Transfection and characterization of plasmodium knowlesi malaria parasites genetically modified to express host intrferon gamma (IFN-g) in baboon (papio anubis)
Munyao, James Muli
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Despite efforts to control malaria, it still remains a major health problem in endemic countries.With the development of transfection technology, it is now possible to determine the structure-function relationship of vaccine candidates. In addition, transgenes relevant in malaria vaccine development can now be expressed and hostparasite interface determined. This study involved characterization of genetically modified P. knowlesi malaria parasites that express host IFN-gamma, in order to develop a baboon (Papio anubis) model for transfection and analysis of host-parasite interface. One olive baboon was infected with wild-type P. knowlesi to generate parasites for transfection. At day 9 post inoculation, the baboon developed acute malaria with a parasitaemia of 7.6% and on day 10 post inoculation it was totally bled out. The blood was processed by centrifugation and 500 ml of top brown layer harvested. This layer contained 0.65 x 109 parasites enriched with mature schizonts. The parasites were resuspended in 300 ml of cytomix, mixed with 50 ~d of plasmids DNA construct. The construct contained pyrimethamine resistant form of dihydrofolate reductase thymidylate synthase (dhfr-ts) from Toxoplasma gondii, IFN-gamma gene under control of P. berghei promoters and enhancer sequences. The mixture was electroporated in a Biorad gene pulsar in a pulse of 2.5 KV, at a capacitance of 25 ~F and a resistance of 200 SZ for 0.9 ms, chilled on ice for 5 minutes and injected into two olive baboons. The baboons were given pyrimethamine orally for drug selection of transfected parasites. This was done 24 hours post inoculation. Pyrimethamine resistant P. knowlesi appeared from day 10 post inoculation. On day 15 post inoculation, blood was collected from the two baboons, and subjected to plasmodipur filtration to remove leucocytes before DNA isolation. The parasites were cultured in vitro for 48 hours, supernatant harvested and analysed by sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for IFN gamma. Antibody response against transfected and passaged parasites in baboon was determined by ELISA from serum collected from baboons. Molecular characterization of isolated DNA from pyrimethamine resistant parasites by PCR revealed presence of selectable marker gene (dhfr-ts) and IFN-gamma gene. Supernatant of in vitro culture of transfected parasites showed release of high levels of IFN-Y, suggesting presence of IFN-gamma gene in transfected parasites. Antibody response against transfected P. knowlesi and passaged transfected parasites, suggested primary response with rise in IgM production at 0.34% and 0.33% parasitaemia respectively. These results showed for the first time that it is possible to genetically modify P. knowlesi to express host IFN-,y and induce immune response in baboon. These studies have developed an in vivo transfection protocol for P. knowlesi in the baboon. This opens possibilities for using the P. knowlesi-baboon model in vaccine development using cuttingedge technology.
- MST-Zoological Sciences