Carboxyl-Terminal Decoy Epitopes in the Capsid Protein of Porcine Circovirus Type 2 Are Immunogenicity-Enhancers That Elicit Predominantly Specific Antibodies in Non-Vaccinated Pigs

Figures

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  Figure 1. Mapping of the linear and minimal epitopes for anti-capsid peptide (peptide P64) mAbs binding. Anti-P64 mAbs bound the linear peptide spanning from residues 205 to 233. The mAb binding was tested by using an iELISA. Peptides contained the sequence of the capsid protein between residues 205 and 233, associated 10-mer peptides, and truncated derivatives (as shown in Table 1). The experiments were performed three times. Data represent the mean ± standard error (SE).

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  Figure 2. The localization of viral proteins of PCV2 on PCV2 (strain: T657)-infected porcine kidney cells. The localization of viral proteins of PCV2 was assessed by indirect immunofluorescence assay (IFA) using anti-PCV2 polyclonal antisera (C–H) and anti-P64 mAbs (I–T) on the Porcine Circovirus Type 2 FA substrate slide (VMRD). Staining with SPF mouse serum was used as the negative control (A,B). Staining with PCV2-infected pig antiserum (C,D), anti-P64 mouse serum (E,F), and anti-VLP mouse serum (G,H) were used as positive controls and to identify all viral proteins of PCV2 (C,D), C-terminus of the capsid protein of PCV2a (E,F), and VLP of PCV2a (G,H), respectively. Left column (A,C,E,G,I,K,M,O,Q,S): Fluorescence microscopy of viral proteins of PCV2 was identified (green). Right column (B,D,F,H,J,L,N,P,R,T): Nuclei were stained with DAPI (blue) and the merge of the images was shown. The scale bar is 20 μm.

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  Figure 3. Location of amino acid residues of designed peptide (P64) and critical binding residues on the capsid protein of PCV2a. (A) The secondary structures of the single capsid protein of PCV2a were represented as a ribbon diagram in one VLP of PCV2a. The residues 228–231 were highlighted in red, the residues 220–227 were labeled in yellow, and other residues 205–219 were labeled in blue (A–F). These color residues represented the peptide P64. (B) The VLP of PCV2a lacks a single capsid protein subunit. (C) 3-D model of the peptide P64 rendered as a solid surface. (D) The secondary structure of the single capsid protein is represented as a ribbon diagram in one VLP. (E) 3-D model of the single capsid protein subunit rendered as a solid surface. (F) The surface map of all capsid proteins subunits in one VLP. The structural model of the capsid protein and the VLP of PCV2a coordinates were retrieved from the Protein Data Bank (PDB) entries for the capsid protein of PCV2a (PDB code: 3JCI) [54], and images were generated using UCSF Chimera version 1.14 from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, USA [55].

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  Figure 4. Screening anti-viral peptide-specific IgGs could differentiate PCV2-vaccinated pigs from non-vaccinated ones. Each group contained three samples from three individuals of different ages (18, 21, and 24 weeks of age, respectively). (A) 42 peptides (capsid peptides and other ORFs peptides), two VLPs (VLP-A, VLP-B), and two inactive PCV2 viruses (VC, VD) were used as coating antigens in the iELISA test. (B) 14 peptides (C-terminus of the capsid peptides) were used as coating antigens in the iELISA test. The data represent the mean ± standard error. Significant p values are indicated as * p < 0.05.

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  Figure 5. Comparison of anti-C-terminus of the capsid peptide-specific IgGs between two groups of pigs at different ages. Among vaccinated (n = 10) and control (n = 10) animals, the anti-PCV2 peptide-specific IgGs were detected in the serum samples from pigs at different ages (3, 6, 9, 12, 15, 18, 21, and 24 weeks of age). The C-terminus of the capsid peptides (P30, P47, P64, P97, P106, and P107) were used as coating antigens in the iELISA test. The peptide P30 had a significantly higher binding interaction with pig sera from the control group than with sera from the vaccine group aged 18 weeks to 24 weeks (A). Further, the others (P47, P64, P97, P106, and P107) also had a similar effect on pig sera aged 15 weeks to 24 weeks (B–F). The data were analyzed by using ANOVA and Tukey’s Studentized Range multiple comparisons test using the SAS Enterprise Guide 7.1® software. The data represent the mean ± standard error. Significant p values are indicated as * p < 0.05.