Immunogenicity of Eight Dormancy Regulon-Encoded Proteins of Mycobacterium tuberculosis in DNA-Vaccinated and Tuberculosis-Infected Mice

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 Rv2626_78-87 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 (CFSE^low) and of peptide-pulsed cells labeled at 10 μM CFSE (CFSE^high). The Rv2626c_78-87 IYYVDANASI (ProImmune Ltd., United Kingdom) peptide (10 μg/ml) was used to pulse the CFSE^high 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 CFSE^high cells/CFSE^low cells in vaccinated mice was compared to the mean ratio of peptide-pulsed CFSE^high /CFSE^low in transferred naïve control mice. Analysis was performed with four naïve and five Rv2626c DNA-vaccinated mice.

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.

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FIG. 1.

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.

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FIG. 2.

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-2^d- and H-2^b-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 Rv2627c_21-40 and Rv2628_11-30, 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 Rv2626c_71-90, 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).

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FIG. 3.

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).

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FIG. 4.

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).

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FIG. 5.

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.

Identification of a K^d-restricted epitope in Rv2626c protein.Sequence analysis according to HLA_BIND revealed that Rv2626c_71-90 contained a predicted K^d 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 Rv2626c_78-87 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).

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FIG. 6.

(a) Intracellular IFN-γ staining of CD8⁺ spleen cells from BALB/c mice vaccinated with Rv2626c DNA and restimulated with K^d-restricted peptide Rv2626c_78-87. 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 K^d-restricted Rv2626c_78-87 peptide, as detected by lysis of peptide-pulsed CFSE^high-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 K^d-restricted Rv2626c_78-87 peptide, as demonstrated by a mean 72% specific lysis of peptide-pulsed, CFSE^high-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.

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FIG. 7.

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.

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 Rv3875_1-20 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), Rv2031c_71-90 (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 Rv3875_1-20 peptide were not different in acutely and persistently infected mice.

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FIG. 8.

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.