Recognition of the different structural forms of the capsid protein determines the outcome following infection with porcine circovirus type 2

Abstract

Porcine circovirus type 2 (PCV2) capsid protein (CP) is the only protein necessary for the formation of the virion capsid, and recombinant CP spontaneously forms virus-like particles (VLPs). Located within a single CP subunit is an immunodominant epitope consisting of residues 169 to 180 [CP(169-180)], which is exposed on the surface of the subunit, but, in the structural context of the VLP, the epitope is buried and inaccessible to antibody. High levels of anti-CP(169-180) activity are associated with porcine circovirus-associated disease (PCVAD). The purpose of this study was to investigate the role of the immune response to monomer CP in the development of PCVAD. The approach was to immunize pigs with CP monomer, followed by challenge with PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV). To maintain the CP immunogen as a stable monomer, CP(43-233) was fused to ubiquitin (Ub-CP). Size exclusion chromatography showed that Ub-CP was present as a single 33-kDa protein. Pigs immunized with Ub-CP developed a strong antibody response to PCV2, including antibodies against CP(169-180). However, only low levels of virus neutralizing activity were detected, and viremia levels were similar to those of nonimmunized pigs. As a positive control, immunization with baculovirus-expressed CP (Bac-CP) resulted in high levels of virus neutralizing activity, small amounts of anti-CP(169-180) activity, and the absence of viremia in pigs following virus challenge. The data support the role of CP(169-180) as an immunological decoy and illustrate the importance of the structural form of the CP immunogen in determining the outcome following infection.

Fig 1

Location of the CP(169–180) epitope within a single CP subunit and VLP. Depicted are the ribbon (A) and surface (B) maps of a single CP subunit. The blue and red residues form the epitope CP(169–180). The blue residues are important for antibody recognition (22). The VLP (C) shows the surface of the VLP with a single CP shown in green, red, and blue. The 173-Tyr residue is colored blue; however, due to it's location and orientation, it is not accessible on the surface of the VLP. The red and blue regions correspond to the same residues identified in panel A. Coordinates for the PCV2 CP(41–233) subunit and VLP were accessed through the RCSB Protein Data Bank (PDB code 3R0R) (2, 11) and loaded into the open source molecular visual program, Chimera (29). Term, terminus.

Fig 2

Study timeline. wks, weeks.

Fig 3

Size exclusion chromatography of CP(43–233). (A) SDS-PAGE gel showing affinity-purified Ub-CP. (B) Standard curve and size estimation for Ub-CP. A monogram of Ub-CP(43–233) eluted on a Sephacryl G-200 column was constructed by measuring total (solid line) and immunoreactive (dashed line) protein. Solid arrows show the location of the peaks for the BSA, Ub-GFP, and lysozyme standards. The dashed arrow shows the predicted peak for Ub-CP. The asterisks identify minor immunoreactive peaks, which may represent CP multimers. The elution volume of each fraction was 2 ml.

Fig 4

Total PCV2 and CP(169–180) oligopeptide antibody response. Total antibody (A) was measured by indirect fluorescent antibody assay (IFA). CP(169–180) immunoreactivity (B) was determined by ELISA using plates coated with BSA-conjugated CP(169–180). Results show the mean value for each group. Means with the same letter are not significantly different (P > 0.05).

Fig 5

PCV2 neutralizing activity. The 50% neutralizing activity (NA₅₀)/ml was determined by performing serum neutralization assays as described in Materials and Methods. The asterisk indicates means that are significantly different (P < 0.05).

Fig 6

PCV2 viremia. Data are shown as the means ± 1 standard deviation. Means with the same letter are not significantly different (P > 0.05).

Fig 7

Structural form of immunogen recognized by the host and relationship to outcome following PCV2 infection.