Recombinant ha

Influenza viruses used in all assays were grown in-house in specific pathogen free (SPF) eggs, harvested, purified, and titered (51 (link)). The A/swine/Mexico/AVX8/2011 H1N2 virus (16 (link)) was provided by Ignacio Mena and Adolfo García-Sastre at Icahn School of Medicine at Mount Sinai. Recombinant HA used for enzyme-linked immunosorbent assays (ELISAs) were obtained from BEI Resources or provided by the Krammer laboratory at Icahn School of Medicine at Mount Sinai. All HA residue numbering is based on FluDB H3 numbering and annotated HA figures were produced using Pymol (Schrödinger). Recombinant HA (A/California/04/2009, with stabilizing mutations E47K or E47G in HA2 (15 (link))) used for negative stain and cryo-EM were produced in-house or kindly provided by Nicholas Wu and Ian Wilson at The Scripps Research Institute.

Nunc MaxiSorp Elisa plates were coated with 1 μg/well PR8 or 0.2 μg/well of recombinant HA (BEI Resources) in sodium carbonate/bicarbonate coating buffer (pH 9.5). Plates were blocked with 1x Blocking Buffer (10x Blocking Buffer, Sigma) plus 2% goat serum (Lampire Biologicals) and washed. The wash buffer used throughout the assay was PBS with 0.1% Tween 20. Plasma or respiratory samples were serially diluted in 1x Blocking Buffer. Wells without virus served as a negative control. HRP-conjugated antibody specific for monkey IgG (Fitzgerald) or IgM (LifeSpan Bioscience) was used to detect bound antibody. Plates were developed with 3,3′,5,5′-Tetramethylbenzidine dihydrochloride (Sigma) and read at 450nm on a BioTek Elx800 Absorbance Microplate Reader. Absorbance for each dilution was calculated by subtracting the OD value obtained for the corresponding non-virus coated wells. Threshold titer was defined as the value that reached 3 times the assay background, i.e. wells that received only sample diluent.