scholarly journals Oral Vaccination of Grass Carp (Ctenopharyngodon idella) with Baculovirus-Expressed Grass Carp Reovirus (GCRV) Proteins Induces Protective Immunity against GCRV Infection

Vaccines ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 41
Author(s):  
Changyong Mu ◽  
Qiwang Zhong ◽  
Yan Meng ◽  
Yong Zhou ◽  
Nan Jiang ◽  
...  

The grass carp reovirus (GCRV) causes severe hemorrhagic disease with high mortality and leads to serious economic losses in the grass carp (Ctenopharyngodon idella) industry in China. Oral vaccine has been proven to be an effective method to provide protection against fish viruses. In this study, a recombinant baculovirus BmNPV-VP35-VP4 was generated to express VP35 and VP4 proteins from GCRV type Ⅱ via Bac-to-Bac baculovirus expression system. The expression of recombinant VP35-VP4 protein (rVP35-VP4) in Bombyx mori embryo cells (BmE) and silkworm pupae was confirmed by Western blotting and immunofluorescence assay (IFA) after infection with BmNPV-VP35-VP4. To vaccinate the grass carp by oral route, the silkworm pupae expressing the rVP35-VP4 proteins were converted into a powder after freeze-drying, added to artificial feed at 5% and fed to grass carp (18 ± 1.5 g) for six weeks, and the immune response and protective efficacy in grass carp after oral vaccination trial was thoroughly investigated. This included blood cell counting and classification, serum antibody titer detection, immune-related gene expression and the relative percent survival rate in immunized grass carp. The results of blood cell counts show that the number of white blood cells in the peripheral blood of immunized grass carp increased significantly from 14 to 28 days post-immunization (dpi). The differential leukocyte count of neutrophils and monocytes were significantly higher than those in the control group at 14 dpi. Additionally, the number of lymphocytes increased significantly and reached a peak at 28 dpi. The serum antibody levels were significantly increased at Day 14 and continued until 42 days post-vaccination. The mRNA expression levels of immune-related genes (IFN-1, TLR22, IL-1β, MHC I, Mx and IgM) were significantly upregulated in liver, spleen, kidney and hindgut after immunization. Four weeks post-immunization, fish were challenged with virulent GCRV by intraperitoneal injection. The results of this challenge study show that orally immunized group exhibited a survival rate of 60% and relative percent survival (RPS) of 56%, whereas the control group had a survival rate of 13% and RPS of 4%. Taken together, our results demonstrate that the silkworm pupae powder containing baculovirus-expressed VP35-VP4 proteins could induce both non-specific and specific immune responses and protect grass carp against GCRV infection, suggesting it could be used as an oral vaccine.

Genes ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1351
Author(s):  
Rui Sun ◽  
Miao Zhang ◽  
Hui Chen ◽  
Yao Wei ◽  
Degang Ning

Oral vaccination is a practical method for the active immunization of farmed fish in the matter of animal welfare and handling costs. However, it always shows insufficient protective immunity, mainly due to antigen degradation in the gastrointestinal tract (GIT). Bacillus subtilis spores have been shown to be able to protect surface-display heterologous antigens against degradation. Neverthless, the spores can germinate in GIT, which causes loss of the antigens with spore coat disassembly. Here, we developed a novel surface display system using the B. subtilis spore coat proteins CotB and CotC as anchors for the heterogenous antigen, and the germination-controlling genes cwlJ and sleB as the ectopic integration sites for the fusion genes. Using this display system, we engineered germination-arrest spores displaying the model antigen Vp7 of grass carp reovirus (GCRV) on their surface. Oral vaccination of the engineered spores could confer immune protection against GCRV in grass carp (Ctenopharyngodon idella) via eliciting adaptive humoral and cellular immune responses. Most importantly, the germination-arrest spores were shown to significantly augment immunogenicity and protection above the engineered spores based on the existing surface display system. Therefore, the presently reported antigen expression strategy opens new and promising avenues for developing oral vaccines for the immunization of farmed fish species.


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