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Vaccine 28 (2010) 63–70

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

oosting with a DNA vaccine expressing ESAT-6 (DNAE6) obliterates therotection imparted by recombinant BCG (rBCGE6) against aerosolycobacterium tuberculosis infection in guinea pigs

appaditya Deya,1, Ruchi Jaina,1, Aparna Kheraa, Vivek Raoa,b, Neeraj Dhara,c,mesh D. Guptad, V.M. Katochd, V.D. Ramanathane, Anil K. Tyagia,∗

Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, IndiaDivision of Mycobacterial Research, The National Institute for Medical Research, The Ridgeweay Mill Hill, London NW71AA, UKLaboratory of Bacteriology, Global Health Institute, Ecole Polytechnique Federale de Lausanne, Lausanne CH1015, SwitzerlandNational JALMA Institute for Leprosy & Other Mycobacterial Diseases, Tajganj, Agra 282001, IndiaDepartment of Clinical Pathology, Tuberculosis Research Center, Chetpet, Chennai 600031, India

r t i c l e i n f o

rticle history:

a b s t r a c t

Owing to its highly immunodominant nature and ability to induce long-lived memory immunity, ESAT-6,

eceived 20 July 2009ccepted 25 September 2009vailable online 15 October 2009

eywords:

a prominent antigen of Mycobacterium tuberculosis, has been employed in several approaches to developtuberculosis vaccines. Here, for the first time, we combined ESAT-6 based recombinant BCG (rBCG) andDNA vaccine (DNAE6) in a prime boost approach. Interestingly, in spite of inducing an enhanced antigenspecific IFN-� response in mice, a DNAE6 booster completely obliterated the protection imparted byrBCG against tuberculosis in guinea pigs. Analysis of immunopathology and cytokine responses suggests

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uberculosisSAT-6rime boost vaccination

involvement of an exagge

. Introduction

Mycobacterium bovis Bacille Calmette Guerin (BCG), the onlyvailable tuberculosis (TB) vaccine for more than 80 years, hasffectively averted the life-threatening forms of TB in children [1].owever, progressive exhaustion of BCG induced immunity withge renders it ineffective against pulmonary TB in adults, thus, lead-ng to loss of millions of human lives annually. Hence, developmentf effective vaccination strategies against TB represents one of therime objectives of TB research.

Comparative genomic analysis has identified more than 16enomic loci called the regions of difference (RD) that were deletedrom BCG genome during its divergence from M. bovis [2,3].he deletion of these genomic loci not only resulted in the co-limination of several virulence-associated genes but also genesncoding several protective antigens, the latter event perhaps con-

ributed to the inadequate protection imparted by BCG [4]. ESAT-6as attracted significant attention from TB investigators as an

mmunodominant secretory protein of Mycobacterium tuberculo-is encoded by RD1 locus. Importantly, while this region is deleted

∗ Corresponding author. Tel.: +91 11 24110970; fax: +91 11 24115270.E-mail addresses: aniltyagi@south.du.ac.in, akt1003@rediffmail.com,

kt.mtb@gmail.com (A.K. Tyagi).1 Joint first authors.

264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2009.09.121

immunity behind the lack of protection imparted by this regimen.© 2009 Elsevier Ltd. All rights reserved.

from the BCG vaccine strains, all the pathogenic members of M.tuberculosis complex are endowed with it [5]. ESAT-6, with a largenumber of B and T cell epitopes, is strongly recognized during theearly phase of M. tuberculosis infection as well as in patients withactive TB and is a potent inducer of long-lasting memory immu-nity against TB [6,7]. The strong immunogenicity of ESAT-6 and itsabsence from BCG make this antigen an attractive target for thedevelopment of new TB vaccine.

To date, several approaches such as recombinant BCG, DNAand subunit vaccines have been employed to develop TB vac-cines based on ESAT-6 [8–10]. Yet, none of the above formulationsalone has successfully improved the protective capabilities of BCG.However, a heterologous prime boost approach involving primaryBCG vaccination and subsequent boosting with a fusion proteincomprised of ESAT-6 and antigen 85B, imparted a superior pro-tection in comparison to BCG [11]. From this, we argued that anESAT-6 based booster vaccine is likely to elicit more efficient sec-ondary immune response, when the primary vaccine namely BCG isenriched with ESAT-6. We have previously demonstrated that rBCGover-expressing antigen ESAT-6 (rBCGE6) induces an enhanced Th1immune response in mice [12] and imparts a comparable protec-

tion, when compared to the parental BCG vaccine in guinea pigsagainst a s.c. M. tuberculosis challenge (unpublished observationsfrom our laboratory). In addition, we have also reported that a DNAvaccine expressing ESAT-6 (DNAE6) imparts notable protection toguinea pigs against a s.c. M. tuberculosis challenge, although, it could

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ot surpass the protective efficacy of BCG [13]. Hence, in the presenttudy, we employed a heterologous prime boost vaccination strat-gy combining the rBCG and DNA vaccines based on ESAT-6 on theremise that it may provide a synergistic enhancement of protec-ion by inducing immune responses against ESAT-6 as well as a vastepertoire of BCG antigens.

. Materials and methods

.1. Bacterial strains and preparation of antigens formmunization

M. bovis BCG (Danish strain) was procured from BCG labora-ories, Chennai, India. rBCGE6 was generated by engineering aycobacteria–Escherichia coli shuttle vector pSD5.pro [14,15] to

ver-express ESAT-6 gene (Rv3875) as described previously [12]. M.uberculosis (H37Rv strain) was kindly provided by Dr. J.S. Tyagi, Allndia Institute of Medical Sciences, New Delhi, India. BCG, rBCGE6nd M. tuberculosis strains were grown to mid-log phase in Mid-lebrook 7H9 media and stocks were prepared as described earlier16]. DNA vaccine expressing ESAT-6 (pAK4-ESAT-6) was prepareds described previously [13].

.2. Immunization of guinea pigs and evaluation of protectivefficacy against M. tuberculosis infection

For protective efficacy studies, two experiments were carriedut (Supplementary Fig. 1a) and in each experiment animals inroups of six were immunized with the following regimens: (i)× 105 CFU of either BCG or rBCGE6 in 100 �l of saline by i.d.

oute, (ii) DNAE6 or vector alone (100 �g in 100 �l of saline) by.m. route thrice at 3 weeks interval, (ii) rBCGE6 once, followed by aooster dose of DNAE6 or vector by i.m. route at 6 weeks (R/D and/V) and (iii) 100 �l of saline by i.d. route (control group). In Exp-I,uinea pigs were challenged at 6 weeks after the last immunizationith 50–100 bacilli of virulent M. tuberculosis H37Rv via respiratory

oute in an aerosol chamber (Inhalation exposure system, Glass-ol Inc., IN, USA) and were euthanized at 10 weeks post-infectionby i.p. injection of thiopentone sodium @ 100 mg/kg body weight,eon Laboratories Ltd., India). In Exp-II, the time interval betweenrimary immunization and challenge was kept constant for all theegimens (12 weeks) and the animals were euthanized at 10 weeksost-infection. In addition to the measurement of bacillary load

n lung and spleen, gross and histopathological changes in variousrgans, extent of pulmonary fibrosis and M. tuberculosis antigenoad were measured as described previously [16]. All the experi-

ental protocols were reviewed and approved by the animal ethicsommittee of the institute.

.3. Immunohistochemistry and image analysis

For in situ localization of IFN-�, TNF-� and M. tuberculosis anti-ens in the lung sections of guinea pigs, IHC staining was carriedut and analysed for area and intensity of staining as describedarlier [16]. Briefly, deparaffinized and re-hydrated lung sectionsere quenched for endogenous peroxidase with 3% hydrogen per-

xide (in methanol) followed by antigen retrieval at 90–100 ◦C for0 min in citrate buffer (pH 6.5). After blocking the non-specificites with 2% BSA and 4% goat sera in PBS, sections were probedith rabbit polyclonal anti-sera against guinea pig IFN-�, TNF-�

kindly provided by Dr. D.N. McMurray, The Texas A&M University

ystem Health Science Center, TX, USA) and Ag85 complex of M.uberculosis (raised in our laboratory) overnight at 4 ◦C. Followingashing with PBST (containing 0.1% Triton-X100 and 0.5% BSA) and

BS three times, sections were treated with horseradish peroxidaseHRP) conjugated goat anti-rabbit antiserum (Jackson laboratories,

8 (2010) 63–70

PA, USA). Finally, the antibody bound antigenic sites were detectedby a colored reaction (brown) using diaminobenzedine as a chro-mogenic substrate for HRP and the slides were counterstained withMayer’s hematoxylin. Negative controls were treated in the samemanner except that primary antibody was replaced with the nor-mal rabbit sera.

2.4. Immunization of mice and assessment of antigen specificcytokine response

For evaluation of immune responses, specific pathogen freefemale BALB/c mice (n = 6) were immunized with either of the fol-lowing: (i) 5 × 105 CFU of either BCG or rBCGE6 in 100 �l of saline bys.c. route, (ii) DNAE6 or vector alone (100 �g in 100 �l of saline) byi.m. route thrice at 3 weeks interval, (iii) rBCGE6 once, followed bya booster dose of DNAE6 and (iv) 100 �l of saline by s.c. route (con-trol group) (Supplementary Fig. 1b). At 4 weeks post-immunization,animals were euthanized and spleens were aseptically removedfor assessment of antigen specific cytokine responses as describedpreviously [17]. Briefly, splenocytes were stimulated in vitro in a96-well cell culture plate (5 × 105 cells/well) with purified recom-binant ESAT-6 antigen (2 �g). Supernatants were collected after90 h for estimation of IFN-� and IL-10 by using cytokine specificcommercial kits (BD OptiaTM, BD Biosciences Pharmingen, SD, USA)according to the manufacturer’s instructions.

2.5. Statistical analysis

Non-parametric Kruskal–Wallis test, followed byMann–Whitney U-test were employed for the comparison ofthe Log10 CFU, score values (for gross pathological lesions), gran-uloma percent, quick score (Q) (for IHC and extent of collagendeposition) across different groups. Differences were consideredstatistically significant, when p < 0.05. Correlation between differ-ent parameters was analysed by Spearman’s Rank Correlation test.These statistical tests were run on SPSS software (Version 10.0,SPSS Inc., IL, USA) and graphs were generated by using Prism 5software (Version 5.01; GraphPad Software Inc., CA, USA).

3. Results

3.1. Influence of ESAT-6 based prime boost vaccination on thebacillary load in lung and spleen

To determine whether the vaccination-induced immunityrestricts multiplication of M. tuberculosis, guinea pigs were infectedwith 50–100 virulent M. tuberculosis bacilli by aerosol route 6 weeksafter the final immunization (Supplementary Fig. 1a). The animalswere euthanized at 10 weeks post-infection and the bacillary loadin the lungs and spleen was measured. Animals immunized withrBCGE6 showed a significantly reduced lung and spleen bacillaryload (by 0.74 Log10 and 2.91 Log10, respectively; p < 0.05), whencompared to the unvaccinated animals (Fig. 1a). However, over-expression of ESAT-6 in rBCG yielded no improvement over BCG,as was evident from a comparable bacillary load in rBCGE6 andBCG immunized animals. The ESAT-6 based DNA vaccine, in con-trast to the protection it had imparted against s.c. M. tuberculosischallenge [13], did not provide any appreciable protection againstthe respiratory route of M. tuberculosis infection, when comparedto the unvaccinated or vector treated animals. Contrary to our

expectations of increased protection, a DNAE6 vaccine boosterfollowing a primary vaccination with rBCGE6 (R/D regimen) com-pletely abrogated the protection imparted by rBCGE6. Animals inthis group exhibited lung and spleen bacillary load comparable tothat observed in case of unvaccinated animals.

B. Dey et al. / Vaccine 28 (2010) 63–70 65

Fig. 1. Influence of ESAT-6 based prime boost regimens on bacillary and antigen load in guinea pig tissues following M. tuberculosis challenge. (a and b) The bacillary load inlung and spleen of guinea pigs (n = 6) at 10 weeks post-M. tuberculosis infection (50–100 bacilli) in Exp-I (a) and Exp-II (b). At the time of euthanasia, lung and spleen wereaseptically removed and homogenized in saline. The homogenates were serially diluted and plated in duplicates on 7H11 medium supplemented with appropriate antibiotics.The CFU were determined and the Log10 CFU are graphically represented by box plot, wherein median values are denoted by horizontal line, the mean is represented by‘+’, inter-quartile range by boxes, and the maximum and minimum values by whiskers. (c) The representative photomicrographs of guinea pig lung sections showing IHCs eeksg ining]a nts 10w

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taining (brown color) for Ag85 complex proteins in pulmonary granulomas at 10 wroup and the extent of staining (Q) was measured [Q = intensity (I) × area (A) of stand the bar depicts median (± inter-quartile range) for each group. Scale bar represehen compared to the BCG group (Mann–Whitney U-test).

As per the protocol followed in Exp-I, animals belonging toBCGE6 and R/D regimens were challenged at different intervalsfter the primary immunization (6 and 12 weeks, respectively),

hus making it difficult to assess whether the observed lack of pro-ection by R/D regimen was due to: (i) a generalized exhaustion ofBCGE6 induced immunity with time or (ii) due to deleterious effectf DNAE6 boosting. In order to dissect the above two mechanisms,e modified the protocol in Exp-II, such that animals belonging

ig. 2. Influence of ESAT-6 based prime boost regimens on gross pathology. The figure diver and spleen of guinea pigs (n = 6) euthanized at 10 weeks post-M. tuberculosis infeize of tubercles, areas of inflammation and necrosis, gross pathological scores were graepicts median (± inter-quartile range) for each group. Missing data points represent theompared to the saline group (Mann–Whitney U-test).

post-infection in Exp-II. For IHC, 4 lung samples were chosen randomly from eachand represented graphically. Each data point represents Q for an individual animal00 �m. *p < 0.05; **p < 0.01, when compared to the unvaccinated animals; �p < 0.05,

to all the regimens were challenged at the same interval post-primary immunization (12 weeks) and an additional control groupwas included with vector DNA as a boosting agent (R/V) instead of

DNAE6 (R/D). In Exp-II, rBCGE6 showed a relatively reduced abilityto restrict hematogenous spread of bacilli to spleen, when com-pared to BCG vaccination (p < 0.05) (Fig. 1b). Moreover, a boosterdose of DNAE6 further accentuated this effect, as was evident froma significantly increased splenic bacillary load in comparison to

epicts representative photographs and graphical depiction of gross scores of lung,ction (50–100 bacilli) in Exp-I. Based on the extent of involvement, number andded from 1 to 4. Each point represents score for an individual animal and the baranimals that succumbed to disease before euthanasia. *p < 0.05 and **p < 0.01, when

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Fig. 3. Influence of ESAT-6 based prime boost regimens on histopathologicalchanges in lung and liver of guinea pigs. The figure depicts representative photomi-crographs of H&E stained lung and liver sections of guinea pigs (n = 6) euthanized at10 weeks post-M. tuberculosis infection (50–100 bacilli) in Exp-I. The lung and livertissues were fixed in 10% buffered formalin and were embedded in paraffin. Sub-sequently, sections of 5 �m thickness were cut on to glass slides and stained withH&E for histopathological examination. Measurement of granuloma % was carriedout by visualizing the tissue sections under a light microscope. Scale bar represents2 mm. Granuloma % were graphically represented by box plot, wherein median val-

6 B. Dey et al. / Vac

BCGE6 as well as R/V immunized animals (p < 0.05). These observa-ions suggest that in addition to a relative decline in the protectivemmunity of rBCGE6 with time, DNAE6 boosting played a definiteole in the abrogation of protection in R/D regimen.

In parallel, we also determined the M. tuberculosis antigen loadn lung tissues by in situ localization of Ag85 complex proteins.ince, these proteins represent some of the predominant antigensocalized in the mycobacterial cell wall and in the secretory frac-ion, their quantitation served as a marker of dead bacilli, bacillaryemnants, secreted antigens as well as the presence of live bacilli inung. M. tuberculosis antigen load has been shown to be closely asso-iated with granulomatous inflammation and pathological damage16]. The bacillary count in lung showed a strong positive corre-ation with antigen load (r = .768, p < 0.001) (Supplementary Table). Importantly, in agreement with the increased bacillary loadbserved in DNAE6 as well as R/D regimen immunized animals, aignificantly higher level of M. tuberculosis antigens was observed inulmonary granulomas (Q = 6, p < 0.05) compared to BCG or rBCGE6accinated animals (Q = 4) (Fig. 1c). Taken together, these obser-ations suggest that immune responses elicited by ESAT-6 basedrime boost regimens were neither able to restrict the bacillaryultiplication nor were efficient in antigen clearance from the site

f infection.

.2. Influence of ESAT-6 based prime boost vaccination onathology

We next assessed the influence of ESAT-6 based prime boostegimens on the pathology in various organs of guinea pigsost-infection. Pathological investigations were carried out atwo different levels: (i) visual scoring of gross lesions and (ii)istopathological measurement of granulomatous inflammation.n comparing the size and number of gross lesions in lung, liver and

pleen, at 10 weeks post-infection, it was observed that the organsf unvaccinated as well as vector treated animals exhibited exten-ive involvement and were characterized by presence of numerousarge tubercles and areas of necrosis (Fig. 2). Animals immunized

ith BCG or rBCGE6 exhibited a significantly reduced size and num-er of lesions in all the organs evaluated, when compared to thenvaccinated animals. In contrast, DNAE6, when used alone or asbooster subsequent to rBCGE6 priming, resulted in severe patho-

ogical damage comparable to that observed in the case of salinend vector treatment (Fig. 2).

Morphometric analysis of lung and liver sections furtherubstantiated the gross pathological observations. As shown inig. 3, the granulomatous inflammation observed in the lungsf unvaccinated guinea pigs typically represented an advancedtage granuloma, wherein, pulmonary parenchyma was effacedy extensive coalescence of multiple necrotic granulomas occu-ying ∼65% area of the lung sections. In addition, ∼35% ofepatic parenchyma was effaced by multi-focal granulomatous

nfiltration in these animals. Immunization with rBCGE6 signifi-antly reduced the granulomatous inflammation in lung to ∼47.5%p < 0.05), which was comparable to BCG vaccination. However,BCGE6 imparted only a partial protection in liver, as was evidentrom a relatively higher granulomatous inflammation (∼17.5%),hen compared to BCG vaccination (0–5%). Commensurate with

he aggravated gross pathology, both DNAE6 and R/D regimensesulted in comparable but extensive involvement of pulmonaryarenchyma with wide spread coalescing granulomas and con-

equent obliteration of airspaces (∼65% and 60%, respectively).owever, the extent of hepatic inflammation in R/D immunizednimals was relatively lower (∼12.5%) in comparison to the DNAE6mmunization (∼45%) (Fig. 3). Thus, the pathological damage wasn coherence with the bacillary load.

ues are denoted by horizontal line, the mean is represented by ‘+’, inter-quartilerange by boxes, and the maximum and minimum values by whiskers. *p < 0.05, whencompared to the saline group (Mann–Whitney U-test).

The detrimental influence of ESAT-6 based prime boost vacci-nation was further substantiated by pathological observations inExp-II, wherein animals immunized with DNAE6 or R/D regimenexhibited a significantly higher pathology in all the organs, whencompared to BCG or rBCGE6 immunized animals (Supplementary

Figs. 2 and 3). In addition, extensive collagen deposition in thelungs of animals immunized with these regimens further sug-gested an irreversible scarification of pulmonary parenchyma inthese animals (Q = 6), which was in stark contrast to BCG or rBCGE6

B. Dey et al. / Vaccine 28 (2010) 63–70 67

Fig. 4. Influence of ESAT-6 based prime boost vaccination on pulmonary fibrosis. The figure depicts representative photomicrographs of Van Gieson stained lung sections ofguinea pigs euthanized at 10 weeks post-M. tuberculosis infection (50–100 bacilli) in Exp-II. The formalin fixed and paraffin embedded lung tissues were cut on to glass slides( n (redE (I) ×a roup.�

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mmunized animals with significantly reduced fibrosis (Q = 3–4,< 0.05) (Fig. 4). Moreover, a significantly increased splenic pathol-gy in animals vaccinated with R/D regimen compared to R/Vegimen further confirmed that the exacerbation of pathology wasue to boosting of ESAT-6 specific immune responses by DNAE6Supplementary Fig. 2).

.3. Cytokine responses elicited by ESAT-6 based prime boostegimens

In order to elucidate the correlation, between the observed exac-rbated pathology in the guinea pigs vaccinated with ESAT-6 basedrime boost regimens and superfluous production of inflammatoryytokines, the lung sections were further examined for the pres-nce of IFN-� and TNF-� by in situ IHC staining (Fig. 5a and b). Thextent of granulomatous pathology showed a strong positive cor-elation with the level of these cytokines (Supplementary Table 1).ommensurate with the increased pathology, lung sections fromnimals immunized with DNAE6 and R/D regimen exhibited a sig-ificantly higher level of IFN-� (Q = 5) and TNF-� (Q = 8), whenompared to BCG as well as rBCGE6 immunized animals. More-ver, IFN-� staining (Fig. 5a) in prime boost vaccinated animals wasound to be comparable to that in unvaccinated animals, however,he TNF-� staining (Fig. 5b) in these animals remained relativelyower in comparison to unvaccinated animals. These observationshus suggest that unwarranted inflammatory responses induced byrime boost vaccination upon M. tuberculosis infection might have

ed to the observed collateral pathological damage and consequentack of protection in these animals.

In order to gain an insight into the immune responses elicited bySAT-6 based prime boost regimens, a similar vaccination experi-ent was carried out in mice (Supplementary Fig. 1b). At 4 weeks

fter the last immunization, mice were euthanized and splenocytesere isolated and stimulated in vitro with purified ESAT-6 protein

s a recall antigen. Immunization with rBCGE6 resulted in a rela-ively increased levels of both IFN-� as well as IL-10 as comparedo BCG vaccination (Fig. 5c and d). However, the ratio of these twoytokines was in favor of IFN-� indicating the elicitation of a Th1iased immune response by rBCGE6 vaccination. A booster dosef DNAE6 following rBCGE6 vaccination further enhanced the lev-ls of IFN-� (Fig. 5c) and IL-10 (Fig. 5d) as compared to both BCGnd rBCGE6 immunizations. Besides, mice receiving three succes-ive doses of DNAE6 exhibited a significantly higher level of these

ytokines as compared to the animals immunized with BCG orBCGE6, but the levels of IFN-� remained relatively lower in com-arison to R/D group. While, qualitatively rBCG, R/D as well asNAE6 regimens elicited a Th1 biased immune response, its mag-itude in case of prime boost regimens was significantly higher.

). For Van Gieson staining, 4 lung samples were chosen randomly from each group.area (A) of staining] and represented graphically. Each data point represents Q forScale bar represents 1 mm. *p < 0.05, when compared to the unvaccinated animals;

4. Discussion

The development of a perfect vaccine against TB remains one ofthe primary goals of TB research. Due to the proven efficiency ofBCG against childhood TB, combined with its remarkable immuno-potentiating properties, several research groups have focused theirefforts on the development of improved vaccination strategiesbased on BCG. Expression of promising immunodominant antigensof M. tuberculosis in BCG, especially those, which are absent fromthe latter, offers an ideal strategy to improve the immunogenic-ity and protective efficacy of BCG [15,18]. Furthermore, boostingBCG or rBCG-induced immunity with appropriate vaccines has alsoemerged as an attractive strategy. In this context, several plas-mid DNA based vaccines have shown substantial promise, whenemployed in a prime boost approach along with BCG [19–21].

We have earlier shown that a DNA vaccine encoding ESAT-6gene of M. tuberculosis provides protection to guinea pigs againsts.c. infection with M. tuberculosis [13]. With the premise to developa more effective vaccination strategy, in this study, we have com-bined this ESAT-6 based DNA vaccine and rBCG vaccine [12] andevaluated the protective efficacy of this heterologous prime boostregimen in the ‘guinea pig’ model of aerosol M. tuberculosis infec-tion. Unexpectedly, a booster dose of DNAE6 not only abrogatedthe protection imparted by rBCGE6, it resulted in a complete lackof protection with exacerbated pathology. The detrimental effectof DNAE6 boosting on hematogenous spread of bacilli to spleen aswell as on extra-pulmonary pathology appeared to stem from theESAT-6 specific immune responses, since boosting with a vectorDNA alone did not influence the protective efficacy of rBCGE6.

Due to lack of immunological reagents for guinea pigs, it is dif-ficult to analyse the immunological basis of exacerbated pathologyand lack of protection observed in animals immunized with ESAT-6based prime boost regimens. However, we can anticipate a possible‘exhaustion’ of memory immunity due to excessive antigenic stim-ulation by these regimens. Several studies in the mouse model haveindicated that initial establishment of memory immune responsesis governed by cumulative history of antigen exposure [22,23].While, a brief period of antigenic stimulation during immunepriming favors commitment to long-lived central memory T cells(TCM), prolonged or excessive antigenic stimulation during prim-ing or successive boosting drives the T cell differentiation pathwaytowards the effector memory T cells (TEM) lineage. Although, theseTEM cells mediate the effector functions, when exposed to anti-

genic stimulation or infection soon after immunization, they mayundergo rapid differentiation into short-lived terminally differen-tiated effector T cells (TEff). Vaccine regimens that over-stimulatethe immune system and result in the production of a large pro-portion of TEff cells would not be effective in long-term, as these

68 B. Dey et al. / Vaccine 28 (2010) 63–70

Fig. 5. Cytokine responses induced in response to vaccination in guinea pigs and mice. (a and b) The representative photomicrographs of guinea pig lung sections showingIHC staining (brown color) for (a) IFN-� and (b) TNF-� in pulmonary granulomas at 10 weeks post-infection in Exp-II. For IHC, 4 lung samples were chosen randomly fromeach group and sections were stained as described in Section 2.3. Extent of staining (Q) was measured [Q = intensity (I) × area (A) of staining] and represented graphically.Each data point represents Q for an individual animal and the bar depicts median (± inter-quartile range) for each group. Scale bar represents 200 �m. *p < 0.05, whenc � p (Ma4 ree pE and tr hen co

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ompared to the unvaccinated animals; p < 0.05, when compared to the BCG grouweeks post-immunization. Mice (n = 6) were euthanized and from each group, th

SAT-6 protein. Supernatant from each pool was harvested after 90 h of incubationepresents the mean concentration (± S.E.) of three pools per group. ***p < 0.001, w

ells have a limited capacity to expand and sustain the mem-ry responses [24]. It is likely that a similar mechanism might beperative in this study as well. Immunization with BCG or rBCGight lead to the generation of seemingly adequate memory T cell

esponses, capable of imparting protection against M. tuberculo-is infection. However, heterologous boosting with DNA vaccinet a short interval following rBCG priming could have resultedn the generation of TEff cells due to excessive antigenic stimula-ion, instead of expanding TCM cell population. Upon M. tuberculosis

nfection, an early consumption of these TEff cells and lack of ade-uate number of memory cells could have resulted in the failuref immune system to control the infection in a sustained manner.similar mechanism might also be responsible for the observed

ack of protection in the case of DNAE6 based homologous prime

nn–Whitney U-test). (c and d) The antigen specific cytokine responses in mice atools of splenocytes were prepared (2 mice in each pool) and cultured with 2 �g ofhe levels of (c) IFN-� and (d) IL-10 were measured by ELISA in duplicates. The datampared to the BCG group (Student’s t-test, two-tailed).

boost regimen. The observed lack of protection by prime boostregimens in this study, is in coherence with the previous studies,which have shown that successive boosting of primary immuneresponses at short intervals may prove to be detrimental owingto the over-exuberant immune responses and thus exhaustion ofmemory immunity [25,26].

The ‘immuno-exhaustion’ hypothesis is further substantiated bythe immune responses observed in the mice study. Mice vaccinatedwith DNAE6 or R/D regimen exhibited a heightened IFN-� response,

when compared to BCG or rBCG immunized animals. This exuber-ant IFN-� response might stem from the TEff cell population andlack of adequate TCM and TEM cell responses might have renderedthese vaccine regimens ineffective. However, further assessmentof T cell phenotype is required to confirm the qualitative nature of

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cells responsible for the observed lack of protection by ESAT-6ased prime boost regimens.

Although, ESAT-6 is an established immunodominant antigen,licitation of an over-exuberant immune response by prime boostegimens involving this antigen may also be attributed to the pres-nce of CpG motifs both in the vector backbone of DNA vaccine13] as well as in the ESAT-6 gene sequence [27]. The immuno-timulatory property of CpG motifs is well known; they stimulatehe innate immune system via TLR-9 receptors and induce theroduction of Th1 cytokines and strong B cell responses [28]. How-ver, in view of the inherent immuno-modulatory properties ofpG motifs and their extensive use in designing DNA vaccines, itas been, rather discouraging to learn from the recent reports thatpG motifs may cause unwarranted inflammation and collateralathological damage following DNA vaccination [29]. Nevertheless,detailed analysis would be required to implicate CpG motifs in thebrogation of protection by DNAE6 and R/D regimens.

Being a localized infection, control of pulmonary TB largelyepends on the orchestration of various cellular components ofhe immune system and a coordinated interplay of several pro-nd anti-inflammatory cytokines at the focus of infection. In thebsence of vaccination, the clinical manifestation of end-stage TBs known to be associated with a strong inflammatory response tohe persistent antigens or bacilli culminating in extensive necro-is and progressive fibrosis [30]. However, an effective vaccine isxpected to fine-tune the immune system to generate an efficientlyegulated and targeted response for an effective antigen and micro-ial clearance, thus minimizing the collateral damage to the host.mmuno-localization of M. tuberculosis antigen 85 complex pro-eins revealed the failure of guinea pigs immunized with ESAT-6ased prime boost regimens to clear antigen depots, consequentlyesulting in an increased granulomatous pathology characterizedy the presence of very high levels of IFN-� and TNF-� in theseissues. Taken together, these observations suggest that the failuref guinea pigs vaccinated with the prime boost regimens to con-rol the unwarranted production of these cytokines results in theollateral pathological damage and consequent lack of protection.

Our study provides an important lesson for future efforts con-erning development of new vaccines against TB. The completeailure of ESAT-6 based prime boost regimens to induce a protectivemmune response demonstrates that a highly immunodominantature of an antigen alone does not guarantee induction of desired

mmune responses in the host and that the immune response iswell choreographed sequence of events guided by several fac-

ors like nature of expression vehicle, level of antigen expression,ormulation of the immunogen, immunization regimen employed,tc. In this study, vaccination with rBCG over-expressing ESAT-6nduced a stronger IFN-� response than BCG but exhibited no signif-cant improvement in the protective efficacy against M. tuberculosisnfection over the BCG vaccine. Literature is replete with such stud-es describing tremendous improvement in the immunogenicity bySAT-6 based vaccines, however, majority of these regimens didot provide protection superior to BCG. For instance, Lang Bownd colleagues demonstrated that expression of antigen ESAT-6,ither in secretory or non-secretory form in BCG, did not result innhanced protective efficacy despite inducing a significantly higherntigen specific humoral and cellular immune responses comparedo the parent BCG strain [31]. In addition, in spite of the induc-ion of a potent IFN-� response, ESAT-6 based DNA vaccine didot exhibit any significant improvement in protection over BCG9,32]. However, the complete lack of protection by DNAE6 vacci-

ation in this study is at variance with our previous observation,herein the ESAT-6 based DNA vaccine conferred a significantrotection against M. tuberculosis infection by s.c. route when com-ared to vector treatment in guinea pigs, although it could noturpass the protective efficacy of BCG vaccine. These differences

8 (2010) 63–70 69

can be attributed to the differences in the methodologies employedfor evaluation of protective efficacy in theses two studies. In ourprevious study, protective efficacy was evaluated at 4 weeks fol-lowing M. tuberculosis infection (105 bacilli) by subcutaneous route[13], however, in the present study, in order to mimic the naturalroute of infection, guinea pigs were infected with 50–100 bacilliby aerosol route and the protective efficacy was evaluated at 10weeks post-infection to assess the long-term effect of vaccination.Based on these observations, it can be speculated that the immuneresponses generated by DNA vaccination although conferred pro-tection against bacillary multiplication till 4 weeks post-infection(s.c. route), the protective effect could not be sustained till 10 weekspost-infection.

In sum, our data demonstrates that antigen ESAT-6 based DNAvaccine does not serve as an effective boosting agent followingprimary immunization with rBCG. More importantly, while het-erologous prime boost strategies have tremendous potential, acareful selection of a booster vaccine is important in order toachieve successful protection. A significant finding by Dietrich et al.,that subunit vaccine based on a fusion protein of ESAT-6 and anti-gen 85B improves BCG induced immunity [11], may still keep theoptimism alive that ESAT-6 based subunit vaccine may indeed actas an efficient boosting agent, as an alternative to DNA vaccine, toimprove the rBCG-induced protective immunity against TB. How-ever, further studies would be required to realize the protectivepotential of such heterologous prime boost regimens.

Acknowledgements

We acknowledge H.K. Prasad, AIIMS, New Delhi, India andPriyanka Chauhan, UDSC, New Delhi, India for critical reading ofthe manuscript and valuable suggestions. Technical assistance of S.Nambirajan, K. Chandran, B. Singh and P. Singh is highly acknowl-edged. BD and RJ are thankful to CSIR, India for research fellowships.This work was supported by a research grant from the Departmentof Biotechnology, India.

Contributors: BD, RJ, AK, VR, ND and AKT conceived and designedthe experiments. BD and RJ conducted the experiments and anal-ysed the data. BD, RJ and AKT wrote the manuscript. VDR oversawthe pathology related experiments. VMK and UDG provided theBSLIII facility and supervised the animal experiments. AKT providedoverall supervision throughout the study.

Conflict of interest statement: The authors have no conflictingfinancial interests.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.vaccine.2009.09.121.

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