Discovery of KC-1: A Novel Porcine Dendritic Cell-Targeting Peptide with Potential Applications in Swine Vaccine Design

Discovery of KC-1: A Novel Porcine Dendritic Cell-Targeting Peptide with Potential Applications in Swine Vaccine Design | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Discovery of KC-1: A Novel Porcine Dendritic Cell-Targeting Peptide with Potential Applications in Swine Vaccine Design Bin Liu, Tian Xia, Chengjie Bian, Yanping Jiang, Wen Cui, Jiaxuan Li, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8110404/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 Dendritic cells (DCs) are key antigen-presenting cells essential for initiating and regulating immune responses. While DC targeting has proven to be an effective strategy for vaccine enhancement, and targeting peptides have been extensively utilized as efficient delivery tools in DC-targeted drug and vaccine development, there remains a notable scarcity of peptides specifically selected through porcine dendritic cell screening platforms. In this study, phage display biopanning was employed to screen a novel DC-targeting peptide, designated KC (KCCYPNQMAAFA). Systematic alanine-scanning mutagenesis identified the N-terminal hexapeptide KC-1 (KCCYPN) as the minimal functional epitope responsible for DC binding. In addition to DCs, KC-1 was also demonstrated selective binding to bone marrow-derived dendritic cells (BM-DCs) and porcine alveolar macrophages (PAMs) but exhibited no interaction with intestinal porcine epithelial (IPI) cells, swine testis (ST) cells, or Vero cells. Further analysis revealed that KC-1 specifically bounds to the N-terminal region (1-126 aa) of SLA-DRB1, which is a key domain of the MHC II β-chain involved in the formation of the peptide-binding groove. Using the PEDV S1 subunit as a model antigen, we further evaluated the immunomodulatory effects of KC-1 on DCs in vitro. The results demonstrated that KC-1-S1 significantly promoted dendritic cell maturation and T cell proliferation, accompanied by increased secretion of key cytokines IL-4, IL-12, and IFN-γ, indicating enhanced activation of both humoral and cellular immune responses with a balanced Th1/Th2 polarization compared to controls. Collectively, these findings establish a theoretical foundation for porcine DC-targeted peptides and provide critical insights for the development of next-generation porcine DC-targeted vaccines. Dendritic cell-targeting peptide Swine Vaccine SLA-DRB1 Phage display Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights A novel porcine dendritic cell-targeting peptide, KC-1, was identified from a phage display library. KC-1 specifically binds to the MHC class II molecule SLA-DRB1 on porcine APCs. Fusion of KC-1 with PEDV S1 enhances DC maturation, antigen presentation, and T-cell activation in vitro. 1. Introduction In recent years, the global swine industry has become a critical pillar for ensuring food security and driving rural economic development. China, in particular, has emerged as the world’s largest producer and consumer of pork, accounting for over half of the global annual output [ 1 ]. However, the intensification of farming practices has led to continuously increasing stocking densities, creating favorable conditions for the spread of major infectious pathogens [ 2 ]. Among swine populations, vaccination remains the most effective and sustainable strategy for controlling infectious diseases. To improve vaccine efficacy, durability, and broad-spectrum protection, researchers have explored various strategies. Among these, DC-targeted vaccine design has garnered increasing attention due to the crucial role of DCs in initiating and regulating immune responses, positioning this approach as a promising avenue for next-generation vaccine development. Dendritic cells (DCs), as the most potent professional antigen-presenting cells in the immune system, are widely distributed at mucosal and cutaneous surfaces—the front lines of pathogen invasion—where they serve as central “immune sentinels” [ 3 , 4 ]. Their core functions involve recognizing pathogen-associated molecular patterns (PAMPs) and danger signals via pattern recognition receptors (such as TLRs), efficiently capturing and processing antigens, and presenting them to naïve T cells via MHC class I and II molecules, thereby activating adaptive immune responses [ 5 – 8 ]. Mature DCs not only act as a bridge linking innate and adaptive immunity, but also coordinate the activity of B cells, NK cells, and other immune effectors through co-stimulatory molecules and cytokines, facilitating integrated humoral and cellular immunity [ 3 ]. Owing to this central immunological role, targeted delivery of antigens to DCs has become a key strategy to enhance vaccine performance. Current DC-targeting approaches typically employ antibodies, ligands, or targeting peptides specific to DC surface receptors to guide antigens, nucleic acids, or drugs to specific DC subsets. [ 9 – 12 ]. Among these approaches, targeting peptides have gradually emerged as an important direction in DC-targeted vaccine design due to their unique advantages over antibodies and ligands. Peptides are small in size, structurally stable, low in immunogenicity, and capable of penetrating cell membranes. They are also easier to synthesize, modify (e.g., PEGylation or D-amino acid substitution), and scale up for production[ 13 , 14 ]. Moreover, techniques such as phage display and receptor docking simulation allow for high-throughput and highly specific identification of functional peptides that bind to DC surface receptors, significantly simplifying the construction of antigen delivery systems [ 15 ]. Many of the identified targeting peptides have been validated to improve the immunogenicity and protective efficacy of vaccines. For instance, the mouse-derived DC-targeting peptide DCpep3 has been shown to enhance antigen uptake and antibody responses when used in a nanoparticle vaccine against porcine circovirus type 2 (PCV2) [ 16 ].. The peptide hr-8, identified from the C-type lectin receptor DEC-205, specifically targets DCs via DEC-205 binding and promotes their maturation by upregulating the surface expression of MHC class II, CD80, and CD86, thereby enhancing antigen presentation and cross-priming of T cells [ 17 ]. Additionally, DP7-C, a cholesterol-modified peptide designed through computational optimization, exhibits dual functionality: it targets DCs while simultaneously enhancing their migration and lymph node homing, acting both as a delivery enhancer and an immunostimulatory adjuvant [ 18 ]. Despite the successful application of DC-targeting peptides in multiple species and their ability to enhance antigen uptake and immune responses, significant interspecies differences exist in the composition of DC subsets, expression of surface receptors, and antigen presentation pathways [ 19 ]. To address this critical need, this study first employed phage display technology to screen high-affinity linear peptides capable of specifically recognizing porcine DCs, and further analyzed their key amino acid motifs and binding sites on DC surface receptors. Based on these findings, the selected targeting peptides were fused with the PEDV S1 subunit, and the fusion proteins’ effects on antigen presentation and DC activation were evaluated. This study is expected to provide novel peptide-based tools and mechanistic insights to facilitate the development of highly efficient and precision-designed DC-targeted vaccines for swine immunization. 2. Materials and Methods 2.1. Materials and reagents Red blood cell lysis buffer and Western blot primary antibody dilution buffer were obtained from Shanghai Beyotime Biotechnology Co., Ltd. (China). Streptavidin-coated magnetic beads and DAPI nuclear stain were purchased from Thermo Fisher Scientific (USA). RPMI-1640 medium and fetal bovine serum (FBS) were supplied by GIBCO (USA). The Ph.D™-12 Phage Display Peptide Library Kit was obtained from New England Biolabs (NEB, USA). Histopaque-1077 lymphocyte separation medium, lipopolysaccharide (LPS), silver staining reagent, and FITC-conjugated dextran were purchased from Sigma-Aldrich (USA). Phage genomic DNA, total RNA, and plasmid extraction kits were obtained from Shanghai Feijie Biotechnology Co., Ltd. (China). SYBR Green qPCR premix was purchased from Roche (USA), and the cell membrane extraction kit was obtained from ActGene (China). KOD DNA polymerase and reverse transcriptase were purchased from Shanghai Biotechnology Co., Ltd. (China). Transfection reagent was obtained from Thermo Fisher Scientific (USA), while T4 DNA ligase and restriction enzymes were purchased from TaKaRa Biotechnology Co., Ltd. (Japan). 2.2 Plasmids, bacterial strains, and cell lines The pMD19-T and pMD19-T Simple cloning vectors were obtained from Dalian TaKaRa Biotechnology Co., Ltd. (China). The recombinant plasmid pMD19-S1, containing the S1 gene of porcine epidemic diarrhea virus (PEDV), as well as the expression vectors pET23a/TG1, pET23a-S1/TG1 (encoding PEDV S1 in E. coli), and pCMV/TG1, were maintained by the Microbiology and Immunology Laboratory, College of Veterinary Medicine, Northeast Agricultural University (China). TG1 competent E. coli cells were purchased from TransGen Biotech (China). The BHK, ST, and IPI cell lines were also preserved by the Microbiology and Immunology Laboratory, College of Veterinary Medicine, Northeast Agricultural University (China). 2.3 Animal Antibody-negative, healthy female 1-month-old piglets were obtained from a certified commercial pig farm in Harbin, China. All animal experiments were performed in accordance with the guidelines of the Animal Ethics Committee of Northeast Agricultural University (approval number: NEAUEC0210337). 2.4 Primary cell isolation and culture PBMCs were isolated from the jugular vein of piglets via density gradient centrifugation. Monocytes were differentiated into DCs by culturing in RPMI-1640 supplemented with 20 ng/mL GM-CSF and 20 ng/mL IL-4. Morphological changes were monitored using light microscopy. On day 7, immature Mo-DCs were harvested and stained with PE-conjugated CD172a and FITC-conjugated MHC II for 30 min, washed thrice with PBS, and analyzed by flow cytometry and fluorescence microscopy. PAMs were obtained from lung lavage fluid of 5–8-week-old pigs. Lungs were flushed with RPMI-1640 containing 1% PBS, filtered through a 70 µm strainer, centrifuged (1,500 rpm, 10 min), treated with red blood cell lysis buffer, and cultured in complete medium (90% RPMI-1640, 10% FBS) at 37°C, 5% CO₂. BM-DCs were generated from femurs and tibiae of 5–8-week-old pigs. Flushed bone marrow cells were filtered, centrifuged, red blood cells lysed, and cultured in complete medium with 20 ng/mL GM-CSF and 20 ng/mL IL-4. Medium was refreshed every 2 days, and immature BM-DCs were harvested on day 5. Rabbit Mo-DCs were prepared from PBMCs isolated via density gradient centrifugation. Cells were seeded at 2 × 10⁶ cells/mL in 12-well plates containing complete medium (RPMI-1640, 10% FBS, 1% penicillin-streptomycin) and incubated at 37°C, 5% CO₂. After 6 h, non-adherent cells were removed, and adherent monocytes were cultured in medium supplemented with 20 ng/mL recombinant human GM-CSF and IL-4. Half of the medium was replaced every 2 days, and immature Mo-DCs were harvested on day 5 for downstream experiments. 2.5 Screen Mo-DCs binding peptides by phage display technique Peptides binding to porcine monocyte-derived dendritic cells (Mo-DCs) were screened using the Ph.D.™-12 phage display peptide library kit (NEB, Beijing, China) according to the manufacturer’s protocol. Immature Mo-DCs (1 × 10⁶ cells/mL) were detached from culture plates with PBS and collected into 1.5 mL centrifuge tubes. The cells were then incubated with 2 × 10¹¹ pfu of the Ph.D.-12 phage library at 4°C for 30 minutes. Following incubation, cells were centrifuged at 2,000 rpm for 3 minutes and resuspended in PBS containing 1% BSA and 0.05% Tween 20. Three rounds of centrifugation and washing were performed to remove unbound phages. Bound phages were eluted using an acidic buffer (pH 2.2) containing 1 mg/mL BSA and 0.2 M glycine-HCl with gentle mixing for 10 minutes, followed by neutralization with 50 µL of Tris-HCl buffer (pH 9.1). After centrifugation at 2,000 rpm for 3 minutes, the supernatant containing eluted phages was collected for subsequent rounds of selection. After four rounds of biopanning, individual plaques were randomly picked, amplified, and the phage genomic DNA was extracted using a phage DNA extraction kit. Sequencing of the extracted phage DNA was performed at Comate Bioscience Co., Ltd. (Jilin, China) using the universal primer 96gⅢ (Supplementary Table 4). 2.6 Peptide synthesis The Porcine -derived dendritic cell (DC) targeting peptides selected in this study, as well as the peptides with alanine mutations or amino acid deletions, were synthesized by Nanjing GenScript Biotechnology Co., Ltd. The peptide sequences and N-terminal modifications are detailed in the Supplementary Table 5 . 2.7 Evaluation of Phage and Peptide Binding by ELISA, Fluorescence Microscopy, and Flow Cytometry PBMCs and Mo-DCs were fixed on 0.03 mg/mL polyline-coated 96-well plates for 30 min at 25°C and blocked with PBS containing 2% BSA for 30 min at 4°C. Phage clones (10¹⁰ pfu/mL) were added and incubated at 4°C for 20 h. PBS and wild-type M13 phage served as negative controls. After three PBST washes, cells were sequentially incubated with mouse anti-M13 polyclonal antibody (1:1,500; LSBiological) for 40 min at 37°C, followed by HRP-conjugated goat anti-mouse IgG (1:4,000; ZSGB Biotech). Color development was performed using TMB substrate, and absorbance was measured at 450 nm. For fluorescence microscopy, Mo-DCs cultured for six days were incubated with FITC-labeled DC-targeting peptides (HS, KC1, MY) or a negative control peptide at 4°C for 30 min, followed by staining with CD86-PE antibody at 37°C in the dark for 30 min. Cells were washed, fixed with 4% paraformaldehyde for 5 min at 37°C, and visualized using a fluorescence microscope (Bio-Rad, USA). For flow cytometry, Mo-DCs (10⁶ cells) were incubated with FITC-labeled peptides (25 µg) at 4°C for 10 min, washed three times, and analyzed using a BD Biosciences cytometer (San Jose, CA, USA). 2.8 The mRNA expression of surface molecules of porcine DCs was tested by qRT-PCR Immature porcine Mo-DCs were collected after incubation with KC-1-S1, NC-S1, S1, and empty protein Tag. Total RNA was extracted using the RNA extraction kit from Shanghai Feijie Biotechnology Co., Ltd., and reverse transcription was performed using the RT-SuperMix kit. Unstimulated Porcine Mo-DCs were used as the control group, with β-actin as the internal reference gene. cDNA samples were adjusted to consistent concentrations. The reaction mixture for SYBR Green real-time RT-PCR was prepared according to the Roche manual for SYBR Green dye. Data collection and analysis were carried out using the real-time PCR detection system (7500 System Software). The primer sequences are listed in the Supplementary Table 4. The relative expression levels of surface molecules were calculated using the relative quantitative 2-ΔΔCT method, as follows: ΔΔCt = (Ct of target gene - Ct of internal reference gene) in the experimental group - (Ct of target gene - Ct of internal reference gene) in the control group. 2.9 Statistical analysis Statistical analysis of the experimental data was performed using GraphPad Prism 5 software. For pairwise comparisons between multiple groups, one-way analysis of variance (ANOVA) was conducted. Statistical significance was determined by different letters, with p < 0.01 indicating significant differences. 3.Results 3.1 Screening and identification of DCs-targeting peptides Porcine monocytes were isolated using previously reported methods [ 20 ] and differentiated into DCs by stimulation with GM-CSF and IL-4, followed by maturation with LPS. The resulting cells exhibited typical dendritic cell characteristics in terms of morphology, surface markers (CD172a, MHC-II, CD80, CD86), and phagocytic activity (Supplementary Figure S1 ). To identify peptides capable of specifically binding to porcine DCs, a 12-mer phage display peptide library was employed for biopanning against porcine Mo-DCs. After four rounds of biopanning, targeted phages were enriched, and 204 monoclonal phage clones were randomly selected for sequencing, yielding 161 distinct peptide sequences (Supplementary Table 1 and Supplementary Figure S2). The binding affinity of the 3 most frequent peptides (HS, KC, and SF) and randomly selected 13 peptides to porcine DCs was detected using phage ELISA (Supplementary Table 2). The results indicated that phages expressing HS, KC, and SF peptides exhibited stronger affinity for porcine Mo-DCs than other 13 peptides (Fig. 1 A). Additionally, fluorescence microscopy and confocal laser scanning microscopy revealed that HS, KC, and SF peptides could bind to porcine Mo-DCs (Fig. 1 B) as well as DCs in piglet intestinal tissue sections (Fig. 1 C). Furthermore, flow cytometry results demonstrated that the peptide KC exhibited a higher targeting affinity for porcine DCs than the peptides HS and SF (Fig. 1 D). These findings indicated that the KC peptide showed the strongest binding affinity for porcine DCs in our study. 3.2 KCCYPN of the KC Peptide Are Critical for Targeted Binding to Porcine DCs KC peptide’s key amino acids involved in binding activity were identified by alanine scanning mutagenesis. The competitive ELISA results demonstrated that four specific amino acid residues (K1, Y4, P5, and N6) within the KC peptide constitute the essential binding sites responsible for its targeted interaction with porcine DCs. In contrast, substitutions at positions C2, C3, Q7, M8, and F11 had minimal impact on binding affinity (Fig. 2 A). Although simultaneous mutations at C2A and C3A did not significantly affect KC binding, complete deletion of these two residues markedly reduced its binding to dendritic cells (DCs) (Fig. 2 B left). Similarly, simultaneous substitutions at Q7, M8, and F11 also had no significant effect on binding. Notably, the truncated peptide KC-1 (KCCYPN), consisting of the N-terminal six amino acids of KC, exhibited stronger binding affinity to monocyte-derived dendritic cells (Mo-DCs) than the full-length peptide (Fig. 2 B right). To exclude possible non-specific binding at high concentrations, the binding affinity of the peptides was further evaluated at low concentrations (1.5, 2.5, and 5 µg/mL). The results demonstrated that KC-1 exhibited stronger binding affinity to porcine dendritic cells than the full-length KC peptide at all tested concentrations (Fig. 2 C). Collectively, these results identify the N-terminal hexapeptide KC-1 as the functional core of KC responsible for selective binding to porcine DCs. 3.3 KC-1 can bind to various immune cells To evaluate the cell-specific binding ability of the targeting peptide KC-1, FITC-conjugated KC-1 peptide was incubated with various cell types, including porcine DCs, PAMs, porcine BM-DCs, rabbit Mo-DCs, IPI, ST cells, and Vero cells. Fluorescence microscopy revealed KC-1 binding to porcine Mo-DCs, PAMs, BM-DCs, and rabbit Mo-DCs, while no detectable fluorescence was observed in IPI, ST, or Vero cells (Fig. 3 A). Flow cytometric analysis further confirmed the binding of KC-1 to porcine DCs, PAMs, porcine BM-DCs, and rabbit Mo-DCs (Fig. 3 B). These results indicate that KC-1 can bind to immune cells derived from different species, demonstrating its potential as a broadly applicable immune cells-targeting peptide. 3.4 SLA-DRB1 as the Target Protein of the KC-1 To identify the molecular target of KC-1, KC-1-Biotin was synthesized by introducing a lysine residue at the C-terminus of KC-1 for biotinylating, with an irrelevant sequence NC-Biotin serving as the negative control. HPLC analysis confirmed > 95% purity for both peptides (Fig. 4 A), and ELISA demonstrated that biotinylating did not compromise KC-1's specific binding to PAMs (Fig. 4 B). Pull-down assays were performed by incubating PAM membrane proteins (Fig. 4 C) with KC-1-Biotin, followed by capture using streptavidin-coated magnetic beads. SDS-PAGE and silver staining of the pull-down eluates revealed a distinct protein band between 25–40 kDa specifically enriched in the KC-1-Biotin group but absent in the negative control (Fig. 4 D). This band was excised for mass spectrometry (MS) analysis, which identified 39 differentially expressed proteins, including 15 membrane proteins (Supplementary Table 3). Among the membrane proteins, the MHC class II β chain (SLA-DRB1) was identified with the highest confidence, while the MHC class I molecule (SLA-1) was likewise detected. Accordingly, SLA-DRB1 and SLA-1 were prioritized as candidate targets of KC-1 for validation. Co-immunoprecipitation (Co-IP) assays demonstrated that KC-1 could interact with SLA-DRB1 and SLA-1 in PAMs and Mo-DCs, the interaction of KC-1 and SLA-DRB1 was effectively blocked by anti-SLA-DRB1 antibodies, however, anti-SLA-1 antibodies could not completely block the interaction of KC-1 and SLA-1 (Fig. 4 E). Therefore, SLA-DRB1 was selected for subsequent experiments. Laser scanning confocal microscopy confirmed pronounced co-localization of KC-1 with SLA-DRB1 on the surface of PAMs and Mo-DCs (Fig. 4 F), further validating their interaction. Collectively, these results confirm SLA-DRB1 was the primary target protein of the targeting peptide KC-1. 3.5 Determination of the Binding Domain of KC-1 to SLA-DRB1 To identify the binding domain of SLA-DRB1 that interacts with the KC-1, molecular docking analysis was performed using HPEPDOCK 2.0 software, resulting in 10 interaction models between KC-1 and SLA-DRB1. Among them, nine models predicted the binding domain of KC-1 to SLA-DRB1 to be between amino acids 38 and 111, while one model predicted the binding domain of the peptide KC-1 to SLA-DRB1 to be between amino acids 130 and 255 (Fig. 5 A). Based on these predictions, SLA-DRB1 was divided into two fragments: SLA-DRB1a (amino acids 1–126) and SLA-DRB1b (amino acids 126–266) (Fig. 5 B). IFA and Western blotting analyses showed that SLA-DRB1, SLA-DRB1a and SLA-DRB1b were successfully expressed (Figs. 5 C– 5 D). Co-IP assays demonstrated that KC-1 interacted with full-length SLA-DRB1 and the SLA-DRB1a (Fig. 5 E). Collectively, these results indicate that the region of SLA-DRB1 interacting with KC-1 is localized within residues 1–126. 3.6 The fusion expression of KC-1 with antigens promoted Porcine DCs Maturation and T Cell Proliferation To evaluate the immunomodulatory effect of the KC-1 on porcine DCs under antigenic stimulation, porcine DCs were incubated for 12 hours with the peptide KC-1, PEDV S1 protein, the fusion proteins KC-1-S1, NC-S1, or LPS. qPCR analysis revealed that treatment with S1, NC-S1, KC-1-S1, and LPS significantly upregulated the expression of costimulatory molecules (CD40, CD80, CD86) and Toll-like receptors (TLR2, TLR6, TLR9), with the highest levels observed in the KC-1-S1 group (Figs. 6 A–B). To further assess whether KC-1 fusion affects antigen presentation capacity, we prepared single-cell suspensions of piglet peripheral blood lymphocytes and conducted T cell proliferation assays. The results revealed that KC-1-S1 significantly enhanced T cell proliferation compared to other treatment groups (Fig. 6 C), indicating that KC-1 fusion facilitates the ability of DCs to activate T cells. Additionally, ELISA analysis showed that KC-1-S1–treated Mo-DCs promoted the secretion of IL-4, IL-12, and IFN-γ (Fig. 6 D), further demonstrating that the KC-1 enhances T cell differentiation. Taken together, KC-1 enhances the antigen-presenting ability of DCs and promotes DC-mediated T cell proliferation and differentiation. 4. Discussion Antigen targeting to DCs has been recognized as an effective strategy to enhance vaccine efficacy [ 21 – 25 ]. Achieving efficient antigen delivery and precise recognition of DCs is key to the development of targeting strategies. Existing studies have shown that screening ligands for DC surface receptors, generating specific antibodies against DC surface molecules (receptors/markers), and developing DC-specific targeting peptides are all feasible methods for antigen delivery [ 26 – 29 ]. However, each of these methods has certain limitations [ 30 ] [ 31 ]. In contrast, targeting peptides [ 32 ], with higher cell membrane permeability and lower immunogenicity [ 15 ], possess unique advantages in targeting strategies [ 33 ]. In this study, a DC-targeting peptide, KC, consisting of 12 amino acids, was identified using phage display technology. As is known, protein–protein interactions are often mediated by a limited number of key amino acid residues [ 34 , 35 ]. Additionally, shorter peptides can not only improve the efficiency of target protein recognition on DCs but also enhance membrane permeability and reduce immunogenicity [ 36 ]. To this end, an alanine scanning approach was employed to identify the functional residues within the DC-targeting peptide KC, which confirmed that the first six N-terminal amino acids of KC constitute the key functional region responsible for DC binding. To determine whether the targeting peptide KC-1 is specific to DCs, this study examined its affinity for different cell types. The results indicate that, in addition to specifically binding to porcine Mo-DCs, KC-1 also binds to porcine BM-DCs and porcine PAMs, but does not bind to IPI or ST cells. This may be because both DCs and macrophages are APCs and share similar surface molecular expression profiles. Therefore, we hypothesized that the targeting peptide KC-1 could bind to all APCs in vivo, and we further investigated the molecular mechanism underlying this interaction. Given that PAMs are readily obtainable and culturable, and possess molecular characteristics representative of APCs [ 37 , 38 ], they were selected in this study for screening KC-1 target proteins by pull down coupled with MS. SLA-DRB1, as the MHC class II β chain, caught our attention with the highest confidence in the results of MS. The interaction between KC-1 and SLA-DRB1 was further confirmed on porcine Mo-DCs. As an MHC class II molecule, SLA-DRB1 is expressed on the surface of all professional APCs [ 39 , 40 ], which provides a molecular explanation for the ability of KC-1 to bind to various types of APCs. Although the MHC class I molecule (SLA-1) also showed high-confidence significant identity in the MS results, its validation outcomes were unsatisfactory. We speculate that it is likely not the primary interacting protein of KC-1 and thus will not pursue further investigation into it. Identifying the precise binding region between KC-1 and SLA-DRB1 is essential for a deeper understanding of the molecular mechanisms by which this targeting peptide modulates the immune function of porcine DCs under antigen stimulation. Previous studies have shown that short targeting peptides often recognize flexible or unstructured regions of surface molecules, which allows them to maintain binding specificity without disrupting native protein functions [ 41 , 42 ]. To this end, this study characterized the binding capacity of KC-1 to different structural domains of SLA-DRB1. Most prediction models indicated that KC-1 primarily binds to the unstructured region (amino acids 1–126) of SLA-DRB1. Experimental validation confirmed these predictions, demonstrating that the binding site of KC-1 is located within the 1–126 amino acid segment of SLA-DRB1. Similar observations have been reported for other MHC-associated ligands, where interactions occur preferentially within unstructured or solvent-exposed domains [ 43 , 44 ]. Since this binding region lies outside the structured domain of SLA-DRB1, we speculate that a KC-1-mediated DC-targeting vaccine would likely not interfere with the native functions of SLA-DRB1 in antigen presentation and immune response activation. Notably, this study confirmed that KC-1 can bind to rabbit Mo-DCs, consistent with previous findings. Previous studies identified a 12-mer peptide “DCpep” using human CD1a + DR bright CD11c bright DCs, which also showed varying degrees of affinity for peripheral blood DCs in multiple species including poultry, dogs, horses, and cats [ 45 ]. The ability of KC-1 to bind DCs across species is likely due to the recognition of conserved structural features of MHC class II molecules or other surface proteins that are shared among mammalian APCs [ 46 ]. Moreover, short targeting peptides often interact with flexible, solvent-exposed regions of these proteins, which tend to be structurally conserved across species [ 47 ]. These findings suggest that KC-1 not only has potential for developing DC-targeted vaccines in pigs but also may be applicable to other species, similar to previous cross-species applications of DC-targeting peptides [ 48 , 49 ]. Since the standalone targeting peptide KC-1 lacks immunogenicity, and considering that the PEDV S1 protein can induce high-titer neutralizing antibodies and promote cellular immune responses [ 50 – 56 ], this study explored its effects on the immune function of antigen-stimulated porcine DCs after fusing it with the model antigen PEDV S1 protein for co-expression. Results demonstrated that compared to recombinant S1 protein, KC-1-S1-incubated Mo-DCs exhibited significantly upregulated surface molecular expression levels, indicating that the targeting peptide KC-1 promotes antigen-stimulated maturation of porcine DCs. DCs activate upon recognizing pathogen-associated molecular patterns via pattern recognition receptors, subsequently migrating to lymph nodes. There, they present antigens and secrete cytokines, driving the differentiation of naive CD4⁺ T cells into distinct subsets such as Th1 and Th2 cells, thereby initiating specific immune responses [ 57 – 59 ]. Based on this, the present study evaluated the T-cell proliferation capacity mediated by DCs stimulated with recombinant proteins using a mixed T-lymphocyte proliferation assay. Results showed that Mo-DCs treated with KC-1-S1 more effectively promoted T-cell proliferation compared to those treated with S1 protein alone. Furthermore, Mo-DCs incubated with KC-1-S1 secreted increased levels of cytokines, with Th1-associated factors IFN-γ and IL-12 significantly exceeding Th2-associated IL-4, suggesting predominant mediation of cellular immune responses. This finding aligns with prior studies demonstrating that co-expression of DCpep and PEDV S1 antigen effectively induces cellular immunity [ 53 ]. This study further demonstrates that the target peptide KC-1 enhances the antigen-presenting capacity of DCs without affecting T cell differentiation. In summary, KC-1, a short peptide derived from the N-terminal six amino acids of KC, specifically targets multiple porcine APC subsets via SLA-DRB1 and enhances DC-mediated antigen presentation and T cell activation. These results provide a concise molecular basis and valuable material for the efficient design of DC-targeted vaccines in pigs and potentially other species. Declarations Funding sources This research was supported by the Technology Support Program of Fourteenth Five Year Plan (2022YFD1800800), the National Natural Science Foundation of China (Nos. 32373048), and Natural Science Foundation of Heilongjiang Province of China (YQ2021C020). Ethics statement The animal study was reviewed and approved by the committee on the Ethics of Animal Experiments of Northeast Agricultural University, Harbin, China. Declaration of competing interest The authors declare that they have no conflict of interest. Copyright and Permissions Statement The authors declare that this manuscript is entirely original and does not contain any material reproduced from other sources, including published works or online content. All figures, tables, and text are the authors’ own work, and no permissions from third parties are required. Author Contribution B.L. and T.X. designed and performed the major experiments, analyzed data, and drafted the initial manuscript.C.J.B. assisted with literature review, experimental protocol organization, and preliminary data compilation.Y.P.J. contributed to workflow optimization and assisted in drafting parts of the Methods section.W.C. assisted in preparing experimental materials and maintaining routine operations in the cell culture facility.J.X.L. contributed to figure preparation, data visualization, and reagent management.Y.J.L. provided discussion support for the study design.L.W. conceived and supervised the study, secured funding, and finalized the manuscript as the corresponding author. Data Availability All data supporting the findings of this study are available within the paper and its Supplementary Information. References Wang L, Li D (2024) - Invited Review - Current status, challenges and prospects for pig production in Asia. Anim Biosci 37(4):742–754 Chepkwony MC, Makau DN, Yoder C, Corzo C, Culhane M, Perez A, Perez Aguirreburualde MS, Nault AJ, Mahero M (2025) A scoping review of knowledge, attitudes, and practices in swine farm biosecurity in North America. Front Vet Sci 12:1507704 Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392(6673):245–252 Colonna M, Pulendran B, Iwasaki A (2006) Dendritic cells at the host-pathogen interface. Nat Immunol 7(2) 117 – 20. Unger WW, van Beelen AJ, Bruijns SC, Joshi M, Fehres CM, van Bloois L, Verstege MI, Ambrosini M, Kalay H, Nazmi K, Bolscher JG, Hooijberg E, de Gruijl TD, Storm G, van Kooyk Y (2012) Glycan-modified liposomes boost CD4 + and CD8 + T-cell responses by targeting DC-SIGN on dendritic cells. J Control Release 160(1):88–95 Chen D, Wang Y, Wang H, Wu Y, Xia S, Zhang M (2015) CD8(+) T activation attenuates CD4(+) T proliferation through dendritic cells modification. Cell Immunol 296(2):138–148 Mazzoccoli L, Liu B (2024) Dendritic Cells in Shaping Anti-Tumor T Cell Response. Cancers (Basel) 16(12) Pittet MJ, Di Pilato M, Garris C, Mempel TR (2023) Dendritic cells as shepherds of T cell immunity in cancer. Immunity 56(10):2218–2230 Kumagai Y, Takeuchi O, Akira S (2008) TLR9 as a key receptor for the recognition of DNA. Adv Drug Deliv Rev 60(7):795–804 Volckmar J, Knop L, Hirsch T, Frentzel S, Erck C, van Ham M, Stegemann-Koniszewski S, Bruder D (2021) Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo. J Vis Exp (168) Krylov VB, Gómez-Redondo M, Solovev AS, Yashunsky DV, Brown AJP, Stappers MHT, Gow NAR, Ardá A, Jiménez-Barbero J, Nifantiev NE (2023) Identification of a new DC-SIGN binding pentamannoside epitope within the complex structure of Candida albicans mannan. Cell Surf 10:100109 Wang Z, He Y, Wang W, Tian Y, Ge C, Jia F, Zhang T, Zhang G, Wang M, Gong J, Huang H, Wang J, Shi C, Yang W, Cao X, Zeng Y, Wang N, Qian A, Jiang Y, Yang G, Wang C (2023) A novel prime and pull strategy mediated by the combination of two dendritic cell-targeting designs induced protective lung tissue-resident memory T cells against H1N1 influenza virus challenge. J Nanobiotechnol 21(1):479 Jalil AT, Abdulhadi MA, Al-Ameer LR, Taher WM, Abdulameer SJ, Abosaooda M, Fadhil AA (2024) Peptide-Based Therapeutics in Cancer Therapy. Mol Biotechnol 66(10):2679–2696 Song Y, Lei L, Cai X, Wei H, Yu CY (2025) Immunomodulatory Peptides for Tumor Treatment. Adv Healthc Mater 14(5):e2400512 Marqus S, Pirogova E, Piva TJ (2017) Evaluation of the use of therapeutic peptides for cancer treatment. J Biomed Sci 24(1):21 Lu Y, Liu ZH, Li YX, Xu HL, Fang WH, He F (2022) Targeted Delivery of Nanovaccine to Dendritic Cells via DC-Binding Peptides Induces Potent Antiviral Immunity in vivo. Int J Nanomed 17:1593–1608 Zheng J, Wang M, Pang L, Wang S, Kong Y, Zhu X, Zhou X, Wang X, Chen C, Ning H, Zhao W, Zhai W, Qi Y, Wu Y, Gao Y (2024) Identification of a novel DEC-205 binding peptide to develop dendritic cell-targeting nanovaccine for cancer immunotherapy. J Control Release 373:568–582 Liu M, Xie D, Hu D, Zhang R, Wang Y, Tang L, Zhou B, Zhao B, Yang L (2023) Situ Cocktail Nanovaccine for Cancer Immunotherapy. Adv Sci (Weinh) 10(31):e2207697 Summerfield A, McCullough KC (2009) The porcine dendritic cell family. Dev Comp Immunol 33(3):299–309 Xia T, Wang N, Tang Y, Gao Y, Gao C, Hao J, Jiang Y, Wang X, Shan Z, Li J, Zhou H, Cui W, Qiao X, Tang L, Wang L, Li Y (2022) Delivery of antigen to porcine dendritic cells by fusing antigen with porcine dendritic cells targeting peptide. Front Immunol 13:926279 Longet S, Lundahl MLE, Lavelle EC (2018) Targeted Strategies for Mucosal Vaccination. Bioconjug Chem 29(3):613–623 Li M, Wang Y, Sun Y, Cui H, Zhu SJ, Qiu HJ (2020) Mucosal vaccines: Strategies and challenges. Immunol Lett 217:116–125 Zhou T, Peng J, Hao Y, Shi K, Zhou K, Yang Y, Yang C, He X, Chen X, Qian Z (2021) The construction of a lymphoma cell-based, DC-targeted vaccine, and its application in lymphoma prevention and cure. Bioact Mater 6(3):697–711 Mestrallet G, Sone K, Bhardwaj N (2022) Strategies to overcome DC dysregulation in the tumor microenvironment. Front Immunol 13:980709 Truex NL, Rondon A, Rössler SL, Hanna CC, Cho Y, Wang BY, Backlund CM, Lutz EA, Irvine DJ, Pentelute BL (2023) Enhanced Vaccine Immunogenicity Enabled by Targeted Cytosolic Delivery of Tumor Antigens into Dendritic Cells. ACS Cent Sci 9(9):1835–1845 Snider DP, Segal DM (1987) Targeted antigen presentation using crosslinked antibody heteroaggregates. J Immunol 139(5):1609–1616 Wille-Reece U, Wu CY, Flynn BJ, Kedl RM, Seder RA (2005) Immunization with HIV-1 Gag protein conjugated to a TLR7/8 agonist results in the generation of HIV-1 Gag-specific Th1 and CD8 + T cell responses. J Immunol 174(12):7676–7683 Shi SH, Yang WT, Yang GL, Zhang XK, Liu YY, Zhang LJ, Ye LP, Hu JT, Xing X, Qi C, Li Y, Wang CF (2016) Lactobacillus plantarum vaccine vector expressing hemagglutinin provides protection against H9N2 challenge infection. Virus Res 211:46–57 Shimizu T, Kawaguchi Y, Ando H, Ishima Y, Ishida T (2022) Development of an Antigen Delivery System for a B Cell-Targeted Vaccine as an Alternative to Dendritic Cell-Targeted Vaccines. Chem Pharm Bull (Tokyo) 70(5):341–350 Matsuda T, Misato K, Tamiya S, Akeda Y, Nakase I, Kuroda E, Takahama S, Nonaka M, Yamamoto T, Fukuda MN, Yoshioka Y (2022) Efficient antigen delivery by dendritic cell-targeting peptide via nucleolin confers superior vaccine effects in mice. iScience 25(11):105324 Iborra S, Izquierdo HM, Martínez-López M, Blanco-Menéndez N, Reis e Sousa C, Sancho D (2012) The DC receptor DNGR-1 mediates cross-priming of CTLs during vaccinia virus infection in mice. J Clin Invest 122(5):1628–1643 Skibska A, Perlikowska R (2021) Signal Peptides - Promising Ingredients in Cosmetics. Curr Protein Pept Sci 22(10):716–728 Kim S, Kim D, Jung HH, Lee IH, Kim JI, Suh JY, Jon S (2012) Bio-inspired design and potential biomedical applications of a novel class of high-affinity peptides. Angew Chem Int Ed Engl 51(8):1890–1894 Sidhu SS (2000) Phage display in pharmaceutical biotechnology. Curr Opin Biotechnol 11(6):610–616 Dai J, Patzke C, Liakath-Ali K, Seigneur E, Südhof TC (2021) GluD1 is a signal transduction device disguised as an ionotropic receptor. Nature 595(7866):261–265 Scodeller P, Asciutto EK (2020) Target Tumors Using Peptides Molecules 25(4) Van Chanh Le Q, Le TM, Cho HS, Kim WI, Hong K, Song H, Kim JH, Park C (2018) Analysis of peptide-SLA binding by establishing immortalized porcine alveolar macrophage cells with different SLA class II haplotypes. Vet Res 49(1):96 Wahyuningtyas R, Lai YS, Wu ML, Chen HW, Chung WB, Chaung HC, Chang KT (2021) Recombinant Antigen of Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2) Promotes M1 Repolarization of Porcine Alveolar Macrophages and Th1 Type Response. Vaccines (Basel) 9(9) Roche PA, Furuta K (2015) The ins and outs of MHC class II-mediated antigen processing and presentation. Nat Rev Immunol 15(4):203–216 Konjević D, Erman V, Bujanić M, Svetličić I, Arbanasić H, Lubura Strunjak S, Galov A (2022) Wild Boar (Sus scrofa)-Fascioloides magna Interaction from the Perspective of the MHC Genes. Pathogens 11(11) Sidhu SS, Koide S (2007) Phage display for engineering and analyzing protein interaction interfaces. Curr Opin Struct Biol 17(4):481–487 Wang Y, Sun D, Laney V, Wang H, Wang LL, Lu ZR (2025) Challenges and opportunities on achieving an adequate delivery efficiency and immunogenicity with peptide-based anticancer vaccines. Adv Drug Deliv Rev 225:115675 Brown JH, Jardetzky TS, Gorga JC, Stern LJ, Urban RG, Strominger JL, Wiley DC (1993) Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 364(6432):33–39 Stern LJ, Brown JH, Jardetzky TS, Gorga JC, Urban RG, Strominger JL, Wiley DC (1994) Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature 368(6468):215–221 Owen JL, Sahay B, Mohamadzadeh M (2013) New generation of oral mucosal vaccines targeting dendritic cells. Curr Opin Chem Biol 17(6):918–924 Olajide OM, Osman MK, Robert J, Kessler S, Toews LK, Thamamongood T, Neefjes J, Wrobel AG, Schwemmle M, Ciminski K, Reuther P (2023) Evolutionarily conserved amino acids in MHC-II mediate bat influenza A virus entry into human cells. PLoS Biol 21(7):e3002182 Ferrante A (2013) For many but not for all: how the conformational flexibility of the peptide/MHCII complex shapes epitope selection. Immunol Res 56(1):85–95 Jia Z, Pan X, Zhi W, Chen H, Bai B, Ma C, Ma D (2022) Probiotics Surface-Delivering Fiber2 Protein of Fowl Adenovirus 4 Stimulate Protective Immunity Against Hepatitis-Hydropericardium Syndrome in Chickens. Front Immunol 13:919100 Yang WT, Li QY, Ata EB, Jiang YL, Huang HB, Shi CW, Wang JZ, Wang G, Kang YH, Liu J, Yang GL, Wang CF (2018) Immune response characterization of mice immunized with Lactobacillus plantarum expressing spike antigen of transmissible gastroenteritis virus. Appl Microbiol Biotechnol 102(19):8307–8318 Hou X, Jiang X, Jiang Y, Tang L, Xu Y, Qiao X, Min L, Wen C, Ma G, Li Y (2018) Oral Immunization against PEDV with Recombinant Lactobacillus casei Expressing Dendritic Cell-Targeting Peptide Fusing COE Protein of PEDV in Piglets, Viruses 10(3) Joshi LR, Okda FA, Singrey A, Maggioli MF, Faccin TC, Fernandes MHV, Hain KS, Dee S, Bauermann FV, Nelson EA, Diel DG (2018) Passive immunity to porcine epidemic diarrhea virus following immunization of pregnant gilts with a recombinant orf virus vector expressing the spike protein. Arch Virol 163(9):2327–2335 Li Q, Peng O, Wu T, Xu Z, Huang L, Zhang Y, Xue C, Wen Z, Zhou Q, Cao Y (2018) PED subunit vaccine based on COE domain replacement of flagellin domain D3 improved specific humoral and mucosal immunity in mice. Vaccine 36(11):1381–1388 Thavorasak T, Chulanetra M, Glab-Ampai K, Teeranitayatarn K, Songserm T, Yodsheewan R, Sae-Lim N, Lekcharoensuk P, Sookrung N, Chaicumpa W (2022) Novel Neutralizing Epitope of PEDV S1 Protein Identified by IgM Monoclonal Antibody. Viruses 14(1) Duong BTT, Thao PTP, Hoa NT, Thu HT, Phuoc MH, Le TH, Van Quyen D (2022) Molecular analysis reveals a distinct subgenogroup of porcine epidemic diarrhea virus in northern Vietnam in 2018–2019. Arch Virol 167(11):2337–2346 Thavorasak T, Chulanetra M, Glab-Ampai K, Mahasongkram K, Sae-Lim N, Teeranitayatarn K, Songserm T, Yodsheewan R, Nilubol D, Chaicumpa W, Sookrung N (2022) Enhancing epitope of PEDV spike protein. Front Microbiol 13:933249 Li M, Wang Y, Wang Y, Li R, Wang S, Ding P, Zhang G (2023) Accurate location of two conserved linear epitopes of PEDV utilizing monoclonal antibodies induced by S1 protein nanoparticles. Int J Biol Macromol 253(6):127276 Huang KY, Yang GL, Jin YB, Liu J, Chen HL, Wang PB, Jiang YL, Shi CW, Huang HB, Wang JZ, Wang G, Kang YH, Yang WT, Wang CF (2018) Construction and immunogenicity analysis of Lactobacillus plantarum expressing a porcine epidemic diarrhea virus S gene fused to a DC-targeting peptide. Virus Res 247:84–93 Gillam F, Zhang J, Zhang C (2018) Hepatitis B core antigen based novel vaccine against porcine epidemic diarrhea virus. J Virol Methods 253:61–69 Yu M, Wang L, Ma S, Wang X, Wang Y, Xiao Y, Jiang Y, Qiao X, Tang L, Xu Y, Li Y (2017) Immunogenicity of eGFP-Marked Recombinant Lactobacillus casei against Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus. Viruses 9(10) Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.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-8110404","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":550063175,"identity":"42607175-a34e-4d0c-ad9c-517ba50d019f","order_by":0,"name":"Bin Liu","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Liu","suffix":""},{"id":550063176,"identity":"fa12b37e-d5c5-418a-beb3-cd39890a41ef","order_by":1,"name":"Tian Xia","email":"","orcid":"","institution":"Heilongjiang Polytechnic","correspondingAuthor":false,"prefix":"","firstName":"Tian","middleName":"","lastName":"Xia","suffix":""},{"id":550063177,"identity":"6230cfa7-f843-4d69-abd2-ed38f872402f","order_by":2,"name":"Chengjie Bian","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chengjie","middleName":"","lastName":"Bian","suffix":""},{"id":550063178,"identity":"a554d159-e76b-44bd-9611-9fb19b2a1d7d","order_by":3,"name":"Yanping Jiang","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yanping","middleName":"","lastName":"Jiang","suffix":""},{"id":550063179,"identity":"5a2bb33a-15ab-40c9-8162-7e9cb4f59ce7","order_by":4,"name":"Wen Cui","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Cui","suffix":""},{"id":550063180,"identity":"63e93f9a-86d4-4c14-8424-e2c0ea46992b","order_by":5,"name":"Jiaxuan Li","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jiaxuan","middleName":"","lastName":"Li","suffix":""},{"id":550063181,"identity":"6a630874-6630-4188-9d19-854debc7e2b4","order_by":6,"name":"Yijing Li","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yijing","middleName":"","lastName":"Li","suffix":""},{"id":550063182,"identity":"b4fc21cf-0f45-4c9d-967f-c04c6b74253b","order_by":7,"name":"Li Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYLCCBwZAgr2x8eEHorUkgLTwHG42liBeC4iQSG8T4CFGtcHxs4dfJBTYyRvcfNjGIMFgJ6fbQEjLmbw0iwSDZMMNtxPbHhQwJBubHSCgxexAjplBgsEBRqCWdgMJhgOJ2whqOf8GrMV+w82DbRI8RGm5kWP8AKglccMNRiK12N94YwYM5OTkmWcSgYFsQIRfJPtzjD98+GNn23f8+MOHHyrs5AhqAQI2pAg0IKwcBJiJTyajYBSMglEwMgEAiI1HevBCmh0AAAAASUVORK5CYII=","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Li","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-11-14 03:53:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8110404/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8110404/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97250935,"identity":"78b3d62f-92ea-46c1-b9e2-f6dd4ee33a57","added_by":"auto","created_at":"2025-12-02 13:15:38","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9300508,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/7c234866252b9b2ba9f42f8d.docx"},{"id":97192566,"identity":"4c3a9bdb-fcda-4c5a-818f-08169554142b","added_by":"auto","created_at":"2025-12-01 20:19:12","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9490,"visible":true,"origin":"","legend":"","description":"","filename":"7f8541f0598c4fdbb0016b4ab41afcb1.json","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/9a626519a38dcd1d112ed320.json"},{"id":97249272,"identity":"3bcb3e19-384b-4b78-a05a-b16e189968ca","added_by":"auto","created_at":"2025-12-02 13:11:53","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4067983,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/55afb6cdf0996d961cd5917c.docx"},{"id":97192577,"identity":"cf5b157a-4314-44bb-82e2-e4c9c04099d6","added_by":"auto","created_at":"2025-12-01 20:19:12","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":200558,"visible":true,"origin":"","legend":"","description":"","filename":"7f8541f0598c4fdbb0016b4ab41afcb11enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/c768d487650970255a38eff8.xml"},{"id":97192571,"identity":"5ca38ecd-7b20-436f-88b0-062342ec144d","added_by":"auto","created_at":"2025-12-01 20:19:12","extension":"eps","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":648,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage1.eps","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/2c0faffba9e39b3d8988bd56.eps"},{"id":97250657,"identity":"29ac2634-7d13-4ae7-82c4-b362338e5abb","added_by":"auto","created_at":"2025-12-02 13:14:52","extension":"eps","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":648,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage1.eps","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/179c2fa81b3977e01b9ecc7d.eps"},{"id":97250900,"identity":"c14a3eb0-8151-4693-a431-de013fd67d56","added_by":"auto","created_at":"2025-12-02 13:15:35","extension":"eps","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":648,"visible":true,"origin":"","legend":"","description":"","filename":"drawingimage1.eps","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/b3ee0e22383867bcde0af9bc.eps"},{"id":97249312,"identity":"527040e0-8a6a-4dd4-8393-2a84d0dab60b","added_by":"auto","created_at":"2025-12-02 13:12:07","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":866870,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/f79cb8d38db689907b0f1618.jpeg"},{"id":97249536,"identity":"9d668708-5d85-4d51-aac7-94ac356727b3","added_by":"auto","created_at":"2025-12-02 13:12:52","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":519272,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/0f48bfc4e0dad589e4f7a34d.jpeg"},{"id":97192578,"identity":"1edfb2e2-9e08-4a10-ac4b-bf76fba6c3d1","added_by":"auto","created_at":"2025-12-01 20:19:12","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1174673,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/b11f8c9820ad69e7e956dd39.jpeg"},{"id":97192583,"identity":"f22ae752-ee4e-41b9-8fca-61d7e536b014","added_by":"auto","created_at":"2025-12-01 20:19:12","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":640696,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/deb0771df34ef2d6d8a82b62.jpeg"},{"id":97192582,"identity":"410e2267-3d78-4578-819c-e8cd57d838d6","added_by":"auto","created_at":"2025-12-01 20:19:12","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1361536,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/8d587856714cf4afc4854700.jpeg"},{"id":97192589,"identity":"73e4e7bc-150b-42b2-a540-0940679d73a0","added_by":"auto","created_at":"2025-12-01 20:19:12","extension":"jpeg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":505825,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/7c41216d1fa0ab22439f493c.jpeg"},{"id":97249439,"identity":"aebc9561-b008-4cec-a6f7-62b1cac80b5d","added_by":"auto","created_at":"2025-12-02 13:12:36","extension":"jpeg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1611157,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/3adbf1212ae9f629fe87319b.jpeg"},{"id":97192591,"identity":"62289ea8-1761-4739-8548-23df0cf96587","added_by":"auto","created_at":"2025-12-01 20:19:13","extension":"jpeg","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2274408,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/46312f1aed02a202b263c80a.jpeg"},{"id":97192579,"identity":"c915d8c4-5afd-4910-a52d-021867fd4876","added_by":"auto","created_at":"2025-12-01 20:19:12","extension":"jpeg","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1114,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/c0aa001f9309954d6124c334.jpeg"},{"id":97249268,"identity":"015cd675-805d-43fb-8656-a0bdaf7e80e0","added_by":"auto","created_at":"2025-12-02 13:11:52","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":111114,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/a61158cafa785f21d2c31380.png"},{"id":97251094,"identity":"66bf00b6-d73f-4cc2-bcaf-8d781f6512e9","added_by":"auto","created_at":"2025-12-02 13:16:02","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":59994,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/112cbb5460ee4f06caf1ff22.png"},{"id":97249342,"identity":"9a9de26d-a279-4071-99a3-6153e2308f71","added_by":"auto","created_at":"2025-12-02 13:12:15","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":263135,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/85fb72159f8bb679d131d511.png"},{"id":97250947,"identity":"09cd6dfa-b905-4231-a9ac-9e1426c0a535","added_by":"auto","created_at":"2025-12-02 13:15:40","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144598,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/4afa1ce5e14bc013e4b3e93d.png"},{"id":97192596,"identity":"4a3709d4-907f-4a68-a648-22c562bf8e9a","added_by":"auto","created_at":"2025-12-01 20:19:13","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":117916,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/2d87d19d24b2dc11abfcd938.png"},{"id":97249271,"identity":"8ff7f099-75b6-4cee-9a9e-b900cf22962c","added_by":"auto","created_at":"2025-12-02 13:11:53","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":97719,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/a0c551112484200011386f99.png"},{"id":97192593,"identity":"f1ca01fc-0a41-41a4-a9a1-35f051391ebb","added_by":"auto","created_at":"2025-12-01 20:19:13","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":359895,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/941ded0191dae31d4ccdf5c6.png"},{"id":97249351,"identity":"4c1f2437-af5e-40a7-8358-1855a8147a85","added_by":"auto","created_at":"2025-12-02 13:12:17","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":499924,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/4a0f392a6a46d08d6a896d56.png"},{"id":97249796,"identity":"f54f8985-24b0-451f-9313-c4121a3ef780","added_by":"auto","created_at":"2025-12-02 13:13:26","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":940,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/025a11de44c309ae32dd340f.png"},{"id":97249339,"identity":"aa8e189c-538d-46c0-b51e-de8742d5a3e9","added_by":"auto","created_at":"2025-12-02 13:12:14","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":195904,"visible":true,"origin":"","legend":"","description":"","filename":"7f8541f0598c4fdbb0016b4ab41afcb11structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/42e7f02824938152f048f9b8.xml"},{"id":97192598,"identity":"d5ebae28-54cc-4f7e-ad9c-94d3b1ec85e2","added_by":"auto","created_at":"2025-12-01 20:19:13","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":209070,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/c8af51a20d03ae45af5f09cc.html"},{"id":97192565,"identity":"b6686a72-0f5a-42c3-8d40-ad806d5237e8","added_by":"auto","created_at":"2025-12-01 20:19:12","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":256967,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening of peptides targeting porcine Mo-DCs.\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003e(A) Phage ELISA was used to assess the binding selectivity of phage clones to porcine Mo-DCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as means ± SD from triplicate experiments. * Indicates P \u0026lt; 0.05 (two-tailed Student’s t-test)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) Fluorescence microscopy detection of the binding of peptides HS, SF, and KC to porcine Mo-DCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKC-FITC, HS-FITC, SF-FITC, and NC-FITC were individually incubated with 4% paraformaldehyde-fixed Mo-DCs, and the binding was observed under a fluorescence microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) Confocal Microscopy Analysis of Peptide Binding to Porcine Intestinal DCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrozen piglet intestinal sections were incubated with PE-anti-CD172a antibody and FITC-labeled peptides (KC, HS, SF, NC), imaged by confocal laser scanning microscopy to observe peptide binding to dendritic cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) Flow cytometry analysis of peptide affinity to porcine DCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePorcine DCs were incubated with FITC-labeled peptides (KC, HS, SF, NC), and peptide binding affinity was analyzed by flow cytometry. The data reflect % abundance for positive populations. Data are presented as mean ± standard deviation from triplicate experiments. Groups labeled with different letters (e.g., a, b, c) are significantly different (p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/66572e87a35b399263d21962.jpg"},{"id":97192567,"identity":"d20ec1e7-9e4e-4f3b-89ac-cbf8460fd069","added_by":"auto","created_at":"2025-12-01 20:19:12","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":190787,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of Key Amino Acids in the KC Peptide Responsible for Binding to Porcine DCs\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003e(A–C) Competitive ELISA analysis of the binding affinities of the KC peptide and its alanine-scanning mutants to porcine DCs.\u003c/strong\u003e\u003cbr\u003e\nPorcine DCs and PBMCs were incubated with various concentrations of the KC peptide and its alanine mutants, followed by absorbance measurement at 450 nm. Data are presented as mean ± standard deviation from triplicate experiments. Groups labeled with different letters (e.g., a, b, c, d) are significantly different (p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/e5c72de3c6d09f9b92cc20b3.jpg"},{"id":97249551,"identity":"35f6749d-b4e7-4303-8884-53db062f4736","added_by":"auto","created_at":"2025-12-02 13:12:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":308989,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular specificity of KC-1 peptide binding.\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003e(A) Fluorescence microscopy detection of the binding of KC-1-FITC peptide to porcine DCs, PAMs, BM-DCs, rabbit Mo-DCs, and non-DC cell lines (IPI, Vero, ST).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKC-1-FITC was incubated with 4% paraformaldehyde-fixed porcine Mo-DCs, PAMs, rabbit Mo-DCs, as well as ST cells, Vero cells, and IPI cells, and the binding was observed using fluorescence microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) Flow cytometry analysis of KC-1 binding affinity to immune cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKC-1-FITC was incubated with porcine Mo-DCs, PAMs, and rabbit Mo-DCs, and flow cytometry was performed to assess the binding affinity of KC-1 to various immune cells.The data reflect % abundance for positive populations. Data are presented as mean ± standard deviation from triplicate experiments. Groups labeled with different letters (e.g., a, b) are significantly different (p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/1accf24d714b65ed05bbde08.jpg"},{"id":97249302,"identity":"d15d2cc0-291c-496b-9518-53a3133f40b5","added_by":"auto","created_at":"2025-12-02 13:12:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":192140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSLA-DRB1 serves as the primary receptor for KC-1 targeting dendritic cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) Synthesis and characterization of KC-1-BIOTIN and NC-BIOTIN.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) ELISA analysis of the binding affinity of KC-1-BIOTIN and NC-BIOTIN to PAMs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePAMs incubated with KC1-BIOTIN or NC-BIOTIN, and absorbance was measured at 450 nm. Data are presented as mean ± standard deviation from triplicate experiments. Groups labeled with different letters (e.g., a, b) are significantly different (p \u0026lt; 0.01).\u003cbr\u003e\n \u003cstrong\u003e(C) Western blotting identification of PAMs membrane proteins.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMembrane proteins of PAMs were extracted using the ExKine™ Total Membrane Protein Extraction Kit. Western blotting analysis was adopted for analyzing the proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) KC-1 targeted protein silver staining results.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSDS-PAGE gels were stained using the Pierce™ mass spectrometry-compatible silver stain kit. \u003cbr\u003e\n \u003cstrong\u003e(E) Analysis of KC-1 Interaction with SLA-DRB1 and SLA-1 by Co-IP.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCo-IP was performed using streptavidin-coated magnetic beads, and the precipitated proteins were analyzed by Western blotting. \u003cstrong\u003e\u003cbr\u003e\n(F) Co-localization results of DC targeting peptide KC-1 and SLA-DRB1 in PAMs and Mo-DCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKC1-FITC was incubated separately with PAMs and Mo-DCs, and observed using confocal microscopy.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/39b03ea54f54f11e9c5cf26d.jpg"},{"id":97192570,"identity":"17630817-bcdb-4582-987f-322904811935","added_by":"auto","created_at":"2025-12-01 20:19:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":255680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrediction and Validation of the Binding Domain of DC-Targeting Peptide KC-1 on SLA-DRB1 Protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) Prediction of binding sites between DC targeted peptide KC-1 and SLA-DRB1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe interaction sites between the targeting peptide KC-1 and SLA-DRB1 were predicted using \u003ca href=\"http://huanglab.phys.hust.edu.cn/hpepdock/\"\u003eHPEPDOCK 2.0\u003c/a\u003e software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e \u003cstrong\u003eSLA-DRB1 Protein Truncation Construction.\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003e(C) IFA detection of SLA-DRB1, SLA-DRB1a, and SLA-DRB1b protein expression in BHK-21 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBHK-21 cells were transfected with plasmids encoding HIS-tagged SLA-DRB1, SLA-DRB1a, and SLA-DRB1b, and the protein expression was observed using fluorescence microscopy.\u003cbr\u003e\n \u003cstrong\u003e(D) Western blotting detection of SLA-DRB1, SLA-DRB1a, and SLA-DRB1b protein expression in BHK-21 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBHK-21 cells were transfected with plasmids encoding HIS-tagged SLA-DRB1, SLA-DRB1a, and SLA-DRB1b, and the protein expression was analyzed using Western blotting.\u003cbr\u003e\n \u003cstrong\u003e(E) Co-IP characterization of the binding domain of KC-1 to SLA-DRB1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBHK-21 cells were transfected with plasmids encoding HIS-tagged SLA-DRB1, SLA-DRB1a, and SLA-DRB1b. Co-IP was performed using streptavidin-coated magnetic beads, and the precipitated proteins were analyzed by Western blotting.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/79a05e2abdc59700c7021a44.jpg"},{"id":97249443,"identity":"fffb4f0d-9a2a-4c2b-b533-60bc2b880da0","added_by":"auto","created_at":"2025-12-02 13:12:37","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":198694,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKC-1 Targeting Peptide Enhances the Immune Function of Antigen-Stimulated Porcine DCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-B) Expression levels of CD40, CD80, CD86, TLR-2, TLR-4, TLR-6, and TLR-9 in Mo-DCs incubated with short peptide KC-1, recombinant proteins (S1, NC-S1, and KC-1-S1), and LPS, measured by qRT-PCR.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnstimulated porcine Mo-DCs were used as the control, and β-actin served as the internal reference gene. Data acquisition and analysis were performed using a real-time PCR detection system (7500 System Software). The relative expression levels of surface molecules were calculated using the relative quantification method (2\u003csup\u003e−ΔΔCT\u003c/sup\u003e). The calculation formula is as follows: ΔΔCt = (Ct\u003csub\u003etarget gene\u003c/sub\u003e − Ct\u003csub\u003ereference gene\u003c/sub\u003e)\u003csub\u003e experimental group \u003c/sub\u003e− (Ct\u003csub\u003etarget gene\u003c/sub\u003e − Ct\u003csub\u003ereference gene\u003c/sub\u003e)\u003csub\u003e control group\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e \u003cstrong\u003eMo-DCs treated with peptide KC-1, recombinant proteins (S1, NC-S1, and KC-1-S1), and LPS were used to stimulate T lymphocyte proliferation in a mixed lymphocyte reaction (MLR).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResponder cells were added at ratios of 1:1, 1:10, or 1:100 and co-cultured with stimulator cells for 72 hours. Proliferation was expressed as the stimulation index (SI), calculated using the formula: Stimulation Index = (OD\u003csub\u003esample\u003c/sub\u003e − OD\u003csub\u003estimulator cells only\u003c/sub\u003e) / (OD\u003csub\u003eresponder cells only \u003c/sub\u003e− OD\u003csub\u003eblank control\u003c/sub\u003e).\u003cbr\u003e\n\u003cstrong\u003e(D) ELISA measurement of cytokine levels (IL-4, IL-12, and IFN-γ) in supernatants from DCs co-cultured with recombinant proteins and T cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDCs were stimulated with KC-1, recombinant proteins (S1, NC-S1, and KC-1-S1), and LPS, then co-cultured with T cells at a ratio of 1:1. Cytokine levels were measured by ELISA.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± standard deviation from triplicate experiments. Groups labeled with different letters (e.g., a, b, c, d, e) are significantly different (p \u0026lt; 0.01)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/67a707421998220663e8bd24.jpg"},{"id":100373316,"identity":"639a2b43-eb74-465a-8b63-a1ed266495ba","added_by":"auto","created_at":"2026-01-16 08:14:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3145921,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/655d68d8-8ade-4f1b-acc0-4027053a486a.pdf"},{"id":97192574,"identity":"f367499d-6e76-4936-8374-252e8b82c4b2","added_by":"auto","created_at":"2025-12-01 20:19:12","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4067983,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8110404/v1/c280904d12e42b030a22a5f9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Discovery of KC-1: A Novel Porcine Dendritic Cell-Targeting Peptide with Potential Applications in Swine Vaccine Design","fulltext":[{"header":"Highlights","content":"\u003cp\u003eA novel porcine dendritic cell-targeting peptide, KC-1, was identified from a phage display library.\u003c/p\u003e\u003cp\u003eKC-1 specifically binds to the MHC class II molecule SLA-DRB1 on porcine APCs.\u003c/p\u003e\u003cp\u003eFusion of KC-1 with PEDV S1 enhances DC maturation, antigen presentation, and T-cell activation in vitro.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eIn recent years, the global swine industry has become a critical pillar for ensuring food security and driving rural economic development. China, in particular, has emerged as the world\u0026rsquo;s largest producer and consumer of pork, accounting for over half of the global annual output [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, the intensification of farming practices has led to continuously increasing stocking densities, creating favorable conditions for the spread of major infectious pathogens [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among swine populations, vaccination remains the most effective and sustainable strategy for controlling infectious diseases. To improve vaccine efficacy, durability, and broad-spectrum protection, researchers have explored various strategies. Among these, DC-targeted vaccine design has garnered increasing attention due to the crucial role of DCs in initiating and regulating immune responses, positioning this approach as a promising avenue for next-generation vaccine development.\u003c/p\u003e\u003cp\u003eDendritic cells (DCs), as the most potent professional antigen-presenting cells in the immune system, are widely distributed at mucosal and cutaneous surfaces\u0026mdash;the front lines of pathogen invasion\u0026mdash;where they serve as central \u0026ldquo;immune sentinels\u0026rdquo; [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Their core functions involve recognizing pathogen-associated molecular patterns (PAMPs) and danger signals via pattern recognition receptors (such as TLRs), efficiently capturing and processing antigens, and presenting them to na\u0026iuml;ve T cells via MHC class I and II molecules, thereby activating adaptive immune responses [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Mature DCs not only act as a bridge linking innate and adaptive immunity, but also coordinate the activity of B cells, NK cells, and other immune effectors through co-stimulatory molecules and cytokines, facilitating integrated humoral and cellular immunity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Owing to this central immunological role, targeted delivery of antigens to DCs has become a key strategy to enhance vaccine performance. Current DC-targeting approaches typically employ antibodies, ligands, or targeting peptides specific to DC surface receptors to guide antigens, nucleic acids, or drugs to specific DC subsets. [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAmong these approaches, targeting peptides have gradually emerged as an important direction in DC-targeted vaccine design due to their unique advantages over antibodies and ligands. Peptides are small in size, structurally stable, low in immunogenicity, and capable of penetrating cell membranes. They are also easier to synthesize, modify (e.g., PEGylation or D-amino acid substitution), and scale up for production[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Moreover, techniques such as phage display and receptor docking simulation allow for high-throughput and highly specific identification of functional peptides that bind to DC surface receptors, significantly simplifying the construction of antigen delivery systems [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Many of the identified targeting peptides have been validated to improve the immunogenicity and protective efficacy of vaccines. For instance, the mouse-derived DC-targeting peptide DCpep3 has been shown to enhance antigen uptake and antibody responses when used in a nanoparticle vaccine against porcine circovirus type 2 (PCV2) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].. The peptide hr-8, identified from the C-type lectin receptor DEC-205, specifically targets DCs via DEC-205 binding and promotes their maturation by upregulating the surface expression of MHC class II, CD80, and CD86, thereby enhancing antigen presentation and cross-priming of T cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, DP7-C, a cholesterol-modified peptide designed through computational optimization, exhibits dual functionality: it targets DCs while simultaneously enhancing their migration and lymph node homing, acting both as a delivery enhancer and an immunostimulatory adjuvant [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite the successful application of DC-targeting peptides in multiple species and their ability to enhance antigen uptake and immune responses, significant interspecies differences exist in the composition of DC subsets, expression of surface receptors, and antigen presentation pathways [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To address this critical need, this study first employed phage display technology to screen high-affinity linear peptides capable of specifically recognizing porcine DCs, and further analyzed their key amino acid motifs and binding sites on DC surface receptors. Based on these findings, the selected targeting peptides were fused with the PEDV S1 subunit, and the fusion proteins\u0026rsquo; effects on antigen presentation and DC activation were evaluated. This study is expected to provide novel peptide-based tools and mechanistic insights to facilitate the development of highly efficient and precision-designed DC-targeted vaccines for swine immunization.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials and reagents\u003c/h2\u003e\u003cp\u003eRed blood cell lysis buffer and Western blot primary antibody dilution buffer were obtained from Shanghai Beyotime Biotechnology Co., Ltd. (China). Streptavidin-coated magnetic beads and DAPI nuclear stain were purchased from Thermo Fisher Scientific (USA). RPMI-1640 medium and fetal bovine serum (FBS) were supplied by GIBCO (USA). The Ph.D\u0026trade;-12 Phage Display Peptide Library Kit was obtained from New England Biolabs (NEB, USA). Histopaque-1077 lymphocyte separation medium, lipopolysaccharide (LPS), silver staining reagent, and FITC-conjugated dextran were purchased from Sigma-Aldrich (USA). Phage genomic DNA, total RNA, and plasmid extraction kits were obtained from Shanghai Feijie Biotechnology Co., Ltd. (China). SYBR Green qPCR premix was purchased from Roche (USA), and the cell membrane extraction kit was obtained from ActGene (China). KOD DNA polymerase and reverse transcriptase were purchased from Shanghai Biotechnology Co., Ltd. (China). Transfection reagent was obtained from Thermo Fisher Scientific (USA), while T4 DNA ligase and restriction enzymes were purchased from TaKaRa Biotechnology Co., Ltd. (Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Plasmids, bacterial strains, and cell lines\u003c/h2\u003e\u003cp\u003eThe pMD19-T and pMD19-T Simple cloning vectors were obtained from Dalian TaKaRa Biotechnology Co., Ltd. (China). The recombinant plasmid pMD19-S1, containing the S1 gene of porcine epidemic diarrhea virus (PEDV), as well as the expression vectors pET23a/TG1, pET23a-S1/TG1 (encoding PEDV S1 in E. coli), and pCMV/TG1, were maintained by the Microbiology and Immunology Laboratory, College of Veterinary Medicine, Northeast Agricultural University (China). TG1 competent E. coli cells were purchased from TransGen Biotech (China). The BHK, ST, and IPI cell lines were also preserved by the Microbiology and Immunology Laboratory, College of Veterinary Medicine, Northeast Agricultural University (China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Animal\u003c/h2\u003e\u003cp\u003eAntibody-negative, healthy female 1-month-old piglets were obtained from a certified commercial pig farm in Harbin, China. All animal experiments were performed in accordance with the guidelines of the Animal Ethics Committee of Northeast Agricultural University (approval number: NEAUEC0210337).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Primary cell isolation and culture\u003c/h2\u003e\u003cp\u003ePBMCs were isolated from the jugular vein of piglets via density gradient centrifugation. Monocytes were differentiated into DCs by culturing in RPMI-1640 supplemented with 20 ng/mL GM-CSF and 20 ng/mL IL-4. Morphological changes were monitored using light microscopy. On day 7, immature Mo-DCs were harvested and stained with PE-conjugated CD172a and FITC-conjugated MHC II for 30 min, washed thrice with PBS, and analyzed by flow cytometry and fluorescence microscopy.\u003c/p\u003e\u003cp\u003ePAMs were obtained from lung lavage fluid of 5\u0026ndash;8-week-old pigs. Lungs were flushed with RPMI-1640 containing 1% PBS, filtered through a 70 \u0026micro;m strainer, centrifuged (1,500 rpm, 10 min), treated with red blood cell lysis buffer, and cultured in complete medium (90% RPMI-1640, 10% FBS) at 37\u0026deg;C, 5% CO₂.\u003c/p\u003e\u003cp\u003eBM-DCs were generated from femurs and tibiae of 5\u0026ndash;8-week-old pigs. Flushed bone marrow cells were filtered, centrifuged, red blood cells lysed, and cultured in complete medium with 20 ng/mL GM-CSF and 20 ng/mL IL-4. Medium was refreshed every 2 days, and immature BM-DCs were harvested on day 5.\u003c/p\u003e\u003cp\u003eRabbit Mo-DCs were prepared from PBMCs isolated via density gradient centrifugation. Cells were seeded at 2 \u0026times; 10⁶ cells/mL in 12-well plates containing complete medium (RPMI-1640, 10% FBS, 1% penicillin-streptomycin) and incubated at 37\u0026deg;C, 5% CO₂. After 6 h, non-adherent cells were removed, and adherent monocytes were cultured in medium supplemented with 20 ng/mL recombinant human GM-CSF and IL-4. Half of the medium was replaced every 2 days, and immature Mo-DCs were harvested on day 5 for downstream experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Screen Mo-DCs binding peptides by phage display technique\u003c/h2\u003e\u003cp\u003ePeptides binding to porcine monocyte-derived dendritic cells (Mo-DCs) were screened using the Ph.D.\u0026trade;-12 phage display peptide library kit (NEB, Beijing, China) according to the manufacturer\u0026rsquo;s protocol. Immature Mo-DCs (1 \u0026times; 10⁶ cells/mL) were detached from culture plates with PBS and collected into 1.5 mL centrifuge tubes. The cells were then incubated with 2 \u0026times; 10\u0026sup1;\u0026sup1; pfu of the Ph.D.-12 phage library at 4\u0026deg;C for 30 minutes. Following incubation, cells were centrifuged at 2,000 rpm for 3 minutes and resuspended in PBS containing 1% BSA and 0.05% Tween 20. Three rounds of centrifugation and washing were performed to remove unbound phages.\u003c/p\u003e\u003cp\u003e Bound phages were eluted using an acidic buffer (pH 2.2) containing 1 mg/mL BSA and 0.2 M glycine-HCl with gentle mixing for 10 minutes, followed by neutralization with 50 \u0026micro;L of Tris-HCl buffer (pH 9.1). After centrifugation at 2,000 rpm for 3 minutes, the supernatant containing eluted phages was collected for subsequent rounds of selection. After four rounds of biopanning, individual plaques were randomly picked, amplified, and the phage genomic DNA was extracted using a phage DNA extraction kit. Sequencing of the extracted phage DNA was performed at Comate Bioscience Co., Ltd. (Jilin, China) using the universal primer 96gⅢ (Supplementary Table\u0026nbsp;4).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Peptide synthesis\u003c/h2\u003e\u003cp\u003eThe Porcine -derived dendritic cell (DC) targeting peptides selected in this study, as well as the peptides with alanine mutations or amino acid deletions, were synthesized by Nanjing GenScript Biotechnology Co., Ltd. The peptide sequences and N-terminal modifications are detailed in the \u003cb\u003eSupplementary Table\u0026nbsp;5\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Evaluation of Phage and Peptide Binding by ELISA, Fluorescence Microscopy, and Flow Cytometry\u003c/h2\u003e\u003cp\u003ePBMCs and Mo-DCs were fixed on 0.03 mg/mL polyline-coated 96-well plates for 30 min at 25\u0026deg;C and blocked with PBS containing 2% BSA for 30 min at 4\u0026deg;C. Phage clones (10\u0026sup1;⁰ pfu/mL) were added and incubated at 4\u0026deg;C for 20 h. PBS and wild-type M13 phage served as negative controls. After three PBST washes, cells were sequentially incubated with mouse anti-M13 polyclonal antibody (1:1,500; LSBiological) for 40 min at 37\u0026deg;C, followed by HRP-conjugated goat anti-mouse IgG (1:4,000; ZSGB Biotech). Color development was performed using TMB substrate, and absorbance was measured at 450 nm.\u003c/p\u003e\u003cp\u003eFor fluorescence microscopy, Mo-DCs cultured for six days were incubated with FITC-labeled DC-targeting peptides (HS, KC1, MY) or a negative control peptide at 4\u0026deg;C for 30 min, followed by staining with CD86-PE antibody at 37\u0026deg;C in the dark for 30 min. Cells were washed, fixed with 4% paraformaldehyde for 5 min at 37\u0026deg;C, and visualized using a fluorescence microscope (Bio-Rad, USA).\u003c/p\u003e\u003cp\u003eFor flow cytometry, Mo-DCs (10⁶ cells) were incubated with FITC-labeled peptides (25 \u0026micro;g) at 4\u0026deg;C for 10 min, washed three times, and analyzed using a BD Biosciences cytometer (San Jose, CA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 The mRNA expression of surface molecules of porcine DCs was tested by qRT-PCR\u003c/h2\u003e\u003cp\u003eImmature porcine Mo-DCs were collected after incubation with KC-1-S1, NC-S1, S1, and empty protein Tag. Total RNA was extracted using the RNA extraction kit from Shanghai Feijie Biotechnology Co., Ltd., and reverse transcription was performed using the RT-SuperMix kit. Unstimulated Porcine Mo-DCs were used as the control group, with β-actin as the internal reference gene. cDNA samples were adjusted to consistent concentrations. The reaction mixture for SYBR Green real-time RT-PCR was prepared according to the Roche manual for SYBR Green dye. Data collection and analysis were carried out using the real-time PCR detection system (7500 System Software). The primer sequences are listed in the Supplementary Table\u0026nbsp;4. The relative expression levels of surface molecules were calculated using the relative quantitative 2-ΔΔCT method, as follows: ΔΔCt = (Ct of target gene - Ct of internal reference gene) in the experimental group - (Ct of target gene - Ct of internal reference gene) in the control group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis of the experimental data was performed using GraphPad Prism 5 software. For pairwise comparisons between multiple groups, one-way analysis of variance (ANOVA) was conducted. Statistical significance was determined by different letters, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 indicating significant differences.\u003c/p\u003e\u003c/div\u003e"},{"header":"3.Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Screening and identification of DCs-targeting peptides\u003c/h2\u003e\u003cp\u003ePorcine monocytes were isolated using previously reported methods [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and differentiated into DCs by stimulation with GM-CSF and IL-4, followed by maturation with LPS. The resulting cells exhibited typical dendritic cell characteristics in terms of morphology, surface markers (CD172a, MHC-II, CD80, CD86), and phagocytic activity (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). To identify peptides capable of specifically binding to porcine DCs, a 12-mer phage display peptide library was employed for biopanning against porcine Mo-DCs. After four rounds of biopanning, targeted phages were enriched, and 204 monoclonal phage clones were randomly selected for sequencing, yielding 161 distinct peptide sequences (Supplementary Table\u0026nbsp;1 and Supplementary Figure S2). The binding affinity of the 3 most frequent peptides (HS, KC, and SF) and randomly selected 13 peptides to porcine DCs was detected using phage ELISA (Supplementary Table\u0026nbsp;2). The results indicated that phages expressing HS, KC, and SF peptides exhibited stronger affinity for porcine Mo-DCs than other 13 peptides (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Additionally, fluorescence microscopy and confocal laser scanning microscopy revealed that HS, KC, and SF peptides could bind to porcine Mo-DCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) as well as DCs in piglet intestinal tissue sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Furthermore, flow cytometry results demonstrated that the peptide KC exhibited a higher targeting affinity for porcine DCs than the peptides HS and SF (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These findings indicated that the KC peptide showed the strongest binding affinity for porcine DCs in our study.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2 KCCYPN of the KC Peptide Are Critical for Targeted Binding to Porcine DCs\u003c/h2\u003e\u003cp\u003eKC peptide\u0026rsquo;s key amino acids involved in binding activity were identified by alanine scanning mutagenesis. The competitive ELISA results demonstrated that four specific amino acid residues (K1, Y4, P5, and N6) within the KC peptide constitute the essential binding sites responsible for its targeted interaction with porcine DCs. In contrast, substitutions at positions C2, C3, Q7, M8, and F11 had minimal impact on binding affinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Although simultaneous mutations at C2A and C3A did not significantly affect KC binding, complete deletion of these two residues markedly reduced its binding to dendritic cells (DCs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB left). Similarly, simultaneous substitutions at Q7, M8, and F11 also had no significant effect on binding. Notably, the truncated peptide KC-1 (KCCYPN), consisting of the N-terminal six amino acids of KC, exhibited stronger binding affinity to monocyte-derived dendritic cells (Mo-DCs) than the full-length peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB right). To exclude possible non-specific binding at high concentrations, the binding affinity of the peptides was further evaluated at low concentrations (1.5, 2.5, and 5 \u0026micro;g/mL). The results demonstrated that KC-1 exhibited stronger binding affinity to porcine dendritic cells than the full-length KC peptide at all tested concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Collectively, these results identify the N-terminal hexapeptide KC-1 as the functional core of KC responsible for selective binding to porcine DCs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3 KC-1 can bind to various immune cells\u003c/h2\u003e\u003cp\u003eTo evaluate the cell-specific binding ability of the targeting peptide KC-1, FITC-conjugated KC-1 peptide was incubated with various cell types, including porcine DCs, PAMs, porcine BM-DCs, rabbit Mo-DCs, IPI, ST cells, and Vero cells. Fluorescence microscopy revealed KC-1 binding to porcine Mo-DCs, PAMs, BM-DCs, and rabbit Mo-DCs, while no detectable fluorescence was observed in IPI, ST, or Vero cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Flow cytometric analysis further confirmed the binding of KC-1 to porcine DCs, PAMs, porcine BM-DCs, and rabbit Mo-DCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These results indicate that KC-1 can bind to immune cells derived from different species, demonstrating its potential as a broadly applicable immune cells-targeting peptide.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4 SLA-DRB1 as the Target Protein of the KC-1\u003c/h2\u003e\u003cp\u003eTo identify the molecular target of KC-1, KC-1-Biotin was synthesized by introducing a lysine residue at the C-terminus of KC-1 for biotinylating, with an irrelevant sequence NC-Biotin serving as the negative control. HPLC analysis confirmed\u0026thinsp;\u0026gt;\u0026thinsp;95% purity for both peptides (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and ELISA demonstrated that biotinylating did not compromise KC-1's specific binding to PAMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Pull-down assays were performed by incubating PAM membrane proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) with KC-1-Biotin, followed by capture using streptavidin-coated magnetic beads. SDS-PAGE and silver staining of the pull-down eluates revealed a distinct protein band between 25\u0026ndash;40 kDa specifically enriched in the KC-1-Biotin group but absent in the negative control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). This band was excised for mass spectrometry (MS) analysis, which identified 39 differentially expressed proteins, including 15 membrane proteins (Supplementary Table\u0026nbsp;3). Among the membrane proteins, the MHC class II β chain (SLA-DRB1) was identified with the highest confidence, while the MHC class I molecule (SLA-1) was likewise detected. Accordingly, SLA-DRB1 and SLA-1 were prioritized as candidate targets of KC-1 for validation. Co-immunoprecipitation (Co-IP) assays demonstrated that KC-1 could interact with SLA-DRB1 and SLA-1 in PAMs and Mo-DCs, the interaction of KC-1 and SLA-DRB1 was effectively blocked by anti-SLA-DRB1 antibodies, however, anti-SLA-1 antibodies could not completely block the interaction of KC-1 and SLA-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Therefore, SLA-DRB1 was selected for subsequent experiments. Laser scanning confocal microscopy confirmed pronounced co-localization of KC-1 with SLA-DRB1 on the surface of PAMs and Mo-DCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), further validating their interaction. Collectively, these results confirm SLA-DRB1 was the primary target protein of the targeting peptide KC-1.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Determination of the Binding Domain of KC-1 to SLA-DRB1\u003c/h2\u003e\u003cp\u003eTo identify the binding domain of SLA-DRB1 that interacts with the KC-1, molecular docking analysis was performed using HPEPDOCK 2.0 software, resulting in 10 interaction models between KC-1 and SLA-DRB1. Among them, nine models predicted the binding domain of KC-1 to SLA-DRB1 to be between amino acids 38 and 111, while one model predicted the binding domain of the peptide KC-1 to SLA-DRB1 to be between amino acids 130 and 255 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Based on these predictions, SLA-DRB1 was divided into two fragments: SLA-DRB1a (amino acids 1\u0026ndash;126) and SLA-DRB1b (amino acids 126\u0026ndash;266) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). IFA and Western blotting analyses showed that SLA-DRB1, SLA-DRB1a and SLA-DRB1b were successfully expressed (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Co-IP assays demonstrated that KC-1 interacted with full-length SLA-DRB1 and the SLA-DRB1a (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Collectively, these results indicate that the region of SLA-DRB1 interacting with KC-1 is localized within residues 1\u0026ndash;126.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.6 The fusion expression of KC-1 with antigens promoted Porcine DCs Maturation and T Cell Proliferation\u003c/h2\u003e\u003cp\u003eTo evaluate the immunomodulatory effect of the KC-1 on porcine DCs under antigenic stimulation, porcine DCs were incubated for 12 hours with the peptide KC-1, PEDV S1 protein, the fusion proteins KC-1-S1, NC-S1, or LPS. qPCR analysis revealed that treatment with S1, NC-S1, KC-1-S1, and LPS significantly upregulated the expression of costimulatory molecules (CD40, CD80, CD86) and Toll-like receptors (TLR2, TLR6, TLR9), with the highest levels observed in the KC-1-S1 group (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;B). To further assess whether KC-1 fusion affects antigen presentation capacity, we prepared single-cell suspensions of piglet peripheral blood lymphocytes and conducted T cell proliferation assays. The results revealed that KC-1-S1 significantly enhanced T cell proliferation compared to other treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), indicating that KC-1 fusion facilitates the ability of DCs to activate T cells. Additionally, ELISA analysis showed that KC-1-S1\u0026ndash;treated Mo-DCs promoted the secretion of IL-4, IL-12, and IFN-γ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), further demonstrating that the KC-1 enhances T cell differentiation. Taken together, KC-1 enhances the antigen-presenting ability of DCs and promotes DC-mediated T cell proliferation and differentiation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAntigen targeting to DCs has been recognized as an effective strategy to enhance vaccine efficacy [\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Achieving efficient antigen delivery and precise recognition of DCs is key to the development of targeting strategies. Existing studies have shown that screening ligands for DC surface receptors, generating specific antibodies against DC surface molecules (receptors/markers), and developing DC-specific targeting peptides are all feasible methods for antigen delivery [\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, each of these methods has certain limitations [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In contrast, targeting peptides [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], with higher cell membrane permeability and lower immunogenicity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], possess unique advantages in targeting strategies [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, a DC-targeting peptide, KC, consisting of 12 amino acids, was identified using phage display technology. As is known, protein\u0026ndash;protein interactions are often mediated by a limited number of key amino acid residues [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Additionally, shorter peptides can not only improve the efficiency of target protein recognition on DCs but also enhance membrane permeability and reduce immunogenicity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. To this end, an alanine scanning approach was employed to identify the functional residues within the DC-targeting peptide KC, which confirmed that the first six N-terminal amino acids of KC constitute the key functional region responsible for DC binding. To determine whether the targeting peptide KC-1 is specific to DCs, this study examined its affinity for different cell types. The results indicate that, in addition to specifically binding to porcine Mo-DCs, KC-1 also binds to porcine BM-DCs and porcine PAMs, but does not bind to IPI or ST cells. This may be because both DCs and macrophages are APCs and share similar surface molecular expression profiles. Therefore, we hypothesized that the targeting peptide KC-1 could bind to all APCs in vivo, and we further investigated the molecular mechanism underlying this interaction.\u003c/p\u003e\u003cp\u003eGiven that PAMs are readily obtainable and culturable, and possess molecular characteristics representative of APCs [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], they were selected in this study for screening KC-1 target proteins by pull down coupled with MS. SLA-DRB1, as the MHC class II β chain, caught our attention with the highest confidence in the results of MS. The interaction between KC-1 and SLA-DRB1 was further confirmed on porcine Mo-DCs. As an MHC class II molecule, SLA-DRB1 is expressed on the surface of all professional APCs [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], which provides a molecular explanation for the ability of KC-1 to bind to various types of APCs. Although the MHC class I molecule (SLA-1) also showed high-confidence significant identity in the MS results, its validation outcomes were unsatisfactory. We speculate that it is likely not the primary interacting protein of KC-1 and thus will not pursue further investigation into it.\u003c/p\u003e\u003cp\u003eIdentifying the precise binding region between KC-1 and SLA-DRB1 is essential for a deeper understanding of the molecular mechanisms by which this targeting peptide modulates the immune function of porcine DCs under antigen stimulation. Previous studies have shown that short targeting peptides often recognize flexible or unstructured regions of surface molecules, which allows them to maintain binding specificity without disrupting native protein functions [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. To this end, this study characterized the binding capacity of KC-1 to different structural domains of SLA-DRB1. Most prediction models indicated that KC-1 primarily binds to the unstructured region (amino acids 1\u0026ndash;126) of SLA-DRB1. Experimental validation confirmed these predictions, demonstrating that the binding site of KC-1 is located within the 1\u0026ndash;126 amino acid segment of SLA-DRB1. Similar observations have been reported for other MHC-associated ligands, where interactions occur preferentially within unstructured or solvent-exposed domains [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Since this binding region lies outside the structured domain of SLA-DRB1, we speculate that a KC-1-mediated DC-targeting vaccine would likely not interfere with the native functions of SLA-DRB1 in antigen presentation and immune response activation.\u003c/p\u003e\u003cp\u003eNotably, this study confirmed that KC-1 can bind to rabbit Mo-DCs, consistent with previous findings. Previous studies identified a 12-mer peptide \u0026ldquo;DCpep\u0026rdquo; using human CD1a\u003csup\u003e+\u003c/sup\u003eDR\u003csup\u003ebright\u003c/sup\u003eCD11c\u003csup\u003ebright\u003c/sup\u003eDCs, which also showed varying degrees of affinity for peripheral blood DCs in multiple species including poultry, dogs, horses, and cats [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The ability of KC-1 to bind DCs across species is likely due to the recognition of conserved structural features of MHC class II molecules or other surface proteins that are shared among mammalian APCs [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Moreover, short targeting peptides often interact with flexible, solvent-exposed regions of these proteins, which tend to be structurally conserved across species [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. These findings suggest that KC-1 not only has potential for developing DC-targeted vaccines in pigs but also may be applicable to other species, similar to previous cross-species applications of DC-targeting peptides [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSince the standalone targeting peptide KC-1 lacks immunogenicity, and considering that the PEDV S1 protein can induce high-titer neutralizing antibodies and promote cellular immune responses [\u003cspan additionalcitationids=\"CR51 CR52 CR53 CR54 CR55\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], this study explored its effects on the immune function of antigen-stimulated porcine DCs after fusing it with the model antigen PEDV S1 protein for co-expression. Results demonstrated that compared to recombinant S1 protein, KC-1-S1-incubated Mo-DCs exhibited significantly upregulated surface molecular expression levels, indicating that the targeting peptide KC-1 promotes antigen-stimulated maturation of porcine DCs. DCs activate upon recognizing pathogen-associated molecular patterns via pattern recognition receptors, subsequently migrating to lymph nodes. There, they present antigens and secrete cytokines, driving the differentiation of naive CD4⁺ T cells into distinct subsets such as Th1 and Th2 cells, thereby initiating specific immune responses [\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Based on this, the present study evaluated the T-cell proliferation capacity mediated by DCs stimulated with recombinant proteins using a mixed T-lymphocyte proliferation assay. Results showed that Mo-DCs treated with KC-1-S1 more effectively promoted T-cell proliferation compared to those treated with S1 protein alone. Furthermore, Mo-DCs incubated with KC-1-S1 secreted increased levels of cytokines, with Th1-associated factors IFN-γ and IL-12 significantly exceeding Th2-associated IL-4, suggesting predominant mediation of cellular immune responses. This finding aligns with prior studies demonstrating that co-expression of DCpep and PEDV S1 antigen effectively induces cellular immunity [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This study further demonstrates that the target peptide KC-1 enhances the antigen-presenting capacity of DCs without affecting T cell differentiation.\u003c/p\u003e\u003cp\u003eIn summary, KC-1, a short peptide derived from the N-terminal six amino acids of KC, specifically targets multiple porcine APC subsets via SLA-DRB1 and enhances DC-mediated antigen presentation and T cell activation. These results provide a concise molecular basis and valuable material for the efficient design of DC-targeted vaccines in pigs and potentially other species.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Technology Support Program of Fourteenth Five Year Plan (2022YFD1800800), the National Natural Science Foundation of China (Nos. 32373048), and Natural Science Foundation of Heilongjiang Province of China (YQ2021C020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study was reviewed and approved by the committee on the Ethics of Animal Experiments of Northeast Agricultural University, Harbin, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCopyright and Permissions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that this manuscript is entirely original and does not contain any material reproduced from other sources, including published works or online content. All figures, tables, and text are the authors’ own work, and no permissions from third parties are required.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eB.L. and T.X. designed and performed the major experiments, analyzed data, and drafted the initial manuscript.C.J.B. assisted with literature review, experimental protocol organization, and preliminary data compilation.Y.P.J. contributed to workflow optimization and assisted in drafting parts of the Methods section.W.C. assisted in preparing experimental materials and maintaining routine operations in the cell culture facility.J.X.L. contributed to figure preparation, data visualization, and reagent management.Y.J.L. provided discussion support for the study design.L.W. conceived and supervised the study, secured funding, and finalized the manuscript as the corresponding author.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang L, Li D (2024) - Invited Review - Current status, challenges and prospects for pig production in Asia. Anim Biosci 37(4):742\u0026ndash;754\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChepkwony MC, Makau DN, Yoder C, Corzo C, Culhane M, Perez A, Perez Aguirreburualde MS, Nault AJ, Mahero M (2025) A scoping review of knowledge, attitudes, and practices in swine farm biosecurity in North America. Front Vet Sci 12:1507704\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBanchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392(6673):245\u0026ndash;252\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eColonna M, Pulendran B, Iwasaki A (2006) Dendritic cells at the host-pathogen interface. Nat Immunol 7(2) 117\u0026thinsp;\u0026ndash;\u0026thinsp;20.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUnger WW, van Beelen AJ, Bruijns SC, Joshi M, Fehres CM, van Bloois L, Verstege MI, Ambrosini M, Kalay H, Nazmi K, Bolscher JG, Hooijberg E, de Gruijl TD, Storm G, van Kooyk Y (2012) Glycan-modified liposomes boost CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T-cell responses by targeting DC-SIGN on dendritic cells. J Control Release 160(1):88\u0026ndash;95\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen D, Wang Y, Wang H, Wu Y, Xia S, Zhang M (2015) CD8(+) T activation attenuates CD4(+) T proliferation through dendritic cells modification. Cell Immunol 296(2):138\u0026ndash;148\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMazzoccoli L, Liu B (2024) Dendritic Cells in Shaping Anti-Tumor T Cell Response. Cancers (Basel) 16(12)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePittet MJ, Di Pilato M, Garris C, Mempel TR (2023) Dendritic cells as shepherds of T cell immunity in cancer. Immunity 56(10):2218\u0026ndash;2230\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKumagai Y, Takeuchi O, Akira S (2008) TLR9 as a key receptor for the recognition of DNA. Adv Drug Deliv Rev 60(7):795\u0026ndash;804\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVolckmar J, Knop L, Hirsch T, Frentzel S, Erck C, van Ham M, Stegemann-Koniszewski S, Bruder D (2021) Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo. J Vis Exp (168)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKrylov VB, G\u0026oacute;mez-Redondo M, Solovev AS, Yashunsky DV, Brown AJP, Stappers MHT, Gow NAR, Ard\u0026aacute; A, Jim\u0026eacute;nez-Barbero J, Nifantiev NE (2023) Identification of a new DC-SIGN binding pentamannoside epitope within the complex structure of Candida albicans mannan. Cell Surf 10:100109\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang Z, He Y, Wang W, Tian Y, Ge C, Jia F, Zhang T, Zhang G, Wang M, Gong J, Huang H, Wang J, Shi C, Yang W, Cao X, Zeng Y, Wang N, Qian A, Jiang Y, Yang G, Wang C (2023) A novel prime and pull strategy mediated by the combination of two dendritic cell-targeting designs induced protective lung tissue-resident memory T cells against H1N1 influenza virus challenge. J Nanobiotechnol 21(1):479\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJalil AT, Abdulhadi MA, Al-Ameer LR, Taher WM, Abdulameer SJ, Abosaooda M, Fadhil AA (2024) Peptide-Based Therapeutics in Cancer Therapy. Mol Biotechnol 66(10):2679\u0026ndash;2696\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong Y, Lei L, Cai X, Wei H, Yu CY (2025) Immunomodulatory Peptides for Tumor Treatment. Adv Healthc Mater 14(5):e2400512\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarqus S, Pirogova E, Piva TJ (2017) Evaluation of the use of therapeutic peptides for cancer treatment. J Biomed Sci 24(1):21\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLu Y, Liu ZH, Li YX, Xu HL, Fang WH, He F (2022) Targeted Delivery of Nanovaccine to Dendritic Cells via DC-Binding Peptides Induces Potent Antiviral Immunity in vivo. Int J Nanomed 17:1593\u0026ndash;1608\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZheng J, Wang M, Pang L, Wang S, Kong Y, Zhu X, Zhou X, Wang X, Chen C, Ning H, Zhao W, Zhai W, Qi Y, Wu Y, Gao Y (2024) Identification of a novel DEC-205 binding peptide to develop dendritic cell-targeting nanovaccine for cancer immunotherapy. J Control Release 373:568\u0026ndash;582\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLiu M, Xie D, Hu D, Zhang R, Wang Y, Tang L, Zhou B, Zhao B, Yang L (2023) Situ Cocktail Nanovaccine for Cancer Immunotherapy. Adv Sci (Weinh) 10(31):e2207697\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSummerfield A, McCullough KC (2009) The porcine dendritic cell family. Dev Comp Immunol 33(3):299\u0026ndash;309\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXia T, Wang N, Tang Y, Gao Y, Gao C, Hao J, Jiang Y, Wang X, Shan Z, Li J, Zhou H, Cui W, Qiao X, Tang L, Wang L, Li Y (2022) Delivery of antigen to porcine dendritic cells by fusing antigen with porcine dendritic cells targeting peptide. Front Immunol 13:926279\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLonget S, Lundahl MLE, Lavelle EC (2018) Targeted Strategies for Mucosal Vaccination. Bioconjug Chem 29(3):613\u0026ndash;623\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi M, Wang Y, Sun Y, Cui H, Zhu SJ, Qiu HJ (2020) Mucosal vaccines: Strategies and challenges. Immunol Lett 217:116\u0026ndash;125\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou T, Peng J, Hao Y, Shi K, Zhou K, Yang Y, Yang C, He X, Chen X, Qian Z (2021) The construction of a lymphoma cell-based, DC-targeted vaccine, and its application in lymphoma prevention and cure. Bioact Mater 6(3):697\u0026ndash;711\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMestrallet G, Sone K, Bhardwaj N (2022) Strategies to overcome DC dysregulation in the tumor microenvironment. Front Immunol 13:980709\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTruex NL, Rondon A, R\u0026ouml;ssler SL, Hanna CC, Cho Y, Wang BY, Backlund CM, Lutz EA, Irvine DJ, Pentelute BL (2023) Enhanced Vaccine Immunogenicity Enabled by Targeted Cytosolic Delivery of Tumor Antigens into Dendritic Cells. ACS Cent Sci 9(9):1835\u0026ndash;1845\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSnider DP, Segal DM (1987) Targeted antigen presentation using crosslinked antibody heteroaggregates. J Immunol 139(5):1609\u0026ndash;1616\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWille-Reece U, Wu CY, Flynn BJ, Kedl RM, Seder RA (2005) Immunization with HIV-1 Gag protein conjugated to a TLR7/8 agonist results in the generation of HIV-1 Gag-specific Th1 and CD8\u0026thinsp;+\u0026thinsp;T cell responses. J Immunol 174(12):7676\u0026ndash;7683\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShi SH, Yang WT, Yang GL, Zhang XK, Liu YY, Zhang LJ, Ye LP, Hu JT, Xing X, Qi C, Li Y, Wang CF (2016) Lactobacillus plantarum vaccine vector expressing hemagglutinin provides protection against H9N2 challenge infection. Virus Res 211:46\u0026ndash;57\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShimizu T, Kawaguchi Y, Ando H, Ishima Y, Ishida T (2022) Development of an Antigen Delivery System for a B Cell-Targeted Vaccine as an Alternative to Dendritic Cell-Targeted Vaccines. Chem Pharm Bull (Tokyo) 70(5):341\u0026ndash;350\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMatsuda T, Misato K, Tamiya S, Akeda Y, Nakase I, Kuroda E, Takahama S, Nonaka M, Yamamoto T, Fukuda MN, Yoshioka Y (2022) Efficient antigen delivery by dendritic cell-targeting peptide via nucleolin confers superior vaccine effects in mice. iScience 25(11):105324\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIborra S, Izquierdo HM, Mart\u0026iacute;nez-L\u0026oacute;pez M, Blanco-Men\u0026eacute;ndez N, Reis e Sousa C, Sancho D (2012) The DC receptor DNGR-1 mediates cross-priming of CTLs during vaccinia virus infection in mice. J Clin Invest 122(5):1628\u0026ndash;1643\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSkibska A, Perlikowska R (2021) Signal Peptides - Promising Ingredients in Cosmetics. Curr Protein Pept Sci 22(10):716\u0026ndash;728\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim S, Kim D, Jung HH, Lee IH, Kim JI, Suh JY, Jon S (2012) Bio-inspired design and potential biomedical applications of a novel class of high-affinity peptides. Angew Chem Int Ed Engl 51(8):1890\u0026ndash;1894\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSidhu SS (2000) Phage display in pharmaceutical biotechnology. Curr Opin Biotechnol 11(6):610\u0026ndash;616\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDai J, Patzke C, Liakath-Ali K, Seigneur E, S\u0026uuml;dhof TC (2021) GluD1 is a signal transduction device disguised as an ionotropic receptor. Nature 595(7866):261\u0026ndash;265\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eScodeller P, Asciutto EK (2020) Target Tumors Using Peptides Molecules 25(4)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVan Chanh Le Q, Le TM, Cho HS, Kim WI, Hong K, Song H, Kim JH, Park C (2018) Analysis of peptide-SLA binding by establishing immortalized porcine alveolar macrophage cells with different SLA class II haplotypes. Vet Res 49(1):96\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWahyuningtyas R, Lai YS, Wu ML, Chen HW, Chung WB, Chaung HC, Chang KT (2021) Recombinant Antigen of Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2) Promotes M1 Repolarization of Porcine Alveolar Macrophages and Th1 Type Response. Vaccines (Basel) 9(9)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoche PA, Furuta K (2015) The ins and outs of MHC class II-mediated antigen processing and presentation. Nat Rev Immunol 15(4):203\u0026ndash;216\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKonjević D, Erman V, Bujanić M, Svetličić I, Arbanasić H, Lubura Strunjak S, Galov A (2022) Wild Boar (Sus scrofa)-Fascioloides magna Interaction from the Perspective of the MHC Genes. Pathogens 11(11)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSidhu SS, Koide S (2007) Phage display for engineering and analyzing protein interaction interfaces. Curr Opin Struct Biol 17(4):481\u0026ndash;487\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang Y, Sun D, Laney V, Wang H, Wang LL, Lu ZR (2025) Challenges and opportunities on achieving an adequate delivery efficiency and immunogenicity with peptide-based anticancer vaccines. Adv Drug Deliv Rev 225:115675\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrown JH, Jardetzky TS, Gorga JC, Stern LJ, Urban RG, Strominger JL, Wiley DC (1993) Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 364(6432):33\u0026ndash;39\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStern LJ, Brown JH, Jardetzky TS, Gorga JC, Urban RG, Strominger JL, Wiley DC (1994) Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature 368(6468):215\u0026ndash;221\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOwen JL, Sahay B, Mohamadzadeh M (2013) New generation of oral mucosal vaccines targeting dendritic cells. Curr Opin Chem Biol 17(6):918\u0026ndash;924\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOlajide OM, Osman MK, Robert J, Kessler S, Toews LK, Thamamongood T, Neefjes J, Wrobel AG, Schwemmle M, Ciminski K, Reuther P (2023) Evolutionarily conserved amino acids in MHC-II mediate bat influenza A virus entry into human cells. PLoS Biol 21(7):e3002182\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFerrante A (2013) For many but not for all: how the conformational flexibility of the peptide/MHCII complex shapes epitope selection. Immunol Res 56(1):85\u0026ndash;95\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJia Z, Pan X, Zhi W, Chen H, Bai B, Ma C, Ma D (2022) Probiotics Surface-Delivering Fiber2 Protein of Fowl Adenovirus 4 Stimulate Protective Immunity Against Hepatitis-Hydropericardium Syndrome in Chickens. Front Immunol 13:919100\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang WT, Li QY, Ata EB, Jiang YL, Huang HB, Shi CW, Wang JZ, Wang G, Kang YH, Liu J, Yang GL, Wang CF (2018) Immune response characterization of mice immunized with Lactobacillus plantarum expressing spike antigen of transmissible gastroenteritis virus. Appl Microbiol Biotechnol 102(19):8307\u0026ndash;8318\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHou X, Jiang X, Jiang Y, Tang L, Xu Y, Qiao X, Min L, Wen C, Ma G, Li Y (2018) Oral Immunization against PEDV with Recombinant Lactobacillus casei Expressing Dendritic Cell-Targeting Peptide Fusing COE Protein of PEDV in Piglets, Viruses 10(3)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJoshi LR, Okda FA, Singrey A, Maggioli MF, Faccin TC, Fernandes MHV, Hain KS, Dee S, Bauermann FV, Nelson EA, Diel DG (2018) Passive immunity to porcine epidemic diarrhea virus following immunization of pregnant gilts with a recombinant orf virus vector expressing the spike protein. Arch Virol 163(9):2327\u0026ndash;2335\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Q, Peng O, Wu T, Xu Z, Huang L, Zhang Y, Xue C, Wen Z, Zhou Q, Cao Y (2018) PED subunit vaccine based on COE domain replacement of flagellin domain D3 improved specific humoral and mucosal immunity in mice. Vaccine 36(11):1381\u0026ndash;1388\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThavorasak T, Chulanetra M, Glab-Ampai K, Teeranitayatarn K, Songserm T, Yodsheewan R, Sae-Lim N, Lekcharoensuk P, Sookrung N, Chaicumpa W (2022) Novel Neutralizing Epitope of PEDV S1 Protein Identified by IgM Monoclonal Antibody. Viruses 14(1)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDuong BTT, Thao PTP, Hoa NT, Thu HT, Phuoc MH, Le TH, Van Quyen D (2022) Molecular analysis reveals a distinct subgenogroup of porcine epidemic diarrhea virus in northern Vietnam in 2018\u0026ndash;2019. Arch Virol 167(11):2337\u0026ndash;2346\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThavorasak T, Chulanetra M, Glab-Ampai K, Mahasongkram K, Sae-Lim N, Teeranitayatarn K, Songserm T, Yodsheewan R, Nilubol D, Chaicumpa W, Sookrung N (2022) Enhancing epitope of PEDV spike protein. Front Microbiol 13:933249\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi M, Wang Y, Wang Y, Li R, Wang S, Ding P, Zhang G (2023) Accurate location of two conserved linear epitopes of PEDV utilizing monoclonal antibodies induced by S1 protein nanoparticles. Int J Biol Macromol 253(6):127276\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHuang KY, Yang GL, Jin YB, Liu J, Chen HL, Wang PB, Jiang YL, Shi CW, Huang HB, Wang JZ, Wang G, Kang YH, Yang WT, Wang CF (2018) Construction and immunogenicity analysis of Lactobacillus plantarum expressing a porcine epidemic diarrhea virus S gene fused to a DC-targeting peptide. Virus Res 247:84\u0026ndash;93\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGillam F, Zhang J, Zhang C (2018) Hepatitis B core antigen based novel vaccine against porcine epidemic diarrhea virus. J Virol Methods 253:61\u0026ndash;69\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu M, Wang L, Ma S, Wang X, Wang Y, Xiao Y, Jiang Y, Qiao X, Tang L, Xu Y, Li Y (2017) Immunogenicity of eGFP-Marked Recombinant Lactobacillus casei against Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus. Viruses 9(10)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dendritic cell-targeting peptide, Swine Vaccine, SLA-DRB1, Phage display","lastPublishedDoi":"10.21203/rs.3.rs-8110404/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8110404/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDendritic cells (DCs) are key antigen-presenting cells essential for initiating and regulating immune responses. While DC targeting has proven to be an effective strategy for vaccine enhancement, and targeting peptides have been extensively utilized as efficient delivery tools in DC-targeted drug and vaccine development, there remains a notable scarcity of peptides specifically selected through porcine dendritic cell screening platforms. In this study, phage display biopanning was employed to screen a novel DC-targeting peptide, designated KC (KCCYPNQMAAFA). Systematic alanine-scanning mutagenesis identified the N-terminal hexapeptide KC-1 (KCCYPN) as the minimal functional epitope responsible for DC binding. In addition to DCs, KC-1 was also demonstrated selective binding to bone marrow-derived dendritic cells (BM-DCs) and porcine alveolar macrophages (PAMs) but exhibited no interaction with intestinal porcine epithelial (IPI) cells, swine testis (ST) cells, or Vero cells. Further analysis revealed that KC-1 specifically bounds to the N-terminal region (1-126 aa) of SLA-DRB1, which is a key domain of the MHC II β-chain involved in the formation of the peptide-binding groove. Using the PEDV S1 subunit as a model antigen, we further evaluated the immunomodulatory effects of KC-1 on DCs in vitro. The results demonstrated that KC-1-S1 significantly promoted dendritic cell maturation and T cell proliferation, accompanied by increased secretion of key cytokines IL-4, IL-12, and IFN-γ, indicating enhanced activation of both humoral and cellular immune responses with a balanced Th1/Th2 polarization compared to controls. Collectively, these findings establish a theoretical foundation for porcine DC-targeted peptides and provide critical insights for the development of next-generation porcine DC-targeted vaccines.\u003c/p\u003e","manuscriptTitle":"Discovery of KC-1: A Novel Porcine Dendritic Cell-Targeting Peptide with Potential Applications in Swine Vaccine Design","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 20:19:07","doi":"10.21203/rs.3.rs-8110404/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"30c27c5a-ee6f-4242-bd64-677a58d24737","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-10T08:39:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-01 20:19:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8110404","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8110404","identity":"rs-8110404","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}