Natural T Cell Epitope Containing Methyl Lysines on Mycobacterial Heparin-Binding Hemagglutinin

Most naturally occurring T cell epitopes presented by class I or class II MHC molecules consist of nonmodified linear peptides. Nevertheless, certain posttranslational modifications of peptide epitopes have been described to be displayed by MHC molecules. These include O- and N-linked glycosylation, acetylation, phosphorylation, deamination, and nitration (for review see Engelhard et al. 1). Arginine methylation of MHC class I–associated peptide epitopes has also been described (2). However, so far, no peptide naturally containing methyl lysines has been described to be presented by either class I or class II MHC molecules. In addition, most described MHC-associated posttranslationally modified peptides originate from viral or self-proteins (1). Their posttranslational modifications rely on the enzymatic activities located in different subcellular compartments of the APCs. No bacterial T cell epitope with a naturally occurring posttranslational modification that is not generated by cellular enzymes has yet been described.

In this article, we describe a methylated T cell epitope present in the mycobacterial heparin-binding hemagglutinin (HBHA). This protein Ag is recognized by peripheral T cells from the majority of subjects latently infected with Mycobacterium tuberculosis (LTBI) but much less by T cells from patients with active tuberculosis (TB) (3−5). As such, it has been proposed as a diagnostic tool for the detection of latent infection with a documented sensitivity and specificity of over 90% (6). In addition, HBHA has been shown to provide protection against M. tuberculosis in mouse models at levels similar to those provided by the Bacille Calmette-Guérin (BCG), the only currently available vaccine against TB (4, 7, 8).

Detailed molecular characterization has revealed that HBHA is a methylated Ag, with an exceptionally complex methylation pattern in the C-terminal region of the protein (9). This region is composed of several lysine-rich repeats (10), which may be mono- or dimethylated (9) as illustrated on Fig. 1A. In fact, native HBHA (nHBHA) produced by the members of the M. tuberculosis complex is a combination of proteins with various degrees of lysine methylation. The major protein contains 25 methyl groups. The methylation is of crucial importance for the immunological and protective properties of HBHA (4). Recombinant HBHA produced in Escherichia coli (rHBHA-Ec) is not methylated and is much less powerful as a diagnostic or vaccine Ag than nHBHA (4, 8). As an alternative to E. coli, fast growing Mycobacterium smegmatis has also been used to produce recombinant HBHA (recombinant HBHA produced in M. smegmatis; rHBHA-Ms), and rHBHA-Ms was found to be methylated by the methyltransferases from M. smegmatis. However, even though some LTBI subjects have been reported to respond to rHBHA-Ms by secreting IFN-γ (11), rHBHA-Ms, like rHBHA-Ec, is not protective in mouse models (8), indicating important biological differences between rHBHA-Ms and nHBHA.

In this study, we analyzed in depth the differences in the immunological properties between nHBHA and rHBHA-Ms, which led us to identify a peptide containing methyl lysines critical for the T cell recognition of HBHA by subjects with LTBI. These results demonstrate that peptides methylated by posttranslational modification can be recognized by human T cells to induce a cellular immune response.

Fourteen LTBI subjects and 18 controls not infected with M. tuberculosis were included in this study approved by the Ethics Committee Erasme-ULB (aggregation number OMO21, study protocol P2007/175), and they all gave their written informed consent. All the subjects were health care workers (HCW) at the Hôpital Erasme in Belgium, a low TB incidence country with no systematic BCG vaccination. Nevertheless, 86% of the LTBI subjects and 50% of the controls were vaccinated more than 20 y before their inclusion in the study. The main demographic and clinical characteristics of the subjects are given in Table I. The noninfected controls were selected among healthy subjects with, at the time of their inclusion in the study, both a negative tuberculin skin test (TST) and the absence of in vitro responses of their PBMC to purified protein derivative (PPD). The LTBI subjects were selected among a cohort of known LTBI HCW, and their detailed clinical characteristics are given in Table II. They were identified as LTBI either recently (<2 y) or several years before their inclusion in the study (up to 25 y, Table II), according to the results of their TST performed annually by the intradermal injection of two tuberculin units of PPD-RT23 (Statens Serum Institute, Copenhagen, Denmark). They were identified following a clearly positive TST result according to the Center for Disease Control guidelines (12) (≥10 mm induration as HCW are exposed to M. tuberculosis; range, 10–40 mm), most often (10/14) with a clear conversion of their TST result from negative to positive, and they all had normal chest radiography and no clinical symptoms. Although QuantiFERON-TB Gold in-Tube (QFT) is not recommended in Belgium as a reference for LTBI screening, it was performed for research purposes for all the LTBI subjects at the time of their inclusion in the study. The technique was applied as recommended by the supplier (Qiagen Benelux, Antwerp, Belgium). All but one of the LTBI subjects had a negative QFT result, confirming the lower sensitivity than previously assumed of the QFT for LTBI (6, 13, 14).

As the hbhA gene from Mycobacterium bovis BCG is 100% identical to that from M. tuberculosis (15), nHBHA was purified from M. bovis BCG (strain 1173P2; World Health Organization) grown in static cultures at 37°C using 175 cm³ Roux flasks containing ∼200 ml of Sauton medium. At stationary phase, the cultures were centrifuged (10,000 × g for 20 min), heat inactivated, and sonicated as described (16). nHBHA was purified by heparin–Sepharose chromatography (16), followed by reverse-phase high-pressure liquid chromatography (3), and then stored at −80°C until further use. rHBHA-Ms was purified from M. smegmatis mc²155 after insertion of the BCG hbhA gene into the M. smegmatis genome and its expression under the control of a strong acetamide-inducible promoter as described (17). rHBHA-Ec was purified from E. coli BL21(DE3) containing pET-HBHA (rHBHA-Ec) as described (18). Both rHBHA-Ms and rHBHA-Ec were purified from centrifuged bacteria with the same protocol as for nHBHA. The three HBHA molecules preparations were quality controlled by SDS-PAGE and Coomassie Brilliant Blue staining and were subjected to mass spectrometry analysis after digestion of their C-terminal domain with endo-Glu, as described (9).

HBHA– IFN-γ release assays (IGRAs) and PPD-IGRAs were performed on PBMC purified from fresh heparinized blood samples and in vitro stimulated during 96 h with the indicated amounts of the different molecular forms of HBHA or with 4 μg/ml PPD as described (6). The IFN-γ concentrations in cell culture supernatants were then measured by ELISA as described (6, 19, 20).

The cell lysate obtained from BCG, recombinant M. smegmatis, or E. coli strains were analyzed by immunoblotting using the three anti-HBHA mAbs. The Ags (200 ng) were loaded onto a 12% polyacrylamide gel, and the proteins were, after electrophoresis, transferred onto three separate nitrocellulose membranes (Hybond-C; Amersham Pharmacia Biotech). After a blocking step (3% BSA-containing PBS–Tween), anti-HBHA mAb 3921E4, 4057D2 (21), or 5F2 (22) was added for 1 h, before the addition of alkaline phosphatase-labeled anti-mouse Ab (1 h, Promega) and staining.

ELISA plates were coated overnight at 4°C with one μg/well of nHBHA, rHBHA-Ms, or rHBHA-Ec. After washing and blocking with PBS–Tween containing 3% BSA, serial 2-fold dilutions of the mAbs in PBS–Tween containing 3% BSA were incubated for 90 min at room temperature. After washing, the plates were further incubated for 1 h at room temperature with peroxidase-labeled anti-mouse Abs, followed, after further washing, by the addition of the tetramethylbenzidine substrate. The reaction was stopped by the addition of 50 μl H₃PO₄ and the absorbance was read at 450 nm.

To map the epitopes recognized by mAb 4057D2, 31 18-aa long peptides with a 6-residue offset, covering the whole HBHA sequence, were synthesized using standard Fmoc/tert-butyl solid phase methods as described (23). They were purified by HPLC, characterized by mass spectrometry, and printed in triplicate at 0.1 mM as a peptide array onto glass slides using a Perkin Elmer BioChip Arrayer 1 (Perkin Elmer, Wellesley, MA) (17). The peptides were in situ methylated using formaldehyde (17), and nHBHA was used as a control and printed at 160 μg/ml. The slides were incubated 1 h at room temperature with mAb 4057D2 before the addition of FITC-linked anti-mouse Abs and reading of the slides in a microarray fluorescence reader as described (17).

Peptide no. 26 was used in its nonmethylated form and as a basis for the synthesis of four differently methylated peptides. The methylated peptides and nonmethylated peptide were purchased from Proimmune with a purity recorded as being between 96 and 100% depending on the peptide. As IFN-γ produced in response to HBHA is recognized as one of the biomarkers of protection against active TB, we evaluated the peptides for recognition by PBMC from LTBI subjects in a 96-h IGRA as described above, the peptides being used at 10 μM.

Statistical analyses were performed using the GraphPad Prism Software version 7.0b for Windows (GraphPad Software, San Diego, CA, www.graphpad.com). Comparisons of IFN-γ secretions obtained with the different forms of HBHA were performed with the nonparametric Kruskal–Wallis test combined with the Dunn multiple comparison, and comparisons obtained for the same subjects with nHBHA and different concentrations of rHBHA-Ms were performed with the nonparametric paired Friedman test. A value of p < 0.05 was considered significant. Correlations between IGRA results obtained with the different molecular forms of HBHA were analyzed by the nonparametric Spearman test.

In contrast to M. tuberculosis or M. bovis BCG, M. smegmatis is a fast growing Mycobacterium. To facilitate HBHA production, the M. tuberculosis hbhA gene was introduced into the M. smegmatis genome and expressed under the control of a strong acetamide-inducible promoter. As shown in Fig. 1B, high levels of HBHA were produced in recombinant M. smegmatis upon acetamide induction, as evidenced by the presence of a major protein with the expected apparent molecular mass of ∼28 kDa visible by Coomassie Brilliant Blue staining after SDS-PAGE. The protein was then purified and subjected to mass spectrometry analysis after digestion of the C-terminal domain with endo-Glu. The resulting spectrum was compared with those of endo-Glu–digested nHBHA and rHBHA-Ec (Fig. 1C). The spectra of rHBHA-Ms (Fig. 1C1) and of nHBHA (Fig. 1C2) showed a similar complex pattern, unlike that of rHBHA-Ec (Fig. 1C3), indicating that similar to nHBHA, rHBHA-Ms had also undergone posttranslational methylation in its C-terminal domain, in contrast to the nonmethylated rHBHA-Ec. However, although the peak of highest intensity for nHBHA was at 4425 d, the highest peak for rHBHA-Ms was at 4355 d. This corresponds to a median difference of ∼5 methyl groups between the two molecular forms, indicating that rHBHA-Ms is less methylated than nHBHA.

We used the standardized nHBHA IGRA previously developed (6) to evaluate the recognition of rHBHA-Ms by blood lymphocytes from LTBI subjects described in Table I and Table II. Using this assay, we compared nHBHA with rHBHA-Ms and rHBHA-Ec, the latter being known to be poorly recognized by human T lymphocytes (4). IFN-γ concentrations secreted by the PBMC from LTBI subjects were significantly lower in response to rHBHA-Ms and rHBHA-Ec than in response to nHBHA (p = 0.0001 and 0.0012, respectively) (Fig. 2A). All three molecular forms of HBHA were used at 2 μg/ml, as defined previously for nHBHA-IGRA (6). Surprisingly, rHBHA-Ms did not induce more IFN-γ than nonmethylated rHBHA-Ec. However, when 25 μg/ml rHBHA-Ms was used in the assay instead of 2 μg/ml, higher amounts of IFN-γ were secreted at 25 μg/ml than at 2 μg/ml rHBHA-Ms (medians, respectively, of 200 and 35 pg/ml at 25 μg/ml and 2 μg/ml rHBHA-Ms) (Fig. 2B). This comparison was possible for 11 out of the 14 LTBI subjects, because of insufficient PBMC recovery for the remaining three subjects. The median IFN-γ secretions induced by 2 μg/ml nHBHA and those induced by 25 μg/ml rHBHA-Ms were no longer significantly different, albeit still generally lower in response to rHBHA-Ms than nHBHA (medians, 200 and 2511 pg/ml for rHBHA-Ms and nHBHA, respectively). However, the IFN-γ concentrations released in response to 25 μg/ml rHBHA-Ms were very heterogenous, and by comparing the IFN-γ responses to 2 and 25 μg/ml rHBHA-Ms, we could identify three different patterns of responses: the PBMC from two LTBI subjects secreted substantial IFN-γ levels only in the presence of high rHBHA-Ms concentrations (200 and 1223 pg/ml for subject no. 7 and 11 in Table II, respectively, indicated by squares in Fig. 2B), whereas the IFN-γ concentrations remained low at both concentrations for the PBMC from five LTBI subjects (no. 1, 5, 8, 9, 14, represented by circles in Fig. 2B), and four subjects (no. 2, 6, 12, 13) secreted significant IFN-γ levels at both concentrations of rHBHA-Ms (triangles in Fig. 2B). Of note, the only individual with a positive QFT result at the time of testing belongs to this third group (i.e., responded to both nHBHA and rHBHA-Ms; no. 13). As indicated in Fig. 2C, the six LTBI subjects who secreted IFN-γ in response to high concentrations of rHBHA-Ms independently of their response to the lower concentration were mostly those with the highest IFN-γ responses to nHBHA. Four of them also secreted IFN-γ in response to rHBHA-Ec, the five LTBI subjects evaluated for their response to rHBHA-Ec among those illustrated on Fig. 2C being represented by a star.

As the optimal concentrations of HBHA to be used in the IGRA appears thus to vary according to the molecular form of the Ag, dose-response curves (from 0 to 25 μg/ml) were established for each of the three forms, comparing the values obtained for LTBI subjects with those obtained for the noninfected controls. The data shown in Fig. 3 confirm that 2 μg/ml is the optimal concentration for nHBHA to provide the best discrimination between LTBI subjects and controls in the HBHA-IGRA, whereas higher concentrations are needed to increase the sensitivity of rHBHA-Ec and rHBHA-Ms to detect LTBI subjects, up to concentrations that, however, also resulted in false positives among the noninfected subjects.

These results thus indicate that even though rHBHA-Ms displays a complex methylation pattern in its C-terminal part, as shown by mass spectrometry analyses, the differences that do exist between these two forms of the Ag may account for their differences in sensitivity for LTBI detection, as even at high concentrations, rHBHA-Ms induced substantial IFN-γ secretions only in 6 of 11 LTBI subjects, when a cut-off value of 100 pg/ml IFN-γ is used. In contrast, all 11 LTBI subjects responded to nHBHA, even at 2 μg/ml. No clear clinical differences were identified between the LTBI subjects responding to both or to only one molecular form of HBHA, nor between those responding only to high rHBHA-Ms concentrations and not to the lower concentrations.

Although the mass spectrometry analyses have revealed quantitative differences in the median numbers of methyl lysines between nHBHA and rHBHA-Ms, these analyses did not allow us to precisely identify the critical methylated peptide that may result in the different antigenic properties of the two forms of HBHA. To further characterize the molecular structures that may help us to understand these differences, we made use of three anti-HBHA mAbs. mAbs 3921E4 and 4057D2 have previously been shown to recognize the methylated C-terminal part of nHBHA (16, 21). Using overlapping peptides spanning the entire HBHA molecule, we identified in this study the epitope recognized by the third mAb, 5F2, just upstream of the C-terminal methylated portion of the protein (Supplemental Fig. 1). Immunoblot analyses showed that, as expected, rHBHA-Ec was only recognized by mAb 5F2 and not by mAb 4057D2, nor by mAb 3921E4 (Fig. 4A, lanes 2). In contrast, nHBHA was recognized by the three mAbs (Fig. 4A, lanes 1), whereas rHBHA-Ms was well recognized by mAbs 5F2 and 3921E4 but not by mAb 4057D2 (Fig. 4A, lanes 3).

To quantify the recognition of the different forms of HBHA by mAbs 3921E4 and 4057D2, the Abs were tested in 2-fold serial dilutions by ELISA on the three molecular forms of HBHA. mAb 3921E4 recognized nHBHA slightly better than rHBHA-Ms, and mAb 4057D2 recognized nHBHA almost 100-fold better than rHBHA-Ms. rHBHA-Ec was recognized by neither mAb 3921E4 nor mAb 4057D2 (Fig. 4B). These observations indicate that the epitope recognized by mAb 4057D2 constitutes an important structural difference between nHBHA and rHBHA-Ms, which might explain the difference in T cell antigenicity by the two forms, as evidenced by the HBHA-IGRAs on LTBI subjects.

Given the importance of the epitope recognized by mAb 4057D2 in the characterization of the molecular difference between nHBHA and rHBHA-Ms, we mapped the epitope using overlapping peptides of HBHA spotted on glass slides and in situ methylated. None of the peptides were recognized by mAb 4057D2 before in situ methylation (data not shown). In contrast, a limited number of peptides corresponding to the C-terminal domain of HBHA were readily recognized by mAb 4057D2 after in situ methylation (Fig. 5A). However, whereas all the methylated peptides corresponding to the C-terminal part of nHBHA (P26 and beyond) were previously reported to be strongly recognized by mAb 3921E4 (17), only one peptide (P26) was strongly recognized by mAb 4057D2. This peptide corresponds to the junction between the middle region of HBHA and its C-terminal, lysine-rich repeat region (Fig. 5B). The peptides downstream of P26 were only weakly recognized by mAb 4057D2 (Fig. 5A). No peptide upstream of P26 was recognized by mAb 4057D2, even after in situ methylation. This recognition pattern of the methylated peptides by mAb 4057D2 suggests that methylated P26 corresponds to at least one of the epitopic differences between rHBHA-Ms and nHBHA.

To determine whether a T cell epitope recognized by lymphocytes from LTBI may be present within methylated P26, we evaluated the capacity of P26 with different methylation patterns to induce IFN-γ secretion by the PBMC from LTBI subjects. Various P26 versions were thus synthesized, varying by the degree of lysine methylation as depicted in Fig. 6, and nHBHA was used as a positive control. The PBMCs from a LTBI subject (no. 12 in Table II), who responded to 2 μg/ml nHBHA and rHBHA-Ms by a similar level of IFN-γ secretion did not secrete significant amounts of IFN-γ in response to any form of P26, regardless of its methylation profile, indicating that the HBHA-induced IFN-γ secretion in this subject was not due to the recognition of P26 (Fig. 6A). In contrast, the PBMC from three other LTBI subjects (no. 4, 7, and 13 on Table II), who responded very poorly to rHBHA-Ms compared with nHBHA, secreted significant IFN-γ concentrations in response to one or several of the methylated peptides, but did not respond or responded poorly to nonmethylated P26 (Fig. 6B–D). Interestingly, one of the subjects (no. 4) responded only significantly to one of the methylated peptides, whereas the two others responded to several of them. The responses to at least one of the methylated peptides were generally as strong as the response to the entire nHBHA Ag. These results thus indicate that the precise methylation of P26 is important for the recognition of HBHA by T cells from some LTBI subjects and that naturally occurring peptides containing methyl lysines are able to induce a T cell response in humans upon bacterial infections, such as infection by M. tuberculosis.

This report describes the first head-to-head comparison, to our knowledge, of the cellular immune responses induced by nHBHA and rHBHA-Ms in humans. Based on previously documented induction by nHBHA of IFN-γ secretion by lymphocytes from LTBI subjects, we selected most subjects (13/14) among either professionally M. tuberculosis–exposed subjects (10/13) and/or people originating from or having recently traveled to a high-TB endemic country (4/14) with a definite diagnosis of LTBI. The diagnosis was based on a positive TST according to the Center for Disease Control criteria, with, in addition, a TST conversion for 10 of 14 subjects. A TST conversion was easy to determine, as the LTBI subjects were all recruited among Belgian HCW who are subjected to a TST every year. We did not use the results of the QFT test as criteria for LTBI diagnosis, as this is not in the official guidelines in Belgium. Moreover, it is well known that in low-endemic countries, QFT is not sensitive enough to identify all LTBI subjects (6, 13, 14).

Our results indicate that, in contrast to previous reports showing that humoral immune responses were similarly induced by nHBHA, and rHBHA-Ms (8), unlike rHBHA-Ec, rHBHA-Ms did not induce stronger IFN-γ secretion by PBMCs from LTBI subjects than the nonmethylated rHBHA-Ec, except for some LTBI subjects when high rHBHA-Ms concentrations were used to stimulate the PBMC. The IFN-γ secretion induced by rHBHA-Ms was heterogenous, which allowed us to classify the LTBI subjects in three different groups according to the IFN-γ responses of their PBMC to nHBHA and to rHBHA-Ms: 1) those with similar IFN-γ secretions induced by both molecular forms of HBHA, 2) those with IFN-γ secretion induced only by high concentrations of rHBHA-Ms, and 3) those with no IFN-γ response to rHBHA-Ms regardless of the concentration used for the in vitro stimulation, but who secreted IFN-γ in response to nHBHA. No obvious epidemiological or clinical differences were identified between these three groups. However, the IFN-γ responders to rHBHA-Ms were all strong IFN-γ responders to nHBHA, as well as to rHBHA-Ec, suggesting that the repertoire of the epitopes targeted by their T lymphocytes is larger than those of the other LTBI subjects and that it includes nonmethylated peptides. The fact that the IFN-γ response of some LTBI subjects was stronger when their PBMCs were incubated with 25 μg/ml nHBHA-Ms instead of 2 μg/ml (group 2) suggests that some potentially methylated epitopes from nHBHA are under-represented in rHBHA-Ms. However, at high concentrations, even some of the noninfected subjects started to respond to rHBHA-Ms. In addition, even with stimulation by high concentrations of rHBHA-Ms, the PBMCs from some LTBI subjects still did not secrete any detectable IFN-γ, although they responded to 2 μg/ml nHBHA (group 3), suggesting that their response relies on the precise methylation pattern that differs between the two forms of HBHA. These observations should be taken into account when different forms of HBHA, such as rHBHA-Ms are proposed for in vitro diagnostic tests in IGRA for the detection of LTBI subjects.

Mass spectrometry analyses have shown that, like nHBHA, rHBHA-Ms also underwent a complex methylation process of its C-terminal region, but they have also revealed quantitative differences between nHBHA and rHBHA-Ms. The median m.w. of the C-terminal domain of rHBHA-Ms is smaller than that of nHBHA, a difference that may correspond to a difference of five methyl groups between the two forms of the molecule. Using different anti-nHBHA mAbs, we noticed that one of them, mAb 4057D2, specifically recognized nHBHA, as demonstrated by immunoblot analysis and confirmed by ELISA, which showed that mAb 4057D2 reacted with nHBHA ∼100-fold better than with rHBHA-Ms. By peptide scan analysis using in situ lysine methylation, we identified P26 as the peptide specifically recognized by mAb 4057D2, but only in its methylated form. This peptide is located at the junction between the N-terminal nonmethylated moiety and the C-terminal methylated region of nHBHA. It contains the first methyl lysine identified in nHBHA (9). M. smegmatis also naturally produces a HBHA homolog (15), but interestingly, the region corresponding to P26 is the most divergent region between the two M. smegmatis and M. tuberculosis, and the first methylated lysine residue in the M. tuberculosis HBHA is replaced by arginine in M. smegmatis (Supplemental Fig. 2). It is therefore conceivable that the M. smegmatis methyltransferases may not or only poorly recognize P26 and may not be able to properly methylate its lysine residues.

The peptide recognized by mAb 4057D2, but only in its methylated form, is also a target for the PBMC of some LTBI subjects, in particular subjects whose PBMC secrete substantial amounts of IFN-γ in response to nHBHA, but not to rHBHA-Ms. In contrast, methylated P26 did not induce IFN-γ secretion by PBMCs from a LTBI subject that respond to nHBHA and rHBHA-Ms equally well. The proper methylation profile of peptide P26 appears thus to be a functionally important difference between nHBHA and rHBHA-Ms, and may be the molecular basis of at least one reason for the lower IFN-γ response induced by rHBHA-Ms compared with nHBHA for most LTBI subjects. Interestingly, the methylation profile of P26 corresponds to the major form of nHBHA (9). Among the tested LTBI subjects who responded to methylated P26, there was heterogeneity. One subject recognized only one of the methylated peptides, whereas the two others recognized several differently methylated peptides, and one of them recognized all the methylated peptides. Whether this reflects different T cell subsets, each recognizing a specifically methylated peptide, or to a single T cell subset with relaxed specificity with respect to the methylation profile remains to be investigated.

The findings reported in this study may have important biological implications, as they may help to understand why rHBHA-Ms provides no protection against M. tuberculosis infection in a murine aerosol challenge model (8), whereas several studies have shown that vaccination with nHBHA provides protection in mice at levels that approach those induced by BCG vaccination (4, 8, 24). In addition, these findings are important in the context of the use of HBHA as a diagnostic tool in HBHA-IGRAs. To optimize the specificity/sensitivity of HBHA-based IGRAs, it appears thus crucial to use the properly methylated form of the Ag.

A limitation of this study is the low number of LTBI subjects included in this head-to-head comparison with the different molecular forms of HBHA. Nevertheless, the study demonstrates that peptides posttranslationally methylated at their lysine residues may be recognized by human T cells and may induce a T cell response upon natural M. tuberculosis infection and that this response is stronger than to the corresponding nonmethylated peptide.

We thank all study participants involved in this study, most being HCW who spontaneously accepted to participate to this study.

The authors have no financial conflicts of interest.