Aluminum hydroxide gel adjuvant

For each group, female Balb/c mice (6–8 weeks of age, n = 4) were immunized 3 times at day 0, 7 and 21 intramuscularly. Spike protein (Sino Biological, Beijing, China) was dissolved in PBS at the final concentration of 0.1 μg/μl. Alum-3M-052 was a mixture that contained 50 μl 2% aluminum hydroxide gel adjuvant (InvivoGen, France) and 2 μg 3M-052 (MedChemExpress, NJ, USA) in 50 ul PBS. For vaccine test group, 50 μl Spike protein solution and 50 μl Alum-3M-052 were mixed at 150 rpm for 30 min by a shaker (Kylin-Bell, Jiangsu, China) at 4°C. For vaccine control group, 50 μl Spike protein solution and 50 μl 2% aluminum hydroxide gel adjuvant were mixed at 150 rpm for 30 min by a shaker at 4°C. For negative control group, 50 μl PBS and 50 μl 2% aluminum hydroxide gel adjuvant were mixed at 150 rpm for 30 min by a shaker at 4°C. The blood samples were collected 1 week following each immunization.

Mice were s.c. injected at the base of the tail with 100 μL of aP and wP vaccine formulations, provided by Sanofi-Pasteur, Marcy l’Etoile (Lyon), which correspond to 1/5 of the human dose but do not correspond to commercial vaccines as they lack additional components like tetanus or diphtheria toxins. The aP vaccine is composed of 20 μg/mL of PT; 6 μg/mL of PRN; 10 μg/mL of Fim2,3; 10 μg/mL of FHA; and 0.66 mg/mL alum; the wP vaccine is made of 30 opacity units/mL whole-cell pertussis and 2.4 mg/mL alum. Control mice were injected with the aluminum hydroxide gel adjuvant (InvivoGen) diluted 16% in PBS.

Allergen challenge of mice has been described previously [56 (link)]. In brief, mice were sensitized to ovalbumin (OVA; Sigma-Aldrich, St. Louis, MO, USA) by intraperitoneal (I.P.) injection of 100 µg OVA in 100 µL Aluminum Hydroxide Gel Adjuvant (InvivoGen, San Diego, CA, USA) on days 0 and 14. At days 21 to 23, mice were anesthetized (ketamine—90–120 mg/kg and xylazine—6–8 mg/kg) and challenged to OVA by intratracheal (I.T.) instillation of 50 µg OVA in 100 µL saline. Control mice were sham-sensitized with aluminum hydroxide gel and challenged to saline by I.T. instillation. Allergen reaction was allowed to develop for 72 h. CLCA1-conditioned media (N-CLCA1; 100 µL; preparation described below) was administered to control or OVA-challenged animals by I.T. instillation 24 h before sacrificing the animals. Control I.T. instillation was performed with mock-conditioned media. All animal experiments complied with the reporting of in vivo experiments guidelines and were carried out in accordance with the United Kingdom Animals Act, 1986, and associated guidelines, and EU Directive 2010/63/EU for animal experiments. All animal experiments were approved by the local Ethics Committee of the Government of Unterfranken/Wurzburg (AZ: 55.2-2532-2-1359-15) and were conducted according to the guidelines of the American Physiologic Society and German Law for the Welfare of Animals.