Identification and immunogenic potential of B cell epitopes of outer membrane protein OmpF of Aeromonas hydrophila in translational fusion with a carrier protein

Abstract

Aeromonas hydrophila, a ubiquitous and virulent bacterial pathogen, affects a variety of fishes, including Labeo rohita. Existing treatment strategies comprise antibiotic therapies and attenuated bacterial strain-based vaccines. No functional subunit vaccine has been available until now. Given their key role in determining pathogenicity, outer membrane proteins have been successfully explored as potential vaccine candidates. We have devised a direct strategy for eliminating non-specific responses by selectively aiming the immune response against specific immunodominant epitopes of the outer membrane protein F (OmpF) of A. hydrophila (AhOmpF). Five putative epitopes of AhOmpF predicted in silico were genetically conjugated with heat labile enterotoxin chain B of E. coli (LTB). Recombinant fusion proteins expressed in E. coli were purified from solubilized inclusion bodies and refolded. The fusion protein retained GM1 ganglioside receptor binding activity of LTB, indicating proper folding. Four of the five fusion proteins were found to be highly immunogenic. Of the four proteins, antisera against the fusion protein (anti-rEpiF1) harboring 66-80 amino acid residues of the OmpF gave maximum cross-reactivity with the targeted rOmpF in enzyme-linked immunosorbent assay (ELISA) and was able to recognize both fusion partners-rOmpF and rLTB-in Western blot. Antibody isotyping of the antisera and cytokine array analysis of the culture supernatants of splenocytes from sensitized mice manifested a mixed Th1/Th2 immune response with a bias toward Th2. Anti-rEpiF1 antibodies were able to bind to the cell membrane of live A. hydrophila cells and agglutinate them. Our results thus suggest that the OmpF epitope (66-80) in fusion with a carrier protein is a promising vaccine candidate against A. hydrophila.

Escherichia coli outer membrane protein F (OmpF): an immunogenic protein induces cross-reactive antibodies against Escherichia coli and Shigella

“…Immunization with OmpF of Yersinia pseudotuberculosis not only resulted in production of high-avidity antibodies, but also stimulated bactericidal activity of peritoneal macrophages (Sidorova et al 2014). Sharma et al suggested that the OmpF epitope (66–80) in fusion with a carrier protein is a promising vaccine candidate against A. hydrophila (Sharma and Dixit 2015). …”

Missing the target: DNAk is a dominant epitope in the humoral immune response of channel catfish (Ictalurus punctatus) to Flavobacterium columnare

“…columnare DNAk protein? Other studies will need to characterize responding B-cell populations and identify specific epitopes and elucidate whether there is a functional role for F. columnare anti-DNAk antibodies in immunity against columnaris disease (Krska et al, 1993;Sharma and Dixit, 2015).…”

Recombinant outer membrane protein C of Aeromonas hydrophila elicits mixed immune response and generates agglutinating antibodies

“…Qualitative analysis of the agglutination ability of rOmpC antisera with live A. hydrophila cells was performed essentially as described by Sharma et al [19]. Cultures (O/ N) of different bacterial strains were grown at 37°C.…”

“…with two booster doses on day 14 and day 28 of primary immunization. Lymphocyte proliferation assay was performed as described essentially by Sharma et al [19]. Briefly, splenocytes isolated from the spleens of the mice immunized with rOmpC (20 lg/mouse), 7 days post second booster (i.e., on day 35), were treated with chilled ammonium chloride (0.9 %) to lyse the red blood cells.…”

“…Expression analysis of the rOmpC was performed essentially as described earlier [18,19]. Briefly, E. coli BL21 (kDE3) cells harboring pETAhompC were grown till A 600 = 0.8 and induced with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG).…”