FIG. 1.

Mice repeatedly inoculated with ETEC mount immune responses to ETEC TPS proteins and are protected from subsequent challenge. (A) Mouse inoculations and challenge timeline. Mice were inoculated with ∼10⁷ CFU of either avirulent strain AAEC191A (▿) or ETEC prototype strain H10407 (▾) on days 0, 14, and 68. Sera and stool stamples were collected on days 0 and 95 prior to challenge, and samples were frozen at −80°C for subsequent ELISA. (B) ELISA determination of total serum antibody to EtpA or EtpB after repeated inoculation with AAEC191A (○) or H10407 (•). ELISA values represent net increases in OD₄₅₀ from pre- to postimmune (**, P = 0.014; *, P = 0.047). (C) ELISA of fecal antibody responses to EtpA (P = 0.012) and EtpB (P = 0.012). All values reflect determination of total IgG, IgA, and IgM. (D) Mouse challenge. Both groups of mice (those previously inoculated with H10407 [•] or AAEC191A [○]) were challenged with H10407 at two different doses: 6 × 10⁷ CFU and 6 × 10³ CFU. After 24 h, mice were sacrificed, and the degree of ileal colonization was determined by plate counting. (*, P = 0.029 and P = 0.016 for the 10⁷ and 10³ CFU challenge groups, respectively, as determined by two-tailed Mann-Whitney test). (For two mice in the 10³ challenge group, no bacteria were recovered. These were arbitrarily assigned values of 1 CFU, the theoretical limit of detection.) Dashed horizontal lines represent the geometric mean values for each group. α, anti.