Preclinical Studies Of Recombinant Fowlpox Virus Live Vaccine Expressing Newcastle Disease Virus HN Gene

Newcastle Disease (ND) is one of the most serious of all avian diseases, which is caused by Newcastle disease virus (NDV). The fusion (F) protein and haemagglutinin-neuraminidase (HN) protein are two major glycoprotein components on its envelope, both of which are target genes expressed in fowlpox virus for vaccine development. In this study, recombinant fowlpox viruses expressing Newcastle disease virus F gene or HN gene and coexpressing F and HN genes were screened by comparison of their protective efficacy. The best one was chosen for further study and pilotscale experiment. This work lays the foundation for clinical trial of recombinant fowlpox virus vector vaccine against ND.1. Protective efficacy of recombinant fowlpox viruses expressing Newcastle disease virus F gene or HN gene and coexpressing F and HN genesTo investigate the effect of maternal antibody to recombinant fowlpox viurs (rFPV) against Newcastle disease, two-week-old commercial layer chickens were immunized with2×104PFU rFPVs expressing fusion gene (rFPV-12LSF) or hemagglutinin-neuraminidase gene (rFPV-12LSHN) of genotype VII Newcastle disease virus (NDV) strain ZJ1and co-expressing F and HN genes (rFPV-12LSFHN) and0.2ml inactivated NDV oil-emulsion vaccine, respectively. At21days post-inoculation, chickens were challenged with106ELD5o of either virulent NDV strain F48E8or ZJ1. The protective efficacies against F48E8of rFPV-12LSF, rFPV-12LSHN, rFPV-12LSFHN and inactivated vaccine were30.3%、73.2%、41.1%and89.3%, while the protective efficacies against ZJ1were35.8%、67.9%、78.6%and100%. A recombinant fowlpox virus expressing chicken IL-2(rFPV-12LSIL-2) did not improve the protective efficacy of rFPV-12LSHN when combined with rFPV-12LSHN. The results indicated that the rFPV-12LSHN may be a effective candidate vaccine against NDV, which lays the foundation for further application of the live vector vaccine.2. Partial biological characteristics of a recombinant fowlpox virus expressing Newcastle disease virus HN geneTo evaluate the effect of transferring gene on biological characteristics fowlpox virus, the rFPV was first subjected to ultrastructural analysis. The result demonstrated that morphology of mature virions, replication pattern, and production of rFPV-12LSHN in infected cells was similar as that of FPV. When11-day-old chicken embryonated egg were inoculated with rFPV by chorioallantoic membrane (CAM) route, typical pock lesions were observed on the CAM, the chicken embryo minimal infecting dose was less than100PFU. The expression of NDV HN gene in rFPV was detected stably by Immunofluorescence assay when rFPV was passed in CEF. SPF chickens vaccinated with one dose of vaccine containing103PFU of rFPV-12LSHN were completely protected from virulent NDV challenge after3weeks vaccination.3. Duration of immunity and boost vaccination of a recombinant fowlpox virus expressing Newcastle disease virus HN gene To evaluate the duration of immunity and the efficacy of boost vaccination of a recombinant fowlpox virus(rFPV-12LSHN) expressing HN gene from a Newcastle disease virus (NDV) isolate(ZJ1). Two-week old SPF chickens vaccinated with one dose of vaccine containing103plaque forming units (PFU) of rFPV-12LSHN were completely protected from virulent NDV after7days vaccination, hemagglutination inhibition (HI) antibody against NDV could be detected as early as7days postvaccination and Immune protection lasted up to18weeks postvaccination. The efficacy of different vaccination schedules was evaluated in SPF chickens, the lowest efficacy (50%) were observed in the group of chickens, which was vaccinated with fowlpox vaccine and followed by rFPV-12LSHN vaccine at4weeks interval.However, the chickens primed and boosted with rFPV-12LSHN at4weeks interval produced significant increase of the HI antibody against NDV (P<0.01)and provided100%protection against virulent NDV challenge. The result indicated that the fowlpox vector—based vaccine for ND could induce an earlier onset of immunity and a long duration of at least18weeks.Furthermore,a second vaccination with rPFV is valuable for induction of optimal immunity..4. Establishment of a plaque forming unit counting method with X-gal staining for detection of recombinant fowlpox virus vaccineA plaque forming unit counting method with X-gal staining was established for detection of recombinant fowlpox virus(rFPV) live vaccine, which genome contained LacZ gene insertion. The secondary chicken embryo fibroblast(CEF) infected with rFPV was grown for72h and fixed in2mL/L glutaraldehyde solution for15min,then incubated in a500ug/mL X-gal staining solution at37℃overnight. The blue-stained plaques were counted under an inverted microscope. The number of plaque forming unit counted with staining was1.6to3.3times more than that without staining.5. Pilotscale production of recombinant fowlpox virus vaccineIn order to provide vaccine for clinical trials, a recombinant fowlpox virus vaccine expressing HN gene from a Newcastle disease virus (NDV) was produced on a pilot scale under Good Manufacturing Practice (GMP). CEFs in10000ml roller bottles were infected with rFPV-12LSHN at a multiplicity of infection (MOI) of approximately0.2PFU/cell and incubated at37℃with rolling at12revs h-1. The rPFVs infected CEFs were harvested when80%of the cells showed cytopathic effcct (CPE), stabilized by the addition of5%sucrose-milk powder and lyophilized. Five lots of vaccine products were manufactured, and the quality of vaccines met the criteria of document requirements.Conclusion1. A rFPV expressing HN gene (rFPV-12LSHN) of NDV was chosen as a effective vaccine candidate against NDV.2. rFPV-12LSHN showed similar replication and production as FPV in CEF.3. The duration of protective immunity by rFPV-12LSHN vaccination was more than18weeks.4. The chickens primed and boosted with rFPV-12LSHN produced significant increase of the HI antibody against NDV.5. A plaque forming unit counting method with X-gal staining for detection of recombinant fowlpox virus vaccine was established.6. Five lots of qualified recombinant fowlpox virus vaccines were produced on a pilot scale under Good Manufacturing Practice.