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We assessed serologic responses to an oral, killed whole-cell enterotoxigenic Escherichia coli plus cholera toxin B-subunit (ETEC-rCTB) vaccine in 73 Egyptian adults, 105 schoolchildren, and 93 preschool children. Each subject received two doses of vaccine or placebo 2 weeks apart, giving blood before immunization and 7 days after each dose. Plasma antibodies to rCTB and four vaccine-shared colonization factors (CFs) were measured by enzyme-linked immunosorbent assay. Immunoglobulin A (IgA) antibodies to rCTB and CFA/I were measured in all subjects, and those against CS1, CS2, and CS4 were measured in all children plus a subset of 33 adults. IgG antibodies to these five antigens were measured in a subset of 30 to 33 subjects in each cohort. Seroconversion was defined as a >2-fold increase in titer after vaccination. IgA and IgG seroconversion to rCTB was observed in 94 to 95% of adult vaccinees, with titer increases as robust as those previously reported for these two pediatric cohorts. The proportion showing IgA seroconversion to each CF antigen among vaccinated children (range, 70 to 96%) and adults (31 to 69%), as well as IgG seroconversion in children (44 to 75%) and adults (25 to 81%), was significantly higher than the corresponding proportion in placebo recipients, except for IgA responses to CS2 in adults. IgA anti-CF titers peaked after one dose in children, whereas in all age groups IgG antibodies rose incrementally after each dose. Independently, both preimmunization IgA titer and age were inversely related to the magnitude of IgA responses. In conclusion, serologic responses to the ETEC-rCTB vaccine may serve as practical immune outcome measures in future pediatric trials in areas where ETEC is endemic.

The immunogenicity of different preparations of an oral inactivated enterotoxigenic Escherichia coli (ETEC) vaccine was evaluated in Swedish volunteers previously unexposed to ETEC infection. The vaccine preparations consisted of recombinant cholera toxin B subunit (CTB) and various amounts of formalin-killed whole bacteria expressing the most prevalent colonization factor antigens (CFAs). Significant immunoglobulin A (IgA) antibody-secreting cell (ASC) responses against CTB and the various CFA components were seen in a majority of volunteers after two doses of ETEC vaccine independent of the vaccine lot given. The IgA ASC responses against CTB were significantly higher after the second than after the first immunization, whereas the CFA-specific IgA ASC responses were almost comparable after the first and second doses of ETEC vaccine. Two immunizations with one-third of a full dose of CFA-ETEC bacteria induced lower frequencies of IgA ASC responses against all the different CFAs than two full vaccine doses, i.e., 63 versus 80% for CFA/I, 56 versus 70% for CS1, 31 versus 65% for CS2, and 56 versus 75% for CS4. The proportion of vaccinees responding with rises in the titer of serum IgA antibody against the various CFA antigens was also lower after immunization with the reduced dose of CFA-ETEC bacteria. These findings suggest that measurements of circulating IgA ASCs can be used not only for qualitative but also for quantitative assessments of the immunogenicity of individual fimbrial antigens in various preparations of ETEC vaccine.

A prototype oral enterotoxigenic Escherichia coli (ETEC) vaccine containing formalin-inactivated whole bacteria expressing colonization factor antigens CFA/I and CFA/II and cholera B subunit (CTB) has been tested for safety and immunogenicity in 20 adult Swedish volunteers. When given in three doses with 2-week intervals the vaccine was found to be safe and to give rise to specific IgA antibody responses in intestinal lavage fluid in most of the volunteers (CFA/I 82%, CFA/II 82% and CTB 91%). The frequencies and magnitudes of these responses, which were already maximal after two doses, were comparable with those previously found in patients convalescing from severe ETEC diarrhoea. All the vaccinated volunteers also responded with antitoxin IgA as well as IgG antibodies in serum, whereas the serum antibody responses against the CFAs were weaker and mainly of the IgA isotype.

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.

A prospective study was performed to evaluate the presence of colonization factor antigens (CFAs) in enterotoxigenic Escherichia coli (ETEC) strains isolated from 1,211 children with diarrhea in Argentina. One hundred nine ETEC strains that were isolated from seven different laboratories in various regions of the country were tested for CFAs by using monoclonal antibodies against CFA/I and E. coli surface antigens CS1, CS2, and CS3 of CFA/II and CS4 and CS5 of CFA/IV; a polyclonal antiserum against CS6 was used. The CFAs searched for were found in 52% of the ETEC strains: 23% of the strains carried CFA/I, 17% carried CFA/IV, and 12% carried CFA/II. All of the CFA/I strains produced heat-stable enterotoxin, and several of them were of the prevalent serotypes O153:H45 and O78:H12. Among the 19 strains expressing CFA/IV, 16 expressed CS5 and CS6 and produced the heat-stable enterotoxin and most were of serotype O128:H21; the remaining 3 strains produced CS6 only. No ETEC strains expressing CS4 were found. Most (11 of 13) of the CFA/II-carrying ETEC strains expressed CS1 and CS3, and 10 of them were of the O6:K15:H16 serotype and produced both heat-labile and heat-stable toxins. As many as 24 of the 109 CFA-negative ETEC strains gave mannose-resistant hemagglutination with erythrocytes from different species; 4 strains had high surface hydrophobicity, suggesting the presence of additional, as yet undefined, colonization factors in up to 25% of the ETEC isolates.

Fecal Escherichia coli isolates from 196 patients with watery diarrhea and 68 healthy individuals (controls) were analyzed in Bangladesh immediately after isolation for the presence of colonization factor antigen (CFA) I or II (CFA/I or CFA/II, respectively) by a mannose-resistant hemagglutination (MRHA) test with six species of erythrocytes and by a slide agglutination test with absorbed CFA/I or CFA/II antisera. The presence of CFAs was confirmed by immunodiffusion analyses done in Sweden. By these methods, it was found that 49 of 69 enterotoxin-producing E. coli strains isolated from patients carried CFA/I or CFA/II, whereas none of the nonenterotoxigenic E. coli isolates or the three toxin-positive strains isolated from healthy individuals carried these adhesins. All E. coli strains retained their MRHA ability after transportation to Sweden followed by one subculture and after storage at -70 degrees C (but not at room temperature) for 1 to 2 years without further subculturing. After 5 to 10 subcultures of the fresh isolates, however, 70% of the initially CFA/I- and 80% of the initially CFA/II-carrying strains analyzed did not hemagglutinate. The efficacy of different methods for detecting CFAs on the fresh isolates was compared with that of immunodiffusion. The sensitivity of MRHA with human blood group A erythrocytes for the detection of CFA/I was high (97%), but the specificity was only 69%. The sensitivity of MRHA with bovine erythrocytes for the detection of CFA/II in Bangladesh was very low but increased considerably when chicken erythrocytes were also used. Whereas both false-positive and false-negative reactions were obtained when absorbed CFA antisera were used for agglutination, antisera against purified CFAs were equally effective as immunodiffusion in identifying CFA/I and CFA/II-carrying strains.

The diarrheal response to an initial and a second infection with Escherichia coli expressing various enterotoxins (the heat-stable toxin [ST] alone or in combination with the heat-labile toxin [LT]) and colonization factor antigens (CFA/I, CFA/II, or E8775-type) was studied in the reversible tie adult rabbit diarrhea model. An initial infection with high doses (1 X 10(10) to 5 X 10(11) bacteria) of the various strains regularly induced diarrhea which was usually self-limiting (only 7 of 85 animals died). The diarrheal response to equally effective doses of different strains producing both ST and LT (ST/LT) did not differ significantly with serotype or colonization factor antigen. ST/LT-producing strains appeared to induce severe disease more regularly than ST-producing strains carrying the same adhesin. Previous infection with CFA/I-carrying, ST/LT-producing E. coli protected all animals reinfected with an otherwise highly diarrheogenic dose of the same strain as well as against challenge with a CFA/I-carrying, ST/LT-producing strain with different O-, K-, and H-antigens. Fecal excretion of bacteria was also significantly reduced in the protected animals, although not completely eliminated. When only one of the two antigens, CFA/I and LT, was shared by the immunizing and rechallenge strains, partial protection was evident consistent with independent antibacterial (anti-CFA) and antitoxic (anti-LT) immune mechanisms. Oral immunization with purified CFA/I significantly reduced fluid secretion in intestinal loops infected with CFA/I-carrying enterotoxigenic bacteria.

In Dhaka, Bangladesh, fresh isolates of Escherichia coli from 197 patients with diarrhea were investigated for production of enterotoxin and possession of colonization factor antigen (CFA) I or II. Enterotoxigenic E. coli (ETEC) was isolated from 34% of the patients, and of the 67 enterotoxin-positive strains, 75% carried CFAs. Among 68 healthy control persons no strains positive for both enterotoxin and CFA were found. The CFAs in general were restricted to certain serotypes of E. coli. In a subgroup of patients, part of an ongoing surveillance study, mixed infection was seen in 23% of those from whom recognized pathogens were identified. There was a tendency to more severe dehydration when the two virulence factors, enterotoxin and CFA, were simultaneously present.

We studied the ability of antisera against different Escherichia coli surface antigens, both alone and in combination with anti-enterotoxin, to decrease fluid secretion induced by intestinal challenge with enterotoxigenic E. coli in rabbits. Antiserum against lipopolysaccharide protected significantly against O group homologous bacteria. Monospecific antisera against pilus-associated colonization factor antigens CFA/I and CFA/II were also effective, giving highly significant protection against enterotoxigenic E. coli strains bearing the corresponding colonization factor antigens. Protection was also observed with Fab fragments of the CFA/I antibodies. Addition of the anti-lipopolysaccharide serum to a protective antiserum against purified heat-labile enterotoxin resulted in an antisecretory effect which slightly exceeded the sum of the effects obtained with each preparation alone. The combination of antiserum against CFA/I or CFA/II with anti-enterotoxin gave protection that equaled the product of the effects obtained with each antiserum alone; i.e. the antisera cooperated synergistically.

A colonization factor antigen (CFA)-deficient mutant has been isolated that is as efficient as the CFA/I-carrying, enterotoxin-producing E. coli strain it was derived from in producing diarrhoea and colonizing the intestine in a rabbit non-ligated intestine model, the RITARD model. Infection with 10(11) mutant bacteria induced diarrhoea in 15 of the 17 rabbits challenged; corresponding diarrhoeal attack rates after challenge with similar doses of the original CFA/I-positive strain and of a non-enterotoxinogenic, non-CFA control strain were 23/24 and 0/15, respectively. Whereas the mean time of excreting the challenge strain in faeces did not differ between the mutant and the CFA/I-positive strain (4.5 and 4.3 days, respectively), it was significantly longer (p less than 0.05) than for the control strain (2.5 days). Similarly, the magnitude of the serum antibody responses to homologous O-antigen was equally high after infection with the mutant and the CFA/I-positive strain and considerably higher than after challenge with the negative control strain. An initial infection in the RITARD model with the CFA-deficient mutant offered highly significant protection against diarrhoea (p less than 0.001) as well as colonization (p less than 0.01) on subsequent challenge with the original CFA/I-positive strain. No significant protection, either against colonization or disease, was induced by initial infection with a similar dose of the control strain.

Many of the enterotoxin-producing Escherichia coli strains causing diarrhea in humans possess one of two types of pili adhesins, the colonization factor antigens CFA/I and CFA/II. We identified two subtypes of CFA/II in two CFA/II bearing Escherichia coli strains (serotypes O6:H16 and O78:H12) by means of immunodiffusion analyses of heated extracts and hemagglutination inhibition tests. In addition to a shared antigenic structure on CFA/II from both strains, a specific antigenic determinant could be demonstrated on CFA/II from the O6:H16 strain. A protective effect against intestinal loop challenge with homologous bacteria in rabbits was noted for antibodies to both the shared and the subtype-specific structures of CFA/II.