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Abstract Single B-cell antibody generation technology is an advanced method that offers several advantages, including rapid production, high efficiency, and high yield. The antibodies generated via this technique retain their natural conformation and are well suited for applications in pathogen diagnosis, disease treatment, and investigations of virus cross-species transmission mechanisms. Our study aimed to establish a platform for generating single B-cell antibodies specifically targeting the foot-and-mouth disease virus (FMDV) 146S antigen in mice. Female BALB/c mice were immunized with inactivated O-type FMDV 146S antigen, and spleen cells were collected for further analysis. Flow cytometric sorting was performed using a biotin-labeled O-type FMDV 146S antigen as a decoy to identify and select CD19 + /CD21/35 + /CD43-/IgM-/Biotin + antigen-specific individual B cells. The gene sequences encoding the variable regions of the heavy and light chains of the murine IgG antibodies were obtained via single-cell nested PCR amplification. Separate constructs were created for the heavy and light chain plasmids to ensure the proper expression of intact IgG antibodies. These plasmids were cotransfected into human embryonic kidney 293T (HEK293T) cells, leading to the successful production and purification of 15 specific monoclonal antibodies (mAbs), 10 which exhibited activity in ELISA tests, and six antibodies that displayed activity in IFA tests. These findings highlight the successful development of a method for generating mouse-derived single B-cell antibodies that target FMDV. This achievement provides a solid foundation for diagnostic techniques and the analysis of antigenic structural variations.

Single B-cell antibody generation technology is an advanced method that offers several advantages, including rapid production, high efficiency, and high yield. The antibodies generated via this technique retain their natural conformation and are well suited for applications in pathogen diagnosis, disease treatment, and investigations of virus cross-species transmission mechanisms. Our study aimed to establish a platform for generating single B-cell antibodies specifically targeting the foot-and-mouth disease virus (FMDV) 146S antigen in mice. Female BALB/c mice were immunized with inactivated O-type FMDV 146S antigen, and spleen cells were collected for further analysis. Flow cytometric sorting was performed using a biotin-labeled O-type FMDV 146S antigen as a decoy to identify and select CD19 + /CD21/35 + /CD43-/IgM-/Biotin + antigen-specific individual B cells. The gene sequences encoding the variable regions of the heavy and light chains of the murine IgG antibodies were obtained via single-cell nested PCR amplification. Separate constructs were created for the heavy and light chain plasmids to ensure the proper expression of intact IgG antibodies. These plasmids were cotransfected into human embryonic kidney 293T (HEK293T) cells, leading to the successful production and purification of 15 specific monoclonal antibodies (mAbs), 10 which exhibited activity in ELISA tests, and six antibodies that displayed activity in IFA tests. These findings highlight the successful development of a method for generating mouse-derived single B-cell antibodies that target FMDV. This achievement provides a solid foundation for diagnostic techniques and the analysis of antigenic structural variations. [ABSTRACT FROM AUTHOR]

Neutralizing antibodies (nAbs) can be used before or after infection to prevent or treat viral diseases. However, there are few efficacious nAbs against classical swine fever virus (CSFV) that have been produced, especially the porcine-originated nAbs. In this study, we generated three porcine monoclonal antibodies (mAbs) with in vitro neutralizing activity against CSFV, aiming to facilitate the development of passive antibody vaccines or antiviral drugs against CSFV that offer the advantages of stability and low immunogenicity. Pigs were immunized with the C-strain E2 (CE2) subunit vaccine, KNB-E2. At 42 days post vaccination (DPV), CE2-specific single B cells were isolated via fluorescent-activated cell sorting (FACS) baited by Alexa Fluor™ 647-labeled CE2 (positive), goat anti-porcine IgG (H + L)-FITC antibody (positive), PE mouse anti-pig CD3ε (negative) and PE mouse anti-pig CD8a (negative). The full coding region of IgG heavy (H) chains and light (L) chains was amplified by reverse transcription-polymerase chain reaction (RT-PCR). Overall, we obtained 3 IgG H chains, 9 kappa L chains and 36 lambda L chains, which include three paired chains (two H + κ and one H + λ). CE2-specific mAbs were successfully expressed in 293T cells with the three paired chains. The mAbs exhibit potent neutralizing activity against CSFVs. They can protect ST cells from infections in vitro with potent IC50 values from 14.43 µg/mL to 25.98 µg/mL for the CSFV C-strain, and 27.66 µg/mL to 42.61 µg/mL for the CSFV Alfort strain. This study is the first report to describe the amplification of whole-porcine IgG genes from single B cells of KNB-E2-vaccinated pig. The method is versatile, sensitive, and reliable. The generated natural porcine nAbs can be used to develop long-acting and low-immunogenicity passive antibody vaccine or anti-CSFV agents for CSF control and prevention. [ABSTRACT FROM AUTHOR]

Human monoclonal antibodies are essential diagnostic and research tools and one of the most promising therapeutics. In the past years, single B cell technologies have evolved and over-come conventional methods' limitations, enabling the isolation of scarce B cell populations with desirable characteristics. In this study, we describe a simple and efficient method to isolate anti-gen-specific plasmablasts and memory B cells from hepatitis B virus vaccinated donors' peripheral blood and consequently amplification of immunoglobulin variable region genes. Amplified immunoglobulin variable region genes were cloned into expression vectors and transfected into a human cell line to produce human recombinant monoclonal antibodies. Major challenges in this protocol were isolation of antigen-specific B cells based on surface markers, recovering mRNA from a single cell for efficient amplification, and cloning the correct insert into a desired expression vector. The essential feature of our protocol was the separation of B cells from peripheral blood mononuclear cells before sorting. We identified antigen-specific binding B cells based on the expression of surface markers CD19, CD27, IgG, and binding to hepatitis B surface antigen. Efficient single-cell reverse transcription and polymerase chain reaction (RT-PCR) were achieved using a random primer mix and Kapa Hifi Hot Start Polymerase. Amplification efficiency differed among heavy and light chain variable regions (highest at heavy chain (68 %) and lowest at lambda light chain (22 %)). After co-transfection of HEK293T/17 with successfully cloned heavy and light chain vectors, 70 % of transfected cells produced recombinant monoclonal antibodies at a concentration ∼ 4 μg/ml and 7 % of them showed binding to HBsAg. Human monoclonal antibodies from peripheral blood have advantages over antibodies of mouse origin or phage display libraries, because of their high specificity and self-tolerance. Using the described protocol, we can generate fully human monoclonal antibodies to any other antigen for application in immunotherapy or basic research., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Human cytomegalovirus (HCMV) can cause severe diseases in fetuses, newborns, and immunocompromised individuals. Currently, no vaccines are approved, and treatment options are limited. Here, we analyzed the human B cell response of four HCMV top neutralizers from a cohort of 9,000 individuals. By single-cell analyses of memory B cells targeting the pentameric and trimeric HCMV surface complexes, we identified vulnerable sites on the shared gH/gL subunits as well as complex-specific subunits UL 128/130/131A and gO. Using high-resolution cryogenic electron microscopy, we revealed the structural basis of the neutralization mechanisms of antibodies targeting various binding sites. Moreover, we identified highly potent antibodies that neutralized a broad spectrum of HCMV strains, including primary clinical isolates, that outperform known antibodies used in clinical trials. Our study provides a deep understanding of the mechanisms of HCMV neutralization and identifies promising antibody candidates to prevent and treat HCMV infection. [Display omitted] • Analysis of HCMV-reactive memory B cells in top neutralizers identifies potent nAbs • Epitope mapping uncovers vulnerable sites on the HCMV Trimer and Pentamer • A gO-targeting nAb prevents infection of fibroblasts, epithelial, and endothelial cells • Cryo-EM structures provide functional understanding of antibody neutralization Vaccines against HCMV are not available, and treatment options are limited. By dissecting the B cell response of HCMV top neutralizers, Zehner et al. reveal key epitopes for the neutralization of HCMV. Structural analyses elucidate HCMV neutralization mechanisms, and highly potent and cross-neutralizing antibodies were identified. These data provide a basis for developing antibody-mediated approaches to effectively prevent and treat HCMV infection. [ABSTRACT FROM AUTHOR]

In vivo VHH discovery approaches have been limited by the lack of methodologies for camelid B cell interrogation. Here, we report a novel application of the Beacon® optofluidic platform to the discovery of heavy-chain-only antibodies by screening alpaca B cells. Methods for alpaca B cell enrichment, culture, IgG2/3 detection, and sequencing were developed and used to discover target-specific VHH from an alpaca immunized with prostate-specific membrane antigen (PSMA) or a second target. PSMA-specific hits were expressed as VHH-Fc and characterized using label-free techniques. Anti-PSMA IgG2/3 titer plateaued on day 153, when on-Beacon IgG2/3 secretion and target binding rates peaked. Of 13 recombinantly expressed VHH-Fc, all but one bound with nanomolar affinity, and five were successfully humanized. Repertoire sequencing uncovered additional variants within the clonal lineages of the validated hits. The establishment of this workflow extends the powerful Beacon technology to enable rapid VHH discovery directly from natural camelid immune repertoires., Competing Interests: M.B.S., J.B., A.B., A.D., J.G., V.K., K.P., F.G., J.W., R.K. and C.A.S. are or were employees of Twist Bioscience Corporation. The authors have applied for a patent on anti-PSMA antibodies and uses thereof., (© The Author(s) 2023. Published by Oxford University Press on behalf of Antibody Therapeutics. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Highlights: [•] Single B cell-based human monoclonal antibody gene PCR cloning method was developed. [•] Plasma autoantibodies against VEGFR2 antigen were detected in glioblastoma patients. [•] Sixty B cells bearing VEGFR2-specific human antibody were obtained by cell sorter. [•] Twenty-two antibody genes were identified using direct human IgG PCR cloning. [•] Two antibody clones showed VEGFR2-specific binding affinity in ELISA and SPR. [ABSTRACT FROM AUTHOR]

Abstract: Recently, because of highly advanced protein engineering technology, beyond the chimeric antibody, highly humanized and fully human antibody development is becoming crucial in the medical field. In the last decade, investigational approaches using clinical samples for fully human antibody production have been performed, but there are still problems with efficiency and accuracy, which should be solved. In the present study, based on novel IgG antibody-measuring ELISA and antibody gene copy number-quantitative PCR, a human single B cell RT-PCR-mediated IgG monoclonal antibody (mAb) gene cloning method was established, and CMVpp65-specific human mAbs were successfully identified. Quantitative PCR for the human IgG mRNA copy number per cell demonstrated that the detection range was 10–250copies/cell. CMVpp65+surfaceIgG+ B cells were collected from melanoma patients who showed high titers of serum anti-CMVpp65 IgG antibody. RT-PCR was successful in 64% (IGH) and 84% (β-actin) of 88 single B cells. Finally, both IGH and IGL gene amplifications in the same cell were successful in 21 single cells, and 18 IgG antibody genes specific for CMVpp65 antigen were cloned. Four of 13 recombinant human single-chain fragment variable (scFv) antibodies showed strong responses to full-length CMVpp65 protein. These results suggested that the current fully human mAb production procedure through antibody-titer screening by ELISA, single B cell RT-PCR-based antibody gene cloning, and the making of scFv recombinant antibody is an efficient method of therapeutic antibody development. [ABSTRACT FROM AUTHOR]

Background: Rapid deployment of technologies capable of high-throughput and high-resolution screening is imperative for timely response to viral outbreaks. Risk mitigation in the form of leveraging multiple advanced technologies further increases the likelihood of identifying efficacious treatments in aggressive timelines., Methods: In this study, we describe two parallel, yet distinct, in vivo approaches for accelerated discovery of antibodies targeting the severe acute respiratory syndrome coronavirus-2 spike protein. Working with human transgenic Alloy-GK mice, we detail a single B-cell discovery workflow to directly interrogate antibodies secreted from plasma cells for binding specificity and ACE2 receptor blocking activity. Additionally, we describe a concurrent accelerated hybridoma-based workflow utilizing a DiversimAb™ mouse model for increased diversity., Results: The panel of antibodies isolated from both workflows revealed binding to distinct epitopes with both blocking and non-blocking profiles. Sequence analysis of the resulting lead candidates uncovered additional diversity with the opportunity for straightforward engineering and affinity maturation., Conclusions: By combining in vivo models with advanced integration of screening and selection platforms, lead antibody candidates can be sequenced and fully characterized within one to three months., (© The Author(s) 2021. Published by Oxford University Press on behalf of Antibody Therapeutics.)

Ectopic lymphoid structure (ELS) can form in the target tissues of patients with chronic inflammatory autoimmune diseases such as rheumatoid arthritis (RA) and Sjögren's syndrome (SS). Although it is still not clear why ELS form only in a subset of patients, it is well known that these structures can acquire features of ectopic germinal centers and contribute actively to the production of autoantibodies. Here, we describe a method to generate recombinant monoclonal antibodies from single ELS + synovial tissue B cells obtained from RA patients. This chapter gives a detailed description of the method beginning from the mononuclear cell preparation from RA synovial tissue, single-cell sort of B cells by flow cytometry, amplification of the immunoglobulin (Ig) genes (both heavy- and light-chain genes) by PCR, and subsequent Ig gene expression vector cloning for full recombinant IgG1 monoclonal antibody (rmAb) production in vitro. The recombinant mAbs generated can be then characterized for (1) analysis of the Ig gene repertoires for clonal studies, (2) immunoreactivity profile, and (3) functional studies both in vitro and in vivo.