Exploitation of stable nanostructures based on the mouse polyomavirus for development of a recombinant vaccine against porcine circovirus 2

<div><p>The aim of this study was to develop a suitable vaccine antigen against porcine circovirus 2 (PCV2), the causative agent of post-weaning multi-systemic wasting syndrome, which causes significant economic losses in swine breeding. Chimeric antigens containing PCV2b Cap protein sequences based on the mouse polyomavirus (MPyV) nanostructures were developed. First, universal vectors for baculovirus-directed production of chimeric MPyV VLPs or pentamers of the major capsid protein, VP1, were designed for their exploitation as vaccines against other pathogens. Various strategies were employed based on: A) exposure of selected immunogenic epitopes on the surface of MPyV VLPs by insertion into a surface loop of the VP1 protein, B) insertion of foreign protein molecules inside the VLPs, or C) fusion of a foreign protein or its part with the C-terminus of VP1 protein, to form giant pentamers of a chimeric protein. We evaluated these strategies by developing a recombinant vaccine against porcine circovirus 2. All candidate vaccines induced the production of antibodies against the capsid protein of porcine circovirus after immunization of mice. The candidate vaccine, Var C, based on fusion of mouse polyomavirus and porcine circovirus capsid proteins, could induce the production of antibodies with the highest PCV2 neutralizing capacity. Its ability to induce the production of neutralization antibodies was verified after immunization of pigs. The advantage of this vaccine, apart from its efficient production in insect cells and easy purification, is that it represents a DIVA (differentiating infected from vaccinated animals) vaccine, which also induces an immune response against the mouse polyoma VP1 protein and is thus able to distinguish between vaccinated and naturally infected animals.</p></div

Electron microscopy of isolated structures.

<p>Negative staining with 2% PTA. Bars represent 100 nm.</p

Model of PCV2 capsid.

<p>(<b>A</b>) Position of selected PCV2 Cap epitopes (colored) on the surface of PCV2 particle (left) or Cap monomer (right). (<b>B</b>) Table of Cap sequences used for construction of individual nanostructure variants.</p

PCV2 virus neutralization by antibodies induced in mice.

<p>PK15 cells were inoculated with a mixture of 50-fold diluted sera collected from immunized pigs and PCV2 virus (final concentration TCID₅₀ = 10⁵). Control cells were infected with the same amount of virus mixed with 50-fold diluted serum from mice immunized with PBS. Infected cells were detected 36 hpi by immunofluorescence assay with specific antiCap PCV2 antibody and the number of Cap PCV2-positive cells (green) was calculated. Columns represent percentage of infected cells relative to control. (<b>A</b>) Graph of neutralizing activity of mouse sera, (<b>B</b>) representative example of immunofluorescence staining of control cells and cells infected with virus incubated with sera of mice immunized by VarC1. Error bars represent standard error of three independent experiments. Bars represent 50 μm.</p

Cellular immune responses induced by candidate vaccines in mice tested <i>in vitro</i>.

<p>(<b>A</b>) Relative representation of Th CD4⁺, Tc CD8⁺ and B lymphocytes CD19⁺ in mouse splenocyte population after <i>in vitro</i> incubation with VarA4, VarB and VarC nanostructures. (<b>B</b>) Activation of Th CD4⁺, Tc CD8⁺ and B lymphocytes CD19⁺ in the mouse splenocyte population after <i>in vitro</i> incubation with VarA4, VarB and VarC nanostructures. (<b>C</b>) IFNγ and IL-4 intracellular level of mouse splenocyte CD45⁺ population after <i>in vitro</i> incubation with VarA4, VarB and VarC nanostructures. Blue and white columns represent controls: splenocytes without added nanostructures (control; blue) and MPyV VLPs without inserted PCV2 epitopes, scaffold only (VP1_Con; white). Error bars represent standard error of the mean, SEM, *P <0.05, ** P<0.005, *** P < 0.0005.</p

Comparison of PCV2-specific antibody responses induced by the VarC candidate vaccine to those induced by commercial Circoflex vaccine.

<p>Two groups of pigs were immunized twice (days 0 and 21) with nanostructure VarC or Circoflex vaccine according to the manufacturer’s instructions or with PBS containing 30% Polygen adjuvant. The levels of antiPCV2 antibodies were measured using a commercial ELISA kit (Ingezim Circo IgG) according to the manufacturer’s instructions. The S/P index—an index expressing the amount of antiPCV2-specific antibodies as a ratio between OD_415nm of the sample / OD_415nm of the control which is part of the ELISA kit. The dashed line represents the border of the S/P index (0.285) between sera that are negative and positive for PCV2-specific antibodies (calculated according to the ELISA kit manual). Error bars represent standard error of the mean, SEM, *P <0.05, ** P<0.005, *** P < 0.0005.</p

Pig serum neutralization assay.

<p>PK15 cells were inoculated with a mixture of 100-fold diluted sera collected from immunized pigs and PCV2 virus (final concentration TCID₅₀ = 10⁵). Control cells were infected with the same amount of the virus mixed with 100-fold diluted serum from a gnotobiotic pig. Infected cells were detected 36 hpi by immunofluorescence assay with antibody against the Cap protein, and the numbers of PCV2-positive cells were calculated. (<b>A</b>) Plot of neutralizing activity of pig sera. The plots represent the percentage of infected cells relative to the control. Cells from 17 optical fields (approx. 1500 infected cells for the control sample) were counted in each experiment. Error bars represent standard error of three independent experiments. (<b>B</b>) Representative fields of counted cells. Control cells (<b>1</b>) and cells infected with the virus neutralized by VarC-induced antibodies (<b>2</b>). Blue—DAPI, green—PCV2 Cap protein. Bars represent 50 μm.</p