Chimeric Porcine Parvovirus VP2 Virus-like Particles with Epitopes of South African Serotype 2 Foot-and-Mouth Disease Virus Elicits Specific Humoral and Cellular Responses in Mice

Abstract

Southern Africa Territories 2 (SAT2) foot-and-mouth disease (FMD) has crossed long-standing regional boundaries in recent years and entered the Middle East. However, the existing vaccines offer poor cross-protection against the circulating strains in the field. Therefore, there is an urgent need for an alternative design approach for vaccines in anticipation of a pandemic of SAT2 Foot-and-mouth disease virus (FMDV). The porcine parvovirus (PPV) VP2 protein can embed exogenous epitopes into the four loops on its surface, assemble into virus-like particles (VLPs), and induce antibodies and cytokines to PPV and the exogenous epitope. In this study, chimeric porcine parvovirus VP2 VLPs (chimeric PPV-SAT2-VLPs) expressing the T-and/or B-cell epitopes of the structural protein VP1 of FMDV SAT2 were produced using the recombinant pFastBac™ Dual vector of baculoviruses in Sf9 and HF cells We used the Bac-to-Bac system to construct the recombinant baculoviruses. The VP2-VLP--SAT2 chimeras displayed chimeric T-cell epitope (amino acids 21-40 of VP1) and/or the B-cell epitope (amino acids 135-174) of SAT FMDV VP1 by substitution of the corresponding regions at the N terminus (amino acids 2-23) and/or loop 2 and/or loop 4 of the PPV VP2 protein, respectively. In mice, the chimeric PPV-SAT2-VLPs induced specific antibodies against PPV and the VP1 protein of SAT2 FMDV. The VP2-VLP-SAT2 chimeras induced specific antibodies to PPV and the VP1 protein specific epitopes of FMDV SAT2. In this study, as a proof-of-concept, successfully generated chimeric PPV-VP2 VLPs expressing epitopes of the structural protein VP1 of FMDV SAT2 that has a potential to prevent FMDV SAT2 and PPV infection in pigs.

Keywords: Epitope; FMDV; PPV; SAT2 serotype; chimeric VLP.

Figure 1

The strategy of inserting SAT2 FMDV VP1 T/B epitopes into PPV VP2 protein and the flow chart of this study. (A) the SAT2 FMDV VP1 T/B epitopes insertion positions in the PPV VP2 protein. (B) This diagram depicts the generation, expression and identification of target genes of recombinant baculoviruses using the Bac-to-Bac expression system and the immune effect of the immunization of the chimeric proteins in mice. Briefly, VP2 or VP2 with SAT2 FMDV T/B epitopes was inserted into pFastBac^TM Dual vector. After DH10Bac transformation and blue and white plaques screening, the recombinant positive shuttle plasmid was transfected into SF9 cells. After observation of the cytopathic changes, recombinant baculovirus were collected and infected with HF cells. The expression and assembly of purified recombination proteins were identified by WB, IFA and TEM, then vaccinated with mice.

Figure 2

Construction of recombinant baculoviruses. (A) PCR amplification of the target gene. lane 1: PCR-amplified VP2 gene; lane 2: PCR-amplified L₂B gene; lane 3: PCR-amplified NT₁L₂B gene; lane 4: PCR-amplified NT₁L₂B₄B gene; lane 5: PCR-amplified N(T₁)₂L₂B₄B gene. M represents DNA DL 2000bp Marker. (B,C) Identification of recombinant shuttle plasmids with PCR. (B) PCR amplification of rBacmidVP2 gene; Lane 1: M13 primer amplified PCR product; lane 2: P1/2 primer amplified PCR product; (C) PCR amplificat the chimeric VP2 genes of rBacmidL₂B, rBacmidNT₁L₂B, rBacmidNT₁L₂B₄B, and rBacmidN(T₁)₂L₂B₄B. Lanes 1, 3, 5, 7: M13 primer amplified PCR products; lanes 2, 4, 6, 8: P3/4, P5/6, P7/8, P9/10 primer of each gene amplified PCR products. M1 and M2 represent DNA DL 10,000 bp or 2000 bp Marker. (D) Morphological changes in Sf9 cells infected with recombinant baculoviruses (400×). Sf9 cells were infected with rBacVP2, rBacL₂B, rBacNT₁L₂B, rBacNT₁L₂B₄B, or rBacN(T₁)₂L₂B₄B. The morphology of Sf9 cells under a microscope; the diameters of the Sf9 cells increased relative to that of the control.

Figure 3

Recombinant proteins were identified by western blotting and an immunofluorescence assay (IFA). (A) The expression of the recombinant proteins in HF cells was detected with western blotting. Porcine parvovirus (PPV) VP2 protein and the SAT2 FMDV B- and T-cell epitopes embedded VP2 proteins were detected. (B) VP2 protein or recombination VP2 proteins in HF cells were incubated with specific antibodies and observed with IFA (400×).

Figure 4

Transmission electron microscopy (TEM) of PPV AV30 and chimeric PPV-SAT2-virus-like particles (VLPs).

Figure 5

A schematic diagram of mice experiment (A) and the specific antibody assessment (B,C). The results shown are the means and standard deviations of triplicate samples (ns (no significance), * p < 0.05, ** p < 0.01, *** p < 0.001 or **** p < 0.0001). ELISA 96-well plates were coated with PPV AV30 virus particles or SAT2 FMDV VP1 protein. Serum was collected at different time points when the mice were inoculated with PBS, P-D, inactivated PPV vaccine, VP2-VLP, L₂B, NT₁L₂B, NT₁L₂B₄B, or N(T₁)₂L₂B₄B. ELISA was used to detect PPV (B), or SAT2 FMDV VP1 specific antibody (C).

Figure 6

Immunization of recombinant proteins increased CD4⁺ and CD8⁺ T-lymphocyte proliferation and cytokine production in mice. each value (pg/mL) in the bar diagram represents the mean ± SEM (n = 7); ns: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared with the control group. (A) Recombinant proteins increased lymphocyte proliferation. RPMI-1640 was used as the negative control, and ConA (5 μg/mL) was used as the positive control. Lymphocyte count (%) was increased significantly in the recombinant protein stimulation groups (* p < 0.05), compared to the control group. The results are presented as the ratio of the OD490 of the stimulated sample to the OD₄₉₀ of the unstimulated sample (stimulation index). (B): CD4⁺ and CD8⁺ T cells from peripheral blood were assayed after immunizations with VP2-VLP, PPV vaccine groups, PBS, P-D groups L₂B, NT₁L₂B, NT₁L₂B₄B or N(T₁)₂L₂B₄B. (C–E): A t-test was used to determine the mean variations in the serum levels of cytokines (IFN-γ, IL-4, and IL-2) among the treatment groups and control groups (ns: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001). (C) The concentrations of IFN-γ were determined in the eight immunized groups. (D) The concentrations of IL-2 in mice were detected after immunizations with recombinant proteins or control. (E) The mice were treated with PBS, P-D, PPV-vaccine, VP2-VLP, or recombinant proteins, respectively, then IL-4 level was determined.