Antibody screening at reduced <scp>pH</scp> enables preferential selection of potently neutralizing antibodies targeting <scp>SARS‐CoV</scp>‐2

Madan, Bharat; Wang, Pengfei; Nair, Manoj S.; Huang, Yaoxing; Souza, Matheus Oliveira de; Acevedo, Sheila N. López; Ho, David D.; Kwong, Peter D.; Shapiro, Lawrence

doi:10.1002/aic.17440

Cited by 5 publications

(7 citation statements)

References 69 publications

(219 reference statements)

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“…These data revealed that the hit selection based on Round 2 ER>5 and VL+ >2 reads was efficient but not perfect. The hit rate was slightly lower compared to our previous studies using human B cell repertoires (72% vs. 90% - 100%), likely due to the larger size of the selected pool for characterization here (~100 vs. ~20 clones), and potentially the differences in B cell tissue origins, cell selection strategies, and sort designs ( 58 , 60 , 61 ). The genuine binding clones from BM and SPL comprised about 5-6% of reads in the BM or SPL clustered populations, further confirming the polarized nature of these antibody libraries ( Tables S4 , S5 ).…”

Section: Resultscontrasting

confidence: 64%

Large-scale antibody immune response mapping of splenic B cells and bone marrow plasma cells in a transgenic mouse model

Pan,

López Acevedo,

Cuziol

et al. 2023

Front. Immunol.

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Molecular characterization of antibody immunity and human antibody discovery is mainly carried out using peripheral memory B cells, and occasionally plasmablasts, that express B cell receptors (BCRs) on their cell surface. Despite the importance of plasma cells (PCs) as the dominant source of circulating antibodies in serum, PCs are rarely utilized because they do not express surface BCRs and cannot be analyzed using antigen-based fluorescence-activated cell sorting. Here, we studied the antibodies encoded by the entire mature B cell populations, including PCs, and compared the antibody repertoires of bone marrow and spleen compartments elicited by immunization in a human immunoglobulin transgenic mouse strain. To circumvent prior technical limitations for analysis of plasma cells, we applied single-cell antibody heavy and light chain gene capture from the entire mature B cell repertoires followed by yeast display functional analysis using a cytokine as a model immunogen. We performed affinity-based sorting of antibody yeast display libraries and large-scale next-generation sequencing analyses to follow antibody lineage performance, with experimental validation of 76 monoclonal antibodies against the cytokine antigen that identified three antibodies with exquisite double-digit picomolar binding affinity. We observed that spleen B cell populations generated higher affinity antibodies compared to bone marrow PCs and that antigen-specific splenic B cells had higher average levels of somatic hypermutation. A degree of clonal overlap was also observed between bone marrow and spleen antibody repertoires, indicating common origins of certain clones across lymphoid compartments. These data demonstrate a new capacity to functionally analyze antigen-specific B cell populations of different lymphoid organs, including PCs, for high-affinity antibody discovery and detailed fundamental studies of antibody immunity.

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Section: Resultscontrasting

confidence: 64%

Large-scale antibody immune response mapping of splenic B cells and bone marrow plasma cells in a transgenic mouse model

Pan,

López Acevedo,

Cuziol

et al. 2023

Front. Immunol.

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“…B cells were isolated from cryopreserved PBMCs of SARS-CoV-2 convalescent donors as previously described [ 16 , 41 , 54 – 56 ]. Briefly, B cells were enriched by CD27+ selection and stimulated in vitro for five days to enhance transcription of antibody genes.…”

Section: Methodsmentioning

confidence: 99%

“…Overlap extension RT-PCR was used to transform separate heavy (VH) and light (VL) variable genes into a single, physically linked VH:VL cDNA amplicons. cDNA amplicons encoding heavy and kappa light chain variable regions were cloned into a yeast display vector for expressing antibody VH:VL genes as Fabs for functional screening via FACS and NGS as previously reported [ 16 , 41 , 55 , 56 ]. Briefly, AWY101 yeast expressing Fabs were cultured in SGCAA media (Teknova) with 2 g/L dextrose (SGDCAA) for 36 hrs at 20°C to induce Fab expression for surface display.…”

Section: Methodsmentioning

confidence: 99%

Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron

et al. 2022

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Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccine-elicited immunity, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring of vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS-CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies.

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“…B cells were isolated from cryopreserved PBMCs of SARS-CoV-2 convalescent donors as previously described [16, 39, 50–52]. Briefly, B cells were enriched by CD27+ selection and stimulated in vitro for five days to enhance transcription of antibody genes.…”

Section: Methodsmentioning

confidence: 99%

“…Overlap extension RT-PCR was used to transform separate heavy (VH) and light (VL) variable genes into a single, physically linked VH:VL cDNA amplicons. cDNA amplicons encoding heavy and kappa light chain variable regions were cloned into a yeast display vector for expressing antibody VH:VL genes as Fabs for functional screening via FACS and NGS as previously reported [16, 39, 51, 52]. Briefly, AWY101 yeast expressing Fabs were cultured in SGCAA media (Teknova) with 2 g/L dextrose (SGDCAA) for 36 hrs at 20°C to induce Fab expression for surface display.…”

Section: Methodsmentioning

confidence: 99%

Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron

Teng

Nazzari

Choe

et al. 2021

Preprint

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Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccines, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring the vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS-CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies.

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Antibody screening at reduced pH enables preferential selection of potently neutralizing antibodies targeting SARS‐CoV‐2

Cited by 5 publications

References 69 publications

Large-scale antibody immune response mapping of splenic B cells and bone marrow plasma cells in a transgenic mouse model

Large-scale antibody immune response mapping of splenic B cells and bone marrow plasma cells in a transgenic mouse model

Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron

Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron

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