Intranasal immunization with protein-linked phosphorylcholine protects mice against a lethal intranasal challenge with streptococcus pneumoniae.

Immunization against phosphorylcholine (PC) linked to a protein protects mice against Streptococcus pneumoniae when used parenterally, and against Salmonella typhimurium when used orally after entrapment in D,L-Lactide-co-Glycolide microspheres. Here, we immunized BALB/c mice intranasally with a serotype 3 S. pneumoniae strain. Immunization was followed by a rise in anti-PC IgA and IgG titers in serum and in pulmonary secretions, but not by any rise in anti ds-DNA antibody nor any glomerular Ig deposition. The survival rates were 91 and 76% in the two groups of mice, respectively. These rates were significantly higher than those in control mice immunized intranasally either with Thyr loaded in microspheres (0%), blank microspheres (22%), free Thyr (17%), and saline (18%). This demonstrates that the mucosal route is effective for vaccination against S. pneumoniae pneumonia with PC linked to a protein carrier. It constitutes another important step forward in the development of the concept that PC can be used as a mucosal immunogen for protection against the different diseases caused by PC-bearing bacteria.

In vivo fate and immune pulmonary response after nasal administration of microspheres loaded with phosphorylcholine-thyroglobulin.

Phosphorylcholine is a widely occurring hapten which is present in the cell wall of many prokaryotes. It is, therefore, an attractive candidate for the development of a vaccine against many bacterial diseases. Poly(D,L-lactide-co-glycolide) microspheres loaded with phosphorylcholine linked to thyroglobulin (PC-Thyr) as protein carrier were prepared. The effect of the protein concentration on antigen encapsulation and release as well as on microsphere morphology has been investigated. When administered intranasally, PC-Thyr-loaded microspheres were taken up by epithelial cells of the nasopharyngeal associated lymphoid tissue and induced a specific IgA and IgG response in pulmonary secretions as well as a strong systemic immune response in BALB/c mice.

Mucosal immunogenicity elicited in mice by oral vaccination with phosphorylcholine encapsulated in poly (D,L-lactide-co-glycolide) microspheres.

Poly(D,L-lactide-co-glycolide) microspheres loaded with phosphorylcholine linked to thyroglobulin (PC-Thyr) as protein carrier were prepared. The entrapment efficiency reached 80% when the initial protein:polymer ratio was 1:8. Ninety-four percent of the loaded microspheres had a diameter < or = 10 microns. The encapsulation process did not alter PC-Thyr absorbance nor PC antigenic reactivity. Oral administration of these microspheres induced a specific IgA response in intestinal, pulmonary and vaginal secretions, as well as a strong specific systemic immune response in female Balb/c mice. This suggests the need to explore further the potential ability of PC-Thyr loaded microspheres to protect against infections caused by PC-bearing microorganisms which invade or colonize different mucosal sites.

Protective immunity against Salmonella typhimurium elicited in mice by oral vaccination with phosphorylcholine encapsulated in poly(DL-lactide-co-glycolide) microspheres.

Encapsulation of vaccines in biodegradable microspheres provides excellent mucosal immunogens with a high potential for immunization against bacterial infections. We tested the protective immunity elicited by intragastric vaccination with phosphorylcholine (PC) encapsulated in poly(DL-lactide-co-glycolide) (DL-PLG) microspheres against Salmonella typhimurium in a mouse model of invasive intestinal infection. We chose PC as the antigen because it was found to elicit an immune response after intestinal exposure of mice to PC-bearing S. typhimurium and because anti-PC immunity protects mice against Streptococcus pneumoniae, another PC-bearing microorganism. Mice were primed intragastrically on days 1, 2, and 3 and boosted on days 28, 29, and 30 with PC (280 microg) coupled to porcine thyroglobulin (PC-thyr) encapsulated in DL-PLG microspheres, free PC-thyr, or blank microspheres. A significant rise in anti-PC immunoglobulin A (IgA) titers, as measured by an enzyme-linked immunosorbent assay, was observed in the intestinal secretions after immunization with PC-loaded microspheres, compared to the titers of mice immunized with free PC-thyr or blank microspheres. This antibody response peaked 14 days after the last boost and correlated with a highly significant resistance to oral challenge by S. typhimurium C5 (P < 10(-3)). Control mice were primed intraperitoneally on day 1 with 15 microg of PC in complete Freund's adjuvant and boosted on days 10, 14, and 20 with the same dose without adjuvant but via the same route. In these mice, the levels of anti-PC IgA in intestinal secretions were equivalent to those of the mice intragastrically immunized with PC-loaded microspheres, but protection was significantly weaker, suggesting that either the IgAs were not functional or that other immune mechanisms are important in protection. Taken together, our results highlight the potential of antigen encapsulation in DL-PLG microspheres for eliciting protective immunity against invasive intestinal bacterial diseases and suggest that a similar strategy could be used against diseases caused by other PC-bearing microorganisms.

Cloning of a genetic determinant from Clostridium difficile involved in adherence to tissue culture cells and mucus.

Our laboratory has previously shown that Clostridium difficile adherence to Caco-2 cells is greatly enhanced after heat shock at 60 degrees C and that it is mediated by a proteinaceous surface component. The experiments described here show that C. difficile could adhere to several types of tissue culture cells (Vero, HeLa, and KB) after heat shock. The type of culture medium (liquid or solid, with or without blood) had little effect on adhesion. To clone the adhesin gene, polyclonal antibodies against C. difficile heated at 60 degrees C were used to screen a genomic library of C. difficile constructed in lambda ZapII. Ten positive clones were identified in the library, one of which (pCL6) agglutinated several types of erythrocytes in the presence of mannose. In Western blots (immunoblots), this clone expressed in Escherichia coli a 40- and a 27-kDa protein; a 27-kDa protein has been previously identified in the surface extracts of heat-shocked C. difficile as a possible adhesin. The clone adhered to Vero, Caco-2, KB, and HeLa cells; the adherence was blocked by anti-C. difficile antibodies, by a surface extract of C. difficile, and by mucus isolated from axenic mice. Furthermore, the clone could attach ex vivo to intestinal mucus isolated from axenic mice. Preliminary studies on the receptor moieties implicated in C. difficile adhesion revealed that glucose and galactose could partially block adhesion to tissue culture cells, as did di- or trisaccharides containing these sugars, suggesting that the adhesin is a lectin. In addition, N-acetylgalactosamine, a component of mucus, and gelatin partially impeded cell attachment.