Research On Safety And Immunogenicity Of BoHV-1 GG~-/TK~-/gD~+ BoHV-1 GG~-/TK~-/gD5~+ In Rabbits

Bovine herpesvirus 1(BoHV-1)infection in cattle named as infectious bovine rhinotracheitis(IBR)is a contagious infectious disease of cattle characterized by fever and upper respiratory and causes significant economic losses to cattle industry in this world.Isolates of BoHV-1 have been classified into three subtypes by restriction endonuclease analysis including BoHV-1.1,BoHV-1.2 and BoHV-1.3.BoHV-1.3 is also called as BoHV-5 and is able to infect different regions of the brain with neurovirulence.The main strategy for control of BoHV-5 mainly relies on vaccines against BoHV-1currently.The main strategy for control of the disease is control and purification by immunization of vaccines.Because the gene-deleted vaccines incorporate immunological markers,they have become a new trend for the new vaccine research and development.Some developed countries have used gene deleted marker vaccines like BoHV-1g E-deleted marker vaccines to successfully implement IBR control and eradication plan.The immunogenicity of marker vaccines existent needs to be further improved and overcome the immunosuppression caused by vaccination.Currently there is no effective marker vaccines based on BoHV-1 against BoHV-1 in China.A double mutant BoHV-1g G~-/TK~-constructed early in our lab.The extracellular domain of g D from BoHV-1 and BoHV-5 were inserted respectively into the site of TK from BoHV-1 g G~-/TK~-.This paper aims to further evaluate the immunogenicity of recombinant vaccines in rabbits and cross-protection against BoHV-5 with providing theoretical basis for development of more effective vaccines and BoHV-1 control.The main researches are as follows:1.Evaluation for safetyThe Japanese white rabbits were vaccinated with virus 4×10~7PFU by nasal inoculation.There is no significant difference between the rectal temperatures from rabbits in vaccinated groups and unvaccinated groups.There was no significant clinical symptoms about the rabbits from BoHV-1 g G~-/TK~-/g D~+group and BoHV-1 g G~-/TK~-/g D5~+group with normal feeding and drinking.There was one rabbit in BoHV-1 g G~-/TK~-group that had white or yellow secretions with normal feeding and drinking at the third,fifth day and seventh day respectively.There was no virus shedding from the six groups after vaccination.2.The study against challenge with wt BoHV-1After 28 days post vaccination(dpv),the Japanese white rabbits were challenged with BoHV-1 4×10~7PFU by nasal inoculation.The rectal temperature in unvaccinated group were above 40℃at the third day.There was no significant difference between the rectal temperatures from rabbits in challenged groups and blank groups.There was one rabbit in BoHV-1 g G~-/TK~-/g D~+group that appeared nasal mucus at the twelfth day with more than 3 days.There was 2 rabbits in BoHV-1 g G~-/TK~-/g D5~+group that appeared nasal mucus at the fifth and seventeenth day respectively with more than 3 days.There were 2rabbits in BoHV-1 g G~-/TK~-group that had more nasal mucus at the fourteenth and seventeenth day respectively with more than 3 days.There were 2 rabbits in unvaccinated group had more nasal secretions at the second and seventh day respectively with more than 3 days.There was no virus shedding from the six groups after challenge with wt BoHV-1.There was no virus detected in organs from six groups.3.The study against challenge with wt BoHV-5After 28 days post vaccination(dpv),the Japanese white rabbits were challenged with BoHV-5 4×10~7PFU by nasal inoculation.The rectal temperature in the unvaccinated group were above 40℃at the tenth day.There was no significant difference between the rectal temperatures from rabbits in challenged groups and blank groups.There was one rabbit in BoHV-1 g G~-/TK~-/g D~+group that appeared nasal mucus at the ninth day for 1 day.There was no rabbit in BoHV-1 g G~-/TK~-/g D5~+group that appeared nasal mucus.There were 2 rabbits in BoHV-1 g G~-/TK~-group that had more nasal mucus at the seventh day with more than 3 days.There were 2 rabbits in unvaccinated group had more nasal secretions at the first and third day respectively with more than 3 days.There was no virus shedding from the six groups after challenge with wt BoHV-1.Virus was detected in lung from one rabbit in BoHV-1 g G~-/TK~-group and there was no virus detected in organs from other groups.4.Immune response(1)Neutralizing antibody titters measured by neutralization experimentThe highest neutralization antibody titters of rabbits in BoHV-1 g G~-/TK~-/g D~+,BoHV-1 g G~-/TK~-/g D5~+and BoHV-1 g G~-/TK~-group were 1:8,1:5 and 0 respectively and the neutralization antibody in recombinant groups could be detected early than BoHV-1g G~-/TK~-group with significant differences(p<0.001).The highest neutralization antibody titters of rabbits in BoHV-1 g G~-/TK~-/g D~+,BoHV-1 g G~-/TK~-/g D5~+and BoHV-1g G~-/TK~-group were 1:38.64,1:38.64 and 1:32 respectively after challenge with wt BoHV-1.There were significant differences between recombinant groups and BoHV-1g G~-/TK~-group(p<0.001).The highest neutralization antibody titters of rabbits in BoHV-1 g G~-/TK~-/g D~+,BoHV-1 g G~-/TK~-/g D5~+and BoHV-1 g G~-/TK~-group were 1:32,1:32 and 1:10 respectively after challenge with wt BoHV-5.There were significant differences between recombinant groups and BoHV-1 g G~-/TK~-group(p<0.001).(2)Antibody titters measured by indirect ELISAAnalysis of serum by indirect ELISA showed that average OD of the highest specific BoHV-1 antibody titters induced in BoHV-1 g G~-/TK~-/g D~+,BoHV-1 g G~-/TK~-/g D5~+and BoHV-1 g G~-/TK~-group were 1.141,0.9598 and 0.8502 respectively.There were significant differences between recombinant groups and BoHV-1 g G~-/TK~-group(p<0.001).The average OD of the highest BoHV-1 antibody titters of rabbits in BoHV-1g G~-/TK~-/g D~+,BoHV-1 g G~-/TK~-/g D5~+and BoHV-1 g G~-/TK~-group were 0.7935、0.7178and 0.6977 respectively after challenge with wt BoHV-1.There was no significant difference between recombinant groups and BoHV-1 g G~-/TK~-group.The average OD of the highest BoHV-5 antibody titters of rabbits in BoHV-1 g G~-/TK~-/g D~+and BoHV-1g G~-/TK~-group were 1.368 and 0.9490 respectively after challenge with wt BoHV-5.There was significant difference between BoHV-1 g G~-/TK~-/g D~+and BoHV-1 g G~-/TK~-group(p<0.001).(3)Detection of specific PBMCs proliferation responseSpecific PBMCs proliferation response results measured by XTT Cell Proliferation Assay Kit showed that average OD of the highest levels of specific PBMCs proliferation response in BoHV-1 g G~-/TK~-/g D~+and BoHV-1 g G~-/TK~-group were 0.2480 and 0.1583respectively after vaccination with significant difference(p<0.001).The average OD of the highest levels of specific PBMCs proliferation response in BoHV-1 g G~-/TK~-/g D~+,BoHV-1 g G~-/TK~-/g D5~+and BoHV-1 g G~-/TK~-group were 0.6667,0.7700 and 0.2560respectively after challenge with wt BoHV-1.There were significant differences between recombinant groups and BoHV-1 g G~-/TK~-group(p<0.001).The average OD of the highest levels of specific PBMCs proliferation response in BoHV-1 g G~-/TK~-/g D~+,BoHV-1 g G~-/TK~-/g D5~+and BoHV-1 g G~-/TK~-group were 0.5290,0.6003 and 0.1183respectively after challenge with wt BoHV-5.There were significant differences between recombinant groups and BoHV-1 g G~-/TK~-group(p<0.001).The results above showed that the recombinant virus inserted g D with the best antigenicity into the site of TK from the BoHV-1 g G~-/TK~-could improve the safety and immunogenicity.