Canine parvovirus VP2 protein expressed in silkworm pupae self-assembles into virus-like particles with high immunogenicity

Abstract

The VP2 structural protein of parvovirus can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. In this study, the VP2 protein of canine parvovirus (CPV) was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4(+) and CD8(+) T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease.

Figure 1. Indirect immune fluorescent antibody assay of VP2 protein expressed in BmN cells infected with recombinant baculovirus.

A, Mock-infected BmN cells, B, Recombinant baculovirus infected BmN cells.

Figure 2. Western blot analysis of VP2 protein expression in the silkworm pupae.

Lane 1, prestained protein marker; lane 2, silkworm pupae infected by recombinant baculovirus; lane 3, silkworm pupae infected by wild type baculovirus; numbers on the left indicate the position of protein size markers.

Figure 3. Electron microscopy of CPV-VLP.

Sample were negatively stained with 2% uranyl acete and observed by electron microscopy. A, Analysis of the particles formed in recombinant baculovirus infected silkworm pupae. B, Particles collected from sucrose gradients (purified VLP). Magnification 40,000×.

Figure 4. Silkworm pupae homogenates hemagglutinate pig erythrocytes.

A, Recombinant baculovirus infected silkworm pupae homogenates. B, Normal silkworm pupae homogenates.

Figure 5. Detection of specific CD4+ and CD8+ Tcell responses.

The Lymphocytes from lymph nodes and spleen were isolated at 3, 6 and 9 days post-immunization. Lymphocytes were recovered and stained with cell surface markers CD3, CD4 and CD8. The stained cells were analyzed by flow cytometry (a,b). A, Representative flow cytometric plots of lymphocytes in lymph nodes and spleen at day 9 post immunization. B, All date are from n = 3 mice in each group and presented as mean values±standard errors(SE). Asterisks indicate significant differences between the experimental groups: *, p<0.05.

Figure 6. Immunogenicity of VLP-VP2 in mice (determination of antibody titers of VLP-VP2 in mice).

Figure 7. Immunogenicity of VLPs in dogs. Serum was analysed by HI.