Third-Generation Antivenomics Analysis of the Preclinical Efficacy Of

Toxicon: X 1 (2019) 100004

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Third-generation antivenomics analysis of the preclinical efficacy of ® T Bothrofav antivenom towards Bothrops lanceolatus venom ∗ Davinia Plaa, Yania Rodrígueza, Dabor Resiereb, Hossein Mehdaouib, José María Gutiérrezc, , ∗∗ Juan J. Calvetea, a Laboratorio de Venómica Evolutiva y Traslacional, Instituto de Biomedicina de Valencia, CSIC, Jaime Roig 11, 46010 Valencia, Spain b Service des Urgences et de Reanimation Polyvalente, Centre Hospitalier Universitaire, Fort-de-France, Martinique, France c Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica

ARTICLE INFO ABSTRACT

® Keywords: Bothrops lanceolatus inflicts severe envenomings in the Lesser Caribbean island of Martinique. Bothrofav , a Bothrops lanceolatus monospecific antivenom against B. lanceolatus venom, has proven highly effective at the preclinical and clinical ® Antivenom levels. Here, we report a detailed third-generation antivenomics quantitative analysis of Bothrofav . With the Snake venom ® ® exception of poorly-immunogenic peptides, Bothrofav immunocaptured all the major protein components. Bothrofav These results, along with previous preclinical and clinical observations, underscore the high neutralizing efficacy Third-generation antivenomics of the antivenom against B. lanceolatus venom. Martinique

Bothrops lanceolatus is an endemic viperid snake in the French and therapeutic antibody molecules present in an antivenom. Thus, overseas Department of Martinique, in the Lesser Caribbean, where it neutralization assays and antivenomics complement each other in the represents the only venomous snake (Campbell and Lamar, 2004). En- characterization of the preclinical efficacy of antivenoms (Gutiérrez venomings by this species are similar to those inflicted by other Bo- et al., 2017). To this end, an antivenomics analysis has been made with throps sp, which are characterized by local effects (edema, pain, he- the venom of B. lanceolatus and the monospecific antivenom in order to ® morrhage, necrosis) and by systemic alterations associated with further characterize the preclinical performance of Bothrofav . hemorrhage and hemodynamic disturbances. In contrast to the majority Third-generation antivenomics (Pla et al., 2017) was applied to ® of Bothrops sp-induced cases, about 30% of people suffering envenom- assess the immunoreactivity of Sanofi Pasteur Bothrofav antivenom ings by B. lanceolatus, if not treated with antivenom, develop severe (batch J8216) against venom from B. lanceolatus, whose proteome has thrombosis that may lead to cerebral, pulmonary, myocardial or me- been previously characterized (Gutiérrez et al., 2008). To this end, the ® senteric infarctions (Thomas et al., 1995; Warrell, 2004; Resiere et al., content of one vial (10 mL) of antivenom was dialyzed against MilliQ

2010). water, lyophilized, and reconstituted in 10 mL of 0.2 M NaHCO3, 0.5 M ® ® A monospecific antivenom against B. lanceolatus venom, Bothrofav NaCl, pH 8.3 (coupling buffer). The monospecific antivenom Bothrofav manufactured by Sanofi-Pasteur, was introduced in Martinique in 1991 produced by Sanofi Pasteur (Lyon, France) is an F(ab′)2 preparation and has proven highly effective in preventing the development of obtained by pepsin digestion and ammonium sulphate fractionation of thrombosis and other systemic disturbances in these envenomings hyperimmune plasma from horses immunized with the venom of B. (Thomas et al., 1995). Preclinical assessment of the neutralizing effi- lanceolatus. The concentration of the antivenom stock solution cacy of two batches of this antivenom has corroborated its efficacy in (207 mg/mL) was determined spectrophotometrically using an extinc- the neutralization of the most important toxic and enzymatic activities tion coefficient for a 1 mg/mL concentration (ε0.1%) at 280 nm of 1.36 of B. lanceolatus venom (Bogarín et al., 1999; Resiere et al., 2018). (mg/mL)−1 cm−1 (Howard and Kaser, 2014). Antivenomics has become a highly valuable tool to assess the ability of The antivenom affinity matrix was prepared in batch. Three mLof antivenoms to recognize specific venoms components previously iden- CNBr-activated Sepharose™ 4B (GE Healthcare, Buckinghamshire, UK) tified by mass spectrometry (Calvete et al., 2018). In addition, anti- were packed in a ABT column (Agarose Bead Technologies, Torrejón de venomics provides a quantitative view of the amount of toxin-binding Ardoz, Madrid) and washed with 10× matrix volumes of cold 1 mM

∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (J.M. Gutiérrez), [email protected] (J.J. Calvete). https://doi.org/10.1016/j.toxcx.2018.100004 Received 6 September 2018; Received in revised form 13 December 2018; Accepted 21 December 2018 Available online 29 December 2018 2590-1710/ © 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). D. Pla et al. Toxicon: X 1 (2019) 100004

Fig. 1. Third Generation Antivenomics. Panel A, reverse-phase HPLC profile of 300 μg of B. lanceolatus venom. The identification of proteins eluting in the various chromatographic peaks were described previously (Gutiérrez et al., 2008). Panels B to I correspond to the non-immunoretained fractions after incubation of im- ® munoaffinity columns containing 10 mg of Bothrofav antivenom with varying amounts (0.3–1.8 mg) of venom. Panels J to L: Specificity controls with mock chromatographic matrix and naïve horse IgG. DISI, disintegrins; DC, disintegrin-like/cysteine-rich fragment; PLA2, phospholipases A2; SP, serine proteinases; CTL, C- type lectin-like proteins; PI and PIII SVMPs, metalloproteinases. See text for details.

HCl, followed by two matrix volumes of coupling buffer to adjust the with 100–300, 600, 900, 1200, 1500 and 1800 μg venom were re- pH to 7.0–8.0. CNBr-activated instead of N-hydroxysuccinimide (NHS)- covered with 5×, 10×, 15×, 20×, 25× and 30× matrix volumes of activated matrix (Pla et al., 2012) was employed because NHS released PBS, respectively. The immunocaptured proteins were eluted, respec- during the coupling procedure absorbs strongly at 280 nm, thus inter- tively, with 5×, 10×, 15×, 20×, 25× and 30× matrix volume of fering with the measurement of the concentration of unbound anti- 0.1 M glycine-HCl, pH 2.7 buffer, and the eluates were brought to bodies remaining in the supernatant of the coupling solution used to neutral pH with 1 M Tris-HCl, pH 9.0. To avoid saturation of the estimate the coupling yield. Eighty mg of antivenom were dissolved in downstream reverse-phase chromatographic analysis, aliquots corre- 2× matrix volume of coupling buffer and incubated with 3 mL of CNBr- sponding to 100–300 μg of initial total venom proteins were con- activated matrix for 4 h at room temperature (∼25 °C). Antivenom centrated in a Savant™ SpeedVac™ vacuum system (ThermoFisher Sci- coupling yield, estimated measuring A280nm before and after incubation entific, Waltham, MA USA) to 40 μL and fractionated by reverse-phase with the matrix, was 26 mg/mL. After the coupling, remaining active HPLC using an Agilent LC 1100 High Pressure Gradient System (Santa ® matrix groups were blocked with 12 mL of 0.1 M Tris-HCl, pH 8.5 at Clara, CA, USA) equipped with a Discovery BIO Wide Pore C18 room temperature for 4 h. Affinity columns, each containing 10.0 mgof (15 cm × 2.1 mm, 3 μm particle size, 300 Å pore size) column and a immobilized antivenom F(ab')2 fragments, were alternately washed DAD detector as above. The column was developed at a flow rate of ® with three matrix volumes of 0.1 M acetate containing 0.5 M NaCl, pH 0.4 mL/min with a linear gradient of 0.1% TFA in MilliQ water 4.0–5.0, and three matrix volumes of 0.1 M Tris-HCl, pH 8.5. This (Merck-Millipore, Darmstadt, Germany) (solution A) and 0.1% TFA in procedure was repeated 6 times. The columns were then equilibrated acetonitrile (solution B), isocratic (5% B) for 1 min, followed by 5–25% with 5 volumes of working buffer (PBS, 20 mM phosphate buffer, B for 5 min, 25–45% B for 35 min, and 45–70% B for 5 min. Eluate was 135 mM NaCl, pH 7.4), incubated with increasing amounts monitored at 215 nm with a reference wavelength of 400 nm. The (100–1800 μg of B. lanceolatus venom proteins dissolved in ½ matrix fraction of non-immunocaptured molecules was estimated as the re- volume of PBS), and the mixtures incubated for 1 h at 25 °C in an orbital lative ratio of the chromatographic areas of the toxin recovered in the shaker. As specificity controls, 400 μL of CNBr-activated Sepharose™ 4B non-retained (NR) and retained (R) affinity chromatography fractions matrix, without (mock) or with 10 mg of immobilized control (naïve) using the equation %NRi = 100-[(Ri/(Ri+NRi)) x 100], where Ri horse IgGs (purified by caprylic acid precipitation from the serumof corresponds to the area of the same protein “i” in the chromatogram of non-immunized horses provided by a local slaughterhouse), were in- the fraction retained and eluted from the affinity column. cubated with 1200 μg of venom and developed in parallel to the im- Fig. 1 depicts the RP-HPLC profiles of the venom fractions that were munoaffinity columns. The non-retained eluates of columns incubated retained and non-retained when passing increasing amounts (ranging

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Table 1 Total and concentration-dependent immunoretained (RET) B. lanceolatus (Martinique) venom proteins by Sanofi-Pasteur antivenom affinity column (10mg ® Bothrofav ). Maximal binding for each RP-HPLC fraction is highlighted in yellow background. “μg TOTAL” was calculated by multiplying the % of the total venom proteome of the RP-HPLC fraction by (n/100), where n = total venom proteins (in mg) used in the antivenomic analysis. “μg RET” was calculated as specified in the manuscript's main text using the equation %NRi = 100-[(Ri/(Ri+NRi)) × 100], where NRi and Ri correspond to the areas of the same protein “i” in the chromatogram of the non-retained and retained fractions eluted from the affinity column.

from 300 to 1800 μg) of B. lanceolatus venom through identical im- proteins, whereas fractions 7 and 9 (serine proteinases), 12 and 13 (C- munoaffinity chromatography columns. With the exception of thefirst type lectin-like proteins), and 15 (P-I metalloproteinase) reached three peaks, comprising small peptides (Gutiérrez et al., 2008), the rest maximal binding to the immobilized F(ab')2 fragments at 1200 μg of the fractions were retained, although to a varied extent, in the im- venom, and fractions 5 (phospholipase A2), 6 and 8 (serine protei- munoaffinity columns. The pathophysiological relevance of these nases), 14 and 15 (P-III metalloproteinases), and 17 (disintegrin-like/ poorly immunogenic peptides has not been addressed in the current cysteine-rich (DC) fragment), saturated their binding sites at study. However, intraperitoneal administration of similar early-eluting 1500–1800 μg of incubated venom (Table 1). When maximum binding peptide fractions from Lachesis venoms induced neither a significant capacity was expressed as the percentage of venom protein contained in change in the mean arterial blood pressure of mice, nor signs of ab- the corresponding chromatographic peak, all fractions, with the ex- normal behavior, or histopathological alterations (Pla et al., 2013). ception of the peptides eluted in fractions 1 through 3, were im- ® Preclinical in vivo evidence showing that the Bothrofav antivenom is munocaptured with efficiency ranging from 61% (fraction 13) to97% quite effective in the neutralization of toxic activities of B. lanceolatus (fraction 17). venom (Resiere et al., 2018) further supports the clinical irrelevance of Ten milligrams of immobilized antivenom F(ab')2 fragments had B. lanceolatus peptide fractions. maximal binding capacity of 843.1 μg total B. lanceolatus venom pro-

Concentration-dependent binding capacities of the antivenom teins (84.3 mg venom/g F(ab')2). Since the protein concentration of this ® column for each venom fraction, expressed as the amount of μg cal- batch of Bothrofav was 207 mg/mL, and the vial contained 10 mL, the culated for the corresponding chromatographic peak, are listed in total amount of venom proteins bound per vial was 174.5 mg. Table 1. RP-HPLC fractions were assigned to venom proteins by com- Considering an average molecular mass for B. lanceolatus toxins of parison with a previous venomics analysis (Gutiérrez et al., 2008). 32 kDa (calculated as ∑ (% i × Mi), where % i is the relative abundance Fractions 4 (disintegrins), and 10 and 11 (C-type lectin-like proteins) of toxin “i” and Mi its molecular mass in Da), 174.5 mg venom equals saturated their antibody combining sites at 900 μg of total venom 5.5 μmoles of venom molecules. Assuming that the two antigen binding

3 D. Pla et al. Toxicon: X 1 (2019) 100004

sites of an F(ab')2 molecule were occupied at maximal antigen binding of this antivenom in envenomings by B. lanceolatus in Martinique ® capacity, a Bothrofav vial contained (5.5/2) μmoles of toxin-binding (Thomas et al., 1995; Resiere, unpublished observations). molecules, or 300 mg F(ab')2 (molar mass, 110 g/mol). This figure ® corresponds to 14.5% [(300/2070) x 100] of the total Bothrofav F Ethical statement (ab')2 molecules. In a previous study, Resiere et al. (2018) determined the neu- This study was performed following standard ethical procedures of ® tralizing ability of Bothrofav antivenom against the lethal effect of B. scientific research in the experimental analyses and in the preparation lanceolatus venom. This allowed us to calculate the antivenom's potency of the manuscript. (P), which corresponds to the amount of venom (mg) completely neutralized per mL of antivenom, using the formula: Acknowledgments

P= [(n 1)/ED50 ]× LD 50 , This work was partly funded by grant BFU2013-42833-P (Ministerio where “n” is the number of Median Lethal Doses (LD50s) used as chal- de Economía y Competitividad, Madrid, Spain) and BFU2017-89103-P lenge dose to determine the antivenom Median Effective Dose, ED50. (Ministerio de Ciencia, Innovación y Universidades, Madrid, Spain) and For the calculation of P, LD50 and ED50 are expressed, respectively, as by Vicerrectoría de Investigación (Universidad de Costa Rica). (mg venom/mouse) and (mL of antivenom that protect 50% of the mouse population injected with n × LD50). In the calculation of P, (n- Transparency document 1) × LD50 is used instead of the total amount of venom, n × LD50, because at the endpoint of the neutralization assay, one LD50 remains Transparency document related to this article can be found online at non-neutralized and causes the death of 50% of mice (WHO, 1981; https://doi.org/10.1016/j.toxcx.2018.100004. Morais et al., 2010). In this particular case, since the ‘challenge dose’ of venom in the lethality assay corresponds to 5 Median Lethal Doses References (LD50), and the LD50 of this venom by the i.v. route is 6 μg venom per gram body weight, the total amount of venom injected in 18–20 g mice Bogarín, G., Romero, M., Rojas, G., Lutsch, C., Casadamont, M., Lang, J., Otero, R., in this test is 114 μg. The ED50 of the antivenom is 12 mg antivenom per Gutiérrez, J.M., 1999. Neutralization, by a monospecific Bothrops lanceolatus anti- mg venom (Resiere et al., 2018), i.e. with an antivenom protein con- venom, of toxic activities induced by homologous and heterologous Bothrops snake venoms. Toxicon 37, 551–557. centration of 207 mg/mL, the ED50, expressed as mL antivenom per Calvete, J.J., Rodríguez, Y., Quesada-Bernat, S., Pla, D., 2018. Toxin-resolved anti- challenge dose of venom (i.e., 5 LD50s = 0.57 mg venom), is 0.033 mL. venomics-guided assessment of the immunorecognition landscape of antivenoms. Therefore, the neutralizing potency of the antivenom can be estimated Toxicon 148, 107–122. Campbell, J.A., Lamar, W.W., 2004. The Venomous Reptiles of the Western Hemisphere. as: Cornell University Press, Ithaca, New York. Gutiérrez, J.M., Sanz, L., Escolano, J., Fernández, J., Lomonte, B., Angulo, Y., Rucavado, P= [(5 1)/0.033 ]× 0.114 A., Warrell, D.A., Calvete, J.J., 2008. Snake venomics of the Lesser Antillean pit vi- = 13.8 mg venom neutralized per mL antivenom pers Bothrops caribbaeus and Bothrops lanceolatus: correlation with toxicological activities and immunoreactivity of a heterologous antivenom. J. Proteome Res. 7, ® The fraction of the toxin-binding Bothrofav F(ab') molecules that 4396–4408. 2 Gutiérrez, J.M., Solano, G., Pla, D., Herrera, M., Segura, Á., Vargas, M., Villalta, M., contribute to its venom lethality neutralization potency can be calcu- Sánchez, A., Sanz, L., Lomonte, B., León, G., Calvete, J.J., 2017. Preclinical evalua- lated by dividing P by the antivenom's maximal total venom proteins tion of the efficacy of antivenoms for snakebite envenoming: state-of-the-art and binding capacity (17.5 mg/mL): challenges ahead. Toxins 9, E163. Howard, G.C., Kaser, M.R., 2014. Making and Using Antibodies: a Practical Handbook, % toxin-binding and neutralizing Bothrofav® F(ab')2 molecules = second ed. CRC Press, Taylor & Francis Group, Boca Raton (FL) 458 pp. ISBN [(13.8 mg/mL) / (17.5 mg/mL)] × 100 = 79%. 9781439869086. Combining this figure with the above antivenomics-derived per- Morais, V., Ifran, S., Berasain, P., Massaldi, H., 2010. Antivenoms: potency or median ® effective dose, which to use? J. Venom. Anim. Toxins Incl. Trop. Dis. 16,191–193. centage of Bothrofav F(ab')2 molecules bearing affinity towards B. Pla, D., Gutiérrez, J.M., Calvete, J.J., 2012. Second generation snake antivenomics: lanceolatus venom toxins (14.49%) indicates that [(79 × 14.5)/ comparing immunoaffinity and immunodepletion protocols. Toxicon 60, 688–699. ® Pla, D., Sanz, L., Molina-Sánchez, P., Zorita, V., Madrigal, M., Flores-Díaz, M., Alape- 100] = 11.5% of Bothrofav F(ab')2 fragments are clinically relevant Girón, A., Núñez, V., Andrés, V., Gutiérrez, J.M., Calvete, J.J., 2013. Snake venomics antivenom molecules (i.e. may contribute to reverse the effects of the of Lachesis muta rhombeata and genus-wide antivenomics assessment of the para- envenoming). specific immunoreactivity of two antivenoms evidence the high compositional and This study illustrates the analytical value of the third-generation immunological conservation across Lachesis. J. Proteom. 89, 112–123. Pla, D., Rodríguez, Y., Calvete, J.J., 2017. Third generation antivenomics: pushing the antivenomics methodology. 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In: Campbell, J.A., Lamar, W.W. (Eds.), The ments. Our antivenomics observations correlate very well with the high Venomous Reptiles of the Western Hemisphere. Cornell University Press, Ithaca, New capacity of this antivenom to neutralize lethal, local and systemic he- York, pp. 709–761. WHO, 1981. Progress in the Characterization of Venoms and Standardization of morrhagic, edema-forming, thrombocytopenic, phospholipase A2 and Antivenoms. WHO Offset Publication No. 58 World Health Organization, Geneva proteinase activities of B. lanceolatus venom (Resiere et al., 2018). Available from: http://apps.who.int/iris/handle/10665/37282. Further, the preclinical observations agree also with the clinical efficacy