Thesis (M.Sc.) - Cairo University - Faculty of Pharmacy - Department of Microbiology and Immunology

Tetanus is one of the major causes of extensive morbidity and mortality in rural areas of developing countries affecting both animals and humans. The commercially available tetanus vaccine is routinely produced through detoxification of the tetanus toxin using formaldehyde followed by purification process, which might show residual formaldehyde presence and some reversion to toxic form upon exposure to variable conditions. In the current study, in an approach to optimize the production conditions and to minimize the risk of crosslinking of unwanted foreign proteins during the detoxification processes; switching the order of the purification and the detoxification processes was tested. The bacterial crude toxin was purified at optimized conditions utilizing 40% ammonium sulfate and incubated for seven days followed by successive dialysis steps. SDS PAGE using both Coomassie brilliant blue and silver staining, as well as HPLC techniques were used to confirm the integrity and the purity of the produced toxins. Detoxification of the purified tetanus toxin was tested by using gradient decreasing concentrations of formaldehyde; where complete detoxification was achieved utilizing 0.08% with five weeks dark incubation at 37{u00B0}C. Another detoxification technique was demonstrated using iodoacetamide buffer as a reducing agent instead of formaldehyde, where complete detoxification was achieved within five hours using dark incubation at 37{u00B0}C, followed by dialysis to remove excessive salts. Comparative analysis was carried out on the newly prepared toxoid versus the traditionally produced one to evaluate the efficacy and safety as well as to monitor the rate and extent of reversion to toxicity. The obtained results proved that iodoacetamide detoxification was faster than the commonly used formaldehyde method, and it produced similar survival rates in animals{u2019} models, also the new preparation was more potent at dilutions (1/5 &1/10) and induced higher protective antibody levels in mice sera

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