Construction And Immunogenicity Of Recombinant Adenovirus Expressing S1 And N Proteins Of Infectious Bronchitis Virus

Avian Infectious Bronchitis (IB) is a highly contagious disease of chickens caused by Infectious Bronchitis Virus (IBV), a member of Coronaviridae. Chickens infected with IBV develop respiratory disease, nephritis and reproductive disorders resulting in coughing, sneezing, rales and reduced egg production and quality. Genomic diversity and the ability of IBV to rapidly change have created different serotypes of the virus which do not cross protect. It is difficult to completely control IBV because the genetic changes of the virus occur far too frequently to develop and provide vaccines of the corresponding serotype. At present, the virus is worldwide in distribution causing huge economic losses to the poultry industry.Vaccination is one of the most effective measures to prevent and control the disease. Although traditional vaccines including attenuated live vaccines and inactivated vaccines have played an important role in control of the prevalence of the disease, there are still some disadvantages such as poor safety, expensiveness, limited effectiveness, or have harmful side effects. Based on the shortages of traditional vaccines, exploiting new vaccines like subunit vaccines, DNA vaccines, live recombinant vaccines, and transgenic plants edible vaccines etc are gradually becoming the hotspot in the field of new vaccine research.In this study, two recombinant human 5 replication defective adenoviruses expressing S1 and N genes respectively were constructed and named as rAd-S1, rAd-N. The results of PCR, Western blot and IFA tests demonstrated that both S1 and N genes were expressed in 293 cells infected with recombinant adenoviruses respectively. The two recombinant adenoviruses were both serially passaged 15 times, and the genetic stability of inserted foreign fragments were confirmed by cloning and sequencing of S1 and N gene from passage 8, 10, 12 and 15 respectively. The growth dynamics indicated that the titers of the two recombinant adenoviruses were as high as 10¹⁰TCID₅₀/mL, and there were no statistically differences between the recombinant and parental adenoviruses.To further study the immunogenicity of the recombinant adenoviruses in chickens, two criterias were introduced. First, serum antibodies specific to S1 and N proteins were measured with an indirect ELISA and Western blot tests. The second one is vaccination-challenge test. Fifty-five SPF chickens were equally divided into five groups named as A, B, C, D and E group. Chickens from A, B and C were experimental groups which were inoculated with rAd-S1, rAd-N, rAd-S1 plus rAd-N (chickens were immunized with rAd-S1 seven days after immunized with rAd-N) through intramuscular injection with virus titres of 10¹⁰TCID₅₀/mL respectively at three days post-hatch. D and E were control groups which were inoculated with IBV LDT3-A vaccine strain and parental adenovirus respectively. The indirect ELISA tests indicated that chickens from group D can produce certain level antibodys at 10, 15 and 20 days and the peak OD630 value is 0.8 at 15 days after inocultated with IBV vaccine strain. Western blot tests indicated that chickens from groups immunized with recombinant adenoviruses can produce a certain level of antibodies at 15 and 20 d.p.i. (days post-innoculation). However, there were no antibodies that could be detected at any time points at all in parental adenovirus inoculated group. All chickens were challenged intranasally with 106EID50 IBV LDL strain at 21 d.p.i. and were observed for another 25 days. The results showed that all chickens from group E inoculated with parental adenovirus showed clinical signs like coughing, sneezing, dyspnea and death at 5 d.p.c. (days post-challenge). Few chickens showed any clinical signs from group D inoculated with IBV vaccine strain. The morbidity from group A, B, C, D and E is 27.3%, 54.5%, 63.6%, 0, 100% respectively, and the mortality from each group is 18.2%, 45.5%, 54.5%, 0 and 81.8% respectively.The results above showed that, compared with the IBV vaccine strain and parental adenovirus, the recombinant adenoviruses expressing IBV S1 and N proteins respectively can induce certain level of antibodies and the recombinant adenovirus expressing S1 protein can provide a higher level of protection than N protein against virulent IBV challenge.