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Snakebite envenomation is an important medical problem in numerous parts of the world causing about 2.7 million envenomations and between 81,000 and 138,000 deaths ayear. Antivenoms (AVs) are time proven effective therapeutics for snakebite envenomation. However, AVs, especially those against elapid neurotoxic venoms (cobras, kraits and mambas), are difficult to produce and are generally of low neutralizing potency. The most lethal component of most elapid venoms is the postsynaptic neurotoxins or the α-neurotoxins, which are responsible for death in most victims. It is generally believed that the low neutralizing potency of the AVs is due to the small molecular sizes, and thus the low immunogenicity, of the α-neurotoxins. Therefore, modifications of the toxins have been made to increase their size, and/or to detoxify them, hoping to improve the toxin's immunogenicity and AV potency. However, these maneuvers have not been applied to commercial AV production. The α-neurotoxins belong to a group of small proteins called three-finger toxins (3FTxs). The 3FTxs contain about 60-77 amino acid residues with four to five disulfide linkages and three anti-parallel β-sheets, which extend from a globular hydrophobic core resembling three fingers. The members of the 3FTxs exhibit a number of important pharmacological activities, e.g., inhibition of neuromuscular transmission and acetyl cholinesterase activities. Recent immunization experiments with a 26 amino acid peptide containing the consensus sequence of the α-neurotoxins, and a mixture of elapid α-neurotoxins using highly effective adjuvants and immunization protocols have resulted in neutralizing antibodies in rabbit and horse, respectively. In the present report using bioinformatics, we show that 23 3FTxs which include α-neurotoxins, cardiotoxins and non-conventional toxins, and the 26 amino acid peptide, were all predicted to contain high to medium score CD4 T-cell epitopes for human and mouse MHC IIs. This information corroborates the results obtained from animal experiments that the α-neurotoxins, in spite of their small sizes and toxicity, are in fact immunogenic. Thus, the uses of effective adjuvants and immunization procedures, rather than chemical/physical modifications of the toxin structures, are crucial to the production of potent AVs against elapid neurotoxic venoms., (Copyright © 2022. Published by Elsevier Ltd.)

Antivenomics is a recently developed powerful method for the study of antivenom antibody profiles when bound to homologous and heterologous snake venoms. The information obtained is useful in gaining an understanding of venom protein immunogenicity, antivenom potency and also for the improvement of antivenom potency and paraspecificity. The preferred method used in this type of study is immunoaffinity chromatography of the venom proteins on an antivenom IgG (or F(ab')2) column where the bound and unbound proteins can be separated and identified. However, there are some parameters of the immunochromatography that can significantly affect the binding of the proteins to the immunoaffinity matrix and lead to imprecise results in antivenom immunoprofiling. The present study demonstrated that the ligand density (mg IgG/ml of the matrix), the buffers used for binding and washing the venom proteins, the amount of venom loaded, the abundance of some venom protein(s) and the eluting buffers can significantly alter the binding of the proteins to the matrix and consequently the conclusions drawn from antivenomics studies. Furthermore, the immunochromatographic procedure can be extended to include the estimation of the relative affinity of venom protein-antibody interactions that can provide additional information useful to antivenomics study., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

An in vitro potency assay of antivenom against Malayan pit viper (Calloselasma rhodostoma, CR) has been developed. The assay is based on the neutralizing activity of the antivenom against the coagulant activity of the venom. The minimum coagulant dose (MCD) of CR venom was 22.12 ± 0.25 μg/ml. The coagulation time induced by 2MCD of the venom was used as the control for calculating the neutralizing activity of each batch of antivenom. The in vitro potency of antivenom, expressed as effective dose (ED), was the antivenom/venom ratio at which the coagulation time was increased three fold of that induced by 2MCD of the venom. Eleven batches of the antivenom were assayed for their lethality neutralizing activity (ED₅₀) by the in vivo assay using mice as well as the developed in vitro assay. The correlation coefficient (r) between the in vitro neutralizing activities (ED) and in vivo neutralizing activities (ED₅₀) was 0.957, (p value < 0.001). This simple and rapid in vitro assay of C. rhodostoma antivenom should be a good alternative method for the assessment of antivenom potency during the immunization program and fractionation process. The assay should be adaptable for use with antivenoms against other similar procoagulant venoms., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This study involved the use of combined stepwise ammonium sulfate (AS) and caprylic acid (CA) fractionation of equine antivenom IgG without intermediate separation of precipitate. Using a microplate and checker board titration format, plasma was treated under 66 conditions with varying concentrations of AS (0-25% saturation) and CA (0-5% v/v). The filtrate of each well was assayed for protein and antibody activity. At about 1.5-4.0% CA without AS, the precipitated plasma gave high specific antibody activity. Twelve precipitation conditions selected from the microplate experiment were studied in detail in tubes. The highest turbidity was with 5% CA alone. The highest antibody recovery of 95.45% was observed at 15% AS with 3.0% CA. The highest specific activity with 3.28 folds purification was observed with 4.0% CA. Thus, AS could reduce the turbidity induced by CA and increase the yield but not the purity of antibody. Size exclusion HPLC showed the antibody to be one single peak with 1.5% of soluble protein aggregate. When all parameters were considered, the optimum fractionation condition appeared to be 3.5% CA alone which gave high specific antibody activity (3.26 folds purification), antibody recovery (93.93%) and low turbidity (0.56% solid). Furthermore, better overall results were observed with one hour than overnight precipitation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Local tissue necrosis due to snake envenoming has resulted in considerable chronic disability. Specific antivenom, though highly useful in systemic poisoning, is not very effective in preventing the local tissue damages which occur within minutes of envenomation. Most of the studies on local tissue necrosis have been done in rodents whose skin structures are significantly different from human skin structure. In this study, pig, which is similar to human in this respect, was used as an experimental model for the study of local tissue damage caused by snake venom. An intradermal (i.d.) injection of Calloselasma rhodostoma (CR) venom induced induration and hemorrhage at the same injection site, the areas of which could be estimated at 30-45 min and at 4h after injection, respectively. Both the hemorrhage and induration were dose-dependent and a total of 24 data points of each assay per pig could be obtained. Catheterization of the jugular vein for gentle serial blood collection could be performed without any hematologic sequelae. Venom injected subcutaneously induced myonecrosis as demonstrated by the increment of serum creatine kinase (CK) level which peaked at 23 h. Furthermore, biopsies at varying distances and depths around the venom injection sites could be made within seconds of injection to study the pathological changes caused by snake venom. These results demonstrated that pig should be a useful animal model for the quantitation, pathogenesis and wound healing studies of snake venom induced local tissue necrosis, and for the search for effective treatment modality.

Although the merit of polyvalent antivenoms is well-recognized, there is still doubt in some medical circles as to the relative efficacy and the propensity to cause adverse reactions of polyvalent (pAV) as compared to monovalent (mAV) antivenoms. Immunochemical and biochemical comparisons of equine polyvalent and monovalent antivenoms prepared under the same immunization protocols were therefore made. These antivenoms were prepared against Naja kaouthia (NK), Ophiophagus hannah (OH) and Bungarus fasciatus (BF) venoms. SDS-PAGE analysis of both types of antivenoms showed similar serum protein profiles. The total amount of immunoglobulin (IgG(T)+IgG) in pAVs was slightly but significantly higher than that of mAVs, while the total serum protein content in mAVs was slightly but significantly higher than that of pAVs. The amounts of total hyperimmune IgG(T), determined by ELISA, were similar in mAVs and pAVs. pAVs contained specific antibodies against the principal NK postsynaptic toxin (NK 3) to the same extent as that observed with the anti-N. kaouthia mAV. The antibodies against OH and BF principal postsynaptic toxins (OH II and BF IX) in pAVs were significantly higher than those of the corresponding anti-O. hannah and anti-B. fasciatus mAVs. These results were in concordance with the comparable in vivo neutralization activities of mAVs and pAV previously reported. The apparent dissociation constant (Kd) of anti-OH II antibody in pAVs was comparable to that of the anti-O. hannah mAVs. The apparent K(d)s of anti-NK 3 and anti-BF IX antibodies in the corresponding mAVs were slightly lower than those in pAVs. The Kd values were all in nM range and were considered to be high affinity binding. It is concluded that pAVs could be prepared with potency and protein contents and thus the propensity to cause adverse reaction that were comparable to those of mAVs.

Experiments have been carried out to find potent inhibitors of hyaluronidases of Naja kaouthia (NK) and Calloselasma rhodostoma (CR) venoms with the aim of reducing local tissue damage and systemic toxicities caused by the venoms. Seven drugs/chemicals known to inhibit hyaluronidases were tested for their activity on venom enzymes. These were: sodium cromoglycate (SC), sodium aurothiomalate (SAT), apigenin, kaemferol, phenylbutazone, oxyphenbutazone and fenoprofen. The results showed that SC or SAT at 10 mM, completely inhibited the enzymes of both venoms. In in vivo experiments, SC or SAT, when incubated with NK venom prior to injection, significantly reduced edema and myonecrosis. In the case of CR venom, hemorrhage, in addition to edema and myonecrosis, was also significantly reduced. In the independent type experiment, SC or SAT were effective if injected within 1 min after the injection of venom. At longer time intervals of 3 and 10 min the inhibitors were effective in reducing some parameters of local tissue necrosis but the extent of inhibition was lower. SC and SAT at 256 and 195 microg/mouse, respectively, significantly prolonged the survival time of mice receiving lethal doses of NK. In the case of CR venoms, the two inhibitors not only prolonged the survival time but also prevented death of mice receiving lethal doses of the venom. The other inhibitors were poorly soluble in water and were studied only on enzyme inhibition and prolongation of survival time; they were mostly ineffective. Thus, SC and SAT when injected immediately at the sites of bites can reduce the systemic and local toxicity of NK and CR venoms. These results suggest that administration of these drugs at the site of venom injection may be useful in reducing venom-induced local tissue damage.

Therapeutic antivenom against snakes was first produced by Albert Calmette in 1894. Since then antivenoms have saved the life of countless snakebite victims. However, there are still many problems associated with antivenom production, for example variable percentage of responder horses, low neutralizing potency of antivenom, the large amount of snake venom needed for immunization and the difficulties encountered in producing potent polyvalent antivenoms. These problems have led to shortage and high cost of antivenom and, in some cases, failure of treatment. In 1997, a new immunization protocol for antivenom production was reported. It involves the injection of venom at low dose (approx. 2mg/horse) emulsified in Complete Freund's adjuvant in low volume (0.1-0.2 ml/site) in a total of 10 sites around the neck area of the horse. This immunization protocol has minimized the local reaction at the injection site thus allowing the use of the potent oil adjuvant. This, together with the increase in total surface area of the droplets, allow a more effective immune response to take place, e.g. enhancing the migration and activation of more antigen presenting cells and lymphocytes. The low dose, low volume multi-site immunization has resulted in dramatic improvements on the antivenom production in terms of amount of venom used for immunization, the time required to reach hyperimmune stage, the percent of responder horses and the potency of the antivenom. Furthermore, this protocol has made it possible to produce potent truly polyvalent antivenoms against several elapid and viperid snakes. This immunization protocol has alleviated various problems associated with antivenom production and has implications for immunization in general.