CpGs Assist The Whole Inactivated H9N2 Influenza Virus In Crossing The Intestinal Epithelial Barriers Via Transepithelial Uptake Of Dendritic Cell Dendrites

The rise and spread of avian influenza virus not only have caused enormous economic losses,but also have seriously increased the risk of a new influenza pandemic.Besides the respiratory tract dissemination,avian influenza viruses also colonized in the intestinal tract of ducks and then disseminated through duck wastes.Vaccination through the oral route can efficiently block viral attachment and subsequent colonization,and then reduce viral shedding from the intestinal tract.Moreover,inactivated vaccines bear the irrefutable advantage of safety,but they seem to be less effective than live attenuated ones for mucosal stimulation.Our previous study demonstrated that CpGs remarkably improved the immunoprotective efficacy of the H9N2 whole inactivated influenza viruses(H9N2 WIV)via oral immunization in ducks by enhancing the level of local and systemic immune responses.However,intestinal mucosa remains a pivotal barrier for influenza oral vaccine delivery and subsequent antigen specific adaptive immune responses,particularly for WIV that are abolished in viral replication.Based on this,we postulated that CpGs also might play a significant role in transepithelial transport of influenza WIV in gut.Therefore,firstly,we explored whether CpGs could assist H9N2 WIV in enhancing both local and systemic immune responses after oral immunization in mice.Secondly,we used DC/Caco-2 coculture system in vitro and intestinal ligated loop model in vivo to study how CpGs provided assistance for transepithelial delivery of H9N2 WIV.Finally,it is clear that maturation of DCs is crucial for the initiation of downstream immune responses.We studied whether CpGs could assist H9N2 WIV in enhancing the maturation of DCs.Our research is divided into the following three parts in detail:1.Effects of CpGs plus H9N2 WIV on both local and systemic immune responses after oral immunization in miceTo analyze the adjuvanticity of CpGs in response to H9N2 WIV,we orally immunized mice with phosphate-buffered saline(PBS),H9N2 WIV alone or combined with CpGs.According to the "Common Mucosal Immune System" theory,we found that the mucosal IgA levels in small intestinal,tracheal,and lung wash were all enhanced.For systemic immunity,the serum antigen-specific IgG,IgG1,and IgG2a/c antibody titers induced by CpGs plus H9N2 WIV were substantially greater than antigen-alone treatment(P<0.01).Besides,serum collected from mice immunized with CpGs plus H9N2 WIV showed powerful ability to inhibit hemagglutination against 4-HA units of reference antigens compared with antigen alone(P<0.01).Furthermore,lymphocytes were isolated from the mesentery lymph nodes(MLNs)and spleen,and restimulated with H9N2 WIV in vitro.We found that the CD69 expression,the proliferative index,and the percentage of CD3+CD4+T cells were all markedly increased in the group of CpGs plus H9N2 WIV compared with that of the antigen alone.The results indicated that oral immunization H9N2 WIV plus CpGs effectively induced local mucosal and systemic immune responses in mice.2.CpGs assist the whole inactivated H9N2 influenza virus in crossing the intestinal epithelial barriers via transepithelial uptake of dendritic cell dendritesIntestinal mucosa remains a pivotal barrier for the oral vaccine absorption of H9N2 WIV.Here,we reported both in vitro and in vivo that CpGs combined with H9N2 WIV were capable of recruiting additional dendritic cells(DCs)to the intestinal epithelial cells(ECs)to form transepithelial dendrites(TEDs)for the uptake of luminal virus and CpGs.Moreover,the mechanism of CpGs was independent of epithelial transcytosis and disruption of the epithelial barriers.Furthermore,Both CD103+ and CD103-intestinal DCs participated in this process.Virus-loaded CD103+ but not CD 103-DCs also quickly migrated into MLNs within 2 h.DC recruitment and TED formation were observed in the terminal ileum and jejunum.However,in Peyer's patches(PPs),DC recruitment was detected in the subepithelial dome,whereas the number of TEDs was very rare in the follicle-associated epithelium(FAE).The engagement of the chemokine CCL20 from the apical ECs and the DCs drove DC recruitment and TED formation.Our findings indicated that CpGs improved the delivery of H9N2 WIV via TEDs of intestinal DCs,and this may be an important mechanism for downstream effective antigen-specific immune responses.3.CpGs assist H9N2 WIV in enhancing the maturation of DCs in in vitro coculture systemIt is clear that maturation of DCs is crucial for the initiation of downstream immune responses.We studied whether CpGs assisted H9N2 WIV in enhancing the maturation of DCs.After incubation of medium,CpGs,and/or H9N2 WIV on the apical side of the Caco-2 monolayer for 24 h,the basolateral DCs and culture supernatants were collected and DCs were analyzed by FACS.The results showed that the expressions of CD40,CD80,CD86,and MHCII were remarkably upregulated in the group of CpGs plus H9N2 WIV compared with that of H9N2 WIV alone.Furthermore,we assessed the functional maturation of DCs by detecting the release of cytokines(interleukin(IL)-10,IL-12p70,and IL-23)from the supernatant of the basolateral side,and the results showed that DCs responded to CpGs plus H9N2 WIV with significant increase in these cytokine secretion compared with viruses alone.Finally,to estimate whether DCs could be as fully functional antigen-presenting cells,another part of the collected DCs was tested for their ability of stimulating allogeneic T cells.Similarly,DCs that were from CpGs plus H9N2 WIV group obviously promoted the proliferation of allogeneic T cells compared with those from H9N2 WIV-only group.However,in above trials,DNase treatment was able to reverse the inductive effect of CpGs.Collectively,these findings indicated that CpGs had the capability of assisting H9N2 WIV in enhancing the maturation of DCs in in vitro coculture system.