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

Abstract

PurposeThe 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 MethodsDNA 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.ResultsTwelve 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.ConclusionDNA 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.

Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein

“…We previously reported that GP DNA vaccines induce Ag-specific antibody responses [ 10 ]. In this study, we investigated whether GP DNA vaccines might induce IgG responses to the native forms of GP expressed on the cell surface.…”

“…A GP DNA vaccine, pcDNA3-GP was previously tested [ 10 ]. It encodes GP of Zaire Ebola virus strain Gabon-94 (GenBank No.…”

“…Mammalian cell-derived recombinant GP was purchased from Y-Biologics (Daejeon, Korea). Escherichia coli -derived GP1 proteins (GP1a, GP1b, GP1c, GP1d, and GP1e) were previously produced and tested [ 10 ].…”

“…DNA vaccines are thought to be useful at generating the native from of viral antigens as they tend to express their proteins in cells in a manner similar to real viral infection. However, DNA vaccines are less effective at producing IgG-secreting hybridomas, possibly due to the presence of un-methylated CpG sequences in the DNA vaccines that stimulate polyclonal B cell activation [ 10 ]. In the Lee et al (2017) study [ 10 ], use of protein boosting immunization was effective at reversing the preference for production of IgM-secreting hybridomas over IgG-secreting hybridomas.…”

“…However, DNA vaccines are less effective at producing IgG-secreting hybridomas, possibly due to the presence of un-methylated CpG sequences in the DNA vaccines that stimulate polyclonal B cell activation [ 10 ]. In the Lee et al (2017) study [ 10 ], use of protein boosting immunization was effective at reversing the preference for production of IgM-secreting hybridomas over IgG-secreting hybridomas. Moreover, multiple immunizations with GP after GP DNA vaccination might be useful at increasing the chance of generating antibodies with higher antigen binding affinity.…”