The Mechanism Of CS-IONzyme As Catalytic Adjuvant To Enhance The Immune Efficacy Of H1N1 Influenza Mucosal Vaccine

To date,the influenza A(H1N1)pdm09 virus which first emerged in 2009 resulted in approximately 575,400 worldwide deaths,posing a huge threat to human health.The nasal cavity is the primary entry site of influenza virus,and the viral infection can be effectively blocked in the first time if mucosal immunity is well established.The whole inactivated virus(WIV)vaccine given via intranasal immunization is a greatly potential strategy.However,mucosal immunization by intranasal delivery with inactivated virus alone is often poorly effective.Iron oxide nanozymes(IONzymes)possess multiple enzyme-like activities,including peroxidase,catalase,and so forth.At acidic pH conditions,IONzyme exhibits a peroxidase(POD)-like activity,which is beneficial to the generation of ROS.Innate immunity of DCs can be enhanced by intracellular ROS levels.Doping Chitosan on the surface of IONzyme can not only increase the enzyme-like activities,but also change the charge to the positive for mucosal delivery of antigens,impling that a CS-IONzyme could be a novel mucosal adjuvant.Therefore,first,we evaluated the ability of mucosal delivery of H1N1 WIV by CS-IONzyme.Second,we determined the effect of CS-IONzyme on the innate immune response of murine DCs in vitro.Finally,we explored the protection efficacity after intranasally immunized H1N1 WIV-CS-IONzyme vaccine in mice.Taken together,our research provides a theoretical basis for the application of CS-IONzyme as a mucosal adjuvant.and is divided into the following three parts in detail:1.Effects of CS-IONzyme on the adhesion ability of H1N1 WIV to nasal mucosa and recruitment of DCsCS-IONzyme was synthesized by using a solvothermal method and then connected with H1N1 WIV for preparing H1N1 WIV-CS-IONzyme vaccine.The mice were intranasally immunized with this vaccine,the confocal laser scanning microscopy(CLSM)images displayed that compared to the H1N1 WIV alone,CS-IONzyme not only increased the adhesion ability and resident time of H1N1 WIV to nasal mucosa,but also recruited DCs into the submucosal region to take up the H1N1 WIV by transepithelial dendrites(TEDs).Further research via flow cytometry and gate strategy found that CS-IONzyme can upregulate the expression levels of CCL20,TLR2,and TLR4 from nasal mucosal epithelial cells.After pretreatment with TLR2 and TLR4 inhibitors,CCL20 expression levels of nasal mucosal epithelium and the number of submucosal DCs significant declined.Taken together,TLR2/4 signaling pathway was required for CS-IONzyme stimulating nasal epithelial cells to secrete CCL20,which induces DCs recruitment and TED formation for H1N1 WIV uptake.2.Effects of CS-IONzyme on the innate immunity of murine DCsTo investigate the effects of CS-IONzyme on innate immunity of murine DCs,DCs were incubated with CS-IONzyme for 24 h,and then DCs and culture supernatants were collected for detecting the expression of phenotype markers,cytokine secretion,phagocytosis,stimulation of CD4+T cell proliferation and DC ROS levels.Compared with control group,CS-IONzyme remarkably up-regulated the expression of MHC?,CD40,CD80,and CD86.Similarly,CS-IONzyme significantly increased the release of cytokines(TNF-?,IL-1?,IL-12p70,IL-6,and IL-10).Morerover.the DCs from CS-IONzyme group strongly enhanced the proliferation of allogeneic T cells compared to that from the control group.Finally,CS-IONzyme effectively enhanced the generation of ROS,and after the pretreatment with ROS scavengers,the phenotype marker expressions(MHC? and CD40)and cytokine secretions(IL-12p70,IL-6,TNF-?.IL-1?,and IL-10)of DCs were suppressed completely,suggesting that CS-IONzyme-activated DCs innate immunity depended on ROS pathway.3.Evaluation of the protective efficacy by H1N1 WIV-CS-IONzyme mucosa delivery vaccineAfter immunizing intransally with H1N1 WIV alone or CS-IONzyme-HIN1 WIV in mice,the changes of body weight,survival rates in lungs were observed,the viral load lesions in lungs was detected.Compared with the H1N1 WIV alone(30%survival rate),the H1N1 WIV-CS-IONzyme group can completely resist the lethal attack of the H1N1 influenza virus(100%survival rate),body weight was slight loss,the lung tissues showed a slight pathologic and histopathological changes,and virus titer in the lung was significantly lower.These data demonstrated that intranasal vaccination with CS-IONzyme-based H1N1 inactivated vaccine protected mice from a lethal influenza challenge.