Rapid isolation and immune profiling of SARS-CoV-2 specific memory B cell in convalescent COVID-19 patients via LIBRA-seq

AbstractB cell response plays a critical role against SARS-CoV-2 infection. However, little is known about the diversity and frequency of the paired SARS-CoV-2 antigen-specific BCR repertoire after SARS-CoV-2 infection. Here, we performed single-cell RNA sequencing and VDJ sequencing using the memory and plasma B cells isolated from five convalescent COVID-19 patients, and analyzed the spectrum and transcriptional heterogeneity of antibody immune responses. Via linking BCR to antigen specificity through sequencing (LIBRA-seq), we identified a distinct activated memory B cell subgroup (CD11chighCD95high) had a higher proportion of SARS-CoV-2 antigen-labeled cells compared with memory B cells. Our results revealed the diversity of paired BCR repertoire and the non-stochastic pairing of SARS-CoV-2 antigen-specific immunoglobulin heavy and light chains after SARS-CoV-2 infection. The public antibody clonotypes were shared by distinct convalescent individuals. Moreover, several antibodies isolated by LIBRA-seq showed high binding affinity against SARS-CoV-2 receptor-binding domain (RBD) or nucleoprotein (NP) via ELISA assay. Two RBD-reactive antibodies C14646P3S and C2767P3S isolated by LIBRA-seq exhibited high neutralizing activities against both pseudotyped and authentic SARS-CoV-2 viruses in vitro. Our study provides fundamental insights into B cell response following SARS-CoV-2 infection at the single-cell level.

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SARS-CoV-2 spike-specific memory B cells express higher levels of T-bet and FcRL5 after non-severe COVID-19 as compared to severe disease

PLoS ONE ◽

10.1371/journal.pone.0261656 ◽

2021 ◽

Vol 16 (12) ◽

pp. e0261656

Author(s):

Raphael A. Reyes ◽

Kathleen Clarke ◽

S. Jake Gonzales ◽

Angelene M. Cantwell ◽

Rolando Garza ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Symptom Onset ◽

Cell Response ◽

Severe Disease ◽

Memory B Cells ◽

Memory B Cell ◽

Specific Memory ◽

Specific Igg ◽

B Cell Response

SARS-CoV-2 infection elicits a robust B cell response, resulting in the generation of long-lived plasma cells and memory B cells. Here, we aimed to determine the effect of COVID-19 severity on the memory B cell response and characterize changes in the memory B cell compartment between recovery and five months post-symptom onset. Using high-parameter spectral flow cytometry, we analyzed the phenotype of memory B cells with reactivity against the SARS-CoV-2 spike protein or the spike receptor binding domain (RBD) in recovered individuals who had been hospitalized with non-severe (n = 8) or severe (n = 5) COVID-19. One month after symptom onset, a substantial proportion of spike-specific IgG+ B cells showed an activated phenotype. In individuals who experienced non-severe disease, spike-specific IgG+ B cells showed increased expression of markers associated with durable B cell memory, including T-bet and FcRL5, as compared to individuals who experienced severe disease. While the frequency of T-bet+ spike-specific IgG+ B cells differed between the two groups, these cells predominantly showed an activated switched memory B cell phenotype in both groups. Five months post-symptom onset, the majority of spike-specific memory B cells had a resting phenotype and the percentage of spike-specific T-bet+ IgG+ memory B cells decreased to baseline levels. Collectively, our results highlight subtle differences in the B cells response after non-severe and severe COVID-19 and suggest that the memory B cell response elicited during non-severe COVID-19 may be of higher quality than the response after severe disease.

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SARS-CoV-2 spike-specific memory B cells express markers of durable immunity after non-severe COVID-19 but not after severe disease

10.1101/2021.09.24.461732 ◽

2021 ◽

Author(s):

Raphael Reyes ◽

Kathleen Clarke ◽

S. Jake Gonzales ◽

Angelene M. Cantwell ◽

Rolando Garza ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Symptom Onset ◽

Cell Response ◽

Severe Disease ◽

Memory B Cells ◽

Memory B Cell ◽

Specific Memory ◽

Specific Igg ◽

B Cell Response

SARS-CoV-2 infection elicits a robust B cell response, resulting in the generation of long-lived plasma cells and memory B cells. Here, we aimed to determine the effect of COVID-19 severity on the memory B cell response and characterize changes in the memory B cell compartment between recovery and five months post-symptom onset. Using high-parameter spectral flow cytometry, we analyzed the phenotype of memory B cells with reactivity against the SARS-CoV-2 spike protein or the spike receptor binding domain (RBD) in recovered individuals who had been hospitalized with non-severe (n=8) or severe (n=5) COVID-19. One month after symptom onset, a substantial proportion of spike-specific IgG+ B cells showed an activated phenotype. In individuals who experienced non-severe disease, spike-specific IgG+ B cells showed increased expression of markers associated with durable B cell memory, including T-bet, FcRL5, and CD11c, which was not observed after severe disease. Five months post-symptom onset, the majority of spike-specific memory B cells had a resting phenotype and the percentage of spike-specific T-bet+ IgG+ memory B cells decreased to baseline levels. Collectively, our results suggest that the memory B cell response elicited during non-severe COVID-19 may be of higher quality than the response after severe disease.

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Inhibition of human antigen-specific memory B cell response in vitro by a diphtheria toxin-related interleukin 2 fusion protein

Cellular Immunology ◽

10.1016/0008-8749(91)90044-c ◽

1991 ◽

Vol 132 (2) ◽

pp. 481-493 ◽

Cited By ~ 4

Author(s):

Alan P. Grailer ◽

Jean C. Nichols ◽

Terry B. Strom ◽

Hans W. Sollinger ◽

William J. Burlingham

Keyword(s):

Fusion Protein ◽

B Cell ◽

Cell Response ◽

Diphtheria Toxin ◽

Interleukin 2 ◽

Memory B Cell ◽

Specific Memory ◽

B Cell Response

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SARS-CoV-2 infection severity is linked to superior humoral immunity against the spike

10.1101/2020.09.12.294066 ◽

2020 ◽

Cited By ~ 3

Author(s):

Jenna J. Guthmiller ◽

Olivia Stovicek ◽

Jiaolong Wang ◽

Siriruk Changrob ◽

Lei Li ◽

...

Keyword(s):

Antibody Response ◽

B Cell ◽

Cell Response ◽

Nucleocapsid Protein ◽

Antigen Specificity ◽

Memory B Cell ◽

Reading Frame ◽

Global Pandemic ◽

Kinetics Of ◽

B Cell Response

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing a global pandemic. The antigen specificity and kinetics of the antibody response mounted against this novel virus are not understood in detail. Here, we report that subjects with a more severe SARS-CoV-2 infection exhibit a larger antibody response against the spike and nucleocapsid protein and epitope spreading to subdominant viral antigens, such as open reading frame 8 and non-structural proteins. Subjects with a greater antibody response mounted a larger memory B cell response against the spike, but not the nucleocapsid protein. Additionally, we revealed that antibodies against the spike are still capable of binding the D614G spike mutant and cross-react with the SARS-CoV-1 receptor binding domain. Together, this study reveals that subjects with a more severe SARS-CoV-2 infection exhibit a greater overall antibody response to the spike and nucleocapsid protein and a larger memory B cell response against the spike.

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Specific memory B cell response in humans upon infection with highly pathogenic H7N7 avian influenza virus

Scientific Reports ◽

10.1038/s41598-020-60048-9 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 2

Author(s):

Brenda Westerhuis ◽

Hinke ten Hulscher ◽

Ronald Jacobi ◽

Josine van Beek ◽

Marion Koopmans ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

B Cell ◽

Avian Influenza Virus ◽

Cell Response ◽

Highly Pathogenic ◽

Memory B Cell ◽

Specific Memory ◽

B Cell Response

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Agonist for Toll-Like Receptor (TLR) 9 Amplifies as Well as Inhibits the Differentiation of FVIII-Specific Memory B Cells into Antibody-Producing Plasma Cells in Vitro and in Vivo.

Blood ◽

10.1182/blood.v112.11.3382.3382 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3382-3382

Author(s):

Peter Allacher ◽

Christina Hausl ◽

Aniko Ginta Pordes ◽

Rafi Uddin Ahmad ◽

Hartmut J Ehrlich ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Memory B Cells ◽

Memory B Cell ◽

Cpg Dna ◽

Cell Responses ◽

Specific Memory ◽

B Cell Responses

Abstract Memory B cells are essential for maintaining long-term antibody responses. They can persist for years even in the absence of antigen and are rapidly re-stimulated to differentiate into antibody-producing plasma cells when they encounter their specific antigen. Previously we demonstrated that ligands for TLR 7 and 9 amplify the differentiation of FVIII-specific memory B cells into anti-FVIII antibody-producing plasma cells at low concentrations of FVIII and prevent the inhibition of memory-B-cell differentiation at high concentrations of FVIII. The modulation of FVIII-specific memory-B-cell responses by agonists for TLR is highly relevant for the design of new immunotherapeutic approaches in patients with FVIII inhibitors because TLR are activated by a range of different viral and bacterial components. Specifically, TLR 7 is triggered by single-stranded RNA derived from viruses and TLR 9 is triggered by bacterial DNA containing unmethylated CpG motifs. We further explored the modulation of FVIII-specific memory-B-cell responses by agonists for TLRs by studying a broad range of concentrations of CpG DNA, a ligand for TLR 9, both in vitro and in vivo using the murine E17 model of hemophilia A. We used CpG-DNA in concentrations ranging from 0.1 to 10,000 ng/ml to study the modulation of FVIII-specific memory-B-cell responses in vitro and verified the specificity of the effects observed by including a blocking agent for TLR 9 and GpC-DNA, a non-stimulating negative control for CpG DNA. Furthermore, we used doses of CpG DNA ranging from 10 to 50,000 ng per dose to study the modulation of FVIII-specific memory-B-cell responses in vivo. E17 hemophilic mice were treated with a single intravenous dose of 200 ng FVIII to stimulate the generation of FVIII-specific memory B cells and were subsequently treated with another dose of FVIII that was given together with CpG DNA. We analyzed titers of anti-FVIII antibodies in the circulation of these mice one week after the second dose of FVIII. Previously we had shown that a single dose of 200 ng FVIII, given intravenously to E17 hemophilic mice, stimulates the formation of FVIII-specific memory B cells but is not sufficient to induce anti-FVIII antibodies that would be detectable in the circulation. Our results demonstrate a biphasic effect of CpG DNA on the re-stimulation of FVIII-specific memory B cells and their differentiation into antibody-producing plasma cells. Both in vitro and in vivo studies show that CpG DNA at high doses inhibits the re-stimulation and differentiation of FVIII-specific memory B cells. However, CpG DNA at low doses amplifies these processes. Amplification and inhibition of memory-B-cell responses are due to specific interactions of CpG DNA with TLR 9. Both effects are blocked by addition of a blocking agent for TLR 9 in vitro. We conclude that triggering of TLR 9 by bacterial DNA has a substantial influence on FVIII-specific memory-B-cell responses. The consequence of TLR 9 triggering can be inhibitory or stimulatory, depending on the actual concentration of the bacterial DNA. Our findings demonstrate the potential modulatory effects of bacterial infections on the regulation of FVIII inhibitor development.

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Single-Cell Analysis of FVIII-Specific Memory B Cells in Murine Hemophilia A.

Blood ◽

10.1182/blood.v110.11.1157.1157 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1157-1157 ◽

Cited By ~ 1

Author(s):

Christina Hausl ◽

Rafi U. Ahmad ◽

Bernhard Baumgartner ◽

Hans Peter Schwarz ◽

Hartmut Ehrlich ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Single Cell ◽

Cd4 T Cells ◽

Single Cell Analysis ◽

Memory B Cells ◽

Cell Analysis ◽

Memory B Cell ◽

Specific Memory

Abstract The elimination of FVIII-specific memory B cells is an essential step in the design of new therapeutic strategies for the induction of immune tolerance in hemophilia A with FVIII inhibitors. Using a mouse model of hemophilia A we recently reported that low dose FVIII stimulates the differentiation of FVIII-specific memory B cells into antibody-secreting plasma cells whereas high dose FVIII inhibits this process. The inhibition of memory-B-cell re-stimulation is irreversible and seems to be due to an induction of apoptosis. Further understanding of the complex interactions that lead to either re-stimulation and differentiation of memory B cells or inhibition and eradication of these cells requires appropriate technologies for single-cell analysis and functional studies. We established a new technology for single-cell analysis and cell sorting of FVIII-specific murine memory B cells. A combination of magnetic bead separation and multi-color flow cytometry enabled us to analyze and purify FVIII-specific memory B cells obtained from hemophilic mice treated with FVIII. In a first step, we depleted undesirable cell populations (IgM+, IgD+, CD11c+, F4/80+, Gr1+ and CD49b+ cells) from total spleen cells by magnetic bead separation. In a second step, we used multicolor flow cytometry to exclude CD4+ T cells and analyze the FVIII-specific memory B cell compartment. This compartment was specified by staining the specific B-cell receptor with FVIII and anti-IgG antibodies. Frequencies of cells in this compartment ranged from 0.1–0.5% of total spleen cells in animals treated with 4 intravenous doses of FVIII, given at weekly intervals. We could not detect any FVIII-specific memory B cells in naïve mice. By means of single cell sorting we isolated FVIII-specific memory B cells for further functional studies. We were able to cultivate FVIII-specific memory B cells in microwell cultures in vitro and differentiate them into antibody-secreting plasma cells. The re-stimulation and differentiation of single-cell sorted memory B cells was strictly dependent on the presence of activated CD4+ T cells. CD4+ T cells obtained from naïve mice did not support the memory response. Furthermore, the re-stimulation and differentiation of memory B cells in the presence of activated CD4+ T cells did not require additional dendritic cells for antigen presentation. Obviously, memory B cells provide sufficient antigen presentation to CD4+ T cells to enable them to trigger the memory response. Our approach for single-cell analysis and purification of FVIII-specific memory B cells provides a new tool for tracking memory B cell populations in vivo and for directly analyzing the regulation of memory B cell function. It opens the field for future studies which should elucidate signals and molecules involved in activation or inhibition and eradication of FVIII-specific memory B cells. These activities will eventually lead to the identification of targets for the design of new treatment strategies for patients with FVIII inhibitors.

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Deletion or Inhibition of Fc Gamma Receptor IIb (CD32) Prevents the Memory B Cell Response to Factor VIII in a Hemophilia A Mouse Model

Blood ◽

10.1182/blood.v118.21.204.204 ◽

2011 ◽

Vol 118 (21) ◽

pp. 204-204 ◽

Cited By ~ 1

Author(s):

Sonja Werwitzke ◽

Marcus von Hornung ◽

Katy Kalippke ◽

Arne Trummer ◽

Arnold Ganser ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Knockout Mice ◽

Cell Response ◽

Hemophilia A ◽

Memory B Cells ◽

Double Knockout ◽

Memory B Cell ◽

Specific Memory ◽

Antibody Secreting Cells

Abstract Abstract 204 The formation of inhibitory antibodies to factor VIII (FVIII) is the foremost complication of replacement therapy in hemophilia A. Patients with inhibitors are treated with very high doses of FVIII, over prolonged periods of time, to induce immune tolerance. Studies in a hemophilia A mouse model demonstrated that very high doses of FVIII can induce apoptosis in FVIII-specific memory B cells and prevent their differentiation into antibody-secreting cells. The Fc gamma receptor IIb (FcgRIIb) is expressed on B cells and mediates inhibitory signals after crosslinking with the B cell receptor. Here, we studied the potential role of this receptor in the regulation of memory B cell response to FVIII. FVIII knockout mice (B6;129S4-F8tm2Kaz/J) were crossed with FcgRIIb knockout mice (B6;129S4-Fcgr2btm1Ttk/J). Comparing F8−/− mice and F8−/−/FcgR2b−/− double knockout mice, the initial anti-FVIII antibody formation was similar after intravenous exposure to 4 weekly doses of 80 or 400 IU/kg. Similar numbers of FVIII-specific antibody-secreting cells were detected in the spleen and bone marrow by ELISPOT. As previously shown, in vitro re-stimulation of memory B cells from spleens of immunized F8−/− mice at doses of 1 to 200 ng/ml induced their differentiation into antibody-secreting cells. Higher doses of 400 to 800 ng/ml prevented differentiation. In F8−/−/FcgR2b−/− double knockout mice, however, formation of antibody-secreting cells was completely inhibited across all FVIII doses tested. Addition of B220-depleted splenocytes from F8−/− mice did not restore memory B cell function in F8−/−/FcgR2b−/− double knockout mice, indicating that the observed effect was not due to dysfunction of follicular dendritic cells or other antigen-presenting cells. Inhibition of FcgRIIb using a monoclonal antibody prevented the FVIII-specific memory B cell response in splenocytes from immunized F8−/− mice. Staining with propidium iodide, annexin V, or anti-caspase 3 indicated increased rates of apoptosis when FcgRIIb was blocked during re-stimulation. In summary, FcgRIIb plays a crucial role for the differentiation of FVIII-specific splenic memory B cells into antibody-secreting cells. Inhibition of FcgRIIb appears to sensitize B cells for apoptosis during re-stimulation with FVIII. This mechanism could potentially facilitate the eradication of FVIII-specific memory B cells during ITI. Disclosures: No relevant conflicts of interest to declare.

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Germ line tumor-associated immunoglobulin VH region peptides provoke a tumor-specific immune response without altering the response potential of normal B cells

Blood ◽

10.1182/blood-2004-01-0105 ◽

2004 ◽

Vol 104 (3) ◽

pp. 752-759 ◽

Cited By ~ 9

Author(s):

Qiang Lou ◽

Raymond J. Kelleher ◽

Alessandro Sette ◽

Jenni Loyall ◽

Scott Southwood ◽

...

Keyword(s):

Immune Response ◽

B Cells ◽

B Cell ◽

Cell Response ◽

Germ Line ◽

T Cell Response ◽

Binding Potential ◽

Variable Regions ◽

B Cell Response

AbstractPrevious studies have suggested that murine T cells are tolerant to epitopes derived from germ line variable regions of immunoglobulin (Ig) heavy (VH) or light chains. This has lead to the prediction that germ line VH-region epitopes found in neoplastic B cells cannot be used to provoke an antitumor immune response. To test these assumptions and address the question of how such a vaccine may alter the normal B-cell response, an antibody-forming B-cell hybridoma (1H6) expressing a conserved germ line VH gene with specificity for dextran was generated and used as a tumor model. Using algorithms for predicting major histocompatibility complex (MHC) binding, potential MHC class I and II binding peptides were identified within the 1H6 VH region, synthesized, and tested for MHC binding and immunogenicity. We show that germ line VH peptides, when presented by dendritic cells, are immunogenic in vitro and provoke a tumor-specific protective immune response in vivo. We conclude that (1) it is possible to induce a T-cell response to germ line VH peptides; (2) such peptides can be used to generate a B-cell tumor-specific vaccine; and (3) a vaccine targeting VH peptides expressed by the dominant dextran-specific B-cell clonotype had no effect upon the magnitude of the normal B-cell response to dextran.

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In situ vaccination with local radiation and intratumoral immunocytokine to elicit a tumor-specific memory B-cell response.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.7_suppl.69 ◽

2017 ◽

Vol 35 (7_suppl) ◽

pp. 69-69

Author(s):

Claire Baniel ◽

Jacquelyn A Hank ◽

Emily I. Guy ◽

Stephen D Gillies ◽

Alan J. Korman ◽

...

Keyword(s):

B Cell ◽

Cell Response ◽

Humoral Response ◽

T Cell Response ◽

Complete Regression ◽

Memory B Cell ◽

Specific Memory ◽

Before And After ◽

B Cell Response

69 Background: In a murine melanoma (MEL) model, we reported an in situ vaccination response to combined radiation (RT) and intra-tumor (IT) injection of anti-GD2 hu14.18-IL2 immunocytokine (IC). This treatment resulted in 71% complete regression of 5-week (~ 200mm3) tumors, a memory T cell response, and augmented response to systemic anti-CTLA-4 antibody (mAb) checkpoint blockade. We hypothesized that mice rendered disease-free (DF) by RT, IT-IC, and anti-CTLA-4 mAb might also exhibit a memory B cell response. Methods: C57BL/6 mice were implanted with 2x106 syngeneic, GD2+ B78 MEL cells and tumors developed for 5 weeks. Mice were treated with 12 Gy RT to this tumor followed by 5 daily IT injections of hu14.18-IL2 d6-10 after RT and IP injection of anti-CTLA-4 d3, 6, and 9 after RT. DF mice and naïve controls were challenged by subcutaneous implantation with 2x106 B78 MEL cells. Peripheral blood was collected from mice before and after B78 challenge and serum was evaluated for presence of tumor-specific mAbs using flow cytometry and ELISA. Results: Seventy-three percent of mice were rendered DF by treatment with RT, IT-hu14.18-IL2, and anti-CTLA-4. All of these (13/13) rejected a rechallange B78 implantation > 1 year later (range d378 – 511), whereas no naïve mice rejected B78 implantation (0/66). IgG from serum of DF mice bound selectively to B78 and parental GD2- B16 MEL cells and the level of this mAb response appeared to increase modestly d14 after B78 challenge. In naïve mice, a modest increase in tumor-specific mAb was identified between non-tumor implanted mice and d35 post-implantation mice (bearing tumors > 200mm3), however this level remained ~ 5 fold below that observed in DF mice prior to B78 rechallenge. In contrast, no appreciable mAb response was observed for unrelated syngeneic GD2+ Panc02 pancreatic tumor cells in serum of DF or naïve mice. Conclusions: We report an endogenous anti-tumor IgG humoral response in DF mice > 1 year after treatment with RT, IT-IC, and anti-CTLA-4 mAb, concurrent with demonstration of long lasting immune protection from re-challenge. Studies are underway to determine whether this response is involved in the therapeutic efficacy of this in situ vaccination regimen.

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