The PiroVac programme consists of thirteen inter-related work packages (WP) each representing a major sub-division of the whole project. An overview of the structure of the work packages is shown in the table below. The Veterinary Research Center in Borstel, the co-ordinating centre, will take responsibility for the overall management of the project including communication with the EU-Commission, financial management, preparation of periodic progress and financial reports as well as the co-ordination of the communication between the participants of the project (WP1). In addition, progress toward the goals of the anticipated project will be reviewed through reports of work package managers and project meetings and assessed by the steering committee.
For a rational and cost effective achievement of the project objectives, in vitro assays have been and will be further developed to cultivate parasites (WP2), to assess strain diversity (WP3) and to achieve and assess attenuation of the parasites and define attenuation markers (WP4, WP5). In addition, candidates for su-bunit vaccines will be identified (WP8, WP9), characterised (WP7, WP10) and screened for their ability to stimulate a recall response in parasite-specific memory CD4 and/or CD8 T-cells.
Lead: Forschungszentrum Borstel, Germany
This section is aimed at overall management of the project at the scientific and financial levels. The co-ordinator will be advised by a steering committee in terms of progress in the project, the dissemination of the activities, evaluation of each work package and prioritisation of milestones. The committee will meet annually.
Lead: Forschungszentrum Borstel, Germany with sub-contacting of other partners
This section is managed by FBZ and is a key activity for the provision of biological resources for use in the other work packages. Vector tick colonies for each of the tick borne pathogens being studied will be established and used to prepare sporozoites of T. lestoquardi , T. uilenbergi , B. ovis and B. sp. (China). In addition cultures of the relevant life cycle stages will be established and used as a source of DNA, RNA and protein for the consortium. These materials will be used by different work packages of the project for: 1. Preparation of cell lines and infected erythrocyte cultures; 2. Preparation of DNA for sequencing; 3. RNA (cDNA) for cDNA expression library generation; 4. Proteome analysis; 5. Identification of immunodominant proteins; 6. Production of recombinant proteins; 7. Preparation of antisera and monoclonal antibodies against recombinant proteins; 8. Attenuation and immunization trials.
Lead: Centro de Investigaciones en Cie ncias Veterinairias y Agronomicas, Argentina
This section is managed by CICVyA but other partners will undertake components of the research. The broad aim is to investigate the level of diversity in T. lestoquardi , T. uilenbergi and B. ovis using a range of different molecular markers including antigen genes, micro- and mini-satellite markers and the orthologues of polymorphic genes identified in malaria. The systems used for detecting diversity will be largely PCR based and so can be readily used by a number of different laboratories in the future.
Lead: Forschungszentrum Borstel, Germany with four other partners
This section is aimed at generating parasite lines that are attenuated for virulence and so can potentially be used as live vaccines. This will be achieved by in vitro passage of the three parasite species with the assessment of virulence of the lines generated being undertaken in WP5. As conventional attenuation involves frequent testing of lines in animals, it is a time consuming and labour intensive process and, to overcome this limitation, markers that define the attenuated state will identified so that attenuation could be defined in vitro. A combination of approaches will be used to identify such markers including testing markers already identified in T. annulata, subtractive cDNA hybridization and microarray analysis.
Lead: Ministry of Agriculture, Israel with five other partners
The in vitro cultured ‘attenuated ‘ lines developed in WP4 will be tested in vivo to determine whether attenuation has been achieved by monitoring a full range of clinical, haematological and parasitological parameters both pre and post challenge with a homologous virulent line. In addition both the humoral and cellular response to immunisation and challenge will be evaluated. If the results show protection against homologous challenge, then the attenuated lines will be further tested against heterologous parasite strains.
Lead: University of Edinburgh, UK
Both T. annulata and T. lestoquardi infect monocytes which are key players in the induction of the innate immune response but we no little about how the parasite alters gene expression in these cells. Similarly we know little about the role NK cells play in the immune response to the parasite. Using expression profiling of infected and uninfected monocytes, the pathways regulated by infection will be identified and the functional profile of NK cells that respond to infection will be defined using reagents and culture systems generated during the project.
Lead: Parco Technologica Padano, Italy
Responses to infection or vaccination are known to vary depending on the breed of host animal and this may be critical in the development of sub unit vaccines. Groups of animals with different susceptibilities will be infected with Theileria or Babesia and the response to infection compared by microarray analysis of host gene expression. This will allow the definition of the interactions between host and parasite and the responses involved in host resistance/susceptibility. Similar analyses will be undertaken using recombinant antigen vaccine candidates as they are identified during the course of the project.
Lead: University of Berne, Switzerland
The proteins on the surface of the schizont are likely to interact with host signalling pathways and so play an important role in the regulation of the host cell and its transformation. In addition the schizont secretome is likely to be important in terms of the parasite antigens presented on the surface of the host cell. Using a combination of identifying orthologues of already characterised Theileria genes and bioinformatic analysis, the secretome and surface antigen gene repertoire of T. lestoquardi will be identified. Additionally proteomic analysis of purified schizonts and their secreted products will be undertaken. Selected antigens will be cloned and expressed for functional analysis in other WPs.
Lead: University of Glasgow, UK
The availability of genome sequences of the two Theileria species and B. ovis will underpin gene discovery and the development of markers for detecting strain diversity. The genome sequences of the three species will be determined and initial annotation undertaken based on comparative analysis with the published genome sequences of closely related species. The assembled sequences will be used to identify orthologues of currently defined vaccine candidates, surface molecules, micro and minisatellites, genes under selection and any species specific genes. Comparative analysis of the non lymphocyte transforming T. uilenbergi with the transforming Theileria genomes, will allow an analysis of genes potentially involved in lymphocyte transformation. If other projects require microarrays for the analysis of gene expression, the genome sequences will be used to design oligonucleotide arrays.
WP10. CD8+ T cell antigens of T. lestoquardi
Lead: Royal Veterinary College, UK
Cytotoxic T-cells play a central role in protective immunity to bovine Theileria species as they recognise parasite antigens presented on MHC Class1 molecules. Both parasite and cytotoxic T lymphocyte cell lines will be generated from animals representing at least four different ovine MHC haplotypes and these will be used to screen for the parasite antigens recognised by the CTL. Using transfection of Class 1 genes into COS cells, the CTL lines will be characterised in terms of the class 1 allele that they recognise. This system with the specific class 1 allele will then be used to screen a parasite cDNA library to define the parasite genes recognised by the CTL. Once identified the antigens will be epitope mapped to provide well characterised vaccine candidate antigens for further evaluation.
Lead: Institut National de la Sante et de la Recherche Medicale, Paris, France with the Centro de Investigaciones en Ciencias Veterinairias y Agronomicas, Argentina
A comparative approach will be used to identify genes which are orthologues of those in other apicomplexa that have been shown or are postulated to play a central role in red blood cell invasion .or growth. Three groups of orthologues will be investigated, firstly those which are putative substrates for cAMP dependent protein kinase A (PKA), secondly orthologues of Plasmodium and Toxoplasma serine and cysteine proteinases and thirdly GPI anchored proteins that are orthologues of those defined in B. bovis and T. annulata . Once orthologues have been identified, they will be expressed as recombinant proteins and antisera raised against them so that invasion blocking assays and cellular localisation studies can be undertaken. In the case of the first group functional studies of recombinant proteins will be undertaken and the role of PKA inhibition on parasite growth determined.
Lead: Bernhard Nocht Institut fur Tropenmedizin, Hamburg, Germany with Intervet Animal Health
The mode of delivery for immunisation of animals with a vaccine can have a significant effect on the resultant immune response and consequently the protective effect. Systems using specific genes derived from invasive bacteria have been developed that allow DNA or recombinant antigens to be delivered into the host or recipients cell cytoplasm so that they can be presented on MHC class1 molecules. A system based on E. coli K12 containing the listeriolysin gene of L. monocytogenes and the invasin gene of Yersinia and a plasmid for the expression of the parasite gene will be used to express candidate vaccine antigens identified in other work packages. The constructs will be optimised for expression, then tested in experimental animals and the humoral and cell mediated responses determined. If the results are promising such experiments could be extended to pilot protection studies.
The aim is to test whether the protection provided by the in vitro attenuated parasite cell lines (generated and tested in WPs 4 and 5) can be enhanced and to test whether improved live vaccine delivery systems can be developed. Candidate vaccine antigens identified in other work packages will be cloned into DNA vaccine constructs and co-delivered with the live vaccines and a range of variables tested to determine if protective immunity can be enhanced. Examples would include the introduction of sub-cellular targeting signals or adjuvant molecules. These different delivery systems will be tested in vivo using appropriate parasite challenge and compared to the performance of the attenuated vaccines delivered on their own. Two of the critical issues for the commercialisation of live vaccines are the need to deliver vaccines frozen and the short half life of such vaccines once thawed. The use of gelatine in the vaccine or lyophilisation will be tested as alternative means of delivery to eliminate the cold chain and prolong vaccine half life.