Epitope-Based Multimeric Subunit Vaccine (ATOMSSUISpenta) Confers Cross-Protection Against Streptococcus suis Infection

Epitope-Based Multimeric Subunit Vaccine (ATOMSSUISpenta) Confers Cross-Protection Against Streptococcus suis Infection | 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 Article Epitope-Based Multimeric Subunit Vaccine (ATOMSSUIS penta ) Confers Cross-Protection Against Streptococcus suis Infection Sun-Young Kim, Fengjia Chen, Woo-Sik Kim, Hyun Jung Ji, Min-Kyu Kim, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7215039/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 Streptococcus suis ( S. suis ) is a zoonotic pathogen that causes significant losses in the swine industry and serious invasive infections in humans. The high serotype variability and genomic diversity of S. suis have substantially limited the development of cross-reactive vaccines. Although recent advances in in silico prediction and database-driven antigen discovery have accelerated the development of protein-based vaccines, several studies have reported inconsistencies between predicted immunogenic profiles and the protective efficacy observed in animal models, emphasizing the importance of integrating computational design with experimental validation. In this study, we selected key antigens of S. suis based on previous experimental reports (HP0197, Fnbp, Sao, ScpB, and SLY) and analyzed their predicted T- and B-cell epitopes. For each antigen, we identified surface-exposed epitope regions (approximately 109–210 amino acids) through structural modeling or available PDB data. These regions were then assembled into a multimeric conjugated vaccine construct (ATOMSSUIS penta ) by optimizing based on predicted immunogenicity, solubility, and allergenicity profiles. As predicted by the in silico design, ATOMSSUIS penta elicited strong humoral immune responses against each of the five component antigens in the mouse model. Notably, the vaccine also induced robust Th1- and Th17-type cellular immune responses, which are known to be essential for effective opsonic and mucosal defense against S. suis infection. In the protection studies, ATOMSSUIS penta conferred significant protection against S. suis serotypes 2, 4, and 9, as demonstrated by improved survival rates and reduced bacterial burdens. These findings highlight the potential of ATOMSSUIS penta as a broadly protective subunit vaccine against S. suis and demonstrate the value of epitope-based multimeric design for targeting antigenically diverse Gram-positive pathogens. Biological sciences/Biotechnology Biological sciences/Computational biology and bioinformatics Biological sciences/Immunology Biological sciences/Microbiology S.suis gram-positive bacteria protein vaccine HP0197 Fnbp Sao ScpB SLY in silico Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Streptococcus suis ( S. suis ) is an important zoonotic pathogen that causes substantial economic losses in the swine industry and poses a serious public health threat due to its ability to cause severe invasive infections in humans 1,2 . S. suis was first identified in 1954 and has been known to cause meningitis, endocarditis, arthritis, and septicemia in post-weaned piglets 3,4 . Although human S. suis infections were initially considered rare and sporadic, first reported in Denmark in 1968 5,6 , a major outbreak occurred in 2005 in Sichuan Province, China, resulting in 215 confirmed cases and 38 deaths across 202 villages 7,8 . Southeast Asia has become the region with the highest incidence of human S. suis infections, largely associated with occupational or dietary exposure to pigs and pork products 9,10 . This unprecedented outbreak underscored the zoonotic potential of S. suis and marked the pathogen as an emerging threat to global public health. To date, 35 serotypes of Streptococcus suis have been identified based on immunogenic differences in their capsular polysaccharide antigens 1 . Although serotype 2 is recognized as the most prevalent and virulent serotype in both pig and human globally, effective control of S. suis infection remains challenging due to the regional variability in circulating serotypes 1,11 . For instance, serotypes 1, 1/2, 2, 3, 5, 7, and 14 are frequently reported in North America; serotypes 1/2, 2, 3, and 6 in South America; serotypes 2, 4, 7, and 9 in Europe; and serotypes 2, 3, and 4 in Asia 11 . The distribution of dominant serotypes continues to evolve over time and across geographic regions, complicating both diagnosis and prevention. Although antibiotics such as tetracyclines, beta-lactams, and macrolides are commonly used to treat S. suis infections in pigs 12,13 , the increasing prevalence of antimicrobial resistance, particularly to tetracyclines, macrolides, and lincosamides, has limited their effectiveness, emphasizing the need for alternative control strategies 13–17 . Consequently, considerable efforts have been directed toward developing cross-protective vaccine platforms capable of targeting multiple serotypes to enhance the global management of S. suis infections. Vaccination is considered a promising strategy to reduce antibiotic use and induce long-term immune memory for preventing infections 18–20 . However, inactivated S. suis vaccines, while widely used in pig farms, exhibit limited efficacy 21,22 . For example, Blouin et al. demonstrated that a formalin-inactivated S. suis serotype 2 vaccine failed to elicit protective antibody responses in piglets, and did not confer passive protection in a mouse challenge model 23 . Similarly, field trials using a licensed formalin-inactivated autogenous vaccine (serotype 7, strain 1750775) showed no induction of measurable immune responses in piglets and failed to transfer maternal immunity 24 . Live-attenuated S. suis vaccines have been investigated, with some temperature-sensitive and conditionally replicating mutants of S. suis serotypes 1/2, 1, 2, and 3, showing protection against homologous strains in mice 25,26 . However, other attenuated strains, including non-encapsulated or serum opacity factor-deficient mutants of serotype 2, failed to induce protective opsonizing antibodies in pigs 27,28 . Indeed, concerns remain regarding their safety in the field due to the potential for reversion to virulence through mutation or recombination. In addition, these serotype specific vaccines offer limited cross-protection due to their serotype-specific nature. As a result, the emergence of infections caused by non-vaccine serotypes may remain a major concern in vaccinated herds. Subunit vaccines provide several advantages over inactivated and live-attenuated vaccines, particularly in terms of safety, specific immune targeting, and standardized manufacturing 28–30 . In principle, subunit vaccines based on conserved antigens across S. suis serotypes can offer serotype-independent cross-protective immunity. Surface antigen one (Sao), suilysin (SLY), muramidase-released protein (MRP), and extracellular factor (EF) are examples of conserved proteins that have been widely studied as subunit vaccine candidates 31–33 . For instance, vaccination with a truncated Sao (Sao-L) showed cross-protective effects against serotypes 1, 2, and 7, suggesting the potential of conserved-antigen-based subunit vaccines as universal strategies against S. suis infections 31 . In recent years, additional surface-exposed proteins such as HP0197, fibronectin-binding protein (Fnbp), and C5a peptidase (ScpB) have also been evaluated for their immunogenicity and protective efficacy in animal models 34–36 . However, despite these promising findings, subunit vaccines face several limitations. Their production costs are generally higher than those of traditional whole-cell vaccines, and a single protein antigen often fails to induce sufficient protective immunity 37 . As a result, multicomponent subunit vaccines combining several antigens, are being actively explored to overcome these limitations and enhance the wide protective immune response 38 . However, the identification of protective antigens capable of eliciting robust and cross-protective immune responses remains a major challenge. To address this, a growing number of studies have employed in silico approaches that integrate computational immunology, structural biology, and systems vaccinology to accelerate antigen discovery and vaccine design 29,39–42 . These in silico pipelines commonly utilize predictive algorithms to identify T cell and B cell epitopes based on peptide binding affinity to MHC molecules, population coverage, and immunogenic potential 40,41,43,44 . In parallel, 3D structural modeling and molecular docking techniques are applied to evaluate surface accessibility, stability, and antigen-antibody interactions of candidate proteins 42,45 . In this study, we developed a novel multimeric subunit vaccine candidate, ATOMSSUIS penta , against S. suis , by integrating bioinformatic tools with known immunologically relevant antigens. The vaccine construct comprises five conserved proteins: HP0197, fibronectin-binding protein (Fnbp), surface antigen one (Sao), C5a peptidase (ScpB), and suilysin (SLY), which were selected based on their sequence conservation, surface localization, and predicted immunogenicity. Using an in silico approach, we analyzed the protein structure, solubility, and T and B cell epitopes to optimize the design of the multimeric antigen. The immunogenic potential of ATOMSSUIS penta was evaluated in a murine infection model, demonstrating its ability to elicit strong humoral and cellular immune responses. Importantly, the vaccine conferred cross-protective efficacy against multiple clinically relevant S. suis serotypes, highlighting its potential as a broadly protective vaccine platform. MATERIALS AND METHODS Ethics Statement All animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea Atomic Energy Research Institute (KAERI; approval no. IACUC-2021-05, IACUC-2022-08, IACUC-2023-07) and all procedures were performed in accordance with the veterinary standards of the KAERI Animal Care Center (RI-BIOMICS SPF Animal Facility). In mice infection model, mice were examined daily for clinical signs, including weight loss and general health condition. Animals that experienced a weight loss exceeding 25% of their baseline body weight, as well as those designated for scheduled tissue collection, were euthanized by carbon dioxide (CO₂) inhalation using a gradually filled chamber. Reagents Tryptic soy broth (TSB) and Luria-Bertani (LB) broth were purchased from Difco (Franklin Lakes, NJ, USA). RPMI-1640 medium, fetal bovine serum (FBS), and penicillin-streptomycin were obtained from Biowest (Nuaillé, France) and HyClone (Logan, UT, USA), respectively. Monophosphoryl lipid A (MPL), dimethyldioctadecylammonium (DDA) bromide, and BCA protein assay kit were sourced from Sigma-Aldrich (Saint Louis, MO, USA). Alhydrogel® was obtained from InvivoGen (San Diego, CA, USA), and Montanide™ ISA 1313 and Montanide™ Gel adjuvants from Seppic (Paris, France). The Live/Dead viability staining kit was purchased from Invitrogen (Carlsbad, CA, USA). GolgiStop, GolgiPlug, fixation/permeabilization solution, and the following antibodies were obtained from BD Biosciences (San Diego, CA, USA): PE-labeled anti-mouse IFN-γ, APC-labeled anti-mouse IL-5, APC-labeled anti-mouse TNF-α, and V450-labeled anti-mouse CD44. Additional antibodies, APC-Cy7-anti-mouse CD3e, Alexa488-anti-mouse CD4, PerCP-Cy5.5-anti-mouse CD8, PE-Cy7-anti-mouse IL-17A, and PE-Cy7-anti-mouse IL-2, were purchased from eBioscience (San Diego, CA, USA). HRP-conjugated anti-mouse IgM, IgG, IgG1, and IgG2a were from Southern Biotech (Birmingham, AL, USA). TMB substrate and stop solution (1N HCl) were obtained from Thermo Fisher Scientific (Waltham, MA, USA), and high-binding 96-well ELISA plates from Corning (Corning, NY, USA). Formaldehyde solution was purchased from JUNSEI (Tokyo, Japan), and H&E staining reagents from Agilent Dako (Santa Clara, CA, USA). Porcine whole blood was sourced from Innovative Research (Sarasota, FL, USA). All other chemical reagents were obtained from Sigma-Aldrich. Structure modeling and validation The 2D structure of ATOMSSUIS penta was modeled using PSIPRED v.4.0 46 and GOR IV 47 . PSIPRED employs position-specific scoring matrices for precise sequence homology identification, while GOR IV utilizes information theory and Bayesian statistics to provide complementary insights. AlphaFold3 48 was used for 3D structure prediction and the resulting model was further refined using the GalaxyRefine webserver 49 . Model validation was conducted using ProSA-web for Z-score validation 50 and Ramachandran plot analysis with PROCHECK to assess stereochemical properties 51 . The final 3D model was visualized with PyMOL3.1 to examine structural details (www.pymol.org). Purification of recombinant S. suis proteins Escherichia coli BL21(DE3) strains harboring pET28a plasmids encoding HP0197, Fnbp, Sao, ScpB, SLY, or the multimeric ATOMSSUISpenta construct were cultured in LB broth at 37 °C until the optical density at 600 nm (OD₆₀₀) reached 0.6-0.8. Protein expression was induced by the addition of 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Biosesang, Seongnam, Republic of Korea), followed by overnight incubation at 16 °C. After induction, bacterial cells were harvested by centrifugation at 7,000 rpm for 20 minutes at 4 °C, resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5; 0.2 M NaCl; 1 mM phenylmethylsulfonyl fluoride), and lysed by sonication for 10min (15s pulse on/off) at 40% amplitude using a Vibra-Cell Ultrasonic Processor VC-505 (Sonics & Materials, Inc., Newtown, CT, USA).. The lysates were clarified by centrifugation at 17,000 rpm for 30 minutes at 4 °C, and the resulting supernatant was applied to a nickel-nitrilotriacetic acid (Ni-NTA; Invitrogen, Carlsbad, CA, USA) affinity chromatography column. The column was washed with binding buffer (20 mM Tris-HCl, pH 7.5; 0.2 M NaCl; 30 mM imidazole), and the bound proteins were eluted with elution buffer containing 500 mM imidazole. Eluted proteins were concentrated, and their concentrations were quantified using a BCA Protein Assay Kit (Sigma-Aldrich).. Immunization and Bacterial Challenge Six-week-old female C57BL/6 mice were purchased from Orient Bio (Seongnam, South Korea). After a one-week acclimatization period, mice (n = 6 per group) were immunized twice at two-week intervals via subcutaneous (s.c.) injection with ATOMSSUIS penta (5, 10, or 20 µg) formulated with MPL (10 µg) and DDA (250 µg). For the protective efficacy studies, immunized mice (n = 6 per group) were challenged two weeks after the booster immunization with either 5 × 10⁷ or 1.5 × 10⁸ colony-forming units (CFU)/mouse of Streptococcus suis BAA-853 (serotype 2). Body weight and survival were monitored daily for 10 days post-challenge (dpc). For histopathological analysis and bacterial load determination, mice were challenged with 1 × 10⁸ CFU/mouse. At 2 dpc, the left lungs and brains were collected for hematoxylin and eosin (H&E) staining, and the right brain, right lung, and entire spleen were harvested for quantification of bacterial burden by CFU counting. Measurement of antibody responses and cytokine levels by ELISA Blood samples were collected from immunized mice two weeks after the booster dose, and serum was obtained by centrifugation at 5,000 rpm for 10 minutes at room temperature. To assess S. suis -specific antibody levels, heat-inactivated S. suis (10⁶ CFU/well) were immobilized onto 96-well ELISA plates. The wells were blocked with 5% skim milk in PBS, and serially diluted sera were added. After a 2-hour incubation, the wells were washed with PBST (0.05% Tween 20 in PBS), followed by the addition of horseradish peroxidase (HRP)-conjugated anti-mouse IgG, IgG1, or IgG2a antibodies. Following an additional 1-hour incubation and subsequent washing, plates were developed using TMB substrate solution, and the reaction was stopped with 2N sulfuric acid. Absorbance was measured at 450 nm using an Epoch 2 microplate reader (BioTek Instruments; Winooski, VT, USA). Spleens were harvested from mice two weeks after the booster immunization, and single-cell suspensions were prepared by mechanical dissociation through a 40 µm cell strainer in RPMI-1640 complete medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. Red blood cells were lysed using RBC lysis buffer for 5 minutes at room temperature. After cell number counting, 1×10 6 splenocytes per well were seeded in 96-well plates and stimulated with ATOMSSUIS penta or individual subunit proteins (20 µg/mL) for 72 hours. Supernatants were collected, and concentrations of IFN-γ, IL-5, and IL-17A were measured using ELISA kits (eBioscience; San Diego, CA, USA) according to the manufacturer’s instructions. Flow cytometry analysis of T cell cytokine profiles Single-cell suspensions were prepared from the spleens of booster-immunized mice as described above. After cell counting, 4 × 10⁶ cells per well were seeded in 48-well plates containing RPMI-1640 complete medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were stimulated with ATOMSSUIS penta or individual subunit proteins (20 μg/mL) in the presence of GolgiPlug and GolgiStop (1:2000 dilution) for 8 hours at 37 °C in a CO₂ incubator. After stimulation, cells were stained with a live/dead fixable viability dye, followed by surface staining with APC-Cy7-conjugated anti-mouse CD3e, Alexa488-conjugated anti-mouse CD4, PerCP-Cy5.5-conjugated anti-mouse CD8, and V450-conjugated anti-mouse CD44 antibodies. The cells were then fixed and permeabilized using a fixation/permeabilization solution for 15 minutes at 4 °C. Subsequently, intracellular cytokines were stained using the following fluorochrome-conjugated antibodies, such as PE-labeled anti-mouse IFN-γ, APC-labeled anti-mouse IL-5, PE-Cy7-labeled anti-mouse IL-17A, PE-Cy7-labeled anti-mouse IL-2, and APC-labeled anti-mouse TNF-α. For multifunctional T cell analysis, Boolean Gating was applied to identify CD4 + and CD8 + T cells co-expressing two or more cytokines among IFN-γ, IL-2, and TNF-α. The stained cells were acquired on a MACSQuant® flow cytometer (Miltenyi Biotec; Bergisch Gladbach, North Rhine‑Westphalia, Germany) and analyzed using FlowJo software (TreeStar Inc.; Ashland, OR, USA). Hematoxylin and eosin (H&E) staining The left lungs and brains of mice were collected and fixed in 4% paraformaldehyde in PBS for 24 hours at room temperature. Tissues were then washed under running tap water for 2 hours, dehydrated sequentially in graded ethanol solutions (70%, 80%, 90%, 95%, and 100%), and cleared with xylene. The samples were embedded in paraffin, sectioned at 5 μm thickness, followed by mounting onto slides by heating at 60 °C for 30 minutes. Paraffin sections were dewaxed with xylene and rehydrated through a descending ethanol series (100%, 95%, 90%, 80%, and 70%), followed by rinsing with tap water. The sections were stained with hematoxylin for 5 minutes and eosin for 30 seconds. After staining, tissues were dehydrated with ethanol (70%, 95%, and 100%) and cleared again with xylene. The slides were sealed with mounting medium, and images were acquired at 40× and 200× magnifications using a Motic EasyScan digital slide scanner (Motic, Xiamen, China). Whole blood killing assay The whole blood killing assay was performed with minor modifications from a previously described protocol 52 . S. suis BAA-853 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and standard serotype strains (serotypes 2, 4, and 9) were provided by the Animal and Plant Quarantine Agency, Republic of Korea. The strains were cultured in tryptic soy broth (TSB) to the exponential growth phase and washed twice with sterile phosphate-buffered saline (PBS). Bacterial suspensions were adjusted to 1 × 10 6 CFU/mL in PBS. For each reaction, 10 µL of diluted bacteria was mixed with 50 µL of PBS, serum from either unimmunized or ATOMSSUIS penta -immunized mice in a 96-well plates. After incubation at 37 °C for 30 minutes, 100 µL of heparinized piglet whole blood was added to each well and further incubated for 1 hour at 37 °C with gentle shaking. Following incubation, the reaction mixtures were serially diluted in a round bottom 96 well plate and spotted onto blood agar plates. After 24 hours of incubation at 37 °C, colony-forming units (CFU) were enumerated. The percentage of bacterial survival was calculated using the following formula: BLAST-based prevalence analysis of selected Streptococcus suis antigen epitopes in GeneBank Genomic Data The prevalence of five candidate antigen epitopes (HP0197, Fnbp, Sao, ScpB, and SLY) was evaluated across 388 S. suis genomes from GeneBank Genomic Data. A genomic database was constructed from all assemblies and indexed using makeblastdb (BLAST+ v2.15.0) 53,54 . Reference protein sequences for both full-length antigens and their conserved "highlight" domains were aligned against the nucleotide database using tblastn 53,54 with an E-value threshold of 1 × 10 -5 . Resulting alignments were processed with a custom Python script using the pandas 55,56 and Biopython libraries 57 . Antigens were defined as present if at least one hit per genome satisfied the following criteria: ≥70% identity and ≥80% query coverage for full-length sequences, or ≥60% identity and ≥50% coverage for highlight domains. These thresholds were selected as described previous Streptococcal studies 58 . Prevalence was calculated as the percentage of genomes containing a positive match. All analysis scripts and a reproducible Docker environment are publicly available on GitHub (https://github.com/leejhhh/suis-antigen-prevalence/tree/main). Statistical Analysis Data are presented as the mean ± standard deviation (SD) from at least three independent replicates per treatment group. Statistical significance was determined using the unpaired Student’s t-test for all group comparisons. Asterisks indicate statistical significance relative to the control group (* P < 0.05, ** P < 0.01, and *** P < 0.001). RESULTS Identification of S. suis protein vaccine candidates To develop a multimeric protein-based vaccine against S. suis , we initially conducted a comprehensive literature review covering the past 30 years of vaccine research involving animal models (mouse or swine). Using deep research algorithms, including the ChatGPT-assisted data mining tools, we identified over 12 potential protein antigens reported to induce protective immunity (Table 1 ). Among the identified candidates, we prioritized proteins containing the LPxTG motif, a well-characterized cell wall-anchoring domain essential for surface localization and immune recognition 59 . Based on this criterion, three major surface-anchored proteins, HP0197, fibronectin-binding protein (Fnbp), and surface antigen one (Sao) were selected due to their frequent identification in multiple serotypes and their previously reported immunogenicity. To enhance the surface coverage of the vaccine, C5a peptidase was included as a fourth LPxTG-containing protein. Although C5a peptidase has not yet been studied extensively in S. suis , it has been shown to be a potent virulence factor and a promising vaccine antigen in other Streptococcus species, including S. agalactiae and S. pyogenes 60–62 . Genomic analyses indicate that homologs of C5a peptidase (ScpB) are also widely conserved in S. suis , suggesting its potential as a cross-protective antigen 63,64 . In addition to the four surface proteins, suilysin (SLY) was selected as the fifth antigen. Although SLY expression is strain-dependent and not conserved among all S. suis isolates, its role as a cholesterol-dependent cytolysin has been associated with strong immunogenicity and pathogenicity 65,66 . Importantly, previous studies demonstrated that streptococcal toxins such as SLY ( S. suis ), erythrogenic toxins (Group A streptococcus), pneumolysin ( S. pneumoniae ) may act as adjuvants, promoting robust immune responses and providing protection through toxin neutralization 67,68 . Therefore, SLY was incorporated not only as a protective antigen but also to enhance the overall immunogenicity of the multimeric vaccine (Table 2 ). Table 1 Summary of Streptococcus suis protein vaccine candidates. Previously reported protein antigens evaluated as vaccine candidates against S. suis were summarized. For each antigen, the corresponding animal model, observed protective immunity, and key references are listed. Protective outcomes include antibody production, survival after lethal challenge, reduction of bacterial burden, and evidence of cross-protection against multiple serotypes. Antigens include surface-exposed proteins (e.g., HP0197, Sao), virulence factors (e.g., suilysin, ScpB), adhesins (e.g., Fnbp, Lmb), enzymes (e.g., SsEno, PrsA), and transporters (e.g., S-ABC). Reported models encompass both murine and porcine systems. Protein Antigen Animal Model Protective Immunity Observed Reference HP0197 (surface antigen) Mice and pigs Immunization with HP0197 provided significant protection in both mice and pig lethal challenge models, improving survival and enhancing opsonophagocytic killing through hyperimmune serum. 99 Sao (Surface Antigen One) Mice and pig Subcutaneous immunization with recombinant Sao protein plus Quil A adjuvant induced strong IgG responses (particularly IgG2a) and 100% survival after lethal S. suis challenge. 31 Fnbp (Fibronectin-binding protein) Mice Induced high levels of FBPS (Fnbp)-specific IgG and IgG2a antibodies. Immunized mice showed 100% survival against lethal S. suis serotype 2 challenge. Significantly reduced bacterial burden in blood and organs after infection. 100 SsPepO (secreted endopeptidase) Mice and pigs SsPepO immunization elicited strong antibody responses and provided significant protection against lethal S. suis serotype 2 challenge in both mice and pig models. 101 Enolase (SsEno) (glycolytic enzyme) Mice The glycolytic enzyme SsEno induced robust antibody responses and conferred complete protection against S. suis serotype 2 in mice, with observed cross-protection against serotypes 7 and 9. 102 Lmb (laminin-binding protein) Mice Vaccination with Lmb generated significant antibody responses and provided partial protection (~ 50% survival) against virulent S. suis serotype 2 in mice. 103 PrsA (peptidyl-prolyl isomerase) Mice Immunization with PrsA elicited high antibody titers and conferred partial protection, with approximately 50% survival in serotype 2 and 66% in serotype 9 S. suis challenges, indicating potential for cross-protection. 104 6PGD (6-phosphogluconate dehydrogenase) Pigs (swine) Vaccination with recombinant 6PGD provided significant protection in piglets, with reduced clinical disease upon S. suis serotype 2 challenge. 105 HtpS (histidine triad protein) Mice Immunization with HtpS (a surface-exposed protein) induced protective immunity, with significantly higher survival versus control in S. suis serotype 2 mouse challenge​. 103 S-ABC (sugar ABC transporter substrate-binding protein) Mice Immunization with S-ABC resulted in high protection, with approximately 87.5% survival against serotype 2 and 100% protection against serotype 9 challenge in mice. Moderate protection (~ 50% survival) was also observed. 103 Sbp (immunogenic membrane protein “Sbp”) Mice Identified via immunoproteomics, Sbp elicited a strong antibody response and significantly protected mice from lethal S. suis serotype 2 infection. Anti-Sbp serum enhanced bacterial clearance in the blood and conferred passive protection. 106 rSLY(P353L) (Genetically modified Suilysin) Mice Immunization with rSLY(P353L) led to reduced inflammatory responses and decreased mortality following S. suis infection. 107 ScpB (C5a peptidase) Mice Immunization with ScpB (a C5a peptidase) induced strong systemic and mucosal IgG and IgA responses, resulting in 100% survival against intranasal GBS challenge and reduced bacterial colonization and dissemination. 108 Table 2 Protein vaccine candidates selected for the construction of ATOMSSUIS penta . This table lists the five protein antigens incorporated into the ATOMSSUIS penta multimeric subunit vaccine. For each antigen, the molecular weight (MW), subcellular localization, predicted or known biological function, prevalence among Streptococcus suis isolates, and corresponding NCBI protein accession numbers are provided. Four of the selected antigens (HP0197, Fnbp, Sao, ScpB) are LPxTG motif-anchored surface proteins, and one (SLY) is a secreted cholesterol-dependent cytolysin. These proteins were chosen based on their reported immunogenicity, structural features, and potential to elicit protective immune responses. Protein MW (kDa) Subcellular Location Function Prevalence Accession # HP0197 (surface antigen) ~ 21 Cell surface (LPxTG anchor) Adhesion to host GAGs; immune evasion High in virulent strains WP_277937340.1 Fnbp (Fibronectin-binding protein) 55 ~ 60 Cell surface (LPxTG anchor) Binds fibronectin; facilitates adhesion/invasion Broadly present WP_014636551.1 Sao (Surface Antigen One) 58 ~ 70 Cell surface (LPxTG anchor) Immunodominant antigen; unknown exact function High in ST1/ST7 strains WP_211840080.1 ScpB (C5a peptidase) 120 ~ 130 Cell surface (LPxTG anchor) C5a peptidase; complement inhibition High in Streptococcus spp. Broadly present in S. suis WP_240208248.1 SLY (Suilysin) ~ 54 Secreted toxin Cholesterol-dependent cytolysin; causes inflammation 70–85% in virulent strains AIG43067.1 Epitope-based selection of immunogenic domains for the construction of the multimeric vaccine To design the multimeric protein vaccine (ATOMSSUIS penta ), immunogenic domains were selected through comprehensive in in silico prediction of T-cell and B-cell epitopes, following by domain filtering based on conservation, solubility, and structural features. T-cell epitopes were initially predicted using the Immune Epitope Database (IEDB), and only those with a conservancy rate above 90% across Streptococcus suis strains were retained. Both MHC class I and II binding epitopes were selected based on their affinity to common human leukocyte antigen (HLA) alleles: HLA-A24:02, HLA-A02:01 for MHC class I; and HLA-DRB109:01 for MHC class II, which are highly prevalent in East Asian populations 52,69 . The top-ranking predicted helper T-cell epitopes were localized to the following regions: residues 144–315 (Fnbp), 159–173 (Sao), 658–672 (ScpB), and 367–381 (SLY). Similarly, the most immunogenic cytotoxic T-cell epitopes were predicted at residues 84–142 (HP0197), 168–191 (Fnbp), 110–271 (Sao), 662–747 (ScpB), and 370–473 (SLY) (Supplementary Table 1; Supplementary Fig. 1). B-cell epitopes were predicted using the ElliPro 70 , and regions scoring above 0.700 were considered. The highest scoring linear B-cell epitopes were identified at residues 104–151 (HP0197), 135–329 (Fnbp), 77–198 (Sao), 668–755 (ScpB), and 388–443 (SLY) (Supplementary Table 2; Supplementary Fig. 1). Overlapping regions enriched for both T- and B-cell epitopes were selected as the immunogenic domains for the final vaccine construct (Table 3 ). Table 3 Immunological Characteristics of Selected Antigenic Domain and Multimeric Protein Vaccine (ATOMSSUIS penta ). Molecular weight, antigenicity, allergenicity, toxicity, and solubility were analyzed by ProtParam ( https://web.expasy.org/protparam/ ), ANTIGENpro ( https://scratch.proteomics.ics.uci.edu/ ), VaxiJen ( https://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html ), AllerTOP ( https://www.ddg-pharmfac.net/allertop_test/ ), ToxinPred( http://crdd.osdd.net/raghava/toxinpred/ ), and Protein-Sol ( https://protein-sol.manchester.ac.uk/ ), respectively. Protein Domain (MW) Antigenicity Allergenicity Toxicity Solubility ANTIGENPro (VaxiJen) AllerTOP ToxinPred Protein-Sol HP0197 (surface antigen) 56–199 (16.9 kDa) 0.7834 (0.3424) Non-allergen Non-toxin 0.855 Fnbp (Fibronectin-binding protein) 126–350 (23.1 kDa) 0.8803 (0.6987) Non-allergen Non-toxin 0.582 Sao (Surface Antigen One) 64–274 (23.7 kDa) 0.8923 (0.6092) Non-allergen Non-toxin 0.883 ScpB (C5a peptidase) 652–762 (12.9 kDa) 0.8198 (0.4746) Non-allergen Non-toxin 0.740 SLY (Suilysin) 366–475 (12.7 kDa) 0.9256 (0.5199) Non-allergen Non-toxin 0.801 ATOMSSUIS penta 90.3 kDa 0.9497 (0.7178) Non-allergen Non-toxin 0.619 The selected immunogenic domains were sequentially linked using flexible GGSGGGSG linkers (Fig. 1 A), and the resulting multimeric construct was evaluated for solubility, toxicity, antigenicity, and structural integrity (Table 3 ). Solubility was predicted using Protein-Sol 71 , toxicity was assessed via ToxinPred 72 . The secondary structure of the designed construct (ATOMSSUIS penta ) was predicted using two widely used algorithms, PSIPRED 46 and GOR IV 47 . The average values of PSIPRED and GOR IV show that, ATOMSSUIS penta is composed of 26.97% α-helix, 47.66% coil, and 25.37% β-strand (Fig. 1 B & 1 C). Tertiary Structure Prediction and Validation of ATOMSSUIS penta The 3D structural coordinates of the ATOMSSUIS penta were generated using AlphaFold3 48 and subsequently refined with the GalaxyRefine server 49 . Structural validation was performed using a Ramachandran plot via the PROCHECK server (Fig. 2 B) 51 . The refined ATOMSSUIS penta models exhibited 95.4% of the residues in the most favored regions, indicating a high-quality stereochemical profile. Structural quality was further assessed using the ProSA server 50 . The ProSA server initially revealed a Z-score of -10.67 for the model (not shown), which improved to -10.87 following refinement, suggesting enhanced overall model reliability (Fig. 2 C). While changes in the domain arrangement had little impact on solubility and toxicity scores, the predicted tertiary structure varied substantially depending on the domain arrangement. Given the uncertain relationship between 3D conformation and immunogenicity, the final domain arrangement was chosen to minimize structural overlap and maximize spatial separation between domains. The finalized configuration of the ATOMSSUIS penta construct was HP0197-Fnbp-Sao-ScpB-SLY (Fig. 2 A). The final ATOMSSUIS penta construct was predicted to be antigenic, non-allergenic and non-toxic. Protein solubility prediction using Protein-Sol 71 yielded a favorable solubility score of 0.619 (Fig. 2 D), indicating potential for soluble expression in E. coli . Antigenicity was assessed using both ANTIGENpro 73 and VaxiJen 74 , resulting in high predictive scores of 0.9497 and 0.7178, respectively (Table 3 ), suggesting strong immunogenic potential. The synthetic gene encoding ATOMSSUIS penta sequence was cloned into an expression vector (pET28a) and successfully expressed in E. coli BL21(DE3). The recombinant protein, with an expected molecular weight of approximately 90kDa, was successfully purified and confirmed by SDS-PAGE analysis. Under standard expression conditions, the yield of purified ATOMSSUIS penta was approximately 12–15 mg per liter of bacterial culture (Fig. 2 E). Comparative analysis of adjuvants for ATOMSSUIS penta vaccine To identify the optimal adjuvant for the ATOMSSUIS penta subunit vaccine, various adjuvants were compared in terms of their ability to induce humoral and cellular immune responses. As shown in Fig. 3 A, all adjuvant groups, including Monophosphoryl lipid A(MPL)/dimethyldioctadecylammonium (DDA), alum hydroxide, ISA1313, and Montanide gel, elicited comparable levels of antigen-specific IgG, indicating no significant difference in the humoral response among groups. However, in the analysis of cellular immunity, notable differences were observed (Fig. 3 B). Specifically, MPL/DDA induced the highest frequencies of both CD4⁺ and CD8⁺ T cells producing IFN-γ, suggesting a stronger cellular immune response compared to the other adjuvants. These findings indicate that while humoral responses were generally consistent across adjuvants, MPL/DDA most effectively promoted T cell-mediated immunity, supporting its selection as the preferred adjuvant for subsequent vaccine formulations. ATOMSSUIS penta vaccination elicits robust antigen-specific humoral immune responses in mice To evaluate the antigenicity of the ATOMSSUIS penta vaccine, BALB/c mice were subcutaneously immunized with 5, 10, or 20 µg of ATOMSSUIS penta formulated with the Th1-promoting adjuvant MPL/DDA on days 0 and 14. Serum samples were collected two weeks after the booster dose to assess the humoral immune response. As shown in Fig. 4 A, all dosage groups displayed significantly elevated levels of ATOMSSUIS penta -specific IgG, IgG2a, and IgM compared to the PBS control. IgG1 was significantly induced only in the 20 µg group, suggesting a Th1-skewed immune response, with limited Th2-type involvement at lower antigen doses. To compare the antigen specificity, sera were further analyzed for IgG, IgG2a, IgG1, and IgM titers against each of the five individual vaccine components (HP0197, Fnbp, Sao, ScpB and SLY). As shown in Fig. 4 B, ATOMSSUIS penta immunization markedly enhanced IgG, IgG2a, and IgG1 responses, particularly against ScpB, HP0197, and Sao. Similarly, antigen-specific IgM responses were elevated for ScpB, Fnbp, HP0197, and Sao. However, antibody responses to SLY remained minimal across all immunoglobulin subclasses. This weak immunogenicity of SLY may be attributed to its relatively small molecular size (113 amino acids), compared to other antigens and is consistent with previous reports indicating that bacterial toxins generally elicit weaker humoral responses 75 . To confirm the induction of S. suis -specific immunity, ELISA plates were coated with whole S. suis serotype 2 cells, and serum antibody titers were quantified. As shown in Fig. 4 C, immunization with 20 µg ATOMSSUIS penta induced significantly higher levels of S. suis -specific IgG, IgG2a, and IgG1 compared to the PBS group. These findings demonstrate that ATOMSSUIS penta elicits a robust systemic humoral immune response not only to the individual antigens but also to the intact bacterial pathogen. ATOMSSUIS penta vaccination promotes Th1/17 biased cellular immune responses in mice To assess vaccine-induced cellular immune responses, BALB/c mice were subcutaneously immunized with 5, 10, or 20 µg of ATOMSSUIS penta formulated with MPL/DDA on days 0 and 14. Two weeks after the booster immunization, splenocytes were harvested and stimulated in vitro with the full-length ATOMSSUIS penta protein (20 µg/mL). As shown in Fig. 5 A, stimulation with the full-length ATOMSSUIS penta protein led to a significant increase in CD4⁺ T cells producing IFN-γ or IL-17 across all dose groups, with the strongest responses observed in the 10 µg group. In contrast, the population of IL-5-producing CD4⁺ T cells remained unchanged, indicating no or weak Th2 response. Additionally, CD8⁺ T cells producing IFN-γ were markedly elevated, supporting a cytotoxic T cell response in a dose-dependent manner. These findings were further validated by quantifying cytokine levels in culture supernatants (Fig. 5 B). IFN-γ and IL-17 were robustly secreted by splenocytes from immunized mice, while IL-5 remained at relatively low levels. These results are consistent with the induction of a Th1- and Th17-skewed immune response by ATOMSSUIS penta . To further explore the antigen-specificity of the T cell responses, splenocytes were stimulated with individual antigens (HP0197, Fnbp, Sao, ScpB or SLY), and intracellular cytokine staining was performed (Fig. 5 C). Notably, CD4⁺ T cells producing IFN-γ or IL-17 were significantly increased in response to all five antigens, particularly ScpB, Fnbp, and SLY. IL-5-producing CD4⁺ T cells showed no substantial change in all groups. In addition, CD8⁺ T cells producing IFN-γ were significantly elevated upon stimulation with each antigen, with ScpB, Fnbp, and SLY again eliciting the strongest responses. Together, these data demonstrate that ATOMSSUIS penta vaccination elicits strong antigen-specific Th1 and Th17 responses, along with activation of cytotoxic CD8⁺ T cells, indicating its potential possibility to induce protective cellular immunity against S. suis . In note, the increased frequencies of both CD4⁺ and CD8⁺ T cells, particularly in response to individual component antigens, suggest that each subunit within ATOMSSUIS penta contributes functionally to T cell activation. These findings are consistent with our in silico predictions of T cell epitopes, supporting the rational design of the multimeric construct. ATOMSSUIS penta vaccination elicits robust multifunctional T cell responses Multifunctional T cells capable of simultaneously producing multiple cytokines such as IFN-γ, TNF-α, and IL-2 are known to play critical roles in protective immunity against bacterial and viral infections, particularly at mucosal sites such as the lung 76,77 , surpassing the efficacy of monofunctional T cells owing to their superior survival potential 78,79 . In addition they can be, prolonged persistence within the memory T cell pool, and capacity to elicit robust effector responses characterized by increased cytokine production and functional versatility 78,80,81 . To evaluate whether ATOMSSUIS penta vaccination induces such multifunctional responses, BALB/c mice were subcutaneously immunized with ATOMSSUIS penta formulated with MPL/DDA. Two weeks after the booster dose, splenocytes were analyzed by intracellular cytokine staining and flow cytometry to assess the production of IFN-γ, TNF-α, and IL-2. As shown in Fig. 6 A, ATOMSSUIS penta immunization significantly increased the frequency of multifunctional CD4⁺ T cells expressing IFN-γ⁺ TNF-α⁺ IL-2⁺ and TNF-α⁺ IL-2⁺ compared to the PBS control. Additional populations of IFN-γ⁺ TNF-α⁺ and IFN-γ⁺ IL-2⁺ CD4⁺ T cells were also elevated, but relatively lower levels. Among single cytokine-producing CD4⁺ T cells, TNF-α⁺ and IL-2⁺ subsets were notably increased (Fig. 6 B), indicating that the vaccine promotes both polyfunctional and monofunctional helper T cell responses. Next, we assessed the multifunctional CD8⁺ T cell responses elicited by ATOMSSUIS penta immunization (Fig. 6 C). Vaccination induced a marked, dose-dependent increase in triple-positive IFN-γ⁺ TNF-α⁺ IL-2⁺ CD8⁺ T cells, with the highest frequencies observed at the 20 µg dose. A similar trend was seen for IFN-γ⁺ IL-2⁺ double-positive CD8⁺ T cells, which also increased significantly in a dose-dependent manner. In contrast, IFN-γ⁺ TNF-α⁺ CD8⁺ T cells showed only a modest elevation, primarily at the 10 µg dose, while TNF-α⁺ IL-2⁺ CD8⁺ T cells remained largely unchanged across all groups. Among the single-cytokine-producing CD8⁺ T cells, a substantial increase was observed only in the IFN-γ⁺ subset, whereas TNF-α⁺ and IL-2⁺ subsets exhibited minimal or no enhancement. These results highlight the ability of ATOMSSUIS penta to induce potent and polyfunctional CD8⁺ T cell responses, characterized predominantly by IFN-γ and IL-2 co-expression. Together with the multifunctional CD4⁺ T cell responses, these findings suggest that ATOMSSUIS penta vaccination promotes a robust and qualitatively superior cell-mediated immune response. This profile, marked by multifunctionality and coordinated cytokine production, highlights its potential to confer durable and effective protection against S. suis infection. ATOMSSUIS penta confers protective response against Streptococcus suis infection in mice To assess the protective efficacy of ATOMSSUIS penta , C57BL/6 mice were subcutaneously immunized with the vaccine formulated with MPL/DDA. Two weeks following the final immunization, mice were challenged with either a mild dose (5 × 10⁷ CFU) for monitoring body weight change or a severe dose (1.5 × 10⁸ CFU) for survival rate of S. suis BAA-853 (serotype 2) infection. As shown in Fig. 7 A, both vaccinated and non-vaccinated mice exhibited weight loss of approximately 12.0 ± 1.56% and 13.4 ± 1.51%, respectively, at 1day post-challenge (dpc). However, mice immunized with ATOMSSUIS penta recovered 90% of the lost weight within 3 days, whereas recovery in the PBS group was markedly delayed, requiring up to 10 days. In the lethal challenge model, all unvaccinated mice succumbed to infection, displaying severe clinical signs such as depression, ruffled hair coat, and labored breathing. In contrast, 5 out of 6 vaccinated mice survived, and only transient and mild clinical signs were observed during the early phase of infection, demonstrating a substantial protective effect. To investigate bacterial dissemination, bacterial burdens were quantified in the brain, lung, and spleen at 2 dpc (Fig. 7 B). Non-immunized mice exhibited high bacterial loads (mean values of 4.46 ± 0.37, 4.90 ± 0.40, and 5.92 ± 0.41 log₁₀ CFU/g in brain, lung, and spleen, respectively). In contrast, ATOMSSUIS penta -immunized mice showed significantly reduced bacterial counts in all examined organs. Histopathological analysis further confirmed the protective effect of vaccination (Fig. 7 C). Unvaccinated mice displayed pronounced histological signs of infection, including hyperemia and dense inflammatory cell infiltration in both brain and lung tissues. In contrast, tissues from vaccinated mice exhibited minimal pathology, with largely preserved architecture, minimal congestion, and reduced immune cell infiltration. Collectively, these data indicate that ATOMSSUIS penta vaccination confers strong protection against S. suis infection by limiting bacterial burden and promoting tissue inflammation, thereby reducing the risk of severe clinical outcomes such as meningitis and pneumonia. ATOMSSUIS penta immunization induces cross-reactive and cross-protective antibody responses against various Streptococcus suis serotypes Although S. suis serotype 2 is the most prevalent and virulent serotype globally, the presence of diverse serotypes across different regions poses a significant challenge to serotype independent vaccine strategies. To evaluate the cross-reactive antibody conferred by ATOMSSUIS penta vaccination, serum IgG responses were assessed against five representative S. suis serotypes (2, 4, 9, 14, and 25). As shown in Fig. 8 A, ATOMSSUIS penta -immunized mice produced high titers of IgG antibodies that strongly recognized S. suis serotypes 2, 4, and 9. However, the antibody reactivity to serotypes 14 and 25 was not significantly different from that of the control group, suggesting limited cross-reactivity to these serotypes. To further determine whether the vaccine-induced antibodies provide functional cross-neutralization, a piglet whole blood killing assay was performed using the same serotypes. As shown in Fig. 8 B, sera from ATOMSSUIS penta -immunized mice significantly enhanced bacterial clearance of serotypes 2, 4, and 9 compared to the PBS control, consistent with the observed IgG binding patterns. No opsonic killing was observed for serotypes 14 and 25. These findings indicate that ATOMSSUIS penta not only elicits serotype 2-specific immune responses but also induces cross-reactive and functionally protective antibody responses against multiple clinically relevant serotypes-most notably serotypes 4 and 9 11,82 . This broad-spectrum reactivity underscores the potential of ATOMSSUIS penta as a promising cross-protective vaccine candidate. Moreover, the observed efficacy validates the robustness of our in silico antigen selection strategy, demonstrating its effectiveness in designing broadly protective subunit vaccines against S. suis and other bacterial target. DISCUSSION Streptococcus suis is a significant zoonotic pathogen that primarily colonizes the upper respiratory tract of pigs but can be transmitted to humans through direct contact with infected animals or consumption of undercooked pork products 83,84 . In recent years, human infections have been increasingly reported across several Asian countries, including China, Vietnam, and Thailand, where pork is a major dietary and pig farming is widespread 9,10 . This growing public health concern underscores the urgent need for effective preventive strategies, including the development of a broadly protective vaccine. The presence of more than 30 serotypes and geographical variations in serotype prevalence poses a major challenge to the development of broadly protective vaccines 85–87 . In this study, we selected protein vaccine candidates based on previously reported in vivo efficacy data and applied established bioinformatic tools to predict immunogenic B and T cell epitopes. Using this bioinformatic analysis data, we rationally designed a multimeric subunit vaccine, ATOMSSUIS penta , composed of five conserved antigens. Immunization with ATOMSSUIS penta elicited robust antigen-specific humoral responses against each component protein. Furthermore, when formulated with the Th1-skewing adjuvant MPL/DDA, it successfully induced cellular immune responses, including Th1 and Th17 subsets, which are known to play crucial roles in protection against S. suis infection. These findings suggest that ATOMSSUIS penta represents a promising universal vaccine approach, overcoming the serotype-specific limitations of conventional vaccine strategies. Universal vaccine strategies often aim to target conserved protein antigens shared across multiple serotypes or clinical isolates 88,89 . However, extensive genetic and antigenic diversity across serotypes and genotypes complicates the identification of universal antigenic targets specifically in Gram-positive bacteria such as Streptococcus suis 33,90 . To overcome this challenge, some studies have attempted to use a mixture of individually purified antigens, but this approach involves a complex GMP production process requiring separate purification of each antigen and necessitates individual evaluation of their immunogenicity and efficacy. Recently, epitope-based vaccine design using bioinformatic tools has gained attention as a rational strategy to reduce antigen size while enhancing specificity and immunogenicity 40,41,43,44 . In this study, we constructed a multimeric epitope antigen (90 kDa) by linking predicted B- and T-cell epitope domains from five conserved S. suis antigens, with epitope selection uniquely based on surface-exposed regions identified through the tertiary structure information of HP0197 (PDB ID: 4FZ4) 91 , Fnbp (PDB ID 5BOB) 92 , ScpB (PDB ID: 8BTY) 93 , SLY (PDB ID: 3HVN) 32 from the PDB database ( http://www.rcsb.org ) or the modelled structure of ATOMSSUIS penta from AlphaFold3 48 . Importantly, in vivo immunization demonstrated that this epitope-based design could elicit strong humoral, cellular, and protective responses against multiple serotypes. While previous in silico designed vaccines have often shown suboptimal efficacy in vivo , our findings highlight the feasibility of a structure-guided multiepitope vaccine approach. This strategy may extend beyond S. suis , offering a platform for the development of cross-protective vaccines against other genetically diverse Gram-positive pathogens, including Staphylococcus aureus and Streptococcus agalactiae . Th1 responses play a crucial role in protection against S. suis infection by enhancing bacterial clearance through cytokine-driven immune cell activation and promoting opsonophagocytosis 94 . These mechanisms are essential in preventing severe disease manifestations such as meningitis, septicemia, and streptococcal toxic shock-like syndrome (STSLS). In our study, ATOMSSUIS penta vaccination induced a strong Th1- and Th17-skewed immune response, consistent with previous reports that highlight the protective roles of these T cell subsets in streptococcal infections 94–96 . Lecours et al. demonstrated that Th1 responses are predominant and critical during S. suis serotype 2 infection, while IL-17-producing Th17 cells have been shown to facilitate bacterial clearance through neutrophil recruitment and the induction of antimicrobial proteins 94 . Despite the immunological profile observed, we were unable to directly confirm the functional efficacy of opsonophagocytic activity due to the lack of a standardized and validated OPKA (Opsonophagocytic Killing Assay) specifically optimized for S. suis . In our previous studies, we successfully applied OPKA to evaluate functional antibody responses against Streptococcus pneumoniae and Streptococcus agalactiae , using well-established assay systems 97,98 . However, these standard OPKA protocols could not be directly applied to S. suis , likely due to differences in bacterial surface structure, phagocytic uptake, and complement susceptibility. This technical limitation hindered direct assessment of vaccine-induced opsonic activity, underscoring the need for development of a S. suis -specific OPKA platform. In this study, we employed a whole blood killing assay, which successfully served as a surrogate method for evaluating opsonic activity. This assay captures the combined effects of functional antibodies, complement, phagocytic immune cells, and other essential blood components present in piglet whole blood, thereby providing an integrated measure of opsonophagocytic killing activity. By adopting this approach, we not only demonstrated the protective potential of ATOMSSUIS penta -induced antibodies, but also introduced a practical and adaptable platform for functional vaccine evaluation in S. suis . This methodological advancement highlights an important contribution of this study and emphasizes the value of establishing standardized assays to assess vaccine-induced immune correlates of protection in S. suis infection models. An emerging focus in vaccine immunology is the role of multifunctional T cells which can capable of simultaneously producing multiple cytokines such as IFN-γ, TNF-α, and IL-2, which are increasingly recognized as correlates of robust and durable protection against infectious diseases, especially at mucosal sites 76,77 . These multifunctional T cells exhibit superior survival and long-term persistence within the memory pool, a feature supported by their elevated expression of survival-associated molecules such as CD127 (IL-7Rα) and Bcl2 78–80 . In addition to enhanced longevity, they contribute to protective immunity by promoting strong effector responses and immunological versatility, and by enabling rapid and effective recall responses upon re-infection 78,80,81 . While multifunctional T cells have been studied in various bacterial infections, their role in mediating protective immunity against S. suis remains poorly understood. In this study, we demonstrate that ATOMSSUIS penta vaccination adjuvanted with MPL/DDA induces a robust multifunctional T cell response, characterized by the co-expression of IFN-γ, TNF-α, and IL-2. The increased frequency of these multifunctional T cells, along with dual positive and monofunctional subsets, suggests that this vaccine not only expands antigen-specific helper T cells but also enhances their functional quality. Such qualitative enhancement of the T cell response is likely a key contributor to the ATOMSSUIS penta ’s protective efficacy and represents a significant advance in the understanding of cellular immunity against S. suis . To build on these findings, further studies should explore whether these cells directly correlate with protection and define their contribution to long-term immune memory. Nevertheless, genome-wide analysis using the S. suis complete genome database (Taxonomy ID:1307, n = 388) revealed that the five selected antigens are not uniformly conserved across clinical isolates. BLASTp analysis (≥ 80% identity) showed that HP0197, Fnbp, Sao, ScpB, and SLY were present in 38.1% (148 strains), 26.3% (102 strains), 66.2% (257 strains), 97.2% (377 strains), and 52.3% (203 strains), respectively. These findings indicate that S. suis antigenic proteins are not highly conserved among clinical isolates, posing a major obstacle to universal vaccine development. Consistent with these genomic observations, our experimental results also suggest that single antigen might not be express in all clinical isolates. Thus, it is unlikely that a single antigen would be sufficient to elicit protective immunity against the diverse S. suis population. To overcome this limitation, the use of multiple antigens in a multimeric construct is essential for broadening immune coverage and enhancing vaccine efficacy across genetically heterogeneous strains. Therefore, future antigen selection strategies should not only focus on immunogenicity but also consider the conservation across clinical strain genome database. Such an approach is essential for designing broadly protective vaccines against genetically variable pathogens. A limitation of the present study is that these genome conservation data were not incorporated during the antigen selection process. Nonetheless, the use of a multimeric antigen composed of multiple distinct proteins appears to have compensated for this variability, achieving broad immune coverage despite the heterogeneity among S. suis strains. In summary, we developed a multimeric subunit vaccine, ATOMSSUIS penta , composed of five conserved S. suis antigens selected based on known immunogenicity and predicted surface exposed B- and T-cell epitopes. The vaccine induced robust antigen-specific humoral and cellular immune responses, including Th1- and Th17-type immunity, and conferred protective efficacy in a murine infection model. Despite limitations such as the lack of a standardized OPKA for S. suis and incomplete consideration of genome-wide antigen conservation during antigen selection, the multimeric design allowed for broad immune coverage across genetically diverse S. suis strains. These findings support the potential of epitope-based multimeric vaccines as a promising platform for the development of broadly protective vaccines against S. suis and potentially other antigenically diverse Gram-positive pathogens. Declarations ACKNOWLEDGMENTS This work was supported in part by the Internal R&D Program of the Korea Atomic Energy Research Institute (KAERI) (523140-24), funded by the Ministry of Science and ICT (MIST); by the Ministry of Food and Drug Safety (22202MFDS171 to K.H.K.); by the National Research Foundation of Korea (NRF) grant funded by the Korean government (RS-2022-00164721 to M.K.); and by the ZODIAC project of the International Atomic Energy Agency (IAEA) in the Asia and the Pacific region (CRP D32039, contract number: 26513 to H.S.S.). CONFLICT OF INTEREST The authors declare that there are no conflicts of interest. Author Contribution S.Y.K., F.C., and W.S.K. contributed equally to this work. H.S.S. and K.B.A. conceived and supervised the project. S.Y.K., F.C., and W.S.K. performed antigen selection, epitope prediction, and structural modeling (Figure 1-3). M.K.K. and H.R.P. conducted the mouse immunization and infection experiments (Figure 4-8). H.J.J. and C.E.L. conducted ELISA and flow cytometry analyses (Figure 5-8). V.W. and K.H.K. assisted in data interpretation and genomic analysis (Figure 1-2). S.Y.K. and F.C. drafted the manuscript. H.S.S. critically revised the manuscript. All authors read and approved the final version of the manuscript. References Feng, Y. et al. Streptococcus suis infection: an emerging/reemerging challenge of bacterial infectious diseases? Virulence 5 , 477-497 (2014). https://doi.org:10.4161/viru.28595 Segura, M., Fittipaldi, N., Calzas, C. & Gottschalk, M. Critical Streptococcus suis Virulence Factors: Are They All Really Critical? Trends Microbiol 25 , 585-599 (2017). https://doi.org:10.1016/j.tim.2017.02.005 Wertheim, H. F., Nghia, H. D., Taylor, W. & Schultsz, C. Streptococcus suis: an emerging human pathogen. Clin Infect Dis 48 , 617-625 (2009). https://doi.org:10.1086/596763 Obradovic, M. R., Segura, M., Segales, J. & Gottschalk, M. Review of the speculative role of co-infections in Streptococcus suis-associated diseases in pigs. Vet Res 52 , 49 (2021). https://doi.org:10.1186/s13567-021-00918-w Perch, B., Kristjansen, P. & Skadhauge, K. Group R streptococci pathogenic for man. Two cases of meningitis and one fatal case of sepsis. Acta Pathol Microbiol Scand 74 , 69-76 (1968). Tang, J. et al. Streptococcal toxic shock syndrome caused by Streptococcus suis serotype 2. PLoS Med 3 , e151 (2006). https://doi.org:10.1371/journal.pmed.0030151 Ye, C. et al. Streptococcus suis sequence type 7 outbreak, Sichuan, China. Emerging infectious diseases 12 , 1203 (2006). Yu, H. et al. Human streptococcus suis outbreak, Sichuan, China. Emerging infectious diseases 12 , 914 (2006). Rayanakorn, A., Goh, B.-H., Lee, L.-H., Khan, T. M. & Saokaew, S. Risk factors for Streptococcus suis infection: A systematic review and meta-analysis. Scientific reports 8 , 13358 (2018). Kerdsin, A. Human Streptococcus suis infections in Thailand: epidemiology, clinical features, genotypes, and susceptibility. Tropical medicine and infectious disease 7 , 359 (2022). Goyette-Desjardins, G., Auger, J.-P., Xu, J., Segura, M. & Gottschalk, M. Streptococcus suis, an important pig pathogen and emerging zoonotic agent—an update on the worldwide distribution based on serotyping and sequence typing. Emerging microbes & infections 3 , 1-20 (2014). Dechene-Tempier, M. et al. Update on the Mechanisms of Antibiotic Resistance and the Mobile Resistome in the Emerging Zoonotic Pathogen Streptococcus suis. Microorganisms 9 (2021). https://doi.org:10.3390/microorganisms9081765 Holmer, I. et al. Antibiotic resistance in porcine pathogenic bacteria and relation to antibiotic usage. BMC Vet Res 15 , 449 (2019). https://doi.org:10.1186/s12917-019-2162-8 Li, L. L. et al. Antimicrobial resistance, serotypes, and virulence factors of Streptococcus suis isolates from diseased pigs. Foodborne Pathog Dis 9 , 583-588 (2012). https://doi.org:10.1089/fpd.2011.1106 Yongkiettrakul, S. et al. Antimicrobial susceptibility of Streptococcus suis isolated from diseased pigs, asymptomatic pigs, and human patients in Thailand. BMC Vet Res 15 , 5 (2019). https://doi.org:10.1186/s12917-018-1732-5 Zhang, C. et al. In vitro antimicrobial susceptibility of Streptococcus suis strains isolated from clinically healthy sows in China. Vet Microbiol 131 , 386-392 (2008). https://doi.org:10.1016/j.vetmic.2008.04.005 Wisselink, H. J., Veldman, K. T., Van den Eede, C., Salmon, S. A. & Mevius, D. J. Quantitative susceptibility of Streptococcus suis strains isolated from diseased pigs in seven European countries to antimicrobial agents licensed in veterinary medicine. Vet Microbiol 113 , 73-82 (2006). https://doi.org:10.1016/j.vetmic.2005.10.035 Buchy, P. et al. Impact of vaccines on antimicrobial resistance. Int J Infect Dis 90 , 188-196 (2020). https://doi.org:10.1016/j.ijid.2019.10.005 Clift, C. & Salisbury, D. M. Enhancing the role of vaccines in combatting antimicrobial resistance. Vaccine 35 , 6591-6593 (2017). https://doi.org:10.1016/j.vaccine.2017.09.053 Micoli, F., Bagnoli, F., Rappuoli, R. & Serruto, D. The role of vaccines in combatting antimicrobial resistance. Nat Rev Microbiol 19 , 287-302 (2021). https://doi.org:10.1038/s41579-020-00506-3 Rieckmann, K., Pendzialek, S.-M., Vahlenkamp, T. & Baums, C. G. A critical review speculating on the protective efficacies of autogenous Streptococcus suis bacterins as used in Europe. Porcine health management 6 , 1-11 (2020). Ghattas, M., Dwivedi, G., Lavertu, M. & Alameh, M.-G. Vaccine technologies and platforms for infectious diseases: Current progress, challenges, and opportunities. Vaccines 9 , 1490 (2021). Blouin, C., Higgins, R., Gottschalk, M. & Simard, J. Evaluation of the antibody response in pigs vaccinated against Streptococcus suis capsular type 2 using a double-antibody sandwich enzyme-linked immunosorbent assay. Can J Vet Res 58 , 49-54 (1994). Corsaut, L. et al. Field Study on the Immunological Response and Protective Effect of a Licensed Autogenous Vaccine to Control Streptococcus suis Infections in Post-Weaned Piglets. Vaccines (Basel) 8 (2020). https://doi.org:10.3390/vaccines8030384 Kebede, M., Chengappa, M. M. & Stuart, J. G. Isolation and characterization of temperature-sensitive mutants of Streptococcus suis: efficacy trial of the mutant vaccine in mice. Vet Microbiol 22 , 249-257 (1990). https://doi.org:10.1016/0378-1135(90)90112-9 Foster, N., Staats, J. J. & Chengappa, M. M. Isolation, characterization and protection studies in mice of a streptomycin-dependent mutant of Streptococcus suis type 1/2. Vet Res Commun 18 , 155-163 (1994). https://doi.org:10.1007/BF01839263 Smith, H. et al. Identification and characterization of the cps locus of Streptococcus suis serotype 2: the capsule protects against phagocytosis and is an important virulence factor. Infection immunity 67 , 1750-1756 (1999). https://doi.org:10.1128/iai.67.4.1750-1756.1999 Baums, C. G. et al. Identification of a novel virulence determinant with serum opacification activity in Streptococcus suis. Infect Immun 74 , 6154-6162 (2006). https://doi.org:10.1128/IAI.00359-06 Hou, Y. et al. Advanced subunit vaccine delivery technologies: From vaccine cascade obstacles to design strategies. Acta Pharmaceutica Sinica B 13 , 3321-3338 (2023). Heidary, M. et al. A comprehensive review of the protein subunit vaccines against COVID-19. Frontiers in microbiology 13 , 927306 (2022). Li, Y. et al. Immunization with recombinant Sao protein confers protection against Streptococcus suis infection. Clin Vaccine Immunol 14 , 937-943 (2007). https://doi.org:10.1128/CVI.00046-07 Xu, L. et al. Crystal structure of cytotoxin protein suilysin from Streptococcus suis. Protein cell 1 , 96-105 (2010). https://doi.org:10.1007/s13238-010-0012-3 Segura, M. Streptococcus suis vaccines: candidate antigens and progress. Expert Rev Vaccines 14 , 1587-1608 (2015). https://doi.org:10.1586/14760584.2015.1101349 Ma, F. et al. Streptococcus suis Serotype 2 Biofilms Inhibit the Formation of Neutrophil Extracellular Traps. Frontiers in cellular infection microbiology 7 , 86 (2017). https://doi.org:10.3389/fcimb.2017.00086 Musyoki, A. et al. Structural and functional analysis of an anchorless fibronectin-binding protein FBPS from Gram-positive bacterium Streptococcus suis. Proceedings of the National Academy of Sciences of the United States of America 113 , 13869-13874 (2016). https://doi.org:10.1073/pnas.1608406113 Lynskey, N. et al. Multi-functional mechanisms of immune evasion by the streptococcal complement inhibitor C5a peptidase. PLoS pathogens 13 , e1006493 (2017). https://doi.org:10.1371/journal.ppat.1006493 Li, J. et al. Evaluation of the immunogenicity and the protective efficacy of a novel identified immunogenic protein, SsPepO, of Streptococcus suis serotype 2. Vaccine 29 , 6514-6519 (2011). https://doi.org:10.1016/j.vaccine.2011.07.010 Weisse, C. et al. Immunogenicity and protective efficacy of a Streptococcus suis vaccine composed of six conserved immunogens. Vet Res 52 , 112 (2021). https://doi.org:10.1186/s13567-021-00981-3 Oli, A. N. et al. Immunoinformatics and vaccine development: an overview. ImmunoTargets and therapy , 13-30 (2020). Shey, R. A. et al. In-silico design of a multi-epitope vaccine candidate against onchocerciasis and related filarial diseases. Scientific reports 9 , 4409 (2019). Meza, B., Ascencio, F., Sierra-Beltrán, A. P., Torres, J. & Angulo, C. A novel design of a multi-antigenic, multistage and multi-epitope vaccine against Helicobacter pylori: an in silico approach. Infection, Genetics and Evolution 49 , 309-317 (2017). Kardani, K., Bolhassani, A. & Namvar, A. An overview of in silico vaccine design against different pathogens and cancer. Expert Review of Vaccines 19 , 699-726 (2020). Potocnakova, L., Bhide, M. & Pulzova, L. B. An introduction to B‐cell epitope mapping and in silico epitope prediction. Journal of immunology research 2016 , 6760830 (2016). Rosa, D. S., Ribeiro, S. P. & Cunha-Neto, E. CD4+ T cell epitope discovery and rational vaccine design. Archivum immunologiae et therapiae experimentalis 58 , 121-130 (2010). Negahdaripour, M. et al. Structural vaccinology considerations for in silico designing of a multi-epitope vaccine. Infection, Genetics and Evolution 58 , 96-109 (2018). McGuffin, L. J., Bryson, K. & Jones, D. T. The PSIPRED protein structure prediction server. Bioinformatics 16 , 404-405 (2000). https://doi.org:10.1093/bioinformatics/16.4.404 Kouza, M., Faraggi, E., Kolinski, A. & Kloczkowski, A. The GOR Method of Protein Secondary Structure Prediction and Its Application as a Protein Aggregation Prediction Tool. Methods Mol Biol 1484 , 7-24 (2017). https://doi.org:10.1007/978-1-4939-6406-2_2 Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630 , 493-500 (2024). https://doi.org:10.1038/s41586-024-07487-w Heo, L., Park, H. & Seok, C. GalaxyRefine: Protein structure refinement driven by side-chain repacking. Nucleic Acids Res 41 , W384-388 (2013). https://doi.org:10.1093/nar/gkt458 Wiederstein, M. & Sippl, M. J. ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res 35 , W407-410 (2007). https://doi.org:10.1093/nar/gkm290 Lovell, S. C. et al. Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins 50 , 437-450 (2003). https://doi.org:10.1002/prot.10286 Nakaoka, H. & Inoue, I. Distribution of HLA haplotypes across Japanese Archipelago: similarity, difference and admixture. Journal of human genetics 60 , 683-690 (2015). Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. Journal of molecular biology 215 , 403-410 (1990). Camacho, C. et al. BLAST+: architecture and applications. BMC bioinformatics 10 , 1-9 (2009). Reback, J. et al. pandas-dev/pandas: Pandas 1.0. 5. Zenodo (2020). McKinney, W. Data structures for statistical computing in Python. SciPy 445 , 51-56 (2010). Cock, P. J. et al. Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics 25 , 1422 (2009). Estrada, A. A., Gottschalk, M., Gebhart, C. J. & Marthaler, D. G. Comparative analysis of Streptococcus suis genomes identifies novel candidate virulence-associated genes in North American isolates. Veterinary research 53 , 23 (2022). Fischetti, V. A. Surface proteins on gram-positive bacteria. Microbiology spectrum 7 , 10.1128/microbiolspec. gpp1123-0012-2018 (2019). Chen, C. C. & Cleary, P. Complete nucleotide sequence of the streptococcal C5a peptidase gene of Streptococcus pyogenes. Journal of Biological Chemistry 265 , 3161-3167 (1990). Gleich-Theurer, U. et al. Human serum induces streptococcal c5a peptidase expression. Infection and immunity 77 , 3817-3825 (2009). Krishnamoorthy, S., Steiger, A. K., Nelson, W. C., Egbert, R. G. & Wright, A. T. An activity-based probe targeting the streptococcal virulence factor C5a peptidase. Chemical Communications 58 , 8113-8116 (2022). Guo, G. et al. Pan‐genome analysis of Streptococcus suis serotype 2 revealed genomic diversity among strains of different virulence. Transboundary and Emerging Diseases 68 , 637-647 (2021). Kerdsin, A. et al. Comparative genome analysis of Streptococcus suis serotype 5 strains from humans and pigs revealed pathogenic potential of virulent, antimicrobial resistance, and genetic relationship. Microbes and Infection , 105273 (2023). Tenenbaum, T., Asmat, T. M., Seitz, M., Schroten, H. & Schwerk, C. Biological activities of suilysin: role in Streptococcus suis pathogenesis. Future microbiology 11 , 941-954 (2016). Li, G. et al. Morin attenuates Streptococcus suis pathogenicity in mice by neutralizing suilysin activity. Frontiers in microbiology 8 , 460 (2017). Barnett, T. C. et al. Streptococcal toxins: role in pathogenesis and disease. Cellular microbiology 17 , 1721-1741 (2015). Wu, M.-C. et al. Recombinant suilysin of Streptococcus suis enhances the protective efficacy of an engineered Pasteurella multocida toxin protein. Research in Veterinary Science 151 , 175-183 (2022). Bae, E. H. & Shin, M. In silico Design of Multi-epitope Vaccines Targeting Iron-regulated lipoproteins of Staphylococcus aureus Using Immunoinformactics. Journal of Bacteriology and Virology 52 , 170-183 (2022). Ponomarenko, J. et al. ElliPro: a new structure-based tool for the prediction of antibody epitopes. BMC bioinformatics 9 , 1-8 (2008). Hebditch, M., Carballo-Amador, M. A., Charonis, S., Curtis, R. & Warwicker, J. Protein-Sol: a web tool for predicting protein solubility from sequence. Bioinformatics (Oxford, England) 33 , 3098-3100 (2017). https://doi.org:10.1093/bioinformatics/btx345 Gupta, S. et al. In silico approach for predicting toxicity of peptides and proteins. PLoS One 8 , e73957 (2013). https://doi.org:10.1371/journal.pone.0073957 Magnan, C. N. et al. High-throughput prediction of protein antigenicity using protein microarray data. Bioinformatics 26 , 2936-2943 (2010). Doytchinova, I. A. & Flower, D. R. VaxiJen: a server for prediction of protective antigens, tumour antigens and subunit vaccines. BMC Bioinformatics 8 , 4 (2007). https://doi.org:10.1186/1471-2105-8-4 Da Costa, R. M., Rooke, J. L., Wells, T. J., Cunningham, A. F. & Henderson, I. R. Type 5 secretion system antigens as vaccines against Gram-negative bacterial infections. NPJ Vaccines 9 , 159 (2024). https://doi.org:10.1038/s41541-024-00953-6 Khakhum, N., Bharaj, P., Walker, D. H., Torres, A. G. & Endsley, J. J. Antigen-specific antibody and polyfunctional T cells generated by respiratory immunization with protective Burkholderia ΔtonB Δhcp1 live attenuated vaccines. npj Vaccines 6 , 72 (2021). O’Neill, A. et al. Mucosal SARS-CoV-2 vaccination of rodents elicits superior systemic T central memory function and cross-neutralising antibodies against variants of concern. EBioMedicine 99 (2024). Lindenstrøm, T. et al. Tuberculosis subunit vaccination provides long-term protective immunity characterized by multifunctional CD4 memory T cells. The Journal of Immunology 182 , 8047-8055 (2009). Turtle, L. et al. Human T cell responses to Japanese encephalitis virus in health and disease. Journal of Experimental Medicine 213 , 1331-1352 (2016). Westerhof, L. M. et al. Multifunctional cytokine production marks influenza A virus‐specific CD4 T cells with high expression of survival molecules. European Journal of Immunology 53 , 2350559 (2023). Pereira Neto, T. A. et al. Multifunctional T cell response in convalescent patients two years after ZIKV infection. Journal of Leucocyte Biology 108 , 1265-1277 (2020). Zhu, J. et al. Streptococcus suis serotype 4: a population with the potential pathogenicity in humans and pigs. Emerging Microbes & Infections 13 , 2352435 (2024). Lunha, K. et al. Serotype Distribution and Pathotypic Characteristics of Streptococcus suis Isolates from Slaughtered Pigs in a High-Density Pig Farming Area in Thailand. Transbound Emerg Dis 2024 , 3186518 (2024). https://doi.org:10.1155/2024/3186518 Guo, P. et al. Network Pharmacology to Unveil the Mechanism of Berberine in the Treatment of Streptococcus suis Meningitis in Humans and Pigs. Toxics 13 (2025). https://doi.org:10.3390/toxics13020138 Frosth, S. et al. Sow vaccination with a novel recombinant protein vaccine protects piglets against Streptococcus suis infection. Vaccine 53 , 127077 (2025). https://doi.org:10.1016/j.vaccine.2025.127077 Li, W., Li, Y. A., Wang, S. & Shi, H. A universal live vaccine platform against multiple serotypes Streptococcus suis based on polyvalent antigen protein. Vaccine 47 , 126700 (2025). https://doi.org:10.1016/j.vaccine.2024.126700 Liu, J. et al. A multi-epitope subunit vaccine providing broad cross-protection against diverse serotypes of Streptococcus suis. NPJ vaccines 9 , 216 (2024). https://doi.org:10.1038/s41541-024-01015-7 Jazayeri, S. D. & Poh, C. L. Development of Universal Influenza Vaccines Targeting Conserved Viral Proteins. Vaccines (Basel) 7 (2019). https://doi.org:10.3390/vaccines7040169 Walker, R. I. Conserved antigens for enteric vaccines. Vaccine 50 , 126828 (2025). https://doi.org:10.1016/j.vaccine.2025.126828 Dolbec, D. et al. Streptococcus suis surface-antigen recognition by antibodies and bacterial elimination is influenced by capsular polysaccharide structure. Front Cell Infect Microbiol 13 , 1228496 (2023). https://doi.org:10.3389/fcimb.2023.1228496 Yuan, Z.-z. et al. Molecular mechanism by which surface antigen HP0197 mediates host cell attachment in the pathogenic bacteria Streptococcus suis. Journal of Biological Chemistry 288 , 956-963 (2013). Zhang, C., Yu, Y., Yang, M. & Jiang, Y. Expression, purification, crystallization and structure determination of the N terminal domain of Fhb, a factor H binding protein from Streptococcus suis. Biochemical and biophysical research communications 466 , 413-417 (2015). Cullen, R. et al. The 1.7 Å crystal structure of the C5a peptidase from Streptococcus agalactiae (ScpB) reveals an active site competent for catalysis. Proteins: Structure, Function, and Bioinformatics 92 , 427-431 (2024). Lecours, M. P. et al. Immune-responsiveness of CD4(+) T cells during Streptococcus suis serotype 2 infection. Sci Rep 6 , 38061 (2016). https://doi.org:10.1038/srep38061 Letendre, C. et al. Streptococcus suis Serotype 2 Infection Impairs Interleukin-12 Production and the MHC-II-Restricted Antigen Presentation Capacity of Dendritic Cells. Front Immunol 9 , 1199 (2018). https://doi.org:10.3389/fimmu.2018.01199 Xing, X. et al. Intranasal Vaccination With Multiple Virulence Factors Promotes Mucosal Clearance of Streptococcus suis Across Serotypes and Protects Against Meningitis in Mice. J Infect Dis 220 , 1679-1687 (2019). https://doi.org:10.1093/infdis/jiz352 Kim, H. W. et al. Opsonophagocytic Antibodies to Serotype Ia, Ib, and III Group B Streptococcus among Korean Infants and in Intravenous Immunoglobulin Products. Journal of Korean medical science 32 , 737-743 (2017). https://doi.org:10.3346/jkms.2017.32.5.737 Kim, G. R. et al. Serotype Distribution and Antimicrobial Resistance of Streptococcus pneumoniae Causing Invasive Pneumococcal Disease in Korea Between 2017 and 2019 After Introduction of the 13-Valent Pneumococcal Conjugate Vaccine. Ann Lab Med 43 , 45-54 (2023). https://doi.org:10.3343/alm.2023.43.1.45 Zhang, A. et al. Identification of a surface protective antigen, HP0197 of Streptococcus suis serotype 2. Vaccine 27 , 5209-5213 (2009). de Greeff, A. et al. Contribution of fibronectin-binding protein to pathogenesis of Streptococcus suis serotype 2. Infection and immunity 70 , 1319-1325 (2002). Li, J. et al. Evaluation of the immunogenicity and the protective efficacy of a novel identified immunogenic protein, SsPepO, of Streptococcus suis serotype 2. Vaccine 29 , 6514-6519 (2011). Feng, Y. et al. Streptococcus suis enolase functions as a protective antigen displayed on the bacterial cell surface. The Journal of infectious diseases 200 , 1583-1592 (2009). Yan, Z. et al. Immunogenicity and Protective Capacity of Sugar ABC Transporter Substrate-Binding Protein against Streptococcus suis Serotype 2, 7 and 9 Infection in Mice. Vaccines 12 , 544 (2024). Jiang, X. et al. Peptidyl isomerase PrsA is surface-associated on Streptococcus suis and offers cross-protection against serotype 9 strain. FEMS Microbiology Letters 366 , fnz002 (2019). Tan, C. et al. Vaccination with Streptococcus suis serotype 2 recombinant 6PGD protein provides protection against S. suis infection in swine. FEMS microbiology letters 296 , 78-83 (2009). Zhou, Y. et al. Evaluation of the protective efficacy of four novel identified membrane associated proteins of Streptococcus suis serotype 2. Vaccine 33 , 2254-2260 (2015). Du, H. et al. The genetically modified suilysin, rSLYP353L, provides a candidate vaccine that suppresses proinflammatory response and reduces fatality following infection with Streptococcus suis. Vaccine 31 , 4209-4215 (2013). Zhang, M. et al. Inhibition of polysome assembly enhances imatinib activity against chronic myelogenous leukemia and overcomes imatinib resistance. Molecular and cellular biology 28 , 6496-6509 (2008). Additional Declarations No competing interests reported. Supplementary Files SuppleFigure01.png Supplementary Figure 1. Structural localization of selected B- and T-cell epitopes in ATOMSSUIS penta vaccine antigens. Predicted CTL (left), HTL (middle), and B cell (right) epitopes were mapped onto the 3D structures of each antigen used in the ATOMSSUIS penta vaccine construct. Structures were modeled using AlphaFold3 and visualized in PyMOL. Three representative epitopes- classified by color as CD8⁺ T cell epitope (red), CD4⁺ T helper epitope (yellow), and B cell epitope (magenta), are highlighted on the surface of each protein. Each column represents a distinct epitope location on the same protein, demonstrating spatial distribution of immunodominant T and B cell epitopes. Surface accessibility and spatial separation of these epitopes were considered during epitope domain selection for multimeric vaccine design. ATOMSSUISpentaMSSupplemntaryTable.docx 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-7215039","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":503221903,"identity":"34b54352-f063-4873-990a-cc89f13ed8aa","order_by":0,"name":"Sun-Young Kim","email":"","orcid":"","institution":"Korea Atomic Energy Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Sun-Young","middleName":"","lastName":"Kim","suffix":""},{"id":503221904,"identity":"a798c323-955e-4601-ac7d-d42291840218","order_by":1,"name":"Fengjia Chen","email":"","orcid":"","institution":"Korea Atomic Energy Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Fengjia","middleName":"","lastName":"Chen","suffix":""},{"id":503221905,"identity":"009ffbb6-d2c4-40e5-9f75-7dd5a92cdb61","order_by":2,"name":"Woo-Sik Kim","email":"","orcid":"","institution":"Korea Research Institute of Bioscience and Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Woo-Sik","middleName":"","lastName":"Kim","suffix":""},{"id":503221906,"identity":"167d4dd2-b1f1-448a-9002-74b10bbb30c1","order_by":3,"name":"Hyun Jung Ji","email":"","orcid":"","institution":"Korea Atomic Energy Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Hyun","middleName":"Jung","lastName":"Ji","suffix":""},{"id":503221907,"identity":"97f4caf8-f727-4010-8ec4-bb8e5598540a","order_by":4,"name":"Min-Kyu Kim","email":"","orcid":"","institution":"Korea Atomic Energy Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Min-Kyu","middleName":"","lastName":"Kim","suffix":""},{"id":503221908,"identity":"7c1c5511-0f0b-4203-a5f8-b167da93b2b4","order_by":5,"name":"Hae Ran Park","email":"","orcid":"","institution":"Korea Atomic Energy Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Hae","middleName":"Ran","lastName":"Park","suffix":""},{"id":503221909,"identity":"2cc47e7a-7753-4b41-9865-409a550884fc","order_by":6,"name":"Charles Euloge Lamien","email":"","orcid":"","institution":"International Atomic Energy Agency","correspondingAuthor":false,"prefix":"","firstName":"Charles","middleName":"Euloge","lastName":"Lamien","suffix":""},{"id":503221912,"identity":"d8e78fe3-6f15-493a-8f76-4b7df443648e","order_by":7,"name":"Viskam Wijewardana","email":"","orcid":"","institution":"International Atomic Energy Agency","correspondingAuthor":false,"prefix":"","firstName":"Viskam","middleName":"","lastName":"Wijewardana","suffix":""},{"id":503221914,"identity":"ce8b5f61-5357-42a5-ac47-b5f3f96e891f","order_by":8,"name":"Kyung-Hyo Kim","email":"","orcid":"","institution":"Ewha Womans University Mokdong Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kyung-Hyo","middleName":"","lastName":"Kim","suffix":""},{"id":503221915,"identity":"d2f1a308-f3f7-4a7c-bd9d-b745f1ddb11f","order_by":9,"name":"Ki Bum Ahn","email":"","orcid":"","institution":"Korea Atomic Energy Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Ki","middleName":"Bum","lastName":"Ahn","suffix":""},{"id":503221916,"identity":"3d0f7933-7ceb-408b-9f6d-73717fa4569f","order_by":10,"name":"Ho Seong Seo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYBACAwST+TCI5AERjA3EaWFLJlkLjzGciVeLOXv7wwcfd9Ta87P3fDb4UXFPhkH68AHGmXtwa7HsOWNsOPPM8cSZPWc3J/acKeZh4EtLYNzwDI/DbuSwSfO2HUswuJG7+QBvWwIPAw+PAeODA/i0pD//DdRib38j5/HBv/9AWvg/ENCSYMbM21bDuEEihzmZtwFsCwPjBnxazpwxlpzZdiBxxpljxsYyxxJ42HjYDA7OwKflePvDDx/b6uz525sfS76pSbDn52F++LAHjxYoOIxgsgExYQ0MDHVEqBkFo2AUjIIRCwADkFHlv1Q14QAAAABJRU5ErkJggg==","orcid":"","institution":"Korea Atomic Energy Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Ho","middleName":"Seong","lastName":"Seo","suffix":""}],"badges":[],"createdAt":"2025-07-25 14:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7215039/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7215039/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89854733,"identity":"09c8d718-fdc6-47df-b95f-48fde6ba2512","added_by":"auto","created_at":"2025-08-25 18:33:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3064853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign of the multimeric subunit vaccine ATOMSSUIS\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003epenta\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e. (A)\u003c/strong\u003e Amino acid sequence of ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e, composed of immunogenic domains derived from five \u003cem\u003eS. suis\u003c/em\u003e antigens. Each colored block represents an epitope-rich domain: HP0197 (light purple), Fnbp (cyan), Sao (orange), ScpB (light green), and SLY (dark purple). White regions indicate flexible linker sequences used for domain connection. \u003cstrong\u003e(B)\u003c/strong\u003e Secondary structure prediction results from PRISPRED and \u003cstrong\u003e(C)\u003c/strong\u003e from GOR IV.\u003c/p\u003e","description":"","filename":"Figure01.png","url":"https://assets-eu.researchsquare.com/files/rs-7215039/v1/ba1476b57bf256cc0e22284f.png"},{"id":89855157,"identity":"e8c9323e-4329-47dc-a4f9-ea8e2a0e7e4a","added_by":"auto","created_at":"2025-08-25 18:41:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6576943,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural analysis and construction of the ATOMSSUIS\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003epenta\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e. (A)\u003c/strong\u003e Predicted tertiary structure of ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e using AlphaFold3. Each domain is color-coded as in Figure 1A, illustrating the spatial organization and separation of the antigenic regions. \u003cstrong\u003e(B)\u003c/strong\u003e Ramachandran plot analysis by PROCHECK . \u003cstrong\u003e(C)\u003c/strong\u003e ProSA server analysis shows the Z-score of the final refined model of ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e. \u003cstrong\u003e(D)\u003c/strong\u003e Solubility prediction of the designed vaccine construct using the Protein-Sol server, compared to the population average across the analyzed datasets. \u003cstrong\u003e(E)\u003c/strong\u003e Recombinant ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e was expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) and purified via affinity chromatography. SDS-PAGE analysis confirmed the expression of the ~90 kDa multimeric protein (indicated by red arrow).\u003c/p\u003e","description":"","filename":"Figure02.png","url":"https://assets-eu.researchsquare.com/files/rs-7215039/v1/d1d49b4c593cb547697fcc30.png"},{"id":89854735,"identity":"8262a5ea-d0de-4b73-b6d0-4c7ace43583c","added_by":"auto","created_at":"2025-08-25 18:33:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":400715,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative analysis of adjuvants for induction of humoral and cellular immune responses by ATOMSSUIS\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003epenta\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e. \u003c/strong\u003eBALB/c mice were subcutaneously immunized with ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e (10 µg) formulated with one of four adjuvants: Monophosphoryl lipid A(MPL)/dimethyldioctadecylammonium (DDA), alum hydroxide, ISA1313, and Montanide gel. Immune responses were evaluated two weeks after the booster immunization. \u003cstrong\u003e(A)\u003c/strong\u003e Total IgG levels specific to ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e were measured by ELISA. All adjuvant groups showed significantly increased antibody responses compared to the PBS control. \u003cstrong\u003e(B)\u003c/strong\u003e Frequencies of IFN-γ-producing CD4\u003csup\u003e+\u003c/sup\u003e T cells were assessed by intracellular cytokine staining. MPL/DDA elicited the strongest Th1-type cellular response. (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001), determined by unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Figure03.png","url":"https://assets-eu.researchsquare.com/files/rs-7215039/v1/7b47438948fb8a50f0cc3ebc.png"},{"id":89855158,"identity":"4b33c774-a9be-4088-8ad9-98495d60606d","added_by":"auto","created_at":"2025-08-25 18:41:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1550705,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHumoral immune responses induced by ATOMSSUIS\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003epenta\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e vaccination. \u003c/strong\u003eC57BL/6 mice (n = 5 per group) were subcutaneously immunized twice, two weeks interval, with 5, 10, or 20 µg of ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e formulated with MPL (10 µg) and DDA (250 µg). \u003cstrong\u003e(A)\u003c/strong\u003e Antigen-specific antibody responses (IgG, IgM, IgG2a, and IgG1) were measured by ELISA using sera collected two weeks after the second immunization. ELISA plates were coated with each of the five individual vaccine components (HP0197, Fnbp, Sao, C5a peptidase and SLY). \u003cstrong\u003e(B)\u003c/strong\u003e Total antibody responses (IgG, IgG2a, and IgG1) against the full-length ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e protein. \u003cstrong\u003e(C)\u003c/strong\u003e Anti-\u003cem\u003eS. suis\u003c/em\u003e serotype 2 whole-cell IgG responses, determined by ELISA using formalin-fixed bacteria as the coating antigen. Data are presented as mean ± SD. Each dot represents an individual mouse. Statistical analysis was performed using unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. Asterisks indicate statistically significant differences compared to the MPL/DDA only group (*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001), determined by unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Figure04.png","url":"https://assets-eu.researchsquare.com/files/rs-7215039/v1/07846154b83d3cb48baf7cc1.png"},{"id":89853758,"identity":"8d9e540b-8c31-406f-ab65-2548d5ec2e50","added_by":"auto","created_at":"2025-08-25 18:17:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1469513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular immune responses induced by ATOMSSUIS\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003epenta\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e vaccination. \u003c/strong\u003eC57BL/6 mice (n = 5 per group) were subcutaneously immunized twice, two weeks apart, with ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e (5, 10, or 20 µg) formulated with MPL (10 µg) and DDA (250 µg). \u003cstrong\u003e(A)\u003c/strong\u003e Two weeks after the final immunization, splenocytes were harvested and stimulated \u003cem\u003ein vitro\u003c/em\u003e with individual vaccine antigens (HP0197, Fnbp, Sao, C5a peptidase or SLY; 20 µg/mL) in the presence of GolgiStop/GolgiPlug. Intracellular cytokine staining was performed to quantify the frequencies of IFN-γ\u003csup\u003e+\u003c/sup\u003e, IL-17\u003csup\u003e+\u003c/sup\u003e, and IL-5\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells, as well as IFN-γ\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells. \u003cstrong\u003e(B)\u003c/strong\u003e Splenocytes from immunized mice were stimulated with the full-length ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e protein (20 µg/mL), and cytokine-producing T cell subsets were analyzed by flow cytometry. \u003cstrong\u003e(C)\u003c/strong\u003e Splenocytes were cultured with ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e for 72 hours, and cytokine secretion (IFN-γ, IL-17, and IL-5) was quantified in the culture supernatants using ELISA. Data are shown as mean ± SD from four or five individual mice per group. Each dot represents a single mouse. Statistical comparisons were made using unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 indicate significant differences compared to the MPL/DDA only control group.\u003c/p\u003e","description":"","filename":"Figure05.png","url":"https://assets-eu.researchsquare.com/files/rs-7215039/v1/c4fe935c4f4b8f64fb532c1f.png"},{"id":89854434,"identity":"ee8bd762-0b44-4216-a2cc-3e47ec18b923","added_by":"auto","created_at":"2025-08-25 18:25:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3033384,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultifunctional T cell responses promoted by ATOMSSUIS\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003epenta\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e vaccination. \u003c/strong\u003eC57BL/6 mice (n = 5 per group) were subcutaneously immunized twice at a two-week interval with ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e (5, 10, or 20 µg) formulated with MPL (10 µg) and DDA (250 µg). Two weeks after the final immunization, splenocytes were harvested and stimulated \u003cem\u003ein vitro\u003c/em\u003e with ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e (20 µg/mL) in the presence of GolgiStop and GolgiPlug. \u003cstrong\u003e(A)\u003c/strong\u003e Gating strategy used to identify multifunctional CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells producing IFN-γ, TNF-α, and IL-2 by intracellular cytokine staining and flow cytometry. \u003cstrong\u003e(B)\u003c/strong\u003e Frequency and functionality of CD4\u003csup\u003e+\u003c/sup\u003e T cells producing one, two, or three cytokines (1\u003csup\u003e+\u003c/sup\u003e, 2\u003csup\u003e+\u003c/sup\u003e, 3\u003csup\u003e+\u003c/sup\u003e) following stimulation with ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e. Pie charts represent the proportion of multifunctional CD4⁺ T cell subsets in each group. \u003cstrong\u003e(C)\u003c/strong\u003e Frequency and functionality of CD8\u003csup\u003e+\u003c/sup\u003e T cells producing IFN-γ, TNF-α, and/or IL-2. Pie charts display the distribution of cytokine-producing CD8⁺ T cell subsets. Data are shown as mean ± SD from five mice per group. Each dot represents an individual animal. Statistical analysis was performed using unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 compared to the MPL/DDA-only control group.\u003c/p\u003e","description":"","filename":"Figure06.png","url":"https://assets-eu.researchsquare.com/files/rs-7215039/v1/9352dd83f8c4ab7fcf763d64.png"},{"id":89854440,"identity":"bbccce37-e9a9-4e04-a134-1c8d72a63244","added_by":"auto","created_at":"2025-08-25 18:25:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":7470673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe protective effect of ATOMSSUIS\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003epenta\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStreptococcus suis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection. \u003c/strong\u003eC57BL/6 mice (n = 6 per group) were subcutaneously immunized twice at a two-week interval with ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e (20 or 50 µg) formulated with MPL (10 µg) and DDA (250 µg). \u003cstrong\u003e(A)\u003c/strong\u003e Two weeks after the final immunization, mice were challenged intraperitoneally with a mild dose (5 × 10\u003csup\u003e7\u003c/sup\u003e CFU) or a lethal dose (1.5 × 10\u003csup\u003e8\u003c/sup\u003e CFU) of \u003cem\u003eS. suis\u003c/em\u003e serotype 2. Body weight changes were monitored for 10 days post-infection. Survival rates were analyzed using the Kaplan–Meier method, and statistical significance was determined using the log-rank (Mantel–Cox) test. \u003cstrong\u003e(B)\u003c/strong\u003e Bacterial burdens in the brain, lung, and spleen were quantified at 2 days post-challenge (dpc) by serial dilution plating and CFU enumeration. \u003cstrong\u003e(C)\u003c/strong\u003e Histopathological changes in lung and brain tissues were examined using hematoxylin and eosin (H\u0026amp;E) staining. Tissues were evaluated for inflammation, edema, necrosis, and immune cell infiltration using a semi-quantitative histology scoring system (0 = no lesion, 1 = mild, 2 = moderate, 3 = severe), and results were averaged per organ. PBS-treated control mice exhibited severe pathological changes, whereas vaccinated groups displayed significantly reduced histological scores. Data are expressed as mean ± SD (n = 6). Statistical comparisons for CFU and histology scores were performed using unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 versus PBS control group.\u003c/p\u003e","description":"","filename":"Figure07.png","url":"https://assets-eu.researchsquare.com/files/rs-7215039/v1/a5916bf584e1185eea16d18b.png"},{"id":89854438,"identity":"d349456e-9edd-4b99-9d7b-0a25bc8610fd","added_by":"auto","created_at":"2025-08-25 18:25:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":641269,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCross-reactive antibody responses induced by ATOMSSUIS\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003epenta\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e immunization. \u003c/strong\u003eC57BL/6 mice (n = 6 per group) were subcutaneously immunized twice at a two-week interval with ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e (20 µg) formulated with MPL (10 µg) and DDA (250 µg). \u003cstrong\u003e(A)\u003c/strong\u003e Two weeks after the second immunization, sera were collected, and IgG antibody levels against five \u003cem\u003eS. suis\u003c/em\u003e serotypes (2, 4, 9, 14, and 25) were measured by ELISA using formalin-inactivated whole-cell antigens. \u003cstrong\u003e(B)\u003c/strong\u003e To evaluate the functional activity of vaccine-induced antibodies, a piglet whole blood killing assay was performed using \u003cem\u003eS. suis\u003c/em\u003e serotype 2 (clinical strain BAA-853), and standard strains of serotypes 2, 4, 9, 14, and 25. Immune sera from vaccinated mice enhanced bacterial clearance against serotypes 2, 4, and 9, but not against serotypes 14 or 25. Data are presented as mean ± SD (n = 6). Each dot represents an individual mouse. Statistical analysis was performed using unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 versus PBS control group.\u003c/p\u003e","description":"","filename":"Figure08.png","url":"https://assets-eu.researchsquare.com/files/rs-7215039/v1/9c97b0ad900ebd76bd1eca45.png"},{"id":92008736,"identity":"98ec9bcc-8f91-4747-8ba8-9f7bde636397","added_by":"auto","created_at":"2025-09-23 15:32:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27898090,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7215039/v1/c289a840-11be-4b1d-b326-ef0e91bd6295.pdf"},{"id":89854436,"identity":"f1f4a653-888e-45ac-b01f-a4d749a0cf04","added_by":"auto","created_at":"2025-08-25 18:25:26","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4205057,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. Structural localization of selected B- and T-cell epitopes in ATOMSSUIS\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003epenta\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e vaccine antigens. \u003c/strong\u003ePredicted CTL (left), HTL (middle), and B cell (right) epitopes were mapped onto the 3D structures of each antigen used in the ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e vaccine construct. Structures were modeled using AlphaFold3 and visualized in PyMOL. Three representative epitopes- classified by color as CD8⁺ T cell epitope (red), CD4⁺ T helper epitope (yellow), and B cell epitope (magenta), are highlighted on the surface of each protein. Each column represents a distinct epitope location on the same protein, demonstrating spatial distribution of immunodominant T and B cell epitopes. Surface accessibility and spatial separation of these epitopes were considered during epitope domain selection for multimeric vaccine design.\u003c/p\u003e","description":"","filename":"SuppleFigure01.png","url":"https://assets-eu.researchsquare.com/files/rs-7215039/v1/56fc1e2131af26397cfd48e2.png"},{"id":89854431,"identity":"9a834ef9-76d5-4e32-8fcf-d22bc6028914","added_by":"auto","created_at":"2025-08-25 18:25:26","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":25705,"visible":true,"origin":"","legend":"","description":"","filename":"ATOMSSUISpentaMSSupplemntaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-7215039/v1/0810adadb232ce9561914b58.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEpitope-Based Multimeric Subunit Vaccine (ATOMSSUIS\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003epenta\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e) Confers Cross-Protection Against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStreptococcus suis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Infection\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003e\u003cem\u003eStreptococcus suis\u003c/em\u003e (\u003cem\u003eS. suis\u003c/em\u003e) is an important zoonotic pathogen that causes substantial economic losses in the swine industry and poses a serious public health threat due to its ability to cause severe invasive infections in humans \u003csup\u003e1,2\u003c/sup\u003e. \u003cem\u003eS. suis\u003c/em\u003e was first identified in 1954 and has been known to cause meningitis, endocarditis, arthritis, and septicemia in post-weaned piglets \u003csup\u003e3,4\u003c/sup\u003e. Although human \u003cem\u003eS. suis\u003c/em\u003e infections were initially considered rare and sporadic, first reported in Denmark in 1968 \u003csup\u003e5,6\u003c/sup\u003e, a major outbreak occurred in 2005 in Sichuan Province, China, resulting in 215 confirmed cases and 38 deaths across 202 villages \u003csup\u003e7,8\u003c/sup\u003e. Southeast Asia has become the region with the highest incidence of human \u003cem\u003eS. suis\u003c/em\u003e infections, largely associated with occupational or dietary exposure to pigs and pork products \u003csup\u003e9,10\u003c/sup\u003e. This unprecedented outbreak underscored the zoonotic potential of \u003cem\u003eS. suis\u003c/em\u003e and marked the pathogen as an emerging threat to global public health.\u003c/p\u003e\u003cp\u003eTo date, 35 serotypes of \u003cem\u003eStreptococcus suis\u003c/em\u003e have been identified based on immunogenic differences in their capsular polysaccharide antigens \u003csup\u003e1\u003c/sup\u003e. Although serotype 2 is recognized as the most prevalent and virulent serotype in both pig and human globally, effective control of \u003cem\u003eS. suis\u003c/em\u003e infection remains challenging due to the regional variability in circulating serotypes \u003csup\u003e1,11\u003c/sup\u003e. For instance, serotypes 1, 1/2, 2, 3, 5, 7, and 14 are frequently reported in North America; serotypes 1/2, 2, 3, and 6 in South America; serotypes 2, 4, 7, and 9 in Europe; and serotypes 2, 3, and 4 in Asia \u003csup\u003e11\u003c/sup\u003e. The distribution of dominant serotypes continues to evolve over time and across geographic regions, complicating both diagnosis and prevention. Although antibiotics such as tetracyclines, beta-lactams, and macrolides are commonly used to treat \u003cem\u003eS. suis\u003c/em\u003e infections in pigs \u003csup\u003e12,13\u003c/sup\u003e, the increasing prevalence of antimicrobial resistance, particularly to tetracyclines, macrolides, and lincosamides, has limited their effectiveness, emphasizing the need for alternative control strategies \u003csup\u003e13\u0026ndash;17\u003c/sup\u003e. Consequently, considerable efforts have been directed toward developing cross-protective vaccine platforms capable of targeting multiple serotypes to enhance the global management of \u003cem\u003eS. suis\u003c/em\u003e infections.\u003c/p\u003e\u003cp\u003eVaccination is considered a promising strategy to reduce antibiotic use and induce long-term immune memory for preventing infections \u003csup\u003e18\u0026ndash;20\u003c/sup\u003e. However, inactivated \u003cem\u003eS. suis\u003c/em\u003e vaccines, while widely used in pig farms, exhibit limited efficacy \u003csup\u003e21,22\u003c/sup\u003e. For example, Blouin et al. demonstrated that a formalin-inactivated \u003cem\u003eS. suis\u003c/em\u003e serotype 2 vaccine failed to elicit protective antibody responses in piglets, and did not confer passive protection in a mouse challenge model \u003csup\u003e23\u003c/sup\u003e. Similarly, field trials using a licensed formalin-inactivated autogenous vaccine (serotype 7, strain 1750775) showed no induction of measurable immune responses in piglets and failed to transfer maternal immunity \u003csup\u003e24\u003c/sup\u003e. Live-attenuated \u003cem\u003eS. suis\u003c/em\u003e vaccines have been investigated, with some temperature-sensitive and conditionally replicating mutants of \u003cem\u003eS. suis\u003c/em\u003e serotypes 1/2, 1, 2, and 3, showing protection against homologous strains in mice \u003csup\u003e25,26\u003c/sup\u003e. However, other attenuated strains, including non-encapsulated or serum opacity factor-deficient mutants of serotype 2, failed to induce protective opsonizing antibodies in pigs \u003csup\u003e27,28\u003c/sup\u003e. Indeed, concerns remain regarding their safety in the field due to the potential for reversion to virulence through mutation or recombination. In addition, these serotype specific vaccines offer limited cross-protection due to their serotype-specific nature. As a result, the emergence of infections caused by non-vaccine serotypes may remain a major concern in vaccinated herds.\u003c/p\u003e\u003cp\u003eSubunit vaccines provide several advantages over inactivated and live-attenuated vaccines, particularly in terms of safety, specific immune targeting, and standardized manufacturing \u003csup\u003e28\u0026ndash;30\u003c/sup\u003e. In principle, subunit vaccines based on conserved antigens across \u003cem\u003eS. suis\u003c/em\u003e serotypes can offer serotype-independent cross-protective immunity. Surface antigen one (Sao), suilysin (SLY), muramidase-released protein (MRP), and extracellular factor (EF) are examples of conserved proteins that have been widely studied as subunit vaccine candidates \u003csup\u003e31\u0026ndash;33\u003c/sup\u003e. For instance, vaccination with a truncated Sao (Sao-L) showed cross-protective effects against serotypes 1, 2, and 7, suggesting the potential of conserved-antigen-based subunit vaccines as universal strategies against \u003cem\u003eS. suis\u003c/em\u003e infections \u003csup\u003e31\u003c/sup\u003e. In recent years, additional surface-exposed proteins such as HP0197, fibronectin-binding protein (Fnbp), and C5a peptidase (ScpB) have also been evaluated for their immunogenicity and protective efficacy in animal models \u003csup\u003e34\u0026ndash;36\u003c/sup\u003e. However, despite these promising findings, subunit vaccines face several limitations. Their production costs are generally higher than those of traditional whole-cell vaccines, and a single protein antigen often fails to induce sufficient protective immunity \u003csup\u003e37\u003c/sup\u003e. As a result, multicomponent subunit vaccines combining several antigens, are being actively explored to overcome these limitations and enhance the wide protective immune response \u003csup\u003e38\u003c/sup\u003e. However, the identification of protective antigens capable of eliciting robust and cross-protective immune responses remains a major challenge. To address this, a growing number of studies have employed \u003cem\u003ein silico\u003c/em\u003e approaches that integrate computational immunology, structural biology, and systems vaccinology to accelerate antigen discovery and vaccine design \u003csup\u003e29,39\u0026ndash;42\u003c/sup\u003e. These \u003cem\u003ein silico\u003c/em\u003e pipelines commonly utilize predictive algorithms to identify T cell and B cell epitopes based on peptide binding affinity to MHC molecules, population coverage, and immunogenic potential \u003csup\u003e40,41,43,44\u003c/sup\u003e. In parallel, 3D structural modeling and molecular docking techniques are applied to evaluate surface accessibility, stability, and antigen-antibody interactions of candidate proteins \u003csup\u003e42,45\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we developed a novel multimeric subunit vaccine candidate, ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e, against \u003cem\u003eS. suis\u003c/em\u003e, by integrating bioinformatic tools with known immunologically relevant antigens. The vaccine construct comprises five conserved proteins: HP0197, fibronectin-binding protein (Fnbp), surface antigen one (Sao), C5a peptidase (ScpB), and suilysin (SLY), which were selected based on their sequence conservation, surface localization, and predicted immunogenicity. Using an \u003cem\u003ein silico\u003c/em\u003e approach, we analyzed the protein structure, solubility, and T and B cell epitopes to optimize the design of the multimeric antigen. The immunogenic potential of ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e was evaluated in a murine infection model, demonstrating its ability to elicit strong humoral and cellular immune responses. Importantly, the vaccine conferred cross-protective efficacy against multiple clinically relevant \u003cem\u003eS. suis\u003c/em\u003e serotypes, highlighting its potential as a broadly protective vaccine platform.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea Atomic Energy Research Institute (KAERI; approval no. IACUC-2021-05, IACUC-2022-08, IACUC-2023-07) and all procedures were performed in accordance with the veterinary standards of the KAERI Animal Care Center (RI-BIOMICS SPF Animal Facility). In mice infection model, mice were examined daily for clinical signs, including weight loss and general health condition. Animals that experienced a weight loss exceeding 25% of their baseline body weight, as well as those designated for scheduled tissue collection, were euthanized by carbon dioxide (CO₂) inhalation using a gradually filled chamber.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReagents\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTryptic soy broth (TSB) and Luria-Bertani (LB) broth were purchased from Difco (Franklin Lakes, NJ, USA). RPMI-1640 medium, fetal bovine serum (FBS), and penicillin-streptomycin were obtained from Biowest (Nuaill\u0026eacute;, France) and HyClone (Logan, UT, USA), respectively. Monophosphoryl lipid A (MPL), dimethyldioctadecylammonium (DDA) bromide, and BCA protein assay kit were sourced from Sigma-Aldrich (Saint Louis, MO, USA). Alhydrogel\u0026reg; was obtained from InvivoGen (San Diego, CA, USA), and Montanide\u0026trade; ISA 1313 and Montanide\u0026trade; Gel adjuvants from Seppic (Paris, France). The Live/Dead viability staining kit was purchased from Invitrogen (Carlsbad, CA, USA). GolgiStop, GolgiPlug, fixation/permeabilization solution, and the following antibodies were obtained from BD Biosciences (San Diego, CA, USA): PE-labeled anti-mouse IFN-\u0026gamma;, APC-labeled anti-mouse IL-5, APC-labeled anti-mouse TNF-\u0026alpha;, and V450-labeled anti-mouse CD44. Additional antibodies, APC-Cy7-anti-mouse CD3e, Alexa488-anti-mouse CD4, PerCP-Cy5.5-anti-mouse CD8, PE-Cy7-anti-mouse IL-17A, and PE-Cy7-anti-mouse IL-2, were purchased from eBioscience (San Diego, CA, USA). HRP-conjugated anti-mouse IgM, IgG, IgG1, and IgG2a were from Southern Biotech (Birmingham, AL, USA). TMB substrate and stop solution (1N HCl) were obtained from Thermo Fisher Scientific (Waltham, MA, USA), and high-binding 96-well ELISA plates from Corning (Corning, NY, USA). Formaldehyde solution was purchased from JUNSEI (Tokyo, Japan), and H\u0026amp;E staining reagents from Agilent Dako (Santa Clara, CA, USA). Porcine whole blood was sourced from Innovative Research (Sarasota, FL, USA). All other chemical reagents were obtained from Sigma-Aldrich.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructure modeling and validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 2D structure of ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e was modeled using PSIPRED v.4.0 \u003csup\u003e46\u003c/sup\u003e and GOR IV \u003csup\u003e47\u003c/sup\u003e. PSIPRED employs position-specific scoring matrices for precise sequence homology identification, while GOR IV utilizes information theory and Bayesian statistics to provide complementary insights. AlphaFold3 \u003csup\u003e48\u003c/sup\u003e was used for 3D structure prediction and the resulting model was further refined using the GalaxyRefine webserver \u003csup\u003e49\u003c/sup\u003e. Model validation was conducted using ProSA-web for Z-score validation \u003csup\u003e50\u003c/sup\u003e and Ramachandran plot analysis with PROCHECK to assess stereochemical properties \u003csup\u003e51\u003c/sup\u003e. The final 3D model was visualized with PyMOL3.1 to examine structural details (www.pymol.org).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePurification of recombinant \u003cem\u003eS. suis\u003c/em\u003e proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003cem\u003e\u0026nbsp;BL21(DE3)\u0026nbsp;\u003c/em\u003estrains harboring pET28a plasmids encoding HP0197, Fnbp, Sao, ScpB, SLY, or the multimeric ATOMSSUISpenta construct were cultured in LB broth at 37 \u0026deg;C until the optical density at 600 nm (OD₆₀₀) reached 0.6-0.8. Protein expression was induced by the addition of 0.2 mM isopropyl \u0026beta;-D-1-thiogalactopyranoside (IPTG; Biosesang, Seongnam, Republic of Korea), followed by overnight incubation at 16 \u0026deg;C. After induction, bacterial cells were harvested by centrifugation at 7,000 rpm for 20 minutes at 4 \u0026deg;C, resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5; 0.2 M NaCl; 1 mM phenylmethylsulfonyl fluoride), and lysed by sonication for 10min (15s pulse on/off) at 40% amplitude using a Vibra-Cell Ultrasonic Processor VC-505 (Sonics \u0026amp; Materials, Inc., Newtown, CT, USA).. The lysates were clarified by centrifugation at 17,000 rpm for 30 minutes at 4 \u0026deg;C, and the resulting supernatant was applied to a nickel-nitrilotriacetic acid (Ni-NTA; Invitrogen, Carlsbad, CA, USA) affinity chromatography column. The column was washed with binding buffer (20 mM Tris-HCl, pH 7.5; 0.2 M NaCl; 30 mM imidazole), and the bound proteins were eluted with elution buffer containing 500 mM imidazole. Eluted proteins were concentrated, and their concentrations were quantified using a BCA Protein Assay Kit (Sigma-Aldrich)..\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunization and Bacterial Challenge\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix-week-old female C57BL/6 mice were purchased from Orient Bio (Seongnam, South Korea). After a one-week acclimatization period, mice (n = 6 per group) were immunized twice at two-week intervals via subcutaneous (s.c.) injection with ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e (5, 10, or 20 \u0026micro;g) formulated with MPL (10 \u0026micro;g) and DDA (250 \u0026micro;g). For the protective efficacy studies, immunized mice (n = 6 per group) were challenged two weeks after the booster immunization with either 5 \u0026times; 10⁷ or 1.5 \u0026times; 10⁸ colony-forming units (CFU)/mouse of \u003cem\u003eStreptococcus suis\u003c/em\u003e BAA-853 (serotype 2). Body weight and survival were monitored daily for 10 days post-challenge (dpc). For histopathological analysis and bacterial load determination, mice were challenged with 1 \u0026times; 10⁸ CFU/mouse. At 2 dpc, the left lungs and brains were collected for hematoxylin and eosin (H\u0026amp;E) staining, and the right brain, right lung, and entire spleen were harvested for quantification of bacterial burden by CFU counting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of antibody responses and cytokine levels by ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples were collected from immunized mice two weeks after the booster dose, and serum was obtained by centrifugation at 5,000 rpm for 10 minutes at room temperature. To assess \u003cem\u003eS. suis\u003c/em\u003e-specific antibody levels, heat-inactivated \u003cem\u003eS. suis\u003c/em\u003e (10⁶ CFU/well) were immobilized onto 96-well ELISA plates. The wells were blocked with 5% skim milk in PBS, and serially diluted sera were added. After a 2-hour incubation, the wells were washed with PBST (0.05% Tween 20 in PBS), followed by the addition of horseradish peroxidase (HRP)-conjugated anti-mouse IgG, IgG1, or IgG2a antibodies. Following an additional 1-hour incubation and subsequent washing, plates were developed using TMB substrate solution, and the reaction was stopped with 2N sulfuric acid. Absorbance was measured at 450 nm using an Epoch 2 microplate reader (BioTek Instruments; Winooski, VT, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSpleens were harvested from mice two weeks after the booster immunization, and single-cell suspensions were prepared by mechanical dissociation through a 40 \u0026micro;m cell strainer in RPMI-1640 complete medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin. Red blood cells were lysed using RBC lysis buffer for 5 minutes at room temperature. After cell number counting, 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e splenocytes per well were seeded in 96-well plates and stimulated with\u0026nbsp;ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e or individual subunit proteins (20 \u0026micro;g/mL) for 72 hours. Supernatants were collected,\u0026nbsp;and concentrations of IFN-\u0026gamma;, IL-5, and IL-17A were measured using ELISA kits (eBioscience; San Diego, CA, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry analysis of T cell cytokine profiles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle-cell suspensions were prepared from the spleens of booster-immunized mice as described above. After cell counting, 4 \u0026times; 10⁶ cells per well were seeded in 48-well plates containing RPMI-1640 complete medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 \u0026mu;g/mL streptomycin. Cells were stimulated with ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e or individual subunit proteins (20 \u0026mu;g/mL) in the presence of GolgiPlug and GolgiStop (1:2000 dilution) for 8 hours at 37 \u0026deg;C in a CO₂ incubator. After stimulation, cells were stained with a live/dead fixable viability dye, followed by surface staining with APC-Cy7-conjugated anti-mouse CD3e, Alexa488-conjugated anti-mouse CD4, PerCP-Cy5.5-conjugated anti-mouse CD8, and V450-conjugated anti-mouse CD44 antibodies. The cells were then fixed and permeabilized using a fixation/permeabilization solution for 15 minutes at 4 \u0026deg;C. Subsequently, intracellular cytokines were stained using the following fluorochrome-conjugated antibodies, such as PE-labeled anti-mouse IFN-\u0026gamma;, APC-labeled anti-mouse IL-5, PE-Cy7-labeled anti-mouse IL-17A, PE-Cy7-labeled anti-mouse IL-2, and APC-labeled anti-mouse TNF-\u0026alpha;. For multifunctional T cell analysis, Boolean Gating was applied to identify CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells co-expressing two or more cytokines among IFN-\u0026gamma;, IL-2, and TNF-\u0026alpha;. The stained cells were acquired on a MACSQuant\u0026reg; flow cytometer (Miltenyi Biotec; Bergisch Gladbach, North Rhine‑Westphalia, Germany) and analyzed using FlowJo software (TreeStar Inc.; Ashland, OR, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematoxylin and eosin (H\u0026amp;E) staining\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe left lungs and brains of mice were collected and fixed in 4% paraformaldehyde in PBS for 24 hours at room temperature. Tissues were then washed under running tap water for 2 hours, dehydrated sequentially in graded ethanol solutions (70%, 80%, 90%, 95%, and 100%), and cleared with xylene. The samples were embedded in paraffin, sectioned at 5 \u0026mu;m thickness, followed by mounting onto slides by heating at 60 \u0026deg;C for 30 minutes. Paraffin sections were dewaxed with xylene and rehydrated through a descending ethanol series (100%, 95%, 90%, 80%, and 70%), followed by rinsing with tap water. The sections were stained with hematoxylin for 5 minutes and eosin for 30 seconds. After staining, tissues were dehydrated with ethanol (70%, 95%, and 100%) and cleared again with xylene. The slides were sealed with mounting medium, and images were acquired at 40\u0026times; and 200\u0026times; magnifications using a Motic EasyScan digital slide scanner (Motic, Xiamen, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole blood killing assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe whole blood killing assay was performed with minor modifications from a previously described protocol \u003csup\u003e52\u003c/sup\u003e. \u003cem\u003eS. suis\u003c/em\u003e BAA-853 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and standard serotype strains (serotypes 2, 4, and 9) were provided by the Animal and Plant Quarantine Agency, Republic of Korea. The strains were cultured in tryptic soy broth (TSB) to the exponential growth phase and washed twice with sterile phosphate-buffered saline (PBS). Bacterial suspensions were adjusted to 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL in PBS. For each reaction, 10 \u0026micro;L of diluted bacteria was mixed with 50 \u0026micro;L of PBS, serum from either unimmunized or ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e-immunized mice in a 96-well plates. After incubation at 37 \u0026deg;C for 30 minutes, 100 \u0026micro;L of heparinized piglet whole blood was added to each well and further incubated for 1 hour at 37 \u0026deg;C with gentle shaking. Following incubation, the reaction mixtures were serially diluted in a round bottom 96 well plate and spotted onto blood agar plates. After 24 hours of incubation at 37 \u0026deg;C, colony-forming units (CFU) were enumerated. The percentage of bacterial survival was calculated using the following formula:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"637\" height=\"75\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBLAST-based prevalence analysis of selected \u003cem\u003eStreptococcus suis\u003c/em\u003e antigen epitopes in GeneBank Genomic Data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe prevalence of five candidate antigen epitopes (HP0197, Fnbp, Sao, ScpB, and SLY) was evaluated across 388 \u003cem\u003eS. suis\u003c/em\u003e genomes from GeneBank Genomic Data. A genomic database was constructed from all assemblies and indexed using makeblastdb (BLAST+ v2.15.0) \u003csup\u003e53,54\u003c/sup\u003e. Reference protein sequences for both full-length antigens and their conserved \u0026quot;highlight\u0026quot; domains were aligned against the nucleotide database using tblastn \u003csup\u003e53,54\u003c/sup\u003e with an E-value threshold of 1 \u0026times; 10\u003csup\u003e-5\u003c/sup\u003e. Resulting alignments were processed with a custom Python script using the pandas \u003csup\u003e55,56\u003c/sup\u003e and Biopython libraries \u003csup\u003e57\u003c/sup\u003e. Antigens were defined as present if at least one hit per genome satisfied the following criteria: \u0026ge;70% identity and \u0026ge;80% query coverage for full-length sequences, or \u0026ge;60% identity and \u0026ge;50% coverage for highlight domains. These thresholds were selected as described previous Streptococcal studies \u003csup\u003e58\u003c/sup\u003e. Prevalence was calculated as the percentage of genomes containing a positive match. All analysis scripts and a reproducible Docker environment are publicly available on GitHub (https://github.com/leejhhh/suis-antigen-prevalence/tree/main).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as the mean \u0026plusmn; standard deviation (SD) from at least three independent replicates per treatment group. Statistical significance was determined using the unpaired Student\u0026rsquo;s t-test for all group comparisons. Asterisks indicate statistical significance relative to the control group (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e\u0026nbsp;P\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003e\u0026nbsp;P\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eIdentification of\u003c/b\u003e \u003cb\u003eS. suis\u003c/b\u003e \u003cb\u003eprotein vaccine candidates\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo develop a multimeric protein-based vaccine against \u003cem\u003eS. suis\u003c/em\u003e, we initially conducted a comprehensive literature review covering the past 30 years of vaccine research involving animal models (mouse or swine). Using deep research algorithms, including the ChatGPT-assisted data mining tools, we identified over 12 potential protein antigens reported to induce protective immunity (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among the identified candidates, we prioritized proteins containing the LPxTG motif, a well-characterized cell wall-anchoring domain essential for surface localization and immune recognition \u003csup\u003e59\u003c/sup\u003e. Based on this criterion, three major surface-anchored proteins, HP0197, fibronectin-binding protein (Fnbp), and surface antigen one (Sao) were selected due to their frequent identification in multiple serotypes and their previously reported immunogenicity. To enhance the surface coverage of the vaccine, C5a peptidase was included as a fourth LPxTG-containing protein. Although C5a peptidase has not yet been studied extensively in \u003cem\u003eS. suis\u003c/em\u003e, it has been shown to be a potent virulence factor and a promising vaccine antigen in other \u003cem\u003eStreptococcus\u003c/em\u003e species, including \u003cem\u003eS. agalactiae\u003c/em\u003e and \u003cem\u003eS. pyogenes\u003c/em\u003e \u003csup\u003e60\u0026ndash;62\u003c/sup\u003e. Genomic analyses indicate that homologs of C5a peptidase (ScpB) are also widely conserved in \u003cem\u003eS. suis\u003c/em\u003e, suggesting its potential as a cross-protective antigen \u003csup\u003e63,64\u003c/sup\u003e. In addition to the four surface proteins, suilysin (SLY) was selected as the fifth antigen. Although SLY expression is strain-dependent and not conserved among all \u003cem\u003eS. suis\u003c/em\u003e isolates, its role as a cholesterol-dependent cytolysin has been associated with strong immunogenicity and pathogenicity \u003csup\u003e65,66\u003c/sup\u003e. Importantly, previous studies demonstrated that streptococcal toxins such as SLY (\u003cem\u003eS. suis\u003c/em\u003e), erythrogenic toxins (Group A streptococcus), pneumolysin (\u003cem\u003eS. pneumoniae\u003c/em\u003e) may act as adjuvants, promoting robust immune responses and providing protection through toxin neutralization \u003csup\u003e67,68\u003c/sup\u003e. Therefore, SLY was incorporated not only as a protective antigen but also to enhance the overall immunogenicity of the multimeric vaccine (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eSummary of\u003c/b\u003e \u003cb\u003eStreptococcus suis\u003c/b\u003e \u003cb\u003eprotein vaccine candidates.\u003c/b\u003e Previously reported protein antigens evaluated as vaccine candidates against \u003cem\u003eS. suis\u003c/em\u003e were summarized. For each antigen, the corresponding animal model, observed protective immunity, and key references are listed. Protective outcomes include antibody production, survival after lethal challenge, reduction of bacterial burden, and evidence of cross-protection against multiple serotypes. Antigens include surface-exposed proteins (e.g., HP0197, Sao), virulence factors (e.g., suilysin, ScpB), adhesins (e.g., Fnbp, Lmb), enzymes (e.g., SsEno, PrsA), and transporters (e.g., S-ABC). Reported models encompass both murine and porcine systems.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProtein Antigen\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAnimal Model\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eProtective Immunity Observed\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHP0197\u003c/b\u003e (surface antigen)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMice and pigs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eImmunization with HP0197 provided significant protection in both mice and pig lethal challenge models, improving survival and enhancing opsonophagocytic killing through hyperimmune serum.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e99\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSao\u003c/b\u003e (Surface Antigen One)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMice and pig\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSubcutaneous immunization with recombinant Sao protein plus Quil A adjuvant induced strong IgG responses (particularly IgG2a) and 100% survival after lethal \u003cem\u003eS. suis\u003c/em\u003e challenge.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e31\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFnbp\u003c/b\u003e (Fibronectin-binding protein)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMice\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInduced high levels of FBPS (Fnbp)-specific IgG and IgG2a antibodies. Immunized mice showed 100% survival against lethal \u003cem\u003eS. suis\u003c/em\u003e serotype 2 challenge. Significantly reduced bacterial burden in blood and organs after infection.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e100\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSsPepO\u003c/b\u003e (secreted endopeptidase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMice and pigs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSsPepO immunization elicited strong antibody responses and provided significant protection against lethal \u003cem\u003eS. suis\u003c/em\u003e serotype 2 challenge in both mice and pig models.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e101\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eEnolase (SsEno)\u003c/b\u003e (glycolytic enzyme)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMice\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eThe glycolytic enzyme SsEno induced robust antibody responses and conferred complete protection against \u003cem\u003eS. suis\u003c/em\u003e serotype 2 in mice, with observed cross-protection against serotypes 7 and 9.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e102\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLmb\u003c/b\u003e (laminin-binding protein)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMice\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eVaccination with Lmb generated significant antibody responses and provided partial protection (~\u0026thinsp;50% survival) against virulent \u003cem\u003eS. suis\u003c/em\u003e serotype 2 in mice.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e103\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePrsA\u003c/b\u003e (peptidyl-prolyl isomerase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMice\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eImmunization with PrsA elicited high antibody titers and conferred partial protection, with approximately 50% survival in serotype 2 and 66% in serotype 9 \u003cem\u003eS. suis\u003c/em\u003e challenges, indicating potential for cross-protection.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e104\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e6PGD\u003c/b\u003e (6-phosphogluconate dehydrogenase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePigs (swine)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eVaccination with recombinant 6PGD provided significant protection in piglets, with reduced clinical disease upon \u003cem\u003eS. suis\u003c/em\u003e serotype 2 challenge.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e105\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHtpS\u003c/b\u003e (histidine triad protein)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMice\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eImmunization with HtpS (a surface-exposed protein) induced protective immunity, with significantly higher survival versus control in \u003cem\u003eS. suis\u003c/em\u003e serotype 2 mouse challenge​.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e103\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS-ABC\u003c/b\u003e (sugar ABC transporter substrate-binding protein)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMice\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eImmunization with S-ABC resulted in high protection, with approximately 87.5% survival against serotype 2 and 100% protection against serotype 9 challenge in mice. Moderate protection (~\u0026thinsp;50% survival) was also observed.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e103\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSbp\u003c/b\u003e (immunogenic membrane protein \u0026ldquo;Sbp\u0026rdquo;)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMice\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIdentified via immunoproteomics, Sbp elicited a strong antibody response and significantly protected mice from lethal \u003cem\u003eS. suis\u003c/em\u003e serotype 2 infection. Anti-Sbp serum enhanced bacterial clearance in the blood and conferred passive protection.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e106\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003erSLY(P353L)\u003c/b\u003e (Genetically modified Suilysin)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMice\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eImmunization with rSLY(P353L) led to reduced inflammatory responses and decreased mortality following \u003cem\u003eS. suis\u003c/em\u003e infection.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e107\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eScpB\u003c/b\u003e (C5a peptidase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMice\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eImmunization with ScpB (a C5a peptidase) induced strong systemic and mucosal IgG and IgA responses, resulting in 100% survival against intranasal GBS challenge and reduced bacterial colonization and dissemination.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003csup\u003e108\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eProtein vaccine candidates selected for the construction of ATOMSSUIS\u003c/b\u003e\u003csub\u003e\u003cb\u003epenta\u003c/b\u003e\u003c/sub\u003e. This table lists the five protein antigens incorporated into the ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e multimeric subunit vaccine. For each antigen, the molecular weight (MW), subcellular localization, predicted or known biological function, prevalence among \u003cem\u003eStreptococcus suis\u003c/em\u003e isolates, and corresponding NCBI protein accession numbers are provided. Four of the selected antigens (HP0197, Fnbp, Sao, ScpB) are LPxTG motif-anchored surface proteins, and one (SLY) is a secreted cholesterol-dependent cytolysin. These proteins were chosen based on their reported immunogenicity, structural features, and potential to elicit protective immune responses.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProtein\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMW (kDa)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSubcellular Location\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFunction\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePrevalence\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAccession #\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHP0197\u003c/b\u003e (surface antigen)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e~\u0026thinsp;21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCell surface\u003c/p\u003e\u003cp\u003e(LPxTG anchor)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAdhesion to host GAGs;\u003c/p\u003e\u003cp\u003eimmune evasion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHigh in virulent strains\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eWP_277937340.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFnbp\u003c/b\u003e (Fibronectin-binding protein)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e55\u0026thinsp;~\u0026thinsp;60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCell surface\u003c/p\u003e\u003cp\u003e(LPxTG anchor)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBinds fibronectin;\u003c/p\u003e\u003cp\u003efacilitates adhesion/invasion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eBroadly present\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eWP_014636551.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSao\u003c/b\u003e (Surface Antigen One)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e58\u0026thinsp;~\u0026thinsp;70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCell surface\u003c/p\u003e\u003cp\u003e(LPxTG anchor)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eImmunodominant antigen;\u003c/p\u003e\u003cp\u003eunknown exact function\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHigh in ST1/ST7 strains\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eWP_211840080.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eScpB\u003c/b\u003e (C5a peptidase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e120\u0026thinsp;~\u0026thinsp;130\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCell surface\u003c/p\u003e\u003cp\u003e(LPxTG anchor)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eC5a peptidase;\u003c/p\u003e\u003cp\u003ecomplement inhibition\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHigh in \u003cem\u003eStreptococcus\u003c/em\u003e spp.\u003c/p\u003e\u003cp\u003eBroadly present in \u003cem\u003eS. suis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eWP_240208248.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSLY\u003c/b\u003e (Suilysin)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e~\u0026thinsp;54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSecreted toxin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCholesterol-dependent cytolysin;\u003c/p\u003e\u003cp\u003ecauses inflammation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e70\u0026ndash;85% in virulent strains\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAIG43067.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEpitope-based selection of immunogenic domains for the construction of the multimeric vaccine\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo design the multimeric protein vaccine (ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e), immunogenic domains were selected through comprehensive in \u003cem\u003ein silico\u003c/em\u003e prediction of T-cell and B-cell epitopes, following by domain filtering based on conservation, solubility, and structural features. T-cell epitopes were initially predicted using the Immune Epitope Database (IEDB), and only those with a conservancy rate above 90% across \u003cem\u003eStreptococcus suis\u003c/em\u003e strains were retained. Both MHC class I and II binding epitopes were selected based on their affinity to common human leukocyte antigen (HLA) alleles: HLA-A24:02, HLA-A02:01 for MHC class I; and HLA-DRB109:01 for MHC class II, which are highly prevalent in East Asian populations \u003csup\u003e52,69\u003c/sup\u003e. The top-ranking predicted helper T-cell epitopes were localized to the following regions: residues 144\u0026ndash;315 (Fnbp), 159\u0026ndash;173 (Sao), 658\u0026ndash;672 (ScpB), and 367\u0026ndash;381 (SLY). Similarly, the most immunogenic cytotoxic T-cell epitopes were predicted at residues 84\u0026ndash;142 (HP0197), 168\u0026ndash;191 (Fnbp), 110\u0026ndash;271 (Sao), 662\u0026ndash;747 (ScpB), and 370\u0026ndash;473 (SLY) (Supplementary Table\u0026nbsp;1; Supplementary Fig.\u0026nbsp;1). B-cell epitopes were predicted using the ElliPro \u003csup\u003e70\u003c/sup\u003e, and regions scoring above 0.700 were considered. The highest scoring linear B-cell epitopes were identified at residues 104\u0026ndash;151 (HP0197), 135\u0026ndash;329 (Fnbp), 77\u0026ndash;198 (Sao), 668\u0026ndash;755 (ScpB), and 388\u0026ndash;443 (SLY) (Supplementary Table\u0026nbsp;2; Supplementary Fig.\u0026nbsp;1). Overlapping regions enriched for both T- and B-cell epitopes were selected as the immunogenic domains for the final vaccine construct (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eImmunological Characteristics of Selected Antigenic Domain and Multimeric Protein Vaccine (ATOMSSUIS\u003c/b\u003e\u003csub\u003e\u003cb\u003epenta\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e).\u003c/b\u003e Molecular weight, antigenicity, allergenicity, toxicity, and solubility were analyzed by ProtParam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), ANTIGENpro (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://scratch.proteomics.ics.uci.edu/\u003c/span\u003e\u003cspan address=\"https://scratch.proteomics.ics.uci.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), VaxiJen (\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), AllerTOP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ddg-pharmfac.net/allertop_test/\u003c/span\u003e\u003cspan address=\"https://www.ddg-pharmfac.net/allertop_test/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), ToxinPred(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crdd.osdd.net/raghava/toxinpred/\u003c/span\u003e\u003cspan address=\"http://crdd.osdd.net/raghava/toxinpred/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and Protein-Sol (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://protein-sol.manchester.ac.uk/\u003c/span\u003e\u003cspan address=\"https://protein-sol.manchester.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), respectively.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eProtein\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDomain\u003c/p\u003e\u003cp\u003e(MW)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAntigenicity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAllergenicity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eToxicity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSolubility\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eANTIGENPro (VaxiJen)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAllerTOP\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eToxinPred\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eProtein-Sol\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHP0197\u003c/b\u003e (surface antigen)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e56\u0026ndash;199\u003c/p\u003e\u003cp\u003e(16.9 kDa)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.7834 (0.3424)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNon-allergen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNon-toxin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.855\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFnbp\u003c/b\u003e (Fibronectin-binding protein)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e126\u0026ndash;350\u003c/p\u003e\u003cp\u003e(23.1 kDa)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.8803 (0.6987)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNon-allergen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNon-toxin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.582\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSao\u003c/b\u003e (Surface Antigen One)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e64\u0026ndash;274\u003c/p\u003e\u003cp\u003e(23.7 kDa)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.8923 (0.6092)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNon-allergen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNon-toxin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.883\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eScpB\u003c/b\u003e (C5a peptidase)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e652\u0026ndash;762\u003c/p\u003e\u003cp\u003e(12.9 kDa)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.8198 (0.4746)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNon-allergen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNon-toxin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.740\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSLY\u003c/b\u003e (Suilysin)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e366\u0026ndash;475\u003c/p\u003e\u003cp\u003e(12.7 kDa)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.9256 (0.5199)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNon-allergen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNon-toxin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.801\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eATOMSSUIS\u003c/b\u003e\u003csub\u003e\u003cb\u003epenta\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e90.3 kDa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.9497 (0.7178)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNon-allergen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNon-toxin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.619\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe selected immunogenic domains were sequentially linked using flexible GGSGGGSG linkers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), and the resulting multimeric construct was evaluated for solubility, toxicity, antigenicity, and structural integrity (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Solubility was predicted using Protein-Sol \u003csup\u003e71\u003c/sup\u003e, toxicity was assessed via ToxinPred \u003csup\u003e72\u003c/sup\u003e. The secondary structure of the designed construct (ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e) was predicted using two widely used algorithms, PSIPRED \u003csup\u003e46\u003c/sup\u003e and GOR IV \u003csup\u003e47\u003c/sup\u003e. The average values of PSIPRED and GOR IV show that, ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e is composed of 26.97% α-helix, 47.66% coil, and 25.37% β-strand (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB \u0026amp; \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTertiary Structure Prediction and Validation of ATOMSSUIS\u003c/b\u003e\u003csub\u003e\u003cb\u003epenta\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003cp\u003eThe 3D structural coordinates of the ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e were generated using AlphaFold3 \u003csup\u003e48\u003c/sup\u003e and subsequently refined with the GalaxyRefine server \u003csup\u003e49\u003c/sup\u003e. Structural validation was performed using a Ramachandran plot via the PROCHECK server (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) \u003csup\u003e51\u003c/sup\u003e. The refined ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e models exhibited 95.4% of the residues in the most favored regions, indicating a high-quality stereochemical profile. Structural quality was further assessed using the ProSA server \u003csup\u003e50\u003c/sup\u003e. The ProSA server initially revealed a Z-score of -10.67 for the model (not shown), which improved to -10.87 following refinement, suggesting enhanced overall model reliability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). While changes in the domain arrangement had little impact on solubility and toxicity scores, the predicted tertiary structure varied substantially depending on the domain arrangement. Given the uncertain relationship between 3D conformation and immunogenicity, the final domain arrangement was chosen to minimize structural overlap and maximize spatial separation between domains. The finalized configuration of the ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e construct was HP0197-Fnbp-Sao-ScpB-SLY (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The final ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e construct was predicted to be antigenic, non-allergenic and non-toxic. Protein solubility prediction using Protein-Sol \u003csup\u003e71\u003c/sup\u003e yielded a favorable solubility score of 0.619 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), indicating potential for soluble expression in \u003cem\u003eE. coli\u003c/em\u003e. Antigenicity was assessed using both ANTIGENpro \u003csup\u003e73\u003c/sup\u003e and VaxiJen \u003csup\u003e74\u003c/sup\u003e, resulting in high predictive scores of 0.9497 and 0.7178, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), suggesting strong immunogenic potential. The synthetic gene encoding ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e sequence was cloned into an expression vector (pET28a) and successfully expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3). The recombinant protein, with an expected molecular weight of approximately 90kDa, was successfully purified and confirmed by SDS-PAGE analysis. Under standard expression conditions, the yield of purified ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e was approximately 12\u0026ndash;15 mg per liter of bacterial culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eComparative analysis of adjuvants for ATOMSSUIS\u003c/b\u003e\u003csub\u003e\u003cb\u003epenta\u003c/b\u003e\u003c/sub\u003e \u003cb\u003evaccine\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify the optimal adjuvant for the ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e subunit vaccine, various adjuvants were compared in terms of their ability to induce humoral and cellular immune responses. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, all adjuvant groups, including Monophosphoryl lipid A(MPL)/dimethyldioctadecylammonium (DDA), alum hydroxide, ISA1313, and Montanide gel, elicited comparable levels of antigen-specific IgG, indicating no significant difference in the humoral response among groups. However, in the analysis of cellular immunity, notable differences were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Specifically, MPL/DDA induced the highest frequencies of both CD4⁺ and CD8⁺ T cells producing IFN-γ, suggesting a stronger cellular immune response compared to the other adjuvants. These findings indicate that while humoral responses were generally consistent across adjuvants, MPL/DDA most effectively promoted T cell-mediated immunity, supporting its selection as the preferred adjuvant for subsequent vaccine formulations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eATOMSSUIS\u003c/b\u003e\u003csub\u003e\u003cb\u003epenta\u003c/b\u003e\u003c/sub\u003e \u003cb\u003evaccination elicits robust antigen-specific humoral immune responses in mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the antigenicity of the ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e vaccine, BALB/c mice were subcutaneously immunized with 5, 10, or 20 \u0026micro;g of ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e formulated with the Th1-promoting adjuvant MPL/DDA on days 0 and 14. Serum samples were collected two weeks after the booster dose to assess the humoral immune response. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, all dosage groups displayed significantly elevated levels of ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e-specific IgG, IgG2a, and IgM compared to the PBS control. IgG1 was significantly induced only in the 20 \u0026micro;g group, suggesting a Th1-skewed immune response, with limited Th2-type involvement at lower antigen doses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo compare the antigen specificity, sera were further analyzed for IgG, IgG2a, IgG1, and IgM titers against each of the five individual vaccine components (HP0197, Fnbp, Sao, ScpB and SLY). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e immunization markedly enhanced IgG, IgG2a, and IgG1 responses, particularly against ScpB, HP0197, and Sao. Similarly, antigen-specific IgM responses were elevated for ScpB, Fnbp, HP0197, and Sao. However, antibody responses to SLY remained minimal across all immunoglobulin subclasses. This weak immunogenicity of SLY may be attributed to its relatively small molecular size (113 amino acids), compared to other antigens and is consistent with previous reports indicating that bacterial toxins generally elicit weaker humoral responses \u003csup\u003e75\u003c/sup\u003e. To confirm the induction of \u003cem\u003eS. suis\u003c/em\u003e-specific immunity, ELISA plates were coated with whole \u003cem\u003eS. suis\u003c/em\u003e serotype 2 cells, and serum antibody titers were quantified. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, immunization with 20 \u0026micro;g ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e induced significantly higher levels of \u003cem\u003eS. suis\u003c/em\u003e-specific IgG, IgG2a, and IgG1 compared to the PBS group. These findings demonstrate that ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e elicits a robust systemic humoral immune response not only to the individual antigens but also to the intact bacterial pathogen.\u003c/p\u003e\u003cp\u003e\u003cb\u003eATOMSSUIS\u003c/b\u003e\u003csub\u003e\u003cb\u003epenta\u003c/b\u003e\u003c/sub\u003e \u003cb\u003evaccination promotes Th1/17 biased cellular immune responses in mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess vaccine-induced cellular immune responses, BALB/c mice were subcutaneously immunized with 5, 10, or 20 \u0026micro;g of ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e formulated with MPL/DDA on days 0 and 14. Two weeks after the booster immunization, splenocytes were harvested and stimulated \u003cem\u003ein vitro\u003c/em\u003e with the full-length ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e protein (20 \u0026micro;g/mL). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, stimulation with the full-length ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e protein led to a significant increase in CD4⁺ T cells producing IFN-γ or IL-17 across all dose groups, with the strongest responses observed in the 10 \u0026micro;g group. In contrast, the population of IL-5-producing CD4⁺ T cells remained unchanged, indicating no or weak Th2 response. Additionally, CD8⁺ T cells producing IFN-γ were markedly elevated, supporting a cytotoxic T cell response in a dose-dependent manner. These findings were further validated by quantifying cytokine levels in culture supernatants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). IFN-γ and IL-17 were robustly secreted by splenocytes from immunized mice, while IL-5 remained at relatively low levels. These results are consistent with the induction of a Th1- and Th17-skewed immune response by ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further explore the antigen-specificity of the T cell responses, splenocytes were stimulated with individual antigens (HP0197, Fnbp, Sao, ScpB or SLY), and intracellular cytokine staining was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Notably, CD4⁺ T cells producing IFN-γ or IL-17 were significantly increased in response to all five antigens, particularly ScpB, Fnbp, and SLY. IL-5-producing CD4⁺ T cells showed no substantial change in all groups. In addition, CD8⁺ T cells producing IFN-γ were significantly elevated upon stimulation with each antigen, with ScpB, Fnbp, and SLY again eliciting the strongest responses. Together, these data demonstrate that ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e vaccination elicits strong antigen-specific Th1 and Th17 responses, along with activation of cytotoxic CD8⁺ T cells, indicating its potential possibility to induce protective cellular immunity against \u003cem\u003eS. suis\u003c/em\u003e. In note, the increased frequencies of both CD4⁺ and CD8⁺ T cells, particularly in response to individual component antigens, suggest that each subunit within ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e contributes functionally to T cell activation. These findings are consistent with our \u003cem\u003ein silico\u003c/em\u003e predictions of T cell epitopes, supporting the rational design of the multimeric construct.\u003c/p\u003e\u003cp\u003e\u003cb\u003eATOMSSUIS\u003c/b\u003e\u003csub\u003e\u003cb\u003epenta\u003c/b\u003e\u003c/sub\u003e \u003cb\u003evaccination elicits robust multifunctional T cell responses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMultifunctional T cells capable of simultaneously producing multiple cytokines such as IFN-γ, TNF-α, and IL-2 are known to play critical roles in protective immunity against bacterial and viral infections, particularly at mucosal sites such as the lung \u003csup\u003e76,77\u003c/sup\u003e, surpassing the efficacy of monofunctional T cells owing to their superior survival potential \u003csup\u003e78,79\u003c/sup\u003e. In addition they can be, prolonged persistence within the memory T cell pool, and capacity to elicit robust effector responses characterized by increased cytokine production and functional versatility \u003csup\u003e78,80,81\u003c/sup\u003e. To evaluate whether ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e vaccination induces such multifunctional responses, BALB/c mice were subcutaneously immunized with ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e formulated with MPL/DDA. Two weeks after the booster dose, splenocytes were analyzed by intracellular cytokine staining and flow cytometry to assess the production of IFN-γ, TNF-α, and IL-2. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e immunization significantly increased the frequency of multifunctional CD4⁺ T cells expressing IFN-γ⁺ TNF-α⁺ IL-2⁺ and TNF-α⁺ IL-2⁺ compared to the PBS control. Additional populations of IFN-γ⁺ TNF-α⁺ and IFN-γ⁺ IL-2⁺ CD4⁺ T cells were also elevated, but relatively lower levels. Among single cytokine-producing CD4⁺ T cells, TNF-α⁺ and IL-2⁺ subsets were notably increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), indicating that the vaccine promotes both polyfunctional and monofunctional helper T cell responses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, we assessed the multifunctional CD8⁺ T cell responses elicited by ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e immunization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Vaccination induced a marked, dose-dependent increase in triple-positive IFN-γ⁺ TNF-α⁺ IL-2⁺ CD8⁺ T cells, with the highest frequencies observed at the 20 \u0026micro;g dose. A similar trend was seen for IFN-γ⁺ IL-2⁺ double-positive CD8⁺ T cells, which also increased significantly in a dose-dependent manner. In contrast, IFN-γ⁺ TNF-α⁺ CD8⁺ T cells showed only a modest elevation, primarily at the 10 \u0026micro;g dose, while TNF-α⁺ IL-2⁺ CD8⁺ T cells remained largely unchanged across all groups. Among the single-cytokine-producing CD8⁺ T cells, a substantial increase was observed only in the IFN-γ⁺ subset, whereas TNF-α⁺ and IL-2⁺ subsets exhibited minimal or no enhancement. These results highlight the ability of ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e to induce potent and polyfunctional CD8⁺ T cell responses, characterized predominantly by IFN-γ and IL-2 co-expression. Together with the multifunctional CD4⁺ T cell responses, these findings suggest that ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e vaccination promotes a robust and qualitatively superior cell-mediated immune response. This profile, marked by multifunctionality and coordinated cytokine production, highlights its potential to confer durable and effective protection against \u003cem\u003eS. suis\u003c/em\u003e infection.\u003c/p\u003e\u003cp\u003e\u003cb\u003eATOMSSUIS\u003c/b\u003e\u003csub\u003e\u003cb\u003epenta\u003c/b\u003e\u003c/sub\u003e \u003cb\u003econfers protective response against\u003c/b\u003e \u003cb\u003eStreptococcus suis\u003c/b\u003e \u003cb\u003einfection in mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the protective efficacy of ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e, C57BL/6 mice were subcutaneously immunized with the vaccine formulated with MPL/DDA. Two weeks following the final immunization, mice were challenged with either a mild dose (5 \u0026times; 10⁷ CFU) for monitoring body weight change or a severe dose (1.5 \u0026times; 10⁸ CFU) for survival rate of \u003cem\u003eS. suis\u003c/em\u003e BAA-853 (serotype 2) infection. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, both vaccinated and non-vaccinated mice exhibited weight loss of approximately 12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56% and 13.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51%, respectively, at 1day post-challenge (dpc). However, mice immunized with ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e recovered 90% of the lost weight within 3 days, whereas recovery in the PBS group was markedly delayed, requiring up to 10 days. In the lethal challenge model, all unvaccinated mice succumbed to infection, displaying severe clinical signs such as depression, ruffled hair coat, and labored breathing. In contrast, 5 out of 6 vaccinated mice survived, and only transient and mild clinical signs were observed during the early phase of infection, demonstrating a substantial protective effect.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo investigate bacterial dissemination, bacterial burdens were quantified in the brain, lung, and spleen at 2 dpc (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Non-immunized mice exhibited high bacterial loads (mean values of 4.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37, 4.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40, and 5.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 log₁₀ CFU/g in brain, lung, and spleen, respectively). In contrast, ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e-immunized mice showed significantly reduced bacterial counts in all examined organs. Histopathological analysis further confirmed the protective effect of vaccination (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Unvaccinated mice displayed pronounced histological signs of infection, including hyperemia and dense inflammatory cell infiltration in both brain and lung tissues. In contrast, tissues from vaccinated mice exhibited minimal pathology, with largely preserved architecture, minimal congestion, and reduced immune cell infiltration. Collectively, these data indicate that ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e vaccination confers strong protection against \u003cem\u003eS. suis\u003c/em\u003e infection by limiting bacterial burden and promoting tissue inflammation, thereby reducing the risk of severe clinical outcomes such as meningitis and pneumonia.\u003c/p\u003e\u003cp\u003e\u003cb\u003eATOMSSUIS\u003c/b\u003e\u003csub\u003e\u003cb\u003epenta\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eimmunization induces cross-reactive and cross-protective antibody responses against various\u003c/b\u003e \u003cb\u003eStreptococcus suis\u003c/b\u003e \u003cb\u003eserotypes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAlthough \u003cem\u003eS. suis\u003c/em\u003e serotype 2 is the most prevalent and virulent serotype globally, the presence of diverse serotypes across different regions poses a significant challenge to serotype independent vaccine strategies. To evaluate the cross-reactive antibody conferred by ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e vaccination, serum IgG responses were assessed against five representative \u003cem\u003eS. suis\u003c/em\u003e serotypes (2, 4, 9, 14, and 25). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e-immunized mice produced high titers of IgG antibodies that strongly recognized \u003cem\u003eS. suis\u003c/em\u003e serotypes 2, 4, and 9. However, the antibody reactivity to serotypes 14 and 25 was not significantly different from that of the control group, suggesting limited cross-reactivity to these serotypes. To further determine whether the vaccine-induced antibodies provide functional cross-neutralization, a piglet whole blood killing assay was performed using the same serotypes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, sera from ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e-immunized mice significantly enhanced bacterial clearance of serotypes 2, 4, and 9 compared to the PBS control, consistent with the observed IgG binding patterns. No opsonic killing was observed for serotypes 14 and 25. These findings indicate that ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e not only elicits serotype 2-specific immune responses but also induces cross-reactive and functionally protective antibody responses against multiple clinically relevant serotypes-most notably serotypes 4 and 9 \u003csup\u003e11,82\u003c/sup\u003e. This broad-spectrum reactivity underscores the potential of ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e as a promising cross-protective vaccine candidate. Moreover, the observed efficacy validates the robustness of our \u003cem\u003ein silico\u003c/em\u003e antigen selection strategy, demonstrating its effectiveness in designing broadly protective subunit vaccines against \u003cem\u003eS. suis\u003c/em\u003e and other bacterial target.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e\u003cem\u003eStreptococcus suis\u003c/em\u003e is a significant zoonotic pathogen that primarily colonizes the upper respiratory tract of pigs but can be transmitted to humans through direct contact with infected animals or consumption of undercooked pork products \u003csup\u003e83,84\u003c/sup\u003e. In recent years, human infections have been increasingly reported across several Asian countries, including China, Vietnam, and Thailand, where pork is a major dietary and pig farming is widespread \u003csup\u003e9,10\u003c/sup\u003e. This growing public health concern underscores the urgent need for effective preventive strategies, including the development of a broadly protective vaccine. The presence of more than 30 serotypes and geographical variations in serotype prevalence poses a major challenge to the development of broadly protective vaccines \u003csup\u003e85\u0026ndash;87\u003c/sup\u003e. In this study, we selected protein vaccine candidates based on previously reported \u003cem\u003ein vivo\u003c/em\u003e efficacy data and applied established bioinformatic tools to predict immunogenic B and T cell epitopes. Using this bioinformatic analysis data, we rationally designed a multimeric subunit vaccine, ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e, composed of five conserved antigens. Immunization with ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e elicited robust antigen-specific humoral responses against each component protein. Furthermore, when formulated with the Th1-skewing adjuvant MPL/DDA, it successfully induced cellular immune responses, including Th1 and Th17 subsets, which are known to play crucial roles in protection against \u003cem\u003eS. suis\u003c/em\u003e infection. These findings suggest that ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e represents a promising universal vaccine approach, overcoming the serotype-specific limitations of conventional vaccine strategies.\u003c/p\u003e\u003cp\u003eUniversal vaccine strategies often aim to target conserved protein antigens shared across multiple serotypes or clinical isolates \u003csup\u003e88,89\u003c/sup\u003e. However, extensive genetic and antigenic diversity across serotypes and genotypes complicates the identification of universal antigenic targets specifically in Gram-positive bacteria such as \u003cem\u003eStreptococcus suis\u003c/em\u003e \u003csup\u003e33,90\u003c/sup\u003e. To overcome this challenge, some studies have attempted to use a mixture of individually purified antigens, but this approach involves a complex GMP production process requiring separate purification of each antigen and necessitates individual evaluation of their immunogenicity and efficacy. Recently, epitope-based vaccine design using bioinformatic tools has gained attention as a rational strategy to reduce antigen size while enhancing specificity and immunogenicity \u003csup\u003e40,41,43,44\u003c/sup\u003e. In this study, we constructed a multimeric epitope antigen (90 kDa) by linking predicted B- and T-cell epitope domains from five conserved \u003cem\u003eS. suis\u003c/em\u003e antigens, with epitope selection uniquely based on surface-exposed regions identified through the tertiary structure information of HP0197 (PDB ID: 4FZ4) \u003csup\u003e91\u003c/sup\u003e, Fnbp (PDB ID 5BOB) \u003csup\u003e92\u003c/sup\u003e, ScpB (PDB ID: 8BTY) \u003csup\u003e93\u003c/sup\u003e, SLY (PDB ID: 3HVN) \u003csup\u003e32\u003c/sup\u003e from the PDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rcsb.org\u003c/span\u003e\u003cspan address=\"http://www.rcsb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) or the modelled structure of ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e from AlphaFold3 \u003csup\u003e48\u003c/sup\u003e. Importantly, \u003cem\u003ein vivo\u003c/em\u003e immunization demonstrated that this epitope-based design could elicit strong humoral, cellular, and protective responses against multiple serotypes. While previous \u003cem\u003ein silico\u003c/em\u003e designed vaccines have often shown suboptimal efficacy \u003cem\u003ein vivo\u003c/em\u003e, our findings highlight the feasibility of a structure-guided multiepitope vaccine approach. This strategy may extend beyond \u003cem\u003eS. suis\u003c/em\u003e, offering a platform for the development of cross-protective vaccines against other genetically diverse Gram-positive pathogens, including \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eTh1 responses play a crucial role in protection against \u003cem\u003eS. suis\u003c/em\u003e infection by enhancing bacterial clearance through cytokine-driven immune cell activation and promoting opsonophagocytosis \u003csup\u003e94\u003c/sup\u003e. These mechanisms are essential in preventing severe disease manifestations such as meningitis, septicemia, and streptococcal toxic shock-like syndrome (STSLS). In our study, ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e vaccination induced a strong Th1- and Th17-skewed immune response, consistent with previous reports that highlight the protective roles of these T cell subsets in streptococcal infections \u003csup\u003e94\u0026ndash;96\u003c/sup\u003e. Lecours et al. demonstrated that Th1 responses are predominant and critical during \u003cem\u003eS. suis\u003c/em\u003e serotype 2 infection, while IL-17-producing Th17 cells have been shown to facilitate bacterial clearance through neutrophil recruitment and the induction of antimicrobial proteins \u003csup\u003e94\u003c/sup\u003e. Despite the immunological profile observed, we were unable to directly confirm the functional efficacy of opsonophagocytic activity due to the lack of a standardized and validated OPKA (Opsonophagocytic Killing Assay) specifically optimized for \u003cem\u003eS. suis\u003c/em\u003e. In our previous studies, we successfully applied OPKA to evaluate functional antibody responses against \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e and \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e, using well-established assay systems \u003csup\u003e97,98\u003c/sup\u003e. However, these standard OPKA protocols could not be directly applied to \u003cem\u003eS. suis\u003c/em\u003e, likely due to differences in bacterial surface structure, phagocytic uptake, and complement susceptibility. This technical limitation hindered direct assessment of vaccine-induced opsonic activity, underscoring the need for development of a \u003cem\u003eS. suis\u003c/em\u003e-specific OPKA platform. In this study, we employed a whole blood killing assay, which successfully served as a surrogate method for evaluating opsonic activity. This assay captures the combined effects of functional antibodies, complement, phagocytic immune cells, and other essential blood components present in piglet whole blood, thereby providing an integrated measure of opsonophagocytic killing activity. By adopting this approach, we not only demonstrated the protective potential of ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e-induced antibodies, but also introduced a practical and adaptable platform for functional vaccine evaluation in \u003cem\u003eS. suis\u003c/em\u003e. This methodological advancement highlights an important contribution of this study and emphasizes the value of establishing standardized assays to assess vaccine-induced immune correlates of protection in \u003cem\u003eS. suis\u003c/em\u003e infection models.\u003c/p\u003e\u003cp\u003eAn emerging focus in vaccine immunology is the role of multifunctional T cells which can capable of simultaneously producing multiple cytokines such as IFN-γ, TNF-α, and IL-2, which are increasingly recognized as correlates of robust and durable protection against infectious diseases, especially at mucosal sites \u003csup\u003e76,77\u003c/sup\u003e. These multifunctional T cells exhibit superior survival and long-term persistence within the memory pool, a feature supported by their elevated expression of survival-associated molecules such as CD127 (IL-7Rα) and Bcl2 \u003csup\u003e78\u0026ndash;80\u003c/sup\u003e. In addition to enhanced longevity, they contribute to protective immunity by promoting strong effector responses and immunological versatility, and by enabling rapid and effective recall responses upon re-infection \u003csup\u003e78,80,81\u003c/sup\u003e. While multifunctional T cells have been studied in various bacterial infections, their role in mediating protective immunity against \u003cem\u003eS. suis\u003c/em\u003e remains poorly understood. In this study, we demonstrate that ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e vaccination adjuvanted with MPL/DDA induces a robust multifunctional T cell response, characterized by the co-expression of IFN-γ, TNF-α, and IL-2. The increased frequency of these multifunctional T cells, along with dual positive and monofunctional subsets, suggests that this vaccine not only expands antigen-specific helper T cells but also enhances their functional quality. Such qualitative enhancement of the T cell response is likely a key contributor to the ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e\u0026rsquo;s protective efficacy and represents a significant advance in the understanding of cellular immunity against \u003cem\u003eS. suis\u003c/em\u003e. To build on these findings, further studies should explore whether these cells directly correlate with protection and define their contribution to long-term immune memory.\u003c/p\u003e\u003cp\u003eNevertheless, genome-wide analysis using the \u003cem\u003eS. suis\u003c/em\u003e complete genome database (Taxonomy ID:1307, n\u0026thinsp;=\u0026thinsp;388) revealed that the five selected antigens are not uniformly conserved across clinical isolates. BLASTp analysis (\u0026ge;\u0026thinsp;80% identity) showed that HP0197, Fnbp, Sao, ScpB, and SLY were present in 38.1% (148 strains), 26.3% (102 strains), 66.2% (257 strains), 97.2% (377 strains), and 52.3% (203 strains), respectively. These findings indicate that \u003cem\u003eS. suis\u003c/em\u003e antigenic proteins are not highly conserved among clinical isolates, posing a major obstacle to universal vaccine development. Consistent with these genomic observations, our experimental results also suggest that single antigen might not be express in all clinical isolates. Thus, it is unlikely that a single antigen would be sufficient to elicit protective immunity against the diverse \u003cem\u003eS. suis\u003c/em\u003e population. To overcome this limitation, the use of multiple antigens in a multimeric construct is essential for broadening immune coverage and enhancing vaccine efficacy across genetically heterogeneous strains. Therefore, future antigen selection strategies should not only focus on immunogenicity but also consider the conservation across clinical strain genome database. Such an approach is essential for designing broadly protective vaccines against genetically variable pathogens. A limitation of the present study is that these genome conservation data were not incorporated during the antigen selection process. Nonetheless, the use of a multimeric antigen composed of multiple distinct proteins appears to have compensated for this variability, achieving broad immune coverage despite the heterogeneity among \u003cem\u003eS. suis\u003c/em\u003e strains.\u003c/p\u003e\u003cp\u003eIn summary, we developed a multimeric subunit vaccine, ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e, composed of five conserved \u003cem\u003eS. suis\u003c/em\u003e antigens selected based on known immunogenicity and predicted surface exposed B- and T-cell epitopes. The vaccine induced robust antigen-specific humoral and cellular immune responses, including Th1- and Th17-type immunity, and conferred protective efficacy in a murine infection model. Despite limitations such as the lack of a standardized OPKA for \u003cem\u003eS. suis\u003c/em\u003e and incomplete consideration of genome-wide antigen conservation during antigen selection, the multimeric design allowed for broad immune coverage across genetically diverse \u003cem\u003eS. suis\u003c/em\u003e strains. These findings support the potential of epitope-based multimeric vaccines as a promising platform for the development of broadly protective vaccines against \u003cem\u003eS. suis\u003c/em\u003e and potentially other antigenically diverse Gram-positive pathogens.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by the Internal R\u0026amp;D Program of the Korea Atomic Energy Research Institute (KAERI) (523140-24), funded by the Ministry of Science and ICT (MIST); by the Ministry of Food and Drug Safety (22202MFDS171 to K.H.K.); by the National Research Foundation of Korea (NRF) grant funded by the Korean government (RS-2022-00164721 to M.K.); and by the ZODIAC project of the International Atomic Energy Agency (IAEA) in the Asia and the Pacific region (CRP D32039, contract number: 26513 to H.S.S.).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.Y.K., F.C., and W.S.K. contributed equally to this work. H.S.S. and K.B.A. conceived and supervised the project. S.Y.K., F.C., and W.S.K. performed antigen selection, epitope prediction, and structural modeling (Figure 1-3). M.K.K. and H.R.P. conducted the mouse immunization and infection experiments (Figure 4-8). H.J.J. and C.E.L. conducted ELISA and flow cytometry analyses (Figure 5-8). V.W. and K.H.K. assisted in data interpretation and genomic analysis (Figure 1-2). S.Y.K. and F.C. drafted the manuscript. H.S.S. critically revised the manuscript. All authors read and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFeng, Y.\u003cem\u003e et al.\u003c/em\u003e Streptococcus suis infection: an emerging/reemerging challenge of bacterial infectious diseases? \u003cem\u003eVirulence\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 477-497 (2014). https://doi.org:10.4161/viru.28595\u003c/li\u003e\n\u003cli\u003eSegura, M., Fittipaldi, N., Calzas, C. \u0026amp; Gottschalk, M. Critical Streptococcus suis Virulence Factors: Are They All Really Critical? \u003cem\u003eTrends Microbiol\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 585-599 (2017). https://doi.org:10.1016/j.tim.2017.02.005\u003c/li\u003e\n\u003cli\u003eWertheim, H. F., Nghia, H. D., Taylor, W. \u0026amp; Schultsz, C. Streptococcus suis: an emerging human pathogen. \u003cem\u003eClin Infect Dis\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 617-625 (2009). https://doi.org:10.1086/596763\u003c/li\u003e\n\u003cli\u003eObradovic, M. R., Segura, M., Segales, J. \u0026amp; Gottschalk, M. Review of the speculative role of co-infections in Streptococcus suis-associated diseases in pigs. \u003cem\u003eVet Res\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 49 (2021). https://doi.org:10.1186/s13567-021-00918-w\u003c/li\u003e\n\u003cli\u003ePerch, B., Kristjansen, P. \u0026amp; Skadhauge, K. Group R streptococci pathogenic for man. Two cases of meningitis and one fatal case of sepsis. \u003cem\u003eActa Pathol Microbiol Scand\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, 69-76 (1968). \u003c/li\u003e\n\u003cli\u003eTang, J.\u003cem\u003e et al.\u003c/em\u003e Streptococcal toxic shock syndrome caused by Streptococcus suis serotype 2. \u003cem\u003ePLoS Med\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, e151 (2006). https://doi.org:10.1371/journal.pmed.0030151\u003c/li\u003e\n\u003cli\u003eYe, C.\u003cem\u003e et al.\u003c/em\u003e Streptococcus suis sequence type 7 outbreak, Sichuan, China. \u003cem\u003eEmerging infectious diseases\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1203 (2006). \u003c/li\u003e\n\u003cli\u003eYu, H.\u003cem\u003e et al.\u003c/em\u003e Human streptococcus suis outbreak, Sichuan, China. \u003cem\u003eEmerging infectious diseases\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 914 (2006). \u003c/li\u003e\n\u003cli\u003eRayanakorn, A., Goh, B.-H., Lee, L.-H., Khan, T. M. \u0026amp; Saokaew, S. Risk factors for Streptococcus suis infection: A systematic review and meta-analysis. \u003cem\u003eScientific reports\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 13358 (2018). \u003c/li\u003e\n\u003cli\u003eKerdsin, A. Human Streptococcus suis infections in Thailand: epidemiology, clinical features, genotypes, and susceptibility. \u003cem\u003eTropical medicine and infectious disease\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 359 (2022). \u003c/li\u003e\n\u003cli\u003eGoyette-Desjardins, G., Auger, J.-P., Xu, J., Segura, M. \u0026amp; Gottschalk, M. Streptococcus suis, an important pig pathogen and emerging zoonotic agent\u0026mdash;an update on the worldwide distribution based on serotyping and sequence typing. \u003cem\u003eEmerging microbes \u0026amp; infections\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 1-20 (2014). \u003c/li\u003e\n\u003cli\u003eDechene-Tempier, M.\u003cem\u003e et al.\u003c/em\u003e Update on the Mechanisms of Antibiotic Resistance and the Mobile Resistome in the Emerging Zoonotic Pathogen Streptococcus suis. \u003cem\u003eMicroorganisms\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e (2021). https://doi.org:10.3390/microorganisms9081765\u003c/li\u003e\n\u003cli\u003eHolmer, I.\u003cem\u003e et al.\u003c/em\u003e Antibiotic resistance in porcine pathogenic bacteria and relation to antibiotic usage. \u003cem\u003eBMC Vet Res\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 449 (2019). https://doi.org:10.1186/s12917-019-2162-8\u003c/li\u003e\n\u003cli\u003eLi, L. L.\u003cem\u003e et al.\u003c/em\u003e Antimicrobial resistance, serotypes, and virulence factors of Streptococcus suis isolates from diseased pigs. \u003cem\u003eFoodborne Pathog Dis\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 583-588 (2012). https://doi.org:10.1089/fpd.2011.1106\u003c/li\u003e\n\u003cli\u003eYongkiettrakul, S.\u003cem\u003e et al.\u003c/em\u003e Antimicrobial susceptibility of Streptococcus suis isolated from diseased pigs, asymptomatic pigs, and human patients in Thailand. \u003cem\u003eBMC Vet Res\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 5 (2019). https://doi.org:10.1186/s12917-018-1732-5\u003c/li\u003e\n\u003cli\u003eZhang, C.\u003cem\u003e et al.\u003c/em\u003e In vitro antimicrobial susceptibility of Streptococcus suis strains isolated from clinically healthy sows in China. \u003cem\u003eVet Microbiol\u003c/em\u003e \u003cstrong\u003e131\u003c/strong\u003e, 386-392 (2008). https://doi.org:10.1016/j.vetmic.2008.04.005\u003c/li\u003e\n\u003cli\u003eWisselink, H. J., Veldman, K. T., Van den Eede, C., Salmon, S. A. \u0026amp; Mevius, D. J. Quantitative susceptibility of Streptococcus suis strains isolated from diseased pigs in seven European countries to antimicrobial agents licensed in veterinary medicine. \u003cem\u003eVet Microbiol\u003c/em\u003e \u003cstrong\u003e113\u003c/strong\u003e, 73-82 (2006). https://doi.org:10.1016/j.vetmic.2005.10.035\u003c/li\u003e\n\u003cli\u003eBuchy, P.\u003cem\u003e et al.\u003c/em\u003e Impact of vaccines on antimicrobial resistance. \u003cem\u003eInt J Infect Dis\u003c/em\u003e \u003cstrong\u003e90\u003c/strong\u003e, 188-196 (2020). https://doi.org:10.1016/j.ijid.2019.10.005\u003c/li\u003e\n\u003cli\u003eClift, C. \u0026amp; Salisbury, D. M. Enhancing the role of vaccines in combatting antimicrobial resistance. \u003cem\u003eVaccine\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 6591-6593 (2017). https://doi.org:10.1016/j.vaccine.2017.09.053\u003c/li\u003e\n\u003cli\u003eMicoli, F., Bagnoli, F., Rappuoli, R. \u0026amp; Serruto, D. The role of vaccines in combatting antimicrobial resistance. \u003cem\u003eNat Rev Microbiol\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 287-302 (2021). https://doi.org:10.1038/s41579-020-00506-3\u003c/li\u003e\n\u003cli\u003eRieckmann, K., Pendzialek, S.-M., Vahlenkamp, T. \u0026amp; Baums, C. G. A critical review speculating on the protective efficacies of autogenous Streptococcus suis bacterins as used in Europe. \u003cem\u003ePorcine health management\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 1-11 (2020). \u003c/li\u003e\n\u003cli\u003eGhattas, M., Dwivedi, G., Lavertu, M. \u0026amp; Alameh, M.-G. Vaccine technologies and platforms for infectious diseases: Current progress, challenges, and opportunities. \u003cem\u003eVaccines\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1490 (2021). \u003c/li\u003e\n\u003cli\u003eBlouin, C., Higgins, R., Gottschalk, M. \u0026amp; Simard, J. Evaluation of the antibody response in pigs vaccinated against Streptococcus suis capsular type 2 using a double-antibody sandwich enzyme-linked immunosorbent assay. \u003cem\u003eCan J Vet Res\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 49-54 (1994). \u003c/li\u003e\n\u003cli\u003eCorsaut, L.\u003cem\u003e et al.\u003c/em\u003e Field Study on the Immunological Response and Protective Effect of a Licensed Autogenous Vaccine to Control Streptococcus suis Infections in Post-Weaned Piglets. \u003cem\u003eVaccines (Basel)\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e (2020). https://doi.org:10.3390/vaccines8030384\u003c/li\u003e\n\u003cli\u003eKebede, M., Chengappa, M. M. \u0026amp; Stuart, J. G. Isolation and characterization of temperature-sensitive mutants of Streptococcus suis: efficacy trial of the mutant vaccine in mice. \u003cem\u003eVet Microbiol\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 249-257 (1990). https://doi.org:10.1016/0378-1135(90)90112-9\u003c/li\u003e\n\u003cli\u003eFoster, N., Staats, J. J. \u0026amp; Chengappa, M. M. Isolation, characterization and protection studies in mice of a streptomycin-dependent mutant of Streptococcus suis type 1/2. \u003cem\u003eVet Res Commun\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 155-163 (1994). https://doi.org:10.1007/BF01839263\u003c/li\u003e\n\u003cli\u003eSmith, H.\u003cem\u003e et al.\u003c/em\u003e Identification and characterization of the cps locus of Streptococcus suis serotype 2: the capsule protects against phagocytosis and is an important virulence factor. \u003cem\u003eInfection immunity\u003c/em\u003e \u003cstrong\u003e67\u003c/strong\u003e, 1750-1756 (1999). https://doi.org:10.1128/iai.67.4.1750-1756.1999\u003c/li\u003e\n\u003cli\u003eBaums, C. G.\u003cem\u003e et al.\u003c/em\u003e Identification of a novel virulence determinant with serum opacification activity in Streptococcus suis. \u003cem\u003eInfect Immun\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, 6154-6162 (2006). https://doi.org:10.1128/IAI.00359-06\u003c/li\u003e\n\u003cli\u003eHou, Y.\u003cem\u003e et al.\u003c/em\u003e Advanced subunit vaccine delivery technologies: From vaccine cascade obstacles to design strategies. \u003cem\u003eActa Pharmaceutica Sinica B\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 3321-3338 (2023). \u003c/li\u003e\n\u003cli\u003eHeidary, M.\u003cem\u003e et al.\u003c/em\u003e A comprehensive review of the protein subunit vaccines against COVID-19. \u003cem\u003eFrontiers in microbiology\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 927306 (2022). \u003c/li\u003e\n\u003cli\u003eLi, Y.\u003cem\u003e et al.\u003c/em\u003e Immunization with recombinant Sao protein confers protection against Streptococcus suis infection. \u003cem\u003eClin Vaccine Immunol\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 937-943 (2007). https://doi.org:10.1128/CVI.00046-07\u003c/li\u003e\n\u003cli\u003eXu, L.\u003cem\u003e et al.\u003c/em\u003e Crystal structure of cytotoxin protein suilysin from Streptococcus suis. \u003cem\u003eProtein cell\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 96-105 (2010). https://doi.org:10.1007/s13238-010-0012-3\u003c/li\u003e\n\u003cli\u003eSegura, M. Streptococcus suis vaccines: candidate antigens and progress. \u003cem\u003eExpert Rev Vaccines\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1587-1608 (2015). https://doi.org:10.1586/14760584.2015.1101349\u003c/li\u003e\n\u003cli\u003eMa, F.\u003cem\u003e et al.\u003c/em\u003e Streptococcus suis Serotype 2 Biofilms Inhibit the Formation of Neutrophil Extracellular Traps. \u003cem\u003eFrontiers in cellular infection microbiology\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 86 (2017). https://doi.org:10.3389/fcimb.2017.00086\u003c/li\u003e\n\u003cli\u003eMusyoki, A.\u003cem\u003e et al.\u003c/em\u003e Structural and functional analysis of an anchorless fibronectin-binding protein FBPS from Gram-positive bacterium Streptococcus suis. \u003cem\u003eProceedings of the National Academy of Sciences of the United States of America\u003c/em\u003e \u003cstrong\u003e113\u003c/strong\u003e, 13869-13874 (2016). https://doi.org:10.1073/pnas.1608406113\u003c/li\u003e\n\u003cli\u003eLynskey, N.\u003cem\u003e et al.\u003c/em\u003e Multi-functional mechanisms of immune evasion by the streptococcal complement inhibitor C5a peptidase. \u003cem\u003ePLoS pathogens\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e1006493 (2017). https://doi.org:10.1371/journal.ppat.1006493\u003c/li\u003e\n\u003cli\u003eLi, J.\u003cem\u003e et al.\u003c/em\u003e Evaluation of the immunogenicity and the protective efficacy of a novel identified immunogenic protein, SsPepO, of Streptococcus suis serotype 2. \u003cem\u003eVaccine\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 6514-6519 (2011). https://doi.org:10.1016/j.vaccine.2011.07.010\u003c/li\u003e\n\u003cli\u003eWeisse, C.\u003cem\u003e et al.\u003c/em\u003e Immunogenicity and protective efficacy of a Streptococcus suis vaccine composed of six conserved immunogens. \u003cem\u003eVet Res\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 112 (2021). https://doi.org:10.1186/s13567-021-00981-3\u003c/li\u003e\n\u003cli\u003eOli, A. N.\u003cem\u003e et al.\u003c/em\u003e Immunoinformatics and vaccine development: an overview. \u003cem\u003eImmunoTargets and therapy\u003c/em\u003e, 13-30 (2020). \u003c/li\u003e\n\u003cli\u003eShey, R. A.\u003cem\u003e et al.\u003c/em\u003e In-silico design of a multi-epitope vaccine candidate against onchocerciasis and related filarial diseases. \u003cem\u003eScientific reports\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 4409 (2019). \u003c/li\u003e\n\u003cli\u003eMeza, B., Ascencio, F., Sierra-Beltr\u0026aacute;n, A. P., Torres, J. \u0026amp; Angulo, C. A novel design of a multi-antigenic, multistage and multi-epitope vaccine against Helicobacter pylori: an in silico approach. \u003cem\u003eInfection, Genetics and Evolution\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 309-317 (2017). \u003c/li\u003e\n\u003cli\u003eKardani, K., Bolhassani, A. \u0026amp; Namvar, A. An overview of in silico vaccine design against different pathogens and cancer. \u003cem\u003eExpert Review of Vaccines\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 699-726 (2020). \u003c/li\u003e\n\u003cli\u003ePotocnakova, L., Bhide, M. \u0026amp; Pulzova, L. B. An introduction to B‐cell epitope mapping and in silico epitope prediction. \u003cem\u003eJournal of immunology research\u003c/em\u003e \u003cstrong\u003e2016\u003c/strong\u003e, 6760830 (2016). \u003c/li\u003e\n\u003cli\u003eRosa, D. S., Ribeiro, S. P. \u0026amp; Cunha-Neto, E. CD4+ T cell epitope discovery and rational vaccine design. \u003cem\u003eArchivum immunologiae et therapiae experimentalis\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 121-130 (2010). \u003c/li\u003e\n\u003cli\u003eNegahdaripour, M.\u003cem\u003e et al.\u003c/em\u003e Structural vaccinology considerations for in silico designing of a multi-epitope vaccine. \u003cem\u003eInfection, Genetics and Evolution\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 96-109 (2018). \u003c/li\u003e\n\u003cli\u003eMcGuffin, L. J., Bryson, K. \u0026amp; Jones, D. T. The PSIPRED protein structure prediction server. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 404-405 (2000). https://doi.org:10.1093/bioinformatics/16.4.404\u003c/li\u003e\n\u003cli\u003eKouza, M., Faraggi, E., Kolinski, A. \u0026amp; Kloczkowski, A. The GOR Method of Protein Secondary Structure Prediction and Its Application as a Protein Aggregation Prediction Tool. \u003cem\u003eMethods Mol Biol\u003c/em\u003e \u003cstrong\u003e1484\u003c/strong\u003e, 7-24 (2017). https://doi.org:10.1007/978-1-4939-6406-2_2\u003c/li\u003e\n\u003cli\u003eAbramson, J.\u003cem\u003e et al.\u003c/em\u003e Accurate structure prediction of biomolecular interactions with AlphaFold 3. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e630\u003c/strong\u003e, 493-500 (2024). https://doi.org:10.1038/s41586-024-07487-w\u003c/li\u003e\n\u003cli\u003eHeo, L., Park, H. \u0026amp; Seok, C. GalaxyRefine: Protein structure refinement driven by side-chain repacking. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, W384-388 (2013). https://doi.org:10.1093/nar/gkt458\u003c/li\u003e\n\u003cli\u003eWiederstein, M. \u0026amp; Sippl, M. J. ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, W407-410 (2007). https://doi.org:10.1093/nar/gkm290\u003c/li\u003e\n\u003cli\u003eLovell, S. C.\u003cem\u003e et al.\u003c/em\u003e Structure validation by Calpha geometry: phi,psi and Cbeta deviation. \u003cem\u003eProteins\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 437-450 (2003). https://doi.org:10.1002/prot.10286\u003c/li\u003e\n\u003cli\u003eNakaoka, H. \u0026amp; Inoue, I. Distribution of HLA haplotypes across Japanese Archipelago: similarity, difference and admixture. \u003cem\u003eJournal of human genetics\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 683-690 (2015). \u003c/li\u003e\n\u003cli\u003eAltschul, S. F., Gish, W., Miller, W., Myers, E. W. \u0026amp; Lipman, D. J. Basic local alignment search tool. \u003cem\u003eJournal of molecular biology\u003c/em\u003e \u003cstrong\u003e215\u003c/strong\u003e, 403-410 (1990). \u003c/li\u003e\n\u003cli\u003eCamacho, C.\u003cem\u003e et al.\u003c/em\u003e BLAST+: architecture and applications. \u003cem\u003eBMC bioinformatics\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1-9 (2009). \u003c/li\u003e\n\u003cli\u003eReback, J.\u003cem\u003e et al.\u003c/em\u003e pandas-dev/pandas: Pandas 1.0. 5. \u003cem\u003eZenodo\u003c/em\u003e (2020). \u003c/li\u003e\n\u003cli\u003eMcKinney, W. Data structures for statistical computing in Python. \u003cem\u003eSciPy\u003c/em\u003e \u003cstrong\u003e445\u003c/strong\u003e, 51-56 (2010). \u003c/li\u003e\n\u003cli\u003eCock, P. J.\u003cem\u003e et al.\u003c/em\u003e Biopython: freely available Python tools for computational molecular biology and bioinformatics. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1422 (2009). \u003c/li\u003e\n\u003cli\u003eEstrada, A. A., Gottschalk, M., Gebhart, C. J. \u0026amp; Marthaler, D. G. Comparative analysis of Streptococcus suis genomes identifies novel candidate virulence-associated genes in North American isolates. \u003cem\u003eVeterinary research\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 23 (2022). \u003c/li\u003e\n\u003cli\u003eFischetti, V. A. Surface proteins on gram-positive bacteria. \u003cem\u003eMicrobiology spectrum\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 10.1128/microbiolspec. gpp1123-0012-2018 (2019). \u003c/li\u003e\n\u003cli\u003eChen, C. C. \u0026amp; Cleary, P. Complete nucleotide sequence of the streptococcal C5a peptidase gene of Streptococcus pyogenes. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e265\u003c/strong\u003e, 3161-3167 (1990). \u003c/li\u003e\n\u003cli\u003eGleich-Theurer, U.\u003cem\u003e et al.\u003c/em\u003e Human serum induces streptococcal c5a peptidase expression. \u003cem\u003eInfection and immunity\u003c/em\u003e \u003cstrong\u003e77\u003c/strong\u003e, 3817-3825 (2009). \u003c/li\u003e\n\u003cli\u003eKrishnamoorthy, S., Steiger, A. K., Nelson, W. C., Egbert, R. G. \u0026amp; Wright, A. T. An activity-based probe targeting the streptococcal virulence factor C5a peptidase. \u003cem\u003eChemical Communications\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e, 8113-8116 (2022). \u003c/li\u003e\n\u003cli\u003eGuo, G.\u003cem\u003e et al.\u003c/em\u003e Pan‐genome analysis of Streptococcus suis serotype 2 revealed genomic diversity among strains of different virulence. \u003cem\u003eTransboundary and Emerging Diseases\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 637-647 (2021). \u003c/li\u003e\n\u003cli\u003eKerdsin, A.\u003cem\u003e et al.\u003c/em\u003e Comparative genome analysis of Streptococcus suis serotype 5 strains from humans and pigs revealed pathogenic potential of virulent, antimicrobial resistance, and genetic relationship. \u003cem\u003eMicrobes and Infection\u003c/em\u003e, 105273 (2023). \u003c/li\u003e\n\u003cli\u003eTenenbaum, T., Asmat, T. M., Seitz, M., Schroten, H. \u0026amp; Schwerk, C. Biological activities of suilysin: role in Streptococcus suis pathogenesis. \u003cem\u003eFuture microbiology\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 941-954 (2016). \u003c/li\u003e\n\u003cli\u003eLi, G.\u003cem\u003e et al.\u003c/em\u003e Morin attenuates Streptococcus suis pathogenicity in mice by neutralizing suilysin activity. \u003cem\u003eFrontiers in microbiology\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 460 (2017). \u003c/li\u003e\n\u003cli\u003eBarnett, T. C.\u003cem\u003e et al.\u003c/em\u003e Streptococcal toxins: role in pathogenesis and disease. \u003cem\u003eCellular microbiology\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1721-1741 (2015). \u003c/li\u003e\n\u003cli\u003eWu, M.-C.\u003cem\u003e et al.\u003c/em\u003e Recombinant suilysin of Streptococcus suis enhances the protective efficacy of an engineered Pasteurella multocida toxin protein. \u003cem\u003eResearch in Veterinary Science\u003c/em\u003e \u003cstrong\u003e151\u003c/strong\u003e, 175-183 (2022). \u003c/li\u003e\n\u003cli\u003eBae, E. H. \u0026amp; Shin, M. In silico Design of Multi-epitope Vaccines Targeting Iron-regulated lipoproteins of Staphylococcus aureus Using Immunoinformactics. \u003cem\u003eJournal of Bacteriology and Virology\u003c/em\u003e \u003cstrong\u003e52\u003c/strong\u003e, 170-183 (2022). \u003c/li\u003e\n\u003cli\u003ePonomarenko, J.\u003cem\u003e et al.\u003c/em\u003e ElliPro: a new structure-based tool for the prediction of antibody epitopes. \u003cem\u003eBMC bioinformatics\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1-8 (2008). \u003c/li\u003e\n\u003cli\u003eHebditch, M., Carballo-Amador, M. A., Charonis, S., Curtis, R. \u0026amp; Warwicker, J. Protein-Sol: a web tool for predicting protein solubility from sequence. \u003cem\u003eBioinformatics (Oxford, England)\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 3098-3100 (2017). https://doi.org:10.1093/bioinformatics/btx345\u003c/li\u003e\n\u003cli\u003eGupta, S.\u003cem\u003e et al.\u003c/em\u003e In silico approach for predicting toxicity of peptides and proteins. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, e73957 (2013). https://doi.org:10.1371/journal.pone.0073957\u003c/li\u003e\n\u003cli\u003eMagnan, C. N.\u003cem\u003e et al.\u003c/em\u003e High-throughput prediction of protein antigenicity using protein microarray data. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 2936-2943 (2010). \u003c/li\u003e\n\u003cli\u003eDoytchinova, I. A. \u0026amp; Flower, D. R. VaxiJen: a server for prediction of protective antigens, tumour antigens and subunit vaccines. \u003cem\u003eBMC Bioinformatics\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 4 (2007). https://doi.org:10.1186/1471-2105-8-4\u003c/li\u003e\n\u003cli\u003eDa Costa, R. M., Rooke, J. L., Wells, T. J., Cunningham, A. F. \u0026amp; Henderson, I. R. Type 5 secretion system antigens as vaccines against Gram-negative bacterial infections. \u003cem\u003eNPJ Vaccines\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 159 (2024). https://doi.org:10.1038/s41541-024-00953-6\u003c/li\u003e\n\u003cli\u003eKhakhum, N., Bharaj, P., Walker, D. H., Torres, A. G. \u0026amp; Endsley, J. J. Antigen-specific antibody and polyfunctional T cells generated by respiratory immunization with protective Burkholderia \u0026Delta;tonB \u0026Delta;hcp1 live attenuated vaccines. \u003cem\u003enpj Vaccines\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 72 (2021). \u003c/li\u003e\n\u003cli\u003eO\u0026rsquo;Neill, A.\u003cem\u003e et al.\u003c/em\u003e Mucosal SARS-CoV-2 vaccination of rodents elicits superior systemic T central memory function and cross-neutralising antibodies against variants of concern. \u003cem\u003eEBioMedicine\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e (2024). \u003c/li\u003e\n\u003cli\u003eLindenstr\u0026oslash;m, T.\u003cem\u003e et al.\u003c/em\u003e Tuberculosis subunit vaccination provides long-term protective immunity characterized by multifunctional CD4 memory T cells. \u003cem\u003eThe Journal of Immunology\u003c/em\u003e \u003cstrong\u003e182\u003c/strong\u003e, 8047-8055 (2009). \u003c/li\u003e\n\u003cli\u003eTurtle, L.\u003cem\u003e et al.\u003c/em\u003e Human T cell responses to Japanese encephalitis virus in health and disease. \u003cem\u003eJournal of Experimental Medicine\u003c/em\u003e \u003cstrong\u003e213\u003c/strong\u003e, 1331-1352 (2016). \u003c/li\u003e\n\u003cli\u003eWesterhof, L. M.\u003cem\u003e et al.\u003c/em\u003e Multifunctional cytokine production marks influenza A virus‐specific CD4 T cells with high expression of survival molecules. \u003cem\u003eEuropean Journal of Immunology\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 2350559 (2023). \u003c/li\u003e\n\u003cli\u003ePereira Neto, T. A.\u003cem\u003e et al.\u003c/em\u003e Multifunctional T cell response in convalescent patients two years after ZIKV infection. \u003cem\u003eJournal of Leucocyte Biology\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 1265-1277 (2020). \u003c/li\u003e\n\u003cli\u003eZhu, J.\u003cem\u003e et al.\u003c/em\u003e Streptococcus suis serotype 4: a population with the potential pathogenicity in humans and pigs. \u003cem\u003eEmerging Microbes \u0026amp; Infections\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 2352435 (2024). \u003c/li\u003e\n\u003cli\u003eLunha, K.\u003cem\u003e et al.\u003c/em\u003e Serotype Distribution and Pathotypic Characteristics of Streptococcus suis Isolates from Slaughtered Pigs in a High-Density Pig Farming Area in Thailand. \u003cem\u003eTransbound Emerg Dis\u003c/em\u003e \u003cstrong\u003e2024\u003c/strong\u003e, 3186518 (2024). https://doi.org:10.1155/2024/3186518\u003c/li\u003e\n\u003cli\u003eGuo, P.\u003cem\u003e et al.\u003c/em\u003e Network Pharmacology to Unveil the Mechanism of Berberine in the Treatment of Streptococcus suis Meningitis in Humans and Pigs. \u003cem\u003eToxics\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e (2025). https://doi.org:10.3390/toxics13020138\u003c/li\u003e\n\u003cli\u003eFrosth, S.\u003cem\u003e et al.\u003c/em\u003e Sow vaccination with a novel recombinant protein vaccine protects piglets against Streptococcus suis infection. \u003cem\u003eVaccine\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 127077 (2025). https://doi.org:10.1016/j.vaccine.2025.127077\u003c/li\u003e\n\u003cli\u003eLi, W., Li, Y. A., Wang, S. \u0026amp; Shi, H. A universal live vaccine platform against multiple serotypes Streptococcus suis based on polyvalent antigen protein. \u003cem\u003eVaccine\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, 126700 (2025). https://doi.org:10.1016/j.vaccine.2024.126700\u003c/li\u003e\n\u003cli\u003eLiu, J.\u003cem\u003e et al.\u003c/em\u003e A multi-epitope subunit vaccine providing broad cross-protection against diverse serotypes of Streptococcus suis. \u003cem\u003eNPJ vaccines\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 216 (2024). https://doi.org:10.1038/s41541-024-01015-7\u003c/li\u003e\n\u003cli\u003eJazayeri, S. D. \u0026amp; Poh, C. L. Development of Universal Influenza Vaccines Targeting Conserved Viral Proteins. \u003cem\u003eVaccines (Basel)\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e (2019). https://doi.org:10.3390/vaccines7040169\u003c/li\u003e\n\u003cli\u003eWalker, R. I. Conserved antigens for enteric vaccines. \u003cem\u003eVaccine\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 126828 (2025). https://doi.org:10.1016/j.vaccine.2025.126828\u003c/li\u003e\n\u003cli\u003eDolbec, D.\u003cem\u003e et al.\u003c/em\u003e Streptococcus suis surface-antigen recognition by antibodies and bacterial elimination is influenced by capsular polysaccharide structure. \u003cem\u003eFront Cell Infect Microbiol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1228496 (2023). https://doi.org:10.3389/fcimb.2023.1228496\u003c/li\u003e\n\u003cli\u003eYuan, Z.-z.\u003cem\u003e et al.\u003c/em\u003e Molecular mechanism by which surface antigen HP0197 mediates host cell attachment in the pathogenic bacteria Streptococcus suis. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e288\u003c/strong\u003e, 956-963 (2013). \u003c/li\u003e\n\u003cli\u003eZhang, C., Yu, Y., Yang, M. \u0026amp; Jiang, Y. Expression, purification, crystallization and structure determination of the N terminal domain of Fhb, a factor H binding protein from Streptococcus suis. \u003cem\u003eBiochemical and biophysical research communications\u003c/em\u003e \u003cstrong\u003e466\u003c/strong\u003e, 413-417 (2015). \u003c/li\u003e\n\u003cli\u003eCullen, R.\u003cem\u003e et al.\u003c/em\u003e The 1.7 \u0026Aring; crystal structure of the C5a peptidase from Streptococcus agalactiae (ScpB) reveals an active site competent for catalysis. \u003cem\u003eProteins: Structure, Function, and Bioinformatics\u003c/em\u003e \u003cstrong\u003e92\u003c/strong\u003e, 427-431 (2024). \u003c/li\u003e\n\u003cli\u003eLecours, M. P.\u003cem\u003e et al.\u003c/em\u003e Immune-responsiveness of CD4(+) T cells during Streptococcus suis serotype 2 infection. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 38061 (2016). https://doi.org:10.1038/srep38061\u003c/li\u003e\n\u003cli\u003eLetendre, C.\u003cem\u003e et al.\u003c/em\u003e Streptococcus suis Serotype 2 Infection Impairs Interleukin-12 Production and the MHC-II-Restricted Antigen Presentation Capacity of Dendritic Cells. \u003cem\u003eFront Immunol\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1199 (2018). https://doi.org:10.3389/fimmu.2018.01199\u003c/li\u003e\n\u003cli\u003eXing, X.\u003cem\u003e et al.\u003c/em\u003e Intranasal Vaccination With Multiple Virulence Factors Promotes Mucosal Clearance of Streptococcus suis Across Serotypes and Protects Against Meningitis in Mice. \u003cem\u003eJ Infect Dis\u003c/em\u003e \u003cstrong\u003e220\u003c/strong\u003e, 1679-1687 (2019). https://doi.org:10.1093/infdis/jiz352\u003c/li\u003e\n\u003cli\u003eKim, H. W.\u003cem\u003e et al.\u003c/em\u003e Opsonophagocytic Antibodies to Serotype Ia, Ib, and III Group B Streptococcus among Korean Infants and in Intravenous Immunoglobulin Products. \u003cem\u003eJournal of Korean medical science\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 737-743 (2017). https://doi.org:10.3346/jkms.2017.32.5.737\u003c/li\u003e\n\u003cli\u003eKim, G. R.\u003cem\u003e et al.\u003c/em\u003e Serotype Distribution and Antimicrobial Resistance of Streptococcus pneumoniae Causing Invasive Pneumococcal Disease in Korea Between 2017 and 2019 After Introduction of the 13-Valent Pneumococcal Conjugate Vaccine. \u003cem\u003eAnn Lab Med\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 45-54 (2023). https://doi.org:10.3343/alm.2023.43.1.45\u003c/li\u003e\n\u003cli\u003eZhang, A.\u003cem\u003e et al.\u003c/em\u003e Identification of a surface protective antigen, HP0197 of Streptococcus suis serotype 2. \u003cem\u003eVaccine\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 5209-5213 (2009). \u003c/li\u003e\n\u003cli\u003ede Greeff, A.\u003cem\u003e et al.\u003c/em\u003e Contribution of fibronectin-binding protein to pathogenesis of Streptococcus suis serotype 2. \u003cem\u003eInfection and immunity\u003c/em\u003e \u003cstrong\u003e70\u003c/strong\u003e, 1319-1325 (2002). \u003c/li\u003e\n\u003cli\u003eLi, J.\u003cem\u003e et al.\u003c/em\u003e Evaluation of the immunogenicity and the protective efficacy of a novel identified immunogenic protein, SsPepO, of Streptococcus suis serotype 2. \u003cem\u003eVaccine\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 6514-6519 (2011). \u003c/li\u003e\n\u003cli\u003eFeng, Y.\u003cem\u003e et al.\u003c/em\u003e Streptococcus suis enolase functions as a protective antigen displayed on the bacterial cell surface. \u003cem\u003eThe Journal of infectious diseases\u003c/em\u003e \u003cstrong\u003e200\u003c/strong\u003e, 1583-1592 (2009). \u003c/li\u003e\n\u003cli\u003eYan, Z.\u003cem\u003e et al.\u003c/em\u003e Immunogenicity and Protective Capacity of Sugar ABC Transporter Substrate-Binding Protein against Streptococcus suis Serotype 2, 7 and 9 Infection in Mice. \u003cem\u003eVaccines\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 544 (2024). \u003c/li\u003e\n\u003cli\u003eJiang, X.\u003cem\u003e et al.\u003c/em\u003e Peptidyl isomerase PrsA is surface-associated on Streptococcus suis and offers cross-protection against serotype 9 strain. \u003cem\u003eFEMS Microbiology Letters\u003c/em\u003e \u003cstrong\u003e366\u003c/strong\u003e, fnz002 (2019). \u003c/li\u003e\n\u003cli\u003eTan, C.\u003cem\u003e et al.\u003c/em\u003e Vaccination with Streptococcus suis serotype 2 recombinant 6PGD protein provides protection against S. suis infection in swine. \u003cem\u003eFEMS microbiology letters\u003c/em\u003e \u003cstrong\u003e296\u003c/strong\u003e, 78-83 (2009). \u003c/li\u003e\n\u003cli\u003eZhou, Y.\u003cem\u003e et al.\u003c/em\u003e Evaluation of the protective efficacy of four novel identified membrane associated proteins of Streptococcus suis serotype 2. \u003cem\u003eVaccine\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 2254-2260 (2015). \u003c/li\u003e\n\u003cli\u003eDu, H.\u003cem\u003e et al.\u003c/em\u003e The genetically modified suilysin, rSLYP353L, provides a candidate vaccine that suppresses proinflammatory response and reduces fatality following infection with Streptococcus suis. \u003cem\u003eVaccine\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 4209-4215 (2013). \u003c/li\u003e\n\u003cli\u003eZhang, M.\u003cem\u003e et al.\u003c/em\u003e Inhibition of polysome assembly enhances imatinib activity against chronic myelogenous leukemia and overcomes imatinib resistance. \u003cem\u003eMolecular and cellular biology\u003c/em\u003e\u003cstrong\u003e28\u003c/strong\u003e, 6496-6509 (2008). \u003c/li\u003e\n\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":"S.suis, gram-positive bacteria, protein vaccine, HP0197, Fnbp, Sao, ScpB, SLY, in silico","lastPublishedDoi":"10.21203/rs.3.rs-7215039/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7215039/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eStreptococcus suis\u003c/em\u003e (\u003cem\u003eS. suis\u003c/em\u003e) is a zoonotic pathogen that causes significant losses in the swine industry and serious invasive infections in humans. The high serotype variability and genomic diversity of \u003cem\u003eS. suis\u003c/em\u003e have substantially limited the development of cross-reactive vaccines. Although recent advances in \u003cem\u003ein silico\u003c/em\u003e prediction and database-driven antigen discovery have accelerated the development of protein-based vaccines, several studies have reported inconsistencies between predicted immunogenic profiles and the protective efficacy observed in animal models, emphasizing the importance of integrating computational design with experimental validation. In this study, we selected key antigens of \u003cem\u003eS. suis\u003c/em\u003e based on previous experimental reports (HP0197, Fnbp, Sao, ScpB, and SLY) and analyzed their predicted T- and B-cell epitopes. For each antigen, we identified surface-exposed epitope regions (approximately 109\u0026ndash;210 amino acids) through structural modeling or available PDB data. These regions were then assembled into a multimeric conjugated vaccine construct (ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e) by optimizing based on predicted immunogenicity, solubility, and allergenicity profiles. As predicted by the \u003cem\u003ein silico\u003c/em\u003e design, ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e elicited strong humoral immune responses against each of the five component antigens in the mouse model. Notably, the vaccine also induced robust Th1- and Th17-type cellular immune responses, which are known to be essential for effective opsonic and mucosal defense against \u003cem\u003eS. suis\u003c/em\u003e infection. In the protection studies, ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e conferred significant protection against \u003cem\u003eS. suis\u003c/em\u003e serotypes 2, 4, and 9, as demonstrated by improved survival rates and reduced bacterial burdens. These findings highlight the potential of ATOMSSUIS\u003csub\u003epenta\u003c/sub\u003e as a broadly protective subunit vaccine against \u003cem\u003eS. suis\u003c/em\u003e and demonstrate the value of epitope-based multimeric design for targeting antigenically diverse Gram-positive pathogens.\u003c/p\u003e","manuscriptTitle":"Epitope-Based Multimeric Subunit Vaccine (ATOMSSUISpenta) Confers Cross-Protection Against Streptococcus suis Infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-25 18:17:21","doi":"10.21203/rs.3.rs-7215039/v1","editorialEvents":[{"type":"communityComments","content":1}],"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":"c62ae0fc-e099-48b3-a477-9ab7e0023dcd","owner":[],"postedDate":"August 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53682723,"name":"Biological sciences/Biotechnology"},{"id":53682724,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":53682725,"name":"Biological sciences/Immunology"},{"id":53682726,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-09-23T15:23:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-25 18:17:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7215039","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7215039","identity":"rs-7215039","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}