Rabbit hemorrhagic disease virus VP60 protein expressed in recombinant swinepox virus self-assembles into virus-like particles with strong immunogenicity in rabbits

Abstract

Rabbit Hemorrhagic Disease (RHD) is an economically significant infectious disease of rabbits, and its infection causes severe losses in the meat and fur industry. RHD Virus (RHDV) is difficult to proliferate in cell lines in vitro, which has greatly impeded the progress of investigating its replication mechanism and production of inactivated virus vaccines. RHDV VP60 protein is a major antigen for developing RHD subunit vaccines. Herein, we constructed a TK-deactivated recombinant Swinepox virus (rSWPV) expressing VP60 protein and VP60 protein coupled with His-tag respectively, and the expression of foreign proteins was confirmed using immunofluorescence assay and western blotting. Transmission electron microscopy showed that the recombinant VP60, with or without His-tag, self-assembled into virus-like particles (VLPs). Its efficacy was evaluated by comparison with available commercial vaccines in rabbits. ELISA and HI titer assays showed that high levels of neutralizing antibodies were induced at the first week after immunization with the recombinant strain and were maintained during the ongoing monitoring for the following 13 weeks. Challenge experiments showed that a single immunization with 10⁶ PFU of the recombinant strain protected rabbits from lethal RHDV infection, and no histopathological changes or antigenic staining was found in the vaccine and rSWPV groups. These results suggest that rSWPV expressing RHDV VP60 could be an efficient candidate vaccine against RHDV in rabbits.

Keywords: VP60 protein; rabbit hemorrhagic disease virus; recombinant swinepox virus; vaccine; virus-like particles.

Figure 1

Construction and characterization of recombinant strains in PK15 cells. (A) Diagram indicating recombinant swinepox virus constructs.TK, thymidine kinase gene; LF, left flank; RF, right flank; EGFP, enhanced green fluorescent protein; P11, vaccinia virus P11 promoter; P28, vaccinia virus P28 promoter; RHDV VP60, rabbit hemorrhagic disease virus VP60 protein; His, Histidine. (B) PCR analysis of the recombinant virus. (C) Immunofluorescence assay detection of the recombinant swinepox virus. The fluorescence of EGFP was observed by confocal microscopy directly, identification of VP60 by IFA assay using an anti-VP60 antibody, respectively. Red and green fluorescence was observed in strain rSWPV-VP60 and rSWPV-VP60-His, but not in strain SWPV-JX20G. The scale bar represents 100 μm. (D) Single-step growth curve. Infected cells were harvested at the indicated hours post-infection. The virus titer was determined by qPCR. Wild-type SWPV JX20G was used as controls. Titers (genome copies/μl) represent the averages of the results of three independent experiments. The titer of the recombinant swinepox virus was highest at 72 hpi.

Figure 2

Analysis of recombinant protein expression. (A) Expression pattern of the recombinant protein as studied by SDS-PAGE. Samples from freeze–thaw supernatant and pellet (lanes 1 and 2, rSWPV-VP60; lanes 3 and 4, rSWPV-VP60-His), respectively, were run in SDS-PAGE gels under reducing conditions. (B) To determine the kinetics of protein expression, 100% confluent PK15 cells were infected with rSWPV-VP60 or rSWPV-VP60-His at an MOI of 5. Equal amounts of total freeze–thaw supernatant, harvested at the indicated time points post-infection, were analyzed by SDS-PAGE, followed by western blotting using the antibodies described in Materials and Methods. A representative blot of the GAPDH as a control protein is also shown. The expression level of VP60 proteins peaked at 120 hpi. (C) TEM images of RHDV VP60 VLPs. Supernatants of infected PK15 cell lysates were filtered and concentrated 10-fold using Amicon Ultra 100 K centrifugation columns and dialyzed in PBS buffer. Samples were negatively stained with 0.5% aqueous uranyl acetate. TEM analysis showed that VP60 proteins assembled into VLPs with diameters of 33–40 nm. Bar = 200 nm.

Figure 3

Humoral immune response to the commercial RHDV vaccine, rSWPV-VP60 or rSWPV-VP60-His in rabbits. (A) Purified VP60-His protein as capture antigen for ELISA. Lane 1: VP60-His protein expressed using the swinepox virus/PK15 system; land 2: the effluent of protein purification; lane 3: the purified VP60-His protein. (B) Anti-VP60 antibody levels were measured by ELISA in serum samples of pre-immune or immunized rabbits using purified recombinant VP60-His. (C) The hemagglutination inhibition (HI) titers of serum samples from immunized rabbits were detected, *p < 0.05, **p < 0.01; #p < 0.05, ##p < 0.01, using one-way ANOVA.

Figure 4

Protective efficacy of the rSWPVs in rabbits. (A) Survival curves of rabbits after a lethal challenge with the RHDV. All the rabbits in 4 groups (6 rabbits in each group) were challenged with RHDV at 13 weeks after immunization and survival rates were recorded for 7 days after the challenge. (B) IHC analysis of lung, liver, and spleen from immunized rabbits. Images of IHC analysis presented that classic RHDV VP60 was predominantly located in the cellular cytoplasm of rabbits immunized with the DMEM, but not in that of rabbits immunized with RHDV Vaccine, rSWPV-VP60, and rSWPV-VP60-His. Magnification ×400, Bar = 50 μm.