Identification of mycobacterial bacterioferritin B for immune screening of tuberculosis and latent tuberculosis infection

Tuberculosis 107 (2017) 119e125

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Identification of mycobacterial bacterioferritin B for immune screening of tuberculosis and latent tuberculosis infection Xinyu Yang a, 1, Jia-bao Wu a, c, 1, Ying Liu a, Yanqing Xiong b, Ping Ji a, Shu-jun Wang a, Yingying Chen a, Guo-ping Zhao c, d, Shui-hua Lu b, **, Ying Wang a, d, * a

Shanghai Jiao Tong University School of Medicine, Shanghai Institute of Immunology, Shanghai, 200025, China Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular Virology of MOE/MOH, Fudan University, 2901 Caolang Rd., Shanghai, 201508, China c Department of Microbiology, School of Life Sciences, Fudan University, Shanghai, 200438, China d Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, 201200, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 January 2017 Received in revised form 14 August 2017 Accepted 20 August 2017

Objectives: It remains necessary and urgent to search for novel mycobacterial antigens to increase the sensitivity and specificity for tuberculosis (TB) diagnosis and latent TB infection (LTBI) screening. Antigens capable of inducing strong immune responses during Mycobacterium tuberculosis (M.tb) infection would be good candidates. Methods: Cellular responses specific to M.tb derived bacterioferritin B (BfrB) were assessed by IFN-g ELISPOT in three human cohorts, including healthy controls (HCs), LTBI population and pulmonary TB (PTB) patients. Its significance in TB diagnosis and LTBI identification was further analyzed. Results: BfrB-specific IFN-g responses in PTB and LTBI groups were significantly higher than that in HCs. However, BfrB-specific IFN-g release was not as strong as that to ESAT-6 or CFP-10 in PTB patients whereas comparable in LTBI cohort with possible complementary properties to ESAT-6 or CFP-10. More interestingly, there were a considerable number of HCs with high BfrB-specific cellular responses. When HCs with high BfrB-specific cellular responses were subgrouped into ESAT-6/CFP-10hi (SFUs ¼ 3, 4, 5) and ESAT-6/CFP-10lo (SFUs < 3) groups, those who belonged to ESAT-6/CFP-10hi group exhibited higher PPD responsiveness than ESAT-6/CFP-10lo group. Conclusions: PTB and LTBI groups exhibit higher BfrB-specific IFN-g responses than HCs. Although BfrB is not as immunodominant as ESAT-6/CFP-10 during acute M.tb infection, comparable BfrB-specific cellular immune responses are observed in LTBI population with the potential to increase the sensitivity for LTBI screening. Moreover, strong BfrB-specific IFN-g release in the healthy cohort is probably cautionary in identifying leaky LTBI from HCs. BfrB might thus be considered as an additional biomarker antigen for LTBI identification. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Tuberculosis Latent tuberculosis infection BfrB Cellular response Immunodiagnosis

1. Introduction Tuberculosis (TB) remains one of the most contagious diseases worldwide. Based on World Health Organization (WHO) 2016 annual report, an estimated 10.4 million people developed TB and 1.5 million died from the disease [1]. TB begins with asymptomatic

* Corresponding author. Shanghai Jiao Tong University School of Medicine, Shanghai Institute of Immunology, Shanghai, 200025, China. ** Corresponding author. E-mail addresses: [email protected] (S.-h. Lu), [email protected] (Y. Wang). 1 Authors contributed equally. http://dx.doi.org/10.1016/j.tube.2017.08.005 1472-9792/© 2017 Elsevier Ltd. All rights reserved.

infection by a low number of Mycobacterium tuberculosis (M.tb). Most of the pathogens can be eliminated by host immune system. Some become dormant in alveolar macrophages leading to a status called latent TB infection (LTBI). With the alteration of metabolic and replication activity of M.tb under certain circumstances, LTBI transforms to active TB with clinical symptoms and spreading capacity [2]. Approximately one third of world population are infected with M.tb, 5%e15% of which suffer from the reactivation of M.tb during their lifetime [1]. Therefore, TB diagnosis and LTBI screening directly influence the global control of TB. At present identification of M.tb infection, including active TB and LTBI, is not satisfactory enough due to the lack of reliable

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methods with rapidity and accuracy. Three conventional methods for TB diagnosis are available in clinic, including acid-fast bacilli (AFB) smear, M.tb sputum culture and X-ray chest radiograph [3]. As a standard method for TB diagnosis, AFB smear with Ziehl-Neelsen (ZN) staining is positive only when there exist more than 5000 bacilli per mL in the sputum [3]. Bacilli culture positivity is a golden criterion for TB diagnosis. But it takes nearly one month with low success even in smear-positive sputum samples. Immunodiagnosis becomes valuable based on M.tb-specific immune responses. Tuberculin purified protein derivative (PPD) based tissue skin test (TST) assay has been used for screening M.tb infection with low cost and quickness for a long time. However, the cross-reaction between PPD derived from mycobacterial bacilli and the Bacillus Calmrin (BCG) vaccine leads to low specificity of TST assay in TB etteeGue diagnosis, which in turn limits its application as well [4]. Recently, immune effector molecules or cells induced by M.tb infection are considered to be reliable biomarkers for TB diagnosis and LTBI screening [5e8]. Interferon-g release triggered by mycobacterial antigens, including 6 kDa early secretary antigenic target (ESAT-6), 10 kDa culture filtrate protein (CFP-10) and TB10.4, are adapted in commercialized diagnostic kits, such as QFT-GIT and T-SPOT.TB, to detect TB and LTBI [9]. However, a recent meta-analysis including nineteen studies claimed pooled sensitivity of 75%e90% as well as specificity of 71%e77% [10], which is not competent enough for diagnosis. In view of low sensitivity of the available methods, it is suggested to add extra antigens to increase the sensitivity and specificity for distinguishing cases of M.tb infection [11]. Bacterioferritin B, known as Rv3841, is a ferritin-like protein identified by whole genome sequencing of M.tb H37Rv strain [12]. It functions to uptake iron and regulate the release of the stored iron [13]. A recent study indicates that after knockdown of lsr-2, which encodes a global transcriptional regulator of M.tb for the adaptation to hypoxia environment [14], there is 5.96 times upregulation of rv3841 expression compared to wildtype M.tb (data unpublished). However, the antigenicity of BfrB after M.tb infection is not well studied. Here we assessed BfrB specific cellular responses in human cohorts, including healthy controls (HCs), LTBI and pulmonary TB (PTB) patients to evaluate its potential as a novel biomarker antigen in TB-related diagnosis.

6 for 6e24 months. All the patients have signed voluntary informed consents. The healthy volunteers (n ¼ 266) have been defined as no medical history, a normal physical examination (including blood test, serum chemistry, chest X-rays) with no disease symptoms. LTBI was defined as positive in T-SPOT.TB assay among HC groups accordingly [11]. This study was approved by the Ethical Committee of Shanghai Jiao Tong University School of Medicine. 2.2. Preparation of M.tb antigens The recombinant plasmids were obtained from NIH Biodefense and Emerging Infection Research Resources Repository (NIAID, NIH, USA), including pMRLB.5 containing rv3841 (Protein BfrB) (NR13278), pMRLB.7 containing rv3875 (Protein ESAT-6) (NR-36431) and pMRLB.46 containing rv3874 (Protein CFP-10) (NR-13297) from M.tb. Target genes were subcloned into pET28a expressing vectors by PCR. The recombinant plasmids were transformed to E.coli BL21 (DE3) strain and the proteins were induced for 4 h with 0.5 mM Isopropyl-b-D-thiogalactoside (IPTG) (Beyotime, Jiangsu, China) at 30  C. Bacteria were lysed and proteins were purified by affinity chromatography with Ni-NTA His-Bind Resin (Qiagen, NRW, Germany) accordingly. Endotoxins were removed from purified antigens using Triton X-114 two-phase separation as previously described [15]. Briefly, Triton X-114 was added to the proteins to a final concentration of 1% and incubated for 30 min at 4  C with constant agitation, followed by 10 min incubation at 37  C and 16,000g centrifugation at 25  C for 10 min. Six cycles of Triton X114 phase separation were performed for sufficient endotoxin depletion. Triton X-114 was removed by dialysis against phosphate buffer saline (PBS). The remaining endotoxin in proteins was detected by Tachyleus Amebocyte Lysate Kits (Gulangyu, Xiamen, China) according to the manufacturer's protocol. Protein concentration was detected by BCA Protein Assay Kit (Pierce, Waltham, MA, USA). 2.3. Peptide synthesis

2. Materials and methods

A total of 19 peptides were synthesized by Sangon Biotech (Shanghai, China). These peptides consisted of 20 amino acid residues in length and covered the entire sequence of ESAT-6 and CFP10 with 10 amino acids overlapping.

2.1. Study subjects

2.4. Peripheral blood mononuclear cell isolation

All PTB patients and healthy volunteers were adults who vaccinated with the BCG Shanghai strain (Shanghai Institute of Biological Products Co. Ltd., Shanghai, China) during childhood. PTB patients (n ¼ 29) were in-patient patients from Shanghai Public Health Clinical Center, including 17 new onset patients and 12 patients undergoing standard treatment, 10 females and 19 males with the age median of 51 (21e83) (Table 1). PTB diagnosis was based on medical history, chest radiograph (X-ray and CT), acid-fast bacilli (AFB) smear and M.tb sputum culture. A standard anti-TB therapy, including isoniazid (INH), rifampicin (RFP), ethambutol (EMB), and pyrazinamide (PZA) was performed on 12 PTB patients, among which 6 patients were treated for 1e6 months and the other

10 mL peripheral blood was collected in tubes containing EDTA. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-hypaque density gradient centrifugation with Lymphoprep™ solution (AXIS-SHIELD Poc AS, Oslo, Norway) at 860g for 20 min at room temperature (RT). The supernatant (plasma) was collected and stored at 80  C. The mononuclear cell layer was carefully transferred to a new 15 mL conical tube and washed twice with RPMI 1640 (GIBCO, Grand Island, USA) by centrifuging at 480g for 10 min at RT. PBMCs were resuspended at a concentration of 2.5  106/mL in RPMI 1640 culture medium containing 10% fetal bovine serum (FBS) (Merck Millipore, Darmstadt, Germany), 100 units/mL penicillin (GIBCO) and 100 mg/mL streptomycin (GIBCO). 2.5. Interferon-gamma (IFN-g) ELISPOT assay

Table 1 Population information.

N Gender (Female/Male) Median age, years (range) N: sample size.

PTB

LTBI

HC

29 10/19 51 (21e83)

59 26/33 46 (18e76)

207 105/102 38 (18e73)

Antigen-specific IFN-g response was detected by an enzymelinked immunospot (ELISPOT) assay according to the manufacturer's instructions (U-CyTech, Utrecht, Netherlands). Briefly, 96well Polyvinylidene fluoride (PVDF) plates (Millipore) were coated with 50 ml anti-human IFN-g coating antibody overnight at 4  C. The wells were blocked with 200 ml blocking buffer for 1 h at

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37  C. 2.5  105 PBMCs in 100 ml culture medium were plated in each well and stimulated with ESAT-6 or CFP-10 peptide pool (2 mg/ mL per peptide), 10 mg/mL tuberculin PPD (Statens Serum Institut, Copenhagen, Denmark), ESAT-6, CFP-10 or BfrB antigens, respectively. Culture medium served as a negative control while 2.5 mg/mL phytohemagglutinin (PHA) treatment (Sigma, MO, USA) as a positive control. After 20 h incubation at 37  C, plates were incubated with 100 ml biotin-labeled detection antibody at 37  C for 1 h and subsequently 100 ml horseradish peroxidase (HRP)-conjugated streptavidin working solution for further 1 h. 100 ml AEC substrate solution was added for 30 min at RT in the dark. Color development was stopped by thoroughly rinsing both sides of PVDF membrane with demineralized water. Plates were dried by air in the dark at RT. The spots were counted by ELISPOT BioReader-4000 (BIO-SYS GmbH, Karben, Germany). The number of antigen-specific IFN-g producing cells was calculated by spot-forming units (SFUs) per 2.5  105 PBMCs with the deduction of SFUs from the paired negative control. Individuals that did not meet the criteria of positive (SFUs > 5) or negative (SFUs < 20) controls were ruled out. 2.6. Statistic analysis Data was represented by mean ± S.E.M. Statistical analyses were performed by GraphPad Prism 5.0 software (GraphPad Software Inc. CA, USA). Statistical differences were assessed by the unpaired Student t-test for the data with gaussian distribution and by MannWhitney test for those with non-gaussian distribution. The cut-off value for each antigen was ascertained using receiver operator characteristic (ROC) curve. ROC curves were constructed by plotting the true positive rate (sensitivity) against the false-positive rate (1specificity). To reduce the possibility of false positivity and false negativity, the optimal cut off values of BfrB-specific SFU level were chosen when the Youden index (YI) value was maximal.

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(18e76). The other 207 subjects were defined as healthy controls (HCs), including 105 females and 102 males with the age median of 38 (18e73). Accordingly, the percentage of LTBI in the health cohort was 22.18%, which is close to the estimation by WHO [16]. There was no significant gender or age preference between LTBI and HC subjects. 3.2. BfrB-specific cellular responses upon M.tb infection in human populations The quantities of BfrB-specific IFN-g producing cells in the periphery of PTB patients, LTBI and HC cohorts were compared by IFNg ELISPOT assay upon stimulation with purified BfrB protein in vitro. ESAT-6 and CFP-10 were used to determine M.tb-specific immune status while PHA and PPD were used as positive controls (Fig. 2A). Our results revealed that the levels of BfrB-specific IFN-g producing cells in PTB (25.03 ± 16.90 SFUs/2.5  105 PBMCs) and LTBI subjects (24.98 ± 39.33 SFUs/2.5  105 PBMCs) were comparable (p ¼ 0.9946). Both of them were significantly higher than that in HC group (11.29 ± 14.81 SFUs/2.5  105 PBMCs, p < 0.0001) (Fig. 2B). Interestingly, the number of BfrB-specific IFN-g producing cells (25.03 ± 16.9 SFUs/2.5  105 PBMCs) was significantly lower than that of ESAT-6 (94.17 ± 105.3 SFUs/2.5  105 PBMCs, p < 0.01) or CFP-10-specific IFN-g producing cells (94.11 ± 109.5 SFUs/2.5  105 PBMCs, p < 0.01) (Fig. 2C), which indicated that BfrB was less immunodominant when compared to ESAT-6 and CFP-10 in PTB patients. No correlations were observed between BfrB and ESAT-6 or CFP-10-triggered IFN-g responses (data not shown). These results indicate that M.tb infection is able to evoke BfrB-specific cellular immune responses whereas BfrB is not as immune dominant as ESAT-6 and CFP-10 under acute infection. 3.3. BfrB-specific cellular response improves the sensitivity of ESAT6 or CFP-10 in LTBI screening

3. Results 3.1. Screening of LTBI subjects from healthy volunteers IFN-g releasing assay targeting synthesized ESAT-6 (E6p) or CFP10 (C10p) peptide pools was performed in 266 healthy volunteers by ELISPOT to screen LTBI subjects from the healthy cohort [11]. LTBI was defined as those with more than 6 SFUs/2.5  105 PBMCs either specific to E6p or C10p (Fig. 1A and B). Among 266 individuals investigated (Table 1), 59 subjects were screened out as LTBI recommended by the manufacturer's instruction of T-SPOT.TB assay, including 26 females and 33 males with the age median of 46

We also compared the response levels between BfrB and ESAT6/CFP-10 in LTBI group. It was shown that LTBI subjects had a comparable BfrB-specific IFN-g releasing response (24.98 ± 39.33 SFUs/2.5  105 PBMCs) when compared to E6p (22.48 ± 28.41 SFUs/ 2.5  105 PBMCs) or C10p-specific (17.48 ± 25.36 SFUs/2.5  105 PBMCs) responses (Fig. 3A), which is different from what was observed in PTB group (Fig. 2C). Since BfrB was supposed to be upregulated under latency, what kind of correlation there existed between BfrB and E6p/C10p-specific cellular responses in LTBI group was further analyzed. LTBI was conventionally defined as those with SFU value greater than 6 in 2.5  105 PBMCs upon either

Fig. 1. Screening of LTBI from the healthy cohort. PBMCs from 266 healthy volunteers were stimulated with EAST-6 peptide pool (E6p) and CFP-10 peptide pool (C10p) for 20 h. No stimulant blank (BL) served as the negative control. The one who was positive against E6p or C10p stimulation was considered as an LTBI subject. Statistic results of E6p-specific (A) and C10p-specific (B) ELISPOT assay in LTBI (n ¼ 59) and HC (n ¼ 207) subjects. The percentage of LTBI in the health cohort was 22.18%.

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Fig. 2. Cellular immune responses targeting BfrB in three groups. (A) Representative results of ELISPOT assay specific to different stimuli. PBMCs from three individuals were stimulated with different antigens for 20 h. No stimulant blank (BL) served as the negative control. PHA and PPD served as the positive controls. (B) Statistic results of BfrB-specific cellular immune responses in PTB (n ¼ 29), LTBI (n ¼ 59) and HC groups (n ¼ 207). (C) Comparison between BfrB-specific and ESAT-6 or CFP-10-specific cellular immune responses in PTB patients. Statistic analysis of ESAT-6, CFP-10 and BfrB-specific IFN-g producing SFUs were performed in PTB patients. **: p < 0.01; ***: p < 0.0001.

Fig. 3. Complementarity of BfrB-specific IFN-g response to ESAT-6 or CFP-10 in LTBI subjects. (A) IFN-g ELISPOT results specific to E6p (n ¼ 58), C10p (n ¼ 58) and BfrB (n ¼ 59) in LTBI subjects. (B) Correlation analysis between BfrB-specific and E6p or C10p-specific responses in LTBI group. Colored plots were subjects with negative E6p response or C10p response but positive BfrB response (n ¼ 2 for E6p and n ¼ 13 for C10p). Plots with the same color from each side represented the same subject.

E6p or C10p stimulation. Accordingly, LTBI subjects could be subgrouped into E6p-positive and E6p-negative groups or C10ppositive and C10p-negative groups with 6 SFUs/2.5  105 PBMCs as a threshold. We found that most of the E6p- or C10p-negative LTBI subjects had high BfrB-specific responses. On the contrary, subjects with high responses to E6p or C10p exhibited low response to BfrB (Fig. 3B and C). However, the correlation coefficients between T cell responses to BfrB and the M. tb-specific antigens

was 0.03 for E6p (p ¼ 0.826) and 0.163 for C10p (p ¼ 0.220) which was not significant, and any possible negative association needs to be validated with more subjects. To investigate the significance of the antithetical pattern between BfrB and ESAT-6/CFP-10 specific responses, we further analyzed the diagnostic sensitivity of BfrB responses in LTBI group. Given 39.0% (23/59) positivity of BfrB-specific cellular immune responses, it is not recommended to use BfrB alone for LTBI

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identification. However, the effects of BfrB on improving sensitivity might be considerable. By adding BfrB, the sensitivity of LTBI screening using ESAT-6 could be improved from 81.4% (48/59) to 84.7% (50/59) while CFP-10 from 59.3% (35/59) to 81.4% (48/59). Two extra LTBI subjects were identified by BfrB response among 11 LTBI subjects unidentified by E6p, while 13 extra subjects were defined out of 24 leaking LTBI by C10p. These results strongly suggest the supplementary diagnostic significance of BfrB responses in LTBI identification. 3.4. High BfrB-specific IFN-g response is correlated with strong PPD responsiveness in HC cohort with E6p/C10p borderline value When we removed 59 LTBI subjects from 266 healthy cohorts, unexpectedly we found that BfrB-specific IFN-g secretion in 207 HCs was dramatically high (11.29 ± 14.81 SFUs/2.5  105 PBMCs) when compared to either E6p (0.42 ± 2.4 SFUs/2.5  105 PBMCs, p < 0.0001) or C10p (0.26 ± 1.97 SFUs/2.5  105 PBMCs, p < 0.0001) specific responses (Fig. 4A). To elucidate the significance of high BfrB-specific IFN-g secretion in HC group, we divided them into E6p/C10p borderline (SFUs of E6p or C10p/2.5  105 PBMCs ¼ 3, 4 or 5) and negative (SFUs of E6p or C10p/2.5  105 PBMCs < 3) groups. BfrB-specific IFN-g secretion was further compared between two subgroups. It was found that E6p/C10p borderline group exhibited higher BfrB-specific IFN-g response (n ¼ 53, 17.98 ± 2.470 SFUs/2.5  105 PBMCs) than negative group (n ¼ 154, 13.35 ± 1.076 SFUs/2.5  105 PBMCs) (p < 0.05) (Fig. 4B). E6p/C10p borderline group was further divided into BfrB-positive and BrfB-negative subjects based on SFU value more than 17.5 (defined in “Methods and Materials”). The percentage of BfrBpositive subjects in ESAT-6/CFP-10 borderline subgroups was 20.75% (11/53). PPD-specific responses were compared between these two groups. Interestingly, BfrB-positive subjects (n ¼ 10) generated more PPD-specific IFN-g responding cells (93.70 ± 45.35 SFUs/2.5  105 PBMCs) than BfrB-negative group (n ¼ 43) (42.77 ± 31.75 SFUs/2.5  105 PBMCs) (p < 0.05) (Fig. 4C). Considering that PPD based TST assay is still valuable to screen out M.tb infection, our results imply that BfrB might serve as an additional antigen to identify latent M.tb infection from the healthy cohort besides ESAT-6/CFP-10. 4. Discussion M.tb is one of the most ancient human pathogen identified with infectious and spreading ability [17] where approximately one third

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of world population are infected. The vast majority of infected individuals become LTBI, among which 5%e15% progress into active TB disease [1]. Accurate diagnosis and precision treatment of active TB as well as identification and prevention of LTBI transforming into active TB are critical for TB control. In the present study, we have authenticated a mycobacterial ferritin-like protein BfrB capable of inducing moderate cellular responses in TB patients and LTBI individuals, suggesting its biomarker potential in LTBI screening. It has been well established that IFN-g, primarily from CD4þ and CD8þ T cells, plays a protective role in defending against M.tb infection and largely influences the disease outcome with other cytokines [18]. Based on this, IGRA is applied to identify M.tb infection in clinic. Antigens currently used in IGRA are ESAT-6, CFP10 and TB10.4, originally identified in short-term culture filtrate of M.tb with strong T cell antigenicity. In spite of the exclusive expression of ESAT-6 and CFP-10 in M.tb without cross-reaction to BCG or most nontuberculous mycobacterium (NTM) strains, there still exist undefined TB subjects both in QFT-GIT and T-SPOT.TB assays [19]. In this study, we found that BfrB specific cellular responses in PTB patients and LTBI individuals were significantly higher than that in HCs, which implies the diagnostic potential of BfrB upon M.tb infection. However, BfrB does not induce IFN-g response as strong as ESAT-6/CFP-10 in PTB patients, indicating that BfrB is not an immunodominant antigen like ESAT-6 or CFP-10 during acute M.tb infection. But in LTBI population, similar BfrB-specific response was observed when compared to ESAT-6 or CFP-10 (Fig. 3A) implying the comparable antigenicity of BfrB under latency. The complementarity of IFN-g responsiveness between BfrB and ESAT-6 or CFP-10 is quite impressive. In fact, there are several reports described the immune dominancy shift of mycobacterial antigens under latent infection. For example, Schuck et al. found that several latency-associated antigens such as Rv1733c, Rv2003, Rv2005c could induce significantly higher T-cell response in LTBI as compared to TB patients [20e22]. These studies, together with our results presented here, strongly suggest that mycobacterial antigens applied in LTBI screening or vaccine development might be different from the present strategy used for active TB. In addition, the sensitivity of ESAT-6 and CFP-10 specific responses is improved separately by BfrB, indicating the supplementary diagnostic significance of BfrB responses in LTBI identification. More interestingly, we observed in the HC group, where LTBIs were excluded, that some with ESAT-6/CFP-10 borderline responses also had higher BfrB-specific IFN-g release level. Considering the possibility of strong immune dominance of BfrB under latency, we

Fig. 4. BfrB-specific cellular responses in LTBI-excluded HC cohort. (A) E6p, C10p and BfrB-specific ELISPOT assays in HC group (n ¼ 207). (B) Comparison of BfrB-specific IFN-g responses between E6p/C10p borderline (n ¼ 53) and negative subjects (n ¼ 154). (C) Comparison of PPD-specific IFN-g SFUs between BfrB-positive and BfrB-negative individuals in E6p/C10p borderline subgroup. *: p < 0.05; ***: p < 0.0001.

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speculate that the individuals with strong BfrB responses might be leaky LTBI unsorted out by ESAT-6/CFP-10. To verify this hypothesis, we simultaneously determined the cellular responses targeting PPD in vitro. Among ESAT-6/CFP-10 borderline subgroup, individuals with high BfrB-specific responses likewise have higher PPD responses when compared to individuals with low responses. PPD is used in TST assay for the screening of previous M.tb infection based on the remaining delayed-type hypersensitivity (DTH) response. It can not be excluded that high BfrB responses in nonLTBI HCs identified by IGRA might be the indicator for the remaining responsiveness to BCG vaccination, since several mycobacterium strains, including M. canettii, M. riyadhense, M. bovis, M. pseudoshottsii etc. contain ferritin-like superfamily member with the sequences more than 90% homology. Nevertheless, based on our results from PPD stimulation we still intend to propose that in addition to ESAT-6/CFP-10, BfrB might serve as an additional and supplemental antigen to identify LTBI subjects from the healthy cohort, especially when those who are low responsive to ESAT-6/ CFP-10. Besides, we will further use a quantitative analysis to determine HLA class I and class II-restricted T cell epitopes based on the strategy reported by Cecilia S. [23] to optimize the values of BfrB in LTBI identification and vaccine development as well. It is quite interesting that BfrB-specific IFN-g responses exert different patterns among three populations investigated, which might reflect the patterns of immunodominant antigens under different situations. As an intracellular protein, BfrB plays key roles in iron storage and supply as well as in protection against ironmediated oxidative stress [24,25]. Hypoxic conditions are generally believed to be one of the environmental features in the granuloma or when the pathogens are dormant inside macrophages. M.tb bacilli slow down their metabolism as well as stop replication, and transit into a dormancy status [26]. With distinct proliferation manner and protein expression profile in latent phase [2], LTBI individuals exert different antigen-specific immune responses owing to the alternative antigen expression profiles at this unique stage of M.tb life cycle. The overexpression of rv3841 during hypoxic conditions [27,28] exactly corresponds with the high BfrB immune response in the latent phase reported here. Based on our observation of the complementary BfrB-specific IFN-g responses to ESAT-6/ CFP-10 in LTBI population, it is supportive that BfrB-specific IFN-g response might have a diagnostic value in combination with ESAT6/CFP-10 for latent M.tb infection screening. Immune profiles targeting BfrB might provide a new clue for discriminating different phases of infection. The exact host-pathogen crosstalk at different stages mediated by BfrB needs further investigation. In summary, BfrB induces comparable cellular immune responses during acute and latent M.tb infection. Since BfrB-specific IFN-g release is significantly higher in LTBI than HC group, this may provide an additional biomarker to distinguish LTBI from HC. In addition, BfrB-specific IFN-g release might be potential in identifying leaky LTBI from the HC cohort. BfrB thus can be considered as a novel target antigen for TB diagnosis and LTBI screening as well as a vaccine candidate against LTBI. Grant support This work was supported by grants from the Key Project Specialized for Infectious Diseases of the Chinese Ministry of Health (2017ZX10301301-001-004 and 2013ZX10003007-003-003) and the National Science Foundation of China (81501361). Conflict of interest All the authors declared no conflict of interest.

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