Snakebite envenomation is considered a neglected tropical disease, affecting tens of thousands of people each year. The recommended treatment is the use of antivenom, which is composed of immunoglobulins or immunoglobulin fragments obtained from the plasma of animals hyperimmunized with one (monospecific) or several (polyspecific) venoms. In this review, the efforts made in the improvement of the already available antivenoms and the development of new antivenoms, focusing on snakes of medical importance from sub-Saharan Africa and Latin America, are described. Some antivenoms currently used are composed of whole IgGs, whereas others use F(ab’)2 fragments. The classic methods of attaining snake antivenoms are presented, in addition to new strategies to improve their effectiveness. Punctual changes in immunization protocols, in addition to the use of cross-reactivity between venoms from different snakes for the manufacture of more potent and widely used antivenoms, are presented. It is known that venoms are a complex mixture of components; however, advances in the field of antivenoms have shown that there are key toxins that, if effectively blocked, are capable of reversing the condition of in vivo envenomation. These studies provide an opportunity for the use of monoclonal antibodies in the development of new-generation antivenoms. Thus, monoclonal antibodies and their fragments are described as a possible alternative for the production of antivenoms, regardless of the venom. This review also highlights the challenges associated with their development.

Snake envenoming is a significant public health problem in underdeveloped and developing countries. In sub-Saharan Africa, it is estimated that 90,000–400,000 envenomations occur each year, resulting in 3,500–32,000 deaths. Envenomings are caused by snakes from the Viperidae (Bitis spp. and Echis spp.) and Elapidae (Naja spp. and Dendroaspis spp.) families. The African continent has been suffering from a severe antivenom crisis and current antivenom production is only sufficient to treat 25% of snakebite cases. Our aim is to develop high-quality antivenoms against the main snake species found in Mozambique. Adult horses primed with the indicated venoms were divided into 5 groups (B. arietans; B. nasicornis + B. rhinoceros; N. melanoleuca; N. mossambica; N. annulifera + D. polylepis + D. angusticeps) and reimmunized two times for antivenom production. Blood was collected, and plasma was separated and subjected to antibody purification using caprylic acid. Plasmas and antivenoms were subject to titration, affinity determination, cross-recognition assays and in vivo venom lethality neutralization. A commercial anti-Crotalic antivenom was used for comparison. The purified antivenoms exhibited high titers against B. arietans, B. nasicornis and B. rhinoceros (5.18 x 10⁶, 3.60 x 10⁶ and 3.50 x 10⁶ U-E/mL, respectively) and N. melanoleuca, N. mossambica and N. annulifera (7.41 x 10⁶, 3.07 x 10⁶ and 2.60 x 10⁶ U-E/mL, respectively), but lower titers against the D. angusticeps and D. polylepis (1.87 x 10⁶ and 1.67 x 10⁶ U-E/mL). All the groups, except anti-N. melanoleuca, showed significant differences from the anti-Crotalic antivenom (7.55 x 10⁶ U-E/mL). The affinity index of all the groups was high, ranging from 31% to 45%. Cross-recognition assays showed the recognition of proteins with similar molecular weight in the venoms and may indicate the possibility of paraspecific neutralization. The three monospecific antivenoms were able to provide in vivo protection. Our results indicate that the anti-Bitis and anti-Naja antivenoms developed would be useful for treating snakebite envenomations in Mozambique, although their effectiveness should to be increased. We propose instead the development of monospecific antivenoms, which would serve as the basis for two polyvalent antivenoms, the anti-Bitis and anti-Elapidae. Polyvalent antivenoms represent an increase in treatment quality, as they have a wider range of application and are easier to distribute and administer to snake envenoming victims. Snake envenoming has long been a serious public health problem in underdeveloped and developing countries. In sub-Saharan Africa, the number of bites can be as high as 400,000 and result in up to 32,000 deaths. As accidents involving snakes are most common among young men in rural areas, complications and amputations can mean the loss of livelihood. Specific antivenoms are the most effective treatment available, but the only African antivenom producer can only supply 25% of the continent’s demand. In this study, we have developed antivenoms against the most important snakes commonly found in Mozambique: Bitis spp. (puff adders), Naja spp. (cobras) and Dendroaspis spp. (mambas). The experimental antivenoms were made by immunizing horses with the specific venoms, then collecting and processing their plasma to purify the antibodies. The experimental antivenoms were compared to the commercially available anti-Crotalic (rattlesnake) antivenom. The antivenoms produced had high titers, showed affinity for the specific venoms, were able to cross-recognized similar venoms and provide in vivo protection. The data in this study indicates that the antivenoms would be effective in treating B. arietans, B. nasicornis, N. melanoleuca and N. mossambica envenomations. We propose the development of monospecific antibodies as a strategy to increase antivenom quality, and as the basis for the production of two polyspecific antivenoms, anti-Bitis and anti-Elapidae.

Bitis arietans is a snake of medical importance found throughout sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. The effects of its venom are characterized by local and systemic alterations, such as inflammation and cardiovascular and hemostatic disturbances, which can lead to victims’ death or permanent disability. To better characterize the inflammatory process induced by this snake’s venom, the participation of eicosanoids and PAF (platelet- activating factor) in this response were demonstrated in a previous study. In addition, edema and early increased vascular permeability followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity were accompanied by the production of the eicosanoids LTB₄, LTC₄, TXB₂, and PGE₂, and local and systemic production of IL-6 and MCP-1. In this context, the present study focused on the identification of inflammatory mediators produced by human macrophages derived from THP-1 cells in response to Bitis arietans venom (BaV), and Kn-Ba, a serine protease purified from this venom. Here, we show that Kn-Ba, and even the less intensive BaV, induced the production of the cytokine TNF and the chemokines RANTES and IL-8. Only Kn-Ba was able to induce the production of IL-6, MCP-1, and IP-10, whereas PGE₂ was produced only in response to BaV. Finally, the release of IL-1β in culture supernatants suggests the activation of the inflammasomes by the venom of Bitis arietans and by Kn-Ba, which will be investigated in more detail in future studies.

Bitis arietans is a venomous snake found in sub-Saharan Africa and in parts of Morocco and Saudi Arabia. The envenomation is characterized by local and systemic reactions including pain, blistering, edema and tissue damage, besides hemostatic and cardiovascular disturbances, which can cause death or permanent disabilities in its victims. However, the action mechanisms that provoke these effects remain poorly understood, especially the activities of purified venom components. Therefore, in order to elucidate the molecular mechanisms that make the Bitis arietans venom so potent and harmful to human beings, this study reports the isolation and biochemical characterization of a snake venom serine protease (SVSP). Solubilized venom was fractionated by molecular exclusion chromatography and the proteolytic activity was determined using fluorescent substrates. The peaks that showed serine protease activity were determined by blocking the proteolytic activity with site-directed inhibitors. In sequence, the fraction of interest was submitted to another cycle of molecular exclusion chromatography. The purified serine protease was identified by mass spectrometry and characterized biochemically and immunochemically. A serine protease of 33 kDa with fibrinogen-degrading and kinin-releasing activities was isolated, described, and designated herein as Kn-Ba. The experimental Butantan Institute antivenom produced against Bitis arietans venom inhibited the Kn-Ba activity. The in vitro activities of Kn-Ba can be correlated with the capacity of the venom to provoke bleeding and clotting disorders as well as hypotension, which are common symptoms presented by envenomed victims. Obtaining satisfactory Kn-Ba inhibition through the experimental antivenom is important, given the WHO’s recommendation of immunotherapy in cases of human accidents with venomous snakes.

Bitis arietans is a snake of medical importance, as it is responsible for more accidents in humans and domestic animals than all other African snakes put together. The accidents are characterized by local and systemic alterations, such as inflammation, cardiovascular and hemostatic disturbances, which can lead victims to death or permanent disability. However, little is known about the envenomation mechanism, especially regarding the inflammatory response, which is related to severe clinical conditions triggered by the venom. Therefore, the aim of the present study was to evaluate the inflammatory response related to the B. arietans envenomation using a peritonitis mice model. By pharmacological interventions and use of mice genetically deficient of the 5-lipoxygenase enzyme (5-LO^−/−) or platelet-activating factor (PAF) receptor (PAFR^−/− the participation of eicosanoids and PAF in this response was also investigated. The obtained results demonstrated that the venom induces an in vivo inflammatory response, characterized by an early increased vascular permeability, followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity, accompanied by the production of the eicosanoids LTB₄, LTC₄, TXB₂ and PGE₂, as well as the local and systemic production of IL-6 and MCP-1. These inflammatory events were attenuated by the pre-treatment with anti-inflammatory drugs that interfere in lipid mediators’ functions. However, 5-LO^−/− mice did not show a reduction of inflammatory response induced by the venom, while PAFR^−/− mice showed a reduction in both the PMN leukocytes number and the local and systemic production of IL-6 and MCP-1. This study demonstrated that the Bitis arietans venom contains toxins that trigger an inflammatory process, which is partially dependent on lipid mediators, and may contribute to the envenomation pathology.