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Genus Calliophis of Asiatic coral snakes: A deficiency of venom cross-reactivity and neutralization against seven regional elapid antivenoms
Toxicon 121 (2016) 130e133
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Short communication
Genus Calliophis of Asiatic coral snakes: A deficiency of venom cross-reactivity and neutralization against seven regional elapid antivenoms Choo Hock Tan a, *, Jia Lee Liew a, Kae Yi Tan b, Nget Hong Tan b a b
Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2016 Received in revised form 26 August 2016 Accepted 6 September 2016 Available online 8 September 2016
Venoms of Calliophis bivirgata and Calliophis intestinalis exhibited moderate binding activities toward Neuro Bivalent Antivenom (Taiwan) but not the other six elapid monovalent or bivalent antivenoms available in the region. All antivenoms failed to neutralize C. bivirgata venom lethality in mice. The findings indicate the need to validate antivenom cross-reactivity with in vivo cross-neutralization, and imply that distinct antigens of Calliophis venoms should be incorporated in the production of a panregional poly-specific antivenom. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Calliophis bivirgata Calliophis intestinalis ELISA Antigen binding Antivenom Cross-neutralization
The genus Calliophis (Family: Elapidae) represents an early lineage of coral snakes that distributes in Asia (Slowinski et al., 2001). The sighting of these snakes is rare; however bites have been reported in humans, with mild local reactions (caused by banded or striped coral snake, Calliophis intestinalis) to uncommon fatal envenomation (by Malayan blue coral snake, Calliophis bivirgata) (Harrison, 1957; Ismail, 2015). The Malayan blue coral snake is classified under Category 2 of medically important venomous snakes by the World Health Organization (WHO, 2010), but there is no specific antivenom clinically available for treating Calliophis envenomation. Thus far, only the venom of C. bivirgata (subspecies: flaviceps) has been characterized functionally and proteomically (Takasaki et al., 1991; Tan et al., 2016a). The venom is lethal in mice with a median lethal dose (LD50) of 0.7 mg/g, and the venom lethality is not cross-neutralized by the major polyvalent antivenom used against neurotoxic elapid envenoming in the Southeast Asian region (Thai Neuro Polyvalent Antivenom, NPAV, raised against Thai king cobra, monocled cobra, banded krait, Malayan krait) (Tan et al., 2016a). Weak neutralization effect was noted in
* Corresponding author. E-mail addresses: [email protected], [email protected] (C.H. Tan). http://dx.doi.org/10.1016/j.toxicon.2016.09.003 0041-0101/© 2016 Elsevier Ltd. All rights reserved.
mice using the Taiwan Neuro Bivalent Antivenom (NBAV, raised against Taiwanese cobra and multi-banded krait), however further validation has been called for to firmly establish the therapeutic significance of the findings (Tan et al., 2016a). Meanwhile, the cross-reactivity of Calliophis venoms against different elapid antivenoms available in the region has not been fully investigated for potential cross-neutralization, which is important from the practical standpoint. In this experiment, venoms of Calliophis bivirgata (Pahang) and Calliophis intestinalis (Penang Island, Kedah, Pahang, Selangor) from the Malayan Peninsula were tested for their immunorecognition by antibodies of seven elapid antivenoms available in the Asia Pacific region: (1) Neuro Bivalent Antivenom, against Naja atra and Bungarus multicinctus of Taiwan (NBAV, batch: FN10101; expiry: April 2017); (2) Sea Snake Antivenom, against Hydrophis schistosus (formerly known as Enhydrina schistosa) of Peninsular Malaya and Notechis scutatus of Australia (SSAV, batch: 0549-08201; expiry: April 2015); (3) Bungarus multicintus Monovalent Antivenom, against the mainland Chinese species (BMMAV, batch: 20150101; expiry: January 2018); (4) Bungarus candidus Monovalent Antivenom, against the Thai species (BCMAV, batch: BC00112; expiry: June 2017); (5) Bungarus fasciatus Monovalent Antivenom, against the Thai species (BFMAV, batch: BK00112; expiry: March 2017); (6)
C.H. Tan et al. / Toxicon 121 (2016) 130e133
131
Fig. 1. Cross-reactivities of Calliophis bivirgata and Calliophis intestinalis venoms against different antivenoms: (a) Neuro Bivalent Antivenom, Taiwan; (b) Bungarus multicinctus Monovalent Antivenom, China; (c) Bungarus candidus Monovalent Antivenom, Thailand; (d) Bungarus fasciatus Monovalent Antivenom, Thailand; (e) Naja kaouthia Monovalent Antivenom, Thailand; (f) Ophiophagus hannah Monovalent Antivenom, Thailand; (g) Sea Snake Antivenom, Australia; (h) Calloselasma rhodostoma Monovalent Antivenom, Thailand. Percentages (%) were values of mean ± S.E.M. (triplicates), representing relative binding activities based on normalized values of absorbance (Abs). C rhodostoma venom served as the negative reference control in all assays testing with elapid antivenoms (aeg); its distinct immunoreactivity from elapid venom was verified using the homologous crotalid antivenom (h).
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C.H. Tan et al. / Toxicon 121 (2016) 130e133
Naja kaouthia Monovalent Antivenom, against the Thai species (NKMAV, batch: NK00514; expiry: October 2019); (7) Ophiophagus hannah Monovalent Antivenom, against the Thai species (OHMAV, batch: LH00112; expiry: June 2017). In brief, the protocol was optimized based on the indirect enzyme-linked immunosorbent assay (ELISA) reported previously from the same laboratory (Tan et al., 2012, 2015c; Yap et al., 2014), conducted in three independent experiments. For consistency and comparative purpose, the coating venoms were standardized to 10 ng venom proteins per well on immunoplate, and all antivenoms were prepared in a 1:6000 dilution from the respective antivenom stocks (20 mg/ml protein concentration) for optimal absorbance based on pilot titration finding. Venoms of the respective species corresponding to the antivenoms (sourced from the same geographical locales of the immunogens) were used as the positive reference controls for binding activity, while the venom of Malayan pit viper, Calloselasma rhodostoma (Malaysia) served as the negative reference control in all assays involving the seven elapid antivenoms. The distinct immunoreactivity of C. rhodostoma (negative reference control) from the elapid venom was demonstrated in a separate assay using the homologous C. rhodostoma Monovalent Antivenom (CRMAV, batch: CR00210; expiry: June 2015). All the elapid antivenoms used were subsequently screened for their in vivo neutralization capability where 2.5 LD50 (median lethal dose) of C. bivirgata venom was pre-incubated with the maximum (physiologically permissible) antivenom dose (200 ml reconstituted antivenom) at 37 C for 30 min prior to injection into the caudal vein of ICR albino mice (n ¼ 2) for each antivenom tested (Wong et al., 2016). The mice were subsequently monitored for toxic signs and fatality over 24 h. The use of laboratory mice and the experiment protocol were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Malaya (ethics code: 2014-09-11/ PHAR/R/TCH). The ELISA result revealed that both C. bivirgata and C. intestinalis venom proteins cross-reacted moderately against NBAV, charting a cross-reactivity of approximately one-half of NBAV binding to N. atra and B. multicinctus venoms, but the binding was significantly higher than that shown by the negative control, C. rhodostoma venom (p < 0.05) (Fig. 1a). The bivalent SSAV and the other five mono-specific elapid antivenoms exhibited essentially no effective binding toward both Calliophis venoms, evidenced by the comparable levels of cross-reactivity toward C. rhodostoma venom (p > 0.05) (Fig. 1beg). The binding activities toward the various antivenoms did not vary significantly between C. bivirgata and C. intestinalis. The moderate cross-reactivity of NBAV against the two coral snake venoms implies common antigenicity shared between the coral snake venoms with N. atra and/or B. multicinctus venoms, and this appears in agreement with the previous reported weak neutralization of C. bivirgata venom by this antivenom (Tan et al., 2016a). By examining the lack of cross-reactivity against BMMAV (Fig. 1b), it is probable that the conserved antigenicity concealed not within B. multicinctus venom but within N. atra venom, which contains a high amount of three-finger cytotoxins as with the C. bivirgata venom (Huang et al., 2015). Of note, the C. bivirgata venom is predominated with three-finger cytotoxins and phospholipases A2 but lacking three-finger neurotoxins (Tan et al., 2016a). The cross-reactivity against NKMAV was however deficient (Fig. 1e), possibly due to the low content of cytotoxins in Thai N. kaouthia venom used as the immunogen in manufacturing NKMAV (Tan et al., 2015d). The lack of cross-reactivity was also observed across all other mono-specific antivenoms against Bungarus species (Fig. 1bed), supporting that Calliophis venoms and Bungarus venoms did not share common antigenicity, despite the high abundance of phospholipases A2 in their venoms and their strict dietary preference on snakes (Rusmili et al., 2014; Tan and
Tan, 2015). This is also applicable to the ophiophagic king cobra (Fig. 1f): the lack of cross-reactivity against OHMAV is unsurprising in view of the deficiency of three-finger cytotoxins in king cobra venom (Tan et al., 2015a). The high content of cytotoxic L-amino acid oxidases in king cobra venom (Tan and Tan, 2016) neither did confer good cross-reactivity of OHMAV against C. intestinalis venom, although the enzyme is present substantially in C. intestinalis venom (unpublished). The Calliophis venoms also did not cross-react against SSAV (Fig. 1g), attributable to the lack of cytotoxins in sea snake and tiger snake venoms (Tan et al., 2015b, 2016b), while the finding also implies that the abundant phospholipases A2 in sea snake and tiger snake venoms do not share antigenicity with these enzymes in Calliophis venoms. Interestingly, the ELISA result also revealed a significantly lower binding activity of SSAV toward the targeted sea snake venom compared to N. scutatus venom (p < 0.05), consistent with the limitation of SSAV neutralization reported recently (Tan et al., 2016b). The antivenom cross-reactivity findings were further validated using an in vivo assay. Against a lethal challenge dose (2.5 LD50) of C. bivirgata venom, all tested antivenoms failed to protect the mice, with the animals noted to die in spasticity (convulsion-like feature with myoclonus followed by muscle spasm, curled front limbs and out-stretched hind limbs) within 1 min post-injection of the venom-antivenom mixtures. NBAV failed to neutralize the venom despite its moderate immunological cross-reactivity in the present study and the weak neutralization reported previously, presumably due to batch-to-batch differences in the antivenom and animals. This observation highlights the importance of supporting immunological binding study with functional or in vivo experiments, and the need for re-validation of batch variability of antivenom efficacy especially in cases of feeble neutralization. Together, the findings revealed that the venoms of the two Calliophis species from Malaysia exhibit distinct antigenicity from other elapid lineages, and the lack of cross-reactivity against the major antivenoms available in the region is evident. The use of non-specific antivenom in the clinical management of Calliophis envenoming is hence not supported even though in vitro findings may appear promising. Further study should be directed toward the incorporation of the unique antigens of Calliophis venoms in the production of an effective poly-specific antivenom envisioned for pan-regional use (Ratanabanangkoon et al., 2016; Williams et al., 2011). Acknowledgment This study was supported by Grants FP028-2014A and RG3522015B. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2016.09.003. References Harrison, J.L., 1957. The bite of blue Malaysian coral snake or ular matahari (Maticora bivirgata). Malay. Nat. J. 11, 130e132. Huang, H.W., Liu, B.S., Chien, K.Y., Chiang, L.C., Huang, S.Y., Sung, W.C., Wu, W.G., 2015. Cobra venom proteome and glycome determined from individual snakes of Naja atra reveal medically important dynamic range and systematic geographic variation. J. Proteomics 128, 92e104. Ismail, A.K., 2015. Snakebite and envenomation management in Malaysia. In: Gopalakrishnakone, P., Faiz, A., Fernando, R., Gnanathasan, C.A., Habib, A.G., Yang, C.-C. (Eds.), Clinical Toxinology in Asia Pacific and Africa. Springer, Netherlands, pp. 71e102. Ratanabanangkoon, K., Tan, K.Y., Eursakun, S., Tan, C.H., Simsiriwong, P., Pamornsakda, T., Wiriyarat, W., Klinpayom, C., Tan, N.H., 2016. A Simple and novel strategy for the production of a pan-specific antiserum against elapid snakes of Asia. PLoS Negl. Trop. Dis. 10, e0004565.
C.H. Tan et al. / Toxicon 121 (2016) 130e133 Rusmili, M.R., Yee, T.T., Mustafa, M.R., Hodgson, W.C., Othman, I., 2014. Proteomic characterization and comparison of Malaysian Bungarus candidus and Bungarus fasciatus venoms. J. Proteomics 110, 129e144. Slowinski, J.B., Boundy, J., Lawson, R., 2001. The phylogenetic relationships of Asian coral snakes (Elapidae: Calliophis and Maticora) based on morphology and molecular characters. Herpetol. J. 57, 233e245. Takasaki, C., Yoshida, H., Shimazu, T., Teruuchi, T., Toriba, M., Tamiya, N., 1991. Studies on the venom components of the long-glanded coral snake, Maticora bivirgata. Toxicon 29, 191e200. Tan, C.H., Fung, S.Y., Yap, M.K., Leong, P.K., Liew, J.L., Tan, N.H., 2016a. Unveiling the elusive and exotic: venomics of the Malayan blue coral snake (Calliophis bivirgata flaviceps). J. Proteomics 132, 1e12. Tan, C.H., Tan, K.Y., Fung, S.Y., Tan, N.H., 2015a. Venom-gland transcriptome and venom proteome of the Malaysian king cobra (Ophiophagus hannah). BMC Genomics 16, 687. Tan, C.H., Tan, K.Y., Lim, S.E., Tan, N.H., 2015b. Venomics of the beaked sea snake, Hydrophis schistosus: a minimalist toxin arsenal and its cross-neutralization by heterologous antivenoms. J. Proteomics 126, 121e130. Tan, C.H., Tan, K.Y., Tan, N.H., 2016b. Revisiting Notechis scutatus venom: on shotgun proteomics and neutralization by the “bivalent” Sea Snake Antivenom. J. Proteomics 144, 33e38. Tan, C.H., Tan, N.H., 2015. Toxinology of snake venoms: the Malaysian context. In: Gopalakrishnakone, P., Inagaki, H., Mukherjee, A.K., Rahmy, T.R., Vogel, C.-W. (Eds.), Snake Venoms. Springer, Netherlands, pp. 1e37. Tan, N.H., Tan, C.H., 2016. Cytotoxicity of snake venoms and toxins: mechanisms and applications. In: Utkin, Y.N., Krivoshein, A.V. (Eds.), Snake Venoms and
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Envenomation: Modern Trends and Future Prospects. Nova Science Publishers, New York, pp. 215e254. Tan, C.H., Tan, N.H., Sim, S.M., Fung, S.Y., Gnanathasan, C.A., 2012. Immunological properties of Hypnale hypnale (hump-nosed pit viper) venom: antibody production with diagnostic and therapeutic potentials. Acta Trop. 122, 267e275. Tan, C.H., Tan, N.H., Tan, K.Y., Kwong, K.O., 2015c. Antivenom cross-neutralization of the venoms of Hydrophis schistosus and Hydrophis curtus, two common sea snakes in Malaysian waters. Toxins (Basel) 7, 572e581. Tan, K.Y., Tan, C.H., Fung, S.Y., Tan, N.H., 2015d. Venomics, lethality and neutralization of Naja kaouthia (monocled cobra) venoms from three different geographical regions of Southeast Asia. J. Proteomics 120, 105e125. WHO, 2010. Guidelines for the Management of Snake-bites. World Health Organization: Regional Office for South-East Asia. Williams, D.J., Gutierrez, J.M., Calvete, J.J., Wuster, W., Ratanabanangkoon, K., Paiva, O., Brown, N.I., Casewell, N.R., Harrison, R.A., Rowley, P.D., O'Shea, M., Jensen, S.D., Winkel, K.D., Warrell, D.A., 2011. Ending the drought: new strategies for improving the flow of affordable, effective antivenoms in Asia and Africa. J. Proteomics 74, 1735e1767. Wong, K.Y., Tan, C.H., Tan, N.H., 2016. Venom and purified toxins of the spectacled cobra (Naja naja) from Pakistan: insights into toxicity and antivenom neutralization. Am. J. Trop. Med. Hyg. 94, 1392e1399. Yap, M.K.K., Tan, N.H., Sim, S.M., Fung, S.Y., Tan, C.H., 2014. Pharmacokinetics of Naja sumatrana (Equatorial spitting cobra) venom and its major toxins in experimentally envenomed rabbits. PLoS Negl. Trop. Dis. 8 (6), e2890. http:// dx.doi.org/10.1371/journal.pntd.0002890.
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Short communication
Genus Calliophis of Asiatic coral snakes: A deficiency of venom cross-reactivity and neutralization against seven regional elapid antivenoms Choo Hock Tan a, *, Jia Lee Liew a, Kae Yi Tan b, Nget Hong Tan b a b
Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2016 Received in revised form 26 August 2016 Accepted 6 September 2016 Available online 8 September 2016
Venoms of Calliophis bivirgata and Calliophis intestinalis exhibited moderate binding activities toward Neuro Bivalent Antivenom (Taiwan) but not the other six elapid monovalent or bivalent antivenoms available in the region. All antivenoms failed to neutralize C. bivirgata venom lethality in mice. The findings indicate the need to validate antivenom cross-reactivity with in vivo cross-neutralization, and imply that distinct antigens of Calliophis venoms should be incorporated in the production of a panregional poly-specific antivenom. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Calliophis bivirgata Calliophis intestinalis ELISA Antigen binding Antivenom Cross-neutralization
The genus Calliophis (Family: Elapidae) represents an early lineage of coral snakes that distributes in Asia (Slowinski et al., 2001). The sighting of these snakes is rare; however bites have been reported in humans, with mild local reactions (caused by banded or striped coral snake, Calliophis intestinalis) to uncommon fatal envenomation (by Malayan blue coral snake, Calliophis bivirgata) (Harrison, 1957; Ismail, 2015). The Malayan blue coral snake is classified under Category 2 of medically important venomous snakes by the World Health Organization (WHO, 2010), but there is no specific antivenom clinically available for treating Calliophis envenomation. Thus far, only the venom of C. bivirgata (subspecies: flaviceps) has been characterized functionally and proteomically (Takasaki et al., 1991; Tan et al., 2016a). The venom is lethal in mice with a median lethal dose (LD50) of 0.7 mg/g, and the venom lethality is not cross-neutralized by the major polyvalent antivenom used against neurotoxic elapid envenoming in the Southeast Asian region (Thai Neuro Polyvalent Antivenom, NPAV, raised against Thai king cobra, monocled cobra, banded krait, Malayan krait) (Tan et al., 2016a). Weak neutralization effect was noted in
* Corresponding author. E-mail addresses: [email protected], [email protected] (C.H. Tan). http://dx.doi.org/10.1016/j.toxicon.2016.09.003 0041-0101/© 2016 Elsevier Ltd. All rights reserved.
mice using the Taiwan Neuro Bivalent Antivenom (NBAV, raised against Taiwanese cobra and multi-banded krait), however further validation has been called for to firmly establish the therapeutic significance of the findings (Tan et al., 2016a). Meanwhile, the cross-reactivity of Calliophis venoms against different elapid antivenoms available in the region has not been fully investigated for potential cross-neutralization, which is important from the practical standpoint. In this experiment, venoms of Calliophis bivirgata (Pahang) and Calliophis intestinalis (Penang Island, Kedah, Pahang, Selangor) from the Malayan Peninsula were tested for their immunorecognition by antibodies of seven elapid antivenoms available in the Asia Pacific region: (1) Neuro Bivalent Antivenom, against Naja atra and Bungarus multicinctus of Taiwan (NBAV, batch: FN10101; expiry: April 2017); (2) Sea Snake Antivenom, against Hydrophis schistosus (formerly known as Enhydrina schistosa) of Peninsular Malaya and Notechis scutatus of Australia (SSAV, batch: 0549-08201; expiry: April 2015); (3) Bungarus multicintus Monovalent Antivenom, against the mainland Chinese species (BMMAV, batch: 20150101; expiry: January 2018); (4) Bungarus candidus Monovalent Antivenom, against the Thai species (BCMAV, batch: BC00112; expiry: June 2017); (5) Bungarus fasciatus Monovalent Antivenom, against the Thai species (BFMAV, batch: BK00112; expiry: March 2017); (6)
C.H. Tan et al. / Toxicon 121 (2016) 130e133
131
Fig. 1. Cross-reactivities of Calliophis bivirgata and Calliophis intestinalis venoms against different antivenoms: (a) Neuro Bivalent Antivenom, Taiwan; (b) Bungarus multicinctus Monovalent Antivenom, China; (c) Bungarus candidus Monovalent Antivenom, Thailand; (d) Bungarus fasciatus Monovalent Antivenom, Thailand; (e) Naja kaouthia Monovalent Antivenom, Thailand; (f) Ophiophagus hannah Monovalent Antivenom, Thailand; (g) Sea Snake Antivenom, Australia; (h) Calloselasma rhodostoma Monovalent Antivenom, Thailand. Percentages (%) were values of mean ± S.E.M. (triplicates), representing relative binding activities based on normalized values of absorbance (Abs). C rhodostoma venom served as the negative reference control in all assays testing with elapid antivenoms (aeg); its distinct immunoreactivity from elapid venom was verified using the homologous crotalid antivenom (h).
132
C.H. Tan et al. / Toxicon 121 (2016) 130e133
Naja kaouthia Monovalent Antivenom, against the Thai species (NKMAV, batch: NK00514; expiry: October 2019); (7) Ophiophagus hannah Monovalent Antivenom, against the Thai species (OHMAV, batch: LH00112; expiry: June 2017). In brief, the protocol was optimized based on the indirect enzyme-linked immunosorbent assay (ELISA) reported previously from the same laboratory (Tan et al., 2012, 2015c; Yap et al., 2014), conducted in three independent experiments. For consistency and comparative purpose, the coating venoms were standardized to 10 ng venom proteins per well on immunoplate, and all antivenoms were prepared in a 1:6000 dilution from the respective antivenom stocks (20 mg/ml protein concentration) for optimal absorbance based on pilot titration finding. Venoms of the respective species corresponding to the antivenoms (sourced from the same geographical locales of the immunogens) were used as the positive reference controls for binding activity, while the venom of Malayan pit viper, Calloselasma rhodostoma (Malaysia) served as the negative reference control in all assays involving the seven elapid antivenoms. The distinct immunoreactivity of C. rhodostoma (negative reference control) from the elapid venom was demonstrated in a separate assay using the homologous C. rhodostoma Monovalent Antivenom (CRMAV, batch: CR00210; expiry: June 2015). All the elapid antivenoms used were subsequently screened for their in vivo neutralization capability where 2.5 LD50 (median lethal dose) of C. bivirgata venom was pre-incubated with the maximum (physiologically permissible) antivenom dose (200 ml reconstituted antivenom) at 37 C for 30 min prior to injection into the caudal vein of ICR albino mice (n ¼ 2) for each antivenom tested (Wong et al., 2016). The mice were subsequently monitored for toxic signs and fatality over 24 h. The use of laboratory mice and the experiment protocol were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Malaya (ethics code: 2014-09-11/ PHAR/R/TCH). The ELISA result revealed that both C. bivirgata and C. intestinalis venom proteins cross-reacted moderately against NBAV, charting a cross-reactivity of approximately one-half of NBAV binding to N. atra and B. multicinctus venoms, but the binding was significantly higher than that shown by the negative control, C. rhodostoma venom (p < 0.05) (Fig. 1a). The bivalent SSAV and the other five mono-specific elapid antivenoms exhibited essentially no effective binding toward both Calliophis venoms, evidenced by the comparable levels of cross-reactivity toward C. rhodostoma venom (p > 0.05) (Fig. 1beg). The binding activities toward the various antivenoms did not vary significantly between C. bivirgata and C. intestinalis. The moderate cross-reactivity of NBAV against the two coral snake venoms implies common antigenicity shared between the coral snake venoms with N. atra and/or B. multicinctus venoms, and this appears in agreement with the previous reported weak neutralization of C. bivirgata venom by this antivenom (Tan et al., 2016a). By examining the lack of cross-reactivity against BMMAV (Fig. 1b), it is probable that the conserved antigenicity concealed not within B. multicinctus venom but within N. atra venom, which contains a high amount of three-finger cytotoxins as with the C. bivirgata venom (Huang et al., 2015). Of note, the C. bivirgata venom is predominated with three-finger cytotoxins and phospholipases A2 but lacking three-finger neurotoxins (Tan et al., 2016a). The cross-reactivity against NKMAV was however deficient (Fig. 1e), possibly due to the low content of cytotoxins in Thai N. kaouthia venom used as the immunogen in manufacturing NKMAV (Tan et al., 2015d). The lack of cross-reactivity was also observed across all other mono-specific antivenoms against Bungarus species (Fig. 1bed), supporting that Calliophis venoms and Bungarus venoms did not share common antigenicity, despite the high abundance of phospholipases A2 in their venoms and their strict dietary preference on snakes (Rusmili et al., 2014; Tan and
Tan, 2015). This is also applicable to the ophiophagic king cobra (Fig. 1f): the lack of cross-reactivity against OHMAV is unsurprising in view of the deficiency of three-finger cytotoxins in king cobra venom (Tan et al., 2015a). The high content of cytotoxic L-amino acid oxidases in king cobra venom (Tan and Tan, 2016) neither did confer good cross-reactivity of OHMAV against C. intestinalis venom, although the enzyme is present substantially in C. intestinalis venom (unpublished). The Calliophis venoms also did not cross-react against SSAV (Fig. 1g), attributable to the lack of cytotoxins in sea snake and tiger snake venoms (Tan et al., 2015b, 2016b), while the finding also implies that the abundant phospholipases A2 in sea snake and tiger snake venoms do not share antigenicity with these enzymes in Calliophis venoms. Interestingly, the ELISA result also revealed a significantly lower binding activity of SSAV toward the targeted sea snake venom compared to N. scutatus venom (p < 0.05), consistent with the limitation of SSAV neutralization reported recently (Tan et al., 2016b). The antivenom cross-reactivity findings were further validated using an in vivo assay. Against a lethal challenge dose (2.5 LD50) of C. bivirgata venom, all tested antivenoms failed to protect the mice, with the animals noted to die in spasticity (convulsion-like feature with myoclonus followed by muscle spasm, curled front limbs and out-stretched hind limbs) within 1 min post-injection of the venom-antivenom mixtures. NBAV failed to neutralize the venom despite its moderate immunological cross-reactivity in the present study and the weak neutralization reported previously, presumably due to batch-to-batch differences in the antivenom and animals. This observation highlights the importance of supporting immunological binding study with functional or in vivo experiments, and the need for re-validation of batch variability of antivenom efficacy especially in cases of feeble neutralization. Together, the findings revealed that the venoms of the two Calliophis species from Malaysia exhibit distinct antigenicity from other elapid lineages, and the lack of cross-reactivity against the major antivenoms available in the region is evident. The use of non-specific antivenom in the clinical management of Calliophis envenoming is hence not supported even though in vitro findings may appear promising. Further study should be directed toward the incorporation of the unique antigens of Calliophis venoms in the production of an effective poly-specific antivenom envisioned for pan-regional use (Ratanabanangkoon et al., 2016; Williams et al., 2011). Acknowledgment This study was supported by Grants FP028-2014A and RG3522015B. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2016.09.003. References Harrison, J.L., 1957. The bite of blue Malaysian coral snake or ular matahari (Maticora bivirgata). Malay. Nat. J. 11, 130e132. Huang, H.W., Liu, B.S., Chien, K.Y., Chiang, L.C., Huang, S.Y., Sung, W.C., Wu, W.G., 2015. Cobra venom proteome and glycome determined from individual snakes of Naja atra reveal medically important dynamic range and systematic geographic variation. J. Proteomics 128, 92e104. Ismail, A.K., 2015. Snakebite and envenomation management in Malaysia. In: Gopalakrishnakone, P., Faiz, A., Fernando, R., Gnanathasan, C.A., Habib, A.G., Yang, C.-C. (Eds.), Clinical Toxinology in Asia Pacific and Africa. Springer, Netherlands, pp. 71e102. Ratanabanangkoon, K., Tan, K.Y., Eursakun, S., Tan, C.H., Simsiriwong, P., Pamornsakda, T., Wiriyarat, W., Klinpayom, C., Tan, N.H., 2016. A Simple and novel strategy for the production of a pan-specific antiserum against elapid snakes of Asia. PLoS Negl. Trop. Dis. 10, e0004565.
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