Omics meets biology: application to the design and preclinical assessment of antivenoms

Abstract

Snakebite envenoming represents a neglected tropical disease that has a heavy public health impact worldwide, mostly affecting poor people involved in agricultural activities in Africa, Asia, Latin America and Oceania. A key issue that complicates the treatment of snakebite envenomings is the poor availability of the only validated treatment for this disease, antivenoms. Antivenoms can be an efficacious treatment for snakebite envenoming, provided they are safe, effective, affordable, accessible and administered appropriately. The shortage of antivenoms in various regions, particularly in Sub-Saharan Africa and some parts of Asia, can be significantly alleviated by optimizing the use of current antivenoms and by the generation of novel polyspecific antivenoms having a wide spectrum of efficacy. Complementing preclinical testing of antivenom efficacy using in vivo and in vitro functional neutralization assays, developments in venomics and antivenomics are likely to revolutionize the design and preclinical assessment of antivenoms by being able to test new antivenom preparations and to predict their paraspecific neutralization to the level of species-specific toxins.

Figure 1

Highly divergent toxin compositions in phylogenetically-close snake taxa. Venom components of four Bothriechis species that inhabit Costa Rica were assigned to protein families, and their abundances were estimated, by using the “snake venomics” analytical strategy. As shown in the corresponding pie charts summarizing protein family abundances (%), the venom of Bothriechis lateralis is dominated by metalloproteinases, whereas small peptides of the vasoactive type are predominant in the venom of B. supraciliaris. On the other hand, B. schlegelii venom contains the highest proportion of phospholipases A₂, while B. nigroviridis venom completely lacks metalloproteinases and presents a high percentage of an unusual phospholipase A₂ recently characterized as a crotoxin-like complex, found for the first time in a non-rattlesnake New World pit viper. In addition, a novel protein type for snake venoms (Kazal-type proteinase inhibitor-like) was found in B. schlegelii and B. supraciliaris, but not in the other two species of this genus. Protein family abbreviations correspond to: SVMP, metalloproteinase; PLA₂, phospholipase A₂; SP, serine proteinase; CRISP, cysteine-rich secretory proteins; CTL, C-type lectins/lectin-like; VEGF, vascular endothelial growth factor; LAO, L-amino acid oxidase; NUCL, nucleotidase; DIS, disintegrin; PEP, small peptide; KAZ, Kazal-type proteinase inhibitor-like. Data adapted from Lomonte et al. [32].

Figure 1

Highly divergent toxin compositions in phylogenetically-close snake taxa. Venom components of four Bothriechis species that inhabit Costa Rica were assigned to protein families, and their abundances were estimated, by using the “snake venomics” analytical strategy. As shown in the corresponding pie charts summarizing protein family abundances (%), the venom of Bothriechis lateralis is dominated by metalloproteinases, whereas small peptides of the vasoactive type are predominant in the venom of B. supraciliaris. On the other hand, B. schlegelii venom contains the highest proportion of phospholipases A₂, while B. nigroviridis venom completely lacks metalloproteinases and presents a high percentage of an unusual phospholipase A₂ recently characterized as a crotoxin-like complex, found for the first time in a non-rattlesnake New World pit viper. In addition, a novel protein type for snake venoms (Kazal-type proteinase inhibitor-like) was found in B. schlegelii and B. supraciliaris, but not in the other two species of this genus. Protein family abbreviations correspond to: SVMP, metalloproteinase; PLA₂, phospholipase A₂; SP, serine proteinase; CRISP, cysteine-rich secretory proteins; CTL, C-type lectins/lectin-like; VEGF, vascular endothelial growth factor; LAO, L-amino acid oxidase; NUCL, nucleotidase; DIS, disintegrin; PEP, small peptide; KAZ, Kazal-type proteinase inhibitor-like. Data adapted from Lomonte et al. [32].

Figure 2

Venomics complements neutralization assays. Major effects of envenomings by the New Guinea small-eyed snake, Micropechis ikaheka, a large and powerfully-built elapid endemic to Papua New Guinea and Indonesian West Papua province, include life-threatening post-synaptic neuromuscular blockade, resulting in respiratory paralysis, PLA₂-mediated myotoxicity, hypotension and cardiovascular abnormalities. The venom proteome of M. ikaheka is dominated by at least 29 D49-phospholipases A₂ (PLA₂) and 14 short and long neurotoxins of the three-finger toxin (3FTx) family [62]. These protein classes represent, respectively, 80% and 9.2% of the total venom proteins. Reverse-phase HPLC allowed the fractionation of M. ikaheka venom (A) into 3FTx- (B) and PLA₂-enriched (C) fractions. In vivo neutralization assays showed that PLA₂ molecules represent the main myotoxic components of M. ikaheka venom. The estimated LD₅₀ for mice of the reverse-phase-isolated 3FTx- (0.22 mg/kg) and PLA₂- (1.62 mg/kg) enriched fractions, indicated that these two toxin classes contribute synergistically to venom lethality (0.62 mg/kg), with the 3FTxs playing a dominant role [62]. Reproduced with permission from reference [62]. Copyright 2014 Elsevier.

Figure 3

Antivenomics complements neutralization assays. The capability of the Costa Rican antivenom, EchiTAb-Plus-ICP^® [81], to reverse the effects of the venoms of African spitting cobras was investigated by neutralization tests [42,77]. EchiTAb-Plus-ICP^® neutralized the PLA₂ and dermonecrotic activities of all of the venoms of African Naja snakes sampled. Lethality induced by venoms of the black-necked spitting cobra (N. nigricollis), the Mozambique spitting cobra (N. mossambica) and the red spitting cobra (N. pallida) was eliminated, but did not prevent the lethal effect of the venoms of the Katian spitting cobra (N. katiensis) and the Nubian spitting cobra (N. nubiae). Antivenomics analysis showed that the antivenom immunocaptured PLA₂ molecules (Peaks 11 and 12 (B) and, to a lesser extent, a 3FTx eluted in Peak 16 (B), but had impaired the capability of the antivenom to immunodeplete a high abundance type-1 α-neurotoxin (α-NTx) of N. nubiae ((A) and (C); Peak 1, 12.6% of the total venom proteome) and N. katiensis (4.4%) venoms correlated with the pre-clinical inability of EchiTAb-Plus-ICP^® antivenom to neutralize the lethality of N. nubiae and N. katiensis venoms. Strikingly, although this lethal α-neurotoxin was originally purified “from the venom of N. nigricollis collected in Ethiopia in 1961” [82], a recent proteomics survey failed to find α-neurotoxin (6,786.7 Da, SwissProt Accession Code P01426) in N. nigricollis venoms [42] (D). African spitting cobras have had a long history of taxonomic uncertainty. Relevant for rationalizing the “α-toxin paradox”, N. nigricollis pallida was elevated to full species status by Branch [83] and Hughes [84]. This was later supported by Wüster and Broadley [85], who, in addition, described a new species, N. nubiae, in populations previously considered to belong to the Katian spitting cobra. α-Neurotoxin is expressed in venoms of N. pallida and N. nubiae (reverse-phase HPLC Peak 1, in (A) and (E), respectively), but is virtually absent from the other African spitting Naja venoms investigated. Thus, the failure of the EchiTAb-Plus-ICP^® antivenom to neutralize the lethal activity of the Nubian and Katian spitting cobra venoms may be due to the absence of α-neurotoxin epitopes in the N. nigricollis venom (D) employed in the immunization mixture to generate this antivenom. The “α-neurotoxin paradox”, resolved by combining neutralization assays and antivenomics, underpins the importance of getting the taxonomy right for the development of a strategy for the improvement of antivenoms.

Figure 3

Antivenomics complements neutralization assays. The capability of the Costa Rican antivenom, EchiTAb-Plus-ICP^® [81], to reverse the effects of the venoms of African spitting cobras was investigated by neutralization tests [42,77]. EchiTAb-Plus-ICP^® neutralized the PLA₂ and dermonecrotic activities of all of the venoms of African Naja snakes sampled. Lethality induced by venoms of the black-necked spitting cobra (N. nigricollis), the Mozambique spitting cobra (N. mossambica) and the red spitting cobra (N. pallida) was eliminated, but did not prevent the lethal effect of the venoms of the Katian spitting cobra (N. katiensis) and the Nubian spitting cobra (N. nubiae). Antivenomics analysis showed that the antivenom immunocaptured PLA₂ molecules (Peaks 11 and 12 (B) and, to a lesser extent, a 3FTx eluted in Peak 16 (B), but had impaired the capability of the antivenom to immunodeplete a high abundance type-1 α-neurotoxin (α-NTx) of N. nubiae ((A) and (C); Peak 1, 12.6% of the total venom proteome) and N. katiensis (4.4%) venoms correlated with the pre-clinical inability of EchiTAb-Plus-ICP^® antivenom to neutralize the lethality of N. nubiae and N. katiensis venoms. Strikingly, although this lethal α-neurotoxin was originally purified “from the venom of N. nigricollis collected in Ethiopia in 1961” [82], a recent proteomics survey failed to find α-neurotoxin (6,786.7 Da, SwissProt Accession Code P01426) in N. nigricollis venoms [42] (D). African spitting cobras have had a long history of taxonomic uncertainty. Relevant for rationalizing the “α-toxin paradox”, N. nigricollis pallida was elevated to full species status by Branch [83] and Hughes [84]. This was later supported by Wüster and Broadley [85], who, in addition, described a new species, N. nubiae, in populations previously considered to belong to the Katian spitting cobra. α-Neurotoxin is expressed in venoms of N. pallida and N. nubiae (reverse-phase HPLC Peak 1, in (A) and (E), respectively), but is virtually absent from the other African spitting Naja venoms investigated. Thus, the failure of the EchiTAb-Plus-ICP^® antivenom to neutralize the lethal activity of the Nubian and Katian spitting cobra venoms may be due to the absence of α-neurotoxin epitopes in the N. nigricollis venom (D) employed in the immunization mixture to generate this antivenom. The “α-neurotoxin paradox”, resolved by combining neutralization assays and antivenomics, underpins the importance of getting the taxonomy right for the development of a strategy for the improvement of antivenoms.