Structure of Fab408 and homology models of Fab403 and Fab410.

<p><b>Left</b>: Overall views of (<b>A</b>) Fab408, with a light blue L chain and yellow H chain; (<b>B</b>) Fab403, with a violet L chain and orange H chain; (<b>C</b>) Fab410, with a dark blue L chain and salmon H chain, displayed with their N-terminal variable region on top and C-terminal constant region at bottom. CDR-L1, CDR-L2, CDR-L3 are displayed in blue, light green, dark green and CDR-H1, CDR-H2, CDR-H3 in red, orange, purple. The extended CDR-H3 in Fab408 is clearly visible. <b>Center</b>: Close-up views of the combining sites, displayed as molecular surfaces with the CDRs colored as on the left panels and labeled. Differences in the sizes of the CDRs and the shapes of the combining surfaces (pocket in Fab408 <i>versus</i> extended surfaces in Fab403 and Fab410) are evident. <b>Right</b>: Distribution of the electrostatic potentials mapped on the Fab molecular surfaces at -3 kT/e (red) to +3 kT/e (blue) (same orientation as on the central panels). Note the electronegative combining site in Fab408 <i>versus</i> the electropositive combining sites in Fab403 (centered around the L-chain CDRs) and Fab410 (centered around the H-chain CDRs).</p

Sequences and numbering of the three anti-EeAChE Fabs.

<p>The light (L, top) and heavy (H, bottom) chains are displayed. The residue numbering and secondary structure elements displayed above the alignment are those of Fab408. Conserved residues are shown on a <i>black</i> background and non-conserved residues on a <i>white</i> background. The CDRs, defined according to <a href="http://www.bioinf.org.uk/abs/" target="_blank"><u>http://www.bioinf.org.uk/abs/</u></a> (cf. Figure S1 in File S1), are highlighted as grey boxes and labeled.</p

Natural scrapie isolates transmission results to TgOvPrP4 ovine transgenic mice.

(1)<p>Animals were inoculated intracerebrally with 2 mg brain tissue.</p>(2)<p>Genotype of sheep or goat (amino acids 136, 141, 154, and 171).</p>(Goat)<p>Goat isolate.</p>(α)<p>Isolates detected in the flock α.</p>(β)<p>Isolates detected in the flock β.</p><p>Natural inoculums were divided into 4 different groups, all originating from active surveillance of TSEs in small ruminants. Group 1a: atypical scrapie cases with <i>prnp</i> alleles associated with increased susceptibility to atypical scrapie (AF₁₄₁RQ or AHQ). Group 1b: atypical scrapie cases without <i>prnp</i> alleles associated with increased susceptibility to atypical scrapie. Group 2: classical usual scrapie cases. Group 3: CH1641-like scrapie cases. Detection of PrP^res/PrP^sc was performed by Western blot or IHC analyses.</p

Effects of variable PrP^c expression levels in TgOvPrP4 mice on TSEs transmission.

<p>Survival periods of PrP^sc positive TgOvPrP4 mice are shown according the level of PrP^c expression (∼0.25, 1.5 or 6× the PrP^c level expressed in a sheep brain control) and to the inoculums; Group 1a: atypical scrapie isolates carrying the AF₁₄₁RQ or AHQ alleles, Group 1b: atypical scrapie isolates without the AF₁₄₁RQ or AHQ alleles, Group 2: usual classical scrapie isolates, Group 3: CH1641-like scrapie isolates, Group 4: experimental isolates (SSBP1, CH1641, BSE^OVINE), Group 5: mouse-adapted strains (BSE, C506M3, 87V). (A) Transmission results. (B) Kaplan-Meier survival curves of PrP^res/PrP^sc positive mice expressing ∼1.5 (blue line) or ∼6× (red line) the PrP^c level expressed in a sheep brain control for each of the 6 different groups. p-values represent log-rank differences in cumulative survival between mice expressing ∼1.5 and ∼6× the PrP^c level expressed in a sheep brain control.</p

Experimental TSEs transmission results to TgOvPrP4 ovine transgenic mice.

(1)<p>Animals were inoculated intracerebrally with 2 mg brain tissue.</p>(2)<p>Genotype of sheep or goat (amino acids 136, 141, 154, and 171).</p><p>Experimental inoculums were divided into 2 different groups. Group 3: small ruminants intracerebrally inoculated with various TSEs. Group 4: experimental strains initially derived from wild-type mice. Detection of PrP^res/PrP^sc was performed by Western blot or IHC analyses.</p

Physical and functional characterization of the purified Fabs (typical experiments).

<p>(<b>A</b>) MALDI-TOF mass spectrometry profiles, showing both the di-charged and mono-charged entities. Note the satisfactory homogeneity in mass of the latter. (<b>B</b>) Electrophoresis patterns obtained by SDS-PAGE <i>in </i><i>non-reducing </i><i>conditions</i> (12.5% PhastGel, left) and native-PAGE (7.5% PhastGel, center) with migration from the cathode (top) toward the anode (bottom), and by isoelectric focusing (pI 3-9 PhastGel, right). The three Fabs are more homogenous in mass than in charge, a feature that likely result from variations in the C-terminus generated by papaine cleavage of the CH chain; yet, all isoforms bind EeAChE equally, as verified by a native-PAGE mobility shift assay (not shown). The neutral average pI of Fab408 and cationic average pIs of Fab403 and Fab410 are evident. (<b>C</b>) Inhibition of native EeAChE (closed symbols, full lines) and N-deglycosylated EeAChE (open symbols, dashed lines) by the three Fabs. The higher residual activity recorded at saturating concentration of Fab408, compared with Fab403 and Fab410, is evident. Removal of the six N-linked glycan chains of EeAChE results in 6.5-fold higher affinity for Fab403 but unaltered residual activity at saturating Fab403 concentration. Data points correspond to the average values of duplicates or mean values of triplicates. Non-linear fitting of the data points used a sigmoidal equation. Mean values of the mass, pI, IC50 and residual activity of each Fab are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077226#pone-0077226-t001" target="_blank">Table 1</a>.</p

Statistical analysis of PrP^c levels influencing survival period of TgOvPrP4 mice inoculated with atypical scrapie.

*<p>PrP^c level set as the baseline category.</p><p>Hazard ratios calculated from model used to identify predictors of survival for mice inoculated with atypical scrapie isolates and illustrating the effect associated with PrP^c sera levels (the mean of each subpopulation were chosen to illustrate the effect of the PrP^c sera level).</p

Levels of transgene expression in transgenic (TgOvPrP4) mice expressing ovine cellular prion protein (PrP^c).

<p>(A) Quantification of seric and cerebral PrP^c levels by ELISA in 47 TgOvPrP4 mice. (B) Repeated measures of PrP^c in the serum for 4 weeks (day D0, D7, D14, D21, D28) in 47 TgOvPrP4 mice representing the 3 subpopulations with distinct PrP^c levels in their brain. Each line represents the average and the standard deviation of 17, 16 and 14 individual mice expressing, respectively, ∼0.25, 1.5 or 6× the PrP^c level expressed in a sheep brain control.</p

Western blot profiles of PrP^res in TgOvPrP4 mice infected with atypical scrapie.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007300#s2" target="_blank">Results</a> are shown before (A) and after (B) PNGase deglycosylation using monoclonal antibodies 4F2 (lanes 1, 5), P4 (lanes 2, 6) (N-terminal), Sha 31 (lanes 4, 8)(core) and 99/97.6.1 (lanes 3, 7)(C-terminal). Western blot profiles with Sha 31 antibody are shown with the curve of chemiluminescence measured along the lanes. Apparent molecular weights were means from 30 mice before PNGase deglycosylation and from 7 mice after PNGase deglycosylation.</p

Schematic representation of PrP^res fragments identified in atypical scrapie.

<p>(A) Diagram illustrating PrP^res A, B and C sequences in atypical scrapie, with location of epitopes recognised by monoclonal antibodies used during the study and estimated cleavages of PrP^res fragments. (B) Diagram of Western blot profiles of PrP^res detected using Sha 31 monoclonal antibody before (1) and after (2) PNGase deglycosylation.</p