Structure analysis of A22-wt and A22-H2093C empty capsids.

<p>(a) The electron density map for A22-wt (left panel) presents the expected histidine side chains for VP2 residue 93 whereas the map for A22-H2093C (right panel) shows the disulphide density at the two-fold axis between pentamers. (b) Side by side ribbon drawings of the recombinant empty capsid protomers and beneath the individual VP1, 2 and 4 proteins. There was no difference between the A22-wt and A22-H2093C capsids on the exterior surface; significant disorder observed on the interior of the A22-H2093C capsids is shown as thickened lines and corresponds to the N terminus of VP1 (residues 1 to 12) and VP4 (residues 15 to 38 and 65 to 80) located near the 3-fold axes. To a lesser extent residues 38–41 in VP2 were also disordered.</p

Rational design to produce stable empty capsids of FMDV A22.

<p>(a) The crystal structure of FMDV serotype A22 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003255#ppat.1003255-Curry1" target="_blank">[2]</a> was analysed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003255#s4" target="_blank">Materials and Methods</a>. This led to the prediction and modelling of a potential disulphide bond between pentamers by mutation to cysteine of VP2 histidine residue 93, located on the α-helix at the two-fold symmetry axis. (b) Western blot analyses of A22-wt and A22-H2093C empty capsids, expressed from recombinant vaccinia viruses and sedimented through 15–45% sucrose gradients. Each gradient was fractionated from the bottom. FMDV capsid proteins were detected using an anti-FMDV strain A22 polyclonal antibody raised in guinea pigs. Crude extracts were left untreated, heated to 56°C for 2 h or acidified to pH 5.2 for 15 min. Following both heat and acid treatments A22-H2093C capsids remained intact and migrated to fraction 4, as before treatment, whereas A22-wt capsids fractured, remaining near the top of the gradient in fraction 10.</p

Virus neutralising antibody titres pre-challenge.

<p>The group mean virus neutralising antibody titres (Log2) for animals inoculated with A22-wt (black line) or A22-H2093C (grey line) are shown from week 0 to week 34. All animals were vaccinated on week 0 and week 3. Blood samples were taken at weekly intervals until week 6 and then on week 22 and finally on week 34 when challenge with live virus was carried out. Error bars represent the standard error of the mean.</p

Virus genome detection.

<p>The quantity of viral genome (Log10/ml) was determined by quantitative real-time PCR in serum samples from each animal on days 0–7 and 9 post-challenge. Individual data for animals vaccinated with (a) A22-wt, (b) A22-H2093C and for non-vaccinated control animals (c). Clinical signs were detected in two of the four animals that had received wild-type empty capsid (FMD140 and FMD143) and in one of the four animals immunised with mutated capsids (FMD144). Both non-vaccinated control animals showed overt clinical signs.</p

Sucrose gradient purification and SDS PAGE analysis of A22-wt and A22-H2093C recombinant capsids.

<p>The extracts from either 2×2125 cm² roller flasks of HEK293T cells co-infected with recombinant vaccinia viruses, vA22-wt+vTF7-3 or vA22-H2093C+vTF7-3 (a) or from 300 ml culture of Sf9 cells infected with recombinant baculovirus virus, bA22-wt or bA22-H2093C (b) were each loaded onto a 36 ml 15–45% sucrose gradient and centrifuged at 21,000 rpm for 22 h at 12°C (SW32 rotor, Beckman). Bands were visualised by illumination of the gradients from the top with a halogen fibre optic light source (Schott). Fractionation of the lower halves of the gradients was in 13 aliquots of 1 ml (fractions 1–13) and 3 aliquots of 2 ml (fractions 14–16) collected from the bottom: 10 µl each from fractions 2 to 15 were analysed on 4–12% acrylamide Novex NuPAGE gels (Invitrogen).</p