IgG ANTIBODIES AGAINST PHOSPHOLIPASE A 2 FROM Crotalus durissus terrificus : CROSS-REACTION WITH VENOMS FROM Bothrops SPECIES FROM ARGENTINA

We examined the ability of IgG anti-crotalic PLA2 to cross-react with Bothrops spp. venoms, from snakes found in the northeast of Argentina. Immunoblotting and ELISA tests showed that IgG anti-crotalic PLA2 recognize antigens of bothropic venoms. Indirect hemolytic activity tests showed that the quantity of antibodies that neutralized 50% of Crotalus durissus terrificus venom (ED50: 2.1 mg IgG anti-crotalic PLA2/100 μg of venom) were also able to neutralize venom from other snakes in the following proportion: 34% of B. alternatus, 18% of B. diporus and 12% of B. jararacussu. Likewise, direct PLA2 activity neutralization tests showed a similar cross-neutralization pattern including 56% of B. alternatus, 29% of B. diporus and 30% of B. jararacussu. In addition, in a myotoxic activity neutralization test, measured by plasma activity of creatine kinase, 35% of B. alternatus venom and 26% of B. diporus venom were neutralized, while no neutralization was detected with B. jararacussu venom. This study presents original data concerning cross-reactions between bothropic venoms from Argentina and IgG anti-crotalic PLA2. Our results suggest that anti-crotalic PLA2 antibodies should not be used to neutralize PLA2 activity of B. alternatus, B. diporus and especially B. jararacussu venoms; nor to enrich commercial antivenoms against these Bothrops species.


INTRODUCTION
The most abundant snake species that inhabit northeastern Argentina are Bothrops diporus (B.d.), Bothrops alternatus (B.a.) and Bothrops jararacussu (B.j.) that belong to the Viperidae family.Crotalus durissus terrificus (C.d.t.) is the only subspecies of the genus Crotalus that is found in this subtropical region of South America (1).The venoms from these viperids contain phospholipases A 2 (PLA 2 ) that hydrolyze phospholipids at the sn-2 position in a calcium-dependent manner, releasing fatty acids and lysophospholipids (2).PLA 2 is the main enzyme responsible for signs and symptoms observed in crotalic envenomation, while it is only partially involved in manifestations of bothropic envenomation (3)(4)(5).
The toxicity of crotalic PLA 2 (C.PLA 2 ) enzyme is potentiated by a small nonenzymatic protein, crotapotin; therefore, both form a well known complex, crotoxin (6,7).This lethal toxin exhibits in vivo a potent neuromuscular and myotoxic activity, and induces in vitro indirect hemolysis when red blood cells are in the presence of egg yolk (8,9).
Snakebites from these species represent a clinical problem in northeastern Argentina and Brazil, and the treatment of choice for victims has been heterologous antiserum administration (13,14).Most commercial antisera are produced in equines and whole venoms are employed for immunizations in order to obtain anti-bothropic (ABS) or anti-crotalic serum (ACS).A specific antiserum is indicated when the snake that bitten the patient is identified (13).However, there are also commercial antisera against venoms from several species of the same genus, or from different genera, in order to increase the neutralization spectrum, thus improving its versatility (15).Over a century ago, Brazil (16) observed that ACS was apparently more efficient than ABS in treating B. jararacussu envenomation.More recently, a mixture of ACS and ABS has been reported to be more effective than ABS alone in neutralizing the myotoxic activity of B. jararacussu venom, suggesting that the addition of antibodies against Crotalus toxic components, such as C.PLA 2 , could be valuable in the treatment of the intoxication (17,18).Moreover, Beghini et al. (19)  In this work, we analyzed the ability of IgG anti-C.PLA 2 to cross-react with components of bothropic venoms, in order to determine the responsibility of these antibodies in the cross-neutralization between ACS and bothropic venoms.A better understanding of cross-reactions may contribute to the development of more versatile antivenoms.

Animals
Male Swiss white mice weighing from 20 to 22 g were supplied by the animal facility of the School of Veterinary Science, UNNE.Mice were housed at 25°C on a 12-hourlight/dark cycle and had free access to food and water.
Male New Zealand white rabbits weighing 3 kg were individually housed with free access to food and water.

IgG anti-C.PLA 2
Rabbit polyclonal antibodies were obtained by immunization with 700 μg of PLA 2 per rabbit mixed with a volume of complete Freund's adjuvant.Boosts were administered on day 7, 14 and 28.Serum antibody levels were monitored by gel immunodiffusion  In order to determine whether IgG anti-C.PLA 2 was able to cross-react with bothropic venoms, the amount of IgG corresponding to ED 50 (158 μg) was pre-incubated with 7.5 μg of each bothropic venom.Then, the mixtures were added to wells in agaroseegg yolk-sheep erythrocyte gels.The hemolytic halos were measured after 20 hours of incubation at 37°C, and the percentage of cross-neutralization was calculated.
Samples of bothropic venoms incubated with PBS were considered positive controls.

Direct PLA 2 Activity Neutralization
PLA 2 activity was assayed by a kinetic method according to Lôbo de Araújo and Radvanyi (22).The substrate was a solution of egg phosphatidylcholine (0.265%) in 10 mM CaCl 2 , 100 mM NaCl, 100 mM phenol red and 7 mM Triton X-100.PLA 2 activity (as fatty acid liberated per min) was measured as changes of absorbance per minute, using a spectrophotometer UV/Visible CamSpec M330® (UK) (at 30°C, λ: 558 nm).The reaction was started by adding venom (7.5 μg) pre-incubated with IgG anti-C.PLA 2 (158 μg, corresponding to ED 50 ) in a final volume of 15 μL.The fatty acids released during the reaction were detected by recording changes in the absorbance per minute.Venom or antibodies pre-incubated with PBS were used as controls.

Myotoxic Activity Neutralization
Creatine kinase (CK) determination was performed in order to detect the maximum liberation in a 24-hour period after venom inoculation (23).Groups of five mice (CF1, In order to have a histological assessment of myotoxicity, tissue samples of injected muscle were taken and fixed with Bouin's solution for 24 to 48 hours.Thereafter, tissue was dehydrated in a graded alcohol series and embedded in paraffin.Sections of 5-mm thick were stained with hematoxylin and eosin.Control muscle tissue was processed similarly (24).

Survival Time
Groups of five animals were intramuscularly inoculated with a challenge dose of two

Statistical Analysis
All experiments were repeated at least three times and the results were expressed as the mean ± SD.The significance of differences between means was assessed by ANOVA followed by Tukey's test for multiple comparisons among groups.Values of p < 0.05 were considered significant.

PLA 2 Cross-reactivity
The reactivity determined by ELISA  Immunobloting was performed in order to identify PLA 2 immunologically related between Crotalus and Bothrops genus.Figure 2A shows SDS-PAGE of crude C.d.t. and bothropic venoms.Bands ranging from 14 to 70 kDa can be observed under reducing conditions.Then, nitrocellulose membranes incubated with IgG anti-C.PLA 2 demonstrated that the reactivity of these specific antibodies was restricted to ~14 kDa components, corresponding to PLA 2s from crotalic and bothropic venoms (Figure 2B).Absorbencies were read at 490 nm.As expected, the greatest reactivity was seen with C.d.t.since its PLA 2 was used in the immunization.

Indirect Hemolytic Activity Neutralization
The amount of antibodies that neutralized 50% of 100 μg of C.d.t.venom was 2.1 ± 0.22 mg.Cross-neutralization of bothropic venoms was express as percentage of inhibition of indirect hemolytic activity (Table 1).

Myotoxic Activity Neutralization
To determine the time of the maximum release of CK for each venom, mice were showed their peaks six hours after injection (Figure 3).

DISCUSSION
The present study was undertaken to assess the cross-reaction of IgG anti-C.PLA 2 against specific bothropic venoms from Argentina, in order to employ it as antivenom or to enrich commercial antisera with these antibodies.
Desiccated C.d.t.venom and bothropic venoms were obtained from the Zoo of Corrientes city.PLA 2 was purified from C.d.t.venom by gel filtration chromatography as described by Rodríguez et al. (21).

ImmunoblottingC
.d.t., B.a., B.d. or B.j. venoms (1 mg.mL -1 ) were separated on 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 200 V for 45 minutes and the proteins were electrophoretically transferred to nitrocellulose membranes (0.45 mm) at 300 mA for one hour.Subsequently, membranes were blocked at room temperature for two hours in a solution of 5% non-fat milk, 0.05% tween-20.After washed three times in TBS (0.01 M Tris-HCl, 0.17 M NaCl, pH 7.6), membranes were incubated overnight with rabbit IgG anti-C.PLA 2 (0.1 mg.mL -1 in TBS).After washing, bound antibodies were detected with goat anti-rabbit IgG peroxidase conjugate (Sigma, USA; 1:1000 in TBS) for one hour at room temperature with shaking.At the end of this incubation, blots were washed, developed with 4-chloro-1naphthol (Sigma, USA; 0.03% in 0.05 M Tris-HCl, pH 7.6, containing 0.03% H 2 O 2 /OPD) and documented.Rabbit pre-immune serum was employed as negative control.Rodrígues JP et al.IgG antibodies against phospholipase A2 from Crotalus durissus terrificus: cross-reaction with venoms from Bothrops species from Argentina.J Venom Anim Toxins incl Trop Dis.2009;15(3):464 Indirect Hemolytic Activity Neutralization Indirect hemolytic activity test was carried out in order to evaluate the IgG anti-C.PLA 2 ability to neutralize the phospholipase activity of C.d.t.venom as well as to cross-neutralize the PLA activity of bothropic venoms.To determine the amount of IgG required to neutralize 50% of C.PLA 2 activity (ED 50 ), 7.5 μg of C.d.t.venom was mixed with different dilutions of IgG anti-C.PLA 2 and then incubated for one hour at 37°C.Aliquots of 15 μL were added to wells in agarose-egg yolk-sheep erythrocyte gels (9).Plates were incubated at 37°C for 20 hours and the hemolytic halos were measured.Control samples included venom incubated without antibodies and antibodies incubated without venom.The degree of inhibition was calculated as percentage of activity, taking as 100% the diameter halo induced by venom alone (7.5 μg) , and ED 50 calculated as milligram of antibodies/100 μg venom able to reduce by 50% the diameter of the hemolytic halo when compared to the effect produced by the venom alone.
18 to 22 g) were intramuscularly injected in the right gastrocnemius with a myotoxic sublethal amount of desiccated crude venom, 1 μg of C.d.t. or 20 μg of bothropic venoms, dissolved in 100 μL of PBS.After 1, 3, 6, 12 and 24 hours of venom injection, mice were intraperitoneally anesthetized with chloral hydrate (300 mg/kg) for blood sample collection.Serum was obtained to analyze the activity of CK with the UV kinetic method (Randox Laboratories, UK) based on the measurement of creatinine formed after the reaction between ADP and phosphocreatine.CK activity was expressed in international units per litter (IU/L).Aiming at determining IgG anti-C.PLA 2 ability to neutralize the myotoxic activity of bothropic venoms, groups of five mice (CF1, 18 to 22 g) received intramuscularly the same challenge amount of bothropic or crotalic venom pre-incubated with IgG anti-C.PLA 2 (420 μg or 21 μg corresponding to ED 50 ) at 37°C for one hour.Control mice were injected with PBS, venom or antibodies alone.According to the time in which each venom induced the maximum release of CK after inoculation, mice were intraperitoneally anesthetized with chloral hydrate (300 mg/kg) for blood sample collection.Then, serum CK level was determined.

LD 50 venom ( 4
μg of C.d.t. or 70 μg of B.a. venom) pre-incubated with IgG anti-C.PLA 2 and continuously observed every hour.Control groups were inoculated with PBS or venom alone.Death was defined as lack of respiratory effort and movement during a 30-second period.An independent observer, blinded to drug injections, determined the time of death (25).

Figure 1 .
Figure 1.Cross-reactivity determined by ELISA.Plates were coated with antigen (5 μg/mL), incubated with rabbit IgG anti-C.PLA 2 antibodies at indicated dilutions and finally with an appropriate IgG-peroxidase conjugate and substrate (OPD).
venom and bothropic venoms to IgG anti-C.PLA 2 .Rodrígues JP et al.IgG antibodies against phospholipase A2 from Crotalus durissus terrificus: cross-reaction with venoms from Bothrops species from Argentina.J Venom Anim Toxins incl Trop Dis.2009;15(3):468 The ability of IgG anti-C.PLA 2 in neutralizing the myotoxic activity was evaluated 1, 3 and 6 hours after inoculation of B.j., B.a. and B.d. or C.d.t.venoms, respectively, corresponding to the maximum release of CK in plasma, as previously detected.

Figure 4
Figure 4 shows that B.a. venom myotoxic activity is better cross-neutralized with 35% neutralization, whereas B.d. showed 26% of plasma CK level reduction.On the contrary, B.j. venom treated with IgG anti-C.PLA 2 showed no significant difference in animals injected with venom alone.Despite the myotoxic activity of B.a. and B.d. venoms was significantly cross-neutralized with IgG anti-C.PLA 2 , the CK activity was high enough to show significant differences in the control group, indicating only a partial neutralization.On the other hand, C.d.t.venom neutralized with IgG anti-C.PLA 2 showed no differences in animals inoculated with PBS, demonstrating complete myotoxic activity neutralization (Figure4).

Figure 5
Figure 5 illustrates the histological changes produced by C.d.t.venom with pronounced myonecrosis while animals treated with venom pre-incubated with IgG anti-C.PLA 2 maintained a normal appearance, and no tissue injury was identified.On the other hand, bothropic venoms produced extensive damage and no protective effect was detected after treatment with IgG anti-C.PLA 2 .

Figure 3 .
Figure 3. Myotoxic activity of C.d.t.venom and bothropic venoms.(A) Effects of C.d.t.venom and (B, C, D) bothropic venoms on mouse skeletal muscle were measured by plasma CK levels (IU/L).Each point represent the mean + SD of five samples.**p < 0.001; compared with the corresponding control group.

Figure 4 .
Figure 4. Neutralization of myotoxic activity.Effects of C.d.t.venom and bothropic venoms on mouse skeletal muscle measured by plasma CK levels (IU/L) and their neutralization by IgG anti-C.PLA 2 .Neutralization was carried out at the time of the maximum liberation of serum CK: one hour (B.j.), three hours (B.a.) or six hours (C.d.t.; B.d.).Each point represent the mean + SD of five samples.# p < 0.001, compared with the control group; * p < 0.01 or ** p < 0.001 compared with the group inoculated with venom alone.

Figure 6 .
Figure 6.Survival of mice treated with C.d.t.venom, B.a. venom or these venoms pre-incubated with IgG anti-C.PLA.Note that animals inoculated with B.a. venom pre-incubated with IgG anti-C.PLA 2 resisted at least for three hours compared with animals inoculated with venom only.
showed that it is possible to neutralize the neuromuscular blockade produced by B.j. and C.d.t.venom Rodrígues JP et al.IgG antibodies against phospholipase A2 from Crotalus durissus terrificus: cross-reaction with venoms from Bothrops species from Argentina.J Venom Anim Toxins incl Trop Dis.2009;15(3):462 with specific antibodies against crotoxin and PLA 2 from Crotalus durissus cascavella.On the other hand, in Argentina, De Roodt et al. (20) observed that ACS had a significant neutralizing capacity on hemorrhagic, necrotizing, procoagulant, proteolytic and lethal activities of bothropic venoms, all focused on proteases.Nevertheless, several toxic features of bothropic venoms related to PLA 2 -such as indirect hemolytic activity -appear not to be adequately neutralized by ACS.Thus, cross-neutralization of the PLA 2 activity of bothropic venoms in Argentina remains unclear up to now.On the other hand, neutralization of B.a. and B.d. venoms with antibodies against C.PLA 2 has not yet been reported.

Enzyme-linked Immunosorbent Assay (ELISA)
Rodrígues JP et al.IgG antibodies against phospholipase A2 from Crotalus durissus terrificus: cross-reaction with venoms from Bothrops species from Argentina.J Venom Anim Toxins incl Trop Dis.2009;15(3):463 and ELISA.Blood was collected from a marginal ear vein, serum was separated by centrifugation and aliquots were stored at -70°C.
Rodrígues JP et al.IgG antibodies against phospholipase A2 from Crotalus durissus terrificus: cross-reaction with venoms from Bothrops species from Argentina.J Venom Anim Toxins incl Trop Dis.2009;15(3):465 of IgG anti-C.PLA 2 against C.d.t., B.a., B.d. and B.j. venoms is shown in Figure 1.As expected, the greatest reactivity was observed with C.d.t.venom since its PLA 2 was used in the immunization process.All bothropic venoms revealed similar behavior with absorbance values around 50% of those obtained with C.d.t venom.

Table 1 .
Indirect hemolytic activity neutralization a Indirect hemolytic activity exhibited by venoms (7.5 μg) expressed in millimeter of hemolytic halo; b hemolytic activity exhibited by venoms (7.5 μg) neutralized with IgG anti-C.PLA 2 (158 μg).Results are expressed as the mean ± SD.Data are representative of three experiments.Direct PLA 2 Activity NeutralizationWe analyzed whether the amount of IgG anti-C.PLA 2 that efficiently neutralize direct PLA 2 activity of C.d.t.venom (ED 50 ) was able to neutralize Bothrops venoms.Table2shows that B.a. venom was best cross-neutralized, while B.d. and B.j. showed similar low percentage of neutralization when IgG anti-C.PLA 2 was employed.

Table 2 .
Direct PLA 2 activity neutralization Rodrígues JP et al.IgG antibodies against phospholipase A2 from Crotalus durissus terrificus: cross-reaction with venoms from Bothrops species from Argentina.J Venom Anim Toxins incl Trop Dis.2009;15(3):469 determined in a 24-hour period.Venoms showed different CK releasing profile.B.j. venom showed the highest release of CK one hour after venom inoculation, B.a. venom presented the peak three hours after, whereas C.d.t. and B.d. venoms Rodrígues JP et al.IgG antibodies against phospholipase A2 from Crotalus durissus terrificus: cross-reaction with venoms from Bothrops species from Argentina.J Venom Anim Toxins incl Trop Dis.2009;15(3):474 Immunoblot assays showed that IgG anti-C.PLA 2 recognized PLA 2 from all Argentinean bothropic venoms.Additionally, ELISA results confirmed the reactivity of IgG anti-C.PLA 2 to bothropic venoms.It is not rare to find considerable crossreactivity among subclasses of venom PLA 2 , since they have an extensive structural homology (8, 19, 26).Although there is a high level of immunochemical crossreactivity among them, bothropic venoms showed lower reactivity than C.d.t.venom, as expected, since C.PLA 2 was used as antigen in rabbit inoculations.Overall, these results are consistent with in vitro and in vivo biological neutralization tests.We demonstrated, by indirect hemolytic activity and direct PLA 2 neutralization tests, that IgG anti-C.PLA 2 partially cross-neutralizes bothropic venoms.The amount of antibodies that efficiently neutralized 50% of C.d.t.venom indirect hemolytic activity, or 77% of direct PLA 2 activity, was not enough for neutralizing bothropic PLA 2 biological activities; B.a. venom was better cross-neutralized than the others whereas a lower level of cross-reaction was obtained with the other bothropic Thus, these results reveal that cross-neutralization of hemorrhagic and lethal activities obtained by other authors with ACS or ACS/ABS mixtures cannot be attributed to a reaction between antibodies against crotoxin and bothropic myotoxins (Lys49).C.d.t.venom presents enzymes with thrombin-like activity; consequently, crossneutralization of hemorrhagic and lethal activities may be explained by the crossreaction between antibodies against crotalic and bothropic thrombin-like enzymes (27).Survival test results were in accordance with those obtained from biological activity neutralization tests.The antivenom did not cross-neutralize the lethal activity of B.a. JP et al.IgG antibodies against phospholipase A2 from Crotalus durissus terrificus: cross-reaction with venoms from Bothrops species from Argentina.J Venom Anim Toxins incl Trop Dis.2009;15(3):475venom, as expected, since it presents other lethal components.However, mice injected with a antivenom-antigen complex exhibited a delay of the survival time compared with animals inoculated with venom alone.Mice treated with B.j. or B.d. venom neutralized with IgG anti-C.PLA 2 did not present significant differences compared with animals inoculated with venom only.This test, like other studies stated before, showed that antibodies against thrombin-like enzymes could play a key role neutralizing the reduction of blood fibrinogen provoked by the enzymes in cross-neutralizations developed with ACS or ABS/ACS mixtures.In conclusion, our results further reveal that the cross-reactivity detected in PLA 2 of bothropic venoms from Argentina to specific IgG antibodies anti-C.PLA 2 does not present the appropriated pharmacological effect required for a complete neutralization of the PLA 2 activity of whole bothropic venoms.Thus, other components of bothropic and crotalic venoms must be studied as molecular targets to generate antibodies, either employed alone or enriching commercial antivenoms with IgG anti-C.PLA antibodies.
venoms, among them, B.j. venom resulted the worst neutralized.These tests were carried out employing specific antibodies anti-C.PLA 2 and agreed with those developed with antivenom against whole C.d.t.venom (20).Myotoxic activity neutralization test demonstrated that IgG anti-C.PLA 2 had a certain degree of protective effect on B.a. venom, in opposition to B.d. and B.j. Mice inoculated with antivenom plus B.a.-antigen complex exhibited a lower releasing of CK compared with mice inoculated with venom only.These tests show that IgG anti-C.PLA do not neutralize neither PLA 2 (Asp49) -responsible for indirect hemolytic activity, edematogenesis and myotoxicity -nor PLA 2 (Lys49) -present in bothropic venoms and particularly powerful in B.j. venom.