Bioinformatics and Multiepitope DNA Immunization to Design Rational Snake Antivenom

Abstract and Introduction

Background: Snake venom is a potentially lethal and complex mixture of hundreds of functionally diverse proteins that are difficult to purify and hence difficult to characterize. These difficulties have inhibited the development of toxin-targeted therapy, and conventional antivenom is still generated from the sera of horses or sheep immunized with whole venom. Although life-saving, antivenoms contain an immunoglobulin pool of unknown antigen specificity and known redundancy, which necessitates the delivery of large volumes of heterologous immunoglobulin to the envenomed victim, thus increasing the risk of anaphylactoid and serum sickness adverse effects. Here we exploit recent molecular sequence analysis and DNA immunization tools to design more rational toxin-targeted antivenom.
Methods and Findings: We developed a novel bioinformatic strategy that identified sequences encoding immunogenic and structurally significant epitopes from an expressed sequence tag database of a venom gland cDNA library of Echis ocellatus, the most medically important viper in Africa. Focusing upon snake venom metalloproteinases (SVMPs) that are responsible for the severe and frequently lethal hemorrhage in envenomed victims, we identified seven epitopes that we predicted would be represented in all isomers of this multimeric toxin and that we engineered into a single synthetic multiepitope DNA immunogen (epitope string). We compared the specificity and toxin-neutralizing efficacy of antiserum raised against the string to antisera raised against a single SVMP toxin (or domains) or antiserum raised by conventional (whole venom) immunization protocols. The SVMP string antiserum, as predicted in silico, contained antibody specificities to numerous SVMPs in E. ocellatus venom and venoms of several other African vipers. More significantly, the antiserum cross-specifically neutralized hemorrhage induced by E. ocellatus and Cerastes cerastes cerastes venoms.
Conclusions. These data provide valuable sequence and structure/function information of viper venom hemorrhagins but, more importantly, a new opportunity to design toxin-specific antivenoms—the first major conceptual change in antivenom design after more than a century of production. Furthermore, this approach may be adapted to immunotherapy design in other cases where targets are numerous, diverse, and poorly characterized such as those generated by hypermutation or antigenic variation.

Random sequencing of expressed genes (ESTs) often provides a rapid and affordable opportunity to gain a comprehensive insight into the biochemical complexities underlying many biological processes. Identifying the most clinically important toxins in venoms to inform the immunotherapeutic treatment of snake bite exemplifies this approach. Snake venom typically contains over 100 inter- and intraspecifically diverse proteins of varying toxicity that are difficult to purify in sufficient amounts for immunization and are often very poorly characterized at the protein sequence and functional levels. Whilst the increasing volume of genomic and transcriptomic data are undoubtedly beneficial here, our current ability to bioinformatically transform these data into novel therapies^[1] is not well developed.

Conventional snake antivenom remains the only effective treatment of snake envenoming, but because of the above problems, current immunization protocols make no attempt to target the immune responses to the most clinically important toxins, but involve hyperimmunization of horses or sheep with whole venom. Variation in representation, immunogenicity, and toxicity of venom proteins means that (i) not all conventional antivenoms contain antibodies to important toxins,^[2] and (ii) dose-efficacy is reduced by antibodies to nontoxic components.^[3,4] This necessitates the administration of large volumes (30 to > 300 ml at 80 mg protein/ml) of antivenoms that increase the risk of early anaphylactoid and late serum sickness adverse reactions.^[5] A systematic approach to select immunoprotective sequences for immunization using molecular sequence data alone would generate novel polyspecific antivenoms of high avidity, thus increasing dose efficacy and reducing toxicity to envenomed victims.

DNA immunization offers a rational approach to the design of toxin-specific immunotherapy, which we have previously demonstrated to induce titres and protective responses appropriate for antivenom production^[6,7] and generate antisera cross-reactive with venoms from phylogenetically distinct viper species and genera.^[8] In light of the above and the sequence conservation of venom toxins, we proposed that a systematic approach to identifying common antigenic epitopes of venom toxins from venom gland EST databases would provide a rational approach to generating interspecific or intergeneric protective antibody responses satisfying the most desirable polyspecific properties of an antivenom.

As proof of principle, a comprehensive EST survey from the venom glands of E. ocellatus, the most medically important snake in West Africa, was undertaken and combined with a bioinformatic approach to identify epitopes of key structural or functional significance. We focused upon the group of snake venom metalloproteinases (SVMPs) responsible for the main, frequently lethal hemorrhagic effect of viper envenoming.^[9] SVMPs are complex targets due to their multifunctional, multidomain nature and broad substrate specificity. SVMPs transcripts are classified by precursors encoding the metalloproteinase domain alone (class PI), metalloproteinase and C-terminal disintegrin domain (class PII), or disintegrin-like and cysteine-rich domains (class PIII).^[10,11] Additional and extensive post-translational diversity of PII and PIII SVMPs also provide a reservoir in whole venom for a diverse array of processed disintegrins that interfere with normal mechanisms of tissue repair and hemostasis.^[12,13] Although their principal effect is the disruption of microvessel architecture by degradation of basement membrane components,^[14] this diversity endows SVMPs with a multiplicity of functions that are difficult to predict from transcriptional data alone. These functions include exacerbation of systemic bleeding by fibrinolytic activities, cleavage (and thus consumption) of coagulation factors, and disruption of platelet aggregation resulting in a variety of tissue-disruptive,^[14] coagulopathic, and haemostasis-disruptive mechanisms^{[15,16,17,18,19]} that contribute to the overall hemorrhagic pathology. Despite determination of the solution structures of several PI and PII SVMPs, the lack of structural information for PIII SVMPs leaves many questions concerning the structure/function relationships unanswered, particularly mechanisms of dimerization^[20,21] and their covalent addition to a C-type lectin domains generating the PIV isoforms.^[22,23]

Here we describe for the first time (i) the design of a synthetic DNA immunization construct (EoSVMP string) containing a string of SVMP epitopes represented across numerous and diverse SVMP isoforms, (ii) the cross-generic and cross-specific antibody responses to multiple SVMPs induced by the EoSVMP string DNA immunogen, and (iii) the in vivo neutralization of venom-induced hemorrhage by the anti-EoSVMP string antibody.

PLoS Med. 2006;3(6):832-844. © 2006 Tang et al. licensee Public Library of Science

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Cite this: Bioinformatics and Multiepitope DNA Immunization to Design Rational Snake Antivenom - Medscape - Jun 01, 2006.