Relevant Mechanism Of Endoplasmic Reticulum Aminopeptidase 1 Activating Antiviral Response By Editing HBcAg Immunopeptides

Background: Antigenic peptides presented by MHC-I are generated inside the cell by complex proteolytic pathways that often begin with the degradation of intracellular proteins by the proteasome and end with the trimming of excess N-terminal amino acids by aminopeptidases.Endoplasmic Reticulum Aminopeptidase 1（ERAP1）is an important molecule in the endogenous antigen processing presentation and is considered to be a key enzyme involved in antigen peptide pruning in the Endoplasmic Reticulum（ER）.ERAP1 can trim the antigen peptide to the appropriate length by trimming the antigen peptide amino segment residues,enabling it to bind to the human histocompatibility complex 1（MHC-I）molecules to form an antigen peptide-MHC-I class molecular complex,then presented to the cell surface and recognized by T cells to produce a specific immune response.Previous studies have shown that the antigenic peptide editing effect of ERAP1 is related to the infection of HIV,HCMV,HPV and other viruses and the body’s immune response to the virus.However,whether ERAP1 is involved in HBV-specific immune response,namely the antigen peptides presentation remains unknown.Methods: In this study,we analyzed peripheral ERAP1 level in chronic hepatitis B（CHB）patients（n=128）and healthy controls（n=44）by ELISA.The ERAP1 in HBV transfected Hep G2.2.15 cells were detected by western blot.The CD8+ T cells were co-cultured with Hep G2.2.15 cells pre-treated by ERAP1 specific inhibitor ERAP1--IN-1 to explore the stimulating ability of Hep G2.2.15 cells to CD8+ T cells.The proliferation of CD8+ T cells was detected by CCK-8（OD 450 nm）.Besides,the peptide repertoire was identified by tandem liquid chromatography–mass spectrometry（LC-MS/MS）.Finally,the binding affinity of 9-mers peptide with MHC-I molecules in Hep G2.2.15 cells was predicted by Net MHCpan-4.1 server.Results: The serum level of ERAP1 in CHB patients were significantly higher than that of HC（8.78±1.82 vs.3.52±1.61,P<0.001）.Furthermore,peripheral ERAP1 level was moderately correlated with HBV DNA levels in CHB patients（r=0.731,P<0.001）.HBV-transfected Hep G2.2.15 cells had substantially increased ERAP1 expression than the germ line Hep G2 cells（P<0.001）.The co-culture of ERAP1 specific inhibitor ERAP1-IN-1 pretreated Hep G2.2.15 cells with CD8+ T cells led to14%-24% inhibition of the proliferation of CD8+ T cells.Finally,liquid chromatography tandem mass spectrometry（LC-MS/MS）test demonstrated that ERAP-IN-1 produce a complete block of 9-mers（30-38,LLDTASALY）derived from HBc Ag.The predictive analysis by Net MHCpan-4.1 server showed that HLA-C*04:01 is a strong binder for the 9-mers peptide in HepG2.2.15 cells.Conclusion: Our results demonstrated that ERAP1 trims HBc Ag to produce 9-mers LLDTASALY peptide for binding onto HLA-C*04:01 in Hep G2.2.15 cells,facilitating potential activation of CD8+ T cells,providing a new method for antiviral therapy in clinical HBV infected patients.