Comparative study of the cytolytic activity of snake venoms from African spitting cobras (Naja spp., Elapidae) and its neutralization by a polyspecific antivenom

Toxicon 58 (2011) 558–564

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Comparative study of the cytolytic activity of snake venoms from African spitting cobras (Naja spp., Elapidae) and its neutralization by a polyspecific antivenom Ileana Méndez a, José María Gutiérrez a, Yamileth Angulo a, Juan J. Calvete b, Bruno Lomonte a, * a b

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José 11501, Costa Rica Instituto de Biomedicina de Valencia, CSIC, Jaume Roig 11, 46010 Valencia, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 July 2011 Received in revised form 26 August 2011 Accepted 30 August 2011 Available online 8 September 2011

Venoms of several Naja species found in Sub-Saharan Africa, and commonly known as "spitting cobras", induce a predominantly cytotoxic pattern of envenomings that may evolve into tissue necrosis and gangrene. Cytotoxic components of their venoms have been identified as members of the three-finger toxin and phospholipase A2 protein families. In this study, an in vitro assay using the myogenic cell line C2C12, was utilized to compare the cytolytic activities of venoms from five species of spitting cobras: Naja nigricollis, Naja katiensis, Naja pallida, Naja nubiae, and Naja mossambica. These venoms were strongly cytotoxic, causing a 50% effect at w1.5 mg/well (15 mg/ml), except for N. katiensis venom, which required nearly twice this amount. Using the cell-based assay, the ability of an equine polyspecific antivenom (EchiTab-Plus-ICP) to neutralize cytotoxicity was assessed. The antivenom completely inhibited the cytotoxic activity of all five venoms, although high antivenom/venom ratios were needed. Neutralization curves displayed the following decreasing order of efficiency: N. nubiae > N. pallida > N. mossambica > N. nigricollis > N. katiensis. Results indicate that neutralizing antibodies toward toxins responsible for this particular effect are present in the antivenom, albeit in low titers. Fucoidan, a natural sulfated polysaccharide known to inhibit the toxic effects of some basic snake venom components, was unable to reduce cytotoxicity of Naja venoms. Results emphasize the need of enhancing the immunogenicity of low molecular mass toxins during antivenom production, as well as to search for useful toxin inhibitors which could complement antivenom therapy. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Snake venom Naja Cytotoxicity Cardiotoxin Antivenom Neutralization C2C12

1. Introduction Human envenomings due to snakebites are a relevant cause of morbidity and mortality in many regions of the world (Chippaux, 1998; Kasturiratne et al., 2008; Warrell, 2010). In Africa, especially in the sub-Saharan region, antivenoms to deal with this neglected public health problem are rarely available, making this continent the most precarious regarding the treatment of this pathology * Corresponding author. Tel.: þ506 2229 0344; fax: þ506 2292 0485. E-mail address: [email protected] (B. Lomonte). 0041-0101/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2011.08.018

(Chippaux, 2010; Gutiérrez et al., 2006). In the last decade, such shortage of antivenoms motivated an initiative by manufacturers from Latin America to develop specific antidotes against the main medically relevant snake species found in sub-Saharan Africa (Laing et al., 2003; Gutiérrez et al., 2005; Stock et al., 2007; Guidolin et al., 2010), thus providing additional therapeutic products to those traditionally manufactured for this region. These newly developed antivenoms have been studied to assess their neutralizing properties in preclinical models (Gutiérrez et al., 2005; Ramos-Cerrillo et al., 2008; Casasola et al., 2009; Segura et al., 2010; Calvete et al., 2010; Guidolin

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et al., 2010), and subsequently, in clinical trials (Chippaux et al., 2007; Abubakar et al., 2010a, 2010b). Although the gold standard to describe the neutralizing potency of antivenoms is their ability to antagonize the lethal action of venoms in animal assays, neutralization of other clinically relevant toxic activities also deserves attention (W.H.O., 1981, 2010b; Theakston and Reid, 1983; Gutiérrez et al., 1990), especially when these effects are associated with critical manifestations of envenomings. The venoms of Naja species inhabiting the sub-Saharan regions induce several systemic, as well as local pathological effects (W.H.O., 2010a). Some species of Naja cause predominantly neurotoxic envenomings, characterized by progressive neuromuscular paralysis that may lead to asphyxia. Other species, such as the "spitting cobras", induce a predominantly cytotoxic pattern of envenoming, which includes progressive and painful swelling of the bite wound, with blistering and bruising, that may evolve into tissue necrosis and gangrene (Warrell, 1996; W.H.O., 2010a). A recent proteomic study analyzed the composition of venoms from five species of African spitting cobras: Naja nigricollis, Naja mossambica, Naja katiensis, Naja pallida, and Naja nubiae (Petras et al., 2011). In agreement with the clinical features described in envenomings by these species, proteomic analyses revealed a very high content of toxins belonging to the cytotoxin/cardiotoxin protein family in all these venoms, ranging from 58 to 73% of their total proteins. Cytotoxins/cardiotoxins are small (6–7 kDa), non-enzymatic, single chain, highly basic proteins, that present the characeristic fold of the "three-finger toxin" (3FTx) superfamily, in similarity to a-neurotoxins of both the short-chain and long-chain types (Fry et al., 2003). The name cardiotoxins stems from their ability to induce systolic cardiac arrest in rodent models, while their potent cytolytic effect upon a wide variety of cells in vitro originates their designation as cytotoxins or cytolysins (Hodges et al., 1987; Rees et al., 1987; Harvey, 1990). Cytotoxins have been implicated in the necrotic tissue damage that may develop in envenomings by Naja species (Warrell, 1995, 1996, 2010). Experimental intramuscular injection of these toxins in mice has been shown to induce overt myonecrosis (Ownby et al., 1993). In addition to cytotoxins, some phospholipases A2 found in Naja venoms are strongly cytolytic (Chwetzoff et al., 1989), and therefore may contribute to the necrotic effects of their venoms. In vitro approaches to assess the ability of venoms and toxins to induce cytolysis are being increasingly used as an alternative to in vivo assays for necrotic activities, aiming to reduce animal suffering, as well as to provide better controlled models to investigate mechanisms of action, or screening for inhibitors (Brusés et al., 1993; Bultrón et al., 1993; Bieber et al., 1994; Lomonte et al., 1994a, 1999; Incerpi et al., 1995; Angulo and Lomonte, 2005; CintraFrancischinelli et al., 2009; Kalam et al., 2011). In the present study, the cytolytic activities of venoms from five species of spitting cobras (Naja) were comparatively assessed by an in vitro assay using the murine myogenic cell line C2C12 as a target. This assay was then utilized to evaluate the ability of an equine therapeutic antivenom to neutralize the cytotoxic components of Naja spp. venoms,

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as well as to screen for the potential inhibition of such toxins by fucoidan, a natural sulfated polysaccharide known to neutralize viperid myotoxic phospholipases A2 (PLA2s) (Angulo and Lomonte, 2003). 2. Materials and methods 2.1. Venoms and antivenom Lyophilized venoms of the African spitting cobras N. nigricollis, N. katiensis, N. pallida, N. nubiae, and N. mossambica, were obtained from Latoxan (France) and kept at 20  C until use. Venom samples were analyzed by SDSPAGE under reducing conditions on 4–20% gradient gels using a Tetra Cell (Bio-Rad). After staining with Coomassie blue R-250, densitometric scanning was performed with a Chemi-Doc imager (Bio-Rad) and the ImageLab software (Bio-Rad) to quantitatively assess the relative distribution of protein bands. The polyspecific EchiTAb-Plus-ICP antivenom (Instituto Clodomiro Picado, University of Costa Rica; batch 4260308PALQ; 6.9 g/dL protein concentration) was used in neutralization experiments. This product is prepared by caprylic acid fractionation of undigested plasma immunoglobulins from horses immunized with a mixture of Echis ocellatus, Bitis arietans, and N. nigricollis venoms, as previously described (Gutiérrez et al., 2005). 2.2. Cell culture The murine myogenic cell line C2C12 (ATCC CRL-1772) was grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 1 mM pyruvic acid, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and amphotericin B (0.25 mg/ml), in a humified atmosphere with 7% CO2, at 37  C. Cells were harvested from subconfluent monolayers grown in 252 cm bottles after detachment by trypsin (1500 U/ml) containing 5.3 mM EDTA, for 4–6 min at 37  C. The resuspended cells were seeded in 96-well microplates, at an approximate initial density of 1–4  104 cells/well, in the same medium. Cells were used in the cytotoxicity experiments after reaching near-confluence, at the myoblast stage. 2.3. Cytotoxicity assay The cytolytic activity of Naja venoms was determined by the release of lactic dehydrogenase (LDH), as described (Lomonte et al., 1999). In brief, serial dilutions of venoms were prepared in assay medium (DMEM supplemented with 1% FCS) and added to the cells growing in 96-well plates, after aspirating their medium, in a final volume of 100 ml/well. All samples were assayed in duplicate wells. Controls for 0% and 100%reference points consisted of assay medium, and 0.1% Triton X-100 in assay medium, respectively. After 2 h of incubation at 37  C, an aliquot of 40 ml of supernatant was collected from each well, and LDH activity was determined using a kinetic assay at 340 nm (Biocon Diagnostik), according to the manufacturer’s instructions. Cytotoxic activity was expressed as Cytolytic Dose 50%

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(CD50), corresponding to the dose of venom that induces 50% cell lysis. 2.4. Neutralization assays On the basis of dose-response curves for cytotoxic activity, a challenge venom dose of 3  CD50 was selected for each species. These venom doses were mixed with variable proportions of the antivenom, or with culture medium alone as a control, and incubated at 37  C for 20 min. Then, the venom/antivenom mixtures were added to the C2C12 cells, in order to determine their cytotoxic activity, as described in the preceding section. An additional control included the addition of antivenom alone to the cultures. In another set of experiments, a screening for the possible neutralizing activity of fucoidan, a natural sulfated polysaccharide extracted from the brown seaweed Fucus vesiculosus (Sigma), toward the different Naja spp. venoms, was performed. Venoms were mixed with a constant amount of fucoidan to obtain a 10:1 fucoidan/venom ratio (w/w), incubated at 37  C for 20 min, and finally assayed as described above. Cultures of C2C12 cells receiving fucoidan alone were included as additional controls. 2.5. Statistics Statistical significance of differences between mean values was determined by ANOVA or Student’s t-test, for multiple or paired comparisons, respectively, using a p value of 0.05. 3. Results and discussion A comparative electrophoretic analysis of the venoms from the five spitting cobra species evidenced their overall similarity in protein composition (Fig. 1). The two most prominent protein bands in these venoms appeared at w15 and w9 kDa, together accounting for nearly 90% of their total proteins, as estimated by densitometry. This is in agreement with the more accurate chromatographic values recently reported for the venom proteomes of these five Naja species (Petras et al., 2011) which demonstrated a marked predominance of 3FTxs, followed by PLA2s, and very few other proteins of higher molecular mass. Under our electrophoretic conditions, the band corresponding to 3FTxs migrated slightly above (w9 kDa) than expected on the basis of their known masses (6–8 kDa), possibly due to their strong intrinsic positive charge, as these are highly basic proteins. Venoms from elapid snake species commonly present a predominance of 3FTxs over PLA2s (Nawarak et al., 2003; Li et al., 2004; Olamendi-Portugal et al., 2008; Petras et al., 2011), although there are exceptions such as the Central American coral snake, Micrurus nigrocinctus, which displays an inverted proportion of these two types of proteins (Fernández et al., 2011). As previously mentioned, proteins in both the 3FTx and PLA2 families of elapid venoms can exert cytotoxic activity, which is associated with the necrotic effects observed in human envenomings by various species of these snakes. In the present work, using an in vitro cell culture model, the

Fig. 1. Electrophoretic analysis of Naja spp. venoms by 4–20% gradient SDSPAGE under reducing conditions. Molecular weight (Mw) markers are indicated at the left, in kDa. Densitometric scanning of the Coommasiestained gel was performed with the ImageLab software (Bio-Rad) and is represented in 3D. The relative amount of the prominent bands at w15 kDa (phospholipases A2; PLA2) and w9 kDa (three-finger toxins; 3FTx) is indicated as a percentage of total proteins, at the bottom.

cytotoxic action of the five Naja venoms was compared. As summarized in Fig. 2A, all these venoms exerted a potent cytolytic action upon the C2C12 cells, as determined by the rapid release of LDH to the medium, at low venom doses, and confirmed by microscopical observation (Fig. 2B). Four of the venoms (N. nigricollis, N. mossambica, N. nubiae, and N. pallida) showed a comparable cytotoxic potency in this assay, with a 50% effect being observed at doses of approximately 1.5 mg/well (15 mg/ml), whereas the venom of N. katiensis departed from the rest, requiring nearly twice the amount (3 mg/well) to induce the same half-maximal effect (Fig. 2). The ability of the equine polyspecific antivenom to inhibit the cytotoxic effect of the five Naja venoms was tested by the in vitro cell assay, as shown in Fig. 3. The cytotoxic activity in the venoms of N. nubiae and N. pallida was completely abrogated at an antivenom/venom ratio of 5 ml/mg, whereas neutralization of the venoms of N. katiensis, N. nigricollis and N. mossambica required 10 ml/ mg (Fig. 3). Differences were evidenced among the neutralization curves of venoms, which were inhibited by the antivenom in the following decreasing order of efficiency: N. nubiae > N. pallida > N. mossambica > N. nigricollis > N. katiensis. In the latter case, since N. katiensis venom had the lowest CD50 of the group, its challenge dose contained twice the absolute amount of protein than the other venoms. This could be related to the finding that it was less efficiently neutralized by the antivenom, because a higher number of toxin molecules would require a higher number of neutralizing antibody molecules. However, this consideration does not rule out other possibilities to

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Fig. 2. Cytotoxic activity of Naja spp. venoms on C2C12 murine myogenic cells. (A) Variable amounts of venoms were added to cell cultures grown in 96-wells, and cytolysis was determined after 2 h of incubation at 37  C, by quantifying the lactic dehydrogenase release to supernatants as described in Materials and Methods. Points represent mean  SD of duplicate cultures. The asterisk indicates a statististically significant (p < 0.05) difference between N. katiensis venom and the other four venoms, at 2 mg/well. Morphological appearance of cell cultures exposed for 2 h at 37  C to assay medium alone (B), or 4 mg of Naja nigricollis venom, which induced a complete cytolysis (C). Magnification: 10.

explain the lower neutralization efficiency recorded for N. katiensis venom. In the case of venoms from the other four species, since their challenge doses were identical, the recorded differences in neutralization curves (Fig. 3) imply that the antivenom must vary in its ability to recognize and inhibit their cytotoxic proteins. A combination of factors such as immunochemical differences among the toxins, arising from their structural variations, and antigenic relatedness to the immunizing components utilized in antivenom manufacture (in this case, from N. nigricollis) may determine the final outcome on neutralization efficiency. It is noteworthy that the venom of N. nigricollis, used in the manufacture of the antivenom studied, was not the most efficiently neutralized, again illustrating the complex nature of factors involved in the immunoneutralization of heterogeneous antigenic mosaics such as snake venoms. Two further conclusions can be derived from the antivenom neutralization experiments presented in Fig. 3. On one hand, the antivenom was clearly able to achieve a complete neutralization of all five venoms, provided that a sufficient amount was used in the assays. On the other hand, the very high ratios of antivenom/venom needed to neutralize the cytotoxic effect of these venoms imply that

neutralizing antibodies toward the toxins responsible for this particular effect are present in low titers. Cytotoxicity in Naja venoms depends mainly on a group of 3FTxs, the cardiotoxins/cytotoxins (Harvey, 1990), together with the possible action of some PLA2s (Chwetzoff et al., 1989). Previous studies have shown that the immunogenicity of 3FTxs, as well as of some PLA2s, tends to be weak in comparison to other types of snake venom components of higher molecular masses (Lomonte et al., 2008; Antúnez et al., 2010; Petras et al., 2011; Fernández et al., 2011). Therefore, present findings reinforce the need to develop improved methods of animal immunization that would ideally induce higher titers of neutralizing antibodies against low molecular mass, clinically relevant toxins in snake venoms (Calvete et al., 2009; Gutiérrez et al., 2009). Preclinical testing of antivenom efficacy is usually performed by assessing the neutralization of lethal activity of venoms (WHO, 2010b). However, in the case of cytotoxic Naja sp. venoms, such assay does not correlate with the clinically predominant picture of envenoming in humans, where local necrosis, and not neurotoxicity, is the main outcome (Warrell, 1995; WHO, 2010a). Indeed, the antivenom neutralizing ability toward cytotoxicity evaluated in

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Fig. 3. Neutralization of the cytotoxic activity of Naja spp. venoms on C2C12 cells by a polyspecific equine antivenom. Venoms were preincubated with the EchiTab-Plus-ICP antivenom at the indicated ratios, and then added to the cultures. The added mixtures contained 3  CD50 (venom dose inducing 50% cytolysis in the experiments presented in Fig. 2). Cytolysis was determined after 2 h of incubation at 37  C, by quantifying the lactic dehydrogenase release to supernatants, as described in Materials and Methods. Points represent mean  SD of duplicate cultures. The asterisk indicates a statistically significant (p < 0.05) difference between the venom of N. nubiae and the other four venoms, at 2.5 ml/mg. The differences between mean values obtained at 5 ml/mg were significant among all venoms, except between N. nubiae and N. pallida, which had been already neutralized at this ratio.

the present study, does not parallel the neutralization of lethality of the five Naja sp. venoms (Petras et al., 2011), in support of the independence of these two effects. Previous studies have assessed the neutralization of local

dermonecrosis as an experimental model for such necrotic effect (Gutiérrez et al., 2005; Petras et al., 2011). Nevertheless, this assay has limitations since its sensitivity is low and, in some cases, mice died at doses that do not induce local necrosis (Petras et al., 2011). Cytotoxic assays in cell culture constitute a valid surrogate model to study the necrotizing effect of these venoms and its neutralization by antivenoms, with the additional advantage of avoiding the use of laboratory animals. In addition to the current use of antibodies to treat envenomings, exploration for novel toxin inhibitors is a relevant task (Gutiérrez et al., 2007; Lomonte et al., 2009). In this regard, it has been reported that cardiotoxins/cytotoxins, as well as basic PLA2s from Naja venoms interact with heparin, a highly sulfated, polyanionic polysaccharide (Patel et al., 1997; Vyas et al., 1997; Lin et al., 1999). Heparin has been shown to inhibit the toxic effects of basic PLA2s from crotalid snake venoms (Melo et al., 1993; Lomonte et al., 1994b, 1994c). A similar neutralizing action as heparin, but with a markedly lower collateral effect toward the coagulation cascade has been recorded for fucoidan, a natural sulfated polysaccharide extracted from the brown seaweed F. vesiculosus (Angulo and Lomonte, 2003; Azofeifa et al., 2008). Therefore, it was of interest to screen for a possible inhibitory effect of fucoidan against the cytotoxic components of spitting cobra venoms, using the present cell-based assay. As shown in Fig. 4, however, fucoidan was unable to reduce to any extent the cytolytic damage inflicted by Naja venoms, even when tested at a high inhibitor/toxin ratio. This clearly indicates that the electrostatic interactions that may occur between the highly cationic 3FTxs and PLA2s of these venoms, and this

Fig. 4. Fucoidan does not neutralize the cytotoxic activity of Naja spp. venoms on C2C12 cells. Venoms were preincubated with either medium alone (light bars) or with fucoidan (dark bars) at 10:1 (w/w) fucoidan/venom ratio, and then added to the cultures. The added mixtures contained 3  CD50 (the venom dose inducing 50% cytolysis in the experiments presented in Fig. 2). Cytolysis was determined after 2 h of incubation at 37  C, by quantifying the lactic dehydrogenase release to supernatants, as described in Materials and Methods. Bars represent mean  SD of duplicate cultures.

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strongly polyanionic polysaccharide require additional spatial elements of specificity to result in the formation of stable, inactive complexes, as shown for other examples of such interactions (Lomonte et al., 1994b; Patel et al., 1997; Lin et al., 1999). More efforts should be devoted in the future to search for useful inhibitors against medically relevant toxins of low immunogenicity, such as the Naja venom components responsible for cytotoxic activity studied in the present work. The in vitro assay here described may be useful to such purpose, as well as to the assessment of the neutralizing ability of conventional therapeutic antivenoms. Conflict of interest Authors declare that they have no conflicts of interest regarding this manuscript. Acknowledgments We thank the technical staff of the Academic Division of Instituto Clodomiro Picado for general laboratory support, as well as the staff of its Industrial Division for providing a sample of EchiTab-Plus-ICP antivenom. Partial financial support from Vicerrectoría de Investigación, University of Costa Rica (VI-741-A9-513) and the ICGEB-CRP Program (COS-08-03) is gratefully acknowledged. References Abubakar, S.B., Abubakar, I.S., Habib, A.G., Nasidi, A., Durfa, N., Yusuf, P.O., Larnyang, S., Garnvwa, J.M., Sokomba, E., Salako, L., Laing, G.D., Theakston, R.D., Juszczak, E., Alder, N., Warrell, D.A.Nigeria-UK EchiTab Study Group, 2010a. Pre-clinical and preliminary dose finding and safety studies to identify candidate antivenoms for treatment of envenoming by saw-scaled or carpet vipers (Echis ocellatus) in northern Nigeria. Toxicon 55, 719–723. Abubakar, I.S., Abubakar, S.B., Habib, A.G., Nasidi, A., Durfa, N., Yusuf, P.O., Larnyang, S., Garnvwa, J.M., Sokomba, E., Salako, L., Theakston, R.D., Juszczak, E., Alder, N., Warrell, D.A.Nigeria-UK EchiTab Study Group, 2010b. Randomised controlled double-blind non-inferiority trial of two antivenoms for saw-scaled or carpet viper (Echis ocellatus) envenoming in Nigeria. PLoS Negl. Trop. Dis. 4 (7), e767. Angulo, Y., Lomonte, B., 2003. Inhibitory effect of fucoidan on the activities of crotaline snake venom myotoxic phospholipases A2. Biochem. Pharmacol. 66, 1993–2000. Angulo, Y., Lomonte, B., 2005. Differential susceptibility of C2C12 myoblasts and myotubes to group II phospholipase A2 myotoxins from crotalid snake venoms. Cell Biochem. Funct. 23, 307–313. Antúnez, J., Fernández, J., Lomonte, B., Angulo, Y., Calvete, J.J., Gutiérrez, J. M., 2010. Antivenomics of Atropoides mexicanus and Atropoides picadoi snake venoms: relationship to the neutralization of toxic and enzymatic activities. J. Venom Res. 1, 8–17. Azofeifa, K., Angulo, Y., Lomonte, B., 2008. Ability of fucoidan to prevent muscle necrosis induced by snake venom myotoxins: comparison of high- and low-molecular weight fractions. Toxicon 51, 373–380. Bieber, A.L., Ziolkowski, C., d’Avis, P.A., 1994. Rattlesnake toxins alter development of muscle cells in culture. Ann. N.Y. Acad. Sci. 710, 126–145. Brusés, J.L., Capaso, J., Katz, E., Pilar, G., 1993. Specific in vitro biological activity of snake venom myotoxins. J. Neurochem. 60, 1030–1042. Bultrón, E., Thelestam, M., Gutiérrez, J.M., 1993. Effects on cultured mammalian cells of myotoxin III, a phospholipase A2 isolated from Bothrops asper (terciopelo) venom. Biochim. Biophys. Acta 1179, 253– 259. Calvete, J.J., Sanz, L., Angulo, Y., Lomonte, B., Gutiérrez, J.M., 2009. Venom, venomics, antivenomics. FEBS Lett. 583, 1736–1743. Calvete, J.J., Cid, P., Sanz, L., Segura, A., Villalta, M., Herrera, M., León, G., Harrison, R.A., Durfa, N., Nasidi, A., Theakston, R.D.G., Warrell, D.A., Gutiérrez, J.M., 2010. Antivenomic assessment of the immunological

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