Clo75

A heparinised whole venous blood sample was taken at 9 months of age prior to administration of the study vaccines. Whole blood was cultured in 100 μL aliquots in 96-well U-bottom plates with a panel of TLR ligands: heat-killed listeria monocytogenes (HKLM) (10⁹ cells/mL) (TLR2 agonist), Escherichia coli K12 lipopolysaccharide protein S (LPS) (1 μg/mL) (TLR4 agonist), flagellin (10 μg/mL) (TLR5 agonist) and CLO75 (10 μg/mL) (TLR7/8 agonist) (all from InvivoGen, San Diego, CA, USA). The TLR ligand concentrations used were selected as optimal in prior titration assays in infants and are in line with our previous published studies in this age group [33 (link)]. RPMI alone was used in the unstimulated control wells. Plates were incubated for 16 h at 37 °C, 5% CO₂, centrifuged at 2000 rpm for 5 min and 50 μL supernatant collected and stored at −20 °C. The Bio-Plex 200 Suspension Array system (Bio-Rad, Hercules, CA, USA) was used to analyse cytokines according to the manufacturer’s instructions (Bio-Rad, Belgium). A 5-plex array (IL-1β, IL-6, IL-10, IL-12(p70), TNF-α) was used. Innate assays were only performed for those infants in the study with sufficient blood volume and quality, and those with low volume, clotted or contaminated blood samples were excluded.

0.1 × 10⁶ PBMCs in 100 µL of culture medium (2% penicillin/streptomycin, 1% l-glutamine, and 10% FCS in RPMI) were stimulated in 96-well round bottom plates for 16 h with the following antigens: purified protein derivative (PPD) (10 µg/mL, Serum Staten Institute, Denmark), heat-killed Listeria monocytogenes (HKLM) (TLR2 agonist; 10⁹/mL, Invivogen), lipopolysaccharide (LPS) (TLR4 ligand; 1 µg/mL, Invivogen), CLO-75 (TLR7/8 agonist; 10 µg/mL, Invivogen); and heat-killed Streptococcus pneumonia (SP) (10⁵ cells/mL), CA (10⁵ cells/mL), or Escherichia coli (EC) (10⁶ cells/mL). PMA/ionomycin (0.1 μg/1 μg/mL, Sigma, UK) was used as a positive control, and RPMI as a negative control. Supernatants were collected after the addition of 100 µL of RPMI and centrifugation at 1,500 rpm for 10 min. Supernatants were stored at −20°C until needed for cytokine analysis.

LPS from Salmonella Minnesota Re 595 was purchased from Calbiochem (Darmstadt, Germany). Pam3CSK4, Poly I:C, flagellin, FSL-1, imiquimod, CLO75, MDP and zymosan were purchased from InvivoGen (San Diego, CA, USA). CpG ODN 1555 (GCTAGACGTTAGCGT) and CpG ODN B2006 (TCGTCGTTTTGTCGTTTTGCTGTT) were synthesized at the FDA core facility (Rockville, MD, USA) and used at the concentration indicated in each individual figure. The purity of each TLR agonists was established using a panel of HEK-BLUE cell lines transfected with individual TLRs (HEK-BLUE-hTLR2, 4, 5, 7 and 9) from InvivoGen (San Diego CA, USA). Commercial lots of Erythropoietin (Procrit) IFNβ-1a (Avonex), and heparin were used.