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Masters Degrees (Biochemistry)

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Browsing Masters Degrees (Biochemistry) by Subject "Alpha macroglobulins."

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    Evaluation of congopain and Oligopeptidase B as anti-disease vaccines for African Trypanosomiasis.
    (2008) Bizaaré, Lorelle Claire.; Coetzer, Theresa Helen Taillefer.
    The protozoan parasite Trypanosoma congolense is one of the aetiological agents of African animal trypanosomiasis that is transmitted by the tsetse fly. The parasite causes nagana in animals and affects livestock throughout sub-Saharan Africa. The toxicity of available drugs and the emergence of drug resistant parasites have affected the treatment of trypanosomiasis. Control of the disease has also been difficult due to ineffective vector control and the potential of trypanosomes to express hundreds of antigenetically distinct proteins on their surface. Vaccination against trypanosomiasis has been thought to be a possible control method. Since a vaccine based on variable surface proteins of the parasite is unlikely, research has been directed towards the identification of invariant pathogenic factors of the parasite as potential targets for therapy. Congopain, the major cysteine protease of T. congolense has been implicated in the pathology of the disease. Antibodies against congopain are known to contribute to the mechanisms of natural resistance to trypanosomiasis known as trypanotolerance by neutralising the pathogenic effects of the enzyme. Oligopeptidase B (OpdB), a trypanosomal serine protease has also been associated as a pathogenic factor of the disease. It is released into the host’s circulation by dead or dying parasites and retains its catalytic activity since it is insensitive to host serum inhibitors. In the present study, the catalytic domain of congopain (C2) and the use of alpha-2-macroglobulin (α2M) as an adjuvant were investigated for their potential use in an anti-disease vaccine. α2-Macroglobulin has been used to varying degrees to target different antigens to cells of the immune system and enhance their immunogenicity. A previous study showed that antibodies raised in rabbits against C2 complexed to α2M gave a higher percentage inhibition than antibodies made using C2 mixed with Freund’s adjuvant. In the present study, goats were immunised with C2 complexed with α2M to confirm the enhanced immunogenicity of C2 and the production of anti-C2 antibodies with superior inhibitory properties. Following immunisation, goats were challenged with T. congolense (strain IL 1180) and showed sustained antibody production during the two month infection period. Goat antibodies made using C2 in complex with α2M inhibited the hydrolysis of hide powder azure by C2 by 96%. Maximum inhibition of the hydrolysis of azocasein was observed to be 63% and hydrolysis of Z-Phe-Arg-AMC by C2 was inhibited by 73%. In order to determine the vaccine potential of OpdB, protein was recombinantly expressed as a glutathione-S-transferase fusion protein in the pGEX expression system and purified by glutathione agarose affinity chromatography and molecular exclusion chromatography. Since a small yield of protein necessitated several rounds of expression and extensive purification, OpdB was subsequently expressed as a His-tagged fusion protein in the pET bacterial expression system. Recombinant protein was easily purified using nickel chelate affinity chromatography. Purified OpdB was used with alum for the immmunisation of mice to produce antibodies capable of inhibiting enzyme activity. Following immunisation, mice were challenged with T. congolense (strain IL 1180) and also showed sustained antibody production following two months infection. Since all mice died, the administration of OpdB conferred no protection; however, anti-OpdB mouse antibodies inhibited 86% of OpdB activity against the substrate Z-Arg-Arg-AMC. In addition immunised mice were observed to survive 40% longer than control mice as they had previously been immunised with OpdB and were able to mount a rapid immune response against this pathogenic factor during infection. In general it could be concluded that immunisation of goats with C2 in complex with α2M produced antibodies with superior inhibitory properties. The immunisation of mice with OpdB and alum also produced inhibitory antibodies and previous administration of OpdB enabled mice to mount a rapid immune response against OpdB during infection. Antibody mediated enzyme inhibition demonstrates the potential use of C2 and OpdB as vaccines that may contribute to the development of an effective anti-disease vaccine.