Transcriptome Characteristics Of Peripheral Blood Mononuclear Cells In Hepatitis B Vaccine Non-Responders Based On Single-Cell Sequencing

Background:After the emergence of hepatitis B vaccine,the prevention of hepatitis B has made a breakthrough progress.The World Health Organization（WHO）has set the goal of"eliminating viral hepatitis by 2030",but some children still develop breakthrough hepatitis B virus infection on the count of non-immune response to hepatitis B vaccine.Therefore,it is very critical to explore the key target of regulating the immune response intensity of hepatitis B vaccine to develop targeted vaccine and eliminate hepatitis B virus infection.However,immune response which was caused by the hepatitis B vaccine is complicated in the body,the interactions of different immune cells regulating the strength of the body’s response to vaccines and the balance of inflammatory reaction induced by vaccines.At present,studies on the transcriptional characteristics of hepatitis B vaccine non-immune response mostly adopt traditional sequencing technology,and the average population information is obtained,so it is difficult to obtain the transcriptional characteristics and key molecules of each immune cell,and the molecular mechanism of hepatitis B vaccine non-immune response has not been fully understood.The single cell RNA sequencing technology makes up for the shortcomings of traditional sequencing techniques,enabling researchers to obtain the transcriptome characteristic of each cell and reveal the key cell and molecular characteristic that determines phenotypic.Objective:To analyze the transcriptional characteristics of cell subtypes in peripheral blood mononuclear cell（PBMC）of patients with high immune response and those without immune response by single-cell RNA sequencing.In order to obtain the transcriptional panorama of peripheral blood immune cells of individuals without immune response and individuals with high immune response after hepatitis B vaccine immunization,so as to provide a data basis for a comprehensive understanding of the differences in transcriptional characteristics between individuals with high immune response and individuals without immune response,reveal key cells and targets regulating the intensity of hepatitis B vaccine immune response,and provide clues to further reveal the difference in the function of immune cells against hepatitis B virus between individuals with high immune response to hepatitis B vaccine and individuals without immune response to hepatitis B vaccine.Method:Volunteers with a history of hepatitis B vaccination and without surface antibodies against hepatitis B virus were recruited.3 people with high immune response and 3 people with no immune response to hepatitis B vaccine matched by gender,age and ethnicity were screened after three doses of hepatitis B vaccine inoculated according to the schedule of 0,1 and 6 months.Peripheral blood was collected to isolate mononuclear cells for single-cell sequencing.After dimensionality reduction,clustering and cell population naming according to the characteristics of transcript in each cell,the expression level and function differences of transcript in each cell population in each group were evaluated by expression enrichment score of predefined gene sets,gene expression difference analysis and differential gene function enrichment analysis.Finally,q RT-PCR was used to verify the expression differences of key genes between two groups.Result:（1）Eighteen cell subtypes were identified from this study,there was no statistical difference in the cell proportion of each subgroup in NR group and HR group.（2）Compared with the HR group,the expression activity scores of HLA-II in each kind of antigen presenting cell from the hepatitis B vaccine NR group were significantly decreased,the expression activity score of HLA-I also decreased significantly besides plasmacytoid dendritic cell（p DC）.The down-regulated genes were significantly enriched in antigen processing and presentation signaling pathways,and the representative genes are HLA-DRB5 and HLA-B.（3）Compared with the HR group,the expression activity score of MAPK gene set in the cluster of naive B from the hepatitis B vaccine NR group was significantly decreased,and the down-regulated genes were significantly enriched in the calcium ion reaction located in the downstream pathway of B cell receptor.Some key molecules,such as IL4R,BLNK,BLK,SCIMP,NFKBIA and JUN were significantly down-regulated.（4）Compared with HR group,CD8⁺Tem,CD8⁺Teff and CD4⁺Teff were the three T cell subsets with the most significant differences in gene expression,and the cell activity score of them in NR group was significantly lower than that in HR group,The differential genes with common low expression in each T cell subpopulation were significantly enriched in oxidative phosphorylation and calcium ion response signaling pathways,and the key gene linking these two pathways was JUN.（5）Compared with the HR group,some cytotoxic genes,including GNLY,NKG7,GZMB,GZMM,KLRC1,KLRD1,PRF1,CST7 and CTSW were highly expressed in CD3^highCD16^lowKLRG1^highNKT cell from the hepatitis B vaccine NR group,the cytotoxicity score of which was significant increase and the up regulated genes were significantly enriched in cytotoxic related pathways.（6）Compared with the HR group,TXNIP was significantly down-regulated in most cell subsets of NR group.Conclusion:（1）In the NR group,the low-expression of the antigen presenting gene sets in antigen presenting cells,the MAPK pathway gene sets in naive B cells and the cell activity gene sets in the three types of effector T cells,and the high expression of cytotoxicity gene sets in CD3^highCD16^lowKLRG1^highNKT was related to no immune response to hepatitis B vaccine.（2）HLA-B and HLA-DRB5 may be the key molecules responsible for the antigen presentation of hepatitis B vaccine.（3）BLNK,BLK,IL4R and SCIMP located in the upstream of MAPK pathway may be the key molecules that determine the immune response strength of Naive B cells to hepatitis B vaccine.（4）HLA-B、TXNIP、JUN is a key molecule that regulates the response intensity of immune cell subsets to hepatitis B vaccine.