Fig 1.
SPOT peptide synthesis scheme.
(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.
Fig 2.
Epitope mapping through the SPOT-synthesis technique.
The relative density values of every single spot were determined and plotted into bar graphs. Black bars correspond to the relative density of each spot after incubation with both primary and secondary antibodies. Grey bars correspond to the relative density of each spot after incubation with only the secondary antibody (negative control). Positive spots are delimited by blue rectangles and their corresponding amino acid sequences (highlighted in clear blue rectangles) are aligned with its respective antigen’s Antigenic index (Jameson-Wolf) and Hydrophobicity plot (Kyte-Doolittle). Spots IDs (below bars) are the same as those described in Fig 1.
Fig 3.
Three dimensional modelling with solvent-accessible surface area (SASA) of all five antigens described in this work.
Reactive epitopes are highlighted in orange. (A) 3D model of Ag1 (Nxh8) based on the crystal structure (PDB ID: 3nds) of Naja nigricollis 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 Naja naja oxiana (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 Bungarus candidus (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, Dendroaspis angusticeps, 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 Naja naja sagittifera (GMQE: 0.82 / Seq. identity: 58.97 / Seq. similarity: 0.50). All images were generated using DeepView (Swiss PDB Viewer) [34].
Fig 4.
Multiepitope DNA string sequence coding for epitopes detected from the selected 3FTx from M. corallinus.
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.
Fig 5.
Multiepitope DNA string sequence coding for epitopes detected from the putative PLA2 from M. corallinus.
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.
Fig 6.
Total IgG antibody titres and neutralising activities of different sera generated through the immunisation experiments performed.
(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) Positive controls: Total IgG titres and neutralising activities for the antielapidic antivenom and for the monospecific anti-M. corallinus horse antiserum. Negative control: 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 Abs492nm ≥ 0.1. (*) Determination of total IgG titres of either the antielapidic antivenom or the monospecific anti-M. corallinus horse antivenom, microtitres plates were coated with 1μg of Micrurus corallinus venom per well.
Fig 7.
RT-PCR from COS-7 cells transfected with all pSECTAG2A constructions.
(M) 1kb Plus DNA ladder. (1) RT-PCR from pSECTAG2A-ag1 transfected cells. (2) RT-PCR from pSECTAG2A-ag2 transfected cells. (3) RT-PCR from pSECTAG2A-ag3 transfected cells. (4) RT-PCR from pSECTAG2A-ag4 transfected cells. (5) RT-PCR from pSECTAG2A-ag5 transfected cells. (6) RT-PCR from pSECTAG2A (empty plasmid) transfected cells.
Fig 8.
Western-blot analysis of extracts from COS-7 cells transfected with pSECTAG2A constructs.
(M) Ponceau-stained low molecular marker (GE Healthcare) transferred to the nitrocellulose membrane. (1) Protein extract from COS-7 cells transfected with pSECTAG2A-ag1. (2) Protein extract from COS-7 cells transfected with pSECTAG2A-ag2. (3) Protein extract from COS-7 cells transfected with pSECTAG2A-ag3. (4) Protein extract from COS-7 cells transfected with pSECTAG2A-ag4. (5) Protein extract from COS-7 cells transfected with pSECTAG2A-ag5. An anti-Micrurus corallinus monospecific horse antiserum was used as primary antibody.