The Baculovirus Expression System Expresses Chimeric RHDV VLPs as Bivalent Vaccine Candidates for Classic RHDV (GI.1) and RHDV2 (GI.2)

Abstract

Background: Rabbit hemorrhagic disease (RHD) is an acute, hemorrhagic and highly lethal infectious disease caused by rabbit hemorrhagic disease virus (RHDV), which causes huge economic losses to the rabbit breeding industry. Moreover, there is limited cross-protection between the two different serotypes of classic RHDV (GI.1) and RHDV2 (GI.2). The shortcomings of traditional inactivated vaccines have led to the development of novel subunit vaccines that can protect against both strains, and the VP60 capsid protein is the ideal antigenic protein. This study focused on developing a bivalent RHDV vaccine that can prevent infection with both GI.1 and GI.2 strains.

Methodology: Baculovirus vectors containing classic RHDV and RHDV2 VP60 were co-transfected with linearized baculovirus into sf9 cells and transferred to baculovirus via homologous recombination of the VP60 gene. Infected sf9 cells were lysed, and after purification via Ni-NTA chromatography, VLPs were observed using transmission electron microscopy (TEM). In order to evaluate the immunogenicity of the chimeric RHDV VLP vaccine in rabbits, the RHDV VP60-specific antibody, IL-4, IFN-γ and neutralizing antibody titers were analyzed in serum using ELISA and HI.

Results: The recombinant baculovirus system successfully expressed chimeric RHDV VLPs with a diameter of 32-40 nm. After immunization, it could produce specific antibodies, IL-4 and IFN-γ. Following the second immunization, neutralizing antibodies, determined using hemagglutination inhibition (HI) assays, were elicited.

Conclusions: These data show that the chimeric RHDV VLP bivalent vaccine for immunized New Zealand rabbits can induce humoral immunity and cellular immunity in vivo, and the immunization effect of the high-dose group is similar to that of the current commercial vaccine.

Keywords: VP60 protein; chimeric RHDV virus-like particle; rabbit hemorrhagic disease virus; recombinant baculovirus expression system; vaccine.

Figure 1

Construction and expression of recombinant VP60 protein. (A) Diagram indicating the structure of the recombinant VP60 protein produced for chimeric RHDV VLPs. ph: AcMNPV polyhedrin promoter, p10: p10 promoter, MCS: multiple cloning sites. Classic RHDV VP60 and RHDV2 VP60 indicate different target proteins. (B) Recombinant baculovirus infection of sf9 cells in P0, P1 and P2 generations. The fluorescence of EGFP was observed via fluorescence microscopy. (C) PCR analysis of the RHDV VP60 gene. M: DNA ladder, lane1 and lane3: sf9 cells not infected with recombinant baculovirus and amplified with classic RHDV and RHDV2 VP60 protein-specific primers, respectively, lane2: classic RHDV VP60 of gene, lane4: RHDV2 VP60 of gene. (D) 10% SDS-PAGE of VP60 protein expression at different times.

Figure 2

Analysis of recombinant RHDV VP60 expression. (A) SDS-PAGE analysis of recombinant RHDV VP60 expression. Lane1 and lane2: sf9 cells; lane3 and lane4: recombinant VP60 protein. (B) Coomassie Brilliant Blue-stained image of the purified VP60 protein. Lane S: cell lysate sample; lane F: fraction of flow through; lane 20 mM250 mM: fractions of wash and elution with imidazole. (C) Determination of VP60 protein expression. Lane1: sf9 cells; lane2: 250 mM imidazole eluate. (D) TEM images of chimeric RHDV VP60 VLPs. TEM analysis showed that VP60 protein assembled into VLPs with diameters of 33–40 nm. Bar = 200 nm.

Figure 3

Comparative analysis of humoral immune responses induced by chimeric RHDV VLPs versus the commercial vaccine in rabbits. (A,B) Anti-VP60 antibody levels measured via indirect ELISA in serum samples of pre-immune and post-immunized rabbits. (C) The hemagglutination inhibition (HI) titers of serum samples from pre-immune and post-immunized rabbits were detected. ns: not significant (p > 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Evaluation of cellular immune responses induced by chimeric RHDV VLPs and the commercial vaccine through cytokine profiling. (A) IFN-γ levels in rabbit serum. (B) IL-4 levels in rabbit serum. ns: not significant (p > 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.