An in vitro α-neurotoxin—nAChR binding assay correlates with lethality and in vivo neutralization of a large number of elapid neurotoxic snake venoms from four continents

Kritsada Pruksaphon; Kae Yi Tan; Choo Hock Tan; Pavinee Simsiriwong; José María Gutiérrez; Kavi Ratanabanangkoon

doi:10.1371/journal.pntd.0008581

Abstract

The aim of this study was to develop an in vitro assay for use in place of in vivo assays of snake venom lethality and antivenom neutralizing potency. A novel in vitro assay has been developed based on the binding of post-synaptically acting α-neurotoxins to nicotinic acetylcholine receptor (nAChR), and the ability of antivenoms to prevent this binding. The assay gave high correlation in previous studies with the in vivo murine lethality tests (Median Lethal Dose, LD₅₀), and the neutralization of lethality assays (Median Effective Dose, ED₅₀) by antisera against Naja kaouthia, Naja naja and Bungarus candidus venoms. Here we show that, for the neurotoxic venoms of 20 elapid snake species from eight genera and four continents, the in vitro median inhibitory concentrations (IC_50s) for α-neurotoxin binding to purified nAChR correlated well with the in vivo LD_50s of the venoms (R² = 0.8526, p < 0.001). Furthermore, using this assay, the in vitro ED_50s of a horse pan-specific antiserum against these venoms correlated significantly with the corresponding in vivo murine ED₅₀s, with R² = 0.6896 (p < 0.01). In the case of four elapid venoms devoid or having a very low concentration of α-neurotoxins, no inhibition of nAChR binding was observed. Within the philosophy of 3Rs (Replacement, Reduction and Refinement) in animal testing, the in vitro α-neurotoxin-nAChR binding assay can effectively substitute the mouse lethality test for toxicity and antivenom potency evaluation for neurotoxic venoms in which α-neurotoxins predominate. This will greatly reduce the number of mice used in toxicological research and antivenom production laboratories. The simpler, faster, cheaper and less variable in vitro assay should also expedite the development of pan-specific antivenoms against various medically important snakes in many parts of the world.

Author summary

Snakebite envenomation is an important public health problem recognized by the World Health Organization (WHO) as a neglected tropical disease affecting about 2 million of poor people of the tropical world. The most effective therapy is the timely administration of efficacious antivenoms which are usually produced in horses. The serum/plasma of horse immunized with snake venoms is purified and tested for its efficacies in neutralizing the target venoms. The neutralization is assayed using mice injected with the venom together with the antivenom. This assay requires about 60 mice for each pair of venom and antivenom. The assay is expensive, laborious, giving highly variable results and is objected on ethical and religious grounds. The present study involves the development of an in vitro assay involving the binding of a snake neurotoxin to a soluble receptor protein called nicotinic acetylcholine receptor. It is shown here that this receptor binding assay gave good correlation with the assay using mice. The test tube assay is simpler, faster, cheaper and less variable when compared with the mouse assay and thus could reduce or even replace the use of life animal. Furthermore, it could expedite the development of effective antivenoms against various venomous snakes in many parts of the world.

Citation: Pruksaphon K, Tan KY, Tan CH, Simsiriwong P, Gutiérrez JM, Ratanabanangkoon K (2020) An in vitro α-neurotoxin—nAChR binding assay correlates with lethality and in vivo neutralization of a large number of elapid neurotoxic snake venoms from four continents. PLoS Negl Trop Dis 14(8): e0008581. https://doi.org/10.1371/journal.pntd.0008581

Editor: Stuart Robert Ainsworth, Liverpool School of Tropical Medicine, UNITED KINGDOM

Received: May 22, 2020; Accepted: July 9, 2020; Published: August 28, 2020

Copyright: © 2020 Pruksaphon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper.

Funding: This study was funded by a research grant (no. IM 2011-01 to KR) from the Chulabhorn Research Institute and a research grant from the University of Malaya grant no. ST032-2019 to CHT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Snakebite envenomation is an important public health problem recognized by the World Health Organization (WHO) as a neglected tropical disease [1]. It has been estimated that 2 million people in the tropical world suffer these envenomations, resulting in about 20,000–94,000 fatalities annually [2]. The only effective treatment is the timely administration of antivenom. However, currently, effective antivenoms are not widely available and/or affordable in many parts of the world, especially in impoverished rural settings of sub-Saharan Africa and parts of Asia and Latin America [3]. A growing awareness on the impact of these envenomations has led to several initiatives by the WHO and diverse stakeholders in order to develop effective strategies for the prevention and control of this disease. A global strategy was developed, under the coordination of the WHO, aimed at reducing the impact of snakebite envenomations [4, 5]. One of the centerpieces of this strategy is the improvement of antivenom supply and access.

Antivenoms are usually produced by immunization of large animals, e.g. horses, donkeys or sheep with venom(s) of snakes inhabiting the country or region in which the antivenom is intended for use. After a few booster immunizations, the serum/plasma of the animals is obtained and fractionated to give either whole IgG or F(ab’)₂ formulations [6]. The resulting preparation is then subjected to various quality control tests before being certified for use in the treatment of snakebite victims. The gold standard in the assessment of the preclinical efficacy of antivenoms is the neutralization of the lethal effect of venoms [6].

The antivenom potency assay requires, initially, the estimation of the Median Lethal Dose (LD₅₀) of the venom(s) under study. This is followed by the neutralization of lethality assay, which is expressed as the Median Effective Dose (ED₅₀) using in vivo murine assay, as recommended by the WHO [6]. In such tests, large number of mice are required. For example, 374 mice were needed per batch to assess venom LD₅₀ and antivenom ED₅₀ against five snake venoms [7], and 2,020 mice were used for testing the efficacy a pan-specific antiserum against 27 elapid venoms [8]. The routine testing of antivenom efficacy in quality control laboratories of manufacturers and regulatory agencies therefore demands a huge number of mice. Moreover, as new therapeutic alternatives are developed, such as new generation ‘synthetic’ antivenoms or chemical inhibitors, the need to validate these novel options requires the testing of their ability to neutralize the lethal effect of venoms [9–11].

There is a general ethical concern regarding this type of animal-based tests, since venoms inflict pain and distress to the animals. In various countries with a strong Buddhist tradition, and where snakebite envenomation is an important medical problem, doing experiments involving the killing of animals is largely prohibited. The 3Rs (Replacement, Reduction and Refinement) rationale in the use of experimental animals urges the development of in vitro tests that would substitute in vivo toxicity assays [12]. Various in vitro assays have been studied with the aim of reducing or replacing the in vivo murine assays. They include, among others, enzyme immunoassays [13–17], inhibition of in vitro coagulant effect [18], and inhibition of phospholipase A₂ activity [19]. Moreover, the use of non-sensate fertilized chick embryo [20–22] and the use of ex vivo pharmacological models, such as isolated chick biventer cervicis [23] or isolated rat hemidiaphragm preparations [24, 25] have been also reported for assessing venom effects and neutralization by antivenoms. In the case of chick embryo, it is useful to assess toxicity of cytotoxic and hemotoxic venoms, but not of neurotoxic venoms since the six days embryo has not developed the target neuronal receptors.

The isolated nerve-muscle preparation has been very useful in the study of neurotoxic and myotoxic activities of venoms and purified toxins, and the ability of antivenoms to neutralize these effects [26–30]. However, few of these studies have correlated the results on nerve-muscle preparations with the in vivo lethality tests [29]. Despite their usefulness to study the action of neurotoxic venoms and toxins, and their neutralization by antivenoms from a research perspective, from a practical standpoint, these assays are technically difficult and time-consuming to set up in quality control laboratories for the routine assessment of antivenom efficacy.

More recently, an in vitro assay has been developed and is based on the high-affinity binding of snake postsynaptic α- neurotoxins to solubilized, purified nicotinic acetylcholine receptor (nAChR) [31, 32]. Since many elapid venoms exert their toxicity by binding to nAChR, hence causing neuromuscular blockade [33], this assay represents an in vitro correlate of the main mechanism of action of α-neurotoxin-rich venoms. The assay has been shown to give good correlation with in vivo estimation of LD₅₀ of venoms and of ED₅₀ of antivenoms when confronted with the venoms of the elapids Naja kaouthia (Thailand) [31], Bungarus candidus (Thailand) and Naja naja (Sri Lanka) [32].

As a follow up of these previous findings, we have expanded the analysis of correlation of this in vitro assay with the lethality of 20 neurotoxic elapid venoms, and also with the assessment of the neutralizing ability of a pan-specific elapid antiserum effective against 20 neurotoxic venoms belonging to 8 genera from 4 continents [34]. Our findings show that these in vitro assays give good correlation with both lethality and neutralization of lethality in vivo tests. This opens the possibility of using these assays in the assessment of antivenom efficacy in the case of neurotoxic venoms whose toxicity is predominantly based on post-synaptically acting α-neurotoxins.

Materials and methods

Chemicals and biochemicals

Chemicals and biochemical were obtained from Sigma Chemicals Co, St. Louis, Missouri, USA, unless otherwise indicated.

Venoms, T. californica nAChR and horse pan-specific antiserum

Lyophilized venoms of Naja siamensis (Thailand), Naja sputatrix (Indonesia), Naja philippinensis (Philippines), Naja atra (Taiwan), Naja melanoleuca (Uganda), Naja nigricollis (Cameroon), Naja haje (Egypt), Naja senegalensis (Mali), Dendroaspis angusticeps (Tanzania), Dendroaspis viridis (Ghana) and Dendroaspis polylepis (Kenya) were purchased from Latoxan (Valence, France). The venoms of Notechis scutatus (Australia), Pseudechis australis (Australia), Oxyuranus scutellatus (Australia) and Laticauda colubrina (Bali, Indonesia) were obtained from Venom Supplies Pty Ltd. (Australia). Bungarus multicinctus venom (China) was from Yiwu City Jiashang Import & Export Co., Ltd., Zhejiang, China. Bungarus candidus (Indonesia) venom was from BioPharma, Bandung, Indonesia.

The venoms of Malayan Peninsula elapids including Naja kaouthia (Malaysia), Naja sumatrana (Seremban, Malaysia), Ophiophagus hannah (Seremban, Malaysia) and Hydrophis schistosus (Penang, Malaysia) were milked from adult snakes in the wild by Dr. Choo Hock Tan. Venoms of the wild caught specimens of Naja oxiana and Naja naja (both from Pakistan) were kind gifts from Dr. Naeem Quraishi. Micrurus nigrocinctus venom (Costa Rica) was provided by Prof. José María Gutiérrez. The venoms of Naja kaouthia (Thailand) pooled from several adult snakes of Thai origin were purchased from Queen Saovabha Memorial Institute (QSMI), The Thai Red Cross Society. The main Naja kaouthia postsynaptic neurotoxin 3 (NK3) was purified according to Karlsson et al [35].

Torpedo californica electroplaque was from Aquatic Research Consultant, California, USA. Nicotinic acetylcholine receptor (nAChR) from T. californica electroplaque was solubilized, extracted and purified as described by Lindstorm et al [36]. The anti-nAChR antisera were generated in rats as described by Ratanabanangkoon et al [31]. The pan-specific antiserum was prepared from horses immunized with 12 elapid toxin fractions/venoms as previously described [8].

Estimation of the Median Lethal Dose (LD₅₀) of neurotoxic venoms

The median lethal dose (LD₅₀) of each of the 24 neurotoxic venoms was determined by intravenous (i.v.) injection in ICR mice (20–30 g, n = 4 per dose). The survival ratio was recorded after 24 h and LD₅₀ was calculated using Probit analysis method, with variation depicted by the 95% confidence limits [37].

In vivo neutralizing activity of horse pan-specific antiserum against various neurotoxic venoms

Neutralization of venom lethality by the pan-specific antiserum in mice was carried out as described previously [8]. Briefly, each venom was prepared in a volume of 50 μl 0.15 M NaCl (saline solution) to give a challenge dose of the venom corresponding to 5 x LD₅₀ (or 2.5 x LD₅₀ or 1.5 x LD₅₀ depending on the venom). In the absence of the antiserum, these doses killed all the injected mice. The venom solution was then incubated with varying dilutions of the pan-specific antiserum using saline as diluent, to give a total volume of 250 μl. After incubation at 37°C for 30 minutes, the venom-antiserum mixtures were injected into the caudal vein of mice (20–30g, n = 4–5). The number of dead/alive mice was recorded after 24 h and ED₅₀ was calculated using Probit analysis method, with variation depicted by the 95% confidence limits. The Median Effective Dose (ED₅₀) of the antiserum against the venom was determined as the volume of antiserum (μl) that protected 50% of the challenged mice tested.

In vitro nAChR binding assay

The basic assay conditions for the binding of purified nAChR to α-neurotoxin NK3 immobilized in microtiter plates (Polystyrene High Binding 3590, Costar), together with the optimal concentrations of reagents, was performed as described previously [31]. This assay involves first the addition of purified, solubilized nAChR to microtiter plates coated with neurotoxin NK3. Under the concentration and conditions used, the nAChR could bind maximally i.e., 100% to the NK3 coated plate. This nAChR binding is inhibited by elapid venoms containing α-neurotoxins. The in vitro Median Inhibitory Concentrations (IC₅₀) of a venom is the concentration at which the nAChR binding was inhibited by 50%. Various concentrations of each venom were incubated with a predetermined optimal concentration of purified nAChR (0.930 μg/ml) at 25°C for 60 minutes. The mixture was then transferred to NK3 coated plates. Any unbound nAChR was washed off with 0.05% Tween 20 in phosphate buffer saline pH 7.2 and the amount of bound nAChR was determined by adding rat-anti-nAChR serum (1: 1,600), goat anti-rat IgG-HRP conjugate (Abcam) (1: 4,000) and TMB/H₂O₂ enzyme substrate (BioFX Laboratories, MN, USA). Absorbances at 450 nm were then recorded with microplate spectrophotometer (Multiskan Go, Thermo Scientific). The result was converted to percent binding of the nAChR to the plate [31, 32]. The concentration of the venom that reduced the nAChR binding by 50% corresponded to the IC₅₀. The IC₅₀ values were reported as mean ± standard deviation (n = 3).

Neutralization of venoms by antiserum using the nAChR binding assay

In the in vitro assay of antiserum neutralization of neurotoxins, a fixed amount of each venom (corresponding to 5 x IC₅₀ or 2.5 x IC₅₀ or 1.5 x IC₅₀) which could completely inhibit the binding of nAChR to the NK3 coated plate is used. The venom was pre-incubated with various volumes (0.078 μl–2.5 μl) of the pan-specific antiserum, and saline solution was added to maintain a constant final volume. Mixtures were incubated at 25°C for 90 min in a total volume of 480 μl. After this incubation, the antibodies, both free and bound to the venom toxins, were removed by ultrafiltration through 100 kDa MWCO ultrafiltration membranes (Amicon). The filtrates (126 μl), containing free α-neurotoxins, were then allowed to react with an optimal amount of nAChR (14 μl) at 25°C for 1 hr. The reaction mixtures were then added to NK3 coated microtiter plates. The amount of bound nAChR was then determined as described above for IC₅₀ determination. The dose-response curves of horse serum volumes versus percent of nAChR binding were constructed. The in vitro neutralizing activities (ED_50s) corresponded to the volumes of horse antiserum at which the nAChR binding was inhibited by 50% compared to wells incubated with non-immune horse serum in place of antiserum. The results were reported as mean ± standard deviation (n = 3).

Miscellaneous procedures

The method described by Lowry et al [38] and the Bicinchoninic acid (BCA) Protein assay Kit (Pierce) were used to determine protein concentration, using bovine serum albumin as standard. GraphPad Prism 5.0 program was used in the calculation of in vitro IC₅₀ and ED₅₀ values and in generating the curves. Correlation analysis was made by linear regression with GraphPad Prism 5.0 software. The correlation coefficient was determined from the linear regression model; R² is the square of the correlation coefficient. An R² of 0.8–1.0 indicates a strong correlation between the two variables. The statistical significance of the correlation test was set at p <0.05.

Ethics approval

The animal experiments in mice were carried out according to the guidelines of the Council for International Organizations of Medical Sciences (CIOMS) and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Malaya (Ethical clearance No. 2016-190607/PHAR/R/TCH).

Results

The contents of α-neurotoxins in the elapid snake venoms studied

The 24 venoms analyzed in this study (Tables 1, 2 and 3) were from neurotoxic snakes of the family Elapidae belonging to 10 genera from 4 continents. Almost all of them are WHO category 1 most medically important snakes in their native countries or regions. Only two snakes, i.e., Ophiophagus hannah and Micrurus nigrocinctus are in WHO category 2 of less medically important snakes, although they have caused fatalities in humans. Hydrophis schistosus is a sea snake, and Laticauda colubrina is a sea krait. Twenty of these neurotoxic venoms have been shown by proteomics, biochemical and/or pharmacological studies to contain α-neurotoxins, being largely devoid of β-neurotoxins (Table 1), whereas others are known to contain α-neurotoxins, β-neurotoxins and other lethal toxins (Table 2) [39–42]. Four venoms (Naja nigricollis, Oxyuranus scutellatus, Dendroaspis angusticeps and Pseudechis australis) contain very low or no α-neurotoxins (Table 3).

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Table 1. In vivo toxicity of Naja spp. and Ophiophagus hannah venoms and neutralization by the pan-specific antiserum.

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Table 2. In vivo toxicity of non-cobra/king cobra venoms and neutralization by the pan-specific antiserum.

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Table 3. In vivo toxicity of venoms containing very low amounts or devoid of α-neurotoxins (no binding to the nAChR in the in vitro assay).

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Lethality assay and the inhibition of nAChR binding by α-neurotoxins of various elapid venoms

Fig 1 shows the results of the inhibition of nAChR binding by α-neurotoxins in Naja philippinensis venom. When the concentration of the venom increases, more nAChR becomes occupied by the α-neurotoxins of the venom, and the binding of nAChR to the NK3 coated plate decreases. The 50% inhibition of nAChR binding (IC₅₀) of N. philippinensis venom, as determined by regression, was 0.095 μg/ml.

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Fig 1. Determination of in vitro Median Inhibitory Concentration (IC₅₀) of N. philippinensis venom in the assay of nAChR binding.

Purified nAChR was incubated with various concentrations of N. philippinensis venom, and the mixture was added to plates coated with the neurotoxin NK3. The plate-bound nAChR was then detected with rat anti-nAChR antibody, followed by the addition of anti-rat IgG-HRP conjugate (see Materials and Methods for details). Results are presented as mean ± S.D. (n = 3).

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For the antiserum neutralization of the in vitro inhibition of nAChR binding, a dose of N. philippinensis venom corresponding to 2.5 x IC₅₀, i.e. 0.237 μg/ml, was used. The venom solutions were incubated with varying volumes of antiserum (0.078 to 2.5 μl), as described in the methods and the binding of nAChR to the NK3 coated plate was then assessed. The volume of the antiserum that resulted in 50% inhibition of nAChR binding is the in vitro ED₅₀ of the antiserum, in this case corresponding to 0.836 μl (Fig 2).

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Fig 2. Determination of in vitro Median Effective Dose (in vitro ED₅₀) of the pan-specific antiserum against N. philippinensis venom.

Venom was incubated with various dilutions of pan-specific antiserum. After an ultrafiltration step, the filtrate was incubated with nAChR, and then added to the plates coated with NK3 neurotoxin. The plate-bound nAChR was detected by addition of rat anti-nAChR antibody (see Materials and Methods for details). Results are presented as mean ± S.D. (n = 3).

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Following these methodologies, the results of the in vivo LD_50s and the in vitro IC_50s of the 20 neurotoxic venoms are shown in Tables 1 (no.1-12) and 2 (no.13-20). The most lethal venom was Hydrophis schistosus (LD₅₀ of 0.07 μg/g), whereas the least potent venoms were Naja sputatrix (Indonesia) and Naja kaouthia (Malaysia) with LD₅₀ of 0.90 μg/g. Bungarus multicinctus from China also possessed potent venom with LD₅₀ at 0.014 μg/g, while that of Bungarus candidus from Indonesia was 0.11 μg/g.

The in vitro inhibition of the nAChR binding to NK3 coated on the microtiter plate, expressed as IC₅₀ values of various elapid venoms are shown in Tables 1 and 2. The Naja haje (Egypt) venom showed the highest activity (IC₅₀ = 0.0653μg/ml), whereas the least active venom was Naja oxiana (Pakistan) (IC₅₀ = 0.7243 μg/ml).

The correlation between the in vivo LD_50s and the in vitro IC_50s for the 20 venoms studied is shown in Fig 3A. The correlation R² is 0.8526 (p < 0.001) which is statistically significant. The 20 neurotoxic venoms tested can be divided into two groups, according to their relative content of α-neurotoxins: (a) One group that contains mainly α-neurotoxins without pre-synaptically active β-neurotoxins (the ‘Naja spp.’ Group). This group consists of 11 venoms from Naja species. In addition, we included the venom of O. hannah within this group owing to its similar pharmacological and toxin profiles as that of other venoms in this group (Table 1). (b) The other group includes venoms that contain highly lethal pre-synaptic β-neurotoxins in addition to α-neurotoxins (the ‘non-Naja spp’ group) (Table 2). The correlation between the in vivo LD_50s and the in vitro IC_50s of the ‘Naja spp.’ venoms gives a correlation R² of 0.912 (p <0.001) which is statistically significant (Fig 3B). The corresponding correlation of the ‘non-Naja spp.’ group is slightly lower at a R² value of 0.718 (p < 0.0079), which is also statistically significant (Fig 3C).

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Fig 3. The correlation plots between the in vivo lethality (LD₅₀) and the in vitro nAChR binding inhibition (IC₅₀) of various neurotoxic venoms.

(3A) all 20 neurotoxic venoms; (3B) 12 ‘Naja spp.’ venoms; (3C) 8 ‘non-Naja spp.’ venoms. The ‘Naja spp.’ venoms are denoted by (●) while the ‘non-Naja spp.’ venoms are denoted by (○). The identities of the ‘non-Naja spp.’ snakes are: #1, M. nigrocinctus; #2, D. viridis; #3, N. scutatus; #4, B. multicinctus; #5, B. candidus; #6, H. schistosus; #7, L. colubrina; #8, D. polylepis.

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The in vivo and in vitro assays of neutralization by pan-specific antiserum against the 20 neurotoxic venoms

Tables 1 and 2 show the in vivo ED_50s, i.e. the neutralization of lethal effect, of the horse pan-specific antiserum against the 20 neurotoxic venoms. The antiserum was shown to neutralize the lethality of all the 20 venoms with different degrees of effectiveness. The in vitro potency assays of the pan-specific antiserum against these 20 venoms (in vitro ED_50s) are shown in Tables 1 and 2. The correlation plot between the in vivo ED_50s and the in vitro ED_50s of the 20 venoms, shown in Fig 4A, gives the R² of 0.689 (p < 0.01) which was statistically significant. When the 20 neurotoxic venoms were divided into the ‘Naja spp.’ and ‘non-Naja spp’ groups, the correlation between the in vivo and in vitro antiserum potency against the ‘Naja spp.’ group was 0.950 (p < 0.001) which is statistically highly significant (Fig 4B). The corresponding correlation for the ‘non-Naja spp.’ group which consists of 6 genera is lower at 0.671 (p < 0.0128) but still remains statistically significant (Fig 4C). Two venoms, those of B. multicintus (#4) and B. candidus (#5) seemed to deviate from the correlation line of other venoms in the plot (Fig 4A and 4C).

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Fig 4. The correlation plots between the in vivo lethality neutralization potency (ED₅₀) and the in vitro inhibition of nAChR binding (ED₅₀) by a pan-specific antiserum.

(4A) all 20 neurotoxic venoms; (4B) 12 ‘Naja spp.’ venoms; (4C) 8 ‘non-Naja spp.’ venoms. The ‘Naja spp.’ venoms are denoted by black triangles while the ‘non-Naja spp.’ venoms are denoted by white triangles. The identities of the ‘non-Naja spp.’ venoms are: #1, M. nigrocinctus; #2, D. viridis; #3, N. scutatus; #4, B. multicinctus; #5, B. candidus; #6, H. schistosus; #7, L. colubrina; #8, D. polylepis.

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In vitro nAChR binding of neurotoxic venoms containing low or no α-neurotoxins

There are 4 neurotoxic venoms (no. 21–24 in Table 3) that failed to inhibit the binding of nAChR to the NK3 coated plate in the in vitro IC₅₀ assay. These venoms were N. nigricollis (Cameroon), P. australis (Australia), O. scutellatus (Australia) and D. angusticeps (Tanzania) (Fig 5). Although the venoms of O. scutellatus and D. angusticeps showed a partial inhibition of about 20% of the nAChR binding, the in vitro IC_50s (and consequently, the in vitro ED_50s of the antiserum) of these four venoms could not be determined. These venoms have been shown by proteomics to contain no (i.e., P. australis and D. angusticeps) or low content of α-neurotoxins (N. nigricollis and O. scutellatus) [57–60] (Table 3). The venom of O. scutellatus or the coastal Taipan, contains approximately 1.5% of short-chain α-neurotoxins [57]. Fig 5 also shows the inhibition of D. polylepis venom, which contains α-neurotoxins [42, 56], and served as a positive control.

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Fig 5. Inhibition of nAChR binding to NK3 coated microtiter plate by various neurotoxic venoms with very low or no α-neurotoxins.

D. polylepis venom is used as a positive control since it contains α-neurotoxins. Results are presented as mean ± S.D. (n = 3).

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Discussion

To reduce the use of mice required for the assessment of venom toxicity (lethality) and antivenom neutralizing ability, the WHO urges the development of alternative in vitro tests that could substitute animal-based assays [6]. We report here an in vitro assay based on the α-neurotoxin-nAChR binding. The post-synaptically acting α-neurotoxins, which bind with high affinity to the nAChR, are abundant in many elapid venoms and play a key role in neurotoxic snakebite envenomation [33, 61]. We showed that this assay is highly useful to assess the ability of neurotoxic venoms in binding to solubilized, purified nAChR, and the capacity of antivenoms to inhibit this binding. By using a large number of venoms from a variety of elapid species, our results show that the two in vitro assays correlated well with the corresponding in vivo tests. Therefore, these in vitro assays could be adapted for research and quality control laboratories as convenient surrogate tests to assess the neurotoxic activity of post-synaptically acting venoms and its neutralization by antivenoms, hence reducing the large scale use of mice in these tests.

In vitro nAChR binding of venom α-neurotoxins and in vivo venom toxicity

It was shown that the IC_50s values of the 20 elapid venoms for the in vitro nAChR binding assay correlate well with the murine LD_50s of these venoms. This correlation was higher in the ‘Naja spp.’ group than that in the ‘non-Naja spp.’ group. The ‘Naja spp.’ group, consisting of eleven Naja spp. and O. hannah venoms, contain α-neurotoxins and cytotoxins, but no β-neurotoxins. The cytotoxins, also known as cardiotoxins, are less toxic than α-neurotoxins, with LD₅₀ values of 1.0–1.75μg/g in mice [45, 49, 62], and are usually involved in local tissue necrosis but not lethality in the victims. The high correlation observed with the ‘Naja spp.’ group is due to the fact that the α-neurotoxins are responsible for both the in vitro nAChR binding and in the in vivo lethality. This is not the case for the ‘non-Naja spp.’ group of venoms, which contain, in addition to α-neurotoxins, highly lethal β-neurotoxins (LD₅₀ about 10 ng/g mice) [63–66] and other lethal toxins which also contribute to lethality in mice.

The scattering of the data points in the correlation plot of the ‘non-Naja spp.’ group is probably attributed to the heterogeneity of the variety of toxins present in the venoms of this group, e.g. α-, β-, κ- neurotoxins in the B. candidus venom; dendrotoxins and fasiculins in the venoms of D. viridis and D. polylepis; and presynaptically-acting neurotoxic PLA₂s in several of these venoms, particularly in those of Bungarus sp. (Table 2). These non-α-neurotoxins play a role in the overall toxicity of the ‘non-Naja spp.’ venoms, hence explaining the lower correlation between LD₅₀ and IC₅₀ values. Nevertheless, the correlation is still significant, underscoring the role of α-neurotoxins in the lethal action of these venoms as well. In contrast, the nAChR binding assay is not useful in the case of venoms which lack, or have very low amount of α-neurotoxins (Table 3).

Regarding the interaction of α-neurotoxins and nAChR in the described in vitro assays, the following aspects deserve consideration:

Some of these elapid venoms contain short and/or long α-neurotoxins which exhibit different nAChR binding kinetics and affinity [67, 68] and could possibly affect the IC₅₀ determination. However, the experimental conditions of the assay using a 60 min pre-incubation time between the venom and nAChR, would allow for complete binding of α-neurotoxins, short or long, to the receptor.
b. Since the nAChR-α-neurotoxins interaction is highly specific and of high affinity, the assay may be used to detect the presence or absence of ‘functional’ α-neurotoxins in the venoms in terms of their ability to bind to nAChR. This allows the distinction between three-finger toxins that bind to the receptor from those that do not bind. For example, proteomics analysis of N. nigricollis venom found short chain α-neurotoxins to be only 0.4% of the venom protein and 73% of three-finger toxins of the cytotoxin type [58]. In agreement, this venom did not show binding to nAChR in our experiments (Fig 5 and Table 3).
The observation that the 20 neurotoxic elapid venoms inhibit the binding of nAChR to the NK3 coated plate indicates that α-neurotoxins of these venoms interact with high affinity to nAChR purified from T. californica. The fact that there is correlation between this in vitro assay using nAChR from ray electric organ, and lethality in mice, highlights the structural similarity between these receptors in these taxa, and the value of using ray receptor for assessing venom post-synaptic neurotoxicity. The immunological similarities between these receptors have been described [69].

Inhibition of in vitro nAChR binding by antiserum and in vivo neutralization of venom toxicity

When assessing the ability of the pan-specific antiserum to neutralize the elapid venoms, a highly significant correlation between the in vivo and in vitro results was found, especially for the ‘Naja spp.’ group. These observations underscore the key role of α-neurotoxins in the overall toxicity of the venoms, and the fact that inhibition of the toxins binding to nAChR abrogates their in vivo lethal activity.

The correlation between the in vitro and in vivo neutralization assays of the ‘non-Naja spp.’ group was lower than that in the case of the ‘Naja spp.’ group venoms. This is probably due to the heterogeneity of the lethal toxins present in these venoms as previously discussed in the lethality assays above. Furthermore, the amount of specific antibodies present in the pan-specific antiserum against these lethal components is likely to vary depending on the cross reaction of the antibodies against the 6 heterologous venoms used in the assay, as discussed below.

Interestingly, the plots of in vivo ED₅₀ vs. in vitro ED₅₀ against the two Bungarus venoms, i.e. B. candidus (Indonesia) and B. multicinctus (China), deviated from the correlation line. When these two Bungarus, venoms were excluded from the analysis, the correlation between the assays was quite high (R² = 0.9046, p < 0.001). Bungarus spp. venoms have a high concentration of the presynaptically-acting PLA₂ heterodimeric β-bungarotoxin [53, 54]. Hence, despite the fact that these venoms also contain α-neurotoxins, β-bungarotoxins are likely to play a dominant role in lethality, although they do not bind to nAChR. This may explain the deviation of these venoms in the correlation curve. For the majority of venoms tested, i.e. those of the ‘Naja spp.’ and ‘non-Naja spp.’ groups, the correlation observed in the neutralization of in vitro nAChR binding and the in vivo lethality support the contention that this in vitro assay could become a useful surrogate test to assess the neutralizing efficacy of antivenoms against these neurotoxic venoms. To the best of our knowledge, this nAChR binding assay is the first in vitro test to show a significant correlation with the in vivo mouse lethality test in the assessment of the neutralizing efficacy of antivenoms against elapid neurotoxic venoms. It would be relevant to expand these studies to other elapid venoms whose toxicity relies predominantly on the action of α-neurotoxins. In contrast, this assay is unsuitable in the case of venoms whose toxicity is based on toxins different from α-neurotoxins, such as those grouped in Table 3.

Advantages of the nAChR binding assay

By significantly reducing the use of mice, the in vitro assay used in this work fits within the 3Rs principles and, therefore, follows the trend proposed by the WHO for preclinical testing of antivenoms [6]. Furthermore, the assay should not encounter ethical and religious restrictions.
The in vitro assays are simple and easy to perform; one researcher or technician can handle dozens of venom and antivenom samples without difficulty. Thus, being high-throughput tests, they allow the assessment of many venom and antivenom samples within a couple of days.
The assays are much cheaper when compared to the in vivo mouse assays, owing to the high cost of mice and their maintenance.
Being an in vitro test, where parameters can be readily controlled, it shows less variability than the in vivo mouse lethality assay.
Although the final preclinical test of antivenom neutralizing efficacy will continue to be the mouse lethality neutralization assay, which is the gold standard of antivenom testing [6], the in vitro assay described may be used for other phases of antivenom production. These include in-process assessment of antivenom efficacy, and testing the samples of sera from immunized horses, in order to define whether a horse has achieved a satisfactory antibody response. These will greatly reduce the number of mice used in antivenom production laboratories. Likewise, this assay will allow the high throughput screening of novel inhibitors and recombinant antibodies against neurotoxic elapid venoms. In addition, the comparative analysis of nAChR binding by different venoms can help in the selection of venom doses to be used in in vivo lethality assays, again reducing the number of mice utilized. Taken together, these advantages will decrease animal suffering and will speed up the development of novel antivenoms and inhibitory substances.

Possible drawbacks of the in vitro nAChR binding assay

A possible drawback of this procedure lies in the fact that the interaction of α-neurotoxins with nAChR has been shown to be prey-selective and hence varies depending on the taxon of origin of the receptor (amphibian, lizard, snake, bird or rodent) [70,71]. Likewise, a venom like N. nigricollis, which showed little affinity for ray nAChR in our study, binds to amphibian receptor [70]. Thus, the selection of the source of nAChR should be carefully considered. Nevertheless, the fact that we observed a high correlation between the nAChR binding assay and the in vivo mouse lethality assay strongly suggests that nAChR from ray electric organ is a suitable model for assessing post-synaptic neurotoxicity of snake venoms.

The major hurdle of these in vitro assays is the need to have solubilized, purified nAChR and the rat anti-nAChR antibodies which are currently not commercially available. However, these reagents can be prepared using standard biochemical techniques [36]. Once these reagents are prepared, they are quite stable and can be used for a large number of samples since each assay requires only nanogram amounts of the receptor and sub-microliter volume of antiserum. A promising alternative is the use of mimotopes and peptides derived from nAChR which bind to α-neurotoxins [70, 72]. The introduction of these synthetic peptides, if shown to bind specifically and with high affinity to α-neurotoxins, in this type of assay will avoid the need to obtain the receptor from rays or eels.

The present in vitro assay is based on the interaction of the venom α-neurotoxins and the nAChR. Thus, the assay does not work for venoms whose toxicity is not based on the action of α-neurotoxins. Proteomic and toxicity score analysis of venoms [73] for identifying the most active neurotoxins will allow the identification of venoms where α-neurotoxins do not play a key role, and for which the in vivo lethality assay has to be used.

Acknowledgments

We are most grateful to Professor Arnold E. Ruoho and Dr Nicholas V. Cozzi from the University of Wisconsin, Madison, to Dr. James Dubbs of the Chulabhorn Research Institute, Professor Nget Hong Tan of the University of Malaya, to Mr Sutat Lapanan of the ANH Scientific Marketing Co., Ltd. and to Dr Chuck Winkler, Aquatic Research Consultant, California, for their valuable suggestions and assistance.

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Figures

Abstract

Author summary

Introduction

Materials and methods

Chemicals and biochemicals

Venoms, T. californica nAChR and horse pan-specific antiserum

Estimation of the Median Lethal Dose (LD50) of neurotoxic venoms

In vivo neutralizing activity of horse pan-specific antiserum against various neurotoxic venoms

In vitro nAChR binding assay

Neutralization of venoms by antiserum using the nAChR binding assay

Miscellaneous procedures

Ethics approval

Results

The contents of α-neurotoxins in the elapid snake venoms studied

Lethality assay and the inhibition of nAChR binding by α-neurotoxins of various elapid venoms

The in vivo and in vitro assays of neutralization by pan-specific antiserum against the 20 neurotoxic venoms

In vitro nAChR binding of neurotoxic venoms containing low or no α-neurotoxins

Discussion

In vitro nAChR binding of venom α-neurotoxins and in vivo venom toxicity

Inhibition of in vitro nAChR binding by antiserum and in vivo neutralization of venom toxicity

Advantages of the nAChR binding assay

Possible drawbacks of the in vitro nAChR binding assay

Acknowledgments

References

Estimation of the Median Lethal Dose (LD₅₀) of neurotoxic venoms