By modulating specific immune responses against antigens, adjuvants are used in many vaccine preparations to enhance protective immunity. The C-terminal domain of the protein P97 (P97c) of Mycoplasma hyopneumoniae, which is the etiologic agent of porcine enzootic pneumonia, has been shown to increase the specific humoral response against an antigen when this antigen is merged with P97c and delivered by adenovectors. However, the immunostimulating mechanism of this protein remains unknown. In the present study, recombinantly expressed P97c triggered a concentration-dependent TLR5 activation and stimulates the production of interleukin-8 from HEK-Blue mTLR5 cells. Circular dichroism spectroscopy and prediction of 3-dimensional conformation exposed a relevant secondary and tertiary structural homology between P97c and flagellin, the known potent TLR5 agonist. P97c adjuvanticity was evaluated by fusing the conserved epitope of the ectodomain matrix 2 protein (M2e) of the influenza A virus to the protein. Mice immunized with P97c-3M2e revealed a high antibody titer against the M2e epitope associated with a mixed Th1/Th2 immune response. Overall, this study identifies a novel agonist of the pattern recognition receptor TLR5 and reveals that P97c is a potential adjuvant through the activation of the innate immune system.

通过调节针对抗原的特异性免疫反应，佐剂被用于许多疫苗制剂中以增强保护性免疫。猪肺炎支原体蛋白 P97 (P97c) 的 C 端结构域是猪地方性动物肺炎的病原体，当该抗原与 P97c 合并并由腺载体递送时，已显示该抗原可增加针对该抗原的特异性体液反应。然而，这种蛋白质的免疫刺激机制仍然未知。在本研究中，重组表达的 P97c 触发了浓度依赖性 TLR5 激活，并刺激 HEK-Blue mTLR5 细胞产生白细胞介素 8。圆二色光谱和 3 维构象的预测揭示了 P97c 和鞭毛蛋白（已知的有效 TLR5 激动剂）之间的相关二级和三级结构同源性。通过将甲型流感病毒胞外域基质 2 蛋白 (M2e) 的保守表位与蛋白质融合来评估 P97c 佐剂性。用 P97c-3M2e 免疫的小鼠显示出针对与混合 Th1/Th2 免疫反应相关的 M2e 表位的高抗体滴度。总的来说，这项研究确定了模式识别受体 TLR5 的一种新型激动剂，并揭示了 P97c 是一种通过激活先天免疫系统的潜在佐剂。