Protection against Invasion of the Mouse Pulmonary Epithelium by a Monoclonal IgA Directed against Shigella flexneri Lipopolysaccharide

Abstract

Bacteria belonging to the genus Shigellu cause a dysenteric syndrome in humans by invading the colonic mucosa.' Systemic as well as mucosal immune responses elicited in infected hosts after natural or experimental infections are directed mainly against the lipopolysaccharide and against a set of proteins associated with invasion and encoded by a 220-kb virulence plasmid.* Despite many studies, the respective role of systemic and mucosal responses in the protection against shigellosis remains to be determined.To study the role of the mucosal immune response directed against the lipopolysaccharide, we generated a hybridoma producing a monoclonal immunoglobulin A (mIgA) antibody directed against a specific determinant of the S. jlexneri serotype. 5 lipopolysaccharide. This hybridoma was obtained by oral immunization of BALB/ c mice with virulent live bacteria and subsequently fusing Peyer's patch lymphoblasts with mouse myeloma cells. The correlation between the presence of mIgAC5 and protection was studied by using the "backpack" model.3 Subcutaneous hybridoma tumors were obtained by injecting mIgAC5 hybridoma cells in the upper back of mice. These tumor-bearing mice were secreting mIgAC5 at the mucosal level. In the absence of a mouse model of intestinal infection by S. jlexneri, a model of invasion of the pulmonary epithelium was used.4 Tumor-bearing mice secreting mIgAC5 into their respiratory tract were challenged intranasally with S. pexneri strains. The level of secretion of interleukin-6 (IL-6), a key member of the cytokine network involved in the host defense against infection, and the intensity of histopathologic damage of lung tissues were measured to evaluate the severity of the infection process in the presence and the absence of mIgAC5. After S. jlexneri 5-intranasal challenge, but not S. j7exneri 2-challenge, levels of IL-6 were significantly lower in mice secreting 356

Inhibition of Salmonella enterica Serovar Typhimurium Motility and Entry into Epithelial Cells by a Protective Antilipopolysaccharide Monoclonal Immunoglobulin A Antibody

“…Despite the recognized importance of anti-LPS SIgA antibodies in preventing the colonization and invasion of epithelial cells by enteric pathogens (3,9,12,27,36,37,42,44,53), the underlying mechanisms by which these antibodies function in mucosal immunity remains unclear. In this study, we have demonstrated that Sal4, an anti-LPS monoclonal IgA that was previously shown to protect mice against oral challenge with serovar Typhimurium bacteria, is a potent inhibitor of serovar Typhimurium flagellum-based motility.…”

Recognition of Gram-positive Intestinal Bacteria by Hybridoma- and Colostrum-derived Secretory Immunoglobulin A Is Mediated by Carbohydrates

“…Cell Lines and Protein Production-Hybridoma cells producing IgAC5 (31) or IgGC20 (32) specific for Shigella flexneri serotype 5a LPS, IgASal4 specific for Salmonella typhimurium LPS (33), and IgAHNK20 specific for the respiratory syncytial virus fusion (F) glycoprotein (34) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 2 mM sodium pyruvate, 10 mM HEPES (pH 7.0), 0.1 mM folic acid, 100 units/ml penicillin, and 100 g/ml streptomycin as described (31,32). Cell cultures were conducted in Celline-350 bioreactors (Intregra Biosciences AG).…”

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

“…One colony was picked and cultured in Luria-Bertani medium (1% Bacto TM tryptone, 0.5% Bacto-yeast extracts (Sigma-Aldrich)) and 1% NaCl for 1 h at 37°C before being spread onto Luria-Bertani agar plates (1.5% Bacto TM Agar in Luria-Bertani medium). Bacteria grown overnight as a lawn were recovered in 0.9% NaCl, and their concentration was determined by spectrophotometry (Ultrospec 300; Pharmacia Biotech) using the following correlation: 1 A 600 corresponds to 5 ϫ 10 8 bacteria/ml (17). Preparation of Rotavirus-The bovine rotavirus (RF strain) was propagated in monkey kidney MA104 cells in presence of trypsin as described previously (18).…”