International Immunopharmacology 11 (2011) 968–975

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International Immunopharmacology

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Immunological adjuvant effect of Boswellia serrata (BOS 2000) on specific antibodyand cellular response to ovalbumin in mice

Amit Gupta ⁎, A. Khajuria, J. Singh, S. Singh, K.A. Suri, G.N. QaziDivision of Pharmacology, Indian Institute of Integrative Medicine (IIIM), Canal Road, Jammu Tawi, Pin code 180001, India

⁎ Corresponding author.E-mail address: amitrrl@yahoo.com (A. Gupta).

1567-5769/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.intimp.2011.02.011

a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 November 2010Received in revised form 2 February 2011Accepted 6 February 2011Available online 1 March 2011

Keywords:Boswellia serrataBOS 2000OvalbuminAlum

In this study, the biopolymeric fraction BOS 2000 from Boswellia serratawas evaluated for its potential abilityas adjuvants on the immune responses to ovalbumin (OVA) in mice. Balb/c mice were immunizedsubcutaneously with OVA 100 μg alone or with OVA 100 μg dissolved in saline containing alum (200 μg) orBOS 2000 (10, 20, 40 and 80 μg) on Days 1 and 15. Two weeks later, OVA specific antibodies in serum;concanavalin A (Con A), OVA stimulated splenocyte proliferation, CD4/CD8/CD80/CD86 analysis in spleencells and its estimation of cytokines (IL-2 and IFN gamma) from cell culture supernatant were measured. OVAspecific IgG, IgG1 and IgG2a antibody levels in serum were significantly enhanced by BOS 2000 (80 μg)compared with OVA control group. Moreover, the adjuvant effect of BOS 2000 (80 μg) on the OVA-specific IgG,IgG1, and IgG2a antibody responses to OVA in mice were more significant than those of alum. BOS 2000significantly enhanced the Con A and OVA induced splenocyte proliferation in the OVA immunized miceespecially at a dose of 80 μg (pb0.001). However, no significant differences were observed among the OVAgroup and OVA/alum group. At a dose of 80 μg (pb0.001), there was a significant increase in the CD4/CD8 andCD80/CD86 analysis in spleen cells and cytokine (IL-2 and IFN-gamma) profile in the spleen cell culturesupernatant was observed. In conclusion, BOS 2000 seems to be a promising balanced Th1 and Th2 directingimmunological adjuvants which can enhance the immunogenicity of vaccine.

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© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Development of newer vaccines include highly purified subunitantigens that are weakly immunogenic in general. Vaccine formula-tions often require adjuvants for increased immunological efficiencyand better vaccination schedules [1,2]. Adjuvants are used forimproving the specific immune response to vaccine antigens and forinduction of immunological memory [3]. The addition of adjuvants tovaccine formulations makes it possible to reduce the amount ofantigen and the number of immunizations needed, while improvingthe magnitude and the duration of the specific immune response.Currently used adjuvants include aluminum hydroxide, aluminumphosphate, calcium phosphate, water-in-oil emulsions, products frombacteria and liposomes. Often there is compromise on level ofadjuvanicity and acceptable level of safety. Other adjuvants such asmono-phosphoryl lipid A, ISCOMS, QS-21—a purified saponin frombark of Quillaja saponaria Molina and Syntex Adjuvant formulation(SAF) are being investigated for development of better and saferadjuvants [2]. Plant based immunomodulators are being considered asone option [4–8]. Previously we have reported various immunomo-dulators of Ayurvedic origin. The extracts and formulations prepared

from Ayurvedic medicinal plants including Withania somnifera,Tinospora cordifolia and Asparagus racemosus demonstrated signifi-cant immunostimulatory activity particularly at humoral level inexperimental systems with or without induced immunosuppression[9–12]. Attempts to organize adjuvants in grouped categories in orderto facilitate adjuvant selection have sometimes been difficult becauseof multiple and overlapping biological effects of many adjuvants.However, this approach can be useful for providing a prospective onthe types of immunostimulators that are available.

Recently, substances for enhancing host defense responses wereisolated from microorganisms, fungi [13] and plants [14,15]. Plant-derived polysaccharides are the best known and most potentimmunomodulatory substances [14] and have been shown to beclinically therapeutic [16]. Their chemical properties and biologicalactivities have been studied extensively. Plant polysaccharides havebeen shown to exhibit anti-inflammatory [17], anti-hypoglycemic[18], anti-bacterial [19] and anti-tumor [20] and anti-complementaryactivities. Indeed, the basic mechanism of the immunostimulatory,anti-tumor, bactericidal and other therapeutic effects of botanicalpolysaccharides are thought to occur via macrophage stimulation andmodulation of the complement system [21]. Furthermore, modulationof these systems can significantly impact both humoral and cellularimmune responses [22].

We propose that BOS 2000 from Boswellia serrata has the potentialof being developed as a potent plant based immune adjuvant. BOS

Fig. 1. Flowsheet of biopolymeric fraction BOS 2000 isolated from Boswellia serrata.

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2000, isolated from B. serrata having anti-arthritic [23], anti-inflammatory [24–26], immunomodulatory [27] and anticanceractivity [28–30] was considered for adjuvant testing in mice. A betterunderstanding of the biology of non-conventional T cell subpopula-tions, T and B cell memory, regulatory T cells and mucosal immunityhas profound implications for a modern approach to adjuvantscreening and development. The future lies in the high throughputscreening of synthetic chemical entities targeting well-characterizedbiological molecules. Used alone or in combination with suchadjuvants will allow stimulation or modulation in a safe and efficientmanner of strong effector, regulatory and memory immune mecha-nisms. The present study was undertaken to validate the immunoad-juvant effects of BOS 2000 against weak antigen ovalbumin.

2. Materials and methods

2.1. Chemicals and reagents

The organic solvent exhausted material (0.5 kg) of the plant B.serrata was used in this study. Ammonium chloride, potassiumbicarbonate, o-phenylenediamine, hydrogen peroxide (H2O2), citrate-phosphate buffer and disodium ethylene diamine tetraacetic acid(EDTA) were purchased from Merck, India. Medium RPMI 1640(Himedia, Mumbai, India), 96-V-well microtitration plates andmicrotissue culture plates (96-U-wells) from Tarson, trypan blue(Microlabs, Mumbai), fetal calf serum (FCS), lipopolysaccharide (LPS,Escherichia coli 055 B5), dimethylsulphoxide (DMSO), bovine serumalbumin (BSA), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl 2,5-dimethyltetrazolium bromide (MTT), Hank's balanced salt solution(HBSS), HEPES, 2-mercaptoethanol, penicillin, streptomycin, strepta-vidin-horse radish peroxidase, rabbit anti-mouse IgG peroxidaseconjugate, goat anti-mouse IgG1 and IgG2a peroxidase conjugate(Sigma), fluoroisothiocyanate (FITC)-labeled anti-CD4/CD80 (B7-1)and phycoerythrin (PE) labeled anti-CD8/CD86 (B7-2) mAbs (BDBiosciences) were used.

2.2. Preparation of biopolymeric fraction BOS 2000

The organic solvent exhausted material (0.5 kg) of the plant B.serrata was air-dried and extracted twice with water using 700 ml ofwater for the first extraction and 200 ml for the subsequent extractions.The combined aqueous extracts were clarified by centrifugation and tothe clear solution (600 ml) alcohol (1.2 l) was added. The light brownsolid, which separated on keeping, was collected by filtration andpurified by dissolving in water and precipitating with alcohol. Thepurification process was repeated once more to get the biopolymericfraction BOS 2000 (Fig. 1) as white solid (70 g).

2.3. Hydrolysis of biopolymeric fraction (BOS 2000)

The biopolymeric fraction BOS 2000 (1.0 g) was suspended in50 ml of aqueous 2 N-TFA and then refluxed (120 °C) for 2.5 h. Thereaction mixture was concentrated under reduced pressure on a filmevaporator and then kept in a desiccator containing NaOH, overnight.Paper chromatography of the hydrolyzed fraction BOS 2000 incomparison with reference monosaccharides revealed the presenceof arabinose, glucose and galactose.

2.4. Quantitative analysis of monosaccharides in the hydrolyzed BOS2000 by HPLC

HPLC grade water was prepared from Milli-Q water purificationsystem. All the four monosaccharides, i.e. D-glucose, D-xylose, D-galactose and D-arabinose were procured from Aldrich chemicals ofpurity ≤98% (HPLC).

2.5. Chromatography

Monosaccharideswere separated and quantified by using ShimadzuHPLC system consisting of Pump LC-10 ATVP, an automatic samplingunit (Autosampler), SIL-10 ADVP, a column oven CTO-10 ASVP, RIdetector and System controller SCL-10 AVP version 5.4. Shimadzu ClassVP software version 6.1 was used for data analysis and data processing.The samples were analyzed at 80 °C on a Phenomenex Rezex RPM-monosaccharide Pb++ (8%) column (300 mm×7.80 mm) by RIdetector using a gradient mobile phase of HPLC grade water.

2.6. Sample preparation

The accurately weighed quantity of the dried hydrolysate of BOS2000 was dissolved in known volume of HPLC grade water. Thesamples were filtered through millipore microfilter (0.45 μm) andthen injected into the HPLC system.

2.7. Preparation of stock solutions and samples

Stock solutions of the pure reference compounds were prepared inHPLC grade water and stored in a refrigerator at 4 °C. From the stocksolutions working solutions for each reference compound wereprepared by dilution with HPLC grade water. These working solutionsof all the reference compounds weremixed together in equal volumesfor further analysis.

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2.8. Quantification

The compounds exhibiting linear response in the calibrationcurves were prepared by using multipoint calibration curve method.Working solutions after mixing were injected in different concentra-tions (2–20 μl). Excellent calibration curves were obtained for D-glucose (r2=0.99891), D-xylose (r2=0.999425), D-galactose(r2=0.0999936) and D-arabinose (r2=0.999976). Calibration curveswere determined on the basis of six levels (2–20 μl) of concentrationof each standard in the mixture.

From HPLC of the hydrolyzed (2 NTFA) biopolymeric fraction incomparison to authentic monosaccharides it was observed that it'stotally endotoxin free fraction and this fraction BOS 2000 is composedof glucose (26.9), arabinose (37.0), and galactose (33.1) in a molarratio of 1.0, 4.2 and 21.4 (Fig. 2).

2.9. Animals

Male adult Balb/C mice 10–12 weeks old and weighing 20–22 gobtained from the animal house of the Indian Institute of IntegrativeMedicine (IIIM), Jammu in groups of 5–10 were employed for thestudy and kept in plastic cages. These were maintained at a roomtemperature of 22±2 °C with 12 h light/dark cycle and free access topellet food (Lipton India limited) and water. The ethical committee ofthe Indian Institute of Integrative Medicine (IIIM) instituted foranimal handling approved all protocols. According to ethical regula-tions on animal research, all animals used in experimental workreceived humane care.

2.10. Antigen-immunization with ovalbumin (OVA)

Animals were immunized subcutaneously with 100 μg OVAformulated with one of the following delivery vehicles: phosphate

Fig. 2. HPLC chromatogram of a) standards and b) BOS 2000.

buffered saline and BOS 2000 (10, 20, 40 and 80 μg) on Day 1. Saline-treated animals were included as controls. A challenging injectionwasgiven two weeks later. Control adjuvant (standard) was alum(200 μg). Alum was formulated with OVA at a ratio of two partsadjuvant to one part antigen immediately prior to administration. Theresultant formulation was thoroughly vortexed before use.

2.11. Measurement of OVA specific IgG, IgG1 and IgG2a antibody titers inthe sera of mice by ELISA

OVA specific IgG, IgG1 and IgG2a antibodies in serum weredetected by an indirect ELISA [31]. In brief, microtiter plate wells werecoated with 100 μl OVA solution (50, 25 and 50 μg/ml) for IgG, IgG1and IgG2a antibodies in 50 mM carbonate–bicarbonate buffer, pH 9.6for 24 h at 4 °C. The wells were washed three times with PBScontaining 0.05 % (v/v) Tween 20(PBS/Tween) and then blocked with5% FCS/PBS at 37 °C for 1 h. After three washings, 100 μl of dilutedserum sample (IgG, 1:400; IgG1, 1:100; IgG2a, 1:200) or 0.5% FCS/PBSas control was added to triplicate wells. The plates were thenincubated for 1 h at 37 °C, followed by three times of washing.Aliquots of 100 μl of rabbit anti-mouse IgG horse radish peroxidase(1:1000), goat anti-mouse IgG1 (1:500) or IgG2a (1:100) peroxidaseconjugate diluted with 0.5% FCS/PBS were added to each plate. Theplates were further incubated for 1 h at 37 °C. After washing, theperoxidase activity was assayed as following: 100 μl of substratesolution (10 mg of o-phenylenediamine and 37.5 μl of 30% H2O2 in25 ml of 0.1 M citrate-phosphate buffer, pH 5.0) was added to eachwell. The plate was incubated for 10 min at 37 °C, and enzymereaction was terminated by adding 50 μl/well of 2 N H2SO4. Theoptical density (OD) was measured in an ELISA reader at 450 nm.

2.12. Splenocyte proliferation assay (ex vivo)

2.12.1. Total lymphocyte isolation from the spleenMice were sacrificed by carbon dioxide anesthesia. The spleens

were excised aseptically and lymphocytes isolated. Briefly, single cellsuspensions were prepared by teasing the tissue between two glassslides and cells were centrifuged at 400×g for 10 min at 4 °C.Erythrocytes present were lysed with red cell lysis buffer (0.5 Mammonium chloride, 10 mM potassium bicarbonate and 0.1 mMdisodium ethylene diamine tetraacetic acid, pH 7.2) for 5 min.Lymphocytes obtained were then washed thrice with PBS. Cellnumber was counted with a hemocytometer by the trypan blue dyeexclusion technique. Cell viability exceeded 95% [32].

To evaluate the effect of BOS 2000 on the proliferation of spleniclymphocytes [33], spleen cell suspension (2×106 cell/ml) waspipetted into 96 well plates (200 μl/well) in the presence of Con A(5 μg/ml) and OVA (5 μg/ml) cultured at 37 °C for 48 h, the plateswere centrifuged at 1400×g, 5 min and the supernatant was collectedfor the estimation of cytokines in cell culture supernatant added with100 μl of fresh complete media in a 96 well plate and again incubatedfor 24 h and then added with 20 μl of MTT solution (5 mg/ml) to eachwell and incubated for 4 h. The plates were centrifuged (1400×g,5 min) and the untransformed MTT was removed carefully bypipetting. To each well, 100 μl of a DMSO working solution (192 μlDMSO with 8 μl 1 M HCl) was added and the absorbance wasevaluated in an ELISA reader at 570 nm after 15 min.

2.13. Analysis of Th1 (IL-2/IFN gamma) and Th2 (IL-4/IL-10) cytokinesin spleen cell culture supernatant

Spleens from Balb/C mice were removed aseptically and singlesplenocyte suspension in RPMI 1640 containing 10% FBS, 1 M HEPES,2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycinwas obtained by passage through a stainless mesh. Red blood cellswere lysed with lysis buffer for 5 min. After washing with PBS, cells

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were cultured in 96-well plates at 2×106 cells/ml and cytokines werethen measured from the supernatants by ELISA. The assay wasperformed according to the manufacturer's instruction with multi-point analysis [34].

Briefly, 100 μl of diluted capture antibody was added to each wellin a 96 well plate and was allowed to adhere overnight for 4 °C. Plateswere washed and then blocked with 1× PBS supplemented with 10%FBS for 1 h at room temperature. After washing, serial dilutions of thestandard and samples were prepared in the plates and were thenincubated for 2 h at room temperature. Then, plates were washed andworking detector solution (including detector antibody and avidin–horse radish peroxidase reagent) was added into each well. Plateswere then sealed and incubated for 1 h at room temperature. Afterwashing, 100 μl of tri-methyl benzidine (TMB) substratewas added intoeach well. Stop solution (2 N H2SO4) was finally added after incubationin the dark for 30 min at room temperature. The absorbancewas read at450 nm. The result was analyzed using softmax program and valueswere determined against the standard provided by the manufacturer.

2.14. Lymphocyte immunophenotyping (CD4/CD8) in spleen

The percentages of T lymphocyte subsets in the spleen weremeasured using flow cytometric analysis (Gupta et al., 2006). Spleensingle cell suspensions were dually labeled with FITC-conjugated andPE-conjugated antibodies.

The spleen (1/3 of the organ) was placed in PBS buffer (withoutMg2+ and Ca2+) stored on ice prior to preparation of single cellsuspensions. Splenic erythrocytes were lysed with red blood celllysing buffer. Cell suspensions were refrigerated at 4 °C pendingstaining with antibodies. For each sample, 2×106 cells were stainedwith conjugated anti-CD4 FITC and anti-CD8a PE antibodies. Afterstaining with antibodies, cells were washed and resuspended in PBSfor flow cytometric analysis. The forward and side scatter gatingapplied for data acquisition on 10,000 events and fraction of cellpopulations representing different phenotypes analyzed using cellquest software.

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2.15. Flow cytometric analysis of co-stimulatory molecules (CD80 andCD86)

Splenic cells from treated and untreated mice were suspended inRPMI-1640 medium after removing the red blood cell by RBC lysisbuffer. Cells (1×107 cells/ml) were washed thrice and incubated for1 h at 37 °C on plastic petri plates. After removing the floating cells (Tand B cells), adherent cells (macrophages) were collected andwashedwith PBS. Thereafter, 2×106/ml of macrophageswas suspended in cellstaining solution (BD Pharmingen) in a 24-well plate. To quantify theexpression of co-stimulatory molecules, macrophages were stainedwith FITC-labeled anti-CD80 (B7-1) and anti-CD86 (B7-2) mAbs onice for 30 min andwashedwith PBS. Ten thousand cells were collectedfor each sample, and the data were analyzed with FACScan flowcytometer [35].

0Control OVA Alum

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OD

OVA (100µg)

Fig. 3. Effect of proposed adjuvant BOS 2000 on OVA-specific IgG, IgG1 and IgG2aantibody titers. Groups of ten male Balb/c mice were immunized s.c. with OVA 100 μgalone or with OVA 100 μg dissolved in saline containing alum (200 μg) or BOS 2000 (10,20, 40 and 80 μg) on Days 0 and 15. Sera were collected two weeks after the lastimmunization. OVA-specific IgG, IgG1 and IgG2a antibodies in the sera were measuredby an indirect ELISA method. The values are presented as mean±S.E. and significantdifferences designated as *Pb0.05, **Pb0.01, ***Pb0.001 (control vs. BOS 2000-treatedgroups; one-way ANOVA followed by Bonferroni multiple comparison test). Forexperimental details, refer to the Materials and methods section.

2.16. Safety studies

Safety of the BOS 2000 with OVA was proven by injecting itsubcutaneously into mice. Balb/c mice were divided into groups of 10animals each and were inoculated with variable doses of BOS 2000and OVA, which had been resuspended in PBS. Control mice, receivedPBS. After injection, mice were observed daily for a period of 8 weeks.Deviations from their normal behavior or reactions at the injectionsite were recorded. Theweight of themicewas taken on Days 0, 24, 28and 60 post-injection.

2.17. Acute toxicity of BOS 2000

Fasting Balb/c mice in groups of 10 each were used for each dose.BOS 2000 dissolved in distilled water was administered in gradeddoses of 10 to 1500 mg/kg i.p. and 10 to 1000 mg/kg per oral. Thegroups of animals administered distilledwater alone served as control.The animals were observed continuously for any gross behavioralchange and mortality for the next 2 weeks.

2.18. Statistical analysis

Data were expressed as mean±S.E. and statistical analysis wascarried out using one-way ANOVA (Bonferroni correction multiplecomparison test).

3. Results

3.1. Effect of BOS 2000 on humoral response against OVA-specificantibody viz. IgG, IgG1 and IgG2a titers

OVA specific IgG, IgG1 and IgG2a antibody levels in the sera weremeasured two weeks after the last immunization, and a significantenhancement in total serum IgG, IgG1 and IgG2a levels was observed(Fig. 3). OVA immunized animals given different doses of BOS 2000showed a dose dependent increase in the secretion of IgG, IgG1 andIgG2a, with a maximum response at 80 μg (6.3, 5.0 and 7.5 foldincrease in IgG, IgG1 and IgG2a) compared with OVA control group.Alum (200 μg) the standard adjuvant taken for comparison andauthentication of the experimentation, increased IgG (2.50 fold) andIgG1 (2.62 fold) titers but poorly elicited IgG2a titer when comparedto the value of OVA control group. Thus, findings indicate that BOS2000 could significantly enhance serum antibody viz. IgG, IgG1 andIgG2a production as compared with alum.

3.2. Effect of BOS 2000 on cell mediated immune response againstsplenocyte proliferation assay (ex vivo)

The effect of BOS 2000 on mitogen (Con A) and OVA stimulatedsplenocyte proliferation in mice immunized with OVA is shown inFig. 4. The results showing that BOS 2000 significantly enhanced thecell mediated immune response were observed. Splenocytes isolatedfrom mice immunized with OVA/BOS 2000, stimulated by Con A orOVA showed a greater proliferative response than that observed for

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10 20 40 80OD

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Fig. 4. Effect of proposed adjuvant BOS 2000 on mitogen and OVA stimulatedsplenocyte proliferation (ex vivo). Splenocytes were prepared two weeks after the lastimmunization and cultured with Con A (5 μg/ml) and OVA (5 μg/ml) for 72 h.Splenocyte proliferation was measured by the MTT method as described and thevalues are presented as mean±S.E. and significant differences were designated as*Pb0.05, **Pb0.01, ***Pb0.001 (control vs. BOS 2000-treated groups; one-way ANOVAfollowed by Bonferroni multiple comparison test). For experimental details, refer to theMaterials and methods section.

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Fig. 5. Th1 (IL-2/IFN-γ) and Th2 (IL-4/IL-10) cytokine profiles of mice immunized withOVA formulated with proposed adjuvant BOS 2000. Lymphocytes were isolated fromspleens of different test groups of mice given BOS 2000. Splenocytes (2×106 cells/well)were stimulated in vitro with Con A (5 μg/ml) and LPS (10 μg/ml) for 48 h ELISA and theinstructions given by the manufacturer measured the productions of Th1 and Th2cytokines in the culture supernatants. The results are presented as mean±S.E. P values:*Pb0.05, **Pb0.01, ***Pb0.001 (control vs. BOS 2000-treated groups; one-way ANOVAfollowed by Bonferroni multiple comparison test). For experimental details, refer to theMaterials and methods section.

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the mice immunized with OVA control group. Moreover, enhancingeffects of BOS 2000 at a dose of 80 μg (1.4 and 2.1 fold increase in ConA and OVA) were more significant than those of OVA control group.Alum (200 μg) the standard adjuvant used in this test model,enhanced proliferative response to OVA (1.89 fold) but there is noincrease in Con A when compared to the control animals. The resultsindicated that BOS 2000 significantly enhanced the splenocyteproliferation assay as compared with alum.

3.3. Effect of BOS 2000 on estimation of Th1 (IL-2/IFN-γ) and Th2 (IL-4/IL-10) cytokines from spleen cell culture supernatant

Significantly higher production of Th1 (IL-2/IFN-γ) and Th2 (IL-4/IL-6/IL-10) cytokines was found in the cell cultures of the miceimmunized with BOS 2000 which are shown in Fig. 5. However,production of IL-2 (6.74 fold increase), IFN-γ (11.5 fold increase), IL-4(4.92 fold increase) and IL-10 (4.19 fold increase) at 80 μg in BOS 2000than those of the mice immunized with OVA control group. However,no significant differences were observed between the OVA controlgroup. Alum (200 μg) the standard adjuvant, inhibited the Th1 (IL-2/IFN-γ) and increased the Th2 (IL-4/IL-10) cytokine level from spleencell culture supernatant. The results indicated that BOS 2000increased the Th1 (IL-2/IFN-γ) and Th2 (IL-4/IL-10) cytokine levelsin spleen cell culture supernatant as compared with alum.

3.4. Effect of BOS 2000 on lymphocyte immunophenotyping (CD4/CD8)in spleen

The effect of BOS 2000 on CD4+ and CD8+ T cells in the spleen cellsfrom the OVA immunized mice were shown in Fig. 6.

a) CD4+ T cells — The proportions of CD4+ T cells in the spleen cellsfrom the mice immunized with 80 μg BOS 2000 and alum werehigher than those from the OVA control group.

b) CD8+ T cells — The proportions of CD8+ T cells in the spleen cellsfrom themice immunizedwithOVAwerenot significantly changedby alum. But the proportion of CD8+ T cells was significantlychanged by 80 μg BOS 2000.

3.5. Effect of BOS 2000 on the expression of CD80 and CD86

BOS 2000 significantly upregulated the expression of both CD80and CD86 on splenic macrophages in a dose-dependent manner. BOS2000 at a dose of 80 μg induced optimum enhancement of 2-fold in

the number of cells expressing CD80/CD86 over the control animals(Fig. 7).

3.6. Safety studies

The mean body weight recorded for the mice injected with thehighest dose of BOS 2000 was 20.26 g at the beginning and 25.36 g atthe end of the experiment, respectively (Table 1). Balb/c mice used fortesting the safety of BOS 2000 remained clinically healthy during theentire period of experiment. Neither local reactions nor alterations inthe animal's behavior were observed. The LD50 of BOS 2000 wasdetermined to be 1350±58.26 mg/kg i.p. in mice. Even at very highdoses BOS 2000 did not exhibit any effect on central nervous system,i.e., stimulant or suppressive.

4. Discussion

A variety of components including mineral salts, microorganism-derived adjuvants, emulsions, cytokines, polysaccharides, and nucleicacid-based adjuvants have been tested for the research or usage innovel vaccine design [36]. However, exacerbating subclinical autoim-mune diseases, in addition to fever and erosion, at the local injectedlesion induced by nature of adjuvants has limited their clinical use[37]. For example, FCA causes inflammation, induration or necrosiswith disseminated granulomas being reported in the lungs, liver,kidneys, heart, lymph nodes and skeletal muscles of rabbits or rats. Forthis reason, until recently, only alum remains the adjuvant approvedfor human use in the USA. Alum-adsorbed vaccines have been shownto elicit essentially Th2 immune responses, and to poorly induce cell-mediated immunity. Meanwhile, cellular immunity now appears

Fig. 6. Flow cytometric analysis of T cell surface antigen CD4 PE and CD8 FITC in splenocytes. Staining of cells with T cell surface markers CD4 (FITC conjugated monoclonal antibody)and CD8 (PE-conjugated monoclonal antibody).

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essential to obtain a more efficient response against virus infectionsby helping achieve total viral elimination. Consequently, it may bedesirable for vaccines against viruses to elicit cell-mediated immunity,as well as producing effective antibodies. Superior and universaladjuvants must be developed that work with almost every antigen,induce both cellular and humoral immune responses (a balanced Th1/Th2 response). The extensive studies on plant based adjuvants such asQS21 saponins from plant Q. saponaria [38], RLJ-NE-299A glycosidesfrom Picrorhiza kurroa [39], and β(1→6)-branched β-(1→3) gluco-hexaose analog [40] indicate that these are excellent candidates toreplace alum as the adjuvant of choice for many vaccines. Particularadvantages offered by plant based proposed adjuvants in inducingcellular in addition to humoral immunity offer excellent safety,tolerability, ease of manufacture and formulation. Thus, adjuvantsbased on plants have enormous potential for use in vaccines againstboth pathogens and cancer [41].

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Fig. 7. Effect of proposed adjuvant BOS 2000 on co-stimulatory molecules CD80 (B7-1)and CD86 (B7-2). Flow cytometric analysis of the expression of co-stimulatory signalmolecules in spleen-derived macrophages. Data represented by percent CD80/CD86positive cell populations are mean±S.E. (n=5); *Pb0.05, **Pb0.01, ***Pb0.001(control vs. BOS 2000-treated groups; one-way ANOVA followed by Bonferronimultiple comparison test). For experimental details, refer to the Materials and methodssection.

Today, with the progress of vaccination, there is an urgent need forpotent adjuvants to accommodate with this situation. An idealadjuvant must promote an appropriate immune response. Aneffective T cell-mediated immunity, which can be shown by thestimulation of lymphocyte proliferation response, plays an importantrole to combat intracellular microbial infections. However, humoralimmunity, mediated by antibodies which are produced by B-lymphocytes, functions by neutralizing and eliminating extracellularmicrobes and microbial toxins [42]. Among the T lymphocytes, helperT cells induce B-lymphocytes to secrete antibodies. Helper T cells canbe divided into two subsets of effector cells, namely Th1 and Th2 cells.The Th1 cells secrete cytokines, such as interleukin-2 (IL-2), tumornecrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), andstimulate the production of IgG2a, IgG2b and IgG3 in mice. They areresponsible for cytotoxic T lymphocyte (CTL) production. On the otherhand, the Th2 subset produces cytokines, such as IL-4, IL-5 and IL-10,and stimulates the production of IgG1 and IgA [43,44]. The Th1response against intracellular pathogens and malignant cells issuperior to the Th2 response.

OVA is commonly used as a model for immunogen specific T and Bcell mediated immune function. BOS 2000 significantly enhanced OVAspecific IgG and its isotypes IgG1 and IgG2a titers were observed inimmunized mice compared with OVA control and standard alumgroup. It is generally known that Con A stimulates T cell proliferation[45,46]. When splenocytes separated frommice immunized with OVAwere exposed to BOS 2000, it is possible that lymphocytes recognizeand receive second signals and proposed adjuvant BOS 2000 couldfacilitate OVA specific lymphocytes to proliferate and differentiateinto effector cells and memory cells [42]. In addition, BOS 2000 couldenhance Con A for stimulating non-specific T lymphocyte prolifera-tion response by helper T cells.

Cell mediated immunity, mediated by T lymphocytes, plays animportant role to combat intracellular infections. Among the Tlymphocytes, helper T cells induce B lymphocytes to secrete antibodiesand cytotoxic T lymphocytes help phagocytes to destroy infectioninduced by pathogen and to kill intracellular microbes. Humoralimmunity, however mediated by antibodies which are produced by Blymphocytes, functions by neutralizing and eliminating extracellularmicrobes and microbial toxins. The capacity to elicit an effective T cellimmunity can be shown by the stimulation of lymphocyte proliferation

Table 1Effect of different doses of BOS 2000 on mouse body weight after different time periods.

S.NO.

Treatment Doses(μg)

Grams

Day 0 Day 7 Day 14 Day 28 Day 60

1 Control – 18.8±1.34 20.4±2.0 22.6±184 23.8±2.44 24.8±3.22 OVA 100 19.6±2.02 21.4±1.8 22.6±2.34 23.6±2.4 25.8±3.53 Alum 200 20.4±1.74 21.3±1.75 22.2±2.68 23.6±2.8 26.4±3.74 BOS 2000 10 18.6±1.88 21.6±2.42 22.6±2.42 23.2±2.6 26.6±3.15 BOS 2000 20 20.7±2.0 21.8±2.52 22.3±2.14 23.4±2.7 25.2±2.86 BOS 2000 40 19.8±1.86 21.8±2.72 22.3±2.2 23.1±2.1 26.6±3.47 BOS 2000 80 19.4±1.8 21.5±1.68 22.4±1.88 23.6±2.9 25.6±2.8

The results show the groups' weight (mean±S.E.) value in grams.

974 A. Gupta et al. / International Immunopharmacology 11 (2011) 968–975

response and cytokine estimation. The results indicated that BOS 2000could significantly increase the activation potential of T and B cells inOVA-immunized mice. BOS 2000 also had a significant stimulatoryeffect on CD4+andCD8+T cells, thereby confirming its general effect onthe cell-mediated immune response. Furthermore, co-stimulatorysignals are the most important secondary signals playing an importantrole in the cell–cell cross-talk for the delivery of the required immuneresponse. CD80 has been found to play key role inmaintaining the stateof immune response,whereas CD86 plays a role inmaintaining immunememory [47]. The expressions of CD80 and CD86 on splenic macro-phages of BOS 2000-treated mice were found significantly enhancedsupporting its potential of activating the antigen presenting cellsthrough co-stimulatory signals that eventually help in the generationof effective immune response by secreting various signal molecules likecytokines and chemokines.

Meanwhile, cytokine measurement also revealed that BOS 2000significantly promoted the production of the Th1 (IL-2/IFN-γ) and Th2(IL-4/IL-6/IL-10) cytokines in OVA-immunized mice. These resultssuggested that BOS 2000, be able to simultaneously stimulate Th1(IgG2a) and Th2 (IgG1) immune responses. In our conditions, BOS2000 enhanced significantly a specific antibody and cellular responseagainst OVA in mice. Acute toxicological studies with biopolymericfraction BOS 2000 were determined by oral administration in micewhere the LD50 was found to be greater than 1500 mg/kg.

In conclusion, two important features of BOS 2000 have beenclearly shown: (1) BOS 2000 is a strong Th1/Th2 adjuvant for the OVAin the mice model and (2) the immunogenicity of the BOS 2000-adjuvanted OVA is better than that of OVA adjuvanted with alum. TheOVA-specific antibody immune response induced by BOS 2000 wasassociated with higher antibody titers than that provided by the alumvaccine and moreover cell-mediated responses were elicited. Fur-thermore, BOS 2000 also significantly enhanced the population of cellsurface marker CD4/CD8 and co-stimulatory molecules CD80/CD86.Thus, BOS 2000 is a potent enhancer of antigen-specific humoral andcell-mediated immune responses, thus showing promise as immuneadjuvant for vaccines against intracellular infectious agents such asviruses, bacteria, protozoa and against cancer cells. Moreover, thedetailed investigations of immune adjuvant activity of BOS 2000 withspecific antigens are in progress. Further immunization and challengestudies with BOS 2000 adjuvanted with other specific antigens willhave to be performed to establish clinically relevant proof of principle.BOS 2000 is well tolerated in mice, and is of no toxicologicalconsequences and may find useful application clinically.

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