti-H3 mabs in plaque reduction assay on MDCK cells.
mAb 7A7 (A), 12D1 (B) and 39A4 (C) neutralize all H3 viruses tested by plaque reduction assay but not representative H1, H4 or H7 viruses. Purified mouse IgG was used for the negative control. The plaque reduction assays were performed multiple times and with each new antibody preparation. Data shown are from a single representative experiment.
Figure 5.
Anti-H3 mAbs protect against H3 virus in vivo.
Mice were given 30mg/kg mAb 7A7, 12D1, 39A4 or isotype control by intraperitoneal injection 1 hour prior, 24 hours post (12D1 only) or 48 hours post (12D1 only) challenge with H3N2 reassortant virus (HK68/PR8). N = 5 per group.
Figure 6.
Treatment with anti-H3 mAbs diminishes lung damage associated with viral pneumonia caused by HK68/PR8 reassortant virus.
(A,B) Untreated (C,D) mice treated with mAb 39A4 (E,F) mice treated with mAb 12D1. 40× magnification.
Figure 7.
Anti-H3 mAbs protect against replication of H3 virus in lungs.
Mice were given 30mg/kg mAb 12D1, 39A4 or isotype control by intraperitoneal injection 1 hour prior to infection with A/Georgia/1981 virus. Data represent lung titers from groups of 5 mice, 2 days post infection.
Figure 8.
Anti-H3 mabs inhibit low-pH induced fusion of HK/68 hemagglutinin with chicken red blood cells. All mAbs are negative for hemagglutinin-inhibition activity. MAb 1A7 is specific for influenza virus NS1 protein.
Figure 9.
MAb 12D1 reacts by western blot with hemagglutinin truncation mutants.
12D1 makes dominant contacts with the HA2 subunit in the region of amino acids 30 to 106 (H3 numbering[21]). Diminished 12D1 binding without diminished GFP expression in the HA2 76–184 and HA2 91–184 truncations along with loss of binding with the HA2 106–184 truncation suggests that the binding epitope lies in the region from amino acids HA2 76–106. These 30 amino acids fall within the membrane distal half of the long alpha-helix of HA2.
Figure 10.
Region of dominant contacts between anti-H3 mab 12D1 and HA2.
mAb 12D1 makes contact in the HA2 region (blue) of the viral hemagglutinin. Evaluation of binding to hemaggluinin truncation mutants demonstrates binding within the region of amino acids 76–106 of HA2 (light blue).