Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth Ernestina; Velázquez-Ramírez, Doireyner Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa Elena; Martínez-Barnetche, Jesús

doi:10.4161/mabs.27435

Cited by 6 publications

(5 citation statements)

References 42 publications

(41 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Fifteen days after immunization, 2 HEL-specific IGHV B cell clones, functionally validated in a previous work, 12 were identified as clones with high relative frequency, but lower Gini coefficients compared to the corresponding high relative frequency clones from control mice, (Fig. 3), suggesting that within the higher frequency range (y axis), antigen-specific clones are the ones having lower Gini coefficient values.…”

Section: Differences In the B Cell Repertoire After An Immune Challengesupporting

confidence: 69%

“…31 As a proof of principle of ImmunediveRsity performance to digitally reconstruct the antibody repertoire with experimental data, we used our previously described IGHV sequence data derived from libraries (IgM and IgG class) from 2 BALB/c mice spleens at 3, 7 and 15 days postimmunization with hen egg lysozyme (HEL) and one mouse inoculated with PBS in each time point as controls. 12 The sequencing metrics of the 18 sequenced libraries are shown on Table S1. As expected, the proportion of somatic hypermutation was higher in the IgG than in the IgM compartment (Fig.…”

Section: Differences In the B Cell Repertoire After An Immune Challengementioning

confidence: 99%

See 1 more Smart Citation

Reconstructing and mining the B cell repertoire with ImmunediveRsity

Cortina-Ceballos

Godoy-Lozano

Sámano-Sánchez

et al. 2015

mAbs

Self Cite

View full text Add to dashboard Cite

(2015) Reconstructing and mining the B cell repertoire with ImmunediveRsity , mAbs, 7:3, 516-524, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Keywords: high-throughput sequencing, Ig repertoire, CDR3, data mining Abbreviations: HEL, hen egg lysozyme; CDRH3, heavy chain complementarity determining region 3; Rep-Seq, repertoire sequencing; SHM, somatic hypermutation.The B cell antigen receptor repertoire is highly diverse and constantly modified by clonal selection. High-throughput DNA sequencing (HTS) of the lymphocyte repertoire (Rep-Seq) represents a promising technology to explore such diversity ex-vivo and assist in the identification of antigen-specific antibodies based on molecular signatures of clonal selection. Therefore, integrative tools for repertoire reconstruction and analysis from antibody sequences are needed. We developed ImmunediveRity, a stand-alone pipeline primarily based in R programming for the integral analysis of B cell repertoire data generated by HTS. The pipeline integrates GNU software and in house scripts to perform quality filtering, sequencing noise correction and repertoire reconstruction based on V, D and J segment assignment, clonal origin and unique heavy chain identification. Post-analysis scripts generate a wealth of repertoire metrics that in conjunction with a rich graphical output facilitates sample comparison and repertoire mining. Its performance was tested with raw and curated human and mouse 454-Roche sequencing benchmarks providing good approximations of repertoire structure. Furthermore, ImmunediveRsity was used to mine the B cell repertoire of immunized mice with a model antigen, allowing the identification of previously validated antigen-specific antibodies, and revealing different and unexpected clonal diversity patterns in the post-immunization IgM and IgG compartments. Although ImmunediveRsity is similar to other recently developed tools, it offers significant advantages that facilitate repertoire analysis and repertoire mining. ImmunediveRsity is open source and free for academic purposes and it runs on 64 bit GNU/Linux and MacOS. Available at: https://bitbucket.org/ImmunediveRsity/immunediversity/

show abstract

Section: Differences In the B Cell Repertoire After An Immune Challengesupporting

confidence: 69%

Section: Differences In the B Cell Repertoire After An Immune Challengementioning

confidence: 99%

Reconstructing and mining the B cell repertoire with ImmunediveRsity

Cortina-Ceballos

Godoy-Lozano

Sámano-Sánchez

et al. 2015

mAbs

Self Cite

View full text Add to dashboard Cite

show abstract

“…The two biggest lineages of the largest heavy chain clonotypes of clonal expansions observed in silico were selected for experimental validation of anti-Influenza virus specificity. The sequence corresponding to the V H region flanked by EcoR I and Nhe I restriction sites were synthesized as gene fragments (Gblocks, IDT) and cloned in the expression vector of human antibody heavy chains pVAJO-CHG1, plasmid coding for human IgG1, as described [ 38 ]. The selected V H sequences were matched with seven different variable region sequences of light chain (VL) (See Additional file 1 ).…”

Section: Methodsmentioning

confidence: 99%

Longitudinal analysis of the peripheral B cell repertoire reveals unique effects of immunization with a new influenza virus strain

Cortina-Ceballos¹,

Godoy-Lozano²,

Téllez-Sosa³

et al. 2015

Genome Med

Self Cite

View full text Add to dashboard Cite

BackgroundDespite the potential to produce antibodies that can neutralize different virus (heterotypic neutralization), there is no knowledge of why vaccination against influenza induces protection predominantly against the utilized viral strains (homotypic response). Identification of structural patterns of the B cell repertoire associated to heterotypic neutralization may contribute to identify relevant epitopes for a universal vaccine against influenza.MethodsBlood samples were collected from volunteers immunized with 2008/2009 trivalent inactivated vaccine (TIV), pandemic H1N1 (pdmH1N1) monovalent inactivated vaccine (MIV) and the 2014/2015 TIV. Neutralization was assessed by hemagglutination and microneutralization test. IgG VH amplicons derived from peripheral blood RNA from pre-immune and 7 days post vaccination were subjected to 454-Roche sequencing. Full reconstruction of the sampled repertoires was done with ImmunediveRsity.ResultsThe TIV induced a predominantly homotypic neutralizing serologic response, while the 09 MIV induced a heterotypic neutralizing seroconversion in 17 % of the individuals. Both the 08/09 and the 14/15 TIV were associated with a reduction in clonotypic diversity, whereas 09 MIV was the opposite. Moreover, TIV and MIV induced distinctive patterns of IGHV segment use that are consistent with B cell selection by conserved antigenic determinants shared by the pre-pandemic and the pandemic strains. However, low somatic hypermutation rates in IgG after 09 MIV immunization, but not after 08/09 and 14/15 TIV immunization were observed. Furthermore, no evidence of the original antigenic sin was found in the same individuals after vaccination with the three vaccines.ConclusionsImmunization with a new influenza virus strain (2009 pdmH1N1) induced unique effects in the peripheral B cell repertoire clonal structure, a stereotyped response involving distinctive IGHV segment use and low somatic hypermutation levels. These parameters were contrastingly different to those observed in response to pre-pandemic and post-pandemic vaccination, and may be the result of clonal selection of common antigenic determinants, as well as germinal center-independent responses that wane as the pandemic strain becomes seasonal. Our findings may contribute in the understanding of the structural and cellular basis required to develop a universal influenza vaccine.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0239-y) contains supplementary material, which is available to authorized users.

show abstract

“…Herein, we suggest the NGS technique based on the antibody transcriptome as an efficient and universal method to deal with various intricate cases, since it can clarify all of the VR gene sequence diversities and gene abundances of individual hybridomas, complemented with specific PCR to amplify exact full-length sequences of the VH and VL. Normally, the clones with the highest amplification frequency are considered to be antigen-specific, i.e., the highly amplified VH and VL genes were paired to construct the rAbs, however subsequent experiments confirmed that the majority of clones were actually antigen-specific [ 42 , 43 ]. However, Bradbury et al hold the opposite viewpoint that the most abundant transcripts of VH and VL found in a hybridoma do not exactly translate to the antibody displaying the desired sensitivity and specificity [ 17 ].…”

Section: Discussionmentioning

confidence: 99%

Characterization of Variable Region Genes and Discovery of Key Recognition Sites in the Complementarity Determining Regions of the Anti-Thiacloprid Monoclonal Antibody

Liu

Guo

Jiao

et al. 2020

IJMS

View full text Add to dashboard Cite

Sequence-defined recombinant antibodies (rAbs) have emerged as alternatives to hybridoma-secreted monoclonal antibodies (mAbs) for performing immunoassays. However, the polyploidy nature of hybridomas often leads to the coexistence of aberrant or non-specific functional variable region (VR) gene transcripts, which complicates the identification of correct VR sequences. Herein, we introduced the use of LC-MS/MS combined with next-generation sequencing to characterize VR sequences in an anti-thiacloprid mAb, which was produced by a hybridoma with genetic antibody diversity. The certainty of VR sequences was verified by the functional analysis based on the recombinant antibody (rAb) expressed by HEK293 mammalian cells. The performance of the rAb was similar to that of the parental mAb, with IC50 values of 0.73 and 0.46 μg/L as measured by ELISAs. Moreover, molecular docking analysis revealed that Ser52 (H-CDR2), Trp98, and Trp93 (L-CDR3) residues in the complementarity determining regions (CDRs) of the identified VR sequences predominantly contributed to thiacloprid-specific recognition through hydrogen bonds and the CH–π interaction. Through single-site-directed alanine mutagenesis, we found that Trp98 and Trp93 (L-CDR3) showed high affinity to thiacloprid, while Ser52 (H-CDR2) had an auxiliary effect on the specific binding. This study presents an efficient and reliable way to determine the key recognition sites of hapten-specific mAbs, facilitating the improvement of antibody properties.

show abstract

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

Abstract: (2014) Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing, mAbs, 6:2, 493-501,

Cited by 6 publications

References 42 publications

Reconstructing and mining the B cell repertoire with ImmunediveRsity

Reconstructing and mining the B cell repertoire with ImmunediveRsity

Longitudinal analysis of the peripheral B cell repertoire reveals unique effects of immunization with a new influenza virus strain

Characterization of Variable Region Genes and Discovery of Key Recognition Sites in the Complementarity Determining Regions of the Anti-Thiacloprid Monoclonal Antibody

Contact Info

Product

Resources

About