Plasmodium falciparum SERA protein transgenics used in the study parasite immunobiology and drug resistance

Genetic and immunological tools have been used to study two protozoan parasites, Plasmodium falciparum and Toxoplasma gondii. Toxoplasma gondii provides an excellent model for the study of protozoan parasite biology. Plasmodium falciparum causes the most severe form of human malaria. Using a genetic approach, we isolated DNA ligands which bound specifically to the surface of P. falciparum infected red blood cells (iRBC), however the binding was transient. A DNA vaccine targeted against P. falciparum was generated using the Serine Repeat Antigen (SERA). The design and development of the plasmids employ genetic and molecular biology techniques. The analysis of the efficacy of the vaccines employs immunological techniques. In mice we were able to generate Th2 type antibody responses to the SERA protein. Gene Gun delivery of the plasmids into the skin resulted in the generation of higher antibody titers to SERA than intramuscular injections which delivered the plasmids to the quadriceps muscles of the mice. The SERA protein was also expressed in the T. gondii parasite through genetic engineering. The dihydrofolate-thymidylate synthase pyrimethamine resistant (DHFRm2m3-TS) selectable marker was used to design a new fusion protein expression system in T. gondii. The SERA protein was fused into the Junction region, located between the enzymatic domains, of the DHFRm2m3-TS protein. This successful genetic expression system led to the study of the functional requirements if any of the junction region as measured by pyrimethamine resistance. Only protozoan parasites and some plants express a bifunctional DHFR-TS enzyme. We determined that the amino terminal portion of the T. gondii junction is unnecessary for optimal protein function. A positive charge at position minus 2 with respect to the start of the TS domain is required either for protein expression or function. A protein containing a truncated junction, with a positive charge at position minus 3, provides partial pyrimethamine resistance. The fusion protein expression system can be used to further study the biology of the DHFR-TS protein, employed to further study the immunobiology of the SERA protein, and can be used to express many foreign proteins in protozoan parasites.