Lineage structure of the human antibody repertoire in response to influenza vaccination

Abstract

The human antibody repertoire is one of the most important defenses against infectious disease, and the development of vaccines has enabled the conferral of targeted protection to specific pathogens. However, there are many challenges to measuring and analyzing the immunoglobulin sequence repertoire, including that each B cell's genome encodes a distinct antibody sequence, that the antibody repertoire changes over time, and the high similarity between antibody sequences. We have addressed these challenges by using high-throughput long read sequencing to perform immunogenomic characterization of expressed human antibody repertoires in the context of influenza vaccination. Informatic analysis of 5 million antibody heavy chain sequences from healthy individuals allowed us to perform global characterizations of isotype distributions, determine the lineage structure of the repertoire, and measure age- and antigen-related mutational activity. Our analysis of the clonal structure and mutational distribution of individuals' repertoires shows that elderly subjects have a decreased number of lineages but an increased prevaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. We have thus shown that global analysis of the immune system's clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.

Fig. 1. Antibody isotype distribution changes after vaccination

(A), antibody isotype composition in PBMCs at visit 1 (before vaccination, top) and visit 3 (28 ± 4 days after vaccination, bottom) averaged for all subjects. (B), percent change of individual’s relative IgM usage in PBMCs from visit 1 to visit 3. The subject IDs were labeled on horizontal axis. (C), comparison of relative change in IgM in different age group and vaccine types. p value was calculated by Mann-Whitney U test (3 samples for TIV receivers of age 8-17, 5 samples for TIV receivers of age 18-32, 4 samples for TIV receivers of age 70-100, 3 samples for LAIV receivers of age 8-17 and 2 samples for LAIV receivers of age 18-32). Red, LAIV receivers; blue, TIV receirvers. Percent change = (IgM reads in visit 1/total reads in visit 1) – (IgM reads in visit 3/total reads in visit 3)

Fig. 2. Informatically defined lineages with influenza specificity

The intra- and inter-lineage structure of all IgG lineages visualized by sequencing the PBs sorted from blood sample collected at visit 2 (7 days after vaccination) from a volunteer in the 70-100 year-old group received TIV (subject 017-043). In this network representation, each cluster of dots connected by lines represents a lineage. Different colors were used to distinguish different lineages. Each dot represents a unique CDR3 protein sequence. Two dots are linked if they differ by one amino acid in the CDR3 region. This is the threshold used when performing the single linkage clustering. The area of a dot is proportional to the number of reads with identical CDR3 protein sequences. Single cell cloned antibodies are labeled with text. Red text indicates antibodies having a high affinity towards one of the virus strains used in the flu vaccine. Black text indicates antibodies with a low affinity towards one of the virus strains used in the flu vaccine or background level of binding towards all three virus strains. 8 of 10 single cell cloned antibodies were found in the 454 sequences, except G04 and A06. All reads from 454 sequencing were used for this plot.

Fig. 3. Age related repertoire diversity and mutation changes

(A), repertoire diversity changes with age as measured by number of lineages in IgG from visit 1 PBMCs. (B and C), before vaccination mutation load as measured by averaging mutations at nucleotide level for IgG (B) and IgM (C) in visit 1 PBMCs respectively. Mutations for each read were defined as the number of mismatches to germline reference in V, D and J regions. (D-G), lineage analysis, performed with 80% nucleotide-sequence identity at the VDJ junctional region, gives measurements of amino acid mutations-per-read at V and J gene segments measured either to the germline reference (D and F) or from the most abundant sequence of the lineage to which each belongs (E and G) for IgG (D and E) and IgM (F and G). X-axes denote the measurement at visit 1, and the Y-axes denote the measurement at visit 3. Elderly patients show a higher number of IgG mutations from the germline (comparing 8-30 year-olds to 70-100 year-olds gives p<0.075 before vaccination and p<0.0044 after; restricting this analysis to TIV-patients alone gives p<0.18 and p<0.017, respectively). 3000-read of subsampling was applied to all panels. All error bars are the standard error. p-values were calculated by Mann-Whitney U test.

Fig. 4. Inter-lineage structure of IgGs in visit 2 PBMCs

Inter-lineage structure of IgGs in visit 2 PBMCs is presented for six randomly selected subjects (A-B, age 8-17; C-D, age 18-32; E-F, age 70-100). Each dot represents a lineage of antibody sequences defined by single linkage clustering with 1 amino acid difference at CDR3 as the threshold. The area of the dot is proportional to the number of reads belonging to this lineage, as indicated in the scale bar. X-axis is the diversity of the lineage which measures number of unique protein sequences (full protein sequence, not just the CDR3 region) within the lineage. Y-axis is the number of mutations at nucleotide level of the lineage averaged over reads. 3000 reads of subsampling was applied.