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Clinical and Experimental Immunology
O R I G I N A L A RT I C L E
doi:10.1111/cei.12309
Potential cross-reactivity of monoclonal antibodies against clinically relevant mycobacteria
K. Flores-Moreno, J. S. Celis-Meneses, D. M. Meneses-Ruiz, A. I. Castillo-Rodal, P. Orduña, B. A. Montiel and Y. López-Vidal
Summary
Nacional Autónoma de México, Facultad de Medicina, Programa de Inmunología Molecular
Tuberculosis is a disease caused by the Mycobacterium tuberculosis complex (MTb). In 2011, global mortality due to tuberculosis was 1·4 million individuals. The only available vaccine is the attenuated M. bovis [bacillus Calmette–Guérin (BCG)] strain, which confers variable protection against pulmonary tuberculosis. Some widely distributed non-tuberculous mycobacteria (NTM), such as M. avium and M. arupense, are also potential pathogens for humans. This work aimed to produce and characterize monoclonal antibodies against the M. bovis BCG Mexico strain of the MTb, M. avium subs. hominissuis and the M. arupense strain from NTM. Hybridomas were produced from splenocytes of BALB/c female mice immunized with radiation-inactivated mycobacteria, and the immunoglobulin (Ig)G2a antibody-producing clones with the highest antigenic recognition were selected. The selected clones, Mbv 2A10 for M. bovis BCG Mexico, Mav 3H1 for M. avium and Mar 2D10 for M. arupense, were used in further studies. Enzyme-linked immunosorbent assay (ELISA) and immune proteomics analyses characterized the clones as having the highest crossreactivity with mycobacteria. Using mass spectrometry, a number of proteins recognized by the monoclonal antibody (mAb) clones were identified. These proteins had roles in metabolic processes, hypoxia, cell cycle and dormancy. In addition, a Clustal W and Immune Epitope Database (IEDB) in-silico analysis was performed in protein sequences that result in the conserved regions within probability epitopes that could be recognized for Mbv2A10 and Mav3H1 clones.
Microbiana. Edificio de investigación. 4to piso, México DF 04510, Mexico. E-mail: [email protected]
Keywords: BCG, mycobacteria
Programa de Inmunología Molecular Microbiana, Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, México City, México
Accepted for publication 21 February 2014 Correspondence: Y. López-Vidal, Universidad
Introduction Mycobacteria are the causal agents of infectious diseases (mycobacteriosis) in several species, including humans. The best-known mycobacterial disease is tuberculosis, which currently affects one-third of the world population and causes 1·4 million deaths annually [1]. The main aetiological agent of tuberculosis is Mycobacterium tuberculosis; this species is a member of the M. tuberculosis complex (MTb). M. bovis, a pathogenic species in humans and cattle, is also part of MTb, and is the source of the only available vaccine, attenuated bacillus Calmette–Guérin (BCG), against severe forms of tuberculosis [2–4]. Among the vaccine strains, 454
epitopes,
monoclonal
antibodies,
non-tuberculous
BCG Mexico has genomic and immune proteomic traits that make it desirable as an effective vaccine against M. tuberculosis [5]. Non-tuberculosis mycobacteria (NTM) are widely distributed [6,7] and are recognized as pathogenic agents [8,9]. Our group has isolated NTM of clinical relevance from water reservoirs used by humans in Mexico City and nearby areas [10]. These NTM isolates consisted of strains from the M. avium complex, regarded as the most pathogenic in humans, causing 70% of NTM-related diseases [8,9,11,12], and M. arupense, which has been isolated from lung, osteoarticular tissue and skin clinical samples [13,14].
© 2014 British Society for Immunology, Clinical and Experimental Immunology, 177: 454–463
Monoclonal antibodies against mycobacteria
Due to the wide distribution and impact upon global public health of mycobacteria, control of related infections requires the development of prophylactic, diagnostic and therapeutic tools. In this regard, the World Health Organization has issued a plan of action to coordinate efforts and significantly reduce tuberculosis by 2015 [15]. This work aimed to produce and characterize monoclonal antibodies (mAbs) against the vaccine strain M. bovis BCG Mexico and environmental isolates of M. avium subsp. hominissuis (M. avium) and M. arupense to evaluate their potential as biomedical tools. Additionally, the proteins recognized by the anti-mycobacterial clones were identified and, using in-silico analysis, allowed us to predict the presence of epitopes and sequences conserved between the proteins recognized by each mAb Mbv 2A10 and Mav 3H1. Finally, we determined that the mAb generated have potential cross-reactivity with different species from the genus Mycobacterium.
Materials and methods Cell culture and mycobacterial preparation for immunization The M. avium and M. arupense strains were obtained from the Programme of Microbial Molecular Immunology and were initially isolated from irrigation and drinking water in Mexico City and nearby areas. Both strains were recovered in 7H10 culture medium (Difco, Detroit, MI, USA) and subsequently subcultured in Sauton medium and 10% albumin–dextrose–catalase (ADC; Becton Dickinson, San Jose, CA, USA)-supplemented Middlebrook 7H9 medium (Difco), respectively, at 37°C under continuous agitation and 5% CO2. The BCG Mexico 1931 strain, provided by the Laboratorios de Biológicos y Reactivos de México S.A. de C.V. (BIRMEX), was cultured in Sauton medium under the same conditions described above for 20 days. The bacterial inoculum for immunization was obtained from cultures in the logarithmic growth stage (10 days for M. arupense and 20 days for M. bovis BCG Mexico and M. avium) and harvested by centrifugation at 5000 g for 10 min. The cell pellets were resuspended twice in isotonic saline solution (ISS). Finally, mycobacteria were inactivated by irradiation with 12 kGy at the Instituto Nacional de Ciencias Nucleares, UNAM. The Sp2/0-Ag14 cell line (ATCC, CRL-1581) was cultured in DMEM (Gibco, Carlsbad, CA, USA) medium supplemented with 10% fetal bovine serum (Gibco), kept at a cell density of 5 × 104 to 5 × 105 cells/ml, and incubated at 37°C, 95% RH and 5% CO2.
Immunization and antibody kinetics Five female BALB/c mice, 6–8 weeks of age, were used for immunization of each mycobacterial strain. Mice were immunized intraperitoneally four times every 15 days with
2 × 106–5 × 107 bacterial inocula in 200 μl of ISS. Five mice were immunized with sterile ISS as a control. Before immunization, a blood sample was taken from the mouse maxillary vein to assess antibodies in sera. Enzyme-linked immunosorbent assay (ELISA) was used to assess the specificity and isotype of the mAbs generated against M. bovis BCG Mexico, M. avium and M. arupense. Briefly, 5 μg/ml of protein extract from the mycobacterium under study was obtained by overnight sonication in carbonate buffer solution at 4°C and plated onto 96-well plates. The samples were blocked with 1% albumin at 37°C, and fivefold dilutions of mouse sera taken at various times after immunization (0, 15, 30, 45 and 60 days) were added. Microplates were incubated at 37°C for 1 h. Horseradish peroxidase (HRP)-linked rabbit anti-mouse IgG2a antibodies were added at a 1 : 2000 dilution to identify immune complexes using chemiluminescent HRP (Millipore, Billerica, MA, USA) as a substrate. All serum samples were run in triplicate, and microplate luminescence was read at 560 nm using a Genius Plus machine and Magellan software.
Clone production and selection of mAbs against mycobacteria BCG Mexico and M. avium clones were obtained from mouse splenocytes, which produced the highest antibody titres. Briefly, 1 ml of polyethylene glycol (PEG) was added drop-wise to 1 × 108 splenocytes and 2 × 107 Sp2/0-Ag14 (ATCC, CRL-1581) cells over 60 s. Dulbecco’s modified Eagle’s medium (DMEM) (4 ml) was then added, and the cells were agitated for 4 min. Ten millilitres of DMEM medium was added, and the cells were incubated in a waterbath at 37°C for 15 min. Finally, 30 ml of supplemented DMEM medium was added. The cell suspension was transferred to a cell culture bottle and incubated at 37°C with 5% CO2 for 24 h. The cells were harvested by centrifugation at 400 g at 37°C for 10 min and resuspended in 10 ml of supplemented DMEM plus 90 ml of hypoxanthine–aminopterin–thymidine (HAT) medium. Cell suspensions were plated in 96-well microplates at 60 μl/well, and the plates were incubated for 8 days under the conditions described above. Hypoxanthine–thymidine (HT) medium at 150 μl/well was added, and the microplates were incubated for another 4 days. After this incubation, 100 μl of supernatant was collected, and positive clones were selected by ELISA. These clones were transferred to 24-well plates in 1 ml of HT medium, which was gradually replaced by DMEM medium. A second screening was performed based on the proliferative capacity and reactivity of the clones. From this screening, only the most reactive clone as determined by ELISA was used for further characterization. Finally, M. arupense mAbs were obtained using the ClonaCell kit (Stemcell cat. no. 03804), according to the manufacturer’s instructions.
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Characterization of mAbs To assess the cross-recognition capability of the mAbs generated against M. bovis BCG Mexico, M. avium and M. arupense, reactivities were determined against different MTb complex strains, such as M. tuberculosis H37Rv, M. microti, M. bovis BCG Mexico, M. bovis BCG Phipps and M. bovis BCG Japan, as well as against NTM strains such as M. avium sp., M. arupense and M. abscessus. Crossreactivity was assessed by ELISA following the procedure described above. The cut-off optical density (OD) was calculated as the mean relative luminescence units (RLU) of negative samples plus two standard deviations.
Isolation of protein extracts and electrophoresis on 2D-polyacrylamide gel electrophoresis (2D-PAGE) gels A bacterial suspension from cultures in the mid-logarithm growth stage was diluted in 20 mM phenylmethylsulphonyl fluoride (PMSF) (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) and subjected to 15 60-s pulses, as reported previously [16]. Protein concentration of the obtained extracts was quantified by the Bradford method. Electrophoresis on two-dimensional (2D) gels was performed in duplicate using gel strips with an immobiliszed pH gradient from 4 to 7 [ReadyStripTM immobilized pH gradient (IPG strips); Bio-Rad Laboratories, Hercules, CA, USA]. Protein aliquots of 100 μg were suspended in a rehydration solution (7 M urea, 2 M thiourea, 1 M dithiothreitol (DTT), 4% CHAPS {3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate}, 1% ampholines and 0·001% bromophenol blue) to a final volume of 180 μl. IPG strips were rehydrated at a pH range of 4–7 for 16 h. Electrophoresis was then performed in a Multiphore II Electrophoresis Unit (Amersham Biosciences, Uppsala, Sweden) at 17°C as follows: 500 V for 30 min, 1000 V for 30 min, 1500 V for 30 min, 2000 V for 30 min and 2500 V for 25 h until 52 000 V/h were completed. After isoelectric focusing, the strip was immersed in an equilibrium buffer [6 M urea, 50 mM Tris-HCl, pH 8·8, 30% glycerol, 2% sodium dodecyl sulphate (SDS)] with 97 mM DTT for 15 min and 203 mM indoleacetic acid (IAA) for 15 min. For the second dimension, the strip was placed on a 12% acrylamide gel in a Hoeffer SE 600 (Pharmacia Biotech, Piscataway, NJ, USA) chamber, and electrophoresis was run with gradual voltage increases of 50 V for 30 min, 100 V for 2 h and 150 V until the electrophoresis was finished. The proteome of M. bovis BCG Mexico, M. avium and M. arupense was obtained by silver staining the 2D gel after electrophoresis. The gel was fixed with penthydrate sodium thiosulphate (0·2 g/l) for 1 min. Silver nitrate (0·2 g/100 ml) was then added, and the gel was developed with sodium carbonate anhydrous (3 g/100 ml). The image was digitalized using a Molecular Imager GS-800TM calibrated 456
densitometer (Bio-Rad Laboratories) and analysed using the Quantity One TM 1-D analysis software (Bio-Rad Laboratories).
Western blot analysis A replicate of the 2D-polyacrylamide gel electrophoresis (2D-PAGE) gel used for proteome determination was transferred to a 0·45-μm pore polyvinylidene difluoride (PVDF) membrane (Hybond-P PVDF Membrane; Amersham) activated previously with methanol (100%) for 10 s and incubated in Bjerrum and Schafer-Nielsen transfer solution for 10 min. The transfer was performed in a Trans-Blot SD chamber (Semi-Dry Electrophoretic Transfer Cell; Bio-Rad Laboratories) at a constant potential difference of 10 V for 1 h. Once the transfer was complete, the membrane was blocked with 4% milk at 4°C overnight. The membrane was then incubated with the selected mAbs against M. bovis BCG Mexico (Mbv 2A10), M. avium (Mav 3H1) or M. arupense (Mar 2D10) at fivefold dilutions. HRP-labelled rabbit IgG2a anti-mouse antibodies were used as secondary antibodies at a 1 : 2000 dilution. The obtained immune complexes were developed by HRP chemiluminescence (Millipore).
Mass spectrometry identification of proteins recognized by mAbs All immunoblot spots identified with mAbs against each mycobacterium were obtained from a Coomassie Brilliant Blue-stained 2D-PAGE gel. The spots were digested as reported by Kinter et al. [17]. Briefly, gel fragments for each sample were washed and destained overnight in a methanol/acetic acid 50%/5% solution at room temperature, and were then dehydrated with acetonitrile and dried by vacuum centrifugation. The proteins were digested with trypsin; the resulting peptides were recovered by successive extractions with ammonium bicarbonate and acetonitrile/ formic acid. The extract was concentrated for mass spectrometry analysis. Finally, the peptide sequence was determined using a hybrid triple linear/quadrupole ion trap mass spectrometer (3200 Q TRAP; Applied Biosystems, Life Technologies Corp., Foster City, CA, USA). The protein sequences obtained by mass spectrometry were searched in the Mascot 52 database from the mycobacterial complex under study.
Epitope search with mAb-recognized proteins In-silico prediction of the proteins identified by the mAbs generated against M. bovis BCG Mexico, M. avium and M. arupense was performed using the Immune Epitope Database (IEDB) bioinformatics software (IEDB; Bioinformatics, Toronto, ON, Canada).
© 2014 British Society for Immunology, Clinical and Experimental Immunology, 177: 454–463
Monoclonal antibodies against mycobacteria Table 1. Cross-reactivity of the Mbv 2A10, Mav 3H1 and Mar 2D10 monoclonal antibody (mAb) clones against mycobacteria in the MTb and NTM complexes. mAbs reactivity (RLU) Complex MTb
NTM
Mycobacteria
Mbv 2A10
Mav 3H1
Mar 2D10
M. tuberculosis H37Rv M. microti M. bovis BCG Phipps M. bovis BCG Japan M. bovis BCG Mexico M. nonchromogenicum M. avium subs. Hominissuis M. abscessus M. arupense
469 558 566 673 2235 377 588 988 ND
218 225 211 218 342 172 426 301 ND
210 179 307 237 261 354 311 396 962
BCG = bacillus Calmette–Guérin; MTb = Mycobacterium tuberculosis complex; Mbv = mAbs for M. bovis BCG Mexico; Mav = mAbs for M. avium; Mar = mAbs for M. arupense; NTM = non-tuberculous mycobacteria; n.d. = not determined.
Multiple alignment of proteins recognized by the Mbv 2A10 and Mav 3H1 mAbs
The Mbv 2A10, Mav 3H1 and Mar 2D10 mAbs showed reactivity against MTb and NTM strains
A multiple sequence alignment of proteins recognized by mAb Mbv 2A10 (glycine dehydrogenase, stress protein Rv2623 and thiosulphate sulphurtransferase CysA2) and those recognized by mAb Mav 3H1 [nitroreductase MSMEG_5246, phosphomethylpyrimidine kinase, acyl (ACP) desaturase, and 3-hydroxyacyl-CoA dehydrogenase] was performed using Clustal W version 2·1 software.
To evaluate the specificity of the mAbs produced by the selected clones, cross-reactivity to antigen extracts from other mycobacteria in the MTb (M. tuberculosis H37Rv, M. microti, BCG Phipps, BCG Japan and BCG Mexico) was determined according to the cut-off threshold (calculated as the mean of the RLU of the negative control plus two standard deviations). Table 1 shows the extent of antigenic recognition (in RLU) by the mAbs generated against BCG Mexico (Mbv 2A10), M. avium (Mav 3H1) and M. arupense (Mar 2D10). All mAbs preferentially recognized protein extracts from the mycobacteria against which they were produced: mAb Mbv 2A10 (2235 UR for BCG Mexico), Mav 3H1 (426·3 UR for M. avium) and Mar 2D10 (962·5 UR for M. arupense). However, all mAbs also showed crossrecognition of MTb and NTM strains, albeit with lesser reactivity (Table 1).
Results Selection of mAbs against BCG Mexico, M. avium and M. arupense Sera from mice immunized at various times (0, 15, 30, 45 and 60 days) with inactivated BCG Mexico, M. avium or M. arupense were collected to evaluate the kinetics of immunoglobulin (Ig)G2a antibody induction after each immunization by ELISA (data not shown). The sera of mice showing the highest antibody titres against each mycobacterium were selected, and the splenocytes from these mice were fused with myeloma Sp2/0-Ag14 cells. From this fusion, 1440 clones producing antibodies against BCG Mexico, 1415 clones producing antibodies against M. avium and 297 clones producing antibodies against M. arupense were obtained. An initial screening was performed by ELISA to evaluate the antibody reactivity of each clone, and the clones with the highest titres were selected: 110 clones (7·6%) for M. bovis BCG Mexico, 98 clones (6·9%) for M. avium and 11 clones (3·7%) for M. arupense. A second screening was performed by assessing the antigen-specific proliferation and reactivity of the antibodies against BCG Mexico and M. avium. Ten clones (0·6%) for BCG Mexico and six clones (0·4%) for M. avium were selected. Finally, ELISA was used to select the most reactive clone to each mycobacterial extract. The selected clones were Mbv 2A10 for BCG Mexico, Mav 3H1 for M. avium and Mar 2D10 for M. arupense.
Proteins related to metabolism, growth regulation, dormancy and ribose transport were recognized by the Mbv 2A10, Mav 3H1 and Mar 2D10 mAbs To identify the proteins recognized by the mAbs produced against BCG Mexico, M. avium and M. arupense, electrophoresis of total protein extracts was performed on two 2D-PAGE gels with a pH range of 4–7, performed simultaneously and with one using an immunoblot. The recognition of immunodominant proteins for each separate antibody was detected (data not shown). Three spots were identified for BCG Mexico, four for M. avium and one for M. arupense. The proteins recognized by immunoblot were identified in the mycobacterial proteome from the gel performed in parallel and were based on molecular weight (MW) and isoelectric point (pI). For BCG Mexico, the recognized proteins were in a pH range of 5–5·5 and a MW range of 30–100 kDa (Fig. 1). For M. avium, the recognized proteins were in a pH range of
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K. Flores-Moreno et al. 4 250 150 100
pl
7
1
75 50 37
2 3
25 20 15
10
Fig. 1. Representative two-dimensional (2D) electrophoresis of total protein extracts from Mycobacterium bovis bacillus Calmette–Guérin (BCG) Mexico. Total protein extracts from M. bovis BCG Mexico were resolved on 2D gels in a pH gradient of 4–7. The spots recognized by monoclonal antibodies against mycobacterium are indicated with a circle and numbered.
4·9–5·9 and a MW range of 25–40 kDa (data not shown). The sole recognized protein for M. arupense had a MW of approximately 30 kDa and pH of 4·3 (data not shown). The identified spots were processed, and their homology with sequences in the Mascot database was identified by mass spectrometry. Among the identified proteins for mAb Mbv 2A10, spot 1 corresponded to M. bovis glycine dehydrogenase, spot 2 corresponded to stress protein Rv2623 from M. tuberculosis H37Rv and spot 3 corresponded to thiosulphate sulphurtransferase CysA2 from M. tuberculosis H37Rv with coverage percentages of 32, 60 and 58%, respectively. Functionally, glycine dehydrogenase and thiosulphate sulphurtransferase CysA2 participate in intermediary metabolism and respiration [18,19]. Stress protein Rv2623 is involved in bacterial growth regulation and is required for mycobacteria to enter dormancy [20,21] (Table 2). With regard to the spots recognized by mAb Mav 3H1, spot 1 corresponded to nitroreductase MSMEG_5246, spot 2 corresponded to a phosphomethylpyrimidine kinase [22], spot 3 corresponded to an acyl [ACP] desaturase [23] and spot 4 corresponded to 3-hydroxyacyl-CoA-dehydrogenase [22], all from M. smegmatis, with coverage percentages of 53, 76, 50 and 84%, respectively (Table 2). Nitroreductase MSMEG_5246 is a protein homologous to M. tuberculosis Acg (Rv2032) in its reduced form and is expressed during 458
hypoxia and dormancy [24]. Phosphomethylpyrimidine kinase participates in thiamine synthesis and metabolism, intermediary metabolism and respiration [22]. Acyl (ACP) desaturase is involved in fatty acid and mycolic acid synthesis [23]; 3-hydroxyacyl-CoA dehydrogenase participates in lipid metabolism and fatty acid oxidation [22] (Table 2). The spot recognized by mAb Mar 2D10 corresponded to a d-ribose-binding periplasmic protein from M. smegmatis with a coverage percentage of 2%. This protein has a role in ribose transport [22] (Table 2). Identification of epitopes in the proteins recognized by Mbv 2A10 and Mav 3H1. The fact that Mbv 2A10 and Mav 3H1 recognized more than one protein suggests the presence of antigenic determinants shared by the recognized proteins, at least under the denaturing conditions present in the immunoblot. To identify these possible epitopes, an in-silico search was conducted using the IEDB software, which allows for database searching of previously identified and characterized epitopes. Only two peptide epitopes were identified in the three proteins recognized by Mbv 2A10, both of which were related to the universal stress family protein, TB31·7 (Table 3). Both sequences have been reported as ligands for the B lymphocyte receptor (BCR). For the proteins recognized by Mav 3H1, one peptide epitope was identified and has been reported as a T lymphocyte receptor (TCR) ligand for the protein 3-hydroxyacyl-CoA dehydrogenase (Table 3). Identification of conserved sequences shared by the proteins recognized by Mbv 2A10 and Mav 3H1. Multiple alignment of the protein sequences recognized by Mbv 2A10 and Mav 3H1 based on in-silico analysis by Clustal W (Fig. 2) and corroborated by T-Coffee (Fig. S1) demonstrated that the three proteins recognized by Mbv 2A10 and the four proteins recognized by Mav 3H1 shared consensus sequences, including the sequences of the epitopes identified using IEDB. The recognition of more than one protein by the mAbs under study could be related to the presence of conserved sequences shared among the proteins (Fig. 2).
Discussion MAbs have been used widely in basic research, diagnostics and clinical therapeutics. The production and characterization of clinically relevant mAbs against the mycobacteria BCG Mexico, M. avium and M. arupense are described in the current study; mAbs selection was performed by identifying the most reactive IgG2a-isotype clones. The IgG2a isotype was sought because these types of antibody play an important role in the T helper type 1 (Th1) adaptive immune response that is protective against Mycobacterium infection [3].
© 2014 British Society for Immunology, Clinical and Experimental Immunology, 177: 454–463
Monoclonal antibodies against mycobacteria Table 2. Identification of proteins recognized by the Mbv 2A10, Mav 3H1 and Mar 2D10 monoclonal antibodies (mAbs) using mass spectrometry.
mAb Mbv 2A10
Spot
Accession no.
Molecular Weight (Da)/pI†
1
gi|31793022
99 452/5·31
2
3
gi|15609760
gi|15607955
31 652/5·45
31 014/5·14
Protein Name function (FC‡) Glycine dehydrogenase
Universal stress protein
Respiration and intermediary metabolism (7)
Regulates growth Indispensable for chronic infection Expressed in hypoxic conditions and oxidative stress (10)
Probable Sulphuric thiosulphate metabolism sulphurtransferase Cyanide CysA2 detoxification Respiration and intermediary metabolism (7)
Gen Locus name 1092983
887442
885449
Mb1863
Mb2656
Mb0838c
Matched peptides Position sequence score 38–73 AVPAGILDTLTDTGAA PGLDSLPPAASEAEALAELR 74–101 ALADANTVAVSM IGQGYYDTHTPPVLLR 173–190 VVVDADVFTQTAAV LATR 191–204 AKPLGIEIVTADLR 205–225 AGLPDGEFFGAIAQL PGASGR 226–239 ITDWSALVQQAHDR 301–315 LVGVSVDSDGTPAYR 384–391 YFDTVLAR 405–412 ANGINLWR 454–467 TSEFLTHPAFTQYR 478–488 ALADKDIALDR 517–530 QHPFAPASDTAGLR 1580–602 DICLIPSSAH GTNAASAALAGMR 602–620 VVVVDCHDNGD VDLDDLR 693–711 TFCIPHGGGG PGVGPVAVR 753–760 MMGAEGLR 761–775 AASLTAITSANYIAR 806–816 LTGITVDDVAK 900–911 EQAAYPLGTAFR 2–25 SSGNSSLGIIVGID DSTAAQVAVR 30–34 DAELR 35–62 KIPLTLVHAVSP EVATWLEVPLPPGVLR 63–69 WQQDHGR 70–77 HLIDDALK 78–85 VVEQASLR 86–109 AGPPTVHSEIV PAAAVPTLVDMSK 110–123 DAVLMVVGC LGSGR 128–138 LLGSVSSGLLR 139–185 HAHCPVVIIH DEDSVMPHPQQAP VLVGVDGSSASELATAI AFDEASR 224–230 LAGWQER 231–238 YPNVAITR 248–252 QLVQR 253–264 SEEAQLVVVGSR 267–286 GGYAGMLVGSVG ETVAQLAR 287–293 TPVIVAR 4–21 CDVLVSATWAES NLHAPK 37–44DHIAGAIK 49–56 TDLQDPVK 58–67 DFVDAQQFSK 68–72 LLSER 100–105 LYGHEK 108–113 LLDGGR 146–158 AFRDEVLAAINVK 165–172 SPDEFSGK 173–200 kILAPAHLPQEQS QRPGHIPGAINVPWSR 201–209 AANEDGTFK 210–216 DEELAK 217–227 LYADAGLDNSK 228–234 ETIAYCR 239–247 SSHTWFVLR 248–256 ELLGHUNVK
© 2014 British Society for Immunology, Clinical and Experimental Immunology, 177: 454–463
31
Global score
Sequence coverage (%)
434
32
733
60
722
58
40 60 90 108 41 89 45 51 35 58 18 24 25 22 7 57 44 39 10 16 30 26 21 61 41 49 60 62
47 56 39 62 95 48 37 61 39 77 29 24 38 46 48 27 43 28 67 14 33 44
459
K. Flores-Moreno et al. Table 2. Continued
mAb Mav 3H1
Spot
Accession no.
Molecular Weight (Da)/pI†
1
gi|118472552
36 220/ 5·19
2
3
4
Mar 2D10
1
gi|118470874
gi|399990000
gi|118468032
gi|118467463
27 975/ 4·81
38 454/5·07
26 301/ 5·23
39 513/ 4·51
Protein Name function (FC‡) Hypothetical protein MSMEG_5246
Phosphomethylpyrimidine kinase
Acyl-(ACP) desaturase
3-hydroxyacyl-CoA dehydrogenase
D-ribose-binding periplasmic protein RbsB
Oxidoreductase activity (–)
Thiamine biosynthesis and metabolism Respiration and intermediary metabolism (–)
Cell growth Mycolic and fatty acids biosynthesis (–)
Glycolysis and fatty acid oxidation Lipid metabolism (–)
Ribose transport (–)
Gen Locus name 4532679
4536806
13426454
4531389
4536608
MSMEG_ 5246
MSMEG_ 0464
MSMEG_ 5619
MSMEG_ 5183
MSMEG_ 4172
Matched peptides Position sequence score 21–31 APSLHNTQPWR 32–40 LIAEDGELK 41–47 LFLDPSR 58–71 EAVMSCGVLLDHLR 72–85 VALAAAGWDTEVQR 114–120 ADAILAR 144–173 LGDGPVHMDTLG EDVREEVAEAAALTESLR 215–224 DFPVAPHSSR 246–257 EDALDAGEGLSK 258–282 VLLECTMSGLATC PVTHVTELHTSR 292–300 DACPQVLVR 301–316 IGLAPALDEVPPPTPR 317–328 RPVDAFLEVRPR 25–46 TFQQLGAYGVGTVT CIVSFDPK 53–78 FVPVEAQVVADQI EAATSAYDLHTVK 79–96 IGMLGTPATIDVVAEGLR 102–112 HIVVDPVLICK 113–127 GQEPGAALDTDTALR 129–145 EILPLATVTTPNLFEAR 146–164 TLSGMDEITTV DDLIEAAR 166–172 IADLGPR 178–194 GGVEFPGSDAVD VLFDR 214–231 VAGAGCTLAAAIT AELAK 232–241 GADVPEAVLR 244–253 EFTTAGIVAR 254–268 VGGNAPFDAVWQGGQ 9–23 ALTLELEPVVAGEMR 64–76 SITDALEILLITK 77–84 DNLAGYHR 123–133 EIDPAANEDVR 168–184 NLQAQITEPVLA SLMGR 185–206 IATDEERHEEF FSNLVSYVLDK 207–218 HRDETVEAIAAR 219–235 AAGLDVIGADIA PYQDK 236–247 VANVAEAGIFDK 258–270 ITAWGLAGEASLK 5–23 DAVAVVTGGASGLGLATTK 25–3849 LLDAGAQVVVIDLK 39–49 GEEVVAELGDR 52–77 FVATDVTDEAG VTEALNVAESLGPVR 78–90 INVNCAGIGNAIK 95–104 NGPFPLDGFR 106–119 VVEVNLIGTFNVIR 128–138 TEPIGPNGEER 139–163 GVIINTASVA AFDGQIGQAAYSASK 164–175 GGVVGMTLPIAR 184–193 VVTIAPGLFK 194–206 TPLLGSLPEEAQK 207–217 SLGAQVPHPAR 218–243 LGDPDEYGA LAQHIIENPMLNGEVIR 353–361 VLDEDLVTK
32 44 27 16 69 10 25
Global score
Sequence coverage (%)
559
53
830
76
485
50
825
84
46
2
31 57 76 20 62 77 17 90 92 55 82 62 33 27 62 7 38 49 25 49 78 26 47 11 38 20 73 87 61 86 101 40 108 71 38 49 35 44 48 44 81 47 93 49
† Molecular weight (Da) and pI were determined using the ProtParam tool, Expasy Bioinformatics Resource Portal. ‡According to BCGList (http://genolist.pasteur.fr/BCGList). FC = functional category. 0 = virulence, detoxification, adaptation; 1 = lipid metabolism; 2 = information pathways; 3 = cell wall and cell processes; 4 = stable RNAs; 5 = insertion sequences and phages; 6 = Pe/PPE; 7 = intermediary metabolism and respiration; 8 = unknown; 9 = regulatory protein; 10 = conserved hypothetical; (–) = not determined. Mbv = mAbs for Mycobacterium bovis bacillus Calmette–Guérin (BCG) Mexico; Mav = mAbs for M. avium; Mar = mAbs for M. arupense.
460
© 2014 British Society for Immunology, Clinical and Experimental Immunology, 177: 454–463
Monoclonal antibodies against mycobacteria Table 3. Epitope prediction of proteins recognized by monoclonal antibodies (mAbs) analysed in silico. Monoclonal antibody Mbv 2A10
Mav 3H1
Protein Universal stress protein family
3-hydroxyacyl-CoA dehydrogenase
Epitopes B cells ID 10168 /PHPQQATDDSGH ID 156380/ MVYGTGGLAYGKVKSAFNLGDD APALHTWSDKTK T cells ID 65596 / TPHPARIGL
Source antigen/organism LMP1 protein/human herpesvirus 4 Outer membrane protein Omp31 Brucella ovis Beta-galactosidase/Escherichia coli
ID = epitope identification key in the Immune Epitope Database and Analysis Resource (IEDB). Mbv = mAbs for Mycobacterium bovis bacillus Calmette–Guérin (BCG) Mexico; Mav = mAbs for M. avium.
Cross-reactivity of the selected mAbs against mycobacteria from the MTb and NTM complexes allowed us to assess the specificity of antigen recognition by the clones. In all cases, there was evidence that the protein extracts from all the assayed mycobacteria were recognized, suggesting the existence of shared antigenic determinants (Table 1). To determine which proteins contained these antigenic determinants, the spots recognized by each mAb clone were identified by immune proteomics, and the respective protein sequences were determined by mass spectrometry. In general, the mAb-recognized proteins were
involved in respiration, metabolism, cell cycle, oxidative stress and dormancy processes [18–24] (Table 2). Interestingly, mAbs Mbv 2A10 and Mav 3H1 recognized three and four spots, respectively. This finding could be due to the fact that the proteins are subject to denaturing conditions on the immunoblot; thus, antigenic determinants that are usually inaccessible in the native protein conformation may be exposed. Another possibility is that the mAbs are able to recognize more than one epitope from different strains [25] or species due to crossreactivity [26].
Fig. 2. Multiple sequence alignment of proteins recognized by monoclonal antibodies 2A10 and 3H1. (a) Alignment of protein sequences recognized by the Mbv monoclonal antibody (mAb): glycine dehydrogenase (glycine), universal stress protein (universal), and thiosulphate sulphurtransferase CysA2 (probable). (b) Alignment of the sequences recognized by the Mav mAb: nitroreductase MSMEG_5246 (hypothetical), phosphomethylpyrimidine kinase (phospho), acyl (ACP) desaturase (acyl) and 3-hydroxyacyl-CoA-dehydrogenase (3-hydrox). Red squares indicate peptide sequences that were identified as epitopes in universal stress protein (a) and in 3-hydroxyacyl-CoA dehydrogenase (b). These peptides are among consensus sequences identified previously in multiple alignments.
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Epitope prediction in the proteins recognized by mAb Mbv 2A10 allowed us to identify one B lymphocyte epitope in the universal stress protein, Rv2623 and one T lymphocyte epitope in 3-hydroxyacyl-CoA dehydrogenase [27]. Sequence identification of these epitopes could be used to produce peptides to be assayed for their effect on the adaptive immune response against the Mycobacterium from which the peptide originated. Conversely, it is noteworthy that the IEBD software did not find epitopes for all the mAb-recognized proteins. The reason for this could be that these epitopes have not yet been reported, or they represent antigen recognition in a denatured state. Finally, determining the epitope sequences using multiple sequence alignment by Clustal W allowed us to identify the conserved motifs present in the epitope sequences that were recognized by both mAbs. We suggest that these epitopes are probable antigenic determinants and identification of the targets is a perspective. In summary, the mAbs generated against M. bovis BCG Mexico, M. avium and M. arupense recognized several mycobacteria from the MTb and NTM complexes. We propose identifying the epitopes recognized for these mAbs and evaluating them as diagnostic tools for clinically relevant mycobacteriosis.
Acknowledgements CONACYT 140998 and DGAPA IN214713-2 supported this study. K. F.-M. is the recipient of a CONACyT México doctoral scholarship (Reg. no. 261862); J. S. C.-M. is the recipient of a CONACyT México master in Science scholarship (Reg. no. 288831); D. M. M.-R is the recipient of a postdoctoral fellowship from DGAPA, UNAM; and P. O. is the recipient of a postdoctoral fellowship from CONACYT (140998).
Disclosures The authors do not have any conflicts of interest to declare.
Author contributions K. F.-M., J. S. C.-M. and B. M.-R. participated in the performance of the experiments; D. M. M. R. and P. O. participated in the analysis results and writing of the paper; A. I. C.-R. and Y. L.-V. participated in the design of the experiments and writing of the paper.
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3 Milstien J. The immunological basis for immunization series, module 5. Tuberculosis. Geneva: World Health Organization, 1993. 4 Liu J, Tran V, Leung AS, Alexander DC, Zhu B. BCG vaccines: their mechanisms of attenuation and impact on safety and protective efficacy. Hum Vaccin 2009; 5:70–8. 5 Orduña P, Cevallos MA, de León SP et al. Genomic and proteomic analyses of Mycobacterium bovis BCG Mexico 1931 reveal a diverse immunogenic repertoire against tuberculosis infection. BMC Genomics 2011; 12:493. 6 Porteous NB, Redding SW, Jorgensen JH. Isolation of nontuberculosis mycobacteria in treated dental unit waterlines. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004; 98:40–4. 7 Katoch VM. Infections due to non-tuberculous mycobacteria (NTM). Indian J Med Res 2004; 120:290–304. 8 Henry MT, Inamdar L, O’Riordain D et al. Nontuberculous mycobacteria in non-HIV patients: epidemiology, treatment and response. Eur Respir J 2004; 23:741–6. 9 Marras T, Chedore P, Ying AM, Jamieson F. Isolation prevalence of pulmonary non-tuberculous mycobacteria in Ontario, 1997– 2003. Thorax 2007; 62:661–6. 10 Castillo-Rodal AI, Mazari-Hiriart M, Lloret-Sánchez LT, Sachman-Ruiz B, Vinuesa P, López-Vidal Y. Potentially pathogenic nontuberculous mycobacteria found in aquatic systems. Analysis from a reclaimed water and water distribution system in Mexico City. Eur J Clin Microbiol Infect Dis 2012; 31:683–94. 11 Falkinham JO III. Epidemiology of infection by nontuberculous mycobacteria. Clin Microbiol Rev 1996; 9:177–215. 12 Tsang AY, Denner JC, Brennan PJ, McClatchy JK. Clinical and epidemiological importance of typing of Mycobacterium avium complex isolates. J Clin Microbiol 1992; 30:479–84. 13 Masaki T, Ohkusu K, Hata H et al. Mycobacterium kumamototense sp. nov. recovered from clinical specimen and the fist isolation report of Mycobacterium arupense in Japan: novel slowly growing nonchromogenic clinical isolates related to Mycobacterium terrae complex. Microbiol Immunol 2006; 50:889–97. 14 Cloud JL, Meyer JJ, Pounder JI et al. Mycobacterium arupense sp. nov., a non-chromogenic bacterium isolated from clinical specimens. Int J Syst Evol Microbiol 2006; 56:1413–8. 15 World Health Organization (WHO). The stop TB strategy. Geneva: WHO; 2006. 16 Rodríguez-Alvarez M, Mendoza-Hernández G, Encarnación S, Calva JJ, López-Vidal Y. Phenotypic differences between BCG vaccines at the proteome level. Tuberculosis 2009; 89:126–35. 17 Kinter CS, Lundie JM, Patel H, Rindler PM, Szweda LI, Kinter M. A quantitative proteomic profile of the Nrf2-mediated antioxidant response of macrophages to oxidized LDL determined by multiplexed selected reaction monitoring. PLoS One 2012; 7:e50016. 18 Eleuterio M, Hutter B, Murugasu-Oei B, Dick T. Oxygen depletion-induced dormancy in Mycobacterium bovis BCG. J Bacteriol 1999; 181:2252–6. 19 Choi GE, Eom SH, Jung KH et al. CysA2: a candidate serodiagnostic marker for Mycobacterium tuberculosis infection. Respirology 2010; 15:636–42. 20 Florczyk MA, McCue LA, Stack RF, Hauer CR, McDonough KA. Identification and characterization of mycobacterial proteins differentially expressed under standing and shaking culture conditions, including Rv2623 from a novel class of putative ATPbindingproteins. Infect Immun 2001; 69:5777–85.
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Monoclonal antibodies against mycobacteria 21 Drumm JE, Mi K, Bilder P et al. Mycobacterium tuberculosis universal stress protein Rv2623 regulates bacillary growth by ATPbinding: requirement for establishing chronic persistent infection. PLOS Pathog 2009; 5:e1000460. 22 Fleischmann RD, Dodson RJ, Haft DH, Merkel JS, Nelson WC, Fraser CM. Direct submission. Bethesda, MD: National Center for Biotechnology Information, NIH; 2006. 23 Gallien S, Perrodou E, Carapito C et al. Ortho-proteogenomics: multiple proteomes investigation through orthology and a new MS-based protocol. Genome Res 2009; 19:128–35. 24 Chauviac FX, Bommer M, Yan J et al. Crystal structure of reduced MsAcg, a putative nitroreductase from Mycobacterium smegmatis and a close homologue of Mycobacterium tuberculosis Acg. J Biol Chem 2012; 287:44372–83. 25 Nakao S, Mori S, Kondo K, Matsumoto K, Yoshikawa H, Kanda T. Monoclonal antibodies recognizing cross-neutralization epitopes in human papillomavirus 16 minor capsid protein L2. Virology 2012; 434:110–7. 26 Petyaev IM, Zigangirova NA, Tsibezov VV, Ross A, Bashmakov YK. Monoclonal antibodies against lipopolysaccharide of Chlamydia trachomatis with cross reactivity to human ApoB. Hybridoma (Larchmt) 2011; 30:131–6. 27 Kim Y, Sette A, Peters B. Applications for T-cell epitope queries and tools in the Immune Epitope Database and Analysis Resource. J Immunol Methods 2011; 374:62–9.
Supporting information Additional Supporting information may be found in the online version of this article at the publisher’s web-site: Fig. S1. Multiple sequence alignment of proteins recognized by monoclonal antibodies 2A10 and 3H1 with T-Coffee. (a) Alignment of protein sequences recognized by the Mbv monoclonal antibody (mAb): glycine dehydrogenase (glycine), universal stress protein (universal) and thiosulphate sulphurtransferase CysA2 (CysA2). (b) Alignment of the sequences recognized by the Mav mAb: nitroreductase MSMEG_5246 (hypothetical), phosphomethylpyrimidine kinase (phospho), acyl (ACP) desaturase (acyl) and 3hydroxyacyl-CoA-dehydrogenase (3-hydrox). Black squares indicate peptide sequences that were identified as epitopes in universal stress protein (a) and in 3-hydroxyacyl-CoA dehydrogenase (b).
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Clinical and Experimental Immunology
O R I G I N A L A RT I C L E
doi:10.1111/cei.12309
Potential cross-reactivity of monoclonal antibodies against clinically relevant mycobacteria
K. Flores-Moreno, J. S. Celis-Meneses, D. M. Meneses-Ruiz, A. I. Castillo-Rodal, P. Orduña, B. A. Montiel and Y. López-Vidal
Summary
Nacional Autónoma de México, Facultad de Medicina, Programa de Inmunología Molecular
Tuberculosis is a disease caused by the Mycobacterium tuberculosis complex (MTb). In 2011, global mortality due to tuberculosis was 1·4 million individuals. The only available vaccine is the attenuated M. bovis [bacillus Calmette–Guérin (BCG)] strain, which confers variable protection against pulmonary tuberculosis. Some widely distributed non-tuberculous mycobacteria (NTM), such as M. avium and M. arupense, are also potential pathogens for humans. This work aimed to produce and characterize monoclonal antibodies against the M. bovis BCG Mexico strain of the MTb, M. avium subs. hominissuis and the M. arupense strain from NTM. Hybridomas were produced from splenocytes of BALB/c female mice immunized with radiation-inactivated mycobacteria, and the immunoglobulin (Ig)G2a antibody-producing clones with the highest antigenic recognition were selected. The selected clones, Mbv 2A10 for M. bovis BCG Mexico, Mav 3H1 for M. avium and Mar 2D10 for M. arupense, were used in further studies. Enzyme-linked immunosorbent assay (ELISA) and immune proteomics analyses characterized the clones as having the highest crossreactivity with mycobacteria. Using mass spectrometry, a number of proteins recognized by the monoclonal antibody (mAb) clones were identified. These proteins had roles in metabolic processes, hypoxia, cell cycle and dormancy. In addition, a Clustal W and Immune Epitope Database (IEDB) in-silico analysis was performed in protein sequences that result in the conserved regions within probability epitopes that could be recognized for Mbv2A10 and Mav3H1 clones.
Microbiana. Edificio de investigación. 4to piso, México DF 04510, Mexico. E-mail: [email protected]
Keywords: BCG, mycobacteria
Programa de Inmunología Molecular Microbiana, Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, México City, México
Accepted for publication 21 February 2014 Correspondence: Y. López-Vidal, Universidad
Introduction Mycobacteria are the causal agents of infectious diseases (mycobacteriosis) in several species, including humans. The best-known mycobacterial disease is tuberculosis, which currently affects one-third of the world population and causes 1·4 million deaths annually [1]. The main aetiological agent of tuberculosis is Mycobacterium tuberculosis; this species is a member of the M. tuberculosis complex (MTb). M. bovis, a pathogenic species in humans and cattle, is also part of MTb, and is the source of the only available vaccine, attenuated bacillus Calmette–Guérin (BCG), against severe forms of tuberculosis [2–4]. Among the vaccine strains, 454
epitopes,
monoclonal
antibodies,
non-tuberculous
BCG Mexico has genomic and immune proteomic traits that make it desirable as an effective vaccine against M. tuberculosis [5]. Non-tuberculosis mycobacteria (NTM) are widely distributed [6,7] and are recognized as pathogenic agents [8,9]. Our group has isolated NTM of clinical relevance from water reservoirs used by humans in Mexico City and nearby areas [10]. These NTM isolates consisted of strains from the M. avium complex, regarded as the most pathogenic in humans, causing 70% of NTM-related diseases [8,9,11,12], and M. arupense, which has been isolated from lung, osteoarticular tissue and skin clinical samples [13,14].
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Monoclonal antibodies against mycobacteria
Due to the wide distribution and impact upon global public health of mycobacteria, control of related infections requires the development of prophylactic, diagnostic and therapeutic tools. In this regard, the World Health Organization has issued a plan of action to coordinate efforts and significantly reduce tuberculosis by 2015 [15]. This work aimed to produce and characterize monoclonal antibodies (mAbs) against the vaccine strain M. bovis BCG Mexico and environmental isolates of M. avium subsp. hominissuis (M. avium) and M. arupense to evaluate their potential as biomedical tools. Additionally, the proteins recognized by the anti-mycobacterial clones were identified and, using in-silico analysis, allowed us to predict the presence of epitopes and sequences conserved between the proteins recognized by each mAb Mbv 2A10 and Mav 3H1. Finally, we determined that the mAb generated have potential cross-reactivity with different species from the genus Mycobacterium.
Materials and methods Cell culture and mycobacterial preparation for immunization The M. avium and M. arupense strains were obtained from the Programme of Microbial Molecular Immunology and were initially isolated from irrigation and drinking water in Mexico City and nearby areas. Both strains were recovered in 7H10 culture medium (Difco, Detroit, MI, USA) and subsequently subcultured in Sauton medium and 10% albumin–dextrose–catalase (ADC; Becton Dickinson, San Jose, CA, USA)-supplemented Middlebrook 7H9 medium (Difco), respectively, at 37°C under continuous agitation and 5% CO2. The BCG Mexico 1931 strain, provided by the Laboratorios de Biológicos y Reactivos de México S.A. de C.V. (BIRMEX), was cultured in Sauton medium under the same conditions described above for 20 days. The bacterial inoculum for immunization was obtained from cultures in the logarithmic growth stage (10 days for M. arupense and 20 days for M. bovis BCG Mexico and M. avium) and harvested by centrifugation at 5000 g for 10 min. The cell pellets were resuspended twice in isotonic saline solution (ISS). Finally, mycobacteria were inactivated by irradiation with 12 kGy at the Instituto Nacional de Ciencias Nucleares, UNAM. The Sp2/0-Ag14 cell line (ATCC, CRL-1581) was cultured in DMEM (Gibco, Carlsbad, CA, USA) medium supplemented with 10% fetal bovine serum (Gibco), kept at a cell density of 5 × 104 to 5 × 105 cells/ml, and incubated at 37°C, 95% RH and 5% CO2.
Immunization and antibody kinetics Five female BALB/c mice, 6–8 weeks of age, were used for immunization of each mycobacterial strain. Mice were immunized intraperitoneally four times every 15 days with
2 × 106–5 × 107 bacterial inocula in 200 μl of ISS. Five mice were immunized with sterile ISS as a control. Before immunization, a blood sample was taken from the mouse maxillary vein to assess antibodies in sera. Enzyme-linked immunosorbent assay (ELISA) was used to assess the specificity and isotype of the mAbs generated against M. bovis BCG Mexico, M. avium and M. arupense. Briefly, 5 μg/ml of protein extract from the mycobacterium under study was obtained by overnight sonication in carbonate buffer solution at 4°C and plated onto 96-well plates. The samples were blocked with 1% albumin at 37°C, and fivefold dilutions of mouse sera taken at various times after immunization (0, 15, 30, 45 and 60 days) were added. Microplates were incubated at 37°C for 1 h. Horseradish peroxidase (HRP)-linked rabbit anti-mouse IgG2a antibodies were added at a 1 : 2000 dilution to identify immune complexes using chemiluminescent HRP (Millipore, Billerica, MA, USA) as a substrate. All serum samples were run in triplicate, and microplate luminescence was read at 560 nm using a Genius Plus machine and Magellan software.
Clone production and selection of mAbs against mycobacteria BCG Mexico and M. avium clones were obtained from mouse splenocytes, which produced the highest antibody titres. Briefly, 1 ml of polyethylene glycol (PEG) was added drop-wise to 1 × 108 splenocytes and 2 × 107 Sp2/0-Ag14 (ATCC, CRL-1581) cells over 60 s. Dulbecco’s modified Eagle’s medium (DMEM) (4 ml) was then added, and the cells were agitated for 4 min. Ten millilitres of DMEM medium was added, and the cells were incubated in a waterbath at 37°C for 15 min. Finally, 30 ml of supplemented DMEM medium was added. The cell suspension was transferred to a cell culture bottle and incubated at 37°C with 5% CO2 for 24 h. The cells were harvested by centrifugation at 400 g at 37°C for 10 min and resuspended in 10 ml of supplemented DMEM plus 90 ml of hypoxanthine–aminopterin–thymidine (HAT) medium. Cell suspensions were plated in 96-well microplates at 60 μl/well, and the plates were incubated for 8 days under the conditions described above. Hypoxanthine–thymidine (HT) medium at 150 μl/well was added, and the microplates were incubated for another 4 days. After this incubation, 100 μl of supernatant was collected, and positive clones were selected by ELISA. These clones were transferred to 24-well plates in 1 ml of HT medium, which was gradually replaced by DMEM medium. A second screening was performed based on the proliferative capacity and reactivity of the clones. From this screening, only the most reactive clone as determined by ELISA was used for further characterization. Finally, M. arupense mAbs were obtained using the ClonaCell kit (Stemcell cat. no. 03804), according to the manufacturer’s instructions.
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Characterization of mAbs To assess the cross-recognition capability of the mAbs generated against M. bovis BCG Mexico, M. avium and M. arupense, reactivities were determined against different MTb complex strains, such as M. tuberculosis H37Rv, M. microti, M. bovis BCG Mexico, M. bovis BCG Phipps and M. bovis BCG Japan, as well as against NTM strains such as M. avium sp., M. arupense and M. abscessus. Crossreactivity was assessed by ELISA following the procedure described above. The cut-off optical density (OD) was calculated as the mean relative luminescence units (RLU) of negative samples plus two standard deviations.
Isolation of protein extracts and electrophoresis on 2D-polyacrylamide gel electrophoresis (2D-PAGE) gels A bacterial suspension from cultures in the mid-logarithm growth stage was diluted in 20 mM phenylmethylsulphonyl fluoride (PMSF) (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) and subjected to 15 60-s pulses, as reported previously [16]. Protein concentration of the obtained extracts was quantified by the Bradford method. Electrophoresis on two-dimensional (2D) gels was performed in duplicate using gel strips with an immobiliszed pH gradient from 4 to 7 [ReadyStripTM immobilized pH gradient (IPG strips); Bio-Rad Laboratories, Hercules, CA, USA]. Protein aliquots of 100 μg were suspended in a rehydration solution (7 M urea, 2 M thiourea, 1 M dithiothreitol (DTT), 4% CHAPS {3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate}, 1% ampholines and 0·001% bromophenol blue) to a final volume of 180 μl. IPG strips were rehydrated at a pH range of 4–7 for 16 h. Electrophoresis was then performed in a Multiphore II Electrophoresis Unit (Amersham Biosciences, Uppsala, Sweden) at 17°C as follows: 500 V for 30 min, 1000 V for 30 min, 1500 V for 30 min, 2000 V for 30 min and 2500 V for 25 h until 52 000 V/h were completed. After isoelectric focusing, the strip was immersed in an equilibrium buffer [6 M urea, 50 mM Tris-HCl, pH 8·8, 30% glycerol, 2% sodium dodecyl sulphate (SDS)] with 97 mM DTT for 15 min and 203 mM indoleacetic acid (IAA) for 15 min. For the second dimension, the strip was placed on a 12% acrylamide gel in a Hoeffer SE 600 (Pharmacia Biotech, Piscataway, NJ, USA) chamber, and electrophoresis was run with gradual voltage increases of 50 V for 30 min, 100 V for 2 h and 150 V until the electrophoresis was finished. The proteome of M. bovis BCG Mexico, M. avium and M. arupense was obtained by silver staining the 2D gel after electrophoresis. The gel was fixed with penthydrate sodium thiosulphate (0·2 g/l) for 1 min. Silver nitrate (0·2 g/100 ml) was then added, and the gel was developed with sodium carbonate anhydrous (3 g/100 ml). The image was digitalized using a Molecular Imager GS-800TM calibrated 456
densitometer (Bio-Rad Laboratories) and analysed using the Quantity One TM 1-D analysis software (Bio-Rad Laboratories).
Western blot analysis A replicate of the 2D-polyacrylamide gel electrophoresis (2D-PAGE) gel used for proteome determination was transferred to a 0·45-μm pore polyvinylidene difluoride (PVDF) membrane (Hybond-P PVDF Membrane; Amersham) activated previously with methanol (100%) for 10 s and incubated in Bjerrum and Schafer-Nielsen transfer solution for 10 min. The transfer was performed in a Trans-Blot SD chamber (Semi-Dry Electrophoretic Transfer Cell; Bio-Rad Laboratories) at a constant potential difference of 10 V for 1 h. Once the transfer was complete, the membrane was blocked with 4% milk at 4°C overnight. The membrane was then incubated with the selected mAbs against M. bovis BCG Mexico (Mbv 2A10), M. avium (Mav 3H1) or M. arupense (Mar 2D10) at fivefold dilutions. HRP-labelled rabbit IgG2a anti-mouse antibodies were used as secondary antibodies at a 1 : 2000 dilution. The obtained immune complexes were developed by HRP chemiluminescence (Millipore).
Mass spectrometry identification of proteins recognized by mAbs All immunoblot spots identified with mAbs against each mycobacterium were obtained from a Coomassie Brilliant Blue-stained 2D-PAGE gel. The spots were digested as reported by Kinter et al. [17]. Briefly, gel fragments for each sample were washed and destained overnight in a methanol/acetic acid 50%/5% solution at room temperature, and were then dehydrated with acetonitrile and dried by vacuum centrifugation. The proteins were digested with trypsin; the resulting peptides were recovered by successive extractions with ammonium bicarbonate and acetonitrile/ formic acid. The extract was concentrated for mass spectrometry analysis. Finally, the peptide sequence was determined using a hybrid triple linear/quadrupole ion trap mass spectrometer (3200 Q TRAP; Applied Biosystems, Life Technologies Corp., Foster City, CA, USA). The protein sequences obtained by mass spectrometry were searched in the Mascot 52 database from the mycobacterial complex under study.
Epitope search with mAb-recognized proteins In-silico prediction of the proteins identified by the mAbs generated against M. bovis BCG Mexico, M. avium and M. arupense was performed using the Immune Epitope Database (IEDB) bioinformatics software (IEDB; Bioinformatics, Toronto, ON, Canada).
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Monoclonal antibodies against mycobacteria Table 1. Cross-reactivity of the Mbv 2A10, Mav 3H1 and Mar 2D10 monoclonal antibody (mAb) clones against mycobacteria in the MTb and NTM complexes. mAbs reactivity (RLU) Complex MTb
NTM
Mycobacteria
Mbv 2A10
Mav 3H1
Mar 2D10
M. tuberculosis H37Rv M. microti M. bovis BCG Phipps M. bovis BCG Japan M. bovis BCG Mexico M. nonchromogenicum M. avium subs. Hominissuis M. abscessus M. arupense
469 558 566 673 2235 377 588 988 ND
218 225 211 218 342 172 426 301 ND
210 179 307 237 261 354 311 396 962
BCG = bacillus Calmette–Guérin; MTb = Mycobacterium tuberculosis complex; Mbv = mAbs for M. bovis BCG Mexico; Mav = mAbs for M. avium; Mar = mAbs for M. arupense; NTM = non-tuberculous mycobacteria; n.d. = not determined.
Multiple alignment of proteins recognized by the Mbv 2A10 and Mav 3H1 mAbs
The Mbv 2A10, Mav 3H1 and Mar 2D10 mAbs showed reactivity against MTb and NTM strains
A multiple sequence alignment of proteins recognized by mAb Mbv 2A10 (glycine dehydrogenase, stress protein Rv2623 and thiosulphate sulphurtransferase CysA2) and those recognized by mAb Mav 3H1 [nitroreductase MSMEG_5246, phosphomethylpyrimidine kinase, acyl (ACP) desaturase, and 3-hydroxyacyl-CoA dehydrogenase] was performed using Clustal W version 2·1 software.
To evaluate the specificity of the mAbs produced by the selected clones, cross-reactivity to antigen extracts from other mycobacteria in the MTb (M. tuberculosis H37Rv, M. microti, BCG Phipps, BCG Japan and BCG Mexico) was determined according to the cut-off threshold (calculated as the mean of the RLU of the negative control plus two standard deviations). Table 1 shows the extent of antigenic recognition (in RLU) by the mAbs generated against BCG Mexico (Mbv 2A10), M. avium (Mav 3H1) and M. arupense (Mar 2D10). All mAbs preferentially recognized protein extracts from the mycobacteria against which they were produced: mAb Mbv 2A10 (2235 UR for BCG Mexico), Mav 3H1 (426·3 UR for M. avium) and Mar 2D10 (962·5 UR for M. arupense). However, all mAbs also showed crossrecognition of MTb and NTM strains, albeit with lesser reactivity (Table 1).
Results Selection of mAbs against BCG Mexico, M. avium and M. arupense Sera from mice immunized at various times (0, 15, 30, 45 and 60 days) with inactivated BCG Mexico, M. avium or M. arupense were collected to evaluate the kinetics of immunoglobulin (Ig)G2a antibody induction after each immunization by ELISA (data not shown). The sera of mice showing the highest antibody titres against each mycobacterium were selected, and the splenocytes from these mice were fused with myeloma Sp2/0-Ag14 cells. From this fusion, 1440 clones producing antibodies against BCG Mexico, 1415 clones producing antibodies against M. avium and 297 clones producing antibodies against M. arupense were obtained. An initial screening was performed by ELISA to evaluate the antibody reactivity of each clone, and the clones with the highest titres were selected: 110 clones (7·6%) for M. bovis BCG Mexico, 98 clones (6·9%) for M. avium and 11 clones (3·7%) for M. arupense. A second screening was performed by assessing the antigen-specific proliferation and reactivity of the antibodies against BCG Mexico and M. avium. Ten clones (0·6%) for BCG Mexico and six clones (0·4%) for M. avium were selected. Finally, ELISA was used to select the most reactive clone to each mycobacterial extract. The selected clones were Mbv 2A10 for BCG Mexico, Mav 3H1 for M. avium and Mar 2D10 for M. arupense.
Proteins related to metabolism, growth regulation, dormancy and ribose transport were recognized by the Mbv 2A10, Mav 3H1 and Mar 2D10 mAbs To identify the proteins recognized by the mAbs produced against BCG Mexico, M. avium and M. arupense, electrophoresis of total protein extracts was performed on two 2D-PAGE gels with a pH range of 4–7, performed simultaneously and with one using an immunoblot. The recognition of immunodominant proteins for each separate antibody was detected (data not shown). Three spots were identified for BCG Mexico, four for M. avium and one for M. arupense. The proteins recognized by immunoblot were identified in the mycobacterial proteome from the gel performed in parallel and were based on molecular weight (MW) and isoelectric point (pI). For BCG Mexico, the recognized proteins were in a pH range of 5–5·5 and a MW range of 30–100 kDa (Fig. 1). For M. avium, the recognized proteins were in a pH range of
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pl
7
1
75 50 37
2 3
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Fig. 1. Representative two-dimensional (2D) electrophoresis of total protein extracts from Mycobacterium bovis bacillus Calmette–Guérin (BCG) Mexico. Total protein extracts from M. bovis BCG Mexico were resolved on 2D gels in a pH gradient of 4–7. The spots recognized by monoclonal antibodies against mycobacterium are indicated with a circle and numbered.
4·9–5·9 and a MW range of 25–40 kDa (data not shown). The sole recognized protein for M. arupense had a MW of approximately 30 kDa and pH of 4·3 (data not shown). The identified spots were processed, and their homology with sequences in the Mascot database was identified by mass spectrometry. Among the identified proteins for mAb Mbv 2A10, spot 1 corresponded to M. bovis glycine dehydrogenase, spot 2 corresponded to stress protein Rv2623 from M. tuberculosis H37Rv and spot 3 corresponded to thiosulphate sulphurtransferase CysA2 from M. tuberculosis H37Rv with coverage percentages of 32, 60 and 58%, respectively. Functionally, glycine dehydrogenase and thiosulphate sulphurtransferase CysA2 participate in intermediary metabolism and respiration [18,19]. Stress protein Rv2623 is involved in bacterial growth regulation and is required for mycobacteria to enter dormancy [20,21] (Table 2). With regard to the spots recognized by mAb Mav 3H1, spot 1 corresponded to nitroreductase MSMEG_5246, spot 2 corresponded to a phosphomethylpyrimidine kinase [22], spot 3 corresponded to an acyl [ACP] desaturase [23] and spot 4 corresponded to 3-hydroxyacyl-CoA-dehydrogenase [22], all from M. smegmatis, with coverage percentages of 53, 76, 50 and 84%, respectively (Table 2). Nitroreductase MSMEG_5246 is a protein homologous to M. tuberculosis Acg (Rv2032) in its reduced form and is expressed during 458
hypoxia and dormancy [24]. Phosphomethylpyrimidine kinase participates in thiamine synthesis and metabolism, intermediary metabolism and respiration [22]. Acyl (ACP) desaturase is involved in fatty acid and mycolic acid synthesis [23]; 3-hydroxyacyl-CoA dehydrogenase participates in lipid metabolism and fatty acid oxidation [22] (Table 2). The spot recognized by mAb Mar 2D10 corresponded to a d-ribose-binding periplasmic protein from M. smegmatis with a coverage percentage of 2%. This protein has a role in ribose transport [22] (Table 2). Identification of epitopes in the proteins recognized by Mbv 2A10 and Mav 3H1. The fact that Mbv 2A10 and Mav 3H1 recognized more than one protein suggests the presence of antigenic determinants shared by the recognized proteins, at least under the denaturing conditions present in the immunoblot. To identify these possible epitopes, an in-silico search was conducted using the IEDB software, which allows for database searching of previously identified and characterized epitopes. Only two peptide epitopes were identified in the three proteins recognized by Mbv 2A10, both of which were related to the universal stress family protein, TB31·7 (Table 3). Both sequences have been reported as ligands for the B lymphocyte receptor (BCR). For the proteins recognized by Mav 3H1, one peptide epitope was identified and has been reported as a T lymphocyte receptor (TCR) ligand for the protein 3-hydroxyacyl-CoA dehydrogenase (Table 3). Identification of conserved sequences shared by the proteins recognized by Mbv 2A10 and Mav 3H1. Multiple alignment of the protein sequences recognized by Mbv 2A10 and Mav 3H1 based on in-silico analysis by Clustal W (Fig. 2) and corroborated by T-Coffee (Fig. S1) demonstrated that the three proteins recognized by Mbv 2A10 and the four proteins recognized by Mav 3H1 shared consensus sequences, including the sequences of the epitopes identified using IEDB. The recognition of more than one protein by the mAbs under study could be related to the presence of conserved sequences shared among the proteins (Fig. 2).
Discussion MAbs have been used widely in basic research, diagnostics and clinical therapeutics. The production and characterization of clinically relevant mAbs against the mycobacteria BCG Mexico, M. avium and M. arupense are described in the current study; mAbs selection was performed by identifying the most reactive IgG2a-isotype clones. The IgG2a isotype was sought because these types of antibody play an important role in the T helper type 1 (Th1) adaptive immune response that is protective against Mycobacterium infection [3].
© 2014 British Society for Immunology, Clinical and Experimental Immunology, 177: 454–463
Monoclonal antibodies against mycobacteria Table 2. Identification of proteins recognized by the Mbv 2A10, Mav 3H1 and Mar 2D10 monoclonal antibodies (mAbs) using mass spectrometry.
mAb Mbv 2A10
Spot
Accession no.
Molecular Weight (Da)/pI†
1
gi|31793022
99 452/5·31
2
3
gi|15609760
gi|15607955
31 652/5·45
31 014/5·14
Protein Name function (FC‡) Glycine dehydrogenase
Universal stress protein
Respiration and intermediary metabolism (7)
Regulates growth Indispensable for chronic infection Expressed in hypoxic conditions and oxidative stress (10)
Probable Sulphuric thiosulphate metabolism sulphurtransferase Cyanide CysA2 detoxification Respiration and intermediary metabolism (7)
Gen Locus name 1092983
887442
885449
Mb1863
Mb2656
Mb0838c
Matched peptides Position sequence score 38–73 AVPAGILDTLTDTGAA PGLDSLPPAASEAEALAELR 74–101 ALADANTVAVSM IGQGYYDTHTPPVLLR 173–190 VVVDADVFTQTAAV LATR 191–204 AKPLGIEIVTADLR 205–225 AGLPDGEFFGAIAQL PGASGR 226–239 ITDWSALVQQAHDR 301–315 LVGVSVDSDGTPAYR 384–391 YFDTVLAR 405–412 ANGINLWR 454–467 TSEFLTHPAFTQYR 478–488 ALADKDIALDR 517–530 QHPFAPASDTAGLR 1580–602 DICLIPSSAH GTNAASAALAGMR 602–620 VVVVDCHDNGD VDLDDLR 693–711 TFCIPHGGGG PGVGPVAVR 753–760 MMGAEGLR 761–775 AASLTAITSANYIAR 806–816 LTGITVDDVAK 900–911 EQAAYPLGTAFR 2–25 SSGNSSLGIIVGID DSTAAQVAVR 30–34 DAELR 35–62 KIPLTLVHAVSP EVATWLEVPLPPGVLR 63–69 WQQDHGR 70–77 HLIDDALK 78–85 VVEQASLR 86–109 AGPPTVHSEIV PAAAVPTLVDMSK 110–123 DAVLMVVGC LGSGR 128–138 LLGSVSSGLLR 139–185 HAHCPVVIIH DEDSVMPHPQQAP VLVGVDGSSASELATAI AFDEASR 224–230 LAGWQER 231–238 YPNVAITR 248–252 QLVQR 253–264 SEEAQLVVVGSR 267–286 GGYAGMLVGSVG ETVAQLAR 287–293 TPVIVAR 4–21 CDVLVSATWAES NLHAPK 37–44DHIAGAIK 49–56 TDLQDPVK 58–67 DFVDAQQFSK 68–72 LLSER 100–105 LYGHEK 108–113 LLDGGR 146–158 AFRDEVLAAINVK 165–172 SPDEFSGK 173–200 kILAPAHLPQEQS QRPGHIPGAINVPWSR 201–209 AANEDGTFK 210–216 DEELAK 217–227 LYADAGLDNSK 228–234 ETIAYCR 239–247 SSHTWFVLR 248–256 ELLGHUNVK
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31
Global score
Sequence coverage (%)
434
32
733
60
722
58
40 60 90 108 41 89 45 51 35 58 18 24 25 22 7 57 44 39 10 16 30 26 21 61 41 49 60 62
47 56 39 62 95 48 37 61 39 77 29 24 38 46 48 27 43 28 67 14 33 44
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K. Flores-Moreno et al. Table 2. Continued
mAb Mav 3H1
Spot
Accession no.
Molecular Weight (Da)/pI†
1
gi|118472552
36 220/ 5·19
2
3
4
Mar 2D10
1
gi|118470874
gi|399990000
gi|118468032
gi|118467463
27 975/ 4·81
38 454/5·07
26 301/ 5·23
39 513/ 4·51
Protein Name function (FC‡) Hypothetical protein MSMEG_5246
Phosphomethylpyrimidine kinase
Acyl-(ACP) desaturase
3-hydroxyacyl-CoA dehydrogenase
D-ribose-binding periplasmic protein RbsB
Oxidoreductase activity (–)
Thiamine biosynthesis and metabolism Respiration and intermediary metabolism (–)
Cell growth Mycolic and fatty acids biosynthesis (–)
Glycolysis and fatty acid oxidation Lipid metabolism (–)
Ribose transport (–)
Gen Locus name 4532679
4536806
13426454
4531389
4536608
MSMEG_ 5246
MSMEG_ 0464
MSMEG_ 5619
MSMEG_ 5183
MSMEG_ 4172
Matched peptides Position sequence score 21–31 APSLHNTQPWR 32–40 LIAEDGELK 41–47 LFLDPSR 58–71 EAVMSCGVLLDHLR 72–85 VALAAAGWDTEVQR 114–120 ADAILAR 144–173 LGDGPVHMDTLG EDVREEVAEAAALTESLR 215–224 DFPVAPHSSR 246–257 EDALDAGEGLSK 258–282 VLLECTMSGLATC PVTHVTELHTSR 292–300 DACPQVLVR 301–316 IGLAPALDEVPPPTPR 317–328 RPVDAFLEVRPR 25–46 TFQQLGAYGVGTVT CIVSFDPK 53–78 FVPVEAQVVADQI EAATSAYDLHTVK 79–96 IGMLGTPATIDVVAEGLR 102–112 HIVVDPVLICK 113–127 GQEPGAALDTDTALR 129–145 EILPLATVTTPNLFEAR 146–164 TLSGMDEITTV DDLIEAAR 166–172 IADLGPR 178–194 GGVEFPGSDAVD VLFDR 214–231 VAGAGCTLAAAIT AELAK 232–241 GADVPEAVLR 244–253 EFTTAGIVAR 254–268 VGGNAPFDAVWQGGQ 9–23 ALTLELEPVVAGEMR 64–76 SITDALEILLITK 77–84 DNLAGYHR 123–133 EIDPAANEDVR 168–184 NLQAQITEPVLA SLMGR 185–206 IATDEERHEEF FSNLVSYVLDK 207–218 HRDETVEAIAAR 219–235 AAGLDVIGADIA PYQDK 236–247 VANVAEAGIFDK 258–270 ITAWGLAGEASLK 5–23 DAVAVVTGGASGLGLATTK 25–3849 LLDAGAQVVVIDLK 39–49 GEEVVAELGDR 52–77 FVATDVTDEAG VTEALNVAESLGPVR 78–90 INVNCAGIGNAIK 95–104 NGPFPLDGFR 106–119 VVEVNLIGTFNVIR 128–138 TEPIGPNGEER 139–163 GVIINTASVA AFDGQIGQAAYSASK 164–175 GGVVGMTLPIAR 184–193 VVTIAPGLFK 194–206 TPLLGSLPEEAQK 207–217 SLGAQVPHPAR 218–243 LGDPDEYGA LAQHIIENPMLNGEVIR 353–361 VLDEDLVTK
32 44 27 16 69 10 25
Global score
Sequence coverage (%)
559
53
830
76
485
50
825
84
46
2
31 57 76 20 62 77 17 90 92 55 82 62 33 27 62 7 38 49 25 49 78 26 47 11 38 20 73 87 61 86 101 40 108 71 38 49 35 44 48 44 81 47 93 49
† Molecular weight (Da) and pI were determined using the ProtParam tool, Expasy Bioinformatics Resource Portal. ‡According to BCGList (http://genolist.pasteur.fr/BCGList). FC = functional category. 0 = virulence, detoxification, adaptation; 1 = lipid metabolism; 2 = information pathways; 3 = cell wall and cell processes; 4 = stable RNAs; 5 = insertion sequences and phages; 6 = Pe/PPE; 7 = intermediary metabolism and respiration; 8 = unknown; 9 = regulatory protein; 10 = conserved hypothetical; (–) = not determined. Mbv = mAbs for Mycobacterium bovis bacillus Calmette–Guérin (BCG) Mexico; Mav = mAbs for M. avium; Mar = mAbs for M. arupense.
460
© 2014 British Society for Immunology, Clinical and Experimental Immunology, 177: 454–463
Monoclonal antibodies against mycobacteria Table 3. Epitope prediction of proteins recognized by monoclonal antibodies (mAbs) analysed in silico. Monoclonal antibody Mbv 2A10
Mav 3H1
Protein Universal stress protein family
3-hydroxyacyl-CoA dehydrogenase
Epitopes B cells ID 10168 /PHPQQATDDSGH ID 156380/ MVYGTGGLAYGKVKSAFNLGDD APALHTWSDKTK T cells ID 65596 / TPHPARIGL
Source antigen/organism LMP1 protein/human herpesvirus 4 Outer membrane protein Omp31 Brucella ovis Beta-galactosidase/Escherichia coli
ID = epitope identification key in the Immune Epitope Database and Analysis Resource (IEDB). Mbv = mAbs for Mycobacterium bovis bacillus Calmette–Guérin (BCG) Mexico; Mav = mAbs for M. avium.
Cross-reactivity of the selected mAbs against mycobacteria from the MTb and NTM complexes allowed us to assess the specificity of antigen recognition by the clones. In all cases, there was evidence that the protein extracts from all the assayed mycobacteria were recognized, suggesting the existence of shared antigenic determinants (Table 1). To determine which proteins contained these antigenic determinants, the spots recognized by each mAb clone were identified by immune proteomics, and the respective protein sequences were determined by mass spectrometry. In general, the mAb-recognized proteins were
involved in respiration, metabolism, cell cycle, oxidative stress and dormancy processes [18–24] (Table 2). Interestingly, mAbs Mbv 2A10 and Mav 3H1 recognized three and four spots, respectively. This finding could be due to the fact that the proteins are subject to denaturing conditions on the immunoblot; thus, antigenic determinants that are usually inaccessible in the native protein conformation may be exposed. Another possibility is that the mAbs are able to recognize more than one epitope from different strains [25] or species due to crossreactivity [26].
Fig. 2. Multiple sequence alignment of proteins recognized by monoclonal antibodies 2A10 and 3H1. (a) Alignment of protein sequences recognized by the Mbv monoclonal antibody (mAb): glycine dehydrogenase (glycine), universal stress protein (universal), and thiosulphate sulphurtransferase CysA2 (probable). (b) Alignment of the sequences recognized by the Mav mAb: nitroreductase MSMEG_5246 (hypothetical), phosphomethylpyrimidine kinase (phospho), acyl (ACP) desaturase (acyl) and 3-hydroxyacyl-CoA-dehydrogenase (3-hydrox). Red squares indicate peptide sequences that were identified as epitopes in universal stress protein (a) and in 3-hydroxyacyl-CoA dehydrogenase (b). These peptides are among consensus sequences identified previously in multiple alignments.
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Epitope prediction in the proteins recognized by mAb Mbv 2A10 allowed us to identify one B lymphocyte epitope in the universal stress protein, Rv2623 and one T lymphocyte epitope in 3-hydroxyacyl-CoA dehydrogenase [27]. Sequence identification of these epitopes could be used to produce peptides to be assayed for their effect on the adaptive immune response against the Mycobacterium from which the peptide originated. Conversely, it is noteworthy that the IEBD software did not find epitopes for all the mAb-recognized proteins. The reason for this could be that these epitopes have not yet been reported, or they represent antigen recognition in a denatured state. Finally, determining the epitope sequences using multiple sequence alignment by Clustal W allowed us to identify the conserved motifs present in the epitope sequences that were recognized by both mAbs. We suggest that these epitopes are probable antigenic determinants and identification of the targets is a perspective. In summary, the mAbs generated against M. bovis BCG Mexico, M. avium and M. arupense recognized several mycobacteria from the MTb and NTM complexes. We propose identifying the epitopes recognized for these mAbs and evaluating them as diagnostic tools for clinically relevant mycobacteriosis.
Acknowledgements CONACYT 140998 and DGAPA IN214713-2 supported this study. K. F.-M. is the recipient of a CONACyT México doctoral scholarship (Reg. no. 261862); J. S. C.-M. is the recipient of a CONACyT México master in Science scholarship (Reg. no. 288831); D. M. M.-R is the recipient of a postdoctoral fellowship from DGAPA, UNAM; and P. O. is the recipient of a postdoctoral fellowship from CONACYT (140998).
Disclosures The authors do not have any conflicts of interest to declare.
Author contributions K. F.-M., J. S. C.-M. and B. M.-R. participated in the performance of the experiments; D. M. M. R. and P. O. participated in the analysis results and writing of the paper; A. I. C.-R. and Y. L.-V. participated in the design of the experiments and writing of the paper.
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Supporting information Additional Supporting information may be found in the online version of this article at the publisher’s web-site: Fig. S1. Multiple sequence alignment of proteins recognized by monoclonal antibodies 2A10 and 3H1 with T-Coffee. (a) Alignment of protein sequences recognized by the Mbv monoclonal antibody (mAb): glycine dehydrogenase (glycine), universal stress protein (universal) and thiosulphate sulphurtransferase CysA2 (CysA2). (b) Alignment of the sequences recognized by the Mav mAb: nitroreductase MSMEG_5246 (hypothetical), phosphomethylpyrimidine kinase (phospho), acyl (ACP) desaturase (acyl) and 3hydroxyacyl-CoA-dehydrogenase (3-hydrox). Black squares indicate peptide sequences that were identified as epitopes in universal stress protein (a) and in 3-hydroxyacyl-CoA dehydrogenase (b).
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