Development of an In Vitro Assay and Demonstration of Plasmodium berghei Liver-Stage Inhibition by TRAP-Specific CD8+ T Cells
Fig 4
Characterization of splenocytes used in the in vitro assays.
(A) Cellular immunogenicity of ChAd63-MVA P. berghei TRAP in C57BL/6 mice. Each data point represents splenocytes from two mice pooled together, with twelve pairs in total that were used in thirteen assays (one pair provided enough cells for two experiments). Cellular immunogenicity was assessed by ICS, after six hours stimulation with a pool of P. berghei TRAP peptides. Vector control mice were vaccinated with ChAd63-MVA luciferase and treated identically to the experimental mice. Results are expressed as the percentage of CD8+ cells, with median and individual data points shown. (B) Prior to addition of the splenocytes into the in vitro assays, samples were enriched for CD8+ cells. Results are expressed as the percentage of CD8+ cells out of total splenocytes, with both median and individual data points shown. (C) In each in vitro assay conducted, CD8+ enriched splenocytes from vector control vaccinated mice were included in the assay, along with wells containing sporozoites only, to act as controls. Results are expressed as the percentage infectivity, with the median shown for each experiment and error bars representing the interquartile range. (D) These controls allowed calculation of the background level of non-specific inhibition. Results are expressed as the percentage inhibition of splenocytes from vector control vaccinated mice compared to sporozoite only wells (no splenocytes), with median and individual data points shown for each experiment. In Exp1 and Exp2 sporozoite only wells were not included and hence the background inhibition could not be calculated.