Malaria vaccines are urgently required. Recombinant, multi-component cocktail vaccines are currently favoured. Using very recent technological advances, this programme revives the concept of attenuated parasites as vaccines. It brings together for the first time leading EU and African groups to develop methodologies allowing for the design and creation of parasites with properties desirable in such a vaccine. The ultimate delivery of such a vaccine may be as sexually incompetent, genetically restricted parasites, or as transmissible self-spreading forms. For whichever route, certain fundamental pre-conditions must be met, and these are the major focus of this proposal. Systems will be developed to provide 1) auxotrophic parasites, 2) sequential unrestricted genetic manipulation of parasites, 3) axenic culture, 4) parasite knockouts for specific genes, and 5) immuno-potentiated parasites through heterologous gene expression. In combination with a dialogue across scientific and public interests, this programme will enable the partnership to provide a clear assessment of the feasibility of vaccination with genetically modified malaria within three years.
Using the particular advantages offered by working with human, primate and rodent malaria parasites, technologies will be developed that permit the rational design and production of live attenuated vaccines. Such technologies are also vital to the general investigation of malaria parasite biology, vaccine research, drug discovery and mechanisms of resistance. This will be achieved by:
1) developing systems for the improved genetic manipulation of Plasmodium based on negative selection that allow the reuse of the limited number of effective selectable markers
2) developing auxotrophic Plasmodium parasites dependent upon an exogenous supply of uracil by targeted deletion of the cloned parasite ura 3 gene
3) developing rationally axenic culture of malaria parasites, based upon detailed measurements of the intracellular environment of the infected erythrocyte
4) developing genetically engineered Plasmodium that is growth-impaired through a strategic reduction in its ability to produce protein
5) initiating a discussion about genetically engineered malaria parasites in Africa that will further debate the application of attenuated parasites and liase with a similar forum in Europe.
Five work packages will be carried out in the three parasite species (see A2 objectives) unless otherwise indicated. Pilot studies will involve the models P. berghei and P. knowlesi.
Work Package 1: Parasites will be made auxotrophic for uracil by deletion of the cloned ura3. Growth and pathogenicity of the mutant parasites will be examined. Ura3 will be used as a selectable marker based upon auxothrophy and can be developed for negative selection protocols. Additional potential auxotrofic markers will be developed during the project with reference to the P. falciparum genome.
Work Package 2: Transgenic parasites will be created that express thymidine kinase (tk). The sensitivity of the recombinant parasites to suicide drugs metabolised by tk will be assessed. Cytidine deaminase will also be assessed. Hypoxanthine phosphoribosyl transferase (hxgprt) will be deleted to generate a mutant parasite amenable to the use of hxgprt as a selectable marker. The existing second selectable marker will be applied to P. knowlesi. Successful markers will be applied to the other systems. Novel vectors will be developed that combine both positive and negative selection activities with unique strategies for the recycling of the vectors.
Work Package 3: The ionic environment of the intra-erythrocytic P. falciparum will be assessed throughout development using state of the art fluorochrome assisted microscopy. The most important ions will be monitored. This knowledge will be applied to the development of more reliable axenic culture systems primarily for P. falciparum but also for the other parasites. The technology could revolutionise genetic manipulation and vaccine production of malaria parasites.
Work Package 4: The translational capacity of the malaria parasite will be greatly reduced by specific and sequential deletion of genes encoding central elements of the translation apparatus. The growth and pathogenicity of the parasites will be assessed. To achieve the potential immune response, temporally and quantitatively controlled transgenic expression of cytokines will be induced and the immunological and pathological consequence of animal infection examined.
Work Package 5: A forum to bring the advances in malaria parasite genetic engineering to the scientific and public health communities in Europe and Africa will be established. This will help shape future directions for implementation.
1) Auxotrophic malaria parasites that can be genetically manipulated.
2) An increased range of selectable markers and strategies for the manipulation of different malaria parasite genomes.
3) A dynamic knowledge of the erythrocytic milieu of malaria parasites and how it might be applied to improve axenic culture.
4) Production of malaria parasites doubly deficient in protein synthesis - are they attenuated?
5) A forum for the dissemination of genetic engineering of malaria parasites and discussion of practical, ethical and clinical problems associated with GMOs in Africa.
Department of Parasitology /Pre-Clinical Laboratories
Tel: +31 71 527 6858
Fax: +31 71 527 6850
E-mail: waters@rullf2. leidenuniv. nl