Screening And Identification Of An H-2K~b-Restricted CTL Epitope Within The Glycoprotein Of Hantaan Virus

【Background】Hantaan virus(HTNV)is the major causative agent of hemorrhagic fever with renal syndrome(HFRS)characterized by fever,hemorrhage,and renal failure.HFRS is a severe,life-threatening illness with high case-fatality,and has always been a serious public health threat in China.Exploration of HTNV specific T cell immune response and antigenic epitope represents a powerful approach to study the area of HTNV specific immune response,immunotherapy,and new vaccines.The current research shows that HTNV nucleocapsid protein harbors most cytotoxic T lymphocyte epitopes and glycoprotein is not only responsible for receptor binding and membrane fusion,but is also considered the main target of neutralizing antibodies.During viral infection,CTLs are important for viral clearance through perforin pathway and death receptor pathway and regulation host immune functions through secreting cytokines.Therefore,research on CTL epitope is critical for development of immune protection and immunotherapy.Recent interest has emerged in the concept of HTNV glycoprotein serving as a potent immunogen to induce T cell responses.Epitopes on HTNV glycoprotein that elicit CD4+ T lymphocyte responses have been extensively studied.However,CD8+ cytotoxic T cell epitopes on the HTNV glycoprotein have not been identified,and specific responses to these immunogens remain largely unknown.Identification of CTL epitope on HTNV glycoprotein is of great significance for the study of new HTNV vaccine.The sequence of HTNV GP was analyzed by algorithms.Then,we selected 15 peptides restricted by H-2K~b to screen CTL epitope on HTNV glycoprotein,and explored their biological activity and characteristics of immunogenicity.Our research could set up a solid foundation for identification of CTL epitope on HTNV glycoprotein.【Methods】To predict the CTL epitope on HTNV GP,the sequence of HTNV GP was evaluated using an algorithm provided by IEDB(Immune epitope database and analysis resource,IEDB).Further,the binding score of each peptide was calculated by BIMAS software(Bio Informatics and Molecular Analysis Section,BIMAS),which is necessary to predict the possibility of peptides bind to H2-K~b.Finally,fifteen H-2K~b restricted peptides from HTNV GP satisfied the above conditions were synthesized.ELISPOT was conducted for mapping the CTL epitopes on HTNV GP.First,splenocytes derived from HTNV infected C57BL/6 were prepared and assayed for their ability to secrete IFN-γ during in vitro restimulation with fifteen predicted peptides.Spleen cells pulsed with NP1 which is a known HTNV NP-specific CTL epitope was used as positive control.Sencond,peptides with positive responses in above experiment were subsequently characterized in CD4-or CD8-depleted splenocytes using anti-CD4-coated Dynalbeads and anti-CD8-coated Dynalbeads.We tried to use CD4+ T cell-depleted splenocytes as effector cells to recognize the CD8+ T cell responsive peptides.On the other hand,CD8+ T cell depletion completely abrogated the IFN-γ response indicating that these peptides were CD8+ T cell epitopes.The level of specific cytotoxicity was detected by cytotoxicity assay.Splenocytes of HTNV-immunized mice were used as effector cells.Target cells used in this study were peptide-pulsed EL-4 cells and HTNV-inoculated macrophages.This performance is for assessing the potential of epitope peptides to stimulate the cytotoxicity of CTL.On the basis of the above work,CTL epitopes were investigated their biological activity in vitro and immunogenicity in vivo.We next assessed whether the antiviral effect of epitope peptides were dependent on the capacity of stimulating PBMC(Peripheral blood mononuclear cell,PBMC)expansion by lymphocyte proliferation assay.We further investigated the production of IFN-γ by CD8+ T cells specific for HTNV GP-specific epitopes by intracellular cytokine staining.To evaluate the immunogenicity of the identified epitopes ex vivo,C57BL/6 mice were subcutaneously immunized four times with the mixture of epitope peptides and Freund’s adjuvant,at 10 day intervals.The mice immunized with NP1 which is NP-specific CTL epitope and inactivated Hantavirus vaccines serve as positive controls,PBS and Freud’s adjuvant as negative controls.The cellular immune response and humoral immune response were evaluated by at 10 d after the final booster immunization with the specific epitope.The cytotoxic activity of splenocytes from immunized mice was detected by cytotoxicity assay.The level of cytokine production was measured by ELISPOT assay.The titer of specific antibody against HTNV NP and GP was detected by ELISA(Enzyme-linked immunoadsordent assay,ELISA),and the titer of neutralizing antibody was measured by Micro neutralization test.Animal protection experiments were evaluated the protection effect induced by CTL epitopes.Immunized mice were received an infectious dose(1×105 pfu/mouse)of HTNV strain 76-118 intramuscularly.To evaluate the protective effect of CTL epitope on HTNV infected mice,immunized mice was sacrificed at three days after infection.ELISA was performed to detect HTNV antigens,and qRT-PCR was performed to detect HTNV nucleic acids in the tissues of immunized mice.【Results】Fifteen octapeptides were selected from the HTNV GP amino acid sequence based on a percentile rank by IEDB.A lower percentile rank indicates higher immune response.The top 15 predicted CTL epitopes restricted by H-2K~b molecular on HTNV GP was selected.The predicted peptides are as follows: GP1(aa39-aa46: SVIGYVEL)、GP2(aa44-aa51: VELPPVPL)、GP3(aa64-aa71: SMDNHQSL)、GP4(aa208-aa215: IVCFFVAV)、GP5(aa420-aa427: VNFVCQRV)、GP6(aa456-aa463: ITSLFSLL)、GP7(aa489-aa496: VTFCFGWV)、GP8(aa499-aa506: PAITFIIL)、GP9(aa797-aa804: ITIRYSRR)、GP10(aa845-aa852: TLLFFGPL)、GP11(aa925-aa932: QSFNTSTM)、GP12(aa987-aa994: VGFTLTCL)、 GP13(aa1102-aa1109: FSGNWIVL)、 GP14(aa1113-aa1120: CVFLLFSL)、GP15(aa1114-aa1121: VFLLFSLV);We also evaluated the binding score of peptide-binding affinity by the BIMAS software to confirm that all peptides were able to bind H-2K~b molecule.A higher binding score means a higher affinity between peptides and H-2K~b molecule.The results of ELISPOT suggested that GP1,GP2,GP3,GP6,GP8 and GP10 induce IFN-γ production in the splenocytes of HTNV-infected mice with a spot magnitude of 225,130,257,461,196 and 162 spot-forming cells(SFC)/106 input cells,respectively.When CD4+ or CD8+ T cell-depleted splenocytes were employed as effector cells,the results showed that one peptide GP6 induced significant numbers of IFN-γ-producing cells(SFC 421)in CD4+ T cell-depleted cultures and limited the response magnitude in CD8+ T cell-depleted cultures.By contrast,another 5 peptides(GP1,GP2,GP3,GP8,and GP10)induced significant numbers of IFN-γ-producing cells in CD8+ T cell-depleted cultures,with a spot magnitude of 216,129,229,183,and 179,respectively.However,they did not induce detectable levels of IFN-γ-producing cells when the CD4+ T cells were depleted.The performance of GP6 are consistent with that of NP1,which is positive epitope control and also induced a significant number of IFN-γ-producing cells when CD4+ T cells were depleted and less than 35 SFC when CD8+ T cells were depleted.Based on this finding,we speculate that GP6 might be an immunodominant CTL epitope.The cell-mediated cytotoxicity assay was performed to evaluate peptide-specific cytotoxic activity of CTLs.The effector cells used in this study were splenocytes derived from HTNV immunized mice with restimulation of GP6 and NP1(a known CTL epitope as positive control)in vitro.The target cells were GP6 and NP1-pulsed EL-4(restricted by H-2K~b molecule)and HTNV infected macrophage.These results indicate that cytotoxicity of effector cells were closely relevant with E/T ratio.The higher E/T ratio follows the stronger cytotoxic effect.However,when the na?ve EL-4,macrophage,and P815(restricted by H-2Dd molecule)as target cells,the nonspecific cytotoxicity of effector cells is extremely low,and cytotoxicity ratio never exceeded 10%.These results suggested that GP6 have the potential to induce the specific cytotoxicity of CTL agaist HTNV infected cells.The results of FCM(Flow cytometry)showed that GP6 peptide plays critical roles in the proliferation of PBMC,and stimulating splenocytes to secrete IFN-γ both from HTNV infected mice.There was a significant difference between experimental and control groups.These suggested that GP6 peptide has good biological activities.To evaluate the immunogenicity of GP6 peptide ex vivo,the humoral and cellular immune response was evaluated in peptide-immunized mice.Our results showed that GP6 peptide can trigger stronger T cell responses,compared with the other groups.Cytotoxicity of splenocytes from mice immunized with GP6 peptide was closely relevant with E/T ratio.The higher E/T ratio follows the stronger cytotoxic effect.However,neither HTNV GP-specific antibody nor neutralizing antibody was produced in mice immunized with GP6 peptide.We further provided evidence of the potential capacity of GP6 to protect mice after HTNV challenge through the animal protection assay.The results of ELISA and qRT-PCR showed that a great quantity of viral antigens and nucleic acids existed in the liver,spleen,and kidney of C57BL/6 mice in the two negative control groups(PBS control and adjuvant control).It was interesting to note that viral antigens and nucleic acids can only be detected in small amounts in the GP6 and NP1 groups.These findings suggests that GP6-specific CTL response could protect animals from HTNV challenge.【Conclusion】In summary,one novel CTL epitope peptide GP6 was identified in this study.Furthermore,we demonstrated that GP6 epitope had good biological activities and that immunization with GP6,HTNV GP-specific CTL epitope,enhanced host’s cellular immunity.All parameters we assessed GP6 immunized group are better than the HFRS inactivated vaccine group.The epitope-specific T cell immune response protected C57BL/6 mice from HTNV challenge.Our study provides a basis for exploring HTNV GP-specific CTL epitopes.