Brefeldin A blocks protein secretion and leads to the accumulation of IFNγ in the Golgi complex (Li et al. 2013). Whole blood samples, from naïve or vaccinated pigs, were stimulated with inactivated PRRSV to measure IFNγ-production in the presence of Brefeldin A. Blood samples without any stimulation served as a control. The four-color flow cytometric assay was developed for the phenotypic characterization of porcine IFNγ-producing lymphocytes. No fluorescence signal was detected in unstimulated cells (Supplementary Figure S1A ). After PRRSV stimulation, naïve porcine blood had a low frequency (0.09% ± 0.03%) of IFNγ-producing cells in CD3- gated lymphocytes (Supplementary Figure S1B). The frequency of IFNγ-producing cells in blood from PRRSVvaccinated pigs (0.42% ± 0.12%) was higher than that observed in naïve porcine blood (Supplementary Figure S1C, left two figures). CD3+ IFNγ-producing cells were further gated by CD4 and CD8 to delineate the percentage of IFNγ-producing in T-helper (CD3+CD4+), cytotoxic T (CD3+CD8+), and Th/memory (CD3+CD4+ CD8+) cell populations (Supplementary Figure S1C right top). CD3- IFNγ-producing cells were further gated by CD4 and CD8 to delineate the percentage of IFNγ-producing in the NK (CD3-CD4-CD8+) cell population (Supplementary Figure S1C right bottom). γδ T cells are another important porcine IFNγ producing T lymphocyte resource. To determine the proportion of γδ T cells contributing to IFNγ-producing after PRRSV stimulation, CD8+ and γδ+ cell populations were gated and defined as γδ T cells in IFNγ-producing lymphocytes (Supplementary Figure S1D).
Whole blood samples were collected at 28 DPV and PBMCs were isolated to perform an ELIspot assay to quantify IFNγ-secreting cells. Only PRRSV-MLVvaccinated pigs developed PRRSV-specific IFNγ-secreting cells at 28 DPV (Fig. 1). As expected, the frequency of PRRSV-specific IFNγ-secreting cells in naïve pigs was negligible. There was no significant difference in the frequency of IFNγ-secreting cells following PBMC stimulation with PRRSV VR2332 or JXA1 strains.
Figure 1. Frequency of IFNγ-secreting cells after vaccination determined by ELIspot assay. Peripheral blood mononuclear cells (PBMCs) from Naïve or vaccinated pigs were collected at 28 DPV and re-stimulated with inactivated VR2332 or JXA1 PRRSV to measure the frequency of IFNγ-secreting cells. Each bar represents the average number of IFNγ-secreting cells per million PBMCs of five pigs ± SEM. NS indicates no statistical difference
Different PBMC T cell subsets were analyzed at 28 DPV. The percentages of all T cell subsets were increased in vaccinated pigs compared with unvaccinated pigs (Table 1). Since T-helper cells, cytotoxic T cells, Th/memory cells, NK cells, and γδ T cells were reported to be responsible for IFNγ generation, we next explored the contribution of the different cell subsets to IFNγ-producing after PRRSV-MLV vaccination. Of the different lymphocyte subsets, T-helper cells accounted for over 30% of total IFNγ-secreting cells (Fig. 2). γδ T cells are T cell subsets unique to porcine blood and are important resources for IFNγ-producing, especially in young pigs. γδ T cells accounted for approximately 13% of IFNγ-producing following PRRSV vaccination (Fig. 2). CTLs and Th/memory cells accounted for 6% and 7% of total IFNγsecreting cells, respectively. NK cells accounted for 3% of total IFNγ-secreting cells.
Table 1. Frequency of T cell subsets in pigs after vaccination. Peripheral blood mononuclear cells (PBMCs) were isolated from blood collected from pigs at 28 DPV
Figure 2. Percentage of different lymphocyte subsets contributing to IFNγ-producing after PRRSV-MLV vaccination (1 million cells were used). Whole blood samples from PRRSV-MLV vaccinated pigs were collected at 28 DPV for flow cytometric assay. Each bar represents the average percentage of IFNγ-secreting cells of five pigs ± SEM
All pigs were challenged with homologous VR2332 (shares 98.9% genome similarity with PRRSV-MLV) or heterologous JXA1 (shares 89.4% genome similarity with PRRSVMLV) at 28 DPV. Different PBMC T cell subsets were also analyzed at 14DPC.Exceptfor T-helper cells(in theVR2332- challenge scenario) and γδ T cells, the percentages of all T cell subsets were decreased in unvaccinated pigs irrespective of whether challenged with VR2332 or JXA1 (Table 2). In contrast, the percentages of all T cell subsets were increased in vaccinated pigs. PBMCs from PRRSV-challenged pigs were stimulated with VR2332 or JXA1 PRRSVs before performing ELIspot assay. At 14 DPC, the IFNγ-secreting cells population increased significantly in PRRSV-MLV vaccinated pigs (MLV + VR2332 and MLV + JXA1 groups) compared with that in unvaccinated pigs (Naïve +VR2332 and Naïve +JXA1 groups) (Fig. 3). There was a higher frequency of IFNγ-secreting cells in VR2332-challenged pigs following PBMCs re-stimulation with homologous VR2332 virus than with heterologous JXA1 virus (within MLV + VR2332 group). Compared to pigs challenged with VR2332, JXA1- challenged pigs developed more IFNγ-secreting cells (MLV + VR2332 group vs. MLV + JXA1 group). However, there was no significant difference in IFNγ-producing following PBMC re-stimulation with VR2332 or JXA1 in this pig group (MLV + JXA1 group).
Table 2. Frequency of T cell subsets in pigs after viral challenge. Peripheral blood mononuclear cells (PBMCs) were isolated from blood collected from pigs at 14 DPC
Figure 3. Frequency of IFNγ-secreting cells analyzed by ELIspot assay after viral challenge. Naïve or PRRSV-MLV vaccinated pigs were challenged with VR2332 or JXA1 PRRSV. PBMCs from PRRSVchallenged pigs were collected at 14 DPC, and re-stimulated with inactivated VR2332 or JXA1 PRRSV before ELIspot assay. Each bar represents the average number of IFNγ-secreting cells per million PBMCs of five pigs ± SEM. Asterisk denotes a statistically significant difference (P < 0.05). NS indicates no statistical difference
To determine how the different cell populations contributing to IFNγ-producing when vaccinated pigs re-encountered PRRSVs, the percentages of IFNγ-secreting cells of different populations were analyzed. T-helper cells still accounted for the largest cell proportion after viral challenge, followed by γδ T cells and Th/memory cells (Fig. 4). The percentages of IFNγ-secreting cells within each cell population were compared following VR2332 or JXA1 challenge. The percentage of Th/memory cells for total IFNγ-secreting cells was significantly higher in VR2332-challenged pigs than in JXA1-challenged pigs. There was no significant difference as for the percentage of other cell populations for total IFNγ-secreting cells after VR2332 or JXA1 virus challenges.
Figure 4. Percentage of different cell populations that contributed to IFNγ-producing after virus challenge as analyzed by flow cytometry. PRRSV-MLV vaccinated pigs were challenged with VR2332 or JXA1 PRRSV. Whole blood samples were collected for flow cytometric analysis at 14 DPC. (A) Representative flow cytometry profile of lymphocytes after viral challenge. CD3+ gated lymphocytes expressing CD4+ (FL3) and CD8+ (FL4) are shown (left). Lymphocytes expressing CD8+ (FL1) and γδ T+ (FL4) are shown (right). (B) Contribution of different cell populations to IFNγ-producing. Each bar represents the average percentage of IFNγ-secreting cells of five pigs ± SEM. Asterisk denotes a statistically significant difference (P < 0.05). NS indicates no statistical difference
To explore the correlation between IFNγ-producing and virus load, serum sample virus titer was measured using real-time PCR. MLV-vaccinated pigs had decreased serum sample virus titers 7 DPC (Fig. 5). On DPC 14, the virus titer in vaccinated pigs was significantly lower than that of unvaccinated pigs, which illustrated the efficacy of vaccination. Compared to the pigs in the heterologous challenge group (MLV + JXA1), the viremia of pigs in the homologous challenge group (MLV + VR2332) was undetectable. No clinical symptoms were exhibited by any vaccinated pigs. By contrast, unvaccinated pigs challenged with VR2332 had a transient fever (2–5 DPC) and no other clinical symptoms were observed. Unvaccinated pigs challenged with JXA1 had lasting fever from 2 to 10 DPC and showed transient clinical symptoms including dyspnea, coughing, and shivering. All pigs that survived to the end of study were terminated at 14 DPC.