Kelley VanBuskirk

Progress Toward Stable Transfection of Human Isolates of Plasmodium Vivax Kelley M. VanBuskirk, Patrick E. Duffy, and Jetsumon Sattabongkot Department of Epidemiology, School of Public Health and Community Medicine, University of Washington Background Malaria caused by the parasite Plasmodium vivax causes serious morbidity and accounts for approximately half of all clinical malaria cases worldwide. Since traditional subunit vaccine approaches have been largely unsuccessful against malaria parasites, novel strategies such as genetic attenuation are being developed. Due to some unique features of its biology, such as dependence on a single molecular pathway for invasion of host red blood cells, P. vivax is an excellent candidate for development as a live, attenuated vaccine. However, genetic targeting by stable transfection of P. vivax has not yet been achieved. P. vivax is less tractable in the laboratory than the better-studied species P. falciparum , and only recently researchers in Thailand have optimized techniques for stable laboratory culture of P. vivax (Udomsangpetch et al, 2007). The aim of this project was to develop and test methods for stable transfection of P. vivax parasites from human infections (Figure 1). Methods ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Results and Discussion A range of transfection conditions were tested. Electroporation constants were relatively high, ranging from 14.0-18.0, suggesting that further optimization of settings may be needed to enhance parasite survival. The highest survival rates, as measured by progression to schizont stage post-transfection, were obtained with the U-033 program of the Amaxa Nucleofector and by pre-loading erythrocytes with DNA prior to parasite invasion. No fluorescent parasites were observed in transfection cultures, possibly due to the low efficiency that is typical of Plasmodium transfection; for instance, efficiency in P. falciparum is estimated to be 1 in 10 6 . Due to an unusually low number of vivax malaria cases at the collaborating clinics and low parasitemia in collected samples, as well as a change in the approved protocol for sample collection, the quantities of available parasites were not as high as expected. To overcome this, we pursued methods of pooling patient samples and enriching parasite concentration within small volumes suitable for transfection. Nycodenz gradients were not found to be optimal for this purpose; in future work Percoll gradients will be tested. Conclusions This project represents preliminary steps toward developing methodology for stable transfection of P. vivax . We determined that the parasite number for transfection must be exceptionally high to overcome the dual challenges of low-efficiency transfection and cultivation of large populations of P. vivax in vitro. Progress has been made toward developing methods of parasite enrichment in vitro for transfection. Transfection conditions were evaluated to select the most promising for parasite survival of electroporation. In addition, the transfection technology was transferred to the Thai lab that hosted this project, facilitating their use of methodology that is underutilized in the global malaria research community due to its technical challenges. Future work will build on these transfection strategies to achieve integration of foreign DNA into the P. vivax genome in order to better understand the biology of the parasite and develop novel ways to control vivax malaria. References Udomsangpetch R, Somsri S, Panichakul T, Chotivanich K, Sirichaisinthop J, Yang, Z, Cui, L, Sattabongkot, J. (2007) Short-term in vitro culture of field isolates of Plasmodium vivax using umbilical cord blood. Parasitol Int 56: 65–69. Acknowledgements The authors would like to thank staff members of the Department of Entomology at AFRIMS, collaborators at the Mae Sod health clinic, and patient volunteers. This work was sponsored by the Thomas J. Francis, Jr. Fellowship in Global Health, the Washington Global Health Alliance and the Department of Global Health, University of Washington. The use of biological samples of human origin was approved by the University of Washington IRB. Figure 2: Plasmodium vivax cultures were grown at 37 º C in anaerobic conditions created within candle jars. Figure 1: Transfection experiments were conducted at AFRIMS in Bangkok and in a clinic-based lab (shown here), in Mae Sot, northwestern Thailand. TABLE I: TRANSFECTION CONDITIONS TESTED AND RESULTS.

Kelley VanBuskirk

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