| H9N2 subtype avian influenza is endemic in all kinds of poultry population throughout China since 1992,resulting in significant economic losses to the poultry industry.During 2010-2013,there appeared the second outbreak of H9N2 avian influenza in China.It is evidenced that the novel G57 genotype of virus is responsible for the epidemic and the current vaccines cannot provide full protection against it.In this study,five H9N2 isolates from vaccinated chickens in Jilin province of China in 2012 are phylogenetically distinguishable from other clades within the Y280-like sublineage and grouped into the G57-like clade.It further showed that there is a definite difference in antigenicity between G57-like and other clades in the Y280-like sublineage,indicating significant antigenic difference,which promotes us to develop a novel H9N2 candidate vaccine in order to mobilize efficacious immunity against the dominant G57 field strains at present.To resist infection caused by the G57 genotype of H9N2 influenza virus widely prevailing in chicken population in China at present,in this study,a novel DC-targeted mucosal vaccine was constructed using L.plantarum NC8 as a delivering vector to specifically direct the HA protein to mucosal DCs.A series of tests in vivo and vitro were performed to analyze the effects of the targeted vaccine on the maturation,migration and differentiation of mucosal DCs,and the potential of inducing T cell proliferation and polarization in order to initially reveal the functionary mechanism of DC-targeted vaccine in mucosal immune and acquired immune response.The hallmark of mucosal immunity and its distinction from adaptive immunity is the local secretion of SIgA.Thus,we measured pulmonary,intestinal and fecal SIgA.The indirect ELISA results showed that the subjects immunized with Lp-HA-DCpep induced a significantly higher SIgA level in BALF,ILF and feces compared with those of Lp-HA and Lp groups throughout the experiment.In particular,there is a significant difference between Lp-HA-DCpep and other groups after challenge,indicating that Lp-HA-DCpep is more effective in activating local mucosal immune response.As to IgG level,it demonstrated that the specific IgG level of Lp-HA-DCpep group was slightly lower than that of Lp-HA group during immunization.However,the IgG level of Lp-HA-DCpep group significantly increased after challenge.In addition,Lp-HA-DCpep secreted significantly higher levels of serum cytokines including IFN-γ,TNF-α,IL-4,IL-6,IL-10,and IL-12p70 than other groups.To confirm the protective efficacy of recombinant Lactobacilli,further IHC detection of virus antigen in lung tissues after challenge was performed.It displayed almost no detectable virus antigen and slight pathological change in Lp-HA-DCpep group,indicating that Lp-HA-DCpep is able to induce more effective protection against the G57 genotype of virus.Overall,Lp-HA-DCpep is more efficient in inducing both local mucosal and adaptive immune response.Increased surface molecule expression and cytokine secretion are characteristics of DC maturation.The results showed that the expressions of MHC II,CD40,CD80,and CD86 were up-regulated by recombinant Lactobacilli as detected by flow cytometry.But the mean fluorescence intensity of MHC II and CD86 molecules for Lp-HA-DCpep is significantly higher than that of other groups,indicating that it can more efficiently induce DC maturation.In the meanwhile,all strains are able to promote DCs to secrete TNF-α,IL-6,IL-10 and IL-12p70 as shown by cytokine concentration in DC supernatants.Furthermore,Lp-HA-DCpep induced a significant increase of TNF-α,IL-6 and IL-10.The existence of L.plantarum in intestine provides the possibility of consistent immune regulation,and intestinal DCs are pivotal in activating natural immune responses.To investigate the ability of Lp-HA-DCpep in activating DCs,the antigen uptake and MLR experiments were performed by the phagocytosis experiment and MLR assay.The results indicated that Lp-HA-DCpep is more efficient in being englobed by DCs determined by FITC-Dextran uptake than other groups and thereby more effective in activating T cells as showed by MLR.These data suggest that although recombinant L.plantarum and L.plantarum itself can up-regulate the surface marker molecules of DCs and promote cytokine secretions,Lp-HA-DCpep shows greater potential in inducing DC maturation,presumably because the antigen modified by DCpep is more easily targeted to DCs and thereby to promote the phenotypic and functional maturation of DCs.TLR4 is the main receptor of bacterial antigen,which further activates NF-κB pathway of DCs.To explore whether recombinant Lactobacilli can induce DC activation,we treated DCs with TLR4 or NF-κB inhibitor before Lp-HA-DCpep treatment.The results showed that both of them can successfully block DC activation by down-regulating expressions of DC surface molecules,indicating that Lactobacillus-induced DC maturation is regulated by the TLR4/NF-κB pathway.The up-regulation of CCR7 on DCs is essential for their migration to the T-cell area of secondary lymphoid organs to initiate adaptive immune response,guided by two CCR7 ligands,including CCL19 and CCL21.It was found that Lp-HA-DCpep significantly up-regulated CCR7 expression in DCs.The in Vitro migratory assay of DCs demonstrated that Lp-HA-DCpep-treated DCs can migrate when exposed to CCL19 or/and CCL21 in the transwell system.Moreover,the migration can be partially blocked by CCL19 or/and CCL21 antibodies.These results indicated that Lp-HA-DCpep can induce DC migration triggered by the CCR7-CCL19/CCL21 axis.The in Vivo migratory assay of DCs showed that the number of migratory DCs(CD3-CD11c+MHC II+ DCs)in MLN and spleen for Lp-HA-DCpep kept significantly accumulating till day 42 post immunization,indicating that Lp-HA-DCpep can recruit much more DCs to migrate towards secondary lymphoid organs.DC activation is accompanied by differentiation towards subsets like CD103+CD11b+ and CD103+CD11b-DCs in MLN and CD8α+ and CD8α-DCs in spleen.It was found that the number of CD103+CD11b+ and CD103+CD11b-DCs in MLN of Lp-HA-DCpep group was significantly higher than that of other groups.In spleen,it showed that Lp-HA-DCpep induced the highest number of CD8α+ DCs on days 21 and 42 post immunization.The number of CD8α-DCs showed an obvious significance between Lp-HA-DCpep and others.In general,Lp-HA-DCpep can efficiently promote the subset differentiation of DCs.As DCs are the only APCs that can activate T cells,we examined the number of T cell subtypes with flow cytometry to measure T cell activation in spleen.The results showed that Lp-HA-DCpep induced much more T helper(Th,CD3+CD4+)cells and cytotoxic T(Tc,CD3+CD8+)cells after immunization than other groups,indicating that Lp-HA-DCpep not only targets to DCs,but also promotes migratory DCs to activate na?ve T cells.To further investigate the polarization of Th and Tc cells,we detected the portions of Th1(CD3+CD4+IFN-γ+),Th2(CD3+CD4+IL-4+),Th17(CD3+CD4+IL-17a+),Treg(CD3+CD4+CD25+Foxp3+),Tc1(CD3+CD8+IFN-γ+),and Tc2(CD3+CD8+IL-4+)cells in spleen.The results showed that Lp-HA-DCpep showed better ability in activating Th1,Th2 and Treg cells than other groups on days 21 and 42 post immunization.In terms of Tc cells,the number of Tc1 cells for Lp-HA-DCpep was significantly higher than that of Lp-HA on days 21 and 42 post immunization.But the number of Tc2 cells showed no difference between Lp-HA-DCpep and Lp-HA groups.To sum up,Lp-HA-DCpep can effectively activate Th and Tc cells,and induce Th cells to polarize towards Th1,Th2 and Treg cells.The effects could be enhanced through repeated immunization and prolonged immune period.All these indicate that the DC-targeted oral mucosal vaccine candidate,Lp-HA-DCpep,provides efficient protection against G57 H9N2 infection,through activating DCs by the TLR-induced NF-κB pathway,promoting DC migration by the CCR7-CCL19/CCL21 axis,thereby enhancing the presentation of immunogen to T and B lymphocytes,resulting in skewing T cells polarization towards Th1,Th2 and Treg cells and evoking more efficient mucosal and adaptive immunity responses.Taken together,the presented oral mucosal vaccine strategy using L.plantarum offers tremendous potential for the development of new influenza vaccines,which illustrates the feasibility and efficacy of antigen targeting to DCs through genetic fusion of vaccines to DC-targeting peptides and provides critical insights as to the ability to activate innate immune cells for oral vaccine platforms. |
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