Sánchez Et Al 2015 Sav Afrique Subsaharienne.pdf

Toxicon 106 (2015) 97e107

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Evaluation of the preclinical efficacy of four antivenoms, distributed in sub-Saharan Africa, to neutralize the venom of the carpet viper, Echis ocellatus, from Mali, Cameroon, and Nigeria
nchez a, 1, Davinia Pla b, ***, 1, María Herrera a, Jean Philippe Chippaux c, d, Laura V. Sa
María Gutie
rrez a, * Juan J. Calvete b, **, Jose Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San Jos
e, Costa Rica
mica Estructural y Funcional, Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain Laboratorio de Veno Institut de Recherche pour le D
eveloppement, UMR MERIT “Mother and Child Facing Tropical Diseases”, Cotonou, Benin d Universit
e Paris Descartes, Sorbonne Paris Cit
e, Facult
e de Pharmacie, Avenue de l'Observatoire, Paris, France a

b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 August 2015 Received in revised form 16 September 2015 Accepted 21 September 2015 Available online 28 September 2015

Snakebite envenoming causes a heavy toll in sub-Saharan Africa in terms of mortality and sequelae. In the West African savannah, the viperid Echis ocellatus is responsible for the vast majority of bites. In the last decades, several new antivenoms have been introduced for the treatment of these envenomings, although the assessment of their preclinical efficacy against the venom of E. ocellatus has been studied only for some of them. This work analyzed comparatively the ability of four antivenoms (FAV Afrique, EchiTAb G, EchiTAB-Plus-ICP®, and Inoserp™ Panafricain) to neutralize lethal, hemorrhagic, and in vitro coagulant activities of the venoms of E. ocellatus from Mali, Cameroon, and Nigeria. In addition, an immunoaffinity chromatography antivenomic protocol was used to assess the ability of the four antivenoms to bind to the proteins of these venoms. Results showed that all the antivenoms were effective in the neutralization of the three effects investigated, and were able to immunocapture, completely or partially, the most abundant components in the E. ocellatus venoms from the geographical origins sampled. Our observations also highlighted quantitative differences between antivenoms in their neutralizing and antivenomics profiles, especially regarding neutralization of in vitro coagulant activity, suggesting that different doses of these antivenoms are probably needed for an effective treatment of human envenomings by this species. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Echis ocellatus Antivenom Hemorrhage Lethality Procoagulant effect Neutralization Antivenomics

1. Introduction Snakebite envenomings constitute a highly relevant, albeit largely neglected, public health problem in sub-Saharan Africa (Warrell, 1995; Chippaux, 2010; WHO, 2007). One of the snake species that inflicts a high number of envenomings, especially in

* Corresponding author.
mica Estructural y Funcional, Insti** Corresponding author. Laboratorio de Veno tuto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain.
mica Estructural y Funcional, *** Corresponding author. Laboratorio de Veno Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain. E-mail addresses: [email protected] (D. Pla), [email protected] (J.J. Calvete), jose.
rrez). [email protected] (J.M. Gutie 1 These authors have contributed equally to this work and should both be considered “first author”. http://dx.doi.org/10.1016/j.toxicon.2015.09.027 0041-0101/© 2015 Elsevier Ltd. All rights reserved.

Western sub-Saharan Africa, is the saw-scaled or carpet viper, Echis ocellatus (Warrell, 1995; WHO, 2010). Envenomings caused by this viperid species are characterized by local tissue damage, i.e. soft tissue necrosis, edema, hemorrhage and blistering, and by systemic manifestations associated mainly with coagulopathies and profuse bleeding which might lead to cerebrovascular accident and cardiovascular shock (Warrell et al., 1974; Warrell, 1995; Abubakar et al., 2010). The only scientifically-validated treatment for snakebite envenoming is the parenteral administration of animal-derived
rrez et al., 2011). Despite the antivenoms (Warrell, 2010; Gutie fact that various antivenom manufacturers produce antivenoms for sub-Saharan Africa (see http://apps.who.int/bloodproducts/ snakeantivenoms/database/), the availability of some of these products is very limited and often discontinuous. In addition, some manufacturers that used to produce antivenoms for sub-

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Saharan Africa have ceased production (Stock et al., 2007; Brown, 2012). This has prompted a crisis in the availability and accessibility of antivenoms for this region, characterized by a vicious cycle that greatly compromises the treatment of this tropical disease in many African countries (Chippaux, 2010; Williams et al., 2011; Brown, 2012). Various international initiatives have emerged in the last decade aimed at developing new antivenoms for Africa; some of these efforts have resulted in the generation of antivenoms manufactured in the United Kingdom, Costa Rica, and
rrez, 2012; Gutie
rrez et al., 2014). In addition, two Mexico (Gutie other antivenoms of demonstrated clinical efficacy and safety, IPSER Afrique (Chippaux et al., 1998) and FAV Afrique (Chippaux et al., 1999), which differ by the presence of E. ocellatus in the immunizing mixture and by the introduction of a chromatographic step of purification for the latter, have been manufactured in France for years, but their production has been discontinued in 1998 and 2014, respectively. One of the aspects that limits the distribution and appropriate use of antivenoms in sub-Saharan Africa is the lack of rigorous knowledge on the spectrum of preclinical efficacy of currently available antivenoms. In the case of Western sub-Saharan Africa, where the most important snake from a medical standpoint is E. ocellatus, there is limited information on the ability of antivenoms to neutralize the main toxic activities induced by the venom of this species. Moreover, there is little information on the variation in the composition of the venom of E. ocellatus from various locations, and whether such variations have an impact in the preclinical neutralizing ability of antivenoms. Owing to the medical impact of this species in the African savannah, there is an urgent need to perform preclinical analysis of antivenom efficacy against the venom of E. ocellatus. This study was designed to assess the preclinical efficacy of four antivenoms, available in Western sub-Saharan Africa, to neutralize the main toxic activities of venoms of E. ocellatus from various geographical origins. Assessment of efficacy has been performed by combining the study of the neutralization of three key toxic activities of the venoms, i.e. lethality, hemorrhagic activity and coagulant activity, with an antivenomics analysis of the ability of these antivenoms to recognize the various components present in these venoms. The integration of these methodological platforms now provides novel evidence on the preclinical efficacy of these antivenoms. 2. Materials and methods 2.1. Venoms Samples of pools of freeze-dried venoms of E. ocellatus from different countries were obtained. The venoms of specimens from Mali and Cameroon were purchased from Latoxan. The venom of specimens from Nigeria was obtained from the Liverpool School of Tropical Medicine, UK. These venoms correspond to pools of many specimens. Venoms were stored at
20  C until used. 2.2. Antivenoms Samples of the following antivenoms were tested in the study: (a) FAV Afrique F(ab0 )2 antivenom, manufactured by Sanofi-Pasteur, France, batch number K8453; (b) EchiTAb-Plus-ICP® IgG antivenom, manufactured by Instituto Clodomiro Picado, Costa Rica, batch number 5370114PALQ; (c) Inoserp™ Panafricain F(ab0 )2 antivenom, manufactured by Inosan Biopharma, S.A., Spain, batch number 2VT08001; and (d) EchiTAb G IgG antivenom, manufactured by Micropharm, UK, batch number EOG 000950. All antivenoms were used within their valid shelf-life. The total protein

concentration of antivenoms was quantified by a modified Biuret method (Parvin et al., 1965). Table 1 summarizes the characteristics of the four antivenoms used. 2.3. Analysis of the preclinical neutralizing profile of antivenoms The protocols previously used in the study of the preclinical efficacy of antivenoms were followed (Segura et al., 2010). The study included the analysis of the neutralization of the three most important toxic activities of E. ocellatus venom, i.e. lethal activity, hemorrhagic activity and in vitro coagulant activity. The protocols which involve the use of mice were approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUA) of the University of Costa Rica. 2.3.1. Lethal activity The Median Lethal Dose (LD50) of each venom, by the intravenous route, was initially determined (Segura et al., 2010). For this, groups of five CD-1 mice (18e20 g body weight) were injected intravenously, in the caudal vein, with various doses of venom, dissolved in 0.12 M NaCl, 0.04 M phosphate, pH 7.2 (PBS), in a volume of 0.2 mL. Deaths occurring during 24 h were recorded, and the value of LD50 was estimated by probits. For the neutralization of lethal activity, mixtures containing a fixed dose of venom and various dilutions of antivenom were prepared, and incubated at 37  C for 30 min. Then, aliquots of 0.2 mL of each mixture, containing a dose of venom corresponding to 5 LD50s, were injected i.v. into groups of five mice. Mixtures corresponded to various ratios of mg venom/mL antivenom (or mg venom/g antivenom protein). A control group of mice was injected with the same dose of venom incubated with PBS instead of antivenom. Deaths were recorded for 24 h and the neutralizing ability of antivenom was expressed as the Median Effective Dose (ED50), i.e. the venom/antivenom ratio at which half of the population of injected mice is protected, as estimated by probits. 2.3.2. Hemorrhagic activity The Minimum Hemorrhagic Dose (MHD) of each venom was
rrez et al., 1985). For this, groups of five initially determined (Gutie CD-1 mice (18e20 g) were injected intradermally, in the abdominal region, with various doses of venom, dissolved in PBS, in a volume of 0.1 mL. Two hours after injection, mice were sacrificed by CO2 inhalation, their skin removed, and the area of the hemorrhagic spot in the inner side of the skin measured. The MHD corresponds to the dose of venom that induces a hemorrhagic spot of 10 mm
rrez et al., 1985). diameter (Gutie For the neutralization of hemorrhagic activity, mixtures containing a fixed dose of venom and various dilutions of antivenom
rrez et al., were prepared, and incubated at 37  C for 30 min (Gutie 1985). Then, aliquots of 0.1 mL of each mixture, containing a dose of venom corresponding to 5 MHDs, were injected intradermally into groups of five mice, as described above. Mixtures corresponded to various ratios of mg venom/mL antivenom (or mg venom/g antivenom protein). A control group of mice was injected with the same dose of venom incubated with PBS instead of antivenom. After 2 h, mice were sacrificed by CO2 inhalation, and the area of the hemorrhagic spot was measured. Neutralizing ability was expressed as the Median Effective Dose (ED50), corresponding to the ratio venom/antivenom at which the diameter of the hemorrhagic spot is reduced by 50% when compared to the diameter of the hemorrhagic spot in mice injected with venom incubated with no antivenom. 2.3.3. Coagulant activity The Minimum Coagulant Dose (MCD) of each venom was


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Table 1 Characteristics of the four antivenoms used in this study. Protein (g/L)a

Antivenom

Manufacturer

Venoms used in immunization

Animal species used in immunization

Active substance

EchiTAb G EchiTAb-Plus-ICP®

Micropharm Instituto Clodomiro Picado

Sheep Horse

IgG IgG

FAV Afrique

Sanofi-Pasteur

Horse

F(ab0 )2

122

Inoserp™ Panafricain

Inosan Biopharma

Echis ocellatus Echis ocellatus, Bitis arietans, Naja nigricollis Bitis gabonica, Bitis arietans, Echis leucogaster, Echis ocellatus, Naja haje, Naja. nigricollis, Dendroaspis polylepis, Dendroaspis viridis, Dendroaspis jamesoni Echis leucogaster, Echis ocellatus, Echis pyramidum, Bitis gabonica, Bitis arietans, Dendroaspis polylepis, Dendroaspis jamesoni, Naja melanoleuca, Naja haje, Naja pallida, Naja nigricollis

Horse

F(ab0 )2

20

a

31 51

Protein concentration was estimated by the Biuret method.

initially determined. For this, various doses of venom, dissolved in 0.1 mL PBS, were added to 0.2 mL of citrated plasma obtained from blood collected from healthy human donors. The clotting time was measured and the MCD was estimated. MCD corresponds to the
et al., dose of venom that induces clotting of plasma in 60 s (Gene 1989). For the neutralization of coagulant activity, mixtures containing a fixed dose of venom and various dilutions of antivenom were
et al., 1989). prepared, and incubated at 37  C for 30 min (Gene Then, aliquots of 0.1 mL of each mixture, containing a dose of venom corresponding to 2 MCDs, were added to 0.2 mL of human citrated plasma, as described. Mixtures corresponded to various ratios of mg venom/mL antivenom (or mg venom/g antivenom protein). A control group included plasma incubated with venom that was previously incubated with PBS instead of antivenom. Clotting times were recorded and neutralization was expressed as Effective Dose (ED), corresponding to the ratio of venom/antivenom in which the clotting time is prolonged three times as compared to the clotting time of plasma incubated with venom alone. 2.4. Antivenomic characterization of the immunoreactivity profile of antivenoms The second generation antivenomics protocol of Pla et al. (2012) was followed. This protocol allows the identification, and a quantitative estimation, of the venom components that are recognized, or not recognized, by a particular antivenom. 2.4.1. Immunoaffinity chromatography-based antivenomics Immunoaffinity antivenom matrix was prepared essentially as previously described (Pla et al., 2012). Briefly, 300 mL of CNBractivated Sepharose™ 4B (Ge Healthcare) were packed in 0.8 mL Pierce® disposable, microcentrifuge spin columns (Thermo Scientific), and washed with 10e15 matrix volumes of cold 1 mM HCl, followed by two matrix volumes of coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) to adjust the pH between 7.0 and 8.0. For antivenom coupling, the CNBr-activated Sepharose columns were incubated (4 h at room temperature) with either 10 mg of EchiTAbPlus-ICP® (ICP, Costa Rica), EchiTab G (Micropharm), FAV Afrique

(Sanofi-Pasteur) or Inoserp™ Panafricain (Inosan Biopharma), each dissolved in ½ matrix volume of coupling buffer. Non-reacting groups were blocked with 200 mL of 0.1 M TriseHCl, pH 8.0 (at 4  C overnight) using an orbital shaker. The concentrations of the antivenom stock solutions and the non-bound antivenom were determined spectrophotometrically using an extinction coefficient of 1.4 for a 1 mg/mL concentration of IgG/F(ab')2 at 280 nm using a 1 cm light path length cuvette. The amounts of coupled antivenom molecules, determined by difference between the initial amount of antivenom incubated with the CNBr-activated matrix and the amount of antivenom that remained in the supernatant after incubation were 9.2 mg (EchiTAb-Plus-ICP®), 10 mg (EchiTab G), 8.3 mg (FAV Afrique), and 7.7 mg (Inoserp™ Panafricain) Before use, the affinity columns were washed alternately at high and low pH, with three volumes of 0.1 M acetate buffer, 0.5 M NaCl, pH 4.0e5.0, and three volumes of 0.1 M TriseHCl buffer, pH 8.5. This treatment was repeated six times and the columns were equilibrated in binding buffer (PBS). For immunoaffinity antivenomics using EchiTAb-Plus-ICP®, EchiTab G and FAV Afrique antivenoms, 100 mg of E. ocellatus crude venom from either Mali, Cameroon or Nigeria, in ½ matrix volume of PBS, were loaded in the columns and incubated in the plugged columns for 1 h at 25  C using an orbital shaker. For immunoaffinity antivenomics against the Inoserp™ Panafricain antivenom, 70 mg of each venom was employed. These conditions correspond to calculated venom:antivenom ratios (g/g) of 1:92 (EchiTAB-Plus-ICP®), 1:100 (EchiTab G), 1:83 (FAV Afrique), and 1:110 (Inoserp™ Panafricain) These venom:antivenom ratios correspond, respectively, to about 15, 16, 21, and 22 antibody molecules of EchiTAB-Plus-ICP®, EchiTab G, FAV Afrique, and Inoserp™ Panafricain per “25 kDa of toxin molecule”. After eluting the non-retained fractions, the columns were thoroughly washed (five times) with PBS, and the immunocaptured proteins were eluted with five column volumes of elution buffer (0.1 M glycine-HCl, pH 2.0), and immediately neutralized with 1 M TriseHCl, pH 9.0. As specificity controls, 300 mL of CNBr-activated Sepharose matrix, with or without 9.2 mg of immobilized control (preimmune) IgG molecules, were incubated with the same amount of venom and developed in parallel to the immunoaffinity columns.


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Immunoaffinity fractions were analyzed by reverse-phase HPLC using a Discovery® BIO Wide Pore C18 (15 cm  2.1 mm, 3 mm particle size, 300 Å pore size) column and an Agilent LC 1100 High Pressure Gradient System equipped with DAD detector and micro-autosampler. The flow-rate was set to 0.4 mL/min and the column developed with a linear gradient of 0.1% TFA in water (solution A) and 0.1% TFA in acetonitrile (solution B), isocratically (5% B) for 1 min, followed by 5e25% B for 5 min, 25e45% B for 35 min, and 45e70% for 5 min. Protein elution was monitored at 214 nm with a reference wavelength of 400 nm. The fraction of non-immunocaptured protein “i” was estimated as the relative ratio of the chromatographic areas of same protein recovered in the non-retained (NRi) and retained (Ri) affinity chromatography fractions using the equation %NRi ¼ 100
[(Ri/(Ri þ NRi))  100] (Pla et al., 2012). However, for SVMPs, which were poorly recovered in the bound fraction owing to the high affinity of the binding, the percentage of non-immunocaptured SVMP“i” (% NRSVMP“i”) was calculated as the ratio between the chromatographic areas of the same SVMP peak recovered in the nonretained fraction (NRSVMP“i”) and in the injected venom (VSVMP“i”), using the equation %NRSVMP“i” ¼ (NRSVMP“i”/ VSVMP“i”)  100. The biochemical identity of the immunocaptured and the non-immunoretained venom components was inferred by comparing the chromatographic profiles to previously characterized venomic analysis of E. ocellatus (Nigeria) venom (Wagstaff et al., 2009). 2.5. Statistical analysis In the case of lethality experiments, results are presented either as LD50 or ED50 with 95% confidence limits. In the cases of hemorrhage and coagulant activities, results are presented as mean ± S.D. In lethality, values are considered significantly different if the 95% confidence limits do not overlap. In the case of hemorrhagic and coagulant activities, the significance of the differences between mean values was determined by ANOVA, followed by Tukey test for the comparison of pairs of means. A value of P < 0.05 was considered significantly different. 3. Results 3.1. Determination of toxic activities Table 2 depicts the results of the quantification of lethal, hemorrhagic and in vitro coagulant activities of the venoms of E. ocellatus from Mali, Cameroon, and Nigeria. The venoms from the three countries exerted lethal, hemorrhagic and coagulant effects, in agreement with previous studies (Segura et al., 2010; RamosCerrillo et al., 2008). The three venoms present similar activity profiles for these effects, although the venom from Cameroon specimens showed slightly lower lethal activity than the other

venoms, and the venom from Nigeria significantly higher in vitro coagulant activity than the venoms of Mali and Cameroon. Therefore, overall the three venoms are similar in their toxicological profile. 3.2. Neutralization of toxic activities by the four antivenoms The preclinical estimation of the neutralizing ability of antivenoms against the effects induced by E. ocellatus venoms is expressed in two ways, i.e. mg of venom neutralized per mL of antivenom, and mg of venom neutralized per g antivenom protein. 3.2.1. Neutralization of lethal activity Table 3 presents the results of the neutralizing ability of antivenoms against lethal activity of E. ocellatus venoms. The four antivenoms are effective in the neutralization of lethality, albeit with different ED50s. When neutralization is expressed as mg venom neutralized per mL antivenom, the highest neutralizing efficacy was observed in EchiTAb-Plus-ICP®, followed by FAV Afrique, EchiTAb G and Inoserp™ Panafricain. On the other hand, when neutralization is expressed as mg venom neutralized per g of antivenom proteins, the highest neutralizing efficacy was observed in EchiTAb-Plus-ICP®, followed by Inoserp™ Panafricain, EchiTAb G and FAV Afrique. 3.2.2. Neutralization of hemorrhagic activity Table 4 presents the results of the neutralizing ability of antivenoms against hemorrhagic activity of E. ocellatus venoms. The four antivenoms are effective in the neutralization of hemorrhagic activity, albeit with different potencies. When neutralization is expressed as mg venom neutralized per mL antivenom, the highest neutralizing efficacy was observed in EchiTAb G and FAV Afrique, followed by EchiTAb-Plus-ICP® and Inoserp™ Panafricain. On the other hand, when neutralization is expressed as mg venom neutralized per g antivenom protein, the highest neutralizing efficacy was observed in EchiTAb G, followed by Inoserp Panafricain, EchiTAb-Plus-ICP and FAV Afrique, with variations depending on the venom being neutralized. 3.2.3. Neutralization of in vitro coagulant activity Table 5 presents the results of the neutralizing ability of antivenoms against in vitro coagulant activity of E. ocellatus venoms. The four antivenoms are effective in the neutralization of hemorrhagic activity, albeit with different potencies. When neutralization is expressed as mg venom neutralized per mL antivenom, the highest neutralizing efficacy was observed in EchiTAb-Plus-ICP®, followed by FAV Afrique, Inoserp™ Panafricain and EchiTAb G. On the other hand, when neutralization is expressed as mg venom neutralized per g antivenom protein, the highest neutralizing efficacy was observed in EchiTAB-Plus-ICP®, followed by Inoserp™ Panafricain, FAV Afrique and EchiTAb G.

Table 2 Lethal, hemorrhagic and in vitro coagulant activities of the venoms of E. ocellatus from three countries in sub-Saharan Africa. Toxic activity Lethality (LD50, mg per mouse) Hemorrhagic (Minimum Hemorrhagic Dose, mg)b Coagulant (Minimum Coagulant Dose, mg)c a

E. ocellatus (Mali)

E. ocellatus (Cameroon)

E. ocellatus (Nigeria)

16.4 (4.1e28.4) 0.26 ± 0.08 1.21 ± 0.03

33.1 (6.9e66.8) 0.28 ± 0.03 1.24 ± 0.02

16.9 (5.5e28.7) 0.26 ± 0.03 0.90 ± 0.02*

*Significantly different (P < 0.05) from the other two venoms. a The Median Lethal Dose (LD50) is expressed in mg venom/mouse (18e20 g); 95% confidence limits are included in parentheses. b The Minimum Hemorrhagic Dose (MHD) corresponds to the dose of venom that induces a hemorrhagic halo of 10 mm diameter in mice 2 h after intradermal injection. Results are presented as mean ± S.D. (n ¼ 5). c The Minimum Coagulant Dose (MCD) corresponds to the dose of venom that induces the clotting of citrated human plasma in 60 s. Results are presented as mean ± S.D. (n ¼ 3).


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Table 3 Neutralization of the lethal effect of venoms of E. ocellatus by four antivenoms. Neutralization is expressed as either mg venom neutralized per mL antivenom or as mg venom neutralized per g of antivenom protein. The 95% confidence limits are expressed in parentheses. Antivenom

Echis ocellatus (Mali)

Neutralization (ED50, mg venom per mL antivenom) FAV Afrique (Sanofi Pasteur) 1.78 EchiTAb G (Micropharm) 0.46 ® EchiTAb-Plus-ICP (Instituto Clodomiro Picado) 4.61 Inoserp™ Panafricain (Inosan Biopharma) 1.13 Neutralization (ED50, mg venom per g of antivenom protein) FAV Afrique (Sanofi Pasteur) 14.6 EchiTAb G (Micropharm) 14.8 EchiTAb-Plus-ICP® (Instituto Clodomiro Picado) 90.4 Inoserp™ Panafricain (Inosan Biopharma) 56.5

Echis ocellatus (Cameroon)

Echis ocellatus (Nigeria)

(1.10e2.59) (0.21e0.73) (3.51e7.55) (0.77e1.56)

2.26 0.98 3.40 0.53

(1.25e3.33) (0.45e1.51) (2.22e5.87) (0.23e0.80)

1.71 0.90 3.13 0.79

(1.13e2.44) (0.44e1.56) (2.32e4.40) (0.43e1.13)

(9.0e21.2) (6.8e23.5) (68.8e148.0) (38.5e78.0)

18.5 31.6 66.7 26.5

(10.2e27.3) (14.5e48.7) (43.5e115.1) (11.5e40.0)

14.0 29.0 61.4 39.5

(9.3e20.0) (14.2e50.3) (45.5e86.3) (21.5e56.5)

Table 4 Neutralization of the hemorrhagic effect of venoms of E. ocellatus by four antivenoms. Neutralization is expressed as either mg venom neutralized per mL antivenom or as mg venom neutralized per g antivenom protein. Results are expressed as mean ± standard deviation. Antivenom

Echis ocellatus (Mali)

Neutralization (ED50, mg venom per mL antivenom) FAV Afrique (Sanofi Pasteur) 4.54 EchiTAb G (Micropharm) 5.08 EchiTAb-Plus-ICP® (Instituto Clodomiro Picado) 3.18 Inoserp™ Panfricain (Inosan Biopharma) 1.56 Neutralization (ED50, mg venom per g of antivenom protein) FAV Afrique (Sanofi Pasteur) 37.2 EchiTAb G (Micropharm) 163.8 ® EchiTAb-Plus-ICP (Instituto Clodomiro Picado) 62.3 Inoserp™ Panafricain (Inosan Biopharma) 78.0

Echis ocellatus (Cameroon)

Echis ocellatus (Nigeria)

± ± ± ±

0.20b* 0.14a 0.30c 0.09d

5.35 4.41 3.04 1.68

± ± ± ±

0.75a 0.44b 0.25c 0.13d

4.16 3.98 2.11 1.07

± ± ± ±

0.39a 0.91ª 0.03b 0.17c

± ± ± ±

1.6d* 4.5a 5.9c 4.5b

43.8 142.3 59.6 84.0

± ± ± ±

6.1d 14.2ª 4.9c 6.5b

34.1 128.4 41.4 53.5

± ± ± ±

3.2b 29.3ª 0.6b 8.5b

*Values with different superscript are significantly different (P < 0.05) between them for a particular venom.

Table 5 Neutralization of in vitro coagulant effect of venoms of E. ocellatus by four antivenoms. Neutralization is expressed either as mg venom neutralized per mL antivenom or as mg venom neutralized per g of antivenom protein. Results are expressed as mean ± standard deviation. Antivenom

Echis ocellatus (Mali)

Neutralization (ED, mg venom per mL antivenom) FAV Afrique (Sanofi Pasteur) 2.56 ± EchiTAb G (Micropharm) 0.22 ± EchiTAb-Plus-ICP® (Instituto Clodomiro Picado) 7.64 ± Inoserp™ Panafricain (Inosan Biopharma) 0.75 ± Neutralization (ED, mg venom per g of antivenom protein) FAV Afrique (Sanofi Pasteur) 21.0 ± EchiTAb G (Micropharm) 7.1 ± EchiTAb-Plus-ICP® (Instituto Clodomiro Picado) 149.8 ± Inoserp™ Panafricain (Inosan Biopharma) 37.5 ±

Echis ocellatus (Cameroon)

Echis ocellatus (Nigeria)

0.01b* 0.01d 0.07a 0.02c

2.85 0.33 12.88 0.96

± ± ± ±

0.04b 0.01d 0.09a 0.02c

1.72 0.20 5.21 0.14

± ± ± ±

0.02b 0.01c 0.03a 0.01d

0.1c* 0.3d 1.4a 1.0b

23.4 10.6 252.5 48.0

± ± ± ±

0.3c 0.3d 1.8a 1.0b

14.1 6.4 102.1 7.0

± ± ± ±

0.2b 0.3c 0.6a 0.5c

*Values with different superscript are significantly different (P < 0.05) between them for a particular venom.

3.2.3.1. Antivenomics analysis. Figs. 1e3 depict the antivenomic analyses of the four antivenoms when confronted with the venoms of E. ocellatus from the three countries included in this study. Each figure presents the RP-HPLC profiles of the venoms, with the identification of the proteins present in the main peaks, which mostly correspond to disintegrins, phospholipases A2 (PLA2s), serine proteinases C-type lectin-like proteins, L-amino acid oxidases and zinc-dependent metalloproteinases (SVMPs) of the PeI and P-III classes, as identified in previous proteomics study of this venom (Wagstaff et al., 2009). Venoms presented qualitatively similar RP-HPLC profiles, with quantitative differences in the heights of the peaks of several components (Figs. 1e3). For each venom, the RP-HPLC profiles of the non-retained venom fractions gathered from each of the four antivenoms affinity column are shown in Figs. 1e3, highlighting the percentages of the nonretained proteins in each fraction, as estimated by the equations described in Materials and methods. In these cases, the higher the percentage, the lower the recognition of the fraction by the

antivenom. The RP-HPLC profiles of the fractions retained in the antivenom affinity columns are shown in Supplementary Figs. S1eS3. The first fractions in the chromatogram, corresponding to small peptides and disintegrins, are poorly recognized by the four antivenoms, in agreement with previous antivenomics studies (Calvete et al., 2010; Pla et al., 2012). The rest of venom fractions, comprising various enzymes and proteins, are immunorecognized by the four antivenoms, albeit to a different extent depending on the venom and on the antivenom (Figs. 1e3). The PLA2 molecules from E. ocellatus from Mali and Cameroon were completely immunorecognized by EchiTAb G (Figs. 1 and 2), but partially immunocaptured by the other three antivenoms, with quantitative differences between them (Figs. 1e3). EchiTAb G and EchiTAb-Plus-ICP® also quantitatively inmunocaptured the PLA2 molecules of E. ocellatus from Nigeria (Fig. 3). EchiTAb G and EchiTAb-Plus-ICP® antivenoms completely immunocaptured the serine proteinases and C-type lectin-like

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Fig. 1. Immunocapturing ability of immobilized antivenoms toward venom proteins of E. ocellatus from Mali. Panels AeE show, respectively, reverse-phase separations of the whole venom components of E. ocellatus from Mali, and the non-immunocaptured venom proteins by immobilized antivenoms FAV Afrique (Sanofi-Pasteur, France), EchiTAb G (Micropharm, UK), EchiTAb-Plus-ICP® (Instituto Clodomiro Picado, Costa Rica), and Inoserp™ Panafricain (Biopharma). Major toxins identified by Wagstaff et al. (2009) are highlighted in panel A: PLA2, phospholipase A2; LAO, L-amino acid oxidase; PI- and PIII-SVMP, snake venom metalloproteinase of class PI and PIII, respectively. Numbers indicate % of non-retained component.

protein peaks, whereas FAV Afrique and Inoserp™ Panafricain immunocaptured these proteins to a partial extent. The four last peaks of the chromatograms of the venoms from Cameroon (Fig. 2) and Nigeria (Fig. 3), containing mainly PeI and P-III SVMPs and Lamino acid oxidase (LAO), were completely immunoretained by EchiTAb-Plus-ICP®, and partially immunocaptured by the other three antivenoms, with quantitative differences among them (Figs. 1e3). SVMPs and LAO molecules present in the venom from Mali were only partially immunocaptured in the immunoaffinity

columns of the four antivenoms (Fig. 1). It was observed that the peaks corresponding to SVMPs bound strongly to the immobilized antivenom antibodies, since they could not be efficiently eluted after application of the glycine acid elution buffer (see asterisks in Supplementary Figs. S1eS3). This is likely to be due to the high affinity of antivenom antibodies to SVMPs in all antivenoms (Calvete et al., 2015). Therefore, the calculated percentage of nonretained SVMPs, uncorrected for possible losses during sample handling and chromatographic analysis, represents a lower limit for


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Fig. 2. Immunocapturing ability of immobilized antivenoms toward venom proteins of E. ocellatus from Cameroon. Panels AeE display, respectively, reverse-phase separations of the whole venom components of E. ocellatus from Cameroon, and the non-immunocaptured venom proteins by immobilized antivenoms FAV Afrique (Sanofi-Pasteur, France), EchiTAb G (Micropharm, UK), EchiTAb-Plus-ICP® (Instituto Clodomiro Picado, Costa Rica), and Inoserp™ Panafricain (Biopharma). Major toxins identified by Wagstaff et al. (2009) are highlighted in panel A: PLA2, phospholipase A2; LAO, L-amino acid oxidase; PI- and PIII-SVMP, snake venom metalloproteinase of class PI and PIII, respectively. Numbers indicate % of non-retained component.

the amount of these proteins not immunorecognized by the antivenoms. Immunoaffinity controls in which venoms were incubated with the CNBr-activated Sepharose matrix, with or without conjugated control IgGs, showed negligible unspecific binding, as previously described (Pla et al., 2012). 4. Discussion This study evaluated the preclinical efficacy of four antivenoms, being distributed in sub-Saharan Africa, against the venom of

E. ocellatus. The efficacy and safety of these antivenoms have been previously analyzed in clinical studies (Chippaux et al., 1998, 2015; Abubakar et al., 2010). However, they had not been previously compared for their preclinical neutralizing efficacy using standard neutralization tests and antivenomics. These antivenoms differ in aspects such as: (a) Venoms used in the immunizing mixtures; (b) type of active substance, i.e. whole IgG purified by caprylic acid precipitation of non-IgG plasma proteins, or F(ab0 )2 fragments purified by pepsin digestion and ammonium sulphate precipitation; and (c) protein concentration (Table 1). These characteristics have

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Fig. 3. Immunocapturing ability of immobilized antivenoms toward venom proteins of E. ocellatus from Nigeria. Panels AeE display, respectively, reverse-phase separations of the whole venom components of E. ocellatus from Nigeria, and the non-immunocaptured venom proteins by immobilized antivenoms FAV Afrique (Sanofi-Pasteur, France), EchiTAb G (Micropharm, UK), EchiTAb-Plus-ICP® (Instituto Clodomiro Picado, Costa Rica), and Inoserp™ Panafricain (Biopharma). Major toxins identified by Wagstaff et al. (2009) are highlighted in panel A: PLA2, phospholipase A2; LAO, L-amino acid oxidase; PI- and PIII-SVMP, snake venom metalloproteinase of class PI and PIII, respectively. Numbers indicate % of non-retained component.

to be taken into account when analyzing the differences observed in the neutralizing activity. An issue that deserves consideration in the preclinical testing of antivenom efficacy has to do with geographical variations of venoms within a single species (Chippaux et al., 1991), especially in those showing wide geographical distribution, such as E. ocellatus. The populations of this species may constitute a species complex along West Africa (Chippaux and Jackson, unpublished observations). In this context, antivenoms might present a higher

neutralizing potency against venoms used in their manufacture than against venoms of snakes from other geographical origins or belonging to distinct populations within the same species complex (see for example S
anchez et al. (2015) for the case of E. ocellatus). As an approximation to this phenomenon of geographic variability and its implications in antivenom preclinical testing, our study assessed the efficacy of antivenoms against venoms of specimens of E. ocellatus collected in Mali, Cameroon and Nigeria. The effects whose neutralization was studied, i.e. lethal,


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hemorrhagic and in vitro coagulant activities, were selected because of their clinical relevance. The neutralization of lethality is considered the gold standard in the preclinical assessment of antivenom efficacy (WHO, 2010). The mechanisms of lethality by E. ocellatus venom in the mouse LD50 test, when using the i.v. route, have not been investigated. However, owing to the potent hemorrhagic and in vitro coagulant effects displayed by these venoms, it can be hypothesized that mice die of systemic hemorrhage and intravascular thrombosis after receiving five LD50s of venom. On the other hand, local and systemic hemorrhage, together with coagulopathies, are the most significant manifestations in envenomings by E. ocellatus (Warrell, 1995; Chippaux et al., 1998; Warrell et al., 1974; Abubakar et al., 2010; Habib, 2013). Hemorrhage is responsible for prominent local tissue damage and for severe systemic manifestations of envenoming, such as cardiovascular shock and cerebral hemorrhage. Coagulopathies, in turn, are the result of the action of procoagulant components, and contribute to the systemic hemorrhagic syndrome. Alterations in clotting laboratory parameters are used to monitor the evolution of envenomings and their treatments (Warrell et al., 1974; Abubakar et al., 2010; Chippaux et al., 2015). Thus, the analysis of the neutralization of these three effects by antivenoms represents a robust approach to evaluate their preclinical efficacy. Our results show that the four antivenoms included in this study are able to neutralize lethal, hemorrhagic and in vitro coagulant activities of the venoms of E. ocellatus from Mali, Cameroon, and Nigeria, although quantitative differences were observed in the values of ED50s for the neutralization of these effects. For instance, EchiTAb-Plus-ICP® showed the highest neutralizing ability towards the lethal and in vitro procoagulant activities, whereas EchiTAb G presented the highest neutralizing ability against hemorrhagic activity. The largest differences in the neutralizing profile of these antivenoms were observed for in vitro coagulant activity, an observation that might have clinical implications owing to the relevance of coagulopathies in these envenomings. Previous preclinical studies with antivenoms EchiTAb G and EchiTAb-Plus-ICP in the neutralization of venom of E. ocellatus from Nigeria described relatively similar results as those reported here (Segura et al., 2010;
nchez et al., 2015). Although extrapolations Casewell et al., 2010; Sa of preclinical findings to the clinical setting have to be done with caution, our observations suggest that different doses of these antivenoms would have to be used in the treatment of envenomings by E. ocellatus, i.e. to halt hemorrhage and, especially, to correct the clotting disturbances. The selection of the appropriate initial doses for these antivenoms demands the development of clinical trials; the information generated in our study should be of value for performing dose-finding studies at the clinical level. The study of the neutralizing profile of antivenoms has been greatly complemented by the antivenomic analysis of immunoreactivity towards venom components. This approach allows the quantitative assessment of the ability of antivenoms to bind to the various venom proteins in an affinity chromatography setting. On the basis of previous proteomics characterization of venoms, antivenomics allows the identification of proteins being totally or partially immunorecognized by antivenom antibodies, as well as of proteins not bound by these antibodies. In this study, and in agreement with previous works with various antivenoms (Calvete
rrez et al., 2014), the four antivenoms were et al., 2010; Gutie largely ineffective in the immunorecognition of low molecular mass peptides, such as tripeptide inhibitors of SVMPs (Wagstaff et al., 2008), and short disintegrins (e.g. ocellatusin [Q14FJ4, Smith et al., 2002]). This is likely to be due to the poor immunogenicity of these low molecular mass venom components. The pathophysiological implications of this observation are unknown, but it is highly likely that peptides and disintegrins do not play a

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key role in the main alterations characteristic of envenomings by E. ocellatus, since some of the antivenoms tested in this study have been known to effectively control bleeding and coagulopathies in clinical studies (Chippaux et al., 1998, 2015; Abubakar et al., 2010). The four antivenoms immunocaptured venom PLA2s, with quantitative variations between them. The role of PLA2s in envenomings by E. ocellatus has not been elucidated, but a cytotoxic Ser49-PLA2 homologue is present in this venom and might induce tissue necrosis (Conlon et al., 2013). The antivenoms also immunorecognized, to a variable extent, the peaks containing serine proteinases and C-type lectin-like proteins. The role played by these components in the pathophysiology of envenoming by E. ocellatus is not clear at present, but is likely to be associated with hemostatic alterations, since a number of serine proteinases display thrombin-like activity and contribute to defibrinogenation (Serrano and Maroun, 2005). In addition, a heterodimeric C-type lectin-like protein, echicetin, isolated from Echis carinatus venom, affects platelet aggregation in vitro and prolongs bleeding time in mice (Peng et al., 1993). It is highly likely that similar proteins are present in E. ocellatus venom (Harrison et al., 2003), and they might induce platelet hypoaggregation. SVMPs comprise a high percentage of the proteome of E. ocellatus (Wagstaff et al., 2009), and are likely to be the most important components in this venom from the pathophysiological standpoint. SVMPs exerting hemorrhagic and procoagulant activities have been isolated from E. ocellatus venom (Howes et al., 2003, 2005) or cloned from the venom gland (Hasson et al., 2003). In general, snake venom hemorrhagic activity is mostly due to P-III
rrez et al., 2005), and the procoagulant activity of SVMPs (Gutie Echis sp venoms depends predominantly on prothrombin activation by monomeric and heterodimeric P-III SVMPs, such as ecarin and carinactivase, respectively (Fortov
a et al., 1983; Yamada et al., 1996). Monomeric and heterodimeric P-III SVMPs are abundant in E. ocellatus venom proteome (Wagstaff et al., 2009) and very likely constitute the key hemorrhagic and procoagulant components of this venom. The variations in the percentage of SVMP venom fraction immunorecognized by the antivenoms may explain the quantitative differences observed in their ability to neutralize hemorrhagic and in vitro coagulant activities. It is noteworthy, however, that antivenoms differ in their neutralization of these two activities; for instance, EchiTAb G shows the highest neutralizing activity against hemorrhage, but the lowest against coagulant effect. This finding may reveal that hemorrhage and coagulation are induced by different SVMPs, and that antivenoms have differences in the concentration or affinity of antibodies against these toxins. However, regardless of these variations, the four antivenoms neutralized the three effects and partially or completely immunorecognized the SVMP peaks. This and previous studies suggest that toxins which are immunorecognized in antivenomic analyses at least to a partial extent are neutralized when mixtures of venom and antivenom are incubated before testing in experimental models (Calvete et al., 2014). This is probably due to the fact that neutralization protocols involve the incubation of venom and antivenom for 30 min before testing in mice or in in vitro assays. In conclusion, the four antivenoms analyzed in this investigation, which differ in their composition, i.e. IgG or F(ab’)2, and in the venom mixtures used in the immunization schemes, are able to neutralize lethal, hemorrhagic and in vitro coagulant activities of the venoms of E. ocellatus from Mali, Cameroon, and Nigeria, albeit showing different neutralizing potencies. The highest variations between antivenoms were observed in the neutralization of in vitro coagulant activity. Furthermore, these antivenoms bind, to a variable extent, the most relevant components of these venoms when

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analyzed by antivenomics. On the other hand, the geographical origin of the venoms does not seem to have a major impact in the neutralizing ability of these antivenoms, since the pattern of neutralization of the toxic effects studied was similar when the venoms from the three countries are compared. On the basis of these preclinical data, it is suggested that the initial dosage of antivenom required to treat human cases of envenoming by E. ocellatus might be different for these four antivenoms, a hypothesis that has to be tested in appropriately designed clinical studies. Moreover, there are other antivenoms being distributed in sub-Saharan Africa whose preclinical efficacy against the venom of E. ocellatus should be investigated in the near future. Ethical statement The protocols which involve the use of mice used in this study followed international guidelines in the use of laboratory animals and were approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUA) of the University of Costa Rica. Conflict of interest statement
María Gutie
rrez work Laura V. S
anchez, María Herrera and Jose at Instituto Clodomiro Picado, Universidad de Costa Rica, where one of the antivenoms analyzed in this study (EchiTAb-Plus-ICP) is manufactured. Acknowledgments

n, Maria
ngela Vargas, Alvaro The authors thank Guillermo Leo
s S
Segura, Andre anchez, Mauren Villalta (Instituto Clodomiro Pic
mica ado), Yania Rodríguez and Libia Sanz (Laboratorio de Veno Estructural y Funcional) for their cooperation in several aspects of this study. Thanks are also due to Robert Harrison (Liverpool School of Tropical Medicine) for the venom of E. ocellatus from Nigeria, and to the manufacturers of the four antivenoms used for providing them for the study. This work was supported by Vicerrectoría de
n, Universidad de Costa Rica (project 741-B4-524), and Investigacio Grants BFU2007e61563 and BFU2010e17373 from the Ministerios
n y Ciencia and Ciencia e
Innovacio
n, Madrid (Spain). de Educacio This study was performed in partial fulfillment of the requirements
nchez at the University of Costa for the M.Sc. degree for Laura V. Sa Rica. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2015.09.027. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.toxicon.2015.09.027. References Abubakar, I.S., Abubakar, S.B., Habib, A.G., Nasidi, A., Durfa, N., Yusuf, P.O., Larnyang, S., Garnvwa, J., Sokomba, E., Salako, L., Theakston, R.D.G., Juszczak, E., Alder, N., Warrell, D.A., 2010. Randomised controlled double-blind non-inferiority trial of two antivenoms for saw-scale or carpet viper (Echis ocellatus) envenoming in Nigeria. PLoS Negl. Trop. Dis. 4, e767. Brown, N.I., 2012. Consequences of neglect: analysis of the sub-Saharan African snake antivenom market and the global context. PLoS Negl. Trop. Dis. 6, e1670.
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