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

背景：毒蛇咬伤(SBE)每年导致13.8万人死亡，40万人致残。抗蛇毒血清(从用混合毒液高度免疫的动物中提纯的免疫球蛋白)是治疗SBE的唯一可靠的疗法，是使用昂贵的百年历史的粗毒免疫马/羊的方案生产的。目前的方案没有试图解释变异的毒液蛋白在设计或制造过程中的免疫原性或毒性。因此，抗蛇毒血清的剂量效应往往很差，这导致大量使用(在印度通常为200-400毫升)来中和病理，往往导致严重的不良反应和本已贫穷的受害者无法负担的费用。此外，由于物种间的毒液多样性，粗制毒液免疫导致针对蛇种的治疗，导致医生在不知道违规蛇种时做出困难的诊断和抗蛇毒选择决定。因此，迫切需要大幅提高抗蛇毒疗法的中和毒液范围和疗效。基本原理：用于治疗神经毒性毒液(一种常见的全球病理，通常会导致快速致命的呼吸麻痹)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较弱，且种类繁多。然而，尽管有这种多样性，对毒素序列数据集的检查表明，神经毒素具有共同的保守特征。该项目将用仅显示神经毒素保守区域的合理设计的合成颗粒来取代抗蛇毒血清生产中使用的粗神经毒性毒液。通过将免疫反应集中在所有神经毒性SSA蛇的毒液中保守的最重要的病理毒素区域，我预计将产生一种抗蛇毒血清，它(I)能够中和整个SSA中的神经毒性蛇毒，无论是哪种，(Ii)高度有效，导致需要较小的抗蛇毒血清剂量才能达到治愈和提高安全性的目的。方法：1首先，我将通过计算和实验研究SSA中最具医学重要性的蛇类神经毒素的编码序列，以确定在所有神经毒素中保守的区域。2然后，识别的区域将被设计用于展示在高免疫原性抗原递送载体(ADV)上，如病毒样颗粒(VLP)或FC融合，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展示外来抗原，因此可以有效地展示和增强免疫系统对已识别的保守神经毒素区域的识别。3我将通过免疫小鼠来测试这些方法，以确定展示神经毒素抗原的ADV的最佳配置，方法是检查(I)免疫反应的程度和(Ii)产生的抗体使用体外分析防止神经毒素活性的能力。然后，展示神经毒素抗原的两种最佳ADV配置将被用于免疫生产抗蛇毒血清的动物(绵羊)，以产生实验性抗蛇毒血清。4最后，我将在体内中和致死性研究之前，在体外证明实验性绵羊产生的抗蛇毒血清的优越效果。通过这些临床前小鼠研究，我将确定ADV产生的抗蛇毒血清是否显示出比现有商业粗制抗蛇毒血清更好的毒液中和潜力。影响：由于该项目将只改进最初的免疫材料，不改变下游抗蛇毒血清的制造工艺或产品配方，我预计ADV产生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.