Testing recombinantly lipidated antigens as oral vaccines against Clostridioides difficile

测试重组脂质化抗原作为艰难梭菌口服疫苗

• 批准号: 2744308
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2744308
• 关键词: Testing; recombinantly; lipidated; antigens; oral

One of the main bottlenecks for vaccine development is identifying suitable adjuvants. The lipid moieties of bacterial lipoproteins have attracted interest owing to their potent immunostimulatory properties. Multiple different fatty acids may be utilised by bacteria to form this lipid moiety, and dipalmitoyl and tripalmitoyl-glyceryl groups are known to be particularly potent.Vaccinologists have exploited the lipoprotein processing machinery of Escherichia coli to lipidate chosen antigens and enhance their potency. Briefly, the antigen gene is cloned downstream of the N-terminal signal peptide of a lipoprotein. This was first demonstrated for meningococcal Factor H binding protein (FHbp) using the signal peptide of Haemophilus influenzae P4 lipoprotein. The lipidated protein elicited bactericidal antibody titres up to 10-fold higher than the non-lipidated form following subcutaneous administration in mice. Pfizer exploited this recombinant lipoprotein to develop the Trumenba vaccine which was licenced in 2014. Other studies have used the signal peptide and adjacent sequence of the meningococcal lipoprotein, Ag473, to create a fusion with the viral HPV protein and with part of toxin A of Clostridioides difficile. These lipoprotein subunit vaccines have shown significantly higher neutralizing antibody titres in mice compared to their non-lipidated counter-parts. For vaccine manufacturing purpose, large scale production of lipoproteins by recombinant expression in E. coli remains a challenge due to typically low yields, incomplete or no lipidation and inherent challenges of purifying membrane-associated proteins from insoluble fractions. Antigens that enter culture filtrates on the other hand, are by default soluble facilitating their purification. We thus set out to identify the optimal signal peptide that can not only direct successful lipidation but also secretion of the acylated fusion protein into the extracellular milieu for facile purification.Testing a range of different peptides of different length to which fluorescent mRaspberry was fused as a reporter, has now led to the identification of the optimal signal peptide. Currently we are testing whether the deletion of 2 specific genes in the chromosome of our E. coli expression strain will facilitate secretion and we are characterising the lipid moiety to confirm the presence of palmitic acids. ObjectiveWith the focus of my group dedicated to testing novel mucosal vaccine platforms for their efficacy against C. difficile infection, the project will be to build on the above platform. There is currently no vaccine available against this deadly gut pathogen as intramuscular toxoid vaccines have failed human trials. Encouragingly we have shown the efficacy of the oral route to direct a mucosal and systemic immune response that affords partial protection from infection with a hypervirulent strain of C. difficile. MethodologyIn this project, using our optimal signal peptide, vaccine candidates will be acylated and purified from culture filtrates, lyophilised and encapsulated and testing for improved immunogenicity and improved protective efficacy in the C. difficile hamster model compared to their non-acylated counterparts. The immunogenicity assays to be conducted will include ELISAs and cellular assays (adherence blocking and toxin neutralisation). Challenge studies will involve monitoring well being (survival from infection) and bacterial load in faecal pellets. Histological analysis will be conducted with help from a Histopathologist. All other experiments are routinely performed in the Griffin lab.

疫苗开发的主要瓶颈之一是确定合适的佐剂。细菌脂蛋白的脂质部分由于其有效的免疫刺激特性而引起人们的兴趣。多种不同的脂肪酸可以被细菌利用来形成这种脂质部分，并且已知二棕榈酰和三棕榈酰甘油基特别有效。疫苗学家已经利用大肠杆菌的脂蛋白加工机制来脂化所选抗原并增强其效力。简言之，将抗原基因克隆到脂蛋白的N-末端信号肽的下游。这是首次证明脑膜炎球菌H因子结合蛋白（FHbp）使用流感嗜血杆菌P4脂蛋白的信号肽。在小鼠皮下给药后，脂化蛋白质引起的杀菌抗体滴度比非脂化形式高10倍。辉瑞利用这种重组脂蛋白开发了Trumenba疫苗，该疫苗于2014年获得许可。其他研究使用脑膜炎球菌脂蛋白Ag 473的信号肽和相邻序列来产生与病毒HPV蛋白和艰难梭菌毒素A的一部分的融合。这些脂蛋白亚单位疫苗在小鼠中显示出比其非脂化对应物显著更高的中和抗体滴度。为生产疫苗，在大肠杆菌中重组表达脂蛋白可大规模生产。大肠杆菌中纯化膜相关蛋白仍然是一个挑战，这是由于典型的低产率、不完全或没有脂化以及从不溶性级分中纯化膜相关蛋白的固有挑战。另一方面，进入培养皿的抗原默认是可溶的，从而促进其纯化。因此，我们着手确定的最佳信号肽，不仅可以直接成功的脂化，但也分泌的酰化融合蛋白到细胞外环境的简易purifiation.Testing一系列不同的肽的不同长度的荧光mRaspberry融合作为一个报告，现在已经导致了最佳信号肽的识别。目前我们正在测试我们的大肠杆菌染色体中2个特定基因的缺失是否。大肠杆菌表达菌株将促进分泌，我们正在表征脂质部分以确认棕榈酸的存在。随着我的小组致力于测试新型粘膜疫苗平台对C. difficile感染，该项目将建立在上述平台上。目前还没有针对这种致命的肠道病原体的疫苗，因为肌内类毒素疫苗在人体试验中失败了。令人鼓舞的是，我们已经显示了口服途径引导粘膜和全身免疫应答的功效，该免疫应答提供了部分保护以免受C的超强毒株的感染。很难方法在本项目中，使用我们的最佳信号肽，将候选疫苗从培养物中酰化和纯化，冻干和封装，并测试改进的免疫原性和改进的C. difficile仓鼠模型与它们的非酰化对应物相比。待进行的免疫原性试验将包括ELISA和细胞试验（粘附阻断和毒素中和）。攻毒研究将包括监测健康状况（感染后存活率）和粪便颗粒中的细菌载量。将在组织学专家的帮助下进行组织学分析。所有其他实验均在Griffin实验室常规进行。