GEM Particle Vaccines For Porcine Circovirus Type2

Porcine circovirus2type (PCV2) is the primary etiologies for postweaning multi-systemic wasting syndrome (PMWS) and this virus could cause swine grow slowly and immune disorder, and further more, co-infection with other diseases, such as porcine multi-disease like dermatitis and nephropathy syndrome (PDNS), porcine reproductive disorders, and so on, which has caused a great lose to hog industry. Vaccination is considered the best method to prevent and control this disease. But the immune effect of domestic traditional inactivated vaccine is not very well for the reason of poor growth on PK-15cell and immune suppression for whole PCV2. So, it’s significant for us to develop safe, effective and low-cost PCV2subunit vaccine.GEM-PA (Gram positive enhancer matrix—protein anchor) display system is a novel surface display technology, and there was many articles has reported of using this system to develop human vaccines in other countries. GEM particles consisted with peptidoglycan of Lactococcal bacterial cell wall, that removed the macromolecule substance such as protein and nucleic acid of the host bacterial after treat with hot acid, could covalently combine with C-terminal domain (PA) of cytohydrolist specifically. When fused with PA, antigen can be loaded on the GEM particles in a high density. There are three distinctive superiority of this system for vaccine developing:Firstly, antigen can be purified with low speed centrifugation, consequently, vaccine can be produced rapidly and cheaply; secondly, GEM which is the agonist for Toll like receptor2can be used as immuneopotentiators to improve immune effect of the target antigen; Thirdly, GEM particles come from food grade lactobacillus, with no residual nucleic acid after treated hot acid, so, vaccine made with GEM particles without any bio-safety risk. 1. Preparation and identification for components of GEM-PA systemPreparation and identification of GEM particles. Collecting new culture of Lactococcal bacterial of MG1363in nutrient medium GM17, resuspended bacteria with0.1mol/L HCl after washed with PBS, and then boiled for30mins. Counting the GEM particles after washing,2.5×109GEM particles were set as1U. The particles were stored at-80℃with a density of10U/ml. Transmission Electron Microscope of the GEM particles showed that particles keep the original shape and size with a smooth surface and uniform empty internal, suggesting the macromolecule substance such as protein and nucleic acid of the host bacterial are all removed.Preparation and identification of protein anchor (PA). Two and three copies of LySMs gene was amplified with a pair of specific primer designed according to the whole genome sequence of Lactococcus MG1363strain in this study. Two prokaryotic expression vectors pET-PA2and pET-PA3were constructed following sequence of LySMs was confirmed. Target gene was successfully expressed in the inclusion body in BL21(DE3) when induced with IPTG, which were named as PA2or PA3.Identification of the combination between GEM particles and protein anchor. Recollecting GEM particles by centrifugation following incubated with500μg PA2or PA3at room temperature for30mins. Then GEM-PA in collection was identified by SDS-PAGE with monoclonal antibody against His tag in recombinant protein. Results showed that both two proteins can bind with the GEM particles, but the binding capacity of PA3is obviously better that of PA2. Transmission electron Microscope of the combination showed that there is a layer floccule around the surface of GEM particles after binding with PA.2. Preparation and identification of PCV2Cap vaccines based on GEM particlesPreparation and identification of recombinant fusion protein. ORF2gene was obtained from PCV2NJ strain. Two prokaryotic expression plasmid pQZ-PA3-Cap and pQZ-Cap-PA3were constructed following sequence of two fused gene was validated, and two fusion protein were expressed with Escherichia coli soluble expressing template CVC1102, which was established by National Center for Veterinary Biological Engineering and Technology. SDS-PAGE result showed that the fusion protein was44.6KD, however, the expression efficiency of Cap-PA3was much lower than PA3-Cap. About60-70%of PA3-Cap fusion protein was soluble form and both two fusion proteins were specific to PCV2positive serum.Identification for the combination between fusion protein and GEM particles.1U GEM particles were recollected by centrifugation following incubated with2ml fusion protein at room temperature for30mins. Then Cap protein in collection was identified with SDS-PAGE, Western-blot, electron microscope observation and immunofluorescence assay. Results showed that both the two fusion protein can bind with GEM particles. Electron microscope observation for GEM-PA3-Cap suggested that there was a protein layer around the surface of GEM particles, and this protein layer can specifically react with PCV2antibody in immunofluorescence assay. Besides, we also establish a method to dissociate the fusion protein from GEM particles, then recovery and quantitation for fusion protein became ture.3. Immunogenicity of the PCV2Cap subunit vaccine based on GEM-PA systemTo test the immunogenicity of the GEM-based PCV2subunit vaccine GEM-PA3-Cap and the most effective adjuvant compatibility of this vaccine,2-3weak-old PCV2negative pigs were grouped and each pig vaccinated with100μg antigens emulsionized with different adjuvant intramuscularly. ELISA antibody titer for each group was detected at three weeks following vaccination to evaluate the humoral immune response. Results indicated that only one group immunized with GEM-PA3-Cap and adjuvant "Y" induced higher antibody level than other groups adjuvanted with " L",206and alhydrogel, also with no adjuvant. Furthermore, immunopotentiator CVC1302can greatly boost the immune response against the vaccine when compared with groups without CVC1302. In conclusion, GEM-based PCV2subunit vaccine GEM-PA3-Cap can induce a better immunology response when adjuvant Y and immunopotentiator CVC1302were added.