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/1
• 负责人: Stuart Ainsworth
• 金额: $ 127.69万
• 依托单位: Liverpool School of Tropical Medicine
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FS03398X%2F1
• 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.

蛇咬伤（SBE）每年造成138，000人死亡，400，000人致残。抗蛇毒血清（从用毒液混合物超免疫的动物中纯化的IgG）是SBE的唯一可靠治疗方法，使用昂贵的，百年历史的用粗毒液免疫马/羊的方案生产。目前的方案没有试图在设计或制造过程中考虑变异毒液蛋白的免疫原性或毒性。因此，抗蛇毒血清的剂量功效往往很差，这导致为了中和病理而服用大量抗蛇毒血清（在印度通常为200-400毫升），这往往导致严重的不良反应，并使已经贫困的受害者支付不起费用。此外，由于物种间毒液的多样性，粗毒液免疫导致蛇物种特异性的治疗，导致医生在未知的攻击蛇物种时不得不做出困难的诊断和抗蛇毒血清选择决定。因此，迫切需要大幅度提高抗蛇毒血清治疗的毒液中和范围和功效。基本原理：用于治疗神经毒性毒液中毒（一种常见的全球性病理学，通常导致快速致命的呼吸麻痹）的抗蛇毒血清特别有效，因为用于免疫的毒液中的神经毒素的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，神经毒素具有共同的保守特征。该项目将取代使用天然神经毒素的抗蛇毒血清生产与合理设计，合成颗粒显示只有保守的区域的神经毒素。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具病理学重要性的毒素的区域，我预期产生一种抗蛇毒血清，其（i）能够中和整个sSA的神经毒性蛇毒液，无论物种如何，和（ii）高度有效，导致需要较小的抗蛇毒血清剂量来实现治愈和提高安全性。方法：1首先，我将通过计算和实验研究编码来自最具医学重要性的sSA蛇的神经毒素的序列，以确定在所有神经毒素中保守的区域。2然后将所确定的区域进行工程改造，以展示在具有固有免疫系统调节特征的高免疫原性抗原递送载体（ADV）上，例如病毒样颗粒（VLP）或Fc融合体。每种方法都可以很容易地操作以展示外源抗原，因此允许免疫系统有效地展示和增强对所鉴定的保守神经毒素区域的识别。3我将通过免疫小鼠以鉴定展示神经毒素抗原的ADV的最佳构型来测试这些方法，通过检查（i）免疫应答的程度和（ii）使用体外测定法产生的抗体防止神经毒素活性的能力来确定。展示神经毒素抗原的ADV的两种最佳构型然后将用于免疫抗蛇毒血清生产动物（绵羊）以产生实验性抗蛇毒血清。4最后，我将证明实验羊产生的抗蛇毒血清在体外的上级功效，在体内中和致死性研究之前。通过这些临床前小鼠研究，我将确定ADV产生的抗蛇毒血清与现有的商业化粗品蛇毒产生的抗蛇毒血清相比是否表现出上级蛇毒中和潜力。含义：由于该项目将仅改进初始免疫材料，而不改变下游抗蛇毒血清生产工艺或产品配方，因此我预计ADV生产的抗蛇毒血清将不需要广泛的监管批准。这将使阳性结果快速转化为临床试验，并在短期至中期内立即减少SBE负担。

项目成果

Analysis of commercially available snake antivenoms reveals high contents of endotoxins in some products

• DOI: 10.1016/j.toxcx.2024.100187
• 发表时间: 2024-02-19
• 期刊: TOXICON-X
• 作者: Solano，Gabriela;Ainsworth，Stuart;Leon，Guillermo
• 通讯作者: Leon，Guillermo

Approaches for implementing society-led community interventions to mitigate snakebite envenoming burden: The SHE-India experience.

• DOI: 10.1371/journal.pntd.0009078
• 发表时间: 2021-03
• 期刊: PLoS neglected tropical diseases
• 影响因子: 3.8
• 作者: Kadam P;Ainsworth S;Sirur FM;Patel DC;Kuruvilla JJ;Majumdar DB
• 通讯作者: Majumdar DB

Exploring the utility of recombinantly expressed snake venom serine protease toxins as immunogens for generating experimental snakebite antivenoms

• DOI: 10.1101/2022.05.07.491032
• 发表时间: 2022-05
• 期刊: bioRxiv
• 作者: Nessrin Alomran;P. Blundell;Jaffer Alsolaiss;E. Crittenden;S. Ainsworth;Charlotte A. Dawson;Rebecca J. Edge;Steven R. Hall;R. Harrison;M. Wilkinson;S. Menzies;N. Casewell

Virus-like particles displaying conserved toxin epitopes stimulate broadly reactive, polyspecific, murine antibody responses capable of snake venom recognition

• DOI: 10.21203/rs.3.rs-1044937/v1
• 发表时间: 2021-11
• 作者: S. Menzies;Charlotte A. Dawson;E. Crittenden;Rebecca J. Edge;Steven R. Hall;Jaffer Alsolaiss;M. Wilkinson;N. Casewell;R. Harrison;S. Ainsworth

Pathology-specific experimental antivenoms for haemotoxic snakebite: The impact of immunogen diversity on the in vitro cross-reactivity and in vivo neutralisation of geographically diverse snake venoms.

• DOI: 10.1371/journal.pntd.0009659
• 发表时间: 2021-08
• 期刊: PLoS neglected tropical diseases
• 影响因子: 3.8
• 作者: Alomran N;Alsolaiss J;Albulescu LO;Crittenden E;Harrison RA;Ainsworth S;Casewell NR
• 通讯作者: Casewell NR