Pre-clinical and preliminary dose-finding and safety studies to identify candidate antivenoms for treatment of envenoming by saw-scaled or carpet vipers (Echis ocellatus) in northern Nigeria.

The aim of this study was to identify candidate antivenoms with specific activity against the venom of the saw-scaled or carpet viper (Echis ocellatus) in northern Nigeria, where bites by this species cause great morbidity and mortality but where effective antivenoms have become scarce and unaffordable. Selected antivenoms were destined to be compared by randomised controlled clinical trials (RCTs). Standard pre-clinical neutralisation assays were carried out in rodents. We included two licensed antivenoms of established clinical efficacy and 6 candidate antivenoms. Although 6 of the tested antivenoms showed promising efficacy, all but 3 were excluded from further study because of inadequate pre-clinical efficacy or because they were unavailable or unaffordable for the anticipated RCTs. Median effective doses (ED(50)) of the remaining three candidate antivenoms suggested that the following doses might neutralise the maximum observed venom yield of 24.8 mg (dry weight) of venom milked from captive E. ocellatus: 10 ml of MicroPharm "EchiTAb G" (ET-G) antivenom; 30 ml of Instituto Clodomiro Picado "EchiTAb-Plus-ICP" (ET-Plus) antivenom; 50 ml of VacSera, Cairo "EgyVac" antivenom. A preliminary clinical dose-finding and safety study of these three antivenoms was carried out in 24 patients with incoagulable blood after E. ocellatus bites who were not severely envenomed. A 3+3 dose escalation design was employed. Initial doses of 10 ml ET-G and 30 ml ET-Plus restored blood coagulability in groups of 6 patients with early mild reactions (pruritus only) in not more than one third of them. EgyVac antivenom did not fulfil efficacy or safety criteria in 12 patients. On the basis of these results, ET-G and ET-Plus were selected for comparison in a RCT.

Neutralization of the haemorrhagic activities of viperine snake venoms and venom metalloproteinases using synthetic peptide inhibitors and chelators.

Envenoming by the West African saw-scaled viper, Echis ocellatus resembles that of most vipers, in that it results in local blistering, necrosis and sometimes life-threatening systemic haemorrhage. While effective against systemic envenoming, current antivenoms have little or no effect against local tissue damage. The major mediators of local venom pathology are the zinc-dependant snake venom metalloproteinases (SVMPs). The high degree of structural and functional homology between SVMPs and their mammalian relatives the matrix metalloproteinases (MMPs) suggests that substrate/inhibitor interactions between these subfamilies are likely to be analogous. In this study, four recently developed MMP inhibitors (MMPIs) (Marimastat, AG-3340, CGS-270 23A and Bay-12 9566) are evaluated in addition to three metal ion chelators (EDTA, TPEN and BAPTA) for their ability to inhibit the haemorrhagic activities of the medically important E. ocellatus venom and one of its haemorrhagic SVMPs, EoVMP2. As expected, the metal ion chelators significantly inhibited the haemorrhagic activities of both whole E. ocellatus venom and EoVMP2, while the synthetic MMPIs show more variation in their efficacies. These variations suggest that individual MMPIs show specificity towards SVMPs and that their application to the neutralization of local haemorrhage may require a synthetic MMPI mixture, ensuring that a close structural component for each SVMP is represented.

Neutralisation of venom-induced haemorrhage by IgG from camels and llamas immunised with viper venom and also by endogenous, non-IgG components in camelid sera.

Envenoming by snakes results in severe systemic and local pathology. Intravenous administration of antivenom, prepared from IgG of venom immunised horses or sheep, is the only effective treatment of systemic envenoming. Conventional antivenoms, formulated as intact IgG, papain-cleaved (Fab) or pepsin-cleaved F(ab')2 fragments, are however ineffective against the local venom effects because of their inability to penetrate the blood/tissue barrier. We have embarked on a new research program to examine (i) whether the unusually small (15 kDa) antigen-binding fragment of camelid heavy chain IgG (V(H)H) can be exploited to neutralise the local effects of envenoming and (ii) whether a novel antivenom to treat both the systemic and local effects of envenoming can be formulated by combining anti-snake venom V(H)H and conventional F(ab')2. In this preliminary study, we demonstrate that camels and llamas respond to immunisation with Echis ocellatus venom with high antibody titres and broad antigen specificity. These encouraging immunological results were matched by the successful elimination of venom-induced haemorrhage by IgG from the venom-immunised camels and llamas. Unexpectedly, we report for the first time that camelid serum contains a non-IgG, highly potent inhibitor of venom-induced haemorrhage.

Effects of three novel metalloproteinases from the venom of the West African saw-scaled viper, Echis ocellatus on blood coagulation and platelets.

Two metalloproteinases, a 24-kDa P-I EoVMP1 and a 56-kDa P-III EoVMP2, have recently been isolated from the venom of the West African saw-scaled viper Echis ocellatus. We now reveal a new 65-kDa haemorrhagic group P-III metalloproteinase which we have designated EoVMP3. The aim of this study was to determine whether these three snake venom metalloproteinases (SVMPs) affect platelets and blood coagulation. EoVMP1 had no effect on the aggregation of washed human platelets, whereas EoVMP2 inhibited collagen-induced platelet aggregation. In contrast, EoVMP3 did not inhibit the aggregation of platelets by collagen but instead activated platelets in the absence of any additional co-factors. All three SVMPs were capable of activating prothrombin to varying degrees and can therefore be described as procoagulants. EoVMP1, EoVMP2 and EoVMP3 share sequence identity with other members of the reprolysin family, but differ greatly in their effects on some of the components that control haemostasis.

Pan-African polyspecific antivenom produced by caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa.

A polyspecific Pan-African antivenom has been produced from the plasma of horses immunized with a mixture of the venoms of Echis ocellatus, Bitis arietans and Naja nigricollis, the three most medically important snakes in sub-Saharan Africa. The antivenom is a whole IgG preparation, obtained by caprylic acid precipitation of non-IgG plasma proteins. The antivenom effectively neutralizes the most important toxic activities of the three venoms used in the immunization in standard assays involving preincubation of venom and antivenom before testing. This antivenom compares favourably with other antivenoms designed for use in Africa with respect to neutralization of the toxins present in the venom of E. ocellatus. Caprylic acid fractionation of horse hyperimmune plasma is a simple, convenient and cheap protocol for the manufacture of high quality whole IgG antivenoms. It constitutes a potentially valuable technology for the alleviation of the critical shortage of antivenom in Africa.

Antigenic relationships and relative immunogenicities of isolated metalloproteinases from Echis ocellatus venom.

The antigenic relationship between snake venom metalloproteinases (SVMPs) was analysed using rabbit antisera raised against the native forms of two SVMPs purified from Echis ocellatus venom. Using enzyme-linked immunosorbent assay (ELISA), western blotting and two-dimensional SDS-PAGE, our findings show that antibodies raised against EoVMP1, a non-haemorrhagic class P-I 24kDa SVMP, and EoVMP2, a haemorrhagic class P-III 56kDa SVMP, demonstrate cross-reactivities which relate to the domain hierarchy observed in class P-I to P-III/IV SVMPs. A third 65kDa P-III metalloproteinase (designated EoVMP3) was also isolated from E. ocellatus venom using hydrophobic interaction, size exclusion and anion exchange chromatography. In comparative immunoassays, EoVMP2 and EoVMP3 bound strongly to the commercial monovalent ovine Fab fragment antivenom EchiTAbtrade mark (raised against the same venom), but EoVMP1 showed no cross-reactivity. This could indicate that antivenoms may lack antibodies to potentially important venom components.

Preclinical testing of three South American antivenoms against the venoms of five medically-important Peruvian snake venoms.

World Health Organization (WHO)-recommended preclinical in vivo and in vitro studies were carried out to compare the efficacy of Brazilian, Peruvian and Colombian antivenoms in neutralizing the venom toxins responsible for the lethal, haemorrhagic, necrotizing, coagulant and defibrinogenating effects of five medically-important Peruvian snake venoms. Overall, the Brazilian antivenom was found to be the most effective followed by the Peruvian and Colombian antivenoms. However, it was concluded that all three antivenoms would be acceptable for use in a randomised clinical trial in envenomed humans in Peru.

The purification and partial characterisation of two novel metalloproteinases from the venom of the West African carpet viper, Echis ocellatus.

Separation of previously uncharacterised Echis ocellatus venom by phenyl-Superose FPLC (Fast Liquid Protein Chromatography) yielded eight protein fractions. Three of these displayed high proteolytic activity when assayed by in vivo and in vitro assays (including enzyme linked immunosorbant assay), and were further separated using Superdex 75 and Mono-Q FPLC. This resulted in the purification of a non-haemorrhagic 24 kDa metalloproteinase (EoVMP1, pI 7.0), and a haemorrhagic 56 kDa metalloproteinase (EoVMP2, pI 5.5). Following tryptic digest, short amino acid sequences of EoVMP1 and EoVMP2 were obtained using Edman degradation. Both sequences displayed homology when aligned with existing snake venom metalloproteinases (SVMPs). The strong homology observed among previously well-characterised SVMPs suggests that principles governing the interaction of substrates and inhibitors are likely to be similar for EoVMP1, EoVMP2 and all members of the reprolysin family.

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.

Molecular cloning of phospholipases A(2) from venom glands of Echis carpet vipers.

Venom toxin-specific antibodies offer a more rational treatment of snake envenoming than conventional antivenom. Here, we describe novel cDNAs encoding phospholipase A(2) (PLA(2)) isoforms from venom gland RNA of Echis pyramidum leakeyi (Epl), Echis sochureki (Es) and Echis ocellatus (Eo). The deduced amino acid sequences of these cDNAs encoded proteins with high overall sequence identity to the viper group II PLA(2) protein family, including the 14 cysteine residues capable of forming seven disulphide bonds that characterize this group of PLA(2) enzymes. Comparison of the PLA(2) sequences from Echis with those from related vipers failed to make significant geographic, taxonomic or PLA(2)-function distinctions between these Echis PLA(2) isoforms. However, their deduced hydrophilicity profiles revealed a conserved tertiary structure that we will exploit, by epidermal DNA immunization, to generate PLA(2)-neutralizing antibodies with polyspecific potential.

Simultaneous GeneGun immunisation with plasmids encoding antigen and GM-CSF: significant enhancement of murine antivenom IgG1 titres.

GeneGun DNA immunisation is a potent means of inducing antibody-dominant immune responses that we are exploiting to generate venom toxin-specific antibodies to improve the therapy of systemic envenoming by snakes. Here, we report that mice immunised with DNA encoding the carboxyl domain (JD9) of a haemorrhagic Zn metalloprotease (Jararhagin) in venom of the South American pit viper, Bothrops jararaca, and a plasmid expressing murine cytokine granulocyte/macrophage-colony stimulating factor (GM-CSF) raised significantly higher antigen-specific IgG1 titres than mice immunised with JD9 DNA alone. Serological responses to GeneGun JD9 DNA immunisation were shown to be dominated by IgG1, an IgG subclass associated with T lymphocyte helper 2 (Th2) immune responses. Further significant enhancement of JD9-specific IgG1 titres was achieved by increasing the number of immunisations. This report illustrates that DNA immunisation protocols to achieve high-titre, venom toxin-specific antibody production are well advanced and encourage the development of a DNA-based approach to antivenom production.

The inhibition of platelet aggregation and blood coagulation by Micropechis ikaheka venom.

Uncoagulable blood and life-threatening bleeding can result from the action of some snake venom toxins on haemostatic components of blood and vessel walls. Although envenoming by Micropechis ikaheka primarily affects neurones and muscle cells causing post-synaptic neuromuscular blockade and rhabdomyolysis, disturbances of haemostasis also occur. Therefore, the present study explored the effects of M. ikaheka venom on platelets and endothelium, which are important components of the haemostatic mechanism. The venom inhibited platelet aggregation in response to ADP and collagen, and also delayed clotting dependent on platelet activation or endothelial cell tissue factor expression. Some of these effects were reduced by the incubation of venom with a phospholipase A2 (PLA2) inhibitor and could be reproduced by a 17 kDa venom fraction containing a PLA2. In addition, an 11 kDa fraction containing a long-chain neurotoxin reduced ADP-induced aggregation. The venom was also found to reduce endothelial cell adherence to vitronectin-, fibronectin- and collagen-coated surfaces. These results suggest that, by inhibiting procoagulant activities of platelets and endothelial cells, a 17 kDa PLA2 plays an important role in the anticoagulant action of M. ikaheka venom.

The effect of jararhagin, a metalloproteinase from Bothrops jararaca venom, on pro-inflammatory cytokines released by murine peritoneal adherent cells.

The release of pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) from murine peritoneal adherent cells (MPAC) was studied after exposure to jararhagin, a metalloproteinase/disintegrin isolated from Bothrops jararaca venom. MPACs were treated with LPS (lipopolysaccharide), jararhagin, or EDTA-inactivated jararhagin for up to 24h. Following incubation, the culture supernatant was assayed by ELISA for the presence of cytokines, while the cells were analysed for viability and cytokine mRNA expression. The cells exposed to native jararhagin released TNF-alpha and IL-1beta after 4 and 24h respectively. When MPACs were exposed to Jararhagin treated with EDTA, TNF-alpha and IL-1beta production was sustained throughout the culture period and IL-6 production was observed. TNF-alpha, IL-6 and IL-1beta mRNA were detected 4h after stimulation with either native or EDTA-treated jararhagin. Addition of jararhagin to LPS stimulated cells resulted in a dramatic decrease in the release of IL-6 and TNF-alpha. RT-PCR showed that this inhibition does not occur at the transcriptional level and further experiments showed that jararhagin degraded soluble cytokines by proteolytic activity. This study suggests that jararhagin induces TNF-alpha, IL-1beta and IL-6 expression, which may be rapidly degraded by its proteolytic activity.

An in vivo but insensate model for the evaluation of antivenoms (ED(50)) using fertile hens' eggs.

The preclinical evaluation of the efficacy of new antivenoms (ED(50)) in animals is required by international regulatory authorities. In vitro testing alone does not provide the end point of lethality of a living system which is essential for an antivenom to prevent. Large numbers of mice are injected with venom/antivenom mixtures and the number of surviving mice is statistically analysed to give an ED(50) value reflecting the efficacy of that antivenom. Our objective was to develop a humane alternative to the conventional rodent ED(50) test using fertile hens' eggs. The egg test combines the advantages of an in vivo system, which gives a clear end point for haemorrhage and lethality, with the certain knowledge that no distress can be experienced by the embryo which is insensate at day 6 of development. Nine different, medically-important venoms and antivenoms were tested by both the rodent and egg ED(50) assays. A good correlation between the two tests was obtained (p<0.01) suggesting that the egg ED(50) method should be considered as an alternative to the rodent ED(50) test for non-neurotoxic venoms. Use of the egg ED(50) test would substantially reduce both the numbers of experimental animals required and also the considerable costs involved in delivering improved clinical treatment of snakebite.

Antibody from mice immunized with DNA encoding the carboxyl-disintegrin and cysteine-rich domain (JD9) of the haemorrhagic metalloprotease, Jararhagin, inhibits the main lethal component of viper venom.

Envenoming by the Brazilian pit viper, Bothrops jararaca, induces extensive local and systemic haemorrhage in humans. The severe and occasionally lethal outcome of envenoming is prevented only by administration of antivenom which is conventionally prepared by hyperimmunization of large animals with an individual venom or a range of venoms. Since snake venoms typically consist of numerous molecules, only some of which are toxic, antivenoms are antigenically crude preparations whose therapeutic value would theoretically be enhanced by restricting antibody specificity to toxic venom molecules. We report here that high-titre IgG antibody from mice immunized by the GeneGun with DNA encoding the carboxy-terminal JD9 domain of Jararhagin, a haemorrhage-inducing metalloprotease in B. jararaca venom, extensively neutralized the main lethal component of B. jararaca venom. This is to our knowledge the first study to apply DNA-based methods to preparation of antivenom; it represents a novel approach with greater immunological specificity and fewer hazards than conventional systems of antivenom production.

Distinct contributions of glycoprotein VI and alpha(2)beta(1) integrin to the induction of platelet protein tyrosine phosphorylation and aggregation.

Platelet activation by collagen depends principally on two receptors, alpha(2)beta(1) integrin (GPIa-IIa) and GPVI. During this activation, the nonreceptor protein tyrosine kinase pp72(syk) is rapidly phosphorylated, but the precise contribution of alpha(2)beta(1) integrin and GPVI to signaling for this phosphorylation is not clear. We have recently found that proteolysis of platelet alpha(2)beta(1) integrin by the snake venom metalloproteinase, jararhagin, results in inhibition of collagen-induced platelet aggregation and pp72(syk) phosphorylation. In order to verify whether the treatment of platelets with jararhagin had any effect on GPVI signaling, in this study we stimulated platelets treated with either jararhagin or anti-alpha(2)beta(1) antibody with two GPVI agonists, an antibody to GPVI and convulxin. Platelet shape change and phosphorylation of pp72(syk) by both GPVI agonists was preserved, as was the structure and function of GPVI shown by (125)I-labeled convulxin binding to immunoprecipitated GPVI from jararhagin-treated platelets. In contrast, defective platelet aggregation in response to GPVI agonists occurred in both jararhagin-treated and alpha(2)beta(1)-blocked platelets. This apparent cosignaling role of alpha(2)beta(1) integrin for platelet aggregation suggests the possibility of a topographical association of this integrin with GPVI. We found that both platelet alpha(2)beta(1) integrin and GPVI coimmunoprecipitated with alpha(IIb)beta(3) integrin. Since platelet aggregation requires activation of alpha(IIb)beta(3) integrin, defective aggregation in the absence of alpha(2)beta(1) suggests that this receptor may provide a signaling link between GPVI and alpha(IIb)beta(3). Our study therefore demonstrates that platelet signaling leading to pp72(syk) phosphorylation initiated with GPVI engagement by either convulxin or GPVI antibody does not depend on alpha(2)beta(1) integrin. However, alpha(IIb)beta(3) integrin may, in this model, require functional alpha(2)beta(1) integrin for its activation.

The use of hens' eggs as an alternative to the conventional in vivo rodent assay for antidotes to haemorrhagic venoms.

One of the tests used routinely for the preclinical assessment of antivenom efficacy is the WHO-approved rodent intradermal skin test for assessing neutralization of venom-induced haemorrhagic activity. This is a useful test as in many viperid venoms haemorrhage is considered to be the principal lethal (pathogenic) venom effect in envenomed humans. The main problems with such an assay are, first, the necessity of using large numbers of experimental rodents (rats or mice) in order to obtain statistically significant results and, second, that the test must result in pain for the animals during the 24 hr assay period. The present study compares the rodent assay with an alternative assay using venom, in both the presence and absence of antidote, applied to a filter paper disc and placed on the highly vascularized yolk sac membrane of chickens' eggs at an early developmental stage. This avoids sensitivity to pain as reflex arcs have not yet developed, and haemorrhage or neutralization/inhibition of haemorrhage can be easily recorded. Preliminary results showed a high level of correlation between the results of the two tests when used to assess the efficacy of an antidote. It is hoped that the new assay will reduce the need for pain-sensitive experimental animals in the future.

Collagen-induced secretion-dependent phase of platelet aggregation is inhibited by the snake venom metalloproteinase jararhagin.

Jararhagin, a 52 kDa metalloproteinase from Bothrops jararaca snake venom, belongs to the family of enzymes with an N-terminal Zn2+-containing enzymatic domain, a disintegrin-like domain and a cysteine-rich C-terminal domain. Both jararhagin and jararhagin C, a 28 kDa-protein from the same venom identical to the disintegrin-like domain of jararhagin, inhibit collagen-induced platelet aggregation. In this study, jararhagin and synthetic linear peptides based on the disintegrin-like domain of jararhagin overlapping with the RGD sequence of venom disintegrins, were shown for the first time to inhibit the release of 5-hydroxytryptamine (5-HT) from platelets preloaded with [14C]5-HT and stimulated with collagen. The normal phosphorylation of the 21-kDa myosin light chain (p21) in response to the stimulation indicated that jararhagin and the peptides did not interfere with platelet shape change. The selective inhibition of the secretion-dependent phase of the platelet response to collagen by the enzyme and its peptides was confirmed by the defective phosphorylation of pleckstrin, a 47-kDa platelet protein (p47) involved in dense granule secretion.

Proteolytic cleavage of the beta1 subunit of platelet alpha2beta1 integrin by the metalloproteinase jararhagin compromises collagen-stimulated phosphorylation of pp72.

Early signaling events in the stimulation of platelets by collagen include the tyrosine phosphorylations of FcR gamma-chain, pp72(syk) and phospholipase Cgamma2. These events are dependent on the main platelet collagen receptor, alpha2beta1 integrin (glycoprotein Ia-IIa complex). We recently found that jararhagin, a 52-kDa snake venom metalloproteinase, selectively inhibits collagen-induced platelet secretion and aggregation in parallel with the cleavage of the beta1 subunit of the alpha2beta1 integrin. The present study demonstrates that jararhagin also interferes with collagen-induced phosphorylation of the protein-tyrosine kinase pp72(syk). This effect is not observed when the platelet aggregation response to collagen is inhibited by two venom RGD-containing disintegrins, contortrostatin and echistatin. These disintegrins inhibit platelet aggregation through their high affinity binding to the platelet alphaIIbbeta3 integrin (glycoprotein IIb-IIIa complex). We also show that mild stimulation by ADP of jararhagin-treated platelets, but not of platelets treated with the RGD-containing disintegrins, restores the collagen-induced platelet aggregation. ADP also restored both pp72(syk) and pleckstrin phosphorylation of jararhagin-treated platelets in response to collagen, presumably via interaction of collagen with ADP-activated alphaIIbbeta3 integrin. Thus, RGD-containing disintegrins do not interfere with agonist-induced pp72(syk) phosphorylation but inhibit aggregation through occupancy of the alphaIIbbeta3 integrin. Conversely, jararhagin affects early platelet signaling events in response to collagen through its effects on the alpha2beta1 integrin without interfering with the function of the alphaIIbbeta3 integrin. Our demonstration that the degradation of the beta1 subunit of alpha2beta1 by jararhagin results in the loss of pp72(syk) phosphorylation, suggests that this subunit is critically involved in collagen-induced platelet signaling.