A thermostable, dry formulation inactivated Hikojima whole cell/cholera toxin B subunit oral cholera vaccine.

The feared diarrheal disease cholera remains an important global health problem. Use of oral cholera vaccine (OCV) from a global stockpile against both epidemic and endemic cholera is a cornerstone in the World Health Organisations (WHOs) global program for "Ending cholera by 2030". Three liquid inactivated whole-cell OCVs (Dukoral®, ShancholTM, and Euvichol-Plus®) are WHO prequalified and have proved to be safe and effective. However, their multicomponent composition and cold-chain requirement increase manufacturing, storage and transport costs. ShancholTM and Euvichol-Plus® OCVs used in WHOs global vaccine stockpile also lack the protective cholera toxin B-subunit (CTB) antigen present in Dukoral®, which results in suboptimal efficacy. WHOs Global Task Force on Cholera Control (GTFCC) has identified a thermostable, dry formulation vaccine as a priority for further OCV development. We describe here the development of such a vaccine, based on a lyophilized mixture of a single strain of formalin-killed Hikojima bacteria together with a low-cost, recombinantly produced CTB. The new vaccine, which is easy and inexpensive to manufacture, could be stored for at least 26 months at 25 °C and for at least 8 months at 40 °C with preservation of cell morphology and with no loss of protective Ogawa and Inaba lipopolysaccharides or CTB. It also proved to be well tolerated and to have equivalent oral immunogenicity in mice as ShancholTM and Dukoral® OCVs with regard to both serum and intestinal-mucosal antibody responses.

A Novel Nonantibiotic, lgt-Based Selection System for Stable Maintenance of Expression Vectors in Escherichia coli and Vibrio cholerae.

Antibiotic selection for the maintenance of expression plasmids is discouraged in the production of recombinant proteins for pharmaceutical or other human uses due to the risks of antibiotic residue contamination of the final products and the release of DNA encoding antibiotic resistance into the environment. We describe the construction of expression plasmids that are instead maintained by complementation of the lgt gene encoding a (pro)lipoprotein glyceryl transferase essential for the biosynthesis of bacterial lipoprotein. Mutations in lgt are lethal in Escherichia coli and other Gram-negative organisms. The lgt gene was deleted from E. coli and complemented by the Vibrio cholerae-derived gene provided in trans on a temperature-sensitive plasmid, allowing cells to grow at 30°C but not at 37°C. A temperature-insensitive expression vector carrying the V. cholerae-derived lgt gene was constructed, whereby transformants were selected by growth at 39°C. The vector was successfully used to express two recombinant proteins, one soluble and one forming insoluble inclusion bodies. Reciprocal construction was done by deleting the lgt gene from V. cholerae and complementing the lesion with the corresponding gene from E. coli The resulting strain was used to produce the secreted recombinant cholera toxin B subunit (CTB) protein, a component of licensed as well as newly developed oral cholera vaccines. Overall, the lgt system described here confers extreme stability on expression plasmids, and this strategy can be easily transferred to other Gram-negative species using the E. coli-derived lgt gene for complementation.IMPORTANCE Many recombinant proteins are produced in bacteria from genes carried on autonomously replicating DNA elements called plasmids. These plasmids are usually inherently unstable and rapidly lost. This can be prevented by using genes encoding antibiotic resistance. Plasmids are thus maintained by allowing only plasmid-containing cells to survive when the bacteria are grown in medium supplemented with antibiotics. In the described antibiotic-free system for the production of recombinant proteins, an essential gene is deleted from the bacterial chromosome and instead provided on a plasmid. The loss of the plasmid becomes lethal for the bacteria. Such plasmids can be used for the expression of recombinant proteins. This broadly applicable system removes the need for antibiotics in recombinant protein production, thereby contributing to reducing the spread of genes encoding antibiotic resistance, reducing the release of antibiotics into the environment, and freeing the final products (often used in pharmaceuticals) from contamination with potentially harmful antibiotic residues.

Construction and preclinical evaluation of mmCT, a novel mutant cholera toxin adjuvant that can be efficiently produced in genetically manipulated Vibrio cholerae.

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.

Development of stable Vibrio cholerae O1 Hikojima type vaccine strains co-expressing the Inaba and Ogawa lipopolysaccharide antigens.

We describe here the development of stable classical and El Tor V. cholerae O1 strains of the Hikojima serotype that co-express the Inaba and Ogawa antigens of O1 lipopolysaccharide (LPS). Mutation of the wbeT gene reduced LPS perosamine methylation and thereby gave only partial transformation into Ogawa LPS on the cell surface. The strains express approximately equal amounts of Inaba- and Ogawa-LPS antigens which are preserved after formalin-inactivation of the bacteria. Oral immunizations of both inbred and outbred mice with formalin-inactivated whole-cell vaccine preparations of these strains elicited strong intestinal IgA anti-LPS as well as serum vibriocidal antibody responses against both Inaba and Ogawa that were fully comparable to the responses induced by the licensed Dukoral vaccine. Passive protection studies in infant mice showed that immune sera raised against either of the novel Hikojima vaccine strains protected baby mice against infection with virulent strains of both serotypes. This study illustrates the power of using genetic manipulation to improve the properties of bacteria strains for use in killed whole-cell vaccines.

Construction of novel vaccine strains of Vibrio cholerae co-expressing the Inaba and Ogawa serotype antigens.

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.

Role of different genes in the CS6 operon for surface expression of Enterotoxigenic Escherichia coli colonization factor CS6.

Coli surface antigen 6, CS6, is one of the most prevalent colonization factors (CFs) associated with Enterotoxigenic Escherichia coli (ETEC) bacteria, the most common cause of diarrhea among infants and children in developing countries. The CS6 operon encodes two structural subunit proteins, CssA and CssB, a chaperon, CssC, and an usher, CssD. Since little is known about the relationship of the genes and their role in expression and surface assembly of CS6, the operon was cloned into a laboratory E. coli strain, and mutants were constructed by creating deletions in each of the genes. Examination of protein expression by different methods, using monoclonal antibodies with specificities for the individual CS6 structural proteins, suggested that the usher (CssD) was not involved in assembly or surface expression of CS6 whereas deletion of the chaperon (CssC) significantly reduced levels of CssA, but not of CssB. Binding studies with CaCo-2 cells demonstrated that there is a combined effect of CssC and CssD on receptor binding, presumably associated with their activities in assembly and transport of the structural subunits. These findings may have important implications for the construction of strains expressing high levels of CS6 on the surface for eventual use in an ETEC vaccine.