Identification Of HLA-E*01 Restricted CTL Epitope Of Hantaan Virus Protein And Its Mechanism In Hemorrhagic Fever With Renal Syndrome

Hemorrhagic fever with renal syndrome(HFRS)caused by Hantaan virus(HTNV)infection is a serious public health problem.Rational prevention and treatment of diseases caused by HTNV is the focus of global public health.The specific CTL immune response restricted by HLA-E is essential to the resistance and elimination of HTNV,which is also related to the occurrence and progression of the disease.Therefore,we synthesized HLA-E/peptide tetramer based on peptide limited by HLA-E*01.The frequency and function of HTNV epitope-specific CTL in HLA-E*01HFRS patients periphral blood mononuclear cell were analyzed by flow cytometry.And its relation with illness,course of disease.In this study,we identified HLA-E *01 restricted CTL epitopes derived from HTNV,and revealed the characteristics of HLA-E*01 restricted HTNV-specific CTL response,providing a new idea and method for clinical prevention,diagnosis and treatment of HFRS,and providing experimental data support for further development of CTL epitope polypeptide vaccine.Therefore,we conducted the following studies:Objective:1.To study the frequency of HLA-E*01 limited HTNV epitope specific CTL and its correlation with the disease.2.To study the correlation between HLA-E*01 limited HTNV epitope specific CTL function detection and disease.3.To study the detection of NT-pro BNP in plasma of hemorrhagic fever with renal syndrome and its application in clinical evaluation.Methods:1.Flow cytometry was used to detect the specific CTL frequency of HLA-E*01epitope-specific CTL in PBMC of patients with HFRS,and the relationship between CTL frequency and disease stage of patients was determined.2.The secretion levels of TNF-α,IFN-γ,Granzyme B and Perforin of HLA-E*01epitope-specific CTL in PBMC of patients with HFRS were detected by flow cytometry combined with intracellular cytokine staining,and their relationship with the disease was also analyzed.3.The level of Granzyme B secreted by HLA-E*01 epitope-specific CTL in PBMC of patients with HFRS was further detected by ELISPOT,and the relationship of Granzyme B secreted by different epitope-specific CTLs and the disease was analyzed.4.Enzyme-linked immunosorbent assay was used to detect the NT-pro BNP level in HFRS patients plasma,and the correlation of its level and the stage of disease was also analyzed.Besides,the relation of secretion level and clinical parameter was also taken into consideration.Results:1.There was no significant difference in the frequency of HLA-E*01 restricted HTNV epitope-specific CTL in PBMC between mild/moderate and severe/critical HFRS patients;The frequency of HLA-E*01 restricted HTNV epitope specific CTL in PBMC of patients with acute HFRS was higher than that of patients with convalescent HFRS.2.In the PBMC of severe/critical HFRS patients,the expression levels of IFN-γ and Granzyme B secreted by HLA-E*01 restricted HTNV epitope-specific CTL were higher than those of mild/moderate HFRS patients,especially Granzyme B expression level.However,there was no significant difference in the expression levels of TNF-α and Perforin induced by HLA-E*01 restricted HTNV epitope-specific CTL in PBMC between severe/critical and mild/moderate HFRS patients.3.All HLA-E*01 restricted HTNV epitope-specific CTL responses of tested HFRS patients secreted Granzyme B.In mild/moderate HFRS patients,HLA-E *01 restricted ILLKALYML、YLRQRQVAL、QLGQRIIVL 和 MMKSCLIAL epitope-specific CTL frequency and secretion level of Granzyme B were higher than those in severe/critical HFRS patients.However,There was no correlation between the frequency of ILWTGMIDL、VILTKTLVI 和 FLLVLESIL epitops-specific CTL and the level of Granzyme B secreted in HFRS patients4.The level of NT-pro BNP in the plasma of HFRS patients is closely related to the course of the patients,especially in the oliguria period of HFRS patients,the level of NT-pro BNP is significantly increased,and the expression of NT-pro BNP in the acute period of patients is significantly higher than that in the recovery period;in addition,the level of NT-pro BNP in Plasma was positively correlated with urea nitrogen and plasma creatinine content,but negatively correlated with platelet,white blood cell count and albumin level.Conclusion:1.The specific CTL frequency of HLA-E*01 restricted HTNV-GP epitope has nothing to do with the disease,but is closely related to the disease stage,When in acute stage,the CTL frequency was relatively high in the acute stage of patients.2.HLA-E*01 restricted HTNV-GP9 peptide epitopes specifically induced the IFN-γand Granzyme B secretions of CTL in severe/critically HFRS patients.However,the expressions of TNF-α and perforin secreted by CTL which was specifically induced by HLA-E*01 restricted HTNV-GP9 peptide epitopes were correlated with patients’ disease with no significance.3.The expression level of NT-pro BNP in plasma of HFRS patients were significantly increased in oliguria stage;Plasma NT-pro BNP level was positively correlated with urea nitrogen and serum creatinine content,but negatively correlated with platelet,white blood cell count and albumin level.