Preclinical Development of Flagellin as a Mucosal Adjuvant in Biodefense Vaccines

描述(由申请人提供)：革兰氏阴性鞭毛蛋白是一种非常有效的先天和获得性免疫反应激活剂。我们和其他人的工作已经证实鞭毛蛋白在小鼠身上是一种有效的佐剂。我们还发现鞭毛蛋白在非人类灵长类动物中是一种有效的佐剂。鞭毛蛋白具有使其成为一种优秀佐剂的特性，包括：效力；粘膜活性；在鞭毛免疫宿主中的有效性；在细菌中易于产生；以及5)能够在不丧失鞭毛蛋白生物活性的情况下将抗原序列插入蛋白质的高变区。我们已经清楚地证明鞭毛蛋白是一种高效的粘膜佐剂，可以在小鼠和非人类灵长类动物中产生针对鼠疫杆菌抗原的强大、特异的抗体反应，并促进对鼠疫杆菌致命的呼吸道攻击的保护。鉴于鞭毛蛋白作为黏膜佐剂在人类中具有巨大的潜力，我们提出了以下目标，以促进鞭毛蛋白人类使用的发展：具体目的1.确定鞭毛蛋白/鼠疫耶尔森菌F1和V融合蛋白作为保护性粘膜疫苗的效力。我们已经构建了一个编码鞭毛蛋白和鼠疫杆菌F1和V抗原的重组蛋白，并证实该融合蛋白具有鞭毛蛋白的生物学活性。我们建议确定融合蛋白是否能促进小鼠和食蟹猴的抗F1和V型免疫球蛋白反应，并能保护小鼠免受鼠疫杆菌CO92的呼吸道攻击。具体目标2.设计和实施符合良好生产规范(GMP)的鞭毛素/鼠疫耶尔森氏菌疫苗的生产工艺，该工艺可以很容易地扩展用于临床前、临床和最终的商业用途。具体目的3.对鞭毛蛋白/鼠疫耶尔森氏菌疫苗进行GLP毒理学研究。一旦该过程被优化，将准备一批工程，在小鼠模型中研究其在GLP条件下的安全性和免疫原性。该批次还将用于特性研究和启动正式的稳定性研究，为临床生产做准备。具体目的4.在良好生产规范(GMP)条件下生产鞭毛素/鼠疫耶尔森氏菌疫苗。在特定目标2中开发的方案将用于在GMP条件下生产克量疫苗。这些目标的完成将为黏膜鞭毛蛋白/鼠疫耶尔森氏菌疫苗在人类的临床评估提供非常坚实的基础。

DESCRIPTION (provided by applicant): Gram-negative flagellin is an extraordinarily potent activator of innate and adaptive immune responses. Our work as well as that of others has established that flagellin is a potent adjuvant in mice. We have also found that flagellin is a potent adjuvant in non-human primates. Flagellin possesses features that make it an excellent adjuvant including: potency; mucosally active; effectiveness in flagellin immune hosts; ease of production in bacteria; and 5) the ability to insert antigen sequences in the hypervariable region of the protein without loss of flagellin bioactivity. We have clearly established that flagellin is a highly effective mucosal adjuvant in the development of a strong, specific antibody response against Y. pestis antigens in mice and non-human primates and also promotes protection against a lethal respiratory challenge with Y. pestis. Since flagellin possesses tremendous potential as a mucosal adjuvant for use in humans, we propose the following aims that will advance the development of flagellin for human use: Specific Aim 1. To determine the efficacy of a flagellin/Y. pestis F1 and V fusion protein as a protective mucosal vaccine. We have generated a recombinant protein encoding flagellin and both the F1 and V antigens of Y. pestis and have established that this fusion protein retains flagellin biological activity. We propose to determine if the fusion protein promotes a robust anti-F1 and V IgG response in mice and cynomolgus monkeys and protection in mice against respiratory challenge with Y. pestis CO92. Specific Aim 2. To design and implement a Good Manufacturing Practice (GMP) compatible production process for a flagellin/Y. pestis vaccine that can be readily scaled for pre-clinical, clinical and ultimately commercial use. Specific Aim 3. To conduct GLP toxicology studies with the flagellin/Y. pestis vaccine. Once the process has been optimized, an engineering lot will be prepared to study its safety and immunogenicity under GLP conditions in a mouse model. This lot will also be used for characterization studies and to initiate formal stability studies in preparation for clinical manufacture. Specific Aim 4. To produce a flagellin/Y. pestis vaccine under Good Manufacturing Practices (GMP) conditions. The protocol developed in Specific Aim 2 will be used to produce gram quantities of the vaccine under GMP conditions. The completion of these aims will provide a very strong foundation for the clinical evaluation of the mucosal flagellin/Y. pestis vaccine in humans.