Exploring the utility of recombinantly expressed snake venom serine protease toxins as immunogens for generating experimental snakebite antivenoms
Exploring the utility of recombinantly expressed snake venom serine protease toxins as immunogens for generating experimental snakebite antivenoms
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DOI：
10.1101/2022.05.07.491032
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发表时间：
2022-05
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通讯作者：
Nessrin Alomran;P. Blundell;Jaffer Alsolaiss;E. Crittenden;S. Ainsworth;Charlotte A. Dawson;Rebecca J. Edge;Steven R. Hall;R. Harrison;M. Wilkinson;S. Menzies;N. Casewell
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作者：
Nessrin Alomran;P. Blundell;Jaffer Alsolaiss;E. Crittenden;S. Ainsworth;Charlotte A. Dawson;Rebecca J. Edge;Steven R. Hall;R. Harrison;M. Wilkinson;S. Menzies;N. Casewell
蛇咬伤是一种被忽视的热带疾病，导致全球高死亡率和发病率。虽然蛇咬伤可导致受害者的各种病理，但血液毒性效应特别常见，通常以出血和/或毒液诱导的消耗性凝血病为特征。尽管基于多克隆抗体的抗蛇毒血清是蛇咬伤的主要救生疗法，但它们与有限的跨蛇物种功效相关，因为蛇毒之间通常存在广泛的毒素变异，包括用作抗蛇毒血清生产的免疫原的那些。这限制了任何抗蛇毒血清在某些地理区域的治疗效用。在这项研究中，我们探讨了使用重组表达的毒素作为免疫原刺激集中，病理特异性，抗体广泛抵消特定毒素与蛇咬伤envenoming的可行性。三种蛇毒丝氨酸蛋白酶（SVSP）毒素，来源于地理上不同的和医学上重要的蝰蛇蛇毒，成功地表达在HEK 293 F哺乳动物细胞和用于小鼠免疫。对所产生的抗体反应的分析显示，安克洛和RVV-V刺激最强的免疫反应，并且针对这些重组SVSP毒素的实验性抗蛇毒血清，以及三种不同免疫原的混合物，广泛识别并表现出对各种天然蛇毒的免疫结合。虽然实验性抗蛇毒血清在毒素免疫原和粗毒液刺激的异常凝血参数中显示出一定程度的减少，特别是减少了纤维蛋白原水平的消耗和凝血酶原时间的延长，但纤维蛋白原降解实验显示它们广泛地保护了毒液和毒素诱导的纤维蛋白原溶解功能活性。总的来说，我们的研究结果进一步加强了使用重组毒液毒素作为补充免疫原的情况下，刺激集中和理想的抗体反应，能够中和毒液诱导的病理作用，因此可能会规避一些限制与目前的蛇咬伤治疗。
Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies.