Caenorhabditis elegans as an expression system for vaccine candidates of parasitic nematodes

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

Parasitic nematode (worm) infections of humans and livestock cause major health, welfare and economic problems worldwide. The efficiency of drugs currently available to control parasitic nematode infections is decreasing due to the emergence of drug-resistant parasites and alternative control approaches are urgently needed. Validation of new drug or vaccine targets of parasitic nematodes is hampered by the difficulty of obtaining sufficient parasite material for testing. Parasite proteins produced artificially in bacteria, yeast or insect cells are often folded incorrectly and/or fail to be modified in the same way as the normal parasite protein. This affects the proteins' ability to stimulate protective immune responses to infection, required for an effective vaccine, and their ability to interact with inhibitors, required for drug design. Development of a system which can synthesise protein in a similar form to the protein present within the worm would, therefore, be advantageous to parasite vaccine and drug research. Parasitic nematodes are closely related to the free-living nematode Caenorhabditis elegans and many of the proteins made in free-living and parasitic nematodes are similar. We have successfully used C. elegans to synthesise a parasite enzyme and shown that the enzyme is active, can interact with inhibitors and is modified correctly. Sufficient protein was expressed for vaccine studies to be carried out. We now propose to use the C. elegans system to make a previously identified, highly effective vaccine candidate of a major sheep parasitic nematode. We will compare the folding and modifications of the C. elegans synthesised protein to the normal parasite protein and test its ability to protect sheep against nematode disease. This work can be developed to generate other important parasite proteins and will be important both to basic research of nematode proteins and to commercial vaccine development. We will link with other researchers with expertise in animal parasite control and with industry (Pfizer) to achieve our aims.

Technical Summary

At present there is no recombinant vaccine available for any parasitic nematode. This is predominantly due to the difficulty of expressing parasite proteins in a correctly folded and/or post-translationally modified form to induce protection. The Haemonchus contortus H11 gut aminopeptidase in native form is one of the most effective parasitic nematode vaccine candidates identified, but recombinant H11 is not protective. Glycan analysis of H11 identified di- and tri-fucosylated structures, not present on mammalian glycoproteins, which are thought to confer strong immunogenicity to the antigen. Similar structures have been identified in the free-living nematode Caenorhabditis elegans. We have recently used C. elegans as an alternative expression system for an H. contortus cysteine protease; the C. elegans expressed enzyme is active and glycosylated and could be purified in sufficient amounts for vaccination studies. The proposed study aims to use the C. elegans system to express previously identified vaccine candidates of H. contortus, focussing initially on H11. H. contortus H11 will be expressed under the control of the promoter of the related C. elegans aminopeptidase T07F10.1. His-tagged protein purified from transgenic worms will be examined biochemically and immunochemically, including deglycosylation and mass spectrometry comparison of C. elegans expressed and native H11. C. elegans expressed and native H11 will be tested in protection trials, the immune response analysed and antigenic epitopes characterised. This work will establish the suitability of C. elegans not only for H11 expression, but for expression of other complex, post-translationally modified parasite proteins. It has important relevance to our understanding of parasite immunity and to commercial development of parasitic nematode vaccines.
 
Description Vaccine testing for parasitic worms currently relies on purifying native proteins from parasite samples collected from animals. We developed an alternative approach using a related free-living nematode C. elegans to express parasite proteins. We showed that the specific protein of interest (H11 vaccine enzyme) was active and modified with sugars in a similar way to the native parasite protein. This approach and the molecular vectors we generated can be applied to a range of proteins and different nematodes. The C. elegans-expressed H11 enzyme was tested in a vaccine trial and although it induced a strong antibody response, it did not protect against infection. Our results indicated that this is an appropriate expression system but combinations of vaccine candidates may be required for protection against these complex parasites.
Exploitation Route Have been used by others to express vaccine candidates from other parasites. This has allowed immunological and biochemical comparisons to determine what protein features are important for inducing protective immunity.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description EU FP7
Amount € 300,000 (EUR)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 04/2011 
End 03/2015
 
Description University of Glasgow and Moredun Research Institute joint PhD studentship
Amount £70,000 (GBP)
Organisation University of Glasgow 
Sector Academic/University
Country United Kingdom
Start 10/2010 
End 10/2013
 
Title C. elegans expression system 
Description Generated and tested plasmid vectors for expression of parasite proteins using the model nematode Caenohabditis elegans in active and post-transcriptionally modified forms 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact Used this tool to express proteins of a number of parasitic nematode species to study function, resulting in collaborative projects and publications. This approach also formed the basis of a collaborative PhD studentship (University funded). The approach is also relevant to 3Rs by potentially reducing the number of animals used in vaccine or drug testing by being able to express proteins in vitro rather than working with native proteins from parasite samples obtained through animals infection. 
 
Description Moredun 
Organisation Moredun Research Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Developed molecular tools for novel expression of parasite proteins and developed methods for gene silencing
Collaborator Contribution Characterised and tested aspects of the recombinant proteins biochemically and immunologically. Provided parasite stages for testing RNAi and shared new data on RNA delivery methods
Impact Publications New collaborative funding (EU PARAVAC) Collaborative PhD studentship
 
Description University of Ghent 
Organisation University of Ghent
Country Belgium 
Sector Academic/University 
PI Contribution To develop techniques for recombinant protein expression and gene silencing for parasitic nematodes
Collaborator Contribution Applying the techniques to other nematode species and immunological analysis of recombinant proteins we produced
Impact Number of collaborative publications
Start Year 2006
 
Description Parasite resistance course 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact 50 students attended the course which involved research talks and practical demonstrations, which sparked discussion and ideas for collaborative projects

Enhanced interaction with different international labs in South America and Europe
Year(s) Of Engagement Activity 2011
URL http://www.cppse.embrapa.br/parasites
 
Description Schools Science week 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact 120 pupils participated in a variety of science workshops and demonstrations which stimulated questions and discussion (eg of cells, parasites, infectious diseases)

Invitations to discuss science and research to other schools
Year(s) Of Engagement Activity 2006,2008,2010