Hemagglutination-inhibition (HAI) assay

The hemagglutination inhibition (HAI) assay was used to assess functional antibodies to the HA that are able to inhibit agglutination of guinea pig erythrocytes. The protocols were adapted from the WHO laboratory influenza surveillance manual⁴⁶. Guinea pig red blood cells are frequently used to characterize contemporary A(H3N2) influenza strains that have developed a preferential binding to alpha (2,6) linked sialic acid receptors^47,48. To inactivate nonspecific inhibitors, sera samples were treated with receptor-destroying enzyme (RDE) (Denka Seiken, Co., Japan) prior to being tested. Briefly, three parts of RDE was added to one part of sera and incubated overnight at 37 °C. RDE was inactivated by incubation at 56 °C for 30 min. RDE-treated sera were diluted in a series of twofold serial dilutions in in v-bottom microtiter plates. An equal volume of each A(H3N2) influenza virus, adjusted to approximately 8 hemagglutination units (HAU)/50 μl in the presence of 20 nM Oseltamivir carboxylate, was added to each well. The plates were covered and incubated at room temperature for 30 min, and then 0.75% guinea pig erythrocytes (Lampire Biologicals, Pipersville, PA, USA) in PBS were added. Red blood cells were washed with PBS, stored at 4 °C, and used within 24 h of preparation. The plates were mixed by gentle agitation, covered, and the RBCs were allowed to settle for 1 h at room temperature. The HAI titer was determined by the reciprocal dilution of the last well that contained non-agglutinated RBCs. Positive and negative serum controls were also included for each plate. All mice were negative (HAI ≤ 1:10) for pre-existing antibodies to currently circulating human influenza viruses prior to vaccination, and for this study sero-protection was defined as HAI titer > 1:40 and seroconversion as a fourfold increase in titer compared to baseline, as per the WHO and European Committee for Medicinal Products to evaluate influenza vaccines⁴⁹. Mice are naïve and seronegative at the time of vaccination, and thus seroconversion and sero-protection rates are interchangeable in this study.