In silico analysis of potential human T Cell antigens from Mycobacterium tuberculosis for the development of subunit vaccines against tuberculosis

2014
Immunological Investigations, 2014; 43(2): 137–159
! Informa Healthcare USA, Inc.
ISSN: 0882-0139 print / 1532-4311 online
DOI: 10.3109/08820139.2013.857353
In silico analysis of potential human
T Cell antigens from Mycobacterium
tuberculosis for the development of
subunit vaccines against tuberculosis
Santhi Devasundaram, Anbarasu Deenadayalan, and
Alamelu Raja
Department of Immunology, National Institute for Research in Tuberculosis (ICMR),
(Formerly Tuberculosis Research Centre), Chetpet, Chennai 600 031, India
In silico analysis was used to predict MHC class I and class II promiscuous epitopes and
potential antigens, from 24 novel T cell antigens of Mycobacterium tuberculosis.
Majority of the antigens (16/24) had high affinity peptides to both MHC class I and
class II alleles and higher population coverage compared to well-proven T cell antigens
ESAT-6, CFP-10 and Ag85B. Among these, highest population coverage were calculated
for three novel T cell antigens Rv0733 (97.24%), Rv0462 (96.9%) and Rv2251 (96.3%).
The prediction results were experimentally tested by in vitro stimulation of these novel
T cell antigens with blood drawn from QuantiFERON-TB Gold In-Tube (QFT-IT)
positive healthy household contacts of tuberculosis patients and pulmonary TB
patients. Significantly higher level interferon-g (IFN-g) was observed, with these
novel T cell antigens, in healthy household contacts compared to pulmonary TB subjects
(p ¼ 0.0001). In silico analysis also resulted in prediction of 36 promiscuous epitopes
from the novel 24 T cell antigens. Population coverage for 4 out of the 36 promiscuous
epitopes was 490% [67 VVLLWSPRS (Rv1324), 42 VVGVTTNPS (Rv1448c), 178
MRFLLSAKS (Rv0242c) and 842 IRLMALVEY (Rv3800c)]. Our results shows that
these novel antigens and promiscuous epitopes identified from our analysis can further
be investigated for their usefulness for subunit vaccine development.
Keywords Epitopes, major histocompatibility complex, promiscuous peptides, T Cell
antigens, tuberculosis
INTRODUCTION
In 2011, 8.7 million new cases of tuberculosis (TB) were estimated (13%
co-infected with HIV) and 1.4 million people died from TB, including almost
one million deaths among HIV-negative individuals (WHO, 2012). Increasing
drug resistance and HIV coinfection worsen the impact of this disease. Bacillus
Calmette-Gueˆrin (BCG) is a prophylactic vaccine for tuberculosis (TB) and
known to protect young children. However it does not efficiently and
consistently protect adults (variable protective efficacy ranges from 0% to
80%), nor does BCG offer protection from establishment of latent TB and
subsequent reactivation (Zvi et al., 2008). Developing an improved vaccine for
Correspondence: Dr. Alamelu Raja, National Institute for Research in Tuberculosis
(ICMR), (Formerly Tuberculosis Research Centre), No. 1, Sathiyamoorthy Road,
Chetpet, Chennai - 600 031, India. E-mail: alameluraja@gmail.com
ImmunolInvestDownloadedfrominformahealthcare.combyNanyangTechnologicalUniversityon05/27/14
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TB, whether a replacement for BCG or a booster to the existing vaccine
(Kao et al., 2012), or a vaccine specifically directed against latent TB, is of
crucial importance in the battle to defeat the disease (Brennan et al., 2007).
Experimental approaches to develop an improved vaccine against TB
have included the use of attenuated mycobacteria, subunit vaccines, and
DNA vaccines. A subunit vaccine, consisting of a few key molecules
of the pathogen, has the advantage of safety when used in immune-
compromised individuals, such as those infected with the HIV, and can be
used alone or to boost immunity in individuals previously immunized with
BCG (Dey et al., 2011).
Extracellular proteins are readily available for immune processing and
subsequent presentation as MHC-bound peptide fragments. They play a key
role in inducing cell-mediated immune responses that provide protection
against pathogens during natural infection (Pal & Horwitz, 1992).
Immunization with extra cellular antigens (Culture filtrate proteins), in
animal models of TB resulted in protective immunity against TB (Sable et al.,
2005). Immunity against mycobacterial infections involve T cell mediated
immune response and CD4þ cells are believed to be the primary subset of
T-lymphocytes involved in the cellular immune response (Talreja et al., 2003).
Multiple lines of evidence indicate that interferon (IFN-g) responses are a
critical component of the host immune defense against tuberculosis (Lahey
et al., 2010). IFN- g induces activation of the infected macrophages, as well as
increased expression of MHC Class I and II proteins on antigen-presenting
cells (McShane et al., 2005). Thus the primary criterion to identify potential
vaccine candidates against TB is their recognition by Th1 cells, the major
players in protective immunity against TB.
In our earlier work (Deenadayalan et al., 2010), we had identified 59 culture
filtrate antigens, from 105 culture filtrate protein fractions, from in vitro
grown culture of M. tuberculosis. These 59 culture filtrate antigens, purified as
a protein fraction, induced significantly higher IFN-g response in healthy
contacts than TB patients and are selected for the present study. Among these,
24 antigens are reported as ‘‘novel T cell antigens’’ and protective immuno-
logical efficiency was not evaluated for each of this antigens. With the help of
Propred, we predicted Promiscuous epitopes from each antigen and their
binding affinity to class I MHC and class II MHC alleles was calculated.
Population coverage tool was used to calculate the percentage of population
coverage. Antigens with highest percentage of binding and population
coverage are considered to be ‘‘potential’’ among other antigen in the present
study. Three antigens (Rv0733, Rv0462 and Rv2251) were found to have
highest percentage of binding and population coverage and are selected for the
present study.
In this light, the ability of novel T cell antigens (Rv0733 communicated as
separate manuscript), Rv0462 and Rv2251 to induce high level of IFN- g was
tested in peripheral blood collected from healthy household contacts (HHC) of
tuberculosis patients and pulmonary tuberculosis patients (PTB). ESAT-6,
CFP-10 and Ag85B (30 kDa) proteins were taken as ‘‘reference antigens’’, for in
silico analysis and in vitro stimulation, which were predicted to be
immunodominant antigens (Kumar et al., 2010; Palma et al., 2007) and are
in Phase I clinical trials (Dissel et al., 2011).
S. Devasundaram et al.138
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Epitopes, fragments of antigen sequences, have the ability to induce
protective immunity against M. tuberculosis infection (Olsen et al., 2000).
Experimental screening of all possible antigenic peptides for each MHC
allele is time consuming, expensive and inefficient. Many bioinformatics
methods exist to predict peptide-MHC binding (Flower, 2008) and able
effectively to discriminate binding from nonbinding peptides. Such methods
include highly sophisticated algorithms like artificial neural networks
(Nielsen et al., 2003) average relative binding (Bui et al., 2005) Hidden
Markov Model (HMM) (Noguchi et al., 2002) and matrix based prediction
methods Singh and Raghava (2001). With the aid of matrix based prediction
method (Propred I and Propred), we listed 36 promiscuous epitopes from the
novel T cell antigens that are yet to be experimentally validated.
MATERIALS AND METHODS
Retrieval of protein sequences of novel T Cell antigens
The protein sequences of 24 novel T cell antigens (termed as ‘‘test antigens’’ in
this manuscript), were retrieved from (http://www.ncbi.nlm.nih.gov/Genbank/)
in FASTA format for amplification and cloning as well for T cell epitopes
prediction.
In Silico analysis of T-cell epitopes prediction and identification of
potential antigens
The 24 novel T-cell antigens were screened for all possible T-cell epitopes by
immuno-informatics algorithm - Propred-I (http://www.imtech.res.in/raghava/
propred1/) and Propred (http://www.imtech.res.in/raghava/propred/). The
ProPred-I and Propred is an on-line server, uses matrices obtained from
BioInformatics & Molecular Analysis Section (BIMAS) and from the litera-
tures, for identifying MHC Class-I and Class II binding regions in the given
antigenic sequences. Propred I implements quantitative matrices for 47 MHC
Class-I alleles which include 40 Human HLA alleles encoded by HLA- A and B
alleles from the test set. Seven alleles (MHC-Db, MHC-Db revised, MHC-Dd,
MHC-Kb, MHC-Kd, MHC-Kk, and MHC-Ld) are from mouse origin and are
not our interest.
Protein sequences of all novel T cell antigens were submitted to Propred I
with threshold value 3, since the sensitivity and specificity of epitope
prediction at this value lies in the range of 66–78% and 80–81%,
respectively. Threshold is a numerical value used to differentiate between
binders and nonbinders. Any peptide frame scoring higher than this
value is predicted as binder or vice versa. Proteasomal and immunoprotea-
somal filters were selected during predictions. Percentage of binding
for each antigen, HLA alleles of mouse origin were excluded, was
calculated by the proportion of alleles a protein binds to that of total
number of alleles.
Propred is a graphical web tool for predicting MHC class II binding regions
in antigenic protein sequences and use matrix based prediction algorithm for
51 HLA-DR alleles. These HLA–DR molecules are encoded by DRB1 and DRB5
genes including HLA DR1 (2 alleles), DR3 (7 alleles), DR4 (9 alleles), DR7
(2 alleles), DR8 (6 alleles), DR11 (9 alleles), DR13 (11 alleles), DR15 (3 alleles)
In silico mycobacterium tuberculosis subunit vaccines 139
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and DR51 (2 alleles). The threshold value is 3%. The predicted epitope
sequence of the protein is displayed as region underlined with ‘‘*’’.
Eg. MTEQQWNFAGIEAAASAIQG
—–*********——
Prediction of population coverage of the novel T Cell antigens
In order to calculate the population coverage of the predicted putative
epitopes, the epitopic sequences with HLA- alleles were submitted to the
population coverage analysis tool housed at the Immuno Epitope Database
(http://tools.immuneepitope.org/tools/population/iedb_input). IEDB tool calcu-
lates fraction of individuals predicted to respond to a given epitope set on the
basis of HLA genotypic frequencies. Promiscuous epitopes from each protein
with their corresponding allele type were selected for the calculation.
All the population included in the site is chosen for our analysis and
included population details are given in http://tools.immuneepitope.org/tools/
population/populationInfo.
Cloning of potential novel T Cell antigens (Rv0462 and Rv2251)
DNA encoding the selected Rv0462 and Rv2251 M. tb genes were PCR amplified
from H37Rv genomic DNA using Phusion High Fidelity DNA polymerase (New
England Biolabs, MA). PCR primers were designed to incorporate specific
restriction enzyme sites 50
and 30
of the gene of interest for directional cloning
into the expression vector pET30a (Novagen, Germany). The 50
(BamHI) and 30
(XhoI) oligos of Rv0462 contains the following sequences 50
(50
GCC GAC GAG
CAC TGG ATC CTT AGG G30
) and 30
(50
CCT CGT CTC GAG CCG CTC AGA
AAT TG 30
). The 50
(KpnI) and 30
(Hind III) oligos of Rv2251 contains the
following sequences 50
(50
G CAG GGT ACC ATG CGC TGG CGC GCA T 30
)
AND 30
(50
GCC CGG CGC TCA TGG AAG CTT CTT GC 30
).
Purified PCR products were digested with restriction enzymes, ligated into
pET30a using T4 DNA ligase (NEB, MA), and transformed into DH5a cells
(Invitrogen, USA). Recombinant pET30a plasmid DNA was recovered from
individual colonies and sequenced to confirm the correctly cloned coding
sequence. The recombinant clones contained an N-terminal six-histidine tag
followed by a thrombin cleavage site and the M. tb gene of interest.
Recombinant plasmid was extracted from E. coli DH5a colonies on an LB
agar media by QIAGEN Plasmid Mini kit (Qiagen, Germany). To confirm the
identity of the construct, purified recombinant plasmids were sequenced by the
Eurofins MWG operon (US).
Purification and western blot analysis of recombinant Rv0462 and Rv2251
protein
Recombinant plasmids (Rv0462 and Rv2251) were transformed into the E. coli
BL21 (DE3) (Invitrogen, USA). Recombinant strains were cultured overnight
at 37
C in LB containing appropriate antibiotics, diluted 1/100 into fresh
culture medium, grown to mid-log phase (OD at 600 nm of 0.5–0.7), and
induced by the addition of 1 mM isopropyl-D-thiogalactoside. Cultures were
grown for an additional 3–4 h, cells were harvested by centrifugation. Bacterial
pellets were disrupted by sonication in 20 mM Tris (pH 8.0), 150 mM NaCl,
1 mM PMSF, followed by centrifugation to fractionate the soluble and insoluble
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material. Recombinant His-tagged protein products were isolated under
denaturing (8 M urea) conditions using Ni-nitrilo triacetic acid metal ion
affinity chromatography according to the manufacturer’s instructions
(Qiagen, Germany).
Amidosulfobetaine-ASB-14 (Sigma Aldrich, USA), a zwitterionic detergent,
used to eliminate Lipopolysaccharides (LPS) contaminations from E. coli
before eluting the protein, followed by washing the column with 10 mM Tris pH
8.0. Protein fractions were eluted with an increasing imidazole gradient and
analyzed by SDS-PAGE. Affinity purified protein fractions were combined
and dialyzed against 20 mM Tris (pH 8.0), concentrated using Amicon Ultra
3-kDa-molecular mass cutoff centrifugal filters (Millipore, MA), and quantified
using a bicinchoninic acid protein assay (Pierce, USA). LPS contamination
was evaluated by the Limulus amoebocyte lysate assay (Lonza Group Ltd.,
Switzerland). All the recombinant proteins used in this study showed
acceptable endotoxin levels 100 EU/mg of protein (Coler et al., 2009).
Antigens were separated by electrophoresis on 12% SDS-PAGE. The
fractionated proteins were electrophoretically transferred onto nitrocellulose
membranes in a transblot unit (Mini Trans-BlotÕ
, Bio-Rad Laboratories, USA).
Membranes were blocked with 1% Alkali-soluble Casein, and then incubated
with HisTag Antibody HRP Conjugate (Novagen, Germany) 1:1000 – 1:2000
(v/v) in blocking solution. Then the blot was developed at room temperature
with Sigma Fast 3, 30
-Diaminobenzidine, the substrate.
Recombinant plasmids for ESAT-6, CFP-10 and Ag85B were obtained from
Colorado state university, Fort Collins, USA. Proteins were overexpressed and
purified according to their instructions.
Study population
The study was approved by the Institutional Ethics Committee of National
Institute for Research in Tuberculosis (NIRT) and informed consent was
obtained from all the persons who were enrolled in this study.
Ten patients with pulmonary TB (PTB) were enrolled at the NIRT clinic.
The subjects of this group had not undergone anti-tuberculosis treatment
when recruited for the study. Their age ranged from 26 to 52 years. All the PTB
patients were positive by sputum smear microscopy.
Ten individuals who shared living quarters with the tuberculosis patient
agreed to join the study as healthy contacts (contacts) whose age ranged
from 28–55 years. These individuals had no history of tuberculosis on the basis
of personal history, physical examination, chest X-ray, and negative acid fast
bacilli sputum smear microscopy. All the ten healthy contacts enrolled in this
study were QFT-IT positive which confirms M. tuberculosis infection and were
considered as a protective population against tuberculosis infection since they
didn’t develop the disease.
Experimental verification of propred predicted potential antigens by
whole blood assay
A whole blood assay was performed by diluting whole blood 1/10 in RPMI-1640
medium (Sigma Chemical Company, USA), supplemented with glutamine
(0.29 g/l), and 1X antimycotic and antibiotic solution, and cultured in 96-well
flat bottom tissue culture plates (Nunc, USA). The diluted blood was
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stimulated, in triplicates, with the recombinant proteins Rv0462 and Rv2251
individually. Culture filtrate protein (CFA), ESAT-6, CFP-10, and Ag85B
(Colorado State University, TB contract) used as a control antigens to compare
the immune responses. A set of three wells did not receive any mycobacterial
antigen/peptide served as a control. Each antigen was added in wells to a final
concentration of 5 mg/ml. The antigen stimulated diluted blood was cultured for
6 days at 37
C in 5% CO2 atmosphere (Hera Cell, Kendro Laboratories,
Germany). After 6 days of incubation cell free supernatants were collected
and secreted IFN-g and TNF- a levels were measured by standard ELISA.
Long-term culture was carried to study the generation of a memory response to
the TB antigens compared to analysis of the immediate effector functions,
which is carried by overnight cultures.
IFN- c and TNF-a measurements
For quantification of IFN-g TNF-a, cell-free culture supernatants were
harvested after 6 days of in vitro stimulation by Rv0462 and Rv2251. Cytokine
production was determined by a double-sandwich ELISA using specific mAb
(BD Biosciences, USA) as per the manufacturer’s instructions. Briefly, 100 ml of
capture antibody (mouse anti human IFN-g monoclonal antibody) at the
recommended concentration was coated in the 96-well flat bottom polystyrene
plates (NUNC Maxisorp, Roskilde, Denmark). After overnight incubation at
4
C, the excess antibodies were washed off using PBSþ 0.05% Tween80.
The sample was added to the plate, incubated for 2 h and then the plates
were washed off. The secondary antibody (biotinylated anti human IFN-g and
TNF-a monoclonal antibody) conjugated with HRP was incubated for 1 h and
the excess antibodies were washed off. Then tetra methyl benzidine (TMB) was
used as substrate and incubated for 30 min and the reaction was arrested by
the addition of 2 N H2SO4. Then the readings were taken at 450 nm using an
ELISA reader (Molecular Devices, Sunnyvale, CA, USA). The detection limit of
the assay ranged from 4.7 to 300 pg/ml. The lowest detection limit of the kit
was 1 pg/ml.
Statistical analysis
Graph Pad prism software (Graph PAD Prism version 6.00 for Windows 7,
GraphPad Software, San Diego, CA, USA, www.graph-pad.com) was used for
data analysis. Unstimulated culture values were subtracted from the protein
stimulation. The actual amount of IFN-g and TNF-a secreted (pg/ml) in
response to each protein was calculated after subtracting the control values.
The levels induced by each protein was compared in the TB patient and
healthy contact group using Mann–Whitney test (Graphpad Software,
Sandiego, CA, USA), and p values 50.05 were considered significant.
RESULTS
Identification of HLA-binding epitopes from 24 novel T Cell antigens of
Mycobacterium tuberculosis
Identification of potent M. tuberculosis antigens that induce cellular immune
responses in host would improve the development of vaccine(s) against
tuberculosis. The immunodominant regions (epitopes) of 24 novel T cell
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antigens were predicted, by submitting their amino acid (FASTA) sequences to
Propred I and Propred. Supplemental Table 1 has total list of class I and class
II epitopes predicted from 24 novel T cell antigens. Pks13 antigen was pre-
dicted to have higher number of class I epitopes (194 epitopes) and
class II epitopes (215 epitopes) among other antigens. Lowest number
of class I epitopes (7 epitopes) was predicted in CFP-10 and lowest number
of class II epitopes (7 epitopes) was predicted for ESAT-6. Standard antigen
ESAT-6 was predicted to have 14 class I epitopes and 7 class II epitopes and
CFP-10 had 6 class I epitopes and 9 class II epitopes, respectively. The ProPred
analysis of the Ag85B showed that this protein was predicted to have 29 class I
epitopes and 49 class II epitopes.
Most of the novel T cell antigens had epitopes that bind to majority of the
40 human class I HLA alleles given in the Propred I. Few class I HLA alleles
predicted to have no epitopes from the novel T cell antigens which are given in
Supplemental Data Table 2. Class II epitopes predicted from these antigens
bind to all 51 class II DRB1 alleles.
Majority of the 24 novel T cell antigens were predicted to have significantly
higher HLA binding affinity than ESAT-6, CFP-10, and Ag85B. Sixteen
antigens (Rv0733, Rv0462, Rv2251, Rv3248c, Fba, Rv1324, Acn, Tal, ProA,
MmsA, Rv2394, Pgi, FabG4, Ald, Rv2721c and Pks13) are having high binding
affinity (more than 90%) to both MHC I and II alleles, were selected for
subsequent population coverage prediction analysis. Binding affinity of ESAT-
6 and CFP-10 was predicted to 87.1% and 82.7%, respectively. Binding affinity
of Ag85B was calculated as 95.9% (Table 1).
The two protein antigens (Rv0462 and Rv2251) selected in this study, for
HLA binding prediction using ProPred, have previously been reported to be
the antigens present in the culture filtrate proteins fractions of M. tuberculosis
(Deenadayalan et al., 2010). The ProPred analysis of the complete sequence
of Rv0462 and Rv2251 showed that these proteins could bind 40(100%) and 39
(97%) of 40 Human class I HLA, respectively, and both antigens bind 51 (100%)
of the 51 HLA–DR alleles included in the ProPred program. These results
reinforce the promiscuous nature of the above proteins for presentation to
T-cells.
Prediction of population coverage by IEDB
A given epitope will elicit a response only in individuals who express an MHC
molecule capable of binding that particular epitope. MHC molecules are
extremely polymorphic and over a thousand different human MHC (HLA)
alleles are known and variation in these alleles can significantly impact
individual responses to vaccination (Kimman et al., 2007). Therefore, we
aimed to identify optimal sets of epitopes, from the given antigens, with
maximal population coverage for different ethnicities. The population coverage
rate of the predicted epitopes of 16 novel T cell antigens were analyzed by
submitting the promiscuous epitopic core sequences with their binding HLA
alleles to IEDB population coverage analysis tool. At least 15 promiscuous
epitopes per protein, with their corresponding alleles were submitted and
percentage of coverage was calculated. This method calculates the fraction of
individuals predicted to respond to a given epitope set on the basis of HLA
genotypic frequencies.
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Table1.NovelT-cellantigensofM.tuberculosisselectedforpredictingdominantepitopesbyPropredmethod.
S.NoProteinname
Gene
number
Mol.
wt(KDa)
No.ofClassIMHC
allelespredicted
(outof40)and
%ofbinding
No.ofClassIIMHC
allelespredicted
(outof51)and
%binding
16kDaEarlySecretoryAntigenicTarget(ESAT-6)Rv387510.4532804282
210kDaCultureFiltrateAntigenCFP-10Rv38741123574486
3Fibronectin-BindingProteinBAg85B(FbpB)-30kdaRv1886c30379251100
4Meromycolateextensionacylcarrierprotein(AcpM)Rv224412.6530752854
5HypotheticalproteinRv2204cRv2204c12.9829724588
6ConservedHypotheticalproteinRv3716cRv3716c14.6330754486
7ConservedHypotheticalproteinRv1558Rv155816.28317751100
8Adenylatekinase(Adk)Rv073319.9136905098
9Bacterioferritin(BfrB)Rv384119.9134854894
10Ribosomerecyclingfactor(Frr)Rv2882c20.35215251100
11ProbableexportedproteinRv1910cRv1910c21.67338251100
12PossiblethioredoxinRv1324Rv132433.4439975098
13Probablefructose-bisphosphatealdolase(Fba)Rv0363c37.8438955098
14SecretedL-alaninedehydrogenase(Ald)(40kDaantigen)(TB43)Rv278040.81399751100
15ProbabletransaldolaseTalRv1448c41.03379251100
16ConservedproteinRv3169Rv316941.14358751100
17Probablegamma-glutamylphosphatereductaseprotein(ProA)Rv2427c45.654010051100
18Probable3-oxoacyl-[acyl-carrierprotein]reductaseFabG4Rv0242c49.944010051100
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19Coronin-InteractingProtein(TB49.2orCIP50)Dihydrolipoamide
dehydrogenaseLpdC
Rv046251.044010051100
20PossibleflavoproteinRv2251Rv225152.25399751100
21PropbableS-adenosyl-L-homocysteinehydrolaseSahHRv3248c54.45389551100
22Probablemethylmalonate-semialdehydedehydrogenaseMmsARv0753c56.1379251100
23Probableglucose-6-phosphateisomerase(Pgi)Rv0946c60.834010051100
24Probablegamma-glutamyltranspeptidaseprecursor(GgtB)Rv239470.73399751100
25Possibleconservedtransmembranealanineandglycine
richproteinRv2721c
Rv2721c76.89399751100
26Probableiron-regulatedaconitatehydratase(ACN)Rv1475c103399751100
27Polyketidesynthase(Pks13)Rv3800c190.524010051100
PropredIandPropred,predictiontoolalgorithm,wasutilizedtopredictepitopes,aswelltocalculatethepercentageofbindingtobothclassIandclassII
MHCmolecules.Among54T-cellantigensidentifiedbyDeenadayalanetal.(2010),only24novelTcellantigenswereconsideredforthisinsilicoanalysis.
Threeprovenimmunodominantantigens(ESAT-6,CFP-10andAg85B)fromM.tuberculosiswasalsoincludedininsilicopredcitonasa‘‘referenceantigen’’
andareunderlinedinthegiventable.Amongthenovel24Tcellantigens,16antigensarepredictedtohavehighpercentageofbinding(490%),
comparedtothe‘‘referenceantigens,’’andaregivenin‘‘boldletters’’inthetable.Onlythese16novelTcellantigensalongwiththe‘‘reference
antigens’’weretakenfurtherfortheanalysisofpromiscuousepitopespredictionandpopulationcoverageanalysis.
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Table2.Promiscuousepitopesfrom16novelTcellantigenswithhighestpopulationcoverage.
S.No
Protein
NameProteinIdStartpositionandaminoacidsequenceofthepotential9merepitope
Theoretical%of
population
coverageof
theantigen
1AdkRv073359VPSDLTNEL,88RSVEQAKAL,37RNIEEGTKL,113FRVSEEVLL,3VLLLGPPGA,18
VKLAEKLGI,143VYRDETAPL
97.24
2TB49.2orCIP50Rv0462305YAIGDVNGL,6VVVLGAGPG,58LVHIFTKDA,16YVAAIRAAQ,53LRNAELVHI,
148LVPGTSLSA,174IIIAGAGAI
96.9
3Rv2251Rv2251177RMITPVGVL,404RGDPIEQWL,387HVYPTGASL,460ATLDPAGIL,92FRAVISLDM,
88VRNDFRAVI
96.3
4Ag85BFbpB-30KdaRv1886c23VVLPGLVGL,141LTSELPQWL,181QQFIYAGSL,43RPGLPVEYL,28LVGLAGGAA,
76VYLLDGLRA,183FIYAGSLSA
95.98
5SahHRv3248c51REYAEVQPL,76VLIETLTAL,227YQFAAAGDL,21FKIADLSLA,88VRWASCNIF,162
MLVLRGMQY,223VLRLYQFAA,294MKGQGARVS,343IIMLEHIKA,400IVLSEGRLL,
419FVMSNSFAN
95.42
6FbaRv0363c57AEFGSGLGV,195GAGEHGKYL,183SPEDFEKTI,133SAVPIDENL,106VRPLLAISA,
249FVFHGGSGS,202YLLAATFGN,135VPIDENLAI
94.78
7Rv1324Rv132480DLLDTLSGL,77VCVDLLDTL,67VVLLWSPRS,59VRSDEVPVV,292VVAGRRNLA,133
FQGLQPADQ
94.66
8AcnRv1475c35KLPYSLKVL,261VVLTVTEML,110GNPDKVNPL,25YRLDAVPNT,123LVIDHSVIA,394
YVGNGSPDS,471VVIAAITSC
93.9
9TalRv1448c150GLPAISAVL,337DLTDVFAVL,42VVGVTTNPS,132WKIVDRPNL,255YRSLKVDGA93.4
10ProARv2427c48LLAHRDQIL,55ILAANAEDL,91AGLRQVAGL,278IAETALPRL,282ALPRLLAAL,186
VQLLSAADR,362MVNASTAFT,254ILLNSKTRR,290LQHAGVTVH
93.39
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11MmsARv0753c373GGFFIGPTL,405RARDYEEAL,71MRFIELVND,76LVNDTIDEL,222VGFVGSSDI,304
IERINNLRV
93.29
12GgtBRv2394203DLFGPAVTL,476DGFILNNQL,4WLRAGALVA,114LGLVEPQSS,393FVRLPGGSL91.6
13PgiRv0946c221KTFSTLETL,273YSVDSAIGL,299FHIIDRHFA,318LLGLIGLWY91.4
14FabG4Rv0242c366GMIGITQAL,420QPVDVAEAI,152LRRGATTAL,178MRFLLSAKS,444
IRVCGQAMI
90.5
15AldRv2780122TADGALPLL,316ATMPYVLEL,14FRVAITPAG,70LLLKVKEPI,204LRQLDAEFC89.9
16Rv2721cRv2721c155ALNAAWDKL,162KLGSSGGVL,337AMVAAWDKL,27VLLAPTVAA,150
FVVRGALNA,259FVGGKVFFS,312IVRFSAADK,638VRPAIHLPL
88.43
17ESAT6Rv387528LLDEGKQSL,61TATELNNAL,64ELNNALQNL,18IQGNVTSIH,69LQNLARTIS87.1
18CFP10Rv387456VRFQEAANK,76IRQAGVQYS82.7
19Pks13Rv3800c708VTTGPVWVL,775TIFAIQIAL,836MLFGEYIRL,1396GIFNELPRL,624LVPLAVSAF,
731YLRNEVFAA,787LRHHGAKPA,841YIRLMALVE,842IRLMALVEY
79.3
PromiscuousT-cellepitopesmakeidealtargetsforvaccinedevelopment.SixteennovelTcellantigensarehavingoneormorepromiscuouspeptidestoboth
classIandclassIIMHCalleles.Promiscuousepitopessequenceofthe16novelTcellantigensisgivenwiththeirstartingaminoacidpositionsintheprefix
andpromiscuousepitiopesforclassIIMHCalleleisunderlinedinthegiventable.Promiscuousepitopessequenceswereusedforcalculatingpercentageof
populationcoverageandonlysequenceswithhigherpercentageofpopulationcoverageisgivenhereothersarenotincludedinthistable.
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The percentage of coverage of each novel 16 antigens were higher than
immunodominant and validated reference antigens (ESAT-6, CFP-10 and
Ag85B protein), except Pks13 (Figure 1). Maximum population coverage rate
(97.24%) was observed for Rv0733 antigen (communicated as separate
manuscript), followed by Rv0462 (96.9%) and Rv2251 (96.3%) (Figure 2 and
Table 2). Thus these two antigens (Rv0462 and Rv225) were selected to
validate our insilico prediction by in vitro whole blood assy. Higher population
coverage of these antigens suggest that they might induce protective immune
response in majority of the population when administered as subunit vaccine.
This approach minimizes the complexity of the vaccine formulation and its
variation to different ethnicity.
Cloning and purification of (Rv0462 and Rv2251 antigens
Amplification of (Rv0462 and Rv2251 gene using specific primers resulted
in a single 1500 bp and 1400 bp fragment (Figures 2a, 2b and 2c) that was
subsequently cloned into pET30/a. Sequencing results confirmed the presence
of the inserted fragment (Rv0462 and Rv2251 gene) in the mentioned vector.
The obtained sequences were searched for homology identity with the NCBI
BLAST software against M. tuberculosis genomic DNA. The results showed
that the sequences were completely identical with the (Rv0462 and Rv2251
sequence.
After the expression of recombinant Rv0462 (rRv0462- 55 kDa) and
recombinant Rv2251 (rRv2251-52 kDa) protein, protein band was detected by
SDS-PAGE analysis (Figure 2d and 2e). SDS-PAGE analysis of the elution
fraction of Ni2þ
-NTA agarose chromatography showed that rRv0462 and
rRV2251 were completely purified. After purification both the proteins were
Rv0733
Rv0462
Rv2251
FbpB
SahH
Fba
Rv1324
Acn
Tal
ProA
MmsA
GgtB
Pgi
FabG4
Ald
Rv2721c
ESAT-6
CFP10
Pks13
50
60
70
80
90
100
Proteins name
%ofcoverage
Figure 1. Percentage of coverage calculated per antigen. Figure 1. Population
coverage of the 24 novel T cell antigens. The Propred predicted epitope sequences,
from the novel T cell antigens, with their HLA binders were submitted to the population
coverage analysis tool of IEDB. Population coverage calculation is made on the basis of
HLA genotypic frequencies and represents number of individuals responding to given set
of pathogen derived epitopes. Populations included, in IEDB web tool, are Australia,
Europe, North Africa, North America, North-East Asia, Oceania, South America, South
East Asia, Others, South-west Asia and Sub-Saharan Africa. Brazilian, Cuban and
Mexican.
Forpersonaluseonly.

Figure 2. (a–c). Cloning, Expression and purification of Rv0462 and Rv2251 and Western
blotting. (a). Amplification of Rv0462 gene with specific primers. Lane 2 indicates a band
of 1500 bp corresponding to the Rv0462 gene plus an additional upstream sequence.
(b) Restriction digestion of recombinant plasmid (pET30 þ Rv0462) with BamHI and
XhoI insert was released with expected size and recombinant plasmid sequence
was confirmed by DNA sequencing. (c). Amplification of Rv2251 gene with specific
primers. Lane 2 indicates a band of 1400 bp corresponding to the Rv2251 gene. Insert
release was not observed but DNA sequencing confirmed the presence of Rv2251 with
universal primer (T7 promoter primer) Lane L shows 10 kb DNA ladder (Thermo Scientific,
USA). (d–f) Expression of recombinant Rv0462 and rRv2251 protein in E. coli BL21.
SDS-PAGE analysis of IPTG induced BL-21 (DE3) containing recombinant plasmids
showed a 56 kDa Rv0462 protein (Figure 2d) and 52 kDa (Figure 2e) Rv2251 protein
and its purity. Figure 2(f) shows Western blot with anti His antibody against Rv0462
and Rv2251. Lane MW indicates molecular weight protein marker, Lane number 1–5
(d) and 1–6 (e) indicates different elutions of the corresponding protein collected
during protein purification.
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immobilized in nitrocellulose membrane and detected by anti-6-His antibody.
Western blot analysis revealed that recombinant proteins were recognised by
anti his antibodies (Figure 2f).
Antigens induced IFN-c and TNF-a secretion assays by whole blood
assay
We evaluated T cell response against these two antigens in terms of production
of IFN-g and TNF-a in healthy household contacts of tuberculosis and PTB
patients (n ¼ 10). After subtracting test – nil (without any stimuli), secreted
levels of IFN-g and TNF-a against antigen stimulation was calculated.
Analysis of the distribution of IFN-g levels showed a significantly high level
of IFN-g level in HHC compared to PTB. The mean levels of IFN-g in HHC, for
recombinant antigens Rv0462 and Rv2251 were 776.8 pg/ml and 898.04 pg/ml,
respectively and in PTB 12.5 pg/ml and 19.8 pg/ml and found to be statistically
significant (p ¼ 0.0004) (Figure 3a).
The mean IFN-g levels were equal in HHC and PTB when stimulated with
ESAT-6 and the mean value was 156.1 pg/ml in HHC and 15.9 pg/ml in PTB.
The mean values of IFN-g was high for CFP-10 (523.1 pg/ml in HHC and
192.2 pg/ml in PTB) and Ag85B (293.1 pg/ml in HHC and in PTB 25.5 pg/ml)
compared to ESAT-6.
When stimulated with Rv0462 and Rv2251, TNF-a levels were high in PTB
(281.8 pg/ml and 494.3 pg/ml, respectively) compared to HHC. TNF-a level was
less in both HHC and PTB when stimulated with ESAT-6, CFP-10 and Ag85B
(ranged from 5–10 pg/ml in HHC and in TB 40–65 pg/ml). No significant
difference was observed in TNF-a level, even with CFA stimulation, between
HHC and PTB (Figure 3b).
Predicted epitopes and alleles of interest and their prevalence
Followed by epitope prediction and in vitro experiments with the potential
antigens, significant role of alleles were also analyzed. Interestingly all the ‘‘24
novel Tcell antigens’’ have epitopes for class I MHC HLA-A*0201, HLA-A*0205
and class II MHC DRB1_0101, DRB1_0102, DRB1_0301, DRB1_0305,
DRB1_0306, DRB1_0307, DRB1_0308 and DRB1_0309 alleles and these
alleles are considered as ‘‘alleles of interest’’ in the present study. Total
numbers of epitopes that bind to the ‘‘alleles of our interest’’ were calculated.
Consistently, DRB1 alleles (class II) were predicted to bind to more numbers of
epitopes, with a median of 217 total epitopes. A total of 90 and 160 class I
epitopes were predicted to bind with HLA-A*0201 and HLA-A*0205 respect-
ively (Figure 4).
Promiscuous peptides are able to bind to multiple MHC molecules and serve
as promising targets for vaccine development (Zhang et al., 2005). To perform
the screening for promiscuous peptides, a score was assigned to each peptide
that indicates the total number of HLA molecules it binds to. Binding scores
ranging from 0 to 35 and a threshold of 14 was fixed for class I HLA binding
epitopes and for class II HLA epitopes binding score of 20 was fixed. In general,
epitopes which are predicted as binders to 10 or more than 10 HLA alleles were
identified as promiscuous epitopes (Sundaramurthi et al., 2012). Totally 37
promiscuous epitopes were predicted from 24 novel T cell antigens, and
sharing affinity with one or more alleles of our interest and their distribution,
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against the alleles of our interest, is given Figure 5 and their sequences is
given in Table 3.
Majority of the promiscuous epitopes have affinity to DRB1 alleles.
Percentage of binding for these promiscuous epitopes was calculated.
The average binding affinity of these promiscuous epitopes was 57%, but
490% affinity was showed by four individual epitopes, 67 VVLLWSPRS
(Rv1324), 42 VVGVTTNPS (Rv1448c), 178 MRFLLSAKS (Rv0242c) and
842 IRLMALVEY (Rv3800c), which were considered as ‘‘highly promiscuous
epitopes.’’
Figure 3. (a) Measurement of IFN-g and TNF-a from whole blood assay supernatants.
Secreted cytokines were analyzed by ELISA to compare IFN-g levels in 10 healthy
household contacts (HHC) and pulmonary TB (PTB) patients with ESAT 6, CFP-10, Ag85B
and test antigens Rv0462 and Rv2251. Culture filtrate antigens (CFA) used as positive
control. * refers to significant value (p ¼ 0.01), ** refers to significant value (p ¼ 0.002) and
*** refers to p value ¼ 0.0004. (b) Secreted cytokines were analyzed by ELISA to compare
TNF-a levels in 10 healthy household contacts (HHC) and pulmonary TB (PTB) patients with
ESAT 6, CFP10, Ag85B and test antigens Rv0462 and Rv2251. Culture filtrate antigens
(CFA) used as positive control. No significant difference was seen between HHC and PTB
with any of the stimuli tested.
Forpersonaluseonly.

DISCUSSION
Synthetic peptide-based vaccines, which are designed to elicit T cell immunity,
are an attractive approach to the prevention or treatment of infectious diseases
and malignant disorders. It is a well established fact that T-cells recognize the
sequences of antigenic proteins in association with appropriate MHC mol-
ecules (Oftung et al., 1997). T-cell epitope mapping allows identification of
immunodominant regions on antigenic proteins. Bioinformatics tools such as
H
LA0201
H
LA0205
D
R
B1_0101
D
R
B1_0102
D
R
B1_0301
D
R
B1_0305
D
R
B1_0306
D
R
B1_0307
D
R
B1_0308
D
R
B1_0309
Alleles of interest
0
10
20
30
Promiscuousepitopes
Figure 5. Promiscuous epitopes predicted by Propred. Promiscuous T-cell epitopes make
ideal targets for vaccine development. Majority of the test antigens having one or more
promiscuous peptides and percentage of binding was calculated. Among these
peptides four were having more than 90% binding towards HLA alleles and considered as
highly promiscuous epitopes.
H
LA-A*0201
H
LA-A*0205
D
R
B1_0101
D
R
B1_0102
D
R
B1_0301
D
R
B1_0305
D
R
B1_0306
D
R
B1_0307
D
R
B1_0308
D
R
B1_0309
0
20
40
60
Alleles of interest
Epitopespredicted
Figure 4. ProPred analysis of HLA–A, B and DR binding predictions for the Mycobacterial
culture filtrate proteins. Each antigen was predicted to have at least one or more
epitopes binding to above mentioned alleles. Total number of epitopes were summed
per alleles and consistently A*0201 and A*0205 were binding higher number of epitopes
from majority of the antigens. DRB1 alleles were predicted to bind large number of
epitopes from all the antigens.
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Table 3. Promiscuous epitopes sequence predicted from overall 24 novel T cell antigens
studied and their amino acid positions.
S. No Gene Name Epitope
sequence
Amino acid
Position
% of binding
1 ESAT-6 LQNLARTIS 69 56
IQGNVTSIH 18 29
2 CFP10 IRQAGVQYS 76 78
VRFQEAANK 56 66
3 Ag85B FIYAGSLSA 183 58
VYLLDGLRA 76 33
4 Adk VPSDLTNEL 59 35
VLLLGPPGA 3 44
VKLAEKLGI 18 75
5 Rv0462 YAIGDVNGL 305 48
LVHIFTKDA 58 71
YVAAIRAAQ 16 57
LRNAELVHI 53 57
6 Rv2251 FRAVISLDM 92 69
7 SahH VLIETLTAL 76 40
YQFAAAGDL 227 37
IIMLEHIKA 343 59
FVMSNSFAN 419 48
8 Fba VRPLLAISA 106 79
FVFHGGSGS 249 48
9 Rv1324 VVLLWSPRS 67 97
FQGLQPADQ 133 65
10 acn LVIDHSVIA 123 46
YVGNGSPDS 394 44
VVIAAITSC 471 77
11 Tal VVGVTTNPS 42 91
WKIVDRPNL 132 46
12 ProA MVNASTAFT 362 63
VQLLSAADR 186 42
13 GgtB WLRAGALVA 4 53
14 Pgi KTFSTLETL 211 42
FHIIDRHFA 299 65
15 FabG4 MRFLLSAKS 178 97
IRVCGQAMI 444 42
16 ald FRVAITPAG 14 48
LLLKVKEPI 70 42
17 Rv2721c VLLAPTVAA 27 65
FVVRGALNA 150 57
IVRFSAADK 312 61
18 pks13 MLFGEYIRL 836 37
IRLMALVEY 842 91
LVPLAVSAF 624 77
Promiscuous T-cell epitopes make ideal targets for vaccine development. Population
coverage of the each peptide is given in this table. Among these four peptides (bold
letters) were having more than 90% binding towards HLA alleles and considered as
‘‘highly promiscuous epitopes’’ among other promiscuous epitopes.
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ProPred have been successfully employed to identify HLA ligands derived from
tumors and endogenous proteins involved in autoimmune diseases (Mustafa
Shaban, 2006).
To experimentally validate our potential antigen prediction, Rv0462 and
Rv2251was over expressed and purified, in E. coli expression system. Purified
antigens were able to stimulate high level of IFN-g in healthy household
contacts compared to ESAT-6, CFP-10 and Ag85B. The strong proliferative
responses and IFN- g secretion induced by these antigens imply that they are
recognized by T cells from protective TB population. In our earlier observation
these two antigens were present in very high significant IFN- g inducing
fractions (as a protein pool). Present observation shows their ability of
inducing IFN-g secretion when stimulated as an individual protein. It also
reveals that our bioinformatics prediction of potential antigens (Rv0462 and
Rv2251) by Propred were reliable and the antigens were able to stimulate
T cells and high level of IFN- g, compared to well characterized standard
antigens of M. tuberculosis. As observed in majority of the studies, (Andrade,
Jr. et al., 2008; Harari et al., 2011; Law et al., 1996) TNF-a level was high in
PTB subjects.
Research reports suggest that blood-based method evaluates the T-cell
response to bacilli antigens, including ESAT-6, CFP-10, and TB7.7 (Kunst,
2006; Mori et al., 2004; Takenami et al., 2013) and immune response to other
intracellular pathogens (Sikora et al., 2013). In majority of the studies, it has
been shown that the optimal time point for detection of IFN- g secreted by
whole blood is day 6 (Hanif et al., 2008; Scholvinck et al., 2004; Weir et al.,
1994). Because expansion of antigen specific IFN-g secreting central memory
T-cells occurs at long-term incubations, 6 days time points were used in our
study. Long-term assays are more sensitive to check diagnostic and vaccine
potential of M. tuberculosis specific antigens. Dilution of the blood (1 in 10)
minimizes the sample consumption (easy to collect from study subjects) and
more number of antigens can be tested. Thus, the whole blood assay, with 1/10
dilution was preferred in this study to evaluate the predicted antigens from
M. tuberculosis.
In our current study healthy household contacts who are in close contact
with TB patients, but remain healthy with no evidence of disease are
viewed as the ‘‘protected’’ population (contacts). Multiple studies provide
evidence that antigens recognized by the ‘‘protected’’ group, but not active
TB patients; can be considered for vaccine development strategies by using
IFN-g response as a protective correlate (Grotzke Lewinsohn, 2005;
Lu et al., 2011; Sampaio et al., 2011; Torres et al., 1998). In our findings
HHC, presumably protected population against TB, produce IFN- g in
response to the Rv0462 and Rv2251 antigens, further suggest that these
antigens could be a target of the human protective immune response
against TB. A type 1 response is dominated by the production of interferon
gamma (IFN- g), which triggers activation of macrophages, enhancing their
microbicidal functions. As these antigens can induce IFN- g, they may also
play a role in the protective immune response against tuberculosis infection.
Despite other cells also secrete IFN-g and TNF-a in very little quantity, the
Th1 cells are shown as predominant subset which secrete the IFN-g and
TNF-a in previous report (Schluger Rom, 1998).
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Epitopes predicted by Propred has been experimentally proved as potent
immunogenic candidates (Mustafa Shaban, 2006) and Propred performs
analysis for each of the alleles independently and computes the binding
strength of all the peptides. In our earlier report (Kumar Raja, 2010) ESAT-6
peptide, Esp6 51YQGVQQKWDATATELNNALQ70, predicted by Propred,
elicited higher CD4þ
response in HHC than TB subjects. Present article also
reports the prediction of the epitope 61TATELNNAL69 from ESAT-6 by
Propred and vaccines based on this subdominant ESAT-651–70 epitope
promoted significant levels of protective immunity, in mice (Olsen et al.,
2000). The promiscuous epitopes, ESAT-669–77 and CFP-1076–84 and 56–64
predicted by the current analysis, were experimentally validated by Mustafa
and Shaban (2006). It strongly confirms that the ProPred predicted
immunodominant epitopes from antigens are reliable for the experimental
validation.
In the design of peptide-based vaccines and diagnostics, the issue of
population coverage in relation to MHC polymorphism is important because
of the fact that different HLA types are expressed at dramatically different
frequencies in different ethnicities. Peptide that functions as T-cell epitope in
one population with certain HLA makeup may not be effective in another
population with a different HLA allelic distribution. To obtain good population
coverage multiple epitopes that specifically bind to various HLA loci that
suffice to cover majority of the population is required. Population coverage
results showed that proteins of our test set have greater coverage with the
least score for Pks13. Though the percentage of coverage for Pks13 is
comparatively less, it may have few or more immunodominant regions; thus,
Pks13 was not excluded during promiscuous epitope prediction.
Mycobacterial peptides are most suitable for subunit vaccine development,
because with single epitopes, the immune response can be generated in
population, against other mycobacterial infections. This approach is based on
the phenomenon of cross-protection, whereby a person infected with a milder
strain of bacteria is protected against a more severe strain of the same bacteria
(Gomase Chitlange, 2010). Hence we suggest that if the promiscuous
epitopes predicted in this study would elicit protective immune response, it can
be included in the vaccine formulation to other mycobacterial infections,
in addition to tuberculosis infection.
The recognition of mycobacterial antigens are unaffected by BCG vaccin-
ation, as well as BCG vaccination can be boosted either by the administration
of the mycobacterial antigens or by DNA encoding antigens. Thus these
epitopes, if experimentally characterized, can enhance protective response in
BCG vaccinated individuals. In general, peptides that show low similarity with
host will elicit effective immune response. Hence promiscuous epitopes that
exhibit low similarity may elicit strong immune response against mycobac-
terial infections. Promiscuous peptides predicted in this study showed only
40 to 50% similarity with host proteins (data not shown) which suggest that
these epitopes may elicit good immune response in the host.
According to WHO, TB is among the leading killers of people living with
HIV and 12% of HIV deaths globally are due to TB. Thirteen million people
living with HIV are at risk of developing TB (http://www.who.int/tb/challenges/
hiv/facts/en/index.html). Some studies reported the association of
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HLA- B*5101(Vijaya Lakshmi et al., 2006) and DRB1*1502 (Raghavan et al.,
2009) alleles with progression of TB in HIV positive individuals in south
Indian population. The frequencies of B51 in the Asian population including
Indians are 59.5% (Vijaya Lakshmi et al., 2005). The promiscuous epitopes
proposed in this study are having affinity to HLA- B*5101 and DRB1*1502 and
can elicit immune response that might protect HIV infected individuals
expressing B51 HLA allele from the development of TB.
The frequency of alleles of our interest (HLA-A*0201, HLA-A*0205, class II
MHC DRB1_0101, DRB1_0102, DRB1_0301, DRB1_0305, DRB1_0306,
DRB1_0307, DRB1_0308 and DRB1_0309) covers majority of the populations
where the incidence of TB is high (WHO Report [http://www.stoptb.org/assets/
images/about/tbl_burden.gif). Twenty two countries listed here account for
80% TB cases worldwide. Among these countries, China, India and Nigeria
are estimated to have high numbers of incidence as well mortality rate.
Alleles of interest, HLA-A*0201 and HLA-A*0205 show high frequency in
Indian population, south and north respectively. Promiscuous epitopes
resulted from present in silico analysis need to be verified by in vitro and
in vivo experiments for their ability to induce IFN-g responses in the host,
similar to their antigen counterpart. The findings from this study may
provide guidance and utilization immunoinformatics to select potential
antigens and epitopes for the vaccine development against mycobacterial
infections. Further it may stimulate in vitro investigations to ascertain the
immunogenicity of these epitopes for designing effective vaccines against
tuberculosis infections.
CONCLUSION
We report three potential novel T cell antigens and four promiscuous epitopes
with higher percentage population coverage, from M. tuberculosis using
immunoinformatics tools. Antigens can be further evaluated for vaccine
development or as a booster vaccine candidate along with BCG. Promiscuous
epitopes resulted from this in silico analysis should be validated by in vitro and
in vivo experiments for their ability to induce immune responses in the host,
similar to the native antigens. The findings from this study may provide
guidance and utilization of these epitopes for the experimental studies aimed
at controlling tuberculosis.
ACKNOWLEDGEMENTS
We thank Indian Council of Medical Research for the Senior Research
fellowship awarded to Santhi Devasundaram. We also acknowledge
Mr. Jagadish Chandrabose Sundaramurthi, Bioinformatics center, National
Institute for Research in Tuberculosis, Chennai for his helpful discussions in
this project.
DECLARATION OF INTEREST
The authors report no conflicts of interest. The authors are responsible for the
content and writing of the paper.
Forpersonaluseonly.

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