Ibrahim Al-Abdulla

First clinical experiences with specific sheep Fab fragments in snake bite. Report of a multicentre study of Vipera berus envenoming.

Objectives. To evaluate the efficacy and safety of specific, ovine Fab fragments in the treatment of envenoming by the common adder, Vipera berus.

A comparison of ovine and equine antivenoms

Commercial antivenoms produced in horses were compared with monospecific antivenoms raised in sheep against Crotalus durissus terrificus, Crotalus atrox, Crotalus adamanteus, Micrurus fulvius fulvius, Naja naja, Naja kaouthia, Echis ocellatus, Vipera lebetina deserti, Vipera berus berus and Vipera ammodytes ammodytes venom. Antibodies raised by immunizing sheep with C. d. terrificus venom were more effective than their equine counterparts in preventing lethal toxicity in mice (ED50), in inhibiting the venoms pharmacological effects (haemolysis, platelet aggregation and coagulation), and in neutralizing phospholipase A2 activity. Comparison of one ovine and three equine F(ab)2 products raised against V. a. ammodytes venom showed that all were at least 95% pure; that all protected mice; and that all contained antibody populations directed against most components of V. a. ammodytes and V. b. berus venoms. The ovine antivenoms generally contained a higher concentration of specific antibodies than the equine products. Finally, the ovine antivenoms raised against E. ocellatus, V. lebetina deserti, V. b. berus, M. f. fulvius and N. naja venoms provided better in vivo protection to mice than the equine antivenoms, but the equine antivenoms to N. kaouthia and C. atrox were more protective than the ovine product.

Development and Evaluation of an Ovine Antibody-Based Platform for Treatment of Clostridium difficile Infection

ABSTRACT Treatment of Clostridium difficile is a major problem as a hospital-associated infection which can cause severe, recurrent diarrhea. The currently available antibiotics are not effective in all cases and alternative treatments are required. In the present study, an ovine antibody-based platform for passive immunotherapy of C. difficile infection is described. Antibodies with high toxin-neutralizing titers were generated against C. difficile toxins A and B and were shown to neutralize three sequence variants of these toxins (toxinotypes) which are prevalent in human C. difficile infection. Passive immunization of hamsters with a mixture of toxin A and B antibodies protected them from a challenge with C. difficile spores in a dose-dependent manner. Antibodies to both toxins A and B were required for protection. The administration of toxin A and B antibodies up to 24 h postchallenge was found to reduce significantly the onset of C. difficile infection compared to nonimmunized controls. Protection from infection was also demonstrated with key disease isolates (ribotypes 027 and 078), which are members of the hypervirulent C. difficile clade. The ribotype 027 and 078 strains also have the capacity to produce an active binary toxin and these data suggest that neutralization of this toxin is unnecessary for the management of infection induced by these strains. In summary, the data suggest that ovine toxin A and B antibodies may be effective in the treatment of C. difficile infection; their potential use for the management of severe, fulminant cases is discussed.

Development and clinical application of immunoassays for European adder (Vipera berus berus) venom and antivenom

An ovine affinity purified Fab antivenom was used in a clinical trial in Sweden to treat European adder (Vipera berus berus) envenoming. Immunoassays were developed to measure V. b. berus venom and antivenom concentrations in clinical samples to help assess the efficacy of treatment. A radioimmunoassay (RIA) was developed, optimized and validated to measure plasma levels of V. b. berus venom and compared with a conventional ELISA. Both showed a similar variation of zero binding in biological samples and the results obtained correlated closely. However, the ELISA was quicker and more sensitive (0.8 compared with 2 micrograms/litre). Before administration of antivenom, V. b. berus venom concentrations in plasma ranged from 10 to 53 micrograms/litre; 12 hr after the Fab infusion, no patient had measurable levels. However, two patients had low venom levels 24 hr after treatment. ELISA and RIA were also developed, optimized and used to measure concentrations of free Fab in plasma. There was a biexponential fall of Fab concentration with a fast distribution phase (t 1/2 = 0.9 hr) and a slower elimination phase (t 1/2 = 18 hr). The amount of Fab excreted in urine was low.

An indirect haemolytic assay for assessing antivenoms

Dilutions of antivenom, venom, human erythrocytes and a phosphatidylcholine suspension, were incubated for 30 min at 37 degrees C. After centrifugation, the liberated haemoglobin was measured spectrophotometrically. The assay was used to assess an ovine antivenom against the venom from the South American rattlesnake, Crotalus durissus terrificus, and an equine Wyeth antivenin (Crotalidae, polyvalent). The ovine antivenom was more than five times as effective as the equine product. It also neutralized venoms from the Western diamondback rattlesnake, Crotalus atrox, and the fer-de-lance, Bothrops atrox. However, antivenoms raised against venoms from other Crotalus and Bothrops species provided little protection against the haemolytic activity of C. d. terrificus venom.

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.

Development of a Cost-effective Ovine Polyclonal Antibody-Based Product, EBOTAb, to Treat Ebola Virus Infection

The highly glycosylated glycoprotein spike of Ebola virus (EBOV-GP1,2) is the primary target of the humoral host response. Recombinant EBOV-GP ectodomain (EBOV-GP1,2ecto) expressed in mammalian cells was used to immunize sheep and elicited a robust immune response and produced high titers of high avidity polyclonal antibodies. Investigation of the neutralizing activity of the ovine antisera in vitro revealed that it neutralized EBOV. A pool of intact ovine immunoglobulin G, herein termed EBOTAb, was prepared from the antisera and used for an in vivo guinea pig study. When EBOTAb was delivered 6 hours after challenge, all animals survived without experiencing fever or other clinical manifestations. In a second series of guinea pig studies, the administration of EBOTAb dosing was delayed for 48 or 72 hours after challenge, resulting in 100% and 75% survival, respectively. These studies illustrate the usefulness of EBOTAb in protecting against EBOV-induced disease.

Production and assessment of ovine antisera for the manufacture of a veterinary adder antivenom

Medically important venomous snakes in Western Europe are Vipera ammodytes, Vipera aspis, Vipera berus and Vipera latastei. Envenomation of dogs and other animals by these snakes receives limited attention despite the relative frequency and potential mortality and morbidity. This reflects, in part, the lack of a dedicated veterinary antivenom. Successful antivenoms are derived from antisera containing high levels of specific polyclonal antibodies that bind to, and neutralise, all the toxins present. This requires a careful choice of immunogen, animals and immunisation schedule. We detected proteomic variation in the venoms of V ammodytes, V aspis, V berus and V latastei by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel electrophoresis. Consequently, we used a mixture containing equal amounts of venom from these species to immunise a flock of sheep. We demonstrate that immunisation resulted in antisera containing high levels of specific antibodies directed against the majority of toxic components found in all four snake venoms using immunoblotting, ELISA and small-scale affinity chromatography assays. The latter shows that all 25 sheep responded quickly and maintained high levels of specific antibodies throughout the two-year period of study. This ensures a consistent starting material for the manufacture of a reproducible veterinary antivenom, ViperaVet. Our next objectives are to purify the antibodies from our antisera and demonstrate their preclinical neutralising efficacy in murine animal studies prior to undertaking a clinical trial in envenomed patients.

Immunological Cross-Reactivity and Neutralisation of European Viper Venoms with the Monospecific Vipera berus Antivenom ViperaTAb

Medically important cases of snakebite in Europe are predominately caused by European vipers of the genus Vipera. The mainstay of snakebite therapy is polyclonal antibody therapy, referred to as antivenom. Here we investigate the capability of the monospecific V. berus antivenom, ViperaTAb®, to cross-react with, and neutralise lethality induced by, a variety of European vipers. Using ELISA and immunoblotting, we find that ViperaTAb® antibodies recognise and bind to the majority of toxic components found in the venoms of the Vipera species tested at comparably high levels to those observed with V. berus. Using in vivo pre-clinical efficacy studies, we demonstrate that ViperaTAb® effectively neutralises lethality induced by V. berus, V. aspis, V. ammodytes and V. latastei venoms and at much higher levels than those outlined by regulatory pharmacopoeial guidelines. Notably, venom neutralisation was found to be superior to (V. berus, V. aspis and V. latastei), or as equally effective as (V. ammodytes), the monospecific V. ammodytes “Zagreb antivenom”, which has long been successfully used for treating European snake envenomings. This study suggests that ViperaTAb® may be a valuable therapeutic product for treating snakebite by a variety of European vipers found throughout the continent.

Snake antivenom trial

WE would like to invite veterinary practitioners to take part in a clinical trial of Europes first dedicated veterinary snake antivenom, ViperaVet. This is directed against the venom of four medically important Vipera species found throughout western Europe, including the adder ( Vipera berus) , Britains only native venomous snake. Between September 1985 and December 2010 the Veterinary Poisons Information Service …