MVA deletion mutants.

<p>MVA deletion mutants used in this study. Different clusters of ORFs were deleted from the mutants, encoding proteins with different functions (mentioned with references). In a few cases, there were no known functions associated with ORFs. The mutants express the 85A or TIP antigens that were inserted at the TK locus. All mutants were made using MVA-BAC recombineering, therefore, all contain BAC DNA and GFP (green fluorescent protein) marker.</p><p>^aTK: Thymidine kinase locus, used as an insertion site for the recombinant antigens.</p><p>^bVACV COP: Vaccinia virus Copenhagen strain.</p><p>^cWR: Vaccinia virus Western Reserve strain.</p><p>^dThe ORF <i>B15R</i> is the <i>MVA184R</i> gene. It is named <i>B15R</i> in VACV WR while it is <i>B16R</i> in VACV COP. This made inconsistency in reporting this ORF as well as the downstream B fragment ORFs in the literature. Here, we report <i>MVA184</i> as the <i>B15R</i>, consistent with our previous work [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128626#pone.0128626.ref022" target="_blank">22</a>], and in accordance with another report <i>[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128626#pone.0128626.ref011" target="_blank">11</a>]</i>. However, it was reported as <i>B16R</i> in another study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128626#pone.0128626.ref013" target="_blank">13</a>]. This ORF encodes IL-1β binding protein [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128626#pone.0128626.ref011" target="_blank">11</a>].</p><p>MVA deletion mutants.</p

<i>In vivo</i> cellular immunogenicity of MVA deletion mutants, lacking fifteen genes, with TIP antigen.

<p>Two groups of female BALB/c mice (n = 10) were immunized (i.m.) with MVA<i>wt</i> with TIP model antigen (MVA-TIP), or MVA deletion mutant (lacking 15 genes) ∆15-MVA-TIP at the dose of 1x10⁶ pfu/ml. <b>Seven days</b> post immunization (top), <i>ex vivo</i> ELISpot was performed to determine the percentage of IFN-γ-secreting CD8⁺ splenocytes in response to <i>in vitro</i> re-stimulation with pb9 peptide <b>(A)</b>, or with MVA vector-specific peptides <b>(B and C)</b> (all three peptides are CD8⁺ T cell specific). <b>28 days</b> (middle), or <b>84 days</b> (bottom) post immunization, five mice were sacrificed and spleens collected for intracellular cytokine staining and flow cytometry to determine the percentage of IFN-γ-secreting CD8⁺ T splenocytes in response to <i>in vitro</i> re-stimulation with pb9 peptide <b>(A)</b>, or with MVA vector-specific peptides <b>(B and C)</b>. These values are presented after subtracting the values of unstimulated cells for every mouse (sample). The mean of each group with the SEM error bars are shown. Data is representative of two independent experiments. * <i>P</i> = 0.0331 using Mann Whitney test.</p

<i>In vivo</i> cellular immunogenicity of MVA deletion mutants, lacking fifteen genes, with 85A antigen.

<p>Four groups of female BALB/c mice (n = 4) were immunized (i.m.) with the respective MVAs at the dose of 2x10⁶pfu/ml. Seven days post immunization, intracellular cytokine staining and flow cytometry was performed to determine the percentage of splenic IFN-γ-secreting T cells in response to <i>in vitro</i> re-stimulation with MVA vector-specific peptide (<b>A</b> and <b>B</b>), with CD8⁺ T cell specific 85A peptide pool <b>(C)</b>, or with CD4⁺ T cell specific 85A peptide pool <b>(D)</b>. Two individual peptides were incubated for 18 hours to determine the CD8⁺<b>(E)</b> or CD4⁺ T <b>(F)</b> cell responses by ELISpot. These values are presented after subtracting the values of unstimulated cells for every mouse (sample). The mean of each group with the SEM error bars are shown. Data is representative of two independent experiments. There was no statistical significant difference in any of MVA mutant groups as compared to MVA85A or MVA-BAC85A groups, using Kruskal-Wallis test with Dunn's multiple comparisons. <b>MVA<i>wt</i>:</b> MVA wild type, <b>MVA85A:</b> MVA expressing TB 85A antigen, <b>MVA-BAC85A:</b> MVA, containing BAC DNA and expressing TB 85A antigen, and <b>∆15-MVA-85A:</b> MVA mutant, containing BAC DNA and expressing TB 85A antigen, with 15 gene deleted (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128626#pone.0128626.t001" target="_blank">Table 1</a>).</p

<i>In vivo</i> cellular immunogenicity of MVA deletion mutants, lacking fifteen genes, with TIP antigen, in a prime-boost regimen.

<p>Two groups of female BALB/c mice (n = 10) were immunized i.m. with 100μg of pSG-TIP DNA, then boosted with i.p. injection of MVA<i>wt</i> with TIP model antigen (MVA-TIP), or MVA deletion mutant (lacking 15 genes) ∆15-MVA-TIP at the dose of 1x10⁷ pfu/ml. <b>7 days</b> (top), or <b>56 days</b> (bottom) post-boost immunization, half of the mice were sacrificed and spleens collected for intracellular cytokine staining and flow cytometry to determine the percentage of IFN-γ-secreting CD8⁺ T splenocytes in response to <i>in vitro</i> re-stimulation with pb9 peptide <b>(A)</b>, or with MVA vector-specific peptides <b>(B and C)</b>. All values are presented after subtracting the values of unstimulated cells for every mouse (sample). The mean of each group with the SEM error bars are shown. Data is representative of two independent experiments. There was no statistical significant difference between the tested groups using Mann Whitney test.</p

Timescale showing BCG persistence in the ears and LNs of <i>id</i>-injected mice up to 12 weeks post immunization.

<p>Log BCG CFU in the ears (estimated by culture) are shown for (<b>a</b>) the high dose group (7000 CFU <i>id</i>); and (<b>b</b>) the low dose group (60 CFU <i>id</i>). Log BCG CFU in the auricular LNs are shown in (<b>c</b>) the high dose group (7000 CFU <i>id</i>); and (<b>d</b>) the mid dose group (60 CFU <i>id</i>). Datasets include individual data points for each mouse; the bars represent the median per group in (<b>a</b>) and (<b>b</b>), and a line connects the means for each group in (<b>c</b>) and (<b>d</b>). * indicates <i>P</i><0.05. Both ears and LNs were homogenized and plated onto 7H11 Middlebrook agar. Log BCG CFU in the ears estimated by culture (<b>e</b>) and BCG genome copies/mHPRT copies estimated by PCR (<b>f</b>) are shown in a timescale for the high dose group, up to 12 weeks post BCG immunization. Quantitative PCR was performed with BCG-specific primers. Individual data points are shown for each mouse with a line connecting the means for each group.</p

Effect of single dose vaccines (subunits MVA85A and Ad85A, and BCG) on an <i>id</i> BCG challenge.

<p>(<b>a</b>) BALB/c mice were immunized <i>id</i> with 1×10⁶ pfu MVA85A or 2×10⁹ vp Ad85A. Control mice (Naïve) received no immunization. Four weeks later all mice were challenged <i>id</i> with 1×10⁵ CFU BCG, contralaterally to the site of vaccination. Ears and LNs were harvested 4 weeks after BCG challenge and processed for CFU quantification. (*<i>P</i><0.05, <i>n</i> = 10 except naïves, <i>n</i> = 5). (<b>b</b>) Corresponding intracellular cytokine staining (ICS) of the local draining LNs. Red bars represent the proportion of IFN-γ-secreting CD4⁺ T cells in response to 85A, blue bars represent the same for CD8⁺ T cells (M, <i>n</i> = 4; Naïve, <i>n</i> = 3; Ad, <i>n</i> = 4. *<i>P</i><0.05). (<b>c</b>) Effect of BCG vaccine compared to subunit MVA85A on an <i>id</i> BCG challenge. BALB/c mice were immunized <i>id</i> with either 1×10⁶ pfu MVA85A or 2.2×10⁴ cfu BCG. “Naïve” and antibiotic-treated (I+R) mice received no immunization. Four weeks later all mice were challenged with 6×10³ CFU BCG, except the BCG control group who received no challenge. In the I+R group, challenge was followed by 4 weeks treatment with isoniazid and rifampicin. Ears and LNs were harvested 4 weeks after BCG challenge and processed for CFU quantification. Log₁₀ BCG CFU individual data points for each mouse are shown. Bars represent the median per group. (<b>c</b>) Ears (**<i>P</i><0.01, “non-significant, ND”, <i>n</i> = 10); (<b>d</b>) LNs (**<i>P</i><0.01, “non-significant, ND”, <i>n</i> = 10).</p

Splenic IFN-γ responses to PPD up to 12 weeks post BCG immunization.

<p>In (<b>a</b>) high dose group (7000 CFU <i>id</i>); (<b>b</b>) mid dose group (60 CFU <i>id</i>) and (<b>c</b>) low dose group (1 CFU <i>id</i>). Datasets include individual data points for each mouse; the bars represent the median value per group. Results expressed as SFC/million splenocytes.</p

The Correlation of IL-10 expression with FoxP3 and TGFβ1 mRNA Expression.

<p>For the A) baseline (r = 0.557 <i>P</i> = 0.030), b) 7 days following vaccination (r = 0.695 P = 0.019) and C) 28 days following vaccination time points (r = 0.554 <i>P</i> = 0.048) IL-10mRNA expression correlates with FoxP3 expression. At the 28 days time point D) IL-10 mRNA also correlates with TGF-β1 mRNA expression (r = 0.642 <i>P</i> = 0.023). The volunteers with sterile protection are indicated by open circles and triangles indicate volunteers who did not enter the challenge study. Correlations were performed using Spearman's one-sided test, n = 9–12.</p

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

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