Bicistronic DNA vaccine against Edwardsiella tarda infection in Labeo rohita: Construction and comparative evaluation of its protective efficacy against monocistronic DNA vaccine

Abstract

DNA-based vaccination or genetic immunization is one of the most promising, effective and prophylactic measures to control aquatic animal diseases. This immunization strategy involves administration of eukaryotic antigen expression vectors (DNA vaccine) into the host that encode for an antigen under the control of a eukaryotic promoter which resulted into elicitation of strong cellular and humoral immune responses. In the present study, a bicistronic DNA vaccine (designated as pGPD+IFN) was constructed which contains an additional immune adjuvant gene (Interferon gamma gene of Labeo rohita) along with a regular antigenic gene (glyceraldehyde-3-phosphate dehydrogenase gene of Edwardsiella tarda) with the purpose to maximize the protective efficacy of the vaccine against E. tarda infection. After construction of the vaccine a pilot study was orchestrated in vitro to ascertain the positive co-expression of the dual genes in vaccine-transfected SSN-1 cell line. Successful co-expression ofGAPDH andIFN-γ genesin the transfected cellwere confirmed byWestern blot and RT-PCR respectively. Further, an in vivo vaccine trial was conducted in which rohu (L. rohita)fingerlings were intramuscularly (I/M) injected (initial and booster immunised) with two DNA constructs one group with pGPD+IFN and the other with pGPD (containing GAPDH gene only) and challenged with E. tarda (1×105CFU/fish) at 35day post-initial vaccination. The protective immune responses were determined in terms of relative percentage survival (RPS), specific antibody production, non-specific immune response and expression kinetics of immune-related iNOS gene. Evaluation of RPS analysis revealed that pGPD+IFN group recordedhighestRPSof63.16%whilethepGPDvaccinatedgroupshowed47.37%whencomparedwith63.33% cumulativemortalityofcontrolgroup.Theresultsregardingrespiratoryburstactivity, myeloperoxidaseactivity as well as antibody titre also showed pGPD+IFN group with highest activities at all the time points. Furthermore,thecurrentstudydisplayedpGPD+IFNgrouphavingsignificant(p < 0.05)upregulationofiNOS gene transcript at 24–48h post-immunization (both initial and booster dose) as well as after challenge. Thus, from this study, we can conclude that the bicistronic vaccine can be an effective immunization strategy in orchestrating a coordinative immune response against E. tarda in L. rohita.