New immunization protocol to produce crotalic antivenom combining Crotalus durissus terrificus venom and its PLA2

Biologicals xxx (2014) 1e9

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New immunization protocol to produce crotalic antivenom combining Crotalus durissus terrificus venom and its PLA2
n Fusco a, b, Juan Pablo Rodríguez a, Pamela Teibler b, Silvana Marun ~ ak b, Luciano Sebastia Ofelia Acosta b, Laura Leiva a, * a
n en Proteínas (LabInPro), Facultad de Ciencias Exactas y Naturales y Agrimensura, Universidad Nacional del Nordeste (UNNE), Laboratorio de investigacio Av. Libertad 5470, Corrientes 3400, Argentina b Facultad de Ciencias Veterinarias, Universidad Nacional del Nordeste (UNNE), Sargento Cabral 2139, Corrientes 3400, Argentina

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 May 2014 Received in revised form 6 September 2014 Accepted 18 September 2014 Available online xxx

Antivenoms are usually obtained by animal immunization with successive inoculations of increasing sublethal amounts of venom, which may impair the animal health. The high lethality of venom requires prolonged immunization plans with small amounts of venom. Thus, we propose an alternative plan that includes a pre-immunization of the animal with phospholipase A2, the main crotoxin component, which is responsible for the whole venom lethality. For comparison, three different immunization schemes were designed: high dose protocol (HDP; 0.5e27 mg of venom), low dose protocol (LDP; 0.1e7 mg of venom) and Mix protocol (MP; preimmunization 0.1e1.2 mg of crotalic PLA2, and then 4.5e8 mg of venom). Antibody titers were determined by ELISA, in blood plasma obtained from the marginal vein of the ear. The neutralizing ability of the different sera obtained by all protocols (HDS, LDS and MS) was tested against the most important pharmacological activities of whole venom: PLA2 activity, myotoxicity, thrombin like activity and lethality. MS showed the best neutralizing efficacy and at the same time, it was obtained by an immunization protocol that takes account of animal health care, since it requires low quantities of venoms in comparison to traditional protocols. © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Keywords: Phospholipase A2 Crotalic envenomation Antivenom production Animal health care

1. Introduction The venom of the South American Rattlesnake, Crotalus durissus terrificus (C.d.t.), is a complex mixture of toxins like crotamine, crotoxin, thrombin-like and convulxin that are responsible of the pathophysiology of snake envenomation [1,2]. It involves a complex series of events that depend on the combined action of these venom components [3,4]. The crotoxin complex (CTX) is a heterodimeric protein composed of a so-called acid subunit crotapotin (CA or CTP), devoid of enzymatic activity and other toxic basic fraction with phospholipase A2 activity (PLA2 or CB) [5,6]. It mainly affects the peripheral transmission at the neuromuscular junction leading to respiratory paralysis and subsequent death of the patient [1,2]. When the complex dissociates into its components, the myotoxic and neurotoxic activities are observed only in the basic

* Corresponding author. Tel.: þ54 379 457996; fax: þ54 379 4473930. E-mail addresses: [email protected], [email protected] (L. Leiva).

fraction (PLA2). This enzyme is much less toxic than the entire complex [2,5,7,8]. Despite advances in pharmacology and therapeutics in different areas of medicine, serotherapy remains the treatment of choice for snakebite envenoming [9e11]. Currently there are different types of antivenom, but essentially they are immunoglobulins concentrates (or antibodies fragments) obtained from producing animals (e.g. horses) with efficient neutralizing capacity of the venom toxic effects. Antivenom production plans are focused on the adaptive immune system stimulation. So, they must start with periodic injections of small amounts of venoms, which then are progressively increased, what considerably lengthens the production time [12,13]. A possible solution to this problem would be the detoxification of toxins through different treatments (radiation, heat or chemical modifications), in order to inoculate higher amounts of toxins, however, not much progress has been made with these methodologies since these process might affect the immunogenicity of toxins [14e17]. The amounts of toxins injected are always low and sublethal, however, they usually produce an organic impact that disturb the

http://dx.doi.org/10.1016/j.biologicals.2014.09.001 1045-1056/© 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Fusco LS, et al., New immunization protocol to produce crotalic antivenom combining Crotalus durissus terrificus venom and its PLA2, Biologicals (2014), http://dx.doi.org/10.1016/j.biologicals.2014.09.001

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animal welfare, and occasionally can complicate the production processes or generate undesirable economic costs. Thus, it is important the investigation of alternative immunization plans aimed to improve these aspects [13,18,19]. To date publications about this purpose are scarce. Chotwiwatthanakun and collaborators (2001) used a mixture of postsynaptic neuropeptide injections prior to inoculation of whole venom in combination with a low dose, multi-site of immunization, and low volumes of inoculation, in order to increase the antibody titer against venom components with low immunogenicity [12]. We have previously isolated and characterized the PLA2 from C.d.t. venom, showing that this enzyme has more attenuated pharmacological activities than those exhibited by the whole venom, particularly its neurotoxicity, which is responsible of venom lethality. PLA2 inoculation into laboratory animals (in high doses, since its low toxicity) allowed us obtaining IgG anti-PLA2 antibodies. Then, we found that these antibodies alone were capable of neutralizing the crude venom lethality, with a higher efficiency in comparison with the commercial antiserum [8]. Accordingly, we observed that PLA2 from C.d.t. venom from the northeastern of Argentina could be an attractive and useful immunogen to be used in the production of crotalic antivenom. Based on these studies, this paper focuses on proposing an alternative immunization plan, centered on the use of crotalic PLA2 as an initial immunogen and subsequently whole venom injections. 2. Materials and methods 2.1. Venom and toxin Desiccated C.d.t. venom was obtained from CEPSAN, Corrientes Argentina. PLA2 was purified from C.d.t. venom by gel filtration chromatography. The procedure was carried out in one step using Sephadex G-75 (100  1 cm) pre-equilibrate with 20 mM glycin, 150 mM NaCl, pH 1.9. Fractions of 0.5 ml/tube were collected at a flow rate of 12 ml/h [8].

Table 1 Immunization procols. Protocol

Low dose High dose (LDP) (HDP)

Days

Whole venom (mg)

1 0.1 15 0.2 30 0.5 45 1 60 2 Bleeding e intermission 90 2 105 3 120 4.5 135 7 Total Venom 20.3 Final bleeding a b c

Whole venom (mg)

Mix (MP)

Adjuvant Via FCA/FIA s.c./i.m.

PLA2 Venom (mg) (mg)

0.5 1 2 4 8

0.1 0.2 0.4 0.8 e

e e e e 8

FCAa FIAb FIA FIA FIA

s.c. s.c. s.c. s.c. s.c.

& & & & &

i.m. i.m. i.m. i.m. i.m.

(r.h.)c (l.h.) (r.h.) (l.h.) (r.h.)

8 12 18 27 80.5

0.8 1.2 e e 3.5

e e 4.5 7 19.5

FIA FIA FIA FIA

s.c. s.c. s.c. s.c.

& & & &

i.m. i.m. i.m. i.m.

(l.h.) (r.h.) (l.h.) (r.h.)

Freund's complete adjuvant. Freund's incomplete adjuvant. Subcutaneous and intramuscular administration. Right or Left hind limb.

venom used in the production of crotalic antivenom raised in rabbits, reported by other authors, and adapted to the experimental conditions of this trial [16,21,22]. On the other hand, for comparison, a third group of animals was inoculated, with markedly lower doses of venom, in a similar amount to that used in the MS protocol. Therefore the second and the third protocols were called HDP (high dose) and LDP (low dose) respectively. Intermediate and final bleedings were developed on day 75 and 150 respectively. The applied schemes are shown in Table 1. 2.4. Antibody detection

2.3. Immunization protocols

2.4.1. Immunoblotting C.d.t. venom (1 mg/ml) was separated on 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 200 V for 45 min and the proteins were electrophoretically transferred onto nitrocellulose membranes (0.45 mm) at 300 mA for 1 h. Subsequently, membranes were blocked at room temperature for 2 h in a solution of 5% non-fat milk, 0.05% tween-20 with shaking. After washed three times in TBS (0.01 M TriseHCl, 0.17 M NaCl, pH 7.6), membranes were incubated overnight, with HDS, LDS or MS in the same dilutions (1/750). After washing, bound antibodies were detected with goat anti-rabbit IgG peroxidase conjugate (Sigma, USA; 1:1000 in TBS) for 1 h at room temperature with shaking. At the end of this incubation, blots were washed, developed with 4chloro-1-naphthol (Sigma, USA; 0.03% in 0.05 M TriseHCl, pH 7.6, containing 0.03% H2O2/OPD) and documented. Rabbit non-immune serum was employed as negative control [23].

According to the Animal Protocol approved by the bioethical committee, a total number of 24 animals were used, divided in four groups, containing six animals per group. One of them were initially injected with a low dose of crotalic PLA2 (0.1 mg; Day 1) and then immunizations were done with increased amounts of enzyme up to day 45 (See Table 1). After that, the first immunization with a high dose of whole venom was performed, before the break; in order to stimulate the adaptive immune system with all those remaining components of venom. After the intermission, two doses of PLA2 and whole venom were injected up to the end of the protocol. Since that the antiserum obtained by this way involved a purified toxin and venom, it was called Mix Serum (MS). In the second group of rabbits, immunization was performed only with whole venom. Doses were chosen according to the amounts of

2.4.2. Enzyme-linked immunosorbent assay (ELISA) Variations of specific antibodies titer against crotalic venom, was measured by ELISA. The dosage was performed 7 days after each inoculation; during intermission and at the end of the protocol, the antibody evaluation titer was assayed 15 days after last immunization. Microtiter plates (96 wells) were coated overnight at 4  C with 100 ml of C.d.t. Venom (5 mg/well) or PLA2 in phosphate-buffered saline (PBS). The plates were washed three times with PBS containing 0.5% Tween 20 (PBS/Tween) and unbound sites were blocked for 1 h at room temperature with 2% bovine casein in PBS. The plates were washed three times with PBS/Tween and used immediately for ELISA. To measure titers, 100 ml of serial dilutions of serum from different immunization protocols (HDS, LDS, MS) were

2.2. Animals Male Swiss white mice weighing 20e22 g were supplied by the animal facility of the Facultad de Ciencias Veterinarias, UNNE. Mice were housed at 25  C on a 12 h light/dark cycle and had free access to food and water. Male New Zealand white rabbits weighing 3 kg were housed individually with free access to food and water. All animals used in this study were maintained and treated under strict ethical conditions in accordance with the “International Animal Welfare recommendations” [20] and the Ethics Committee of Animal of Facultad de Ciencias Veterinarias e UNNE.

Please cite this article in press as: Fusco LS, et al., New immunization protocol to produce crotalic antivenom combining Crotalus durissus terrificus venom and its PLA2, Biologicals (2014), http://dx.doi.org/10.1016/j.biologicals.2014.09.001

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added to the plates and incubated for 1 h at 37  C. The plates were then washed and incubated for 1 h with 100 ml of a goat anti-rabbit IgGeperoxidase conjugate (Sigma, 1:10.000 in PBS), followed by further washing. The substrate solution for the peroxidase assay (H2O2/OPD) was added and the enzymatic reaction allowed proceeding for 15 min at room temperature in the dark. The reaction was stopped with 50 ml of H2SO4 3N and the absorbance was read at 492 nm with a SpectraMax 340 multi-well plate reader [24]. 2.5. Indirect hemolysis activity neutralization The technique described by Gutierrez and collaborators was used with little modifications. Briefly, venom and antisera were mixed in different proportions and then incubated for 30 min at 37  C. Then, samples of the mixtures were added to wells made on a gel containing 0.8% agarose with erythrocytes, egg yolk and CaCl2. Plates were incubated at 37  C for 20 h, after that, diameters of the hemolytic halos were measured [25]. The PLA2 neutralizing ability of the antisera was expressed as ED100 defined as the amount of antibodies (mg of IgG protein) needed to reduce by 100% the hemolytic activity of 1 mg of whole venom. IgG protein quantification on antivenoms were performed using a quantitative radial immunodifusion assay commercial kit (MP Biomedicals). 2.6. Myotoxic activity neutralization Aiming to determine the antiserum ability to neutralize the myotoxic activity of crotalic venom, 8 mg of venom pre-incubated with the amount of serum corresponding to ED100, (see above: indirect hemolytic activity neutralization test), were injected into gastrocnemius muscle of mice (CF1, 18e22 g, n ¼ 5). Animals were intraperitoneally anesthetized with chloral hydrate (300 mg/kg), 1, 3, 6 and 24 h after inoculation, in order to take serum samples to analyze the Creatine Kinase activity (UV kinetic method; Randox Laboratories, UK). Positive control mice were injected with 2 mg im (note that 8 mg is a high dose that induces death in a short time, surviving up to 6 hs, therefore 2 mg is enough to show muscle damage). Negative control mice were injected with PBS or antibodies alone. CK activity was expressed as international units per litter (IU/L). In order to have a histological assessment of myotoxicity, tissue samples of injected muscle were taken and fixed with Bouin's solution for 24e48 h. 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 muscles tissues were processed similarly [26]. 2.7. In vitro TLE activity neutralization The neutralization of the thrombin-like activity of venom was tested using a Wiener Lab Fibrintimer 2® coagulometer (Germany). Different amount of venom (0.19e75 mg) were incubated with HDS, LDS or MS for 30 min at 37  C and, then, 75 mL of the sample was added to 75 mL of human plasma. The venom e antiserum mixture was incubated at 37  C and the time until clot formation was measured. Venom solutions incubated with PBS were used as control samples. Complete inhibition was assumed when no clot formation occurred within 10 min and zero inhibition when the clotting time was identical to that of the control sample [24]. 2.8. Lethality neutralization It was determined by incubating different proportions of venom and antiserum or PBS for 30 min at 37  C. Then 0.1 ml of the

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mixtures containing 8 mg of venom (4 LD50) were injected intraperitoneally in groups of four mice (20e22 g body weight). Deaths were recorded during 48 h. Neutralization activity was expressed as effective dose 100% (ED100), defined as the amount of antiserum (mg of IgG) needed to block the lethal potency of the venom [27]. 2.9. Medical veterinary control The Department of Diagnostic Imaging at the Hospital de Clínicas, Facultad de Ciencias Veterinarias, conducted all X-ray and ultrasound studies. Ultrasound studies were performed on a Mindray Vet5 Color Doppler Color with a multifrequency transducer (5e8 HZ). X-ray studies were done on a high frequency radiology equipment DINAR PAF 100TP. Blood biochemistry was analyzed according to the colorimetric methodologies that are described ahead. Total protein: Biuret reaction (Wiener Lab 1999736); Albumin: Bromocresol green (Wiener Lab 1009327); Urea: urease UV cinetic (Wiener Lab 1810322); Cholesterol: CHOD-PAP (Wiener Lab 1009610); Aspartate aminotransferase (AST): IFCC optimized (37  C) (Wiener Lab 1009619); Alanine aminotransferase (ALT): IFCC optimized (37  C) (Wiener Lab 1762360); Alkaline phosphatase ALP 405 (Wiener Lab 1361401). All these determinations were performed using commercial kits from Weiner Laboratory, calibrated with standers troll SeE 2 (Wiener Lab 1937553). Globulin was estimated as the difference between total protein and albumin contents. Serum assays were made in triplicated on samples, 75 and 150 days after the first inoculation. Serum from non-immunized animals was used as internal controls. 2.10. 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. 3. Results 3.1. Immunoassays The reactivity between antisera from different protocols raised in rabbits and C.d.t. venom was evaluated by immunoblotting. Fig. 1 shows that all serum antibodies strongly recognized C.d.t. venom components. Rabbits were bled several times during the immunization protocol and a curve of evolution of specific antibody titers was built using ELISA (Fig. 2). Major differences can be observed in the titer of the antibodies produced by the three protocols, between days 45 and 120. As expected, the HDS showed the highest titer on days 60e75, while the lowest antibodies titter was recorded on LDS, in the same time period. MS exhibited intermediate values and, at the end, all sera reached the same titer. In order to compare the effectiveness of the antisera produced, we next discriminated antibodies against crotalic PLA2 and against the whole venom, measuring antibodies titer by ELISA in two different moments of the immunization progress: on days 75 and 150. Fig. 3 shows that the best way to rise specific antibodies against PLA2 is using the MP, where the rabbits were subjected to a pre-immunization with the isolated enzyme. It can be observed that the antibody titer achieved in this case (105), is significantly higher than those obtained by the HDP (104) or the LDP (103). Differences are also evident, though not so marked, on day 150. MS exhibited a titer nearby 106 and the other two showed a similar

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According to that, results showed in Table 2 indicate that, at the end of the plan, MS was the most effective, requiring only 59 mg of IgG to neutralize 1 mg of C.d.t. venom. Moreover, this evidence was also verified on day 75. In order to neutralize the same amount of venom, it was necessary to duplicate the quantity of HDS or almost quadruplicate the amount of antibodies, in the case of LDS. 3.3. Myotoxic activity

Fig. 1. The reactivity of antivenoms produced in rabbits against C.d.t. venom was measured by immunoblotting. Fifteen micrograms of venom was resolved in 12% SDSPAGE then transferred onto nitrocellulose membranes. From left to right: Mm: molecular markers. C.d.t.: crotalic whole venom. Lanes 1, 2 and 3 corresponding to MS, HDS and LDS antiserum respectively.

titer around 105. It is also noteworthy that the MS showed no significant differences between antibodies titers against whole venom or PLA2; however, it can be seen that the reactivity of sera obtained by HDP or LDP is different against whole venom or the enzyme. 3.2. Indirect hemolytic activity neutralization The ED100 for crotalic antisera (HDS, LDS, MS) were calculated according to the amount of IgG (mg) that abolished the hemolytic activity of whole venom. The ability to produce erythrocytes hemolysis exhibited by the crotalic venom, in the presence of an exogenous source of phospholipids, is direct responsibility of its PLA2. Thus, since this enzyme has been used as antigen in the immunization process, and ELISA demonstrated high titers against PLA2, it is expected that MS possesses a high neutralizing capacity.

The capacity of HDS, LDS and MS, on Day 150, to neutralize the myotoxic activity was evaluated in mice 1, 3, 6 and 24 h after i.m. inoculation of C.d.t. venom, by serum CK activity. Fig. 4 indicates that the venom neutralized by MS, showed no differences in comparison with control animals, demonstrating complete myotoxic activity neutralization. Although the myotoxic activity of C.d.t. venoms was significantly neutralized by all antisera, only MS completely abolished the venom activity. In order to evaluate the extent of damage, histological observations of gastrocnemius muscle samples obtained after exposure to C.d.t. venom incubated with PBS (2 mg; control) or serum (8 mg þ the amount of antibodies corresponding to each DE100), were developed. Abundant interfibrillar material and destroyed muscle fibers can be observed in mice treated with whole venom. Myonecrosis was observed in all venom-treated samples at every time, however, it was more intense at 6 h of exposition (Fig. 5E and F). Animals inoculated with venom preincubated with HDS or LDS revealed moderate inflammatory infiltrate, predominantly polymorphonuclear neutrophil and mononuclear cells (Fig. 5C and D), however, venom neutralized by MS did not evidence these alterations, and retained a normal appearance, like those treated with PBS (Fig. 5B and A, respectively). Histological observations were in agreement with the values obtained by the CK assay, all sera decreased muscle damage but only MS blocked the venom miotoxicity. Hemorrhage was not detected in any case, but a vascular congestion was evidenced. 3.4. Lethal activity neutralization Lethality neutralization capacities of sera from different protocols are shown in Table 2. MS demonstrate to be the best in neutralizing the lethal activity of the venom, followed by HDS and LDS. 3.5. In vitro TLE activity neutralization tests An important toxic activity exhibited by this venom is depicted by the ability of thrombin like enzymes to clot fibrinogen. The presence of antibodies against TLE was assayed on HDS, LDS and MS measuring the clotting time of citrated plasma. Results shown in Table 2, provide evidence that the three sera were capable to neutralize the crotalic TLE activity, requiring low or moderate amounts of IgG from the rabbits serum to prevent the fibrin mesh formation. Taking into account the total amount of venom inoculated in producing animals, MS turned out to be less effective than HDS, as expected, however, the former resulted to be significantly more effective that LDS (p > 0.01); this feature is striking, considering the fact that both received similar amounts of whole venom. 3.6. Blood clinical analysis

Fig. 2. Variations of specific antibodies titer against crotalic venom, measured by ELISA. The dosage was performed 7 days after each inoculation, during intermission and at the end of the protocol, the antibody evaluation titer was assayed 15 days after last immunization. Low dose protocol (C; LDP), High dose protocol (-; HDP), Mix protocol (:; MP).

A significant increase in serum protein concentration was observed during the production of HDS. On the other hand, this disruption was not observed on the other two antisera. Furthermore, serum enzymes studied remained within the normal range

Please cite this article in press as: Fusco LS, et al., New immunization protocol to produce crotalic antivenom combining Crotalus durissus terrificus venom and its PLA2, Biologicals (2014), http://dx.doi.org/10.1016/j.biologicals.2014.09.001

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Fig. 3. Variations of specific antibodies titer against crotalic venom (-) and crotalic PLA2 (C) determined by ELISA. The dosage was performed during intermission (Day 75) and at the end of the protocol (Day 150).

during immunization (Table 3). When corresponded, serum albumin was used as a marker of kidney damage and chronic liver dysfunction, in all rabbits employed.

Table 2 Neutralization of venom activities.

Mix High dosis Low dosis

Lethalitya 150 days

Coagulant activityb 150 days

75 Days

150 Days

ED100d

ED100d

ED100d

ED100d

54.9 ± 0.4 83.4 ± 0.2 95.0 ± 0.3

23 ± 2 16 ± 1 32 ± 3

124 ± 3 273 ± 6 532 ± 5

59 ± 5 177 ± 3 180 ± 7

Indirect hemolytic activityc

a Lethal activity neutralization. Expressed as the amount of IgG (mg) needed to neutralize 1 mg of venom. b TLE Activity neutralization tests. Expressed as the amount of IgG (mg) needed to neutralize 1 mg of venom. c Indirect hemolytic activity neutralization of C.d.t. venom. Expressed as the amount of IgG (mg) needed to neutralize 1 mg of venom. d Results are given as means ± SD, n ¼ 4.

3.7. X-ray studies and ultrasound correlations Animals submitted to HDP exhibited some systemic damage, detected on day 150 by ultrasound studies (see Fig. 6). A reduction of the liver size and the presence of biliary sludge were observed, suggesting that the inoculated venom generated hepatotoxicity. On the other hand, the presence of nodules was detected on the spleen and distention of the splenic vein and the renal pelvis. The other producing animals groups did not show any kind of alterations. In all cases X-ray studies did not show abnormalities.

Please cite this article in press as: Fusco LS, et al., New immunization protocol to produce crotalic antivenom combining Crotalus durissus terrificus venom and its PLA2, Biologicals (2014), http://dx.doi.org/10.1016/j.biologicals.2014.09.001

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4. Discussion

Fig. 4. Myotoxic activity neutralization. Effects of C.d.t. venom on mouse skeletal muscle measured by plasma CK levels (IU/L) and their neutralization by different antisera. Each point represents the mean ± SD of five samples. *: Values compared with the control group. #: Values compared with the group inoculated with venom alone, (p < 0.01).

There are very few works aimed to innovate the immunization process, focusing on the selection of toxins to obtain more efficient antivenoms. Some authors tried to improve the adjuvant used in the immunization protocol or the traditional schemes of inoculation; some others have studied how to increase the versatility of the purification process of antibodies [28e31]. Contrary, some researchers employed detoxified venoms, obtained by chemical or physical treatment, however not much progresses have been made [15,16]. Similarly, de Andrade and collaborators chose different animals to those used routinely as chickens to produce IgY anti crotalic or bothropic venoms [32]. The aim of this work was to obtain a polyclonal antiserum against C.d.t. venom, through an alternative protocol by preinjecting crotalic PLA2 and then C.d.t. crude venom. At the best of our knowledge, there are not original works in this research line with the exception of the study developed by Chotwiwatthanakun et al., who produced polyvalent antivenom by pre-immunizing animals with postsynaptic neurotoxins and three different elapid venoms [12]. For comparative purpose, in this work, a group of animals, taken as controls, was immunized using doses related to those reported

Fig. 5. Histopathological changes induced by C.d.t. venom (8 mg; 6 h after injection) or venom neutralized by LDS, MS, or HDS. A: Control injected with PBS. B: Animals treated with venom neutralized by MS; Note that no pathological changes can be observed. C-D: Mice treated with venom neutralized by HDS and LDS respectively. E-F: Note the myonecrosis characterized by densely clumped myofibrils in different stages of aggregation produced by C.d.t. venom.

Please cite this article in press as: Fusco LS, et al., New immunization protocol to produce crotalic antivenom combining Crotalus durissus terrificus venom and its PLA2, Biologicals (2014), http://dx.doi.org/10.1016/j.biologicals.2014.09.001

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Fig. 6. Organs ultrasound studies of rabbits undergoing HDP. A: Kidney with dilated renal pelvis. B: Liver with distended gallbladder with biliary sludge. C & D: Spleen with nodular echogenic and hipogenic areas, respectively.

earlier to produce rabbit antivenoms, named HDP. It required increasing amounts of protein, to raise neutralizing antibodies against the venom toxins. Conversely, LD protocol was developed, in other group of rabbits, with substantially lower venom doses, in which the protein load was equal to that provided in the MP. A striking feature of the alternative plan that we propose in this paper, is finding that animals can tolerate up to 8 mg of whole venom, corresponding to the first injection. This was most likely due to the protective effect of IgG anti-PLA2, in rabbits, able to neutralize the harmful effects of the venom CTX. To demonstrate this, we next investigated the presence of antibodies against Table 3 Clinical blood analysis evaluated during the immunization process. Antiserum Albumin Total protein IgG (g/dl) First perioda HDS 4.2 ± 0.4 LDS 4.0 ± 0.2 MS 4.4 ± 0.1 Second periodb HDS 4.4 ± 0.2 LDS 3.4 ± 0.5 MS 4.2 ± 0.3 c RS 2.7 e 4.6

Creatinine ASAT

ALAT

(mg/dl)

(UI/L)

(g/dl)

(g/dl)

7.9 ± 0.2 6.8 ± 0.5 7.1 ± 0.3

3.4 ± 0.2 1.41 ± 0.3 41 ± 0.8 18 ± 5 3.2 ± 0.4 0.88 ± 0.4 20 ± 0.4 26 ± 2 3.1 ± 0.3 0.99 ± 0.3 28 ± 0.5 30 ± 1

8.0 7.5 7.2 5.4

± 0.3 ± 0.4 ± 0.2 e 7.8

4.5 4.3 2.9 1.5

± 0.4 ± 0.3 ± 0.5 e 2.8

1.18 1.22 1.05 0.08

± ± ± ±

0.5 0.3 0.2 1.8

(UI/L)

30 19 27 12

± 0.2 ± 0.4 ± 0.3 e 67

37 25 42 14

±4 ±6 ±6 e 113

a Blood samples were collected from rabbits on the intermission and at the end of the immunization protocols. Measured values on day 75 and 150 respectively. b Blood samples were collected from rabbits on the intermission and at the end of the immunization protocols. Measured values on day 75 and 150 respectively. c Reference Score.

crotalic PLA2 and whole venom using ELISA. As expected, MS presented a high reactivity against PLA2, showing high titers of antibodies anti-PLA2. We observed a progressive increment of the antibodies titer against C.d.t. venom during the immunization process; in opposition to the evolution detected by Chotwiwatthanakun and collaborators (2001) who observed a sudden increase of antibodies against neurotoxins. This difference could be explained on the basis that we used venom of a single species as immunogen, unlike the mixture of three different venoms, with probable differences in immunogenicity. On the other hand, it is widely known that C.d.t. venom has an additional difficulty in order to be used as an immunogen, which deserves to be analyzed: its inhibitory effect on immune response. C.d.t. venom engages the anti-SRBC antibody production [33] and reduces the humoral immune response to classical soluble antigens [34], and this effect was attributed to CTX [35]. It was observed that also affects cellular immunity; Sampaio and coworkers, through a broad line of studies, have shown that C.d.t. venom and its CTX inhibit macrophage phagocytosis, affect lymphocyte recirculation in secondary lymphoid organs and produce lipid mediators that trigger an anti-inflammatory response [2,36e41]. Given this immunosuppressive effect of the venom, in this paper, we propose an option that avoids the use of large amounts of it, alleviating the toxic impact on antibody-producing cells. In order to study the effectiveness of the antisera produced by different protocols, we evaluated the neutralizing capacity of MS, HDS and LDS on the toxic activities of the venom. MS showed the greatest effectiveness to neutralize the indirect hemolytic activity of the venom, both after the first intermission of the protocol and at

Please cite this article in press as: Fusco LS, et al., New immunization protocol to produce crotalic antivenom combining Crotalus durissus terrificus venom and its PLA2, Biologicals (2014), http://dx.doi.org/10.1016/j.biologicals.2014.09.001

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the end of it. This pharmacological activity is strikingly dependent on the catalytic activity of crotalic PLA2, and it is known related to lethality. It was found that mice tolerated a venom dose as high as 4LD50, neutralized by MS, while those who received only whole venom died within a 6 h period. On the other hand, it was necessary a greater antisera:venom ratio to achieve the same survival when controls sera (HDS or LDS) were used. The coagulant activity neutralization test of C.d.t. venom reveled that all antisera were capable to abolish this pharmacological property, however MS showed an intermediate effectiveness. This plan was developed with only three whole venom immunizations (19.5 mg) that provided TLE antigen, in opposition to HD or LD protocols that required nine injections (80.5 and 20.3 respectively). HDS showed to be the best antiserum to neutralize the coagulant activity of C.d.t. venom, as expected, since that 80.5 mg represents a fourfold increase in the total quantity of venom inoculated, in comparison to those used in MP or LP. This latter proved to be very ineffective, something predictable, since the low amount of venom injected to obtain it. The efficacy of MS can be explained considering that animals subjected to MP received, on day 60, a high dose of venom for the first time; it probably induced a high humoral immune response against the remaining venom toxins, including the TLE. The fact that MS can neutralize this pharmacological activity of the whole venom is remarkable, since clotting disorders [42,43], and afibrinogenaemia [44] have been reported in crotalic accidents, what exacerbates the venom lethality. Myotoxicity was evaluated by biochemical and histological approaches. Since the histological evaluation was only qualitative, CK release was used as a quantitative biochemical marker of skeletal muscle damage. These results showed that HDS, LDS and MS were able to significantly reduce the venom myotoxic activity, however, only MS achieved a complete neutralization, inducing plasma CK releases similar to those obtained in animals inoculated with PBS. Many studies have demonstrated that C.d.t. venom generates kidney [45e49] and liver damage [50,51]. In order to assess the general welfare of the producing animals at the end of the immunization process, clinical, biochemical, ultrasound and radiographic studies were develop. The biochemical parameters evaluated were not significantly different among the animal groups; even in comparison to reference values. The same observation was also observed by Angulo et al. who showed no biochemical alterations on animals immunized with Bothropic venoms [52]. However, our studies showed organic changes evaluated by imaging studies, specifically in those animals undergoing HDP. On the other hand, animals submitted to MP showed normal ultrasound and x-ray images. Thus, these studies of high sensitivity, can detect subclinical alterations that could evolve and then trigger irreversible changes that ultimately affect the animal health; that is why this kind of studies are worth developing. Usually, antivenoms are produced in animals by immunization with whole venoms or isolated venom components, and this approach is likely to continue. However, many instances of this process like animal selection, immunization and bleeding or clinical control are performed following a rather empirical and poorly-controlled process [13]. In conclusion, the design of a new immunization protocol that combined periods of purified toxins immunization and whole venom inoculation, allowed us to produce, an efficient crotalic antisera paying attention to animal health care and avoiding large amounts of venom injection. In this way we add a valuable contribution to the difficult and complex process of antivenom production. Acknowledgments We thank to Laura Rey from CEPSAN, for her technical assistance and venom provision; and also to Patricia Goycochea and her

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Please cite this article in press as: Fusco LS, et al., New immunization protocol to produce crotalic antivenom combining Crotalus durissus terrificus venom and its PLA2, Biologicals (2014), http://dx.doi.org/10.1016/j.biologicals.2014.09.001

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Please cite this article in press as: Fusco LS, et al., New immunization protocol to produce crotalic antivenom combining Crotalus durissus terrificus venom and its PLA2, Biologicals (2014), http://dx.doi.org/10.1016/j.biologicals.2014.09.001