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1 ORIGINAL ARTICLE /j x PPE protein (Rv3425) from DNA segment RD11 of Mycobacterium tuberculosis: a potential B-cell antigen used for serological diagnosis to distinguish vaccinated controls from tuberculosis patients H. Zhang 1, J. Wang 1, J. Lei 1, M. Zhang 1,Y.Yang 1, Y. Chen 2 and H. Wang 1 1 State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, Shanghai and 2 No. 6 Hospital of Zhengzhou, Henan, China ABSTRACT Proteins encoded by a 9.5-kb DNA segment, termed the region of difference (RD), of Mycobacterium tuberculosis have been demonstrated to be important in bacterial virulence, vaccine development and the design of diagnostic reagents. This study evaluated the immunogenic properties of Rv3425, a member of the PPE family of proteins, encoded by an open reading frame found in RD11 of M. tuberculosis, in comparison with two other well-known antigens, the early secreted antigen target 6 (ESAT-6) and the 10-kDa culture filtrate protein (CFP-10). RT-PCR demonstrated that Rv3425 mrna is expressed in liquid culture by M. tuberculosis H37Rv. When tested in a conventional ELISA in the form of a Histagged recombinant protein, Rv3425 revealed a statistically significant antigenic distinction between healthy bacille Calmette Guérin (BCG)-vaccinated controls and tuberculosis (TB) patients (p <0.0001). The anti-igg response to recombinant Rv3425 was almost equal to that for CFP-10, and was higher than that for ESAT-6. The results highlight the immunosensitive and immunospecific nature of Rv3425, which shows promise for use in the serodiagnosis of TB. Keywords Antigen, diagnosis, Mycobacterium tuberculosis, PPE family protein, region of difference, Rv3425 protein Original Submission: 22 December 2005; Revised Submission: 26 April 2006; Accepted: 18 June 2006 Clin Microbiol Infect 2007; 13: INTRODUCTION Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a major public health problem worldwide, with 8 million new cases and 2 3 million deaths annually, despite the use of the bacille Calmette-Guérin (BCG) vaccine and effective antibiotics [1]. Indeed, the problem has been exacerbated by the increasing emergence of multidrug-resistant strains and co-infection with human immunodeficiency virus (HIV) [2 4]. The increasing threat of this disease has emphasised the need for basic and applied research aimed at developing new drugs, improving diagnosis and vaccines, and gaining a better understanding of host parasite interactions. Currently, diagnosis of TB relies mainly on clinical examination and Corresponding author and reprint requests: H. Wang, State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, 220 Handan Road, Shanghai , China hhwang@fudan.edu.cn radiographical findings, confirmed by sputum smear microscopy and bacterial culture. However, culture of M. tuberculosis requires up to 6 8 weeks, so that confirmation of the diagnosis often depends on sputum smear microscopy, which has a sensitivity of only 50 60% [5]. Several rapid diagnostic techniques have been investigated in recent years to determine their ability to improve the diagnosis of TB. These techniques include the detection of M. tuberculosis in clinical specimens by PCR, as well as immune reactions based on the cell-mediated immune response (CMI) or on humoral immune responses. In-vitro tests based on CMI make possible the early detection of individuals with latent infection, and are of great importance for contact tracing and screening of high-risk groups in a setting of low endemicity [6,7]. However, this method is not an ideal alternative to culture and microscopy in developing countries, as large proportions of the populations in such countries are likely to harbour latent infection with Ó 2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases

2 140 Clinical Microbiology and Infection, Volume 13 Number 2, February 2007 M. tuberculosis [8]. A serological test would be an attractive alternative diagnostic method; such a test would be rapid, easy to perform and userfriendly, and could be implemented easily in the conditions found in developing countries as, unlike CMI-based assays, it does not require viable cell cultures. Considerable progress has been made in the identification of suitable antigens, of which the most frequently studied is the 38-kDa antigen [9 11]. The sensitivity of this antigen has been reported to be 16 80% in various studies, depending on the smear status of the patient and the patient population used. Other antigens studied have included Mtb81 (malate synthase) [12], MTB48 [13], a-crystallin (14 kda) [10], MTC28 [10], thiol peroxidase [14], MPT51 [10], MPT32 [15], ICDI [16], ICDII [16], Ag85A, Ag85B [10,17] and Rv3369 [18]. Finally, ESAT-6 and CFP-10, two antigens that have been used for CMI-based tests, have also been evaluated as targets of humoral immune responses [10,11,19]. About 10% of the genome of M. tuberculosis codes for proteins belonging to the PE and PPE families [20], which are glycine-rich and essentially exclusive to M. tuberculosis [21]. Few studies support the notion that PE and PPE proteins are of functional importance [20,22], and it is widely speculated that they could be responsible for generating antigenic variation [23 27]. However, little has been documented concerning the effect that the PPE family of proteins, each of which is unique in its protein sequence and possible structure, may have on the immune system, with the exception of Rv2430c [28], Rv2608 [29] and Rv2108 [30]. On the basis of comparative genomics of BCG vaccines by whole genome DNA microarray analysis [31] and antigen index calculations by Lasergene Navigator (DNA STAR) software, the present study used an open reading frame (ORF), Rv3425, which is missing from BCG and all virulent Mycobacterium bovis strains tested, and which codes for a PPE family protein. The importance of this protein in eliciting immune responses was then evaluated with human sera obtained from two well-defined groups of patients: those with pulmonary TB, and those with extra-pulmonary disease. Human sera from clinically healthy individuals were used as controls to compare the immunological responses to the protein. The responses obtained were compared with those obtained with two other well-known antigens, ESAT-6 and CFP-10. MATERIALS AND METHODS RNA extraction and RT-PCR RNA was extracted from 10 9 M. tuberculosis H37Rv cells, cultured in Middlebrook 7H9 medium, supplemented with albumin dextrose complex, using a Qiaquick total RNA extraction kit (Qiagen, Hilden, Germany), and was dissolved in 50 ll of nuclease-free water and stored at )70 C until required. First-strand synthesis was carried out with avian myeloblastosis virus reverse transcriptase, followed by heat denaturation to inactivate the enzyme. Subsequent secondstrand synthesis was performed with Tfl polymerase. The amplification product was visualised by electrophoresis in an agarose 1% w v gel. Before cdna synthesis, the RNA was treated with RNase-free DNase I (Boehringer, Mannheim, Germany) at 37 C for 20 min. RT-PCR was performed using the ThermoScript RT-PCR system (Invitrogen, Carlsbad, CA, USA), with the specific primers Rv3425-F (5 -ATGCATC- CAATGATACCAGC) and Rv3425-R (5 -CTACCCGCCCCTG- TAGATCTG), according to the manufacturer s instructions. Cloning, expression and purification of the recombinant protein Rv3425 and ESAT-6 and CFP-10 The ORFs corresponding to Rv3425 (528 bp), ESAT-6 (Rv3875, 288 bp) and CFP-10 (Rv3874, 303 bp) were amplified by PCR from the genomic DNA of H37Rv. BamHI and HindIII restriction sites were incorporated at the 5 -end of the forward and reverse primers, respectively, for Rv3425, ESAT-6 and CFP-10. The primers and parameters for thermal cycle amplification are shown in Table 1. The amplicons comprising the full-length Rv3425, ESAT-6 and CFP-10 ORFs were cloned at the BamHI and HindIII sites of the cloning vector puc18. The clones were confirmed by sequencing, using the T7 promoter primer, on a Prism 377 DNA sequencer (Applied Biosystems, Warrington, UK), and were then sub-cloned into the expression vector pet32a (Invitrogen) with six N-terminal histidine sequence tags. The constructs generated were then transformed into the BL21-CodonPlus (DE3)-RP strain of Escherichia coli (Novagen, Madison, WI, USA) for expression. The over-expressed Histagged recombinant Rv3425 and ESAT-6 proteins were purified from the insoluble inclusion bodies contained in 1 L of isopropyl-b-d-thiogalactopyranoside-induced batch cultures by affinity chromatography using a His-Bind Column (Novagen) in the presence of 8 M urea, according to the manufacturer s recommendations, while CFP-10 was purified under native conditions. The purified recombinant proteins were dialysed against 20 mm Tris-HCl, ph 7.5, containing 100 mm NaCl and glycerol 3% v v, and were quantified using Bradford s reagent (Bio-Rad, Hemel Hempstead, UK). Study population In total, 110 serum samples were examined in this study. The sera were obtained from patients in three distinct groups: (i) 50 patients (median age 44.3 years; range years; 71.4% males) with a diagnosis of pulmonary TB, based on clinical signs and symptoms (fever, cough and productive sputum), a

3 Zhang et al. Response to a PPE antigen of M. tuberculosis 141 Table 1. PCR primers and thermal cycle parameters for amplification of open reading frames Primer Sequence PCR parameters ESAT-6 Forward GAC ggatcc ATG ACA GAG CAG CAG CAG TGG AAT TTC 94 C for 5 min Reverse GAC aagctt CTA TGC GAA CAT CCC AGT GAC G Then 30 cycles: 94 C for 1 min 66 C for 1 min; 72 C for 45 s Then 72 C for 5 min CFF-10 Forward GAC ggatcc ATG GCA GAG ATG AAG ACC GAT GCC 94 C for 5 min Reverse GAC aagctt TCA GAA GCC CAT TTG CGA GGA CAG Then 30 cycles: 94 C for 1 min; 66 C for 1 min; 72 C for 45 s Then 72 C for 5 min Rv3425 Forward GAC ggatcc ATG CAT CCA ATG ATA CCA GC 94 C for 5 min Reverse GAC aagctt CTA CCC GCC CCT GTA GAT CTG Then 30 cycles: 94 C for 1 min; 65 C for 1 min; 72 C for 45 s Then 72 C for 5 min Amplicon size 288 bp 303 bp 528 bp suggestive chest X-ray, and the presence of acid-fast bacilli in sputum smears; (ii) 32 patients (median age 42.2 years; range years; 64.7% males) with a diagnosis of an extra-pulmonary form of TB, e.g., of the lymph nodes (neck, axillae and groin), pericardium, spine, thoracic spine, pleura, mammae, joints or peritoneum, based on clinical signs and symptoms and the demonstration of acid-fast bacilli in biopsies of the affected tissues; and (iii) 28 healthy individuals (median age 19.1 years; range years; 53% males) who had received M. bovis BCG vaccination during childhood. Since this study was performed on a protein from the PPE family, members of which are unique to mycobacteria, cross-reactivity to this protein would not be expected, and it was therefore considered unnecessary to include control subjects with other bacterial infections in the present study. All patient serum samples were obtained from No. 6 Hospital of Zhengzhou, Henan Province, China. Blood samples were drawn from TB patients shortly after diagnosis and initiation of treatment, while control sera were drawn from students in Fudan University who had received BCG vaccination during childhood. Donors in this group answered a questionnaire concerning BCG vaccination, travel history, contact with TB patients and occupational exposure to mycobacteria. Subsequently, donors with known prior exposure, or who were considered to be at risk of prior exposure, were excluded. All donors were aged >18 years and gave informed consent; the study was also approved by the Ethics Committee, Ministry of Health, China. Only HIV-seronegative individuals were included in the study. Immunosorbent assays ELISAs were performed to check the B-cell immune response in humans to the Rv3425 recombinant protein and to the other well-known antigens, ESAT-6 and CFP-10. In brief, 96-well microtitre plates (Nalge Nunc International, Rochester, NY, USA) were coated with 500 ng 100 ll of either control antigens or Rv3425, incubated overnight at 4 C, washed three times with phosphate-buffered saline, and blocked with 100 ll of blocking buffer (bovine serum albumin 2% w vin phosphate-buffered saline) for 2 h at 37 C. The plates were then washed three times with PBST wash buffer (Tween % v vin1 phosphate-buffered saline). The sera from TB patients belonging to different clinical groups were diluted 200-fold in blocking buffer, according to the results of the initial standardisation experiments (results not shown). Diluted serum (50 ll) was then added to antigen-coated wells and incubated for 1 h at 37 C. The plates were thoroughly washed with PBST and then incubated with anti-human IgG horseradish peroxidase (Sigma, Poole, UK) at 37 C for 1 h. Horseradish peroxidase activity was detected with a chromogenic substrate, o-phenylenediamine tetrahydrochloride (Sigma), dissolved (1 ll ml) in citrate phosphate buffer (ph 5.4) and H 2 O 2. The reaction was terminated by the addition of 0.5 M H 2 SO 4, and absorbance values were measured at 492 nm in an ELISA reader (Bio-Rad). Each ELISA was repeated at least twice. Data analysis The ELISA results were analysed using cut-off values equal to the mean optical density for the healthy control serum samples plus three standard deviations. Any sample exhibiting absorbance above the cut-off value was considered to be positive. For statistical analysis, the differences between groups of TB patients and healthy controls were calculated by the independent-samples t-test using the Statistics Package for Social Science (SPSS Inc., Chicago, IL, USA), with p <0.05 considered to be significant. RESULTS Expression of the hypothetical PPE ORF Rv3425 at the mrna level In order to verify whether the putative ORF Rv3425 represents a functional gene, total RNA from M. tuberculosis H37Rv was analysed by RT- PCR with a pair of Rv3425-specific primers that were selected to produce a 528-bp amplicon.

4 142 Clinical Microbiology and Infection, Volume 13 Number 2, February 2007 When the PCR products were analysed by agarose gel electrophoresis, an amplicon of the expected size was revealed. No PCR products were detected in the absence of reverse transcriptase. These data indicate that Rv3425 is expressed during exponential growth in vitro. Expression and purification of M. tuberculosis Rv3425 and ESAT-6 and CFP-10 To evaluate the antigenic ability of Rv3425, the corresponding gene was expressed in E. coli BL21-CodonPlus(DE3)-RP cells and purified as a 6 His-tag fusion protein. Purified Rv3425 protein was fractionated by electrophoresis on a polyacrylamide 12% w v gel. A single 37.5-kDa protein (including the mass of the N-terminal fusion domain of pet32a) was observed by staining the gel with Coomassie Brilliant Blue dye. There was no expression of the protein in uninduced cells. The recombinant protein was largely present in the insoluble fraction, and therefore the purification was carried out under denaturing conditions from insoluble fractions in the presence of 8 M urea, with a yield of 6.5 mg of protein/l of culture. The over-expressed N-terminal His-tagged Rv3425 protein was purified to >98% homogeneity on a nickel affinity column, and was confirmed by HPLC analysis. Similarly, recombinant ESAT-6 and CFP-10 were purified to 95 98% homogeneity, with a yield of 20.4 mg/l of culture. The recombinant proteins were dialysed overnight and used for immunoreactivity analyses. Reactivity of recombinant PPE protein Rv3425 with patient sera Humoral immune responses directed against Rv3425 were compared among TB patients and BCG-vaccinated healthy controls (Fig. 1a). The recombinant protein was used to screen the infected and healthy sera by ELISA, with antihuman IgG horseradish peroxidase as conjugate. The N-terminal fusion protein of pet32a was negative in the ELISA assay. Most of the sera from pulmonary TB and extra-pulmonary TB patients showed a statistically significant (p <0.0001) response against recombinant Rv3425 protein when compared with the healthy controls. Since negligible antibody responses were obtained in the healthy control group (Fig. 1a), it is likely that Absorbance at 492 nm (a) (b) Absorbance at 492 nm (c) Absorbance at 492 nm Rv3425 IgG HC PTB Extra-PTB Rv3874 IgG HC PTB Extra-PTB Rv3875 IgG HC PTB Extra-PTB Fig. 1. The humoral immune responses directed against the recombinant proteins Mtb Rv3425 (a) and control antigens ESAT-6 (b) and CFP-10 (c) were compared among different categories of patients and healthy controls. Horizontal lines on each graph indicate the cut-off absorbance, which was determined by the mean optical density for the healthy control serum samples plus three standard deviations. HC, healthy Mycobacterium bovis bacille Calmette Guérin-vaccinated controls; PTB, pulmonary tuberculosis patients; Extra-PTB, extra-pulmonary tuberculosis patients.

5 Zhang et al. Response to a PPE antigen of M. tuberculosis 143 Rv3425 is expressed during the course of M. tuberculosis infection, and that it may be associated with disease manifestation and progression. Immunodominant nature of Rv3425 Since most of the patients infected with TB showed strong humoral responses against Rv3425 compared with the healthy controls, it was of interest to compare the responses to other antigens, and also to evaluate whether Rv3425 is immunodominant. Therefore, two well-known immunodominant antigens of M. tuberculosis, ESAT-6 (Rv3874) and CFP-10 (Rv3875), were also tested (Fig. 1b,c). In order to compare Rv3425 to ESAT-6 and CFP-10, the data presented in Fig. 1 were recalculated as percentages of positive sera (Table 2). Although only 36.0% of individuals with pulmonary TB and 34.4% of extra-pulmonary TB cases showed a strong IgG antibody response to ESAT-6, a high percentage of individuals with pulmonary TB (70.0%) and extrapulmonary TB (59.4%) recognised Rv3425 (Table 2). In comparison, 78.0% of individuals with pulmonary TB and 65.6% of extra-pulmonary TB cases showed a strong IgG response to CFP-10. Thus, both pulmonary and extra-pulmonary TB patients showed an Rv3425-specific response equivalent to that for CFP-10 and higher than that for ESAT-6. Thus, the results demonstrate the immunodominant nature of the product of the hypothetical PPE ORF Rv3425. DISCUSSION Serological diagnosis of TB has traditionally been regarded as unreliable, mostly because of the low sensitivity and specificity of the tests. Apart from the intrinsic complexity of the immunological and pathophysiological characteristics of TB (a chronic disease that induces predominantly cellular immunity and weak humoral immunity), one possible explanation for the general lack of success with most serological tests is the complex mixture of crude antigens used in these tests [32,33]. However, several highly purified M. tuberculosis recombinant antigens have now been described and evaluated as serological markers for the disease. Some of these antigens have been evaluated extensively and have been shown to have relatively high sensitivity and specificity for the identification of patients with pulmonary TB [34 41]. These findings have revived the concept that serological tests are feasible for the diagnosis of TB. Comparative hybridisation experiments using DNA microarrays have revealed that ORFs absent from M. bovis may be of considerable practical utility. Diagnostic tests available currently, e.g., the tuberculin skin test, are unable to distinguish between individuals infected with M. tuberculosis and those vaccinated with BCG. This distinction may be possible with an assay based on antigens derived from an M. tuberculosis-specific gene [31]. The PE and PPE families, two large unrelated protein families, occupy c. 10% of the coding capacity of the M. tuberculosis genome, and it has been hypothesised that these proteins represent the principal source of antigenic variation in M. tuberculosis [20]. The Rv3425 ORF selected for the present study is a member of subgroup 3 of the PPE family, and is located in RD11 [28,31]. The proteins in this group are unrelated, except for the presence of the common PPE domain, and show good possibilities for diagnostic use in a clinical setting. The main objective of the present study was to evaluate the immune features of the rrv3425 protein in comparison with ESAT-6 and CFP-10, both of which have been used for the diagnosis of TB, with sensitivities of 30 60% and specificities of >96% [10,11,15,18,19]. Table 2. IgG seroreactivity of recombinant proteins in sera from tuberculosis patients and healthy controls Pulmonary tuberculosis patients Extra-pulmonary tuberculosis patients Healthy controls Recombination proteins No. examined No. positive a % No. examined No. positive a % No. examined No. positive a % CFP ESAT Rv a Positive sera, defined as cases for which the optical density (OD) generated by the patient serum was greater than the mean OD of the healthy control sera plus three standard deviations.

6 144 Clinical Microbiology and Infection, Volume 13 Number 2, February 2007 The comparative immunoreactivity of Rv3425, ESAT-6 and CFP-10 clearly distinguished between healthy subjects and TB patients (p <0.0001). The negligible antibody response obtained in the BCG-vaccinated healthy control group suggests that Rv3425, ESAT-6 and CFP-10 can be used for diagnosis of M. tuberculosis infection, even in geographical areas in which BCG vaccination is used routinely. The high sensitivity of Rv3425 for the serological diagnosis of TB patients, which is sufficient for use alone in routine tests for the diagnosis of this disease, is of great interest. Few recombinant proteins have been reported to perform this well. Among the molecules described previously, only the 38-kDa antigen and CFP-10 have sensitivity equal to that of Rv3425 for the diagnosis of pulmonary TB in the absence of co-infection with HIV [10]. Although the lung is the primary site of disease in 80 84% of TB cases, extra-pulmonary TB has become more common with the advent of HIV infection [42]. Extra-pulmonary TB remains an important diagnostic and therapeutic problem. Efforts to control the disease are currently hampered by the lack of effective tools for the detection of infected individuals, although new diagnostic tests are being developed [43]. Bacteria in extra-pulmonary TB cases can be present in low numbers at inaccessible sites [42]. The present study demonstrated that the PPE protein Rv3425 was recognised by sera from both pulmonary TB and extra-pulmonary TB cases, and that it is an immunodominant B-cell target antigen with apparent diagnostic potential. Since Rv3425 has the conserved motif of the PPE gene family of M. tuberculosis, it is likely that other members of the same family also serve the same function in the bacterium. Rv3426 and Rv3429, two close homologues of Rv3425 in M. tuberculosis, are also present in RD11, and their immunological potential is currently being studied. It is inevitable that there is a homologue in M. bovis, because Rv3425 is a member of the PPE family, but the immunological response was negative with healthy BCG-vaccinated individuals. It is interesting to speculate on the possible use of Rv3425 and other immunodominant antigens for vaccine development, and further studies are in progress to investigate the CMI response to Rv3425, with the aim of evaluating its use in the cellular immunity-based immunodiagnosis of TB. ACKNOWLEDGEMENTS The authors would like to thank W. Xiaolei for invaluable discussions. We also thank the No. 6 Hospital of Zhengzhou, Henan, and the Hospital of Fudan University for providing us with the sera from TB patients and healthy controls. J. Zhe is gratefully acknowledged for critical reading of the manuscript. 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