Abstract
Recent advances in the next-generation sequencing of B-cell receptors (BCRs) enable the characterization of humoral responses at a repertoire-wide scale and provide the capability for identifying unique features of immune repertoires in response to disease, vaccination, or infection. Immunosequencing now readily generates 103–105 sequences per sample; however, statistical analysis of these repertoires is challenging because of the high genetic diversity of BCRs and the elaborate clonal relationships among them. To date, most immunosequencing analyses have focused on reporting qualitative trends in immunoglobulin (Ig) properties, such as usage or somatic hypermutation (SHM) percentage of the Ig heavy chain variable (IGHV) gene segment family, and on reducing complex Ig property distributions to simple summary statistics. However, because Ig properties are typically not normally distributed, any approach that fails to assess the distribution as a whole may be inadequate in (1) properly assessing the statistical significance of repertoire differences, (2) identifying how two repertoires differ, and (3) determining appropriate confidence intervals for assessing the size of the differences and their potential biological relevance. To address these issues, we have developed a technique that uses Wilcox’ robust statistics toolbox to identify statistically significant vaccine-specific differences between Ig repertoire properties. The advantage of this technique is that it can determine not only whether but also where the distributions differ, even when the Ig repertoire properties are non-normally distributed. We used this technique to characterize murine germinal center (GC) B-cell repertoires in response to a complex Ebola virus-like particle (eVLP) vaccine candidate with known protective efficacy. The eVLP-mediated GC B-cell responses were highly diverse, consisting of thousands of clonotypes. Despite this staggering diversity, we identified statistically significant differences between non-immunized, vaccine only, and vaccine-plus-adjuvant groups in terms of Ig properties, including IGHV-family usage, SHM percentage, and characteristics of the BCR complementarity-determining region. Most notably, our analyses identified a robust eVLP-specific feature—enhanced IGHV8-family usage in B-cell repertoires. These findings demonstrate the utility of our technique in identifying statistically significant BCR repertoire differences following vaccination. More generally, our approach is potentially applicable to a wide range of studies in infection, vaccination, auto-immunity, and cancer.
Highlights
Establishing a diverse repertoire of antibody responses is central to humoral immunity acquired through natural infection and vaccination [1,2,3]
Inclusion of pICLC resulted in heightened day 10 (D10) draining lymph nodes (dLN) cellularity and sustained germinal center (GC) formation on D21—a time point typically used for murine Ebola virus-like particle (eVLP) boosting (Figures 2A,B)
We focused on D10 eVLP-mediated responses, which allowed us to characterize the early vaccine GC B-cell seeding events and to increase the overall sequencing breadth for our repertoire analysis owing to the higher absolute GC B-cell cellularity compared to D21
Summary
Establishing a diverse repertoire of antibody responses is central to humoral immunity acquired through natural infection and vaccination [1,2,3]. Most of our understanding of GC B cells and affinity maturation is based on the results of studies that have used simple model antigens, such as haptens, which contain a single epitope for BCR binding [5] In such cases, affinity maturation leads to reduced diversity and increased clonality of the GC B-cell repertoire, eventually resulting in a small number of distinct clonotypes, i.e., groups of clonally related B cells, and the emergence of B-cell clones shared across different individuals. Affinity maturation leads to reduced diversity and increased clonality of the GC B-cell repertoire, eventually resulting in a small number of distinct clonotypes, i.e., groups of clonally related B cells, and the emergence of B-cell clones shared across different individuals Such studies have yielded deep insights into affinity maturation, these simple model antigens differ greatly from more complex vaccine antigens, such as recombinant proteins, virus-like particles, and whole-pathogen vaccines, which may contain a multitude of epitopes for BCR binding. It remains unclear whether the oligoclonal responses to model antigens are pertinent to real-world vaccines
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
