Identification of a conserved linear B-cell epitope in the Staphylococcus aureus GapC protein

Accepted Manuscript Identification of a conserved linear B-cell epitope in the Staphylococcus aureus GapC protein Mengyao Wang, Yuhua Wei, Wei Yu, Lizi Wang, Lu Zhai, Xiaoting Li, Xintong Wang, Hua Zhang, Zhenyue Feng, Liquan Yu, Yongzhong Yu, Jinzhu Ma, Yudong Cui PII:

S0882-4010(17)31360-8

DOI:

10.1016/j.micpath.2018.03.007

Reference:

YMPAT 2823

To appear in:

Microbial Pathogenesis

Received Date: 1 November 2017 Revised Date:

27 February 2018

Accepted Date: 5 March 2018

Please cite this article as: Wang M, Wei Y, Yu W, Wang L, Zhai L, Li X, Wang X, Zhang H, Feng Z, Yu L, Yu Y, Ma J, Cui Y, Identification of a conserved linear B-cell epitope in the Staphylococcus aureus GapC protein, Microbial Pathogenesis (2018), doi: 10.1016/j.micpath.2018.03.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1 2

Identification of a conserved linear B-cell epitope in the

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Staphylococcus aureus GapC protein

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4 Mengyao Wang 1, Yuhua Wei 2, Wei Yu 1, Lizi Wang 1, Lu Zhai 1, Xiaoting Li 2,

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Xintong Wang 1, Hua Zhang 1, Zhenyue Feng 1, Liquan Yu 1, Yongzhong Yu 1, Jinzhu

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Ma 1, Yudong Cui 1,2*

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1. College of Life Science and Technology, Heilongjiang Bayi Agricultural

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University, Daqing 163319, China,

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2. College of Animal Science and Technology, Heilongjiang Bayi Agricultural

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University, Daqing 163319, China.

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* Corresponding author: College of Life Science and Technology, Heilongjiang Bayi

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Agricultural University, Daqing 163319, China.

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E-mail address: [email protected] (Y. Cui).

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Abstract

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The GapC protein of Staphylococcus aureus (S. aureus) is a surface protein that is

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highly conserved among Staphylococcus strains, and it can induce protective humoral

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immune responses. However, B-cell epitopes in S. aureus GapC have not been

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reported. In this study, we generated a monoclonal antibody (mAb2A9) targeting S.

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aureus GapC. Through a passive immunity test, mAb2A9 was shown to partially

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protect mice against S. aureus infection. We screened the motif 236PVATGSLTE243 that

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is recognized by mAb2A9 using a phage-display system. The motif sequence exactly

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matched amino acids 236–243 of the S. aureus GapC protein. Then, we identified the

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key amino acids in the motif using site-directed mutagenesis. Site-directed

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mutagenesis revealed that residues P236, G240, L242, and T243 formed the core of

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the

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surface of S. aureus, and it induced a protective humoral immune response against S.

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aureus infection in immunized mice. Overall, our results characterized a conserved

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B-cell epitope, which will be an attractive target for designing effective epitope-based

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vaccines against S. aureus infection.

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PVATGSLT243 motif. In addition, this epitope was proven to be located on the

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Keywords: B-cell epitope, GapC protein, Staphylococcus aureus

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1. Introduction S. aureus is a significant pathogen that causes various infections in humans,

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including life-threatening sepsis, endocarditis and pneumonia [1]. S. aureus is also

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one of the most important contagious bacteria causing bovine mastitis, which is

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considered to be a common, complicated and economically unbearable disease

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worldwide [2]. Particularly, the incidence of S. aureus mastitis in China is relatively

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high [3, 4]. Historically, the main method of preventing S. aureus infections has been

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antibiotic therapy. However, the excessive use of antibiotics has led to an increase in

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drug-resistant strains. Since an epidemic of methicillin-resistant S. aureus was first

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reported in the UK in 1961, it has become a global focus [5]. Presently, many S.

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aureus strains have high resistance rates to vancomycin [6, 7], erythromycin,

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tetracycline, and gentamicin [8]. However, an effective method of controlling S.

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aureus infections has not been found. Therefore, developing new immunotherapies is

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being investigated extensively. One strategy is to develop effective monoclonal

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antibodies (mAbs) and vaccines against S. aureus [9, 10].

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Currently, several S. aureus surface proteins have been used as recombinant

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vaccine components. They have been shown to protect against S. aureus infections

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[11, 12]. One of these proteins is GapC, which possesses glyceraldehyde 3-phosphate

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dehydrogenase (GAPDH) activity. GAPDH is a key enzyme in the glycolytic pathway,

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and

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glyceral-dehyde-3-phosphate into 1,3 bisphosphoglycerate. GAPDH is also associated

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reversibly

catalyzes

the

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oxidative

phosphorylation

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ACCEPTED MANUSCRIPT with protein binding, cell signaling in many organisms, and immune evasion in

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bacteria [13–15]. S. aureus has two GAPDH homologues. They have been termed

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GapA (also known as GapC in bovine mastitis isolates) and GapB. GapA has been

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characterized as a functional GAPDH protein that is important for pathogenesis

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during S. aureus infections [16, 17]. GapC has been demonstrated to play a role in

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adhesion and internalization into bovine mammary epithelial (MAC-T) cells [17, 18].

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Moreover, GapC is present on the surface of all S. aureus strains [16], it shares

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considerable homology at both the DNA and amino acid levels in different

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Staphylococcus species [19]. Especially, GapC induces an effective humoral immune

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response against S. aureus, suggesting that GapC is a good candidate for use as an

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immunodominant antigen in vaccines to prevent S. aureus infections [20].

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An antigen is a substance that the immune system treats as an exogenous agent

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that stimulates the body to produce an immune response. Antigens are usually large,

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but only certain portions of antigens, called epitopes, can induce the generation of

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specific antibodies and bind to their corresponding specific antibodies. B-cell epitopes

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are defined as regions on the surface of natural antigens that bind to B-cell receptors

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or recognize specific antibodies [21], they are essential for the induction of protective

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antibody responses [22]. Until now, the B-cell epitopes on S. aureus GapC have not

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been well characterized. Thus, in this study, we used molecular cloning techniques to

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express the S. aureus GapC protein in Escherichia coli. (E. coli) Recombinant GapC

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was purified and used to immunize BALB/c mice to generate a specific monoclonal

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antibody (mAb2A9) using a cell hybridization technique. We identified an epitope

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ACCEPTED MANUSCRIPT recognized by mAb2A9 using a phage-displayed, random 12-peptide library. The key

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amino acids in the epitope motif. Then, we further proved that the epitope is located

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on the cell surface and that the peptide epitope can induce a humoral immune

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response that protects against S. aureus infection.

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2. Materials and Methods

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2.1. Plasmids, cell line, and bacterial strains

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S. aureus strain Newman, a capsular type 5 strain, was stored in our laboratory

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and grown in tryptic soy broth (Difco, Becton Dickinson, Sparks, MD, USA) at 37 °C.

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To express the full-length GapC protein of S. aureus, the full-length gapC gene of S.

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aureus (1,008 bp, GenBank accession number AY356529) was amplified by

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polymerase chain reaction as described in our previous study [23] using the primers

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listed in Table 1. The resulting amplicon was cloned into the pET-32a(+) plasmid,

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resulting in the plasmid pET-32a (+)/gapC, which contains a gene encoding a

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histidine-tagged GapC-thioredoxin (TrxA) fusion protein. E. coli DH5a (Invitrogen,

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Carlsbad, CA, USA) was used for cloning purposes. Recombinant GapC was

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expressed in E. coli BL21 (DE3) (Novagen, Madison, WI, USA). Mutated versions of

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the epitope motifs were cloned into the pGEX-6p-1 plasmid and expressed in BL21

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(DE3) cells. The myeloma cell line SP2/0 was maintained in Roswell Park Memorial

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Institute 1640 medium supplemented with 10% fetal bovine serum (HyClone, USA)

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and 1% penicillin-streptomycin. All cells were maintained at 37 °C in 5% CO2.

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2.2. Anti-GapC mouse serum

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ACCEPTED MANUSCRIPT Female BALB/c (H-2d) mice (SPF, 6–8 weeks, 18–22 g) were provided by the

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Changchun Institute of Biological Products (Changchun, China). Animal experiments

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were performed in accordance with animal ethics guidelines and approved by the

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Ethics Committee of the Experimental Animal Center, Heilongjiang Bayi Agricultural

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University. Criteria for determining when the animals should be sacrificed humanely

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included weight loss, weakness, an inability to obtain feed or water, signs of severe

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organ dysfunction, and no response to treatment. For all immunizations, 50 g of the

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GapC fusion protein was blended with the same volume of Freund’s complete

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adjuvant, then injected subcutaneously into the female BALB/c mice and followed by

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two boosts with the same dose at 2-week intervals. Passive control mice were

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immunized with the same volume of PBS blended with the adjuvant. Two weeks after

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the final boost, the BALB/c mice were anesthetized and sacrificed humanely, and

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their blood was collected. Anti-GapC mouse serum was isolated from coagulated

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blood.

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2.3. Expression of Recombinant GapC

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The recombinant plasmid pET-32a (+)/gapC was transformed into E. coli strain

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BL21 (DE3). After the cells were cultivated to an optical density at 600 nm (OD600) of

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0.6–0.8 in LB medium at 37 °C, 0.1 mM isopropyl-b-D-1-thiogalactopyranoside

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(IPTG) was added to the medium to induce protein expression for 4 h. The cells were

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then harvested by centrifugation and resuspended in Tris-NaCl buffer (pH 8.8). The

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cells were disrupted using ultrasonication, the supernatant containing soluble

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recombinant GapC was collected. This protein, with 6× histidine tag, was purified by

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ACCEPTED MANUSCRIPT a histidine-affinity chromatography purification system (CTB, China) according to the

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manufacturer’s instructions. The purity and yield of recombinant GapC were analyzed

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by SDS-PAGE and Western blot.

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2.4. Enzyme-linked immunosorbent assay (ELISA)

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Purified GapC was coated onto microplates (96-well) in 0.05 M carbonate buffer (pH

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9.6) overnight at 4 °C. The plates were blocked with 5% nonfat milk for 1.5 h at

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37 °C. The nonfat milk in the wells was discarded, then the wells were washed three

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times with PBS containing 0.05% Tween-20 (PBST). Then, the plates were incubated

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with anti-GapC serum for 1 h at 37 °C. After washing with PBST, a secondary

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anti-mouse IgG horseradish peroxidase (HRP)-linked antibody (OriGene, Rockville,

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MD, USA) was added, followed by incubation for 1 h at 37 °C. The plates were

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washed, then incubated with 3, 3′, 5, 5′-tetramethylbenzidine dihydrochloride

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(Sigma-Aldrich, St. Louis, MO, USA) as a chromogenic substrate. The reaction was

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terminated with 2 M H2SO4. The OD450 value of each well was read using a

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microplate reader (Bio-Rad, Hercules, CA, USA).

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2.5. Preparation and characterization of mAb2A9

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Murine mAbs targeting GapC were generated by the standard hybridoma method

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described in earlier studies [24, 25]. Then, the culture supernatant from the

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hybridomas was analyzed by an ELISA and western blotting. After three rounds of

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screening, strongly positive hybridoma cell lines capable of stably secreting the

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antibodies were screened. mAb2A9 was selected for the next step. mAb2A9 was

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purified using a HiTrapTM Protein G HP column (GE Healthcare, Chicago, IL, USA)

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ACCEPTED MANUSCRIPT according to the manufacturer’s recommendations. The IgG subclass was identified

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using the Rapid Antibody Isotyping Kit (GE Healthcare). The specificity of mAb2A9

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was determined by an indirect ELISA. Recombinant FnbpA, Trap, Mntc, and IsdB of

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S. aureus and GapC from Streptococcus dysgalactiae were incubated overnight in 96

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well-plates at 4 °C as described above. mAb2A9 was used as the primary antibody.

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An anti-mouse IgG HRP-linked antibody was used as the secondary antibody. The

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effect of mAb2A9 on the GAPDH activity of recombinant GapC was assayed using a

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GAPDH activity assay kit (COMIN, Suzhou, China). GapC was incubated with

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glyceraldehyde 3-phosphate and NAD+ in a final volume of 1 ml of assay buffer

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(40mM triethanolamine, 50mM Na2HPO4, 0.2mM EDTA). mAb2A9 was added,

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incubated with the mixture at 37 °C for 10 min. The reduction of NAD+ to NADH

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was monitored spectrophotometrically at OD340, it reflected the level of the GAPDH

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activity. The OD340 was monitored at 20 s (A1) and 5 min and 20 s (A2). Activity

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calculation was based on a molar absorption coefficient of 6.22×103 L/mol/cm for

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NADH at 340 nm. Protein concentration was determined using the quartz cuvette.

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GAPDH activity (nmol/min/mg protein) = [ΔA × Vtotal ÷ (× d) × 109] ÷ (Vsample × Cpr)

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÷ T. [where ΔA  A1−A2; Vtotal is the total reaction volume; d is the light path of the

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cuvette (1 cm); Vsample is the sample volume in the reaction; Cpr is the sample protein

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concentration].

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2.6. Passive immunization of mice and lethal challenge

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The passive immunity assay involved 30 mice allocated to three groups of 10

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each. Two of the groups were immunized intravenously with anti-GapC serum or

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ACCEPTED MANUSCRIPT mAb2A9. The third group received the SP2/0 supernatant (the control group).

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Twenty-four hours after injection, the immunized and control animals were

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challenged intraperitoneally with 0.1 ml (5×108 colony-forming units [CFU]/mouse)

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of the S. aureus Newman strain, which was confirmed to be an LD 100 dose of these

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bacteria in a pre-test. The mice were monitored for mortality for 10 days after

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challenge.

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2.7. Screening a random phage-displayed 12 peptide library

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A random Ph.D.-12 phage display peptide library (New England Biolabs,

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Beverly, MA, USA) was screened with mAb2A9 according to a previous study [26].

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Positive phage clones were selected randomly after three rounds of biopanning, and

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their reactivity with mAb2A9 was verified by a sandwich ELISA. mAb2A9 (10 g/ml)

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or a bovine serum albumen negative control (100 l/well) were incubated in 96-well

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plates overnight at 4 °C. After washing with Tris-buffered saline containing 0.5%

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Tween-20, the purified phage particles of the positive clones were incubated in the

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96-well plates. After washing, an HRP-conjugated anti-M13 antibody was added as a

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probe (New England Biolabs). The single-stranded phage DNAs of the strongly

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positive phage clones were extracted and sequenced with the sequencing primer

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5′–TGAGCGGATAACAATTTCAC–3′ according to the manufacturer’s instructions.

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The phage peptide sequences were deduced from the DNA sequences. Aligned with

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the S. aureus GapC sequence using the MEGALIGN program in DNASTAR.

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2.8. Site-directed mutagenesis assay

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Each amino acid residue of the epitope was substituted with alanine to determine

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ACCEPTED MANUSCRIPT the crucial amino acids of the epitope. A series of complementary oligonucleotides

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encoding the wild-type and mutated versions of the epitope were synthesized by

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Comate Bioscience Co., Ltd. (Jilin, China). Cloned into the BamHI and XhoI multiple

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cloning sites of the pGEX-6p-1 vector to produce recombinant plasmids (Table 2).

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The recombinant plasmids were transformed into competent E. coli BL21 (DE3) cells

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to express the GapC epitopes fused to a GST tag. The GST fusion proteins were

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detected by 12% SDS-PAGE and western blotting using mAb2A9 as the primary

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antibody.

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2.9. Confocal laser-scanning microscopy analysis

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S. aureus (Newman) was grown in tryptic soy broth medium (Difco) at 37 °C for

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5 h. Then centrifuged at 8,000 × g for 3 min. The supernatant was discarded, and the

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sediment was washed three times with sterile PBS. The cells were diluted to the

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appropriate concentration, 20 μl was spotted onto microslides, soaked overnight in

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75% ethanol. After air-dried, the cells were fixed with ice-cold acetone at −20 °C for

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30 min. After washing the cells for 30 min with PBST (0.5% Tween-20), the slides

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were blocked with 5% skimmed milk at room temperature for 2 h. After washing with

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PBST for 30 min, anti-GapC serum, mAb2A9 (100 g/ml), or SP2/0 supernatant

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(negative control) were used as the primary antibodies. After washing, a fluorescein

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isothiocyanate-conjugated goat anti-mouse antibody (Bioss, China) was added, and

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incubated at 4 °C for 5 h. The cells were rinsed with deionized water. Then covered

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with glycerol buffer (glycerol/PBS = 3/1). A Leica (Wetzlar, Germany) TCS-SP8

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confocal laser-scanning microscope was used to examine the cells.

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2.10. Analysis of the opsonophagocytic killing of S. aureus by anti-epitope

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peptide serum Mouse peritoneal macrophages was taken from BALB/c mice (female,

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6-week-old) 72 h after the intraperitoneal injection of sterile fluid thioglycollate

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medium (1 ml/mouse). The concentration of macrophages was adjusted to 1×105

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cells/ml using Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal

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bovine serum, 1 ml of the medium per well was added to the six-well plates. Cultured

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at 37 °C in 5% CO2 for 24 h. S. aureus Newman was washed and adjusted to a

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concentration of 2×105 CFU/ml using DMEM containing 10% fetal bovine serum

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after washing with sterile PBS. 1ml of the S. aureus Newman suspension was added

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to 50 µl of anti-GapC serum, anti-GapC (243–246) epitope peptide serum, anti-GST

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tag protein serum, or anti-PBS serum (the negative control). The mixtures were

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incubated at 37 °C for 30 min, the cellular supernatants in the six-well plates that

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contained macrophages were discarded. Incubated with the mixtures at 37 °C for 90

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min. After the incubation, the supernatant was discarded, the cells were washed three

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times with Hank’s balanced salt solution (HyClone). The cells were lysed by adding 1

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ml of pre-cooled deionized water at 4 °C for 20 min. Finally, the lysate was plated on

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tryptic soy agar to determine the number of CFUs.

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2.11. Positive immunization of mice and lethal challenge

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The positive immunity protective assay involved 40 mice allocated to four

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groups of 10 each. The groups were immunized intramuscularly with 50 g of GapC,

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the GST-epitope peptide fusion protein, GST-tag protein, HIS-tag protein or PBS

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ACCEPTED MANUSCRIPT emulsified with 50 l of Freund's adjuvant (Sigma-Aldrich, St. Louis, MO, USA).

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Complete Freund’s adjuvant was used for the first immunization, incomplete Freund’s

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adjuvant was used for the second. Two weeks after the second injection, the

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immunized and control mice were challenged intraperitoneally with 0.1 ml of S.

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aureus Newman (5 × 108 CFUs/mouse). Survival rates were monitored at daily for 15

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days after the challenge.

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2.12. Statistical analysis

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Experimental data were analyzed with the Student’s t-test. Statistical significance

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was marked as ‘*’ when p < 0.05 and ‘**’ when p < 0.01, both p < 0.05 and p < 0.01

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were regarded as statistically significant. Data are shown as means ± standard

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deviations.

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3. Results

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3.1. Expression and purification of recombinant GapC

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Recombinant GapC was expressed in E. coli BL21 (DE3) harboring the pET-32a

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(+)/gapC plasmid using IPTG induction, it was purified by a histidine-affinity

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chromatography purification system. SDS-PAGE showed that the molecular weight of

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recombinant GapC was approximately 55 kDa, which is consistent with the

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theoretical molecular mass of GapC (Fig. 1). In addition, western blotting revealed

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that recombinant GapC was recognized by anti-GapC polyclonal antisera, thereby

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confirming its identity.

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3.2. Characterization of mAb2A9

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We generated mAbs using hybridoma technology. mAbs targeting different

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ACCEPTED MANUSCRIPT epitopes of GapC were screened using ELISA assays. One of the cell lines, referred to

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as mAb2A9, was selected for further study, as it stably secreted anti-GapC antibody at

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a high titer for more than 15 passages. The antibody titers of the culture supernatant

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and ascites fluid were 1:3.2 × 104 and 1:2.56 × 105, respectively. mAb2A9 was

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determined to belong to the IgG1 subclass and the  chain type (Fig. 2A). Western

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blotting revealed that purified GapC was recognized by mAb2A9, but not by the

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control SP2/0 ascites fluid, suggesting that GapC contains a specific epitope that is

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recognized by mAb2A9 (Fig. 2B).

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The reactivity of mAb2A9 with recombinant GapC, FnbpA, Trap, MntC, and

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IsdB of S. aureus and GapC of S. dysgalactiae was determined by indirect ELISAs

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(Fig. 2C). The effect of mAb2A9 on the GAPDH activity of recombinant GapC was

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assayed as described above, the GAPDH activity (nmol/min/mg protein) was deduced

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from the OD340. The results indicate that mAb2A9 inhibited the GAPDH activity of S.

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aureus GapC (Fig. 2D).

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To evaluate the immunoprotection of mAb2A9, mice were immunized passively

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with mAb2A9, then challenged with a lethal dose of S. aureus (Newman). Mice

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immunized with purified mAb2A9, anti-GapC serum, or SP2/0 ascites fluid were

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challenged intraperitoneally with S. aureus 24 h after the antibody transfer. The results

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showed that mice in the control group died within 20 h, but the mice immunized with

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mAb2A9 survived 15 h longer than those immunized with the SP2/0 supernatant after

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challenge (Fig. 2E). These results indicate that mAb2A9 offers a certain degree of

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immunoprotection against S. aureus Newman infection.

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3.3. Phage-displayed epitope biopanned by mAb2A9 After three rounds of biopanning with mAb2A9, screened phages were well

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enriched, the yield of positive phage clones increased significantly (Table 3). Thirty

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clones selected randomly by mAb2A9 were analyzed by a sandwich ELISA. 12 of

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them showed specific reactivity to mAb2A9 (Fig. 3). The single-stranded phage

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DNAs extracted from the 12 ELISA-confirmed positive clones were sequenced and

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their amino acid sequences were deduced. The amino acid sequences of the 12 clones

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showed the consensus motif PVATGSLT (Table 4). The motif PVATGSLT is located

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at amino acids 236–243 of the GapC protein. These results suggest that the motif

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3.4. Precise definition of the epitope

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PVATGSLT243 is an epitope of the S. aureus GapC protein.

To verify the epitope precisely, we extended the analysis to include one amino

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acid before and after the motif PVATGSLT. A series of mutated recombinant proteins

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GST-VPVATGSLTE, GST-APVATGSLTE, GST-VAVATGSLTE, GST-VPAATGSLTE,

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GST-VPVAAGSLTE,

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GST-VPVATGSATE, GST-VPVATGSLAE, and GST-VPVATGSLTA, were expressed

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in E. coli BL21(DE3) with a GST-tag (via pGEX-6P-1). Western blotting results

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showed that the P236A, G240A, L242A, and T243A mutations completely abrogated

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the reactivity of the epitope with mAb2A9 (Fig. 4). These results confirmed that the

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motif 236PVATGSLT243 is an authentic epitope in the GapC protein of S. aureus, at the

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same time, it represents the minimal reactivity unit of the continuous epitope

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recognized by mAb2A9.

GST-VPVATASLTE,

GST-VPVATGALTE,

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3.5. Alignment of the epitope sequence on GapC from different Staphylococcus

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species To identify whether the selected epitope is conserved, the epitope sequences of S.

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aureus strains and other Staphylococcus species were aligned using the BLAST tool.

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The results indicated that the motif

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epitope in the GapC proteins of S. aureus stains as well as other Staphylococcus

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species (Table 5).

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3.6. Analysis of the secondary and tertiary structures of the epitope The

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PVATGSLT243 represents a highly conserved

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PSIPRED

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COUDES

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and

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(http://www.rcsb.org/pdb/home/home.do) [29] methods were used to analyze the

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secondary and tertiary structures of GapC or the

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The major secondary structure of amino acids 236–243 is a coil. The coil domain is

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located at amino acids 236–241, a small -turn domain is located at amino acids 242

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and 243 (Fig. 5A). The position of the epitope in GapC three-dimensional structure is

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marked in red (Fig. 5B), the result indicates that amino acids 236–243 are exposed on

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the surface of the molecule, which is consistent with the location of a protective

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epitope.

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3.7. The epitope recognized by mAb2A9 is located on the cell surface

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PVATGSLT243 epitope (Fig. 5).

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S. aureus Newman cells were observed by laser scanning confocal microscopy.

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mAb2A9 and anti-GapC serum were used as the primary antibody. A fluorescein

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isothiocyanate-conjugated goat anti-mouse antibody (which fluoresces green) was

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used as the secondary antibody. After the analysis, a homogeneous dispersion of

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ACCEPTED MANUSCRIPT obvious green fluorescence was detected on the S. aureus cell surface in the darkfield

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image when anti-GapC serum or mAb2A9 was used as the primary antibody, the same

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bacterial morphology was observed in the brightfield image (Fig. 6). The results prove

324

that the B-cell epitope 236PVATGSLT243, which is recognized by mAb2A9, is exposed

325

on the surface of the bacteria. These results are consistent with the results of the above

326

structural analysis.

327

3.8. Anti-epitope peptide serum promotes the phagocytic activity of macrophages

328

We measured the serum opsonophagocytic activity mediated by immune sera

M AN U

SC

RI PT

321

236

PVATGSLT243 peptide. Briefly, we

from mice immunized with the epitope

330

incubated the S. aureus Newman strain with anti-epitope serum for 30 min, washed

331

the cells, then added them to macrophages. After incubation, the macrophages were

332

washed and lysed, the number of phagocytosed bacteria was quantified by plating on

333

tryptic soy agar. Anti-GapC serum and negative serum were used as positive and

334

negative controls, respectively. The results showed that the level of phagocytosis

335

mediated by the anti-236PVATGSLT243 epitope was significantly higher than that of the

336

anti-GST serum, negative control and blank control. The results further prove that the

337

epitope 236PVATGSLT243 is a functional epitope.

338

3.9. The protective effect of GapC and the epitope peptide in a lethal S. aureus

339

sepsis model

340

AC C

EP

TE

D

329

To evaluate the immune responses of the epitope

236

PVATGSLT243, mice

341

immunized with purified GapC, the GST-epitope peptide, GST, HIS, or PBS were

342

challenged intraperitoneally with S. aureus on day 14 after the last immunization. The

16

ACCEPTED MANUSCRIPT 343

antibody titers of the immunized mice were detected by an indirect ELISA. The

344

antibody titer of full-length GapC was 1:128,000, the epitope peptide was 1:32,000.

345

These results show that the B-cell epitope

346

Then, we challenged the mice intraperitoneally with S. aureus (5 × 108 CFUs/mouse).

347

The results showed that the recombinant GapC group had the highest survival rate

348

(70%), while the group immunized with the epitope 236PVATGSLT243 had the second

349

highest survival rate (30%); these survival rates were much higher than those of the

350

groups immunized with GST or PBS (Fig 8). These results suggest that GapC

351

vaccination generated a protective effect against S. aureus infection in a sepsis model.

352

The 236PVATGSLT243 B-cell epitope also induces a humoral immune response against

353

S. aureus infection, which is consistent with previous results.

M AN U

SC

RI PT

PVATGSLT243 is highly immunogenic.

D

354

Discussion

TE

355

236

S. aureus is one of the major pathogenic bacteria in humans and animals. In recent

357

years, S. aureus has become prevalent and there is a lack of new classes of antibiotics

358

to treat infections. Therefore, immunotherapeutic approaches against S. aureus have

359

been investigated extensively [9]. Previous studies have shown that developing

360

epitope vaccines is an attractive strategy for combating infectious diseases [30, 31].

361

Epitope-based vaccines can induce intense and broad-spectrum humoral and cellular

362

immune responses [32, 33]. B-cell epitopes are important for inducing protective

363

humoral immune responses [22]. Therefore, screening B-cell epitopes is an important

364

step in epitope-based vaccine development. In addition, effective mAbs have been

AC C

EP

356

17

ACCEPTED MANUSCRIPT proposed as attractive antibody-based therapeutic reagents because they have a variety

366

of preferred features, such as low-immunogenicity, good stability, homogeneity and

367

so on [34, 35]. Therefore, preparing mAbs against S. aureus surface antigens,

368

identifying B-cell epitopes on the antigens have important scientific significance and

369

practical value.

RI PT

365

S. aureus expresses a series of surface proteins that play important roles in

371

nutrient transport, cellular metabolism and virulence-related functions [19]. S. aureus

372

GapC is a conserved surface protein with GAPDH activity, it possess several

373

properties that contribute to the evasion of host defenses, adhesion and invasion [16,

374

17, 36]. GapC homologues in other Gram-positive bacteria, such as Streptococcus,

375

have also been found on the cell surface, are described as proteins associated with

376

virulence because of their ability to bind several host proteins or to confer resistance

377

against reactive oxygen species produced by host phagocytic cells. Their potential use

378

as vaccines to prevent bovine mastitis has been demonstrated [36-38]. Therefore, the

379

function and immune efficacy of GapC make it a good candidate for the development

380

of a preventive vaccine. However, the B-cell epitope of the S. aureus GapC had not

381

been investigated previously. In consideration of the pivotal role of GapC in inducing

382

a protective humoral immune response against S. aureus, we chose to study GapC and

383

identify its B-cell epitope.

AC C

EP

TE

D

M AN U

SC

370

384

We generated mAbs against S. aureus GapC using the hybridoma technique. One

385

of the cell clones, 2A9, stably secreted a high titer antibody that belonged to the IgG1

386

subclass and the κ chain type. Purified mAb2A9 recognized the GapC protein of S.

18

ACCEPTED MANUSCRIPT aureus, but not S. dysgalactiae GapC or other recombinant S. aureus proteins,

388

suggesting the reactivity between mAb2A9 and S. aureus GapC was extremely

389

specific. Additionally, we further proved that mAb2A9 inhibited the GAPDH activity

390

of recombinant GapC. Furthermore, after S. aureus challenge, the survival time of

391

mice that were immunized passively with mAb2A9 was significantly higher than that

392

of controls, demonstrating that mAb2A9 is an effective mAb that provides immune

393

protection against S. aureus infection.

SC

RI PT

387

Subsequently, we identified the immunodominant B-cell epitope on GapC by

395

screening the Ph.D.-12 Phage Display Peptide Library. The epitope 236PVATGSLT243

396

is the minimal binding unit recognized by mAb2A9. Residues P236, G240, L242 and

397

T243 were proven to be the core of this epitope by a site-directed mutagenesis

398

analysis. The B-cell epitope

399

the epitope sequences of GapC from various Staphylococcus strains, including

400

coagulase-negative staphylococci, which have become the predominant pathogens of

401

mastitis in cows in numerous countries in recent years [39], using the Basic Local

402

Alignment

403

conservation of the epitope suggests that it might be a potential component of an

404

epitope-based vaccine against S. aureus. Moreover, an indirect immunofluorescence

405

assay and a bioinformatics analysis of the secondary and tertiary structures of GapC

406

indicated that the epitope

407

bacterial surface. Furthermore, an opsonophagocytic assay showed that the B-cell

408

epitope antisera promoted the ability of macrophages to phagocytose S. aureus. Lastly,

PVATGSLT243 was shown to be conserved by aligning

EP

TE

D

236

M AN U

394

Tool

(https://blast.ncbi.nlm.nih.gov/Blast.cgi).

The

high

AC C

Search

236

PVATGSLT243 recognized by mAb2A9 is exposed on the

19

ACCEPTED MANUSCRIPT 409

we evaluated the protective efficacy of the B-cell epitope peptide in a murine sepsis

410

model. The results showed that the epitope peptide provided a protection rate of 30%,

411

suggesting that it induced a humoral immune response against S. aureus infection. In summation, the B-cell epitope

236

PVATGSLT243 is a conserved epitope that is

RI PT

412

exposed on the bacterial surface. Active immunization with the epitope peptide

414

markedly increased the survival rate of mice challenged with S. aureus. Therefore,

415

this epitope is a highly conserved immunodominant epitope that should be studied

416

further as a potential epitope-based vaccine against not only S. aureus, but also other

417

Staphylococcus strains.

418

420

Competing interests

The authors declare that they have no competing interests.

D

419

TE

421 422

M AN U

SC

413

Acknowledgements

This work was supported by the Natural Science Foundation of Heilongjiang

424

Province of China (grant no. ZD2016004) and the Research Innovation Program for

425

College Graduates of Heilongjiang Bayi Agricultural University (grant no.

426

YJSCX2017-Y62).

AC C

427

EP

423

428

References

429

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567

Table 1. Primers used in this study. Sequence (5′–3′)

G1

CGCGGATCCATGGCAGTAAAAGTAGCAA

G2

CCCAAGCTTTTTAGAAAGTTCAGCTAAG

Description

SC

The forward primer of GapC, plus Bam HI site The reverse primer of GapC, plus Hind III site

Note: BamH I and Hind III restriction enzyme sites were introduced at the 5′ end of the primers as indicated by the underlined sequences.

M AN U

568 569

Primer

RI PT

563

570

Table 2. The oligonucleotides encoding the truncated GapC amino-terminus and

572

alanine-scanning peptides.

D

571

Name

235

WT235-244S

5’-gatccGTTCCTGTAGCTACAGGTTCATTAACTGAAc-3’

WT235-244R

5’-tcgagTTCAGTTAATGAACCTGTAGCTACAGGAACg-3’

V235A-S

5’-gatccGCGCCTGTAGCTACAGGTTCATTAACTGAAc-3’

V235A-R

5’-tcgagTTCAGTTAATGAACCTGTAGCTACATTCGCg-3’

P236A-S

5’-gatccGTTGCGGTAGCTACAGGTTCATTACTGGAAc-3’

P236A-R

5’-tcgagTTCAGTTAATGAACCTGTAGCTACCGCAACg-3’

V237A-S

5’-gatccGTTCCTGCGGCTACAGGTTCATTAACTGAAc-3’

V237A-R

5’-tcgagTTCAGTTAATGAACCTGTAGCCGCAGGAACg-3’

T239A-S

5’-gatccGTTCCTGTAGCTGCGGGTTCATTAACTGAAc-3’

T239A-R

5’-tcgagTTCAGTTAATGAACCCGCAGCTACAGGAACg-3’

G240A-S

5’-gatccGTTCCTGTAGCTACAGCGTCATTAACTGAAc-3’

G240A-R

5’-tcgagTTCAGTTAATGACGCTGTAGCTACAGGAACg-3’

S241A-S

5’-gatccGTTCCTGTAGCTACAGGTGCGTTAACTGAAc-3’

S241A-R

5’-tcgagTTCAGTTAATGAACCTGTAGCTACAGGAACg-3’

L242A-S

5’-gatccGTTCCTGTAGCTACAGGTTCAGCGACTGAAc-3’

L242A-R

5’-tcgagTTCAGTCGCTGAACCTGTAGCTACAGGAACg-3’

T243A-S

5’-gatccGTTCCTGTAGCTACAGGTTCATTAGCGGAAc-3’

T243A-R

5’-tcgagTTCCGCTAATGAACCTGTAGCTACAGGAACg-3’

VPVATGSLTE244 244

235

244

APVATGSLTE

AC C

VAVATGSLTE

EP

235

235

VPAATGSLTE

244

235

VPVAAGSLTE244

235

244

235

244

235

244

235

244

VPVATASLTE

VPVATGALTE

VPVATGSATE

VPVATGSLAE

The sequences of oligonucleotides

TE

Coding motifs

27

ACCEPTED MANUSCRIPT 235

VPVATGSLTA244

573 574

E244A-S

5’-gatccGTTCCTGTAGCTACAGGTTCATTAACTGCGc-3’

E244A-R

5’-tcgagCGCAGTTAATGAACCTGTAGCTACAGGAACg-3’

Note: The mutational sites are shown in lowercase letters. The mutated amino acids are shown in bold and underlined.

575

Cycles

mAb 2A9 (mg/L)

Washing (%TBST)

Input (pfu)

1 2 3

100 50 30

0.1 0.3 0.5

1.0×1011 1.0×1011 1.0×1011

RI PT

Table 3. Enrichment of positive phage clones by biopanning of the Ph.D.-12 library. Output(pfu)

Yield

2.8×105 2.4×108 1.2×109

2.8×10-6 2.4×10-3 1.2×10-2

SC

576

TBST, Tris-buffered saline containing 0.1% , 0.3 % or 0.5% Tween-20.

579

Table 4. Amino acid sequences of the Ph.D.-12 phage-displaying peptides from the strongly

580

positive phage clones as obtained through bio-panning.

581

R R

S

E

S P P

A R Q A A Q R H D C D C A A

TE

S S

G R D G G D R V T G T G V V

EP

S

P T T P P T T W

AC C

1 2 3 4 5 6 7 8 9 10 11 12 Consensus GapC

Amino acid sequence of the insert T I T T T T I E Q T Q T T T

D

Phage

M AN U

577 578

A G H A A H G W A S A S G G

G W R G G R W V S G S G S S

P L L P P L L T R L R L L L

S T A S S A T S L H L H T T

S D S S D N A R A R E

Note: Consensus amino acid motifs are shown in bold and underlined.

582 583

Table 5. Alignment of the sequences surrounding the epitope-coding region on the GapC

584

protein from different strains of Staphylococcus species.

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ACCEPTED MANUSCRIPT Epitope motif

Staphylococcus aureus (ORN58982) Staphylococcus aureus (SCU36715) Staphylococcus aureus (CYC45357) Staphylococcus aureus (SGR52366) Staphylococcus aureus (CXP79263) Staphylococcus aureus (SGV91609) Staphylococcus aureus (SHC13630) Staphylococcus aureus (SBA95846) Staphylococcus aureus mrsa252 (BX571856) Staphylococcus aureus USA300 (ABD21603) Staphylococcus argenteus (SGW47323) Staphylococcus carnosus (KOR4139) Staphylococcus capitis (BAW91423) Staphylococcus chromogenes MU 970 (KDP13817) Staphylococcus equorum subsp. Equorum (KKI55086) Staphylococcus epidermidis (CUY00364) Staphylococcus gallinarum (KIR10623)

GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL

Staphylococcus lutrae (ARJ51303)

GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL

Staphylococcus saccharolyticus (PAK56875) Staphylococcus saprophyticus (SCS35867)

GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL

Staphylococcus schleiferi (AKS71947)

GGAQRVPVATGSLTELTVVL

Staphylococcus schweitzeri (CDR26320) Staphylococcus simiae (EHJ08914) Staphylococcus xylosus (OEK87827)

GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL GGAQRVPVATGSLTELTVVL

587

EP

D

Staphylococcus pseudintermedius (KZK20974)

Note: The GenBank accession numbers of the strains are indicated in parentheses. The homologous amino acid residues of the epitope motif are in bold.

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585 586

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GGAQRVPVATGSLTELTVVL

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Staphylococcus microti (KIX90321)

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Staphylococcus intermedius (PCF87640)

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Species

588

Figure legends

589

Fig. 1. Purification and detection of the GapC fusion protein by 12% SDS-PAGE and

590

western blotting using an anti-GapC serum antibody. Lane M, protein marker; lane 1,

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E. coli BL21 with pET32a(+) prior to IPTG induction; lane 2, E. coli BL21 with

592

pET32a(+) after IPTG induction; lane 3, E. coli BL21 with pET32a(+)/gapC prior to 29

ACCEPTED MANUSCRIPT IPTG induction; lane 4, E. coli BL21 with pET32a(+)/gapC after IPTG induction;

594

lane 5, the induced and purified GapC protein; lane 6, the GapC fusion protein was

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confirmed by western blotting using anti-GapC serum.

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Fig. 2. Characterization of mAb2A9. (A) mAb2A9 was determined to belong to the

597

IgG1 class and the chain type using a mouse mAb isotyping kit. (B) The binding of

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mAb2A9 to GapC was detected by western blotting. (C) The specificity of mAb2A9

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toward recombinant FnbpA, Trap, Mntc, and IsdB of S. aureus and GapC from S.

600

dysgalactiae was determined by indirect ELISAs. (D) Effect of mAb2A9 on the

601

GAPDH activity of GapC. (E) Passive immunization with mAb2A9 induces an

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immunoreaction against S. aureus infection.

603

Fig. 3. Detection of the binding activities of 12 strongly positive phage clones to

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mAb2A9 that were selected by a sandwich ELISA. Wild-type M13 phage was used as

605

the negative control and an E. coli ER2738 culture supernatant was used as the blank

606

control. Bovine serum albumen-coated wells were used to exclude cross-activity.

607

Fig. 4. The reactivity of the GST-epitope fusion proteins, in which each amino acid of

608

the epitope (235VPVATGSLTE244) was mutated to alanine, with mAb2A9, as

609

determined by western blotting. The PAGE-PAGE gel of these mutated peptides was

610

used as the reference.

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Fig. 5. Secondary and tertiary structures of the

612

major secondary structure of the epitope

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the epitope in the GapC tertiary structure (red).

614

Fig. 6. Location of the

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236

236

PVATGSLT243 epitope. (A) The

236

PVATGSLT243 is a coil. (B) Location of

PVATGSLT243 epitope on S. aureus (Newman) cells. The

30

ACCEPTED MANUSCRIPT cells were observed by confocal laser-scanning microscopy. (A–C) Primary antibodies

616

were anti-GapC serum, mAb2A9, and the SP2/0 supernatant. Fluorescein

617

isothiocyanate-conjugated goat anti-mouse antibody (which fluoresces green) was

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used as the secondary antibody.

619

Fig. 7. Opsonophagocytic activity mediated by immune sera from mice immunized

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with the epitope 236PVATGSLT243 peptide. PBS-washed S. aureus (2 × 105 CFUs) was

621

incubated with anti-serum for 30 min, washed, and then added to macrophages. The

622

negative control was incubated with anti-PBS serum and no serum was included in

623

the blank control. Statistical significance was measured using a Student’s t-test (*, p <

624

0.05; **, p < 0.01).

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Fig. 8. Validation of the protective effects of the B-cell epitope peptide in an S. aureus

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sepsis model. BALB/c mice (n = 10) were immunized with GapC or the GST-epitope

627

peptide, and the mice were intraperitoneally infected with S. aureus Newman (5 × 108

628

CFUs). Survival rates were monitored for 15 days.

SC

M AN U

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EP AC C

629

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615

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ACCEPTED MANUSCRIPT 630

Fig. 1

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631

SC

632

AC C

EP

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M AN U

633

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ACCEPTED MANUSCRIPT

Fig. 2 B

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A

635 636 C

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D

SC

634

638

AC C

EP

E

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637

639

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ACCEPTED MANUSCRIPT

Fig. 3

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640

SC

641 642

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Fig. 4

D

643

AC C

EP

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644 645

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ACCEPTED MANUSCRIPT

Fig. 5

646 647

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A

M AN U

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B

AC C

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648 649

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ACCEPTED MANUSCRIPT 650

Fig. 6

SC

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651

AC C

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652 653

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ACCEPTED MANUSCRIPT

Fig. 7

SC

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655 656

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Fig. 8

AC C

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EP

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657

37

ACCEPTED MANUSCRIPT Dear Editors:

Our manuscript entitled “Identification of a conserved linear B-cell epitope in the Staphylococcus aureus GapC protein” (YMPAT_2017_1213), which we wish to be

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considered for publication in “ELSEVIER”. In this work, we generated a protective anti-GapC monoclonal antibody 2A9. It showed strong specificity to Staphylococcus aureus GapC. We identified a linear B-cell epitope 236PVATGSLT243 and analyzed the core amino acids. The epitope could induce a protective humoral immune response

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against S. aureus infection in immunized mice. So we think it might be a promising epitope for a epitope vaccine.

please don’t hesitate to contact me. Thank you and best regards. Yours sincerely,

Name: Yudong Cui

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Corresponding author:

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We deeply appreciate your consideration of our manuscript. If you have any queries,

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E-mail: [email protected]