Table Of ContentAlbanetal.BMCInfectiousDiseases2013,13:42
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RESEARCH ARTICLE Open Access
Identification of mimotopes of Mycobacterium
leprae as potential diagnostic reagents
Silvana M Alban1, Juliana Ferreira de Moura2, João Carlos Minozzo3, Marcelo Távora Mira4
and Vanete Thomaz Soccol1*
Abstract
Background: Anearly diagnostic testfor detecting infectionin leprosy is fundamental for reducing patients’
sequelae. The currently used lepromin is not adequatefor disease diagnosis and, so far, noantigen to be used in
intradermoreactionhas proved to be sensitive and specific for thatpurpose. Aiming atidentifying new reagents to
be used in skin tests, candidate antigens were investigated.
Methods: Randompeptide phage display libraries were screened by using antibodies from leprosy patients in
order to identify peptides as diagnostic reagents.
Results: Seven different phage cloneswere identifiedusing purified antibodies pooled from sera of leprosy
patients. When theclones were tested with serum samples byELISA, threeof them, 5A, 6A and 1B, allowed
detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed
byselected phage clones were chemically synthesized.A pilot study was undertaken to assess theuse of peptides
inskin tests. The intradermal challenge with peptides in animals previously sensitized withMycobacterium leprae
induced a delayed-type hypersensitivity with peptide 5A (2/5)and peptide 1B (1/5). Inpositive controls, therewas a
3/5reactivity for lepromin and a 4/5 reactivity of thesensitizedanimals withsolubleextract ofM. leprae.
Conclusions: The preliminarydata suggestthat maybe possible to develop reagents with diagnostic potential
based onpeptide mimotopes selected by phage display using polyclonal human antibodies.
Background Leprosy presents a wide range of clinical manifestations
Leprosy is a chronic granulomatous infectious disease determined by the immune response of the individual
caused by M. leprae that affects the skin and the periph- against the bacillus. Tuberculoid leprosy patients show a
eral nervous system [1]. Thisdisease constitutes a public response that limits pathogen growth and result in few
health problem in countries such as Brazil [2], where lesions containing rare (or absence of) bacilli however,
large numbers of patients with sequelae as a result of nerve damage is often present. Lepromatous leprosy
neural damages are still observed every year [3]. Upon patients are susceptible to disseminated infection. Skin
the introduction of multidrug therapy in 1981 by the lesions are numerous and there is an uncontrolled
WHO, the prevalence of leprosy has been dramatically proliferation of leprosy bacilli. Such clinical presentations
reduced, but over the last 5 years, more than 200,000 correlate to the level of cell-mediated immunity (CMI)
new cases have been detected each year [4]. These more against M. leprae. Antibody responses are greater in lep-
recent data indicate that the transmission of leprosy still romatouspatients,indicatingthathumoralimmunitydoes
goes on [5], which makes it clear that new strategies - notcontributetohostdefense[6].
besides treatment - are necessary to eliminate the The delayed-type hypersensitivity reaction (DTH) in
diseaseasapublic healthproblem. skintestsisconsideredamanifestationofthecell-mediated
immunity [2]. In leprosy, cells of M. leprae from different
originsandsubmittedtodifferentprocessesofpurification
*Correspondence:[email protected] are the basis for different types of preparations employed
1EngenhariadeBioprocessoseBiotecnologia,UniversidadeFederaldo in skin tests. Among those preparations, the most
Paraná,Curitiba81531-990,Brasil
frequently used is lepromin, which corresponds to a
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Albanetal.BMCInfectiousDiseases2013,13:42 Page2of9
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suspension of heat-killed bacilli [7]. As M. leprae is lysis,thesolublefractionwasseparatedbycentrifugationat
not cultivated in vitro, cells for lepromin preparation 10000×g for 20 minutes at 4(cid:1)C, and the protein concen-
can only be obtained from naturally infected human tration was determined using the Quant-iT Protein Assay
tissues (lepromas) or from experimentally infected kit(Invitrogen,California-USA).
armadillos [8].
Two types ofpositive skinreactions aredescribed after Patientserumsamples
the intradermal injection of 0.1 mL of lepromim: an Serum samples of 10 paucibacillary patients (PB), 23
early reaction (Fernandez Reaction), read after 48–72 - multibacillary patients (MB), and 26 household contacts
hours; and a late reaction (Mitsuda Reaction), read after wereobtained.ThesampleswerecollectedattheHospital
3–4 weeks [9]. The Mitsuda reaction is measured as in- de Dermatologia Sanitária (Piraquara, Paraná-Brazil),
duration and provides a measure of the individual’s CentroRegionaldeEspecialidades-Barão(Curitiba,Paraná-
ability to mount a granulomatous response against Brazil), Fundação Pró-Hansen (Curitiba, Paraná-Brazil),
mycobacterial antigens [7]. Positive Mitsuda reactions are andatSociedadeFilantrópicaHumanitas(SãoJerônimoda
seen in the vast majority of contacts and unexposed Serra, Paraná-Brazil). Among the leprosy patients, 14 had
individuals, as well as in tuberculoid leprosy patients; in not yet started the treatment by the time of blood collec-
leprosy patients with lepromatous disease no response is tion, and 19 were under treatment (days - 4 months).
observed. Therefore, reactivity to lepromin can not used Serum of 30 patients with pulmonary tuberculosis (TB) -
fordiagnosis,buthasaprognostic value[9].InBrazil,the under treatment (days - 3 months) - was obtained at the
CentrodeProduçãoe Pesquisa deImunobiológicos(CPPI) CentroRegionaldeEspecialidades-Barão(Curitiba,Paraná-
is the only supplier of lepromin antigen for the Brazilian Brazil)andattheHospitalRegionaldaLapa‘SãoSebastião’
Department of Health, and its production is based on (Lapa, Paraná-Brazil). Leprosy and TB patients were
lepromas obtained from lepromatous leprosy patients. diagnosed and classified by a specialized physician at
This is a difficult process because the production of the Health Units. Serum of 30 endemic controls was
the antigen depends on access to rare lepromas that obtained from volunteers with no history of infection by
may be contaminated with other species of mycobac- leprosy and tuberculosis. In all cases, drawing of blood
teria [10]. In addition, the strategy is associated with was carried out with informed consent and approved by
the risk of manipulation and inoculation of human the Research Ethics Committee of Federal University of
biological material. Paraná (Comitê de Ética em Pesquisa, Setor de Ciências
An alternative for replacing whole cells or purified daSaúde,UniversidadeFederaldoParaná).
fractions of M. leprae is to use synthetic peptides. Previ-
ous studies have demonstrated delayed-type hypersensi- Immunoglobulinsanti-M.lepraeandanti-M.tuberculosis
tivity responses to peptides in skin tests [11,12]. Aiming IgG of the sera pool from MB patients were obtained by
at finding alternative diagnostic supplies for leprosy, precipitation with ammonium sulfate followed by chro-
peptides binding antibody from multibacillary leprosy matography using protein G-agarose [14]. The anti-M.
patients were selected from phage displayed peptide leprae IgGs were recovered from immunoblots. For that,
libraries and assessed in their capacity to induce cell the M. leprae protein extract was resolved by SDS-
responseinguinea-pigssensitizedwith M.leprae. PAGE in gradient polyacrylamide gel 10-20%; after
having been transferred to the PVDF membrane, IgGs
Methods binding antigen immobilized in membrane were eluted
Antigens with 0.1 M glycine, 0.15 M NaCl, pH 2.8 at room
The M. tuberculosis H37Rv (ATCC 27294) strain was temperature for 30 minutes. Anti-M. leprae IgGs were
obtainedfromtheInstitutoAdolfoLutz,SãoPaulo-Brazil. dialyzed against PBS after neutralization with 1 M Tris–
ThecellsweregrowninMiddlebrook7H10for14–21days HCl, pH 9.0, and the protein concentration was
at37(cid:1)C,followedbysuspensionin0.9%NaClandinactiva- determined by the Bradford method [15]. The same
tion at 100(cid:1)C for 15 minutes. Irradiated M. leprae whole process was repeated using a sera pool from tuberculosis
cells isolated from armadillo liver were kindly supplied patients and M. tuberculosis protein in order to obtain
by Dr. J. S. Spencer from Colorado State University anti-M.tuberculosisantibodies.
(Fort Collins-USA) through the National Institute of
Allergy and Infectious Diseases/National Institutes of Screeningofrandompeptidephagedisplaylibraries
Health under contract N01-AI-25469. Bacilli in 0.9% Linear (X15 and X8CX8) and constrained(XCX4CX and
NaCl containing 100 μg/mL phenylmethylsulfonyl fluoride XCX8CX) random peptide libraries were obtained from
(PMSF)and2mMethylenediaminetetraaceticacid(EDTA) J. Scott, Simon Fraser University (Burnaby BC-Canada).
were broken by sonication (Sonopuls HD 2200, Bandelin, Peptides are expressed on the surface of protein pVIII of
Berlin-Germany) four cycles of 15 minutes [13]. Following phage f88.4. Two panning strategies were used. In
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strategy I, the phages of libraries not bound to anti-M. Aldrich, Missouri-USA) as chromogen for measuring ab-
tuberculosisimmunoglobulinsimmobilizedinimmunotube sorbanceat492nm.Themostreactiveclones(absorbance
were used in the first round of panning with anti-M. atleasttwiceashighasthenegativecontrol)wereselected
leprae immunoglobulins. In strategy II, the phages of li- for DNA sequencing and subsequent identification of the
braries were directly used in the first round of panning peptidesequenceinsertedintothephages.
with anti-M. leprae immunoglobulins. Panning wascarried
out according to the previously given description [16]. Determinationofpeptidesequences
Briefly, one immunotube (Nunc, Roskilde-Denmark) was Single-stranded DNA was prepared out of phage clones
coated with anti-M. leprae immunoglobulin in 100 mM using QIAprep Spin M13 kit (Qiagen, Hilden-Germany).
NaHCO , pH 8.6 at 5 μg/1.5 mL and incubated overnight Peptide sequences of the positive phage clones were
3
(cid:1)
at 4 C. The immunotube was washed with TBST-0.05% determined by the BigDye Terminator v3.1 kit (Applied
(TBS Tween 20–0.05%), filled with blocking solution Biosystems, California-USA) using the reverse primer
(cid:1) 0 0
(TBST-0.05%,3%BSA)andincubatedfor2hoursat37 C. 5-TCGGCAAGCTCTTTTAGG-3.
Forthefirstroundofpanning,1.5×1011phagesoflibraries
X15 and XCX8CX, and 2.5×1010 phages of libraries Phage-ELISA
X8CX8 and XCX4CX were incubated in TBST-0.05% with Reactivity of the phage clones most reactive in the pres-
theimmobilizedantibodies.AfterthewashingswithTBST- enceof humanserumwasmeasuredbyELISAaccording
0.5%,theboundphageswereelutedwith1.5mL0.1Mgly- to description given above. Microtiter plates were coated
cine, pH 2.2, 1 mg/mL BSA. After neutralization with 2 M with anti-phage antibody and incubated with 2×1010
Tris–HCl, pH 9.0, the eluted phages were amplified by phages/mL followed by the addition of sera from leprosy
infectingacultureofE.coliK91.Afterincubationovernight patients, tuberculosis patients, household contacts of
(cid:1)
at 37 C, the supernatant was obtained and precipitated leprosy patients, and endemic controls, diluted 1:100.
with PEG/NaCl. In the remaining rounds, 2×1011 ampli- The detection of the reaction was performed with
fied phages of the previous cycle were incubated with IgG peroxidase conjugated anti-human antibody and OPD
anti-M. leprae. The procedures were equal to those as chromogen.
applied to the first round except for the fact that the
concentration of the antibody immobilized to the Syntheticpeptides
immunotube was 1 μg and 0.5 μg in the second and Peptides were synthesized using solid phase 9-
subsequent rounds, respectively. fluorenilmetoxycarbonyl (Fmoc) chemistry by PepTron
(Daejon-Korea). Peptide purity was equal to or higher
Immunologicalscreeningofphageclones than 70%.
After four rounds of panning, individual colonies
containing phages were picked at random and grown Guineapigsanddelayed-typehypersensitivityresponses
(cid:1)
overnight at 37 C in a 96-well microtiter plates in LB The experiments with these animals followed the
medium containing 20 μg/mL tetracycline. The plates guidelinesofBrazilianCollegeofAnimalExperimentation
were centrifuged (1600×g, 20 minutes) and supernatant (Colégio Brasileiro de Experimentação Animal - COBEA)
containing phages was analyzed regarding its binding to and were approved by the institutional committee of
IgG from leprosy patients by ELISA. Microtiter plates Federal University of Paraná.
(Falcon, BD Biosciences, California-USA) were coated by Three groups of five 250–300 g outbred female guinea
incubating 50 μL anti-phage antibody (Sigma-Aldrich, pigs (Cavia porcellus) were used for the skin test. The
Missouri-USA) diluted 1:800 in 100 mM NaHCO , antigens were diluted in 0.9% NaCl. The Mitsuda
3
pH 8.6, overnight at 4(cid:1)C. The plates were washed with lepromin (4x107 bacilli/mL) provided by the Centro de
PBST-0.05% and blocked with 2% skimmed milk powder Produção e Pesquisa de Imunobiológicos (Piraquara,
in PBST-0.05% for 1 hour at 37(cid:1)C. The supernatant of Paraná-Brazil) was used as positive control, and 0.9%
each phage clone was added to each microtiter well. As a NaCl was used as negative control. Five animals (group
negativecontrol,thesupernatantoftheE.coliK91culture I) were sensitized by subcutaneous inoculation of 200 μg
was used. After incubation for 2 hours at 37(cid:1)C, the plate [17] of M. leprae in Freund’s incomplete adjuvant
waswashedandincubatedwith100μg/mLIgGfor1hour (Sigma-Aldrich, Missouri-USA). As control, five animals
at 37(cid:1)C. After washing, peroxidase conjugated anti- (group II) received 0.9% NaCl in Freund’s incomplete
human antibody (Sigma-Aldrich, Missouri-USA) - diluted adjuvant subcutaneously. Control group III consisted
(cid:1)
1:10000 in a blocking solution for 1 hour at 37 C - of non-sensitized animals. After 30 days, each animal
was added. After washing, bound antibodies were received, intradermally, 0.1 mL of the antigens: peptides
monitored for determining the peroxidase activity using (10 and 2 μg), peptide pool (10 and 2 μg), Mitsuda
ο-phenylenediamine dihydrochloride (OPD) (Sigma- lepromin (positive control), soluble extract of M. leprae
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(10μg,positivecontrol)and0.9%NaCl(negativecontrol). antibodies are purified from total IgG well as most of
The diameter of the skin reaction was measured after 24, the extraneous IgGs active against other antigens are
48,and72hours,21and28days. removed. Lipoarabinomannan (LAM) is a major antigen
of M. leprae that is recognized by immunoglobulins in
serafromleprosypatients[18]andrunsasabroaddiffuse
Bioinformatics
bandfrom30–40kDaasshowninFigure1.
The peptide sequences were analyzed for sequence simi-
Two panning strategies were used. In one of them, the
larity using FASTA program on M. leprae sequence data
phage pool with anti-M. leprae antibodies, was depleted
(http://genolist.pasteur.fr/Leproma/).
of reactive phages with anti-M. tuberculosis antibodies.
Four roundsof affinity selection were performed and the
Results and discussion amplified phage pool of each selection round was
In ordertoidentify mimotopes ofmycobacterialantigens, assessed by ELISA (Figure 2). In both strategies, an in-
peptidelibrariesdisplayedbyphagewereselectedbyaffin- crease of reactivity was observed with each round of
ity using M. leprae antigen-specific antibodies. The anti- panning. However, the best result was detected between
bodies were obtained from a pool of sera from MB thephages andtheIgGobtainedinstrategy II.
patients. The IgGs were purified by ammonium sulfate The phage pool of the last selection round was then
precipitationandaffinitychromatographyfollowedbyelu- chosen to isolate individual positive clones. Each phage
tion from membranes containing M. leprae antigens clone was assessed – by ELISA – regarding its ability to
bound. The total IgGs from MB patients recognized bind to IgG of multibacillary patients. In strategy I, 24
proteins in M. leprae especially below 50 kDa (Figure 1B, out of 400 clones analyzed showed recognition against
lane 1). The same profile of reactivity was seen with patients’ antibodies, and 6 different peptide sequences
antigen-specific IgGs (Figure 1B, lane 2), however, the were identified. In strategy II, 36 out of the 342 clones
antibody preparation method represent an important were recognized for binding to antibodies against
purification and concentration step, as relatively specific leprosy and all expressed an identical sequence of 15
aminoacids.
The respective phage clones carrying the identified
peptidesequenceswerethenassessedagainst seraoflep-
rosy patients and controls by ELISA (Figure 3). Reactive
control samples werethe same regardless ofthe assessed
clones, which could indicate an unspecific reaction
betweenserumandphages.
0,6
0,5
m
n
2
9 0,4
4
at
e 0,3
c
n
a
orb 0,2
s
b
A 0,1
0
Negativo 1 round 2 round 3 round 4 round
control
Figure1SDS-PAGEandWesternblotanalysisoftotalextract
ofM.leprae.A.TwentymicrogramsoftotalextractofM.leprae Strategy I Strategy II
wereseparatedby10-20%gradientSDS-PAGEandthegelwas Figure2Reactivityofphagespoolsselectedbyaffinity
stainedwithsilvernitrate(lane2).Molecularweightmarkerisshown strategiesateachselectionroundbyELISA.Microtiterplatewas
inthelane1.B.TheproteinsweretransferredtoPVDFmembrane coatedwithanti-phageantibody(1:800)andincubatedwith2×1010
andblotswereprobedwith1μg/mLofMBpatients’totalIgG phages/mLfollowedbytheadditionofIgG(100μg/mL).The
(lane1),60ng/mLofMBpatients’antigen-specificIgG(lane2)and detectionofthereactionwasperformedwithperoxidase
1μg/mLofPBpatient’totalIgG(lane3).Detectionofthereaction conjugatedanti-humanantibodyandOPDaschromogen.Valuesof
wasperformedwithanti-humanIgG(Fc-specific)-peroxidase absorbanceat492nmaremeansofduplicates.Wildphagewas
antibody(1:30000)andchemiluminescence. usedasnegativecontrol.
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Phage Sera
clone Paucibacillary Multibacillary
1A
2A
3A
4A
5A
6A
1B
Phage Sera
clone Tuberculosis
1A
2A
3A
4A
5A
6A
1B
Phage Sera
clone Household Contacts
1A
2A
3A
4A
5A
6A
1B
Phage Sera
clone Endemic Control
1A
2A
3A
4A
5A
6A
1B
Figure3ReactivityofphageclonesbyELISAwithseraofpatientsandcontrols.Microtiterplateswerecoatedwithanti-phageantibody
(1:800)andincubatedwith2×1010phages/mLfollowedbytheadditionofsera(1:100).Thedetectionofthereactionwascarriedoutwith
peroxidaseconjugatedanti-humanantibodyandOPDaschromogen.Eachcellinthetablerepresentsanindividual.Theanalysiswasperformed
induplicateandthehatchedcellsrepresentpositivesamples.Thecut-offvaluewascalculatedastheaverageoftheopticaldensityplustwice
thestandarddeviationobtainedwithendemiccontrols.
Seven phage clones were selected and, among them, The search for similarity of peptide sequences in
clones 5A, and 6A, clones isolated by strategy I, and 1B, Leproma database showed that the peptides selected
clone isolated by strategy II, were detected in a higher mimic mainly proteins in M. leprae (Table 1). However,
number of leprosy patients when compared to the nega- the LAM antigen, from which mimotopes can be
tive controls. Even fortheselected clones,alowsensitiv- isolated, would not be detected by FASTA approach.
ity was observed in the detection of leprosy patients. Further studies could reveal if those peptides represents
Each serum sample was also tested with wild phage by mimotopes of the same antigen or of different antigens.
ELISA in order to evaluate the cross reaction of the Comparison of selected mimotopes and the native anti-
serum with the phage without the insert. The results gen sequence could lead to a better understanding of
show that the reaction of the phage clones was stronger molecular mechanisms that participate in the immune
than that of the wild phage with the positive sera response and the design of peptides for diagnostic
samples against the phage clones (data not shown), indi- purposes[19].
cating that the reactivity happened exclusively between Since a humoral response depends on Thelper cell re-
the antibodies and the peptides expressed on the surface sponse, the peptides identified were assessed regarding
ofthephages. their ability to induce a delayed-type hypersensitivity
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Table1SimilaritysearchbetweenpeptidesequenceandM.lepraesequencedatabase
Peptide Size Potentialantigens
(amino M.lepraeSangerID Description
acids)
5A 15 ML2028 Secretedantigen85-BfbpB
ML0781 Conservedhypotheticalprotein
ML1135 ProbableprotoporhyrinogenoxidasehemKhomolog
ML0041 Possiblesecretedprotease
ML0053c Possibleconservedtransmembraneprotein
ML2031 Conservedhypotheticalprotein
ML0213 PossiblecellcycleproteinMesJ
ML2307c ProbabletranscriptionalregulatoryproteinWhiB-likewhiB4
ML2137 Conservedhypotheticalprotein
ML1177c ProbableconservedliproproteinlprD
6A 12 ML2263c ProbablenaphthoatesynthasemenB
ML2028 Secretedantigen85-BfbpB
ML2030 Probableresuscitation-promotingfactorrpfC
ML0053c Possibleconservedtransmembraneprotein
ML0679 Hypotheticalprotein
ML0468c Possibleconservedintegralmembraneprotein
ML0213 PossiblecellcycleproteinMesJ
1B 15 ML2028 Secretedantigen85-BfbpB
ML0097 Secretedantigen85-AFbpA
ML2485c PossibleRNAmethyltransferase
ML1195 Isoleucyl-tRNAsynthetaseIleS
ML2324 2-isopropylmalatesynthaseleuA
ML2409c PossiblecytochromeC-typebiogenesisproteinccsA
ML2597 ProbableconservedMceassociatedprotein
ML0455c Conservedhypotheticalprotein
ML1037c Conservedhypotheticalprotein
ML0052c Conservedhypotheticalprotein
ML0130c Probablemethyltransferase
ML0369c Conservedhypotheticalprotein
PotentialantigenscorrespondstothebestscoresobtainedforeachpeptidesequenceasaquerysequenceagainstM.lepraedatabaseusingFASTAsearch.
response in guinea-pigs sensitized with M. leprae. The with similar sizes - 16.5 mm and 15.5 mm respectively -
use of guinea-pigs constitutes the animal model used in whereas for peptide 5A, only the 10 μg dose generated a
delayed-type hypersensitivity tests with M. leprae response. The two animals that did not respond to
antigens, according to description presented previously Mitsuda lepromin in 48 hours produced a response
[17,20-22]. The intradermal challenge with peptides in after 72 hours (induration) or 21 days (induration)
animals previously sensitized with M. leprae induced after the application of the antigen. The detection of
delayed-type hypersensitivity. The maximum reading response with Mitsuda lepromin and the soluble extract
was observed in 48 hours and, therefore, that time was ofM.lepraeindicatethattheanimalsweresensitized.The
considered in the description ofthe results. No reactions skin reactions observed were erythematous; only one
were detected inanimals sensitized with adjuvant aswell of the animals immunized with M. leprae, besides the
as in non-sensitized animals. In the group sensitized erythema, also developed a papule as a result of
with M. leprae, a DTH reaction was observed with pep- applying peptide 5A.
tide 5A (2/5), peptide 1B (1/5), Mitsuda lepromin (3/5), The lack of cellular response in other animals is prob-
and with M. leprae (4/5) (Figure 4). For peptide 1B, the ably due to the influence of genetic factors involving
skin reaction was observed for both 2 and 10 μg doses genes MHC in the responses to peptides [11,23]. Thus,
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with larger number of patients. The selection of
25
mimotopes was based on seroreactivity and therefore
m) 20 these mimetic peptides correspond to B cell epitopes.
m The strategy of the study was to test whether these
er ( peptides would be able to act as T cell epitopes. Sero-
met 15 logical and cellular assays, as proposed initiated this
a
di study may determine whether a peptide can comprise a
a 10
m serological or Tcell test reagent. Tests for humoral im-
e
h mune response benefit lepromatous patients while tests
yt 5
Er for evaluation of cellular response will aid in the diagno-
sisoftuberculoidpatients.
0
Lepromin is probably the only skin test antigen that
5A 6A 1B Pool M ML C
reflects the individual’s ability to generate a granuloma-
Guinea Antigen tous response to mycobacterial antigens, as opposed to
Pig 5A 6A 1B Pool M ML C the 48–72 hours delayed-type hypersensitivity response
1 - - + - -(*) - -
found with tuberculin and other skin tests [7]. In the
2 - - - - + + -
lepromin test, the Fernandez reaction is similar to the
3 + - - - + + -
4 - - - - -(**) + - reaction produced by tuberculin, since it assesses the
5 + - - - + + - delayed-typehypersentivity oftheindividualtoM.leprae
soluble antigens [22]. In this aspect, the present study
Figure4Delayed-typehypersensitivityresponseinducedby
peptidesinguinea-pigs.Guinea-pigsweresensitizedwith200μg proposes the use of peptides in skin tests to assess the
M.lepraeinFreund’sincompleteadjuvantand-after30days-were cellular response, similar to the one captured by the
inoculatedintradermallywithpeptidesandcontrols.Eachgroup Mantoux test used in tuberculosis. Besides in vivo stud-
containedfiveanimals.Theresultin48hoursafterapplicationof
ies, the peptides identified can be assessed in the future
10μgpeptides,Mistsudalepromin(M)with4×106bacilliand10μg regarding their ability to induce INF-γ production by
solubleextractofM.leprae(ML)isindicated.Thepeptidepool
containequimolaramountsofthethreepeptides.Thediameterof peripheral blood mononuclear cells of patients, aiming
thereactioncorrespondstotheaverageofhorizontalandvertical to verify whether those reagents allow the detection of
diametersoftheerythema.0.9%NaCl(C)wasusedasnegative the paucibacillary disease. However, the skin tests are
control.*Theanimalrespondedafter72hours.**Theanimal more feasible and useful as compared to INF-γ produc-
respondedafter21days.
tion assays,since the latter isdependent onthe access to
advanced lab facilities and trained personnel, which
the mixture of peptides able to recognize different forms limits its use in countries with limited resources [30], a
ofdiseaseordifferentgeneticaspectscouldhelptoover- proposal advocated in the present study. Recently, the
come problems of genetic restriction to individual development of a user-friendly assay to detect multiple
peptides. Other factors that need to be evaluated are the cytokines [31] which can make INF-γ detection assays
dosage and the constitution of the antigen as, for ex- more accessibleandeasiertoperformwasreported.
ample, the conjugation with proteins or modification of Synthetic peptides prove to be very attractive to be
the peptide structure. Differences were found in the used in diagnostic tests or for monitoring the degree of
DTH response regarding the dosage of the inoculated exposure to leprosy within different communities, or
peptide [11,24]. Other studies have tested peptides in even an adjunct to leprosy control programs [32], since
the form of peptide-protein conjugation proteins [25] or they are easy to produce with reproducibility, as well as
as peptides modified by the addition of fatty acids and high quality and purity control [33]. In addition, syn-
acetylation[26]. thetic peptides can be easily obtained at a low produc-
Differences in reactivity between phage clones and the tion cost through chemical synthesis. Comparatively,
corresponding free peptide have been described previ- crude antigens originating from M. leprae are complex
ously [27,28]. When in phage, the sequence could to as- mixtures that carry proteins of humans or animal tissues
sume a favorable position to act as binder of antibodies intheircomposition,whichmay posebiologicalrisks.
[29] and therefore this conformation could disappear
when used as synthetic peptides. Likewise, the conform- Conclusions
ation of the peptide expressed on the phage surface may Peptides mimotopes were isolated from phage displayed
discourage recognition by antibodies. Thus, other pep- libraries by screening purified antibodies derived from
tide sequences identified but not included in DTH tests leprosy patients’ sera. Our strategy was to evaluate the
will be evaluated in future studies. The decision was to ability of mimotopes to induce cellular response in DTH
limit the tests to the phage clones that were reactive tests. The selected sets of peptides are also a starting
Albanetal.BMCInfectiousDiseases2013,13:42 Page8of9
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participatedintheexecutionofexperiment.MTMparticipatedinanalyzing
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thedataandwritingthemanuscript.VTSconceivedthestudy,participated
A,KaveriSV,Saint-RemyJM,GranierC:Peptidedecoysselectedbyphage
indesigningthestudy,dataanalysisandwritingofthemanuscript.Allthe
displayblockinvitroandinvivoactivityofahumananti-FVIIIinhibitor.
authorsreadandapprovedthisfinalmanuscript.
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Spencer,UniversityofColorado,forprovidingtheM.lepraecells;DrJamieK.
MimotopesselectedwithaneutralizingantibodyagainstureaseBfrom
Scott,SimonFraserUniversity,forhavingprovidedthepeptideslibraries
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displayedinphages.ThepresentresearchwassupportedbytheConselho
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supportedbyfundsgrantedbyCAPES(CoordenaçãodeAperfeiçoamentode 20. MehraV,BloomBR:Inductionofcell-mediatedimmunityto
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Paraná,Curitiba81531-990,Brasil.2DepartamentodePatologiaBásica, 22. BrennanPJ:Skintestdevelopmentinleprosy:progresswithfirst-
UniversidadeFederaldoParaná,Curitiba81531-990,Brazil.3Centrode generationskintestantigens,andanapproachtothesecond
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doi:10.1186/1471-2334-13-42
Citethisarticleas:Albanetal.:Identificationofmimotopesof
Mycobacteriumlepraeaspotentialdiagnosticreagents.BMCInfectious
Diseases201313:42.
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