Ply sequence with localization of BALB/c and C57BL/6 T cell-immunogenic regions and predicted HLA-DR binding regions.

<p>Immunodominant epitopes of both BALB/c and C57BL/6 mice are indicated with a #, and subdominant epitopes with a *. ¶ shows the Epi-bars that are <i>in silico</i> predicted for HLA-DR-binding epitopes in humans. Mutated aa residues of PlyD1 are squared and positions with allelic variation as described by Jeffries <i>et al</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193650#pone.0193650.ref008" target="_blank">8</a>] are underlined. Colors, cyan, green, red and purple, indicate domains 1, 2, 3, and 4 of Ply, respectively.</p

3D modelling of T cell epitopes in Ply for BALB/c and C57BL/6 mice.

<p>Yellow spheres represent the C-ß atoms of the ID epitope and orange spheres represent the C-ß atoms of the subD epitopes. The dotted circle represents the overlap region between both mouse strains. Colors of the protein structure display the different domains of Ply: cyan indicates domain 1 (amino acids 1–21, 58–147,198–243,319–342), green indicates domain 2 (amino acids 22–57,343–359), red indicates domain 3 (amino acids 148–197,244–318), and purple indicates domain 4 (amino acids 360–469).</p

Identified immunodominant and subdominant T cell regions in PlyD1.

<p>Identified immunodominant and subdominant T cell regions in PlyD1.</p

Determination of ID and subD PlyD1 T cell epitopes.

<p>Splenocytes from mice immunized with PlyD1, PlyD1+Al(OH)₃ or PBS placebo were tested, freshly in BALB/c mice and after thawing in C57BL/6 mice, against PlyD1 and individual peptides from positively tested smart pools. ID regions are peptides with highest proliferation, subD regions are epitopes with subsequent proliferative levels. Proliferation is expressed as mean SI with standard deviation per group. * p value < 0.05 compared to the placebo group. # p value < 0.05 comparing PlyD1 with and without Al(OH)₃.</p

Serological responses after 3 immunizations with PlyD1.

<p>BALB/c and C57BL/6 mice were immunized 3 times with PlyD1, with or without aluminum hydroxide, or with PBS placebo. Ply-specific antibody titers are shown for total IgG (A), IgG1 (B), IgG2a for BALB/c mice and IgG2c for C57BL/6 mice, respectively (C). Neutralizing capacity of antibodies against hemolytic activity of Ply was determined using HIMA (D). Geometric means of (reciprocal) titers at 50% ODmax with standard deviations are displayed per group.</p

Antibody titers of mice after immunization with <i>N. meningitidis</i> P1.5-1,2-2 OMVs.

<p>Mice were immunized with <i>N. meningitidis</i> P1.5-1,2-2 OMVs and antigen-specific titers of IgG, IgG1, IgG2a/c, IgG2b, and IgG3 in sera were determined with ELISA. Also total IgE in sera of mice immunized with PBS or <i>N. meningitidis</i> P1.5-1,2-2 OMVs was measured with ELISA. Results for C57BL/6, TLR2−/−, and TRIF-deficient (TRIFdef) mice are shown in panel A, results for C3H/HeOuJ and C3H/HeJ mice are shown in panel B. Levels of serum bactericidal antibodies after immunization with <i>N. meningitidis</i> P1.5-1,2-2 OMVs are shown in panel C. Data are expressed as means of log₁₀ titers for 6 mice per group, except levels of serum bactericidal antibodies in C57BL/6 and TLR2−/− mice (n = 12). Error bars represent S.E.M. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01.</p

Cytokines produced by spleen cells after restimulation with FHA.

<p>Spleen cells from C57BL/6, TLR2−/−, TRIF-deficient (TRIFdef) mice (A), or C3H/HeOuJ and C3H/HeJ mice (B) immunized with PBS or whole cell pertussis vaccine were incubated for 4 days with 500 ng/ml FHA Cytokines IL-2, IL-4, IL-5, IL-10, IL-13, IL-17, and IFN-γ were determined in the supernatant with Luminex. Data are expressed as means of three mice per group (PBS), or six mice per group (whole cell pertussis vaccine), error bars represent S.E.M. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01.</p

Cytokines produced by spleen cells after restimulation with P1.5-1,2-2 PorA peptides.

<p>Spleen cells from C57BL/6, TLR2−/−, or TRIF-deficient (TRIFdef) mice immunized with PBS or <i>N. meningitidis</i> P1.5-1,2-2 OMVs were incubated for 4 days with peptide 11–24/25 (A) or peptide 4–52/53 (B). Spleen cells from C3H/HeOuJ and C3H/HeJ mice immunized with PBS or <i>N. meningitidis</i> P1.5-1,2-2 OMVs were incubated for 4 days with peptide pool 6 (C). Cytokines IL-4, IL-5, IL-10, IL-13, IL-17, and IFN-γ were determined in the supernatant with Luminex. Data are expressed as means of three mice per group (PBS), or six mice per group (<i>N. meningitidis</i> OMVs), error bars represent S.E.M. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01.</p

Proliferation of spleen cells after restimulation with antigen or peptide.

<p>Spleens were taken from the mice after immunizations and spleen cells were incubated for 4 days with medium, antigen, or peptides, after which [³H]thymidine incorporation was determined. Spleen cells of C57BL/6, TLR2−/−, and TRIF-deficient (TRIFdef) mice immunized with PBS (3 mice per group) or <i>N. meningitidis</i> P1.5-1,2-2 OMVs (6 mice per group) were restimulated with 1 µM of peptides 11–24/25 and 4–52/53 (A). Spleen cells of C3H/HeOuJ and C3H/HeJ mice immunized with PBS (3 mice per group) or <i>N. meningitidis</i> P1.5-1,2-2 OMVs (6 mice per group) were restimulated with peptide pool 6 (1 µM of each peptide, B). Spleen cells of C57BL/6, TLR2−/−, TRIF-deficient, C3H/HeOuJ, and C3H/HeJ mice immunized with PBS (3 mice per group) or whole cell pertussis vaccine (<i>B. pertussis</i>, 6 mice per group) were restimulated with 500 ng/ml of FHA antigen (C). Data are expressed as means of stimulation indices.</p

Serum cytokine levels shortly after vaccination.

<p>Two and four hours after immunization blood samples were taken from all mice and cytokine levels in the sera were analyzed with Luminex. Results for <i>N. meningitidis</i> OMVs are shown in panel A, results for whole cell pertussis vaccine are shown in panel B. There were 3 mice per group for the animals that received PBS and 6 mice per group for <i>N. meningitidis</i> OMVs and whole cell pertussis vaccine. The data are expressed as means, error bars represent S.E.M. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.</p