2.6. Mouse Vaccination and Challenge

(a) Vaccine formulation and immunization. Formulated vaccines contained 10 µg of P. aeruginosa antigens/dose of PHA beads, mixed with Alhydrogel^® (aluminum hydroxide adjuvant 2%, 25 µL/dose; InvivoGen San Diego, CA, USA) in 200 µL volume incubated for 1 h at room temperature with gentle rotation for adsorption prior to vaccination. Vaccines were mixed with Alhydrogel (InvivoGen) immediately before injection. Animal experiments were approved by the Boston Children’s Hospital IACUC (protocol number 20-12-4326R). FVB/N mice (6–8 weeks old, female) were obtained from the Jackson Laboratories and maintained for both vaccination periods and challenge experiments in the animal facility of Boston Children’s Hospital. Mice were SC vaccinated with Alhydrogel alone (negative control), PHA + Alhydrogel, Ag-PHA + Alhydrogel and PHA-PopB + Alhydrogel suspended in saline. There were 12 mice per group and vaccination was conducted once every two weeks for 6 weeks with a total of 3 vaccinations (Day 0, 14, and 28).

(b) Mouse sera collection. Mouse sera were collected by retro-orbital blood collection on Day 49 of the experiment in serum separator tubes (BD microtainer serum separator tubes). Serum samples were aliquoted and stored at −80 °C until use.

(c) Challenge experiment. For the preparation of the inocula, P. aeruginosa strain N13 was plated from a frozen bacterial stock on tryptic soy agar (TSA) and grown overnight at 37 °C. Bacteria were suspended in 10 mL PBS (Invitrogen) until OD_{650 nm} = 0.55. Bacteria we diluted further ¼ in PBS to prepare the inocula. Inocula were verified by serial dilution and plating for CFU enumeration. Mice (8 mice/group) were anesthetized with ketamine/xylazine and intranasal challenge was performed by the administration of 20 μL of the inoculum, applying 10 μL to each nostril (2 × 10⁶ CFU/dose). Mice were monitored for 7 days, and survival percentage was measured. Moribund mice were euthanized.