Vaccine: X 14 (2023) 100330 Contents lists available at ScienceDirect Vaccine: X journal homepage: www.elsevier.com/locate/jvacx Immune responses following neonatal vaccination with conserved F4 fragment of VtaA proteins from virulent Glaesserella parasuis adjuvanted with CAFÒ01 or CDA Sergi López-Serrano a,b,c,⇑, Yasser S. Mahmmod d,e, Dennis Christensen f, Thomas Ebensen g, Carlos A. Guzmán g, Fernando Rodríguez a,b,c, Joaquim Segalés a,b,h, Virginia Aragón a,b,c a Unitat mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra 08193, Catalonia, Spain b Institut de Recerca i Tecnologia Agroalimentàries, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra 08193, Catalonia, Spain c WOAH Collaborating Centre for the Research and Control of Emerging and Re-Emerging Swine Diseases in Europe (IRTA-CReSA), 08193 Bellaterra, Catalonia, Spain d Department of Animal Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt e Section of Veterinary Sciences, Health Sciences Division, Al Ain Men’s College, Higher Colleges of Technology, Al Ain 17155, United Arab Emirates f Department of Infectious Disease Immunology, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen, Denmark g Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany h Departament de Sanitat i Anatomia animals. Facultat de Veterinària. Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra 08193, Catalonia, Spain a r t i c l e i n f o a b s t r a c t Article history: Glaesserella parasuis is a Gram-negative bacterium that colonizes the upper airways of swine, capable of Received 8 March 2023 causing a systemic infection called Glässer’s disease. This disease is more frequent in young post-weaning Received in revised form 7 June 2023 piglets. Current treatments against G. parasuis infection are based on the use of antimicrobials or inacti- Accepted 7 June 2023 vated vaccines, which promote limited cross-protection against different serovars. For this reason, there Available online 10 June 2023 is an interest in developing novel subunit vaccines with the capacity to confer effective protection against different virulent strains. Herein, we characterize the immunogenicity and the potential benefits of Keywords: neonatal immunization with two different vaccine formulations based on the F4 polypeptide, a conserved Glaesserella parasuis Swine vaccination immunogenic protein fragment from the virulence-associated trimeric autotransporters of virulent G. Bacterial infection parasuis strains. With this purpose, we immunized two groups of piglets with F4 combined with cationic Vaccine adjuvant CAFÒ01 or cyclic dinucleotide CDA. Piglets immunized with a commercial bacterin and non- Colonization immunized animals served as control groups. The vaccinated piglets received two doses of vaccine, at Immunization 14 days old and 21 days later. The immune response induced against the F4 polypeptide varied depend- ing on the adjuvant used. Piglets vaccinated with the F4+CDA vaccine developed specific anti-F4 IgGs, biased towards the induction of IgG1 responses, whereas no anti-F4 IgGs were de novo induced after immunization with the CAFÒ01 vaccine. Piglets immunized with both formulations displayed balanced memory T-cell responses, evidenced upon in vitro re-stimulation of peripheral blood mononuclear cells with F4. Interestingly, pigs immunized with F4+CAFÒ01 controlled more efficiently a natural nasal colo- nization by a virulent serovar 4 G. parasuis that spontaneously occurred during the experimental proce- dure. According to the results, the immunogenicity and the protection afforded by F4 depend on the adjuvant used. F4 may represent a candidate to consider for a Glässer‘s disease vaccine and could con- tribute to a better understanding of the mechanisms involved in protection against virulent G. parasuis colonization. Ó 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Introduction Glaesserella (formerly Haemophilus) parasuis is a Gram-negative ⇑ Corresponding author at: present address at: Infection Biology Laboratory, gammaproteobacterium member of the Pasteurellaceae family that Department of Medicine and Life Sciences (MELIS), Universitat Pompeu Fabra, Barcelona Biomedical Research Park (PRBB), 08003 Barcelona, Spain. colonizes the upper respiratory tract of pigs early after birth [1]. G. E-mail address: sergi.lopez@upf.edu (S. López-Serrano). parasuis comprises strains that can be pathogenic to pigs, espe- https://doi.org/10.1016/j.jvacx.2023.100330 2590-1362/Ó 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). S. López-Serrano, Y.S. Mahmmod, D. Christensen et al. Vaccine: X 14 (2023) 100330 cially in the post-weaning period. At that age, around 4–8 weeks of Material and methods life, susceptible piglets may develop invasive G. parasuis infection leading to Glässer’s disease, which is characterized by fibrinous Ethics regulation polyserositis, arthritis and meningitis. Glässer’s disease causes serious economic and welfare problems in the swine industry Animal experiments were conducted in AM Animalia (La Vall de [2]. G. parasuis infection can be treated using antimicrobials, but Bianya, Girona, Spain) according to the ARRIVE guidelines, the increasing concern about the use of these compounds makes approved by the AM Animalia Ethics Committee for Animal Exper- vaccination the main alternative tool to control the impact of this imentation with number CEEA 20/20-P1 and CEEA 20/20-P2 in infection [3]. compliance with the EU directive 63/2010, the Spanish legislation Neonate vaccination is an effective approach for the protection (RD 53/2013) and the Catalan law 5/1995 and decree 214/1997. against infectious diseases that are common in the early stages of life [4]. However, newborn animals have an immature immune Experimental vaccines preparation system, and protective immunity in these early stages of life can be difficult to achieve due to the immune system’s skew towards The conserved F4 antigen from VtaAs consisting of the sequence stimulation of Th2 and Treg responses, with limited Th1 and anti- AGPTGNSELKGITSIANGNDATKANGAKITLSAGSTD KTVNVNDAKITN body responses [5,6]. Moreover, neonate immunity is also affected VAAGTADTDAVNVSQLNTKAAASKTEVEAGKNVKVTSKTGANGQNIYN by other factors such as maternal immunity components acquired VSVSGDLSDITSISNGDTKVSLGKDKQGNPVVNMNGARITNVGDGSAE via colostrum that, in the case of pigs, can modulate the immune GDIVNVRQLNKVVSSVNTGFNQLSRDIGRSARGS was produced as a maturation of the offspring and interfere with vaccination [7–9], His-tagged recombinant protein by induction of the expression and the microbiota that colonize their mucosae [10,11]. plasmid pASK-IBA33plus-F4 in E. coli BL21. Briefly, transformed Current vaccines against Glässer’s disease consist of autogenous bacteria were grown overnight at 37 °C in LB broth supplemented or commercial bacterins, which confer protection against homolo- with 100 lg ampicillin and induced in the stationary phase with gous or a narrow heterologous range of serovars [12]. The lack of AHT 0.2 lg/mL to allow the optimal expression of F4. For purifica- cross-protection afforded by these vaccines urges to look for tion, bacterial cultures were pelleted and later resuspended in sal- broad-spectrum strategies that could protect against heterologous ine sodium phosphate buffer with 1 lM PefablocÒSC (Sigma- virulent serovars. In this respect, subunit vaccines consisting of Aldrich, Madrid, Spain) and disrupted with a Branson 450 Digital surface-exposed proteins or protein domains exclusively con- Sonifier (Branson Ultrasonics Corporation, Brookfield, CT, USA) served in pathogenic strains of G. parasuis represent an attractive using 50 pulses of one second in an amplitude of 20%. Purification alternative. Among possible candidates, the virulent-associated tri- of His-F4 was performed with His-Spin Trap columns (GE Health- meric autotransporters (VtaA), a family of outer-membrane pro- care Life Sciences, Chicago, IL, USA) with the help of 20 mM of imi- teins of G. parasuis involved in adhesion to extracellular proteins dazole and later dialyzed against PBS. F4 purity was confirmed by and phagocytosis resistance by alveolar macrophages, may be of protein gel electrophoresis and Coomassie blue staining and later interest [13,14]. Comparison of the amino acid sequence among quantified with the Pierce BCA Protein Assay Kit (ThermoFisher, the VtaAs from strains with different degrees of virulence allowed Madrid, Spain). Purified F4 was stored at 20 °C until use. the identification of a surface-exposed fragment, named F4, within CAFÒ01 and CDA were provided by the Statens Serum Institut the group 1 and 2 VtaAs from the Nagasaki strain (serovar 5) that is (Copenhagen, Denmark) and the Helmholtz-Zentrum für Infektions- highly conserved in virulent G. parasuis [15]. Recent work per- forschung (Braunschweig, Germany), respectively. Experimental formed in our laboratory has additionally shown that sow immu- vaccine formulations were prepared and administered in seven nization with F4 emulsified with Carbopol Polymer adjuvant not pigs for each group (Fig. 1), injecting 100 lg of the immunogen only induced anti-F4 specific immune responses but also modu- in a total volume of 500 lL per dose intramuscularly in the neck lated the immunity traits in their offspring with an increase of cir- of the piglets. Two additional piglets were immunized with the culating TGF-b [16]. PorcilisÒ Glässer vaccine, a commercial product composed of inac- Here we explored the vaccine potential of the F4 protein frag- tivated G. parasuis serovar 5 adjuvanted with dl-a-tocopheryl acet- ment in neonate pigs, testing the efficacy of two novel adjuvants: ate, as controls for the in vivo assay, following the the Cationic Adjuvant Formulation 01 (CAFÒ01) and the bis-(3,5)- recommendations of the manufacturer (MSD Animal Health, Sala- cyclic dimeric adenosine monophosphate (CDA), specifically manca, Spain). designed to stimulate different immune pathways. CAFÒ01 is a liposome-based mixture of the cationic surfactant N,N’-dimethyl- N,N’-dioctadecylammonium (DDA) and the synthetic glycolipid Experimental design analogue to the Mycobacterium tuberculosis cord factor a,a’-treha lose 6,60 -dibehenate (TDB), and it was chosen in this study due The study design is displayed in Fig. 1. Twenty-three to its ability to promote Th1/Th17-like responses [17,18]. Further- Landrace  Duroc piglets of about five days-of-age were included more, CAFÒ01 has been shown to be efficient in the induction of in the study. Piglets were obtained from 8 sows between parities immunity in neonates [19–22]. CDA is a monocyclic dinucleotide 3–5 with variable antibody values against G. parasuis according naturally secreted by Listeria monocytogenes, which was selected to the commercial Ingezim–Haemophilus ELISA (Ingenasa, Madrid, due to its ability to efficiently activate STING, the ‘‘Stimulator of Spain) and low OD values measured by an in-house ELISA against Interferon Genes”, triggering antibody production as well as bal- the F4 fragment (see F4-specific antibody detection). Animals were anced Th1/Th2/Th17-like responses and induction of cytotoxic transported to the experimental facilities of AM Animalia (La Vall CD8+ lymphocytes [23,24]. Recent in vitro and in vivo studies sug- de Bianya, Girona, Spain), where they were housed in one room, gested that CDA is also a suitable candidate for the development of and distributed into four pens, one group in each pen. Each group neonatal vaccines [25]. included piglets from all the sows from which they were weaned. In this vaccination study, we assessed the humoral and cell- Animals were fed ad libitum during all the experiment and were mediated immune responses in neonate piglets following immu- treated with 5 mg/kg of ceftiofur (Naxcel porcino, Zoetis S.L.U., nization with the two adjuvanted F4 formulations. In addition, Spain) during the first week of the study to prevent undesirable the effect of the vaccines on the natural colonization by virulent bacterial diseases. After seven days of acclimation, animals were G. parasuis was also examined. intramuscularly vaccinated in the left flank of the neck on day 12 2 S. López-Serrano, Y.S. Mahmmod, D. Christensen et al. Vaccine: X 14 (2023) 100330 serially diluted in blocking solution or used undiluted. After a 1 h of incubation at 37 °C, wells were incubated with a goat anti- porcine IgG HRP-conjugated antibody (Sigma-Aldrich, Madrid, Spain) diluted 1:10,000. For IgG1 and IgG2 specific detection, a mouse anti-pig IgG1 or IgG2 (both from Bio-Rad Laboratories, Her- cules, CA, USA) were used diluted 1:2,000, followed by a goat anti- mouse IgG conjugated with HRP (Sigma-Aldrich, Madrid, Spain) diluted 1:10,000. To detect specific IgM and IgA responses, goat anti-porcine IgA or IgM HRP conjugated antibodies (both from AbD Serotec, Oxford, UK) were both used diluted 1:1,000. Positive reactions in all the ELISAs were developed using the 3,3,3,5- tetramethylbenzidine (TMB) substrate (Sigma-Aldrich, Madrid, Spain) and the reactions were stopped with 1 N sulfuric acid. Plates were then read in a Power Wave XS spectrophotometer (Biotech, Winooski, VT, USA) at 450 nm. PBMC isolation and specific IFN-c ELISPOT Peripheral blood mononuclear cells (PBMCs) were isolated from 10 mL of EDTA-treated blood of D0 control pigs (prior to PBS or PorcilisÒ Glässer injection) to determine the presence of F4 specific IFN-c secretory T-cells by ELISPOT at the starting point. Additional IFN-c ELISPOT assays were later performed using PMBCs from all Fig. 1. Schematic representation of the in vivo study procedure and group animals at 21 and 38 days postvaccination (D21 and D38). PBMC distribution of the experimental vaccines. Piglets from 3 to 4 days of age from 8 isolation was performed under a density gradient centrifugation sows were transported to the animal facilities and left for acclimation for one week. using Histopaque 1077 (Sigma-Aldrich, Madrid, Spain), followed Each group included piglets collected from every sow at the beginning of the study. by an osmotic shock to remove red blood cells. Concentration of After acclimation, piglets were twice immunized 21 days apart. Samples of clotted PBMCs was adjusted and 5  105 cells/well were plated in flat- blood, EDTA-treated blood and nasal swabs were collected on days 0 (prior to vaccination), 21 (after the first immunization, prior to boosting) and 38 (17 days bottomed 96-well tissue culture plates with RPMI 1640 medium after boosting). NV: non-vaccinated. Figure created with BioRender.com. (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS) (Euroclone, Milan, Italy), 1% glutamine (Gibco Life Technologies, Madrid, Spain), 1% penicillin-streptomycin (Gibco of life (Day zero of the study; D0) and twenty-one days later, pig- Life Technologies, Madrid, Spain) and 0.05 mM of b- lets were boosted with a second dose of each vaccine (D21), this mercaptoethanol (Sigma-Aldrich, Madrid, Spain). PBMCs were time in the right flank of the neck. Whole blood (EDTA), sera and freshly used to perform an IFN-c Enzyme-Linked ImmunoSpot nasal swabs were taken on study days D0, D21 and D38 (17 days Assay (ELISPOT), following a protocol described before [28], with after boosting). Sera obtained by centrifugation (10 min at minor modifications. Briefly, high binding 96-well plates (Costar 860  g) of clotted-blood tubes were aliquoted and stored at Corning Incorporated, New York, NY, USA) pre-coated with 80 °C. Nasal swabs were resuspended in 500 lL of PBS and stored 250 ng of porcine anti-IFN-c per well (Clone P2G10 Mouse IgG1, at 80 °C until use. BD PharmingenTM, San José, CA USA) were used to plate PBMCs. PBMCs were stimulated for 48 h at 37 °C and 5% CO2 with F4 (2 DNA extraction and G. parasuis detection by PCR lg/mL) to detect the F4 specific IFN-c secretory cells. Phytohemag- glutinin (10 lg/mL, Sigma-Aldrich, Madrid, Spain) and RPMI were Two hundred microlitres of the resuspended nasal swabs were used as positive and negative control, respectively. After 48 h of processed with the MagMax Pathogen kit (Life Technologies, incubation, plates were washed to remove cells and stained with Madrid, Spain) according to the manufacturer’s instructions. The 25 ng of biotinylated anti-IFN-c antibody (Clone P2C11 Mouse presence of virulent and non-virulent G. parasuis colonizing strains IgG2a, BD PharmingenTM, San José, CA USA) and developed with was monitored by PCR using two primer sets that differentially HRP-streptavidin (Invitrogen Life technologies. Madrid, Spain) fol- amplify their vtaA leader sequences (LS-PCR) [26]. Four lL of the lowed by insoluble TMB (Merck Life Science, Madrid, Spain). DNA extracted from the nasal swabs were used as a template for Resulting spots were counted under the microscope (Stereoscopic the reaction. Purified DNA from the virulent Nagasaki (serovar 5) Zoom Microscope SMZ800, Nikon Instruments Inc., Chiyoda, and the non-virulent SW114 (serovar 3) strains were included as Japan). In the analysis, background spots obtained in the control controls. Molecular serotyping was performed by a serotype- wells (stimulated with RPMI alone) from each animal, were sub- specific PCR using the DNA purified from nasal swabs as template tracted to the F4-stimulated ones and expressed by 106 cells for [27], to assess the serovars of naturally colonizing G. parasuis. statistical analysis. G. parasuis and F4-specific antibody detection Immune phenotyping and cytokine detection The commercial Ingezim-Haemophilus ELISA (Ingenasa, In parallel to the IFN-c ELISPOT, PBMCs were plated on flat- Madrid, Spain) was used for the detection of total antibodies bottomed 96-well cell culture plate (SPL Biosciences, Gyeongido, against G. parasuis, following the manufacturer’s indications. In Korea) for immunophenotyping and cytokine detection. For that addition, an in-house ELISA was used for detection of antibodies purpose, PBMCs were incubated for 48 h with 2 lg/mL of F4 as against F4 [16]. Briefly, high binding plates were coated overnight specific stimulus. RPMI and 10 lg/mL of concanavalin A (Sigma- at 4 °C with 500 ng of F4 per well. After washes, wells were blocked Aldrich, Madrid, Spain) were used as negative and positive controls with 1% casein in phosphate-buffered saline (PBS) with 0.05% for the assay, respectively. Supernatants were collected and stored Tween 20 (PBS-Tw20). Sera and nasal swab resuspensions were at 80 °C for cytokine analysis. PBMCs were harvested and washed 3 S. López-Serrano, Y.S. Mahmmod, D. Christensen et al. Vaccine: X 14 (2023) 100330 Table 1 List of antibodies used for the detection of surface markers. Marker Host Antibody Target Clone Fluorochrome Brand Dilution isotype CD3e Mouse IgG2a Pig BB23-8E6-8C8 PE-Cy7 BD Pharmingen 1:200 CD4 Mouse IgG2b Pig 74-12-4 Alexa Fluor 647 BD Pharmingen 1:200 CD8a Mouse IgG2a Pig 76-2-11 FITC BD Pharmingen 1:200 CD27 Mouse IgG1 Pig B30C7 Stained with APC-Cy7 BIO RAD 1:100 (sec 1:400) secondary antibody CD154 Mouse IgG1 Human, 5c8 In-house conjugated with Mix n’ BIOxCELL 1:100 (CD40L) Monkey Stain CF405L (Biotium, Fremont, CA, USA) with FACS buffer (0.5% FBS-PBS). After washes, cells were surface The significant independent variables from the univariable stained for 45 min at room temperature with a mixture of labelled analysis were then offered to a multivariable mode and a manual antibodies at the dilution indicated in Table 1 in FACS buffer. After backward elimination was implemented, to obtain a final model staining, cells were washed again and analyzed in a MACSQuant 10 that exclusively included variables with a p value < 0.05, consid- Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany), acquiring ered as significant. The p value and the regression coefficient (b) twenty-thousand events per sample to perform the analysis. with a 95% confidence interval (95% CI) were reported for each Unstained cells, stained samples with viability marker (Live-or- variable. All these statistical analyses were conducted using the R DyeTM 405/545 Fixable Viability Staining Kit, Biotium, Fremont, version 3.3.3 software (R Core Team, 2015). CA, USA), isotypes for each antibody subclass, and Fluorescence Significance of the proportions of colonized animals among Minus One (FMO) stained samples were included as controls to experimental groups was calculated with the Comparison of pro- adjust the analysis and discard false positive results. Analysis of portions calculator v20.206. https://www.medcalc.org/calc/com- the results was carried out with FLOWLOGIC software v7.3 (Inivai parison_of_proportions.php (MedCalc Software Ltd, Ostend, Technologies, Melbourne, Australia). Dead cells were excluded Belgium), which uses the ‘‘N-100 Chi-squared test as recommended from the analysis according to the pattern obtained by the fixable by Campbell [30] and Richardson [31]. The confidence interval was cell-viability staining mentioned above. The gating strategy can be calculated according to Altman et al. [32]. Graphs were plotted referred in supplementary Figure S1. using Prism v9 (GraphPad Software, San Diego, CA, USA). Harvested supernatants from in vitro PBMC stimulation were used for IFNa, IFN-c, IL-1b, IL-4, IL-6, IL-8 (CXCL8), IL-10, IL-12/ IL-23p40 and TNF-a determination using InvitrogenTM Cytokine & Results Chemokine 9-Plex Porcine ProcartaPlexTM Immunoassay kit (Thermo Fisher, Madrid, Spain), following manufacturer’s indica- Natural colonization by a virulent G. parasuis strain was modulated by tions. Supernatants were analyzed using a MagPixÒ xMAPÒ Ana- F4-vaccination lyzer (Luminex Corporation, Austin, TX, USA). Cytokine concentrations were determined using the xPONENTÒ software. A natural nasal colonization by virulent serovar 4 G. parasuis TGF-b detection was individually evaluated in same supernatants was detected by PCR at D0 (before immunization started) in four by using the TGF-b Human Matched Antibody Pair kit from Invitro- unvaccinated control animals (Fig. 2). Further PCR analyses evi- gen (Carlsbad, CA, USA), following manufacturer’s instructions. denced that the colonization spread to the rest of the piglets and pens, as was observed on day 21, when nasal colonization was common in all the groups (6/7 [86%] in PBS, 7/7 [100%] in F4 Statistical analysis and modelling +CAFÒ01, 5/7 [71%] in F4+CDA and 1/2 [50%] in PorcilisÒ Glässer). Interestingly, at day 38 (17 days after vaccine boosting), the colo- Before conducting any statistical analysis, data were screened nization by serovar 4 virulent G. parasuis was significatively for unlikely, extreme, or missing values and no data were excluded reduced in the F4+CAFÒ01 immunized pigs (p = 0.02), with a 71% based on these premises. First of all, a descriptive analysis was car- of reduction, from 100% (7/7) at D21 to 28.5% (2/7) at D38 ried out with the main variables of interest: experimental groups (Fig. 2). The reduction of the virulent/serovar 4 G. parasuis presence (F4+CAFÒ01, F4+CDA, PBS and PorcilisÒ Glässer), colonization with was not that evident in the rest of the groups, with a reduction of virulent G. parasuis at D38 (yes or no), sow origin (n = 8 sows), sow 16.6% in control non-immunized pigs PBS-NV (from 86% (6/7) to parity (3, 4 and 5), total anti-G. parasuis antibody ratio (doubtful, 71% (5/7)); and a 20% reduction for the F4+CDA vaccinated piglets positive and negative values by the Ingezim-Haemophilus ELISA (from 71% (5/7) to 57% (4/7)), respectively (Fig. 2). kit), and anti-F4 IgG levels found in sows by the above described F4 ELISA. Different statistical models of linear regression were additionally run for the following continuous outcome variables: F4+CDA induced significantly higher anti-F4 specific IgG1 antibodies IgG F4 D38/anti-F4 IgG levels in sow, ELISPOT Nagasaki D21, pro- than F4+CAFÒ01 portion of T helper (CD4+) F4, T-memory (CD4+CD8+), Central T- memory (CD4+CD8+CD27+) and Effector T-memory T (CD4+CD8 The kinetics of anti-F4 specific IgG induction were evaluated by +CD27-) cells, and IFN-a and TGF-b levels found in supernatants ELISA using F4-coated plates and sera from all animals prior to after 48 h of stimulation with the F4 fragment. The outcome vari- immunization (D0), after the first immunization (D21) and after ables that showed a skew pattern were, therefore, transformed by boosting (D38). As shown in Fig. 3A, at D38, animals vaccinated taking their natural logarithm or log10 and a univariable analysis with F4+CDA showed significantly higher antibody levels than was done to test the unconditional associations between outcome the PBS-NV control pigs (p < 0.01). Similar results were found for and different explanatory variables of interest. At this initial the anti-F4 IgG1 immunoglobulin isotype (Fig. 3B), but not for screening, explanatory variables with p < 0.25 were included in the anti-F4 specific IgG2 (Fig. 3C) and IgM (Fig. 3D). Individual multivariable linear regression models according to Dohoo et al. kinetics of specific anti-F4 IgGs can be found in supplementary [29]. Figure S2. 4 S. López-Serrano, Y.S. Mahmmod, D. Christensen et al. Vaccine: X 14 (2023) 100330 Fig. 2. Nasal colonization of the piglets by virulent G. parasuis during the vaccination experiment detected by LS-PCR from nasal swabs collected at 0-, 21- and 38-days post- vaccination. Bars represent the percentage of positive animals to virulent strains within the group in the different timepoints. Above each bar is depicted the number of positive animals out of the total. Positive signals detected by LS-PCR were later confirmed to belong to serovar 4 by G. parasuis serotyping PCR. Before immunization (D0), the F4-ELISA also detected low levels of IFN-c secreting cells (Fig. 5). Interestingly, two piglets in the CDA of anti-F4 IgGs (Fig. 3A), mostly of the IgG2 isotype (Fig. 3C), which group did not get colonized at any time of the study and showed reduced their presence by D21 in all animals, indicating their mater- increase in both IFN-c and IgG responses comparing D38 and nal origin. Except for the pigs immunized with F4+CAFÒ01, the over- D21, while a third piglet cleared the colonization by D38 and also all levels of F4 anti-IgG2 antibodies tended to increase after D21 showed an increase in IFN-c and IgG responses. On the other hand, (Fig. 3C), most probably reflecting the natural colonization with the the net increase of F4 specific antibodies in some piglets of the G. parasuis strain (serovar 4) together with the vaccination in those unvaccinated group indicates that the presence of F4-specific vaccinated groups. Variable values of total anti – G. parasuis were anti-IgG or IFN-c secreting cells are potential indicators of colo- found at 0 DPV in all the groups. Only the two animals immunized nization by virulent G. parasuis (Fig. 5). with the bacterin vaccine PorcilisÒ Glässer showed a rise of total anti For the analysis of the cell-mediated immune response at D38, – G. parasuis antibodies on 38 DPV (supplementary Figure S3). the experimental groups were split according to their nasal colo- nization status on this timepoint. Evaluation of PBMCs after in vitro restimulation with F4 revealed some trends in T helper cells Vaccination and colonization affected T-cell specific F4 responses (CD3+CD4+), memory T cells (CD3+CD4+CD8+), effector memory T cells (CD3+CD4+CD8+CD27-) and central memory T cells (CD3 Specific secretion of IFN-c by F4-stimulated PBMCs was already +CD4+CD8+CD27+) in the pigs immunized with the F4 combina- detected by ELISPOT in piglets before vaccination (D0), a fact that tions (Fig. 6). Thus, the animals vaccinated with F4+CAFÒ01 might indicate early colonization by G. parasuis and/or maternal showed a reduction of the percentage of all of the above cell sub- transfer of F4-specific T-cells to their offspring. Conversely to what sets in the colonized animals with respect to the uncolonized ones. was found for maternal anti-F4 antibodies, the number of F4-IFN-c Colonization status did not influence the proportion of those cell specific spots notably increased in all groups by D21, indepen- subsets in the F4+CDA immunized pigs. On the other hand, the dently of the treatment received and coinciding with the peak of non-vaccinated control group showed higher proportions of the virulent G. parasuis detection in their nasal cavities (Fig. 4). After cell subsets in colonized piglets (Fig. 6). CD154 signal was not the second vaccination, the levels of F4-specific IFN-c secretion detected in any of the samples. proportionally decreased in all animals, including those in the con- Albeit not statistically significant, the amount of TGF-beta and trol group. The individual kinetics of IFN-c secreting cells are avail- IL-8 found in the supernatant of PBMCS after F4-in vitro stimula- able in supplementary Figure S4. In fact, colonization status at D38 tion was higher for the F4+CAFÒ01 pigs than for the rest of the was significantly associated with the IFN-c results in the ELISPOT groups (Fig. 7). at D21, using bacterin as stimulus (supplementary Figure S5); i.e., the piglets with higher IFN-c response against the whole bac- terium at D21, had higher probability to harbor virulent G. parasuis Maternal immunity and piglets’ colonization affected the in their nasal cavities at D38 (22.00 [-17.95–61.95], p = 0.0021). immunological outcome A closer look at the individual responses for anti-F4 IgG and IFN-c secretion revealed that even though the strongest values of The multivariable statistical model performed to evaluate pos- specific antibodies were found in the pigs vaccinated with F4 sible relations among variables showed a significant association +CDA, these results did not completely correlate with the number between the amount of IFN-c secreting cells from the piglets and 5 S. López-Serrano, Y.S. Mahmmod, D. Christensen et al. Vaccine: X 14 (2023) 100330 G. parasuis G. parasuis ** p<0.01 Fig. 3. F4-specific antibodies detected in sera from pigs immunized with either F4+CDA, F4+CAFÒ01, PBS or PorcilisÒ Glässer along the experimental procedure from days 0-, 21- and 38-days post-vaccination. Anti-F4 specific antibody kinetics: Total IgG (A), IgG1 (B) and IgG2 (C). ODs obtained for each pig are plotted with symbols, averages and standard are also plotted. Individual piglets colonized at each time point by virulent G. parasuis are represented with filled symbols, whereas non-colonized animals are represented with empty symbols. Statistical significance (*) and p values are indicated accordingly. their sow of origin. According to the results, the sow origin affected D21, and the colonization by virulent G. parasuis detected 17 days the IFN-c secreting PBMC in the piglets with a p value of 1.36e-08, later with a p value of 0.0021. and specifically the sow’s anti-F4 IgGs were positively associated The sow parity negatively affected the expression of memory F4 with the IFN-c secreting cells of the offspring before boosting specific Th helper cells (p = 0.02), central memory T cells (p = 0.02) (D21) with a p value of 0.02556. Moreover, the final model showed and effector memory T cells (p = 0.04). Similarly, TGF-b secretion a significant positive association between the secreted IFN-c on by piglets’ stimulated PBMCs was positively associated with the 6 S. López-Serrano, Y.S. Mahmmod, D. Christensen et al. Vaccine: X 14 (2023) 100330 identified parameters of maternal influence together with the nasal colonization with virulent G. parasuis as the sources of this high individual variability. This latter fact had a higher impact on the analyzed immune parameters, as observed in the non- G. parasuis G. parasuis vaccinated group. Regardless of these facts, the experimental vac- cine formulations were capable to trigger different immune responses in the piglets, an important circumstance considering the young age of the animals and the bacterial colonization, fact that occurs often under natural conditions [1]. The piglets immunized with the CDA combination elicited an efficient induction of anti-F4 IgG1 antibodies by D38 (17 days after boosting), indicating a Th2 skewed response. In addition, two F4 +CDA vaccinated animals did not become colonized during the experiment, displaying a congruent humoral and cellular immune response. Similar humoral systemic responses were previously Fig. 4. F4-specific IFN-c secreting cells measured by ELISPOT. PBMCs (5x105) reported with other antigens from diverse pathogens adjuvanted collected at different times during the study were plated and stimulated for 48 h with 2 lg/mL of F4. Individual piglets colonized at each timepoint by virulent G. with STING agonists (CDA or cyclic di-GMP) in mice [33,34]. How- parasuis are represented with filled symbols, whereas non-colonized animals are ever, in our case, the delivery system of the vaccine formulation represented with empty symbols. Averages and standard deviations found in each might also have affected the development of a proper mucosal group are also plotted. On day 0, 8 piglets (that later belonged to PBS-NV and response. A different delivery system of administration may have PorcilisÒ Glässer groups) are represented in the same bar, as all piglets were naïve at this timepoint. been more efficient in terms of both humoral and cell-mediated immune responses, as reported in pigs intramuscularly vaccinated with a Mycoplasma hyopneumoniae bacterin adjuvanted with anti-F4 specific antibodies found in the respective sow of origin liposome-encapsulated CDA [35]. At this point, it is important to (p < 0.05). note that cyclic dinucleotide adjuvants, like CDA, stimulate effi- cient mucosal responses when administered by either nasal [36,37] or oral routes [38] rather than intramuscularly. This might Discussion be instrumental to achieve both efficient protection against infec- tion and reduction of horizontal transmission. According to our The immunity elicited by both F4-adjuvanted formulations pre- results, F4-specific systemic antibodies might not correlate with sented in this study was highly variable within groups, and we a putative protection against natural colonization by virulent G. Fig. 5. F4-specific immune responses, antibodies (above) and IFN-c secreting T-cells (below) found in individual pigs injected with either PBS, F4+CAFÒ01, F4+CDA, or PorcilisÒ Glässer. Danti-F4 IgG result from subtracting the values obtained on day 21 (post-priming) to the results of day 38 (post-boost). DIFN-c-SC is the result of the subtraction of the spots obtained after boost D38 to the results after priming D21. Nasal colonization by G. parasuis serovar 4 is indicated at the bottom of the figure. 7 S. López-Serrano, Y.S. Mahmmod, D. Christensen et al. Vaccine: X 14 (2023) 100330 G. parasuis G. parasuis Non-colonized Colonized Non-colonized Colonized Non-colonized Colonized Non-colonized Colonized Fig. 6. Proportion of T cell populations found on in vitro F4-stimulated PBMCs collected on day 38 and analyzed by flow cytometry. Results are expressed after gating of twenty thousand events acquired on total PBMCs. Individual piglets colonized at this timepoint by virulent G. parasuis are represented with filled symbols, whereas non- colonized animals are expressed with empty symbols. Averages and standard deviations found in each group are also plotted. parasuis, in agreement to the results observed with antigens from response without interaction with maternally-derived antibodies. similar bacteria [39–41]. According to our results, CDA seems to help overcoming the pres- The piglets vaccinated with F4+CAFÒ01, on the contrary, did not ence of maternally derived antibodies, at least partially, suggesting display a significant humoral response against the immunogen. an alternative way of immune stimulation. From this perspective, Only two animals showed a low F4 antibody signal that might be previous studies in mice attributed the enhanced antibody attributed to the colonization by G. parasuis. Generalized lack of response to an autonomous activation of B cells through the STING antibody response can be explained by the interference of mater- signaling pathway [47]. nal antibodies in the maturation of B cells helped by the follicular In the case of the CAFÒ01 formulation, we also cannot rule out T cells (Tfh) in the germinal centers of the lymph nodes as sug- the effect of the antigen adsorption into the liposome formulation. gested by Vono et al. [42]. On the other hand, one can also think Indeed, a recent study demonstrated that changes of the electro- that the early T cell priming redirects to mucosal tissues and the static interactions between antigens and CAFÒ01 can lead to differ- systemic immunity in this case was poorly induced [43]. In agree- ent immune responses [48]. Thus, the authors associated efficient ment with this idea and previous observations with CAFÒ01, F4 Th1/Th17-type responses to higher adsorption rates of the antigen +CAFÒ01 vaccination significatively reduced the nasal colonization into the vaccine formulation, in inverse correlation with antibody by G. parasuis [28,44,45]. A reduction of G. parasuis nasal coloniza- titers. Of note, the generation of opsonizing antibodies is consid- tion after vaccination has been also shown with another subunit ered crucial for protection against Glässer’s disease [49,50] and vaccine, TbpB, using different adjuvants and administration routes maternally-derived immunity plays an important role protecting [46]. The clearance observed in our study might indicate a primed the offspring by passive transfer via colostrum during their first mucosal response with a mechanism still to be determined as a weeks of life [51]. result of the adjuvant effect characterized by a Th17-type response Despite the limitations of this study, the situation offered the elicited by both adjuvants. opportunity to evaluate the effectivity of the different vaccine In previous neonatal studies performed in mice, CAFÒ01 and combinations under a natural exposure to the pathogen, under CDA adjuvants proved a complete efficacy and immunogenicity conditions closer to a conventional farm. Since a relatively low sys- [21,25]. However, the animals used in the mentioned studies temic humoral response as observed after F4+CAFÒ01 immuniza- belonged to naïve mothers and, therefore, a proper maturation of tion did not correlate with the protective efficacy, the putative the germinal centers was possible leading to an efficient humoral mucosal response may be useful against the colonization of patho- 8 S. López-Serrano, Y.S. Mahmmod, D. Christensen et al. Vaccine: X 14 (2023) 100330 G. parasuis G. parasuis Fig. 7. Cytokine profiles obtained by ProcartaPlexTM immunoassay and TGF-b ELISA from the supernatants of F4- stimulated PBMCs on day 38 from all pigs. Individual pigs colonized on this time point by virulent G. parasuis are represented with filled symbols, while non-colonized animals are expressed with empty symbols. Averages in bars and standard deviations found in each group are also plotted. genic respiratory bacteria. Therefore, it would be of interest to secreted TGF-b by the in vitro stimulated PBMCs and the level of address in future studies the mucosal humoral and cell-mediated specific anti-F4 IgGs in the sow of origin. This association of the response in the upper respiratory tract of the piglets after vaccina- TGF-b levels together with an increase anti-F4 antibodies are in tion with CAFÒ01 or CDA adjuvanted antigens. line with the ones published in a previous study of maternal vacci- The multivariable model indicated correlation between sow nation, where TGF-b in sera and specific antibodies against F4 anti- parity and amount of T helper and memory T cells in the piglets, gen were upregulated in the offspring of F4-vaccinated sows [16]. as reported after a cell-subset assessment in sows of diverse pari- This feature, together with a slight increase of Th17-like response ties [52]. Likewise, the model showed an association between cytokines, might indicate an intrinsic immunogenicity of the vac- 9 S. López-Serrano, Y.S. Mahmmod, D. Christensen et al. Vaccine: X 14 (2023) 100330 cine protein that requires further attention. Although not yet work. The work of Núria Galofré-Milà in synthesizing the F4 described, it cannot be ruled out that the processing and presenta- immunogen and all the laboratory procedures she carried out is tion of F4-protein might result in a microenvironment favored greatly acknowledged. The contribution of Diego Pérez from the towards a Treg or Th17 polarization, since cytokines related to Animal Field Studies staff of IRTA-CReSA is also greatly acknowl- these responses were described recently for G. parasuis [53]. edged, together with Miguel Blanco-Fuertes and Carlos Neila- In summary, the immune responses observed in vaccinated and Ibáñez for their help. The authors want finally to thank Néstor non-vaccinated piglets were affected by the adjuvant included in Gómez Trias from the Biochemistry Department of The Veterinary the vaccine but also by the sow and the natural colonization by a Faculty of UAB for his willingness to lend us the sonifier used for virulent strain of G. parasuis. The F4 conserved fragment currently the immunogen preparation. represents a good marker for the detection of colonization/infec- tion, either by PCR detecting the F4 gene or measuring specific Appendix A. Supplementary data F4 antibodies, making the F4 conserved fragment not only a vac- cine candidate but also an indicator to include for diagnostics of Supplementary data to this article can be found online at immunity against G. parasuis in pigs. 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