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Virus-like particles of chimeric recombinant porcine circovirus type 2 as antigen vehicle carrying foreign epitopes

Viruses (2014) 6(12) 4839-4855
DOI: 10.3390/v6124839
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Abstract

Virus-like particles (VLPs) of chimeric porcine circovirus type 2 (PCV2) were generated by replacing the nuclear localization signal (NLS; at 1–39 aa) of PCV2 capsid protein (Cap) with classical swine fever virus (CSFV) T-cell epitope (1446–1460 aa), CSFV B-cell epitope (693–716 aa) and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The abilities to form PCV2 VLPs were confirmed by transmission electron microscopy. Immunogenicities of the three recombinant proteins were evaluated in mice. Our Results indicated that Cap protein NLS deletion or substitution with CSFV epitopes did not affect the VLPs assembly. Three chimeric Cap proteins could form VLPs and induce efficient humoral and cellular immunity against PCV2 and CSFV in mice. Results show that PCV2 VLPs can be used as an efficient antigen carrier for delivery of foreign epitopes, and a potential novel vaccine.

Figures

  • Figure 1. Schematic diagrams of the recombinant transfer vector (pFBD, pFBD-3-Cap-T, pFBD-3-Cap-B and pFBD-3-Cap-TB). (A) Schemes of chimeric Cap protein genes. NLS, the nuclear localization signal of Cap protein; the black boxes, the CSFV B-cell epitope (693–716 aa); the twill boxes, the CSFV T-cell epitope (1446–1460 aa); the white boxes, the gene of Cap; (B) Scheme of each recombinant transfer vector. The eGFP expression cassette was driven by the p10 promoter (PP10); PPH, the polyhedrin promoter of baculovirus; poly(A), polyadenylation signal.
  • Table 1. Primers used in this study.
  • Table 2. Experimental design.
  • Figure 2. Western-blot analysis of recombinant proteins expression in Sf9 cells. Lane 1: cell lysates of normal Sf9 cells as negative control; Lane 2: cell lysates of Ac-Cap; Lane 3: cell lysates of Ac-Cap-T; Lane 4: cell lysates of Ac-Cap-B; Lane 5: cell lysates of Ac-Cap-TB. Primary antibody is the mouse anti-cap MAb and secondary antibody is the goat anti-mouse IgG-HRP.
  • Figure 3. Confocal microscopy analysis of recombinant proteins expression in Sf9 cells. Sf9 cells were infected with recombinant viruses (Ac-Cap, Ac-Cap-T, Ac-Cap-B and Ac-Cap-TB), respectively. At 48 h post-infection, cells were fixed by methanol/acetone (1:1) and were analyzed by IFA using the anti-cap MAb as primary antibody and CY3-conjugated goat anti-mouse IgG as secondary antibody. Scale bar indicates 5µm.
  • Figure 4. Analysis of chimeric Cap particles by electron microscopy. Electron microscopy of negatively stained purified chimeric Cap particles (A) recombinant protein Cap-T (B) recombinant protein Cap-B (C) recombinant protein Cap-TB. Scale bar indicates 100 nm.
  • Figure 5. Cap-specific antibody responses in mice detected by indirect ELISA. (A) IgGs specific to Cap were found in serum samples at different times by indirect ELISA; (B) IgGs isotypes specific to Cap were detected by indirect ELISA on the day 40 post primary immunization. Data represent the mean ± SEM. Different letters (a,b) indicate a statistically significant difference between the different experimental groups (p < 0.05).
  • Figure 6. Detection of PCV2-specific neutralizing antibodies in sera. Neutralizing antibodies against PCV2 WH strain were measured in serum samples at day 14 and 40 dpi. The neutralizing antibody titers were calculated and expressed as the log2 of the reciprocal of the highest serum dilution that was able to completely block PCV2-infection in PK-15 cells. Data represent the mean ± SEM. Different letters (a,b) indicate a statistically significant difference between the different experimental groups (p < 0.05).

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APA

Zhang, H., Qian, P., Liu, L., Qian, S., Chen, H., & Li, X. (2014). Virus-like particles of chimeric recombinant porcine circovirus type 2 as antigen vehicle carrying foreign epitopes. Viruses, 6(12), 4839–4855. https://doi.org/10.3390/v6124839

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