| Reticuloendotheliosis virus (REV) is a member of gammaretrovirus with a variety of strains, whichcauses an oncogenic, immunosuppressive and runting syndrome in multiple avian hosts includingchickens, ducks, turkeys and some other bird species. REV causes immunosuppression in infectedchickens, resulting in an increased susceptibility to concurrent or secondary bacterial and viralinfections and poor immune responses to other vaccines. REV fragments could integrate into thegenome of Marek’s disease virus (MDV) and increase its virulence when co-infecting with MDV. REVis widely distributed around the world and also highly prevalent in China, causing severe damages toour poultry industry. The oncogenicity and the immunosuppressive ability of REVs, their co-infectionwith other infectious viruses and their presence as contaminants in poultry biologics warrantdevelopment of a suitable vaccine. However, no commercially effective vaccine is available forpreventing this disease until now.The pathogenicity of REV HLJR0901strain in different ages of specific-pathogen-free (SPF)chickens was investigated, and the infection model of REV HLJR0901strain was established. Theinfected chickens were monitored weekly after infection for signs of illness and pathological changes.Decline in body weight, atrophy in bursa and thymus, tumefaction in liver, spleen and proventriculuswere observed after REV infection in1-day-old SPF chickens. The chickens infected with REV at5-week-old showed no or mild clinical signs. However, both the early infected chickens and the lateinfected chickens showed viremia and high proviral loads in multiple organs including bursa, thymus,liver, spleen and proventriculus. Additionally, the antibody responses to AIV and NDV vaccines weresignificantly inhibited by REV early infection compared with the control chickens. The detailedinfection model of REV in SPF chickens could be used to evaluate the efficacy of potential vaccines andantiviral agents.For the development of the recombinant subunit vaccine against REV, the gp90protein wasexpressed in yeast Pichia pastoris. The construction of recombinant Pichia strains and the expressionconditions were optimized in order to enhance the gp90production. The results showed that the REVgp90protein was secretory expressed in P. Pastoris successfully and SMD1168was the optimal hoststrain for gp90expression than the GS115and KM71strains. An increase in gp90copy number canimprove the yield of gp90protein in P. pastoris. The combined usage of both constitutive promoterGAP and inducible promoter AOX1further enhanced the gp90expression by41.1%. The results alsoindicated that co-expression of gp90protein with the yeast chaperones PDI and Ubi4reduced theproteolytically degradation of the gp90protein. In addition, the fermentation culture was optimized, and28°C and pH6.0were found to be the optimal conditions for gp90expression. The gp90productionwas further improved by adding1%casanimo acids in the fermentation medium. Following large-scalefermentation, the average gp90production level from strain SMD1168/GA90reached up to200mg/L inflask cultures, and1.0g/L after fermentation. The recombinant gp90protein could react with thegp90-specific MAb and induced high levels of REV antibody in immunized chickens, which showed that the yeast-expressed gp90protein had good antigenicity.To evaluate the efficacy and safety of the recombinant gp90as a subunit vaccine against REV,3-week-old SPF chickens as well as the SPF and commercial layer breeders were immunized with thegp90protein. The results showed that the minimum immunizing dose of the recombinant gp90proteinwas20μg; the optimal immunizing procedure was two shots at3week intervals, and the protectiveantibody duration was six months. The minimum antibody titer that could confer protection from REVinfection was2800as detected by ELISA. The immunization assay further showed that the recombinantsubunit vaccine induced high levels of antibody responses in SPF and commercial breeders, and thematernal antibody of the new-born chicks protected them from REV-induced viremia and runtingsyndrome. The results demonstrated that the chickens immunized with the recombinant gp90protein didnot show any abnormal clinical signs, and the immunization of the subunit vaccine made no influenceon the egg production rate of the breeder chickens. Overall, the yeast-expressed recombinant gp90protein was capable of inducing high levels of protective immune responses in both SPF chickens andlayer breeders with good safety.The DNA vaccines expressing the gp90protein of REV was constructed and also evaluated in SPFchickens in this study. The results showed that the DNA vaccines containing gp90gene induced bothhumoral and cellular immune responses and were efficacious in conferring protection against REVchallenge in chickens. Further, codon optimization and woodchuck hepatitis virus posttranscriptionalregulatory element (WPRE) were very powerful approaches in improving the immunogenicity of theDNA vaccines. Immunization of100μg of the optimized plasmid pCAGWoptigp90provided87%protection against REV challenge, lower than that conferred by40μg recombinant gp90protein whichconferred full protection against REV-induced viremia. Additionally, a DNA prime-protein booststrategy produced higher levels of humeral and cellular immune respinses in chickens than did either ofthe component parts of this vaccine alone. These findings highlight the potential value of combinationusage of both DNA and subunit vaccines for the prevention of REV infection. |
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