Clin Exp Vaccine Res.  2017 Jul;6(2):135-145. 10.7774/cevr.2017.6.2.135.

Preferential production of IgM-secreting hybridomas by immunization with DNA vaccines coding for Ebola virus glycoprotein: use of protein boosting for IgG-secreting hybridoma production

• Lee SH
• Han BS
• Choe J
• Sin JI

• KMID: 2391574
• DOI: http://doi.org/10.7774/cevr.2017.6.2.135

Abstract

PURPOSE
The goal of this study was to investigate the utility of DNA vaccines encoding Ebola virus glycoprotein (GP) as a vaccine type for the production of GP-specific hybridomas and antibodies.
MATERIALS AND METHODS
DNA vaccines were constructed to express Ebola virus GP. Mice were injected with GP DNA vaccines and their splenocytes were used for hybridoma production. Enzyme-linked immunosorbent assays (ELISAs), limiting dilution subcloning, antibody purification methods, and Western blot assays were used to select GP-specific hybridomas and purify monoclonal antibodies (MAbs) from the hybridoma cells.
RESULTS
Twelve hybridomas, the cell supernatants of which displayed GP-binding activity, were selected by ELISA. When purified MAbs from 12 hybridomas were tested for their reactivity to GP, 11 MAbs, except for 1 MAb (from the A6-9 hybridoma) displaying an IgG2a type, were identified as IgM isotypes. Those 11 MAbs failed to recognize GP. However, the MAb from A6-9 recognized the mucin-like region of GP and remained reactive to the antigen at the lowest tested concentration (1.95 ng/mL). This result suggests that IgM-secreting hybridomas are predominantly generated by DNA vaccination. However, boosting with GP resulted in greater production of IgG-secreting hybridomas than GP DNA vaccination alone.
CONCLUSION
DNA vaccination may preferentially generate IgM-secreting hybridomas, but boosting with the protein antigen can reverse this propensity. Thus, this protein boosting approach may have implications for the production of IgG-specific hybridomas in the context of the DNA vaccination platform. In addition, the purified monoclonal IgG antibodies may be useful as therapeutic antibodies for controlling Ebola virus infection.

Keyword

Ebola virus; Hybridoma; DNA vaccines; Glycoprotein; Antibody production

MeSH Terms

Animals
Antibodies
Antibodies, Monoclonal
Antibody Formation
Blotting, Western
Clinical Coding*
DNA*
Ebolavirus*
Enzyme-Linked Immunosorbent Assay
Glycoproteins*
Hemorrhagic Fever, Ebola
Hybridomas*
Immunization*
Immunoglobulin G
Immunoglobulin M
Mice
Vaccination
Vaccines, DNA*
Antibodies
Antibodies, Monoclonal
DNA
Glycoproteins
Immunoglobulin G
Immunoglobulin M
Vaccines, DNA

Figure

• 

  Fig. 1 Production of glycoprotein (GP) DNA vaccines (A), immunization schemes using GP DNA vaccines (B), and the evaluation of GP-specific antibody production by GP DNA vaccination (C, D). (A) pcDNA3-GP encoding Zaire Ebola virus strain Gabon-94 virion spike GP was constructed as described in the “Materials and Methods.” (B, C) BALB/c mice (n = 5/group) were injected by intramuscular-electroporation with pcDNA3-GP (50 µg/mouse) at 0, 1, and 3 weeks. The mice were bled at 1 week following the final injection and sera were collected (B). The sera were reacted with recombinant Ebola virus GP (0.5 µg/mL) in an enzyme-linked immunosorbent assay (C). The optical density (OD) values were measured at 405 nm. The values and bars represent OD values and standard deviation, respectively. (D) Recombinant Ebola virus GP (3 µg) was separated on a 15% sodium dodecyl sulfate–polyacrylamide gel. The right arrow indicates a 140-kDa GP. *p < 0.05 compared with the negative control.

• 

  Fig. 2 Production of monoclonal antibodies–secreting hybridomas by Ebola virus glycoprotein (GP) DNA vaccination. BALB/c mice were injected by intramuscular-electroporation with pcDNA3-GP (50 µg/muse) at 0, 1, and 3 weeks. The mice were sacrificed at 1, 2, and 3 weeks following the final injection and the splenocytes were used for fusion (as shown in Fig. 1B). Among a total of 960 hybridomas, 12 (A6, A13, C21, C22, E53, G3, G29, G44, J10, M65, N47, and N52) with reactivity to Ebola virus GP in an enzyme-linked immunosorbent assay (secondary Ab, horseradish peroxidase [HRP]–conjugated anti-mouse IgG [H+L]) were selected. These hybridomas were subcloned with the limiting dilution subcloning method. The resulting monoclonal hybridoma clones were designated A6-9, A13-9, C21-2, C22-2, E53-2, G3-2, G29-5, G44-2, J10-2, M65-16, N47-9, and N52-7. For enzyme-linked immunosorbent assay, cell supernatants from the 12 monoclonal clones were reacted with GP, followed by detection with HRP-conjugated anti-mouse IgG (H+L) (A), HRP-conjugated anti-mouse IgA and HRP-conjugated IgM (B), and HRP-conjugated anti-mouse IgG (Fc-specific) (C) secondary antibodies. The positive control was GP-specific sera (1:10 dilution) from Fig. 1C, whereas the negative control was naive sera (1:10 dilution). (D) Cell supernatants (20 µL) were loaded onto a 12% sodium dodecyl sulfate–polyacrylamide gel, followed by Western blot assay using HRP-conjugated anti-mouse IgG (H+L). OD, optical density.

• 

  Fig. 3 The structure of the whole Ebola virus glycoprotein (GP) and the amino acid sequence numbers of recombinant GPs spanning GP1 region. The signal peptide (SP), the putative receptor-binding domain, mucin-like region, furin cleavage site and trans-membrane region (TMR) are shown. GP1a, GP1b, GP1c, and GP1d are recombinant proteins spanning the putative receptor-binding domains of GP1, whereas GP1e is a recombinant protein spanning the mucin-like region of GP1. The amino acid sequence numbers are also displayed.

• 

  Fig. 4 Production of recombinant GP1a-e (A), the binding activity of monoclonal antibodies (MAbs) in cell supernatants to the recombinant GP1a and GP1e (B), purification of IgG and IgM (C), and their binding to the recombinant GP1a and GP1e (D). (A) The recombinant proteins GP1a, GP1b, GP1c, GP1d, and GP1e were produced as described in the “Materials and Methods.” GP1a, GP1b, GP1c, GP1d, and GP1e (2 µg) were loaded on an 8% sodium dodecyl sulfate–polyacrylamide gel. (B) We assessed the binding activity of the MAbs in the hybridoma cell supernatants to the recombinant GP1a and GP1e by enzyme-linked immunosorbent assay. Horseradish peroxidase (HRP)–conjugated anti-mouse IgG (H+L) was used as the secondary antibody (Ab). (C) Hybridoma cells that produced MAbs were cultured, and the cell supernatants were collected for Ab purification. Some hybridoma cells (C21-2, C22-2, G44-2, and M65-16) were injected into nude mice for ascites fluid production. IgG was purified from cell supernatants using the protein G-resin column (for A6-9). IgM was purified either from ascites fluids using the HiTrap IgM purification kit (for C21-2, C22-2, G44-2, and M65-16) or from cell supernatants using the protein L resin columns (for A13-9, E53-2, G3-2, G29-5, J10-2, N47-9, and N52-7). The purified Abs (2 µg) were run on a 12% sodium dodecyl sulfate–polyacrylamide gel for Brilliant Blue R250 staining. (D) The purified IgG and IgM (2 µg/mL) Abs were reacted with GP1a and GP1e in an enzyme-linked immunosorbent assay using HRP-conjugated anti-IgG (H+L) as the secondary antibody. The positive control was GP-specific sera (1:10 dilution) from Fig. 1C, whereas the negative control was naive sera (1:10 dilution). OD, optical density.

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  Fig. 5 Evaluation of glycoprotein (GP)-specific IgG subclass (A) and its lowest concentration for GP binding (B). (A) Purified IgG (2 µg/mL) from clone A6-9 was reacted with GP1e, followed by incubation with horseradish peroxidase (HRP)–conjugated anti-IgG1, HRP-conjugated anti-IgG2a, HRP-conjugated anti-IgG2b and HRP-conjugated anti-IgG3 by enzyme-linked immunosorbent assay (ELISA), to determine its IgG subclass. (B) IgG from clone A6-9 (2 µg/mL) was serially diluted by 2-fold, and then reacted with GP1e in parallel with control IgG (2 µg/mL) to determine its lowest concentration for GP binding using ELISA. OD, optical density.

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  Fig. 6 Effects of booster-injections with glycoprotein (GP) DNA vaccines vs. GP on the production of IgM- and IgG-secreting hybridomas. BALB/c mice were immunized by intramuscular-electroporation (IM-EP) with pcDNA3-GP (50 mg/mouse) at 0 and 4 weeks. The mice received a booster-injection at 12 weeks with either GP DNA vaccines by IM-EP or with GP by intravenous delivery. (A, B) One week after booster injection with GP DNA vaccines, the mice were sacrificed and their splenocytes were used for hybridoma production. Among the numerous hybridomas, six were selected, which had cell supernatants with optical density (OD) values at least 6× higher than those of the negative control in an enzyme-linked immunosorbent assay (ELISA) with horseradish peroxidase (HRP)–conjugated anti-mouse IgG (H+L). Cell supernatants from the six hybridomas were next reacted with GP in an ELISA using HRP-conjugated anti-mouse IgM. The OD values of the six hybridomas are shown (A). Cell supernatants from the six hybridomas were tested for the presence of IgG and IgM by Western blot assay as in Fig. 2D (B). (C, D) Experiments were performed as in Fig. 6A on splenocytes harvested 3 days after intravenous injection with GP. Subsequently, 12 hybridomas were selected. The OD values of the 12 hybridomas are shown (C). (D) Cell supernatants (20 µL) from the 12 hybridomas were loaded onto a 12% sodium dodecyl sulfate–polyacrylamide gel, followed by Western blot assay using HRP-conjugated anti-mouse IgG (H+L). The positive control was GP-specific sera (1:10 dilution) from Fig. 1C, whereas the negative control was naive sera (1:10 dilution).

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