Profiling B cell immunodominance after SARS-CoV-2 infection reveals antibody evolution to non-neutralizing viral targets.

Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we used single-cell sequencing to profile SARS-CoV-2-reactive B cells in 38 COVID-19 patients. Using oligo-tagged antigen baits, we isolated B cells specific to the SARS-CoV-2 spike, nucleoprotein (NP), open reading frame 8 (ORF8), and endemic human coronavirus (HCoV) spike proteins. SARS-CoV-2 spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients several months post symptom onset. With severe acute infection, substantial populations of endemic HCoV-reactive antibody-secreting cells were identified and possessed highly mutated variable genes, signifying preexisting immunity. Finally, MBCs exhibited pronounced maturation to NP and ORF8 over time, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal antibody adaptation to non-neutralizing intracellular antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.

Germinal Center B Cells Replace Their Antigen Receptors in Dark Zones and Fail Light Zone Entry when Immunoglobulin Gene Mutations are Damaging.

Adaptive immunity involves the development of bespoke antibodies in germinal centers (GCs) through immunoglobulin somatic hypermutation (SHM) in GC dark zones (DZs) and clonal selection in light zones (LZs). Accurate selection requires that cells fully replace surface B cell receptors (BCRs) following SHM, but whether this happens before LZ entry is not clear. We found that most GC B cells degrade pre-SHM receptors before leaving the DZ, and that B cells acquiring crippling mutations during SHM rarely reached the LZ. Instead, apoptosis was triggered preferentially in late G1, a stage wherein cells with functional BCRs re-entered cell cycle or reduced surface expression of the chemokine receptor CXCR4 to enable LZ migration. Ectopic expression of the anti-apoptotic gene Bcl2 was not sufficient for cells with damaging mutations to reach the LZ, suggesting that BCR-dependent cues may actively facilitate the transition. Thus, BCR replacement and pre-screening in DZs prevents the accumulation of clones with non-functional receptors and facilitates selection in the LZ.

Effectiveness of Angle-Based Minimally Invasive Glaucoma Surgery after Laser Trabeculoplasty: An Analysis of the IRIS® Registry (Intelligent Research in Sight).

OBJECTIVE: Angle-based minimally invasive glaucoma surgery (ab-MIGS) has grown substantially, although long-term efficacy is poorly understood. We analyze ab-MIGS effectiveness with and without preceding laser trabeculoplasty (LTP). DESIGN: Retrospective cohort study. SUBJECTS: Eyes undergoing ab-MIGS (Canaloplasty, Goniotomy, Trabectome, and iStent) with and without prior laser trabeculoplasty (< 2 years preceding MIGS) were identified in the IRIS® Registry (Intelligent Research in Sight) 2013 to 2018. METHODS: Propensity score matching (PSM) was undertaken to define the following 4 cohorts: (1) standalone ab-MIGS, no prior LTP vs. (2) standalone ab-MIGS, with prior LTP; and (3) ab-MIGS + phacoemulsification, no prior LTP vs. (4) ab-MIGS + phacoemulsification, with prior LTP. MAIN OUTCOME MEASURES: Failure was defined as subsequent glaucoma reoperation after ab-MIGS (either MIGS or traditional glaucoma surgery). Time-to-event outcome and incidence rates were calculated using survival analysis, and adjusted hazard ratios (aHRs) were generated using multivariate Cox proportional hazards models. Medication data were not available for analysis. RESULTS: A total of 164 965 unique MIGS procedures were performed, from 2013 to 2018. After PSM, we identified 954 eyes undergoing standalone ab-MIGS and 7522 undergoing ab-MIGS + phacoemulsification. For eyes undergoing standalone ab-MIGS, those with prior LTP (n = 477) were more likely to undergo reoperation vs. those without LTP (n = 477) at 6 and 12 months. In multivariable models, those with prior LTP were more likely to undergo reoperation over the 36-month period vs. those without prior LTP (aHR, 1.53; CI, 1.15-2.04; P = 0.004). For eyes undergoing ab-MIGS + phacoemulsification, those with prior LTP (n = 3761) were more likely to undergo reoperation vs. those without LTP (n = 3761) at 12, 24, and 36 months. In multivariable models, those with prior LTP were more likely to undergo reoperation over the 36-month period vs. those without prior LTP (aHR, 1.53 CI, 1.15-2.04; P = 0.004). CONCLUSIONS: Prior LTP may be associated with a higher chance of subsequent glaucoma surgery following ab-MIGS, either with or without concurrent phacoemulsification. These findings have important implications for understanding who may benefit most from ab-MIGS, and for guiding patient and surgeon treatment expectations. FINANCIAL DISCLOSURES: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Distinct B cell subsets give rise to antigen-specific antibody responses against SARS-CoV-2.

Discovery of durable memory B cell (MBC) subsets against neutralizing viral epitopes is critical for determining immune correlates of protection from SARS-CoV-2 infection. Here, we identified functionally distinct SARS-CoV-2-reactive B cell subsets by profiling the repertoire of convalescent COVID-19 patients using a high-throughput B cell sorting and sequencing platform. Utilizing barcoded SARS-CoV-2 antigen baits, we isolated thousands of B cells that segregated into discrete functional subsets specific for the spike, nucleocapsid protein (NP), and open reading frame (ORF) proteins 7a and 8. Spike-specific B cells were enriched in canonical MBC clusters, and monoclonal antibodies (mAbs) from these cells were potently neutralizing. By contrast, B cells specific to ORF8 and NP were enriched in naïve and innate-like clusters, and mAbs against these targets were exclusively non-neutralizing. Finally, we identified that B cell specificity, subset distribution, and affinity maturation were impacted by clinical features such as age, sex, and symptom duration. Together, our data provide a comprehensive tool for evaluating B cell immunity to SARS-CoV-2 infection or vaccination and highlight the complexity of the human B cell response to SARS-CoV-2.