Direction Targeting Of PCV2Capsid Protein To DEC-205Elicits Both Hummoral And Cellular Immune Responses In Mice

Porcine circovirus(PCV) is a major causative agent for inducingimmunosuppression in porcinehostsand has beenassociated with PostweaningMultisystemic Wasting Syndrome (PMWS), porcine reproductive failure, porcinedermatopathy and nephropathy syndrome (PDNS), which has been a great threat toswine industries around the world. Current commercial vaccine against PCV2can beclassified as inactive, chimeric virus vaccine, and subunit vaccine. These currentcommercially vaccine shows ability at reducing clinical diseases, however, vaccinationdoes not completely prevent the spread and infection of PCV2. Moreover, theinactivated and attenuated vaccine contains genetic material which may lead tovirulence recovery. Therefore, a safe and high immunogenic vaccine is needed for thecontrol of PCV2spread. The Cap protein is the major structural protein encoded byORF2of PCV2and can be reacted with neutralizing monoclonal antibodies andneutralizing swine sera. Recent studies have focused on the main structure andimmunogenic Cap protein and successfully expressed in Escherichia coli).In this study we generated a novel fusion protein using Cap protein fused withantibodies that target to dendritic cells (DCs). DCs are antigen presenting cells (APCs)with strong capacities for capturing antigen, triggering and regulating immuneresponses. DEC-205is a type I C-type lectin receptor, expressed by DCs that belongs tothe mannose receptor (MR) family. In DCs, DEC-205plays a critical role in antigeninternalization, processing and presentation. Strategy of targeting antigen to DEC-205has been successfully performed, such as gag (the major protective antigen of Humanimmunodeficiency virus, HIV), ED Ⅲ (the major structure protein of Denguevirus).Herein we generate a novel recombinant subunit vaccine with highimmunogenicity by adding a single chain antibody fragment(scFv) against DEC-205tothe N terminal of Cap protein inE. coli. BALB/c mice were immunized to evaluate thespecific immune responses induced by scFvCD205-Cap. In addition, a preliminarystudy about antigen mechanism of direction targeting of PCV2capsid protein to thedendritic cell by a multilectin receptor DEC-205was carried out. Ourresults indicate thatthe hybrid vaccine may be a promising and safe candidate vaccine for protectionofPCV2infection.The main content of the work and results as shownbelow: ①The construction and purification ofscFvDEC205-CapThe scFvDEC205-Capgene fragments were inserted into prokaryotic expressionvector pGEX6P-1.The recombinant fusion protein scFvCD205-Cap was expressedaccording to the AraC-araBAD promoter-Regulated T7Expression system and inducedby adding arabinose at a final concentration of2g/L for4h at37℃.The target proteinwas purified followed the procedure ofsonication, inclusion bodies denaturation, affinitychromatography purification and protein refolding. The targetproteinswerecharacterizedby SDS-PAGE and Western blot.②Specific immune responses induced by recombinant scFvDEC205-CapTheELISAresults showed that: specific IgG antibody against Cap protein afterprimer immunization was not increased for all groups. After the boost immunization,the IgG titers were greatly increased. Groups administratedwithscFvDEC205-Capshowed higher hummoral responses (1:6400)same as those treatedwith inactivated PCV2(1:12800), which were higher than mice treated with Cap protein(1:1600, p＜0.05). The negative group were treated with GST and scFvDEC205showed nospecific anti-Cap antibody responses. All of the above results indicated thatthe recombinant protein could elicit higher hummoral responses in mice compared toCap and GST. The IFA results showed that the group of scFvDEC205-Cap and Capcould protect PK15cell from PCV2infection, the neutralization titer was1:16and1:12after boost immunization, respectively; whereas no neutralization activity was detectedin scFvDEC205and GST groups. The serum neutralization titer induced by inactivatedPCV2was higher than scFvDEC205-CAP up to1:20. The animal immunization resultssuggested that the recombinant protein scFvDEC205-Cap could induce strongerimmune responses in Balb/c mice compared with Cap alone, which couldbe comparable to PCV2inactivated vaccine. Since this new fusion subunit vaccinepossess APCs target function, and could stimulate stronger immune responses withoutgenetic material.Firstly, compared to the live vaccine and inactivated vaccine, subunitvaccine do not contain any unsafe factors; secondly, this new type of subunitvaccinecarrying anti DEC-205monoclonal antibody, with higher immunogenicitycompared to the subunit vaccine compared with the traditional subunit vaccine.③preliminary study about antigen mechanism of direction targeting ofPCV2capsid protein to the dendritic cell by a multilectin receptor DEC-205The flow cytometry results implied that, the recombinant protein couldstimulate bone marrow derived dendritic cells(BMDCs)tomaturity under vitro conditions, and the binding efficiency between scFvDEC205-Cap and BMDCswas higher than Cap (p<0.05).Similarly, theenzyme linked immunosorbent assay(ELISA) and enzyme linked immunospot assay (ELISPOT)results showed thatT cellcould secreteIL-17, IL-4and IFN-γstimulated by scFvDEC205-Cap&DCs, and thesecretion of IFN-γwas obviously higher than IL-4. The antigen present pathwayinhibiting results suggested that all inhibit groups showed low IFN-γ secretion abilitycompared to non-treated group (p<0.05).These results indicated that the TAP, HSP90and proteasome were mayinvolved in scFvDEC205-Cap antigen present mechanism.In conclusion, direction targeting of PCV2capsid protein to the dendritic cell by amultilectin receptor CD205elicits bothhummoral and cellular immune responses inmice compared with Cap alone. These results willprovidetheory and experimentinformation for vaccine design for a novel PCV2vaccine or other virus vaccine.