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The concept of directing specific effector B cells not only to mucosal tissues in general but to a selected part of the gastrointestinal tract is of potential importance when constructing vaccines against pathogenic microorganisms affecting different parts of the intestine, e.g., Vibrio cholerae, enterotoxigenic Escherichia coli, and rotavirus in the small intestine and Shigella spp. and Clostridium difficile in the large intestine. In this study, we wanted to gain detailed information about the anatomical distribution of antibodies and antibody-secreting cells (ASC) in different parts of the intestinal tract of primates following oral and rectal immunizations compared to that after intradermal immunization. For this purpose, cynomolgus monkeys (Macaca fascicularis) were immunized with cholera toxin (CT) (List Biological Laboratories, Inc., Campbell, Calif.) three to five times, 3 to 6 weeks apart. Intragastric vaccination was performed by administering 50 µg of CT in 5 ml of a 2.8% bicarbonate-1.1% citric acid buffer (pH 7.0) (CT buffer) through a baby-feeding tube into the stomach. Rectal vaccination was performed by instilling 50 µg of CT in 1 ml of CT buffer for 5 min through a tube between two inflated ballons placed in the rectum 2 to 5 cm from the anus. Intradermal injections of 2 µg of CT were given in 0.2 ml of phosphate-buffered saline. Seven days after the final immunization, animals were sacrificed by an intracardiac injection of a lethal dose of thiopental sodium (500 mg) (Pentothal Natrium; Abbott S.p.A.-Campoverde LT.). The small and large intestines were collected and divided into duodenum, ileum, jejunum, ascending colon, transverse colon, descending colon, and rectum. The preparation of mononuclear cell suspensions and the extraction of immunoglobulins from tissue were performed exactly as described previously (4). The numbers of specific ASC were analyzed in a CT-specific enzyme-linked immunospot assay as described previously (3) and are expressed as the means and standard errors of the mean of specific ASC. Specific antibody titers in tissue extracts were analyzed by a GM1-CT enzyme-linked immunosorbent assay (4) and were estimated as the interpolated sample dilution giving an absorbance of 0.4 above the background level (6, 7). Enzyme-linked immunosorbent assay data are expressed as the specific antibody titer in 1 mg of tissue per ml. Pearson's correlation coefficient (r) was determined with Microsoft Excel 97.

Specific ASC in the intestine after oral, rectal, and intradermal immunizations. We immunized monkeys three to five times orally (three animals), rectally (two animals), or intradermally (three animals) with CT and evaluated the numbers of specific ASC in anatomically distinct parts of the intestine.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   CT-specific IgA-ASC and IgG-ASC in the intestinal tract following oral and rectal immunizations with CT. Data are expressed as means plus the standard errors of the mean of CT-specific IgA-ASC (A) and IgG-ASC (B) titers measured 7 days after the last booster immunization. Black bars represent oral immunizations (n = 3) and white bars represent rectal immunizations (n = 2). DU, duodenum; JE, jejunum; IL, ileum; AC, ascending colon; TC, transverse colon; DC, descending colon; R, rectum.

Specific intestinal tissue antibody levels after oral, rectal, or intradermal immunizations. We also analyzed the anti-CT antibody levels in saponin-extracted tissue from the small and large intestines of an additional set of monkeys receiving oral, rectal, or intradermal immunization (three animals per group). As with the ASC responses, oral immunization proved to be the best way of inducing high levels of specific antibodies in the small intestine, and the levels of specific antibodies in different parts of the small intestine followed the same pattern as the CT-specific ASC, with high titers of specific IgA and IgG in the duodenum and lower levels in the jejunum and ileum (Fig. 2). Oral immunization also induced specific IgG (but not IgA) in the ascending colon.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   CT-specific antibody concentration in the intestinal mucosa after oral, rectal, and intradermal immunizations with CT. Data are expressed as the means plus the standard errors of the mean of the IgA (A) and IgG (B) titers obtained in the protein extract from 1 mg of intestinal tissue per ml taken 7 days after the last immunization. Black bars represent oral immunizations (n = 3), white bars represent rectal immunizations (n = 3), and grey bars represent intradermal immunizations (n = 3). DU, duodenum; JE, jejunum; IL, ileum; AC, ascending colon; TC, transverse colon; DC, descending colon; R, rectum.

Relation of immune responses to vascularization. This distribution of specific antibodies and ASC within the intestinal tract corresponds closely to the vascularization and lymphatic drainage patterns which distinctly separate the small intestine and upper part of the large intestine from the middle and lower parts of the large intestine. In both humans and macaques, the partition between the two systems takes place in the middle of the transverse colon. Thus, the blood supply through the small intestine, the ascending colon, and proximal parts of the transverse colon depends largely on the superior mesenteric artery and vein, whereas the distal part of the transverse colon, the descending colon, and the rectum are vascularized mainly by the inferior mesenteric artery and vein. The lymphatic drainage follows a similar pattern.