Inhibition of invasive salmonella by orally administered IgA and IgG monoclonal antibodies

Abstract

Non-typhoidal Salmonella enterica strains, including serovar Typhimurium (STm), are an emerging cause of invasive disease among children and the immunocompromised, especially in regions of sub-Saharan Africa. STm invades the intestinal mucosa through Peyer's patch tissues before disseminating systemically. While vaccine development efforts are ongoing, the emergence of multidrug resistant strains of STm affirms the need to seek alternative strategies to protect high-risk individuals from infection. In this report, we investigated the potential of an orally administered O5 serotype-specific IgA monoclonal antibody (mAb), called Sal4, to prevent infection of invasive Salmonella enterica serovar Typhimurium (STm) in mice. Sal4 IgA was delivered to mice prior to or concurrently with STm challenge. Infectivity was measured as bacterial burden in Peyer's patch tissues one day after challenge. Using this model, we defined the minimal amount of Sal4 IgA required to significantly reduce STm uptake into Peyer's patches. The relative efficacy of Sal4 in dimeric and secretory IgA (SIgA) forms was compared. To assess the role of isotype in oral passive immunization, we engineered a recombinant IgG1 mAb carrying the Sal4 variable regions and evaluated its ability to block invasion of STm into epithelial cells in vitro and Peyer's patch tissues. Our results demonstrate the potential of orally administered monoclonal IgA and SIgA, but not IgG, to passively immunize against invasive Salmonella. Nonetheless, the prophylactic window of IgA/SIgA in the mouse was on the order of minutes, underscoring the need to develop formulations to protect mAbs in the gastric environment and to permit sustained release in the small intestine.

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).