Development a cross-protective subunit cocktail vaccine against diverse serotypes of Glaesserella parasuis infection in pigs

Development a cross-protective subunit cocktail vaccine against diverse serotypes of Glaesserella parasuis infection in pigs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development a cross-protective subunit cocktail vaccine against diverse serotypes of Glaesserella parasuis infection in pigs Fengyang Li, Yan Gong, Ziheng Li, Zhen Wang, Zengshuai Wu, Di Zhang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8593719/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Glässer's disease caused by Glaesserella parasuis (GPS) is a severe disease that leads to huge economic losses in the swine industry worldwide due to the poor cross-protective efficacy of existing vaccines. In this study, a multi-antigen cocktail subunit vaccine against GPS infection was developed. By in-silico analyzing antigenicity and comparison of immunoprotective efficacy and induction of antibody titers in a murine model, the component of the cocktail vaccine was determined to be VacJ, PtsG and GAPDH, with Gel-01 as the optimal adjuvant. Immunization of the cocktail vaccine induced robust humoral and cellular immune responses both in vitro and in vivo . Importantly, the vaccine not only conferred effective cross-protection against lethal-dose of GPS4 and GPS5 infections in mice, but also demonstrated favorable immune protective efficacy against GPS infection in piglets. Moreover, their immune sera significantly inhibited the growth of GPS4 and GPS5. Overall, these results suggested that the subunit cocktail vaccine established in this study is a promising agent for the prevention and control of porcine Glässer's disease. Subunit vaccine Glaesserella parasuis Glässer’s disease PtsG GAPDH VacJ Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Porcine Glässer's disease is a swine-specific bacterial infection that caused by Glaesserella parasuis (GPS) and characterized by meningitis, polyserositis, arthritis, and pneumonia [ 1 ]. GPS primarily affects young pigs aged from 2 weeks to 4 months, particularly those at 5–8 weeks of age. Epidemiological investigations have demonstrated that the incidence rate of this disease generally ranges from 10% to 15%, and in severe outbreaks, the mortality rate can escalate to 50%, thereby posing a severe threat to the health of piglets and fattening pigs [ 2 ]. To date, 15 serotypes of GPS have been identified, among which serotypes 4 and 5, are the predominant circulating strains in China, followed by serotypes 12, 13, and 14 [ 2 ]. Notably, GPS frequently engages in co-infections with other viral and bacterial pathogens, especially respiratory pathogens including porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), Streptococcus suis (SS) and Actinobacillus pleuropneumoniae (APP), leading to increased morbidity and mortality and substantial losses to swine industries that hinders sustainable development [ 3 – 5 ]. Currently, vaccination remains the primary strategy for preventing swine Glässer's disease, with available vaccines predominantly categorized into inactivated vaccines and subunit vaccines. Among these, inactivated vaccines have been commercialized and demonstrate favorable protective efficacy against same serotype. However, due to the diverse prevalent serotypes of GPS, commercial inactivated vaccines exhibit limited or even no cross-protection against different serotypes [ 1 , 6 ]. Additionally, they have many problems such as insufficient safety, short protection time, and poor immune effect. Therefore, it is urgent to develop a vaccine with robust cross-protection against multiple serotypes of GPS infections. Subunit vaccines are composed of immunoprotective components derived from microbial secretory proteins or outer membrane proteins and offer advantages of high safety and minimal side effects [ 7 , 8 ]. Of note, only two bivalent subunit vaccines (SS-GPS and PCV2-GPS, both of which use the phenylalanine ammonia-lyase family protein PalA as the primary antigen) were approved for market use [ 6 ]. A variety of subunit vaccines based on different antigens such as pili subunit PilA [ 9 ], transferrin-binding proteins TbpA and TbpB [ 10 – 12 ], trimeric autotransporter VtaA [ 13 ], polyamine transport protein PotD [ 14 ], lipoprotein VacJ [ 15 , 16 ], and glyceraldehyde-3-phosphate dehydrogenase GAPDH [ 17 , 18 ], have been assessed in laboratory. However, using these antigens alone have been proven to provide only partial protection or no cross-protection against GPS infection. Thus, identification of novel antigens with high immunogenicities and combination of two or more of these antigens should be employed to improve the immune efficacy of subunit vaccines. In this study, a multi-antigen subunit cocktail vaccine was successfully developed after systematic screening of in-silico immunogenicity, protective efficacy, optimal adjuvant and antigen ratio in a murine model of GPS infection. This vaccine exhibited prominent immunogenicity, as it elicited robust humoral and cellular immune responses both in vitro and in vivo , and conferred effective cross-protection against GPS4 and GPS5 infections in mice. Notably, the vaccine also demonstrated favorable immune protective efficacy against GPS infection in piglets. Collectively, these results suggested that the subunit cocktail vaccine established in this study is a promising agent for the prevention and control of porcine Glässer's disease. Materials and methods Bacterial strains and growth conditions All strains used in this study are listed in Additional file 1. E. coli strains were cultured in Luria-Bertani (LB; Becton Dickinson) liquid medium or on LB solid agar plates containing ampicillin (100 µg/mL) or kanamycin (50 µg/mL). GPS moderate virulent serotype 4 (GPS4) and high virulent serotype 5 (GPS5) strains were kindly provided by Prof. Lei Wang (Henan Institute of Science and Technology, Xinxiang, China). GPS strains were cultivated in Brain Heart Infusion (BHI; Hopebio) medium supplemented with 5% horse serum and 20 µg/mL NAD (BHI+) or plated on BHI + agar plates. All cultures were incubated at 37 o C with 5% CO 2 if not specified. Antigen selection In this study, the antigenicity of major antigens of GPS were predicted using VaxiJen V2.0 ( https://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html ) [ 19 ]. The proteins sequences of these antigens were retrived from NCBI database ( https://www.ncbi.nlm.nih.gov/genbank/ ). The proteins with good antigenicity (≥ 0.4) were chose for further anslysis. Finally, seventeen GPS antigens including PotD (GenBank: ACL31786.1), MnSOD (GenBank: ACL32450.1, 618 bp), GPD (GenBank: ACL32465.1), YgiW (GenBank: ACL33049.1), Omp26 (GenBank: ACL32860.1), QseB (GenBank: ACL33513.1), GAPDH (GenBank: ACL31707.1), TEX (GenBank: ACL32154.1), PalA (GenBank: ACL31779.1), ApbE (GenBank: ACL31801.1), PtsG (GenBank: ACL33546.1), HutZ (GenBank: ACL32674.1), KefA (GenBank: ACL32066.1), VacJ (GenBank: ACL33700.1), ClpP (GenBank: ACL33474.1), CRP (GenBank: ACL33503.1), and QueA (GenBank: ACL32317.1) were selected. DNA manipulation The transmembrane regions and the signal peptides of the fourteen antigens were predicted using the TMHMM ( https://services.healthtech.dtu.dk/services/TMHMM-2.0/ ) and SignalP ( https://services.healthtech.dtu.dk/services/SignalP-5.0/ ), respectively [ 20 , 21 ]. Fragments without transmembrane regions and the signal peptides were amplified by PCR from the genomic DNA extracted using a gDNA isolation kit (Qiagen). The PCR products were digested with BamHI/XhoI (NEB) restriction enzymes and ligated into pET28a or pET32a vector using a Rapid DNA ligation kit (Roche Diagnostics). Inserted DNA sequences were confirmed by DNA sequencing and double digestion by BamHI/XhoI (NEB) restriction enzymes. All plasmids and primers used in this study are listed in Additional file 1 and Additional file 2, respectively. Protein purification The constructed plasmids were transformed into E. coli BL21 (DE3). When grown to OD600 = 0.6–0.8, 0.5 mM of IPTG was added to induce protein expression for 16 h at 16 o C. Bacterial cells were disrupted by sonication and the supernatants were collected after centrifugation at 12,000 ×g for 30 min at 4 o C. Protein purification was performed using high-affinity Ni-NTA resin (GeneScript) by gravity at a flow rate of 1 mL/min. Proteins were eluted using 500 mM imidazole and dialyzed subsequently against washing buffer (50 mM NaH 2 PO 4 pH 8.0, 300 mM NaCl, 20 mM imidazole) at 4 o C. Protein concentration was measured by a BCA kit (Thermo Scientific) and stored in aliquots at -80 o C. Prediction of tertiary structures, molecular docking and immune simulation The tertiary structures of PtsG, VacJ, PalA and GAPDH were predicted by AlphaFold2. The binding affinity between each antigen and immune receptors including TLR2 (PDB ID: 2z7x), MHC II (PDB ID: 5jlz), and swine leukocyte antigen SLA-1 (PDB ID: 3qq3) were assessed by molecular docking using the ClusPro 2.0 server ( https://cluspro.bu.edu/home.php ) [ 22 ]. The docking results were visualized with Pymol and the interaction residues between the docked chains were analyzed using PDBsum ( https://www.ebi.ac.uk/thornton-srv/databases/pdbsum/ ) [ 23 ]. The immune responses profile of each antigen were simulated use the C-ImmSim server ( https://kraken.iac.rm.cnr.it/C-IMMSIM/index.php ) [ 24 ]. The time steps of the three injections in the simulation phase were set to 1, 42, and 84, respectively. The standard time of one injection was 8 h, corresponding to a 14-day gap between immunizations. The simulation volume is 50 and the simulation step is 1050. All other parameters set to their default values. Immunization and challenge of mice and piglets For immunization of mice, two hundred and ten of 6-weeks old healthy female ICR mice (body weight 18 ± 2 g, purchased from Changsheng Biotechnology Co., Ltd., China) were randomly divided into different groups (10 mice/group) and immunized with 100 µg antigens mixed with or without Gel-01 adjuvant (10%; SEPPIC, France), PBS, PBS plus Gel-01, inactivated vaccine (Kefuning, China) by multi-point subcutaneous injection on the mice back, respectively. The immunization was administered every two weeks for a total of two doses. For immunization of piglets, six of 4-weeks old healthy piglets (purchased from Zybio, China) were randomly divided into two groups (3 piglets/group) and immunized with 1.5 mg antigens mixed with Gel-01 adjuvant (10%; SEPPIC, France) and PBS plus Gel-01 by intramuscular injection in neck, respectively. The immunization was administered every three weeks for a total of two doses. Body temperature, mental status and feeding behaviors of piglets were recorded everyday after the first immunization for one week. Blood was collected from the tail vein of mice at 14 and 28 days after first dose or from anterior vena cava of piglets at 21 and 35 days after first dose to detect specific antibody levels by ELISA. Alternatively, blood was collected from the tail vein of mice at 0, 2, 4, 6, 8, 9, 10, and 11 weeks after the second dose to monitor the antibody levels by ELISA. After two weeks of the second dose, the mice were challenged with lethal dose of GPS4 (4×10 9 CFU) and GPS5 (2.4×10 9 CFU) by intraperitoneal injection. Animal death and body weight was recorded every 24 h for one week. Similarly, the piglets were challenged with GPS5 (4×10 10 CFU) by intraperitoneal and intranasal injection after two weeks of the second dose. Animal death and clinical symptoms were recorded every 24 h for one week. Both piglets and mice were euthanized using CO 2 or if they lost 20% of maximum body weight for two consecutive days, were immobile, or were found moribund. ELISA An indirect ELISA was used to detect the specific antibody levels in animal serum. In brief, the antigens were diluted separately with coating buffer (0.1 mol/L Na 2 CO 3 -NaHCO 3 , pH 9.6) to 1 µg/mL and coated on ELISA plate by 100 µL/well at 4 o C overnight. After washing, 100 µL of 5% BSA was added to each well and sealed at 37 o C for 1 h. Then, the sera were diluted 10 times and added to ELISA plate (100 µL/well). After incubation at 37 o C for 1 h, goat anti-mouse IgG HRP (1:5000) was added to each well (100 µL/well) and incubated for another 1 h. The reaction was visualized by addition of TMB reagent (100 µL) and terminated by 2M H 2 SO 4 (50 µL). Absorbance was detected at OD450 nm. Whole blood killing GPS4 and GPS5 from the midlog growth phase were collected, centrifuged, washed twice by PBS and adjusted to 2×10 9 CFU. Bacterial suspensions (10 µL) were mixed with 150 µL of mice blood with or without immunization and incubated for 2 h at 37 o C. Te mixture was serially diluted and plated on BHI + agar plates for cell counting. Bacterial survival was calculated as follows: (1 - recovered CFU/CFU in the original inoculum) × 100%. Western blot Identification and the antigenicity of the purified proteins VacJ, PtsG, GAPDH, and PalA were assessed by SDS-PAGE and Western blot using anti-His antibody and GPS4 or GPS5 antibody-positive serum, respectively. The proteins were boiled in SDS sample buffer at 95 o C for 10 min and then separated on 12.5% SDS-PAGE gels. After electrophoresis, gels were transferred to the PVDF membrane (Millipore) and blocked with 3% BSA overnight. After washing in TBST, the membranes were incubated with rabbit anti-His (1:3000; Abconal, AE068) or GPS antibody-positive serum at room temperature for 2 h. After washing in TBST, the membranes were incubated with goat anti-rabbit HRP-IgG (1:5000; Abconal, AS014) for another 1 h at room temperature. The targeted proteins were visualized using ECL reagent (Millipore) under a luminescent imaging system (Tanon 5200 Multi). Flow cytometry After 2 weeks of the second immunization, the mice were euthanized by inhalation of CO 2 . Mice spleen were collected, cut into pieces and digested in HBSS containing gelatinase A (100 U/mL) and DNase ( 20 µg/mL). Following washing with PBS, tissue samples were exposed to ice-cold RBC lysis buffer for 5 min. The cell pellets were gathered by centrifugation and suspended in fluorescent washing buffer (ThermoFisher) to a concentation of 1×10 7 /mL. Then, cells were labeled with fluorescent antibodies, including PerCP/Cy5.5 anti-mouse CD45 (Biolegend, 103132), FITC anti-mouse CD3 (Biolegend, 100203), APC anti-mouse CD4 (Biolegend, 100515), PE-Cy7 anti-mouse CD8 (Biolegend, 100722), APC anti-mouse CD19 (Biolegend, 152409) antibodies, and incubated in the dark for 30 min. After washing with PBS, cells were analyzed using a flow cytometer (CytoFLEX). Data were processed using FlowJo software (Version 10.4). Histopathological analysis and bacterial loads in organs The Histopathological analysis was performed as described previously [ 25 ]. Briefly, lung, liver, and spleen tissues were fixed with 4% paraformaldehyde solution. After dehydration via graded ethanol, the tissues were cleared with xylene, embedded in paraffin and sliced (3–5 µm) using a microtome. Then, the slices were mounted on slides, deparaffinized, rehydrated, and stained with Hematoxylin-Eosin (HE). After dehydration, slides were coverslipped for microscopic examination. To analysis bacterial loads in organs, equal weights of lung, liver, spleen, and blood samples were collected and homogenized with PBS (1 g/3 mL). Then, the samples were serial diluted and spotted on BHI + agar plates. The number of viable bacteria were counted after overnight inculation at 37 o C. Lymphocyte proliferation assay Spleens were aseptically harvested from 6-week-old ICR mice euthanized by cervical dislocation, and single-cell suspensions were prepared after red blood cell lysis. Cells were resuspended in complete RPMI 1640 medium containing 10% inactivated fetal bovine serum and 1% penicillin-streptomycin. Isolated lymphocytes were seeded in a 96-well plate at a density of 5×10 6 and treated with either PBS or VA + PT + GA (10 µg/well, 1:1:1). After incubation at 37 o C for 48 hours, cell viability was assessed using a CCK-8 kit (Beyotime, China) according to the manufacturer’s instructions. Absorbance was detected at 450 nm. Alternatively, total RNA was isolated from different groups of cells from a 6-well plate under a same procedure of stimulation. RNA isolation and qRT-PCR Total RNA was extracted using a tissue RNA isolation kit according to the manufacturer’s instructions (Invitrogen, USA). The extracted RNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Scientific) and its quality was assessed by PCR and gel electrophoresis. cDNA is synthesized from 1 µg of purified RNA using Prime Scrip RT Master Mix Kit (TaKaRa, China). Quantitative PCR was then performed with gene-specific primers using TB Green PCR Master Mix (TaKaRa, China) on a real-time PCR system (q225, Kubo Instruments, China). Data was analyzed using the 2 −ΔΔCT method. Amplification curves are analyzed to determine cycle threshold (Ct) values, with target gene expression normalized to reference gene ( GAPDH ) for relative quantification. Primers are listed in Additional file 2. Statistical analysis The experimental data were statistically analyzed using GraphPad Prism (version 9.0). Data are presented as mean ± SD from three independent replicates. Differences between the mean values of normally distributed data were assessed using one-way and two-way ANOVA (Dunnett’s test). P -value < 0.05 was considered to be significant and indicated by "*". P -value < 0.01 was considered to be extremely significant and indicated by "**.” Results Selection of GPS candidate antigens To identify antigens for a effective subunit vaccine for GPS infection, we first selected seventeen potential or characterized antigens of GPS and evaluated their antigenicity using the VaxiJen V2.0 server at threshold of 0.4. Except PotD, GPD, and CRP, all other fourteen antigens retrived scores of higher than 0.4 (Additional file 3). So, these fourteen antigens were chose for further analysis. Next, the coding sequences of these antigens were cloned in either pET28a or pET32a expression vectors for protein expression (Additional file 4A). The expression profiles of the His-tagged recombinant proteins were analyzed by SDS-PAGE. Clear and single band was observed in the SDS-PAGE gel (Additional file 4B), suggesting all of these proteins were successfully purified with high purity. Subsequently, the immunoprotective effect of the recombinant proteins were preliminary evaluated in a murine infection model. Mice were subjected to a two-step immunization regimen, followed by challenge with GPS4 and GPS5 at 14 days after the secondary immunization (Fig. 1 A). The results showed that the levels of specific antibodies in the mice’s serum were significantly increased after immunization at 14 and 28 days compared with the control group (PBS + Adjuvant) (Fig. 1 B). Notebly, half of the antigens induced significant higher antibody levels after the second immunization than those after the first immunization. These findings indicate that these antigens have good immunogenicity that can trigger a strong humoral immune response after immunization in mice. Moreover, immunization of mice with PtsG, GAPDH, VacJ and PalA increased mice survival rate to 100%, 60%, 80% and 60%, respectively, upon infection with GPS4 compared with control group, while the survival rates for the immunization with other antigens were all below 60% (Fig. 1 C). Upon infection with GPS5, immunization of mice with PtsG, GAPDH, VacJ, MnSOD and PalA increased mice survival rate to 60%, 60%, 100%, 60% and 60%, respectively, compared with control group, while the survival rates for the immunization with other antigens were all below 40% (Fig. 1 D). Collectively, immunization with PtsG, GAPDH, VacJ and PalA conferred to better immunoprotective effect than other antigens in mice against both GPS4 and GPS5 infection. Furthermore, the tertiary structures of PtsG, GAPDH, VacJ and PalA and their binding affinities with host immune receptors TLR2, MHC II and swine leukocyte antigen (SLA I) were docked to assess the capability to induce immune response. Molecular docking results demonstrated that all the four antigens exhibited good binding affinity to both murine and porcine immune receptors (Fig. 2 A; Additional file 5), suggesting a strong capability to activate innate immune cells. Correspondingly, a broad and robust activation of the immune system post-injection was observed by immune simulation using the C-ImmSim server (Additional file 6). Simulated immunization of these antigens not only increased the total number of T cells and B cells, but also upregulated cytokine responses and the level of antibodies, indicating these antigens are capable of inducing strong immune responses in silico . Notably, detection of the antigenicity of the purified proteins illustrated that these antigens can be recognized by both GPS4- and GPS5-positive antiserums (Additional file 7; Fig. 2 B), suggesting that they could possibly confer to cross-protection against both GPS4 and GPS5 infection in mice. Based on the observations above, these four antigens were chose for the construction of cocktail vaccine. Development and optimization of antigens for cocktail vaccine To further improve the immunoprotective effect of the antigens, we randomly combined two or three antigens into different groups and immunized mice with Gel-01 adjuvant. As shown in Fig. 3 A- 3 D, the levels of specific antibodies in the mice’s serum were significantly increased after immunization at both 14 and 28 days compared with the control group. Of note, although the level of PalA-specific antibodies increased significantly after the secondary immunization compared with that after the primary immunization, the magnitude of this increase was lower than that of the specific antibodies against the other three antigens (Fig. 3 D). Furthermore, compared with the control group, immunization regimens including VA + PT (VacJ and PtsG), VA + PT + GA (VacJ, PtsG and GAPDH), and VA + PT + PA (VacJ, PtsG and PalA) all conferred over 50% protection in mice following GPS4 challenge, with the VA + PT + GA exhibiting the optimal efficacy at 80% (Fig. 3 E). Similarly, all the above groups provided more than 60% protection after GPS5 challenge, among which VA + PT + GA achieved complete protection (100%) (Fig. 3 F). Notably, the protective efficacy of VA + PT + GA immunization was significantly superior to that of the commercial inactivated vaccine group, which only conferred 20% and 40% protection against GPS4 and GPS5 challenges, respectively (Fig. 3 E). These results indicate VacJ, PtsG and GAPDH was the optimal assembly of antigens of cocktail vaccine. Screening of optimal immune adjuvants Adjuvants are critical in improving vaccine efficacy, so we further screened the optimal adjuvants to identify those that best enhance the immunogenicity and protective efficacy of the cocktail vaccine. To this end, antigen assemble of VacJ, PtsG and GAPDH was emulsified with a number of commonly used adjuvants including Montanide ™ ISA201, ISA206, ISA563, Gel-01, aluminum hydroxide, and white oil (OIW) and immunonized mice. In terms of antibody responses, all adjuvant groups exhibited significantly higher antibody levels than the control group, regardless of primary or secondary immunization (Fig. 4 A). Following the primary immunization, the traditional aluminum hydroxide adjuvant group induced the highest titers of GAPDH- and VacJ-specific antibodies compared to other adjuvant groups; however, these antibody levels declined after the secondary immunization, with a similar trend observed in the induction of PtsG-specific antibodies. In contrast, although the ISA201 and Gel-01 adjuvant groups did not elicit the highest antibody levels relative to other adjuvant groups, they showed increased GAPDH- and PtsG-specific antibody titers after the secondary immunization. Moreover, across all adjuvant-immunized groups, a certain level of protective efficacy against both GPS4 and GPS5 challenges was observed in mice. Notably, the ISA201 and Gel-01 adjuvant groups exhibited the most robust protective effects, with survival rates of 100% and 80% respectively following GPS4 challenge, and 80% and 100% respectively upon GPS5 challenge (Fig. 4 B). Analysis of murine body weight changes revealed no significant difference in weight changes between the two groups following GPS4 challenge. However, after GPS5 challenge, mice in the ISA201 adjuvant group exhibited significantly more pronounced weight changes compared to those in the Gel-01 adjuvant group (Fig. 4 C). Consistently, mice immunized with Gel-01 exhibited lower bacterial loads in the lung and liver compared to those in the ISA201 adjuvant group upon GPS5 challenge (Fig. 4 D). Overall, these results indicate that Gel-01 was the optimal adjuvant of the cocktail vaccine. Immunization of the cocktail vaccine enhanced both cellular and humoral immune responses Next, the effect of the cocktail vaccine on host cellular and humoral immune responses was evaluated both in vitro and in vivo . We observed that the splenic index in mice was significantly increased following vaccine immunization compared to the control group (Fig. 5 A). Splenic lymphocytes were isolated from mice, and stimulation with VA + PT + GA was found to not only significantly enhanced lymphocyte proliferation but also upregulated the transcriptional levels of cytokine genes Il2 , Il4 and Ifnγ (Fig. 5 B-C). Considering the roles of these cytokines in lymphocyte differentiation and antibody production, we subsequently analyzed the proportion of B and T lymphocytes in the spleen and antibody titers in the peripheral blood post-immunization. Flow cytometric analysis revealed no significant increase in the overall B cell population in immunized mice relative to controls; however, serum antibody levels exhibited a highly significant elevation as early as 2 weeks post-immunization, gradually peaking between 4 to 6 weeks (Fig. 5 D-E). Subsequent to this peak, a gradual decline was observed; however, antibody levels remained substantially elevated, with such high titers persisting for a minimum of 11 weeks. Furthermore, results from the whole-blood bactericidal assay revealed that serum treatment from the vaccine-immunized group significantly reduced the colony counts of both GPS4 and GPS5 (Fig. 5 F), indicating that the antibodies elicited by the cocktail vaccine exhibited bactericidal activity against GPS4 and GPS5. In terms of T cells, flow cytometric analysis showed that vaccine immunization not only induced a significant increase in the total number of T cells but also a higher ratio of CD3 + CD4 + /CD3 + CD8 + T cells (Fig. 5 G), suggesting a possible Th2 type immune response elicited by vaccine. Collectively, these findings confirmed the vaccine's capacity to induce sustained humoral and cellular immunity in response to vaccine immunization. Cocktail vaccination reduced pathological lesions and alleviated inflammatory responses in mice Major organs including liver, lungs and spleen of mice post-challenge were collected for histopathological analysis. Gross necropsy examination showed that, mice challenged with GPS4 or GPS5 exhibited significantly swollen lungs with extensive hemorrhage, congestion, and reddish-brown inflammatory foci compared with the control group (PBS + Adjuvant) (Fig. 6 A). The lungs in the inactivated vaccine group displayed mild enlargement and partial reddish-brown inflammatory foci, whereas only a few petechiae and congestion spots were observed in the cocktail vaccine-immunized group, with no lung swelling. Similar findings were noted in the spleen and liver of the mice. Histopathological examination showed that challenged with GPS led to drastic pathological changes in the lungs of the mice, including large amounts of inflammatory exudate, thickening and collapse of alveolar walls, dramatic atrophy of alveoli, extensive infiltration of red blood cells and inflammatory cells (Fig. 6 B). Immunization with the commercial inactivated vaccine alleviated the pathological changes to a certain degree, while the cocktail vaccine-immunized group displayed only faint histopathological lesions, with only limited red blood cell infiltration and focal alveolar wall thickening. Comparable pathological profiles were observed in the spleen and liver of the mice. Analysis of bacterial loads in these organs and blood revealed that immunized with the cocktail vaccine significantly reduced the total number of both GPS4 and GPS5 in lungs and blood, but not spleen and liver compared to the control group (Fig. 6 C). Moreover, the transcription levels of pro-inflammatory cytokine TNF-α, but not IL-1β and IL-6, was significantly reduced in lung by immunization with the cocktail vaccine after GPS4 challeng compared to the control group, while the transcriptional level of IL-6, but not TNF-α and IL-1β, was significantly reduced following GPS5 challenge (Fig. 6 D). Taken together, these results demonstated that the cocktail vaccine can effectively protect mice against GPS infection by reducing organ lesions and alleviating inflammatory responses. Cocktail vaccination protected piglets from GPS infection Given that pigs constitute the target animals for GPS infection, subsequent investigations were conducted to assess the immunoprotective efficacy of the cocktail vaccine in piglets (Fig. 7 A). Vaccination was delivered via cervical intramuscular injection. Within 24 h post-vaccination, a transient elevation in body temperature was observed in the vaccinated piglets, yet this increase was not statistically significant compared to the control group (Additional file 8). After 24 h, the body temperature of the vaccinated piglets gradually decreased, eventually converging with that of the control group piglets. In terms of antibody responses, high levels of antigen-specific antibodies were elicited following the primary immunization, with titers significantly higher than those in the control group (Fig. 7 B). After the secondary immunization, although no significant changes were observed in the antibody levels against VacJ and PtsG, the antibody titer against GAPDH increased notably compared to that of the first immunization. These results demonstrated that the cocktail vaccine can induce a favorable humoral immune response in piglets. Two weeks after the 2nd immunization, piglets were challenged with GPS5 and clinical symptoms were observed for one week. Without vaccination, GPS5 challenge resulted in a dramatic increase in body temperature, which reached the highest at 40.2 o C after 4 days post challenge (Additional file 9; Fig. 7 C). Moreover, the piglets were accompanied by heavy breathing, nasal discharge, depression, and a tendency to lie down. Piglets in the immunized group also showed elevated body temperature, which reached the highest at 40 o C after 2days post challenge, reduced and remained stable thereafter (Additional file 9; Fig. 7 C). The piglets also exhibited breathing difficulties, nasal discharge, and depression after challenge; however, these symtoms were gradually disappeared after two days. Notebly, the body temperature of the immunized group was significantly lower than that of the control group at four days after challenge (Fig. 7 C). Gross pathological observations following GPS5 challenge revealed that piglets in the control group exhibited substantial fibrinous exudation within the thorax, accompanied by severe adhesions among the heart, lungs, and thoracic wall, which were typical characteristics of pleurisy (Additional file 10). The lungs were characterized by enlargement and hemorrhage, with a pale yellow membranous fibrinous exudate present on their surface; well-demarcated congested or hemorrhagic regions were detected in the anterior-inferior segments of the apical, cardiac, and diaphragmatic lobes, as well as in the accessory lobe of both lungs (Additional file 10; Fig. 7 D). Cross-sectional examination of the lungs demonstrated heterogeneous pigmentation and multiple necrotic foci within the lesioned areas. Similar pathological changes were also observed in liver and spleen. In contrast, the immunized group showed significantly attenuated overall pathological changes compared to the control group upon necropsy. No overt features of pleurisy or peritonitis were noted, and either an absence or only a minimal quantity of fibrinous exudate was observed on the lung surface or within the abdominal cavity. Additionally, there were no significant adhesions between the visceral organs and the thoracic or abdominal walls. Consistent with these findings, the HE-stained sections further showed that GPS5 challenge induced widened alveolar interstitium, alveolar atrophy, and presence of inflammatory exudate in the lungs; the boundary between the red and white pulp in the spleen was obscure with infiltration of inflammatory cells; hepatocytes were swollen and lost their cord-like arrangement in the liver (Fig. 7 E). In contrast, these pathological manifestations were markedly alleviated in the immunized group. Moreover, although there was no significant difference in the bacterial load in the liver, vaccine immunization significantly reduced the bacterial loads in the blood, lungs, and spleen of piglets compared with the control group (Fig. 7 F). Discussion Porcine Glässer's disease, an infectious disease caused by GPS, imposes a severe threat to swine health owing to its high mortality rate and incurs substantial economic losses in the global swine industry. Currently, vaccination stands as the most effective preventive measure against this disease; nevertheless, existing vaccines are commonly plagued by critical limitations including short protective durations and poor cross-protection efficacy across different GPS serotypes [ 2 , 6 ]. In this study, an effective subunit cocktail vaccine against porcine Glässer's disease was successfully developed. Immunization of the vaccine not only conferred effective cross-protection against GPS4 and GPS5 infections in mice, but also demonstrated favorable immune protective efficacy against GPS infection in piglets, suggesting it can be used as a promising vaccine against porcine Glasser's disease in future. GPS possesses dozens of virulence factors, many of which are membrane-associated or secreted antigens and are frequently regarded as the antigen candidates for subunit vaccine development. Among the seventeen GPS antigens selected in this study, a subset comprises well-characterized GPS virulence factors, including OMP26, YgiW, VacJ, PalA, GAPDH and MnSOD. Cumulative evidence from relevant studies has demonstrated that immunization with recombinant forms of these antigens elicited substantial immunoprotection across diverse animal models. For example, in a guinea pig model, recombinant VacJ and OMP26 conferred 67% and 83% protection against infection with GPS5 strain SH0165, respectively [ 15 ]. In murine models, recombinant GAPDH and PalA achieved protective rates of 75% and 80% against SH0165 challenge, respectively [ 18 , 26 ]. Notably, previous studies have reported that recombinant MnSOD and YgiW also exhibited cross-immunoprotective activity in mice [ 27 , 28 ]. However, the cross-immunoprotective efficacy of these two recombinant proteins was suboptimal (both below 60%) in this study (Fig. 1 C and D). Besides well-characterized virulence factors, we also incorporated virulence factors with uncharacterized immunogenicity, including PtsG, ApbE, ClpP and QseB, as well as the proteins that are significantly upregulated during GPS infection, including Tex, HutZ, KefA, and QueA, all of which have been proposed as potential candidate antigens for GPS vaccine development [ 29 , 30 ]. Unexpectedly, only recombinant PtsG displayed robust cross-immunoprotective efficacy in murine models, whereas the immunoprotective efficacy of the remaining proteins was consistently below 60%. PtsG is a vital protein in the bacterial glucose phosphotransferase system (PTS Glc ) and mainly in charge of glucose transport as part of the enzyme II complex. PTS Glc not only transports glucose, but also regulates various cellular activities including virulence, biofilm formation and metabolism [ 31 – 33 ]. For example, deletion of ptsG in Borrelia burgdorferi were unable to cause infection in mice [ 34 ]. PtsG as well as other genes that involved in glycolysis are required for the replication of Salmonella Typhimurium in macrophages within the "Salmonella-containing vacuole" (SCV) [ 35 ]. In GPS, deletion of ptsG led to dramatic changes in metabolic and transcriptomic pathways, some of which were associated with bacterial virulence and biofilm formation [ 31 ]. However, conclusive experimental evidence addressing whether PtsG regulates the virulence of GPS and whether it qualifies as a antigen for GPS subunit vaccine remains elusive to date. In the present study, we demonstrated, for the first time, that PtsG possessed robust immunogenicity. Immunization of mice with recombinant PtsG alone or in combination with VacJ and GAPDH conferred effective protection against GPS challenge, thereby identifying PtsG as a promising candidate antigen for GPS vaccine development. In the context of suboptimal cross-protection against multiple serotypes following immunization with a single antigen, the strategy of combining two or more antigens represents an effective approach to enhance the protective efficacy [ 36 ]. Many studies have demonstrated that the combined use of distinct antigens is more effective in disease prevention compared to the use of a single antigen alone. For example, the SARS-CoV-2 multi-antigen vaccine that co-delivers S, M, and N antigens elicits more robust immune responses and superior protective efficacy compared to the single antigen [ 37 ]. Chen et al. demonstrated that co-administration of multi-antigen combinations afforded complete protection and elicited significantly higher titers of neutralizing antibodies than immunization with single antigen which provided only partial protection against vaccinia virus infection [ 38 ]. In this study, the multi-antigen combination VA + PT + GA exhibited potent cross-immunoprotective activity against distinct serotypes of GPS, conferring 80% protection against GPS4 and 100% protection against GPS5 (Fig. 3 A-E). Importantly, the immunoprotective performance of the cocktail vaccine was superior to that of any individual antigen component. The multi-antigen strategy not only expands the serotype coverage of the subunit vaccine but also enhances its cross-protective efficacy, addressing the limitation of narrow serotype protection associated with single-antigen vaccines. Furthermore, although the protective efficacy of the antigen combination VA + PT + PA was lower than that of the VA + PT + GA combination, it still conferred considerable protection against GPS4 and GPS5 infections (60% and 80%, respectively), suggesting GAPDH is more important than PalA for protection against GPS infection. GAPDH is a glycolytic enzyme that catalyzes the conversion of glyceraldehyde-3-phosphate to 1, 3 diphosphoglycerate [ 39 ]. Moreover, GAPDH is regarded as a suitable vaccine candidate for protection against bacterial diseases due to its role in microbial virulence [ 40 ]. As a vaccine component, the immunoprotective efficacy of GAPDH has been confirmed in several bacterial pathogens, such as Listeria monocytogenes , Mycobacterium marinum , Streptococcus pneumoniae and GPS [ 17 , 18 , 41 , 42 ]. Specifically, immunization with recombinant GAPDH was found to be protective both in murine and porcine model of GPS5 infection [ 18 ], but the immnoprotective efficacy aginst other serotypes of GPS infection was not evaluated. PalA is a peptidoglycan-associated outer membrane lipoprotein that shows highly homology to the P6 protein of Haemophilus influenzae [ 43 ]. P6 is a protective antigen against H. influenzae induced meningitis [ 44 ]. Notably, previous studies indicated that PalA showed deleterious affects on the Apx toxinbased subunit vaccine against APP infection by exacerbation of respiratory symptoms, higher mortality rate and severe lung damage [ 43 ]. Thus, it has been proposed that PalA is an immunogenic but not a protective antigen for the development of effective subunit vaccines against infections by APP [ 45 ]. On the other hand, immunization with recombinant PalA was found to be protective in murine model of GPS5 infection [ 26 ]. Indeed, both of the two commercial bivalent subunit vaccines SS-GPS and PCV2-GPS utilized PalA as the primary antigen [ 6 ]. The drastically opposing protective effects of PalA across different bacterial genera needs to be further investigated. As a type of immune enhancer, adjuvants help reduce the number of immunizations and the amount of antigens, overcome antigen competition in combined vaccines and, importantly, modulate the type of induced immunity [ 46 ]. Moreover, many studies have confirmed that the type of adjuvant used in vaccine affect the immune response and protection efficacy of the vaccine against homologous challenge [ 47 ]. Consequently, we further conducted adjuvant screening following the confirmation of the antigen componants for the subunit cocktail vaccine. The results demonstrated that Montanide ™ Gel-01 and ISA201 exerted the most robust adjuvant activity in potentiating the immune responses induced by the subunit vaccine (Fig. 4 A-D). Montanide™ Gel-01 is an aqueous polymeric adjuvant designed for aqueous type vaccines, while Montanide™ ISA201 is a mineral oil based adjuvant that has been developed for the manufacture of Water-in-Oil-in-Water (W/O/W) emulsions [ 48 ]. In line with our results, it’s demonstrated that the adjuvant effect of Gel-01 was superior to that of mineral oil and aluminum hydroxide adjuvants for trivalent inactivated GPS serovars 4, 5 and 12 vaccines against Glässer's disease in terms of antibody titers and protective efficacy [ 49 ]. Similar conclusions were also observed for ISA201 adjuvanted foot-and-mouth disease vaccine [ 50 , 51 ]. Considering significant difference in body weight changes and bacterial loads in organs of mice after GPS5 challenge, Gel-01 was selected as the optimal adjuvant for the subunit cocktail vaccine in this study. The level of host immune response following immunization is one of the key indicators for evaluating immunogenicity of vaccines. The cocktail vaccine developed in this study induced robust humoral and cellular immune responses after immunization in mice (Fig. 5 C). Interestingly, the proportion of CD4⁺ T cells after vaccine immunization was significantly higher than that of CD8⁺ T cells, and the induction of the transcriptional level of the Th2 cytokine IL-4 was dramatically stronger than that of the Th1 cytokine IFN-γ (Fig. 5 B and 5 G). IL-4 contributes to the proliferation and differentiation of B cells to produce IgG and may directly act on naïve CD4⁺ T cells during T-cell activation to induce Th2 cell differentiation [ 52 ]. Thus, these results suggested that the cocktail vaccine may be more inclined to induce a Th2 immune response rather than a Th1 immune response. In conclusion, a subunit cocktail vaccine was successfully developed against GPS infection in this study. The vaccine showed superior immunogenicity than inactivated vaccine and induced robust humoral and cellular immune responses both in vitro and in vivo . Importantly, immunization of the vaccine not only conferred effective cross-protection against GPS4 and GPS5 infections in mice, but also demonstrated favorable immune protective efficacy against GPS infection in piglets. These results suggested that the subunit cocktail vaccine established in this study is a promising agent for the prevention and control of porcine Glässer's disease. Declarations Ethics approval and consent to participate Animal experiments in mice were approved by the Institutional Animal Care and Use Committee of Jilin University (Approval No: KT202507001), while experiments in piglets were approved by the the Institutional Animal Care and Use Committee of Jilin Zhengye Biologics Co., Ltd (Approval No: SC-2501-001). All animal experiments were conducted in accordance with Guidance on the Chinese Laboratory Animal Administration Act 1988. All mice were housed in the laboratory animal room and maintained on a 14/10 h light-dark cycle with food and water ad libitum . Informed consent was obtained from all individual participants included in the study. Consent to publication Not applicable. Competing interest The authors report there are no competing interests to declare. Funding This work was supported by the National Key Research and Development Project Program of China (No. 2022YFD1800905) and National Natural Science Foundation of China (No. 32102670). Author Contribution **Fengyang Li:** Conceptualization, Formal analysis, Methodology, Visualization, Funding acquisition, Supervision, Writing–original draft, Writing–review, and editing. **Yan Gong:** Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization. **Ziheng Li, Zhen Wang, Zengshuai Wu, Di Zhang, Peng Zhang, Zhichao Lu, Hong Chu, Kaixin Zhang:** Investigation. **Na Li:** Data curation, Resources. **Liancheng Lei:** Conceptualization, Methodology, Funding acquisition, Project administration, Resources, Supervision, Writing–review, and editing. Acknowledgments We thank Prof. Lei Wang (Henan Institute of Science and Technology, Xinxiang, China) for providing GPS strains. Data Availability The raw data used in this article and the supplementary materials are available in figshare ( https://doi.org/10.6084/m9.figshare.30571994 ). References Macedo N, Rovira A, Torremorell M (2015) Haemophilus parasuis: infection, immunity and enrofloxacin. 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Additionalfile3.docx Additional file 3: Prediction of antigenicity of the selected seventeen antigens using VaxiJen V2.0 server. Additionalfile4.tif Additional file 4: Antigen cloning and purification. (A) Cloning of the coding sequences of the selected antigens by PCR. M, marker; 1, va (687 bp); 2, pt (366 bp); 3, pa (399 bp); 4, ga (1020 bp); 5, potD (981 bp); 6, mnsod (639 bp); 7, ygiw (705 bp); 8, gpd (1503 bp); 9, abpE (987 bp); 10, queA (1086 bp); 11, tex (2300 bp); 12, Hutz (540 bp); 13, qseB (672 bp); 14, crp (672 bp); 15, omp26 (804 bp); 16, clpp (582 bp); 17, Hutz (540 bp); 18, kefA (1365 bp). (B) Purification of the selected antigens. M, marker; 1, before IPTG induction; 2, after IPTG induction; 3, supernatant; 4, cell pellets; 5, flow-through; 6, elution by 20 mM imidazole; 7, elution by 50 mM imidazole; 8, elution by 500 mM imidazole. Additionalfile5.tif Additional file 5: Tertiary structures and molecular docking of VacJ, PtsG, and PalA with TLR2, MHC II and SLA I. (A) Predicted tertiary structures of VacJ, PtsG, GAPDH, and PalA by AlphaFold2. (B) Molecular docking of each antigen with immune receptors TLR2, MHC II and SLA I using the ClusPro 2.0 server. The dashed box denotes the docking region of the complex and the specific amino acid residues involved in docking. Additionalfile6.tif Additional file 6: Immune simulation of VacJ, PtsG, GAPDH, and PalA by C-ImmSim server. (A) The immunoglobulin levels; (B) B cell population; (C) Tc cell population; (D) TH cell population; (E) Cytokine levels in response to vaccination. Additionalfile7.tif Additional file 7: Identification of purified recombinant His-tagged antigens Vacj, PalA, GAPDH, and Ptsg by Western blot using anti-His tag primary antibody. M, marker. PA, PalA, 34.5 kDa. PT, PtsG, 30 kDa. VA, Vacj. GA, 43 kDa, GAPDH, 41 kDa. Additionalfile8.tif Additional file 8: Changes in body temperature upon immunization with or without the cocktail vaccine. Body temperatures were recorded every 12h for a total of five days. Additionalfile9.docx Additional file 9: Clinical symptoms of the piglets after challenge by GPS5. Additionalfile10.tif Additional file 10: Gross necropsy observations of the thorax and enterocoelia after GPS5 challenge in piglets. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8593719","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":580064828,"identity":"71bedf48-e7dc-434b-8ddf-69d31cccaa88","order_by":0,"name":"Fengyang Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYBAC9gYog4+Z+YDBAxDrAAEtPDAFbMxsCQYJpGlh4DFgIE4Le+/h1zw1d+za2Hk+FCS2Mcjx3Uhg/FyATwvPuTRrnmPPktuYeTcYALUYS95IYJaegUeLvUSOmTEP2+FkNqiWxA03EtiYefDZIv8GqOUfSAvPA5CWesJaJHiMH/O2HbYDKQNpSTAgqIUnx4xxbt9hoDI2A4OEcxKGM888bJbGq4X9jPGHN98O2/PzH35m8KHMRp7vePLBz/i0AAGbFFBBYgOQYcDAIAEUYGzAr4GBgfnjD2DIgRgPCCkdBaNgFIyCkQkAUZxGz6L8R7kAAAAASUVORK5CYII=","orcid":"","institution":"Jilin University","correspondingAuthor":true,"prefix":"","firstName":"Fengyang","middleName":"","lastName":"Li","suffix":""},{"id":580064829,"identity":"1fc241e6-44e0-49ec-94b0-0a292296118e","order_by":1,"name":"Yan Gong","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Gong","suffix":""},{"id":580064830,"identity":"8e1327c2-9aa9-4812-b746-006df5de5395","order_by":2,"name":"Ziheng Li","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Ziheng","middleName":"","lastName":"Li","suffix":""},{"id":580064831,"identity":"bc644238-91e4-4a9c-97e8-f9610f437a63","order_by":3,"name":"Zhen Wang","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Wang","suffix":""},{"id":580064832,"identity":"9ce97d29-7d50-44c8-b043-5c22ac5d0ec0","order_by":4,"name":"Zengshuai Wu","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Zengshuai","middleName":"","lastName":"Wu","suffix":""},{"id":580064833,"identity":"227ed533-b09a-4b7b-afd0-f7c222f3297b","order_by":5,"name":"Di Zhang","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Di","middleName":"","lastName":"Zhang","suffix":""},{"id":580064834,"identity":"5f162997-f31c-491f-ad18-e00e94431662","order_by":6,"name":"Peng Zhang","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Zhang","suffix":""},{"id":580064835,"identity":"0e7cc2a1-2248-448e-b238-2c1f98dacb9a","order_by":7,"name":"Zhichao Lu","email":"","orcid":"","institution":"Yangtze University","correspondingAuthor":false,"prefix":"","firstName":"Zhichao","middleName":"","lastName":"Lu","suffix":""},{"id":580064836,"identity":"f838d930-8a29-41e4-88b6-6832579d5c23","order_by":8,"name":"Hong Chu","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Chu","suffix":""},{"id":580064837,"identity":"566d287c-dddf-47b4-bf8c-f2b35dd01219","order_by":9,"name":"Kaixin Zhang","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Kaixin","middleName":"","lastName":"Zhang","suffix":""},{"id":580064838,"identity":"6719b574-5121-4735-8539-be368647b9c1","order_by":10,"name":"Na Li","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Na","middleName":"","lastName":"Li","suffix":""},{"id":580064839,"identity":"6bd9dc9f-d6c2-428b-8e5d-1edf5d06f4dc","order_by":11,"name":"Liancheng Lei","email":"","orcid":"","institution":"Jilin University","correspondingAuthor":false,"prefix":"","firstName":"Liancheng","middleName":"","lastName":"Lei","suffix":""}],"badges":[],"createdAt":"2026-01-13 15:23:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8593719/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8593719/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101397959,"identity":"83a0abf7-a231-4d50-b503-15e9a32639de","added_by":"auto","created_at":"2026-01-29 09:38:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11137798,"visible":true,"origin":"","legend":"\u003cp\u003eSelection of the antigens with high immunoprotective efficacy in mice. (A) Schematic representation of the immunization program of mice. (B) Detection of the production of antigen-specific antibodies in mice serum at 14 and 28 days after immunization by ELISA. Different groups of mice (n=10) were immunized with 100 μg antigens mixed with 10% Gel-01 adjuvant by multi-point subcutaneous injection. The immunization was administered every two weeks for a total of two doses. (C-D) Survival curve of mice upon challenge by GPS4 (C) and GPS5 (D). Mice were challenged with GPS4 (4×10\u003csup\u003e9\u003c/sup\u003e CFU) and GPS5 (2.4×10\u003csup\u003e9\u003c/sup\u003e CFU) by intraperitoneal injection after two weeks of the second dose of immunization. Animal death was recorded every 24 h for one week. Data were expressed as mean ± SD and analyzed using two-way ANOVA (B). ns, no significance, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/10a0775e9ef150df9de825af.png"},{"id":101397793,"identity":"48f1ab39-92e8-4aca-97d1-bd8f908c4dd4","added_by":"auto","created_at":"2026-01-29 09:37:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":16569963,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking of selected antigens with mouse TLR2, MHC II and SLA I. (A) The dashed box denotes the docking region of the complex and the specific amino acid residues involved in docking. (B) Detection of the antigenicity of the purified antigens with GPS4- and GPS5-positive antiserums. M, marker (Thermo Scientific, 26616). Expected size: VacJ, 43 kDa; GAPDH, 41 kDa; PtsG, 30 kDa; PalA, 35 kDa.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/9758daf33a92735a96ad7ec8.png"},{"id":101398233,"identity":"8de034de-44f6-4904-9454-1d95f3210f74","added_by":"auto","created_at":"2026-01-29 09:40:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7965328,"visible":true,"origin":"","legend":"\u003cp\u003eScreening of optimal antigen combination and ratio for the cocktail vaccine. (A-D) Detection of the production of antibodies specific to PtsG (A), VacJ (B), GAPDH (C), PalA (D) in mice serum at 14 and 28 days after immunization by ELISA. Different groups of mice (n=10) were immunized with 100 μg antigen combinations mixed with 10% Gel-01 adjuvant by multi-point subcutaneous injection. The immunization was administered every two weeks for a total of two doses. (E) Survival curve of mice upon challenge with GPS4 (4×10\u003csup\u003e9\u003c/sup\u003e CFU) and GPS5 (2.4×10\u003csup\u003e9\u003c/sup\u003e CFU) by intraperitoneal injection after two weeks of the second dose of immunization. Animal death was recorded every 24 h for one week. Data were expressed as mean ± SD and analyzed using two-way ANOVA (A-D, F). ns, no significance, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/58179e0f4407963dce98a24c.png"},{"id":101397727,"identity":"92d25aa5-64d8-48c9-b170-599a02700588","added_by":"auto","created_at":"2026-01-29 09:36:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":11854711,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening of optimal immune adjuvants.\u003c/strong\u003e (A) Detection of the production of antigen-specific antibodies in mice serum at 14 and 28 days after immunization\u003cstrong\u003e \u003c/strong\u003eby ELISA. Mice (n=10) were immunized with 100 μg antigens (PtsG:VacJ:GAPDH=1:1:1) mixed with different adjuvants including Montanide\u003csup\u003eTM\u003c/sup\u003e ISA201, ISA206, ISA563, Gel-01, aluminum hydroxide, and white oil (OIW). The immunization was administered every two weeks for a total of two doses. (B-C) Survival curve (B) and body weight changes (C) of mice after challenge with GPS4 (4×10\u003csup\u003e9\u003c/sup\u003e CFU) and GPS5 (2.4×10\u003csup\u003e9\u003c/sup\u003e CFU) by intraperitoneal injection. Animal death and body weight was recorded every 24 h for one week. (D) Assessment of bacterial loads in key organs of mice after GPS4 and GPS5 challenge. Data were expressed as mean ± SD and analyzed using two-way ANOVA (A, C, D). ns, no significance, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/7209db9af6e3c38268b9f558.png"},{"id":101398271,"identity":"343a435e-3917-40c3-bb33-2cea80c28f1c","added_by":"auto","created_at":"2026-01-29 09:40:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":24005885,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of host cellular and humoral immune responses following immunization of the cocktail vaccine. (A) Assessment of spleen index at 28 days after immunization. Three representative photographs are shown. (B-C) Measurement of lymphocyte proliferation by CCK-8 assay (B) and the transcriptional levels of \u003cem\u003eIl2\u003c/em\u003e, \u003cem\u003eIl4\u003c/em\u003e and \u003cem\u003eIfnγ\u003c/em\u003e by qRT-PCR (C) after incubation with VA+PT+GA (10 μg, 1:1:1) for 48 h. (D) Analysis of B cells by flow cytometry at 28 days after immunization. (E) Detection of the production of antigen-specific antibodies in mice serum by ELISA. The antibody levels were monitored at 0, 2, 4, 6, 8, 9, 10, 11 weeks after the second immunization. (F) Bactericidal analysis of the antigen-specific antibodies against GPS4 (above) and GPS5 (bellow). (G) Analysis of the proportion of CD3\u003csup\u003e+\u003c/sup\u003e, CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells by flow cytometry. Data were expressed as mean ± SD and analyzed using unpaired t test (A-D, F-G) and one-way ANOVA (E). ns, no significance, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/52ef2a68687da158c2402da6.png"},{"id":101398515,"identity":"6f681d12-85dc-480b-b5db-c7a2a7c54175","added_by":"auto","created_at":"2026-01-29 09:41:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":81657257,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of pathological lesions and inflammatory responses in immunized mice following GPS challenge. (A-B) Gross necropsy (A) and histopathological examination (B) of spleen, lung and liver of mice with or without immunization. Spleen, top left; lung, top right; liver, bottom. NC, PBS+Adjuvant, no GPS challenge; Challenge, GPS4 or GPS5 challenge; Vaccine, immunized with the cocktail vaccine; Inactivated, immunized by inactivated vaccine. (C) Assessment of bacterial loads in spleen, lung, liver and blood of mice after GPS4 and GPS5 challenge. (D) Detection of transcription levels of proinflammatory cytokines in lung tissue after GPS4 and GPS5 challenge. Data were represented as fold change. Data were expressed as mean ± SD and analyzed using unpaired t test (C) and two-way ANOVA (D). ns, no significance, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, and ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/40da53830272b62da6123bf1.png"},{"id":101397960,"identity":"2fa2b71f-c250-4323-bc7d-d58676a46083","added_by":"auto","created_at":"2026-01-29 09:38:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":13915978,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of the immunoprotective effect of the cocktail vaccine in piglets. (A) Schematic representation of the immunization program of piglets. (B) Detection of antigen-specific antibodies in piglets serum at 21 and 35 days after immunization by ELISA. Different groups of piglets (n=3) were immunized with 1.5 mg antigens mixed with 10% Gel-01 adjuvant by intramuscular injection in neck. (C) Body temperature of piglets upon challenge by GPS5 (4×10\u003csup\u003e10\u003c/sup\u003e CFU) via intraperitoneal and intranasal injection after two weeks of the second dose. Animal death and clinical symptoms were recorded every 24 h for one week. (D-E) Gross necropsy (D) and histopathological examination (E) of spleen, lung and liver of piglets with or without immunization. (F) Assessment of bacterial loads in spleen, lung, liver and blood of piglets after GPS5 challenge. Data were expressed as mean ± SD and analyzed using two-way ANOVA (B, C, F). ns, no significance, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/bec244bc994e73b89af51eca.png"},{"id":102298356,"identity":"8c44a536-25a0-4883-9872-0c36a4ca343f","added_by":"auto","created_at":"2026-02-10 10:37:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":97857825,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/e0c4c0dc-c960-4c85-9b9d-994c5ead7608.pdf"},{"id":101398581,"identity":"493caf6e-b026-434c-a517-26107f1f73a2","added_by":"auto","created_at":"2026-01-29 09:42:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20605,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: Bacterial strains and plasmids used in this study.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/a5adb494d429ef72849c7c0e.docx"},{"id":101348785,"identity":"b0dd78b7-5ca6-42d5-ab1a-8159a4ac60b5","added_by":"auto","created_at":"2026-01-28 18:05:01","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2: Primers used in this study.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Additionalfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/75ba5c2a790fe70809056e2c.docx"},{"id":101398176,"identity":"a3e6ceb8-e106-4bf2-a2b2-f8e7205e1279","added_by":"auto","created_at":"2026-01-29 09:39:57","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17809,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 3: Prediction of antigenicity of the selected seventeen antigens using VaxiJen V2.0 server.\u003c/p\u003e","description":"","filename":"Additionalfile3.docx","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/0e4a061bd8588c228f45d525.docx"},{"id":101398062,"identity":"ed593782-5a9f-4b2b-9e58-8cd8a7551748","added_by":"auto","created_at":"2026-01-29 09:39:23","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":8303436,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 4: Antigen cloning and purification. (A) Cloning of the coding sequences of the selected antigens by PCR. M, marker; 1, \u003cem\u003eva\u003c/em\u003e (687 bp); 2, \u003cem\u003ept\u003c/em\u003e (366 bp); 3, \u003cem\u003epa\u003c/em\u003e (399 bp); 4, \u003cem\u003ega\u003c/em\u003e (1020 bp); 5, \u003cem\u003epotD\u003c/em\u003e(981 bp); 6, \u003cem\u003emnsod\u003c/em\u003e (639 bp); 7, \u003cem\u003eygiw\u003c/em\u003e (705 bp); 8, \u003cem\u003egpd\u003c/em\u003e(1503 bp); 9, \u003cem\u003eabpE\u003c/em\u003e (987 bp); 10, \u003cem\u003equeA\u003c/em\u003e (1086 bp); 11, \u003cem\u003etex\u003c/em\u003e(2300 bp); 12, \u003cem\u003eHutz\u003c/em\u003e (540 bp); 13, \u003cem\u003eqseB\u003c/em\u003e (672 bp); 14, \u003cem\u003ecrp\u003c/em\u003e(672 bp); 15, \u003cem\u003eomp26\u003c/em\u003e (804 bp); 16, \u003cem\u003eclpp\u003c/em\u003e (582 bp); 17, \u003cem\u003eHutz\u003c/em\u003e(540 bp); 18, \u003cem\u003ekefA\u003c/em\u003e (1365 bp). (B) Purification of the selected antigens. M, marker; 1, before IPTG induction; 2, after IPTG induction; 3, supernatant; 4, cell pellets; 5, flow-through; 6, elution by 20 mM imidazole; 7, elution by 50 mM imidazole; 8, elution by 500 mM imidazole.\u003c/p\u003e","description":"","filename":"Additionalfile4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/9e338e04cd28688a18c380c9.tif"},{"id":101398481,"identity":"99b5868d-512c-4493-8516-22ff51c73a55","added_by":"auto","created_at":"2026-01-29 09:41:47","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":9262984,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 5: Tertiary structures and molecular docking of VacJ, PtsG, and PalA with TLR2, MHC II and SLA I. (A) Predicted tertiary structures of VacJ, PtsG, GAPDH, and PalA by AlphaFold2. (B) Molecular docking of each antigen with immune receptors TLR2, MHC II and SLA I using the ClusPro 2.0 server. The dashed box denotes the docking region of the complex and the specific amino acid residues involved in docking.\u003c/p\u003e","description":"","filename":"Additionalfile5.tif","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/5d414b6d7b6860f6016ea9d4.tif"},{"id":101398385,"identity":"c9e32563-c596-488a-a1c2-aa5a2e1e7eb0","added_by":"auto","created_at":"2026-01-29 09:41:16","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":8305304,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 6: Immune simulation of VacJ, PtsG, GAPDH, and PalA by C-ImmSim server. (A) The immunoglobulin levels; (B) B cell population; (C) Tc cell population; (D) TH cell population; (E) Cytokine levels in response to vaccination.\u003c/p\u003e","description":"","filename":"Additionalfile6.tif","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/2dc1868771fbd8c6879ea3ba.tif"},{"id":102294838,"identity":"795cc71c-0a85-4a1a-80cd-e72e75390828","added_by":"auto","created_at":"2026-02-10 09:59:52","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1705624,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 7: Identification of purified recombinant His-tagged antigens Vacj, PalA, GAPDH, and Ptsg by Western blot using anti-His tag primary antibody. M, marker. PA, PalA, 34.5 kDa. PT, PtsG, 30 kDa. VA, Vacj. GA, 43 kDa, GAPDH, 41 kDa.\u003c/p\u003e","description":"","filename":"Additionalfile7.tif","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/6270160a49b356de443ac95c.tif"},{"id":101398275,"identity":"6a2bbd67-e7ed-4864-b871-f954e32d47fc","added_by":"auto","created_at":"2026-01-29 09:40:37","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":917624,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 8: Changes in body temperature upon immunization with or without the cocktail vaccine. Body temperatures were recorded every 12h for a total of five days.\u003c/p\u003e","description":"","filename":"Additionalfile8.tif","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/bc05f008024417e58df11018.tif"},{"id":101397783,"identity":"7f269d0e-f916-4e32-9384-82d640805039","added_by":"auto","created_at":"2026-01-29 09:37:03","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":19169,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 9: Clinical symptoms of the piglets after challenge by GPS5.\u003c/p\u003e","description":"","filename":"Additionalfile9.docx","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/8452f8d5df2545c35be7767a.docx"},{"id":101751356,"identity":"ba98888e-d32b-45fb-b9c2-149bddf17cad","added_by":"auto","created_at":"2026-02-03 10:19:38","extension":"tif","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":22274916,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 10: Gross necropsy observations of the thorax and enterocoelia after GPS5 challenge in piglets.\u003c/p\u003e","description":"","filename":"Additionalfile10.tif","url":"https://assets-eu.researchsquare.com/files/rs-8593719/v1/21f33889a4c46c891cab6bf2.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development a cross-protective subunit cocktail vaccine against diverse serotypes of Glaesserella parasuis infection in pigs","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePorcine Gl\u0026auml;sser's disease is a swine-specific bacterial infection that caused by \u003cem\u003eGlaesserella parasuis\u003c/em\u003e (GPS) and characterized by meningitis, polyserositis, arthritis, and pneumonia [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. GPS primarily affects young pigs aged from 2 weeks to 4 months, particularly those at 5\u0026ndash;8 weeks of age. Epidemiological investigations have demonstrated that the incidence rate of this disease generally ranges from 10% to 15%, and in severe outbreaks, the mortality rate can escalate to 50%, thereby posing a severe threat to the health of piglets and fattening pigs [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. To date, 15 serotypes of GPS have been identified, among which serotypes 4 and 5, are the predominant circulating strains in China, followed by serotypes 12, 13, and 14 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Notably, GPS frequently engages in co-infections with other viral and bacterial pathogens, especially respiratory pathogens including porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), \u003cem\u003eStreptococcus suis\u003c/em\u003e (SS) and \u003cem\u003eActinobacillus pleuropneumoniae\u003c/em\u003e (APP), leading to increased morbidity and mortality and substantial losses to swine industries that hinders sustainable development [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, vaccination remains the primary strategy for preventing swine Gl\u0026auml;sser's disease, with available vaccines predominantly categorized into inactivated vaccines and subunit vaccines. Among these, inactivated vaccines have been commercialized and demonstrate favorable protective efficacy against same serotype. However, due to the diverse prevalent serotypes of GPS, commercial inactivated vaccines exhibit limited or even no cross-protection against different serotypes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Additionally, they have many problems such as insufficient safety, short protection time, and poor immune effect. Therefore, it is urgent to develop a vaccine with robust cross-protection against multiple serotypes of GPS infections.\u003c/p\u003e \u003cp\u003eSubunit vaccines are composed of immunoprotective components derived from microbial secretory proteins or outer membrane proteins and offer advantages of high safety and minimal side effects [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Of note, only two bivalent subunit vaccines (SS-GPS and PCV2-GPS, both of which use the phenylalanine ammonia-lyase family protein PalA as the primary antigen) were approved for market use [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. A variety of subunit vaccines based on different antigens such as pili subunit PilA [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], transferrin-binding proteins TbpA and TbpB [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], trimeric autotransporter VtaA [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], polyamine transport protein PotD [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], lipoprotein VacJ [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and glyceraldehyde-3-phosphate dehydrogenase GAPDH [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], have been assessed in laboratory. However, using these antigens alone have been proven to provide only partial protection or no cross-protection against GPS infection. Thus, identification of novel antigens with high immunogenicities and combination of two or more of these antigens should be employed to improve the immune efficacy of subunit vaccines.\u003c/p\u003e \u003cp\u003eIn this study, a multi-antigen subunit cocktail vaccine was successfully developed after systematic screening of \u003cem\u003ein-silico\u003c/em\u003e immunogenicity, protective efficacy, optimal adjuvant and antigen ratio in a murine model of GPS infection. This vaccine exhibited prominent immunogenicity, as it elicited robust humoral and cellular immune responses both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, and conferred effective cross-protection against GPS4 and GPS5 infections in mice. Notably, the vaccine also demonstrated favorable immune protective efficacy against GPS infection in piglets. Collectively, these results suggested that the subunit cocktail vaccine established in this study is a promising agent for the prevention and control of porcine Gl\u0026auml;sser's disease.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains and growth conditions\u003c/h2\u003e \u003cp\u003eAll strains used in this study are listed in Additional file 1. \u003cem\u003eE. coli\u003c/em\u003e strains were cultured in Luria-Bertani (LB; Becton Dickinson) liquid medium or on LB solid agar plates containing ampicillin (100 \u0026micro;g/mL) or kanamycin (50 \u0026micro;g/mL). GPS moderate virulent serotype 4 (GPS4) and high virulent serotype 5 (GPS5) strains were kindly provided by Prof. Lei Wang (Henan Institute of Science and Technology, Xinxiang, China). GPS strains were cultivated in Brain Heart Infusion (BHI; Hopebio) medium supplemented with 5% horse serum and 20 \u0026micro;g/mL NAD (BHI+) or plated on BHI\u0026thinsp;+\u0026thinsp;agar plates. All cultures were incubated at 37\u003csup\u003eo\u003c/sup\u003eC with 5% CO\u003csub\u003e2\u003c/sub\u003e if not specified.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntigen selection\u003c/h3\u003e\n\u003cp\u003eIn this study, the antigenicity of major antigens of GPS were predicted using VaxiJen V2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html\u003c/span\u003e\u003cspan address=\"https://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The proteins sequences of these antigens were retrived from NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/genbank/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/genbank/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The proteins with good antigenicity (\u0026ge;\u0026thinsp;0.4) were chose for further anslysis. Finally, seventeen GPS antigens including PotD (GenBank: ACL31786.1), MnSOD (GenBank: ACL32450.1, 618 bp), GPD (GenBank: ACL32465.1), YgiW (GenBank: ACL33049.1), Omp26 (GenBank: ACL32860.1), QseB (GenBank: ACL33513.1), GAPDH (GenBank: ACL31707.1), TEX (GenBank: ACL32154.1), PalA (GenBank: ACL31779.1), ApbE (GenBank: ACL31801.1), PtsG (GenBank: ACL33546.1), HutZ (GenBank: ACL32674.1), KefA (GenBank: ACL32066.1), VacJ (GenBank: ACL33700.1), ClpP (GenBank: ACL33474.1), CRP (GenBank: ACL33503.1), and QueA (GenBank: ACL32317.1) were selected.\u003c/p\u003e\n\u003ch3\u003eDNA manipulation\u003c/h3\u003e\n\u003cp\u003eThe transmembrane regions and the signal peptides of the fourteen antigens were predicted using the TMHMM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/TMHMM-2.0/\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/TMHMM-2.0/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and SignalP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/SignalP-5.0/\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/SignalP-5.0/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), respectively [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Fragments without transmembrane regions and the signal peptides were amplified by PCR from the genomic DNA extracted using a gDNA isolation kit (Qiagen). The PCR products were digested with BamHI/XhoI (NEB) restriction enzymes and ligated into pET28a or pET32a vector using a Rapid DNA ligation kit (Roche Diagnostics). Inserted DNA sequences were confirmed by DNA sequencing and double digestion by BamHI/XhoI (NEB) restriction enzymes. All plasmids and primers used in this study are listed in Additional file 1 and Additional file 2, respectively.\u003c/p\u003e\n\u003ch3\u003eProtein purification\u003c/h3\u003e\n\u003cp\u003eThe constructed plasmids were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3). When grown to OD600\u0026thinsp;=\u0026thinsp;0.6\u0026ndash;0.8, 0.5 mM of IPTG was added to induce protein expression for 16 h at 16\u003csup\u003eo\u003c/sup\u003eC. Bacterial cells were disrupted by sonication and the supernatants were collected after centrifugation at 12,000 \u0026times;g for 30 min at 4\u003csup\u003eo\u003c/sup\u003eC. Protein purification was performed using high-affinity Ni-NTA resin (GeneScript) by gravity at a flow rate of 1 mL/min. Proteins were eluted using 500 mM imidazole and dialyzed subsequently against washing buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e pH 8.0, 300 mM NaCl, 20 mM imidazole) at 4\u003csup\u003eo\u003c/sup\u003eC. Protein concentration was measured by a BCA kit (Thermo Scientific) and stored in aliquots at -80\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\n\u003ch3\u003ePrediction of tertiary structures, molecular docking and immune simulation\u003c/h3\u003e\n\u003cp\u003eThe tertiary structures of PtsG, VacJ, PalA and GAPDH were predicted by AlphaFold2. The binding affinity between each antigen and immune receptors including TLR2 (PDB ID: 2z7x), MHC II (PDB ID: 5jlz), and swine leukocyte antigen SLA-1 (PDB ID: 3qq3) were assessed by molecular docking using the ClusPro 2.0 server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cluspro.bu.edu/home.php\u003c/span\u003e\u003cspan address=\"https://cluspro.bu.edu/home.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The docking results were visualized with Pymol and the interaction residues between the docked chains were analyzed using PDBsum (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/thornton-srv/databases/pdbsum/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/thornton-srv/databases/pdbsum/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The immune responses profile of each antigen were simulated use the C-ImmSim server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://kraken.iac.rm.cnr.it/C-IMMSIM/index.php\u003c/span\u003e\u003cspan address=\"https://kraken.iac.rm.cnr.it/C-IMMSIM/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The time steps of the three injections in the simulation phase were set to 1, 42, and 84, respectively. The standard time of one injection was 8 h, corresponding to a 14-day gap between immunizations. The simulation volume is 50 and the simulation step is 1050. All other parameters set to their default values.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunization and challenge of mice and piglets\u003c/h2\u003e \u003cp\u003eFor immunization of mice, two hundred and ten of 6-weeks old healthy female ICR mice (body weight 18\u0026thinsp;\u0026plusmn;\u0026thinsp;2 g, purchased from Changsheng Biotechnology Co., Ltd., China) were randomly divided into different groups (10 mice/group) and immunized with 100 \u0026micro;g antigens mixed with or without Gel-01 adjuvant (10%; SEPPIC, France), PBS, PBS plus Gel-01, inactivated vaccine (Kefuning, China) by multi-point subcutaneous injection on the mice back, respectively. The immunization was administered every two weeks for a total of two doses. For immunization of piglets, six of 4-weeks old healthy piglets (purchased from Zybio, China) were randomly divided into two groups (3 piglets/group) and immunized with 1.5 mg antigens mixed with Gel-01 adjuvant (10%; SEPPIC, France) and PBS plus Gel-01 by intramuscular injection in neck, respectively. The immunization was administered every three weeks for a total of two doses. Body temperature, mental status and feeding behaviors of piglets were recorded everyday after the first immunization for one week. Blood was collected from the tail vein of mice at 14 and 28 days after first dose or from anterior vena cava of piglets at 21 and 35 days after first dose to detect specific antibody levels by ELISA. Alternatively, blood was collected from the tail vein of mice at 0, 2, 4, 6, 8, 9, 10, and 11 weeks after the second dose to monitor the antibody levels by ELISA. After two weeks of the second dose, the mice were challenged with lethal dose of GPS4 (4\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU) and GPS5 (2.4\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU) by intraperitoneal injection. Animal death and body weight was recorded every 24 h for one week. Similarly, the piglets were challenged with GPS5 (4\u0026times;10\u003csup\u003e10\u003c/sup\u003e CFU) by intraperitoneal and intranasal injection after two weeks of the second dose. Animal death and clinical symptoms were recorded every 24 h for one week. Both piglets and mice were euthanized using CO\u003csub\u003e2\u003c/sub\u003e or if they lost 20% of maximum body weight for two consecutive days, were immobile, or were found moribund.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eELISA\u003c/h3\u003e\n\u003cp\u003eAn indirect ELISA was used to detect the specific antibody levels in animal serum. In brief, the antigens were diluted separately with coating buffer (0.1 mol/L Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e-NaHCO\u003csub\u003e3\u003c/sub\u003e, pH 9.6) to 1 \u0026micro;g/mL and coated on ELISA plate by 100 \u0026micro;L/well at 4\u003csup\u003eo\u003c/sup\u003eC overnight. After washing, 100 \u0026micro;L of 5% BSA was added to each well and sealed at 37\u003csup\u003eo\u003c/sup\u003eC for 1 h. Then, the sera were diluted 10 times and added to ELISA plate (100 \u0026micro;L/well). After incubation at 37\u003csup\u003eo\u003c/sup\u003eC for 1 h, goat anti-mouse IgG HRP (1:5000) was added to each well (100 \u0026micro;L/well) and incubated for another 1 h. The reaction was visualized by addition of TMB reagent (100 \u0026micro;L) and terminated by 2M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (50 \u0026micro;L). Absorbance was detected at OD450 nm.\u003c/p\u003e\n\u003ch3\u003eWhole blood killing\u003c/h3\u003e\n\u003cp\u003eGPS4 and GPS5 from the midlog growth phase were collected, centrifuged, washed twice by PBS and adjusted to 2\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU. Bacterial suspensions (10 \u0026micro;L) were mixed with 150 \u0026micro;L of mice blood with or without immunization and incubated for 2 h at 37\u003csup\u003eo\u003c/sup\u003eC. Te mixture was serially diluted and plated on BHI\u0026thinsp;+\u0026thinsp;agar plates for cell counting. Bacterial survival was calculated as follows: (1 - recovered CFU/CFU in the original inoculum) \u0026times; 100%.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eIdentification and the antigenicity of the purified proteins VacJ, PtsG, GAPDH, and PalA were assessed by SDS-PAGE and Western blot using anti-His antibody and GPS4 or GPS5 antibody-positive serum, respectively. The proteins were boiled in SDS sample buffer at 95\u003csup\u003eo\u003c/sup\u003eC for 10 min and then separated on 12.5% SDS-PAGE gels. After electrophoresis, gels were transferred to the PVDF membrane (Millipore) and blocked with 3% BSA overnight. After washing in TBST, the membranes were incubated with rabbit anti-His (1:3000; Abconal, AE068) or GPS antibody-positive serum at room temperature for 2 h. After washing in TBST, the membranes were incubated with goat anti-rabbit HRP-IgG (1:5000; Abconal, AS014) for another 1 h at room temperature. The targeted proteins were visualized using ECL reagent (Millipore) under a luminescent imaging system (Tanon 5200 Multi).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eAfter 2 weeks of the second immunization, the mice were euthanized by inhalation of CO\u003csub\u003e2\u003c/sub\u003e. Mice spleen were collected, cut into pieces and digested in HBSS containing gelatinase A (100 U/mL) and DNase ( 20 \u0026micro;g/mL). Following washing with PBS, tissue samples were exposed to ice-cold RBC lysis buffer for 5 min. The cell pellets were gathered by centrifugation and suspended in fluorescent washing buffer (ThermoFisher) to a concentation of 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e/mL. Then, cells were labeled with fluorescent antibodies, including PerCP/Cy5.5 anti-mouse CD45 (Biolegend, 103132), FITC anti-mouse CD3 (Biolegend, 100203), APC anti-mouse CD4 (Biolegend, 100515), PE-Cy7 anti-mouse CD8 (Biolegend, 100722), APC anti-mouse CD19 (Biolegend, 152409) antibodies, and incubated in the dark for 30 min. After washing with PBS, cells were analyzed using a flow cytometer (CytoFLEX). Data were processed using FlowJo software (Version 10.4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological analysis and bacterial loads in organs\u003c/h2\u003e \u003cp\u003eThe Histopathological analysis was performed as described previously [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Briefly, lung, liver, and spleen tissues were fixed with 4% paraformaldehyde solution. After dehydration via graded ethanol, the tissues were cleared with xylene, embedded in paraffin and sliced (3\u0026ndash;5 \u0026micro;m) using a microtome. Then, the slices were mounted on slides, deparaffinized, rehydrated, and stained with Hematoxylin-Eosin (HE). After dehydration, slides were coverslipped for microscopic examination. To analysis bacterial loads in organs, equal weights of lung, liver, spleen, and blood samples were collected and homogenized with PBS (1 g/3 mL). Then, the samples were serial diluted and spotted on BHI\u0026thinsp;+\u0026thinsp;agar plates. The number of viable bacteria were counted after overnight inculation at 37\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLymphocyte proliferation assay\u003c/h2\u003e \u003cp\u003eSpleens were aseptically harvested from 6-week-old ICR mice euthanized by cervical dislocation, and single-cell suspensions were prepared after red blood cell lysis. Cells were resuspended in complete RPMI 1640 medium containing 10% inactivated fetal bovine serum and 1% penicillin-streptomycin. Isolated lymphocytes were seeded in a 96-well plate at a density of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e and treated with either PBS or VA\u0026thinsp;+\u0026thinsp;PT\u0026thinsp;+\u0026thinsp;GA (10 \u0026micro;g/well, 1:1:1). After incubation at 37 \u003csup\u003eo\u003c/sup\u003eC for 48 hours, cell viability was assessed using a CCK-8 kit (Beyotime, China) according to the manufacturer\u0026rsquo;s instructions. Absorbance was detected at 450 nm. Alternatively, total RNA was isolated from different groups of cells from a 6-well plate under a same procedure of stimulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation and qRT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using a tissue RNA isolation kit according to the manufacturer\u0026rsquo;s instructions (Invitrogen, USA). The extracted RNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Scientific) and its quality was assessed by PCR and gel electrophoresis. cDNA is synthesized from 1 \u0026micro;g of purified RNA using Prime Scrip RT Master Mix Kit (TaKaRa, China). Quantitative PCR was then performed with gene-specific primers using TB Green PCR Master Mix (TaKaRa, China) on a real-time PCR system (q225, Kubo Instruments, China). Data was analyzed using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. Amplification curves are analyzed to determine cycle threshold (Ct) values, with target gene expression normalized to reference gene (\u003cem\u003eGAPDH\u003c/em\u003e) for relative quantification. Primers are listed in Additional file 2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe experimental data were statistically analyzed using GraphPad Prism (version 9.0). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from three independent replicates. Differences between the mean values of normally distributed data were assessed using one-way and two-way ANOVA (Dunnett\u0026rsquo;s test). \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be significant and indicated by \"*\". \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01 was considered to be extremely significant and indicated by \"**.\u0026rdquo;\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSelection of GPS candidate antigens\u003c/h2\u003e \u003cp\u003eTo identify antigens for a effective subunit vaccine for GPS infection, we first selected seventeen potential or characterized antigens of GPS and evaluated their antigenicity using the VaxiJen V2.0 server at threshold of 0.4. Except PotD, GPD, and CRP, all other fourteen antigens retrived scores of higher than 0.4 (Additional file 3). So, these fourteen antigens were chose for further analysis. Next, the coding sequences of these antigens were cloned in either pET28a or pET32a expression vectors for protein expression (Additional file 4A). The expression profiles of the His-tagged recombinant proteins were analyzed by SDS-PAGE. Clear and single band was observed in the SDS-PAGE gel (Additional file 4B), suggesting all of these proteins were successfully purified with high purity. Subsequently, the immunoprotective effect of the recombinant proteins were preliminary evaluated in a murine infection model. Mice were subjected to a two-step immunization regimen, followed by challenge with GPS4 and GPS5 at 14 days after the secondary immunization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The results showed that the levels of specific antibodies in the mice\u0026rsquo;s serum were significantly increased after immunization at 14 and 28 days compared with the control group (PBS\u0026thinsp;+\u0026thinsp;Adjuvant) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Notebly, half of the antigens induced significant higher antibody levels after the second immunization than those after the first immunization. These findings indicate that these antigens have good immunogenicity that can trigger a strong humoral immune response after immunization in mice. Moreover, immunization of mice with PtsG, GAPDH, VacJ and PalA increased mice survival rate to 100%, 60%, 80% and 60%, respectively, upon infection with GPS4 compared with control group, while the survival rates for the immunization with other antigens were all below 60% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Upon infection with GPS5, immunization of mice with PtsG, GAPDH, VacJ, MnSOD and PalA increased mice survival rate to 60%, 60%, 100%, 60% and 60%, respectively, compared with control group, while the survival rates for the immunization with other antigens were all below 40% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Collectively, immunization with PtsG, GAPDH, VacJ and PalA conferred to better immunoprotective effect than other antigens in mice against both GPS4 and GPS5 infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, the tertiary structures of PtsG, GAPDH, VacJ and PalA and their binding affinities with host immune receptors TLR2, MHC II and swine leukocyte antigen (SLA I) were docked to assess the capability to induce immune response. Molecular docking results demonstrated that all the four antigens exhibited good binding affinity to both murine and porcine immune receptors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA; Additional file 5), suggesting a strong capability to activate innate immune cells. Correspondingly, a broad and robust activation of the immune system post-injection was observed by immune simulation using the C-ImmSim server (Additional file 6). Simulated immunization of these antigens not only increased the total number of T cells and B cells, but also upregulated cytokine responses and the level of antibodies, indicating these antigens are capable of inducing strong immune responses \u003cem\u003ein silico\u003c/em\u003e. Notably, detection of the antigenicity of the purified proteins illustrated that these antigens can be recognized by both GPS4- and GPS5-positive antiserums (Additional file 7; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), suggesting that they could possibly confer to cross-protection against both GPS4 and GPS5 infection in mice. Based on the observations above, these four antigens were chose for the construction of cocktail vaccine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment and optimization of antigens for cocktail vaccine\u003c/h2\u003e \u003cp\u003eTo further improve the immunoprotective effect of the antigens, we randomly combined two or three antigens into different groups and immunized mice with Gel-01 adjuvant. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, the levels of specific antibodies in the mice\u0026rsquo;s serum were significantly increased after immunization at both 14 and 28 days compared with the control group. Of note, although the level of PalA-specific antibodies increased significantly after the secondary immunization compared with that after the primary immunization, the magnitude of this increase was lower than that of the specific antibodies against the other three antigens (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Furthermore, compared with the control group, immunization regimens including VA\u0026thinsp;+\u0026thinsp;PT (VacJ and PtsG), VA\u0026thinsp;+\u0026thinsp;PT\u0026thinsp;+\u0026thinsp;GA (VacJ, PtsG and GAPDH), and VA\u0026thinsp;+\u0026thinsp;PT\u0026thinsp;+\u0026thinsp;PA (VacJ, PtsG and PalA) all conferred over 50% protection in mice following GPS4 challenge, with the VA\u0026thinsp;+\u0026thinsp;PT\u0026thinsp;+\u0026thinsp;GA exhibiting the optimal efficacy at 80% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Similarly, all the above groups provided more than 60% protection after GPS5 challenge, among which VA\u0026thinsp;+\u0026thinsp;PT\u0026thinsp;+\u0026thinsp;GA achieved complete protection (100%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Notably, the protective efficacy of VA\u0026thinsp;+\u0026thinsp;PT\u0026thinsp;+\u0026thinsp;GA immunization was significantly superior to that of the commercial inactivated vaccine group, which only conferred 20% and 40% protection against GPS4 and GPS5 challenges, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These results indicate VacJ, PtsG and GAPDH was the optimal assembly of antigens of cocktail vaccine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eScreening of optimal immune adjuvants\u003c/h2\u003e \u003cp\u003eAdjuvants are critical in improving vaccine efficacy, so we further screened the optimal adjuvants to identify those that best enhance the immunogenicity and protective efficacy of the cocktail vaccine. To this end, antigen assemble of VacJ, PtsG and GAPDH was emulsified with a number of commonly used adjuvants including Montanide\u003csup\u003e\u0026trade;\u003c/sup\u003e ISA201, ISA206, ISA563, Gel-01, aluminum hydroxide, and white oil (OIW) and immunonized mice. In terms of antibody responses, all adjuvant groups exhibited significantly higher antibody levels than the control group, regardless of primary or secondary immunization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Following the primary immunization, the traditional aluminum hydroxide adjuvant group induced the highest titers of GAPDH- and VacJ-specific antibodies compared to other adjuvant groups; however, these antibody levels declined after the secondary immunization, with a similar trend observed in the induction of PtsG-specific antibodies. In contrast, although the ISA201 and Gel-01 adjuvant groups did not elicit the highest antibody levels relative to other adjuvant groups, they showed increased GAPDH- and PtsG-specific antibody titers after the secondary immunization. Moreover, across all adjuvant-immunized groups, a certain level of protective efficacy against both GPS4 and GPS5 challenges was observed in mice. Notably, the ISA201 and Gel-01 adjuvant groups exhibited the most robust protective effects, with survival rates of 100% and 80% respectively following GPS4 challenge, and 80% and 100% respectively upon GPS5 challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Analysis of murine body weight changes revealed no significant difference in weight changes between the two groups following GPS4 challenge. However, after GPS5 challenge, mice in the ISA201 adjuvant group exhibited significantly more pronounced weight changes compared to those in the Gel-01 adjuvant group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Consistently, mice immunized with Gel-01 exhibited lower bacterial loads in the lung and liver compared to those in the ISA201 adjuvant group upon GPS5 challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Overall, these results indicate that Gel-01 was the optimal adjuvant of the cocktail vaccine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eImmunization of the cocktail vaccine enhanced both cellular and humoral immune responses\u003c/h2\u003e \u003cp\u003eNext, the effect of the cocktail vaccine on host cellular and humoral immune responses was evaluated both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. We observed that the splenic index in mice was significantly increased following vaccine immunization compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Splenic lymphocytes were isolated from mice, and stimulation with VA\u0026thinsp;+\u0026thinsp;PT\u0026thinsp;+\u0026thinsp;GA was found to not only significantly enhanced lymphocyte proliferation but also upregulated the transcriptional levels of cytokine genes \u003cem\u003eIl2\u003c/em\u003e, \u003cem\u003eIl4\u003c/em\u003e and \u003cem\u003eIfnγ\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). Considering the roles of these cytokines in lymphocyte differentiation and antibody production, we subsequently analyzed the proportion of B and T lymphocytes in the spleen and antibody titers in the peripheral blood post-immunization. Flow cytometric analysis revealed no significant increase in the overall B cell population in immunized mice relative to controls; however, serum antibody levels exhibited a highly significant elevation as early as 2 weeks post-immunization, gradually peaking between 4 to 6 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E). Subsequent to this peak, a gradual decline was observed; however, antibody levels remained substantially elevated, with such high titers persisting for a minimum of 11 weeks. Furthermore, results from the whole-blood bactericidal assay revealed that serum treatment from the vaccine-immunized group significantly reduced the colony counts of both GPS4 and GPS5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), indicating that the antibodies elicited by the cocktail vaccine exhibited bactericidal activity against GPS4 and GPS5. In terms of T cells, flow cytometric analysis showed that vaccine immunization not only induced a significant increase in the total number of T cells but also a higher ratio of CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e /CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), suggesting a possible Th2 type immune response elicited by vaccine. Collectively, these findings confirmed the vaccine's capacity to induce sustained humoral and cellular immunity in response to vaccine immunization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCocktail vaccination reduced pathological lesions and alleviated inflammatory responses in mice\u003c/h2\u003e \u003cp\u003eMajor organs including liver, lungs and spleen of mice post-challenge were collected for histopathological analysis. Gross necropsy examination showed that, mice challenged with GPS4 or GPS5 exhibited significantly swollen lungs with extensive hemorrhage, congestion, and reddish-brown inflammatory foci compared with the control group (PBS\u0026thinsp;+\u0026thinsp;Adjuvant) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The lungs in the inactivated vaccine group displayed mild enlargement and partial reddish-brown inflammatory foci, whereas only a few petechiae and congestion spots were observed in the cocktail vaccine-immunized group, with no lung swelling. Similar findings were noted in the spleen and liver of the mice. Histopathological examination showed that challenged with GPS led to drastic pathological changes in the lungs of the mice, including large amounts of inflammatory exudate, thickening and collapse of alveolar walls, dramatic atrophy of alveoli, extensive infiltration of red blood cells and inflammatory cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Immunization with the commercial inactivated vaccine alleviated the pathological changes to a certain degree, while the cocktail vaccine-immunized group displayed only faint histopathological lesions, with only limited red blood cell infiltration and focal alveolar wall thickening. Comparable pathological profiles were observed in the spleen and liver of the mice. Analysis of bacterial loads in these organs and blood revealed that immunized with the cocktail vaccine significantly reduced the total number of both GPS4 and GPS5 in lungs and blood, but not spleen and liver compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Moreover, the transcription levels of pro-inflammatory cytokine TNF-α, but not IL-1β and IL-6, was significantly reduced in lung by immunization with the cocktail vaccine after GPS4 challeng compared to the control group, while the transcriptional level of IL-6, but not TNF-α and IL-1β, was significantly reduced following GPS5 challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Taken together, these results demonstated that the cocktail vaccine can effectively protect mice against GPS infection by reducing organ lesions and alleviating inflammatory responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eCocktail vaccination protected piglets from GPS infection\u003c/h2\u003e \u003cp\u003eGiven that pigs constitute the target animals for GPS infection, subsequent investigations were conducted to assess the immunoprotective efficacy of the cocktail vaccine in piglets (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Vaccination was delivered via cervical intramuscular injection. Within 24 h post-vaccination, a transient elevation in body temperature was observed in the vaccinated piglets, yet this increase was not statistically significant compared to the control group (Additional file 8). After 24 h, the body temperature of the vaccinated piglets gradually decreased, eventually converging with that of the control group piglets. In terms of antibody responses, high levels of antigen-specific antibodies were elicited following the primary immunization, with titers significantly higher than those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). After the secondary immunization, although no significant changes were observed in the antibody levels against VacJ and PtsG, the antibody titer against GAPDH increased notably compared to that of the first immunization. These results demonstrated that the cocktail vaccine can induce a favorable humoral immune response in piglets.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTwo weeks after the 2nd immunization, piglets were challenged with GPS5 and clinical symptoms were observed for one week. Without vaccination, GPS5 challenge resulted in a dramatic increase in body temperature, which reached the highest at 40.2\u003csup\u003eo\u003c/sup\u003eC after 4 days post challenge (Additional file 9; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Moreover, the piglets were accompanied by heavy breathing, nasal discharge, depression, and a tendency to lie down. Piglets in the immunized group also showed elevated body temperature, which reached the highest at 40\u003csup\u003eo\u003c/sup\u003eC after 2days post challenge, reduced and remained stable thereafter (Additional file 9; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The piglets also exhibited breathing difficulties, nasal discharge, and depression after challenge; however, these symtoms were gradually disappeared after two days. Notebly, the body temperature of the immunized group was significantly lower than that of the control group at four days after challenge (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Gross pathological observations following GPS5 challenge revealed that piglets in the control group exhibited substantial fibrinous exudation within the thorax, accompanied by severe adhesions among the heart, lungs, and thoracic wall, which were typical characteristics of pleurisy (Additional file 10). The lungs were characterized by enlargement and hemorrhage, with a pale yellow membranous fibrinous exudate present on their surface; well-demarcated congested or hemorrhagic regions were detected in the anterior-inferior segments of the apical, cardiac, and diaphragmatic lobes, as well as in the accessory lobe of both lungs (Additional file 10; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Cross-sectional examination of the lungs demonstrated heterogeneous pigmentation and multiple necrotic foci within the lesioned areas. Similar pathological changes were also observed in liver and spleen. In contrast, the immunized group showed significantly attenuated overall pathological changes compared to the control group upon necropsy. No overt features of pleurisy or peritonitis were noted, and either an absence or only a minimal quantity of fibrinous exudate was observed on the lung surface or within the abdominal cavity. Additionally, there were no significant adhesions between the visceral organs and the thoracic or abdominal walls. Consistent with these findings, the HE-stained sections further showed that GPS5 challenge induced widened alveolar interstitium, alveolar atrophy, and presence of inflammatory exudate in the lungs; the boundary between the red and white pulp in the spleen was obscure with infiltration of inflammatory cells; hepatocytes were swollen and lost their cord-like arrangement in the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). In contrast, these pathological manifestations were markedly alleviated in the immunized group. Moreover, although there was no significant difference in the bacterial load in the liver, vaccine immunization significantly reduced the bacterial loads in the blood, lungs, and spleen of piglets compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePorcine Gl\u0026auml;sser's disease, an infectious disease caused by GPS, imposes a severe threat to swine health owing to its high mortality rate and incurs substantial economic losses in the global swine industry. Currently, vaccination stands as the most effective preventive measure against this disease; nevertheless, existing vaccines are commonly plagued by critical limitations including short protective durations and poor cross-protection efficacy across different GPS serotypes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In this study, an effective subunit cocktail vaccine against porcine Gl\u0026auml;sser's disease was successfully developed. Immunization of the vaccine not only conferred effective cross-protection against GPS4 and GPS5 infections in mice, but also demonstrated favorable immune protective efficacy against GPS infection in piglets, suggesting it can be used as a promising vaccine against porcine Glasser's disease in future.\u003c/p\u003e \u003cp\u003eGPS possesses dozens of virulence factors, many of which are membrane-associated or secreted antigens and are frequently regarded as the antigen candidates for subunit vaccine development. Among the seventeen GPS antigens selected in this study, a subset comprises well-characterized GPS virulence factors, including OMP26, YgiW, VacJ, PalA, GAPDH and MnSOD. Cumulative evidence from relevant studies has demonstrated that immunization with recombinant forms of these antigens elicited substantial immunoprotection across diverse animal models. For example, in a guinea pig model, recombinant VacJ and OMP26 conferred 67% and 83% protection against infection with GPS5 strain SH0165, respectively [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In murine models, recombinant GAPDH and PalA achieved protective rates of 75% and 80% against SH0165 challenge, respectively [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Notably, previous studies have reported that recombinant MnSOD and YgiW also exhibited cross-immunoprotective activity in mice [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, the cross-immunoprotective efficacy of these two recombinant proteins was suboptimal (both below 60%) in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and D). Besides well-characterized virulence factors, we also incorporated virulence factors with uncharacterized immunogenicity, including PtsG, ApbE, ClpP and QseB, as well as the proteins that are significantly upregulated during GPS infection, including Tex, HutZ, KefA, and QueA, all of which have been proposed as potential candidate antigens for GPS vaccine development [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Unexpectedly, only recombinant PtsG displayed robust cross-immunoprotective efficacy in murine models, whereas the immunoprotective efficacy of the remaining proteins was consistently below 60%. PtsG is a vital protein in the bacterial glucose phosphotransferase system (PTS\u003csup\u003eGlc\u003c/sup\u003e) and mainly in charge of glucose transport as part of the enzyme II complex. PTS\u003csup\u003eGlc\u003c/sup\u003e not only transports glucose, but also regulates various cellular activities including virulence, biofilm formation and metabolism [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. For example, deletion of \u003cem\u003eptsG\u003c/em\u003e in \u003cem\u003eBorrelia burgdorferi\u003c/em\u003e were unable to cause infection in mice [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. PtsG as well as other genes that involved in glycolysis are required for the replication of \u003cem\u003eSalmonella\u003c/em\u003e Typhimurium in macrophages within the \"Salmonella-containing vacuole\" (SCV) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In GPS, deletion of \u003cem\u003eptsG\u003c/em\u003e led to dramatic changes in metabolic and transcriptomic pathways, some of which were associated with bacterial virulence and biofilm formation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, conclusive experimental evidence addressing whether PtsG regulates the virulence of GPS and whether it qualifies as a antigen for GPS subunit vaccine remains elusive to date. In the present study, we demonstrated, for the first time, that PtsG possessed robust immunogenicity. Immunization of mice with recombinant PtsG alone or in combination with VacJ and GAPDH conferred effective protection against GPS challenge, thereby identifying PtsG as a promising candidate antigen for GPS vaccine development.\u003c/p\u003e \u003cp\u003eIn the context of suboptimal cross-protection against multiple serotypes following immunization with a single antigen, the strategy of combining two or more antigens represents an effective approach to enhance the protective efficacy [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Many studies have demonstrated that the combined use of distinct antigens is more effective in disease prevention compared to the use of a single antigen alone. For example, the SARS-CoV-2 multi-antigen vaccine that co-delivers S, M, and N antigens elicits more robust immune responses and superior protective efficacy compared to the single antigen [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Chen et al. demonstrated that co-administration of multi-antigen combinations afforded complete protection and elicited significantly higher titers of neutralizing antibodies than immunization with single antigen which provided only partial protection against vaccinia virus infection [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In this study, the multi-antigen combination VA\u0026thinsp;+\u0026thinsp;PT\u0026thinsp;+\u0026thinsp;GA exhibited potent cross-immunoprotective activity against distinct serotypes of GPS, conferring 80% protection against GPS4 and 100% protection against GPS5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-E). Importantly, the immunoprotective performance of the cocktail vaccine was superior to that of any individual antigen component. The multi-antigen strategy not only expands the serotype coverage of the subunit vaccine but also enhances its cross-protective efficacy, addressing the limitation of narrow serotype protection associated with single-antigen vaccines.\u003c/p\u003e \u003cp\u003eFurthermore, although the protective efficacy of the antigen combination VA\u0026thinsp;+\u0026thinsp;PT\u0026thinsp;+\u0026thinsp;PA was lower than that of the VA\u0026thinsp;+\u0026thinsp;PT\u0026thinsp;+\u0026thinsp;GA combination, it still conferred considerable protection against GPS4 and GPS5 infections (60% and 80%, respectively), suggesting GAPDH is more important than PalA for protection against GPS infection. GAPDH is a glycolytic enzyme that catalyzes the conversion of glyceraldehyde-3-phosphate to 1, 3 diphosphoglycerate [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Moreover, GAPDH is regarded as a suitable vaccine candidate for protection against bacterial diseases due to its role in microbial virulence [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. As a vaccine component, the immunoprotective efficacy of GAPDH has been confirmed in several bacterial pathogens, such as \u003cem\u003eListeria monocytogenes\u003c/em\u003e, \u003cem\u003eMycobacterium marinum\u003c/em\u003e, \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e and GPS [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Specifically, immunization with recombinant GAPDH was found to be protective both in murine and porcine model of GPS5 infection [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], but the immnoprotective efficacy aginst other serotypes of GPS infection was not evaluated. PalA is a peptidoglycan-associated outer membrane lipoprotein that shows highly homology to the P6 protein of \u003cem\u003eHaemophilus influenzae\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. P6 is a protective antigen against \u003cem\u003eH. influenzae\u003c/em\u003e induced meningitis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Notably, previous studies indicated that PalA showed deleterious affects on the Apx toxinbased subunit vaccine against APP infection by exacerbation of respiratory symptoms, higher mortality rate and severe lung damage [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Thus, it has been proposed that PalA is an immunogenic but not a protective antigen for the development of effective subunit vaccines against infections by APP [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. On the other hand, immunization with recombinant PalA was found to be protective in murine model of GPS5 infection [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Indeed, both of the two commercial bivalent subunit vaccines SS-GPS and PCV2-GPS utilized PalA as the primary antigen [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The drastically opposing protective effects of PalA across different bacterial genera needs to be further investigated.\u003c/p\u003e \u003cp\u003eAs a type of immune enhancer, adjuvants help reduce the number of immunizations and the amount of antigens, overcome antigen competition in combined vaccines and, importantly, modulate the type of induced immunity [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Moreover, many studies have confirmed that the type of adjuvant used in vaccine affect the immune response and protection efficacy of the vaccine against homologous challenge [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Consequently, we further conducted adjuvant screening following the confirmation of the antigen componants for the subunit cocktail vaccine. The results demonstrated that Montanide\u003csup\u003e\u0026trade;\u003c/sup\u003e Gel-01 and ISA201 exerted the most robust adjuvant activity in potentiating the immune responses induced by the subunit vaccine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). Montanide\u0026trade; Gel-01 is an aqueous polymeric adjuvant designed for aqueous type vaccines, while Montanide\u0026trade; ISA201 is a mineral oil based adjuvant that has been developed for the manufacture of Water-in-Oil-in-Water (W/O/W) emulsions [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In line with our results, it\u0026rsquo;s demonstrated that the adjuvant effect of Gel-01 was superior to that of mineral oil and aluminum hydroxide adjuvants for trivalent inactivated GPS serovars 4, 5 and 12 vaccines against Gl\u0026auml;sser's disease in terms of antibody titers and protective efficacy [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Similar conclusions were also observed for ISA201 adjuvanted foot-and-mouth disease vaccine [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Considering significant difference in body weight changes and bacterial loads in organs of mice after GPS5 challenge, Gel-01 was selected as the optimal adjuvant for the subunit cocktail vaccine in this study.\u003c/p\u003e \u003cp\u003eThe level of host immune response following immunization is one of the key indicators for evaluating immunogenicity of vaccines. The cocktail vaccine developed in this study induced robust humoral and cellular immune responses after immunization in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Interestingly, the proportion of CD4⁺ T cells after vaccine immunization was significantly higher than that of CD8⁺ T cells, and the induction of the transcriptional level of the Th2 cytokine IL-4 was dramatically stronger than that of the Th1 cytokine IFN-γ (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). IL-4 contributes to the proliferation and differentiation of B cells to produce IgG and may directly act on na\u0026iuml;ve CD4⁺ T cells during T-cell activation to induce Th2 cell differentiation [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Thus, these results suggested that the cocktail vaccine may be more inclined to induce a Th2 immune response rather than a Th1 immune response.\u003c/p\u003e \u003cp\u003eIn conclusion, a subunit cocktail vaccine was successfully developed against GPS infection in this study. The vaccine showed superior immunogenicity than inactivated vaccine and induced robust humoral and cellular immune responses both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Importantly, immunization of the vaccine not only conferred effective cross-protection against GPS4 and GPS5 infections in mice, but also demonstrated favorable immune protective efficacy against GPS infection in piglets. These results suggested that the subunit cocktail vaccine established in this study is a promising agent for the prevention and control of porcine Gl\u0026auml;sser's disease.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e Animal experiments in mice were approved by the Institutional Animal Care and Use Committee of Jilin University (Approval No: KT202507001), while experiments in piglets were approved by the the Institutional Animal Care and Use Committee of Jilin Zhengye Biologics Co., Ltd (Approval No: SC-2501-001). All animal experiments were conducted in accordance with Guidance on the Chinese Laboratory Animal Administration Act 1988. All mice were housed in the laboratory animal room and maintained on a 14/10 h light-dark cycle with food and water \u003cem\u003ead libitum\u003c/em\u003e. Informed consent was obtained from all individual participants included in the study.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interest\u003c/h2\u003e \u003cp\u003eThe authors report there are no competing interests to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Key Research and Development Project Program of China (No. 2022YFD1800905) and National Natural Science Foundation of China (No. 32102670).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e**Fengyang Li:** Conceptualization, Formal analysis, Methodology, Visualization, Funding acquisition, Supervision, Writing\u0026ndash;original draft, Writing\u0026ndash;review, and editing. **Yan Gong:** Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization. **Ziheng Li, Zhen Wang, Zengshuai Wu, Di Zhang, Peng Zhang, Zhichao Lu, Hong Chu, Kaixin Zhang:** Investigation. **Na Li:** Data curation, Resources. **Liancheng Lei:** Conceptualization, Methodology, Funding acquisition, Project administration, Resources, Supervision, Writing\u0026ndash;review, and editing.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe thank Prof. Lei Wang (Henan Institute of Science and Technology, Xinxiang, China) for providing GPS strains.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw data used in this article and the supplementary materials are available in figshare ( https://doi.org/10.6084/m9.figshare.30571994 ).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMacedo N, Rovira A, Torremorell M (2015) Haemophilus parasuis: infection, immunity and enrofloxacin. 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Fac Rev 10:71\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Subunit vaccine, Glaesserella parasuis, Glässer’s disease, PtsG, GAPDH, VacJ","lastPublishedDoi":"10.21203/rs.3.rs-8593719/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8593719/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGl\u0026auml;sser's disease caused by \u003cem\u003eGlaesserella parasuis\u003c/em\u003e (GPS) is a severe disease that leads to huge economic losses in the swine industry worldwide due to the poor cross-protective efficacy of existing vaccines. In this study, a multi-antigen cocktail subunit vaccine against GPS infection was developed. By \u003cem\u003ein-silico\u003c/em\u003e analyzing antigenicity and comparison of immunoprotective efficacy and induction of antibody titers in a murine model, the component of the cocktail vaccine was determined to be VacJ, PtsG and GAPDH, with Gel-01 as the optimal adjuvant. Immunization of the cocktail vaccine induced robust humoral and cellular immune responses both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Importantly, the vaccine not only conferred effective cross-protection against lethal-dose of GPS4 and GPS5 infections in mice, but also demonstrated favorable immune protective efficacy against GPS infection in piglets. Moreover, their immune sera significantly inhibited the growth of GPS4 and GPS5. Overall, these results suggested that the subunit cocktail vaccine established in this study is a promising agent for the prevention and control of porcine Gl\u0026auml;sser's disease.\u003c/p\u003e","manuscriptTitle":"Development a cross-protective subunit cocktail vaccine against diverse serotypes of Glaesserella parasuis infection in pigs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 18:04:56","doi":"10.21203/rs.3.rs-8593719/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1976f40a-3fd9-4157-99fc-3c13077c00e6","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T18:23:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-28 18:04:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8593719","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8593719","identity":"rs-8593719","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}