Modulation of nasopharyngeal innate defenses by viral coinfection predisposes individuals to experimental pneumococcal carriage

Glennie, S; Gritzfeld, J F; Pennington, S H; Garner-Jones, M; Coombes, N; Hopkins, M J; Vadesilho, C F; Miyaji, E N; Wang, D; Wright, A D; Collins, A M; Gordon, S B; Ferreira, D M

Depletion and purification of antibodies from nasal wash and sera samples. Nasal wash samples from 10 subjects were pooled and IgG and IgA were depleted by anti-human IgG and anti-human IgA agarose (Sigma-Aldrich, Gillingham, UK), respectively. Samples were incubated with agarose and flow-through fractions were used in pneumococcal adherence assays. Dot blot assays were performed to confirm antibody depletion (data not shown). Serum samples from seven subjects were used for anti-PspC IgG purification. First, purified total IgG was obtained from individual samples by HiTrap protein G affinity column (GE Healthcare, Little Chalfont, UK). Purified samples were then loaded in CNBr-activated Sepharose coupled with recombinant purified PspC. Coupling (1 mg of ligand PspC to 200 mg of sepharose) and anti-PspC purification was performed as per the manufacturer's instructions. To confirm anti-PspC IgG purification, ELISAs were performed using plates coated with PspC as previously described. 17 Bacterial culture for in vitro assays. D39 pneumococci were grown in Todd Hewitt broth supplemented with 0.5% yeast extract (THY) at 37 1C in 5% CO 2 until optical density 0.4-0.5 was reached. Bacteria were either used immediately for flow binding assays or resuspended in THY 10% glycerol and stored at À 80 1C for adherence assays. Samples were washed with PBS and incubated in 100 ml of goat antifactor H (1/200) (Calbiochem) for 45 min at 37 1C before wash and incubation in 100 ml of FITC-conjugated anti-goat (1/500) (Sigma-Aldrich) at 4 1C in the dark for 30 min. Samples were washed twice and resuspended in 500 ml of PBS and 4% paraformaldehyde and stored at 4 1C before acquisition using a BD LSR II flow cytometer. A total of 20,000 bacterial events were acquired and samples were gated relative to a negative control containing no anti-FH. Anti-PspC IgG binding was evaluated by incubating bacterial pellets with individually purified human anti-PspC IgG samples for 45 min at 37 1C before detection using anti-human IgG FITC antibody (1/10,000, Sigma-Aldrich). As FH binding to S. pneumoniae is often biphasic 48 with strongly positive and weakly negative populations of bacteria, results are presented as fluorescence index (percentage of positive bacteria multiplied by the geometric mean fluorescence index in arbitrary units).