A first-generation oral inactivated whole-cell enterotoxigenic Escherichia coli (ETEC) vaccine, comprising formalin-killed ETEC bacteria expressing different colonization factor (CF) antigens combined with cholera toxin B subunit (CTB), when tested in phase III studies did not significantly reduce overall (generally mild) ETEC diarrhea in travelers or children although it reduced more severe ETEC diarrhea in travelers by almost 80%. We have now developed a novel more immunogenic ETEC vaccine based on recombinant non-toxigenic E. coli strains engineered to express increased amounts of CF antigens, including CS6 as well as an ETEC-based B subunit protein (LCTB. A), and the optional combination with a nontoxic double-mutant heat-labile toxin (LT) molecule (dmLT) as an adjuvant.Two test vaccines were prepared under GMP: (1) A prototype E. coli CFA/I-only formalin-killed whole-cell. +. LCTB. A vaccine, and (2) A "complete" inactivated multivalent ETEC-CF (CFA/I, CS3, CS5 and CS6 antigens) whole-cell. +. LCTB. A vaccine. These vaccines, when given intragastrically alone or together with dmLT in mice, were well tolerated and induced strong intestinal-mucosal IgA antibody responses as well as serum IgG and IgA responses to each of the vaccine CF antigens as well as to LT B subunit (LTB). Both mucosal and serum responses were further enhanced (adjuvanted) when the vaccines were co-administered with dmLT. We conclude that the new multivalent oral ETEC vaccine, both alone and especially in combination with the dmLT adjuvant, shows great promise for further testing in humans. 

The approach of inducing protective immunity against cholera by oral vaccination with killed whole Vibrio cholerae cells is effective, but the complexity of current cholera vaccines makes them difficult and relatively expensive to manufacture, especially if recombinant cholera toxin B subunit is included in the formulation. In an effort to simplify the composition of a new generation of oral cholera vaccines we have generated a novel non-toxigenic candidate vaccine strain of V. cholerae O1 that stably expresses both the Ogawa and Inaba serotype antigens on its surface. This was done by introducing a functional wbeT gene without a functional promoter into the chromosome of an O1 Inaba strain. The resulting low levels of expression of the wbeT gene product allowed for the desired partial serotype switching. This strain (MS1342) can potentially replace the three virulent strains used in currently manufactured cholera vaccines. Oral immunization of mice with formalin-killed MS1342 bacteria gave rise to Ogawa-specific, Inaba-specific and cross-reactive serum antibodies that were detectable both by lipopolysaccharide (LPS)-specific ELISAs and as vibriocidal antibodies that are considered to predict protective efficacy. These responses as well as intestinal mucosal IgA anti-LPS antibody responses were fully comparable with those obtained by immunization with the internationally licensed oral cholera vaccine Dukoral ®. We propose that such a strain may form the basis of a single strain killed whole cell cholera vaccine protecting against cholera caused by either the Inaba or Ogawa serotype of V. cholerae O1. 

There is an urgent need for new adjuvants that are effective with mucosally administered vaccines. Cholera toxin (CT) is the most powerful known mucosal adjuvant but is much too toxic for human use. In an effort to develop a useful mucosal adjuvant we have generated a novel non-toxic mutant CT molecule that retains much of the adjuvant activity of native CT. This was achieved by making the enzymatically active A subunit (CTA) recalcitrant to the site-specific proteolytic cleavage ("nicking") required for toxicity, which was found to require mutations not only in the two residues rendering the molecule resistant to trypsin but also in neighboring sites protecting against cleavage by Vibrio cholerae proteases. This multiple-mutated CT (mmCT) adjuvant protein could be efficiently produced in and purified from the extracellular medium of CT-deleted V. cholerae. The mmCT completely lacked detectable enterotoxicity in an infant mouse model and had >1000-fold reduced cAMP inducing activity compared to native CT in a sensitive mammalian target cell system. It nonetheless proved to have potent adjuvant activity on mucosal and systemic antibody as well as cellular immune responses to mucosally co-administered antigens including oral cholera and intranasal influenza vaccines. We conclude that mmCT is an attractive novel non-toxic mucosal adjuvant for enhancing immune responses to co-administered mucosal vaccines

Protocols are described for the induction of strong, consistent serum and mucosal antibody responses to Vibrio cholerae O1 or O139 lipopolysaccharide (LPS) following intranasal or oral immunization of adult mice with viable or formalin-killed bacteria. A simplified two-dose schedule for intranasal immunization has been identified, whereby viable bacteria elicit strong serum responses and, most importantly, also induce significant, sustained intestinal IgA responses. Using higher doses of bacteria it was also possible to generate consistently high intestinal and serum anti-LPS responses by the oral route. The efficacy of these immunization schedules was not dependent on co-administration of adjuvant. Gut responses were estimated using two sampling techniques involving the collection of fresh faecal pellets or the preparation of intestinal tissue extracts. The significant correlation between these estimates validates the more convenient approach of measuring intestinal responses using faecal pellet extracts, which allows repeated sampling from the same animals. V. cholerae O1 and O139 were similarly immunogenic by either mucosal route. More intensive immunization schedules for administration of formalin-killed bacteria have also been defined. Using these regimes it was possible to generate serum and gut antibody responses comparable to those elicited by viable V. cholerae. The established immunization protocols will allow evaluation of the systemic and mucosal immunogenicity of new vaccine formulations.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and deaths among children in developing countries and the major cause of traveller's diarrhea. Since surface protein colonization factors (CFs) of ETEC are important for pathogenicity and immune protection is mainly mediated by locally produced IgA antibodies in the gut, much effort has focused on the development of an oral CF-based vaccine. We have recently described the development of recombinant strains over-expressing CFA/I; the most prevalent CF among human clinical ETEC isolates. Here, non-toxigenic recombinant E. coli strains over-expressing Coli surface antigen 2 (CS2), CS4, CS5, and CS6, either alone, or each in combination with CFA/I were constructed by cloning the genes required for expression and assembly of each CF into expression vectors harboring a strong promoter. Immunological assays showed that recombinant strains expressing single CFs produced those in significantly larger amounts than did corresponding naturally high producing reference strains. Recombinant strains co-expressing CFA/I together with another CF also expressed significantly larger amounts of both CFs compared with the corresponding references strains. Further, when tested in mice, oral immunization with formalin-killed recombinant bacteria co-expressing one such double-expression CF pair, CFA/I. +. CS2, induced specific serum IgG. +. IgM and fecal IgA antibody responses against both CFs exceeding the responses induced by immunizations with natural reference strains expressing CFA/I and CS2, respectively. We conclude that the described type of recombinant bacteria over-expressing major CFs of ETEC, alone or in combination, may be useful as candidate strains for use in an oral whole-cell CF-ETEC vaccine.