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.

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.