Induction of a robust immunity response against novel duck reovirus in ducklings using a subunit vaccine of sigma C protein

Abstract

Novel duck reovirus (NDRV) disease emerged in China in 2011 and continues to cause high morbidity and about 5.0 to 50% mortality in ducklings. Currently there are no approved vaccines for the virus. This study aimed to assess the efficacy of a new vaccine created from the baculovirus and sigma C gene against NDRV. In this study, a recombinant baculovirus containing the sigma C gene was constructed, and the purified protein was used as a vaccine candidate in ducklings. The efficacy of sigma C vaccine was estimated according to humoral immune responses, cellular immune response and protection against NDRV challenge. The results showed that sigma C was highly expressed in Sf9 cells. Robust humoral and cellular immune responses were induced in all ducklings immunized with the recombinant sigma C protein. Moreover, 100% protection against lethal challenge with NDRV TH11 strain was observed. Summary, the recombinant sigma C protein could be utilized as a good candidate against NDRV infection.

Figure 1

Schematic representation of the structure of pFB-1-sigma C (A) and amino acid sequence alignment of the sigma C protein of NDRVs compared with those of ARVs and MDRVs. (B). Notes: pFB-1, pFastBac1 vector; P_PH, the polyhedron promoter of the baculovirus; Poly-A, polyadenylation signal.

Figure 2. Identification and purification of recombinant sigma C protein.

(A) Characterization of baculovirus infected cells. Sf9 cells were infected with the recombinant sigma C baculovirus (Bac-sigma C), wild-type baculovirus (Bac-wt), and blank control (Blank). The arrows indicate larger cell size formation in Sf9 cells infected with Bac-wt and Bac-sigma C. (B) Immunofluorescence assay (IFA) of Bac-sigma C infected cells. After 48 h post-infection, cells were analyzed by IFA using the mouse antiserum of sigma C as primary antibody and FITC-conjugated goat anti-mouse IgG antibody as secondary antibody (original magnification, 100x). (C) Western blot analysis of recombinant sigma C protein in Bac-sigma C infected cells using anti-sigma C serum (i) and anti-His monoclonal antibody (ii). Lane M, molecular weight markers; Lane 1: Sf9 cells infected with Bac-wt; Lane 2: Sf9 cells infected with Bac-sigma C. (D) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified sigma C fusion protein. Lane M, molecular weight markers; Lane 1: non-treated Sf9 cells; Lane 2: Sf9 cells infected with Bac-wt; Lane 3: Sf9 cells infected with Bac-sigma C; Lane 4: 2 μg purified sigma C protein.

Figure 3. Antibody response in ducklings immunized with recombinant sigma C protein.

(A) Time point of duckling experiments. (B) Sigma C-specific antibody levels detected by indirect enzyme-linked immunosorbent assay (ELISA) analysis. (C) Neutralization antibody levels detected by serum neutralization assay. Each data represents the mean ± SD. ^*p < 0.05, ^**P < 0.01 vs. PBS group. ^#p < 0.05 vs. NDRV-TH11 group.

Figure 4. Cellular immune response in ducklings immunized with sigma C.

(A) The dynamic changes of lymphocyte proliferation in immune response test (A₅₇₀ value). The lymphocyte proliferation response was measured on 2 weeks and 4 weeks after the first immunization. Each data represents the mean ± SD. ^**P < 0.01 vs. Bac-sigma C group stimulated with DMEM alone. ^##p < 0.01 vs. NDRV-TH11 group stimulated with DMEM alone. (B) The interferon gamma (IFN-γ) concentration in serum harvested from immunized ducklings was measured by enzyme-linked immunosorbent assay (ELISA). (C) The interleukin 4 (IL-4) concentration in serum harvested from immunized ducklings was measured by ELISA. Data are the mean ± SD. ^*P < 0.05, **p < 0.01 vs. PBS group.

Figure 5. Weight loss and survival rate in ducklings after lethal challenge.

(A) Weight change was calculated for 14 d after virus challenge and is presented as the mean ± SD percentage of the original body weight. (B) Survival curves after challenge. The statistical significance of differences in mortality between groups was determined using the Kaplan-Meier method, and analyzed with a Log-rank (Mantel-Cox) test. For Bac-sigma C vs. PBS, P < 0.05; for Bac-sigma C vs. Bac-wt, p < 0.05; and for Bac-wt vs. NDRV-TH11, p > 0.05.

Figure 6. Quantification of viral loads in different tissues of euthanized ducklings using real-time quantitative RT-PCR (qRT-PCR).

Samples were collected from ducklings challenged with NDRV-TH11 strain at 14 dpc, and their viral copies were then detected by qRT-PCR. (A) Viral loads in blood. (B)Viral loads in the spleen. (C) Viral loads in the liver. The dotted line marks the positive cut-off. The statistical significance of differences in the virus copies between vaccinated and control groups were determined using One-Way ANOVA and Tukey’s multiple comparison tests between groups. (**p < 0.01 vs. PBS group).