Snake Antivenom Production Research Articles

Pilot-scale evaluation of a dynamic body-feed filtration system for primary clarification of snake antivenoms produced by the caprylic acid method

The performance of dynamic body-feed filtration (DBF) in the removal of bulky solids produced during the manufacturing of snake antivenoms using the caprylic acid method was evaluated. For this purpose, diatomites with different filterability properties were compared in a bench-scale study to assess their effectiveness in removing the precipitated material formed after the addition of caprylic acid to equine hyperimmune plasma. C1000 diatomite at a concentration of 90 g/L of precipitated plasma showed the best performance. Then, the process was scaled up to three batches of 50 L of hyperimmune horse plasma. At this pilot scale, 108 ± 4% of the immunoglobulins present following plasma precipitation were recovered after DBF. The antivenoms generated using this procedure met quality specifications. When compared to open filtration systems commonly used at an industrial scale by many antivenom manufacturers, DBF has a similar yield and produces filtrates with comparable physicochemical characteristics. However, DBF ensures the microbiological quality of the primary clarification in a way that open systems cannot. This is because: 1) DBF is performed in a single-use closed device of depth filters which prevents microbial contamination, and 2) DBF removes bulky material in few minutes instead of the more than 24 h needed by open filtration systems, thus reducing the risk of contamination. It was concluded that DBF is a cost-effective, easily validated, and GMP-compliant alternative for primary clarification following caprylic acid precipitation of plasma in snake antivenom production.

Evaluation of candidate monovalent Bitis arietans antivenom using eggs from hyperimmunized layer chickens in Zaria Kaduna State, Nigeria

Aim: This research sought to experiment on a cheaper alternative to snake antivenom production using hyperimmunized chicken eggs. Methods: Twenty-two Swiss albino mice were used to determine the median lethal dose (LD50) and lethality neutralization assay. Twenty-layer chickens, aged 17-weeks-old were divided into two groups of 10 chickens each. Based on body weight, group A were hyperimmunized with 1/5th LD50 of crude Bitis arietans venom in adjuvant (Montanide™ ISA 201) at two-weeks interval over eight-weeks period; while group B were administered normal saline. Using polyethylene glycol 6000, immunoglobulin Y was extracted from 15 pooled egg yolks. Twelve Wistar rats were used for the haemorrhagic and anti-haemorrhagic assays. The haemolytic activity of the venom and anti-haemolytic effect of the candidate antivenom was determined in vitro using caprine erythrocytes. Results: The LD50 of the pooled Bitis arietans venom was 0.9 mg/kg. Agar gel immunodiffusion test confirmed the presence of precipitin antibodies in the candidate antivenom. EchiTab plus-ICP® neutralized lethal effect of all dilutions and the stock (10 mg/ml) venom of Bitis arietans while the candidate antivenom was limited to 3log2 dilution of the stock venom (1.25 mg/ml). In the haemorrhagic assay the venom exhibited haemorrhagic activity at 2.5, 5 and 10 mg/ml and the highest anti-haemorrhagic activity of the candidate antivenom at 10mg/ml. The candidate antivenom exhibited significantly higher (p < 0.05) anti-haemolytic activity against the venom of Bitis arietans at 10 mg/ml. Conclusion: The antibodies have inhibitory properties against the lethal, haemorrhagic and haemolytic properties of Bitis arietans venom.

Evaluating Antivenom Efficacy against Echis carinatus Venoms-Screening for In Vitro Alternatives.

In India, polyvalent antivenom is the mainstay treatment for snakebite envenoming. Due to batch-to-batch variation in antivenom production, manufacturers have to estimate its efficacy at each stage of IgG purification using the median effective dose which involves 100–120 mice for each batch. There is an urgent need to replace the excessive use of animals in snake antivenom production using in vitro alternatives. We tested the efficacy of a single batch of polyvalent antivenom from VINS bioproducts limited on Echis carinatus venom collected from three different locations—Tamil Nadu (ECVTN), Goa (ECVGO) and Rajasthan (ECVRAJ)—using different in vitro assays. Firstly, size-exclusion chromatography (SEC-HPLC) was used to quantify antivenom–venom complexes to assess the binding efficiency of the antivenom. Secondly, clotting, proteolytic and PLA2 activity assays were performed to quantify the ability of the antivenom to neutralize venom effects. The use of both binding and functional assays allowed us to measure the efficacy of the antivenom, as they represent multiple impacts of snake envenomation. The response from the assays was recorded for different antivenom–venom ratios and the dose–response curves were plotted. Based on the parameters that explained the curves, the efficacy scores (ES) of antivenom were computed. The binding assay revealed that ECVTN had more antivenom–venom complexes formed compared to the other venoms. The capacity of antivenom to neutralize proteolytic and PLA2 effects was lowest against ECVRAJ. The mean efficacy score of antivenom against ECVTN was the greatest, which was expected, as ECVTN is mainly used by antivenom manufacturers. These findings pave a way for the development of in vitro alternatives in antivenom efficacy assessment.

Influence of sexual dimorphism on venom composition in Bothrops asper and Crotalus simus (Serpentes: Viperidae) and its potential implications on the snake antivenom production

This work is aimed to bring insights on the potential sexual dimorphism differences on the venom composition of Bothrops asper and Crotalus simus to expand the knowledge of the venom variability that might improve the antivenom design. Biological characterization of venoms of each sex in both species did not show significant qualitative differences. Considerations on the sexual venom variations in these species are not relevant for choosing the snake donors for venom production.

An optimized method of IgG’s precipitation with ammonium sulphate from hyper immune horse plasma for snake anti venom production

An optimized method of IgG’s precipitation with ammonium sulphate from hyper immune horse plasma for snake anti venom production

Improvement of Naja haje snake antivenom production using gamma radiation and a biotechnological technique

This study aimed to improve antivenom production using gamma radiation and calcium phosphate nanoparticles (CPN) as adjuvant. Toxicological and immunological properties of the Naja haje venom before and after exposure to radiation were estimated. The immune response of immunized rabbits was evaluated. Toxicity of irradiated venom was reduced six times as compared to native venom. Moreover, no change in antigenic reactivity between native and irradiated venoms. Serum titer produced with irradiated venom loaded on CPN showed highest titer and neutralization capacity as compared to other sera. Thus, irradiation and nanoparticles adjuvant could be a new approach for snake antivenom production.

Cloning and sequencing of three-finger toxins from the venom glands of four Micrurus species from Mexico and heterologous expression of an alpha-neurotoxin from Micrurus diastema

The three-finger toxins (3FTxs) represent an extremely diverse protein family in elapid venoms, where the short chain α-neurotoxins are the most relevant toxin group from the clinical point of view. Essentially, the 3FTxs variability and the low proportions of α-neurotoxins in the venoms of North American coral snakes make it difficult to obtain effective elapid antivenoms against the envenomation symptoms caused mainly by these α-neurotoxins. In this work, thirty 3FTx transcript sequences were obtained from the venom glands of four coral snake species from Mexico (M. diastema, M. laticollaris, M. browni and M. tener). The transcripts were mined using a forward oligonucleotide based on the highly conserved signal peptide from the 3FTxs, and four of these transcripts, named MlatA1, B.D, B.E and D.H, encoded for short-chain α-neurotoxins. Additionally, one isoform of the D.H α-neurotoxin transcript was identified in the venom of M. diastema. The mature α-neurotoxin coded in the D.H transcript was heterologously expressed, and it was found soluble (4.2 mg/l) in the cytoplasm of a bacterial system. The recombinant D.H (rD.H) had an IC50 value of 31.5 ± 4.4 nM on nicotinic acetylcholine receptors of the muscular type expressed in rhabdomyosarcoma cells (TE671). The rDH also had an LD50 of 0.15 mg/kg mice, and it was evaluated as a potential immunogen in New Zealand rabbits. The protective capacity of rabbit sera was tested against two native coral snake α-neurotoxins, and against rD.H. One of the anti-rD.H rabbit sera was able to neutralize the lethality of all three neurotoxins when tested in groups of CD1 mice. This work contributes to the increasing understanding of the high diversity of 3FTxs, and shows that recombinant coral snake α-neurotoxins are promising supplements for hyperimmunization protocols for coral snake antivenom production.

Prognosis of Hepatic Amyloidosis Status and Insufficiency in Snake Antivenom Producing Horses

Hyperimmunization of horses with specific antigens to harvest antisera is associated with high incidence of liver amyloidosis. The histopathologic examination is the gold standard method for diagnosis of the hepatic amyloidosis. The aim of the present study was to determine whether the clinical manifestations and serum values of appropriate chemical constituents due to liver injury, provide the prognostic criteria for liver amyloidosis and insufficiency. it was endeavored to determine the usage duration and survival in the horses, applied for polyvalent snake antivenom products. Thirty-seven Iranian, mixed breed male horses used for snake antivenom production were recruited in this study. All of the clinical characteristics of liver disease were assessed and the appropriate serum biochemical parameters were screened between two hyperimmunization stages. The results revealed that weakness, progressive weight-loss and chronic diarrhea were the warning signs for end of the period of using a horse in snake antivenom production. However, moderate to severe colic, often, was the sign of imminent death due to acute hepatic swelling and rupture. However, Serum biochemical findings were not suitable and useful for the assessment of outcome compared with the clinical manifestations. In this study, the authors have attempted to introduce the clinical and serum biochemical findings that are useful for prediction of the liver amyloidosis and insufficiency and, to determine the usage duration and survival in these horses. Consequently, a preliminary scoring guideline for assessing the issue was suggested.

Cross neutralization of coral snake venoms by commercial Australian snake antivenoms

Context: Although rare, coral snake envenomation is a serious health threat in Brazil, because of the highly neurotoxic venom and the scarcely available antivenom. The major bottleneck for antivenom production is the low availability of venom. Furthermore, the available serum is not effective against all coral snake species found in Brazil. An alternative to circumvent the lack of venom for serum production and the restricted protection of the actually available antivenom would be of great value. We compared the Brazilian coral snake and mono and polyvalent Australian antivenoms in terms of reactivity and protection.Methods: The immunoreactivity of venoms from 9 coral snakes species were assayed by ELISA and western blot using the Brazilian Micrurus and the Australian pentavalent as well as monovalent anti-Notechis, Oxyuranus and Pseudechis antivenoms. Neutralization assays were performed in mice, using 3 LD50 of the venoms, incubated for 30 minutes with 100 μL of antivenom/animal.Discussion: All the venoms reacted against the autologous and heterologous antivenoms. Nevertheless, the neutralization assays showed that the coral snake antivenom was only effective against M. corallinus, M. frontalis, M. fulvius, M. nigrocinctus and M. pyrrhocryptus venoms. On the other hand, the Australian pentavalent antivenom neutralized all venoms except the one from M. spixii. A combination of anti-Oxyuranus and Pseudechis monovalent sera, extended the protection to M. altirostris and, partially, to M. ibiboboca. By adding Notechis antivenom to this mixture, we obtained full protection against M. ibiboboca and partial neutralization against M. lemniscatus venoms.Conclusions: Our findings confirm the limited effectiveness of the Brazilian coral snake antivenom and indicate that antivenoms made from Australian snakes venoms are an effective alternative for coral snake bites in South America and also in the United States were coral snake antivenom production has been discontinued.

Evaluation of health status of horses immunized with snake venom and montanide adjuvants, IMS 3012 (nanoparticle), ISA 206 and ISA 35 (emulsion based) during polyvalent snake antivenom production: Hematological and biochemical assessment

Several biochemical and hematological changes in horses are observed during production of snake antivenom. Although conventional adjuvants like Freund's (Complete and Incomplete) are good immunopotentiators, they produce considerable local reactions in animals. Variety of commercial adjuvants, like montanide adjuvants, having high immunopotentiation and showing lesser side effects are available. The prime objective during antivenom production is to strike a balance between safety of immunized horses and efficacy of the product. In our earlier work, efficacy of montanide group of adjuvants in antivenom production has already been established. The aim of the present work was to assess the safety parameters in horses, viz.: biochemical and hematological, during production of snake antivenom. In the present study, 33 new horses were randomly divided into four groups and hyperimmunized using mixture of snake venoms, viz.: Cobra venom, Russell's viper venom, Krait venom and Echis venom along with montanide adjuvants, IMS 3012, ISA 206, ISA 35 and Incomplete Freund's adjuvant as a control adjuvant; through subcutaneous route at intervals of two weeks. During the immunization period, biochemical and hematological parameters were monitored at 0th, 14th, 21st, 30th and 42nd weeks. The mean hemoglobin values dropped slightly during initial immunization but subsequently regained to normal levels. The mean serum total protein values and globulin levels showed an increment in all the four groups, compared to day zero, vice-versa a slight drop was observed in albumin levels. No significant changes were observed in serum creatinine, bilirubin, alkaline phosphatase and blood urea nitrogen values. Finally, we conclude that montanide adjuvants could be a safer alternative to the conventional adjuvants for primary phase of immunization in antivenom production.

Immune Response Towards Snake Venoms

The immune response involves a complex repertoire of innate and adaptive responses to foreign agents in the organism. The present review focuses on the immune response to snake venoms, including those occurring in snakebite accidental envenomation, experimental vaccination and animal hyperimmunization for snake antivenom production. The following aspects are considered: (a) the structural characteristics of snake toxins and their relationship to immunogenicity, (b) the effects that factors such as administration route, venom dose, type of adjuvant, and individual and species characteristics of the immunized animal have on the immune response, (c) the initial venom-induced inflammatory response, (d) the process by which specific antibodies towards individual toxins are produced, and (e) the techniques currently used to evaluate the antibody response. Understanding the immune response to snake venoms is highly relevant for improving antivenom production and for gaining a more complete view of snakebite envenoming.

Development and improvement in the snake antivenom production

Development and improvement in the snake antivenom production

Economic evaluation of snake antivenom production in the public system

Snake antivenom, an expensive animal product, is presently the only effective treatment for the consequences of snakebite. In Latin America, antivenoms are mainly produced by public institutions with frequent shortages of the necessary supply. Here, we present an economical analysis of the factors affecting production cost, assuming a basic processing batch of 100 L hyperimmune plasma. Three annual production volumes were considered for two typical production technologies. The components of cost were classified as fixed, variable and semi-variable. We found that in all stages of production, fixed cost represents the major contribution to total cost, and is given essentially by manpower cost, particularly for low production volumes. Our estimation shows that antivenom cost can vary from US$ 2.4 to US$ 25 per 10 mL vial, depending on the production volume, the plasma processing technology used and the titer achieved during the immunization stage. We conclude that interested laboratories and authorities of countries with population at risk should consider the possibility of a joint production to improve the process efficiency, lower the product unitary cost and obtain the necessary supply for their own demand or that of other countries in need.