Effects Of Influenza Vaccine On BCR CDR3 Diversity In Peripheral Blood Memory B Cells

Objective:This study was based on high-throughput sequencing technology to analyze the characteristics of IGH-CDR3 sequences of memory B cells in peripheral blood of 5 healthy volunteers before and after seasonal influenza vaccine vaccination.Provide new theoretical basis and research ideas for the research of vaccine immune effect and the development of new vaccines.Methods:1.5 volunteers were recruited in this study.All were injected intramuscularly with seasonal influenza vaccine(Hualan Biological),5ml of peripheral blood was collected before and 28 days after the vaccine injection,and the serum was separated and detected by indirect ELISA Changes of influenza virus-specific antibodies in volunteer peripheral blood;2.50 ml of peripheral blood was collected before and 28 days after the vaccine injection.Total B cells were sorted out by magnetic beads and cultured for 6 days in vitro(IL2 + Cp G + H7N9).Elispot was used to detect the frequency of H3 stalk memory B cell frequency;3.Collect 50 ml of peripheral blood before the vaccine injection and 28 days after the vaccine injection.Magnetic beads were used to isolate the memory B cells and RNA was extracted.The analysis software was used to compare and analyze the sequence obtained by sequencing.The analysis content is as follows:(1)The diversity of CDR3 sequence and its distribution and abundance;(2)IGHV,IGHJ,IGHD gene family access and pairing distribution to observe the preference of gene segment access;(3)CDR3 overlap rate between samples;(4)Compare the relationship between CDR3 and influenza virus hemagglutinin antigen.Results:1.After being vaccinated the levels of anti-H1,H3 influenza virus-specific antibodies in the peripheral blood of the five volunteers increased by 2.682 times and 2.096 times compared to before the vaccination.2.After the seasonal influenza vaccination,the frequency of influenza virus hemagglutinin H3 memory B cells in the peripheral blood of volunteers was 7.36 times higher than before the vaccination.3.Effects of seasonal influenza vaccine on BCR CDR3 diversity in peripheral blood memory B cells3.1 Length distribution of BCR H-CDR3 length distribution After vaccination,the length of H-CDR3 amino acids in the three volunteers showed a standard bell-shaped distribution with 16 AA,and the other two volunteers showed a skewed distribution with 14 AA and 17 AA.3.2 Frequency distribution of BCR H chain V region genes After vaccination,the BCR H-CDR3 bank of peripheral blood memory B cells all used V genes IGHV1-18,IGHV1-3,IGHV1-46,IGHV1-69,IGHV1-8,IGHV5-51 at high frequency,and the frequency of use was ≥3 %.Statistical analysis of the frequency of V-family high frequency genes(≥2%)before and after inoculation revealed that the gene IGHV1-8(P = 0.043)had a statistically significant decrease compared to the frequency before inoculation.3.3 Frequency distribution of BCR H chain D region genes After vaccination,the D-genes IGHD1-26,IGHD2-15,IGHD3-10,IGHD2-21,IGHD6-19,IGHD3-16,IGHD6-13,and IGHD3 were taken from peripheral blood memory B-cell B-CR H-CDR3 banks.-22,taking frequency ≥3%.Statistical analysis of the frequency of D-family high-frequency genes(≥2%)before and after inoculation revealed that IGHD1-1(P = 0.043)had a statistically significant increase compared to the frequency before inoculation.3.5 Frequency distribution of BCR H chain J region genes Analysis of the IGHJ gene accession of the BCR H-CDR3 repertoire of memory B cells before and after vaccination in 5 volunteers found that all samples were taken IGHJ-4 gene(≥50%)at high frequency,and the frequency of IGHJ gene accession before and after vaccination showed no significant difference.3.6 BCR H chain V region and J region gene combination Through the analysis of H-CDR3 repertoire acquisition of a total of 10 samples before and after vaccination of 5 volunteers,it was found that the advantageous pairing access(≥2%)includes IGHV1-18-IGHJ4,IGHV1-46-IGHJ4,IGHV1-3-IGHJ4,IGHV5-51-IGHJ4,IGHV1-69-IGHJ4,IGHV6-1-IGHJ4,IGHV4-4-IGHJ4,IGHV3-23-IGHJ4.The IGHJ region frequently expressed the IGHJ4 family,while the IGHV region highly expressed the IGHV1 and IGHV3 families.We conducted statistical analysis on the frequency of VJ high-frequency paired genes before and after vaccination,and the results showed that only the changes in IGHV1-18-IGHJ4,IGHV1-8-IGHJ4,IGHV3-11-IGHJ4(P = 0.043)were statistically significant.3.7 Shared clones Among the volunteers,the shared clones proportion of four volunteers before and after vaccination was greater than 3%,and the other’s was relatively low(1.62%),and the overlap rate between different volunteers was less than 1%.Five volunteers had 593 common amino acids in memory B-cell BCR H-CDR3 before influenza vaccine and 37 shared amino acids after vaccination,suggesting a high correlation between shared clones and vaccine antigens.3.8 Amino acid sequence analysis of the first 10 high-frequency clones of the memory B cell BCR H-CDR3 Analysis of the amino acid sequences amplified by the first 10 high-frequency clones of the BCR CDR3 bank of peripheral blood memory B cells before and after vaccination revealed that the sequences shared before and after vaccination were the amino acid sequences of YMDVW,WFDYW,YFDYW,and WFDPW.Three of the volunteers shared the YFDYW amino acid sequence after vaccination.Conclusion:1.Seasonal influenza vaccine can effectively increase the frequency of influenza-specific memory B cells;2.The frequency of access to IGHV,IGHD,and VJ paired gene families of peripheral blood memory B-cell BCR H-CDR3 repertoires in volunteer peripheral blood cells after seasonal influenza vaccination changes,among which IGHV1-8,IGHV1-18-IGHJ4 and IGHV1-8-The frequency of IGHJ4 gene acquisition is reduced,and the frequency of IGHD1-1 and IGHV3-11-IGHJ4 gene acquisition is significantly increased,which may be related to the epitope of influenza vaccine.3.The number of common clones in the memory B cell BCR H-CDR3 repertoires of the five volunteers decreased after the seasonal influenza vaccination,suggesting a high correlation between the common clones and the vaccine antigen.4.After vaccination,YFDYW shared amino acid sequences appeared between different volunteers,suggesting that the emergence of shared sequences may be related to vaccination.