ABSTRACT
Hypoxia and low concentrations of nitric oxide have been reported to upregulate in vitro gene expression of 48 proteins of the dormancy (DosR) regulon of Mycobacterium tuberculosis. These proteins are thought to be essential for the survival of bacteria during persistence in vivo and are targeted by the immune system during latent infection in humans. Here we have analyzed the immunogenicity of eight DosR regulon-encoded antigens by plasmid DNA vaccination of BALB/c and C57BL/6 mice, i.e., Rv1733c, Rv1738, Rv2029c (pfkB), Rv2031c/hspX (acr), Rv2032 (acg), Rv2626c, Rv2627c, and Rv2628. Strong humoral and/or cellular Th1-type (interleukin-2 and gamma interferon) immune responses could be induced against all but one (Rv1738) of these antigens. The strongest Th1 responses were measured following vaccination with DNA encoding Rv2031c and Rv2626c. Using synthetic 20-mer overlapping peptides, 11 immunodominant, predicted major histocompatibility complex class II-restricted epitopes and one Kd-restricted T-cell epitope could be identified. BALB/c and (B6D2)F1 mice persistently infected with M. tuberculosis developed immune responses against Rv1733c, Rv2031c, and Rv2626c. These findings have implications for proof-of-concept studies in mice mimicking tuberculosis (TB) latency models and their extrapolation to humans for potential new vaccination strategies against TB.
Tuberculosis (TB) remains a global health problem, killing millions of people each year. The only TB vaccine available today, live-attenuated Mycobacterium bovis Bacillus Calmette-Guérin (BCG), is effective against the severe childhood forms of tuberculosis, miliary TB and TB meningitis, which generally develop within 2 years after infection. Unfortunately, BCG demonstrates variable efficacy against the pulmonary forms of TB in young adults. Interference of environmental mycobacteria and genetic variations, in both host populations and the BCG vaccine strains used, explains the variable efficacy of BCG to some extent, although other factors may also be involved (19).
It is estimated that one-third of the world's population is latently infected with Mycobacterium tuberculosis. Active postprimary tuberculosis can occur following endogenous reactivation of such a latent infection or following exogenous reinfection. A defective immune system (human immunodeficiency virus infection, immunosuppressive treatment, malnutrition, stress, and ageing) generally lies at the basis of endogenous reactivation of TB disease and increased susceptibility to exogenous reinfection (10). In high-incidence countries, the contribution of exogenous reinfection is thought to be more important than that in low-incidence countries (9), but in populations that have immigrated from high- to low-risk areas, a combination of both reactivation and recent transmission are responsible for the high-incidence rates in these communities (1, 3).
In vitro cultures of “stressed” M. tuberculosis, grown under conditions of nutrient starvation (4), hypoxia (35, 38), or low pH (20) are thought to mimic, at least in part, the in vivo conditions of dormant tubercle bacilli persisting in lung granulomas. Well documented are the 48 gene products of the dormancy regulon controlled by DosR (Rv3133c), the expression of which is specifically upregulated by in vitro conditions of hypoxia or at low concentrations of nitric oxide (NO) (44). Similar transcriptional adaptations have also been reported for M. tuberculosis isolated from gamma interferon (IFN-γ)-activated mouse macrophages (37) and from persistently infected mouse lung tissue (40). More recently, the study of artificial granulomas of encapsulated bacteria grown in semidiffusible hollow fibers implanted subcutaneously into mice has given a comprehensive view of the dormancy-associated transcriptional modifications, pointing again to the induction of DosR and at least 20 other proteins encoded by the DosR regulon (28).
The best-known member of the DosR regulon is the 16-kDa alpha-crystallin homologue (Rv2031c, hspX) encoded by acr. In 1998, it was shown that acr transcription was strongly induced by mildly hypoxic conditions and that it was required for in vivo growth in mouse bone marrow-derived macrophages and human THP-1 cells (46). The HspX protein is highly immunogenic for B cells, as reflected by the presence of antibodies in about 70% of smear-positive and 50% of smear-negative patients with pulmonary tuberculosis and also in many healthy subjects latently infected after household exposure to tuberculosis (12, 26, 29, 32). With respect to T-cell immunity, Caccamo et al. have reported Th1-type CD4+ and CD8+ T cells recognizing epitopes of hspX in tuberculosis patients (7, 8). On the other hand, Vekemans et al. showed that neonatal BCG does not induce IFN-γ responses to hspX, whereas the vaccine is clearly capable of inducing immune responses to other TB antigens, such as the mycolyl transferases (42).
Recently we have observed preferential T-cell recognition of DosR regulon-encoded antigens by Mantoux-positive individuals with latent TB compared to patients with TB disease, suggesting that these immune responses are part of the protective immune response to M. tuberculosis (12, 30). In contrast, and extending Vekemans' results, we found that T-cell responses against DosR regulon-encoded antigens were very low in BCG-vaccinated mice and humans (M. Y. Lin, A. Geluk, M. Verduyn, A. Friggen, K. L. Franken, K. van Meijgaarden, S. Smith, H. Dockrell, M. Voskuil, F. Verreck, K. Huygen, T. H. M. Ottenhoff, and M. R. Klein, submitted for publication).
In order to characterize the T-cell response of mice against this novel group of M. tuberculosis antigens in more detail as well as study whether the poor immune responses to latency antigens following BCG vaccination are caused by an inherent lack of immunogenicity or rather by a deficient expression by the vaccine, we analyzed immune responses in mice vaccinated with plasmid DNA encoding these proteins. We have previously reported that DNA vaccination is a powerful and easy method for screening immune potential and identifying immunodominant major histocompatibility complex (MHC) class I- and II-restricted epitopes of TB vaccine candidates (13, 17, 25, 33). BALB/c and C57BL/6 mice were vaccinated with DNA plasmids carrying eight dormancy regulon-encoded proteins, e.g., Rv1733c, Rv1738, Rv2029c, Rv2031c, Rv2032, Rv2626c, Rv2627c, and Rv2628. These eight proteins were selected from a series of 25 DosR regulon-encoded proteins on the basis of their strong stimulation of T-cell responses in a group of latently infected humans (30). Antibody production and Th1 cytokine secretion were analyzed, and using synthetic overlapping 20-mer peptides, we could map T-cell epitopes for five of these proteins. Immune responses against DosR regulon-encoded proteins were also analyzed in mice that were acutely or persistently infected with M. tuberculosis.
MATERIALS AND METHODS
Mice.BALB/c, C57BL/6 (B6), and (C57BL/6 × DBA/2)F1 [(B6D2)F1] mice were bred in the Animal Facilities of the Pasteur Institute in Brussels, Belgium, from breeding couples originally obtained from Bantin & Kingman (United Kingdom). All animals were 6 to 8 weeks old at the start of the experiments.
Plasmid vaccination.The genes encoding eight full-length latency DosR regulon-encoded antigens from M. tuberculosis H37Rv, Rv1733c, Rv1738, Rv2029c (pfkB), Rv2031c (hspX, acr), Rv 2032 (acg), Rv2626c, Rv2627c and Rv2628, were recombined into a Gateway (Invitrogen)-adapted pV1J.ns-tPA vector. Plasmid DNA encoding the early secreted antigen Ag85A (Rv3804c) was included in the study for comparison (25). In the pV1J.ns-tPA vector (pDNA), the genes of interest are expressed under the control of the promoter of the IE1 antigen from cytomegalovirus including intron A and preceded by the signal sequence of human tissue plasminogen activator (25). Mice were anesthetized with ketamine-xylazine and injected four times intramuscularly in both quadriceps muscles with 2 × 50 μg of pDNA at 3-week intervals. For intracellular IFN-γ staining, BALB/c mice were vaccinated with 2 × 50 μg of DNA encoding Rv2626c on weeks 0, 3, and 16 and tested on week 19. For in vivo cytotoxic T lymphocyte (CTL) assays, BALB/c mice were vaccinated with 2 × 50 μg of DNA encoding Rv2626c on weeks 0, 3, 16, and 23 and tested on week 24.
Induction of acute and persistent M. tuberculosis infection.Luminescent M. tuberculosis H37Rv was grown as a surface pellicle on synthetic Sauton medium as described before (40). Bacteria were harvested after 2 weeks and aliquots were stored frozen at −70° until use. The mycobacterial load in the lung and spleen of infected mice was quantified by plating on Middlebrook 7H11 agar supplemented with oleic acid-albumin-dextrose-catalase or using a bioluminescence assay (for the determination of relative light units [RLU]) (16). For acute infection, BALB/c and (B6D2)F1 mice were infected intravenously with 105 CFU of M. tuberculosis and sacrificed 4 weeks later. Persistent M. tuberculosis infection was induced by low-dose intratracheal infection as previously described by Arriaga et al. (2) or by intravenous infection followed by short-term chemotherapy as previously described by Scanga et al. (36). Briefly, BALB/c mice were instilled intratracheally with 103 CFU of M. tuberculosis H37Rv, resulting in a persistent infection and a prolonged survival time of at least 1 year, in contrast to a median survival time of 4 months when mice were infected with 105 CFU by the intravenous route (34). Alternatively, (B6D2)F1 mice were infected intravenously with 105 CFU of M. tuberculosis H37Rv and treated from week 4 to week 12 with a combined antibiotic treatment of isoniazid (INH; 0.1 g/liter) and pyrazinamide (PZA; 8 g/liter) in the drinking water.
Recombinant M. tuberculosis antigens.Recombinant proteins were produced as previously described (22, 30). Briefly, nucleotide sequences of selected M. tuberculosis H37Rv genes were obtained from TubercuList (http://genolist.pasteur.fr/TubercuList ). Genes were amplified by PCR from genomic DNA of M. tuberculosis H37Rv and cloned by Gateway Technology (Invitrogen, San Diego, CA) in pDEST17, a bacterial expression vector containing an N-terminal hexahistidine tag for rapid purification with nickel-chelating resin. The proteins were overexpressed in Escherichia coli BL21(DE3) and purified as previously described. Sequencing was performed to confirm the identity of the cloned DNA fragments. Size and purity were checked by gel electrophoresis and Western blotting with anti-His antibodies (Invitrogen). Residual endotoxin levels were determined with a Limulus amoebocyte lysate assay (Cambrex) and were found to be below 50 IU/mg recombinant protein.
Synthetic peptides.Synthetic 20-mer peptides, overlapping by 10 amino acid residues, were synthesized as previously described (23). Peptides were initially dissolved in dimethyl sulfoxide, and stock solutions were subsequently prepared in RPMI 1640 culture medium at 1 mg/ml. Aliquots were stored frozen at −20° until use.
Antibody ELISA.Sera from naïve, DNA-immunized, and TB-infected mice were collected by tail bleeding. Levels of total immunoglobulin G (IgG) antibodies specific for TB latency antigens were determined by an enzyme-linked immunosorbent assay (ELISA) on individual sera, using recombinant latency antigens for coating (400 ng/well), a secondary peroxidase-labeled rat anti-mouse κ light chain monoclonal antibody (LO-MK-1; Experimental Immunology Unit, Université Catholique de Louvain, Brussels, Belgium), and orthophenyldiamine (Sigma) for revelation. Data are presented as the optical density at 492 nm (OD492) for a serum dilution of 1:400. At this dilution, sera from all naïve mice had OD levels between 0.100 and 0.150.
Cytokine production.Plasmid DNA-vaccinated mice were sacrificed 3 weeks after the fourth immunization. Spleens from three individual mice were removed aseptically and homogenized by gentle disruption in a Dounce homogenizer, and cells were adjusted to 4 × 106 white blood cells/ml in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 5 × 10−5 M 2-mercaptoethanol, and antibiotics. Cells were incubated at 37°C in round-bottom, 96-well microtiter plates in a humidified CO2 incubator and stimulated with purified recombinant latency antigens (5 μg/ml) or synthetic peptides (10 μg/ml). Spleens from individual mice were tested for response against whole protein, and spleens were pooled for peptide testing. Culture supernatants were harvested after 24 h for interleukin-2 (IL-2) assays and after 72 h for IFN-γ assays, when peak values of the respective cytokines can be measured. Supernatants were stored frozen at −20°C until testing. Experiments were performed twice, and data from one experiment are reported.
IL-2 and IFN-γ assays.IL-2 activity was measured in duplicate using an IL-2-dependent CTLL-2 bioassay, and IL-2 levels were expressed as mean counts per minute (cpm) of incorporated [3H]thymidine. In this assay, a standard IL-2 preparation of 600 pg/ml corresponded to 15,000 cpm and the detection limit was 30 pg/ml (17). IFN-γ activity was quantified by sandwich ELISA using coating antibody R4-6A2 and biotinylated detection antibody XMG1.2 (both BD Pharmingen). The detection limit of the IFN-γ ELISA was 5 pg/ml. Antigen-specific cytokine levels were considered positive when values were more than fivefold higher than those of unstimulated cells.
Intracellular IFN-γ measurement using flow cytometry.Intracellular IFN-γ measurement using flow cytometry was performed as described before (14, 40). In short, spleen cells (2.5 × 106 cells/ml) were cultured in 48-well tissue culture plates in RPMI 1640 medium (Gibco) supplemented with penicillin and streptomycin, 2 mM l-glutamine, 5 × 10−5 M 2-mercaptoethanol, 10% FCS, and 10 μg/ml of Rv262678-87 IYYVDANASI (ProImmune Ltd., United Kingdom) peptide. Cells were maintained in 5% CO2 at 37°C. After 6 days of culture, living cells were harvested on a Ficoll gradient and IFN-γ production was analyzed by intracellular staining and fluorescence-activated cell sorter analysis after a second restimulation of 24 h in the presence or absence of peptide. Brefeldin A (Sigma) was added to the cells at a concentration of 10 μg/ml during the last 4 h of culture, and then cells were harvested, washed once in staining buffer, and surface stained. One million cells in 100 μl of staining buffer (phosphate-buffered saline [PBS], 0.1% NaN3, and 5% FCS) were incubated with fluorescein isothiocyanate-conjugated monoclonal rat anti-mouse CD8 antibodies (clone 53-6.7; Pharmingen) for 30 min at 4°C. Cells were washed twice in staining buffer and fixed in PBS containing 3% (wt/vol) paraformaldehyde for 30 min. Paraformaldehyde-fixed cells were washed once with staining buffer and then permeabilized in staining buffer supplemented with 0.1% (wt/vol) saponin. Cells were incubated for 20 min at 4°C with phycoerythrin-conjugated rat anti-mouse IFN-γ antibodies (clone XMG1.2; Pharmingen) diluted in staining buffer supplemented with 0.1% saponin. Cells were washed twice in staining buffer containing saponin and finally resuspended in PBS containing paraformaldehyde. Samples were acquired on a flow cytometer (FACSCalibur; BD Biosciences, Mountain View, CA) calibrated with CaliBRITE beads (Becton Dickinson), and fluorescence was analyzed using CellQuest software. For each sample, 6 × 104 lymphocytes were gated on the basis of their characteristic forward- and side-scatter profiles.
In vivo CTL activity assessment by adoptive transfer of CFSE-labeled target cells.In vivo cytotoxic activity assessment was performed as described before (33). Briefly, 1 week after the fourth vaccination with DNA encoding Rv2626c, BALB/c mice were adoptively transferred with 20 × 106 cells of a 1:1 mix of unpulsed syngeneic terget cells labeled at 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSElow) and of peptide-pulsed cells labeled at 10 μM CFSE (CFSEhigh). The Rv2626c78-87 IYYVDANASI (ProImmune Ltd., United Kingdom) peptide (10 μg/ml) was used to pulse the CFSEhigh cells. Twenty-four hours later, adoptively transferred mice were sacrificed, spleens were removed and homogenized, erythrocytes were depleted, and cells were washed and resuspended in PBS for acquisition on a FACSCalibur cytofluorometer. To evaluate the percentage of specific lysis, the ratio of peptide-pulsed CFSEhigh cells/CFSElow cells in vaccinated mice was compared to the mean ratio of peptide-pulsed CFSEhigh /CFSElow in transferred naïve control mice. Analysis was performed with four naïve and five Rv2626c DNA-vaccinated mice.
RESULTS
Th1-type cytokine secretion in response to eight DosR regulon-encoded antigens in plasmid DNA-vaccinated BALB/c and C57BL/6 mice.As shown in Fig. 1, positive levels of antigen-specific IFN-γ could be induced in spleen cells from mice vaccinated with DNA encoding five out of eight DosR regulon-encoded proteins. DNA encoding Rv2626c was the most immunogenic of the eight plasmids tested in both BALB/c (3,094 ± 1,075 pg/ml) and B6 (4,552 ± 1,336 pg/ml) mice. In BALB/c mice, vaccination with Rv2626c DNA induced IFN-γ levels that were comparable to those induced in response to Rv3804c (2,527 ± 1,710 pg/ml) encoding the secreted mycolyl transferase Ag85A, which has well-documented vaccine potential (25). Mice vaccinated with DNA encoding Rv2031c (hspX) also produced high levels of IFN-γ; DNA encoding this antigen was more immunogenic in BALB/c mice (1,529 ± 716 pg/ml) than in B6 mice (796 ± 839 pg/ml). Intermediate responses (between 600 and 800 pg/ml) were induced following vaccination with DNA plasmids encoding Rv2032, Rv2627c, and Rv2628, whereas IFN-γ responses to Rv1733c, Rv1738, and Rv2029 were very low (below 250 pg/ml) in both mouse strains. The production of the other major Th1-type cytokine, IL-2, was also highest in mice vaccinated with DNA encoding Rv2626c and Rv2031c, and as for IFN-γ, Rv2031c vaccination induced higher responses in BALB/c mice than in B6 mice.
IFN-γ and IL-2 levels in 72-h and 24-h spleen cell culture supernatant from BALB/c (white bars) and C57BL/6 (black bars) mice vaccinated with plasmid DNA and stimulated with the corresponding antigen (5 μg/ml) 3 weeks after the fourth DNA vaccination. Results represent mean ± standard deviation (error bars) values of three mice tested individually in each group.
Antibody production in response to eight DosR regulon-encoded antigens in plasmid DNA-vaccinated BALB/c and C57BL/6 mice.As shown in Fig. 2, significant antigen-specific antibody production could be induced in mice vaccinated with six out of eight DosR regulon-encoded proteins. Antibody responses to Rv1733c and Rv2029c were positive, indicating that the absent Th1 T-cell responses to these antigens were not caused by expression defects in the plasmid. Therefore, only one plasmid, i.e., the one encoding Rv1738, was found to induce neither antibodies nor T-cell responses.
Antigen-specific antibodies in sera from naïve (open symbols) and DNA-vaccinated (closed symbols) BALB/c (dots) and C57BL/6 (squares) mice. Results represent mean ± standard deviation (error bars) of OD492 values of serum diluted 1:400 (three mice tested individually in each group).
Identification of immunodominant H-2d- and H-2b-restricted epitopes of five latency-associated antigens.We have previously shown that plasmid DNA vaccination is a powerful tool for the identification of CD4+ and CD8+ epitopes on mycobacterial antigens (15, 17, 33). Cells from DNA-vaccinated BALB/c and C57BL/6 mice were stimulated with synthetic, overlapping 20-mer peptides spanning the entire protein sequence of Rv1733c-, Rv2029c-, Rv2031c-, Rv2626c-, Rv2627c-, and Rv2628-encoded antigens, and supernatants were tested for IFN-γ and IL-2 content. A number of immunodominant epitopes (IFN-γ levels above 250 pg/ml) could be identified for five DosR regulon-encoded antigens (Table 1). Complete epitope-mapping results are shown for Rv2031c, Rv2626c, and Rv2628 (Fig. 3, 4, and 5). As expected from the low T-cell responses to whole Rv1733c antigen, no immunodominant peptides could be identified for this antigen in plasmid-vaccinated mice (data not shown). In general, BALB/c mice reacted to more peptides and with higher magnitudes than did B6 mice. All three peptides recognized by DNA-vaccinated B6 mice, i.e., p8 and p13 of Rv2626c and p6 from Rv2627c were also recognized by DNA-vaccinated BALB/c mice. All identified peptide sequences contained predicted MHC class II epitopes according to the predictive program TSites (18). Stimulation with some of the peptides, such as Rv2627c21-40 and Rv262811-30, induced clearly higher responses than did stimulation with the total recombinant antigen, which may have been caused by difficulties in antigenic processing of these NH2-terminal hexahistidine-tagged proteins. All IFN-γ-inducing peptides also stimulated positive IL-2 responses, except for the peptide of Rv2626c71-90, which stimulated only a positive production of the former cytokine. We have previously observed that this dissociation of IFN-γ and IL-2 response reflects the activation of CD8+ T cells, and on this basis, we have been able to define MHC class I-restricted T-cell epitopes on the mycolyl transferase Ag85A (15) and on the phosphate-binding protein PstS-3 (Rv928) (33).
IFN-γ and IL-2 levels in 72-h and 24-h spleen cell culture supernatant (pool of three mice per group) of BALB/c (black bars) and C57BL/6 (white bars) mice vaccinated with plasmid DNA encoding Rv2031c and stimulated with 14 synthetic 20-mer peptides overlapping by 10 amino acids (10 μg/ml) 3 weeks after the fourth DNA vaccination (p14 spanning amino acids 125 to 144).
IFN-γ and IL-2 levels in 72-h and 24-h spleen cell culture supernatant (pool of three mice/group) of BALB/c (black bars) and C57BL/6 (white bars) mice vaccinated with plasmid DNA encoding Rv2626c and stimulated with 14 synthetic 20-mer peptides (10 μg/ml) 3 weeks after the fourth DNA vaccination. (p14 spanning amino acids 124 to 143).
IFN-γ and IL-2 levels in 72-h and 24-h spleen cell culture supernatant (pool of three mice/group) of BALB/c (black bars) and C57BL/6 (white bars) mice vaccinated with plasmid DNA encoding Rv2628 and stimulated with 11 synthetic 20-mer peptides (10 μg/ml) 3 weeks after the fourth DNA vaccination.
Predicted MHC class II epitopes on IFN-γ inducing synthetic 20-mer peptides of five DosR regulon-encoded antigensa
Identification of a Kd-restricted epitope in Rv2626c protein.Sequence analysis according to HLA_BIND revealed that Rv2626c71-90 contained a predicted Kd motif spanning amino acids 79 to 87, YYVDANASI (underlining indicates anchor motifs), with a very high predictive score of 4,800 (48.22% of maximal score). The score of this peptide according to the prediction program SYFPEITHI was 26 (68.42% of the maximal score). Intracellular IFN-γ staining of spleen cells of Rv2626c DNA-vaccinated BALB/c mice stimulated with a synthetic Rv2626c78-87 peptide IYYVDANASI showed that this was indeed an MHC class I-restricted peptide capable of inducing a strong IFN-γ response in 29% of peptide-amplified CD8+ T cells (Fig. 6a).
(a) Intracellular IFN-γ staining of CD8+ spleen cells from BALB/c mice vaccinated with Rv2626c DNA and restimulated with Kd-restricted peptide Rv2626c78-87. Spleen cells from four naïve mice (a and b) and four Rv2626c DNA-vaccinated (c and d) mice were pooled and stimulated in vitro for 6 days without or with the peptide, Ficoll purified, and cultured for another 24 h without (a and c) or with the peptide (b and d). Following brefeldin treatment, cells were stained with fluorescein isothiocyanate-labeled anti-CD8+ and phycoerythrin-labeled anti-IFN-γ antibodies. The values in the rectangles represent the percentages of total gated CD8+ T cells expressing IFN-γ. (b) In vivo cytotoxic T-cell response against Kd-restricted Rv2626c78-87 peptide, as detected by lysis of peptide-pulsed CFSEhigh-labeled syngeneic target cells using cytofluorometry. The figure shows one representative animal of the group of naïve mice (n = 4) (left panel) and of the group of mice vaccinated with Rv2626c (n = 5) (right panel). The mean ± standard deviation of percent specific lysis in the vaccinated group is indicated at the top.
Vaccination of BALB/c mice with DNA encoding Rv2626c DNA also induced a very strong in vivo cytotoxic T-cell response against this Kd-restricted Rv2626c78-87 peptide, as demonstrated by a mean 72% specific lysis of peptide-pulsed, CFSEhigh-labeled target cells (Fig. 6b).
Immune response against DosR regulon-encoded antigens in BALB/c mice persistently but not acutely infected with M. tuberculosis.In order to analyze the immunogenicity of the DosR regulon-encoded antigens during persistent M. tuberculosis infection, BALB/c mice were infected intratracheally with 103 CFU of M. tuberculosis, as originally described by Arriaga et al. (2). Four mice were sacrificed 9 months later, and antibody levels against the eight DosR regulon-encoded proteins and against Ag85A were analyzed by ELISA. The number of M. tuberculosis bacteria recovered at this time point was 3.23 ± 0.10 log10 milli-RLU (4.70 ± 0.46 log10 CFU) in spleen and 4.35 ± 0.47 log10 milli-RLU (5.85 ± 0.35 log10 CFU) in lung. As shown in Fig. 7, positive antibody responses could be detected against Rv2031c and Rv2626c at this late time point, whereas serum antibody levels of BALB/c mice infected intravenously with 2 × 105 CFU 1 month before were only slightly higher than serum levels of naïve BALB/c mice.
Total IgG antibodies against eight DosR regulon-encoded antigens or Ag85A in serum from naïve mice (open circles), in sera from mice infected intravenously with 105 CFU of M. tuberculosis 1 month before (open squares) or in persistently infected mice infected intratracheally with 103 CFU 9 months before (solid circles). Results represent mean ± standard deviation (error bars) values (OD492 values) of three (naïve/month 1) and four (month 9) mice tested individually in each group at 1:400 serum dilution.
DosR-specific IFN-γ production was also compared in the spleens of these persistently and acutely infected BALB/c mice (Table 2). Acutely infected BALB/c mice demonstrated strong responses to mycolyl transferase Ag85A. In contrast, little to no response (<250 pg/ml) to five of the eight DosR regulon-encoded proteins was detected, and weak responses, tenfold lower in magnitude than that against Rv3804c, were observed against Rv1733c and Rv2626c. Spleen cells from persistently infected BALB/c mice demonstrated threefold-decreased responses to Rv3408c but significantly increased responses to Rv1733c and Rv2626c (fourfold increases). Moreover and confirming the antibody response, T cells from persistently infected BALB/c mice also reacted strongly against Rv2031c. The magnitude of the IFN-γ response against the three DosR regulon-encoded proteins was comparable to the magnitude of the IFN-γ response against mycolyl transferase Ag85A at this time point.
DosR-specific IFN-γ responses in BALB/c mice actively or persistently infected with M. tuberculosisa
Spleen cell IFN-γ response against DosR regulon-encoded Rv2031c and Rv2626c in (B6D2)F1 mice acutely or persistently infected with M. tuberculosis.As an alternative way to induce persistent TB infection, (B6D2)F1 mice were infected by the intravenous route with 105 CFU of M. tuberculosis and subsequently administered a combined INH-PZA antibiotic treatment, as previously described by Scanga et al. (36). Bacterial load in spleen from intravenously infected (B6D2)F1 mice decreased from 5.74 ± 0.13 log10 CFU at 4 weeks postinfection (p.i.) to below detection level (2.0 log10) at the end of the 8-week treatment. CFU counts in lungs decreased from 6.67 ± 0.10 log10 CFU to 3.1 ± 0.67 log10 CFU after completion of chemotherapy.
As shown in Fig. 8, at 4 weeks p.i., elevated IFN-γ responses were found in spleen in response to the early secreted antigen 85B (Rv1886c) protein and the immunodominant Rv38751-20 epitope of ESAT-6, but not in response to Rv2031c and weakly in response to Rv2626c. In contrast, after the completion of chemotherapy, significantly increased IFN-γ responses were detected in response to Rv2031c (2,732 ± 496 at week 12 versus 112 ± 65 pg/ml at week 4), Rv2031c71-90 (2,165 ± 949 versus 111 ± 78), and Rv2626c (3,008 ± 1,681 versus 740 ± 355). As observed in the previous experiment for the Ag85A-specific response in BALB/c mice, T-cell responses of (B6D2)F1 mice against Ag85B were about threefold lower in persistently infected animals than that in acutely infected animals. ESAT-6-specific T-cell responses against immunodominant Rv38751-20 peptide were not different in acutely and persistently infected mice.
Spleen cell IFN-γ responses of (B6D2)F1 mice, 4 weeks after intravenous infection with 105 CFU of M. tuberculosis (open bars) or at the end of 8 weeks of INH-PZA therapy (black bars) in response to recombinant proteins (5 μg/ml) or their immunodominant peptides (10 μg/ml). Culture supernatants were harvested after 72 h of culture. Data represent mean ± standard deviation (error bars) values (pg/ml) of four mice tested individually.
DISCUSSION
Bacterial and host factors that induce and maintain latent M. tuberculosis infection and those that induce the reactivation of TB disease are ill defined. Dormancy can be induced in vitro by nutrient starvation (4), hypoxia (37, 38), or low pH (20), while the maintenance of latent infection in an in vivo murine infection model requires IFN-γ, tumor necrosis factor (TNF-α), and NO (21). CD4+ T cells and TNF-α are essential in this maintenance, as demonstrated by the increased risk for reactivation of TB in human immunodeficiency virus-coinfected and anti-TNF-α-treated individuals. In a mouse model of latent TB, it was furthermore shown that CD8+ T cells are important for controlling persistent M. tuberculosis infection (41). Wayne and Sohaskey demonstrated that the provision of O2 to hypoxic cultures could result in the resuscitation of dormant tubercle bacilli into an active state, even after prolonged periods of latency (45). More recently, it was shown that O2 depletion and low, nontoxic concentrations of nitric oxide modulate the expression of 48 gene products of the DosR (Rv3133c) regulon (44). Although not formally proven, these proteins are thought to be necessary for the survival of M. tuberculosis during latent phases of infection. We have shown that selected DosR regulon-encoded antigens are efficiently recognized by human T cells from M. tuberculosis-exposed individuals. Of particular relevance, we found a preferential recognition of these antigens by individuals with latent infection compared to individuals with tuberculosis disease (12, 30), suggesting that T-cell recognition of these antigens can contribute to control of latent infection.
Very little is known so far about the potential immunogenicity of these DosR regulon-encoded antigens in mice, a useful model species for the study of TB vaccines. In order to examine the immune potential of latency antigens in the mouse, we immunized BALB/c and C57BL/6 mice with DNA plasmids encoding eight proteins of the M. tuberculosis DosR regulon and analyzed antigen-specific Th1-type cytokine secretion and antibody production. Two proteins consistently induced very strong T- and B-cell responses, notably Rv2031c and Rv2626c. Rv2031c or hspX is a prototype latency antigen, reported to be present in the thickened cell wall of M. tuberculosis in the late stationary phase (11). It is not clear how hspX contributes to dormancy, but it was reported that Rv2031c is required for in vitro growth in macrophages (46) and that the deletion of the hspX gene causes increased bacterial growth in vivo (24).
Increased expression of the cystathionine β-synthase domain containing Rv2626c has also been documented in the context of latency, viz., using comparative proteome analysis of in vitro-grown mycobacteria (27, 35) and during in vivo transition from growth to persistence in lungs from immunocompetent mice (39). Interestingly, BCG-vaccinated mice react very weakly against Rv2031c and Rv2626c (or to any other of the eight DosR latency antigens tested for that matter) (Lin et al., submitted), although genes with identical sequences are present in the BCG genome. The upregulation of Rv2031c and Rv2626c in dormant M. bovis BCG requires the same DosR response regulator (Rv3133c) as does M. tuberculosis (5, 6), and it is possible that the weak latency-specific responses induced following BCG are the result of a lack of in vivo triggering of DosR upon the administration of this attenuated vaccine, which does not replicate or persist in the mouse lungs, even after intravenous inoculation. Indeed, we could demonstrate strong B- and T-cell responses directed against Rv2031c and Rv2626c proteins in two models of persistent M. tuberculosis infection, i.e., low-dose intratracheal infection and high-dose intravenous infection followed by combined INH-PZA chemotherapy.
Synthetic, overlapping 20-mer peptides spanning Rv1733c, Rv2029c, Rv2031c, Rv2626c, Rv2627c, and Rv2628 were used to identify immunodominant H-2d- and H-2b-restricted epitopes in plasmid DNA-vaccinated mice. The identification of T-cell epitopes is an important tool for the analysis of immune responses. Synthetic peptides are more easily manufactured than recombinant proteins, and potential problems of endotoxin contamination or residual E. coli contaminants, which may induce nonspecific cytokine production, can be avoided using peptides. Also, problems of antigenic processing, well documented in M. tuberculosis-infected macrophages (31), can be overcome to some extent by using synthetic peptides. More epitopes could be defined in DNA-vaccinated BALB/c mice than in DNA-vaccinated B6 mice, and furthermore, all epitopes recognized in B6 mice were also recognized, and generally more strongly, in BALB/c mice, suggesting a permissive recognition. T-cell epitopes of Rv2031c have previously been mapped by Vordermeier et al. (43) in H-2b and H-2k haplotype mice, using peptide immunizations in incomplete Freund adjuvant. Two peptides defined by Vordermeier, spanning amino acids 31 to 40 and 71 to 91 could also be recognized by T cells from mice that had been presensitized with either killed or live M. tuberculosis. The latter peptide is identical to the one identified in the present study in DNA-vaccinated BALB/c (H-2d) mice, and that was recognized by persistently infected (B6D2)F1 mice at the end of a 2-month chemotherapy.
In total, 11 predicted MHC class II-restricted IFN-γ- and IL-2-inducing Th1 epitopes and one Kd-restricted epitope (of Rv2626c) could be identified in DNA-vaccinated mice. Besides producing IFN-γ, CD8+ T cells from Rv2626c DNA-vaccinated mice demonstrated strong in vivo cytolytic activity against Rv2626c78-87-pulsed, CFSE-labeled target cells. Spleen cells from persistently but not from recently infected (B6D2)F1 mice also recognized Rv2626c and this Kd-restricted epitope. As CD8+ T cells may be particularly relevant in the control of latent M. tuberculosis infection (41), the monitoring of these Rv2626c-specific CD8+ T-cell responses in experimental models of latent and reactivation TB may be an easy tool for determining the protective potential of this DosR regulon-encoded antigen.
In conclusion, we have shown that Rv2031c and Rv2626c are promising, immunogenic latency-associated antigens and that more extensive testing of their vaccine potential in experimental models of latent TB is highly warranted. Also, their combination with antigens preferentially expressed at the onset of M. tuberculosis infection, such as the mycolyl transferase Rv3804c, eventually in prime-boost strategies with BCG, needs further analysis.
ACKNOWLEDGMENTS
This work was partially supported by grants G.0266.00, G.0376.05, and 1.5.026.07 from the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen and by the Damiaanaktie Belgium. V.R. holds a FRIA bursary. L.Z. received a scholarship from the China Scholarship Council. M.Y.L. is supported by the Foundation of Medical Microbiology Leiden, The Netherlands. This project was also funded partly by a grant from the Bill & Melinda Gates Foundation through the Grand Challenges in Global Health Initiative (http://www.grandchallengesgh.org ) (grant no. 37885, “Preclinical and Clinical Evaluation of Post-Exposure TB Vaccine”) and by the European Commission, within the 6th Framework Programme, contract no. LSHP-CT-2003-503367. The study was also partly funded by ISA Pharmaceuticals B.V., Bilthoven, The Netherlands.
The text represents the authors' views and does not necessarily represent a position of the European Commission, who will not be liable for the use made of such information.
FOOTNOTES
- Received 20 July 2006.
- Returned for modification 25 August 2006.
- Accepted 15 November 2006.
- Copyright © 2007 American Society for Microbiology