Genetic measurement of memory B-cell recall using antibody repertoire sequencing

Abstract

Annual influenza vaccinations aim to protect against seasonal infections, and vaccine strain compositions are updated every year. This protection is based on antibodies that are produced by either newly activated or memory B cells recalled from previous encounters with influenza vaccination or infection. The extent to which the B-cell repertoire responds to vaccination and recalls antibodies has so far not been analyzed at a genetic level-which is to say, at the level of antibody sequences. Here, we developed a consensus read sequencing approach that incorporates unique barcode labels on each starting RNA molecule. These labels allow one to combine multiple sequencing reads covering the same RNA molecule to reduce the error rate to a desired level, and they also enable accurate quantification of RNA and isotype levels. We validated this approach and analyzed the differential response of the antibody repertoire to live-attenuated or trivalent-inactivated influenza vaccination. Additionally, we analyzed the antibody repertoire in response to repeated yearly vaccinations with trivalentinactivated influenza vaccination. We found antibody sequences that were present in both years, providing a direct genetic measurement of B-cell recall.E very year, influenza viruses cause the deaths of an average of 36,000 individuals in the United States alone (1). Although the immunological memory created through vaccination can confer decade-long protection against a particular viral strain, antigenic drift in the original strain and the occurrence of distinct viral strains can enable the virus to evade the immune system (2). As a result, influenza vaccination formulations have to be reevaluated, adjusted, and administered annually to best match the annual influenza strain. Vaccine-induced immunity against influenza is primarily antibody-based, and as such, it relies on the activation of naive B cells or the reactivation (recall) of memory B cells to produce high levels of antibody specific to the vaccine strain. Prior studies approached recall memory responses by measuring plasma antibody levels and specificity or sequencing antibody loci of isolated B cells, with one study concluding that the response to influenza vaccination is pauciclonal (i.e., composed of only a few distinct clones) (3, 4). However, this study and others were limited in the number of B cells that they were able to analyze and not able to show that the same clone recurs during recall. The strength of the recall response, the isotype distribution, and the clonal relationship to others have been unclear.Recently, methods to sequence antibody repertoires of whole organisms and human blood samples were developed and applied to investigate several features of B-cell repertoires (5, 6). This approach has been used to investigate a variety of phenomena, including effects of influenza vaccination, residual disease in leukemia, effects of immune suppression, and differences between memory and naive B-cell compartments (5-11).Analyzing vaccine recall response requires ...

Phylogenetic analysis of the human antibody repertoire reveals quantitative signatures of immune senescence and aging

“…2A). Interestingly, the abnormally expanded "superlineages" continued to make up a similar fraction of the total repertoire at days 7 and 28 after vaccination as they did before vaccination at day 0, indicating that the size of any vaccine response [e.g., release of recalled IgG plasmablasts around day 7 (11,24,25)] was minor compared with the weight of the superlineages. Note that no CDR3 sequences were shared between superlineages, which provides confidence that superlineages did not arise from contamination.…”

“…The Stanford Institutional Review Board approved protocols, and participants gave informed consent. The BCR repertoire sequencing process was a minor variation on a previously published protocol (11). Briefly, RT was carried out on 500 ng of total RNA from peripheral blood mononuclear cells using primers annealing to the IGH-constant region (Table S2); secondstrand synthesis was carried out using IGH variable-region primers (Table S3).…”

“…However, previous work was limited by small numbers of elderly individuals analyzed (8), did not analyze in detail the composition of the oligoclonal lineages (8), or did not study the effect of applying an immune stimulus such as a vaccine (10). Furthermore, previous B-cell repertoire studies have focused only on isolated aspects of B-cell mutation counts, V/D/J-gene use levels, lineage sizes, and convergent CDR3 sequences (8,(10)(11)(12)(13) without analyzing the character of mutations and without using the full phylogenetic information inherent in the IGH repertoire to quantify dissimilarities between immune systems.…”

Highly sensitive and unbiased approach for elucidating antibody repertoires

“…Being DNA-based, HTGTS-Rep-seq also bypasses a major limitation of RNA-based methods for certain applications by quantitatively capturing the frequency of Ig rearrangements in a population regardless of their expression level or whether they are productive or nonproductive. Current means to address biases due to multiplex PCR or varying expression levels between cells include the use of universal identifiers (25,36,37) or single cell methods (38), but HTGTS-Rep-seq can accurately identify a population repertoire profile without the additional cost or steps of synthesizing primers with random barcodes, or sorting for single cells.…”

High-resolution antibody dynamics of vaccine-induced immune responses

“…Recent advances in next-generation sequencing (NGS) (2) have enabled any DNA-encodable assay to produce massive amounts of data. Indeed, NGS has enabled unprecedented views into the immune repertoire, as its immune receptor diversity is genetically encoded within a complex collection of lymphocytes (3)(4)(5)(6)(7)(8).…”