Bivalent DNA vaccine induces significant immune responses against infectious hematopoietic necrosis virus and infectious pancreatic necrosis virus in rainbow trout

Abstract

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens of salmon and trout. An active bivalent DNA vaccine was constructed with the glycoprotein gene of Chinese IHNV isolate Sn1203 and VP2-VP3 gene of Chinese IPNV isolate ChRtm213. Rainbow trout (5 g) were vaccinated by intramuscular injection with 1.0 µg of the bivalent DNA vaccine and then challenged with an intraperitoneal injection of IHNV, IPNV, or both, at 30 and 60 days post-vaccination (d.p.v.). High protection rates against IHNV were observed, with 6% and 10% cumulative mortality, respectively, compared with 90-94% in the mock-vaccinated groups. IPNV loads (531-fold and 135-fold, respectively) were significantly reduced in the anterior kidneys of the vaccinated trout. Significant protection against co-infection with IHNV and IPNV was observed, with cumulative mortality rates of 6.67% and 3.33%, respectively, compared with 50.0% and 43.3%, respectively, in the mock-vaccinated groups. No detectable infective IHNV or IPNV was recovered from vaccinated trout co-infected with IHNV and IPNV. The bivalent DNA vaccine increased the expression of Mx-1 and IFN-γ at 4, 7, and 15 d.p.v, and IgM at 21 d.p.v., and induced high titres (≥160) of IHNV and IPNV neutralizing antibodies at 30 and 60 d.p.v.

Figure 1

In vitro and in vivo expression of both antigen genes from the pCh-IHN/IPN DNA vaccine. An immunofluorescence antibody test confirmed the expression of both antigen genes in vitro (a). EPC cells transfected with pCh-IHN/IPN were incubated with an IHNV-glycoprotein-specific rabbit polyclonal antibody and a Cy3-conjugated goat anti-rabbit-IgG secondary antibody or a mouse anti-IPNV-VP2 polyclonal antibody and a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse-IgG antibody. EPC cells transfected with the pcDNA3.1 vector and treated identically were used as the negative control. Western blotting of muscle samples from vaccinated rainbow trout (n = 5), collected at 3, 7, and 15 days post-vaccination, detected the expression of both antigen genes in vivo (b). Muscle samples from pcDNA3.1-mock-vaccinated trout were used as the negative controls. β-Actin was used as the reference protein.

Figure 2

Cumulative percentage mortality (CPM) curves for pCh-IHN/IPN-vaccinated rainbow trout challenged with IHNV strain Sn1203 at 30 or 60 d.p.v. Rainbow trout injected with plasmid pcDNA3.1 (vector) were used as the negative controls. Duplicate groups of 30 fish were challenged with an intraperitoneal injection of 10² plaque-forming units of IHNV Sn-1203 per fish. No mortality was observed in the sham-infected control group, and no significant differences were observed in mortality between any replicates within any treatment group.

Figure 3

Quantitative reverse transcription–PCR determination of IPNV load using VP1 gene expression in the anterior kidneys of vaccinated rainbow trout (n = 5) challenged with IPNV strain ChRtm213 at 30 or 60 d.p.v. IPNV loads were measured at 15 days post challenge. Rainbow trout injected with plasmid pcDNA3.1 alone or with PBS were used as the negative controls. EF-α was used to normalize the expression of the IPNV VP1 gene. Individual VP1 gene expression levels (a,c) and average expression levels (b,d) are shown separately. Differences were analysed, and different symbols above the bars indicate significant differences (P < 0.05).

Figure 4

Evaluation of the protection afforded by the bivalent DNA vaccine against co-infection with IHNV and IPNV. Cumulative percentage mortality curves (a) and flow-cytometric quantification of IHNV and IPNV in the tissues of rainbow trout (n = 5) challenged with IHNV and IPNV at 30 and 60 d.p.v. (b,c) Dual protection afforded by the vaccine against co-infection. Q1: IPNV-infected cells; Q2: dual-infected cells; Q3: uninfected cells; Q4: IHNV-infected cells. The average proportions of virus-infected cells are shown in the histograms (d). Rainbow trout injected with plasmid pcDNA3.1 were used as the negative controls. *Significantly different (P < 0.05).

Figure 5

Fold changes in the expression of immune-related genes induced by the combined DNA vaccine in rainbow trout (n = 5). β-Actin was used to normalize the transcription of each gene in anterior kidney samples from rainbow trout at 1, 4, 7, 15, and 21 days post-vaccination (d.p.v.). The fold changes in their expression were calculated relative to their expression in the pcDNA3.1-vaccinated group. The differences were analysed, and different symbols above the bars indicate significant differences (P < 0.05).

Figure 6

Specificity of NAb serum for IHNV and IPNV. Virus-infected cells were incubated with NAb-containing serum (from vaccinated trout), a rabbit polyclonal antibody directed against rainbow trout IgM Fc, and a fluorescently labelled secondary antibody. Sera from trout vaccinated with pcDNA3.1 were used as the negative controls. NC: negative control.