Antigens DHG, SagA, and PpiC were produced and purified as described previously (Supplementary Materials) [8 (link), 11 (link), 12 (link)]. After the purification procedure, rSagA and rPpiC were used for conjugation at 5 mg/mL. DHG was covalently coupled to the proteins as described by Lees et al, using the cyanylating reagent 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP; Sigma-Aldrich) at 100 mg/mL in acetonitrile [29 (link)]. A solution of 1 mg of DHG in 100 µL of ultrapure water was slowly mixed with 10 µL of CDAP. After 30 seconds, 15 µL of 0.2 M trimethylamine was added. Final coupling was done by adding 1 mg of protein to the mixture, and the reaction was incubated overnight at room temperature. The glycoconjugates (DHG-SagA and DHG-PpiC) were cleaned up with an Amicon ultrafiltration device with a 100-kDa membrane (Merck-Millipore). The correct conjugation process was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (Supplementary Materials). Sugar and protein content were determined by hexose and Bradford assays to establish the polysaccharide to protein ratio in the glycoconjugates (Supplementary Materials).