Effective equine immunization protocol for production of potent poly-specific antisera against Calloselasma rhodostoma, Cryptelytrops albolabris and Daboia siamensis.

Snake envenomation has been estimated to affect 1.8 million people annually with about 94,000 deaths mostly in poor tropical countries. Specific antivenoms are the only rational and effective therapy for these cases. Efforts are being made to produce effective, affordable and sufficient antivenoms for these victims. The immunization process, which has rarely been described in detail, is one step that needs to be rigorously studied and improved especially with regard to the production of polyspecific antisera. The polyspecific nature of therapeutic antivenom could obviate the need to identify the culprit snake species. The aim of this study was to produce potent polyspecific antisera against 3 medically important vipers of Thailand and its neighboring countries, namely Cryptelytrops albolabris "White lipped pit viper" (CA), Calleoselasma rhodostoma "Malayan pit viper" (CR), and Daboia siamensis "Russell's viper" (DS). Four horses were immunized with a mixture of the 3 viper venoms using the 'low dose, low volume multi-site' immunization protocol. The antisera showed rapid rise in ELISA titers against the 3 venoms and reached plateau at about the 8th week post-immunization. The in vivo neutralization potency (P) of the antisera against CA, CR and DS venoms was 10.40, 2.42 and 0.76 mg/ml, respectively and was much higher than the minimal potency limits set by Queen Soavabha Memorial Institute (QSMI). The corresponding potency values for the QSMI monospecific antisera against CA, CR and DS venoms were 7.28, 3.12 and 1.50 mg/ml, respectively. The polyspecific antisera also effectively neutralized the procoagulant, hemorrhagic, necrotic and nephrotoxic activities of the viper venoms. This effective immunization protocol should be useful in the production of potent polyspecific antisera against snake venoms, and equine antisera against tetanus, diphtheria or rabies.

The impact of a low dose, low volume, multi-site immunization on the production of therapeutic antivenoms in Thailand.

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.