Identification And Evaluation Of Immune Protective Effect Of HLA-A*02-restricted Hantaan Virus Glycoprotein CTL Epitopes

Hantaan virus(HTNV)infections can cause severe hemorrhagic fever with renal syndrome(HFRS)in humans,with a case-fatality rate as high as 15%.Cytotoxic T lymphocyte(CTL)responses contribute to the elimination of intracellular virus and closely related to the occurrence,development and prognosis of the disease.To date,most HTNV-specific CTL epitopes identified are distributed on nucleocapsid protein(NP),and only few study focus on HTNV glycoprotein(GP)-specific CTL epitopes,which limits our understanding of CTL responses against HTNV infection in humans.In our previous study,seven HTNV-GP epitopes were demonstrated to bind to HLA-A*0201 molecules with high affinity and were successfully used to synthesize HLA-A*0201/nonapeptide tetramers.Using these tetramers,we detected HTNV-GP epitope-specific CTLs in peripheral blood mononuclear cells(PBMCs)of HLA-A*02~+HFRS patients.In this study,we enlarged the sample size and found that the frequencies of each epitope-specific CTL were higher in patients with milder HFRS during acute phase,which indicated that the epitopes may induce protective CTL responses after HTNV infection.The results of IFN-?enzyme-linked immunospot assay further confirmed that each HTNV-GP epitope could elicit epitope-specific IFN-?-producing CTL responses in PBMCs of HFRS patients.In an HTNV challenge trial,we selected nonapeptides VV9(aa8-aa16,VMASLVWPV),SL9(aa996-aa1004,SLTECPTFL)and LL9(aa358-aa366,LIWTGMIDL),which showed higher HLA-A*0201 binding affinity and higher frequencies of epitope-specific CTLs in patients with mild/moderate severity,to immunize HLA-A2.1/K~b transgenic mice.Significant inhibition of HTNV replication characterized by lower levels of antigens and HTNV RNA loads was observed in major target organs(liver,spleen and kidneys)of HLA-A2.1/K~b transgenic mice pre-vaccinated with each nonapeptide.Importantly,LL9exhibited the best ability to induce protective CTL responses and showed a prominent effect on the kidneys of the mice,potentially preventing kidney injury after HTNV infection.Taken together,based on the identification of HLA-A*02-restricted HTNV-GP epitopes,we demonstrated the immune protective effect of HTNV-GP epitope-specific CTLs in HFRS for the first time and confirmed that HTNV GP-derived HLA-A*02-restricted epitopes could induce protective effect by reducing HTNV antigen level and RNA load in major organs of HLA-A2.1/K~b transgenic mice challenged with HTNV,which may advance the design of safe and effective CTL-based HTNV peptide vaccines for humans.