An Alphavirus Replicon-vectored Plasmid DNA-based Vaccine Elicits Protective Immunity Against Classical Swine Fever In Pigs

Classical swine fever (CSF) is a highly contagious and often fatal disease of pigs caused byClassical swine fever virus (CSFV), which, together with Bovine viral diarrhea virus 1 (BVDV-1),BVDV-2, Border disease virus (BDV) and Pestivirus of Giraffe, belongs to the genus Pestiviruswithin the family Flaviviridae.Vaccination has been an important measure to prevent CSF. Live attenuated vaccines,including Chinese strain (C-strain), are still in use in many countries. But a great disadvantage ofthese conventional vaccines is that vaccinated animals cannot be discriminated from those infectedwith wild-type CSFV. It is urgent to develop DIVA (differentiating infected from vaccinatedanimals) or marker vaccines. Most of the candidate marker vaccines are based on the E2glycoprotein, which has been shown to be a protective immunogen. Among the marker vaccines areE2 subunit vaccines produced with recombinant baculovirus system, live virus vector vaccinesbased on recombinant Porcine adenovirus, Pseudorabies virus or Swinepox virus expressing E2gene, E2 gene-based DNA plasmid vaccines, E^rns gene-deleted viruses derived from CSFVinfectious cDNA clone, and chimeric viruses replacing CSFV E^rns gene with the counterpart ofBVDV, etc. These vaccines do not contain or express E^rns glycoprotein, which facilitatesdifferentiation of infected pigs from vaccinates based on E^rns-ELISA. But up to now, most of thesevaccines have been put into practical applications due to suboptimal safety or efficacy.Recently, self-replicating RNA (RNA replicons) have emerged as an important strategy toenhance the efficacy of nucleic acid vaccines for cancer immunotherapy and viral disease controls.RNA replicon vaccines derived from alphaviruses, such as Semliki Forest virus (SFV), Sindbisvirus (SIN), or Venezuelan equine encephalitis virus (VEEV), are self-replicating and self-limiting,and may be administrated as either RNA or DNA, which is then transcribed into RNA replicons intransfected cells or in vivo. RNA replicons eventually cause apoptosis of transfected cells. These vectors therefore do not raise concerns associated with naked DNA vaccines of integration into thehost genome. RNA replicon vaccines, on the other hand, are able to induce both humoral andcellular immune responses. So they have been widely used to develop replicon vaccines. This paperis aimed to develop a safer and more efficacious SFV replicon-based vaccine against CSF andevaluate its efficacy in pigs.CMV immediately early promoter and SV40 polyadenylation signal were amplified frompEGFP-N1 by PCR. The PCR products were cloned into pSFV1, a eukaryotic expression vectorderived from SFV RNA replicon, generating a new replicative DNA expression vector, pSFV1CS.This modified expression vector does not need in vitro transcription, which makes it to be easier touse than its parent vector pSFV1.To confirm if pSFV1CS can be used as vaccine vector to develop a candidate CSFV DNAvaccine, the E2 gene of CSFV from pcDST (pcDNA3.1-derived plasmid harboring the E2 gene ofCSFV Shimen strain) was cloned into pSFV1CS, resulting in a recombinant plasmid designated aspSFV1CS-E2. The E2 gene expression was verified in pSFV1CS-E2-transfected BHK-21 cells, asdemonstrated by indirect immunofluorescence assay.To verify the potential of pSFV1CS-E2 as a candidate marker vaccine against CSFV, we testits efficacy in pigs. Ten 8-week-old pigs were randomly assigned to 2 groups. Pigs in group 1 (n=5) were immunized intramuscularly three times with 600μg of the DNA vaccine pSFV1CS-E2 at3-week intervals, and pigs in group 2 served as control were immunized with the control vectorpSFV1CS (n=5). All pigs were challenged with highly virulent Shimen strain 4 weeks after thelast boost. Pigs immunized with pSFV1CS-E2 developed low-level neutralizing antibody againstCSFV before challenge, and the antibody titers increased rapidly upon challenge. The immunizedpigs did not display clinical signs except short-time fever, and no pathological changes wereobserved at necropsy and fewer viral antigens were detected in occasional tissues. On the contrary,pigs immunized with the control vector did not develop antibody titers before or after challenge,and showed severe clinical signs and lasting viremia. Three of the 5 control pigs died of illness. Allcontrol pigs showed severe pathological lesions typical of virulent CSFV infection at necropsy, andabundant viral antigens were detected in all tissues tested.To further evaluate the efficacy of the DNA vaccine with lower dosage and fewer inoculations,pigs were immunized twice with 100μg of pSFV1CS-E2 (n=5) or control plasmid pSFV1CS (n=3), respectively. Pigs immunized with pSFV1CS-E2 developed high titers of specific neutralizing antibodies against CSFV following the booster, and the antibody titers increased rapidly uponchallenge 6 weeks post-booster. The immunized animals showed no clinical symptoms exceptshort-term fever and low-level viremia, whereas the control pigs immunized with the controlplasmid did not produce detectable antibody titers before challenge, and showed obvious clinicalsigns following challenge, and 2 pigs died of illness on 10 to 11 days post-challenge. All controlanimals developed extended viremia as detected by nested RT-PCR and real-time RT-PCR. Severepathologic lesions typical of CSFV infection were observed at necropsy.These results show that the DNA-based vaccine vectored by the SFV replicon can be used as apotential candidate marker vaccine against CSF.