Harnessing the Cross-Neutralisation Potential of Existing Antivenoms for Mitigating the Outcomes of Snakebite in Sub-Saharan Africa.

Over 32,000 individuals succumb to snake envenoming in sub-Saharan Africa (sSA) annually. This results from several factors, including a lack of antivenom products capable of neutralising the venoms of diverse snake species in this region. Most manufacturers produce polyvalent antivenoms targeting 3 to 16 clinically important snake species in sSA. However, specific products are unavailable for many others, especially those with a restricted geographic distribution. While next-generation antivenoms, comprising a cocktail of broadly neutralising antibodies, may offer an effective solution to this problem, given the need for their clinical validation, recombinant antivenoms are far from being available to snakebite victims. One of the strategies that could immediately address this issue involves harnessing the cross-neutralisation potential of existing products. Therefore, we assessed the neutralisation potency of PANAF-Premium antivenom towards the venoms of 14 medically important snakes from 13 countries across sSA for which specific antivenom products are unavailable. Preclinical assays in a murine model of snake envenoming revealed that the venoms of most snake species under investigation were effectively neutralised by this antivenom. Thus, this finding highlights the potential use of PANAF-Premium antivenom in treating bites from diverse snakes across sSA and the utility of harnessing the cross-neutralisation potential of antivenoms.

Critical aspects on traditional antivenom production processes and their optimization by factorial analysis.

Most antivenoms are produced by techniques developed over 50 years ago, with minor modifications. Herein we revise the core of traditional antivenom production processes aiming to optimize key determinants for both consistent antivenom production and the best balance between F(ab')2 quality and recovery. Factorial design analysis revealed that pepsin digestion of 1:3 saline diluted equine plasma for 60 min under pH: 3.20, 37 °C temperature and a 1:15 pepsin to protein ratio conditions, allowed to achieve maximal IgG to F(ab')2 conversion with minimal protein aggregate formation. Further downstream processing by salting out with ammonium sulfate was also studied by factorial analysis. The influence of ammonium sulfate (AS) concentration, temperature (T) and the albumin to total plasma protein ratio plasma (Alb:P) were assayed, revealing that both AS, T and their interaction have a significant impact in F(ab')2 quality and recovery. Taking into account the existing compromise between F(ab')2 monomer recovery and quality two alternative conditions were selected: 14 g/dl AS at 56 °C and, alternatively 16 g/dl AS at 30 °C. Reasonable yields (42%) and product quality (2.5% of aggregates) without significant changes in production cost of traditional methodologies was achieved under the optimized conditions found.

Management of Snakebites in the Upper Extremity.

Encounters with venomous snakes can lead to substantial morbidity or mortality if managed inappropriately. Medical management is the mainstay of treatment, but surgery may be necessary in rare cases. The hand surgeon should be well versed in the types of venomous snakes, the mechanism of action of venom, and the management of these injuries, given the frequency of hand envenomation. The indications for surgery are not well established and rely on clinical judgment and practitioner's experience. An understanding of previously reported outcomes and an algorithmic approach to treatment will help improve patient care and avoid complications.

Black Mamba Death: Venom Versus Antivenom?

We present the case of a male adult who was admitted to an emergency department after having sustained envenomation from a black mamba (Dendroaspis polylepis). According to the available history, a single fang hooked his right index finger, post venom extraction. After administering antivenom in the accident and emergency department, further vials were transfused in the intensive care unit. An urticarial rash was noted, which was thought to be related to the antivenom. The victim remained in a coma for 3 days, after which he was declared dead. A medicolegal postmortem examination was performed 4 days after death because of logistical reasons. The complexities of differentiating acute envenomation from black mamba versus early acute reactions to polyvalent antivenom administration are highlighted in this case study.

ImmunoCAP cellulose displays cross-reactive carbohydrate determinant (CCD) epitopes and can cause false-positive test results in patients with high anti-CCD IgE antibody levels.

BACKGROUND: Cross-reactive carbohydrate determinants (CCDs) in plants and insect venoms are a common cause of irrelevant positive test results during in vitro allergy diagnosis. We observed that some CCD-positive sera show nonspecific IgE binding even with CCD-free recombinant allergens when using the Phadia ImmunoCAP platform. OBJECTIVE: We investigated whether cellulose used as an allergen carrier in ImmunoCAP harbors residual N-glycans, causing nonspecific background binding in CCD-positive sera. METHODS: IgE binding to 6 samples of blank ImmunoCAPs coupled to either streptavidin (SA-CAP-1 or 2) or nonallergenic maltose-binding protein (MBP; MBP-CAP-1 to 4) and binding to a panel of 4 recombinant allergens were compared in CCD-positive sera before and after inhibition with a CCD inhibitor (MUXF3-human serum albumin). RESULTS: Of 52 CCD-positive sera (bromelain, 1.01-59.6 kilounits of antigen per liter [kUA/L]) tested on SA-CAP-1, 35 (67%) showed IgE binding of greater than 0.35 kUA/L (0.41-4.22 kUA/L). Among those with anti-CCD IgE levels of greater than 7.0 kUA/L, 90% (26/29) were positive. IgE binding to SA-CAP-1 correlated with IgE binding to bromelain (r = 0.68) and was completely abolished by serum preincubation with the CCD inhibitor (n = 15). Binding scores with SA-CAP-2 and MBP-CAP-1 to MBP-CAP-4 were generally lower but strongly correlated with those of SA-CAP-1 and bromelain. IgE reactivity of 10 CCD-positive sera (14.0-52.5 kUA/L) with the recombinant allergens rPhl p 12, rFel d 1, rAra h 2, and rPru p 3 was positive to at least 1 allergen in 8 of 10 (0.36-1.63 kUA/L) and borderline in 2 of 10 (0.21-0.25 kUA/L). Binding correlated with antibody binding to bromelain (r = 0.61) and to all blank ImmunoCAPs (r > 0.90) and could be completely blocked by the CCD inhibitor. Overall, mean background binding to cellulose CCDs corresponded to 2% to 3% of the reactivity seen with bromelain. CONCLUSIONS: Cellulose used as a solid-phase allergen carrier can contain varying amounts of CCDs sufficient to cause false-positive test results up to 2 kUA/L with nonglycosylated recombinant allergens in patients with high levels of anti-CCD IgE antibodies.

Snake Bite Envenomation in a Tertiary Care Centre.

Background: In India, it is estimated that up to 20,000 people die annually from snake bites. The present study was carried to out to estimate the snake bite related epidemiology, predictors of severity, relationship between type of snake, clinical severity, complications, outcome and usage pattern of polyvalent anti snake venom (ASV) in a tertiary care center. Methods: All indoor patients admitted in our institute with definitive history of bite by a snake, with or without presence of fang marks, Evidence of cellulitis, acute onset of neurotoxicity or bleeding diathesis were serially recruited in the study. Results: The majority of cases were in the range of 21- 40 years (54.7%). There were 82.8% males (53/64), 17.2% females (11/64) and 60.9% (39/64) bites were during day time. Upper limb bites were seen in 34% (22/64) of the patients and lower limb bites in 54% (35/64), and axial body bites in 6%. There were 43.8% (28/64) vasculotoxic bites, 34.4% (22/64) neurotoxic bites and 20.3% (14/64) non-poisonous bites. Viper was the most common (9%) identified snake, followed by krait (5%). References from Rural Health Centers were 57.8% (57/64), 11% were from Primary health centers and rest from private sector. Anti snake venom (ASV) was received by 68.75% (44/64) patients before reaching tertiary care. Local swelling was present in 90.6% (58/64) patients, Systemic bleeding was seen in 35.9% (23/64), and Neuromuscular weakness in 35.9% (23/64) patients. Complications like Respiratory paralysis developed in 18.75% (12/64), Acute kidney injury in 12% (8/64), DIC in 9% (6/64), and hepatic involvement in 7% (5/64) of snake bite patients. Blood transfusion was required in 20.3% (13/64) p<0.001), 18.75% (12/64) required Mechanical ventilation (p=0.001), 4 received hemodailysis and 4 required ionotropic support (p<0.001). Improvement was seen in 57.8% (37/64), morbidity during hospital stay was seen in 39% (25/64) and 2 patients expired (3%). ASV was received within 4 hours in 67% (42/64) patients, 22.5% (14/64) received ASV between 4 to 24 hours and remaining after 24 hours (p=0.016). Total ASV requierment was 24.05 vials in patients who improved and 34.4vials in patients in Morbid group and 29.0 vials in mortality group (p>0.05). The SSS score amongst improved was 4.76 ± 2.46 whereas among morbid, it was 8.48 ± 1.75 and amongst expired, it was 8.5 ± 0.707 (p<0.05). Conclusions: Patients requiring various supportive treatments like blood transfusion, Inotropes, Haemodialysis and Mechanical ventilation, had a statistically significant correlation with poor outcome. Early administration of ASV that is within 4 hours was, associated with better outcome. The total amount of ASV (in vials) had no a significant correlation with outcome. Snakebite Severity Score correlates significantly with early recovery in vasculotoxic snake bites (p=0.03).

MP-4 Contributes to Snake Venom Neutralization by Mucuna pruriens Seeds through an Indirect Antibody-mediated Mechanism.

Mortality due to snakebite is a serious public health problem, and available therapeutics are known to induce debilitating side effects. Traditional medicine suggests that seeds of Mucuna pruriens can provide protection against the effects of snakebite. Our aim is to identify the protein(s) that may be important for snake venom neutralization and elucidate its mechanism of action. To this end, we have identified and purified a protein from M. pruriens, which we have named MP-4. The full-length polypeptide sequence of MP-4 was obtained through N-terminal sequencing of peptide fragments. Sequence analysis suggested that the protein may belong to the Kunitz-type protease inhibitor family and therefore may potentially neutralize the proteases present in snake venom. Using various structural and biochemical tools coupled with in vivo assays, we are able to show that MP-4 does not afford direct protection against snake venom because it is actually a poor inhibitor of serine proteases. Further experiments showed that antibodies generated against MP-4 cross-react with the whole venom and provide protection to mice against Echis carinatus snake venom. This study shows that the MP-4 contributes significantly to the snake venom neutralization activity of M. pruriens seeds through an indirect antibody-mediated mechanism.

What killed Karl Patterson Schmidt? Combined venom gland transcriptomic, venomic and antivenomic analysis of the South African green tree snake (the boomslang), Dispholidus typus.

BACKGROUND: Non-front-fanged colubroid snakes comprise about two-thirds of extant ophidian species. The medical significance of the majority of these snakes is unknown, but at least five species have caused life-threatening or fatal human envenomings. However, the venoms of only a small number of species have been explored. METHODS: A combined venomic and venom gland transcriptomic approach was employed to characterise of venom of Dispholidus typus (boomslang), the snake that caused the tragic death of Professor Karl Patterson Schmidt. The ability of CroFab™ antivenom to immunocapture boomslang venom proteins was investigated using antivenomics. RESULTS: Transcriptomic-assisted proteomic analysis identified venom proteins belonging to seven protein families: three-finger toxin (3FTx); phospholipase A2 (PLA2); cysteine-rich secretory proteins (CRISP); snake venom (SV) serine proteinase (SP); C-type lectin-like (CTL); SV metalloproteinases (SVMPs); and disintegrin-like/cysteine-rich (DC) proteolytic fragments. CroFab™ antivenom efficiently immunodepleted some boomslang SVMPs. CONCLUSIONS: The present work is the first to address the overall proteomic profile of D. typus venom. This study allowed us to correlate the toxin composition with the toxic activities of the venom. The antivenomic analysis suggested that the antivenom available at the time of the unfortunate accident could have exhibited at least some immunoreactivity against the boomslang SVMPs responsible for the disseminated intravascular coagulation syndrome that caused K.P. Schmidt's fatal outcome. GENERAL SIGNIFICANCE: This study may stimulate further research on other non-front-fanged colubroid snake venoms capable of causing life-threatening envenomings to humans, which in turn should contribute to prevent fatal human accidents, such as that unfortunately suffered by K.P. Schmidt.

Cross-reactivity and inhibition myotoxic effects induced by Bothrops snake venoms using specific polyclonal anti-BnSP7 antibodies.

Polyclonal antibodies raised in Balb-c mice against BnSP-7, a Lys-49 phospholipase A2, were used to measure cross reactivity against other snake venoms. Using ELISA, these antibodies were able to recognize PLA2s isoforms present in venoms of botropic snakes at 1:6400, 1:3200 and 1:100 ratios (w/w). These antibodies highly recognized proteins of low molecular weight (∼14,000) from crude snake venom Bp and Bm by Western Blotting. PLA2 these venoms, by alignment of primary structures demonstrated high identity with BnSP-7 PLA2, especially in the C-terminal region. However, the crude snake venom Bd and Bj, showed low recognition. The PLA2 activity of Bothrops pauloensis, Bothrops moojeni venoms or BpPLA2-TXI was inhibited significantly when anti-BnSP-7 antibodies were incubated at 1:10 and 1:20 ratios (venoms or toxin:anti-BnSP-7, w/w), respectively. The myotoxic effect induced by the same venoms was also reduced significantly at 1:1, 1:10 and 1:20 ratios, by decreased creatine kinase levels. The anti-PLA2 polyclonal antibodies effectively recognized PLA2s from Bothrops pauloensis and Bothrops moojeni venoms, and neutralized specific catalytic and myotoxic activity.

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.

Antibodies against Venom of the Snake Deinagkistrodon acutus.

Snake venom protein from Deinagkistrodon acutus (DA protein), one of the major venomous species in Taiwan, causes hemorrhagic symptoms that can lead to death. Although horse-derived antivenin is a major treatment, relatively strong and detrimental side effects are seen occasionally. In our study, yolk immunoglobulin (IgY) was purified from eggs, and DA protein was recognized using Western blotting and an enzyme-linked immunosorbent assay (ELISA), similar to therapeutic horse antivenin. The ELISA also indicated that specific IgY antibodies were elicited after the fifth booster, plateaued, and lasted for at least 3 months. To generate monoclonal single-chain variable fragment (scFv) antibodies, we used phage display technology to construct two libraries with short or long linkers, containing 6.24 × 10(8) and 5.28 × 10(8) transformants, respectively. After four rounds of biopanning, the eluted phage titer increased, and the phage-based ELISA indicated that the specific clones were enriched. Nucleotide sequences of 30 individual clones expressing scFv were analyzed and classified into four groups that all specifically recognized the DA venom protein. Furthermore, based on mass spectrometry, the scFv-bound protein was deduced to be snake venom metalloproteinase proteins. Most importantly, both IgY and mixed scFv inhibited the lethal effect in mice injected with the minimum lethal dosage of the DA protein. We suggest that together, these antibodies could be applied to the development of diagnostic agents or treatments for snakebite envenomation in the future.

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

BACKGROUND: Snakebite envenomation inflicts a high burden of mortality and morbidity in sub-Saharan Africa. Antivenoms are the mainstay in the therapy of envenomation, and there is an urgent need to develop antivenoms of broad neutralizing efficacy for this region. The venoms used as immunogens to manufacture snake antivenoms are normally selected considering their medical importance and availability. Additionally, their ability to induce antibody responses with high neutralizing capability should be considered, an issue that involves the immunization scheme and the animal species being immunized. METHODOLOGY/PRINCIPAL FINDINGS: Using the lethality neutralization assay in mice, we compared the intrageneric neutralization scope of antisera generated by immunization of horses with monospecific, bispecific/monogeneric, and polyspecific/monogeneric immunogens formulated with venoms of Bitis spp., Echis spp., Dendroaspis spp., spitting Naja spp. or non-spitting Naja spp. It was found that the antisera raised by all the immunogens were able to neutralize the homologous venoms and, with a single exception, the heterologous congeneric venoms (considering spitting and non-spitting Naja separately). In general, the polyspecific antisera of Bitis spp, Echis spp, and Dendroaspis spp gave the best neutralization profile against venoms of these genera. For spitting Naja venoms, there were no significant differences in the neutralizing ability between monospecific, bispecific and polyspecific antisera. A similar result was obtained in the case of non-spitting Naja venoms, except that polyspecific antiserum was more effective against the venoms of N. melanoleuca and N. nivea as compared to the monospecific antiserum. CONCLUSIONS/SIGNIFICANCE: The use of polyspecific immunogens is the best alternative to produce monogeneric antivenoms with wide neutralizing coverage against venoms of sub-Saharan African snakes of the Bitis, Echis, Naja (non-spitting) and Dendroaspis genera. On the other hand, a monospecific immunogen composed of venom of Naja nigricollis is suitable to produce a monogeneric antivenom with wide neutralizing coverage against venoms of spitting Naja spp. These findings can be used in the design of antivenoms of wide neutralizing scope for sub-Saharan Africa.

Snakebites in Cameroon: Tolerance of a Snake Antivenom (Inoserp™ PAN-AFRICA) in Africa in Real-Life Conditions.

Snakebite envenomation (SBE) is a public health issue in sub-Saharan countries. Antivenom is the only etiological treatment. Excellent tolerance is essential in managing SBE successfully. This study aimed to evaluate tolerance of InoserpTM PAN-AFRICA (IPA). It was conducted on fourteen sites across Cameroon. IPA was administered intravenously and repeated at the same dose every two hours if needed. Early and late tolerance was assessed by the onset of clinical signs within two hours and at a visit two weeks or more after the first IPA administration, respectively. Over 20 months, 447 patients presenting with a snakebite were included. One dose of IPA was administered to 361 patients and repeated at least once in 106 patients. No significant difference was shown between the proportion of adverse events in patients who received IPA (266/361, 73.7%) and those who did not (69/85, 81.2%) (p = 0.95). Adverse reactions, probably attributable to IPA, were identified in four (1.1%) patients, including one severe (angioedema) and three mild. All these reactions resolved favorably. None of the serious adverse events observed in twelve patients were attributed to IPA. No signs of late intolerance were observed in 302 patients. Tolerance appears to be satisfactory. The availability of effective and well-tolerated antivenoms would reduce the duration of treatment and prevent most disabilities and/or deaths.

The role of venom proteomics and single-domain antibodies for antivenoms: Progress in snake envenoming treatment.

Single-domain antibodies (sdAbs) hold promise for developing new biopharmaceuticals to treat neglected tropical diseases (NTDs), including snakebites, which are severe and occur frequently. In addition, limitations of conventional snakebite treatments, especially in terms of local action, and the global antivenom crisis incentivize the use of this biotechnological tool to design next-generation snakebite antivenoms. Conventional antivenoms for snakebite treatment are usually composed of immunoglobulin G or F(ab')2 fragments derived from the plasma of immunized animals. sdAbs, the smallest antigen-binding fragments, are derived from the variable domains of camelid heavy-chain antibodies. sdAbs may have some advantages over conventional antivenoms for local toxicity, such as better penetration into tissues due to their small size, and high solubility and affinity for venom antigens due to their unique antigen-binding loops and ability to access cryptic epitopes. We present an overview of current antivenom therapy in the context of sdAb development for toxin neutralization. Furthermore, strategies are presented for identifying snake venom's major toxins as well as for developing antisnake toxin sdAbs by employing proteomic tools for toxin neutralization.

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.

Isolation, characterization and antigenic cross-reactivities of the major hemorrhagin from Cryptelytrops purpureomaculatus venom.

The major hemorrhagin from C. purpureomaculatus (mangrove pit viper) venom was purified to homogeneity and termed Maculatoxin. Maculatoxin has a molecular weight of 38 kDa as determined by SDS-PAGE. It is an acidic protein (pI= 4.2) and exhibited proteolytic and hemorrhagic activities (MHD10 = 0.84 microg in mice) but was not lethal to mice at a dose of 1 microg/g. The hemorrhagic activity of Maculatoxin was completely inactivated by EDTA and partially inhibited by ATP and citrate. The N-terminal sequence of Maculatoxin (TPEQQRFPPTYIDLGIFVDHGMYAT) shares a significant degree of homology with the metalloprotease domain of other venom hemorrhagins. Indirect ELISA showed anti-Maculatoxin cross reacted with protein components of many snake venoms. In the double-sandwich ELISA, however, anti-Maculatoxin cross-reacted only with venoms of certain species of the Trimeresurus (Asia lance-head viper) complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.

[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.

A study on the use of caprylic acid and ammonium sulfate in combination for the fractionation of equine antivenom F(ab')(2).

This study involved the fractionation of equine antivenom F(ab')(2) by combined stepwise ammonium sulfate (AS) and caprylic acid (CA) precipitation without intermediate separation of precipitate. Using a microplate format, 55 conditions with combinations of AS (0-20% saturation) and CA (0-5% v/v), were tested. AS significantly reduced the turbidity raised by CA. High specific antibody activity was observed in the area containing 2-5% CA and 10-20% AS. From these results, 12 precipitation conditions were selected for detailed quantitative studies. Two combinations, one with 4% CA and 15% AS and another with 5% CA and 20% AS, gave the highest fold-purification (1.79 and 1.83) with antibody recoveries at 68% and 59%, respectively. These combinations offered a benefit over CA alone in reducing the turbidity and in increasing the purity but not the recovery of antibody. The conditions giving more favorable overall results were with 2% CA alone and another with a combination of 1.5% CA and 10% AS. These preparations of F(ab')(2) were homogeneous and without protein aggregate under size-exclusion HPLC. Lastly, 1 h precipitation showed better results than those of overnight precipitation. These results could be useful for the production of therapeutic antivenoms.