Evaluation of trained immunity as a potential strategy for a universal Streptococcus suis vaccine using immunogenic proteins in a murine model 

Abstract

Background

Universal vaccine against Streptococcus suis (S. suis) remains a challenge due to the high number of serotypes/strains, the absence of cross-protection among them and the existence of different immune escape strategies. In this study, the immunomodulatory potential of live and inactivated porcine derived Bacillus Calmette-Guérin (dpB) in combination with specific immunogenic proteins S. suis is evaluated in murine model.

Results

Results revealed different immune responses depending on dpB formulation. Live-dpB administered intravenously induced consistent results with trained immunity, including elevated proinflammatory cytokines and enhanced phagocytosis activity, reflecting stronger innate immune activation. However, inactivated dpB administered intravenously twice, enhanced adaptive responses post-S. suis vaccination showing increased IFN-γ levels in plasma and higher spot forming units in splenic ELISpot assays.

Discussion

These findings suggest complementary roles for live and inactivated dpB in innate and adaptive immunity. This approach may represent an initial step towards improving vaccine efficacy against S. suis, combining targeted innate with adaptive immunity. Further research is needed to optimize combinations of immunomodulators with traditional S. suis antigens.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-025-05228-3.

Introduction

New approaches for vaccines have become a priority in scientific research in recent years, particularly in the context of the global rise in antibiotic resistance, which is a growing public health concern worldwide [1]. The imperative need for prophylactic tools to reduce antimicrobial therapies has led to intensive efforts to develop effective vaccines [2]. Despite significant progress, the development of universal and safe vaccines for certain pathogens remains a challenge. For example, in the swine industry, diseases such as African swine fever (ASF) [3], porcine reproductive and respiratory syndrome (PRRS) [4] and streptococcosis [5] have proven particularly difficulties. Their etiological pathogens exhibit high genetic variability, complex host interactions, and/or immune evasion strategies, which have presented significant challenges to vaccine development [6, 7]. The difficulties to produce effective vaccines against these pathogens (and others with great impact on animal health) underscores the need to explore alternative approaches for disease control, including novel ways of improving the immune response [8].One promising approach is based on the trained immunity, a phenomenon in which innate immune cells develop an enhanced response to secondary infections after an initial stimulus with an immunomodulator [9], which has led to new possibilities for enhancing host defence against infectious diseases, particularly in the absence of effective adaptive immunity-based vaccines [10–12].

In response to this issue, strategies involving immunomodulators have emerged, with Bacillus Calmette-Guérin (BCG), the vaccine against tuberculosis, standing out as a key example, followed by other mycobacteria and β- glucans [13–15] .To date, BCG has demonstrated positive effects as a treatment in autoimmune diseases [16], and bladder cancer [17], as well as the protection or reduction of the severity of viral, bacterial and fungal infections in studies conducted in murine models to explore its importance in human health. However, evidence supporting this effects in veterinary diseases remains limited [18–22].

Particularly, research in trained immunity in swine pathologies is very limited, but Byrne et al. study already elicited the “non-specific” BCG effect in pig monocytes, showing an increase of IL-1β and TNF-α gene expression when exposed to LPS-stimulation [23]. Other experimental studies have been conducted using heat-inactivated Mycobacterium tuberculosis against Salmonella Choleraesuis [24] resulting in beneficious effects and protection. Based on the results in other animal models and its effectiveness against different pathogens [25], trained immunity may constitute a potential tool for the control of porcine infectious diseases [26].

S. suis is considered one of the most important pathogens affecting pig production, causing severe economic losses [5, 27].The main symptoms in subacute cases are meningitis and septicaemia [28]. Additionally, this pathogen is a zoonotic agent worldwide, meaning that humans can become infected either during the handling or slaughtering of pigs, through the consumption of undercooked pork products [29] or throughout interaction with wildlife such as wild boar [30].

Moreover, the emergence of antibiotic-resistant S. suis to lincosamides, macrolides, tetracyclines and sulphonamides in swine, has been reported with rates raising up to 85% [31–34]. This growing resistance, taken together with its demonstrated ability for horizontal AMR gene transfer [35], strengthens the need for effective S. suis vaccination strategies [36].

The capsular polysaccharide (CPS) is the primary virulence factor of S. suis, which protects the bacterium from phagocytosis and immune recognition [37]and hinders the host’s immune cells from effectively targeting the pathogen, reducing the efficacy of vaccines. To date, there are 29 serotypes of S. suis widely distributed in pigs and, at least, 11 of them are associated with human infections. Serotypes 2 and 9 are the most prevalent and frequently associated with infections both in pigs and humans worldwide [5, 38].

One of the primary obstacles in developing an effective vaccine for S. suis is probably this high variability of serotypes and sequence types [39] (even high genetic variation among strains within the same serotype has been described [40]).Some experimental vaccines have been explored including subunit vaccines [41], conjugated vaccines [42, 43] and DNA vaccines [39]. However, to date, some vaccines or antigens evaluated have shown protection against a limited number of serotypes [44–47]. This underscores the importance of exploring alternative approaches or enhancing existing vaccines to achieve broader protection against S. suis serotypes/strains.

In this situation, autogenous vaccines (bacterins) have increased their use because of its low cost of production and its ability to be formulated according to the strains circulating in each farm [48]. However, field studies that evaluated the protective capacity of autogenous vaccines have showed contradictory findings [49–52].

In the current context of the S. suis vaccine-related challenges, tools based on trained immunity could represent an interesting resource not only by itself inducing innate immunity training but also as a complementary tool to immunomodulate acquired immunity-based vaccines to achieve better levels of protection.

This study evaluates the effectiveness and protection of a BCG-isolate derived from porcine (dpB), both live and inactivated, to elicit trained immunity against S. suis in the C57/BL6 experimental murine model, as well as its potential impact on the modulation of immune response after S. suis immunogenic proteins vaccination on the response through ex vivo analysis.

Material and methods

Animals

C57BL/6 female mice (n = 56) aged 42–48 days of life were obtained from Charles River Laboratories (Wilmington, Massachusetts, USA). The cages were equipped with Altromin-LASQCdiet^® Rod 14-H (Altromin Lage, Germany) for feeding. Both food and water were available ad libitum. Wheeled houses for environmental enrichment were also included (Ref: K3327 + K3250, Sodispan Research, Madrid, Spain). The animals were marked with an ear tagger for individual identification (in some cases, it was not necessary since mice were easily identified by white areas on their tails). DpB administration procedures were carried out in the Biosafety Level 2 + (BSL2+) area at the VISAVET Health Surveillance Centre (Complutense University of Madrid).

Experimental design

The animals were divided into seven groups, each consisting of eight individuals (Table 1). Groups G1 to G5 received stimulation with an immunomodulator (either dpB or inactivated dpB) through two different routes of administration, oral or intravenous. In certain groups, G3 and G5, a second dose of the same immunomodulator was administered 20 days later. A blood sample took place 34 days after the first dose of dpB in BD^®Microtainer^® LH Blood Collection tubes (Becton Dickinson Biosciences, Madrid, Spain). Additionally, except for the negative control group (G7), all groups were vaccinated intraperitoneally with S. suis proteins 41 days after the first dose of the immunomodulator. The animals were sampled and euthanized by cervical dislocation 22 days after vaccination for ex vivo challenge assessments (Fig. 1).

Table 1.

Experimental groups from the study with the stimulation carried out, the number of doses administered and whether they were vaccinated with S. suis intraperitoneal vaccine or not

Group	Stimulation	Dose	Vaccine
G1	DpB IV	1	S. suis IP
G2	Inactivated dpB IV	1	S. suis IP
G3	Inactivated dpB IV	2	S. suis IP
G4	Inactivated dpB oral	1	S. suis IP
G5	Inactivated dpB oral	2	S. suis IP
G6	Saline solution IV	1	S. suis IP
G7	Saline solution IV	1	Saline solution IP

Fig. 1.

Experimental design showing immunization times, number of doses, vaccination with S. suis, as well as the timing of bleeding for each of the experimental groups: live intravenous dpB + S. suis vaccine (group 1), inactivated intravenous dpB + S. suis vaccine (group 2), inactivated dpB 2 doses IV + S. suis vaccine (group 3), inactivated dpB via oral + S. suis vaccine (group 4), inactivated dpB via oral 2 doses + S. suis vaccine (group 5), S. suis vaccine (group 6), negative control group (group 7). IV: intravenous, IP: intraperitoneally

DpB preparation

The strain used in this study was a BCG-isolate derived from porcine (dpB) which shows a high homology with Danish CCUG 27,863, obtained from swine and subsequent formulation, has already showed immunostimulatory properties [53]. A vial of frozen strain was inoculated in a starter liquid culture, incubated at 37 °C in aerobiosis for 5 weeks. At the end of the culture, the growth was collected with a pipette, centrifuged, washed with Phosphate Buffered Saline (PBS) (Sigma-Aldrich, Misuri, USA), given a treatment of physical breakage with glass beads, and diluted with PBS until a homogeneous product was at approximately 1 McFarland [equivalent to approximately 10⁶ colony forming units (CFU)/mL] according to commercial patrons employed (M-IDENT^®-MacFARLAND, Laboratorios Microkit, Valdemorillo, Spain). Once the inoculum was ready, a plate count was performed to determine the concentration of the prepared inoculum, as well as after the inoculation of dpB in the mice. The final dose for inoculation obtained was 2 × 10⁵ CFU/mL.

For inactivated dpB preparation, a liquid culture of live dpB was inactivated by heat, centrifuged, washed and resuspended in saline. The solution was sonicated and filtrated to remove larger particles. Afterwards, thimerosal was added at 1%. The product was plated and incubated at 37 °C for 48 h to check sterility.

Streptococcus suis immunogenic proteins preparation

First, soluble S. suis antigen was carried out by growing the S. suis isolate (strain 1023 serotype 9) [54] in BHI medium (150 rpm, 37 °C, 18 H) to obtain a stationary phase culture. The culture was centrifuged at 8000 rpm, 20 min at 4 °C, the supernatant was decanted, and the pellet was washed twice with 25 mM Tris − 50 mM NaCl buffer, pH 8.1. After the last wash, the pellet was collected and the bacteria were disrupted by resuspending the pellet in 25 mM Tris buffer pH 8.1 with protease inhibitors (Protease Inhibitor Cocktail, Sigma- Aldrich, Missouri, United States) and mixing with glass beads (≤ 106 μm) previously activated with 5.8 M HCl. Then, 5 cycles of 1 min of vortexing and 30 s on ice were performed, followed by another 5 cycles of 10 s of sonication at a power of 30 Watts (Branson Sonifier B-12) for 10 s on wire. The homogenate obtained was centrifuged at 8000 rpm, 20 min at 4 °C and the supernatant was collected. This solution was subjected to FPLC ion exchange chromatography (Mono Q anion exchange) using 25 mM Tris pH 8.1 as elution buffer with a gradient from 0 to 1 M NaCl. Fractions eluted between 0.1 and 0.7 M were analysed by dot-blot using a mixture of three monoclonal antibodies obtained from mice and produced in the Microbiology Unit of Carlos III Institute (Spain) that recognize three S. suis proteins (Dihydrolipoamide dehydrogenase, high-affinity zinc uptake system protein and enolase). The dot-blot positive fractions were subsequently analysed by SDS PAGE and Western blot, determining that the proteins of interest, among others, were found in the fractions eluted at a concentration between 0.5 and 0.6 M NaCl, fractions that were mixed and constituted the soluble antigen selected for the vaccination of the animals.

S. suis proteins preparation was carried out by emulsifying the adjuvant [ISA 61 VG (water in oil), Seppic, Coubevoie, France] with the soluble antigen in a 60/40 ratio (60% adjuvant, 40% solubilized protein) and was plated and incubated at 37 °C for 48 h to check sterility. An inoculum of 200 µL containing 25 µg of antigen was prepared for each mouse.

S. suis proteins sample preparation for proteomic analysis

Firstly, 200 µL of the sample was taken and diluted in denaturing buffer + 2% SDS. After reduction with TCEP and chloroacetamide, samples were processed with the Opentrons OT-2 robot in the presence of MagReSyn^® HILIC microparticles (ReSyn Biosciences) and digested at 37 °C overnight using trypsin and Lys-C enzymes [55].

For nano-Liquid Chromatography coupled to Electrospray Ionization Tandem Mass Spectrometry (nanoLC-ESI-MS/MS) analysis, 500 ng of each sample was individually analysed using an Ultimate 3000 nano HPLC system (Thermo Fisher Scientific) coupled online to an Orbitrap Exploris™ 240 mass spectrometer (Thermo Fisher Scientific) in a 120 min gradient. Data acquisition was performed using a data-dependent top-20 method.

Proteomic data analysis of S. suis proteins

Raw data files were processed using the Proteome Discoverer 2.5.0.400 software (Thermo Scientific), and a database search was carried out using Mascot (v2.8.0) search engine against Streptococcus suis GZ1 UniProt database (27th November 2024, 2,037 sequences) containing the most common laboratory contaminants (cRAP database with 70 sequences).

DpB administration, S. suis vaccination, and sampling

DpB both alive and inactivated were intravenously administered (100 µL) using a syringe with a 30G x ½’’ needle through the caudal vein of the tail (groups 1, 2, 3) (Fig. 1). A restrainer was used to immobilize the mouse while administering dpB. The tail was pre-warmed slightly using a heat lamp for enhanced vasodilation. Oral administration of dpB is performed using a 200 µL pipette, supplying a total of 125 µL in 50 µL increments while restraining the animals.

The first blood sampling took place 34 days after the first dpB dose. For this bleeding, a maximum of 200 µL of blood per animal was obtained with a 23G needle from the submandibular vein. Once the procedure was completed, pressure was applied until the animals stop bleeding, and fluids were replaced with IP physiological saline. Subsequently, blood was centrifugated to obtain plasma for the determination of cytokines.

S. suis vaccine was administered intraperitoneally with a 23G x 1’’ (200 µL) in all the experimental groups except the negative control (G7) (Fig. 1). Twenty-two days after vaccination, blood collection was carried out prior to slaughter of the animals by cardiac puncture with a 25G needle prior anesthetized with a combination of 100 mg/kg of Ketamidor 100 mg/mL (Richter Pharma AG, Wells, Austria) and 10 mg/kg of Xilagesic 20 mg/mL intraperitoneally (Calier, S.A, Madrid, Spain) (Fig. 1). A total blood volume of maximum 400–450 µL was obtained. In addition, immediately after cervical dislocation slaughter, spleens from all animals were collected and placed in 10 mL complete RPMI (penicillin/streptamycin and L-glutamine) with no foetal bovine serum (FBS) (Merk, Darmstadt, Germany) to enzyme-linked immunospot.

Blood stimulation and cytokine measurements

The prepared soluble S. suis antigen was used at a concentration of 300 µg/mL. The stimulation consists of placing 100 µL of blood obtained by the end of the experiment from each animal in wells of a 96 V-bottom plate and 15 µL of antigen. Thereafter, an incubation was carried out at 37 °C overnight in a humidity box. The next day, the plates were centrifuged at 550–700 g for 10–15 min and then the plasma was collected and frozen at -80 °C until processing.

Cytokines were evaluated both in plasma obtained by the end of the experiment from blood stimulated as explained before, and in plasma samples from 34 days after the first dpB dose with no stimulation. Cytokines measured were interleukin 1β (IL-1β), interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), and anti-inflammatory as interleukin 10 (IL-10) employing ProcartaPlex™ kits Thermo Fisher Scientific (Massachussetts, United States) and Bio-Plex 200 System (Bio-Rad, Alcobendas, Madrid).

Phagocytosis assay

The phagocytosis assay was evaluated from the last sampling of the experiment. It involved incubating 320 µL of whole blood from the animals with 160 µL of a live S. suis inoculum at a concentration of 1 × 10⁶ CFU/mL and carefully mixing the solution using a pipette. This results in a final concentration of approximately 3.3 × 10⁵ CFU of S. suis/mL blood. Additionally, a negative control was prepared by substituting the blood with PBS, and immediately (time 0) serial tenfold dilutions were performed in PBS and plated in duplicate on blood agar (100 µL) using a Drigalski spreader and incubated at 37 °C.

Subsequently, both the blood samples and the negative controls were placed on a rotative shaker with gentle agitation and incubated at 37 °C for 2 h. It is during this incubation time that neutrophils and macrophages should recognize S. suis and phagocytose it. After incubation (time 1), the same dilution and plating procedure described below was performed to assess bacterial survival.

After 24 h of incubation, the CFUs were counted to evaluate the phagocytosis response of innate immune cells against S. suis. If necessary, the incubation was extended up to 48 h for accurate counting.

IFN-γ enzyme-linked immunospot

To isolate spleen mononuclear cells, after euthanizing the mice in a biological safety cabinet under aseptic conditions, the spleen was extracted and placed in a 50 mL screw-cap tube with 10 mL of RPMI 1640 medium with L-glutamine (Lonza Group Ltd, Basel Switzerland) supplemented with non-essential amino acids 1mM, pyruvate 1mM, 100 IU/mL penicillin, and 100 µg/mL streptomycin (complete RPMI or cRPMI). Next, the spleen was placed in a 70 μm nylon filter (Falcon), fragmented it with scissors, and completely dissociated by passing it through the filter using the plunger of a sterile syringe.

Subsequently, the cell suspension was repeatedly washed using RPMI with 5% fetal bovine serum (FBS), centrifuging between washes to maximize cell recovery. For erythrocyte lysis, a commercial ammonium chloride solution (Gibco, Grand Island, NY, United States) was used. Finally, the cells were maintained at 4 °C in RPMI supplemented with 10% FBS for 2 h while cell counting, and viability staining were performed.

The Mouse IFN-γ ELISPOT set (Becton Dickinson Biosciences, Madrid, Spain) was used according to the manufacturer’s instructions, with the following modifications: Spleen cell suspensions were adjusted to a density of 5 × 10⁵ cells per well. For cellular activation to stimulate IFN-γ production, concanavalin A at 20 µg/mL was used as a non-specific mitogen, RPMI medium with 5% FBS was used as a negative control, and S. suis soluble antigen was used (22.5 µg/mL) as specific stimuli. After 17 h of incubation at 37 °C, the ELISPOT plates were washed, and captured IFN-γ was detected by adding a biotinylated detection antibody. Finally, the addition of streptavidin and a 10-minute incubation with AEC Substrate Reagent (Becton Dickinson Biosciences, Madrid, Spain) led to the formation of spots. The number of spots was analyzed using an ELISPOT reader (AID Fluorospot, Autoimmun Diagnostika GmbH, Strassberg, Germany). Mean values of triplicates cell cultures were considered. The IFN-γ response were determined as stimulated minus non-stimulated values.

Statistical analysis and graphic creation

Differences between groups for cytokines, phagocytosis values and ELISPOT results were evaluated using a Mann-Whitney test with pos-hoc p-value adjusted by Bonferroni. The Spearman’s rank correlation coefficient (r_s) was used to assess the relationship between the different factors. Statistically significant differences in all tests were considered when p-value was < 0.05. Analyses were performed in IBM SPSS Statistics for Windows, version 287.0. 1. 1 (IBM Corp, Armonk, N.Y, USA).

Figures 2–4 and 5 were created using GraphPad Prism 10 software.

Results

Proteomic data analysis of S. suis vaccine

Proteins identified with a q-value < 0.01 were considered present (Additional file 1). A total of 734 proteins were identified. It was selected as the Streptococcus suis vaccine because it contained enolase, dihydrolipoyl dehydrogenase, and high-affinity zinc uptake system protein (Additional file 1). These three proteins had previously been identified as immunogenic proteins [54].

Intravenous route and live format of dpB are the most effective strategies to induce a significantly higher IFN-γ, IL-10 and TNF-α response by itself

The results from the first blood sampling reflected a cytokine response in animals that had only been dpB-inoculated (once or twice) but had not yet been S. suis-stimulated (vaccination or ex-vivo whole blood stimulation). The results showed a higher IFN-γ (Fig. 5A), IL-10 (Fig. 5B) and TNF-α (Fig. 5C) cytokine levels (pg/mL) in the group immunized with live dpB intravenously (group 1) compared to the other groups. Significant differences were observed for these cytokines in comparison to the groups receiving one dose (group 4) or two doses (group 5) of inactivated dpB orally, as well as the untreated control group (group 6) (Mann-Whitney (M-W), p < 0.05). In the case of IL-6 (Fig. 5D), the live intravenous dpB group exhibited significant differences when compared to all other groups (M-W, p < 0.05). For IL-1β (Fig. 5E), no significant differences were observed, as the values obtained for this cytokine were close to zero in all the groups.

Fig. 5.

Dot plot showing pre-S. suis vaccination cytokine concentrations (pg/mL) in plasma for each of the experimental groups [1–7], being the cytokines evaluated INF-γ (A), IL-10 (B), TNF-α (C), IL-6 (D), IL-1β (E). Groups 6–7 were included in the same as in this point of the experiment their treatment is the same

IV-two doses of inactivated dpB may impact on the immunomodulation of S. suis vaccine-response

After S. suis vaccine and subsequent stimulation of the blood samples with the S. suis antigen (second blood sampling, performed in the sacrifice day), cytokine levels were measured, revealing different results compared to the previous sampling (Fig. 4). In this case, the group with the highest cytokine levels for all markers was the one immunized twice with inactivated dpB IV (G3). However, significant differences were only observed for IFN-γ (Fig. 4A) when compared to the groups immunized with oral inactivated dpB with one dose (group 2) (M-W, p = 0.004) and two doses followed by S. suis vaccination (group 5) (M-W, p = 0.022), as well as the group vaccinated only with S. suis (M-W, p < 0.001) (group 6) and the untreated control group (M-W, p < 0.001) (group 7). For IL-1β (Fig. 4B), higher significant values were obtained for the two-dose IV inactivated dpB group (G3) when compared with the IV live dpB animals (G1) (M-W, p = 0.035).

Fig. 4.

Dot plot showing post- S. suis vaccination and infection cytokine concentrations (pg/mL) in plasma for each of the experimental groups [1–6], being the cytokines evaluated INF-γ (A), IL-1β (B), TNF-α (C), IL-6 (D), IL-10 (E)

Animals immunized with dpB intravenously may present a more effective phagocytic response against S. suis

Phagocytosis results are represented as CFU/mL, which indicates the capacity of neutrophils and monocytes in the animals’ blood to eliminate bacteria, in which animals with lower CFU/mL counts were those with a more prepared immune response against the infection. In general, no significant differences were observed between the experimental groups. However, the group immunized with live dpB IV (group 1) showed lower CFU/mL (approaching significance, M-W, p = 0.099) compared to the untreated control group. On the other hand, both groups of animals immunized with inactivated dpB via the oral route showed the highest CFU/mL counts among all groups (however, for group 4 only three samples were analysed due to the limited plasma volume) (Fig. 3).

Fig. 3.

Dot plot representation of phagocytosis results in CFU of S. suis/mL in blood from animals in groups from 1–7 when stimulated with S. suis antigen. Missing results are due to the limited plasma volume, which did not allow the analysis of all parameters in some animals. Finally, a Spearman test was carried on in all the parameters and negative correlation was observed between the phagocytosis results and cytokines as IFN-γ, (rs=-0.34 -, p = 0.21) and IL-10, (rs=-0.31, p = 0.044)

Stronger IFN-γ ELISPOT response was induced by two IV doses of inactivated dpB plus S. suis vaccination

After stimulation of spleen cell suspensions with S. suis soluble antigen, the results were represented as the number of spots analysed by the ELISPOT reader. Animals in G3 (two doses of IV inactivated dpB + S. suis vaccine) exhibited a significantly higher number of spots compared to animals immunized with oral inactivated dpB, both in the one-dose group (M-W, p = 0.020) and the two-dose group (M-W, p = 0.025), as well as compared to the negative control group (G7) (M-W, p = 0.027) (Fig. 2).

Fig. 2.

Dot plot representation of the number of spots in the IFN-γ enzyme-linked immunospot counted in groups from 1–7 when stimulated with S. suis antigen in spleen cells

On the other hand, a significant positive correlation was detected between the IFN-γ enzyme-linked immunospot in spleen cell suspensions and IFN-γ levels in plasma (r_s=0.52, p < 0.001).

Discussion

This study was conducted to evaluate the effects of both live and inactivated dpB administrated through different routes, on the immune response to S. suis in a murine model, focusing on the impact of these immunomodulators on the response induced by a specific vaccine against S. suis. The findings of this study highlight the distinct roles of live and inactivated dpB in modulating the immune response, particularly regarding cytokine production and phagocytic activity.

In the initial sampling (prior S. suis vaccination), animals immunized intravenously with live dpB displayed elevated proinflammatory cytokine levels (IFN-γ, IL-6 and TNF-α), suggesting sustained innate immune activation and a trained immunity process in the innate immune cells [14, 15]. This medium-term innate immunity could be explained by a training of innate bone marrow progenitor cells (“central trained immunity”) [14].

However, after the specific S. suis vaccination and subsequent ex vivo stimulation with S. suis antigen, animals that received two intravenous doses of inactivated dpB intravenously were the ones showing the highest cytokine levels, particularly IFN-γ, compared to the other groups. The elevated IFN-γ production in the two-dose inactivated dpB group could suggest an enhanced response between the dpB-induced immunity and the specific S. suis vaccine, promoting a more targeted adaptive response to S. suis. While IFN-γ is not directly associated with trained immunity, it is a key cytokine in both innate and adaptative immunity against bacteria, cancer or viruses [56]. However, some studies support the idea that macrophages may have a direct production of IFN-γ, which may also elicit local and systemic signals reinforcing microbial response [57]. Furthermore, this IFN-γ production by macrophages is associated with control of bacterial growth [58].

Thus, the combination of inactivated dpB and specific vaccination may enhance IFN-γ production, providing a targeted immune response beneficial for combating S. suis with a greater involvement of T lymphocytes. However, we cannot rule out that this IFN-γ is part of an innate immune response and is produced by differentiation of trained macrophages.

In contrast, the phagocytic results revealed that animals immunized with live dpB presented the highest phagocytic response, as indicated by lower CFU/mL counts. Phagocytosis, as a measure of innate immune function, represents the immediate, unspecific response to pathogens, and, even differences were not significative, live dpB could be compatible a stronger trained immunity response in neutrophils and macrophages, facilitating early bacterial clearance [14, 59]. In this context, the high levels of IFN-γ observed in the two-dose inactivated dpB group appear to represent an adaptative, specific response facilitated by the S. suis vaccine, rather than an immediate, non-specific control of the pathogen.

It is worth mentioning the presence of the protein enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11) which is a protein with significant immunogenic potential in the S. suis vaccine, has demonstrated to be an important protective antigen against S. suis infection [60]. The synergistic effects observed might reflect a dual mechanism, where dpB primes the immune system through trained immunity and enhances the development of antigen-specific responses, while the enolase in the S. suis vaccine directs and amplifies the adaptive response [9].

These findings underscore the complementary roles of live and inactivated dpB in the immune modulation. Live dpB appears to promote a rapid, non-specific phagocytic response through trained immunity, which is advantageous for initial control of extracellular pathogens like S. suis [61]. Meanwhile, inactivated dpB, particularly with a two-dose regimen, enhances the capacity of immune cells to produce IFN-γ specifically in response to S. suis antigens, supporting a Th1-oriented adaptive response critical for bacterial control [56].

Among limitations of this study, it is worth highlighting the importance of animal model used. Although these results may differ from what would happen in field studies in swine model, our study highlights the usefulness of murine model to evaluate ability of mycobacteria to induce trained immunity against swine pathogens. Besides, no S. suis challenge was carried out in vivo so no clinical signs or mortality were evaluated, however, welfare was conserved in these animal models and preliminary results in these study in terms of trained/adaptative immunity are valid for being taken in consideration in further studies [62]. Moreover, it should also be noted that several outcomes did not reach statistical significance, which is expected in preliminary exploratory designs with limited sample sizes (related to research with animals which requires strict compliance with animal welfare regulations, including the 3Rs principle and limitations in the number of animals that can be used). Nevertheless, these early patterns are important to guide subsequent targeted experiments in swine and to refine hypotheses on how dpB may modulate both innate and adaptive responses.

Conclusion

In conclusion, this preliminary study shows that live and inactivated dpB induce different immunomodulatory effects in the murine model. Live dpB tended to promote a more immediate, non-specific response reflected by enhanced phagocytic activity, while two doses of inactivated two were associated with stronger IFN-γ production in response to the specific S. suis vaccine, suggesting an enhanced vaccine-specific adaptative activation. Although the statistical significance of several findings were limited, the patterns observed indicate that dpB, particularly in its inactivated form, may influence the quality of subsequent vaccine induced responses against S. suis. This results, while preliminary, may provide valuable initial evidence for the potential use of mycobacterial immunomodulators as complementary tools to improve immune responses against S. suis. Further research into such combination strategies could reveal valuable insights for managing zoonotic bacterial infections with complex immune requirements.

Authors’ contributions

Conception and design (LSM, TGS, MPS, LD) , analysis and interpretation of the data (LSM, SC, LZ, TGS, IM, APD, MDF, AB, MAR, MD), drafting of the manuscript (LSM, LZ, MPS, MD, LD), revising and editing the manuscript (LSM, SC, LZ, MPS, TGS, IM, APD, MDF, AB, MAR, MD, LD).

Funding

LSM holds a PhD funding (reference CT15/23) from the Universidad Complutense de Madrid and Banco Santander. LZ holds a PTA2021-021020-I funded by MICIU/AEI /10.13039/501100011033 and FSE+. This research was funded by PID2020-112966RB-I00 project financed by MCIN/AEI/10.13039/501100011033 (TAIV-suis).

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

Animal care and procedures were performed by following the guidelines of good experimental practices according to Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes (amended by the Regulation (EU) 2019/1010) and Spanish laws (RD 53/2013). The protocol was approved by the Community of Madrid Ethics Committee (reference PROEX 133.1/23).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.