Multivalent S2-subunit Vaccines Provide Broad Protection Against Clade 1 Sarbecoviruses (preprint)

The continuing emergence of immune evasive SARS-CoV-2 variants and the previous SARS-CoV-1 outbreak have accentuated the need for broadly protective sarbecovirus vaccines. Targeting the conserved S2-subunit of SARS-CoV-2 is a particularly promising approach to elicit broad protection. Here, expanding on our previous work with S2-based vaccines, we developed a nanoparticle vaccine displaying multiple copies of the SARS-CoV-1 S2 subunit. This vaccine alone, or as a cocktail with a SARS-CoV-2 S2 subunit vaccine, protected transgenic K18-hACE2 mice from challenges with Omicron subvariant XBB as well as several sarbecoviruses identified as having pandemic potential including the bat sarbecovirus WIV1, BANAL-236, and a pangolin sarbecovirus. Challenge studies in Fc-g receptor knockout mice revealed that antibody-based cellular effector mechanisms played a role in protection elicited by these vaccines. These results demonstrate that our S2-based vaccines provide broad protection against clade 1 sarbecoviruses and offer insight into the mechanistic basis for protection.

Mutability and hypermutation antagonize immunoglobulin codon optimality (preprint)

The efficacy of polyclonal antibody responses is inherently linked to paratope diversity, as generated through V(D)J recombination and somatic hypermutation (SHM). These processes arose in early jawed vertebrates; however, little is known about how immunoglobulin diversity, mutability, and hypermutation have evolved in tandem with another more ubiquitous feature of protein-coding DNA - codon optimality. Here, we explore these relationships through analysis of germline IG genes, natural V(D)J repertoires, serum VH usage, and monoclonal antibody (mAb) expression, each through the lens of multiple optimality metrics. Strikingly, proteomic serum IgG sequencing showed that germline IGHV codon optimality positively correlated with VH representation after influenza vaccination, and in vitro, codon deoptimization of mAbs with synonymous amino acid sequences caused consistent expression loss. Germline V genes exhibit a range of codon optimality that is maintained by functionality, and inversely related to mutability. SHM caused a load-dependent deoptimization of IGH VDJ repertoires within human tonsils, bone marrow, and lymph nodes (including SARS-CoV-2-specific clones from mRNA vaccinees), influenza-infected mice, and zebrafish. Comparison of natural mutation profiles to true random suggests the presence of selective pressures that constrain deoptimization. These findings shed light on immunoglobulin evolution, providing unanticipated insights into the antagonistic relationship between variable region diversification, codon optimality, and antibody secretion; ultimately, the need for diversity takes precedence over that for the most efficient expression of the antibody repertoire.

Cross neutralization of emerging SARS-CoV-2 variants of concern by antibodies targeting distinct epitopes on spike (preprint)

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have arisen that exhibit increased viral transmissibility and partial evasion of immunity induced by natural infection and vaccination. To address the specific antibody targets that were affected by recent viral variants, we generated 43 monoclonal antibodies (mAbs) from 10 convalescent donors that bound three distinct domains of the SARS-CoV-2 spike. Viral variants harboring mutations at K417, E484 and N501 could escape most of the highly potent antibodies against the receptor binding domain (RBD). Despite this, we identified 12 neutralizing mAbs against three distinct regions of the spike protein that neutralize SARS-CoV-2 and the variants of concern, including B.1.1.7 (alpha), P.1 (gamma) and B.1.617.2 (delta). Notably, antibodies targeting distinct epitopes could neutralize discrete variants, suggesting different variants may have evolved to disrupt the binding of particular neutralizing antibody classes. These results underscore that humans exposed to wildtype (WT) SARS-CoV-2 do possess neutralizing antibodies against current variants and that it is critical to induce antibodies targeting multiple distinct epitopes of the spike that can neutralize emerging variants of concern.

Integration of Transcriptome and Cell Surface Protein Expression by LinQ-View Identifies Unique Immune Subpopulations (preprint)

Single cell RNA sequencing is a powerful tool for characterizing the molecular and cellular heterogeneity of immune cells during their development and activation. Multimodal advances in single cell sequencing have enabled the simultaneous quantification of cell surface protein expression alongside unbiased transcriptional profiling. Here we present LinQ-View, a toolkit designed for multimodal single cell data visualization and analysis that links transcriptional and cell surface protein expression profiling data. Further, we propose a quantitative metric for cluster purity of CITE-seq data, enabling effective determination of clustering algorithms and their parameters, and finally demonstrate the utility of our toolkit through seamless integration with standard single cell analysis workflows on several public datasets. Through comparison to existing multimodal methods, we demonstrate that LinQ-View generates more accurate cell clusters and is highly efficient, even with massive datasets. LinQ-View is specialized in handling CITE-seq data with routine numbers of surface protein features (e.g. less than 50), by preventing variations in a single surface protein feature from affecting results. Finally, we utilized this method to integrate single cell transcriptional and protein expression data from COVID-19-infected patients and influenza-immunized subjects, revealing antigen-specific B cell subsets and previously unknown T cell subsets post-infection and vaccination.

COVID-19 Infection Induces Substantial Memory B Cell Maturation to Non-Neutralizing Viral Targets (preprint)

Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we utilized single-cell sequencing to profile SARS-CoV-2-reactive B cell subsets in 42 COVID-19 patients. We isolated thousands of B cells in multiple distinct subsets specific to the SARS-CoV-2 spike, endemic coronavirus (HCoV) spikes, nucleoprotein (NP), and open reading frame 8 (ORF8). Spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients 1.5–5 months post-infection. With severe acute infection, we identified substantial populations of endemic HCoV-reactive antibody-secreting cells with highly mutated variable genes, indicative of preexisting immunity. Finally, MBCs exhibited maturation to NP and ORF8 over time relative to spike, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal considerable antibody adaptation to non-neutralizing antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.Trial Registration Number: This clinical trial was registered at ClinicalTrials.gov with identifier NCT04340050, and clinical information for patients included in the study is detailed in Table S1–S3.Funding: This project was funded in part by the National Institute of Allergy and Infectious Disease (NIAID); National Institutes of Health (NIH) grant numbers U19AI082724 (P.C.W.), U19AI109946 (P.C.W.), U19AI057266 (P.C.W.), the NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS) grant numbers HHSN272201400005C(P.C.W.). N.W.A. was supported by the Multi-disciplinary Training program in Cancer Research (MTCR) - NIH T32 CA009594. A.J. and R.P.J were supported by federal funds from the NIAID, NIH, and Department of Health and Human Services under Contract HHSN272201700060C. F.K and F.A. were funded by the NIAID CEIRS contractHHSN272201400008C, Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract 75N93019C00051 and the generous support of the JPB foundation, the Open Philanthropy Project (#2020-215611) and other philanthropic donations. Y.K. and P.H.were funded by the Research Program on Emerging and Re-emerging Infectious Disease grant (JP19fk0108113) and the Japan Program for Infectious Diseases Research and Infrastructure (JP20fk0108272) from the Japan Agency for Medical Research and Development (AMED), NIAID CEIRS contract HHSN272201400008C, and CIVIC contract 75N93019C00051. D.F., C.N, Y.D., and P.D.H, were supported by NIAID contracts HHSN272201700060C and 75N93019C00062. M.S.D. and E.S.W. were supported by NIH grants R01 AI157155 and F30 AI152327, respectively.Conflict of Interest: Several antibodies generated from this work are being used by Now Diagnostics in Springdale, AR for the development of a diagnostic test. M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, and Carnival Corporation, and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions.Ethical Approval: All studies were performed with the approval of the University of Chicago institutional review board IRB20-0523 and University of Chicago, University of Wisconsin-Madison, and Washington University in St. Louis institutional biosafety committees. Informed consent was obtained after the research applications and possible consequences of the studies were disclosed to study subjects.

Pediatric Helmet CPAP Pilot Study

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

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.

Clinical predictors of donor antibody titer and correlation with recipient antibody response in a COVID-19 convalescent plasma clinical trial (preprint)

BackgroundConvalescent plasma therapy for COVID-19 relies on the transfer of anti-viral antibody from donors to recipients via plasma transfusion. The relationship between clinical characteristics and antibody response to COVID-19 is not well defined. We investigated predictors of convalescent antibody production and quantified recipient antibody response in a convalescent plasma therapy clinical trial. MethodsMultivariable analysis of clinical and serological parameters in 103 confirmed COVID-19 convalescent plasma donors 28 days or more following symptom resolution was performed. Mixed effects regression models with piecewise linear trends were used to characterize serial antibody responses in 10 convalescent plasma recipients with severe COVID-19. FindingsMean symptom duration of plasma donors was 11.9{+/-}5.9 days and 7.8% (8/103) had been hospitalized. Antibody titers ranged from 0 to 1:3,892 (anti-receptor binding domain (RBD)) and 0 to 1:3,289 (anti-spike). Multivariable analysis demonstrated that higher anti-RBD and anti-spike titer were associated with increased age, hospitalization for COVID-19, fever, and absence of myalgia (all p<0.05). Fatigue was significantly associated with anti-RBD (p=0.03) but not anti-spike antibody titer (p=0.11). In pairwise comparison among ABO blood types, AB donors had higher anti-RBD titer than O negative donors (p=0.048) and higher anti-spike titer than O negative (p=0.015) or O positive (p=0.037) donors. Eight of the ten recipients were discharged, one remains on ECMO and one died on ECMO. No toxicity was associated with plasma transfusion. After excluding two ECMO patients and adjusting for donor antibody titer, recipient anti-RBD antibody titer increased on average 31% per day during the first three days post-transfusion (p=0.01) and anti-spike antibody titer by 40.3% (p=0.02). InterpretationAdvanced age, fever, absence of myalgia, fatigue, blood type and hospitalization were associated with higher convalescent antibody titer to COVID-19. Despite variability in donor titer, 80% of convalescent plasma recipients showed significant increase in antibody levels post-transfusion. A more complete understanding of the dose-response effect of plasma transfusion among COVID-19 patients is needed to determine the clinical efficacy of this therapy. Trial RegistrationNCT04340050 FundingDepartment of Surgery University of Chicago, National Institute of Allergy and Infectious Diseases (NIAID) Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract 75N93019C00051