Alhydrogel adjuvant 2

Female 6-to-8-week-old C57BL/6 mice were immunized subcutaneously three times at 2-week intervals with 200 μL of the vaccine formulation. The animals were administered either AuNP-EscC, AuNP-Eae, AuNP-EscC + AuNP-Eae, or unconjugated AuNPs. The vaccine formulations contained 10 μg of protein (5 μg of each protein for the combination vaccine) along with 10 μg of detoxified cholera toxin B subunit (Sigma, Cream Ridge, NJ, USA) and 500 μg Alhydrogel^® adjuvant 2% (InvivoGen, San Diego, CA, USA) as the adjuvants. Control mice were given unconjugated AuNPs with the same concentration of adjuvants. A total of 24 mice received each vaccine formula. For antibody titer assessment, whole blood was obtained retro-orbitally using microvette tubes without anticoagulant 1 week before the first vaccination (baseline titers) and 2 weeks after the last boost (immune titers). Sera was isolated by allowing the whole blood to clot at room temperature (RT) for 30 min, followed by centrifugation at 5000× g for 5 min. Sera were collected and stored at −80 °C until use. For fecal IgA titers, fecal samples were collected following the same chronology and resuspended in PBS to a final concentration of 100 mg/mL, homogenized by vortexing, and centrifuged to remove debris. Supernatants were collected and stored at −80 °C.

Animal studies were carried out in accordance with the University of Pittsburgh's IACUC guidelines for the use and care of laboratory animals. Seven to eight week old Balb/cJ female mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Female mice were immunized via intramuscular (i.m.) injection with 50 mcl of vehicle (Naïve and PBS), stabilized RSV prefusion protein (PreF; 10 mcg; Calder Biosciences) alone, or formulated with Advax-SM™ (PreF/Advax-SM; 1 mg/mouse; Vaxine Pty Ltd, Bedford Park, Australia) or Alum (PreF/Alum; 2 mg/mL). Specifically, Alhydrogel adjuvant 2%, an aluminum hydroxide wet gel suspension from InvivoGen, was used in these studies. Advax-SM is composed of microparticles of polyfructofuranosyl-_D-glucose (delta inulin) combined with CpG55.2-ODN (5′ATCGACTCTCGAGCGTTCTC-3′), which was synthesized by GeneDesign (Osaka, Japan). The immunized mice were boosted 3 weeks later with their respective vaccine formulations. At 9 weeks post-prime, mice were challenged intranasally (i.n) with RSV L19 (5 × 10⁵ pfu/gm) and culled at 4 or 8 days post-infection (dpi) using 100% isoflurane and cervical dislocation. RSV L19 was propagated and viral titers quantified as previously described (25 (link)). All animal studies were approved by the University of Pittsburgh Institutional Animal Care and Use Committee; protocol #20047209.

8-week-old female CD1 mice (Charles River Laboratories, Inc., Wilmington, MA, USA) were immunized intraperitoneally with purified F. tularensis subspecies holarctica LVS LPS (NR-2627) (BEI Resources, Manassas, VA, USA) coupled to BSA using the Imject™ EDC BSA Spin Kit (Thermo Scientific, Waltham, MA, USA) to improve immunogenicity. Immunizations were performed with and without Alhydrogel^® adjuvant 2% (Invivogen, San Diego, CA, USA). Mice in both conditions were immunized with 10 μg of F. tularensis LPS-BSA subcutaneously and a further 10 μg given at 6 and 8 weeks post initial immunization. Boosts of 25 μg of F. tularensis LPS-BSA were given at weeks 11 and 13 post immunization. An indirect ELISA was used as outlined below to determine antibody titers to LPS in mouse immune serum. Mice were immunized with a final dose of 5 μg of purified LPS-BSA three days prior to spleen harvest. Fusions were performed using P3x63Ag.653 fusion partner and hybridoma cells produced using standard techniques [54 (link)]. Supernatant was collected from hybridoma cells and mAbs purified using recombinant protein A affinity chromatography.