Biological Activity And Long-term Immune Response Of Chimeric Hantaan Virus-like Particles

【Background】Infection of Hantaan virus(HTNV)usually causes hemorrhagic fever with renal syndrome(HFRS).There is no specific effective therapy so that vaccines will be the useful method to control the virus infection.VLPs usually emerge in natural virus life cycles,which consist of viral structural protein.The formation of VLPs can be achieved by simply expressing one or several factors like structural proteins,and can be genetically altered to insert foreign gene and display desired molecular on the surface,they are widely used in researches of vaccines and mechanism of virus infection.The VLP approach appears promising and advantageous over many other structural forms of vaccines.VLPs have been found to provide high immunogenic potency and safety in protecting against various pathogens.As an adjuvant,granulocyte-macrophage colony stimulating factor(GM-CSF)or CD40 ligand(CD40L)incorporated into VLP was able to enhance the immune responses.We have already constructed the HTNV VLPs by expressing the nucleoprotein(NP)and glycoprotein(GP)conjugated GM-CSF or CD40 L in eukaryotic cells.In this study,we explored the biological activity and long-term immune responses of HTNV VLPs in order to provide evidence for the further application of HTNV VLPs in clinic.【Methods】We separated the bone marrow cells of the C57BL/6 mice and incubated with HTNV VLP、VLP-GM-CSF or VLP-CD40 L seperately,and then detected the numbers of F4/80+ CD11b+ cells、CD11c+ cells and the expression level of MHC-Ⅱ、CD40、CD86 expressing on the bone marrow cells by flow cytometry in order to estimate the role of VLP in promoting the bone marrow cells activation.Then,we separated the lymphocytes from wild-type C57BL/6 mice or the mice immunized with HTNV VLP、VLP-GM-CSF or VLP-CD40 L and detected the numbers of CD4+ CD25+、CD8+ CD25+ T lymphocytes and B220+ CD69+ B lymphocytes by flow cytometry in order to estimate the role of HTNV VLPs in promoting the T、B lymphocytes activation.C57BL/6 mice were grouped and separately immunized with three types of HTNV VLPs or HTNV inactivated vaccines for 6 months.6 months after the primary immunization,all the mice were immunized with the same amount HTNV VLPs or HTNV inactivated vaccines once again.The effect of of humoral immunity was evaluated by indirect enzyme-linked immunoadsordent assay(ELISA)to detect the NP specific antibodies and by micro-neutralization assay to detect the neutralizing antibodies.At the same time,the cellular immunity effect was evaluated by detecting the level of the cytokines(IFN-γ and IL-4)by enzyme-linked immunospot assay(ELISPOT)and the cytotoxic T lymphocyte assay.The mice were challenged with HTNV before and after boost.Then we detected the antigen by sandwich ELISA,the virus nucleic acid by quantitative reverse transcription-polymerase chain reaction(RT-qPCR),and observed the pathological changes of heart、liver、spleen、lung、kidney and brain by HE staining.【Results】FCM results showed that three HTNV VLPs increased the numbers of F4/80+ CD11b+ cells、CD11c+ cells and the expression of MHC-Ⅱ、CD40、CD86 on bone marrow cells,which indicating that HTNV VLPs promoted bone marrow cells differentiate into APCs and induce APCs maturation.Mean while,three HTNV VLPs activated T lymphocytes and B lymphocytes in vitro and in vivo.VLP-GM-CSF had an obvious role in promoting the bone marrow cells to differentiate into APC and enhancing the activity of T lymphocytes in vitro and in vivo.VLP-CD40 L enhanced the activity of B lymphocytes obviously in vitro and in vivo.After long-term immune response,there were little HTNV specific antibodies and a little neutralizing antibodies detected in HTNV inactivated vaccine immunized group.There were much more HTNV NP specific antibodies and neutralizing antibodies in HTNV VLPs immunized mice than HTNV inactivated vaccine immunized mice.The antibodies in VLP-GM-CSF and VLP-CD40 L immunized mice were more than that of HTNV VLP immunized mice.The antibodies of all the groups were increased after boost.We detected the levels of IFN-γ and IL-4 by ELISPOT assay after 6 months.There was no difference in IFN-γ between the HTNV inactivated vaccine immunized group and the saline control group.The level of IFN-γ of three HTNV VLPs immunized groups were higher than that of the HTNV inactivated vaccine immunized group.IFN-γ was increased in all groups after boost.There was no difference in IL-4 levels of all groups before and after boost.The results of CTL killing assay were consistent with the results of IFN-γ detected by the ELIPOT.6 months later,all the immunized mice were challenged with HTNV.The quantity of HTNV antigen and nucleic acid were higher in the saline control group and the HTNV inactivated vaccine immunized group.The pathological changes appeared in livers、kidneys and lungs,such as inflammatory cell infiltration in the two groups.It indicated that HTNV inactivated vaccine had no protection effect for HTNV infected mice after long-term immune response.On the contrary,there were low antigen and nucleic acid and no pathological changes in the HTNV VLPs immunized groups.After boost,HTNV inactivated vaccine and HTNV VLPs both had protective effect for HTNV challenged mice.【Conclusion】In this study we explored the biological activities and long-term immune responses of the three HTNV VLPs.The results showed that HTNV VLPs had good effect in vitro and in vivo.It indicated that the HTNV inactivated vaccine did not have long-term immune protection for the mice challenged with HTNV.HTNV VLPs had good long-term immune protection effect,among them VLP-GM-CSF and VLP-CD40 L had a better effect than VLP.HTNV VLPs can protect mice against virus challeng after long-term immune response,which indicated that chimeric HTNV VLPs had a great potential against HTNV infection.