Increased Expression of Mycobacterium tuberculosis 19 kDa Lipoprotein Obliterates the Protective Efficacy of BCG by Polarizing Host Immune Responses to the Th2 Subtype

Authors


Dr A. K. Tyagi, Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India. E-mail: akt1000@hotmail.com

Abstract

Mycobacterium tuberculosis can not only neutralize immune effector functions, but also has the ability to modulate host-signalling cascades involved in the development of these responses. The 19 kDa antigen (Rv3763), a lipoprotein of M. tuberculosis, elicits high levels of interleukin (IL)-12 from macrophages in addition to its powerful immunomodulatory properties, leading to suppression of antigen-presentation signalling cascades. The present study was aimed at analysing the effect of overexpression of this antigen on the immunostimulatory properties of M. bovis Bacille Calmette–Guérin (BCG). We have constructed a recombinant BCG strain (rBCG19N) producing higher levels of the 19 kDa antigen in both the cytoplasmic (approximately eightfold) and extracellular (approximately fivefold) fractions as compared to the wildtype BCG. Immunization of mice with rBCG19N elicited high levels of interferon-gamma (IFN-γ) and relatively low levels of IL-10 against the purified 19 kDa antigen. However, in response to total BCG sonicate, mice immunized with rBCG19N produced significantly high levels of IL-10 with relatively very low levels of IFN-γ. This polarization of the host immune responses towards T-helper 2 subtype resulted in complete abrogation of the protective efficacy of BCG, when rBCG19N was used as a live vaccine against M. tuberculosis challenge in guinea pigs.

Introduction

With approximately one-third of the world's population infected asymptomatically with Mycobacterium tuberculosis, a very large reservoir of infection already exists. Owing to the HIV-TB nexus and development of multidrug-resistant strains of M. tuberculosis, the cases of active TB infection are increasing globally [1]. In order to block further transmission and reactivation of TB in the already infected population, it is necessary to develop better intervention strategies that require a thorough understanding of host–pathogen interactions. A wealth of information exists about the immunodominant nature of several M. tuberculosis antigens [2, 3]. By virtue of their immunodominant nature and capacity to activate the T-helper 1 (Th1) type of host immunity, the culture filtrate proteins (CFP) of M. tuberculosis have been frequently employed to develop new candidate vaccines against TB [4–6].

The 19 kDa lipoprotein, a CFP of M. tuberculosis, is capable of eliciting potent, humoral and cell-mediated immune responses in addition to stimulating a recall memory response in immunized animals [7–9]. Purified 19 kDa antigen induces high levels of inflammatory cytokine interleukin (IL)-12 from macrophages, thereby leading to the activation of Th1 type of immune responses in the host. However, recent studies have also implicated this antigen in several immunomodulatory functions of M. tuberculosis, leading to the downregulation of immune effector mechanisms of the host [10–12]. In spite of its capacity to induce IL-12 and prime memory T cells in vitro, candidate mycobacterial vaccines based on this antigen have largely been unsuccessful [13, 14]. Expression of this antigen in the saprophytic mycobacterial species –M. smegmatis and M. vaccae– proved detrimental to the protective efficacy of the live bacteria themselves, indicating that this antigen extensively modulates the host immune system. In contrast, however, recombinant Bacille Calmette–Guérin (BCG) strain overexpressing this antigen when used as a heat-killed preparation was observed to impart as much protection as the wildtype BCG strain (wtBCG) [14]. This suggested that the detrimental effect of the 19 kDa antigen might be manifested only in saprophytic species of mycobacteria and not in slow-growing species like BCG. Alternatively, it is possible that while a transient expression of the 19 kDa antigen may be sufficient to obliterate the protective efficacy of a saprophytic species (lacking this antigen endogenously and capable of imparting low levels of protection to animals), a powerful immunogen like BCG may require prolonged overexpression of the antigen for manifestation of similar detrimental effects. Thus, this study was aimed at understanding the effect of overexpression of the 19 kDa lipoprotein on the protective efficacy of live recombinant BCG.

We developed a recombinant BCG strain (rBCG19N) expressing the 19 kDa antigen at (approximately fivefold to eightfold) higher levels than BCG. We demonstrate that rBCG19N exacerbated Th2 responses in mice against the BCG antigens with high levels of IL-10 production. Commensurate with this shift towards Th2 immunity in mice, we observed that rBCG19N completely lost its ability to protect guinea pigs challenged with M. tuberculosis H37Rv. Our results thus show that overexpression of the 19 kDa antigen abrogates the protective efficacy of a live BCG vaccine.

Materials and methods

Bacterial culture and transformation. Escherichia coli DH5α, M. smegmatis LR222 and M. bovis BCG (Danish strain) were cultured and transformed, as described earlier [15–17].

DNA manipulations. All restriction endonucleases and the DNA-modifying enzymes such as T4 DNA ligase, Klenow DNA polymerase and T4 polynucleotide kinase (New England Biolabs, Beverly, MA, USA) were used according to the recommended protocols. All polymerase chain reaction (PCR) amplifications were carried out using Pfu DNA polymerase (Stratagene GmbH, La Jolla, CA, USA) according to the manufacturer's recommendations.

Construction of the expression vector pSD5.19N. The 19 kDa gene along with its ribosomal binding site and promoter region was PCR amplified using the plasmid pSMT319, which carries the 19 kDa gene along with its native expression signals, as a template. Amplification was carried out by using gene-specific primers 19pUP (GAT CGG CGT CGT CGA AAT C) and 19p3 (TTA GGA ACA GGT CAC CTC G). The blunt-ended PCR product was cloned in EcoRV-digested pSD5 [18], resulting in pSD5.19N.

Analysis of expression of the 19 kDa antigen in recombinant M. bovis BCG. For analysis of expression, pSD5.19N was electroporated into M. bovis BCG, as described before [15–17]. Transformants were selected on Middlebrook 7H10 medium supplemented with 0.5% glycerol, 1× OADC Middlebrook enrichment and 25 µg/ml kanamycin. The M. bovis BCG transformants were grown in Middlebrook 7H9 medium supplemented with 1× ADC enrichment, 0.5% glycerol, 0.2% Tween-80 and 25 µg/ml kanamycin. Expression of the 19 kDa antigen was monitored both in the cell-free extracts and in the culture supernatants by immunoblot analysis using the monoclonal antibody TB23 against 19 kDa lipoprotein [19]. Thereafter, the membrane was washed thrice with PBS supplemented with 0.05% Tween-20 (PBST) and then incubated for 1 h with HRP-conjugated goat anti-rabbit immunoglobulin G (IgG; 1 : 2500; Jackson Immuno Research Laboratories, Westgrove, PA, USA). The membrane was then washed thrice with PBST and the expression was analysed by using diaminobenzidine and H2O2. Extent of overexpression of the 19 kDa antigen in rBCG19N was measured by subjecting the blots to densitometric analysis by using the NIH image software version 1.52 (NIH image by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). Expression levels in the wildtype BCG strain were used as the basal values for comparisons.

Preparation of antigens.M Wild type BCG and recombinant BCG (rBCG) cultures were grown in 7H9 broth, as described earlier [15–17]. Bacterial cells were harvested by centrifugation at 4000 × g for 10 min and antigens were prepared for immunization, as described earlier [19]. Before immunization, the bacterial colony-forming units (CFU) were determined by plating 10-fold serial dilutions of a mildly sonicated cell suspension on Middlebrook 7H10 plates containing kanamycin (25 µg/ml) and 1× OADC.

Recall antigens used in the study. For its use as a recall antigen, the 19 kDa antigen was purified as a His-Tag fusion protein from recombinant E. coli cultures by Ni2+-NTA metal-affinity chromatography [18]. For preparation of the BCG sonicate, wtBCG was grown in 7H9 medium as described above, to an A600 of 2.0–3.0, harvested and sonicated at 4 °C [15]. The sonicate was then centrifuged at 8000 × g for 15 min and proteins in the soluble fraction devoid of cells and cellular debris were used at various concentrations as a recall antigen.

Animals and immunization protocols. Pathogen-free BALB/c mice (6–8 weeks old) obtained from National Center for Laboratory Animals Studies (NCLAS), Hyderabad, were used in the study. Mice in groups of six were immunized intravenously with live 106 CFU of either rBCG19N or wtBCG. The immune responses elicited by wtBCG or rBCG19N on immunization of mice were compared at 4, 8 and 12 weeks post immunization.

Antigen-specific cytokine assays. Proliferation assays were set up using splenocytes prepared from spleens pooled from mice belonging to the same group [20]. Splenocytes were stimulated with either purified 19 kDa antigen or crude BCG sonicate at concentrations ranging from 0.03 µg/ml to 3 mg/ml. Supernatants were harvested after 72 h of culture for the measurement of antigen-specific interferon-gamma (IFN-γ), IL-10 and IL-13 levels using cytokine-specific kits (R&D Systems, Minneapolis, MN, USA). Purified mouse IFN-γ, IL-10 and IL-13 at concentrations ranging from 0.025 to 1 ng/ml were used for generating a standard curve.

Antibody isotyping analysis. Sera from mice belonging to the same group was pooled and analysed for antigen-specific reactivity by enzyme-linked immunosorbent assay. The antibody responses were measured against purified 19 kDa antigen as well as against crude BCG sonicate (500 ng/well). The levels of 19 kDa antigen-specific and BCG sonicate-specific IgG1 and IgG2a antibodies in the various groups of mice were also determined in sera obtained at 4, 8 and 12 weeks post immunization by using the mouse antibody isotyping kit (Gibco BRL, Gaithersburg, MD, USA).

Analysis of protective efficacy of BCG strains in guinea pigs. Random bred guinea pigs (Hartley strain) weighing 200–400 g were purchased either from Pasteur Institute, Conoor, India or from NCLAS, Hyderabad, India. The animals were immunized intradermally in three groups of 10 animals each, with either normal saline or live BCG-Danish strain (1 × 106 CFU) or live rBCG19N (1 × 106 CFU). Eight weeks after immunization, five animals from each of the three groups were subcutaneously challenged with 7.5 × 105 CFU (Challenge dose-1) and the remaining five animals from each group were challenged with 5 × 104 CFU (Challenge dose-2) of M. tuberculosis H37Rv.

All the animals were euthanized at 8 weeks post challenge. Postmortem examination of the guinea pigs was carried out immediately after euthanasia. The gross pathological tissue damage at the site of infection, liver, lung and spleen was scored, as described by Mitchison [21]. The spleen from an individual animal was homogenized in 5 ml of sterile water. Tenfold serial dilutions of the spleen homogenate was plated in duplicates on Löwenstein–Jensen slopes and incubated at 37 °C for 4–6 weeks. The CFU obtained per spleen and the mean splenic CFU for animals belonging to the same group was calculated.

For histopathological analysis, lungs of the animals were also collected and stored in 10% formalin. Portions of these organs (2 cm × 2 cm) were processed for histopathological analysis. The sections were stained with haematoxylin and eosin and the extent of granuloma and type of cellular infiltration in the tissue were microscopically assessed, as described previously [22, 23].

Statistical analysis. All statistical analyses of the data were performed by using the Student's unpaired t-test. A P-value of <0.05 was considered as statistically significant.

Results

Cloning and expression of the 19 kDa antigen in M. bovis BCG

In an attempt to understand the effect of overexpression of the 19 kDa antigen on the protective efficacy of BCG, we constructed a genetically modified BCG strain, rBCG19N. A DNA fragment of approximately 650 bp carrying the complete ORF of 19 kDa antigen along with its ribosome-binding site and promoter region was PCR amplified and cloned into EcoRV-digested pSD5 [16] resulting in pSD5.19N, as shown in Fig. 1A. pSD519N was then electroporated into BCG and the transformants, selected as described in Materials and methods, were designated as rBCG19N.

Figure 1.

Cloning and expression of the 19 kDa antigen in Mycobacterium bovis Bacille Calmette–Guérin (BCG). Panel A shows the strategy employed for cloning. The gene encoding 19 kDa antigen of M. tuberculosis was polymerase chain reaction amplified using specific primers and cloned into EcoRV-digested pSD5 to generate pSD519N. Relevant restriction enzyme sites of the vector are shown. Ori M and p15A represent origins of DNA replication from mycobacteria and Escherichia coli, respectively. Knr represents the kanamycin-resistance marker and Ter3 and Ter4 represent transcriptional terminators in pSD5. (B) Plasmid pSD5.19N was used to transform M. bovis BCG. Equal amounts of proteins (50 µg) from cell-free extracts and the concentrated supernatants of mid-log phase broth cultures of wtBCG and rBCG19N were subjected to 12.5% SDS–PAGE, transferred onto nitrocellulose membrane and subjected to immunoblot analysis using monoclonal antibody TB23 against 19 kDa lipoprotein, as described in the Materials and methods. The fold induction in the expression of the 19 kDa antigen in rBCG strain was quantitated by densitometric analysis. Values at the bottom of the blot depict the signal intensity for each immunoreactive band, measured as described in the Materials and methods.

Expression of the 19 kDa antigen in rBCG19N was analysed in the cell-free extracts as well as in the culture supernatants using the monoclonal antibody TB23 (Fig. 1B). The levels of 19 kDa antigen in rBCG19N were observed to be approximately eightfold and approximately 4.8-fold higher in the cytosolic and extracellular fractions, respectively, in comparison to the levels of this antigen in wtBCG (Fig. 1B).

Murine immune responses

Cellular (T cell) and humoral responses to mycobacterial antigens were monitored in mice immunized with either rBCG19N or wtBCG at 4, 8 and 12 weeks post immunization. In response to purified 19 kDa antigen, the splenocytes from mice immunized with rBCG19N secreted very high levels of IFN-γ and low levels of IL-10 (Fig. 2A). At 4 weeks post immunization, approximately 2.5-fold higher IFN-γ was secreted by mice immunized with rBCG19N in comparison to mice immunized with wtBCG (P < 0.05). However, IFN-γ secretion was comparable in both groups at 8 and 12 weeks post immunization (Fig. 2A). In contrast, the mice immunized with rBCG19N secreted relatively very low levels of IL-10 (2–2.5 times lower) in comparison to mice immunized with wtBCG (P < 0.05).

Figure 2.

Immune responses in mice immunized with wildtype Bacille Calmette–Guérin (wtBCG) (open bars) or rBCG19N (shaded bars) against purified 19 kDa antigen and BCG sonicate. The levels of antigen-specific cytokines secreted by splenocytes of immunized mice (open bars – wildtype immunized mice and shaded bars – rBCG19N-immunized mice) in response to either purified 19 kDa antigen (A) or BCG sonicate antigens (B) were measured at various time points post immunization as described in the Materials and methods. The levels of interferon-gamma (IFN-γ) and interleukin (IL)-10 induced after 72 h of incubation with 1 µg/ml of either antigen were estimated by using the commercial mouse IFN-γ and IL-10 enzyme-linked immunosorbent assay (ELISA) kits. Purified recombinant IFN-γ and IL-10 at twofold dilutions from 1 ng/ml were used as the standards for estimating the concentration in the test samples. Values represented are mean concentration of cytokine (in ng/ml) released in two separate experiments carried out in triplicates + SD. Panels C and D depict the mean ratios of immunoglobulin G2a (IgG2a) and IgG1 subtypes in a 1 : 500 dilution of sera against purified 19 kDa antigen and BCG sonicate, respectively. The levels of the individual isotypes were determined by ELISA; absorbance values measured at 450 nm were used to calculate the ratios and are represented as mean values for three individual reactions in triplicate wells.

In response to total BCG sonicate, the secretion of IFN-γ and IL-10 showed a different pattern. The overall IFN-γ secretion by rBCG19N and wtBCG-immunized mice was relatively very low (Fig. 2B). Mice immunized with rBCG19N produced significantly higher levels of IL-10 (1.3-fold to sevenfold) in comparison to mice immunized with wtBCG (Fig. 2B; P < 0.05). Also, mice immunized with rBCG19N produced higher levels of IL-13 (twofolds) in comparison to mice immunized with wtBCG (data not shown). These observations suggested that overexpression of the 19 kDa antigen in BCG shifted the host immune responses to Th2 type against BCG sonicate.

This pattern of T-cell subset activation was confirmed by the antibody isotype profiles in the two groups of mice. Against purified 19 kDa antigen, the rBCG19N-immunized mice exhibited significantly elevated IgG2a/IgG1 ratios when compared with IgG2a/IgG1 ratios from wtBCG-immunized mice at all time points (P < 0.02), indicating an increased Th1 response in case of rBCG19N-immunized mice in comparison to wtBCG-immunized mice (Fig. 2C). However, when the BCG sonicate-specific responses were analysed, the IgG2a/IgG1 ratios were higher in case of wtBCG-immunized mice when compared with the ratios from rBCG19N-immunized mice (P < 0.02, at 4 and 8 weeks post immunization) (Fig. 2D), suggesting essentially a Th2 response against BCG sonicate in rBCG19N-immunized mice.

Evaluation of protective efficacy of rBCG19N in guinea pigs

The effect of overexpression of the 19 kDa antigen on the protective efficacy of BCG was evaluated in guinea pigs. Guinea pigs were immunized intradermally either with rBCG19N or with wtBCG and subsequently challenged with two different doses of M. tuberculosis (7.5 × 105 CFU/animal, CD-1 and 5 × 104 CFU/animal, CD-2).

Virulence scores

Scores were assigned to guinea pigs based on the extent of infection and the distribution of lesions in different organs such as lungs, spleen, liver and lymph nodes [21]. Gross pathology and tissue damage in rBCG19N-immunized guinea pigs was comparable to that observed in sham-immunized animals. The total organ scores at the higher dose of challenge was 57.4 ± 8.3 in case of rBCG19N-immunized guinea pigs and 61.6 ± 7.5 in case of sham-immunized guinea pigs (Fig. 3A). At the lower dose of challenge (5 × 104) also, similar extent of tissue damage was observed in case of rBCG19N-immunized (55.8 ± 6.8) and sham-immunized guinea pigs (65.4 ± 7.2). Guinea pigs immunized with wtBCG were quite well protected against both doses of M. tuberculosis challenge and had very little traces of infection in various organs (scores: 29.5 ± 5.5 and 34 ± 5.1 in case of CD-1 and CD-2, respectively). This twofold enhancement in tissue damage in case of rBCG19N and sham-immunized guinea pigs in comparison with wtBCG-immunized guinea pigs was found to be statistically significant (P < 0.02).

Figure 3.

Extent of infection in the various groups of immunized guinea pigs challenged with two doses (CD-1 and CD-2) of Mycobacterium tuberculosis H37Rv at 8 weeks post challenge. (A) Virulence scores for guinea pigs. At the time of necropsy, depending upon the extent of infection and gross pathological damage seen in the various organs (lung, liver, spleen and lymph nodes), scores were assigned to each animal. The total score for each animal was obtained by adding the scores obtained for individual organs. Mean total scores + SEM for different groups are depicted on the y-axis. ** represents P-values <0.02 CD-1 and CD-2 represent the two challenge doses employed in the study (7.5 × 105 CFU and 5 × 104 CFU, respectively). (B) Bacterial load in spleens of animals was determined by plating 10-fold serial dilutions of the spleen homogenates in duplicates on LJ slopes. The mean log10CFU values for different groups are depicted on the y-axis. * represents P-values <0.05 and ** represents P-values <0.02.

Bacillary load in spleens

Bacillary loads in the spleens of guinea pigs from various groups exhibited a similar pattern as was observed with virulence scores (Fig. 3B). At higher dose of M. tuberculosis challenge (animal: CD-1), the highest bacillary load was observed in spleens from sham-immunized guinea pigs (log10CFU = 4.2 ± 0.5). Guinea pigs immunized with wtBCG were protected and had about 1 log lower bacillary load in their spleens (log10CFU = 3.4 ± 0.3, P < 0.05). However, guinea pigs immunized with rBCG19N exhibited splenic CFU (log10CFU = 4.2 ± 0.45), which was comparable to the bacillary load from sham-immunized guinea pigs.

Even at a 15-fold lower dose of challenge (5 × 104 CFU/animal: CD-2), a similar pattern of infection was observed in guinea pigs from various groups. rBCG19N failed to protect guinea pigs against M. tuberculosis challenge. Guinea pigs immunized with rBCG19N exhibited bacillary load in their spleens (log10CFU = 3.9 ± 0.5), which was comparable to the bacillary load in the spleens of sham-immunized animals (log10CFU = 4.15 ± 0.23). As observed at higher dose, guinea pigs immunized with wtBCG exhibited markedly reduced splenic bacillary load (log10CFU = 2.7 ± 0.35, P < 0.05, Fig. 3B).

Histopathological analysis

Lung sections from guinea pigs belonging to the various groups were subjected to histopathological analysis in order to determine the extent of tissue damage (Fig. 4). At both the doses of M. tuberculosis challenge (CD-1 and CD-2), sham- and rBCG19N-immunized guinea pigs exhibited comparable extent of granulomatous tissue in the lungs. Lungs from wtBCG-immunized guinea pigs, on the other hand, exhibited much lower percentage of granuloma formation. Microscopic examination of the granulomas in rBCG19N- and sham-immunized guinea pigs revealed a mixed population of macrophages and lymphocytes. In contrast, granulomas in wtBCG-immunized guinea pigs were more compact and comprised exclusively of lymphocytes (Fig. 4).

Figure 4.

Histopathological analysis of lung sections from immunized guinea pigs challenged with two doses (CD-1 and CD-2) of Mycobacterium tuberculosis H37Rv. Sections of lung from animals in various groups were stained with haematoxylin and eosin as described in the Materials and methods. The sections were observed under a magnification of ×40 for the presence of granuloma. Tissue section from an unvaccinated and unchallenged animal (normal) was used as a reference for normal tissue histology.

Discussion

Several immunodominant antigens of M. tuberculosis have been shown to prime the Th1-type T cells, capable of IFN-γ secretion in the host [2, 24–28]. The 19 kDa antigen by virtue of its lipoproteinaceous nature has been shown to activate the innate immune mechanisms resulting in the production of copious amounts of Th1-promoting cytokine IL-12, in activated macrophages [29]. The 19 kDa antigen is actively recognized by the host-activated T cells by virtue of numerous CD4 and CD8 epitopes and also induces memory recall responses in the host [7, 8].

However, overexpression of this antigen in the nonpathogenic mycobacterial species namely M. vaccae and M. smegmatis resulted in the abrogation of the limited protection conferred by these mycobacterial species [13]. This reduction in protective efficacy of saprophytic mycobacteria was not observed when a mixture of purified 19 kDa antigen and mycobacteria were administered into mice, indicating that the capability of the 19 kDa lipoprotein to modulate the host immune system was dependent on the mode of its presentation to the host [13]. In contrast to the results observed in the cases of saprophytic mycobacteria, overexpression of the 19 kDa antigen in BCG did not alter the ability of BCG to protect mice against M. tuberculosis challenge [14]. These results indicated that the ability of 19 kDa antigen to modulate host immune responses leading to abrogation of protective efficacy of the recombinant mycobacteria was limited only to the saprophytic species like M. smegmatis and M. vaccae and did not extend to the vaccine strain BCG. However, it is crucial to consider here that in the protection studies employing saprophytic mycobacteria, live, replicating bacteria were used for immunization of mice. In contrast, in the studies involving rBCG (overexpressing the 19 kDa antigen), where no detrimental effect was observed, heat-killed bacilli were used for immunization. Thus, it is possible that the limited kinetics of antigen overexpression (transient overexpression) in the case of heat-killed rBCG strain might not have been sufficient enough for modulation of protective efficacy of a very potent immunogen such as BCG. In order to address this issue, we employed a live recombinant BCG strain to study the influence of overexpression of the 19 kDa antigen on the ability of BCG to elicit protective immune responses against M. tuberculosis challenge.

We engineered BCG to overexpress the 19 kDa antigen at very high levels by transforming it with an extrachromosomal vector pSD519N carrying the gene encoding the 19 kDa antigen under the transcriptional control of its native promoter. In comparison with the wtBCG strain, the rBCG strain (rBCG19N) exhibited enhanced levels of the 19 kDa antigen in the cytoplasm (approximately eightfold) as well as in the extracellular fraction (approximately fivefold). Overexpression of the 19 kDa antigen in rBCG19N resulted in significant modulation of murine immune responses. The responses elicited by rBCG strain against the purified 19 kDa antigen were predominantly Th1, as characterized by increased production of IFN-γ and IgG2a responses. However, against BCG sonicate, immune responses in the rBCG-immunized mice indicated a complete shift to the Th2 type, as characterized by the production of copious amounts of IL-10. This was further substantiated by the antibody isotype profile induced in rBCG19N-immunized mice in response to BCG sonicate.

The elicitation of Th1 response against purified 19 kDa antigen on immunization with rBCG19N is in accordance with earlier studies on immunization with recombinant mycobacteria overexpressing this antigen [10]. However, in this study, we show that in addition to this antigen-specific immune response, the overexpression of this antigen elicits a powerful Th2 response against the repertoire of BCG proteins (BCG sonicate). BCG sonicate is a complex mixture of immunodominant antigens, and responses to any individual antigen (for example the 19 kDa antigen) would be masked by the overwhelming responses of other more immunodominant antigens of the mixture. Moreover, earlier studies have shown that immunization with the 19 kDa antigen declines macrophage activation by lowering the levels of cytokines produced by the host phagocytes [12]. Infection with recombinant M. smegmatis expressing the M. tuberculosis 19 kDa antigen has been demonstrated to induce significantly lower levels of a variety of cytokines in the macrophages as compared to infection with the wildtype M. smegmatis[12]. The overall downregulatory property associated with this antigen in the context of mycobacteria would thus completely obscure the elevated Th1 phenotype observed with purified antigen as a recall immunogen.

In addition, we observed that immunization of guinea pigs with the wtBCG reduced the splenic bacillary load of M. tuberculosis by more than 10-fold; the levels of mycobacterial colonization in spleens of rBCG19N-immunized animals were comparable to those in the sham-immunized animals indicating the abrogation of protection conferred by the BCG backbone itself against M. tuberculosis challenge. Thus, immunization of guinea pigs with live rBCG overexpressing the 19 kDa antigen in our study resulted in entirely different immune responses when compared with the observations made by Yeremeev et al. [14] as a result of immunization with heat-killed rBCG strain overexpressing this antigen. The live recombinant BCG provides sustained expression of the 19 kDa antigen in the host, which may be responsible for these observed differences. These observations suggest that live organisms may serve as a better model for evaluating the effect of overexpression of an antigen on the protective efficacy of BCG.

Active downregulation of host immunity or activation of an immune response that is beneficial for the pathogen (Th2 in case of mycobacteria) is an effective mechanism that a pathogen can utilize for its survival in the host. Sustained high levels of IL-10 causing subversion of the host immune system towards Th2-type immune responses against mycobacterial antigens (BCG sonicate) could be responsible for reduced protective efficacy of rBCG19N. A careful analysis of the molecular mechanisms involved in subversion of host immune responses by mycobacterial products like the 19 kDa antigen could thus provide better insights into the mechanism of M. tuberculosis pathogenesis and aid in the development of better intervention strategies.

Acknowledgments

This work was supported by a financial grant from the Department of Biotechnology, Government of India. VR, ND and RS are thankful to UGC for senior research fellowships. AK is thankful to CSIR for a fellowship. We are thankful to Dr Douglas B. Young, Imperial College, London, UK for providing us with the plasmid pSMT319 and monoclonal antibody TB23. Bindu Nair, Manisha Jain, Jaya Gopinath, S. Nambirajan, K. Chandran and M. Asokan are acknowledged for their technical assistance. Rajiv Chawla is acknowledged for the efficient preparation of the manuscript.

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