A Heterologous Multiepitope DNA Prime/Recombinant Protein Boost Immunisation Strategy for the Development of an Antiserum against Micrurus corallinus (Coral Snake) Venom.

BACKGROUND:Envenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage. METHODS AND FINDINGS:In this work we describe the mapping, by the SPOT-synthesis technique, of potential B-cell epitopes from five putative toxins from M. corallinus, which were used to design two multiepitope DNA strings for the genetic immunisation of female BALB/c mice. Results demonstrate that sera obtained from animals that were genetically immunised with these multiepitope constructs, followed by booster doses of recombinant proteins lead to a 60% survival in a lethal dose neutralisation assay. CONCLUSION:Here we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity

Expressed sequence tags (ESTs) from the salivary glands of the tick Amblyomma cajennense (Acari: Ixodidae)

The neotropical tick Amblyomma cajennense is a significant pest to domestic animals, the most frequently human-biting tick in South America and the main vector of Brazilian spotted fever (caused by Rickettsia rickettsii), a deadly human disease. The purpose of this study is to characterize the adult A. cajennense salivary gland transcriptome by expressed sequence tags (ESTs). We report the analysis of 1754 clones obtained from a cDNA library, which reveal mainly transcripts related to proteins involved in the hemostatic processes, especially proteases and their inhibitors. Remarkably, five types of possible serine protease inhibitors were found, including a molecule with a distinguished structure that contains repeats of the active motif of hirudin inhibitors. Besides, other components that may be active over the host immune system or acting as defensins against infecting microorganisms were also described, including a molecule similar to insect venom allergens. The conjunction of components from this transcriptome suggests a diverse strategy of A. cajennense tick during feeding, but emphasized in the coagulation syste

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

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 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

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