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

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

CAF01 induced dose sparing measured by virus neutralization titers.

<p>Virus neutralization antibody titers from mice (n = 11–12/group) 14 days following the second IM immunization with inactivated polio vaccine (TP) (with or without adjuvant). Neutralizing antibody titers against poliovirus serotypes 1–3 are shown. The individual titers for each mouse are plotted and the bar represents the mean neutralizing antibody titer. SEM of estimated log₂ values are shown, *, p<0.05, **, p<0.01, ***, p<0.001 as indicated in the graph using one-way ANOVA and Tukeys post-test for multiple comparisons. Indicated dosage units in the experiments correspond to Type 1 D-antigen units.</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

IM+ID administration of CAF01 induces intestinal IgA.

<p><b>A</b>. Mice were vaccinated twice at two weeks intervals. Two weeks after the second immunization with the indicated TP vaccines IgA and IgG isotypes was analyzed in fecal samples. The fecal samples were pooled within each experimental group. <b>B</b>. Serum IgG against TP two weeks post vaccination 2. The vaccination groups are indicated (N = 4). <b>C</b>. Virus 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 = 8 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

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

Cellular immune responses induced after immunization in CAF01 with or without a T helper epitope.

<p>IFN-γ ELISPOT responses against (A) Vif₁₀₁ CTL epitope and (B) PADRE Th epitope of splenocytes from HLA-A*0201 transgenic HHD mice after 10 days of s.c. immunization with Vif₁₀₁ and PADRE (grey bars, n = 5) or with Vif₁₀₁ alone (black bars, n = 5) and 5 days <i>in vitro</i> prestimulation with individual epitopes. The background of an unimmunized mouse is substracted. Specific ⁵¹Cr-release from target cells preloaded with Vif₁₀₁ after incubation with effector splenocytes from individual mice immunized with (C) Vif₁₀₁ and PADRE (grey, n = 5) or (D) with Vif₁₀₁ alone (black, n = 5). The percentage of specific lysis was calculated as 100×(experimental release-spontaneous release)/(total release-spontaneous release). Background lysis from an unimmunized mouse is shown (open circles). Significant levels are considered >10% lysis at a 50∶1 ratio of effector:target (E:T) cells. One representative experiment out of three. SFU, spot forming units.</p

Cellular immune responses are induced to a similar level after immunization with IFA and CAF01.

<p>IFN-γ ELISPOT responses against (A) HLA-A2-restricted CD8 T cell epitope Vif₁₀₁ and (B) PADRE Th epitope of splenocytes from HLA-A*0201 transgenic HHD mice after 10 days of s.c. immunization with Vif₁₀₁ and PADRE in either IFA (grey bars, n = 5) or CAF01 adjuvants (black bars, n = 5) and 5 days <i>in vitro</i> prestimulation with individual epitopes. The background of an unimmunized mouse is substracted. Specific ⁵¹Cr-release from target cells preloaded with Vif₁₀₁ after incubation with effector splenocytes from Vif₁₀₁ and PADRE immunized mice adjuvanted with (C) IFA (grey, n = 5) or (D) CAF01 (black, n = 5). The percentage of specific lysis was calculated as 100×(experimental release-spontaneous release)/(total release-spontaneous release). Background lysis from an unimmunized mouse is shown (open circles). Significant positive levels are considered for >10% lysis at a 50∶1 ratio of effector:target (E:T) cells. One representative experiment out of three. SFU, spot forming units.</p

Proliferative CD8⁺ T cell responses induced after immunization with IFA and CAF01.

<p>The percentage of proliferating CD3+CD8⁺ splenocytes (having reduced CFSE dye) derived from HLA-A*0201 transgenic HHD mice 10 days after s.c. immunization with a peptide mix consisting of 10 peptides adjuvanted with (A) IFA or (B) CAF01. The background proliferation of non-stimulated (NS) cells, incubated with media alone is shown. The epitope peptides were (from left to right): NS, Gag₁₅₀, Gag₄₃₃, Env₆₇, Pol₆₀₆, Vpu₆₆, Vif₁₀₁, Vif₂₃, Gag₂₉₈ (Th), Env₅₇₀ (Th) and PADRE (Th).</p

TB bacterial burden in mice upon vaccination.

<p>Mice were challenged by the aerosol route with virulent M. tuberculosis A) 6 weeks and B) 52 weeks after the last immunization. Six weeks post challenge, mice were sacrificed and the bacterial burden (CFU) measured in the lungs (expressed as log10 CFU). As a positive control group, a group of mice received a BCG vaccination ten weeks before challenge.</p