The demonstrated variable efficacy of the only licensed TB vaccine Mycobacterium bovis bacillus CalmetteeGue´rin (M. bovis BCG) encourages the need for new vaccine candidates against TB. Antigen specific cellular immune response is often considered imperative during Mycobacterium tuberculosis (M. tuberculosis) infection and antigens that are strongly associated with the latent phase of infection are drawing increasing attention for anti-TB vaccine development. Here, we investigated the phenotypic and functional profiles of two novel mycobacterial antigens Rv2251 and Rv2721c during T cell recall response via multi-color flow cytometry.

1. T cell recall response of two hypothetical
proteins (Rv2251 and Rv2721c) from
Mycobacterium tuberculosis in healthy
household contacts of TB e Possible subunit
vaccine candidates
D. Santhi, Alamelu Raja*
Department of Immunology, National Institute for Research in Tuberculosis (ICMR), (Formerly
Tuberculosis Research Centre), No.1, Mayor Sathyamoorthy Road, Chetpet, Chennai 600 031, India
Accepted 28 June 2016
Available online 9 July 2016
KEYWORDS
M. tuberculosis;
Latent and active
tuberculosis;
T cell response;
Whole blood culture;
Multicolur flow
cytometry
Summary The demonstrated variable efficacy of the only licensed TB vaccine Mycobacte-
rium bovis bacillus CalmetteeGue´rin (M. bovis BCG) encourages the need for new vaccine can-
didates against TB. Antigen specific cellular immune response is often considered imperative
during Mycobacterium tuberculosis (M. tuberculosis) infection and antigens that are strongly
associated with the latent phase of infection are drawing increasing attention for anti-TB vac-
cine development. Here, we investigated the phenotypic and functional profiles of two novel
mycobacterial antigens Rv2251 and Rv2721c during T cell recall response via multi-color flow
cytometry. Healthy household contacts of TB (latent/HHC) and active pulmonary TB (PTB) pa-
tients were recruited to investigate the difference in antigen specific T cell recall response.
These two antigens induced expansion of CD45RAÀ
CCR7þ
central memory subtypes and
CD45RAÀ
CCR7À
effector memory cells in latent population which suggests their possible asso-
ciation with HHC. Rv2251 and Rv2721c antigen specific IFN-g, TNF-a and IL-2 response was also
significantly high in HHC when compared to the PTB (p < 0.005, p < 0.05 and p < 0.05 respec-
tively). The frequency of multifunctional T cells also was high in HHC compared to the PTB with
statistical significance only for the antigen Rv2251. Often, the dominant Th1 immune response
in HHC is correlated with the protection against the active TB disease. Collectively, we report
the first insights into Rv2251 and Rv2721c antigen specific immune response in human donors of
TB and provide the immunologic rationale for selecting them for vaccine development against
TB.
ª 2016 Published by Elsevier Ltd on behalf of The British Infection Association.
* Corresponding author. Fax: þ91 (044) 2836 2528.
E-mail address: alamelur@nirt.res.in (A. Raja).
http://dx.doi.org/10.1016/j.jinf.2016.06.012
0163-4453/ª 2016 Published by Elsevier Ltd on behalf of The British Infection Association.
www.elsevierhealth.com/journals/jinf
Journal of Infection (2016) 73, 455e467

2. Introduction
Despite the availability of the only licensed vaccine BCG,
tuberculosis (TB) is still the leading microbial cause of
death.1
Though, the administration of BCG conferred pro-
tection against TB from its first oral administration in
1921, it later showed little or no protection during large
field trials.2
BCG might diminish the most severe forms of
TB, such as TB meningitis in children. But, it has had varying
impacts on the occurrence of pulmonary TB in adults, which
represents the transmissible form of this disease. Hence, an
improved vaccine as a replacement to BCG or a subunit
boosting vaccine to administer after BCG vaccination is
needed. Subunit vaccine approaches hold a number of ad-
vantages, like increased safety, stability and ability to
boost prior BCG immunization.3
Pools of proteins obtained
from Mycobacterium tuberculosis, the causative agent of
tuberculosis, are often screened for subunit vaccine devel-
opment and several promising candidates have been
targeted.
The role of CD4þ
Th1-cells in TB is best understood and
many researchers concluded that anti-TB immunity is pre-
dominantly mediated by CD4þ
Th1 cells.4,5
To date, many
mycobacterial antigens have been screened for their ability
to induce cellular immune responses with the goal of sub-
unit vaccine application. These include Ag85 complex,6
ESAT-6,7
CFP-10,8
Rv0577,9
HspX5
etc., but their T cell re-
sponses are not homogenous.10
We hypothesize that, one
of the reasons for variation in T cell response to these anti-
gens might be due to the absence of specific MHC alleles
amongst different ethnicities for presentation of these anti-
genic peptides. This hypothesis is well supported by our
earlier published work that showed comparatively low pop-
ulation coverage and MHC binding for ESAT-6 and CFP-10
peptides relative to other screened antigens.11
Speculations
about the reasons for the differences in BCG efficacy also
linked the role of genetic differences in different ethnic-
ities.12
In this light, we have selected two mycobacterial an-
tigens, predicted to have high MHC binding affinity and high
percentage of population coverage in lieu of immunodomi-
nant antigens ESAT-6 and CFP-10. We evaluated T cell im-
mune responses with the aim of subunit vaccine targetings.
These two antigens are Rv2251 (Possible flavoprotein)
and Rv2721c (Possible conserved transmembrane alanine
and glycine rich protein). Rv2251 was predicted to be an
outer membrane protein13
and the Rv2721c was identified
by mass spectrometry in the membrane fractions of M.
tuberculosis.14
In general, membrane localized proteins
are targeted for the vaccination studies since they are
readily available for immune processing within the host.15
In the field of TB, we can consider latently infected individ-
uals who remain healthy as protected against active TB dis-
ease due to their ability to control the infection, unlike
active TB patients. Their cellular immune response would
reflect the type of immunity responsible for their efficient
disease control and serve as a good experimental model.16
Thus, we are interested in evaluating these two antigens’
specific T cell responses to make a better correlation either
with protection or pathology. This human based in vitro
study would minimize the variations with other existing an-
imal based in vitro TB models. Our analysis is bringing the
first insight into Rv2251 and Rv2721c antigens’ specific im-
mune response in human models.
Materials and methods
In vitro cloning of Rv2251 and Rv2721c, over-
expression and purification
The recombinant plasmid encoding Rv2251 antigen was
obtained by in vitro cloning.11
The same methodology was
adapted for cloning and purification of Rv2721c. Briefly, en-
coding gene sequences of Rv2721c were amplified from M.
tuberculosis H37Rv genomic DNA with gene specific primers
by Phusion High Fidelity DNA polymerase (New England Bio-
labs, MA). The amplified Rv2721c gene and pRSET-A plasmid
DNA were digested with restriction enzymes Sac-I and Hind-
III (NEB, MA). The digested gene and plasmid DNA were
ligated by T4 DNA ligase (NEB, MA) and transformed into a
cloning strain of Escherichia coli (E. coli) DH5a. Positive re-
combinants confirmed by DNA sequencing were then trans-
formed into the E. coli BL21 (DE3) (Invitrogen, USA) strain
for protein induction and purification. The purified
Rv2721c antigen was used for in vitro blood stimulation
along with recombinant Rv2251 antigen.
Study participants
Informed written consent was obtained from all the donors
and the study was approved by an Institutional Ethical
committee of the National Institute for Research in Tuber-
culosis, Chennai, India. Thirteen milliliters (mL) peripheral
blood samples were collected from a total of thirty nine
individuals. Among the thirty nine donors, twenty two were
latently infected individuals in our case healthy household
contacts (HHCs) of TB and seventeen had active pulmonary
tuberculosis (PTB). The criteria for HHC recruitment was
based on sharing the living shelter minimum for three
months with at least one sputum positive active TB patients
(index TB case) who are naive for anti-tubercular therapy.
HHCs had more than ten hours per day of close contact with
adult smear-positive PTB patients. They were identified by
visiting the households of adult smear positive pulmonary
TB patients, who were enrolled for treatment at the
Government Thiruvatteeswarar Hospital of Thoracic Medi-
cine, Otteri, Chennai, Tamil Nadu, India. HHCs had no
active TB symptoms which were ruled out by chest X-ray
and smear microscopy. PTB individuals were selected based
on positive sputum microscopy results of three sputum
samples.
The infection state of all the study participants was
assessed by the Interferon gamma release assay (IGRA). No
active TB symptoms were found in HHCs which was
confirmed by negative sputum smear microscopy. PTB study
participants who had symptoms of immunosuppressive dis-
ease such as HIV infection were excluded from this study.
Interferon gamma release assay (IGRA)
QuantiFERON-TB Gold In-Tube (QFT-GIT) kit (Cellestis,
Qiagen) contains three pre-coated tubes coated with TB-
456 D. Santhi, A. Raja

3. antigens (ESAT-6, CFP-10, and TB7.7) as a test, phytohe-
magglutinin (PHA) as a positive control, the nil antigen
(coated with saline) as a negative control. Out of thirteen
milliliters (mL) of collected blood, one mL was added to
each tube of the kit and incubated for 16e24 h at 37
C, 5%
atmospheric CO2. The supernatant was collected after
centrifugation of the three tubes and stored at À80
C until
the assay was done. The test results were interpreted as
per kit guidelines, using software provided by the
manufacturer.
Whole blood culture with Rv2251 and Rv2721 from
the blood of HHCs and PTB
The collected blood was diluted 1:1 with RPMI1640 (Sigma
Aldrich, St. Louis, MO, USA) medium with penicillin/
streptomycin (100 U/100 mg/mL), L-glutamine (2 mM),
and HEPES (10 mM) and distributed as two mL into tissue
culture plates. Then, the cultures were stimulated with
M. tuberculosis ESAT-6 (E6), CFP-10 (C-10), Rv2251 and
Rv2721c antigens at the final concentration of 5 mg/mL as
determined previously.16
Phytohemagglutinin (PHA) was
used as mitogen control, at the final concentration of
1 mg/mL, to test the proliferating capacity of the cells
from donors. The recombinant plasmids of immunodomi-
nant antigens ESAT-6 and CFP-10 were received from Colo-
rado State University (CSU), USA. Diluted blood without
any added antigen was set as an unstimulated culture con-
trol. To all the stimulations, purified co-stimulatory mole-
cules CD49d/CD28 (BD biosciences, San Diego, CA, USA)
were added at a final concentration of 0.5 mg/mL. These
culture plates were incubated for 4 h at 37
C, 5% CO2
and removed for the addition of Brefeldin A (10 mg/mL)
then continued the incubation for 12 h strictly. Upon
completion of 16 h incubation, plates were removed for
cell harvesting with PBS. The harvested cells were pelleted
down by centrifuging at 2600 rpm for 10 min and then
treated with a BD FACS lysing solution (BD, San Diego,
CA, USA) to lyse RBCs as per manufacturer’s instruction.
The cells were fixed using BD cytofix/cytoperm buffer
and cryopreserved with 10% DMSO at À80
C until intracel-
lular staining was done.
Surface markers and intracellular cytokine staining
Fixed cultured cells were rapidly thawed from À80
C and
washed with PBS. These cells were stained for T cell
surface markers and intracellular markers after single cell
suspensions were made. The following antibodies were
used for surface staining at several different panels: APC-
anti-CD69 (clone FN 50), PerCP 5.5 - anti-CD3 (clone
UCHT1), APC-Cy7-CD4 (clone RPA-T4), PE-Cy7-CD8 (clone
RPA-T8), FITC-CD197 (CCR7) (clone 3D12), APC-C45RA
(clone HI100) at the final concentration of 5 ml/1 million
cells. The following antibodies were used for intracellular
staining: FITC-IFN-g (clone B27), PE-TNF-a (clone MAb11),
APC-IL-2 (clone 5344.111), FITC-IL-17A (clone N49-653), PE-
IL-22 (clone BG/IL-22) and APC-IL-21 (clone 3A3-N2.1). All
antibodies were purchased from BD Pharmingen, USA or
BioLegend, USA. All staining was done at 4
C for 30 min in
the dark followed by washing with PBS for surface staining
and with perm wash buffer after intracellular staining.
FACSCanto II flow cytometer with FACSDiva software,
version 6 (Becton Dickinson and Company, Cockeysville,
MD) was used for acquisition of stained single cell suspen-
sions. Acquired data was analyzed in Flow Jo software
(TreeStar). All data is depicted as the percentage of CD4þ
T
cells expressing cytokine(s).
Positive and negative boundaries were defined by setting
Fluorescence minus one (FMO) controls for all the used
antibodies and for all the positive results obtained, FMO
control is shown in supplementary figures. CompBeads
purchased from BD Biosciences, USA and stained with the
all used fluorochrome monoclonal antibodies as compensa-
tion control.
Data analysis
Statistical analyses were performed using Graphpad Prism
software version 5 (GraphPad software, CA, USA). Inter-
group comparisons were performed using the nonpara-
metric Mann Whitney U and with Holm’s correction for
multiple comparisons. For all the analysis, differences were
considered as significant if p value was less than 0.05.
Results
Table 1 summarizes the clinical characteristics of the
healthy household contacts (HHC) and the active TB pa-
tients (PTB). QFT-IT test confirmed the mycobacterial
infection status of all the study participants who were pos-
itive (3.5) for QFT-IT. The cellular response, was similar in
both HHC and PTB, !0.5 IU/mL, in the mitogen tube of
QFT-IT kit showed no defects in immune response in all
study subjects. All PTB subjects were naive to anti-TB
treatment at the time of enrolling in our study. Percentage
of CD4þ
cells in both HHC and PTB ranged between 20% and
65% and expression of early T cell activation marker CD69þ
was similar also between the two groups (Supplementary
Fig. 1) showing no defects in immune cell responsiveness
in all the study subjects. Fluorescence minus One (FMO)
for all the results obtained (memory T cell and Th1 analysis)
is given in Supplementary Fig. 2.
Cloning, expression and purification of Rv2721c
The encoding gene sequence of Rv2721c (2.1 kb) was
amplified from M. tuberculosis H37Rv with gene specific
primers (forward primer sequence (Sac I) is 50
CTG GGA
GAG CTC GTG AAC GGG CAG AG 30
and Reverse primer
sequence (Hind-III) 50
CC GCG TGC AAG CTT TCA ATC CGC
CC30
) by Phusion high fidelity DNA polymerase which
yielded a single 2100 bp fragment at an optimal tempera-
ture of 60.8
C (Fig. 1a, lane 4). The plasmid DNA pRSET-
A was digested with Sac I and Hind-III to produce cohesive
termini (Fig. 1b). Digested plasmid pRSET-A and Rv2721c
gene were ligated by T4 DNA ligase and transformed into
E. coli DH5a. Then, these recombinant colonies (taken as
template) were screened for presence of Rv2721c gene by
colony PCR with gene specific primers and PCR
T-cell response of Rv2251 and Rv2721c 457

4. amplification to confirm the presence of the insert gene
Rv2721c (Fig. 1c). Colonies with amplification of Rv2721c
were marked as “positive recombinant colonies”.
Plasmid DNA was isolated from positive recombinant
colonies and subjected to restriction digestion with the
same set of enzymes used for cloning (Sac-I and Hind-III).
Release of Rv2721c (insert release) from the recombinant
plasmid DNA confirms the presence of gene of interest
(Fig. 1d). The presence of the Rv2721c gene in recombinant
plasmid DNA was also confirmed by DNA sequencing.
Sequencing result showed 100% homology identity with
the original gene sequence when searched against M.
Table 1 Demographic details of all study participants.
Characteristic of study participants Healthy household contacts (HHC) Active pulmonary TB individuals (PTB)
Sample size 22 17
Mean age (yr) (range) 39 (27e55) 41 (25e56)
Sex
Female, N (%) 12 (54%) 8 (47%)
Male, N (%) 10 (45%) 9 (55%)
Sputum smear positivity, N (%) 0 (0) 17 (100)
Semear grade, þþþ, N (%) 0 (0) 8 (50)
Semear grade, þþ, N (%) 0 (0) 5 (27)
Semear grade, þ, N (%) 0 (0) 4 (22)
QFT-GIT
Positive, N (%) 22 (100) 17 (100)
Negative, N (%) 0 (0) 0 (0)
Indeterminate, N (%) 0 (0) 0 (0)
CD4 count e range in % 20e65 21e62
Figure 1 In vitro cloning, over-expression and purification of Rv2721c from H37Rv genome. a) Rv2721c gene, 2100 bp, was ampli-
fied by PCR. Lane 1e7 shows amplification at various temperatures ranging from 55
C to 70
C. Maximum amplification with no non-
specific amplification was considered as optimal and 60.8
C was selected. Lane “M” is 10 kb DNA ladder. b) pRSET-A plasmid DNA
was digested with Sac-I and Hind-III restriction enzyme and complete linearization was obtained at 37
C for 4 h s (lane 2), lane 1
shows undigested plasmid pRSET-A. Lane “M” is 10 kb DNA ladder. c) PCR analysis with recombinant colonies showed the amplifi-
cation of Rv2721c (2.1 kb) which confirmed the presence of gene. Lane M is 10 kb DNA ladder, lane “1” is positive control (H37Rv
genomic DNA) and lane 2e12 various colonies screened. d) The release of Rv2721c (insert release) from the positive recombinant
colonies confirms the presence of Rv2721c gene. Lane 1 and 2 shows the insert release and 3 is negative control. Lane M is 10 kb DNA
ladder. e) IPTG induction of positive recombinant colonies. Lane UI denotes culture aliquoted before adding IPTG and lane 1e3
indicates expression of Rv2721c at 25
C, 30
C and 37
C. f) Rv2721c protein was purified by His-tag Ni-NTA affinity chromatography
and lane 4e8 shows purified protein fractions. M e indicates protein ladder. g) Western blot with anti-His antibody confirms the
recombinant Rv2721c.
458 D. Santhi, A. Raja

5. tuberculosis genomic DNA in NCBI BLAST. Positive recombi-
nant plasmids were transformed into E. coli expression sys-
tem BL21 DE3 and protein expression was tested at various
temperatures (25
C, 30
C and 37
C) (Fig. 1e). Cultures
were grown at 37
C until reaching OD600 nm Z 0.5, induced
with 1 mM Isopropyl b-D-1-thiogalactopyranoside (IPTG) and
the eluted fraction of Rv2721 was analyzed by SDS-PAGE
(Fig. 1f). The recombinant Rv2721c was confirmed by West-
ern blot with His-Tag Antibody (Novagen, Germany)
(Fig. 1g).
Antigen specific central memory phenotype
CD45RAL
CCR7D
in healthy mycobacterial infected
individuals
Gating strategy for the selection of CD4þ
cells from whole
blood cells to analyze surface and intracellular T cell
markers is given in Fig. 2a. The most commonly used sur-
face markers to define memory T cell subsets are CD45RA,
a protein tyrosine phosphatase regulating src-family ki-
nases, and the chemokine receptor CCR7.17e20
Based on
the expression of these surface markers, memory subsets
are classified as naive cells (CD45RAþ
CCR7þ
) central mem-
ory (CD45RAÀ
CCR7þ
), effector memory (CD45RAÀ
CCR7À
),
and terminal effector cells or CD45RAþ
cells (CD45RAþ
CCR7À
). Expression levels of these phenotypic markers in
our cultured cells were measured at baseline (unstimulated
control) and mycobacterial antigen stimulations by poly-
chromatic flow cytometry. Antigen specific T cell response
was calculated by subtracting the unstimulated values
from the test (Test-nil). Density plot is given to represent
the memory T cell population in our antigens stimulations
as mentioned19e21
and representative plots from HHC and
PTB are given in Fig 2b.
In our 16 h whole blood cultures, CD45RAÀ
CCR7À
effector memory types were predominantly present
compared to other memory subtypes in both HHC and
PTB. The percentage of central memory subtypes (CD45RAÀ
CCR7þ
) was high in HHC compared to the PTB with all anti-
gen stimulations except for CFP-10. ManneWhitney U test
analysis showed highest significance for Rv2721c
(p Z 0.0004) and followed by Rv2251 (p Z 0.002) when
compared to PTB. The levels of significance for these two
novel antigens were comparatively higher than the immu-
nodominant antigen ESAT-6. Based on these observations,
we conclude that the antigen specific memory cells are
readily detectable and high in healthy household contacts.
The level of significance was low for the standard immuno-
dominant antigens ESAT-6 and CFP-10 as these antigens
specific memory cells were also present in PTB (Fig.2c).
Like central memory cells, effector memory T cell
subtypes (CD45RAÀ
CCR7À
) were also high in HHC against
all the antigen stimulation used when compared to active
TB, with the exception of CFP-10. But, the significance
was observed only for Rv2251 and Rv2721 (p 0.01)
Fig. 2d. The other two memory cell types, naive (CD45RAþ
CCR7þ
) and terminal effector cells (CD45RAþ
CCR7À
) re-
vealed no significant in differences between HHC and
PTB, with any of the stimulations used Fig. 2e and f.
Antigen specific single positive Th1 cytokine
secreting cells were higher than multifunctional T
cells in healthy household contacts
Th1 polarized immune responses are considered important
for anti-TB immunity. Thus, the circulating levels of Th1
immunomodulatory cytokines (IFN-g, TNF-a and IL-2) spe-
cific to Rv2251 and Rv2721 antigens in addition to immu-
nodominant antigens ESAT-6 and CFP-10 were measured. As
observed by several studies, the circulating levels of Th1
cytokines were high in HHCs compared to PTB.
Elevated levels of CD4þ
IFN-gþ
was observed in HHC for
Rv2251 and Rv2721 antigen stimulations (p 0.005) and
with ESAT-6 (p 0.05) showing the presence of these anti-
gen specific IFN-g secreting cells in circulation of HHC. In
PTB, the percentage of CD4þ
IFN-gþ
cells were detectable
but less than HHCs. However, except CFP-10 stimulation
had a high frequency of IFN-g cells in PTB Fig. 3a and b.
Like CD4þ
IFN-g, the frequency of CD4þ
TNF-a cell was
high in HHC compared to PTB with statistical significance
only for the novel test antigens Rv2251 and Rv2721c
(p 0.05). In PTB, Rv2251 and Rv2721c specific expansion
of CD4þ
TNF-aþ
cell was minimal when compared to
ESAT-6 and CFP-10 specific CD4þ
TNF-aþ
Fig. 3c and d. Like-
wise, the percentage of IL-2 secreting T cells were high in
HHC than PTB and the antigen specific recall response
was found to be significant (p 0.05) in Rv2251 and
Rv2721c antigen stimulations over other stimulations where
no statistical significance was found Fig. 3e and f.
T cells that coexpress IFN-g, TNF-a and IL-2 are desig-
nated polyfunctional T cells (PFT) with a possible role in
protection against TB. The responding CD4þ
T cells were
classified as triple positive (IFN-gþ
/IL-2þ
/TNF-aþ
), double
positive (IFN-gþ
/IL-2þ
, IFN-gþ
/TNF-aþ
or TNF-aþ
/IL-2þ
)
or single positive (IFN-gþ
, IL-2þ
or TNF-aþ
). Boolean gate
platform in FlowJo analysis software was used to calculate
proportions of PFT and are given in Fig. 4a. As shown in
Fig. 4b the Rv2251 had potency to induce high levels of
poly functional T cells in HHCs compared to the PTB
(p 0.05). Although Rv2721c specific poly functional T cells
were high in HHC, this difference was not statistically sig-
nificant. ESAT-6 and CFP-10 poly functional T cells were
detectable in both HHC and PTB with no significant increase
in either of the study groups. Among the double positive
cells, CD4þ
that co-expresses IFN-gþ
and TNF-aþ
were
high in HHCs with Rv2251 and Rv2721c antigen stimulations
(p 0.05).
Variable antigen specific Th17 responses
Among the analyzed Th17 cytokines, IL-22 was predomi-
nantly present in our 16 h whole blood cultures. Baseline
values did not vary between HHC and PTB for IL-17 and IL-
21, whereas IL-22 was present high in PTB at baseline (data
not shown). Elevated levels of IL-17 were observed in HHC
after all antigen stimulations except for CFP-10 and to
some extent in PTB Supplementary Fig. 3a. A similar type of
recall response was also observed with IL-21 levels in HHC
in all stimulations except for Rv2721c Supplementary
T-cell response of Rv2251 and Rv2721c 459

6. 460 D. Santhi, A. Raja

7. Fig. 3b, but both of these cytokines did not vary signifi-
cantly between the groups.
IL-22 levels were high in PTB compared to HHC for all the
antigen stimulations except for Rv2251 Supplementary
Fig. 3c. Comparatively high levels of IL-22 were observed
for both the immunodominant antigens ESAT-6 and CFP-10
in PTB. Th17 cells secreting both IL-22 and IL-17 (IL-17/
22) cytokines were high in HHC for all antigens’ stimulation
Figure 2 Antigen specific CD4D memory T cell subtypes in HHC and PTB. a) In vitro stimulated whole blood was stained and
acquired in flow cytometry. Single cells were selected by taking FSC-H vs FSC-A and then lymphocyte populations were analyzed for
the expression of CD3þ
, a specific T cell marker (1 to 3). CD3þ
cells were analyzed for the expression of CD4 and CD8 (4) to analyse
the expression of memory cell markers. b) Representative flow diagram of memory markers CD45RA and CCR7 from HHC and PTB is
given. CD4þ
cells were classified according to the expression of CD45RA and CCR7 into various memory cell subtypes. The respec-
tive percentage of naive cells (CD45RAþ
CCR7þ
) central memory (CD45RAÀ
CCR7þ
), effector memory (CD45RAÀ
CCR7À
), and ter-
minal effector cells (CD45RAþ
CCR7À
) were determined. The expansion of central memory cells was high in HHC compared to PTB
against Rv2251 and Rv2721c antigens when compared to other antigens stimulations. PHA mitogen control showed response in both
HHC and PTB indicating their immune reactivity is not compromised. The percentage of central memory (c), effector memory (d),
naive cells (e) and terminal effector cells (f) found in 16 h whole blood culture from 22 HHC and 17 PTB subjects are represented as
scatter plots and line in the middle depicts median. Central memory and effector memory population were found to be higher in
HHC against Rv2251 and Rv2721c stimulations with statistical significance of p 0.005 for Rv2251 and for Rv2721c and ESAT-6 p
0.02 by ManneWhitney U test with Holm’s correction. p values less than 0.05 was considered as significant.
Figure 3 Intracellular staining for Th1 cytokines expression. CD4þ
T cells were gated as described before. Intracellular Th1
cytokines in circulation of healthy household contacts (n Z 22) and pulmonary TB subjects (n Z 17). Frequency of IFN-g secreting
CD4þ
cells (a), TNF-a secreting CD4þ
cells (c) and IL-2 secreting CD4þ
cells (e) against all antigenic stimulation is shown and repre-
sentative flow diagram from HHC and PTB is given. Box whisker plot showing the percentage of cells secreting IFN-g (b), TNF-a (d)
and IL-2 (e) from HHC and PTB is given. The whisker graph illustrates minimum to maximum range and middle line in the box in-
dicates median value. p values less than 0.05 was considered as significant.
T-cell response of Rv2251 and Rv2721c 461

8. compared to PTB. And in a few PTB patients Th17 dual cells
were absent completely with no statistical differences.
Supplementary Fig. 3d.
Discussion
Circulating antigen specific T cells in a protected popula-
tion of TB would reflect the possible dominant immune
response necessary for effective disease control and are
well measured by antigen specific recall responses. This
approach has been used for the selection of potential
antigens for anti-TB vaccine development.5
Effective TB
vaccines are often aimed at evoking T cell-mediated im-
mune responses, since that confers protective immunity
to TB.22
To assess the T-lymphocyte recall responses, assays
based on peripheral blood mononuclear cells (PBMC) and
whole blood (WB) were widely used with the similar
outcome. But, WB assays hold an advantage as compara-
tively less blood samples are required than PBMC assays.
Hence, we preferred to use WB assays in our study and
diluted the blood sample to minimize volume use as well as
to screen many antigens with the same blood sample.
Various blood sample dilutions of 1:1,23
1:2,16
1:5 and
1:1024
were used by different studies. Though, a few re-
ports have mentioned that peripheral blood might not
necessarily address the immune response at the site of
infection,25
recent evidence has shown that disease activity
at the site of the infection may be reflected in peripheral
blood. Blood serves as a reservoir of trafficking immune
cells that travel to and from sites of active disease and
lymphoid organs. As it is readily accessible, it could be an
appropriate test tissue in humans for studying immune
mechanisms26e28
and to evaluate vaccine efficacy in terms
of T cell response.29e31
But, for a better understanding of
antigen or vaccine mediated protective immunity to M.
tuberculosis, a closer look at the immunological events in
the lung is also essential.
The difference between protection and the immunopa-
thology of any infectious disease is conferred by the quality
of memory responses.32
The quality of two selected anti-
gens Rv2251 and Rv2721c in eliciting memory responses
are demonstrated first in our results. They induced high
levels of central and effector memory phenotypes, predom-
inantly in healthy M. tuberculosis infected individuals. Our
observation suggests that these two antigens could be
possibly associated with latently infected subjects, who
Figure 3 Continued
462 D. Santhi, A. Raja

9. are presumed to be protected against active TB disease,
and provides the immunological rationale for evaluating
these antigens for vaccine development against TB. Central
memory subtypes are highly correlated as mediators of pro-
tection (high proliferative capacity and rapidly transform to
effector cells upon re-exposure to antigen) and their pres-
ence is often found with latent TB rather than active TB.21
The same was observed in our study. Emerging reports sug-
gest the necessity of effector memory subtypes in addition
to central memory cells for effective infection control.33
This is well documented from our study where the levels
of effector memory cells were high in HHCs showing their
possible role in protection against active TB disease. Our
observations open a new path to explore the effector cyto-
kines secreted by Rv2251 and Rv2721c specific memory
cells. The two standard antigens ESAT-6 and CFP-10 specific
memory cells were also detected at significant levels in the
active TB population showing their lack of uniqueness to
the latent TB population. Hence, protection against TB
from these standard antigens might not be guaranteed.
Rv2251 and Rv2721c antigens specific Th1 response IFN-
g, TNF-a and IL-2 were also dominant in latently infected
subjects. Resistance to intracellular pathogens and
macrophage activation is thought to be mediated by IFN-
g.34
The role of TNF-a which synergizes with IFN-g for intra-
cellular pathogen elimination, is supported by other
studies.35
TNF-a is also important in orchestrating granu-
loma formation, the hallmark of tuberculosis, and is needed
for active disease control. IFN-g is often used as a marker of
protective immunity against M. tuberculosis infection.36e38
The marked increase in Th1 immune response against these
two antigens supports our hypothesis for these antigens as
promising vaccine candidates against TB. Our results
demonstrated minimal expansion of antigen specific TNF-
a cells in PTB with Rv2251 and Rv2721c, in contrast to
ESAT-6 and CFP-10, which shows that these antigens are
not predominantly detected by active TB patients hence,
these antigens could be latently associated. Compared to
the test antigens (Rv2251 and Rv2721c), CD4þ
IFN-gþ
levels
were minimal against the standard antigen ESAT-6 stimula-
tion. This was already observed with diluted whole blood16
and PBMC39
when ESAT-6 was used at 5 mg/mL with an
agreement to our observation.
We showed that frequency of Rv2251 and Rv2721c
specific poly functional T cells were high in HHC compared
to other antigen stimulations with statistical significance
Figure 3 Continued
T-cell response of Rv2251 and Rv2721c 463

10. Figure 4 Mycobacterial antigen specific poly functional CD4D
Th1 cells. Whole blood cells were cultured with ESAT-6 (E6),
CFP-10 (C-10) and Rv2251, Rv2721c mycobacterial antigens and percentage of poly functional T cells were determined. a) Boolean
464 D. Santhi, A. Raja

11. only for the former antigen. This could be attributed to the
elevated levels of IFN-g and TNF-a with these two antigenic
stimulations. These multifunctional T cells are capable of a
broader repertoire of T cell functions, hence are associated
with enhanced protection.40
In humans, high levels of cyto-
kine secretion by triple-positive antiviral T cells have been
shown rather than single positive cells.41
Thus, the qualities
of T cells induced by these two antigens could be superior in
producing of Th1 cytokines, which could be correlated with
protective immunity and the development of a preventive
vaccine.
The frequency of Rv2251 and Rv2721c specific Th1
cytokine T cell profiles also supports evidence for the T
cell co-expressing either TNF-aþ
or TNF-aþ
/IFN-gþ
. The
maintenance of this population in the late stage of infec-
tion was associated with enhanced control of bacterial
growth.42
Interestingly, these two antigens’ specific double
positive cells that secrete IFN-gþ
/TNF-aþ
were high in
latent study subjects. The role of these IFN-gþ
/TNF-aþ
secreting cells as a latent marker and as a correlate of pro-
tection during PCV2 vaccination43
imply the possible poten-
tial of these two antigens to elicit protective immunity.
New evidence supporting the role of Th17 cells in
vaccine mediated immunity against TB are emerging. Our
study implied the detection of IL-17A in both groups, unlike
other studies where insufficient or absent IL-17A was
reported.44
Though, the statistical differences were not sig-
nificant, our results showed high antigen specific Th17 re-
sponses in HHC suggesting that, Th17 response might be
associated with latent study subjects. Distinctly IL-22
levels, despite belonging to Th17 group of cells, were
high in PTB, but during Rv2251 and Rv271c stimulations.
Comparative high levels of IL-22 in PTB were further sup-
ported by our analysis on Th17 dual cells where a majority
of the Th17 dual cells were secreting IL-22 rather than
secreting both IL-17 and IL-22. This suggestion of a distinct
Th17 response to Rv2251 and Rv2721c antigens are to be
studied further for better understanding.
Antigen specific Th1 response was observed also in
peripheral blood of active TB patients and used for TB
diagnosis.45e47
Increase in Th1 cell response during MVA85A
(modified Vaccinia Ankara virus expressing antigen 85A)
vaccine trial did not confer protection against M. tubercu-
losis infection. But, MVA85A vaccine efficacy trial was car-
ried in infants aged 4e6 months and their immune system is
still immature, but it could protect adults against PTB.48
These reports suggested Th1 response alone might not be
sufficient for controlling TB infection and contradict the hy-
pothesis of correlating antigen specific Th1 response in pe-
ripheral blood for protection. Notably, these observations
used ESAT-6, CFP-10 and Ag85A standard antigens of M.
tuberculosis to assess peripheral blood Th1 response. This
supports our observation that ESAT-6 and CFP-10 specific
T cell response is not unique to latency, but also to TB,
hence novel antigens of M. tuberculosis that are highly spe-
cific to latency might be needed for assessing their immune
status and to correlate it with protection against active TB
disease.
The lack of reliable correlates to immune protection
during TB infection or a biomarker to predict vaccine
efficacy poses challenges for TB vaccine development. For
optimal protection against TB CD4þ
T cells, effector cyto-
kines such as IFN-g and TNF-a are requisite, but are not an
exclusive component of protective immunity.49e51
Major
histocompatibility complex (MHC) class II-restricted CD4þ
T cells play an essential role in protective immunity
against M. tuberculosis. Identification of immunogenic T
cell epitopes is also essential for the design of peptide/
protein-based vaccines. Our earlier in silico analysis pre-
dicted high class I and II MHC binding affinity (97% and
100%, respectively) and a high percentage of population
coverage for these two novel antigens which were compar-
atively higher than the standard antigens, ESAT-6 and CFP-
10.11
Taken together, the first comprehensive analysis of
mycobacterial antigens Rv2251 and Rv2721c from human
donors showed dominant central and effector memory
subtypes, with Th1 dominant response alluding to its
importance for vaccine development. The frequency and
phenotypes of memory cells against these antigens are well
documented in our study. Assessing the quality of these
antigens specific memory cells for their effector cytokines
and whether the T cell responses against these two antigens
is sufficient to control the TB infection are yet to be
studied. Further evidence from large study populations,
animal based experiments to evaluate the efficacy of these
two antigens, and tolerance assessments would shed more
light on the potential of these two antigens in protection
against the TB infection.
Acknowledgments
The authors wish to thank all the study subjects who
participated in this study. Authors also thank the clinicians
of Govt. Thiruvatteeswarar Hospital of Thoracic
Medicine (GTHTM) hospital, Otteri, Chennai, India. Santhi
Devasundaram expresses her gratitude to Indian Council of
Medical Research (ICMR), New Delhi, India for providing
Senior Research Fellowship. ESAT-6, CFP-10 recombinant
proteins were kind gift from Colorado State University. The
authors wish to thank Dr. Subash Babu, National Institutes
of HealthdNIRTdInternational Center for Excellence in
Research, Chennai, India for allowing to use their FACS
facility.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.jinf.2016.06.012.
gating strategy to assess the co-expression of Th1 cytokines is given. b) The frequency of triple positive IFN-g/IL-2/TNF-a and dual
positive (IFN-g/IL-2, IFN-g/TNF-a or TNF-a/IL-2) Th1 cytokines in percentages are shown as bar graphs. p values were calculated by
ManneWhitney U test and p values less than 0.05 was considered as significant.
T-cell response of Rv2251 and Rv2721c 465

12. Competing interests
The authors declare that they have no competing interests.
References
1. McShane H. Tuberculosis vaccines: beyond bacille Calmet-
teeGuerin. Philosophical Trans R Soc Lond Ser B Biol Sci
2011;366(1579):2782e9.
2. Baily GV. Tuberculosis prevention trial, Madras. Indian J Med
Res 1980;72(Suppl.):1e74.
3. Dietrich J, Andersen C, Rappuoli R, Doherty TM, Jensen CG,
Andersen P. Mucosal administration of Ag85B-ESAT-6 protects
against infection with Mycobacterium tuberculosis and boosts
prior bacillus CalmetteeGuerin immunity. J Immunol 2006;
177(9):6353e60.
4. Kaufmann SH. How can immunology contribute to the control
of tuberculosis. Nat Rev Immunol 2001;1:20e30.
5. Bertholet S, Ireton GC, Kahn M, Guderian J, Mohamath R,
Stride N, et al. Identification of human T cell antigens for
the development of vaccines against Mycobacterium tubercu-
losis. J Immunol 2008;181(11):7948e57.
6. Horwitz MA, Lee BW, Dillon BJ, Harth G. Protective immunity
against tuberculosis induced by vaccination with major extra-
cellular proteins of Mycobacterium tuberculosis. Proc Natl
Acad Sci USA 1995;92:1530e4.
7. Weinrich Olsen A, van Pinxteren LA, Meng Okkels L, Birk
Rasmussen P, Andersen P. Protection of mice with a tubercu-
losis subunit vaccine based on a fusion protein of antigen 85b
and esat-6. Infect Immun 2001;69:2773e8.
8. Luo Y, Wang B, Hu L, Yu H, Da Z, Jiang W, et al. Fusion protein
Ag85B-MPT64 (190-198)-Mtb8.4 has higher immunogenicity
than Ag85B with capacity to boost BCG-primed immunity
against Mycobacterium tuberculosis in mice. Vaccine 2009;
27:6179e85.
9. Byun EH, Kim WS, Kim JS, Jung ID, Park YM, Kim HJ, et al.
Mycobacterium tuberculosis Rv0577, a novel TLR2 agonist, in-
duces maturation of dendritic cells and drives Th1 immune
response. FA Seb J 2012;26:2695e711.
10. Arlehamn CS, Sidney J, Henderson R, Greenbaum JA,
James EA, Moutaftsi M, et al. Dissecting mechanisms of immu-
nodominance to the common tuberculosis antigens ESAT-6,
CFP10, Rv2031c (hspX), Rv2654c (TB7.7), and Rv1038c
(EsxJ). J Immunol 2012;188(10):5020e31.
11. Devasundaram S, Deenadayalan A, Raja A. In silico analysis of
potential human T Cell antigens from Mycobacterium tuber-
culosis for the development of subunit vaccines against tuber-
culosis. Immunol Invest 2014;43(2):137e59.
12. Kollmann TR. Variation between populations in the innate im-
mune response to vaccine adjuvants. Front Immunol 2013;4:
81. http://dx.doi.org/10.3389/fimmu.2013.00081.
13. Song H, Sandie R, Wang Y, Andrade-Navarro MA,
Niederweis M. Identification of outer membrane proteins of
Mycobacterium tuberculosis. Tuberculosis (Edinb) 2008;
88(6):526e44.
14. de Souza GA, Leversen NA, Malen H, Wiker HG. Bacterial pro-
teins with cleaved or uncleaved signal peptides of the general
secretory pathway. J Proteomics 2011;75(2):502e10.
15. Kunnath-Velayudhan S, Salamon H, Wang HY, Davidow AL,
Molina DM, Huynh VT, et al. Dynamic antibody responses to
the Mycobacterium tuberculosis proteome. Proc Natl Acad
Sci USA 2010;107(33):14703e8.
16. Kumar M, Meenakshi N, Sundaramurthi JC, Kaur G, Mehra NK,
Raja A. Immune response to Mycobacterium tuberculosis spe-
cific antigen ESAT-6 among south Indians. Tuberculosis 2010;
90(1):60e9.
17. Takahashi A, Hanson MG, Norell HR, Havelka AM, Kono K,
Malmberg KJ, et al. Preferential cell death of CD8þ effector
memory (CCR7eCD45RA-) T cells by hydrogen peroxide-
induced oxidative stress. J Immunol 2005;174(10):6080e7.
18. D’Asaro M, Dieli F, Caccamo N, Musso M, Porretto F, Salerno A.
Increase of CCR7- CD45RAþ CD8 T cells (T(EMRA)) in chronic
graft-versus-host disease. Leukemia 2006;20(3):545e7.
19. Mojumdar K, Vajpayee M, Chauhan NK, Singh A, Singh R,
Kurapati S. Loss of CD127 increased immunosenescence of
T cell subsets in HIV infected individuals. Indian J Med Res
2011;134(6):972e81.
20. Dintwe OB, Day CL, Smit E, Nemes E, Gray C, Tameris M, et al.
Heterologous vaccination against human tuberculosis modu-
lates antigen-specific CD4þ T-cell function. Eur J Immunol
2013;43(9):2409e20.
21. Lindestam Arlehamn CS, Gerasimova A, Mele F, Henderson R,
Swann J, Greenbaum JA, et al. Memory T cells in latent Myco-
bacterium tuberculosis infection are directed against three
antigenic islands and largely contained in a CXCR3þCCR6þ
Th1 subset. PLoS Pathog 2013;9(1):e1003130.
22. Flynn JL, Chan J. Immunology of tuberculosis. Annu Rev Im-
munol 2001;19:93e129.
23. Kumar NP, Sridhar R, Banurekha VV, Jawahar MS, Nutman TB,
Babu S. Expansion of pathogen-specific T-helper 1 and T-help-
er 17 cells in pulmonary tuberculosis with coincident type 2
diabetes mellitus. J Infect Dis 2013;208(5):739e48.
24. Deenadayalan A, Maddineni P, Raja A. Comparison of whole
blood and PBMC assays for T-cell functional analysis. BMC
Res Notes 2013;6:120.
25. Tully G, Kortsik C, Hohn H, Zehbe I, Hitzler WE, Neukirch C,
et al. Highly focused T cell responses in latent human pulmo-
nary Mycobacterium tuberculosis infection. J Immunol 2005;
174:2174e84.
26. Beamer GL, Flaherty DK, Vesosky B, Turner J. Peripheral
blood gamma interferon release assays predict lung responses
and Mycobacterium tuberculosis disease outcome in mice.
Clin Vaccine Immunol 2008;15:474e83.
27. Berry MP, Graham CM, McNab FW, Xu Z, Bloch SA, Oni T, et al. An
interferon-inducible neutrophil-driven blood transcriptional
signature in human tuberculosis. Nature 2010;466:973e7.
28. Cliff JM, Kaufmann SH, McShane H, van Helden P, O’Garra A.
The human immune response to tuberculosis and its treat-
ment: a view from the blood. Immunol Rev 2015;264:88e102.
29. Harris SA, Meyer J, Satti I, Marsay L, Poulton ID, Tanner R,
et al. Evaluation of a human BCG challenge model to assess
antimycobacterial immunity induced by BCG and a candidate
tuberculosis vaccine, MVA85A, alone and in combination. J
Infect Dis 2014;209:1259e68.
30. Xu Y, Liu W, Shen H, Yan J, Yang E, Wang H. Recombinant
Mycobacterium bovis BCG expressing chimaeric protein of
Ag85B and ESAT-6 enhances immunostimulatory activity of hu-
man macrophages. Microbes Infect 2010;12:683e9.
31. Lahey T, Sheth S, Matee M, Arbeit R, Horsburgh CR, Mtei L,
et al. Interferon gamma responses to mycobacterial antigens
protect against subsequent HIV-associated tuberculosis. J
Infect Dis 2010;202:1265e72.
32. Prezzemolo T, Guggino G, La Manna MP, Di Liberto D, Dieli F,
Caccamo N. Functional signatures of human CD4 and CD8 T
cell responses to Mycobacterium tuberculosis. Front Immunol
2014;5:180.
33. Farber DL, Ahmadzadeh M. Dissecting the complexity of the
memory T cell response. Immunol Res 2002;25(3):247e59.
34. Fenton MJ, Vermeulen MW, Kim S, Burdick M, Strieter RM,
Kornfeld H. Induction of gamma interferon production in hu-
man alveolar macrophages by Mycobacterium tuberculosis.
Infect Immun 1997;65(12):5149e56.
35. Kannanganat S, Ibegbu C, Chennareddi L, Robinson HL,
Amara RR. Multiple-cytokine-producing antiviral CD4 T cells
466 D. Santhi, A. Raja

13. are functionally superior to single-cytokine-producing cells. J
Virol 2007;81(16):8468e76.
36. Beveridge NE, Fletcher HA, Hughes J, Pathan AA, Scriba TJ,
Minassian A, et al. A comparison of IFN gamma detection
methods used in tuberculosis vaccine trials. Tuberculosis
2008;88(6):631e40.
37. Pollock L, Basu Roy R, Kampmann B. How to use: interferon
gamma release assays for tuberculosis. Archives Dis Child
Educ Pract Ed 2013;98(3):99e105.
38. Thillai M, Pollock K, Pareek M, Lalvani A. Interferon-gamma
release assays for tuberculosis: current and future applica-
tions. Expert Rev Respir Med 2014;8(1):67e78. http:
//dx.doi.org/10.1586/17476348.2014.852471.
39. Cardoso FL, Antas PR, Milagres AS, Geluk A, Franken KL,
Oliveira EB, et al. T-cell responses to the Mycobacterium
tuberculosis-specific antigen ESAT-6 in Brazilian tuberculosis
patients. Infect Immun 2002;70(12):6707e14.
40. Seder RA, Darrah PA, Roederer M. T-cell quality in memory
and protection: implications for vaccine design. Nat Rev Im-
munol 2008;8:247e58.
41. Precopio ML, Betts MR, Parrino J, Price DA, Gostick E,
Ambrozak DR, et al. Immunization with vaccinia virus induces
polyfunctional and phenotypically distinctive CD8(þ) T cell
responses. J Exp Med 2007;204:1405e16.
42. Nemeth J, Winkler HM, Karlhofer F, Selenko-Gebauer N,
Graninger W, Winkler S. T cells co-producing Mycobacterium
tuberculosis-specific type 1 cytokines for the diagnosis of
latent tuberculosis. Eur cytokine Netw 2010;21(1):34e9.
43. Koinig HC, Talker SC, Stadler M, Ladinig A, Graage R,
Ritzmann M, et al. PCV2 vaccination induces IFN-
gamma/TNF-alpha co-producing T cells with a potential role
in protection. Vet Res 2015;46:20.
44. Matthews K, Wilkinson KA, Kalsdorf B, Roberts T, Diacon A,
Walzl G, et al. Predominance of interleukin-22 over
interleukin-17 at the site of disease in human tuberculosis.
Tuberculosis 2011;91(6):587e93.
45. Jafari C, Ernst M, Kalsdorf B, Greinert U, Diel R, Kirsten D,
et al. Rapid diagnosis of smear-negative tuberculosis by bron-
choalveolar lavage enzyme-linked immunospot. Am J Respir
Crit Care Med 2006;174:1048e54.
46. Nemeth J, Winkler HM, Zwick RH, Muller C, Rumetshofer R,
Boeck L, et al. Peripheral T cell cytokine responses for diag-
nosis of active tuberculosis. PLoS One 2012;7:e35290.
47. Goletti D, Vincenti D, Carrara S, Butera O, Bizzoni F,
Bernardini G, et al. Selected RD1 peptides for active tubercu-
losis diagnosis: comparison of a gamma interferon whole-
blood enzyme-linked immunosorbent assay and an enzyme-
linked immunospot assay. Clin Diagn Lab Immunol 2005;12:
1311e6.
48. Tameris MD, Hatherill M, Landry BS, Scriba TJ, Snowden MA,
Lockhart S, et al. Safety and efficacy of MVA85A, a new tuber-
culosis vaccine, in infants previously vaccinated with BCG: a
randomised, placebo-controlled phase 2b trial. Lancet 2013;
381:1021e8.
49. Kaveh DA, Carmen Garcia-Pelayo M, Hogarth PJ. Persistent
BCG bacilli perpetuate CD4 T effector memory and optimal
protection against tuberculosis. Vaccine 2014;32:6911e8.
50. Serbina NV, Lazarevic V, Flynn JL. CD4(þ) T cells are required
for the development of cytotoxic CD8(þ) T cells during Myco-
bacterium tuberculosis infection. J Immunol 2001;167:
6991e7000.
51. Cooper AM. Cell-mediated immune responses in tuberculosis.
Annu Rev Immunol 2009;27:393e422.
T-cell response of Rv2251 and Rv2721c 467