Generation of Monoclonal Antibodies Against Human Recombinant Interferon Beta Using Genetic Immunization with Simultaneous Expression of IgM and IgG Isotypes

Abstract

Monoclonal antibodies (MAbs) against human recombinant interferon beta (hrIFNbeta) were generated by genetic immunization (GI). In order to test two viral promoters frequently used in mammalian expression plasmid vectors, mice were inoculated four times by intramuscular injection, without adjuvant, with 100 microg of either pcDNA 3.1hrIFNbeta or pZeoSV2IFNbeta containing the entire human interferon beta gene and under the control of, respectively, human cytomegalovirus (HCMV) immediate-early promoter or early SV-40 enhancer/promoter. Only serum samples from mice immunized with pZeoSV2IFNbeta were positive to anti-hrIFNbeta. The spleens of the immunized mice were fused with myeloma Sp2/0 cells and the hybridoma clones generated screened by an in house enzyme-linked immunosorbent assay (ELISA). Fourteen MAbs were selected as reactive with hrIFNbeta. Western blot analysis was performed and only one recognized the 18 kDa isoform (non-glycosylated) of hrIFNbeta. All MAbs were subjected to antibody isotype characterization with a commercial ELISA and showed unusual profile with simultaneous expression of both IgM and IgG2a isotypes. This observation is further supported by RT-PCR amplification of the IgM CH4 domain using total RNA from hybridomas.

DNA immunization as a technology platform for monoclonal antibody induction

“…One interesting finding from literature is that although the delivery approach may be critical for the induction of high-level immune responses for human vaccine development, different DNA delivery approaches have been similarly successful in producing mAbs against a wide range of target antigens. Table 3 lists the mAbs elicited by the gene gun approach; 22,24,[31][32][33][34]37,43,55,57,58 needle injection, including intramuscular 19,20,23,38,41,[44][45][46][48][49][50] or intradermal 21,35,42 injection; and electroporation following intramuscular or intradermal injection. 27,28,39,52,53,56 One unique but less-studied approach is hydrodynamic intravenous delivery.…”

“…67 Another research group used a similar DNA prime-protein boost approach to generate a higher antibody titer and higher quality mAbs than those observed with protein immunization alone. 36 33 Intracellular (PED/PEA-15) 57 Intracellular (annexin-V) 58 Single transmembrane (CAR) 22 Two-transmembrane (P2X7) 24 GPI anchored enzyme 43 Intracelluar (BCL-6) 31 Intracelluar (MALT1) 32 Single transmembrane (MHCI-related gene A) 34 Parasite lipoprotein 55 Viral envelop (HIV gp120) 37 IM Bacteria toxin (Helicobacter pylori vacuolating cyto toxin) 38 Seven transmembrane, GPCR (TSHR) 19 Viral envelop (HGV E2) 48 Seven transmembrane, GPCR (TSHR) 20 Viral non-structure (Dengue NS1) 41 Viral envelop (H5N1) 49 Secretory protein, enzyme (prostate-specific antigen) 45 Viral surface (HBV preS2/S) 50 Seven transmembrane, GPCR (TSHR) 23 Secretory protein, cytokine (CKLF1) 44 Secretory protein, cytokine (Interferon beta) 46…”

“…Hybridoma fusions using cells from DNA-immunized animal hosts without a final boost have been reported by several groups. 38,41,44,46,55 However, the overall fusion efficiency was low, and the resulting antibodies had low binding affinity, with IgM as the dominant isotype. One study compared the ability of a final protein boost with no final protein boost with respect to hybridoma generation and concluded that despite significant antibody responses in the immunized animals, the fusion of mouse spleen cells yielded a low number of and lowquality hybridomas unless the mice were given a boost 3-5 days before fusion.…”

In vivoElectroporation and Non-protein Based Screening Assays to Identify Antibodies Against Native Protein Conformations

“…(5,6) It is well documented that gene delivery and inducing antibodies to conformational epitopes are achieved via genebased vaccination for the native form of the protein. (5)(6)(7)(8) The in vivo electroporation is known to result in a ''danger signal'' in the injection site, recruiting antigen presentation cells as well as a strong milieu of cytokines that elicit immune responses. (9) A final boost with either proteins or cells expressing the antigen has improved the titers dramatically.…”