Recombinant cathepsin L-like cysteine proteases for species-specific diagnosis of animal African trypanosomiasis.

Abstract

African trypanosomiasis is a disease caused by protozoan parasites i.e. Trypanosoma spp. in livestock and humans and affects 37 sub-Saharan countries. Animal African trypanosomiasis (AAT) is known as nagana and African human trypanosomiasis (HAT) as sleeping sickness. Trypanosoma congolense, T. vivax and T. brucei brucei cause AAT which is an economic burden and hampers agricultural development in Africa. The parasite escapes the host’s immune response by switching the genes coding for the variable surface glycoproteins, resulting in new variable antigen types. This has made it unlikely to develop a vaccine against the disease and therefore many studies now focus on non-variant trypanosome antigens as potential diagnostic and drug targets. Trypanosomal cysteine proteases, such as the cathepsins B and L, have been identified and validated as potential diagnostic targets. They are expressed throughout the parasite life cycle and are essential for the survival of the parasite. The mammalian host produces an antibody response against trypanosomal cysteine proteases which do not affect the survival of the parasite, however, the antibodies are believed to play a role in trypanotolerance by neutralising the effects of the enzyme. Antigen-based ELISA is a good tool for accurate diagnosis of AAT, but also relies on good antibodies. The overall aim of this study was to produce antibodies against the cathepsin-L-like protease of T. b. brucei (TbbCATL) which can be used for specific diagnosis of T. b. brucei infections. This included polyclonal antibodies as well as single chain variable fragments (scFvs) using phage display. The protease, TbbCATL, was recombinantly expressed in E. coli for the first time as a 61 kDa protease (including the GST tag) using the pGEX-4T expression vector. The homologues from T. congolense (TcoCATL) (29 kDa) and T. vivax (TviCATL) (28 kDa and 32 kDa) were also recombinantly expressed using the P. pastoris yeast expression system and were shown to hydrolyse the Z-Phe-Arg-AMC substrate and to be inhibited by E-64. Antibodies against whole TbbCATL were produced in chickens and together with anti-TcoCATL peptide antibodies and anti-TviCATL antibodies, were evaluated in a western blot to determine possible cross-reactivity. Whereas the anti-TbbCATL antibodies were specific for TbbCATL, the anti-TcoCATL peptide and anti-TviCATL antibodies cross-reacted with TbbCATL. The production of scFvs was optimised using TviCATL as the panning antigen against the Nkuku® phage display library. The TviCATL-specific phages were enriched after the fourth round of panning and a total of seven clones gave high signals when analysed by monospecific ELISA. Future work will include recombinant expression and purification of the selected TviCATL-specific scFvs for testing in diagnostic assays as well as panning with the TbbCATL antigen. This study laid the groundwork for evaluating TbbCATL as a diagnostic target for AAT