Snake Venomics of African Spitting Cobras: Toxin Composition and

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Snake Venomics of African Spitting Cobras: Toxin Composition and Assessment of Congeneric Cross-Reactivity of the Pan-African EchiTAb-Plus-ICP Antivenom by Antivenomics and Neutralization Approaches lvaro Segura,§ María Herrera,§ Mauren Villalta,§ Daniela Solano,§ Daniel Petras,†,‡ Libia Sanz,† A § Mariangela Vargas, Guillermo Leon,§ David A. Warrell,|| R. David G. Theakston,^ Robert A. Harrison,^ Nandul Durfa,# Abdulsalam Nasidi,# Jose María Gutierrez,*,§ and Juan J. Calvete*,† †

Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (CSIC), Jaume Roig 11, 46010 Valencia, Spain Hochschule Darmstadt, Fachbereich Chemie und Biotechnologie, Darmstadt, Germany § Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San Jose, Costa Rica Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom ^ Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool, United Kingdom # Federal Ministry of Health, Abuja, Nigeria

)

‡

ABSTRACT: Venomic analysis of the venoms of Naja nigricollis, N. katiensis, N. nubiae, N. mossambica, and N. pallida revealed similar compositional trends. The high content of cytotoxins and PLA2s may account for the extensive tissue necrosis characteristic of the envenomings by these species. The high abundance of a type I R-neurotoxin in N. nubiae may be responsible for the high lethal toxicity of this venom (in rodents). The ability of EchiTAb-Plus-ICP antivenom to immunodeplete and neutralize the venoms of African spitting cobras was assessed by antivenomics and neutralization tests. It partially immunodepleted 3FTx and PLA2s and completely immunodepleted SVMPs and CRISPs in all venoms. The antivenom neutralized the dermonecrotic and PLA2 activities of all African Naja venoms, whereas lethality was eliminated in the venoms of N. nigricollis, N. mossambica, and N. pallida but not in those of N. nubiae and N. katiensis. The lack of neutralization of lethality of N. nubiae venom may be of medical relevance only in relatively populous areas of the Saharan region. The impaired activity of EchiTAb-Plus-ICP against N. katiensis may not represent a major concern. This species is sympatric with N. nigricollis in many regions of Africa, although very few bites have been attributed to it. KEYWORDS: African spitting cobra, Naja nigricollis, Naja katiensis, Naja pallida, Naja mossambica, Naja nubiae, EchiTAb-Plus-ICP antivenom, snake venomics, antivenomics, venom neutralization assays

’ INTRODUCTION Envenoming by snakes is an important public health problem in many tropical and subtropical countries and has been aptly described as a disease of poverty.1 A recently published study estimated at least 421 000 cases of envenoming and 20 000 deaths yearly,2 although these figures, based on hospital returns or incomplete central databases, are likely underestimates because many snakebite victims prefer to attend traditional healers and may die at home unrecorded. The true global incidence of snakebite may be as high as 1.8-2.5 million envenomings resulting in 94 000-125 000 deaths2,3 with an unknown percentage surviving with permanent physical disability caused by local r 2010 American Chemical Society

tissue necrosis. In sub-Saharan Africa alone, there are an estimated 1 000 000 bites each year, resulting in 500 000 envenomations and 3500-32 000 fatalities annually.4,5 Because most victims are subsistence farmers and children living in isolated villages, the socioeconomic impact of their disability is considerable. However, despite this, snakebite has not received the attention it deserves, despite being appropriately categorized by the World Health Organization as a Neglected Tropical Disease.6 Received: October 16, 2010 Published: December 20, 2010 1266

dx.doi.org/10.1021/pr101040f | J. Proteome Res. 2011, 10, 1266–1280

Journal of Proteome Research Since antivenom is the only specific antidote to snake venom, and its timely administration is critical to reverse all systemic manifestations of envenoming, thereby preventing mortality and reducing the incidence of permanent tissue damage, one of the key aspects of international collaborative programmes is to improve the effectiveness, quantity, optimization of clinical use, and affordability of antivenoms.1-12 The severe shortage (“crisis”) of commercially available effective antivenoms in Africa,13 due to the high cost of such animal serum-derived products and difficulties in distribution, has provided an opportunity for the unscrupulous marketing of inappropriate imported antivenoms that has proved clinically disastrous.14,15 This sad situation has prompted a committment from manufacturers in other parts of the world to provide antivenoms for Africa.10-12,16 Thus, in addition to laboratories traditionally producing antivenoms for Africa, such as EgyVac (Egypt), Sanofi-Pasteur (France) and South African Vaccine Producers (South Africa),17 other manufacturers, such as MicroPharm Ltd. (UK),18 Instituto Bioclon (Mexico),10 Instituto Clodomiro Picado (Costa Rica)12,19 and Instituto Butantan (Brazil),20 have recently developed new antivenoms against the African snake venoms that have the greatest medical importance. Echis ocellatus (savannah-dwelling carpet viper) causes the majority of cases of severe envenoming throughout the savannah region of West Africa.11,21 A number of other species, mainly from the genera Echis, Bitis (Viperidae), Naja and Dendroaspis (Elapidae), are also important in different parts of the continent.21 In standard preclinical tests in mice, the monospecific E. ocellatus Fab, F(ab')2 fragment, and whole IgG antivenoms (MicroPharm), polyspecific EchiTAb-Plus-ICP (Instituto Clodomiro Picado, CR) and EgyVac (Egypt), both prepared using venoms of E. ocellatus, Bitis arietans, and Naja nigricollis,11,12 and polyspecific (Echis, Bitis, Naja, Dendroaspis) Antivipmyn Africa,10 all neutralized E. ocellatus venom, albeit with varying potency.10-12 PanAfrican EchiTAb-Plus-ICP and Antivipmyn Africa also exhibited para-specific neutralization of lethality and other toxic (hemorrhagic, coagulant, and necrotizing) activities of the venoms sampled.10,12 EchiTAb G and EchiTAb-Plus-ICP antivenoms proved clinically effective and safe in a powerful trial performed in Nigeria in patients who presented with incoagulable blood, indicative of systemic envenoming by E. ocellatus in Kaltungo, northeastern Nigeria.11,22 In addition to preclinical neutralization tests, we have developed a complementary proteomic protocol, termed antivenomics,23-29 to explore the potential spectrum of coverage of an antivenom by assessing its capability to immunodeplete individual venom components from homologous and heterologous venoms. In previous studies12,23 we have used in vivo neutralization assays and antivenomics to assess neutralization efficacy and immunological cross-reactivity of EchiTAb-Plus-ICP toward the venoms of seven species of sub-Saharan snakes of the family Viperidae (e.g., E. ocellatus (Nigeria), E. leucogaster (Mali), E pyramidum leakeyi (Kenya), Bitis arietans arietans (from Ghana and Nigeria), B. gabonica, B. rhinoceros, and B. nasicornis (undisclosed origin)). The antivenom showed a high degree of crossreactivity against all venoms tested, although a small number of venom proteins (primarily PLA2s, disintegrins, and Kunitz-type inhibitor) were only partially immunodepleted from the venoms. The immunizing mixture designed for the production of EchiTAb-Plus-ICP includes the venom of Naja nigricollis,19 a spitting cobra that provokes severe envenoming in sub-Saharan Africa.21 The efficacy of this antivenom to neutralize the lethal, dermonecrotic and myotoxic activities of N. nigricollis venom has

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been demonstrated.19 However, it is not known whether this antivenom is also capable of neutralizing the venoms of other species of African spitting cobras whose venom causes similar clinical features. Here, we have investigated the toxin composition of venoms from African spitting cobras and have assessed the ability of this antivenom to recognize venom components of homologous and heterologous Naja venoms by antivenomics and to neutralize their toxicological activities by standard laboratory tests.

’ EXPERIMENTAL SECTION Venoms and Antivenom

The venoms of Naja nigricollis, black necked spitting cobra, (from Tanzania, Togo, Cameroon, Nigeria), N. katiensis, Malian or Katian spitting cobra, (Burkina Faso), N. pallida, red spitting cobra, (Kenya), N. nubiae, Nubian spitting cobra, (North Africa), and N. mossambica, Mozambique spitting cobra, (Tanzania) were used. Except for Nigerian N. nigricollis venom, which was pooled from adult specimens kept at the Liverpool School of Tropical Medicine, all the venoms were purchased from Latoxan (Valence, France). All venoms were lyophilized and stored at 20 C until used. The polyspecific EchiTAb-Plus-ICP antivenom was manufactured by caprylic acid fractionation of the plasma of four horses that had been immunized with a mixture (at a weight ratio of 1:1:1.33) of the venoms of Echis ocellatus, Bitis arietans and Naja nigricollis from Nigeria.19 The particular antivenom batch used in this preclinical study (Batch 4260308PALQ) had the following composition: protein concentration 69.6 g/L, sodium chloride 7.6 g/L, phenol 1.86 g/L, and pH 6.78. The antivenom batch passed all the quality control requirements at the Quality Control Laboratory of Instituto Clodomiro Picado, University of Costa Rica. Isolation and Characterization of Venom Proteins

Venom proteins were separated by reverse-phase HPLC as described30 using a Teknokroma Europa C18 (0.4 cm 25 cm, 5 mm particle size, 300 Å pore size) column and an Agilent LC 1100 High Pressure Gradient System equipped with DAD detector and micro-Autosampler. The flow-rate was set to 1 mL/min and the column was developed with a linear gradient of 0.1% TFA in water (solution A) and acetonitrile (solution B), isocratically (10% B) for 10 min, followed by 10-25% B for 20 min, 25-45% B for 120 min, and 45-70% for 20 min. Protein detection was carried out at 215 nm with a reference wavelength of 400 nm. Isolated fractions were subjected to N-terminal sequence analysis (using a Procise instrument, Applied Biosystems, Foster City, CA) following the manufacturer's instructions. Amino acid sequence similarity searches were performed against the available databanks using the BLAST program31 implemented in the WUBLAST2 search engine at http://www.bork.embl-heidelberg.de. Molecular masses of the purified proteins were determined by SDS-PAGE (on 12 or 15% polyacrylamide gels), and by electrospray ionization (ESI) mass spectrometry using an Applied Biosystems QTrap 2000 mass spectrometer32 operated in Enhanced Multiple Charge mode in the range m/z 600-1700. Protein bands of interest were excised from a Coomassie Brilliant Blue-stained SDS-PAGE and subjected to automated in-gel digestion, tryptic peptide mass fingerprinting (recorded with an Applied Biosystems Voyager-DE Pro TOF instrument), and CID-MS/ MS (using an Applied Biosystem's QTrap 2000 instrument) as 1267

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Journal of Proteome Research

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described.24-30 Production spectra were interpreted manually or using the online form of the MASCOT program at http://www. matrixscience.com against a private database containing viperid protein sequences deposited in the Swiss-Prot/TrEMBL database plus the previously assigned peptide ion sequences from snake venomics projects carried out in our laboratories.24-30 MS/MS mass tolerance was set to ( 0.6 Da. Carbamidomethyl cysteine and oxidation of methionine were fixed and variable modifications, respectively. The relative abundances (expressed as % of the total venom proteins) of the different protein families was estimated as described.29 Antivenomics: Immunodepletion of Venom Proteins by the Polyspecific EchiTAb-Plus-ICP Antivenom

To assess the immunodepleting ability of the EchiTAb-PlusICP antivenom, 10 μL of a 24 μg/μL venom solution were mixed with 320 μL of the antiserum and 170 μL of 100 mM sodium phosphate, 150 mM NaCl, pH 7.4, and incubated with gentle stirring overnight at room temperature (∼23 C). IgG concentration was determined spectrophotometrically using an extinction coefficient (e) of 1.4 for a 1 mg/mL IgG concentration at 280 nm using a 1 cm light path length cuvette.33 To deplete immunocomplexes, the sample was centrifuged for 15 min at 12 000 g in an Eppendorf centrifuge (Eppendorf Iberica, Madrid, Spain), and the supernatant was incubated at room temperature for 1 h in a 1 mL Spin-Column filled with 500 μL of Protein G Sepharose 4 Fast Flow (GE Healthcare; binding capacity of ∼18 mg human IgG/ml drained medium). The nonbound fraction was submitted to reverse-phase separation as above. As control of specificity, venom samples were subjected to the same procedure except that nonimmune equine IgG instead of EchiTAb-Plus-ICP antivenom was included in the reaction mixture. Western Blot Analysis

For assessing the immunoreactivity of the EchiTAb-Plus-ICP antivenom toward venom proteins by Western blot analysis, the reverse-phase HPLC chromatographic fractions were electrophoresed in SDS-PAGE (12 or 15%) polyacrylamide gels under nonreduced conditions followed by electrotransfer to Hybond-P membrane (GE-Healthcare) using a Bio-Rad Semy-Dry minitransfer cell operated at 200 V during 120 min. Transfer efficiency was evaluated by staining the nitrocellulose membranes with Ponceau-S Red. Membranes were then destained in water, incubated in 1% bovine serum albumin in 0.12 M NaCl, 0.04 M sodium phosphate, pH 7.2 (blocking buffer), for 30 min at room temperature to block nonspecific reactive sites, and incubated overnight at 4 C with antivenom diluted 1:4000 or 1:500 (v/v) in blocking buffer. After washing 4 times with blocking buffer containing 0.05% Tween-20, the membranes were incubated for 2 h at room temperature with alkaline phospatase-conjugated antihorse IgG (Sigma) diluted 1:2000, washed 4 times as above, and developed using ECL PLUS Western Blotting Detection Reagents (GE-Healthcare). Toxic and Enzymatic Activities of Venoms

Lethality. Lethal activity of venoms was assessed by the intravenous (i.v.) route34 in CD-1 mice (18-20 g) of both sexes. Groups of five mice were injected with various doses of the venoms, dissolved in 0.2 mL of 0.14 M NaCl, 0.04 M phosphate, pH 7.2 (PBS). Deaths occurring within 48 h were recorded and the Median Lethal Dose (LD50) was estimated by the Spearman Karber procedure.35

Figure 1. Geographical distribution of citotoxic Naja species in Africa. Physical map of Africa highlighting the geographical ranges and sampling localities of the Naja species whose venoms were investigated in this work: N. nigricollis (green); N. katiensis (yellow); N. pallida (red); N. nubiae (blue); and N. mossambica (magenta). Distribution data are indicative and are based the WHO database (http://apps.who.int/ bloodproducts/snakeantivenoms/database).

Dermonecrosis. Local skin necrosis, dermonecrosis, was assessed as described by Theakston and Reid34 but using mice. Groups of five CD-1 mice (18-20 g) of both sexes were injected, intradermally in the ventral abdominal region, with various doses of the venoms, dissolved in 0.1 mL of PBS. Seventy-two hours after injection, animals were killed by CO2 inhalation, the skins were removed, and the area of the necrotic lesion in the inner side of the skin was measured. The Minimum Necrotizing Dose (MND) was defined as the amount of venom that induced a necrotic lesion of 5 mm diameter.19,34 Phospholipase A2. PLA2 activity was assessed of egg yolk phospholipids, as described by Gutierrez et al. (1986).36 Released fatty acids were extracted and titrated according to Dole (1956).37 Activity was expressed as μEq fatty acid/mg protein/min. Neutralization of Venom Activities

Neutralization of venom activities by the antivenom was assessed by incubating, for 30 min at 37 C, a constant amount of venom with various dilutions of antivenom.38,39 Controls included venom solutions incubated with PBS instead of antivenom. After incubation, an aliquot of the mixture, containing one “challenge dose” of venom, was tested in the corresponding assay systems described above. The challenge doses of venom used in this study were: Three LD50s for lethality, one MND for dermonecrosis, and 5 μg venom for PLA2 activity. Neutralization was expressed as Median Effective Dose (ED50), corresponding to the ratio μL antivenom/mg venom in which the activity of venom was reduced to 50%.39 All experiments involving the use of animals were approved by the Institutional Committee for the Use of Laboratory Animals (CICUA) of the University of Costa Rica. 1268



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Figure 2. Reverse-phase HPLC separation of Naja venom proteins. Panels A-E display, respectively, the reverse-phase separation of the proteins from the venoms of N. nigricollis (Tanzania), N. katiensis (Burkina Faso), N. pallida (Kenya), N. nubiae (North Africa), and N. mossambica (Tanzania). Fractions were collected manually and characterized by N-terminal sequencing, ESI mass spectrometry, and SDS-PAGE (Table 1). Peaks labeled with an asterisk correspond to type I R-neurotoxins. Peaks rising above the dashed line correspond to toxins comprising >5% of total venom proteins. Insets, SDS-PAGE showing the protein composition of the corresponding reverse-phase HPLC separated venom protein fractions run under nonreduced (upper panels) and reduced (lower panel) conditions. Molecular mass markers (in kDa) are indicated at the left of the figure. Selected protein bands were excised and proteins identified by tryptic peptide mass fingerprinting and CID-MS/MS of selected doubly- or triply charged peptide ions. The results are listed in Table 1.

’ RESULTS AND DISCUSSION Venom Proteomes of African Spitting Cobras

The proteins from the venoms of N. nigricollis (from Tanzania, Nigeria, Togo, and Cameroon), N. katiensis (Burkina Faso),

N. pallida (Kenya), N. nubiae (North Africa), and N. mossambica (Tanzania) (Figure 1) were separated by reverse-phase HPLC (Figure 2A-E; Figure 3). Most isolated proteins could be assigned to known protein families by N-terminal sequencing and 1269



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Figure 3. Reverse-phase HPLC separation of the venom proteins from N. nigricollis venoms. Panels A-C display, respectively, the reverse-phase separation of the proteins from the venoms of N. nigricollis from Nigeria, Togo, and Cameroon. HPLC peaks containing proteins exhibiting the same elution time, N-terminal sequence, and molecular mass are labeled with the same numbers, which correspond to the numbering of Figure 1A. For details of the proteins consult Table 1.

molecular mass determination by electrospray-ionization (ESI) mass spectrometry (Table 1). A few chromatographic peaks contained N-terminal blocked proteins and were identified following MS/MS fragmentation of tryptic peptides generated by in-gel digestion of their SDS-PAGE bands (inserts in panels A-E of Figure 2, Table 1). The five Naja venom proteomes investigated share toxins belonging to 2 major (3FTx, PLA2) and a minor (PIII-SVMP) protein families (Table 2). In addition, an endonuclease was found in the venoms of N. nigricollis, N. katiensis, N. pallida, and N. mossambica; a cysteine-rich secretory protein (CRISP) was identified in N. nigricollis and N. katiensis venoms; and the venom of N. nigricollis from Nigeria, Togo, and Cameroon, but not from Tanzania, contained also nawaprin (Tables 1 and 2; peak 13N in panels A-C of Figure 3). Notwithstanding these taxa-specific or geographic differences, the venom proteomes of African spitting Naja display very similar chromatographic profiles (Figures 2 and 3) and compositional trend (Tables 1 and 2). Thus, all venoms investigated are comprised by 22-32 proteins, although in each case only 6-8 toxins (particularly 3FTxs and PLA2s) have relative abundances of >5% of the total venom proteins (Figure 2). A closer comparison of the venom proteomes show that this set of major toxins is made up of similar molecules in the five African spitting Naja venoms. In particular,

each venom contains 3FTx molecules similar to N. mossambica toxins P01452 isolated in peak 9 (N-terminal sequence LKCNKLIPIAYKTCP; molecular mass 6.7 kDa); and P01467 (peak 17) (N-terminal sequence LKCNQLIPPFWKTCP; molecular mass 6818 Da); and N. katiensis P01470 (peak 13) (N-terminal sequence LKCNRLIPPFWKTCP; molecular mass 6886 Da) (Table 1). The venoms of N. nigricollis, N. katiensis, and N. pallida also contain a 3FTx that possesses N-terminal (XQCTQQKPPFYMNCP) similarity with N. atra Q9W6W6 (Table 1, Nn8, Nk7, Np5). In addition, each venom also contains one (N. nigricollis, N. katiensis, and N. pallida), two (N. nubiae), or three (N. mossambica) homologous PLA2 molecules, which share N-terminal sequence and very similar molecular masses (Table 1). On the other hand, the most striking departure from the above outlined toxin profile is the high abundance (∼12% of the venom proteins) of the type I R-neurotoxin-like protein Nnu-1 (Figure 2D) in the venom of N. nubiae. Venom represents a trophic adaptive trait. The relevance of knowing in detail the composition of the venom proteomes of African spitting cobras (Naja) to understanding the clinical symptoms caused by their bites is discussed below. This knowledge may also contribute to a broader picture of the functional biogeography of this medically important group of snakes. Furthermore, 1270



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Table 1. Assignment of the Reverse-Phase Fractions from the Venoms of Naja nigricollis (Nn, Tanzania), N. katiensis (Nk, Burkina-Faso), N. pallida (Np, Kenya), N. nubiae (Nnu, North Africa), and N. mossambica (Nm, Tanzania), Isolated as in Figure 2A-E, Respectively, to Protein Families by N-Terminal Edman Sequencing and Collision-Induced Fragmentation by nESI-MS/MS of Selected Peptide Ions from In-Gel Digested Protein Bands Separated by SDS-PAGE (Insets in Corresponding Panels of Figure 2)a HPLC Fraction Nn Nk Np Nnu Nm 1 1 2 2T

1 2

1

2

peptide ion N-terminal sequence

molecular mass

m/z

z

MS/MS-derived sequence

protein family

LNCHNQMSAQPPTTT

7067.3

3 FTx ∼ P01432 (N. m. mossambica) (I)

L(D/N)CHNQMSAQPPTTT LRCYSCGRNGCRDIV

7009.1 7210.3

3 FTx ∼ P01432 (N. m. mossambica) (I) 3 FTx 3 FTx

LRCYSCGRNGCRDIVA

7068.3

1

MICHNQQSSQPPTTK

6787.1

3 FTx ∼ P25675 (N. haje) (I)

2

LRCYNQQSSQSN

7111.6

3 FTx ∼ P10455 (Laticauda colubrina) (I)

3

LTCLICPEKYCNKVH

7069.8

3 FTx ∼ P29179 (N. naja naja) (I)

3N

LRCYSCGRNGCRDIVTCSE

6588.4, 6773.2

3 FTx 3 FTx ∼ P24780 (N. naja naja) (C)

4

3

LTCLICPEKYCNKVH

7499.6

EGKNICYKMMMVSNK LTCLICPEKYCNKVH

7594.2 7443.9


2

LTCLICPEKYCNKIH

7456.1


LTCLICPEKYCNKIH

7471.6


3

LTCLICPEKYCNKVH

7422.6

3 FTx ∼ P29179 (N. naja naja) (I) 3 FTx ∼ P29179 (N. naja naja) (I)

4 2 4 4N 5N 3 5

4

5 6N 6 7

5

4

3 FTx ∼ Q9W6W6 (N. atra) C) 3 FTx ∼ Q9W6W6 (N. atra) C)

LTCVKEKSIFYVTKE

6880.4

3 FTx ∼ P82463 (N. kaouthia) (M)

QQCTQKKPPFYMNCP

7790.8

3 FTx ∼ Q9W6W6 (N. atra) (C)

7175.2

XQCTQQKPPFYMNCP

7210.1

5

XQCTQQKPPFYMNCP LKCNKLIPIAYKTCP

7251.6 7191.1, 6751.6

3 FTx ∼ P01452 (N. m. mossambica) (C)

6

KICKQQALQFPICT

6896.2

3 FTx ∼ P01420 (N. haje annulifera) (I)

8 9

10

ND 6878.9

XQCTQQKPPFYMNCP

7

12

QQCTQKKPPFYMNCP RQCTQKKPPFYMNCP

3 FTx ∼ P01420 (N. haje annulifera) (I)

7

8


3 FTx ∼ Q9W6W6 (N. atra) (C)

9

7

7452.1

6904.2

6

10N 11 9


LKCNKLIPIAYKTCP

7250.4

5

10

7477.7

LKCYKQQALQFICT

7

6

7426.8

XQCTQQKPPFYMNCP

4 8

9

LTCLICPEKYCNKVH LKCNKLIPIAYKTCP

LKCNKLIPIAYKTCP

7281.8


LKCNKLIPIAYKTCP

6750.6


LKCNKLIPIAYKTCP

7310.3


LQCVKYYTIFGVTPV

7284.1

3 FTx ∼ Q53B49 (Ophiophagus hannah) (I)

LKCNKLIPIAYKTCP

6707.8

3 FTx P01452 (N. m. mossambica) (C)

LKCNKLIPIAYKTCP LKCNKLIPIAYKTCP

6734.9 6753.4

3 FTx ∼ P01452 (N. m. mossambica) (C) 3 FTx ∼ P01452 (N. m. mossambica) (C)

LKCNKLIPIAYKTCP

6740.3 3 FTx ∼ P01452 (N.m. mossambica) (C)

LKCNKLIPIAYKTCPAGMNIC 6237.3 8

LKCNKLIPIAYKTCP

6210.8

NEKSGSCPDMSMPIPPL

5286.8

LKCNQLIPPFWKTCP

6818.5


LKCKKLIPLFWKTCP

6784.3


14C

LKCNRLIPPFWKTCP LKCNQLIPPFWKTCP

6872.6 6819.6

3 FTx ∼ P01470 (N. m. mossambica) (C) 3 FTx P01467 (N. m. mossambica) (C)

14

NLYQFKNMIHCTVPS

13252.6

PLA2 ∼P00605 (N. nigricollis)

14N

NLYQFKNMIHCTVPS

13277.6


15N

NLYQFKNMIHCTVPS

13257.2


NLYQFKNMIHCTVPS

13336.6

PLA2 ∼P00605/ P14556

NLYQFKNMIHCTVPS

13212.3

PLA2 P14556 (N. pallida)

13N 13

11 10 10

12 11

Nawaprin [P60589] (N. nigricollis)

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Table 1. Continued HPLC Fraction

peptide ion

Nn Nk Np Nnu Nm 14T

11

15T

12

13

N-terminal sequence NLYQFKNMIHCTVPS

molecular mass

m/z

z

MS/MS-derived sequence

protein family PLA2 P14556 (N. nigricollis)

13323.8

11 NLYQFKNMIHCTVPS

13203.6

PLA2 P00602 (N. m. mossambica)

12 NLYQFKNMIHCTVPS

13243.2

PLA2 ∼ P00604 (N. m. mossambica)

13 NLYQFKNMIHCTVPS

13287.2

PLA2 P00604 (N. m. mossambica)

6814.6


LKCKKLIPLFSKTCP 13 LKCNRLIPPFWKTCP

6886.3


15N

LKCNRLIPPFWKTCP

6886.3


15T 15 14

LKCNRLIPPFWKTCP LKCNQLIDPFWKTCP

6886.3 6817.6

3 FTx P01470 (N. m. mossambica) (C) 3 FTx ∼ P01470 (N. m. mossambica) (C)

LKCNQLIPPFFKTCP

6723.4


14 LKCNLHIPPFW(E/K)TCP

7050.6


7051.6


15

13 12

14 16

LKCNLHIPLFS(E/K)TCP 13 14 15

LKCNQLIPPFWKTCP

6843.4


15 LKCNQLIPPFWKTCP

6833.9


17

LKCKKLIPLFSKTCP

6814.6


17N 18

LKCKKLIPLFSKTCP LKCNQLIPPFFKTCP

6883.6 6686.6

3 FTx ∼ P25517 (N. m. mossambica) (C) 3 FTx ∼ P01469 (N. m. mossambica) (C)

19

- 15 16

17 LKCNQLIPPFWKTCP

6817.4


LKCNQLIPPFWKTCP

6818.4


20

LKCNQLIPPFWKTCP

6 kDa


21

LKCNQLIPPFWKTCP

6 kDa


22

19 16 -

19 LVXDSFQGHCPQFFLR

30.5 kDa

20 24

20

TVTPQQDXYLXAKKY

55 kDa

24

20

DVYFNSESNRRKYMQ

25 kDa

N.D.

98 kDa

25 26

24 20 21 21

27 28

25 22 22

735.8

2

JPVYSAYVYNPGK

655.9

3

(531.1)SFQGHCPQFFJR

686.9 708.6

2 2

WMVEPE(602.2) YJEFYVVPDDR

Endonuclease ∼ O73911 (Gallus gallus)

SVMP ∼ ADG02948 (N. atra) CRISP ∼ P84807 (N. haje haje) unknown

23 TNTPEQDRMLQAKKY

48 kDa

570.8

2

DSCFTJNQR

SVMP ∼ Q10749 (N. m. mossambica)

TNTPEQDRMLQAKKY

48 kDa

570.8

2

DSCFTJNQR


TNTPEQD

67 kDa 868.2

2

AJIVTPPVCpGNYFVER SVMP ∼ Q10749 (N. m. mossambica)

24 TNTPQQXRYLQAKKY

50 kDa


Fractions labelled “nN”, “nT”, and “nC” correspond, respectively, to isoforms found in venoms of N. nigricollis from Togo, Nigeria, and Cameroon (HPLC profiles displayed in Fig.3). In MS/MS-derived sequences, J = Ile or Leu. Unless other stated, for MS/MS analyses, cysteine residues were carbamidomethylated; Cp, propionamide cysteine. Molecular masses in Daltons were determined by ESI-MS and those in kDa were calculated from SDS-PAGE of reduced samples. ND, not determinbed. (I), type I R-neurotoxin-like proteins; (C), cytotoxin (cardiotoxin)-like protein; M, muscarinic toxin-like protein.

a

identifying evolutionary trends and assessing the cross-immunoreactivity among congeneric and conspecific populations from different geographic origins could provide important guidance in the design of improved novel taxon-wide antivenoms.7,25-29 The radiation of the African spitting cobras appears to date back to the early Miocene, about 16 million years ago (MYA).40 Major cladogenic events, dated at ∼13-14 MYA, separated N. katiensis from the nigricollis-mossambica group and N. nubiae from N. pallida.40 The venom toxin multigene families of this clade have been subjected to positive Darwinian selection41 over this period and a birth-anddeath model best describes the evolution of both, the 3FTx and the PLA2 multigene families.42,43 Thus, the remarkably high degree of overall inter- and intraspecific preservation of the toxin arsenal among the African spitting cobras (Figure 4) despite the huge geographic range of this clade (Figure 1) is evidence of the evolutionary success of this venom formulation. This also raises the possibility that an antivenom raised against the venom of a single spitting Naja species may exhibit paraspecific neutralization of the venom of other African spitting cobra species (see below).

Correlating Venom Composition and Pathological Effects of Envenoming

Snake venom PIII-metalloproteinases (PIII-SVMPs) are zincdependent multidomain enzymes which degrade some plasma proteins that are crucial for hemostasis and the extracellular matrix surrounding blood vessels, leading to local and systemic hemorrhage, and to coagulopathy.44-46 PIII-SVMPs are present in the venoms of Viperidae, Elapidae, Colubroidea and Atractaspidinae, supporting the view of an early recruitment event predating the radiation of the advanced (Caenophidia) snakes.47,48 The proportion of SVMPs in elapid venoms is much lower than in viperid venoms,30,49 resulting in the limited investigation on SVMPs in elapid venoms,50,51 in contrast to the extensive studies performed on viper metalloproteinases.44,45 The scarcity of SVMPs in venoms of African spitting cobras suggests that the role of these enzymes in the pathophysiology of envenomings is likely to be minor, despite the fact that local tissue damage, hemorrhage and complement depletion have been reported following bites by Naja nigricollis.21,52 The pathological significance of SVMPs in 1272



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Figure 4. Conservation of venom composition among African spitting cobras. Detail of the phylogenetic tree of African spitting cobras (adapted from W€uster et al.40), highlighting estimated dates of divergence of the Naja taxa studied in this work and the remarkable high degree of conservation of the overall composition of their venoms (Table 2) despite the huge geographic range of this clade (Figure 1).

Table 2. Overview of the Relative Occurrence of Toxins (in Percentage of the Total HPLC-Separated Proteins) of Different Families in the Venoms of African Spitting Naja Speciesa % of total venom proteins protein family 3FTx Type I R-neurotoxin-like

Naja nigricollis (Nigeria) (32)

Naja katiensis (28)

Naja pallida (22)

Naja nubiae (22)

Naja mossambica (25)

73.3

67.1

67.7

70.9

69.3

0.4

4.4

2.8

12.6

1.6

Muscarinic toxin-like

10c

150 ( 14

N. pallida

17 (13-25)

0.89 (0.68-1.31)

>50c

180 ( 9

a

Median Lethal Dose (LD50) was determined by the i.v. route in 18-20 g mice; 95% confidence limits are included in parentheses. b MND: Minimum Necrotizing Dose: Dose of venom inducing a necrotic lesion of 5 mm diameter 72 h after venom injection. c In these cases, no necrotic lesions were observed at the highest sublethal dose injected.

suggested that waprins may play a part in the offensive constitution of venoms,59 their biological functions remain elusive. Although the relevance of the low abundance venom components deserves detailed research, it seems reasonable to assume that the major pathological effects are exerted by the more abundant toxin families. This study demonstrated that the three finger toxins (3FTx) and cytotoxic PLA2 molecules60 comprise the major components of the five spitting cobra venom proteomes, accounting, respectively, for 67-73% and 22-30% of the total venom proteins. Elapidae venom group I PLA2 molecules are evolutionary derived from nontoxic pancreatic-type PLA2 and exert a multiplicity of toxic activities, including neurotoxic, myotoxic, and antiplatelet activities.61,62 In the case of N. pallida, a cytotoxic PLA2, named nigexine, has been described,60 and PLA2s exerting myotoxic, cardiotoxic and anticoagulant activities have been isolated from the venom of N. nigricollis.63-66 Since some of these PLA2s induce cytotoxicity in cell culture, they may play a role in the dermonecrotic and myonecrotic activities described for this venom.21,52,67 All venoms sampled here showed a similar PLA2 activity (Table 4), in agreement with the similar proportion of this enzyme in their proteomes. The 3FTx of elapids form a large multigene superfamily of nonenzymatic 60-74 amino-acid-residue polypeptides characterized by the presence of three finger-like β strands emerging from a globular core stabilized by four invariant disulfide linkages.68 Despite their overall structural similarity, the 3FTxs differ in their biological activities.68 A cladistic analysis revealed that 3FTxs fall

into monophyletic functional clades:42,47 type I (short-chain) and type II (long-chain) are postsynaptic R-neurotoxins that antagonize R1 (R1 and R7) neuromuscular nicotinic acetylcholine receptor (AChR) subtypes. These neurotoxins interfere with cholinergic transmission at various postsynaptic sites in the peripheral and central nervous systems. A large number of the 3FTxs also exhibit general cytolytic effects (i.e., disruption of the membrane bilayer forming pores in the cellular surface) and, therefore, they are also referred to as cytolysins or cytotoxins. The major group of 3FTxs found in the five African spitting cobra venom proteomes are the cytotoxins (Tables 1 and 2), although these venoms contain some well-known neurotoxic 3FTxs, notably Naja nigricollis toxin-R which is likely to be a major cause of lethality in the snakes' rodent prey. This neurotoxin was purified from the venom of N. nigricollis “collected in Ethiopia in 1961”69 and maintained at the Pasteur Institute (France). The fact that we did not find this protein (P01426; expected N-terminal sequence: LECHNQQSSQPPTT, and calculated isotope-averaged molecular mass, 6786.7 Da) in the venoms of N. nigricollis from Tanzania, Nigeria, Togo, and Cameroon, strongly suggests the occurrence of geographic variation in the toxin composition of this wide-ranging species or misidentification of the species from which the R-neurotoxin was isolated (Figure 1). Only N. nubiae possesses a relatively high content (12.6%) of a type I Rneurotoxin (Figure 1, HPLC peak 1; Tables 1 and 2). Such a cocktail of toxins explains the clinical effects of envenoming by African spitting cobras. Thus, bites by these species (N. nigricollis, 1274



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Figure 5. Immunodepletion of proteins from Naja nigricollis (Tanzania) venom by the EchiTAb-Plus-ICP antivenom. (A) Reverse-phase separation of the proteins from the venom of adult N. nigricollis (Tanzania) recovered after antivenomic treatment of the crude venom with an 8-fold molar excess of total EchiTAb-Plus-ICP IgGs over total venom proteins. (B and E) Ponceau red-stained nitrocellulose membrane showing electroblotted venom proteins; (C and D) replicas of the nitrocellulose membrane showed in (B) probed with a 1:4000 (v/v) dilution of the PanAfrican EchiTAb-Plus-ICP antivenom (C) and with a 1:1000 (v/v) dilution of the ICP polyvalent antivenom (D); (F) replica of the nitrocellulose membrane showed in E probed with 1:500 (v/v) dilutions of the PanAfrican EchiTAb-Plus-ICP antivenom.

N. katiensis, N. pallida, N. mossambica, N. nigricinta, N. nubiae, N. ashei) produce a distinctive clinical syndrome unlike that caused by other elapid snakes: severe local tissue damage (cytotoxicity), local necrosis without clear evidence of neurotoxicity.21,70 The venom proteomes of spitting cobras are consistent with this pathophysiological profile: the high content of cytotoxins and PLA2s are likely to be responsible for the extensive tissue necrosis, that characterize these envenomings. Thus, in the murine model used here, venoms of N. nigricollis, N. katiensis and N. mossambica induced dermonecrosis (Table 4). In the case of the venoms of N. pallida and N. nubiae, dermonecrosis was not observed at the highest sublethal doses tested (Table 4). Owing to their abundance in these venoms, it is hypothesized that cytotoxins play a key role in this local necrotizing effect, since they induce cell damage in vitro and necrosis in vivo.71-73 Moreover, at the experimental level, cardiotoxins reproduce the ocular effects (chemosis, blepharitis and corneal opacification) described in cases in which venom was spat into the eyes.74 Spitting cobras have been reported to possess less toxic venoms (LD50 15-24 μg) than known neurotoxic species (i.e., N. melanoleuca, N. haje, and N. nivea) (LD50 6-12 μg), although N. nubiae venom exhibits toxicity (LD50 8.3 μg) comparable to that of the neurotoxic nonspitters.75 Our own data summarized in Table 4 are in line with these reports. We hypothesize that the distinct feature of envenoming by N. nubiae may be ascribed to the high content (12.6% of the total venom proteins) of type I Rneurotoxin-like protein Nnu-1 (Figure 2D, Table 2) in the venom of this species. Neutralizing Capability of the Pan-African EchiTAb-Plus-ICP Antivenom

Antivenomics and Western blotting. In a previous antivenomic study,23 we showed that the EchiTAb-Plus-ICP antivenom,

produced by immunizing horses with a mixture of the venoms of Echis ocellatus, Bitis arietans, and Naja nigricollis from Nigeria,19 recognized the majority of homologous venom components but also reacted with venoms from other medically relevant subSaharan saw-scaled vipers (E. leucogaster and E. pyramidum leakeyi) and Bitis species (B. gabonica, B. rhinoceros, and B. nasicornis) not included in the immunization mixture. Preclinical analyses in mice also demonstrated the effectiveness of EchiTAb-Plus-ICP in the neutralization of the lethal and toxic activities of the above-mentioned homologous and heterologous Echis and Bitis venoms.12 The combination of antivenomics and preclinical neutralization of venom-induced pathology assays permit a detailed evaluation of the predicted clinical efficacy of antivenoms; the outcome of these laboratory assays was recently corroborated by results of a clinical trial performed in Nigeria on patients envenomed by E. ocellatus.11,22 To complete the analysis of the immunoreactivity of the EchiTAb-Plus-ICP antivenom, we have now assessed the antigen recognition spectrum, the immunological cross-reactivity, and the in vivo venom-neutralization efficacy of the EchiTAbPlus-ICP antivenom toward the venoms of N. nigricollis from different geographic origins, and from another four African spitting cobras not included in the immunization mixture, N. katiensis, N. pallida, N. mossambica, and N. nubiae, by combination of antivenomics, Western blot analysis, and neutralization assays in mice. For the antivenomic analysis the highest antivenom:venom ratio that could be used was 1.3 μL antivenom/μg of venom proteins (= 92 μg IgG/μg venom proteins). Increasing the concentration of antivenom in the reaction mixture greatly increased the nonspecificity of the antivenomic assay. Assuming an average molecular mass of 10 kDa for Naja toxins (calculated from Table 2 as [(0.7 7 kDa) þ (0.25 13.2 kDa) þ (0.03 60 kDa) = 10 kDa]), the concentration of antivenom used 1275



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Table 5. Neutralization of Toxic and PLA2 Activities by EchiTAb-Plus-ICP Antivenoma lethal activity

dermonecrotic activity

PLA2 activity

μL antivenom/mg

μL antivenom/ 3

μL antivenom/ mg

μL antivenom/ 1

μL antivenom/mg

venom

LD50s

venom

MND

venom

μL antivenom/ 5 μg venom

venom

b

N. katiensis

>1754

>142

N.

1515 (1064-2174)

100 (70-143)

604 ( 96

60 ( 10

1637 ( 163

8.2 ( 0.8

1223 ( 202

61 ( 10

1166 ( 4

5.8 ( 0.0

mossambica N. nigricollis

781 (493-1042)

54 (34-72)

645 ( 162

65 ( 16

N. nubiae

>6024b

>181

NDc

ND

1094 ( 1

890 ( 35

4.5 ( 0.2 5.5 ( 0.0

N. pallida

2632 (1923-5000)

134 (98-255)

ND

ND

1065 ( 60

5.3 ( 0.3

a Neutralizing activity is expressed as Median Effective Dose (ED50), defined as the ratio μL antivenom/mg venom or μL antivenom/ challenge dose of venom in which the activity of venom was reduced by 50% (see details in the Experimental Section). In the case of lethality, the 95% confidence limits are presented in parentheses. In the case of dermonecrosis and PLA2 activity results are presented as mean ( SD (n = 3). b In these cases, all mice died at the highest antivenom/venom ratio tested. c ND: In these cases, venoms did not induce a necrotic lesion at the highest sublethal dose tested (see Table 4). Consequently, neutralization of the effect could not be determined.

corresponded to an 8-fold molar ratio of IgG molecules over venom toxins. This antivenom:venom ratio is of the same order of magnitude as the median effective dose (ED50) (∼124 μg antivenom/μg venom) of a Pan African antivenom (73 μL (9.5 mg)/mouse), which neutralized 5xLD50 of N. nigricollis (and N. pallida) venom. This equine IgG antivenom was raised by the Colombian Instituto Nacional de Salud against 13 venoms of Echis, Bitis, and Naja species,76 but its production has been discontinued. The antivenom:venom ratio used in the antivenomic protocol is also comparable to the ED50 outlined in this work (Table 4) against challenge with 3xLD50 of N. mossambica (106 μg antivenom), N. nigricollis (55 μg antivenom), and N. pallida (184 μg antivenom), but departs from the doses required to neutralize the lethality of 3xLD50 of N. nubiae (>422 μg antivenom) and N. katiensis (>123 μg antivenom) venoms. Consistent with these data the venom of N. katiensis was the least inmunodepleted by the antivenom (Table 3). On the other hand, the low immunodepletion level (16%) of the major neurotoxin Nnu-1 (Table 3) may explain the inability of the antivenom to neutralize the lethal activity of N. nubiae venom. The results from the antivenomic analysis (illustrated in Figure 5A for the venom of N. nigricollis from Tanzania, and summarized in Table 3 for all venoms) indicated that although EchiTAb-Plus-ICP contains antibodies targeting major 3FTx and PLA2 molecules, these toxins were only partially immunodepleted. On the other hand, strikingly, the minor toxins (CRISP, SVMPs) were quantitatively removed from all the venoms. The existence of a correlation between molecular mass of venom proteins and their immunodepletion by antivenom IgGs, regardless of their relative abundance in the venom, is not surprising since the surface area accessible to the immune system is directly proportional to the molecular mass of the protein. Hence, the contact area of an antigen-antibody complex varies between 600 and 1200 A2 and involves about 14-21 surfaceexposed residues,77 and thus a small protein binds simultaneously fewer IgG molecules than a higher molecular mass protein does. However, the immunodepletion capability of an IgG molecules is also correlated to its affinity for the antigen. To assess whether (i) the minor CRISP and SVMPs induced a strong immune response or (ii) the polyvalent antivenom contains paraspecific anti-CRISP and anti-SVMPs antibodies raised against Echis and Bitis toxins included in the immunizing mixture,19 the crossreactivity of immunoblotted Naja toxins toward the

EchiTAb-Plus-ICP antivenom and the ICP polyvalent antivenom, was investigated. PIII-SVMPs and CRISP molecules comprise, respectively, 40 and 1.5% of Echis ocellatus venom proteins,78 and 23 and 2% of the Bitis gabonica proteome79 and were effectively immunodepleted from these venoms by the EchiTAb-Plus-ICP.23 The ICP polyvalent antivenom is produced at the Instituto Clodomiro Picado from the plasma of horses immunized with a mixture of the venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys;80 it does not recognize CRISP molecules but displays strong paraspecific immunodepleting activity toward PIII-SVMPs.24,27,81,82 The results of the immunoblotting experiments (Figure 5B-F) clearly showed that the two antivenoms strongly recognized the high molecular mass toxins, indicating that this reactivity must be due to cross-reactivity with homologous toxins from Echis ocellatus and/or Bitis gabonica venoms. The immunoblotting analysis also confirmed the presence of antibodies specific to the 3FTxs and PLA2 molecules (Figure 5F) in the EchiTAb-PlusICP antivenom. Neutralization Assays in Mice

In vivo assays assessed the ability of the EchiTAb-Plus-ICP antivenom to neutralize lethality, dermonecrosis, and PLA2 activity. Neutralization of lethality is the gold standard for the assessment of antivenom potency.83 We included the dermonecrosis assay because of the predominance of local necrosis in the clinical picture of spitting cobra envenoming.21 The PLA2 assay was included because of the abundance of this highly bioactive enzyme in the Naja venom proteomes studied, and because toxic PLA2s have been previously reported for these venoms.60,63-66 EchiTAb-Plus-ICP antivenom effectively neutralized the lethal, necrotizing and PLA2 activities of N. nigricollis venom (Table 5). This result agrees with previous observations19 and was expected as this venom is present in the immunization mixture.19 Regarding paraspecific protection by the antivenom, the lethal effects of the N. mossambica and N. pallida venom were neutralized by EchiTAb-Plus-ICP. This antivenom did not neutralize lethality induced by venoms of N. nubiae and N. katiensis, even at the highest antivenom dose tested (Table 5). The dermonecrotic activity of the N. katiensis and N. mossambica venoms was effectively neutralized by the antivenom (Table 5). This assay could not be studied for N. pallida and N. nubiae venoms since no dermonecrosis was observed at the highest sublethal doses tested. PLA2 activity was neutralized in all venoms, with ED50s 1276


Journal of Proteome Research ranging from 890 ( 35 μL antivenom/mg venom (N. nigricollis) to 1637 ( 163 μL antivenom/mg venom (N. katiensis) (Table 5). Paraspecific neutralization of lethality of several cobra venoms from Africa has been also reported by a monospecific anti-N. melanoleuca antivenom.75 The picture of antivenom effectiveness that emerges from our preclinical study is that the pathology associated with venoms of N. nigricollis and N. mossambica is effectively neutralized by the EchiTAb-Plus-ICP antivenom, a result consistent with the close phylogenetic kinship of these species.40 The venom of N. pallida is also neutralized, albeit at higher antivenom/venom ratios. The lack of neutralization of lethality of N. nubiae venom may be of medical relevance only in relatively populous areas of the Southeastern part of the Saharan region: Egypt, Nile Valley of northeastern Sudan and Eritrea (Figure 1). EchiTAb-Plus-ICP antivenom neutralized the dermonecrotic and PLA2 activities of N. katiensis venom but not its lethality. N. katiensis was the least immunodepleted venom in our antivenomic experiments. These results indicate that horses immunized with the venom of N. nigricollis developed a poor immune response against N. katiensis venom toxins. To improve the coverage of this antivenom for the treatment of envenoming by spitting cobras in sub-Saharan Africa, it is thus necessary to enhance the immune response against the venom of N. katiensis. This goal could be achieved by (a) increasing the immune response against N. nigricollis venom, with the possible parallel increment in paraspecific protection against N. katiensis, or (b) incorporating the venom of N. katiensis in the immunizing mixture to produce the antivenom. Further studies are required to assess the validity of these strategies and their clinical/logistic justification. Thus, the public health significance of the reduced efficacy of EchiTAb-Plus-ICP against N. katiensis venom requires careful consideration. While this species is distributed in several sub-Saharan countries, often sympatric with N. nigricollis, (Figure 1), very few bites have been reported.

’ CONCLUDING REMARKS AND PERSPECTIVES Proteomic analysis of the venoms of five species of African spitting cobras revealed similar toxin profiles characterized by the predominance of 3FTxs (mostly cytotoxins) and PLA2 molecules, and the minor presence of SVMPs, CRISPs, endonucleases and nawaprin. On the basis of this composition, it is likely that the major cytotoxins and PLA2s are responsible for the predominant toxic effects induced by these venoms (i.e., local tissue necrosis). However, the occurrence of a neurotoxic component comprising 12% of the total venom proteins in N. nubiae venom is in agreement with this venom having the highest mouse lethality among those included in this study. EchiTAb-Plus-ICP reacted with (immunoblotting assay) and immunodepleted (antivenomic assay) the major and minor homologous, and heterologous, venom components, albeit with different affinity and to varying extent in the different venoms investigated. The antivenom effectively neutralized the lethal, dermonecrotic and PLA2 activities of the venoms of N. nigricollis from Nigeria, and although not assessed in detail in this study, most probably also the highly similar venoms of this widely distributed species in Tanzania, Togo, and Cameroon. EchiTAbPlus-ICP proved also its effectiveness in neutralizing the lethal, dermonecrotic and PLA2 activities of N. mossambica venom, the lethality and PLA2 activities of the venom of N. pallida, and the PLA2 activity of the venoms of N. nubiae and N. katiensis. However, it failed to neutralize the lethal effect of the latter

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two venoms. Efforts may be required to improve the neutralizing efficacy of this antivenom against the venom of N. katiensis since, althoug this species seems to inflict very few bites, it is distributed in various countries in west sub-Saharan Africa, where it is sympatric with N. nigricollis. On the other hand, the demonstrated preclinical effectiveness of the EchiTAb-Plus-ICP antivenom against the venoms of N. nigricollis, N. mossambica, and N. pallida should prompt the design of clinical trials aimed at assessing the efficacy of this antivenom in human envenomings by these spitting cobras in Africa, thus complementing ongoing trials involving patients of systemic envenoming by E. ocellatus in Nigeria.11,22

’ AUTHOR INFORMATION Corresponding Author

*For the toxinological aspects of the study, contact Jose María Gutierrez, Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San Jose, Costa Rica. Phone: 5062229-3135. Fax: 506-2292-0485. E-mail: [email protected]. cr. For issues concerning proteomics, contact Juan J. Calvete, Instituto de Biomedicina de Valencia, C.S.I.C., Jaume Roig 11, 46010 Valencia, Spain. Phone: 34 96 339 1778. Fax: 34 96 369 0800. E-mail: [email protected].

’ ACKNOWLEDGMENT This study was supported by Vicerrectoría de Investigacion, Universidad de Costa Rica (project 741-A9-003), CRUSA-CSIC (project 2009CR0021), Ministerio de Innovacion y Ciencia (Madrid, Spain) (grants BFU2007-61563 and BFU2010-17373), PROMETEO/2010/005 from the Generalitat Valenciana, and the EchiTAb Study Group in partnership with the Federal Ministry of Health, Republic of Nigeria.

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