Preparation And Immunogenicity Evaluation Of Multi-epitopes Immunogen And Epitope Chimeric Virus-like Particles Of Foot-and-mouth Disease Virus Serotype O In Escherichia Coli

Foot-and-mouth disease(FMD)is a highly contagious disease of cloven-hoofed animals including cattle,pigs,sheepand many wildlife species.It can cause enormous economic losses when incursions occur into countries which are normally disease free.In addition,it has long-term effects within countries where the disease is endemic due to reduced animal productivity and the restrictions on international trade in animal roducts.The current control measures to prevent the spread of the Foot-and-mouth disease include restriction of animal movements,decontamination of affected premises,slaughtering of diseased and sick-contact animals,and regular mass vaccination of animals in some endemic regions.Especially in developing countries,vaccine immunity is still the main means of controlling foot-and-mouth disease.China currently implements a comprehensive prevention and control technology that combines compulsory immunization and culling.However,there are many limitations.For instance,cultivation of virulent FMD virus(FMDV)in the manufacturing units poses a risk of escape from production sites.Vaccines may sometimes contain traces of FMD viral non-structural proteins(NSPs),therefore,interfering with the NSP-based serological differentiation infected from vaccinated animals(DIVA).Poor stability,low temperature are needed during transport and storage.Moreover,vaccines are unable to eliminate virus from carrier animals.To address the shortcomings of inactivated vaccines,many efforts are currently devoted to develop novel vaccines.The epitope vaccine became one of the new candidate vaccines.In this study,two B cell epitopes and one T cell epitope on structural protein VP1,and one T cell epitope on the non-structural protein 3A of the serotype O FMDV were selected as the target sequence.And one amino acid mutation was introduced in one of the B cell epitopes.Four epitopes are arranged in different combinations and multiple tandem repeats.The artificially synthesized three nucleotide sequences were inserted into plasmid p ET28(a)to construct recombinant plasmids,respectively.The recombinant multi-epitope proteins were expressed in E.coli BL21(DE3).Recombinant proteins were expressed as inclusion bodies.Purified,renatured and cyclized recombinant proteins immunized animals.The recombinant multi-epitope proteins r P2 stimulated to produce slightly higher specific antibody levels than the synthetic peptide vaccine in guinea pigs,and induced a strong cellular immune response.However,when preforming a challenge test on pigs,r P2 only protects two pigs.At the same time,phage MS2 was used as a virus-like particle platform to display exogenous polypeptides.Nucleotide sequence containing amino acids 131-160 of FMDV VP1 inserted into the MS2 phage coat protein gene sequence.The chimeric sequence was inserted into the vector p ET28(a)to construct a recombinant plasmid.Chimeric protein was expressed and self-assembled into virus-like particles in vitro.The chimeric virus-like particles induced to produce higher specific antibody titers than synthetic peptide vaccines,and also stimulated higher cellular immune response than tandem repeat proteins.1.Preparation and immunogenicity analysis of recombinant multi-epitope antigen of serotype O FMDVAccording to the domestic epidemic porcine serotype O Mya98 lineage O/MYA98/BY/2010 strain,we select position 124-167(including the GH loop and its two flanking sequences)and position 200-213 on FMDV VP1 as the B cell epitope.Choosing position 21-40 on VP1 and position 21-35 on non-structural protein 3A as T-cell epitopes,and mutations at position P158 of 124-167 are replaced by cysteine(P158C).The four epitope sequences were arrayed in different combinations and tandem repeats.Codons of three sequences were optimized and artificially synthesized.Recombinant vectors were constructed and transformed into E.coli BL21(DE3)for expression.Three recombinant proteins were expressed as inclusion bodies.Three proteins were purified,renatured and cyclized.Western blotting and indirect ELISA assays were performed to verify the immunoreactivity of recombinant proteins.Three recombinant proteins and synthetic peptide vaccines were immunized in guinea pigs.Multi-epitope protein r P2 with cyclization and two tandem repeats stimulated to produce more specific antibodies than synthetic peptide vaccines,but there is no significant difference between them.Recombinant protein r P2 induced higher antibody levels,more lymphocyte proliferation and cytokine concentrations(IL-2 and IFN-?)than the recombinant protein r P1 and r P3.Among four different kinds of adjuvants,the ISA 50V2 adjuvant emulsified with recombinant multi-epitope protein r P2 was the best choice.The mixture of ISA50V2 and r P2 was immunized in swine,which stimulated to produce the same levels of specific antibody with synthetic peptide vaccines.But only 2 pigs out of 5 were resistant to virus challenge,and other three pigs developed clinical symptoms.Two of which delayed the appearance of blisters,and symptoms appeared on the 8th and 10 th after challenge.Tandem recombinant proteins provide only partial protection,which does not achieve the intended purpose.We need to further analyze and find ways to solve these problems.2.Construction and immunogenicity analysis of phage MS2-mediated display of pig serotype O FMDV G-H Loop VLPsIn this experiment,the phage MS2 was used as a vector platform to display foreign epitopes.The amino acid sequence 131-160 of FMDV VP1 was inserted into the amino acids 15-16 of the A-B loop of phage MS2 by RT-PCR and SOE-PCR.The recombinant plasmid was constructed,and recombinant proteins were expressed in Escherichia coli BL21(DE3).The chimeric protein was purified by PEG8000 and gel-filtered,and the purity reached to 90%.Chimeric proteins were physically characterized by DLS and TEM,and it was confirmed that the insertion of 30 amino acids din not affect the assembly of the MS2 coat protein.Chimeric proteins self-assemble to form chimeric virus-like particles in vitro.DOT-ELISA further showed that the epitope of FMDV was well displayed on the surface of MS2 VLPs.Chimeric virus-like particles r P-CP-EP131-160 and tandem multi-epitope protein r P–(EP131-160)3 immunized mice,and chimeric virus-like particles induced higher specific antibody levels and stronger T cell responses than tandem multi-epitope proteins.Similar results were obtained in guinea pig immunization trials.Whether the chimeric virus-like particles could stimulate to produe the same or higher antibody levels as synthetic peptide vaccines in swine,and could provide complete protection when challenged,all these need to be further confirmed by experiments.