Construction And Evaluation On Immune Effect Of Biomimetic Cell-derived Vaccine Delivery System Against Spring Viremia Of Carp

Spring viremia of carp(SVC)is a highly infectious and lethal aquatic animal disease,which can cause massive and explosive death of cyprinid fishes.Spring viremia of carp virus(SVCV)has been detected in many areas of China in recent years.How to prevent SVC is an urgent problem to be solved.Fishery vaccines plays an irreplaceable role in the prevention and control of aquatic diseases.However,the development of SVC vaccine is still in the laboratory stage.With the in-depth study on the mechanism of vaccine immunity,the design and development of multi-component vaccine composed of antigens,immune stimulants and delivery systems has become a hot direction of vaccine research.Biomimetic nanomaterials and target factors can be used to construct efficient and safe vaccine delivery systems.To solve these problems,we constructed a targeted bionic nanovaccine system for effective protection and efficient delivery of SVCV antigenic substances by wrapping chitosan-DNA nanoparticles in red blood cell membrane(RBCM)and embellishing them with mannose,and tested the synergistic mechanism of the vaccine system in immune response through in vitro and in vivo experiments.1.Construction of targeted red blood cell membrane coated chitosan-borne DNA vaccine systemAfter the expanded culture of engineering bacteria,a plasmid pc DNA3.1-G was prepared by using the plasmid carrier,and the electrostatic adsorption between chitosan and plasmid was used to form spherical nanoparticles CS-G,and the zebrafish RBCM was combined with CS-G to generate RBCM coated chitosan-borne nanoparticles CS-G@M by physical extrusion method.Next,the biomimetic nanoparticles were functionalized with mannose.Then a targeted RBCM coated chitosan-borne DNA vaccine system CS-G@M-M was constructed.Subsequently,the characterization of the vaccine system was analyzed using transmission electron microscopy,BCA kit and dynamic light scattering(DLS).The results showed that the targeted RBCM coated chitosan-borne DNA vaccine system had been successfully constructed.The particle size of CS-G@M-M was about 204 nm.CS-G@M-M has a core-shell structure and has good stability.2.Safety evaluation of targeted bionic nanovaccine systemEpithelioma papulosum Cyprinid cell line(EPC)and carp macrophages were incubated with the vaccine systems.Meanwhile,zebrafish was immunized by intraperitoneal injection.The comprehensive safety evaluation of the vaccine system was carried out by cell viability detection,tissue section observation and growth performance detection.Our research showed that after the macrophages and EPC cells were treated in the vaccine system with a concentration not higher than 20 μg/m L for 24 h,the cell viability of both cells could reach over 90% at the cellular level.At the individual level,zebrafish were immunized with 4 μg/g vaccine systems.Within 60 days,the immunized zebrafish showed no abnormal behavior compared with untreated zebrafish,and the main tissues and organs of the fish such as brain,intestine,spleen,liver and kidney showed no abnormality or injury.The growth and development of zebrafish were not affected by intraperitoneal injection.It was confirmed that our vaccine system had high biocompatibility in a certain concentration range,and would not cause obvious toxic and side effects on fish cells and individuals.3.Study on immune effect of targeted bionic nanovaccine systemAfter intraperitoneal injection of zebrafish,the immune effect of the vaccine system was evaluated by enzyme-linked immunoadsorption(ELISA)and fluorescence quantitative PCR combined with SVCV challenge test.The results showed that after immunization for the same time,compared with the zebrafish treated with no vaccine and the same dose of CSG@M,CS-G@M-M could significantly increase the level of antibodies and immune-related physiological indexes in serum of immunized zebrafish,and significantly enhance the m RNA expression level of immune-related genes;After SVCV challenged zebrafish at different time of immunization,the relative protection rates of CS-G@M group at 3 d,21 d and 70 d were 38.7%,49.3% and 34.7%,respectively,while those of CS-G@M-M group were 63.3%,85.3% and 51.3%,respectively.The results showed that CS-G@M-M vaccine system has a long-term and highly effective preventive effect on SVCV.4.Study on the mechanism of immune synergismAfter the vaccine systems were labeled with lip-philic fluorescent dye Di D,immunofluorescence,in vivo imaging and ELISA techniques were used to study the delivery,distribution and metabolism of the targeted bionic vaccine system in antigen presenting cells and immune tissues in fish.The results showed that the uptake of CS-G@M-M by carp macrophages was apparently higher than that of CS-G@M and the control group at the cellular level.At individual level,the intake of CS-G@M-M in kidney,spleen and hindgut tissues of zebrafish was also obviously higher than that of CS-G@M and control group.In addition,CS-G@M and CS-G@M-M can be detected in spleen,kidney,hindgut and other immune organs 6 h after immunization,and CS-G@M-M and CS-G@M have a longer duration of action in fish.At the same time,the targeted bionic nanovaccine system can significantly stimulate the activation of antigen presenting cells and induce mucosal immune response and systemic immune response.The results showed that the targeted bionic nanovaccine system could enhance the immune effect of the vaccine by enhancing the presentation of antigen presenting cells and prolonging the duration of the vaccine.In summary,this article builds a targeted RBCM coated chitosan-borne DNA vaccine system(CS-G@M-M)with good stability and biocompatibility,its synergistic mechanism is through extending vaccine immune function duration,increasing vaccine on efficiency of antigen presenting cells,and producing strong mucosal immune and systemic immune responses,so as to realize the efficient prevention and treatment of SVC.The research results can provide high efficiency,low cost and easy to popularize fishery vaccines for the industry,and also provide theoretical support for the research and application of other aquatic animal vaccines,which is of great value to the healthy and sustainable development of fishery and the green and safe production of aquatic products.