Development Of Mucosal Vaccine Of Influenza Virus Using Manganese Modified Nanozyme Adjuvant

Influenza is a highly contagious disease caused by influenza virus that poses a serious threat to human and animal health.The nasal cavity is the main portal for influenza virus to invade the host,and mucosal immunity can effectively prevent the virus from invading and spreading.However,due to the existence of mucosal barrier,the intranasal immune effect of whole inactivated virus(WIV)vaccine alone is often ineffective.Therefore,it is necessary to develop a novel mucosal adjuvant combined with inactivated virus vaccine to improve the efficacy of nasal mucosal immunization.Previous studies have shown that Iron oxide nanozyme(IONzyme)could be applied as mucosal adjuvant,while its efficacy still needs to be improved.In this study,a new mucosal immune adjuvant(Mn-PEI-IONzyme)was designed by modifying the surface of IONzyme with Manganese(Mn)which had multienzyme mimetic activities and immune properties,and with Polyethylenimine branched(PEI)which was positively charged,safe and stable.The safety and immunological efficacy of Mn-PEI-IONzyme were evaluated.1.Preparation and characterization of Manganese modified iron oxide nanozymeIn this study,Mn-PEI-IONzyme was prepared by hydrothermal synthesis,the synthesis parameters were optimized and the characteristics were determined.Firstly,the molecular weight of PEI and the modified doses of Mn and PEI were screened in the preparation process.The observation of Transmission electron microscope(TEM)and the determination of peroxidase(POD)-like activity showed that PEI with molecular weight of 25 kDa and modification amount of 0.1 g,and Mn with 0.5 g were selected.The results of TEM and Scanning electron microscopy(SEM)showed that Mn-PEI-IONzyme was about 160 nm,and the morphology was regular and spherical.Energy dispersive spectroscopy(EDS),X-ray diffraction(XRD),and X-ray photoelectron spectroscopy(XPS)indicated that Mn and PEI.were successfully doped on IONzyme surface.The POD-like activity of Mn-PEI-IONzyme was the highest.The Zeta potential showed that IONzyme was negatively charged while MnPEI-IONzyme was positively charged.The results of safety evaluation in mice showed that Mn-PEI-IONzyme maintained the body weight and no pathological changes in the organs of mice.In addition,there was no significant difference in blood biochemical and hematological indexes compared with the control group.The results indicate that Mn-PEIIONzyme has a good biocompatibility.2.Effects of Mn-PEI-IONzyme on activation and maturation of dendritic cellsTo investigate the effects of Mn-PEI-IONzyme on the activation and maturation of murine Dendritic cells(DCs),DCs were incubated with Mn-PEI-IONzyme for 24 h,and then DCs and culture supernatants were collected for detecting the expression of phenotype,activation,and migration markers,cytokine secretion,proliferation of CD4+T cell,phagocytosis,and Reactive oxygen species(ROS)levels.Compared with control group,the results showed that Mn-PEI-IONzyme remarkably up-regulated the expression of MHCII,CD40,CD80,and CD86,activation marker CD69 and migration marker CCR7,Similarly,Mn-PEI-IONzyme significantly increased the release of cytokines(TNF-α,IL-1β,IL-12p70,IL-6,and IL-10).Mixed lymphocyte reaction test showed that DCs from Mn-PEI-IONzyme group strongly enhanced the proliferation of allogeneic T cells compared to that from the control group.The ability of DCs to phagocytose FITC-Dextran was significantly decreased after Mn-PEI-IONzyme stimulation.Mn-PEI-IONzyme was found to be present in DCs lysosomes by TEM.Mn-PEI-IONzyme effectively enhanced the generation of ROS in DCs.However,after the pretreatment with ROS scavengers,the ROS and the phenotype marker expressions(MHCII and CD40)of DCs were suppressed significantly.These results indicate that Mn-PEI-IONzyme induces activation and maturation of DCs through ROS pathway.3.Evaluation of the immune efficacy by mucosa delivery influenza vaccine based on Mn-PEI-IONzyme adjuvant.The immune efficacy of H1N1 WIV+Mn-PEI-IONzyme vaccine was determined 28 days after intranasal immunization of mice.In nasal,tracheal,and lung wash samples,H1N1 WIV+Mn-PEI-IONzyme group was found to induce much higher IgA specific antibody titers compared to H1N1 WIV alone.Furthermore,we also found that serum antigen-specific IgG,IgG1,IgG2a/c antibody titers,the Hemagglutination inhibition(HI)titers,and the percentage of CD3+CD4+splenic T cells induced by H1N1 WIV+Mn-PEI-IONzyme were significantly higher than antigen-alone immunization.After 28 days of immunization,the mice were challenged with 106 EID50/50μL of H1N1 virus.The changes of body weight and survival rates were monitored.Lung lesions were observed and viral load in lungs was detected.The results showed that the challenged mice from the group of H1N1 WIV alone showed a remarkable weight loss,the survival rate of the mice was 40%.However,the mice immunized with H1N1 WIV+Mn-PEI-IONzyme recovered after a slight loss of body weight and survived to the end of the experimental period,demonstrated a 100%protective efficacy.H1N1 WIV+Mn-PEI-IONzyme immunized mice showed a slight pathologic and histopathological changes.Moreover,the virus titer in the lung from the group of H1N1 WIV+Mn-PEI-IONzyme was significantly lower than that from the H1N1 WIV group.These results demonstrate that intranasal vaccination with Mn-PEI-IONzyme-based influenza vaccine strongly induce mucosal and systemic immune responses in mice and protected mice from a lethal influenza challenge.In summary,Mn-PEI-IONzyme has a good biocompatibility,promotes the activation and maturation of DCs.As a mucosal adjuvant,Mn-PEI-IONzyme can significantly enhance the immune efficacy of H1N1 whole inactivated virus vaccine.