Neutralization of Vipera and Macrovipera venoms by two experimental polyvalent antisera: A study of paraspecificity

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Author's personal copy Toxicon 57 (2011) 1049–1056

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Neutralization of Vipera and Macrovipera venoms by two experimental polyvalent antisera: A study of paraspecificity Irving G. Archundia a, Adolfo R. de Roodt b, Blanca Ramos-Cerrillo a, Jean-Philippe Chippaux c, Laura Olguín-Pérez d, Alejandro Alagón a, Roberto P. Stock a, * a

Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Cuernavaca, Morelos 62210, Mexico Laboratorio de Toxinopatología, Centro de Patología Experimental y Aplicada, Facultad de Medicina, Universidad de Buenos Aires, Uriburu 950, 5
Piso, CP 1427, Buenos Aires, Argentina c Institut de Recherche pour le Développement, UMR 216, Mère et enfant face aux infections tropicales, 08 BP 841, Cotonou, Benin d Instituto Bioclon S.A. de C.V. Calzada de Tlalpan 4687, Col. Toriello Guerra C.P. 14050, Distrito Federal, Mexico b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 January 2011 Received in revised form 7 April 2011 Accepted 12 April 2011 Available online 21 April 2011

We conducted an extensive study of neutralization of lethality of 11 species and one subspecies of snakes of the genus Vipera, and of five species of Macrovipera, by two experimental equine antisera. One antiserum was a trivalent preparation raised against the venoms of Vipera aspis aspis, Vipera berus berus and Vipera ammodytes ammodytes; the other was a pentavalent preparation that also included venoms of Vipera (now Montivipera) xanthina and Macrovipera lebetina obtusa. We measured specific neutralization of lethality against all venoms included in the immunization schemes, and paraspecific neutralization against the venoms of Vipera ammodytes montandoni, Vipera (Montivipera) bornmuelleri, Vipera latastei, Vipera (Mo.) latifii, Vipera (Mo.) lotievi, Vipera (Daboia) palaestinae, Vipera (Mo.) raddei and Vipera seoanei, as well as against Macrovipera (D.) deserti, Macrovipera lebetina cernovi, Macrovipera lebetina turanica and Macrovipera schweitzeri. We found an important degree of paraspecific protection within each genera (omitting recent reclassification) that was quite independent of both the lethal potency of the venoms and their geographic origin. This information may be of use to clinicians charged with the treatment of Vipera or Macrovipera envenomations with non-specific antivenoms. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Vipera Macrovipera Daboia Montivipera Antivenom Paraspecific neutralization

1. Introduction The genus Vipera is widespread throughout Europe, Western and Central Asia. It is a genus in constant revision and recognizes some two dozen species and a number of subspecies (Barbanera et al., 2009; David and Ineich, 1999; Garrigues et al., 2005; Joger et al., 2007; Lenk et al., 2001; Stümpel and Joger, 2009; Thorpe et al., 2007; Ursenbacher et al., 2006, 2008; Wüster, 1998; Wüster et al., 2008). The

* Corresponding author. Tel.: þ52 777 329 0829. E-mail address: [email protected] (R.P. Stock). 0041-0101/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2011.04.009

genus Macrovipera extends from Eastern Europe to Western and Central Asia, as well as Mediterranean Africa (David and Ineich, 1999). Between 1999 and 2008, several genus-level name changes have occured, most notably the transfer of some species of Vipera and Macrovipera to the genera Daboia and Montivipera (usefully summarized in WHO, 2010, Reptile database, 2010, and references therein). The epidemiology of snake bite by several European Vipera is known, especially in West, North and Central Europe, whereas information on morbidity and mortality due to Vipera and Macrovipera in Eastern Europe and Asia is more patchy and scarce (Chippaux, 1998, 2006). Table 1 summarizes estimations of incidence of snake bites in Europe including

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Table 1 Epidemiology of snake bite in Europe (including Russia and Turkey) from the literature between 1980 and 2010. Includes the period surveyed, the population covered, the incidence (/100,000) of bites reported as such and symptomatic cases. Country

Period

Population (x100.000)

Cases

Incidence (/100.000) Bites

Andorra Bosnia-Herzegovina Croatia Croatia Finland France France France France France France France France France France France Germany Greece Herzegovina Italy Italy Norway Norway Poland Poland Portugal Russia Spain Spain Spain Spain Spain Sweden Sweden Sweden Switzerland Switzerland Switzerland Turkey Turkey Turkey Turkey Turkey United Kingdom United Kingdom United Kingdom

1984–1988 1983–2006 1982–2002 1980–1995 1995–2000 1990 1992–1997 1999–2008 1976–1981 1973–1981 1980–1987 2008 1978–1983 1997–2001 1996–2008 1973–1984 1995–2006 1988–2003 1997–2002 1995–2001 1980–1984 1999–2003 1998–2003 2000–2008 1971–1980 1986–1988 2009 1997–2006 1987–1988 1980–1987 1991–2003 1976–1989 1985–1989 1995 1975 1967–1983 1983–2002 1973–2004 2000–2003 2003–2004 2001–2003 2004–2005 1995–2004 1982–1990 1983–1987 1982–1992

0.5 45 50 8.5 4.5 177 30 8 4.6 3.5 1.5 620 540 200 70 3 22 2.6 3.5 575 565 45 45 25 340 100 170 4 45 400 400 1.75 84 89 52 62 70 3 19 14 8.5 10.5 680 51 15 480

Turkey. Although the incidence is not high, especially when comparing to the tropical and sub-tropical world, severe envenomations often require antivenom and there is little data for a number of European nations. A few Vipera venoms have been studied in some depth, notably Vipera aspis, Vipera ammodytes ammodytes and Vipera berus, and several of their toxins and activities characterized (for example, see Calderón et al., 1993; Carlsson et al., 2004; Jan et al., 2007; Lang Balija et al., 2005; Ramazanova et al., 2008). However, less is known of Central Asian members of the genus (Bernadsky et al., 1986; Latifi, 1984; Sanz et al., 2008; Weinstein and Minton, 1984). Vipera and Macrovipera venoms have been described as strongly inflammatory and necrotizing, and in some instances Vipera bites result in manifest neurotoxicity (Ferquel et al., 2007; Westerström et al., 2010). Coagulopathy is rare, at least in V. berus bites (Warrell, 2005).

20 389 542 439 130 117 231 268 67 16 80 979 2247 37 174 137 78 147 71 1541 2329 291 318 44 346 285 500 7 5 11915 482 36 426 231 136 113 161 99 45 79 14 21 550 72 15 134

8.00 0.36 3.23 4.81 0.66 1.28 2.43 6.67 0.69

3.81 0.30

0.38 0.82 1.29 0.20

0.33 0.52 2.88 2.52 0.53 1.02 3.35 1.92 0.51 3.4 1.58 0.83 0.04 0.19 3.42 0.27 3.53 3.38 0.43 0.31 1.13 1.18 0.19 0.10 0.95 2.94 0.18 0.06

3.72 0.093 1.47 2.60 2.62 0.12 1.03 0.59 2.82 1.00

Reference

Envenomations

1.01 2.31 0.11 0.1 0.99 0.8 2.57 0.55 0.81 0.08

0.16 0.20 0.03

Gonzalez, 1991 Curi c et al., 2009 Luksi c et al., 2006 Radoni c et al., 1997 Grönlund et al., 2003 Audebert et al., 1992 Harry et al., 1999 Boels et al., 2010 Blettery et al., 1984 Boles et al., 1982 Claud et al., 1989 de Haro et al., 2010 Lagraulet and Pays, 1984 CAP Lille, 2001 de Haro et al., 2009 Chippaux et al., 1995 Felgenhauer et al., 2009 Frangides et al., 2006 Bubalo et al., 2004 Barelli et al., 2002 Pozio, 1988 Aakvik et al., 2004 (N) Lein et al., 2004 Magdalan et al., 2010 Szyndlar, 1981 Gonzalez, 1991 Karbovskyy, 2010 Fonseca Aizpurua et al., 2007 Anglés et al., 1991 Gonzalez, 1991 Ballesteros et al., 2006 Blanco Bruned et al., 1993 Karlson-Stiber and Persson, 1994 Karlson-Stiber et al., 2006 Persson and Irestedt, 1981 Stahel et al., 1985 Meier et al., 2003 Petite, 2005 Açikalin et al., 2008 Al et al., 2010 Ertem et al., 2005 Köse, 2007 Casaretli and Ozkan, 2010 Reading et al., 1995 Hawley, 1988 Reading, 1996

Paraspecificity (also known as cross-neutralization) refers to the capacity of an antivenom to neutralize the venom of species not included in the immunization scheme of the animals used for antivenom production at therapeutically useful doses, i.e. not excessively beyond those necessary for specific neutralization. It has been studied within some genera, and sometimes extends beyond a genus (Casasola et al., 2008; Christensen, 1959; Gutiérrez et al., 2010; Khow et al., 1997; Ramos-Cerrillo et al., 2008; Segura et al., 2010; Tan et al., 1994). Paraspecificity is determined by animal experimentation, notably by neutralization of venom lethality in mice, and extrapolation of these results to clinical envenomation is to be undertaken with caution (WHO, 2010). Nevertheless, systematic information of the bona fide spectrum of paraspecific neutralization of lethality may be of use to treating clinicians in cases where the offending snake is not identified, or in cases

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where the offending species is identified but not included in the immunization protocol; the severity of envenomation, the resources available and other considerations, i.e. the expected safety of the antivenom and the danger of sequelae even when symptomatic treatment would suffice to prevent death, must guide the choice to use antivenom in the absence of clinical validation of antivenom efficacy for particular species (WHO, 2010). Paraspecific neutralization has not been studied in any detail in either Vipera or Macrovipera, and very little is known in general about the neutralization of some European species of Vipera such as Vipera latastei, Vipera seoanei or Vipera ammodytes montandoni or Asian Vipera such as Vipera (Mo.) raddei, Vipera (Mo.) latifii, Vipera (Mo.) bornmuelleri or Vipera lotievi. In this investigation we generated two polyvalent experimental equine antisera to study the paraspecific spectrum of protection afforded by each against a collection of 12 Vipera and 5 Macrovipera venoms. The aims of the study were to establish whether paraspecific neutralization exists, its extent and the potency of paraspecific versus specific neutralization within and between each genus. 2. Materials and Methods 2.1. Venoms All venoms (Latoxan, Valence, France) were purchased as lyophilized solids and were certified as to the origin of the snakes. Before use, they were dissolved in sterile 0.15 M NaCl at a concentration of 2.5 mg/ml. 2.2. Antisera The trivalent antiserum was raised by immunizing three horses with a mixture of equal quantities of V. ammodytes ammodytes, Vipera aspis aspis and Vipera berus berus venoms. The pentavalent antiserum was raised by immunization of three horses with a mixture consisting of equal quantities of V. ammodytes ammodytes, Vipera aspis aspis, V. berus berus and Vipera (Mo.) xanthina venoms (70% of total venom) and Macrovipera lebetina obtusa (30%). The immunization scheme was the same for both groups, and consisted of 12 fortnightly immunizations starting with an initial dose of 500 mg/horse of each venom mixture emulsified with Complete Freund’s Adjuvant (CFA, Rockland, PA), followed by doses of 1, 1, 1.5, 2, 2.5, 3, 3.0, 3, 6, 12 and 12 mg of each venom mixture alternating Incomplete Freund’s Adjuvant (IFA, Rockland) and Alum (Imject, Thermo Scientific) until the 2.5 mg dose. The remaining boosts were without adjuvant. All immunizations were subcutaneous and antibody titers were monitored regularly by ELISA. Each experimental antiserum used in the study consisted of equivolumetric pools of the sera of the three horses in each group. 2.3. Animals Determinations of hemorrhagic and defibrinogenating doses were performed with mice of the CF1 strain (18–22 g) and rats (Wistar, 180–220 g), provided by the Animal

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Facility Laboratorio Gulcos (Pte. Perón, Buenos Aires). Animals were fed with commercial rodent food and water ad libitum and kept under controlled environmental conditions, with dark–light cycles of 12 h. For lethal potency and neutralization of lethality, 18–20 g mice (CD-1, Harlan Mexico) were used. All animal experimentation was carried out in accordance with the Guide for the Care and Use of Laboratory Animals (U.S. National Research Council, 2002.) For in vivo experiments of hemorrhage and defibrinogenation, animals were sedated with 10 mg acepromazine þ200 mg ketamine, intraperitoneally in mice and intramuscularly in rats. 2.4. Epidemiological survey Epidemiological data were obtained from medical literature, mainly from hospitals or poison control centers. Depending on the source, the number of snake bites and/or envenomations were available. When the number of inhabitants was not stated in the articles, demographic information for the period was searched in official national documents or through the United Nations database (http:// www.un.org/esa/population). The results are expressed as incidence per 100 000. 2.5. Determination of lethal potency Different doses of each venom were injected intravenously in CD-1 mice (5 mice per dose) according to conventional techniques (WHO, 2010). The number of deaths 48 h after injection was recorded and the lethal potency calculated as Median Lethal Dose (LD50), the dose of venom in mg/mouse that causes a statistical mortality of 50%. The plot of mortality versus venom dose was analyzed by nonlinear regression (Casasola et al., 2008). 2.6. Measurement of hemorrhagic activity Hemorrhagic activity was determined as described by Theakston and Reid (1983). To determine Minimal Hemorrhagic Dose (MHD), different doses of each venom were injected intradermally in Wistar rats (6.25–400 mg), or in mice (1.6–100 mg) using at least 3 points per dose level. After 24 h, the skin was excised and the major perpendicular diameters of the hemorrhagic haloes were measured from the dermal face with a digital caliper. The MHD was defined as the amount of venom (mg) that produces a mean hemorrhagic halo of 1 cm of diameter. All experiments were done in triplicate. 2.7. Determination of procoagulant activity Pro-coagulation was studied in normal human plasma and in bovine fibrinogen, according to Theakston and Reid (1983) with some modifications. Briefly, normal plasma or bovine fibrinogen (20 g/L in NaCl 0.15 M) was treated with different doses of venom and the time of clot formation was recorded. The minimal venom dose that produced an evident clot in 60 s (at 37
C) was considered as the minimum coagulant dose in plasma (MCD-P) or in

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fibrinogen (MCD-F). The doses were expressed in mg and are the mean of triplicate determinations. 2.8. Defibrinogenation in vivo The ability of venoms to cause blood incoagulability in vivo was measured according to Theakston and Reid (1983). Briefly, mice were injected intraperitoneally with increasing doses of venom (starting with 1 mg and up to 60 mg for Vipera and 100 mg for Macrovipera venoms). Animals were bled and the blood samples allowed to clot for 30 min and inspected for fragile or absent blood clot. The minimum defibrinogenating dose (MDD) is defined as the minimum amount of injected venom that inhibits normal coagulation of fresh blood from the experimental animals. 2.9. Neutralization of lethality Different doses of antivenom were incubated with five median lethal doses (LD50) of each venom for 30 min at 37
C. After incubation, samples were injected intravenously (i. v.) in mice (n ¼ 5 per dose level). The number of deaths 48 h after injection was recorded and the median Effective Doses (ED50) were calculated as the antivenom dose in microliters that protected 50% of the mice. Antivenom potency was calculated using the formula Potency ¼ [(n-1)/ED50]  LD50, where n-1 represents the number of lethal doses of the challenge minus one. The LD50 subtracted from the total challenge dose (n) represents the dose that was theoretically responsible for the death of half the mice, i.e. the calculation based on the total challenge minus one represents the actual quantity of venom that would be otherwise responsible for 100% mortality and was therefore neutralized by the antivenom (Ministerio de Saúde, 1996.) As the ED50 is expressed in ml and the LD50 in mg, the final result is mg/ml (or mg/ml), indicating the milligrams of venom neutralized by 1 mL of antivenom. Results were analyzed by non-linear regression.

Table 2 Species, geographic provenance and median lethal dose (LD50 in mg/mouse, 18–20 g mice) of all venoms used in this study. Venom

Origin

LD50 (95% c.i.)

Vipera ammodytes ammodytes V. ammodytes montandoni V. aspis aspis V. berus berus Montivipera bornmuelleri V. latastei Mo. latifii Mo. lotievi Daboia palaestinae Mo. raddei V. seoanei Mo. xanthina D. deserti Macrovipera lebetina cernovi Ma. lebetina obtusa Ma. lebetina turanica Ma. schweitzeri

Croatia Bulgaria France Russia Lebanon Spain Iran Russia Israel Turkey Portugal Turkey Algeria Turkmenistan Azerbaijan Russia Greece

8.4 7.0 15.6 9.4 12.6 15.1 8.0 12.15 8.4 3.87 9.7 12.2 21.2 17.6 30.1 20.5 23.5

(7.5–9.5) (6.1–8.1) (15.0–16.2) (7.6–11.7) (–) (–) (–) (12.14–12.16) (5.1–13.9) (3.86–3.88) (7.5–12.7) (–) (20.9–21.6) (12.9–24.0) (26.4–34.3) (17.9–23.3) (23.4–23.6)

(-) indicates a confidence interval of 600 mg). In our hands, neither subspecies of Vipera ammodytes elicited hemorrhage in mice (i.e. sublethal doses failed to elicit hemorrhage) whereas all other venoms were hemorrhagic in this model, with potencies ranging between 3.7 and 35.7 mg. In general, mice were more sensitive to the hemorrhagic effects on a venom weight basis (de Roodt et al., 2000, 2003). In vivo defibrinogenation was not observed with any of the venoms tested, that is, even the blood from animals treated with doses well above the LD50 clotted normally. No procoagulation effects on normal human plasma or bovine fibrinogen were detected, even at very high venom doses in vitro.

2.10. Statistics Results are presented as mean  standard deviation (SD) or with the 95% confidence intervals (c.i.) in parentheses. When necessary, Student’s t test was used for comparisons. Data were analyzed using the combined Prism 4.0 software package (GraphPad, CA, USA).

Table 3 Hemorrhagic potency (as MHD, mg), procoagulant potency in plasma (as MCD-P, in mg) and fibrinogen (as MCD-F, in mg) and defibrinogenating potency in vivo (as MDD, in mg) of selected venoms. Venom

Hemorrhage

Coagulation

MHD (mg)

in vitro (MCD, mg) in vivo (MDD, Plasma Fibrinogen mg)

Rat

3. Results 3.1. Lethal potency of Vipera and Macrovipera venoms With the exception of the venoms of Vipera aspis and V. seoanei, all Vipera venoms were significantly more lethal than Macrovipera venoms. The most potent was that of V. (Mo). raddei (3.87 mg) and the one with lowest potency was that of V. aspis (15.6 mg). All LD50 values are summarized in Table 2. In the case of Macrovipera, the most lethal was that of Ma. l. cernovi (17.6 mg) and the least was that of Ma. l. obtusa (30.1 mg).

V. a. ammodytes V. a. montandoni V. aspis aspis V. berus berus Mo. xanthina Ma. lebetina obtusa Ma. schweitzeri

Mouse

373  40 –

–

–

–

>600

–

–

–

29 15.8  2.5 – 15 7.7  2.5 – 27 9.5  2.2 – 6.4 3.7  0.8 –

– – – –

– – – –

107  6.1 35.7  6.4 –

–

–

130 73 110 83.3

   

–

MHD, Minimal Hemorrhagic Dose; MCD, Minimal Coagulating Dose one plasma (-P) or fibrinogen (-F); MDD, Minimal Defibrinogenating Dose. (–) No activity.

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3.3. Neutralization of lethality of Vipera and Macrovipera venoms

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in vivo in mice challenged intraperitoneally with up to several LD50 of venom. Interestingly, although venoms failed to induce clot formation in plasma even at very high doses (up to 400 mg in 0.4 ml of plasma), subsequent treatment with strongly procoagulant Bothrops venom failed to induce coagulation, suggesting non-specific degradation and/or inhibition of coagulation factors, although in vivo no anticoagulant effects were observed. In general, hemorrhagic potency was low by comparison to other viper venoms e.g. species of Bothrops, Crotalus, Echis, etc., and this is consistent with the clinical picture of at least some Vipera envenomations (Warrell, 2005). The trivalent antiserum against V. a. ammodytes, V. aspis aspis and V. berus berus paraspecifically neutralized all Vipera venoms tested (range 0.35–1.72 mg/ml) with potencies that went from lower to greater than specific neutralization (specific potency range 0.47–0.86 mg/ml). Neutralization spanned all groups of Vipera defined by Garrigues et al. (2005) by molecular phylogeny indicating that, at least in mice, the venom elements responsible for lethality are antigenically conserved and widespread through species/subspecies. The lowest paraspecific neutralization potency was against V. a. montandoni (0.35 mg/ml) although full (apparent) asymptomatic protection was achieved. In quite a few cases paraspecific potency was significantly greater than specific neutralization, and not necessarily related to geographic proximity between species, i.e. V. latastei venom from Spain was very efficiently neutralized at 1.72 mg/ml while V. seoanei venom from Portugal was neutralized with a potency of about a third (0.58 mg/ml); both were [paraspecifically] neutralized more efficiently than the venoms of V. berus (0.47 mg/ml) and V. a. ammodytes (0.49 mg/ml). Neutralization was also largely independent of lethal potency, i.e. V. (Mo.) raddei (the most lethal venom) was neutralized with the same potency as V. seoanei (nearly three times less lethal). Paraspecific neutralization of V. (Mo.) xanthina by the trivalent antiserum was the second lowest (0.44 mg/

As shown in Table 4, all Vipera venoms were neutralized by both the trivalent and the pentavalent antisera. For the trivalent antiserum, the range of specific neutralization potency was from 0.47 mg/ml (Vipera berus) to 0.86 mg/ml (V. aspis) whereas paraspecific neutralization within Vipera ranged from 0.35 mg/ml (V. a. montandoni) to 1.72 mg/ml (V. latastei). No significant neutralization of lethality of the venom of Ma. l. obtusa was observed at the maximum dose tested (300 mL) and no other Macrovipera venoms were tested with this antiserum. In the case of the pentavalent one, specific neutralization of lethality ranged from 0.82 mg/ml (V. berus) to 2.05 mg/ml (Ma. l. obtusa). All other Vipera venoms were paraspecifically neutralized, from 0.38 mg/ml (V. a. montandoni) to 2.15 mg/ml (V. latastei). The pentavalent antiserum neutralized all Macrovipera venoms with potencies between 0.50 mg/ml (D. deserti) and 3.63 mg/ml (Ma. l. cernovi). In general, neutralization potencies for Vipera venoms were higher with the pentavalent antiserum than with the trivalent with the exceptions of V. (Mo.) bornmuelleri (although the confidence intervals are wide and overlap), V. seoanei and D. palaestinae. 4. Discussion In our hands Vipera venoms were significantly more lethal than Macrovipera venoms, with LD50 values which were low by comparison to many vipers studied to date. All venoms tested caused subcutaneous hemorrhage in rats, and all but the two subspecies of V. ammodytes (ammodytes and montandoni) were also hemorrhagic in mice. None of the venoms tested exhibited procoagulant effects on normal human plasma (indicating activation of thrombin and/or other coagulation factors) or bovine fibrinogen (indicating thrombin-like activity), nor did they cause defibrinogenation

Table 4 Median effective dose (as ED50 in ml) and neutralization potency (in mg neutralized per ml of antivenom) of all venoms by the trivalent and pentavalent experimental antisera. Venom

Trivalent antiserum

Pentavalent antiserum

ED50 (95% c.i.)

Mg/ml (95% c.i.)

ED50 (95% c.i.)

mg/ml (95% c.i.)

Vipera ammodytes ammodytes V. aspis aspis V. berus berus V. ammodytes montandoni Montivipera bornmuelleri V. latastei Mo. latifii Mo. lotievi Daboia palaestinae Mo. raddei V. seoanei Mo. xanthina Macrovipera lebetina obtusa Ma. lebetina turanica Ma. lebetina cernovi D. deserti Ma. schweitzeri

67.9 (60.2–76.6)* 72.9 (58.1–91.6)* 79.3 (74.3–84.7)* 80.1 (72.9–88.1) 47.0 (27.7–80.0) 35.1 (30.4–40.5) 69.8 (69.8–69.9) 81.4 (51.8–127.8) 68.7 (68.6–68.9) 26.6 (24.8–28.5) 67.3 (59.3–76.3) 110.1 (76.9–157.6) NN ND ND ND ND

0.49 0.86 0.47 0.35 1.07 1.72 0.46 0.60 0.49 0.58 0.58 0.44 NA NA NA NA NA

33.4 54.2 45.9 73.1 104.7 28.1 34.4 61.2 86.6 15.8 92.4 56.9 58.7 73.1 19.4 169.1 47.4

1.01 1.15 0.82 0.38 0.48 2.15 0.93 0.79 0.39 0.98 0.42 0.86 2.05 1.12 3.63 0.50 1.98

(0.44–0.56) (0.68–1.07) (0.44–0.51) (0.32–0.38) (0.63–1.82) (1.49–1.99) (0.46–0.46) (0.38–0.94) (0.49–0.49) (0.54–0.62) (0.51–0.65) (0.31–0.63)

(29.1–38.4)* (49.1–59.8)* (39.5–53.4)* (58.3–91.6) (72.1–152.2) (24.2–32.5) (28.9–41.0) (44.3–84.6) (85.8–87.5) (13.4–18.7) (79.5–107.4) (55.8–58.1)* (37.4–92.3)* (63.7–83.8) (19.1–19.6) (126.9–225.1) (34.5–65.0)

(0.88–1.15) (1.04–1.27) (0.70–0.95) (0.31–0.48) (0.33–0.70) (1.86–2.50) (0.78–1.11) (0.57–1.1) (0.38–0.39) (0.83–1.16) (0.36–0.59) (0.84–0.87) (1.30–3.22) (0.98–1.29) (3.59–3.59) (0.38–0.67) (1.45–2.72)

*Specific neutralization. NN, not neutralized at the highest dose tested; ND, not determined; NA, not applicable; mg/ml, potency of neutralization as mg of venom neutralized per ml of antiserum (see Materials and Methods).

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ml). The trivalent antiserum did not neutralize the venom of Ma. l. obtusa at the highest dose tested (20% survival with 300 ml of antiserum) and we did not assay the other Macrovipera venoms with it. While all Vipera venoms were neutralized by both the trivalent and pentavalent antisera, most specific and paraspecific neutralization potencies were higher for the pentavalent antiserum (specific potency range 0.82– 1.15 mg/ml; paraspecific range 0.38–2.15 mg/ml). As with the trivalent antiserum, geographic proximity of the species tested was not clearly related to efficiency of neutralization (for example, compare again V. latastei and V. seoanei and, to a lesser extent V. (Mo.) bornmuelleri from Lebanon to D. palaestinae from Israel). Three species (bornmuelleri, palaestinae and seoanei) were neutralized less potently with the pentavalent than with the trivalent antiserum, although in the case of V. (Mo.) bornmuelleri the wide confidence intervals overlap. In the other two cases, potency was lower but only slightly (from 0.49 to 0.39 mg/ml for D. palaestinae and from 0.58 to 0.42 mg/ml for V. seoanei.) This could reflect an antagonistic effect of the extra venoms added in the pentavalent immunizing mixture, or immunologic variability in the horses against particular venom antigens; in any case, the differences observed are in the limits of significance. The inclusion of the venom of V. (Mo.) xanthina nearly doubled neutralization potency (now specific) to 0.86 mg/ml from the value of the trivalent (0.44 mg/ml), and may be responsible for the improved protection against the other venoms. In general, it is taken as fact that increased polyvalency in antivenoms entails diminished potency against any one venom in particular, although few studies have addressed this question directly (see, for example, Raweerith and Ratanabanangkoon, 2005, for some Asian elapids, and Dos-Santos et al., 2010, for some American vipers). In the case of the genus Vipera, our results show that the opposite is true; addition of one more species of Vipera (V. (Mo.) xanthina) and, may be, Ma. l. obtusa significantly increased neutralization potency, both specific and paraspecific, for the majority of Vipera venoms. For the trivalent antivenom, the average specific potency was 0.61  0.22 mg/ml, whereas for the pentavalent it was 0.99  0.17 mg/ ml, excluding V. (Mo.) xanthina which was specific in one case and paraspecific in the other. Considering all Vipera venoms (excluding V. (Mo.) xanthina), average trivalent potency was 0.70  0.40 mg/ml (median 0.58) and pentavalent potency was 0.86  0.51 mg/ml (median 0.82). Inclusion of two additional (Ma. l. obtusa and V. (Mo.) xanthina) venoms in the immunization mixture of the pentavalent antiserum conferred, as expected, protection against the Macrovipera venom and, interestingly, also against the venoms of the other four Macrovipera species tested. The specific neutralization potency was of 2.05 mg/ml, and the lowest paraspecific potency was against the venom of D. deserti (0.50 mg/ml); the other species were neutralized with potencies comparable to, or well above, those for most Vipera venoms (range 1.12–3.63 mg/ml). Our study cannot discriminate whether neutralization of Macrovipera venoms is solely due to the inclusion

of M. l. obtusa, but it is nonetheless a reasonable hypothesis considering that the trivalent antiserum did not neutralize it. In Europe, there are four antivenoms produced against Vipera. Two of them are monovalent against V. berus (Vipera TabÒ, BTG, UK, http://www.btgplc.com; Viper Venom AntitoxinÒ, Biomed, Poland, http://www.biomed. com.pl). There is a trivalent preparation against V. aspis, V. ammodytes and V. berus (ViperFavÒ, Sanofi Pasteur, France; Pepin-Covatta et al., 1998) in use for about a decade (de Haro, 2003; Petite, 2005) and a polyvalent antivenom against V. ammodytes, V. aspis, V. berus, Ma. lebetina, V. (Mo.) xanthina and Vipera ursinii (Viper Venom AntiserumÒ, Institute of Immunology, Croatia, http://www.imz.hr). Two ampoules of the Zagreb antivenom are generally used in V. berus envenomation cases in Great Britain (Warrell, 2005), while 1–4 vials of ViperFavÒ are used in France (de Haro, 2003) mostly for V. aspis bites. Knowledge of the extent and constraints of paraspecific neutralization may be of use in medical situations where venom from the species of attacking snake is not included in the spectrum of specific neutralization of a given antivenom. Reliable characterization of paraspecificity requires that actual production animals (generally, but not only, horses) be used in the generation of experimental antisera, that relevant in vivo effects be measured (as opposed to crossreactivity by ELISA or other in vitro tests), and that immunization procedures ensure mature and stable antibody responses for reproducible antivenom production in terms of the paraspecific spectrum of neutralization. Our results suggest that an antivenom raised against the venoms of a few Vipera may be paraspecifically useful in numerous envenomations in a vast swath of the Old World extending from Spain to Central Asia. Furthermore, an antivenom including Ma. lebetina obtusa may extend coverage to several other species of this important and relatively little studied genus. Author contributions A.A. and R.P.S. designed research. A.A., A.R.d.R, B.R.-C, I.G.A. and J.-P.C. conducted research. A.d.R., I.G.A., J.-P.C., L.O.-P. and R.P.S analyzed data. J.-P.C. and R.P.S. wrote the paper. Conflict of Interest None declared. Acknowledgments We wish to acknowledge the technical assistance of Carlos Olvera and Claudia Moctezuma. This project was partially funded by a collaborative research grant to R.P.S. and A.A. from Instituto Bioclon of Mexico and by a grant from FONCICyT-CONACyT (Mexico) 93608. References Aakvik, R., Refstad, S., Ringstad, L.G., Jacobsen, D., 2004. Hoggormbitt – forekomst og behandling. Tidsskr. Nor. Laegeforen. 124, 1779–1781.

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