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(约32,000人死亡，每年96,000人残疾)，因为目前的多特异性抗蛇毒血清效力较弱。在像英国这样只有一种毒蛇的地区，单特异性抗蛇毒(从用一种蛇毒免疫的马/羊的血液中提纯的抗体(ABY))通常是高效和安全的。然而，撒哈拉以南非洲有几个毒蛇群体，因此临床上首选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会对多特异性抗蛇毒血清的效果产生负面影响。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(100+蛋白质/毒液)的数量和多样性就越多，在毒液免疫的动物中产生的不同ABY的数量就越多。这意味着针对任何一种蛇的毒液的总ABY的比例很小-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，迫切需要研究解决这种疗法的缺陷。我们将使用一种创新的策略，极大地扩大抗蛇毒蛇种的疗效。因此，我们首先在实验室基质(色谱柱)上添加一种来自现有产品的“基本”抗蛇毒血清，该产品安全且负担得起，但表现出不充分的多特异性效力。接下来，我们将来自所有具有重要医学意义的地区性蛇的毒液单独添加到柱子中，然后收集所有未被抗蛇毒血清结合的蛋白质，并确定它们的身份。那些被发现是致病的(许多毒液蛋白是无毒的)被分离出足够数量的疫苗，并与用于制备基础抗蛇毒血清的毒液混合。然后用“蛇毒+补充毒素”混合物免疫一组新的绵羊，以合理地产生抗蛇毒血清，极大地扩大蛇类的效力。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：从而覆盖所有具有重要医学意义的撒哈拉以南非洲蛇的蛇毒效应。我们将使用一套体外和体内测试来证实这两种新的抗蛇毒蛋白结合了导致这些病理的毒液蛋白，并中和了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的多特异性抗蛇毒血清进行比较。在这些信息的指导下，我们接下来将开发一种单一的泛非洲抗蛇毒血清，以第一个综合征抗蛇毒血清为基础，从所有非洲蛇毒中分离出非结合蛋白，用于对一组新的绵羊进行毒液+补充免疫。我们还将测试由两种综合征抗蛇毒血清以不同比例混合而成的替代泛非抗蛇毒血清的效力，并对表现出非洲蛇毒蛋白最大结合的比率进行效力测试。我们还将开发/测试能够以非冷链存储液体抗蛇毒血清的方案-极大地扩大这些改进的抗蛇毒血清的分布潜力。我们还将开发/测试方案，以增加抗蛇毒ABY的数量和结合强度的高毒性蛇毒蛋白，在刺激强大的Aby反应。在这里，在同类唯一的研究，我们将提供新的非冷链抗蛇毒血清泛非洲的功效，并具有无与伦比的剂量-功效，确保该产品构成的不良反应风险很小，而且是负担得起的。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进行的最具成本效益的投资。

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.