Preliminary Study On The Bivalent Multi-epitope Immunogen Against Foot-and-Mouth Disease Virus Type A And O Based On Different Built-in Adjuvants

Foot and mouth disease(FMD)is a highly infectious disease caused by foot and mouth disease virus(FMDV)that mainly infects cloven-hoofed animals such as cattle,pigs,sheep and goats and can cause serious economic losses.At present,the application of inactivated vaccine against FMD is still the main means to prevent and control FMD.However,in view of the hidden dangers of inactivated vaccine in the process of manufacture,it is urgent to develop a new safe and effective subunit vaccine of FMD.In the field of FMD epitope vaccine research,how to present FMDV neutralizing epitopes to the immune system more effectively,so as to stimulate the immune response against FMD infection more efficiently,is the main direction of epitope vaccine research at present.In this study,three kinds of built-in adjuvants were selected,namely hepatitis B core antigen(HBcAg),the carboxyl terminal peptide binding region of mycobacterium tuberculosis heat shock protein 70(mHSP70C)and heparin-binding hemagglutinin adhesin(HBHA).In addition,the coding genes of the B cell epitope(VP1 134-161 aa and 200-213aa)of type A and O FMDV representative strains or the T cell epitope(3A 21-35aa)of FMDV non-structural protein 3A were inserted into the major immunogenic region(MIR)of truncated HBcAg,and different recombinant antigens rHBc/AO and rHBc/AOT were obtained in prokaryotic expression system.After affinity chromatography and urea gradient dialysis,virus-like particles(VLPs)containing FMDV epitopes were efficiently self-assembled in vitro.In addition,the bivalent multi-epitope gene of type A and O FMDV were fused with the coding genes of mHSP70 C and HBHA respectively,and the HAO and HBHA genes were constructed into prokaryotic expression vector pET-28 a and the recombinant proteins AO-HSP,HAO,HBHA and HAOHBHA were successfully expressed and purified in prokaryotic system.The immune effects of rHBc/AO,rHBc/AOT VLPs and a series of multi-epitope recombinant proteins AO-HSP,HAO-HBHA were evaluated in mice model.Both rHBc/AO and rHBc/AOT VLPs can stimulate the production of specific neutralizing antibodies against type A and O FMDV.And both of them could significantly stimulate the Th1 type cellular immune response,while inactivated vaccine was more likely to stimulate the Th2 type cellular immune responses.At the same time,compared with rHBc/AO VLPs,rHBc/AOT VLPs can produce a higher level of Th2 type cellular immune responses.The proliferation level of splenic lymphocytes and the antibody level of HAO-HBHA immunization group were significantly higher than that of HAO+HBHA mixed immunization group,HAO and HAO +206 adjuvant immunization group.In sheep model,HAO-HBHA immunized group can produce the same level of FMDV specific antibody as inactivated vaccine group.It is concluded that the epitopes of multiple serotypes of FMDV was inserted into the MIR region of HBc to improve the broad-spectrum of this type of VLPs vaccine.In addition,mHSP70 C and HBHA also have advantages in enhancing the immunogenicity of FMDV epitope-based vaccines,especially HBHA has a good application prospect in improving the level of FMDV specific immune responses.