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

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人致残），因为目前的多特异性抗蛇毒血清效果不佳。在像英国这样只有一种毒蛇的地区，单特异性抗蛇毒血清（从用一种蛇毒免疫的马/羊的血液中纯化出来的抗体）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，因此临床更倾向于使用由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，用于制造抗蛇毒血清的蛇毒之间的遗传差异越大，蛇毒蛋白的数量和种类就越多（100多个蛋白质/毒液），并且在毒液免疫的动物中产生的不同的幼虫数量也越多。这意味着针对任何一种蛇的毒液的总抗体比例很小，因此需要更多的多特异性抗蛇毒血清来实现临床治愈。这大大增加了抗蛇毒血清引起的不良反应，并往往使面临最大风险的贫困社区负担不起治疗费用。因此，迫切需要研究来解决这一治疗缺陷。我们将采用一种创新的策略，极大地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，该抗蛇毒血清来源于一种安全且价格合理的现有产品，但其多特异性功效不足。接下来，我们分别将所有在该地区具有重要医学意义的蛇的毒液添加到这一栏中，并收集所有未与抗蛇毒血清结合的蛋白质，并确定它们的身份。那些被发现具有致病性的（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”的混合物对一组新的羊进行免疫，以合理地产生抗蛇毒血清，大大扩展了蛇类的功效。我们将使用这种方法首先生产新的抗蛇毒血清，以治疗(i)组织破坏和出血，或（ii）瘫痪病理综合征：从而覆盖所有具有重要医学意义的撒哈拉以南非洲蛇的毒害影响。我们将使用一套体外和体内试验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，并在小鼠中中和它们的致命和组织破坏性作用，并将这些结果与基础抗蛇毒血清和现有多特异性抗蛇毒血清的结果进行比较。有了这些信息作为指导，我们下一步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，用于对一组新绵羊进行“毒液+补充”免疫。我们还将测试另一种泛非洲抗蛇毒血清的功效，这种抗蛇毒血清是由两种综合征抗蛇毒血清以不同比例混合而成的，并测试非洲蛇毒蛋白质结合最大的比例的功效。我们还将制定/测试能够非冷链储存液体抗蛇毒血清的方案，从而大大扩大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，以增加抗蛇毒血清与高毒性蛇毒蛋白的数量和结合强度，这些蛋白在刺激婴儿有效反应方面表现不佳。在此，在同类研究中，我们将提供具有泛非洲功效的新型非冷链抗蛇毒血清，并具有无与伦比的剂量功效，确保产品产生的不良反应风险很小，而且价格合理。这些抗蛇毒血清将是撒哈拉以南非洲和其他地区政府迄今可获得的最具成本效益的蛇咬伤管理投资。

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.