Production and Characterization of Chemically Inactivated Genetically Engineered Clostridium difficile Toxoids.

A recombinant Clostridium difficile expression system was used to produce genetically engineered toxoids A and B as immunogens for a prophylactic vaccine against C. difficile-associated disease. Although all known enzymatic activities responsible for cytotoxicity were genetically abrogated, the toxoids exhibited residual cytotoxic activity as measured in an in vitro cell-based cytotoxicity assay. The residual cytotoxicity was eliminated by treating the toxoids with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide. Mass spectrometry and amino acid analysis of the EDC-inactivated toxoids identified crosslinks, glycine adducts, and ß-alanine adducts. Surface plasmon resonance analysis demonstrated that modifications resulting from the chemical treatment did not appreciably affect recognition of epitopes by both toxin A- and B-specific neutralizing monoclonal antibodies. Compared to formaldehyde-inactivated toxoids, the EDC/N-hydroxysuccinimide-inactivated toxoids exhibited superior stability in solution with respect to reversion of cytotoxic activity.

Reduction of diesters of 1,2-diols. Regioselective C-O bond cleavage of the anionic forms.

The electrochemical reduction of benzoate diesters of glycols has been studied in acetonitrile and N,N-dimethylformamide as solvents. The reductions occur in two closely spaced one-electron steps, and it was found that the dianion diradicals decompose by one of two routes, depending on the substituents on the ethylene moiety: cleavage of two benzoates to produce alkene or formation of benzil by way of a postulated cyclic intermediate to produce also the dianion of the diol. These correspond to cleavage of the R-OC(O)Ar bonds and the RO-C(O)Ar bonds, respectively. When the radical formed by the former cleavage is a primary or secondary radical, the reaction is too slow to compete with the latter reaction that produces benzil. However, when that radical is either tertiary or benzylic, the former cleavage reaction is fast and no benzil is detected. The dianions of p-cyano- and p-nitrobenzoate esters are rather stable on the voltammetric time scale. However, the addition of lithium ions results in detectable formation of 4,4'-dicyanobenzil from four different p-cyanobenzoate diesters.

Microencapsulated subunit vaccine approach to enterotoxigenic Escherichia coli and other mucosal pathogens.

Infections of the intestinal, urogenital, and respiratory tracts are serious health problems worldwide from both a morbidity and mortality perspective. Mucosal pathogens attach to surfaces of mucosa as a prerequisite for colonization and subsequent pathogenesis. By expressing various surface adhesins (colonization factors, CF) they are able to bind to specific mucosal receptors. Enterotoxigenic Escherichia coli (ETEC) can express numerous CF that allow them to attach to a variety of hosts. Mucosal immunity directed against pathogenic microorganisms is critical in host protection with secretory IgA being particularly important in preventing microoganisms from colonizing host cells. M cells likewise have an important immunological function in the small intestines by binding and transporting antigens to lymphocytes and macrophages thus enhancing the immune response. The use of subunit vaccines, such as antigen encapsulated microspheres, can act to effectively deliver specific antigens so as to optimize their immunological response. With the threat of bioterrorism becoming a reality in recent years, the miroencapsulation of antigens from potential bioterrorist agents may be an effective method of delivery so as to induce a level of protection in at risk individuals. The encapsulation of ETEC colonization factors in microspheres and their subsequent administration in small animals and humans has been conducted for many years. Evidence suggests that this type of delivery system for ETEC antigens may enhance their immunogenicity and provide protection against this microorganism.