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

Abstract

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.

Comparison of the intrageneric neutralization scope of monospecific, bispecific/monogeneric and polyspecific/monogeneric antisera raised in horses immunized with sub-Saharan African snake venoms

“…was generated by immunization with N . senegalensis venom, as per previous observations with antisera produced in rabbits [ 12 ]. The monospecific anti-non-spitting Naja antiserum was able to neutralize lethality of all neurotoxic Naja venoms, with variable ED 50 s ( Fig 6 ).…”

“…The venom of N . nigricollis was used to formulate the monospecific immunogen of spitting (cytotoxic) Naja spp., following previous observations using rabbit antisera [ 12 ]. The antibody response induced by this venom was able to neutralize the lethality induced by all the venoms of spitting Naja assessed in this study, albeit with different ED 50 s, having low efficacy against the venom of N .…”

“…Values of Naja spp. venoms from Gómez et al (2023) [ 12 ], and Dendroaspis spp. venoms from Gómez et al (2024) [ 13 ].…”

Intrageneric cross-reactivity of monospecific rabbit antisera against venoms of mamba (Elapidae: Dendroaspis spp.) snakes

“…The experimental protocol used by Gómez and colleagues ( Gómez et al, 2022 , 2023 ) was followed to assess the cross-reactivity and cross-neutralization of the antisera, using a combination of antibody titers quantified by ELISA, Western blot, and neutralization of the lethal activity to assess the intrageneric cross-reactivity of monospecific rabbit sera against homologous and heterologous venoms. Cross-reactivity was evidenced in all the anti- Dendroaspis antisera against the four mamba venoms studied, showing an antigenic similarity between venoms, yet quantitative differences were noticed ( Fig.…”

African polyvalent antivenom can maintain pharmacological stability and ability to neutralise murine venom lethality for decades post-expiry: evidence for increasing antivenom shelf life to aid in alleviating chronic shortages

“…Mice were then injected intravenously with mixtures containing a challenge dose of venom dissolved in sterile 0.9% NaCl USP injection solution and variable dilutions of antivenom, which were premixed and incubated at 37°C for 30 min (volume of injection was 0.2 mL). 34 Challenge doses were three times the previously determined median lethal dose (LD 50 ) for the venoms of N. nigricollis (3× LD 50 =55.2 µg/mouse) 35 and D. polylepis (3× LD 50 =1.14 µg/mouse) 36 and five times for the venom of B. arietans (5× LD 50 =110 µg/mouse). 37 The rationale for using 3× LD 50 challenge doses for N. nigricollis and D. polylepis, rather than the conventional 5× LD 50 , was a refinement due to experience with these particular venoms demonstrating the use of 5× LD 50 resulted in poor resolution of dose groups outcomes.…”