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.

撒哈拉以南非洲的蛇咬伤受害者在治疗上被忽视（每年约32，000人死亡; 96，000人残疾），因为目前的多特异性抗蛇毒血清疗效较弱。在像英国这样只有一种毒蛇的地区，单特异性抗蛇毒血清（从用单一蛇毒免疫的马/羊的血液中纯化的抗体（abys））通常是非常有效和安全的。然而，在撒哈拉以南非洲有几种毒蛇，因此临床上首选用几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接种会对多特异性抗蛇毒血清的功效产生负面影响。因此，其毒液用于抗蛇毒血清生产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），并且在毒液免疫的动物中产生的不同abys的数量就越多。这意味着，针对任何一种蛇的毒液的总阿比的比例很小-因此需要更多的多特异性抗蛇毒血清小瓶来实现临床治愈。这大大增加了抗蛇毒血清引起的不良反应，往往使风险最大的贫困社区负担不起治疗费用。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对蛇类的功效。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，该抗蛇毒血清来源于现有的产品，该产品安全且价格合理，但表现出不充分的多特异性功效。接下来，我们将来自所有地区医学上重要的蛇的毒液单独添加到柱中，收集所有不被抗蛇毒血清结合的蛋白质并确定其身份。那些被发现是致病的（许多毒液蛋白是无毒的）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”混合物免疫一组新的绵羊，以合理地产生具有大大扩展的蛇种效力的抗蛇毒血清。我们将使用这种方法首先生产新的抗蛇毒血清来治疗（i）组织破坏和出血，或（ii）瘫痪病理综合征：从而涵盖所有医学上重要的撒哈拉以南非洲蛇的毒液中毒影响。我们将使用一套体外和体内试验来证实这两种新的抗蛇毒血清结合引起这些病理的毒液蛋白，并中和它们在小鼠中的致死和组织破坏作用，并将这些结果与基础和现有的多特异性抗蛇毒血清进行比较。有了这些信息作为指导，我们接下来将开发一种单一的泛非洲抗蛇毒血清，使用第一种综合征抗蛇毒血清作为基础，从所有非洲蛇毒中分离出非结合蛋白，用于一组新绵羊的“毒液+补充剂”免疫。我们还将测试一种替代泛非洲抗蛇毒血清的效力，这种抗蛇毒血清是将两种综合征抗蛇毒血清以不同比例混合而成的，并对表现出最大结合非洲蛇毒蛋白的比例进行效力测试。我们还将开发/测试能够实现液体抗蛇毒血清非冷链储存的方案，从而极大地扩大这些改良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的数量和结合强度，以刺激强有力的aby反应。在这里，在同类研究中，我们将提供新的非冷链抗蛇毒血清，具有泛非洲功效，并具有无与伦比的剂量功效，确保产品几乎没有不良反应风险，并且价格合理。这些抗蛇毒血清将是撒哈拉以南非洲和其他地区政府有史以来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.