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Immune responses against enterotoxigenic Escherichia coli (ETEC) were examined in Bangladeshi adults with naturally acquired disease and compared to responses in age-matched Bangladeshi volunteers who had been orally immunized with a vaccine consisting of inactivated ETEC bacteria expressing different colonization factor antigens (CFs) and the B subunit of cholera toxin. B-cell responses in duodenal biopsy samples, feces, intestinal washings, and blood were determined. Because most of the patients included in the study were infected with ETEC expressing CS5, immune responses to this CF were studied most extensively. Vaccinees and patients had comparable B-cell responses against this antigen in the duodenum: the median numbers of antibody-secreting cells (ASC) were 3,300 immunoglobulin A (IgA) ASC/10 7 mononuclear cells (MNC) in the patient group ( n = 8) and 1,200 IgA ASC/10 7 MNC in the vaccinees ( n = 13) (not a significant difference). Similarly, no statistically significant differences were seen in the levels of duodenal B cells directed against enterotoxin among vaccinees and patients. A comparison of the capacities of the various methods used to assess mucosal immune responses revealed a correlation between numbers of circulating B cells and antibody levels in saponin extracts of duodenal biopsy samples ( r = 0.58; n = 13; P = 0.04) after vaccination. However, no correlation was seen between blood IgA ASC and duodenal IgA ASC after two doses of vaccine. Still, a correlation between numbers of CF-specific B cells in blood sampled from patients early during infection and numbers of duodenal B cells collected 1 week later was apparent ( r = 0.70; n = 10; P = 0.03).

We have examined whether oral immunization of adult Swedish volunteers with a prototype enterotoxigenic Escherichia coli vaccine would induce antigen-specific T-cell responses in blood. Volunteers were given one to three doses of the whole-cell component of the vaccine, which consisted of formalin-inactivated bacteria expressing the fimbrial colonization factor antigens I and II. Following immunization, in vitro stimulation of blood mononuclear cells with the colonization factor antigens resulted in modest proliferative responses which were accounted for mainly by CD4+ T cells and, to a lesser extent, by CD8+ T cells. A main finding of this study was that a majority of the orally immunized volunteers had circulating T cells capable of producing large quantities of gamma interferon following in vitro exposure to either of the colonization factor antigens. No interleukin 2 production could be detected in the cell cultures. These results suggest that oral immunization of humans induces the migration of specific mucosal T immunocytes from the intestine into peripheral blood.

The roles of the subcomponents of colonization factor antigen II, the coli surface antigens CS1, CS2, and CS3, as colonization factors and protective antigens was studied in a nonligated rabbit intestine model (RITARD). Infection with enterotoxigenic Escherichia coli (ETEC) carrying CS3 alone or CS1 plus CS3 induced diarrhea in most (80%) of the rabbits, whereas nonenterotoxigenic strains expressing CS1 or CS2 rarely induced diarrhea. Strains carrying CS1, CS2, or CS3 alone were all shed in stools for a significantly longer period than normal fecal flora-type E. coli. Initial infection with ETEC positive for CS1 plus CS3 induced significant protection against disease caused by reinfection with a highly diarrheagenic dose of the homologous strain; rabbits previously infected with serotype-heterologous, nontoxigenic bacteria carrying CS1 only were also protected against this challenge, whereas no such protection was induced by serogroup-homologous E. coli carrying CS2 only. Animals previously infected with CS1-, CS3-, or CS1-plus-CS3-positive bacteria excreted the CS1-plus-CS3 challenge strain for a significantly shorter period than did "nonimmunized" rabbits, whereas initial infection with bacteria carrying CS2 only did not result in such reduced shedding. Monoclonal antibodies against CS1, CS2, or CS3 all protected against experimental infection with ETEC carrying the corresponding CS factor. These results suggest that all the subcomponents of colonization factor antigen II are colonization factors and may induce anticolonization immunity.

Cholera toxin (CT)-specific antibody responses of the immunoglobulin E (IgE) isotype in the sera of adult patients suffering from infection with eitherVibrio choleraeO1,V. choleraeO139, or enterotoxigenicEscherichia coli(ETEC) were analyzed and compared with those in the sera of volunteers immunized with a bivalent B subunit O1/O139 whole-cell cholera vaccine. A significant IgE response to CT was observed in 90% of the patients withV. choleraeO1 infection (18 of 20;P= V. choleraeO139 infection (19 of 20;P= V. choleraeO1 showed CT-specific IgE responses (P= 0.004). In contrast, Swedish volunteers immunized with the oral cholera vaccine showed no IgE responses to CT (Pvalue not significant). During the study period, total IgE levels in the sera of the diarrheal patients, the North American volunteers, and the Swedish cholera vaccinees alike remained unchanged. However, the total IgE levels in the sera of patients and healthy Bangladeshi controls were on average 89-fold higher than those in the sera of the healthy Swedish volunteers and 34-fold higher than those in the sera of the North American volunteers.

Vaccine antigen-specific antibody-secreting cell (ASC) responses in peripheral blood of healthy adult volunteers were studied after oral immunization with a prototype enterotoxigenic Escherichia coli (ETEC) vaccine by means of the enzyme-linked immunospot technique. Three doses of vaccine consisting of formalin-killed ETEC bacteria expressing fimbrial colonization factor antigens I and II (CFA/I and CFA/II) in combination with purified cholera toxin B subunit (CTB) were given 2 weeks apart. The ASC responses were detected 7 days after each immunization. Immunoglobulin A (IgA) was the predominant isotype produced by CFA/I- as well as CFA/II-specific ASCs. Moderate CFA/I- and CFA/II-specific IgM-secreting ASC (IgM-ASC) responses were also seen, whereas IgG-ASC responses to either of the CFAs were negligible. The ASC responses to CTB, on the other hand, comprised both IgA- and IgG-ASCs, with few if any specific IgM-ASCs. Almost 90% of the volunteers developed CFA-specific ASC responses after vaccination. Maximal CFA-specific ASC responses were usually observed after a single dose or two doses of vaccine. A third dose of vaccine did not result in increased but rather resulted in decreased magnitudes of CFA-specific ASC responses. Furthermore, it was found that CTB did not function as a mucosal adjuvant, since CFA-specific ASC responses were not enhanced by the simultaneous administration of CTB. These results suggest that two oral doses of ETEC vaccine induce a strong mucosal immune response, as reflected by the presence of large numbers of antigen-specific mucosal B cell immunoblasts in the blood.