Vaccine formulations and immunization schedule

Animal studies were performed in accordance with the United States National Research Council Guide for the Care and Use of Laboratory Animals. The animal protocol was approved by the Institutional Animal Care and Use Committee at The University of Texas at Austin.

OVA as an antigen was adsorbed on Alhydrogel® by mixing them at a 1:4 ratio (OVA: Alhydrogel®, w/w) in normal saline and stirring at 4°C overnight. To determine the binding isotherm of OVA to Alhydrogel®, normal saline-diluted Alhydrogel® suspension and OVA were mixed to reach a final aluminum concentration of 0.25 mg/mL and OVA concentrations ranging from 0.025 to 4.5 mg/mL. After stirring for 16 h at 4°C, the adjuvant-protein mixtures in suspension were centrifuged at 14,000 g for 5 min at room temperature. The protein concentration in the supernatants was determined using the BCA Protein Assay kit. The amount of protein absorbed onto the Alhydrogel® was calculated by subtracting the amount of protein in the supernatant from the amount of the initially added protein. The adsorption efficiency of OVA to Alhydrogel® reached ∼100% when the Alhydrogel® to OVA weight ratio was equal or above 1:1 (data not shown). However, at the weight ratio of 4:1, the resultant OVA/Alhydrogel® preparation was most stable, having the least precipitation after 2 weeks of storage at 4^oC (data not shown). BALB/c mice (6–8 weeks, female, Charles River Laboratories, Wilmington, MA) were randomized, lightly anesthetized with a ketamine and xylazine mixture, and then immunized intranasally by applying the OVA/Alhydrogel® liquid suspension to the nostrils of the mice using a fine pipet tip (20 μL total, 10 μL per nostril) 3 times, 2 weeks apart. Mice in control groups were intranasally dosed with OVA alone, OVA mixed with MPLA (OVA/MPLA), or left untreated. The doses of OVA, Alhydrogel®, and MPLA were 5, 20 and 5 μg/mouse/dose, respectively.

The 3 × M2e-HA2 antigen was adsorbed onto Alhydrogel® by mixing the protein in a normal saline solution and the Alhydrogel® in suspension at a 1:10 ratio (w/w) and stirring overnight at 4°C. The adsorption efficiency of 3 × M2e-HA2 to Alhydrogel® at such a ratio was determined to be 97.7 ± 0.1%. BALB/c mice (6–8 weeks, female) were immunized intranasally with the 3 × M2e-HA2/Alhydrogel® in suspension 3 times, 2 weeks apart. Mice in the control groups were intranasally dosed with 3 × M2e-HA2 alone, 3 × M2e-HA2 mixed with MPLA (3 × M2e-HA2/MPLA), or left untreated. The doses of the 3 × M2e-HA2, Alhydrogel®, and MPLA were 5, 50, and 5 μg/mouse/dose, respectively.

The size and size distribution of the Alhydrogel® and the antigen-adsorbed Alhydrogel® were measured using a Sympatec HELOS laser diffraction instrument equipped with a R3 lens (Sympatec GmbH, Germany). Antigen was mixed with MPLA at a 1:1 ratio (w/w) in normal saline and stirring at 4°C overnight, and the particle size and size distribution of the MPLA and antigen/MPLA were determined using a Malvern Zeta Sizer Nano ZS (Westborough, MA). In both animal studies, mice were killed 2 weeks after the last immunization to collect blood and spleen. Nasal wash and BAL samples were collected as described previously using 500 μL of sterile PBS.⁴¹ Blood samples were centrifuged to collect sera. Spleens were used to prepare splenocyte suspensions from individual mouse as described previously.⁴²