Identification of Human T Cell Antigens for
the Development of Vaccines against
Mycobacterium tuberculosis
This information is current as
of June 13, 2013.
Sylvie Bertholet, Gregory C. Ireton, Maria Kahn, Jeffrey
Guderian, Raodoh Mohamath, Nicole Stride, Elsa M.
Laughlin, Susan L. Baldwin, Thomas S. Vedvick, Rhea N.
Coler and Steven G. Reed
J Immunol 2008; 181:7948-7957; ;
http://www.jimmunol.org/content/181/11/7948
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The Journal of Immunology
Identification of Human T Cell Antigens for the Development
of Vaccines against Mycobacterium tuberculosis1
Sylvie Bertholet,2,3 Gregory C. Ireton,2 Maria Kahn, Jeffrey Guderian, Raodoh Mohamath,
Nicole Stride, Elsa M. Laughlin, Susan L. Baldwin, Thomas S. Vedvick, Rhea N. Coler,
and Steven G. Reed
T
uberculosis (TB)4 is a chronic infectious disease caused
by Mycobacterium tuberculosis (Mtb) and is one of the
leading causes of mortality due to infectious disease
worldwide (1). Nearly one-third of the world’s population is infected with Mtb, with ⬃25 million people actively infected and 8.8
million new cases arising each year (2). Upon infection with Mtb,
active disease develops in ⬃10% of subjects within 1–2 years of
the initial exposure. The remainder of those infected with Mtb
enter a state of latent infection, which can reactivate at a later
stage, particularly in the elderly or in immunocompromised
individuals.
The high mortality associated with Mtb infection occurs despite
the widespread use of a live, attenuated TB vaccine Mycobacterium bovis, bacillus Calmette-Guérin (BCG). BCG appears to be
effective at preventing disease in newborns and toddlers, but not
pulmonary tuberculosis in adults (3, 4). The variable efficacy afforded by BCG vaccination and the absence of a TB vaccine pro-
Infectious Disease Research Institute, Seattle WA 98104
Received for publication March 19, 2008. Accepted for publication September 25,
2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health (NIH) Grants AI-044373
and AI-067251 (to S.G.R.), Infectious Disease Research Institute, and by the NIH TB
Vaccine Testing and Research Materials Contract N01-AI-40091 (to Colorado State
University).
2
S.B. and G.C.I. contributed equally to this study.
3
Address correspondence and reprint requests to Dr. Sylvie Bertholet, Infectious
Disease Research Institute, 1124 Columbia Street, Suite 400, Seattle WA 98104.
E-mail address: sbertholet@idri.org
4
Abbreviations used in this paper: TB, tuberculosis; BCG, bacillus Calmette-Guérin;
GrB, granzyme B; ICS, intracellular cytokine staining; Mtb, Mycobacterium tuberculosis; PPD, purified protein derivative.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
www.jimmunol.org
tective in adults have been the primary rationale for our approach
to identify immunodominant Mtb Ags that could be used in a subunit vaccine to boost immune responses leading to improved protection. There is a new urgency for a TB vaccine as the World
Health Organization recently reported alarming rates of “multidrug
resistant” and “extensively drug-resistant” TB (5), mostly because
of improper observance of a lengthy and costly drug regimen
treatment.
Protective immunity to TB is conferred by Th1 CD4 and effector
CD8 T cells (6), and it is recognized that an effective TB vaccine
requires the generation of a T cell-mediated immune response.
Thus far, many of the dominant Mtb T cell Ags have been associated with proteins expressed by Mtb growing in macrophages,
up- or down-regulated under hypoxic conditions, required for reactivation, membrane associated, secreted, or represented by virulence factors such as PE/PPE or EsX (7, 8). Several promising
subunit vaccine candidates have been developed using Ags among
these protein classes (reviewed in Refs. 9, 10).
Choosing the right adjuvant or delivery platform is critical to the
success of a subunit vaccine. In this study, we used the oligodeoxynucleotide CpG, a TLR9 ligand, as an adjuvant based on its
reported ability to potentiate Th1 CD4 and CD8 T cell responses
(reviewed in Ref. 11).
Our approach to vaccine Ag discovery has been to identify relevant human T cell Ags that elicit dominant Th1 responses and to
evaluate these Ags in appropriate adjuvant formulations in animal
models. Taking advantage of the published genome sequence of
Mtb (12), we used bioinformatic selection criteria to identify genes
of interest, including protein secretion, induction during growth in
macrophages, up- or down-regulation in response to low oxygen or
carbon source, and/or selection of members in the EsX or PE/PPE
families. Recombinant proteins were made for 94 Mtb genes and
evaluated with human PBMCs from healthy purified protein derivative (PPD)⫺ and PPD⫹ individuals for IFN-␥ responses. A
subset of 49 Ags adjuvanted with CpG oligonucleotides was tested
Downloaded from http://www.jimmunol.org/ by guest on June 13, 2013
Development of a subunit vaccine for Mycobacterium tuberculosis (Mtb) depends on the identification of Ags that induce appropriate T cell responses. Using bioinformatics, we selected a panel of 94 Mtb genes based on criteria that included growth in
macrophages, up- or down-regulation under hypoxic conditions, secretion, membrane association, or because they were members
of the PE/PPE or EsX families. Recombinant proteins encoded by these genes were evaluated for IFN-␥ recall responses using
PBMCs from healthy subjects previously exposed to Mtb. From this screen, dominant human T cell Ags were identified and 49
of these proteins, formulated in CpG, were evaluated as vaccine candidates in a mouse model of tuberculosis. Eighteen of the
individual Ags conferred partial protection against challenge with virulent Mtb. A combination of three of these Ags further
increased protection against Mtb to levels comparable to those achieved with bacillus Calmette-Guérin vaccination. Vaccine
candidates that led to reduction in lung bacterial burden following challenge-induced pluripotent CD4 and CD8 T cells, including
Th1 cell responses characterized by elevated levels of Ag-specific IgG2c, IFN-␥, and TNF. Priority vaccine Ags elicited pluripotent
CD4 and CD8 T responses in purified protein derivative-positive donor PBMCs. This study identified numerous novel human T
cell Ags suitable to be included in subunit vaccines against tuberculosis. The Journal of Immunology, 2008, 181: 7948 –7957.
The Journal of Immunology
prophylactically in mice using the Mtb aerosol challenge model.
Ab and cytokine responses to the vaccine Ags were characterized.
Materials and Methods
Cloning and purification of target Ags
Human PBMC assay
PBMCs were purified from heparinized blood obtained from 7 PPD⫺ and
18 PPD⫹ healthy subjects. Informed consent was obtained from all the
subjects and the study was approved by Western Institutional Review
Board (Seattle, WA). PBMCs were plated in triplicate at 2–2.5 ⫻ 105
cells/well and cultured with medium, 10 g/ml PHA, 10 g/ml Mtb lysate,
or 10 g/ml of each recombinant protein for 72 h. Supernatants were harvested and analyzed for IFN-␥ by a double-sandwich ELISA using specific
mAb (eBioscience), following the manufacturer’s protocol. Values above
mean background (medium) ⫾ 3 SD were considered positive.
Immunization, challenge, and CFU
Female C57BL/6 mice, 5–7 wk old, were purchased from Charles River
Laboratories. All mice were maintained in Infectious Disease Research
Institute (IDRI) animal care facility under specific pathogen-free conditions and were treated in accordance with the regulations and guidelines of
the IDRI Animal Care and Use Committee. Mice were immunized (s.c.)
three times, 3 wk apart, with 6 or 8 g protein in PBS and 25 g of
CpG1826 (Coley Pharmaceutical Group) in a volume of 100 l, except for
the BCG group, which received a single dose of 5 ⫻ 104 CFU via the
intradermal route (Pasteur strain, Sanofi Pasteur). Four weeks after the last
immunization, groups of seven mice were challenged by low-dose aerosol
exposure with Mtb H37Rv strain (American Type Culture Collection no.
35718) using a University of Wisconsin–Madison aerosol exposure chamber calibrated to deliver 50 –100 bacteria into the lungs. Vaccine efficacy
was determined 4 wk postchallenge by harvesting lungs and spleen from
the infected mice, homogenizing the tissues in 0.05% PBS-Tween 80, plating 5-fold serial dilutions on 7H10 agar plates (Molecular Toxicology) for
bacterial growth, and counting CFU 2 wk later. Values ranging between 10
and 100 colonies were used to calculate the bacterial burden per organ and
expressed as log10 values. Vaccine-induced protection is expressed as
(mean log10 CFUsaline ⫺ mean log10 CFUvaccine).
Ab ELISA
Animals were bled 1 wk after the last immunization and serum IgG1 and
IgG2c Ab titers were determined. Nunc-Immuno PolySorb plates were
coated for 4 h at room temperature with 2 g/ml of recombinant protein in
0.1 M bicarbonate buffer, blocked overnight at 4°C with 0.05% PBSTween 20/1% BSA, washed with 0.05% PBS-Tween 20, incubated for 2 h
at room temperature with sera at a 1/50 dilution and subsequent 5-fold
serial dilutions, washed, and incubated for 1 h with anti-IgG1-HRP or
anti-IgG2c-HRP 1/2000 in 0.05% PBS-Tween 20/0.1% BSA. Plates were
washed and developed using SureBlue tetramethylbenzidine substrate
(Kirkegaard & Perry Laboratories). The enzymatic reaction was stopped
with 1 N H2SO4, and plates were read within 30 min at 450 nm with a
reference filter set at 650 nm using a microplate ELISA reader (Molecular
Devices) and SoftMax Pro5. Endpoint titers were determined with GraphPad Prism 4 software with a cutoff of 0.1.
ELISPOT
MultiScreen 96-well filtration plates (Millipore) were coated with 10
g/ml rat anti-mouse IFN-␥ or TNF capture Ab (eBioscience) and incubated overnight at 4°C. Plates were washed with PBS, blocked with RPMI
1640 and 10% FBS for at least 1 h at room temperature, and washed again.
Splenocytes were plated in duplicate at 2 ⫻ 105 cells/well and stimulated
with medium, 3 g/ml Con A, 10 g/ml PPD, or 10 g/ml of each recombinant protein for 48 h at 37°C. The plates were subsequently washed
with PBS and 0.1% Tween 20 and incubated for 2 h with a biotin-conjugated rat anti-mouse IFN-␥ or TNF secondary Ab (eBioscience) at 5 g/ml
in PBS, 0.5% BSA, and 0.1% Tween 20. The filters were developed using
the Vectastain ABC avidin peroxidase conjugate and Vectastain AEC substrate kits (Vector Laboratories) according to the manufacturer’s protocol.
The reaction was stopped by washing the plates with deionized water,
plates were dried in the dark, and spots were counted on a automated
ELISPOT reader (CTL series 3A analyzer, Cellular Technology) and analyzed with ImmunoSpot (CTL Analyzers).
Flow cytometry
Splenocytes from immunized animals or PBMCs from healthy PPD⫹ subjects were plated at 1–2 ⫻ 106 cells/well in 96-well V-bottom plates and
stimulated with anti-CD28/CD49d (1 g/ml each, eBioscience) and recombinant proteins (20 g/ml) for 6 –12 h at 37°C in the presence of GolgiStop
(eBioscience). The cells were then fixed for 10 min with Cytofix/Cytoperm
(BD Biosciences), washed in 0.1% PBS-BSA, incubated with Fc block
(anti-CD16/CD32, eBioscience) for 15 min at 4°C, stained with fluorochrome-conjugated mAb anti-CD3, CD8, CD44, IFN-␥, TNF, IL-2, granzyme B (GrB, eBioscience) and CD4 (Caltag Laboratories) in 1⫻ Perm/
Wash buffer (BD Biosciences) for 30 min at 4°C, washed twice in Perm/
Wash buffer, suspended in PBS, and analyzed on a modified three-laser
LSRII flow cytometer (BD Biosciences). Viable lymphocytes were gated
by forward and side scatter, and 20,000 CD3/CD8 events were acquired for
each sample and analyzed with BD FACSDiva software (version 5.0.1, BD
Biosciences).
Statistical analysis
Student’s t test and standard one-way ANOVA followed by Dunnett’s
multiple comparison test were used for statistical analysis; p-values of 0.05
or less were considered significant.
Results
Ag selection, expression, and purification
Mtb genes selected for testing human immune responses and as
possible vaccine candidates included 1) those genes that were required for growth in macrophages as defined by Sassetti and Rubin
(13) 2) those that were up- or down-regulated in response to oxygen and carbon limitation (14), 3) mycobacterial-specific genes
within known immunogenic classes EsX and PE/PPE as based in
TuberCulist (genolist.pasteur.fr/TubercuList/index.html), and 4)
those identified as secreted by two-dimensional PAGE and mass
spectrometry analysis (www.mpiib-berlin.mpg.de/2D-PAGE)
and/or containing putative secretion signals. All targets were subjected to N-terminal signal sequence analysis and membrane spanning region using the SignalP (www.cbs.dtu.dk/services/SignalP)
and TMpred (www.ch.embnet.org/software/TMPRED_form.html)
programs. Predicted proteins were chosen containing less than
three transmembrane regions and a molecular mass between 6 and
70 kDa and low (⬍30%) sequence homology within the human
genome. DNAs encoding selected Mtb genes were PCR amplified
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DNAs encoding selected Mtb genes were PCR amplified from HRv37
genomic DNA using Pfx DNA polymerase (Invitrogen). PCR primers were
designed to incorporate specific restriction enzyme sites 5⬘ and 3⬘ of the
gene of interest for directional cloning into the expression vector pET28a
(Novagen). Purified PCR products were digested with restriction enzymes,
ligated into pET28a using T4 DNA ligase (NEB), and transformed into
XL10G cells (Stratagene). Recombinant pET28a 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 Mtb gene of
interest.
Recombinant plasmids were transformed into the Escherichia coli BL21
derivative Rosetta2(DE3)pLysS (Novagen). Recombinant strains were cultured overnight at 37°C in 2⫻ yeast tryptone containing appropriate antibiotics, diluted 1/25 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, and bacterial pellets were stored at
⫺20°C. Bacterial pellets were thawed and 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 material. Recombinant His-tagged
protein products were isolated under native (soluble recombinant proteins)
or denaturing (8 M urea) conditions using Ni-nitrilotriacetic acid metal ion
affinity chromatography according to the manufacturer’s instructions (Qiagen). 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 10-kDa-molecular mass cutoff centrifugal filters (Millipore), and
quantified using a bicinchoninic acid protein assay (Pierce). LPS contamination was evaluated by the Limulus amoebocyte lysate assay (Cambrex).
All the recombinant proteins used in this study showed residual endotoxin
levels ⬍100 EU/mg of protein.
7949
7950
NOVEL Mycobacterium tuberculosis VACCINE Ags
Table I. Categories of selected Mtb Ags
Secreted/Membrane
Rv0054
Rv0068
Rv0125
Rv0129c
Rv0153c
Rv0164
Rv0390
Rv0410
Rv0455c
Rv0496
Rv0577
Rv0733
Rv0831c
Rv0909
Rv0934
Rv1411
Rv1511
Rv1626
Rv1860
Rv1886c
Rv1926c
Rv1932
Rv1980c
Rv1984c
Rv2031
Rv2220
Rv2873
Rv2875
Rv3029c
Rv3246
Rv3310
Rv3628
Rv3804c
Rv3841
Rv3881
PE/PPE
EsX
Database
Rv0915
Rv1196
Rv1789
Rv1818c
Rv2608
Rv3478
Rv0287
Rv1793
Rv3020
Rv3619
Rv3620c
Rv3874
Rv3875
Rv0952
Rv1009
Rv1174
Rv1211
Rv1253
Rv1270c
Rv1288
Rv1884c
Rv2389c
Rv2450
Rv2520c
Rv3204
Rv3211
Rv3407
Rv3876
Human PBMC cytokine responses to Mtb Ags
Candidate Ags were initially prioritized based on their abilities to
stimulate PBMCs from PPD⫹ (but not PPD⫺) donors to secrete
IFN-␥. The donors used for the screening were tuberculin skin
test-positive, disease-free, and healthy. Ags from the RD1 region
that are deleted in M. bovis BCG, such as ESAT-6 (Rv3875), were
able to elicit responses from these PBMCs. Out of 94 proteins
tested, 68 (72%) elicited positive IFN-␥ responses by PBMCs from
PPD⫹, but not PPD⫺ donors (Fig. 1A). Responses to the proteins
were compared with those elicited by Mtb lysate in PPD⫹ (2.79 ⫾
0.25) and PPD⫺ (0.05 ⫾ 0.12) and PHA (4.40 ⫾ 0.27 and 2.59 ⫾
0.47, respectively). Of the PPD⫹ donors, 100% recognized Mtb
lysate (data not shown) and a set of 28 proteins (Fig. 1B). Another
set of 49 Ags was recognized by 67–90% of the subjects, and,
overall, all the Ags tested were recognized by at least 44% of the
PPD⫹ donors. Of the 28 Ags recognized by all donors, 7 were
PE/PPE or EsX proteins, 6 were hypoxia-associated, 6 were identified by database searches, 5 were secreted/membrane proteins,
and 4 were associated with Mtb survival in macrophages.
Protection afforded by Mtb Ags adjuvanted with CpG
Recombinant Mtb proteins inducing the highest levels of IFN-␥ in
the PBMC assays and percentage of positive responders were then
tested for protective efficacy in a mouse model of TB. The number
of viable bacilli (CFU) in the lungs of C57BL/6 mice immunized
with Mtb proteins adjuvanted with CpG was determined 4 wk after
aerosol challenge with a low dose of virulent Mtb H37Rv or Erdman strains. Twenty Mtb Ags were tested at IDRI in 7 independent experiments using H37Rv for challenge, 22 Mtb Ags were
tested at Colorado State University under National Institutes of
Health TB Vaccine Testing and Research Material contract in four
independent experiments using the Erdman strain for challenge,
and 6 Mtb Ags were tested by both laboratories. No differences in
Rv0523c
Rv0655
Rv0716
Rv1099
Rv1397c
Rv1410c
Rv1569
Rv1589
Rv1590
Rv1908
Rv3541c
Rv3587
Rv3611
Rv3614
Hypoxia
Rv0363c
Rv0570
Rv1240
Rv1246c
Rv1738
Rv1813c
Rv2032
Rv2558
Rv2623
Rv2624c
Rv2626c
Rv2801c
Rv2866
Rv3044
Rv3129c
Rv3133
Rv3810
protection outcome were observed when using Mtb H37Rv or Erdman (data not shown), therefore indicating that both strains can
be used interchangeably in this study. Each experiment included
unvaccinated (saline) and BCG control groups, along with 8 –10
groups of different Ags with CpG (seven mice per group). Mean
lung log10 CFU in the various experiments ranged for the unvaccinated groups from 5.76 to 6.71 and from 4.30 to 5.82 for BCGvaccinated mice (data not shown). Variations are often important
in the aerosol model of TB, rendering direct comparison of CFU
difficult due to differences in infection levels between experiments.
Therefore, an accepted way of comparing the relative efficacy of a
vaccine is to calculate the mean difference of CFU observed between unvaccinated and vaccinated groups.
Out of 48 proteins formulated with CpG, 30 produced no reduction in lung CFU, and 12 induced a small decrease in bacterial
counts (0.1– 0.3 log10). Six Ags (three secreted (Rv0577, Rv1626,
and Rv2875), two PE/PPE (Rv2608 and Rv3478), and one hypoxic
protein (Rv3044)) elicited ⬎0.3 log10 reduction in lung CFU (Table II). Interestingly, the two PE/PPE proteins, Rv3478 and
Rv2608, in combination with CpG resulted in the greatest decrease
in viable Mtb bacilli (0.66 and 0.58 log10, respectively). Rv0577,
Rv1626, Rv2875, and Rv3044 reduced lung CFU by 0.36, 0.32,
0.44, and 0.43 log10, respectively. As a comparison, three injections of Rv1886 (Ag85B, part of TB vaccines currently in clinical
trials) ⫹ CpG or one injection of 104 viable BCG reduced lung
CFU by 0.20 and 0.78 log10, respectively. CpG adjuvant alone did
not reduce lung CFU (⬍0.1 log10). Reduction in CFU ⬎0.2 log10
were reproducible and generally statistically significant ( p ⬍ 0.05)
by one-way ANOVA followed by Dunnett’s multiple comparison
test (data not shown). Protection observed with weaker Ags was
more variable from experiment to experiment and, due to the high
number of animals that would have been required, experiments
were not powered to detect statistical significance for CFU reduction ⬍0.2 log10.
These results are very promising, as the levels of protection
against Mtb infection achieved with two of the Ags (adjuvanted
with CpG) were close to those obtained with the more complex
BCG vaccine.
Immunogenicity of vaccine candidate Ags
Experiments were conducted to investigate the T and B cell responses induced against the vaccine candidates. C57BL/6 mice
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from H37Rv strain genomic DNA, cloned into the expression vector pET28a, and transformed into E. coli for recombinant protein
expression. His-tagged protein products were isolated, affinity purified, identified on SDS-PAGE gel (data not shown), and tested
for residual endotoxin. Mtb genes that failed in PCR, had low or no
detectable expression, failed to purify to ⬎90% homogeneity, or
contained ⬎100 EU/mg were discarded. A total of 94 recombinant
proteins were used in this analysis (Table I).
Growth in
Macrophages
The Journal of Immunology
7951
were immunized three times 3 wk apart with Ag and CpG. Agspecific Ab titers and cytokine responses were determined 1 wk
and 3 wk, respectively, after the last boost.
At the Ab level, higher IgG2c responses were measured in response to 17 Ags, 18 Ags showed mixed IgG2c/IgG1 Abs, and 1
Ag induced higher IgG1 (Table III). Rv0577, Rv1886, Rv2608,
Rv2875, Rv3044, Rv3478, and Rv3619 induced log10 IgG2c Ab
titers ranging from 4.2 to 5.9 and log10 IgG1 titers ranging from
3.8 to 5.7 (Fig. 2A), while no IgG2c or IgG1 to Rv1626 could be
detected (data not shown). The Ag-specific IgG2c responses were
Table II. Vaccine-induced protection against Mtb
CFU Reduction (Log10) ⫾ SEMa
0.1–0.3
⬍0.1
Rv0164b
Rv0410
Rv0455
Rv0655
Rv0952
Rv1211
Rv1270
Rv1410
Rv1590
Rv1738
Rv1818
Rv1884
Rv1926
Rv1984
Rv2032
Rv2389
Rv2450
Rv2623
Rv2626
Rv2801
Rv2866
Rv2945
Rv3029
Rv3133
Rv3204
Rv3407
Rv3541
Rv3620
Rv3628
Rv3810
CpG
(⫺0.09 ⫾ 0.05)
Rv0496
Rv0733
Rv0831
Rv1411
Rv1569
Rv1789
Rv1813
Rv1860
Rv1886
Rv2220
Rv3020
Rv3619
0.11 ⫾ 0.08
0.23 ⫾ 0.10
0.13 ⫾ 0.06
0.11 ⫾ 0.11
0.12 ⫾ 0.05
0.15 ⫾ 0.16
0.14 ⫾ 0.14
0.19 ⫾ 0.07
0.20 ⫾ 0.04
0.25 ⫾ 0.11
0.17 ⫾ 0.07
0.24 ⫾ 0.05
⬎0.3
Sc
S
S
S
M
P
H
S
S
S
E
E
Rv0577
Rv1626
Rv2608
Rv2875
Rv3044
Rv3478
BCG
0.36 ⫾ 0.07
0.32 ⫾ 0.07
0.58 ⫾ 0.16
0.44 ⫾ 0.18
0.43 ⫾ 0.06
0.66 ⫾ 0.15
0.78 ⫾ 0.07
Sc
S
P
S
H
P
a
Reduction of viable bacteria (CFU) in the lungs of immunized animals compared to saline controls 4 wk after a low-dose aerosol challenge with M.
tuberculosis H37Rv and/or Erdman strains. Data represent the mean of two to three independent experiments ⫾ SEM for Ags inducing ⬎0.1 log reduction.
Antigens inducing ⬍0.1 log reduction in lung CFU in the first screening were considered not protective and were not repeated for ethical concerns to
reduce the number of animals used in these studies.
b
Mice were immunized s.c. three times, 3 wk apart, with 8 g Mtb Ags (Rv no.) ⫹ 25 g CpG, or intradermally once with 5 ⫻ 104 BCG.
c
Ag category: S, secreted; P, PE/PPE; E, EsX; M, growth in macrophages; H, regulated under hypoxic growth.
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FIGURE 1. Levels of IFN-␥ released by Ag-stimulated human PBMCs were measured in vitro. PPD⫹ and PPD⫺ PBMCs were incubated for 72 h in medium,
PHA (10 g/ml), Mtb lysate (10 g/ml), or Mtb recombinant proteins (50 g/ml). Ags are grouped according to the selection criteria: m, membrane-associated;
S, secreted proteins; P, PE/PPE; E, EsX; M, macrophage growth required; H, hypoxic response; D, database searches. A, IFN-␥ in supernatants was measured by
sandwich ELISA. Mean (meanAg ⫺ meanmedium) ⫾ SEM are shown for PPD⫹ (n ⫽ 18) and PPD⫺ (n ⫽ 7) PBMCs. B, Percentage of PPD⫹ positive responders.
7952
NOVEL Mycobacterium tuberculosis VACCINE Ags
Table III. Immunogenicity of Mtb Ags
Reduction in CFU of ⬎0.3 Log10a
b
Ag
IFN-␥
Rv0577
Rv1626
Rv2608
Rv2875
Rv3044
Rv3478
523 ⫾ 8
20 ⫾ 21
798 ⫾ 11
428 ⫾ 172
331 ⫾ 161
453 ⫾ 4
b
Reduction in CFU of 0.1–0.3 Log10
c
TNF
IgG
Ag
IFN-␥
TNF
IgG
388 ⫾ 297
268 ⫾ 117
175 ⫾ 105
137 ⫾ 60
57 ⫾ 1
149 ⫾ 73
0.98
1.19*
1.09
1.05*
1.05*
1.03
Rv0496
Rv0733
Rv0831
Rv1411
Rv1569
Rv1789
Rv1813
Rv1860
Rv1886
Rv2220
Rv3020
Rv3619
68 ⫾ 52
NDd
24 ⫾ 12
187 ⫾ 36
ND
ND
388 ⫾ 103
ND
590 ⫾ 106
ND
48 ⫾ 27
604 ⫾ 184
24 ⫾ 5
ND
24 ⫾ 8
ND
ND
ND
32 ⫾ 13
ND
102 ⫾ 37
ND
20 ⫾ 16
1261 ⫾ 319
1.21*
ND
1.19*
1.00
ND
ND
1.18*
ND
1.00
ND
1.18*
1.13*
92 ⫾ 31
4⫾4
2⫾1
7⫾4
ND
32 ⫾ 12
ND
6⫾2
12 ⫾ 8
5⫾7
ND
ND
72 ⫾ 33
ND
7 ⫾ 10
1.02
0.97
1.14*
0.95
ND
1.31*
ND
1.25*
1.94*
2.10*
ND
ND
1.13*
1.09
1.08
Reduction in CFU of ⬍0.1 Log10
163 ⫾ 87
ND
24 ⫾ 12
ND
100 ⫾ 83
12 ⫾ 4
8⫾4
ND
28 ⫾ 24
24 ⫾ 16
155 ⫾ 72
1600 ⫾ 372
41 ⫾ 64
344 ⫾ 210
28 ⫾ 16
94 ⫾ 58
ND
44 ⫾ 24
ND
14 ⫾ 5
0⫾0
ND
ND
4⫾8
32 ⫾ 16
10 ⫾ 2
ND
12 ⫾ 21
70 ⫾ 49
ND
1.17*
ND
1.06
ND
1.03
1.00
1.01
ND
2.12*
1.23
0.90*
1.01
1.08
1.09
1.14*
Rv2389
Rv2450
Rv2623
Rv2626
Rv2801
Rv2866
Rv2945
Rv3029
Rv3133
Rv3204
Rv3407
Rv3541
Rv3620
Rv3628
Rv3810
39 ⫾ 49
148 ⫾ 104
21 ⫾ 12
95 ⫾ 30
ND
104 ⫾ 56
ND
116 ⫾ 29
24 ⫾ 8
8 ⫾ 13
ND
ND
184 ⫾ 44
16 ⫾ 8
44 ⫾ 56
a
Ags are displayed based on efficacy at reducing lung bacterial burden.
Spot-forming units (SFU) per million cells ⫾ SD. Mice were immunized s.c. three times, 3 wk apart, with Mtb Ags (Rv no.) ⫹ CpG. Cytokine responses to the Ags were
determined by ELISPOT 3 wk after the last injection.
c
IgG2c/IgG1 ratio, ⴱ, p ⬍ 0.05, Student’s t test.
d
ND, not done.
b
predominant for all the proteins but two (Rv0577, Rv1886), as
indicated by an IgG2c/IgG1 ratio ⬎1 (Fig. 2B). Differences in
Ag-specific IgG2c vs IgG1 Ab titers were found statistically significant ( p ⬍ 0.05) for Rv2875, Rv3044, and Rv3619 using Student’s t test.
FIGURE 2. Ab responses to Mtb recombinant Ags were measured in
sera isolated from mice vaccinated with Rv0577, Rv2875, Rv1886,
Rv2608, Rv3478, Rv3619, Rv3620, Rv1813, and Rv3044 ⫹ CpG. Sera
were tested for Ag-specific IgG1 and IgG2c Ab. A, Mean of reciprocal
dilution ⫾ SEM, and (B) IgG2c/IgG1 ratios are shown. ⴱ, p ⬍ 0.05,
Student’s t test. Data shown are representative of two independent
experiments.
FIGURE 3. IFN-␥ responses to Mtb recombinant Ags were determined
in vaccinated mice. Splenocytes were prepared from animals immunized
with Rv0577, Rv1626, Rv1813, Rv1886 (85B), Rv2875, Rv2608, Rv3619,
Rv3620, Rv3044, and Rv3478 ⫹ CpG or saline, 3 wk after the last injection, and tested for IFN-␥ in vitro recall responses. Cells were plated in
duplicate at 2 ⫻ 105 cells/well and cultured with medium, Con A (3 g/
ml), PPD (10 g/ml), or each recombinant Ag (10 g/ml) for 48 h. Frequencies of IFN-␥-secreting cells (spot-forming units, SFU) were determined by ELISPOT. Means ⫾ SD (n ⫽ 5 mice) shown are representative
of two independent experiments.
Downloaded from http://www.jimmunol.org/ by guest on June 13, 2013
Rv0164
Rv0410
Rv0455
Rv0655
Rv0952
Rv1211
Rv1270
Rv1410
Rv1590
Rv1738
Rv1818
Rv1884
Rv1926
Rv1984
Rv2032
The Journal of Immunology
At the cytokine level, all the Mtb Ags associated with ⬎0.3
log10 reduction in lung bacterial burden induced high levels of
IFN-␥- (Table III and Fig. 3) and/or TNF- producing cells (Table
III and Fig. 4). Mtb Ags associated with 0.1– 0.3 log10 reduction in
CFU induced mixed levels of IFN-␥- and/or TNF-producing cells,
with half of the Ags tested showing high levels of one or both
cytokines. Finally, Mtb Ags associated with ⬍0.1 log10 reduction
in CFU induced lower frequencies of IFN-␥- and/or TNF production, with notable exceptions, including Rv1884, Rv1984, and
Rv3620. Splenocytes from immunized mice responded to in vitro
stimulation with Con A, as well as to various levels with PPD
(Figs. 3 and 4) and Mtb lysate (data not shown). Splenocytes from
saline and CpG control groups showed positive cytokine responses
upon in vitro stimulation with Con A, and background levels only
in response to antigenic stimulation (data not shown).
CD4 and CD8 T cell expression of multiple cytokines and effector molecules in response to antigenic stimulation were further
determined at the single cell level by intracellular cytokine staining
(ICS) and flow cytometry using a modified three-laser LSRII
FACS analyzer. Cytokine profiles were analyzed on a limited subset of Mtb Ags, identified as partially protective at the time of the
study, and compared with the ones obtained with Rv1886. Expression of IFN-␥, TNF, and/or IL-2 by CD3/CD4/CD44high T cells
(Ag experienced, Fig. 5A) and CD3/CD4/CD44low T cells (naive,
data not shown) was determined. CD44high, but not CD44low, CD4
T cells from immunized mice produced two or more cytokines in
response to in vitro antigenic stimulation. Rv1886 showed the
greatest CD4 T cell response, with cells staining positive predominantly for IFN-␥/TNF and TNF/IL-2. Rv3619 was the next best,
followed by Rv3620, Rv2608, Rv3478, and Rv1813. Cells staining
positive for the three cytokines were seen in response to Rv3478,
Rv3619, and Rv3620, that is, in three of the six vaccines tested.
CD4/CD44high and CD4/CD44low T cells from saline control mice
showed only background levels of cells positive for multiple
cytokines.
Effector memory CD8 T cells were identified by ICS as CD3/
CD44highCD8 cells staining positive for IFN-␥ and/or TNF
upon Ag stimulation in vitro, and they were further analyzed for
their content in GrB (Fig. 5B). Rv1813 showed the greatest
number of CD8 T cells positive for either or both cytokines,
followed by Rv3620, Rv3619, and Rv2608. Cell responses to
Rv3478 were the lowest. More than 50% of the cells positive
for IFN-␥ and/or TNF also contained GrB, a protein of the lytic
machinery of CTLs.
Taken together, these results show that these novel Mtb Ags
adjuvanted with CpG induced potent Th1-type CD4 and CD8 effector T cell immune responses in C57BL/6 mice.
Combination of multiple Ags increased protection against
challenge with Mtb
We hypothesized that combining multiple Ags would lead to increased reduction in bacterial burden following Mtb infection. We
used Rv2608, Rv1813, and Rv3620, which were the first Ags
we identified as inducing partial protection against Mtb. Mice were
injected three times 3 wk apart with single Ags ⫹ CpG, a mixture
of the three Ags ⫹ CpG, CpG alone, or BCG. The number of
viable bacilli (CFU) in the lung of mice from the different immunized groups was compared (Fig. 6A). In this experiment, Rv1813
and Rv2608 adjuvanted with CpG reduced the number of lung
bacteria by 0.30 log10 ( p ⬍ 0.01) and 0.25 log10 ( p ⬍ 0.05),
respectively, while immunization with Rv3620 ⫹ CpG was ineffective (0.05 log10, p ⬎ 0.05). The weak protection we observed
with Rv3620 in our initial experiment that led to its inclusion in the
combination was not confirmed in this or subsequent experiments.
Nevertheless, with 0.67 log10 reduction ( p ⬍ 0.01), the effect of
the three Ags combined was greater than that seen with any single
Ag and comparable to vaccination with BCG (0.71 log10, p ⬍
0.01).
FIGURE 5. Ag-specific CD4 and CD8 T cell cytokine responses were analyzed in vaccinated mice. Splenocytes, isolated from animals immunized with
Rv1813, Rv3620, Rv2608, Rv3619, Rv3478, or Rv1886 ⫹ CpG or saline, were plated at 2 ⫻ 106 cells/well and cultured with medium or each recombinant
Ag (20 g/ml) for 12 h in the presence of GolgiStop. A, CD4 T cells were identified by ICS based on CD3 and CD4 expression and further gated as CD44high
(Ag experienced) or CD44low (naive). The percentages of cells expressing two cytokines or more of IFN-␥, TNF, and IL-2 were determined. Means (n ⫽
3) are shown. B, CD8 Ag-specific T cells were identified by ICS as CD3/CD44high/CD8 cells also positive for IFN-␥ and/or TNF, and further analyzed for
their content in GrB. Numbers of cytokine⫹ (IFN/TNF) and cytokine⫹GrB⫹ per million splenocytes were determined. Means ⫾ SD (n ⫽ 3 mice) shown
are representative of two independent experiments.
Downloaded from http://www.jimmunol.org/ by guest on June 13, 2013
FIGURE 4. TNF responses to Mtb recombinant Ags were determined in
vaccinated mice. Splenocytes were prepared from animals immunized with
Rv0577, Rv1626, Rv1813, Rv1886 (85B), Rv2875, Rv2608, Rv3619,
Rv3620, Rv3044, and Rv3478 ⫹ CpG or saline, 3 wk after the last injection, and tested for TNF in vitro recall responses. Cells were plated in
duplicate at 2 ⫻ 105 cells/well and cultured with medium, Con A (3 g/
ml), PPD (10 g/ml), or each recombinant Ag (10 g/ml) for 48 h. Frequencies of TNF-secreting cells (spot-forming units, SFU) were determined by ELISPOT. Means ⫾ SD (n ⫽ 5 mice) shown are representative
of two independent experiments.
7953
7954
NOVEL Mycobacterium tuberculosis VACCINE Ags
receiving the three Ags compared with animals injected with
Rv3620 only. Additional T-dependent help provided by the other
two Ags might explain this difference, as Rv3620 is a small (10
kDa) protein. Ag-specific IgG1 was also detected (data not
shown). IgG2c/IgG1 ratios were ⬎1 for all groups showing Agspecific Abs, consistent with a predominant Th1 response. Mice
immunized with CpG alone did not show IgG2c- or IgG1-specific
responses to either Ag. In contrast, IFN-␥ and TNF responses were
essentially restricted to Rv2608 and diminished in the group receiving the Ag combination compared with the single Ag (Fig. 6,
C and D). The reduced cytokine responses in the group receiving
the three Ags ⫹ CpG can at least partially be attributed to the fact
that these animals were immunized with less protein (6 vs 8 g for
the single Ag groups) to use a dose of ⬍20 g Ag total per injection. We observed in other infection models that immunizing
mice with ⬎20 g of Ag was detrimental (R. Coler, unpublished
observations).
Human T cell responses to vaccine candidate Ags
Immunization with the selected Ag combination resulted in significant production of Ag-specific IgG2c Abs (Fig. 6B). IgG2c
titers to Rv3620 were increased by 1.3 log10 ( p ⬍ 0.05) in mice
FIGURE 7. Human PPD⫹ CD4 T
cells respond to recombinant Mtb Ag
stimulation. PBMCs from six healthy
PPD⫹ subjects with diverse HLA
types were incubated for 12 h in medium, PPD (10 g/ml), or recombinant Ags (20 g/ml) in the presence
of anti-CD28/CD49d and GolgiStop.
T cells were identified by ICS based
on CD3 expression and further gated
as CD4/CD45RO memory T cells.
Percentages of CD4 T cells expressing IFN-␥, TNF, or the two cytokines
in response to a panel of Mtb Ags are
shown.
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FIGURE 6. The efficacy of a combination of three Ags adjuvanted with
CpG was addressed. Mice were immunized s.c. three times, 3 wk apart, with
25 g CpG, 8 g single Ag ⫹ CpG, 6 g of each Ag ⫹ CpG (3 Ags⫹CpG),
or once intradermally with 5 ⫻ 104 BCG. Four weeks after the last boost, mice
were challenged with Mtb H37Rv. A, The number of viable bacteria (CFU
log10) in the lungs was determined 4 wk postchallenge. One-way ANOVA
followed by Dunnett’s multiple comparison test was used for statistical analysis. ⴱ, p ⬍ 0.05 and ⴱⴱ, p ⬍ 0.01. B, Mice were bled 1 wk after the last boost
and sera were tested for Ag-specific IgG2c Ab. Means of reciprocal dilution ⫾
SD (n ⫽ 3 mice) are shown. C and D, Splenocytes were isolated from the
vaccinated animals 3 wk after the last boost, plated in duplicate at 2 ⫻ 105
cells/well, and cultured with medium, Con A (3 g/ml), PPD (10 g/ml), or
each recombinant Ag (10 g/ml) for 48 –72 h. C, IFN-␥ levels measured by
ELISA in 72 h supernatants. D, Frequencies of TNF-secreting cells (spotforming units, SFU) determined by ELISPOT. Means ⫾ SD (n ⫽ 3 mice)
shown are representative of two independent experiments.
Human PBMCs from six healthy PPD⫹ subjects with documented prior exposure to Mtb were HLA typed (Pudget Sound
Blood Center, Seattle, WA): D160 (HLA-A2/3, B14/44, DR4/
13, DQ1/7), D204 (HLA-A3/68, B27/35, DR7/15, DR51/53,
DQ6/9), D336 (HLA-A2/3, B7/44, DR4/13, DQ1/7), D365
(HLA-A1/2, B7/8, DR2/3, DQ1/2), D366 (HLA-A2/24, B60,
DR4, DQ3), and D368 (HLA-A2/29, B44, DR15/4, DQ0602/
0301). CD4 and CD8 T cell cytokine responses to PPD and the
nine most protective Ags were analyzed by ICS at the single
cell level. We also looked at responses to Rv1813 and Rv3620
because of their possible additive effect when used in combination with Rv2608. Memory T cells were gated on expression
of CD3/CD4/CD45RO or CD3/CD8/CD45RO molecules, and
the data reflect the percentage of cells staining positive for
IFN-␥, TNF, or the two cytokines after background (media control) removal. PPD and PMA⫹Ionomycin stimulation induced
CD4 T cells staining positive for IFN-␥, TNF, and/or the two
cytokines in all subjects (Fig. 7). CD4 T cell responses to the
different Mtb Ags were more variable among donors, with
mixed profiles consisting most of the time of cells positive for
a single cytokine (IFN-␥ or TNF), as well as cells expressing
both cytokines. For CD8 T cells, PMA⫹Ionomycin stimulation
The Journal of Immunology
7955
FIGURE 8. Human PPD⫹ CD8 T
cells respond to recombinant Mtb Ag
stimulation. PBMCs from six healthy
PPD⫹ subjects with diverse HLA
types were incubated for 12 h in medium, PPD (10 g/ml), or recombinant Ags (20 g/ml) in the presence
of anti-CD28/CD49d and GolgiStop.
T cells were identified by ICS based
on CD3 expression and further gated
as CD8/CD45RO memory T cells.
Percentages of CD8 T cells expressing IFN-␥, TNF, or the two cytokines
in response to a panel of Mtb Ags are
shown.
Discussion
With the goal of identifying Ags for use in a human TB vaccine,
we performed the most comprehensive analysis to date of human
T cell Ags of Mtb. From selected genome mining, we chose ⬃100
potential candidates to be expressed as recombinant proteins and
evaluated for IFN-␥ recall responses using PBMCs from healthy
PPD⫹ subjects. Based on PBMC results, a subset of 48 Ags was
selected to be administered to mice in a CpG formulation, with 18
of them conferring partial protection against challenge with virulent Mtb H37Rv and/or Erdman strains. A combination of three of
these Ags further increased protection against TB to levels comparable to those achieved with BCG vaccination. Induction of predominant Th1 CD4 responses by vaccine Ags were observed in
mice with reduced lung bacterial burden.
Recall secretion of IFN-␥ by pathogen-exposed cells is a relevant marker for cellular immune responses in Mtb infections, and
it has been used as a rationale for Ag selection for potential vaccines (reviewed in Refs. 8, 15). With 94 Mtb proteins evaluated for
antigenicity on PPD⫹ donor PBMCs, our study considerably expands the number of Mtb proteins tested so far. Most of the proteins (79) were widely recognized as indicated by positive IFN-␥
production by PBMCs from ⬎70% of the subjects. Of the 79 proteins, 28 were recognized by all PPD⫹ donors tested. HLA typing
of a subset of donors indicated a wide heterogeneity in MHC class
I and II alleles, suggesting that these Ags contain epitopes recognized by a wide range of different HLA molecules. The promiscuous recognition of Ags by individuals with a wide variety of
ethnic origins is of major importance in the context of human
vaccines.
We and others have demonstrated the feasibility of using protein
subunit vaccines against leishmaniasis (16) and TB (17, 18) with
vaccines currently in clinical trials. Unlike DNA vaccines, protein-
based vaccines are immunogenic in small animal models, nonhuman primates, and humans. Properly adjuvanting Mtb proteins,
however, is the key to the induction of strong in vivo immune
responses. In this study, we used CpG as an adjuvant based on its
reported ability to potentiate Th1 CD4 and CD8 T cell responses
(11, 19). To favor the selection of potent Ags, we chose a relatively
low dose of CpG as an adjuvant. Under these conditions, we observed only a modest reduction in lung bacterial counts (0.20
log10) following immunization with Rv1886 (Ag85B), previously
reported to have protective capacity (18), lower than some of the
novel Ags reported herein.
We identified 17 previously uncharacterized Ags that were
able to induce partial protection against Mtb in mice. Most of
the Ags were either secreted or membrane-associated. Rv2608
and Rv3478 belong to the family of PE/PPE proteins, the members of which have been shown to induce Th1 responses (20,
21) and confer protection against Mtb (22–24). The PE/PPE
proteins have also been shown to have higher levels of polymorphism among Mtb clinical isolates variants (25). However,
no genetic variation was observed for Rv2608 or Rv3478 within
recently published MDR and XDR genomes (www.broad.
mit.edu/annotation/genome/mycobacterium_tuberculosis_spp.9/
ToolsIndex.html). Rv3020 and Rv3619 are members of the EsX
family of virulence factors, among which several potential vaccine candidates have been described (26 –30). Finally, Rv1813
and Rv3044 are proteins expressed by Mtb in conditions of low
oxygen and are associated with latent growth of the bacteria
(14). Identifying novel vaccine Ags within families of proteins
known to induce potent immune responses further validates the
bioinformatic approach. In this study, adjuvanting recombinant
proteins with CpG proved effective at reducing bacterial burden, further extending results obtained with CpG-adjuvanted
culture filtrate proteins (31) or BCG (32). Combining multiple
Mtb Ags led to increased vaccine efficacy, extending observations from us and others using combination of Ags (33) or fusion proteins like Mtb72f, Ag85B-ESAT6, and Ag85B-TB10
(17, 18, 27, 34). Fusion proteins comprised of our newly identified priority candidates are currently in development.
Downloaded from http://www.jimmunol.org/ by guest on June 13, 2013
induced positive responses for IFN-␥, TNF, and the two cytokines in all subjects, while PPD induced IFN-␥ and IFN-␥ ⫹
TNF, but not TNF alone (Fig. 8). Most of the CD8 T cell responses to Mtb Ags were IFN-␥ or/and IFN-␥ ⫹ TNF. Overall,
each Ag was recognized by subjects with a diversity of HLA by
either CD4 or/and CD8 T cells.
7956
Acknowledgments
We thank Irina Zharkikh, Elena Kristalinskaia, Valerie Reese, Tara
Evers, Kevin Durgan, Laura Appleby, and Jackie Whittle for technical
expertise, and Ajay Bhatia and Randall Howard for valuable
discussions.
Disclosures
The authors have no financial conflicts of interest.
References
1. World Health Organization. 2007. Global Tuberculosis Control: Surveilance,
Planning, Financing. World Health Organization, Geneva.
2. Maartens, G., and R. J. Wilkinson. 2007. Tuberculosis. Lancet 370:
2030 –2043.
3. Andersen, P., and T. M. Doherty. 2005. The success and failure of BCG: implications for a novel tuberculosis vaccine. Nat. Rev. Microbiol. 3: 656 – 662.
4. Doherty, T. M., and P. Andersen. 2005. Vaccines for tuberculosis: novel concepts
and recent progress. Clin. Microbiol. Rev. 18: 687–702.
5. World Health Organization and International Union Against Tuberculosis and
Lung Disease Global Project on Anti-Tuberculosis Drug Resistance Surveillance
2002–2007. 2008. Anti-tuberculosis drug resistance in the world: fourth global
report. World Health Organization, Geneva.
6. Flynn, J. L., and J. Chan. 2001. Immunology of tuberculosis. Annu. Rev. Immunol. 19: 93–129.
7. Andersen, P., and T. M. Doherty. 2005. TB subunit vaccines: putting the pieces
together. Microbes Infect. 7: 911–921.
8. Sable, S. B., M. Kalra, I. Verma, and G. K. Khuller. 2007. Tuberculosis subunit
vaccine design: the conflict of antigenicity and immunogenicity. Clin. Immunol.
122: 239 –251.
9. Andersen, P. 2007. Tuberculosis vaccines: an update. Nat. Rev. Microbiol. 5:
484 – 487.
10. Kaufmann, S. H., S. Baumann, and A. Nasser Eddine. 2006. Exploiting immunology and molecular genetics for rational vaccine design against tuberculosis.
Int. J. Tuberc. Lung Dis. 10: 1068 –1079.
11. Klinman, D. M. 2006. Adjuvant activity of CpG oligodeoxynucleotides. Int. Rev.
Immunol. 25: 135–154.
12. Cole, S. T., R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris,
S. V. Gordon, K. Eiglmeier, S. Gas, C. E. Barry 3rd, et al. 1998. Deciphering the
biology of Mycobacterium tuberculosis from the complete genome sequence.
Nature 393: 537–544.
13. Sassetti, C. M., and E. J. Rubin. 2003. Genetic requirements for mycobacterial
survival during infection. Proc. Natl. Acad. Sci. USA 100: 12989 –12994.
14. Sherman, D. R., M. Voskuil, D. Schnappinger, R. Liao, M. I. Harrell, and
G. K. Schoolnik. 2001. Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding ␣-crystallin. Proc. Natl. Acad. Sci. USA 98:
7534 –7539.
15. Skeiky, Y. A., and J. C. Sadoff. 2006. Advances in tuberculosis vaccine strategies. Nat. Rev. Microbiol. 4: 469 – 476.
16. Coler, R. N., and S. G. Reed. 2005. Second-generation vaccines against leishmaniasis. Trends Parasitol. 21: 244 –249.
17. Reed, S., and Y. Lobet. 2005. Tuberculosis vaccine development; from mouse to
man. Microbes Infect. 7: 922–931.
18. Weinrich Olsen, A., L. A. van Pinxteren, L. Meng Okkels, P. Birk Rasmussen,
and P. Andersen. 2001. Protection of mice with a tuberculosis subunit vaccine
based on a fusion protein of antigen 85b and esat-6. Infect. Immun. 69:
2773–2778.
19. Speiser, D. E., P. Baumgaertner, V. Voelter, E. Devevre, C. Barbey, N. Rufer,
and P. Romero. 2008. Unmodified self antigen triggers human CD8 T cells with
stronger tumor reactivity than altered antigen. Proc. Natl. Acad. Sci. USA 105:
3849 –3854.
20. Chaitra, M. G., R. Nayak, and M. S. Shaila. 2007. Modulation of immune responses in mice to recombinant antigens from PE and PPE families of proteins of
Mycobacterium tuberculosis by the Ribi adjuvant. Vaccine 25: 7168 –7176.
21. Chakhaiyar, P., Y. Nagalakshmi, B. Aruna, K. J. Murthy, V. M. Katoch, and
S. E. Hasnain. 2004. Regions of high antigenicity within the hypothetical PPE
major polymorphic tandem repeat open-reading frame, Rv2608, show a differential humoral response and a low T cell response in various categories of patients with tuberculosis. J. Infect. Dis. 190: 1237–1244.
22. Dillon, D. C., M. R. Alderson, C. H. Day, D. M. Lewinsohn, R. Coler, T. Bement,
A. Campos-Neto, Y. A. Skeiky, I. M. Orme, A. Roberts, et al. 1999. Molecular
characterization and human T-cell responses to a member of a novel Mycobacterium tuberculosis mtb39 gene family. Infect. Immun. 67: 2941–2950.
23. Skeiky, Y. A., P. J. Ovendale, S. Jen, M. R. Alderson, D. C. Dillon, S. Smith,
C. B. Wilson, I. M. Orme, S. G. Reed, and A. Campos-Neto. 2000. T cell expression cloning of a Mycobacterium tuberculosis gene encoding a protective
antigen associated with the early control of infection. J. Immunol. 165:
7140 –7149.
24. Vipond, J., R. Vipond, E. Allen-Vercoe, S. O. Clark, G. J. Hatch, K. E. Gooch,
J. Bacon, T. Hampshire, H. Shuttleworth, N. P. Minton, et al. 2006. Selection of
novel TB vaccine candidates and their evaluation as DNA vaccines against aerosol challenge. Vaccine 24: 6340 – 6350.
25. Fleischmann, R. D., D. Alland, J. A. Eisen, L. Carpenter, O. White, J. Peterson,
R. DeBoy, R. Dodson, M. Gwinn, D. Haft, et al. 2002. Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains. J. Bacteriol.
184: 5479 –5490.
26. Brandt, L., M. Elhay, I. Rosenkrands, E. B. Lindblad, and P. Andersen. 2000.
ESAT-6 subunit vaccination against Mycobacterium tuberculosis. Infect. Immun.
68: 791–795.
27. Dietrich, J., C. Aagaard, R. Leah, A. W. Olsen, A. Stryhn, T. M. Doherty, and
P. Andersen. 2005. Exchanging ESAT6 with TB10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine: efficient protection and ESAT6-based
sensitive monitoring of vaccine efficacy. J. Immunol. 174: 6332– 6339.
28. Al-Attiyah, R., A. S. Mustafa, A. T. Abal, A. S. El-Shamy, W. Dalemans, and
Y. A. Skeiky. 2004. In vitro cellular immune responses to complex and newly
defined recombinant antigens of Mycobacterium tuberculosis. Clin. Exp. Immunol. 138: 139 –144.
29. Hogarth, P. J., K. E. Logan, H. M. Vordermeier, M. Singh, R. G. Hewinson, and
M. A. Chambers. 2005. Protective immunity against Mycobacterium bovis induced by vaccination with Rv3109c: a member of the esat-6 gene family. Vaccine
23: 2557–2564.
30. Olsen, A. W., P. R. Hansen, A. Holm, and P. Andersen. 2000. Efficient protection
against Mycobacterium tuberculosis by vaccination with a single subdominant
epitope from the ESAT-6 antigen. Eur. J. Immunol. 30: 1724 –1732.
31. Wedlock, D. N., M. A. Skinner, G. W. de Lisle, H. M. Vordermeier,
R. G. Hewinson, R. Hecker, S. van Drunen Littel-van den Hurk, L. A. Babiuk,
and B. M. Buddle. 2005. Vaccination of cattle with Mycobacterium bovis culture
filtrate proteins and CpG oligodeoxynucleotides induces protection against bovine tuberculosis. Vet. Immunol. Immunopathol. 106: 53– 63.
32. Freidag, B. L., G. B. Melton, F. Collins, D. M. Klinman, A. Cheever, L. Stobie,
W. Suen, and R. A. Seder. 2000. CpG oligodeoxynucleotides and interleukin-12
improve the efficacy of Mycobacterium bovis BCG vaccination in mice challenged with M. tuberculosis. Infect. Immun. 68: 2948 –2953.
33. Kalra, M., A. Grover, N. Mehta, J. Singh, J. Kaur, S. B. Sable, D. Behera,
P. Sharma, I. Verma, and G. K. Khuller. 2007. Supplementation with RD antigens
enhances the protective efficacy of BCG in tuberculous mice. Clin. Immunol. 125:
173–183.
34. Skeiky, Y. A., M. R. Alderson, P. J. Ovendale, J. A. Guderian, L. Brandt,
D. C. Dillon, A. Campos-Neto, Y. Lobet, W. Dalemans, I. M. Orme, and
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Strong Th1 CD4 responses and cytokines like IFN-␥ and TNF
are critical for protection against TB (35–39), while Ab responses
are not considered to be involved in antimycobacterial adaptive
immunity. In this study, we used the Ag-specific IgG2c/IgG1 ratio
as an indication of a predominant Th1 (⬎1), mixed Th1/Th2 (⫽1),
or Th2 (⬍1) response induced by vaccination. We show that vaccine-induced protection against challenge with Mtb in mice was
associated with a Th1 response characterized by predominant serum IgG2c and high levels of IFN-␥ and TNF. High frequencies of
IFN-␥- or TNF-producing cells, however, did not always correlate
with lower lung bacterial counts, as exemplified by Rv1884 or
Rv1984, suggesting that other factors are important to control the
infection in mice, and extending observations by others (40 – 42).
The ability of a vaccine to induce pluripotent T cell responses has
been associated with increased protection in a mouse model of
Leishmania; such a mechanism has also been proposed for M.
bovis BCG vaccine (43). All the vaccine candidates induced effector memory CD4 T cells expressing at least two, if not all three,
of the cytokines among IL-2, IFN-␥, and TNF, with Rv3478 and
Rv3619 stimulating individual T cells to produce all three cytokines. Most importantly, when tested on PBMCs isolated from six
healthy subjects with confirmed exposure to Mtb, and who received chemotherapy, all the vaccine candidate Ags induced CD4
and/or CD8 T cell responses by PMBCs from subjects with a diversity of HLA. Cytokine profiles were a mix of single and double
IFN-␥ ⫹ TNF-expressing CD4 T cells, and predominantly IFN-␥and IFN-␥ ⫹ TNF-expressing CD8 T cells. This suggests that
immune responses to these novel Ags naturally occur in individuals with diverse HLA types exposed to and cured of Mtb infection. This is of particular interest in the context of a vaccine that
could be used to boost an existing immune response induced by
infection. Because most of these Ags are also found in the BCG
vaccine, they might be used in a BCG prime/protein boost regimen
to improve upon BCG vaccination alone. We are currently expanding our approach by testing multiple Ag combinations in different
adjuvant formulations for protection in different animal models
of TB.
NOVEL Mycobacterium tuberculosis VACCINE Ags
The Journal of Immunology
35.
36.
37.
38.
39.
S. G. Reed. 2004. Differential immune responses and protective efficacy induced
by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked
DNA or recombinant protein. J. Immunol. 172: 7618 –7628.
Cooper, A. M., D. K. Dalton, T. A. Stewart, J. P. Griffin, D. G. Russell, and
I. M. Orme. 1993. Disseminated tuberculosis in interferon gamma gene-disrupted
mice. J. Exp. Med. 178: 2243–2247.
Flynn, J. L., J. Chan, K. J. Triebold, D. K. Dalton, T. A. Stewart, and
B. R. Bloom. 1993. An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection. J. Exp. Med. 178: 2249 –2254.
Bean, A. G., D. R. Roach, H. Briscoe, M. P. France, H. Korner, J. D. Sedgwick,
and W. J. Britton. 1999. Structural deficiencies in granuloma formation in TNF
gene-targeted mice underlie the heightened susceptibility to aerosol Mycobacterium tuberculosis infection, which is not compensated for by lymphotoxin. J. Immunol. 162: 3504 –3511.
Botha, T., and B. Ryffel. 2003. Reactivation of latent tuberculosis infection in
TNF-deficient mice. J. Immunol. 171: 3110 –3118.
Denis, B., A. Lefort, R. M. Flipo, F. Tubach, M. Lemann, P. Ravaud, D. Salmon,
X. Mariette, and O. Lortholary. 2008. Long-term follow-up of patients with tu-
7957
40.
41.
42.
43.
berculosis as a complication of tumour necrosis factor (TNF)-␣ antagonist therapy: safe re-initiation of TNF-␣ blockers after appropriate anti-tuberculous treatment. Clin. Microbiol. Infect. 14: 183–186.
Cowley, S. C., and K. L. Elkins. 2003. CD4⫹ T cells mediate IFN-␥-independent
control of Mycobacterium tuberculosis infection both in vitro and in vivo. J. Immunol. 171: 4689 – 4699.
Elias, D., H. Akuffo, and S. Britton. 2005. PPD induced in vitro interferon ␥production is not a reliable correlate of protection against Mycobacterium tuberculosis. Trans. R. Soc. Trop. Med. Hyg. 99: 363–368.
Majlessi, L., M. Simsova, Z. Jarvis, P. Brodin, M. J. Rojas, C. Bauche, C. Nouze,
D. Ladant, S. T. Cole, P. Sebo, and C. Leclerc. 2006. An increase in antimycobacterial Th1-cell responses by prime-boost protocols of immunization does not
enhance protection against tuberculosis. Infect. Immun. 74: 2128 –2137.
Darrah, P. A., D. T. Patel, P. M. De Luca, R. W. Lindsay, D. F. Davey,
B. J. Flynn, S. T. Hoff, P. Andersen, S. G. Reed, S. L. Morris, et al. 2007.
Multifunctional TH1 cells define a correlate of vaccine-mediated protection
against Leishmania major. Nat. Med. 13: 843– 850.
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