Balasch et al. Porcine Health Management (2019) 5:2 https://doi.org/10.1186/s40813-018-0112-7 RESEARCH Open Access Immune response development after vaccination of 1-day-old naïve pigs with a Porcine Reproductive and Respiratory Syndrome 1-based modified live virus vaccine Monica Balasch1* , Maria Fort1, Lucas P. Taylor2, Ivan Díaz5, Enric Mateu3,4 and Jay G. Calvert2 Abstract Background: The development of the innate and adaptive immune responses to Porcine reproductive and respiratory syndrome virus (PRRSV) after vaccination of 1 day-old pigs with a PRRSV-1 based modified live virus (MLV) vaccine by intramuscular (IM) and intranasal (IN) routes was characterised, before and after challenge with a heterologous PRRSV-1 isolate at 18 weeks post-vaccination. Twenty-five PRRSV-seronegative piglets were used. At 1 day of age, pigs were administered with a single dose of vaccine via the IM (n = 10) or the IN route (n = 10). Control group (n = 5) received saline solution. After vaccination, pigs were bled at days 3, 7, 28, 56, 83, 113 and 125. Levels of cytokines IL-10, IL-8, IFN-α (measured by ELISA tests of serum), TNF-α and IFN-γ (measured by ELISA and ELISPOT, respectively, from stimulated peripheral blood mononuclear cells), and serum neutralising antibodies (NA) to the vaccine strain, were measured. Results: The induction of IL-10 was rare, indicating that IL-10 mediated immunomodulation/immune dysfunction was not a feature of this vaccine or of the challenge virus. IL-8 was detected in only two pigs following vaccination, but in the majority of pigs after challenge, indicating that their ability to produce an innate immune response was not impaired. TNF-α was not detected in any vaccinated pigs until day 83. After challenge, only a minority of pigs produced TNF-α. IFN-α was detected in all vaccinated pigs following vaccination, indicating the potential for development of an effective Th1 adaptive immune response. IFN-γ-secreting cells were detected in all vaccinated pigs after vaccination. NA to the vaccine strain were first detected at day 56 in pigs vaccinated by both routes, and remained at similar levels until challenge. After challenge, a boost in NA was observed. The efficacy of the vaccine was demonstrated by reduction of viraemia and nasal shedding after challenge. Conclusions: The administration of a PRRSV-1 based MLV vaccine to 1 day-old piglets was able to induce an immune response characterised by: (1) undetectable or low levels of IL-10, IL-8 and TNF-α, (2) an increase in IFN-α expression within the first seven days, (3) a gradual increase in the number of antigen-specific IFN-γ-secreting cells, and (4) induction of detectable NA. After challenge with a heterologous strain, there was a rapid boost in NA titres, indicating a priming effect of the vaccine. Keywords: Porcine reproductive and respiratory syndrome virus, Cell-mediated immunity, Serum Neutralising antibodies, Modified live virus vaccine * Correspondence: monica.balasch@zoetis.com 1 Zoetis Manufacturing & Research Spain S.L., Ctra. Camprodon s/n, Finca La Riba, 17813 Vall de Bianya, Girona, Spain Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Balasch et al. Porcine Health Management (2019) 5:2 Page 2 of 13 Background The ability of the immune system to fight against in- The development of an adaptive immune response to fectious agents is conditioned by a sufficient degree of Porcine reproductive and respiratory syndrome virus functional maturation of the immune system [13]. It has (PRRSV) depends on both humoral and cellular compo- been demonstrated that the ability of monocytes and neutro- nents [1]. The innate immune system is the first mechan- phils from young pigs to produce pro-inflammatory cyto- ism of defence to prevent viral invasion and replication kines is reduced compared to those from adult pigs: the most and influences the adaptive response of the immune sys- pronounced changes in cytokine production occurred at tem. Adequate early activation of the innate immune sys- weaning [13]. It should therefore be expected that the ability tem is critical to initiate generation of protective adaptive of the immune system of a pre-weaning pig to respond to a immunity to achieve complete viral clearance [2]. How- MLV vaccine could be reduced relative to older animals. ever, PRRSV induces a slow and weak innate response [1]. The objective of this study was to define the profile of The quantities of important cytokines secreted in pigs in- development of innate and adaptive immunity to PRRSV fected by PRRSV, like IFN-α or IFN-γ, appeared to be sig- after vaccination of 1-day-old pigs with a PRRSV-1 nificantly lower than in pigs infected with Influenza A based MLV vaccine (Suvaxyn PRRS MLV), and after virus or Porcine respiratory coronavirus [3–5]. The devel- challenge with a virulent heterologous field isolate of opment of neutralising antibodies (NA) is also known to PRRSV-1. One day old piglets were selected as they rep- occur later in the course of the infection although resent the “worse-case scenario” (youngest pigs) allowed non-neutralising antibodies appear by 7–14 days post-in- by the indications of the vaccine used. Two routes of oculation [6]. In that way, both humoral and cell-mediated vaccine administration, intramuscular (IM) and intrana- specific immunity are delayed, compromising clearance of sal (IN), were evaluated. Challenge was delayed until 18 the virus [7]. weeks after vaccination to ensure satisfactory duration Serum NA also play an important role in the protec- of immunity. Innate immunity was measured by evaluat- tion of animals against clinical disease. Passive transfer ing the level of anti-inflammatory and pro-inflammatory of NA to pregnant sows (titres 1/16) can protect them cytokines (IL-10, IL-8, TNF-α and IFN-α), humoral im- against reproductive failure by blocking transplacental munity by measuring the development of NA, and infection [8]. Using the same antibody transfer system, a cell-mediated immunity by evaluating the development titre of 1/8 or higher protected piglets against the devel- of IFN-γ- secreting cells. opment of viraemia, with sterilising immunity being ob- tained at titres of 1/32 [9]. These results suggest that a Methods vaccine capable of inducing NA titres of 1/32 against a Experimental design given PRRSV strain should prevent the clinical disease Twenty-five piglets born from PRRSV-seronegative sows produced by it and could be an important tool in the were used, thus the potential impact of maternally de- control of PRRSV [10]. It has to be taken into ac- rived antibodies was not assessed in this study. The ani- count, however, that these studies used a homologous mals were allocated to treatments following a completely challenge model. Despite the significant role that NAs randomised design. At 1 day of age, two groups of 10 seem to play in protection, their effectiveness might pigs were administered a single 2 mL dose of vaccine via be limited against heterologous isolates, as demon- the IM (T02) or the IN (T03) route. Five pigs from the strated by the limited ability of PRRSV hyperimmune control group (T01) received 2 mL intramuscular and sera to effectively neutralise a variety of heterologous 2 mL intranasal of saline solution. After vaccination, strains [11]. pigs were bled at days 3, 7, 28, 56, 83, 113 and 125. The nature of the protection induced by PRRSV MLV Animals were housed by treatment with one pen per vaccines against heterologous challenges is controversial. treatment in the vaccination phase. Prior to challenge, Although it is widely accepted that heterologous protec- animals were re-housed and comingled in one room. tion is rather limited and strain dependent [12], con- At 18 weeks post-vaccination (126 days), pigs were trasting models of the immune response have been challenged IN with the PRRSV-1 isolate Olot/91. Levels of proposed for different PRRSV strains: one based on the cytokines interleukin-10 (IL-10), interleukin-8 (IL-8), development of NA with low IFN-γ responses, the other interferon-alpha (IFN-α) and tumor necrosis factor-alpha based on effective IFN-γ responses with a poor develop- (TNF-α), number of interferon-gamma (IFN-γ) secreting ment of NA [12]. cells, and levels of NA to the vaccine strain, were mea- Taken together, it is highly recommended to define the sured at different time points after vaccination, and 6 profile of humoral and cell-mediated immunity induced days after challenge. Also, PRRSV viraemia and nasal by a given MLV vaccine in order to predict its ability to shedding were determined by RT-qPCR 1 day prior to provide strong protection in front of challenge with challenge and at days 3, 6, 8 and 10 after challenge. heterologous strains. Ten days post-challenge, pigs were euthanised. Balasch et al. Porcine Health Management (2019) 5:2 Page 3 of 13 Throughout the experimental period the animals were previously reported [15] at days 3 and 7. Briefly, plates monitored daily for clinical observations. were coated with anti-pig IFN-α (mAb K9, R&D sys- tems) at 1.1 Ug/mL; 50 μl of each sample and biotinyl- Vaccination ated pig IFN-α (mAb F17, R&D systems) were added. Newborn piglets were used at 24 ± 12 h of age. The com- Positive reactions were revealed using Streptavidin- mercial vaccine Suvaxyn PRRS MLV contains a deriva- Horseradish (Thermo Fisher Scientific) and soluble TMB tive of the PRRSV-1 field isolate 96 V198, which has (Merck Millipore). IFN-α concentrations were calculated been attenuated by serial passage on BHK21 cells engi- according to the ODs obtained from the serial dilutions neered to express the porcine CD163 PRRS receptor. of the recombinant porcine IFN-α protein (R&D sys- This vaccine was used, at minimum immunising dose tems), from 250 to 4 U/mL. (2.2 log10 CCID50/dose), for T02 and T03 groups. At day 0, piglets in T02 were injected IM in the right side of Peripheral blood mononuclear cell (PBMC) cultures the neck. Piglets in T03 were administered IN, delivering PBMC cultures were obtained from heparinised blood 1.0 mL in each nostril. Piglets in T01 received saline so- samples by density-gradient centrifugation with Histopa- lution (2 mL IM and 2 mL IN). que 1077 (Sigma). To measure TNF-α responses, PBMC were seeded at a density of 1 × 106 cells per well in Challenge 96-well plates and were mock-stimulated or stimulated At 18 weeks post-vaccination (126 days), pigs were chal- with either PRRSV 96 V198 (vaccine strain) at a multi- lenged IN with the PRRSV-1 isolate Olot/91 [14], at a plicity of infection (moi) of 0.1, or 10 mg/ml of PHA. dose of 105.8 CCID50/pig. The challenge virus is pas- After 24 h incubation, cell culture supernatants were col- saged only on primary porcine alveolar macrophages in lected and frozen at − 80 °C until used. A TNF-α com- order to ensure retention of virulence. The degree of mercial ELISA test (Porcine TNF-α ELISA Kit, Thermo ORF7 and ORF5 nucleotide identity between the vaccine Fisher Scientific) was performed as above. To calculate virus and the challenge virus is 93.0 and 89.6%, respect- PRRSV-specific quantity of TNF-α, responses in un- ively. Although both viruses belong to subtype 1 of stimulated wells were subtracted from responses in PRRSV-1, they differ significantly in sequence. virus-stimulated wells. Results were expressed as pg/mL. Sampling ELISPOT IFN-γ All pigs were bled before vaccination. After vaccination, PBMC were used to evaluate the frequencies of specific pigs were bled at days 3, 7, 28, 56, 83, 113 and 125. After IFN-γ-secreting cells (IFN-γ-SC) against the vaccine challenge (day 126), pigs were bled and nasal swabs were strain by ELISPOT IFN-γ as reported elsewhere [16]. taken at days 3, 6, 8 and 10 (study days 129, 132, 134 The ELISPOT assay was developed using commercial and 136). mAbs (Porcine IFN-γ P2G10 and biotin P2C11, BD Bio- Blood samples collected before vaccination (day 0) and sciences Pharmingen). PBMC were stimulated with the before challenge (day 125) were tested by PRRSV ELISA PRRSV strain 96 V198 at a moi of 0.1; the stimulating (PRRS × 3, IDEXX) following manufacturer’s instructions. vaccine strain had been previously titrated in a Blood was collected in appropriate tubes to obtain BHK-CD163 expressing cell line by end-point limiting serum (PRRSV NA assay, PRRSV RT-qPCR and cytokine dilution and it was adjusted to the corresponding moi ELISA tests for IL-10, IL-8 and IFN-α), and whole blood just prior to conducting the test. Unstimulated and in heparinised tubes (IFN-γ ELISPOT and TNF-α PHA-stimulated cells (10 μg/mL) were used as negative ELISA). Nasal swabs were placed in 1 mL of PBS. and positive controls, respectively. All tests were done in quadruplicate. Reactions were developed by adding Cytokine analysis 3-amino-9-ethylcarbazole as substrate. PRRSV-specific Sera frequencies of IFN-γ-SC were calculated by subtracting Cytokines IL-8 and IL-10 in sera were measured by counts of spots in unstimulated wells from counts in ELISA using commercial pairs of monoclonal antibodies PRRSV-stimulated wells and were expressed as number (mAb) and according to the manufacturer’s instructions of responding cells in 5 × 106 PBMC. (Porcine IL-8 and IL-10 ELISA Kits, Thermo Fisher Scientific). The cut-off point of each ELISA was calcu- PRRSV serum neutralisation assay lated as the mean + 3SD OD (optical density) of negative Inactivated serum samples were serially diluted two-fold controls. Cytokine concentrations were calculated by (1:2 to 1:4096) in 96-well plates. PRRSV strain 96 V198 using the linear regression formula from ODs of the vaccine virus suspension (125 μl) containing 800 cytokine standards provided by the manufacturer. Re- CCID50/mL was added to each well and plates were in- sults were expressed as pg/mL. IFN-α was measured as cubated for 1 h at 36–38 °C. A BHK21-CD163 expressing Balasch et al. Porcine Health Management (2019) 5:2 Page 4 of 13 cell suspension [17] containing 2.5-3 × 105 cells/mL was data were analysed using a general linear repeated mea- prepared and 100 μL aliquoted to each well in a new plate. sures mixed model with fixed effects: treatment, time Fifty microliters of each serum-virus mix were transferred point, and treatment by time point interaction; and ran- to the plate containing cells. The mixture was incubated dom effects: animal within treatment, which is the ani- at 36–38 °C and 5% CO2 for five days. A direct immuno- mal term. fluorescence assay technique using FITC-conjugated Prior to statistical analysis the RT-qPCR was trans- monoclonal antibody SDOW-17 (Rural Technologies Inc) formed using an appropriate logarithm transformation. was performed. The NA titre was determined as the The transformed data was analysed using a general lin- inverse of the last dilution of serum that inhibited the ear repeated measured mixed model. Pairwise treatment FA signal. comparisons were made at each time point if the treat- ment or treatment by time point interaction effect was PRRSV RT-qPCR significant (P ≤ 0.05). Treatment least squares mean and RNA was purified from serum and nasal swab samples 95% confidence intervals were back-transformed for pres- using the Biosprint 96 DNA Blood kit. Viraemia was entation. Negative samples were given a value of 50 PRRSV measured by means of a Reverse Transcription (RT) RNA copies/mL (1.7 log10 PRRSV RNA copies/mL), which qPCR. In brief, the purified viral RNA was used as tem- corresponds to a half of the quantification limit of the tech- plate, reverse transcribed at 50 °C for 30 min, and dena- nique (100 PRRSV RNA copies/mL). tured at 95 °C for 5 min. The PCR program of reactions Viraemia and nasal shedding were analysed with a consisted of 40 cycles of denaturation at 95 °C for 20 s general linear repeated measures mixed model with a and annealing at 53 °C for 40 s. The qRT-PCR was con- logit link with fixed effects: treatment, time point, and ducted in a 7500 Real-Time PCR System thermal cycler. treatment by time point interaction, and random effects: The oligonucleotide primers and dual-labeled probe pen, block within pen, and animal within block, pen, used for amplification were 5’-GCACCACCTCACCC and treatment, which is the animal term. AGAC- 3′ (forward, final concentration 0.5 μM); 5’- Linear combinations of the parameter estimates were CAGTTCCTGCGCCTTGAT- 3′ (reverse, final concen- used in a priori contrasts after testing for a significant tration 0.5 μM); and 5′-6-FAM-CCTCTGCTTGCAAT (P ≤ 0.05) treatment effect or treatment by time point CGATCCAGAC- BHQ1–3′ (dual-labeled probe, final interaction. Comparisons were made between treatments concentration 0.6 μM), which correspond to base pair at each time point. The 5% level of significance (P ≤ positions 14,792–14,809, 14,851–14,868, and 14,819– 0.05) was used to assess statistical differences. Least 14,842, respectively, of the EU prototype strain Lelystad squares means (back transformed), standard errors, and (Genbank accession number M96262). The amplicon 95% confidence intervals of the means were calculated consists of a 77-bp fragment from ORF7. for each treatment and time point. If the model did not The genome equivalents (RNA copy number per 5 μL) converge, Fisher’s Exact were used for the analysis. were interpolated from the RNA standard curve for this assay and adjusted (RNA copy number per 1 mL of sam- Results ple) according to the sample dilution. Clinical observations Eight weeks after vaccination an infectious urinary Data analysis process, characterised by cystitis, urinary bladder rup- Data summaries and analyses were performed with a ture and peritonitis, occurred in the pigs allocated into centralised data management system (SAS/STAT User’s the vaccinated groups (T02 and T03). One pig from T02 Guide Version 9.3 or higher, SAS Institute, Cary, NC). and two pigs from T03 died. All pigs were treated with Only post-challenge data (once animals were comingled) enrofloxacin (7.5 mg/kg IM). Another pig, belonging to were analysed. Pre-challenge data was summarised with T02, died during the bleeding process at D56. The rest descriptive statistics, since the experimental unit (the of the pigs remained healthy during the post-vaccination pen) was not replicated. Post-challenge data was ana- period. After challenge, no clinical signs associated with lysed, since animals had been commingled and the ex- PRRSV infection were observed in pigs of any of the perimental unit was the individual animal. Prior to treatment groups. statistical analysis, results were transformed, where ne- cessary, using an appropriate logarithm transformation. Viraemia and nasal shedding For tests that had a single result after being moved All pigs were non-viraemic before vaccination and be- into challenge housing, the transformed data were ana- fore challenge. After challenge, all control animals (T01) lysed using a general linear mixed model with fixed ef- became viraemic at Day 129 (3 days after challenge), co- fects: treatment. For tests that had multiple results after inciding with the peak of viraemia. The length of vir- being moved into challenge housing, the transformed aemia was at least 10 days, since all control pigs were Balasch et al. Porcine Health Management (2019) 5:2 Page 5 of 13 still viraemic at the end of the study. In pigs vaccinated Control pigs (T01) did not produce measurable IL-10 by the IM route (T02), viraemia was detected in 8/10 during the vaccination phase. After challenge, IL-10 was pigs, and in 2 pigs it was not detected throughout the detected in one out of 5 control pigs. In pigs vaccinated post-challenge period. The peak of viraemia was also at by the IM route (T02), IL-10 was not produced after 3 days after challenge, although the virus load was more vaccination. After challenge, IL-10 was detected in one than 2.0 log lower than in control pigs (Table 1). At the out of 8 pigs. In pigs vaccinated by the IN route (T03), end of the study, 10 days after challenge, all IM vacci- IL-10 was produced in 1 out of 10 pigs after vaccination nated pigs but one had already cleared the virus from at Day 3. However, IL-10 was not detected in any pig at blood. In pigs vaccinated by IN route (T03), viraemia Day 7. After challenge, IL-10 was detected in 2 out of 6 was detected in at least one time point in all animals in tested pigs. Differences on IL-10 levels at Day 132 were the post-challenge period. The peak of viraemia was also not statistically significant between vaccinated and con- at 3 days after challenge, although the virus load was trol groups, nor between vaccinated groups. more than 1.0 log lower than in control pigs (Table 1). At the end of the study, 10 days after challenge, all IN Il-8 vaccinated pigs but two had already cleared the virus IL-8 was measured after vaccination (Days 3 and 7), and from blood. 6 days after challenge, Day 132 (Table 3, Fig. 2). Control Viral loads (Table 1) were significantly lower in IM pigs (T01) did not produce measurable IL-8 during the vaccinated than in control pigs at days 3, 6, 8 and 10 vaccination phase. After challenge, IL-8 was detected in 3 after challenge; also, viral loads were significantly lower out of 5 control pigs. In pigs vaccinated by the IM route in IN vaccinated than in control pigs at days 6, 8 and 10 (T02), IL-8 was not produced after vaccination at Day 3. after challenge. The percentage of pigs viraemic was sig- However, IL-8 was produced in 1 out of 10 pigs at Day 7. nificantly lower at days 8 (p = 0.0047) and 10 (p = 0.0210) After challenge, IL-8 was detected in 3 out of 8 pigs. In after challenge in both IM and IN vaccinated groups, pigs vaccinated by the IN route (T03), IL-8 was not pro- compared to non-vaccinated pigs. duced after vaccination at Day 3. However, IL-8 was pro- The viral load detected in nasal swabs (Table 2) was duced in 1 out of 10 pigs at Day 7. After challenge, IL-8 significantly lower in IM vaccinated than in control pigs was detected in 6 out of 8 pigs. Differences on IL-8 levels at days 3 and 6 after challenge; viral loads were not sig- at Day 132 were not statistically significant between vacci- nificantly lower in IN vaccinated than in control pigs nated and control groups, nor between vaccinated groups. after challenge (Table 2). The percentage of shedding pigs was significantly lower in the IM vaccinated group, IFN-α compared to the control group, at 3 days after challenge. IFN-α was measured after vaccination at Days 3 and 7 No significant differences were detected between the IN (Table 3, Fig. 3). Control pigs (T01) did not produce meas- vaccinated group and control group, or between the vac- urable IFN-α during the vaccination phase, except in one cinated groups. out of 5 pigs, in which low amounts of IFN-α were de- tected at Day 3. At Day 7, IFN-α was not detected in any Cell mediated immunity control pig. In pigs vaccinated by the IM route (T02), Il-10 IFN-α was produced in all pigs at Day 3 and at Day 7. In IL-10 was measured after vaccination (Days 3 and 7), pigs vaccinated by the IN route (T03), IFN-α was pro- and 6 days after challenge, Day 132 (Table 3, Fig. 1). duced in 9 out of 10 pigs at Day 3, and in all pigs at Day 7. Table 1 Mean (±SD) viral load in serum after challenge and percentage of viraemic pigs Treatment Day of study 125/DC-1 129/DC + 3 132/DC + 6 134/DC + 8 136/DC + 10 Viral % viraemic Viral load % viraemic Viral load % viraemic Viral load % viraemic Viral load % viraemic load pigs pigs pigs pigs pigs Mean PRRSV T01 ND 0% 6.01 ± 0.3 100% 4.72 ± 0.1 100% 4.21 ± 0.3 100% 5.32 ± 0.8 100% viral load T02 ND 0% 3.57 ± 0.6 62.5% 2.90 ± 0.5 50% 1.80 ± 0.1 12.5% 2.11 ± 0.5 12.5% T03 ND 0% 4.74 ± 0.7 87.5% 3.80 ± 0.4 87.5% 2.15 ± 0.3 25% 1.86 ± 0.1 25% Contrast P value T01 vs T02 0.0059 NS 0.0054 NS 0.0016 0.0047 0.0087 0.0047 T01 vs T03 NS NS 0.0458 NS 0.0011 0.0210 0.0096 0.0210 T02 vs T03 NS NS NS NS NS NS NS NS Values are expressed as log10 PRRSV RNA copies per mL of serum. DC day of challenge, ND non-detectable (below limit of detection of the technique), NS non-significant Balasch et al. Porcine Health Management (2019) 5:2 Page 6 of 13 Table 2 Mean (±SD) viral load in nasal swabs after challenge and percentage of shedder pigs Treatment Day of study 125/DC-1 129/DC + 3 132/DC + 6 134/DC + 8 136/DC + 10 Viral load % shedder Viral load % shedder Viral load % shedder Viral load % shedder Viral load % shedder pigs pigs pigs pigs pigs Mean PRRSV T01 ND 0% 5.56 ± 0.7 100% 4.53 ± 0.5 100% 2.07 ± 0.3 20% 2.10 ± 0.4 20% viral load T02 ND 0% 2.21 ± 0.6 12.5% 2.64 ± 0.4 37.5% ND 0% 2.03 ± 0.3 25% T03 ND 0% 3.72 ± 0.6 62.5% 3.19 ± 0.4 62.5% 2.06 ± 0.2 25% 2.52 ± 0.3 37.5% Contrast P value T01 vs T02 0.0019 0.0047 0.0119 NS NS NS NS NS T01 vs T03 NS NS NS NS NS NS NS NS T02 vs T03 NS NS NS NS NS NS NS NS Values are expressed as log10 PRRSV RNA copies per mL. DC day of challenge, ND non-detectable (below limit of detection of the technique), NS non-significant Table 3 Cytokine measure geometric mean (±SD) results Treatment Day of study 3 7 28 83 125 (DC-1) 132 (DC + 6) IL-10 T01 ND ND 4.8 ± 1.0 (1/5) T02 ND ND 5.2 ± 1.7 (1/8) T03 ND ND 10.1 ± 6.4 (2/8) P value T01 vs T02 NS T01 vs T03 NS T02 vs T03 NS IL-8 T01 ND ND 48.1 ± 22.9 (3/5) T02 ND ND 33.8 ± 12.7 (3/8) T03 ND ND 69.6 ± 26.2 (6/8) P value T01 vs T02 NS T01 vs T03 NS T02 vs T03 NS IFN-α T01 ND 1.9 ± 0.0 (1/5) T02 9.8 ± 6.1 (10/10) 32.8 ± 11.8 (10/10) T03 9.5 ± 9.7 (9/10) 14.1 ± 9.9 (10/10) TNF-α T01 ND ND (1/5)a ND 62.7 ± 7.6 (1/5) ND 29.1 ± 1.5 a T02 ND ND ND ND (1/8) ND 41.9 ± 1.6 (1/8) T03 ND ND ND ND (2/8)a ND 78.2 ± 2.9 (1/8) P value T01 vs T02 NS T01 vs T03 NS T02 vs T03 NS IFN-γ T01 1.6 ± 0.0 (5/5) 1.6 ± 0.0 (4/5) 0.0 ± 0.0 (3/4) 8.1 ± 0.5 (3/4) T02 22.1 ± 1.1 (9/10) 80.5 ± 8.2 (8/8) 166.5 ± 7.1 (8/8) 133.5 ± 5.7 (8/8) T03 51.3 ± 4.1 (8/9) 77.2 ± 8.2 (8/8) 102.2 ± 4.3 (8/8) 155.7 ± 6.6 (8/8) P value T01 vs T02 0.0003 0.0033 T01 vs T03 0.0108 0.0011 T02 vs T03 NS NS a Although some pigs had values above the cut-off of the technique, the mean value was below the cut-off Results from IL-10, IL-8, IFN-α and TNF-α ELISA tests are expressed as pg/mL of serum. Results from IFN-γ ELISPOT are expressed as number of IFN-γ- secreting cells /5 × 105 PBMC. In brackets, the number of positive animals per treatment and time point. DC day of challenge, ND non-detectable (below limit of detection of the technique), NS non-significant; blank cell indicates not measured Balasch et al. Porcine Health Management (2019) 5:2 Page 7 of 13 Fig. 1 Evolution of IL-10 levels (red line indicates limit of detection: 7.8 pg/mL) TNF-α After challenge, TNF-α was detected in one out of 8 TNF-α was measured during the vaccination phase pigs. In pigs vaccinated by the IN route (T03), TNF-α (Days 3, 7, 28, 83 and 125), and 6 days after chal- was not produced after vaccination, except for 2 ani- lenge, Day 132 (Table 3, Fig. 4). Control pigs (T01) mals at Day 83. After challenge, TNF-α was detected did not produce measurable TNF-α during the vaccin- in one out of 8 pigs. Differences on TNF-α levels at ation phase, except in one out of 5 pigs, in which Days 125 and 132 were not statistically significant be- low amounts of TNF-α were detected at Day 7, and tween vaccinated and control groups, nor between another pig at Day 83. Just before challenge and after vaccinated groups. challenge, it was not detected in any pig. In pigs vac- cinated by the IM route (T02), TNF-α was not pro- IFN-γ duced after vaccination, except for 1 animal that IFN-γ-SC were measured during the vaccination phase produced very low amounts of TNF-α at Day 83. (Days 28, 83 and 125), and 6 days after challenge, Day Fig. 2 Evolution of IL-8 levels (red line indicates limit of detection: 31.25 pg/mL) Balasch et al. Porcine Health Management (2019) 5:2 Page 8 of 13 Fig. 3 Evolution of IFN-α levels (Limit of detection: 3.9 pg/mL) 132 (Table 3, Fig. 5). Control pigs (T01) produced Humoral immunity very low IFN-γ secreting cells during the post-vaccination All pigs were seronegative to PRRSV antibodies detected period. After challenge, there was a mild increase in by ELISA before vaccination. Before challenge, at day the number of IFN-γ-SC. In pigs vaccinated by the 125, control pigs (T01) remained seronegative (mean S/P IM route (T02), the number of IFN-γ-SC increased ratio 0.011), while vaccinated pigs (T02 and T03) had ser- from 22 to 166 during the post-vaccination period, oconverted. The geometric mean antibody titres were and were 133 after challenge. In pigs vaccinated by 1.681 for T02 and 1.103 for T03. the IN route (T03), the number of IFN-γ-SC in- NA were measured during the vaccination phase (Days creased from 51 to 102 during the post-vaccination 28, 56, 83, 113 and 125), and 6 days after challenge, Day period, and to 155 after challenge. Differences in the 132 (Table 4, Fig. 6). Control pigs (T01) had low to un- number of IFN-γ-SC at Days 125 and 132 were statis- detectable NA titres throughout the study. All vacci- tically significant between vaccinated (both IM and nated pigs developed some level of NA after vaccination IN) and control groups, but not between the vacci- and before challenge. In pigs vaccinated by the IM route nated groups. (T02), NA were first detected at Day 56 after vaccination Fig. 4 Evolution of TNF- α levels (red line indicates limit of detection: 62.5 pg/mL) Balasch et al. Porcine Health Management (2019) 5:2 Page 9 of 13 Fig. 5 Evolution of the number of IFN-γ secreting cells, expressed as number of IFN-γ producing cells /5 × 105 PBMC (geometric mean of 4.9), and were kept at very similar response [3, 4], and a delayed adaptive immune response levels until challenge. After challenge, NA titres went [1, 6, 15]. The objective of this study was to characterise from 4.6 (Day 125, day before challenge) to 14.7 (Day the development of the innate, humoral and cell medi- 132, 6 days after challenge). In pigs vaccinated by the IN ated immune responses to PRRSV after vaccination of route (T03), NA were first detected at Day 56 after vac- 1-day-old naïve pigs with a PRRSV-1 based MLV vaccine cination (geometric mean of 4.5), and were kept at very by the IM and IN routes, before and after challenge with similar levels until challenge. After challenge, NA titres a PRRSV-1 isolate at 18 weeks post-vaccination. Levels went from 2.3 (Day 125, day before challenge) to 16.1 of cytokines IL-10, IL-8, IFN-α, TNF-α and IFN-γ, as (Day 132, 6 days after challenge). Differences in NA ti- well as levels of NA, were measured. tres at Day 132 were statistically significant between vac- IL-10 is an anti-inflammatory cytokine which has been cinated and control groups. Differences in NA titres at linked to regulatory T cell activity and to decreased cell- Day 125 were statistically significant between pigs vacci- mediated immune responses, by inhibiting the induction nated by the IM route and control pigs, but not between of pro-inflammatory cytokines such as IFN-γ and TNF-α the IN vaccinated group and the control group. [18]. It has been hypothesised that in some strains of PRRSV the induction of IL-10 pushes the immune re- Discussion sponse towards a relatively ineffective induction of IFN-γ PRRSV is known to interact with the immune system of specific response [19]. In this study, the induction of de- the host and to modulate the immune response after in- tectable levels of IL-10 was rare, both after vaccination fection. PRRSV induces a slow and weak innate immune and after challenge. During the post-vaccination phase, a Table 4 Serum NA test geometric mean (±SD) results Treatment Day of study 28 56 83 113 125 (DC-1) 132 (DC + 6) SN antibody inverse titre T01 ND ND ND ND ND ND T02 ND 4.9 ± 3.5 3.1 ± 3.3 3.9 ± 4.0 4.6 ± 1.4 14.7 ± 4.4 T03 ND 4.5 ± 3.9 2.3 ± 3.0 2.7 ± 2.7 2.3 ± 0.7 16.1 ± 4.8 Contrast P value T01 vs T02 0.0149 <.0001 T01 vs T03 NS <.0001 T02 vs T03 NS NS Titres are expressed as the inverse of the highest dilution at which NA activity is detected. DC day of challenge, ND non-detectable (below limit of detection of the technique), NS non-significant Balasch et al. Porcine Health Management (2019) 5:2 Page 10 of 13 Fig. 6 Evolution of SN titers (inverse titer). Positive ≥2 single pig (from the IN-vaccinated group) showed induc- good safety profile of the vaccine (no systemic or local tion of a low level of IL-10 (10.9 pg/mL). After challenge reactions observed after vaccination; data not shown). with the Olot/91 strain, four pigs were positive for IL-10 TNF-α is involved in systemic inflammation and acute with levels up to 346.6 pg/mL. The near absence of meas- phase reaction as a pro-inflammatory cytokine. It is pro- urable IL-10 induction by the vaccine virus indicates that duced mainly by activated macrophages, although it can be vaccination did not induce any significant response attrib- produced by other cell types. Being an endogenous pyro- utable to this regulatory cytokine, either in the period of gen, TNF-α is able to induce fever, apoptotic cell death, innate response (day 3) or in later stages of the trial. IL-10 cachexia, inflammation, and to enhance viral replication mediated immunomodulation/ immune dysfunction was [23]. In the present study, TNF-α was not detected in any not a general feature of this vaccine strain, as demon- vaccinated pigs before challenge. After challenge, only a mi- strated by induction of a high number of IFN-γ-SC. This nority of pigs in all three groups produced detectible levels is a positive feature of the vaccine strain, since IL-10 has of TNF-α, confirming the inhibitory effect of PRRSV on the been related to an inhibition of the development of an ef- induction of pro-inflammatory cytokines. fective immune response against PRRSV [10]. IFN-α is a type I interferon produced by infected cells, IL-8 is a pro-inflammatory cytokine that induces and mediates the first line of defense against viral infec- chemotaxis in target cells, primarily neutrophils and tion (innate antiviral cytokine). Interaction of IFN-α with other granulocytes, causing them to migrate towards the receptors on target cells establishes a potent antiviral site of infection [20]. Usually macrophages detect anti- state. In addition, IFN-α serves as an important link be- gen first, and are the first cells to release IL-8 to recruit tween innate and adaptive immunity, and can enhance other cells. Some studies have linked an IL-8 response to the development of a strong T helper type 1 (Th1) in- enhanced resistance to PRRSV infection [21, 22]. In this flammatory response [1]. IFN-α induction has been re- study IL-8 was detected in only two pigs following vac- ported to be weak or absent for some PRRSV strains. cination, and not at all in the non-vaccinated group, sug- The active inhibition of IFN-α production, and inhib- gesting that the vaccine did not generate intense ition of various IFN-induced cellular gene products, has inflammatory reactions. Young pigs have a reduced abil- been attributed to specific viral proteins and implicated ity to produce IL-8 in response to an immune stimula- as a primary cause of the impaired immune responses to tion, compared to adults [13]. However, IL-8 was the virus [1, 24]. In this study, IFN-α was detected in all detected in the majority of vaccinated and non-vacci- vaccinated pigs, regardless of route. IFN-α levels in- nated pigs after challenge, indicating that the ability of creased between days 3 and 7 following vaccination, pigs to produce IL-8 was not impaired after challenge. while remaining low in the non-vaccinated control There were no significant differences between the vacci- group. These results would be consistent with the devel- nated and non-vaccinated groups. The lack of induction opment of antiviral responses. Consistent induction of of this pro-inflammatory cytokine is consistent with the IFN-α secretion shortly after vaccination indicates the Balasch et al. Porcine Health Management (2019) 5:2 Page 11 of 13 potential for development of an effective Th1 adaptive neutralizing antibodies alone, and includes an important immune response dominated by IFN-γ, which was dem- cell-mediated component [1]. In this study, an almost onstrated by frequencies of IFN-γ-SC in response to the complete clearance of viraemia was achieved 10 days virus well above 100 per million. after challenge, in 80% of the animals vaccinated by IFN-γ (type II interferon) is a pro-immune cytokine the IM route. All these animals had detectable levels that has an ability to inhibit viral replication directly, but of NA and IFN-γ-SC, consistent with humoral and its most important function is immunomodulatory. cell-mediated immunity both contributing to the con- IFN-γ is the primary cytokine that defines Th1 cells: trol of infection. Th1 cells secrete IFN-γ, as do natural killer (NK) cells. It Viraemia and nasal shedding results confirmed that is considered to be correlated with protection, although vaccine protected pigs using both administration routes. IFN-γ response in PRRSV infected pigs appears to be All animals belonging to the control group were still vir- delayed [7, 25], and it is considered to be weak, com- aemic at 10 days after challenge; in contrast, only 1/8 IM pared to other swine viruses such as Aujeszky’s Disease vaccinated pigs and 2/8 IN vaccinated pigs were vir- virus [1]. In the current study, IFN-γ-SC were detected aemic at day 10, indicating that the immunity induced in all vaccinated pigs, increased steadily in number be- by the vaccine was able to clear the challenge virus from tween days 28 and 125 post-vaccination, and remained blood. Specifically, it was noted that those animals hav- high after challenge (means of 133 and 156 cells/5 × 105 ing a high NA titre at challenge were able to clear vir- PBMC in the IM and IN treatments, respectively), con- aemia in 6–8 days, or even did not become viraemic. firming that vaccination can positively modulate IFN-γ One IM-vaccinated animal with a low NA titre at chal- gene expression and sustain IFN-γ secretion when cells lenge did not become viraemic, but had the highest are re-stimulated [7]. The number of IFN-γ-SC in IFN-γ response. NA and IFN-γ responses were mea- non-vaccinated pigs remained low. sured against the vaccine strain only. Results would As mentioned, IL-8 and TNF-α are pro-inflammatory likely be different (lower) against a heterologous virus, cytokines induced in some PRRSV infections. In this such as the challenge virus used in this study. However, study IL-8 and TNF-α were either not detected, or de- it is important to establish homologous baseline perform- tected in only in a minority of animals after vaccination. ance of the vaccine before considering the effects on However, both innate and adaptive immune responses heterologous cross-reaction on these immunoassays. The could be clearly demonstrated following vaccination by ability of the vaccine to protect against this heterologous the production of IFN-α and subsequent development of challenge virus is evident from the viraemia results. The IFN-γ-SC. Induction of IL-8 or TNF-α by the vaccine magnitude of the induction of NA and IFN-γ-SC (using could have been harmful to 1-day-old pigs. The near homologous antigen) will serve as an important internal absence of these pro-inflammatory cytokines may control in future studies with other challenge viruses. contribute to the observed safety of this vaccine in Differences (other than minor) in the development of pre-weaning pigs. the immune response or in the efficacy of the vaccine All pigs became effectively vaccinated, as indicated by were not observed between pigs vaccinated by the IM or the seroconversion (by ELISA) of all pigs from both vac- IN routes. Since the mechanism of development of pro- cinated groups. Levels of NA after vaccination were in tection is not known for this vaccine, it was of interest the expected range for a PRRSV MLV vaccine. Neutralis- to try both routes of administration. It could be specu- ing antibodies to the vaccine strain were first detected at lated that both routes result in vaccine virus replication Day 56 in pigs vaccinated by either the IN or IM route, and development of a systemic immune response under and were sustained at similar levels until challenge. After these conditions. challenge, a boost in NA was observed in both vacci- It has been demonstrated that 1 day-old pigs can have nated groups, while no evidence of a response was ob- a reduced ability to respond to immunological stimula- served in the non-vaccinated group. Interestingly, no tion compared to pigs of weaning age [13]. The present differences were seen for IN and IM vaccinated pigs. At study has demonstrated that piglets vaccinated at 1 day challenge (Day 126) NA levels were significantly higher of age can develop innate, humoral and cell-mediated in IM-vaccinated pigs than in the non-vaccinated control immune responses that are sufficient to protected them group, and at Day 132 both IM and IN-vaccinated from a wild type PRRSV challenge up to at least 16 groups were significantly higher than the control group. weeks after vaccination. One explanation for this appar- In piglets, titres above 1/8 have been correlated with ent contradiction is the live nature of this vaccine. The protection against homologous reinfection [6]. In the vaccine virus actively replicates for weeks following vac- present study NA titres prior to challenge did not reach cination, without causing disease, allowing time for the this value. However, it is generally acknowledged that immune system of the neonatal pig to reach a greater protection against PRRSV infection is not mediated be level of maturity before eliminating the vaccine virus. Balasch et al. Porcine Health Management (2019) 5:2 Page 12 of 13 In conclusion, the administration of a PRRSV-1 based biometrics analysis; EM, ID and JC reviewed the manuscript. All authors read MLV to 1-day-old naïve piglets, derived from PRRS-free and approved the final manuscript. parent stock, was able to induce innate, cell mediated Ethics approval and humoral immune responses characterised by: (1) The study protocol was approved by the Zoetis Manufacturing & Research undetectable or low levels of IL-10, IL-8 and TNF-α in Spain S.L. Animal Welfare committee prior to the start of the experiment, vaccinated pigs, (2) a mean 7-fold (IN) or 13-fold (IM) and was reported to the competent authorities. increase in IFN-α expression within the first 7 days, ob- Consent for publication served in all vaccinated pigs, (3) a gradual increase in Not applicable the number of antigen-specific IFN-γ-SC, from near zero to ≥100 cells/5 × 105 PBMC between days 28 post-vac- Competing interests cination and challenge, observed in all vaccinated pigs, With the exception of EM and ID, all other authors of this research article are employees from the sponsor company. and (4) induction of detectable NA in all vaccinated pigs by Day 56. After challenge with a heterologous field strain, there was a rapid boost in NA titres that was not Publisher’s Note observed in the non-vaccinated control group, indicating Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. a priming effect of the vaccine. Author details 1 Zoetis Manufacturing & Research Spain S.L., Ctra. Camprodon s/n, Finca La Conclusions Riba, 17813 Vall de Bianya, Girona, Spain. 2Zoetis Inc, 333 Portage St, The administration of a PRRSV-1 based MLV to Kalamazoo, MI 49007, USA. 3UAB, Centre de Recerca en Sanitat Animal 1-day-old naïve piglets is able to induce innate, cell me- (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain. 4Departament de Sanitat i Anatomia Animals, diated and humoral immune responses characterised by: Facultat de Veterinària, UAB, 08193 Bellaterra, Barcelona, Spain. 5IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat  Undetectable levels of IL-10, IL-8 and TNF-α Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.  Seven- (IN) to 13-fold (IM) increase in IFN-α Received: 9 October 2018 Accepted: 20 December 2018 expression  Gradual increase of IFN-γ-SC  NA induction References 1. Murtaugh MP, Xiao Z, Zuckermann F. 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