Table 1.
Amino acid sequences from the HA-epitopes used in the VC4-flagellin construct; aa positions from their respective consensus IV are also indicated.
Table 2.
Schematic outline of Experiments I and II.
Table 3.
Sampling schedule for experiments I and II.
Fig 1.
Viral RNA load in nasal swabs by RT-qPCR.
(A) Mean of genomic equivalent copies (GEC) per mL obtained from nasal swabs from seronegative pigs (Experiment I) collected at 0, 5 and 7 dpi (B) and from nasal swabs from seropositive animals (Experiment II) collected at 0, 5, 7, 11 and 14 dpi. Group A (unvaccinated animals) is represented by grey bars and Group B (pCDNA3.1(+)-VC-4-flagellin vaccinated group) by black bars. Dpi, days post-inoculation. Dashed lines indicate the detection limit of the assays: 1.24 log10GEC/mL. Error bars indicate the mean ± SEM.
Fig 2.
Serum antibody HA-specific IgG titers detected in sera and BALFs samples by ELISA test.
Mean of serum IgG antibody levels detected at 0, 20 PVD, 35 PVD, and 7 DPI of Groups A and B (A) against HA from A/California/04/09(H1N1)pdm09, and (B) against HA from A/Aichi/2/1968(H3N2) are represented. Mean of BALFs IgG antibody levels detected in pigs sacrificed at 7 and 14 dpi of Groups A and B (C) against HA from A/California/04/09(H1N1)pdm09, and (D) against HA from A/Aichi/2/1968(H3N2). Grey circles/bars refer to group A (unvaccinated group), and black squares/bars refer to group B (pCDNA3.1(+)-VC4-flagellin vaccinated group). OD, optical density. PVD, post-vaccination days and DPI, days post-inoculation. Error bars indicate the mean ± SEM. Statistically significant differences between vaccine treatment groups (P value <0.05) are marked with *: P<0.05, **: P<0.01.
Fig 3.
HI activity against pH1N1 from seronegative pigs (Experiment I) and against H3N2 from seropositive pigs (Experiment II).
HI titers obtained with sera from unvaccinated (Group A) and vaccinated (Group B) pigs against (A) the pH1N1 from SIV-seronegative pigs (Experiment I) and (B) the SwH3N2 from SIV-seropositive pigs (Experiment II). Grey circles refer to group A (unvaccinated group) and black squares depict group B (pCDNA3.1(+)-VC-4-flagellin vaccinated group). HI, hemagglutination inhibition. DPI, days post-inoculation. Dashed lines indicate the threshold to obtain a “seroprotective” titer (HI≥40) which has been used as a criterion of immunogenicity in a vaccine. Error bars indicate the mean ± SEM and statistically significant differences between vaccine treatment groups are marked with **: P<0.01.
Table 4.
Pathological microscopic score for all the animals from Experiment II based on BIP-compatible lesions.
BIP was assessed by a semi-quantitative scoring (0–3, indicating lack of, mild, moderate or severe pneumonia lesions, respectively).
Fig 4.
Antibody HA-specific IgA titers detected in BALFs samples from Experiment II.
Mean of BALFs IgA antibody levels detected in pigs sacrificed at 7 and 14 dpi of Groups A and B (A) against HA from A/California/04/09(H1N1)pdm09, and (B) against HA from A/Aichi/2/1968(H3N2). Grey bars refer to group A (unvaccinated group), and black bars refer to group B (pCDNA3.1(+)-VC4-flagellin vaccinated group). OD, optical density. PVD, post-vaccination days and DPI, days post-inoculation. Error bars indicate the mean ± SEM.
Fig 5.
Seroneutralization (SN) titers detected in BALF samples from Experiment II by seroneutralization assay.
Mean of seroneutralization titers detected at 7 and 14 dpi of Groups A and B against the A/swine/Spain/003/2010 H3N2 IV challenged virus. Grey bars refer to group A (unvaccinated group) and black bars depict group B (pCDNA3.1(+)-VC-4-flagellin vaccinated group). SN, seroneutralization. DPI, days post-inoculation. Error bars indicate the mean ± SEM.
Fig 6.
Flow cytometry from PBMCs isolated at 35 PVD (Experiment II).
Mean of the percentages of T cells CD4, CD8 and CD4-CD8 DP from unvaccinated (Group A) and vaccinated (Group B). Grey bars refer to group A (unvaccinated group) and black bars depict group B (pCDNA3.1(+)-VC-4-flagellin vaccinated group). Error bars indicate the mean ± SEM and statistically significant differences between vaccine treatment groups are marked with ***: P<0.001.