The infectivity of the Hepa1-6 cell line.

<p>40 000 <i>P</i>. <i>berghei</i> 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.</p

Characterization of splenocytes used in the <i>in vitro</i> assays.

<p>(A) Cellular immunogenicity of ChAd63-MVA <i>P</i>. <i>berghei</i> 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 <i>P</i>. <i>berghei</i> 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 <i>in vitro</i> 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 <i>in vitro</i> 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.</p

Inhibition of liver-stage parasites by <i>P</i>. <i>berghei</i> TRAP-specific CD8⁺ T cell enriched splenocytes.

<p>(A) Representative example of the flow cytometry plots, from ‘Experiment 3’ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119880#pone.0119880.t001" target="_blank">Table 1</a>). (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.</p

Correlation of the percentage inhibition with the effector to target ratio.

<p>(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.</p

The gating strategy used to define infected hepatoma cells.

<p>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.</p

Outline and characteristics of splenocytes used in the thirteen <i>in vitro</i> assays performed.

<p>^a PbTRAP-specific CD8⁺ cells: infected hepatocytes.</p><p>^b Sporozoite only wells were not included; number of infected hepatocytes was based on the median infectivity of 2.76%.</p><p>Outline and characteristics of splenocytes used in the thirteen <i>in vitro</i> assays performed.</p

Immunodominant responses to PfUIS3.

<p>(A) BALB/c, (B) C57BL/6 or (C) HLA-A2 tg mice (n = 4 per strain) were vaccinated i.m. with 1x10⁸ infectious units (ifu) ChAd63-PfUIS3 followed eight weeks later by 1x10⁶ plaque forming units (pfu) MVA-PfUIS3. Two weeks post-MVA boost, mice were sacrificed and splenocytes isolated to perform an <i>ex vivo</i> IFNγ ELISpot. Splenocytes were stimulated with either an overlapping peptide pool to PfUIS3 or individual peptides (20aa each, overlapping by ten). Both median and individual data points are shown. For (A) BALB/c and (B) C57BL/6, CD4⁺ and CD8⁺ epitopes were also determined (right panel). Two weeks post-ChAd63 (n = 4 per strain), splenocytes were isolated and incubated with the appropriate peptide for six hours prior to ICS staining. Box plots show the percentage IFNγ⁺ of CD4⁺ or CD8⁺ cells, with whiskers representing the maximum and minimum.</p

Polyfunctionality of CD8⁺ T cells induced by ChAd63-MVA vaccination in BALB/c mice.

<p>BALB/c mice (n = 4) were vaccinated with ChAd63-MVA (A) PfUIS3, (B) PfLSA1 or (C) PfLSAP2, as previously described. Two weeks post-ChAd63 prime and one-week post-MVA boost blood was taken and cellular immunogenicity assessed by ICS, after stimulation for six hours with an overlapping peptide pool to the appropriate antigen. Two weeks post-MVA boost mice were sacrificed, spleens harvested and cellular immunogenicity again assessed by ICS. The proportion of cells at each time-point expressing one, two or three cytokines is shown. The bar chart indicates which cytokines were produced, whilst the slices of the pie chart indicate the proportion of cells producing one (purple), two (orange) or three (black) cytokines.</p

Image_1_CXCR3+ T Follicular Helper Cells Induced by Co-Administration of RTS,S/AS01B and Viral-Vectored Vaccines Are Associated With Reduced Immunogenicity and Efficacy Against Malaria.JPEG

<p>A malaria vaccine strategy targeting multiple lifecycle stages may be required to achieve a high level of efficacy. In two Phase IIa clinical trials, we tested immunogenicity and efficacy of RTS,S/AS01B administered alone, in a staggered regimen with viral-vectored vaccines or co-administered with viral-vectored vaccines. RTS,S/AS01B induces high titers of antibody against sporozoites and viral-vectored vaccines ChAd63 ME-TRAP and MVA ME-TRAP induce potent T cell responses against infected hepatocytes. By combining these two strategies, we aimed to improve efficacy by inducing immune responses targeting multiple parasite antigens. Vaccination with RTS,S/AS01B alone or in a staggered regimen with viral vectors produced strong immune responses and demonstrated high levels of protection against controlled human malaria infection. However, concomitant administration of these vaccines significantly reduced humoral immunogenicity and protective efficacy. Strong Th1-biased cytokine responses induced by MVA ME-TRAP were associated with a skew in circulating T follicular helper cells toward a CXCR3⁺ phenotype and a reduction in antibody quantity and quality. This study illustrates that while a multistage-targeting vaccine strategy could provide high-level efficacy, the regimen design will require careful optimization.</p

Local and systemic adverse events (AEs) possibly, probably or definitely related to vaccination shown as percentage of volunteers affected.

<p>(a) Post ChAd63 CS 5×10⁹ vp; (b) Post ChAd63 CS 5×10¹⁰ vp; (c) Post MVA CS 2×10⁸ pfu.</p