Abstract: Cholera remains the cause of high rates of morbidity and mortality in poor-sanitation areas in the developing world (20). Vibrio cholerae, the etiologic agent of cholera, is a gram-negative prototrophic bacterium able to persist for long periods of time in the environment and reemerge as a fully virulent pathogen for humans (14, 26). Live oral cholera vaccines seem the most promising for elicitation of multifactorial and long-lasting immunity after a single dose (32). However, the implicit release of living bacteria into the environment continues to be a cause of concern worldwide. The El Tor Ogawa live cholera vaccine candidate strain 638 was recently demonstrated to be well tolerated and immunogenic in Cuban volunteers (5), as was CVD103HgR in North American volunteers (32). Inactivation of the thyA gene has been proposed as a biological containment tool for microorganisms intended to be released into the environment (23). The thyA gene codes for thymidylate synthase (TS), the enzyme responsible for the catalytic conversion of dUMP into dTTP (21). Bacterial strains bearing deletions within the thyA gene are auxotrophic for thymine or thymidine and are not expected to proliferate in the environment, where free pyrimidines are absent. Previous to this work, undefined mutants of V. cholerae with thymidine requirements had been selected by trimethoprim resistance. For example, CVD102, a spontaneous thyA-defective derivative of CVD101, was poorly excreted by humans and minimally immunogenic (19). Further experiments demonstrated the CVD102 colonization defect to be unrelated to the thyA mutation, and similar thyA mutants of CVD101 colonized well in the infant mouse cholera model (2). The construction of a thyA-defined mutant of V. cholerae has not been reported previous to this work. The present paper describes cloning and nucleotide sequencing of the thyA gene from V. cholerae and the construction of a thyA-defined mutant derived from our vaccine candidate strain 638 (V. cholerae O1, El Tor Ogawa, ΔCTXΦ, hap::celA). The resultant mutant, 638T, was unable to proliferate in thymidine-free minimal medium unless it was complemented by plasmid pVT1 carrying the V. cholerae thyA gene. The environmental survival of 638T was also examined and demonstrated to be diminished with respect to that of 638. Additionally, this mutant was able to colonize in the infant mouse cholera model and was also immunogenic in rabbits, suggesting that although limited in proliferation, the vaccine candidate effectively stimulates the mucosal immune system to induce a serological response.

Abstract: Cholera toxin (CT)-specific antibody responses of the immunoglobulin E (IgE) isotype in the sera of adult patients suffering from infection with either Vibrio cholerae O1, V. cholerae O139, or enterotoxigenic Escherichia coli (ETEC) were analyzed and compared with those in the sera of volunteers immunized with a bivalent B subunit O1/O139 whole-cell cholera vaccine. A significant IgE response to CT was observed in 90% of the patients with V. cholerae O1 infection (18 of 20; P = <0.001) and 95% of the patients with V. cholerae O139 infection (19 of 20; P = <0.001). Similarly, the majority of the patients with ETEC diarrhea (83%; 13 of 15) showed a positive IgE response to CT. Eight of 10 North American volunteers (80%) orally challenged with V. cholerae O1 showed CT-specific IgE responses (P = 0.004). In contrast, Swedish volunteers immunized with the oral cholera vaccine showed no IgE responses to CT (P value not significant). During the study period, total IgE levels in the sera of the diarrheal patients, the North American volunteers, and the Swedish cholera vaccinees alike remained unchanged. However, the total IgE levels in the sera of patients and healthy Bangladeshi controls were on average 89-fold higher than those in the sera of the healthy Swedish volunteers and 34-fold higher than those in the sera of the North American volunteers.

Abstract: The antimicrobial susceptibility and the presence of a heat-stable toxin were researched into 100 non-01 Vibrio cholerae strains sent by 7 different health centers to the National Reference Laboratory of Acute Diarrheal Diseases in "Pedro Kouri" Tropical Medicine Institute. The presence of 20% toxigenic non-01 Vibrio cholerae was detected, a figure substantially higher than that reported in other geographic areas, except for endemic areas. This result will make it possible to set epidemiological alert in Cuba because these strains can be infected by CTX phages (element transporting genes that encode for choleric toxin) which will give such strains an epidemic potential similar to that of the etiologic agent of cholera. The identified strains could be studied as possible cholera vaccine candidates.

Abstract: Cholera vaccines developed by the deletion of CTX genes fromVibrio cholerae induce a residual reactogenicity in up to 10% of vaccinees. A novel cytotonic agent named secreted CHO cell elongating protein (S-CEP) was purified from culture supernatants of CVD 103-HgR (Levine et al., Lancet ii:467–470, 1988). Five fractionation steps yielded electrophoretically pure S-CEP with anMr of 79,000. A partially purified preparation caused fluid accumulation in the sealed infant mouse model. The amino terminus bore a unique sequence with strong homology to a cytotonic toxin of El Tor V. cholerae.

Abstract: The effectiveness of dot immunoanalysis for evaluating the dynamics of the synthesis of O-antigen, cholera toxin, neuraminidase, adhesin CFA1 in the process of the reactor cultivation of V. cholerae used for the production of oral chemical cholera vaccine is shown. The established regularities of the synthesis of the protective antigens of V. cholerae in the process of scaled-up cultivation are discussed.

Abstract: Because concurrent infections with geohelminth parasites might impair the immune response to oral vaccines, we studied the vibriocidal antibody response to the oral cholera vaccine CVD 103-HgR in children infected with Ascaris lumbricoides and investigated the effect of albendazole pretreatment on the postvaccination response. Children with ascariasis were randomized to receive either 2 sequential doses of 400 mg of albendazole or placebo. After the second dose, CVD 103-HgR was given, and serum vibriocidal antibody levels were measured before and 10 days after vaccination. Postvaccination rates of seroconversion were greater in the treatment group that received albendazole (P=.06). Significantly greater rates of seroconversion and geometric mean titer were observed in the albendazole group in subjects with non-O ABO blood groups. A significant association was observed between vibriocidal seroconversion rates and treatment group, suggesting that A. lumbricoides infections impair the immune response to oral cholera vaccine, particularly in subjects of non-O blood groups.