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
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
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
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
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
Department of Parasitology /Pre-Clinical Laboratories
Tel: +31 71 527 6858
Fax: +31 71 527 6850
E-mail: waters@rullf2. leidenuniv. nl
- Med Biotech Laboratories - Medical Biotechnology, Kampala, Uganda
- Universitätsklinikum Heidelberg - Abteilung Parasitologie /Hygiene-Institut,
- University of Ibadan - Postgraduate Institute For Medical Research and
Training College of Medicine, Ibadan, Nigeria
- Biomedical Primate Research Centre - Department of Parasitolgy, NL-Rijswijk,