Expression And Immunogenicity Of Rabbit Hemorrhagic Disease Virus-Like Particles Carrying B Cell Epitopes Form Food-And-Mouth Disease Virus

Rabbit hemorrhagic disease(RHD) is a highly contagious and fatal disease caused by Rabbit hemorrhagic disease virus(RHDV). The capsid protein(VP60) is the exclusive structure protein and can make animals produce neutralised antybody and directly related to immune response. Some research showed that VP60protein could assembly into virus-like particles (VLPs) which was morphologically similar to native RHDV but was not RNA packaged in vitro, without disrupting the formation of VLPs. The virus-like particles have been shown to be highly immunogenic and themselves can be used as adjuvant. Other studies indicated that calicivirus VLPs could be excellent candidates to induce a potent humoral or cellular immune responses to foreign antigens inserted in their particulate structure which were presented by the antigen-presenting cells by means of endocytosis or endocytic, and therefore to be a vaccine vector.Foot-and-mouth disease (FMD) is an acute, febrile and highly contagious disease by foot and mouth disease virus (FMDV). O-type foot-and-mouth disease virus(FMDV) has five B cell epitopes at least, including epitopes from the VP1141to160(G-H loop) and the C terminal amino acid residues200to213from the baseline of the epitope. Both of the epitopes are the most important B cell epitopes, and are the most important antigenic sites of FMDV.And the amino acid residues141to160of FMDV VP1is also contain at least one T cell epitope, which can induce specific T-cell response.Based on these theories, we reported the generation of six recombinant chimeric RHDV-VLPs incorporating a B cell epitope corresponding to aa200-213from FMDV VP1, or a immunocompetence tandem fragment FB [(FMDV VP1141～160aa)-GS-(200-213aa)]of foot-and-mouth disease virus. The foreign epitopes were inserted at three different locations:1) at C-terminal of VP60protein which is predicted to be exposed to the surface of the VLPs;2) at N-terminal of VP60protein, which is predicted to be buried in the internal face of the VLPs;3) at a novel insertion site between amino acid positions306and307aa of VP60protein, which is predicted to be located within an exposed loop at the P2subdomain of VP60protein. We analyzed influences of the six kinds of insertions on the assembly of VLPs, and the immunogenic potential of these six chimeric poreins in vitro and in vivo. The main experiments and results are as follows:1. Four fragments of the target genes were amplified by PCR with four pairs of specific primers designed to amplify VP60gene of RHDV WanFu strain in rabbits. The target genes were encoding into pMD19-T Vector and then choned into eukaryotic expression vector. Then the six recombinant plasmids weire inserted the B cell eptiopes from FMDV. The six recombinant recombinant transfer vectors were transformed into Escherichia coli DH10Bac, and the target genes were integrated into Bacmid by screening with blue-white method and PCR. The results showed that six recombinant shuttle vectors were constructed, which named as CF,306F, NF, CFB,306FB and NFB, respectively.2.The recombinant shuttle vectors were transfected into monolayer Sf9cells with lipofectamine2000. The siginifican cytopathic effect (CPE) was found in Sf9cell culture infected with recombinant shuttle vectors after3-5days incubation. Recombiant baculovirus were harvested. The RNA was extracted with RNAout Kit method and the target genes were determined by RT-PCR. The positive virus stocked and expressed. The expressed chimeric proteins were assayed by IFA, HA, SDS-PAGE and Western-blot. The results showed:the generation of the six recombinant baculovirus were assayed by RT-PCR; Those six chemeric proteins expressed in Sf9cells by IFA; the six chimeric proteins were successfully expressed in Bac-to-Bac baculovirus expression system, and the moecular weight was about65kDa by SDS-PAGE; Western-blot ananysis with monoclonal antibody of RHDV (A3C) and sera against O-type FMDV showed that six chimeric proteins had good VP60-specificity and peptide-specificity; only NF and NFB have hemagglutinating of the six chimeric proteins.3. After being primary purificated, the chimeric proteins were observated by Electronmicroscopic. The six chimeric proteins were mixed seperately and emulsified1:1with Freund’s adjuvant, and the ICR mice were immunized with these vaccine, meanwhile set RHDV subunit vaccine and O-type FMDVsynthetic peptide vaccine as positive control, and sterile PBS as negative control. The titer of antibody against VP60and anti-FMDV VP1antibody were measured by indirect ELISA measured at Od,7d,14d,21d,28d,35d and42d. The results showed:All the chimeric proteins correctly assembled into virus-like particles (VLPs) which was morphologically and structurelly similar to native RHDV. All of the six chimeric proteins can induce strong anti-VP60humoral immunes and moderate anti-peptide humoral immunes by means of subcutaneous immunization. And the chimeric protein NF has the highest level of epitope-specific antibody. The peptide-specific antibody could still maintain moderate level even if the foreign fragments inserted up to126bp. In this study, the level of antibodies been not affected by adjuvant; and the two doses (50ug and25ug) also did not affect the level of peptide-specific antibody.We have analyzed the potential of virus like particles(VLPs) from rabbit hemorrhagic virus(RHDV) as a delivery system for foreign B cell epitopes. To accomplish this goal, we generated chimeric RHDV VLPs including two kinds of B cell epitopes derived from O-type foot-and-mouth disease virus(FMDV) VP1protein. The epitopes were inserted in the capsid protein(VP60) of RHDV at the C-terminal, N-terminal and the site between amino acid positions306and307of VP60. The results revealed that the six recombinant shuttle vectors were constructed and successfully expressed in Bac-to-Bac baculovirus expression system; western-blot ananysis showed that six chimeric proteins had good VP60-specificity and peptide-specificity; all constructions can correctly assembly into VLPs by the analysis of Electron microscopy. The animal experiments showed that RHDV-VLPs can induce strong anti-VP60humoral immunes and moderate anti-peptide humoral immunes and keep moderate immunogenicity even being inserted126bp exogenous fragment. All above results had laid an important theoretical foundation for the possibility of RHDV VLPs as multivalent vaccines vector, and these results will be helpful to research the RHDV VLPs display system.