Inhibition of Middle East respiratory syndrome coronavirus infection by anti-CD26 monoclonal antibody

Abstract

We identified the domains of CD26 involved in the binding of Middle East respiratory syndrome coronavirus (MERS-CoV) using distinct clones of anti-CD26 monoclonal antibodies (MAbs). One clone, named 2F9, almost completely inhibited viral entry. The humanized anti-CD26 MAb YS110 also significantly inhibited infection. These findings indicate that both 2F9 and YS110 are potential therapeutic agents for MERS-CoV infection. YS110, in particular, is a good candidate for immediate testing as a therapeutic modality for MERS.

Fig 1

CD26 expression and binding of MERS-CoV S1-Fc in parental Jurkat cells and CD26 Jurkat transfectants. (A) Representative histograms showing results of staining of Jurkat cells stably transfected with the full-length human CD26 (JKT-hCD26WT) or vector control (JKT-Mock) with Alexa Fluor 488-labeled anti-CD26 MAb 2F9 (5 μg/ml; black).Gray histograms show results of staining with an isotype control (Alexa Fluor 488-labeled mouse IgG [msIgG-488]; 5 μg/ml). Results representative of three different experiments are shown. (B) Representative histograms showing results of staining with Alexa Fluor-labeled MERS-CoV S1-Fc (5 μg/ml; black) using JKT-Mock or JKT-hCD26WT. Gray histograms show staining with Alexa Fluor 488-labeled recombinant human Fc (Fc-488) as an isotype control. Results representative of three different experiments are shown.

Fig 2

Anti-CD26 MAb 2F9 inhibits binding of MERS-CoV S1-Fc. (A) Representative histograms showing results of staining with MERS-CoV S1-Fc in the presence of various clones of anti-CD26 MAbs or control mouse IgG (left). JKT-hCD26WT cells were incubated with the indicated anti-CD26 MAb (mouse MAb 4G8, 1F7, 14D10, 2F9, 16D4B, or 9C11 or humanized MAb YS110) (red) or control IgG (black) (each 10 μg/ml) for 30 min at 4°C. After being washed, cells were stained with Alexa Fluor 488-labeled MERS-CoV S1-Fc (5 μg/ml). Gray histograms show results of staining with Fc-488 as an isotype control. Mean fluorescence intensities (MFI) of Alexa Fluor 488-labeled MERS-CoV S1-Fc are indicated in the bar graph (right). Results representative of three different experiments are shown as mean MFI. Error bars indicate standard errors of the means (SEMs) (two-tailed Student's t test; * or **, P < 0.05 versus control IgG). (B to D) MFI of staining with Alexa Fluor 488-labeled MERS-CoV S1-Fc in the presence of various concentrations of the anti-CD26 MAb 2F9 (B), 1F7 (C), or YS110 (D) (red) or control msIgG (black). JKT-hCD26WT cells were incubated with the indicated concentrations of the anti-CD26 MAbs or control IgG for 30 min at 4°C. After being washed, cells were stained with Alexa Fluor 488-labeled MERS-CoV S1-Fc (5 μg/ml). Results of three different experiments are shown as mean MFI ± SEMs (two-tailed Student's t test; *, P < 0.05 versus corresponding control IgG). The black and red bars at 0 μg/ml of preincubated control IgG or anti-CD26 MAbs were plotted using the same data.

Fig 3

Preincubation with MERS-CoV S1-Fc partially inhibits binding of MERS-CoV S1-Fc. (A) Representative histograms showing results of staining with MERS-CoV S1-Fc in the presence of various concentrations of unlabeled MERS-CoV S1-Fc or control Fc (left). JKT-hCD26WT cells were incubated with the indicated concentrations of unlabeled MERS-CoV S1-Fc (dashed) or control Fc (black) for 30 min at 4°C. After being washed, cells were stained with Alexa Fluor 488-labeled MERS-CoV S1-Fc (5 μg/ml). Gray histograms show staining with the isotype control (Fc-488). MFI of Alexa Fluor 488-labeled MERS-CoV S1-Fc are indicated in the bar graph (right). Results representative of three different experiments are shown as mean MFI. Error bars indicate SEMs (two-tailed Student's t test; *, P < 0.05 versus corresponding control Fc). The black and dark-gray bars at 0 μg/ml of preincubated MERS-CoV S1-Fc or control Fc were plotted using the same data. (B) Representative histograms showing staining with the anti-CD26 MAb 14D10 in the presence of various concentrations of MERS-CoV S1-Fc or control Fc. The experiments were conducted as for panel A. Gray histograms show staining with the isotype control (msIgG-488).

Fig 4

Binding regions of ADA (adenosine deaminase 1) in CD26 are involved in the binding of MERS-CoV S1-Fc to human CD26. (A) Representative histograms showing results of the binding of ADA to JKT-Mock (left) or JKT-hCD26WT (right). JKT-Mock or JKT-hCD26WT was incubated with 10 μg/ml of Alexa Fluor 488-labeled ADA or ADA2 (CECR1) as a fluorescence control. Data are representative of three independent experiments, with similar results being obtained. (B) MFI for staining with Alexa Fluor 488-labeled ADA in the presence of various concentrations of the anti-CD26 MAb 2F9 (dark gray), 1F7 (light gray), YS110 (gray), or control msIgG (black). JKT-hCD26WT cells were incubated with the indicated concentrations of anti-CD26 MAbs or control IgG for 30 min at 4°C. After being washed, cells were stained with Alexa Fluor 488-labeled ADA (10 μg/ml). Alexa Fluor 488-labeled ADA2 was used as a fluorescence control, with MFI being <10. Results representative of three different experiments are shown as mean MFI. Error bars indicate SEMs (two-tailed Student's t test; *, P < 0.05 versus corresponding control IgG). (C, panel a) Representative histograms showing results for binding of ADA (left) or the anti-CD26-MAb 14D10 (right) to Jurkat cells stably transfected with human CD26 with a deletion of the ADA binding region (JKT-hCD26-ADA⁻). Gray histograms show Alexa Fluor 488-labeled ADA2 or msIgG-488 as a fluorescence control. (b) Representative histograms showing results for staining with MERS-CoV S1-Fc of JKT-hCD26-ADA⁻. JKT-hCD26-ADA⁻ cells were stained with Alexa Fluor 488-labeled MERS-CoV S1-Fc (black) at the indicated concentrations. Gray histograms show results for staining with Fc-488 as an isotype control. Data are representative of three independent experiments, with similar results being obtained.

Fig 5

2F9 fully inhibits binding to MERS-CoV S1-Fc of CD26 in the presence of exogenous ADA. (A and B) Representative histograms showing results for staining with MERS-CoV S1-Fc (A) or 2F9 (B) in the presence of various concentrations of exogenous ADA (dashed) or PBS (black) as a solvent control (vehicle) (left). JKT-hCD26WT cells were incubated with the indicated concentrations of exogenous ADA or corresponding concentrations of diluted PBS for 30 min at 37°C. After being washed, cells were stained with Alexa Fluor 488-labeled MERS-CoV S1-Fc or 2F9 (each 5 μg/ml). Gray histograms show results for staining with Fc-488 or msIgG-488 as an isotype control. MFI for Alexa Fluor 488-labeled MERS-CoV S1-Fc or 2F9 are indicated in the bar graphs (right). Results representative of three different experiments are shown as mean MFI. Error bars indicate SEMs (two-tailed Student's t test; *, P < 0.05 versus corresponding vehicle). The black and gray bars at 0 μg/ml of preincubated ADA or vehicle were plotted using the same data. (C) Representative histograms showing results for staining with MERS-CoV S1-Fc in the presence of various concentrations of exogenous ADA with the addition of 2F9 (dashed). JKT-hCD26WT cells were incubated with the indicated concentrations of exogenous ADA or corresponding concentrations of diluted PBS for 30 min at 37°C, followed by additional incubation with 2F9 (10 μg/ml) or control msIgG (10 μg/ml) for 30 min at 4°C. After being washed, cells were stained with Alexa Fluor 488-labeled MERS-CoV S1-Fc (5 μg/ml). The dashed histogram in the left panel shows results for staining with MERS-CoV S1-Fc in the presence of PBS with the addition of control msIgG. Gray histograms show staining with Fc-488 as an isotype control. Results representative of three different experiments are shown.

Fig 6

Characterization of regions of CD26 that bind to MERS-CoV S1-Fc through the use of CD26 deletion or human/rat swap mutants. Representative histograms show results for staining for MERS-CoV S1-Fc, 2F9, or YS110. CD26 cDNAs with full-length human CD26 (a), the indicated deletion (b through f), human/rat (H/R) swap mutants (h through j), or vector control (g) were cotransfected with GFP-expressing plasmids to COS-1 cells. After 24 h of transfection, cells were stained with Alexa Fluor 647-labeled MERS-CoV S1-Fc, 2F9, or YS110 (each 5 μg/ml). Gray histograms show results of staining with Fc-647 or msIgG-647 as an isotype control. The histograms for Alexa Fluor 647 were obtained by gating for GFP-positive cells among all acquired cells. Results of three different experiments are shown as percent positive cells in full-length CD26 (bar graphs, bottom). Error bars indicate SEMs (two-tailed Student's t test; *, P < 0.05 versus mock; **, P < 0.05 versus corresponding mutants; ***, P < 0.05 versus corresponding rat full-length CD26). Vertical dashed lines in the histograms indicate borders between negative and positive intensities. In the diagrams of CD26, TM indicates a transmembrane region of human CD26, and amino acids (aa) derived from human (H) or rat (R) are represented in white or dark-gray boxes, respectively.

Fig 7

Schematic diagram of human CD26 profiling the predicted contact areas of anti-CD26 MAbs 2F9, 1F7, YS110, and MERS-CoV S1. 2F9 recognizes between 248 and 449 aa, including the ADA binding regions, and 1F7 and YS110 recognize between 248 and 358 aa, excluding the ADA binding regions. MERS-CoV-contacting residues of human CD26 are indicated by asterisks; information was obtained from recently published data (17, 18). TM indicates a transmembrane region of human CD26 (black box), and the extracellular domain of CD26 is located at the C-terminal residues of TM.

Fig 8

Inhibition of MERS-CoV infection by the anti-CD26 MAb 2F9. Huh-7 cells were preincubated with normal mouse IgG, various anti-CD26 MAbs (4G8, 1F7, 2F9, 16D4B, 9C11, or 14D10), humanized anti-CD26 MAb (YS110), or anti-CD26 goat polyclonal antibody (pAb) at a concentration of 40 μg/ml for 0.5 h prior to MERS-CoV virus inoculation (1 h), all at room temperature. Mock-incubated cells (control) were used as controls. Following incubation at 37°C for 8 h, infected cells were detected by immunofluorescence using anti-SARS-CoV NSP4 antibodies that are cross-reactive for MERS-CoV, and infection was quantified as the number of anti-SARS-CoV NSP4-positive cells. Two independent experiments were performed, and data from one representative experiment are shown. Error bars indicate SEMs (two-tailed Student's t test; *, **, or ***, P < 0.05 versus control.