Aguirre et al. Porcine Health Management (2022) 8:47 Porcine Health Management https://doi.org/10.1186/s40813-022-00289-4 RESEARCH Open Access In the presence of non-neutralising maternally derived antibodies, intradermal and intramuscular vaccination with a modified live vaccine against porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) induce similar levels of neutralising antibodies or interferon-gamma secreting cells Laia Aguirre1*, Yanli Li1, Massimiliano Baratelli2, Gerard Martín-Valls1, Martí Cortey1, Joel Miranda2, Marga Martín1 and Enric Mateu1 Abstract The purpose of this study was to compare the immune response generated by the intramuscular and the intradermal vaccination route against the porcine reproductive and respiratory syndrome virus (PRRSV). Piglets from a seronegative and a seropositive farm were selected (n = 28 piglets per farm), and each group was divided into two groups and vaccinated after weaning with modified live vaccine Unistrain® PRRS (Laboratorios Hipra Amer, Spain) by the intramuscular (IM) or the intradermic (ID) route. For the following 6 weeks, animals were weekly bled to assess the humoral response by PRRSV-specific antibody ELISA and viral neutralisation test. At 0-, 3-, 4- and 6 weeks post-vaccination, peripheral mononuclear blood cells (PBMC) from eight animals per group were recovered to analyse cellular response by IFN-γ ELISPOT and lymphoproliferation. Serum IL-12 was also quantified by ELISA. Seroconversion was first detected 14 days post-vaccination (dpv) for both IM and ID routes, and peaked at 35 dpv (both IM groups and ID seropositive) or 42 dpv (ID seronegative). At 3 weeks after vaccination, 6/27 (22.22%) animals from negative origin had not seroconverted, and neutralising titres were significantly lower at 35 dpv compared to the seropositive origin (mean log2 titres of 1.36 and 4.25 respectively) Also, it was 10 times more probable for them to have high levels of IL-12 a week after vaccination than for animals of seropositive origin. Cellular immune response analysed by lymphoproliferation and IFN-γ ELISPOT was already present at 21 dpv and until 42 dpv, with no significant differences between groups except for a higher lymphoproliferation at 35 dpv in the IM seropositive group (Kruskal-Wallis, p < 0.05). These results indicate that the intradermal route induces an *Correspondence: Laia Aguirre laia.aguirre@uab.cat Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 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 in a credit line to the data. Aguirre et al. Porcine Health Management (2022) 8:47 Page 2 of 9 immune response equivalent to the classical intramuscular route even in presence of non-neutralising maternal immunity, which in this study has proven to facilitate seroconversion after vaccination with an heterologous strain. Keywords Porcine reproductive and respiratory syndrome virus, Vaccine, Intramuscular, Intradermal, Maternally derived antibodies Background are effective for immunizing children with MDA against The porcine reproductive and respiratory syndrome poliovirus [12, 14]. (PRRS) is one of the main causes of economic loss in In PRRS, the immune response of vaccination by ID swine worldwide. Control of the infection is usually route has been proven to be similar to the intramuscu- done by a combination of virus monitoring, biosecu- lar (IM) route [6–9] when pigs were not having specific rity restrictions, herd management measures and vac- MDA. However, much less is known about the efficacy of cination. Although vaccination most often targets sows, the ID route in the presence of MDA. The main objec- piglet vaccination is increasingly being considered to tive of the present study was to compare the immune protect weaners in farms that have endemic problems in responses generated by a PRRSV live attenuated vaccine nurseries. administered by either the ID or the IM routes in pig- Vaccination of piglets against PRRS virus (PRRSV) lets with high levels of specific MDA or in seronegative is usually performed during suckling or immediately piglets. For this purpose, 4-week old piglets from a sero- after weaning. In endemically infected farms, most pig- positive and a seronegative farm were vaccinated with lets present high levels of maternally derived antibod- an heterologous strain from the one used in the farm, ies (MDA) against PRRSV which may interfere with the by either ID or IM route and weekly followed until 10 development of an active immunity after vaccination weeks of age. PRRSV-specific antibody ELISA and viral [1, 2]. The general mechanisms of blocking by MDA for neutralisation test were done to assess humoral response, viruses are diverse (reviewed by Niewiesk [3]) and most and cellular response was tested by IFN-γ ELISPOT and often do not involve neutralization of the antigen but the lymphoproliferation. Innate immunity was measured by interaction of MDA with B-cell receptors. However, the ELISA of cytokines with anti-inflammatory (IL-10), pro- exact mechanism behind maternal blocking in PRRS is inflammatory (IL-12) or anti-viral (IFN-α) role. not fully elucidated and might involve neutralization as well. Results Different strategies can be used to avoid the eventual Clinical follow-up interference caused by maternal antibodies on vaccine No adverse reactions to vaccination were recorded. Two immunisation. For instance, one common approach is to animals from the seropositive group died prior to vacci- administer repeated doses of the vaccines (commonly two nation, one because of an internal haemorrhage (prob- doses separated by a 3-4-week period) close to the age ably because of a fight) and the other one because of at which MDA are expected to wane to compensate for diarrhoea. One additional animal from group ID-POS the eventual detrimental effect of the MDA on the first was withdrawn from the study on arrival at the experi- dose. Another option is to deliver the antigen to anatomi- mental facilities because of a lameness produced during cal sites where the concentration of MDA is lower than the load and transport. At the beginning of the study, the concentration in the muscle so there is less chance of there were 12 animals in ID-POS, 13 in IM-POS and interference with the vaccination. The intradermal (ID) 14 animals in IM-NEG and ID-NEG. During the study, route has several advantages such as the reduction of the three animals were euthanised because of the develop- antigen needed to induce an immune response [4] and ment of bacterial meningitis (two from ID-NEG and one the potential of targeting a higher diversity of dendritic from IM-NEG), and a piglet from the IM-POS group was cells (reviewed by Combadiere and Liard [5]). Of special found dead, which necropsy was compatible with menin- interest are Langerhans cells, that are particularly able gitis. No other incidents were observed in the rest of the to induce cytotoxic T cells [13] but are less efficient in animals during the 42-day follow-up. inducing responses from B cells. On the other hand, in humans, CD14+ dermal dendritic cells are especially able Vaccine-induced viremia to prime naïve B cells. This diversity of antigen-present- Figure 1 shows the evolution of vaccine-induced viremia ing cells in the skin can be advantageous for achieving in the different groups. After vaccination animals were effective immune responses after vaccination even when tested weekly to evaluate the presence of PRRS virus in reduced amounts of antigen are available. Several studies blood. In all groups, vaccination produced a viremia at 7 proved that reduced doses of dermally-delivered antigens days post-vaccination (dpv) (from 43 to 91% of the vac- cinated animals, depending on the group). After 21 dpv Aguirre et al. Porcine Health Management (2022) 8:47 Page 3 of 9 Fig. 1 Proportion of PCR-positive pigs for PRRSV at different time-points after vaccination. Differences between groups were non-significant the proportion of viremic animals dropped significantly until day 28 dpv. It is worth noting that the ID-POS group and after 35 dpv, only 1 or 2 animals/group were positive. presented the highest average S/P value at the end of the No significant differences between groups were observed study, significantly different from that of the ID-NEG regarding the proportion of positive animals. Regarding group. Differences between POS and NEG IM-vaccinated Ct-values, significant differences were observed at 7 dpv animals were non-significant. between ID-POS and the IM-NEG with lower Ct values Sera obtained on the day of vaccination could neutral- for the latter (Supplementary material S1). ise the vaccine used in sows (titres of 2.75 ± 1.42 log2 and 3.2 ± 2.1 log2 in IM and ID, respectively, non-significant) Development of antibodies but they were devoid of neutralisation capacity against The cohort of 2-week old pigs tested in the seroposi- the vaccine administered to piglets. The development tive herd (n = 132) showed S/P values (ELISA) ranging of neutralising antibody titres induced by the adminis- between 0.44 and 2.24 (Supplementary material S2). The tration of the vaccine was firstly detected at 28 dpv. At 28 selected animals had S/P values from 1.45 to 2.24. 35 dpv, significant differences were noticed between the Those S/P values were in the quartile 75% of the distri- groups that were vaccinated in the presence of MDA and bution. At 4 weeks of age, when the vaccination was the seronegative ones (Fig. 3, p < 0.05), with a mean titre performed, animals in subgroup IM-POS had an S/P of of 4.25 log2 and 1.36 log2 respectively. The main differ- 1.22 ± 0.28 versus 1.16 ± 0.23 for ID-POS (non-significant ence was in the IM vaccinated groups, in which animals differences). As expected, animals in group NEG were of the seropositive origin presented significantly higher seronegative at the moment of vaccination. titres compared to animals from the negative origin Figure 2 shows the evolution of average S/P values (Fig. 3, p < 0.01). per group after vaccination. Vaccination produced a clear seroconversion in the seronegative animals (group Lymphoproliferation NEG) that was noticeable by 14 dpv. Average S/P values PRRS-specific lymphocyte proliferation was clear by 21 increased until 35 dpv in the IM-NEG group, and until dpv in some animals, which was more evident at 28 dpv 42 dpv in the ID-NEG group. In the POS groups sero- and present in most animals at 35 dpv. Some differences conversion was also observed, with increasing S/P ratios in the proliferative responses were noticed at 35 dpv Aguirre et al. Porcine Health Management (2022) 8:47 Page 4 of 9 Fig. 2 Evolution of the S/P ratios in the different groups as determined by ELISA. Statistically significant differences are indicated by different superscript letters Fig. 3 Distribution of virus neutralisation titres in the different groups. Only statistically significant differences (Kruskal-Wallis, p < 0.05 are indicated, * p < 0.05; ** p < 0.01). Each dot represents one examined individual favouring the IM-NEG group (Kruskal-Wallis, p < 0.05), several animals from different groups and peaked at 42 but they disappeared at 42 dpv (Fig. 4). dpv, with no significant differences between groups at any age (Fig. 5). IFN-γ secreting cells ELISPOT results were negative for all the animals at 0 dpv. Virus-specific responses were detected at 21 dpv in Aguirre et al. Porcine Health Management (2022) 8:47 Page 5 of 9 Fig. 4 Results of the proliferation assays. The graphs depict the proportion of proliferating PBMC in cell cultures stimulated with the vaccine virus (21, 28 and 42 dpv, from left to right) at multiplicity of infection 0.1 once the spontaneous proliferation in mock-stimulated cultures was subtracted. Asterisks indicate the statistically significant differences (p < 0.05). Each dot represents an individual Fig. 5 Evolution of the virus-specific IFN-γ responses as determined by ELISPOT. The graph depicts the frequencies of virus-specific IFN-γ secreting cells at 21, 28 and 42 dpv for the different groups. The bar indicates the median of each group. Differences between groups were non-significant. Each dot represents an individual Cytokine production animals having the highest IL-12 levels at 14 dpv, 6 were IFN-α. Levels of IFN-α in serum at 7 dpv ranged between the ones having elevated IL-10 at the same sampling time. 300 and 700 pg/mL, with no significant differences between groups (IM-NEG: 435 ± 83 pg/ml, ID-NEG: Discussion 420 ± 79; ID-POS: 454 ± 86; ID-POS: 481 ± 86). Interference of MDA with vaccination is a major con- IL-10. At 7 dpv, levels of IL-10 were undetectable or cern when young piglets are to be vaccinated. In PRRSV very low (< 64 pg/ml) in all animals with no differences endemic farms, circulation of the virus often starts in between groups. At 14 dpv values higher than 100 pg/ml the farrowing units because of the existence of vertical of IL10 could be detected only in sera of NEG animals, transmission from sows to newborns, or very soon after 4 in the ID subgroup (164-1,849 pg/ml) and 2 in the IM weaning since MDA wane between 4 and 5 weeks of age subgroup (106–553 pg/ml). Of these, at 21 dpv 2 animals (reviewed by Pileri and Mateu [10]). Given the fast spread still showed elevated IL10 levels in the ID-NEG group of the virus in naïve populations, vaccination of piglets (249–505 pg/ml) and one in the IM-NEG group (273 pg/ must be administered at weaning or even before, imply- ml). In culture supernatants, IL-10 concentration was ing that most vaccinated piglets will have MDA. very low and with no significant differences. The intradermal administration of vaccines is an IL-12. At 7 dpv it was 10 times more likely to be IL-12 increasingly interesting alternative for vaccination, positive (9/28 vs. 1/23; p = 0.033) for animals of the sero- being less invasive and more respectful of animal wel- negative origin compared to the seropositive ones. At fare [11], and that could diminish interference with 14 dpv, all animals but three showed high levels of IL-12 MDA as shown for the human poliovirus vaccine admin- in serum (195.1-3,821.6 pg/ml). Interestingly, of the 10 istered to children [12]. The skin has a rich diversity of Aguirre et al. Porcine Health Management (2022) 8:47 Page 6 of 9 antigen-presenting cells that are highly efficient for cap- the present case, Ct values of POS animals at 7 dpv were turing and transporting antigens to the draining lymph similar or higher to those of NEG animals. However, it nodes. In the case of PRRSV, the intradermal administra- is worth noting that there were more PCR-positive pigs tion of MLV PRRSV vaccines has proven to be as effec- at 7 dpv in the POS (IM and ID) compared to the NEG tive as the IM administration in naïve MDA-free pigs groups. Nevertheless, it has to be stressed that the differ- [6–9, 15]. The vaccine tested in the present study showed ent genetics in POS and NEG farms (DanBred x Pietrain that both routes were equally effective in developing and Duroc x Landrace, respectively) might have had an immunity against PRRS in absence of MDA, which is in impact on the immune response. line with what showed by the above authors; moreover, Regarding the cell-mediated responses, the presence the vaccine was demonstrated to be able to produce simi- of MDA did not significantly interfere with the develop- lar levels of immunity by both routes even in presence of ment of proliferative or IFN-γ responses. In other stud- high maternal non-neutralising antibodies. ies, it has been shown that vaccine blocking by MDA The mechanisms by which MDA interference occurs usually affects the development of humoral responses but are not fully elucidated. Renson et al. [1] showed that not the development of cell-mediated immunity [21, 22]. homologous neutralising MDA titres of about 1:10 may In the present case, the development of vaccine- produce some interference with the development of induced viremia was similar in all groups following a pat- immunity in piglets. However, although neutralising anti- tern described in other previous works [1, 23]. As shown bodies (NA) may have a role in blocking the replication of before, some animals did not develop detectable viremia the MLV virus, there is strong evidence in other viruses while others persisted as positive for several weeks. One like measles virus favouring the hypothesis that blocking possible element involved in this different behaviour by MDA may involve the inhibition of B-cell responses by could have been the IFN-α response. Renson et al. [1] the cross-linking between B cell receptor with the Fcγ- reported a potential correlation of this phenomenon with receptor IIB by a vaccine–antibody complex, regardless decreased response to the vaccine. This was not our case of the neutralising capacity of the antibodies involved [3, since all animals had comparable levels of this cytokine 16, 17]. in serum. The design of the present study could have certainly Interestingly, the results showed that seronegative overlooked the case of animals with high homologous animals had a higher probability of having high levels NA titres, since the scenario selected was that where of IL-12 in serum after vaccination than seropositive seropositive piglets did not have NA against the vaccine ones. This suggests a differential targeting of TLRs in virus. Since NA against PRRSV usually have a narrow animals with or without antibodies against the vaccine breadth of neutralisation [18], the capability of MDA to virus. Most of the animals with the highest IL-12 levels neutralise a particular vaccine virus will depend on what in serum were the ones with high IL-10 levels. A pos- vaccine was used in the sows and on their previous con- sible explanation could be in the homeostatic action of tact with other PRRSV strains. Therefore, piglets being IL-10 to counterbalance excessive IL-12 production. It is vaccinated with an heterologous strain is not an unsual known that IL-10 is a potent regulator of IL-12 transcrip- scenario and would account for situations that can be tion [24]. encountered in the field, including the vaccination of the These results raise questions concerning the role of offspring of sows vaccinated with a different vaccine, or MDA in PRRS vaccination. The present results would simply, of seropositive unvaccinated sows. The interfer- indicate that in cases where the MDA were subneutral- ence of MDA antibodies resulting from vaccination with ising (i.e., heterologous vaccines in sows and piglets), different vaccines should be evaluated in the future. humoral immunity induced by MLV vaccination could The most remarkable results of the present study were even be enhanced. Further research should be done in on these animals of seropositive origin, which presented this area to figure out what were the mechanisms lead- a stronger humoral response despite having high levels ing to this difference including the possible enhancement of MDA. This was clear for both S/P values (particularly of the vaccine virus replication and also whether vaccine- for ID-POS) and virus NA titres of IM-POS group, sig- antibody complexes result in different stimulation of nificantly higher than IM-NEG. This phenomenon of antigen-presenting cells. increased response to vaccination in individuals with sub-neutralising levels of antibodies has been proven Conclusion in humans that were vaccinated with an experimental In conclusion, intradermal and intramuscular vacci- attenuated vaccine against the dengue virus [19, 20]. In nations have been proven as equivalent administra- that case, the greater immune response in individuals tion routes for the immunity parameters evaluated in with antibodies was attributed to increased infection of this experimental study. The presence of non-neutral- macrophages mediated by virus-antibody complexes. In ising antibodies at the time of vaccination resulted in Aguirre et al. Porcine Health Management (2022) 8:47 Page 7 of 9 enhanced humoral response without being detrimental and PRRSV vaccine viraemia. Heparinized blood samples to the development of cell-mediated responses. were additionally taken from 8 animals/group at 0-, 21-, 28-, and 42 dpv. The heparinized samples were used to Materials and methods obtain peripheral mononuclear blood cells (PBMC) by Animals and experimental design gradient density centrifugation using Histopaque-1077® The study was approved by the Committee for Ethics in (Sigma-Aldrich) and Sepmate™ tubes (Stemcell Technol- Human and Animal Experimentation of the Universitat ogies, Saint-Égrève, France). The resulting PBMC were Autònoma de Barcelona (number CEEAH 5357) and by frozen in Cryostor® CS10 (Merck) and stored in liquid the Generalitat de Catalunya (FUE-2020-01836411). nitrogen until further analysis. PBMC were recovered Pigs were recruited from two different herds; in the as described elsewhere [26] and viability was checked first, high levels of PRRSV-specific non-neutralising by trypan blue staining (0.4%). Only samples with > 90% MDA were detected (group POS) whereas in the second viability were used. no PRRSV antibodies were detected (group NEG). Group POS were DanBred x Pietrain crossbred animals. The Serological analysis farm was positive stable according to the revised AASV Serum samples were tested for the presence of PRRSV scheme of classification as PRRSV was not detected at specific antibodies by HerdCheck® PRRS X3 Ab test weaning [25]. Sows were vaccinated with Porcilis™ PRRS Idexx ELISA. (MSD Animal Health) every 3 months, last vaccination Viral neutralisation tests were performed in sera from was implemented approximately two months before far- 0-, 21-, 28-, 35-, and 42 dpv using the vaccine strain. For rowing. At two weeks of age, 132 piglets were randomly group POS animals, samples of 0 dpv were also tested selected from the same farrowing batch and bled to against the vaccine strain used for the sows in the ori- determine the levels of MDA by ELISA (PRRS X3 Ab, gin farm (Porcilis). The neutralisation tests were per- Idexx). The absence of PRRSV circulation in the group formed following the procedure described by Yoon et al. was confirmed by RT-qPCR (VetMax PRRS EU&NA 2.0 [27] with minor modifications. Briefly, sera were inacti- RT-qPCR, ThermoFisher, Madrid, Spain). The 28 animals vated at 56 °C for 30 min and diluted from 1:2 to 1:256 with the highest S/P ratios as determined by the test were in Minimum Essential Medium with non-essential amino selected for the experiment. Group NEG animals (n = 28) acids, sodium pyruvate, 100 IU/mL penicillin and 100 µg/ were Duroc x Landrace pigs obtained from a historically mL streptomycin. Equal volumes (100 µl:100 µl) of the PRRSV-free farm and its status was confirmed as well by diluted serum and the vaccine strain adjusted at 2,000 ELISA. In both cases, animals were weaned at 3 weeks TCID50/mL were mixed and incubated overnight at 4 °C. of age and transported to the experimental facilities Then, 100 µL of the serum-virus mixture was transferred located in the UAB. There, they were ear-tagged and ran- to MARC-145 monolayers in 96 well-plates. Negative domly divided into two subgroups that were housed in (only medium) and virus controls (virus at working dilu- physically separated boxes. One subgroup was assigned tion and at 200, 20, and 2 TCID50/mL) were also included the intramuscular (IM) vaccination and the other was in the plate, as well as a negative and positive serum assigned the intradermal (ID) vaccination. Thus, the final (post-vaccination serum of a sow) as controls. Plates were design contained 4 subgroups: IM-POS, IM-NEG, ID- read after 6 days of incubation. The neutralisation titres POS, and ID-NEG according to the serological status and were assessed by the development of the cytopathic effect the route of administration of the vaccine. Pigs were left and confirmed by immunofluorescence using the mono- to acclimate for a week before the administration of the clonal antibody 1CH5 (Eurofins, Madrid). The neutrali- vaccine. sation titre was the log2 of the reciprocal of the highest At 4 weeks of age, animals were bled and the PRRSV-1 dilution without cytopathic effect and without significant vaccine was administered (Unistrain® PRRS, Laboratorios fluorescence. Hipra). The IM vaccination was performed by the injec- tion of 2 ml of the vaccine in the neck muscles; the ID Quantification of PRRSV RNA vaccination was performed by delivering 0.2 ml of the Quantitative PCR for the quantification of PRRSV1 in vaccine in the neck using a needle-free device (Hipra- serum was performed as previously reported [28]. RNA dermic® 3.0). Each dose of the vaccine contained 1 × 104.3 extraction was performed with the BioSprint 96 One- TCID50 as titrated in MARC-145 cells. For-All-Vet kit (Qiagen), according to a BS96 Vet 100 protocol. Briefly, 100 µl serum was mixed with 300 µl Sampling and sample processing RLT, 300 µl Isopropanol, 25 µl MagAttract Suspension G Animals were monitored for 42 days after vaccination. and 2.7 µl carrier RNA. Then, 700 µL mixed sample was Blood samples were obtained weekly from all animals to applied to the S-Block plate. The kit BS96 Vet 100 pro- determine the development of PRRSV-specific antibodies tocol for RNA isolation was then followed. Finally, RNA Aguirre et al. Porcine Health Management (2022) 8:47 Page 8 of 9 was eluted in 80 µl nuclease-free water and the amount of Table 1 Type and date of samples analysed for cytokines PRRSV genome determined. Cytokine Sample Date IL-10 Serum 7, 14, 21 dpv Lymphoproliferation Culture supernatant 21 dpv Isolated PBMC were stained with CellTrace™ Violet IL-12 Serum 7, 14, 21 dpv (ThermoFisher) following the manufacturer’s instruc- IFN-α Serum 7 dpv tions. Briefly, 1 × 105 PBMC were dispensed on 96-well round-bottomed plates and the vaccine strain was added were calculated by subtracting the counts in the mock- at a multiplicity of infection (MOI) of 0.1 (cultures were stimulated wells from the counts obtained in virus-stim- done in triplicate). In parallel, phytohemagglutinin- ulated wells for each sample. Results were expressed as stimulated cultures (PHA, 10 µg/mL) or mock-stimu- the number of responding cells per 1 × 106 PBMC. lated cultures (RPMI medium) were used as positive and negative controls respectively. Plates were incubated for Cytokine ELISA in culture supernatant and serum 5 days at 37 °C in a 5% CO2 atmosphere. Then, cell cul- ELISA for cytokines IL-10, IL-12 and IFN-α were per- tures were recovered and centrifuged at 300 g for 10 min. formed in PBMC culture supernatants and/or serum. The resulting pellet was resuspended in PBS and anal- Cell culture supernatants were obtained from PBMC cul- ysed in a Cytoflex flow cytometer (Beckman Coulter, CA, tures produced in the same conditions as the ELISPOT. USA). Flow cytometer files were analysed using the FCS Supernatants were collected after overnight incubation Express Flow Cytometry 6 (de Novo Software). For each of the PBMC and frozen at − 80 °C until further analysis. sample, the PRRSV-specific proliferation was calculated Details of the samples analysed are shown in Table 1. as follows: percentage of cell proliferation in the PRRSV Antibody pairs and kits used are shown in supplemen- stimulated cultures - percentage of cell proliferation in tary materials (S3). Optimal work concentrations of each the negative control. A relative proliferation index was antibody were previously determined using the reference also calculated as the proportion of cell proliferation in standards provided by each kit. Cytokine concentra- PRRSV-stimulated culture versus the proportion of cell tions in the supernatants were estimated by calculating proliferation in the mock-stimulated cultures. a regression formula obtained from the results produced by a serial dilution of the standard provided by the kit. IFN-γ ELISPOT IFN-γ ELISPOT was performed as described by Zucker- Statistical analysis man et al. [29] with minor modifications. Briefly, Costar Data were analysed by StatsDirect v.3.3.5 and GraphPad 3590 plates (Corning) were coated with 50 µL of the 9.2 software. Differences between groups (continuous monoclonal antibody P2G10 against porcine IFN-γ (ref. variables) were tested by ANOVA (Tukey for multiple 559,961; BD Pharmingen, NJ, USA) at 5 µg/mL diluted comparisons). χ2 (2xK) was used to compare the propor- in carbonate-bicarbonate buffer (0.15 M pH 9.5). After tion of responses from the different groups. overnight incubation at 4 °C, plates were washed five times with PBS and blocked for 1 h with RPMI contain- Supplementary Information ing 10% foetal calf serum. Following the removal of the The online version contains supplementary material available at https://doi. org/10.1186/s40813-022-00289-4. blocking solution, 50 µL/well of PBMC were dispensed at 1 × 105 PBMC/well (negative control and virus stimu- Supplementary Material 1 lated wells) and 5 × 104 PBMC/well (PHA-stimulated Supplementary Material 2 wells). PRRSV-stimulated wells were added to the vac- Supplementary Material 3 cine strain at a MOI of 0.1. Mock-stimulated cultures (culture medium) and PHA-stimulated wells (10 µg/mL) were added as negative and positive controls, respec- Authors’ contributions Laboratory work was performed by LA, YL, GMV and EM. Sample collection tively. Plates were incubated overnight at 37 °C with 5% was done by GMV, MC and LA. Study design was performed by MM, EM, MB CO2. After washing, the biotinylated detection antibody and JM. EM, GMV, MM and LA analysed results and wrote the manuscript. JM P2C11 (ref. 559,958; BD Pharmingen) was added at and MB reviewed the manuscript. All authors read and approved the final manuscript. 0.5 µg/mL diluted in PBS with 0.4% bovine serum albu- min. After a 1 h incubation at 37 °C, 50 µL of streptavi- Funding din-peroxidase was added at 0.5 µg/mL (ThermoFisher) This study was funded by Laboratorios Hipra, S. A., 17170 Amer, Girona (Spain). and the reaction was developed by adding insoluble TMB Availability of data and materials (50 µL/well). Spots were counted in a Leica stereoscope The datasets used and/or analysed during the current study are available from after a 10-minute incubation in the dark. Samples were the corresponding author on reasonable request. tested in triplicate. The frequencies of responding cells Aguirre et al. Porcine Health Management (2022) 8:47 Page 9 of 9 Declarations 12. Snider CJ, Zaman K, Estivariz CF, Yunus M, Weldon WC, Wannemuehler KA, et al. Immunogenicity of full and fractional dose of inactivated poliovirus vac- Ethics approval cine for use in routine immunisation and outbreak response: an open-label, The study protocol was approved by the Committee for Ethics in Human and randomised controlled trial. Lancet. 2019;393:2624–34. Animal Experimentation of the Universitat Autònoma de Barcelona (number 13. Klechevsky E, Morita R, Liu M, Cao Y, Coquery S, Thompson-Snipes LA, et al. CEEAH 5357) and by the Generalitat de Catalunya (FUE-2020-01836411). Functional Specializations of Human Epidermal Langerhans Cells and CD14 + Dermal Dendritic Cells. Immunity. 2008;29:497–510. Consent for publication 14. Mashunye TR, Ndwandwe DE, Dube KR, Shey M, Shelton M, Wiysonge CS. Not applicable. Fractional dose compared with standard dose inactivated poliovirus vaccine in children: a systematic review and meta-analysis. Lancet Infect Dis Elsevier Competing interests Ltd. 2021;21:1161–74. Massimiliano Baratelli and Joel Miranda are employees of Laboratorios Hipra. 15. Ferrari L, Martelli P, Saleri R, De Angelis E, Cavalli V, Bresaola M, et al. Lympho- cyte activation as cytokine gene expression and secretion is related to the Author details porcine reproductive and respiratory syndrome virus (PRRSV) isolate after 1 Departament de Sanitat i Anatomia Animals, Universitat Autònoma de in vitro homologous and heterologous recall of peripheral blood mono- Barcelona (UAB), 08193 Cerdanyola del Vallès, Spain nuclear cells (PBMC) from pigs. Vet Immunol Immunopathol Elsevier B V. 2 HIPRA, Amer (Girona), Girona, Spain 2013;151:193–206. 16. Edwards KM. Maternal antibodies and infant immune responses to vaccines. Received: 14 July 2022 / Accepted: 11 October 2022 Vaccine Elsevier Ltd. 2015;33:6469–72. 17. Kim D, Huey D, Oglesbee M, Niewiesk S. Insights into the regulatory mechanism controlling the inhibition of vaccine-induced seroconversion by maternal antibodies. Blood. 2011;117:6143–51. 18. Martínez-Lobo FJ, Díez-Fuertes F, Simarro I, Castro JM, Prieto C. Porcine Repro- ductive and Respiratory Syndrome Virus isolates differ in their susceptibility References to neutralization. Vaccine. 2011;29:6928–40. 1. Renson P, Fablet C, Andraud M, Normand V, Lebret A, Paboeuf F, et al. 19. Villar L, Dayan GH, Arredondo-García JL, Rivera DM, Cunha R, Deseda C, et al. Maternally-derived neutralizing antibodies reduce vaccine efficacy against Efficacy of a Tetravalent Dengue Vaccine in Children in Latin America. N Engl porcine reproductive and respiratory syndrome virus infection. Vaccine. J Med. 2015;372:113–23. 2019;37:4318–24. 20. Hadinegoro SR, Arredondo-García JL, Capeding MR, Deseda C, Chotpitayasu- 2. Fablet C, Renson P, Eono F, Mahé S, Eveno E, Le Dimna M, et al. Maternally- nondh T, Dietze R, et al. Efficacy and Long-Term Safety of a Dengue Vaccine in derived antibodies (MDAs) impair piglets’ humoral and cellular immune Regions of Endemic Disease. N Engl J Med. 2015;373:1195–206. responses to vaccination against porcine reproductive and respiratory 21. Gans HA, Yasukawa LL, Alderson A, Rinki M, DeHovitz R, Beeler J, et al. syndrome (PRRS). Vet Microbiol. Elsevier B.V.; 2016;192:pp. 175–80. Humoral and cell-mediated immune responses to an early 2-dose measles 3. Niewiesk S. Maternal antibodies: Clinical significance, mechanism of interfer- vaccination regimen in the United States. J Infect Dis. 2004;190:83–90. ence with immune responses, and possible vaccination strategies. Front 22. Gans H, Yasukawa L, Rinki M, DeHovitz R, Forghani B, Beeler J, et al. Immune Immunol. 2014;5:1–15. responses to measles and mumps vaccination of infants at 6, 9, and 12 4. Schnyder JL, Garcia Garrido HM, De Pijper CA, Daams JG, Stijnis C, Goorhuis months. J Infect Dis. 2001;184:817–26. A, et al. Comparison of equivalent fractional vaccine doses delivered by 23. Martínez-Lobo FJ, De Lome LC, Díez-Fuertes F, Segalés J, García-Artiga C, intradermal and intramuscular or subcutaneous routes: A systematic review. Simarro I, et al. Safety of porcine reproductive and respiratory syndrome Travel Med Infect Dis Elsevier Ltd. 2021;41:102007. modified live virus (MLV) vaccine strains in a young pig infection model. Vet 5. Combadiere B, Liard C. Transcutaneous and intradermal vaccination. Hum Res. 2013;44:1–14. Vaccin. 2011;7:811–27. 24. D’Andrea A, Aste-Amezaga M, Valiante NM, Ma X, Kubin M, Trinchieri G. Inter- 6. Stadler J, Naderer L, Beffort L, Ritzmann M, Emrich D, Hermanns W, et al. leukin-10 inhibits human lymphocyte IFN-gamma production by suppress- Safety and immune responses after intradermal application of Porcilis PRRS in ing natural killer cell stimulatory factor/interleukin-12 synthesis in accessory either the neck or the perianal region. PLoS ONE. 2018;13:1–17. cells. JExpMed. 1993;178:1041–8. 7. Madapong A, Saeng-chuto K, Chaikhumwang P, Tantituvanont A, Saardrak 25. Holtkamp DJ, Torremorell M, Corzo CA, Linhares L, Almeida DC, Yeske MN. K, Pedrazuela Sanz R, et al. Immune response and protective efficacy of P, et al. Proposed modifications to porcine reproductive and respiratory intramuscular and intradermal vaccination with porcine reproductive and syndrome virus herd classification. J Swine Heal Prod. 2021;29:261–70. respiratory syndrome virus 1 (PRRSV-1) modified live vaccine against highly 26. Li Y, Mateu E, Díaz I. Impact of Cryopreservation on Viability, Phenotype, and pathogenic PRRSV-2 (HP-PRRSV-2) challenge, either alone or in co. Vet Micro- Functionality of Porcine PBMC. Front Immunol. 2021;12:1–11. biol. 2020;244. 27. Yoon J, Joo HS, Goyal SM, Molitor TW. A Modified Serum Neutralization Test 8. Martelli P, Gozio S, Ferrari L, Rosina S, De Angelis E, Quintavalla C, et al. Efficacy for the Detection of Antibody to Porcine Reproductive and Respiratory of a modified live porcine reproductive and respiratory syndrome virus Syndrome Virus in Swine Sera. J Vet Diagnostic Investig. 1994;6:289–92. (PRRSV) vaccine in pigs naturally exposed to a heterologous European (Italian 28. Martínez E, Riera P, Sitjà M, Fang Y, Oliveira S, Maldonado J. Simultaneous cluster) field strain: Clinical protection and cell-mediated immunity. Vaccine. detection and genotyping of porcine reproductive and respiratory syndrome 2009;27:3788–99. virus (PRRSV) by real-time RT-PCR and amplicon melting curve analysis using 9. Martelli P, Cordioli P, Alborali LG, Gozio S, De Angelis E, Ferrari L, et al. Protec- SYBR Green. Res Vet Sci. 2008;85:184–93. tion and immune response in pigs intradermally vaccinated against porcine 29. Zuckermann FA, Husmann RJ, Schwartz R, Brandt J, Mateu De Antonio E, reproductive and respiratory syndrome (PRRS) and subsequently exposed to Martin S. Interleukin-12 enhances the virus-specific interferon gamma a heterologous European (Italian cluster) field strain. Vaccine. 2007;25:3400–8. response of pigs to an inactivated pseudorabies virus vaccine. Vet Immunol 10. Pileri E, Mateu E. Review on the transmission porcine reproductive and respi- Immunopathol. 1998;63:57–67. ratory syndrome virus between pigs and farms and impact on vaccination. Vet Res BioMed Central. 2016;47:1–13. 11. Temple D, Escribano D, Jiménez M, Mainau E, Cerón JJ, Manteca X. Effect of the needle-free “intra dermal application of liquids” vaccination on the Publisher’s Note welfare of pregnant sows. Porc Heal Manag Porcine Health Management. Springer Nature remains neutral with regard to jurisdictional claims in 2017;3:1–7. published maps and institutional affiliations.