Lps eb vaccigrade

8 weeks old female C57BL/6 mice were ordered from The Jackson Laboratory and housed in pathogen-free conditions at the core animal facility at the University of Pittsburgh Medical Center with the approval from the University of Pittsburgh Institutional Animal Care and Use Committee. 40μg recombinant RBD plus 5μg LPS-EB VacciGrade^™ (InvivoGen) was given to isoflurane anesthetized mice in sterile PBS (50μL) intranasally on day 0 and day 7 and sacrificed 2 weeks after the last immunization for spleen and lung harvesting.

For vaccine preparation, OVA was dissolved in endotoxin-free water and mixed or emulsified with the different commercial adjuvants according to the manufacturer’s instructions. Briefly, LPS-EB Vaccigrade™ (LPS), Chitosan Vaccigrade™ (Chitosan), and Quil-A^® (InvivoGen) were mixed with the antigen at working concentrations of 25, 100, and 10 µg per mouse, respectively, in physiological solution. The other emulsion adjuvants were prepared by agitation (1400 rpm at RT) for at least 30 min. Imject™ Alum adjuvant (Alum, Thermo Scientific) was added to the antigen suspension at a final volume ratio of 1:1, and the same procedure was used to prepare the emulsion of the antigen with complete and incomplete Freund’s adjuvant (CFA/IFA, Sigma-Aldrich). AddaVax™ (InvivoGen) and Montanide™ ISA 51 and Montanide™ ISA 720 (Seppic) were emulsified at a 50/50 (v/v) ratio with the antigen dissolved in water.