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Diarrheal episodes with enterotoxigenic Escherichia coli (ETEC) were prospectively monitored during the first 2 years of life in a cohort of 235 infants from Leon, Nicaragua. ETEC was an etiological finding in 38% (310 of 808) of diarrheal episodes and in 19% (277 of 1,472) of samples taken as asymptomatic controls at defined age intervals (P = <0.0001). The majority of diarrheal episodes (80%) occurred before 12 months of age. The major ETEC type was characterized by colonization factor CFA I and elaboration of both heat-labile enterotoxin and heat-stable enterotoxin (ST). The proportion of E. coli strains with CFA I was significantly higher in cases with diarrhea (P = 0.002). The second most prevalent type showed putative colonization factor PCFO166 and production of ST. The prevalence of PCFO166 was approximately 20%, higher than reported before. Children with a first CFA I episode contracted a second ETEC CFA I infection 24% of the time, compared with 46% for ETEC strains of any subtype. Most of the ETEC episodes were of moderate severity, and only 5% (15 of 310) were characterized as severe. In conclusion, our results give valuable information for the planning of intervention studies using ETEC vaccines.

Enterotoxigenic Escherichia coli (ETEC) strains were isolated from travelers or military personnel who developed diarrhea after visiting Nepal or who were deployed to Thailand, Indonesia, or the Philippines. ETEC isolates were examined for colonization factor antigen (CFA). CFAs were identified on 59% (40 of 68) of the isolates examined. The lack of a detectable CFA on 41% (28 of 68) of the isolates is of concern for the development of an effective ETEC vaccine.

We describe, for the first time, the production of monoclonal antibodies (MAbs) against coli surface antigen 6 (CS6) of enterotoxigenic Escherichia coli (ETEC) and their use for characterization and diagnosis of CS6. Two MAbs, MAbs CS6-20:11:9 and CS6-2A:14, were produced by immunizing mice with purified CS6 or CS6-containing bacterial extracts. The MAb specificity was demonstrated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunoelectron microscopy, which showed that the MAbs bound to CS6-expressing bacteria as well as to purified CS6 and CS6 structural subunits but not to CS6-negative bacteria or other purified ETEC colonization factors. By using bacterial recombinants, i.e., strains with a complete CS6 operon or parts thereof, it was found that both MAbs were specific for CssB, one of the two structural subunits of CS6. Although the MAbs bound specifically to the entire surface of CS6-expressing bacteria, no structure of CS6 could be identified. The usefulness of the MAbs for the detection of CS6 was evaluated in an inhibition ELISA and in a dot blot test. Ninety-two ETEC strains with known colonization factors were analyzed, and all CS6-positive strains were identified by either assay with MAb CS6-2A:14, whereas MAb CS6-20:11:9 failed to identify two CS6-positive strains; in no instance was any CS6-negative strain identified by either of the MAbs. Parallel analyses of 48 strains with a gene probe specific for the other structural subunit of CS6, i.e., CssA, and the MAb-based assays gave identical results, suggesting the simultaneous presence of both subunits.

In 17 healthy volunteers, we studied the dynamics of secretory immunoglobulin A (SIgA) release in the proximal small intestine (distal duodenum and proximal jejunum), using a modified triple-lumen perfusion technique. Secretory data were related to the current phase of the migrating motor complex (MMC), as recorded by manometry. In both segments, luminal SIgA output changed cyclically, with peak outputs occurring at the end of the MMC cycle (late phase II). These MMC-related changes were due to two factors: cyclic inflow of SIgA from more proximal sites, and a cyclic release of SIgA from the test segment that correlated in time with an increase in epithelial fluid secretion. Cyclic intestinal SIgA release may thus be another secretory component of the MMC. The data are compatible with the hypothesis that motility and SIgA release are linked by motility-activated chloride secretion from the intestinal crypts.

Samples (1,318) of enterotoxigenic Escherichia coli (ETEC) isolated in 1994-1995 from children with diarrhea from Nepal, Indonesia, Peru, and Thailand were examined for colonization factor antigen (CFA) and coli surface (CS) antigens. Fifty-five percent of 361 heat-labile and heat-stable (LT-ST), 14% of 620 LT-only, and 48% of 337 ST-only ETEC had CFA/CS antigens. LT-ST ETEC strains were predominantly in the CFA II group, and ST only strains were in the CFA IV group. Additional studies are needed to identify ETEC strains that do not have CFA/CS antigens.

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of childhood and travellers' diarrhoea. The ability of ETEC to adhere to the intestinal epithelium of the host is an important virulence determinant, and adhesion is mediated by proteinaceous surface appendages called colonization factors.

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine by means of several antigenically distinct colonization factors (CFs). Several of these CFs have very significant amino acid sequence similarity or identity, particularly in the N-terminal end. We have previously shown that a monoclonal antibody (MAb) raised against the subunits of colonization factor antigen I (CFA/I) fimbriae, which reacts with a peptide corresponding to the 25 N-terminal amino acids of such subunits, can inhibit attachment to intestinal cells of ETEC expressing heterologous as well as homologous CFs, with related amino acid sequences. In this study we have, by means of Pepscan analysis, determined the sequence of the MAb-specific linear epitope to be 15IDLLQ19. Parenteral immunization of rabbits with an N-terminal 25-mer synthetic peptide of CFA/I fimbrial subunit, either covalently coupled to bovine serum albumin or uncoupled, induced high titers of specific antibodies against this peptide as well as against CFA/I fimbriae. Increased titers against several heterologous CF fimbriae with a related N-terminal sequence were also induced, whereas no increase was seen against fimbriae with an unrelated sequence. Neither antisera against the coupled peptide nor antisera against the uncoupled peptide inhibited binding of CF-expressing bacteria to the human intestinal cell line Caco-2 in spite of high titers. The difference in the inhibitory capabilities of the antipeptide sera and the MAb might be due to slightly different epitope specificities. Thus, whereas the antipeptide sera bound to several continuous epitopes in the N-terminal end, none of them reacted specifically with the epitope 15IDLLQ19.

There is a need for an effective vaccine that can protect against cholera caused by either Vibrio cholerae O1 or by the new pandemic serotype O139 Bengal. An oral bivalent B subunit-O1/O139 whole cell (B-O1/O139 WC) cholera vaccine has been prepared by adding formalin-killed O139 vibrios to the recently licensed oral recombinant B-O1 WC vaccine. When tested in Swedish volunteers, this B-O1/O139 WC vaccine was found to be safe and immunogenic. Two vaccine doses given 2 weeks apart induced statistically significant, P < 0.05, mucosal IgA antibody responses in intestinal lavage fluid against cholera toxin in all of nine vaccinees and against both O1 and O139 vibrios in seven of nine cases. The intestinal responses were associated with similar high frequencies of intestine-derived antibody-secreting cell responses in peripheral blood to the different antigens. A third dose of vaccine given after 5-6 weeks did not result in any further increased response. All of 12 vaccinees responded with significant IgA and IgG antitoxin responses in serum associated with significant vibriocidal antibody titre rises against O1 vibrios in 10 cases (83%) and against O139 vibrios in eight vaccinees (67%). The frequencies and magnitudes of the serological responses to the B subunit and O1 WC components were similar to those induced by the B-O1 WC vaccine. Thus, the O139 component of the vaccine induced intestinal and systemic antibacterial immune responses in the majority of the vaccinees, and its addition to the vaccine did not interfere with the immunogenicity of the B subunit or O1 WC components.

Colonization factor antigens (CFAs) mediate attachment of enterotoxigenic Escherichia coli (ETEC) to the intestinal mucosa and induce protective immunity against ETEC diarrhea. ETEC strains (n = 111) isolated from North Indian children from 1985 to 1989 were examined for CFAs and putative colonization factors (PCFs). CFA/IV was the most common factor (26%), followed by coli surface antigen 17 (CS17) (19%), CFA/I (14%), PCFO166 (7%), and CFA/II (5%), while 24% of the isolates were negative for CFAs and PCFs. Among the strains producing heat-stable and heat-labile toxin (ST+LT+ strains), the STaI gene was strongly associated with the absence of known CFAs and PCFs, making the STaI+LT+ isolates an interesting target for the identification of previously undescribed factors. Repetitive sequence--based polymerase chain reaction revealed that the CS17+ strains, although clonally related, represented endemically circulating strains with a diversity greater than that of the CFA/I+ strains, which showed a substantial clonal clustering.

We have characterized 11 isolates of Vibrio cholerae O139 Bengal with regard to properties deemed to be relevant for development of a vaccine against O139 cholera. For most strains two colony variants, A and B, which are nonhemolytic and hemolytic, respectively, were detected on blood agar. The A and B variants were associated with high- and low-level production of soluble hemagglutinin-protease, respectively. However, on Luria-Bertani agar both types formed opaque colonies, which has been shown to be associated with capsule formation. Interestingly, under the stationary tube-shaken flask culture conditions in yeast extract-peptone water medium which were used to stimulate the production of cholera toxin (CT) and toxin-coregulated pili, B variants constitutively produced CT and TcpA, two ToxR-regulated proteins, at 28 and 37 degrees C, whereas the production of these proteins by A variants was downregulated at the higher temperature. One of the strains, 4260B, having a well-exposed O antigen and capsule and the capacity to produce large amounts of TcpA, CT, and mannose-sensitive hemagglutinin pili but minimal amounts of the proteolytic soluble hemagglutinin, was selected to produce antibacterial antisera and as a challenge strain in protection studies using the rabbit ileal loop model. Rabbit antisera to live, heat-killed, or formalin-killed O139 vibrios or to purified O139 lipopoly-saccharide (LPS) as well as monoclonal antibodies (MAbs) to O139 LPS agglutinated all O139 isolates. However, when A and B variants of strain 4260 were tested for sensitivity to vibriocidal activity of these antibody preparations, only the B variant was killed. All of the antisera against live or killed O139 vibrios conferred passive protection against fluid accumulation induced by the challenge strain. The protective effects of the antisera were correlated to anti-LPS antibody titers rather than to titers against whole bacteria that had been grown for toxin-coregulated pilus expression. This protection was considerably higher than that conferred by antisera to classical, EI Tor, or recombinantly produced (classical) CT or CTB. Furthermore, MAbs to O139 LPS and CTB-CT exhibited a strong synergistic protection against O139 challenge irrespective of the level of sensitivity of challenge strains to O139 LPS MAbs in vibriocidal assays in vitro.

The binding of ETEC strains expressing different colonization factors to the human enterocyte-like cell line Caco-2 and to isolated human enterocytes were determined. Strains expressing CFA/I, CS2, CS4+CS6, CS5+CS6, CS7, CFA/III+CS6 and PCFO166 adhered well to both types of cells whereas bacteria producing CS1, CS6 only, or CS17 did not adhere to either Caco-2 cells or to jejunal human enterocytes, suggesting that similar binding structures are present in both cell types. However, in some instances, binding of bacteria to the two types of cells differed, e.g. bacteria expressing CS3 or PCFO9 bound well to human enterocytes but not to Caco-2 cells. Conversely, bacteria producing PCFO20 or PCFO159 only adhered to Caco-2 cells and not to jejunal enterocytes. With few exceptions, this inability to bind to human enterocytes was the same for cells from all parts of the small intestine. This study contradicts previous reports suggesting that the binding structures of Caco-2 cells are identical to those of human enterocytes.

Enterotoxigenic Escherichia coli (ETEC) bind to enterocytes in the small intestine by means of antigenically distinct colonization factors (CFs). By immunizing with isolated subunits of CFA/I fimbriae we have previously produced monoclonal antibodies (MAbs) that cross-react immunologically in vitro with several CFs. Two of these MAbs [S(subunit)-CFA/ I 17:8 and S-CFA/I 5:6] were found to significantly inhibit the binding of ETEC strains expressing either homologous or heterologous CFs, i.e. CFA/I and CS4, to isolated human jejunal enterocytes. The two MAbs also conferred passive protection against fluid accumulation in rabbit ileal loops caused by CFA/I-as well as CS4-expressing ETEC strains. Immunoelectron microscopy studies showed that both MAbs bound specifically to CFA/I as well as to CS4 fimbriae expressed on bacteria. These results indicate the possibility to induce anti-CF antibodies that can protect against ETEC infection caused by bacteria expressing not only homologous but also heterologous CFs, by immunizing with fimbrial subunits.

Putative colonization factor PCFO20 was recently identified in an enterotoxigenic Escherichia coli (ETEC) strain of serogroup O20 isolated from a child with diarrhea in Argentina. The gene encoding the structural subunit of PCFO20 fimbriae, fotA, was cloned from strain ARG-2 in the expression phage vector lambda ZAP Express. One positive clone, pGV29, that carried a 3.3-kb fragment was identified on the basis of fimbrillin production by using a monospecific rabbit anti-PCFO20 serum. Nucleotide sequencing of a 1.3-kb Sau3A-ClaI fragment of the subclone pGV292 containing the region coding for PCFO20 fimbrillin revealed two open reading frames of which one was complete. A western blot (immunoblot) showed that the cloned protein, FotA, migrated like the PCFO20 fimbrial subunit protein did. Fimbriae were not detected on the surface of E. coli host bacteria containing pGV292 or pGV29, suggesting that the genes needed for assembly of PCFO20 fimbriae are lacking in both clones. The fotA gene encodes a 20,574-Da prefimbrillin protein which contains a 21-amino-acid signal sequence; the mature protein has a size of 18.1 kDa. The subunit protein FotA was found to be more homologous to the subunit of porcine 987P than to any fimbrial subunit produced by human ETEC. Alignments of the amino acid sequences of the two proteins indicate that they are partly identical, with an overall similarity of 82%. FotA fimbrillin was shown to be transported and assembled by the fimbria assembly machinery in porcine ETEC strain 987. PCFO20 and 987P may have evolved from a common ancestral gene. They are immunologically related but have affinity for different host cell receptors, since PCFO20-producing bacteria do not bind to neonatal piglet enterocytes.

To determine the protective efficacy (PE) of three doses of oral B subunit-killed whole cell (BS-WC) or killed whole cell-only (WC) vaccines against cholera, a clinical trial was conducted among 62285 children over 2 years and adult women in rural Bangladesh. During 5 years of follow-up, there were 144 cases of cholera in the BS-WC group (PE = 49%; P < 0.001), 150 in the WC group (PE = 47%; P < 0.001), and 283 in the K12 group. Protection by each vaccine was evident only during the first three years of follow-up; long-term protection of young children was observed only against classical but not El Tor cholera; 3-year protection against both cholera biotypes occurred among older persons, but at a higher level against classical cholera.

In January and February 1992, an assessment was conducted of the safety and immunogenicity of two doses of a new oral cholera vaccine prepared from the recombinant B subunit of the toxin and from killed whole cells (rBS/WC) in 1,165 individuals between the ages of 12 months and 64 years in Barranquilla, Colombia. This was a randomized, double-blind placebo-controlled study. Participants received two doses of either the vaccine or a placebo (killed Escherichia coli K12) over a two-week interval. Few symptoms were detected during the three days following administration of the initial dose and even fewer following the second. Sera obtained upon administration of the first dose and two weeks after administration of the second were tested for Vibrio cholerae 01 Inaba vibriocidal antibodies and antitoxins. Geometric mean titers (GMT) of vibriocidal antibodies were found to increase two-fold in subjects receiving the vaccine. In the paired samples taken from vaccinated subjects, two-fold or greater increases were observed in 44% and four-fold or greater increases were observed in 34%, as compared to similar increases in 9.2% and 2.2% of the sera taken from those receiving the placebo (P < 0.05). The GMTs of IgG and IgA antitoxins, as determined by ELISA, increased by factors of 4 and 3.2, respectively, in those receiving the vaccine, as compared to factors of 1.1 and 1.1 in those given the placebo (P < 0.001 for IgG, P < 0.01 for IgA). Approximately 80% of the paired samples from the vaccinated group showed an increase of both IgG and IgA antitoxins > or = 1.5, as compared to only about 20% of those in the placebo group (P < 0.000001). Belonging to the O blood group did not significantly affect the immune response. Children under age four tended to show a weaker vibriocidal antibody response and a stronger antitoxin response than older subjects. The two doses of oral vaccine were found to be safe and without attributable side-effects. The vibriocidal antibody and antitoxin responses were similar to those obtained previously with the conventional oral killed whole cell B subunit cholera vaccine.

Optimal conditions to process, concentrate and store intestinal lavage fluid were studied in samples collected from volunteers before and after oral immunization with a prototype vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhoea. Total IgA and specific IgA antibody titres against enterotoxin and colonization factor antigen were determined in 22 lavage samples which were either enzyme-inhibited or heat-inactivated and then subjected to different long-term storage conditions. Samples were analysed within 1 month of collection and also after 3, 6 and 24 months of storage. Total IgA concentrations and specific IgA antibody levels were higher in lavage samples treated with enzyme inhibitors (soybean trypsin inhibitor and phenylmethylsulfonyl fluoride) than in those heat-inactivated. Similarily, concentration of the lavage fluid by freeze-drying was superior to concentration against polyethylene glycol. Specific antibody titres remained elevated after storage for at least 6 months but declined after 2 years in frozen compared with freeze-dried samples.

Background: Since the urea breath test (UBT) indirectly detects gastric Helicobacter pylori infection by measuring urease activity, the possibility of false-positive results due to other urease-producing bacteria cannot be excluded. Previous studies have shown that increased 14CO2 activity in early breath samples could be attributed to urea hydrolysis in the oropharynx. For that reason, reliable assessment of H. pylori status is hampered for at least 20 min after administration of a 14C-urea drink., Methods: To overcome this problem we have developed a modified breath test in which 111kBq 14C-urea is supplied in a gelatin capsule, which prevents release of 14C before reaching the stomach. Our modified 14C UBT was evaluated in 100 healthy volunteers, and results were compared with those from enzyme-linked immunosorbent assay serology., Results: The study showed a 99% concordance between the two noninvasive tests. When a biometric method for determination of cut-off values between positive and negative UBT results with the smallest possible arbitrariness was used, the calculated statistical probability of a false diagnosis was lowest in the 10-min breath sample (0.20%), and 100% sensitivity and specificity was achieved. Our capsule method was also compared with the urea drink method and was found more reliable because no overlapping in 14CO2 activity occurred between H. pylori-positive and -negative subjects, whereas conventional breath testing showed overlapping during the whole 30-min test period. Our study also showed that a fatty test meal lowers the 14CO2 excretion the first 20 min and may adversely affect the accuracy of a rapid UBT., Conclusions: Supplying the 14C-urea in a capsule obviates the problem of false-positive results in early breath samples and makes it possible to diagnose H. pylori infection with 99.8% reliability from a single 10-min breath sample, without the use of a test meal of adjustments for assumed individual CO2 production.

Vibrio cholerae serogroup O139, now considered to be the second organism capable of causing epidemic severe dehydrating cholera, contains a capsular polysaccharide which makes it difficult for it to be used in the conventional vibriocidal antibody assay optimized for V. cholerae O1. After modification of the procedure, which involved the use of specific bacterial strains, a lower bacterial inoculum, and increased amounts of complement, the vibriocidal antibody responses to V. cholerae O139 were measured in acute- and convalescent-phase sera from 33 V. cholerae O139-infected and 18 V. cholerae O1-infected patients and in single serum samples from 20 healthy control subjects. The responses in these individuals to V. cholerae O1 strains were also determined. Significant elevations in the homologous antibody response were found only in the convalescent-phase sera from both groups of patients with cholera. These findings may explain the basis for the lack of heterologous protection between the two serogroups of V. cholerae. Healthy controls had higher background levels of vibriocidal antibody to V. cholerae O1 than to V. cholerae O139.

Infection caused by enterotoxigenic Escherichia coli (ETEC) poses a serious health problem to children in developing countries. Colonization of the small intestinal mucosa by ETEC strains is mediated by antigenically specific fimbriae, also known as colonization factor antigens (CFA). The importance of this study arises from reports that active and passive immunization with ETEC strains harboring CFAs induced protective immunity against diarrhea in animal models with preformed antibodies. In humans, ETEC containing CFA/I, II, III and IV have been identified. The aim of this study was to define CFAs of ETEC isolated in Alexandria, Egypt. One hundred and seven ETEC isolates from 132 human residents in Alexandria, Egypt were isolated during a birth cohort study. ETEC isolates were screened for heat labile (LT) and heat stable (ST) toxins using a 32P oligonucleotide hybridization probe and a GM1 ELISA. These isolates were examined using monoclonal antibodies against CFA/I, II, III, IV, and against the putative colonization antigens PCF0159 and PCF0166, CS 7 and CS 17. CFAs were found in 48% of ETEC strains. CFA/I was found in 18% of the strains, CFA/II in 10% and CFA/IV in 14%. CFA III was not found. All fifteen strains expressing CFA/IV expressed CS6 and produced ST. CFA/IV was not found in non-ST producing strains, while CFA/I was absent in ST-only producing strains.

A 'double-blind', randomized, placebo controlled study of an oral inactivated whole cell plus recombinant B subunit (WC/rBS) cholera vaccine was conducted during February-March 1992 in Peru in 346 military recruits, 307 (89%) of whom received 2 oral doses of vaccine or Escherichia coli K12 placebo, 2 weeks apart. Paired serum samples were obtained from 155 (50%) of the recipients of 2 doses. An epidemic of cholera took place between doses. No difference in cholera attack rates was detected between vaccine and placebo recipients after one dose (8% versus 14%). Seroconversion (4-fold or higher increase in vibriocidal antibody titres) was detected in 90% and 80% of vaccine and placebo recipients, respectively, with low pre-existing vibriocidal titres (< 0.01). The anti-cholera toxin seroconversion rate among those with low pre-existing titres was higher in vaccinated subjects (97%) than in placebo recipients (68%) (P < 0.01). Administration of 2 doses of WC/rBS vaccine concomitantly with natural V. cholerae O1 infection enhanced the serum anti-cholera toxin response. The immune response to the whole cell component of the vaccine was reduced by high pre-existing vibriocidal antibody titres.

Background/aims: The immunologic reactivity of the gastric mucosa is poorly understood. The origin and dynamics of immunoglobulin A (IgA) occurring in the gastric lumen were investigated in healthy, Helicobacter pylori-negative volunteers., Methods: Gastroduodenal manometric motility recordings were combined with gastric luminal perfusion, enabling calculation of gastric acid output and analysis of the total IgA output., Results: Acid output and total IgA correlated with the migrating motility complexes (MMC). The gastric IgA release showed maximal values in association with gastric motility phase III (maximal motor activity) and lowest values during phases I and II (none or irregular motor activity). The IgA output correlated with neither swallowed saliva (as indicated by amylase in the gastric perfusate) nor duodenogastric reflux (as indicated by gastric occurrence of bilirubin and/or duodenally infused PEG4000). Stimulation of gastric acid secretion by sham feeding during phase II-like motor activity (n = 6) induced a rapid and transient doubling of IgA output. There was no significant correlation between gastric acid secretion and gastric IgA release., Conclusion: Substantial amounts of IgA are released into the human stomach, most likely originating from the gastric mucosa. The up-regulation of IgA release in association with the activity front of the MMC and anticipatory to food intake suggests a neuroendocrine control of gastric mucosal immune responses.

Escherichia coli adhesins are virulence factors in intestinal and extra-intestinal infections, but their role in normal intestinal colonization has not been defined. We investigated the intestinal adherence of E. coli with Dr hemagglutinin, S fimbriae, CFA/I or CFA/II, using freshly isolated ileal or colonic enterocytes and cells from the human colonic cell line HT-29. E. coli with S-fimbrial adhesins (Sfa I or Sfa II), P or type 1 fimbriae, adhered in a non-polarized manner, and in similar numbers to colonic and ileal enterocytes. S fimbriae of the variety Sfa II (originating from a meningitis isolate), mediated a stronger binding than Sfa I (of uropathogenic origin). Strains expressing Dr hemagglutinin adhered preferentially to the brush borders, slightly better to colonic than ileal enterocytes. Strains expressing CFA/I or II adhered to colonic and ileal enterocytes, although brush border adherence was predominantly observed with ileal cells. Binding to HT-29 cells paralleled binding to colonic enterocytes for all adhesin specificities except CFA/I. The results suggest that Dr hemagglutinin, P-, type 1- and S-fimbrial adhesins mediate binding to both colonic and ileal enterocytes. These specificities may contribute to the establishment of E. coli in the intestinal microflora, which precedes their spread to extra-intestinal sites.

Every year since its introduction in 1991, there have been epidemics of cholera in Lima, Peru. Vaccination is one approach to the control of cholera. A pilot study was conducted to assess the safety and immunogenicity of a whole cell plus recombinant B subunit (WC/rBS) cholrea vaccine in Lima, Peru. Five hundred and forty-one volunteers aged 2-65 years received two doses two weeks apart of WC/rBS vaccine or Escherichia coli K12 placebo administered in bicarbonate buffered water. Symptoms were monitored on all subjects and blood was collected from 102 persons before the first dose and two weeks after the second dose. Mild post-vaccination gastrointestinal symptoms were reported with equal frequency for both the vaccine and placebo recipients. Among 51 vaccines, 49% had a twofold or greater increase in serum vibriocidal titers (GMT = 78; range < 1:10 to 1:5120); and 92% and 82% developed a twofold or greater serum anti-cholera toxin IgG and IgA response, respectively. Persons with elevated prevaccination vibriocidal titers had a decreased response to the WC/rBS. Age and blood group did not affect the immune response. The WC/rBS vaccine was safe and immunogenic in a group of native Peruvians.

Serum antibody responses against Escherichia coli heat-labile enterotoxin (LT) and colonization factor antigens (CFAs) I and II were studied in 84 children < 5 years old living in two communities. These villages differed in the quality of their drinking water. Children from both communities developed significantly increased antibody titers against LT and CFA/II but not against CFA/I during 3 months of follow-up. The magnitude of the anti-LT response was significantly higher in children from Zaiman than in those from Las Dolores. Antibody titers rose to maximum levels during the second year of age and reached relatively constant levels in children aged 2-5 years, probably due to repeated exposure to enterotoxigenic E. coli strains. Antibody levels of 30 children were followed for 2 years; increases in anti-LT and anti-CFA titers varied in the different age groups.

Monoclonal antibodies (MAbs) against membrane preparations of Helicobacter pylori were produced. One MAb was found to be specific for H. pylori, because it did not react with a number of other bacterial species, including Helicobacter felis and Campylobacter jejuni. This MAb reacted with a 30-kDa protein found in outer membrane preparations of H. pylori. The protein was also detected on the cell surface on intact bacteria when analyzed by immunoelectron microscopy. To facilitate the identification of H. pylori isolates after culturing of biopsies, an immunodot blot assay based on the reaction of this MAb was developed. This assay was found to be highly specific for H. pylori. Sixty-six clinical isolates typed as H. pylori by conventional biochemical tests were found to be positive, whereas no other bacterial species tested gave a positive result. By this method, reliable and rapid identification of H. pylori could be accomplished.

We have attempted to characterize the binding specificity of the coli surface 3 (CS3) subcomponent of colonization factor antigen II of enterotoxigenic Escherichia coli, by means of an immunoblot method in which the binding of fimbriated bacteria to sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated rabbit intestinal cell membranes was evaluated. Isolated CS3 fibrillae as well as bacteria expressing CS3 on their surface bound to several intestinal cell membrane structures, i.e., structures present in the electrophoretic front and in the 30- to 35-kDa range and, most prominently, 120- to 140-kDa structures. Delipidization and protein digestion of the rabbit brush borders revealed that CS3 bound to structures of a proteinaceous nature. Sodium meta-periodate oxidation of the intestinal cell membranes abolished all their CS3 binding activity, indicating that CS3 bound to carbohydrate moieties of glycoproteins. The binding of CS3 to the separated intestinal proteins could also be inhibited by preincubation with the lectin derived from Maackia amurensis, indicating that CS3 bound to galactoproteins in the rabbit intestine. Inhibition experiments using equimolar amounts of various gangliosides demonstrated that GM1, asialo-GM1, and GM2 inhibited the binding of CS3 equally well, whereas GM3 was not as effective. These results suggested that the critical CS3 binding epitope consisted of the carbohydrate sequence GalNAc beta 1-4Gal. This was supported by electron microscopic experiments showing that this disaccharide, O linked to bovine serum albumin via a spacer, localized around CS3-positive bacteria but not at all around corresponding CS3-negative mutants. Furthermore, CS3-expressing bacteria recognized this neoglycoprotein when it was immobilized on nitrocellulose. The GalNAc beta 1-4Gal disaccharide has also been implicated as a binding structure for other pathogenic bacteria such as enteropathogenic E. coli and Pseudomonas aeruginosa.

Background: As most of the clinical laboratories in Israel do not use toxigenicity testing as a routine procedure in cases of Escherichia coli gastroenteritis, current information regarding clinical and epidemiological aspects of infection by enterotoxigenic E. coli (ETEC) is lacking. This survey aimed at retrieving that information by conducting routine enterotoxigenicity tests in a clinical laboratory for patients suspected of E. coli gastroenteritis., Methods: The survey population included 415 children < or = 2.5 years old with gastroenteritis, during the second half of 1992. Fresh first stool cultures were checked daily for toxigenicity by ELISA. Thereafter the serotypes, and the colonization factor antigens (CFAs) were evaluated for the ETEC strains., Results: in 86 of the 415 children (20.7%) we found ETEC. Half these strains would have not been reported as pathogenic without this routine toxigenicity test. CFA were defined in 48 (55.8%) of the ETEC strains. The most common serogroups were O-153, O-6, O-126, and O-128.

The occurrence of Campylobacter and enterotoxigenic E. coli (ETEC) was studied in faecal samples from Tanzanian children (< 5 years of age), adolescents and adults (only Campylobacter) with and without diarrhoea. The Campylobacter strains isolated were tested for subspecies, enterotoxigenicity and serotype. Out of 394 children with diarrhoea 18% were infected with Campylobacter and 20% with ETEC. In 278 samples tested for Campylobacter and 136 tested for ETEC from asymptomatic children the corresponding numbers were 12 and 5%, respectively. In children < 18 months with diarrhoea Campylobacter was noted in 22% and ETEC in 18%, whereas the figures were 11 and 4% respectively in asymptomatic children. In the age group 18 months to 5 years Campylobacter was demonstrated in 2% of the children with diarrhoea and 27% had ETEC, while the figures were 15 and 8% for asymptomatic children. Among adults the prevalence of Campylobacter-positive samples was 1% both for symptomatic and asymptomatic individuals. There were no seasonal differences in the prevalences of both Campylobacter and ETEC either in the symptomatic or the asymptomatic group. Campylobacter jejuni was the dominating Campylobacter species among both symptomatic and asymptomatic individuals. C. jejuni strains from patients with diarrhoea were significantly more often enterotoxigenic than were C. coli strains. The serotype pattern regarding Campylobacter was in general similar for symptomatic and asymptomatic individuals. We conclude that Campylobacter and ETEC are common causes of bacterial diarrhoea in Tanzanian children, and that Campylobacter infections are more important in children younger than 18 months, than in older ones.

We have demonstrated the feasibility of studying antigen-specific immune responses in a variety of mucosal tissues in humans after vaccination and during infection. In this respect, we have documented the usefulness of both oral cholera and ETEC vaccines for assessing in functional terms specific subpopulations of B- and T-cell immunocytes during an immune response initiated and/or expressed in human mucosal tissues. Circulating specific IgA antibody-secreting cells in blood appear to reflect recent or ongoing antigen exposure of mucosal surfaces. This implies that the detection of such cells in blood, the most accessible lymphoid compartment in humans, represents the simplest way to assess the immunogenicity of mucosal vaccines and to supplement the diagnostic and monitoring of active mucosal infections. Our studies indicate that while the concept of an integrated mucosal immune network is clearly operational in humans (at least in regards to induction of secretory antibody responses), its generalization appears somewhat simplistic as illustrated by the compartmentalization of immune responses initiated in certain mucosal organs such as the small intestine and the tonsils. Finally, the potential of the cholera toxin B subunit as a carrier for delivery of chemically or genetically linked foreign epitopes for induction of disseminated mucosal immune responses raises hope for the development of broadly applicable vaccines to control mucosal infections.

The cholera epidemic in South America has reinforced the need for safe and effective oral vaccines. In a randomised, double-blind, placebo-controlled efficacy trial among 1563 Peruvian military recruits we have investigated the protective efficacy of an oral inactivated whole-cell/recombinant-B-subunit (WC/rBS) cholera vaccine. Participants were given two oral doses of cholera vaccine or Escherichia coli K12 placebo, with an interval of 7-14 days. 1426 (91%) subjects received the two prescribed doses and were followed up for a mean of 18 weeks (median 21 weeks). After vaccination, Vibrio cholerae O1 El Tor Ogawa was isolated from 17 subjects with diarrhoea. 16 of the cholera cases occurred 2 weeks or longer after the second dose of vaccine (14 placebo recipients, 2 vaccinees). We also detected 14 symptomless infections (11 [7 placebo recipients, 4 vaccinees]) 2 weeks or longer after the second dose. The vaccine had significant protective efficacy against cholera (86% [95% CI 37-97], p < 0.01) but not against symptomless infection (42% [-96 to 85]). All cholera cases were in people of blood group O, who made up 76% of the study population (p < 0.01). Two doses of WC/rBS vaccine, given 1 to 2 weeks apart, provide rapid, short-term protection against symptomatic cholera in adult South Americans, who are predominantly of blood group O. Long-term efficacy studies in Peruvian adults and children are under way.

Enterotoxigenic Escherichia coli binds to enterocytes in the small intestine by means of antigenically distinct colonization factors (CFs), usually termed colonization factor antigens (CFAs), coli surface antigens (CS), or putative colonization factor antigens (PCFs). To explore the immunological relationship between different CFs, we dissociated CFA/I fimbriae into subunits and produced monoclonal antibodies (MAbs) against these subunits. We selected three MAbs that cross-reacted immunologically with a number of different, whole purified CFs in a dot blot test and with the corresponding subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of the MAbs, i.e., subunit CFA/I 17:8 (S-CFA/I 17:8), reacted more strongly with subunits of CFA/I than with whole purified fimbriae. This MAb cross-reacted with whole purified fimbriae and subunits of CS4, PCFO166, CS1, and CS2. Moreover, it bound strongly to a peptide of 25 amino acids corresponding to the N-terminal end of CFA/I. The other two MAbs, i.e., S-CFA/I 5:6 and S-CFA/I 8:11, cross-reacted with CS1, CS2, CS4, PCFO166, and CS17 fimbriae but reacted only slightly or not at all with the CFA/I peptide. MAbs S-CFA/I 17:8 and S-CFA/I 5:6 were shown to inhibit hemagglutination by bacterial strains that express either CFA/I, CS1, or CS4. In addition, the binding of enterotoxigenic E. coli strains expressing CFA/I, CS2, CS4, and PCFO166 to enterocyte-like cell-line Caco-2 was inhibited by both MAbs. These results show that several antigenically different CFs have common epitopes and that among these at least one is located in the N-terminal end of the subunit protein. Moreover, antibodies against the common epitopes seem to block binding of the bacterial strains that express different CFs to both erythrocytes and Caco-2 cells.

The capacity of peroral immunization with either two or three doses of B subunit-whole cell (B-WC) cholera vaccine to induce immunological memory was examined in Swedish volunteers by testing the immune responses to a single dose of B-WC vaccine given 10 months after the initial immunization. Antibody responses in serum and antibody-secreting cell (ASC) responses in peripheral blood were studied, since these responses seem to reflect the gut mucosal IgA immune responses after oral immunization with B-WC vaccine. Previously immunized volunteers responded to a single dose of B-WC vaccine more frequently and with higher levels of IgA and IgG antitoxin antibodies as well as vibriocidal antibodies in serum than did previously unvaccinated controls. The IgA-ASC responses to cholera toxin B subunit were also higher in primed volunteers than in controls. Two doses of B-WC vaccine were as effective as three doses in inducing immunological memory for cholera immunity. A new B-WC cholera vaccine based on recombinant B subunit had the same capacity as the first generation of B-WC vaccine to induce immunological memory for cholera antitoxin immunity.

Vibrio cholerae O1, which exists as two biotypes, classical and El Tor, expresses fimbrial antigens called toxin-coregulated pili (TCP) and mannose-sensitive hemagglutinin (MSHA) pili, respectively. We have raised rabbit antisera and monoclonal antibodies against these fimbrial antigens and prepared Fab fragments which possess specific antibodies directed against the respective fimbrial antigens from these antisera. The protective effect of these antibody preparations was studied in the infant mouse cholera model. Antibodies against TCP were able to protect baby mice against challenge with V. cholerae O1 of the classical but not of the El Tor biotype. Similar but reverse biotype differences in protection against challenge with classical and El Tor vibrios were observed when antibodies against MSHA pili were used. The protective effect of V. cholerae O1 antilipopolysaccharide (anti-LPS) antibodies, both alone and in combination with antifimbrial antibodies, was also evaluated. We showed that antibodies to the LPS component also prevented infections with V. cholerae O1. Moreover, our results indicate that antibodies against TCP or MSHA pili and against LPS cooperate at least additively, and possible even synergistically, in protecting baby mice against challenge with group O1 vibrios. These results indicate that TCP and MSHA pili as well as LPS play an important role in the pathogenesis of experimental cholera. We could also demonstrate that antibacterial immunity preventing colonization is biotype specific. Our results might be used for the generation of new oral cholera vaccines including both TCP and MSHA fimbrial antigens.

A polynucleotide probe comprising the gene encoding a major structural subunit protein of coli surface antigen 6 (CS6) of enterotoxigenic Escherichia coli (ETEC) was developed. Eighty-nine ETEC isolates were examined in parallel with the probe in a colony hybridization assay and in a recently developed polyclonal-antibody-based inhibition enzyme-linked immunosorbent assay (ELISA). The two assays showed a high level of concordance in the detection of CS6-positive ETEC (kappa = 0.84, P < 0.00001). Thus, 36 of the 89 ETEC isolates were identified as CS6-positive by both assays. Six strains that were negative for other colonization factor antigens were positive with the CS6 probe but negative in the ELISA, suggesting lack of surface CS6 expression in these strains. One strain was probe negative but positive in the ELISA, while the remaining 46 strains were negative in both assays. The phenotypic and genotypic assays will prove useful in vaccine-oriented studies of ETEC disease.

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 fimbrial colonization factor antigen I (CFA/I) and coli surface antigen 4 (CS4) of enterotoxigenic Escherichia coli (ETEC) are antigenically different, but have closely related N-terminal amino acid sequences. We have studied the capacity of purified CFA/I and CS4 fimbriae respectively to prime and boost immune responses against the homologous and heterologous CFAs in parenterally immunized mice. Based on initial characterization of the kinetics of the primary and secondary CFA/l immune responses in serum as well as antibody-secreting cell (ASC) responses in spleen, two doses with different combinations of the purified CFAs were given 7 weeks apart. It was shown, using either assay, that CFA/l could both prime and boost immune responses against CS4, and, conversely, that CS4 could prime and boost immune responses against CFA/l. These findings suggest the presence of immunorecessive epitopes, or epitopes that are not surface-exposed on whole fimbriae, that are shared between CFA/l and CS4 and which can expand B-cell clones with specificity for heterologous fimbriae.

The concept of a common mucosal immune system, through which specific antigen-activated lymphocytes from the gut can disseminate immunity both along the intestinal tract and to various other mucosal and glandular tissues, has generated much current interest in the possibility of developing oral vaccines, not only for enteric infections but also for infections in the respiratory and urogenital tracts. However, to date it has proven difficult in practice to stimulate strong mucosal IgA immune responses by either parenteral or oral-mucosal administration of most antigens, and experience with soluble protein antigens has, on the whole, been disappointing. A notable exception in this regard is cholera toxin (CT) and in humans more than in other species, its nontoxic B subunit pentamer moiety (CTB). Based on this, CTB has become an important component in recently developed oral vaccines against cholera as well as against diarrhea caused by enterotoxigenic Escherichia coli producing CT-like heat-labile enterotoxin(s). Since the strong immunogenicity of CT and CTB can, to a large extent, be explained by their ability to bind to receptors on the intestinal mucosal surface, there has recently been much interest in approaches using CTB as an oral delivery carrier system for other vaccine-relevant antigens, and much progress has been made in preparing immunogenic hybrid proteins by coupling various protein or peptide antigens chemically or genetically to CTB. Indeed, in several systems, oral administration of such hybrid antigens has been found to markedly potentiate both intestinal and extraintestinal IgA immune responses against the CTB-coupled antigens and also to elicit substantial circulating antibody responses. Besides the mucosal immunopotentiating effect of either CT or CTB owing to their similar capacity as oral antigen-delivery vehicles, CT, but in most systems tested not CTB, also has strong adjuvant properties for stimulating mucosal IgA immune responses to admixed (not coupled) unrelated antigens after oral immunization. This adjuvant activity appears to be closely linked to the ADP-ribosylating action of CT (and specifically of its A subunit) leading to enhanced cyclic AMP formation in the affected cell, and efforts to eliminate the enterotoxic activity without losing adjuvanticity have so far not met with success.

The ability to colonize the small intestine is essential for enterotoxigenic Escherichia coli (ETEC) to cause diarrhea. Several colonization factor antigens (CFAs) and putative colonization factors (PCFs) have been described for ETEC. However, there are still many ETEC strains isolated from patients with diarrhea which do not possess any of these antigens. To identify CFAs in ETEC lacking the above-mentioned antigens, we exploited the ability of ETEC to adhere to tissue-cultured cells from an enterocyte-like cell line, Caco-2. An ETEC strain producing heat-labile toxin and heat-stable toxin of serotype O20:K27:H- (ARG-2) that was isolated from a child with diarrhea in Argentina and bound to Caco-2 cells was studied in further detail. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of this strain revealed a band of 25 kDa when bacteria were grown at 37 degrees C that was missing when the same strain was cultured at 20 degrees C. Furthermore, electron microscopy examination revealed the presence of fimbriae on the surfaces of cells of this strain when cells were grown at 37 degrees C but not at 20 degrees C. Rabbit antiserum raised against purified fimbriae reacted with the 25-kDa protein in immunoblotting and bound specifically to the fimbriae, as shown by immunoelectron microscopy. The presence of fimbriae, adhesion to Caco-2 cells, and the 25-kDa band seen in the SDS-PAGE were all simultaneously lost by single-insertion mutations. The N-terminal amino acid sequence of the protein subunit of the fimbriae showed no relation with those of the known colonization factors of ETEC. Furthermore, the fimbriae of the ARG-2 strain did not cross-react immunologically with any of the previously described adhesive factors in human ETEC when specific antisera against colonization factor antigens and putative colonization factors were used. Moreover, a specific antiserum raised against the fimbriae in ARG-2 did not react with ETEC carrying known colonization factors. We propose to name these new fimbriae PCFO20.

The recent spread of cholera to Latin America, together with the persistent burden of this disease in Asia and Africa, have stimulated efforts to evaluate new cholera vaccines in field settings. Although the standard experimental paradigm for vaccine field trials is well established, the success of these trials will also depend on suitable consideration of the epidemiology of cholera and of cholera vaccination in the setting under study. Epidemiological studies done in Bangladesh emphasize the importance of appreciating the poorly predictable, multifocal occurrence of cholera in estimating a probable incidence of cholera for a field trial. They also underscore how the filtering effect of enrolling subjects into a prospective trial can dramatically reduce the available population for study, and can yield a study sample whose expected risk of cholera differs markedly from that for the source population. Finally, the data highlight the subtle effects that the mode of surveillance and the choice of an outcome definition can have upon protective efficacy, and emphasize the need for subgroup analyses that address the distinctive variations in vaccine protection that may occur in subjects differing in age and in ABO blood groups, and in subjects exposed to classical versus El Tor cholera.

A newly formulated, oral, inactivated whole cell plus recombinant B subunit (WC/rBS) cholera vaccine was evaluated in US military personnel. In the first study, 74 subjects were given two doses 14 days apart. In the second study, 186 subjects were randomized into four groups; two groups received vaccine with either full (4 g) or half (2 g) strength bicarbonate buffer, and two groups received either full or half strength buffer without vaccine. Mild gastrointestinal symptoms were associated with full buffer (P = .02) but not with the vaccine. In the first study, 36% of all subjects and 55% with low prevaccination titers (< 1:40) had a > or = 2-fold rise in vibriocidal antibody level; > 80% of subjects developed a 4-fold rise in anti-cholera toxin (CT) titers. Post-vaccination IgA and IgG anti-CT titers were approximately 1.5-fold higher among persons receiving full strength buffer (P = .05). The WC/rBS vaccine is safe and immunogenic in North Americans, although some mild gastrointestinal symptoms occur with the high concentration of buffer necessary to protect the B subunit from gastric acid denaturation. Prior immunity to cholera conferred by parenteral vaccine decreased vibriocidal antibody response.

It is now almost axiomatic that vaccines against enteric infections must be able to stimulate the gut lymphoid tissue to be efficacious and that this goal is usually better achieved by administering immunogens orally rather than parenterally. On the basis of the notion of a common mucosal immunologic system that provides immune reactivity not only at the site of antigen deposition but also at remote mucosal sites, there is much interest in developing oral vaccines against infections in the respiratory and urogenital tracts. Recent studies indicate that oral administration of small amounts of protein antigens that are chemically or genetically linked to intestinal binding carrier molecules (such as the B subunit of cholera toxin) can evoke vigorous mucosal and extramucosal immune responses. The apparent compartmentalization of systemic and mucosal immune responses may explain not only why parenteral vaccines are just partly effective in protecting against mucosal pathogens but also why currently employed immunoanalytical methods do not provide accurate information regarding mucosal immune status. Following the developments of oral vaccines against infections due to Vibrio cholerae and enterotoxigenic Escherichia coli, two archetypes of enteropathogens, we have devoted extensive efforts to gaining insight into the humoral and cellular events involved in the development of protective immunity in the human intestinal tract. We have developed preparative and analytical methods for studying vaccine-induced intestinal and extraintestinal immune responses in humans. These include techniques for collecting intestinal fluid and lymphoid cells as well as procedures to quantitate secretory antibodies and lymphokines secreted by activated intestinal immunocytes. These developments should serve the dual purpose of facilitating analyses of human mucosal immune responses in general and assessing the immunogenicity of enteric vaccines in particular.

Monoclonal antibodies (MAbs) against five putative colonization factors (PCFs), i.e., colonization factor antigen (CFA)/III, coli surface antigen (CS)7 and CS17, PCFO159, and PCFO166 of enterotoxigenic Escherichia coli (ETEC), were produced. Hybridomas (one each) producing specific antibodies against the respective PCFs were selected. All the MAbs reacted with the corresponding fimbriae but not with CFA/I, CFA/II, or CFA/IV or the heterologous PCFs in bacterial agglutination and enzyme-linked immunosorbent assays (ELISAs). In immunoelectron microscopy these MAbs bound along the fimbriae, and they also reacted with the corresponding subunits in immunoblots. The five MAbs were used to evaluate the prevalence of CFA/III, CS7, CS17, PCFO159, and PCFO166 in ETEC strains isolated from children with diarrhea in Argentina. One hundred five ETEC isolates negative for CFA/I, CFA/II, and CFA/IV were tested in slide agglutination or in a dot blot test for spontaneously agglutinating strains; positive results were confirmed by inhibition ELISAs. It was found that 27% of the CFA-negative ETEC strains carried one of the PCFs. The sensitivity of slide agglutination with these MAbs was similar to that with specific polyclonal antisera; however, the specificity was higher. PCFO166 was found in 9.5% of the strains tested, mainly in ETEC of serogroup O78 producing heat-stable toxin alone. CS17 and CS7 were identified in 6.7 and 5.7%, respectively, of strains producing heat-labile toxin only, most of which belonged to serogroup O114. PCFO159 was found in 3.8% of the isolates tested, whereas CFA/III was detected in only one ETEC strain.

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.

Different immunization schedules for oral B subunit-whole cell (B-WC) cholera vaccine were evaluated in Swedish volunteers to obtain information for recommendations of vaccine use in non-endemic areas. Two peroral doses of B-WC vaccine were as effective as three doses in inducing IgA and IgG antitoxin as well as vibriocidal antibody responses in serum. Administration of two vaccine doses either at 7, 14 or 28-42 day intervals resulted in comparable antitoxin responses in serum, whereas a 3-day immunization interval resulted in significantly lower titre increases. Vibriocidal antibody responses were comparable after the different time intervals tested (3-42 days). The B-WC vaccine can be effectively administered together with a cheap, commercially available sodium bicarbonate powder dissolved in water to protect the vaccine from gastric acid.

Regulatory proteins control the expression of the fimbrial colonization factor antigens CFA/I and CS4 of enterotoxigenic Escherichia coli (ETEC). To examine the mechanism behind lack of expression of these antigens in spontaneous CFA-negative mutants, we mobilized a recombinant plasmid harbouring the cfaD gene, which encodes a positive regulator of CFA/I and CS4 expression, into such derivatives. In electron microscopy, the induced surface structures were morphologically identical to the fimbriae of the CFA/I+ and CS4+ wild type strains. Immunogold labelling with monoclonal antibodies showed that the distribution of CFA/I and CS4 specific epitopes along the induced fimbriae was indistinguishable from that of the wild type strains. The percentage of fimbriated cells was consistently higher in the cfaD transformants than in the corresponding wild type strains. The present work reports on the efficiency of the cloned cfaD gene in restoring and enhancing the production of morphologically intact CFA/I and CS4 fimbriae.

The role of some well-characterized putative colonization factors (PCFs) in enterotoxigenic Escherichia coli (ETEC), i.e. PCFO159, PCFO166, CS7, CS17 and CFA/III, for colonization of the bacteria in the intestine was studied in a non-ligated rabbit intestine model (RITARD). Intestinal administration of 10(11) organisms of the various strains only resulted in very mild symptoms with loose stools during a few days in most of the animals. Strains expressing PCFO159, CS7, CS17 and CFA/III were shed in the stool for a significantly longer period than PCF/CS-negative ETEC. However, the mean time of shedding PCFO166 positive organisms did not significantly exceed that of non-fimbriated E. coli. All strains that colonized rabbit intestine, as assessed by prolonged fecal excretion, also gave rise to high serum antibody responses against the homologous fimbriae whereas non-colonizing strains failed to induce such responses. This study strongly suggests that several of the recently described PCFs, e.g. PCFO159, CS7, CS17 and CFA/III are colonizing factors and strong immunogens.

An ELISA for determination of antibodies to V. cholerae TCP was developed. Since purified TCP preparations contained detectable amounts of LPS (as shown by ELISA and immunoelectron microscopy with anti-LPS polyclonal serum), a capture ELISA was used. In this test the plate was coated with anti-TCP monoclonal antibody followed by incubation with TCP fimbriae. By this procedure no LPS bound to the solid phase as shown by the loss of reactivity with anti-LPS serum. The capture ELISA allowed sensitive and specific determination of TCP antibodies in sera of rabbits immunized with classical but not El Tor V. cholerae strains. There was good agreement between results in the TCP ELISA and reactivity with the TcpA band in immunoblot analyses when antisera raised against classical and El Tor vibrios were studied.