Production of Monoclonal Antibodies Against the 8 kDa Subunit ofEchinococcus granulosusAntigen B (EgAgB8/2) Using DNA Immunization

Abstract

Cystic echinococcosis (CE), an endemic cosmopolitan zoonotic helminthic disease caused by the larval stage of Echinococcus granulosus, lacks reliable diagnostic tools that fulfill the criteria of high sensitivity and specificity. Antigen B (AgB), a thermostable lipoprotein that constitutes a considerable fraction of the cystic hydatid fluid (HF), is being considered as a suitable source for vaccination and immunodiagnosis of CE due to its high specificity. Genetic immunization was used to immunize BALB/c mice with the second subunit of antigen B (EgAgB8/2) for the production of monoclonal antibodies (MAb). Fusion products between the spleen cells and myeloma cells produced six MAbs of the following isotypes: IgG2a (two clones), IgG2b (three clones), and IgM (one clone). The MAbs were tested for their specificity to crude sheep hydatid fluid (CSHF) versus other antigens prepared from other helminthic parasites including Toxocara canis, Acanthocheilonema viteae, Fasciola hepatica, Schistosoma mansoni, and Taenia. Five MAbs reacted with E. granulosus antigens, one showed cross reactivity with S. mansonia antigens, and one showed a high reactivity with E. granulosus but was cross reactive with all helminthic antigens tested. Using SDS-PAGE and immunoblotting under reducing conditions, all MAbs identified the four AgB subunits with molecular weights of 8, 16, 24, and 36 kDa. Further work on the specificity and sensitivity of these MAbs as well as their use in detecting circulating parasite antigens and in antigen purification will be assessed in future studies.

DNA immunization as a technology platform for monoclonal antibody induction

“…Abbreviation: enzyme-linked immunosorbent assay, ELISA; fluorescence-activated cell sorting, FACS; immunohistochemistry, IHC. Extracellular domain Single transmembrane [33][34][35][36][37] Fragment/subunit Any type 25,38,39,55 Mini-gene insert Any type 40 Novel format, vaccibody Secretory 56 Immunogen-transmembrane domain fusion Viral non-structure 41 Immunogen-Fc fusion Intracellular 57,58 Abbreviation: glycophosphatidylinositol, GPI.…”

“…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.…”

Potent monoclonal antibodies againstClostridium difficiletoxin A elicited by DNA immunization

“…3,8,9 Frequently, such antibodies are also highly functional in their expected biological activities, such as blocking the infection of highly virulent HIV-1, SARS-CoV, or influenza viruses to targeted cells. 8,10,11 Parallel to such progress, DNA immunization has also been proposed as a useful technology to produce monoclonal antibodies (mAb) [12][13][14] to decrease the need for protein and peptide antigens for immunization. This is significant progress to circumvent the need of production and purification of difficult and complex proteins while ensuring effective induction of antigen-specific antibody responses against native conformation.…”