Immune response assessment following influenza exposure

Two nasal swabs (one per nostril) were taken daily following infection with wt H1N1 and immunization with LAIV and following subsequent challenge with wt H1N1. The swabs were placed into 2 ml of virus transport medium comprising tissue culture medium 199 (Sigma-Aldrich, UK) supplemented with 25 mM Hepes, 0.035% sodium bicarbonate, 0.5% BSA, penicillin, streptomycin and nystatin, vortexed, centrifuged to remove debris and stored at −80°C for subsequent virus titration. Serum and heparin anti-coagulated blood samples were collected at the start of the study (prior to LAIV immunization or wt H1N1 pre-exposure), before challenge and four dpc at post-mortem. Heparin blood samples were diluted 1:1 in PBS before density gradient centrifugation at 1,200 × g for 30 min over Histopaque® 1.083 g/ml (Sigma-Aldrich, UK). PBMC were harvested from the interface, washed and red blood cells lysed with Red Blood Cell Lysis Buffer (BioLegend, UK), washed again and cryopreserved in FCS (Gibco) with 10% DMSO (Sigma-Aldrich, UK). Broncho-alveolar lavage (BAL) and tracheobronchial lymph nodes (TBLN) were processed as previously described (38 (link)).