Abstract

THE IMMUNOCHEMICAL REACTIVITY AND NEUTRALIZING CAPACITY OF POLYVALENT Vipera (EUROPEAN) ANTIVENOM ON ENZYMATIC AND TOXIC ACTIVITIES IN THE VENOMS OF CROTALIDS FROM ARGENTINA

A.R. de ROODT, J. A. DOLAB, L. SEGRE, C. SIMONCINI,

S. E. HAJOS, T. FERNANDEZ, J. C. DOKMETJIAN, S. LITWIN,

C. ACCATTOLI, J. C. VIDALCORRESPONDENCE TO: J. C. VIDAL – Area de Iologia, Museo Argentino de Ciencias Naturales "Bernardino Rivadavia", Av. Angel Gallardo, 470 1405, Buenos Aires, Argentina. Phone/Fax: 54 1 303 2492

1 Instituto Nacional de Producción de Biológicos ANLIS "Dr. Carlos G. Malbrán", Av. Vélez Sarsfield 563, 1281-Buenos Aires, Argentina, 2 Cátedra de Inmunología, Facultad de Farmacia y Bioquímica, University of Buenos Aires, 3 Area de Iologia, Museo de Ciencias Naturales "Bernardino Rivadavia," (CONICET), Buenos Aires, Argentina.

Cross-reactivity and cross-neutralization between antivenoms and venoms from snake species belonging to the same Genus or Family is frequently observed (8,9,12,22,27-30). This may be related to the high degree of similarity in the primary, secondary, and tertiary structures of homologous venom proteins, such as phospholipases A2 (3,33) and metalloproteinases (4), even from taxonomically unrelated species.

On the other hand, cross-reactivity and cross-neutralization studies have been used as taxonomic tools (2) to ascertain the level of contribution of some venom components to the physiopathology of envenomation (8) These studies could even be critically important for therapeutic use in emergencies, such as snakebites by foreign specimens in zoos, serpentariums, or for people who keep snakes as pets.

Immunochemical cross-reactivity between antivenoms and venoms has been reported to be relatively high for crotalids and viperids, lower for viperids and elapids, and very poor for crotalids and elapids (24). Thus, high amounts of commercial Vipera ammodytes antivenom from Behringwerke (Mardeburg, Germany) were reported to protect mice against B. alternatus, B. neuwiedii, and B. jararaca venoms, but were ineffective in neutralizing the lethal potency of C. d. terrificus venom (10). In contrast, polyvalent Behringwerke antivenom (raised against Bitis, Echis, Dendroaspis, and African Naja venoms) protected mice against the lethal potency of Crotalus atrox venom, while Wyeth polyvalent antivenom (raised against several Crotalus and Bothrops venoms) neutralized the hemorrhagic activity of Bitis arietans venom (20).

In this study, the immunochemical reactivity and neutralizing capacity of commercial Vipera antivenom were tested on the enzymatic and toxic activities present in the venoms of several crotalid species from Argentina.

VENOMS: The venoms of C. d. terrificus, B. alternatus, B. jararaca, B. jararacussu, B. moojeni, and B. neuwiedii were obtained from healthy adult specimens kept at the serpentarium of the Instituto Nacional de Producción de Biológicos A. N. L. I. S. "Dr. Carlos G. Malbrán" (Buenos Aires, Argentina). The snakes were milked and the venoms were collected in cold sterile Petri dishes, desiccated under vacuum, and kept in tightly closed vials at 4ºC. B. ammodytoides venom was a gift from Dr. E. Gould, Fundación de Estudios Biológicos (Buenos Aires, Argentina). Venom solutions were prepared in 0.15 M NaCl just before use and sterilized by filtration.

Crotoxin complex was purified from C. d. terrificus venom as previously described (23). The purified preparation had a specific phospholipase A2 activity of 25 ± 3 units mg protein. SDS-PAGE under non-reducing conditions showed two bands with mobilities corresponding to molecular weights of 9.5 ± 0.3 kD (sub-unit A) and 14.5 ± 0.2 kDa (sub-unit B). The crotoxin preparation was equilibrated in 0.15 M NaCl by chromatography on Sephadex G-25 and sterilized by filtration.

ANTIVENOM: Horse "Immunoserum contra Venena Viperarum Europearum", Series 160 (Exp. 2/98) from Imunolsky zabod Laboratories (Zagreb, Croatia) was kindly provided by Prof. Dr. Franc Gubensek, Dept. of Biochemistry and Molecular Biology, Josef Stefan Institute (Ljubljana, Slovenia). According to the manufacturers, 1.0 ml of antivenom neutralizes 100 LD50 of V. ammodytes or V. aspis venom, and 50 LD50 of V. berus, V. lebetina, or V. xanthina venoms.

DOUBLE IMMUNOPRECIPITATION (DIP): This was performed in Petri dishes with a 3.0 mm thick layer of agarose (Ultrapure, Sigma Chemical Co.) with 10.0 ml of venom solutions (0.5 mg/ml) in the peripheral wells and 10.0 ml of Vipera antivenom in the central well. Diffusion was allowed to proceed for 72 h at 25ºC in a humid chamber (27,28). DIP was stained with amidoblack (Sigma Chemical Co.).

SDS-PAGE AND WESTERN BLOT: SDS-PAGE in non-reducing conditions was performed on an acrylamide 12.5/2.5 (T/C) gel, with a vertical unit Miniprotean II (Bio Rad), using the discontinuous buffer system described by Laemmli (16). After electrophoretic separation, the bands were stained with Coomassie Brilliant Blue R250 (Bio Rad), or transferred to a nitrocellulose membrane, as described by Towbin (32). The molecular weight markers used were those included in the Sigma VII-S kit.

LD50: Mice (CF1 strain, 18-22 g, n=6 per dose level) were injected intravenously with different concentrations of B. alternatus or C. d. terrificus venoms in 0.15 M NaCl. The LD50 value was calculated by regression using Prisma software (GraphPad Software, Inc. San Diego, CA). The LD50 for C. d. terrificus venom and crotoxin via intraperitoneal was determined by the procedure described by Meier and Theakston (21).

NEUTRALIZATION OF LETHAL POTENCY: Mice (CF1 strain, 18-22 g, n = 5) were injected intravenously with 25 mg (7.1 LD50) of C. d. terrificus venom or with 100 mg (4.5 LD50) of B. alternatus venom preincubated for 45 min at 37ºC in 300 ml of 0.15 M NaCl (positive controls). The other groups (n = 5 each) were injected with the same amounts of venoms preincubated for 45 min at 37ºC with Vipera antivenom at 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 ml per mg venom in 300 ml of 0.15 M NaCl. The ED50 was estimated from plotting the number of surviving animals against the volume (microliters) of antivenom.

HEMORRHAGIC AND NECROTIC ACTIVITY OF B. ALTERNATUS VENOM: Six mice (CF1 strain, 25 ± 2 g) were injected intradermically with 100 mg (hemorrhagic) or 50 mg (necrotic) of B. alternatus venom preincubated for 45 min at 37º C in 150 ml of 0.15 M NaCl (positive controls). Three groups of mice (n = 6 each) were injected intradermically with the same amounts of venom preincubated for 45 min at 37ºC with 25.0, 50.0, and 100.0 ml of antivenom in a final volume of 150 ml. The hemorrhagic areas were measured 3 h after injection, as described by Ferreira et al. (11,12). The necrotic areas were measured 72 hours after injection, as described by Theakston and Reid (31).

INDIRECT HEMOLYSIS AND PHOSPHOLIPASE A2 ACTIVITY: Indirect hemolysis was determined using the procedure described by Al-Abdhula et al.(1). Mixtures containing 0.5 ml of egg yolk, 0.5 ml of washed and pelleted human erythrocytes, and 100 ml of 0.1 M CaCl2 in 10.0 ml of 0.15 M NaCl, 10 mM phosphate buffer pH 7.2 were incubated for 60 min at 37ºC with 0.15 M NaCl (negative controls), 1 % Triton X-100 (positive control), 100 or 200 mg of venom preincubated with either 0.15 M NaCl or Vipera antivenom at 0.5 and 1.0 ml per mg of venom in 0.15 M NaCl. The reaction was stopped with 1.0 ml of 10% EDTA and the mixtures were centrifuged for 30 min at 1500 r.p.m. The absorbances at 550 nm of the supernatants were recorded. The differences due to preincubation with antivenom were expressed as percentage of those obtained with 100 and 200 mg of venom.

Phospholipase A2 activity of the crotoxin complex before and after incubation with Vipera antivenom at 1.0 and 2.0 ml per mg of toxin was measured by automatic titration of the fatty acids released from a sample of egg yolk using a pH-Stat (Radiometer). One unit of phospholipase A2 activity was defined as the consumption of 1.0 mEquiv of NaOH (or the release of 1.0 mEquiv of fatty acids) per min at 25ºC.

STATISTICAL ANALYSIS: The results are presented as Mean ± S.D values. The Student t-test was used for the comparison between means and variances. The comparison between groups was made using the Student t-test and the Wilcoxon’s method.

Cross reactivity between Vipera antivenom and all the crotalid venoms tested was measured using DIP, in which several weak precipitation archs were observed. ELISA showed positive for all Bothrops venoms using dilutions of Vipera antivenom from 1:128000 (B. neuwiedii) up to 1:64000 (B. moojeni). The detection of C. d. terrificus venom and crotoxin required dilutions of the antivenom from 1: 64000 to 1: 32000 and from 1: 32000 to 1: 16000, respectively.

The Western blots of the different venoms used are shown in Figure 1. Using B. neuwiedii and B. jararacussu venoms, Vipera antivenom produced a band with molecular weight of about 67.4 kD. Except for B. jararaca and B. moojeni venoms, all the other bothropic venoms exhibited one to three strongly stained bands with molecular weights between 56.5 and 60.0 kD, as well as between 38.0 and 53.0 kD. Bands of about 34.0 and 38.0 kD were strongly stained with B. neuwiedii and B. jararaca venoms and less stained with B. alternatus venom. Except for B. jararacussu, all the Bothrops venoms showed one or more strongly stained bands of about 20-24 kD. Stained bands between 18.0 and 19.0 kD were observed with B. neuwiedii, B. jararaca, B. jararacussu, and B. moojeni. Using the venom of C. d. terrificus, strong stained bands of 57.3-59.7 kD, 45-46 kD and weakly stained bands were observed in the region with molecular weights of 39.0, 31.0, 27.0, 20.0, and 14.3 kD. Despite the different contents in phospholipase A2, only C. d. terrificus venom exhibited a band of 14.3 kD.

The LD50 of C. d. terrificus venom in mice was 0.175 ± 0.03 mg/g (intravenous) and 0.33 ± 0.1 mg/g (intraperitoneal). The intraperitoneal LD50 of crotoxin was 0.13 ± 0.09 mg/g. The intravenous LD50 of B. alternatus venom in mice was 1.1 ± 0.2 mg/g.

After the intravenous injection of 1.24 mg/g of C. d. terrificus venom (7.1 LD50), all the animals died within 20 min. When the same amount of venom preincubated with Vipera antivenom at 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 ml per mg was injected intravenously, all the animals died, although the survival time was longer (up to 6 h with 5.0 ml per mg venom).

A poor neutralizing effect of the antivenom was again observed after the intraperitoneal injection of 4.5 LD50 of C. d. terrificus venom. Only one in five mice survived after the injection of this amount of venom preincubated with Vipera antivenom at 5.0 ml per mg. According to Meier and Theakston (21), plots of the results obtained after intraperitoneal injection of different doses of C. d. terrificus venom preincubated with either 0.15 M NaCl or Vipera antivenom at 2.0 or 4.0 ml per mg suggest that the antivenom produces a decrease in the lethal potency of about 16% (p = 0.05).

According to Meier and Theakston, plots of results obtained after intraperitoneal injection of pure crotoxin preincubated with 0.15 M NaCl or Vipera antivenom (at 2.0 ml per mg toxin) are shown in Figure 2. The apparent LD50 of the antivenom-treated toxin (0.47 ± 0.16 mg/g) was higher than that of untreated toxin (0.13 ± 0.09mg/g), suggesting a significant decrease (about 60%, p<0.01) in lethal potency.

Conversely, Vipera antivenom afforded effective protection against the lethal action of 4.5 LD50 of B. alternatus venom. The calculated ED50 was 1.25 ± 0.25 ml of antivenom per mg of venom, an amount only 4-5 times higher than that obtained with the homologous antivenom.

As shown in Table 1, preincubation of B. alternatus venom with Vipera antivenom resulted in neutralization of its hemorrhagic and necrotic activities.

Preincubation of all the Bothrops venoms used in this study with 2.0 ml of Vipera antivenom per mg of venom resulted in complete inhibition of their proteolytic activity on gelatin.

The inhibitory effect of Vipera antivenom on the procoagulant activity of all the venoms studied is shown in Table 2. When compared to the crude venoms, preincubation with Vipera antivenom at 1.0 ml per mg of venom increased the time for clot formation 2-4 times with B. ammodytoides, B alternatus, B. neuwiedii, and B jararacussu, 6 times with B.moojeni, and more than 6 times with B. jararaca and C. d. terrificus venoms.

The results of indirect hemolysis assay are shown in Table 3. Preincubation with 0.5 ml of Vipera antivenom per mg of venom resulted in a poor inhibition of indirect hemolysis with B. ammodytoides and B. neuwiedii venoms, and inhibitions ranging between 25 and 50% with the other venoms. Preincubation with 1.0 ml of antivenom per mg of venom did not increase inhibition with B. ammodytoides and B. jararaca venoms, somewhat increased inhibition with B. moojeni, B. jararacussu, B. neuwiedii, and C. d. terrificus venoms, and produced complete inhibition with B. alternatus venom.

Phospholipase A2 activity of crotoxin, as measured in the pH-Stat with 10.0, 20.0, and 40.0 mg of toxin increased linearly with the toxin concentration. The calculated specific activity was 25.0±3.0 mEquiv. minmg protein. Preincubation with Vipera antivenom at 1.0 and 2.0 ml per mg of crotoxin was 20% (p < 0.05) and 45% (p < 0.01) inhibition, respectively.

Both DIP and ELISA indicate a strong immunochemical cross-reactivity between Vipera antivenom and all the venoms used in this study. The concentrations of Vipera antivenom required for the detection of the crotalid venoms using ELISA were only 2-3 times higher than those using homologous antivenoms. These results confirm the high degree of immunochemical cross-reactivity between antivenoms raised against venoms of the Family Viperidae and venoms of the Family Crotalidae (10,20), suggesting that Vipera antivenom could inhibit some of the biological activities of the crotalid venoms.

Hemorrhagic, necrotic, and procoagulant activities play an important role in the physiopathology of envenomation by Bothrops species (14,15,17,25,26) and venoms from Vipera species have a procoagulant activity and also produce hemorrhage and necroses (19).

The hemorrhagic activity of both viperid and crotalid venoms is caused by metalloproteinases, known generically as "hemorrhagins" (4,18). Bjarnasson and Fox (4) have classified these metalloproteinases into four groups, and the hemorrhagins from viperid and crotalid venoms can be found in most of them: LEBETASE from V. lebetina, HF1 from B. jararaca, and MPA from B. moojeni in group I; HT-1, HT-2, and HT-3 from V. ammodytes, HMP from V. berus, HR1, HR2, and HR3 from V. palestinae, BOTHROPASINE from B. jararaca, NHFa and NHFb from B. neuwiedii venom, in group II; HT-1 from V. aspis, HF2, HF3, and JARARRHAGIN from B. jararaca venoms in group IV.

Hemorrhagic and necrotic activities of B. alternatus and B. ammodytoides venoms appear to be associated with fractions of molecular weights between 40 and 70 kD, while the procoagulant activity appears to be associated mainly with fractions of molecular weights between 20 and 40 kD (6). Western blots (Figure 1) showed a strong recognition of venom components in those range of molecular weights, and preincubation with Vipera antivenom inhibited the hemorrhagic, necrotic, and procoagulant activities of B. alternatus venom. This may explain the significant neutralizing capacity of Vipera antivenom against the lethal potency of B. alternatus venom with an estimated ED50 of 1.25 ± 0.25 ml per mg of venom (only 4 to 5 times higher than that of the homologous antivenom).

In all the bothropic venoms studied, the hemorragic and proteolytic activities on gelatin appear to be highly correlated (r= 0.6) (7), and preincubation with Vipera antivenom at 2.0 ml per mg of Bothrops venoms resulted in complete inhibition of the proteolytic activity on gelatin.

The indirect hemolysis assay (related to phospholipase A2 activity) showed that preincubation with 1.0 ml of Vipera antivenom per mg of venom produced complete inactivation of B. alternatus venom, 45% to 66% inactivation with B. moojeni and B. jararacussu venoms, and only a poor inhibition (14%) with B. ammodytoides and B. jararaca venoms (Table 3). Cross-inactivation of indirect hemolysis may be explained due to viperid and crotalid venoms contain Group II phospholipases A2 (5) and also due to a high degree of similarity between the in primary, secondary, and tertiary structures of this large family of homologous proteins (3).

This work was partially supported by grants from the Fundación "FUNMECACET" and the Ministry of Health. The authors are indebted to Prof. Dr. F. Gubensek for his encouragement and suggestions. J. C. Vidal is Career Investigator of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.

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