African animal trypanosomosis or nagana is a tsetse fly-transmitted disease, caused by Trypanosoma congolense, T. vivax and to a lesser extent T. brucei brucei. The disease causes major losses in revenue in many livestock-producing African countries. The available control methods, including chemotherapeutic drugs and insecticidal spraying, have become environmentally unacceptable. Antigenic variation displayed by the parasites has hindered vaccine development efforts. In this context, rather than focusing solely on the parasite itself, efforts in vaccine development have shifted towards targeting pathogenic factors released by the parasites during infection. Congopain, the major cysteine protease of T. congolense, has been shown to act as a pathogenic factor in the disease process. Analysis of the immune response of trypano-tolerant cattle revealed that these animals have the ability to control congopain activity in vivo. Therefore, congopain is an attractive vaccine candidate. To test the protective potential of congopain, immunisation studies had been conducted in cattle using the baculovirus-expressed catalytic domain of congopain (C2) in RWL, a saponin-based proprietary adjuvant from SmithKline-Beecham. Immunised animals were partially protected against a disease caused by an infection with T.congolense. Unfortunately, subsequent attempts to reproduce these results were disappointing. It was hypothesised that this failure could be due to the different expression system (P. pastoris) used to produce the antigen (C2), or the different adjuvant, ISA206 (Seppic), used, thus hinting towards an epitope presentation problem. Congopain had been shown to dimerise at physiological pH in vitro. Sera from trypano-tolerant cattle preferentially recognised the dimer conformation, advocating for protective epitopes to be dimer associated. For that reason, the present study aimed at improving the antigenicity of congopain through firstly, the elucidation of the protective epitopes associated with the dimer, secondly, the determination of the 3-D structure of the protease in order to map protective epitopes to later design mimotopes, and thirdly improve the delivery of congopain to the immune cells while maintaining the conformation of the protease by using a molecular adjuvant, BiP. A dimerisation model was proposed, identifying the amino acid residues forming the dimerisation motif of congopain. In the present study, particular amino acid residues located in the dimerisation motif were mutated by PCR-based site-directed mutagenesis to generate mutants with different dimerisation capabilities. The congopain mutants were expressed in yeast and their dimerisation capability was assessed by PhastGel® SDS-PAGE. The mutations altered both the electrophoretic mobility of the mutants and their enzymatic characteristics compared to wild-type congopain. This advocated for the involvement of these amino acid residues in the dimerisation process, although they seem not to be the only partakers. Wild-type C2 and mutant forms of C2 were heterologously expressed in P. pastoris and purified to crystallisation purity levels. Crystallisation of these proteins is currently underway, but the results are still unknown. While awaiting the crystallisation results, in silico homology modelling was employed to gain insight into the 3-D structure, using cruzipain crystal structure as a template. The modelled 3-D structure of congopain followed the common framework of cathepsin L-like cysteine proteases. Due to time constraints and awaiting the crystal-derived 3-D structure, the 3-D model of congopain was not exploited to design mimotopes with the potential to provide protection against the disease. As it was shown that protective epitopes are likely to be dimer-specific, maintaining the native conformation of congopain is essential for stimulating a protective immune response in animals. Chemically formulated adjuvants usually contain high salt concentration, at acidic or basic pH, thus might change the conformation of the protease. Adjuvants capable of efficiently delivering the antigen to immune cells while maintaining the conformation of the protease were sought. Proteins belonging to the HSP70 family are natural adjuvants in higher eukaryotes. A protein belonging to the HSP70 family was previously identified in T. congolense lysates and is homologous to mammalian BiP. Congopain was genetically fused with T. congolense BiP in order to improve antigen delivery and production of congopain activity-inhibiting antibodies. The chimeric proteins were successfully expressed in both bacteria and yeasts. The low yields of recombinantly expressed chimeras in yeast and problems associated with renaturation and purification of bacteria-expressed chimeras prevented immunisation studies in mice. However, the groundwork was laid for producing BiP-congopain chimeras for use in an anti-disease vaccine for African trypanosomosis.

Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.