The Mechanism Of Induction Of Nasal Immunity By Whole Inactivated Avian Influenza Virus In Mice

Avian influenza(AI)has caused great losses in china and world.AI is divided into the highly pathogenic avian influenza and low pathogenic avian influenza.Although the morbidity and mortality rates of low pathogenic avian influenza are lower,the widespread presence of viruses raise the possibility of viral recombinant.The nasal cavity is the main portal for influenza viral invasion.Previous studies reported that nasal immunization can effectively induce local mucosal immune response to directly cut off the virus infection pathway.However,because of the nasal mucosa barrier,the whole inactivated influenza virus(WIV)induced mucosal immunity weakly after nasal immunization.Recently,CpG oligodeoxynucleotides(CpG ODN),a known adjuvant,can remarkably enhance the mucosal and systemic immune responses for WIV with a tendency toward Thl type response.However,we do not lose sight of the fact that nasal mucosa barrier is a key impediment for influenza WIV uptake and subsequent antigen-specific adaptive immune responses.Therefore,first,we explored whether CpG ODN could facilitate H9N2 WIV to trigger the local mucosal and systemic immune responses.Second,in vitro,CpG ODN and/or H9N2 WIV were directly interacted with DCs,and then phenotypes,cytokines,and CD4~+ T cell proliferation were detected by using flow cytometry,ELISA,and mixed lymphocyte reaction assay(MLRs),respectively.Finally,in vitro DCs/Calu-3 epithelial cell co-culture model and in vivo mice nasal cavity perfusion model,using flow cytometry and confocal microscopy,we studied how CpG ODN recruited nasal submucosal DCs and induced transepithelial dendrites(TEDs)formation for luminal H9N2 WIV uptake.Furthermore,we investigated whether the viruses-loaded DCs could quickly migrate into the draining cervical lymph nodes(CLNs)for antigen presentation.We also discussed the DC subsets and receptors involved in above process.These might be a novel mechanism for the optimal adaptive immune responses.Our research is divided into the following three parts in detail:1.Effects of CpG ODN combined with H9N2 WIV on mucosal and systemic immunity after intranasal immunization in miceAfter immunizing intranasally with H9N2 WIV alone or in combination with CpG ODN in mice,antigen-specific antibody IgG(total IgG,IgGl,and IgG2a/c)and hemagglutination inhibition(HI)titers in serum,and secretory IgA in nasal,tracheal and lung lavage fluid,were determined.Beside,the activation,subtypes,and proliferation of splenic lymphocytes were also detected.Our results showed that,after 28 days,the local secretion IgA antibodies in the nasal,tracheal and lung lavage fluid from CpG ODN plus H9N2 WIV were much higher than H9N2 WIV alone.Furthermore,we found the similar changes in IgG(including their subtype),HI titer,the activation,subtypes,and proliferation of splenic lymphocytes.However,H9N2 WIV alone did not effectively enhanced these above immune indexes.Altogether,these results demonstrated that intranasal vaccination with CpG ODN plus H9N2 WIV effectively induced local mucosal and systemic immune responses.2.Effects of CpG ODN plus H9N2 WIV on DC activation and maturation in the monocultureDCs is a bridge between innate immune responses and adaptive immune responses.DC activation and maturation is a key step for subsequent antigen-specific immune responses.Therefore,firstly,DCs were generated from GM-CSF-induced bone marrow progenitor cells,and then identified by morphology,purity and function.Subsequently,CpG ODN plus H9N2 WIV were directly interacted with DCs for 24 h,and then DCs and basolateral supernatants were collected for detecting phenotype and cytokines.For MLRs,the collected DCs were cocultured with CD4~+ T cells,and the T cell proliferation were detected.In monoculture,H9N2 WIV and/or CpG ODN remarkably up-regulated the expression of CD40 and CD80 compared with medium.Similarly,CpG ODN plus H9N2 WIV significantly increased the release of cytokines(IL-12p70 and IL-10).Finally,the DCs from H9N2 WIV plus CpG ODN group strongly enhanced the proliferation of allogeneic T cells(DC:T cell ratios,1:1;1:5)compared to that from H9N2 WIV-only group.Taken together,these results implied that CpG ODN facilitated H9N2 WIV to enhance DC maturation in monoculture.3.Effects of CpG ODN on the DC recruitment and TED formationIn vitro and in vivo,we showed that CpG ODN provided assistance for H9N2 WIV in recruiting DCs to the nasal epithelial cells(ECs),and forming transepithelial dendrites(TEDs)to capture luminal viruses.CD103~+ DCs were the main participants.Chemokine CCL20 from nasal ECs played a key role in driving DC recruitment and TED formation.Viruses-loaded DCs quickly migrated into the draining cervical lymph nodes(CLNs)for antigen presentation.In addition,the competence of CpG ODN was independent of direct epithelial transport via transcellular or paracellular pathway.Taken together,our data demonstrated that CpG ODN enhanced the transport of H9N2 WIV via TEDs of nasal DCs.