High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies

Abstract

Defining human B cell repertoires to viral pathogens is critical for design of vaccines that induce broadly protective antibodies to infections such as HIV-1 and influenza. Single B cell sorting and cloning of immunoglobulin (Ig) heavy- and light-chain variable regions (V(H) and V(L)) is a powerful technology for defining anti-viral B cell repertoires. However, the Ig-cloning step is time-consuming and prevents high-throughput analysis of the B cell repertoire. Novel linear Ig heavy- and light-chain gene expression cassettes were designed to express Ig V(H) and V(L) genes isolated from sorted single B cells as IgG1 antibody without a cloning step. The cassettes contain all essential elements for transcriptional and translational regulation, including CMV promoter, Ig leader sequences, constant region of IgG1 heavy- or Ig light-chain, poly(A) tail and substitutable V(H) or V(L) genes. The utility of these Ig gene expression cassettes was established using synthetic V(H) or V(L) genes from an anti-HIV-1 gp41 mAb 2F5 as a model system, and validated further using V(H) and V(L) genes isolated from cloned EBV-transformed antibody-producing cell lines. Finally, this strategy was successfully used for rapid production of recombinant influenza mAbs from sorted single human plasmablasts after influenza vaccination. These Ig gene expression cassettes constitute a highly efficient strategy for rapid expression of Ig genes for high-throughput screening and analysis without cloning.

Fig. 1

Schematic diagram for generation of linear full-length Ig heavy- and light-chain genes. Shown is a schematic diagram for the assembly by overlapping PCR of linear full-length Ig heavy-chain gene (A), Ig kappa light-chain gene (B) and lambda light-chain gene (C) expression cassettes. Sequences in the Ig leader region at the 3′ end of the C fragment overlapping with the sequences at the 5′ end of the V_H, V_κ and V_λ fragments are indicated. Sequences at the 5′ end of the H fragment, K fragment and L fragments overlapping with the sequences at the 3′ end of the corresponding V_H, V_κ and V_λ fragments are also indicated. The same forward and reverse primers (CMV-F262 and BGH-R1235, Supplementary Table 7) used in the overlapping PCR for all Ig heavy-chain genes, Ig kappa light-chain genes and lambda light-chain genes are indicated with arrows.

Fig. 2

Expression of 2F5 V_H and V_L. In panel A, lane M: DNA ladders marked in kilobases (kb) next to the lane, lanes 1-8, respectively: DNA fragments C (705 bp), H (1,188 bp), K (569 bp), 2F5 V_H (489 bp) and V_L (370 bp) as well as the full-length Ig heavy- (2,339 bp) and light-chain (1,595 bp) gene expression cassettes generated by PCR and analyzed on a 1% agarose gel. Arrows indicate the expected DNA fragments. Panel B shows the results of Western blots of commercial mAb 2F5 (lane 1), supernatant harvested from 293T cells transfected with plasmids expressing 2F5 Ig genes (lane 2), with the linear full-length 2F5 Ig heavy- and light-chain gene cassettes (lanes 3) and mock-transfected 293T cells (lane 4). Igs on the blots were detected by either an anti-human heavy-chain specific antibody or an anti-human kappa light-chain specific antibody as indicated at the bottom of the blots. The arrows with short notations indicate the possible composition of antibody heavy-chains (HC) and light-chains (LC). Panel C is a comparison of the amounts of Ig secreted from 293T cells transiently transfected with either linear full-length 2F5 heavy- and light-chain Ig gene constructs (1ug of each) or plasmids (1ug of each) expressing the 2F5 heavy- or light-chain Ig genes as indicated. Average amounts (n=6) of IgG secreted in the transfected 293T cells are shown on the y axis and were determined by comparison to a standard curve generated using known concentrations of IgG1. Panel D shows the measurement of antibody binding by ELISA to the following antigens: HIV-1 Env MPER epitope peptide, HIV-1 gp41 and HIV-1 gp140 or scrambled SP62 peptides as negative controls. Supernatants from the 293T cells transfected with each DNA construct (indicated on the x axis) were assayed by ELISA for binding to HIV-1 antigens and the results were compared to the purified mAb 2F5 at 1.25 μg/ml.

Fig. 2

Expression of 2F5 V_H and V_L. In panel A, lane M: DNA ladders marked in kilobases (kb) next to the lane, lanes 1-8, respectively: DNA fragments C (705 bp), H (1,188 bp), K (569 bp), 2F5 V_H (489 bp) and V_L (370 bp) as well as the full-length Ig heavy- (2,339 bp) and light-chain (1,595 bp) gene expression cassettes generated by PCR and analyzed on a 1% agarose gel. Arrows indicate the expected DNA fragments. Panel B shows the results of Western blots of commercial mAb 2F5 (lane 1), supernatant harvested from 293T cells transfected with plasmids expressing 2F5 Ig genes (lane 2), with the linear full-length 2F5 Ig heavy- and light-chain gene cassettes (lanes 3) and mock-transfected 293T cells (lane 4). Igs on the blots were detected by either an anti-human heavy-chain specific antibody or an anti-human kappa light-chain specific antibody as indicated at the bottom of the blots. The arrows with short notations indicate the possible composition of antibody heavy-chains (HC) and light-chains (LC). Panel C is a comparison of the amounts of Ig secreted from 293T cells transiently transfected with either linear full-length 2F5 heavy- and light-chain Ig gene constructs (1ug of each) or plasmids (1ug of each) expressing the 2F5 heavy- or light-chain Ig genes as indicated. Average amounts (n=6) of IgG secreted in the transfected 293T cells are shown on the y axis and were determined by comparison to a standard curve generated using known concentrations of IgG1. Panel D shows the measurement of antibody binding by ELISA to the following antigens: HIV-1 Env MPER epitope peptide, HIV-1 gp41 and HIV-1 gp140 or scrambled SP62 peptides as negative controls. Supernatants from the 293T cells transfected with each DNA construct (indicated on the x axis) were assayed by ELISA for binding to HIV-1 antigens and the results were compared to the purified mAb 2F5 at 1.25 μg/ml.

Fig. 2

Expression of 2F5 V_H and V_L. In panel A, lane M: DNA ladders marked in kilobases (kb) next to the lane, lanes 1-8, respectively: DNA fragments C (705 bp), H (1,188 bp), K (569 bp), 2F5 V_H (489 bp) and V_L (370 bp) as well as the full-length Ig heavy- (2,339 bp) and light-chain (1,595 bp) gene expression cassettes generated by PCR and analyzed on a 1% agarose gel. Arrows indicate the expected DNA fragments. Panel B shows the results of Western blots of commercial mAb 2F5 (lane 1), supernatant harvested from 293T cells transfected with plasmids expressing 2F5 Ig genes (lane 2), with the linear full-length 2F5 Ig heavy- and light-chain gene cassettes (lanes 3) and mock-transfected 293T cells (lane 4). Igs on the blots were detected by either an anti-human heavy-chain specific antibody or an anti-human kappa light-chain specific antibody as indicated at the bottom of the blots. The arrows with short notations indicate the possible composition of antibody heavy-chains (HC) and light-chains (LC). Panel C is a comparison of the amounts of Ig secreted from 293T cells transiently transfected with either linear full-length 2F5 heavy- and light-chain Ig gene constructs (1ug of each) or plasmids (1ug of each) expressing the 2F5 heavy- or light-chain Ig genes as indicated. Average amounts (n=6) of IgG secreted in the transfected 293T cells are shown on the y axis and were determined by comparison to a standard curve generated using known concentrations of IgG1. Panel D shows the measurement of antibody binding by ELISA to the following antigens: HIV-1 Env MPER epitope peptide, HIV-1 gp41 and HIV-1 gp140 or scrambled SP62 peptides as negative controls. Supernatants from the 293T cells transfected with each DNA construct (indicated on the x axis) were assayed by ELISA for binding to HIV-1 antigens and the results were compared to the purified mAb 2F5 at 1.25 μg/ml.

Fig. 2

Expression of 2F5 V_H and V_L. In panel A, lane M: DNA ladders marked in kilobases (kb) next to the lane, lanes 1-8, respectively: DNA fragments C (705 bp), H (1,188 bp), K (569 bp), 2F5 V_H (489 bp) and V_L (370 bp) as well as the full-length Ig heavy- (2,339 bp) and light-chain (1,595 bp) gene expression cassettes generated by PCR and analyzed on a 1% agarose gel. Arrows indicate the expected DNA fragments. Panel B shows the results of Western blots of commercial mAb 2F5 (lane 1), supernatant harvested from 293T cells transfected with plasmids expressing 2F5 Ig genes (lane 2), with the linear full-length 2F5 Ig heavy- and light-chain gene cassettes (lanes 3) and mock-transfected 293T cells (lane 4). Igs on the blots were detected by either an anti-human heavy-chain specific antibody or an anti-human kappa light-chain specific antibody as indicated at the bottom of the blots. The arrows with short notations indicate the possible composition of antibody heavy-chains (HC) and light-chains (LC). Panel C is a comparison of the amounts of Ig secreted from 293T cells transiently transfected with either linear full-length 2F5 heavy- and light-chain Ig gene constructs (1ug of each) or plasmids (1ug of each) expressing the 2F5 heavy- or light-chain Ig genes as indicated. Average amounts (n=6) of IgG secreted in the transfected 293T cells are shown on the y axis and were determined by comparison to a standard curve generated using known concentrations of IgG1. Panel D shows the measurement of antibody binding by ELISA to the following antigens: HIV-1 Env MPER epitope peptide, HIV-1 gp41 and HIV-1 gp140 or scrambled SP62 peptides as negative controls. Supernatants from the 293T cells transfected with each DNA construct (indicated on the x axis) were assayed by ELISA for binding to HIV-1 antigens and the results were compared to the purified mAb 2F5 at 1.25 μg/ml.

Fig. 3

Measurement of the reactivity of recombinant antibodies. Panel A compares the reactivity of recombinant antibody produced in 293T cells by transfection of 7B2 Ig genes and mAb 7B2 produced by the EBV-transformed 7B2 B cell line. Supernatants of 293T cells transfected with linear rH70 Ig genes and supernatants from mock transfections were used as negative controls. Panel B compares the reactivity of the recombinant antibody produced by transfection with linear G8 Ig genes with antibody produced by the EBV-transformed G8 B cell line and with purified mAb G8. Supernatant from 293T cells transfected with linear rH0045 Ig genes with unknown specificity and from mock-transfection were used as negative controls.

Fig. 3

Measurement of the reactivity of recombinant antibodies. Panel A compares the reactivity of recombinant antibody produced in 293T cells by transfection of 7B2 Ig genes and mAb 7B2 produced by the EBV-transformed 7B2 B cell line. Supernatants of 293T cells transfected with linear rH70 Ig genes and supernatants from mock transfections were used as negative controls. Panel B compares the reactivity of the recombinant antibody produced by transfection with linear G8 Ig genes with antibody produced by the EBV-transformed G8 B cell line and with purified mAb G8. Supernatant from 293T cells transfected with linear rH0045 Ig genes with unknown specificity and from mock-transfection were used as negative controls.

Fig. 4

Changes in plasmablast populations induced by vaccination with Fluzone. Shown is the frequency of plasmablasts (located in the upper right and defined as CD19⁺, CD20low-neg, CD3^-, CD14^-, CD16^-, CD235^-, CD38^hi and CD27^hi) in PB collected at day 0, 7 and 21 after vaccination from a subjecte vaccinated with Fluzone^R 2007-2008 and analyzed by flow cytometry.

Fig. 5

Reactivity of recombinant human mAbs from single plasma cells after Fluzone® 2007-2008 vaccination. Shown are the results of ELISA assays for detection of the reactivity of the antibodies derived from the individual Ig heavy- and light-chain gene pairs isolated from sorted single plasma cells (first 9 bars in the x-axis) or a negative control Ig pair (- Ab Ctl) and mock transfection control (Mock Tx.). Antibody reactivity to the inactivated influenza viruses (Panel A), with H1 A/Solomon islands HA (Panel B) and H3 A/Wisconsin HA (Panel C) are shown. Serum samples collected from the vaccinee at day 0 and 21 (grey columns) were used as positive controls in these assays. Data are representative of 2 independent experiments.

Fig. 6

Serum antibody responses to Fluzone vaccination. Serum samples were collected from the vaccinee at day 0 and 21 days after vaccination with Fluzone and assayed against a panel of influenza antigens as indicated on the x-axis. Shown is the reactivity of serum samples at 1:800 dilution to the indicated antigens. Data are representative 2 independent experiments.