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NEUTRALIZATION OF DIFFERENT ACTIVITIES OF VENOMS FROM NINE SPECIES OF BOTHROPS SNAKES BY BOTHROPS JARARACA ANTIVENOM MOHICA L. F>?ItR>~w, AxA M. MOURA-nA-SILVA and I. MOTA' Laboratory of Immunopathology, Instituto Butantan,
Sao Paulo, Av . Vital Brazil, 1500 05504 Säo Paulo, Hra~l (Received 1l Afay 1992 ; accepted 11 July 1992) .
M. L. F~RREmA, A. M. MOURA-DA-SILVA and I. MOTA. Neutralization of different activities of venoms from nine species of Bothrops snakes by Bothrops jararaca antivenom. Toxicon 30, 1591-1602, 1992 .-Antivenoms are the usual treatment in ca .4es of systemic envenoming by Bothrops snakes . However, the neutralization of each venom component by the antivenom is not well established . Bothrops jararaca antivenom, produced in rabbits, recognizes the venoms of nine different Bothrops species with high ELISA antibody titres . Western blot analysis showed that almost all antigens present on both homologous and heterologous venoms are recognized. Neutralization tests were performed using whole antivenom or its IgG fraction. The antivenom was able to neutralize the haemorrhagic, coagulant and necrotizing activities of the heterologous venoms in the same antivenom/venom proportion as for the homologous venom. Myotoxic activity was only partially neutralized . Neutralization of the proteolytic activity of heterologous venoms required higher amounts of antivenom than for the homologous venom. Phospholipase and oedema-inducing activities were completely neutralized only in the homologous system . INTRODUCTION
Ix BRAZIL, 90% of snakebites are caused by Bothrops species. Mortality has been estimated as 2.4% and may even be as high as 8% when no antivenom is given (WORLD HEALTH ORGANIZATION, 1981). Oedema, haemorrhage and necrosis are the most serious local manifestations in these cases, causing prolonged or permanent disability . Several components isolated from Bothrops venoms, such as haemorrhagic factors (MANDELBAUM et al., 1976, 1984), enzymes disturbing blood coagulation (NAHA3 et al., 1979; HOFMANN and Box, 1987), proteolytic enzymes (ASSAKURA et al., 1985 ; TANIZARI et al., 1989) and myotozins (GUTmtREZ et al., 1984; 1989 ; HoiKSI-BRANDEHURl.îO et al., 1988; MOURA-DA-SILVA et al., 1991a) are responsible for the clinical symptoms of bothropic envenoming . The usual treatment for snakebites is serum therapy. Antisera are produced by immunization of horses with venoms from a limited number of Bothrops species, the aim 'Author to whom correspondence should be addressed . (591
1592
M. L. FERREIRA et al.
being to neutralize all Bothrops venoms . The efficacy of the serum is usually assayed by assessing its capacity to neutralize the lethality of a reference venom in experimental mice. According to the WORLD HEALTH ORGANIZATION (1981), commercial antivenoms should also be tested for their capacity to neutralize specific venoms activities such as haemorrhage, necrosis and coagulation. This approach is important in the case of Bothrops antivenom, since the species of this genus cause severe local injuries . Also important in relation to serum therapy in Bothrops envenoming is that the venoms of the different species may differ in their pharmacological properties . Several studies have shown that Bothrops venoms share many antigens, and antiserum against the venom of one species is capable of neutralizing the lethal effects of the other venoms (ARAIV'IE.S and BRANDÂO, 1949; VILLARROEL et al., 1978/79; DIAS-nA-SILVA et al., 1989). However, individual antigens have also been reported in Bothrops venoms (VILLARROEL et al., 1974 ; MOURA-DA-$II,VA et al ., 1990x) and some have recently been characterized as myotoxins (MOIJRA-DA-SII.VA et al., 1991x) . Although some data are available on the cross-neutralization of Bothrops haemorrhagic factors (MANDELBAUM and A3SAKURA, 1988), coagulant activity (RosEIVPEL.n and KELEN, 1966) and myotoxins (MOURA-DA-SILVA et al ., 1991 b), these data include only a limited number of components. This does not allow evaluation of the efficacy of the antivenom in neutralizing the local effects of the different venoms . The aim of this study is to evaluate the neutralization of different activities of the venoms obtained from nine species of Bothrops by B. jararaca antivenoms to better understand the mode of action of homologous and heterologous antivenoms . MATERIALS AND METHODS Yenoms Venons of B. alternates, B. atrox, B. erythromelas, B. cotiarq B. jararacq B. jararaceste, B. mookni, B. neuwiedl and B. pradoi were supplied by Instituto Butantan (SÜo Paulo, Brazil) . Thirty to 40 snakes of each species were milked, the venom samples pooled, desiccated and stored at 4°C until used. Animals
Outbred male mice (18-22 g) and New Zealand white rabbits (3.5 kg) were obtained from the Instituto Butantan. Antisera
Anti-B . jararaca serum was obtained by immunization of rabbits. One milligram of B. jararaca venom dissolved in 1 ml of saline was emulsified with 1 ml of Freund's complete adjuvant. Five-hundred microlittns of the mixture was injected i.m . into each of the hind legs of three rabbits. One month later, the animals were injected i.d . three times at weekly intervals with 2001tg of B. jararaca venom in saline. They were bled 10 days after the last injection. Sera were stored at -20°C until used. The neutralizing ability was estimated in leg of venom neutralizing/ld/antiserum . The potency of the rabbit antiserum was estimated by comparing its ELISA titre and its neutralizing ability with those of the antibothropic standard serum produced by Instituto Hutantan . Mtlbody assay (ELISA)
Sera were titrated for their antibody content against different venoms (1 pg/well) coated on ELISA plates (Hemobag, Surgical Products, Campings, S Paulo, Hrazil). The ELISA technique was done as described by T't~tcsrox et al. (1987). Plates were read using an ELISA reader (Multiskan spectrophotometer, Eflab, Helsinki, Finland), and the titres were determined as the reciprocal of the highest dilution that causes an absorbance greater than 0.100 at 492 nm, as non-specific reactions were observed below this value. Electrophoresis and Western blotting
Venom antigens were separated according to the method of Lwr?~nn .i (1970) under non-reducing conditions. The proteins were then stained by Coomassie blue or transblotted onto nitrocellulose papers (Towgnv et al.,
Serum Neutralization of Bothrops Venoms
(593
1979) and developed by addition of serum samples diluted 100-fold in PBS (phosphate buffeted saline O.15 M, pH 7.5) plus 5% defatted milk . After washing, the strips were incubated for 1 hr with peroxidase labelled antirabbit IgG and stained by addition of 4~hloro-l-naphthol plus H=Or Mutine IgG (150,000 mol. wt), bovine serum albumin (68,000 mol. wt), ovalbumin (43,000 mol. wt), trypsinogcn (24,000 mol. wt), and alphalactalbutnin (14,000 mol. wt) were used as mol. wt markers. Isolatiote of rabbit IgGs The IgG fraction of B.jararaca antivenom was isolated using a FPLC system (Pharmacia, Uppsala, Sweden) by protein A amity chromatography . Serum (1 ml) was filtered and centrifuged at 10,000 rev/rain immediately before fractionation, and applied to the protein-A Superose HR 10/2 column equilibrated with 50 mM Tris-HCl buffer, pH 8.0. Proteins bound to the column were eluted with 0.l M citrate buffer, pH 3.0, and the fractions immediately neutralized with Tris 1 M. The optical density of the eluant was monitored at 280 mn. The flow rate was 2 ml/min and 2ml fractions were collected. The fractions eluted with pH 3.0 were pooled, dialysed against saline and concentrated by positive pressure filtration to the original serum volume . The fraction bound to the column showed a single band in SDS-PAGE of mol. wt close to 150,000 under non-reducing conditions and contained all the anti-venom antibody activity of the serum as assayed by ELISA. Haemorrlurgic activity Haemorrhagic activity was determined according to Koxno et al. (1960) with the modifications introduced by OwrreY et al. (1984). Briefly, mice were shaved in We backs and injected i.d. with 50 pl of the test solutions. After 2hr, the mice were killed with ether and the dorsal skin removed. The haemorrhagic lesion was measured as described by Koxno et al. (1960) . Results are expressed as the product of the diameters multiplied by a factor from I to 4, corresponding to the intensity of the colour of the spot (mm'/Pg of venom) . Quarttitation ojrrecrosis Necrosis was quantitated following i.d. injection of SO pl of the test solution into the shaved backs of mice . After 72 hr, the mice were killed with ether and the akin removed. The necrotic area was measured and is expressed as described for haemoahagic activity. In this case the factor from 1 to 4 corresponded to the depth of the lesion on the skin . Results are expressed as mm~/ltg of venom. Oedema-inducing activity The oedema inducing activity of the venoms was assayed according to the method of Y~tuwe et al. (1976) . Results are expressed in mglttg of venom. Coagulant activity Coagulant activity was determined using ciliated horse plasma as substrate and measured as described by N~+s et al. (1979). Results are expressed as the reciprocal of venom concentration able to clot the plasma in 1 sec. Phospholipasc As activity The method of M~tttrnrrtl (1965) was used for evaluation of phosphoGpase activity with slight modifications. Egg-yolk was suspended in Tris-HCl buffer, pH 8.0, to an initial absorbance of 1.0 at 740 nm . One hundred micrograms of each venom sample was added to 3 ml of this suspetrsion . Phoapholipase activity was assessed as the rate of a linear decrease in O.D. over an incubation period of 5-15 min. One unit of phospholipase Az activity torraponds to a decrease of 0.001 in absorbance per min. Activity is expressed as U/mg of venom. Myofoxic activity Mite were injected i.m . (thigh muscle) with 200111 of the test solutions. After 3 hr, blood was collected from the ophthalmic plexus under light ether anaesthesia. Sera from three mite were separated, pooled and immediately assayed for CK activity (Nat-CK assay system, Merck do Brasil S.A ., Siio Paulo, Brazil), as described by the manufacturers (G~tnN Socmrr oa C~.rxrc~t. Ct~easrs, 1977). One unit corresponds to the amount of enzyme that hydrolyses 1 umole of creative per min at 25°C . Myotoxic activity is expressed as U/mg of venom.
1594
M . L . FERREIRA et al. TAatE 1 . ELISA AN77BODY T11RE4 OF B. jararaca ANTIVENOUt AOAINS'r NINE DIFFERENT' VENOIN3 OF BOthrOpJ SNAKP3
Antigens (venoms of) B.jararaca (homologous system) B. alternatus B. atrox B. cotiara B. erythromelas B . jararacussu B . ntoojeni B . rteawiedi B . pradoi
ELISA antibody titres" Test l
Test 2
Test 3
256,000
128,000
128,000
128,000 256,000 256,000 128,000 256,000 128,000 256,000 128,000
128,000 64,000 64,000 32,000 32,000 64,000 32,000 64,000
128,000 128,000 64,000 64,000 64,000 128,000 64,000 64,000
" ELISA antibody titres were determined as the reciprocal of the maximal dilution that causes an absorbance higher than 0.100 at 492 nm . Each test was performed with a different preparation of serum from different rabbits . Proteolytic activity Proteolytic activity was estimated using cavein as substrate as described by are expressed in U/mg of venom.
MANDELBAUM
et al. (1982) . Results
Serum neutralization of venom activitieJ The ability of B. jararaca antivenom to neutralize different venoms activities from the nine BothropJ species was estimated by preincubating venom samples wiW an excess of antiserum (5 or 20 klJpg of venom) for 1 hr at 37°C . Following incubation, the mixtures were centrifuged in a microcentrifuge (Centrifuge 5414, Beckman) for 5 min and the supernatants were assayed for each activity. As we observed that normal rabbit serum was able to inhibit some activities (necrosis, phospholipase and myotoxicity) or to induce others (oedema and proteolytic), we used the IgG fraction instead of whole serum when assaying these activities . Results are expressed as the percentage of neutrali78tion taking as 100% the values obtained when venom was incubated with saline. All experiments were repeated three times. RESULTS
Antisera to B. jararaca venom obtained from three different rabbits were assayed separately . ELISA antibody titres against the homologous and the heterologous venoms did not vary between the three antisera. Comparison of the ELISA antibody titres (Table 1) obtained when B. jararaca antiserum was assayed against the homologous and heterologous antigens showed no significant differences (since variation of the ELISA titres for a same sample was two-fold or less, only differences above this value were considered significant) . These antisera were then pooled and tested for specificity by Western blotting. Figure 1 shows the venoms from nine species of Bothrops fractionated by SDS-PAGE and stained by Coomassie blue (1) or revealed by the B. jararaca antivenom (2). Almost all proteins stained by Coomassie blue were also revealed by Western blotting with B. jararaca antivenom. Some bands with mol. wt in the region of 15,000 were stained by Coomassie blue in the venoms of B. alternates, B. atrox, B. erythromelas and B. jararaca, failing to react with B. jararaca antivenom (Fig. 1). The efficacy of B. jararaca antivenom in neutralizing the toxic activities of the homologous or heterologous venoms of the Bothrops species was then evaluated.
Serum Neutralization of Bothrops Venoms
159 5
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1
2
A
1
2
B
1
'C
2
1
2
D
12
E
1
2
F
1
G
2
1
H
2
1
I
2
FIG. 1. WE47ERN BLOTTING ANALYSE4 OF BOthrOpS VENOMS HY B . jararaca ANT'IVENOM . Venoms of B. alternates (A), B . atrox (B), B . cotiara (C), B. erythromelas (D), B. jararaca (E), B. jararacussu (F), B . moojeni (G), B. neuwiedi (H), and B . pradoi (I) were fractionated by SDS-PAGE and stained by Coomassie blue (1) or revealed by Western blotting with B . jararaca antivenom (2) as described in Material and Methods. Numbers on the left indicate the migration of mol . wt markers.
Neutralization tests were performed using as a standard the homologous system (B . venom/antivenom) . The evaluation of the neutralizing capacity of B. jararaca antivenom was estimated after pre-incubation of venom with an excess of antibodies (5 ~1 of antivenom/F~g of venom) . Coagulant and haemorrhagic activities of B. jararaca venom were completely neutralized by the antivenom (Table 2). This was certainly due to the antibodies, as normal rabbit serum did not significantly inhibit either activity . However, during experiments to test the ability of the serum to neutralize oedema, phospholipase, proteolytic and necrotizing activities the normal rabbit serum used as control interfered with the tests. Therefore experiments were performed using the IgG fraction isolated from B. jararaca antivenom, in the same antiserum/antigen proportion as with immune serum. Necrosis and oedema induced by the venom were inhibited completely by 5 ~tl of IgG per ~g of venom (Table 2). Some residual proteolytic and phospholipase activities were observed using this proportion . However, complete inhibition was obtained using 20 ~1 of IgG per ~g of venom. The neutralizing capacity of B. jararaca antivenom was then assessed against the other Bothrops venoms (heterologous system) . The same amount of the different venoms was used for assaying neutralization of each activity . Tests were not performed with the venoms of B. alternates, B. atrox, B. cotiara, B. erythromelas and B. jararaca due to their low myotoxic activity (1VIOURA-nA-SILVA et al., 19906) . The haemorrhagic activity of 10 Fig of all the venoms was completely inhibited by B. jararaca antivenom (5/1 : antivenom/venom ratio) . Neutralization of the necrotizing activity of 40 ~g of heterologous venoms was also almost complete . Residual necrotizing activity was observed after pre-incubation of the antivenom with B. moojeni, B. neuwiedi and B. pradoi venoms . The coagulant activity of 10 ~g of each venom was almost completely neutralized after incubation with 50 ~1 of B. jararaca antivenom. However, using this same antiserum/ jararaca
0 ND ND ND
0
16.815.5 15.411 .9
Haemorrhagic mm= /pg
0 ND ND ND
0
469123 5861119
Coagulant U/mg
0.2610.07 0
0 0
21517 8010
0 0
0.3410.06 0
0
Necroti~ng mm/~Pg
22010 0
0
Phospholipasic U/mg
70.0115.5 0
ND ND 0
55 .2110.3 82.6112.4
Oedematogenic mg/ug
0.13710.020 0.30310.054 0.28910.108 0.17910.065 0.04610.001 0.23710.064 0.03210.018
Proteolytic U/mg
0 49 .2117.7 30 .1114.0 28 .4114.4 0 37 .613 .9 25 .711 .E
Myotoxic U/mg
ND : Not determined . Different activities of B. jararaca venom pre-incubated with normal rabbit serum (NRS), immune rabbit serum (IRS), normal rabbit IgG (NIgG), immune rabbit IgG (IIgG) or saline for 1 hr at 37°C. Activities were assayed as described in Materials and Methods, and are expressed as the mean of throe determinations 1S.D . 'Myotoxic activity was standardized with B. jararacwsu venom, since B . jararaca venom is almost devoid of myotoxicity.
NRS alone Venom+saline Venom+NRS Venom+IRS NIgG alone Venom+NIgG Venom+IIgG
Samples
Activities
TABIE 2. NEUTRAr.IZAi'ION oF B. jararaca vexo~ ecrwrt~ms aY B. jararaca ex~metvoe~
Serum Neutralization of Bothrops Venons
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antigen proportion, venoms of B. alternatus, B. moojeni and B, atrox still clotted horse plasma after 1 to 2 min ofincubation. However, when the proportion of 20/ 1 was used the supernatants were unable to induce clotting up to 5 min . Bothrops erythromelas and B. pradoi venoms possess high coagulant activities . Clotting times of 10 pg of these venoms are, respectively, 10 and 12 sec. Pre-incubation with the antivenom in the proportion of 5/ 1 increased these clotting times to 17 and 39 sec, respectively . When the antivenom/venom ratio was increased to 20/1, B. pradoi supernatants became ineffective in clotting horse plasma, but B. erythromelas venom still retained some coagulant activity, clotting the horse plasma in 1.45 min (Table 3). Bothrops jararacussu venom was used as standard in the neutralization of myotoxic activity because little increase in serum creatine kinase levels was observed after injection of B.jararaca venom . Normal rabbit serum partly inhibits myotoxic activity . Normal IgG did not interfere with the test. However, using 5 pl of antiserum IgG/hg of venom, inhibition of B. jararacussu myotoxicity was only partial (48%) (Table 3). The proportion of 20/1 was not used since the adequate volume to inject i.m. is below 200 pl and the minimal amount of B.jararacussu venom that induces measurable myotoxicity is 40 pg. In the same conditions, B. jararaca antivenom partially neutralized the myotoxic activity of B. moojeni, B. neuwiedi and B. pradoi venoms (Table 3). Thé proteolytic and oedema-inducing activities of the heterologous venoms were partially neutralized by immune IgG in the 5/1 IgG/venom ratio. Neutralization in both cases was not complete even when using 20 pl of immune IgG/pg of venom. Neutralization of the proteolytic activity was higher in the ratio of 20/ 1 than 5/ 1, but the neutralization of oedema-inducing activity was very similar in both proportions . In spite of this the maximal neutralization of the oedema-inducing activity was almost complete using the 5/1 proportion . Phospholipase activity of 101eg of the venoms was significantly neutralized by the IgG fraction of B. jararaca antivenom only in the homologous system, using both 5/1 and 20/1 antivenom/venom ratios (Table 3). DISCUSSION
Antivenoms have been traditionally tested according to their capacity to neutralize the lethal effects of snake venoms in experimental animals . However, it has recently been observed that neutralization of lethality does not necessarily correlate with neutralization of the specific pharmacological activities of the venoms (WORLD HEALTH ORGANIZATION, 1981). Antivenoms are usually very efficient in neutralizing the systemic effects of the venoms such as lethality, but fail to decrease the extent of the local effects (MINTON, 1954; McCoLLOUCH and GENARO, 1970 ; GuTmRREZ et al., 1981) . The cause of this difference in the effectiveness of the antivenoms has been related to a rapid development of local effects after snakebite while the systemic events resulting in lethality would develop more slowly (GiJ1TERREZ et al., 1987). However, it cannot be excluded that this may be due to lower amounts of antibodies able to neutralize the toxins responsible for the local effects . Furthermore, it is also possible that the antivenoms may not have access to the venom at the site of injection. These considerations are also important as some venoms are not included in the immunization pools; antigenic variability is extensive even in venoms of closely related species (CHIPPAUX et al., 1991) . The results in this paper, however, show considerable antigenic similarity among the nine Bothrops venoms used (e.g. there is a high level of cross-neutralization of the haemorrhagic, coagulant, myotoxic and necrotizing activities) . However, phospholipases, proteolytic enzymes and oedema-inducing
Serum NeutraliTation of Bothropa Venoms
1599
factors appear to be heterogeneous, interacting with heterologous antibodies with low affinity . As a result the degree of cross-neutralization is decreased. Serum of rabbits immunized with B. jararaca venom showed similar ELISA antibody titres against both homologous and heterologous venoms . Antibodies recognized almost all antigens of the nine different venoms when analysed by Western blotting. Using mouse anti-B. jararaca serum, antibodies against the 20,000-30,000 mol. wt antigens were not detected and there was a very weak response against the 15,000 mol. wt antigens (Moulun~-Su va et al., 1990a) . This problem was overcome using rabbit antiserum to B. jararaca venom. However, 15,000 mol . wt antigens in the venoma of B. alternates, B. atrox, B. erythrotnelas and B. jararaca were still not recognized by rabbit B. jararaca antivenom. The high level of antigenic cross-reactivity observed among these venoma is the main factor responsible for the cross-neutralization of lethality already described for venoms of Bothrops snakes (ARAIVIF3 and B1urmAo, 1949 ; Vu,l.~exoBl. et al., 1978/79; Dl~s-n~-Su.v~ et al., 1989) and the cross-neutralization of haemorrhagic, coagulant and necrotizing activities reported here. Haemorrhagic factors seem to share antigenic properties even in venoms of snakes from different genera or geographic areas (M>?ss et al., 1988 ; MANDSLHAUM et al., 1989; M~eTtxEZ et al., 1989). A high degree of immunological crossreactivity also occurs with the coagulant factors (RosElvl~.n and K$1.>~v, 1966; Gaza et al., 1989). Among snakes of the same genus, neutralization of both activities appears to be complete, whereas cross-neutralization of haemorrhagic and coagulant factors from different families of snakes seems to be only partial (Koxx~l.lx and T~oltslu, 1989 ; Cl,~us and M»s, 1989). After incubation with an excess of B. jararaca antivenom some residual necrotizing activity was detected in B. tnoojeni, B. neewiedi and B. pradoi venoms. These venoms possess high necrotizing activity and the residual activity found in these cases may be due to an insufficient amount of antibodies present in B. jararaca antivenom. The residual coagulant activity observed in B. erythrornelas venom may be due to the different proportions of coagulant enzymes present in this venom in comparison with B. jararaca venom (N~lns et al., 1979). Although B.jararaca venom is low in basic myotoxins (Moulsw-n~-SII,VA et al., 1991a), there was still sufficient activity to induce anti-myotoxin antibodies in rabbits, capable of partial neutralizing of the myotoxicity of B. jararacussu, B. moojeni, B. neuwiedi and B. pradoi venoms . In this test, the maximal proportion of antiserum/antigen that could be injected into mice was 5/1, and the partial neutralization of the highly myotoxic venoms may be due to the small amount of antibody and not to its specificity. Neutralization of proteolytic activity by B, jararaca antivenom varied depending on whether homologous or heterologous venoms were used. Proteolytic activity of B. jararaca venom was completely neutralized by the antivenom in the antivenom/venom ratio of 5/1, whereas neutralization of proteolytic activity of heterologous venoms was significantly reduced only after incubation with antiserum in the ratio of 20/1 . Even under such conditions, B. erythrornelas and B. atrox venoms were only poorly neutralized by B. jararaca antivenom. Since B. jararaca venom is highly proteolytic, it is reasonable to expect that the inefficiency of B.jararaca antivenom in neutralizing the proteolytic activity of heterologous venoms may be due to structural differences in professes. The B. jararaca antivenom may recognize the epitopes of the heterologous professes with lower affinity, causing insufficient neutralization. Neutralization of phoapholipase activity of heterologous venoms was not significant even in the 20/ 1 antivenom/venom ratio. It is therefore suggested that venom phospholipases are variable and that the B. jararaca venom phospholipasea share very few, if any, TaX 30 :1Y-E
160 0
M. L . FERREIRA et al.
epitopes with the other Bothrops venom phospholipases. In support of this, B. jararaca antivenom failed to recognize the 15,000 mol. wt antigens of B. alternates, B. atrox and B. erythromelas venoms, which probably represent the acidic phospholipases. In B. jararacussu, B. moojeni, B. neuwiedi and B. pradoi venoms, this effect was not observed, probably due to the strong recognition of myotoxins, with similar mol. wt to the acidic phospholipases. It is important that B. jararaca antivenom did not recognize the 15,000 mol. wt antigen of the homologous venom but was efficient in neutralizing the homologous phospholipases activity. Cross-neutralization of phospholipases was also not observed in the rattlesnake venoms (NAm et al., 1980). Antiserum against a phospholipase isolated from the venom of Crotales scutulatus salvini failed to neutralize phospholipase activity of some other Crotales venoms . The residual oedema-inducing activity observed in B. atrox, B. cotiara, B. erythromelas, B. moojeni, B. neuwiedi and B. pradoi venoms after incubation with B. jararaca antivenom in the proportion of 20/1 may be due to antigenic differences in proteolytic and phospholipase enzymes of Bothrops venoms . However, different oedema-inducing factors may be present in such venoms. In conclusion, antigenic variability of venoms of the genus Bothrops appears to be related mostly to proteolytic, phospholipase and oedema-inducing activities. Apart from investigating the ineffectiveness of antivenoms to neutralize the local venom effects due to their rapid development, it is also necessary to evaluate whether antivenoms possess antibodies against such components. In this initial study only the possibility of immunologically related epitopes contributing to the cross-neutralization of the individual activities of Bothrops venoms, using an excess of antibodies, has been examined . The efficacy of the in vivo administration of antivenoms against heterologous venoms is now under investigation. Acknowledgements-This work was supported by a grant from FAPESP (process 90/1879-2), M . L. F. was the recipient of a FAPESP fellowship (process 90/2465-7) . REFERENCES AxAxIrs, J . B . and BxAxuxo, C. H . (1949) Antigenos e antioorpos botr8picos . Mem. Inst. Butantan Zl, 255-260. AssAxuItA,, M . T ., Rtaci~ A. P., AsrEtert, M . C . A . and MA~Et.BAUes, F. R . (1985) Isolation of the major proteolytic enzyme from the venom of the snake Bothrops moojcni (caissaca) . Toxicon 23, 691-706 . CHIPPAUX, J . P., Wn .L(Ards, V . and WtmE, J . (1991) Snake venom variability: methods of study, results and interpretation . Toxicon 29, 1279-1303 . CLAUS, I . and Mme, D . (1989) Cross-neutralization of thrombin-like enzymes in snake venoms by polyvalent antivenoms . Toxicon 27, 1397-1399 . Dus-DA-SILVA, W., Gurnot.nv, R ., RAw, L, HIOAS~, H . G ., CAxICArI, C . P., MoxAIS, J. F., LuaA, M. L ., YAIIAOUCHI, I . K., NtsIISwwA, A. K ., Snel(Ano, M . A ., MARCELINO, J . R., Pwro, J. R. and SArrtns, M . J . (1989) Cross-reactivity of horse monovalent antivenoms to venoms of ten Bothrops species. Man . Inst . Butantan 51,153-168. Gee, J . A., RoY, A ., ROlA3, G., Gurti~exez, J . M. and CeanAS, L. (1989) Comparative study on coagulant, defibrinating, fibrinolytic and fibrinogenolytic activities of Costa Rican crotalinae snake venoms and their neutralization by a polyvalent antivenom. Toxicon 27, 841-848 . Gt?tvtAx $OCIE(Y of Ct.QnCAI. G~srs (1977) Standard method for determination of creatine ki 1183e activity--revised draft of 1976 . J. Clin . Chan . Biochem . Berlin 1S, 255-259 . Guxi>~ez, J . M ., CxAVES, F., Bowvos, R., CeanAS, L., ROTAS, E ., AxaoYO, O. and POATILLA, E. (1981) Neutraliz8tion de los efectos locales dal veneno de Bothrops aspen por un antiveneno polivalente . Toxicon 19, 493-500 . Gur~z, J. M ., Owrw, C. L. and On>~.I., G . V . (1984) Isolation of a myotoxin from Bothrops aspen venom : partial characterization and action on skeletal muscle. Toxicon 22, 115-128 . G rmenn~ J. M., Ra(AS, G . and CStuAS, L . (1987) Ability of a polyvalent antivenom to neutralize the venom of l achesis muta melanocephala, a new Costa Rican subspecies of the bushmaster . Toxicon 23, 713-720 . Gur~enezz, J. M., CxAVm, F., G», J . A., LOltON1E, B., CAeucxo, Z . and Scltosuvss:v, K . (1989) Myonecrosis
Serum Neutralization of Bothrops Venoms
1601
induced in mice by a basic myotoxin isolated from the venom of the snake Bothrops manmijer (jumping viper) from Costa Rica. Toxicon 27, 735-745. Hot+etwxN, H. and Hors, C . (1987) Blpod coagulation induced by the venom of Bothrops atrox . 2. Identification, purification and properties of two factor X activators . Biochemistry 26, 780-787 . Hoasl-BaArtnnuROO, M . L, Qumtoz, L. S., SAxro-N~ro, H ., Ronluot~-Sn~oxt, L. and GxIt.IO, J . R. (1988) Fractionation of Bothrops jaroracuxru snake venom: partial chemical characterisation of bothropstoxin. Toxicon 26, 615-627. Korroo, H ., Koxno, S., IrcezAWA, L, MuxArA, R. and OIISAxA, A. (1960) Studien of the quantitative method for determination of hemorrhagic activity of habu snake venom . Jpn. J. Med. Scl . Biol. 13, 43-51 . KoxxAUr, F . and TAeoI~[A, E . (1989) Cross-reactivity of mono- and polyvalent antivenom with Viperidae and Crotalidae snake venoms. Toxicon 27, 1135-1142. LAe~a-I, U . K . (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T,. Natio~e 227, 680-685. MwrmeLaAUI~r, F. R. and Ag3A1CUSA, M . T . (1988) Antigenic relationship of hemorrhagic factors and professes isolated from the venomn of three species of Bothrops snakes. Toxkon 26, 279-385 . MAtvnet,aeuu, F. R., R>ac~., A . P. and AssAeulu, M . T. (1976) Some physical and biochemical characteristics of HF2, ône of the hemorrhagic factors in the venom of Bothrops fararaca . In: Animal, Plant and Microbial Toxins, Vol . 1, pp. 111-121 (t~IISAxA, A., HAYASFa, K. and SAWAY, Y., Eds) . London: Pleaum Prow. Mwxnet,aAtnt, F. R., Retclu ., A . P. and AssAtculu, M . T. (1982) Isolation and characterization of a proteolytic enzyme from the venom of Bothrops jararaca (jarataca) . Toxicon 20, 955-972 . MANDELBAUèl, F . R., AssASUxA, M . T . and R>aclfl., A . P. (1984) Characterization of two hemorrhagic factors isolated from the venom of Bothrops neuwiedi (jararaca pintada) . Toxicon 22, 193-206 . Mwrmer-aAUr~t, F . R ., SEAAANO, S. M . T ., SAxuaAnA, J . K ., RAxoPr., H . A . and AssARUIeA, M . T . (1989) Immunological comparison of hemorrhagic principles present in venoms of the Crotalinae and Viperinae subfamilies . Toxicon 27, 169-177 . MAaIrn-rn, G . V . (1%5) The action of phospholipase A~ on lipoproteins . Biochim . biophys . seta 9d9, 554-558. MAxrn~z, R A., HuAt~a, S. Y . and Peliez, J . C . (1989) Antigenic relationships of fractionated western diamondback rattlesnake (Crotahrs atrox) hemorrhagic toxins and other rattlesnake vesoms as indicated by monoclonal antibodies . Toxicon 27, 239-245 . McCoLwvatl, M . C. and Gi~AAO, J . F . (1970) Treatment of venomous snake bite in the United States. Clip . Toxicol. 3, 48387 . McBS, D., Pot~n.rsArtx, S . and TersroLne, W . V. (1988) Snake venom hemorrhagins : neutralization by commercial antivenoms . Toxicon 26, 453-458 . MnvroN, S . A . (1954) Polyvalent antivenin in the treatment of experimental snake venom poisoning. Am . J. trop . Med. 3, 1077-1081 . MOURA-DA-SILVA, A . M ., D'IssreIaaLnrA, M . R., NlstmtewA, A . K ., Bxonstcvx, C . L, Doe SAxros, M . C ., FuxrAno, M. F. D ., DtAS-DA-$1LVA, W . and MorA, I . (1990a) Antigenic cross-reactivity of venoms obtained from snakes of genus Bothrops. Toxicon 28, 181-188 . MOUxA-DA-SII.VA, A. M ., CASnaso, D . F. and TAriIZAgI, M . M. (19906) Differences in distribution of myotoxic proteins in venoma from different Bothrops species . Toxicon 28, 1293-1301 . MOURA-DA-SILVA, A. M ., Desamrm, H ., LAUVa, G . and THEA6SrON, R. D . G . (1991a) Isolation and comparison of myotoxins isolated from venoms of different species of Bothrops snakes . Toxicon 29, 713-723 . MOURA-DA-SII.VA, A. M ., CAauoso, D. F., T"nn7. "cr, M . M . and MorA, I. (1991b) Neutralization of myotoxic activity of Bothrops venoms by antiaera to purified myotoxins and crude venoma . Toxicon 29, 1471-1480. NAxAS, L ., KAllIOUTI, A. S . and HARR()S, M . A. R . (1979) Thrombin-like and factor X activator components of Bothrops snake venoms. Thromb . Haetnott . 41, 314-328. NAat, C ., NAm, B . C . and ELUOrr, W . H. (1980) Immunological comparison of phospholipase A z preaeat in rattlesnake (genus Crotahts) venoms . Toxicon 18, 675-680 . NAT
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NEUTRALIZATION OF DIFFERENT ACTIVITIES OF VENOMS FROM NINE SPECIES OF BOTHROPS SNAKES BY BOTHROPS JARARACA ANTIVENOM MOHICA L. F>?ItR>~w, AxA M. MOURA-nA-SILVA and I. MOTA' Laboratory of Immunopathology, Instituto Butantan,
Sao Paulo, Av . Vital Brazil, 1500 05504 Säo Paulo, Hra~l (Received 1l Afay 1992 ; accepted 11 July 1992) .
M. L. F~RREmA, A. M. MOURA-DA-SILVA and I. MOTA. Neutralization of different activities of venoms from nine species of Bothrops snakes by Bothrops jararaca antivenom. Toxicon 30, 1591-1602, 1992 .-Antivenoms are the usual treatment in ca .4es of systemic envenoming by Bothrops snakes . However, the neutralization of each venom component by the antivenom is not well established . Bothrops jararaca antivenom, produced in rabbits, recognizes the venoms of nine different Bothrops species with high ELISA antibody titres . Western blot analysis showed that almost all antigens present on both homologous and heterologous venoms are recognized. Neutralization tests were performed using whole antivenom or its IgG fraction. The antivenom was able to neutralize the haemorrhagic, coagulant and necrotizing activities of the heterologous venoms in the same antivenom/venom proportion as for the homologous venom. Myotoxic activity was only partially neutralized . Neutralization of the proteolytic activity of heterologous venoms required higher amounts of antivenom than for the homologous venom. Phospholipase and oedema-inducing activities were completely neutralized only in the homologous system . INTRODUCTION
Ix BRAZIL, 90% of snakebites are caused by Bothrops species. Mortality has been estimated as 2.4% and may even be as high as 8% when no antivenom is given (WORLD HEALTH ORGANIZATION, 1981). Oedema, haemorrhage and necrosis are the most serious local manifestations in these cases, causing prolonged or permanent disability . Several components isolated from Bothrops venoms, such as haemorrhagic factors (MANDELBAUM et al., 1976, 1984), enzymes disturbing blood coagulation (NAHA3 et al., 1979; HOFMANN and Box, 1987), proteolytic enzymes (ASSAKURA et al., 1985 ; TANIZARI et al., 1989) and myotozins (GUTmtREZ et al., 1984; 1989 ; HoiKSI-BRANDEHURl.îO et al., 1988; MOURA-DA-SILVA et al., 1991a) are responsible for the clinical symptoms of bothropic envenoming . The usual treatment for snakebites is serum therapy. Antisera are produced by immunization of horses with venoms from a limited number of Bothrops species, the aim 'Author to whom correspondence should be addressed . (591
1592
M. L. FERREIRA et al.
being to neutralize all Bothrops venoms . The efficacy of the serum is usually assayed by assessing its capacity to neutralize the lethality of a reference venom in experimental mice. According to the WORLD HEALTH ORGANIZATION (1981), commercial antivenoms should also be tested for their capacity to neutralize specific venoms activities such as haemorrhage, necrosis and coagulation. This approach is important in the case of Bothrops antivenom, since the species of this genus cause severe local injuries . Also important in relation to serum therapy in Bothrops envenoming is that the venoms of the different species may differ in their pharmacological properties . Several studies have shown that Bothrops venoms share many antigens, and antiserum against the venom of one species is capable of neutralizing the lethal effects of the other venoms (ARAIV'IE.S and BRANDÂO, 1949; VILLARROEL et al., 1978/79; DIAS-nA-SILVA et al., 1989). However, individual antigens have also been reported in Bothrops venoms (VILLARROEL et al., 1974 ; MOURA-DA-$II,VA et al ., 1990x) and some have recently been characterized as myotoxins (MOIJRA-DA-SII.VA et al., 1991x) . Although some data are available on the cross-neutralization of Bothrops haemorrhagic factors (MANDELBAUM and A3SAKURA, 1988), coagulant activity (RosEIVPEL.n and KELEN, 1966) and myotoxins (MOURA-DA-SILVA et al ., 1991 b), these data include only a limited number of components. This does not allow evaluation of the efficacy of the antivenom in neutralizing the local effects of the different venoms . The aim of this study is to evaluate the neutralization of different activities of the venoms obtained from nine species of Bothrops by B. jararaca antivenoms to better understand the mode of action of homologous and heterologous antivenoms . MATERIALS AND METHODS Yenoms Venons of B. alternates, B. atrox, B. erythromelas, B. cotiarq B. jararacq B. jararaceste, B. mookni, B. neuwiedl and B. pradoi were supplied by Instituto Butantan (SÜo Paulo, Brazil) . Thirty to 40 snakes of each species were milked, the venom samples pooled, desiccated and stored at 4°C until used. Animals
Outbred male mice (18-22 g) and New Zealand white rabbits (3.5 kg) were obtained from the Instituto Butantan. Antisera
Anti-B . jararaca serum was obtained by immunization of rabbits. One milligram of B. jararaca venom dissolved in 1 ml of saline was emulsified with 1 ml of Freund's complete adjuvant. Five-hundred microlittns of the mixture was injected i.m . into each of the hind legs of three rabbits. One month later, the animals were injected i.d . three times at weekly intervals with 2001tg of B. jararaca venom in saline. They were bled 10 days after the last injection. Sera were stored at -20°C until used. The neutralizing ability was estimated in leg of venom neutralizing/ld/antiserum . The potency of the rabbit antiserum was estimated by comparing its ELISA titre and its neutralizing ability with those of the antibothropic standard serum produced by Instituto Hutantan . Mtlbody assay (ELISA)
Sera were titrated for their antibody content against different venoms (1 pg/well) coated on ELISA plates (Hemobag, Surgical Products, Campings, S Paulo, Hrazil). The ELISA technique was done as described by T't~tcsrox et al. (1987). Plates were read using an ELISA reader (Multiskan spectrophotometer, Eflab, Helsinki, Finland), and the titres were determined as the reciprocal of the highest dilution that causes an absorbance greater than 0.100 at 492 nm, as non-specific reactions were observed below this value. Electrophoresis and Western blotting
Venom antigens were separated according to the method of Lwr?~nn .i (1970) under non-reducing conditions. The proteins were then stained by Coomassie blue or transblotted onto nitrocellulose papers (Towgnv et al.,
Serum Neutralization of Bothrops Venoms
(593
1979) and developed by addition of serum samples diluted 100-fold in PBS (phosphate buffeted saline O.15 M, pH 7.5) plus 5% defatted milk . After washing, the strips were incubated for 1 hr with peroxidase labelled antirabbit IgG and stained by addition of 4~hloro-l-naphthol plus H=Or Mutine IgG (150,000 mol. wt), bovine serum albumin (68,000 mol. wt), ovalbumin (43,000 mol. wt), trypsinogcn (24,000 mol. wt), and alphalactalbutnin (14,000 mol. wt) were used as mol. wt markers. Isolatiote of rabbit IgGs The IgG fraction of B.jararaca antivenom was isolated using a FPLC system (Pharmacia, Uppsala, Sweden) by protein A amity chromatography . Serum (1 ml) was filtered and centrifuged at 10,000 rev/rain immediately before fractionation, and applied to the protein-A Superose HR 10/2 column equilibrated with 50 mM Tris-HCl buffer, pH 8.0. Proteins bound to the column were eluted with 0.l M citrate buffer, pH 3.0, and the fractions immediately neutralized with Tris 1 M. The optical density of the eluant was monitored at 280 mn. The flow rate was 2 ml/min and 2ml fractions were collected. The fractions eluted with pH 3.0 were pooled, dialysed against saline and concentrated by positive pressure filtration to the original serum volume . The fraction bound to the column showed a single band in SDS-PAGE of mol. wt close to 150,000 under non-reducing conditions and contained all the anti-venom antibody activity of the serum as assayed by ELISA. Haemorrlurgic activity Haemorrhagic activity was determined according to Koxno et al. (1960) with the modifications introduced by OwrreY et al. (1984). Briefly, mice were shaved in We backs and injected i.d. with 50 pl of the test solutions. After 2hr, the mice were killed with ether and the dorsal skin removed. The haemorrhagic lesion was measured as described by Koxno et al. (1960) . Results are expressed as the product of the diameters multiplied by a factor from I to 4, corresponding to the intensity of the colour of the spot (mm'/Pg of venom) . Quarttitation ojrrecrosis Necrosis was quantitated following i.d. injection of SO pl of the test solution into the shaved backs of mice . After 72 hr, the mice were killed with ether and the akin removed. The necrotic area was measured and is expressed as described for haemoahagic activity. In this case the factor from 1 to 4 corresponded to the depth of the lesion on the skin . Results are expressed as mm~/ltg of venom. Oedema-inducing activity The oedema inducing activity of the venoms was assayed according to the method of Y~tuwe et al. (1976) . Results are expressed in mglttg of venom. Coagulant activity Coagulant activity was determined using ciliated horse plasma as substrate and measured as described by N~+s et al. (1979). Results are expressed as the reciprocal of venom concentration able to clot the plasma in 1 sec. Phospholipasc As activity The method of M~tttrnrrtl (1965) was used for evaluation of phosphoGpase activity with slight modifications. Egg-yolk was suspended in Tris-HCl buffer, pH 8.0, to an initial absorbance of 1.0 at 740 nm . One hundred micrograms of each venom sample was added to 3 ml of this suspetrsion . Phoapholipase activity was assessed as the rate of a linear decrease in O.D. over an incubation period of 5-15 min. One unit of phospholipase Az activity torraponds to a decrease of 0.001 in absorbance per min. Activity is expressed as U/mg of venom. Myofoxic activity Mite were injected i.m . (thigh muscle) with 200111 of the test solutions. After 3 hr, blood was collected from the ophthalmic plexus under light ether anaesthesia. Sera from three mite were separated, pooled and immediately assayed for CK activity (Nat-CK assay system, Merck do Brasil S.A ., Siio Paulo, Brazil), as described by the manufacturers (G~tnN Socmrr oa C~.rxrc~t. Ct~easrs, 1977). One unit corresponds to the amount of enzyme that hydrolyses 1 umole of creative per min at 25°C . Myotoxic activity is expressed as U/mg of venom.
1594
M . L . FERREIRA et al. TAatE 1 . ELISA AN77BODY T11RE4 OF B. jararaca ANTIVENOUt AOAINS'r NINE DIFFERENT' VENOIN3 OF BOthrOpJ SNAKP3
Antigens (venoms of) B.jararaca (homologous system) B. alternatus B. atrox B. cotiara B. erythromelas B . jararacussu B . ntoojeni B . rteawiedi B . pradoi
ELISA antibody titres" Test l
Test 2
Test 3
256,000
128,000
128,000
128,000 256,000 256,000 128,000 256,000 128,000 256,000 128,000
128,000 64,000 64,000 32,000 32,000 64,000 32,000 64,000
128,000 128,000 64,000 64,000 64,000 128,000 64,000 64,000
" ELISA antibody titres were determined as the reciprocal of the maximal dilution that causes an absorbance higher than 0.100 at 492 nm . Each test was performed with a different preparation of serum from different rabbits . Proteolytic activity Proteolytic activity was estimated using cavein as substrate as described by are expressed in U/mg of venom.
MANDELBAUM
et al. (1982) . Results
Serum neutralization of venom activitieJ The ability of B. jararaca antivenom to neutralize different venoms activities from the nine BothropJ species was estimated by preincubating venom samples wiW an excess of antiserum (5 or 20 klJpg of venom) for 1 hr at 37°C . Following incubation, the mixtures were centrifuged in a microcentrifuge (Centrifuge 5414, Beckman) for 5 min and the supernatants were assayed for each activity. As we observed that normal rabbit serum was able to inhibit some activities (necrosis, phospholipase and myotoxicity) or to induce others (oedema and proteolytic), we used the IgG fraction instead of whole serum when assaying these activities . Results are expressed as the percentage of neutrali78tion taking as 100% the values obtained when venom was incubated with saline. All experiments were repeated three times. RESULTS
Antisera to B. jararaca venom obtained from three different rabbits were assayed separately . ELISA antibody titres against the homologous and the heterologous venoms did not vary between the three antisera. Comparison of the ELISA antibody titres (Table 1) obtained when B. jararaca antiserum was assayed against the homologous and heterologous antigens showed no significant differences (since variation of the ELISA titres for a same sample was two-fold or less, only differences above this value were considered significant) . These antisera were then pooled and tested for specificity by Western blotting. Figure 1 shows the venoms from nine species of Bothrops fractionated by SDS-PAGE and stained by Coomassie blue (1) or revealed by the B. jararaca antivenom (2). Almost all proteins stained by Coomassie blue were also revealed by Western blotting with B. jararaca antivenom. Some bands with mol. wt in the region of 15,000 were stained by Coomassie blue in the venoms of B. alternates, B. atrox, B. erythromelas and B. jararaca, failing to react with B. jararaca antivenom (Fig. 1). The efficacy of B. jararaca antivenom in neutralizing the toxic activities of the homologous or heterologous venoms of the Bothrops species was then evaluated.
Serum Neutralization of Bothrops Venoms
159 5
43--
1
2
A
1
2
B
1
'C
2
1
2
D
12
E
1
2
F
1
G
2
1
H
2
1
I
2
FIG. 1. WE47ERN BLOTTING ANALYSE4 OF BOthrOpS VENOMS HY B . jararaca ANT'IVENOM . Venoms of B. alternates (A), B . atrox (B), B . cotiara (C), B. erythromelas (D), B. jararaca (E), B. jararacussu (F), B . moojeni (G), B. neuwiedi (H), and B . pradoi (I) were fractionated by SDS-PAGE and stained by Coomassie blue (1) or revealed by Western blotting with B . jararaca antivenom (2) as described in Material and Methods. Numbers on the left indicate the migration of mol . wt markers.
Neutralization tests were performed using as a standard the homologous system (B . venom/antivenom) . The evaluation of the neutralizing capacity of B. jararaca antivenom was estimated after pre-incubation of venom with an excess of antibodies (5 ~1 of antivenom/F~g of venom) . Coagulant and haemorrhagic activities of B. jararaca venom were completely neutralized by the antivenom (Table 2). This was certainly due to the antibodies, as normal rabbit serum did not significantly inhibit either activity . However, during experiments to test the ability of the serum to neutralize oedema, phospholipase, proteolytic and necrotizing activities the normal rabbit serum used as control interfered with the tests. Therefore experiments were performed using the IgG fraction isolated from B. jararaca antivenom, in the same antiserum/antigen proportion as with immune serum. Necrosis and oedema induced by the venom were inhibited completely by 5 ~tl of IgG per ~g of venom (Table 2). Some residual proteolytic and phospholipase activities were observed using this proportion . However, complete inhibition was obtained using 20 ~1 of IgG per ~g of venom. The neutralizing capacity of B. jararaca antivenom was then assessed against the other Bothrops venoms (heterologous system) . The same amount of the different venoms was used for assaying neutralization of each activity . Tests were not performed with the venoms of B. alternates, B. atrox, B. cotiara, B. erythromelas and B. jararaca due to their low myotoxic activity (1VIOURA-nA-SILVA et al., 19906) . The haemorrhagic activity of 10 Fig of all the venoms was completely inhibited by B. jararaca antivenom (5/1 : antivenom/venom ratio) . Neutralization of the necrotizing activity of 40 ~g of heterologous venoms was also almost complete . Residual necrotizing activity was observed after pre-incubation of the antivenom with B. moojeni, B. neuwiedi and B. pradoi venoms . The coagulant activity of 10 ~g of each venom was almost completely neutralized after incubation with 50 ~1 of B. jararaca antivenom. However, using this same antiserum/ jararaca
0 ND ND ND
0
16.815.5 15.411 .9
Haemorrhagic mm= /pg
0 ND ND ND
0
469123 5861119
Coagulant U/mg
0.2610.07 0
0 0
21517 8010
0 0
0.3410.06 0
0
Necroti~ng mm/~Pg
22010 0
0
Phospholipasic U/mg
70.0115.5 0
ND ND 0
55 .2110.3 82.6112.4
Oedematogenic mg/ug
0.13710.020 0.30310.054 0.28910.108 0.17910.065 0.04610.001 0.23710.064 0.03210.018
Proteolytic U/mg
0 49 .2117.7 30 .1114.0 28 .4114.4 0 37 .613 .9 25 .711 .E
Myotoxic U/mg
ND : Not determined . Different activities of B. jararaca venom pre-incubated with normal rabbit serum (NRS), immune rabbit serum (IRS), normal rabbit IgG (NIgG), immune rabbit IgG (IIgG) or saline for 1 hr at 37°C. Activities were assayed as described in Materials and Methods, and are expressed as the mean of throe determinations 1S.D . 'Myotoxic activity was standardized with B. jararacwsu venom, since B . jararaca venom is almost devoid of myotoxicity.
NRS alone Venom+saline Venom+NRS Venom+IRS NIgG alone Venom+NIgG Venom+IIgG
Samples
Activities
TABIE 2. NEUTRAr.IZAi'ION oF B. jararaca vexo~ ecrwrt~ms aY B. jararaca ex~metvoe~
Serum Neutralization of Bothrops Venons
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1598
M . L . FERREIRA et al.
antigen proportion, venoms of B. alternatus, B. moojeni and B, atrox still clotted horse plasma after 1 to 2 min ofincubation. However, when the proportion of 20/ 1 was used the supernatants were unable to induce clotting up to 5 min . Bothrops erythromelas and B. pradoi venoms possess high coagulant activities . Clotting times of 10 pg of these venoms are, respectively, 10 and 12 sec. Pre-incubation with the antivenom in the proportion of 5/ 1 increased these clotting times to 17 and 39 sec, respectively . When the antivenom/venom ratio was increased to 20/1, B. pradoi supernatants became ineffective in clotting horse plasma, but B. erythromelas venom still retained some coagulant activity, clotting the horse plasma in 1.45 min (Table 3). Bothrops jararacussu venom was used as standard in the neutralization of myotoxic activity because little increase in serum creatine kinase levels was observed after injection of B.jararaca venom . Normal rabbit serum partly inhibits myotoxic activity . Normal IgG did not interfere with the test. However, using 5 pl of antiserum IgG/hg of venom, inhibition of B. jararacussu myotoxicity was only partial (48%) (Table 3). The proportion of 20/1 was not used since the adequate volume to inject i.m. is below 200 pl and the minimal amount of B.jararacussu venom that induces measurable myotoxicity is 40 pg. In the same conditions, B. jararaca antivenom partially neutralized the myotoxic activity of B. moojeni, B. neuwiedi and B. pradoi venoms (Table 3). Thé proteolytic and oedema-inducing activities of the heterologous venoms were partially neutralized by immune IgG in the 5/1 IgG/venom ratio. Neutralization in both cases was not complete even when using 20 pl of immune IgG/pg of venom. Neutralization of the proteolytic activity was higher in the ratio of 20/ 1 than 5/ 1, but the neutralization of oedema-inducing activity was very similar in both proportions . In spite of this the maximal neutralization of the oedema-inducing activity was almost complete using the 5/1 proportion . Phospholipase activity of 101eg of the venoms was significantly neutralized by the IgG fraction of B. jararaca antivenom only in the homologous system, using both 5/1 and 20/1 antivenom/venom ratios (Table 3). DISCUSSION
Antivenoms have been traditionally tested according to their capacity to neutralize the lethal effects of snake venoms in experimental animals . However, it has recently been observed that neutralization of lethality does not necessarily correlate with neutralization of the specific pharmacological activities of the venoms (WORLD HEALTH ORGANIZATION, 1981). Antivenoms are usually very efficient in neutralizing the systemic effects of the venoms such as lethality, but fail to decrease the extent of the local effects (MINTON, 1954; McCoLLOUCH and GENARO, 1970 ; GuTmRREZ et al., 1981) . The cause of this difference in the effectiveness of the antivenoms has been related to a rapid development of local effects after snakebite while the systemic events resulting in lethality would develop more slowly (GiJ1TERREZ et al., 1987). However, it cannot be excluded that this may be due to lower amounts of antibodies able to neutralize the toxins responsible for the local effects . Furthermore, it is also possible that the antivenoms may not have access to the venom at the site of injection. These considerations are also important as some venoms are not included in the immunization pools; antigenic variability is extensive even in venoms of closely related species (CHIPPAUX et al., 1991) . The results in this paper, however, show considerable antigenic similarity among the nine Bothrops venoms used (e.g. there is a high level of cross-neutralization of the haemorrhagic, coagulant, myotoxic and necrotizing activities) . However, phospholipases, proteolytic enzymes and oedema-inducing
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factors appear to be heterogeneous, interacting with heterologous antibodies with low affinity . As a result the degree of cross-neutralization is decreased. Serum of rabbits immunized with B. jararaca venom showed similar ELISA antibody titres against both homologous and heterologous venoms . Antibodies recognized almost all antigens of the nine different venoms when analysed by Western blotting. Using mouse anti-B. jararaca serum, antibodies against the 20,000-30,000 mol. wt antigens were not detected and there was a very weak response against the 15,000 mol. wt antigens (Moulun~-Su va et al., 1990a) . This problem was overcome using rabbit antiserum to B. jararaca venom. However, 15,000 mol . wt antigens in the venoma of B. alternates, B. atrox, B. erythrotnelas and B. jararaca were still not recognized by rabbit B. jararaca antivenom. The high level of antigenic cross-reactivity observed among these venoma is the main factor responsible for the cross-neutralization of lethality already described for venoms of Bothrops snakes (ARAIVIF3 and B1urmAo, 1949 ; Vu,l.~exoBl. et al., 1978/79; Dl~s-n~-Su.v~ et al., 1989) and the cross-neutralization of haemorrhagic, coagulant and necrotizing activities reported here. Haemorrhagic factors seem to share antigenic properties even in venoms of snakes from different genera or geographic areas (M>?ss et al., 1988 ; MANDSLHAUM et al., 1989; M~eTtxEZ et al., 1989). A high degree of immunological crossreactivity also occurs with the coagulant factors (RosElvl~.n and K$1.>~v, 1966; Gaza et al., 1989). Among snakes of the same genus, neutralization of both activities appears to be complete, whereas cross-neutralization of haemorrhagic and coagulant factors from different families of snakes seems to be only partial (Koxx~l.lx and T~oltslu, 1989 ; Cl,~us and M»s, 1989). After incubation with an excess of B. jararaca antivenom some residual necrotizing activity was detected in B. tnoojeni, B. neewiedi and B. pradoi venoms. These venoms possess high necrotizing activity and the residual activity found in these cases may be due to an insufficient amount of antibodies present in B. jararaca antivenom. The residual coagulant activity observed in B. erythrornelas venom may be due to the different proportions of coagulant enzymes present in this venom in comparison with B. jararaca venom (N~lns et al., 1979). Although B.jararaca venom is low in basic myotoxins (Moulsw-n~-SII,VA et al., 1991a), there was still sufficient activity to induce anti-myotoxin antibodies in rabbits, capable of partial neutralizing of the myotoxicity of B. jararacussu, B. moojeni, B. neuwiedi and B. pradoi venoms . In this test, the maximal proportion of antiserum/antigen that could be injected into mice was 5/1, and the partial neutralization of the highly myotoxic venoms may be due to the small amount of antibody and not to its specificity. Neutralization of proteolytic activity by B, jararaca antivenom varied depending on whether homologous or heterologous venoms were used. Proteolytic activity of B. jararaca venom was completely neutralized by the antivenom in the antivenom/venom ratio of 5/1, whereas neutralization of proteolytic activity of heterologous venoms was significantly reduced only after incubation with antiserum in the ratio of 20/1 . Even under such conditions, B. erythrornelas and B. atrox venoms were only poorly neutralized by B. jararaca antivenom. Since B. jararaca venom is highly proteolytic, it is reasonable to expect that the inefficiency of B.jararaca antivenom in neutralizing the proteolytic activity of heterologous venoms may be due to structural differences in professes. The B. jararaca antivenom may recognize the epitopes of the heterologous professes with lower affinity, causing insufficient neutralization. Neutralization of phoapholipase activity of heterologous venoms was not significant even in the 20/ 1 antivenom/venom ratio. It is therefore suggested that venom phospholipases are variable and that the B. jararaca venom phospholipasea share very few, if any, TaX 30 :1Y-E
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epitopes with the other Bothrops venom phospholipases. In support of this, B. jararaca antivenom failed to recognize the 15,000 mol. wt antigens of B. alternates, B. atrox and B. erythromelas venoms, which probably represent the acidic phospholipases. In B. jararacussu, B. moojeni, B. neuwiedi and B. pradoi venoms, this effect was not observed, probably due to the strong recognition of myotoxins, with similar mol. wt to the acidic phospholipases. It is important that B. jararaca antivenom did not recognize the 15,000 mol. wt antigen of the homologous venom but was efficient in neutralizing the homologous phospholipases activity. Cross-neutralization of phospholipases was also not observed in the rattlesnake venoms (NAm et al., 1980). Antiserum against a phospholipase isolated from the venom of Crotales scutulatus salvini failed to neutralize phospholipase activity of some other Crotales venoms . The residual oedema-inducing activity observed in B. atrox, B. cotiara, B. erythromelas, B. moojeni, B. neuwiedi and B. pradoi venoms after incubation with B. jararaca antivenom in the proportion of 20/1 may be due to antigenic differences in proteolytic and phospholipase enzymes of Bothrops venoms . However, different oedema-inducing factors may be present in such venoms. In conclusion, antigenic variability of venoms of the genus Bothrops appears to be related mostly to proteolytic, phospholipase and oedema-inducing activities. Apart from investigating the ineffectiveness of antivenoms to neutralize the local venom effects due to their rapid development, it is also necessary to evaluate whether antivenoms possess antibodies against such components. In this initial study only the possibility of immunologically related epitopes contributing to the cross-neutralization of the individual activities of Bothrops venoms, using an excess of antibodies, has been examined . The efficacy of the in vivo administration of antivenoms against heterologous venoms is now under investigation. Acknowledgements-This work was supported by a grant from FAPESP (process 90/1879-2), M . L. F. was the recipient of a FAPESP fellowship (process 90/2465-7) . REFERENCES AxAxIrs, J . B . and BxAxuxo, C. H . (1949) Antigenos e antioorpos botr8picos . Mem. Inst. Butantan Zl, 255-260. AssAxuItA,, M . T ., Rtaci~ A. P., AsrEtert, M . C . A . and MA~Et.BAUes, F. R . (1985) Isolation of the major proteolytic enzyme from the venom of the snake Bothrops moojcni (caissaca) . Toxicon 23, 691-706 . CHIPPAUX, J . P., Wn .L(Ards, V . and WtmE, J . (1991) Snake venom variability: methods of study, results and interpretation . Toxicon 29, 1279-1303 . CLAUS, I . and Mme, D . (1989) Cross-neutralization of thrombin-like enzymes in snake venoms by polyvalent antivenoms . Toxicon 27, 1397-1399 . Dus-DA-SILVA, W., Gurnot.nv, R ., RAw, L, HIOAS~, H . G ., CAxICArI, C . P., MoxAIS, J. F., LuaA, M. L ., YAIIAOUCHI, I . K., NtsIISwwA, A. K ., Snel(Ano, M . A ., MARCELINO, J . R., Pwro, J. R. and SArrtns, M . J . (1989) Cross-reactivity of horse monovalent antivenoms to venoms of ten Bothrops species. Man . Inst . Butantan 51,153-168. Gee, J . A., RoY, A ., ROlA3, G., Gurti~exez, J . M. and CeanAS, L. (1989) Comparative study on coagulant, defibrinating, fibrinolytic and fibrinogenolytic activities of Costa Rican crotalinae snake venoms and their neutralization by a polyvalent antivenom. Toxicon 27, 841-848 . Gt?tvtAx $OCIE(Y of Ct.QnCAI. G~srs (1977) Standard method for determination of creatine ki 1183e activity--revised draft of 1976 . J. Clin . Chan . Biochem . Berlin 1S, 255-259 . Guxi>~ez, J . M ., CxAVES, F., Bowvos, R., CeanAS, L., ROTAS, E ., AxaoYO, O. and POATILLA, E. (1981) Neutraliz8tion de los efectos locales dal veneno de Bothrops aspen por un antiveneno polivalente . Toxicon 19, 493-500 . Gur~z, J. M ., Owrw, C. L. and On>~.I., G . V . (1984) Isolation of a myotoxin from Bothrops aspen venom : partial characterization and action on skeletal muscle. Toxicon 22, 115-128 . G rmenn~ J. M., Ra(AS, G . and CStuAS, L . (1987) Ability of a polyvalent antivenom to neutralize the venom of l achesis muta melanocephala, a new Costa Rican subspecies of the bushmaster . Toxicon 23, 713-720 . Gur~enezz, J. M., CxAVm, F., G», J . A., LOltON1E, B., CAeucxo, Z . and Scltosuvss:v, K . (1989) Myonecrosis
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induced in mice by a basic myotoxin isolated from the venom of the snake Bothrops manmijer (jumping viper) from Costa Rica. Toxicon 27, 735-745. Hot+etwxN, H. and Hors, C . (1987) Blpod coagulation induced by the venom of Bothrops atrox . 2. Identification, purification and properties of two factor X activators . Biochemistry 26, 780-787 . Hoasl-BaArtnnuROO, M . L, Qumtoz, L. S., SAxro-N~ro, H ., Ronluot~-Sn~oxt, L. and GxIt.IO, J . R. (1988) Fractionation of Bothrops jaroracuxru snake venom: partial chemical characterisation of bothropstoxin. Toxicon 26, 615-627. Korroo, H ., Koxno, S., IrcezAWA, L, MuxArA, R. and OIISAxA, A. (1960) Studien of the quantitative method for determination of hemorrhagic activity of habu snake venom . Jpn. J. Med. Scl . Biol. 13, 43-51 . KoxxAUr, F . and TAeoI~[A, E . (1989) Cross-reactivity of mono- and polyvalent antivenom with Viperidae and Crotalidae snake venoms. Toxicon 27, 1135-1142. LAe~a-I, U . K . (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T,. Natio~e 227, 680-685. MwrmeLaAUI~r, F. R. and Ag3A1CUSA, M . T . (1988) Antigenic relationship of hemorrhagic factors and professes isolated from the venomn of three species of Bothrops snakes. Toxkon 26, 279-385 . MAtvnet,aeuu, F. R., R>ac~., A . P. and AssAeulu, M . T. (1976) Some physical and biochemical characteristics of HF2, ône of the hemorrhagic factors in the venom of Bothrops fararaca . In: Animal, Plant and Microbial Toxins, Vol . 1, pp. 111-121 (t~IISAxA, A., HAYASFa, K. and SAWAY, Y., Eds) . London: Pleaum Prow. Mwxnet,aAtnt, F. R., Retclu ., A . P. and AssAtculu, M . T. (1982) Isolation and characterization of a proteolytic enzyme from the venom of Bothrops jararaca (jarataca) . Toxicon 20, 955-972 . MANDELBAUèl, F . R., AssASUxA, M . T . and R>aclfl., A . P. (1984) Characterization of two hemorrhagic factors isolated from the venom of Bothrops neuwiedi (jararaca pintada) . Toxicon 22, 193-206 . Mwrmer-aAUr~t, F . R ., SEAAANO, S. M . T ., SAxuaAnA, J . K ., RAxoPr., H . A . and AssARUIeA, M . T . (1989) Immunological comparison of hemorrhagic principles present in venoms of the Crotalinae and Viperinae subfamilies . Toxicon 27, 169-177 . MAaIrn-rn, G . V . (1%5) The action of phospholipase A~ on lipoproteins . Biochim . biophys . seta 9d9, 554-558. MAxrn~z, R A., HuAt~a, S. Y . and Peliez, J . C . (1989) Antigenic relationships of fractionated western diamondback rattlesnake (Crotahrs atrox) hemorrhagic toxins and other rattlesnake vesoms as indicated by monoclonal antibodies . Toxicon 27, 239-245 . McCoLwvatl, M . C. and Gi~AAO, J . F . (1970) Treatment of venomous snake bite in the United States. Clip . Toxicol. 3, 48387 . McBS, D., Pot~n.rsArtx, S . and TersroLne, W . V. (1988) Snake venom hemorrhagins : neutralization by commercial antivenoms . Toxicon 26, 453-458 . MnvroN, S . A . (1954) Polyvalent antivenin in the treatment of experimental snake venom poisoning. Am . J. trop . Med. 3, 1077-1081 . MOURA-DA-SILVA, A . M ., D'IssreIaaLnrA, M . R., NlstmtewA, A . K ., Bxonstcvx, C . L, Doe SAxros, M . C ., FuxrAno, M. F. D ., DtAS-DA-$1LVA, W . and MorA, I . (1990a) Antigenic cross-reactivity of venoms obtained from snakes of genus Bothrops. Toxicon 28, 181-188 . MOUxA-DA-SII.VA, A. M ., CASnaso, D . F. and TAriIZAgI, M . M. (19906) Differences in distribution of myotoxic proteins in venoma from different Bothrops species . Toxicon 28, 1293-1301 . MOURA-DA-SILVA, A. M ., Desamrm, H ., LAUVa, G . and THEA6SrON, R. D . G . (1991a) Isolation and comparison of myotoxins isolated from venoms of different species of Bothrops snakes . Toxicon 29, 713-723 . MOURA-DA-SII.VA, A. M ., CAauoso, D. F., T"nn7. "cr, M . M . and MorA, I. (1991b) Neutralization of myotoxic activity of Bothrops venoms by antiaera to purified myotoxins and crude venoma . Toxicon 29, 1471-1480. NAxAS, L ., KAllIOUTI, A. S . and HARR()S, M . A. R . (1979) Thrombin-like and factor X activator components of Bothrops snake venoms. Thromb . Haetnott . 41, 314-328. NAat, C ., NAm, B . C . and ELUOrr, W . H. (1980) Immunological comparison of phospholipase A z preaeat in rattlesnake (genus Crotahts) venoms . Toxicon 18, 675-680 . NAT
