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Sexually transmitted diseases (STDs) are a major health problem in both industrialized and developing countries. Induction of a local mucosal immune response including the development of specific secretory immunoglobulin A (IgA) antibodies is important for protection against microorganisms that invade via mucosal surfaces (9, 19). One of the portals of entry for sexually transmitted pathogens such as the human immunodeficiency virus and herpes simplex virus is the mucus surface of the rectum. To provide specific protection against such pathogens, it is of great interest to develop immunization schemes that can induce specific immune responses including a strong IgA antibody response in the rectum (6, 10).

The aim of the present study was to assess the rectal immunization route for its ability to induce specific antibody-secreting cell (ASC) responses in suspensions of mononuclear cells (MNCs) from rectal tissues as well as from peripheral blood of healthy volunteers after rectal administration of cholera vaccine containing cholera toxin B subunit (CTB). CTB-specific antibodies in rectal secretions were also collected and analyzed along with antitoxin antibodies in serum.

Subjects and immunization. The study was performed with due informed consent and ethical committee approval on eight healthy volunteers (three women), aged 20 to 44 years, who received three rectal immunizations with an inactivated B subunit-whole cell cholera vaccine, which is normally administered orally. The immunizations were given 2 weeks apart. The vaccine, containing 1.0 mg of recombinantly produced CTB and 10¹¹ heat- and formalin-killed vibrios per 3-ml dose (SBL Vaccin, Stockholm, Sweden) (12), was administered by means of a rubber tube, 3 mm in diameter, inserted approximately 5 cm beyond the anus. After administration of the vaccine, the volunteers remained in horizontal position for 30 min.

Collection of specimens. Rectal biopsies (eight persons), rectal secretions (five persons), and blood specimens (eight persons) were collected before the first immunization (day 0) and 7 days after the third vaccine dose. The rectal biopsies were obtained using a rigid sigmoidoscope and a standard flexible endoscope biopsy forceps (Olympus, Solna, Sweden). On each occasion, four to eight pinched biopsy samples 2 mm in diameter, were collected from rectum approximately 8 to 10 cm from the anus. Rectal secretions were collected before pinch biopsies. After insertion of the sigmoidoscope, each of four polywick tampons (2 by 25 mm; Polyfiltronics Inc., Rockland, Mass.), composed of a mixture of synthetic fibers and cellulose, was grasped with the forceps and carefully placed onto a relatively clean mucosal surface in the rectum approximately 12 to 15 cm from the anus. After 5 min, the tampons were collected with the forceps, and each tampon was placed in an Eppendorf tube. To extract proteins from the tampon, 200 µl of a buffer solution, containing enzyme inhibitors supplemented in 0.1% bovine serum albumin at concentrations previously specified (13), was added. Thereafter, the tubes were centrifuged at 10,000 × g for 2 min at 4°C in order to drive the fluid from the tampon. Supernatants were collected, pooled, and stored at 20°C until analyzed. For determination of circulating vaccine-specific ASC responses, 20 ml of heparinized venous blood was collected from all volunteers immediately before the first immunization and then 7 days after the last immunization. Serum specimens were obtained on the same occasions.

Detection of total and specific Ig-secreting cells. Intestinal MNCs were isolated from the rectal biopsies using an enzymatic dispersion technique as previously described (20). A pool of four to eight biopsy samples from each individual yielded a mean of 2.7 × 10⁵ viable MNCs (range, 0.9 × 10⁵ to 5.9 × 10⁵). MNCs from heparinized venous blood were isolated by standard gradient centrifugation on Ficoll-Isopaque (Pharmacia, Uppsala, Sweden). Rectal and peripheral blood MNC suspensions were assayed for numbers of total IgA- and IgG-secreting cells and CTB-specific IgA and IgG ASCs by a two-color micromodification (4) of the original enzyme-linked immunospot method (3, 22). Total Ig and CTB-specific Ig ASCs were expressed per 10⁵ MNCs in the rectum and per 10⁶ MNCs in peripheral blood. Vaccinees who had 5 CTB-specific ASCs per 10⁵ MNCs in their rectal biopsy samples after vaccination were considered responders when no ASCs, i.e., <2.5 CTB-specific ASCs per 10⁵ MNCs, could be detected prior to immunization. When the preimmune specimens (one case) contained >2.5 CTB-specific ASCs per 10⁵ MNCs, a more than twofold increase in CTB-specific ASCs between pre- and postvaccination samples was considered a vaccine response. The corresponding figure for a response in peripheral blood was set at a postvaccination value of 5 CTB-specific Ig ASCs per 10⁶ MNCs (5).

Antibody determinations. The content of total IgA1 in rectal secretions was determined with an enzyme-linked immunosorbent assay (ELISA) method as previously described (24). Specific IgA (IgA1) antibody responses to cholera toxin in rectal secretions were measured by a GM1 ELISA method (23). The antibody titer was determined as the interpolated dilution of the specimen giving an absorbance value at 405 nm of 0.4 above background. The specific IgA antitoxin activities in rectal secretions were determined by dividing the IgA ELISA antibody titer by the total IgA concentration (micrograms per milliliter) of the sample to adjust for variations in the IgA content in specimens collected from different persons and on various days. A greater than twofold increase in the mean IgA antibody titer/total IgA between pre- and postimmunization specimens was regarded as a response (1). When preimmune specimens were missing (two cases), volunteers were considered responders if their postvaccination IgA antitoxin titer/total IgA exceeded by 2 standard deviations the geometric mean IgA antitoxin titer/total IgA in rectal secretions from seven other nonimmunized individuals.

Local immune responses in the rectum. The frequencies of total IgA and IgG secreting cells were similar in rectal biopsy samples obtained before and after three rectal administrations of CTB (Table 1). Prior to immunization, no CTB-specific IgA or IgG ASCs were found in the rectum, except for one individual who had 5 IgG ASCs per 10⁵ MNCs. The vaccination induced substantial increases in CTB-specific IgA ASCs in the rectum in each of the eight volunteers, with a geometric mean fold increase in ASCs of more than 14-fold (Table 1; Fig. 1a). Increases in CTB-specific IgG ASCs were also seen in five (63%) of the volunteers after vaccination, and the geometric mean fold rise in ASCs for these responders was more than fivefold (Table 1; Fig. 1a).

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Levels of CTB-specific IgA and IgG ASCs in the rectum (a) and peripheral blood (b) of healthy volunteers 7 days after three rectal administrations of recombinant CTB. The geometric mean numbers of vaccine-specific ASCs + 1 standard error of the mean are shown.

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View larger version (11K): [in this window] [in a new window]   FIG. 2.   Intestinal IgA (IgA1) antitoxin levels in rectal secretions collected from five volunteers before (Pre) and 7 days after (Post) three rectal administrations of recombinant CTB. The dotted line denotes the geometric mean IgA antitoxin titer/total IgA in rectal secretions from seven nonimmunized individuals. Preimmune specimens were missing for two vaccinees.

Immune responses in blood. Monitoring of different homing receptors on circulating ASCs indicates that especially ASCs of the IgA isotype, assayed approximately 7 days after oral or rectal vaccination, almost exclusively represent cells of the intestinal immune system (15). Prior to immunization, the number of circulating CTB-specific IgA and IgG ASCs were negligible (1 ASC × 10⁶ MNCs). All of the eight volunteers responded with increased numbers of CTB-specific IgA ASCs in blood after three rectal administrations of CTB (Table 2), with a geometric mean fold increase of more than 19-fold (Fig. 1b). Increased levels of circulating IgG ASCs against CTB were also found in four (50%) of the volunteers after vaccination (Table 2; Fig. 2), and the mean fold increase in ASCs for these responders was more than 27-fold. There were also significant increases in serum IgA antitoxin titers in six (75%) of eight volunteers after three rectal doses of CTB, and seven (88%) of the vaccinees developed IgG antitoxin responses too (Table 2). Among responders, these increases in antitoxin were 7.6-fold for IgA and 4.4-fold for IgG.