Development of an In Vitro Assay and Demonstration of Plasmodium berghei Liver-Stage Inhibition by TRAP-Specific CD8+ T Cells
Fig 3
A schematic representation of the murine in vitro T cell killing assay.
C57BL/6 mice were vaccinated with a ChAd63-MVA prime-boost regimen using vaccines expressing P. berghei TRAP. Six days post-MVA boost, Hepa1-6 cells were labeled with the membrane dye Vybrant DiD and seeded into a 96-well flat bottom plate. The following day salivary glands were dissected from P. berghei GFP infected mosquitoes, sporozoites isolated and 40 000 added per well to the Hepa1-6 cell cultures, followed by centrifugation at 500xg for five minutes. Subsequently, spleens from vaccinated mice were enriched for CD8+ cells via negative depletion prior to addition to the sporozoite infected Hepa1-6 cell cultures approximately three hours post-infection. Splenocytes were also harvested from vector control vaccinated mice and treated in the same way, to determine the effect of non-specific killing. Plates were then incubated for approximately 24 hours prior to trypsin treatment to harvest cells for acquisition on the flow cytometer.