IgTreeZ, A Toolkit for Immunoglobulin Gene Lineage Tree-Based Analysis, Reveals CDR3s Are Crucial for Selection Analysis.

Somatic hypermutation (SHM) is an important diversification mechanism that plays a part in the creation of immune memory. Immunoglobulin (Ig) variable region gene lineage trees were used over the last four decades to model SHM and the selection mechanisms operating on B cell clones. We hereby present IgTreeZ (Immunoglobulin Tree analyZer), a python-based tool that analyses many aspects of Ig gene lineage trees and their repertoires. Using simulations, we show that IgTreeZ can be reliably used for mutation and selection analyses. We used IgTreeZ on empirical data, found evidence for different mutation patterns in different B cell subpopulations, and gained insights into antigen-driven selection in corona virus disease 19 (COVID-19) patients. Most importantly, we show that including the CDR3 regions in selection analyses - which is only possible if these analyses are lineage tree-based - is crucial for obtaining correct results. Overall, we present a comprehensive lineage tree analysis tool that can reveal new biological insights into B cell repertoire dynamics.

The Relationship Between Convergent IGH Signatures and Severity of COVID-19 Patients by Next-Generation Sequencing of B-Cell Repertoire.

OBJECT: To reveal convergent IGH signatures and the association with severity of coronavirus disease 2019 (COVID-19) patients. METHOD: A total of 25 COVID-19 inpatients were classified into three clinical conditions: mild, severe, and critical. We analyzed convergent IGH signatures by ImmuHub® B-cell receptor (BCR) profiling system. RESULTS: IGH singleton frequency in patients is significantly lower than that of healthy donors (HDs). The clonality index of IGH in patients is significantly higher than that in HDs. Nevertheless, no significant difference was observed among the three groups. The difference in IGH clonality (top five clones) between post- and pretreatment was significant in the improvement and deterioration groups. Three common public motifs were shared by all COVID-19 patients: ARDYGG, RWYFDY, and YYYYGMDV. CONCLUSION: B cells could recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and produce clonal expansion. Patients who had better outcomes after treatment had higher IGH clonality. Three common public motifs-ARDYGG, RWYFDY, and YYYYGMDV-might be used for vaccine development (ChiCTR2000029626).

Impact of Host Immune Status on Discordant Anti-SARS-CoV-2 Circulating B Cell Frequencies and Antibody Levels.

Individuals with pre-existing chronic systemic low-grade inflammation are prone to develop severe COVID-19 and stronger anti-SARS-CoV-2 antibody responses. Whether this phenomenon reflects a differential expansion of antiviral B cells or a failure to regulate antibody synthesis remains unknown. Here, we compared the antiviral B cell repertoire of convalescent healthcare personnel to that of hospitalized patients with pre-existing comorbidities. Out of 277,500 immortalized B cell clones, antiviral B cell frequencies were determined by indirect immunofluorescence screening on SARS-CoV-2 infected cells. Surprisingly, frequencies of SARS-CoV-2 specific clones from the two groups were not statistically different, despite higher antibody levels in hospitalized patients. Moreover, functional analyses revealed that several B cell clones from healthcare personnel with low antibody levels had neutralizing properties. This study reveals for the first time a key qualitative defect of antibody synthesis in severe patients and calls for caution regarding estimated protective immunity based only on circulating antiviral antibodies.

Unbiased interrogation of memory B cells from convalescent COVID-19 patients reveals a broad antiviral humoral response targeting SARS-CoV-2 antigens beyond the spike protein.

Patients who recover from SARS-CoV-2 infections produce antibodies and antigen-specific T cells against multiple viral proteins. Here, an unbiased interrogation of the anti-viral memory B cell repertoire of convalescent patients has been performed by generating large, stable hybridoma libraries and screening thousands of monoclonal antibodies to identify specific, high-affinity immunoglobulins (Igs) directed at distinct viral components. As expected, a significant number of antibodies were directed at the Spike (S) protein, a majority of which recognized the full-length protein. These full-length Spike specific antibodies included a group of somatically hypermutated IgMs. Further, all but one of the six COVID-19 convalescent patients produced class-switched antibodies to a soluble form of the receptor-binding domain (RBD) of S protein. Functional properties of anti-Spike antibodies were confirmed in a pseudovirus neutralization assay. Importantly, more than half of all of the antibodies generated were directed at non-S viral proteins, including structural nucleocapsid (N) and membrane (M) proteins, as well as auxiliary open reading frame-encoded (ORF) proteins. The antibodies were generally characterized as having variable levels of somatic hypermutations (SHM) in all Ig classes and sub-types, and a diversity of VL and VH gene usage. These findings demonstrated that an unbiased, function-based approach towards interrogating the COVID-19 patient memory B cell response may have distinct advantages relative to genomics-based approaches when identifying highly effective anti-viral antibodies directed at SARS-CoV-2.

Deep Sequencing of B Cell Receptor Repertoires From COVID-19 Patients Reveals Strong Convergent Immune Signatures.

Deep sequencing of B cell receptor (BCR) heavy chains from a cohort of 31 COVID-19 patients from the UK reveals a stereotypical naive immune response to SARS-CoV-2 which is consistent across patients. Clonal expansion of the B cell population is also observed and may be the result of memory bystander effects. There was a strong convergent sequence signature across patients, and we identified 1,254 clonotypes convergent between at least four of the COVID-19 patients, but not present in healthy controls or individuals following seasonal influenza vaccination. A subset of the convergent clonotypes were homologous to known SARS and SARS-CoV-2 spike protein neutralizing antibodies. Convergence was also demonstrated across wide geographies by comparison of data sets between patients from UK, USA, and China, further validating the disease association and consistency of the stereotypical immune response even at the sequence level. These convergent clonotypes provide a resource to identify potential therapeutic and prophylactic antibodies and demonstrate the potential of BCR profiling as a tool to help understand patient responses.

Next-Generation Sequencing of T and B Cell Receptor Repertoires from COVID-19 Patients Showed Signatures Associated with Severity of Disease.

We profiled adaptive immunity in COVID-19 patients with active infection or after recovery and created a repository of currently >14 million B and T cell receptor (BCR and TCR) sequences from the blood of these patients. The B cell response showed converging IGHV3-driven BCR clusters closely associated with SARS-CoV-2 antibodies. Clonality and skewing of TCR repertoires were associated with interferon type I and III responses, early CD4+ and CD8+ T cell activation, and counterregulation by the co-receptors BTLA, Tim-3, PD-1, TIGIT, and CD73. Tfh, Th17-like, and nonconventional (but not classical antiviral) Th1 cell polarizations were induced. SARS-CoV-2-specific T cell responses were driven by TCR clusters shared between patients with a characteristic trajectory of clonotypes and traceability over the disease course. Our data provide fundamental insight into adaptive immunity to SARS-CoV-2 with the actively updated repository providing a resource for the scientific community urgently needed to inform therapeutic concepts and vaccine development.