FIG 1.

Timeline depicting the intranasal vaccination strategy. Illustration highlights the timeline of the experiments used to test the efficacy and mechanism of protection of the different AuNP protein candidates. BALB/c (n = 10) mice were intranasally immunized with three doses of 50-μl formulations in 2-week intervals. Two weeks after receiving the last immunization, serum and fecal samples were obtained to evaluate humoral responses. Seven days after sample collection, animals were challenged with 9 × 10⁹ cells of EHEC O157:H7 via gavage. Individual intestinal segments were collected 3 or 5 days after infection and processed for CFU enumeration. Two weeks after receiving the last immunization, the intestinal epithelial layer (IEL) was isolated and stimulated in the presence of each antigen for 5 days. After stimulation, samples were analyzed by a multiplex cytokine array. Small-intestine lavage samples from immunized and naive animals were obtained 2 weeks after receiving the last immunization to assess secretory IgA levels.