SummaryTo determine the role of humoral mucosal immune response in protection against shigellosis, we have obtained a monoclonal dimeric immunoglobulin A (IgA) antibody specific for Shigella flexneri serotype 5a lipopolysaccharide (mlgA) and used a routine pulmonary infection model that mimics the lesions occurring in natural intestinal infection. Adult BALB/c mice challenged with 10 7 S. flexneri organisms developed a rapid inflammatory response characterized by polymorphonuclear cell infiltration around and within the bronchi and strong systemic interleukin 6 response. Implantation of hybridoma cells in the back of mice, resulting in the development of a myeloma tumor producing mlgA in the serum and subsequently secretory mlgA in local secretions, or direct intranasal administration of these antibodies, protected the animals against subsequent intranasal challenge with S. flexneri serotype 5a. Absence of histopathological lesion and significant decrease in bacterial load of the lungs and of systemic interleukin 6 response were the three major criteria of protection. This protection was shown to be serotype-specific and dependent on local concentration of mlgA. These data demonstrate that mucosal antibodies directed against a single polysaccharidic surface epitope of Shigella can protect against the disease.

Oral administration of rabbit secretory IgA (sIgA) to adult BALB/c mice induced IgA ؉ , IgM ؉ , and IgG ؉ lymphoblasts in the Peyer's patches, whose fusion with myeloma cells resulted in hybridomas producing IgA, IgM, and IgG1 antibodies to the secretory component (SC). This suggests that SC could serve as a vector to target protective epitopes into mucosal lymphoid tissue and elicit an immune response. We tested this concept by inserting a Shigella flexneri invasin B epitope into SC, which, following reassociation with IgA, was delivered orally to mice. To identify potential insertion sites at the surface of SC, we constructed a molecular model of the first and second Ig-like domains of rabbit SC. A surface epitope recognized by an SC-specific antibody was mapped to the loop connecting the E and F ␤ strands of domain I. This 8-amino acid sequence was replaced by a 9-amino acid linear epitope from S. flexneri invasin B. We found that cellular trafficking of recombinant SC produced in mammalian CV-1 cells was drastically altered and resulted in a 50-fold lower rate of secretion. However, purification of chimeric SC could be achieved by Ni 2؉ -chelate affinity chromatoraphy. Both wild-type and chimeric SC bound to dimeric IgA, but not to monomeric IgA. Reconstituted sIgA carrying the invasin B epitope within the SC moiety triggers the appearance of seric and salivary invasin B-specific antibodies. Thus, neo-antigenized sIgA can serve as a mucosal vaccine delivery system inducing systemic and mucosal immune responses.

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