Identifying functional anti-Staphylococcus aureus antibodies by sequencing antibody repertoires of patient plasmablasts

Abstract

Infection by Staphylococcus aureus is on the rise, and there is 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.

Elevated IgA Plasmablast Levels in Subjects at Risk of Developing Rheumatoid Arthritis

“…Similarly, the size and frequency of clonal families in Ab+ At-Risk subjects was lower than what we have observed for acute infection and vaccination responses (29, 59), and was more in line with chronic autoimmune activation ((23) and unpublished observations). Whether plasmablasts in at-risk subjects are induced by a sudden trigger (such as viral or bacterial infection) or progress steadily towards autoreactivity over time due to low-level stimulation of the gingival and pulmonary mucosa is not yet clear.…”

Microscale Technologies for High-Throughput Analysis of Immune Cells

“…Previous studies have shown that when scRT-PCR is combined with antigen baiting 80-90 % of sorted B cells encoded antigen-specifi c antibodies [ 69 , 70 ]. Remarkably, this combination of techniques has been successfully applied to identify potent broadly neutralizing HIV-specifi c human monoclonal antibodies [ 69 , 71 -74 ], and anti-Staphylococcus aureus antibodies from infected patients [ 75 ]. Additionally, human monoclonal antibodies specifi c for transglutaminase 2 have been isolated and cloned from antigen baited IgA + gut plasma cells obtained from Celiac disease patient using scRT-PCR [ 70 ].…”

Mass Cytometry Analysis Shows That a Novel Memory Phenotype B Cell Is Expanded in Multiple Myeloma

“…Single B cells were identified by their scatter (FSC/SSC) characteristics, CD19, and CD20 expression and sorted into RT-PCR buffer in 96-well plates according to the gating strategy in Figure 4A and Supplementary Figure 8. Ig genes were amplified and sequenced as previously described (7,8). …”

“…To some extent this may be due to the limited resolving power of standard cell analysis methods such as flow cytometry and the heterogeneity of peripheral blood cells. However, recent advances in cell phenotyping technology, particularly cytometry by time-of-flight (CyTOF), which supports 40-label analysis (4–6), and next generation sequencing (NGS) of immunologlobulin (Ig) genes (7,8) have greatly increased the possible depth of analysis at the single-cell level and expanded the resolving power of immune phenotyping tremendously. This suggests that it may be possible to detect disease-related immune signatures in human peripheral blood using these methods.…”