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Protection against the lethal effects of Crotalus durissus terrificus (South American rattlesnake) venom in animals immunized with crotoxin
Toslcon Vol. 28, No . 12, pp. 1491-14%, 1990. Printed ia Great Britain.
0041-0101/90 53.00 + .00 ® 1990 Pergamon Pres pk
PROTECTION AGAINST THE LETHAL EFFECTS OF CROTALUS DURISSUS TERRIFICUS (SOUTH AMERICAN RATTLESNAKE) VENOM IN ANIMALS IMMUNIZED WITH CROTOXIN T. V. FRErrns, C. L. FoR~s-Dens and C. R. DINIz Funtiaçäo F.zequiel Dias, Belo Horizonte (MG), Brazil (Accepted for publication 5 June 1990)
T. V. FRErr~s, C. L. FoRZ~s-Dens and C. R. Dnvlz. Protection against the lethal effects of Crotalus durissus terr~cus (South American rattlesnake) venom in animals immunized with crotoxin . Toxicon 28, 1491-1496, 1990.-Mice and rabbits were immunized against crotoxin (the neurotoxic component isolated from Crotalus durissus terrificus venom) using small amounts of antigen in a water-in-oil emulsion . Following boosting (three times at 21-day intervals) a high titre of antibodies against crotoxin was obtained . Crotoxin immunoglobulin G antibody recognizes whole venom antigen at a level comparable with that of crotoxin antigen, using the ELISA method for antibody detection. The antibodies generated by crotoxin were capable of providing 100% protection against challenge with 11 and 50 i.p . LDsp doses of whole venom in mice . When 100 i.p . LD P doses of whole venom were injected survival was 77 .8% .
INTRODUCTION
FOLLOWING bites by Crotalus durissus terrificus it is estimated that about 74% of untreated systemically envenomated victims die; with antivenom therapy the mortality falls to about 12% (WHO, 1981). The high mortality is generally attributed to the neurotoxic effects of the venom coupled with renal complications (HnwcooD, 1982; Aa~toRIM and MELLO, 1954; AMORIM, 1971 ; DANZIG and ABELS, 1961). Recent studies have questioned the efficacy of antivenom therapy for the treatment of severe rattlesnake venom poisoning (Snssncx et al., 1977). SCHnsFFER et al. (1988) studied the binding of antivenom (Wyeth, Crotalidae, Polyvalent) to venom fractions from crotalid and viperid snakes and demonstrated that high ELISA antibody titres were observed for many of the high mol. wt fractions in C. adamanteus, C. atrox and Bothrops atrox venoms . A unique elution pattern was observed with C. d. terrificus venom, with low ELISA titres, suggesting that antivenom neutralization of venom toxins may be poor . FRI:TTAS et al. (1989), using mice and rabbits immunized with venom incorporated into liposomes, detected an increase in the level of antibody response with a high protective effect against whole venom. SANTOS et al . (1988), using mice immunized against the phospholipase A2 from crotoxin, demonstrated that the antiserum was able to neutralize most of the lethal effects of C. d. terrificus venom. 1491
1492
T. V. FRETTAS et al.
Because of its high toxicity, crotoxin, the principal neurotoxic component of South American rattlesnake venom, has never been used successfully for animal immunization . Immunodetection of snake venom using enzyme-linked immunosorbent assay (ELISA) has been widely used (PLraH et al., 1979; COULTER et al., 1980; PUGH and THEAKSTON, 1980; PUGH et al., 1980 ; SUTHERLAND, 1980 ; IiHALIWAL et al., 1983; Ho et al., 1986; WARRELh et al., 1986). In most of these studies polyclonal antisera raised against crude venom were used for the detection of whole venom. Ho et al. (1986) reported that the use of single venom components as antigens may reduce the extent of cross-reactivity between closely related species. In the present study the protective effect of antibodies raised against a single venom component, crotoxin, was assessed in terms of protection against the lethal effects of whole venom.
MATERIALS AND METHODS Animals Adult female CFZ mice (18-20 g; Fundaçáo Ezequiel Dias, Belo Horizonte, Brazil), and adult female half-breed white rabbits (2 .0-2 .5 kg) received water and food ad /ibitum under controlled environmental conditions . Antigens C. d. terr/ficus venom from 50 adult snakes (Fundaçäo Ezequiel Dias herpetarium) was pooled . Venom was lyophilized and stored at -20°C before use. The venom had an i.p. I.D w of 1 .8 ug/20 g mouse (95% confidence limits 1.5-2.2 teg) . Crotoxin purified from the whole venom (Sc.m-re and F~er.-CotauT, 1938 as modified by SExt et al., 1980), had an i.p . cow of 1 .24 pg/20 g mouse (1 .00-1 .47 pg). t,n, ° values were calculated by probit analysis (FtNivt:r, 1971). Phospholipase A2 and crotapotin were purified from crotoxin using the method of Rut~~v et al. (1971) and subsequently eluted using two-step gradients. The phospholipase activities of crude venom, crotoxin and its purified components were evaluated by the egg yolk coagulation assay (ViDet. and S1~OPPAHI, 1971). One phospholipase unit (PU) is defined as the amount of enzyme which increases by one minute the wagulability of an egg yolk suspension when compared to the wntrol (saline) . Immunization and challenge schedule Animals were immunized with crotoxin using the method of GODIN(l (1986) . Twenty mice and two rabbits were injected with a water-in-oil emulsion produced by mixing four volumes of Freund's wmplete (priming) or incomplete (boosters) adjuvant (Difw Laboratories, Detroit, U.S.A.) with one volume of an aqueous solution of crotoxin (0 .2 ml/mouse and 2.0 tnl in four sites/rabbit) . Animals were boosted three times at 21-day intervals. In We mouse an immunization schedule of 6.25 Rg of crotoxin in the emulsion was used for priming and for subsequent booster i.p . injections . This amount was equivalent to five i.p. t-D~ doses of crotoxin . The final booster was 9.40 /tg of crotoxin ín an aqueous solution. Control groups (20 mice) were injected with the above solution without antigen. Mice were bled weekly for 14 weeks from the tail vein . Whole blood samples (1 : 50 dilutions in phosphate buffered saline wntaining 0.05% Tween) were stored at -20°C before assay. The indirect ELISA method was used for detection of antibodies against crude venom, crotoxin, phospholipase Ai and aotapotin. On the 65th day, 10 mice were bled by axillary vein puncture. Sera were pooled and stored at -20°C. The remaining mice wntinued to be bled weekly for specific antibody detoction and were challenged i.p . with whole venom doses of either 11 r.n~ (20 pgl20 g mouse), 6 days after the final injection or 50 t.D~ (90 pgl20 g mouse), 21 days after the final injection or 100 tDSO (180 /íg/20 g mouse), 36 days after the final injection (Fig. 1) . Survivors were recorded 24 hr after each challenge. Control groups of 10 female mice of the same weight received the same doses of C. d. terrhcus venom at each challenge (Table 1) . A similar schedule was adopted for rabbit immunization with s.c . injections of 47 pg of crotoxin in the emulsion and 63 /tg of crotoxin in aqueous solution. Protective e (jects Hyperimmune sera obtained from mice and rabbits immunized against crotoxin were tested for precipitating antibodies using Ouchterlony (double diffusion) analysis . Undiluted and diluted sorum (I :2, 1 :4, 1 :8, I : 16, 1 : 32) were analysed for antibodies against C. d. tenifecus venom and crotoxin . The protective effects of rabbit hyperimmune sera were evaluated by the En~ estimation (numbers of mice alive 24 hr following injections of five i.p . t.D~ doses of C. d. terr~eus venom with different amounts of rabbit antiserum) .
Immunization with Crotozin
1493
assay Whole blood samples from mice were assayed by ELISA using the indirect method (~I%AR.4rON et al., 1977) with the modifications described by Ho et al . (1986). Hemobag plates (Hemobag Produtos Cirurgicos Ltda, Campinas, Brazil) were coated with 100 pl of a 5 ixg/ml solution of either C. d. terr~cus venom, crotozin, phospholipase A~ or crotapotin pct well. Peroxidase conjugated anti-mouse immunoglobulin diluted 1/1000 (Sigma Co ., U .S.A .) was used as the enzyme marker. Ortho-phenylcnediamine (Sigma Co ., U .S.A .) in the presence of H ZOZ was used as substrate . After 20 min of incubation at room temperature, the reaction was stopped by adding H,S04 (30% v/v). Absorbance was measured at 492 nm. Antibody
RESULTS
The ELISA antibody titres induced by i.p. injections of crotoxin in mice during the immunization period of 62 days were higher in crotoxin antibody than in whole venom, phospholipase Az or crotapotin antibodies (Fig. 1). Higher antibody response were obtained to whole venom and phospholipase AZ than to crotapotin (Fig. 1) despite the fact that crotoxin (which is a component of crude venom composed of crotapotin and phospholipase A~ had been used as immunogen. The component of crotoxin with phospholipase activity produced a higher antibody response than crotapotin which does not possess that activity . The phospholipase activity of crotoxin and its purified components crotapotin and phospholipase AZ were respectively 20, 0 and 50 PU/mg of protein using the egg yolk coagulability assay. The protective effect induced by immunization with crotoxin in mice correlated well with ELISA antibody titre. The antibodies generated by crotoxin were capable of
FIG. 1 . ANI7BODY RBSPON3E IN MI(x DURING LP . GIMUNIZATION WITH CR01'OXIN AND FREUND's ennrveNr, I+OLLOWED BY CHALIBNGHS GF Crotalur dwissus terrifecus veNOSt . Whole venom antibody response, D----D ; crotoxin antibody response fir ; phospholipase A 2 antibody response ~~C: crotapotin antibody response ~--i . A to D-Immunization with crotoxin (A, 6 .25 pg/Freund complete adjuvant; B, C, 6 .25 ~g/Freund incomplete adjuvant; D, 9 .4 l+g/~e) . I to K~hallenge with whole venom (I, 11 Ln,° doses (20 ßg/20 g mouse); J, 50 Ln~ doses (90 ßg/20 g mouse) ; K, 100 Ln m doses (180 pg/20 g mouse)). Each point represents the mean ELISA absorbance values from 20 mice (immunization schedule) and 10 mice (challenge schedule) . The variation coeHICient ((standard deviation) mean) x 100) for each point with A~ nm > 0 .200, was about 15% .The mouse blood dilution was 1 : 50 . Crotales duriasus terrificus venom and crotoxin had an i .p. ~w dose of 1 .8 (1 .52-2 .2) pg/20 mouse and 1 .24 (1 .00-1 .47) pg/20 g mouse, respectively.
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Immunization with Crotoxin
1495
providing 100% protection against challenges of 11 and 50 i.p . LD S° doses of whole venom (Table 1) . Following challenge with 11 ~w doses of whole venom, a large increase in antibody titre was observed . At that dosage, the crude venom boosted the antibody level. Following challenge with 50 r n~ doses of whole venom a slight decrease in antibody titre was observed . Twenty four hours after challenge all mice were alive, but one animal died after 48 hr (this animal had an ELISA absorbance of 0 .022, 48 hr post-challenge). Seven out of nine mice survived challenge with 100 I,n~ doses of whole venom. The two animals which died had similar ELISA titres to the survivors. Using double diffusion analysis, sera and purified IgG from the two crotoxin-immunized rabbits showed a weak precipitin line at a 1 : 2 dilution when tested against crotoxin and whale venom. The En~ values of the sera from immunized rabbits were 2214 serum/mouse (12-3811) and 3311 serum/mouse (240 k1) respectively . Antibody levels of the hyperimmune sera, evaluated by double diffusion analysis, did not reflect the protective effect in either mice or rabbits. DISCUSSION
According to HURN and CIiANTLER (1980), antisera obtained using crude preparations are often more highly immunagenic than purer materials . However, since venoms are complex mixtures of different antigenic components, a multitude of potential antigenic sites are presented to the animal's immune system . Under these conditions only the most antigenic determinants may stimulate significant antibody production, and they may not be associated with a toxin's active site . SCHAEFFER et al. (1988) used ELISA for testing size-selected fractions of whole crotalid venoms for binding to antivenom (Wyeth, Crotalidae Polyvalent) and found a minimal titre of antibodies to the low molecular weight venom components . These observations led us to investigate the efficacy of crotoxin in protecting experimental animals against venom lethality. The choice of immunization schedule enabled the use of a highly toxic antigen with a dramatic decrease in lethality. The mechanism of action of adjuvants which permits slow release of antigen in a highly aggregated form, probably explains the resistance of animals to such high doses of crotoxin. As a result, a high protective effect against whole venom was obtained in both mice and rabbits immunized against crotoxin . Antibodies raised against crotoxin appear to recognise the whole venom antigen at levels comparable to the crotoxin antigen. The level of protection against 100 i.p . Ln~ doses of whole venom (36 days after the final booster dose of crotoxin) in mice was 78%,compared with 90% reported by SANTbS et al. (1988) following challenge with 16 s.c . LD P doses of C. d. terrificus venom 21 days after the final booster injection of phospholipase AZ in mice. The >~w of the sera from two rabbits immunized with crotoxin were 2211 serum/mouse (12-3811) and 33 pl serum/mouse (24~01~1), compared to 7811 serum/mouse (69-8211) obtained by Fxi?rrns et al. (1989) from one rabbit immunized with C. durissus venom incorporated into liposomes. The antibody levels elicited by crotoxin were not enough to produce a strong precipitin line in the double diffusion analysis . However, the protection afforded by those antibodies suggests that they must have a high afïtnity to the toxic components of whole venom. WnxxELI, et al. (1986) showed that the response of coagulopathy to initial treatment was most rapid in patients given Twyford antivenom, compared to other hyperimmune serum raised against Calloselasma rhodostoma venom. The Twyford antivenom has its titer of anti-procoagulant (antiArvin/Ancrod) increased four-fold during manufacture.
1496
T. V. FREITAS et al.
These data suggest that immunization with crotoxin and/or the addition
of anti-crotoxin
as a supplement to polyclonal antivenom should be considered in order to obtain a hyperimmune serum with a high level
of protection
against
C. d. terrficus
venom.
Acknowledgements-We thank Mr R. Mnc~, and Mrs B. C. B. DA FoxsECe for excellent technical assistance; the authors are grateful to Dr R. D. G. Tt~AKSTON for his valuable suggestions during the preparation of this manuscript . This research was partially supported by Conselho National de Desenvolvimento Cientifico e Tecnologico (CNPeil, Financiadora de Estudes e Projetos (FINEP) and Commission of We European Communities (CEC). REFERENCES AMORIM, M. F. (1971) Intermediate nephron nephrosis in human and experimental crotalic poisoning. In: Venomous Animals and their Venoms . Vol. II, pp . 319-343 (BucrmtL, W. and BUCICLEY, E., Eds). New York: Academic Press. AMOAtAt, M. F. and MII.i .o, O. (1954) Intermediate nephron nephrosis from snake poisoning in man. Am. J. Path . 3, 47999. CouLrelt, A. R., HARxts, R. D. and 5u1T~rtLANn, S. K. (1980) Enzyme immunoassay for the rapid clinical identification of snake venom. Med. J. Aust . 1, 43335. DANZIG, L. E. and Aie:LS, G. H. (1961) Haemodialysis of acute renal failure following rattlesnake bite, with recovery . J. Am . Med. Ass. 175, 136-137. DI3ALIWAL, J. S., LrM, T. W. and SuxutrAIteN, K. D. (1983) A double antibody sandwich MICRO-ELISA kit for the rapid diagnosis of snake bite. Southeast Asian J. Trop . Med. Pub. Hth 14, 367-373. FtNNt=.v, D. J. (1971) . Probit Analysis. 3rd. ed . Cambridge: Cambridge University Press. FxatTAS, T. V., TAVAxFS, A. P., TFmAtcsroN, R. D. G., LAtxo, G. and New, R. R. C. (1989) Use of liposomes for protective immunisation against Crotales durissus (Tropical rattlesnake) venom. Toxicon 27, 341-347. Gootxc, J. W. (1986) Production of monoclonal antibodies . In : Monoclonal Antibodies : Principles and Practice, Ch . 3, pp . 59-103 . London: Academic Press. HAWOOOD, J. B. (1982) Physiological and pharmacological effects of rattlesnake venoms. In : Rattlesnake Venom: Their Actions and Treatment, pp. 121-162 fTu, A. T., Ed .) . New York: Marcel Dekker . Ho, M., WAtexELL, M. J., WAxxsLL, D. A., BroweLL, D. and VoLtEIy A. (1986) A critical reappraisal of the use of enzyme-linked immunosorbent assays in the study of snake bite. Toxicon 24, 211-221. HuxN, B. A. L. and GtArrrtaa, S. M. (1980) Production of reagent antibodies . Mcth . Enzymol. 70, 104-143. PuGa, R. N. H. and TtIEAxsroN, R. D. G. (1980) Incidence and mortality of snake bite in Savanna Nigeria. Lancet II, 1181-1183 . Pvoa, R. N. H., TaeAtcsTON, R. D. G. and REtn, H. A. (1979) Bites by the carpet viper in the Niger Valley. Lancet II, 625-627. Puoa, R. N. H., TtIEAxsroN, R. D. G. and REtn, H. A. (1980) Epidemiology of human encounters with the spitting cobra, Naja nigricollis, in the Malumfashi area of northern Nigeria. Ann. Trop . Med. Parasitol. 74, 523-530. RuasAt~N, K., BttFlTaertrT, H. and HAHnERMANN, E. (1971) Biochemistry and pharmacology of the crotoxin complex. Naunyn-Schmiedebergs Arch. Pharmacol. 270, 274-288. SASeecI~, M. S., CbNNINOrwu, E. R. and Furs, C. T. (1977) A study of the treatment of pit viper envenomation in 45 patients. J. Trauma. 17, 569-573 . SANTas, M. C., DINIZ, C. R., WHrIAIO:R Pact->ECO, M. A. and blas DA Save, W. (1988) Phospholipase Az injection in mice induces immunity against the lethal effects of Crotales durissus terr~cus venom. Toxicon 26, 207-213. ScaAeKreIt, R. C., Jtt, RANDALL, H., Roc, J. and CAxISON, R. W. (1988) Enzyme-linked immunosorbent assay (ELISA) of size-selected crotalid venom antigens by Wyeth's Polyvalent Antivenom. Toxicon 26, 67-76. St:ICI, C., VinAL, J. C. and BAxxto, A. (1980) . Purification of gyroxin from a South American rattlesnake (Crotales darissus terrificus) venom. Toxicon 18, 235-247. St.orrA, K. H. and Fx~tvt~st.-CorrxaT, H. L. (1938) Schalangengifte, III : Mitteilung: Reinigung und krysta11i7ation des Klapperschangengiftes . Ber. Dtch Chem. Ges. 71, 1076-1081. SUTHERLAND, S. K. (1980) Venom and Antivenom research. Med. J. Aust. 2, 246-250. TI~AjcsroN, R. D. G., LLOYD-JONES, M. J. and RErD, H. A. (1977) MICRO-ELISA for detecting and assaying snake venom and venom antibody . Lancet II, 639-641 . VIRAL, J. C. and STOPPANI, A. O. M. (1971) Isolation and purification of two phospholipases A from Bothrops venom. Arch. Biochem . Biophys . 145, 543-556. WARRELL, D. A., LOOAREESUWAN, S., Tt~AttsroN, R. D. G., Prm,L>ps, R. E., GtANTaevArnca, P., VIItAVAN, C., $UPANARANOND, W., KAttswANO, J., Ho, M., HuTTON, R. A. and Vetcao, S. (1986) Randomized comparative trial of three monospecific antivenoms for bites by the Malayan pit viper (Callostlasma rhodostoma) in Southern Thailand: Clinical and laboratory correlations. Am. J. Trop . Med. Hyg . 35, 1235-1247. WORLD HEALTH ORGANIZATION (1981) Progress in the characterization of venoms and standardization of antivenoms. WHO Offset Publication no . 58 . Geneva : World Health Organization.
0041-0101/90 53.00 + .00 ® 1990 Pergamon Pres pk
PROTECTION AGAINST THE LETHAL EFFECTS OF CROTALUS DURISSUS TERRIFICUS (SOUTH AMERICAN RATTLESNAKE) VENOM IN ANIMALS IMMUNIZED WITH CROTOXIN T. V. FRErrns, C. L. FoR~s-Dens and C. R. DINIz Funtiaçäo F.zequiel Dias, Belo Horizonte (MG), Brazil (Accepted for publication 5 June 1990)
T. V. FRErr~s, C. L. FoRZ~s-Dens and C. R. Dnvlz. Protection against the lethal effects of Crotalus durissus terr~cus (South American rattlesnake) venom in animals immunized with crotoxin . Toxicon 28, 1491-1496, 1990.-Mice and rabbits were immunized against crotoxin (the neurotoxic component isolated from Crotalus durissus terrificus venom) using small amounts of antigen in a water-in-oil emulsion . Following boosting (three times at 21-day intervals) a high titre of antibodies against crotoxin was obtained . Crotoxin immunoglobulin G antibody recognizes whole venom antigen at a level comparable with that of crotoxin antigen, using the ELISA method for antibody detection. The antibodies generated by crotoxin were capable of providing 100% protection against challenge with 11 and 50 i.p . LDsp doses of whole venom in mice . When 100 i.p . LD P doses of whole venom were injected survival was 77 .8% .
INTRODUCTION
FOLLOWING bites by Crotalus durissus terrificus it is estimated that about 74% of untreated systemically envenomated victims die; with antivenom therapy the mortality falls to about 12% (WHO, 1981). The high mortality is generally attributed to the neurotoxic effects of the venom coupled with renal complications (HnwcooD, 1982; Aa~toRIM and MELLO, 1954; AMORIM, 1971 ; DANZIG and ABELS, 1961). Recent studies have questioned the efficacy of antivenom therapy for the treatment of severe rattlesnake venom poisoning (Snssncx et al., 1977). SCHnsFFER et al. (1988) studied the binding of antivenom (Wyeth, Crotalidae, Polyvalent) to venom fractions from crotalid and viperid snakes and demonstrated that high ELISA antibody titres were observed for many of the high mol. wt fractions in C. adamanteus, C. atrox and Bothrops atrox venoms . A unique elution pattern was observed with C. d. terrificus venom, with low ELISA titres, suggesting that antivenom neutralization of venom toxins may be poor . FRI:TTAS et al. (1989), using mice and rabbits immunized with venom incorporated into liposomes, detected an increase in the level of antibody response with a high protective effect against whole venom. SANTOS et al . (1988), using mice immunized against the phospholipase A2 from crotoxin, demonstrated that the antiserum was able to neutralize most of the lethal effects of C. d. terrificus venom. 1491
1492
T. V. FRETTAS et al.
Because of its high toxicity, crotoxin, the principal neurotoxic component of South American rattlesnake venom, has never been used successfully for animal immunization . Immunodetection of snake venom using enzyme-linked immunosorbent assay (ELISA) has been widely used (PLraH et al., 1979; COULTER et al., 1980; PUGH and THEAKSTON, 1980; PUGH et al., 1980 ; SUTHERLAND, 1980 ; IiHALIWAL et al., 1983; Ho et al., 1986; WARRELh et al., 1986). In most of these studies polyclonal antisera raised against crude venom were used for the detection of whole venom. Ho et al. (1986) reported that the use of single venom components as antigens may reduce the extent of cross-reactivity between closely related species. In the present study the protective effect of antibodies raised against a single venom component, crotoxin, was assessed in terms of protection against the lethal effects of whole venom.
MATERIALS AND METHODS Animals Adult female CFZ mice (18-20 g; Fundaçáo Ezequiel Dias, Belo Horizonte, Brazil), and adult female half-breed white rabbits (2 .0-2 .5 kg) received water and food ad /ibitum under controlled environmental conditions . Antigens C. d. terr/ficus venom from 50 adult snakes (Fundaçäo Ezequiel Dias herpetarium) was pooled . Venom was lyophilized and stored at -20°C before use. The venom had an i.p. I.D w of 1 .8 ug/20 g mouse (95% confidence limits 1.5-2.2 teg) . Crotoxin purified from the whole venom (Sc.m-re and F~er.-CotauT, 1938 as modified by SExt et al., 1980), had an i.p . cow of 1 .24 pg/20 g mouse (1 .00-1 .47 pg). t,n, ° values were calculated by probit analysis (FtNivt:r, 1971). Phospholipase A2 and crotapotin were purified from crotoxin using the method of Rut~~v et al. (1971) and subsequently eluted using two-step gradients. The phospholipase activities of crude venom, crotoxin and its purified components were evaluated by the egg yolk coagulation assay (ViDet. and S1~OPPAHI, 1971). One phospholipase unit (PU) is defined as the amount of enzyme which increases by one minute the wagulability of an egg yolk suspension when compared to the wntrol (saline) . Immunization and challenge schedule Animals were immunized with crotoxin using the method of GODIN(l (1986) . Twenty mice and two rabbits were injected with a water-in-oil emulsion produced by mixing four volumes of Freund's wmplete (priming) or incomplete (boosters) adjuvant (Difw Laboratories, Detroit, U.S.A.) with one volume of an aqueous solution of crotoxin (0 .2 ml/mouse and 2.0 tnl in four sites/rabbit) . Animals were boosted three times at 21-day intervals. In We mouse an immunization schedule of 6.25 Rg of crotoxin in the emulsion was used for priming and for subsequent booster i.p . injections . This amount was equivalent to five i.p. t-D~ doses of crotoxin . The final booster was 9.40 /tg of crotoxin ín an aqueous solution. Control groups (20 mice) were injected with the above solution without antigen. Mice were bled weekly for 14 weeks from the tail vein . Whole blood samples (1 : 50 dilutions in phosphate buffered saline wntaining 0.05% Tween) were stored at -20°C before assay. The indirect ELISA method was used for detection of antibodies against crude venom, crotoxin, phospholipase Ai and aotapotin. On the 65th day, 10 mice were bled by axillary vein puncture. Sera were pooled and stored at -20°C. The remaining mice wntinued to be bled weekly for specific antibody detoction and were challenged i.p . with whole venom doses of either 11 r.n~ (20 pgl20 g mouse), 6 days after the final injection or 50 t.D~ (90 pgl20 g mouse), 21 days after the final injection or 100 tDSO (180 /íg/20 g mouse), 36 days after the final injection (Fig. 1) . Survivors were recorded 24 hr after each challenge. Control groups of 10 female mice of the same weight received the same doses of C. d. terrhcus venom at each challenge (Table 1) . A similar schedule was adopted for rabbit immunization with s.c . injections of 47 pg of crotoxin in the emulsion and 63 /tg of crotoxin in aqueous solution. Protective e (jects Hyperimmune sera obtained from mice and rabbits immunized against crotoxin were tested for precipitating antibodies using Ouchterlony (double diffusion) analysis . Undiluted and diluted sorum (I :2, 1 :4, 1 :8, I : 16, 1 : 32) were analysed for antibodies against C. d. tenifecus venom and crotoxin . The protective effects of rabbit hyperimmune sera were evaluated by the En~ estimation (numbers of mice alive 24 hr following injections of five i.p . t.D~ doses of C. d. terr~eus venom with different amounts of rabbit antiserum) .
Immunization with Crotozin
1493
assay Whole blood samples from mice were assayed by ELISA using the indirect method (~I%AR.4rON et al., 1977) with the modifications described by Ho et al . (1986). Hemobag plates (Hemobag Produtos Cirurgicos Ltda, Campinas, Brazil) were coated with 100 pl of a 5 ixg/ml solution of either C. d. terr~cus venom, crotozin, phospholipase A~ or crotapotin pct well. Peroxidase conjugated anti-mouse immunoglobulin diluted 1/1000 (Sigma Co ., U .S.A .) was used as the enzyme marker. Ortho-phenylcnediamine (Sigma Co ., U .S.A .) in the presence of H ZOZ was used as substrate . After 20 min of incubation at room temperature, the reaction was stopped by adding H,S04 (30% v/v). Absorbance was measured at 492 nm. Antibody
RESULTS
The ELISA antibody titres induced by i.p. injections of crotoxin in mice during the immunization period of 62 days were higher in crotoxin antibody than in whole venom, phospholipase Az or crotapotin antibodies (Fig. 1). Higher antibody response were obtained to whole venom and phospholipase AZ than to crotapotin (Fig. 1) despite the fact that crotoxin (which is a component of crude venom composed of crotapotin and phospholipase A~ had been used as immunogen. The component of crotoxin with phospholipase activity produced a higher antibody response than crotapotin which does not possess that activity . The phospholipase activity of crotoxin and its purified components crotapotin and phospholipase AZ were respectively 20, 0 and 50 PU/mg of protein using the egg yolk coagulability assay. The protective effect induced by immunization with crotoxin in mice correlated well with ELISA antibody titre. The antibodies generated by crotoxin were capable of
FIG. 1 . ANI7BODY RBSPON3E IN MI(x DURING LP . GIMUNIZATION WITH CR01'OXIN AND FREUND's ennrveNr, I+OLLOWED BY CHALIBNGHS GF Crotalur dwissus terrifecus veNOSt . Whole venom antibody response, D----D ; crotoxin antibody response fir ; phospholipase A 2 antibody response ~~C: crotapotin antibody response ~--i . A to D-Immunization with crotoxin (A, 6 .25 pg/Freund complete adjuvant; B, C, 6 .25 ~g/Freund incomplete adjuvant; D, 9 .4 l+g/~e) . I to K~hallenge with whole venom (I, 11 Ln,° doses (20 ßg/20 g mouse); J, 50 Ln~ doses (90 ßg/20 g mouse) ; K, 100 Ln m doses (180 pg/20 g mouse)). Each point represents the mean ELISA absorbance values from 20 mice (immunization schedule) and 10 mice (challenge schedule) . The variation coeHICient ((standard deviation) mean) x 100) for each point with A~ nm > 0 .200, was about 15% .The mouse blood dilution was 1 : 50 . Crotales duriasus terrificus venom and crotoxin had an i .p. ~w dose of 1 .8 (1 .52-2 .2) pg/20 mouse and 1 .24 (1 .00-1 .47) pg/20 g mouse, respectively.
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1494
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Immunization with Crotoxin
1495
providing 100% protection against challenges of 11 and 50 i.p . LD S° doses of whole venom (Table 1) . Following challenge with 11 ~w doses of whole venom, a large increase in antibody titre was observed . At that dosage, the crude venom boosted the antibody level. Following challenge with 50 r n~ doses of whole venom a slight decrease in antibody titre was observed . Twenty four hours after challenge all mice were alive, but one animal died after 48 hr (this animal had an ELISA absorbance of 0 .022, 48 hr post-challenge). Seven out of nine mice survived challenge with 100 I,n~ doses of whole venom. The two animals which died had similar ELISA titres to the survivors. Using double diffusion analysis, sera and purified IgG from the two crotoxin-immunized rabbits showed a weak precipitin line at a 1 : 2 dilution when tested against crotoxin and whale venom. The En~ values of the sera from immunized rabbits were 2214 serum/mouse (12-3811) and 3311 serum/mouse (240 k1) respectively . Antibody levels of the hyperimmune sera, evaluated by double diffusion analysis, did not reflect the protective effect in either mice or rabbits. DISCUSSION
According to HURN and CIiANTLER (1980), antisera obtained using crude preparations are often more highly immunagenic than purer materials . However, since venoms are complex mixtures of different antigenic components, a multitude of potential antigenic sites are presented to the animal's immune system . Under these conditions only the most antigenic determinants may stimulate significant antibody production, and they may not be associated with a toxin's active site . SCHAEFFER et al. (1988) used ELISA for testing size-selected fractions of whole crotalid venoms for binding to antivenom (Wyeth, Crotalidae Polyvalent) and found a minimal titre of antibodies to the low molecular weight venom components . These observations led us to investigate the efficacy of crotoxin in protecting experimental animals against venom lethality. The choice of immunization schedule enabled the use of a highly toxic antigen with a dramatic decrease in lethality. The mechanism of action of adjuvants which permits slow release of antigen in a highly aggregated form, probably explains the resistance of animals to such high doses of crotoxin. As a result, a high protective effect against whole venom was obtained in both mice and rabbits immunized against crotoxin . Antibodies raised against crotoxin appear to recognise the whole venom antigen at levels comparable to the crotoxin antigen. The level of protection against 100 i.p . Ln~ doses of whole venom (36 days after the final booster dose of crotoxin) in mice was 78%,compared with 90% reported by SANTbS et al. (1988) following challenge with 16 s.c . LD P doses of C. d. terrificus venom 21 days after the final booster injection of phospholipase AZ in mice. The >~w of the sera from two rabbits immunized with crotoxin were 2211 serum/mouse (12-3811) and 33 pl serum/mouse (24~01~1), compared to 7811 serum/mouse (69-8211) obtained by Fxi?rrns et al. (1989) from one rabbit immunized with C. durissus venom incorporated into liposomes. The antibody levels elicited by crotoxin were not enough to produce a strong precipitin line in the double diffusion analysis . However, the protection afforded by those antibodies suggests that they must have a high afïtnity to the toxic components of whole venom. WnxxELI, et al. (1986) showed that the response of coagulopathy to initial treatment was most rapid in patients given Twyford antivenom, compared to other hyperimmune serum raised against Calloselasma rhodostoma venom. The Twyford antivenom has its titer of anti-procoagulant (antiArvin/Ancrod) increased four-fold during manufacture.
1496
T. V. FREITAS et al.
These data suggest that immunization with crotoxin and/or the addition
of anti-crotoxin
as a supplement to polyclonal antivenom should be considered in order to obtain a hyperimmune serum with a high level
of protection
against
C. d. terrficus
venom.
Acknowledgements-We thank Mr R. Mnc~, and Mrs B. C. B. DA FoxsECe for excellent technical assistance; the authors are grateful to Dr R. D. G. Tt~AKSTON for his valuable suggestions during the preparation of this manuscript . This research was partially supported by Conselho National de Desenvolvimento Cientifico e Tecnologico (CNPeil, Financiadora de Estudes e Projetos (FINEP) and Commission of We European Communities (CEC). REFERENCES AMORIM, M. F. (1971) Intermediate nephron nephrosis in human and experimental crotalic poisoning. In: Venomous Animals and their Venoms . Vol. II, pp . 319-343 (BucrmtL, W. and BUCICLEY, E., Eds). New York: Academic Press. AMOAtAt, M. F. and MII.i .o, O. (1954) Intermediate nephron nephrosis from snake poisoning in man. Am. J. Path . 3, 47999. CouLrelt, A. R., HARxts, R. D. and 5u1T~rtLANn, S. K. (1980) Enzyme immunoassay for the rapid clinical identification of snake venom. Med. J. Aust . 1, 43335. DANZIG, L. E. and Aie:LS, G. H. (1961) Haemodialysis of acute renal failure following rattlesnake bite, with recovery . J. Am . Med. Ass. 175, 136-137. DI3ALIWAL, J. S., LrM, T. W. and SuxutrAIteN, K. D. (1983) A double antibody sandwich MICRO-ELISA kit for the rapid diagnosis of snake bite. Southeast Asian J. Trop . Med. Pub. Hth 14, 367-373. FtNNt=.v, D. J. (1971) . Probit Analysis. 3rd. ed . Cambridge: Cambridge University Press. FxatTAS, T. V., TAVAxFS, A. P., TFmAtcsroN, R. D. G., LAtxo, G. and New, R. R. C. (1989) Use of liposomes for protective immunisation against Crotales durissus (Tropical rattlesnake) venom. Toxicon 27, 341-347. Gootxc, J. W. (1986) Production of monoclonal antibodies . In : Monoclonal Antibodies : Principles and Practice, Ch . 3, pp . 59-103 . London: Academic Press. HAWOOOD, J. B. (1982) Physiological and pharmacological effects of rattlesnake venoms. In : Rattlesnake Venom: Their Actions and Treatment, pp. 121-162 fTu, A. T., Ed .) . New York: Marcel Dekker . Ho, M., WAtexELL, M. J., WAxxsLL, D. A., BroweLL, D. and VoLtEIy A. (1986) A critical reappraisal of the use of enzyme-linked immunosorbent assays in the study of snake bite. Toxicon 24, 211-221. HuxN, B. A. L. and GtArrrtaa, S. M. (1980) Production of reagent antibodies . Mcth . Enzymol. 70, 104-143. PuGa, R. N. H. and TtIEAxsroN, R. D. G. (1980) Incidence and mortality of snake bite in Savanna Nigeria. Lancet II, 1181-1183 . Pvoa, R. N. H., TaeAtcsTON, R. D. G. and REtn, H. A. (1979) Bites by the carpet viper in the Niger Valley. Lancet II, 625-627. Puoa, R. N. H., TtIEAxsroN, R. D. G. and REtn, H. A. (1980) Epidemiology of human encounters with the spitting cobra, Naja nigricollis, in the Malumfashi area of northern Nigeria. Ann. Trop . Med. Parasitol. 74, 523-530. RuasAt~N, K., BttFlTaertrT, H. and HAHnERMANN, E. (1971) Biochemistry and pharmacology of the crotoxin complex. Naunyn-Schmiedebergs Arch. Pharmacol. 270, 274-288. SASeecI~, M. S., CbNNINOrwu, E. R. and Furs, C. T. (1977) A study of the treatment of pit viper envenomation in 45 patients. J. Trauma. 17, 569-573 . SANTas, M. C., DINIZ, C. R., WHrIAIO:R Pact->ECO, M. A. and blas DA Save, W. (1988) Phospholipase Az injection in mice induces immunity against the lethal effects of Crotales durissus terr~cus venom. Toxicon 26, 207-213. ScaAeKreIt, R. C., Jtt, RANDALL, H., Roc, J. and CAxISON, R. W. (1988) Enzyme-linked immunosorbent assay (ELISA) of size-selected crotalid venom antigens by Wyeth's Polyvalent Antivenom. Toxicon 26, 67-76. St:ICI, C., VinAL, J. C. and BAxxto, A. (1980) . Purification of gyroxin from a South American rattlesnake (Crotales darissus terrificus) venom. Toxicon 18, 235-247. St.orrA, K. H. and Fx~tvt~st.-CorrxaT, H. L. (1938) Schalangengifte, III : Mitteilung: Reinigung und krysta11i7ation des Klapperschangengiftes . Ber. Dtch Chem. Ges. 71, 1076-1081. SUTHERLAND, S. K. (1980) Venom and Antivenom research. Med. J. Aust. 2, 246-250. TI~AjcsroN, R. D. G., LLOYD-JONES, M. J. and RErD, H. A. (1977) MICRO-ELISA for detecting and assaying snake venom and venom antibody . Lancet II, 639-641 . VIRAL, J. C. and STOPPANI, A. O. M. (1971) Isolation and purification of two phospholipases A from Bothrops venom. Arch. Biochem . Biophys . 145, 543-556. WARRELL, D. A., LOOAREESUWAN, S., Tt~AttsroN, R. D. G., Prm,L>ps, R. E., GtANTaevArnca, P., VIItAVAN, C., $UPANARANOND, W., KAttswANO, J., Ho, M., HuTTON, R. A. and Vetcao, S. (1986) Randomized comparative trial of three monospecific antivenoms for bites by the Malayan pit viper (Callostlasma rhodostoma) in Southern Thailand: Clinical and laboratory correlations. Am. J. Trop . Med. Hyg . 35, 1235-1247. WORLD HEALTH ORGANIZATION (1981) Progress in the characterization of venoms and standardization of antivenoms. WHO Offset Publication no . 58 . Geneva : World Health Organization.