柯萨奇病毒A16型抗原与呼吸道黏膜上皮组织内先天淋巴细胞的相互关系及其疫苗应用价值

Relationship between coxackievirus A16 antigen and innate lymphoid cells in respiratory tract epithelia and immunological analysis of its vaccine development

柯萨奇病毒A16型(coxsakievirus A16,CA16)是引起手足口病的主要病原,但其发病及相关免疫机制尚不清楚,导致其疫苗研发面临诸多困难。众多研究表明,呼吸道黏膜上皮细胞及其相关免疫细胞尤其是天然淋巴细胞(innate lymphoid cell,ILC),在很大程度上促进病毒感染中天然免疫和适应性免疫反应的激活。然而,ILC在CA16感染诱导的免疫反应中如何发挥作用仍未知晓。为揭示CA16感染过程中ILC与病原的相互关系及发挥的作用,首先利用定量反转录-聚合酶链反应(quantitative reverse transcription-polymerase chain reaction,qRT-PCR)检测CA16感染的人支气管上皮细胞16HBE和以特别配制的佐剂导入CA16灭活抗原的小鼠支气管上皮细胞CP-M175中对ILC具有激活效应的重要基因表达情况。然后,将特别配制的CA16灭活疫苗通过鼻腔免疫BALB/c小鼠,分析呼吸道黏膜组织中ILC与病毒抗原之间的相互关系。体内外实验结果均显示,CA16活病毒和灭活的病毒抗原能诱导ILC活化所需信号通路分子的表达增加,且ILC分泌的细胞因子表达增加。免疫荧光和共聚焦显微镜观察结果亦证实,病毒抗原与ILC1/ILC3分别存在共定位现象。比较4种途径/程序免疫的小鼠体内抗CA16中和抗体和特异性细胞免疫应答情况,发现采用特别配制的CA16灭活疫苗免疫小鼠能诱导产生较好的抗病毒免疫应答。结果提示,CA16抗原能通过激活ILC及其相关信号通路诱导产生有效的免疫反应。

Coxackievirus A16(CA16)is a major pathogen of hand,foot and mouth disease.Because of the unclear pathogenesis and immunomechanism of CA16,the development of a CA16vaccine has encountered numerous difficulties.Many studies have reported that epithelial cells and associated immune cells in respiratory tract mucosa,especially innate lymphoid cells(ILCs),are largely contributed to the activation of innate and adaptive immune responses during viral infection.It is still unknown that whether ILCs play a similar role in the immune response induced by CA16infection.To reveal the potential interactions between ILCs and CA16infection,the expression levels of genes related to ILC activation in CA16-infected human tracheal epithelial cells(16HBE)and mouse respiratory tract epithelial cells(CP-M175)stimulated by adjuvants formulated CA16-inactivated antigen were firstly determined.Then,a formulated experimental CA16-inactivated vaccine was used to immunize BALB/c mice through nasal route,and the relationship between ILCs and viral antigens in the respiratory mucosa was analyzed.Both CA16live virus and inactivated virus particles could induce upregulation of various molecules required for the activation of ILCs and stimulate transcription and expression of cytokines,which were secreted by activated ILCs,no matter in vitro and in vivo.Moreover,immunofluorescence and confocal microscopic results showed that the viral antigen was co-localized with ILC1/ILC3.Finally,the anti-CA16neutralizing antibodies and specific cellular immune response in mice of four different immunization pathways/immunization procedures were tested.The results suggested that the mice immunized with the formulated CA16-inactivated vaccine could induce an effective antiviral immune response in vivo.In conclusion,CA16antigen can induce an effective immune response by activating ILCs.