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The Journal of Immunology, 2005, 175: 5298-5305.
Copyright © 2005 by The American Association of Immunologists

Cloning of the Gene Encoding a Protective Mycobacterium tuberculosis Secreted Protein Detected In Vivo during the Initial Phases of the Infectious Process1

Sandeep Mukherjee2,*, Suely S. Kashino2,{dagger}, Yanni Zhang{ddagger}, Nada Daifalla*, Virmondes Rodrigues, Jr§, Steven G. Reed*,¶ and Antonio Campos-Neto3,{dagger}

* Infectious Disease Research Institute, Seattle, WA 98104; {dagger} The Forsyth Institute, Boston, MA 02115; {ddagger} Department of Process Science, Corixa Corporation, Seattle, WA 98101; § Medical School of Triangulo Mineiro, Uberaba, Minas Gerais, Brazil; and Department of Pathobiology, University of Washington, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The existence of therapeutic agents and the bacille Calmette-Guérin (BCG) vaccine have not significantly affected the current tuberculosis pandemic. BCG vaccine protects against serious pediatric forms of tuberculosis but not against adult pulmonary tuberculosis, the most common and contagious form of the disease. Several vaccine candidates, including Mycobacterium tuberculosis recombinant proteins formulated in newer adjuvants or delivered in bacterial plasmid DNA have recently been described. An attractive source of vaccine candidates has been M. tuberculosis Ags present in culture supernatants of the initial phases of the bacterial growth in vitro. In this study we describe an Ag discovery approach to select for such Ags produced in vivo during the initial phases of the infection. We combined RP-HPLC and mass spectrometry to identify secreted or shed M. tuberculosis proteins eliminated in animal urine within 14 days after the infection. A peptide containing sequence homology with a hypothetical M. tuberculosis protein was identified and the recombinant protein produced in Escherichia coli. The protein was recognized by Ab (IgG2a and IgG1) and T cells (Th1) of mice infected with M. tuberculosis and by lymphoid cells from healthy donors who had a positive purified protein derivative skin test but not from tuberculosis patients. Moreover, this Ag induced protection in mice against M. tuberculosis at levels comparable to protection induced by BCG vaccine. These results validate the Ag discovery approach of M. tuberculosis proteins secreted or shed in vivo during the early phases of the infection and open new possibilities for the development of potential vaccine candidates or of markers of active mycobacterial multiplication and therefore active disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tuberculosis remains a major infectious cause of morbidity and mortality worldwide (1). The incidence of the disease remains high and is increasing in many parts of the world due in part to its association with HIV infection (2). Despite the existence of specific antimicrobial agents and the widespread application of the bacille Calmette-Guérin (BCG)4 vaccine, it is estimated that one-third of the world population is infected with Mycobacterium tuberculosis (3), with 8 million newly diagnosed cases of tuberculosis and up to 2.5 million deaths occurring each year (4). Treatment of tuberculosis is complex due to the necessity of several medications over extended periods of time and the existence of resistant strains. BCG vaccine is the only available vaccine, which has been in use since the early 1920s. However, BCG only protects children from disseminated tuberculosis, but does not prevent pulmonary disease (5, 6), the most common and contagious form of tuberculosis. Moreover, the poor efficacy of BCG vaccine has varied considerably in clinical trials in geographically distinct populations (5, 7). Finally, because BCG is a viable vaccine it can cause disseminated disease in immune-compromised individuals (8, 9).

Considering the scale of the disease burden it is obvious that the development of a more effective vaccination strategy is urgently required and major efforts have been dedicated to subunit vaccines. Much effort has been spent on the Ags from culture filtrate (CF) proteins derived from in vitro cultivated M. tuberculosis. This has been an attractive source of Ags, primarily because CF protein has been shown to induce protection when used as vaccines in various animal models of tuberculosis (10, 11, 12, 13, 14). In addition, the ability of CF proteins to stimulate the proliferation and cytokine production from T cells of infected mice, guinea pigs, and PBMC from purified protein derivative (PPD)-positive human donors (15, 16, 17) has led to the conclusion that the characterization of CF proteins is an important asset for the development of recombinant subunit vaccine against tuberculosis.

Using either biochemical fractionation or screening of M. tuberculosis expression libraries to select for vaccine candidates present in CF, interesting Ags have been discovered over the past years (15, 16, 17, 18, 19, 20, 21, 22, 23). However, CF is a complex mixture composed of more than 200 proteins (24) produced in vitro, which may not necessarily represent the pattern of proteins secreted by M. tuberculosis during its growth in vivo under the selective pressure of the host resistance defense mechanisms. Therefore, the discovery of CF proteins produced in vivo, particularly during the initial phases of the infectious process, can be an interesting strategy to identify vaccine candidates.

To test this hypothesis, we used RP-HPLC and mass spectrometry (MS) to identify M. tuberculosis proteins shed by the bacteria and eliminated in animal urine during the early phase of the infectious process (10–14 days after challenge). A peptide containing sequence homology with a hypothetical M. tuberculosis protein was identified and the recombinant protein was produced in Escherichia coli. Lymphoid cells from both PPD-positive individuals and mice infected with M. tuberculosis recognized this protein. In addition, immunization of mice with the protein in combination with MPL-SE (the adjuvant containing monophosphoryl lipid A and QS-21) induced protection against a challenge with virulent M. tuberculosis similar to protection induced by BCG.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

C57BL/6 mice were obtained from Charles River Breeding Laboratories. The mice were maintained under pathogen-free conditions and used at 8 wk of age.

Bacteria, mice infections, and electrophoresis analysis

Virulent M. tuberculosis H37Rv strain (35718; American Type Culture Collection) suspended in PBS Tween 80 (0.05%) was delivered i.v. at 5–10 x 106 CFU/mouse. After infection, urine was collected daily from day 10 to 14 from infected mice and for four consecutive days from a group of noninfected mice. Urine samples were centrifuged immediately after collection, sterile filtered in 0.2-µm pore size filters, and stored in a –80°C freezer until use. For comparative analyses, 15 µl of urine of noninfected as well as from infected mice were submitted to SDS-PAGE (4–12% gradient gel) followed by silver staining. For protection experiments mice were challenged via the respiratory route using ~200 aerosolized CFU of M. tuberculosis H37Rv.

Mass spectrometry

Peptide mixtures were initially concentrated using a Capillary LC column filled with C18 resin (100-µm internal diameter, 12 cm long). Peptides were eluted using a gradient of 2% per minute increase from 5 to 65% buffer B (80% acetronitrile/0.2% acetic acid in water). The Capillary LC column was online connected with LCQ Ion Trap MS. Eluted peptides from Capillary LC column were introduced into the LCQ Ion Trap MS (Thermo Finnigan) by electrospray ionization interface (Cytopeia) and analyzed by data-dependent MS and MS/MS scan. The collision-induced dissociation spectra (tandem mass spectra, MSMS) generated during the experiment were searched against mouse and M. tuberculosis protein databases using Sequest software to identify possible sequence matches (25, 26).

M. tuberculosis Ags

CF antigenic preparation was obtained from 2-wk-old cultures of M. tuberculosis H37Rv strain grown in defined medium as described (24). CF protein was centrifuged at 2000 x g for 20 min, and supernatant was sterilized by passing through 0.2-µm filters. CF protein was concentrated with an Amicon 3 Centriprep concentrator to 1/100 of original volume, and protein content was determined with a bicinchoninic acid protein assay (Pierce). The recombinant Ag used in these studies, named U2, was produced as described (27). This protein has a deduced molecular mass (6.4 kDa) and could only be expressed as a homotrifusion, i.e., fusion of three copies of the same gene in tandem. Briefly, three sets of oligonucleotide PCR primers containing different restriction enzymes (NdeI and HindIII, HindIII and BamHI, and BamHI and EcoRV; New England Biolabs) were used separately to amplify the full-length open reading frame of U2 from genomic DNA of the virulent Erdman strain of M. tuberculosis. The resultant PCR product was double-digested with the respective enzymes, ligated (T4 DNA ligase; Stratagene), and cloned into the pET-17b vector using E. coli XL1-Blue competent cells (Stratagene). The expression vector was subsequently used to transform E. coli BL-21 (DE3) pLysS for optimal expression (Novagen; EMD Biosciences). The gene encoding the recombinant trifusion protein was induced in Luria-Bertani medium with 0.25 mM IPTG (isopropyl {beta}-D-thiogalactoside) for 3 h at 37°C, and the recombinant protein was purified from the inclusion bodies using the one-step QIAexpress Ni-NTA agarose matrix (Qiagen) in the presence of 8 M urea, as previously described (27). Yields of 8–10 mg of protein per liter of E. coli culture were achieved. Ag85 complex was provided by Dr. J. Belisle and Dr. K. M. Dobos (National Institutes of Health contract nos. HHSN266200400091C/ADB and NO1-AI-40091, Tuberculosis Vaccine Testing and Research Material contract). PPD was prepared as described (28).

Generation of rabbit anti-U2 antiserum

The purified recombinant protein (100 µg) was mixed with 100 mg of muramyl dipeptide, brought up to 1 ml with PBS, and emulsified with 1 ml of IFA. The emulsion was injected at multiple s.c. sites into a female New Zealand rabbit (R&R Rabbitry). The rabbit was given two s.c. boosters (100 µg of Ag in IFA) 3 wk apart. One week after the final boost the rabbit was bled and serum collected.

Western blot

Purified recombinant U2 protein (40 ng), CF protein (1 µg), as well whole bacteria lysate were fractionated by electrophoresis on 12% SDS-PAGE gel and transferred to nitrocellulose membrane (BioRad). The blot was blocked overnight at 4°C with PBS-0.1% Tween 20 containing 5% nonfat dry milk and subsequently incubated with polyclonal rabbit anti-U2 antiserum (1/1000) for 1 h. Before using in the immunoblot, the rabbit antiserum was preadsorbed with E. coli whole cell extract for 1 h at 37°C. Following several rinses in TBS-0.1% Tween 20, Staphyloccocus aureus protein A labeled with HRP at 1/20,000 dilution (Pierce) was added for 30 min. After additional washings, bound conjugates were detected using ECL system (ECL; Amersham Biosciences) and proteins were visualized by exposing the blot to autoradiography film (BioMax; Kodak).

IgG isotype ELISA

Mice were bled before and 8 wk after infection and sera were stored at –20°C until use. The specific serum IgG isotype Ab response was measured by conventional ELISA. Wells of ELISA plates (Costar) were coated with U2 at a concentration of 100 ng/well. Sera were added at 2-fold serial dilutions followed by washes and addition of biotinylated isotype-specific secondary Abs (rabbit anti-mouse IgG1 or IgG2a; BD Pharmingen). Wells were then washed and incubated with streptavidin-conjugated HRP (SAV-HRP; Zymed Laboratories) after which substrate and chromogen were added and absorbance was read on an ELISA plate reader (Dynatech Laboratories) at 490 nm.

Cytokine and proliferation assays

Spleen cells from mice immunized with the recombinant protein or infected with M. tuberculosis were obtained by conventional procedures, and then centrifuged over Ficoll-Hypaque to remove red cells, granulocytes, and dead cells. Mononuclear cells (2 x 105 cells per well) were cultured for 72 h at 37°C and 5% CO2 in the presence of medium, U2, Ag85, or CF. Supernatants were harvested and analyzed for IFN-{gamma} and IL-4 by a double sandwich ELISA using specific mAb (BD Pharmingen). For human assays, PBMC were isolated by gradient centrifugation and used in proliferation assays (20). PBMC was obtained from 22 healthy PPD-negative individuals, from 10 healthy PPD-positive individuals, and from 11 patients with pulmonary tuberculosis registered at the University Hospital, Medical School of Triângulo Mineiro, Uberaba, Minas Gerais, Brazil. None of the healthy PPD-positive volunteers had histories of active tuberculosis and had recent negative chest radiographs. Therefore, these donors were assumed to have latent infection. PBMC (2 x 105 well) were incubated in 96-well round-bottom plates (Costar) in medium only (RPMI 1640 with 10% pooled human AB serum and gentamicin (50 µg/ml), or in medium containing U2 Ag at various concentrations. Plates were cultured for 5 days at 37°C in 5% CO2 and were pulsed with 1 µCi of [3H]thymidine (Amersham) for an additional 18 h. Cells were harvested onto filter mats and counted using a Matrix 9600 scintillation counter (Packard Instrument).

Statistical analysis

Data are presented as mean and SD with the exception of the protection data, which are presented as mean and SEM. Statistical significance was determined by the Student t test for comparison of two groups. Multigroup comparisons were performed using one-way ANOVA multiple comparisons among mean values, with Tukey’s HSD test at overall {alpha} = 0.05 for all pairwise comparisons. Values of p < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Ag discovery

The discovery and characterization of M. tuberculosis Ags secreted or shed in vivo during the initial phase of infection (as early as 10–14 days postinfection) was conducted by direct search of such proteins excreted in the urine of infected mice. The hypothesis for this strategy was based on the fact that during the multiplication phase of the mycobacteria, secreted or shed proteins produced during the infectious process could reach the animals’ circulation and subsequently be eliminated in the urine. The search for these Ags was done by comparative SDS-PAGE of urine obtained from infected and control animals. Fig. 1 illustrates the general protocol of Ag discovery and Fig. 2 shows the actual migration pattern obtained from urine of C57BL/6 mice infected with M. tuberculosis compared with urine of noninfected syngeneic control mice. Several unique bands were present in the urine of infected mice, which were excised from the gels and in-gel tryptic digested and used for protein identification by micro LC ESI-MS. The search for M. tuberculosis and mouse protein databases using the Sequest software revealed that several bands generated sequences with a high degree of homology with mouse proteins. However, a band of ~6.5 kDa showed high homology with a hypothetical M. tuberculosis protein present in the genome database released by The Institute for Genomic Research (<http://www.tigr.org>), primary locus Rv0909 (GenBank Accession no. CAB08507). This protein was symbolically named urine protein 2 or simply U2.



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  		FIGURE 1. Schematic representation of Ag discovery strategy of M. tuberculosis proteins secreted or shed in vivo during the initial phase of the infectious process.

 


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  		FIGURE 2. PAGE of mouse urine. Approximately 15 µl of urine from either normal C57BL/6 mice or M. tuberculosis-infected C57BL/6 mice were submitted to gel electrophoresis (4–20% gradient gel) followed by silver stain. Numbers on the left indicate the molecular mass of the markers (MWM) in kilodaltons. Arrows and numbers on the right point to protein bands unique to the urine of infected mice.

 
Cloning of the gene, protein expression, and characterization of U2

The open reading frame of the full-length gene was amplified by PCR with 5'- and 3'-specific oligonucleotides and cloned into the pET17-b expression vector. After initial attempts to express the 6.5-kDa protein in E. coli failed, we obtained excellent expression by creating a construct of three copies of the gene fused to each other in tandem (27). Expression of the recombinant trifusion was consistently achieved at yields ranging from 8 to 10 mg/L of E. coli culture. Coomassie blue staining of gels (Fig. 3) performed under reduction conditions revealed the presence of distinct induced protein migrating at the 25-kDa molecular mass position, which is slightly higher than expected of the trimer U2 molecule (~22 kDa, after addition of the His-tag sequence and the spacer amino acids to the monomer molecule). Western blot analyses with anti His-tag Ab confirmed that the overexpressed protein was correct (data not shown). To validate U2 as a genuine M. tuberculosis protein (in contrast to hypothetical), Western blot analyses were conducted using a polyclonal rabbit anti-U2 antiserum and crude antigenic preparations of M. tuberculosis Ag (whole bacterial cell extract and CF preparation). Fig. 4 indicates that the antiserum recognizes a band of 6.5 kDa in both the mycobacterial lysate and CF preparations (Fig. 4A, lanes 1 and 2) as well as, as expected, the homofusion recombinant protein (Fig. 4A, lane 3). In addition, the anti-U2 antiserum recognizes a band of ~35 kDa in both mycobacterial antigenic preparations. The 6.5-kDa band matches the predicted molecular mass of U2 thus suggesting that this molecule is a M. tuberculosis protein that is actively produced and secreted or shed during the bacterial growth. However, the nature of the strong 35-kDa band present in both the bacterial lysate and CF preparations is not known. It is possible that the U2 aggregates with itself, or is part of a larger protein complex or a building block of a larger protein originated by post-translational modification. Although this has not been investigated in detail, we have found that this band is resistant to treatment with 8 M urea and is soluble in strong organic solvents like chloroform thus suggesting that U2 might associate with bacterial lipid moieties (data not shown). Alternatively, this band could be a contamination from the E. coli host cells. However this alternative is less likely to be the case because the rabbit anti-U2 antiserum was extensively adsorbed with E. coli, and no bands were seen in control gels using nontransformed E. coli extract as Ag (data not shown).



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  		FIGURE 3. Expression and purification of U2. Recombinant U2 homotrimer containing six His-tag in the N terminus of the molecule was expressed in E. coli BL21 (DE3) pLysS transformed with pET-17b containing three U2 gene copies in tandem. After induction with isopropyl {beta}-D-thiogalactoside (IPTG) the E. coli cells were lysed followed by purification of U2 by affinity chromatography using Ni-NTA agarose matrix. Purity of the recombinant protein was analyzed by Coomassie blue stained SDS-PAGE (15%). E. coli lysate from noninduced culture (lane 1), IPTG-induced cultures (lanes 2–4) at 1, 2, and 3 h, respectively, and purified U2 trimer (lane 5) are shown. The numbers on the left indicate molecular mass of the markers (MWM) in kilodaltons. Arrow points to the recombinant molecule.

 


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  		FIGURE 4. Identification of native U2 in whole M. tuberculosis cells and in the CF proteins. A, The identification of U2 as a genuine M. tuberculosis protein was performed by Western blot analyses. Crude whole bacterial cell lysate (lane 1), CF of M. tuberculosis (lane 2), and purified recombinant Ag (lane 3) were electrophoresed under reducing conditions in a 4–20% gradient gel and transferred to nitrocellulose membrane followed by probing with a rabbit anti-U2 antiserum. Reactivity was detected with peroxidase-labeled goat anti-rabbit Ig and developed using ECL reagent. Numbers on the left are the molecular mass of the markers (MWM). Arrow points to a 6.5-kDa band, which was calculated (B) using the molecular mass (standard MW curve) obtained for the 4–20% gel.

 
Immunological recognition of U2

To evaluate the potential use of U2 as a possible vaccine candidate, experiments were initially performed to verify the recognition of this molecule by serum Ab and lymphoid cells of M. tuberculosis-infected mice as well as by PBMC from various PPD-positive and PPD-negative skin test individuals including healthy subjects and patients with pulmonary tuberculosis. C57BL/6 mice were bled before and 8 wk after infection to obtain serum samples. Anti-U2 Ab responses of the IgG1 and IgG2a isotypes were measured by ELISA. Subsequent to the second bleeding the mice were sacrificed and spleen lymphocytes were obtained and tested for the production of IFN-{gamma} and IL-4 cytokine after stimulation with U2. Fig. 5A shows that the sera from infected mice contain high titers of specific anti-U2 Abs of both IgG1 and IgG2a isotypes. No anti-U2 Ab could be detected in the sera obtained from mice previous to infection (data not shown). Because production of IgG2a is dependent of Th1 responses, these results suggest that during infection U2 stimulates this phenotype of T cells. Moreover, Fig. 5B shows that U2, similarly to CF Ags and Ag85 complex (mycobacterial Ags known to induce potent T cell response and protection against challenge with M. tuberculosis), stimulates in a dose response manner spleen cells of infected mice to produce IFN-{gamma}. No IL-4 production could be detected in any culture supernatants (data not shown). Therefore, these results further suggest that infection of mice with M. tuberculosis generates a predominant Th1 response to U2.



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  		FIGURE 5. Immunological recognition of U2 by mice infected with M. tuberculosis. A, Anti-U2 Ab responses of the IgG1 and IgG2a isotypes were measured by ELISA in sera obtained from C57BL/6 mice bled before and 8 wk after infection. No anti-U2 Ab was detected in the sera obtained from the mice before infection (data not shown). B, Immediately after the second bleeding, mice were sacrificed and their spleen cells were obtained and tested for cytokine production by sandwich ELISA in the culture supernatants harvested 72 h after the initiation of the cultures. IFN-{gamma} was detected, in a dose dependent manner, upon stimulation with U2, CF Ags, and Ag85 complex. The concentration IFN-{gamma} in control stimulated cultures (0 µg of Ag) was below the sensitivity of the ELISA. Pairwise comparisons of IFN-{gamma} levels for CF, U2, and Ag85, as done by Student’s t test (adjusted for variance inequalities), showed no significant differences between the means obtained for U2, CF, and Ag85b at 2 µg/ml (p > 0.08). However at 10 µg/ml, the mean value of IFN-{gamma} production after stimulation with either U2 or CFP was significantly higher than that obtained after stimulation with Ag85b (p < 0.005). No IL-4 could be detected in any supernatant (data not shown). Error bars are the SD of the results obtained from four infected mice.

 
The recognition of the recombinant molecule by human PBMC is depicted in Fig. 6. PBMC from 9 of 10 healthy, PPD-positive skin test individuals (14–18 mm of induration reactions) with no previous history of vaccination with BCG were stimulated by U2 and PPD to proliferate and to produce IFN-{gamma}. In contrast, PBMC from 11 patients with pulmonary tuberculosis with strong skin test PPD-positive reactions (>18 mm of induration), although proliferated and produced IFN-{gamma} upon in vitro stimulation with PPD, did not respond to stimulation with U2. Moreover, neither proliferation nor IFN-{gamma} production was observed with PBMC from 22 healthy PPD-negative individuals stimulated with either PPD or U2.



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  		FIGURE 6. Recognition of U2 by human PBMC. Proliferative response and IFN-{gamma} production were evaluated in PBMC obtained from 22 healthy PPD-negative skin test donors (A), from 11 PPD-positive skin test tuberculosis patients (B), and from 10 PPD-positive skin test healthy volunteers (C). Cultures were either nonstimulated (Medium) or stimulated with U2 (5 µg/ml) or with PPD (2 µg/ml). Proliferation was measured by [3H]thymidine incorporation at day 6 and is expressed in cpm. IFN-{gamma} concentration is expressed in picograms per milliliter and was determined by sandwich ELISA using culture supernatants harvested at day 3 after the initiation of the cultures. Symbols represent the results obtained for each individual and bars are the average values obtained for each group. Paired t tests were used to compare mean values of IFN-{gamma} levels as well as cpm between nonstimulated (Medium) and U2-stimulated cultures within the tuberculosis patient group and within the PPD-positive skin test healthy volunteers. No significance (*, p > 0.08) was found among the tuberculosis patients group. In contrast among the PPD-positive skin test healthy controls, the differences between the mean values from control cultures compared with U2-stimulated cultures were highly significant (**, p < 0.002).

 
These results suggest that the recombinant U2 has the potential of being a subunit vaccine candidate because this molecule has epitopes readily recognized by T cells from both M. tuberculosis-infected mice and from healthy individuals presumably sensitized with M. tuberculosis.

Induction of protection against challenge with M. tuberculosis by immunization with U2

Preliminary immunogenicity experiments using U2 formulated with the adjuvant MPL-SE (Corixa) indicated that this formulation induced strong cellular immune response to U2 primarily of the CD4+ Th1 phenotype (data not shown). MPL-SE is a formulation of monophosphoryl lipid A with squalene oil emulsion, which has been used in vaccine formulations in several ongoing human clinical trials including those for malaria, hepatitis B, AIDS, and leishmaniasis. In addition it has been shown that MPL-SE modulates primarily CD4 Th1 response to protein Ags (29). In view of these observations, protection experiments were next performed in mice challenged with virulent M. tuberculosis H37Rv. For this purpose, C57BL/6 mice were immunized in the footpad three times, 3 wk apart, with 10 µg of U2 formulated with MPL-SE (25 µg). As internal controls, groups of mice were also immunized with saline and MPL-SE alone. As positive controls, mice were vaccinated once with BCG. Thirty days after the last immunization, the mice were challenged with M. tuberculosis H37Rv and bacteriological burden (CFU) was measured in mice lungs and spleen 3 wk after the challenge. Fig. 7 illustrates the results and indicates that immunization with U2 caused significant CFU reduction in both lungs and spleen as compared with control nonimmunized animals. Although the CFU reduction in both organs was slightly smaller (particularly in spleen) than that induced by vaccination with BCG, these results confirm that U2 can potentially be a component of a subunit vaccine against tuberculosis.



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  		FIGURE 7. Vaccination of mice against tuberculosis with U2. C57BL/6 mice were injected three times (3-wk interval) in the footpad with 10 µg of U2 mixed with 25 µg of MPL-SE or once (s.c.) with 5 x 104 CFU of BCG or with saline. One month after the last immunization, the animals were challenged with M. tuberculosis H37Rv, and CFU in the spleen and lungs were enumerated 3 wk later. This is one representative experiment of three separate experiments with essentially the same results. Error bars are SE of the CFU given by five animals per group. The overall ANOVA indicated highly significant differences between the mean values for CFU (lung and spleen) obtained from mice immunized with either BCG or MPL-SE with U2 compared with the mean values of the CFU obtained from mice inoculated with saline or with MPL-SE plus saline, respectively (p < 0.0001). No statistical difference (p > 0.05) was found for the mean values of CFU from mice inoculated with saline compared with the CFU obtained from mice immunized with MPL-SE plus saline. Pairwise comparisons were done by Tukey’s test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Over the past 10 years major efforts have been dedicated to develop novel vaccine candidates against tuberculosis and evaluate them in a variety of animal models. These efforts were fueled by the publication of the M. tuberculosis genome in 1998 and have resulted in the discovery of numerous novel vaccine candidates using different and efficient Ag selection strategies. These vaccines include protein or peptide vaccines in newer adjuvants, DNA vaccines, recombinant BCG vaccines, or other living vaccine vectors expressing immunodominant mycobacterial Ags, and naturally or rationally attenuated strains of Mycobacterium.

Several of these vaccine candidates have been validated using assays involving T cell responses from individuals who have a history of old or chronic sensitization with M. tuberculosis (PPD-positive individuals including both health persons and patients with pulmonary tuberculosis). At this phase of the infectious process, it is likely that most of the M. tuberculosis antigenic repertoire, which includes some protective Ags and a great number of molecules that are irrelevant for vaccine development, have been recognized by the host immune system. Therefore, this readout is not biased to select protective Ags. In other words, the host pool of T cells are recognizing Ags that are both actively produced and secreted or shed during the early implantation and establishment of the infectious process as well as vaccine irrelevant somatic Ags and Ags released from bacterial due to cell death during the later phases of the infection. If on the one hand, it is generally accepted that secreted Ags produced early on during the growth phase of the mycobacterial in vitro (CF) contains protective molecules (15, 16, 17, 18, 19, 20, 21, 22), on the other hand, the ability of mycobacterial somatic Ags to confer protection is a controversial issue. This notion is based on classical experiments that demonstrated that immunization of mice with dead M. tuberculosis bacteria results in the induction of a potent immune response to a vast number of Ags and low levels of short-term protective immunity (30, 31, 32). However, more recently this notion has been re-examined because protection can be achieved in mice or guinea pigs immunized with several somatic M. tuberculosis or M. bovis Ags formulated with newer adjuvants (33, 34, 35). Nonetheless, the premise that protective CF Ags are selectively produced in vivo guided us to undertake a direct search for these Ags released during the early phases of the infectious process. We opted for an approach that could identify and characterize these Ags in biological fluids of the infected host. Specifically, our premise was that M. tuberculosis CF proteins or their breakdown products (peptides) produced in vivo could reach the animals’ blood circulation and subsequently be excreted in the urine. The mouse model was chosen because during the first 10–14 days that follow infection, the microorganisms are actively multiplying in the lungs, spleen, and liver (36, 37), which is a situation that favors the hypothesis. This effort has led to the identification and preliminary immunological characterization of a mycobacterial Ag (U2) with protective properties, thus validating this approach of Ag discovery and vaccine development using M. tuberculosis CF proteins produced in vivo.

The success of this approach was made possible because of modern technologies that allow sequencing of proteins at fentomole levels and because of the availability of the entire sequence of the M. tuberculosis genome. The ultra-high sensitivity of MS/MS in sequencing proteins was used to sequence silver-stained polyacrylamide gel fragments containing protein bands on SDS-PAGE gel unique to the urine of M. tuberculosis-infected C57BL/6 mice and absent in the urine of normal uninfected syngeneic mice. Some of these bands contained sequences with a strong homology to mouse protein sequences in the database, which included properdine factor D, orosomucoid, prothrombin precursor, PGH2-D-isomerase, C3 convertase activator, tyrosine protein kinase receptor, cathepsin E, {alpha}1-antitrypsin, serum albumin, glycan-1, {alpha}-amylase, and IL-2R {alpha}-chain. The presence of autologous proteins in the urine of these mice could be a result of glomerular and/or tubular dysfunction caused by the infectious process. However, the sequence of a band of ~6.5 kDa revealed strong match with a predicted protein encoded by an open reading frame of the M. tuberculosis genome. Unfortunately, the initial attempts to express the gene encoding this protein in both E. coli and M. smegmatis expression systems failed. The problem was circumvented by the construction a homotrifusion (i.e., fusions of three copies of the U2 gene in tandem). This simple improvisation resulted in the excellent expression of the immunologically reactive recombinant protein. Western blot analyses using a specific rabbit anti-recombinant protein clearly revealed that U2 was a M. tuberculosis protein that is detected in both bacterial extract and culture filtrate of these organisms. Surprisingly though, no obvious signal peptide encoding sequences is associated with the U2 gene. Therefore, is likely that U2 is not a typical secreted protein and must be exported to the mycobacterial extracellular compartment by an alternative mechanism, e.g., been chaperoned by other molecules. Indeed our results suggest that U2 might be associated with Mycobacterium lipids both intracellularly and in the CF compartments of the microbial growth, thus supporting the possibility that U2 is exported to the extracellular compartment of the Mycobacterium associated with lipid molecules.

The evaluation of U2 as a potential vaccine candidate was accomplished by experiments conducted in mice and by studies done with human PBMC. The mouse model was used to investigate the recognition of this molecule by lymphoid cells of mice previously exposed to viable M. tuberculosis. Human cells were used to evaluate the ability of T cells from healthy PPD-positive skin test individuals and from patients with pulmonary tuberculosis to recognize U2, i.e., to validate this Ag for human use. The mouse experiments showed that sensitization of the animals with viable mycobacteria generates anti-U2-specific Th1 immune response. This conclusion is supported by both the high titers of specific IgG2a Abs generated during the infection as well as by the production of high concentrations of IFN-{gamma} and no IL-4 by spleen cells obtained from infected mice and stimulated in vitro with the recombinant Ag. These observations point to U2 as a possible vaccine candidate because during infection this molecule stimulates strong immune response of the protective phenotype. In addition, the results obtained with human cells further support this idea and suggest that U2 is a ubiquitous and potent Mycobacterium immunogen. PBMC from 9 of 10 healthy PPD-positive skin test donors with no previous history of vaccination with BCG proliferated upon stimulation with U2 and produced IFN-{gamma}. In contrast PBMC from 11 patients with pulmonary tuberculosis and with strong positive PPD skin test (>18 mm of induration) practically did not respond to stimulation with U2. Although the numbers of positive PPD skin test individuals (healthy controls and patients with pulmonary tuberculosis) so far tested are relatively small, and assuming that a positive PPD skin test in healthy individuals reflects some degree of protection, these results point to possible association of resistance to tuberculosis and responsiveness to U2. Consequently U2 qualifies as a promising vaccine candidate.

Indeed, protection experiments performed in mice support this possibility. These experiments showed that mice immunized with U2 formulated with MPL-SE were able to control the progression of the infectious process caused by virulent M. tuberculosis to levels comparable to or only slightly lower than that induced by vaccination with BCG. However, considering the fact that U2 is a small molecule (6.5 kDa), hence with potentially few epitopes, the level of protection that was achieved with this molecule is consequently considerably high when compared with the protection induced by BCG vaccination. Altogether, these and the immunogenicity studies indicate that U2 can potentially be an interesting component of a subunit vaccine against tuberculosis.

In conclusion, our results support the premise of the approach proposed in these studies, i.e., the use of secreted M. tuberculosis Ags detected in vivo during the early stages of the disease to clone genes encoding protective Ags. Moreover such Ags can alternatively be tested in Ag detection assays as possible markers of active mycobacterial multiplication and therefore active disease. These studies are in progress.


   Acknowledgment
 
We thank Dr. Ralph Kent for the statistical analysis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the National Institutes of Health Grants AI 43528 (to A.C.-N.) and AI 44373 (to S.G.R.). This work also received financial support from the United Nations Development Programme/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases Contract OD/TS-0400211 (to A.C.-N.). Back

2 S.M. and S.S.K. contributed equally to this work. Back

3 Address correspondence and reprint requests Dr. Antonio Campos-Neto, The Forsyth Institute, 140 The Fenway, Boston, MA 02115-3799. E-mail address: acampos{at}forsyth.org Back

4 Abbreviations used in this paper: BCG, bacille Calmette-Guérin; PPD, purified protein derivative; CF, culture filtrate; MPL, monophosphoryl lipid A; MS, mass spectrometry. Back

Received for publication January 11, 2005. Accepted for publication August 10, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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