Rational design of rapidly translatable, highly antigenic and novel recombinant immunogens to address deficiencies of current snakebite treatments

背景：每年有13.8万人死于蛇咬伤，40万人致残。抗蛇毒血清（从混合毒液高度免疫的动物中纯化的IgG）是SBE唯一可靠的治疗方法，它是使用昂贵的、有百年历史的原始毒液免疫马/羊的方法制造的。目前的方案没有尝试在设计或制造过程中解释变异毒液蛋白的免疫原性或毒性。因此，抗蛇毒血清的剂量效力往往较差，这导致需要大量注射（在印度通常为200-400毫升）来中和病理，这往往导致严重的不良反应，并使已经贫困的受害者负担不起费用。此外，由于物种间毒液的多样性，原始毒液免疫导致治疗是蛇的物种特异性，导致医生不得不做出困难的诊断和抗蛇毒血清选择的决定，当冒犯蛇的种类是未知的。因此，迫切需要大幅提高抗蛇毒血清治疗的毒液中和范围和疗效。理由：用于治疗神经毒性中毒（一种通常导致快速致命呼吸麻痹的常见全球病理）的抗蛇毒血清效果特别弱，因为用于免疫的毒液中的神经毒素免疫原性弱且多样性大。然而，尽管存在这种多样性，毒素序列数据集的检查表明，神经毒素具有普遍保守的特征。该项目将用合理设计的合成颗粒取代抗蛇毒血清生产中使用的粗制神经毒素，这些合成颗粒只显示神经毒素的保守区域。通过将免疫反应集中在所有神经毒性sSA蛇的毒液中保存的最重要的病理毒素区域，我期望产生一种抗蛇毒血清，它(I)能够中和整个sSA的神经毒性蛇毒，无论物种如何；（ii）高效，从而减少治疗所需的抗蛇毒血清剂量，提高安全性。方法：1首先，我将通过计算和实验研究来自sSA最重要的医学蛇的神经毒素编码序列，以确定所有神经毒素中保守的区域确定的区域随后将被设计用于高免疫原性抗原递送载体（ADVs），如病毒样颗粒（vlp）或Fc融合物，它们具有固有的免疫系统调节特性。每种方法都可以很容易地显示外来抗原，因此可以有效地显示和增强免疫系统对已确定的保守神经毒素区域的识别我将通过免疫小鼠来测试这些方法，以确定显示神经毒素抗原的ADVs的最佳配置，通过检查(I)免疫反应的程度和（ii）使用体外试验产生的抗体阻止神经毒素活性的能力来确定。显示神经毒素抗原的两种最佳ADVs结构将用于免疫抗蛇毒血清制造动物（羊）以产生实验性抗蛇毒血清。最后，我将在体内中和致死率研究之前，在体外证明实验性羊生成的抗蛇毒血清的优越功效。通过这些临床前小鼠研究，我将确定与现有的商业、原始蛇毒生产的抗蛇毒血清相比，ADV生成的抗蛇毒血清是否具有更好的毒液中和潜力。影响：由于该项目只会改进初始免疫材料，下游抗蛇毒血清制造工艺或产品配方不会发生变化，我预计av生成的抗蛇毒血清将不需要广泛的监管批准。这将使积极结果迅速转化为临床试验，并在中短期内立即减轻SBE负担。

Background: Snakebite envenomation (SBE) kills 138,000 and maims >400,000 people annually. Antivenom (IgG purified from animals hyper-immunized with mixtures of venoms) is the only assured therapy for SBE, is manufactured using expensive, century-old protocols of immunizing horses/sheep with crude venoms. Current protocols make no attempt to account for variant venom protein immunogenicity or toxicity during design or manufacture. Consequently, antivenoms often have poor dose-efficacy, which results in the administration of large volumes (often 200-400 ml in India) to neutralize pathology, often leading to severe adverse reactions and unaffordable costs for already impoverished victims. Furthermore, due to inter-species venom diversity, crude venom immunisation results in therapy that is snake species-specific, resulting in physicians having to make difficult diagnostic and antivenom-selection decisions when the offending snake species is unknown. There is therefore an urgent and compelling need to drastically improve the venom-neutralizing scope and efficacy of antivenom therapy. Rationale: Antivenoms for treating neurotoxic envenoming (a common global pathology often resulting in rapid fatal respiratory paralysis) are especially weakly-effective because of the weak immunogenicity and large diversity of the neurotoxins in the venoms used for immunisation. However, despite this diversity, examination of toxin sequence datasets demonstrates that neurotoxins possess commonly conserved features. This project will replace the use of crude neurotoxic venoms in antivenom manufacture with rationally engineered, synthetic particles displaying only the conserved regions of neurotoxins. By focusing the immune response to regions of only the most pathology-important toxins that are conserved in venoms of all the neurotoxic sSA snakes, I anticipate generating an antivenom which is (i) able to neutralize neurotoxic snake envenoming throughout sSA, regardless of species, and (ii) highly potent, resulting in smaller antivenom doses being needed to effect cure and improved safety. Approach: 1 First, I will computationally and experimentally investigate sequences encoding neurotoxins from the most medically important snakes of sSA to identify regions that are conserved among all neurotoxins.2 Identified regions will then be engineered for display on highly immunogenic antigen delivery vehicles (ADVs) such as Virus Like Particles (VLPs) or Fc fusions, which have inherent immune system modulating characteristics. Each approach can be easily manipulated to display foreign antigens, therefore allowing efficient display and enhanced recognition of the identified conserved neurotoxin regions by the immune system.3 I will test these approaches by immunizing mice to identify optimal configurations of ADVs displaying neurotoxin antigens, determined by examining (i) the extent of immune-responses and (ii) the ability of the antibodies generated to prevent neurotoxin activity using in vitro assays. The two optimal configurations of ADVs displaying neurotoxin antigens will then be used to immunise antivenom manufacturing animals (sheep) to generate experimental antivenom. 4 Finally, I will demonstrate the superior efficacy of the experimental sheep-generated antivenom in vitro, prior to in vivo neutralisation of lethality studies. Through these pre-clinical murine studies, I will determine whether the ADV generated antivenom exhibits superior venom neutralisation potential compared to existing commercial, crude venom produced antivenoms. Implications: As this project will improve the initial immunizing material only, with no changes to downstream antivenom manufacturing processes or product formulation, I anticipate that ADV-generated antivenoms will not require extensive regulatory approval. This will allow rapid translation of positive results into clinical trials and an immediate reduction in SBE burden in the short to medium term.