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

合理设计可快速翻译、高抗原性和新型重组免疫原，以解决当前蛇咬伤治疗的缺陷

• 批准号: MR/S03398X/2
• 负责人: Stuart Ainsworth
• 金额: $ 23.12万
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FS03398X%2F2
• 关键词: Rational; design; rapidly; translatable; highly

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.

背景：蛇咬伤（SBE）每年杀死138,000人，> 40万人杀死。抗蛇毒（IgG（IgG）用毒液混合物过度免疫的动物纯化）是SBE的唯一保证治疗，是使用昂贵的，具有百年历史的用来使马/绵羊用粗毒液免疫的羊/绵羊制造的。当前的方案无需考虑在设计或制造过程中考虑变异的毒蛋白免疫原性或毒性。因此，抗蛇毒的剂量效能感通常很差，这导致施用大量（通常在印度200-400毫升）来中和病理学，通常会导致严重的不良反应和已经贫穷的受害者负担不足的成本。此外，由于物种间的毒液多样性，原油毒液免疫导致蛇类特异性的治疗，导致医生在未知时必须做出困难的诊断和抗蛇毒式选择决策。因此，迫切需要迫切需要大幅度提高抗毒液疗法的毒液中和效果。理由：治疗神经毒性蛋白（一种常见的全球病理通常导致致命呼吸瘫痪的常见病理）特别有效，因为在用于免疫的毒液中的免疫原性和神经毒素的巨大多样性较弱。然而，尽管多样性，对毒素序列数据集的检查表明，神经毒素具有通常保守的特征。该项目将用一个合理设计的合成颗粒仅显示神经毒素的保守区域，取代在抗蛇毒制造中使用粗神经毒性毒液的使用。通过将免疫反应集中在仅在所有神经毒性SSA蛇的毒液中保守的最重要的毒素区域的免疫反应，我预计会产生一种抗蛇毒，该抗蛇毒能够中和神经毒性的蛇在整个SSA中都能在整个SSA中，无论是在物种上，无论是高度有效的，都需要在较小的动物中效应，并在较小的动物中效应，并构成了对较小的剂量的效果。方法：1首先，我将在计算和实验上研究编码来自SSA最重要的蛇的神经毒素的序列，以识别在所有神经毒素中保守的区域。2鉴定的区域将在高度免疫原性的抗原递送工具（ADVS）（例如病毒粒子（VLPS）（vlps）或模型的模型中，用于高度免疫原性抗原递送工具（ADVS）或模型的模型，该模型固有的构成构成了固有的自身固体或已固体的自身固体。可以轻松地操纵每种方法以显示外国抗原，因此可以通过免疫系统进行有效的表现和增强对所识别的神经毒素区域的认识。3我将通过免疫小鼠免疫以识别显示神经毒素抗原的最佳构型来测试这些方法，从而确定（II）均能确定（i）的（i）均具有（i）的（i）的（i）的（I）。使用体外测定。然后，显示神经毒素抗原的ADV的两种最佳构型将用于免疫抗蛇毒制造动物（绵羊）以生成实验性抗蛇毒抗性。 4最后，我将证明在体外进行致命性研究之前，实验绵羊生成的抗蛇毒的体外功效。通过这些临床前的鼠研究，我将确定与现有的商业，粗毒液产生的抗蛇毒物相比，ADV产生的抗蛇毒的中和潜力是否具有优势的毒液中和潜力。含义：由于该项目只能改善最初的免疫材料，而不会更改下游抗蛇毒制造工艺或产品配方，因此我预计Adv-Inerage-eneragent-Interagent of Adv-eneragent of Adv-Ineragent of Adv-Ineragent-near-Interagen-Interagent of Adv-Ineragent-Inerage-eneragen-eneragen od-near的抗抗蛇毒物质将不需要广泛的监管批准。这将允许将积极结果快速转化为临床试验，并在短期内立即减轻SBE负担。