Immunogenicity Of A Recombinant Adenovirus Co-expressing Porcine Reproduction And Respiratory Syndrome Virus GP5 Protein And Classical Swine Fever Virus E2 Protein

Porcine reproductive and respiratory syndrome (PRRS), characterized by severe reproductive failure in sows and respiratory distress in piglets and growing pigs, is caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRS is one of OIE-listed diseases and is now considered to be one of the most important diseases in countries with intensive swine industries.Classical swine fever (CSF) is a highly contagious and lethal disease of pigs caused by Classical swine fever virus (CSFV), which is one of OIE-listed diseases.Adenovirus vectors, particularly those constructed from human adenovirus serotype 5 (Ad5), have been shown to be an excellent delivery system to express genes of interest for vaccine development. The Ad5 recombinant virus is often replication-defective due to a large deletion in the early transcription region1 (E1) of the genome. These replication-defective Ad5 viruses can grow only in certain cells, like 293 cells, that are able to complement the E1 protein of the adenovirus. High levels of expression are achieved in the Ad5 vector system when foreign genes are under the control of constitutive promoters like the CMV promoter. Adenoviruses have shown great promise as vectors for recombinant vaccine development.In recent studies, an 18-amino-acid-long 2A sequence of either aphtoviruses or cardioviruses, members of Picornaviridae family, was used to enable the expression of two proteins from one cistron. Fusion constructs harboring the 2A sequences allow the separation of the proteins by a ribosomal skipping mechanism, which leads to impairment of a peptide bond without stopping the translation.In this study, we constructed a recombinant adenovirus rAdV-GP52AE2, using a replication-defective Ad5 as a delivery vector, co-expressing the GP5 protein of highly pathogenic PRRSV and the E2 protein of CSFV, and foot-and-mouth disease virus (FMDV) 2A peptide was used as a linker between GP5 and E2 proteins to allow automatic self-cleavage of the polyprotein. The GP5 and E2 genes were expressed as demonstrated by immunofluorescence assay (IFA) and Western blotting. Immunization of mice resulted in CSFV-neutralizing antibodies titer of 1:128 and PRRSV-neutralizing antibodies titer of 1:16. The lymphoproliferative responses were detected by CCK-8 and the stimulation index of CFSV-specific and PRRSV-specific lymphocytes in rAdV-GP52AE2 group was significantly higher than that of the negative control group. The results show that rAdV-GP52AE2 can induce both effective humoral and cell-mediated immune responses in mice. The protective efficacy of the recombinant against CSF was evaluated in immunized rabbits, which were protected from fever induced by challenge with C-strain.Our study provided supporting evidence for the use of FMDV 2A to develop a novel multi-gene engineering vaccine.