Brief introduction of current technologies in isolation of broadly neutralizing HIV-1 antibodies

Sun, Zehua; Yan, Lei; Tang, Jiansong; Qian, Qian; Lenberg, Jerica; Zhu, Dandan; Wan, Lei; Wu, Kao; Wang, Yilin; Lu, Shiqiang

doi:10.1016/j.virusres.2017.10.011

Cited by 11 publications

(11 citation statements)

References 170 publications

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“…Other traditional methods of generating mAbs, such as Epstein-Barr virus-transformation and phage-display libraries, are limited by being unstable, time consuming or inefficient (14,15). With the advent of single-cell reverse transcription PCR (RT-PCR) technologies, the amplification of full-length immunoglobulin gene (Ig) fragments from single B cells by utilizing nested PCR has allowed the bypass of many of these limitations and this technology has been shown to provide a versatile tools for generation of new mAbs; this is an important advancement in mAbs production (16,17). Prior to the amplification of Ig-encoding genes, antigen-specific memory B cells must be stained with fluorescently-labelled antigen baits and then sorted.…”

Section: Introductionmentioning

confidence: 99%

Development of Whole-Porcine Monoclonal Antibodies with Potent Neutralization Activity against Classical Swine Fever Virus from Single B Cells

Dong

et al. 2019

ACS Synth. Biol.

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Classical swine fever (CSF) is a highly contagious swine disease found worldwide that has caused devastating economic losses. However, there are few efficacious mAbs against the CSF virus (CSFV) that can be used for treatment because most mAbs against CSFV are derived from mouse hybridoma cells and these murine mAbs have disadvantages of inefficient effector functions elicitations and high immunogenicity in vivo. Accordingly, we characterized whole-porcine anti-CSFV neutralizing mAbs (NAbs) isolated directly from single B cells sorted from a CSFV-vaccinated pig using the fluoresceinated conserved linear neutralizing epitope of the CSFV E2 protein and fluorophore conjugated goat anti-pig IgG.Immunoglobulin (Ig) genes were isolated via nested PCR, and two porcine mAbs termed HK24 and HK44 were produced. We determined that these mAbs can bind to E2 protein and recognize sites within this major antigenic epitope. In addition, we found that mAbs HK24 and HK44 exhibit potent neutralizing activity against CSFV, and they can protect PK-15 cells from infections in vitro with potent IC 50 values of 9.3 μg/ml and 0.62 μg/ml, respectively.Notably, we demonstrated that these two mAbs can be used as novel reagents for detecting virus infection. These data suggest that our results not only provide a method for efficiently obtaining mAbs against CSFV but also offer promising mAb candidates for development of antibody-based diagnostic and antiviral agents.

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Section: Introductionmentioning

confidence: 99%

Development of Whole-Porcine Monoclonal Antibodies with Potent Neutralization Activity against Classical Swine Fever Virus from Single B Cells

Dong

et al. 2019

ACS Synth. Biol.

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“…Classical serology approaches, based on ELISA and viral neutralization assays, offer a wealth of information but require a relatively large set of biological and viral reagents (3)(4)(5). For rational and structure-based vaccine design efforts it is also necessary to isolate specific monoclonal antibodies (mAbs) elicited by a vaccine/pathogen (6)(7)(8)(9)(10)(11), yet the isolation of antigenspecific B-cells requires fluorescently labeled probes and access to advanced cell sorting equipment (12)(13)(14). Individual monoclonal antibodies are subsequently produced and subjected to further binding, structural and functional evaluation to assess epitope specificity, affinity, and activity (i.e.…”

Section: Main Textmentioning

confidence: 99%

From Structure to Sequence: Identification of polyclonal antibody families using cryoEM

Anatanasijevic

Bowman

Kirchdoerfer

et al. 2021

Preprint

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One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity- determining regions and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next generation sequencing of immune repertoires we were able to specifically identify clonal family members, synthesize the monoclonal antibodies and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design.

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“…In addition to the hybridoma technique, B cell immortalization and microneutralization, [ 111 ] large‐scale screening of libraries by phage, ribosome, yeast, or bacterial display are widely applied in antibody isolation. [ 112,113,61,114 ] Using these technologies, we and others have successfully developed functional antibodies against various targets.…”

Section: Strategies Of Functional Antibody Discovery From Antibody Rementioning

confidence: 99%

Deep Mining of Human Antibody Repertoires: Concepts, Methodologies, and Applications

Tian

et al. 2020

Small Methods

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of V-J genes in the immunoglobulin light chains variable regions (V L), during the early stages of B cell maturation in the bone marrow; [1] 2) junctional diversity resulting from exonuclease trimmings and the random addition of palindrome (P) or non-template encoded (N) nucleotides in the process of rearrangement; [2] and 3) B cell proliferation after antigen stimulation in order to produce high-affinity antibodies and eliminate the antigen through subsequent class switch recombination (CSR) and somatic hypermutation (SHM). [3,4] It is estimated that there are over 10 13 BCRs in the human antibody repertoire. [5] This astronomical size prevents a global analysis of the antibody repertoire using conventional DNA sequencing. However, advancements in highthroughput sequencing (HTS) for Ig-Seq have improved the ability to characterize the antibody repertoire on a large scale. [6] Over a decade of development, a variety of new technologies and approaches have improved Ig-Seq methods, resulting in improved understanding of B-cell immunology and accumulation of massive antibody sequence data. In this review, an overview of Ig-Seq and advanced methodologies applied to HTS of antibody repertoires will be discussed. We will describe the wide applications of Ig-Seq in basic and clinical immunology, and strategies for functional antibody discovery from antibody repertoires. Finally, we will provide our perspective on the future directions and challenges in human antibody repertoire deep mining. 2. Overview of Ig-Seq Pipeline Although multiple strategies have been used for studying antibody repertoires, each strategy follows a similar workflow including sample collection, library preparation and sequencing, data cleaning, sequence alignment, and high-level processing (Figure 1). For sample collection, peripheral blood mononuclear cells (PBMCs) and total B cells have been generally used because of their accessibility. [9-11] However, because antibody repertoire properties are divergent between antigen-specific B cells and plasma cells, [12] specific B cell populations or B cells located in targeted tissues should be enriched for more precise studies. Considering that as few as 2% of total B cells are circulating in The ability of the human adaptive immune system to respond to antigens relies upon the tremendous diversity of T cell receptors (TCR) and B cell receptors (BCR). The entirety of an individual's BCRs, often referred to as an antibody repertoire, shapes the humoral immune system. Therefore, technologies to identify and characterize antibody repertoires are critical for understanding fundamental aspects of the development and maintenance of the humoral immune system. Recently, innovative methodologies and technologies devoted to high-throughput sequencing of antibody repertoires (Ig-Seq) have broadened the understanding of humoral immunity. This review provides an overview of the Ig-Seq pipeline from sample collection, library preparation, and sequencing, to data cleaning, sequence alignment, and highlevel processing....

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Brief introduction of current technologies in isolation of broadly neutralizing HIV-1 antibodies

Cited by 11 publications

References 170 publications

Development of Whole-Porcine Monoclonal Antibodies with Potent Neutralization Activity against Classical Swine Fever Virus from Single B Cells

Development of Whole-Porcine Monoclonal Antibodies with Potent Neutralization Activity against Classical Swine Fever Virus from Single B Cells

From Structure to Sequence: Identification of polyclonal antibody families using cryoEM

Deep Mining of Human Antibody Repertoires: Concepts, Methodologies, and Applications

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