in silico analysis of potential human t cell antigens from mycobacterium tuberculosis for the...

2014

Immunological Investigations, 2014; 43(2): 137–159! Informa Healthcare USA, Inc.ISSN: 0882-0139 print / 1532-4311 onlineDOI: 10.3109/08820139.2013.857353

In silico analysis of potential humanT Cell antigens from Mycobacteriumtuberculosis for the development ofsubunit 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

(p¼ 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-Guerin (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: [email protected]

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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 antigensThe 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 ofpotential antigensThe 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 antigensIn 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 (50GCC 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 Rv2251proteinRecombinant 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-Blot�, Bio-Rad Laboratories, USA).

Membranes were blocked with 1% Alkali-soluble Casein, and then incubated

with His�Tag 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 populationThe 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 bywhole blood assayA 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 5mg/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 measurementsFor 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 analysisGraph 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 values50.05 were considered significant.

RESULTS

Identification of HLA-binding epitopes from 24 novel T Cell antigens ofMycobacterium tuberculosisIdentification 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 IEDBA 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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echn

olog

ical

Uni

vers

ity o

n 05

/27/

14Fo

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(2010),

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‘‘re

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ntig

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’’a

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un

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ive

nta

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.A

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s,16

an

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icte

dto

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hp

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ind

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(490%

),c

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nin

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the

tab

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16

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‘‘re

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s’’

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furt

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ea

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so

fp

rom

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uo

us

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latio

nc

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rag

ea

na

lysi

s.


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om in

form

ahea

lthca

re.c

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y N

anya

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echn

olog

ical

Uni

vers

ity o

n 05

/27/

14Fo

r pe

rson

al u

se o

nly.

Tab

le2

.P

rom

isc

uo

us

ep

ito

pe

sfr

om

16

no

ve

lT

ce

lla

ntig

en

sw

ith

hig

he

stp

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ula

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nc

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rag

e.

S.N

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osi

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na

nd

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ino

ac

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pito

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The

ore

tic

al

%o

fp

op

ula

tio

nc

ov

era

ge

of

the

an

tig

en

1A

dk

Rv0733

59

VP

SDLT

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88

RSV

EQ

AK

AL,

37

RN

IEEG

TKL,

113

FRV

SEEV

LL,

3V

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PP

GA

,18

VK

LAEK

LGI,

143

VY

RD

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PL

97.2

4

2TB

49.2

or

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50

Rv0462

305

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IGD

VN

GL,

6V

VV

LGA

GP

G,

58

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IFTK

DA

,16

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AA

IRA

AQ

,53

LRN

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HI,

148

LVP

GTS

LSA

,174

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GA

GA

I96.9

3R

v2251

Rv2251

177

RM

ITP

VG

VL,

404

RG

DP

IEQ

WL,

387

HV

YP

TGA

SL,

460

ATL

DP

AG

IL,

92

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VIS

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,88

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ND

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VI

96.3

4A

g85B

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B-3

0K

da

Rv1886c

23

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LVG

L,141

LTSE

LPQ

WL,

181

QQ

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AG

SL,

43

RP

GLP

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L,28

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LAG

GA

A,

76

VY

LLD

GLR

A,

183

FIY

AG

SLSA

95.9

8

5Sa

hH

Rv3248c

51R

EY

AEV

QP

L,76

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ETL

TAL,

227

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AG

DL,

21FK

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,88

VR

WA

SCN

IF,

162

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MQ

Y,223

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LYQ

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,294

MK

GQ

GA

RV

S,343

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HIK

A,400

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EG

RLL

,419

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SNSF

AN

95.4

2

6Fb

aR

v0363c

57

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SGLG

V,

195

GA

GEH

GK

YL,

183

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FEK

TI,

133

SAV

PID

EN

L,106

VR

PLL

AIS

A,

249

FVFH

GG

SGS,

202

YLL

AA

TFG

N,

135

VP

IDEN

LAI

94.7

8

7R

v1324

Rv1324

80

DLL

DTL

SGL,

77

VC

VD

LLD

TL,67

VV

LLW

SPR

S,59

VR

SDEV

PV

V,292

VV

AG

RR

NLA

,133

FQG

LQP

AD

Q94.6

6

8A

cn

Rv1475c

35

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YSL

KV

L,261

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LTV

TEM

L,110

GN

PD

KV

NP

L,25

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LDA

VP

NT,

123

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HSV

IA,394

YV

GN

GSP

DS,

471

VV

IAA

ITSC

93.9

9Ta

lR

v1448c

150

GLP

AIS

AV

L,337

DLT

DV

FAV

L,42

VV

GV

TTN

PS,

132

WK

IVD

RP

NL,

255

YR

SLK

VD

GA

93.4

10

Pro

AR

v2427c

48

LLA

HR

DQ

IL,

55

ILA

AN

AED

L,91A

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QV

AG

L,278

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L,282

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AA

L,186

VQ

LLSA

AD

R,

362

MV

NA

STA

FT,

254

ILLN

SKTR

R,

290

LQH

AG

VTV

H93.3

9


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re.c

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y N

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ng T

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Uni

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ity o

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/27/

14Fo

r pe

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nly.

11

Mm

sAR

v0753c

373

GG

FFIG

PTL

,405

RA

RD

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ND

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222

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9

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tBR

v2394

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476

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114

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13

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iR

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221

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TL,

273

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DSA

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299

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RH

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318

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91.4

14

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G4

Rv0242c

366

GM

IGIT

QA

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VD

VA

EA

I,152

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GA

TTA

L,178

MR

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AK

S,444

IRV

CG

QA

MI

90.5

15

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122

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GA

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,316

ATM

PY

VLE

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PA

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70

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VK

EP

I,204

LRQ

LDA

EFC

89.9

16

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Rv2721c

155

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162

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150

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259

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GK

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638

VR

PA

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3

17

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28

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EG

KQ

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61

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AL,

64

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NA

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L,18

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NV

TSIH

,69

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LAR

TIS

87.1

18

CFP

10

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56

VR

FQEA

AN

K,

76

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AG

VQ

YS

82.7

19

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13

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708

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GP

VW

VL,

775

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IQIA

L,836

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GEY

IRL,

1396

GIF

NELP

RL,

624

LVP

LAV

SAF,

731

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NEV

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LRH

HG

AK

PA

,841

YIR

LMA

LVE,

842

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ALV

EY

79.3

Pro

mis

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arg

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Ca

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rom

isc

uo

us

ep

ito

pe

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qu

en

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of

the

16

no

ve

lTc

ell

an

tig

en

sis

giv

en

with

the

irst

art

ing

am

ino

ac

idp

osi

tio

ns

inth

ep

refix

an

dp

rom

isc

uo

us

ep

itio

pe

sfo

rc

lass

IIM

HC

alle

leis

un

de

rlin

ed

inth

eg

ive

nta

ble

.Pro

mis

cu

ou

se

pito

pe

sse

qu

en

ce

sw

ere

use

dfo

rc

alc

ula

tin

gp

erc

en

tag

eo

fp

op

ula

tio

nc

ove

rag

ea

nd

on

lyse

qu

en

ce

sw

ith

hig

he

rp

erc

en

tag

eo

fp

op

ula

tio

nc

ove

rag

eis

giv

en

he

reo

the

rsa

ren

ot

inc

lud

ed

inth

ista

ble

.


Imm

unol

Inv

est D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y N

anya

ng T

echn

olog

ical

Uni

vers

ity o

n 05

/27/

14Fo

r pe

rson

al u

se o

nly.

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 antigensAmplification 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

Rv0

733

Rv0

462

Rv2

251

Fbp

BS

ahH

Fba

Rv1

324

Acn Tal

Pro

AM

msA

Ggt

BP

giF

abG

4A

ldR

v272

1cE

SA

T-6

CF

P10

Pks

13

50

60

70

80

90

100

Proteins name

% o

f cov

erag

e

Figure 1. Percentage of coverage calculated per antigen. Figure 1. Populationcoverage 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 populationcoverage analysis tool of IEDB. Population coverage calculation is made on the basis ofHLA genotypic frequencies and represents number of individuals responding to given setof pathogen derived epitopes. Populations included, in IEDB web tool, are Australia,Europe, North Africa, North America, North-East Asia, Oceania, South America, SouthEast Asia, Others, South-west Asia and Sub-Saharan Africa. Brazilian, Cuban andMexican.


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Figure 2. (a–c). Cloning, Expression and purification of Rv0462 and Rv2251 and Westernblotting. (a). Amplification of Rv0462 gene with specific primers. Lane 2 indicates a bandof 1500 bp corresponding to the Rv0462 gene plus an additional upstream sequence.(b) Restriction digestion of recombinant plasmid (pET30þRv0462) with BamHI andXhoI insert was released with expected size and recombinant plasmid sequencewas confirmed by DNA sequencing. (c). Amplification of Rv2251 gene with specificprimers. Lane 2 indicates a band of 1400 bp corresponding to the Rv2251 gene. Insertrelease was not observed but DNA sequencing confirmed the presence of Rv2251 withuniversal 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 plasmidsshowed a 56 kDa Rv0462 protein (Figure 2d) and 52 kDa (Figure 2e) Rv2251 proteinand its purity. Figure 2(f) shows Western blot with anti His antibody against Rv0462and Rv2251. Lane MW indicates molecular weight protein marker, Lane number 1–5(d) and 1–6 (e) indicates different elutions of the corresponding protein collectedduring 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 bloodassayWe 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 (n¼ 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 (p¼ 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 prevalenceFollowed 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 healthyhousehold contacts (HHC) and pulmonary TB (PTB) patients with ESAT 6, CFP-10, Ag85Band test antigens Rv0462 and Rv2251. Culture filtrate antigens (CFA) used as positivecontrol. * refers to significant value (p¼ 0.01), ** refers to significant value (p¼ 0.002) and*** refers to p value¼ 0.0004. (b) Secreted cytokines were analyzed by ELISA to compareTNF-a levels in 10 healthy household contacts (HHC) and pulmonary TB (PTB) patients withESAT 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 PTBwith any of the stimuli tested.


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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

HLA02

01

HLA02

05

DRB1_01

01

DRB1_01

02

DRB1_03

01

DRB1_03

05

DRB1_03

06

DRB1_03

07

DRB1_03

08

DRB1_03

09

Alleles of interest

0

10

20

30

Pro

mis

cuou

s ep

itope

s

Figure 5. Promiscuous epitopes predicted by Propred. Promiscuous T-cell epitopes makeideal targets for vaccine development. Majority of the test antigens having one or morepromiscuous peptides and percentage of binding was calculated. Among thesepeptides four were having more than 90% binding towards HLA alleles and considered ashighly promiscuous epitopes.

HLA-A

*020

1

HLA-A

*020

5

DRB1_01

01

DRB1_01

02

DRB1_03

01

DRB1_03

05

DRB1_03

06

DRB1_03

07

DRB1_03

08

DRB1_03

090

20

40

60

Alleles of interest

Epi

tope

s pr

edic

ted

Figure 4. ProPred analysis of HLA–A, B and DR binding predictions for the Mycobacterialculture filtrate proteins. Each antigen was predicted to have at least one or moreepitopes binding to above mentioned alleles. Total number of epitopes were summedper alleles and consistently A*0201 and A*0205 were binding higher number of epitopesfrom majority of the antigens. DRB1 alleles were predicted to bind large number ofepitopes from all the antigens.


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Table 3. Promiscuous epitopes sequence predicted from overall 24 novel T cell antigensstudied and their amino acid positions.

S. No Gene Name Epitopesequence

Amino acidPosition

% of binding

1 ESAT-6 LQNLARTIS 69 56IQGNVTSIH 18 29

2 CFP10 IRQAGVQYS 76 78VRFQEAANK 56 66

3 Ag85B FIYAGSLSA 183 58VYLLDGLRA 76 33

4 Adk VPSDLTNEL 59 35VLLLGPPGA 3 44VKLAEKLGI 18 75

5 Rv0462 YAIGDVNGL 305 48LVHIFTKDA 58 71YVAAIRAAQ 16 57LRNAELVHI 53 57

6 Rv2251 FRAVISLDM 92 69

7 SahH VLIETLTAL 76 40YQFAAAGDL 227 37IIMLEHIKA 343 59FVMSNSFAN 419 48

8 Fba VRPLLAISA 106 79FVFHGGSGS 249 48

9 Rv1324 VVLLWSPRS 67 97FQGLQPADQ 133 65

10 acn LVIDHSVIA 123 46YVGNGSPDS 394 44VVIAAITSC 471 77

11 Tal VVGVTTNPS 42 91WKIVDRPNL 132 46

12 ProA MVNASTAFT 362 63VQLLSAADR 186 42

13 GgtB WLRAGALVA 4 53

14 Pgi KTFSTLETL 211 42FHIIDRHFA 299 65

15 FabG4 MRFLLSAKS 178 97IRVCGQAMI 444 42

16 ald FRVAITPAG 14 48LLLKVKEPI 70 42

17 Rv2721c VLLAPTVAA 27 65FVVRGALNA 150 57IVRFSAADK 312 61

18 pks13 MLFGEYIRL 836 37IRLMALVEY 842 91LVPLAVSAF 624 77

Promiscuous T-cell epitopes make ideal targets for vaccine development. Populationcoverage of the each peptide is given in this table. Among these four peptides (boldletters) 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.


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in silico analysis of potential human t cell antigens from mycobacterium tuberculosis for the...

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