Snake Venomics and Antivenomics of Cape Cobra (Naja nivea) from South Africa: Insights into Venom Toxicity and Cross-Neutralization Activity.

Naja nivea (Cape Cobra) is endemic to southern Africa. Envenoming by N. nivea is neurotoxic, resulting in fatal paralysis. Its venom composition, however, has not been studied in depth, and specific antivenoms against it remain limited in supply. Applying a protein decomplexation approach, this study unveiled the venom proteome of N. nivea from South Africa. The major components in the venom are cytotoxins/cardiotoxins (~75.6% of total venom proteins) and alpha-neurotoxins (~7.4%), which belong to the three-finger toxin family. Intriguingly, phospholipase A2 (PLA2) was undetected-this is a unique venom phenotype increasingly recognized in the African cobras of the Uraeus subgenus. The work further showed that VINS African Polyvalent Antivenom (VAPAV) exhibited cross-reactivity toward the venom and immunorecognized its toxin fractions. In mice, VAPAV was moderately efficacious in cross-neutralizing the venom lethality with a potency of 0.51 mg/mL (amount of venom completely neutralized per milliliter of antivenom). In the challenge-rescue model, VAPAV prevented death in 75% of experimentally envenomed mice, with slow recovery from neurotoxicity up to 24 h. The finding suggests the potential para-specific utility of VAPAV for N. nivea envenoming, although a higher dose or repeated administration of the antivenom may be required to fully reverse the neurotoxic effect of the venom.

Equatorial Spitting Cobra (Naja sumatrana) from Malaysia (Negeri Sembilan and Penang), Southern Thailand, and Sumatra: Comparative Venom Proteomics, Immunoreactivity and Cross-Neutralization by Antivenom.

The Equatorial Spitting Cobra (Naja sumatrana) is a medically important venomous snake species in Southeast Asia. Its wide geographical distribution implies potential intra-specific venom variation, while there is no species-specific antivenom available to treat its envenoming. Applying a protein-decomplexing proteomic approach, the study showed that three-finger toxins (3FTX), followed by phospholipases A2 (PLA2), were the major proteins well-conserved across N. sumatrana venoms of different locales. Variations were noted in the subtypes and relative abundances of venom proteins. Of note, alpha-neurotoxins (belonging to 3FTX) are the least in the Penang specimen (Ns-PG, 5.41% of total venom proteins), compared with geographical specimens from Negeri Sembilan (Ns-NS, 14.84%), southern Thailand (Ns-TH, 16.05%) and Sumatra (Ns-SU, 10.81%). The alpha-neurotoxin abundance, in general, correlates with the venom's lethal potency. The Thai Naja kaouthia Monovalent Antivenom (NkMAV) was found to be immunoreactive toward the N. sumatrana venoms and is capable of cross-neutralizing N. sumatrana venom lethality to varying degrees (potency = 0.49-0.92 mg/mL, interpreted as the amount of venom completely neutralized per milliliter of antivenom). The potency was lowest against NS-SU venom, implying variable antigenicity of its lethal alpha-neurotoxins. Together, the findings suggest the para-specific and geographical utility of NkMAV as treatment for N. sumatrana envenoming in Southeast Asia.

On characterizing the Red-headed Krait (Bungarus flaviceps) venom: Decomplexation proteomics, immunoreactivity and toxicity cross-neutralization by hetero-specific antivenoms.

The Red-headed Krait (Bungarus flaviceps) is a medically important venomous snake species in Southeast Asia, while there is no specific antivenom available for its envenoming. This study investigated the venom composition through a decomplexation proteomic approach, and examined the immunoreactivity as well as cross-neutralization efficacy of two hetero-specific krait antivenoms, Bungarus candidus Monovalent Antivenom (BcMAV) and Bungarus fasciatus Monovalent Antivenom (BfMAV), against the venom of B. flaviceps from Peninsular Malaysia. A total of 43 non-redundant proteoforms belonging to 10 toxin families were identified in the venom proteome, which is dominated by phospholipases A2 including beta-bungarotoxin lethal subunit (56.20 % of total venom proteins), Kunitz-type serine protease inhibitors (19.40 %), metalloproteinases (12.85 %) and three-finger toxins (7.73 %). The proteome varied in quantitative aspect from the earlier reported Indonesian (Sumatran) sample, suggesting geographical venom variation. BcMAV and BfMAV were immunoreactive toward the B. flaviceps venom, with BcMAV being more efficacious in immunological binding. Both antivenoms cross-neutralized the venom lethality with varying efficacy, where BcMAV was more potent than BfMAV by ~13 times (normalized potency: 38.04 mg/g vs. 2.73 mg/g, defined as the venom amount completely neutralized by one-gram antivenom protein), supporting the potential utility of BcMAV for para-specific neutralization against B. flaviceps venom.

Elucidating the Venom Diversity in Sri Lankan Spectacled Cobra (Naja naja) through De Novo Venom Gland Transcriptomics, Venom Proteomics and Toxicity Neutralization.

Inadequate effectiveness of Indian antivenoms in treating envenomation caused by the Spectacled Cobra/Indian Cobra (Naja naja) in Sri Lanka has been attributed to geographical variations in the venom composition. This study investigated the de novo venom-gland transcriptomics and venom proteomics of the Sri Lankan N. naja (NN-SL) to elucidate its toxin gene diversity and venom variability. The neutralization efficacy of a commonly used Indian antivenom product in Sri Lanka was examined against the lethality induced by NN-SL venom in mice. The transcriptomic study revealed high expression of 22 toxin genes families in NN-SL, constituting 46.55% of total transcript abundance. Three-finger toxins (3FTX) were the most diversely and abundantly expressed (87.54% of toxin gene expression), consistent with the dominance of 3FTX in the venom proteome (72.19% of total venom proteins). The 3FTX were predominantly S-type cytotoxins/cardiotoxins (CTX) and α-neurotoxins of long-chain or short-chain subtypes (α-NTX). CTX and α-NTX are implicated in local tissue necrosis and fatal neuromuscular paralysis, respectively, in envenomation caused by NN-SL. Intra-species variations in the toxin gene sequences and expression levels were apparent between NN-SL and other geographical specimens of N. naja, suggesting potential antigenic diversity that impacts antivenom effectiveness. This was demonstrated by limited potency (0.74 mg venom/ml antivenom) of the Indian polyvalent antivenom (VPAV) in neutralizing the NN-SL venom. A pan-regional antivenom with improved efficacy to treat N. naja envenomation is needed.

A Neurotoxic Snake Venom without Phospholipase A2: Proteomics and Cross-Neutralization of the Venom from Senegalese Cobra, Naja senegalensis (Subgenus: Uraeus).

The Senegalese cobra, Naja senegalensis, is a non-spitting cobra species newly erected from the Naja haje complex. Naja senegalensis causes neurotoxic envenomation in Western Africa but its venom properties remain underexplored. Applying a protein decomplexation proteomic approach, this study unveiled the unique complexity of the venom composition. Three-finger toxins constituted the major component, accounting for 75.91% of total venom proteins. Of these, cardiotoxin/cytotoxin (~53%) and alpha-neurotoxins (~23%) predominated in the venom proteome. Phospholipase A2, however, was not present in the venom, suggesting a unique snake venom phenotype found in this species. The venom, despite the absence of PLA2, is highly lethal with an intravenous LD50 of 0.39 µg/g in mice, consistent with the high abundance of alpha-neurotoxins (predominating long neurotoxins) in the venom. The hetero-specific VINS African Polyvalent Antivenom (VAPAV) was immunoreactive to the venom, implying conserved protein antigenicity in the venoms of N. senegalensis and N. haje. Furthermore, VAPAV was able to cross-neutralize the lethal effect of N. senegalensis venom but the potency was limited (0.59 mg venom completely neutralized per mL antivenom, or ~82 LD50 per ml of antivenom). The efficacy of antivenom should be further improved to optimize the treatment of cobra bite envenomation in Africa.

Quantitative proteomics of Naja annulifera (sub-Saharan snouted cobra) venom and neutralization activities of two antivenoms in Africa.

Envenomation by Naja annulifera (snouted cobra), a non-spitting African cobra, can result in local tissue damage and fatal paralysis but a species-specific antivenom treatment is currently lacking. In this study, we investigated the quantitative proteome of N. annulifera venom, incorporating HPLC and LC-MS/MS to elucidate the venom toxicity. The immunoreactivities and in vivo neutralization activities of two hetero-specific antivenom products (Premium Serums Pan Africa polyvalent antivenom, PANAF and VINS African polyvalent antivenom, VAPAV) against the venom were subsequently examined. N. annulifera venom comprises 10 toxin families, with three-finger toxin (3FTx) being the most abundantly expressed (~78%). Within 3FTx, cytotoxin is the most dominant form and made up three-quarter of the venom bulk (~74%), whereas alpha-neurotoxins constitute <4% of the total venom proteins. Phospholipase A2 was undetected in the venom proteome, consistent with the unusual absence of PLA2 from the venoms of cobras in the Uraeus subgenus. In ELISA, PANAF and VAPAV showed comparable immunoreactivity toward the protein antigens of N. annulifera venom. These antivenoms, despite being raised against hetero-specific venoms, were capable of cross-neutralizing the lethal effect of N. annulifera venom in mice, with PANAF being marginally more potent.

Proteomic insights into short neurotoxin-driven, highly neurotoxic venom of Philippine cobra (Naja philippinensis) and toxicity correlation of cobra envenomation in Asia.

The Philippine cobra, Naja philippinensis, is a WHO Category 1 venomous snake of medical importance responsible for fatal envenomation in the northern Philippines. To elucidate the venom proteome and pathophysiology of envenomation, N. philippinensis venom proteins were decomplexed with reverse-phase high-performance liquid chromatography, and protein fractions were subsequently digested with trypsin, followed by nano-liquid chromatography-tandem mass spectrometry analysis and data mining. Three-finger toxins (3FTX, 66.64% of total venom proteins) and phospholipases A2 (PLA2, 22.88%) constitute the main bulk of venom proteome. Other proteins are present at low abundances (<4% each); these include metalloproteinase, serine protease, cobra venom factor, cysteine-rich secretory protein, vespryn, phosphodiesterase, 5' nucleotidase and nerve growth factor. In the three-finger toxin family, the alpha-neurotoxins comprise solely short neurotoxins (SNTX, 44.55%), supporting that SNTX is the principal toxin responsible for neuromuscular paralysis and lethality reported in clinical envenomation. Cytotoxins (CTX) are the second most abundant 3FTX proteins in the venom (21.31%). The presence of CTX correlates with the venom cytotoxic effect, which is more prominent in murine cells than in human cells. From the practical standpoint, SNTX-driven neuromuscular paralysis is significant in N. philippinensis envenomation. Antivenom production and treatment should be tailored accordingly to ensure effective neutralization of SNTX. BIOLOGICAL SIGNIFICANCE: The venom proteome of Naja philippinensis, the Philippine cobra, is unravelled for the first time. Approximately half the protein bulk of the venom is made up of short neurotoxins (44.55% of the total venom proteins). As the only alpha-neurotoxins present in the venom, short neurotoxins are the causative toxins of the post-synaptic blockade and fast-onset neuromuscular paralysis in N. philippinensis envenomation. A substantial amount of cytotoxins (21.31%) was also detected in N. philippinensis venom, supporting that the venom can be cytotoxic although the effect is much weaker in human cells compared to murine cells. The finding is consistent with the low incidence of local tissue necrosis in N. philippinensis envenomation, although this does not negate the need for monitoring and care of bite wound in the patients.

Distinctive Distribution of Secretory Phospholipases A2 in the Venoms of Afro-Asian Cobras (Subgenus: Naja, Afronaja, Boulengerina and Uraeus).

The protein abundances of phospholipases A2 in cobra venom proteomes appear to vary among cobra species. To determine the unique distribution of snake venom phospholipases A2 (svPLA2) in the cobras, the svPLA2 activities for 15 cobra species were examined with an acidimetric and a colorimetric assay, using egg yolk suspension and 4-nitro-3-octanoyloxy benzoic acid (NOBA) as the substrate. The colorimetric assay showed significant correlation between svPLA2 enzymatic activities with the svPLA2 protein abundances in venoms. High svPLA2 activities were observed in the venoms of Asiatic spitting cobras (Naja sputatrix, Naja sumatrana) and moderate activities in Asiatic non-spitters (Naja naja, Naja atra, Naja kaouthia), African spitters (subgenus Afronaja), and forest cobra (subgenus Boulengerina). African non-spitting cobras of subgenus Uraeus (Naja haje, Naja annulifera, Naja nivea, Naja senegalensis) showed exceptionally low svPLA2 enzymatic activities. The negligible PLA2 activity in Uraeus cobra venoms implies that PLA2 may not be ubiquitous in all snake venoms. The svPLA2 in cobra envenoming varies depending on the cobra species. This may potentially influence the efficacy of cobra antivenom in specific use for venom neutralization.

Elucidating the biogeographical variation of the venom of Naja naja (spectacled cobra) from Pakistan through a venom-decomplexing proteomic study.

Naja naja is a medically important species that is distributed widely in South Asia. Its venom lethality and neutralization profile have been reported to vary markedly, but the understanding of this phenomenon has been limited without a comprehensive venom profile for the Pakistani N. naja. This study set to investigate the venom proteome of Pakistani N. naja applying reverse-phase HPLC, SDS-PAGE, mass spectrometry and data-mining approaches. The venom enzymatics and antigen binding activities were also studied. A total of 55 venom proteins comprising 11 toxin families were identified, with three-finger toxins (75.29%) being the predominant component, followed by phospholipase A2 (14.24%) and other proteins (<5%). The enzyme activities of most of the venom components were also detected in this work. The high abundance of long neurotoxins (LNTX, 21.61%) in the Pakistani N. naja venom is varied from that reported for N. naja venoms from other geographical origins. The venom exhibited high immunoreactivity toward Naja kaouthia monovalent antivenom (NKMAV), which was raised against the LNTX-predominated heterologous Thai N. kaouthia venom. Together, the findings show that the Pakistani N. naja venom is predominated by LNTX, and this unique property correlates with its high lethality and effective neutralization by the heterologous NKMAV. BIOLOGICAL SIGNIFICANCE: This study reveals the compositional details of the venom proteome of Pakistani spectacled cobra (Naja naja). The protein subtypes, proteoforms, and relative abundances of individual proteins were comprehensively revealed in this study, following a venom decomplexing proteomic approach. The Pakistani cobra venom is unique among the rest of the N. naja venom composition reported thus far, as it contains a high abundance of alpha-neurotoxins (predominated by long neurotoxins); these are highly potent post-synaptic neuromuscular blockers that cause paralysis and are principal toxins that account for the high lethality of the venom (LD50=0.2µg/g in mice). In contrast, previous reports showed that the N. naja venoms of India and Sri Lanka had a lower content of neurotoxins and a relatively higher value of LD50. The Pakistani cobra venom demonstrated sufficient immunoreactivity toward three antivenom products manufactured outside Pakistan (including the Indian product VINS), however the potency of antigen binding was the highest toward Naja kaouthia monovalent antivenom, a heterologous antivenom raised against a long neurotoxin-predominated venom of the Thai monocled cobra. From the practical standpoint, the findings indicate that the treatment of N. naja envenomation in Pakistan may be improved by the production of a locale-specific antivenom, in which the antivenom produced contains more antibodies that can target and react more specifically with the highly abundant lethal neurotoxins in the Pakistani N. naja venom.

Venom proteome of the yellow-lipped sea krait, Laticauda colubrina from Bali: Insights into subvenomic diversity, venom antigenicity and cross-neutralization by antivenom.

The venom proteome of Laticauda colubrina (Bali, Indonesia) was elucidated by nano-ESI-LCMS/MS of the venom reverse-phase HPLC fractions. Altogether 31 distinct forms of proteins were identified and clustered into three toxin families: three-finger toxin (3FTX, 66.12% of total venom proteins), phospholipase A2 (PLA2, 33.26%) and cysteine-rich secretory protein (CRiSP, 0.05%). The 3FTX were α-neurotoxins (five long neurotoxins, LNTX, 48.87%; two short neurotoxins, SNTX, 16.94%) and a trace amount of two cytotoxins (CTX, 0.31%). PLA2 were present with a large diversity of homologues (≥20 forms), however none was annotated to the lethal proteoform reported previously. The venom is highly lethal in mice (LD50=0.10µg/g) and this is driven primarily by the SNTX and LNTX (LD50=0.05-0.13µg/g), since the PLA2 proteins were non-lethal up to 2µg/g (20-time the venom LD50). The SNTX and LNTX were effectively cross-neutralized by the heterologous Sea Snake Antivenom (SSAV, Australian product) (potency=0.27mg toxin per ml antivenom, and 0.40mg/ml, respectively), corroborating the cross-neutralization of the whole venom (potency=1.09mg/ml) and its antigenic immunoreactivity toward SSAV. Furthermore, compared with earlier studies, the present work reveals geographical variation of venom composition for L. colubrina which may have implication for the evolution and conservation of the species. BIOLOGICAL SIGNIFICANCE: Laticauda colubrina (yellow-lipped sea krait) is a widely distributed, semi-aquatic venomous snake species. The venom proteome at the level of protein family is unsophisticated and consistent with its restricted prey selection. Nonetheless, the subproteomic findings revealed geographical variability of the venom for this widely distributed species. In contrast to two previous reports, the results for the Balinese L. colubrina venom showed that LNTX Neurotoxin a and Neurotoxin b were co-existent while the PLA2 lethal subtype (PLA-II) was undetected by means of LCMS/MS and by in vivo assay. This is an observable trait of L. colubrina considered divergent from specimens previously studied for the Philippines and the Solomon Islands. The stark geographical variation might be reflective of trophic adaptation following evolutionary arms race between the snake and the prey (eels) in different localities. The preferred trait would likely propagate and remain significant within the geographical population, since the strong behaviour of site fidelity in the species would have minimized gene flow between distant populations. Meanwhile, the in vivo neutralization study verified that the efficacy of the heterologous Sea Snake Antivenom (Australian product) is attributable to the cross-neutralization of SNTX and LNTX, two principal lethal toxins that made up the bulk of L. colubrina venom proteins. The findings also implied that L. colubrina, though could be evolutionarily more related to the terrestrial elapids, has evolved a much streamlined, neurotoxin- and PLA2-predominated venom arsenal, with major antigenicity shared among the true sea snakes and the Australo-Papuan elapids. The findings enrich our current understanding of the complexity of L. colubrina venom and the neutralizing spectrum of antivenom against the principal toxins from this unique elapid lineage.

Venomics of Naja sputatrix, the Javan spitting cobra: A short neurotoxin-driven venom needing improved antivenom neutralization.

The venom proteome of Naja sputatrix (Javan spitting cobra) was elucidated through reverse-phase HPLC, nano-ESI-LCMS/MS and data mining. A total of 97 distinct protein forms belonging to 14 families were identified. The most abundant proteins are the three-finger toxins (3FTXs, 64.22%) and phospholipase A2 (PLA2, 31.24%), followed by nerve growth factors (1.82%), snake venom metalloproteinase (1.33%) and several proteins of lower abundance (<1%) including a variety of venom enzymes. At subproteome, the 3FTx is dominated by cytotoxins (48.08%), while short neurotoxins (7.89%) predominate over the long neurotoxins (0.48%) among other neurotoxins of lesser toxicity (muscarinic toxin-like proteins, 5.51% and weak neurotoxins, 2.26%). The major SNTX, CTX and PLA2 toxins were isolated with intravenous median lethal doses determined as 0.13, 1.06 and 0.50µg/g in mice, respectively. SABU, the Indonesia manufactured homologous tri-specific antivenom could neutralize the CTX and PLA2 fraction with moderate potency (potency=0.14-0.16mg toxin per ml antivenom). The SNTX, however, was very poorly neutralized with a potency level of 0.034mg/ml, indicating SNTX as the main limiting factor in antivenom neutralization. The finding helps elucidate the inferior efficacy of SABU reported in neutralizing N. sputatrix venom, and supports the call for antivenom improvement. BIOLOGICAL SIGNIFICANCE: The Javan spitting cobra, Naja sputatrix is by itself a unique species and should not be confused as the equatorial and the Indochinese spitting cobras. The distinction among the spitting cobras was however unclear prior to the revision of cobra systematics in the mid-90's, and results of some earlier studies are now questionable as to which species was implicated back then. The current study successfully profiled the venom proteome of authenticated N. sputatrix, and showed that the venom is made up of approximately 64% three-finger toxins (including neurotoxins and cytotoxins) and 31% phospholipases A2 by total venom proteins. The findings verified that the paralyzing components in the venom i.e. neurotoxins are predominantly the short-chain subtype (SNTX) far exceeding the long-chain subtype (LNTX) which is more abundant in the venoms of monocled cobra and Indian common cobra. The neurotoxicity of N. sputatrix venom is hence almost exclusively SNTX-driven, and effective neutralization of the SNTX is the key to early reversal of paralysis. Unfortunately, as shown through a toxin-specific assay, the immunological neutralization of the SNTX using the Indonesian antivenom (SABU) was extremely weak, implying that SABU has limited therapeutic efficacy in treating N. sputatrix envenomation clinically. From the practical standpoint, actions need to be taken at all levels from laboratory to production and policy making to ensure that the shortcoming is overcome.

Venom and Purified Toxins of the Spectacled Cobra (Naja naja) from Pakistan: Insights into Toxicity and Antivenom Neutralization.

Geographical variations of snake venoms can result in suboptimal effectiveness of Indian antivenoms that are currently used in most South Asian countries. This study investigated the toxicity and neutralization profile of the venom and toxins from Pakistani spectacled cobra, Naja naja, using VINS polyvalent antivenom (VPAV, India), Naja kaouthia monovalent antivenom (NKMAV, Thailand), and neuro bivalent antivenom (NBAV, Taiwan). Cation-exchange and reverse-phase high-performance liquid chromatography fractionations followed by toxin identification through liquid chromatography-mass spectrometry (MS)/MS indicated that the venom comprised mainly of postsynaptic neurotoxins (NTXs) (long neurotoxins [LNTXs], 28.3%; short neurotoxins [SNTXs], 8%), cytotoxins (CTXs) (31.2%), and acidic phospholipases A2 (12.3%). NKMAV is the most effective in neutralizing the lethal effect of the venom (potency = 1.1 mg venom/mL) and its LNTX (potency = 0.5 mg toxin/mL), consistent with the high content of LNTX in N. kaouthia venom. VPAV was effective in neutralizing the CTX (potency = 0.4 mg toxin/mL), in agreement with the higher CTX abundance in Indian cobra venom. All the three antivenoms were weak in neutralizing the SNTX (potency = 0.03-0.04 mg toxin/mL), including NBAV that was raised from the SNTX-rich Taiwanese cobra venom. In a challenge-rescue experiment, envenomed mice were prevented from death by a maximal dose of VPAV (intravenous 200 µL) but the recovery from paralysis was slow, indicating the need for higher or repeated doses of VPAV. Our results suggest that optimal neutralization for Pakistani N. naja venom may be achieved by improving the formulation of antivenom production to enhance antivenom immunoreactivity against long and SNTXs.