Wagstaff Simon C, Laing Gavin D, Theakston R David G, Papaspyridis Christina, Harrison Robert A
Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, United Kingdom.
PLoS Med. 2006 Jun;3(6):e184. doi: 10.1371/journal.pmed.0030184.
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.
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.
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.
蛇毒是一种潜在致命且复杂的混合物，包含数百种功能各异的蛋白质，难以纯化，因此难以进行特性描述。这些困难阻碍了毒素靶向疗法的发展，传统抗蛇毒血清仍由用全毒液免疫的马或羊的血清制备。尽管抗蛇毒血清能挽救生命，但其包含的免疫球蛋白库具有未知的抗原特异性和已知的冗余性，这就需要向中毒者大量输送异源免疫球蛋白，从而增加了类过敏反应和血清病不良反应的风险。在此，我们利用最近的分子序列分析和DNA免疫工具来设计更合理的毒素靶向抗蛇毒血清。
我们开发了一种新的生物信息学策略，从非洲医学上最重要的蝰蛇——锯鳞蝰毒液腺cDNA文库的表达序列标签数据库中鉴定出编码免疫原性和结构重要表位的序列。聚焦于导致中毒者严重且常致命出血的蛇毒金属蛋白酶（SVMPs），我们鉴定出七个表位，预测这些表位存在于这种多聚体毒素的所有异构体中，并将其设计成单一的合成多表位DNA免疫原（表位串）。我们将针对该表位串产生的抗血清的特异性和毒素中和效力与针对单一SVMP毒素（或结构域）产生的抗血清或通过传统（全毒液）免疫方案产生的抗血清进行了比较。如计算机模拟预测的那样，SVMP表位串抗血清包含对锯鳞蝰毒液及其他几种非洲蝰蛇毒液中多种SVMP的抗体特异性。更重要的是，该抗血清能交叉特异性中和锯鳞蝰和角蝰毒液诱导的出血。
这些数据提供了蝰蛇毒出血毒素的有价值的序列和结构/功能信息，但更重要的是提供了设计毒素特异性抗蛇毒血清的新机会——这是一个多世纪以来抗蛇毒血清设计的首次重大概念性变革。此外，这种方法可能适用于其他靶点众多、多样且特性不佳的情况的免疫治疗设计，例如由高突变或抗原变异产生的靶点。
