Evaluation of humoral and cellular immune responses induced by a cocktail of recombinant African swine fever virus antigens fused with OprI in domestic pigs

Figures

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  Fig. 1. Schematic diagram of six fusion proteins, denoted as OPMT, OPET, OCET, PM, PE and CE. OprI, complete sequence of bacterial lipoprotein I; p30, complete sequence of p30; p54-1 and p54-2 respectively represents (Met1-Thr29) and (Ser53-Leu184) of p54; p72-E1, p72-E2, p72-E3 and p72-E4 respectively represents (Gln364-Pro395), (Ala518-Lys552), (Tyr179-Leu210) and (Leu242-Pro273) of p72; △pE248R, pE248R (Met1-Lys198); △CD2v, CD2v (Asp17-Tyr206); △pEP153R, pEP153R (Asn49-Lys158); TT-P2, a universal CD4+ T cell epitope; thick yellow line, linker 1 “ (GGGGS)3”; thin yellow line, linker 2 “PG”

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  Fig. 2. Identification of purified recombinant fusion proteins by SDS-PAGE and western blotting. A SDS-PAGE analysis of purified recombinant proteins. Lane M, molecular weight markers; lane 1, OPMT; lane 2, OPET; lane 3, OCET; lane 4, PM; lane 5, PE; lane 6, CE. Western blotting confirmation of purified recombinant proteins with anti-polyhistidine mAb (B) or anti-ASFV swine serum (C) as primary antibody and corresponding secondary antibodies. Lane contents are the same as in panel (A)

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  Fig. 3. Cytokines secreted by DCs after stimulation. TNF-α (A) and IL-12p70 (B) levels in culture supernatants following stimulation of BMDCs with each recombinant protein (1 or 5 μg/mL), LPS (0.1 µg/mL) or medium (control) for 24 h. All data are displayed as mean ± SD (n = 3); NS = P > 0.05; *P < 0.05, **P < 0.01, ***P < 0.001

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  Fig. 4. Antigen-specific IgG detection. IgG responses to p30 (A), p54 (B), p72 (C), pE248R (D), CD2v (E) and pEP153R (F) in the sera from each immunized pig at 0, 14, 21, 35 and 42 dpv were detected by indirect ELISAs, with serum dilutions of 1:100 and goat anti-pig IgG-HRP dilutions of 1:10,000. The results are expressed as OD450 (mean ± SD). NS = P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001

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  Fig. 5. Lymphocyte proliferation in PBMCs from immunized pigs after in vitro stimulation with inactivated ASFV. A The percentage of CFSE-low lymphocytes (represented by P2) detected by flow cytometry in PBMCs from immunized pigs at 42 dpv after staining with CFSE and incubation with inactivated ASFV, medium (negative control) or concanavalin A (positive control). Group 1 and group 2 respectively represent pigs immunized with O-Ags-T formulation or Ags formulation. B Calculated percentages of proliferative lymphocytes based on CFSE-low lymphocytes in PBMCs from three separate experiments. The graphs show mean results with error bars indicating the SD; **P < 0.01; ***P < 0.001

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  Fig. 6. IFN-γ-producing T cells in PBMCs from immunized pigs at 42 dpv. The percentage of IFN-γ-secreting CD4+ T cells (A) and IFN-γ-secreting CD8+ T cells (B) in PBMCs from immunized pigs at 42 dpv after in vitro stimulation with inactivated ASFV, medium (negative control) or a cell activation cocktail (positive control). The graphs show mean results with error bars indicating the SD. NS = P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001

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  Fig. 7. Neutralization of ASFV infection by the sera from immunized pigs at 42 dpv. A The ASFV genome copy number in PAMs infected by ASFV (MOI = 0.01) preincubated with heat-inactivated pre-immune sera, immune sera (both at 1:5 dilution) or equal volume of medium (negative control), respectively. B The percentage of ASFV neutralization by the sera from pigs immunized with O-Ags-T formulation or Ags formulation at 42 dpv. The mean results with standard error are shown. NS = P > 0.05; ***P < 0.001

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  Fig. 8. Indirect immunofluorescence identification of PAMs infected with ASFV. PAMs infected with ASFV (MOI = 0.01) preincubated with heat-inactivated immune sera (42 dpv) from group 1 (O-Ags-T formulation), group 2 (Ags formulation) and PBS group or medium (negative control) were subjected to indirect immunofluorescence assay, with anti-p30 mAb as primary antibody and goat anti-mouse IgG-TRITC as secondary antibody. DAPI indicates the staining of the nucleus. The lower panel represents the merge of the 2 channels above

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  Fig. 9. Inhibition of ASFV infection in PBMCs from immunized pigs at 42 dpv. A Copy number of ASFV genome in PBMCs from non-immunized pigs or immunized pigs at 42 dpv infected with ASFV (MOI = 0.01). B ASFV inhibition percentages in PBMCs from pigs immunized with O-Ags-T formulation or Ags formulation at 42 dpv. The mean results with standard error are shown. NS = P > 0.05; ***P < 0.001