Development Of Genetically Engineered Vaccine Against Pseudorabies Virus Novel Variants

Pseudorabies, caused by Pseudorabies virus(PRV), is an acute febrile infectious disease in many livestock. Pseudorabies, resulted in tremendous economic lost in pig industry, causes high mortality in piglets and reproductive failure in sow and boar Vaccination is the most critical method in Pseudorabies control. Since 2011, novel PRV variant emerged in China and caused significant economic lost in pig industry. Commercial vaccines with classic PRV strains showed compromised protection efficacy, failed to stop the PRV spread and pandemic in China. In this research, gI, gE, TK were deleted from a PRV novel variant CH/HNSMX/2012, resulted in an attenuated live vaccine strain rSMXΔgI/gEΔTK. The safety and the efficacy tests of rSMXΔgI/gEΔTK were carried out in various animal models. This work provided the foundation for further live vaccine development. 1. Pseudorabies virus prevalent strain isolation and virulence studyTotally 16 PRV strains were isolated from 85 samples; plaque assay indicated that all 16 strains share similar plaque area, which were significantly larger than Ea strain. gC genes of all 16 strains were sequenced; multiple sequence alignment and phylogeny analysis suggested that all 16 strains isolated in this study were highly homologous with recently published PRV variants, showed 99.4% homology with Chinese classic PRV strains and 93.39% homologous homology with Europe and US classic PRV strains.The virulence of PRV CH/HNSMX/2012, a representative strain of PRV variant, was evaluated in mice and piglet. Inoculate the Balb/c mice with continuously dose gradient(100.0-106.0 TCID50) and calculate the LD50. The LD50 of CH/HNSMX/2012 and Ea, a representative of classic PRV strain, are 19.5 and 154.2 TCID50 respectively. CH/HNSMX/2012 showed significantly lower lethal dose than Ea in Balb/c mice. To evaluate the virulence of CH/HNSMX/2012 toward piglet, 60 day-old piglets were inoculated with 106.0, 107.0, 108.0 TCID50 CH/HNSMX/2012 intranasally. Post inoculation, 3 piglets in 106 TCID50 group showed neural symptoms, all piglets in 107.0 and 108.0 TCID50 showed severe neural symptoms and ended up dead. CH/HNSMX/2012 could lead to severe neural symptoms in 60 day-old piglets and showed strong virulence toward pig. 2. PRV attenuated strain construction and valiationAccording to PRV genome sequence, recombinant arms flanking gI/gE and TK deletion regions were designed and recombinant transfer vectorswere constructed. Through co-transfect the plasmid and genome of CH/HNSMX/2012 in PK-15 cell, recombinant PRV strain were generated and purified with EGFP marker. Purified gene knockout strain rSMXΔgI/gEΔTK was validated by RFLP and Southern Blot. rSMXΔgI/gEΔTK showed similar growth curve and maximum titer, compared with wild type strains, but plaque area decreased significantly. Continuously passage proved that rSMXΔgI/gEΔTK was genetically stable through passage. 3. Safety test of PRV attenuated strainThe safety of rSMXΔgI/gEΔTK was validated in mice, rabbit, sheep, piglet and pregnant sow.The dose gradient of rSMXΔgI/gEΔTK(101.0-107.0 TCID50) was used to inoculate Balb/c mice, no mice present any clinical symptoms and no death occurred. The dose gradient of rSMXΔgI/gEΔTK(102.0-7.0 TCID50) was used to inoculate rabbit, no rabbit present any clinical symptoms and no death occurred. Sheep were inoculated with 107.0 TCID50 rSMXΔgI/gEΔTK intramuscularly, no sheep exhibited any symptoms and no death occurred. Sentinel sheep maintained PRV antibody negative. Two groups of Newborn piglets were inoculate with 107.0 TCID50 rSMXΔgI/gEΔTK via intramuscular and intranasal respectively, no clinical symptoms were observed with normal growth property, and sentinel animal in each group maintained PRV antibody negative. Two group of pregnant sows were inoculate with 107.0 TCID50 rSMXΔgI/gEΔTK via intramuscular and intranasal respectively, no clinical symptoms were observed with normal reproductive property. rSMXΔgI/gEΔTK is safe toward mice, rabbit, sheep and pig. Therefore, it is qualified as live vaccine candidate. 4. Protection efficacy evaluation of rSMXΔgI/g EΔTKrSMXΔgI/gEΔTK protection efficacy was evaluated through immunization and challenge experiment. 28 day-old piglets were immunized 1 time intramuscularly with 106.0 TCID50 r SMXΔgI/gEΔTK, or Merial commercialized PRV vaccine(Bartha-K61) or 1mL DMEM. Blood samples were collected in 0d, 28 d and 49 d post vaccination. Serum neutralization test(SNT) indicated that rSMXΔgI/gEΔTK elicited higher neutralization antibody than Bartha-K61. Bartha-K61 vaccine elicited antibody showed compromised neutralization activity against CH/HNSMX/2012. Challenge experiment was carried out in 28 days post vaccination with 107.0TCID50 CH/HNSMX/2012 via intranasal rout. Post challenge, unimmunized group showed most severe neural and respiratory symptoms, and end up 100% mortality. rSMXΔgI/gEΔTK group only showed transient fever with no neural symptoms. Bartha-K61 group showed high fever and severe respiratory symptoms, as well as 3 piglets in Bartha-K61 group showed neural symptoms. Bartha-K61 group stopped body weight growth in 1 week post challenge. rSMXΔgI/gEΔTK could effectively elicit neutralization antibody and fully protect piglet from fatal challenge, but not Bartha-K61 vaccine.