Barlow, K. Theakston Snakebite venom inhibition, Warrell Glycemic load and satiety, Griffiths E. albolabris— a surprising Restful recovery services since neither of inuibition venoms were included in the Snakebitr mixture used to generate this EAV Figs 4B and S4. Snakebite venom inhibition Snakeebite of the eight venoms, the procedure ibhibition of groups of five mice 4—5 weeks old, 18—20 g, male, CD-1 mice, Charles River, UK; maintained as described above receiving an iv tail injection of either: i 5 x LD 50 dose of venom only, or 5 x LD 50 dose of venom preincubated for 30 mins at 37°C with μl 5 mg of ii EAV 1, iii EAV 2 or iv SAIMR polyvalent as a positive control antivenom. SVSP activity was measured using a chromogenic substrate S, Cambridge Bioscience.

Snakebite venom inhibition -

Snake venom is hemotoxic — destructive to the ability of blood to clot — and can cause the destruction of fibrinogen, an essential protein that enables blood to clot and stop excessive bleeding.

Snake-venom enzymes also can cause abnormally fast clotting, which can lead to heart attack, stroke and damage to the body's organs.

Both reactions are inhibited by the therapy. Vance G. Nielsen , professor and vice chair for research in the Department of Anesthesiology at the UA College of Medicine — Tucson, has confirmed that, if given soon enough after a snake bite, the carbon monoxide-iron-based therapy directly can inhibit snake venom's ability to block blood clotting in laboratory animals for as long as an hour.

Nielsen also demonstrated for the first time in the test tube that the therapy blocks snake venom's ability to cause abrupt clotting. Time is of the essence following exposure to rattlesnake venom because without fibrinogen, blood does not clot and the risk of internal bleeding increases, resulting in serious health consequences such as blood entering the brain or intestines.

In addition, abnormally fast clotting in the blood vessels can deplete clotting factors and cause excessive bleeding or the clots can block blood vessels, causing lethal loss of blood flow to tissue. Nielsen has found that the therapy works against the venom of more than three dozen species of snakes throughout the world.

Nielsen is working toward developing the treatment to work in humans. To further advance the research, he is seeking commercial backing and is working with Tech Launch Arizona , the UA office that commercializes inventions stemming from University research, to protect the intellectual property of the treatment and strategize ways to get it into the hands of health professionals.

He also is collaborating with Dr. A positive control venom with known serine protease activity was also used for standardisation across assays 1 μg; B. arietans [ 43 ] , alongside a PBS no venom negative control.

Each sample was added in triplicate to a well microplate Greiner. The plate was then incubated for 3 min at 37°C in a FLUOstar Omega microplate reader BMG Labtech. Next, 15 μl of buffer mM Tris, pH 8. Finally, 15 μl of 6 mM chromogenic substrate S, Cambridge Bioscience at a final concentration of 2 mM was added.

The enzymatic reaction was then monitored kinetically for 60 cycles 30 sec. each; ~30 min total using a FLUOstar Omega microplate reader BMG Labtech , with a temperature setting of 37°C and using a wavelength of nm.

Mean measures of absorbance were plotted against time to compare venom activity with baseline negative controls and positive control readings.

The rate of substrate consumption was calculated by subtracting the 0 min readings from the 5 min readings and then dividing by 5 min. Finally, the reduction percentage for all antivenom samples was calculated by subtracting the mean of the relevant negative control readings from the venom readings.

The resulting triplicate readings were then plotted with standard error of the means SEMs. To quantify snake venom metalloproteinase activity and inhibition by the EAVs, we used a fluorescent kinetic enzymatic assay [ 44 ]. For determining baseline venom activity, we replaced antivenom with PBS, to act as venom only controls.

A positive control venom with known metalloproteinase activity was also used for standardisation across assays 1 μg; E.

ocellatus [ 44 ] , alongside a PBS no venom negative control. Ten microlitres of each sample was then added in triplicate to a well microplate Greiner followed by the addition of 90 μl of the substrate 10 μl of the substrate [supplied as a 6.

The assay was then read kinetically for 1 hour at 25°C using a FLUOstar Omega microplate reader BMG Labtech and an excitation wavelength of nm and an emission wavelength of nm. Mean measures of absorbance were then plotted against time to compare venom activity with baseline negative controls and positive control readings.

For quantification, we calculated areas under the curves AUCs and the standard error of mean AUCs readings for each sample in the 0—40 min interval, this time point was chosen as the time where all fluorescence curves had reached a plateau maximum fluorescence.

We then subtracted the mean of the relevant negative control readings from the venom readings and calculated the reduction percentage for all antivenom samples and plotted the triplicate readings with SEMs. To quantify venom coagulotoxicity and inhibition by the EAVs, we used a kinetic absorbance-based coagulation assay [ 45 ].

The positive control used was 1 μg of E. ocellatus venom based on its potent procoagulant effect in this assay [ 44 ], and the negative control was plasma with the addition of PBS instead of venom. During sample incubation, bovine plasma sterile filtered, Biowest, Nuaille, France was defrosted and centrifuged for 4 min at x g to remove precipitate prior to use.

Following incubation, 10 μl of each venom-antivenom mixture was added to a well microplate Greiner , followed by the addition of 20 μl of 20 mM CaCl 2 prepared fresh for each assay , and finally 20 μl of the citrated bovine plasma. The plates were loaded using a multidrop pipetting robot Multidrop Labsystems and resulting clotting data were captured kinetically for 2 hours using a FLUOstar Omega microplate reader BMG Labtech at OD nm and 25°C.

For quantification, we calculated the AUCs and the standard error of the mean AUCs. We then subtracted the mean of the relevant negative control readings from the venom readings and calculated the reduction percentage of venom activity for all datasets with SEMs.

To assess the preclinical efficacy of the EAVs, we used modified versions of established murine models of envenoming [ 44 ] and selected a sub-set of the venoms used as immunogens, along with two additional venoms not used as immunogens, to assess the breadth of antivenom paraspecific efficacy against haemotoxic snakebite.

As an essential prerequisite to preclinically assessing antivenom efficacy, World Health Organization-recommended protocols were used to determine the median murine lethal dose LD 50 of various snake venoms via the intravenous route [ 44 ].

For these studies, we selected four venoms used as immunogens for generating both EAVs Bothrops asper , Calloselasma rhodostoma , E. ocellatus and Daboia russelii and two venoms used as immunogens only for generating EAV 2 B.

arietans and Echis carinatus. We also used venom from two species of snake not used as venom immunogens for either EAVs to assess their paraspecific neutralisation capabilities, specifically Vipera ammodytes captive bred and Lachesis muta Brazil. Existing LD 50 data was available for six of the eight venoms used Table 2 and, for each of these six venoms, the described LD 50 data had previously been applied to the same venom samples as those used in this study in other studies previously conducted by the authors [ 43 , 44 , 46 ] Therefore, LD 50 experiments were only performed for venom samples from C.

rhodostoma and L. muta , as these had not previously been utilised for in vivo research. All experimental animals 4—5 weeks old, 18—20 g, male, CD-1 mice, Charles River, UK were housed in groups of five with environmental enrichment, water and food ad libitum and their health monitored daily during acclimatisation.

Each animal received an iv tail injection of varying doses of venom in μl PBS, and experimental animals were monitored for 6 hours after injection, with the number of surviving mice in each group recorded.

Experimental animals were euthanised on ethical grounds via rising concentrations of CO 2 if they exhibited signs of humane end points that are predicators of lethality e. To assess the efficacy of the EAVs at protecting experimental animals from the lethal effects of envenoming, we used a modified version of the median effective dose ED 50 assay [ 48 ].

For each of the eight venoms, the procedure consisted of groups of five mice 4—5 weeks old, 18—20 g, male, CD-1 mice, Charles River, UK; maintained as described above receiving an iv tail injection of either: i 5 x LD 50 dose of venom only, or 5 x LD 50 dose of venom preincubated for 30 mins at 37°C with μl 5 mg of ii EAV 1, iii EAV 2 or iv SAIMR polyvalent as a positive control antivenom.

As detailed above, experimental animals were monitored for signs of suffering for 6 hours, with euthanasia applied via the implementation of the described humane endpoints where necessary, and the number of deaths, time of euthanasia and survivors in each group recorded throughout the experiment.

This monitoring window was selected to capture protection against acute venom toxicity, and applied based on animal ethical grounds to reduce the duration of severe suffering imposed on experimental animals during envenoming experiments.

Thereafter, Kaplan—Meier survival graphs were used to visualise the relative preclinical protection provided by the antivenoms against each of the venoms. All statistical analyses were performed using Prism v8 software GraphPad.

To assess the specificity of the immune response towards the venom immunogens over the course of the immunisation period, serum samples were collected every four weeks and assessed via ELISA to quantify antibody binding levels. Both sheep responded in a highly similar manner to the different immunogen mixtures see Table 1 for details of immunogens , with rapid increases in antibody binding levels observed following primary immunisation, followed by a short plateau in antibody titres and then secondary increases Fig 1.

For both immunised animals, serum samples collected at the end of the immunisation schedule week 42 exhibited the highest antibody binding titres Fig 1. These samples were subsequently subjected to IgG extraction for preparation of EAV 1 and EAV 2.

A Responses of EAV 1 against immunogen mixture I containing seven venoms and B responses of EAV 2 against immunogen mixture II containing 12 venoms. Data points represent means of duplicate readings and error bars represent standard deviation SD of the duplicate measurements.

To visualise the immunological recognition of the two EAVs against the venom proteins found in the 12 venoms used in the immunisation process, we subjected each venom and each venom immunogen mixture to reduced SDS-PAGE gel electrophoresis and western blotting.

As anticipated, the reduced SDS-PAGE profiles illustrated considerable inter-specific variation in the molecular weights and relative abundances of the different proteins found in the various venoms Fig 2A.

Immunoblotting experiments with each of the EAVs revealed extensive immunological recognition of the proteins found in each of the snake venoms and the two immunising mixtures, despite EAV 1 being generated with only seven of the 12 venoms used as immunogens to generate EAV 2 Fig 2C and 2D.

However, despite these broad similarities, these experiments revealed that EAV 1 exhibited some reductions in binding intensity compared with EAV 2, and also did not recognise the low molecular weight components i.

typus and R. subminiatus venoms to the same extent as EAV 2. As anticipated, both EAVs exhibited increases in venom recognition compared with the SAIMR polyvalent antivenom positive control, and for which only B.

arietans is an immunogen; Fig 2B and the normal horse and sheep negative controls Fig 2E and 2F. A Venoms were separated by reduced SDS-PAGE gel electrophoresis and visualised by Coomassie blue staining. The same venom samples were transferred to nitrocellulose membranes for immunoblotting, and incubated with , dilutions of SAIMR polyvalent antivenom positive control B , EAV 1 C , EAV 2 D , normal horse IgG E and normal sheep IgG F both negative controls.

PM indicates protein marker. Next, we used end-point titration ELISAs to quantify the binding between the EAVs and the various venom immunogens. Comparisons of the binding titres of each EAV revealed highly comparable levels to each of the individual venoms used as immunogens Figs 3A and S1 , despite five additional venoms being added to the immunogen mixture used to raise EAV 2.

An exception to this was that EAV 2 exhibited considerably highly titres to the venom of R. subminiatus than EAV 1 Figs 3A and S1. This finding is consistent with the presence of this venom only being in the immunising mixture used to raise EAV 2, though, interestingly, no such differences were observed with the other four venoms unique to this immunogen mixture i.

arietans , E. carinatus , Trimeresurus albolabris and Crotalus atrox. Secondly, and perhaps surprisingly, we observed substantial increases in immunological binding to immunogen mixture II with EAV 1, rather than EAV 2 Figs 3A and S2A.

When comparing the levels of immunological cross-reactivity from the venom perspective, we observed considerable variation in binding levels, with six venoms exhibiting high titres, five with more moderate levels of binding and perhaps the most noticeable finding being that only low levels of binding were observed between C.

rhodostoma venom and both EAVs Figs 3A and S1. A A summary of the immunological binding observed by end-point titration ELISA. The data displayed represents the mean optical density following the addition of primary antibodies at dilution.

See S1 and S2A Figs for full binding profiles generated using fivefold titrations. B A summary of the immunological binding observed by avidity ELISA. The data displayed represents the mean percentage reduction in antibody binding levels for each venom measured in the presence of 4M and 0M control ammonium thiocyanate.

See S2B and S3 Figs for full binding profiles at increasing concentrations of the chaotrope M. For both sets of experiments, the primary antibodies consisted of EAV 1 and EAV 2, the commercial SAIMR polyvalent antivenom positive control, and normal sheep and horse IgG as negative controls.

Error bars represent SD of duplicate measurements. To determine the strength of these antibody-venom protein binding interactions we performed avidity ELISAs, which consisted of quantifying venom-antibody binding levels in the presence of increasing concentrations of a chaotropic agent ammonium thiocyanate, NH 4 SCN; M that disrupts antibody-antigen binding.

The results revealed that both EAVs exhibit highly comparable, and potent, binding to the two venom mixtures and to each individual venom used as an immunogen, even in the presence of up to 4M ammonium thiocyanate Figs 3B , S2B and S3.

The exception to this was the venom of C. Comparisons between EAV 1 and EAV 2 revealed very little difference in avidities across the individual venoms and the venom immunogen mixtures, though in general EAV 2 exhibited slightly lower reductions in antibody binding in the presence of the chaotrope compared with EAV 1 Figs 3B , S2B and S3.

While high levels of immunological binding appear to be a prerequisite for antivenom efficacy, immunological binding does not necessarily result in toxin neutralisation. To assess the capability of the EAVs to neutralise venom functional activities related to haemotoxicity, we employed three different in vitro functional screening assays, which specifically related to SVSP, SVMP and coagulotoxic venom activities.

The results of our kinetic chromogenic SVSP assay revealed that seven of the 12 venoms tested, and both of the venom immunogen mixtures, exhibited considerable SVSP activity as evidenced by substantial increases in the rate of substrate cleavage compared to the negative control Figs 4A and S4.

These venoms included three used as venom immunogens for both EAVs B. asper , Bothrops jararaca , E. ocellatus and four used only to raise EAV 2 B. carinatus , T. albolabris and C. Consequently, we noted that venom immunogen mixture II exhibited increased SVSP activity compared with mixture I Figs 4A and S2C.

The other venoms had little to no SVSP activity at the concentration tested, and thus were not used in subsequent neutralisation experiments using the EAVs. asper or B. arietans venom, and only resulted in minor reductions against B. jararaca Figs 4B and S4.

Contrastingly, EAV 1 only exhibited high levels of SVSP inhibition against B. arietans and T. albolabris— a surprising result since neither of these venoms were included in the immunogen mixture used to generate this EAV Figs 4B and S4.

B Inhibition of venom serine protease activities by the EAVs displayed as the percentage reduction of venom only activities from A. Also see S2C and S4 Figs. C The metalloproteinase SVMP activity of individual venoms and the venom immunogen mixtures displayed as the area under the curve AUCs resulting from cleavage of a fluorescent substrate over time.

D Inhibition of venom metalloproteinase activities by the EAVs displayed as the percentage reduction of venom only activities from C. See also S2D and S5 Figs. E The coagulopathic activity of individual venoms and the venom immunogen mixtures displayed as the AUCs resulting from increases in absorbance stimulated by clotting over time.

F Inhibition of coagulopathic venom activities by the EAVs displayed as the percentage reduction of venom only activities from E.

See also S2E and S6 Figs. For all data shown, data points represent means of triplicate readings, and error bars represent standard error of the mean SEMs. The enzymatic SVMP activities of the venoms used in this study were quantified via a kinetic, fluorescent assay.

The majority of the 12 venom immunogens exhibited detectable SVMP activity, with the exceptions being those from R. subminiatus , T. albolabris and D. russelii Figs 4C and S5. The latter finding is perhaps surprising given that a potent Factor X activating SVMP is known to be present in D.

russelli venom [ 13 ], suggesting that this result may perhaps be a reflection of limitations in substrate specificity.

Detailed comparisons between both EAVs revealed highly similar percentage reductions in SVMP activity for six of the nine venoms tested B. asper , D. typus , Deinagkistrodon acutus , B. carinatus and C. atrox , while EAV 1 exhibited modest increases in inhibitory potency against B.

jararaca ocellatus venom Interestingly, the SAIMR polyvalent control antivenom outperformed both EAVs in inhibiting B. arietans and C. atrox venoms, though the former of these two venoms is used as an immunogen during the generation of this product. This control antivenom also exhibited comparable SVMP neutralising capabilities to the EAVs against D.

acutus and D. typus venoms, though it was much less effective against B. asper and E. ocellatus Figs 4D and S5. While there was little difference between the levels of inhibition observed between EAV 2 and the SAIMR polyvalent control antivenom, EAV 1 outperformed both comparators, resulting in percentage reductions of To assess the coagulopathic activity of the venom immunogen mixtures and their constitutive venoms, we used an absorbance-based plasma clotting assay.

Nine of the 12 venom immunogens and both venom mixtures exhibited potently procoagulant activities with an additional venom C. atrox exhibiting moderate procoagulant activity, as evidenced by increases in kinetic profiles compared with the negative control Figs 4E and S6.

Neither B. arietans nor T. albolabris exhibited potent coagulopathic activity in this assay at the concentration tested. Neutralisation experiments with the two EAVs revealed marked inhibition of the coagulopathic toxins found in the majority of the coagulopathic venom immunogens Figs 4F and S6.

With the exception of C. subminiatus and E. carinatus Figs 4F and S6. atrox , EAV 2 also lacked inhibitory potency against the venom of E. Both venom immunogen mixtures were also found to be potently procoagulant, and this venom activity was effectively neutralised by both EAV 1 and EAV 2, although inhibition was greatest with EAV 1, irrespective of the venom immunogen mixture However, despite neither EAV by itself affecting normal coagulation, the addition of EAV 1 in the presence of the two venom immunogen mixtures resulted in a delay of clotting, and therefore net anticoagulant activity S2E Fig.

The reason for this remains unclear, but might indicate potent inhibition of procoagulant venom toxins, but ineffective inhibition of anticoagulant venom toxins, as seen elsewhere [ 43 ].

Contrastingly, EAV 2 effectively inhibited venom immunogen stimulated coagulation to control levels, though this same phenomenon was observed for both EAVs against many of the individual venom immunogens S6 Fig.

The SAIMR polyvalent antivenom was ineffective against all of the individual venoms and venom immunogen mixtures tested, likely as the result of no procoagulant snake venoms being used as immunogens during the production of this product Fig 4F.

Following promising evidence of in vitro immunological cross-reactivity and venom neutralisation with the two EAVs, we next assessed their preclinical efficacy against a subset of haemotoxic venoms using a variation of the World Health Organization-recommended assay for assessing antivenom efficacy, the murine median effective dose [ 49 ].

We selected eight venoms for preclinical assessment; four that were included in both venom immunogen mixtures B. asper , C. rhodostoma , E. ocellatus and D. russelii , two that were only included in the immunogen mixture used to generate EAV 2 B.

arietans and E. carinatus and, to assess whether the breadth of antibodies generated by the diverse immunogen mixtures stimulated broad paraspecific efficacy, two haemotoxic venoms that were not used in either immunogen mixture V.

ammodytes and L. muta Table 2. Prior to performing antivenom efficacy experiments, the LD 50 of L. muta and C. rhodostoma venoms were determined, resulting in LD 50 s of 6. The LD 50 s for the remaining species were sourced from previously published studies Table 2. Our dose-matched pilot in vivo findings demonstrated that, despite a reduction in the number of venoms used in the immunogen mixture 7 vs 12 , EAV 1 outperformed EAV 2 in terms of preclinical efficacy superior vs four venoms; equipotent vs two venoms; inferior vs one venom Fig 5.

Indeed, EAV 1 effectively prevented venom-induced lethality for the duration of the experiment against three of the four venoms used as immunogens B. asper , E. russelii , and two of the four venoms tested that were not present in the immunising mixture B. arietans and V.

ammodytes in this model Fig 5. Contrastingly, none of the venoms present in the immunogen mixture used to raise EAV 2 were fully neutralised i. asper , B. carinatus venom Fig 5. The result with this latter venom proved to be the only example of superior preclinical efficacy exhibited by EAV 2 over EAV 1 four vs one survivor, respectively.

russelii— arguably the two most medically-important species tested Fig 5. Both EAVs exhibited comparable, limited protection against the lethal effects of C. rhodostoma venom two of five experimental animals survived the duration of the experiment , which correlates with our earlier observations of reduced immunological cross-reactivity to the toxins found in this venom see Fig 3 , and suggests that higher therapeutic doses than tested here are likely required to provide full protection.

Identical and contrasting efficacy observations were also observed in respect of the remaining two venoms tested, neither of which was used as an immunogen to generate either EAV; both antivenoms failed to provide any protection against the lethal effects of L.

muta venom, though both EAVs exhibited potent paraspecific preclinical efficacy against V. ammodytes Fig 5. To place these findings into context, we compared our preclinical efficacy data against that obtained using our antivenom control, SAIMR polyvalent, which is manufactured using venom from a variety of African vipers and elapids as immunogens.

As anticipated, both EAVs outperformed SAIMR polyvalent, with only EAV 1 proving inferior against the lethal effects of E. carinatus venom, while EAV 2 was only outperformed in experiments using E.

ocellatus and B. arietans venoms Fig 5. Groups of five mice were challenged intravenously with 5 x LD 50 doses of each venom purple lines or venom preincubated for 30 min at 37°C with EAV 1 blue or EAV 2 red , or the SAIMR polyvalent control antivenom green.

All experimental animals were monitored for 6 hours and survival times recorded. There are a number of major obstacles that need to be tackled to ensure the effective treatment of tropical snakebite victims.

These include, but are not limited to, addressing the poor affordability, lack of availability and the often limited cross-species neutralising potency of commercial antivenom [ 19 , 50 ].

Currently, all existing antivenoms are manufactured for specific geographical regions, typically parts of a continent, or specific countries within a continent, leading to a highly fragmented drug market [ 3 , 50 ]. Ultimately, the venoms used as immunogens for antivenom production dictate the breadth of snake species efficacy of those products, and it has been suggested that simply adding additional venoms to the immunogen mixture to increase paraspecific efficacy could be potentially counter-productive by diluting those antibodies specific to the toxins delivered during the bite by any particular snake [ 11 , 19 , 32 ].

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