1 Introduction
There has been an extensive research on the development of new vaccine adjuvants, but aluminum salts remain the only adjuvant registered for human beings and most veterinary vaccines, primarily because aluminum salts prove to be safe (Rivera et al., 2003). Unfortunately, vaccines adjuvanted with aluminum have a number of limitations (Tizard, 1996), such as the short duration of the antibody response, the poor cell mediated immunity and the formation of IgE antibodies, which may result in hypersensitive reactions. Recent researches suggest that the aluminum vaccine may be involved in a muscle ailment in human beings (Malakoff, 2000). Therefore, searching for an ideal vaccine adjuvant is still a challenge.
This study mainly focuses on the evaluation of a traditional Chinese medicine Cochinchina momordica seeds for its adjuvant properties. Seeds are from the plant Momordica cochinchinensis (Lour.) Spreng, which is a perennial vine and grows mainly in Southeast Asian countries such as Vietnam, Laos, Cambodia, Burma, the Philippines, Malaysia, Bangladesh and Thailand, and many provinces in southern China such as the provinces of Guangxi, Sichuan, Hubei, Hunan, Guizhou, Yunnan, Guangdong, Anhui, etc. (Xu, 1996). The local people, in the winter, collect fruits, and separate them from seeds which are then dried for medicinal purposes such as the treatment of inflammatory swelling, scrofula, tinea, diarrhea as well as suppurative skin infections such as sore, carbuncles, furuncles and boils in human beings and animals (Gao, 2005; Zheng et al., 1992). An ancient Chinese medical literature Kai Bao Materia Medica in the Song Dynasty (793 AD) described that the seeds had a therapeutic effect on the acute mastitis and anusitis in humans (Gao, 2005). The drug is now included in both Chinese Pharmacopeia (Chinese Pharmaceutical Codex Evaluation Committee, 2005) and Chinese Veterinary Pharmacopeia (Chinese Veterinary Pharmaceutical Codex Evaluation Committee, 2000). Chemical analysis shows that the C. mormodica seeds contain fatty acid, saponin, protein, α-spinasterol, oleanolic acid, momordica acid, etc. (Xu, 1996; Gao, 2005; Shang et al., 2002; Shang et al., 2000; Ding et al., 2005). Recent investigations prove that a crude liquid extract from the seeds has anti-tumor functions (Tien et al., 2005; Tsoi et al., 2005; Wong et al., 2004). Iwamnoto et al. (1985) isolated two kinds of saponins called momordica saponins I and II from the seeds. According to his observation, momordica saponin I is gypsoside, which is the tri-terpenoid saponin containing disaccharide chains, and momordica saponin II is mostly the quillaic acid. Some saponins with tri-terpenoid structures exhibit vaccine adjuvant effects, such as the saponins extracted from the bark of Quillaja saponaria Molina (Dalsgaard, 1978) or those from the root of Panax ginseng C.A. Meyer (Rivera et al., 2003; Hu et al., 2003; Riveria et al., 2003). But no report has been found up to now regarding to its immunological effects on both humans and animals. However, based on the information from the above mentioned, it can be assumed that the seeds may have the properties of an adjuvant that augments the immune response to vaccine antigens. To prove this hypothesis, an extract from C. momordica seeds (ECMS) was prepared, and then its adjuvant effect on the immune response in mice immunized with ovalbumin (OVA) in combination with ECMS was evaluated. Since aluminum hydroxide gel (Al-gel) is a common adjuvant in most commercially available vaccines, this adjuvant was used as a reference in the experiment.
2 Materials and methods
2.1 Preparation of ECMS
Thirty kilograms of dried C. momordica seeds were purchased from a market of traditional Chinese herbs in Anhui Province, China, and the seeds were appraised by Zhejiang Institute of Veterinary Drug Control. The drugs were divided into three batches with each containing 10 kg of seeds. Seeds were crushed or ground into powder, which was then dissolved in 50% ethanol for 24 hours. The powder and ethanol mixture was put into a round-bottom flask. The mixture was refluxed three times at 90°C with each reflux taking two hours. The remaining ethanol was removed by a R502B rotary evaporator (Shenko Tech Co Ltd, Shanghai, China). The extract was then dissolved in water and washed with diethyl ether to remove the ethersoluble substance. After that, the saponin fraction was extracted with water-saturated n-butanol. The butanol-soluble fraction was purified by passing through a chromatography column with resin D101A (Hai Guang Chemical Co Ltd, Tianjin, China). A purified ECMS was obtained by evaporating the liquid eluted from the column.
2.2 Haemolysis of ECMS
Haemolytic activity of ECMS was assayed by incubating with red blood cells (RBC) from swine, sheep and cattle. In short, blood was washed three times with saline solution at 1 000 r/min for 10 min. Thereafter, the RBC was suspended in saline solution at a concentration of 0.5%. The RBC suspension was incubated with equal volumes of saline solution containing ECMS or Quil A (NOR-VET ApS, Denmark) at concentrations ranging from 1 to 500 μg/mL at 37°C for one hour. After that, samples were centrifuged and the optical density (OD) value of the supernatant was measured at 405 nm.
2.3 Adjuvant effect of ECMS
2.3.1 Preparation of ECMS solution
ECMS was dissolved in distilled water to make a solution containing ECMS of 1 mg/mL, and it was then filtered through a filter membrane with 0.2 μm pores. The solution was analysed for endotoxin by a gel-clot Limulus amebocyte lysate assay (bath No. 050260, Zhanjiang A & C Biological Ltd, Zhanjiang, China). The endotoxin level in the ECMS solution was less than 0.5 endotoxin unit per mL.
2.3.2 Immunisation and sample collection
Thirty 5-week-old female mice were purchased from the National Experimental Rodents Center, Shanghai, China, and provided with a specific pathogen-free condition. The mice were randomly divided into 5 groups with 6 mice in each group. Each mouse in all the test groups was subcutaneously injected with a 0.89% saline containing 10 μg of ovalbumin (OVA, bath No. 0200902, Bio-tech Co. Ltd, Shanghai, China) and 0, 10, 50, 100 μg of ECMS or 50 μg of aluminum hydroxide gel (Al-gel, bath No. 20050726, Biological Medicine Factory, Animal Husbandry Industry Co Ltd, Jiangxi, China). Booster immunization was conducted three weeks after the first immunization. The details are shown in
Table 1. Two weeks after the booster, blood samples were collected from the retro-orbital plexus to separate the serum, which was stored at -20°C until use, and the spleen cells were isolated for proliferation assay.
2.3.3 Measurement of OVA-specific IgG
An indirect enzyme-linked immunosorbent assay (ELISA) was conducted to analyse the titres of anti-OVA antibodies in serum mainly as described by Hu et al. (2003). Briefly, after coating flat-bottomed 96-well plates (Gong Dong Medical Plastic Factory, Zhejiang, China) with OVA in 0.05 mol carbonate buffer, pH 9.6 (5 μg/mL), the plate was washed with 0.01 mol phosphate buffer solution (PBS) containing 0.05% of Tween 20 (PBS-T) (pH 7.4) and obturated to culture in (PBS) containing 1% of calf serum (pH 7.4) for one hour at 37°C. After that, twofold serial dilution of the serum was made in PBS with 0.5% calf serum (PBS-C) and the plates were cultured for two hours at 37°C. Subsequently, 92 horseradish peroxidase conjugated goat anti-mouse IgG (Chemicon International, Inc, Temecula California USA) in PBS with 0.5% of calf serum (1:500, v/v) was added to the plates (100 μL per well), and cultured for two hours at 37°C. After being rewashed, the plates were cultured for 15 min at 37°C with TMB (Sigma Chemical, St. Louis, MO, USA) substrate (100 μL per well). The reaction was stopped by adding 50 μL of 2 mol H2SO4 to each well. The OD was read on an automatic ELISA plate reader (Dialab, GMBH, Austria) at 450 nm. The cut-off OD value was set as described by Frey et al. (Du, 1994). The antibody titre was defined as the highest dilution that gave a reading above the cut-off value.
2.3.4 Measurement of OVA-specific IgG1 and IgG2a
A flat-bottomed 96-well plate was coated as described above. After the coating, 100 μL of diluted serum (1:800) was added to each well of the plate and cultured for one hour at 37°C. After the plate was washed, 100 μL of biotin conjugated goat anti-mouse IgG1 or IgG2a diluted 1:600, (Santa Cruz Biotechnology Inc, California, USA) was added to each well of the plate, which was then cultured for one hour at 37°C. Then, the plate was rewashed and 100 μL of horseradish peroxidase conjugated anti-biotin (BD Biosciences, Pharmingen, USA) diluted at 1:4 000 in PBS-C was added to each well to have another incubation for one hour at 37°C. After another washing cycle with PBS-T, the plates were incubated for 15 min at 37°C with TMB substrate (100 μL per well). The reaction ended by adding 50 μL of 2 mol H2SO4 to each well. The OD of the plate was read on an automatic ELISA plate reader at 450 nm.
2.3.5 Splenocyte proliferation assay
Spleen cells were harvested with aseptic procedures from the mice treated as mentioned above. The cells were suspended in RPMI-1640 (Gibco, Bio-cult, Glasgow, UK) containing 300 mg glutamine/L, 2 g sodium bicarbonate/L, 10% calf serum and 50 μg getamycin/mL. Cell suspension was adjusted to 5x106 viable cells/mL by using the Trypan Blue exclusion test (Frey et al., 1998). Splenocyte proliferation was assayed as described previously (Concha et al., 1996). Briefly, the assays were carried out in a flat-bottom 96-well microplate. Wells containing 5x105 cells in 100 μL medium were cultured in 5% CO2 at 37°C. Concanavalin A (Con A, Sigma Chemical, St. Louis, MO, USA) at 5 μg/mL or lipopolysaccharide (LPS, Sigma Chemical, St. Louis, MO, USA) at 5 μg/mL or OVA at 25 μg/mL in the well was used as stimulators. Wells without mitogens were used as controls. All the tests were carried out in triplicate. After incubation with Con A or LPS for 44 h, incubation with OVA for 92 h, 50 μL of MTT (Amresco, Cleveland Ohio, USA) solution (2 mg/mL) was added to each well, and incubated for another 4 h. The plates were then centrifuged (400 g, 5 min) and the untransformed MTT was removed carefully by a pipette. To each well, 150 μL of a DMSO working solution (144 μL of DMSO with 6 μL 1 mol HCl) was added and incubated for 15 min at 37°C to dissolve crystallisable formazan. The plates were read by an ELISA reader at 570 nm. The stimulation index (SI) was calculated based on the following formula:
SI =OD value for mitogen-cultrues/OD value for non-stimulated cultures.
2.4 Statistical analysis
Comparisons between mouse groups with respect to the levels of serum antibody and stimulation index were made using Duncan’s multiple range test. Probabilities less than 0.05 were considered statistically significant.
3 Results
3.1 Production of ECMS from C. momordica seeds
The amount of ECMS extracted from 3 extractions of C. momordica seeds at 10 kg at a time was 20.58 g, 22.10 g and 19.63 g, respectively. The average recovery rate of ECMS from the seeds was 2.08 g/kg. The extract was a yellowish powder with a special fragrance.
3.2 Hemolytic properties
The minimum hemolytic concentration of ECMS on RBC of swine, sheep and cattle was around 125 μg/mL while Quil A was at 4 μg, 8 μg and 16 μg/mL, respectively (
Fig. 1).
3.3 Local reaction at the injection site and effects of ECMS on the body weight gain of mice
No local reaction was found in all mice after injection.
Table 2 shows that subcutaneous injection of OVA (10 μg) mixed with various amounts of ECMS or 50 μg of Al-gel in mice did not significantly influence the body weight gain as compared with injection of OVA only.
3.4 Adjuvant properties of ECMS in mice
3.4.1 Effect of ECMS on OVA-specific IgG response
There was a dose-dependent relation between ECMS and IgG titers (
Fig. 2). The titers of OVA-specific IgG were positively proportional to the amount of ECMS co-administered. The titer in group 3, 4 or 5 injected with OVA solution containing 50 μg or 100 μg of ECMS or 50 μg of Al-gel was significantly higher than that of the control (Group 1) (
P<0.01). The IgG titer in group 4 injected with OVA solution containing 100 μg of ECMS was considerably high.
Six Balb/c mice in each group were immunized subcutaneously at a 3-week interval with10 μg of OVA mixed with 0 (Group 1), 10 μg (Group 2), 50 μg (Group 3), 100 μg (Group 4) of ECMS or 50 μg of Al-gel (Group 5). Blood samples were collected two weeks after the booster for separation of serum, which was used to determine specific-OVA IgG titers by using an ELISA. Bars with different letters show significant differences between groups (P<0.05).
3.4.2 Effect of ECMS on OVA-specific IgG1 and IgG2a responses
The IgG1 level in the serum from Group 4 injected with OVA solution containing 100 μg of ECMS was significantly higher than that of the control (Group 1) and Group 2 (
P<0.05), but no significantly increased IgG1 level in the sera from other groups was found when compared with that from the control (
P>0.05) (
Fig. 3). The IgG2a levels in sera from mice injected with OVA solution containing 10 μg (Group 2) or 50 μg (Group 3) or 100 μg (Group 4) of ECMS or 50 μg of Al-gel (Group 5) were significantly higher than that of the control (Group 1) (
P<0.05) with the highest IgG2a level recorded in Group 3.
3.4.3 Effect of ECMS on splenocyte proliferation
Figure 4 indicates that co-administration of OVA with ECMS significantly increased the splenocyte proliferative responses to Con A, LPS or OVA as compared with that co-administered with Al-gel or the control (saline group) (
P<0.05).
4 Discussion
An important finding in this research is the adjuvant effect of ECMS in mice. When co-administered with ECMS, OVA as a model antigen induced significantly higher specific antibody production than the OVA alone did (
Fig. 2). Such adjuvant effect was dose-dependent: when ECMS was at a lower dose (10 μg) no adjuvant effect was found, while when the dose of ECMS increased to 50 μg or 100 μg, OVA-specific IgG titer significantly increased. Such a dose-dependent mode was also found in other saponins such as ginsenosides (Rivera et al., 2003). When compared to Al-gel, ECMS showed a similar adjuvant potency as the mice produced almost the same antibody level when they were immunized with 10 μg of OVA adjuvanted with 50 μg of ECMS or the same amount of Al-gel (
Fig. 2).
Our study also demonstrated that ECMS and Al-gel can promote the production of both IgG1 and IgG2a while Al-gel favored the production of IgG1 and ECMS favored the production of IgG2a (
Fig. 3). In mice, cytokines such as interleukin-4 (IL-4), IL-5 and IL-10 produced by Th2 lymphocytes improved the production of IgG1 while IL-2, tumor necrosis factor-
β (TNF-
β) and interferon-
γ (IFN-
γ) produced by Th1 lymphocytes improved the production of IgG2a (Soren et al., 2000; Cherwinski et al., 1987). For the immunity against infectious diseases, Th1 response primarily targets the intracellular pathogens such as virus and some bacteria while Th2 response is mainly responsible for the extracellular pathogens such as most bacteria and parasites (Soren et al., 2000; Cherwinski et al., 1987). Thus, when a vaccine is designed to activate a higher Th1 than Th2 immune response, the ECMS is a more suitable adjuvant than Al-gel.
The splenocyte proliferative responses varied with the mitogen used. Both Con A- and LPS-induced proliferations were enhanced in the ECMS group. The enhanced lymphocyte response to Con A indicated that T lymphocytes were activated while the enhanced response to LPS indicated that B lymphocytes were activated. Although the splenocyte response to OVA antigenic stimulation in all the three groups was lower, the proliferation in the ECMS group was statistically higher than that in both saline and Al-gel groups. This result paralleled the increased antibody response detected in the mice injected with OVA co-administered with ECMS.
Six Balb/c mice in each group were immunized sub cutaneously at a 3-week interval with10 μg of OVA mixed with 0 (Group 1), 10 μg (Group 2), 50 μg (Group 3), 100 μg (Group 4) of ECMS or 50 μg of Al-gel (Group 5). Blood samples were collected two weeks after the booster for separation of serum, which was used to determine specific-OVA IgG1 and IgG2a levels by using an ELISA. Bars with different letters mean a significant difference between groups (P<0.05).
Six Balb/c mice in each group were immunized subcutaneously at a 3-week interval with10 μg of OVA mixed with saline or 50 μg of ECMS or Al-gel. Splenocytes were collected two weeks after the booster, which was used to assay cell proliferative responses to Con A and LPS and OVA by using a MTT method as described in the text. Data were expressed as stimulation index (SI) and bars with different letters means a significant difference between groups (P<0.05)
Results from this study demonstrate that ECMS is safe. Although Quil A has been reported to have adjuvant effect stimulating both humoral and cellular branches of the immune system (Windmill and Lee, 1999), its use as an adjuvant is limited due to its highly hemolytic activity (Sjølander and Barr, 1998). The ECMS used in this study showed lower hemolytic effect on RBC of swine, sheep and cattle than Quil
A. No local reactions and negative effects on the body weight gain were found after injection of OVA mixed with various amount of ECMS in mice as compared with injection of OVA only.
Although this study reports the adjuvant effect of the ECMS in mice experiments, the ECMS used as a co-adjuvant for commercially available oil-adjuvant vaccines for cattle has been evaluated. Those experiments show that the addition of ECMS to oil-adjuvanted vaccines may improve the immune responses of dairy cattle to the vaccination against foot and mouth disease.
In summary, when co-administered with ECMS in Balb/c mice, OVA can induce significantly higher production of IgG, IgG1 and IgG2a, but favor IgG2a, and enhance splenocyte proliferative responses to Con A, LPS or OVA. The ECMS has low hemolytic activity and is safe for local injection. Therefore, the ECMS presented in this paper deserves further studies to evaluate its use in the development of new vaccines.
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