BACKGROUND Engineered vaccine proteins incorporating both antigen and adjuvant components are constructed with the aim of combining functions to induce effective protective immunity. Bacterial flagellin is a strong candidate for an engineered vaccine scaffold as it is known to provide adjuvant activity through its TLR5 and inflammasome activation. Moreover, polymerized flagellin filaments can elicit a more robust immunoglobulin response than monomeric flagellin, and the multimeric antigen form can also promote T cell-independent antibody responses. Here, we aim to produce and test a covalently stabilized polymerized flagellar filament, providing additional immune efficacy through stabilization of its polymeric filament structure, as well as stabilization for long-term storage. RESULTS Computational modeling of monomer packing in flagellin filaments helped identify amino acids with proximity to neighboring flagella protofilaments. Paired cysteine substitutions were made at amino acids predicted to form inter-monomer disulfide cross-links, and these substitutions were capable of forming flagella when transfected into a flagellin-negative strain of Salmonella enterica subspecies Typhimurium. Interestingly, each paired substitution stabilized different helical conformational polymorphisms; the stabilized filaments lost the ability to transition between conformations, reducing bacterial motility. More importantly, the paired substitutions enabled extensive disulfide cross links and intra-filament multimer formation, and in one of the three variants, permitted filament stability in high acidic and temperature conditions where wild-type filaments would normally rapidly depolymerize. In addition, with regard to potential adjuvant activity, all crosslinked flagella filaments were able to induce wild-type levels of epithelial NF-κB in a cell reporter system. Finally, bacterial virulence was unimpaired in epithelial adherence and invasion, and the cysteine substitutions also appeared to increase bacterial resistance to oxidizing and reducing conditions. CONCLUSIONS We identified amino acid pairs, with cysteine substitutions, were able to form intermolecular disulfide bonds that stabilized the resulting flagellar filaments in detergent, hydrochloric acid, and high temperatures while retaining its immunostimulatory function. Flagellar filaments with disulfide-stabilized protofilaments introduce new possibilities for the application of flagella as a vaccine adjuvant. Specifically, increased stability and heat tolerance permits long-term storage in a range of temperature environments, as well as delivery under a range of clinical conditions.

中文翻译：

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交联鞭毛作为稳定的疫苗佐剂支架。

背景技术构建了融合了抗原和佐剂成分的工程疫苗蛋白，其目的是结合功能以诱导有效的保护性免疫。细菌鞭毛蛋白是工程疫苗支架的强力候选者，因为它已知通过其TLR5和炎性体激活提供佐剂活性。此外，聚合的鞭毛蛋白丝可以引发比单体鞭毛蛋白更强大的免疫球蛋白应答，并且多聚体抗原形式还可以促进非T细胞依赖性抗体应答。在这里，我们的目标是生产和测试共价稳定的聚合鞭毛丝，通过稳定其聚合丝结构以及长期保存的稳定性提供额外的免疫功效。结果鞭毛蛋白丝中单体堆积的计算模型有助于鉴定与邻近鞭毛原丝接近的氨基酸。在预计会形成单体间二硫键的氨基酸处进行成对的半胱氨酸替代，当这些替代品被转染到鼠伤寒沙门氏菌鼠伤寒沙门氏菌阴性鞭毛蛋白阴性菌株中时，它们就能够形成鞭毛。有趣的是，每个成对的取代稳定了不同的螺旋构象多态性。稳定的细丝失去了在构象之间过渡的能力，从而降低了细菌的运动性。更重要的是，配对取代可实现广泛的二硫键交联和丝内多聚体形成，并且在三个变体之一中，允许在高酸性和高温条件下使长丝保持稳定，在这种条件下，野生型长丝通常会迅速解聚。另外，关于潜在的佐剂活性，所有交联的鞭毛丝都能够在细胞报告系统中诱导野生型水平的上皮NF-κB。最后，细菌毒力在上皮粘附和侵袭中没有受到损害，并且半胱氨酸替代也似乎增加了细菌对氧化和还原条件的抵抗力。结论我们鉴定了具有半胱氨酸取代的氨基酸对，它们能够形成分子间二硫键，从而稳定了去污剂，盐酸和高温下产生的鞭毛丝，同时保留了其免疫刺激功能。带有二硫键稳定原丝的鞭毛丝为鞭毛作为疫苗佐剂的应用提供了新的可能性。特别地，增加的稳定性和耐热性允许在一定温度范围内长期保存，以及在一定范围的临床条件下递送。