CAF01 induction of cellular immunity.

<p><b>A and B</b>. Mice were vaccinated twice at two weeks intervals with IPV (TP) 2DU (white bars), IPV 20DU (grey bars) or IPV 2DU + CAF01 (black bars). Striped bars indicate non-vaccinated mice. At week 2 (A) or week 5 (B) post vaccination 2, PBMCs were stimulated with IPV for 72 hours and secretion of the indicated cytokines was analyzed by MSD. The cytokines are indicated in the figure. Indicated dosage units in the experiments correspond to Type 1 D-antigen units. All mice were vaccinated via the IM route with inactivated polio vaccine (TP).</p

Polyfunctional vaccine specific T cells recruited to the lungs after infection.

<p>Mice immunized as described in the experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039909#pone-0039909-g001" target="_blank">figure 1</a> were infected by the aerosol route with virulent M.tb Erdman seven weeks after the last H4-IC31® booster immunization. Six weeks after infection lung lymphocytes from three pools consisting of two half lungs per pool were used from each group for intracellular cytokine analysis by flow cytometry. Cells were stimulated with the indicated antigens derived from H4 (Ag85B and TB10.4; A) or ESAT-6 (B) as specified in the graph. Bars represent the proportions of lung CD4 T cell subsets producing different cytokines in response to stimulation as indicated on the X-axis. Background levels obtained in media-stimulated samples has been deducted. *, p<0.05, **, p<0.01, ***, p<0.001, proportion of cytokine producing CD4 T cells subsets compared to non-vaccinated controls using one-way ANOVA and Tukey’s post-test for multiple comparisons. This experiment was repeated once with similar results.</p

Boosting BCG with H4-IC31® induces polyfunctional memory and effector CD4 T cells.

<p>Mice were immunized as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039909#pone-0039909-g001" target="_blank">figure 1A</a>, and parallel with the ELISA analysis, splenocytes and PBMCs (pooled from n = five mice/group) obtained two weeks after the first H4-IC31® immunization (A) and PBMCs (pooled from n = 15 mice/group) obtained six weeks after the second immunization (B) were used for intracellular cytokine analysis by flow cytometry. Cells were stimulated with TB10.4 (upper panels) and Ag85B (lower panels). Bars represent the proportions of CD4 T cell subsets producing different cytokines as indicated on the X-axis. Background levels obtained in media-stimulated samples has been subtracted. This experiment was repeated once with similar results.</p

CAF01 increase the Antibody binding titers.

<p><b>A</b>. The serum IgG against TP or the individual virus serotypes was measured by indirect ELISA using trivalent IPV or the MPs as the antigen. The Ab titer was measured by the reaction of a series of 10-fold dilution of sera with the antigen. The Ab titration from the sera of 4 mice are shown for the vaccine groups indicated. <b>B</b>. The serum IgG1, IgG2a, IgG2b and IgG2c against TP was measured by indirect ELISA using trivalent IPV as the antigen. (N = 6–8). <b>C</b>. Serum IgG against TP 5 and 8 weeks post the second vaccination in the vaccination groups indicated (N = 6). Indicated dosage units in the experiments correspond to Type 1 D-antigen units. All mice were vaccinated via the IM route with inactivated polio vaccine (TP).</p

Percentages of CD4 T cells producing cytokines after first and second H4-IC31-boost and after infection.

a<p>The relative proportion of the responding CD4 T cells producing combinations of IFNγ, TNFα and/or IL-2 as listed when stimulated with either <i>in vitro</i> TB10.4 or Ag85B, assessed by intracellular cytokine staining and flowcytometry.</p

Boosting BCG with H4-IC31® improves BCG-derived protection.

<p>Six weeks after infection the level of protection was assessed in the lungs (upper panel) and spleens (lower panel) of infected mice. Each data-point represents the log₁₀ CFU value of individual mice (N = 29 for non-vaccinated controls and n = 9−10 for the vaccination groups). Means and SEM for each group is indicated. *, p<0.05, **, p<0.01, ***, p<0.001, using one-way ANOVA and Newman-Keuls post test for multiple comparisons.</p

The IPV-CAF01 formulation.

<p><b>A</b>. The Z_avg hydrodynamic diameter of IPV3 (2237 DU/ml), IPV TP (400∶80∶320 DU), CAF01 (2500DDA/500TDB) and IPV (8DU)-CAF01 (2500DDA/500TDB). All data show the average of 3 measurements. <b>B</b>. Analysis of IPV content, as D-unit activity, of the indicated formulations. <b>C</b>. The Zeta potential of IPV, CAF01 and IPV-CAF01 formulations. <b>D</b>. The degree of association between CAF01 and IPV was determined by measuring the IPV content in the pellet or supernatant fraction by DU ELISA. The content of IPV in similar vaccine without CAF01 was also measured (“IPV1-3 content”).</p

Comparing 2 versus 3 vaccinations.

<p>Virus neutralization antibody titers from mice (n = 4/group) 14 days following the second or the third IM immunization with inactivated polio vaccine (TP) (with or without adjuvant). Neutralizing antibody titers against poliovirus serotype 1–3 are shown. The individual titers for each mouse are plotted and the bar represents the mean neutralizing antibody titer. SEM of estimated log₁₀ values from N = 4 mice per group *, p<0.05, **, p<0.01, ***, p<0.001 are indicated in the graph using one-way ANOVA and Tukey's post-test for multiple comparisons. Indicated dosage units in the experiments correspond to Type 1 D-antigen units.</p

Correlates of protection six weeks after infection.

<p>A, the pulmonary responses obtained using ICS and flow cytometry six weeks after infection shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039909#pone-0039909-g003" target="_blank">figure 3</a> after stimulation with ESAT-6 (A) or the H4-vaccine antigens Ag85B- and TB10.4 (B) were correlated to the corresponding mean log₁₀ CFU value shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039909#pone-0039909-g004" target="_blank">figure 4</a>. Points represent mean percentage and SEM (vertical) of CD4 T cells producing IFNγ/TNFα (A) or IFNγ/TNFα/IL-2 (B) in response to stimulation with indicated antigens from 3 pools of two lungs plotted on the y-axis and mean and SEM (horizontal) log₁₀ CFU values of mice lungs (N = 29 for non-vaccinated controls and n = 9−10 for the vaccination groups) on the x-axis. Each point represents one group in the order BCG/H4-IC31, BCG, H4-IC31, and non vaccinated from left. *, p<0.05, **, p<0.01, using Pearson’s product-moment correlation coefficient (r) and correlation test.</p

H4-IC31® boost BCG-primed immune responses and memory.

<p>Mice that received BCG were immunized at week 0 and the H4-IC31® and BCG/H4-IC31® groups were subsequently immunized with H4 in IC31® at 19 and 22 weeks after initiating the experiment (A). The amount of IFNγ produced by splenocytes and PBMCs (pooled from five mice per group) in response to Ag85B and TB10.4 stimulation was evaluated by ELISA. The responses were evaluated two weeks after the first H4-IC31® immunization (B), and memory responses were evaluated six weeks after the second immunization in PBMCs pooled from n = 15 mice per group (C). Bars represent means and standard deviations of triplicate 72 hour culture-supernatants. Stimulation with media alone as well as ESAT-6 as a negative control antigen resulted in IFNγ secretion below 300 pg/ml (data not shown). This experiment was repeated once with similar results.</p