Antigenic and immunogenic properties of recombinant proteins consisting of two immunodominant African swine fever virus proteins fused with bacterial lipoprotein OprI

Abstract

Background: African swine fever (ASF) is a highly fatal swine disease, which threatens the global pig industry. There is no commercially available vaccine against ASF and effective subunit vaccines would represent a real breakthrough.

Methods: In this study, we expressed and purified two recombinant fusion proteins, OPM (OprI-p30-modified p54) and OPMT (OprI-p30-modified p54-T cell epitope), which combine the bacterial lipoprotein OprI with ASF virus proteins p30 and p54. Purified recombinant p30 and modified p54 expressed alone or fused served as controls. The activation of dendritic cells (DCs) by these proteins was first assessed. Then, humoral and cellular immunity induced by the proteins were evaluated in mice.

Results: Both OPM and OPMT activated DCs with elevated expression of relevant surface molecules and proinflammatory cytokines. Furthermore, OPMT elicited the highest levels of antigen-specific IgG responses, cytokines including interleukin-2, interferon-γ, and tumor necrosis factor-α, and proliferation of lymphocytes. Importantly, the sera from mice vaccinated with OPM or OPMT neutralized more than 86% of ASF virus in vitro.

Conclusions: Our results suggest that OPMT has good immunostimulatory activities and immunogenicity in mice, and might be an appropriate candidate to elicit immune responses in swine. Our study provides valuable information on further development of a subunit vaccine against ASF.

Keywords: African swine fever virus; Immune response; Immunomodulation; OprI; Recombinant fusion proteins; Vaccine.

Fig. 1

Construction and expression of recombinant fusion proteins. A All five constructs, denoted as p30, modified p54, PM, OPM and OPMT, were inserted into expression plasmid pET30a (+) and expressed in E. coli. B SDS-PAGE analysis of purified recombinant proteins. Lane M, molecular weight markers; lane 1, p30; lane 2, modified p54; lane 3, PM; lane 4, OPM; lane 5, OPMT. Proteins bands were visualized by Coomassie staining

Fig. 2

Immunoblot analysis of the recombinant proteins. A The recombinant proteins were subjected to western-blotting with 1:5000 diluted anti-polyhistidine monoclonal antibody as primary antibody and 1:5000 diluted goat anti-mouse peroxidase conjugated secondary antibody. B Western blotting confirmation of recombinant proteins using 1:300 diluted anti-ASFV swine serum as primary antibody and 1:5000 diluted goat anti-pig peroxidase conjugated secondary antibody. Lane M: molecular weight markers; lane 1, p30; lane 2, modified p54; lane 3, PM; lane 4, OPM; lane 5, OPMT; lane 6, bovine serum albumin

Fig. 3

Expression of costimulatory and antigen-presenting molecules by DCs induced by recombinant proteins. BMDCs (10⁶/well) were incubated with p30 + modified p54, PM, OPM or OPMT (10 μg/mL each) for 24 h at 37 °C under 5% CO₂. RPMI-1640 complete medium and LPS (0.1 µg/mL) served as negative and positive controls, respectively. After staining with anti-CD11c, anti-CD40, anti-CD80, anti-CD86 or anti-MHC-II fluorescent antibodies, the cells were gated on the CD11c-positive population to analyze expression of CD40, CD80, CD86 and MHC-II by flow cytometry. The percentages of CD40 (A), CD80 (B), CD86 (C) and MHC-II (D) positive cells in each group were calculated. The graph bars represent mean ± SD (n = 3); ns = P > 0.05; *** P < 0.001

Fig. 4

Cytokine production in BMDCs induced by recombinant proteins. BMDCs (10⁶ cells/well) were stimulated with p30 + modified p54, PM, OPM, OPMT (10 μg/mL each) or LPS (0.1 µg/mL) for 24 h at 37 °C under 5% CO₂. The quantities of TNF-α (A) and IL-12p70 (B) in the culture supernatant were determined by commercial ELISA kits. All data are displayed as mean ± SD (n = 3); ns = P > 0.05; *P < 0.05; ***P < 0.001

Fig. 5

Levels of IgG to p30 and p54 detected by indirect ELISAs. Five BALB/c mice/group were immunized by subcutaneous route with each recombinant protein (30 μg) or an equal volume of PBS. Blood samples were obtained at 0, 7, 21 and 28 dpv. IgG levels to p30 (A) and p54 (B) of each group at serum dilutions of 1:100 were measured by indirect ELISAs and are expressed as OD₄₅₀ (mean ± SD, n = 5). Characterization of p30-specific (C) and p54-specific (D) IgG isotype profiles of sera (diluted in twofold series) from immunized mice at 28 dpv by indirect ELISAs. The titer of a given serum sample is defined as the reciprocal value of the highest dilution yielding a positive result and displayed as mean ± SD (n = 3); ns = P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 6

Cytokine levels and T lymphocyte proliferation in splenocytes from mice that received recombinant proteins or PBS. IL-2 (A), IFN-γ (B) and TNF-α (C) produced in splenocytes from mice immunized with each recombinant protein or PBS were detected using commercial ELISA kits. Each value represents the mean ± SD of cytokine production in splenocytes from three individual mice. T lymphocyte proliferation of immunized mice was assessed by CCK8 assays and is presented as a stimulation index (D). The graphs show mean results with error bars indicating the SD (n = 3); ns = P > 0.05; **P < 0.01; ***P < 0.001

Fig. 7

Intracellular cytokine expression in splenocytes from mice that received recombinant proteins or PBS. (A) Splenocytes from mice in each group were isolated at 28 dpv and stimulated with p30 + modified p54 (10 μg/mL) for 40 h and incubated with monensin (1.7 μg/mL) for another 8 h at 37 °C under 5% CO₂. After staining with APC-conjugated anti-CD3, FITC-conjugated anti-CD8α and PE-conjugated anti-IFN-γ antibodies, the cells were gated to select CD3⁺ T lymphocyte (represented by p2) and the percentages of IFN-γ⁺ and CD8⁺ T cells in CD3⁺ T lymphocyte were analyzed by flow cytometry. (B) Calculated percentages of IFN-γ⁺ CD8⁺ T cells from three separate experiments. Chart shows means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 8

Neutralization of ASFV infection by sera from immunized and non-immunized mice. CN/SC/19 was pre-incubated with heat-inactivated pre-immune sera, immune sera (both at 1:5 dilution) or an equal volume of medium and used to infect PAMs. Viral production was measured at 5 days after infection by plaque assay. A Numbers of lysis plaques were counted and presented as means ± SD (n = 3). B Neutralization percentages of sera from mice that received each recombinant protein were calculated. Chart shows average of sera from three mice in each group with standard error; ns = P > 0.05; *P < 0.05; ***P < 0.001