Fig 1.
The gating strategy used to define infected hepatoma cells.
Cells were gated by size, followed by exclusion of doublets. Viable cells were selected based on the absence of DAPI staining. Hepatoma cells were labeled with the Vybrant DiD membrane dye prior to seeding, thus enabling identification with DiD fluorescence. Finally, the hepatoma cells that were GFP positive were selected. Uninfected hepatoma cells were run as a control to guide gating of the population of GFP+ cells.
Fig 2.
The infectivity of the Hepa1-6 cell line.
40 000 P. berghei GFP sporozoites were added per well with (n = 69) or without (n = 6) centrifugation. To determine whether dead or aborted sporozoites maintained expression of GFP, sporozoites were heat-killed for 20 minutes at 95°C prior to infection of Hepa1-6 cells (n = 6). 24 hours post-infection cells were harvested and run on a flow cytometer. (A) Representative example of the flow cytometry plots. (B) Data from multiple experiments was pooled and results are expressed as the percentage of GFP positive viable Hepa1-6 cells. The effect of centrifugation on infectivity was assessed using the Mann Whitney test, *** p = 0.0001.
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.
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.
Table 1.
Outline and characteristics of splenocytes used in the thirteen in vitro assays performed.
Fig 5.
Inhibition of liver-stage parasites by P. berghei TRAP-specific CD8+ T cell enriched splenocytes.
(A) Representative example of the flow cytometry plots, from ‘Experiment 3’ (see Table 1). (B) Results are expressed as the percentage infectivity, with the median shown for each experiment and error bars representing the interquartile range. A statistically significant difference was evident overall between wells containing splenocytes from PbTRAP compared to vector control vaccinated mice, p = 0.0479 (Wilcoxon matched-pairs signed rank test). (C) Results are expressed as the percentage inhibition compared to the wells containing cells from vector control vaccinated mice (equal number of CD8+ enriched splenocytes from ChAd63-MVA luciferase vaccinated mice), with median and individual data points shown for each experiment. If the percent inhibition was negative, it was considered to be zero.
Fig 6.
Correlation of the percentage inhibition with the effector to target ratio.
(A) Results are expressed as the percent inhibition compared to mock-vaccinated control wells (equal number of CD8+ enriched splenocytes from ChAd63-MVA luciferase vaccinated mice). Across all thirteen experiments there was no significant correlation between E:T ratio and inhibition (Spearman r = 0.35, p = 0.24), although a trend can be observed. Full circles represent those experiments that fit this trend, whilst empty circles represent those that did not. Experiments were then divided into data that fitted the E:T pattern (full circles) and those that did not (empty circles) and a graph (B) of the infectivity measured in wells containing only Hepa1-6 cells and sporozoites are shown. Statistical difference was assessed using the Mann Whitney test, **** p<0.0001. (C) Correlation of the E:T ratio with the percentage inhibition (n = 9 experiments), excluding the outliers. Spearman r = 0.82, p = 0.011.