Effect Of RGD-Modified PCV2 VLP On Antibody Production Level And The Mechanism In Interaction With Cellular Proteins

Porcine circovirus type 2(PCV2),one of the most important pathogens and prevail worldwide in pigs,causes porcine circovirus diseases(PCVD)that bring huge economic losses in swine industry.At present,vaccination is an effective and efficient way to fight against PCV2 infection.The novel PCV2 virus-like particles(VLPs)formed by PCV2 capsid proteins as a promising subunit vaccine with its advantages of safety and high potency has already been used in farms.However,attempts to use mass vaccination do not eradicate PCV2 from farms.To realize the goal of disease control by effective vaccination,to date further research is necessary.Arg-Gly-Asp(RGD),a common peptide motif responsible for cell adhesion,is well recognized and bound by integrins,and has been utilized by lots of pathogenic agents.Studies indicated RGD modified VLPs or virus could enhance cell adhesion,viral shedding,and immune respond,while it has not been applied on PCV2.In order to study the effect of incorporated RGD motif on the invasion and immunogenicity of PCV2-VLP,loop CD of N terminal truncated PCV2 capsid protein was inserted or replaced by RGD motif.Then chimeric PCV2 capsid proteins were expressed in E.coil and assembled to VLPs in vitro.Subsequently,formed VLPs were observed via transmission electron microscope(TEM)and the traits of chimeric VLPs when invading IPEC-J2 cells were determined by indirect immunofluorescence(IFA).What’more,mice were immunized with chimeric VLPs according to the protocol.Both humoral and cellular immune responds were elicited and tested by ELISA.Furthermore,co-immunoprecipitation(Co-IP)and mass spectrometric(MS)analysis were applied to investigate the interactions between the cell surface receptors and the RGD motif.Our results showed that two chimeric PCV2 capsid proteins iRGD,one copy of RGD motif was inserted into the loop CD of N terminal truncated PCV2 capsid protein,and rRGD,two copies of RGD with a GS linker replaced the eight amino acids of loop CD,were successfully expressed and purified.iRGD-VLP was successfully formed in vitro and capable to invade IPEC-J2 and PK15 cell whereas have distinct distribution comparing with WT-VLP.However,rRGD could not assemble into VLP.ELISA showed that when compared with original PCV2-VLP,mice immunized with iRGD-VLP produce more specific antibodies(IgM and IgG)agasint PCV2.Besides,iRGD-VLP could induce spleen to secrete high level of IFN-γ in mice.Moreover,We observed that RGD insertion facilitated cell entry of PCV2-VLPs,and RGD modified PCV2-VLPs had stronger bond with heparitin sulfate recepter,actin associated proteins and Nucleolin meanwhile specifically retargeting some host cellular proteins(e.g.vinculin,CD44 and other proteins associated with viral invasion).These results were contributed to explain the effect of enhanced immunogenicity caused by RGD insertion.Taken together,this study indicated that the RGD modified PCV2-VLPs facilitated cell entry,and significantly increased the level(and quality)of the PCV2-VLPs immunogenicity,compared to original PCV2-VLPs.Hence,RGD modified PCV2-VLPs could be a novel highly immunogenic PCV2 vaccine candidate and RGD modification had potential as an approach for quality improvement of virus like particles vaccine.