High-resolution antibody dynamics of vaccine-induced immune responses.

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.

Effects of aging, cytomegalovirus infection, and EBV infection on human B cell repertoires.

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 CMV is implicated as a driver of clonal T cell proliferations in some aging humans, but whether CMV or EBV infection contributes to alterations in the B cell repertoire with age is unclear. We have used high-throughput DNA sequencing of IGH gene rearrangements to study the BCR repertoires over two successive years in 27 individuals ranging in age from 20 to 89 y. 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 Ig 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, whereas CMV infection correlates with the proportion of highly mutated Ab 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.

The effects of somatic hypermutation on neutralization and binding in the PGT121 family of broadly neutralizing HIV antibodies.

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.

Using Machine Learning to Identify Intravenous Contrast Phases on Computed Tomography.

PURPOSE: The purpose of the present work is to demonstrate the application of machine learning (ML) techniques to automatically identify the presence and physiologic phase of intravenous (IV) contrast in Computed Tomography (CT) scans of the Chest, Abdomen and Pelvis. MATERIALS AND METHODS: Training, testing and validation data were acquired from a dataset of 82,690 chest and abdomen CT examinations performed at 17 different institutions. Free text in DICOM metadata was utilized as weak labels for semi-supervised classification training. Contrast phase identification was approached as a classification task, using a 12-layer CNN and ResNet18 with four contrast-phase output. The model was reformulated to fit a regression task aimed to predict actual seconds from time of IV contrast administration to series image acquisition. Finally, transfer learning was used to optimize the model to predict contrast presence on CT Chest. RESULTS: By training based on labels inferred from noisy, free text DICOM information, contrast phase was predicted with 93.3% test accuracy (95% CI: 89.3%, 96.6%) . Regression analysis resulted in delineation of early vs late arterial phases and a nephrogenic phase in between the portal venous and delayed excretory phase. Transfer learning applied to Chest CT achieved an AUROC of 0.776 (95% CI: 0.721, 0.832) directly using the model trained for abdomen CT and 0.999 (95% CI: 0.998, 1.000) by fine-tuning. CONCLUSIONS: The presence and phase of contrast on CT examinations of the Abdomen-pelvis accurately and automatically be ascertained by a machine learning algorithm. Transfer learning applied to CT Chest achieves high precision with as little as 100 labeled samples.

Human responses to influenza vaccination show seroconversion signatures and convergent antibody rearrangements.

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 with those expressing influenza-specific monoclonal antibodies. 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.

Genovo: de novo assembly for metagenomes.

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 .