| Objective:1.Screening and obtaining the level of hepatitis B virus surface antibody(HBsAb)in the healthy volunteers after the hepatitis B virus(HBV)vaccine was vaccination,and analyzed the composition of human leukocyte antigen(HLA),and analyzed the composition characteristics and changes of the TCR β CDR3 repertoire before vaccination,after the second vaccination,after the third vaccination,and during follow-up monitoring.2.The composition characteristics and dynamic changes of TCR β CDR3 repertoire before and after HBV vaccine response of each individual with ultra-high and extremely low HBsAb levels,as well as the differences of HLA sites and CDR3 repertoire between ultrahigh and extremely low groups,are compared and analyzed in detail,so as to provide basic data and new research ideas and technologies for the characteristics of T cell response after recombinant HBV vaccine and HBV infection,and the possible mechanism of ultra-high and extremely low HBsAb levels,At the same time,HLA loci and TCR β CDR3 sequence with significant characteristics found in the experiment are provided to IMgt and other public databases for sharing.Methods:1.From the 721 student volunteers in Zhuhai Campus of Zunyi Medical University,49 volunteers who fully met the conditions for recombinant HBV vaccination were selected and vaccinated according to the standard vaccination procedures.After the second vaccination,5 volunteers with ultra-high HBsAb level(Group H)(HBsAb>10000 m IU/m L)and 5volunteers with extremely low HBsAb level(group L)(HBsAb<10 m IU/m L)were selected according to the HBsAb level.The peripheral blood of volunteers was collected before the vaccination,after the second vaccination,after the third vaccination and during the followup monitoring period.2.The mononuclear cells(PBMCs)in each peripheral blood sample(10m L)were isolated,the total RNA was extracted and reverse transcribed into c DNA.The human TCR β CDR3 repertoire was amplified by multiplex PCR.After the library was established,the samples were recovered,and the sequence of human TCR β CDR3 repertoire was sequenced by Illumina Solexa HTS Technology(completed by BGI).3.After the third vaccination,another 2 m L of peripheral blood was collected for HLA typing(completed by Beijing Boao Jingdian Biotechnology Co.,Ltd.using PCR-SBT method).4.After the original sequence of each sample obtained by HTS is compared with NCBI database,the user-defined script in R language is used for processing,and then the data of each sample is homogenized to the same library size to obtain the TCR β CDR3 sequence that can be used for analysis(completed by Hangzhou immuquad Biotechnology Co.,Ltd.);The characteristics of TCR β CDR3 repertoire diversity,access and pairing of V and J gene families,CDR3 length,AA access and CDR3 region overlap before and after vaccination in volunteers with ultra-high and extremely low HBsAb levels were compared and analyzed;The characteristics and differences of HLA locus composition between the two groups were compared and analyzed.Results:1.HBsAb level: 10 volunteers were hepatitis B and five Yin Yin before vaccination.In group H,after the second vaccination,the level of HBsAb was ultra-high(HBsAb>10000m IU/m L);After 4 years of vaccination,the level of HBsAb remained high(HBsAb>1000m IU/m L)except H5(HBsAb=309.01 m IU/m L);In group L,after the second vaccination,the level of HBsAb was extremely low(HBsAb< 10 m IU/m L);After the third vaccination,except L2(HBsAb=435.6 m IU/m L),the level of HBsAb was low(HBsAb<100 m IU/m L);After 4 years of vaccination,except L2(HBsAb=51.65 m IU/m L),they all decreased to extremely low HBsAb level(HBsAb<10 m IU/m L).2.HLA typing: in group H,3 of 5 HLA-B loci expressed HLA-B*40:01;Three out of five HLA-C loci expressed HLA-C*03:04;Three out of five HLA-DQ loci expressed HLADQB1*03:01;Three of the five HLA-DP loci expressed HLA-DPB1*05:01.In group L,3 of5 HLA-A loci expressed HLA-A*02:07,and the paired alleles were mainly HLA-B*33:03;Three of the five HLA-B loci expressed HLA-B*46:01 and HLA-B*58:01 respectively;Three of the five HLA-C loci expressed HLA-C*01:02,and their paired alleles were mainly HLAC*03:02;Four of the five HLA-DP loci expressed HLA-DPB1*05:01,and their paired alleles were mainly HLA-DPB1*13:01.3.Diversity of CDR3 repertoire: in group H,the diversity increased significantly after the second and third injections,and decreased significantly after 4 years of Vaccination;In group L,the diversity increased significantly after the second injection,increased after the third injection,and decreased after 4 years,but there was no significant difference.4.Access and pairing of TRBV-TRBJ gene family: compared with group L,the access frequency of TRBV15,TRBV29-1 and TRBJ1-4 in group H was significantly higher than that before vaccination;TRBV12-3-TRBJ 1-5 was the significant dominant pairing in group h after the second and third vaccinations.5.Amino acid(AA)intake frequency and length distribution: Group H and group l had similar intake frequency of AA in TCR β CDR3 repertoire before vaccination,after the second vaccination,after the third vaccination and 4 years after vaccination.They all took glutamine(Q),glutamate(E),tyrosine(Y),serine(S),threonine(T),glycine(G)and alanine(A)at high frequency.The length distribution of CDR3 was similar and close to normal distribution,It is mainly composed of 8-18 amino acids.6.CDR3 overlap: in each individual in group H,there were 75 CDR3 sequences that were not detected or low-frequency cloned and amplified before vaccination.There were common motifs: "Y-NEQ" and "G-DTQ" in these 75 CDR3 sequences.Among them,"T-NEQ","DTQ" and CATSRVAGETQYF were consistent with those reported,and the reported motifs " NTE "," ETQ "," GETQ " and " GNEQ " also existed in the experimental results.Conclusions:1.Four years after HBV vaccination,the HBsAb level of ultra-high HBsAb volunteers decreased with time,but remained at a high level,suggesting that there has always been a high proportion of HBV vaccine related memory B cells/plasma cells in their bodies.2.The ultra-high HBsAb level after HBV vaccination may be related to the efficient presentation of HBV vaccine related antigens by HLA-B*40:01,HLA-C*03:04,HLADQB1*03:01 and allele pairing with HLA-DPB1*05:01-DPB1*02:01/02:02/03:01;The extremely low HBsAb level after HBV vaccination may be related to the presentation efficiency of HBV vaccine related antigens by HLA-A*33:03,HLA-A*02:07,HLAB*46:01,HLA-B*58:01,HLA-C*01:02,HLA-C*03:02 and allele pairing with HLADPB1*05:01-DPB1*13:01.The composition and pairing of HLA is the entry point for further research on the mechanism of individuals with ultra-high and extremely low HBsAb level after HBV vaccination.3.After the second and third doses of HBV vaccine,the diversity of TCR β CDR3 repertoire increased,which may be related to the interaction or synergy between T cells and B cells.There may be differences in the effect of T cells on assisted B cells between individuals with ultra-high and extremely low HBsAb levels.In the CDR3 receptor of individuals with ultrahigh HBsAb levels,the common motifs "Y-NEQ" and "DTQ" and CATSRVAGETQYF sequences,may play an important role in the T-cell response mechanism of HBV vaccine.The maintenance of ultra-high HBsAb level is related to memory B cells/plasma cells,It may also be associated with these T cell helper cells sharing high-frequency clones. |
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