Maintenance Of Ultra-high And Extremely Low HBsAb Levels And The Characterization Of Corresponding IgG-H CDR3 Repertoire After HBV Vaccination

Objective:We screened healthy volunteers with ultra-high and extremely low Hepatitis B virus surface antibody(HBsAb)levels after hepatitis B virus(HBV)vaccination.The level and changes of HBsAb were monitored before vaccination,after the second vaccination,after the third vaccination and four years after HBV vaccination,and the composition and dynamic characteristics of Ig G-H CDR3 repertoire at the corresponding time points were analyzed.The changes of HBsAb level and the composition characteristics of corresponding Ig G-H CDR3 repertoire of each individual with ultra-high and extremely low HBsAb levels before and after HBV vaccination were compared and analyzed in detail,and the mechanism of ultra-high and extremely low HBsAb levels of B cells after HBV vaccination was discussed.In addition,this topic provides basic data and new research ideas for finding high HBsAb level technology and prevention and treatment of HBV infection through vaccines.And this topic provides the Ig G-H CDR3 sequences with significant characteristics found in this experiment to public databases such as NCBI and IMGT for sharing.Methods:1.Sample collection:48 volunteers who fit the conditions of HBV vaccination in this experiment were selected from 721 student volunteers in Zhuhai campus of Zunyi Medical University,and were vaccinated according to the standard HBV vaccination procedure.According to the level of HBsAb,5 volunteers with ultra-high HBsAb level(group H)(HBsAb>10000 m IU/m L)and 5 volunteers with extremely low HBsAb level(group L)(HBsAb<10 m IU/m L)were selected after the second vaccination.The peripheral blood samples of volunteers were collected before vaccination(T1),after the second vaccination(T2),after the third vaccination(T3)and four years after HBV vaccination(T4).2.Detection of HBsAb level:Five hepatitis B markers were detected by ELISA method at T1.3m L serum was obtained from 10 volunteers at T2 and T3,and 2m L plasma was obtained from 9 volunteers at T4.HBsAb levels were detected by chemiluminescence method.Detection of the proportion of B cells,memory B cells and plasma cells:The peripheral blood of 10 volunteers with 10m L at T2 were collected and detected by flow cytometry.3.The principle and process of BCR-H CDR3 repertoire construction:The total RNA of peripheral blood mononuclear cells(PBMCs)of each sample was extracted and then reverse transcribed into c DNA.According to the preparation technology of human BCR-H CDR3 repertoire,the BCR-H CDR3 repertoire of each sample was amplified by multiple PCR,and the BCR-H CDR3 repertoire was sequenced by Illumina Hi SeqⅩTen sequencing platform high throughput sequencing(HTS)technology(sequencing was completed by BGI).4.Sequencing data processing and analysis:the BCR-H CDR3 repertoire database data of each sample obtained by sequencing are provided to the NCBI database for comparison,and the aligned sequences are further clustered using R custom scripts.Clustering rules:The same IGHV and IGHJ gene segment and the same CDR3 AA length.And there is no more than one mismatch for every 12 amino acids(data comparison and clustering are completed by Hangzhou Immu Quad~?Biotechnologies,LLC).The characteristics of the clustered data were analyzed:diversity of CDR3 repertoire,usage and pairing characteristics of IGHV-IGHJ gene,CDR3 length,amino acid usage,overlap and comparative analysis of anti-HBV antibody sequences,somatic hypermutation and insertion and deletion characteristics of BCR repertoire.IBM SPSS Statistics 22 software was used to analyze the data before and after HBV vaccination and between the two groups.Mann-Whitney U test and Kruskal-Wallis test were used for statistical analysis,and Spearman correlation coefficient was used for correlation analysis.All the data were analyzed by double-tail test,and the test level wasα=0.05."*"stands for P<0.05,"**"stands for P<0.01 and"***"stands for P<0.001.Results:1.HBsAb level:All volunteers were negative for five markers of hepatitis B at T1;5volunteers showed ultra-high HBsAb level and 5 volunteers showed extremely low HBsAb level at T2;the HBsAb level of 3 volunteers in group L increased above the protective level at T2;the HBsAb level of 10 volunteers decreased,but the level of HBsAb in group H remained at a high level at T4.The proportion of B cells,memory B cells and plasma cells:The proportion of memory B cells in group H was slightly higher than that in group L at T2.2.The diversity of CDR3 repertoire:The diversity of group H decreased at T2 and increased at T3,and there was statistical difference compared with that T1;the change trend of group L was similar to that of group H,but there was no statistical difference.3.The usage and pairing of IGHV and IGHJ gene:Compared with group L,the frequency of taking IGHV1-18,IGHV2-26,IGHV2-5,IGHV3-21,IGHV3-23,IGHV3-33,IGHV3-7,IGHV3-74,IGHV3-9 and IGHV7-27 in group H after twice vaccination was higher than that T1.There was no significant difference in the usage of IGHJ gene.Both group H and group L used IGHJ4 with high frequency.The pairing frequency of IGHV1-18-IGHJ4,IGHV4-4-IGHJ4 and IGHV6-1-IGHJ4 in group H was significantly higher than that in group L.4.CDR3 length distribution and amino acid usage:The length of CDR3 was similar at T1,T2 and T3,mainly with 12-15 amino acids,while T4,significant cloning and amplification of longer sequences appeared in group H and group L,mainly 14-16 amino acids.In amino acid usage,Arg(R),Asp(D),Tyr(Y),Ser(S),Gly(G)and Ala(A)were used in each sample in group H and group L.5.CDR3 overlap:The related cluster of HBV vaccine in each sample of group H mainly showed low frequency or non-existence at T1,but the clone amplification was obvious at T2 and T3,while the clone amplification of group L was consistent at T1 and T3.The sequences contained in the HBV vaccine-related clusters screened in group H were compared with HBV-related sequences in the reported literature,and common conserved motifs with HBV-related sequences were found in multiple samples:"YGLDV","DAFD","YGSGS","GAFDI"and"NWFDP".6.Somatic hypermutation:The average mutation rate in group H was slightly higher than that in group L at T3,and the nucleotides number of mutations in group H"151-200"and">200"increased significantly,while in group L,the nucleotides number of"151-200"increased significantly only at T3.Insertions and deletions:There was no significant difference in the average number of nucleotides between group H and group L.Conclusions:1.Four years after HBV vaccination,the HBsAb level of volunteers with ultra-high HBsAb level remained at a high level,and the volunteers with extremely low HBsAb level could not produce sustained protective antibodies.And the results showed that volunteers with ultra-high HBsAb levels stimulated significant clonal proliferation of corresponding B cells after vaccination.2.Volunteers with ultra-high HBsAb levels have a certain preference for IGHV gene usage and IGHV-IGHJ pairing;most of the overlapping clusters have high clonal expansion after HBV vaccination,and low frequency presence or absence before vaccination;and experienced more somatic hypermutations.All suggest mechanisms associated with different HBsAb levels after vaccine response,and these overlapping clusters in volunteers with ultra-high HBsAb levels may be associated with high HBsAb levels.