Study On Targeted Carbon Nanotubes-based Vaccine Delivery System Against Spring Viremia Of Carp

Spring viremia of carp(SVC)is an acute,contagious and lethal disease to cyprinids,caused huge economic loss in cyprinids farming industry.SVC is designated as a notifiable disease by the Office International des Epizooties(OIE).Vaccine has probably became an efficient way to resolve SVCV infection problem.So far,vaccine via intramuscular injection is considered as a prophylactic measure that exhibited good immunoprotective.However,it is labor intensive,costly and not feasible to vaccinate large numbers of the fish.Bath immunization is convenient and suitable for vaccinating fish in different scale.Therefore,bath immunization becomes an attractive option to prevent aquatic diseases.However,the efficacy of bath immunization is not ideal due to biological barriers.The study of efficient transport technology of nanocarrier vaccine is one of the safest and effective means to solve the high-efficiency immune protection of vaccines in the aquaculture industry.In this study,we constructed a targeted carbon nanotubes-base vaccine deliver system(SWCNTs-MG)which can recognize the signature receptor(mannose)of antigen-presenting cells(APCs).After fish vaccinated with SWCNTs-MG via intramuscular injection and bath administration,its immune effect on the prevention of SVC has been evaluated;the targeted immune mechanism of SWCNTs-MG has been measured by immunofluorescence techniques and in vivo imaging systems.Furthermore,the immune program was optimized in the aspects of immune dose,immersion time and immune density.The results obtained in this work were as follows:1.Construction of targeted carbon nanotubes based vaccine delivery systemRecombinant SVCV glycoprotein(G)was prepared by fermentation and purification techniques.Meantime,the structure modification and identification of CNTs and mannose were carried out.Then,a targeted carbon nanotubes-based vaccine delivery system(SWCNTs-MG)was constructed by chemical synthesis.After SWCNTs-MG was incubated with head kidney macrophages of carp,Epithelima Popuasum cuprini(EPC)cells and common carp,respectively,the safety of SWCNTs-MG was evaluated by cell viability assay,histological observation and so on.The results showed that targeted carbon nanotubes based vaccine delivery system were successfully constructed,the percentage content of G and mannose in SWCNTS-MG were 40.2% and 3.4%,respectively.After head kidney macrophages and EPC cells incubated with 40 ?g/mL SWCNTs-MG for 24 h,the survival rate of both kind of cells reached more than 80%.After common carp immersed with 60 mg/L SWCNTs-MG for 24 h,no damage or abnormality was found in fish brain,gill,intestine,kidney,liver and spleen,moreover,within 60 d after immersion immunization,there was no lesion or abnormality in vaccinated carp compared with control group.2.Evaluation of immune effect induced by targeted carbon nanotubes-based vaccine delivery systemCommon carp(1.0 ± 0.2 g)were vaccinated with G,MG,SWCNTs-G and SWCNTs-MG via bath immunization and intramuscular injection,respectively.Then the immune response were analyzed by enzyme-linked immunosorbent assay(ELISA),quantity real-time PCR(qRT-PCR)and virus challenge.The results showed that after fish vaccinated with SWCNTs-MG via bath or injection administration,its antibody levels were significantly elicited(P < 0.05);immune-related enzyme activities(complement C3,SOD,ACP,AKP)were significantly increased(P < 0.05);immune related genes expression(TNF-?,IL-10,Cxcr 1,Cxca,IFNg2 b,IgM,CD4 and MHC-?)were significantly increased(P < 0.05),when compared with control,G,MG and SWCNTs-MG at the same immunization dose/concentration.Moreover,after carps challenged with SVCV,fish immunized with naked G at the highest concentration(30 mg/L)or dose(12 ?g/fish)obtained the relative percentage survival(RPS)of 19.2% and 34.6%,respectively.Meanwhile,SWCNTs-MG immunized fish showed a much higher survival rate than the G vaccinated fish,which correspond to RPS of 63.5% and 73.1%,respectively.3.Study of targeted immune mechanism of vaccine delivery systemFluorescence labeling technology was used to label G,MG,SWCNTs-G and SWCNTs-MG with FITC,respectively.Targeted transport mechanism of the vaccine system in antigen-presenting cell and fish tissues will be measured by and immunofluorescence techniques and in vivo imaging systems.The results showed that: in cell levels,the uptake of SWCNTs-MG in cyprinoid head kidney macrophage were much higher than other vaccine formulations(G,MG and SWCNTs-G)(P < 0.01);in tissue levels,the intake of SWCNTs-MG in fish kidney and spleen were much higher than other vaccines(G,MG and SWCNTs-G)(P < 0.01).Furthermore,within 6 h immersed vaccination,SWCNTs-MG could cross into fish body and present to internal tissues through gill,surface and intestine.4.Study on the optimization of the immune schemeThe immune scheme was optimized from the vaccine concentrations(30,40,and 50 mg/L),immune density(8,24 and 48 fish/L)and immersion time(6,12,and 24 h).different immune program were evaluated by serum antibody titer assay and SVCV challenge.The results indicated that the highest protection rate(84.38%)was observed in the immune scheme of 40 mg/L,8 fish/L and 12 h;the lowest protection rate of 51.04% was observed in 50 mg/L SWCNTs-MG immersed with 48 fish/L for 6 h.Comprehensively,the optimal immune scheme(protection rate of 83.33%)is 30 mg/L SWCNTs-MG,24 fish/L and 12 h.In summary,the targeted carbon nanotubes-based vaccine delivery system was successfully constructed.With more vaccine enter into fish body,and stronger targeted presenting ability to APCs,targeted vaccine system could significantly enhance the immune effect in common carp against SVC.This study not only provide a theoretical basis and research template for the application of targeted nanovaccine system in aquatic animals,but also play an important role in supporting development of healthy aquaculture and ensuring the safety of aquatic products and ecology.