Diversity of Micrurus Snake Species Related to Their Venom Toxic Effects and the Prospective of Antivenom Neutralization

Background

Micrurus snake bites can cause death by muscle paralysis and respiratory arrest, few hours after envenomation. The specific treatment for coral snake envenomation is the intravenous application of heterologous antivenom and, in Brazil, it is produced by horse immunization with a mixture of M. corallinus and M. frontalis venoms, snakes that inhabit the South and Southeastern regions of the country. However, this antivenom might be inefficient, considering the existence of intra- and inter-specific variations in the composition of the venoms. Therefore, the aim of the present study was to investigate the toxic properties of venoms from nine species of Micrurus: eight present in different geographic regions of Brazil (M. frontalis, M. corallinus, M. hemprichii, M. spixii, M. altirostris, M. surinamensis, M. ibiboboca, M. lemniscatus) and one (M. fulvius) with large distribution in Southeastern United States and Mexico. This study also analyzed the antigenic cross-reactivity and the neutralizing potential of the Brazilian coral snake antivenom against these Micrurus venoms.

Methodology/Principal Findings

Analysis of protein composition and toxicity revealed a large diversity of venoms from the nine Micrurus species. ELISA and Western blot assays showed a varied capability of the therapeutic antivenom to recognize the diverse species venom components. In vivo and in vitro neutralization assays indicated that the antivenom is not able to fully neutralize the toxic activities of all venoms.

Conclusion

These results indicate the existence of a large range of both qualitative and quantitative variations in Micrurus venoms, probably reflecting the adaptation of the snakes from this genus to vastly dissimilar habitats. The data also show that the antivenom used for human therapy in Brazil is not fully able to neutralize the main toxic activities present in the venoms from all Micrurus species occurring in the country. It suggests that modifications in the immunization scheme, with the inclusion of other venoms in the antigenic mixture, should occur in order to generate effective therapeutic coral snake antivenom.     

Viper and Cobra Venom Neutralization by Alginate Coated Multicomponent Polyvalent Antivenom Administered by the Oral Route

Background

Snake bite causes greater mortality than most of the other neglected tropical diseases. Snake antivenom, although effective in minimizing mortality in developed countries, is not equally so in developing countries due to its poor availability in remote snake infested areas as, and when, required. An alternative approach in this direction could be taken by making orally deliverable polyvalent antivenom formulation, preferably under a globally integrated strategy, for using it as a first aid during transit time from remote trauma sites to hospitals.

Methodology/Principal Findings

To address this problem, multiple components of polyvalent antivenom were entrapped in alginate. Structural analysis, scanning electron microscopy, entrapment efficiency, loading capacity, swelling study, in vitro pH sensitive release, acid digestion, mucoadhesive property and venom neutralization were studied in in vitro and in vivo models. Results showed that alginate retained its mucoadhesive, acid protective and pH sensitive swelling property after entrapping antivenom. After pH dependent release from alginate beads, antivenom (ASVS) significantly neutralized phospholipaseA₂ activity, hemolysis, lactate dehydrogenase activity and lethality of venom. In ex vivo mice intestinal preparation, ASVS was absorbed significantly through the intestine and it inhibited venom lethality which indicated that all the components of antivenom required for neutralization of venom lethality were retained despite absorption across the intestinal layer. Results from in vivo studies indicated that orally delivered ASVS can significantly neutralize venom effects, depicted by protection against lethality, decreased hemotoxicity and renal toxicity caused by russell viper venom.

Conclusions/Significance

Alginate was effective in entrapping all the structural components of ASVS, which on release and intestinal absorption effectively reconstituted the function of antivenom in neutralizing viper and cobra venom. Further research in this direction can strategize to counter such dilemma in snake bite management by promoting control release and oral antivenom rendered as a first aid.     

Pre-clinical assays predict pan-African Echis viper efficacy for a species-specific antivenom

Background

Snakebite is a significant cause of death and disability in subsistent farming populations of sub-Saharan Africa. Antivenom is the most effective treatment of envenoming and is manufactured from IgG of venom-immunised horses/sheep but, because of complex fiscal reasons, there is a paucity of antivenom in sub-Saharan Africa. To address the plight of thousands of snakebite victims in savannah Nigeria, the EchiTAb Study Group organised the production, testing and delivery of antivenoms designed to treat envenoming by the most medically-important snakes in the region. The Echis saw-scaled vipers have a wide African distribution and medical importance. In an effort to maximise the clinical utility of scarce antivenom resources in Africa, we aimed to ascertain, at the pre-clinical level, to what extent the E. ocellatus-specific EchiTAbG antivenom, which was designed specifically for Nigeria, neutralised the lethal activity of venom from two other African species, E. pyramidum leakeyi and E. coloratus.

Methodology/Principal Findings

Despite apparently quite distinctive venom protein profiles, we observed extensive cross-species similarity in the immuno-reactivity profiles of Echis species-specific antisera. Using WHO standard pre-clinical in vivo tests, we determined that the monospecific EchiTAbG antivenom was as effective at neutralising the venom-induced lethal effects of E. pyramidum leakeyi and E. coloratus as it was against E. ocellatus venom. Under the restricted conditions of this assay, the antivenom was ineffective against the lethal effects of venom from the non-African Echis species, E. carinatus sochureki.

Conclusions/Significance

Using WHO-recommended pre-clinical tests we have demonstrated that the new anti-E. ocellatus monospecific antivenom EchiTAbG, developed in response to the considerable snakebite-induced mortality and morbidity in Nigeria, neutralised the lethal effects of venoms from Echis species representing each taxonomic group of this genus in Africa. This suggests that this monospecific antivenom has potential to treat envenoming by most, perhaps all, African Echis species.     

Protective Effect of Tetracycline against Dermal Toxicity Induced by Jellyfish Venom

Background

Previously, we have reported that most, if not all, of the Scyphozoan jellyfish venoms contain multiple components of metalloproteinases, which apparently linked to the venom toxicity. Further, it is also well known that there is a positive correlation between the inflammatory reaction of dermal tissues and their tissue metalloproteinase activity. Based on these, the use of metalloproteinase inhibitors appears to be a promising therapeutic alternative for the treatment of jellyfish envenomation.

Methodology and Principal Findings

Tetracycline (a metalloproteinase inhibitor) has been examined for its activity to reduce or prevent the dermal toxicity induced by Nemopilema nomurai (Scyphozoa: Rhizostomeae) jellyfish venom (NnV) using in vitro and in vivo models. HaCaT (human keratinocyte) and NIH3T3 (mouse fibroblast) incubated with NnV showed decreases in cell viability, which is associated with the inductions of metalloproteinase-2 and -9. This result suggests that the use of metalloproteinase inhibitors, such as tetracycline, may prevent the jellyfish venom-mediated local tissue damage. In vivo experiments showed that comparing with NnV-alone treatment, tetracycline pre-mixed NnV demonstrated a significantly reduced progression of dermal toxicity upon the inoculation onto rabbit skin.

Conclusions/Significance

It is believed that there has been no previous report on the therapeutic agent of synthetic chemical origin for the treatment of jellyfish venom-induced dermonecrosis based on understanding its mechanism of action except the use of antivenom treatment. Furthermore, the current study, for the first time, has proposed a novel mechanism-based therapeutic intervention for skin damages caused by jellyfish stings.     

Preclinical evaluation of caprylic acid-fractionated IgG antivenom for the treatment of Taipan (Oxyuranus scutellatus) envenoming in Papua New Guinea

Background

Snake bite is a common medical emergency in Papua New Guinea (PNG). The taipan, Oxyuranus scutellatus, inflicts a large number of bites that, in the absence of antivenom therapy, result in high mortality. Parenteral administration of antivenoms manufactured in Australia is the current treatment of choice for these envenomings. However, the price of these products is high and has increased over the last 25 years; consequently the country can no longer afford all the antivenom it needs. This situation prompted an international collaborative project aimed at generating a new, low-cost antivenom against O. scutellatus for PNG.

Methodology/Principal Findings

A new monospecific equine whole IgG antivenom, obtained by caprylic acid fractionation of plasma, was prepared by immunising horses with the venom of O. scutellatus from PNG. This antivenom was compared with the currently used F(ab'')₂ monospecific taipan antivenom manufactured by CSL Limited, Australia. The comparison included physicochemical properties and the preclinical assessment of the neutralisation of lethal neurotoxicity and the myotoxic, coagulant and phospholipase A₂ activities of the venom of O. scutellatus from PNG. The F(ab'')₂ antivenom had a higher protein concentration than whole IgG antivenom. Both antivenoms effectively neutralised, and had similar potency, against the lethal neurotoxic effect (both by intraperitoneal and intravenous routes of injection), myotoxicity, and phospholipase A₂ activity of O. scutellatus venom. However, the whole IgG antivenom showed a higher potency than the F(ab'')₂ antivenom in the neutralisation of the coagulant activity of O. scutellatus venom from PNG.

Conclusions/Significance

The new whole IgG taipan antivenom described in this study compares favourably with the currently used F(ab'')₂ antivenom, both in terms of physicochemical characteristics and neutralising potency. Therefore, it should be considered as a promising low-cost candidate for the treatment of envenomings by O. scutellatus in PNG, and is ready to be tested in clinical trials. Author Summary Snake bite envenoming represents an important public health hazard in Papua New Guinea (PNG). In the southern lowlands of the country the majority of envenomings are inflicted by the taipan, Oxyuranus scutellatus. The only currently effective treatment for these envenomings is the administration of antivenoms manufactured in Australia. However, the price of these products in PNG is very high and has steadily increased over the last 25 years, leading to chronic antivenom shortages in this country. As a response to this situation, an international partnership between PNG, Australia and Costa Rica was initiated, with the aim of generating a new, low-cost antivenom for the treatment of PNG taipan envenoming. Horses were immunised with the venom of O. scutellatus from PNG and whole IgG was purified from the plasma of these animals by caprylic acid precipitation of non-immunoglobulin proteins. The new antivenom, manufactured by Instituto Clodomiro Picado (Costa Rica), was compared with the currently available F(ab'')₂ antivenom manufactured by CSL Limited (Australia). Both were effective in the neutralisation of the most relevant toxic effects induced by this venom, although the whole IgG antivenom showed a higher efficacy than the F(ab'')₂ antivenom in the neutralisation of the coagulant activity.     

Venom of the crotaline snake Atropoides nummifer (jumping viper) from Guatemala and Honduras: comparative toxicological characterization,isolation of a myotoxic phospholipase A2 homologue and neutralization by two antivenoms

A comparative study was performed on the venoms of the crotaline snake Atropoides nummifer from Guatemala and Honduras. SDS-polyacrylamide gel electrophoresis, under reducing conditions, revealed a highly similar pattern of these venoms, and between them and the venom of the same species from Costa Rica. Similar patterns were also observed in ion-exchange chromatography on CM-Shephadex C-25, in which a highly basic myotoxic fraction was present. This fraction was devoid of phospholipase A₂ activity and strongly reacted, by enzyme-immunoassay, with an antiserum against Bothrops asper myotoxin II, a Lys-49 phospholipase A₂ homologue. A basic myotoxin of 16 kDa was isolated to homogeneity from the venom of A. nummifer from Honduras, showing amino acid composition and N-terminal sequence similar to those of Lys-49 phospholipase A₂ variants previously isolated from other crotaline snake venoms. Guatemalan and Honduran A. nummifer venoms have a qualitatively similar toxicological profile, characterized by: lethal; hemorrhagic; myotoxic; edema-forming; coagulant; and defibrinating activities, although there were significant quantitative variations in some of these activities between the two venoms. Neutralization of toxic activities by two commercially-available antivenoms in the region was studied. Polyvalent antivenom produced by Instituto Clodomiro Picado was effective in the neutralization of: lethal; hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A₂ activities, but ineffective against edema-forming activity. On the other hand, MYN polyvalent antivenom neutralized: hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A₂ activities, albeit with a lower potency than Instituto Clodomiro Picado antivenom. MYN antivenom failed to neutralize lethal and edema-forming activities of A. nummifer venoms.     

P-I Snake Venom Metalloproteinase Is Able to Activate the Complement System by Direct Cleavage of Central Components of the Cascade

Background

Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom.

Results

Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity.

Conclusion

We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complement system by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation.     

Venom of the Brazilian Spider Sicarius ornatus (Araneae,Sicariidae) Contains Active Sphingomyelinase D: Potential for Toxicity after Envenomation

Background

The spider family Sicariidae includes two genera, Sicarius and Loxosceles. Bites by Sicarius are uncommon in humans and, in Brazil, a single report is known of a 17-year old man bitten by a Sicarius species that developed a necrotic lesion similar to that caused by Loxosceles. Envenomation by Loxosceles spiders can result in dermonecrosis and severe ulceration. Sicarius and Loxosceles spider venoms share a common characteristic, i.e., the presence of Sphingomyelinases D (SMase D). We have previously shown that Loxosceles SMase D is the enzyme responsible for the main pathological effects of the venom. Recently, it was demonstrated that Sicarius species from Africa, like Loxosceles spiders from the Americas, present high venom SMase D activity. However, despite the presence of SMase D like proteins in venoms of several New World Sicarius species, they had reduced or no detectable SMase D activity. In order to contribute to a better understanding about the toxicity of New World Sicarius venoms, the aim of this study was to characterize the toxic properties of male and female venoms from the Brazilian Sicarius ornatus spider and compare these with venoms from Loxosceles species of medical importance in Brazil.

Methodology/Principal Findings

SDS-PAGE analysis showed variations in the composition of Loxosceles spp. and Sicarius ornatus venoms. Differences in the electrophoretic profiles of male and female venoms were also observed, indicating a possible intraspecific variation in the composition of the venom of Sicarius spider. The major component in all tested venoms had a Mr of 32–35 kDa, which was recognized by antiserum raised against Loxosceles SMases D. Moreover, male and female Sicarius ornatus spiders'' venoms were able to hydrolyze sphingomyelin, thus showing an enzymatic activity similar to that determined for Loxosceles venoms. Sicarius ornatus venoms, as well as Loxosceles venoms, were able to render erythrocytes susceptible to lysis by autologous serum and to induce a significant loss of human keratinocyte cell viability; the female Sicarius ornatus venom was more efficient than male.

Conclusion

We show here, for the first time, that the Brazilian Sicarius ornatus spider contains active Sphingomyelinase D and is able to cause haemolysis and keratinocyte cell death similar to the South American Loxosceles species, harmful effects that are associated with the presence of active SMases D. These results may suggest that envenomation by this Sicarius spider has the potential to cause similar pathological events as that caused by Loxosceles envenomation. Our results also suggest that, in addition to the interspecific differences, intraspecific variations in the venoms composition may play a role in the toxic potential of the New World Sicarius venoms species.     

Neutralizing Antibodies Induced by Recombinant Virus-Like Particles of Enterovirus 71 Genotype C4 Inhibit Infection at Pre- and Post-attachment Steps

Background

Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease, which has been prevalent in Asia–Pacific regions, causing significant morbidity and mortality in young children. Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection. However, how neutralizing antibodies inhibit infection in vitro remains unclear.

Methods/Findings

In the present study, we explored the mechanisms of neutralization by antibodies against EV71 virus-like particles (VLPs). Recombinant VLPs of EV71 genotype C4 were produced in insect cells using baculovirus vectors. Immunization with the VLPs elicited a high-titer, EV71-specific antibody response in mice. Anti-VLP mouse sera potently neutralized EV71 infection in vitro. The neutralizing antibodies in the anti-VLP mouse sera were found to target mainly an extremely conserved epitope (FGEHKQEKDLEYGAC) located at the GH loop of the VP1 protein. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment.

Conclusions

Collectively, our findings represent a valuable addition to the understanding of mechanisms of EV71 neutralization and have strong implications for EV71 vaccine development.     

Effective Equine Immunization Protocol for Production of Potent Poly-specific Antisera against Calloselasma rhodostoma,Cryptelytrops albolabris and Daboia siamensis

Snake envenomation has been estimated to affect 1.8 million people annually with about 94,000 deaths mostly in poor tropical countries. Specific antivenoms are the only rational and effective therapy for these cases. Efforts are being made to produce effective, affordable and sufficient antivenoms for these victims. The immunization process, which has rarely been described in detail, is one step that needs to be rigorously studied and improved especially with regard to the production of polyspecific antisera. The polyspecific nature of therapeutic antivenom could obviate the need to identify the culprit snake species. The aim of this study was to produce potent polyspecific antisera against 3 medically important vipers of Thailand and its neighboring countries, namely Cryptelytrops albolabris "White lipped pit viper" (CA), Calleoselasma rhodostoma “Malayan pit viper” (CR), and Daboia siamensis “Russell’s viper” (DS). Four horses were immunized with a mixture of the 3 viper venoms using the ‘low dose, low volume multi-site’ immunization protocol. The antisera showed rapid rise in ELISA titers against the 3 venoms and reached plateau at about the 8th week post-immunization. The in vivo neutralization potency (P) of the antisera against CA, CR and DS venoms was 10.40, 2.42 and 0.76 mg/ml, respectively and was much higher than the minimal potency limits set by Queen Soavabha Memorial Institute (QSMI). The corresponding potency values for the QSMI monospecific antisera against CA, CR and DS venoms were 7.28, 3.12 and 1.50 mg/ml, respectively. The polyspecific antisera also effectively neutralized the procoagulant, hemorrhagic, necrotic and nephrotoxic activities of the viper venoms. This effective immunization protocol should be useful in the production of potent polyspecific antisera against snake venoms, and equine antisera against tetanus, diphtheria or rabies.     

Anti-V3 Monoclonal Antibodies Display Broad Neutralizing Activities against Multiple HIV-1 Subtypes

Background

The V3 loop of the HIV-1 envelope (Env) glycoprotein gp120 was identified as the “principal neutralizing domain” of HIV-1, but has been considered too variable to serve as a neutralizing antibody (Ab) target. Structural and immunochemical data suggest, however, that V3 contains conserved elements which explain its role in binding to virus co-receptors despite its sequence variability. Despite this evidence of V3 conservation, the ability of anti-V3 Abs to neutralize a significant proportion of HIV-1 isolates from different subtypes (clades) has remained controversial.

Methods

HIV-1 neutralization experiments were conducted in two independent laboratories to test human anti-V3 monoclonal Abs (mAbs) against pseudoviruses (psVs) expressing Envs of diverse HIV-1 subtypes from subjects with acute and chronic infections. Neutralization was defined by 50% inhibitory concentrations (IC₅₀), and was statistically assessed based on the area under the neutralization titration curves (AUC).

Results

Using AUC analyses, statistically significant neutralization was observed by ≥1 anti-V3 mAbs against 56/98 (57%) psVs expressing Envs of diverse subtypes, including subtypes A, AG, B, C and D. Even when the 10 Tier 1 psVs tested were excluded from the analysis, significant neutralization was detected by ≥1 anti-V3 mAbs against 46/88 (52%) psVs from diverse HIV-1 subtypes. Furthermore, 9/24 (37.5%) Tier 2 viruses from the clade B and C standard reference panels were neutralized by ≥1 anti-V3 mAbs. Each anti-V3 mAb tested was able to neutralize 28–42% of the psVs tested. By IC₅₀ criteria, 40/98 (41%) psVs were neutralized by ≥1 anti-V3 mAbs.

Conclusions

Using standard and new statistical methods of data analysis, 6/7 anti-V3 human mAbs displayed cross-clade neutralizing activity and revealed that a significant proportion of viruses can be neutralized by anti-V3 Abs. The new statistical method for analysis of neutralization data provides many advantages to previously used analyses.     

Immune Response to Snake Envenoming and Treatment with Antivenom; Complement Activation,Cytokine Production and Mast Cell Degranulation

Background

Snake bite is one of the most neglected public health issues in poor rural communities worldwide. In addition to the clinical effects of envenoming, treatment with antivenom frequently causes serious adverse reactions, including hypersensitivity reactions (including anaphylaxis) and pyrogenic reactions. We aimed to investigate the immune responses to Sri Lankan snake envenoming (predominantly by Russell''s viper) and antivenom treatment.

Methodology/Principal Findings

Plasma concentrations of Interleukin (IL)-6, IL-10, tumor necrosis factor α (TNFα), soluble TNF receptor I (sTNFRI), anaphylatoxins (C3a, C4a, C5a; markers of complement activation), mast cell tryptase (MCT), and histamine were measured in 120 Sri Lankan snakebite victims, both before and after treatment with antivenom. Immune mediator concentrations were correlated with envenoming features and the severity of antivenom-induced reactions including anaphylaxis. Envenoming was associated with complement activation and increased cytokine concentrations prior to antivenom administration, which correlated with non-specific systemic symptoms of envenoming but not with coagulopathy or neurotoxicity. Typical hypersensitivity reactions to antivenom occurred in 77/120 patients (64%), satisfying criteria for a diagnosis of anaphylaxis in 57/120 (48%). Pyrogenic reactions were observed in 32/120 patients (27%). All patients had further elevations in cytokine concentrations, but not complement activation, after the administration of antivenom, whether a reaction was noted to occur or not. Patients with anaphylaxis had significantly elevated concentrations of MCT and histamine.

Conclusions/Significance

We have demonstrated that Sri Lankan snake envenoming is characterized by significant complement activation and release of inflammatory mediators. Antivenom treatment further enhances the release of inflammatory mediators in all patients, with anaphylactic reactions characterised by high levels of mast cell degranulation but not further complement activation. Anaphylaxis is probably triggered by non allergen-specific activation of mast cells and may be related to the quality of available antivenom preparations, as well as a priming effect from the immune response to the venom itself.     

In Vitro Neutralization of Low Dose Inocula at Physiological Concentrations of a Monoclonal Antibody Which Protects Macaques against SHIV Challenge

Background

Passive transfer of antibodies can be protective in the simian human immunodeficiency virus (SHIV) – rhesus macaque challenge model. The human monoclonal antibody IgG1 b12 neutralizes human immunodeficiency type 1 (HIV-1) in vitro and protects against challenge by SHIV. Our hypothesis is that neutralizing antibodies can only completely inactivate a relatively small number of infectious virus.

Methods And Findings

We have used GHOST cell assays to quantify individual infectious events with HIV-1_SF162 and its SHIV derivatives: the relatively neutralization sensitive SHIV_SF162P4 isolate and the more resistant SHIV_SF162P3. A plot of the number of fluorescent GHOST cells with increasing HIV-1_SF162 dose is not linear. It is likely that with high-dose inocula, infection with multiple virus produces additive fluorescence in individual cells. In studies of the neutralization kinetics of IgG1 b12 against these isolates, events during the absorption phase of the assay, as well as the incubation phase, determine the level of neutralization. It is possible that complete inactivation of a virus is limited to the time it is exposed on the cell surface. Assays can be modified so that neutralization of these very low doses of virus can be quantified. A higher concentration of antibody is required to neutralize the same dose of resistant SHIV_SF162P3 than the sensitive SHIV_SF162P4. In the absence of selection during passage, the density of the CCR5 co-receptor on the GHOST cell surface is reduced. Changes in the CD4 : CCR5 density ratio influence neutralization.

Conclusions

Low concentrations of IgG1 b12 completely inactivate small doses of the neutralization resistant SHIV _SF162P3. Assays need to be modified to quantify this effect. Results from modified assays may predict protection following repeated low-dose shiv challenges in rhesus macaques. It should be possible to induce this level of antibody by vaccination so that modified assays could predict the outcome of human trials.     

Consequences of neglect: analysis of the sub-Saharan African snake antivenom market and the global context

Background

The worldwide neglect of immunotherapeutic products for the treatment of snakebite has resulted in a critical paucity of effective, safe and affordable therapy in many Third World countries, particularly in Africa. Snakebite ranks high among the most neglected global health problems, with thousands of untreated victims dying or becoming permanently maimed in developing countries each year because of a lack of antivenom—a treatment that is widely available in most developed countries. This paper analyses the current status of antivenom production for sub-Saharan African countries and provides a snapshot of the global situation.

Methods

A global survey of snake antivenom products was undertaken in 2007, involving 46 current and former antivenom manufacturers. Companies producing antivenom for use in sub-Saharan Africa were re-surveyed in 2010 and 2011.

Results

The amount of antivenom manufactured for sub-Saharan Africa increased between 2007 and 2010/11, however output and procurement remained far below that required to treat the estimated 300,000–500,000 snakebite victims each year. Variable potency and inappropriate marketing of some antivenoms mean that the number of effective treatments available may be as low as 2.5% of projected needs. Five companies currently market antivenom for sale in Africa; three others have products in the final stages of development; and since 2007 one has ceased production indefinitely. Most current antivenom producers possess a willingness and capacity to raise output. However inconsistent market demand, unpredictable financial investment and inadequate quality control discourage further production and threaten the viability of the antivenom industry.

Conclusion

Financial stimulus is urgently needed to identify and develop dependable sources of high-grade antivenoms, support current and emerging manufacturers, and capitalise on existing unutilised production capacity. Investing to ensure a consistent and sustainable marketplace for efficacious antivenom products will drive improvements in quality, output and availability, and save thousands of lives each year.     

Protection in macaques immunized with HIV-1 candidate vaccines can be predicted using the kinetics of their neutralizing antibodies

Background

A vaccine is needed to control the spread of human immunodeficiency virus type 1 (HIV-1). An in vitro assay that can predict the protection induced by a vaccine would facilitate the development of such a vaccine. A potential candidate would be an assay to quantify neutralization of HIV-1.

Methods and Findings

We have used sera from rhesus macaques that have been immunized with HIV candidate vaccines and subsequently challenged with simian human immunodeficiency virus (SHIV). We compared neutralization assays with different formats. In experiments with the standardized and validated TZMbl assay, neutralizing antibody titers against homologous SHIV_SF162P4 pseudovirus gave a variable correlation with reductions in plasma viremia levels. The target cells used in the assays are not just passive indicators of virus infection but are actively involved in the neutralization process. When replicating virus was used with GHOST cell assays, events during the absorption phase, as well as the incubation phase, determine the level of neutralization. Sera that are associated with protection have properties that are closest to the traditional concept of neutralization: the concentration of antibody present during the absorption phase has no effect on the inactivation rate. In GHOST assays, events during the absorption phase may inactivate a fixed number, rather than a proportion, of virus so that while complete neutralization can be obtained, it can only be found at low doses particularly with isolates that are relatively resistant to neutralization.

Conclusions

Two scenarios have the potential to predict protection by neutralizing antibodies at concentrations that can be induced by vaccination: antibodies that have properties close to the traditional concept of neutralization may protect against a range of challenge doses of neutralization sensitive HIV isolates; a window of opportunity also exists for protection against isolates that are more resistant to neutralization but only at low challenge doses.     

Neutralization of toxic and enzyme activities of 4 venoms from snakes of Guatemala and Honduras by the polyvalent antivenin produced in Costa Rica

We studied the ability of the polyvalent antivenom produced in Costa Rica to neutralize lethal, hemorrhagic, edema-forming, proteolytic, hemolytic, hyaluronidase and fibrinolytic activities of the venoms of Bothrops asper and B. nummifer from Honduras, and of Agkistrodon bilineatus and Crotalus durissus durissus from Guatemala. Neutralizing ability of antivenom was expressed as ED50 (effective dose 50%), defined as the antivenom/venom ratio at which the activity of the venom is reduced 50%. Antivenom is highly effective in the neutralization of lethal, hemorrhagic, hemolytic, hyaluronidase, and caseinolytic activities of B. asper, B. nummifer, and C. d. durissus venoms. In the case of B. nummifer venom, neutralization of fibrinolytic effect was only partial, whereas this activity was adequately neutralized when studying the venoms of B. asper and C. d. durissus. The venom of A. bilineatus was adequately neutralized by the antivenom, with the only exception of hemolytic effect that was reduced only partially. However, in quantitative terms, a relatively large volume of antivenom was required to neutralize some effects induced by A. bilineatus venom. Regarding edema-forming activity, antivenom neutralized efficiently the venoms of B. asper and A. bilineatus, whereas that of B. nummifer was neutralized only partially; on the other hand, edema induced by the venom of C. d. durissus was not neutralized at all. Immunochemical results indicate a close immunological relationship between venoms of B. asper, B. nummifer and C. d. durissus collected in Honduras and Guatemala with those of the same species collected in Costa Rica. Interspecies comparison, however, showed variation between venoms obtained from different species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological Approaches That Slow Lymphatic Flow As a Snakebite First Aid

Background

This study examines the use of topical pharmacological agents as a snakebite first aid where slowing venom reaching the circulation prevents systemic toxicity. It is based on the fact that toxin molecules in most snake venoms are large molecules and generally first enter and traverse the lymphatic system before accessing the circulation. It follows on from a previous study where it was shown that topical application of a nitric oxide donor slowed lymph flow to a similar extent in humans and rats as well as increased the time to respiratory arrest for subcutaneous injection of an elapid venom (Pseudonaja textilis, Ptx; Eastern brown snake) into the hind feet of anaesthetized rats.

Methodology/Principal Findings

The effects of topical application of the L-type Ca²⁺ channel antagonist nifedipine and the local anesthetic lignocaine in inhibiting lymph flow and protecting against envenomation was examined in an anaesthetized rat model. The agents significantly increased dye-measured lymph transit times by 500% and 390% compared to controls and increased the time to respiratory arrest to foot injection of a lethal dose of Ptx venom by 60% and 40% respectively. The study also examined the effect of Ptx venom dose over the lethal range of 0.4 to 1.5 mg/kg finding a negative linear relationship between increase in venom dose and time to respiratory arrest.

Conclusions/Significance

The findings suggest that a range of agents that inhibit lymphatic flow could potentially be used as an adjunct treatment to pressure bandaging with immobilization (PBI) in snakebite first aid. This is important given that PBI (a snakebite first aid recommended by the Australian National Health and Medical research Council) is often incorrectly applied. The use of a local anesthetic would have the added advantage of reducing pain.     

Clinical Effects and Antivenom Dosing in Brown Snake (Pseudonaja spp.) Envenoming — Australian Snakebite Project (ASP-14)

Background

Snakebite is a global health issue and treatment with antivenom continues to be problematic. Brown snakes (genus Pseudonaja) are the most medically important group of Australian snakes and there is controversy over the dose of brown snake antivenom. We aimed to investigate the clinical and laboratory features of definite brown snake (Pseudonaja spp.) envenoming, and determine the dose of antivenom required.

Methods and Finding

This was a prospective observational study of definite brown snake envenoming from the Australian Snakebite Project (ASP) based on snake identification or specific enzyme immunoassay for Pseudonaja venom. From January 2004 to January 2012 there were 149 definite brown snake bites [median age 42y (2–81y); 100 males]. Systemic envenoming occurred in 136 (88%) cases. All envenomed patients developed venom induced consumption coagulopathy (VICC), with complete VICC in 109 (80%) and partial VICC in 27 (20%). Systemic symptoms occurred in 61 (45%) and mild neurotoxicity in 2 (1%). Myotoxicity did not occur. Severe envenoming occurred in 51 patients (38%) and was characterised by collapse or hypotension (37), thrombotic microangiopathy (15), major haemorrhage (5), cardiac arrest (7) and death (6). The median peak venom concentration in 118 envenomed patients was 1.6 ng/mL (Range: 0.15–210 ng/mL). The median initial antivenom dose was 2 vials (Range: 1–40) in 128 patients receiving antivenom. There was no difference in INR recovery or clinical outcome between patients receiving one or more than one vial of antivenom. Free venom was not detected in 112/115 patients post-antivenom with only low concentrations (0.4 to 0.9 ng/ml) in three patients.

Conclusions

Envenoming by brown snakes causes VICC and over a third of patients had serious complications including major haemorrhage, collapse and microangiopathy. The results of this study support accumulating evidence that giving more than one vial of antivenom is unnecessary in brown snake envenoming.