Hemagglutination Inhibition Assay for Vaccine Antibodies

HAI titres were used to determine vaccine-specific antibody titres based on a standard WHO protocol, as previously described^{57 (link)}. Briefly, plasma samples were treated with receptor-destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) by adding one-part plasma to three-parts RDE and incubating at 37 °C overnight. The next day, RDE was inactivated by incubating the samples at 56 °C for 1 h. Then, the samples were serially diluted with phosphate-buffered saline (PBS) in 96-well V-bottom plates (Nalge Nunc International Corporation, Rochester, NY, USA), and 4 HA-units each of H1N1 (California strain), H3N2 (Switzerland strain), or influenza-B virus (Phuket strain) was added to each well. After 30 min at room temperature, 50 µl of 0.5% turkey red blood cells (Rockland Immunochemicals, Philadelphia, PA, USA) suspended in PBS with 0.5% bovine serum albumin (BSA) was added to each well, and the plates were manually agitated. After an additional 30 min at room temperature, the plasma titres were read. Negative and positive control plasma samples for each virus were used for reference. “Sero-protection” was defined as an HAI titre >1:40, and a “sero-response” was defined as a minimum four-fold increase in antibody titre, 30 days post-vaccination³³ .