Construction, Characterization And Immunogenicity Of Recombinant Swinepox Viruses Expressing Protective Antigens Of Streptococcus Suis Type2

Streptococcus suis type2(SS2) is a major swine pathogen responsible for a wide range of diseases, including meningitis, arthritis, endocarditis, pneumonia, and septicaemia with sudden death. Moreover, SS2is an agent of zoonosis that has the potential to afflict those who are in close contact with infected pigs or pork-derived products. Swinepox virus (SPV) infects only swine. Natural SPV infections are typically mild and occasionally accompanied by localized skin lesions that heal naturally. SPV is well suited for the development of recombinant vaccines, because of its biological and clinical safety, large packaging capacity for recombinant DNA, and its ability to induce solid protective immunity. In this study, we constructed several recombinant SPVs expressing protective antigens of SS2, using SPV as the delivery vehicle. Furthermore, we assessed immunogenicity of these recombinant viruses.1. The isolation of SPV and the serosurvey of swinepoxA strain of SPV was isolated from the pigs that showed typical evidence of the pox infection. Virus isolations were performed with the mixtures of encrusted cutaneous material and biopsied skin lesions. Three DNA fragments including unique sequence of SPV were amplified and sequenced, nucleotide sequence homology was91%,96%and99%respectively, when compared to those of the SPV17077-99strain. Furthermore, to investigate the epidemic status of swinepox, serosurvey of swinepox was conducted by agar gel diffusion precipitation test on1004adult swine serum samples collected from Jiangxi, Anhui, Hubei, Jiangsu, Guangdong and Shanghai. The results indicated that SPV infection was rarely present and only40(=4percent) of these serum samples contained anti-SPV antibodies. However, these positive samples were distributed in all six regions surveyed.2. Construction and characterization of recombinant SPVs expressing lacZ geneTo test the activity of vaccinia virus (VV) late promoters P11and P28in the recombinant SPV, a vector was developed. We used this system to produce two recombinant viruses: rSPV-Z11and rSPV-Z28, and characterized replication and expression of the recombinant virus. In this system, the366bp non-coding region between the SPV20gene and the SPV21gene in the SPV genome was used as a novel insertion locus for generating recombinant SPV, and a dominant selection procedure based on lacZ gene expression was developed for recombinant virus isolation. Two recombinant viruses were identified by PCR and SDS-PAGE, and their replication were compared with the wild-type virus. We also assessed genetic stability of recombinant viruses. The results demonstrated that lacZ gene expression from two recombinant viruses was stable over ten passages and the generated viral titer and plaque area was comparable to wild-type SPV. From these observations, we concluded that the recombinant viruses had good genetic stability and the partial deletion of the intergene region was dispensable for growth in PK-15cells. Furthermore, the activity of promoters P11and P28was very strong in the recombinant SPV and suitable to the production of recombinant vaccines.3. Replication and expression of a recombinant SPV expressing green fluorescent protein in two murine cell linesTo explore the potential of the mouse as an animal model for the immunological evaluation of SPV-based vaccines, a recombinant virus expressing green fluorescent protein (GFP), designated rSPV-G28, was produced, and we characterized replication and expression of the recombinant virus in two murine cell lines, LLC and Hepal-6. Our results showed that murine cells were susceptible to infection by SPV and supported expression of GFP, and SPV viral DNA was replicated in LLC cells and infectious virus was recovered. These results suggest that the mouse may have potential as an animal model for the immunological evaluation of SPV-based vaccines.4. Construction and immunogenicity of a recombinant SPV expressing truncated MRP of SS2To explore the potential of the SPV as vector for Streptococcus suis vaccines, a vector system was developed for the construction of a recombinant SPV carrying bacterial genes. Using this system, a recombinant virus expressing truncated muramidase-released protein (MRP) of SS2, designated rSPV-MRP, was produced and identified by PCR, western blotting and immunofluorescence assays. After immunized intramuscularly with rSPV-MRP, SS2inactive vaccine (positive control), wild-type SPV (negative control) and PBS (blank control) respectively, all CD1mice were challenged with a lethal dose or a sublethal dose of SS2highly virulent strain ZY05719. Immunization with rSPV-MRP resulted in60%survival and significantly protected mice against a lethal dose of the highly virulent SS2strain, compared with the negative control (P<0.05). Our data indicate that animals immunized with rSPV-MRP had a significantly reduced bacterial burden in all organs examined, compared to negative controls (P<0.05). Antibody titers of the rSPV-MRP-vaccinated group were significantly higher (P<0.001), when compared to negative controls. Antibody titers were also significantly higher in the vaccinated group at all time points post-vaccination (P<0.001), compared with the positive controls. The results demonstrated that the rSPV-MRP provided mice with significant protection from systemic SS2infection. It suggested that SPV recombinants have the potential as S. suis vaccines for the use in pigs.5. Development of recombinant SPVs coexpressing SLY and MRP of SS2: immunogenicity and efficacy studies in CD1mice modelWe developed a vector system for the construction of a recombinant SPV carrying foreign genes, using GFP gene as a dominant selection marker. We used this system to produce two recombinant viruses expressing protective antigens of SS2, rSPV-SLY and rSPV-SLY-MRP. CD1mice were immunized intramuscularly three times with rSPV-SLY, rSPV-SLY-MRP, SS2inactive vaccine (positive control), wild-type SPV (negative control), and PBS (blank control). Two weeks after the last immunization, all mice were challenged with lethal or sublethal doses of the highly virulent SS2strain ZY05719. Sera were examined for the specific antibody titers using enzyme-linked immunosorbent assay (ELISA). Specific antibody titers of mice vaccinated with recombinant viruses increased dramatically at day7post-vaccination, and continued to increase after the boost. Antibody titers were significantly higher (P<0.001), when compared to negative controls. Antibody titers were also significantly higher at all time points post-vaccination (P<0.05), compared with the positive controls. Our data indicate that animals immunized with recombinant viruses had a significantly reduced bacterial burden in all organs examined, compared to negative control (P<0.05). Immunization with rSPV-SLY resulted in70%survival, and immunization with rSPV-SLY-MRP resulted in100%survival. Both rSPV-SLY (P<0.05) and rSPV-SLY-MRP (P<0.001) significantly protected mice against a lethal dose of the highly virulent SS2strain, compared with the negative control. Our results suggest that rSPV-SLY and rSPV-SLY-MRP provide mice with significant protection from systemic SS2infection and can take as vaccine candidates of SS2.6. Development of recombinant SPVs coexpressing ECE, MRP and SLY of SS2: immunogenicity and efficacy studies in CD1mice modelWe generated three recombinant SPVs:rSPV-ECE、rSPV-ECE-SLY and rSPV-ECE-MRP-SLY, using SPV as the delivery vehicle. CD1mice were immunized intramuscularly three times with rSPV-ECE、rSPV-ECE-SLY, rSPV-ECE-MRP-SLY, SS2inactive vaccine (positive control), wild-type SPV (negative control), and PBS (blank control). Two weeks after the last immunization, all mice were challenged with lethal or sublethal doses of the highly virulent SS2strain ZY05719. Sera were examined for the specific antibody titers from all groups using ELISA. Specific antibody titers of mice vaccinated with recombinant SPVs increased dramatically at day7post-vaccination, and continued to increase after the boost. Antibody titers were significantly higher (P<0.001), when compared to negative controls. Antibody titers were also significantly higher at all time points post-vaccination (P<0.05), compared with the positive controls. Our data indicate that animals immunized with recombinant SPVs had a significantly reduced bacterial burden in all organs examined, compared to negative control (P<0.05). Immunization with rSPV-ECE resulted in60%survival, and immunization with rSPV-ECE-SLY or rSPV-ECE-MRP-SLY resulted in100%survival. Our results suggest that rSPV-ECE、rSPV-ECE-SLY and rSPV-ECE-MRP-SLY provide mice with significant protection from systemic SS2infection and can take as vaccine candidates of SS2.