In this case, the genetic sequences of the immunoglobulin variable domains single-chain variable fragments, scFvs were fused with the DNA sequence of the phage gene III, allowing the variable domains to be expressed on the surface of fd bacteriophages, thereby conveniently linking genotype with phenotype through the phage.
When an antigen of interest is available, antibody phage display technology enables the selection and isolation of high affinity scFv binders To date, more than 10 monoclonal antibodies derived from phage display experiments have successfully entered the market for a diverse range of therapeutic indications. In , Hamers-Casterman et al. Later on, further research confirmed that the variable domain of these antibodies V H H alone, with a size of about 12—15 kDa, is still functional, turning it into the smallest antibody fragment with antigen binding capacity Notably, these single-domain antibodies also known as nanobodies possess important structural differences in the antigen binding region compared to conventional antibodies, which may provide them with an ability to recognize cryptic hidden epitopes Due to their specific antigen binding capacity, together with their high solubility, stability, and ability to penetrate rapidly into deep tissue, among other favorable characteristics 78 , several therapeutic candidates based on single-domain antibodies have recently been under clinical investigation.
Of note, last year, caplacizumab, an anti-von Willebrand factor, became the first nanobody-derived therapy to gain regulatory approval, and it is now on the market HCAbs were also discovered in in cartilaginous fish i. From these antibodies, variable new antigen receptor VNAR domains similar to V H Hs in terms of structure and function were described Transgenic mice carrying human immunoglobulin loci were developed in as an innovative approach for obtaining human antibodies using hybridoma technology 83 — This method allows for the generation of fully human monoclonal antibodies with the advantage of maintaining the processes of natural recombination and affinity maturation that occur in vivo Along with the different display techniques, the use of transgenic mice circumvents many of the drawbacks of therapeutic murine, chimeric, and humanized antibodies, such as suboptimal pharmacokinetics and unfavorable immunogenicity, mainly due to the fully or partial heterologous nature of the latter mentioned antibody formats.
Other transgenic animals have also been developed for the same purpose, including rats, rabbits, and calves 89 , It is also worth mentioning that in the late s, other display technologies, different from phage and bacterial display, were developed, such as yeast surface display 91 , ribosome display 92 , and mRNA display Within these types of technologies, CIS-display and mammalian cell surface display appeared last, in 94 and 95 , respectively.
Nowadays, the current molecular biology tools together with the above-mentioned biotechnological progress have made it possible to design and express antibody-based proteins in a vast repertoire of molecular formats. Conventional formats already in use as antitoxins, either experimentally or in the clinic, include whole IgG, F ab' 2 , Fab, diabody, scFv, and V H H Besides these formats, in fields different from envenoming therapy, antibody engineering has led to diverse multivalent and multispecific constructs 97 , For example, ALX is a trimeric nanobody against respiratory syncytial virus currently under development Also, bispecific antibodies showing neutralizing capacity in vitro and in vivo against filoviruses have been reported Additionally, other binding proteins may be investigated for their potential to neutralize toxins Favorably, the field of antivenom research now has an opportunity to build on top of decades of progress on design and engineering of biotherapeutic agents to generate high affinity toxin-neutralizing molecules with unprecedented neutralizing capacity, low immunogenicity, and desirable pharmacokinetics , Despite the advancements that have occurred in the field of biotechnology since Calmette's first steps towards the introduction of antiserum therapy as a treatment for animal envenoming, to this date, antiserum remains the only effective treatment against envenomings caused by venomous animals 2.
Many barriers are likely to have contributed to this, including the neglected character of the problem, the complexity of developing an alternative treatment, and the low economic incentive for companies to develop treatments against envenomings. This may possibly help create the necessary awareness, political will, and incentives to help researchers develop novel therapies against snakebite and other envenomings 54 , Nevertheless, over the last many years, academic research groups across the world have been attempting to use the last decades of biotechnological advancements to improve current or develop novel treatments against animal envenomings.
Many of the avenues that have been, or still are being pursued toward the development of alternative therapies to current antivenom treatment, include many different types of monoclonal antibodies 96 and several types of non-antibody-based molecules, such as oligonucleotide aptamers , , nanoparticles , peptides , naturally occurring protein inhibitors — , and small molecule inhibitors — Varespladib and batimastat are examples of small molecule inhibitors originally developed against indications outside the field of snakebite envenoming that were later shown to inhibit toxic effects from phospholipases A 2 and snake venom metalloproteinases, respectively , , Both peptides, naturally occurring non-antibody proteins, and nanoparticles have also been shown to have neutralizing capacities against snake venoms However, none of these molecules have ever reached the clinic, and they fall outside of the scope of this review.
Within the scope of this review, the development of novel envenoming therapies based on monoclonal antibodies is being pursued using many of the technologies presented in previous sections of this review. One technology that has been employed numerous times to discover monoclonal antibodies against animal toxins is hybridoma technology 1. In the field of envenoming, it was first used in by Bahraoui et al. In this study, mice were immunized with toxin II, spleen cells were fused with myeloma cells, and the resulting hybridomas were tested for secretion of toxin-binding monoclonal antibodies.
The obtained antibodies were tested for their ability to prevent lethality after lethal amounts of toxin were preincubated with each antibody and injected intracerebroventricularly in mice. One monoclonal antibody neutralized toxin doses as high as 50 LD 50 s Other groups have since then employed murine hybridomas for discovery of monoclonal antibodies against many other animal toxins , Similar to the transition from the use of monoclonal antibodies of animal origin to human origin in other antibody research fields, human monoclonal antibodies have also gained increasing interest within antivenom research.
To the best of our knowledge, to this date, only one example of the discovery of human IgGs against snake toxins using transgenic mice has been reported. In , transgenic mice were used to discover human IgGs against the metalloproteinase HR1a from Protobothrops flavoviridis a pit viper from Ryukyu Islands of Japan by Morine et al.
In that study, hybridoma cell fusions were screened for production of toxin-binding IgGs, and of these, 80 antibodies were identified as HR1a-reactive. The IgGs were tested for their ability to inhibit proteolytic and hemorrhagic activity in vitro , where some showed the ability to partially inhibit both toxic effects Another technology that has been utilized by different research groups to discover monoclonal antibodies of different origin against toxins from snake , spider , scorpion , and bee venoms is antibody phage display.
Antibody phage display technology was first used for the discovery of monoclonal antibody fragments against animal toxins in by Meng et al. The authors used an scFv library generated from spleen cells of mice that had been immunized with crotoxin obtained from the snake Crotalus durissus terrificus.
The affinity matured library was used to discover scFvs with specificity to crotoxin. The scFvs were tested in lethality assays in mice upon preincubation with lethal doses of Mojave toxin, demonstrating the ability of the scFvs to provide prolonged survival in mice. Since the first use of antibody phage display technology in toxinology, this discovery methodology has been employed by several groups within the field, and some groups have reported the discovery of V H H monoclonal antibody fragments from phage display libraries generated from both non-immunized and immunized llamas against animal toxins , The first report on the use of antibody phage display technology for generating a human monoclonal antibody fragment against a snake toxin was made by Lafaye et al.
Here, scFvs from a human semi-synthetic antibody phage display library were discovered against crotoxin, and these antibodies were demonstrated to bind the toxin in ELISA experiments.
Following four rounds of selection, an scFv was affinity matured using directed evolution. In a subsequent lethality assay, where the most promising affinity matured scFvs were incubated with either toxin or whole venom prior to intravenous injection into mice, one scFv demonstrated the ability to prevent lethality of 2 LD 50 s of venom and toxin.
In , Funayama et al. A combination of two of the resulting scFvs was reported to inhibit myotoxic effects in vivo and prolonged survival of mice in lethality assays, where venom and scFvs were preincubated prior to administration.
Since these first discoveries of human monoclonal antibody fragments against animal toxins, phage display technology has been used to discover human monoclonal antibody fragments against toxins from other snakes — , scorpions , , and bees , To the best of our knowledge, no human monoclonal antibody fragment has yet been discovered against a spider toxin using phage display selection.
In , Laustsen et al. Moreover, with their study, Laustsen et al. Using phage display technology, research groups have attempted to take antibody discovery a step further by engineering monoclonal antibody fragments to be specific to more than one toxin. This phenomenon is referred to as antibody cross-reactivity, which in relation to animal toxin neutralization is a desirable antibody property, as animal venoms are complex mixtures of toxins of both high and low homology Being able to use only one monoclonal antibody to target two or more toxins will help lower the total number of monoclonal antibodies needed for a recombinant antivenom based on oligoclonal antibodies, which in turn will improve developability and cost of manufacture , , — In this regard, Pucca et al.
Similarly, in the work of Roncolato et al. Silva et al. The gene was mutated in selected residues of the CDR3 region, where upon a new antibody library was constructed and used to select binders against other toxins from C.
A resulting scFv displayed neutralizing abilities against 13 neurotoxins present in the venoms of nine different species of Mexican scorpions In combination, the many reports on the discovery of a different types of monoclonal antibodies against a multitude of different toxins from venomous animals demonstrate that increased interest and application of newer biotechnological approaches and techniques are building in the field of envenoming therapy research.
Although many of these developments are yet to enter the clinical setting, the future perspectives for this field are improving. It should, however, be noted that recombinant antivenoms based on oligoclonal antibodies may possess somewhat different therapeutic properties than traditional antivenoms based on polyclonal heterologous antibodies. Oligoclonal antivenoms are much simpler in composition, making them less likely to exhibit cross-neutralization properties to the extent of having paraspecificity cross-reactivity to venoms that were not part of the development or manufacturing process for an antivenom.
As many medically relevant venomous animals possess up to dozens of medically important toxins, the possibility of engineering the cross-reactivity of monoclonal antibodies, as well as oligoclonal mixtures thereof, may be key to successful recombinant antivenom design , With the renewed focus on snakebite envenoming as a Category A Neglected Tropical Disease by the WHO, there is a hope that the development of much needed therapies against both snakebite and other animal envenomings will become increasingly incentivized for researchers worldwide.
Among the scientific and technological fields that are expected to gain increased interest, the development of standardized approaches for rational engineering of cross-reactivity for both individual monoclonal antibodies and oligoclonal antibody mixtures is likely to gain traction, as this is an essential parameter for creating broadly-neutralizing recombinant antivenoms that can be used against multiple species Also, manufacturing strategies for low cost production of recombinant antivenoms will need to be further developed.
Moreover, the field of antivenom development has only recently seen the introduction of systematic and holistic strategies for developing recombinant antivenoms 51 , , , , and these strategies need to be both strengthened and further tested in the laboratory setting. Finally, the entire field of envenoming diagnostics has seen very little innovation for decades, and an opportunity exists for implementing both bio and nanotechnologies for the development of novel diagnostic tools and devices that can help stratify envenoming cases and quantitatively monitor pathogenesis of envenoming FC was in charge of drawing the figures.
MP and AL designed the review, wrote part of the manuscript, and provided revisions. JB gave his valuable and professional suggestions and revised the manuscript. All authors read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. JB — passed away during the revision of this article. This work is dedicated to his memory in gratitude for all his discoveries within recombinant antivenoms based on human monoclonal antibodies.
National Center for Biotechnology Information , U. Journal List Front Immunol v. Front Immunol. Published online Jul Manuela B. Pucca , 1, 2 Felipe A. Barbosa , 3 and Andreas H. Felipe A. Andreas H. Author information Article notes Copyright and License information Disclaimer. Laustsen kd. This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology.
Received Apr 23; Accepted Jun The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. This article has been cited by other articles in PMC.
Abstract Each year, millions of humans fall victim to animal envenomings, which may either be deadly or cause permanent disability to the effected individuals.
Keywords: envenoming therapy, antivenom, antibodies, antiserum, hybridoma technology, phage display, recombinant antivenom, antivenom history. Introduction The discovery of serum therapy has paved the way for many human therapies, including envenoming therapy.
Open in a separate window. Figure 1. The Origin of Serum Therapy The use of serum therapy began in when Emil von Behring and Shibasaburo Kitasato published their groundbreaking paper on tetanus immunity 3. Figure 2. Important Discoveries Within Antibody Generation and Characterization Although Behring and Kitasato were the pioneers of serum therapy, Paul Ehrlich was the scientist responsible for the first large-scale production of antiserum. Figure 3. The History of Antivenom Therapies The history of antivenom begins with the work of the French physician Albert Calmette in the late Nineteenth century.
The History of Antibody-Based Therapies From the s to the present, numerous breakthroughs in the fields of molecular biology, biochemistry, and immunology have laid the foundation for the development of antibody-based therapies. Advances in Antivenom Research Despite the advancements that have occurred in the field of biotechnology since Calmette's first steps towards the introduction of antiserum therapy as a treatment for animal envenoming, to this date, antiserum remains the only effective treatment against envenomings caused by venomous animals 2.
Future Perspectives With the renewed focus on snakebite envenoming as a Category A Neglected Tropical Disease by the WHO, there is a hope that the development of much needed therapies against both snakebite and other animal envenomings will become increasingly incentivized for researchers worldwide. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments JB — passed away during the revision of this article. Because they do not want to waste the precious treatment, some doctors feel pressured to wait and see if a bite-victim shows symptoms of envenomation before administering antivenom.
However, the power of the treatment can be compromised by waiting. Although the World Health Organization includes snakebite antivenom on its List of Essential Medicines, the world is experiencing shortages of antivenom. The populations hardest hit by the shortages tend to live and work in rural areas where highly venomous snakes are endemic, especially in less-developed nations with housing that allows for easier access by venomous snakes.
Hospitals currently face a multifaceted antivenom problem. Antivenom can be very expensive, a problem that is compounded when the product goes unused before its expiration date. Many clinics do not have sufficient training in selecting the correct antivenom or administering the treatment. The challenges do not stop there: patients can suffer serious allergic reactions to antivenom, and medical supervision during treatment is important.
New monoclonal antibody antivenoms that cause fewer allergic reactions are being developed. However, because the CroFab product uses only a fragment of the cultured antibody, it causes fewer serious allergic reactions than older serum-based, whole antibody antivenoms. Antivenom is one of those treatments that most of us never think about—until we suddenly and very desperately need it.
Contemporary antivenoms made under strict controls are very effective. Yet, they remain out of reach for many victims who most need them. Refining scheme resulted in the completely pure, aggregate- and pepsin-free active principle with overall yield advantageously comparable to others so far reported.
Suitability for larger scale production, as well as estimation of its cost-effectivenes, should be determined through additional study, together with stability, pre-clinical and clinical efficacy of the final product prepared according to optimised procedure.
List of identified proteins is given in S1 Table. Proteins are denoted by numbers as in S1 Fig. Other protein spots were assigned based on PMF spectra overlapping or remained unidentified.
Abstract Antivenoms from hyperimmune animal plasma are the only specific pharmaceuticals against snakebites. Author summary Animal plasma-derived antivenoms constitute the most important therapy against snakebite envenoming. Introduction Antivenoms prepared from hyperimmune animal plasma, mostly equine or ovine, are the only specific therapeutics for rapid counteracting post-snakebite pathophysiological manifestations.
Snake venom, plasma pools, animals and reagents Crude venom of V. Pepsin digestion optimisation Preliminary optimisation of pepsin digestion was done using a model IgG substrate—highly pure IgG sample eIgG isolated from HHP by protein A based affinity chromatography. Diafiltration steps IgG-enriched supernatant following caprylic acid precipitation was diafiltrated into water or saline using Vivaspin device Sartorius, Germany with a kDa molecular weight cut-off MWCO polyethersulfone membrane.
Pepsin activity The enzymatic activity of pepsin was measured spectrophotometrically on Multiskan Spectrum instrument Thermo Fischer Scientific, USA using haemoglobin as substrate. Protein concentration determination Throughout the isolation procedure total protein concentration was estimated spectrophotometrically by use of the Eq 2 [ 32 ], 2 where Ehresmann's factor " f " for equine IgG of 0.
Production process yields and sample purity calculation Concentrations determined by ELISA assays were used for yield and purity calculations. Download: PPT. Fig 1.
Preliminary determination of optimal caprylic acid concentration for precipitation step of the purification protocol. Fig 2. The assessment of purification steps by size-exclusion chromatography. Table 1. Purities and yields of the intermediates and the final product obtained by developed downstream processing protocol.
Pepsin characterisation Commercial pepsin preparation involved in the manufacturing procedure had 7 times lower total protein concentration in comparison to the one derived from the weighted mass.
Fig 4. Verification of pepsin removal by the final polishing procedure. Optimisation of pepsin digestion Preliminary screening of digestion conditions. Pepsin digestion on IgG obtained by caprylic acid precipitation. Fig 6. Fig 7. Table 2. Characterisation of the unbound fractions following incubation of F ab 2 preparation containing pepsin with UNOsphere Q stationary phase under variuos pH conditions.
Final polishing step. Efficacy of flow-through chromatographic final polishing in pepsin removal. Protective efficacies of IgG and F ab' 2 preparations. Table 3. Discussion The production of immunotherapeutics has always been a struggle of finding balance between retaining the potency of the product and reducing the appearance of its side effect-inducing properties.
Fig 9. Flow sheet of downstream processing steps with corresponding samples and performance rationales. Supporting information. S1 Fig. S2 Fig. Presentation of purification strategy with main research activities, goals and outcomes.
S1 Table. List of proteins detected in the final F ab' 2 sample. References 1. World Health Organisation WHO Guidelines for the production control and regulation of snake antivenom immunoglobulins. Toxicon Afr J Biotechnol 9: View Article Google Scholar 7. Rojas G, Jimenez JM, Gutierrez JM Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production.
Arch Biochem Biophys Biologicals View Article Google Scholar Al-Abdulla I, Casewell NR, Landon J Long-term physicochemical and immunological stability of a liquid formulated intact ovine immunoglobulin-based antivenom.
Raweerith R, Ratanabanangkoon K Fractionation of equine antivenom using caprylic acid precipitation in combination with cationic ion-exchange chromatography. J Immunol Methods J Immun Methods J Biotechnol Clin Pharmacokinet Eur J Pharm Biopharm Guo J, Zhang S, Carta G Unfolding and aggregation of a glycosylated monoclonal antibody on a cation exchange column. Part I. Chromatographic elution and batch adsorption behavior. J Chromatogr A Guo J, Carta G Unfolding and aggregation of a glycosylated monoclonal antibody on a cation exchange column.
Part II. Protein structure effects by hydrogen deuterium exchange mass spectrometry. Ryle AP The porcine pepsins and pepsinogens. Methods Enzymol Anal Biochem J Pharmaceut Biomed These lifesaving products would then be more affordable to poor people and health authorities in developing countries where the highest incidences of snakebites occur 9 , Furthermore, pan-specific antivenoms with wide para specificity can be useful in cases where the culprit snake is not identified or captured, and consequently species identification of the snake cannot be made.
In this context, we have previously produced an experimental pan-specific equine antiserum that is capable of neutralizing 27 neurotoxic venoms from homologous and heterologous snake species inhabiting Asia and Africa. This should result in the production of antibodies with a variety of paratopes against the diverse toxin epitopes, and consequently, exhibit wide para-specificity.
These toxin fractions contained all the toxic components of the venoms, mostly presynaptic and postsynaptic neurotoxins and cytotoxins, but were devoid of the high molecular mass, highly immunogenic non-toxic proteins 11 , In the present study, we demonstrated that this pan-specific antiserum also neutralized nine additional neurotoxic venoms of elapids from Central America, Africa, and Australia, including sea snakes and sea kraits. Altogether, 36 neurotoxic venoms from 4 continents have been shown to be neutralized by the antiserum.
The 10 neurotoxic venoms hereby tested are shown in Table 1. The list includes venoms of the coral snake Micrurus nigrocinctus , the most medically important elapid in Central America, the yellow-lipped sea krait Laticauda colubrina , and the beaked sea snake Hydrophis schistosus distributed from Australian waters to the Arabian Sea. Other venoms tested include those of the tiger snake Notechis scutatus , the king brown snake Pseudechis australis and the coastal taipan Oxyuranus scutellatus , which are classified within WHO Category 1 most medically important snakes from Australia and Papua New Guinea.
In addition, neutralization of venoms of the African species black mamba Dendroaspis polylepis , the green mamba Dendroaspis angusticeps , the western green mamba Dendroaspis viridis , and the Senegalese cobra Naja senegalensis was assessed. From the median lethal dose LD 50 results, the coastal taipan O. Of the ten venoms studied, nine of them, including those from the two sea snakes, the Central American coral snake and the Australian snakes were cross-neutralized, and so were those of two African mambas D.
Only the green mamba D. The antiserum most effectively neutralized the venom of N. The P value of antiserum against the sea krait L. Thus the results showed that 9 out of ten neurotoxic venoms were neutralized by the pan-specific antiserum; only the venom of D. Table 2 depicts the major toxic components described for these venoms, with the exception of N. The proteomics toxin profiles show the major toxic lethal components of each of these 10 venoms. Neurotoxicity caused by P.
The toxins were present at very low level that probably explained its non-detection in the proteomic study 15 , and in our in vitro assay based on T. All nine venoms with available proteomic information contained phospholipases A 2 PLA 2 s , some of which are basic PLA 2 that contribute to presynaptic neurotoxicity in O. Besides, the myotoxic PLA 2 was also found abundantly in the sea snake H.
The lethal toxins present in the nine venoms were presumably neutralized by the pan-specific antiserum, as evidenced by neutralization results. As such, the pan specific antiserum was not tested for neutralization of these activities associated with high molecular mass components.
In addition, fasciculins, members of the 3FTx family which induce fasciculations by inhibiting acetylcholinesterase, were found in D.
Dendrotoxins, which have homology to Kunitz-type proteinase inhibitors and block voltage-dependent potassium channels, are typical of mamba venoms, with highest concentration in the venom of D. Both dendrotoxins and fasciculins were probably not neutralized by the pan-specific antiserum since these toxins are not present in the immunogen mix. This explains why the lethality of this venom was neutralized by the pan-specific antiserum even though the dendrotoxins were unlikely neutralized.
From its proteome, fourty-two different proteins were detected, among which 3FTxs were the most abundant, followed by the Kunitz-type proteinase inhibitor family. None of the venom HPLC fractions was lethal to mice at the doses tested. Thus, it was proposed that the lethality of the venom was due to the synergistic action of various components, such as fasciculins and dendrotoxins, and probably other synergistically-acting toxins It is not surprising that the pan-specific antiserum did not neutralize the lethal effects of the venom since the toxins of the venom were not present in the immunogen mix, and simultaneous neutralization of various synergistic acting toxins are required in order to neutralize the lethality of the venom.
The antiserum most likely contains antibodies against these components in this spitting cobra venom probably due to the presence of similar toxins in the venoms used in the immunizing mix. In the case of N. This venom was effectively neutralized by the pan-specific antiserum, underscoring that these lethal toxins were immunorecognized by the antibodies.
Nevertheless, caution should be exerted when extrapolating data from mouse experiments to the human situation when studying venom-induced neurotoxicity Table 3 shows the proteomics toxin profiles of the 16 heterologous venoms previously shown to be neutralized by the pan-specific antiserum.
Venoms from species of Bungarus , e. These toxins, except for cytotoxins, are highly lethal in mice and are known to be the cause of death in elapid envenomations. On the basis of our observations, they were likely neutralized by the antibodies in the pan-specific antiserum. Thus, the pan-specific antiserum neutralized most, if not all, the potentially lethal toxins in the 25 heterologous neurotoxic venoms tested, hence stressing the value of using fractions enriched with various lethal toxins of several venoms in the immunization process.
Table 4 presents the toxin profiles of these 12 venoms. Table 4 shows the number of isoforms of each type of these toxins in these 12 venoms. These large numbers of toxin isoforms are likely to contain numerous epitopes of these lethal toxins. It is therefore conceivable that the immune system of the horses generated a diverse set of paratopes against all the isoforms of these lethal toxins, hence explaining the ability of the antiserum to neutralize the lethal activity of 36 neurotoxic elapid venoms 25 heterologous and 11 homologous venoms from 10 snake genera.
It is evident that the experimental antiserum showed very wide para-specificity against numerous neurotoxic venoms. The following considerations may form the bases for explaining this phenomenon:. For some venoms, e. Bungarus spp. They adopt a planar structure similar to a 3-finger configuration and are referred to as three-finger toxins 3FTxs Thus all these toxins share structural and functional homology. Some of the epitopes from homologous toxins are conserved for structural and functional reasons.
Because of the high sequence identity, some of these epitopes are expected to be structurally similar, though not identical, and thus explain some degree of immunochemical cross reactivity of antisera 47 , 48 , This could be a reason for the low cross-neutralization of monospecific antivenoms usually observed 5. Moreover, it has been shown that a single antibody could adopt different conformations of its paratope to bind different epitopes, thus enhancing its antigenic coverage These interactions, albeit with lower affinity should, through cross-linking and lattice formation 52 , result in antisera with higher avidity leading to more effective neutralization of diverse heterologous neurotoxic venoms This is crucial because, due to steric hindrance, no more than two antibody molecules can interact simultaneously with one toxin molecule Supplementary Fig.
Whether or not the proposed bases for the wide para-specificity of our antiserum are correct, the results of these studies show that this is the widest cross-neutralizing antiserum ever reported against neurotoxic snake venoms from wide geographical distribution. Our results represent a proof of concept that an antiserum with wide spectrum of cross-neutralization against elapid venoms can be raised.
The genus-wide analysis of venom composition and toxicity of these venoms to identify the lethal toxins 24 followed by use of the combined toxin fractions to immunize horses, is likely to result in widely para-specific antiserum against these snake venoms. As shown in Tables 1 , 2 and 3 , the antiserum could neutralize lethality of 25 heterologous venoms, but its neutralizing potency against some of them is rather low.
This becomes a problem especially when dealing with species that inject a large volume of venom in a bite. However, the potency of the antiserum can be improved by a concentration process during plasma fractionation. Since horse hyperimmune sera have an average protein concentration at After such concentration process, the present horse pan-specific antiserum could have higher neutralizing activity against the lethality of many neurotoxic venoms.
This may not only increase the potency against the venoms tested, but also provide neutralization of additional elapid venoms. There is a growing interest in the development of recombinant antivenoms 55 , This involves, for example, the preparation of animal- or human-derived monoclonal antibodies against the lethal components of venoms. Proofs of concept of this strategy have been published 57 , One major requirement of this approach is that the major lethal toxin s of the venom must be identified and used for antibody selection.
When more than one toxin is relevant in a particular venom, there is a need to generate additional antibodies for a successful neutralization. Since these antibodies are produced against one or few toxins, a challenging issue for this strategy is to ensure the neutralization of heterologous toxins present in other venoms.
It should be possible to further increase the para-specificity of the antiserum by including additional venom toxin fractions in the immunization mix. For example, inclusion of toxin fractions of some African mamba venoms D. By carefully selecting the venoms and fractions to be added to the immunizing mix it should be possible to expand the scope of coverage of neurotoxic venoms, ideally to neutralize the most important elapid venoms in the world. One of the four pillars of this strategy is to ensure safe and effective treatments, particularly referring to antivenoms, which represent the only scientifically-validated therapy for these envenomings.
As shown in this work, a pan-specific antivenom against neurotoxic venoms would be a powerful therapeutic tool to save lives of people suffering these envenomings in different parts of the world, by neutralizing a wide spectrum of neurotoxic snake venoms which otherwise require region- or species-specific antivenoms for treatment.
The antiserum exhibited a wide para-specificity by neutralizing at least 36 neurotoxic venoms of snakes of 10 genera from four continents. The pool of diverse toxin antigens in the immunogen mix enabled the production of diverse antibody paratopes, which facilitate the interaction of the antibodies with the epitopes of various neurotoxins from homologous as well as heterologous snake venoms. Dendroaspis polylepis, D. Hydrophis schistosus venom was provided by Dr. CH Tan, and Micrurus nigrocinctus venom was provided by Prof.
These two venoms were obtained from several specimens kept in captivity M.
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