Vibrio cholerae 638 is a living candidate cholera vaccine strain attenuated by deletion of the CTX⌽ prophage from C7258 (O1, El Tor Ogawa) and by insertion of the Clostridium thermocellum endoglucanase A gene into the hemagglutinin/protease coding sequence. This vaccine candidate was previously found to be well tolerated and immunogenic in volunteers. This article reports a randomized, double-blind, placebo-controlled trial conducted to test short-term protection conferred by 638 against subsequent V. cholerae infection and disease in volunteers in Cuba. A total of 45 subjects were enrolled and assigned to receive vaccine or placebo. The vaccine contained 10 9 CFU of freshly harvested 638 buffered with 1.3% NaHCO 3 , while the placebo was buffer alone. After vaccine but not after placebo intake, 96% of volunteers had at least a fourfold increase in vibriocidal antibody titers, and 50% showed a doubling of at least the lipopolysaccharide-specific immunoglobulin A titers in serum. At 1 month after vaccination, five volunteers from the vaccine group and five from the placebo group underwent an exploratory challenge study with 10 9 CFU of ⌬CTX⌽ attenuated mutant strain V. cholerae 81. Only two volunteers from the vaccine group shed strain 81 in their feces, but none of them experienced diarrhea; in the placebo group, all volunteers excreted the challenge strain, and three had reactogenic diarrhea. An additional 12 vaccinees and 9 placebo recipients underwent challenge with 7 ؋ 10 5 CFU of virulent strain V. cholerae 3008 freshly harvested from a brain heart infusion agar plate and buffered with 1.3% NaHCO 3 . Three volunteers (25%) from the vaccine group and all from the placebo group shed the challenge agent in their feces. None of the 12 vaccinees but 7 volunteers from the placebo group had diarrhea, and 2 of the latter exhibited severe cholera (>5,000 g of diarrheal stool). These results indicate that at 1 month after ingestion of a single oral dose (10 9 CFU) of strain 638, volunteers remained protected against cholera infection and disease provoked by the wild-type challenge agent V. cholerae 3008. We recommend that additional vaccine lots of 638 be prepared under good manufacturing practices for further evaluation.
Vibrio cholerae 638 (El Tor, Ogawa), a new CTXΦ-negative hemagglutinin/protease-defective strain that is a cholera vaccine candidate, was examined for safety and immunogenicity in healthy adult volunteers. In a double-blind placebo-controlled study, no significant adverse reactions were observed in volunteers ingesting strain 638. Four volunteers of 42 who ingested strain 638 and 1 of 14 who received placebo experienced loose stools. The strain strongly colonized the human small bowel, as evidenced by its isolation from the stools of 37 of 42 volunteers. V. cholerae 638, at doses ranging from 4 × 107 to 2 × 109 vibrios, elicited significant serum vibriocidal antibody and anti-Ogawa immunoglobulin A antibody secreting cell responses.
In recent clinical assays, our cholera vaccine candidate strain, Vibrio cholerae 638 El Tor Ogawa, was well tolerated and immunogenic in Cuban volunteers. In this work we describe the construction of 638T, a thymidine auxotrophic version of improved environmental biosafety. In so doing, the thyA gene from V. cholerae was cloned, sequenced, mutated in vitro, and used to replace the wild-type allele. Except for its dependence on thymidine for growth in minimal medium, 638T is essentially indistinguishable from 638 in the rate of growth and morphology in complete medium. The two strains showed equivalent phenotypes with regard to motility, expression of the celA marker, colonization capacity in the infant mouse cholera model, and immunogenicity in the adult rabbit cholera model. However, the ability of this new strain to survive environmental starvation was limited with respect to that of 638. Taken together, these results suggest that this live, attenuated, but nonproliferative strain is a new, promising cholera vaccine candidate.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 O...
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