Table 1.
ST strains and derivatives used in this study.
Table 2.
Anti-ST and control mAbs used in this study.
Fig 1.
Orally administered Sal4 IgA blocks STm invasion into mouse Peyer’s patches.
Adult BALB/c mice were challenged by gavage with a one-to-one mixture of STm O5 and O4 strains STm (4 x 107 CFU total) co-administered with Sal4 IgA or an isotype control. Peyer’s patches were collected ~24 h later and assessed for bacterial loads. (A) Competitive indices and (B) total CFUs of AR05 and AR04 STm. Shown are the combined results of five independent experiments with at least 4 mice per group. Each symbol represents an individual mouse. Statistical significance evaluated for each concentration over the isotype control, as determined by Kruskal-Wallis test and Dunn’s post-hoc test.
Fig 2.
PeA3 IgA blocks wildtype STm invasion in vitro and in vivo.
(A) A 1:1 mixture of AR04 and AR05 STm strains was treated with Sal4 IgA, PeA3 IgA, or isotype control antibody and applied to HeLa cells. Monolayers were then treated with gentamicin to eliminate extracellular bacteria and HeLa cells were lysed. The remaining cell lysate was enumerated for CFUs (n = 3 experiments, each done in triplicate). Panels B, C: Adult BALB/c mice were challenged by gavage with a 1:1 mixture of STm AR04 and AR05 O5 (4 x 107 CFU total) in the present of 30 μg/mL (B) or 10 μg/mL (C) of indicated mAb. The mice were euthanized 24 h later and Peyer’s patches were assessed for bacterial loads. Shown are the combined results of two independent experiments with at least 4 mice per group. Statistical significance evaluated for each concentration over the isotype control, as determined by one-way ANOVA with either Dunnett’s (A, B) or Tukey’s (C) post-hoc tests.
Fig 3.
Sodium bicarbonate and protease inhibitors improve Sal4 IgA prophylactic activity.
Adult BALB/c mice were gavaged with Sal4 IgA (50 μg) in (A) 3% NaHCO3 or (B) a protease inhibitor cocktail 20 min or 1 min before STm challenge. The mice were euthanized 24 h later and Peyer’s patches were assessed for bacterial loads, as a readout of bacterial invasion. Shown are the results of three independent experiments with at least 5 mice per group. Each symbol represents an individual mouse. Statistical significance compared to the isotype control at each time point, as determined by unpaired Student’s t-test.
Fig 4.
Comparison between Sal4 dimeric IgA (dIgA) and secretory IgA (SIgA) preparations in mouse model of STm infection.
Adult BALB/c mice were challenged by gavage with a one-to-one mixture of STm O5 and O4 strains (4 x 107 CFU total). Dimeric IgA (dIgA) or secretory IgA (SIgA) forms of Sal4 were (A) premixed with the bacterial inoculum or (B) administered ~1 min prior to STm challenge. The mice were euthanized 24 h later and Peyer’s patches were assessed for bacterial loads. Shown are the results of two independent experiments with 4 mice per group. Statistical significance evaluated for each concentration over the isotype control, as determined by one-way ANOVA and Tukey’s post-hoc test.
Fig 5.
In vitro functionality of Sal4 IgG.
(A) Effect of Sal4 IgG on STm motility in soft agar. STm was stab-inoculated into 0.3% LB agar containing 15 μg/mL of IgA control, Sal4 IgA, IgG control, or Sal4 IgG antibody. Plates were incubated at 37°C and the diameter of bacterial swimming was measured every hour for 6 h (n = 3 experiments each done in triplicate). (B) A one-to-one mixture of AR05 and AR04 STm (107 CFUs total) were incubated for 10 min with 15 μg/mL of IgA control, Sal4 IgA, IgG control, or Sal4 IgG antibody before being applied to HeLa cell monolayers (MOI ~10), as described in the Materials and Methods. After 1 h, the HeLa cells were lysed and the CFUs were enumerated. Asterisks indicate significant reduction in wildtype STm motility (A) or invasion (B) over the respective isotype control, as determined by unpaired Student’s t-test (n = 2 experiments done in triplicate; P < 0.05).
Fig 6.
Sal4 IgG fails to inhibit STm invasion following oral infection in vivo, but can be partially rescued upon stabilization.
BALB/c females were orally challenged with a competitive index of wildtype (AR05) and oafA mutant (AR04) STm (4 x 107 CFUs) either (A) pre-incubated with 30 μg/mL of antibody in PBS or (B) administered antibody prior to STm challenge at the indicated time points with sodium bicarbonate and protease inhibitors. 24 hours (p.i.) mice were euthanized and Peyer’s patches were harvested and enumerated for CFUs and competitive indices. Statistical significance evaluated for each group over the isotype control, as determined by unpaired Student’s t-test (n = at least 5 mice per group).