Participants were enrolled sequentially in three steps preceded by a safety review (Fig. 1). They were randomized find more (1:2:2:2:2:2:2, block size 4 [step 1], 7 [step 2] and 5 [step 3]) using a central internet randomization system (SBIR) to receive a two-dose primary vaccination series with one of six investigational vaccine formulations (GlaxoSmithKline Vaccines) or a single dose of the 23-valent pneumococcal polysaccharide vaccine (23PPV; Pneumovax23™, Sanofi Pasteur

MSD) followed by placebo (150 mM NaCl) ( Fig. 1; supplementary methods). All vaccines and the placebo were administered intramuscularly into the deltoid region of the non-dominant arm. Two investigational vaccines contained 10 or 30 μg of dPly alone (dPly-10 and dPly-30, respectively). Two other formulations contained Lumacaftor in vitro both dPly and PhtD, each at a dose of 10 μg (dPly/PhtD-10) or 30 μg (dPly/PhtD-30). The remaining two formulations contained the 10 PHiD-CV PS-conjugates (serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F) [18], in combination with 10 or 30 μg of both dPly and PhtD (PHiD-CV/dPly/PhtD-10 and PHiD-CV/dPly/PhtD-30).

Production of PhtD and dPly is described in supplementary methods. The control group received one dose of 23PPV, containing 25 μg of each capsular polysaccharide for pneumococcal serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, and placebo (150 mM NaCl) as a second dose. Participants from the dPly/PhtD-10 and dPly/PhtD-30 groups were invited to participate in the booster vaccination study, to receive a booster dose 5–9 months after completion of the two-dose primary series. Solicited local and general symptoms were recorded during the 7-day post-vaccination period and unsolicited adverse events (AEs) during the 31-day post-vaccination period. Symptom intensity was graded on a scale of 1 (mild) to 3 (severe). Grade 3 symptoms were defined as follows: for redness or swelling, a diameter >50 mm; for fever, oral temperature >39.5 °C; and for all

other events, preventing normal activity. Serious adverse events (SAEs) were recorded throughout the duration of each study, and were defined as any medical occurrence that resulted in death, disability or incapacity, was life-threatening, required Electron transport chain hospitalization, or any congenital anomaly or birth defect in the descendants of a study participant. Blood samples for immunogenicity assays were collected before primary and booster vaccination, and 1 month after each dose. 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, Ply (non-detoxified) and PhtD (supplementary methods), with assay cut-offs of 112 LU/mL for anti-PD, 599 LU/mL for anti-Ply and 391 LU/mL for anti-PhtD.