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Cross Neutralization of Afro-Asian Cobra and Asian Krait Venoms by a Thai Polyvalent Snake Antivenom (Neuro Polyvalent Snake Antivenom)

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Cross Neutralization of Afro-Asian Cobra and Asian Krait Venoms by a Thai Polyvalent Snake Antivenom (Neuro Polyvalent Snake Antivenom) Cross Neutralization of Afro-Asian Cobra and Asian Krait Venoms by a Thai Polyvalent Snake Antivenom (Neuro Polyvalent Snake Antivenom) Poh Kuan Leong1, Si Mui Sim1, Shin Yee Fung2, Khomvilai Sumana3, Visith Sitprija3, Nget Hong Tan2* 1 Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, 2 CENAR and Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, 3 Queen Saovabha Memorial Institute, Bangkok, Thailand Abstract Background: Snake envenomation is a serious public health threat in the rural areas of Asian and African countries. To date, the only proven treatment for snake envenomation is antivenom therapy. Cross-neutralization of heterologous venoms by antivenom raised against venoms of closely related species has been reported. The present study examined the cross neutralizing potential of a newly developed polyvalent antivenom, termed Neuro Polyvalent Snake Antivenom (NPAV). NPAV was produced by immunization against 4 Thai elapid venoms. Principal Findings: In vitro neutralization study using mice showed that NPAV was able to neutralize effectively the lethality of venoms of most common Asiatic cobras (Naja spp.), Ophiophagus hannah and kraits (Bungarus spp.) from Southeast Asia, but only moderately to weakly effective against venoms of Naja from India subcontinent and Africa. Studies with several venoms showed that the in vivo neutralization potency of the NPAV was comparable to the in vitro neutralization potency. NPAV could also fully protect against N. sputatrix venom-induced cardio-respiratory depressant and neuromuscular blocking effects in anesthetized rats, demonstrating that the NPAV could neutralize most of the major lethal toxins in the Naja venom. Conclusions/Significance: The newly developed polyvalent antivenom NPAV may find potential application in the treatment of elapid bites in Southeast Asia, especially Malaysia, a neighboring nation of Thailand. Nevertheless, the applicability of NPAV in the treatment of cobra and krait envenomations in Southeast Asian victims needs to be confirmed by clinical trials. The cross-neutralization results may contribute to the design of broad-spectrum polyvalent antivenom. Citation: Leong PK, Sim SM, Fung SY, Sumana K, Sitprija V, et al. (2012) Cross Neutralization of Afro-Asian Cobra and Asian Krait Venoms by a Thai Polyvalent Snake Antivenom (Neuro Polyvalent Snake Antivenom). PLoS Negl Trop Dis 6(6): e1672. doi:10.1371/journal.pntd.0001672 Editor: Janaka de Silva, University of Kelaniya, Sri Lanka Received February 6, 2012; Accepted April 22, 2012; Published June 5, 2012 Copyright: ß 2012 Leong 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. Funding: This study was funded by research grants UM-HIR J20001-73803 and UMRG 088/09HTM from the University of Malaya. 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. * E-mail: [email protected] Introduction envenomations are generally characterized by neurotoxic enven- oming [5]. The global figure of snake envenoming cases has been estimated Antivenom therapy is the only effective treatment for snake to be greater than 1.8 million annually, with an annual death toll envenomation. Monovalent antivenoms are raised with venom of more than 90,000. Most of the snake envenoming cases occurs from one particular species and hence generally only effective in in South Asia and Southeast Asia (estimated 720,000 cases, 53,000 the treatment of envenomation caused by the particular species. fatality), followed by Africa (estimated 420,000 cases, 32,000 Because of the difficulties in accurate diagnosis of the biting fatality) [1], and the main biting species are snakes from the species, polyvalent antivenoms that offer paraspecific protection Elapidae and Viperidae families. Among members of the Elapidae against several venomous snake bites have also been developed family, the cobras and kraits are the main causes of snake and become commercially available. It has been argued that envenoming [2,3]. There are about 34 species of cobras belonging monovalent antivenoms are generally more effective than polyva- to 7 genera (Aspidelaps, Boulengerina, Hemachatus, Naja, Ophiophagus, lent antivenoms, though this has not been firmly established. At Pseudohaje and Walterinnesia). The genus Naja distributed extensively present, several types of polyvalent antivenoms against Afro-Asian across large regions of the Africa (13 species) and Asia (12 species) venomous snakes are available in the market, produced mainly by [4]. Ophiophagus hannah or commonly known as the king cobra, is Asian or African commercial pharmaceutical firms or government the only member of the Ophiophagus genus and is found only in institutions [5,6]. There is, however, a lack of rigorous evaluation Asia. Bungarus (the kraits), are represented by 12 species and their of the paraspecific protective actions of these commercially distribution is confined to the Indian subcontinent, Southeast Asia, available polyvalent antivenoms. Recently, Thai Red Cross as well as Southern China and Taiwan [4]. Cobra and krait Society produced a new polyvalent antivenom that offers www.plosntds.org 1 June 2012 | Volume 6 | Issue 6 | e1672 Cross Neutralization of Cobra Venoms Author Summary 31st, 2014), a purified F(ab9)2 obtained from serum of equines hyperimmunized specifically against the venom of Thai N. kaouthia. Snake envenomation is a serious public health threat in Both of these antivenoms are produced by Queen Saovabha the rural areas of Asia and Africa. To date, the only proven Memorial Institute (QSMI), the Thai Red Cross Society from treatment for snake envenomation is antivenom therapy. Bangkok, Thailand. For neutralization studies, both antivenoms Owing to the difficulties in the diagnosis of the biting were reconstituted in the same manner: 10 mL of normal saline species, there is a need to develop polyvalent antivenoms was added to 1 vial of the freeze-dried antivenom. According to that could cross-neutralize venoms of medically important the attached fact sheet, 1 mL of the NPAV antivenom is able to venomous snakes in the various regions. Recently, Thai neutralize the following amount of snake venoms: 0.6 mg each of Red Cross Society from Thailand has developed a new N. kaouthia and B. fasciatus venoms, 0.4 mg of B. candidus and polyvalent antivenom for treatment of cobra and krait 0.8 mg of O. hannah venoms; while 1 mL of the NKMAV can venoms. The polyvalent antivenom, termed ‘‘Neuro Poly- neutralize 0.6 mg of N. kaouthia venom. valent Snake Antivenom (NPAV),’’ is raised against venoms of two Thai cobras and two Thai kraits. Our results indicated that the polyvalent antivenom can effectively Animals neutralize venoms from many Southeast Asian cobras, Albino mice (ICR strain, 20–25 g) and male Sprague Dawley kraits and king cobra but is less effective against Indian rats (250–300 g) were supplied by the Laboratory Animal Centre, cobra venoms. Studies using anesthetized rats showed Faculty of Medicine, University of Malaya. The animals were that NPAV can effectively protect against cobra venom- handled according to the guidelines given by CIOMS on animal induced cardio-respiratory depressant and neuromuscular experimentation [8]. All experiments involving animals were blocking effects, confirming that the antivenom can approved by the Animal Care and Use Committee (ACUC) of the effectively neutralize the major lethal toxins of common University of Malaya (Ethical clearance letter No. PM/03/03/ cobra venoms. This new antivenom may find potential 2010/FSY(R)). application in the treatment of elapid bites in Southeast Asia, especially Malaysia, a neighboring nation of Thailand. Determination of protein content Protein content was determined by Bradford method [9]. All measurements were performed in triplicate. Bovine serum protection against neurotoxic envenomations by elapids in Thai- albumin (Sigma, USA) was use to generate a standard curve. land. This polyvalent antivenom, termed Neuro Polyvalent Snake Antivenom (abbreviated as NPAV) is raised against venoms of four Chromatographic and electrophoretic profiling of the medically important cobras and kraits in Thailand, i.e. Naja kaouthia (Thai monocellate cobra), Ophiophagus hannah (king cobra), antivenom Bungarus candidus (Malayan krait) and Bungarus fasciatus (banded SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was krait). In this paper, we report evaluation of the cross-neutralizing conducted according to the method of Studier [10], using the potential of NPAV against heterologous venoms of common Afro- Bio-Rad broad-range prestained SDS-PAGE standards (6.5– Asian cobras (Naja spp. and Ophiophagus hannah) and Asian kraits 200 kDa) and 15 mL of each antivenom sample (3 mg/mL) was (Bungarus spp.) We also compared the efficacy of the polyvalent loaded in the gel (12.5%). High performance gel filtration antivenom versus the relevant monovalent antivenom. The results chromatography of the reconstituted antivenom (100 mL, will provide preliminary information as to whether the polyvalent 10 mg/mL) was performed using a Superdex 200 HR 10/30, antivenom could find therapeutic application for cobra
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