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Identification and characterization of CD4+ T cell epitopes on manganese transport protein C of Staphylococcus aureus
Microbial Pathogenesis 112 (2017) 30e37
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Identification and characterization of CD4þ T cell epitopes on manganese transport protein C of Staphylococcus aureus Wei Yu, Lizi Wang, Mengyao Wang, Shuo Liu, Wanyu Li, Xintong Wang, Xiaoting Li, Simiao Yu, Di Yao, Jinzhu Ma, Liquan Yu, Jing Chen, Zhenyue Feng, Yudong Cui* College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 July 2017 Received in revised form 26 August 2017 Accepted 19 September 2017 Available online 20 September 2017
Manganese transport protein C (MntC) of Staphylococcus aureus represents an excellent vaccinecandidate antigen. The important role of CD4þ T cells in effective immunity against S. aureus infection was shown; however, CD4þ T cell-specific epitopes on S. aureus MntC have not been well identified. Here, we used bioinformatics prediction algorithms to evaluate and identify nine candidate epitopes within MntC. Our results showed that peptide M8 emulsified in Freund's adjuvant induced a much higher cellproliferation rate as compared with controls. Additionally, CD4þ T cells stimulated with peptide M8 secreted significantly higher levels of interferon-g and interleukin-17A. These results suggested that peptide M8 represented an H-2d (I-E)-restricted Th17-specific epitope. © 2017 Published by Elsevier Ltd.
Keywords: Staphylococcus aureus Manganese transport protein C CD4þ T cell Epitopes
1. Introduction Staphylococcus aureus is the most common pathogenic bacteria associated with food poisoning and purulent infection in humans and is capable of causing multi-organ infection, pneumonia, pseudomembranous colitis, and endocarditis. In severe cases, S. aureus can cause life-threatening systemic diseases, such as septicemia and sepsis [1,2]. In the past, diseases caused by S. aureus infection were treated clinically with vancomycin and methicillin antibiotics [3]; however, drug-resistant strains have appeared due to the extensive use of antibiotics. Facing the growing prevalence of drug-resistant S. aureus strains, it is particularly important to determine new antibacterial targets for effective treatment. Metal ions are often closely related to protein/enzyme structure and function [4e6]. Upon S. aureus pathogen invasion of a host, its growth, reproduction, and overall virulence require a certain concentration of iron, manganese, zinc, and other trace elements of metals [7]. Immune responses against pathogenic bacteria include limiting the availability of transition metal ions and are referred to as nutritional immunity [8,9]. This process affects S. aureus proliferation and survival by interfering with the uptake of iron. Previous studies
* Corresponding author. College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, 163319, China. E-mail address: [email protected] (Y. Cui). https://doi.org/10.1016/j.micpath.2017.09.045 0882-4010/© 2017 Published by Elsevier Ltd.
focused on attenuated S. aureus virulence in relation to iron [10e13]; however, other reports also indicated important relationships between S. aureus virulence and manganese concentration [7,14e16]. Manganese is primarily an important cofactor of S. aureus superoxide dismutase, which is involved in attenuating reactive oxygen species and oxidative stress [15,18e20]. The manganese transport-adenosine triphosphate (ATP)-binding cassette (MntABC) transport complex is the primary pathway involved in S. aureus uptake of manganese and comprises an ATP-binding protein (MntA), a membrane protein (MntB), and a manganese-binding protein (MntC). Manganese is initially captured by MntC from the extracellular environment, followed by transport via MntB [16], with ATP hydrolysis via MntA necessary for metal-ion release and transport [22,23]. MntC contains a manganese-binding site [17], and in animal models of S. aureus systemic infection, this protein expressed on the cell-membrane surface [21]. High levels of the MntC protein are detected during S. aureus infection [24,25], with Anderson et al. reporting MntC conservation in the S. aureus genome and expression during both the early stages of infection and 1- to 6-h post-infection. Additionally, CD1 mice immunized against a purified-MntC variant and exposed to S. aureus Reynolds and Staphylococcus epidermidis 047 showed significantly lower levels of bacteria in the blood relative to those observed in controls, indicating that the MntC-specific epitope was capable of producing immune protection against both types of bacteria and confirming the efficacy of a MntC monoclonal
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antibody [26,27]. Moreover, Zou et al. identified three immunodominant B cell epitopes of MntC (MntC113e136, MntC209e232, and MntC263e286) that generated higher levels of antibodies, which promoted Th2-cell differentiation and provided effective immune protection and elevated phagocytic cytotoxicity against methicillinresistant S. aureus infection in vitro [28]. An MntC vaccine is capable of simultaneously inducing the humoral and cellular immune responses. Cellular immune responses play a major role against S. aureus infection; however, the efficacy of a MntC-specific vaccine for cellular immune responses has not been studied. The cellular immune response is mainly regulated by CD4þ and CD8þ T cells, with CD4þ T cells playing an important role in clearing pathogenic bacteria [29e31]. Epitopes are divided into subclasses comprising immunodominant, subdominant, and invisible epitopes. Because immunodominant epitopes activate the immune response, we explored CD4þ T cellspecific immunodominant epitopes representing effective S. aureus antigens. Here, we investigated the efficacy of using MntC-specific epitopes to engage the cellular immune response against S. aureus infection. Using bioinformatics methods, we successfully determined MntCspecific CD4þ T cell immunodominant epitopes and subsequently confirmed their ability to mediate CD4þ T cell differentiation and immunologically protect mice against S. aureus infection. 2. Materials and methods 2.1. Purification of recombinant MntC The mntc gene sequence was acquired from GenBank (https:// www.ncbi.nlm.nih.gov/genbank/) and amplified by polymerase chain reaction (PCR). Genomic DNA of S. aureus Newman was accompanied by the following primers: F, 50 -CGCGGATCCACTGGTGGTAAACAAAGCAGTGATA-30 ; and R, 50 -CCCAAGCTTTTATTTCATGCTTCCGTGTACAGTT-30 . The PCR products were cloned into the pMD18T-Easy vector and transformed into Escherichia coli DH5a cells. Single colonies were isolated and enlarged in liquid Ampþ Luria-Bertani cultures. Plasmids were acquired and digested by the restriction enzymes BamHI and HindIII, and target fragments were cloned into the pET-28a expression vector that had been restriction digested using the same enzymes for subsequent expression in E. coli BL21 (DE3) cells (Tiangen, Beijing, China). Isopropyl-bD-1-thiogalactopyranoside (IPTG; 0.1 mM; Biosharp, Hefei, China) was added to the bacterial culture after a 4-h incubation at 37 C in order to induce the expression of recombinant MtnC. Cells were harvested by centrifugation, resuspended, and ultrasonicated, and recombinant MntC was acquired as a His-tagged protein. The His-tagged protein was purified by nickel-affinity chromatography (Novagen, Darmstadt, Germany). The suspension was supplemented with a Ni2þ-chelating agent, followed by addition of buffer [50 mM Tris, 250 mM NaCl, and 20 mM imidazole (pH 8.8)] to remove contaminants and injection onto the column. Recombinant MntC was eluted from the column using a buffer gradient consisting of 50 mM Tris, 250 mM NaCl, and imidazole at concentrations ranging from 20 mM to 500 mM. The protein was dialyzed against phosphate-buffered saline (PBS) for 24 h. Purified protein samples were analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot and separated into aliquots for storage at 70 C. 2.2. Prediction of MntC CD4þT cell epitopes and synthesis of candidate peptides SYFPEITHI (http://www.syfpeithi.de/bin/MHCServer.dll/Epitope Prediction.htm) [32], MHCPred (http://www.ddg-pharmfac.net/
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mhcpred/MHCPred/) [33], and IEDB (http://www.iedb.org/) [34] were used to identify candidate H-2d (I-A and I-E)-restricted CD4þ T cell epitopes on the MntC protein, and ProPred (http:// www.imtech.res.in/raghava/propred/) [35] was used to predict human leukocyte antigen (HLA)-restricted CD4þ T cell epitopes. The top 10 predicted peptides were chosen for secondary structure prediction using PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred) [36]. The nine highest scoring H-2d-restricted epitopes were selected for identification, with all selected epitopes synthesized and purified by Shanghai Apeptide Corporation, Ltd. (Shanghai, China) at purities >95%. Peptides were stored at 70 C. 2.3. Immunization with MntC or peptides and challenge infection Specific-pathogen-free BALB/c mice (6e8-week-old females) were purchased from Changchun Institute of Biological Products (Changchun, China) and supplied with water and food ad libitum according to the Regulations for the Administration of Affairs Concerning Experimental Animals. All experiments were approved by the Animal Ethics Committee of HeiLongJiang BaYi Agricultural University. The mice were injected intramuscularly with 50 mg MntC or synthetic peptides with 100 mL complete Freund's adjuvant (CFA; Sigma-Aldrich, St. Louis, MO, USA). Immunizations were administered 2 weeks later using MntC or synthetic peptides with incomplete Freund's adjuvant (IFA; Sigma-Aldrich). PBS immunization was administered as a negative control. Two-weeks later, the immunized BALB/c mice in the challenge group were infected intraperitoneally with S. aureus Newman [5 108 colony forming units (CFUs) based on a lethal dose calculated from previous experiments], and survival rates were monitored 14-days post-challenge. 2.4. Preparation of antigen-presenting cells (APCs) and CD4þ T-cells The mice were humanely sacrificed, spleens were harvested under aseptic conditions, and single-cell suspensions were harvested through a 200-mm nylon membrane. Briefly, splenocytes were washed three times with RPMI-1640 (HyClone, Logan, UT, USA) after treatment with erythrocyte-lysing buffer, followed by incubation for 24 h at 37 C in 5% CO2. Cells were resuspended to 1 108 cells/mL in RPMI-1640 complete medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 U/mL streptomycin, and 10 mg/mL mitomycin C (Sigma-Aldrich), followed by washing with RPMI-1640 three times. The supernatant was discarded, and APCs were collected and diluted to 1 105 cells/mL. Specific splenocytes obtained from mice vaccinated with MntC or the respective epitopes were harvested 7 days after the last immunization. Isolation of specific CD4þ T cells following dilution to 5 105 cells/mL was performed using immunomagnetic beads (OctoMACS; Miltenyi Biotec, Bergisch Gladbach, Germany). 2.5. Proliferation assay and cytokine-profile analysis Analysis of specific CD4þ T cell proliferation was performed by seeding 100 mL of CD4þ T cells (5 105 cells/well) and APCs (1 105 cells/well) into 96-well flat-bottom culture plates (Falcon, Montreal, Canada) in complete medium containing 1 mg of synthetic peptide at 37 C in 5% CO2. PBS-stimulated cells were used as negative controls. After 1 day of ex vivo antigen stimulation, assessment of CD4þ T cell proliferation was performed using a cell counting kit-8 (CCK-8; Sigma-Aldrich) according to manufacturer instructions (10 mL of CCK-8 reagent was added to the culture medium and incubated for an additional 3 h). Absorbance was determined at 450 nm using an enzyme-linked immunosorbent
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assay (ELISA) reader (Bio-Rad, Hercules, CA, USA). All cytokines, including interferon (IFN)-g, interleukin (IL)-17, and IL-4 in the CD4þ T lymphocyte culture supernatant were measured using commercial ELISA kits (Dakewei, Beijing, China) according to manufacturer instructions.
2.6. Specific T cell bulk culture Spleens of mice were harvested 7 days after the final immunization, and lymphocytes were isolated using a mouse lymphocyteseparation kit (Dakewei). Isolated lymphocytes were pulsed with MntC protein (0.5 mM) or peptides (5 mM) and stimulated with 5 U/ mL IL-2 (PeproTech, Rocky Hill, NJ, USA) in RPMI-1640 complete medium. Half of the medium was removed upon turning yellow and replaced with fresh RPMI-1640. Lymphocytes were collected and cultured in RPMI-1640 containing 20 U/mL IL-2 on day 5. Lymphocytes were harvested and analyzed at specific times.
2.7. Cell cytokine staining for flow cytometry
Fig. 1. Expression and purification of recombinant MntC. SDS-PAGE analysis of expressed and purified recombinant MntC. Lane M: prestained protein ladder; Lane 1, E. coli BL21(DE3) lysate with pET-28a before induction; Lane 2: E. coli BL21(DE3) lysate with pET-28a after IPTG induction; Lane 3: E. coli BL21(DE3) lysate with pET-28a/MntC before induction; Lane 4: E. coli BL21(DE3) lysate with pET-28a/MntC after IPTG induction; Lane 5: purified MntC.
Cultured lymphocytes were harvested, and labeled CD4þ T cells were separated by OctoMACS (Miltenyi Biotec). CD4þ T cells were incubated with 5 mM M8 in RPMI-1640 for 6 h in the presence of phorbol myristate acetate (PMA; 50 ng/mL; Sigma-Aldrich), ionomycin (1 mM; Sigma-Aldrich), and Golgistop (1 mL/1.5 mL cell culture medium; BD Pharmingen, San Diego, CA, USA). Cells were then washed and labeled with anti-IFN-g-phycoerythrin (PE; eBioscience, San Diego, CA, USA), anti-IL-4-PE (eBioscience), or anti-IL-17A-fluorescein isothiocyanate (eBioscience) in 1 permeabilization buffer (eBioscience). Approximately 1 105 cells were acquired by flow cytometry (CytoFLEX A00-11102; Beckman Coulter, Brea, CA, USA).
Fig. 2. Predicted secondary structure of MntC and the sites of the nine synthetic peptide. (A) MntC amino acid sequence (AA), helical (H), coiled (C), b-strand (E), and b-turn (t) structures are annotated. (B) Sites of the nine synthetic peptides in the MntC tertiary structure.
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For major histocompatibility complex (MHC)-restricted determination assays, lymphocytes were initially incubated with specific MHC antibodies (eBioscience) for 30 min and then pulsed with peptides for 5 h after washing. Shortly thereafter, cells were labeled with anti-CD4-PE (BD Biosciences), quantified using flow cytometry (CytoFLEX; Beckman Coulter), and data were analyzed using CytExpert software (Beckman Coulter).
2.8. Statistical analysis Data are expressed as the mean ± standard error of the mean and compared using a two-tailed Student's t-test. The results were analyzed using Origin Pro (v8.0; OriginLab, Northampton, MA, USA)
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and SPSS software (v12.0; SPSS, Inc., Cary, NC, USA). A P-value <0.05, <0.01, or <0.001 was considered statistically significant. 3. Results 3.1. MntC expression and purification Recombinant MntC protein was expressed in E. coli BL21 (DE3) cells and extracted from the soluble fraction after induction with 0.1 mM IPTG. After nickel-affinity purification by, SDS-PAGE analysis showed that the recombinant MntC proteins corresponded to the predicted molecular mass (34 kDa) (Fig. 1) and confirmed removal of most of the contaminants. 3.2. Prediction of MntC epitopes
Table 1 Predictive outcomes of peptides derived from S. aureus MntC (nine candidate of epitopes). Synthetic peptides
Sequence
Actual Mr
Purity (%)
M1 M2 M3 M4 M5 M6 M7 M8 M9
NGKLKVVTTNSILYDMAKNV DPHEYEVKPKDIKKLTDADV GNGWFEKALEQAGKSLKDKK VIAVSKDVKPIYLNGEEGNK QDPHAWLSLDNGIKYVKTIQ DIPKEQRAMITSEGAFKYFS INTEKQGTPEQMRQAIEFVK KHKLKHLLVETSVDKKAMES TKGDSYYKMMKSNIETVHGS
2250.76 2382.75 2276.69 2215.65 2368.80 2360.76 2389.83 2363.92 2318.71
95.88% 96.80% 96.64% 96.86% 96.95% 95.06% 95.99% 96.33% 97.35%
(11e30) (46e65) (76e95) (96e115) (118e137) (175e194) (207e226) (227e246) (269e288)
The PSIPRED and COUDES tools were used to analyze MntC secondary structure, and helical (H), extended strand (E), coil (C), and b-turn (t) structures were annotated (Fig. 2). Our analysis revealed the presence of several potential N-endopeptidase sites (N) typically found in domains associated with the bacterial cell wall and known for enhancing antigen-processing for MHC presentation. A synthetic peptide dataset was used to evaluate the binding affinity of the epitopes to the MHC-II molecule. SYFPEITHI, MHCPred, and IEDB bioinformatics software were used to predict peptide-binding affinities to mouse H-2d (I-A and I-E), and ProPred
Fig. 3. Proliferative responses and cytokines secretion of CD4þ T cells from MntC-immunized mice. At 1 week after the final immunization with MntC or PBS, spleen lymphocytes were isolated from BALB/c mice, and CD4þ T cells were incubated with 1 mg of each synthetic peptide þ mitomycin C-treated naive syngeneic feeder cells for 1 day at a ratio of 5:1, respectively. (A) Proliferation response assay. SI was measured by CCK-8 incorporation, which was measured with a microplate reader, experiments were done in triplicate. The levels of (B) IFN-g, (C) IL-17A and (D) IL-4, secreted from CD4þ T-cells were determined by ELISA. The solid bars represent the mean ± standard deviation. Significant differences were assessed relative to the negative-control group (PBS) and indicated by *P < 0.05, **P < 0.01, and ***P < 0.001.
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was used to predict peptide-binding affinities to human HLA-DR, -DP, and -DQ. For each method, peptides were tested and ranked by their IC50 scores against epitopes exhibiting higher binding affinity. Because of the different prediction programs used, we summarized the predictions. According to the binding affinities to the MHC II molecule and incorporation based on highly conserved amino acid sequences and secondary structures, nine peptides of S. aureus MntC were synthesized (Table 1). The positions of the candidate epitopes in MntC three-dimensional structure (PDB ID: 4K3V) were noted, and analysis for solvent accessibility suggested that most of the residues within MntC exhibited high degrees of solvent accessibility. 3.3. CD4þ T cell proliferation and cytokine production Evaluation of antigen recognition by selected MntC epitopes M1 through M9 indicated correspondingly low synthetic peptideinduced proliferative responses to PBS-immunized CD4þ T cells. By contrast, CD4þ T cells from MntC-immunized mice showed selective and significant proliferation indices to the M2, M8, and M9 peptides relative to that observed in PBS-immunized CD4þ T-cells. Antigen-specific cell responses were evaluated by ELISA, with cytokine levels from CD4þ T cells were measured following stimulation with the synthetic peptides. Our results showed that CD4þ T cells stimulated with the synthetic peptides produced significantly more IFN-g, IL-17A, and IL-4 (Fig. 3), and that upon stimulation with M8 and M9, IL-17A and IL-4 secretion increased significantly relative to groups stimulated with other peptides. Furthermore, M8 stimulation resulted in the highest IFN-g secretion level observed from all peptides. These findings provided evidence that the M8 and M9 epitopes derived from MntC were recognized by CD4þ T cell populations. To confirm M8 and M9 as specific CD4þ T cell epitopes, CD4þ T cells were extracted from mice immunized with M8 or M9 and evaluated by cell-proliferation and cytokine-secretion assays. Our results clearly indicated that peptide-specific T cell proliferation was detected in mice immunized with M8 and M9, but not in mice immunized with PBS. Additionally, these CD4þ T cells exhibited significantly higher levels of secreted IFN-g and IL-17A as compared with those observed from T cells derived from mice stimulated with PBS (Fig. 4). Furthermore, the IL-17A levels secreted by CD4þ T cells from M8-stimulated mice were higher than those from M9stimulated mice, indicating that the M8 peptide represented the immunodominant epitope recognized by CD4þ T cells. These results suggested that immunization with M8 increased the potential of splenocytes to induce cytokine responses and polarize toward Th1/ Th17 subsets.
which MHC-II molecule subtype binds to the novel CD4þ T cell epitope. As shown in Fig. 6, the H-2d (I-Ek) antibody efficiently blocked M8-specific proliferation of CD4þ T cells, whereas MHC-I and H-2d (I-Ad) antibodies were unable to block these CD4þ T cell responses. These results confirmed M8 as an H-2d (I-Ek) CD4þ T cell epitope, but not a CD8þ T cell epitope.
3.6. The protective effect of MntC or M8 in a lethal S. aureus sepsis model Mice were immunized with MntC, M8, or PBS þ adjuvant, and their protective effects against S. aureus infection were evaluated in a sepsis model. We found that mice vaccinated with the MntC antigen exhibited higher survival rates (60% survival) as compared with those vaccinated with M8 (20% survival) or PBS (10% survival) (Fig. 7). These findings showed that MntC vaccination generated a protective effect against S. aureus infection in a sepsis model.
3.4. Flow cytometric analyses to determine the percentage of specific Th1/Th17 cells Having established that immunodominant Th1 and Th17 responses could be induced by the MntC protein or a novel CD4þ T cell epitope (M8), we investigated the characteristics of these epitope-specific Th1 and Th17 cells. M8-specific lymphocytes cultured in vitro were harvested, and CD4þ T cells were isolated and analyzed by intracellular cytokine staining. As shown in Fig. 5, M8stiluated cells exhibited an increased ability to polarize toward Th1 and Th17 cells as compared with PBS-stimulated cells. These results suggested that immunization with M8 elicited an immune response associated with Th1 and Th17 polarization. 3.5. MHC-restriction analysis An MHC-antibody blocking assay was performed to confirm
Fig. 4. Identification of CD4þ T cell epitopes on the MntC protein. CD4þ T cell proliferation in mice immunized with peptides M8 or M9, respectively. At 1 week after the final immunization with peptide M8 or M9, mouse splenic lymphocytes were isolated. (A) SI was measured by CCK-8 incorporation, which was assessed using a microplate reader. The levels of (B) IFN-g, (C) IL-17A and (D) IL-4 were determined by ELISA. The solid bars represent the mean ± standard deviation. Significant difference was compared with the negative control group (PBS) was indicated by *P < 0.05, **P < 0.01 and ***P < 0.001.
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Fig. 5. Phenotypic analysis of CD4þ T cells following immunization with the M8 epitope. (A) M8-specific lymphocytes cultured in vitro were harvested, and CD4þ T cells were isolated from lymphocytes and stimulated with the M8 epitope for 6 h in the presence of PMA, ionomycin, and Golgistop. Cells were then washed and labeled for flow cytometry. (B) Statistical analysis of Th1, Th17, and Th2 responses in M8-immunized mice. The gray bars represent the proportion of double-positive cells. Significant differences were indicated by *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 6. MHC-restriction analysis of the M8 epitope. (A) Anti-MHC-I, -MHC-II (I-Ad), and -MHC-II (I-Ek) antibodies were added to M8-immunized splenic lymphocytes and incubated for 30 min, respectively. Following incubation, the M8 epitope was used to stimulate the cells for an additional 5 h, with no peptide added to the negative control. (B) Cells were labeled with anti-CD4-PE, and lymphocyte proliferation was quantified by flow cytometry for statistical analysis. Significant differences were indicated by *P < 0.05 and **P < 0.01.
4. Discussion S. aureus is a ubiquitous pathogen associated with high infection rates [2], and screening and defining S. aureus-specific antigens are key to vaccine development. Until now, S. aureus vaccines have primarily relied upon immunization conferred by vaccination with various forms of recombinant S. aureus proteins. Although the MntC protein represents a valid antigenic molecule providing efficacious immunization against S. aureus infection, previous studies also reported its ability to induce humoral immune responses [27,28]. However, the ability of MntC to induce specific cell-based immunologic responses had not been investigated, and given the pivotal
role of T cells in the prevention of S. aureus infection [29,37], they were used as the model for the study. In recent years, the roles of antigen epitopes in immune responses have been gradually accepted, with increased study of pathogenic bacterial and viral antigen epitopes. A conventional method for identifying T cell epitopes (pepscan) involving the synthesis of multiple overlapping peptides spanning the full length of the target antigen and tests for immunogenicity using T cell assays have also been developed; however, the shortcomings of these approaches include their being time consuming and costly. Bioinformatics programs can predict which peptides are more likely to contain T cell epitopes, thereby greatly reducing the number of
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indicated a lower protection rate relative to that conferred by the M8 peptide. Given the disparity between the immunogenic response to the M8 epitope and the MntC protein in mice, we hypothesized that the relationship between T cell and B cell epitopes is more suitable for a specific peptide rather than an entire protein sequence. In conclusion, we identified a CD4þ T cell epitope on the S. aureus MntC protein capable of inducing Th1 and Th17 responses, resulting in a synthetic M8 peptide that represents an H-2d (I-E)restricted epitope. These data support the potential development of an epitope-based S. aureus-specific vaccine. Competing interests The authors declare that they have no competing interests. Acknowledgements
Fig. 7. Validation of the protective effects of the M8 peptide in an S. aureus sepsis model. BALB/c mice (n ¼ 10) were immunized with the MntC or M8 epitope with FA, and mice were intraperitoneally infected with S. aureus Newman (5 108 CFUs). Survival rates were monitored for 14 days. The significance of the protective immunity generated by the MntC and M8 epitope relative to that observed in the control was measured using a log-rank test. Asterisks represent statistically significant differences.
candidate sequences [40]. MHC-II-binding-prediction methods are categorized into two main groups: quantitative and qualitative. Qualitative matrices determine binding status (whether a peptide is a “binder” or “non-binder”) based on the predicted score (according to position-specific binding profiles), with SYFPEITHI one example of this software. Quantitative approaches also predict the strength of binding and include programs, such as MHCPred and ProPred [32,33]. IEDB software utilizes the gene ontology for biomedical investigations, as well as several additional ontologies to represent immune epitope-mapping experiments [34]. Previously, our research group successfully identified CD4þ T cell epitopes associated with S. aureus iron-regulated surface determinant protein B precursor (IsdB) and Streptococcus glyceraldehyde-3phosphate dehydrogenase, cytosolic [38,39], and developed epitope vaccines, including multiple epitopes capable of generating adequate immune protection. This study expanded those findings into the search for additional S. aureus-specific antigen epitopes. Antigen-epitope specificity related to T cell response plays an important role in understanding immune responses against S. aureus infection and the development of vaccines designed based on those epitopes. Here, we predicted candidate MntC epitopes using the bioinformatics software SYFPEITH, MHCPred, ProPred, and IEDB. We also predicted HLA-restricted epitopes to identify HLA-binding epitopes. Interestingly, we determined that M8 was an H-2d (I-E)-restricted epitope, with ProPred results also showing that M8 might be able to combine with HLA, thereby encouraging the need for future studies. We then tested their efficacy in vitro based on stimulation of CD4þ T cell proliferation and cytokine secretion. According to in vivo results, we found that mice immunized with a synthetic M8 peptide induced Th1 and Th17 differentiation. Our cumulative results suggested that the cell response mediated by M8 vaccination was significantly higher than that elicited by other synthetic peptides, indicating it as the immunodominant epitope. Furthermore, we verified these findings by flow cytometric analyses cytokine-detection results. BLAST analysis of the immunodominant-epitope sequences revealed that high levels of conservation conserved among various S. aureus strains and specific to the MntC protein. Subsequent evaluation of the MntC protein as a vaccine against S. aureus
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Identification and characterization of CD4þ T cell epitopes on manganese transport protein C of Staphylococcus aureus Wei Yu, Lizi Wang, Mengyao Wang, Shuo Liu, Wanyu Li, Xintong Wang, Xiaoting Li, Simiao Yu, Di Yao, Jinzhu Ma, Liquan Yu, Jing Chen, Zhenyue Feng, Yudong Cui* College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang Province, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 July 2017 Received in revised form 26 August 2017 Accepted 19 September 2017 Available online 20 September 2017
Manganese transport protein C (MntC) of Staphylococcus aureus represents an excellent vaccinecandidate antigen. The important role of CD4þ T cells in effective immunity against S. aureus infection was shown; however, CD4þ T cell-specific epitopes on S. aureus MntC have not been well identified. Here, we used bioinformatics prediction algorithms to evaluate and identify nine candidate epitopes within MntC. Our results showed that peptide M8 emulsified in Freund's adjuvant induced a much higher cellproliferation rate as compared with controls. Additionally, CD4þ T cells stimulated with peptide M8 secreted significantly higher levels of interferon-g and interleukin-17A. These results suggested that peptide M8 represented an H-2d (I-E)-restricted Th17-specific epitope. © 2017 Published by Elsevier Ltd.
Keywords: Staphylococcus aureus Manganese transport protein C CD4þ T cell Epitopes
1. Introduction Staphylococcus aureus is the most common pathogenic bacteria associated with food poisoning and purulent infection in humans and is capable of causing multi-organ infection, pneumonia, pseudomembranous colitis, and endocarditis. In severe cases, S. aureus can cause life-threatening systemic diseases, such as septicemia and sepsis [1,2]. In the past, diseases caused by S. aureus infection were treated clinically with vancomycin and methicillin antibiotics [3]; however, drug-resistant strains have appeared due to the extensive use of antibiotics. Facing the growing prevalence of drug-resistant S. aureus strains, it is particularly important to determine new antibacterial targets for effective treatment. Metal ions are often closely related to protein/enzyme structure and function [4e6]. Upon S. aureus pathogen invasion of a host, its growth, reproduction, and overall virulence require a certain concentration of iron, manganese, zinc, and other trace elements of metals [7]. Immune responses against pathogenic bacteria include limiting the availability of transition metal ions and are referred to as nutritional immunity [8,9]. This process affects S. aureus proliferation and survival by interfering with the uptake of iron. Previous studies
* Corresponding author. College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, 163319, China. E-mail address: [email protected] (Y. Cui). https://doi.org/10.1016/j.micpath.2017.09.045 0882-4010/© 2017 Published by Elsevier Ltd.
focused on attenuated S. aureus virulence in relation to iron [10e13]; however, other reports also indicated important relationships between S. aureus virulence and manganese concentration [7,14e16]. Manganese is primarily an important cofactor of S. aureus superoxide dismutase, which is involved in attenuating reactive oxygen species and oxidative stress [15,18e20]. The manganese transport-adenosine triphosphate (ATP)-binding cassette (MntABC) transport complex is the primary pathway involved in S. aureus uptake of manganese and comprises an ATP-binding protein (MntA), a membrane protein (MntB), and a manganese-binding protein (MntC). Manganese is initially captured by MntC from the extracellular environment, followed by transport via MntB [16], with ATP hydrolysis via MntA necessary for metal-ion release and transport [22,23]. MntC contains a manganese-binding site [17], and in animal models of S. aureus systemic infection, this protein expressed on the cell-membrane surface [21]. High levels of the MntC protein are detected during S. aureus infection [24,25], with Anderson et al. reporting MntC conservation in the S. aureus genome and expression during both the early stages of infection and 1- to 6-h post-infection. Additionally, CD1 mice immunized against a purified-MntC variant and exposed to S. aureus Reynolds and Staphylococcus epidermidis 047 showed significantly lower levels of bacteria in the blood relative to those observed in controls, indicating that the MntC-specific epitope was capable of producing immune protection against both types of bacteria and confirming the efficacy of a MntC monoclonal
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antibody [26,27]. Moreover, Zou et al. identified three immunodominant B cell epitopes of MntC (MntC113e136, MntC209e232, and MntC263e286) that generated higher levels of antibodies, which promoted Th2-cell differentiation and provided effective immune protection and elevated phagocytic cytotoxicity against methicillinresistant S. aureus infection in vitro [28]. An MntC vaccine is capable of simultaneously inducing the humoral and cellular immune responses. Cellular immune responses play a major role against S. aureus infection; however, the efficacy of a MntC-specific vaccine for cellular immune responses has not been studied. The cellular immune response is mainly regulated by CD4þ and CD8þ T cells, with CD4þ T cells playing an important role in clearing pathogenic bacteria [29e31]. Epitopes are divided into subclasses comprising immunodominant, subdominant, and invisible epitopes. Because immunodominant epitopes activate the immune response, we explored CD4þ T cellspecific immunodominant epitopes representing effective S. aureus antigens. Here, we investigated the efficacy of using MntC-specific epitopes to engage the cellular immune response against S. aureus infection. Using bioinformatics methods, we successfully determined MntCspecific CD4þ T cell immunodominant epitopes and subsequently confirmed their ability to mediate CD4þ T cell differentiation and immunologically protect mice against S. aureus infection. 2. Materials and methods 2.1. Purification of recombinant MntC The mntc gene sequence was acquired from GenBank (https:// www.ncbi.nlm.nih.gov/genbank/) and amplified by polymerase chain reaction (PCR). Genomic DNA of S. aureus Newman was accompanied by the following primers: F, 50 -CGCGGATCCACTGGTGGTAAACAAAGCAGTGATA-30 ; and R, 50 -CCCAAGCTTTTATTTCATGCTTCCGTGTACAGTT-30 . The PCR products were cloned into the pMD18T-Easy vector and transformed into Escherichia coli DH5a cells. Single colonies were isolated and enlarged in liquid Ampþ Luria-Bertani cultures. Plasmids were acquired and digested by the restriction enzymes BamHI and HindIII, and target fragments were cloned into the pET-28a expression vector that had been restriction digested using the same enzymes for subsequent expression in E. coli BL21 (DE3) cells (Tiangen, Beijing, China). Isopropyl-bD-1-thiogalactopyranoside (IPTG; 0.1 mM; Biosharp, Hefei, China) was added to the bacterial culture after a 4-h incubation at 37 C in order to induce the expression of recombinant MtnC. Cells were harvested by centrifugation, resuspended, and ultrasonicated, and recombinant MntC was acquired as a His-tagged protein. The His-tagged protein was purified by nickel-affinity chromatography (Novagen, Darmstadt, Germany). The suspension was supplemented with a Ni2þ-chelating agent, followed by addition of buffer [50 mM Tris, 250 mM NaCl, and 20 mM imidazole (pH 8.8)] to remove contaminants and injection onto the column. Recombinant MntC was eluted from the column using a buffer gradient consisting of 50 mM Tris, 250 mM NaCl, and imidazole at concentrations ranging from 20 mM to 500 mM. The protein was dialyzed against phosphate-buffered saline (PBS) for 24 h. Purified protein samples were analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot and separated into aliquots for storage at 70 C. 2.2. Prediction of MntC CD4þT cell epitopes and synthesis of candidate peptides SYFPEITHI (http://www.syfpeithi.de/bin/MHCServer.dll/Epitope Prediction.htm) [32], MHCPred (http://www.ddg-pharmfac.net/
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mhcpred/MHCPred/) [33], and IEDB (http://www.iedb.org/) [34] were used to identify candidate H-2d (I-A and I-E)-restricted CD4þ T cell epitopes on the MntC protein, and ProPred (http:// www.imtech.res.in/raghava/propred/) [35] was used to predict human leukocyte antigen (HLA)-restricted CD4þ T cell epitopes. The top 10 predicted peptides were chosen for secondary structure prediction using PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred) [36]. The nine highest scoring H-2d-restricted epitopes were selected for identification, with all selected epitopes synthesized and purified by Shanghai Apeptide Corporation, Ltd. (Shanghai, China) at purities >95%. Peptides were stored at 70 C. 2.3. Immunization with MntC or peptides and challenge infection Specific-pathogen-free BALB/c mice (6e8-week-old females) were purchased from Changchun Institute of Biological Products (Changchun, China) and supplied with water and food ad libitum according to the Regulations for the Administration of Affairs Concerning Experimental Animals. All experiments were approved by the Animal Ethics Committee of HeiLongJiang BaYi Agricultural University. The mice were injected intramuscularly with 50 mg MntC or synthetic peptides with 100 mL complete Freund's adjuvant (CFA; Sigma-Aldrich, St. Louis, MO, USA). Immunizations were administered 2 weeks later using MntC or synthetic peptides with incomplete Freund's adjuvant (IFA; Sigma-Aldrich). PBS immunization was administered as a negative control. Two-weeks later, the immunized BALB/c mice in the challenge group were infected intraperitoneally with S. aureus Newman [5 108 colony forming units (CFUs) based on a lethal dose calculated from previous experiments], and survival rates were monitored 14-days post-challenge. 2.4. Preparation of antigen-presenting cells (APCs) and CD4þ T-cells The mice were humanely sacrificed, spleens were harvested under aseptic conditions, and single-cell suspensions were harvested through a 200-mm nylon membrane. Briefly, splenocytes were washed three times with RPMI-1640 (HyClone, Logan, UT, USA) after treatment with erythrocyte-lysing buffer, followed by incubation for 24 h at 37 C in 5% CO2. Cells were resuspended to 1 108 cells/mL in RPMI-1640 complete medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 U/mL streptomycin, and 10 mg/mL mitomycin C (Sigma-Aldrich), followed by washing with RPMI-1640 three times. The supernatant was discarded, and APCs were collected and diluted to 1 105 cells/mL. Specific splenocytes obtained from mice vaccinated with MntC or the respective epitopes were harvested 7 days after the last immunization. Isolation of specific CD4þ T cells following dilution to 5 105 cells/mL was performed using immunomagnetic beads (OctoMACS; Miltenyi Biotec, Bergisch Gladbach, Germany). 2.5. Proliferation assay and cytokine-profile analysis Analysis of specific CD4þ T cell proliferation was performed by seeding 100 mL of CD4þ T cells (5 105 cells/well) and APCs (1 105 cells/well) into 96-well flat-bottom culture plates (Falcon, Montreal, Canada) in complete medium containing 1 mg of synthetic peptide at 37 C in 5% CO2. PBS-stimulated cells were used as negative controls. After 1 day of ex vivo antigen stimulation, assessment of CD4þ T cell proliferation was performed using a cell counting kit-8 (CCK-8; Sigma-Aldrich) according to manufacturer instructions (10 mL of CCK-8 reagent was added to the culture medium and incubated for an additional 3 h). Absorbance was determined at 450 nm using an enzyme-linked immunosorbent
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assay (ELISA) reader (Bio-Rad, Hercules, CA, USA). All cytokines, including interferon (IFN)-g, interleukin (IL)-17, and IL-4 in the CD4þ T lymphocyte culture supernatant were measured using commercial ELISA kits (Dakewei, Beijing, China) according to manufacturer instructions.
2.6. Specific T cell bulk culture Spleens of mice were harvested 7 days after the final immunization, and lymphocytes were isolated using a mouse lymphocyteseparation kit (Dakewei). Isolated lymphocytes were pulsed with MntC protein (0.5 mM) or peptides (5 mM) and stimulated with 5 U/ mL IL-2 (PeproTech, Rocky Hill, NJ, USA) in RPMI-1640 complete medium. Half of the medium was removed upon turning yellow and replaced with fresh RPMI-1640. Lymphocytes were collected and cultured in RPMI-1640 containing 20 U/mL IL-2 on day 5. Lymphocytes were harvested and analyzed at specific times.
2.7. Cell cytokine staining for flow cytometry
Fig. 1. Expression and purification of recombinant MntC. SDS-PAGE analysis of expressed and purified recombinant MntC. Lane M: prestained protein ladder; Lane 1, E. coli BL21(DE3) lysate with pET-28a before induction; Lane 2: E. coli BL21(DE3) lysate with pET-28a after IPTG induction; Lane 3: E. coli BL21(DE3) lysate with pET-28a/MntC before induction; Lane 4: E. coli BL21(DE3) lysate with pET-28a/MntC after IPTG induction; Lane 5: purified MntC.
Cultured lymphocytes were harvested, and labeled CD4þ T cells were separated by OctoMACS (Miltenyi Biotec). CD4þ T cells were incubated with 5 mM M8 in RPMI-1640 for 6 h in the presence of phorbol myristate acetate (PMA; 50 ng/mL; Sigma-Aldrich), ionomycin (1 mM; Sigma-Aldrich), and Golgistop (1 mL/1.5 mL cell culture medium; BD Pharmingen, San Diego, CA, USA). Cells were then washed and labeled with anti-IFN-g-phycoerythrin (PE; eBioscience, San Diego, CA, USA), anti-IL-4-PE (eBioscience), or anti-IL-17A-fluorescein isothiocyanate (eBioscience) in 1 permeabilization buffer (eBioscience). Approximately 1 105 cells were acquired by flow cytometry (CytoFLEX A00-11102; Beckman Coulter, Brea, CA, USA).
Fig. 2. Predicted secondary structure of MntC and the sites of the nine synthetic peptide. (A) MntC amino acid sequence (AA), helical (H), coiled (C), b-strand (E), and b-turn (t) structures are annotated. (B) Sites of the nine synthetic peptides in the MntC tertiary structure.
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For major histocompatibility complex (MHC)-restricted determination assays, lymphocytes were initially incubated with specific MHC antibodies (eBioscience) for 30 min and then pulsed with peptides for 5 h after washing. Shortly thereafter, cells were labeled with anti-CD4-PE (BD Biosciences), quantified using flow cytometry (CytoFLEX; Beckman Coulter), and data were analyzed using CytExpert software (Beckman Coulter).
2.8. Statistical analysis Data are expressed as the mean ± standard error of the mean and compared using a two-tailed Student's t-test. The results were analyzed using Origin Pro (v8.0; OriginLab, Northampton, MA, USA)
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and SPSS software (v12.0; SPSS, Inc., Cary, NC, USA). A P-value <0.05, <0.01, or <0.001 was considered statistically significant. 3. Results 3.1. MntC expression and purification Recombinant MntC protein was expressed in E. coli BL21 (DE3) cells and extracted from the soluble fraction after induction with 0.1 mM IPTG. After nickel-affinity purification by, SDS-PAGE analysis showed that the recombinant MntC proteins corresponded to the predicted molecular mass (34 kDa) (Fig. 1) and confirmed removal of most of the contaminants. 3.2. Prediction of MntC epitopes
Table 1 Predictive outcomes of peptides derived from S. aureus MntC (nine candidate of epitopes). Synthetic peptides
Sequence
Actual Mr
Purity (%)
M1 M2 M3 M4 M5 M6 M7 M8 M9
NGKLKVVTTNSILYDMAKNV DPHEYEVKPKDIKKLTDADV GNGWFEKALEQAGKSLKDKK VIAVSKDVKPIYLNGEEGNK QDPHAWLSLDNGIKYVKTIQ DIPKEQRAMITSEGAFKYFS INTEKQGTPEQMRQAIEFVK KHKLKHLLVETSVDKKAMES TKGDSYYKMMKSNIETVHGS
2250.76 2382.75 2276.69 2215.65 2368.80 2360.76 2389.83 2363.92 2318.71
95.88% 96.80% 96.64% 96.86% 96.95% 95.06% 95.99% 96.33% 97.35%
(11e30) (46e65) (76e95) (96e115) (118e137) (175e194) (207e226) (227e246) (269e288)
The PSIPRED and COUDES tools were used to analyze MntC secondary structure, and helical (H), extended strand (E), coil (C), and b-turn (t) structures were annotated (Fig. 2). Our analysis revealed the presence of several potential N-endopeptidase sites (N) typically found in domains associated with the bacterial cell wall and known for enhancing antigen-processing for MHC presentation. A synthetic peptide dataset was used to evaluate the binding affinity of the epitopes to the MHC-II molecule. SYFPEITHI, MHCPred, and IEDB bioinformatics software were used to predict peptide-binding affinities to mouse H-2d (I-A and I-E), and ProPred
Fig. 3. Proliferative responses and cytokines secretion of CD4þ T cells from MntC-immunized mice. At 1 week after the final immunization with MntC or PBS, spleen lymphocytes were isolated from BALB/c mice, and CD4þ T cells were incubated with 1 mg of each synthetic peptide þ mitomycin C-treated naive syngeneic feeder cells for 1 day at a ratio of 5:1, respectively. (A) Proliferation response assay. SI was measured by CCK-8 incorporation, which was measured with a microplate reader, experiments were done in triplicate. The levels of (B) IFN-g, (C) IL-17A and (D) IL-4, secreted from CD4þ T-cells were determined by ELISA. The solid bars represent the mean ± standard deviation. Significant differences were assessed relative to the negative-control group (PBS) and indicated by *P < 0.05, **P < 0.01, and ***P < 0.001.
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was used to predict peptide-binding affinities to human HLA-DR, -DP, and -DQ. For each method, peptides were tested and ranked by their IC50 scores against epitopes exhibiting higher binding affinity. Because of the different prediction programs used, we summarized the predictions. According to the binding affinities to the MHC II molecule and incorporation based on highly conserved amino acid sequences and secondary structures, nine peptides of S. aureus MntC were synthesized (Table 1). The positions of the candidate epitopes in MntC three-dimensional structure (PDB ID: 4K3V) were noted, and analysis for solvent accessibility suggested that most of the residues within MntC exhibited high degrees of solvent accessibility. 3.3. CD4þ T cell proliferation and cytokine production Evaluation of antigen recognition by selected MntC epitopes M1 through M9 indicated correspondingly low synthetic peptideinduced proliferative responses to PBS-immunized CD4þ T cells. By contrast, CD4þ T cells from MntC-immunized mice showed selective and significant proliferation indices to the M2, M8, and M9 peptides relative to that observed in PBS-immunized CD4þ T-cells. Antigen-specific cell responses were evaluated by ELISA, with cytokine levels from CD4þ T cells were measured following stimulation with the synthetic peptides. Our results showed that CD4þ T cells stimulated with the synthetic peptides produced significantly more IFN-g, IL-17A, and IL-4 (Fig. 3), and that upon stimulation with M8 and M9, IL-17A and IL-4 secretion increased significantly relative to groups stimulated with other peptides. Furthermore, M8 stimulation resulted in the highest IFN-g secretion level observed from all peptides. These findings provided evidence that the M8 and M9 epitopes derived from MntC were recognized by CD4þ T cell populations. To confirm M8 and M9 as specific CD4þ T cell epitopes, CD4þ T cells were extracted from mice immunized with M8 or M9 and evaluated by cell-proliferation and cytokine-secretion assays. Our results clearly indicated that peptide-specific T cell proliferation was detected in mice immunized with M8 and M9, but not in mice immunized with PBS. Additionally, these CD4þ T cells exhibited significantly higher levels of secreted IFN-g and IL-17A as compared with those observed from T cells derived from mice stimulated with PBS (Fig. 4). Furthermore, the IL-17A levels secreted by CD4þ T cells from M8-stimulated mice were higher than those from M9stimulated mice, indicating that the M8 peptide represented the immunodominant epitope recognized by CD4þ T cells. These results suggested that immunization with M8 increased the potential of splenocytes to induce cytokine responses and polarize toward Th1/ Th17 subsets.
which MHC-II molecule subtype binds to the novel CD4þ T cell epitope. As shown in Fig. 6, the H-2d (I-Ek) antibody efficiently blocked M8-specific proliferation of CD4þ T cells, whereas MHC-I and H-2d (I-Ad) antibodies were unable to block these CD4þ T cell responses. These results confirmed M8 as an H-2d (I-Ek) CD4þ T cell epitope, but not a CD8þ T cell epitope.
3.6. The protective effect of MntC or M8 in a lethal S. aureus sepsis model Mice were immunized with MntC, M8, or PBS þ adjuvant, and their protective effects against S. aureus infection were evaluated in a sepsis model. We found that mice vaccinated with the MntC antigen exhibited higher survival rates (60% survival) as compared with those vaccinated with M8 (20% survival) or PBS (10% survival) (Fig. 7). These findings showed that MntC vaccination generated a protective effect against S. aureus infection in a sepsis model.
3.4. Flow cytometric analyses to determine the percentage of specific Th1/Th17 cells Having established that immunodominant Th1 and Th17 responses could be induced by the MntC protein or a novel CD4þ T cell epitope (M8), we investigated the characteristics of these epitope-specific Th1 and Th17 cells. M8-specific lymphocytes cultured in vitro were harvested, and CD4þ T cells were isolated and analyzed by intracellular cytokine staining. As shown in Fig. 5, M8stiluated cells exhibited an increased ability to polarize toward Th1 and Th17 cells as compared with PBS-stimulated cells. These results suggested that immunization with M8 elicited an immune response associated with Th1 and Th17 polarization. 3.5. MHC-restriction analysis An MHC-antibody blocking assay was performed to confirm
Fig. 4. Identification of CD4þ T cell epitopes on the MntC protein. CD4þ T cell proliferation in mice immunized with peptides M8 or M9, respectively. At 1 week after the final immunization with peptide M8 or M9, mouse splenic lymphocytes were isolated. (A) SI was measured by CCK-8 incorporation, which was assessed using a microplate reader. The levels of (B) IFN-g, (C) IL-17A and (D) IL-4 were determined by ELISA. The solid bars represent the mean ± standard deviation. Significant difference was compared with the negative control group (PBS) was indicated by *P < 0.05, **P < 0.01 and ***P < 0.001.
W. Yu et al. / Microbial Pathogenesis 112 (2017) 30e37
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Fig. 5. Phenotypic analysis of CD4þ T cells following immunization with the M8 epitope. (A) M8-specific lymphocytes cultured in vitro were harvested, and CD4þ T cells were isolated from lymphocytes and stimulated with the M8 epitope for 6 h in the presence of PMA, ionomycin, and Golgistop. Cells were then washed and labeled for flow cytometry. (B) Statistical analysis of Th1, Th17, and Th2 responses in M8-immunized mice. The gray bars represent the proportion of double-positive cells. Significant differences were indicated by *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 6. MHC-restriction analysis of the M8 epitope. (A) Anti-MHC-I, -MHC-II (I-Ad), and -MHC-II (I-Ek) antibodies were added to M8-immunized splenic lymphocytes and incubated for 30 min, respectively. Following incubation, the M8 epitope was used to stimulate the cells for an additional 5 h, with no peptide added to the negative control. (B) Cells were labeled with anti-CD4-PE, and lymphocyte proliferation was quantified by flow cytometry for statistical analysis. Significant differences were indicated by *P < 0.05 and **P < 0.01.
4. Discussion S. aureus is a ubiquitous pathogen associated with high infection rates [2], and screening and defining S. aureus-specific antigens are key to vaccine development. Until now, S. aureus vaccines have primarily relied upon immunization conferred by vaccination with various forms of recombinant S. aureus proteins. Although the MntC protein represents a valid antigenic molecule providing efficacious immunization against S. aureus infection, previous studies also reported its ability to induce humoral immune responses [27,28]. However, the ability of MntC to induce specific cell-based immunologic responses had not been investigated, and given the pivotal
role of T cells in the prevention of S. aureus infection [29,37], they were used as the model for the study. In recent years, the roles of antigen epitopes in immune responses have been gradually accepted, with increased study of pathogenic bacterial and viral antigen epitopes. A conventional method for identifying T cell epitopes (pepscan) involving the synthesis of multiple overlapping peptides spanning the full length of the target antigen and tests for immunogenicity using T cell assays have also been developed; however, the shortcomings of these approaches include their being time consuming and costly. Bioinformatics programs can predict which peptides are more likely to contain T cell epitopes, thereby greatly reducing the number of
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indicated a lower protection rate relative to that conferred by the M8 peptide. Given the disparity between the immunogenic response to the M8 epitope and the MntC protein in mice, we hypothesized that the relationship between T cell and B cell epitopes is more suitable for a specific peptide rather than an entire protein sequence. In conclusion, we identified a CD4þ T cell epitope on the S. aureus MntC protein capable of inducing Th1 and Th17 responses, resulting in a synthetic M8 peptide that represents an H-2d (I-E)restricted epitope. These data support the potential development of an epitope-based S. aureus-specific vaccine. Competing interests The authors declare that they have no competing interests. Acknowledgements
Fig. 7. Validation of the protective effects of the M8 peptide in an S. aureus sepsis model. BALB/c mice (n ¼ 10) were immunized with the MntC or M8 epitope with FA, and mice were intraperitoneally infected with S. aureus Newman (5 108 CFUs). Survival rates were monitored for 14 days. The significance of the protective immunity generated by the MntC and M8 epitope relative to that observed in the control was measured using a log-rank test. Asterisks represent statistically significant differences.
candidate sequences [40]. MHC-II-binding-prediction methods are categorized into two main groups: quantitative and qualitative. Qualitative matrices determine binding status (whether a peptide is a “binder” or “non-binder”) based on the predicted score (according to position-specific binding profiles), with SYFPEITHI one example of this software. Quantitative approaches also predict the strength of binding and include programs, such as MHCPred and ProPred [32,33]. IEDB software utilizes the gene ontology for biomedical investigations, as well as several additional ontologies to represent immune epitope-mapping experiments [34]. Previously, our research group successfully identified CD4þ T cell epitopes associated with S. aureus iron-regulated surface determinant protein B precursor (IsdB) and Streptococcus glyceraldehyde-3phosphate dehydrogenase, cytosolic [38,39], and developed epitope vaccines, including multiple epitopes capable of generating adequate immune protection. This study expanded those findings into the search for additional S. aureus-specific antigen epitopes. Antigen-epitope specificity related to T cell response plays an important role in understanding immune responses against S. aureus infection and the development of vaccines designed based on those epitopes. Here, we predicted candidate MntC epitopes using the bioinformatics software SYFPEITH, MHCPred, ProPred, and IEDB. We also predicted HLA-restricted epitopes to identify HLA-binding epitopes. Interestingly, we determined that M8 was an H-2d (I-E)-restricted epitope, with ProPred results also showing that M8 might be able to combine with HLA, thereby encouraging the need for future studies. We then tested their efficacy in vitro based on stimulation of CD4þ T cell proliferation and cytokine secretion. According to in vivo results, we found that mice immunized with a synthetic M8 peptide induced Th1 and Th17 differentiation. Our cumulative results suggested that the cell response mediated by M8 vaccination was significantly higher than that elicited by other synthetic peptides, indicating it as the immunodominant epitope. Furthermore, we verified these findings by flow cytometric analyses cytokine-detection results. BLAST analysis of the immunodominant-epitope sequences revealed that high levels of conservation conserved among various S. aureus strains and specific to the MntC protein. Subsequent evaluation of the MntC protein as a vaccine against S. aureus
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