FR flagellin activates the innate immune response in FljB neutralizing mice.

<p>CCL20 levels (pg/ml) in sera of FljB-hyperimmunized mice after inoculation with 4.8 µg of FR, FljB or <i>Vibrio vulnificus</i> (Vvul or FlaB) flagellins. Positive control, 71 pg of CCL20. Negative control, two naïve-pooled mice. *, differences in the CCL20 amounts induced by FR and Vvul were statistically significant with respect to FljB, and the higher CCL20 expression induced by FR with respect to Vvul was also statistically significant at the p≤0.05 level.</p

Cross-reaction of the anti-MA and –STF flagellin antisera with these flagellins.

<p>Cross-reactivity of anti-FljB (A), anti-F (B) and anti-FR (C) antiserum with MA (F, FR) and STF (FljB, FliC) flagellins (200 ng/well). IgG antibody binding was measured by ELISA. A) *, statistical significance differences, at the p≤0.05 level, between all FljB/FliC STF flagellins and F and FR MA flagellins at each dilution point. B) *, statistical significance differences, at the p≤0.05 level, between F and other flagellins at each dilution point. ◊,FljB_Δ180–400. ○, FljB_Δ220–320. □,FljB. ???, FliC. ▴, FR. •, F.</p

<i>In vitro</i> cytokine production in Caco-2 cells stimulated by MA (F, FR) and STF (FljB) flagellins.

<p>A) Human IL8 secretion by Caco-2 cells stimulated with FljB (black bars), F (grey bars) and FR (white bars) flagellins. B) Human CCL2 secretion by Caco-2 cells stimulated with FljB, F and FR at 1000 ng/ml. IL8 and CCL2 levels are shown in pg/ml. Differences in cytokine expression were not statistically significant at the p≤0.05 level. PBS, phosphate-buffered saline.</p

Neutralizing anti-flagellin antisera inoculated by the retro-orbital route do not block heterologous flagellins.

<p>CCL20 levels (pg/ml) in sera of mice inoculated by the retro-orbital route with hyperimmune anti-flagellin antiserum 1 h before injection with FljB or FR. A range of amounts of hyperimmune antiserum from 1 to 100 µl were used for retro-orbital inoculation. Mice were stimulated 1 h after serum inoculation with 50 ng of FljB or FR. <b>Black bars</b>, mice inoculated with anti-FR antiserum and further stimulated with FljB. <b>Grey bars</b>, mice inoculated with anti-FljB antiserum and further stimulated with FljB. <b>White bars</b>, mice inoculated with anti-FljB antiserum and further stimulated with FR. Differences in cytokine expression were not statistically significant at the p≤0.05 level.</p

Comparison of the flagellin amino acid sequences of MA (FR and F) and STF (FljB).

<p>Boxed areas indicate putative TLR5 binding domains. Grey sequences show hypervariable domains. *,denotes identical amino acids. :, denotes related amino acids.</p

Antibody mediated cross-neutralization of MA and STF flagellins measured by the IL8 secretion by Caco-2 cells.

<p>Caco-2 cells were incubated with a premix of 3 µl (grey bars) or 0 µl (black bars) of anti-flagellin hyperimmune serum with MA or STF flagellins. The flagellins (abscissa) indicated were pre-incubated with anti-FljB antiserum (A), anti-FR antiserum (B), and anti-F antiserum (C). (D) Panel showing the IgG titration profile of anti-FljB (▵) and anti-FR (▴).*, statistical significance differences at the p≤0.05 level.</p

Production of the PCV2-Cap protein in <i>Sf9</i> cells grown in suspension and infected by a conventional TB(-) or by a TB (+) baculovirus expressing the ORF2 from porcine circovirus gene.

<p>A) Coomassie blue staining of SDS-PAGE gels resolving protein extracts obtained from cells infected with TB(-) or TB(+) baculoviruses and collected from 0 to 120 hpi. The same samples were submitted to a Western blot using a monoclonal antibody against the PCV2-Cap antigen. B) Infected cells viability at different hpi with TB(-) and TB(+) baculoviruses expressing the PCV2-Cap protein, analyzed by Trypan blue staining. This panel represents the mean values of three different experiments. C) Calculation of PCV2-Cap protein productivity in <i>Sf9</i> cells infected by TB(-) and TB(+) baculoviruses at the optimal production time in each case (24 hpi for TB(-) and 48 hpi for the TB(+) baculovirus).</p

Schematic representation of the two constructs used to generate recombinant baculoviruses expressing the recombinant proteins RHDV-VP60 and PCV2-Cap.

<p>A) Expression cassette used to express the recombinant proteins under the control of the <i>polh</i> promoter in a conventional TB(-) baculovirus. B) Expression cassette used to express the recombinant proteins in a TB(+) baculovirus containing all genetic regulatory elements, including the transactivation elements encoded by <i>Ac-ie-01</i> sequence, the enhancer sequence <i>hr1</i>, and the combination of promoters <i>p6</i>.<i>9</i> and <i>p10</i>.</p

Production of the RHDV-VP60 protein in <i>Sf9</i> cells grown in suspension and infected by a conventional TB(-) or by a TB (+) baculovirus expressing the VP1 and VP2 genes from RHDV genogroup 1.

<p>A) Coomassie blue staining of SDS-PAGE gels resolving protein extracts obtained from infected cells with TB(-) or TB(+) baculoviruses and collected from 0 to 96 hpi. The same samples were submitted to a Western blot using a monoclonal antibody against the RHDV-VP60 antigen. B) Infected cells viability at different hpi with TB(-) and TB(+) baculoviruses expressing the RHDV-VP60 protein, analyzed by Trypan blue staining. This panel represents the mean values of three different experiments. C) Calculation of VP60 protein productivity in <i>Sf9</i> cells infected by TB(-) and TB(+) baculoviruses at the optimal production time in each case (72 hpi for TB(-) and 96 hpi for the TB(+) baculovirus).</p

VLPs formed after infection of <i>Sf9</i> cells with a conventional TB(-) or a TB (+) baculovirus expressing the ORF2 from porcine circovirus.

<p>Extracts from infected cells at the optimal production times with each baculovirus were processed for VLP purification. The protein patterns of the purified VLPs obtained with each baculovirus are shown in the inserts (Coomassie blue stained). Samples were observed by Electron microscopy using negative staining. The figure shows the VLPs at two magnifications. VLPs obtained with the two baculoviruses presented identical sizes and shapes but were more abundant when produced by the TB(+) virus. The micrographs are representative of the fields analyzed.</p