Salmonella vaccine H683-pC1 stimulates elevated anti-CFA/I fimbriae Ab titers and protects against i.p. ETEC challenge. (a) BALB/c mice (5/group) were orally immunized twice on days 0 and 28 with 5 × 10⁹ CFUs of S. Typhimurium H683-pC1 and H647, respectively. Elevated serum IgG and mucosal IgA anti-CFA/I fimbriae Ab responses were induced. Depicted are the mean ± SEM from two independent experiments. (b) At wk 6, serum IgG subclass analysis was performed, and statistical differences in the endpoint Ab titers were determined. *P < 0.05, **P < 0.01, ***P < 0.001 versus IgG1; ^¶¶P < 0.01 versus IgG2a. (c)–(e) Comparison of different routes of challenges with wt ETEC H10407 among CFA/I fimbriae-immunized BALB/c mice. At 6-wk postprimary immunization, mice were challenged with wt ETEC H10407 (c) orally with 5 × 10⁸ or 5 × 10⁹ CFUs, (d) i.n. with 5 × 10⁸ or 5 × 10⁹ CFUs, or (e) i.p. with 5 × 10⁷ CFUs. Mice survival was monitored for two wks. Results are from two independent experiments; survival fractions obtained from vaccinated mice were compared to H647-immunized mice, and significance was determined; *P < 0.05.

Evaluation of the protective immunity of the detoxed CFA/I fimbriae. (a)–(d) Endpoint Ab titers were induced to detoxed CFA/I fimbriae. BALB/c mice (7/group) were i.m. immunized with (a) 60, (b) 100, (c) 150, or (d) 0 μg CFA/I fimbriae on days 0 and 14. Serum IgG and mucosal IgA anti-CFA/I fimbriae endpoint Ab titers were measured at 0, 2, and 3-wk postprimary immunization. IgG subclass responses were determined at wk 3, and *P < 0.05, **P < 0.01, and ***P < 0.001 versus IgG1 titers. (e) Assessment of the protective efficacy against i.p. ETEC challenge was determined. CFA/I fimbriae-immunized mice (a)–(c) and sPBS-dosed mice (d) were i.p. challenged with 5 × 10⁷ CFUs of wt ETEC H10407 at 4-wk postprimary immunization. Mouse survival rates were observed for 14 days. The survival rates of 60 μg, 100 μg, and 150 μg CFA/I fimbriae-immunized mice were 100% (7/7), 71.4% (5/7), and 42.9% (3/7), respectively, versus 14.3% (1/7) for sPBS-dosed mice. Survival fractions obtained from vaccinated mice were compared to sPBS-dosed mice, and significance was determined: *P < 0.05.

Evaluation of protective immunity induced by a low dose of CFA/I fimbriae. BALB/c mice (6–8/group) were i.m. immunized with (a) 30, (b) 10, and (c) 0 μg detoxed CFA/I fimbriae with or without CT on day 0. (a, b) Elevated serum IgG and mucosal IgA anti-CFA/I Ab fimbriae responses were induced by 2 wks after immunization (c), but not by control mice. Depicted is the mean ± SD as **P < 0.01 and ***P < 0.001 versus 10 μg CFA/I fimbriae-immunized mice. (d) Assessment of the protective efficacy by the low-dose CFA/I fimbriae-immunized mice. The CFA/I fimbriae-immunized and sPBS-dosed mice (a)–(c) were i.p. challenged with 5 × 10⁷ CFUs of wt ETEC H10407 at 4 wks after immunization. Mouse survival rates were determined for 14 days. Both 30 μg CFA/I + 2.5 μg CT-immunized mice and 10 μg CFA/I + 2.5 μg CT-immunized mice survived (8/8); the survival rates for 30 μg CFA/I- and 10 μg CFA/I-immunized mice were 87.5% (7/8) and 62.5% (5/8), respectively; the 2.5 μg CT-immunized mice showed 25% (2/8) survival; and the sPBS-dosed mice all succumbed to challenge (0/6). Survival fractions obtained from 30 μg CFA/I + 2.5 μg CT-immunized mice and 10 μg CFA/I + 2.5 μg CT-immunized mice were compared to sPBS-dosed mice and were determined, *P < 0.05 and ^†P < 0.05, respectively.

ETEC colonization in CFA/I fimbriae-immunized mice. Determination of ETEC colonization in mouse spleen, liver, lungs, and kidneys subsequent to ETEC challenge at (a) 4, (b) 8, and (c) 16 hr by CFA/I fimbriae-immunized (10 μg CFA/I fimbriae + 2.5 μg CT) or PBS-dosed mice. Depicted is the mean ± SD for differences in bacterial CFUs between CFA/I-dosed and sPBS-dosed mice; *P < 0.05 and **P < 0.01.

The sera from the CFA/I-immunized mice are effective in killing wt CFA/I⁺ ETEC H10407 but not K99⁺ ETEC B41. The sera of the 60 μg CFA/I-immunized mice were compared to those from sPBS-dosed mice (Figure 1) for bactericidal activity serially diluted (a, e) 2-, (b, f) 4-, (c, g) 8-, and (d, h) 16-fold. Depicted are the percentages of the survival bacterial CFU relevant to the initial bacterial CFU before supplement of sera (time = 0 min). At 20, 40, and 60 min after incubation, the bacterial survival rates were statistically calculated between anti-CFA/I sera and the -sPBS sera via Student's t-test: *P < 0.05, **P < 0.01, and ***P < 0.001. Values are the mean ± SEM (n = 3).