Targeted Alteration of Antibody-Based Immunodominance Enhances the Heterosubtypic Immunity of an Experimental PCV2 Vaccine

Figures

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  Figure 1. Selection of an immunogenic marker: Evaluation of the antigenicity of the Neospora caninum SRS2 protein by: A. Kyle Doolittle hydropathy plot—a negative value indicates hydrophilic residues; B. Jameson Wolfe antigenicity index; C. Emini surface probability plot. B and C: the height of the vertical bar is proportional to the predicted antigenicity of the sequence. A positive value indicates higher immunogenicity. Horizontal bar: amino acid positions in the sequence selected for analysis. Solid dark line: peptide sequence 324 QSSEKRDGEQVNKGKPP 348 selected as the marker in the vaccine construct.

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  Figure 2. Rescue of the recombinant rPCV2-Vac. (A) PK-15 cells transfected with the rPCV2-Vac construct and stained with a PCV2 specific polyclonal antibody (C) PK-15 cells transfected with the rPCV2-Vac construct and stained with a Neospora caninum specific polyclonal antibody. (B) and (D) Un-transfected PK-15 cells stained with the PCV2 or Neospora caninum antibodies respectively. Apple green florescence is indicative of a specific signal. White bar–Scale of 50 µM. Image obtained at 20x magnification.

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  Figure 3. Anti-PCV2 IgG responses: Mean signal to positive (S/P) ratios of sera collected on days 0, 14 and 28 post vaccination (DPV) and on days 9- and 21-days post-challenge (DPC), as measured by a PCV2 specific commercial ELISA. X axis: time points of serum collection, Y axis: sample to positive (S/P) ratio, dotted line: commercial vaccine, solid line: rPCV2-Vac, dashed line: unvaccinated. Error bars indicate the standard deviation, * significantly different from the unvaccinated control, p ≤ 0.05, Students t test. Significant differences were not detected between the rPCV2-Vac and commercial vaccine.

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  Figure 4. Virus neutralization responses: Mean virus neutralization titers measured by a rapid fluorescent focus reduction assay. Values for days 14 and 28 post vaccination (DPV) are depicted. No significant activity was detected in the sera of the unvaccinated control group or at day 0. X axis-virus neutralization titers against PCV2a, PCV2b or PCV2d. Y axis–% virus neutralization, horizontal lines: rPCV2-Vac, slanted lines: commercial vaccine, solid bar: unvaccinated. Error bars indicate the standard deviation, a-significantly different from the unvaccinated control, b-significantly different from the commercial vaccine group, p ≤ 0.05, Students t test.

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  Figure 5. Antibody responses to the mutated epitopes: Loss of immunodominant effects due to mutation of epitopes (A) and (B) as assessed qualitatively by surface plasmon resonance. 20 µM of purified IgG was tested for all experimental antisera. X axis—Time in seconds, Y axis—Response measured in µRU (µ response units). (A). Responses to a peptide encoding the wildtype epitope (A,B). Responses to a peptide encoding wildtype epitope (B). Slashed line-anti-serum to the wildtype virus, dotted line: anti-serum to the commercial vaccine, solid line-anti-serum to the rPCV2-Vac, connected triangles: anti-serum from the unvaccinated group.

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  Figure 6. Antibody responses to the marker: Mean optical density values of sera collected on days 0, 14 and 28 post vaccination (DPV) (N = 9), as measured by an ELISA specific to an antigenic peptide selected from the Neospora caninum SRS2 protein. X axis: time points of serum collection, Y axis: mean optical density (O.D) value, solid line: rPCV2-Vac, dotted line: commercial vaccine, dashed line: unvaccinated group. Error bars indicate the standard deviation, a: significantly different from the unvaccinated control, b: significantly different from the commercial vaccine group; p ≤ 0.05; Students t test.

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  Figure 7. Replication of the challenge virus. Quantification of the heterologous PCV2d challenge virus at 9 and 21 days post challenge by a PCV2d subtype specific qPCR. X axis: experimental groups and time of serum collection, Y axis: log10 viral copy numbers per mL of serum. Interval bars: 95% confidence interval of the means. * Significantly different from the unvaccinated control group; p ≤ 0.05; Students t test.

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  Figure 8. Lesion scores in tissues. Assessment of lesion scores resulting from viral challenge is represented as the sum of the scores for each tissue (A–E). Gross lung lesions were scored from 0%–100% to represent the % area of affected lung. Microscopic lesions were scored with a scale of 1–4; where 1 = single follicle or focus staining 2 = rare to scattered staining, 3 = moderate staining 4 = strong widespread staining. X axis: groups, Y axis: scores, dots: values for the individual pigs, horizontal bar with the large circle: group mean, bars: 95% confidence interval of the means, a: Significantly different from the unvaccinated control, b: Significantly different from the commercial vaccine group, (p < 0.05) by a Mann–Whitney U test.