Construction And Evaluation Of Porcine Epidemic Diarrhea Virus,Porcine Deltacoronavirus Vaccines Based On Recombinant Adenoviral Vector

Porcine epidemic diarrhea virus(PEDV)and porcine deltacoronavirus(PDCoV)are porcine enteric pathogenic alpha and delta coronaviruses,respectively,which can cause severe watery diarrhea,vomiting,dehydration and even death of piglets,causing significant economic losses to the global pig industry.Most commercially available PEDV vaccines are developed based on the GI genotype strains,which have poor immune protection against the currently dominant GII genotype strains.PDCoV has the potential to spread across species,while there is no commercially available vaccine.Therefore,the development of safe and effective PEDV and PDCoV vaccines is urgent.Spike proteins(S),structural proteins encoded by PEDV and PDCoV,are responsible for recognizing host cell receptors to initiate virus invasion,in which S1 domains contain rich neutralizing epitopes,so S and S1 proteins are the main targets for the research and development of coronavirus vaccines.Human adenovirus type 5(Ad5)vector has been widely used in the research and development of a variety of pathogen vaccines due to its advantages of large capacity,high safety,and strong stimulation of systemic and mucosal immune responses.Therefore,PEDV and PDCoV vaccines based on recombinant adenoviral vector may be promising vaccines.In this study,four novel PEDV replication-deficient Ad5 vector vaccines were constructed to express codon optimized S proteins(Ad-XT-t PA-Sopt and Ad-PJ-t PA-Sopt)and S1 proteins(Ad-XT-ori SIP-S1opt and Ad-PJ-ori SIP-S1opt)of GIIa and GIIb strains,and two novel PDCoV replication-deficient Ad5 vector vaccines were constructed to express codon optimized S protein(Ad-PD-t PA-Sopt)and S1 protein(Ad-PD-ori SIP-S1opt).The immunogenicity of the recombinant adenoviruses was evaluated in BALB/c mice by one or two intramuscular injections with doses of 10~8,10~7 and 10~6 Infectious Units(IFU)/mouse.In addition,the immune effects of Ad-XT-t PA-Sopt and Ad-PD-t PA-Sopt with 10~8 IFU/mouse were also evaluated by mixed immunizations of a prime intramuscular injection followed by boost oral gavage administration,and by two oral gavage immunizations.Finally,the immunogenicity of Ad-XT-t PA-Sopt and Ad-PD-t PA-Sopt combined vaccine was evaluated in mice in this study.The results show that:1.Shuttle plasmids p DC-XT-t PA-Sopt and p DC-PJ-t PA-Sopt can be used for expression of PEDV S proteins in vitro,and p DC-XT-ori SIP-S1opt,p DC-XT-t PA-S1opt,p DC-PJ-ori SIP-S1opt and p DC-PJ-t PA-S1opt can be used for expression of PEDV S1 proteins in vitro.And shuttle plasmids p DC-PD-t PA-Sopt and p DC-PD-ori SIP-S1opt can be used for expression of PDCoV S and S1proteins in vitro,respectively.2.The mice immunized with PEDV recombinant adenoviruses by intramuscular injection produced strong humoral and cellular immune responses which were dose-and time-dependent.Ad-XT-t PA-Sopt and Ad-PJ-t PA-Sopt induced higher neutralizing antibody titers than Ad-XT-ori SIP-S1opt and Ad-PJ-ori SIP-S1opt,respectively(P<0.05).3.Compared with intramuscular injection routes,mice immunized with Ad-XT-t PA-Sopt by mixed and oral gavage administrations produced not high levels of small intestinal Ig A antibodies(P>0.05)and lower neutralizing antibody titers(P<0.05),and the content of CD8~+T cells was lower in mice immunized by oral gavage administration(P<0.05).4.Ad-XT-t PA-Sopt and Ad-PD-t PA-Sopt produced similar neutralizing antibody titers as PEDV and PDCoV inactivated vaccines by intramuscular injection,respectively(P>0.05).5.The mice immunized with PDCoV recombinant adenoviruses by intramuscular injection produced strong humoral and cellular immune responses which were dose-and time-dependent.Ad-PD-t PA-Sopt produced higher neutralizing antibody titers than Ad-PD-ori SIP-S1opt(P<0.05).6.Compared with intramuscular injection routes,mice immunized with Ad-PD-t PA-Sopt by oral gavage administration produced not high levels of small intestinal Ig A antibodies(P>0.05)and lower contents of neutralizing antibodies and CD8~+T cells(P<0.05),and the contents of neutralizing antibodies and CD8~+T cells were similar in mice immunized by mixed administration(P>0.05).7.Ad-XT-t PA-Spot and Ad-PD-t PA-Spot produced similar neutralizing antibody levels between combined and single immunizations in mice(P>0.05).In conclusion,both the PEDV and PDCoV vaccines based on recombinant adenoviral vector by intramuscular injection produced robust humoral and cellular immune responses in mice,suggesting that they would be promising vaccine candidates.