Screening Of Severe Markers And Analysis Of Immune Characteristics Induced By Vaccine In Patients With SARS-CoV-2 Infection

PurposeThere are differences in the severity of symptoms of novel coronavirus(SARS-CoV-2)infection.Severe symptoms seriously endanger people’s life safety,and there is no specific drug at present.Therefore,screening of severe biomarkers is of great significance for the prevention and scientific diagnosis and treatment of COVID-19 infection.Immune response induced following SARS-CoV-2 infection can produce serum biomarkers.At the same time,vaccination is an important measure to prevent severe cases.To clarify the characteristics of the immune cell gene expression profile and its regulatory network of COVID-19 infection and vaccination can provide theoretical support for the association of COVID-19 infection,pathogenesis of severe cases and biomarkers,and also provide scientific basis for the screening of COVID-19 vaccine and optimization of immune strategies.Methods1.With the approval and informed consent of the Ethics Committee of Shunde Hospital of Guangzhou University of Traditional Chinese Medicine,111 cases of COVID-19 infection were collected,including 14 asymptomatic infected persons,12 mild infected persons,34 ordinary infected persons,18 severe infected persons,33 rehabilitated persons,and 20 healthy controls;2.Using the Q440 multifactor protein chip to detect the expression level of 440 protein factors in serum,the difference factors were preliminarily screened through group comparison,and the differences between groups of routine test items were analyzed.3.Clinical information and blood samples from 15 infected patients at three time points of admission,remission,and discharge were collected,and the severity related factors were verified which obtained through preliminary screening in the longitudinal space-time dimension to verify whether the changes in their expression levels are consistent with the change trend in the treatment and remission process,and further screen and determine severity related markers.4.Enzyme linked immunosorbent assay(ELISA)to verify the selected protein factors as well GO enrichment for biological function analysisas was used.5.With the approval and informed consent of the Ethics Committee of Shunde Hospital.Guangzhou University of Traditional Chinese Medicine,12 peripheral blood samples were collected from individuals receiving inactivated vaccines on the day of inoculation(D0),the 28th day(D28),and the 42nd day(D42).Peripheral blood mononuclear cells(PBMC)were sequenced for single cell RNA sequencing(scRNA-seq),T cell receptor(TCR),and B cell receptor(BCR).According to the second dose,12 vaccine recipients were divided into high IgG group and low IgG group,the differences in immune response between the two groups were compared.6.We obtained the single cell transcriptome sequencing,TCR and BCR sequencing results of 102 samples of COVID-19 infected individuals(not vaccinated)in the public database,and integrated them with the data of 12 vaccinators,Clustering analysis and visual display of cells were performed using Uniform Manifold Approximation and Projection(UMAP)and T-distributed neighbor embedding(T-SNE)algorithms.Based on the differential expression of genes in different types of cells,single cell regulatory network inference and clustering(SCENIC)were used to analyze the activity of transcription factors(TF)in different cell subsets,and the immune response characteristics of T/NK cells,TCR,BCR,myeloid cells,as well as related signal pathway changes were analyzed to compare the difference of immune response between COVID-19 infection and vaccination.Results1.Screening of COVID-19 severe factors1.1 Compared with the non severe group,the white blood cell count,neutrophil count,neutrophil lymphocyte ratio(NLR),alanine aminotransferase(ALT),aspartate aminotransferase(AST),and C-reactive protein(CRP)expression in the COVID-19 severe group significantly increased,Lymphocytes and eosinophils were significantly decreased in the severe group.1.2 The protein chip results showed that the expression of differentially expressed factors such as TRAIL R1,IGFBP-1,IGFBP-4,VCAM-1,sFRP-3,FABP2,Transferin,GDF15,IL-1F7,IL-5Rα,and CD200 significantly increased(P<0.01).The expression of differentially expressed factors such as IL-1F7 and IL-5Rα were significantly reduced(P<0.01).1.3 The expression level of GDF15 was positively correlated with the severity of SARS-CoV-2 infection.1.4 The diagnostic value of GDF15 through the subject’s work characteristic curve,whose area under the curve is 0.89.The data were validated by ELISA,and the correlation was(R=0.87,P<2.2e-16).2.Analysis of single cell sequencing immune characteristics induced by COVID-19 vaccine2.1 A total of 771577 high-quality single cells were obtained in the COVID-19 infection and vaccination groups.These cells can be clustered into 19 main cell types,including 6 T cell groups(naive T,active T,Treg γδ T,MAIT,and pro T),3 NK cell populations(CD16+NK,CD56+NK,and pro NK),3 B cell populations(immature B,memory B,and plasma cells),6 myeloid cell populations(CD14+monocytes,CD16+monocytes,cDCl,cDC2,pDC,and platelets),and 1 hematopoietic stem cell cluster(HSC).2.2 The results showed that SARS-CoV-2 infection resulted in more significant alterations to the cell proportion and PBMC transcriptome compared to vaccination.2.3 The IFN-α and NF-κB signaling pathways were hyperactivated by SARS-CoV-2 infection,while vaccination only caused a subtle upregulation of IFN-a and deregulation of NF-κB signaling pathways.Additionally,both vaccination and SARS-CoV-2 infection can reshape T and B cell receptors(TCR and BCR)repertoires through preference of VJ genes,although changes to the repertoire were more pronounced after infection than after Ⅳ.2.4 The cellular communication between vaccination and SARS-CoV-2 infection also differed greatly,with the IL-15RA_IL-15receptor being selectively activated after vaccination,potentially contributing to vaccine-induced immune responses.Collectively,this study provides a systematic comparison between vaccination and SARS-Cov-2 infection-induced immune responses and identifies potential novel targets for development of more effective COVID-19 vaccines in the future.ConclusionGDF15 could be used as a biomarker associated with the disease severity of COVID-19.This study provides a systematic comparison between immune responses induced by SARS-CoV-2 infection and vaccination,uncovers the immune response signatures of the major cell types of SARS-CoV-2 infection and vaccinees,and provides new insights for further understanding of COVID-19 initiation and development and for future development of more effective potential targets for COVID-19.