Trigger factor assisted self-assembly of canine parvovirus VP2 protein into virus-like particles in Escherichia coli with high immunogenicity

Abstract

Canine parvovirus (CPV) has been considered to be an important pathogen, which can cause acute infectious disease in canids. Although current vaccines are effective in preventing CPV infection, safety problems still remain unsolved. In this study, a subunit vaccine against CPV based on virus-like particles (VLPs) with good safety and immunogenicity is reported. Soluble CPV VP2 protein was produced by co-expression of chaperone trigger factor (Tf16) in Escherichia coli (E.coli), and assembled into CPV VLPs which could be affected by NaCl and pH. At 250 mM NaCl pH 8.0, the VLPs co-expressed with Tf16 had similar size (25 nm) and shape with the authentic virus capsid under the transmission electron microscopy (TEM), which is also in accordance with the dynamic light scattering (DLS) data. Immunization with these particles could induce high-titer hemagglutination inhibition (1:12288) and neutralizing antibodies (1:6144) in guinea pigs. Splenic cells of them could secrete IFN-γ and IL-4 after stimulation by CPV. Thus, the VLPs produced by the new approach with high yield and immunogenicity could be a potential candidate for CPV vaccine.

Keywords: Canine parvovirus; Escherichia coli; Immunogenicity; Trigger factor 16; Virus-like particle.

Fig. 1

Expression and purification of CPV VP2 protein co-expressed with Tf16. CPV VP2 protein were analyzed by 12% SDS-PAGE and Western blot using an anti-CPV polyclonal antibody a SDS-PAGE analysis of the VP2 protein expression. Lane M, protein molecular weight (MW) markers; Lane 1, pET30a vector control; Lane 2, pET30a-VP2 cells before induction of IPTG; Lane 3, induced pET30a-VP2 cells; Lane 4, induced pET30a-VP2 cell lysate; Lane 5, inclusion body of induced pET30a-VP2 cells; Lane 6, pET30a-VP2-Tf16 cells before induction of IPTG and L-Arabinose; Lane 7, induced pET30a-VP2-Tf16 cells; Lane 8, induced pET30a-VP2-Tf16 cell lysate; Lane 9, inclusion body of induced pET30a-VP2-Tf16 cells. b SDS-PAGE analysis of purification of VP2 protein co-expressed with chaperone Tf16. Lane 1, cells before induction; Lane 2, induced cells lysate; Lane 3, flow buffer; Lane 4, wash buffer; Lane 5, purified VP2 protein. c Western-blot analysis of purified VP2 protein with an anti-CPV polyclonal antibody. Lane1, purified VP2 protein; Lane 2, induced cell lysate; Lane 3, cells before induction. The MW of VP2 protein is approximately 70 kDa. The Tf16 is approximately 56 kDa

Fig. 2

Assembly and characterization of CPV VLPs under different conditions. a DLS results of VLPs assembled at pH 7.0 and 8.0. b DLS results of VLPs assembled in different concentrations of NaCl (150, 250, 400 mM). c TEM result of VLPs co-expressed with Tf16. Bar = 200 nm d TEM result of VLPs without co-expression of Tf16. Bar = 200 nm e DLS result of VLPs without co-expression of Tf16

Fig. 3

Anti-CPV antibody responses in guinea pigs. Groups of guinea pigs (n = 5) were immunized with 50 μg, 30 μg and 10 μg of CPV VLPs, commercial CPV vaccine and PBS. Serum samples were collected once a week until 42 dpi for the detection of neutralization and HI antibody titers. a Neutralization antibody titers of guinea pigs. b HI antibody titers of guinea pigs. The values are expressed as means ± SEM. The statistical significance of antibody titer differences between vaccinated and PBS control guinea pigs were analyzed by one-way ANOVA statistical analysis (*p < 0.05, **p < 0.01, and ***p < 0.001)

Fig. 4

Analysis of cytokine production secreted from the spleen lymphocytes of guinea pigs. Spleens of guinea pigs from each group were removed at 42 dpi. The culture supernatants of cells stimulated by CPV were collected after 72 h and detected by ELISA kits. a the concentration of guinea pig IFN-γ in each group. b the concentration of guinea pig IL-4 in each group. The values are expressed as means ± SEM. The statistical significance of cytokine concentration differences between vaccinated and PBS control guinea pigs were analyzed by one-way ANOVA statistical analysis (*p < 0.05, **p < 0.01, and ***p < 0.001)