Swine T-Cells and Specific Antibodies Evoked by Peptide Dendrimers Displaying Different FMDV T-Cell Epitopes

Figures

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  Figure 1. Time course of specific IgG antibodies elicited by B2T constructions combining different T-cell epitopes. Each point represents the mean of sera from the indicated group at different days pi (n = 4; except for B2 and PBS groups in which n = 2) ± SD. Data on day 121 pi corresponds to two pigs per group. Arrow points the day of peptide boost.

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  Figure 2. VNTs are influenced by the presence and orientation of the T-cell epitopes in the dendrimer. Sera samples were collected after one dose of each dendrimeric peptide (pre-boost) panels (A–C) and after peptide boost [panels (D–F). Each point represents the VNT of one single pig and the mean of each group is shown with a horizontal bar. Dotted line depicts the detection limit.

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  Figure 3. Sera from pigs immunized with dendrimers can neutralize a panel of different FMDVs type O topotypes. Sera recovered from animals immunized with the different peptides after the first (day 21 pi or pre-boost) (A) and the second immunization (day 28 pi or post-boost) (B), were tested to neutralize a panel of different type O FMDVs. Individual columns represent the mean of each group (n = 4) ± SD. Values are expressed as the reciprocal log10 of the last serum dilution that neutralized 100 TCID50 of each FMDV. Statistically significant differences are indicated by one (*) or two (**) asterisks for p < 0.05 or p < 0.005, respectively.

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  Figure 4. IgG1 and IgG2 profiles elicited by B2T constructions combining different T-cell epitopes. Anti-B peptide IgG1 and IgG2 profiles in sera from pigs collected at 21 days pi with: B2T-3A (A), B2T-3D (B), B2TT-3A3D (C), and B2TT-3D3A (D). Each point represents the value of a duplicate of an individual animal. Endpoint titers are expressed as the reciprocal of serum dilution (log10) giving the absorbance recorded in control wells (sera collected at day 0) + 2 x SD. Each symbol represents the IgG1 and IgG2 titers (X and Y values respectively) for an individual pig. In the lower right quadrant, the percentage of pigs with titers above 3 log10 is shown. No individual spontaneous reactivity was observed in the titers determined at day 0.

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  Figure 5. T-cell responses elicited by B2TT-3A3D and B2TT-3D3A unveil 3D peptide as an immunodominant T-cell epitope. PBMCs were isolated from individual animals of each group, at different days pi. Cells were in vitro stimulated for 48 h with dendrimers and the frequency of IFN-γ producing cells was determined by ELISPOT. (A) Frequency of IFN-γ secreting cells in the groups of pigs immunized as indicated, in response to the homologous dendrimer and the different constructions indicated. Each line corresponds to an individual animal and arrows show the day of the boost. (B) IFN-γ production was stimuli-specific and sequence-dependent. The values corresponded to PBMC recovered at day 183 and are presented as IFN-γ spots per 106 cells). Each column represents the mean (n = 4) ± SD.

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  Figure 6. CD4+ and CD8β+ specific T-cell responses elicited by different dendrimeric peptides in PBMCs from immunized animals. Intracellular cytokine staining was performed in overnight dendrimer stimulated PBMCs from immunized animals at day 21 pi. (A) PBMCs were stained and analyzed for surface CD4 or CD8β expression (representative plot from pig #49 is shown). (B) CD4+ or CD8β+ populations from each pig were gated and further analyzed for IFN-γ expression. IFN-γ-producing cells are shown as percent of total CD4+ or CD8β+ T cells.

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  Figure 7. Depletion of CD4+ T-cells abolishes IFN-γ production of peptide-stimulated PBMCs. Cells from a B2T-3D immunized pig (pig #77) were magnetically sorted using an anti-CD4 mAb. (A) FACS analysis was performed to determine the CD4 staining quality of initial pre-sorted PBMCs and each purified fraction after MACS fractioning. (B) Pre-sorted PBMCs and fractioned positive fraction (CD4+), negative fraction (CD4−) and reconstituted fraction (CD4+/CD4−) were plated in triplicates and stimulated either with B2T-3D or T-3D peptides for 48 h and IFN-γ production was determined by ELISPOT. The values are presented as IFN-γ spots per 106 cells (spots from mock-stimulated cells are subtracted). Each column represents the mean of a triplicate ± SD. Statistically significant differences between the positive fraction (CD4+) and the rest of the groups are indicated by three asterisks (***) for p < 0.001.

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  Figure 8. CD4+2E3- T-cells are the major subset producing IFN-γ. Intracellular cytokine staining was performed in overnight dendrimer stimulated-PBMCs isolated from immunized pigs at day 14 pi. (A) PBMCs were stained and analyzed for surface CD4 and 2E3 expression (representative plot from pig #49 is shown). (B) CD4+ 2E3+ and CD4+2E3− populations were gated and further analyzed for IFN-γ expression. IFN-γ-producing cells are shown as percent of total CD4+2E3+ or CD4+2E3− T cells.

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  Figure 9. Multifunctionality of CD4 T cells from immunized pigs upon stimulation with homologous dendrimer. Intracellular cytokine staining of PBMCs was performed following overnight in vitro stimulation with homologous dendrimers. (A) CD4 staining and expression was analyzed (representative plot of animal #49 is shown). (B) CD4+ T cells were gated and analyzed for production of a single cytokine (IFN-γ−TNF-α+ and IFN-γ+TNF-α− subsets, in upper left and bottom right quadrants) or of both cytokines (IFN-γ+TNF-α+ subset in upper right quadrant). Cytokine-producing cells are shown as percent of total CD4+ T cells.