METHODS FOR THE DIAGNOSIS OF BURULI ULCER

Abstract

The present invention relates to the diagnosis of Buruli ulcer. The inventors investigated the potential role of local natural antibodies in the recognition of mycolactone produced by M. ulcerans, and confirmed the presence of such antibodies in humans. In particular, the present invention relates to a method of diagnosing Buruli ulcer in a subject comprising detecting anti-mycolactone immunoglobulin (anti-mycolactone IgG) in a biological sample obtained from said subject.

Claims

1. Method of diagnosing and treating Buruli ulcer in a subject comprising detecting anti-mycolactone immunoglobulin (anti-mycolactone IgG) in a biological sample obtained from said subject, and if anti-mycolactone IgG is detected, then administering one or more antibiotics to the subject.

2. The method of claim 1 wherein the sample is a skin tissue.

3. The method of claim 1 wherein the detection and quantification of anti-mycolactone IgG in the sample is performed by ELISA.

4. The method of claim 1 wherein the detection and quantification of anti-mycolactone IgG is performed with a kit or device comprising at least a mycolactone or fragments thereof; at least one solid support wherein the mycolactone or fragments thereof is deposited on the support and a detection antibody, wherein the detection antibody is specific for the anti-mycolactone IgG in the sample of the subject and the detection antibody produces a detectable signal.

Description

FIGURES

[0040] FIG. 1: Detection of cutaneous IgG binding to mycolactone. Each well of an ELISA Maxisorp plate was coated with 3 ng mycolactone, and the immunoglobulin concentrations of cutaneous tissues samples were normalized before their addition to the plate. Antibodies binding to mycolactone on the plate were recognized by HRP-conjugated secondary antibodies. A) Kinetics of the recognition of the mycolactone present in cutaneous tissue by cutaneous IgG (n=5 for each mouse strain). B) Kinetics of cutaneous IgG1, IgG2a, IgG3 and IgG2b recognition of mycolactone in cutaneous tissues from FVB/N and C57Bl/6 mice (n=3 to 5 mice per mouse strain) at each time point in clinical M. ulcerans infection (healthy, redness, ulcer, necrosis or healing). The detection limit was an absorbance of 0.1. The histograms show the means±SD. * p<0.5 (Mann-Whitney U test).

[0041] FIG. 2: Neutralization of mycolactone by cutaneous immunoglobulin from FVB/N mice infected with M. ulcerans. CD4+ lymphocytes were stimulated with PMA/ionomycin. Mycolactone was used at a concentration of 4 ng/mL. Immunoglobulins purified from the skin of A) FVB/N mice or B) C57Bl/6 mice infected with M. ulcerans were used at a ratio of 10 immunoglobulin molecules per molecule of mycolactone. Left panel: IL-2 quantification, Right panel: IFNγ quantification. The data shown correspond to one experiment performed in triplicate (mean±SD).

[0042] FIG. 3: Detection of cutaneous immunoglobulins (IgG) binding to mycolactone in the lesions of patients diagnosed with Buruli ulcer. Wells were coated with mycolactone, and antibodies binding to mycolactone in the plate were recognized by HRP-conjugated secondary antibodies. Biopsy specimens from patients with suspected and/or PCR-confirmed Buruli ulcer were provided by the CDTUB of Pobè (Benin). The detection limit was an absorbance of 0.150. ** p<0.05 (Mann Whitney U test). Buruli ulcer group n=15, control group n=19.

EXAMPLE

[0043] Material & Methods

[0044] Ethics Statement for Animal Experiments and Use of Human Tissues

[0045] All animal experiments were performed in accordance with national guidelines (articles R214-87 to R214-90 of the French “rural code”) and European guidelines (directive 2010/63/EU of the European Parliament and of the Council of Sep. 22, 2010 on the protection of animal used for scientific purposes). All protocols were approved by the ethics committee of the Pays de la Loire region, under protocol no. APAFIS8904. Mice were housed in specific pathogen-free conditions in the animal house of the Angers University Hospital, France (agreement A 49 007 002). The use of biopsy samples from patients for research purposes was approved by the research committee of government of Benin (Ministry of Health, Republic of Benin, agreement number 2893).

[0046] M. ulcerans Strain and Inoculation

[0047] Mycobacterium ulcerans strain 01G897 was originally isolated from patients from French Guiana (18). A bacterial suspension was prepared, as previously described (19, 8), and its concentration was adjusted to 1×104 acid-fast bacilli/mL for inoculation (50 μL) into the tails of six-week-old female consanguineous C57Bl/6 and FVB/N mice (Janvier, Le Genest Saint Isle, France).

[0048] Mycolactone

[0049] The Malaysian M. ulcerans 1615 strain was cultured on solid 7H10 medium supplemented with 10% OADC (oleic acid, dextrose, catalase; Difco, Becton-Dickinson) at 30° C. for 45 days. Mycolactone A/B was then purified from whole bacteria, as previously described (20).

[0050] Mycolactone was diluted to a concentration of 3 mg/mL in absolute ethanol and stored in the dark, in amber glass tubes, at −20° C.

[0051] Mouse Tissue Preparation and Serum Sampling

[0052] Skin samples from clinically infected mice (displaying redness, edema, ulcer, necrosis or healing) were excised (we removed one centimeter of cutaneous tissue from around the lesion) and crushed in PBS supplemented with Complete EDTA-free cocktail (Roche), with a TissueRuptor (Qiagen). The resulting suspensions were centrifuged at 3,500×g for 10 minutes at 4° C. The supernatants were stored at −80° C. Blood samples were collected by retro-orbital puncture at each clinical stage of infection, before tail excision. The blood was centrifuged at 1,500×g for 10 minutes to isolate serum, which was recovered and stored at −80° C.

[0053] Preparation of Human Biopsy Tissues

[0054] Skin biopsy specimens were provided by the CDTLUB of Pobé (Benin). The biopsy specimens were crushed in PBS supplemented with protease inhibitors (Complete EDTA-free cocktail, Roche), with a TissueRuptor (Qiagen), as described for mouse tissues. The resulting suspensions were centrifuged at 3,500×g for 10 minutes at 4° C. The resulting supernatants were stored at −80° C.

[0055] Quantitative PCR Analysis

[0056] Tail skins from infected mice were excised and immediately placed into RNAlater (Qiagen) and stored at −20° C. Skin tissues were crushed and homogenized with a TissueRuptor (Qiagen) and total RNA was then purified with the RNeasy fibrous tissue midi kit (Qiagen). The first-strand cDNA was synthesized from 750 ng RNA with the M-MLV reverse transcriptase (Invitrogen). Quantitative PCR was performed to quantify the levels of IgM, IgA and IgG mRNA. Specific gene expression was calculated by the relative expression method (using actin as the calibrator). The sequences of the primers and probes used are provided in Supplementary table 1.

[0057] Quantification of Proteins and Immunoglobulins

[0058] Total protein levels were determined with a colorimetric assay (Protein Assay Dye Reagent), according to the manufacturer's instructions, with Dye Reagent Concentrate Refill (Bio-Rad 5000006) and a standard curve (bovine gamma globulin, kit 1, 50000001, Bio-Rad). Mouse tissue samples were normalized to identical protein concentrations before testing. IgA, IgM and IgG were quantified in crushed tissue samples and mouse sera by ELISA kit from eBioscience, according to the manufacturer's recommendations (Mouse IgA Ready-SET-Go, 88-50450; Mouse IgM Ready-SET-Go, 88-50470 and Mouse IgG Ready-SET-Go, 88-50400).

[0059] Immunoglobulins Purification

[0060] Tail skins from infected mice were excised and crushed with metallic beads and a TissueRuptor (Qiagen) in an equal volume of PBS supplemented with proteases inhibitor (Complete EDTA-free cocktail Roche) and protein A/G IgG Binding Buffer (Thermo Fisher Scientific). The resulting suspensions were centrifuged at 8,000×g for 45 minutes at 4° C. Ig were then purified with a Nab™ protein A/G spin column (Thermo Fisher Scientific) followed by a Nab™ protein L spin column (Thermo Fisher Scientific), according to the manufacturer's instructions.

[0061] Detection of Antibodies Directed Against M. ulcerans Lysate by ELISA

[0062] M. ulcerans lysate was prepared as previously described (8). M. ulcerans lysate (0.5 μg), diluted in 100 μL of sodium bicarbonate buffer (50 mM; pH 9.6), was immobilized in 96-well ELISA plates (Thermo Fisher Scientific®, Nunc-Immuno™ Plates, Maxisorp 456537) by overnight incubation at 4° C. The coated plates were washed four times with 0.05% Tween 20 in PBS and were then saturated by incubation with 5% skim milk powder in PBS for 2 h at room temperature. The plates were washed a further four times and were then incubated with crushed tissue samples with normalized protein contents in 1% skim milk powder in PBS for 2 h at room temperature. After four washes, antibodies directed against M. ulcerans lysate were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies diluted 1:500 in 1% skim milk powder in PBS. The plates were incubated with the secondary antibodies (Supplementary table 1) for 1 h at room temperature and were then washed four times. The SureBlue™ TMB Microwell Peroxidase Substrate (KPL) was used for secondary antibody detection, and 1 M H2SO4 was added to stop the reaction. Absorbance was measured at λ=450 nm (with a reference at λ=570 nm) (Thermo Fisher Scientific®, Multiskan Ascent); results are expressed in optical density units.

[0063] Detection of Mycolactone-Binding Antibodies

[0064] ELISA was performed to detect antibodies directed against mycolactone present in human tissues, mouse cutaneous tissues and mouse sera. Mycolactone A/B (3 ng) in absolute ethanol was immobilized in 96-well plates (Thermo Fisher Scientific®, Nunc-Immuno™ Plates, Maxisorp 456537), by evaporating off the ethanol; coated plates were stored at −20° C. in dark. They were incubated overnight at 4° C. with 5% skim milk in PBS. The protein concentrations of mouse tissue samples were normalized, mouse serum samples were 10-fold diluted and human samples diluted two-fold. The plates were washed three times in 0.05% Tween 20 in PBS and were then incubated with diluted samples for 2 h at room temperature. The plates were washed four times and incubated with HRP-conjugated secondary antibodies (Goat anti-mouse IgG1, Goat anti-mouse IgG2a, Goat anti-mouse IgG2b, Goat anti-mouse IgG3, Goat anti-mouse IgG/IgA/IgM (H+L) (diluted 1:1000 in 1% skimmed milk in PBS for 2 h at room temperature (supplementary table 1). Bound antibodies were revealed by using the SureBlue™ TMB Microwell Peroxidase Substrate (KPL) was used for secondary antibody detection, and 1 M H2SO4 was added to stop the reaction. Absorbance was measured at 450 nm and is expressed in optical density units.

[0065] Neutralizing Activity Assay

[0066] CD4+ T cells were isolated from the spleens of C57Bl/6 mice by magnetic sorting (MACS technology kit 130-104-454), according to the manufacturer's instructions (Miltenyi Biotec). The purity of CD4+ T-cell preparations was determined by flow cytometry, with PE-conjugated anti-CD4 monoclonal antibody (eBioscience) and FITC-conjugated anti-CD3E monoclonal antibody (eBioscience). CD4+ T cells (200,000 cells/well; 100 μL per well) were used to seed 96-well plates. Purified IgG from infected tissue was diluted in RPMI 1640 (Lonza) supplemented with 10% FCS (Eurobio), 2 mM glutamine, 10 U/mL streptomycin, and 100 U/mL penicillin (Lonza). In some conditions, these were mixed with 8 ng/mL mycolactone A/B in a 10:1 ratio in a tube and incubated at 37° C. with continuous stirring for 45 minutes. Fetal calf serum was added to the preparation at a final concentration of 10%, followed by 10 ng/mL PMA and 1 nM ionomycin. 100 μL per well of this preparation was then added to CD4+ T cells. Cells were incubated for 6 h at 37° C. under an atmosphere containing 5% CO2, and supernatants were collected and stored at −20° C. IL-2 and IFN-γ were quantified by ELISA (eBioscience), according to the manufacturer's instructions (Mouse IL-2 Ready-SET-Go: 88-7024 and Mouse IFN-γ Ready-SET-Go: 88-7314 respectively).

[0067] Immune Cell Isolation and Flow Cytometry Analysis

[0068] Tail skin was excised from three mice. Tissues were digested with Multi Tissue Dissociation kit 1 from Miltenyi Biotec (reference 130-110-201) according to the manufacturer's instructions. A four-color staining method was used to identify B-cell subsets: CD45+, CD19+, B220+ and CD138+ cells were labeled with APC cyanidine7 anti-CD45 (BD Biosciences), phycoerythrin anti-CD19 (BD Biosciences), [phycoerythrin-cyanidine 7] anti-CD45R/B220 (BD Biosciences) and BB515anti-CD138 (BD Biosciences) antibodies; dead cells were excluded from the analysis by staining with 7AAD (Miltenyi Biotec). Flow cytometry analysis was performed on a MACSQuant analyzer. Results were analyzed with FlowLogic software.

[0069] Immunohistology

[0070] Tails from infected mice were excised and immediately fixed by incubation in 4% paraformaldehyde for 24 h. Tissues were then embedded in paraffin, and cut into 5 mm-thick sections. Hematoxylin-phloxine-saffron (HPS) staining was performed according to the manufacturer's protocol. Immunohistochemical staining was performed with an anti-CD138 polyclonal antibody (Thermo Fisher Scientific ref. #36-2900) diluted 1:250 according to the manufacturer's protocol.

[0071] Statistical Analysis

[0072] The data, presented as means and SE (standard error), were analyzed with GraphPad Prism 5.0 software (GraphPad Software, San Diego, Calif., USA). Different clinical stages in each mouse strain were compared in Kruskal-Wallis tests with Dunn's Multiple Comparison test. FVB/N and C57Bl/6 mice were compared, at each clinical stage, in Mann-Whitney U tests. Buruli ulcer patients and controls were analyzed in Mann-Whitney U tests.

[0073] Results

[0074] Production of Skin Immunoglobulins During M. ulcerans Infection

[0075] We investigated the local humoral response during M. ulcerans infection, including the spontaneous healing process, by evaluating immunoglobulin gene expression through analyses of mRNA levels in cutaneous tissue samples. The FVB/N mouse model (which displays spontaneous healing) was compared with the C57Bl/6 mouse (which displays no such healing). Relative mRNA levels for IgM, IgA and IgG remained stable in both models during early stages of infection (Data not shown). However, the levels of mRNA for all three immunoglobulin isotypes increased significantly in FVB/N mice at the ulcerative stage (day 45), reaching a peak during the healing stage (day 75). The levels of mRNA encoding these three isotypes were significantly higher (p-value<0.05 for IgM and IgG and p-value<0.01 for IgA, Mann-Whitney U test) in FVB/N mice at the healing stage than in C57Bl/6 mice at the necrotic stage. We characterized the immunoglobulin profiles of infected tissues by estimating IgM, IgA and IgG concentrations by ELISA. Consistent with the mRNA data, total immunoglobulin levels increased in the cutaneous tissues of FVB/N mice throughout M. ulcerans infection (Data not shown). However, despite the absence of clear changes in local levels of immunoglobulin mRNA in C57B1l/6 mice, we observed an increase in tissue Ig concentration throughout M. ulcerans infection in these mice (Data not shown). Differences in IgM and IgG concentrations were detected between the two mouse strains. Indeed, IgG levels in FVB/N mice were twice higher than in C57Bl/6 mice (p-value <0.01, Mann-Whitney U test) during both the redness and ulcerative stages, and were 1.6 times higher (p-value <0.05, Mann-Whitney U test) during the healing process than during the necrotic stage, whereas IgM levels were higher in C57Bl/6 mice from the ulcerative stage (2.7 times higher, p-value <0.01, Mann-Whitney U test) until necrosis (7.8 times higher, p-value <0.01, Mann-Whitney U test). These differences, evident from the earliest stages, could reflect elevated circulating immunoglobulins. However, no significant differences in the levels of these immunoglobulins were detected between the serum samples of the two mouse strains during the early stages of infection (Data not shown). Thus, the course of M. ulcerans infection is associated with a local humoral response, with the following immunoglobulin profiles: IgM>IgG>IgA in C57Bl/6 mice and IgG>IgM>IgA in FVB/N mice. There thus seems to be a specific humoral immunity signature of the healing process, in which IgG predominates.

[0076] Recognition of M. ulcerans Lysate by Iocal IgG Antibodies

[0077] We investigated the specificity of this local immunoglobulin production, by evaluating their capacity to recognize the whole M. ulcerans lysate in an ELISA assay. IgG was the only isotype of immunoglobulin able to bind M. ulcerans lysate components (Data not shown). This recognition became stronger with successive stages of infection in both FVB/N and C57Bl/6 mice, and was maximal during the healing and necrosis stages (p-value <0.01, Mann-Whitney Test). We then evaluated the ability of IgG subclasses to recognize M. ulcerans components (Data not shown). IgG1 bind at high levels to the M. ulcerans lysate components in both mouse models. IgG1 binding was detected early in the redness stage in FVB/N mice and later, at the edema stage in C57Bl/6 mice (p-value <0.01 for the difference between the two mouse models at the redness stage, Mann-Whitney U test). By contrast, IgG3 recognized M. ulcerans lysate components very weakly, but similarly in the two mouse strains. The major difference between FVB/N and C57Bl/6 mouse concerned the IgG2a/b subclasses. IgG2a recognized M. ulcerans components at all stages of infection (from the ulcerative to the healing stage) in FVB/N mice, but seemed to be absent in C57Bl/6 mice. The last subclass, IgG2b, recognized M. ulcerans lysate significantly more strongly in C57Bl/6 mice, from the ulcerative to the necrotic stage, than in FVB/N mice at equivalent stages (p-value <0.05, Mann-Whitney U test). In conclusion, local humoral responses differed between FVB/N mice (healing model) and C57Bl/6 mice (not able to heal). This difference mostly concerned two specific immunoglobulin subclasses: (i) IgG2a, which was produced only in the healing model, and (ii) IgG2b, which recognized M. ulcerans components strongly only in the C57Bl/6 model. These results suggest a potential role for IgG2a in controlling M. ulcerans infection in the FVB/N healing model.

[0078] Recognition of Mycolactone by Local Skin Antibodies

[0079] The ability of FVB/N mice to heal spontaneously after M. ulcerans infection could be explained, in part, by the production of antibodies recognizing the bacterial toxin, mycolactone. We investigated the specificity of these local antibodies for mycolactone in skin tissues by performing ELISA (FIGS. 1A and 1B). IgM produced locally in both mouse strains were unable to recognize mycolactone. IgA, which is known to protect tissues (ie. epithelial cells) against bacterial toxins (Janeway 2001), bound mycolactone at low levels only during the ulcerative stage in FVB/N mice. As observed for M. ulcerans lysate, IgG recognized mycolactone at high levels in both mouse models throughout infection. The level of mycolactone recognizing by IgG was significantly higher in C57Bl/6 mice at the necrotic stage than in FVB/N mice at the healing stage (p-value <0.05, Mann-Whitney U test). We then analyzed IgG subtypes at each stage of infection. From the edema stage to the ulcerative stage, IgG1 appeared to be the principal subclass of mycolactone-binding antibody in FVB/N mice, whereas IgG2b seemed to be present exclusively in C57Bl/6 mice (p-value <0.01 for IgG1 in FVB/N versus C57Bl/6 mice, p-value <0.01 for IgG2b in the ulcerative stage in C57Bl/6 mice relative to FVB/N mice, Mann-Whitney U test). During the last stage of infection (necrosis vs. healing), IgG1 antibody seemed to be the principal isotype at work in the FVB/N model, whereas IgG2b remained the major subclass of antibody binding the toxin in C57Bl/6 mice (p-value <0.05 versus FVB/N mice, Mann-Whitney U test). The mycolactone-binding profile of IgG3 was similar in both mouse models. Finally, as for the recognition of M. ulcerans lysate, the major difference concerned the IgG2a subclass, which appears to be specific to FVB/N mice. This subclass bound the toxin from the ulcerative stage to the healing stage. The lack of IgG2a detection in assays performed on C57Bl/6 mice may be explained by a mutation of the IgG2a gene in these mice (27). For exclusion of a strain-specific phenotype and confirmation of the importance of this protein in the healing process and in mycolactone binding, we performed assays in another mouse model displaying no spontaneous healing (BALB/c mice) in which the IgG2a gene is present and functional. We found that the IgG2a antibody produced by BALB/c mice did not recognize mycolactone (Data not shown). In parallel, the systemic humoral recognition of mycolactone was assessed in FVB/N and C57Bl/6 mice, but no systemic antibodies binding the toxin were detected (data not shown). Taken together, these results reveal different mycolactone recognition profiles between mouse models of healing and necrotic infection. IgG2b appears as the principal subclass able to recognize M. ulcerans toxin in C57Bl/6 mice, but appears to be unable to control infection efficiently. By contrast, IgG2a was specific to the spontaneous healing model (FVB/N mice) and may be involved in the control of M. ulcerans infection associated with spontaneous healing.

[0080] Neutralization of Mycolactone by Antibodies Present in the Skin

[0081] In this context, we assessed whether these antibodies could not only recognized but also neutralized mycolactone, by evaluating cytokines production by T lymphocytes as an indicator of the immunomodulatory property of mycolactone. To this end, we compared the IL-2 and IFNγ production by T cells after the stimulation of these cells and their incubation with mycolactone alone or mycolactone previously incubated with IgG purified from the skin of FVB/N and C57Bl/6 mice. Contact between T cells and the toxin greatly decreased IL-2 and IFNγ production as compared to the positive control (T cells only stimulated) in both mouse models (FIGS. 2A and 2B). By contrast, the production of these two cytokines was similar to the positive control for T cells in contact with mycolactone previously incubated with IgG purified from the skin of FVB/N mice, but not for T cells in contact with mycolactone previously incubated with IgG purified from C57Bl/6 mice. Taken together, these results show that IgG produced by FVB/N mice skin during the healing stage can neutralize the toxin of M. ulcerans. This neutralization may be involved in the healing process.

[0082] Local Emergence of Antibody-Producing Cells During M. ulcerans Infection

[0083] Consistent with the rational of the establishment of a local humoral immune response during spontaneous healing, we tried to demonstrate the physiological relevance of this process by investigating the presence of antibody-producing B cells in vivo. Using four-color staining, we analyzed B cell populations from FVB/N mice, and identified three populations: (P1) cells with a CD45+, CD19+, B220+ phenotype corresponding to B cells, (P2) CD45+, CD19+, B220int cells corresponding to plasmablasts, and (P3) CD45+, CD19−, B220int, CD138+ cells, corresponding to the murine markers of the last stage of B-cell maturation, plasma cells (Tellier & Nutt. 2017). The total number of lymphoid cells (CD45+) increased 37-fold as compare to the control skin during the spontaneous healing process (Data not shown). Consequently, the total number of B cells increased, but the proportions of the various subsets remained constant, except for the plasma cell subset, which increased in proportion from 0.07 to 0.54% of total lymphoid cells during the first few steps of the spontaneous healing process (Day 55), whereas no such increase was observed in uninfected skin (control) (Data not shown). The presence of this specific B-cell subtype was confirmed by histological analysis (Data not shown). The proportion of B1-like B cells (CD45+, CD19+, B220int, CD43+), a specific subset which has been shown to produce antibodies specifically in the skin (28) increased during spontaneous healing, reaching 0.07% of total lymphoid cells (Data not shown). The proportion of these antibody-producing cells decreased when the lesion appears to be completely healed (D75), but remained higher than in control skin. Finally, we have shown that antibody-producing cells strongly increase in the skin during the spontaneous healing process, supporting the role of the local humoral response in this phenomenon, through the production of anti-mycolactone antibodies.

[0084] Recognition of Mycolactone by Local IgG Purified from Skin Biopsy Specimens from Buruli Ulcer Patients

[0085] In addition to this detailed characterization of anti-mycolactone immunoglobulins in mice, we also assessed the levels of these IgGs in Buruli ulcer patients. We used ELISA to detect anti-mycolactone antibodies in skin samples from patients (either diagnosed or not diagnosed as Buruli ulcer patients) provided by the CDTUB of Pobé (Benin). In 73% of PCR-confirmed Buruli ulcer patients, the most severe form of the disease had been diagnosed: an ulcerative lesion (which may be associated with other forms, such as edema or plaques). Mycolactone was recognized by local antibodies recovered from the lesions of 60% of patients with PCR-confirmed Buruli ulcer (9/15) (all presenting an ulcer), whereas mycolactone was not detected in the biopsy specimens of all but one of the control patients (not diagnosed with Buruli ulcer; p-value<0.05, Mann-Whitney U test; FIG. 3). This particular patient has since then developed squamous cell carcinoma, a disease that is known to appear in some cases after Buruli ulcer lesions (29, 30). We cannot, therefore, exclude the possibility that this patient may have already had a lesion due to M. ulcerans infection. Finally, there may be several reasons for the lack of mycolactone-binding IgG detection in 40% of patients with PCR-confirmed Buruli ulcer (6/15) such as for instance the type of lesion, the sampling site (distance from the site of infection), and the treatment duration. Finally, we demonstrate here, for the first time to our knowledge, that the human organism can generate an effective humoral response involving the production of antibodies able to recognize mycolactone during M. ulcerans infection.

[0086] Discussion

[0087] Buruli ulcer is a neglected tropical disease and remains the third most common mycobacterial disease worldwide. This debilitating skin disease is caused by M. ulcerans, which produces a lipid-like toxin, mycolactone, the main virulence factor of the bacillus. Without treatment, lesions can escalate into chronic skin ulcers. However, these severe lesions can spontaneous heal, as observed in 5% of patients cases, suggesting that the host may be able to develop strategies for counteracting the effects of M. ulcerans. Despite the development of animal models, the mechanisms of the spontaneous healing process remain unclear. We previously showed (i) the absence of a systemic immune cell response signature and (ii) a weak involvement of the local cellular immune response in the switch from acute to chronic infection (8). These results highlight the local consequences of the disease, as observed with other approaches. Indeed, recent histological studies have shown that B cells accumulate in clusters around the site of M. ulcerans infection (10). B-cell infiltrates have been observed in chronic inflammatory skin conditions, including cutaneous leishmaniosis and atopic dermatitis (33, 34). The debate about the pro- or anti-inflammatory role of skin B cells is still ongoing, yet these cells have been shown to be involved in the resolution of skin inflammation in a mouse model of psoriasis-like inflammation (35). Furthermore, in addition to producing antibodies locally, B cells have been shown to play a role in the process of wound healing (36).

[0088] In this context, we used our mouse model of spontaneous healing to investigate the humoral response at all stages of M. ulcerans infection, including spontaneous healing. We demonstrated the presence of antibody-producing B cells during infection and their increase in number during infection, peaking during the early stages of spontaneous healing (the transition from ulceration to healing). We detected immunoglobulins able to recognize the lipid toxin of M. ulcerans, mycolactone, throughout all stages of infection. Interestingly, these immunoglobulins were found in the skin of infected mice, but not in their sera. We also provide the first demonstration of the presence of these immunoglobulins in biopsy specimens from Buruli ulcer patients. No anti-mycolactone antibodies have ever been detected in serum from patients. Collectively, these results highlight the existence of a distinct humoral signature in response to M. ulcerans infection, with the skin-specific production of antibodies against mycolactone.

[0089] The specific local production of immunoglobulins able to recognize M. ulcerans components, including mycolactone in particular, may contribute to the control of infection observed during spontaneous healing. We investigated this possibility, by evaluating the ability of IgG subclasses to recognize M. ulcerans components. We tagged, for the first time, a specific subclass of immunoglobulin, IgG2a, which is known to diffuse readily in the skin (37). This subclass reported as highly effective on neutralizing bacterial exotoxins, such as diphtheria toxin, enterotoxin B or Bacillus anthracis-associated toxin (38), was produced only in the spontaneous healing model and seems to be the signature of this model in terms of mycolactone recognition. Our results also showed that IgG isolated from the skin of FVB/N mice that was able to recognize M. ulcerans components neutralized similarly the toxic activity of mycolactone. No other mycolactone-neutralizing antibodies have been identified in other mouse models.

[0090] The neutralization of bacterial toxins is an important part of the humoral immune response to bacterial infections (38). Our results suggest therefore that mycolactone neutralization may be the key to the spontaneous healing process of Buruli ulcer. Indeed, this phenomenon may block mycolactone activity in various ways: it would probably be more difficult for mycolactone linked to an antibody to gain access to or inhibit known intracellular targets, such as the Sec61 translocon (i) if the antibody-mycolactone complex cannot cross membranes or (ii) if the antibody hampers the fixation of the mycolactone. It is therefore reasonable to assume that (iii) neutralizing antibodies may help to eliminate the toxin by targeting it to phagocytes cells.

[0091] The existence of anti-mycolactone antibodies opens up exciting new perspectives for innovations in diagnosis, treatment and vaccine development responding to the scientific challenge issued by the World Health Organization. Indeed, there is currently no simple diagnostic tool suitable for use in the rural areas of developing countries. This situation is particularly regrettable, as the early stages of Buruli ulcer can be treated locally, whereas the treatment of later stages requires extensive surgery in larger hospitals, with longer periods of hospitalization, at much greater expense. The development of a new diagnostic tool, such as a test based on monoclonal antibody production, might be more appropriate for these endemic regions.

REFERENCES

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