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

Despite the availability of commercial vaccines which can effectively prevent clinical signs, porcine circovirus type 2 (PCV2) continues to remain an economically important swine virus, as strain drift, followed by displacement of new subtypes, occurs periodically. We had previously determined that the early antibody responses to the PCV2 capsid protein in infected pigs map to immunodominant but non-protective, linear B cell epitopes. In this study, two of the previously identified immunodominant epitopes were mutated in the backbone of a PCV2b infectious clone, to rationally restructure the immunogenic capsid protein. The rescued virus was used to immunize 3-week-old weanling piglets, followed by challenge with a virulent heterologous PCV2d strain. As expected, immunodominant antibody responses to the targeted epitopes were abrogated in vaccinated pigs, while a broadening of the virus neutralization responses was detected. Vaccinated pigs were completely protected against challenge viral replication, had reduced microscopic lesions in lymphoid organs and gained significantly more body weight when compared to unvaccinated pigs. Thus, the experimental PCV2 vaccine developed was highly effective against challenge, and, if adopted commercially, can potentially slow down or eliminate new strain creation.

Establishment of a Rep′ protein antibody detection method to distinguish natural infection with PCV2 from subunit vaccine immunization

Introduction. PCV2 is a DNA virus that exists widely in pigs and has caused great economic losses to the pig industry worldwide. In the existing commercial PCV2 enzyme-linked immunosorbent assay (ELISA) kits both natural infection with PCV2 and vaccine immunization produce results that are positive for PCV2 Cap antibodies and therefore they cannot diagnose PCV2 infection in immunized pig farms. Aim. To establish a PCV2 non-structural protein antibody detection method that distinguishes between antibodies resulting from natural prior exposure (infection) and those induced by subunit vaccine immunization. Methodology. Based on the non-structural Rep′ protein, we established an indirect ELISA (iELISA) using sera from guinea pigs and piglets. Results. The results for iELISA for guinea pig serum showed that animals vaccinated with a whole-virus inactivated PCV2 vaccine had 100 % (10/10) Cap antibody positivity and 100 % (10/10) Rep′ antibody positivity. Guinea pigs vaccinated with a recombinant subunit PCV2 vaccine had 100 % (10/10) Cap antibody positivity, while no (0/10) guinea pigs were Rep′ antibody-positive. The combined detection results for the Rep′ iELISA and a PCV2 Antibody Test kit (Commercial) showed that pigs vaccinated with a whole-virus inactivated PCV2 vaccine or PCV2 SD/2017 had 100 % (5/5) Cap antibody positivity and 100 % (5/5) Rep′ antibody positivity. Pigs vaccinated with a recombinant subunit PCV2 vaccine had 100 % (5/5) Cap antibody positivity, while no (0/10) pigs were Rep′ antibody-positive. Conclusion. This paper describes an effective iELISA method that can distinguish natural infection with PCV2 (Cap and Rep positive) or inoculation with a whole-virus inactivated vaccine (Cap and Rep positive) from subunit vaccine immunization (Cap-positive, Rep-negative). These comparative assays could be very useful in the control of PCV2 in pig herds.

Cloning and Expression of the Tibetan Pig Interleukin-23 Gene and Its Promotion of Immunity of Pigs to PCV2 Vaccine

Vaccines against Porcine circovirus type 2 (PCV2) have been studied intensely and found to be effective in decreasing mortality and improving growth in swine populations. In this study, interleukin-23 (IL-23) gene was cloned from peripheral blood mononuclear cells (PBMCs) of Tibetan pigs and inserted into a eukaryotic VR1020 expression vector-VRIL23. Coated with chitosan (CS), the VRIL23-CS was intramuscularly injected into 3-week-old piglets with PCV2 vaccine. The blood was collected after vaccination at 0, 1, 2, 4, 8, and 12 weeks, respectively, to detect the immunological changes. The IgG2a and specific PCV2 antibodies were detected using ELISA, and blood CD4+ and CD8+ T cells were quantified by flow cytometry. Quantitative fluorescence PCR was used to evaluate the expression of immune genes. The results indicate that leukocytes, erythrocytes, and CD4+ and CD8+ T cells increased significantly in the blood of VRIL23-CS inoculated piglets in comparison with the control (p < 0.05) and so did the IgG2a and PCV2 antibodies. In addition, the expressions of Toll-like receptor (TLR) 2, TLR7, cluster of differentiation (CD) 45, IL-15, IL-12, signal transducer and activator of transcription (STAT)1, STAT2, STAT3, STAT4, and B-cell lymphoma (Bcl)-2 genes were also obviously higher in the VRIL23-CS inoculated pigs at different time points (p < 0.05). Overall, the results demonstrated that VRIL23-CS can enhance the comprehensive immune responses to PCV2 vaccine in vivo and has the promising potential to be developed into a safe and effective adjuvant to promote the immunity of pig against PCV disease.

The Adjuvant Activity ofEpimediumPolysaccharide-Propolis Flavone Liposome on Enhancing Immune Responses to Inactivated Porcine Circovirus Vaccine in Mice

Objectives.The adjuvant activity ofEpimediumpolysaccharide-propolis flavone liposome (EPL) was investigated in vitro and in vivo.Methods.In vitro, the effects of EPL at different concentrations on splenic lymphocytes proliferation and mRNA expression of IFN-γand IL-6 were determined. In vivo, the adjuvant activities of EPL, EP, and mineral oil were compared in BALB/c mice through vaccination with inactivated porcine circovirus type 2 (PCV2) vaccine.Results.In vitro, EPL promoted lymphocytes proliferation and increased the mRNA expression of IFN-γand IL-6, and the effect was significantly better than EP at all concentrations. In vivo, EPL significantly promoted the lymphocytes proliferation and the secretion of cytokines and improved the killing activity of NK cells, PCV2-specific antibody titers, and the proportion of T-cell subgroups. The effects of EPL were significantly better than EP and oil adjuvant at most time points.Conclusion.EPL could significantly improve both PCV2-specific cellular and humoral immune responses, and its medium dose had the best efficacy. Therefore, EPL would be exploited in an effective immune adjuvant for inactivated PCV2 vaccine.