Two groups received a formulation containing 10 or 30 μg of each dPly and PhtD (dPly/PhtD-10 and dPly/PhtD-30). Two further groups received a formulation containing the PS-conjugates of PHiD-CV (serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F) and 10 or 30 μg of each dPly and PhtD (PHiD-CV/dPly/PhtD-10 and PHiD-CV/dPly/PhtD-30). All investigational vaccines were adjuvanted with aluminum phosphate. The fifth group received the licensed PHiD-CV [20]. All vaccines were manufactured by GlaxoSmithKline Vaccines. No other vaccines were

co-administered. Solicited FG4592 and unsolicited adverse events (AEs) were recorded by the participant’s parents in paper diary cards that were returned to the investigator at the next study visit. Solicited local and general symptoms were recorded within seven days post-vaccination and unsolicited AEs within

31 days post-vaccination. Symptom intensity was graded on a scale of 1 (mild) to 3 (severe). Serious adverse events (SAEs), defined as any medical occurrence that resulted in death, disability or incapacity, was life-threatening, or required hospitalization, were recorded over the whole Fludarabine study period. Blood samples were collected pre-vaccination, one month post-dose 2, and pre- and one month post-booster. Serum samples were stored at −20 °C until analysis at GlaxoSmithKline’s laboratory, Rixensart, Belgium and SGS laboratory, Wavre, Belgium. Antibodies were quantified using an in-house multiplex assay coated with protein D (PD), non-detoxified pneumolysin (Ply) and PhtD, with a cut-off of 112 LU/mL for PD, 599 LU/mL for Ply and 391 LU/mL for PhtD. These cut-offs were based on the lower limit of quantification [21], the global

variability of the assay at the highest dilution and the lower limit of linearity. Serotype-specific anti-capsular antibodies against the 10 PS-conjugates and two cross-reactive serotypes (6A, 19A) were measured using a GlaxoSmithKline 22F-inhibition enzyme-linked immunosorbent assay (ELISA), with a cut-off of 0.05 μg/mL. An antibody concentration of 0.2 μg/mL measured by the 22F-ELISA is equivalent to the antibody concentration of 0.35 μg/mL measured by the non-22F ELISA of the World Health Organization reference laboratory [22]. Opsonophagocytic activity (OPA) for the above-mentioned antibodies was measured secondly by a pneumococcal killing assay with a cut-off opsonic titer of 8, described previously [23]. Safety and reactogenicity analyses were performed on the total vaccinated cohort (TVC), comprising all toddlers with at least one vaccine dose administration documented. To assess the impact of each protein formulation on the incidence of grade 3 fever (primary objective), the dPly/PhtD-10 and dPly/PhtD-30 groups were pooled, as were the PHiD-CV/dPly/PhtD-10 and PHiD-CV/dPly/PhtD-30 groups, and group differences (pooled dPly/PhtD minus PHiD-CV or pooled PHiD-CV/dPly/PhtD minus PHiD-CV) were calculated.