Cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive chronic respiratory viral disease of pigs that is responsible for major economic losses to the swine industry worldwide. The efficacy of parenteral administration of widely used modified live virus PRRS vaccine (PRRS-MLV) against genetically divergent PRRSV strains remains questionable. Therefore, we evaluated an alternate and proven mucosal immunization approach by intranasal delivery of PRRS-MLV (strain VR2332) with a potent adjuvant to elicit cross-protective immunity against a heterologous PRRSV (strain MN184). Mycobacterium tuberculosis whole cell lysate (Mtb WCL) was chosen as a potent mucosal adjuvant due to its Th1 biased immune response to PRRS-MLV. Unvaccinated pigs challenged with MN184 had clinical PRRS with severe lung pathology; however, vaccinated (PRRS-MLV+ Mtb WCL) pigs challenged with MN184 were apparently healthy. There was a significant increase in the body weight gain in vaccinated compared to unvaccinated PRRSV challenged pigs. Vaccinated compared to unvaccinated, virus-challenged pigs had reduced lung pathology associated with enhanced PRRSV neutralizing antibody titers and reduced viremia. Immunologically, an increased frequency of Th cells, Th/memory cells, γδ T cells, dendritic cells, and activated Th cells and a reduced frequency of T-regulatory cells were detected at both mucosal and systemic sites. Further, reduced secretion of immunosuppressive cytokines (IL-10 and TGF-β) and upregulation of the Th1 cytokine IFN-γ in blood and lungs were detected in mucosally vaccinated, PRRSV-challenged pigs. In conclusion, intranasal immunization of pigs with PRRS-MLV administered with Mtb WCL generated effective cross-protective immunity against PRRSV.

Fig. 1

Mucosal vaccination with adjuvant rescued pigs from clinical PRRS with reduced gross lung pathology to a virulent PRRSV challenge. Pigs were unvaccinated (n = 9) or vaccinated (n = 9) with PRRS-MLV+ Mtb WCL and challenged with PRRSV MN184 on day post-immunization (DPI) 21 and then three pigs from each group were euthanized on day post-challenge (DPC) 15, 30, and 60. (A) Body weight of pigs was monitored on every third day post-challenge for 4 wks. Percentage body weight gain of individual pig was calculated by considering the weight of the pig at DPC 0 as 100%. (B) A representative lung picture of an unvaccinated or vaccinated and PRRSV challenged and then euthanized at DPC 15 is shown. (C) Gross lung lesion scores present in all the pig lung lobes at DPC 15, 30 and 60 were scored using a standard procedure. Each data point represents the average body weight gain from nine and six pigs ± SEM at DPC 15 and 30, respectively, and each bar represents the average lung lesion score from three pigs ± SEM. Asterisk denotes a statistically significant difference (P < 0.05) between unvaccinated vs. vaccinated and PRRSV challenged pigs.

Fig. 2

Reduced viremia and PRRSV titer in mucosally vaccinated and MN184 challenged pigs. Experimental details were as described in legend to Fig. 1. Serum collected at indicated DPC were analyzed for (A) total PRRS viral load; (B) PRRS viral titer; by a standard immunofluorescence assay. Each bar represents the average of six pigs ± SEM at DPC 15 and 30, and three pigs ± SEM at DPC 60. Asterisk denotes a statistically significant difference (P < 0.05) between unvaccinated vs. vaccinated and PRRSV challenged pigs.

Fig. 3

Enhanced PRRSV specific neutralizing antibody titers and reduced immunosuppressive cytokines in serum of mucosally vaccinated and MN184 challenged pigs. Experimental details were as described in legend to Fig. 1. Serum samples collected at indicated DPC were analyzed for (A) anti-PRRSV specific neutralizing antibody titers by standard immunofluorescence assay, (B) IL-10, and (C) TGF-β by ELISA. Each bar or data point in the graph represents the average VN titer or amount of cytokines from three pigs ± SEM. Asterisk denotes a statistically significant difference (P < 0.05) between unvaccinated vs. vaccinated and PRRSV challenged pigs.

Fig. 4

Mucosally vaccinated and PRRSV challenged pig immune cells secreted increased recall Th1 and suppressed immunosuppressive cytokines. Experimental details were as described in legend to Fig. 1, and the data from only DPC 15 pigs is shown. (A) PBMC was restimulated in the presence of killed MN184 antigens and the IFN-γ-secreting cells were analyzed by the ELISPOT assay. Supernatants harvested from lung MNC and PBMC cultures restimulated using killed MN184 antigens or PRRSV recombinant matrix (M3′) protein were analyzed for cytokines: (B) IL-10; (C) IL-6 by ELISA. Each bar represents the average number of IFN-γ-secreting cell spots or amount of cytokines from three pigs ± SEM. Asterisk denotes a statistically significant difference (P < 0.05) between unvaccinated vs. vaccinated and PRRSV challenged pigs.

Fig. 5

Protective anti-PRRSV specific humoral and cell-mediated immune responses to PRRS-MLV were mediated by Mtb WCL. Pigs (three each) were inoculated intranasally with mock, PRRS-MLV, or PRRS-MLV+ Mtb WCL and then vaccinated pigs were challenged with MN184 at DPI 21 and euthanized at DPC 30. (A) Serum collected at indicated DPC were analyzed for PRRSV specific VN titers by a standard immunofluorescence assay. Supernatants harvested from lung MNC and PBMC cultures restimulated using killed MN184 Ags were analyzed for cytokines: (B) IFN-γ; (C) IL-12; (D) IL-10 by ELISA. Each bar represents the average values from three pigs ± SEM. Asterisk denotes a statistically significant difference (P < 0.05) between PRRS-MLV vs. PRRS-MLV+ Mtb WCL received and PRRSV challenged pigs.