This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Zhejiang University. All animal experiments were approved by the Animal Welfare and Ethics Committee at the College of animal science of Zhejiang University with reference number ZJU20230315.

Six to eight-week-old wild-type C57BL/6 mice were purchased from the Center of Comparative Medicine at Yangzhou University. Three to four-week-old healthy piglets were purchased from Kairong biotechnology company (Suzhou, China). All pigs were detected negative for antibodies against PRV-gB and gE antigen, PRRSV, ASFV and CSFV and nucleic acid of PRV, PRRSV, PCV2, CSFV and ASFV. Human embryonic kidney cells (HEK293T) and PCV-free PK-15 cells (ATCC-CCL-33) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, America) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, America). Classical PRV vaccine strain Bartha-K61 (GenBank accession no. JF797217.1) was gifted by Prof. Song Gao at Yangzhou University. Virulent PRV type II strain DX and an attenuated PRV vaccine strain HD/c (gE/TK deletion) was kept in our laboratory (21). All the viruses were propagated in PK-15 cells, titrated, aliquoted in stocks and stored in -80°C in our lab.

A total of ninety female C57BL/6 mice were divided into two groups, with 45 mice in each group. One group was intramuscularly and subcutaneously immunized with 1×10⁵ TCID⁵⁰ PRV Bartha K61 in 100μl DMEM (BK61 imm). This dose of Bartha K61 was relatively unlethal to C57BL/6 mice. Another group was mock immunized with 100μl phosphate-buffered saline (PBS). At indicated time-points after immunization, five mice from each group (n=25) were sacrificed, spleens and lymph nodes were harvested for single cell isolation and quantification of viral loads, respectively. Serum samples were collected by tail vein at days 7, 14, and 28 post immunization to determine the antibody titers. At day 30 after immunization, all the remaining mice from both groups (n=20 per group) were challenged with 5×10³ TCID⁵⁰ virulent PRV DX strain. At indicated time-points after challenge, mice from each group (n=4-5) were sacrificed, spleens were harvested for single cell preparation and viral load quantification again. Five mice from each group were spared for recording the survival rate after challenge.

For pig study, ten piglets were divided into two groups. One group (n=5) was immunized intramuscularly at the neck with 2 ml of 10^7.5 TCID₅₀/ml PRV HD/c. The other group was immunized with equal volume of DMEM medium. Periphery blood from each pig was collected at 7, 14 and 21 days after immunization. The isolation of PBMCs and intracellular straining of cytokines in T cells were performed and analyzed by flow cytometry as our previous studies (32, 33).

Single-cell suspensions from spleen and lymph nodes were prepared as previously described (33, 34). Briefly, tissues were mechanically disrupted and pushed through a 70 μm nylon cell strainer and the cells were resuspended in 10 ml PBS containing 2% fetal bovine serum (FBS). Red blood cells were removed from single cell suspensions by hypotonic lysis with Gey’s solution. Cells were counted using a hemocytometer and the final cell concentration was adjusted to 2 × 10⁷ cells/mL before cell surface staining.

Bone marrow-derived dendritic cells (BMDCs) were derived from mouse bone marrow progenitors using GM-CSF and IL-4 (PeproTech, Rocky Hill, NJ) at a final concentration of 10 ng/mL and 2 ng/mL, respectively, as previously described (35). At day 6 of culture, BMDCs were harvested and pulsed with inactivated PRV Bartha K61 (iBK61) or infected with live Bartha K61 (BK61) at a multiplicity of infection (MOI) of 3 for 18 h or indicated length of time. iBK61- or BK61-pulsed BMDCs were washed twice and used as stimulators to activate T cells isolated from PRV-immunized and unimmunized mice.

For DC immunization, BMDCs harvested at day 6 of culture were plated in six-well plate with 6.1×10⁶ cells per well, live PRV Bartha K61 was added up to 1×10⁶ TCID₅₀ per mL (MOI=0.5) and infected for overnight, then BK61-infected BMDCs were harvested and washed twice, and a number of 2.17×10⁶ cells was sampled and lysed to confirm the infection rate by IFA on PK-15 cells. 5×10⁵ BK61-infected BMDCs (around 1×10^4.6 TCID₅₀ PRV) were used to immunize mice. A total of eighty female C57BL/6 mice were divided into four groups (DMEM, BK61, DC alone and BK61-DCs, respectively), with 20 mice in each group. Group 1 and 2 were intramuscularly immunized with DMEM and 1×10⁵ TCID⁵⁰ PRV Bartha K61 each mouse (BK61), respectively. Group 3 was intraperitoneally immunized with 5×10⁵ uninfected BMDCs each mouse (DC alone). Group 4 was intraperitoneally immunized with 5×10⁵ BK61-infected BMDCs each mouse containing around 1×10^4.6 TCID₅₀ PRV Bartha K61 (BK61-DCs). At indicated time-points after immunization, mice were sacrificed, spleens were harvested for single cell isolation. At day 30 after immunization, five mice from each group were challenged and the survival rate of each group was recorded.

Anti-mouse monoclonal antibodies (mAb) against CD8α (53–6.7), CD44 (1M7), CD62L (MEL14), CD127 (A7R34), KLRG1 (2F1/KLRG1), B220 (RA36B2), CD4 (GK 1.5), TCRβ (H57-597), NK1.1 (PK136), TCRγδ (GL-3) and CD69 (H1.2F3) with different fluorochrome conjugate, were purchased either from BioLegend or BD Bioscience (San Diego, CA). Surface marker staining of cells was carried out as our previous reports (36). For detecting the early activation of distinct immune cells after immunization, 2×10⁶ cells were plated into 96-well V-bottom plates and incubated with 2.4G2 Fcγ RII/RIII blocking mAb for 15 min, then stained for 30 min on ice with 50 μL antibody cocktail containing fluorochrome-conjugated mAbs against CD69, CD8α B220, CD4, TCRβ, NK1.1 and TCRγδ. Afterward, cells were washed and resuspended in 200 μL PBS for flow cytometric analysis.

For defining memory T cells, 2×10⁶ splenocytes were plated and stained for 30 min on ice with 50 μL antibody cocktail containing different fluorochrome-labeled mAbs against markers TCRβ, CD8α, CD44, CD62L, CD127, KLRG1 and CD4. Thereafter, cells were washed and resuspended in 200 μL PBS for flow cytometric analysis. Flow cytometry was performed with a FACS CantoII (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Tree Star, Ashland, OR).

2×10⁶ cells were plated into 96 round-bottom-well plates and stimulated with or without live BK61 at MOI of 1 or inactivated BK61-pulsed DCs for 18h. Brefeldin A (Sigma, St. Louis, MO) was added for the last 6 h of co-culture at a final concentration of 10 μg/ml. After incubation, cells were harvested and stained for cell surface makers TCRβ, CD8α, and CD4 as above-mentioned protocol, and then fixed with 4% paraformaldehyde for 10 min and permeabilized twice by Fixation/Permeabilization solution (BD Biosciences) following the manufacturer’s protocol. Subsequently, cells were stained with APC-conjugated anti-IFN-γ (XMG1.2, Biolegend), FITC-conjugated anti-TNF-α (MP6-XT22, Biolegend) and PE-conjugated anti-IL-2 mAbs (JES6-5H4, Biolegend). Flow cytometry was performed as described above.

Statistical analyses were performed using Prism software 5.0 (GraphPad, La Jolla, CA). When comparing experimental values from two groups of mice, two-tailed student’s t-tests were used. When comparing experimental values from multiple groups, one-way ANOVA Bonferroni post-tests were used. Statistically significant differences are noted (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05).

Attenuated live vaccines was shown to elicit potent cellular immune responses in mice or human after immunization that significant increase of CD44^hiCD62L^-, CD44^hiCD62L⁺ or IFN-γ-producing T cells was observed (9, 37–39). Therefore, we anticipated that vigorous activation of distinct lymphocytes should occur after PRV LAV vaccination. Thus, we detected the expression of CD69 and CD44 on lymphocyte subsets to evaluate the very early activation and antigen-experienced effector and memory T cells in mice after immunization (40, 41). Surprisingly, vaccination with PRV Bartha K61 (BK61) did not induce the upregulation of CD69 on NK cells, B cells, γδ T cells, and CD4 and CD8 T cells at day 4, 7 and 14 after immunization (dpi) ( Figures 1A, B ). Instead, PRV BK61 immunization even led to the reduction of CD69-positive CD4 and CD8 T cells at 4 dpi ( Figure 1B ). To further confirm if there is late activation of T cells or memory T cell development, we detected the CD44 expression on T cells. As shown in Figures 1C, D , PRV BK61 immunization did not increase the percentage of CD44^hi-postive effector and memory CD4 and CD8 T cells at any time-point after vaccination, compared to the un-immunized mice. These results suggested that PRV BK61 induced no detectable phenotypic activation of NK cells, B cells and T cells in mice.

To further determine whether PRV BK61 vaccination induce PRV-specific T cells response, we examined the frequency of IFN-γ-producing T cells upon ex vivo restimulation of the BK61-primed T cells with live BK61 through intracellular cytokine staining (ICS). As shown in Figures 2A, B , we detected more IFN-γ-producing CD4 and CD8 T cells in the BK61-immunized mice, compared to the unimmunized control, but the frequencies of both IFN-γ⁺CD4⁺ and IFN-γ⁺CD8⁺ T cells were very low (less than 0.5%). Further analysis of the kinetics of PRV-specific T cells showed that both IFN-γ⁺CD4⁺ and IFN-γ⁺CD8⁺ T cells peaked at 14 dpi in terms of percentage but the magnitude of IFN-γ⁺CD4⁺ T cells was higher than that of IFN-γ⁺CD8⁺ T cells and PRV-specific CD4 T cells were detected earlier (4 dpi) than PRV-specific CD8 T cells ( Figure 2B ). These results indicated that PRV BK61 induced a dominant CD4 Th1 response in mice with low magnitude.

Among activated T cells, polyfunctional T cells that are able to simultaneously produce multiple cytokines (IFN-γ, TNF-α and IL-2) have been shown to correlate with protective immunity, and are equivalent to central memory T cells (42, 43). We further analyzed the functionalities of those PRV-specific T cells by ICS of multiple cytokines though their frequency was low. As shown in Figures 3A, B , compared to the unimmunized mice, BK61-immunized mice have more PRV-specific IFN-γ-producing CD4 T cells, which also simultaneously co-expressed TNF-α (IFN-γ⁺TNF-α⁺) or IL-2 (IFN-γ⁺IL-2⁺) or TNF-α and IL-2 (IFN-γ⁺TNF-α⁺IL-2⁺), with higher proportion in IFN-γ single positive (61.2%) and IFN-γ⁺TNF-α⁺IL-2⁺ CD4 T cells (19.8%) ( Figures 3A, B ). In addition, there were also slightly more IFN-γ-negative TNF-α⁺, IL-2⁺ and TNF-α⁺IL-2⁺ CD4 T cells in BK61-immunized mice ( Figure 3A ). These results demonstrated that BK61 immunization induced multi-functional CD4 T cells. Further analysis of the kinetics of PRV-specific CD4 T cells showed that all three subsets of double-cytokine-producing CD4 T cells peaked at 14 dpi and then decreased by 28 dpi ( Figure 3C ). In contrast, very few of IFN-γ⁺TNF-α⁺CD8 T cells were induced in BK61-immunized mice. These cells showed similar kinetics to that of cytokine-producing CD4 T cells but no triple cytokine-producing CD8 T cells were detected after immunization ( Figures 3D, E ). These results showed that PRV BK61-primed CD4 but not CD8 T cells showed characteristics of multi-functional T cells.

It has been reported that PRV vaccine-induced T-cell immunity played a critical role in early protection against PRV in pigs and repeated vaccination are required clinically for long-term protection (28, 29), suggesting that PRV attenuated vaccine may induce weak memory T cells response. Therefore, we examined the secondary response of PRV-specific T cells after challenge with PRV DX strain. Both BK61-immunized and unimmunized mice were challenged at 28 dpi or left unchallenged and IFN-γ- and TNF-α-producing T cells were detected at 7 dpc upon restimulation with BK61. As shown in Figure 4 , when comparing the percentage and kinetic change of total IFN-γ-producing CD4 T cells, there was no significant difference between the immunized and unimmunized mice after DX challenge ( Figures 4A–C ). However, when comparing the percentage of IFN-γ⁺TNF-α⁺ CD4 T cells, BK61-immunized mice have more IFN-γ⁺TNF-α⁺ CD4 T cells than the unimmunized mice at 4 and 7 days after DX challenge ( Figures 4D–F ), which were detected as early as 4 dpc ( Figure 4F ), suggesting BK61 immunization had induced antigen-specific memory CD4 T cells, thus have rapid secondary response after challenge. Similarly, we also analyzed BK61-induced memory CD8 T cells by comparing the percentage of IFN-γ⁺TNF-α⁺ CD8 T cells between the two groups after challenge. We found no significant increase in secondary CD8 T cells response, compared to primary CD8 T cells response in terms of percentage ( Figures 4F–H ). Of note, the percentages of both PRV-specific secondary CD4 and CD8 T cells were very low though BK61-primed T cells were pre-existed before challenge. These results indicated that BK61 immunization mainly induced a weak memory response of CD4 T cells.

Previous studies showed that antigen-specific CD4 and CD8 T cells response can be rapidly induced and potently enhanced by dendritic cell (DC) immunization (33, 44). To further demonstrate whether the weak T cells response induced by BK61 is an intrinsic property of PRV BK61. We employed DC immunization to enhance BK61-induced T cells response. BMDCs were pulsed with live or inactivated PRV BK61 at MOI of 0.5 (DC-BK61 or DC-iBK61) and then used to immunize mice. PRV-specific CD4 and CD8 T cells were examined and compared after immunization as aforementioned protocol. As shown in Figures 5A, B , PRV-specific IFN-γ-producing CD4 and CD8 T cells response were significantly enhanced at 7 dpi by DC-BK61 immunization as DC-BK61-immunized mice have the highest percentage (1%) of IFN-γ-producing CD4 and CD8 T cells, compared to the other three groups (DMEM, DC alone and BK61). However, by 14 dpi, this difference was no longer observed between BK61 and DC-BK61 group ( Figures 5C, D ). To further test whether DC-BK61-enhanced T cell response was translated into better protection against PRV DX challenge. The survival rates of each group were compared after challenge. As a result, DC vaccination with BK61 or inactivated BK61 did not provide better protection than BK61 immunization ( Figure 5F ). These results indicated that although the weak T cells response induced by BK61 were transiently enhanced by dendritic cell vaccination, the eventual protective immunity was not increased, suggesting that there is an intrinsic drawback for PRV BK61 in inducing cellular immunity to some extent.

PRV BK61 strain originally contains deletions of the US2, gE, gI, and US9 genes. To test whether other gene-deleted attenuated PRV strain also induce similar T cell responses in mice, we examined PRV-specific IFN-γ-producing T cells elicited by PRV HD/c strain, a gE/TK-deleted PRV variant strain constructed in our lab (21). As shown in Figure 6 , PRV HD/c immunization induced similar kinetics and magnitude of IFN-γ-producing CD4 and CD8 T cells as well as IFN-γ⁺TNF-α⁺IL-2⁺CD4 T cells before 14 dpi and more IFN-γ⁺CD4 T cells from 21 to 35 dpi, compared to the BK61 immunization, suggesting that the magnitude of T cell response (less than 0.5%) may be determined by the PRV LAVs itself but not by the difference of gene deletions in the LAVs. To further confirm whether attenuated PRV vaccine induce similar magnitude of T cells responses in pigs as well, we used HD/c to immunize piglets and examined the kinetics and magnitude of IFN-γ-expressing T cells in PBMCs of the immunized pigs. As shown in Figure 7 , HD/c immunization barely induced phenotypic activation (CD69⁺) of CD4 T cells at 21 dpi and CD8 T cells at 7 dpi with the percentages less than 0.6% in the PBMCs ( Figures 7A, B ). PRV-specific IFN-γ⁺ or IFN-γ⁺TNF-α⁺ CD4 T cells and CD8 T cells were significantly elicited in the PBMCs of pigs after PRV HD/c immunization but with low frequency (less than 0.6%) as well ( Figures 7C–F ). No PRV-specific triple cytokine-producing T cells were detected in the immunized pigs (data not shown). These results demonstrated that attenuated PRV vaccine strain also induced weak Th1 immunity in pigs, similar to that observed in mice, which may be an intrinsic property of PRV LAVs.