Functional Application of Snake Venom Proteomics in In Vivo Antivenom Assessment.

Reverse-phase high-performance liquid chromatography is commonly employed as a decomplexing strategy in snake venom proteomics. The chromatographic fractions often contain relatively pure toxins that can be assessed functionally for toxicity level through the determination of their median lethal doses (LD50). Further, antivenom efficacy can be evaluated specifically against these venom fractions to understand the limitation of the antivenom as the treatment for snake envenomation. However, methods of toxicity assessment and antivenom evaluation vary across laboratories; hence there is a need to standardize the protocols and parameters, in particular those related to the neutralizing efficacy of antivenom. This chapter outlines the important in vivo techniques and data interpretation that can be applied in the functional study of snake venom proteomes.

Toxin-resolved antivenomics-guided assessment of the immunorecognition landscape of antivenoms.

Snakebite envenoming represents a major issue in rural areas of tropical and subtropical regions across sub-Saharan Africa, South to Southeast Asia, Latin America and Oceania. Antivenoms constitute the only scientifically validated therapy for snakebite envenomings, provided they are safe, effective, affordable, accessible and administered appropriately. However, the lack of financial incentives in a technology that has remained relatively unchanged for more than a century, has contributed to some manufacturers leaving the market and others downscaling production or increasing the prices, leading to a decline in the availability and accessibility for these life-saving antidotes to millions of rural poor most at risk from snakebites in low income countries. The shortage of antivenoms can be significantly alleviated by optimizing the use of current antivenoms (through the assessment of their specific and paraspecific efficacy against the different medically relevant homologous and heterologous snake venoms) and by generating novel polyspecific antivenoms exhibiting broad clinical spectrum and wide geographic distribution range. Research on venoms has been continuously enhanced by advances in technology. Particularly, the last decade has witnessed the development of omics strategies for unravelling the toxin composition of venoms ("venomics") and to assess the immunorecognition profile of antivenoms ("antivenomics"). Here, we review recent developments and reflect on near future innovations that promise to revolutionize the mutually enlightening relationship between evolutionary and translational venomics.

Venomous Snake Bite in India - Why do 50,000 Indians Die Every Year?

Snakebite is an occupational hazard causing considerable morbidity and mortality worldwide, particularly so in tropical countries like India. An estimated 50,000 Indians die due to venomous snakebite every year, seventy percent of whom are males between the ages of 20 to 50 years. Along with the associated morbidity and mortality, snakebite leads to a significant financial burden on the victim, both by way of hospital bills and labour hours lost. Snakebite is also a cause for considerable psychological stress among survivors. Most snakebites are eminently treatable and curable. Given a concerted thrust from all concerned , this menace could surely be curtailed considerably over the next few years.

Evaluation of health status of horses immunized with snake venom and montanide adjuvants, IMS 3012 (nanoparticle), ISA 206 and ISA 35 (emulsion based) during polyvalent snake antivenom production: hematological and biochemical assessment.

Several biochemical and hematological changes in horses are observed during production of snake antivenom. Although conventional adjuvants like Freund's (Complete and Incomplete) are good immunopotentiators, they produce considerable local reactions in animals. Variety of commercial adjuvants, like montanide adjuvants, having high immunopotentiation and showing lesser side effects are available. The prime objective during antivenom production is to strike a balance between safety of immunized horses and efficacy of the product. In our earlier work, efficacy of montanide group of adjuvants in antivenom production has already been established. The aim of the present work was to assess the safety parameters in horses, viz.: biochemical and hematological, during production of snake antivenom. In the present study, 33 new horses were randomly divided into four groups and hyperimmunized using mixture of snake venoms, viz.: Cobra venom, Russell's viper venom, Krait venom and Echis venom along with montanide adjuvants, IMS 3012, ISA 206, ISA 35 and Incomplete Freund's adjuvant as a control adjuvant; through subcutaneous route at intervals of two weeks. During the immunization period, biochemical and hematological parameters were monitored at 0th, 14th, 21st, 30th and 42nd weeks. The mean hemoglobin values dropped slightly during initial immunization but subsequently regained to normal levels. The mean serum total protein values and globulin levels showed an increment in all the four groups, compared to day zero, vice-versa a slight drop was observed in albumin levels. No significant changes were observed in serum creatinine, bilirubin, alkaline phosphatase and blood urea nitrogen values. Finally, we conclude that montanide adjuvants could be a safer alternative to the conventional adjuvants for primary phase of immunization in antivenom production.

Assessment of snake antivenom purity by comparing physicochemical and immunochemical methods.

Purity is a characteristic that, together with effectiveness and safety, must be tested to determine the quality of biopharmaceutical products. In therapeutic immunoglobulins, such as human intravenous immunoglobulin (IVIG), purity is evaluated on the basis of physicochemical properties, and is usually assessed by chromatography and electrophoresis. However, in the case of antivenoms these methods fail to discriminate between antibodies towards venom antigens, which constitute the active substance, and antibodies towards non-venom antigens, which are the major impurities in most of the current formulations. The assessment of this aspect of purity requires the use of the immunochemical methods. In this study, it was demonstrated that antivenoms showing physicochemical purity higher than 90% might present immunochemical purity lower than 40%. It is proposed that a comprehensive analysis of antivenom purity should combine physicochemical and immunochemical parameters. In addition, these results are crucial to decide the more appropriate strategies to improve antivenom purity. Taking into account that the current methods of antivenom purification remove most non-antibodies proteins, we propose that efforts must be primarily directed to the improvement of immunization protocols to enhance the antibody response towards venom components in hyperimmunized animals, and secondarily, in the realm of immunoglobulin purification technology.

[Role of antivenoms in the treatment of snake envenomation]. / Place de l'immunothérapie dans le traitement actuel des envenimations ophidiennes.

The production of antivenoms, which were long deemed ineffective, dangerous and difficult to use, has improved dramatically. These antibodies (immunoglobulin G) are now fragmented, purified and controlled for their quality, leading to significantly better safety and facilitating their emergency use. Envenomation can result in various syndromes depending on the snake species: Viperidae venoms are highly inflammatory, hemorrhagic and necrotising, while Elapidae venoms can cause fatal respiratory paralysis. However, some Viperidae venoms can lead to asphyxiation similar to that observed in Elapidae envenomation while, conversely, Elapidae bites may be complicated by hemorrhage or necrosis, thus complicating etiologic diagnosis. Symptomatic treatment is complex, often insufficient, and frequently associated with adverse events. In contrast, antivenoms neutralize the venom and accelerate its clearance, thus providing an etiological treatment for envenomation, particularly in remote healthcare facilities in developing countries. Current formulations consist of polyvalent antivenoms covering most of the venomous species present in a specific region. The main limitation is their high cost, and the priority should be to develop new treatment strategies, including more affordable antivenoms, especially in developing countries where they are most needed.

[Assessment of the availability and use of antivenom in the district of Bamako, Mali, West Africa]. / Évaluation de la disponibilité et l'utilisation du sérum antivenimeux dans le district de Bamako, Mali, Afrique de l'Ouest.

The aim of this study is to describe the difficulties related to problems of supply and use of antivenom serum (SAV) in the district of Bamako. A retrospective study over a span of five years (January 1998-December 2002) and an interview with the staff of various facilities were conducted. The study included 2 wholesalers of pharmaceuticals, 20 private pharmacies, and 2 hospital pharmacies as they were involved in antivenom trades. A market-driving ability survey of driving practice was conducted in 37 community health centers (CHCs) and 4 dispensaries because they performed antivenom treatments during the study period. A total of 3,318 doses of antivenom were bought, including 84.4% by the People Pharmacy of Mali (PPM), a public organization, and 15.6% by Laborex, a private company. These were out of stock in 1999. Three kinds of SAV were ordered: the polyvalent IPSER Africa (1,200 vials or 36.2%), FAV Africa (318 vials or 9.6%), and Sii anti-snake venom polyvalent serum (1,800 vials or 54.2%). Orders from PPM involved IPSER Africa (Pasteur Mérieux Serum & Vaccines) and Sii anti-snake venom polyvalent serum (Serum Institute of India), and those from Laborex involved IPSER Africa and FAVAfrica (Aventis Pasteur). Onehalf of private pharmacies (54.3%) had made at least one order of SAV. The PPM lost 50% of 2,000 vials of SAV in 1996 due to the expiration of vials that were bought. Private pharmacies lost 3.6% of stocks due to expiration. Prices varied depending on the type of service and the point of sale. Costs of vials were 19,440-35,000 CFA francs (29.6-53.4 euros) for Sii antivenom and 50,200-63,000 CFA francs (76.5-96.1 euros) for FAV Africa antivenom. In CHCs, 59.5% of prescribers were unaware of the indications and methods of proper administration of the SAV, 32.3% ignored the existence of SAV, and 30.9% were skeptical about its effectiveness in treatment of envenomation by snakebite.

Antisnake venom production crisis--who told us it was uneconomic and unsustainable?

The world of antisnake venom production is currently a gloomy place to visit. It is described as being in crisis, characterized by shortages, producers leaving the market, high prices, and unsustainability. It has been reduced to a pauper-like status, doomed to relying on charitable handouts for resolution. The worrying aspect of this is that little work has been done to establish the true economics and return on antisnake venom if provided by private companies. Fortunately, it is amenable to economic analysis, and in this manner, a rational approach to further development and distribution can be obtained. This article proposes a model antisnake venom (ASV) production unit and shows the likely economics and return based on the production of various volumes of ASV. It estimates the costs for the key components of the unit, which are production equipment and staffing. A profit and loss account and balance sheet are constructed for the unit, and the effects of ASV volume and neutralizing titres are demonstrated. It is our contention that ASV production can be sustained at affordable prices in the developing world. We recommend that any solution to the ASV shortage must take into account the most cost efficient method(s) of production.

The global snakebite crisis--a public health issue misunderstood, not neglected.

The global problem of venomous snakebite continues to attract attention despite it being described as a "neglected" issue. The current focus of the World Health Organization (WHO) remains anti-snake venom quality, although "availability and sustainability" of supply are consistently described as the key issues. Sustainability of antivenom supply has been elusive, with cost and pricing in developing countries being cited as the major reasons. The current WHO approach fails to explore the cost issue, but rather focuses on quality improvements, which may well adversely affect the costs of a product already perceived to be 'unaffordable.' The reference to cost and price indicates a marketing-based perspective may well give more relevant solutions to the snakebite crisis. This paper introduces a marketing model to examine global snakebite and to identify if the current approach is relevant and effective. The "4 Ps" model examines if the correct products are available, whether sufficient information exists concerning estimated market size, whether the assumptions frequently made about the costs of the product are correct and fully understood, if the product is promoted properly, and whether the method by which the product reaches the end user is optimum. The resulting analysis demonstrates that the current approach is characterized by a misunderstanding of the nature of the global snakebite problem. Further, a lack of implementation of key solutions, such as training doctors in developing countries with relevant protocols, has inevitably led to a lack of improvement in the snakebite arena over the last 30 years.

Time for an alternative perspective: the eternal problem of supply and quality of anti snake venom in the developing world--"it's the economy, stupid".

The "crisis in anti snake venom supply" has been an enduring problem. Despite the frequency with which it appears in the literature, it remains unquantified and an enigma. If there is a serious shortage of anti snake venom (ASV), why has this not been resolved? Anti snake venoms are produced, and yet many suppliers are described as leaving the market. There appears to be a problem in the call for highly effective, high-quality, and cheap anti venoms that contributes to this result of suppliers leaving the market. Private companies are tasked with achieving adequate shareholder returns and by doing so ensure continued supply. Efforts should therefore target a means of lowering production cost by introducing whole immunoglobulin G (IgG) antivenoms with greater antibody yields, reducing the drive to eliminate adverse reactions, for which there are other more cost-effective treatments, as well as a means of introducing good manufacturing processes, with care based on demonstrable need. In order to ensure sustainability of supply, a private company supplier providing a whole IgG antivenom that effectively neutralizes venom is the most credible option. The need for ASV in areas of shortage mandates the need for clear decisions regarding the type of ASV and the recognition that the market requires acceptable returns for producers if supply is to be sustainable. This paper reviews the economic realities of ASV production and suggests a pragmatic, sustainable approach to the problem of supplying ASV to developing countries.

Human heterophilic antibodies against equine immunoglobulins: assessment of their role in the early adverse reactions to antivenom administration.

The presence of human heterophilic antibodies against horse immunoglobulins (HHA-HI) was determined by ELISA in sera from healthy volunteers and from patients who received equine antivenom for therapy of snake bite envenoming. These patients were selected from two independent clinical studies: one in Colombia in which patients received antivenom constituted by whole IgG (n=25); and the other in Brazil where an antivenom constituted by F(ab')(2) fragments was administered (n=31). Results show that healthy volunteers have antibodies, mainly of the IgG class, able to react with whole equine IgG. Additionally, patients have IgG antibodies that react both with whole equine IgG and F(ab')(2) fragments. In both clinical studies, no significant differences were observed in the HHA-HI titres between the patients who presented early adverse (anaphylactoid) reactions and those who did not develop them. In addition, no variation in titre was observed in samples collected before and after antivenom administration. These results do not support the hypothesis that the incidence of early adverse reactions to antivenom administration correlates with the titre of HHA-HI in the serum of patients. Nevertheless, participation of these antibodies as part of a multifactorial pathogenic mechanism associated with these reactions cannot be ruled out.

Comparison of mouse and rabbit model for the assessment of strong PGM-containing oil-based adjuvants.

Peptidoglycan monomer (PGM) is an adjuvant active molecule with potential for use in human and veterinary vaccine. PGM's action is short-lived in mammals hence its effects might be limited. Novel PGM-containing oil-based formulations have been developed recently by incorporation of PGM into Montanide ISA720 and ISA206 adjuvants with the aim to prolong and improve immunostimulating activities of PGM. In the present work we studied the efficacy of such novel adjuvant formulations using two different antigens, ovalbumin and snake venom, respectively. Novel formulations were also tested in two experimental models, mice and rabbits. In rabbits the incorporation of PGM into oil-based adjuvants led to overall improvement of antigen-specific IgG response. However, in the mouse model, under experimental conditions used, it was not possible to distinguish differences in antigen-specific IgG response among several strong oil-based adjuvant formulations.

A new Pan African polyspecific antivenom developed in response to the antivenom crisis in Africa.

Currently there is a crisis in the supply of antivenom for treatment of snake bite in sub-Saharan Africa. Commercial pressures have resulted in the reduction or even cessation of production of antivenom by European manufacturers while continued production of antivenom in Africa has been threatened by the privatisation of the only remaining company based in Africa. As a consequence, there has been an increase in snake bite morbidity and mortality in many African countries. Two Latin American antivenom manufacturers have agreed to produce antivenom suitable for Africa, using venoms from the species which are of the greatest medical importance in sub-Saharan Africa. Preclinical in vivo assays of neutralising potency demonstrated that a new Pan African antivenom produced in Colombia compared favourably with the existing commercial monospecific and polyspecific antivenoms. This new antivenom, and a similar product being manufactured in Costa Rica, are now candidates for clinical testing at an appropriate site in Africa.

The development and use of immunotherapy in Africa.

The immunotherapy was recently developed due to the improvement of purification techniques of antivenoms and results of the research in toxicology and pharmacology. The utilisation of highly purified IgG fragments leads to a better tolerance and a higher efficacy. Snake envenomations constitute in Africa, as in many tropical countries, an important public health problem. The annual incidence of snakebites reaches 1 million and the annual mortality is about 20,000 deaths. Less than 25% of the antivenom needs are effectively covered and, probably in most of envenomations, used at insufficient doses. The treatment of snakebites would be improved by better knowledge on snakebite epidemiology, standardisation of treatment and training medical staff, and development of new financial procedures for antivenom supply.

Immunodiagnosis of snake venom poisoning.

Uncertainty as to the species diagnosis remains a serious problem in the management of snake venom poisoning. This is particularly so in areas inhabited by numerous species, the venoms of which elicit similar pharmacological effects and clinical symptoms and against which para-specific cross-neutralizing antivenom is not available. Attempts have been made to eliminate some of this ambiguity through the development of various immunodiagnostic tests. Tests based on ELISA are sensitive, specific and even quantitative and adaptable to field application. In the development of diagnostic tests for use in developing countries, however, practical consideration must be given to speed, cost, simplicity in terms of equipment and expertise, and stability to the climate and storage conditions. This may dictate further modification or selection of more suitable alternative methodologies. Furthermore, the test may have to allow more flexibility in accommodating local species distributions and to address probable complications of heterophile antibodies in test samples from rural people.

Clinical laboratory: enzyme immunoassay for the rapid clinical identification of snake venom.

The treatment of snakebite could be simplified if the identity of the offending snake was more frequently known. A positive identification, which allows the use of a specific monovalent antivenom, probably occurs in less than 20% of cases. Recently published methods of venom detection (RIA and ELISA) take at least three hours to complete. We have developed a sandwich enzyme immunoassay (EIA) which is capable of detecting 0.5 ng of crude snake venom in about 90 minutes or 2 ng of crude venom in about 30 minutes. This substantial reduction in incubation times, while still retaining the sensitivity required, was due to the use of protein A purified rabbit IgG antivenom from hyperimmune serum and the enzyme horseradish peroxidase (HRPO). A rapid identification of the offending snake by this method may reduce the use of large-volume polyvent antivenoms, thus avoiding the clinical and economic disadvantages of such preparations. Other advantages would be an increased understanding of the clinical syndrome produced by the individual species of snake, and accumulation of data about the incidence of envenoming attributed to specific snakes.