Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper.

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in rural populations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000 to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced by hyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyper immune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebite envenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have produced recombinant single chain variable fragment scFv against the venom of the pit viper Bothrops asper at high levels expressed transiently and stably in transgenic plants and in vitro cultures that is reactive to BaP1 (a metalloproteinase from B. asper venom). The yield from stably transformed plants was significantly (p > 0.05) higher than the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stable transformation were: transgenic callus 62 µg/g (±2); biomass from cell suspension cultures 83 µg/g (±0.2); culture medium from suspensions 71.75 mg/L (±6.18). The activity of scFvBaP1 was confirmed by binding and neutralization of the fibrin degradation induced by BnP1 toxins from B. neuwiedi and by Atroxlysin Ia from B. atrox venoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to be used in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms to be used in human therapy against snakebites.

Production and preclinical assessment of camelid immunoglobulins against Echis sochureki venom from desert of Rajasthan, India.

Snakebite is a significant cause of death and disability in subsistent farming populations of rural India. Antivenom is the most effective treatment of envenoming and is manufactured from IgG of venom-immunised horses. Because of complex fiscal reasons, the production, testing and delivery of antivenoms designed to treat envenoming by the most medically-important snakes in the region has been questioned time to time. In this study, we report successful immunisation of dromedaries (Camelus dromedarius) against the venom of Indian saw-scaled Viper- Echis carinatus sochureki. This study assessed the specificity and potential of camels immunised with venom of medically most important snake of Western India, the saw-scaled viper (Echis c. sochureki). Using WHO standard pre-clinical in vivo tests the neutralisation of the venom responsible for the lethal, haemorrhagic, coagulant and local necrotizing activities were measured, since these are the most significant effects that characterize envenoming by this species. The anti-venom was found significantly effective in the neutralisation of all these effects tested and thus, revealed further an immunological perspective, that camel IgG anti-venom (monospecific) would be as efficacious as specific equine anti-venoms or even of better choice in treating snake specific envenoming.

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.

Maintaining Coral Snakes (Micrurus nigrocinctus, Serpentes: Elapidae) for venom production on an alternative fish-based diet.

American Elapid snakes (Coral Snakes) comprise the genera Leptomicrurus, Micruroides and Micrurus, which form a vast taxonomic assembly of 330 species distributed from the South of United States to the southern region of South America. In order to obtain venom for animal immunizations aimed at antivenom production, Coral Snakes must be kept in captivity and submitted periodically to venom extraction procedures. Thus, to maintain a snake colony in good health for this purpose, a complete alternative diet utilizing an easily obtained prey animal is desirable. The development of a diet based on fish is compared to the wild diet based on colubrid snakes, and assessed in terms of gain in body weight rate (g/week), longevity (weeks), venom yield (mg/individual), venom median lethal dose (LD50) and venom chromatographic profiles. The animals fed with the fish-based diet gained more weight, lived longer, and produced similar amount of venom whose biological and biochemical characteristics were similar to those of venom collected from specimens fed with the wild diet. This fish-based diet appears to be suitable (and preferable to the wild diet) to supply the nutritional requirements of a Micrurus nigrocinctus snake collection for the production of antivenom.

A new Pan African polyspecific antivenom developed in response to the antivenom crisis in Africa.

Currently there is a crisis in the supply of antivenom for treatment of snake bite in sub-Saharan Africa. Commercial pressures have resulted in the reduction or even cessation of production of antivenom by European manufacturers while continued production of antivenom in Africa has been threatened by the privatisation of the only remaining company based in Africa. As a consequence, there has been an increase in snake bite morbidity and mortality in many African countries. Two Latin American antivenom manufacturers have agreed to produce antivenom suitable for Africa, using venoms from the species which are of the greatest medical importance in sub-Saharan Africa. Preclinical in vivo assays of neutralising potency demonstrated that a new Pan African antivenom produced in Colombia compared favourably with the existing commercial monospecific and polyspecific antivenoms. This new antivenom, and a similar product being manufactured in Costa Rica, are now candidates for clinical testing at an appropriate site in Africa.

Assessment of an ovine antivenom raised against venom from the desert black cobra (Walterinnesia aegyptia).

The desert black cobra (Walterinnesia aegyptia) is an elapid widely distributed throughout the deserts of Saudi Arabia and currently available antivenoms are ineffective in the treatment of its envenoming. Walterinnesia aegyptia venom was assessed for several of its physicochemical, enzymatic and biological characteristics. An antivenom was raised in sheep using a low-dose immunization schedule and digested with papain to provide Fab fragments. The antivenom neutralized all of the above enzymatic and biological activities and provided good protection in mice (ED50 0.25 g/kg), whereas the commercial polyspecific products showed only partial neutralization and did not protect mice.