pCMV-sHAPQ does not protect against lethal ASFV challenge.

<p>(A) Surviving kinetics of pCMV, pCMV-pCMV-PQ and pCMV-sHAPQ immunized pigs after lethal challenge with ASFV (10⁴ UHA of the E75 isolate). (B) Viremia kinetics of these same individuals after ASFV challenge (days 3, 5 and 7 post infection). Results are represented as the logarithm of HAU₅₀/ml serum (mean and standard deviation from each group are shown).</p

pCMV-UbsHAPQ protects against lethal ASFV challenge.

<p>(A) PBMCs from pigs immunized twice (2x) or 4 times (4x) with pCMV-UbsHAPQ or with pCMV, were <i>in vitro</i> stimulated with media (negative control), with the E75 ASFV isolate (10⁵ HAU₅₀/ml), with a mix of the p54, p30 and HA ASFV proteins (6 μg/ml of each one) or with each one of these proteins individually. Values shown for each immunization group correspond to the average number of specific IFNγ-secretory cells detected per million PBMCs. Standard deviations found within each group are also represented. (B) Immunized pigs were challenged with a lethal dose of ASFV (10⁴ UHA of the E75 isolate) and survival record were plotted. Groups immunized with pCMV-UbsHAPQ showed statistically significant differences (*) when compared with the control group (p = 0.0068).</p

pCMV-sHAPQ induces specific T-cell responses in pigs.

<p>PBMCs obtained before ASFV challenge from pigs immunized with pCMV, pCMV-PQ or pCMV-sHAPQ (immunized three or four times), were <i>in vitro</i> stimulated with media (negative control), with the E75 ASFV isolate (10⁵ HAU₅₀/ml), with a mix of the p54, p30 and HA ASFV proteins (6 μg/ml of each one) or with each one of these proteins individually. Values shown correspond to the average number of IFNγ-secretory cells detected per million of PBMCs. Standard deviations found within each group are also represented.</p

Immunization with VIN1-HA1 partially protects pigs <i>in vivo</i> against heterologous challenge with pH1N1.

<p>Influenza viral RNA quantification in BAL was performed by RT-qPCR at 6 dpi, corresponding to necropsy day. Bars indicated positive samples in genome equivalent copies (GEC) per ml of BAL. The detection limit in the assay was 3 log₁₀ GEC/ml.</p

Amino acid sequences from the peptides used for immunization compared to the homologue sequence of the HA receptor recognition domain of the challenging strain (pH1N1) and the HA purified proteins used for the serologic tests.

<p>In bold type, the amionacids differences between sequences are represented. Differences between the pH1N1 virus and the H1-peptide (NF-34) in homologous positions within the HA receptor recognition domain are marked. Aminoacid differences in the two H5-HK derived peptides (LE-35.1/2) are also represented.</p

Surviving pigs show a rapid recovery from the ASFV-provoked leukopenia and a dramatic expansion of CD8⁺ T-cells.

<p>(A) Total nucleated-cell counting using whole blood samples taken at different days after ASFV challenge. (B) Number of CD8⁺ T-cells found at different times post-infection from these same blood samples after PBMC separation and CD8⁺ surface specific staining using the specific anti-CD8 monoclonal antibody. Values represented in both panels correspond to the average number of cell per mm³ of pig blood. Standard deviation shown corresponds to those found within the pCMV-control group (dashed line) and those found for the three surviving pigs, pre-immunized pCMV-UbsHAPQ (solid line). No significant differences were observed in the cell counts (nor in the total, nor the specific CD8⁺ T-cell counts), between non-surviving pigs, independent of the DNA plasmid used.</p

pCMV-sHAPQ induces specific antibody responses in pigs.

<p>14 pigs were divided into three groups receiving either: the pCMV-sHAPQ plasmid (6 pigs), the pCMV-PQ plasmid (4 pigs) or the pCMV empty plasmid as controls for the assay (4 pigs). Four pigs from each group were immunized three times and two extra pigs from the pCMV-sHAPQ immunized-group received a fourth dose of this same plasmid. (A) Sera collected 15 days after each DNA vaccine administration were used to follow by ELISA the kinetics of the specific anti-p30 antibodies induced after DNA immunization. (B) Sera collected 15 days after the last DNA vaccination was used to ELISA-titrate the anti-p30 antibodies induced. Data shown correspond to average O.D values and standard deviations obtained per each immunization group. (C) Confirmatory Western-blot using ASFV infected cell extracts as antigen. Immunoreactive bands correspond to the specific recognition of the immunodominant p30 protein (arrow) and to the diverse p54 isoforms (arrow head) found in these extracts, ranging the last ones between 22 and 27 KD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040942#pone.0040942-Rodriguez4" target="_blank">[65]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040942#pone.0040942-Alcaraz2" target="_blank">[66]</a>. Figure shown corresponds to the results obtained with a representative serum (1∶100 dilution) obtained 15 days after the third immunization with pCMV (a), pCMV-PQ (b) or pCMV-sHAPQ (c). The anti-p30 monoclonal antibody (d) and the specific polyclonal anti-p54 antibody (e) confirm the specificity of the reactions.</p

Identification of protective SLAI restricted 9-mer T-cell peptides.

<p>PBMCs from surviving pigs, pre-immunized with pCMV-UbsHAPQ (obtained at day 25 after ASFV lethal challenge), were <i>in vitro</i> stimulated with 53 9-mer peptides predicted as potential CTL epitopes. Data represented in grey boxes corresponds to the specific responses found for the two individual sHA epitopes: F3, and A6. Data represented in open boxes corresponds to the values obtained after <i>in vitro</i> stimulation with peptide-coated autologous SLAI⁺/SLAII⁻ skin fibroblasts.</p

VIN1-peptide cocktail acts as a potent immunogen and the elicited sera reacts with different hemagglutinin subtypes and against VIN1-peptides.

<p>(A) Sera from individuals were obtained 15 days after each immunization and were tested for binding to a mixture of the VIN1-peptides (serum dilution 1∶100) by ELISA. (B) Sera from individual pigs were obtained 15 days after the third immunization and were serially diluted and tested for binding to each single peptide by ELISA and (C) Sera described in B) were tested for binding to H5- or H1- recombinant hemagglutinin by ELISA.</p

VIN1-sera recognize distinct viral subtypes.

<p>Indirect immunofluorescence of either H5N1, SwH3N2 or SwH1N1-infected MDCKs cells at 16 hpi using as primary antibody the serum from one pig (representative of the group), immunized three times with VIN1 peptides.</p