Rev. Biol. Trop. 36 (2B): 511-517, 1988.

Production oí monovalent anti-Bothrops asper antivenom: development oí immune response in horses and neutralizing ability

José María Gutiérrez, Fernando Chaves, Errnila Rojas, Jeannette Elizondo, Claudio Avila and Luis Cerdas. Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.

(Rec. 13-IV-1988. Acep. 21-VIl-1988)

Abstract: A monovalent antivenom was produced by immunizing two horses with venom of the pit viper Bothrops asper (Ophidia: VipeIidae). Although development of the immune response against four tolde and enzymatic activities of the venom was similar in both horses during the fmt two thirds of the immu­ nization schedule, antibody response in one of the horses reached much higher levels in the last part oC the immunization. Immunoelectrophoretic analysis indicates that there were precipitating antibodies in the sera of these horses during all the stages of immunization. However, immunoprecipitation did not correlate with the ability of sera to neutralize toxic activities of B. asper venom. Monovalent antivenom was more effective than the commercially available polyvalent antivenom in the neutralization of Bothrops asper venom. On the other hand, despite the fact that it neutralizes lethal and hemorrhagic activities of the venoms of Lachesia muta and Crotalus durissus durissus, it was less effective than polyvalent antivenom in these neutralizations. Moreover, it does not neutralize defibrinating activity induced by these two venoms, whereas it neutralizes this effect in the case of B. asper venom. It is proposed that monovalent antivenom may be highly effective in the case of envenomations induced by Bothrops asper venom; its use in treating accidents by L. muta and C. durissus would be indicated only if polyvalent antivenom is not available. Results also demonstrate that it is important to monitor antibody response individually in horses being irnmunized fOl antivenom production, due to the conspicuous variability in the response of different animals.

Bothrops asper is responsible for the large majority of snakebite cases in Central America forrning activity (Lomonte 1985; Gutiérrez (Bolaños 1982). Current1y, the polyvalent et al. 1986). antivenom produced at the Instituto Clodo­ In the past years there has been a significant miro Picado in Costa Rica is being used in ,increment in the production of polyvalent several Central American countries to treat antivenom in Costa Rica. If this trend accidents induced by crotaline snakes. 1his continues, there is a potential problem with antivenom is produced in horses by immuniza­ venom supply, since although the reserves of tion with a mixture of equal parts of the B. asper venom are high, those of L. muta venoms of Bothrops asper, Lachesis muta and and e durissus venoms have decreased recent1y Crotalus durissus durissus (Bolaños and Cerdas due to the difficulty in collecting these snakes 1980). Previous studies have demonstrated that in the field. 1his fact, together with the polyvalent antivenom is high1y effective in the observation that the majority of accidents are neutralization of toxic and enzymatic activities inflicted by B. asper, prompted us to produce a of Central American crotaline venoms (Gutié­ monovalent anti-B. asper antivenom in order rrez et al. 1981, 1985, 1987a. Gené et al. to study its neutralizing capacity against 1985, Rojas et al. 1987). The only effect homologous and heterologous crotaline which is poorly neutralized is the edema- venoms.

511 512 REVISTA DE BIOLOGIA TROPICAL

On the other hand, there is a need of more Neutralizatian ai hemarrhage and protealysís: studies dealing with the development of The were determined by experiments with immune response in horses l!sed to produce preincubation of venom and antivenom, as antivenoms. In this work we followed the described by Gutiérrez et al (1985). Neutra­ humoral immune response against toxic and lization was expressed as effective dose 50% enzymatic activities of B. asper venom in (ED50), defmed as the antivenom/venom horses utilized to produce this monovalent ratio that reduces in a 50% the activity of antivenom. venom·alone.

Neatralizatian al indirect hema/ysís: The MATERIAL AND METHODS technique described by Gutiérrez et al. (1988) was used. Venom and antivenom were mixed Venams: The venoms of the snakes Bathrops in different proportions and incubated for 30 asper, Lachesis muta stenaphrys and Crotalus minutes at 370C. Then, samples of the mixtures durissus durissus were obtained from specimens were added to wells made on a gel containig collected in different regions of Costa Rica and 0.8% agarose with erythrocytes, egg yolk maintained at the Instituto Clodomiro Picado. and CaC12. Plates were incubated at 370C Once obtained, venoms were frozen, lyophilized for 20 hours after which the diameter of and kept at -200C. hemolytic at 370C for 20 hours after which the diameter of hemolytic halos were measured. Neutralization was expressed as effective dose Immunizatian schedule and plasma iractiana­ 50%, defined as the antivenom/venom ratio tian: Two adult horses, which had not been that reduces in a 50% the diameter of the halo previously injected with snake venoms, were induced by venom alone. immunized with venom obtained from adult Bathrops asper specimens collected in the Neutralizatían ai edema: The technique of Atlantic region of Costa Rica and kept at the Yamakawa et al. (1976) modified by Gutié­ Instituto Clodomiro Picado. The immunization rrez et al. (1986) was used. Groups of four schedule is shown in Table 1. At different mice (20-22 g) were injected with various times along immunization, blood samples volumes of antivenom (100 pI, 200 pI and were obtained from the jugular vein imme­ 400 ti!) intravenously. Five minutes later, diately before venom injection: sera were animals were injected subcutaneously in the separated, distributed in aliquots and right foot pad with a dose of venom equivalent maintained at -200C until used. At the end to 6 mínimum edema-forming doses dissolved of immunization, horses were bled and plasma in 50 pI of phosphate-buffered saline solution, fractionated by ammonium sulfate precipita­ pH 7.2. The left foot pad was injected with tion. In sorne experiments, the polyvalent 50 pI of saline solution in the same conditions. antivenom produced in Costa Rica (Bolaños Edema was evaluated 6 hours after injection and Cerdas 1980) was used for comparative as described elsewhere (Gutiérrez et al 1986). purposes. Neutralization ai defibrinatíng activity: The Neutralizatian ai lethality: It was determined procedure of Theakston and Reid (1978), by incubating different proportions of venom modified by Gutiérrez et al. (1987), was and antivenom for 30 minutes at 370 C. followed. Several mixtures of venom and Then 0.5 mI of the mixtures (containing antivenom were incubated at 370C for 30 4 LD50 of venom) were injected intrape­ minutes. Then, 0.2 mI (containing two ritoneally in groups of four mice (16-18 g minimum defibrinating doses of venom) were body weight). Deaths were recorded during injected intravenously in the tail vein of groups 48 hours and data were analyzed according of four mi ce (10-22 g). After one hour, mice to the Spearman-Karber method (W.H.O. were anesthetized with ether and bled by 1981). Neutralization was expressed as effective cardiac puncture. After incubation during dose 50% (ED50), defined as the antivenom/ one hour at room temperature, the formation venom ratio which protects 50% of the po­ of dots was observed. Neutralization was pulation. expressed as effective dose, defined as the GUTIERREZ et al.: Monovalent anti-Bothrops asper antivenom 513 lowest antivenom/venom ratio wbich neutra­ antivenom forrned 11 bands against the same venom (Fig. 2). lized defibrinating activity in all mice tested.

Immunoelectrophoresis: Venoms were separat­ Neutralizing capacity o[ antivenom: The final ed by electrophoresis on 1 % agarose gels in monovalent antivenom was tested for its ability 0.025 M barbital buffer, pH 8.2 (Ouchterlony to neutralize lethal, hemorrhagic, defibrinat­ and Nilsson 1978). After electrophoresis, ing and edema-forrning activities of the venom two channels were made in the gel and filled of Bothrops asper, Lachesis muta stenophrys with either polyvalent or monovalent and Crotalus durissu& durissus. Table 3 and ativenom. After 24 hours of diffusion at room fig. 2 show that monovalent antivenom was temperature in a humid chamber, gels were bighIy effective in neutralizing toxic activities washed several times with saline solution and of B. asper venom, with the exception of stained with Amidoblack 10B. The number of edema-forrning effect which was onIy partially precipitin arcs was recorded. For comparison, neutralized. On the other hand, monovalent samples of polyvalent antivenom were tested antivenom was partially effective in neutralizing simultaneously with each sample of monovalent the venoms of L. muta and C. durissus, being antisera. more effective against the first one (Table 3, Fig. 3). Interestingly, monovalent antivenom RESULTS neutralized the lethal effect of the three venoms tested. When compared with the Development o[ immune response: Antibody polyvalent antivenom produced in Costa Rica production against B. asper venom components at the Instituto Clodomiro Picado, the reached higher levels in horse No. 1 than in monovalent antivenom was more effective in horse No. 2, although during the first two the neutralization of B. asper venom, having tbirds of the immunization schedule both a lower neutralizing ability against the venoms horses had a relatively similar response (Fig. 1). of L. muta and C. durissus (Table 3). In agreement with previous publications (Gené When confronted with L. muta and C. et al. 1985, Gutiérrez et al. 1985) neutraliza­ durissus venoms by immunoelectrophoresis, tion of hemorrhage and proteolysis was more monovalent antivenom formed many precipitin effective than neutralization of lethality and arcs, corroborating the high degree of cross­ indirect hemolysis. The development of reactivity between these venoms and that of antibody response against lethal, hemorrhagic, B. aspero Nevertheless, there were bands result­ and indirect hemolytic effects showed a similar ant from the reaction between venoms and time-course. However, response against polyvalent antivenom that were absent when proteolytic activity presented a different final monovalent antivenom was tested against pattern, since the striking difference observed heterologous venoms (Fig. 4). between the two ho!ses regarding neutralization of lethality, hemorrhage and hemolysis was DISCUSSION not as pronounced in the case of of proteolysis (Fig. 1). Despite the fact that many centers in the world produce antivenoms (Chippaux and Analysis of immunoelectrophoretic results Goyffon 1983), there are very few studies indicates the presence of precipitating anti­ related to the development of immune response bodies against several venom components in animals injected with snake venoms Of even at the earliest times of immunization toxins. In tbis work, antibody production (Fig. 2, Table 3). For instance, after the tbird against components responsible of four toxic injection (corresponding to 0.083 mg venom), and enzymatic activities of B. asper venom sera from horses 1 and 2 forrned seven and was monitored. Results indicate that there eight precipitin bands, respectively, when was a remarkably different response in the confronted against B. asper venom. At the two horses studied, since although neutralizing end of the immunization, monovalent antisera ability was similar during the first stages of the formed 11 precipitin arcs when confronted immunization schedule, horse No. 1 developed with B. asper venom. In the same conditions, a much higher antibody response at later stages. the commercially avaílable polyvalent Interestingly, this difference was not associated 514 REVISTA DE BIOLOGIA TROPICAL

A B 12

500 10 400 8 --So 300 --So >< >< o 6 -b� 200 1�Lf) - o 4 o· I 100 /o .0- 2 �..d ó o-o'" / / 20 100 140 180 0- __ -o" 20 60 100 140 180 days days O e 180 100

14 0 80

---ro 60 I ----:ro 100 / >< ><0 ,o IÓ o I -I�� 40 _I�LO I o' 60 I I 20 , I 20

, 20 60 100 180 140 20 60 100 140 180 days days

Fig. l. Time-course of the development of antibody response in two horses immunized with Bothrops asper venom against four toxic and enzymatic activities of the venom. Neutralizing ability is expressed as (l/ED50) x 10', where ED50 is defined as the antivenom/venom ratio which neutralizes venom activity in a 500/0 o. The immunization schedule is shown in Table 1. (A) Development of antibody response against hemorrhagic activity; (B) Development of antibody response against indirect hemolytic activity; (e) Development of anti­ body response against lethal activity; (D) Development of antibody response against proteolytic activity. (e--e) horse No. 1, (0 -- 0) horse No. 2. with a different clinical response of both horses neutralization of indirect hemolysis in vitro to venom injections. Since individual horses correlates significantly with neutralization of respond so differentIy to the immunization, lethality. Since this is a simple and inexpensive it is important to monitor antibody response assay, antibody response in individual horses in each animal during immunization. This could be easily evaluated by studying the would help in the selection of horses with neutralization of indirect hemolysis, thereby a good antibody titer, in order to bleed them avoiding the large scale use of mice. and obtain a pool of plasma with high Antibody response (expressed as ED50) neutralizing ability. Such individual monitoring raised earlier and reached higher levels against could be performed by in vitro techniques hemorrhagic toxins and proteases than against such as the one described by Gutiérrez et al. phospholipases A2 and lethal toxins. This find­ (1988) who showed that, in the case of the ing parallels previous results in that polyvalent polyvalent antivenom produced in Costa Rica, antivenom is more efective in the neutralization GUTIERREZ et al.: Monovalent anti-Bothrops asper antivenom 515

TABLE 1 p Immunization schedule used in the production of ,� Monovalent anti- Bothrops asper antivenom A +

DayNo. Venom injected (mg) * Adjuvant m

1 0.083 Incomplete Freund 10 0.083 Sodium alginate Sodium alginate 20 0.083 m 30 0.12 Sodium alginate 40 0.23 Sodium algínate 50 0.35 Sodium alginate 8 + 60 0.50 Sodium algínate 7WJLt 70 0.70 Sodium alginate 80 1.0 Sodium alginate p 90 1.5 Sodium alginate 100 3.0 Sodium algínate 110 5.0 Sodium alginate 120 10.0 Sodium alginate m 130 15.0 Sodium alginate 140 30.0 Complete Freund 150 30.0 Sodium alginate e + 160 50.0 Sodium alginate

* Injections were made subcutaneously. p of proteolysis and hemorrhage thlln in the neu­ Fig. 2. Immunoelectrophoresis of sera obtained from horses immunized with B. asper venom at different tralizlltion of indirect hemolysis and lethality stages in the immunization.For comparative purposes, (Bolaños and Cerdas 1980, Gutiérrez et al. eommercíal polyvalent antivenom was al so eonfronted 1981, 1985, 1987a, Gené et al. 1985). Thus wíth B. asper venom. (A) Serum from horse No. 1 hemorrhagic and proteolytic components are eolleeted at day 20 of immunizatíon; (B) Serum from more immunogenic, due perhaps to their high horse No. 1 eolleeted at day 60 of inmunization; and (C) Serum from horse N01 eolleeted at day 90 of molecular weight which contrast with the re1a­ immunization. Commercial polyvalent antivenom was tively low molecular weight of phospholipases placed in ehannels (P) whereas samples of monovalent A2 (Tu 1977). antisera were plaeed in ehannels (M). Notiee the presenee of many precipitin ares even at a very early Immunoelectrophoresis was more sensitive stage in the immunization proeess. than neutralization assays in detecting antibody TABLE 2 response since even at the earliest venom inoculations there were several precipitin bands Immunoelectrophoretic analysis of the antibody response in horses immunized with Bothrops asper venom when venom was confronted with antisera. However, immunoprecipitation was not usefuI Dose of venom injeeted Number of precípitin ares in predicting the neutralizing ability of antisera (mg) 10 days before in immunoeleetrophoresis* as the number of precipitin bands was similar in sera from both horses at a11 times tested, Horse No. 1 Horse No. 2 8 in contrast to neutralizing ability which was 0.083 7 0.12 8 7 higher in horse No. 1. 0.23 7 7 Monovalent anti-B. asper antivenom has a 0.35 8 8 higher neutralizing capacity when tested 1.0 8 8 1.5 7 8 against B. asper venom than polyvalent anti­ 3.0 8 9 venom (Table 3). Moreover, it cross reacts 10.0 12 11 significantly with the venoms of L. muta 30.0 12 11 50.0 11 and C. durissus, as revealed by irnmuno­ 11 electrophoresis and neutralization assays. This * For comparison, eommereially-available polyvalent type of neutralization against heterologous antivenom formed 11 precipitin ares when eonfronted venoms has been also observed with other wíth B. asper venom in identical experimental eondi­ monovalent anti-Bothrops antivenoms (Siles et tions. 516 REVISTA DE BIOLOGIA TROPICAL

1) 100, l' A + 0. \ \ l· 80 I \ \ \ '\ . \ I \c � 60 'i\ I \ - 1 ;r: _ ------1 ''i- \.- - r- jJ \ B + \ .�_. ·-2 - . _. _

20

100 200 �OO p �I AV/6 MEO e +

Fig. 3. Neutralization of edema-forming activity of three snake venoms by monovalent anti B. asper Fig. 4. Immunoelectrophoresis of polyvalent and antivenom. In order to express neutralization of edema monovalent antivenoms against the venoms of on percentage basis, swelling induced by venoms alone Bothrops asper Crotalus durissus durissus (B) and was considered as 100% edema. (.-- .) Neutrali­ (A), Lachesis muta stenophrys (C). Channels labelled as (P) zation of durissus venom; -- Neutraliza­ C. (o --- o) correspond to polyvalent antivenom and those labelled tion of L. muta venom; -- Neutralization of (6, 6,) as (M) correspond to monovalent antivenom. B. asper venom. In the cases of L. muta and B. asper, antivenom reduced edema-forming activity significan­ tJy (P 0.05) at all doses tested. < induced by e d durissus at any of the doses tested (Lomonte 1985, Rojas et al. 1987). TABLE 3 In conclusion, it is proposed that although Comparison o[ rhe ability afmar/ovalent ond polyvolent antivenoms ro neutralize monovalent anti B. asper antivenom ís highly lethality, hemorrhage and difibrination induced by Ihe venoms o[ Bothrops aspero Lachesis muta and Crotalus durissus durissus. effective in the neutralization of B. asper

Neu tralizing ability venom, its use in treating accidents by L. (ED50 ) * muta and e d durissus seems to be indicated Venom Toxic activity Monovalent Polyvalent onIy if polyvalent antivenom is not available. antivenom antivenom

Bothrops asper Lethality 216 333** Hemorrhage 70 135*** Defibrination 400 600**** ACKNOWLEDGEMENTS

Lachesis muta Lethality 333 400** Hemorrhage 250 85*** We thank Abel Robles, Jorge Alvarado, Al­ Defibrination >4000 600**** fredo Vargas and Javier Núñez for their help Crota/us durissus Lethality 1390 666** dun'ssus Hemorrhage 840 150*** in the laboratory work, and Rocío Monge Defibrination >4000 400**"'* for her assistance. We are also grateful to Y. Expressed as effective dose 50% (ratio of ¡JI antivenom/mg venom in which the effect is neutralized 50%). Kozuka fot his collaboration in the photo­ According to Bolaños and Cerdas (1980), graphic work. This work was supported by the According to Glltiérrez et al. (1985). International Foundation for Science Gené, J.A. (llnpublished data). (Research Grant Agreement No.Fj883-2). J. M. Gutiérrez and F. Chaves were recipients al. 1978/79). However, despite the fact that of a research career award from the Costa monovalent anti-B asper antivenom was Rican National Scientific and Technological effective in neutralizing lethality and hemo­ Research Council (CONICIT). rrhage of L. muta and e d durissus venoms, it was ineffective against defibrinating activity (Table 3). Regarding edema, monovalent and RESUMEN polyvalent antivenoms have a similar neutraliz­ ing ability (Gutiérrez et al. 1986). Surprisingly, En el Instituto Clodomiro Picado se produ­ neither antivenom was capable to reduce edema jo un suero monovalente contra el veneno de GUTIERREZ et al.: Monovalent anti-Bothrops asper antivenom 517

la serpiente Bothrops asper (terciopelo). Du­ Gutiérrez, 1. M., G. Rojas, B. Lomonte, J. A. Gené rante las primeras etapas del proceso los caba­ & L. Cerdas. 1986. Comparative study of the edema-fOIming activity of Costa Rican snake llos inmunizados mostraron un título similar venoms and its neutralization by a polyvalent en lo que respecta a la neutralización del vene­ antivenom. Comp. Biochem. Physiol. 85C: 171- no; sin embargo, uno de ellos mostró un título 175. muy superior en la última fase de la inmuniza­ ción. El suero monovalente fue más efectivo Gutiérrez, 1. M., G. Rojas & L. Cerdas, 1987. Ability of a polyvalent antivenom to neutralize the venom que el suero polivalente comercial para neutra­ of Lachesis muta melanocephala, a new Costa lizar el veneno de B. aspero Por otra parte, el Ric�n subspecies of the bushmaster. Toxicon suero monovalente fue menos efectivo que el 25: 713-720. polivalente para neutralizar las actividades le­ Gutiérrez, J. M., C. Avila, E. Rojas & L. Cerdas. 1988. tal y hemorrágica de los venenos de Lachesis An alternative in vitro method fOI testing the muta (cascabel muda) y Crotalus durissus potency of the polyvalent antivenom produced (cascabel). Más aún, el monovalente no neu­ in Costa Rica. Toxicon (in press). tralizó la actividad desfibrinante de estos dos Lomonte, B. 1985. Edema-forming activity of venenos. Se propone que el suero monovalen­ bushmaster (Lachesis muta stenophrys) and te anti-B asper es altamente efectivo en la Central American rattlesnake (Crotalus durissus neutralización del veneno homólogo, en tanto durissus) venoms and neutraiization by a poly­ que su empleo en el tratamiento de envenena­ valent antivenom. Toxicon 23: 173-176. mientos por L. muta y durissus se justifica e Ouchterlony, O. & L. A. Nilsson. 1978. Immunodif­ sólo cuando el suero polivalente no esté dispo­ fusion and immunoelectrophoresis, p. 191. In nible. D. M. Weir (ed.). Handbook of Experimental Immunology, Vol. 1. Immunochemistry. Blackwell Scientific, Oxford.

Rojas, G., 1. M. Gutiérrez, J. A. Gené, M. Gómez & L. Cerdas. 1987. Neutralización de las activida­ REFERENCES des tóxicas y enzimáticas de cuatro venenos de serpientes de Guatemala y Honduras por el anti­ Bolaños, R. 1982. Las serpientes venenosas de Cen­ veneno polivalente producido en Costa Rica. troamérica y el problema de ofidismo. Primera Rev. Biol. Trop. 35: 59-67. parte. Aspectos zoológicos, epidemiológicos y bio­ médicos. Rev. Cost. Cienc. Med. 3: 165-184. Siles, M., R. Rolin, F. Zelante & R. Guidolin. 1978/ 79. Evidencia�ao em camundongos da soroneu­ Bolaños, R. & L. Cerdas. 1980. Producción y control traliza�ao para especifica entre venenos e anti­ de sueros antiofídicos en Costa Rica. Bol. Of. venenos botrópicos. Mem. Inst. Butantan 42/43: Sanit. Panam. 88: 189-196. 337-344.

Chippaux, J. P. & M. Goyffon. 1983. Producers of Theakston, R. D. G. & H. A. Reid. 1978. Development antivenomous sera. Toxicon 21: 739-752. of simple standard assay proceduUls fur the charac­ terization of. snake venoms. Bull. World Health Gené, 1. A., M. Gómez, 1. M. Gutiérrez & L. Cerdas. Org. 61: 949-956. 1985. Neutralization of hyaluronidase and indirect hemolytic activities of Costa Rican snake venoms Tu, A. T. 1977. Venoms: Chemistry and Molecular by a polyvalent antivenom. Toxicon 23: 1015- Biology. Wiley, New York. 1018. World Health Organization. 1981. Progress in the Gutiérrez, J. M., F. Chaves, R. Bolaños, L. Cerdas, E. characterization of venoms and standardization Rojas, O. Arroyo & E. Portilla. 1981. Neutraliza­ of antivenoms. WHO Offset Publication No. 58. ción de los efectos locales del veneno de Bothrops asper por un antiveneno polivalente. Toxicon 19: Yamakawa, M., M. Nozaki & Z. Hokama 1976. Frac­ 493-500. tionation of sakishima-habu (Trimeresurus elegans) venom and lethal, hemorrhagic and edema-forming Gutiérrez, J. M., J. A. Gené, G. Rojas & L. Cerdas. activities of the fractions, p. 97-109 In A. Ohsaka, 1985. Neutralization of proteolytic and hemor­ K. Haysahi & Y. Sawai (eds.). Animal, Plant and rhagic activities of Costa Rican snake venoms by Microbial Toxins. Vol 1. Plenum Press, New a polyvalent antivenom. Toxicon 23: 887-893. York.