PPE Antigen Rv2430c of Mycobacterium tuberculosis Induces a Strong B-Cell Response

In silico analysis of Rv2430c.In silico pattern search analysis of the PPE family was carried out to classify the products of the various ORFs into three subgroups. Products of ORFs with ≤200 amino acids and belonging to the third subgroup of the PPE family were further analyzed using protein analysis software (Protean version 4.0, Lasergene Navigator; DNASTAR Inc., Madison, Wis.) to calculate their antigenic indexes.

RNA extraction and reverse transcriptase (RT) PCR.RNA was extracted from 10⁹ H₃₇Rv cells, cultured in vitro in Middlebrook 7H9 medium supplemented with albumin-dextrose complex according to the Qiaquick total RNA extraction kit (Qiagen Inc.) instructions, dissolved in 50 μl of nuclease-free water, and stored at −70°C until further use. First-strand synthesis was carried out using avian myeloblastosis virus reverse transcriptase. This was followed by heat denaturation to inactivate the enzyme. Subsequent second-strand synthesis was performed using Tfl polymerase. The PCR product was visualized by electrophoresis in a 1% agarose gel.

Expression and purification of the recombinant protein encoded by Rv2430c.Genomic DNA of H37Rv was extracted using the Genome Extraction kit provided by Epicentre Technologies, as described earlier (26). The Rv2430c gene was PCR amplified from the genomic DNA of H₃₇Rv using upstream (5′-GGATCCATGCATTTCGAAGCGTAC-3′) and downstream (5′-AAGCTTCTAAGTGTCTGTACGCGATGA-3′) primers. BamHI and HindIII sites were incorporated in the 5′ and 3′ ends of the primers, respectively. The purified fragment was ligated into the pGEMT-Easy vector (Promega Inc.), and the recombinant clone carrying the Rv2430c insert was confirmed by DNA sequencing (ABI Prism 377 DNA Sequencer; PE Biosystems). The insert was then subcloned as a BamHI and HindIII fragment into the PQE30 expression vector (Qiagen Inc.) to generate the plasmid construct PQERv2430c carrying Rv2430c as an N-terminal histidine-tagged fusion. PQERv2430c was transformed into the host M15pREP4 strain of Escherichia coli. A single colony of E. coli strain M15pREP4 harboring PQERv2430c was inoculated into 5 ml of Luria-Bertani broth with the appropriate antibiotics (100 μg of ampicillin/ml and 25 μg of kanamycin/ml) and grown overnight at 37°C with constant agitation; 100 μl of this overnight culture was inoculated into 5 ml of Luria-Bertani broth with the appropriate antibiotics and grown to an optical density at 590 nm of 0.6, at which time expression was induced with 1 mM IPTG. A separate aliquot of uninduced culture was kept as a control. Cells were harvested 3 h postinduction, suspended in 1× SDS sample buffer, and denatured by being heated at 100°C for 10 min. The recombinant protein was purified to homogeneity with the QIA Expressionist kit (Qiagen Inc.).

Serological characterization of the recombinant protein.ELISAs were performed in 96-well microtiter plates (Corning Costar) coated with the recombinant Rv2430c protein. After overnight incubation at 4°C, the plates were washed three times with phosphate-buffered saline (PBS) buffer and blocked with 200 μl of blocking buffer (PBS containing 1% bovine serum albumin) for 1 h at 37°C. The plates were then washed three times with PBS-Tween wash buffer (0.05% Tween 20 in 1× PBS, pH 8.0) and incubated for 1 h at 37°C with human sera (1: 200 dilution in blocking buffer). The plates were washed with PBS-Tween and further incubated with either anti-human immunoglobulin G (IgG)-horseradish peroxidase (HRP) or anti-human IgM-HRP (Sigma). HRP activity was detected using a chromogenic substance, o-phenylenediamine tetrahydrochloride (Sigma), in citrate-phosphate buffer (pH 5.4) and 1 μl of H₂O₂ (Qualigens, India)/ml. The reactions were terminated using 1 N H₂SO₄, and the absorbance values were measured at 492 nm in an ELISA reader (Bio-Rad).

Study population.Serum samples were obtained from 92 TB patients who had reported to the Mahavir Hospital and Research Centre, Hyderabad, India, and 10 clinically healthy donors. The 92 patients belonged to three well-defined categories. Category 1 (n = 32) comprised patients who had contracted the pathogen for the first time and had no history of TB treatment. Category 2 (n = 30) comprised patients with relapsed TB, i.e., who were treated earlier for TB but the symptoms resurfaced after completion of the treatment. Category 3 (n = 30) comprised patients with extrapulmonary TB in which the disease was confirmed by tissue biopsy. In the cases of category 1 and category 2 patients, diagnosis was confirmed by examination of the sputum (acid-fast bacillus smear positive). Clinically healthy donors were vaccinated with Mycobacterium bovis BCG. The study was carried out after approval from the Institute Bioethics Committee.

Statistical analysis.Student's t test was used for analysis of statistical significance (P value). Graphpad Quickcalcs (online t test calculator [http://www.graphpad.com/quickcalcs/ttest1.cfm ]) was used for this purpose.

The product of the hypothetical PPE ORF Rv2430c has a high antigenicity profile score and is expressed at the mRNA level.In silico analyses of the PPE ORF products, ≤200 amino acids in length, belonging to subgroup 3 of the PPE family (7) were carried out. Rv2430c and Rv3425 displayed major antigenic stretches (Fig. 1) with peak values of ≥3.0. However, an analysis of the microarray expression data (24) identified Rv2430c as one of the overexpressed genes in an IdeR mutant of M. tuberculosis, and it was thereby implicated in pathogenesis. Rv2430c was thus selected for our study. Rv2430c was further subjected to detailed analysis to predict its likely structure through various algorithms. The Predict Protein server (http://www.embl-heidelberg.de/predictprotein/ ) gave a very low score for Rv2430c, pointing to the unlikely possibility that this ORF encoded a transmembrane protein (data not shown). To check whether the hypothetical Rv2430c indeed represented a functional gene, the mRNA extracted from in vitro-cultured H₃₇Rv cells was used as a template for reverse transcription followed by PCR. The RT-PCR product was fractionated on a 1% agarose gel. A 597-bp band was observed upon staining with ethidium bromide, indicating that the ORF was expressed at the mRNA level in the liquid cultures of M. tuberculosis (Fig. 2A). The ORF Rv2430c was expressed as a His-tagged fusion protein in E. coli (Fig. 2B) and used for immunological studies.

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FIG. 1.

In silico analysis of PPE ORFs. Only the ORFs belonging to subgroup 3 and encoding products ≤200 amino acids in length were subjected to analysis. The presence of stretches of high antigenic index are shown. The x axis indicates the ORF product length in amino acids, while the y axis represents the antigenic index.

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FIG. 2.

(A) Transcription of hypothetical PPE ORF Rv2430c. RNA extracted from the virulent H37Rv laboratory strain of M. tuberculosis was used in an RT-PCR. A 597-bp RT-PCR product was observed after electrophoresis in a 1% agarose gel (lane 1). Lane M is the 100-bp DNA molecular size marker run alongside. The arrow on the right indicates the position of the 597-bp PCR product. (B) Expression and purification of the recombinant PPE protein. The recombinant protein was expressed in strain M15pREP4 of E. coli and was purified to homogeneity using the NiNTA protein purification kit. Lane 1, marker; lane 2, purified protein. The arrow on the right indicates the position of the 23-kDa protein.

The recombinant PPE protein Rv2430c displays strong B-cell responses during infection with TB.Having shown that the ORF encoding RV2430c of M. tuberculosis was expressed at the mRNA level, experiments were designed to evaluate the immune response of TB patients to recombinant Rv2430c PPE protein. For this, the recombinant Rv2430c protein was used to screen the TB patient sera by ELISA, using anti-human IgG-HRP and anti-human IgM-HRP as conjugates. The humoral immune responses directed against the recombinant protein by patients with TB and healthy controls were compared. The data (Fig. 3) reveal that the sera of all the infected patients mounted significantly higher antibody responses against Rv2430c than those of the healthy controls (P < 0.0001). Since negligible antibody responses were obtained in the healthy-control group (Fig. 3), it is likely that this protein is expressed during the course of M. tuberculosis infection, and it may be associated with disease manifestation and progression.

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FIG. 3.

The recombinant Rv2430c PPE protein elicits strong antibody responses in M. tuberculosis-infected patients as opposed to healthy controls. ELISA reactivities of IgG anti-Rv2430c antibodies were assayed in sera of either M. tuberculosis-infected patients or healthy controls (P < 0.0001). Symbols, patient population.

Immunodominant nature of Rv2430c.Since all the patients infected with TB revealed strong humoral responses against Rv2430c compared to the healthy controls, it was of interest to compare the antibody responses in various clinical categories and also to evaluate whether Rv2430c is immunodominant. For this, we chose recombinant Hsp10 of M. tuberculosis, which is a well-known immunodominant antigen of M. tuberculosis (28). Figure 4 clearly shows that strong antibody responses were elicited against Rv2430c in all three study groups (category 1, category 2, and category 3). Rv2430c elicited statistically significant immune responses compared to Hsp10 (P < 0.003) in patients with fresh infections (Fig. 4A). Patients belonging to categories 2 and 3 exhibited Rv2430c-specific antibody equivalent to that for Hsp10 (Fig. 4B and C). Since the PPD antigen of M. tuberculosis is also used to diagnose TB infection (2, 15, 29), we compared the immunopotentiality of Rv2430c over PPD only in cases of fresh infection (Fig. 4A). The results clearly indicated that the PPE protein Rv2430c is far more immunogenic and could elicit a stronger B-cell response than PPD. Sera from the fresh-infection category responded better against the Rv2430c antigen than against PPD (P < 0.0001) (Fig. 4A).

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FIG. 4.

PPE Rv2430c protein shows strong reactivities to sera from all three patient categories. Reactivities to both recombinant Rv2430c and Hsp10 of M. tuberculosis in the three categories of patients were estimated by ELISA. The patients belonging to categories 2 (B) and 3 (C) displayed similar antibody responses to both antigens. However, the antibody responses of category 1 patients (A) to Rv2430c were higher than those to Hsp10 (P < 0.003) or PPD (P < 0.0001).

In order to compare the serological sensitivities of Rv2430c and Hsp10, the data presented in Fig. 4 were recalculated as percentages of individuals showing absorbances above 0.65 at 492 nm. Although only 28.12% of individuals with fresh infections mounted strong antibody responses of the IgG type to Hsp10, a very high percentage of individuals (62.5%) recognized Rv2430c (Fig. 5A). Similar conclusions could be drawn when IgM antibodies were assayed (Rv2430c versus Hsp10, 71 versus 37.5%) (Fig. 5B). From these results, it is apparent that Rv2430c shows better reactivity vis-a-vis Hsp10 to sera from patients with fresh infection than to those of relapsed or extrapulmonary-TB patients. Our results, therefore, convincingly demonstrate the immunodominant nature of the product of the hypothetical PPE ORF Rv2430c.

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FIG. 5.

Serological sensitivities of PPE Rv2430c and Hsp10 as a function of percentages of individuals. (A) The results shown in Fig. 4 were recalculated as percentages of individuals showing A₄₉₂ values of >0.65. The anti-IgG responses against Rv2430c or Hsp10 were compared for all three categories of patients studied. A higher percentage of individuals belonging to category 1 showed stronger reactivity to Rv2430c than to Hsp10, but for the other two categories, the values were comparable. (B) Percentage of individuals in category 1 showing anti-IgM antibody A₄₉₂ of >0.5. A higher percentage of individuals showed anti-IgM antibody A₄₉₂ of >0.5 against Rv2430c than against Hsp10.