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Cross-neutralisation of Australian brown snake, taipan and death adder venoms by monovalent antibodies
Vaccine 28 (2010) 798–802
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Cross-neutralisation of Australian brown snake, taipan and death adder venoms by monovalent antibodies Geoffrey K. Isbister a,b,c,∗ , Margaret A. O’Leary a , Jessica Hagan b , Kearney Nichols b , Tammy Jacoby d , Kathleen Davern d , Wayne C. Hodgson e , Jennifer J. Schneider b a
Department of Clinical Toxicology and Pharmacology, Calvary Mater Newcastle Hospital, Newcastle, New South Wales, Australia School of Biomedical Science, University of Newcastle, Newcastle, New South Wales, Australia Tropical Toxinology Unit, Menzies School of Health Research, Charles Darwin University, Darwin, Australia d Western Australian Institute of Medical Research, Perth, Western Australia, Australia e Monash Venom Group, Department of Pharmacology, Monash University, Victoria, Australia b c
a r t i c l e
i n f o
Article history: Received 3 September 2009 Received in revised form 27 September 2009 Accepted 12 October 2009 Available online 29 October 2009 Keywords: Antivenom Cross-neutralisation Snake venom
a b s t r a c t An understanding of the cross-neutralisation of snake venoms by antibodies is important for snake antivenom development. We investigated the cross-neutralisation of brown snake (Pseudonaja textilis) venom, taipan (Oxyuranus scutellatus) venom and death adder (Acanthophis antarcticus) with commercial antivenoms and monovalent anti-snake IgG, using enzyme immunoassays, in vitro clotting and neurotoxicity assays. Each commercial antivenom bound all three venoms, and neutralised clotting activity of brown snake and taipan venoms and neurotoxicity of death adder venom. The ‘in-house’ monovalent anti-snake venom IgG raised against procoagulant brown snake and taipan venoms, did not neutralise the neurotoxic effects of death adder venom. However, they did cross-neutralise the procoagulant effects of both procoagulant venoms. This supports the idea of developing antivenoms against groups of snake toxins rather than individual snake venoms. Crown Copyright © 2009 Published by Elsevier Ltd. All rights reserved.
1. Introduction An understanding of cross-neutralisation of snake venoms by antibodies raised against different snake venoms is important for the development of monovalent and polyvalent antivenoms, used for the treatment of snake envenoming. With the current shortage of antivenoms in the rural tropics, most importantly in Africa [1], the ability to make large quantities of antivenom that is effective against many different species would be greatly beneficial. In addition, an examination of cross-neutralisation of the toxic effects of venoms by antibodies raised against different snakes will give insight into the similarity of venom components and potentially the biology and evolution of snake venoms. We have previously shown that there is cross-neutralisation of brown snake (Pseudonaja textilis) and tiger snake (Notechis scutatus) venoms by CSL tiger snake antivenom (TSAV) and brown snake antivenom (BSAV), respectively [2]. We suggested that this was most likely due to the commercial antivenoms, which are labelled as being “monovalent”, really being polyvalent and containing anti-
∗ Corresponding author at: Newcastle Mater Hospital, Edith St, Waratah, NSW 2298, Australia. Tel.: +612 4921 1211; fax: +612 4921 1870. E-mail addresses: [email protected], [email protected] (G.K. Isbister).
bodies to both snake venoms. However, the use of a specific ‘in house’ monovalent antibody suggested that there was at least partial cross-neutralisation between brown snake venom and tiger snake venom [2]. The most clinically important toxins found in Australasian elapid venoms are procoagulant toxins, which cause plasma to clot in vitro [3], and neurotoxins, which act presynaptically or postsynaptically [4]. Venoms from Australian brown snakes (Pseudonaja spp.), tiger snakes (Notechis spp.) and taipans (Oxyuranus spp.) contain potent prothrombin activators that have similar structures [5]. This structural similarity may be responsible for the cross-neutralisation of the venom-mediated clotting effects by monovalent antibodies. However, the neutralisation of other venom properties has not been shown definitively with monovalent antibodies. Venoms from these three groups of snakes also contain pre-synaptic neurotoxins and there appears to be crossneutralisation between these neurotoxins, at least between notexin in Notechis spp. venom and textilotoxin in P. textilis venom [6]. In contrast, death adder (Acanthophis spp.) venom contains mainly neurotoxins [7] and no procoagulant toxins, so is potentially useful for examining cross-neutralisation when compared to procoagulant Australian snake venoms. To further explore the cross-neutralisation of Australian snake venoms, we examined the cross-neutralisation of three representative venoms of Australian elapids by their respective commercial
0264-410X/$ – see front matter. Crown Copyright © 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.10.055
G.K. Isbister et al. / Vaccine 28 (2010) 798–802
antivenoms (CSL Ltd.) and polyclonal monovalent antibodies raised against each of the venoms. 2. Materials and methods Eastern or common brown snake (P. textilis) venom, Taipan (Oxyuranus scutellatus) venom and Death Adder (Acanthophis antarcticus) venom were purchased from Venom Supplies (Tanunda, South Australia). Tetramethylbenzidine (TMB), 2,2 Azino-bis 3-ethylbenzothiazoline-6-sulfonic acid) [ABTS], bovine serum albumin (BSA) and rabbit anti-horse IgG (whole molecule) peroxidase conjugate were obtained from Sigma. All chemicals used were of analytical grade. The following commercial antivenoms were obtained from CSL Ltd.—brown snake antivenom (BSAV, batch # 0559-10201), death adder antivenom (DAAV, batch # 0557-07301) and taipan antivenom (TAV, batch # 0548-05601). All of the rabbit IgG antibodies were biotinylated using the EZ-Link biotinylation kit (Pierce) and Sulfo-NHS-LC biotin reaction system as described by the manufacturer. Fresh frozen human plasma was obtained from the Australian Red Cross. Aliquots (10 mL) were thawed at 37 ◦ C then spun at 2500 rpm for 10 min prior to use. Calcium (60 L 0.2 M CaCl2 per mL) was added immediately before use. Clotting studies were carried out in a medium of Tris buffered saline containing 0.5% BSA. All procedures were carried out at room temperature unless indicated otherwise. Animal experiments were approved by the animal ethics committee at the University of Western Australia for rabbit antibodies production and Monash University (SOBS-B) animal ethics committee for in vitro neurotoxicity studies. 2.1. Preparation of rabbit polyclonal monovalent antibodies Lyophilised brown snake venom, taipan venom and death adder venom were reconstituted in physiological saline at 1–2 mg/mL. Venom (80–400 g) was emulsified with an equal volume of complete Freund’s adjuvant (CFA) and injected subcutaneously into multiple sites on the hind legs of New Zealand white rabbits. Each rabbit was injected with venom from only one snake species. Four weeks after the first injection, rabbits were given a booster dose at the same concentration in incomplete Freund’s adjuvant (IFA). Two weeks after this booster, 10–20 mL of blood was collected from each rabbit. Rabbits were then boosted with venom in aqueous solution every 4 weeks and bled 2 weeks after each injection. The presence of reactive venom antibodies was tested by an indirect enzymelinked immunosorbent assay (ELISA) with the respective venom used as coating antigen. Polystyrene microtitre plates were coated with 50 L of venom (5 g/mL) per well in coating buffer (0.1 M carbonate buffer, pH 9.7) overnight at 4 ◦ C. After washing three times in wash buffer (PBS, pH 7.3 containing 0.05% TWEEN20), wells were blocked with 200 L blocking solution (2% BSA in PBS) for 1 h at room temperature. After one wash, 50 L of suitably diluted rabbit serum in sample buffer (0.1% BSA in PBS pH 7.3) was titrated by using two-fold serial dilutions and incubated for 1 h at room temperature. Plates were washed three times again followed by the addition of 50 L of anti-rabbit secondary HRP-conjugated antibody to detect the bound anti-snake venom antibodies. Enzymatic activity was measured with the addition of peroxide substrate solution (50 L per well of ABTS and 0.001% hydrogen peroxide in 0.01 M citrate buffer, pH 4.5). Activity was assessed after 15 min by absorbance measured at 405 nm with Victor 3 microplate reader (PerkinElmer). Rabbit serum samples from hyper-immunised rabbits, with antibody titres higher than 50,000 as determined by ELISA, were subject to antibody purification. Serum was diluted 1:5 in PBS and
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sterile filtered. The dilute serum was then purified by affinity purification by being passed through a Sepharose-Protein G column using a BioRad BioLogic liquid chromatography system. Upon elution of IgG antibodies using 0.1 M glycine, pH 2.7, fractions containing IgG as determined by absorbance at 280 nm, were immediately neutralised with 2 M Tris, pH 9.0 and dialysed against PBS for 24 h. For biotinylation, 1–2 mg of purified rabbit polyclonal IgG in PBS had the appropriate volume of 10 mM of Sulfo-NHS-Biotin solution added to give a 20-fold molar excess of biotin. The mixtures were incubated for 1 h on ice or 2 h at room temperature followed by dialysis against PBS to remove free biotin. Success of biotinylation of each rabbit polyclonal IgG was assessed by ELISA and Western blot against the respective venoms using streptavidin-HRP as a detecting agent. 2.2. Binding Studies: measurement of venoms with and without prior incubation with antivenoms and specific antibodies To investigate the binding to venom of commercial antivenoms (BSAV, TAV, DAAV) compared to specifically raised polyclonal monovalent rabbit IgG to each of the venoms (anti-brown snake venom, anti-death adder venom, anti-taipan venom) we used a competitive sandwich ELISA with pre-incubated mixtures of the anti-snake venom antibodies and venoms. The washing solution was 0.02% TWEEN 20 in PBS, the blocking solution 0.5% BSA in PBS and 96-well Greiner High Binding plates were used. Venom and antivenom mixtures were made up from 200 L of 10 ng/mL venom in blocking solution with 50 mU antivenom (or 13 g monovalent anti-snake IgG) in 200 L PBS and allowed to stand for 60 min before applying to wells. Wells were coated with monovalent anti-snake venom IgG (100 L of 1–2 g/mL in carbonate buffer) for 1 h at room temperature then at 4 ◦ C overnight. The plates were washed, blocking solution applied for 1 h and then washed again. The venom and antivenom/antibody mixture (100 L) was applied in triplicate for 1 h, the plates washed, and 100 L of 0.5 g/mL biotinylated anti-snake venom IgG in blocking solution applied. After a further hour, the plates were washed again, and a solution of 100 L of 2 g/mL streptavidin-horseradish peroxidase in blocking solution was applied. After a final wash, 100 L of TMB was added, followed by 50 L of 1 M H2 SO4 . Plates were then read at 450 nm on a BioTek ELx808 plate reader. Standard curves were fitted to a sigmoidal dose-response curve. 2.3. Clotting studies We investigated the ability of commercial antivenoms and the polyclonal anti-snake venom IgG to prevent venom-induced clotting using a previously described method [8]. In brief, 10 ng of venom and 50 mU of antivenom or 13 g of anti-snake venom IgG were mixed in a total volume of 100 L and placed in the wells of a 96-well microtitre plate at 37 ◦ C in a BioTek ELx808 (BioTek Instruments, Inc., Winooski, VT, USA) plate reader. Plasma (100 L) was then added to each well simultaneously with a multichannel pipette and the optical density monitored at 340 nm for up to 20 min. The lag time before the sharp rise in optical density upon clot formation was recorded as the clotting time. The concentration of venom in the clotting studies was 100 ng/mL of plasma because this is in the upper range of venom concentrations in brown snake envenomed patients we have previously measured [9]. Similarly, the amount of CSL antivenom used was equivalent to 500 mU/mL of plasma based on expected and measured concentrations in patients to be 250 mU/mL, 1500 mU/mL and 3000 mU/mL respectively for the administration of 1 vial of BSAV, DAAV and TAV [9,10].
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2.4. In vitro neurotoxicity studies Studies using commercial monovalent BSAV and DAAV, as well as in-house polyclonal anti-snake venom IgG to brown snake venom and death adder venom, were undertaken to examine whether they were able to neutralise the neurotoxic effects of common death adder venom. Chicks (4–10-day-old males) were killed by CO2 inhalation and exsanguination, and the two biventer cervicis muscles were removed from the back of the neck. Each muscle was attached to a wire tissue holder and placed in a 5 mL organ bath filled with physiological salt solution of the following composition (mM): NaCl, 118.4; NaHCO3 , 25; glucose, 11.1; KCl, 4.7; MgSO4 , 1.2; KH2 PO4 , 1.2 and CaCl2 , 2.5. The organ baths were bubbled with carbogen (95% O2 , 5% CO2 ) and maintained at a temperature of 33–34 ◦ C under a resting tension of 1 g. Motor nerves were indirectly stimulated every 10 s (0.2 ms duration) at supramaximal voltage using a Grass S88 stimulator. The tissues were equilibrated for 10–15 min after which d-tubocurarine (10 M) was added, and the subsequent abolition of twitches confirmed the selective stimulation of the motor nerves. The tissues were then washed repeatedly until twitch height was restored. CSL DAAV (3 U/mL), CSL BSAV (3 U/mL), anti-death adder venom IgG (150 g/mL) and anti-brown snake venom IgG (150 g/mL) were added to the bath and incubated for 10 min. Death adder venom (10 g/mL) was then added and left in contact with the tissue for 60 min.
2.5. Data analysis Data were analysed by non-linear regression using Prism 5.0 to fit the curve with the most appropriate model, including for standard curves. The in vitro neurotoxicity data were analysed by ANOVA using Prism 5.0 with statistical significance indicated by P < 0.05.
3. Results High titre anti-snake venom rabbit IgG were produced to each venom and assessed with ELISA and Western blot. The concentration of these antibodies was 5 mg/mL. Commercial antivenoms are formulated as 17% equine protein or 170 mg/mL, but the proportion of this which are active antibodies is not given.
Fig. 1. Suppression of binding of venom to an anti-snake venom IgG-coated plates by pre-incubated mixtures of each of the three venoms (brown snake venom, 2 ng, death adder venom, 2 ng, and taipan venom, 2 ng) with 50 mU of commercial antivenoms (BSAV, DAAV, TAV) or 13 g of ‘in-house’ anti-brown snake venom (antiBSV) IgG, anti-death adder venom (anti-DAV) IgG or anti-taipan venom (anti-TV) IgG.
3.2. Clotting studies Taipan venom resulted in a longer clotting time than brown snake venom consistent with it being known to be less potent [11]. All three commercial antivenoms were able to neutralise clotting activity of both brown snake venom and taipan venom (Tables 1 and 2). The anti-brown snake venom IgG was also able to neutralise clotting activity of both venoms. Although anti-taipan venom IgG was completely effective in neutralising the clotting effect of taipan venom, it was only partially effective in neutralising the clotting effect of brown snake venom, even at twice the concentration shown (Table 1, Fig. 2). The anti-death adder venom IgG had minimal effect on either. 3.3. In vitro neurotoxicity Death adder venom (1 g/mL) abolished indirect (i.e. nervemediated) twitches of the chick biventer cervicis nerve-muscle preparation (Fig. 3). This inhibitory effect was prevented by the prior addition of CSL DAAV (3 U/mL, n = 4), CSL BSAV (3 U/mL, n = 4) and anti-DA IgG antibodies (150 g/mL, n = 4) but not by the prior
Table 1 Clotting times for brown snake venom in plasma alone, with the three commercial antivenoms and the three in-house anti-snake venom IgGs. Antibodies
3.1. Binding studies. All three commercial antivenoms (BSAV, TAV, DAAV) prevented brown snake venom binding to the anti-brown snake venom IgG-coated plate, taipan venom binding to anti-taipan venom IgGcoated plate and death adder venom binding to the anti-death adder venom IgG-coated plate (Fig. 1). In contrast only anti-brown snake venom IgG interacted with brown snake venom, anti-taipan venom IgG with taipan venom and anti-death adder venom IgG with death adder venom, preventing the venom binding to the corresponding anti-snake venom IgG-coated plate (Fig. 1). For example, only anti-death adder venom IgG pre-mixed with death adder venom prevented the death adder venom binding to the antideath adder venom IgG-coated plates, but anti-death adder venom IgG did not prevent brown snake venom or taipan venom from binding to their respective anti-snake venom IgG-coated plates. However, there was some interaction between anti-taipan venom IgG and brown snake venom which partially prevented brown snake venom from binding to the anti-brown snake venom IgGcoated plate (Fig. 1).
Clotting time (s)
Amount (mU) Mass (g) Nil 0 Anti-brown snake venom IgG – Anti-death adder venom IgG – Anti-taipan venom IgG – BSAV 50 DAAV 50 TAV 50
0 13 13 17 69 35 74
140 >900 170 215 >900 >900 >900
Table 2 Clotting times for taipan venom in plasma alone, with the three commercial antivenoms and the three ‘in-house’ anti-snake venom IgGs. Antibodies
Clotting time (s)
Amount (mU) Mass (g) Nil 0 Anti-brown snake venom IgG – Anti-death adder venom IgG – Anti-taipan venom IgG – BSAV 50 DAAV 50 TAV 50
0 13 13 17 69 35 74
305 >900 335 >900 >900 >900 >900
G.K. Isbister et al. / Vaccine 28 (2010) 798–802
Fig. 2. Clotting curves for brown snake venom (dotted lines) and taipan venom (thick lines) with a range of concentrations of the anti-taipan venom IgG showing that anti-taipan venom IgG can completely neutralise taipan venom-induced clotting but only partially effect brown snake venom-induced clotting.
Fig. 3. Effect of death adder venom (1 g/mL, n = 4) alone or in the presence of CSL death adder antivenom [DAAV] (3 U/mL, n = 4), CSL brown snake antivenom [BSAV] (3 U/mL, n = 4), anti-death adder venom IgG [DA IgG] (150 g/mL, n = 4) or anti-brown snake venom IgG [BS IgG] (150 g/mL, n = 3) on indirect twitches of the chick biventer cervicis nerve-muscle preparation. (*p < 0.05, significantly different from venom alone, ANOVA).
addition of anti-brown snake venom IgG antibodies (150 g/mL, n = 3) [Fig. 3]. 4. Discussion The study confirms that the Australian commercial antivenoms (CSL Ltd.) that are labelled as “monovalent” are in fact mixtures (polyvalent) and all three antivenoms used in this study were able to bind to all three venoms, neutralise the procoagulant effects of taipan venom and brown snake venom, and prevent the neurotoxic effects of death adder venom. More interesting were the results between the anti-snake IgG and different venoms, because these antibodies were monovalent, having been raised in rabbits given only single venom for immunisation. Using the monovalent IgG antibodies, there was no cross-neutralisation between the mainly neurotoxic death adder venom and the mainly procoagulant brown snake venom, based on binding studies, clotting and neurotoxicity assays. However, there was cross-neutralisation between the procoagulant venoms, brown snake venom and taipan venom, shown by the binding and clotting studies.
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Anti-brown snake venom IgG was unable to prevent the neurotoxic effects of death adder venom and conversely anti-death adder venom IgG was unable to neutralise the clotting effects of both brown snake venom and taipan venom. This is consistent with the known effects of these venoms and the clinical effects of envenoming by the respective snakes. Death adder envenoming causes isolated neurotoxicity and it is the only snake to cause this pattern of effects in Australia [12]. Death adder venom contains no known procoagulant toxins. Although brown snake venom is known to contain pre-synaptic and post-synaptic neurotoxins, the largest component of the venom is a potent prothrombin activator [3,13]. In human patients, isolated venom-induced consumption coagulopathy is the major feature of envenoming [14]. It is therefore not surprising that anti-brown snake venom IgG and anti-death adder venom IgG show no or very little crossneutralisation with the death adder venom and brown snake venom, respectively. In contrast, there was cross-neutralisation between taipan venom and anti-brown snake venom IgG and conversely partial cross-neutralisation between brown snake venom and anti-taipan venom IgG. This cross-neutralisation is likely to be due to the close similarity of the brown snake venom and taipan venom prothrombin activators [5]. We have previously shown a similar cross-neutralisation between anti-brown snake venom IgY and tiger snake venom [2]. Even though the prothrombin activator in tiger snake venom lacks the human factor Va-like subunit present in brown snake and taipan venoms, the human factor Xa-like parts of the prothrombin activators in all three venoms share close sequence homology [5]. Envenoming by all three corresponding snakes causes venom-induced consumptive coagulopathy [15]. This cross-neutralisation between venoms for the procoagulant effect has some important implications for antivenom design and manufacture, and supports the recent approach of toxin specific immunotherapy [16,17]. These investigators identified important epitopes that would be representative of the multimeric snake venom metalloproteinases in Echis ocellatus venom and used these to make a single synthetic multiepitope DNA immunogen. Antibodies were then raised to this immunogen or epitope string which were shown to not only neutralise the haemorrhagic effect of E. ocellatus, but also cross-neutralise the haemorrhagic effect of Cerastes cerastes venom, another viper that contains similar haemorrhagic metalloproteinases [17]. The presence of cross-neutralisation between the prothrombin activators in Australasian elapid venoms suggests that antibodies raised to an epitope string developed from the prothrombin activators in these venoms could potentially be used to neutralise the procoagulant effects of all Australasian elapid venoms with prothrombinase activity. A limitation of the study was the lower protein concentration of the ‘in-house’ anti-snake IgG making it difficult to completely neutralise some effects. This may be the reason that anti-taipan venom IgG only partially neutralised the clotting effect of brown snake venom and a more concentrated solution in the well would have caused complete neutralisation. In the neurotoxicity experiments large amounts of both anti-death adder venom IgG and anti-brown snake venom IgG were used, but there was no cross-neutralisation of death adder venom-induced neurotoxicity with the anti-brown snake venom IgG.
Acknowledgements Funding: GKI is supported by an NHMRC Clinical Career Development Award ID300785. Conflicts of interest: None.
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References [1] Theakston RDG, Warrell DA. Crisis in snake antivenom supply for Africa. Lancet 2000;356:2104. [2] O’Leary MA, Schneider JJ, Krishnan BP, Lavis C, McKendry A, Ong LK, et al. Cross-neutralisation of Australian brown and tiger snake venoms with commercial antivenoms: cross-reactivity or antivenom mixtures? Toxicon 2007;50:206–13. [3] Isbister GK. Procoagulant snake toxins: laboratory studies, diagnosis, and understanding snakebite coagulopathy. Semin Thromb Hemost 2009;35:93–103. [4] Kuruppu S, Smith AI, Isbister GK, Hodgson WC. Neurotoxins from AustraloPapuan elapids: a biochemical and pharmacological perspective. Crit Rev Toxicol 2008;38:73–86. [5] St Pierre L, Masci PP, Filippovich I, Sorokina N, Marsh N, Miller DJ, et al. Comparative analysis of prothrombin activators from the venom of Australian elapids. Mol Biol Evol 2005;22:1853–64. [6] Pearson JA, Tyler MI, Howden ME. Immunological relationships between the subunits of textilotoxin and rabbit antisera raised against textilotoxin and some snake venoms. Toxicon 1991;29:375–8. [7] Wickramaratna JC, Hodgson WC. A pharmacological examination of venoms from three species of death adder (Acanthophis antarcticus, Acanthophis praelongus and Acanthophis pyrrhus). Toxicon 2001;39:209–16. [8] O’Leary MA, Isbister GK. A turbidimetric assay for the measurement of clotting times of procoagulant venoms in plasma. J Pharmacol Toxicol Methods 2009 Jul 14. [Epub ahead of print] doi:10.1016/j.vascn.2009.06.004.
[9] O’Leary MA, Isbister GK, Schneider JJ, Brown SG, Currie BJ. Enzyme immunoassays in brown snake (Pseudonaja spp.) envenoming: detecting venom, antivenom and venom-antivenom complexes. Toxicon 2006;48:4–11. [10] Isbister GK, Halkidis L, O’Leary MA, Whitaker R, Cullen P, Mulcahy R, et al. Human anti-snake venom IgG antibodies in a previously bitten snake-handler, but no protection against local envenoming. Toxicon 2009. Aug 5. [Epub ahead of print]. [11] Isbister GK, Woods D, Alley S, O’Leary MA, Seldon M, Lincz L. Endogenous thrombin potential as a novel method for the characterization of procoagulant snake venoms. In: Haematology Society of Australia and New Zealand Annual Conference. 2008. [12] Currie BJ. Snakebite in tropical Australia: a prospective study in the “Top End” of the Northern Territory. Med J Aust 2004;181:693–7. [13] Birrell GW, Earl S, Masci PP, de Jersey J, Wallis TP, Gorman JJ, et al. Molecular diversity in venom from the Australian brown snake, Pseudonaja textilis. Mol Cell Proteom 2005. [14] Isbister GK, O’Leary MA, Schneider JJ, Brown SG, Currie BJ. Efficacy of antivenom against the procoagulant effect of Australian brown snake (Pseudonaja sp.) venom: in vivo and in vitro studies. Toxicon 2007;49:57–67. [15] Isbister GK, Duffull SB, Brown SG. Failure of antivenom to improve recovery in Australian snakebite coagulopathy. QJM 2009;102:563–8. [16] Wagstaff SC, Sanz L, Juarez P, Harrison RA, Calvete JJ. Combined snake venomics and venom gland transcriptomic analysis of the ocellated carpet viper, Echis ocellatus. J Proteom 2009;71:609–23. [17] Wagstaff SC, Laing GD, Theakston RD, Papaspyridis C, Harrison RA. Bioinformatics and multiepitope DNA immunization to design rational snake antivenom. PLoS Med 2006;3:e184.
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Cross-neutralisation of Australian brown snake, taipan and death adder venoms by monovalent antibodies Geoffrey K. Isbister a,b,c,∗ , Margaret A. O’Leary a , Jessica Hagan b , Kearney Nichols b , Tammy Jacoby d , Kathleen Davern d , Wayne C. Hodgson e , Jennifer J. Schneider b a
Department of Clinical Toxicology and Pharmacology, Calvary Mater Newcastle Hospital, Newcastle, New South Wales, Australia School of Biomedical Science, University of Newcastle, Newcastle, New South Wales, Australia Tropical Toxinology Unit, Menzies School of Health Research, Charles Darwin University, Darwin, Australia d Western Australian Institute of Medical Research, Perth, Western Australia, Australia e Monash Venom Group, Department of Pharmacology, Monash University, Victoria, Australia b c
a r t i c l e
i n f o
Article history: Received 3 September 2009 Received in revised form 27 September 2009 Accepted 12 October 2009 Available online 29 October 2009 Keywords: Antivenom Cross-neutralisation Snake venom
a b s t r a c t An understanding of the cross-neutralisation of snake venoms by antibodies is important for snake antivenom development. We investigated the cross-neutralisation of brown snake (Pseudonaja textilis) venom, taipan (Oxyuranus scutellatus) venom and death adder (Acanthophis antarcticus) with commercial antivenoms and monovalent anti-snake IgG, using enzyme immunoassays, in vitro clotting and neurotoxicity assays. Each commercial antivenom bound all three venoms, and neutralised clotting activity of brown snake and taipan venoms and neurotoxicity of death adder venom. The ‘in-house’ monovalent anti-snake venom IgG raised against procoagulant brown snake and taipan venoms, did not neutralise the neurotoxic effects of death adder venom. However, they did cross-neutralise the procoagulant effects of both procoagulant venoms. This supports the idea of developing antivenoms against groups of snake toxins rather than individual snake venoms. Crown Copyright © 2009 Published by Elsevier Ltd. All rights reserved.
1. Introduction An understanding of cross-neutralisation of snake venoms by antibodies raised against different snake venoms is important for the development of monovalent and polyvalent antivenoms, used for the treatment of snake envenoming. With the current shortage of antivenoms in the rural tropics, most importantly in Africa [1], the ability to make large quantities of antivenom that is effective against many different species would be greatly beneficial. In addition, an examination of cross-neutralisation of the toxic effects of venoms by antibodies raised against different snakes will give insight into the similarity of venom components and potentially the biology and evolution of snake venoms. We have previously shown that there is cross-neutralisation of brown snake (Pseudonaja textilis) and tiger snake (Notechis scutatus) venoms by CSL tiger snake antivenom (TSAV) and brown snake antivenom (BSAV), respectively [2]. We suggested that this was most likely due to the commercial antivenoms, which are labelled as being “monovalent”, really being polyvalent and containing anti-
∗ Corresponding author at: Newcastle Mater Hospital, Edith St, Waratah, NSW 2298, Australia. Tel.: +612 4921 1211; fax: +612 4921 1870. E-mail addresses: [email protected], [email protected] (G.K. Isbister).
bodies to both snake venoms. However, the use of a specific ‘in house’ monovalent antibody suggested that there was at least partial cross-neutralisation between brown snake venom and tiger snake venom [2]. The most clinically important toxins found in Australasian elapid venoms are procoagulant toxins, which cause plasma to clot in vitro [3], and neurotoxins, which act presynaptically or postsynaptically [4]. Venoms from Australian brown snakes (Pseudonaja spp.), tiger snakes (Notechis spp.) and taipans (Oxyuranus spp.) contain potent prothrombin activators that have similar structures [5]. This structural similarity may be responsible for the cross-neutralisation of the venom-mediated clotting effects by monovalent antibodies. However, the neutralisation of other venom properties has not been shown definitively with monovalent antibodies. Venoms from these three groups of snakes also contain pre-synaptic neurotoxins and there appears to be crossneutralisation between these neurotoxins, at least between notexin in Notechis spp. venom and textilotoxin in P. textilis venom [6]. In contrast, death adder (Acanthophis spp.) venom contains mainly neurotoxins [7] and no procoagulant toxins, so is potentially useful for examining cross-neutralisation when compared to procoagulant Australian snake venoms. To further explore the cross-neutralisation of Australian snake venoms, we examined the cross-neutralisation of three representative venoms of Australian elapids by their respective commercial
0264-410X/$ – see front matter. Crown Copyright © 2009 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.10.055
G.K. Isbister et al. / Vaccine 28 (2010) 798–802
antivenoms (CSL Ltd.) and polyclonal monovalent antibodies raised against each of the venoms. 2. Materials and methods Eastern or common brown snake (P. textilis) venom, Taipan (Oxyuranus scutellatus) venom and Death Adder (Acanthophis antarcticus) venom were purchased from Venom Supplies (Tanunda, South Australia). Tetramethylbenzidine (TMB), 2,2 Azino-bis 3-ethylbenzothiazoline-6-sulfonic acid) [ABTS], bovine serum albumin (BSA) and rabbit anti-horse IgG (whole molecule) peroxidase conjugate were obtained from Sigma. All chemicals used were of analytical grade. The following commercial antivenoms were obtained from CSL Ltd.—brown snake antivenom (BSAV, batch # 0559-10201), death adder antivenom (DAAV, batch # 0557-07301) and taipan antivenom (TAV, batch # 0548-05601). All of the rabbit IgG antibodies were biotinylated using the EZ-Link biotinylation kit (Pierce) and Sulfo-NHS-LC biotin reaction system as described by the manufacturer. Fresh frozen human plasma was obtained from the Australian Red Cross. Aliquots (10 mL) were thawed at 37 ◦ C then spun at 2500 rpm for 10 min prior to use. Calcium (60 L 0.2 M CaCl2 per mL) was added immediately before use. Clotting studies were carried out in a medium of Tris buffered saline containing 0.5% BSA. All procedures were carried out at room temperature unless indicated otherwise. Animal experiments were approved by the animal ethics committee at the University of Western Australia for rabbit antibodies production and Monash University (SOBS-B) animal ethics committee for in vitro neurotoxicity studies. 2.1. Preparation of rabbit polyclonal monovalent antibodies Lyophilised brown snake venom, taipan venom and death adder venom were reconstituted in physiological saline at 1–2 mg/mL. Venom (80–400 g) was emulsified with an equal volume of complete Freund’s adjuvant (CFA) and injected subcutaneously into multiple sites on the hind legs of New Zealand white rabbits. Each rabbit was injected with venom from only one snake species. Four weeks after the first injection, rabbits were given a booster dose at the same concentration in incomplete Freund’s adjuvant (IFA). Two weeks after this booster, 10–20 mL of blood was collected from each rabbit. Rabbits were then boosted with venom in aqueous solution every 4 weeks and bled 2 weeks after each injection. The presence of reactive venom antibodies was tested by an indirect enzymelinked immunosorbent assay (ELISA) with the respective venom used as coating antigen. Polystyrene microtitre plates were coated with 50 L of venom (5 g/mL) per well in coating buffer (0.1 M carbonate buffer, pH 9.7) overnight at 4 ◦ C. After washing three times in wash buffer (PBS, pH 7.3 containing 0.05% TWEEN20), wells were blocked with 200 L blocking solution (2% BSA in PBS) for 1 h at room temperature. After one wash, 50 L of suitably diluted rabbit serum in sample buffer (0.1% BSA in PBS pH 7.3) was titrated by using two-fold serial dilutions and incubated for 1 h at room temperature. Plates were washed three times again followed by the addition of 50 L of anti-rabbit secondary HRP-conjugated antibody to detect the bound anti-snake venom antibodies. Enzymatic activity was measured with the addition of peroxide substrate solution (50 L per well of ABTS and 0.001% hydrogen peroxide in 0.01 M citrate buffer, pH 4.5). Activity was assessed after 15 min by absorbance measured at 405 nm with Victor 3 microplate reader (PerkinElmer). Rabbit serum samples from hyper-immunised rabbits, with antibody titres higher than 50,000 as determined by ELISA, were subject to antibody purification. Serum was diluted 1:5 in PBS and
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sterile filtered. The dilute serum was then purified by affinity purification by being passed through a Sepharose-Protein G column using a BioRad BioLogic liquid chromatography system. Upon elution of IgG antibodies using 0.1 M glycine, pH 2.7, fractions containing IgG as determined by absorbance at 280 nm, were immediately neutralised with 2 M Tris, pH 9.0 and dialysed against PBS for 24 h. For biotinylation, 1–2 mg of purified rabbit polyclonal IgG in PBS had the appropriate volume of 10 mM of Sulfo-NHS-Biotin solution added to give a 20-fold molar excess of biotin. The mixtures were incubated for 1 h on ice or 2 h at room temperature followed by dialysis against PBS to remove free biotin. Success of biotinylation of each rabbit polyclonal IgG was assessed by ELISA and Western blot against the respective venoms using streptavidin-HRP as a detecting agent. 2.2. Binding Studies: measurement of venoms with and without prior incubation with antivenoms and specific antibodies To investigate the binding to venom of commercial antivenoms (BSAV, TAV, DAAV) compared to specifically raised polyclonal monovalent rabbit IgG to each of the venoms (anti-brown snake venom, anti-death adder venom, anti-taipan venom) we used a competitive sandwich ELISA with pre-incubated mixtures of the anti-snake venom antibodies and venoms. The washing solution was 0.02% TWEEN 20 in PBS, the blocking solution 0.5% BSA in PBS and 96-well Greiner High Binding plates were used. Venom and antivenom mixtures were made up from 200 L of 10 ng/mL venom in blocking solution with 50 mU antivenom (or 13 g monovalent anti-snake IgG) in 200 L PBS and allowed to stand for 60 min before applying to wells. Wells were coated with monovalent anti-snake venom IgG (100 L of 1–2 g/mL in carbonate buffer) for 1 h at room temperature then at 4 ◦ C overnight. The plates were washed, blocking solution applied for 1 h and then washed again. The venom and antivenom/antibody mixture (100 L) was applied in triplicate for 1 h, the plates washed, and 100 L of 0.5 g/mL biotinylated anti-snake venom IgG in blocking solution applied. After a further hour, the plates were washed again, and a solution of 100 L of 2 g/mL streptavidin-horseradish peroxidase in blocking solution was applied. After a final wash, 100 L of TMB was added, followed by 50 L of 1 M H2 SO4 . Plates were then read at 450 nm on a BioTek ELx808 plate reader. Standard curves were fitted to a sigmoidal dose-response curve. 2.3. Clotting studies We investigated the ability of commercial antivenoms and the polyclonal anti-snake venom IgG to prevent venom-induced clotting using a previously described method [8]. In brief, 10 ng of venom and 50 mU of antivenom or 13 g of anti-snake venom IgG were mixed in a total volume of 100 L and placed in the wells of a 96-well microtitre plate at 37 ◦ C in a BioTek ELx808 (BioTek Instruments, Inc., Winooski, VT, USA) plate reader. Plasma (100 L) was then added to each well simultaneously with a multichannel pipette and the optical density monitored at 340 nm for up to 20 min. The lag time before the sharp rise in optical density upon clot formation was recorded as the clotting time. The concentration of venom in the clotting studies was 100 ng/mL of plasma because this is in the upper range of venom concentrations in brown snake envenomed patients we have previously measured [9]. Similarly, the amount of CSL antivenom used was equivalent to 500 mU/mL of plasma based on expected and measured concentrations in patients to be 250 mU/mL, 1500 mU/mL and 3000 mU/mL respectively for the administration of 1 vial of BSAV, DAAV and TAV [9,10].
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2.4. In vitro neurotoxicity studies Studies using commercial monovalent BSAV and DAAV, as well as in-house polyclonal anti-snake venom IgG to brown snake venom and death adder venom, were undertaken to examine whether they were able to neutralise the neurotoxic effects of common death adder venom. Chicks (4–10-day-old males) were killed by CO2 inhalation and exsanguination, and the two biventer cervicis muscles were removed from the back of the neck. Each muscle was attached to a wire tissue holder and placed in a 5 mL organ bath filled with physiological salt solution of the following composition (mM): NaCl, 118.4; NaHCO3 , 25; glucose, 11.1; KCl, 4.7; MgSO4 , 1.2; KH2 PO4 , 1.2 and CaCl2 , 2.5. The organ baths were bubbled with carbogen (95% O2 , 5% CO2 ) and maintained at a temperature of 33–34 ◦ C under a resting tension of 1 g. Motor nerves were indirectly stimulated every 10 s (0.2 ms duration) at supramaximal voltage using a Grass S88 stimulator. The tissues were equilibrated for 10–15 min after which d-tubocurarine (10 M) was added, and the subsequent abolition of twitches confirmed the selective stimulation of the motor nerves. The tissues were then washed repeatedly until twitch height was restored. CSL DAAV (3 U/mL), CSL BSAV (3 U/mL), anti-death adder venom IgG (150 g/mL) and anti-brown snake venom IgG (150 g/mL) were added to the bath and incubated for 10 min. Death adder venom (10 g/mL) was then added and left in contact with the tissue for 60 min.
2.5. Data analysis Data were analysed by non-linear regression using Prism 5.0 to fit the curve with the most appropriate model, including for standard curves. The in vitro neurotoxicity data were analysed by ANOVA using Prism 5.0 with statistical significance indicated by P < 0.05.
3. Results High titre anti-snake venom rabbit IgG were produced to each venom and assessed with ELISA and Western blot. The concentration of these antibodies was 5 mg/mL. Commercial antivenoms are formulated as 17% equine protein or 170 mg/mL, but the proportion of this which are active antibodies is not given.
Fig. 1. Suppression of binding of venom to an anti-snake venom IgG-coated plates by pre-incubated mixtures of each of the three venoms (brown snake venom, 2 ng, death adder venom, 2 ng, and taipan venom, 2 ng) with 50 mU of commercial antivenoms (BSAV, DAAV, TAV) or 13 g of ‘in-house’ anti-brown snake venom (antiBSV) IgG, anti-death adder venom (anti-DAV) IgG or anti-taipan venom (anti-TV) IgG.
3.2. Clotting studies Taipan venom resulted in a longer clotting time than brown snake venom consistent with it being known to be less potent [11]. All three commercial antivenoms were able to neutralise clotting activity of both brown snake venom and taipan venom (Tables 1 and 2). The anti-brown snake venom IgG was also able to neutralise clotting activity of both venoms. Although anti-taipan venom IgG was completely effective in neutralising the clotting effect of taipan venom, it was only partially effective in neutralising the clotting effect of brown snake venom, even at twice the concentration shown (Table 1, Fig. 2). The anti-death adder venom IgG had minimal effect on either. 3.3. In vitro neurotoxicity Death adder venom (1 g/mL) abolished indirect (i.e. nervemediated) twitches of the chick biventer cervicis nerve-muscle preparation (Fig. 3). This inhibitory effect was prevented by the prior addition of CSL DAAV (3 U/mL, n = 4), CSL BSAV (3 U/mL, n = 4) and anti-DA IgG antibodies (150 g/mL, n = 4) but not by the prior
Table 1 Clotting times for brown snake venom in plasma alone, with the three commercial antivenoms and the three in-house anti-snake venom IgGs. Antibodies
3.1. Binding studies. All three commercial antivenoms (BSAV, TAV, DAAV) prevented brown snake venom binding to the anti-brown snake venom IgG-coated plate, taipan venom binding to anti-taipan venom IgGcoated plate and death adder venom binding to the anti-death adder venom IgG-coated plate (Fig. 1). In contrast only anti-brown snake venom IgG interacted with brown snake venom, anti-taipan venom IgG with taipan venom and anti-death adder venom IgG with death adder venom, preventing the venom binding to the corresponding anti-snake venom IgG-coated plate (Fig. 1). For example, only anti-death adder venom IgG pre-mixed with death adder venom prevented the death adder venom binding to the antideath adder venom IgG-coated plates, but anti-death adder venom IgG did not prevent brown snake venom or taipan venom from binding to their respective anti-snake venom IgG-coated plates. However, there was some interaction between anti-taipan venom IgG and brown snake venom which partially prevented brown snake venom from binding to the anti-brown snake venom IgGcoated plate (Fig. 1).
Clotting time (s)
Amount (mU) Mass (g) Nil 0 Anti-brown snake venom IgG – Anti-death adder venom IgG – Anti-taipan venom IgG – BSAV 50 DAAV 50 TAV 50
0 13 13 17 69 35 74
140 >900 170 215 >900 >900 >900
Table 2 Clotting times for taipan venom in plasma alone, with the three commercial antivenoms and the three ‘in-house’ anti-snake venom IgGs. Antibodies
Clotting time (s)
Amount (mU) Mass (g) Nil 0 Anti-brown snake venom IgG – Anti-death adder venom IgG – Anti-taipan venom IgG – BSAV 50 DAAV 50 TAV 50
0 13 13 17 69 35 74
305 >900 335 >900 >900 >900 >900
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Fig. 2. Clotting curves for brown snake venom (dotted lines) and taipan venom (thick lines) with a range of concentrations of the anti-taipan venom IgG showing that anti-taipan venom IgG can completely neutralise taipan venom-induced clotting but only partially effect brown snake venom-induced clotting.
Fig. 3. Effect of death adder venom (1 g/mL, n = 4) alone or in the presence of CSL death adder antivenom [DAAV] (3 U/mL, n = 4), CSL brown snake antivenom [BSAV] (3 U/mL, n = 4), anti-death adder venom IgG [DA IgG] (150 g/mL, n = 4) or anti-brown snake venom IgG [BS IgG] (150 g/mL, n = 3) on indirect twitches of the chick biventer cervicis nerve-muscle preparation. (*p < 0.05, significantly different from venom alone, ANOVA).
addition of anti-brown snake venom IgG antibodies (150 g/mL, n = 3) [Fig. 3]. 4. Discussion The study confirms that the Australian commercial antivenoms (CSL Ltd.) that are labelled as “monovalent” are in fact mixtures (polyvalent) and all three antivenoms used in this study were able to bind to all three venoms, neutralise the procoagulant effects of taipan venom and brown snake venom, and prevent the neurotoxic effects of death adder venom. More interesting were the results between the anti-snake IgG and different venoms, because these antibodies were monovalent, having been raised in rabbits given only single venom for immunisation. Using the monovalent IgG antibodies, there was no cross-neutralisation between the mainly neurotoxic death adder venom and the mainly procoagulant brown snake venom, based on binding studies, clotting and neurotoxicity assays. However, there was cross-neutralisation between the procoagulant venoms, brown snake venom and taipan venom, shown by the binding and clotting studies.
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Anti-brown snake venom IgG was unable to prevent the neurotoxic effects of death adder venom and conversely anti-death adder venom IgG was unable to neutralise the clotting effects of both brown snake venom and taipan venom. This is consistent with the known effects of these venoms and the clinical effects of envenoming by the respective snakes. Death adder envenoming causes isolated neurotoxicity and it is the only snake to cause this pattern of effects in Australia [12]. Death adder venom contains no known procoagulant toxins. Although brown snake venom is known to contain pre-synaptic and post-synaptic neurotoxins, the largest component of the venom is a potent prothrombin activator [3,13]. In human patients, isolated venom-induced consumption coagulopathy is the major feature of envenoming [14]. It is therefore not surprising that anti-brown snake venom IgG and anti-death adder venom IgG show no or very little crossneutralisation with the death adder venom and brown snake venom, respectively. In contrast, there was cross-neutralisation between taipan venom and anti-brown snake venom IgG and conversely partial cross-neutralisation between brown snake venom and anti-taipan venom IgG. This cross-neutralisation is likely to be due to the close similarity of the brown snake venom and taipan venom prothrombin activators [5]. We have previously shown a similar cross-neutralisation between anti-brown snake venom IgY and tiger snake venom [2]. Even though the prothrombin activator in tiger snake venom lacks the human factor Va-like subunit present in brown snake and taipan venoms, the human factor Xa-like parts of the prothrombin activators in all three venoms share close sequence homology [5]. Envenoming by all three corresponding snakes causes venom-induced consumptive coagulopathy [15]. This cross-neutralisation between venoms for the procoagulant effect has some important implications for antivenom design and manufacture, and supports the recent approach of toxin specific immunotherapy [16,17]. These investigators identified important epitopes that would be representative of the multimeric snake venom metalloproteinases in Echis ocellatus venom and used these to make a single synthetic multiepitope DNA immunogen. Antibodies were then raised to this immunogen or epitope string which were shown to not only neutralise the haemorrhagic effect of E. ocellatus, but also cross-neutralise the haemorrhagic effect of Cerastes cerastes venom, another viper that contains similar haemorrhagic metalloproteinases [17]. The presence of cross-neutralisation between the prothrombin activators in Australasian elapid venoms suggests that antibodies raised to an epitope string developed from the prothrombin activators in these venoms could potentially be used to neutralise the procoagulant effects of all Australasian elapid venoms with prothrombinase activity. A limitation of the study was the lower protein concentration of the ‘in-house’ anti-snake IgG making it difficult to completely neutralise some effects. This may be the reason that anti-taipan venom IgG only partially neutralised the clotting effect of brown snake venom and a more concentrated solution in the well would have caused complete neutralisation. In the neurotoxicity experiments large amounts of both anti-death adder venom IgG and anti-brown snake venom IgG were used, but there was no cross-neutralisation of death adder venom-induced neurotoxicity with the anti-brown snake venom IgG.
Acknowledgements Funding: GKI is supported by an NHMRC Clinical Career Development Award ID300785. Conflicts of interest: None.
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