Culture supernatants prepared from reactogenic strains of Vibrio cholerae cause a decrease in the transcellular epithelial resistance of T84 intestinal cells. This decrease correlates with the presence of hemagglutinin/ protease but not with the presence of other potential accessory toxins or proteases. These data suggest a possible role for hemagglutinin/protease in reactogenicity, although other factors may also contribute.The potentially life-threatening disease cholera is caused by toxigenic strains of the gram-negative organism Vibrio cholerae. The hallmark symptom of cholera, profuse, watery diarrhea, is caused primarily by cholera toxin (CT). The genes encoding CT, ctxAB, are carried on a transducing phage, CTX⌽, that integrates into the V. cholerae genome (29). The "core" element of the phage genome carries four genes in addition to ctxAB : cep, orfU, ace, and zot (29). Both zot and orfU are known to be essential for phage morphogenesis (29). In addition, the recombinant products of the zot and ace genes have also been associated with changes in intestinal tissue conductance, suggesting that these genes encode accessory toxins of V. cholerae (6,27,28).The development of a safe and effective vaccine to protect against cholera is a multifaceted problem. An ideal cholera vaccine must confer long-lasting protective immunity, be inexpensive, and be easy to use. A vaccine strategy employing live attenuated V. cholerae strains should meet each of these requirements (15, 17). However, production of a safe, attenuated strain has been problematic. Shortly after the discovery of the genes zot and ace, one would have predicted that a core deletion, resulting in mutants unable to produce CT, zonula occludens toxin (Zot), and accessory cholera enterotoxin (Ace), would produce ideal vaccine candidate strains. However, even with the core element deleted, strains CVD110, CVD111, and CVD112 were still "reactogenic," causing residual side effects in volunteer recipients, including mild diarrhea, nausea, vomiting, abdominal cramps, and fever (21, 23). Similarly, vaccine strains with deletions of the entire integrated CTX⌽ element (attRS deletions) are also mildly reactogenic (5, 24). These data indicate that these strains encode additional reactogenic factors encoded at loci other that the integrated CTX⌽ prophage. Curiously, the reactogenic effect of these undefined factors is absent from vaccine candidate strains that have additional defects in motility (5,11,24). This paper describes experiments using transcellular epithelial resistance (TER) across polarized T84 epithelial cells to monitor potential reactogenic factors in various vaccine strains. We show that an activity associated with the zot gene is not detected in this system. However, we find that a decrease in TER correlates with the presence of the genes for production of hemagglutinin/protease (HA/protease), indicating that HA/protease may be a significant contributor to the reactogenicity of some V. cholerae vaccine strains.Addition of supernatant fluids to pol...