Cross-reactivity, antivenomics, and neutralization of toxic activities of Lachesis venoms by polyspecific and monospecific antivenoms

Background Bothrops, Crotalus and Lachesis represent the most medically relevant genera of pitvipers in Central and South America. Similarity in venom phenotype and physiopathological profile of envenomings caused by the four nominal Lachesis species led us to hypothesize that an antivenom prepared against venom from any of them may exhibit paraspecificity against all the other congeneric taxa. Methods To assess this hypothesis, in this work we have applied antivenomics and immunochemical methods to investigate the immunoreactivity of three monovalent antivenoms and two polyvalent antivenoms towards the venoms from different geographic populations of three different Lachesis species. The ability of the antivenoms to neutralize the proteolytic, hemorrhagic, coagulant, and lethal activities of the seven Lachesis venoms was also investigated. Results A conspicuous pattern of immunorecognition and cross-neutralization for all effects was evident by the polyspecific antivenoms, indicating large immunoreactive epitope conservation across the genus during more than 10 million years since the Central and South American bushmasters diverged. Conclusions Despite the broad geographic distribution of Lachesis, antivenoms against venoms of different species are effective in the neutralization of congeneric venoms not used in the immunization mixture, indicating that they can be used equivalently for the clinical treatment of any lachesic envenoming. General significance This study demonstrates that antivenoms raised against venom of different Lachesis species are indistinctly effective in the neutralization of congeneric venoms not used in the immunization mixture, indicating that antivenoms against conspecific venoms may be used equivalently for the clinical treatment of envenomings caused by any bushmaster species.


Introduction
Nicaragua, Costa Rica and Panama; L. melanocephala is found in the Pacific versant of southwestern Costa Rica, and the extreme western regions of Panama; L. acrochorda inhabits both the Atlantic and Pacific versants of western Panama and into northwestern Colombia, on the Atlantic coast, where it extends southward into the Cauca and Magdalena rivers valleys, and along the Pacific versant of Colombia into northwestern Ecuador. L. muta is the most widely distributed species of the genus, including the equatorial forest east of the Andes, from Colombia, eastern Ecuador, Peru, northern Bolivia, eastern and southern Venezuela to Guyana, Surinam, French Guiana and most of northern Brazil [10]. Two subspecies of L. muta are reported: L. m. muta and L. m. rhombeata, with an exclusive distribution of the latter subspecies in the Atlantic forest of eastern-center of Brazil. Based on morphology, some authors consider that there are populations of L. m. rhombeata in the Amazonia basin [11].
The high conservation of the overall composition of Central and South American bushmaster venoms and their qualitatively similar pathophysiological profile observed in clinical settings [14,17,[27][28][29], suggested that antivenoms generated against any conspecific Lachesis venom may exhibit paraspecific protection against the toxic activities of all other Lachesis species [24,25]. The aim of the present work was to assess this hypothesis. To this end, we carried out a comparative study of the cross-reactivity, neutralization of toxic activities and immunoaffinity antivenomic profiles towards a panel of Lachesis venoms of two commercial polyspecific antivenoms (BCL), manufactured at Instituto Clodomiro Picado, Costa Rica, against a mixture of L. stenophrys, Bothrops asper, and Crotalus simus venoms, and antivenom produced at Instituto Vital Brazil, Brazil, against venoms from L. m. rhombeata and five bothropic species (BL), and experimental monospecific antivenoms AL, AB, and AC, generated, respectively, against venoms of adult Costa Rican L. stenophrys, B. asper, and C. simus.

Ethics statement
All the procedures involving the use of animals in this study were approved by the Institutional Committee for the Care

Snake venoms and antivenoms
Lachesis venoms were obtained from different geographic areas of Central and South America. Venom from L. stenophrys (Central American bushmaster) was pooled from more than 25 adult (>5 years old) snakes maintained in the herpetarium of Instituto Clodomiro Picado (ICP, San José, Costa Rica). Venom from L. melanocephala (black-headed bushmaster) was pooled from two adult specimens maintained at Instituto Nacional de Biodiversidad (San José, Costa Rica). L. muta muta (South American bushmaster) venoms pooled from adult specimens from Colombia, Peru, and Cascalheria and Tucurui regions of Brazil were kindly provided by Dr. María de Fatima D. Furtado (Instituto Butantan, São Paulo, Brazil). Samples of L. muta rhombeata (Atlantic forest bushmaster) venom pooled from adult specimens were a generous gift from Dr. María de Fatima D. Furtado of Instituto Butantan and from Instituto Vital Brazil (IVB, Niterói, Rio de Janeiro, Brazil). All venoms were lyophilized and stored at -20˚C until used.
Commercial polyspecific BCL antivenom (batch 4800611POLQ) was manufactured by the Industrial Division of Instituto Clodomiro Picado (San José, Costa Rica) from the plasma of horses hyperimmunized with a mixture of venoms of Costa Rican Bothrops asper, Crotalus simus and Lachesis stenophrys [30,31], and consists of whole IgGs purified by caprylic acid fractionation [32]. BL antivenom (batch 125901) from Instituto Vital Brazil (Niterói, RJ, Brazil) was produced in horses hyperimmunized with a mixture of venoms from L. m. rhombeata and a mixture of five bothropic species, B. jararaca (50%), B. jararacussu (12.5%), B. moojeni (12.5%), B. alternatus (12.5%) and B. neuwiedi (12.5%), and consists of purified F(ab') 2 fragments generated by digestion with pepsin of ammonium sulfate-precipitated IgG molecules [33]. Experimental monospecific AB, AC, and AL antivenoms were prepared by the Industrial Division of Instituto Clodomiro Picado from plasma of horses subjected to a single round of immunization with venoms of Costa Rican adult B. asper (from the Pacific and Caribbean versants of Costa Rica), adult C. simus, and adult L. stenophrys, respectively, as described [33]. These monospecific antivenoms are also whole IgG preparations prepared by caprylic acid precipitation [33]. BCL and BL antivenoms are used therapeutically in the clinical management of Lachesis envenomings in Central America and Brazil, respectively. Monospecific AC, AB and AL antivenoms were developed for experimental use. For in vitro and in vivo assays the protein concentration of antivenoms was adjusted to 50 mg/mL.

Two-dimensional electrophoresis
L. stenophrys venom proteins were separated by two-dimensional electrophoresis (2DE) using an Ettan IPGphor III instrument (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). For isoelectric focusing, 300-350 μg of total venom proteins in 200 μL DeStreak Rehydration Solution (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) including 10 mM DTT and 0.5% IPG buffer pH 3-10 NL (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were loaded on a 11 cm IPG strip, pH 3-10 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and then focused using the following electrophoretic conditions: 500 V for 30 min, 1000 V for 30 min and 5000 V for 80 min. After isoelectric focusing, SDS-PAGE was performed under reducing conditions in 4-15% Criterion TGX precast 11 cm gels (Bio-Rad, USA). An unstained protein molecular weight marker (Fermentas) was included in the analysis. Gels were stained using Bio-Safe Coomassie Stain (Bio-Rad, USA) or PlusOne Silver Staining Kit (GE Healthcare AB, Uppsala, Sweden) following the manufacturer´s instructions, and images were taken with Chemidoc XRS imaging system (BioRad, USA). Spot identification was done using the collaborative bioimage informatics platform Icy [34] and quantified as relative density percentage using ImageJ software [35].

Western blot analysis
2DE gels of 350 μg L. stenophrys venom proteins were transferred to polyvinylidene fluoride (PVDF) membranes at 50 mA in a Criterion Blotter instrument (Bio-Rad, USA) overnight. To assess transfer efficiency, PVDF membranes were previsualized by reversible Ponceau-S Red staining. Unoccupied membrane protein-binding sites were blocked with 2% casein in TBS-T (Tris-buffered saline with Tween 20, pH 7.6) for 30 min at room temperature, and the membranes were incubated for 1 h with 1/1000 dilution of antivenoms in TBS-T containing 1% casein. After five washing steps (5 min each) with TBS-T, the membranes were incubated for 1 h at room temperature with rabbit anti-horse IgG-peroxidase conjugate (1:15000 dilution; Sigma-Aldrich, St. Louis, USA). Purified antibodies from non-immunized horses were used as control. After washing off unbound secondary antibodies, the immunoreactive spots were visualized using a chemiluminescence substrate (Invitrogen, USA). Images were taken with Chemidoc XRS imaging system (BioRad, USA) and protein spots of interest were analyzed using ImageJ software.

Protein identification by MALDI-TOF-TOF MS
2DE protein spots were excised and subjected to reduction (10 mM dithiothreitol), alkylation (50 mM iodoacetamide), and overnight in-gel digestion with sequencing grade trypsin (Sigma), in 50 mM ammonium bicarbonate at 37˚C. Tryptic peptide digests were extracted in 50% acetonitrile containing 1% trifluoroacetic acid (TFA), and analyzed by MALDI-TOF-TOF MS using an AB4800-Plus Proteomics Analyzer (Applied Biosystems). To this end, tryptic digests were mixed with an equal volume of α-cyano-hydroxycinnamic acid saturated in 50% acetonitrile, 0.1% TFA, and 1 μL spotted onto an Opti-TOF 384-well plate, dried, and analyzed in positive reflector mode. TOF MS spectra were acquired using 500 shots at a laser intensity of 3000. TOF/TOF fragmentation spectra were acquired (500 shots at a laser intensity of 3900) for the ten most intense precursor ions. External calibration in each run was performed with CalMix standards (ABSciex) spotted onto the same plate. Fragmentation spectra were searched against the UniProt/SwissProt database (taxonomy: Serpentes) using the ProteinPilot v.4 and the Paragon algorithm (ABSciex) at !95% confidence, or manually interpreted and the deduced sequences BLASTed against the NCBI (http://blast.ncbi.nlm.nih.gov) non-redundant database for protein class assignment by similarity.

Antivenomics
The immunoreactivity of poly-and monospecific antivenoms towards the different Lachesis venoms was assessed using a second-generation antivenomics approach [36]. To prepare the antivenom affinity column, 200 μL of NHS-activated Sepharose 4 Fast Flow (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) matrix was packed in a Pierce centrifuge column and washed with 15 matrix volumes of cold 1 mM HCl followed by two matrix volumes of 0.2 M NaHCO 3 , 0.5 M NaCl, pH 8.3 (coupling buffer) to adjust the pH of the column to 7.0-8.0. Antivenoms were dialysed against MilliQ water, lyophilised, and reconstituted in coupling buffer. The concentration of the antivenom stock solutions was determined spectrophotometrically using an extinction coefficient of 1.36 for a 1 mg/mL concentration of Fab at 280 nm using a 1 cm light pathlength cuvette. Twenty milligrams of polyspecific BCL antivenom, 15 mg of BL antivenom, and 35-50 mg of monospecific AC, AB and AL antivenoms were dissolved in a half matrix volume of coupling buffer and incubated with the matrix for 4 h at room temperature. Antivenom coupling yield was estimated measuring the non-bound antivenom by quantitative band densitometry of SDS-PAGE (MetaMorph software, MDS Analytical Technologies) using as standard for the linear range the pre-coupled antivenom. After the coupling, any remaining active groups were blocked with 200 μL of 0.1 M Tris-HCl, pH 8.0 at 4˚C overnight using an orbital shaker. The affinity column was washed alternately at high and low pH, with three volumes of 0.1 M acetate buffer, 0.5 M NaCl, pH 4.0-5.0 and three volumes of 0.1 M Tris-HCl buffer, pH 8.5. This treatment was repeated six times and the column was equilibrated in binding buffer (20 mM phosphate, 135 mM NaCl, pH 7.4, PBS). For the immunoaffinity assay, 200 μg of venoms from L. stenophrys (Costa Rica), L. melanocephala (Costa Rica), L. m. rhombeata (Recife, Brazil), and L. m. muta from Colombia, Peru and Brazil (Tucurui and Cascalheira regions), dissolved in 1⁄2 matrix volume of PBS, were loaded and incubated for 1 h at room temperature with the affinity matrix, followed by incubation in an orbital shaker overnight at 4˚C. As specificity controls, 200 μL of Sepharose 4 Fast Flow matrix, without or with 8.5 mg of immobilized pre-immune IgGs, were incubated with venom and developed in parallel to the immunoaffinity columns. Non-retained fractions were collected with 5 matrix volumes of PBS, and the immunocaptured proteins were eluted with 5 matrix volumes of elution buffer (0.1 M glycine-HCl, pH 2.0) and neutralised with 150 μL 1 M Tris-HCl, pH 9.0. The non-retained and the immunocaptured venom fractions were lyophilized, reconstituted in 40μl of MilliQ water, and fractionated by reverse-phase HPLC using a Discovery BIO Wide Pore C 18 (15 cm x 2.1 mm, 3 μm particle size, 300 Å pore size) column using an Agilent LC 1100 High Pressure Gradient System equipped with a DAD detector and microauto sampler. The column was developed at a flow rate of 0.4 mL/min and proteins eluted with a linear gradient of 0.1% TFA in MilliQ water (solution A) and 0.1% TFA in acetonitrile (solution B): isocratic at 5% solution B for 1 min, followed by 5-25% solution B for 5 min, 25-45% solution B for 35 min, and 45-70% solution B for 5 min. Protein was detected at 215 nm with a reference wavelength of 400 nm.

Neutralization of venom activities
Polyspecific and monospecific antivenoms were assessed for their ability to neutralize the lethal, hemorrhagic, coagulant and proteolytic activities of venoms. The protein concentration of all antivenoms was adjusted to 50 mg/mL, as determined using a NanoDrop 2000 (Thermo Fischer Scientific, DE, USA). For the neutralization assays, a fixed dose of venom ("challenge dose"), dissolved in PBS, was incubated with various dilutions of antivenom. Controls including venom solutions incubated with PBS instead of antivenom were used. The venom/antivenom mixtures and controls were incubated for 30 min at 37˚C and then tested in the experimental systems described below and detailed in previous publications [37,38]. Neutralizing ability was expressed as Median Effective Dose (ED 50 ), defined as the μL antivenom/mg venom ratio in which the activity of venom was reduced by 50% [39]. In the case of coagulant activity, neutralization was expressed as Effective Dose (ED), defined as the antivenom/venom ratio in which the clotting time of plasma was prolonged three times when compared with clotting time of plasma incubated with venom alone [40].
All the in vivo experiments were performed in CD-1 mice, and the protocols were approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUA) of the University of Costa Rica. Lethality was assessed by the intraperitoneal route in 16-18 g mice and a challenge dose corresponded to 3 Median Lethal Doses (LD 50 ) was used for the neutralization tests [37]. An arbitrary level of 500 μL antivenom/mg venom was selected to evaluate the efficacy of antivenoms for neutralizing lethality. Only this antivenom/venom ratio was used owing to the scarcity of some venoms and also for reducing the number of mice used.  Table 1.

Statistical analyses
The results of neutralization assays of venom activities were compared by ANOVA, followed by Tukey test for specific comparisons between means of pairs of groups. A p value <0.05 was

Results and discussion
Venom represents a trophic adaptive trait that plays key roles in the organismal ecology and evolution of advanced snakes [43,44]. The well-documented geographic variability of snake venoms at all taxonomic levels [45] may contribute to the snake's capability to adapt to different ecological niches, but at the same time imposes an added difficulty to the production of antidotes to counteract the toxic activities of snakebite envenomings. The parenteral administration of an effective antivenom constitutes the mainstay in the treatment of snakebite envenomings [46,47]. Defining the geographic boundaries of the efficiency of an antivenom, even against disjunct populations of the same nominal species, has implications for its rational and efficient use. In this regard, unveiling the immunological profile of an antivenom towards the landscape of congeneric venom phenotypic variation provides the necessary knowledge-informed ground for assessing whether it is clinically justified the generation of a new antivenom for a specific geographic region, or if the deployment of an existing antivenom to a new geographical setting can be recommended. This is particularly relevant for widely distributed species, such as those comprising the genus Lachesis, which can be found in disjunct habitats ranging from the Caribbean coast of Central America to the Atlantic rainforest of Brazil (see Fig 1 in [24]). The combination of venom neutralization tests and antivenomics constitutes a powerful toolbox for evaluating an antivenom's preclinical efficacy [48][49][50][51]. Using this platform we have investigated the capability of two therapeutic polyvalent antivenoms and three experimental monospecific antivenoms to neutralize the hemorrhagic, coagulant, proteolytic and lethal activities of homologous and heterologous Lachesis venoms.

Immunoreactivity profile of Lachesis antivenoms by ELISA and 2DE immunoblotting analysis
Initial assessment of the immunoreactivity of the commercial polyspecific BCL and BL antivenoms, and the experimental monospecific B, C and L antivenoms, against antigens present in the venoms of Costa Rican L. stenophrys and L. melanocephala, Brazilian L. m. rhombeata (Recife) and L. m. muta from different geographic locations (Colombia, Peru, and Brazil [Cascalheira and Tucurui]) were done by ELISA and 2DE immunoblotting analysis.
No significant differences were found in the levels of specific antibodies against Lachesis venoms present in the BCL antivenom, and the AB and AL antivenoms (S1 Fig). The highest titer corresponded to the binding of BL antivenom to L. stenophrys, L. m. muta (Colombia), L. m. muta (Cascalheira), and L. m. rhombeata (Recife) venoms, whereas the titer of this antivenom against venoms from L. melanocephala, L. m. muta (Peru) and L. m. muta (Tucurui) was indistinguishable from that of the BCL antivenom (S1 Fig). Monospecific AC antivenom exhibited the lowest reactivity against the seven Lachesis venoms analyzed (S1 Fig). The spectrum of L. stenophrys toxins immunorecognized by the poly-and monospecific antivenoms was investigated by 2DE and immunoblot analysis. Fig 1A displays a 2DE reference map and the MALDI-TOF-TOF MS protein assignments are listed in S1 Table. In concordance with ELISA results, Western blot analyses revealed extensive protein spot recognition by all the five antivenoms (Fig 1B and S2 Table), particularly for spots in the range of 25-35 kDa (serine proteinases, SVSPs) and 14-16 kDa (phospholipases A 2 (PLA 2 ) and Galactose-binding lectin). Polyspecific BL and monospecific AB antivenoms showed also strong immunoreactivity towards protein spots of apparent molecular mass 55-80 kDa, which were identified as snake venom metalloproteinases of class PIII (PIII-SVMP) and L-amino acid oxidase (LAO) molecules. However, all the antivenoms showed weak  immunostaining of spot 64 containing the major SVMP of class PI (PI-SVMP). Weak immunorecognition of PI-SVMPs has been also reported for other antibothropic antivenoms [52,53].

Immunoreactivity profile of antivenoms by immunoaffinity-based antivenomics
Second generation antivenomics [36] was applied to complement the ELISA and Western blot analyses of the immunoreactivity of the lachesic antivenoms towards the panel of Lachesis venoms used for this study. Figs 2-8 display the immunoaffinity chromatography-based antivenomic profiles of commercial BCL and BL polyspecific antivenoms and monospecific B, L, and C experimental antivenoms towards the venoms of L. stenophrys and L. melanocephala from Costa Rica, L. muta muta from Colombia, Peru, and the Brazil regions of Cascalheria, Tucurui, and L. muta rhombeata from Recife, Brazil. The results show impaired immunocapturing ability of the early eluting chromatographic fractions comprising bradykinin-potentiating-like peptides (BPP-like) by all the antivenoms. Although together these fractions account for about 1/3 by weight of total venom components, previous investigations have shown that the intraperitoneal administration of an amount of BPP-like peptides contained in 10-24 LD 50 s of venom induced neither a significant change in the mean arterial blood pressure of mice, nor signs of abnormal behavior, or histopathological alterations in heart and lungs [25]. These observations strongly suggest that, despite being a major venom component, the BPP-  retained (B, D, F, H, and J) fractions recovered, respectively, from the affinity columns of immobilized BCL, BL, AL, AB, and AC antivenoms. Panels K and L, non-retained and retained venom fractions by immobilized equine control immunoglobulins. BPP, bradykinin-potentiating peptide; DISI, disintegrin; VEGF, vascular endothelial growth factor; PLA 2 , phospholipase A 2 ; Gal, galactose-binding lectin; SVSP, snake venom serine proteinase; LAO, L-amino acid oxidase; PI and PIII, snake venom metalloproteinase (SVMP) of class PI and PIII, respectively.
https://doi.org/10.1371/journal.pntd.0005793.g002 like peptides by themselves may not represent a serious clinical concern in the treatment of Lachesis envenomings. The interpretation of these results has to consider that certain antigens may become denatured during reverse-phase separation and, henceforth, some conformational epitopes might be lost.
Except for the BPPs, both polyspecific antivenoms efficiently immunocaptured all the components from L. stenophrys (Fig 2), L. m. muta (Colombia) (Fig 4) and L. m. rhombeata (Fig 8) venoms. In addition, the BL antivenom immunocaptured the venom components of L. m. muta from the Brazilian localities Cascalheira (Fig 6) and Tucurui (Fig 7). The apparent low recovery of PI-and PIII-SVMPs (eluting from the RP-HPLC column at 40-42 min) in the immunoaffinity captured fractions of the BCL and BL affinity columns (Figs 2-8, panels B and D, respectively) may be ascribed to the high affinity of these venom proteins for the antivenom molecules, as has been demonstrated in a previous work [25].
Monospecific antivenoms showed significantly more limited immunorecognition profiles than BCL and BL antivenoms toward venoms of all Lachesis taxa investigated. The three  Although a comparison of the levels of immune recognition gathered from antivenomics with the in vivo neutralization capacity of an antivenom is not straightforward, since both experiments involve radically different protocols, in our experience, an immunocapturing capability of !25% of total viperid venom proteins correlates with a good outcome in in vivo neutralization tests [48-51]. As a whole, the antivenomics evidence reinforce our view that both polyspecific BCL and BL antivenoms are likely to be effective in the neutralization of heterologous congeneric venoms, thus supporting their use for the treatment of Lachesis envenomings throughout the range of distribution of these snakes. In addition, the fact that antivenoms BCL, and particularly BL, are more effective than the monospecific AL antivenom, even against the homologous L. stenophrys venom, seems to indicate that the inclusion of botropic venoms in the immunization mixture aided in the generation of antibodies exhibiting paraspecificity against Lachesis toxins. This combination of immunogens seems to be a more appropriate formulation than a single venom for the treatment of envenomings by Lachesis species.  Neutralization of enzymatic and toxic activities of Lachesis venoms Standard neutralization assays were performed to assess the extent of neutralization of proteolytic, hemorrhagic, procoagulant and lethal activities [39][40][41]. Despite the fact that all antivenoms were standardized for having a protein concentration of 50 mg/mL, two aspects need to be considered when comparing the values of neutralization experiments: (a) Most antivenoms are made of whole IgG molecules, whereas one of them is made of F(ab') 2 fragments; hence, for the same amount of protein, the number of molecules present in an IgG antivenom is 1.5 times lower than in a F(ab') 2 antivenom; and (b) only an unknown proportion of all IgGs or F (ab') 2 fragments are specific against venom components. Hence, quantitative conclusions drawn by comparing the neutralizing abilities of different type of antivenoms should be regarded as gross estimates.

Proteolytic activity
The BCL and the BL therapeutic antivenoms, and the monospecific AL antivenom effectively neutralized the proteolytic activity of venoms from the 7 Lachesis taxa investigated ( Table 2). The BL antivenom showed higher neutralization activity than the other antivenoms used in this study ( Table 2). The AC monospecific antivenom was only able to neutralize the proteolytic activity of L. melanocephala venom ( Table 2). The AB monospecific antivenom was unable to neutralize the proteolytic activity of any of the venoms ( Table 2).

Hemorrhagic activity
The BCL and BL antivenoms, and the monospecific L antivenom effectively neutralized the hemorrhagic activity of all the Lachesis venoms studied ( Table 3). The BL antivenom showed the highest neutralization capacity of the hemorrhagic activity than any of the other antivenoms used in this study ( Table 3). The monospecific AB antivenom was only able to neutralize the hemorrhagic activity of L. stenophrys and L. melanocephala venoms, whereas the monospecific AC antivenom was unable to neutralize the hemorrhagic activity of any of the venoms (Table 3).

Coagulant activity
The BCL and BL polyspecific antivenoms, and the monospecific L antivenom effectively neutralized the coagulant activity of Lachesis venoms from the seven bushmaster taxa sampled ( Table 4). The BL antivenom showed the highest coagulant neutralization activity than any of the other antivenoms use in this study (Table 4). On the other hand, neither the AB nor the AC monospecific antivenoms were able to neutralize the coagulant activity of any of the Lachesis venoms used in this study (Table 4). These data agree with a previous work showing the inefficacy of monospecific bothropic antivenom in the neutralization of the coagulation activity of L. m. muta venom [54].

Lethal activity
At the antivenom/venom ratio of 500 μL antivenom/mg venom, the BCL and BL polyspecific antivenoms and the monospecific L antivenom, effectively neutralized the lethal activity of the  seven Lachesis venoms investigated ( Table 5). The monospecific AB antivenom only neutralized the lethal activity of L. stenophrys, L. muta muta (Cascalheira) and L. muta rhombeata (Recife) ( Table 5), while the monospecific AC antivenom was unable to neutralize the lethal activity of any of the Lachesis venoms studied at the ratio of 500 μL antivenom/mg venom (Table 5).

Concluding remarks
Snakes from the Lachesis genus cause severe envenomings in humans and are widely distributed in a variety of habitats ranging from the Caribbean coast of Central America to the Atlantic rainforest of Brazil. Based on the high conservation of the overall protein composition of Lachesis venoms and their qualitatively similar pathophysiological profile observed in experimental envenomings and clinical settings we have suggested that antivenoms generated against any conspecific Lachesis venom may exhibit paraspecific protection against the toxic activities of all other Lachesis species. Combining immunochemical methods, second generation antivenomics, and venom neutralization tests we have unveiled the efficacy of two therapeutic polyvalent antivenoms and three experimental monospecific antivenoms to recognize the complete proteomes and neutralize the hemorrhagic, coagulant, proteolytic and lethal activities from three different Lachesis species from different geographic populations. The results demonstrate that antivenoms raised by immunizing horses with the venoms of different Lachesis species are effective in the neutralization of congeneric venoms  not used in the immunization mixture, indicating that they could be used equivalently for the clinical treatment of any lachesic envenoming. Owing to the similar clinical presentations of envenomings by Lachesis sp. and Bothrops sp., the use of polyvalent antivenoms which include the Lachesis component is therefore recommended in Latin America.