In this study, we analyzed whether attachment of Vibrio cholerae vaccine strains to human intestinal epithelial cells can induce an interleukin-8 (IL-8) response. The IL-8 transcripts were detected by PCR amplification of reverse-transcribed mRNA, and the gene product secretion was measured by an enzyme-linked immunosorbent assay. Infection of monolayers of the undifferentiated HT29-18N2 cell line with reactogenic (JBK70 and 81) and nonreactogenic (CVD103HgR and 638) vaccine strains of V. cholerae resulted in markedly higher IL-8 expression by epithelial cells exposed to reactogenic strains than by cells exposed to the nonreactogenic strains. Additionally, epithelial cells produced IL-8 transcripts following stimulation with cholera vaccine strains in a concentration-dependent manner. These results represent a new insight into the inflammatory component of reactogenicity and could be used as a predictive marker of vaccine reactogenicity prior to human testing.Cholera is traditionally considered to be a noninflammatory diarrheal disease caused by Vibrio cholerae serogroups O1 and O139 (10). Several groups of investigators have constructed a wide range of live attenuated V. cholerae O1 and O139 strains by deleting the CTX genes in order to immunize against cholera (2,5,9,11,16,19). However, many of these strains have shown residual adverse properties (reactogenicity) in volunteer studies (1,12,22,23). The cellular basis of reactogenicity is not clear, but there is some evidence of an intestinal inflammatory response. It was postulated that close proximity or contact of bacterial cells to the apical surface of the intestinal epithelium causes reactogenicity, due to the induction of a local inflammatory response; this theory was based on the lower reactogenicity observed for nonmotile mutants of V. cholerae compared with parental motile vaccine strains in volunteer studies (15). Furthermore, oral vaccination with CVD110, a reactogenic strain, produced copious amounts of lactoferrin and increased interleukin-8 (IL-8) levels in the stool of volunteers, while a nonreactogenic strain (CVD103HgR) did not (21). The proinflammatory cytokine IL-8 is a potent chemoattractant for polymorphonuclear leukocytes and T lymphocytes (17). Polymorphonuclear leukocytes, which are found in large numbers in inflammatory diarrheas (6, 20), can induce chloride secretion similar to that seen in toxigenic secretory diarrhea illnesses (14) and could play an important role in the diarrhea seen with live attenuated cholera vaccine strains. On the other hand, epithelial cells infected with several invasive and some noninvasive enteric pathogens produced an IL-8 response (8). However, the induction of proinflammatory signals by the attachment of V. cholerae strains to intestinal epithelial cell lines has not been investigated. Here, we examined the impact of attachment of reactogenic and nonreactogenic live cholera vaccine strains to human intestinal epithelial cells on the IL-8 response by using the undifferentiated HT29-18N2 cell line (7), a c...

The ability of the RD (rhabdomyosarcoma) and MRC-5 cell-lines to detect enteroviruses in 33 clinical samples (cerebrospinal fluid, stools and throat swabs) was evaluated. The samples had previously tested enterovirus-positive by traditional tube-culture and had been frozen after their initial processing. By traditional tube-culture, 100 and 85 % of samples were positive for enterovirus in RD and MRC-5 cells, respectively. By rapid shell-vial assay, 94 and 45·5 % were positive after 48 h incubation in RD and MRC-5 cells, respectively. RD cells supported growth of all enterovirus serotypes, whereas MRC-5 cells were not able to detect any of the three coxsackieviruses that were found (one coxsackievirus A9 and two coxsackievirus B5). The shell-vial assay with RD cell-lines may be a useful tool for rapid diagnosis of enteroviral infection.

No abstract

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...

No abstract