SPOT peptide synthesis scheme.

<p>(Black circles) Blank spots. (Cyan circles) Spots from Ag1 (Nxh8— 3FTx). (Red Circles) Spots from Ag2 (Nxh 7/3/1— 3FTx). (Yellow circles) Spots from Ag3 (3Ftx). (Blue circles) Spots from Ag4 (3Ftx). (Green circles) Spots from Ag5 (PLA₂). (Highlighted spot sequences) Signal peptides, which were not considered for multiepitope gene design.</p

Multiepitope DNA string sequence coding for epitopes detected from the putative PLA₂ from <i>M</i>. <i>corallinus</i>.

<p>Cysteine codons were exchanged by serine codons to avoid the formation of disulphide bond-mediated protein multimerisation. Epitopes were separated by a six residues linker. Restriction sites (red sequences) were inserted between epitopes to allow further DNA manipulation when required.</p

RT-PCR from COS-7 cells transfected with all pSECTAG2A constructions.

<p>(M) 1kb Plus DNA ladder. (1) RT-PCR from pSECTAG2A-<i>ag1</i> transfected cells. (2) RT-PCR from pSECTAG2A-<i>ag2</i> transfected cells. (3) RT-PCR from pSECTAG2A-<i>ag3</i> transfected cells. (4) RT-PCR from pSECTAG2A-<i>ag4</i> transfected cells. (5) RT-PCR from pSECTAG2A-<i>ag5</i> transfected cells. (6) RT-PCR from pSECTAG2A (empty plasmid) transfected cells.</p

Three dimensional modelling with solvent-accessible surface area (SASA) of all five antigens described in this work.

<p>Reactive epitopes are highlighted in orange. (A) 3D model of Ag1 (Nxh8) based on the crystal structure (PDB ID: 3nds) of <i>Naja nigricollis</i> toxin alpha (GMQE: 0.80 / Seq. identity: 54.10 / Seq. similarity: 0.48). (B) 3D model of Ag2 (Nxh7/3/1) based on the NMR structure (PDB ID: 1nor) of neurotoxin II from <i>Naja naja oxiana</i> (GMQE: 0.78 / Seq. identity: 40.35 / Seq. similarity: 0.48). (C) 3D model of Ag3 (3FTx) based on the crystal structure (PDB ID: 2h8u) of Bucain, a cardiotoxin from the Malayan Krait <i>Bungarus candidus</i> (GMQE: 0.89 / Seq. identity: 57.63 / Seq. similarity: 0.51). (D) 3D model of Ag4 (3FTx) based on the crystal structure (PDB ID: 4iye) of the green mamba, <i>Dendroaspis angusticeps</i>, ρ-Da1a toxin (GMQE: 0.75 / Seq. identity: 40.00 / Seq. similarity: 0.40). (E) 3D model of Ag5 (PLA₂) based on the crystal structure (PDB ID: 1yxh) of a phospholipase A₂ from <i>Naja naja sagittifera</i> (GMQE: 0.82 / Seq. identity: 58.97 / Seq. similarity: 0.50). All images were generated using DeepView (Swiss PDB Viewer) [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004484#pntd.0004484.ref034" target="_blank">34</a>].</p

Multiepitope DNA string sequence coding for epitopes detected from the selected 3FTx from <i>M</i>. <i>corallinus</i>.

<p>Cysteine codons were exchanged by serine codons to avoid the formation disulphide bond-mediated protein multimerisation. Epitopes were separated by a six residues linker. Epitopes 1 and 2 are from Ag1, while Epitopes 3, 4 and 5 are from Ags 2, 3 and 4, respectively. Restriction sites (red sequences) were inserted between epitopes to allow further DNA manipulation when required.</p

Total IgG antibody titres and neutralising activities of different sera generated through the immunisation experiments performed.

<p>(A-i) Total IgG titres and neutralising activity of pooled sera from mice genetically immunised with the complete cDNA coding sequences of all five selected antigens. (A-ii) Total IgG titres and neutralising activity of pooled sera from mice subjected to a heterologous cDNA prime-recombinant multiepitope protein boost immunisation regimen. (A-iii) Total IgG titres and neutralising activity of sera from mice immunised with only the recombinant proteins. (B-i) Total IgG titres and neutralising activity of sera from mice genetically immunised with the multiepitope DNA strings. (B-ii) Total IgG titres and neutralising activity of sera from mice subjected to a heterologous multiepitope DNA prime-recombinant multiepitope protein boost immunisation regimen. (B-iii) Total IgG titres and neutralising activity of sera from mice immunised with only the multiepitope recombinant proteins. (C) <b><i>Positive controls</i>:</b> Total IgG titres and neutralising activities for the antielapidic antivenom and for the monospecific anti-<i>M</i>. <i>corallinus</i> horse antiserum. <b><i>Negative control</i>:</b> Total IgG titres and neutralising activities for the physiological saline solution. ELISA assays were performed in duplicate and titres were specified as the last dilution of the sample whose Abs_492nm ≥ 0.1. (*) Determination of total IgG titres of either the antielapidic antivenom or the monospecific anti-<i>M</i>. <i>corallinus</i> horse antivenom, microtitres plates were coated with 1μg of <i>Micrurus corallinus</i> venom per well.</p

Epitope mapping through the SPOT-synthesis technique.

<p>The relative density values of every single spot were determined and plotted into bar graphs. <b><i>Black bars</i></b> correspond to the relative density of each spot after incubation with both primary and secondary antibodies. <b><i>Grey bars</i></b> correspond to the relative density of each spot after incubation with only the secondary antibody (<b><i>negative control</i></b>). Positive spots are delimited by <b><i>blue rectangles</i></b> and their corresponding amino acid sequences (<b><i>highlighted in clear blue rectangles</i></b>) are aligned with its respective antigen’s <b><i>Antigenic index</i></b> (Jameson-Wolf) and <b><i>Hydrophobicity plot</i></b> (Kyte-Doolittle). Spots IDs (below bars) are the same as those described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004484#pntd.0004484.g001" target="_blank">Fig 1</a>.</p

Western-blot analysis of extracts from COS-7 cells transfected with pSECTAG2A constructs.

<p>(M) Ponceau-stained low molecular marker (GE Healthcare) transferred to the nitrocellulose membrane. (1) Protein extract from COS-7 cells transfected with pSECTAG2A-<i>ag1</i>. (2) Protein extract from COS-7 cells transfected with pSECTAG2A-<i>ag2</i>. (3) Protein extract from COS-7 cells transfected with pSECTAG2A-<i>ag3</i>. (4) Protein extract from COS-7 cells transfected with pSECTAG2A-<i>ag4</i>. (5) Protein extract from COS-7 cells transfected with pSECTAG2A-<i>ag5</i>. An anti-<i>Micrurus corallinus</i> monospecific horse antiserum was used as primary antibody.</p