Oral application of freeze-dried yeast particles expressing the PCV2b Cap protein on their surface induce protection to subsequent PCV2b challenge in vivo

Abstract

Porcine circovirus type 2 (PCV2) is now endemic in every major pig producing country, causing PCV-associated disease (PCVAD), linked with large scale economic losses. Current vaccination strategies are based on the capsid protein of the virus and are reasonably successful in preventing PCVAD but fail to induce sterile immunity. Additionally, vaccinating whole herds is expensive and time consuming. In the present study a "proof of concept" vaccine trial was employed to test the effectiveness of powdered freeze-dried recombinant Saccharomyces cerevisiae yeast stably expressing the capsid protein of PCV2b on its surface as an orally applied vaccine. PCV2-free pigs were given 3 doses of vaccine or left un-vaccinated before challenge with a defined PCV2b strain. Rectal temperatures were measured and serum and faeces samples were collected weekly. At the end of the study, pigs were euthanized, tissue samples taken and tested for PCV2b load by qPCR and immunohistochemistry. The peak of viraemia in sera and faeces of unvaccinated pigs was higher than that of vaccinated pigs. Additionally more sIgA was found in faeces of vaccinated pigs than unvaccinated. Vaccination was associated with lower serum concentrations of TNFα and IL-1β but higher concentrations of IFNα and IFNγ in comparison to the unvaccinated animals. At the end of the trial, a higher viral load was found in several lymphatic tissues and the ileum of unvaccinated pigs in comparison to vaccinated pigs. The difference between groups was especially apparent in the ileum. The results presented here demonstrate a possible use for recombinant S. cerevisiae expressing viral proteins as an oral vaccine against PCV2. A powdered freeze-dried recombinant S. cerevisiae used as an oral vaccine could be mixed with feed and may offer a cheap and less labour intensive alternative to inoculation with the additional advantage that no cooling chain would be required for vaccine transport and storage.

Keywords: Freeze-dried; IFNα; IFNγ; ORF2; Oral; PCV2b; Yeast.

Fig. 1

Mean PCV2 copy number detected in the serum of pigs in different groups over the 12 weeks of the study. DNA was isolated from serum collected from each pig on a weekly basis and the number of PCV2 copies was determined by qPCR and comparison to known standards. Arrow indicates time-point of last vaccination and challenge. Significant differences between groups are depicted by *p < 0.05; **p < 0.01.

Fig. 2

Mean PCV2 copy number detected in the faeces of pigs in different groups over the 12 weeks of the study. DNA was isolated from faeces samples collected from each pig on a weekly basis and the number of PCV2 copies was determined by qPCR and comparison to known standards. Arrow indicates time-point of last vaccination and challenge. Significant differences between groups are depicted by *p < 0.05; **p < 0.01.

Fig. 3

Mean PCV2 antibody titre in the serum of pigs in different groups over the 12 weeks of the study. Diluted serum samples collected from each pig on a weekly basis were tested for the presence of anti-PCV2 antibodies by ELISA and comparison to known standards. Arrow indicates time-point of last vaccination and challenge.

Fig. 4

Mean IgA antibody titre in the faeces of pigs in different groups over the 12 weeks of the study. Faeces samples collected from each pig on a weekly basis were diluted 8-fold (w/v) in PBS, 0.05% Tween 20 and supernatant containing antibodies was harvested. The antibody concentration was determined by ELISA and comparison to known standards. Arrow indicates time-point of last vaccination and challenge. Significant differences between groups are depicted by *p < 0.05.

Fig. 5

Mean inflammatory cytokine levels in the serum of pigs in different groups over the 12 weeks of the study. Diluted serum samples collected from each pig on a weekly basis was tested for the presence of TNFα (A), IL-1β (B), IFNα (C) and IFNγ (D) by cytometric bead array and comparison to known standards. Arrow indicates time-point of last vaccination and challenge. Significant differences between groups are depicted by *p < 0.05; **p < 0.01.

Fig. 6

PCV2 copies in the mesenteric lymph node (A), jejunal lymph node (B), tonsil (C) and ileum (D) as well as IHC scores of pigs in the vaccinated and unvaccinated groups at week 12 of the study. Tissue was collected from each pig post mortem and DNA was isolated from a known mass. The number of PCV2 copies was determined by qPCR and comparison to known standards. Tissue was collected post mortem and sectioned in paraffin after which sections were stained for the capsid protein of PCV2. Examples of positive (Ei) and isotype control staining (Eii) are shown. IHC scoring (F) and overall scoring (G) are shown for pigs in the vaccinated and unvaccinated groups for all tissues collected.