Snakebite envenoming by the monocled cobra (Naja kaouthia) is a major health burden in Southeast Asia, driven largely by the tissue-damaging effects of cytotoxin-II (CTX-II). This study identifies a human light-chain monoclonal antibody, clone E1, with dose-dependent binding and good selectivity for CTX-II using phage display biopanning. While pairing E1 with a heavy-chain domain to form an scFv (clone E1-H2) slightly optimized binding at high concentrations, it significantly reduced protein solubility and expression yield compared to the standalone domain. The antibody demonstrated broad, genus-specific recognition across various Asiatic and African cobras while showing no cross-reactivity with non-cobra elapid or viperid venoms. These results suggest that the smaller, more stable architecture is better suited for accessing epitopes on compact toxins like CTX-II. Consequently, the E1 VL domain antibody is a promising, high-stability candidate for developing regional point-of-care diagnostics for cobra envenoming.

Short-chain α-neurotoxins (SNTXs) are the principal neurotoxic components in several Asiatic cobras, including Naja atra , Naja philippinensis , and N. samarensis . Although structurally conserved, SNTXs exhibit marked antigenic variation that can limit the effectiveness of regional antivenoms used for snakebite envenoming in Asia. Here, we evaluated the immunoreactivity of the Philippine Cobra Antivenom (PCAV) and three regional products— Naja kaouthia Monovalent Antivenom (NkMAV), Neuro Bivalent Antivenom (NBAV), and Indonesian SABU—against a panel of cobra venoms and purified α-neurotoxins. PCAV bound strongly to homologous N. philippinensis SNTX but showed weak cross-reactivity with SNTXs from N. kaouthia , N. sputatrix , and N. atra , and minimal recognition of marine elapid SNTXs and long-chain α-neurotoxins (LNTXs) of Monocled Cobra as well as King Cobra. Conversely, NkMAV and NBAV recognized mainland Asian SNTXs more broadly but reacted poorly with the Philippine cobra toxin. Hierarchical clustering of normalized immunoreactivity delineated two major SNTX antigenic subtypes corresponding to Philippine versus continental Asian lineages. Peptide sequence analysis unmasked two distinct loop-II motifs ( 28 WWS–TII 37 and 28 RWR–YRT 37 ) associated with these divergent immunotypes. Phylogenetic reconstruction suggests that the Philippine cobras retain an ancestral SNTX motif, while Sundaic and East Asian cobras have diversified to employ another major SNTX form. Epitope prediction further revealed differences in surface exposure and accessibility that help explain the limited cross-neutralization across antivenoms. These findings demonstrate a clear antigenic dichotomy among Asian cobra SNTXs, which underlies species-specific antivenom effectiveness and highlights the need to incorporate representative SNTX variants into immunogen formulations to improve regional antivenom coverage.

High molecular weight proteins are present abundantly in viperid venoms. The amino acid sequence can be highly variable, contributing to the structure and function diversity of snake venom protein. However, this variability remains poorly understood in many species. The study investigated the venom protein variability in a distinct clade of Asian pit vipers (Trimeresurus species complex) through comparative proteomics, applying gel electrophoresis (SDS-PAGE), liquid chromatography-tandem mass spectrometry (LCMS/MS), and bioinformatic approaches. The proteomes revealed a number of conserved protein families, within each are variably expressed protein paralogs that are unrelated to the snake phylogeny and geographic origin. The expression levels of two major enzymes, i.e., snake venom serine proteinase and metalloproteinase, correlate weakly with procoagulant and hemorrhagic activities, implying co-expression of other functionally versatile toxins in the venom. The phospholipase A2 (PLA2) abundance correlates strongly with its enzymatic activity, and a unique phenotype was discovered in two species expressing extremely little PLA2. The commercial mono-specific antivenom effectively neutralized the venoms' procoagulant and hemorrhagic effects but failed to inhibit the PLA2 activities. Instead, the PLA2 activities of all venoms were effectively inhibited by the small molecule inhibitor varespladib, suggesting its potential to be repurposed as a highly potent adjuvant therapeutic in snakebite envenoming.

Background: Although antivenom is the standard treatment for snakebite envenoming, its efficacy may be impacted by geographic variation in venom composition, emphasizing the need for region-specific antivenom development. Methods: We report a case of snakebite envenoming, in which the patient was bitten on the hand by a captive Malayan pit viper (Calloselasma rhodostoma) with typical clinical manifestations following. Antivenom (produced in Thailand) was administered at 33 and 39 h post-bite. Venom from the causative individual snake was collected for compositional analysis via SDS-PAGE. Enzymatic activity of the venom was evaluated through the degradation of casein and phospholipid substrates, along with the assessment of enzymatic inhibition by two regionally specific antivenoms produced in Vietnam (AV. Cr. VN.) and Thailand (AV. Cr. TL.). Results: The patient showed good recovery, with complete normalization by day 7. SDS-PAGE profiling of the venom revealed five major enzymes, with SVSP, SVMP and PLA2 being the most abundant (16.7%, 40.11% and 26.11%, respectively). Antivenom inhibition tests revealed remaining casein percentages of 67.43% (AV. Cr. VN) and 59.35% (AV. Cr. TL). Blood agar assays indicated that phospholipase activity was reduced to 21.01% by AV. Cr. VN. and 23.30% by AV. Cr. TL. Conclusions: Our results show that the Vietnamese antivenom generated greater inhibitory activity against proteinases compared to the Thai product, underscoring the importance of using regionally specific antivenoms that are more effective against the venom profiles of locality-matched snake populations.

Monoclonal antibodies are commonly expressed using mammalian cell lines due to their required post-translational modifications. However, this process can become costly and requires investment in specialized equipment and facilities. The use of eukaryotic cell Leishmania tarentolae offers a cost-effective alternative while still allowing N-linked glycosylation to occur. The focus of this study explores the use of L. tarentolae as a platform for expressing a monoclonal IgG antibody against the long-chain neurotoxin (LNTX) from the venom of the Monocled Cobra, Naja kaouthia. The monoclonal antibody was isolated from a human naïve phage display library and expressed as single-chain fragment variable (scFv) in Escherichia coli with good specificity against the LNTX of Naja kaouthia. The scFv was then converted to the scFv-Fc format and full-length IgG for expression in L. tarentolae. The IgG expression was achieved using a 2A peptide-based bicistronic vector system with good expression yield. The expressed IgG demonstrated homogeneity in size and minimal degradation. Therefore, L. tarentolae can be considered a possible alternative platform for the expression of human scFv-Fc and IgG antibodies.

Snakebite envenoming (SBE) is a priority Neglected Tropical Disease listed by the World Health Organization. South Asia is heavily affected, and virtually all countries in the region import polyvalent antivenom products from India for clinical use. The imported antivenoms, however, have suboptimal effectiveness due to geographical venom variation. Recently, a domestic bivalent product, named Pakistani Viper Antivenom (PVAV) has been developed specifically for Pakistani vipers, Echis carinatus sochureki and Daboia russelii. As a bivalent viperid antivenom, it is unknown yet if PVAV exhibits higher immunological binding and neutralization activities against viper venoms from distant locales compared with polyvalent antivenoms manufactured in India. This study thus examined the preclinical efficacy of PVAV against venoms of Western Russell's Vipers and Saw-scaled Viper subspecies from selected locales in the Indian subcontinent. PVAV generally outperformed the commonly used VINS polyvalent antivenom (VPAV, manufactured in India) in binding toward venoms, and showed superior or comparable neutralization efficacy against the venom procoagulant and hemorrhagic effects of Saw-scaled Vipers as well as Russell's Vipers from Pakistan and Sri Lanka. Based on normalized potency values, PVAV is far more potent than VPAV in neutralizing the lethality of all viper venoms, except that of the Indian Russell's Viper. The study shows conserved antigenicity of toxins responsible for major toxicity across these viperid venoms, and suggests the feasible production of a viper-specific antivenom with higher potency and broader geographical utility for the region.

OBJECTIVE:There is very limited published experience on mangrove pit viper envenomation in the medical literature. This study aims to analyze the clinical characteristics, treatment modalities and outcomes of patients presenting to Selangor middle zone cluster Hospitals in Malaysia with confirmed mangrove pit viper bites. METHODS:We conducted a retrospective observational study, reviewing medical records of patients treated for mangrove pit viper bites between July 1, 2020 to June 30, 2023. Data on patient demographics, clinical characteristic, laboratory findings, treatment modalities and clinical outcomes were collected and analyzed. RESULTS:A total of 25 patients were included in this study. The majority of the patients were male (n = 23, 92%) with the mean age of 38.7 ± 17.6 years. Most frequent anatomical region involved is foot (n = 12, 48%). Common clinical presentation included localized pain (n = 24, 96%), swelling (n = 22, 88%) and fang mark (n = 22, 88%). Systemic symptoms were less common, with 1 patient exhibiting coagulopathy with clinical bleeding at 28 h post bite. Antivenom was administered to 68% (n = 17) of the patients. The majority of the patients (n = 23, 92%) recovered without significant morbidity while 8% (n = 2) of the patients developed skin infection that required antibiotic therapy. No fatalities were reported. CONCLUSION:Mangrove pit viper envenomation encountered in these regions predominantly causes local symptoms while systemic symptoms were less common. This study provides a glimpse to the clinical characteristics and management of mangrove pit viper envenomation, coagulopathy may be delayed due to characteristic of the snake venom and patient's preexisting illness. Further research is needed to enhance our understanding of this snakebite envenomation.

Echis ocellatus is one of the commonest snakes responsible for envenomation in Nigeria. Antivenom is the only effective treatment, but the country suffers from a limited supply of effective antivenom. This study therefore aimed to explore the feasibility of effective, mono-specific antibodies production through immunization in rabbits using the venom of Echis ocellatus from Nigeria. The World Health Organization guide on antivenom production was employed in the immunization and the resultant antibodies were purified using protein A agarose column chromatography. Antibody titer reached a high plateau by 2-month immunization, and SDS PAGE of the sera suggests the presence of intact immunoglobulins accompanied with the heavy (50 kDa) and light (25 kDa) chains. The venom has an intravenous LD50 of 0.35 mg/kg in mice, and the venom lethality at a challenge dose of 2 LD50 was effectively neutralized by the antibodies with a potency value of 0.83 mg venom per g antibodies. The antibodies also neutralized the procoagulant activity of the venom with an effective dose (ED) of 13 +/- 0.66 mu l, supporting its use for hemotoxic envenomation. The study establishes the feasibility of developing effective, mono-specific antibodies against the Nigerian Carpet viper.

Snakebite is a major global health concern, for which antivenom remains the only approved treatment to neutralise the harmful effects of the toxins. However, some medically important toxins are poorly immunogenic, resulting in reduced efficacy of the final product. Boosting the immunogenicity of these toxins in the commercial antivenom immunising mixtures could be an effective strategy to improve the final dose efficacy, and displaying snake antigens on Virus-like particles (VLPs) is one method for this. However, despite some applications in the field of snakebite, VLPs have yet to be explored in methods that could be practical at an antivenom manufacturing scale. Here we describe the utilisation of a “plug and play” VLP system to display immunogenic linear peptide epitopes from three finger toxins (3FTxs) and generate anti-toxin antibodies. Rabbits were immunised with VLPs displaying individual consensus linear epitopes and their antibody responses were characterised by immunoassay. Of the three experimental consensus sequences, two produced antibodies capable of recognising the consensus peptides, whilst only one of these could also recognise native whole toxins. Further characterisation of antibodies raised against this peptide demonstrated a sub-class specific response, and that these were able to elicit partially neutralising antibody responses, resulting in increased survival times in a murine snakebite envenoming model.

In Southeast Asia, the Malayan Pit Viper (Calloselasma rhodostoma) is a venomous snake species of medical importance and bioprospecting potential. To unveil the diversity of its toxin genes, this study de novo assembled and analyzed the venom gland transcriptome of C. rhodostoma from Malaysia. The expression of toxin genes dominates the gland transcriptome by 53.78% of total transcript abundance (based on overall FPKM, Fragments Per Kilobase Million), in which 92 non-redundant transcripts belonging to 16 toxin families were identified. Snake venom metalloproteinase (SVMP, PI > PII > PIII) is the most dominant family (37.84% of all toxin FPKM), followed by phospholipase A(2) (29.02%), bradykinin/angiotensin-converting enzyme inhibitor-C-type natriuretic peptide (16.30%), C-type lectin (CTL, 10.01%), snake venom serine protease (SVSP, 2.81%), L-amino acid oxidase (2.25%), and others (1.78%). The expressions of SVMP, CTL, and SVSP correlate with hemorrhagic, anti-platelet, and coagulopathic effects in envenoming. The SVMP metalloproteinase domains encode hemorrhagins (kistomin and rhodostoxin), while disintegrin (rhodostomin from P-II) acts by inhibiting platelet aggregation. CTL gene homologues uncovered include rhodocytin (platelet aggregators) and rhodocetin (platelet inhibitors), which contribute to thrombocytopenia and platelet dysfunction. The major SVSP is a thrombin-like enzyme (an ancrod homolog) responsible for defibrination in consumptive coagulopathy. The findings provide insight into the venom complexity of C. rhodostoma and the pathophysiology of envenoming.

Snakebite envenoming is a medical emergency requiring urgent and specific treatment. Unfortunately, snakebite diagnostics are scarce, time-consuming and lacking specificity. Hence, this study aimed to develop a simple, quick and specific snakebite diagnostic assay using animal antibodies. Anti-venom horse immunoglobulin G (IgG) and chicken immunoglobulin Y (IgY) were produced against the venoms of four major medically important snake species in Southeast Asia, i.e., the Monocled Cobra (Naja kaouthia), Malayan Krait (Bungarus candidus), Malayan Pit Viper (Calloselasma rhodostoma), and White-lipped Green Pit Viper (Trimeresurus albolabris). Different capture:detection configurations of double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) were constructed using both immunoglobulins, and the horse IgG:IgG-HRP configuration was found to be most selective and sensitive in detecting the corresponding venoms. The method was further streamlined to develop a rapid immunodetection assay, which is able to produce a visual color change within 30 min for discrimination between different snake species. The study shows it is feasible to develop a simple, quick and specific immunodiagnostic assay using horse IgG, which can be derived directly from antisera prepared for antivenom production. The proof-of-concept indicates it is a sustainable and affordable approach in keeping with on-going antivenom manufacturing activities for specific species in the region.

Objectives: To evaluate the safety and efficacy of expired lyophilized snake antivenom of Thai origin during a medical emergency in 2020/2021 in Lao People's Democratic Republic. Methods: Observational case series of patients with potentially life-threatening envenoming who consented to the administration of expired antivenom between August 2020 and May 2022. Results: A total of 31 patients received the expired antivenom. Malayan pit vipers (Calloselasma rhodostoma) were responsible for 26 (84%) cases and green pit vipers (Trimeresurus species) for two cases (6%). In three patients (10%) the responsible snake could not be identified. Of these, two presented with signs of neurotoxicity and one with coagulopathy. A total of 124 vials of expired antivenom were administered. Fifty-nine vials had expired 2-18 months earlier, 56 vials 19-36 months and nine vials 37-60 months before. Adverse effects of variable severity were observed in seven (23%) patients. All 31 patients fully recovered from systemic envenoming. Conclusions: Under closely controlled conditions and monitoring the use of expired snake antivenom proved to be effective and safe. Discarding this precious medication is an unnecessary waste, and it could be a valuable resource in ameliorating the current shortage of antivenom. Emergency use authorization granted by health authorities and preclinical testing of expired antivenoms could provide the support and legal basis for such an approach.

Snakebite envenoming is a neglected tropical disease prevalent in South Asia. In Pakistan, antivenoms are commonly imported from India despite the controversy over their effectiveness. To solve the problem, the locals have developed the Pakistani Viper Antivenom (PVAV), raised against Sochurek's Saw-scaled Viper (Echis carinatus sochureki) and Russell's Viper (Daboia russelii) of Pakistani origin. This study is set to evaluate the composition purity, immuno-specificity and neutralization efficacy of PVAV. Chromatographic and electrophoretic profiling coupled with proteomic mass spectrometry analysis showed PVAV containing high-purity immunoglobulin G with minimum impurities, notably the absence of serum albumin. PVAV is highly immuno-specific toward the venoms of the two vipers and Echis carinatus multisquamatus, which are indigenous to Pakistan. Its immunoreactivity, however, reduces toward the venoms of other Echis carinatus subspecies and D. russelii from South India as well as Sri Lanka. Meanwhile, its non-specific binding activities for the venoms of Hump-nosed Pit Vipers, Indian Cobras and kraits were extremely low. In the neutralization study, PVAV effectively mitigated the hemotoxic and lethal effects of the Pakistani viper venoms, tested in vitro and in vivo. Together, the findings suggest the potential utility of PVAV as a new domestic antivenom for the treatment of viperid envenoming in Pakistan.

BACKGROUND:Philippine Cobra Antivenom (PCAV) is the only snake antivenom manufactured in the Philippines. It is used clinically to treat envenoming caused by the Philippine Spitting Cobra (Naja philippinensis). While PCAV is effective pharmacologically, it is crucial to ensure the safety profile of this biologic that is derived from animal plasma.METHODS:This study examined the composition purity of PCAV through a decomplexation proteomic approach, applying size-exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tandem mass spectrometry liquid chromatography-tandem mass spectrometry (LC-MS/MS).RESULTS:SDS-PAGE and SEC showed that the major protein in PCAV (constituting ∼80% of total proteins) is approximately 110 kDa, consistent with the F(ab')2 molecule. This protein is reducible into two subunits suggestive of the light and heavy chains of immunoglobulin G. LC-MS/MS further identified the proteins as equine immunoglobulins, representing the key therapeutic ingredient of this biologic product. However, protein impurities, including fibrinogens, alpha-2-macroglobulins, albumin, transferrin, fibronectin and plasminogen, were detected at ∼20% of the total antivenom proteins, unveiling a concern for hypersensitivity reactions.CONCLUSIONS:Together, the findings show that PCAV contains a favorable content of F(ab')2 for neutralization, while the antibody purification process awaits improvement to minimize the presence of protein impurities.

Introduction: Most elapid snakes produce venoms that contain alpha-neurotoxins (α-NTXs), which are proteins that cause post-synaptic blockade and paralysis in snakebite envenoming. However, existing elapid antivenoms are known for their low potency in neutralizing the neurotoxic activity of α-NTXs, while the immunological basis has not been elucidated. Methods: In this study, a structure-based major histocompatibility complex II (MHCII) epitope predictor of horse ( Equus caballus ), complemented with DM-editing determinant screening algorithm was adopted to assess the immunogenicity of α-NTXs in the venoms of major Asiatic elapids ( Naja kaouthia, Ophiophagus hannah, Laticauda colubrina, Hydrophis schistosus, Hydrophis curtus ). Results: The scoring metric M 2 R, representing the relative immunogenic performance of respective α-NTXs, showed all α-NTXs have an overall low M 2 R of <0.3, and most of the predicted binders feature non-optimal P1 anchor residues. The M 2 R scores correlate strongly (R 2 = 0.82) with the potency scores (p-score) generated based on the relative abundances of α-NTXs and the neutralization potency of commercial antivenoms. Discussion: The immunoinformatic analysis indicates that the inferior antigenicity of α-NTXs is not only due to their small molecular size but also the subpar immunogenicity affected by their amino acid composition. Structural modification with conjugation and synthetic epitope as immunogen may potentially enhance the immunogenicity for improved antivenom potency against α-NTXs of elapid snakes.