Proteomics and antivenomics of Papuan black snake (Pseudechis papuanus) venom with analysis of its toxicological profile and the preclinical efficacy of Australian antivenoms.

The Papuan black snake (Pseudechis papuanus Serpentes: Elapidae) is endemic to Papua New Guinea, Indonesian Papua and Australia's Torres Strait Islands. We have investigated the biological activity and proteomic composition of its venom. The P. papuanus venom proteome is dominated by a variety (n≥18) of PLA2s, which together account for ~90% of the venom proteins, and a set of low relative abundance proteins, including a short-neurotoxic 3FTx (3.1%), 3-4 PIII-SVMPs (2.8%), 3 cysteine-rich secretory proteins (CRISP; 2.3%) 1-3 l-amino acid oxidase (LAAO) molecules (1.6%). Probing of a P. papuanus cDNA library with specific primers resulted in the elucidation of the full-length nucleotide sequences of six new toxins, including vespryn and NGF not found in the venom proteome, and a calglandulin protein involved in toxin expression with the venom glands. Intravenous injection of P. papuanus venom in mice induced lethality, intravascular haemolysis, pulmonary congestion and oedema, and anticoagulation after intravenous injection, and these effects are mainly due to the action of PLA2s. This study also evaluated the in vivo preclinical efficacy of Australian black snake and polyvalent Seqirus antivenoms. These antivenoms were effective in neutralising the lethal, PLA2 and anticoagulant activities of P. papuanus venom in mice. On the other hand, all of the Seqirus antivenoms tested using an antivenomic approach exhibited strong immunorecognition of all the venom components. These preclinical results suggest that Australian Seqirus1 antivenoms may provide paraspecific protection against P. papuanus venom in humans. SIGNIFICANCE PARAGRAPH: The toxicological profile and proteomic composition of the venom of the Papuan black snake, Pseudechis papuanus, a large diurnal snake endemic to the southern coast of New Guinea and a handful of close offshore islands, were investigated. Intravenous injection of P. papuanus venom in mice induced intravascular hemolysis, pulmonary congestion and edema, anticoagulation, and death. These activities could be assigned to the set of PLA2 molecules, which dominate the P. papuanus venom proteome. This study also showed that Australian Seqirus black snake or polyvalent antivenoms were effective in neutralising the lethal, PLA2 and anticoagulant activities of the venom. These preclinical results support the continued recommendation of these Seqirus antivenoms in the clinical management of P. papuanus envenoming in Australia, Papua New Guinea or Indonesian Papua Province.

Neutralization of the neuromuscular inhibition of venom and taipoxin from the taipan (Oxyuranus scutellatus) by F(ab')2 and whole IgG antivenoms.

The neuromuscular junction activity of Oxyuranus scutellatus venom and its presynaptic neurotoxin, taipoxin, and their neutralization by two antivenoms were examined in mouse phrenic nerve-diaphragm preparations. The action of taipoxin was also studied at 21°C. The efficacy of the antivenoms was also assessed in an in vivo mouse model. Both antivenoms were effective in neutralizing the neuromuscular blocking activity in preincubation-type experiments. In experiments involving independent addition of venom and antivenoms, neutralization depended on the time interval between venom addition and antivenom application. When taipoxin was incubated for 5, 10 or 20min at 21°C, and antivenom added and temperature increased to 37°C, neutralization was achieved only when the toxin was incubated for 5 or 10min. The neutralization by the two antivenoms in an in vivo model showed that both whole IgG and F(ab')2 antivenoms were effective in neutralizing lethality. Our findings highlight the very rapid action of taipan venom at the nerve terminal, and the poor capacity of antivenoms to revert neurotoxicity as the time interval between venom or taipoxin application and antivenom addition increased. Additionally the disparity between molecular masses of the active substances of the two antivenoms did not result in differences in neutralization.

Combined venom gland cDNA sequencing and venomics of the New Guinea small-eyed snake, Micropechis ikaheka.

The venom arsenal of the New Guinea small-eyed snake, Micropechis ikaheka, was investigated by a joint cDNA sequencing and venomics approach. Twenty-seven full-length DNA sequences encoding novel venom proteins were recovered in this study. Using this cDNA dataset we achieved locus-specific resolution for 19 out of the approximately 50 reverse-phase- and SDS-PAGE-separated venom proteins. The venom proteome of M. ikaheka is dominated by at least 29 D49-phospholipase A2 (PLA2) and 14 short and long neurotoxins of the three-finger toxin (3FTx) family. These protein classes represent, respectively, 80% and 9.2% of the total venom proteins. Two PIII-metalloproteinase (SVMP) molecules (7.6%), three CRISP isoforms (1.8%), and a single Kunitz-type inhibitor, vespryn, 5'-nucleotidase, serine proteinase and LAO molecules, none of which represents more than 0.7% of the total venom proteome, complete the protein arsenal of M. ikaheka. In concordance with clinical observations, this venom composition points to a central role for post-synaptically-acting neurotoxic toxins in the envenomation strategy developed by this species. PLA2 molecules represent the main myotoxic components of M. ikaheka venom. In addition, the estimated LD50 for mice of the reverse-phase-isolated 3FTx (0.22 mg/kg) and PLA2 (1.62 mg/kg) enriched fractions, strongly suggests that these two toxin classes contribute synergistically to venom lethality, with the 3FTxs playing a dominant role. The high structural and functional conservation exhibited by M. ikaheka and Australian elapid venoms may underlay the positive clinical outcomes of envenoming resulting from bites by M. ikaheka that have been documented through the use of bioCSL polyvalent antivenom. BIOLOGICAL SIGNIFICANCE: The poorly understood venom proteome of the New Guinea small-eyed snake, Micropechis ikaheka, a large and powerfully built elapid endemic to Papua New Guinea and Indonesian West Papua province, was investigated through a combined venomics and venom gland transcriptomics approach. Although M. ikaheka accounts for only a small proportion of snakebites on the mainland, 40% of snakebites on Karkar Island are attributed to bites by this snake. Major effects of envenomings include life-threatening post-synaptic neuromuscular blockade resulting in respiratory paralysis, myotoxicity, severe bleeding, hypotension and cardiovascular abnormalities. We have investigated the contribution of 3FTxs and PLA2 molecules in venom lethality, myotoxicity, and cardiovascular function. Our work provides important correlations between venom composition and its pharmacological activity. In conjunction with the antivenomics work reported in the companion paper, our study may contribute to improve treatment outcomes for snakebite victims of M. ikaheka.

Antivenomic characterization of two antivenoms against the venom of the taipan, Oxyuranus scutellatus, from Papua New Guinea and Australia.

Antivenoms manufactured by bioCSL Limited (Australia) and Instituto Clodomiro Picado (Costa Rica) against the venom of the taipan snakes (Oxyuranus scutellatus) from Australia and Papua New Guinea (PNG), respectively, were compared using antivenomics, an analytical approach that combines proteomics with immunoaffinity chromatography. Both antivenoms recognized all venom proteins present in venom from PNG O. scutellatus, although a pattern of partial recognition was observed for some components. In the case of the Australian O. scutellatus venom, both antivenoms immunorecognized the majority of the components, but the CSL antivenom showed a stronger pattern of immunoreactivity, which was revealed by the percentage of retained proteins in the immunoaffinity column. Antivenoms interacted with taipoxin in surface plasmon resonance. These observations on antivenomics agree with previous neutralization studies.

Preclinical efficacy of Australian antivenoms against the venom of the small-eyed snake, Micropechis ikaheka, from Papua New Guinea: an antivenomics and neutralization study.

There is no specific antivenom for the treatment of envenoming by the small-eyed snake, Micropechis ikaheka, a dangerous fossorial species endemic to Papua New Guinea, Irian Jaya (West Papua) and neighbouring islands. This study evaluated one marine (sea snake) and four terrestrial (tiger snake, brown snake, black snake and polyvalent) antivenoms, manufactured in Australia by bioCSL Limited, for their ability to immunoreact ('antivenomic' analysis) and neutralize enzymatic and toxic activities of M. ikaheka venom. All antivenoms neutralized lethality of the venom and attenuated, dose-dependently, myotoxic activity. The polyvalent antivenom also neutralized cardiotoxic activity. In contrast, antivenoms were ineffective in the neutralization of phospholipase A2 (PLA2) and anticoagulant activities. Antivenomics outcomes were in concordance with neutralization tests, for chromatographic peaks corresponding to α-neurotoxins of the three finger family, responsible for lethality, were quantitatively retained in the immunoaffinity columns, whereas peaks corresponding to PLA2s were immunocaptured only to a partial extent. The ability of antivenoms to neutralize lethal, i.e. neurotoxic, and myotoxic activities of M. ikaheka venom, which represent the most relevant clinical manifestations of envenoming, suggests that these antivenoms may provide paraspecific protection in humans, although the poor neutralization of PLA2 supports the need for well-designed clinical studies to not only determine which antivenoms are most appropriate for treatment of M. ikaheka envenoming, but to also fully describe the syndrome of envenoming caused by this beautiful, but lethal species. BIOLOGICAL SIGNIFICANCE: Snakebite by the small-eyed snake, Micropechis ikaheka, in Papua New Guinea can be life-threatening. The predominant clinical features in this envenoming are neurotoxicity and systemic myotoxicity. Although it accounts for only a small proportion of snakebites on the mainland, 40% of snakebites on Karkar Island are attributed to bites by the Ikaheka snake. However, no specific antivenom is available for the treatment of M. ikaheka envenoming in Papua New Guinea. This study evaluated a panel of Australian bioCSL antivenoms for their paraspecific immunoreaction and neutralization of the toxic activities of M. ikaheka venom. All antivenoms exhibited strong immunorecognition of α-neurotoxins of the 3FTx family and neutralized the lethal, i.e. neurotoxic, and myotoxic activities of M. ikaheka venom. However, these antivenoms exhibited poor neutralization of PLA2 and anticoagulant activities. This study suggests that the Australian antivenoms may provide paraspecific protection against M. ikaheka venom in humans, a hypothesis that demands studies aimed at assessing whether these antivenoms neutralize neurotoxicity and myotoxicity in the clinical setting.

Snake venomics of two poorly known Hydrophiinae: Comparative proteomics of the venoms of terrestrial Toxicocalamus longissimus and marine Hydrophis cyanocinctus.

The venom proteomes of Toxicocalamus longissimus and Hydrophis cyanocinctus, a fossorial and a marine species, respectively, of the Hydrophiinae genus of Elapidae, were investigated by Edman degradation of RP-HPLC isolated proteins, and de novo MS/MS sequencing of in-gel derived tryptic peptide ions. The toxin arsenal of T. longissimus is made up of 1-2 type-I PLA(2) molecules, which account for 6.5% of the venom proteins, a minor PIII-SVMP (1.4% of the venom toxins), and ~20 members of the 3FTx family comprising 92% of the venom proteome. Seventeen proteins (5 type-I PLA(2)s and 12 3FTxs) were found in the venom of H. cyanocinctus. Three-finger toxins and type-I PLA(2) proteins comprise, respectively, 81% and 19% of its venom proteome. The simplicity of the H. cyanocinctus venom proteome is highlighted by the fact that only 6 venom components (3 short-chain neurotoxins, two long-chain neurotoxins, and one PLA(2) molecule) exhibit relative abundances >5%. As expected from its high neurotoxin abundance, the LD(50) for mice of H. cyanocinctus venom was fairly low, 0.132µg/g (intravenous) and 0.172µg/g (intraperitoneal). Our data indicate that specialization towards a lethal cocktail of 3FTx and type-I PLA(2) molecules may represent a widely adopted trophic solution throughout the evolution of Elapidae. Our results also points to a minimization of the molecular diversity of the toxin arsenal of the marine snake Hydrophis cyanocinctus in comparison to the venom proteome of its terrestrial relatives, and highlight that the same evolutionary solution, economy of the toxin arsenal, has been convergently adopted by different taxa in response to opposite selective pressures, loss and gain of neurotoxicity.

Comparative proteomic analysis of the venom of the taipan snake, Oxyuranus scutellatus, from Papua New Guinea and Australia: role of neurotoxic and procoagulant effects in venom toxicity.

The venom proteomes of populations of the highly venomous taipan snake, Oxyuranus scutellatus, from Australia and Papua New Guinea (PNG), were characterized by reverse-phase HPLC fractionation, followed by analysis of chromatographic fractions by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. Proteins belonging to the following seven protein families were identified in the two venoms: phospholipase A(2) (PLA(2)), Kunitz-type inhibitor, metalloproteinase (SVMP), three-finger toxin (3FTx), serine proteinase, cysteine-rich secretory proteins (CRISP), and coagulation factor V-like protein. In addition, C-type lectin/lectin-like protein and venom natriuretic peptide were identified in the venom of specimens from PNG. PLA(2)s comprised more than 65% of the venoms of these two populations. Antivenoms generated against the venoms of these populations showed a pattern of cross-neutralization, corroborating the immunological kinship of these venoms. Toxicity experiments performed in mice suggest that, at low venom doses, neurotoxicity leading to respiratory paralysis represents the predominant mechanism of prey immobilization and death. However, at high doses, such as those injected in natural bites, intravascular thrombosis due to the action of the prothrombin activator may constitute a potent and very rapid mechanism for killing prey.

Ending the drought: new strategies for improving the flow of affordable, effective antivenoms in Asia and Africa.

The development of snake antivenoms more than a century ago should have heralded effective treatment of the scourge of snakebite envenoming in impoverished, mostly rural populations around the world. That snakebite still exists today, as a widely untreated illness that maims, kills and terrifies men, women and children in vulnerable communities, is a cruel anachronism. Antivenom can be an effective, safe and affordable treatment for snakebites, but apathy, inaction and the politicisation of public health have marginalised both the problem (making snakebite arguably the most neglected of all neglected tropical diseases) and its solution. For lack of any coordinated approach, provision of antivenoms has been pushed off the public health agenda, leading to an incongruous decline in demand for these crucial antidotes, excused and fed by new priorities, an absence of epidemiological data, and a poor regulatory framework. These factors facilitated the infiltration of poor quality products that degrade user confidence and undermine legitimate producers. The result is that tens of thousands are denied an essential life-saving medicine, allowing a toll of human suffering that is a summation of many individual catastrophes. No strategy has been developed to address this problem and to overcome the intransigence and inaction responsible for the global tragedy of snakebite. Attempts to engage with the broader public health community through the World Health Organisation (WHO), GAVI, and other agencies have failed. Consequently, the toxinology community has taken on a leadership role in a new approach, the Global Snakebite Initiative, which seeks to mobilise the resources, skills and experience of scientists and clinicians for whom venoms, toxins, antivenoms, snakes and snakebites are already fields of interest. Proteomics is one such discipline, which has embraced the potential of using venoms in bio-discovery and systems biology. The fields of venomics and antivenomics have recently evolved from this discipline, offering fresh hope for the victims of snakebites by providing an exciting insight into the complexities, nature, fundamental properties and significance of venom constituents. Such a rational approach brings with it the potential to design new immunising mixtures from which to raise potent antivenoms with wider therapeutic ranges. This addresses a major practical limitation in antivenom use recognised since the beginning of the 20th century: the restriction of therapeutic effectiveness to the specific venom immunogen used in production. Antivenomic techniques enable the interactions between venoms and antivenoms to be examined in detail, and if combined with functional assays of specific activity and followed up by clinical trials of effectiveness and safety, can be powerful tools with which to evaluate the suitability of current and new antivenoms for meeting urgent regional needs. We propose two mechanisms through which the Global Snakebite Initiative might seek to end the antivenom drought in Africa and Asia: first by establishing a multidisciplinary, multicentre, international collaboration to evaluate currently available antivenoms against the venoms of medically important snakes from specific nations in Africa and Asia using a combination of proteomic, antivenomic and WHO-endorsed preclinical assessment protocols, to provide a validated evidence base for either recommending or rejecting individual products; and secondly by bringing the power of proteomics to bear on the design of new immunising mixtures to raise Pan-African and Pan-Asian polyvalent antivenoms of improved potency and quality. These products will be subject to rigorous clinical assessment. We propose radically to change the basis upon which antivenoms are produced and supplied for the developing world. Donor funding and strategic public health alliances will be sought to make it possible not only to sustain the financial viability of antivenom production partnerships, but also to ensure that patients are relieved of the costs of antivenom so that poverty is no longer a barrier to the treatment of this important, but grossly neglected public health emergency.

Preclinical evaluation of caprylic acid-fractionated IgG antivenom for the treatment of Taipan (Oxyuranus scutellatus) envenoming in Papua New Guinea.

BACKGROUND: Snake bite is a common medical emergency in Papua New Guinea (PNG). The taipan, Oxyuranus scutellatus, inflicts a large number of bites that, in the absence of antivenom therapy, result in high mortality. Parenteral administration of antivenoms manufactured in Australia is the current treatment of choice for these envenomings. However, the price of these products is high and has increased over the last 25 years; consequently the country can no longer afford all the antivenom it needs. This situation prompted an international collaborative project aimed at generating a new, low-cost antivenom against O. scutellatus for PNG. METHODOLOGY/PRINCIPAL FINDINGS: A new monospecific equine whole IgG antivenom, obtained by caprylic acid fractionation of plasma, was prepared by immunising horses with the venom of O. scutellatus from PNG. This antivenom was compared with the currently used F(ab')(2) monospecific taipan antivenom manufactured by CSL Limited, Australia. The comparison included physicochemical properties and the preclinical assessment of the neutralisation of lethal neurotoxicity and the myotoxic, coagulant and phospholipase A(2) activities of the venom of O. scutellatus from PNG. The F(ab')(2) antivenom had a higher protein concentration than whole IgG antivenom. Both antivenoms effectively neutralised, and had similar potency, against the lethal neurotoxic effect (both by intraperitoneal and intravenous routes of injection), myotoxicity, and phospholipase A(2) activity of O. scutellatus venom. However, the whole IgG antivenom showed a higher potency than the F(ab')(2) antivenom in the neutralisation of the coagulant activity of O. scutellatus venom from PNG. CONCLUSIONS/SIGNIFICANCE: The new whole IgG taipan antivenom described in this study compares favourably with the currently used F(ab')(2) antivenom, both in terms of physicochemical characteristics and neutralising potency. Therefore, it should be considered as a promising low-cost candidate for the treatment of envenomings by O. scutellatus in PNG, and is ready to be tested in clinical trials.