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A new vaccine adjuvant (BOS 2000) a potent enhancer mixed Th1/Th2 immune responses in mice immunized with HBsAg

A new vaccine adjuvant (BOS 2000) a potent enhancer mixed Th1/Th2 immune responses in mice immunized with HBsAg
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  Vaccine 25 (2007) 4586–4594 A new vaccine adjuvant (BOS 2000) a potent enhancer mixed Th1/Th2immune responses in mice immunized with HBsAg Anamika Khajuria a , ∗ , Amit Gupta a , Fayaz Malik  a , Surjeet Singh a , Jaswant Singh a ,B.D. Gupta b , K.A. Suri b , Pankaj Suden a , V.K. Srinivas c , Krishna Ella c , G.N. Qazi a , b a  Division of Pharmacology, Indian Institute of Integrative Medicine (CSIR, Jammu), Jammu Tawi 180001, India b  Division of Natural Products Chemistry, Indian Institute of Integrative Medicine (CSIR, Jammu), Jammu Tawi 180001, India c  Bharat Biotech International Limited Genome Valley, Turkapally, Shameerpet Mandal, Hyderabad 500078, A.P., India Received 24 January 2007; received in revised form 21 March 2007; accepted 26 March 2007Available online 23 April 2007 Abstract Adjuvants in vaccines are immune stimulants that play an important role in the induction of effective and appropriate immune responsesto vaccine component. In search of a potent vaccine adjuvant, the water-soluble biopolymeric fraction BOS 2000 from  Boswellia serrata was evaluated for desired activity. We investigated the ability of BOS 2000 to enhance HBsAg specific immune responses. The effect wasdetermined in the form of protective anti-HBsAg titers, neutralizing antibodies (IgG1 and IgG2a), spleen cell lymphocyte proliferation byusing MTT assay, Th1 (IFN-  and TNF-  ) and Th2 (IL-4) cytokines as well as T-lymphocyte subsets (CD4/CD8) and intracellular cytokines(IFN-   /IL-4), these responses were highest in BOS 2000 immunized mice. Alum induced only a modest enhancement of antibody responses.Reducing the dose of adjuvant by 18.1-fold in comparison to alum, total IgG and its subtypes (IgG1 and IgG2a) antibodies titer in serumwas significantly enhanced. Analysis of HBsAg specific cytokines revealed that alum was associated with a predominantly IL-4 response. Incontrast,BOS2000wasassociatedwithproductionofbothIFN-  andIL-4.WeconcludethatBOS2000isapotentenhancerofantigen-specificTh1 and Th2 immune responses in comparison to alum with Th2 limitation and is a promising adjuvant for vaccine applications.© 2007 Elsevier Ltd. All rights reserved. Keywords: Boswellia serrata ; Hepatitis B (HBsAg) surface antigen; Humoral and cellular 1. Introduction Vaccination remains the most cost-effective biomedicalapproach for the control of infectious diseases. Subunit vac-cines have a long track record of safety, but are often poorlyimmunogenic and require addition of adjuvants to enhancetheir efficacy. Currently, move towards the development of safer adjuvants for subunit vaccines has created a majorneed of vaccine adjuvants capable of boosting cellular (Th1)immunity but without unacceptable toxicity. Surprisingly,despite the description of over 100 adjuvants in the sci-entific literature, alum remains the only adjuvant approved ∗ Corresponding author at: Regional Research Laboratory, Canal Road,JammuTawi180001,India.Tel.:+919419123976;fax:+91548607/546383.  E-mail address: (A. Khajuria). for human use in the USA. Unfortunately limitation behindalum is that it has no effect on cellular immunity. Thereis a major unmet need for a safe and efficacious adjuvantcapable of boosting cellular and humoral immunity. Theextensive studies on plant based adjuvants such as QS21saponins from plant  Quillaja saponaria  [1] and glycosidesfrom  Picrorhiza kurroa  [2] indicate that these are excellentcandidatestoreplacealumastheadjuvantofchoiceformanyvaccines. Particular advantages offered by plant based pro-posed adjuvants in inducing cellular in addition to humoralimmunity offer excellent safety, tolerability, ease of man-ufacture and formulation. Thus, adjuvants based on plantshave enormous potential for use in vaccines against bothpathogens and cancer [3]. Some of the features involved in adjuvant selection are: the antigen, the species to be vac-cinated, the route of administration, the likelihood of side 0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.vaccine.2007.03.051   A. Khajuria et al. / Vaccine 25 (2007) 4586–4594  4587 effects and the requirement for a cell-mediated or humoralantibody response [4,5]. Ideally, adjuvants should promote an appropriate immune response (Th1 or Th2), be stablewith long shelf life, biodegradable, cheap to produce and notthemselves immunogenic [6].Developing efficient adjuvants for human vaccines thatelicit broad and sustained immune responses at systemic ormucosal levels remains a formidable challenge for the vac-cine industry. Conventional approaches in the past have beenlargelyempiricalandatbestpartiallysuccessful.Importantly,recent advances in our understanding of the immune system,most particularly with respect to early proinflammatory sig-nals,areleadingtotheidentificationofnewbiologicaltargetsfor vaccine adjuvants. BOS 2000, isolated from  Boswelliaserrata  having anti-arthritic [7,8], anti-inflammatory [9,10], immunomodulatory[11]andanticanceractivity[12–14]was considered for adjuvant testing in mice. A better understand-ingofthebiologyofnon-conventionalTcellsubpopulations,T and B cell memory, regulatory T cells and mucosal immu-nity has profound implications for a modern approach toadjuvant screening and development. The future lies in thehigh throughput screening of synthetic chemical entities tar-geting well-characterized biological molecules. Used aloneor in combination with such adjuvants will allow stimula-tion or modulation in a safe and efficient manner of strongeffector, regulatory and memory immune mechanisms.Ingeneral,polysaccharides[15–17]elicitaT-independent immuneresponse,characterizedbylackofmemoryandpoorimmunogenicity.BOS2000hasovercomethepoorimmuno-genicity of polysaccharides and has led to development of vaccine adjuvant because of its immunomodulatory activityand has exhibited anti-arthritic activity also [7]. It is possi- ble to induce a T-dependent immune response against theseantigens. The present study was undertaken to validate theimmunoadjuvanteffectsofBOS2000againstspecificantigenHBsAg. 2. Materials and methods 2.1. Animals Study was conducted on male BALB/c mice (18–22g).The institutional animal Ethics committee of the RegionalResearch Laboratory (CSIR, Jammu) approved all protocols.The animals were bred and maintained under standard hus-bandry conditions; viz. humidity, temperature (25 ± 2 ◦ C)and photoperiod of 12h. Commercial pellet diet and waterwere given  ad libitum . 2.2. Chemicals and reagents Ninety-six wells V shaped microtitration plates, micro-tissue culture plates (96U wells) and plastic wares fromNunc, USA. RPMI-1640 medium (Himedia, India) sup-plemented with 10% fetal calf serum, 2mM glutamine,penicillin 100IU/ml, streptomycin 100IU/ml, 1mM sodiumpyruvate and 2-mercaptoethanol (Sigma) was used forcell culture. 3-[(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl 2,5-dimethyltetrazoliumbromide(MTT),bovineserumalbumin,streptavidin-horse radish peroxidase, rabbit anti-mouse IgGperoxidase conjugate, HRP-conjugated swine anti-rabbitimmunoglobulin, biotinylated goat anti-mouse IgG1 andIgG2a conjugate, phorbol 12-myristate 13-acetate (PMA)and ionomycin were purchased from Sigma Chemicals,USA. Fluorosceinisothiocyanate (FITC)-labeled CD3 andIFN-   anti-mouse monoclonal antibody, phycoerytherin(PE)-labeled CD4, CD8 and IL-4 anti-mouse monoclonalantibody were purchased from BD pharmingen. 2.3. Antigen The hepatitis B surface antigen (HBsAg) used was therecombinant product and purified from  Pichia pastoris  atBharat Biotech International Limited Hyderabad, India. Thisantigenwasusedeitheralonewithoutadjuvant(referredtoasHBsAg) or adjuvanted with aluminium hydroxide as in theconventional marketed one (referred to as alum+HBsAg) orwith BOS 2000 (referred to as BOS 2000+HBsAg) in thisstudy. 2.4. Preparation of biopolymeric fraction (BOS 2000) The organic solvent exhausted material (0.5kg) of theplant  B.serrata wasthriceextractedwithmethanol(3 × 1.5l)at room temperature in a percolator. The marc 175g was airdriedandextractedtwicewithwaterusing700mlofwaterforthe first extraction and 200ml for the subsequent extraction.Thecombinedaqueousextractwasclarifiedbycentrifugationandtotheclearsolution(600ml)alcohol1.2lwasadded.Thelight brown solid, which separated on keeping, was collectedbyfiltrationandpurifiedbydissolvinginwaterandprecipitat-ing with alcohol. The purification process was repeated oncemore to get the biopolymeric fraction BOS 2000 as whitesolid (70g). 2.5. Hydrolysis of biopolymeric fraction (BOS 2000) The biopolymeric fraction BOS 2000 (1.0g) was sus-pended in 50ml of aqueous 2N-TFA and then refluxed(120 ◦ C) for 2.5h. The reaction mixture was concentratedunder reduced pressure on a film evaporator and then kept ina desiccator containing NaOH, overnight. Paper chromatog-raphy of the hydrolysed fraction BOS 2000 in comparisonwith reference monosaccharides revealed the presence of arabinose, glucose and galactose. 2.6. Quantitative analysis of monosaccharides in thehydrolysed BOS 2000 by HPLC  HPLC grade water was prepared from Milli-Q waterpurification system. All the four monosaccharides, i.e.  4588  A. Khajuria et al. / Vaccine 25 (2007) 4586–4594 d -glucose,  d -xylose,  d -galactose and  d -arabinose were pro-cured from Aldrich chemicals of purity ≤ 98% (HPLC). 2.7. Chromatography Monosaccharides were separated and quantified by usingshimadzu HPLC system consisting of Pump LC-10 AT VP ,an automatic sampling unit (Autosampler), SIL-10 AD VP ,a Column oven CTO-10 AS VP, RI   detector and Systemcontroller SCL-10 A VP  version 5.4. Shimadzu Class  VP software version 6.1 was used for data analysis and dataprocessing. The samples were analyzed at 80 ◦ C on a Phe-nomenex Rezex RPM-monosaccharide Pb++ (8%) column(300mm × 7.80mm) by RI detector using a gradient mobilephase of HPLC grade water. 2.8. Sample preparation The accurately weighed quantity of the dried hydrolysateofBOS2000wasdissolvedinknownvolumeofHPLCgradewater.Thesampleswerefilteredthroughmilliporemicrofilter(0.45  m) and then injected into the HPLC system. 2.9. Preparation of stock solutions and samples Stock solutions of the pure reference compounds wereprepared in HPLC grade water and stored in a refrigeratorat 4 ◦ C. From the stock solutions working solutions for eachreference compound were prepared by dilution with HPLCgrade water. These working solutions of all the referencecompoundsweremixedtogetherinequalvolumesforfurtheranalysis. 2.10. Quantification The compounds exhibiting linear response in the calibra-tion curves were prepared by using multipoint calibrationcurve method. Working solutions after mixing were injectedin different concentrations (2–20  l). Excellent calibra-tion curves were obtained for  d -glucose ( r  2 =0.99891), d -xylose ( r  2 =0.999425),  d -galactose ( r  2 =0.0999936) and d -arabinose ( r  2 =0.999976). Calibration curves were deter-mined on the basis of six levels (2–20  l) of concentration of each standard in the mixture.From HPLC of the hydrolysed (2 NTFA) biopolymericfraction in comparison to authentic monosaccharides it wasobserved that biopolymeric fraction BOS 2000 is composedof glucose (26.9), arabinose (37.0), galactose (33.1) (Fig. 1)in the molar ratio of 1.0, 4.2 and 21.4. 2.11. Mouse studies Male BALB/c mice were immunized intraperitoneallywith HBsAg 20  g dissolved in phosphate buffered solu-tion (PBS) containing alum (0.5mg/ml) in a final volumeof 1ml. All experiments were performed using 20  g of HBsAg.Thedoseresponseexperimentswerecarriedoutwithvarious doses of BOS 2000 (10, 20, 40 and 80  g) againstHBsAg immunized on days 0 and 15. Phosphate bufferedsolution-treated animals were included as controls. A chal-lenging injection was given on day 15. Sera and splenocyteswerecollected2weeksafterthesecondinjectionforHBsAg-specific antibody titers were obtained by ELISA and spleencell proliferation. 2.12. Measurement of HBsAg-specific antibody HBsAg-specific antibodies in serum were measured inindividualserumsamplesbyELISA.Microtiterplates(Nunc,USA) were coated with HBsAg (2  g/ml in 50mM carbon-ate buffer, pH 9.6) overnight at 37 ◦ C. For measurement of IgG, coated plates were incubated with pooled sera of testsamplesandrabbitanti-mouseIgGperoxidaseconjugateandHRP-conjugated swine anti-rabbit immunoglobulin (Sigma,USA). The enzyme reaction was visualized by incubationwith substrate buffer, the reaction was stopped after 10minby the addition of 50  l 2M H 2 SO 4  and the absorbance at450nm was measured. All washings were done with PBScontaining0.05%Tween(PBS-T).Serumsamplesandrabbitanti-mouse IgG peroxidase conjugated (Sigma, USA) weredilutedinPBS-T.Titerswereexpressedinmilli-internationalunits per ml (mIU/ml). 2.13. Estimation of IgG1 and IgG2a in sera by ELISA HBsAg-specificIgG1andIgG2aantibodiesinserumweredetected by an indirect ELISA. In brief, microtiter platewells (Nunc) were coated with HBsAg solution (2.5 and5  g/ml) for IgG1 and IgG2a antibodies, respectively, in50mM carbonate–bicarbonate buffer, pH 9.6) for 24h at4 ◦ C. The wells were washed three times with PBS contain-ing 0.05% Tween 20 (PBS/Tween), and then blocked with5% FCS/PBS at 37 ◦ C for 1h. After three washings, 100  lof diluted serum sample or 0.5% FCS/PBS as control wereadded to triplicate wells. The plates were then incubatedfor 1h at 37 ◦ C, followed by three times of washing. Next,horseradish peroxidase conjugated antibody against IgG1 orIgG2a was added to the wells, and incubated for 1h at 37 ◦ C.After washing, the peroxidase activity was assayed as fol-lows:100  lofsubstratesolution(TMBH 2 O 2 )wasaddedtoeach well. The plate was incubated for 10min at 37 ◦ C, andenzyme reaction was terminated by adding 50  l/well of 2NH 2 SO 4 .Theopticaldensity(OD)wasmeasuredinanELISAreader at 450nm. 2.14. Viability assay The viability of cells was determined by the standard try-pan blue exclusion assay. Approximately 2 × 10 6 cells/mlwere treated with two-fold diluted concentration from 10 to80  g/mlofBOS2000andcontrolswith0.1%ofethanolfor3daysat37 ◦ Cinahumidifiedatmospherecontaining5%CO 2 .   A. Khajuria et al. / Vaccine 25 (2007) 4586–4594  4589Fig. 1. HPLC chromatogram of (A) standards and (B) BOS 2000. A visual count was then made of the number of live (white)versus dead (blue) cells using a hemocytometer followingstaining by trypan blue (0.4% in normal saline–0.9% NaCl)and the percentage of live versus dead cells determined. 2.15. Splenocyte proliferation assay Spleen collected from the HBsAg-immunized mice underaseptic conditions, in Hank’s balanced salt solution (HBSS,Sigma), was minced using a pair of scissors and passedthrough a fine steel mesh to obtain a homogeneous cellsuspension, and the erythrocytes were lysed with ammo-nium chloride (0.8%, w/v). After centrifugation (380 × g at 4 ◦ C for 10min), the pelleted cells were washed threetimes in PBS, and resuspended in complete medium [RPMI1640 supplemented with 12mM HEPES (pH 7.1), 0.05mM2-mercaptoethanol, 100IU/ml penicillin, 100  g/ml strep-tomycin, and 10% FCS]. Cell numbers were counted witha haemocytometer by trypan blue dye exclusion technique.Cell viability exceeded 95% [18]. Briefly, splenocytes were seeded into three to four wells of a 96-well flat-bottommicrotiterplate(Nunc)at2 × 10 6 cells/mlin100  lcompletemedium, thereafter HBsAg (final concentration 0.5  g/ml),or medium were added giving a final volume of 200  l. Theplates were incubated at 37 ◦ C in a humid atmosphere with5% CO 2 . After 72h, 50  l of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) solution (5mg/ml)was added to each well and incubated for further 4h. The  4590  A. Khajuria et al. / Vaccine 25 (2007) 4586–4594 plates were centrifuged (1400 × g , 5min) and the untrans-formed MTT was removed carefully by pipetting 200  l of aDMSO (Sigma, USA) working solution (192  l DMSO with8  l 1N HCl) was added to each well, and the absorbancewas evaluated in an ELISA reader at 570nm after 15min. 2.16. Cytokine measurements by ELISA Splenocytes were seeded into three to four wells of a 96-well flat-bottom microtiter plate (Nunc) at 2 × 10 6 cells/ml.Cells were incubated with or without HBsAg (0.5  g/ml)for 72h at 37 ◦ C in 5% CO 2 . The culture supernatants wereharvested for the detection of cytokines (IFN-  , TNF-   andIL-4) by ELISA [19,20], according to the protocol of the manufacturer (R&D Systems, USA). 2.17. Spleen T-cell subtyping The expression of CD3 FITC (clone 17A2), CD4 (cloneL3T4) and CD8 (clone Ly-2) on cellular surface wasevaluated by monitoring the fluorimetric changes of the cor-responding FITC/PE conjugated monoclonal antibody witha flow cytometer. Splenic lymphocytes (2 × 10 6 cells/ml)were cultured in the presence of BOS 2000 (10–80  g/ml)in a 96 well plate. After 3 days, splenic lymphocytes werestained with 100  l of blocking buffer (PBS containing 1%bovine serum albumin and 0.1% NaN 3 ) containing 5  l of FITC conjugated anti-CD3 and followed by simultaneousstaining of 5  l of PE anti-CD4 and CD8 mAb and incu-bated for 30min at 4 ◦ C. Analysis was carried out by theFACScan flow cytometer (BD, LSR) using the Cell Questsoftware. 2.18. Intracellular cytokine detection by flowcytometry Splenocytes (2 × 10 6 cells/ml) were incubated for 24hwith or without HBsAg (0.5  g/well) for antigen-specificre-stimulation. After incubation, the cells were washed withPBS and then stained with the indicated surface markers,as described above. The detection of intracellular cytokinestaining was as previously described [21]. Briefly, samples were stimulated with 20ng/ml PMA plus 500ng/ml iono-mycin (Sigma, USA) for 4h at 37 ◦ C, the last 2h in thepresence of monensin (2  M; Sigma). After fixation in 4%formaldehyde for 20min, cells were stained intracellularlywith FITC-conjugated anti-mouse IFN-   antibody (cloneXMG1.2) and/or PE-conjugated anti-mouse IL-4 antibody(clone 11B) in the presence of 0.5% saponin for cell perme-abilization (Sigma, USA). 2.19. Statistical analysis Data were expressed as mean ± S.E.M. and statisticalanalysis was carried out using one-way ANOVA (Bonferronicorrection multiple comparison test). Fig. 2. Dose-dependent increase of anti-HBsAg antibody titer in serum byadjuvant BOS 2000. Groups of BALB/c mice ( n =10) were immunizedintraperitoneally with 20  g HBsAg alone or associated with variable dosesof BOS 2000. Serum anti-HBsAg antibody (IgG) titer in BALB/c mice aftertwo immunizations on day 0 and 15 and bled out at 2 weeks post immu-nization. Anti-HBsAg titer was determined by ELISA Kit. *** P <0.001significant against the groups immunized by BOS 2000 (80  g) adjuvantedwith20  gHBsAg.** P <0.01significantagainstthegroupsofmiceimmu-nized with 20  g HBsAg containing BOS 2000 (40  g) and ** P <0.05significant against the groups of mice immunized with 20  g HBsAg con-taining BOS 2000 (10 and 20  g) and alum (0.5mg/ml). 3. Results 3.1. Adjuvant effective dose of BOS 2000 (anti-HBsAgtiter) The serum anti-HBsAg IgG titer was assayed by ELISAand titers obtained after challenging immunization are pre-sented in Fig. 2. The dose response curve for BOS 2000 reveals that the production of anti-HBsAg antibodies wasstrongly enhanced in mice treated with a dose of 80  g BOS2000 in comparison with HBsAg alone or associated withalum. Those vaccines containing adjuvant especially BOS2000inHBsAgthatelicitthehighestanti-HBsAgtiterswouldbepredictedtomaintainthesetitersatprotectivelevelsforthelongestperiodsoftime.Moreover,antibodytitersinducedbyBOS 2000 adjuvanted HBsAg after two injections were stillhigher than those elicited by alum-adjuvanted vaccine. 3.2. Adjuvant (BOS 2000) mediated elevation of antigen-specific immunoglobulin subtypes (IgG1 and  IgG2a) in mice sera The HBsAg-specific IgG1 and IgG2a antibody titers inthe serum were measured by indirect ELISA as shown inFig. 3. The serum IgG (IgG1 and IgG2a) titer in HBsAg-immunized mice was significantly enhanced by BOS 2000(80  g). Alum (0.5mg/ml) and BOS 2000 (80  g) signifi-cantly enhanced the serum IgG1 titers in HBsAg-immunizedmice. Significant enhancements in HBsAg-specific serumIgG2a titers were observed in BOS 2000 (80  g) immu-nized mice compared with control group. Moreover, IgG2a
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