Using Caenorhabditis elegans as a novel expression system for the generation of recombinant Teladorsagia circumcincta vaccine candidates

Longhi-Browne, Cassandra W. (2014) Using Caenorhabditis elegans as a novel expression system for the generation of recombinant Teladorsagia circumcincta vaccine candidates. PhD thesis, University of Glasgow.

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Abstract

Teladorsagia circumcincta is a common abomasal parasite of sheep in temperate regions and is one of the major causes of parasitic gastroenteritis (PGE) in growing lambs. Control of infection is achieved using anthelmintic drugs; however, this practice is rapidly becoming unsustainable due to widespread anthelmintic resistance within the T. circumcincta population. Sheep can acquire protective immunity against this parasite; immunity involves local and systemic antibodies and immune cells which can impair worm growth and fecundity and lead to expulsion of the parasites from the abomasum. Vaccination against this parasite is therefore a feasible option of control. A recent study showed that a recombinant vaccine cocktail containing 8 T. circumcincta antigens significantly reduced the faecal egg count and worm burdens of immunised sheep, compared to an adjuvant-only control group. However, the recombinant antigens induced a suboptimal antibody response to the recombinant antigens. This suggests that differencies between the native antigens and their recombinant versions may exist, possibly due to variations in structure and/or post-translational modifications (PTMs). The main aim of this work was to use a novel expression system, the free living nematode Caenorhabditis elegans, to generate alternative recombinant versions of two of the T. circumcincta antigens used in the 8-antigen vaccine, cathepsin F (Tci-CF-1) and monocyte Migration Inhibitory Factor (Tci-MIF-1). This was achieved by micro-injection of C. elegans worms with plasmids containing the cDNA sequences of Tci-cf-1 and Tci-mif-1 followed by purification of recombinant Tci-CF-1 and Tci-MIF-1 from the transformed worms. Immune recognition, enzyme activity and biological effects on sheep cells of the recombinant antigens were characterised. The results show that immunisation-induced antibodies bind to native Tci-CF-1 purified from T. circumcincta L4 ES, whereas infection-induced antibodies were unable to bind the recombinant Tci-CF-1 versions. Further characterisation of recombinant Tci-CF-1 versions expressed in C. elegans or Pichia pastoris showed that in order to be enzymically active, these proteins require cleavage of the pro-peptide by an exogenous enzyme and that some differences were present in the glycosylation of the recombinant versions and native Tci-CF-1. Characterisation of both recombinant Tci-MIF-1 versions showed that although both are enzymically active, neither showed a significant inhibitory effect on the migration of sheep monocytes or on the activation of sheep macrophages in vitro compared to unstimulated controls. It is speculated that Tci-MIF-1 may be involved in T. circumcincta larval development rather than host immunosuppression.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Teladorsagia circumcincta, Caenorhabditis elegans, vaccine antigens, recombinant vaccine, recombinant protein expression, parasitology
Subjects: Q Science > Q Science (General)
Q Science > QR Microbiology > QR180 Immunology
S Agriculture > SF Animal culture > SF600 Veterinary Medicine
Colleges/Schools: College of Medical Veterinary and Life Sciences
Funder's Name: UNSPECIFIED
Supervisor's Name: Britton, Dr. Collette and McNeilly, Dr. Tom and Nisbet, Dr. Alasdair
Date of Award: 2014
Depositing User: Mrs Cassandra W. Longhi-Browne
Unique ID: glathesis:2014-5547
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Sep 2014 15:28
Last Modified: 16 Oct 2017 08:55
URI: http://theses.gla.ac.uk/id/eprint/5547

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