Adjuvant Effect And The Underlying Mechanism Of Salmonella Flagellin In Subunit Vaccine Of H7N9 Avian Influenza

Since the first reported case of human infection with a novel avian influenza A(H7N9)virus in March 2013 in China,there have been five epidemics,resulting in 1567 infections and a mortality rate of nearly 40%.The highly pathogenic H7N9 avian influenza virus,which broke out in the fifth epidemics,poses a serious threat to poultry trade and live poultry markets.Meanwhile,the number of infected people has also increased sharply and the scope of infection has been further expanded,which has aroused widespread concern at home and abroad that these viruses are the subtypes most likely to cause the next influenza pandemic.Therefore,a safe and effective vaccine against this subtype is urgently needed.Compared with traditional vaccines,subunit vaccines confer a higher level bio-security,and reduced vaccine production times.However,the immunogenicity of subunit vaccine is weak,and an appropriate adjuvant is required to enhance its immunogenicity.Toll-like receptor(TLR)agonists are considered potential candidate adjuvants because they are capable of recognizing pathogen-associated molecular patterns and initiating an immune response.Flagellin is a TLR5 ligand.Its adjuvant activity has been continuously verified in a variety of pathogens.However,whether it can induce antigen-specific cellular immune response to subunit vaccine of H7N9 influenza remains unclear,while virus-specific cellular immune responses play a critical role in virus clearance during influenza infection.Because of differences in the immune systems of mammals and poultry,the adjuvant effect and mechanism of flagellin in poultry is not entirely clear,such as the activation of innate immune system and the immune response types(Thl or Th2)induced by flagellin fused with HA1-2 antigen.Flagellin has been deemed as a potent adjuvant candidate.However,its high antigenicity and potential for causing inflammatory injury might restrict its clinical application.The potent antigenicity of flagellin and severe systemic inflammation induced by high-dose flagellin indicates that immunity to flagellin might affect its potency and induce side effects,as an adjuvant.Thus,efforts must be made to reduce potential adverse effects induced by flagellin while maintaining its adjuvanticity.Developing modified flagellin adjuvants with unique properties is a major challenge in manipulating immune responses.Flagellin has been well characterized for its adjuvant activity owing to its TLR5 and NLRC4 binding sites located in a domain.Activation of MyD88-dependent pathways and inflammasomes,respectively,promotes the expression of various cytokines,to further activate the immune and non-immune cells.Macrophages,located in various tissues,are the first line of defense against infection of innate immune system.RNA-seq is the rapid high-throughput sequencing platform,and used in comprehensive acquisition of almost all transcripts and gene sequences of a specific cell or tissue in a certain state through which can reflect the expression and regulation of genes in cells at the overall level.Therefore,it can be used as an important means to study the interaction between flagellin and macrophages,so as to explore the new molecular mechanism of flagellin adjuvant.1.The adjuvant effect of flagellin in subunit vaccine of H7N9 avian influenza in miceIn this study,we evaluate the adjuvant activity of the fusion protein HA1-2-FliC in vivo and in vitro.HA1-2 antigen of avian influenza A(H7N9)was used as a control.Compared with HA 1-2 treated group,we found that flagellin significantly increased the expression levels of cytokines and chemokines(IL-1β,IL-6,TNF-α,CXCL2 and CXCL10)in BMDCs and spleen lymphocytes in vitro,suggesting that flagellin activated immune responses of these immune cells.Then,the intraperitoneal administration experiment was used to assess their effects on the immunogenicity of the recombinant HA1-2 subunit vaccine of H7N9 influenza.The fusion protein HA1-2-FliC could induce significantly higher HA 1-2-specific IgG and hemagglutination inhibition titers than HA 1-2 alone at 12 days post-boost.Meanwhile,the HA1-2-FliC induced higher IgG2a and IgG1 levels,which is indicative of a mixed Thl/Th2-type profile.Consistent with this,significant levels,and equal numbers,of IFN-γ-and IL-4-producing cells were detected after HA1-2-FliC vaccination.Moreover,the marked increase in CD69 expression and the proliferative index with the HA1-2-FliC vaccines indicated that FliC adjuvanted vaccine candidate effectively induced antigen-specific cellular immune responses.Taken together,our findings indicate that the HA1-2-FliC vaccine candidate promote the activation of immune cells,and elicit effective and HA1-2-specific humoral and cellular immune responses,offering significant promise for the development of a successful recombinant HA1-2 subunit vaccine for H7N9 influenza.2.The adjuvant effect of flagellin in subunit vaccine of H7N9 avian influenza in chickensIn this study,we determined flagellin-induced expression profiles of cytokines and chemokines in different types of avian immune cells in vitro and ex vivo.We found that flagellin significantly increased the expression levels of CXCL inflammatory chemokines(CXCLil and CXCLi2)and CCL chemokines(MIP-1β and MCP-3)in avian macrophage HD 11 cells.In addition,HA1-2-FliC induced significant upregulation of cytokines(IL-1β,IL-6,IL-18 and IFN-γ)and chemokines(CXCLi1,CXCLi2 and MIP-1β)in ex vivo splenic lymphocytes and peripheral blood mononuclear cells(PBMCs),suggesting that flagellin promoted immune responses of avian cells in vitro.We also evaluated specific humoural and cellular immune responses induced by HA1-2-FliC and found that chickens immunised intramuscularly with HA1-2-FliC showed significantly higher HA 1-2-specific IgG titers in serum.Furthermore,HA1-2-FliC potentiated cellular immune responses,as reflected by an increase in CD4+and CD8+T cells and proliferation of PBMCs.Significantly higher levels of IFN-γ and IL-4 in PBMCs from chickens vaccinated with HAl-2-FliC further indicated that FliC promoted a balanced Th1/Th2 immune response.SPF chickens were immunized intranasally to evaluate the effect of flagellin as a mucosal adjuvant for the H7N9 avian influenza subunit vaccine.The results showed that immunization with HA1-2-FliC candidate vaccine significantly increased the levels of HA1-2-specific IgG in serum and HA1-2-specific IgA in nasal and bronchoalveolar lavage fluids.Furthermore,the results of virus challenge showed that chickens receiving FliC adjuvant vaccine exhibited robust immune responses leading to a significant reduction in viral loads of throat and cloaca compared to chickens receiving only HA1-2.In conclusion,we demonstrated that the use of the flagellin as an adjuvant potentiated immunogenicity of influenza subunit vaccine HA1-2 in vitro and in vivo.These findings provide a basis for the development of H7N9 influenza HA1-2 subunit vaccines for chickens.3.The adjuvant effect of modified flagellin FliCΔD2D3 in subunit vaccine of H7N9 avian influenzaHere,we constructed an improved fusion protein,HA1-2-FliCAD2D3,with HA1-2 fused to the FliCΔD2D3(lacking the hypervariable-region domains D2 and D3 of FliC).HA1-2-FliCΔD2D3 exhibited efficient immunoreactivity and TLR5 agonist efficacy,and promoted innate immune-response activation in mouse macrophages,peripheral blood mononuclear cells,and splenocytes,based on cytokine-and chemokine-expression profiles.BALB/c mice immunized with HA1-2-FliCΔD2D3 showed significantly lower systemic inflammatory responses comparing with HA1-2-FliC in the early stage of immunity with significantly down-regulated serum-cytokine levels(IL-6,IL-12p40,TNF-αand IL-23p19).After 12 days of boost vaccination,HA1-2-FliCΔD2D3 highly reduced flagellin-specific antibody production,without affecting HA1-2-specific antibody production and cellular immune responses.Enhanced IFN-y/IL-4 generation suggested that HA1-2-FliCΔD2D3 maintained balanced Thl/Th2 immune responses.Furthermore,virus challenge was performed in a chicken model.The results showed that chickens receiving FliCΔD2D3 adjuvant vaccine induced high levels of serum antibody responses,and exhibited a significant reduction of viral loads in throat and cloaca compared to chickens receiving only HA 1-2.In conclusion,we constructed the H7N9 influenza subunit vaccine candidate HA1-2-FliCΔD2D3,with reduced immunogenicity against FliC and lower adverse events.The improved adjuvant FliCΔD2D3 can potentially help in developing safe and effective universal influenza vaccines for humans.4.The preliminary study of IFN-β production induced by flagellinIn this study,transcriptome sequencing(RNA-seq)was used to analyze the expression level of RAW264.7 induced by flagellin compared with unstimulated cells.The sequencing report showed that a total of 2204 differentially expressed genes(DGEs)were screened between the two groups,including 1114 up-regulated genes and 1090 down-regulated genes.The KEGG Pathway showed the number of DGEs enriched in immune-related pathways was the largest.The significant DGEs in GO analysis mainly focused on metabolism,positive regulation,immune response and defense biological processes.Notably,flagellin significantly increased the expression of IFN-β in RAW264.7 cells.The results of RNA-seq were verified by qRT-PCR and ELISA.Moreover,it was demonstrated that flagellin increased expression of IFN-β in BMMs and BMDCs.To further exclude the effect of other factors in this study,the FliC-FljB-and trypsin-treated FliC were added as the control groups in the stimulation of RAW264.7 cells.The results showed that neither FliC-FljB-nor trypsin-treated FliC could increase the expression of IFN-β,which further proved FliC itself has the ablility to promote the up-regulation of IFN-β.Based on the structural characteristics of flagellin,His-tagged fusion proteins FliC,N-FliC,C-FliC and FliCΔD2D3 were constructed and expressed in prokaryotes system,which had good immunoreactivity,and FliC and FliCΔD2D3 had good TLR5 ligand activity.After stimulating RAW264.7 cells,FliC,C-FliC and FliCΔD2D3 proteins significantly increased the expression of IL-1β and IFN-β.The N-FliC was consistent with the negative control,indicating that the key domain of flagellin promoting the up-regulation of IFN-β in RAW264.7 was C-FliC,which was consistant with IL-1β.These findings provide new ideas for further study of the mechanisms of flagellin adjuvant.