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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Chuanminshen violaceum polysaccharides improve the immune responses of foot-and-mouth disease vaccine in mice

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Feng Haibo a,∗,1 , Jing Fan b,1 , Hong Qiu a , Zhenhua Wang c , Zhiqiang Yan a , Lihua Yuan a , Lu Guan a , Xiaogang Du d , Zhenhui Song a , Xingfa Han c , Juan Liu a a

Department of Veterinary Medicine, Southwest University, Rongchang, Chongqing 402460, PR China Sichuan Industrial Institute of Antibiotics, Chengdu University, Chengdu, Sichuan 610051, PR China c Department of Animal and Veterinary Science, Chengdu Vocational College of Agricultural Science and Technology, WenJiang, Sichuan 611130, PR China d Applied Biophysics and Immune Engineering Laboratory, College of Life and Science, Sichuan Agricultural University, Ya’an, Sichuan 625014, PR China

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Article history: Received 11 January 2015 Received in revised form 27 March 2015 Accepted 8 April 2015 Available online xxx

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Keywords: Chuanminshen violaceum polysaccharide FMDV Adjuvant

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1. Introduction

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Water-soluble polysaccharides from Chuanminshen violaceum (CVPS) were evaluated for their physicochemical properties, monosaccharide composition, and adjuvant potential to specific cellular and humoral immune responses in a mouse model of foot-and-mouth disease virus (FMDV) vaccination. The average molecular weight (Mw) of the CVPS was 968.31 kDa. The monosaccharide components of the CVPS was rhamnose, arabinose, fucose, mannose, glucose, and galactose with a relative mass of 6.29%, 21.87%, 16.59%, 12.54%, 13.07%, and 28.05%, respectively. Administering CVPS as an adjuvant significantly enhanced the phagocytic capacity of peritoneal macrophages, splenocyte proliferation, and the activity of NK cells and CTL as well as increased FMDV-specific IgG and IgG subclass antibody titers. Moreover, CVPS increased the expression of IL-2, IFN-␥, and IL-4 in CD4+ T cells and IFN-␥ expression in CD8+ T cells. Additionally, CVPS enhanced CD40+ , CD80+ , and CD86+ expression on DCs. Moreover, CVPS upregulated MHC-I/II, TLR-2/4 mRNA levels. In contrast, CVPS downregulated TGF-␤ mRNA expression and the frequency of CD4+ CD25+ Foxp3+ Treg cells. Taken together, these results indicate that administering CVPS as an adjuvant enhances both cellular and humoral immune responses via the TLR-2 and TLR-4 signalling pathways, thereby promoting DC maturation and suppressing TGF-␤ expression and Treg frequency. © 2015 Published by Elsevier B.V.

Foot-and-mouth disease (FMD) is an acute, febrile, contagious, and economically devastating viral disease of cloven-hoofed animals, such as cattle, swine, and sheep, that has spread throughout most parts of the world [1,2]. The FMD virus (FMDV) spreads rapidly among susceptible animals and is therefore listed by the World Organization for Animal Health (OIE) as one of the world’s most important animal diseases [3]. Widespread vaccination with inactivated foot-and-mouth disease virus (FMDV) is currently the

Abbreviations: ConA, concanavalin A; CFSE, carboxyfluorescein succinimidyl ester; CTL, cytotoxic T lymphocyte; CVPS, Chuanminshen violaceum polysaccharide; DCs, dendritic cells; FMDV, foot-and-mouth disease virus; IL-2, interleukin-2; IL-4, interleukin-4; IFN-␥, interferon-␥; IR, infrared; LPS, lipopolysaccharide; NK, natural killer cells; PM, peritoneal macrophage; PI, propidium iodide; TGF-␤, transforming growth factor-␤; TLRs, toll-like receptors. Q2 ∗ Corresponding author. Tel.: +86 83 52886138; fax: +86 23 46751732. E-mail addresses: [email protected], [email protected] (F. Haibo). 1 These authors contributed equally to this work.

only practical means of controlling the epidemic in most developing countries [4]. However, these vaccination strategies often fail to confer protection [5–7]. Higher vaccine doses or increased numbers of treatments, different administration routes (e.g., intradermal versus intramuscular administration), accelerated dosing schedules, and the use of adjuvants have been considered as important strategies for improving protection rates [8–12]. Increasing evidence suggests that adjuvants improve current vaccine efficacies against infectious diseases [13]. Unfortunately, more potent adjuvants are often associated with increased toxicity, as exemplified by Freund’s complete adjuvant (FCA). Chinese herbal medicine (CHM) and ingredients with immune-enhancing effects have been studied. Therefore, many investigators have successfully used medicinal herbs as adjuvants to vaccines [14,15]. Chinese herbal medicinal adjuvants have many advantages, including their relative abundance, reliable efficacy, fewer side effects, and lower toxicity. Chuanminshen violaceum, which is a well-tolerated, non-toxic, commonly used Chinese medical herb, is traditionally used as a tonic to strengthen the body and nourish the spleen and lung. This

http://dx.doi.org/10.1016/j.ijbiomac.2015.04.044 0141-8130/© 2015 Published by Elsevier B.V.

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traditional Chinese medicine was also used as a treatment for a variety of pathological conditions, including infirmity, apositia and cough. Approximately 28% of the raw C. violaceum plant is composed of polysaccharides (CVPS) [16]. CVPS strengthens specific and non-specific humoral immunity, stimulates T cell proliferation and exhibits an anti-mutagenic effect [17–19]. In the present paper, we evaluated the physicochemical properties, monosaccharide composition and the adjuvant effects of CVPS. Our data indicate that CVPS efficiently enhances the immunogenicity of FMDV vaccines by improving DC maturation and decreasing the Treg frequency.

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2. Materials and methods

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2.1. Reagents and cell line

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Propidium iodide (PI), concanavalin A (ConA), and RPMI-1640 medium were purchased from Sigma Chemical Co. (Saint Louis, Missouri, USA). The alum adjuvant was kindly provided by the North China Pharmaceutical Group Corporation (NCPC, Hebei, China). The cell counting kit-8 (CCK-8) was purchased from the Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Goat anti-mouse IgG, IgG1, IgG2a, and IgG2b peroxidase conjugates were purchased from Santa Cruz Biotechnology Inc. (California, USA). Fetal calf serum (FCS) was provided by Invitrogen Gibco Biotechnology Inc. (California, USA). Fluorescent-labelled anti-mouse monoclonal antibodies, specifically, CD8-PE, CD4-PE, IL-4-PE, CD4FITC, IL-2-FITC, IFN-␥-FITC, CD11c-FITC, CD40-PE, CD80-PE, and CD86-PE were obtained from eBiosciences (San Diego, CA). Carboxyfluorescein diacetate succinimidyl ester (CFSE) was purchased from Fanbo Biochemicals (Beijing, China). An FMDV O-serotyped inactivated vaccine in oil emulsion was acquired from Lanzhou Veterinary Research Institute China Animal Husbandry Industry Co., Ltd. (No. 2014001, Lanzhou, China). K562 cell lines were provided by Shanghai Xinyu Biological Technology Co., Ltd. (Shanghai, China). The cells were maintained in the logarithmic growth phase in RPMI 1640 medium supplemented with 100 IU/mL penicillin, 2 mM l-glutamine (Sigma), 10% fetal bovine serum, and 100 ␮g/mL streptomycin in an incubator set at 37 ◦ C and 5% CO2 .

2.2. Extraction and purification of polysaccharide CVPS The Chuanminshen violaceum (CV) was collected in Cangxi county, Sichuan province, China in October 2013. The samples were dried at 50 ◦ C and ground into powder with a mixer, and filtered through a 80 mesh sieve. Dried powder (500 g) was repeatedly refluxed with petroleum ether at a temperature of 60–90 ◦ C and then with anhydrous ethanol for 3 h, to remove colored ingredients and lipids. The residue was centrifuged at 5000 rpm for 10 min and dried at room temperature. The residue was extracted with boiling water three times under reflux at 80 ◦ C for 4 h. The aqueous extract was filtered through filter paper. The filtrate was concentrated using a rotary evaporator under reduced pressure, centrifuged at 5000 rpm for 15 min and dried at room temperature. The dried filtrate was extracted with distilled water at 90 ◦ C for 3 h, and filtered using vacuum filtration. The residue was re-extracted three times according to the same condition. The filtrates solution was concentrated under reduced pressure. The filtrates were then precipitated with ethanol at 95% of working concentration at 4 ◦ C for 12 h. The fractions were then dissolved in distilled water, and protein was eliminated using the Sevag method [20]. The fractions were dialyzed against distilled water for 72 h. The fractions were purified by a macroporous adsorption resin column to eliminate pigment, and Sephadex G-100 and Sephadex G-50 columns to remove other

impurities. Finally, the aqueous polysaccharide fraction of CVPS was obtained. 2.3. Characterization of CVPS 2.3.1. Physicochemical property analysis The physicochemical properties of CVPS were characterized using the following methods: color observation, solubility test, iodination reaction, ␣-naphthol reaction, phenol–sulfuric acid test, uronic acid carbazole reaction, Fehling’s test, full wavelength scanning, Coomassie brilliant blue reaction, and FeCl3 reaction [21]. 2.3.2. Analysis contents of carbohydrate, uronic acid and protein Total sugar content was measured using the Anthrone–sulfuric acid method with glucose as a standard [20]. The uronic acid content was assayed using the carbazole–sulfuric acid method using glucuronic acid as a standard. The protein content was determined using the method of Bradford [22], with bovine serum albumin as a standard. 2.3.3. Infrared spectroscopy analysis of CVPS An infrared (IR) spectra analysis was used to investigate the organic functional groups of the CVPS, and the IR spectra of CVPS were recorded within 4000–400 cm−1 using a FTIR spectroscopy (FTIR-8400S, Shimadzu Co., Japan). The purified CVPS was dried and ground with KBr powder and pressed into pellets for FTIR measurement [23]. 2.3.4. Analysis of monosaccharide composition of CVPS The monosaccharide composition of CVPS was determined using gas chromatography (GC) analysis because this is a recognized method for the quantification of neutral sugars. CVPS (20 mg) was hydrolyzed with 2 M trifluro acetic acid (TFA) at 110 ◦ C for 7 h to hydrolyze and release component monosaccharides. The digested solution was dried by evaporation and 4 mL of distilled water and 60 mg of NaBH4 were added to the dried solids. Subsequently, the mixture was acidified by incubating it with acetic acid for 30 min. The mixture was then evaporated at 60 ◦ C until it was a dry solid. Four mL of 0.1% HCl–MeOH (v/v) was added to the dried solids, and the mixture was evaporated to a dry solid again. The dried products were prepared for acetylation [24]. The acetylation was carried out using 1:1 pyridine–acetic anhydride in a water bath at 85 ◦ C for 1 h. The monosaccharide composition of CVPS was determined using the GC–MS alditol acetates of standard monosaccharides (d-fructose, d-xylose, dglucose, d-galactose, l-rhamnose, d-arabinose, and d-mannose) with inositol as the internal standard. Each monosaccharide was prepared and subjected to GC–MS analysis, separately, but using the same procedure. The operation was performed using the following conditions: the column temperature was initially 120 ◦ C, held for 4 min, increased to 200 ◦ C at a rate of 5 ◦ C/min, held for 4 min, then increased to 250 ◦ C at a rate of 5 ◦ C/min; detector temperature was 280 ◦ C; inlet temperature; 250 ◦ C; The N2 carrier gas rate was 20 mL/min. 2.3.5. Molecular weight determination of CVPS The average molecular weight of CVPS was determined using gel permeation chromatography (GPC) on a column (60 cm × 1.6 cm) of Sephadex G-100. The column was eluted using ultrapure water and the flow rate was 0.6 mL/min. The average molecular weight was detected by calibration curve, and the curve was established using the Dextran standards (known molecular weights: 11,600; 48,600; 80,900; 147,600; 273,000; 667,800; 1,185,000).

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2.4. Endotoxin detection

stimulation cultures divided by the absorbance value for the nonstimulated cultures.

The prepared CVPS was dissolved in saline at 50 mg/mL. The diluted preparations were sterilized using pasteurization and pyrogen tests were used to detect the presence of endotoxins. Solutions with endotoxin concentrations that met the standard of the Chinese Veterinary Pharmacopoeia (less than 0.5 EU/mL) [25] were stored at 4 ◦ C for subsequent experimental tests. 2.5. Animals and immunization Female imprinting control region (ICR) mice (Grade II, 5 weeks old) weighing 18–22 g were purchased from the Sichuan Laboratory Animal Centre (SLAC) Co., Ltd. (Sichuan, China). The mice were housed in polypropylene cages with sawdust bedding in hygienically controlled environments. Fifty mice were randomly divided into five groups: a naive group, a FMDV group, a FMDV plus CVPS group, a FMDV plus alum group, and a CVPS group. On day 0 and 14, the animals were immunized subcutaneously in the hind limb with the vehicles listed in Table 1. 2.6. Antigen-specific antibody measurements

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The FMDV-specific IgG titres and isotypes were determined using an indirect enzyme-linked immunosorbent assay as previously described [26,27]. Briefly, the wells of a polyvinyl 96-well microtitre plate were coated with 100 ␮L of FMDV solution and incubated overnight at 4 ◦ C. After three washes, the wells were blocked with 5% skimmed milk and incubated at 37 ◦ C for 1 h. After washing three times, 100 ␮L of serum (diluted serially for IgG titre analysis or diluted 1:50 in PBS 5% skimmed milk for isotype analysis) was subsequently added into each well, and the mixture was incubated at 37 ◦ C for 1 h. Horseradish peroxidase-conjugated antibodies against IgG, IgG1, IgG2a, or IgG2b were incubated for 1 h at 37 ◦ C. After washing, the peroxidase activity was assayed as follows: the substrate solution (10 mg O-phenylenediamine and 37.5 ␮L of 30% H2 O2 in 25 mL of 0.1 M citrate–phosphate buffer, pH 5.0) was added into each well and the mixture was incubated for 10 min at 37 ◦ C. The reaction was stopped with 2 M H2 SO4 . The optical density (OD) was measured using a microplate absorbance reader at 450 nm. The data are expressed as the mean sample ODs minus the mean control ODs.

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2.7. Splenocyte proliferation assay

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On day 14, after the booster immunization, the spleens of the FMDV-immunized ICR mice were collected under aseptic conditions. Single splenocyte suspensions were prepared and a splenocyte proliferation assay was performed according to previously described protocols [27]. T cell proliferation was then determined using a CCK-8 assay (Beyotime, Haimen, China) according to the manufacturer’s instructions. The ODs were determined at 450 nm using a microplate reader (Bio-Red). The data are expressed as the stimulation index (SI), which was calculated based on the following formula: SI = the absorbance value for the antigen

Table 1 Animal grouping. Group

Vaccine

Naive FMDV FMDV + CVPS FMDV + alum CVPS

Naive 200 ␮L FMDV vaccine 200 ␮L FMDV vaccine 200 ␮L FMDV vaccine

Adjuvant

0.5 mg CVPS 200 ␮g alum 0.5 mg CVPS

2.8. Phagocytosis assay The mice were injected intraperitoneally with 1.0 mL of 2% starch solution on day 3 after the first immunization. After 24 h, 1.0 mL of 1% chicken red blood cell solution was also injected intraperitoneally. After 30 min, a drop of peritoneal fluid was stained by Wright’s stain and was observed under a high-powered microscope. The results are expressed as the phagocytic cell percentage (PP) and the phagocytic index (PI). PP was defined as the percentage of peritoneal macrophages (PM) that ingested one or more chicken red blood cells. PI was defined as the average number of chicken red blood cells ingested per PM, which was calculated by dividing the total ingested chicken red blood cell number by the total PM number (200 cells). 2.9. In vivo CTL assay An in vivo cytotoxic assay was performed as previously described [28]. Briefly, splenocytes were isolated from naive ICR mouse spleens after removing the erythrocytes and divided them into two equal portions. One portion was pulsed with 50 ␮g of FMDV for 4 h and then labeled with a high concentration of CFSE (2.5 ␮M, CFSEhigh cells) which served as the target cells [29]. The other portion was labelled with CFSE at a low concentration (0.25 ␮M, CFSElow cells) without the FMDV pulse and was utilized as the non-target control. The control and target cells were mixed at a 1:1 ratio and were injected into the immunized mice via the tail vein at 2 × 107 total cells per mouse on day 14 after the second immunization. The mice were sacrificed after 4 h to isolate their splenocytes, which were then analysed on a FACS Calibur analyser (BD Biosciences, USA). Specific lysis was calculated using the following formula: ratio = percentage CFSElow /percentage CFSEhigh . Percentage specific lysis = [1 − (ratio unprimed/ratio primed) × 100]. 2.10. In vitro natural killer (NK) assay The cytotoxic activity of NK cells was measured as previously described [30,31]. Briefly, splenocytes that were prepared as described above were used as the effector cells and were seeded in 96-well microtiter plates at 1 × 106 cells/mL. K562 cells labelled with CFSE were used as target cells, which were added at 2 × 104 cells/mL in 100 ␮L of RPMI 1640 complete medium to obtain a T/E ratio of 1:50. The plates were then incubated for 4 h at 37 ◦ C in a 5% CO2 atmosphere. Then, 2.5 ␮L of propidium iodide (PI) solution (100 ␮g/mL) was added into each well and the plate was incubated for another 10 min. The viability of the CFSE + PI + cells was determined using FACS Calibur. Three types of control measurements were performed, which included target cell control, blank control, and effector cell control measurements. 2.11. Target gene quantification by real-time PCR Total RNA was extracted from splenocytes on day 3 after the second vaccination using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The total RNA (intact rRNA 28s/18s) was tested using formaldehyde agarose gel electrophoresis. Additionally, total RNA concentrations were quantified at 260 nm using a ND1000 nano drop spectrophotometer (Nano Drop Technologies, Wilmington, USA). First-strand cDNA was reverse transcribed using a PrimeScript® RT reagent kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Dalinan, China), according to the manufacturer’s instructions.

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Table 2 The primer sequences used for real-time quantitative RT-PCR. Target gene

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Product size

Accession No.

MHCI

Sense Antisense

GCCCAAGAAGTGGATTACGGAG TCCAGAACCATTTGGCGACC

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NM 008209

MHCII

Sense Antisense

CTGTCTGGATGCTTCCTGAGTTT CAGCTATGTTTTGCAGTCCACC

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NM 207105.3

TLR-2

Sense Antisense

AGAAGATGACACCGACGAGGC AGTCTTCCGCACGGCATCTC

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NM 173394

TLR-4

Sense Antisense

TACCTGGAATGGGAGGACAATC CAGGTCCAAGTTGCCGTTTC

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NM 021297

TGF-␤

Sense Antisense

ACCTGCAAGACCATCGACAT GGTTTTCTCATAGATGGCGT

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NM 011577.1

␤-Actin

Sense Antisense

CGGTTCCGATGCCCTGAGGCTC CAGCAACAGCAAGGCGAA

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NM 007393

Relative target gene quantification was conducted on a CFX96 real-time PCR detection system (Bio-Rad) using SYBR green. The primer sequences of the target and reference genes are presented in Table 2. The PCR cycling conditions were as follows: initial denaturation at 95 ◦ C for 1 min; followed by 40 cycles of denaturation at 95 ◦ C/10 s; annealing at 58–61 ◦ C (seen in Table 1 for different genes)/25 s; and a final melting curve analysis to monitor the PCR product purity. The transcript copy numbers were normalized against the copy numbers of ␤-actin transcripts for each sample. The relative quantification of each gene was determined using a standard curve method for both the target genes and endogenous references, and this quantification was expressed as fold change relative to mRNA levels in the control groups.

2.12. Intracellular cytokine staining For the intracellular cytokine assay, mice were sacrificed 14 d after the second immunization. Splenic T cell suspensions were prepared to 0.5 × 106 cells/200 ␮L and were stimulated with FMDV (5 ␮g/mL) in 96-well plates for 6 h at 37 ◦ C under a 5% CO2 atmosphere. Monensin (2 ␮g/mL) was subsequently added for 4 h, and the mixture was then washed three times with PBS/10% FCS. The cells were blocked with 1 ␮L of Fc␥ mAb (0.5 ␮g/mL) for 30 min at 4 ◦ C and fixed with 4% paraformaldehyde at 4 ◦ C for 15 min. The cells were permeabilized with 0.1% saponin at 4 ◦ C for 10 min. After washing once with PBS, the cells were stained with IgG isotype controls, or double stained for 30 min at 4 ◦ C with anti-CD4FITC and anti-IL-4-PE, anti-CD4-FITC and anti-IL-2-PE, anti-CD4-PE and anti-IFN-␥-FITC, anti-CD8-PE and anti-IFN-␥-FITC, or anti-CD4FITC. The cells were analysed using a FACS Calibur and the Cellquest Prosoftware (BD Bioscience, USA). The CD4+ CD25+ Foxp3+ Treg cell frequency was tested using a mouse regulatory T cell staining kit according the manufacturer’s instructions (eBioscience, San Diego, CA).

2.14. Statistical analysis

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Data analysis was performed using SPSS software (SPSS, Version 11.5, SPSS Inc., Chicago, IL), and analysed with a two-sided Student’s t-test; P-value differences of less than 0.05 were considered to be statistically significant.

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2.13. DC surface costimulatory molecules staining The mice were sacrificed on day 3 after the first immunization, and single-cell suspensions were prepared from the spleens and concentrated to 2 × 106 cells/200 ␮L. Cell suspensions were blocked with 2 ␮L of Fc␥ mAb (0.5 ␮g/mL) for 30 min at 4 ◦ C. After rinsing once with PBS, the cells were stained with isotype controls or double stained with anti-CD11c-FITC and anti-CD40-PE, anti-CD11c-FITC and anti-CD80-PE, or anti-CD11c-FITC and antiCD86-PE. The fluorescence intensities were measured using a FACS Calibur and analysed using the Cell Questpro software (BD Biosciences, USA).

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The physicochemical properties of CVPS are shown in Table 3. The CVPS solution was milky. The solubility test indicated that the CVPS was water soluble. The results of the phenol–sulfuric acid, ␣-naphthol, iodination, Fehling’s, carbazole, FeCl3 , and Coomassie brilliant blue tests suggested that the extractions were polysaccharides and contained some uronic acid, but did not contain starch, proteins, reducing sugar, or polyphenol. The full wavelength scanning analysis also confirmed that the CVPS had no proteins. 3.2. Chemical properties of polysaccharide In the present study, the average molecular weight of CVPS was 9.6831 × 105 Da, according to the calibration curve with standard dextrans. The uronic acid content was 7.2%, using the carbazole–sulfuric acid method. The total sugar content of the CVPS was 91.32%, using the Anthrone–sulfuric acid method, and the polysaccharide samples were not contaminated with proteins. The CVPS was hydrolyzed with TFA and a GC analysis of the hydrolysates was performed using the precolumn-derivatization techniques with aldononitrile acetate to identify the component monosaccharides released from the polysaccharide. The CVPS contained a neutral monosaccharide composition of rhamnose,

Method

CVPS

Color observation Solubility test Iodination reaction ␣-Naphthol reaction Phenol–sulfuric acid test Uronic acid carbazole reaction Fehling’s test Peak at UV 280 nm FeCl3 reaction Coomassie brilliant blue reaction

Milky (−)a (−) (+)b (+) (+) (−) (−) (−) (−)

a b

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Table 3 The physicochemical properties of the CVPS. 306

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Negative. Positive.

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Fig. 1. FTIR spectra of CVPS.

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arabinose, fucose, mannose, glucose, and galactose with a relative mass of 6.29%, 21.87%, 16.59%, 12.54%, 13.07%, and 28.05%, respectively.

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3.3. FTIR spectroscopy

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The FTIR spectra of the CVPS in the ranges of are illustrated in Fig. 1. The spectra of CVPS revealed obvious characteristic absorption peaks of the polysaccharide. The broadly stretched peak at around 3424.86 cm−1 corresponded to the O–H stretching vibrations, and the band at around 2936.93 cm−1 had the characteristic absorption of a weak C–H stretching vibrations. The absorption peak at around 1631.24 cm−1 was attributed to the C O asymmetric stretching vibration. In addition, in the region of 1000–1200 cm−1 , the CVPS had a specific absorption band which was dominated by ring vibrations overlapped with the (C–O–C) glycosidic band vibrations and (C–OH) stretching vibrations of side groups. The absorption peaks at 1022.64 cm−1 , 1079.96 cm−1 and 1149.52 cm−1 suggested that the CVPS was a pyranose form of sugar [32]. The absorptions peaks at around 846.68 cm−1 and 912.17 cm−1 revealed that the polysaccharide contained both ␣and ␤-type glycosidic bond in their structure [33].

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3.4. The effect of CVPS on the humoral response

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Fourteen days after the second immunization, serum was collected from each animal and the FMDV-specific IgG, IgG1, IgG2a, and IgG2b antibodies were measured using indirect ELISAs to

evaluate the effect of CVPS on the humoral immune response. Fig. 2A shows that the FMDV-specific IgG OD values were significantly higher in the mice immunized with FMDV + CVPS compared with those immunized with FMD vaccine alone or FMDV + alum (P < 0.05). Moreover, all of the IgG isotypes tended to be higher in mice that were co-administered CVPS compared with the control group mice (Fig. 2B). These findings indicate that CVPS significantly enhanced FMDV-specific antibody production in the immunized mice. 3.5. The effect of CVPS on splenocyte proliferation To measure the effect of the CVPS treatment on the cellular immune response, lymphocytes were isolated from the immunized mice on day 14 after their second immunization. The effects of CVPS on splenocyte proliferation in the immunized mice are shown in Fig. 3. The SI of proliferation in response to FMDV was increased in mice administered CVPS compared with the FMDV group mice (P < 0.05). The results suggest that using CVPS as an adjuvant enhances splenocyte proliferation. 3.6. The CVPS effects on PM phagocytic function The effect of CVPS on PM phagocytosis is listed in Table 4. Compared with the FMDV group, both PP and PI fluorescence were significantly higher in the FMDV plus CVPS group (P < 0.01) and the CVPS alone group (P < 0.01). Therefore, CVPS significantly enhanced the phagocytic function of PM in the immunized mice.

Fig. 2. The effect of CVPS on FMDV-specific IgG and IgG isotypes in the FMD-immunized mice. Serum samples were collected on day 14 after the final immunization. FMDV-specific IgG (A), IgG1, IgG2a, and IgG2b (B) levels were measured using ELISA, as described in the text. The values are presented as the mean ± S.D. (n = 10). Significant differences between the FMDV groups are designated as *P < 0.05 and **P < 0.01.

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3.9. The effect of CVPS on NK cell activity NK cells are functionally similar to cytotoxic T lymphocytes and NK cells are part of the first line of innate defence against cancer and virus-infected cells [34]. The NK cell activity assay is a routine method for analysing cellular immune response in vitro. Therefore, we measured the cytotoxic activity of splenocytes against NK cell-sensitive K562 cells using FACS. As shown in Fig. 6, CVPS significantly enhanced the NK cell killing activity in the FMDV-immunized mice. These findings indicate that CVPS promotes NK cell lytic activity. 3.10. The effects of CVPS on DC maturation

Fig. 3. The effect of CVPS on splenocyte proliferation in the FMDV-immunized mice. Single splenocytes were isolated 14 days after the final immunization. Splenocyte proliferation was measured via the WST-8 method, as described in the text, and shown as a stimulation index. The values are presented as the mean ± S.D. (n = 10). Significant differences with the FMDV groups were designated as *P < 0.05 and **P < 0.01.

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The cytokine profile of the immune response is an important indicator of the CD4+ T helper–bias. Single mouse splenocytes were isolated on day 14 after the second immunization and were cocultured with the FMDV antigen. To measure the effect of CVPS on the FMDV-specific Th cell response, cytokine expression in CD4+ cells was examined through intracellular staining. The findings revealed that CVPS induced the highest level of IL-2, IL-4, and IFN-␥ expression in antigen-specific CD4+ T cells (Fig. 4A, C and E). Moreover, a robust antigen-specific IFN-␥ response in CD8+ T cells from mice immunized with the FMD vaccine plus CVPS was also observed (Fig. 4G). IFN-␥ production in the CD8+ T cells from the mice immunized with FMDV plus CVPS was significantly higher than that in the other groups. These findings suggest that using CVPS as an adjuvant enhances both the Th1 and Th2 responses.

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3.8. The effect of the CVPS on CTLs

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DC maturation and activation are crucial and the earliest events in immune regulation that highlight the subsequent adaptive immunity [35,36]. Thus, we evaluated the effects of CVPS on DCs in mice after the first immunization. Costimulatory molecules (CD40, CD80, and CD86) were detected by FACS, and the mRNA levels of MHC molecules (MHCI/II) were determined using real-time PCR. The mice immunized with the FMDV vaccine plus CVPS induced the highest levels of CD40+ , CD80+ , and CD86+ expression on DCs compared with the other groups (Fig. 7). At the same time, MHC-I and MHC-II mRNA expression was upregulated in the FMDV + CVPS groups compared with the FMDV group (Fig. 7G). These results suggest that CVPS activates DCs and induces their maturation. 3.11. The effect of CVPS on TLR-2, TLR-4, TGF-ˇ, and Treg cells To further evaluate how CVPS modulates immune response as an adjuvant, TLR-2, TLR-4, and TGF-␤ mRNA levels were determined using real-time PCR and the Treg frequency was analysed using FACS on day 3 after the first immunization. As shown in Fig. 8, CVPS dramatically increased TLR-2 and TLR-4 expression, decreased TGF-␤ expression, and lowered the frequency of CD25+ Foxp3+ Treg cells in the mice that were immunized with FMDV vaccine plus CVPS compared with the other groups. These data suggest that CVPS enhances the immune response by increasing TLR-2 and TLR-4 expression and by inhibiting TGF-␤ expression and the Treg cell frequency. 4. Discussion

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To further determine whether CVPS influences CTL responses, the specific cytotoxic response was tested in vivo on day 14 after the second immunization. As shown in Fig. 5, antigen-specific lysis of target cells in mice immunized with FMDV plus CVPS reached 64.6%, whereas it was 15.8% in the FMDV alone group. Compared with the mice immunized with the FMDV vaccine alone, the mice immunized with FMDV plus CVPS significantly increased the FMDV-specific cytotoxic responses. These data suggest that CVPS increases antigen-specific CTL activities, which is consistent with the above finding regarding the high IFN-␥ expression levels in the CD8+ T cells (Fig. 4G).

Table 4 Q7 The effect of CVPS on peritoneal macrophage (PM) functions. Group Naive FMDV FMDV + CVPS FMDV + alum CVPS

PMs observed (number)

PP

200 200 200 200 200

0.534 0.583 0.757 0.622 0.539

PI ± ± ± ± ±

0.11 0.09 0.05** 0.05 0.18

0.84 0.89 1.37 0.95 0.86

± ± ± ± ±

0.03 0.11 0.23** 0.31 0.12

An ideal vaccine should elicit strong humoral and cellular immune responses. The humoral immune response of B cells is a specific antigen–antibody reaction [37], whereby a strong antibody response may prevent FMDV from entering the host and neutralizing the virus in the serum. In the present study, co-administration of CVPS with the FMD vaccine significantly increased IgG and IgG titres, which suggests that CVPS helps the FMDV vaccine induce strong humoral immune responses to confer protection against FMDV. These observations are similar to previously reported studies. For example, Liang et al. reported that Taishan Robinia pseudoacacia polysaccharides (TRPPS) act as an adjuvant and can promote the humoral responses to ND vaccine [38]. Zhang also confirmed that Ganoderma lucidum polysaccharide (GLP) increased the ND antibody titer, indicating that GLP can significantly enhance the humoral immunity in response to ND vaccine [39]. Cellular immune responses play a central role in the host defence system against infections by accelerating the clearance of pathogens and producing cytokines for regulating immune responses [40]. The splenocyte proliferation response is an indicator of cellular immunity in animals. Abula et al. demonstrated that co-administering the Siberian solomonseal rhizome polysaccharide (SRPS) with the ND vaccine in chickens dramatically promoted the levels of lymphocyte proliferation, suggesting that

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Fig. 4. Antigen-specific cytokine production analysis in T cells by FACS. T cells isolated from the spleen of ICR mice after the final booster dose were stimulated with FMDV for 4 h in vitro. The cells were double-stained with anti-CD4 plus anti-IL2 (A), anti-CD4 plus anti-IL4 (C), anti-CD4 plus anti-IFN-␥ (E), or anti-CD8 plus anti-IFN-␥ (G) and then analysed using FACS. The percentage of IL-2 in the total CD4+ cells, IL-4 in the total CD4+ cells, IFN-␥ in the total CD4+ cells, and IFN-␥ in the total CD8+ cells are shown in (B), (D), (F), and (H), respectively. Significant differences between the FMDV groups are designated as *P < 0.05 and **P < 0.01.

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Fig. 5. FMDV-specific cytotoxicity analysis in vivo by FACS. 1 × 107 CFSE-labelled target cells at a CFSEhigh to CFSElow of 1:1 ratio from naive ICR mice were IV injected into syngeneic mice boosted with FMDV on day 14 after the second immunization. After 4 h, the mice were sacrificed and the CFSE-labelled cells were analysed for ratio changes in the CFSEhigh (M2) and CFSElow (M1) target cell populations. The percentage of specific lysis was determined for each group as described in Section 2.

Fig. 6. NK cells activity analysis by FACS. Splenocytes were prepared and assayed for NK cell activity using FACS. The percentage of non-specific lysis was determined for each group as described in Section 2.

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Fig. 7. Dendritic cell maturation and MHC-I and MHC-II mRNA expression analysis. Total splenocytes were isolated from the spleens of ICR mice on day 3 after the first immunization. Double staining for CD40+ CD11c+ (A), CD80+ CD11c+ (C), and CD86+ CD11c+ (E) in the cells was performed. The percentages of double-positive cells in the dot plots are shown in the upper right corner of the quadrant, and the gates were set on the CD11c+ cells or events. The percentage of CD40+ cells in the total CD11c+ cells, CD80+ cells in the total CD11c+ cells, and CD86+ cells in the total CD11c+ cells are shown in (B), (D), and (F), respectively. MHC-I and MHC-II mRNA expression (G) was detected using real-time quantitative RT-PCR. The relative mRNA expression levels are presented as the mean ± S.E. (n = 10). Significant differences between the FMDV groups were designated as *P < 0.05 and **P < 0.01.

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Fig. 8. Treg frequency and TLR-2, TLR-4, and TGF-␤ mRNA expression. Splenocytes were isolated from ICR mice on day 3 after the first immunization. The frequencies of CD25+ , Foxp3+ , and CD4+ cells were measured using FACS (A and B). RNA was extracted from the splenocytes of immunized mice 3 d after the first immunization. TLR-2, TLR-4, and TGF-␤ mRNA expression (C and D) was detected using real-time quantitative RT-PCR. The relative mRNA expression levels are presented as the mean ± S.E. (n = 10). Significant differences between the FMDV groups are designated as *P < 0.05 and **P < 0.01. The gel analysing the PCR products is shown as a representative of three independent experiments.

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SRPS enhanced cellular immunity [41]. In the present study, CVPS significantly increased the splenocyte proliferation response in immunized mice, which indicates that CVPS promotes cellular immune responses to the FMD vaccine. T lymphocytes and their subpopulations (CD4+ and CD8+ T cells) play a crucial role in eradicating viruses and preventing viral persistence, and the number of CD4+ and CD8+ T cells is an index for the immune response in the body [42]. If the proportion of CD4+ and CD8+ T cells subpopulations increase, the immune responses will also be promoted. CD8+ T cells mediate pathogen clearance and activated CD8+ T cells accomplish various functions, including recognizing and killing infected host cells and secreting antiviral cytokine such as IFN-␥ and TNF-␣ [43]. CD4+ T cells proliferate, acquire a Th1 or Th2 phenotype, and secret cytokines that regulate the activation of the immune responses and sustain cytotoxic T cell responses [44]. In general, Th1 cells secrete high amounts of IFN-␥, IL-2, TNF-␣, and IL-12, which stimulate lymphocyte proliferation and division, facilitate DC maturation and are correlated with the induction of cell-mediated immunity [45,46]. IL-2 drives immunity towards Th1-biased responses to improve cell-mediated responses [47]. Th2 cells secrete high IL-4, IL-5, and IL-10 amounts, which enhance antibody production to help Th2-based immune responses [48]. An ideal adjuvant can induce both Th1 and Th2

responses to initiate protective immunity against certain infectious diseases. In the current study, co-administering CVPS with the FMD vaccine induced high IFN-␥, IL-2, and IL-4 levels in CD4+ T cells and high levels of IFN-␥ in CD8+ T cells (Fig. 4), which indicates that CVPS improves both Th1- and Th2-based immune responses. Many other studies have also shown that Chinese herbal polysaccharides were better at enhancing cytokine release and inducing a balanced Th1/Th2 response, which was consistent with our experimental results [49–51]. Specific and non-specific cytotoxic responses have been previously demonstrated as key factors in protecting against infectious diseases and cancer [52]. NK cells play a major role in the rejection of tumours and virus-infected cells. NK cell killing activity is relatively non-specific and kills cells by inducing apoptosis. However, CTL killing activity is antigen-specific and requires the recognition of antigens/MHCs on the target cell [53]. CTL and NK cell activity assays are commonly used to measure levels of cellular immune responses. Wang and Yang et al. reported that rats treated with a water-soluble polysaccharide from the roots of Salvia miltiorrhiza Bunge showed significantly increased NK cell and CTL activity [54]. In this study, co-administering CVPS with the FMDV vaccine significantly increased the killing activity of CTLs and enhanced NK cell function in CVPS-treated mice (Figs. 5 and 6), which indicates

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that CVPS improves the specific and non-specific cytolytic activity against autologous infected cells and viruses. DCs, as antigen presenting cells, play a key role in the immune system by sensing foreign antigens and triggering the adaptive immune response. DC maturation and activation are the earliest crucial events in immune regulation and initiating adaptive immunity [55]. Mature DCs are characterized by their decreased antigen uptake and processing, high surface MHC-I and MHC-II expression, and a high capacity to prime T cells [56]. The expression of co-stimulatory molecules (CD40, CD80, CD86) on DCs is typically correlated with T lymphocyte differentiation and the upregulation of CD80, CD86, CD40, which reinforces T cell activity [57,58]. Engel et al. confirmed that protein-bound polysaccharide-K (PSK) extract from Trametes versicolor mushrooms can enhance cellular and humoral immune responses to OVA vaccine in mice via activation of DCs and lead to their maturation [59]. Du et al. in 2012 and Zhang et al. in 2010 reported that Astragalus polysaccharide acts as an adjuvant and can promote humoral immune and cellular responses in mice, and can encourage DCs to maturation characterized by higher CD80, CD40, CD86, MHC molecules expression [28,60]. In the present study, CVPS significantly increased the mRNA expression of MHC molecules as an adjuvant to the FMDV vaccine. At the same time, CVPs significantly enhanced the costimulatory molecule expression (CD40, CD80, and CD86). Therefore, CVPS facilitates the maturation of DCs when used as an adjuvant. This may be one of the ways in which CVPS promotes immunity. Recent studies have demonstrated that Foxp3, the forkhead/winged helix transcription factor, is crucial for the development and function of CD4+ CD25+ Treg cells which have a regulatory role in immunologic suppression [61–64]. Blockade or reduction of CD4+ CD25+ Treg cells enhances both humoral and cellular responses and provides a balanced immunity after vaccination [65,66]. Transforming growth factor-␤ (TGF-␤) is an immunosuppressive factor and may be responsible for the impaired regulatory function of CD4+ CD25+ Treg cells [67]. Du et al. in 2011 observed that APS served as an adjuvant and decreased the frequency of CD4+ CD25+ Treg cells, down-regulated the mRNA expression of TGF-␤, evoked both humoral and cellular responses and maintains balanced immunity in HBV vaccination [15]. In our study, co-administration of CVPS with the FMDV vaccine significantly decreased TGF-␤ mRNA expression and the frequency of CD4+ CD25+ Foxp3+ cells (Fig. 8). These results show that CVPS increases the immune response by inhibiting negative signals. Toll-like receptors (TLRs) are a crucial class of patternrecognition receptors that are mostly expressed in innate immune cells such as DCs, NK cells, macrophages, and mast cells [68]. TLR-2 and TLR-4 are important innate immune signalling molecules that play a central role in both innate and adaptive immune responses [69]. Engel et al. reported that protein-bound polysaccharide-K (PSK) from Trametes versicolor mushrooms can activate Toll-like receptor 2 (TLR2) and promote humoral and cellular immune responses to OVA vaccine in mice [59]. Du et al. found that APS act as an adjuvant for the hepatitis B subunit vaccine, and they increased both humoral and cellular immune responses by activating the TLR4 signalling pathway [15]. In the present study, we found that CVPS significantly increased TLR-2 and TLR-4 mRNA expression in total splenocytes in vivo (Fig. 8), which indicates that CVPS likely improves the activation of the innate immune system through the TLR-2 and TLR-4 signalling pathways. In conclusion, CVPS can be used as an effective adjuvant for enhancing both humoral and cellular responses to the foot-andmouth disease vaccine by inducing DC maturation, activating the innate signalling pathway, and inhibiting negative signals. These data provide a scientific basis for the potential application of CVPS as a novel adjuvant for vaccine design.

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