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Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India
Correspondence
R. Nayak
nayak{at}mcbl.iisc.ernet.in
Received 8 August 2007
Accepted 13 May 2008
) and low interleukin-4 responses. Three nonameric peptides of Rv3812 and two of Rv3018c elicited a strong T-cell response in an MHC-restricted manner. An epitope-specific response was demonstrated by the lysis of peptide-pulsed antigen-presenting cells, release of perforin and IFN- production. Experimentally, these peptides bound with high affinity to MHC H-2Kd and showed low dissociation rates of peptide–MHC complexes. This study suggests that the identified T-cell epitopes may contribute to immunity against tuberculosis if included in a vaccine.
Abbreviations: BCG, Bacillus Calmette–Guérin; CTL, cytotoxic lymphocyte; DT50, time for 50 % of molecules to decay; ELISPOT, enzyme-linked immunosorbent spot assay; HLA, human leukocyte antigen; IFN-, gamma interferon; IL, interleukin; LDH, lactate dehydrogenase; MHC, major histocompatibility complex; PPD, purified protein derivative; SI, stimulation index; TB, tuberculosis.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Protective immunity to Mycobacterium tuberculosis infection is incompletely understood. It is evident that a coordinated response of the cellular arm of the immune system, involving both CD4+ and CD8+ T lymphocytes, plays an important role in immunity to TB by secreting gamma interferon (IFN-) (Orme et al., 1993; Silva et al., 1996). In addition, CD8+ T cells mediate their effects through cytokine production and direct lysis of infected macrophages, and control of bacterial multiplication through the release of cytotoxic granules such as granulysin and perforin (Cooper & Flynn, 1995; Flynn et al., 1992; Kaufmann, 1995; Stenger et al., 1999; Tascon et al., 1998). Therefore, immunity to TB, and thus the development of a vaccine, relies on the identification, formulation and delivery of protective T-cell antigens in a manner that will generate prolonged memory responses.
The sequencing of the genome of M. tuberculosis revealed two large families of proteins, PE and PPE, which are highly acidic, glycine-rich proteins and constitute 10 % of the coding capacity of the genome (Cole et al., 1998). Microarray analysis has suggested that variable expression of certain PE_PGRS protein-encoding genes occurs under conditions that mimic in vivo pathogenesis, such as nutrient depletion, low pH and oxidative stress (Betts et al., 2002; Fisher et al., 2002; Saviola et al., 2003; Voskuil et al., 2004). Some of the PE_PGRS proteins have been found to be associated with the cell wall (Delogu et al., 2004) and influence interactions with other cells (Brennan et al., 2001). These two multigene families are of potential interest from an immune response point of view, as they could function as a source of antigenic variation for M. tuberculosis in order to evade the host immune response (Cole et al., 1998) and as cell-surface antigens (Banu et al., 2002). However, little is known about the immunogenicity of these two classes of protein and only a few proteins encoded by the rv3873, mtb39 and rv0915c genes have been shown to be T-cell antigens (Dillon et al., 1999; Okkels et al., 2003; Skeiky et al., 2000).
Two of the PE_PGRS protein-encoding genes have been shown to be expressed by pathogenic Mycobacterium marinum in granulomas of infected frogs, and M. marinum mutants containing deletions in these genes replicate poorly in cultured macrophages and show decreased persistence in frog granulomas (Ramakrishnan et al., 2000). One of these genes has a homologue, rv3812, in M. tuberculosis. Using a signature-tagged mutagenesis approach, it has been shown that inactivation of PPE46 (Rv3018c) leads to attenuation of M. tuberculosis in a murine model (Camacho et al., 1999).
Using an immunoinformatics approach (Chaitra et al., 2005), a set of major histocompatibility complex (MHC) class I-binding peptides has been identified from two proteins encoded by the rv3812 and rv3018c genes (Chaitra et al., 2005). This study led to the identification of putative T-cell epitopes from the Rv3812 and Rv3018c proteins. In an earlier study, mice were injected subcutaneously with recombinant proteins encoded by the rv3018c and rv3812 genes of M. tuberculosis strain H37Rv, and for the first time, we demonstrated that the Rv3018c and Rv3812 proteins of the PE/ PPE family are T-cell antigens (Chaitra et al., 2007b).
DNA vaccination is a potent method that can engender both humoral and cellular immune responses in a variety of murine and primate disease models (Gurunathan et al., 2000). In addition, DNA vaccines mimic the effects of live attenuated vaccines in their ability to induce MHC class I-restricted CD8+ T-cell responses (Ulmer et al., 1993).
In the present work, we analysed the immunogenicity of DNA vaccine constructs carrying the genes encoding the Rv3812 and Rv3018c proteins of M. tuberculosis and demonstrated proliferative responses of the splenic lymphocytes to synthetic peptides derived from these two proteins. Peptide-specific T cells from DNA-immunized animals were shown to produce IFN-.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Recombinant plasmids and antigens. The ORFs corresponding to M. tuberculosis genes rv3812 and rv3018c were PCR-amplified from the genomic DNA of strain H37Rv, and cloned into pET14b and pET33b vectors, respectively. His-tagged Rv3812 and Rv3018c proteins were expressed in Escherichia coli BL21 and purified by His-tag affinity chromatography on a Ni2+-nitrilotriacetate column under denaturing conditions as described by Razeghifard (2004). The proteins were analysed by SDS-PAGE and confirmed by Western blotting with detection by anti-His antibody (data not shown). In vivo expression of the proteins was checked by Western blotting with detection by anti-BCG antibody (data not shown).
The protein sequences of Rv3812 and Rv3018c were analysed in silico, employing BIMAS (Parker et al., 1994) and SYFPEITHI (Rammensee et al., 1999) algorithms, for binding of all overlapping nonameric peptides generated from these proteins to class I human leukocyte antigen (HLA) and class I mouse MHC. Based on this prediction, five peptides were chosen from Rv3812 and Rv3018c (Chaitra et al., 2005). These were 490NYIPQQLAL498, 260NLLVTGFDT268 and 61EPQTVSNQL69 from Rv3812, and 71AYVPYVAWL79 and 254AQLLTEFAI262 from Rv3018. The peptides were synthesized by Peptron and dissolved in 500 µl DMSO, diluted in 0.9 % NaCl to a concentration of 10 mM, and stored at –70 °C.
Stable expression of Rv3812 and Rv3018c in mammalian cells.
The full-length rv3812 and rv3018c genes were cloned into mammalian expression vector pFLAG-CMV4 (Sigma-Aldrich) using procedures described previously (Chaitra et al., 2007a). Stable P815 cell lines expressing Rv3812 or Rv3018c were generated, and expression of the recombinant protein was confirmed using procedures described previously (Chaitra et al., 2007a). Plasmid DNA was amplified in E. coli DH5
, purified using a plasmid purification kit (Qiagen) and redissolved in PBS.
Immunization. BALB/c mice were obtained from and maintained in the Central Animal Facility, Indian Institute of Science. All experiments were performed according to institutional ethical committee guidelines. Six mice (8–12 weeks old) were immunized intramuscularly with 100 µg recombinant plasmid DNA in 100 µl, together with three mice immunized with control plasmid DNA (pFLAG alone), and boosted with the same amount of DNA on days 21 and 36. Spleens were isolated from groups of six mice 2 weeks after the last injection.
In vitro proliferation assay and cytokine assay.
Splenocytes (5x105) from plasmid DNA-immunized mice were cultured in RPMI 1640 in 96-well plates in the presence of 20 µg purified protein derivative (PPD) ml–1 (Spam Diagnostics), 20 µg M. tuberculosis cell lysate ml–1 or 20 µg purified recombinant Rv3812 or Rv3018 ml–1, or with irradiated P815 cells, pre-pulsed with the synthetic peptides (10 µg ml–1 for 2 h), at 37 °C. For blocking experiments, the splenocytes were mixed with anti-CD8 mAb (10 µg ml–1; eBioscience) or anti-MHC I mAb (10 µg ml–1; eBioscience). Plates were incubated for 96 h at 37 °C. [3H]Thymidine (1 µCi per well; Perkin-Elmer) was added for the last 16 h of incubation and incorporation was measured with a scintillation spectrometer. Supernatants from parallel cultures were harvested after 72 h and assayed for the presence of IFN- and interleukin (IL)-4 using antibody pairs anti-mouse IFN- (clone XMG1.2)/biotinylated anti-mouse IFN- (clone R4-6A2) and anti-mouse IL-4 (clone 11B11)/biotinylated anti-mouse IL-4 (clone BVD6-24G2) (all from eBioscience), respectively, according to the manufacturer's instructions. Standard curves were generated with mouse recombinant IFN- and IL-4. The detection limits of the IFN- and IL-4 assays were 20 and 10 pg ml–1, respectively.
In vitro expansion of antigen-specific T cells. Antigen-specific T cells were expanded in vitro as described previously (Chaitra et al., 2007a). The antigen-specific T cells were further used for enzyme-linked immunosorbent spot assay (ELISPOT) and cytotoxic lymphocyte (CTL) assays.
Intracellular cytokine assay.
After the expansion of antigen-specific T cells from the immunized mice for 6 days, the cells were restimulated with the peptides (10 µg ml–1). The expanded T cells were cultured in vitro with gamma-irradiated (50 Gy) P815 cells (1x104) pulsed with peptides, in a total volume of 200 µl RPMI 1640 complete medium for 6 h. Brefeldin A (10 µg ml–1; Sigma) was added to the culture and cells were incubated at 37 °C in 5 % CO2 for 6 h. At the end of 6 h, cells were harvested by washing three times with PBS and stained for surface CD8/CD4 and intracellular IFN- expression using fluorochrome-conjugated mAbs. Cells were stained for CD8/CD4 with FITC-conjugated rat anti-mouse CD8/CD4 mAbs (clones 53-6.7 and RM4-5; eBioscience) by adding 0.5 µg antibody to 1x106 cells followed by incubation at room temperature for 30 min. Cells were washed, fixed with 4 % paraformaldehyde and permeabilized with 0.1 % saponin. Cells were incubated with phycoerythrin-conjugated anti-IFN- mAb (clone XMG1.2; eBioscience) for 30 min at 4 °C, washed and analysed on a cytofluorometer (FACSCalibur; BD). FITC-conjugated rat IgG2a (eBioscience) and phycoerythrin-conjugated rat IgG1 were used as isotype controls for CD8 and IFN- antibodies, respectively. Analysis was carried out using CellQuest software. The predicted frequency of CD8+/CD4+ IFN-+ cells was determined by subtracting the percentage of unsensitized CD8+/CD4+ IFN-+ cells from the percentage of antigen-sensitized CD8+/CD4+ IFN-+ cells.
ELISPOT assay. An ELISPOT assay was carried out as described by Daftarian et al. (2006). P815 cells, used as antigen-presenting cells, were pre-pulsed with 1 pg peptide ml–1 for 2 h at 37 °C and then added to antigen-specific T cells.
MHC I–peptide binding and stabilization assays. Binding assays were performed as described elsewhere (Zhou et al., 1992) using transporter associated with antigen processing (TAP)-deficient RMAS-Kd cells expressing H2-Kd. In the RMAS stabilization assay (Zhou et al., 1992), cells were cultured at 25 °C for 16 h and then incubated at 25 °C for 1 h with peptide at a concentration of 100 µM, followed by incubation at 37 °C for 2 h. The cells were washed to remove unbound peptide and the incubation continued at 37 °C for 0, 1, 2, 4, 6 and 12 h, followed by washes at 4 °C in RPMI 1640. For the zero time-point determination, cells were washed immediately at 4 °C after the 37 °C incubation. Each sample was stained for H-2Kd on the cell surface, as described above. The stability of peptide–MHC complexes was expressed as the time (h) required for 50 % of the molecules to decay (DT50).
CTL assay. A CTL assay was performed using a non-radioactive method based on release of lactate dehydrogenase (LDH) from target cells. Briefly, antigen-specific splenocytes were collected from an in vitro-expanded culture and washed once with RPMI 1640. Viable cells were purified using a Ficoll density gradient and resuspended in RPMI 1640. P815 target cells were incubated with the peptides (10 µg ml–1) for 2 h at 37 °C. After washing, 5x103 cells in 100 µl were added to 100 µl of various numbers of effector cells that had been plated in 96-well plates to obtain target : effector cell ratios of 1 : 20, 1 : 40 and 1 : 100. The medium or the target cells alone were used as the low-level control (spontaneous LDH release). For the high-level control (maximum LDH release), 2 % Triton X-100 was added to the target cells. The cells were incubated for 12 h and assayed for LDH release using a cytotoxicity detection kit (LDH) (Roche Applied Science). The percentage of cell-mediated cytotoxicity was determined by the following equation:
cytotoxicity (%)={[(effector target cell mix–effector cell control)–low-level control]/(high-level control–low-level control)}x100
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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secretion from T cells and provided protection against challenge with M. tuberculosis (Olsen et al., 2000). Interestingly, the level of protection afforded by the peptide vaccine was equivalent to the level of protection observed after immunization with the complete ESAT-6 antigen (Olsen et al., 2000). These results suggest that some of the IFN--inducing peptides could replace complete antigens in subunit vaccines. To identify the T-cell epitopes of Rv3812 and Rv3018c that induce strong IFN- responses, we used a DNA immunization strategy with in vitro assays for detecting peptide-specific T-cell responses.
Peptide–MHC class I (H2-Kd) binding and stabilization by Rv3812 and Rv3018c peptide–MHC complexes
Based on our previous in silico analysis, we identified a set of peptides exhibiting good HLA or MHC binding ability that were also shown to form stable structures after replacement of the resident peptide in the crystal structure of the HLA molecule (Chaitra et al., 2005). In order to validate the in silico MHC I-binding prediction, peptides from Rv3812 and Rv3018c with known mouse MHC I-binding motifs were studied for their ability to upregulate expression of MHC class I molecules on RMAS-Kd cells. RMAS-Kd cells are TAP-deficient cells, in which low levels of unstable MHC I are expressed on the cell surface at 37 °C. However, incubation of cells at low temperature enhances transport, stability and cell-surface expression of these MHC molecules (Ljunggren et al., 1990). Culturing TAP-deficient cells in the presence of MHC class I-binding synthetic peptides increases class I surface expression. This upregulation was measured using an anti-MHC class I mAb, which is structure dependent. Peptide binding was dose dependent with optimum binding at a peptide concentration of 100 µM (data not shown). It has been demonstrated that the capacity of a peptide to bind and stabilize MHC I molecules is directly correlated with its ability to induce specific CTL responses (Zhou et al., 1992). Therefore the stability of peptide–MHC complexes was measured by following MHC class I surface expression on peptide-pulsed RMAS-Kd cells over a 0–12 h period at 37 °C. Stability was expressed as DT50 (Fig. 1
). The binding and stabilization of the peptides was tested in comparison with a positive control peptide, LLFGYPVYV (Ogata et al., 2003). The Rv3812 peptides aa 490–498 and aa 260–268 could stabilize the MHC complex on the cell surface for more than 6 h, whereas aa 61–69 could stabilize the complex only for about 4 h (Fig. 1). Rv3108c peptides aa 254–262 and aa 71–79 could stabilize the H-2Kd complex for more than 6 h. This indicated that the peptides are able to stay on the cell surface for a considerable time, increasing the chances of circulating T cells recognizing the MHC-bound peptide.
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) (P<0.005). There was very low stimulation of splenocytes from mice immunized with control DNA (pFLAG vector alone) (Fig. 2a, b). Similarly, peptides derived from both proteins were able to stimulate the in vivo DNA-primed splenocytes (Fig. 2c, d). The results showed that higher responses in proliferation assays were obtained with aa 490–498 (SI=12; P<0.003) and aa 260–268 (SI >9; P<0.006) of Rv3812, and aa 71–79 (SI >9; P<0.001) of Rv3018c, whereas the remaining peptides elicited a lower response (Fig. 2c, d). We used stable cell lines expressing the full-length Rv3812 or Rv3018c protein for restimulation. The results were not qualitatively different from that of purified protein. Hence, in further experiments, purified proteins were used for restimulation.
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and IL-4, respectively, by the responding lymphocyte population. The mean levels of IFN- induced in response to in vitro stimulation with Rv3812 were much higher than those induced by stimulation with Rv3018c (P<0.004) (Fig. 3).
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). Addition of anti-CD8 antibody to in vitro-restimulated cultures resulted in a significant reduction in the proliferation of responding splenocytes (P<0.02 and P<0.022 for Rv3812 and Rv3018c peptides, respectively) (Fig. 2b).
The peptides that showed strong proliferation responses also showed strong IFN- responses (Fig. 3). In both proliferation and IFN- assays, peptides were recognized, demonstrating that the corresponding epitopes were generated in vitro as a result of natural processing of Rv3812 and Rv3018c in vivo followed by presentation. IL-4 secreted by all of the stimulated cells was either below the detection level or was zero (data not shown). Selected peptides from Rv3812 and Rv3018c stimulated the primed T lymphocytes to express higher IFN- levels to create a Th1-favoured response. Preferential induction of Th1-type cytokines is interesting as this may lead to the activation of macrophages to kill intracellular bacteria, perhaps via the production of nitric oxide (Raupach & Kaufmann 2001), and may also promote the activation and proliferation of specific CD8+ CTLs (O'Donnell et al., 1994; Young et al., 2002).
Antigen-specific T-cell lines show proliferation in vitro following stimulation by peptides of Rv3812 and Rv3018c proteins
To confirm that Th1 cells are the major cells responding to Rv3812 and Rv3018c, peptide-specific T-cell lines were established from splenocytes from DNA-immunized mice after primary stimulation of splenocytes with recombinant proteins. All of the T-cell lines responded to Rv3812 and Rv3018c in proliferation assays (Table 1). When tested with peptides, the results confirmed the dominant recognition of peptides by T cells. The frequency of IFN--secreting splenocytes was enumerated by ELISPOT analysis of T lymphocytes from spleens 24 h after stimulation with Rv3812 or Rv3018c protein or with synthetic peptides. Splenocytes from naive mice did not respond to stimulation with proteins and were at background levels, whereas splenocytes from DNA-immunized mice showed a marked increase in the number of cells producing IFN- following in vitro stimulation with the protein or peptides. Rv3812 and Rv3018c proteins induced 474 (±75) and 381 (±41) spot-forming cells per 106 antigen-specific splenocytes, respectively (Table 1). Strong IFN- responses in PPD-positive healthy subjects and inhibition of growth of mycobacteria in macrophages stimulated with IFN- suggest that IFN- plays an important role in mediating protective immunity in humans (Kampmann et al., 2000; Kawakami et al., 1994). The fact that there were peptide-specific Th1-cell responses, as judged by IFN- secretion, suggests that the corresponding peptides may be relevant to protective immune responses in humans. Consistent with these data, IFN- mRNA was induced at 6 h after stimulation with either of the proteins (data not shown). Tumour necrosis factor alpha and inducible nitric oxide synthase mRNA levels were also higher at about 24 h after stimulation with the proteins, indicating that these proteins activate the bactericidal activity of macrophages (Chan et al., 1995). The level of IL-10 mRNA was either low or unchanged up to 24 h of stimulation (data not shown).
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. Rv3812 induced production of IFN- from CD4+ T cells (19.2±2.1 %), as well as CD8+ T cells (16.5±3.7 %). CD4+ T cells contributed to IFN- production in all mice tested whose splenocytes secreted significant levels of IFN-, with a similar number also displaying measurable frequencies of CD8+ T cells (Table 1).
Cytotoxic activity of antigen-specific T-cell lines
T-cell lines from mice immunized with rv3812 or rv3018c DNA that responded to the proteins in IFN- assays were tested for cytotoxic activity against P815 cells endogenously expressing Rv3812 or Rv3018c protein or P815 cells pulsed with synthetic peptides. The results revealed that all T-cell lines were cytotoxic for P815 cells pulsed with Rv3812 or Rv3018c protein. When individual peptides were examined, cytotoxic activity seemed to be associated with IFN- secretion. Rv3812 peptides (aa 260–268 and aa 490–498) induced comparatively higher IFN- secretion and cytotoxic activity in T-cell lines than aa 61–69 (Fig. 4a). Rv3018c aa 254–262 showed higher cytotoxic activity compared with aa 71–79 (Fig. 4b).
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Together with different cytokines, leukocyte attractants are released in inflammatory foci and play a major role in directing the host response against invading pathogens, including mycobacteria (Riedel & Kaufmann, 1997). It is known that Th1-associated cytokines (such as IFN- and tumour necrosis factor alpha) promote tissue responses induced by mycobacterial antigens present in PPD (Qiu et al., 2001). Our findings indicate that the chemokines MIG (monokine induced by IFN-) and MCP-1 (monocyte chemotactic factor) are efficiently and persistently produced in response to mycobacterial antigen stimulation (data not shown), and are chemotactic for activated T lymphocytes in vitro and involved in the recruitment of the predominant innate immunity effector cells found in M. tuberculosis infections, namely monocytes and granulocytes.
These immunomodulatory activities of Rv3812 and Rv3018c on splenocytes, inducing a Th1-type immune response, may be helpful in the development of a therapeutic vaccine for TB, especially for TB caused by multidrug-resistant M. tuberculosis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-interferon in an immuno-compromised mouse model. Kekkaku 69, 607–613.[Medline]
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, Rv3812 aa 260–268; 
, Rv3812 aa 490–498; 
, Rv3018c aa 71–79; 
, Rv3812 aa 61–69; 
, Rv3018c aa 254–262; 
, HTLV-1 Tax-positive positive-control peptide. (b) The ability of the peptides to stabilize the empty MHC class I molecules on the cell surface of TAP-deficient cells is shown. Stability is expressed as DT50 (h). HTLV 1, human T-lymphotropic virus type 1.
