Applications of high-throughput single B-cell sequencing to accelerate rational vaccine design

Understanding the antibody repertoire response to vaccination is critical for the rational design and evaluation of experimental vaccines. Immune receptors comprise two chains encoded by separate mRNA strands and thus conventional NextGen sequencing fails to identify the native pairings encoded by individual lymphocytes. To overcome this limitation, we are applying recent technical advances in high-throughput sequencing of complete antibodies (i.e., paired heavy and light chain sequencing) to generate a quantitative understanding of experimental vaccine performance and to accelerate vaccine design. We apply repertoire-based metrics of vaccine-elicited antibodies to evaluate and select promising candidate immunogens for inducing HIV-1 Envelope-specific VRC01-class antibodies. The VRC01 class of broadly neutralizing antibodies have been observed in multiple individuals and targets the HIV CD4 binding site via a common recognition motif that requires specific features in both heavy and light chains (e.g., VH1-2 heavy chain V-gene and a short, £5 amino acid light chain CDR3). We are using paired heavy and light chain sequencing to quantify the performance of various candidate HIV immunogens for inducing VRC01-class broadly neutralizing HIV antibodies in transgenic mouse models. We are also elucidating the ontogeny of antibodies in vaccinated and naturally infected human subjects and animal models via interrogation of paired heavy and light chain antibody sequences and antibody synthesis/testing of promising clones, including experimental influenza vaccine trials and a Phase I Ebola vaccine trial. These next-generation immunoanalytic approaches are providing detailed molecular feedback regarding experimental vaccine performance to accelerate vaccine design efforts against pathogens of major public health importance

Supplemental Material for Edskes et al., 2018

Supplemental Methods_figures_tables.pdf - Provides a detailed description of the culture conditions, induction of <i>Hermes</i> transposition, selection of colonies carrying a transposition, extraction of cellular DNA, PCR amplification and isolation of the junction points between transposon and chromosomal insertion site, next-generation sequencing of these sites, and analysis of the data by visual display and by counting insertions per open reading frame. <br><br> Exon Intron Counts.xlsx - Gives the insertions in every yeast open reading frame, distinguishing exons from introns where appropriate.<br><br>Sorted Hits.xlsx - Gives prominent hits sorted by functional group, “TY gag-pol Counts.xlsx comparing insertions in the Ty retrotransposons at different locations in the genome. <br><br>Count Insertions in ORFs and Introns.txt - The Python program used for counting insertions. <br><br>LUGsIGV-InsertDistributions.pptx - Is a slide show of insert distributions in each of 500 genes for which insertions were recovered more frequently in [ure-o] than in [URE3] cultures

Protein conformation templating mechanism.

<p>The in-register parallel amyloid architecture naturally suggests a mechanism for transfer of conformation information from molecules in the filament to those joining the filament. H-bonding or hydrophobic favorable interactions among identical side chains require in-register alignment for the interactions. This directs the monomer joining the end of the filament to have its folds/turns at the same residues as previous molecules in the filament. Different prion variants have folds/turns at different locations, but each is faithfully propagated by this mechanism. Modified from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004584#ppat.1004584.ref006" target="_blank">6</a>].</p

Btn2p sequesters prion amyloid filaments, curing the prion.

<p>Btn2p gathers filaments of Ure2p amyloid (the [URE3] prion) to a single site in the cell, possibly the endosome. If the prion has a low seed number then even the normal levels of Btn2p are sufficient to sequester nearly all of the seeds, so that daughter cells without seeds, and therefore cured of the prion, are often produced.</p

Glycan Masking Focuses Immune Responses to the HIV-1 CD4-Binding Site and Enhances Elicitation of VRC01-Class Precursor Antibodies

This work is licensed under a Creative Commons Attribution Non-Commercial-No Derivatives 4.0 International License.An important class of HIV-1 broadly neutralizing antibodies, termed the VRC01 class, targets the conserved CD4-binding site (CD4bs) of the envelope glycoprotein (Env). An engineered Env outer domain (OD) eOD-GT8 60-mer nanoparticle has been developed as a priming immunogen for eliciting VRC01-class precursors and is planned for clinical trials. However, a substantial portion of eOD-GT8-elicited antibodies target non-CD4bs epitopes, potentially limiting its efficacy. We introduced N-linked glycans into non-CD4bs surfaces of eOD-GT8 to mask irrelevant epitopes and evaluated these mutants in a mouse model that expressed diverse immunoglobulin heavy chains containing human IGHV1-2∗02, the germline VRC01 VH segment. Compared to the parental eOD-GT8, a mutant with five added glycans stimulated significantly higher proportions of CD4bs-specific serum responses and CD4bs-specific immunoglobulin G+ B cells including VRC01-class precursors. These results demonstrate that glycan masking can limit elicitation of off-target antibodies and focus immune responses to the CD4bs, a major target of HIV-1 vaccine design