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In rural Bangladesh, family contacts of patients with cholera were studied prospectively to examine whether protection against colonization and disease due to Vibrio cholerae O1 was associated with circulating antibodies to V. cholerae. Family contacts (1,071) of 370 patients with cholera were visited daily for 10 days, cultured for V. cholerae, and queried about diarrhea. Sera collected on days 1 and 21 were assayed for vibriocidal antibodies, IgG and IgA antibodies to cholera toxin, and IgG antibodies to lipopolysaccharide (LPS). Vibriocidal titers of greater than or equal to 20 present in 50% of contacts by 20 years of age were associated with protection against both colonization and disease. An elevated level of IgG antitoxin was not associated with protection against colonization or disease but was the most sensitive indicator of recent symptomatic cholera and of immune response to the oral immunogen B subunit. IgG antibody to LPS and IgA antitoxin were of little value in predicting colonization or disease.

The development of antibodies to Escherichia coli heat-labile enterotoxin (LT) and its relationship to diarrhoea was studied in two Swedish battalions (one in the winter, WB, and one in the summer, SB) of the United Nations Peace-keeping Force in Cyprus. Whereas only 6-15% of the soldiers had detectable levels of anti-LT antibodies in their sera before leaving Sweden, as many as 69% of the SB and 43% of the WB soldiers who developed diarrhoea in Cyprus acquired significant titres of enterotoxin antibodies in their sera (acute phase serum). During the summer, many soldiers without diarrhoea also obtained anti-LT antibodies, whereas no such response was observed in healthy soldiers during the winter. Civilian Turkish Cypriots had positive sera in high frequency (60-66%) during both study periods. Parallel titration of positive sera against E. coli LT and cholera toxin indicated that the enterotoxin-neutralizing antibodies registered were induced by bacteria producing enterotoxin identical or closely related to LT.

Fifteen patients hospitalized with acute, watery diarrhea and with enterotoxigenic Escherichia coli (ETEC) detected from stool samples were studied to evaluate the extent to which natural ETEC diarrhea induces local and systemic antibody responses to E. coli heat-labile toxin (LT), homologous lipopolysaccharide (LPS), and colonization factors (CFA/I and CFA/II). Specific IgA and IgG antibodies to LT, CFA I and II, and each patient's homologous LPS were determined by ELISA in serum, saliva, breastmilk, and intestinal lavage fluid. The majority of patients had greater than a twofold rise in local levels of IgA antibodies in the intestine: 80% of LT+ patients responded to LT, 63% of CFA+ patients responded to CFA, and 78% of all toxin-positive patients responded to the LPS of their infecting strain. Local antibody responses in the intestine were associated with responses in breastmilk and saliva, but relationships were not clear-cut, and the usefulness of these secretions as proxy measures of local intestinal antibody production remains unclear. Antibody responses in serum also occurred in most patients and were significantly more frequent in cases than in controls. This study demonstrates that natural ETEC disease results in local IgA responses to LT, CFA, and LPS in the gut and also in immune responses in breastmilk, saliva, and serum.

The effect of Giardia muris infection on the diarrheal response and gut mucosal antibody response to cholera toxin was examined in mice. The results obtained showed that the fluid accumulation in intestinal loops exposed to cholera toxin was increased in mice infected with a low number (5 X 10(4) ) of G. muris cysts compared with the response in noninfected mice. This effect was associated with a marked reduction in absorption of oral rehydration fluid from the intestine. In contrast, mice infected with a high dose (2 X 10(5) ) of cysts showed a marked decrease in fluid accumulation in response to the toxin. This decrease might be related to the finding that both G. muris and Giardia lamblia trophozoites can bind significant amounts of cholera toxin. Evidence is presented which suggests that the gut mucosal antibody response, mainly immunoglobulin A but also immunoglobulin G, to an immunization course with perorally administered cholera toxin was depressed in mice infected with G. muris. The reduction in antibody levels was particularly evident when the primary immunization was made very early after infection. The serum antitoxin antibodies to the oral immunization with cholera toxin were, however, not affected. Likewise, the delayed-type hypersensitivity response against sheep erythrocytes in animals primed subcutaneously with sheep erythrocytes was not modified during the course of G. muris infection.

The association between undernutrition and the risk of colonization and disease with Vibrio cholerae O1, concentrations of salivary IgA and the serologic response to infection and to orally administered cholera B subunit were examined prospectively in a family study in Bangladesh. Children ages 1 to 8 years who were family contacts of patients hospitalized with culture-confirmed cholera were visited within 24 hours of the hospitalization and daily for 10 days, queried for the presence of diarrhea and cultured for V. cholerae O1. On Day 1 each child was weighed and saliva was collected to measure total IgA. On Days 1 and 21 blood was taken to assess vibriocidal and antitoxin titers, and on Days 1 and 2 B subunit or placebo was given orally as part of a trial to look for a toxin-blocking effect. Of 412 children enrolled in the study 35% (143) became infected with V. cholerae O1 and 49% (70) of these developed diarrhea. Undernutrition, defined in a child as weight less than 70% of the Harvard reference weight-for-age, was not associated with colonization, disease or the duration or severity of cholera. Moreover well-nourished children did not differ from undernourished children in their concentrations of salivary total IgA, initial serum antitoxin or vibriocidal antibodies or in their serologic response to colonization, disease or B subunit. The immune system in its response to cholera appears to be quite resistant to nutritional insults. The good antitoxin response to B subunit among undernourished children is of particular importance in considering the use of future oral cholera vaccines in areas where such undernutrition is common.

The occurrence of specific antibodies to Vibrio cholerae lipopolysaccharide in serum, milk, and saliva of Pakistani women from a very low socioeconomic group was studied before and after a single subcutaneous cholera vaccination. Before immunization all women had low levels of specific antibodies in serum, primarily of IgM class, and in many cases cholera IgA angibodies were found in milk and saliva as well, indicating earlier natural exposure. The vaccination consistently induced a marked rise in serum antibody titer, and notably also produced significant titer increases in 70% of the milk and in 45% of the saliva samples. Whereas the serum antibodies induced were predominantly of the IgG class, secretory IgA was responsible for most of the titer increase in the secretions. The results indicate that parenteral cholera vaccination can boost local secretory IgA antibody responses in intestinally primed individuals.

A soluble hemagglutinin/protease from Vibrio cholerae has been suggested to be a putative virulence factor and protective antigen. However, clinical cholera infection gave rise to detectable serum antibody responses to soluble hemagglutinin in only 2 of 10 Bangladeshi patients or 1 of 17 cholera-infected North American volunteers. A gut mucosal immunoglobulin A antibody response to soluble hemagglutinin was seen in 4 of 8 Bangladeshi patients, but in 0 of 10 North American volunteers. These responses were much weaker than those to cholera toxin or lipopolysaccharide.

The immunoglobulin subclass distribution of cholera antitoxin antibody responses in serum was studied in Swedish volunteers after different routes of immunization with a cholera B subunit-whole cell vaccine (B + WCV) and in Bangladeshi patients convalescing from cholera disease. Both oral and parenteral immunization induced antitoxin antibodies of all the different IgG subclasses (IgG1, IgG2, IgG3 and IgG4) whilst the IgA antibodies were restricted to the IgA1 subclass. A single oral dose of B + WCV induced proportionally higher levels of IgG4 antitoxin in previously cholera-immunized volunteers than in a matched group who had not been cholera-vaccinated before, suggesting that repeated immunization preferentially stimulate formation of IgG4 antibodies. The IgG and IgA subclass distribution of antitoxin antibodies in orally vaccinated Swedes closely resembled that in Bangladeshi cholera patients.

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.

Serological responses to relevant enterotoxinogenic Escherichia coli (ETEC) antigens were studied in 85 Swedish travellers to subtropical and tropical areas. Serum samples were collected from the travellers before and then after 3-5 weeks, i.e. within a week after return to Sweden, when a faecal specimen was also taken. 40% of the participants had traveller's diarrhoea during their visit abroad, while 21% reported "loose stools" and 39% had no such problems. ETEC was rarely isolated from the stool of any of the travellers on their return to Sweden (6%). Salmonellae, Shigellae or Campylobacter jejuni were isolated from 13 (15%) of the travellers, 12 of whom were healthy when the specimens were collected and 4 of whom had been healthy during travel as well. Significant serum antibody responses to E. coli heat-labile enterotoxin (LT) and colonization factor antigens CFA I or II were seen in 33% and 20% of the travellers, respectively. Anti-LT responses were comparable in participants with traveller's diarrhoea or "loose stools" and in the healthy ones, whereas anti-CFA responses were more frequent in those with symptomatic infections. Of 34 Swedes in a non-travelling control group none responded to LT and one to CFA I or CFA II.

An in vitro culture technique with synthesis of 14C-labeled protein has been used to study immunoglobulin and specific-antibody formation by spleen, mesenteric lymph nodes, Peyer's patches, and small intestine of rabbits, which were immunized twice subcutaneously with Vibrio cholerae lipopolysaccharide (LPS) and enterotoxin; saline-injected rabbits served as controls. Newly synthesized immunoglobulin G (IgG), IgA, and IgM were quantitated by liquid scintillation after their isolation by means of affinity chromatography from columns with immunoglobulin class-specific antibodies coupled to Sepharose. Specific antibodies were similarly measured after purification from gels derivatized with LPS or cholera toxin. The isolated antibodies had full biological activity as studied in protection tests. The immunization increased the overall IgM synthesis in the spleen. It also enhanced the production of IgA and IgG in Peyer's patches and of IgA in intestine. Significant synthesis of radiolabeled antibodies against both V. cholerae LPS and enterotoxin was found in spleen as well as in Peyer's patches of immunized animals. Titration with an enzyme-linked immunosorbent assay (ELISA) showed significant levels of IgG as well as IgA antibodies in incubation medium from all the studied tissues, whereas specific IgM was only found for spleen and mesenteric lymph nodes. Simultaneous tissue incubations at 37 degrees C and in an ice bath indicated that the major part of the antibodies registered with the ELISA represented de novo synthesis.

The protective effect of enternal and parenteral immunization with cholera toxin antigen against experimental cholera in rabbits was studied by using the small-bowel loop technique. Subcutaneous injection of crude toxin as well as purified toxin or toxoids gave rise to significant protection against toxin challenge. The enhanced resistance to toxin was found to correspond to a many-fold higher magnitude of protection against challenge with live vibrios. In the primary response the protection increased during the first month. Booster immunization gave rise to a further increased immunity which, however, declined rapidly. Multiple oral or repeated intraintestinal antigen administrations also induced protective antitoxic immunity although of less magnitude than that obtained by parenteral immunization. Enteral and, to a lesser extent, parenteral immunization gave rise to increased antitoxic antibody titers and immunoglobulin levels in intestinal washings and mucosa scraping. Immunoglobulin G (IgG) and IgG antitoxins predominated, but after enteral immunization total IgA and specific IgA antibodies occasionally reached levels similar to those for IgG. In serum, significantly increased antibody levels (IgG) were only recorded after parenteral immunization. Both the primary binding and the neutralizing antitoxin titers showed a stayistically significant correlation with the degree of protection against toxin challenge; however, for the neutralizing antibodies this correlation was not without exceptions. No relation to protection was found for intestinal antibodies. The results of the present study indicate that enternal as well as parenteral immunization with toxin antigen can give rise to effective cholera immunity. After enternal immunization, the protection appears to be medicated by locally synthesized antibodies. After parenteral vaccination both serum-derived and locally produced antibodies seem to be effective.

The enzyme-linked immunosorbent assay (ELISA) principle of Engvall and Perlmann, which employs antigen-coated tubes and enzyme-labeled anti-immunoglobulins, was elaborated for use in cholera serology. Immunoglobulin class-specific determinations of primary binding titers of antibodies to cholera exotoxin and endotoxin were more sensitive than were neutralization and vibriocidal tests. It was notable that rather high ELISA titers of immunoglobulin M (IgM) antibodies to exotoxin, which lacked neutralizing capacity were registered, whereas corresponding levels of immunoglobulin G (IgG) antibodies were effectively neutralizing. A modified technique permitting measurement of the binding rate of antibody to the solid-phase antigen was introduced as a possible tool for antibody avidity estimation. Such measurements indicated a wide avidity difference between the IgG and the IgM anti-exotoxin antibodies, which could explain their different neutralizing capacities. The observation that increasing binding rate of the IgG antibodies during the course of the primary immune response could compensate for decreasing antibody amount with regard to neutralizing capacity of serum also indicated the importance of antibody avidity for toxin neutralization. Inhibition with soluble antigen permitted quantitative determination of antigen; levels of exotoxin 0.09 mug/ml and of endotoxin to 1.3 mug/ml were measured.