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Grass carp (Ctenopharyngodon idella) is subject to a hemorrhagic disease caused by grass carp reovirus (GCRV), which can lead to mass mortality in grass carp culture, causing significant economic loss. Vaccination is the most promising strategy for the prevention of infectious diseases. Immersion vaccination is considered the most effective disease prevention method for juvenile fish because it can be implemented on many fish at once and administered without causing stress. However, immune responses by immersion vaccination are markedly less robust due to the skin barrier and insufficient antigen uptake. The display of heterologous proteins on the cell surface has been explored as a delivery system for viral antigens in veterinary and human vaccine studies. To improve the efficacy of the immersion vaccine, the major capsid protein (VP7) of GCRV was co-displayed with Aeromonas hydrophila outer membrane protein a (OmpA) and major adhesion protein (Mah) on the outer membrane surface of nonpathogenic Escherichia coli BL21 using the anchoring motif of ice-nucleation protein (Inp). The immune responses and protection efficiency against GCRV infection via both the injection and immersion routes were evaluated. The results indicated that the activities of anti-oxidant enzymes (ACP, AKP, SOD and T-AOC), as well as the expression of immune-related genes (TNF-α , IL-1β , MHCI and IgM) and specific VP7 antibody levels, were strongly increased in the grass carp from 7 to 21 days post-injection inoculation in a dose dependent manner. The cumulative mortality rates of injection-vaccinated groups were much lower than those of the control group after the GCRV challenge, and the relative percent survival (RPS) was greater than 80 %. Vitally, the surface co-display of vp7-Mah protein conferred marked protection to grass carp against GCRV infection after immersion administration (RPS >50 %); this was consistent with the production of high level of specific serum antibodies, non-specific immune responses, and the expression of immune-related genes. Moreover, the invasion analysis further showed that surface co-display of the vp7-Mah protein indeed significantly improved the invasion of E. coli BL21 (DE3) in vitro. Altogether, this study demonstrated that surface display GCRV core antigen vaccine system accompanied by invasion component from aquatic pathogenic microorganism is an effective prophylactic against GCRV viral diseases via the immersion administration approach. • A recombinant surface display vaccine prevented grass carp hemorrhagic disease. • Grass carp immersed in a vaccine with surface display of vp7-Mah elicited robust immunity against GCRV. • Surface display of the vp7-Mah improved the invasion of immersed vaccine. • Surface display-based immersion vaccines have prospects for application in aquaculture. [ABSTRACT FROM AUTHOR]

Infection with Grass carp reovirus (GCRV) is becoming unprecedentedly widespread in grass carp ( Ctenopharyngodon idella ) aquaculture industry, yet the management of GCRV infection still remains a challenge. Therefore, it is of importance to develop effective means against GCRV. As a delivery system of viral antigens, surface displaying of heterologous proteins on bacteria using anchoring motifs has successfully been implemented in human and veterinary vaccines research. In this study, a novel vaccine (BL21/InpN/vp7) was developed based on surface displaying a major capsid protein (vp7) of GCRV using the anchoring motif of N-terminal unique domain of ice-nucleation protein (InpN) on Escherichia coli BL21 (DE3) vaccine. Then the grass carp were immunized by surface displaying BL21/InpN/vp7 vaccine against GCRV using both intraperitoneal injection and bath immunization and their immune responses were tested. The results revealed that some non-specific immune parameters (acid phosphatase (ACP), alkaline phosphatase (AKP) and total antioxidant capacity (T-AOC)) were strongly increased in grass carp post injection inoculation (vp7 dose ranged from 10 to 20 μg). The specific antibody levels against GCRV and the transcriptional of immune-related genes ( TNF-α , IL-1β , MHCI and IgM ) were also significantly enhanced in grass carp by injection inoculation (vp7 dose ranged from 5 to 20 μg). On the other hand, only the highest dose of bath vaccination significantly induced the production of specific antibody and up-regulated transcriptions of several immune-related genes ( IgM and MHCI ) in grass carp. The lower cumulative mortality of grass carp in vaccinated groups after GCRV challenge clearly demonstrated that surface displayed vp7 vaccine could protect fish against GCRV infection. The relative percentage survival (RPS) value in injection vaccinated group (88.89%) was much higher compared to bath group (18.89%), which was in consistent with the production of specific serum antibodies, non-specific immune response and immune related genes expression. To sum up, our results indicated the surface display of heterologous antigenic proteins on E. coli BL21 (DE3) using the anchoring motif of ice-nucleation protein may provide a promising approach to the vaccine development of aquatic animals and suggested its potential to be used as vaccine to fight against GCRV infection. [ABSTRACT FROM AUTHOR]

Grass carp reovirus (GCRV) is one of the most pathogenic aquareovirus and can cause lethal hemorrhagic disease in grass carp ( Ctenopharyngodon idella ). However, management of GCRV infection remains a challenge. Therefore, it is necessary to find effective means for the control of its infection. The uses of bacterial ghost (BG, non-living bacteria) as carriers for DNA delivery have received considerable attentions in veterinary and human vaccines studies. Nevertheless, there is still no report about intramuscular administration of bacterial ghost-based DNA vaccines in fish. In the current study, a novel vaccine based on Escherichia coli DH5α bacterial ghost (DH5α-BG), delivering a major capsid protein gene ( vp 7) of grass carp reovirus encoded DNA vaccine was developed to enhance the efficacy of a vp7 DNA vaccine against GCRV in grass carp. The grass carp was injected intramuscularly by different treatments -i) naked pcDNA- vp 7 (containing plasmid 1, 2.5 and 5 μg, respectively), ii) DH5α-BG/pcDNA- vp 7 (containing plasmid 1, 2.5 and 5 μg, respectively) and iii) naked pcDNA, DH5α-BG or phosphate buffered saline. The immune responses and disease resistance of grass carp were assessed in different groups, and results indicated that the antibody levels, serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, acid phosphatase (ACP) activity and alkaline phosphatase (AKP) activity and immune-related genes were significantly enhanced in fish immunized with DH5α-BG/pcDNA- vp 7 vaccine (DNA dose ranged from 2.5 to 5 μg). In addition, the relative percentage survival were significantly enhanced in fish immunized with DH5α-BG/pcDNA- vp 7 vaccine and the relative percentage survival reached to 90% in DH5α-BG/pcDNA- vp 7 group than that of naked pcDNA- vp 7 (42.22%) at the highest DNA dose (5 μg) after 14 days of post infection. Moreover, the level of pcDNA- vp 7 plasmid was higher in DH5α-BG/pcDNA- vp 7 groups than naked pcDNA- vp 7 groups in muscle and kidneys tissues after 21 days. Overall, those results suggested that DH5α bacterial ghost based DNA vaccine might be used as a promising vaccine for aquatic animals to fight against GCRV infection. [ABSTRACT FROM AUTHOR]

The uses of single walled carbon nanotubes (SWCNTs) as carriers for DNA delivery have received considerable attention in cell studies. DNA vaccination of fish has been shown to elicit durable transgene expression, but no reports exist on intramuscular administration of SWCNTs-DNA vaccine electrostatic complexes which prepared through non-covalent conjugation. In this study, we injected grass carp intramuscularly with a plasmid vector containing a major capsid protein gene ( vp7 ) of grass carp reovirus as a) naked pcDNA- vp7 , b) SWCNTs-pcDNA- vp7 , c) empty plasmid vector, or phosphate buffered saline. After intramuscular administration, the ability of the different immune treatments to induce transgene expression was analyzed. The results indicated that higher levels of transcription and expression of the vp7 gene could be detected in muscle tissues of grass carp 28 days intramuscular injection in SWCNTs-pcDNA- vp7 treatment groups compare with naked pcDNA- vp7 treatment groups. Moreover, the serum respiratory burst activity, complement activity, lysozyme activity, superoxide dismutase activity, immune-related genes, antibody levels and relative percentage survival were significantly enhanced in fish immunized with SWCNTs-pcDNA- vp7 vaccine. The data in this study suggested that SWCNTs were promising carriers for plasmid DNA vaccine and might be used to vaccinate fish by intramuscular approach. [ABSTRACT FROM AUTHOR]

To reduce the economic losses caused by diseases in aquaculture industry, more efficient and economic prophylactic measures should be urgently investigated. In this research, the effects of a novel functionalized single-walled carbon nanotubes (SWCNTs) applied as a delivery vehicle for recombinant Aeromonas hydrophila vaccine administration via bath or injection in juvenile grass carp were studied. The results showed that SWCNT as a vector for the recombinant protein aerA, augmented the production of specific antibodies, apparently stimulated the induction of immune-related genes, and induced higher level of survival rate compared with free aerA subunit vaccine. Furthermore, we compared the routes of bath and intramuscular injection immunization by SWCNTs-aerA vaccine, and found that similar antibody levels induced by SWCNTs-aerA were observed in both immunization routes. Meanwhile, a similar relative percentage survival (approximately 80%) was found in both a 40 mg/L bath immunization group, and a 20 μg injection group. The results indicate that functionalized SWCNTs could be a promising delivery vehicle to potentiate the immune response of recombinant vaccines, and might be used to vaccinate juvenile fish by bath administration method. [ABSTRACT FROM AUTHOR]

Grass carp reovirus (GCRV), the most pathogenic aquareovirus, can cause fatal hemorrhagic disease in fingerling and yearling grass carp. Vaccination by injection is by far the most effective method of combating disease. However it is labor intensive, costly and not feasible to vaccinate large numbers of the fish. Thus, an efficient and economic strategy for the prevention of GCRV infection becomes urgent. Here, functionalized single-walled carbon nanotubes (SWCNTs) as carrier were used to manufacture SWCNTs-VP7 subunit vaccine with chemical modification. Different developmental stages of grass carps were immunized by VP7/SWCNTs-VP7 subunit vaccine against GCRV by intramuscular injection and bath immunization. The results indicate that better immune responses of grass carp immunized with the SWCNTs-VP7 subunit vaccine were induced in comparison with VP7 subunit vaccine alone. Immunization doses/concentrations are significantly reduced (about 5–8 times) to prevent GCRV infection in different developmental stages of grass carp with injection or bath treatment when SWCNTs carrier was used. A good immune protective effect (relative percentage survival greater than 95%) is observed in smaller size fish (0.2 g) with SWCNTs-VP7 bath immunization. In addition, serum respiratory burst activity, complement activity, lysozyme activity, superoxide dismutase activity, alkaline phosphatase activity, immune-related genes and antibody levels were significantly enhanced in fish immunized with vaccine. This study suggested that functionalized SWCNTs was the promising carrier for recombinant subunit vaccine and might be used to vaccinate fish by bath approach. [ABSTRACT FROM AUTHOR]

Hemorrhagic disease caused by grass carp reovirus (GCRV) is considered to be one of the most serious threats to grass carp, resulting in significant economic loss in aquaculture industry. Currently, vaccination is the most effective method to against GCRV infection and improve the production of grass carp. In the study, the antigen epitope of GCRV-II was screened, and its protective effects on grass carp against GCRV-II infection using carbon nanotubes as a carrier were assessment. The vp4 gene of GCRV-II was divided into four segments and respectively expressed in Escherichia coli. The potential antigen epitope was screened by the enzyme linked immunosorbent assay (ELISA). Afterwards, the VP4 and VP4-3 were conjugated with single-walled carbon nanotubes (SWCNTs) to vaccinate grass carp, and the protective effects were assessed by checking immune related parameters (specific serum antibodies, enzyme activities and immune related genes). The results indicate that better immune responses of grass carp immunized with the SWCNTs-VP4 subunit vaccine were induced in comparison with naked VP4 subunit vaccine. Besides, compared to the SWCNTs-VP4 groups, the immune related parameters showed higher levels in SWCNTs-VP4-3 treatment groups. The survival of grass carp treated with SWCNTs-VP4-3 was 76.7% during 10 days post infection, while that were 58.3% and 0% for the SWCNTs-VP4 and control groups, respectively. These results indicated that SWCNTs-VP4-3 can be a plausible candidate for preventing and controlling GCRV-II among grass carp. • Grass carp immersed with SWCNTs-VP4 and SWCNTs-VP4-3 confers strong immunity against GCRV-II. • SWCNTs as a promising vehicle can enhance immune effect of subunit vaccine. • The antigen epitope of GCRV-II was screened. [ABSTRACT FROM AUTHOR]