Cross neutralization of common Southeast Asian viperid venoms by a Thai polyvalent snake antivenom (Hemato Polyvalent Snake Antivenom)

Accepted Manuscript Title: Cross Neutralization of Common Southeast Asian Viperid Venoms by a Thai Polyvalent Snake Antivenom (Hemato Polyvalent Snake Antivenom) Author: Poh Kuan Leong Choo Hock Tan Si Mui Sim Shin Yee Fung Khomvilai Sumana Visith Sitprija Nget Hong Tan PII: DOI: Reference:

S0001-706X(13)00367-7 http://dx.doi.org/doi:10.1016/j.actatropica.2013.12.015 ACTROP 3262

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

6-9-2013 20-12-2013 21-12-2013

Please cite this article as: Leong, P.K., Tan, C.H., Sim, S.M., Fung, S.Y., Sumana, K., Sitprija, V., Tan, N.H.,Cross Neutralization of Common Southeast Asian Viperid Venoms by a Thai Polyvalent Snake Antivenom (Hemato Polyvalent Snake Antivenom), Acta Tropica (2013), http://dx.doi.org/10.1016/j.actatropica.2013.12.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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*Graphical Abstract (for review)

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*Highlights (for review)

Highlights: The cross-neutralization capacity of Hemato polyvalent antivenom (HPAV) was assessed

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HPAV was able to effectively neutralize lethality of Southeast Asia viperid venoms HPAV also neutralized the venoms’ procoagulant and hemorrhagic activities

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HPAV could prevent the D. siamensis venom-induced nephrotoxiciy

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*Manuscript

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Cross Neutralization of Common Southeast Asian Viperid Venoms by a Thai Polyvalent

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Snake Antivenom (Hemato Polyvalent Snake Antivenom)

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Poh Kuan Leonga, Choo Hock Tana, Si Mui Sima, Shin Yee Fungb, Khomvilai Sumanac, Visith

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Sitprijac, Nget Hong Tanb*

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a

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Malaysia, b Department of Molecular Medicine, Faculty of Medicine, University of Malaya,

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Kuala Lumpur, Malaysia, c Queen Saovabha Memorial Institute, Rama IV Road, Bangkok,

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Thailand.

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Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur,

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*Corresponding author:

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Dr. Nget Hong Tan

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Department of Molecular Medicine

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University of Malaya, Kuala Lumpur, Malaysia

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Email: [email protected]

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Abstract

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Snake envenomation is a serious public health threat in many rural areas of Asia and Africa.

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Antivenom has hitherto been the definite treatment for snake envenomation. Owing to a lack of

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local production of specific antivenom, most countries in these regions fully depend on foreign

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supplies of antivenoms. Often, the effectiveness of the imported antivenoms against local

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medically important species has not been validated. This study aimed to assess cross-neutralizing

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capacity of a recently developed polyvalent antivenom, Hemato Polyvalent Snake Antivenom

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(HPAV), against venoms of a common viper and some pit vipers from Southeast Asia.

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Neutralisation assays showed that HPAV was able to effectively neutralize lethality of the

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common Southeast Asian viperid venoms examined (Calloselasma, Crytelytrops, Popeia, and

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Daboia sp.) except for Tropidolaemus wagleri venom. HPAV also effectively neutralized the

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procoagulant and hemorrhagic activities of all the venoms examined, corroboratively supporting

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the capability of HPAV in neutralizing viperid venoms which are principally hematoxic. The

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study also indicated that HPAV fully prevented the occurrence of hematuria and proteinuria in

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mice envenomed with Thai D. siamensis venom but was only partially effective against venoms

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of Myanmar D. siamensis. Thus, HPAV appears to be useful against its homologous venoms

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and venoms from Southeast Asian viperids including several medically important pit vipers

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belonging to the Trimeresurus complex. Nevertheless, the effectiveness of HPAV as a

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paraspecific antivenom for treatment of viperid envenomation in Southeast Asian region requires

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further assessment from future clinical trials.

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Keywords: Cross neutralization; Hemato polyvalent antivenoms; Southeast Asian viper and pit

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vipers

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1.

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Snake envenomation has been a serious yet often neglected global medical threat. According to

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the highest estimates, 1.8 million envenomings with an annual death toll of 94,000 occur from

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snakebites worldwide (Kasturiratne et al., 2008). Asia (1.2 million cases and 58,000 fatalities),

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Africa (420,000 cases and 32,000 fatalities) and America (210,000 cases and 2,000 fatalities) are

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the three worst affected continents. Most of the snake envenomation cases are inflicted by snakes

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from the Elapidae and Viperidae families (Warrell, 2010). The Viperidae family comprises of

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four major subfamilies, i.e. Azemiopinae (Fea’s viper), Causinae (night adders), Viperinae (true

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vipers) and Crotalinae (pit vipers) (Vidal et al., 2007). Members of the Viperinae and Crotalinae

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account for most of the mortality and morbidity of viperid envenomings (Chippaux, 2010). The

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Viperinae subfamily, whose members are known as the ‘Old World’ true vipers, consists of 12

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genera and more than 60 species distributed widely across Asia, Africa and Europe; whereas the

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Crotalinae subfamily, also known as the ‘pit vipers’, is represented by 18 genera and more than

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150 species. Pit vipers are found only in Asia and the Americas. Venoms of the viperid snakes

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are generally highly hematoxic and most of the envenomings inflicted by the viperids are often

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characterised by hemorrhage and tissue necrosis (Gutiérrez et al., 2006).

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Introduction

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Antivenom treatment has hitherto been the definitive treatment for snake envenomation. In

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recent years, owing to high production costs and low profit margin, many antivenom

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manufacturers have discontinued the production of antivenoms (Theakston and Warrell, 2000;

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Williams et al., 2011), resulting in a critical global shortage of antivenoms. Such shortage

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problem could possibly worsen in future if more manufacturers put a halt to the antivenom

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production due to economic constraints. The concept of producing a ‘Pan-Asian or Pan-African

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polyvalent antivenom’ has therefore been proposed to overcome the antivenom shortage

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problems (Williams et al., 2010).

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In South Asia and Southeast Asia, only a few nations (notably India and Thailand) have the

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capacity to produce antivenoms against the venoms of local medically important venomous

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snakes. Countries without local antivenom producers have to resort to importing antivenoms

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manufactured in foreign countries. Unfortunately, there are little vigorous scientific

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investigations to validate the effectiveness of the imported antivenoms to neutralize venoms of

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the local snakes. The aim of the present studies is to investigate the cross-neutralization

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capacities of a new polyvalent antivenom (Hemato polyvalent antivenom, HPAV) produced by

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Thai Red Cross Society against venoms of some common viperids from Southeast Asia. HPAV

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is raised against the venoms of Calloselasma rhodostoma (Malayan pit viper), Cryptelytrops

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albolabris (white-lipped pit viper) and Thai Daboia siamensis (Russell’s viper). In addition to

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neutralization against venom lethality, the capability of HPAV to neutralize the procoagulant,

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hemorrhagic, necrotic as well as nephrotoxic effects (where appropriate) of the venoms were also

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examined. The findings will provide valuable information towards the development of a regional

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broad-spectrum polyvalent antivenom.

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2.

Materials and Methods

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2.1 Venoms and antivenoms

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Venoms of Calloselasma rhodostoma (Malaysia), Calloselasma rhodostoma (Indonesia),

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Cryptelytrops albolabris, Daboia siamensis (Myanmar), Daboia siamensis (Thailand),

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Cryptelytrops purpureomaculatus, Tropidolaemus wagleri, were purchased from Latoxan

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(Valence, France). Venom of Popeia popeorum was obtained from Liverpool School of Tropical

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Medicine. Two antivenoms were used in the studies: (a) Hemato Polyvalent Snake Antivenom

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(HPAV) (Lyophilised; Batch no. 0020107; Exp. Date November 6th, 2013), a purified F(ab’)2

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obtained from sera of horses hyperimmunized against a mixture of three venoms: C. rhodostoma

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(Malayan pit viper), C. albolabris (white-lipped pit viper) and D. siamensis (Russell’s viper), all

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of Thai origin; (b) C. rhodostoma monovalent antivenom (CRMAV) (Full name: Malayan pit

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viper antivenin; Lyophilised; Batch no. 0120406; Exp. Date November 2nd, 2014), a purified

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F(ab’)2 obtained from sera of horses hyperimmunized against the venom of C. rhodostoma. Both

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of these antivenoms were produced by Thai Red Cross Society of the Queen Saovabha Memorial

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Institute (QSMI), Bangkok, Thailand. For neutralization studies, both antivenoms were

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reconstituted in the same manner: 10 mL of normal saline was added to 1 vial of the freeze-dried

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antivenom. According to the attached product leaflet, 1 mL of the HPAV antivenom is able to

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neutralize the following amount of snake venoms: 1.6 mg of C. rhodostoma, 1.7 mg of C.

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albolabris and 0.6 mg of D. russelii (now known as D. siamensis) venoms; while 1 mL of the

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CRMAV can neutralize 1.6 mg of C. rhodostoma venom.

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2.2 Animals

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Albino mice (ICR strain, 20-30 g) were supplied by the Laboratory Animal Centre, Faculty of

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Medicine, University of Malaya. The animals were handled according to the guidelines given by

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CIOMS on animal experimentation (Howard-Jones, 1985). All experiments involving animals

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were approved by the Animal Care and Use Committee (ACUC) of the University of Malaya

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(Ethical clearance letter No. 2013-06-07/MOL/R/FSY).

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2.3 Determination of protein content

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Protein content was determined by Bradford (1976) method. All measurements were performed

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in triplicate. Bovine serum albumin (Sigma, USA) was used as standard.

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2.4 Chromatographic and electrophoretic profiling of the antivenoms

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SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition was conducted

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according to the method of Studier (1973), using the Bio-Rad broad-range prestained SDS-

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PAGE standards (6.5-200 kDa) and 15 µL of each antivenom sample (3 mg/mL) was loaded in

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the gel (12.5% gel). High performance gel filtration chromatography of the reconstituted

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antivenom (100 µL, 14-19 mg/mL) was performed using a Superdex 200 HR 10/30, 13 µm SEC

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10 X 300 mm (GE Healthcare, Sweden). Elution buffer was 100 mM sodium phosphate, 0.15 M

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NaCl, pH 7.4 at a flow rate of 0.75 mL/min. Protein was monitored by absorbance measurement

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at 280 nm. The column was calibrated using the following protein standards obtained from Bio-

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Rad (Bio-Rad Gel filtration Standard): thyroglobulin (670 kDa), γ-globulin (158 kDa),

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ovalbumin (44 kDa) and myoglobin (17 kDa).

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2.5 Mass Spectrometry for protein identification

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The protein sample excised from the SDS-PAGE electrophoretic band of the antivenom was

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digested with chymotrypsin and peptides were extracted according to standard method (Bringans

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et al., 2008). Peptides were analyzed by matrix-assisted laser desorption/ionisation-time of

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flight/time of flight (MALDI-TOF/TOF) mass spectrometry using a 41800 Proteomics Analyzer

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(AB Sciex, USA). Spectra were analyzed to identify protein of interest using Mascot sequence

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matching software (Matrix Science, USA) with Ludwig NR Database.

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2.6 Protection against venom lethality: Experiments with preincubation of venom and

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antivenom prior to injection and experiments with independent administration of venom and

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antivenom.

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Median lethal dose, LD50, of the venom was determined by intravenous (caudal veins) or

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intramuscular (caudal thigh muscles) injection into ICR mice (20-30 g, n = 4 for each dosage).

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The survival ratio was recorded after 48 h to determine the LD50 value (Ramos-Cerrillo et al.,

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2008). Experiments with preincubation of venom and antivenom prior to injection assay was

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conducted as described by Ramos-Cerrillo et al. (2008). Briefly, a challenge dose of the venom

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dissolved in saline was pre-incubated with various dilutions of the reconstituted antivenom at 37°

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C for 30 min. The venom-antivenom mixture was subsequently centrifuged at 10000 x g and the

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supernatant was injected intravenously into the mice (20-30 g, n=4). In experiments with

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independent administration of venom and antivenom, the assay was carried out by intramuscular

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injection of venom into the mice, followed by intravenous injection of 200 μL of appropriately

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diluted reconstituted antivenom at 10 min post envenomation. Generally, the challenge dose used

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was 5 LD50. However, if 200 μL of the reconstituted antivenom (maximum volume allowed for

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intravenous injection in a mouse in our protocol) failed to give full protection to the mice, a

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lower challenge dose of 2.5 LD50 or 1.5 LD50 was used instead. All the lethality neutralization

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tests were conducted with Hemato polyvalent snake antivenom (HPAV); however, C.

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rhodostoma monovalent antivenom (CRMAV) has been included in the assay specific for C.

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rhodostoma venom for efficacy comparison between the polyvalent and the monovalent

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antivenoms. Neutralizing potency of the antivenom was expressed as ED50 (the amount of

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reconstituted antivenom in µL or the ratio of mg venom/mL reconstituted antivenom that gives

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50% survival of the animals tested) as well as in term of ‘neutralization potency’ (P, the amount

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of venom that is completely neutralized by a unit volume of antivenom) calculated according to

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Morais et al. (2010). The neutralization potency is theoretically unaffected by the challenge dose

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and also serves as an indicator for comparing ‘neutralizing capability’ of (a) one antivenom

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against various venoms; or (b) various antivenoms against one venom (Leong et al., 2012).

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2.7 Neutralization of procoagulant, hemorrhagic and necrotic activities

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Procoagulant activity was assessed by adding 100 µL venom of various concentrations in saline

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to 200 μL of fibrinogen solution (2 g/L) as described by Theakston and Reid (1983), and human

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plasma (recalcified by the addition of CaCl2 (0.2M, 20 μL) to 1 mL of 2x diluted plasma

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incubated at 37° C, using a method modified from Bogarín et al. (2000). Hemorrhagic and

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necrotic activity was assessed by intradermal venom injection into the dorsal skin of ICR mice

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(20-30 g, n = 3) as described by Gutierrez et al. (1985). The animals were euthanized with

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urethane 90 min (hemorrhagic activity assay) or three days (necrotic activity assay) after venom

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exposure and the skins were removed. Minimal hemorrhagic dose, MHD, was defined as the

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amount of venom that induces a skin hemorrhagic lesion of 10 mm diameter; while minimal

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necrotic dose, MND, was defined as the amount of venom that induces a skin necrotic lesion of 5

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mm diameter.

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For neutralisation assays, various doses of antivenom (HPAV, Hemato polyvalent snake

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antivenom, or CRMAV, C. rhodostoma monovalent antivenom) were pre-incubated with a

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constant amount of venom challenge dose (2 MCD-F, 2 MHD and 2 MND, for procoagulant,

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hemorrhagic and necrotic activity assays, respectively) at 37° C for 30 min prior to intradermal

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injection into the animals (hemorrhagic and necrotic activity assays) or, addition of venom to

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fibrinogen solution/ human plasma (procoagulant assay). For neutralization of hemorrhagic and

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necrotic activities, neutralization was expressed as median effective dose (ED50), defined as the

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amount of reconstituted antivenom in µL or the ratio of mg venom/mL reconstituted antivenom

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in which the venom activity was reduced by 50%. For neutralization of procoagulant activity,

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neutralization was expressed as effective dose (ED), defined as the amount of antivenom in µL

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or the ratio of mg venom/mL antivenom in which the clotting time was prolonged three times

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compared to that of fibrinogen or human plasma incubated with venom.

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In vivo protective effect of Hemato polyvalent snake antivenom (HPAV) against nephrotoxic effects induced by Thai and Myanmar D. siamensis venoms

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In vivo neutralization of venom’s nephrotoxicity assay was assessed by a method as described by

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Tan et al. (2012). A control group of mice (20-30 g, n = 4) received intramuscular injection of

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physiological saline into the caudal thigh muscles. Two envenomed groups of mice (n = 4 each

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group) received intramuscular injection of 1/3 i.m. LD50 of Thai and Myanmar Daboia siamensis

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venoms, respectively. Two treatment groups (n = 4 each group) received intravenous injection of

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200 μL HPAV at 10 minutes after the injection of Thailand and Myanmar Daboia siamensis

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venoms, respectively. Urine samples from all groups were collected pre-envenomation and at 4 h

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post-envenomation. The urine samples were screened for hematuria and proteinuria, using Roche

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Combur 10-test® M strips (Roche, Germany). Blood was collected from the mice under urethane

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anesthesia (1.4 mg/g) for urea and creatinine analysis by an independent pathology laboratory

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service center. Kidneys were harvested for histopathological study following euthanasia (by

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cervical dislocation) of the mice.

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2.9 Statistical analysis

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LD50 of the venoms and ED50 of antivenoms are expressed as means with 95% confidence

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intervals (C.I.). LD50, ED50 (median effective dose) and the 95% confidence intervals (C.I.) were

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calculated using the probit analysis method of Finney (1952) with the BioStat 2009 analysis

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software (AnalystSoft Inc., Canada).

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3. Results

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3.1 Protein composition of the antivenoms

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The protein content of the reconstituted HPAV and CRMAV were determined to be 19.82 ± 1.39

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g/L and 14.53 ± 0.74 g/L, respectively. Size-exclusion chromatographic analysis revealed

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slightly higher F(ab’)2 content (95.45%) as well as lower quantity of dimers (0.3%) and low

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molecular weight proteins (4.24%) in HPAV compared to that of CRMAV (87.03%; 1.58% and

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11.39% accordingly) (Figure not shown). However, no high molecular weight aggregates was

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detected. The electrophoretic patterns of reducing SDS-PAGE，however, revealed a distinct

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protein band with molecular weight of 45 kDa in HPAV (Fig 1). The band was identified as

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horse albumin by MALDI-TOF/TOF analysis (Table 1). H’ and L chains appeared as two

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electrophoretic bands indicative of proteins with ~25 and ~22 kDa, respectively.

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3.2 Protection against venom lethality by HPAV in experiments with preincubation of venom

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and antivenom prior to injection

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Table 2 shows the results of protection against venom lethality by HPAV in experiments with

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preincubation of venom and antivenom prior to injection assay. The polyvalent antivenom was

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able to confer protection against all of the viperid venoms examined, except for the venom of

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Tropidolaemus wagleri. The neutralizing potency

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mg/mL) to very high (10 mg/mL). The neutralization potencies of HPAV against C. rhodostoma,

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C. albolabris and D. siamensis venoms are much higher than the values stated in the product

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insert provided by the manufacturer. It should be noted, however, that the definition of the

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neutralization potency listed in the product insert has not been given.

(P), however, ranged from moderate (1

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3.3 Protection against venom lethality by HPAV in experiments with independent administration

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of venom and antivenom

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The results of the neutralization of lethal effects of two medically important Southeast Asian pit

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viper venoms by HPAV, when the venom and antivenom were administered independently, are

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shown in Table 3. HPAV effectively neutralized the two pit viper venoms. Although the i.m.

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LD50 values determined for the venoms were generally much higher than the corresponding i.v.

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LD50 values, the neutralizing potencies (P) of HPAV, determined by independent administration

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of venom and antivenom, were comparable to the corresponding potencies (P) of the antivenom

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when the venom and antivenom were preincubated prior to injection..

venoms by the antivenoms

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3.4 Neutralization of procoagulant, hemorrhagic and necrotic activities of selected viperid

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Table 4 shows the neutralization of procoagulant and hemorrhagic activities of 4 viperid venoms

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by HPAV (polyvalent antivenom) and CRMAV (C. rhodostoma monovalent antivenom). The

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polyvalent antivenom (HPAV) was able to effectively neutralize the procoagulant activities of all

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the venoms examined, including that of D. siamensis tested specifically on human plasma.

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Coagulation induced by D. siamensis venom was observed in the plasma but not the fibrinogen

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solution. The neutralizing effect of procoagulant activity by the polyvalent antivenom (HPAV)

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was generally stronger than that by the monovalent antivenom (CRMAV). HPAV also

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neutralized the hemorrhagic activities of the various venoms tested, while CRMAV was

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completely ineffective against hemorrhagic activities of the heterologous venoms (Table 4).

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The two antivenoms were also effective in the neutralization of necrotic activity of C.

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rhodostoma venom (Table 5). Neutralization of necrotic activities of other viperid venoms was

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not investigated because of the low necrotic activities of the venoms: at the LD50 dose,

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intradermal injection of the venom into the dorsal skin of the mice did not yield observable

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necrotic lesion.

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3.5 Comparison of neutralizing potencies of polyvalent and monovalent antivenoms

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Table 5 shows the comparison of the neutralizing potencies of the polyvalent antivenom HPAV

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and the monovalent antivenom CRMAV, against various toxic activities induced by Malaysian C.

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rhodostoma venom. The results showed that HPAV was more effective in neutralizing the

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lethality, procoagulant, hemorrhagic as well as the necrotic activities of the venom.

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3.6 In vivo protective effect of HPAV against nephrotoxic effects induced by Thai and Myanmar D. siamensis venoms

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Table 6 shows the renal abnormality (hematuria and proteinuria, 4 h post-injection) induced by

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i.m. injection of sublethal (1/3 i.m. LD50) dose of Daboia siamensis venoms. Intramuscular LD50

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values of the Thai and Myanmar D. siamensis were 0.25 μg/g and 0.3 μg/g, respectively. The

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control group (n = 4) showed negative hematuria with trace amount of protein in urine.

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Hematuria and proteinuria were observed in all animals injected with both D. siamensis venoms.

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The blood urea, creatinine levels as well as kidney histology, however, did not show remarkable

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abnormalities (data not shown).

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

22

13

Page 15 of 34

Table 6 shows also the results of in vivo neutralization of venom nephrotoxicity using HPAV.

2

HPAV effectively prevented the occurrence of hematuria and proteinuria (p < 0.005) in Thai D.

3

siamensis venom-challenged group. The same dose of HPAV could prevent the occurrence of

4

proteinuria but moderate hematuria still occurred in the challenge groups envenomed with

5

Myanmar D. siamensis venom.

ip t

1

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6

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7

Ac ce p

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

14

Page 16 of 34

1

4.

Discussion

2

4.1 Physicochemical properties of the antivenoms

4

The amount of F(ab’)2 in HPAV was ~95%, which meets the World Health Organization’s

5

recommendation on major active substance composition in antivenom (>90%) (World Health

6

Organization, 2010). However, the amount of F(ab’)2 in CRMAV (~87%) was slightly below the

7

recommended values. Impurities such as unwanted serum products (albumin), low molecular

8

weight digested products (Fc fragments) and digestive enzyme residues are known to be a major

9

cause of antivenom-induced hypersensitivity reactions (Theakston et al., 2003). In this study,

10

albumin was detected in Hemato polyvalent snake antivenom (HPAV) by SDS-PAGE analysis.

11

According to World Health Organization (World Health Organization, 2010), the albumin

12

content in antivenoms should ideally be less than 1% of the total protein content. Further study

13

on this should be carried out to verify the significance of the albumin presence in HPAV, and

14

whether there is batch variation.

15 16 17

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3

Ac ce p

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

4.2 Comparison of neutralizing potency between the monovalent antivenom (CRMAV) and polyvalent antivenom (HPAV)

18

There has been much debate over the relative superiority of monovalent antivenoms and

19

polyvalent antivenoms. Ahmed et al. (2008) suggested that the monovalent antivenoms exhibit

20

better potency and are less likely to cause adverse reactions. However, these findings differ from

21

reports by other authors (Chippaux, 2010; Raweerith and Ratanabanangkoon, 2005). Our

22

comparative study on two antivenoms produced by the same manufacturer using the same venom

23

source of C. rhodostoma showed that the neutralizing potency of the polyvalent antivenom 15

Page 17 of 34

HPAV is generally higher than that of the monovalent antivenom CRMAV, in terms of

2

neutralization of lethality, procoagulant, hemorrhagic and necrotic activities. Based on the results

3

of the physicochemical analysis (Bradford protein assay, SDS-PAGE and size-exclusion HPLC),

4

the higher neutralizing potency was most probably due to the presence of relatively higher

5

amount of antibodies in the polyvalent antivenom. In addition, the presence of synergistic cross-

6

neutralizing components in the antivenom may also play a role in enhancing the neutralizing

7

potency of the antivenom.

us

cr

ip t

1

8

10

4.3 Neutralization of lethality, procoagulant and hemorrhagic activities of selected Asiatic pit

an

9

viper venoms by the antivenoms

HPAV is raised against the venoms of one true viper (D. siamensis) and two pit vipers (C.

12

rhodostoma and C. albolabris) in Thailand. Our results showed that HPAV was able to

13

effectively neutralize the lethality of all the Asiatic common pit viper venoms examined except

14

for T. wagleri venom. The polyvalent antivenom was also able to neutralize the procoagulant and

15

hemorrhagic activities of the several pit viper venoms tested. The fibrinogen-clotting effect of

16

most viperid venoms is mediated by thrombin-like serine proteases, except that Daboia sp.

17

venoms has been known to induce coagulation of plasma through its specific action on factor V

18

and factor X instead of the fibrinogen (Chippaux, 2006). In contrast, the monovalent CRMAV

19

was less effective in the neutralization of the procoagulant activity of heterologous venoms, and

20

not effective against the hemorrhagic activity of these venoms, indicating that the antigenic

21

properties of procoagulant enzymes and hemorrhagins of C. rhodostoma venom differ

22

substantially from the other Asiatic pit vipers. It should be noted that C. albolaris, C.

23

purpureomaculatus, P. popeorum and T. wagleri were previously classified under the same

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11

Ac ce p

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

16

Page 18 of 34

genus of Trimeresurus (Malhotra and Thorpe, 2004), therefore it is not surprising that venoms

2

from these species share similar antigenic properties (with the exception of T. wagleri venom).

3

Our results are consistent with the earlier report that the immunoglobulins raised against

4

Cryptelytrops (Trimeresurus) albolabris venom’s component generally exhibited broad cross-

5

reactivity against heterologous venoms from the Trimeresurus complex (Tan et al., 1994). The

6

failure of HPAV to neutralize T. wagleri (Wagler’s pit viper) venom is not surprising as the

7

venom of T. wagleri is known to be rather atypical with its principal toxins being low molecular

8

mass neurotoxic peptides, and the venom is generally feebly procoagulant and low in

9

hemorrhagic activity (Gutiérrez et al., 2006; Sánchez et al., 2003). In fact, Wagler’s pit viper has

cr

us

an

10

ip t

1

been reclassified as a member of the genus Tropidolaemus (Hoge et al., 1978/79).

M

11

4.4 Neutralization of necrotic activity of C. rhodostoma venom

13

Local tissue necrosis is a common clinical feature of pit viper venom envenoming (Chippaux,

14

2006). However, the necrotic activity assay employed in this study was not sufficiently sensitive

15

to measure the necrotic effects of most of the venoms examined, except for C. rhodostoma

16

venom. Although both HPAV and CRMAV could effectively neutralize the necrotic effect of the

17

venom, the higher potency of HPAV suggests that the other two immunizing venoms used in the

18

preparation of HPAV may contain proteins that share common antigenic properties with the

19

necrotizing toxin(s) of C. rhodostoma venom.

20

Generally with in vitro neutralization method (venom and antivenom are preincubated prior to

21

injection), the ability of antivenoms to neutralize venom-induced tissue necrosis does not

22

necessary imply that the antivenom can clinically effectively alleviate local necrosis in

23

envenomation (Gutiérrez et al., 2006). However, in the experiments with independent

te

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12

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

17

Page 19 of 34

administration of venom and antivenom, HPAV injected 10 min post i.m. venom injection

2

prevented the development of necrotic lesion in the experimentally envenomed mice. Earlier,

3

Tan et al. (2011) also reported similar alleviation of H. hypnale venom-induced local necrosis by

4

HPAV. The finding, nevertheless, was confined to experimental envenoming in mice and hence

5

requires verification from clinical trials.

ip t

1

8

4.5 Neutralization of lethality, procoagulant, hemorrhagic activity and nephrotoxicity of Daboia

us

7

cr

6

siamensis venoms

In the procoagulant activity assay, coagulation induced by D. siamensis venom was observed in

10

the plasma but not the fibrinogen solution. This is expected as Daboia sp. venoms has been

11

known to induce coagulation by its specific action on factor V and factor X instead of the

12

fibrinogen (Chippaux, 2006).

M

d te

13

an

9

14

The venom of Thai D. siamensis is the only true viper venom included in the immunizing

15

mixture of HPAV. Russell’s viper has been divided into two distinct species, D. russelii and D.

16

siamensis; the former distributes across India, Sri Lanka, Pakistan and Bangladesh whilst the

17

latter, inhabits Myanmar, Thailand, China and Taiwan (Wüster et al., 1992). According to

18

Wüster et al. (1992), the venoms of the two Daboia species exhibit considerable variations in

19

their toxin composition as well as clinical manifestations of envenomation (Wüster, 1998).

20

Coagulopathy and renal failure are widespread, but neurotoxicity and myotoxicity have been

21

exclusively reported in bites inflicted by the D. russelii in Sri Lanka and India (Wüster, 1998).

22

Our results demonstrated that the polyvalent antivenom HPAV is at least effective to neutralize

23

the lethality, coagulopathic and hemorrhagic effects of the species D. siamensis, common in

Ac ce p

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

18

Page 20 of 34

1

Indo-China of the Southeast Asia, supporting its use in Thailand and potential benefit for

2

neighbouring countries where local antivenom for D. siamensis is lacking.

3

In addition, D. siamensis bite is known to lead to acute kidney injury (AKI) (Kanjanabuch and

5

Sitprija, 2008), a common and lethal complication. Complex mechanisms have been suggested

6

for contributing to venom-induced AKI, including direct nephrotoxic action of the venom, and

7

indirect effects like renal hemodynamic disturbance, systemic inflammatory response,

8

immunological reaction, consumptive coagulopathy, bleeding diathesis, intravascular hemolysis,

9

rhabdomyolysis, fibrin aggregation as well as thrombotic micro-angiopathy that is believed to be

10

responsible for venom-induced multiorgan injuries (Chaiyabutr and Sitprija, 1999; Isbister, 2010;

11

Kanjanabuch and Sitprija, 2008; Sitprija and Chaiyabutr, 1999; Suntravat et al., 2011; Thamaree

12

et al., 2000) . Even in surviving victims, it may potentially develop into chronic kidney disease

13

demanding long term renal replacement therapy which is a very heavy economic burden for

14

people in the under-developed countries. It is therefore relevant to assess the ability of HPAV in

15

neutralizing the nephrotoxic effect of Daboia siamensis from the affected region. In this study,

16

nephrotoxic effect of Daboia sp. venoms was essentially manifested by the development of

17

significant proteinuria and hematuria in mice. The absence of abnormalities in blood urea and

18

creatinine as well as kidney histology was possibly due to a short monitoring time (4 h) and

19

concurrent renal cell recovery, suggesting that higher range of imaging modalities e.g. electronic

20

microscopy may be required for investigating subtle changes at cellular level. The neutralization

21

of nephrotoxicity of homologous Thai D. siamensis venom by HPAV was evident, supporting

22

the clinical use of the antivenom product in patients envenomed by the Thai species. However,

23

HPAV failed to completely prevent nephrotoxicity induced by the Myanmar Daboia sp. venom,

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4

Ac ce p

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

19

Page 21 of 34

although it was able to reduce the severity of renal functional defect induced by the venom. The

2

findings imply that the nephrotoxic components in the venoms of Daboia may vary in term of

3

antigenic properties due to geographical differences, and it may be beneficial to expand the

4

scope of immunogens using D. siamensis of different SEA countries to improve the neutralizing

5

capacity of nephrotoxicity

ip t

1

8

4.6 Potency comparison: Experiments with preincubation of venom and antivenom prior to

us

7

cr

6

injection versus experiments with independent administration of venom and antivenom It is interesting to note that i.m. LD50 values of the pit viper venoms are generally much higher

10

than the corresponding i.v. LD50. This is in contrast to the results of our previous study on elapid

11

venoms, where the venoms examined exhibited i.m. LD50 comparable to the i.v. LD50 (Leong et

12

al., 2012). This is presumably due to the differences in pharmacokinetics and bioavailability as a

13

result of the differences in protein composition of the venom (unpublished observations).

14

Despite the differences in LD50 values, the neutralization potency of HPAV determined by

15

independent administration of venom and antivenom (where venom was injected intramuscularly,

16

followed by intravenous injection of HPAV) is comparable to that determined by experiments

17

with preincubation of venom and antivenom prior to injection assay (venom-HPAV mixture

18

injected intravenously). This suggests that the standard neutralization assay that involves

19

preincubation of venom and antivenom prior to injection does provide a good indication of the

20

HPAV effectiveness in the in vivo situation. Nevertheless, it should be noted that antivenoms that

21

are effective in animal models may show varied efficacy in human victims (Warrell et al., 1980).

22

Therapeutic benefits of HPAV against Southeast Asia viperid envenomation therefore awaits

23

validation through carefully designed clinical trials. In the future, an integrative approach largely

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

20

Page 22 of 34

1

driven with proteomics tools incorporating various immunological assays should be employed to

2

elucidate the immunoreactivity profiles of antivenoms against individual toxins.

3 4

5.

5

Hemato polyvalent antivenom (HPAV), which is raised against venoms of Thai C. rhodostoma,

6

D. siamensis and C. albolabris, was found to be effective in neutralizing the lethality of venoms

7

of C. rhodostoma from Malaysia and Indonesia, and medically important Southeast Asiatic pit

8

vipers belonging to the Trimeresurus complex, as well as Daboia siamensis from Thailand and

9

Myanmar. These results suggest that the polyvalent antivenom may be used as basis for the

10

design of a broad-spectrum polyvalent antivenom against viper and pit viper envenomation in

11

Southeast Asia. However, HPAV’s failure to neutralize the lethal effect of T. wagleri venom and

12

its incomplete neutralization of the nephrotoxic effects of Myanmar D. siamensis venom (at 200

13

uL antivenom per mouse) indicate the limitation of HPAV as a broad-spectrum polyvalent

14

antivenom in the treatment of bites inflicted by these species concerned.

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Conclusions

15

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

16

Acknowledgements

17

This

18

UM.C/625/1/HIR/MOHE/E20040-20001 from the Ministry of Higher Education Malaysia, and

19

PV 069/2011B, PV023/2012A from University of Malaya. The authors are grateful to Idaman

20

Pharma for assisting in the import of the antivenoms, and to Queen Saovabha Memorial Institute

21

for supplying the antivenoms.

work

was

supported

by

UM

High

Impact

Research

Grant

UM-MOHE

22 23

21

Page 23 of 34

References

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Ahmed, S.M., Ahmed, M., Nadeem, A., Mahajan, J., Choudhary, A., Pal, J., 2008. Emergency treatment of a snake bite: Pearls from literature. J. Emerg. Trauma Shock 1, 97-105.

4 5 6 7

Bogarín, G., Morais, J. F., Yamaguchi, I. K., Stephano, M. A., Marcelino, J. R., Nishikawa, A. K., Guidolin, R., Rojas, G., Higashi, H. G., Gutiérrez, J. M., 2000. Neutralization of crotaline snake venoms from Central and South America by antivenoms produced in Brazil and Costa Rica. Toxicon 38, 1429-1441.

8 9

Bradford, M., 1976. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

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Bringans, S., Eriksen, S., Kendrick, T., Gopalakrishnakone, P., Livk, A., Lock, R., Lipscombe, R., 2008. Proteomic analysis of the venom of Heterometrus longimanus (Asian black scorpion). Proteomics 8, 1081-1096.

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Chaiyabutr, N., Sitprija, V., 1999. Pathophysiological effects of Russell's viper venom on renal function. J. Nat. Toxins 8, 351-358.

15

Chippaux, J.P., 2006. Snake venoms and envenomations. Krieger Publishing Company, Florida.

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Chippaux, J.P., 2010. Guidelines for the production, control and regulation of snake antivenom immunoglobulins. Biol. Aujourdhui 204, 10-16.

18

Finney, D., 1952. Probit Analysis. Cambridge University Press, Cambridge.

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Gutierrez, J.M., Gene, J.A., Rojas, G., Cerdas, L., 1985. Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom. Toxicon 23, 887-893.

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Gutiérrez, J.M., Theakston, R.D.G., Warrell, D.A., 2006. Confronting the neglected problem of snake bite envenoming: The need for a global partnership. PLoS Med. 3, e150.

24 25

Hoge, A.R., Romano-hoge, S.A.R.W.L, 1978/79. Poisonous snakes of the world. Part 1: check list of the pitvipers, Viperoidea, Viperidae, Crotalinae. Mem. Inst. Butantan 42/43, 179–310.

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Howard-Jones, N., 1985. A CIOMS ethical code for animal experimentation. WHO Chron. 39, 51-56.

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Isbister, G.K., 2010. Snakebite doesn't cause disseminated intravascular coagulation: coagulopathy and thrombotic microangiopathy in snake envenoming. Semin. Thromb. Hemost. 36, 444-451.

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Kanjanabuch, T., Sitprija, V., 2008. Snakebite nephrotoxicity in Asia. Semin. Nephrol. 28, 363372.

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Kasturiratne, A., Wickremasinghe, A.R., de Silva, N., Gunawardena, N.K., Pathmeswaran, A., Premaratna, R., Savioli, L., Lalloo, D.G., de Silva, H.J., 2008. The global burden of snakebite: A literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Med. 5, e218.

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Leong, P.K., Sim, S.M., Fung, S.Y., Sumana, K., Sitprija, V., Tan, N.H., 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, e1672.

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Malhotra, A., Thorpe, R.S., 2004. A phylogeny of four mitochondrial gene regions suggests a revised taxonomy for Asian pitvipers (Trimeresurus and Ovophis). Mol. Phylogenet. Evol. 32, 83-100.

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Morais, V., Ifran, S., Berasain, P., Massaldi, H., 2010. Antivenoms: potency or median effective dose, which to use? J. Venom. Anim. Toxins Incl. Trop. Dis. 16, 191-193.

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Ramos-Cerrillo, B., de Roodt, A.R., Chippaux, J.-P., Olguín, L., Casasola, A., Guzmán, G., Paniagua-Solís, J., Alagón, A., Stock, R.P., 2008. Characterization of a new polyvalent antivenom (Antivipmyn® Africa) against African vipers and elapids. Toxicon 52, 881-888.

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Raweerith, R., Ratanabanangkoon, K., 2005. Immunochemical and biochemical comparisons of equine monovalent and polyvalent snake antivenoms. Toxicon 45, 369-375.

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S nchez, . ., Ram rez, .a.S., al n, .A., L pez, ., Rodr guez-Acosta, A., Pérez, J.C., 2003. Cross reactivity of three antivenoms against North American snake venoms. Toxicon 41, 315320.

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Sitprija, V., Chaiyabutr, N., 1999. Nephrotoxicity in snake envenomation. J. Nat. Toxins 8, 271277.

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Studier, F.W., 1973. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79, 237-248.

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Suntravat, M., Yusuksawad, M., Sereemaspun, A., Perez, J.C., Nuchprayoon, I., 2011. Effect of purified Russell's viper venom-factor X activator (RVV-X) on renal hemodynamics, renal functions, and coagulopathy in rats. Toxicon 58, 230-238.

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Tan, C.H., Leong, P.K., Fung, S.Y., Sim, S.M., Ponnudurai, G., Ariaratnam, C., Khomvilai, S., Sitprija, V., Tan, N.H., 2011. Cross neutralization of Hypnale hypnale (hump-nosed pit viper) venom by polyvalent and monovalent Malayan pit viper antivenoms in vitro and in a rodent model. Acta Trop. 117, 119-124.

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Tan, C.H., Tan, N.H., Sim, S.M., Fung, S.Y., Jayalakshmi, P., Gnanathasan, C.A., 2012. Nephrotoxicity of hump-nosed pit viper (Hypnale hypnale) venom in mice is preventable by the paraspecific Hemato polyvalent antivenom (HPA). Toxicon 60, 1259-1262.

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Tan, N.-H., Choy, S.-K., Chin, K.-M., Ponnudurai, G., 1994. Cross-reactivity of monovalent and polyvalent Trimeresurus antivenoms with venoms from various species of Trimeresurus (lanceheaded pit viper) snake. Toxicon 32, 849-853.

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Thamaree, S., Sitprija, V., Leepipatpaiboon, S., Witayalertpunya, S., Thaworn, N., 2000. Mediators and renal hemodynamics in Russell's viper envenomation. J. Nat. Toxins 9, 43-48.

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Theakston, R.D., Reid, H.A., 1983. Development of simple standard assay procedures for the characterization of snake venom. Bull. World Health Organ. 61, 949-956.

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Theakston, R.D., Warrell, D.A., 2000. Crisis in snake antivenom supply for Africa. Lancet 356, 2104.

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Theakston, R.D., Warrell, D.A., Griffiths, E., 2003. Report of a WHO workshop on the standardization and control of antivenoms. Toxicon 41, 541-557.

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Vidal, N., Delmas, A.S., David, P., Cruaud, C., Couloux, A., Hedges, S.B., 2007. The phylogeny and classification of caenophidian snakes inferred from seven nuclear protein-coding genes. C. R. Biol. 330, 182-187.

4

Warrell, D.A., 2010. Guidelines for the management of snake-bite. SEARO, New Delhi.

5 6 7

Warrell, D.A., Warrell, M.J., Edgar, W., Prentice, C.R., Mathison, J., 1980. Comparison of Pasteur and Behringwerke antivenoms in envenoming by the carpet viper (Echis carinatus). Br. Med. J. 280, 607-609.

8 9 10 11 12 13 14 15

Williams, D.J., Gutierrez, J.M., Calvete, J.J., Wüster, 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, 1735-1767.

16 17

World Health Organization, 2010. WHO Guidelines for the production, control and regulation of snake antivenom immunoglobins. WHO, Geneva.

18 19

Wüster, W., 1998. The genus Daboia (Serpentes: Viperidae): Russell's viper. Hamadryad 23, 3340.

20 21

Wüster, W., Otsuka, S., Malhotra, A., Thorpe, R.S., 1992. Population systematics of Russell's viper: a multivariate study. Biol. J. Linn. Soc. 47, 97-113.

26 27 28 29 30

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Williams, D., Gutiérrez, J.M., Harrison, R., Warrell, D.A., White, J., Winkel, K.D., Gopalakrishnakone, P., 2010. The Global Snake Bite Initiative: an antidote for snakebite. Lancet 375, 89-91.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

31 32 33

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Page 26 of 34

1

Legend for Figure

2 3

Figure 1: Reducing SDS-PAGE (12.5% gel) of Hemato polyvalent snake antivenom (HPAV) and Calloselasma rhodostoma monovalent antivenom (CRMAV).

5

The reconstituted antivenom (15 μL 3 mg/ml) was loaded on the middle (CRMAV)

6

and right lane (HPAV), respectively. Prestained molecular weight markers mixture

7

was loaded on the left lane (M, molecular weight in kDa). The digested heavy chain

8

(H’), light chains (L) and albumin were indicated by black arrowheads.

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4

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Ac ce p

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

25

Page 27 of 34

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Figure 1

Page 28 of 34

Table 1

Table 1: Identification of protein family of the 45 kDa protein band by MALDI-TOF/TOF analysis

ALBU_HORSE (P35747)

LYEIARRHPY SRRHPEYAVSVLL GFQNALIVRY

Number of matched peptide

Sequence coverage %

3

5%

ip t

Matched peptide sequence

us

cr

Protein family name and Accession Number

The 45 kDa protein band was a distinct band appeared in the SDS-PAGE (reducing) of HPAV. For the MALDI analysis, the spectra were analyzed to identify protein of interest using Mascot

an

sequence matching software with Ludwig NR Database. Mascot score was 136 (Mascot score of

Ac ce p

te

d

M

30 is considered as significant, p < 0.05)

1

Page 29 of 34

ip t

Table 2

cr

Table 2: Neutralization of lethality of medically important Southeast Asian viperid venoms by Hemato polyvalent snake

Venom

M an

us

antivenom (HPAV): Experiments with preincubation of venom and antivenom prior to injection.

i.v. LD50 (µg/g)

ED50

(µL per mouse /

challenging dose)

South/Southeast Asia

HPAV ED50 (mg/mL)

P (mg/mL)

1.48 (0.92-2.56)

22.47/5 LD50

9.09 (5.88-14.29)

7.27

Calloselasma rhodostoma (Indonesia)

1.35 (0.78-2.06)

11.20/5 LD50

12.5 (7.69-20.00)

10.00

Cryptelytrops albolabris

0.5 (0.40-0.63)

11.24/5 LD50

4.55 (2.94-6.67)

3.64

1.1 (0.75-1.68)

21.55/5 LD50

5.26 (2.04-14.28)

4.21

2.0 (1.61-2.48)

21.81/5 LD50

8.33 (5.56-12.5)

6.66

Tropidolaemus wagleri

1.50 (1.37-1.64)

NE/2.5 LD50

NE

NE

Daboia siamensis (Thailand)

0.13 (0.10-0.15)

11.24/5 LD50

1.27 (0.84-1.92)

1.02

Daboia siamensis (Myanmar)

0.34 (0.08-0.81)

35.36/5 LD50

5.00 (2.38-11.0)

4.00

Ac

Popeia popeorum

ce pt

Cryptelytrops purpureomaculatus

ed

Calloselasma rhodostoma (Malaysia)

NE: Not effective at maximum volume of antivenom (200 µL) used. For LD50 and ED50, values in brackets are 95% CI. P is potency of the antivenom. Challenge dose 2.5 or 5.0 LD50 of venom in 50 µL was premixed with 200 µL of HPAV and incubated for 30 min. The mixture was then injected into mice (n=4, 20-25 g) and monitored for 48 h.

1

Page 30 of 34

ip t

Table 3

cr

Table 3: Comparison of lethality-neutralizing potencies of Hemato polyvalent snake antivenom (HPAV) between neutralization experiments with preincubation of venom and antivenom prior to injection versus experiments with

M an

us

independent administration of venom and antivenom.

Neutralization potency

Neutralization potency i.m.LD50

i.v.LD50

independent administration of

with preincubation of venom

(µg/g of

(µg/g of

and antivenom prior to

mouse)

venom and antivenom

mouse)

injection**

ed

Species

ED50 (mg/mL)

Calloselasma

(21.64-36.93)

(Malaysia) Cryptelytrops

15.67

(13.02-18.85)

(7.69-12.5)

3.25

a

10.02

(6.60-15.22)

6.01

b

ED50 (mg/mL) 1.48

9.09

(0.92-2.56)

(5.88-14.29)

1.1

5.26

(0.75-1.68)

(2.04-14.28)

P (mg/mL)

7.27

4.21

Ac

purpureomaculatus

P (mg/mL)

9.75

ce pt

28.27

rhodostoma

determined by experiment

determined by experiment with

For LD50 and ED50, values in brackets are 95% CI. P is potency of the antivenom. Challenge dose a1.5 LD50 or b2.5 LD50 of venom was intramuscularly injected into the mice, followed by intravenous injection of 200 μL of appropriately diluted reconstituted antivenom at 10 min post envenomation. *:

estimated value, as the maximum volume of antivenom (200 µL) only achieved 50% protection of the animals.

** Data derived from Table 2

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Page 31 of 34

Table 4

Table 4: Neutralization of procoagulant and hemorrhagic activities of selected venoms by Hemato polyvalent snake antivenom (HPAV) and Calloselasma rhodostoma monovalent antivenom (CRMAV).

ip t

6.7 ± 0.3

12.90 ± 0.37

NE

9.8 ± 0.3

NE

14.81 ± 0.25

2.71 ± 0.05

6.1 ± 0.2

NE

70.82 ± 18.71*

17.6 ± 1.1

NE

127.13 ± 21.5*

te

MHD (μg/mouse) : 11.4 ± 0.4

8.3 ± 1.0

M

: 29.7 : NA

2.73 ± 0.02

d

MCD-P (mg/L) MCD-F (mg/L)

5.17 ± 0.15

an

Calloselasma rhodostoma (Indonesia) MCD-F (mg/L) : 5.6 MHD (μg/mouse) : 20.2 ± 0.7 Cryptelytrops albolabris MCD-F (mg/L) : 54.6 MHD (μg/mouse) : 6.3 ± 0.4 Cryptelytrops purpureomaculatus MCD-F (mg/L) : 61.6 MHD (μg/mouse) : 7.6 ± 0.2 Daboia siamensis (Thailand)

us

Venom

Neutralization of hemorrhagic activityb ED50 (mg/mL) HPAV CRMAV

cr

Neutralization of procoagulant activitya ED (mg/mL venom) HPAV CRMAV

a

Ac ce p

NE: Not effective. NA: No activity was detected. Pro-coagulant activity is defined as the amount of venom that clots the fibrinogen solution

(MCD-F) or plasma (MCD-P) in 60 s. b

Hemorrhagic activity is expressed in terms of MHD (μg/mouse), defined as the amount of

venom that induces a skin hemorrhagic lesion of 10 mm diameter. *

Coagulation assay was performed on human plasma sample.

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Page 32 of 34

Table 5

Table 5: Comparison of neutralizing potency between the Hemato polyvalent snake antivenom (HPAV) and the Calloselasma rhodostoma monovalent antivenom (CRMAV) against the lethal, procoagulant, hemorrhagic and necrotic activities induced by C.

Venom toxic properties

Neutralization by HPAV

ip t

rhodostoma (Malaysia) venom.

Neutralization by CRMAV

Lethality i.v. LD50 = 1.48 (0.92-2.56) μg/g Procoagulant activitya

an

ED = 7.47 ± 0.02

MCD-F = 12.06 ± 0.33 mg/L

M

Hemorrhagic activityb MHD = 24.0 ± 0.9 μg/mouse

te

MND = 28.7 ± 2.6 μg/mouse

ED50 = 8.2 ± 0.5 mg/mL

ED = 5.17 ± mg/mL 193.5 ± 24.2 μL/mg

ED50 = 7.0 ± 2.4 mg/mL

d

Necrotic activityc

ED50 = 4.00 (1.87- 8.33)

us

ED50 = 9.09 (5.88-14.29)

cr

(mg venom/mL antivenom) (mg venom/mL antivenom)

Ac ce p

ED50 = 36.3 ± 5.0 mg/mL

ED50 = 8.0 ± 0.7 mg/mL

Challenge dose (5 LD50) of venom in 50 µL was premixed with 200 µL of HPAV and incubated for 30 min. The mixture was then injected into mice (n=4, 20-25 g) and monitored for 48 h. For LD50 and ED50, values in brackets are 95% CI. a

Pro-coagulant activity is expressed in terms of MCD-F (minimal coagulant dose);

Hemorrhagic activity is expressed in terms of MHD (minimum hemorrhagic dose).

c

b

Necrotic

activity is expressed in terms of MND (minimal necrotic dose).

1

Page 33 of 34

Table 6

Table 6: In vivo neutralization of nephrotoxic activity of Daboia siamensis venoms by Hemato polyvalent snake antivenom (HPAV)

Envenomed (n=4) HPAV-treated

ip t

Trace (1.00 ± 0.00)

Protein (+)

Negative

Erythrocytes (+) Protein (+)

1.75 ± 0.25

1.60 ± 0.71

Erythrocytes (+)

3.75 ± 0.25

3.5 ± 0.50

Protein (+) Erythrocytes (+)

Trace

Trace

Negative*

0.25 ± 0.25*

M

(n=4)

(Myanmar)

cr

(n=4)

(Thailand)

D. siamensis

us

Control

D. siamensis

Urinalysis Parameter

an

Group

Proteinuria is described based on the following scale: negative (<10 mg/dL), trace (10 mg/dL),

d

1+ (30 mg/dL), 2+ (100 mg/dL), 3+ (300 mg/dL), and 4+ (1,000 mg/dL or greater). Hematuria is described based on the following scale: negative (< 10 rbcs/μL), trace (~10

te

rbcs/μL), 1+ (~25 rbcs/μL), 2+ (~50 rbcs/μL), 3+ (~150 rbcs/μL) and 4+ (≥250 rbcs/μL).

Ac ce p

Results are presented as mean of score ± S.E.M. * The difference between the envenomed group and treated group is statistically significant (P<0.005)

1

Page 34 of 34