Reagents.

Purified FHA for immunizations was purchased from Kaketsuken (Japan) and was free of detectable endotoxin as tested using the Limulus amebocyte lysate (LAL) test (Lonza). Purified proteins FHA, Prn, and PT for ELISAs were from List Biologicals. Boostrix (GlaxoSmithKline) was purchased from the Wake Forest School of Medicine (WFSM) pharmacy. BcfA was overproduced in E. coli and purified essentially as described previously (16), with size exclusion chromatography as an additional step. Residual LPS was removed using MustangQ filters or polymyxin B agarose. Endotoxin was quantified and was ≤150 pg/μg protein (∼4 EU/30-μg injection). Alhydrogel, polymyxin B agarose, and fetal bovine serum (FBS) were from Sigma-Aldrich (St. Louis, MO). ELISA kits were from eBioscience, and antibodies for flow cytometry were from eBioscience (Thermo Fisher), BD Biosciences (San Jose, CA), or Tonbo Biosciences (San Diego, CA). RPMI was from Lonza (Walkersville, MD). Biotinylated secondary antibodies were from BioLegend (San Diego, CA). F1/V fusion protein was provided by Steven Mizel, Wake Forest University Health Sciences (WFUHS). Ovalbumin protein, SIINFEKL peptide, and anti-TLR2 antibody were from InvivoGen (San Diego, CA). MagPlex C magnetic microspheres were from Luminex Corporation.