High-Throughput Mapping of B Cell Receptor Sequences to Antigen Specificity.

B cell receptor (BCR) sequencing is a powerful tool for interrogating immune responses to infection and vaccination, but it provides limited information about the antigen specificity of the sequenced BCRs. Here, we present LIBRA-seq (linking B cell receptor to antigen specificity through sequencing), a technology for high-throughput mapping of paired heavy- and light-chain BCR sequences to their cognate antigen specificities. B cells are mixed with a panel of DNA-barcoded antigens so that both the antigen barcode(s) and BCR sequence are recovered via single-cell next-generation sequencing. Using LIBRA-seq, we mapped the antigen specificity of thousands of B cells from two HIV-infected subjects. The predicted specificities were confirmed for a number of HIV- and influenza-specific antibodies, including known and novel broadly neutralizing antibodies. LIBRA-seq will be an integral tool for antibody discovery and vaccine development efforts against a wide range of antigen targets.

Anti-CD20 therapy corrects a CD8 regulatory T cell deficit in multiple sclerosis.

OBJECTIVE: To determine the effect of long-term anti-CD20 B-cell-depleting treatment on regulatory T cell immune subsets that are subnormal in untreated MS patients. METHODS: 30 clinically stable MS patients, before and over 38 months of ocrelizumab treatment, were compared to 13 healthy controls, 29 therapy-naïve MS, 9 interferon-ß-treated MS, 3 rituximab-treated MS, and 3 rituximab-treated patients with other autoimmune inflammatory diseases. CD8, CD28, CD4, and FOXP3 expression in peripheral blood mononuclear cells was quantitated with flow cytometry. RESULTS: CD8+ CD28- regulatory cells rose from one-third of healthy control levels before ocrelizumab treatment (2.68% vs 7.98%), normalized by 12 months (13.5%), and rose to 2.4-fold above healthy controls after 18 months of ocrelizumab therapy (19.0%). CD4+ FOXP3+ regulatory cells were lower in MS than in healthy controls (7.98%) and showed slight long-term decreases with ocrelizumab. CD8+ CD28- and CD4+ FOXP3+ regulatory T cell percentages in IFN-ß-treated MS patients were between those of untreated MS and healthy controls. INTERPRETATION: Long-term treatment with ocrelizumab markedly enriches CD8+ CD28- regulatory T cells and corrects the low levels seen in MS before treatment, while slightly decreasing CD4+ FOXP3+ regulatory T cells. Homeostatic enrichment of regulatory CD8 T cells provides a mechanism, in addition to B cell depletion, for the benefits of anti-CD20 treatment in MS.

Discovery and Characterization of a Pan-betacoronavirus S2-binding antibody.

Three coronaviruses have spilled over from animal reservoirs into the human population and caused deadly epidemics or pandemics. The continued emergence of coronaviruses highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using LIBRA-seq, we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these antibodies, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryo-EM structure of 54043-5 bound to the pre-fusion S2 subunit of the SARS-CoV-2 spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses, including ADCC and ADCP. In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.

Can Technology-Based Social Memory Aids Improve Social Engagement? Perceptions of a Novel Memory Aid for Persons With Memory Concerns.

Social withdrawal and isolation are frequently experienced among people with cognitive impairment, Alzheimer's disease, and Alzheimer's disease related dementias. Few assistive technologies exist to support persons with memory concerns' (PWMC) continuing social engagement. This study aimed to understand PWMC and family caregivers' initial perspectives on the feasibility and utility of a wearable technology-based social memory aid. We recruited 20 dyads, presented the memory aid, and conducted semi-structured interviews from June to August 2020 over Zoom video conferencing. Interviews were transcribed and analyzed using thematic analysis. Overall, participants anticipated the technology could reduce socializing-related stress now and in the future for both members of the care dyad. However, certain features of the memory aid (e.g., visitors must have the app), could limit utility, and participants provided recommendations to enhance the tool. Our findings will inform future technology-enabled social memory aid development for PWMC and family caregivers.

An interlaboratory study to evaluate the utility of gas chromatography-mass spectrometry and gas chromatography-infrared spectroscopy spectral libraries in the forensic analysis of fentanyl-related substances.

Synthetic opioids such as fentanyl account for over 71,000 of the approximately 107,000 overdose deaths reported in the United States in 2021. Fentanyl remains the fourth most identified drug by state and local forensic laboratories, and the second most identified drug by federal laboratories. The unambiguous identification of fentanyl-related substances (FRS) is challenging due to the absence or low abundance of a molecular ion in a typical gas chromatography-mass spectrometry (GC-MS) analysis and due to a low number of fragment ions that are similar among the many potential isomers of FRS. This study describes the utility of a previously reported gas chromatography-infrared (GC-IR) library for the identification of FRS within a blind, interlaboratory study (ILS) involving seven forensic laboratories. Twenty FRS reference materials, including those with isomer pairs in the library, were selected based on either their presence in the NIST library and/or some similarity of the mass spectra information produced. The ILS participants were requested to use the Florida International University (FIU) GC-MS and GC-IR libraries supplied by FIU to search for matches to their unknown spectra generated from in-house GC-MS and GC-IR analysis. The laboratories reported improvement in the positive identification of unknown FRS from ~75% using GC-MS alone to 100% correct identification using GC-IR analysis. One laboratory participant used solid phase IR analysis, which produced spectra incompatible with the vapor phase GC-IR library to generate a good comparison spectrum. However, this improved when searched against a solid phase IR library.

Functional HIV-1/HCV cross-reactive antibodies isolated from a chronically co-infected donor.

Despite prolific efforts to characterize the antibody response to human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) mono-infections, the response to chronic co-infection with these two ever-evolving viruses is poorly understood. Here, we investigate the antibody repertoire of a chronically HIV-1/HCV co-infected individual using linking B cell receptor to antigen specificity through sequencing (LIBRA-seq). We identify five HIV-1/HCV cross-reactive antibodies demonstrating binding and functional cross-reactivity between HIV-1 and HCV envelope glycoproteins. All five antibodies show exceptional HCV neutralization breadth and effector functions against both HIV-1 and HCV. One antibody, mAb688, also cross-reacts with influenza and coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We examine the development of these antibodies using next-generation sequencing analysis and lineage tracing and find that somatic hypermutation established and enhanced this reactivity. These antibodies provide a potential future direction for therapeutic and vaccine development against current and emerging infectious diseases. More broadly, chronic co-infection represents a complex immunological challenge that can provide insights into the fundamental rules that underly antibody-antigen specificity.

Efficient discovery of SARS-CoV-2-neutralizing antibodies via B cell receptor sequencing and ligand blocking.

Although several monoclonal antibodies (mAbs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been approved for coronavirus disease 2019 (COVID-19) therapy, development was generally inefficient, with lead generation often requiring the production and testing of numerous antibody candidates. Here, we report that the integration of target-ligand blocking with a previously described B cell receptor-sequencing approach (linking B cell receptor to antigen specificity through sequencing (LIBRA-seq)) enables the rapid and efficient identification of multiple neutralizing mAbs that prevent the binding of SARS-CoV-2 spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The combination of target-ligand blocking and high-throughput antibody sequencing promises to increase the throughput of programs aimed at discovering new neutralizing antibodies.

An antibody targeting the N-terminal domain of SARS-CoV-2 disrupts the spike trimer.

The protective human antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) focuses on the spike (S) protein, which decorates the virion surface and mediates cell binding and entry. Most SARS-CoV-2 protective antibodies target the receptor-binding domain or a single dominant epitope ("supersite") on the N-terminal domain (NTD). Using the single B cell technology called linking B cell receptor to antigen specificity through sequencing (LIBRA-Seq), we isolated a large panel of NTD-reactive and SARS-CoV-2-neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies against the NTD supersite were commonly encoded by the IGHV1-24 gene, forming a genetic cluster representing a public B cell clonotype. However, we also discovered a rare human antibody, COV2-3434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface (TI) and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited the cell-to-cell spread of the virus in culture, and conferred protection in human angiotensin-converting enzyme 2-transgenic (ACE2-transgenic) mice against the SARS-CoV-2 challenge. This study provides insight into antibody targeting of the S protein TI region, suggesting this region may be a site of virus vulnerability.

Single-cell profiling of the antigen-specific response to BNT162b2 SARS-CoV-2 RNA vaccine.

RNA-based vaccines against SARS-CoV-2 have proven critical to limiting COVID-19 disease severity and spread. Cellular mechanisms driving antigen-specific responses to these vaccines, however, remain uncertain. Here we identify and characterize antigen-specific cells and antibody responses to the RNA vaccine BNT162b2 using multiple single-cell technologies for in depth analysis of longitudinal samples from a cohort of healthy participants. Mass cytometry and unbiased machine learning pinpoint an expanding, population of antigen-specific memory CD4+ and CD8+ T cells with characteristics of follicular or peripheral helper cells. B cell receptor sequencing suggest progression from IgM, with apparent cross-reactivity to endemic coronaviruses, to SARS-CoV-2-specific IgA and IgG memory B cells and plasmablasts. Responding lymphocyte populations correlate with eventual SARS-CoV-2 IgG, and a participant lacking these cell populations failed to sustain SARS-CoV-2-specific antibodies and experienced breakthrough infection. These integrated proteomic and genomic platforms identify an antigen-specific cellular basis of RNA vaccine-based immunity.

Simultaneous Immunization with Multiple Diverse Immunogens Alters Development of Antigen-Specific Antibody-Mediated Immunity.

Vaccination remains one of the most successful medical interventions in history, significantly decreasing morbidity and mortality associated with, or even eradicating, numerous infectious diseases. Although traditional immunization strategies have recently proven insufficient in the face of many highly mutable and emerging pathogens, modern strategies aim to rationally engineer a single antigen or cocktail of antigens to generate a focused, protective immune response. However, the effect of cocktail vaccination (simultaneous immunization with multiple immunogens) on the antibody response to each individual antigen within the combination, remains largely unstudied. To investigate whether immunization with a cocktail of diverse antigens would result in decreased antibody titer against each unique antigen in the cocktail compared to immunization with each antigen alone, we immunized mice with surface proteins from uropathogenic Escherichia coli, Mycobacterium tuberculosis, and Neisseria meningitides, and monitored the development of antigen-specific IgG antibody responses. We found that antigen-specific endpoint antibody titers were comparable across immunization groups by study conclusion (day 70). Further, we discovered that although cocktail-immunized mice initially elicited more robust antibody responses, the rate of titer development decreases significantly over time compared to single antigen-immunized mice. Investigating the basic properties that govern the development of antigen-specific antibody responses will help inform the design of future combination immunization regimens.

Feasibility of a Health Coaching and Home-Based Rehabilitation Intervention With Remote Monitoring for COPD.

BACKGROUND: Pulmonary rehabilitation is an effective treatment for patients with COPD, but patient uptake and adherence to the current offering of center-based pulmonary rehabilitation is modest due to transportation, access, poverty, and frailty, and even more so in the context of the COVID pandemic. Home-based options have been proposed and were found noninferior to center-based rehabilitation; however, there is a lack of home-based programs, and more understanding is needed. We aimed to test the feasibility, uptake, and adherence to a home-based program for COPD rehabilitation with health coaching. METHODS: We conducted a randomized trial with a wait-list controlled design to evaluate the effects of a home-based program with health coaching on breathlessness in subjects with moderate to severe COPD unable to attend the regular pulmonary rehabilitation program. The 8-week intervention consisted of video-guided exercises to be done 6 times a week and captured with a computer tablet. Health coaching was done weekly over the telephone to review subject activity and symptoms and to provide an opportunity for the subject to define their weekly goals. The primary outcomes were uptake, adherence, and Chronic Respiratory Questionnaire (CRQ) Dyspnea Domain. Secondary outcomes were self-management abilities and CRQ Emotions-Mastery-Fatigue. RESULTS: 154 subjects with moderate to severe COPD were randomized. Subject adherence was 86% to the proposed 6-times a week exercise routine. There (P = .062) was no significant difference in breathlessness (CRQ dyspnea). There was a significant improvement in self-management abilities (P < .001). The results of the qualitative interviews showed high levels of acceptability of the program. CONCLUSIONS: The tested home-based rehabilitation program with health coaching was feasible, highly acceptable, showed a high degree of adherence, and improved self-management abilities. This study offers seminal information for home-based rehabilitation programs to design alternative options of rehabilitation to individuals with COPD that cannot attend to the well-established center-based pulmonary rehabilitation. (ClinicalTrials.gov registration NCT02557178.).

Efficient discovery of potently neutralizing SARS-CoV-2 antibodies using LIBRA-seq with ligand blocking.

SARS-CoV-2 therapeutic antibody discovery efforts have met with notable success but have been associated with a generally inefficient process, requiring the production and characterization of exceptionally large numbers of candidates for the identification of a small set of leads. Here, we show that incorporating antibody-ligand blocking as part of LIBRA-seq, the high-throughput sequencing platform for antibody discovery, results in efficient identification of ultra-potent neutralizing antibodies against SARS-CoV-2. LIBRA-seq with ligand blocking is a general platform for functional antibody discovery targeting the disruption of antigen-ligand interactions.

Single-Cell Profiling of the Antigen-Specific Response to BNT162b2 SARS-CoV-2 RNA Vaccine.

RNA-based vaccines against SARS-CoV-2 are critical to limiting COVID-19 severity and spread. Cellular mechanisms driving antigen-specific responses to these vaccines, however, remain uncertain. We used single-cell technologies to identify and characterized antigen-specific cells and antibody responses to the RNA vaccine BNT162b2 in longitudinal samples from a cohort of healthy donors. Mass cytometry and machine learning pinpointed a novel expanding, population of antigen-specific non-canonical memory CD4 + and CD8 + T cells. B cell sequencing suggested progression from IgM, with apparent cross-reactivity to endemic coronaviruses, to SARS-CoV-2-specific IgA and IgG memory B cells and plasmablasts. Responding lymphocyte populations correlated with eventual SARS-CoV-2 IgG and a donor lacking these cell populations failed to sustain SARS-CoV-2-specific antibodies and experienced breakthrough infection. These integrated proteomic and genomic platforms reveal an antigen-specific cellular basis of RNA vaccine-based immunity. ONE SENTENCE SUMMARY: Single-cell profiling reveals the cellular basis of the antigen-specific response to the BNT162b2 SARS-CoV-2 RNA vaccine.

Cross-reactive coronavirus antibodies with diverse epitope specificities and Fc effector functions.

The continual emergence of novel coronaviruses (CoV), such as severe acute respiratory syndrome-(SARS)-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. From a recovered SARS-CoV donor sample, we identify and characterize a panel of six monoclonal antibodies that cross-react with CoV spike (S) proteins from the highly pathogenic SARS-CoV and SARS-CoV-2, and demonstrate a spectrum of reactivity against other CoVs. Epitope mapping reveals that these antibodies recognize multiple epitopes on SARS-CoV-2 S, including the receptor-binding domain, the N-terminal domain, and the S2 subunit. Functional characterization demonstrates that the antibodies mediate phagocytosis-and in some cases trogocytosis-but not neutralization in vitro. When tested in vivo in murine models, two of the antibodies demonstrate a reduction in hemorrhagic pathology in the lungs. The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies.

Potent neutralization of SARS-CoV-2 variants of concern by an antibody with an uncommon genetic signature and structural mode of spike recognition.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease (COVID-19). Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs.

Modulating lung immune cells by pulmonary delivery of antigen-specific nanoparticles to treat autoimmune disease.

Antigen-specific particles can treat autoimmunity, and pulmonary delivery may provide for easier delivery than intravenous or subcutaneous routes. The lung is a "hub" for autoimmunity where autoreactive T cells pass before arriving at disease sites. Here, we report that targeting lung antigen-presenting cells (APCs) via antigen-loaded poly(lactide-co-glycolide) particles modulates lung CD4+ T cells to tolerize murine experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Particles directly delivered to the lung via intratracheal administration demonstrated more substantial reduction in EAE severity when compared with particles delivered to the liver and spleen via intravenous administration. Intratracheally delivered particles were associated with lung APCs and decreased costimulatory molecule expression on the APCs, which inhibited CD4+ T cell proliferation and reduced their population in the central nervous system while increasing them in the lung. This study supports noninvasive pulmonary particle delivery, such as inhalable administration, to treat autoimmune disease.

The feasibility of verbal and virtual reality exposure for youth with academic performance worry.

With exposure emerging as a key ingredient in anxiety treatment for childhood anxiety disorders (CADs), expansion of exposure techniques is a promising avenue for improving treatment efficacy. The present study examined use of imaginal exposure (IE), a technique understudied in the treatment of CADs. Specifically, the study tested whether two forms of exposure to worries (verbal IE and virtual reality exposure therapy, VRET) would be effective and acceptable forms of exposure with youth. Twenty youth with fears of academic failure completed both types of worry exposure, presented in randomized order. Regardless of order of presentation, both verbal IE and VRET elicited moderate anxiety that decreased to mild over the span of the exposures. Both were found to be acceptable by youth and neither was associated with negative side effects. Youth found VRET to be slightly more interesting and novel, but noted that verbal IE was more realistic and individualized. The present study supports the use of standalone worry exposure as an effective and acceptable treatment for general worries in youth and suggests VRET could be more effective with improved realism.

Cross-reactive coronavirus antibodies with diverse epitope specificities and extra-neutralization functions.

The continual emergence of novel coronavirus (CoV) strains, like SARS-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. Coronavirus spike (S) proteins share common structural motifs that could be vulnerable to cross-reactive antibody responses. To study this phenomenon in human coronavirus infection, we applied a high-throughput sequencing method called LIBRA-seq (Linking B cell receptor to antigen specificity through sequencing) to a SARS-CoV-1 convalescent donor sample. We identified and characterized a panel of six monoclonal antibodies that cross-reacted with S proteins from the highly pathogenic SARS-CoV-1 and SARS-CoV-2 and demonstrated a spectrum of reactivity against other coronaviruses. Epitope mapping revealed that these antibodies recognized multiple epitopes on SARS-CoV-2 S, including the receptor binding domain (RBD), N-terminal domain (NTD), and S2 subunit. Functional characterization demonstrated that the antibodies mediated a variety of Fc effector functions in vitro and mitigated pathological burden in vivo . The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies that may be useful for preventing potential future coronavirus outbreaks.

Design of biodegradable nanoparticles to modulate phenotypes of antigen-presenting cells for antigen-specific treatment of autoimmune disease.

Current therapeutic options for autoimmune diseases, such as multiple sclerosis (MS), often require lifelong treatment with immunosuppressive drugs, yet strategies for antigen-specific immunomodulation are emerging. Biodegradable particles loaded with disease-specific antigen, either alone or with immunomodulators, have been reported to ameliorate disease. Herein, we hypothesized that the carrier could impact polarization of the immune cells that associate with particles and the subsequent disease progression. Single injection of three polymeric carriers, 50:50 poly (DL-lactide-co-glycolide) (PLG) with two molecular weights (Low, High) and poly (DL-lactide) (PLA), loaded with the disease-specific antigen, proteolipid protein (PLP139-151), were investigated for the ability to attenuate clinical scores in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. At a low particle dose, mice treated with PLA-based particles had significantly lower clinical scores at the chronic stage of the disease over 200 days post immunization, while neither PLG-based particles nor OVA control particles reduced the clinical scores. Compared to PLG-based particles, PLA-based particles were largely associated with Kupffer cells and liver sinusoidal endothelial cells, which had a reduced co-stimulatory molecule expression that correlated with a reduction of CD4+ T-cell populations in the central nervous system. Delivery of PLA-based particles encapsulated with higher levels of PLP139-151 at a reduced dose were able to completely ameliorate EAE over 200 days along with inhibition of Th1 and Th17 polarization. Collectively, our study demonstrates that the carrier properties and antigen loading determine phenotypes of immune cells in the peripheral organs, influencing the amelioration of both acute and chronic stages of autoimmunity.

Development and Feasibility of a Home Pulmonary Rehabilitation Program With Health Coaching.

BACKGROUND: Pulmonary rehabilitation (PR) is an effective intervention for COPD. However, traditional center-based PR programs suffer from low uptake. Home-based PR is a viable solution, but few studies have shown the effectiveness of remote PR, as there is a scarcity of systems that can be easily adopted in clinical practice. The aim of this report is to communicate the development and feasibility of a home PR program that includes commercially available technology that allows the PR health coach to follow the patient through his or her PR process and to present the design of a prospective clinical trial. METHODS: We developed a home PR system that includes a computer tablet, an activity monitor, and an oximeter connected to a cloud server. The home PR consists of 12 min of walking and 6 full-body exercises, to be completed 6 d/week, plus weekly telephone calls with the PR health coach. Two pilot studies were conducted in subjects with moderate-to-severe COPD. The first aimed to fine-tune the system development (N = 3), and the second tested the program feasibility of the 8-week program (N = 12). RESULTS: In pilot study 1, PR monitoring data from the subjects' home PR sessions were transmitted to the health coach application successfully. On a 10-point scale, participants rated the system as helpful (median = 8, interquartile range 8-9) and simple to use (median = 10, interquartile range 9-10). In pilot study 2, adherence ± SD for prescribed use was 87 ± 0.24%. Overall, participants gave the home PR system a rating of 6.2 ± 0.94 on a 7-point scale. CONCLUSIONS: A home PR program was developed that integrated health coaching and a home PR system that facilitated remote monitoring. Pilot testing indicated that the program is well-developed and feasible in a population of individuals with COPD. (ClinicalTrials.gov registration NCT02999685.).