| Porcine epidemic diarrhea(PED)is an acute and highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV),with a mortality rate up to 90%.The research results in this laboratory showed that the antibody levels produced by the PED-TGE vaccine based on nano-oil adjuvant(hereinafter referred to as nano adjuvant)were much better than those of commercial vaccines.In order to evaluate the immune effect and the mechanism of the nano-adjuvant through comparing with traditional oil adjuvant,and the ovalbumin(OVA)and PEDV were selected as antigen in this experiment to prepare OVA emulsified by nano-adjuvant(Nano-OVA),PEDV emulsified by nano-adjuvant(Nano-PEDV)and OVA emulsified by 201 adjuvant(201-OVA),PEDV emulsified by 201 adjuvant(201-PEDV).This study provided a preliminary evaluation of the immune effect induced by the nano-adjuvant and provided a theoretical basis for its clinical application.Firstly,FITC-labeled ovalbumin(FITC-OVA)was used as the model antigen.In order to determine whether or not the different adjuvants effected the residence time of the antigens and draining lymph nodes,Nano-OVA or 201-OVA were respectively injected into C57BL/6 mice subcutaneously through the foot pad.The results showed that compared with 201-OVA and contrast,Nano-OVA could extend antigen residence time,distribute widely and mediate more cells targeting to mice’s popliteal lymph nodes observed by small animal imaging and immunofluorescence.In order to explore the effects of the phagocytosis,cross-presentation of antigens by DC2.4 cells and activation of DC2.4 cells mediated by nano-adjuvant,Nano-OVA or 201-OVA were respectively co-incubated with DC2.4 cells.The flow cytometry results showed that the levels of FITC-OVA~+%and PE-25-D1.16~+%of DC2.4 cells were extremely significantly higher in the Nano-OVA group(p<0.001),comparing with that in 201-OVA group.The qPCR results showed that the transcriptional levels of CD86 and IL-6 m RNA were extremely significantly higher in DC2.4 cells treated with Nano-OVA than that in DC2.4 cells with 201-OVA treatment(p<0.01)and the transcriptional level of IL-12p40 m RNA was significantly higher in DC2.4 cells treated with Nano-OVA than that in DC2.4 cells treated by 201-OVA(p<0.05).To further explore the ability of PEDV emulsifying with nano-adjuvant to induce adaptive immune response,the representative epidemic virus strain of PEDV G2(LNCT)was selected as the immunogen.This part was taking mice as the animal model:In order to explore the effect of the maturation of DC2.4 cells induced by nano-adjuvant,Nano-PEDV or 201-PEDV were separately co-incubated with DC2.4cells.The results of flow cytometry showed that compared with 201-PEDV and contrast,Nano-PEDV induced DC2.4 cell maturation through up-regulating CD86 all the way and up-regulating CD80 after long-term incubation.To further investigate the maturation(CD80 expression)of different dendritic cells(DCs)in the spleen of mice induced by nano-adjuvant,Nano-PEDV or 201-PEDV were respectively subcutaneously injected into foot pads of C57BL/6 mice.The flow cytometry results showed that after 38 hours of immunization,Nano-PEDV could not induce the maturation of CD11b~+CD11c~+DCs and CD11c~+DCs in the spleen of mice by upregulating the level of CD80 compared to 201-PEDV and contrast;and compared with 201-PEDV,Nano-PEDV couldn’t induce the maturation of CD8α~+CD11c~+by upregulating the level of CD80.On the contrary,the flow cytometry results showed that compared with 201-PEDV and contrast group,the mean fluorescence intensity of PE-CD80 in the Nano-PEDV group significantly enhanced(p<0.001)at 42 days of immunization.In order to explore the effect of nano-adjuvant on the activation of dendritic cells differentiated from bone marrow(BMDCs)and spleen cells in mice,Nano-PEDV or 201-PEDV were respectively co-incubated with mice’s BMDCs and spleen cells,the qPCR results identified that compared with 201-PEDV and contrast,Nano-PEDV could better elevate the transcriptional level of IL-12p40 which is helpful for cellular immunity response rather than IL-6 that mediated humoral immunity in mice’s BMDCs and spleen cells.In order to explore the immune response types and cytokines induced by nano-adjuvant of mice at 42 days of immunization,Nano-PEDV or 201-PEDV were respectively subcutaneously inoculated into C57BL/6 mice through the back.Compared with 201-PEDV and control group,the mean fluorescence intensity of IFN-γ~+/CD8~+T cells in Nano-PEDV group in spleen cells extremely significantly improve(p<0.01),the mean fluorescence intensity of IFN-γ~+/CD4~+T cells in Nano-PEDV group in spleen cells extremely significantly increase(p<0.001),the mean fluorescence intensity of IFN-γ~+/CD8~+T cells in Nano-PEDV group in LNs enhance(p<0.05),the mean fluorescence intensity of IFN-γ~+/CD4~+T cells and IL-4~+/CD4~+T cells in LNs;IFN-γ~+/CD4~+T cells in blood have no significant difference(p>0.05).The ELISA results showed that compared to 201-PEDV and contrast,the levels of IFN-γand TNF-αsecreting by mice which inoculated with Nano-PEDV were higher in spleens indicated that Nano-PEDV can better induce cellular immune responses,while the improvement of humoral immunity is not mediated by the secreting levels of IL-21 and IL-4.In order to evaluate the level of neutralizing antibody induced by nano-adjuvant in mice,the neutralizing antibody test proved that the level of neutralizing antibody was significantly enhanced by vaccinating nano-adjuvant vaccine relative to 201 adjuvant vaccine.Next,piglets were used as animal models.In order to explore the ability of activation in piglet BMDCs by nano-adjuvant,the piglet BMDCs were respectively threated with Nano-PEDV or 201-PEDV.The flow cytometry results showed that the mean fluorescence intensity of MHC-I was extremely significantly higher(p<0.001)on piglet BMDCs that threated with nano-adjuvant than that treated with 201-adjuvant,but there was no difference(p>0.05)about MHC-II molecule between them.The qPCR results testified that compared with 201-PEDV and control group,the transcriptional levels of IL-12p40,IL-1βand TNF-αin piglet BMDCs were significantly higher in the Nano-PEDV group,rather than IL-6;compared with the contrast,Nano-PEDV and 201-PEDV could both promote the transcription of TLR9to some extent,but there was no difference between them.To sum up,nano-adjuvant can be used as adjuvant of virus vaccine to enhance the immune responce of the body,because of its superior production ability of neutralizing antibody and CTL immune response proved by mice.Nano-adjuvant exerts the immune function mainly through extending antigen residence time,targeting more lymphocytes,promoting the phagocytosis of antigens by DCs and last stimulating the maturation of DCs for a long time.What caused the mechanism of cellular immunity mediated by nano-adjuvant were the high expression of MHC-Ⅰ,TNF-αand IFN-γproteins and high transcription of IL-12. |
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