Polymeric IgA Is Superior to Monomeric IgA and IgG Carrying the Same Variable Domain in Preventing Clostridium difficile Toxin A Damaging of T84 Monolayers

Abstract

The two exotoxins A and B produced by Clostridium difficile are responsible for antibiotic-associated enterocolitis in human and animals. When added apically to human colonic carcinoma-derived T84 cell monolayers, toxin A, but not toxin B, abolished the transepithelial electrical resistance and altered the morphological integrity. Apical addition of suboptimal concentration of toxin A made the cell monolayer sensitive to toxin B. Both toxins induced drastic and rapid epithelial alterations when applied basolaterally with a complete disorganization of tight junctions and vacuolization of the cells. Toxin A-specific IgG2a from hybridoma PCG-4 added apically with toxin A alone or in combination with toxin B abolished the toxin-induced epithelial alterations for up to 8 h. The Ab neutralized basolateral toxin A for 4 h, but not the mixture of the two toxins. Using an identical Ab:Ag ratio, we found that recombinant polymeric IgA (IgAd/p) with the same Fv fragments extended protection against toxin A for at least 24 h in both compartments. In contrast, the recombinant monomeric IgA counterpart behaved as the PCG-4 IgG2a Ab. The direct comparison between different Ig isotype and molecular forms, but of unique specificity, demonstrates that IgAd/p Ab is more efficient in neutralizing toxin A than monomeric IgG and IgA. We conclude that immune protection against C. difficile toxins requires toxin A-specific secretory Abs in the intestinal lumen and IgAd/p specific for both toxins in the lamina propria.

Reconstituted Human Polyclonal Plasma-derived Secretory-like IgM and IgA Maintain the Barrier Function of Epithelial Cells Infected with an Enteropathogen

“…Similar in vitro models have been used previously to compare the neutralizing function of antigen-specific monoclonal IgG and IgA/SIgA (31,39,40). Although one has to acknowledge that the Caco-2 cell line may not recapitulate all functions of normal intestinal epithelial cells, their pattern of cytokines following exposure to S. flexneri was consistent with that of other cell lines and that found in vivo (41,42).…”

Human Plasma-derived Polymeric IgA and IgM Antibodies Associate with Secretory Component to Yield Biologically Active Secretory-like Antibodies

“…Fractionation of pIgA and mIgA Present in Plasma-derived Preparations-Size exclusion chromatography is a robust technique to separate pIgA from mIgA recovered from hybridoma cell supernatants (15) and from cell clones engineered to produce IgA (26). Two different resins and column sizes were used in the present study.…”

Antigen Binding to Secretory Immunoglobulin A Results in Decreased Sensitivity to Intestinal Proteases and Increased Binding to Cellular Fc Receptors

“…A combination of equimolar amounts of purified pIgA and hSC (IgAPCG-4) or mSC (IgA7D9 and IgAC5) for 1 h at room temperature resulted in the formation of covalent heterodimer complexes, as indicated by the detection of shifted SC to the position of pIgA (lanes 3, 6, and 9) when the protein was analyzed under nonreducing conditions. As reported previously, the percentage of covalence varied from one pIgA to another (11,25) as reflected by the appearance of free SC migrating as a band of 80 kDa. When loaded onto a molecular sieving column run in PBS, co-elution of SC and pIgA confirmed that the two proteins were indeed associated in the form of covalent and noncovalent SIgA complexes (15) (data not shown).…”

“…100 ng of C. difficile toxin A was mixed with 50 ng of either pIgAPCG-4 or SIgAPCG-4 permitting neutralization in in vitro assays (11). 60 ng of purified rotavirus was incubated with 100 ng of either pIgA7D9 or SIgA7D9 in PBS for 2 h at room temperature, corresponding to ϳ160 Ab molecules/virus (19).…”

“…Source of Protein-Chimeric IgAPCG-4 specific for C. difficile toxin A was produced as described (11). Ascites fluid (30 ml) was obtained from BALB/c mice inoculated intraperitoneally with 2 ϫ 10 6 IgA7D9 hybridoma cells (anti-VP6 rhesus monkey rotavirus) (12).…”