C57BL/6 female mice received a primary immunization and three boosts with compounds 2 to 5 on days 0, 21, 28, and 35. The compounds 2 to 5/PBS solutions and compound 1/CFA emulsion (1:1 volume ratio) were prepared fresh before each immunization. Compounds 2 to 5 were dissolved in PBS directly and then vortexed for 2 min. Mice in each group (10 mice per group) were immunized subcutaneously at the base of their tails with 150 μg of compound in 50 to 100 μl of PBS.

All immunized and control mice were challenged intranasally with the GAS strain M1, with a predetermined dose on the 61st day after the primary immunization. The throat swab was obtained on days 1 to 3 after the mice were challenged with the GAS bacteria. All throat swabs were streaked on Columbia base agar plates containing 2% defibrinated horse blood incubated at 37°C overnight. The plates were stored in a 4°C room for later determination of GAS colonization. Nasal shedding was determined on days 1 to 3 after challenge by pressing the nares of each mouse onto the surface of the prepared Columbia blood agar (CBA) plates 10 times (triplicate CBA plates per mouse per day), and exhaled particles were streaked out. On days 2 and 3 after challenge, five mice from each group were euthanized to harvest the NALT and spleen. The NALT and spleen samples were homogenized in PBS first and then plated in serial dilutions on Columbia base agar plates containing 2% defibrinated horse blood (37°C, overnight) to assess bacterial burden.

In the second vaccination experiment, C57BL/6 female mice received a primary immunization and one boost with compound 5 and the controls on days 0 and 14. The compound 5 solutions (150 μg of compound 5 in 100 μl of PBS) and compound 1/AS04 emulsion (5 μg of MPLA-SM/5 μl of DMSO mixed with 150 μg of compound 1/45 μl of PBS according to the manufacturer MPLA-SM VacciGrade’s instruction) were prepared freshly before each immunization. Compound 5 was dissolved in PBS directly and then vortexed for 2 min. Mice in each group (six mice per group) were immunized subcutaneously at the base of their tails with the formulation bearing150 μg of appropriate antigen.

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