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Development of process to produce polyvalent IgY antibodies anti-African snake venom
Toxicon 52 (2008) 293–301
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Toxicon journal homepage: www.elsevier.com/locate/toxicon
Development of process to produce polyvalent IgY antibodies anti-African snake venomq Cla´udia Maria Costa de Almeida a, Cla´udia Letı´cia da Silva b, Humberto Pena Couto c, Rita de Ca´ssia Mothe´ Escocard b, David Gitirana da Rocha b, Lynna de Paula Sentinelli b, Thereza Liberman Kipnis b, Wilmar Dias da Silva b, * a ´rias, Universidade Estadual do Norte FluminensedDarcy Ribeiro, ´ rio de Sanidade Animal, Centro de Cieˆncias e Tecnologias Agropecua Laborato Campos dos Gottacazes, RJ, Brazil b ´rio de Biologia do Reconhecer, Centro de Biocieˆncias e Biotecnologia, Universidade Estadual do Norte FluminensedDarcy Ribeiro, Laborato Av. Alberto Lamego, 2000 Parque California, CEP 28015-620, Campos dos Gottacazes, RJ, Brazil c ´rias, Universidade Estadual do Norte FluminensedDarcy Ribeiro, ´rio de Zootecnia e Nutriça ˜o Animal, Centro de Cieˆncias e Tecnologias Agropecua Laborato Campos dos Gottacazes, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 January 2008 Received in revised form 28 May 2008 Accepted 29 May 2008 Available online 13 June 2008
Polyvalent anti-Bitis and anti-Naja antivenom IgY antibodies were prepared using B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca, and N. mossambica venoms to immunize chickens. Blood and eggs were collected before and during the 10-month immunization period; the sera and yolk extracts were then prepared and assayed for the presence of antivenom antibodies by ELISA and Western blot methods. ELISA Antivenom antibody titers, referred to as U-ELISA/ml of serum or egg yolk extracts, absent in pre-immunization sera or yolk, increased sharply during the 4 weeks after immunization, reaching a plateau thereafter. Yolk extracts with high antivenom titers, as detected by ELISA were used to isolate and purify IgY. Purified IgY preparations recognized venom protein bands from 10 to 20 kDa to 60 and 70 kDa, as shown by Western blot. Recovery of antivenom antibodies from the whole yolk was over 80%. Final preparations exhibited high antivenom activity (>100,000 U-ELISA/ml) as well as efficacy in neutralizing venom lethality (1440 mg of IgY neutralize 62.2 LD50 of venom), and were free of toxic products, pyrogen or bacterial and fungal contaminations. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: IgY Snake venom Snake bite Envenoming
1. Introduction The snake species usually found in Guine´, S. Tome´, Angola and Mozambique belong to the Elapidae and Viperidae families (Manaças, 1981–1982). Elapidae includes the genus Aspidelaps, Elapsoidea, Hemachatus, Naja, Pseudohajc, and Dendroaspis, comprising around 20 species. Their venoms are essentially neurotoxic. The local effects of bites from these snakes are usually minimal, restricted to small q Ethical statement: The experiments with chickens were approved by the ‘‘Ethical Committee of Animal WelfaredUENF’’. * Corresponding author. Tel./fax: þ55 22 2726 1591. E-mail address: [email protected] (W.D. da Silva). 0041-0101/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2008.05.022
fang puncture wounds, exceptionally with tender local swelling, blistering and superficial necrosis (Reid, 1964; Viravan et al., 1986). Early systemic symptoms of envenoming are vomiting, ‘‘heaviness’’ of the eyelids, blurred vision, fasciculation, paraesthesiae around the mouth, hyperacusis, headache, dizziness, vertigo, hypersalivation, congested conjunctivae and ‘‘gooseflesh’’. Ptosis and external ophthalmoplegia appear soon after the bite, followed minutes or hours later by paralysis of the face, jaws, tongue, vocal cords, neck muscles and (bulbar) muscles of deglutition with pooling of secretions in the pharynx. The intercostals and diaphragm become paralyzed, leading to abdominal breathing, respiratory distress, with agitation, tachycardia, sweating, and central cyanosis (Warrell, 1996). Viperidae
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includes the genus Causus, Bitis, Vipera, Atheris, and Atractaspis, with also around 20 species. Their venoms are rich in various enzymes, mainly proteinases. Swelling and bruising may develop at the site of the bite within a few minutes or hours. Swelling spreads rapidly and sometimes involves the whole of the bitten limb and trunk. Regional lymphatic vessel and lymph node involvement is common. Blistering and necrosis may develop at the site of the bite. Hemostatic abnormalities are clinically manifested by persistent bleeding from the fang puncture wounds and other partially healed wounds, suggesting coagulopathy. The blood clot alterations are sometimes systemically reflected as epistaxis, hematemesis, cutaneous ecchymoses, petechiae or discoid ecchymoses, hemoptisis, and subconjuntival, retroperitoneal, subarachnoid, intracranial hemorrhages (Warrell, 1996). These venom effects can be completely and acutely reversed by specific antivenoms, if the patients or bitten animals are properly treated. Snake venom poisoning is one disease process that has been successfully treated with specific polyclonal antibodies. This therapeutic tool has been used since the pioneering work of Sewall (1887) and Calmette (1894). Conventional antivenoms are prepared by immunizing large animals, usually horses, with individual venom or a range of different venoms obtained from several snakes to eliminate intraspecific variation (Theakston, 1996). In order to minimize the expected adverse reactions, upon injection into the bitten victims, the IgG immunoglobulins are partially purified, enzymatically cleaved by pepsin, and the resulting F(ab0 )2 fragments are concentrated and tested for their antivenom neutralizing properties, and submitted to quality control, before release for use in human beings. As antivenoms prepared in horses are very expensive for use to treat domestic animals bitten by venomous snakes, alternative animals and methods are being tried. Previous studies in our laboratory have demonstrated that hens (Gallus gallus) immunized with Bothrops spp. and Crotalus durissus terrificus crude venoms, produce IgY antibody specific for these venoms, which can then be recovered from the egg yolks and purified (Almeida et al., 1998). The resulting IgY preparations were endowed with the ability to combine with venom components used to immunize the hens, and neutralize their toxic activities. The present study aimed to improve procedures to produce, on a large scale, IgY-specific antibodies to Bitis spp. and Naja spp. venoms to be used to treat domestic animals and eventually human patients bitten by African snake venoms. Before being released for use, these antivenoms will be submitted to pre-clinical trials. Based on the simplicity of the entire procedure, the low price of hens and, especially, the high capacity of the resulting antivenoms to neutralize the venoms’ lethality, IgY antivenoms are indicated to treat animals bitten by snakes, especially in developing countries.
Laborato´rio de Biologia do Reconhecer (LBR)/Centro de Biocieˆncias e Biotecnologia (CBB)/Universidade Estadual do Norte FluminensedDarcy Ribeiro (UENF). Hens were used to produce IgY antivenoms, Swiss outbred mice were used to determine venom lethality potency and the neutralizing potency of antivenoms, and rabbits were used to produce anti-IgY antiserum. Animal care was provided by expert personnel, in compliance with the relevant laws and institutional guidelines. 2.2. Crude venoms from African snakes The Bitis arietans, Bitis nasicornis, Bitis rhinoceros, Naja melanoleuca, and Naja mossambica snake venoms were purchased from Venom Supplies Pty Ltd (Tanunda, Australia). 2.3. Reagents and supplies Freund’s complete adjuvant (FCA), incomplete Freund’s adjuvant (IFA), and rabbit anti- IgY antiserum were prepared in our Laboratory. The following reagents were purchased as indicated: Bovine serum albumin (BSA), goat anti-rabbit IgG peroxidase conjugate, 3,30 -diaminobenzidine tetrahydrochloride (DAB) (Sigma Aldrich Chemical Co., St Louis, MO, USA). All other reagents were of analytical grade as otherwise indicated. Polystyrene ELISA plates were utilized (Microlon Medium Binding, Greiner, Germany). 2.4. Determination of venom lethality The lethal potencies of the venoms (LD50) were determined by i.p. injection of Swiss outbreed mice (18–20 g) using eight mice per group. Mortalities were recorded after 48 h, and LD50 was calculated using the Bliss (1934) method. 2.5. Immunization schedule of chickens
2. Material and methods
Groups of eight chickens were immunized intramuscularly in the breast region at two or three sites with 20 mg of African snake venoms, alone or mixed as indicated in FCA: Group #1, B. arietans; Group #2, B. nasicornis plus B. rhinoceros; Group #3, N. mossambica; Group #4, N. melanoleuca plus N. mossambica; Group #5, non immunized. Three weeks later, the injections were repeated with the venoms in IFA. Three boosters were given with the venoms in 0.15 M NaCl by the same route, also at 3-week intervals. Blood samples and eggs were collected before immunization to be used as negative controls either in immunochemical assays or in immunoprotection tests. Eggs were collected every day from each immunized chick and refrigerated at 4 C. Egg yolks were separated from the albumin and stored at 20 C.
2.1. Animals
2.6. Antibody purification
Rhode Island Red female chickens (1.1–1.5 kg body mass), Swiss outbreed (18–20 g) mice, and rabbits (0.5–1.0 kg) were maintained in the animal facility of the
2.6.1. Isolation and partial purification Yolks from 30 eggs of the same immunized group of hens were diluted 10-fold with distilled water, pH 5.5.
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The suspensions were incubated overnight at 4 C. The supernatants containing the IgY were collected by centrifugation (10,000 g at 4 C for 30 min), the temperature was adjusted to 20 C and subjected to 29% saturated ammonium sulfate precipitation. After centrifugation (10,000 g at 25 C for 30 min), the precipitated proteins were recovered, dissolved in 0.15 M NaCl and dialyzed against this same solution. Protein contents were adjusted to 10 mg/ml and analyzed by SDS–PAGE (15%) and Western blotted using rabbit serum anti-IgY as the first antibody. Anti-snake venom antibodies were quantified by the ELISA method, and their lethality neutralizing efficacies were assessed by in vitro/in vivo assays using Swiss outbred mice as the animal test. IgY immunoglobulins were similarly prepared and analyzed from pre-immunized egg yolks. 2.7. Characterization of egg antivenom IgY antibodies 2.7.1. SDS–PAGE and Western blot analyses Western blot analysis was carried out, according to the previously described method (Towbin et al., 1979). Crude B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca, and N. mossambica snake venoms (2 mg/ml) were treated with SDS–PAGE sample buffer under reducing conditions and resolved in 15% polyacrylamide gel (Laemmli, 1970) before electroblotting onto PVDF (Immobilon P), after treating membranes with methanol. The membrane, after treating again with methanol at 37 C for 15 min, was blocked with blocking buffer (0.01 M PBS, pH 7.4, with 5% low fat milk) at room temperature for 30 min. After washing with wash buffer (blocking buffer containing 0.1% Tween-20), the membrane was incubated with 5.0 mg of rabbit anti-chicken IgY (whole molecule) diluted in the same blocking buffer and incubated for 1.0 h at room temperature on a horizontal shaker. The membrane was washed with blocking buffer and incubated with 1:2000 dilution of peroxidase-conjugated goat anti-rabbit IgG for 1.0 h at room temperature. After washing, the membrane was placed in peroxidase chromogenic substrate solution (0.1 M citric acid plus 0.2 M sodium diphosphate, 5.0 mg of DAB (0.05%) and 3 ml of H2O2). The reaction was terminated by washing with distilled water. 2.7.2. Evaluation of antibody activity: ELISA method Polystyrene ELISA plates (96 wells) were coated with 1.0 mg of native snake venom in 50 ml coating buffer (0.1 M carbonate bicarbonate, pH 9.6) kept overnight at 4 C. The wells were washed once with PBS buffer containing 0.05% Tween-20. The wells were next blocked for 1.0 h at room temperature with 150 ml PBS buffer plus 1.0% gelatin. The wells were again washed thrice with 300 ml washing buffer. Serial dilutions of IgY preparations (1:1000 to 1:320,000) in PBS buffer plus 1.0% gelatin buffer containing 0.05% Tween-20 were prepared and 50 ml of each were added to individual wells and the plates were incubated at 37 C for 45 min. The wells were washed five times with the same washing buffer. Rabbit peroxidase-conjugated anti-chicken IgY (whole molecule), diluted (1:800) in PBS buffer plus 1.0% gelatin buffer containing 0.05% Tween-20 (50 ml), was added to each well.
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The plates were incubated for 45 min at 37 C. After five washes with the washing buffer, 50 ml of substrate buffer (0.1 M citric acid, plus 0.2 M sodium diphosphate, 5.0 ml H2O, 5.0 mg OPD, 5ml of H2O2) were added and incubated at room temperature for 10–15 min. The reaction was terminated with 50 ml of 3 N sulfuric acid. Absorbance was recorded at 492 nm using an ELISA plate reader. IgY from eggs collected before immunization was always used as a negative control. Wells free of venom were used as blanks. The IgY dilution, giving an optical density of close to 0.2, was used to calculate the U-ELISA per milliliter of undiluted IgY solution. One U-ELISA is defined as the smallest amount of antibody giving an OD of 0.2 under conditions of ELISA assay. 2.7.3. Neutralization of venom lethality by IgY antivenom antibodies The neutralizing efficacy of IgY antivenom antibodies, produced along the immunization procedure, was evaluated by incubating samples of B. arietans venom (600 mg) corresponding to 62.5 LD50, for 1 h at 37 C, with IgY antivenom antibodies (1440 mg) prepared from egg yolks collected before immunization (control) or at the 3rd and 6th weeks after immunization. The mixtures were i.p. injected into groups of eight Swiss mice (18–20 g), and deaths were recorded over 72 h. 3. Results 3.1. LD50 of the venoms Groups of eight Swiss mice (18–20 g) were injected i.p. with 500 ml of 0.15 M NaCl containing different concentrations of the snake venoms and the death/survival ratios were recorded along 72 h of observation. Results depicted in Table 1 show that, as expected, the lethality of venoms varies, with B. arietans venom being the most toxic. 3.2. SDS–PAGE analysis of snake venoms B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca, and N. mossambica snake venoms, submitted to 15% SDS– PAGE under reducing conditions, were resolved into venom components with different apparent molecular masses, ranging from 14 to 97 kDa. Nine protein bands were resolved for B. arietans (well #2), six for B. nasicornis (well #3), six for B. rhinoceros (well #4), five for N. melanoleuca (well #5) and seven for N. mossambica (well #6), as depicted in Fig 1. Fig. 1 also demonstrates that some prominent protein bands were randomly distributed over the electrophoretic profiles. A single protein band with apparent molecular mass ranging from 24-36 kDa is observed only in B. arietans venom. 3.3. Antivenom IgY antibody 3.3.1. Characterization and quantification IgY antibody by ELISA method Groups of laying hens were intramuscularly primed with 0.5 ml of 0.15 M NaCl containing 20 mg of venom or mixtures of venoms emulsified in equal volumes of
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Table 1 LD50 of the African snake venoms Snake venom
LD50 (in mg of dried venom)
Statistical analysisa
Naja melanoleuca
13.43
Naja mossambica
22.40
Bitis arietans
0.96
Bitis gabonica
95.28
Bitis nasicornis
123.67
Precision factor Standard deviation Superior confidence limit (95%) Inferior confidence limit (95%) Precision factor Standard deviation Superior confidence limit (95%) Inferior confidence limit (95%) Precision factor Standard deviation Superior confidence limit (95%) Inferior confidence limit (95%) Precision factor Standard deviation Superior confidence limit (95%) Inferior confidence limit (95%) Precision factor Standard deviation Superior confidence limit (95%) Inferior confidence limit (95%)
a
¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼
0.29691 4.725761E-02 14.84792 11.94401 0.3565223 7.941066E-02 27.14655 18.83186 0.2807443 6.506576E-02 1.106156 0.8197657 0.2914934 5.322902E-02 105.0646 82.22396 0.2716146 7.666159E-02 142.0168 99.77381
Deaths were recorded over the following 48h, and the LD50 was estimated by ‘‘Full Probits Analysis’’ (Bliss, 1934).
complete Freund’s adjuvant, and boosted 2 weeks later first with the same amounts of the venoms in incomplete Freund’s adjuvant, followed every week by four injections with the venoms in 0.15 M NaCl. Blood samples were harvested during the first week of immunization while eggs were collected along the entire 6 months of the experiment. Antibody titration was performed in serum samples and in egg extracts by the indirect ELISA method: Group #1, B. arietans; Group #2, B. nasicornis plus B. rhinoceros; Group #3, N. mossambica; Group #4, N. melanoleuca plus N. mossambica; Group #5, non-immunized. Antibodies
Fig. 1. Electrophoretic separation on SDS–PAGE (15%) of African snake venoms. 1, M.W. markers: 97 kDa; 66 kDa; 45 kDa; 36 kDa; 24 kDa; 14 kDa (from top to bottom). 2, Bitis arietans; 3, Bitis nasicornis 4, Bitis rhinoceros; 5, Naja melanoleuca; 6, Naja mossambica.
against the native snake venom started to appear in the serum 2 weeks after immunization, reaching a plateau at about 35–45 days, persisting with small individual variations for at least 8 months (data not shown). Transfer of these antivenom antibodies to egg yolk coincided with their appearance in serum. Presence of antibodies in sera and in corresponding egg yolks, collected at 6 weeks after immunization, are depicted in Fig. 2A and in B, respectively. Although the antibody titers are variable according to the venom used for immunization, the amounts of antibodies produced were consistently high. The antibody formation, although consistent in all immunized hens, was not uniform even among hens immunized with the same venom or a mixture of venoms, as depicted in Fig. 3A and B. 3.3.2. Purification of IgY antivenom antibodies The protocol used, as described in Section 2, includes the following steps: dilution of egg yolks in pH 5.5 distilled water, removal of the insoluble lipids in the upper supernatant, precipitation of IgY immunoglobulins with 29% ammonium sulfate, removal of soluble proteins by sucking out through a filter paper followed by also washing the IgY rich precipitates with ammonium sulfate at the same concentration. After extensive dialysis against sterile 0.15 M NaCl solution, protein contents were determined, and the protein concentration adjusted to 10.0 mg/ml. Antivenom antibodies were evaluated by ELISA method, and the presence of IgY protein was analyzed by both SDS–PAGE and Western blot methods. Individual batches of IgY, prepared from the same immunized hen groups after dialysis against 0.15 M NaCl, were adjusted to 10 mg/ml of total protein concentration and subjected to SDS–PAGE and Western blot analyses under reducing conditions. SDS–PAGE analysis shows, in addition to small faint protein bands, two typical protein bands with an apparent MW ranging from 65.1 kDa to 18.0 kDa (Fig. 4A). These two bands were identified as heavy and light chains by
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Fig. 2. Antibody profiles obtained by ELISA method in sera (serially diluted 1:100 to 1: 3200) (A) or in egg yolk IgY preparations (serially diluted 1: 500 to 1:64000) (B) from hens immunized with B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca, and N. mossambica African snake venoms. Results are representative of three independent experiments.
polyclonal rabbit anti-egg IgY immunoglobulin, employing Western blot analysis (Fig. 4B). 3.3.3. Increase in IgY antivenom antibodies along the immunization procedure In order to evaluate the production of IgY antivenom antibodies along the immunization procedure, two IgY batches of yolks were recovered from eggs collected after the 3rd and 9th weeks of immunization. IgY was isolated, analyzed and quantified by the methods described. As can be seen in Fig. 5A and B, the antibody titers, referred to in terms of U-ELISA, for both IgY anti-Bitis and for anti-Naja venoms, were significantly higher in IgY preparations recovered after the 9th immunization booster. 3.3.4. Neutralization of venom lethality Fig. 6 shows that mice injected i.p. with 62.2 LD50 of B. arietans venom and pretreated with 1440 mg of IgY purified from egg yolks collected at the 6th week after immunization were fully protected (100% survival at 72 h of observation). In contrast, mice injected with mixtures containing the same venom and antivenom ratio, but with IgY purified from egg yolks collected at the 3rd week after immunization, were only partially protected (20% survival at 24 h and 0% survival at 72 h). All mice injected with venom pretreated with IgY purified from egg yolks collected before immunization (control) died within 48 h of observation.
3.3.5. General features of polyvalent anti-Bitis and anti-Naja IgY antivenoms The IgY antivenom method developed on a small scale was adjusted for use with larger yolk volumes. Data on the ‘‘Polyvalent IgY anti-Bitis and anti-Naja antivenoms’’ are included in Table 2. Western blot analysis of these antivenoms shows that IgY anti-Bitis (Fig. 7A) and anti-Naja (Fig. 7B) venoms recognize three and one protein bands, respectively. 4. Discussion Although not officially reported, some species belonging to the genera Naja, Dendroaspis and Bitis, such as N. nivea, D. polylepis and B. arietans (Chapman 1968), are potentially dangerous causes of bites in Africa. B. arietans is reported to be responsible for a larger number and more severe bites than all other species taken together (Broadley, 1968). Based on these fragile data, it was decided to develop a process of production and adapt quality control methods to prepare antivenoms containing specific antibodies to the toxic components present in B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca and N. mossambica snake venoms. It was expected that immunization with these venoms could produce antivenoms to treat animals and, eventually, human patients bitten by African snakes. Even with this limitation in place, attention was given to ensuring specificity and efficacy of neutralizing lethality, with
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Fig. 4. Identification of IgY in purified preparation of egg yolk anti-B. arietans antivenom. Five micrograms of purified IgY were resolved on 10% reducing SDS–PAGE and revealed by staining with Coomassie brilliant blue (left) or by Western blot, using a rabbit polyclonal specific anti-whole chicken IgY as the first antibody (right). Heavy (1) and Light (2) chains. This figure is representative of several independent experiments.
Fig. 3. Antibody profiles obtained by the ELISA method in serum of individual hens immunized with B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca, and N. mossambica African snake venoms. Results are representative of three independent experiments. Comparison between sera collected on the 3rd (lighter bars) or 9th (darker bars) weeks after immunization. Each bar represents the mean of triplicate values. Results are representative of two or three independent experiments.
minimal adverse reaction risks to ensure the qualities necessary for intravenous infusion of the antivenoms. Attention was also paid to the production of antivenoms of low cost, another requirement of paramount importance, especially in developing countries. Structurally, IgY, like IgG, is composed of two light and two heavy chains. The former contains one variable and one constant domain, while the latter contains one variable and four constant domains. The molecular weight of IgY is approximately 167 kDa and the isoelectric point varies between 5.7 and 7.6. Chicken antibodies are actively transferred from the blood to the eggs, IgY to yolk and IgM and
IgA to the egg white, a process that is mediated by specific receptors. IgY is found in the egg yolk at a variable concentration of between 10 and 20 mg/ml. Chicken IgY presents several advantages in comparison with mammalian IgG: it presents a low cost, high yield, avoids animal bleeding, does not cross react with rheumatoid factors or human anti-mouse antibodies and does not activate the mammalian complement system (Bauwens et al., 1988; Carlander, 2002; Chacana et al., 2004). With small individual variations, the antibody production in IgY egg yolks started at 15 days after the first injection of the venoms, progressively increasing along the immunization procedure, and attaining a plateau after the last booster, which was maintained thereafter (Fig. 2A). Two weeks after immunization, transference of venom-specific antibodies to the egg yolks was already observed (Fig. 2B). Lack of complete genetic homogeneity may explain the individual variations in the antibody titers among different groups of chickens (Fig. 3A and B). IgY antivenom antibody titers increased significantly after the venom injections, reaching maximal values 9 weeks later
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Table 2 General characteristics of IgY antivenom antibodies preparations: Batch #01 Dataa
Anti-Bitis venom
Anti-Naja venoms
Number of eggs collected Number of eggs used Egg yolk obtained (ml) Protein content of purified IgY (mg/ml) Final volume of purified IgY (ml) U-ELISA value (106)
4538 367 3650 105.07 210.5 7.138
2367 149 1500 167.5 88.5 5.705
a
All data refer to the mean.
efficacy of neutralizing lethality of the IgY (Fig. 6). Since anti-Bitis IgY recognizes three (Fig. 7A) and anti-Naja recognizes only one (Fig. 7B) of the several protein bands revealed by SDS–PAGE of venoms (Fig. 1), presumably Western blot detection of these bands includes the main toxic venom components. The neutralizing potency of IgY anti-B. arietans is particularly efficient, as shown by the capacity of 1440 mg IgY to
Fig. 5. Increase in IgY antivenom antibodies along the immunization procedure. IgY was quantified by the ELlSA method and the titers referred to as UELISA. IgY preparations: 1, B. arietans; 2, Bitis mix (B. nasicornis þ B. rhinoceros; 3, Naja mix (N. melanoleuca þ N. mossambica). Each preparation was obtained from 40–60 egg yolks. Top A: The eggs were collected on the 3rd and 9th weeks after immunization. Each bar represents the mean of triplicate values. Results are representative of two or three independent experiments.
(Fig. 5A and B). Higher antibody titers in IgY prepared with yolk from eggs collected after the 9th week of immunization, as compared with IgY prepared with yolk collected after the 3rd immunization, was reflected the increased
Fig. 6. Survival curve of Swiss mice (18–20 g), following injection of B. arietans venom pretreated with egg yolk IgY immunoglobulins. Groups of mice (6 per group) were injected i.p. with 33.3 mg (34 LD50) of venom that had been pre-incubated at 37 C for 60 min with 330 mg of purified IgY. Non-immune IgY (control); IgY anti-B. arietans venom prepared from egg yolks collected after the 3rd week of immunization; IgY anti-B. arietans venom prepared from egg yolks collected after the 6th week of immunization. Survival rates was determined after 72 h of observation.
Fig. 7. Western blot analysis of African crude venoms using IgY antivenoms as the first antibody. Five micrograms of crude snake venoms were resolved on non-reducing 15% SDS–PAGE, electroblotted onto PVDF (Immobilon P), and probed with IgY antivenom antibodies. (A) B. arietans venom, revealed by 1:800 dilution of IgY anti-polyvalent Bitis-Venom prepared with egg yolks collected at 6 weeks after immunization. (B) N. melanoleuca plus N. mossambic venoms revealed by 1:800 dilution of IgY anti-polyvalent BitisVenom prepared with egg yolks collected at 6 weeks after immunization.
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antibodies to African snake venoms allowed the preparation of larger batches of potent, specific, antibodies with high antibody titers and potent lethality neutralizing capabilities, as indicated in antivenoms profiles (Table 2). Chickens are available in most countries throughout the world. They are inexpensive to purchase and maintain and, as we have previously demonstrated using Brazilian snake venoms (Almeida et al., 1998); they also produce lower cost and effective antivenoms specific to African snake venoms. Rhode Island Red chickens were chosen to produce IgY antivenms, based on several of their intrinsic physiological qualities. Firstly, they can be easily maintained in confined spaces. The chickens, before immunization, start laying eggs at the age of 4 weeks, continuing after immunization, until 10 months later. Secondly, they are capable of mounting effective immune responses to the protective vaccines and become immunized against the usual infective diseases that affect chickens. Thirdly, each chicken produces around 20 eggs/month, corresponding approximately to 200 ml of egg yolk. Since the IgY concentration in egg yolk is around 10 mg/ml, each chicken produces 2000 mg of IgY. Therefore, 2 g of egg IgY corresponds approximately to an IgY content of 300 ml of mammalian serum or 600 ml of blood (Akita and Nakai, 1992). Compared with horses that produce approximately 10 ml of serum per kilogram (Thalley and Carroll, 1990), the use of IgY antivenoms to treat envenomed domestic animals is the cheapest alternative. Before the release of these antivenoms to treat domestic animals bitten by snakes, the characteristics for efficacy and safety for the IgY antivenoms obtained by the described method will be evaluated in simulated pre-clinical tests using goats as the animal test. Antivenoms will be administered before or after venom injection and local and systemic signals of envenoming will be compared with non-treated envenomed control animals. Table 2 summarizes the main characteristics of the two batches of IgY antivenom antibodies. Fig. 8A and B show that IgY antivenom antibodies recovered from eggs collected after the 9th week of immunization were significantly higher as compared with IgY prepared with yolk collected after the 3rd immunization.
Fig. 8. Increase in IgY antivenom antibody titers prepared from egg yolk IgY, collected after 6 weeks, compared with IgY antivenoms prepared from eggs collected at 3 weeks after immunization. (A) IgY anti-polyvalent Bitis-Venom; IgY anti-polyvalent Bitis-Venom. (B) IgY anti-polyvalent Naja-Venom.
neutralize 62.2 LD50 of the venom. According the official requirements for the antivenoms to be used to treat victims of snake bitten, 1.0 ml of antivenom containing 1.0–1.2 mg of total proteins must neutralize 5 LD50 of the venom (World Health Organization, 1981). The different neutralizing capacities of the two IgY preparations may be explained in part by the observation of more antibodies in the first IgY preparation, as compared with the second preparation. High affinity antibodies, commonly generated later on during the second immune response (Eisen and Siskind, 1964), may also be responsible for the high potency exhibited by first IgY preparation. The method of production of IgY
Acknowledgments The authors thank: CNPqdBrazil, for providing a Research Grant (Proc. No: 490048/2005- 6) and ICFellowships (David Gitirana and Lynna de Paula Sentinelli); FENORTE TECNORTE for the fellowship to Cla´udia Letı´cia da Silva (Proc. No.E15/021668/04); Mr. Fernando Ce´sar Lopes, Laboratory Technician (LBR/CBB/UENF), for helping with the organization of the Chicken Animal House Facility and the Animal Care Technicians, Mrs. Maria Joana Chagas Atayde, Mr. Maurı´cio de Oliveira Atayde and Mr. Fa´bio Conceiça˜o de Oliveira (Biote´rio /CBB/UENF). Dr. Nicola Conran (UNICAMP, Campinas, Sa˜o Paulo) for English revision. Conflict of interest The authors have no financial conflict of interest to declare.
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References Akita, E.M., Nakai, S., 1992. Isolation and purification of immunoglobulins from egg yolk. J. Food. Sci. 57, 620–634. Almeida, C.M., Kanashiro, M.M., Rangel Filho, F.B., Mata, M.F., Kipnis, T.L., Dias da Silva, W., 1998. Development of snake antivenom antibodies in chickens and their purification from yolk. Vet. Rec 143, 579–584. Bauwens, R.M., Devos, M.P., Kint, J.A., De Leenheer, A.P., 1988. Chicken egg yolk and rabbit serum compared as sources of antibody for radioimmunoassay of 1,25- dihydroxyvitamin D in serum or plasma. Clin. Chem. 34, 2153–2154. Bliss, C.I., 1934. The method of probits. Science 79, 38–39. Broadley, C.M., 1968. The venemous snakes in Central and South Africa. In: Bu¨cherl, W., Buckley, E., Deulofeu, V. (Eds.), Venomous Animals and Their Venoms, Vol. I. Academic Press, New York London, p. 433. Calmette, A., 1894. Lı´mmunisation artificelle des animaux contre le venin des serpents et la therapeutic experimentale de morsures venimeuses. C.R. Soc. Biol. 46, 120–124. Carlander, D., 2002. Avian IgY antibody: In vitro and in vivo. Acta Universitatis Upsaliensis, Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1119, 1–53. Chacana, P.A., Terzolo, H.R., GutierrezCalzado, E., Schader, R., 2004. Tecnologı´a IgY o aplicaciones de los anticuerpos de yema de huevo de gallina. Rev. Med. Vet. (Bs. As.) 85 (5), 179–189. Chapman, D.S., 1968. Symptomology, pathology and treatment of bites of venomous snakes of Central and South Africa. In: Bu¨cherl, W., Buckley, E., Deulofeu, V. (Eds.), Venomous Animals and Their Venoms, Vol. I. Academic Press, New York London, pp. 463–528.
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Eisen, H.N., Siskind, G.W., 1964. Variations on affinities of antibodies during the immune response. Biochemistry 3, 996–1008. Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680–685. Manaças, S., 1981-1982. Ofı´deos vennosos da Guine´, S. Tome´, Angola e Moçambique. Garcia de Orta, Ser. Zool., Lisboa 10(1-2). 13-46. Reid, H.A., 1964. Cobra bites. Br. Med. J. ii, 540–545. Sewall, H., 1887. Experiments on the preventive inoculation of rattlesnake venom. J. Physiol. Lond 8, 203–210. Thalley, B.T., Carroll, S., 1990. Rattlesnake and scorpion antivenoms from the egg yolks of immunized hens. Biotechnology 8, 934–938. Theakston, R.D.G., 1996. Clinical features of envenoming from snake bites. In: Bon, C., Goyffon, M. (Eds.), Envenomings and their Treatments. Fondation Marcel Me´rrieux, pp. 117–126. Towbin, H., Staechelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrilamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354. Viravan, C., Veeravat, U., Warrell, M.J., Theakston, R.D.G., Warrell, D.A., 1986. ELISA confirmation of acute and past envenoming by the monocellate Thai cobra (Naja kaouthia). Am. J. Trop. Med. Hyg 35, 173–182. Warrell, D.A., 1996. Clinical features of envenoming from snake bites. In: Bon, C., Goyffon, M. (Eds.), Envenomings and their Treatments. Fondation Marcel Me´rrieux, pp. 64–76. World Health Organization, 1981. Progress in the Characterization of Venoms and Standardization of Antivenoms (WHO Offset Publication No. 58). WHO, Geneva.
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Toxicon journal homepage: www.elsevier.com/locate/toxicon
Development of process to produce polyvalent IgY antibodies anti-African snake venomq Cla´udia Maria Costa de Almeida a, Cla´udia Letı´cia da Silva b, Humberto Pena Couto c, Rita de Ca´ssia Mothe´ Escocard b, David Gitirana da Rocha b, Lynna de Paula Sentinelli b, Thereza Liberman Kipnis b, Wilmar Dias da Silva b, * a ´rias, Universidade Estadual do Norte FluminensedDarcy Ribeiro, ´ rio de Sanidade Animal, Centro de Cieˆncias e Tecnologias Agropecua Laborato Campos dos Gottacazes, RJ, Brazil b ´rio de Biologia do Reconhecer, Centro de Biocieˆncias e Biotecnologia, Universidade Estadual do Norte FluminensedDarcy Ribeiro, Laborato Av. Alberto Lamego, 2000 Parque California, CEP 28015-620, Campos dos Gottacazes, RJ, Brazil c ´rias, Universidade Estadual do Norte FluminensedDarcy Ribeiro, ´rio de Zootecnia e Nutriça ˜o Animal, Centro de Cieˆncias e Tecnologias Agropecua Laborato Campos dos Gottacazes, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 January 2008 Received in revised form 28 May 2008 Accepted 29 May 2008 Available online 13 June 2008
Polyvalent anti-Bitis and anti-Naja antivenom IgY antibodies were prepared using B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca, and N. mossambica venoms to immunize chickens. Blood and eggs were collected before and during the 10-month immunization period; the sera and yolk extracts were then prepared and assayed for the presence of antivenom antibodies by ELISA and Western blot methods. ELISA Antivenom antibody titers, referred to as U-ELISA/ml of serum or egg yolk extracts, absent in pre-immunization sera or yolk, increased sharply during the 4 weeks after immunization, reaching a plateau thereafter. Yolk extracts with high antivenom titers, as detected by ELISA were used to isolate and purify IgY. Purified IgY preparations recognized venom protein bands from 10 to 20 kDa to 60 and 70 kDa, as shown by Western blot. Recovery of antivenom antibodies from the whole yolk was over 80%. Final preparations exhibited high antivenom activity (>100,000 U-ELISA/ml) as well as efficacy in neutralizing venom lethality (1440 mg of IgY neutralize 62.2 LD50 of venom), and were free of toxic products, pyrogen or bacterial and fungal contaminations. Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: IgY Snake venom Snake bite Envenoming
1. Introduction The snake species usually found in Guine´, S. Tome´, Angola and Mozambique belong to the Elapidae and Viperidae families (Manaças, 1981–1982). Elapidae includes the genus Aspidelaps, Elapsoidea, Hemachatus, Naja, Pseudohajc, and Dendroaspis, comprising around 20 species. Their venoms are essentially neurotoxic. The local effects of bites from these snakes are usually minimal, restricted to small q Ethical statement: The experiments with chickens were approved by the ‘‘Ethical Committee of Animal WelfaredUENF’’. * Corresponding author. Tel./fax: þ55 22 2726 1591. E-mail address: [email protected] (W.D. da Silva). 0041-0101/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2008.05.022
fang puncture wounds, exceptionally with tender local swelling, blistering and superficial necrosis (Reid, 1964; Viravan et al., 1986). Early systemic symptoms of envenoming are vomiting, ‘‘heaviness’’ of the eyelids, blurred vision, fasciculation, paraesthesiae around the mouth, hyperacusis, headache, dizziness, vertigo, hypersalivation, congested conjunctivae and ‘‘gooseflesh’’. Ptosis and external ophthalmoplegia appear soon after the bite, followed minutes or hours later by paralysis of the face, jaws, tongue, vocal cords, neck muscles and (bulbar) muscles of deglutition with pooling of secretions in the pharynx. The intercostals and diaphragm become paralyzed, leading to abdominal breathing, respiratory distress, with agitation, tachycardia, sweating, and central cyanosis (Warrell, 1996). Viperidae
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includes the genus Causus, Bitis, Vipera, Atheris, and Atractaspis, with also around 20 species. Their venoms are rich in various enzymes, mainly proteinases. Swelling and bruising may develop at the site of the bite within a few minutes or hours. Swelling spreads rapidly and sometimes involves the whole of the bitten limb and trunk. Regional lymphatic vessel and lymph node involvement is common. Blistering and necrosis may develop at the site of the bite. Hemostatic abnormalities are clinically manifested by persistent bleeding from the fang puncture wounds and other partially healed wounds, suggesting coagulopathy. The blood clot alterations are sometimes systemically reflected as epistaxis, hematemesis, cutaneous ecchymoses, petechiae or discoid ecchymoses, hemoptisis, and subconjuntival, retroperitoneal, subarachnoid, intracranial hemorrhages (Warrell, 1996). These venom effects can be completely and acutely reversed by specific antivenoms, if the patients or bitten animals are properly treated. Snake venom poisoning is one disease process that has been successfully treated with specific polyclonal antibodies. This therapeutic tool has been used since the pioneering work of Sewall (1887) and Calmette (1894). Conventional antivenoms are prepared by immunizing large animals, usually horses, with individual venom or a range of different venoms obtained from several snakes to eliminate intraspecific variation (Theakston, 1996). In order to minimize the expected adverse reactions, upon injection into the bitten victims, the IgG immunoglobulins are partially purified, enzymatically cleaved by pepsin, and the resulting F(ab0 )2 fragments are concentrated and tested for their antivenom neutralizing properties, and submitted to quality control, before release for use in human beings. As antivenoms prepared in horses are very expensive for use to treat domestic animals bitten by venomous snakes, alternative animals and methods are being tried. Previous studies in our laboratory have demonstrated that hens (Gallus gallus) immunized with Bothrops spp. and Crotalus durissus terrificus crude venoms, produce IgY antibody specific for these venoms, which can then be recovered from the egg yolks and purified (Almeida et al., 1998). The resulting IgY preparations were endowed with the ability to combine with venom components used to immunize the hens, and neutralize their toxic activities. The present study aimed to improve procedures to produce, on a large scale, IgY-specific antibodies to Bitis spp. and Naja spp. venoms to be used to treat domestic animals and eventually human patients bitten by African snake venoms. Before being released for use, these antivenoms will be submitted to pre-clinical trials. Based on the simplicity of the entire procedure, the low price of hens and, especially, the high capacity of the resulting antivenoms to neutralize the venoms’ lethality, IgY antivenoms are indicated to treat animals bitten by snakes, especially in developing countries.
Laborato´rio de Biologia do Reconhecer (LBR)/Centro de Biocieˆncias e Biotecnologia (CBB)/Universidade Estadual do Norte FluminensedDarcy Ribeiro (UENF). Hens were used to produce IgY antivenoms, Swiss outbred mice were used to determine venom lethality potency and the neutralizing potency of antivenoms, and rabbits were used to produce anti-IgY antiserum. Animal care was provided by expert personnel, in compliance with the relevant laws and institutional guidelines. 2.2. Crude venoms from African snakes The Bitis arietans, Bitis nasicornis, Bitis rhinoceros, Naja melanoleuca, and Naja mossambica snake venoms were purchased from Venom Supplies Pty Ltd (Tanunda, Australia). 2.3. Reagents and supplies Freund’s complete adjuvant (FCA), incomplete Freund’s adjuvant (IFA), and rabbit anti- IgY antiserum were prepared in our Laboratory. The following reagents were purchased as indicated: Bovine serum albumin (BSA), goat anti-rabbit IgG peroxidase conjugate, 3,30 -diaminobenzidine tetrahydrochloride (DAB) (Sigma Aldrich Chemical Co., St Louis, MO, USA). All other reagents were of analytical grade as otherwise indicated. Polystyrene ELISA plates were utilized (Microlon Medium Binding, Greiner, Germany). 2.4. Determination of venom lethality The lethal potencies of the venoms (LD50) were determined by i.p. injection of Swiss outbreed mice (18–20 g) using eight mice per group. Mortalities were recorded after 48 h, and LD50 was calculated using the Bliss (1934) method. 2.5. Immunization schedule of chickens
2. Material and methods
Groups of eight chickens were immunized intramuscularly in the breast region at two or three sites with 20 mg of African snake venoms, alone or mixed as indicated in FCA: Group #1, B. arietans; Group #2, B. nasicornis plus B. rhinoceros; Group #3, N. mossambica; Group #4, N. melanoleuca plus N. mossambica; Group #5, non immunized. Three weeks later, the injections were repeated with the venoms in IFA. Three boosters were given with the venoms in 0.15 M NaCl by the same route, also at 3-week intervals. Blood samples and eggs were collected before immunization to be used as negative controls either in immunochemical assays or in immunoprotection tests. Eggs were collected every day from each immunized chick and refrigerated at 4 C. Egg yolks were separated from the albumin and stored at 20 C.
2.1. Animals
2.6. Antibody purification
Rhode Island Red female chickens (1.1–1.5 kg body mass), Swiss outbreed (18–20 g) mice, and rabbits (0.5–1.0 kg) were maintained in the animal facility of the
2.6.1. Isolation and partial purification Yolks from 30 eggs of the same immunized group of hens were diluted 10-fold with distilled water, pH 5.5.
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The suspensions were incubated overnight at 4 C. The supernatants containing the IgY were collected by centrifugation (10,000 g at 4 C for 30 min), the temperature was adjusted to 20 C and subjected to 29% saturated ammonium sulfate precipitation. After centrifugation (10,000 g at 25 C for 30 min), the precipitated proteins were recovered, dissolved in 0.15 M NaCl and dialyzed against this same solution. Protein contents were adjusted to 10 mg/ml and analyzed by SDS–PAGE (15%) and Western blotted using rabbit serum anti-IgY as the first antibody. Anti-snake venom antibodies were quantified by the ELISA method, and their lethality neutralizing efficacies were assessed by in vitro/in vivo assays using Swiss outbred mice as the animal test. IgY immunoglobulins were similarly prepared and analyzed from pre-immunized egg yolks. 2.7. Characterization of egg antivenom IgY antibodies 2.7.1. SDS–PAGE and Western blot analyses Western blot analysis was carried out, according to the previously described method (Towbin et al., 1979). Crude B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca, and N. mossambica snake venoms (2 mg/ml) were treated with SDS–PAGE sample buffer under reducing conditions and resolved in 15% polyacrylamide gel (Laemmli, 1970) before electroblotting onto PVDF (Immobilon P), after treating membranes with methanol. The membrane, after treating again with methanol at 37 C for 15 min, was blocked with blocking buffer (0.01 M PBS, pH 7.4, with 5% low fat milk) at room temperature for 30 min. After washing with wash buffer (blocking buffer containing 0.1% Tween-20), the membrane was incubated with 5.0 mg of rabbit anti-chicken IgY (whole molecule) diluted in the same blocking buffer and incubated for 1.0 h at room temperature on a horizontal shaker. The membrane was washed with blocking buffer and incubated with 1:2000 dilution of peroxidase-conjugated goat anti-rabbit IgG for 1.0 h at room temperature. After washing, the membrane was placed in peroxidase chromogenic substrate solution (0.1 M citric acid plus 0.2 M sodium diphosphate, 5.0 mg of DAB (0.05%) and 3 ml of H2O2). The reaction was terminated by washing with distilled water. 2.7.2. Evaluation of antibody activity: ELISA method Polystyrene ELISA plates (96 wells) were coated with 1.0 mg of native snake venom in 50 ml coating buffer (0.1 M carbonate bicarbonate, pH 9.6) kept overnight at 4 C. The wells were washed once with PBS buffer containing 0.05% Tween-20. The wells were next blocked for 1.0 h at room temperature with 150 ml PBS buffer plus 1.0% gelatin. The wells were again washed thrice with 300 ml washing buffer. Serial dilutions of IgY preparations (1:1000 to 1:320,000) in PBS buffer plus 1.0% gelatin buffer containing 0.05% Tween-20 were prepared and 50 ml of each were added to individual wells and the plates were incubated at 37 C for 45 min. The wells were washed five times with the same washing buffer. Rabbit peroxidase-conjugated anti-chicken IgY (whole molecule), diluted (1:800) in PBS buffer plus 1.0% gelatin buffer containing 0.05% Tween-20 (50 ml), was added to each well.
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The plates were incubated for 45 min at 37 C. After five washes with the washing buffer, 50 ml of substrate buffer (0.1 M citric acid, plus 0.2 M sodium diphosphate, 5.0 ml H2O, 5.0 mg OPD, 5ml of H2O2) were added and incubated at room temperature for 10–15 min. The reaction was terminated with 50 ml of 3 N sulfuric acid. Absorbance was recorded at 492 nm using an ELISA plate reader. IgY from eggs collected before immunization was always used as a negative control. Wells free of venom were used as blanks. The IgY dilution, giving an optical density of close to 0.2, was used to calculate the U-ELISA per milliliter of undiluted IgY solution. One U-ELISA is defined as the smallest amount of antibody giving an OD of 0.2 under conditions of ELISA assay. 2.7.3. Neutralization of venom lethality by IgY antivenom antibodies The neutralizing efficacy of IgY antivenom antibodies, produced along the immunization procedure, was evaluated by incubating samples of B. arietans venom (600 mg) corresponding to 62.5 LD50, for 1 h at 37 C, with IgY antivenom antibodies (1440 mg) prepared from egg yolks collected before immunization (control) or at the 3rd and 6th weeks after immunization. The mixtures were i.p. injected into groups of eight Swiss mice (18–20 g), and deaths were recorded over 72 h. 3. Results 3.1. LD50 of the venoms Groups of eight Swiss mice (18–20 g) were injected i.p. with 500 ml of 0.15 M NaCl containing different concentrations of the snake venoms and the death/survival ratios were recorded along 72 h of observation. Results depicted in Table 1 show that, as expected, the lethality of venoms varies, with B. arietans venom being the most toxic. 3.2. SDS–PAGE analysis of snake venoms B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca, and N. mossambica snake venoms, submitted to 15% SDS– PAGE under reducing conditions, were resolved into venom components with different apparent molecular masses, ranging from 14 to 97 kDa. Nine protein bands were resolved for B. arietans (well #2), six for B. nasicornis (well #3), six for B. rhinoceros (well #4), five for N. melanoleuca (well #5) and seven for N. mossambica (well #6), as depicted in Fig 1. Fig. 1 also demonstrates that some prominent protein bands were randomly distributed over the electrophoretic profiles. A single protein band with apparent molecular mass ranging from 24-36 kDa is observed only in B. arietans venom. 3.3. Antivenom IgY antibody 3.3.1. Characterization and quantification IgY antibody by ELISA method Groups of laying hens were intramuscularly primed with 0.5 ml of 0.15 M NaCl containing 20 mg of venom or mixtures of venoms emulsified in equal volumes of
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Table 1 LD50 of the African snake venoms Snake venom
LD50 (in mg of dried venom)
Statistical analysisa
Naja melanoleuca
13.43
Naja mossambica
22.40
Bitis arietans
0.96
Bitis gabonica
95.28
Bitis nasicornis
123.67
Precision factor Standard deviation Superior confidence limit (95%) Inferior confidence limit (95%) Precision factor Standard deviation Superior confidence limit (95%) Inferior confidence limit (95%) Precision factor Standard deviation Superior confidence limit (95%) Inferior confidence limit (95%) Precision factor Standard deviation Superior confidence limit (95%) Inferior confidence limit (95%) Precision factor Standard deviation Superior confidence limit (95%) Inferior confidence limit (95%)
a
¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼
0.29691 4.725761E-02 14.84792 11.94401 0.3565223 7.941066E-02 27.14655 18.83186 0.2807443 6.506576E-02 1.106156 0.8197657 0.2914934 5.322902E-02 105.0646 82.22396 0.2716146 7.666159E-02 142.0168 99.77381
Deaths were recorded over the following 48h, and the LD50 was estimated by ‘‘Full Probits Analysis’’ (Bliss, 1934).
complete Freund’s adjuvant, and boosted 2 weeks later first with the same amounts of the venoms in incomplete Freund’s adjuvant, followed every week by four injections with the venoms in 0.15 M NaCl. Blood samples were harvested during the first week of immunization while eggs were collected along the entire 6 months of the experiment. Antibody titration was performed in serum samples and in egg extracts by the indirect ELISA method: Group #1, B. arietans; Group #2, B. nasicornis plus B. rhinoceros; Group #3, N. mossambica; Group #4, N. melanoleuca plus N. mossambica; Group #5, non-immunized. Antibodies
Fig. 1. Electrophoretic separation on SDS–PAGE (15%) of African snake venoms. 1, M.W. markers: 97 kDa; 66 kDa; 45 kDa; 36 kDa; 24 kDa; 14 kDa (from top to bottom). 2, Bitis arietans; 3, Bitis nasicornis 4, Bitis rhinoceros; 5, Naja melanoleuca; 6, Naja mossambica.
against the native snake venom started to appear in the serum 2 weeks after immunization, reaching a plateau at about 35–45 days, persisting with small individual variations for at least 8 months (data not shown). Transfer of these antivenom antibodies to egg yolk coincided with their appearance in serum. Presence of antibodies in sera and in corresponding egg yolks, collected at 6 weeks after immunization, are depicted in Fig. 2A and in B, respectively. Although the antibody titers are variable according to the venom used for immunization, the amounts of antibodies produced were consistently high. The antibody formation, although consistent in all immunized hens, was not uniform even among hens immunized with the same venom or a mixture of venoms, as depicted in Fig. 3A and B. 3.3.2. Purification of IgY antivenom antibodies The protocol used, as described in Section 2, includes the following steps: dilution of egg yolks in pH 5.5 distilled water, removal of the insoluble lipids in the upper supernatant, precipitation of IgY immunoglobulins with 29% ammonium sulfate, removal of soluble proteins by sucking out through a filter paper followed by also washing the IgY rich precipitates with ammonium sulfate at the same concentration. After extensive dialysis against sterile 0.15 M NaCl solution, protein contents were determined, and the protein concentration adjusted to 10.0 mg/ml. Antivenom antibodies were evaluated by ELISA method, and the presence of IgY protein was analyzed by both SDS–PAGE and Western blot methods. Individual batches of IgY, prepared from the same immunized hen groups after dialysis against 0.15 M NaCl, were adjusted to 10 mg/ml of total protein concentration and subjected to SDS–PAGE and Western blot analyses under reducing conditions. SDS–PAGE analysis shows, in addition to small faint protein bands, two typical protein bands with an apparent MW ranging from 65.1 kDa to 18.0 kDa (Fig. 4A). These two bands were identified as heavy and light chains by
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Fig. 2. Antibody profiles obtained by ELISA method in sera (serially diluted 1:100 to 1: 3200) (A) or in egg yolk IgY preparations (serially diluted 1: 500 to 1:64000) (B) from hens immunized with B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca, and N. mossambica African snake venoms. Results are representative of three independent experiments.
polyclonal rabbit anti-egg IgY immunoglobulin, employing Western blot analysis (Fig. 4B). 3.3.3. Increase in IgY antivenom antibodies along the immunization procedure In order to evaluate the production of IgY antivenom antibodies along the immunization procedure, two IgY batches of yolks were recovered from eggs collected after the 3rd and 9th weeks of immunization. IgY was isolated, analyzed and quantified by the methods described. As can be seen in Fig. 5A and B, the antibody titers, referred to in terms of U-ELISA, for both IgY anti-Bitis and for anti-Naja venoms, were significantly higher in IgY preparations recovered after the 9th immunization booster. 3.3.4. Neutralization of venom lethality Fig. 6 shows that mice injected i.p. with 62.2 LD50 of B. arietans venom and pretreated with 1440 mg of IgY purified from egg yolks collected at the 6th week after immunization were fully protected (100% survival at 72 h of observation). In contrast, mice injected with mixtures containing the same venom and antivenom ratio, but with IgY purified from egg yolks collected at the 3rd week after immunization, were only partially protected (20% survival at 24 h and 0% survival at 72 h). All mice injected with venom pretreated with IgY purified from egg yolks collected before immunization (control) died within 48 h of observation.
3.3.5. General features of polyvalent anti-Bitis and anti-Naja IgY antivenoms The IgY antivenom method developed on a small scale was adjusted for use with larger yolk volumes. Data on the ‘‘Polyvalent IgY anti-Bitis and anti-Naja antivenoms’’ are included in Table 2. Western blot analysis of these antivenoms shows that IgY anti-Bitis (Fig. 7A) and anti-Naja (Fig. 7B) venoms recognize three and one protein bands, respectively. 4. Discussion Although not officially reported, some species belonging to the genera Naja, Dendroaspis and Bitis, such as N. nivea, D. polylepis and B. arietans (Chapman 1968), are potentially dangerous causes of bites in Africa. B. arietans is reported to be responsible for a larger number and more severe bites than all other species taken together (Broadley, 1968). Based on these fragile data, it was decided to develop a process of production and adapt quality control methods to prepare antivenoms containing specific antibodies to the toxic components present in B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca and N. mossambica snake venoms. It was expected that immunization with these venoms could produce antivenoms to treat animals and, eventually, human patients bitten by African snakes. Even with this limitation in place, attention was given to ensuring specificity and efficacy of neutralizing lethality, with
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Fig. 4. Identification of IgY in purified preparation of egg yolk anti-B. arietans antivenom. Five micrograms of purified IgY were resolved on 10% reducing SDS–PAGE and revealed by staining with Coomassie brilliant blue (left) or by Western blot, using a rabbit polyclonal specific anti-whole chicken IgY as the first antibody (right). Heavy (1) and Light (2) chains. This figure is representative of several independent experiments.
Fig. 3. Antibody profiles obtained by the ELISA method in serum of individual hens immunized with B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca, and N. mossambica African snake venoms. Results are representative of three independent experiments. Comparison between sera collected on the 3rd (lighter bars) or 9th (darker bars) weeks after immunization. Each bar represents the mean of triplicate values. Results are representative of two or three independent experiments.
minimal adverse reaction risks to ensure the qualities necessary for intravenous infusion of the antivenoms. Attention was also paid to the production of antivenoms of low cost, another requirement of paramount importance, especially in developing countries. Structurally, IgY, like IgG, is composed of two light and two heavy chains. The former contains one variable and one constant domain, while the latter contains one variable and four constant domains. The molecular weight of IgY is approximately 167 kDa and the isoelectric point varies between 5.7 and 7.6. Chicken antibodies are actively transferred from the blood to the eggs, IgY to yolk and IgM and
IgA to the egg white, a process that is mediated by specific receptors. IgY is found in the egg yolk at a variable concentration of between 10 and 20 mg/ml. Chicken IgY presents several advantages in comparison with mammalian IgG: it presents a low cost, high yield, avoids animal bleeding, does not cross react with rheumatoid factors or human anti-mouse antibodies and does not activate the mammalian complement system (Bauwens et al., 1988; Carlander, 2002; Chacana et al., 2004). With small individual variations, the antibody production in IgY egg yolks started at 15 days after the first injection of the venoms, progressively increasing along the immunization procedure, and attaining a plateau after the last booster, which was maintained thereafter (Fig. 2A). Two weeks after immunization, transference of venom-specific antibodies to the egg yolks was already observed (Fig. 2B). Lack of complete genetic homogeneity may explain the individual variations in the antibody titers among different groups of chickens (Fig. 3A and B). IgY antivenom antibody titers increased significantly after the venom injections, reaching maximal values 9 weeks later
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Table 2 General characteristics of IgY antivenom antibodies preparations: Batch #01 Dataa
Anti-Bitis venom
Anti-Naja venoms
Number of eggs collected Number of eggs used Egg yolk obtained (ml) Protein content of purified IgY (mg/ml) Final volume of purified IgY (ml) U-ELISA value (106)
4538 367 3650 105.07 210.5 7.138
2367 149 1500 167.5 88.5 5.705
a
All data refer to the mean.
efficacy of neutralizing lethality of the IgY (Fig. 6). Since anti-Bitis IgY recognizes three (Fig. 7A) and anti-Naja recognizes only one (Fig. 7B) of the several protein bands revealed by SDS–PAGE of venoms (Fig. 1), presumably Western blot detection of these bands includes the main toxic venom components. The neutralizing potency of IgY anti-B. arietans is particularly efficient, as shown by the capacity of 1440 mg IgY to
Fig. 5. Increase in IgY antivenom antibodies along the immunization procedure. IgY was quantified by the ELlSA method and the titers referred to as UELISA. IgY preparations: 1, B. arietans; 2, Bitis mix (B. nasicornis þ B. rhinoceros; 3, Naja mix (N. melanoleuca þ N. mossambica). Each preparation was obtained from 40–60 egg yolks. Top A: The eggs were collected on the 3rd and 9th weeks after immunization. Each bar represents the mean of triplicate values. Results are representative of two or three independent experiments.
(Fig. 5A and B). Higher antibody titers in IgY prepared with yolk from eggs collected after the 9th week of immunization, as compared with IgY prepared with yolk collected after the 3rd immunization, was reflected the increased
Fig. 6. Survival curve of Swiss mice (18–20 g), following injection of B. arietans venom pretreated with egg yolk IgY immunoglobulins. Groups of mice (6 per group) were injected i.p. with 33.3 mg (34 LD50) of venom that had been pre-incubated at 37 C for 60 min with 330 mg of purified IgY. Non-immune IgY (control); IgY anti-B. arietans venom prepared from egg yolks collected after the 3rd week of immunization; IgY anti-B. arietans venom prepared from egg yolks collected after the 6th week of immunization. Survival rates was determined after 72 h of observation.
Fig. 7. Western blot analysis of African crude venoms using IgY antivenoms as the first antibody. Five micrograms of crude snake venoms were resolved on non-reducing 15% SDS–PAGE, electroblotted onto PVDF (Immobilon P), and probed with IgY antivenom antibodies. (A) B. arietans venom, revealed by 1:800 dilution of IgY anti-polyvalent Bitis-Venom prepared with egg yolks collected at 6 weeks after immunization. (B) N. melanoleuca plus N. mossambic venoms revealed by 1:800 dilution of IgY anti-polyvalent BitisVenom prepared with egg yolks collected at 6 weeks after immunization.
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antibodies to African snake venoms allowed the preparation of larger batches of potent, specific, antibodies with high antibody titers and potent lethality neutralizing capabilities, as indicated in antivenoms profiles (Table 2). Chickens are available in most countries throughout the world. They are inexpensive to purchase and maintain and, as we have previously demonstrated using Brazilian snake venoms (Almeida et al., 1998); they also produce lower cost and effective antivenoms specific to African snake venoms. Rhode Island Red chickens were chosen to produce IgY antivenms, based on several of their intrinsic physiological qualities. Firstly, they can be easily maintained in confined spaces. The chickens, before immunization, start laying eggs at the age of 4 weeks, continuing after immunization, until 10 months later. Secondly, they are capable of mounting effective immune responses to the protective vaccines and become immunized against the usual infective diseases that affect chickens. Thirdly, each chicken produces around 20 eggs/month, corresponding approximately to 200 ml of egg yolk. Since the IgY concentration in egg yolk is around 10 mg/ml, each chicken produces 2000 mg of IgY. Therefore, 2 g of egg IgY corresponds approximately to an IgY content of 300 ml of mammalian serum or 600 ml of blood (Akita and Nakai, 1992). Compared with horses that produce approximately 10 ml of serum per kilogram (Thalley and Carroll, 1990), the use of IgY antivenoms to treat envenomed domestic animals is the cheapest alternative. Before the release of these antivenoms to treat domestic animals bitten by snakes, the characteristics for efficacy and safety for the IgY antivenoms obtained by the described method will be evaluated in simulated pre-clinical tests using goats as the animal test. Antivenoms will be administered before or after venom injection and local and systemic signals of envenoming will be compared with non-treated envenomed control animals. Table 2 summarizes the main characteristics of the two batches of IgY antivenom antibodies. Fig. 8A and B show that IgY antivenom antibodies recovered from eggs collected after the 9th week of immunization were significantly higher as compared with IgY prepared with yolk collected after the 3rd immunization.
Fig. 8. Increase in IgY antivenom antibody titers prepared from egg yolk IgY, collected after 6 weeks, compared with IgY antivenoms prepared from eggs collected at 3 weeks after immunization. (A) IgY anti-polyvalent Bitis-Venom; IgY anti-polyvalent Bitis-Venom. (B) IgY anti-polyvalent Naja-Venom.
neutralize 62.2 LD50 of the venom. According the official requirements for the antivenoms to be used to treat victims of snake bitten, 1.0 ml of antivenom containing 1.0–1.2 mg of total proteins must neutralize 5 LD50 of the venom (World Health Organization, 1981). The different neutralizing capacities of the two IgY preparations may be explained in part by the observation of more antibodies in the first IgY preparation, as compared with the second preparation. High affinity antibodies, commonly generated later on during the second immune response (Eisen and Siskind, 1964), may also be responsible for the high potency exhibited by first IgY preparation. The method of production of IgY
Acknowledgments The authors thank: CNPqdBrazil, for providing a Research Grant (Proc. No: 490048/2005- 6) and ICFellowships (David Gitirana and Lynna de Paula Sentinelli); FENORTE TECNORTE for the fellowship to Cla´udia Letı´cia da Silva (Proc. No.E15/021668/04); Mr. Fernando Ce´sar Lopes, Laboratory Technician (LBR/CBB/UENF), for helping with the organization of the Chicken Animal House Facility and the Animal Care Technicians, Mrs. Maria Joana Chagas Atayde, Mr. Maurı´cio de Oliveira Atayde and Mr. Fa´bio Conceiça˜o de Oliveira (Biote´rio /CBB/UENF). Dr. Nicola Conran (UNICAMP, Campinas, Sa˜o Paulo) for English revision. Conflict of interest The authors have no financial conflict of interest to declare.
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