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Clinical Immunology (2007) 125, 173–183

Supplementation with RD antigens enhances theprotective efficacy of BCG in tuberculous miceMamta Kalra a,1, Ajay Grover a, Neena Mehta a, Jaspreet Singh a,Jaspreet Kaur a, Suraj B. Sable a, Digambar Behera b, Pawan Sharma c,Indu Verma a,⁎, G.K. Khuller a

a Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, Indiab Department of Pulmonary Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, Indiac Immunology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi-110 064, India

Received 23 February 2007; accepted with revision 17 July 2007Available online 4 September 2007

⁎ Corresponding author. Fax: +91 017E-mail address: induvermabio@red

1 Present address: Department of BiOklahoma State University, Centre for74107, USA.

1521-6616/$ – see front matter © 200doi:10.1016/j.clim.2007.07.007

Abstract Different combinations of ESAT-6, CFP-10, CFP-21, MPT-64, encoded by RD1 and RD2of Mycobacterium tuberculosis were evaluated on the basis of antigenicity in PPD positive TBcontacts and immunogenicity in C57BL/6J mice immunized with the combination of all four RDantigens. The peripheral blood mononuclear cells of TB contacts showed maximum recognition inresponse to the combination of ESAT-6+MPT-64 in terms of predominant lymphoproliferation,IFN-γ levels and the number of responders. On the contrary, the combination of ESAT-6+CFP-21+MPT-64 was found to be most immunogenic based on both T-cell and antibody responses inimmunized mice. Prophylactic potential of the selected combinations was assessed assupplementation vaccines to BCG against intravenous challenge with M. tuberculosis in mice.BCG supplementation with the selected combinations resulted in significantly greater protectionas compared to BCG alone against experimental tuberculosis and thus appears to be a promisingapproach to enhance the protective efficacy of the existing vaccine.© 2007 Elsevier Inc. All rights reserved.

KEYWORDSBCG supplementation;Multicomponent vaccine;RD antigens

Introduction

Tuberculosis [TB] has reemerged over the past few years asthe most dreaded disease of all microbial infections. Myco-bacterium bovis BCG is the only vaccine currently available

2 744401.iffmail.com (I. Verma).ochemistry and Microbiology,Health Sciences, Tulsa, OK-

7 Elsevier Inc. All rights reserved

against TB for human use. The large percentage of world’stotal population is today vaccinated with BCG, making itone of the most widely used vaccines. However, the vaccinehas failed to confer total and permanent immunity in adults[1]. Consequently, one-third of the world’s populationpresently harbors latent tuberculosis infection that leadsto 8–12 million new cases of active TB each year [2]. Thesecases in turn result in 2–3 million deaths annually due tothe lack of early and accurate diagnosis and the limitationsof the current therapeutic regimen. The scenario furtherworsened by the onset of HIV epidemic and the emergence

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174 M. Kalra et al.

of multidrug resistant strains warrants serious considerationfor the development of improved and more reliable anti-TBvaccine.

Deciphering of the whole genome sequence of Myco-bacterium tuberculosis [3] allied with comparative andfunctional genomic analyses has led to the identification of16 genomic regions that are absent from BCG and most ofthe environmental mycobacteria [4,5]. These DNA se-quences, designated as Regions of Difference (RD1–16) areknown to encode many putative molecules relevant fordesigning improved diagnostic and prophylactic strategies[4,5,6]. Few well characterized proteins like ESAT-6, CFP-10, CFP-21 and MPT-64 are encoded by RD1 and RD2 of M.tuberculosis genome. Loss of RD1 has been implicated to bethe primary deletion that led to the attenuation of M. bovisand generation of vaccine strain BCG [7]. However, con-tinued in vitro passaging subsequently led to more deletionsfrom the genome of BCG resulting in over-attenuation of thevaccine [8,9]. The loss of genomic regions during in vitropassaging is believed to delete not only the virulencefactors but also certain key protective antigens, henceresulting in impotency of BCG [9,10]. Therefore, supple-menting BCG with the missing immunodominant RD antigensappears to be a promising strategy to restore its prophy-lactic potential.

It is therefore worthwhile to investigate the role ofimmunodominant RD antigens as novel vaccine candidates.To ensure efficient recognition in heterogeneous popula-tions of varied haplotypes multicomponent formulations ofRD antigens should be evaluated. However, combining onlywell defined antigens, which do not interfere immu-nologically with each other, is desirable. Recent reportssuggest the existence of antigenic competition betweenvarious components of multivalent vaccines which results inreduced immunogenicity and protective efficacy of immu-nodominant mycobacterial antigens [11,12]. Hence, when ithas become important to design multicomponent antigenicpreparations, it is even more crucial to evaluate theindividual components of the cocktail and their all possiblecombinations not only for mutual potentiation/synergismbut also for interference in the immunological perfor-mance. In view of the above facts, the present study wasplanned to screen various combinations of four immunodo-minant RD antigens i.e. ESAT-6, CFP-10, CFP-21 and MPT-64on the basis of antigenicity and immunogenicity. Even-tually, immunoprophylactic potential of the best combina-tions was evaluated as subunit supplementation vaccines toBCG.

Materials and methods

Animals

Four to five weeks old female C57BL/6J (H-2b) mice (weigh-ing 15–20 g) obtained from National Centre for LaboratoryAnimal Sciences, National Institute of Nutrition (NIN),Hyderabad, were used in the study. Animals were housed innegative pressure regulated animal isolators and were fed onthe standard pellet diet (Hindustan Lever Ltd., Mumbai) andwater ad libitum. The study was approved by the Institu-tional Animal Ethics Committee.

Study population

Healthy medical, paramedical and laboratory subjects(n=11; 7 males [mean age, 30±5] and 4 females [meanage, 28±4]) working at Postgraduate Institute of MedicalEducation and Research, Chandigarh, India, who were indirect contact with TB patients or their biological samples/cultures for more than 6 months, were recruited as hospitalcontacts. All TB contacts had no previous history of TB andwere BCG vaccinated and HIV seronegative. Seventy threepercent of TB contacts showed ESAT-6-specific lymphoproli-feration (stimulation index (SI) N3) and all TB contacts werepositive for Siebert purified protein derivative of tuberculin(PPD) with a median induration response of 20 mm andminimum induration readings of 15 mm in the Mantoux test.Blood samples were withdrawn from the study subjects byvenipuncture with prior consent. The study was approved bythe Institutional Ethics Committee.

Antigens

Native ESAT-6, CFP-10, CFP-21 and MPT-64 were purifiedfrom the culture filtrate (RvCFP) of M. tuberculosis H37Rv byanion exchange chromatography and preparative SDS-PAGEfollowed by electroelution as described elsewhere [13].Purity and apparent molecular mass were determined bySDS-PAGE analysis and absolute identity was established byELISA using MoAbs or by LC-MS-MS [13]. Monoclonal/mono-specific antibodies HYB76-8 (anti-ESAT-6), K8493 (anti-CFP-10), K8483 (anti-CFP-21) and L24B4 (anti-MPT-64) were akind gift by I. Rosenkrands, Statens Serum Institute, Copen-hagen, Denmark.

For immunogenicity and protection studies, recombinantESAT-6, CFP-10 and CFP-21 were purified from the clonesgenerated and maintained in our laboratory using Ni-NTA affi-nity chromatography [14]. Recombinant MPT-64 was obtainedfrom Dr. Karen Dobos (Colorado State University, Colorado).

PBMC proliferation assay

Mononuclear cells were isolated from the peripheral bloodof donors by Ficoll-Hypaque density centrifugation asdescribed elsewhere [13]. PBMC (2×105) were seeded in96-well flat-bottom sterile tissue culture plates (GreinerBio-One, Germany) in 0.2 ml of complete RPMI-1640supplemented with 100 IU/ml penicillin, 50 μg/ml strepto-mycin, 1 mM L-glutamine (all from Sigma, St. Louis, MO),25 mM HEPES (Fluka, Switzerland), 1 mM sodium pyruvate(SRL, Mumbai, India), 5×10−5 M mercaptoethanol, and 10%heat inactivated autologous serum. Cells were cultured induplicate in either medium alone, or in the presence ofdifferent combinations of four antigens (2 μg/ml each) orPPD/RvCFP (2 μg/ml) at 37 °C in a humidified atmospherecontaining 5% CO2. Phytohemagglutinin (PHA, 1 μg/ml)(Sigma, St. Louis, MO) was used as a positive control. After5 days, cells were pulsed with [3H] thymidine (0.25 μCi/well,Bhabha Atomic Research Centre, Mumbai, India) and har-vested onto glass fiber filters after 18–22 h. The incorpo-rated radioactivity was measured and stimulation indices(SI) were calculated by dividing mean counts per minute inantigen-stimulated wells by mean counts per minute in un-stimulated wells.

175Experimental BCG supplementation vaccine

Estimation of IFN-γ levels from PBMCculture-supernatants

The concentration of IFN-γ released in response to in vitrostimulation with different combinations in PBMC culturesupernatants was determined using a commercial ELISA withspecific pairs of monoclonal antibodies (Opt EIA™ BD Phar-mingen Inc., San Diego, CA, USA) as per manufacturer’s spe-cifications. The detection limit of the assay was 2.35 pg/ml.

Immunization of animals

Mice (4–5 per group) were immunized with the combinationof ESAT-6, CFP-10, CFP-21 and MPT-64 (10 μg each/dose/animal) emulsified in dimethyldioctadecyl ammonium bro-mide (DDA, 250 μg per dose) coadjuvanted with monopho-sphoryl lipid A (25 μg/dose). Both MPL and DDA emulsionswere prepared as described previously [15] and mixed witheither PBS or the four M. tuberculosis antigens. The antigenformulation was given subcutaneously on the back of animalsas three biweekly doses. Immune responses were studied4 weeks post immunization.

Splenocyte proliferation assay

Lymphoproliferative responses were determined by tritiatedthymidine uptake assay as described earlier [16]. Immunizedmice were sacrificed and spleens were removed aseptically.The splenocytes were isolated and RBCs were lysed using0.84% sterile NH4Cl solution. After 3 washings, the splenicmononuclear cells were finally suspended in RPMI-1640medium supplemented with antibiotics and 10% heatinactivated (v/v) fetal calf serum (Sigma, St. Louis, MO,USA). The cells were cultured in the presence of eithermedium alone or mixtures of RD antigens (2 μg/ml each) orRvCFP/PPD (2 μg/ml each). Concanavalin-A (1 μg/ml) servedas a positive control for cell viability. After incubation for4 days, tritiated thymidine was added and cells wereharvested to determine the amount of incorporated radio-activity as described for human studies.

Estimation of IFN-γ and IL-4 levels from splenocyteculture supernatants

Supernatants of the splenocyte cultures were collected after96 h of incubation for measuring IFN-γ and IL-4 levels inducedin response to various antigenic combinations. The cytokineswere estimated by ELISA using commercially available kits(Opt EIA™ Set BD Pharmingen, CA, USA) following manufac-turers’ instructions. The detection limits of the assays forIFN-γ and IL-4 were 31.3 pg/ml and 7.8 pg/ml respectively.

Detection of IL-12 (p40 levels)

Peritoneal exudates cells (PEC) were obtained by flushing theperitoneal cavities of C57BL/6J mice repeatedly with 5 ml ofRPMI-1640. The cells were washed thrice and seeded in 96well tissue culture plates at a density of 2×105 cells/ml.Antigen combinations (2 μg/ml each), RvCFP (2 μg/ml) andLPS (5 μg/ml) were added for stimulation. After 24 h of

incubation at 37 °C in 5% CO2 supernatants were assayed forthe levels of IL-12 (p40) using commercially available ELISAkit (Opt EIA™ set, BD Pharmingen, USA). The detection limitof the assay was 15.6 pg/ml.

IgG isotyping

IgG1 and IgG2b titers were determined in the sera ofimmunized and control mice by indirect ELISA. Briefly, thewells of ELISA plates were coated with different combina-tions of RD antigens at a concentration of 2 μg/ml incarbonate buffer (pH 9.6). Following overnight incubation at4 °C, plates were washed thrice with PBS containing 0.05%Tween-20 (PBS-T) and blocked with 3% BSA at 37 °C for 2h.Sera of immunized and control animals were added at 1:100dilution in PBS-T having 1% BSA. After 2 h, plates werewashed and 1:100 diluted goat anti-mouse IgG1 and IgG2b(Sigma, St. Louis, MO, USA) were added. Finally, the wellswere incubated with horseradish peroxidase labeled anti-goat IgG at 37 °C for 30 min. The reaction was developedusing o-phenylenediamine in citrate buffer (pH-5.0) andabsorbance was read at 492 nm.

Infection of mice and CFU enumeration

Mice were immunized with experimental vaccines as des-cribed above. BCG was given subcutaneously as a single doseof 106 CFUs/animal at the time of first subunit vaccination. Insupplementation group, BCG was administered as a separatesubcutaneous injection given side by side with the first doseof subunit vaccine followed by two biweekly boosters of thesubunit vaccine only. Animals were challenged intravenouslywith M. tuberculosis H37Rv (1×105 bacilli) 8 weeks after thefirst immunization. Infected animals were sacrificed 4 weeksafter the challenge and lungs and spleens were removedaseptically. The organs were homogenized and appropriateserial dilutions were spread on Middlebrook 7H11 agar platessupplemented with oleic acid–albumin–dextrose–catalase(OADC). To selectively inhibit the growth of the residual BCGbacteria in the test organs, 2-thiophenecarboxylicacidhydrazide (2 μg/ml) was added to media. Colonies werecounted after 2–3 weeks of incubation at 37 °C and bacterialcounts per organ were determined.

Statistical analyses

Two-way comparison between test and control group wascarried out using Student’s t-test. Multiple comparisonsamongst different groups were performed by analysis ofvariance (ANOVA). The statistical analyses were made usingSigmaStat software version 3.1. A p-value of b0.05 wasconsidered significant.

Results

Antigenicity studies in PPD positive TB contacts

Considering TB contacts to represent a model of immunity totuberculosis, different possible combinations of four RDantigens ESAT-6, CFP-10, CFP-21 and MPT-64 were evaluatedfor their antigenicity in PPD positive contacts to select thebest combination exhibiting favorable immunological inter-

Figure 1 (a) Lymphocyte proliferation and (b) interferon(IFN)-γ responses of healthy tuberculosis contacts (n=11) afterin vitro stimulation with different combinations of ESAT-6,CFP-10, CFP-21 and MPT-64. The median induration response ofstudy subjects in tuberculin skin test was 20 mm. Each symbolrepresents one individual and short horizontal bars represent themedian values. The median counts per minute (cpm) of cultureswithout antigen was 789. The median stimulation indices (SI) inresponse to phytohaemagglutinin (PHA), culture filtrate of My-cobacterium tuberculosis H37Rv (RvCFP) and purified proteinderivative of tuberculin (PPD) were 27.64 (interquartile range(IQR) 16.05–62.4), 12.34 (IQR 3.84–26.4) and 8.5 (IQR 6.20–21.6), respectively. The median IFN-γ response of cultureswithout antigen was 18.4 pg ml−1. The IFN-γ levels released intolymphocyte culture supernatants in response to in vitrostimulation with PHA, RvCFP and PPD were 1910.00 (IQR950.50–2860.00), 428.50 (IQR 182.25–1675.00) and 235.00(IQR 72.5–1260.00) pg ml−1, respectively. E6 — ESAT-6, C10 —CFP-10, C21 — CFP21, M64 — MPT-64. Each combination is alsorepresented by a number given above the respective column.

176 M. Kalra et al.

actions among its various components to generate an optimalimmune response. Peripheral blood mononuclear cellresponses of healthy BCG vaccinated, PPD positive (Medianinduration 20 mm) contacts were monitored in terms oflymphocyte proliferation and IFN-γ induction.

Fig. 1a describes the lymphocyte proliferation resultsexpressed as stimulation indices (SI). Median SI values andpercentage recognition taking SI N3.0 as cut off [17,18] werefound to be high for the combinations ESAT-6+CFP-10+CFP-21+MPT-64 (Median 15.54; interquartile range (IQR) 3.81–21.72, percentage recognition 72.7%), CFP-10+CFP-21+MPT-64 (Median 15.0; IQR 3.40–19.74, percentage recognition72.7%), ESAT-6+MPT-64 (Median 13.41; IQR 6.613–22.43,percentage recognition 90.9%) and CFP-10+CFP-21 (Median13.36; IQR 3.38–20.80, percentage recognition 72.7%),however, the differences were not found to be statisticallysignificant. The combinations ESAT-6+CFP-10 (Median 3.75,IQR 2.18–12.43) and ESAT-6+CFP-10+MPT-64 (Median 3.48,IQR 2.63–17.71) showed lower proliferative responses ascompared to other combinations.

The IFN-γ levels released in response to each combinationare depicted in Fig. 1b. Mean IFN-γ levels of unstimulatedwells plus three times standard deviation was selected as cutoff for calculating the percentage recognition of variousantigen combinations [19,20] thus giving a very high pro-bability that any response above this level was antigenspecific. Maximum IFN-γ was detected for combination ofESAT-6 +MPT-64 (Median 236 pg ml− 1, IQR 130.75–1429.00 pg ml−1, percentage recognition 81.8%) followedby CFP-10+CFP-21 (Median 116 pg ml− 1, IQR 42.75–720.00 pg ml−1, percentage recognition 63.6%), CFP-10+CFP-21 + MPT-64 (Median 94 pg ml− 1 IQR 54.25–1067.5 pg ml−1, percentage recognition 72.7%) and thecombination of ESAT-6+CFP-10+CFP-21+MPT-64 (Median103 pg ml−1, IQR 46.7–902.50 pg ml−1, percentage respon-ders 72.7%) although differences were not statisticallysignificant. Similar to proliferation responses, the medianIFN-γ levels for the combination of ESAT-6+CFP-10 (Median49.00 pg ml−1, IQR 39.6–193.25 pg ml−1) and ESAT-6+CFP-10+MPT-64 (Median 48.00 pg ml−1, IQR 45.25–563.5 pg ml−1)were found to be the lowest of all other combinations.

In view of the lack of significant differences amongdifferent combinations, the selection of the best combina-tion based on antigenicity studies was made taking medianSIN10 and median IFN-γN200 pg/ml as arbitrary cut offs[13,21]. Although, four combinations showed median SIvalues above 10 but levels of IFN-γ were above the cut offfor the combination of ESAT-6+MPT-64 only (Median SI 13.41,Median IFN-γ 236 pg ml−1). This combination also hadmaximum number of responders on the basis of bothlymphocyte proliferation (90.9%) as well as IFN-γ level(81.8%) estimation. Thus, based on recognition in humanimmune subjects, the combination of ESAT-6 and MPT-64 wasselected for further evaluation in protection experiments.

Immunogenicity studies in C57BL/6J mice

To assess the immunogenic potential of M. tuberculosis spe-cific RD antigens, C57BL/6J mice were immunized with thecombination of all four RD antigens i.e. ESAT-6+CFP-10+CFP-21+MPT-64 emulsified in DDA co-adjuvanted with MPL. Fourweeks post immunization, cellular and humoral immune

parameters were studied in response to various combinationsof the selected RD antigens in in vitro assays.

Fig. 2a describes the capacity of RD antigens to inducelymphocyte proliferation as measured by thymidine uptakeassay. Counts per minute in immunized group were found tobe significantly higher than those in the control group irres-pective of the antigenic combination used (pb0.0001).However, within the immunized group, the mean counts per

177Experimental BCG supplementation vaccine

minute of CFP-21+MPT-64 (5792.44±1176.46) were found tobe the maximum followed by the combinations ESAT-6+CFP-21+MPT-64 (Mean 4299.00±337.8), ESAT-6+CFP-10+CFP-21+MPT-64 (Mean 4053.20±441.0) and CFP-10+CFP-21+MPT-64 (Mean 3998.2±534.5). As observed in human studies thelymphoproliferative responses of the combinations ESAT-6+CFP-10 and ESAT-6+CFP-10+MPT-64 were lower than rest of thecombinations and significant difference was observed withrespect to combinations 6, 9, 10 and 11 (Fig. 2a).

As observed in case of stimulation indices, the levels ofIFN-γ produced by the in vitro stimulation of splenocytes ofimmunized group were found to be significantly higher thanthose observed for the control group (pb0.0001). However,with in the immunized group, the highest IFN-γ levels wereelicited by the combination of ESAT-6+CFP-21+MPT-64 (Fig.2b). The mean IFN-γ (750.5±102.4 pg ml−1) of this com-bination was found to be significantly higher (pb0.001) thanall other combinations except combination 6, CFP-21+MPT-64 (620.8±126.5 pg ml−1).

The levels of IL-12 (p40) and IL-4 representing theactivation of Th1 and Th2 immunity are shown in Figs. 3aand b. Levels of IL-12 (p40) induced in response to in vitrostimulation of peritoneal exudate cells of C57BL/6J micewith different combinations of RD antigens are shown in

Fig. 3a. Maximum IL-12 (p40) production was evident inresponse to the combination of ESAT-6+ CFP-21+MPT-64 (Mean372.0±36.0 pg ml−1) followed by ESAT-6+CFP-10+CFP-21+MPT-64 (Mean 176.5± 24.2 pg ml−1), CFP-21+MPT-64(168.5 ± 7.76 pg ml− 1) and ESAT-6 + CFP-21 (163.1 ±15.2 pg ml−1). The IL-12 (p40) levels of the combination ESAT-6+CFP-21+MPT-64 were found to be significantly higher ascompared to all other combinations (pb0.001). Moderate levelsof IL-4 were also observed in response to all combinations withmaximum amount induced by the combination ESAT-6+CFP-10+MPT-64 (pb 0.001 compared to all other combinations)followed by ESAT-6+CFP-21+MPT-64 (pb0.001 with respect toall other combination except combination 8) (Fig. 3b). Thelymphocytes of control animals failed to produce detectablelevels of IL-4 irrespective of the combination used.

Titers of antigen-specific IgG1 and IgG2b isotypes wereevaluated in the sera of immunized as well as control mice byindirect ELISA to study the humoral responses. As depicted inFig. 4, there was marked production of IgG2b in response toall antigenic formulations. Substantial levels of IgG1 werealso detected in the sera of immunized mice, however, thesewere lower than the IgG2b. Notably, the extent of IgG1 andIgG2b induction was found to be equivalent in case of thecombination of ESAT-6+CFP-10+MPT-64 (ratio IgG2b/IgG10.94) and ESAT-6+CFP-21+MPT-64 (ratio IgG2b/IgG1 1.24),indicating a balanced Th1 and Th2 response. The levels of RDantigens specific antibodies in the sera of control mice weresignificantly lower than immunized mice (pb0.0001).

Thus, comparing all the immunological parameters, thecombination of ESAT-6+CFP-21+MPT-64 appears to containoptimal combination of antigens to activate both Th1 as well

Figure 2 (a) Splenocyte proliferative and (b) IFN-γ responsesof mice immunized with the combination of ESAT-6+CFP-10+CFP-21+MPT-64 using DDA-MPL as adjuvants in response to invitro stimulation with different combinations of RD antigens4 weeks post immunization. The results are expressed as mean±S.D. of counts perminute and IFN-γ (pgml−1) of four animals. Thecounts per minute (mean±S.D.) of cultures without antigen were534±84 and themean counts perminute of Con-A, RvCFPand PPDwere 12550±680, 8860±580 and 7652±428 respectively. Themean IFN-γ responses with ConA, RvCFP and PPD were5482 pg ml−1, 2150 pg ml−1 and 1760 pg ml−1, respectively.IFN-γ levels in unstimulated wells (cultures without antigen)were below 31.3 pg/ml. E6 — ESAT-6, C10 — CFP-10, C21 —CFP21, M64 — MPT-64. Each combination is also represented by anumber given above the respective column. Symbols representthe statistical significance calculated by ANOVA applyingpairwise multiple comparison procedures (Tukey's test). Com-parison of all groups showed that differences between differentantigenic combinations were statistically significant (pb0.001)in case of both SI and IFN-γ. The statistical significance of topthree combinations for each parameter is depicted. ***pb0.001w.r.t. combinations 1, 2, 3, 4, 5, 7,8 and pb0.05 w.r.t. combina-tions 10, 11. **pb0.001 w.r.t. combinations 1, 8 and pb0.05 w.r.t.combinations 5, 7. *pb0.01 w.r.t. combination 1,8. +++pb0.001w.r.t. all combinations except combination 6. ++pb0.001 w.r.t.combinations 1, 2, 3, 4, 5, 10, 11 and pb0.01 w.r.t. combination8. +pb0.001 w.r.t. combinations 1, 4; pb0.01 w.r.t. combination11 and pb0.05 w.r.t. combinations 2, 3.

178 M. Kalra et al.

as Th2 arms of cellular immunity and was further evaluatedfor protection studies.

Immunoprophylaxis studies with selectedcombinations

Protective efficacies of the experimental vaccines consti-tuted on the basis of antigenicity studies in humans andimmunogenicity studies in mice were evaluated both as in-dependent subunit vaccines and as supplementation vac-cines to BCG. The immunoprophylactic potential wasassessed by estimating the colony forming units (CFU), inthe lungs and spleen of mice 4 weeks after intravenouschallenge with M. tuberculosis H37Rv. The log10 CFUrecovered from the organs in different groups are shown inTable 1. All vaccinated animals showed significantly lessbacillary load as compared to unvaccinated group both at thelevel of spleen and lungs. The log 10 CFU in BCG immunizedanimals was found to be comparable to the mice immunizedwith the combination of ESAT-6+CFP-21+MPT-64. Thus,indicating that level of protection imparted by the lattercombination was the same as that shown by BCG. However,the combination ESAT-6+MPT-64 selected on the basis ofhuman studies imparted less protection than BCG as well as

Figure 3 Levels of (a) IL-12 (p40) and (b) IL-4 elicited byvarious combinations of RD antigens in culture supernatants ofperitoneal exudates cells and splenocytes respectively of miceimmunized with the combination of ESAT-6+CFP-10+CFP-21+MPT-64 at 4 weeks p.im. The IL-12 (p40) production inunstimulated wells (cultures without antigen) was below15.6 pg ml−1. Mean IL-12 (p40) responses of LPS and RvCFPwere found to be 740 pg ml−1 and 450 pg ml−1 respectively. IL-4production in unstimulated wells (cultures without antigen) wasbelow 7.8 pg ml−1. The results are expressed as mean cytokinelevels (pg ml−1) ± standard deviation observed in antigenstimulated wells of four animals. The IL-4 responses of RvCFPand PPD were 108 pg/ml and 94 pg/ml, respectively. E6 — ESAT-6, C10 — CFP-10, C21 — CFP21, M64 — MPT-64. Each combinationis also represented by a number given above the respectivecolumn. Symbols represent the statistical significance calcu-lated by one-way ANOVA on ranks applying pairwise multiplecomparison procedures (Tukey’s test). Comparison of all groupsshowed that differences between different antigenic combina-tions were statistically significant (pb0.001) in case of bothIL-12 p40 and IL-4 levels. The statistical significance of top threecombinations for each parameter is depicted. ***pb0.001 w.r.t. allcombinations. **pb0.001 w.r.t. combinations 1, 3, 4, 5, 7, 8 andpb0.05 w.r.t. combinations 6, 10. *pb0.001 w.r.t. combination1, 5 and pb0.05 w.r.t. combinations 3, 4, 7, 8. +++pb 0.001 w.r.t.all combinations. ++pb0.001 w.r.t. combinations except combina-tion 8. +pb0.001 w.r.t. combinations 1, 2, 3, 4, 5, 7 and pb0.01

ESAT-6+CFP-21+MPT-64 selected on the basis of immuno-genicity studies.

Interestingly, mice receiving both BCG and either of thesupplementation vaccines showed higher reduction inbacillary load as compared to BCG alone in terms of numberof CFU recovered from both lungs and spleen (Table 1). Thereduction in CFU was found to be significant at the level ofspleen only in BCG+ESAT-6+MPT-64 immunized group(pb0.05) and in lungs (pb0.01) as well as spleen (pb0.001)of BCG+ESAT-6+CFP-21+MPT-64 immunized animals. Thus,protection imparted by BCG supplemented with RD antigen-combinations was significantly higher than unsupplementedBCG.

Discussion

Improving the existing BCG vaccine is one of the keystrategies to develop better prophylaxis against tuberculo-sis. Several attempts have been made to modify immuno-genicity or antigenicity of BCG by generating recombinantstrains expressing cytokines [22], pore forming listeriolysin[23], immunodominant antigens [24] or additional antigensmissing from its existing repertoire [25]. Still others docu-ment the use of prime boost regimes to augment the immu-noprotective potential of BCG [26]. In this study we plannedto enhance the efficacy of BCG vaccine by co-administrationof a multicomponent supplementation subunit vaccine com-posed of optimally combined immunodominant RD antigens.Earlier studies conducted in our lab on ESAT-6, CFP-10, CFP-21 and MPT-64 revealed their preferential recognition by PPDpositive contacts and memory immune individuals over TBpatients and community controls based on T-cell responses

w.r.t. combinations 6, 10.

Figure 4 IgG1 and IgG2b titers detected against variouscombinations of RD antigens by ELISA in the sera of miceimmunized with the combination of ESAT-6+CFP-10+CFP-21+MPT-64 four weeks post immunization. The results areexpressed as mean O.D. at 492 nm, where variation betweenduplicate wells was below 10%. The mean O.D.±S.D. of wellswithout antigen were 0.072±0.002 and 0.058±0.006 for IgG2band IgG1 respectively. E6 — ESAT-6, C10 — CFP-10, C21 —CFP21, M64 — MPT-64. Each combination is also represented bya number given above the respective column.

Table 1 Colony forming units (CFU) of M. tuberculosisH37Rv, 4 weeks post infection in lungs and spleen of C57BL/6Jmice (5–7 animals/group) vaccinated with BCG and/ordifferent experimental subunit vaccines comprising thecombinations of RD antigens selected on the basis ofantigenicity studies in humans and immunogenicity studiesin mice

Vaccine formulation Total CFU/organ(Mean log10 units±S.D.)

Lung Spleen

Unvaccinated (naïve) 5.64±0.085 5.44±0.17PBS–DDA–MPL 5.48±0.23 5.36±0.29BCG 4.55±0.34⁎ 4.33±0.16⁎ESAT-6+MPT-64 4.78±0.26⁎ 4.65±0.28⁎ESAT-6+CFP-21+MPT-64 4.54±0.17⁎ 4.46±0.36⁎BCG+ESAT-6+MPT-64 4.11±0.34⁎ 3.90±0.28⁎⁎+

BCG+ESAT-6+CFP-21+MPT-64 3.87±0.24⁎⁎++ 3.63±0.14⁎⁎+++

CFU — Colony forming units; DDA — Dimethyldioctadecyl ammo-nium bromide; MPL — Monophosphoryl Lipid A; BCG — BacilleCalmette–Guerin; PBS — Phosphate buffered saline.ANOVAwas performed for comparison among all groups (pb0.001for both the target organs). Statistical significance for comparingtwo groups was calculated using unpaired t-test. ⁎pb0.001,⁎⁎pb0.0001 w.r.t. naive controls; +++pb0.001, ++pb0.01, +pb0.05w.r.t. BCG immunized group.

179Experimental BCG supplementation vaccine

[13,18]. In addition, we observed that the combination of allabove mentioned RD antigens showed significantly higherlymphocyte proliferation, IFN-γ production and percentagerecognition as compared to the individual proteins in im-mune population comprising TB contacts and treated TBpatients (memory immune) (unpublished data). Hence, wespeculated that combining M. tuberculosis complex specificRD antigens would be a novel approach to constitute a mul-ticomponent subunit vaccine.

However, combining only well defined antigens that donot interfere with each other immunologically is desirable. Ithas been documented that the construction of multi-epitopevaccines can result in both potentiating and suppressive in-teractions between the individual components [27]. Immu-nodominant antigens like ESAT-6 and Ag85B have been shownto exhibit protective efficacies equivalent to BCG using va-rious adjuvants in subunit vaccine formulations [28–30]. Butseveral multicomponent vaccines containing ESAT-6, Ag85Band other immunodominant antigens with proven protectiveefficacies have not been reported to surpass BCG inexperimental challenge against M. tuberculosis [18,30,31].One of the reasons for this could be an immunological inter-ference among various antigens used to constitute the multi-valent vaccines. On the same line in a recent study, Romanoet al. have reported that competition between Ag85A andPstS-3, two known protective vaccine candidates, when usedto develop a combination vaccine resulted in induction ofimpaired CD4+ and antibody responses by PstS-3 [11,12].Furthermore, antibody responses elicited by Ag85A afterimmunization with the fusion molecule were also low as

compared to the levels obtained following a single plasmidimmunization [12]. Hence, when it has become important todesign multicomponent antigenic preparations, it is evenmore crucial to evaluate the individual components of thecocktail and all possible combinations for their immunologi-cal compatibility. Keeping in view the increasing need ofcombination vaccines, the present study evaluated 11possible combinations of well characterized RD1 (ESAT-6and CFP-10) and RD2 (CFP-21 and MPT-64) antigens.

In the quest to search suitable vaccine candidates vastarrays of mycobacterial components are being studied world-wide either on the basis of their “antigenicity” governed bythe ability to be recognized by T-lymphocytes in in vitroassays during early stage of animal or human infection[32,33] or by the “immunogenicity” reflecting their capacityto induce T-cell response following immunization in animalmodels [15,16]. With the availability of supportive as well asopposing evidences from the literature, the conflict ofselecting vaccine candidates on the basis of antigenicity orimmunogenicity remains unresolved. Since “antigenicity”determines the recognition of a molecule among geneticallydifferent populations and “immunogenicity” governs themagnitude of immune response elicited by that moleculefollowing immunization, the generation of multicomponentvaccines addresses both aspects equally [34]. Thus, weplanned to screen various combinations of four immunodo-minant RD antigens on the basis of antigenicity as well asimmunogenicity.

Antigen specific lymphocyte proliferation and IFN-γresponses elicited by the PBMC of PPD positive TB contactsin the present and earlier study from our lab [18] were foundto be low as compared to those documented in other reports[28, 35]. This may be due to suboptimal concentration of RDantigens used for in vitro stimulation in the present study as

180 M. Kalra et al.

dose dependent increase in IFN-γ secretion has beenpreviously reported with the same antigens [28]. In view ofhigh T-cell proliferative response, remarkable IFN-γ induc-tion and maximum number of percentage responders (Figs.1a and b), the combination of ESAT-6+MPT-64 was demon-strated to be the best antigenic combination of RD proteinsupon screening in healthy PPD positive contacts. However,the prevailing hypothesis of screening vaccine candidates inhealthy tuberculosis contacts has been opposed by theemerging concept of selecting prophylactically significantmolecules on the basis of induction of protective immuneresponses following immunization [15,16]. We thereforemonitored Th1 and Th2 responses in splenocytes andperitoneal exudates cells isolated from C57BL/6J (H-2bhaplotype) mice immunized with the combination of ESAT-6+CFP-10+CFP-21+MPT-64 using DDA-MPL as adjuvants.

It is interesting to note that in both human and animalstudies the responses observed against the combination ofESAT-6+CFP-10 were less than majority of combinations(Figs. 1–3) and also ESAT-6 or CFP-10 alone (data not shown).Contrary to our observations with human subjects, vanPinxteren et al. showed an additive effect upon stimulationof human PBMC with ESAT-6+CFP-10 [35]. In our study thecombination of the two antigens rather showed reducedproliferative and cytokine responses in both the models(Figs. 1–3). This may be attributed to mutual immunologicalinterference in terms of competition for antigen processingand presentation owing to similar biochemical and immuno-logical properties [21,35]. In addition, the decreased pro-liferative response might be the consequence of the cytolyticproperties of ESAT-6 and CFP-10 as indicated by previousstudies [7]. ESAT-6 and CFP-10 have been documented toform 1:1 tight complex that has implications in pathogenesisand in interacting with the host cells [36,37]. However, theirability to cause cytolysis and possible function in dissemina-tion and pathogenesis awaits further demonstrations.Although in our study ESAT-6 and CFP-10 were not used as acomplex but Okkels and Andersen recently demonstratedusing western–western immunoblotting that individuallypurified ESAT-6 and CFP-10 proteins interact with eachother when incubated together [38]. Our observations arealso supported by one of the recent study that suggests thatthe natural complex of ESAT-6:CFP-10 is less amenable toantigen processing leading to a lower T cell response ascompared with the individual proteins [39].

In immunogenicity studies, the combination of RD2 anti-gens only, i.e. CFP-21+MPT-64 was found to release cyto-kines to substantial levels. However, the addition of ESAT-6 tothe combination of CFP-21 and MPT-64 showed a stimulatoryeffect in terms of enhancement of IFN-γ and IL-12 (Figs. 2band 3a) suggesting the influence of antigens present in amulticomponent preparation upon each others immunologi-cal performance. The immunostimulatory role of ESAT-6 hasbeen demonstrated lately in which the fusion of ESAT-6 withthe membrane protein P71 of Mycoplasma hyopneumoniae,resulted in increased production of IFN-γ by P71 than in theabsence of ESAT-6 [40]. Hence, ESAT-6 has always remainedthe antigen of choice in a number of multicomponent vaccineformulations [18,30,31].

Though, cell mediated immune response characterized byactivation of Th1 subsets and cytotoxic effector cells liescentral to protection against tuberculosis but latest reports

also highlight the role of antibodies mediated resistance toinfectious diseases [41–45]. In our study all the combinationsshowed relatively higher titers of IgG2b than IgG1 reflectinga bend towards Th1 type (Fig. 4). This is in agreement withthe observations of various workers who have demonstratedall the four selected proteins ESAT-6, CFP-10, CFP-21 andMPT-64 to be prominent T-cell antigens (Th1 type) on thebasis of lymphoproliferative responses and induction of IFN-γ[18,21,28,29,35,46]. However, we also observed moderatelyhigh levels of IgG1 immunoglobulins for all the combinations.The presence of IgG1 antibodies supports the activation ofTh2 subsets and thus explains the induction of IL-4 releasedupon stimulation with different combinations of RD antigens(Figs. 3b and 4). The combinations ESAT-6+CFP-10+MPT andESAT-6+CFP-21+MPT-64 for which the levels of IL-4 werefound to be higher than all other combinations, the ratio ofIgG2b/IgG1 was approximately equal to one indicatingbalanced Th1 and Th2 responses.

The requirement of Th1 and Th2 cytokine balance gover-ned by IFN-γ, IL-10, and IL-4 to prevent tissue destruction hasrecently been suggested by a model used to predict cellmediated immune regulatory mechanisms during human TB[47] and the vaccines inducing balanced Th1 and Th2response following immunization have been hypothesizedto be most effective [16,48]. Immunization with the combi-nation of ESAT-6+CFP-21+MPT-64 resulted in optimal activa-tion of both Th1 and Th2 responses (Figs. 2–4) and henceimparted significant protection against experimental TBinfection which was comparable to BCG (Table 1). In ourattempt to improve BCG by restoring the missing immuno-dominant antigens we observed that mice receiving RDantigens as supplementation vaccines to BCG were betterprotected against M. tuberculosis H37Rv challenge as com-pared to BCG alone (Table 1). Protection observed in case ofBCG supplementation may be attributed to the expansion ofthe antigenic repertoire of BCG and increase in its ability toprime varied subsets of immune cells to mount an optimallyeffective immune response against tuberculosis infection. Ina preliminary experiment to study the immune responses inmice receiving supplemented BCG, we observed highly signi-ficant increase in splenocyte proliferation and IFN-γ produc-tion following supplementation of BCG with RD antigens ascompared to alone BCG or the subunit vaccine [unpublisheddata].

In addition, in a recent study conducted on similar lines,the authors demonstrated that BCG and Ag85-ESAT-6/DDAwhen injected as separate formulations similar to ourapproach but close enough to allow drainage into the samelymph node shows higher immune activation and hencegreater reduction in bacterial burden in lungs as compared toBCG vaccinated and unvaccinated mice [49]. Besides, thepresence of depot forming cationic vehicle as DDA potenti-ates the adjuvant effect of BCG which on its own is a mode-rate adjuvant. Further, the authors report that although BCGis essential for initial priming, however, it can be replacedwith strong adjuvant like MPL in the subsequent boosters.Thus, the use of DDA/MPL in conjunction with BCG in thepresent study could also be responsible for enhanced pro-tection observed in BCG supplemented groups.

Few recent reports have documented the addition of DNAencoding RD1 antigens to enhance the vaccine potential ofBCG. Recombinant BCG (r-BCG) with whole RD1 segment

181Experimental BCG supplementation vaccine

incorporated in its genome secreting ESAT-6 as well as CFP-10showed increased protection [25]. But r-BCG vaccineshowed higher virulence in immunocompromised mice ascompared to wild type BCG. Further, mice vaccinated withBCG secreting high levels of Ag85B-ESAT-6 fusion proteinwere found to be significantly better protected in the lungagainst aerosol infection with M. tuberculosis [50]. Ourresults, supported by the above findings suggest that theexisting vaccine could be improved by adding immunodomi-nant and protective proteins to its antigenic repertoire. It ishowever, essential to have critical evaluation of individualantigens being incorporated to the BCG especially inreference to the RD antigens that are known to be asso-ciated with the virulence. Although RD1 has been demon-strated to be primarily responsible for the expression ofvirulence trait in M. tuberculosis but recent studies reflectthat the role of other RDs in this context cannot be com-pletely ruled out. Sherman et al. [51] revealed that RD1knock out strains of M. tuberculosis are more virulent thanBCG suggesting the possible role of other RD genes invirulence. Hence, supplementation of missing antigens toBCG as an adjunctive subunit vaccine is comparatively safeas compared to the generation of recombinant strains ex-pressing RD proteins.

The present study aimed at evaluating an immunizationstrategy where RD antigens based novel TB subunit vaccinecan be administered as a part of pre-exposure neonatalvaccine along with BCG followed by boosting with subunitvaccine. However, RD antigens based multicomponent sub-unit vaccines can also have implications for development ofbooster vaccines to BCG. Boosting BCG with the subunitvaccine based on missing RD antigens is speculated toincrease anti-TB immunity by expanding the repertoire ofM. tuberculosis specific strongly reactive T-cells. A studyby Derric et al. supports this hypothesis demonstrating thatboosting BCG vaccinated mice after 1 year with the DNAvaccine expressing ESAT-6-Ag85B fusion protein substantiallyaugmented protection in the lungs with significant reductionin bacterial burden as compared to BCG without boostingafter 15 and 18 months of challenge [52]. The authors des-cribe that the booster effect of a DNA vaccine expressingAg85B containing fusion protein is evident as this is the majormycobacterial immunogen. However, despite its absencefrom M. bovis BCG genome, the booster effect of ESAT-6could be due to its ability to amplify overall anti-tuberculosisimmunity in BCG immune animals by activating additionalanti-mycobacterial T cells. Nevertheless, to conclude uponthe potential of RD antigens as a component of boostervaccine to expand the anti-mycobacterial T-cell repertoiregenerated by BCG priming requires experimental validation.The use of RD antigens in prime-boost strategy with supple-mented BCG appears to be an ideal future strategy for cons-tituting the anti-TB vaccine as it will be able to overcomethree major limitations of BCG i.e. short term memory,interference with environmental mycobacteria and theabsence of immunoprotective antigens.

Acknowledgments

This work was funded by the grants from the Department ofBiotechnology and Indian Council of Medical Research, Govt.

of India, New Delhi. Mamta Kalra was recipient of the SeniorResearch Fellowship from University Grants commission,New Delhi. We acknowledge Dr. Karen Dobos for providingrecombinant MPT-64 as part of NIH, NIAID Contract No.HHSN266200400091C, entitled “Tuberculosis Vaccine Testingand Research Materials,”which was awarded to the ColoradoState University.

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