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Systemic vaccination does not effectively induce a protective immune response to Streptococcus pneumoniae infections in infants, elderly people and immunosuppressed patients, who are highly susceptible to this pathogen. As mucosal immune responses develop early in life and still function well in the elderly, mucosal vaccination (that is, by oral, intratracheal or intranasal routes) may be an alternative approach to current strategies.

The immune response to pneumococcal polysaccharide type 14 (PPS-14), induced by native PPS-14 was compared with the response induced by PPS-14 conjugated with CRM197 (PPS-14-CRM197). In our animal model, immunization with PPS-14-CRM197 gave a significant enhancement of anti-PPS-14 serum titres for IgM and IgG, but not for IgA. Also an increase in total number of anti-PPS-14 antibody-secreting cells was found. Using immunohistochemical techniques, a different distribution pattern of specific antibody-containing cells in spleen section after immunization with PPS-14-CRM197 was observed. Furthermore, a higher number of IFN-gamma producing cells was found after immunization with PPS-14-CRM197, as compared with immunization with PPS-14. This enhanced IFN-gamma production may be the cause for enhanced IgG response observed after immunization with PPS-14-CRM197. The specific immune response was less affected by splenectomy in animals immunized with PPS-14-CRM197 than with PPS-14. However, an age-related response to the native as well as the conjugated form of the PPS-14 was observed, since no effect of conjugation with CRM197 was seen in the onset of the immune response to PPS-14 in young animals. In conclusion, our results affirm the hypothesis that conjugation of polysaccharides changes the characteristics of the antigen towards a thymus-dependent antigen.

The role of the spleen in the rat mucosal immune response was investigated to three structural different pneumococcal polysaccharides, type 3, 4, and 14. Following immunization with pneumococcal polysaccharides, a larger amount of free antigen was found in several lymphoid tissues and an increased trapping of immune complexes was seen in follicles of splenectomized animals, as compared to control animals. Thus, clearance of the polysaccharides seems to be less effective after splenectomy. An increase in specific IgA antibody-containing cells (ACC) was found in mesenteric lymph nodes, villi and Peyer's patches in splenectomized rats. Apparently, splenectomy and subsequent decreased clearance of the antigen causes a prolonged stay of the antigen in the system and therefore specific ACC can be induced in different lymphoid tissues. After splenectomy the specific IgM and IgG antibody titers in serum decreased significantly for pneumococcal polysaccharides types 4 and 14, but not for type 3. Furthermore, the serum IgA antibody titers against the three types of polysaccharides under study were not affected. After elimination of macrophages in the spleen by treatment with dichloromethylene diphosphonate liposomes no ACC against type 14 were evoked in the marginal zone of the spleen, and again, an increase was observed in specific IgA ACC in mucosa-associated lymphoid tissues. The IgA antibody titers were also enhanced. In conclusion, IgA responses against pneumococcal polysaccharides can be elicited in absence of the spleen, i.e. at mucosal sites or in the draining lymph nodes. Furthermore, polysaccharide-specific IgA responses are enhanced after reduction of splenic phagocytic functions.

Gut mucosal immune responses to bacterial polysaccharide antigen in rats were investigated in vivo. Rats were immunized with pneumococcal polysaccharide type 3 (PPS-3) via different routes, i.e. in the Peyer's patch (iPP), in the colon (ic), in the peritoneal cavity (ip), and intravenously (iv). The development of specific antibody-forming cells (AFC) and their isotypes in the intestinal mucosa, gut-associated lymphoid tissue (GALT), mesenteric lymph nodes (MLN) and spleen were studied by immunohistochemistry. Furthermore, the serum antibody levels were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that iPP immunization evoked high numbers of anti-PPS-3 AFC of the IgA isotype in the mucosa of the small intestine and in the PP. On the contrary, the ic route did not elicit a mucosal response, though a few AFC were found in the MLN and spleen. Following ip priming, a specific IgA response was found, especially in MLN and spleen, and a low response was detected in the villi. A high response was found in the parathymic lymph nodes (PTLN). Iv immunization gave rise to the development of AFC in the spleen, particularly of the IgM isotype. We failed to induce mucosal responses to PPS-3 antigen in the colon, irrespective of the route of immunization.

Four immunization routes were investigated to induce an immune response against three structurally different types of pneumoccoccal polysaccharide (PPS) in the rat. In particular, the contribution of the IgA isotype in these immune responses was studied. Six days after administration of PPS type 3, 4 or 14, the localization of specific antibody-containing cells (ACC) in different lymphoid tissues and the antibody titres in serum were studied. All four routes induced anti-PPS ACC in the spleen. After intraduodenal, intravenous and especially intraperitoneal administration of PPS, many IgA-specific anti-PPS ACC were also found in parathymic and mesenteric lymph nodes and in the lamina propria of intestinal tissue. Several anti-PPS ACC were found in Peyer's patches, located peripheral of the B-cell areas. The intratracheal immunization elicited only a local immune response, in bronchus-associated lymphoid tissues and paratracheal lymph nodes. The localization of these anti-PPS ACC was influenced by the route of immunization. After all four investigated routes, specific antibodies were found in serum against PPS. However, some remarkable differences between PPS-3, 4 and 14 were found in the magnitude of the immune response and the distribution of the isotypes. Both route of immunization and structure of the PPS have a profound influence on the immune responses in rats.

Two different methods have been used to study immune responses in the rat to Streptococcus pneumoniae type 3 and type 4 capsular polysaccharides (PPS). First, for simultaneous detection of the specificity and isotype of anti-PPS antibody-containing cells (ACC) in cryostat sections of lymphoid tissue, a double immunocytochemical method was developed. This method is a combination of a three-step immunoperoxidase method to demonstrate specific anti-PPS ACC as bright red cells and a two-step immunophosphatase method to detect the isotype of ACC as blue cells. Double positive cells appear violet. Using this staining procedure, the detection of antigen was also possible. Second, to study the anti-PPS response in serum, an ELISA procedure was modified. In this ELISA, polyvinylchloride microtiter plates are coated directly with type-specific pneumococcal polysaccharide. After intraperitoneal (i.p.) immunization of rats with PPS-3 or PPS-4, both antigen (PPS) and specific ACC could be detected. Specific ACC were found in the spleen and mesenteric lymph nodes. In the spleen, the specific ACC were found in the red pulp, marginal zone, outer PALS, and follicles. Most of these ACC were IgM-positive and to a lesser extent IgG-positive and IgA-positive. However, specific ACC in mesenteric lymph nodes were predominantly of the IgA isotype, with only few IgM or IgG positive cells. The anti-PPS response in serum, as measured by the ELISA, consisted mainly of IgM antibodies with small amounts of IgG and IgA. Both methods were found to be valuable in studies of immune responses against bacterial polysaccharides.