Research On Construction And Immunological Properties Of Hantaan Virus Chimeric Virus Like Particle

【Background】A safe and effective hantaan virus (HTNV) vaccine is highly desirable since HTNVcauses an acute and often fatal disease (hemorrhagic fever with renal syndrome, HFRS) inChina. The HFRS inactivated vaccine has many shortcomings such like the poorimmunogenicity to elicit neutralizing antibodies and cell-mediated immunity, though ithas been used and plays a positive role in the prevention of HFRS occurrence andepidemic. Since HFRS inactivated vaccine is less effective and live attenuated HTNV isnot accepted as a vaccine due to safety concerns, HTNV virus-like particle (VLP) offer anattractive safe alternative because they lack the viral genome yet they are perceived by theimmune system as a virus particle. It has been reported that adding immunostimulatorysignals to VLP would enhance their efficacy as well. To accomplish this, we generatedchimeric HTNV VLP containing glycosylphosphatidylinositol (GPI)-anchoredgranulocyte-macrophage colony-stimulating factor (GM-CSF) or CD40ligand (CD40L)and investigated their biological activity in vitro and immunogenicity in vivo. Taken together, we have set up a solid foundation for development of HFRS genetic engineeringvaccine.【Methods】Plasmids, respectively containing cDNAs encoding HTNV76-118M, S and mouseGPI-GM-CSF, CD40L, were digested to obtain DNA fragments of M, S, GM-CSF andCD40L respectively, then these fragments were respectively ligated into the pFastBacTMDual vectors with the polyhedrin (polh) promoter. All the recombinant DNA constructswere confirmed by restriction enzyme digestion and DNA sequencing, then subsequentlytransformed Escherichia coli DH10BacTMcells to isolate recombinant bacmid,respectively. All the recombinant bacmids were confirmed by PCR amplification, thensubsequently transfected into Sf9insect cells to produce rBVs (rBV-M, rBV-S,rBV-GM-CSF, rBV-CD40L). IFA was used to determine the expression of rBVs.For HTNV VLP, Sf9insect cells were coinfected with rBV-M and rBV-S. ForVLP-GM-CSF, Sf9insect cells were coinfected with rBV-M, rBV-S and rBV-GM-CSF.For VLP-CD40L, Sf9insect cells were coinfected with rBV-M, rBV-S and rBV-CD40L.Electronic microscopy was firstly used to examine the production of VLP in sf9cells andculture supernatant, then the VLP was further purified through a20-60%discontinuoussucrose gradient. ELISA, SDS-PAGE, WB, Co-IP were used to identify the VLP.FCM was used to examine the functional characteristic of VLP-GM-CSF andVLP-CD40L through detection of bone marrow cell proliferation, bone marrow celldifferentiation and B-cell activation. C57BL/6mice were equally divided into sevengroups with eight mice per group (three experimental groups and four control groups). Thethree experimental groups were subcutaneously injected with purified VLP，VLP-GM-CSF or VLP-CD40L, and the four control groups were subcutaneously injectedwith purified VLP with soluble recombinant mouse GM-CSF or CD40L (VLP+GM-CSF,VLP+CD40L), the HFRS inactivated vaccine or PBS, respectively. ELISA and Microneutralization test were used to detect the HTNV NP-or GP-specific antibody andneutralizing antibody. ELISPOT was used to detect the cytokines secreted by splenocyte.Cytotoxicity assay was used to detect the level of specific toxicity from splenocyte. Animal protection experiments in our research were HTNV antigens in the tissuesdetection by ELISA, HTNV nucleic acid in the tissues detection by qRT-PCR andhistological analysis by hematoxylin and eosin (HE) staining.【Results】After enzyme digestion, the results of Agarose gel electrophoresis suggested that wesuccessfully constructed the rBV transfer vectors. The sequencing results confirmed theconstruction. The results of PCR suggested that we successfully construct the rBVbacmids. The results of IFA suggested that all the target proteins are found to be expressedin sf9insect cells.HTNV VLP were produced by sf9insect cells coinfected with rBV-M and rBV-S,HTNV VLP-GM-CSF were produced by sf9insect cells coinfected with rBV-M, rBV-Sand rBV-GM-CSF, HTNV VLP-CD40L were produced by sf9insect cells coinfected withrBV-M, rBV-S and rBV-CD40L. We collected the cells coinfected with rBVs to detect theproduction of VLP by electron microscope, the particles sizes ranged from approximately80to220nm in diameter. We then harvested VLP from the culture supernatants. The VLPwere routinely tested for integrity and homogeneity by electron microscope, the particlessizes ranged from approximately80to220nm in diameter. We then harvested VLP fromthe culture supernatants and purified them successfully by using sucrose gradientultracentrifugation. After purification, the purified VLP were also analyzed by SDS-PAGEand western blot. From the results of SDS-PAGE and WB, all the VLP showed the proteinbands that were similar to the molecular weights of Gn, Gc, NP, GM-CSF, CD40Lrespectively. The results of ELISA and Co-IP suggested that GM-CSF or CD40L is exactlyincorporated into the HTNV VLP.The results of FCM suggested that all the VLP play critical roles in the proliferationand differentiation of bone marrow cells, activation of B cells. VLP-GM-CSF had strongerbiological activities than VLP-CD40L and VLP (p<0.05). The results of ELISA and Microneutralization test suggested that all the VLP can induce the C57BL/6to produce theHTNV NP-or GP-specific antibody and neutralizing antibody. The antibody titers ofVLP-CD40L and VLP-GM-CSF groups were higher than VLP, inactivated vaccine and PBS groups. The VLP-CD40L group was the highest (p <0.05). The results of ELISPOTsuggested that splenocytes from VLP-CD40L or VLP-GM-CSF immunized micestimulated with HTNV purified NP or GP demonstrate a significantly higher numbers ofIFN-γ and IL-2spots compared to the VLP, inactivated vaccine and VLP+GM-CSF orVLP+CD40L groups. The highest number derived from the VLP-CD40L group (p <0.05).The results also suggested that IL-4and IL-10levels do not change remarkably for any ofthe immunization groups (p>0.05). The results of cytotoxicity assay suggested that thecytotoxicity of splenocytes from mice immunized with VLP is enhanced in accordancewith the E/T ratio, which is the most significant at the ratio of100:1. Among theexperimental groups, splenocytes from mice immunized with VLP-CD40L orVLP-GM-CSF group stimulated with HTNV purified NP or GP showed higher specificcytotoxic activity compared to the VLP group, inactivated vaccine and VLP+GM-CSF orVLP+CD40L groups. The highest specific cytotoxic activity occurred in the VLP-CD40Lgroup (p <0.05).The results from ELISA and qRT-PCR suggested that the HTNV specific antigensand nucleic acids are detected in the livers, spleens and kidneys of C57BL/6mice in thePBS group. However HTNV specific antigens and nucleic acids could not be detected inthe livers, spleens and kidneys of C57BL/6mice from all experimental groups orinactivated hantavirus vaccine group. HTNV specific antigens and nucleic acids could notbe detected from other tissues of C57BL/6mice in all groups. The results fromhistopathological analysis by HE staining suggested that no significant alterations arefound in the tissues from all the groups except spleens of PBS group. The resultssuggested some pathological changes such as diffuse infiltration of lymphocytes, diffusehemorrhage and increase of white pulp in the spleens of naive group, but not in othergroups.【Conclusion】In this study, we have demonstrated for the first time that membrane-bound forms ofthe immunostimulatory molecules GM-CSF and CD40L could be incorporated intoHTNV VLP in a functionally active form when expressed in insect cells coinfected with rBVs expressing HTNV GP, NP, and GPI-anchored GM-CSF or CD40L. Furthermore, wedemonstrated that these GM-CSF and CD40L molecules incorporated into VLPmaintained their biological activities and that immunization with chimeric HTNV VLPprotected C57BL/6mice from HTNV challenge. We also found that GM-CSF and CD40Lincorporation into HTNV VLP induced high levels of humoral and cellular immuneresponses and protected the C57BL/6mice from HTNV challenge. This studydemonstrated that the surfaces of VLP can be decorated with biologically active molecules,as well as the potential application of HTNV VLP-GM-CSF or HTNV VLP-CD40L as anew type of HTNV vaccine. Further studies are needed to investigate the process ofincorporation and to increase these levels of incorporation into VLP as well as todetermine how immune responses generated.