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

Pneumococcal adherence and internalization assays. D39 bacterial pellets were washed with PBS containing Ca 2 þ Mg 2 þ (Sigma-Aldrich) before being resuspended and diluted to 2 Â 10 6 CFU ml À 1 in EMEM with no antibiotics. Bacterial suspensions were added to wells containing D562 cells (500 ml per well), plates were gently shaken, and 20 ml was obtained from each well for dilution and initial CFU quantification on blood agar plates (Oxoid, Basingstoke, UK). The 24well plates were then incubated at 37 1C 5% CO 2 for 3 h. Wells were washed 5 times with PBS Ca 2 þ Mg 2 þ to remove unbound bacteria and cells were either lysed with 1% saponin for 10 min (for adherence) or treated with 100 mg ml À 1 of ampicillin in EMEM (1 ml per well) for 3 h at 37 1C 5% CO 2 before lysing (for internalization). Recovered bacteria were quantified by serial dilution on blood agar plates. Plates were incubated at 37 1C 5% CO 2 overnight and initial and recovered CFUs were counted. Averages of initial D39 CFU values were used to create a correction factor so that the recovered CFUs for both strains could be compared. All conditions were performed in duplicate and experiments were performed at least twice. For each experiment, the average recovered CFUs from the control condition (adherence of D39 untreated bacteria to noninflamed cells) was used to calculate fold change for all remaining conditions. Anti-PspC antibody epitope mapping. Overlapping peptide arrays containing 15-mer peptides with a frameshift of four residues corresponding to the amino acid sequence of PspC3 (GenBank accession no. EF424119) were synthesized in a slide support (CelluSpots, Intavis, Cologne, Germany). Peptide arrays were incubated individually with 29 sera samples collected from 18 subjects inoculated with pneumococcus or from mice immunized with 3 doses of 5 mg of PspC3 containing 50 mg of Alum as previously described. 49 Concentration of pneumococcal-specific antibodies was standardized so that each sample used for incubation contained 10 mg ml À 1 of PspC IgG. Detection was performed using alkaline phosphatase-conjugated anti-mouse IgG and anti-human IgG (Sigma-Aldrich). MTT 0.12M (Methylthiazolyldiphenyl-tetrazolium bromide), BCIP 0.16M (5-Bromo-4-chloro-3-indolyl phosphate), and MgCl2 1M in citrate-buffered saline pH 7.0 was used for development.