Protective capacity of PPV-PYCS prime followed by booster with replication competent VACV vector expressing CS.

<p>Groups of BALB/c mice (5 per group) were immunized with different amounts of PPV-PYCS [PPV(10 µg) and PPV(50 µg)] or non-vaccinated and two weeks later animals were boosted with the replication competent VACV vector from the WR strain (referred as VV-PYCS) or with PPV-PYCS. Thirteen days later, animals were challenged with sporozoites and the amount of parasite rRNA in the liver of each animal was estimated 42 h after the challenge, as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034445#s4" target="_blank">Materials and Methods</a>. Results are expressed as the mean with standard deviation. Statistical values were determined by one-way ANOVA; <i>P</i> values, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. Lane (–), non-vaccinated.</p

PPV-PYCS/MVA-PYCS prime/boost improves cytokine secretion by memory CD8⁺ T-cells.

<p>Groups of BALB/c mice (4 per group) were immunized in prime/boost as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034445#pone-0034445-t001" target="_blank">Table 1</a> and 53 days post boost splenocytes were processed for ICS as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034445#s4" target="_blank">Materials and Methods</a>. Phenotypic differentiation of CD8⁺ T-cells based on memory markers CD127 (V450) and CD62L (FITC). Each quadrant represents different memory population of CD8 cells with its respective percentages. The distribution of antigen specific CD8+ T-cells secreting cytokines in response to CS peptide stimulation, within the different memory population is also shown. The total CD8+ T-cell response is indicated by the black boxes. The boxes on left indicate the responses towards CS peptide stimulation while the ones on right represent its respective RPMI controls.</p

Protection against malaria after prime/boost with PPV-PYCS/MVA-PYCS protocol.

<p>Groups of BALB/c mice (5 per group) were immunized with different amounts of PPV-VLPs [PPV(10 µg) and PPV(50 µg)] and two weeks later animals were boosted with the replication restricted MVA-PYCS or the replication competent VV-PYCS. A positive control group primed with an influenza virus recombinant expressing the CD8+ T cell epitope and booster with VV-PYCS, a protocol previously shown to induce high protection against the parasite, was included <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034445#pone.0034445-Li1" target="_blank">[10]</a>. Thirteen days later, animals were challenged with sporozoites and the amount of parasite rRNA in the liver of each animal was estimated 42 h after the challenge, as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034445#s4" target="_blank">Materials and Methods</a>. Results are expressed as the mean with standard deviation. Statistical values were determined by one-way ANOVA; <i>P</i> values, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

The CS-specific CD8+ T-cell priming effect of PPV-PYCS is markedly enhanced by boosting with replication competent VACV vector expressing CS.

<p>Different groups of BALB/c mice (5 per group) were immunized s.c with 10 or 50 µg of PPV-PYCS [PPV(10 µg) and PPV(50 µg) respectively] or with VACV vectors in prime/boost protocols. Fourteen days after the boost CS-specific IFN-γ secreting cells for the plasmodial epitope SYVPSAEQI in splenocytes were measured by ELISPOT as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034445#s4" target="_blank">Materials and Methods</a>. The results are expressed as the mean of triplicate assays using cultured pooled splenocytes. Statistical values were determined by one-way ANOVA; P values, *P<0.05, **P<0.01, ***P<0.001.</p

Dosage of different vaccination regimes: A summary of priming and boosting agents used in the study with the corresponding dosages.

<p>All agents were administered s.c except for DNA-PYCS which was injected intradermally.</p>1<p>PPV refers to PPV-VLPs containing the CD8 epitope of <i>P.yoelii</i> CS protein.</p>2<p>nrPPV refers to non-recombinant PPV-VLPs.</p

Polyfunctionality of CD8+ T-cells is improved by heterologous prime/boost.

<p>Groups of BALB/c mice (4 per group) were immunized in prime/boost as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034445#pone-0034445-t001" target="_blank">Table 1</a> and 53 days post boost splenocytes were processed for ICS as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034445#s4" target="_blank">Materials and Methods</a>. Memory CD8 T-cells were differentiated into single, double or triple positive cells secreting IFN-γ (PeCy7), TNF-α (PE) and IL-2 (APC). Polyfunctionality is indicated by the pie chart.The data was then analyzed using SPICE software. Data are expressed as mean ± s.e. (n = 4 mice per group). Statistical values were determined by a novel approach previously described (33). P values were calculated between PPV/MVA-PYCS and PPV/PPV groups. **<i>p</i><0.01; ***<i>p</i><0.001.</p