| 【Background】Hantavirus infection is manifested as two different forms of severe febrile diseases,hemorrhagic fever with renal syndrome (HFRS) and Hantavirus pulmonary syndrome(HPS). Most HFRS infections occur in East Asia and Eastern Europe coursed by Hantaanvirus (HTNV), Seoul virus (SEOV) and Puumala virus (PUUV).In China, Hantaviruses result in most severe morbidity and mortality. Inactivatedvaccines have been produced by using rodent brain and cell cultures in China. Althoughthe protective efficacy of several inactivated vaccine have been confirmed, there remains cellular immunity. Moreover, there still some problems concerning their production andhuman use. There is an urgent need to develop new vaccines to prevent the epidemic ofHFRS in China.Hantaviruses are enveloped viruses with a genome that consists of threesingle-stranded, negative sense RNA segments designated S (small), M (medium), and L(large). The S RNA encodes the nucleocapsid (N) protein. The M RNA encodes apolyprotein that is co-translationally cleaved to yield the envelope glycoproteins Gn andGc. Both GP and NP play important role in elicit protective immune response duringHantavirus infection. The Gn and Gc are presumed to be the major elements involved ininduction of neutralizing antibodies during Hantavirus infection. Nevertheless, theimmunogenicity of GP is feeble, the antibody elicited by GP appeared later, the antibodytiter is low. Cellular immunity is believed to be associated with the Hantavirus NP. It issuggested that NP contains antigenic sites involved in CTL responses, which have a grateinfluence on elicit cellular immune response. Experiments show that immunization withNP induces a protective immune response which can increase the survival of animalsfollowing challenge with a lethal dose of Hantavirus. Therefore, utilization of both theglycoprotein and NP components for vaccine development could be a very promisingapproach.Our lab has constructed a series of prokaryotic and eukaryotic expression vectorcontaining HTNV chimeric genes (GnS0.7and GnS0.7) in previous studies. Data showsthat the fusion protein expressed by chimeric genes could elicit humoral immunity andpart of cellular immunity. During our research the adenovirus expression system shows thebest result, but there are still problems in fusion proteins express level and eliciting betterimmune response. Aiming at these problems, we try to optimize the expression vector andimprove the form of interest genes, joint application of molecular adjuvant to elevateantigen expression level and immune response level. The purpose of this study lies here.【Methods】Based on the CAG promoter and HSP70C molecular adjuvant, we take two strategiesto increase the humoral and cellular immune response. Adenovirus vectors containing HTNV structural protein and CTL were constructed through different combinations waysand different expression methods. The humoral and cellular immune response wereassessed by detect mice immunized with recombinant adenoviruses.(1) In consideration of the spatial conformation of GP, fusion expression coulddestruction immunity of GP; we take advantage of ECMV IRES to independently expressGP and NP.(2) Joint application of CTL epitopes (NP127-141aa, NP331-345aa and NP421-429aa)and HSP70C to increase cellular immunity.【Results】Part1Construction and identification of the immunological properties of adenovirusvector containing HTNV GP, IRES, NP and HSP70C1. Adenovirus vector containing HTNV GP, IRES, NP and HSP70C was constructed.Recombinant adenoviruses were obtained by infected HEK293A cells and namedrAd-GnH-IRES-S0.7H, rAd-GcH-IRES-S0.7H, rAd-GnH-IRES-S0.7, rAd-GcH-IRES-S0.7, rAd-Gn-IRES-S0.7H, rAd-Gc-IRES-S0.7H, rAd-Gn-IRES-S0.7and rAd-Gc-IRES-S0.7. The interest proteins of different recombinant adenoviruses were identified by IFA.Specific fluorescence was observed in cells after infection.2. C57BL/6mice were immunized with recombinant adenoviruses. Adeno-lacZ, PBSand inactivated vaccine immunized mice were set as controls. Humoral immunity levelwere assessed by ELISA and cell micro-neutralization test. Results showed that alladenovirus could elicit humoral immune responses incorporation of neutralizingantibodies and GP and NP specific antibodies. Adenovirus incorporation of HSP70C elicitshigher geometric mean titer (GMT)(glycoprotein and NP-specific) than others. Amongadenovirus containing Gn, rAd-GnH-IRES-S0.7H elicit highest GMT (glycoprotein andNP-specific), which were95.1and320.0. Among adenovirus containing Gc, miceimmunized with rAd-GcH-IRES-S0.7H elicit highest GMT (glycoprotein and NP-specific),which were80.0and380.5. Adenovirus incorporation of HSP70C also elicits higherneutralizing antibodies GMT than others. The neutralizing antibodies GMT of miceimmunized with rAd-GnH-IRES-S0.7H (30.3) and rAd-GcH-IRES-S0.7H (40.0) were higher than that mice immunized with inactivated vaccine (p<0.05).3. During CTL assay, the spleno6cytes of mice immunized withrAd-GnH-IRES-S0.7H and rAd-GcH-IRES-S0.7H showed highest specific Tcell-mediated cytotoxicity and the cytotoxicity were higher than inactivated vaccine atdifferent E/T ratio (p<0.05). ELISPOT results showed that mice were immunized withrecombinant adenoviruses which containing HSP70C exhibited higher frequency ofsplenic CD8+T cells secreting IFN-(p<0.05). rAd-GnH-IRES-S0.7H andrAd-GcH-IRES-S0.7H elicited higher frequency of splenic CD8+T cells secreting IFN-than vaccine during stimulated with different peptides（p<0.05）.4. Immunized mice were challenged by HTNV76-118, virus antigens and nucleicacid was detected by ELISA and RT-PCR. The amount of HTNV antigens and nucleicacids in organs of mice immunized with recombinant adenoviruses were lower thanimmunized with Adeno-lacZ and PBS. It is suggested that all recombinant adenovirusesimmunized groups show protectiveness against HTNV infection. Mice immunized withrAd-GnH-IRES-S0.7H and rAd-GcH-IRES-S0.7H exhibited the lowest HTNV antigensand nucleic acids capacity equal to the vaccine (p>0.05).Part2Construction and identification of the immunological properties of adenovirusvector containing HTNV chimeric genes and CTL1. Adenovirus vector containing HTNV chimeric genes (GnS0.7and GnS0.7) andCTL was constructed. After infected HEK293A cells, recombinant adenoviruses wereobtained and named rAd-GnS0.7-CTLH, rAd-GnS0.7-CTL, rAd-GcS0.7-CTLH andrAd-GcS0.7-CTL. The interest proteins of different recombinant adenoviruses wereidentified by IFA. Specific fluorescence was observed in cells after infection.2. C57BL/6mice were immunized with recombinant adenoviruses. Adeno-lacZ, PBSand inactivated vaccine immunized mice were set as controls. Humoral immunity levelwere assessed by ELISA and cell micro-neutralization test. Results showed that alladenovirus could elicit humoral immune responses incorporation of neutralizingantibodies and GP and NP specific antibodies. Adenovirus incorporation of HSP70C elicitshigher geometric mean titer (GMT)(glycoprotein and NP-specific) than others. Among adenovirus containing Gn, rAd-GnS0.7-CTLH elicit highest GMT (glycoprotein andNP-specific), which were80.0and320.0. Among adenovirus containing Gc, miceimmunized with rAd-GcS0.7-CTLH elicit highest GMT (glycoprotein and NP-specific),which were113.1and452.5. Adenovirus containing HSP70C also elicits higherneutralizing antibodies GMT than others. The neutralizing antibodies GMT of miceimmunized with rAd-GnS0.7-CTLH (26.4) and rAd-GcS0.7-CTLH (30.3) were higherthan that mice immunized with inactivated vaccine (p<0.05).3. During CTL assay, the splenocytes of mice immunized with rAd-GnS0.7-CTLHand rAd-GcS0.7-CTLH showed higher specific T cell-mediated cytotoxicity thanrAd-GnS0.7-CTLH and rAd-GcS0.7-CTLH. The cytotoxicity of rAd-GnS0.7-CTLH washigher than inactivated vaccine at E/T ratio of100:1(p<0.05). The cytotoxicity ofrAd-GcS0.7-CTLH was higher than inactivated vaccine at E/T ratio of10:1and100:1(p<0.05). ELISPOT results showed that mice were immunized with recombinantadenoviruses which containing HSP70C exhibited higher frequency of splenic CD8+Tcells secreting IFN-(p<0.05). rAd-GcS0.7-CTLH elicited higher frequency of splenicCD8+T cells secreting IFN-than vaccine during stimulated with different peptides(p<0.05). Besides stimulated with Gn2peptides pool rAd-GnS0.7-CTLH elicited higherfrequency of splenic CD8+T cells secreting IFN-than vaccine (p<0.05). Set15aaoverlapping peptide containing CTL epitopes as stimulator, data shows that NP48andNP60exhibit the best effects. It is suggested that the amino acid sequence in NP48andNP60play an important role in elicit CTL response.4. Immunized mice were challenged by HTNV76-118, virus antigens and nucleicacid was detected by ELISA and RT-PCR. The amount of HTNV antigens and nucleicacids in organs of mice immunized with recombinant adenoviruses were lower than whichimmunized with Adeno-lacZ and PBS. Suggested that all recombinant adenovirusesimmunized groups show protectiveness against HTNV infection. Mice immunized withrAd-GnS0.7-CTLH and rAd-GcS0.7-CTLH exhibited the lowest HTNV antigens andnucleic acids capacity equal to the vaccine (p>0.05).【Conclusion】 During this study, based on the CAG promoter and HSP70C molecular adjuvant, we take twostrategies to promote humoral and cellular immune response. We optimize the expression ofinterest genes in adenovirus expression system. A series of experiments in vivo and in vitroshows that take advantage of ECMV IRES to independently express GP and NP(fused toHSP70C respectively) could elevate humoral and cellular immune response. Joint applicationof CTL epitopes and HSP70C also could increase humoral and cellular immunity. Data showsthat both strategies acquired success, in most respects (humoral and cellular immune response)even exceed vaccine. By contrast, the former strategies elicit better humoral immunity and thelatter strategies elicit better cellular immune response. In future studies, we will integrateapplication of both strategies to acquire better immune response, which will provide theory,experiment and material basis for HTNV vaccine research. |
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