Deciphering the involvement of innate immune factors in the development of the host response to PRRSV vaccination

Abstract

The natural response of pigs to porcine reproductive and respiratory syndrome virus (PRRSV) infections and vaccinations needs to be altered so that better protection is afforded against both homologous and heterologous challenges by this pathogen. To address this problem, real-time gene expression assays were coupled with cytokine Elispot and protein analyses to assess the nature of the anti-PRRSV response of pigs immunized with modified live virus (MLV) vaccine. Although T helper 1 (Th1) immunity was elicited in all vaccinated animals, as evidenced by the genesis of PRRSV-specific interferon-gamma secreting cells (IFNG SC), the overall extent of the memory response was variable and generally weak. Peripheral blood mononuclear cells (PBMC) isolated from these pigs responded to PRRSV exposure with a limited increase in their expression of the Th1 immune markers, IFNG, tumor necrosis factor-alpha and interleukin-15 (IL15), and a reduction in the quantity of mRNAs encoding the innate and inflammatory proteins, IL1B, IL8 and IFNA. Efforts to enhance Th1 immunity, by utilizing an expression plasmid encoding porcine IFNA (pINA) as an adjuvant, resulted in a temporary increase in the frequency of PRRSV-specific IFNG SC but only minor changes overall in the expression of Th1 associated cytokine or innate immune marker mRNA by virus-stimulated PBMC. Administration of pINA, however, did correlate with decreased IL1B secretion by cultured, unstimulated PBMC but had no effect on their ability to release IFNG. Thus, while exogenous addition of IFNA during PRRSV vaccination has an impact on the development of a Th1 immune response, other alterations will be required for substantial boosting of virus-specific protection.

Fig. 1

Kinetics of the development of cell-mediated immune response of individual pigs to PRRSV MLV vaccination. Two litters of 6-week-old pigs (n = 19) were immunized with PRRS MLV vaccine and boosted 8 weeks later. At the indicated times post-primary immunization, the frequency of virus-specific IFNG SC in their PBMC was determined using an ELISPOT assay as described in Section 2.

Fig. 2

Comparison of the IFNG SC and IFNA SC responses of individual pigs to vaccination with PRRSV. PBMC were isolated from the pigs described in the legend to Fig. 1 during their peak IFNG response to PRRSV (weeks 4 and 5 post-primary vaccination) and the frequencies of virus-specific IFNG SC in these populations were determined via an ELISPOT assay. The frequency of innate IFNA SC in the pigs’ PBMC populations was measured at 5 weeks after the booster immunization (week 13 post-primary vaccination) by using an ELISPOT assay. The average IFNG response was compared to the IFNA response on an individual basis for members of litter A (circles) and B (diamonds). Regression analyses confirmed that these two types of immune functions were correlated on a pig by pig basis with R = 0.6 at a P ≤ 0.001 level.

Fig. 3

Comparison of individual pig IFNG SC and immune marker gene expression at 5 weeks post-MLV vaccination. (A) The frequencies of virus-specific IFNG SC in pig PBMC populations, cultured for 48 h in vitro with PRRS viral strain VR-2332, at 5.5 weeks post-PRRSV MLV immunization were determined using an ELISPOT assay. (B) Immune gene expression profiles using real time PCR of cDNA prepared from bulk PBMC cultures, stimulated in vitro with PRRSV for 48 h, and measured as described in Section 2. Results represent the relative ratio of C_t values of the indicated genes at 5.5 weeks to 0 weeks post-PRRSV MLV immunization. Squares are color-coded to denote either up- or down-regulation of gene expression.

Fig. 4

Temporal comparison of immune marker gene expression in activated PBMC from pigs vaccinated with PRRS MLV. Immune marker gene expression was measured by real time PCR for cDNA prepared from PBMCs stimulated in vitro with PRRSV for 48 h, as described in Section 2. Results represent the relative ratio of averaged C_t values obtained for each marker, with cDNA prepared at the indicated time points post-PRRSV MLV immunization of the nine pigs described in Fig. 3, to the pre-vaccination value. Squares are color-coded to denote either up- or down-regulation, with statistical differences (P ≤ 0.05) between the values derived at a given time and at 0 weeks post-vaccination indicated by the letter “s”.

Fig. 5

Effect of co-administration of IFNA plasmids on kinetics of the development of the cell-mediated immune response of pigs to PRRS MLV vaccine. Groups of pigs were vaccinated with PRRS MLV vaccine in the presence of DDAB alone, or co-administered with IFNA expression plasmid, pINA, or with control pANI plasmid. Following vaccination blood PBMC were harvested, restimulated 24 h with PRRSV in vitro and IFNG SC responses quantitated using an ELISPOT assay. Each value represents the mean response of 5 animals ± standard error of the mean (SEM). Significant differences (P < 0.05) between the frequencies of virus-specific IFNG SC in the peripheral blood of animals receiving the pINA plasmid vs. the other two groups are represented by an asterisk.

Fig. 6

Temporal comparison of immune marker gene expression in activated PBMC from pigs vaccinated with PRRSV alone or in the presence of adjuvant pINA or pANI. PBMC were prepared from swine vaccinated with PRRS MLV vaccine only, or MLV co-administered with IFNA expression plasmid, pINA, or with control pANI plasmid. PBMC were restimulated 24 h with PRRSV, pelleted and stored at −70 °C until RNA and cDNA were prepared. Immune marker gene expression was analyzed as described in Section 2. Results represent the relative ratio of C_t values obtained for PBMC isolated at 2 or 4 weeks post-PRRSV MLV immunization for 5 pigs/group compared to data acquired prior to vaccination. Squares are color-coded to denote either up- or down-regulation of the indicated genes. Significant differences (P < 0.05) between the values derived at either 2 or 4 weeks post-vaccination were calculated relative to week 0 data and noted by the letter “s”.

Fig. 7

Cytokine secretion by PBMC from pigs vaccinated with PRRSV alone or in the presence of adjuvant pINA or pANI. Groups of 5 pigs were immunized with PRRS MLV vaccine in the presence of DDAB alone or containing plasmid pINA or pANI, and PBMC isolated at 2 weeks after immunization. After a 48 h culture without virus stimulation, cell-free supernatants were harvested and the amount (pg/ml) of IFNG, IL1B, IL6 and IL10 determined using a specific ELISA as noted in Section 2. Results for each pig are shown separately and the average value for each group designated by a horizontal line.