The truncated open reading frames of LukE, SplB, and SspB were polymerase chain reaction–amplified, restriction-digested, and cloned in frame with a His-tag in pET28a (Novagen). The resulting plasmid was expressed in Escherichia coli (DE3, BL21; Invitrogen). The proteins were chromatography-purified from E. coli using the His-Bind Kit (Novagen). The constructs for purification of HlaH35L and LukS-PV were provided by J. B. Wardenburg (Washington University, St. Louis). HlaH35L, cloned in pET24b with a His-tag, was purified from E. coli (BL21), as described (20). LukS-PV, cloned in pGEX with a glutathione S-transferase (GST) tag, was purified from E. coli (BL21), and the GST removed, as described (20). Endotoxin was removed using an endotoxin removal kit (Pierce). Mice were vaccinated with one of three vaccines: HlaH35L, 4S (LukE, LukS-PV, SplB, and SspB), or 5S (4S + HlaH35L). Vaccines were prepared by adding 10 μg of each antigen, adjuvanted with Al(OH)3 (Alhydrogel; Brenntag) at a final concentration of 0.1% in a total volume of 200 μl. Vaccinations were administered subcutaneously 5 and 2 weeks before infection. For the serum adoptive transfer experiments, naïve mice received 150 μl of serum from Al(OH)3, LukE, 4S, or HlaH35L-vaccinated mice via retro-orbital injection 1 day before infection. For the T cell adoptive transfer experiments, T lymphocytes were isolated by negative selection using the Pan T cell Isolation Kit II or the CD8+ T cell Isolation Kit II (Miltenyi Biotec). One day before infection, each recipient mouse received 8 × 106 T cells or PBS in a volume of 200 μl by retro-orbital injection.

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