Evaluation Of The Cell-mediated Immunity Induced By Alphavirus Replicon-based DNA Vaccines Against Classical Swine Fever

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious and lethal disease of pigs, which is characterized by fever, hemorrhages and mortality. It is one of OIE-listed diseases (Diseases Notifiable to the Office International des Epizooties, OIE). CSF outbreaks often lead to severe economic losses and impair the trade of pigs and pork products. In China, a compulsive vaccination policy is pursued to prevent the disease. At present, the most frequently used vaccine is the Chinese lapinized attenuated vaccine, i.e. C-strain, which can induce complete protection against CSF. A major disadvantage of this vaccine is that the antibodies induced by the vaccine cannot be distinguished from those induced by wild-type CSFV infection. It is important to develop novel marker vaccines capable of inducing rapid protection combined with the possibility to differentiate infected from vaccinated animals (DIVA).Now many marker vaccines against CSFV are being developed. We previously developed a DNA vaccine (pSFV1CS-E2) encoding the E2 protein of CSFV delivered by an alphavirus replicon. Immunized pigs were completely protected against virulent CSFV challenge in spite of low level specific antibody titers prior to challenge. This observation implies that cell-mediated immunity (CMI) may play an important role in the protective immunity of CSF. This study is aimed to study the CMI induced by the alphavirus replicon vectored vaccines, and develop the strategy to enhance the immunogenicity of the DNA vaccine.We constructed a recombinant plasmid expressing the E2 protein of CSFV (pSC11-E2) based on the vaccine virus vector pSC11. HuTKˉ143B cell cultures infected with vaccinia virus WR stain were transfected with the recombinant plasmid. The recombinant virus was screened by plaque assay on TKˉcells in the presence of 5-bromo-2-deoxyuridine (BrdU) and screened by the blue plaques assay. Then the recombinant virus was identified by PCR, immuno?uorescence assay and western blot assay. Furthermore, the recombinant viruses were inoculated into mice and could induce E2 antibody in the immunized mice. The recombinant virus can be used to boost the alphavirus replicon based DNA vaccine; it can also be used to study the CMI induced by the alphavirus replicon-based DNA vaccines.In this study, the CMI induced by pSFV1CS-E2 and its derivative pSFV1CS-E2-UL49 encoding a fusion protein of CSFV E2 and pseudorabies virus (PRV) VP22 was evaluated in mouse model by lymphoproliferation assays based on CFSE or WST-8, intracellular cytokine staining, and cytokine ELISA. The results showed that both vaccines induced CSFV-specific lymphoproliferative responses and cytokine production, and pSFV1CS-E2-UL49 induced stronger lymphoproliferative responses and higher cytokine levels than pSFV1CS-E2.The vaccine pSFV1CS-E2 could induce CSFV-specific CMI, and pSFV1CS-E2-UL49 induced stronger CMI. However, they could just induce low level antibody titers. In order to enhance the immunogenicity of the alphavirus replicon based DNA vaccines, using prime-boost vaccination strategy, BALB/c mice were primed with the alphavirus replicon vectored DNA vaccine pSFV1CS-E2-UL49, then homologously boosted with pSFV1CS-E2-UL49 or heterologously boosted with the recombinant adenovirus (rAdV-E2) expressing E2 protein or with the baculovirus-produced recombinant E2 protein (rE2) in adjuvant. The humoral and cellular immune responses elicited by homologous or heterologous prime-boost vaccination regimens were assessed. The results showed that high-level antibody titers were elicited in the mice boosted with rAdV-E2 or rE2 protein, whereas the antibody titers were below positive threshold in the mice homologously boosted with pSFV1CS-E2-UL49. Stronger CD8+ and CD4+ T cell proliferation responses were detected by CFSE lymphoproliferation assay in the mice heterologously boosted with rAdV-E2, and higher stimulation indexes were also detected by WST-8 lymphoproliferation assay. Furthermore, more IFN-γproduction was induced in the mice heterologously boosted with rAdV-E2.In order to study the CMI induced by the alphavirus replicon based DNA vaccines and prime-boost vaccination strategy in pigs, a novel lymphoproliferation assay was developed. Porcine peripheral blood mononuclear cells (PBMCs) were stained with an intracellular fluorescent dye, CFSE, and stimulated with varying amounts of ConA, and plated in 3 replicate cultures for 3, 5, or 7 days. Then CD4+ cell and CD8+ cells were labeled with fluorescence-labeled antibodies and analyzed with flow cytometry (FCM). The method could be used to assess the proliferation of porcine PBMCs.These findings suggest that the alphavirus replicon-delivered DNA vaccines are capable of inducing CMI, and PRV VP22 was able to enhance the immunogenicity of the co-delivered antigen, while DNA prime-recombinant adenovirus boost could enhance the humoral and cellular immune responses significantly.