A key step in developing engineered B cells for therapeutic purposes is evaluation in immunocompetent, large-animal models. Therefore, we developed methods to purify, expand, and differentiate non-human primate (NHP; rhesus macaque) B cells. After 7 days in culture, B cells expanded 10-fold, differentiated into a plasma cell phenotype (CD38, CD138), and secreted immunoglobulin G. Using single-cell sequencing and flow cytometry, we verified the presence of plasma cell genes in differentiated NHP B cells and unearthed less-recognized markers, such as CD59 and CD79A. In contrast with human cells, we found that the immune checkpoint molecule CD274 (PD-L1) and major histocompatibility complex (MHC) class I molecules were upregulated in NHP plasma cells in the transcriptional data. Lastly, we established the conditions for efficient transduction of NHP B cells with adeno-associated virus (AAV) vectors, achieving a delivery rate of approximately 60%. We envision that this work will accelerate proof-of-concept studies using engineered B cells in NHPs.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

The most advanced P. falciparum circumsporozoite protein-based malaria vaccine, RTS,S/AS01 (RTS,S), confers partial protection but with antibody titers that wane relatively rapidly, highlighting the need to elicit more potent and durable antibody responses. Here, we elucidate crystal structures, binding affinities and kinetics, and in vivo protection of eight anti-NANP antibodies derived from an RTS,S phase 2a trial and encoded by three different heavy-chain germline genes. The structures reinforce the importance of homotypic Fab-Fab interactions in protective antibodies and the overwhelmingly dominant preference for a germline-encoded aromatic residue for recognition of the NANP motif. In this study, antibody apparent affinity correlates best with protection in an in vivo mouse model, with the more potent antibodies also recognizing epitopes with repeating secondary structural motifs of type I β- and Asn pseudo 310 turns; such insights can be incorporated into design of more effective immunogens and antibodies for passive immunization.

Seasonal influenza vaccines elicit antibody responses that can prevent infection, but their efficacy is reduced in the elderly. While a subset of elderly individuals can still mount sufficient vaccine-induced antibody responses, little is known about the properties of the vaccine-induced antibody repertoires in elderly as compared to young responders. To gain insights into the effects of aging on influenza vaccine-induced antibody responses, we used flow cytometry and a cell-barcoding method to sequence antibody heavy and light chain gene pairs expressed by individual blood plasmablasts generated in response to influenza vaccination in elderly (aged 70-89) and young (aged 20-29) responders. We found similar blood plasmablast levels in the elderly and young responders seven days post vaccination. Informatics analysis revealed increased clonality, but similar heavy chain V(D)J gene usage in the elderly as compared to young vaccine responders. Although the elderly responders exhibited decreased antibody sequence diversity and fewer consequential mutations relative to young responders, recombinant antibodies from elderly responders bound a broader range of influenza strain HAs. Thus elderly influenza vaccine responders mount plasmablast responses with restricted diversity but with an increased breadth of binding across influenza strains. Our results suggest that the ability to generate plasmablast responses encoding cross-strain binding antibodies likely represents a mechanism important to vaccine responses in the elderly.
Published by Elsevier Inc.

Infection by Staphylococcus aureus is on the rise, and there is a need for a better understanding of host immune responses that combat S. aureus. Here we use DNA barcoding to enable deep sequencing of the paired heavy- and light-chain immunoglobulin genes expressed by individual plasmablasts derived from S. aureus-infected humans. Bioinformatic analysis of the antibody repertoires revealed clonal families of heavy-chain sequences and enabled rational selection of antibodies for recombinant expression. Of the ten recombinant antibodies produced, seven bound to S. aureus, of which four promoted opsonophagocytosis of S. aureus. Five of the antibodies bound to known S. aureus cell-surface antigens, including fibronectin-binding protein A. Fibronectin-binding protein A-specific antibodies were isolated from two independent S. aureus-infected patients and mediated neutrophil killing of S. aureus in in vitro assays. Thus, our DNA barcoding approach enabled efficient identification of antibodies involved in protective host antibody responses against S. aureus.
Published by Elsevier Inc.

We developed a DNA barcoding method to enable high-throughput sequencing of the cognate heavy- and light-chain pairs of the antibodies expressed by individual B cells. We used this approach to elucidate the plasmablast antibody response to influenza vaccination. We show that >75% of the rationally selected plasmablast antibodies bind and neutralize influenza, and that antibodies from clonal families, defined by sharing both heavy-chain VJ and light-chain VJ sequence usage, do so most effectively. Vaccine-induced heavy-chain VJ regions contained on average >20 nucleotide mutations as compared to their predicted germline gene sequences, and some vaccine-induced antibodies exhibited higher binding affinities for hemagglutinins derived from prior years' seasonal influenza as compared to their affinities for the immunization strains. Our results show that influenza vaccination induces the recall of memory B cells that express antibodies that previously underwent affinity maturation against prior years' seasonal influenza, suggesting that 'original antigenic sin' shapes the antibody response to influenza vaccination.
Published by Elsevier Inc.