INTIMP-03207; No of Pages 7 International Immunopharmacology xxx (2014) xxx–xxx

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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp

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Xiaoyan Su, Zengyang Pei ⁎, Songhua Hu

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Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, PR China

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a r t i c l e

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Article history: Received 24 January 2014 Received in revised form 25 February 2014 Accepted 10 March 2014 Available online xxxx

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Keywords: Ginsenoside Re Adjuvant effect Rabies Th1/Th2

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Ginsenoside Re as an adjuvant to enhance the immune response to the inactivated rabies virus vaccine in mice

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The inactivated rabies virus vaccine (RV) is a relatively expensive vaccine, prone to failure in some cases. Ginsenoside Re (Re) is a saponin isolated from Panax ginseng, and has an adjuvant property. Here the adjuvant effect of Re to improve the immune response to the RV is evaluated in mice. ICR mice were immunized with saline, 2.50 mg/kg Re, 20 μl RV, 100 μl RV, or 20 μl of RV adjuvanted with Re (1.25, 2.50 or 5.00 mg/kg). Different time points after boosting, we measured serum antibodies in blood samples and separated splenocytes to detect lymphocyte proliferation and the production of IL-4, IL-10, IL-12, and IFN-γ in vitro. We also compared immunizations containing 20 μl RV and 20 μl RV adjuvanted with Re (5.00 mg/kg) for the expression of CD4+ and CD8+ T-cell subsets at different time points. Results indicated that co-administration of Re significantly enhanced serum antibody titers, increased the CD4+:CD8+ ratio, and enhanced both proliferation responses and IL-4, IL-10, IL-12 and IFN-γ secretions. Both Th1 and Th2 immune responses were activated. The supplementation of the Re (5.00 mg/kg) to 20 μl of RV significantly amplified serum antibody responses and Th1/Th2 responses inducing similar protection as did 100 μl of RV. This suggests that Re could be used to reduce the dose, and therefore the cost, of the RV to achieve the same effective protection. Re merits further studies for use with vaccines of mixed Th1/Th2 immune responses. © 2014 Published by Elsevier B.V.

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1. Introduction

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The rabies virus causes a fatal illness characterized by encephalopathy and generalized paresis [1]. It remains a significant threat to human and animal health throughout much of the world [2,3]. Globally, more than 55,000 people die from the infection of the virus each year, with about 99% of those deaths occurring in the developing countries, mainly in Asia and Africa [4,5]. Recent studies conducted in China reported approximately 3000 human deaths from rabies annually, which make China become second only to India worldwide in the number of rabies-related death [6,7]. The domestic dog plays a pivotal role in rabies transmission which accounts for more than 95% of human rabies cases in China [6]. Consequently, the vaccination of dogs against rabies is believed to be one of the most effective approaches for the control of the disease and its transmission to humans [8,9]. Research has shown that the vaccination of 70% of dogs should be sufficient to prevent epidemics and eliminate endemic rabies infections [10]. However, given the fact that there are approximately 200 million dogs in China [6,11] and no definitive dog

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⁎ Corresponding author at: Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, PR China. Tel.: + 86 571 86971347. E-mail address: [email protected] (Z. Pei).

rabies surveillance system [12], the implementation of such a level of vaccination coverage seems some way off [13]. Most of the commercially available rabies vaccines currently in use for dog prophylaxis in China are inactivated cell-culture vaccines. Compared to many live vaccines, the inactivated rabies vaccine (RV) is generally safe and convenient for use and storage but it suffers from a significant failure rate. Even for dogs that are RV immunized, it is known that the rabies vaccination does not protect humans and animals from rabies in all cases and vaccine failure sometimes occurs where the vaccine is unable to induce a long-lasting protective immune response in some cases. For example, a retrospective study in North America suggested that Alaskan dogs receiving a single injection of RV failed to achieve antibody titers above 0.5 IU/ml in 27% of cases on day 60, 24% on day 180, and 33% on day 360 after the immunization [14]. Minke et al. [15] analyzed serum samples of laboratory dogs having received commercially available RV and found that only 67% of dogs had sufficient antibody titers required for immune protection immunity on day 28. Thereafter the proportion of dogs with protective antibody titers dropped significantly, with only 7% remaining on day 120 after vaccination. Therefore, though increasing dog vaccination coverage rates remains an important future target for China, in the meantime improving the immune effectiveness of rabies vaccines for individually immunized dogs could be a more realistic short term goal to aid in the control rabies transmission between immunized and unimmunized dogs and thereafter to prevent dog-bite acquired rabies in humans.

http://dx.doi.org/10.1016/j.intimp.2014.03.008 1567-5769/© 2014 Published by Elsevier B.V.

Please cite this article as: Su X, et al, Ginsenoside Re as an adjuvant to enhance the immune response to the inactivated rabies virus vaccine in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.03.008

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A commercially available rabies vaccine of Rabvac™ 3 Rabies was used in this study (Zoetis Inc., USA). It is a monovalent inactivated rabies vaccine, containing the Street Alabama Dufferin/HCP-SAD strain at ≥ 1.5 IU/dose (106.3 FAID50/ml). The virus had been cultivated in cell line FKCU and was inactivated with beta-propiolactone. Re was extracted from the stem and leaf of P. ginseng C. A. Meyer, which had been purchased from Hongjiu Ginseng Industry Co., Ltd. (Jilin, China) as a white powder with 98% purity and a molecular weight of 947. The Re was first dissolved in dimethylsulfoxide (DMSO), diluted with physiological saline solution (1000 μg/ml), and sterilized by passing it through a 0.22 μm filter. The endotoxin level in the above solutions was less than 0.5 endotoxin unit (EU)/ml as determined by a gel-clot Limulus amebocyte lysate assay (Zhanjiang A&C Biological Ltd., Zhanjiang, China). FITCconjugated anti-mouse CD4 (GK1.5) and anti-mouse CD8a (53-6.7) antibodies, combined with phycoerythrin-conjugated antibodies were purchased from eBioscience (San Diego, CA, USA).

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2.2. Mice and immunization

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2.2.1. Mice Female ICR mice (18-22 g) were obtained from Shanghai Laboratory Animal Center and randomly assigned to experimental groups generally consisting of between six to twenty mice in each group. They were housed in polypropylene cages with sawdust bedding in a hygienically controlled environment. The temperature was controlled at 24 ± 1 °C and humidity at 50 ± 10%. Feed and water were supplied ad libitum.

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2.2.3. Immunization experiment 2 Sixty-four ICR mice were randomly distributed into two groups of 32. Each of the animals was subcutaneously immunized on days 0, 7 and 21 with 20 μl of RV (group 1) or 20 μl of RV adjuvanted with Re of 5.00 mg/kg (group 2). At two, four, eight and sixteen weeks after the boost, blood samples from each group (n = 8 at each time period), were randomly collected for measurement of rabies-specific serum antibodies titers.

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2.2.2. Immunization experiment 1 Forty-two ICR mice were randomly distributed into seven groups of six. Each mouse was subcutaneously injected on day 0 and again on day 21 with saline (group 1), 2.50 mg/kg Re (group 2), 100 μl RV (group 3), 20 μl RV (group 4), 20 μl RV adjuvanted with Re of either 1.25 mg/kg (group 5), 2.50 mg/kg (group 6), or 5.00 mg/kg (group 7). Two weeks after boosting, six blood samples from each group were collected to measure the rabies-specific serum antibody (Ab) titers. Splenocytes were prepared to determine the cellular proliferation and production of IL-4, IL-10, IL-12, and IFN-γ.

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All procedures related to the animals and their care conformed to the in- 139 ternationally accepted principles as found in the Guidelines for Keeping 140 Experimental Animals issued by the government of China. 141

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2.2.4. Immunization experiment 3 Forty ICR mice were randomly distributed into two groups of 20. Each of the animals was subcutaneously immunized on day 0, again on day 21 with 20 μl RV (group 1) or 20 μl RV adjuvanted with Re of 5.00 mg/kg (group 2). Two and four weeks after the boost, blood samples from each group (n = 10 at each time period) were randomly collected for immunofluorescence analysis of CD4+ and CD8+ T-cell subsets.

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2.3. Measurement of serum antibodies

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Serum samples were analyzed to measure the rabies-specific antibodies using a commercially available ELISA kit (Zoetis Inc., USA) according to the manufacturer's instructions. Positive and negative control sera were provided in the kit. Briefly, serum samples (1:100) were incubated in plates pre-coated with the rabies virus antigen at 37 °C for 1 h. After four washes, horseradish peroxidase-conjugated protein A was added and incubated at 37 °C for another 1 h. The plate was washed again followed by the addition of TMB substrate. The optical density of the plate was recorded at 450 nm. Sera titers were expressed as equivalent units per ml (EU/ml) corresponding to international units by using the values obtained by the WHO reference serum.

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2.4. Lymphocyte proliferation assay

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Spleens were collected from the ICR mice under aseptic conditions. They were placed in Hank's balanced salt solution (Sigma), minced, and passed through a fine steel mesh to obtain a homogeneous cell suspension. After centrifugation (380 ×g at 4 °C for 10 min), the pelleted cells were washed three times in PBS and resuspended in a complete medium (RPMI 1640 supplemented with 0.05 mM 2mercaptoethanol, 100 UI/ml penicillin, 100 μg/ml streptomycin, and 10% heat inactivated FCS). Cell numbers were then counted with a hemocytometer by the trypan blue dye exclusion technique. Splenocytes were seeded into a 96-well flat-bottom micro-titer plate at 5.0 × 106 cell/ml in 100 μl of complete medium. Concanavalin A (ConA; final concentration = 5.00 μg/ml), LPS (final concentration = 7.50 μg/ml), or medium was then added to a final volume of 200 μl. The plates were incubated at 37 °C in a humid atmosphere with 5% CO2 for 2 days. All tests were carried out in triplicate. Cell proliferation was evaluated using the MTT method. In a typical evaluation, 2.5 μl of MTT solution (2 mg/ml) was added to each well 4 h before the end of

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In the development of suitable vaccine protocols and antigens, little attention has been paid to the potential of new vaccine adjuvants to enhance the host's immune response. Yet, in most cases, vaccines require the addition of an adjuvant to induce a protective and long-lasting immune response [16]. In addition, rabies vaccines derived from cultured cells are costly to produce and, in some cases, prohibitively expensive to purchase [17]. Our aim is to investigate the addition of Re as an adjuvant with the aim of improving the RV's effect and to examine the potential for Re addition to reduce the required RV immunization dose and thus reduce the cost of vaccination. The root of Panax ginseng C. A. Meyer as a traditional Chinese medicine has been used for at least 2000 years [18], and is believed to be a tonic to stimulate the body resistance against infections [19]. Ginseng saponins (ginsenosides) are believed to be the main pharmacologically active constituents of the plant [19,20]. Ginsenoside Re (Re) is one of the constituents, isolated from the root as well as the stem and leaf of P. ginseng [19,20]. Interest in Re for medical and veterinary vaccines has recently increased due to its many advantages, such as ready availability, low cost, high effectiveness, and low risk of side effects and toxicity. Studies of Re suggest that it possesses a broad range of biological activities. These include antidiabetic effects in obese mice, beneficial effects on cardiac function by suppressing electromechanical alternans (EMA) in rats, as well as significant antioxidant and antihyperlipidemic efficacies in diabetic rats [21,22]. Recent investigations on Re have demonstrated its adjuvant abilities to boost both cellular (Th1) and humoral (Th2) immune responses [23,24]. We previously found that Re also enhanced the immune responses to the model antigen ovalbumin (OVA) [25] and the influenza vaccine (H3N2) in mice [26]. Despite the effects of Re as an adjuvant has been assessed in several vaccines, to the author's knowledge, the immune effects of Re as an adjuvant to the widely used veterinary rabies vaccines have not been investigated. The present study aims to determine whether Re can act as an adjuvant to enhance the immune response to the inactivated rabies virus vaccine in mice.

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Please cite this article as: Su X, et al, Ginsenoside Re as an adjuvant to enhance the immune response to the inactivated rabies virus vaccine in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.03.008

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Table 1 Primers and their parameters used for RT-PCR.

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Forward: 5′-GAGACTCTTTCGGGCTTTTCG-3′ Reverse: 5′-CAGGAAGTCTTTCAGTGATGTGG-3′ Forward: 5′-CCAGTTTTACCTGGTAGAAGTGATG-3 Reverse: 5′-CTTGCTCTTATTTTCACAGGGGAG-3′ Forward: 5′-TTGCTGGTGTCTCCACTCATG-3 Reverse: 5′-GTCACAGGTGAGGTTCACTGTTTC-3′ Forward: 5′-GCTTTGCAGCTCTTCCTCATG-3′ Reverse: 5′-CTTCCACATCTATGCCACTTGAG-3′ Forward: 5′-AGCGGTTCCGATGCCCT-3′ Reverse: 5′-AGAGGTCTTTACGGATGTCAACG-3′

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2.7. Statistical analysis

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Splenocytes were seeded into a 24-well flat-bottom micro-titer plate at 5 × 106 in 2 ml of complete medium. ConA (final concentration = 5.00 μg/ml) was then added and the plates were incubated at 37 °C in a humid atmosphere with 5% CO2. After 24 h of treatment, cells were harvested by centrifugation (380 ×g at 4 °C for 10 min), washed with ice-cold PBS, and subjected to RNA extraction. Splenocytes (1 × 107) were lysed in 1 ml of RNAiso™ Plus reagent (Takara, China) and the total RNA was isolated according to the manufacturer's protocol. Total RNA was used and reverse transcription was performed by mixing 1 μg of RNA with 5 μl of iScript reagent (Bio-Rad) in a DEPC-treated tube. Nuclease-free water was added to a final volume of 20 μl. The reaction condition for reverse transcription was performed according to the manufacturer's protocol. Real-time quantitative PCR (RT-PCR) was performed on the ABI 7500 (PE Applied Biosystems, USA) using SYBR premix Taq™ (Takara, China). The housekeeping gene β-actin was used to correct for minor variations [27]. Primers used in this study were described in Table 1. 6-Carboxy-X-rhodamine (ROX; Molecular Probes, Eugene, USA) was used as a positive internal reference [28]. Given that SYBR Green I is not specific for the PCR product and binds to primer dimers formed nonspecifically during all PCR reactions, an optimum temperature was necessary to obtain the specific product for analysis, as previously described [29]. Each cytokine analyzed by SYBR Green I was examined by this melting temperature profile. The optimum temperature was the one that gave the maximum reading for the specific product when the nonspecific product could no longer be detected. The following experimental run protocol was used: denaturation program (95 °C for 2 min), amplification and quantification program repeated 40 times (95 °C for 15 s, 65 °C for 34 s), and melting curve program (95 °C for 15 s, 60 °C for 1 min). Amplification was carried out in a total volume of 20 μl containing 2 μl of cDNA template, 1 μl of form primer, 1 μl of reverse primer, 6 μl of nuclease-free water, 0.4 μl of ROX, and 10 μl of SYBR premix Taq™ (2×; Takara, China). The relative quantification between samples was achieved by the 2 − ΔΔCt method [30], and calculated by the software REST 2005 (provided by Eppendorf Company, German). It is reported as the nfold difference relative to the target gene mRNA expression of the calibrator group (group of mice immunized with saline).

Peripheral blood lymphocytes were isolated by centrifugation over lymphocyte separation medium. The mouse red blood cells were removed by lysis with an ACK lysis buffer. Immunofluorescence analyses of CD4+ and CD8+ T-cell subsets derived from immunized animals were performed with a Becton–Dickinson FACStar for fluorescenceactivated cell sorting (FACS). Single-cell suspensions were stained for 30 min at 4 °C with PE-conjugated anti-mouse CD4 (clone GK 1.5, eBioscience) or FITC-conjugated anti-mouse CD8a (clone 53-6.7, eBioscience) MAbs or buffer alone as described previously. After staining, the light scattering properties of a total of 5000 cells were analyzed through flow cytometry. Viable lymphocytes, as determined by their forward and 90 degrees light scattering properties, were then gated for analysis.

Data are expressed as the mean ± standard deviation (mean ± S.D.). An independent-sample t-test or one-way ANOVA followed by post hoc Bonferroni's multiple comparison test was used as appropriate to compare the parameters between groups. P values less than 0.05 were considered statistically significant.

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2.6. FACS analyses

3.1. Effect of Re on serum rabies antibodies

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incubation. The plates were centrifuged (1400 ×g, 5 min) and untransformed MTT was carefully removed by pipetting. To each well, 150 μl of a DMSO working solution (192 μl of DMSO with 8 μl of 1 N HCl) was added. The absorbance was evaluated using an ELISA reader at 570 nm, with a 630 nm reference, after 15 min. The stimulation index (SI) was calculated based on the following formula: SI = absorbance value for mitogen cultures divided by the absorbance value for nonstimulated cultures.

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Serum rabies antibodies were measured using a commercially ELISA kit to determine the adjuvant effect of Re on humoral immune responses. Fig. 1A shows that 20 μl of RV induced significantly lower RVspecific antibody titers (1:100) than 100 μl of RV (1:100) (P b 0.001). However, antibody titers were significantly higher in mice immunized with 20 μl of RV + any of the dose ranges of Re (from 1.25 to 5.00 mg/kg) than with 20 μl of RV alone. The highest antibody titer was found in the group adjuvanted with 5.00 mg/kg of Re. In the mice immunized with 20 μl of RV adjuvanted with Re at a dose of 5.00 mg/kg, RV-specific antibody titers had risen to levels where there was no significant difference between this and the 100 μl of RV alone group (P = 0.058) (Fig. 1A). Moreover, to determine the adjuvant effect of Re on humoral immune responses at different time periods, serum rabies antibodies were measured at two, four, eight, and sixteen weeks after the boost. Whereas the results of the antibody titers induced by the coadministration of RV with 5.00 mg/kg Re (co-admin) group and that of the RV alone group both reduced over time, the co-admin group's titer level continued to remain significantly higher than that of the RV alone group at all time points (Fig. 1B).

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As shown in Fig. 2, Re supplements (1.25, 2.50 or 5.00 mg/kg) to 20 μl of RV significantly enhanced the splenocyte proliferative responses to LPS compared with mice immunized with 20 μl RV alone. In the 5.00 mg/kg Re group, (but not in the 1.25 or the 2.50 mg/kg Re groups), there was a significantly enhanced splenocyte proliferative responses to ConA as compared with mice immunized with 20 μl RV alone (P = 0.010). Although there were no significant differences between the 1.25 or the 2.50 mg/kg co-admin group and the 20 μl RV alone group, the splenocyte proliferative responses to ConA was higher in co-admin groups than the 20 μl RV alone group. Both the 2.50 and 5.00 mg/kg Re groups evidenced a significantly higher increased of splenocyte proliferative responses to LPS as compared with mice immunized with 100 μl RV group (P = 0.010 and P b 0.001, respectively). Similarly, the splenocyte responses to ConA for the mouse groups immunized with 20 μl of RV with Re additions of any of the

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Please cite this article as: Su X, et al, Ginsenoside Re as an adjuvant to enhance the immune response to the inactivated rabies virus vaccine in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.03.008

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3.3. Effect of Re on mRNA expression of IL-4, IL-10, IL-12, and IFN-γ by splenocytes

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3.4. Effect of Re on CD4+ and CD8+ expression on peripheral blood

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To investigate the effect of Re on CD4+ and CD8+ expression on murine peripheral blood, peripheral blood lymphocytes were collected

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Rabies remains a very important public health problem in China and vaccination is believed to be the most efficacious and valuable tool in the prevention of the disease. It is an urgent requirement to investigate a novel adjuvant to improve the potency of current veterinary rabies vaccines based on previous findings that the vaccination does not protect animals from rabies in some cases. The present study demonstrated that Ginsenoside Re acted as an adjuvant to enhance the immune response to the inactivated rabies virus vaccine in mice. Compared with the control (mice given 20 μl of RV alone), the co-administration of Re with 20 μl RV vaccine induced significantly higher serum antibody response, increased the CD4+:CD8+ ratio, increased splenocyte proliferation in response to ConA and LPS, and increased IL-4, IL-10, IL-12 and IFN-γ production by splenocytes.

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To investigate the effect of Re on cytokine mRNA expression, RNA was isolated from mouse splenocytes at 2 weeks after inoculation. Cytokine mRNA was then quantified by real-time PCR (Fig. 3). Adjuvant addition resulted in enhanced levels of each cytokine. Both the 1.25 and 2.50 mg/kg Re groups induced a significant increase of IL-4 and IFN-γ expression as compared with mice immunized with 20 μl RV alone. Supplement of 2.50 mg/kg of Re significantly enhanced the expression of IL10 mRNA, (but not for IL-12 mRNA), when compared to the 20 μl RV alone group. The highest cytokine (IL-4, IL-10, IFN-γ, and IL-12) mRNA expression levels were found in the group given 5.00 mg/kg of Re. This 5.00 mg/kg of Re group exhibited a similar cytokine (IL-4, IL-10, IFN-γ, and IL-12) mRNA expression level as did the 100 μl of RV group (P N 0.05).

from mouse splenocytes at two and four weeks after inoculation. Expression of CD4+ and CD8+ T-cell subsets was then analyzed by flow cytometry (Fig. 4). The percentage of CD4+ lymphocytes was significantly higher (P b 0.001) in the co-administration of RV with Re (co-admin) group at two weeks compared with the RV administered alone (RV alone) group. However, the percentage of CD8+ cells was markedly lower in the co-admin group than that in the RV alone group at four weeks (P = 0.002). This was reflected in a higher CD4+:CD8+ ratio in the co-admin group (4.14, 5.7) in comparison with the RV alone group (3.50, 3.64) at two and four weeks, respectively.

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measured dosages (1.25, 2.50 or 5.00 mg/kg) now all showed values without any significant difference from the 100 μl RV alone group. Results of this study suggested that both T- and B-cells were activated by Re.

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Fig. 1. Effect of Re on serum rabies antibodies. Serum antibody titers were analyzed by ELISA. (A) Mice (n = 6 in each group) were subcutaneously injected on day 0 and again on day 21 with saline, 2.50 mg/kg of Re, 100 μl of RV, 20 μl of RV, or 20 μl of RV adjuvanted with Re (1.25, 2.50 or 5.00 mg/kg). The mice were bled two weeks after the last immunization. (B) Mice (n = 32 in each group) immunized by 20 μl RV or 20 μl RV adjuvanted with Re (5.00 mg/kg) on day 0, 7 and 21. The mice were bled two, four, eight, and sixteen weeks after the last immunization. The values are presented as the mean ± S.D. Bars with different lowercase letters are statistically different (P b 0.05), *P b 0.05.

Fig. 2. Effect of Re on lymphocyte proliferation. Mitogen-stimulated proliferation of splenocytes isolated from mice (n = 6 in each group) subcutaneously injected on day 0 and again on day 21 with saline, 100 μl of RV, 20 μl of RV, and 20 μl of RV adjuvanted with Re (1.25, 2.50 or 5.00 mg/kg). Splenocytes were prepared two weeks after the last immunization and cultured with ConA, LPS, or RPMI 1640. Splenocyte proliferation was measured by the MTT method as described in the text, and shown as a stimulation index. The values are presented as the mean ± S.D. Bars with different lowercase letters are statistically different for each treatment (P b 0.05), and bars with same uppercase letter ‘A’ are not statistically different for each treatment (P N 0.05).

Please cite this article as: Su X, et al, Ginsenoside Re as an adjuvant to enhance the immune response to the inactivated rabies virus vaccine in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.03.008

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times of experiment 2 should have remained consistent with those of the immunization times of experiments 1 and 3. We regret this experimental error but, as the results remain valid if we compare the data between the co-administration group and the RV alone group within Fig. 1B, we have retained and included them in our experimental data. That Re acts as an adjuvant to the amplified serum antibody response of antigens in mice is consistent with previous studies. When co-administered with the model antigen ovalbumin (OVA) in ICR mice, Re, at a dose range of 50 μg, promoted significantly higher IgG and IgG isotype responses than did OVA alone [25]. Song et al. also found that Re acts as an adjuvant to enhance serum specific IgG, IgG1, IgG2a and IgG2b responses in HI titers of the inactivated influenza vaccine (H3N2) in mice [26]. Qu et al. have reported an enhanced specific anti-rROP18 immunoglobulin with high levels of IgG antibody in mice injected with recombinant Toxoplasma gondii ROP18 protein antigen [31]. Su et al. also observed that Re significantly promotes IgG response and nuclear factor-kappa B expression in C3H/HeB mice injected with OVA [24]. The capacity to elicit an effective cellular immunity can be measured by lymphocyte proliferation. The lymphocyte proliferation assay depends on the mitogen used. ConA stimulates T-cell proliferation, whereas LPS stimulates B-cell proliferation [32]. Results of this study showed that different doses of Re (1.25, 2.5 or 5.00 mg/kg) significantly

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The rabies virus specific antibody plays a vital role in the protection of individual against rabies virus infection. In this study, Re (1.25, 2.5 or 5.00 mg/kg) to 20 μl of RV led to a dose-dependent increase of serum antibody levels, as indicated in Fig. 1A. The serum antibody response elicited by the RV was also found to be dose dependent. Mice immunized with 100 μl of the RV had significantly higher serum antibody level than mice immunized with 20 μl of the RV. However, supplementation of the Re (5.00 mg/kg) to 20 μl of RV significantly amplified serum antibody responses (Fig. 1A) and Th1/Th2 responses (Figs. 2 and 3) inducing similar protection as did 100 μl of RV. Rabies is an intracellular pathogen against which effective vaccines derived from tissue cultures do exist. However, such preparations are expensive [33]. Our result suggests that Re could be used to reduce the dose, and therefore the cost, of the RV to achieve the same effective protection. Moreover, we observed that the co-administration of Re (5.00 mg/kg) enhanced the maintenance of long-lasting serum antibody titers to the RV in this study (Fig. 1B). In light of the fact that a yearly prophylactic shot of inactivated rabies vaccines is currently being used to immunize dogs to control rabies in China, a longer duration of protective antibody titers is preferable to make the rabies control program more effective. These findings indicated that Re may be a promising adjuvant to the veterinary rabies vaccine by providing higher antibody levels and longlasting protection. Here we must note that ideally the immunization

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Fig. 3. Effect of Re on mRNA expression of IL-4, IL-10, IL-12 and IFN-γ by splenocytes. Splenocytes were prepared 2 weeks after the last immunization and cultured with ConA (5.00 μg/ml) for 48 h, then collected to determine the IL-4, IL-10, IL-12, and IFN-γ levels by RT-PCR. The values are presented as the mean ± S.D. Bars with different lowercase letters are statistically different for each cytokine (P b 0.05), and bars with same uppercase letter ‘A’ are not statistically different for each cytokine (P N 0.05).

Fig. 4. Expression of CD4+ and CD8+ in peripheral blood lymphocytes. Cells were incubated with FITC-labeled anti-mice CD4+ and PE-labeled anti-mice CD8+ and analyzed by flow cytometry. The numbers in the quadrants represent percentages. The values are represented mean ± S.D. *P b 0.05.

Please cite this article as: Su X, et al, Ginsenoside Re as an adjuvant to enhance the immune response to the inactivated rabies virus vaccine in mice, Int Immunopharmacol (2014), http://dx.doi.org/10.1016/j.intimp.2014.03.008

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We would like to thank the medical staff at the veterinary teaching hospital of Zhejiang University for their assistance during the study. This study was supported by Specialized Research Fund for the Doctoral Program of Higher Education of China (SRFDP, No. 20110101120089) and the National Natural Science Foundation of China (NSFC, No. 31201960).

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[1] Dietzschold B, Schnell M, Koprowski H. Pathogenesis of rabies. Curr Top Microbiol Immunol 2005;292:45–56. [2] Meslin FX, Fishbein DB, Matter HC. Rationale and prospects for rabies elimination in developing countries. Curr Top Microbiol Immunol 1994;187:1–26. [3] Dreesen DW. A global review of rabies vaccines for human use. Vaccine 1997;15: S2–6. [4] Knobel DL, Cleaveland S, Coleman PG, Fèvre EM, Meltzer MI, Miranda ME, et al. Reevaluating the burden of rabies in Africa and Asia. Bull World Health Organ 2005;83(5):360–8. [5] Rupprecht CE. A tale of two worlds: public health management decisions in human rabies prevention. Clin Infect Dis 2004;39(2):281–3. [6] Hu RL, Fooks AR, Zhang SF, Liu Y, Zhang F. Inferior rabies vaccine quality and low immunization coverage in dogs (Canis familiaris) in China. Epidemiol Infect 2008;136(11):1556–63. [7] Wang X, Lou J. Two dynamic models about rabies between dogs and human. J Biol Syst 2008;16(04):519–29. [8] Bögel K, Meslin FX. Economics of human and canine rabies elimination: guidelines for programme orientation. Bull World Health Organ 1990;68(3):281–91. [9] Cleaveland S, Kaare M, Tiringa P, Mlengeya T, Barrat J. A dog rabies vaccination campaign in rural Africa: impact on the incidence of dog rabies and human dogbite injuries. Vaccine 2003;21(17–18):1965–73. [10] Davlin SL, VonVille HM. Canine rabies vaccination and domestic dog population characteristics in the developing world: a systematic review. Vaccine 2012;30(24):3492–502. [11] Tang X, Luo M, Zhang S, Fooks AR, Hu R, Tu C. Pivotal role of dogs in rabies transmission, China. Emerg Infect Dis 2005;11(12):1970–2. [12] Wu X, Hu R, Zhang Y, Dong G, Rupprecht CE. Reemerging rabies and lack of systemic surveillance in People's Republic of China. Emerg Infect Dis 2009;15(8):1159–64. [13] Zhang J, Jin Z, Sun GQ, Zhou T, Ruan S. Analysis of rabies in China: transmission dynamics and control. PLoS One 2011;6(7):e20891. [14] Sage G, Khawplod P, Wilde H, Lobaugh C, Hemachudha T, Tepsumethanon W, et al. Immune response to rabies vaccine in Alaskan dogs: failure to achieve a consistently protective antibody response. Trans R Soc Trop Med Hyg 1993;87(5):593–5. [15] Minke JM, Bouvet J, Cliquet F, Wasniewski M, Guiot AL, Lemaitre L, et al. Comparison of antibody responses after vaccination with two inactivated rabies vaccines. Vet Microbiol 2009;133(3):283–6. [16] Reed SG, Bertholet S, Coler RN, Friede M. New horizons in adjuvants for vaccine development. Trends Immunol 2009;30(1):23–32. [17] Lodmell DL, Ray NB, Ulrich JT, Ewalt LC. DNA vaccination of mice against rabies virus: effects of the route of vaccination and the adjuvant monophosphoryl lipid A (MPL). Vaccine 2000;18(11–12):1059–66. [18] Li CP, Li RC. An introductory note to ginseng. Am J Chin Med 1973;1(2):249–61. [19] Song X, Hu S. Adjuvant activities of saponins from traditional Chinese medicinal herbs. Vaccine 2009;27(36):4883–90. [20] Kiefer D, Pantuso T. Panax ginseng. Am Fam Physician 2003;68(8):1539–42. [21] Cho W, Chung WS, Lee SK, Leung AW, Cheng CH, Yue KK. Ginsenoside Re of Panax ginseng possesses significant antioxidant and antihyperlipidemic efficacies in streptozotocin-induced diabetic rats. Eur J Pharmacol 2006;550(1–3):173–9. [22] Wang YG, Zima AV, Ji X, Pabbidi R, Blatter LA, Lipsius SL. Ginsenoside Re suppresses electromechanical alternans in cat and human cardiomyocytes. Am J Physiol Heart Circ Physiol 2008;295(2):H851–9. [23] Rivera E, Hu S, Concha C. Ginseng and aluminium hydroxide act synergistically as vaccine adjuvants. Vaccine 2003;21(11–12):1149–57. [24] Su F, Yuan L, Zhang L, Hu S. Ginsenosides Rg1 and Re act as adjuvant via TLR4 signaling pathway. Vaccine 2012;30(27):4106–12. [25] Sun J, Hu S, Song X. Adjuvant effects of protopanaxadiol and protopanaxatriol saponins from ginseng roots on the immune responses to ovalbumin in mice. Vaccine 2007;25(6):1114–20. [26] Song X, Chen J, Sakwiwatkul K, Li R, Hu S. Enhancement of immune responses to influenza vaccine (H3N2) by ginsenoside Re. Int Immunopharmacol 2010;10(3):351–6. [27] Overbergh L, Valckx D, Waer M, Mathieu C. Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR. Cytokine 1999;11(4):305–12. [28] Yin JL, Shackel NA, Zekry A, McGuinness PH, Richards C, Putten KV, et al. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for measurement of cytokine and growth factor mRNA expression with fluorogenic probes or SYBR Green I. Immunol Cell Biol 2001;79(3):213–21.

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enhanced splenocyte proliferation induced by ConA, LPS and rabies virus antigen (Fig. 2), suggesting that both T- and B-cells were activated by Re. Moreover, the enhanced lymphocyte responses to Con A or LPS stimulation (Fig. 2) paralleled the increased serum antibody responses detected in the mice injected with RV plus different doses of Re (Fig. 1) in the present study. In order to induce antibody production, triggered B lymphocytes are required for clone expansion. Therefore, the increased titer of serum antibody in ICR mice injected with RV plus Re (Fig. 1), at least in part, may be attributed to the activation of B-cells. T-cell dependent immune response, including T-cells and T-cell related cytokines are also involved in B cell activations and the production of the specific antibody. Immunity to different infectious agents requires distinct types of immune responses. Defense against intracellular pathogens tends to involve Th1 type immune responses, while resistance to extracellular pathogens is often associated with humoral responses dominated by production of IL-4 and IL-5 (Th2 type) [34]. As an intracellular pathogen, rabies virus particle binds cell-surface receptor and release virus necleocapsid into the cytoplasm with transcription of the five viral genes [35]. Ideally, a rabies vaccine should be able to prime a Th1/Th2 balanced and efficient immune response to protect animals from rabies virus infections. Aluminum adjuvant is the most often used commercial adjuvant for veterinary rabies vaccines nowadays. The major limitations of aluminum adjuvant include its inability to elicit cell-mediated Th1 or cytotoxic T lymphocyte (CTL) responses that are required to control most intracellular pathogens, including those that cause tuberculosis, malaria, leishmaniasis, leprosy, and AIDS [36,37]. Previous studies reported that Re results in mixed Th1/Th2 responses in different mouse models [24,26]. Similarly, the present study demonstrated that Re significantly promotes the expression of Th1-type related cytokines (IFN-γ and IL-12) and Th2-type related cytokines (IL-4 and IL-10). IFN-γ, a key cytokine secreted by Th1-CD4 and Th1-CD8 lymphocytes as well as natural killer cells, is essential for the activation of macrophages which can destroy intracellular pathogens by producing NO and O3 [38]. IL-12 is a powerful inducer of the differentiation of naïve T-helper cells to Th1-type cells that generally produce specific sets of cytokines, such as IFN-γ and TNF-β. On the other hand, IL-4 plays an important role in Th2 differentiation and potently induces IL10, IL-5 and IL-9 production [34]. Our results suggested that both Th1 and Th2 immune responses were stimulated. Thus, when mixed Th1/Th2 responses is needed for a vaccination, the supplement of Re in vaccine is indicated. For rabies, the supplement of Re with the vaccine may be particularly beneficial. To further investigate the effect of Re on T cells, the expressions of CD4+ and CD8+ T-cell subsets were analyzed by flow cytometry. The percentage of CD4+ lymphocytes was significantly higher and the percentage of CD8+ lymphocytes was lower in the co-administration of RV (20 μl) with Re (5.00 mg/kg) group than the RV (20 μl) alone group. And the percentage of CD8+ cells was markedly lower in the co-admin group than that in the RV alone group at four weeks (Fig. 4). T-cell depletion studies showed the importance of CD4+ T cells in generating neutralizing, protective antibodies in mice after RV infection. For instance, Nunberg et al. showed that selective loss of the CD4+ T-cell subset markedly suppressed antibody response in street rabies virus (SRV)-infected SJL/J and BALB/cByJ mice [39]. They also believed that depletion of CD8+ T cells had no measurable effect on host resistance to SRV infection. Our results suggested that the increased production of serum antibody may be attributed to the higher percentages of CD4+ cells. In conclusion, the co-administration of Re with the RV in mice significantly amplified serum antibodies responses, and increased the CD4+: CD8+ ratio, lymphocyte proliferation, and the production of IL-4, IL-10, IL-12, and IFN-γ by splenocytes. Both Th1 and Th2 immune responses were activated. Considering the adjuvant effect of Re demonstrated in this study and the fact that some herbal preparation containing Re has been licensed for injection in humans, Re warrants further studies

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[29] Morrison TB, Weis JJ, Wittwer CT. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 1998;24(6):954–8. [30] Pfaffl MW. A new mathematical model for relative quantification in real-time RTPCR. Nucleic Acids Res 2001;29(9):e45. [31] Qu D, Han J, Du A. Enhancement of protective immune response to recombinant Toxoplasma gondii ROP18 antigen by ginsenoside Re. Exp Parasitol 2013;135(2):234–9. [32] Tizard I. Veterinary immunology: an introduction. 5th ed. Philadelphia: WB Saunders; 1992. [33] Dorfmeier CL, Lytle AG, Dunkel AL, Gatt A, McGettigan JP. Protective vaccine-induced CD4(+) T cell-independent B cell responses against rabies infection. J Virol 2012;86(21):11533–40. [34] Constant SL, Bottomly K. Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu Rev Immunol 1997;15(1):297–322.

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[35] Lafon M, Megret F, Lafage M, Prehaud C. The innate immune facet of brain. J Mol Neurosci 2006;29(3):185–94. [36] Edelman R. The development and use of vaccine adjuvants. Mol Biotechnol 2002;21(2):129–48. [37] Gottwein JM, Blanchard TG, Targoni OS, Eisenberg JC, Zagorski BM, Redline RW, et al. Protective anti-Helicobacter immunity is induced with aluminum hydroxide or complete Freund's adjuvant by systemic immunization. J Infect Dis 2001;184(3):308–14. [38] Feria-Romero IA, Chávez-Rueda K, Orozco-Suárez S, Blanco-Favela F, CalzadaBermejo F, Chávez-Sánchez L, et al. Intranasal anti-rabies DNA immunization promotes a Th1-related cytokine stimulation associated with plasmid survival time. Arch Med Res 2011;42(7):563–71. [39] Perry LL, Lodmell DL. Role of CD4+ and CD8+ T cells in murine resistance to street rabies virus. J Virol 1991;65(7):3429–34.

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