Construction And Evaluation Of An Adenovirus/alphavirus Replicon Chimeric Vectored Vaccine Against Classical Swine Fever

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is one of themost devastating epizootic diseases of pigs worldwide. At present, vaccination is still an importantmeasure for prevention and control of CSF. Efficacious and safe attenuated vaccines have played a keyrole in CSF control, but this kind of vaccine has some disadvantages. First of all, the currently used CSFvaccine production process usually meet with some problems, for example, the production of vaccinesby the use of calf testicle cells and calf sera is often contaminated with bovine viral diarrhea virus(BVDV) or its antibody, which seriously influences the CSF vaccine potency and quality. Secondly,improper immune inoculation procedure may cause immune failure. In addition, antibodies against liveattenuated vaccines do not allow differentiation of infected from vaccinated animals (DIVA principle).Therefore, researchers make efforts to develop new-type CSF marker vaccine.In this study, we first constructed a recombinant human adenovirus type5expressing the CSFV E2gene (rAdV-E2) and evaluated its efficacy in rabbits and pigs. The results showed E2protein wasexpressed in rAdV-E2-infected HEK293cells. The rabbits and the pigs immunized with the rAdV-E2developed high-level CSFV-specific neutralizing antibodies. The rAdV-E2-immunized rabbits werecompletely protected from fever induced by infection with C-strain, and the rAdV-E2-immunized pigswere protected from lethal challenge with highly virulent Shimen strain, however, a few pigs showedshort-term fever and occasional pathological changes following virulent challenge.We further evaluated the efficacy of the heterologous prime-boost immunization approach (primewith pSFV1CS-E2and boost with rAdV-E2, at three-week intervals) in pigs. The results showed thatthe pigs (n=5) receiving pSFV1CS-E2/rAdV-E2heterologous prime-boost immunization developedsignificantly higher titers of CSFV-specific neutralizing antibodies and comparable CD4and CD8Tcell proliferation. When challenged with virulent CSFV Shimen strain, the pigs of the heterologousprime-boost group did not show clinical symptoms or viremia, which were observed in one of the5pigsimmunized with rAdV-E2alone and in all the5control pigs immunized with wild-type adenovirus. Theresults demonstrate that the heterologous DNA prime and recombinant adenovirus boost strategy caninduce full protective immunity.Later, we used the safe, efficient adenoviral vector to deliver the alphavirus replicon vector andconstructed an adenovirus/alphavirus replicon chimeric virus expressing the E2protein of CSFV. First,the alphavirus replicon vector expressing the E2gene was cloned into the adenovirus shuttle vector. Arecombinant adenovirus plasmid was constructed through homologous recombination in bacteria, andthe linearzied plasmid was transfected into HEK293cells by liposome to package adenovirus/alphavirusreplicon vector chimeric virus rAdV-SFV-E2. The recombinant chimeric virus was identified by PCR,IFA and Western blot and evaluated in pigs. PCR results confirmed that the exogenous gene can existstably in the recombinant chimeric virus. IFA and Western blot confirmed that the exogenous gene wasexpressed in rAdV-SFV-E2infected cells. Pigs immunized with rAdV-SFV-E2(n=5) developed robust humoral and cell-mediated responses to CSFV and were completely protected from subsequent lethalCSFV infection clinically and virologically. The level of immunity and protection induced byrAdV-SFV-E2was comparable to that provided by the currently used live attenuated vaccine, C-strain.By contrast, both the conventional alphavirus replicon-vectored vaccine pSFV1CS-E2and conventionaladenovirus-vectored vaccine rAdV-E2provided incomplete protection.In order to further verify whether rAdV-SFV-E2can be used as an ideal CSFV marker vaccine, wecomprehensively evaluated the safety and efficacy of the chimeric virus with respect to differentimmunization times and doses, pre-existing antibodies to the adenovirus vector, maternal antibodyinterference and combination immunization with PRV vaccine. The results showed that the minimalimmunization dose is6.25×105TCID50in pigs. A single immunization provided complete protectionagainst lethal CSFV challenge, though specific neutralizing antibodies were undetectable beforechallenge. The chimeric virus can overcome CSF maternal antibody interference and immunization withthe chimeric virus did not affect re-immunization of adenovirus-vectored vaccines. When chimeric viruswas co-administered with pseudorabies vaccine, all immunized pigs developed high-level specificantibodies against CSFV and PRV compared to either vaccine administered alone.The chimeric vector-based vaccine represents the first gene-based vaccine that is able to confersterile immunity and complete protection against CSFV. The novel vaccination strategy may also bevaluable in vaccine development against other pathogens.