Formation And Oral Immunisation Of Biomaterial Microparticles Containing A Plasmid Vaccine Against Lymphocystis Disease Virus In Japanese Flounder (Paralichthys Olivaceus)

Lymphocystis disease, caused by lymphocystis disease virus (LCDV), is a kind of infectious disease of many fish. The pathogen was suggested as an iridovirus based on histopathological and morphological evidences. Outbreaks of lymphocystis disease with high mortalities have been reported in cultured Japanese flounder in Northern culture of China and caused significant economic losses in some marine net-cage farms. Therefore, in order to control LCDV, it is essential to find a vaccine and vaccinate fish by optimal approach.DNA vaccines are plasmid DNAs encoding specific proteins that can be expressed in cells of an inoculated host. The plasmids used are eukaryotic expression vectors, which contain the necessary elements for expression in eukaryotic cells. DNA vaccines are considered to be a promising form of vaccine for the control of infectious diseases and cancers since they offer many advantages over other conventional vaccines such as peptide or attenuated live pathogens. For instance, DNA vaccines are relatively safe and can be administered repeatedly without adverse effects. In addition, DNA vaccines are comparatively easy to produce on a large scale and are able to yield products with high purity and stability. Most importantly, effective DNA vaccine delivery systems, such as direct intradermal administration of DNA vaccines via gene gun to professional antigen presenting cells (APCs), have been well established. Experts in the research field now believe that the first step in the success of nucleic acids as a biopharmaceutical drug could come in terms of genetic immunization (DNA vaccines) which only needs transient expression of the encoding protein unlike long term expression in gene therapy. Plasmid DNA encoding for a variety of antigens (proteins) have been demonstrated in animal models of infectious diseases, cancers and allergies.Nucleic acid-based immunotherapy is a new treatment option for many complex disorders including a variety of infectious diseases and cancers. More importantly, DNA-based vaccines could have increased advantages over recombinant proteins and peptides in treatments of viral and parasitic infectious diseases as well as cancers. The reproducible and robust clinical efficacy of naked pDNA in fish trials has been demonstrated by intramuscular, intravenous, or intradermal injection. However, for fry in the aquaculture industry, injection approach is inappropriate because of the difficulty in operation. In addition, injection may harm the skin of fish, and bacterium and/or parasite would infect fish and cause higher mortality rate. The lower efficacy of orally administered pDNAs may be attributable to the inactivation of pDNA caused by the nucleases, lipases and peptidases present in the gastrointestinal tract. The pDNA encapsulated in microparticles can be protected from degradation in the gut, and a controlled release of pDNA could be achieved. In order to overcome the problems described above, three kinds of microparticles, including alginate microspheres loaded with DNA vaccine, PLGA microcapsules loaded with DNA vaccine and chitosan microspheres loaded with DNA, were prepared to vaccinate Japanese flounder.Oral alginate microcapsules loaded with pDNA against fish lymphocystis disease viruses (LCDV) were prepared with a modified oil containing water (W/O) emulsification method. Yield, loading percent and encapsulation efficiency of alginate microcapsules were 90.5%, 1.8% and 92.7%, respectively. Alginate microspheres prepared had size diameters of less than 10μm, and their shape was spherical. As compared to sodium alginate, a remarkable increase of DNA-phosphodiester and DNA-phosphomonoester bonds was observed for alginate microcapsules loaded with pDNA by Fourier transform infrared (FTIR) spectroscopic analysis. Agarose gel electrophoresis showed a little supercoiled pDNA was transformed to open circular and linear pDNA during encapsulation. The cumulative release of pDNA in alginate microcapsules was≤10% in pH 2.0 acidic media, and it was less than 6.5% in pH 9.0 alkaline media in 12 h. RT-PCR and immunofluorescence image indicated that pDNA expressed RNA and fluorescence protein in tissues of fish 10–90 days after oral administration. An indirect enzyme-linked immunosorbent assay (ELISA) showed that immune response of sera was positive (O.D≥0.3) from week 3 to week 16 for fish vaccinated with alginate microcapsules loaded with pDNA, in comparison with fish vaccinated with naked pDNA.Poly(DL-lactide-co-glycolide) (PLGA) microcapsules, loaded with plasmid DNA (pDNA) against lymphocystis disease virus (LCDV), were prepared by modified water in oil in water (W/O/W) double emulsion method in our laboratory. Encapsulation efficiency, loading percent and diameter of microcapsules were 78-88%, 0.5-0.7% and less than 10μm, respectively. In simulated gastric fluid (SGF), less than 10% of pDNA was released from microcapsules in 12 h, and about 6.5% of pDNA was released in 12 h in simulated intestinal fluid (SIF). The content of the supercoiled of pDNA in microcapsules and control was 80% and 89% respectively, which indicated that a little supercoiled pDNA degradation occurred during encapsulation. RT-PCR showed that lots of RNA containing information of MCP gene existed in all tissues of fish vaccinated with microcapsules 10-90 days after oral administration. Immunofluorescence images displayed that major capsid protein (MCP) of LCDV was expressed in tissues of fish vaccinated with pDNA-loaded microcapsules. In addition, indirect enzyme-linked immunosorbent assay (ELISA) showed that the immune responses of sera were positive (O.D≥0.3) from week 1 to week 24 for fish vaccinated with microcapsules, in comparison with fish vaccinated with naked pDNA.Chitosan microcapsules containing a plasmid vaccine (pDNA) against lymphocystis disease virus were prepared through an emulsion-based methodology. The yield, drug loading and encapsulation efficiency of microcapsules were 93.6%, 0.3% and 94.5%, respectively. Scanning electron microscopy (SEM) showed that pDNA-loaded microcapsules yielded a spherical shape with smooth surfaces. The disproportion of supercoiled to open circle and linear pDNA suggested that high transfection efficiencies of pDNA in microcapsules were retained. In simulated gastrointestinal tract environment, the release rate of pDNA encapsulated in acidic media was faster than that in alkaline media. The release profile at PBS buffer (PH 7.4) displayed that pDNA-loaded chitosan microcapsules had a sustained release up to 42 days after intestinal imbibition. In addition, the results of RT-PCR and immunofluorescence images indicated that the pDNA expressed antigen protein in tissues of fish 10-90 days after oral administration.Our results suggested that pDNA-loaded alginate microcapsules and PLGA microcapsules as well as chitosan microcapsules were promising carriers for pDNA vaccine. The three pDNA-loaded microcapsules have potential for drug delivery applications due to their ease of operation, low cost and notable immunisation efficacy.