Summary B cells produce a diverse antibody repertoire by undergoing gene rearrangements. Pathogen exposure induces the clonal expansion of B cells expressing antibodies that can bind the infectious agent. To assess human B cell responses to trivalent seasonal influenza and monovalent pandemic H1N1 vaccination, we sequenced gene rearrangements encoding the immunoglobulin heavy chain, a major determinant of epitope recognition. The magnitude of B cell clonal expansions correlates with an individual’s secreted antibody response to the vaccine and the expanded clones are enriched for those expressing influenza-specific mAbs. Additionally, B cell responses to pandemic influenza H1N1 vaccination and infection in different people show a prominent family of convergent antibody heavy chain gene rearrangements specific to influenza antigens. These results indicate that microbes can induce specific signatures of immunoglobulin gene rearrangements and that pathogen exposure can potentially be assessed from B cell repertoires.
Broadly neutralizing HIV antibodies (bnAbs) are typically highly somatically mutated, raising doubts as to whether they can be elicited by vaccination. We used 454 sequencing and designed a novel phylogenetic method to model lineage evolution of the bnAbs PGT121–134 and found a positive correlation between the level of somatic hypermutation (SHM) and the development of neutralization breadth and potency. Strikingly, putative intermediates were characterized that show approximately half the mutation level of PGT121–134 but were still capable of neutralizing roughly 40–80% of PGT121–134 sensitive viruses in a 74-virus panel at median titers between 15- and 3-fold higher than PGT121–134. Such antibodies with lower levels of SHM may be more amenable to elicitation through vaccination while still providing noteworthy coverage. Binding characterization indicated a preference of inferred intermediates for native Env binding over monomeric gp120, suggesting that the PGT121–134 lineage may have been selected for binding to native Env at some point during maturation. Analysis of glycan-dependent neutralization for inferred intermediates identified additional adjacent glycans that comprise the epitope and suggests changes in glycan dependency or recognition over the course of affinity maturation for this lineage. Finally, patterns of neutralization of inferred bnAb intermediates suggest hypotheses as to how SHM may lead to potent and broad HIV neutralization and provide important clues for immunogen design.
The adaptive immune system confers protection by generating a diverse repertoire of antibody receptors that are rapidly expanded and contracted in response to specific targets. Next-generation DNA sequencing now provides the opportunity to survey this complex and vast repertoire. In the present work, we describe a set of tools for the analysis of antibody repertoires and their application to elucidating the dynamics of the response to viral vaccination in human volunteers. By analyzing data from 38 separate blood samples across 2 y, we found that the use of the germ-line library of V and J segments is conserved between individuals over time. Surprisingly, there appeared to be no correlation between the use level of a particular VJ combination and degree of expansion. We found the antibody RNA repertoire in each volunteer to be highly dynamic, with each individual displaying qualitatively different response dynamics. By using combinatorial phage display, we screened selected VH genes paired with their corresponding VL library for affinity against the vaccine antigens. Altogether, this work presents an additional set of tools for profiling the human antibody repertoire and demonstrates characterization of the fast repertoire dynamics through time in multiple individuals responding to an immune challenge.next-generation sequencing | influenza | immunology T he immune system is able to rapidly sense and respond to a vast array of invading organisms. Its arsenal contains systems that are immediately effective against commonly seen patterns (innate immunity) and systems that are capable of responding to novel invaders (adaptive immunity). Given the acute nature and diversity of infections, the immune system must be capable of rapid recognition of a pathogen, amplification of the response, and subsequent contraction of the response after the resolution of the infection. Adaptive immune responses rely on the continuous selection and amplification of specific clones from an enormous library of immune receptors (antibodies and T cell receptors). Specifically, stimulation of B-cell immunity results in the synthesis of antibodies that are secreted into the blood stream or into the mucosa as well as the programming of B memory cells that play a crucial role in the generation of rapid protective responses upon reinfection.Currently, many immunology studies depend on characterizing lymphocyte subsets (e.g., assaying cell-surface receptors) and the ability to correlate them to encoded genetic information (1). 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 (3-8).The present study set out to dissect the rapid dynamics of the complete human peripheral antibody response against a controlled immune challenge (vaccination), without the a priori notion of cell state markers or functions...
Elderly humans show decreased humoral immunity to pathogens and vaccines, yet the effects of aging on B cells are not fully known. Chronic viral infection by cytomegalovirus (CMV) is implicated as a driver of clonal T cell proliferations in some aging humans, but whether CMV or Epstein-Barr virus (EBV) infection contributes to alterations in the B cell repertoire with age is unclear. We have used high-throughput DNA sequencing of immunoglobulin heavy chain (IGH) gene rearrangements to study the B cell receptor repertoires over two successive years in 27 individuals ranging in age from 20 to 89 years. Some features of the B cell repertoire remain stable with age, but elderly subjects show increased numbers of B cells with long CDR3 regions, a trend toward accumulation of more highly mutated IgM and IgG immunoglobulin genes, and persistent clonal B cell populations in the blood. Seropositivity for CMV or EBV infection alters B cell repertoires, regardless of the individual's age: EBV infection correlates with the presence of persistent clonal B cell expansions, while CMV infection correlates with the proportion of highly mutated antibody genes. These findings isolate effects of aging from those of chronic viral infection on B cell repertoires, and provide a baseline for understanding human B cell responses to vaccination or infectious stimuli.
Next-generation sequencing technologies produce a large number of noisy reads from the DNA in a sample. Metagenomics and population sequencing aim to recover the genomic sequences of the species in the sample, which could be of high diversity. Methods geared towards single sequence reconstruction are not sensitive enough when applied in this setting. We introduce a generative probabilistic model of read generation from environmental samples and present Genovo, a novel de novo sequence assembler that discovers likely sequence reconstructions under the model. A nonparametric prior accounts for the unknown number of genomes in the sample. Inference is performed by applying a series of hill-climbing steps iteratively until convergence. We compare the performance of Genovo to three other short read assembly programs in a series of synthetic experiments and across nine metagenomic datasets created using the 454 platform, the largest of which has 311k reads. Genovo's reconstructions cover more bases and recover more genes than the other methods, even for low-abundance sequences, and yield a higher assembly score. Supplementary Material is available at www.liebertoinline.com/cmb .
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