title: A new approach to generate a safe double-attenuated Plasmodium liver stage vaccine.
abstract: Recently it has been shown in rodent malaria models that immunisation with genetically attenuated Plasmodium parasites can confer sterile protection against challenge with virulent parasites. For the mass production of live attenuated Plasmodium parasites for vaccination, safety is a prerequisite. Knockout of a single gene is not sufficient for such a strategy since the parasite can likely compensate for such a genetic modification and a single surviving parasite is sufficient to kill an immunised individual. Parasites must therefore be at least double-attenuated when generating a safe vaccine strain. Genetic double-attenuation can be achieved by knocking out two essential genes or by combining a single gene knockout with the expression of a protein toxic for the parasite. We generated a double-attenuated Plasmodium berghei strain that is deficient in fatty acid synthesis by the knockout of the pdh-e1α gene, introducing a second attenuation by the liver stage-specific expression of the pore-forming bacterial toxin perfringolysin O. With this double genetically attenuated parasite strain, a superior attenuation was indeed achieved compared with single-attenuated strains that were either deficient in pyruvate dehydrogenase (PDH)-E1 or expressed perfringolysin O. In vivo, both single-attenuated strains resulted in breakthrough infections even if low to moderate doses of sporozoites (2,000-5,000) were administered. In contrast, the double genetically attenuated parasite strain, given at moderate doses of 5,000 sporozoites, did not result in blood stage infection and even when administered at 5- to 20-fold higher doses, only single and delayed breakthrough infections were observed. Prime booster immunisation with the double genetically attenuated parasite strain completely protected a susceptible mouse strain from malaria and even a single immunisation conferred protection in some cases and lead to a markedly delayed onset of blood stage infection in others. Importantly, premature rupture of the parasitophorous vacuole membrane by liver stage-specific perfringolysin O expression did not induce host cell death and soluble parasite proteins, which are released into the host cell cytoplasm, have the potential to be processed and presented via MHC class I molecules. This, in turn, might support immunological responses against Plasmodium-infected hepatocytes.
authors.affiliation: Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Nagel
authors.forename: Monica
authors.initials: M
authors.lastname: Prado
authors.forename: Anna
authors.initials: A
authors.lastname: Heitmann
authors.forename: Susanne
authors.initials: S
authors.lastname: Tartz
authors.forename: Thomas
authors.initials: T
authors.lastname: Jacobs
authors.forename: Christina
authors.initials: C
authors.lastname: Deschermeier
authors.forename: Susanne
authors.initials: S
authors.lastname: Helm
authors.forename: Rebecca
authors.initials: R
authors.lastname: Stanway
authors.forename: Volker
authors.initials: V
authors.lastname: Heussler
mesh_terms: D000140:Acidosis, Lactic; D000818:Animals; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics; D001769:Blood / Q000469:parasitology; D004195:Disease Models, Animal; D005260:Female; D055786:Gene Knockout Techniques; D020043:Genes, Essential; D017125:Genes, Protozoan; D008288:Malaria / Q000276:immunology / Q000517:prevention & control; D017780:Malaria Vaccines / Q000008:administration & dosage / Q000009:adverse effects* / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D010962:Plasmodium berghei / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity*; D030481:Pyruvate Dehydrogenase (Lipoamide) / Q000172:deficiency; D016019:Survival Analysis; D014613:Vaccines, Attenuated / Q000008:administration & dosage / Q000009:adverse effects / Q000235:genetics / Q000276:immunology
chemical_list: D001427:Bacterial Toxins; D017780:Malaria Vaccines; D014613:Vaccines, Attenuated; D030481:Pyruvate Dehydrogenase (Lipoamide)