Intrageneric cross-reactivity of monospecific rabbit antisera against venoms of the medically most important Naja spp. African snakes.

Background: Envenomations by African snakes represent a high burden in the sub-Sahara region. The design and fabrication of polyspecific antivenoms with a broader effectiveness, specially tailored for its use in sub-Saharan Africa, require a better understanding of the immunological features of different Naja spp. venoms of highest medical impact in Africa; and to select the most appropriate antigen combinations to generate antivenoms of wider neutralizing scope. Methodology/principal findings: Rabbit-derived monospecific antisera were raised against the venoms of five spitting cobras and six non-spitting cobras. The effects of immunization in the animal model were assessed, as well as the development of antibody titers, as proved by immunochemical assays and neutralization of lethal, phospholipase A₂ and dermonecrotic activities. By the end of the immunization schedule, the immunized rabbits showed normal values of all hematological parameters, and no muscle tissue damage was evidenced, although alterations in aspartate aminotransferase (AST) and alkaline phosphatase (ALP) suggested a degree of hepatic damage caused mainly by spitting cobra venoms. Immunologic analyses revealed a considerable extent of cross-reactivity of monospecific antisera against heterologous venoms within the spitting and no-spitting cobras, yet some antisera showed more extensive cross-reactivity than others. The antisera with the widest coverage were those of anti-Naja ashei and anti-N. nigricollis for the spitting cobras, and anti-N. haje and anti-N. senegalensis for the non-spitting cobras. Conclusions/significance: The methods and study design followed provide a rationale for the selection of the best combination of venoms for generating antivenoms of high cross-reactivity against cobra venoms in sub-Saharan Africa. Results suggest that venoms from N. ashei, N. nigricollis within the spitting cobras, and N. haje and N. senegalensis within the non-spitting cobras, generate antisera with a broader cross-reactivity. These experimental results should be translated to larger animal models used in antivenom elaboration to assess whether these predictions are reproduced. Author summary: The African elapids pose a major potential to induce clinically important envenomations and severe sequelae after a snakebite accident. Within the genus Naja, different radiation events have occurred rendering the African cobras with diverse features in their venom composition. Commonly divided into spitting and non-spitting cobras, their venom is characterized as either cytotoxic or neurotoxic, respectively, based on the pathophysiology of envenomation. In this study, an experimental protocol was developed based on the generation of monospecific antisera in rabbits immunized with venoms of spitting and non-spitting African cobras. Cross-reactivity of monospecific antisera was assessed by immunochemical analyses and by neutralization of the lethal, phospholipase A₂ (PLA₂) and dermonecrotic activities. Results showed a large extent of intrageneric cross-reactivity by all antisera, yet few exceptions were noticed. The venoms that generated antisera with the highest cross-neutralization ability among the spitting cobras were Naja nigricollis and N. ashei, whereas for the non-spitting cobras were N. haje and N. senegalensis. These results provide evidence for the selection of the best combination of immunogens for antivenom production with the widest neutralization scope within the African cobras. Additionally, the results obtained in the rabbit model should be translated to a larger animal model used for antivenom fabrication to assess whether these predictions are confirmed. [ABSTRACT FROM AUTHOR]