Antibody and T cell responses following vaccination with OVA targeted to MHC class II molecules or CCR1/3/5.

<p>(a,b) Supernatants of 293E cells transfected with the indicated plasmids were tested for secreted proteins in ELISA (a) and examined by Western blotting with anti-OVA mAb under reducing (+ME) or non-reducing (-ME) conditions (b). Vaccine proteins are indicated below lanes. (c-f) Mice were immunized once i.d. with 25 µg DNA/EP, as indicated. (c-e) Sera were assayed for total IgG (c), IgG1 (d) or IgG2a (e) against OVA. (f) Splenocytes collected at day 14 post immunization were stimulated <i>in vitro</i> with OVA protein or controls as indicated, and analyzed by an IFNγ EliSpot. *indicates p<0.008 and **p<0.002.</p

Characterization of fusion vaccine proteins.

<p>a) Schematic overview of homodimeric vaccine proteins. The fusion proteins consists of HA antigen connected to a targeting unit via a shortened Ig hinge and a dimerizing human γ3 CH3 domain and Ig hinge. As targeting units we used a scFv directed against the MHC class II molecule I-E^d (αMHCII-HA), or the mouse chemokine MIP-1α (MIP-1α-HA). For non-targeted controls, a scFv directed against the hapten NIP (αNIP-HA), or a mutated MIP-1α (MIP-1α(C11S)-HA), replaced functional targeting units. b) Supernatants of transfected 293E cells were examined by Western blotting with anti-HA mAb under reducing (-ME) or non-reducing (+ME) conditions. Vaccine proteins are indicated below lanes, and MW by arrows. c) Binding of vaccine proteins to anti-C_H3 mAb in Sandwich ELISA, followed by detection with an anti-HA mAb. d) Binding of vaccine proteins to MHCII I-E^d-transfected L cell fibroblasts. Vaccine proteins were detected by anti-HA mAb. e) Supernatants of 293E cells transfected with MIP-1α-HA or the mutated counterpart (C11S) were examined for chemotaxis. Recombinant human MIP-1α(rLD78β) was included as positive control. Chemotactic index is shown. f, g) Binding of vaccine proteins to CD11b⁺ BALB/c splenocytes.</p

Antibodies in sera after intradermal DNA vaccination.

<p>(a-g) Mice were immunized once i.d. with 25 µg DNA/EP as indicated (n = 6/group), and assayed for total IgG (a), IgG1 (b), IgG2a (c), IgG2b (d) and IgG3 (e) against PR8 in ELISA (mean+/-SEM). f) Hemagglutination-inhibition (HI) titers (mean+/-SEM) in sera. g) Sera from day 14 were assayed in a micro neutralization assay (PR8 virus). Dotted line indicates threshold for positive neutralization (50%). (h,i) Mice were immunized twice i.d. at days 0 and 50 as indicated by arrows (↑), and sera assayed in ELISA against PR8 for induced IgG1 (h) and IgG2a (i) (mean +/- SEM).</p

Targeted DNA fusion vaccines enhance immune responses after intramuscular delivery.

<p>Mice were vaccinated once i.m. with 25 µg DNA/electroporation (EP) as indicated (n = 6/group). (a-c) Serum samples were assayed for total IgG (a), IgG1 (b) and IgG2a (c) against PR8 in ELISA (mean+/-SEM). (d-f) Three weeks after vaccination, splenocytes were harvested and stimulated <i>in vitro</i> with either class II restricted HA peptides [d, (SVSSFERFEIFPK) or e, (HNTNGVTAACSHEG)], a class I restricted HA peptide [f, (IYSTVASSL)], or a control peptide (GYKDGNEYI). Frequencies of IFNγ-producing cells were evaluated by EliSpot. The control peptide did not elicit responses beyond that observed for NaCl. Horizontal lines indicate sample means.</p

Frequency of IFNγ secreting PBMC measured in ELISpot assays after stimulation with inactivated pdm09 virus.

<p>(A, D), uCD4i, (B, E), and uCD8i (C, F) epitope library. Top row represents differences among cases and controls (A, B, C), bottom row represents differences between asymptomatic and symptomatic cases (D, E, F). Inlet in figure A zooms on the range of cell frequencies presented on a linear scale (p values, Wilcoxon-Mann-Whitney test).</p

Cell frequency fold change among controls, asymptomatic and symptomatic cases after the pdm09 virus stimulation.

<p>(A) total NK cells, (B) CD107a⁺ NK cells, (C) CCR7⁺ NK cells, (D) IFNγ⁺ NK cells, (E) radar graph of specific NK cell populations characterized by the surface markers CD16 and CD56, and positive for CD107a, CCR7 or IFNγ expression. Dotted lines in graph E represent the fold change level (p values, Dunn’s Kruskal-Wallis post hoc test in D, factorial ANOVA in E).</p

Effector functions of CD4⁺ and CD8⁺ T cells.

<p>Median cell frequency fold changes of (A) CD4⁺, (B) CD8⁺ T cells after stimulation with the pdm09 virus, and median CD8⁺ T cell frequency fold change after stimulation with (C) uCD8i, (D) CMV epitopes. Dotted lines in graphs represent the fold change level (p values, Dunn’s Kruskal-Wallis post hoc test).</p

Humoral responses against A(H1N1)pdm09 virus.

<p>Humoral responses against A(H1N1)pdm09 virus in acute (one time point) and convalescent patients (3 and 32 weeks following disease onset) plotted according to the disease severity. A) HI titers in acute (n = 27) and convalescent patients (n = 19 and 15). The dotted line represents an HI titer of 40. B) Microneutralization (MN) titers in acute (n = 27) and convalescent patients (n = 19 and 15). The dotted line represents an MN titer of 80. C) Serum IgG concentration in acute (n = 25) and convalescent patients (n = 19 and 15). ○ = acute samples, ● = convalescent samples □ = single samples in the convalescent group. Disease severity is defined as mild (out-patients), moderate (hospitalized ≤ 2 days) or severe (hospitalized > 2 days). ^#p≤ 0.05 (Mann Whitney test (HI and IgG) unpaired t-test (MN)).</p

CD4⁺ T-cell cytokine responses in moderately and severely ill acute patients.

<p>PBMC from acute patients (n = 24) were stained for intracellular cytokines and the percentage of CD4⁺ T-cells secreting either single (A and B) or multiple (C and D) cytokines were measured by flow cytometry. Each symbol represents the response of one individual with bars depicting the mean and SEM percentage of CD4⁺ T-cells. ⁺p<0.05 (Student’s t test). Gating strategy is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143281#pone.0143281.s001" target="_blank">S1 Fig</a>. Disease severity is defined as moderate (hospitalized ≤ 2 days) or severe (hospitalized > 2 days).</p

T-cell responses measured by IFN-γ Elispot assay after stimulation with universal or A(H1N1)pdm09 specific peptides.

<p>A) T-cell responses against universal influenza epitopes in a subset of acute patients (n = 11). B) T-cell responses against A(H1N1)pdm09 specific epitopes in a subset of acute patients (n = 11). C) T-cell responses against universal influenza epitopes in a subset of convalescent patients (n = 5) with paired samples for 3 and 32 weeks. ○ = acute samples, ● = convalescent samples. Disease severity is defined as mild (out-patients), moderate (hospitalized ≤ 2 days) or severe (hospitalized > 2 days). The bars are plotted as median with range. The T-cell responses were directed against epitopes from internal (i = NP, M1, PA, PB and NS) or external (e = HA and NA) influenza antigens. CD4 = CD4i + CD4e and CD8 = CD8i + CD8e. *p≤ 0.05 (Wilcoxon matched-pair signed-rank test). PBMC were also shown to respond to stimulation with live influenza virus (data not shown).</p