Recombinant ha protein

Balb/c mice (n=6) were immunized subcutaneously with 5 µg of HA antigens, 0.5 mm [II] and their combination. Booster was done at D60, and sera were collected at D14, D28 and D75. 4 different influenza antigens; Recombinant HA protein from flu A/Brisbane/59/2007 (H1N1)(NR‐28607), HA from influenza A/New Caledonia/20/99 (H1N1) (NR‐48873), HA Protein from flu Virus A/St. Petersburg/100/2011 (H1N1) (NR‐34588) and Recombinant HA protein from flu A/California/04/2009 (H1N1) (NR‐15749) were obtained from BEI Resources. Peptides are mixed for 30 min as explained in the manuscript and mixed with the antigens at room temperature to formulate the vaccines before immunizations.

Immunolon 4 HBX microtiter 96-well strips (Thermo Fisher, Waltham, MA, USA) were coated with 200 ng/well of recombinant HA protein from BEI Resources, Manassas, VA, USA NIAID (A/California/04/2009, NR-15749; A/PR/8/34, NR-19240; A/Brisbane/59/07, NR-28607; A/New Caledonia/20/99, NR-48873) in bicarbonate/carbonate buffer overnight at 4 °C. Plates were blocked with 2% bovine serum albumin (BSA) in PBS and incubated for two hours at RT. Sera was serial diluted two-fold in PBS with 1% BSA starting with a 1:100 dilution in 100 μL per well. After two hours of incubation at RT, plates were washed 4× with phosphate buffered saline with Tween 20 (PBST) and 2× with PBS before incubation with 1:5000 goat anti-mouse-HRP antibody (sc-47724; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) in 1% BSA at RT for one hour. Plates were washed 4× with PBST and 2× with PBS prior to development with 1-Step Ultra TMB-ELISA (Thermo Fisher, Waltham, MA, USA). Development was stopped with the addition of 2M sulfuric acid. The OD450 was measured using SpectraMax i3x Multi-Mode microplate reader (Molecular Devices, San Jose, CA, USA). Responses ≥2 times the negative control absorbance were considered positive and used to calculate the geometric mean titer (GMT).