Novel immuno-proteomic strategies to develop a polyspecific, non-cold chain liquid snake antivenom with unparalleled sub-Saharan African efficacy

新型免疫蛋白质组学策略，用于开发具有无与伦比的撒哈拉以南非洲功效的多特异性、非冷链液体蛇抗蛇毒血清

• 批准号: MR/L01839X/1
• 负责人: Robert Harrison
• 金额: $ 84.81万
• 依托单位: Liverpool School of Tropical Medicine
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FL01839X%2F1
• 关键词: Novel; immuno; proteomic; strategies; develop

Snakebite victims in Sub-Saharan Africa are therapeutically neglected (~32,000 deaths; 96,000 disabled yearly) because of the weak efficacy of current polyspecific antivenoms. In areas like Britain with a single venomous snake species, monospecific antivenom (antibodies (abys) purified from the blood of horses/sheep immunised with a single snake venom) are typically highly effective and safe. However, there are several venomous snake groups in sub-Saharan Africa, and polyspecific antivenoms, manufactured with venoms from several snakes, are therefore clinically preferred.However, using many venoms for immunisation negatively impacts upon the efficacy of polyspecific antivenoms. Thus, the greater the genetic difference between the snakes whose venoms are used in antivenom manufacture, the more numerous and diverse the venom proteins (100+ proteins/venom), and the greater the number of distinct abys generated in the venom-immunised animals. This means that the proportion of total abys targeting the venom of any one snake is small - and consequently more vials of polyspecific antivenom are needed to achieve clinical cure. This significantly increases antivenom-induced adverse effects and often makes treatment unaffordable to the impoverished communities at greatest risk. There is therefore an urgent, compelling need for research to resolve this therapy deficit.We will use an innovative strategy that greatly expands antivenom snake-species efficacy. Thus, we first add a 'base' antivenom, derived from an existing product that is safe and affordable but exhibits inadequate polyspecific efficacy, onto a laboratory matrix (a chromatography column). We next individually add venoms from all regional medically-important snakes to the column, and all proteins that are not bound by the antivenom are collected and their identity determined. Those found to be pathogenic (many venom proteins are not toxic) are isolated in sufficient amounts for immunisation and mixed with the venoms used to prepare the base antivenom. A new group of sheep is then immunised with the 'venom+supplement toxins' mixture to rationally generate antivenom with greatly expanded snake-species efficacy. We will use this approach to first generate new antivenoms to treat either the (i) tissue destruction & bleeding, or the (ii) paralysis pathology syndromes: thereby covering the effects of envenoming by all medically-important sub-Saharan African snakes. We will use a suite of in vitro & in vivo tests to confirm that the two new antivenoms bind the venom proteins causing these pathologies and neutralise their lethal and tissue-destructive effects in mice, and compare these results with that of the base and existing polyspecific antivenoms. With this information as a guide, we will next develop a single pan-African antivenom using the first syndromic antivenom as the base to isolate non-binding proteins from all the African snake venoms for 'venom+supplement' immunisation of a new group of sheep. We will also test the efficacy of an alternate pan-African antivenom created by mixing abys of the two syndromic antivenoms in different ratios, and efficacy-testing the ratio that exhibits maximal binding of proteins from the African snake venoms.We will also develop/test protocols enabling non-cold chain storage of liquid antivenom - greatly expanding the distribution potential of these improved antivenoms. We will also develop/test protocols to increase the amount and binding strength of antivenom abys to highly toxic venom proteins that are poor at stimulating potent aby responses.Here, in the only research of its kind, we will deliver new non-cold chain antivenom with pan-African efficacy, and with an unparalleled dose-efficacy ensuring the product poses little adverse-effect risk and is affordable. These antivenoms will represent the most cost-effective investment in snakebite management ever available to governments in sub-Saharan Africa, and elsewhere.

由于目前多特异性抗蛇毒血清的功效较弱，撒哈拉以南非洲地区的蛇咬伤受害者在治疗上受到忽视（每年约 32,000 人死亡；96,000 人致残）。在像英国这样只有单一毒蛇物种的地区，单特异性抗蛇毒血清（从用单一蛇毒免疫的马/羊的血液中纯化出来的抗体（abys））通常非常有效和安全。然而，撒哈拉以南非洲有多个毒蛇类群，因此临床上优选用多种蛇的毒液制成的多特异性抗蛇毒血清。然而，使用多种毒液进行免疫会对多特异性抗蛇毒血清的功效产生负面影响。因此，用于制造抗蛇毒血清的蛇之间的遗传差异越大，毒液蛋白质的数量和多样性就越多（100+蛋白质/毒液），并且在毒液免疫动物中产生的不同深渊的数量也就越多。这意味着针对任何一种蛇的毒液的总深渊比例很小，因此需要更多瓶多特异性抗蛇毒血清才能实现临床治愈。这显着增加了抗蛇毒血清引起的不良反应，并且常常使风险最大的贫困社区无力承担治疗费用。因此，迫切需要进行研究来解决这种治疗缺陷。我们将使用一种创新策略，大大扩展抗蛇毒血清的功效。因此，我们首先将一种“基础”抗蛇毒血清添加到实验室基质（色谱柱）上，该“基础”抗蛇毒血清源自安全且价格实惠但多特异性功效不足的现有产品。接下来，我们将所有区域性医学上重要的蛇的毒液单独添加到柱中，收集所有未与抗蛇毒血清结合的蛋白质并确定其身份。那些被发现具有致病性的毒液蛋白（许多毒液蛋白无毒）被分离出足够的量用于免疫，并与用于制备基础抗蛇毒血清的毒液混合。然后用“毒液+补充毒素”混合物对一组新的羊进行免疫，以合理地产生抗蛇毒血清，从而大大扩展了蛇种的功效。我们将使用这种方法首先产生新的抗蛇毒血清来治疗（i）组织破坏和出血，或（ii）麻痹病理综合征：从而涵盖所有具有医学重要性的撒哈拉以南非洲蛇的毒液影响。我们将使用一系列体外和体内测试来确认这两种新的抗蛇毒血清与引起这些病理的毒液蛋白结合并中和它们对小鼠的致命和组织破坏作用，并将这些结果与基础和现有多特异性的结果进行比较抗蛇毒血清。以此信息为指导，我们接下来将开发一种单一的泛非洲抗蛇毒血清，以第一种综合症抗蛇毒血清为基础，从所有非洲蛇毒中分离出非结合蛋白，用于对一组新的羊进行“毒液+补充剂”免疫。我们还将测试通过以不同比例混合两种综合症抗蛇毒血清而创建的替代泛非洲抗蛇毒血清的功效，并测试与非洲蛇毒蛋白表现出最大结合的比例。我们还将开发/测试协议能够实现液体抗蛇毒血清的非冷链存储——极大地扩展了这些改进的抗蛇毒血清的分销潜力。我们还将开发/测试方案，以增加抗蛇毒血清 abys 的数量和与高毒性毒液蛋白的结合强度，这些蛋白不能刺激有效的 aby 反应。在这里，在此类唯一的研究中，我们将提供新的非冷链抗蛇毒血清具有泛非洲功效，并且具有无与伦比的剂量功效，确保该产品几乎没有不良反应风险并且价格实惠。这些抗蛇毒血清将成为撒哈拉以南非洲和其他地区政府迄今为止在蛇咬伤管理方面最具成本效益的投资。

项目成果

The paraspecific neutralisation of snake venom induced coagulopathy by antivenoms.

• DOI: 10.1038/s42003-018-0039-1
• 发表时间: 2018
• 期刊: Communications biology
• 影响因子: 5.9
• 作者: Ainsworth S;Slagboom J;Alomran N;Pla D;Alhamdi Y;King SI;Bolton FMS;Gutiérrez JM;Vonk FJ;Toh CH;Calvete JJ;Kool J;Harrison RA;Casewell NR
• 通讯作者: Casewell NR

Incorporating the 3Rs (Refinement, Replacement and Reduction of animals in research) into the preclinical assessment of snake venom toxicity and antivenom efficacy

将 3R（研究中动物的改进、替换和减少）纳入蛇毒毒性和抗蛇毒血清功效的临床前评估

• 发表时间: 2017
• 作者: Bolton F. M.