Construction And Immunogenicity Of Chimeric Virus Like Particles(VLPs) And DC-targeting Recombinant Fusion Proteins Of Foot-and-mouth Disease Virus

Foot-and-mouth disease?FMD?is an acute,virulent and highly contagious disease caused by foot-and-mouth disease virus?FMDV?,leading to severe economic losses and social impact.Although conventional FMD inactivated vaccines play a vital role in current FMD prevention and control,series of problems still exist,such as the virus sometimes escapes from vaccine production facilities due to incomplete inactivation.Therefore,there is an urgent need to develop novel genetic engineering vaccine for the prevention and control of FMDV in future.Virus-like particles?VLP?,which are similar to natural virus particles and have higher immunogenicity and security,have been broadly applied in development and applications of vaccine.In related studies,however,the viral capsid of serotype O is more acid sensitive than other types of FMDV and can lead to inefficient self-assemble,and even no self-assemble.Therefore,a chimeric capsid protein gene P12A?O-VP1?of FMDV were formed by the antigenic structural protein gene VP1 from serotype O and segments of the viral capsid proteins gene?VP2,VP3 and VP4?from serotype A.The synthesized gene products encoding chimeric P12A?O-VP1?and proteinase gene 3C was digested with restriction enzyme and cloned into the pFastBac?Dual vector under the control of the polyhedron promoter.The resulting transfer plasmids were named pFBDual-P12A3C?O-VP1?and was transformed into Escherichia coli to generate the recombinant bacmid rBacmid-P12A3C?O-VP1?.Subsequently,the positive bacmids were transfected into Sf9 insect cells to produce recombinant baculoviruses rBac-P12A3C?O-VP1?.The results of immunofluorescence assay?IFA?and Western-blot showed chimeric P12A?O-VP1?gene can be expressed in Sf9 and have good immunogenicity.Transmission electron microscopy revealed that the chimeric P12A?O-VP1?protein could be cleaved by the 3C protease and self-assemble to VLPs that were morphologically similar to empty particles from FMDV.Furthmore,the guinea pigs was immunized by intramuscular injection of expressed VLPs with the ISA-206 adjuvant at an adjuvant:antigen ratio of 1:1.The results showed the chimeric VLPs elicited significant humoral and cellular immune responses of anti-FMDV serotype O and could protected four of the five guinea pigs against challenge with 100 50%guinea pig infectious doses(GPID₅₀)of the virulent FMDV strain.This study provide new way and foundations for future design and development of FMDV VLPs vaccine.In recnet years,it has become an novel design strategy of vaccine that using dendritic cell to improve immunogenicity of vaccine through targeted antigen to dendritic cell.DC-SIGN?DC-specific ICAM-grabbing non-integrin?,also known as CD209,is a C-type lectin receptors that is mostly expressed on DCs.The combination of antigens and DC-SIGN ligands using chemical cross-linking or genetic engineering methods will specifically target antigens to DC and improve its immune effect.In this study,the major antigen protein VP1 of FMDV strain AF/72 were co-expressed with HIV membrane glycoprotein gp120 and HCV envelope glycoprotein E2 by using insect-baculovirus expression system,respectively,and two recombinant fusion proteins VP1-gp120 and VP1-E2 were produced.Furthmore,the guinea pigs was immunized by intramuscular injection of expressed recombinant fusion proteins with the ISA-206 adjuvant at an adjuvant:antigen ratio of 1:1.The immunogenicity among two recombinant fusion proteins and VP1 proteins were compared and the results showed that VP1-gp120 and VP1-E2 could elicited significant specific anti-FMDV IgG and IFN-?and stimulated specific proliferation of DC.More importantly,the ability of VP1-gp120 and VP1-E2 to induce specific anti-FMDV IgG were significant higher than separately expressed VP1protein.Therefore,the design of recombinant fusion protein showed in this study could improve immunogenicity of antigen and provide new way for future study of subunit vaccine.