| Gibel carp(C.auratus gibelio),an important cultured freshwater fish,has great market value and development potential worldwide,especially in China,but its production is often affected by diseases.Cyprinid herpesvirus 2(Cy HV-2)outbreaks worldwide,and its high infection rate and high mortality rate have brought significant economic losses to the aquaculture industry.At present,effective drugs against Cy HV-2 infection have not yet been identified,and therefore there is a pressing need to develop effective prevention and treatment strategies.Vaccination has been shown to be the most effective and environmentally friendly countermeasure against viral infection.Most of the fishery vaccines approved for marketing in China are vaccinated by injection,which not only requires a large number of the experienced labor force but also has a high cost and will cause stress stimulation to fish.Therefore,it is urgent to develop a vaccine with high relative percent survival(RPS)that is easy to vaccinate.In this study: 1)Developed an oral vaccine against Cy HV-2 by using Saccharomyces cerevisiae cell surface display technology;2)Detected the immune response of gibel carp after oral yeast vaccination;3)Evaluated d the protective effect of the vaccine in gibel carp.The research details are as follows:1.Preparation of S.cerevisiae surface display vaccinesBased on the reported genomic sequence of Cy HV-2,we selected ORF25 analytically as the antigen,then the obtained fragment was inserted into p YD1.After PCR verification,a surface display vaccine of S.cerevisiae EBY100/p YD1-ORF25 was constructed to analyze its expression efficiency by immunofluorescence.The results show that ORF25 can express stably and efficiently on the yeast surface.2.Safety testing of yeast vaccine and detection of its induced immune responseImmunizations were subsequently administered to gibel carps,300 gibel carp were randomly divided into three groups(control,vaccine,and booster vaccine)to test the safety and immunogenicity of the vaccine.The results showed that gibel carps survived for 30 days after the vaccine.Histological analysis of intestinal sections showed that no pathological changes and no significant difference in the aspect ratio of intestinal folds.After immunization,the amount of innate immune enzymes,such as complement 3(C3),lysozyme(LZM),and total superoxide dismutase(TSOD),increased in the intestine mucus and serum,and the recombinant yeast vaccine could effectively upregulate antiviral and immune-related genes(mx1、lgp2、ifn-γ2、stat2、irf3、irf7、tnf-α、il-1β、il-2、il-4、il-10)in foregut,midgut,hindgut,spleen,and head and kidney,especially immunoglobulin genes(igt,igm,and igd),were significantly higher in both intestinal and systemic tissues of the booster vaccine group than in the vaccine group,and after the booster,these genes were rapidly upregulated in the short term.The above results indicated that this recombinant yeast oral vaccine was safe and well immunogenic,which could effectively induce innate and adaptive immune responses in the systemic tissues and intestinal mucosa,and the effect of the booster vaccination was better than that of single vaccination.3.Detection of yeast vaccine protective capacityTo explore the protective efficacy of the recombinant yeast vaccine,we constructed an infection model of gibel carp.Basis on the detection of the viral loads and intestinal pathological changes after Cy HV-2 infection in three groups,the results indicated that the vaccine could enhance the antiviral capacity of gibel carp by inhibiting viral replication,alleviating intestinal damage,protecting the intestinal villus morphology,and increasing the number of goblet cells.The calculated relative protection after a single vaccination with this recombinant yeast vaccine was 17%,and it was as high as 66.7% after secondary vaccination.In summary,this study is the first time to produce a safe oral S.cerevisiae vaccine by the surface display of the Cy HV-2 ORF25,and proved that the vaccine had good immunogenicity and protective efficacy.These results may provide new research thoughts and strategies for controlling Cy HV-2 in gibel carp and lay a foundation for the innovations of therapeutic vaccines against Cy HV-2. |
