Hypoxia Induces an Immunodominant Target of
Tuberculosis Specific T Cells Absent from Common BCG
Vaccines
Hannah Priyadarshini Gideon1, Katalin Andrea Wilkinson1,2, Tige R. Rustad3, Tolu Oni1,4, Heinner Guio4,
Robert Andrew Kozak5, David R. Sherman3, Graeme Meintjes1, Marcel A. Behr5, Hans Martin
Vordermeier6, Douglas Brownlee Young2,4, Robert John Wilkinson1,2,4*
1 Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Observatory, South Africa, 2 MRC National Institute for Medical Research, Mill Hill,
London, United Kingdom, 3 Seattle Biomedical Research Institute, Seattle, Washington, United States of America, 4 Division of Medicine and Center for Molecular
Microbiology and Infection, Imperial College London, London, United Kingdom, 5 McGill University Health Centre, Montreal, Canada, 6 Veterinary Laboratories Agency,
Weybridge, United Kingdom
Abstract
M. tuberculosis (MTB) species-specific antigenic determinants of the human T cell response are important for
immunodiagnosis and vaccination. As hypoxia is a stimulus in chronic tuberculosis infection, we analyzed transcriptional
profiles of MTB subject to 168 hours of hypoxia to test the hypothesis that upregulation by hypoxia might result in gene
products being recognized as antigens. We identified upregulation of two region of difference (RD) 11 (Rv2658C and
Rv2659c), and one RD2 (Rv1986) absent from commonly used BCG strains. In MTB infected persons, the IL-2 ELISpot
response to Rv1986 peptides was several times greater than the corresponding IFN-c response to the reference
immunodominant ESAT-6 or CFP-10 antigens. The IL-2 response was confined to two epitopic regions containing residues
61–80 and 161–180. The biggest population of IL-2 secreting T cells was single cytokine positive central memory T cells. The
IL-2 response to live MTB bacilli lacking Rv1986 was significantly lower than the response to wild type or mutant
complemented with Rv1986. In addition, the IL-2 response to Rv1986 was significantly lower in HIV-TB co-infected persons
than in HIV uninfected persons, and significantly increased during antiretroviral therapy. These findings demonstrate that
Rv1986 is an immunodominant target of memory T cells and is therefore of relevance when considering the partial efficacy
of currently used BCG vaccines and provide evidence for a clinical trial comparing BCG strains.
Citation: Gideon HP, Wilkinson KA, Rustad TR, Oni T, Guio H, et al. (2010) Hypoxia Induces an Immunodominant Target of Tuberculosis Specific T Cells Absent
from Common BCG Vaccines. PLoS Pathog 6(12): e1001237. doi:10.1371/journal.ppat.1001237
Editor: Vojo Deretic, University of New Mexico, United States of America
Received April 22, 2010; Accepted November 22, 2010; Published December 23, 2010
Copyright: ß 2010 Gideon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a joint the Bill and Melinda Gates Foundation/Wellcome Trust Grand Challenges in Global Health award (reference 37882).
Additional support came from Wellcome Trust awards to GM and RJW (081667, 072070, 084323, 088316), by the European and Developing Countries Clinical
Trials Partnership to MXR (060613) and by the European Union (Sante/2006/105-061). KAW, DBY and RJW also receive programme support from the Medical
Research Council of the United Kingdom. HPG received a bursary from the Lutheran World Federation and the University of Cape Town. HMV is funded by the
Department for Environment, Food & Rural Affairs, UK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: r.j.wilkinson@imperial.ac.uk
proteins (ESAT-6 and CFP-10) that are highly immunogenic [5,6]:
restoration of these genes into BCG improves its vaccine efficacy
[7]. Assays of the T cell interferon (IFN)-c secretion in response to
the combination of ESAT-6 and CFP-10 (interferon-c release
assays, IGRA) have been developed that have operational
advantages and improve the specificity and possibly sensitivity of
tuberculosis immunodiagnosis [8].
The availability of the complete sequence of M. tuberculosis also
permitted further genomic characterization of various BCG strains
[9,10,11]. It became apparent that, against a background of
accumulating single nucleotide polymorphisms, BCG underwent
sequential genomic deletions that thereby characterize various
strains. The strains most commonly in use such as BCG Glaxo,
Danish and Pasteur have most deletions. This led to the proposal
that one of the reasons behind the partial vaccine efficacy of BCG
was that it had become too attenuated to successfully mimic
natural MTB infection [12]. Some empirical evidence in humans
Introduction
Mycobacterium tuberculosis remains a formidable health problem as
it is estimated to infect one-third of the world’s population and
causes around 1.5 million deaths per year [1]. Control is largely
based around the partially effective vaccine Mycobacterium bovis
Bacille Calmette Guérin (BCG) and on the early detection and
treatment of infected persons with active or latent disease [2].
Study of the antigens of M. tuberculosis is therefore a priority both
to improve vaccination via the selection of protective antigens, and
to define immunodiagnostic candidates that enhance the specificity and sensitivity of the widely used purified protein derivative
(PPD) based tuberculin skin test (TST). A significant landmark in
both respects was the discovery that a M. tuberculosis genomic
region designated region of difference (RD) 1 is deleted from all
strains of BCG and thereby partially accounts for the avirulence of
the vaccine [3,4]. RD1 encodes a pair of co-regulated secreted
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Rv1986 of M. tuberculosis
It has been shown in vitro that the up regulation of dosR
regulated genes represents an early somewhat transient response to
hypoxia: upregulation of a larger group of genes characterizes the
hypoxic time course occurs in cultures subject to 4–7 days (as
opposed to 2 hours) hypoxia [25], of which 230 are defined as the
enduring hypoxic response (EHR). We hypothesized that these
EHR and other hypoxia-induced genes would be worthy of
consideration as antigens, especially those that were speciesspecific. We therefore undertook a study of the immunogenicity of
M. tuberculosis specific genes induced by hypoxia and other wellcharacterized antigens (ESAT-6, CFP-10, Acr1) in humans with
active and latent tuberculosis.
Author Summary
Mycobacterium tuberculosis (the cause of tuberculosis) can
persist for many years in humans without causing disease
but has the potential to reactivate. One of the conditions
the bacterium must survive in these circumstances is
hypoxia. In order to do so, the bacterium uses a
characteristic set of genes that help alter its metabolism.
It follows that the products of such genes may encode
protein antigens that can be recognized by the immune
response. We therefore analyzed gene response patterns
of tuberculosis subject to prolonged hypoxia as a guide to
the discovery of new antigens that might be useful as
vaccines or diagnostic agents. Amongst the genes most
strongly increased by low oxygen levels, one was
identified (known as Rv1986) that is missing from most
strains of the tuberculosis vaccine Mycobacterium bovis
BCG. When we analyzed human immune responses to this
protein in tuberculosis infected people our experiments
showed it was particularly well recognized by cells that
produce a chemical messenger (cytokine) called interleukin-2. Interleukin-2 is important for long-term immunological memory. The BCG vaccine is only partially effective and
our experiments therefore suggest one of the reasons
could be that an important immunological target is
missing from many strains. Further evaluation of BCG
strains in which Rv1986 is present or absent is therefore
warranted in the hope that this might improve the efficacy
of existing or new tuberculosis vaccines.
Results
Antigen selection
Cross-reference of genes with the greatest fold induction in
hypoxic culture over 7 days [25] with sequence databases revealed
two species-specific (Rv2658c and Rv2659c, both RD11 encoded)
and one partially species-specific gene (Rv1986, RD-2 encoded).
The fold induction and sigA normalized transcript intensity over a
time course of 168 hrs hypoxia for these genes (and of Acr1, CFP10 and ESAT-6) are shown in Table 1. Interestingly whilst the fold
induction for the RD1 encoded genes fell, the normalized intensity
remained at a similar absolute level to that of both EHR and the
dosR regulated Acr1 gene.
Interferon-c ELISpot analysis of active and latent
tuberculosis
favoring this hypothesis is provided by the finding that BCG Japan
induced greater cytotoxic and T helper 1 responses in infants than
Danish BCG [13]. The largest difference between BCG Japan and
BCG Danish is the presence of RD2 in the former but not the
latter.
The discovery of immunodominant antigens in M. tuberculosis
has hitherto largely been based on dominance in antibody
responses that are neither the basis of protection against
tuberculosis nor of IGRA. A more rational approach might be
to relate what is highly expressed by bacilli in vivo or in vitro (and
thereby potentially available as an antigen) as recently investigated
in bovines [14]. In humans there has been investigation of proteins
encoded by genes of the dosR regulon that is induced in axenic
culture by hypoxia and by nitric oxide [15], stresses that are
considered relevant to bacilli in nature [16,17,18]. Analysis of
selected dosR regulated proteins confirmed the immunodominance
of a-crystallin 1 (Acr1) encoded by Rv2031 [19,20,21], as well as
potentially infection stage specific antigens [19,22,23,24].
Interferon-c (IFN-c) ELISpot was performed using PBMC from
40 persons with active (20) or latent (20) tuberculosis, and IL-2
ELISpot on 13 and 14 persons in each category. Immunodominance was assessed both quantitatively (median SFC/106 PBMC)
and by frequency of response (.20 SFC/106 PBMC). CFP-10 and
ESAT-6 were co-dominant for the IFN-c response by both
methods (Figure 1A and B). The largest SFC response in latent
disease was to CFP-10 (102 SFC/106 PBMC, IQR 38-444). With
the exception of ESAT-6 all other responses were significantly
lower (p#0.007). The largest response in active disease was to
ESAT-6 (172 SFC/106 PBMC, IQR 47–423). With the exception
of CFP-10 all other responses were significantly lower (p#0.002).
Although peptide pool Rv2659c-2 was preferentially recognized
by latently infected persons (6 SFC/106 PBMC, IQR 1–28 versus
0 SFC/106 PBMC, IQR 0–7, p = 0.028) these responses were very
modest. When analyzed by proportions, no pool was preferentially
recognized by either clinical group (Figure 1B). The most frequent
response in the combined group (latent plus active) was to CFP-10
Table 1. M. tuberculosis transcript levels in bacilli exposed to 7 days hypoxia in vitro.
Log base2 Hypoxia/Log
Rv#
Gene name
4 hrs
1 day
(a)
Median transcript intensity normalized to SigA
4 days
(b)
7 days
4 hrs
1 day
4 days
7 days
4.33
Rv3875
ESAT-6
20.34
22.17
21.88
21.40
5.97
4.22
3.49
Rv3874
CFP-10
0.14
21.20
20.56
21.03
7.42
2.3
1.97
4.04
Rv2659c
Rv2659c
1.49
3.56
3.43
3.29
0.14
1.66
1.47
0.72
Rv2658c
Rv2658c
2.15
4.68
4.93
4.29
0.18
1.69
1.25
0.66
Rv2031c
Acr1
5.34
4.02
4.95
4.22
4.09
1.84
0.63
1.40
Rv1986
Rv1986
1.10
3.77
3.60
3.06
0.21
2.38
2.10
1.35
(a) Shows the fold induction over hypoxic time course with reference to phase of aerobic cultures.
(b) Shows the median transcript intensity normalized to SigA over hypoxic time course.
doi:10.1371/journal.ppat.1001237.t001
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Figure 1. Interferon-c and IL-2 ELISpot response to peptide pools. Panel A shows the spot forming cells (SFC) in patients with latent (L) and
active (A) tuberculosis. The strongest response in latent disease was to CFP-10. With the exception of ESAT-6 all other responses were significantly
lower (p#0.007). The strongest response in active disease was to ESAT-6. With the exception of CFP-10 all other responses were significantly lower
(p#0.002). Peptide pool Rv2659c-2 was preferentially recognized by latently infected persons (p = 0.03). Panel B shows the proportion of responders
(defined by a response of $20 SFC/106 PBMC above background). No pool was preferentially recognized by either clinical group. The most frequent
response in the combined group (latent plus active) was to CFP-10: with the exception of ESAT-6 the proportion subjects responding to the other
antigens was lower in every case (p#0.002). Panel C shows IL-2 spot forming cells (SFC). The strongest response in both active and latent disease was
to Rv1986-2. All other responses in both latent (p#0.0007) and active infection (p#0.02) were significantly lower. Peptide pool Rv2658c was
preferentially recognized by actively infected persons (p = 0.042). Panel D shows the proportion of responders. No pool was preferentially recognized
by either clinical group. The most frequent response in the combined group (latent plus active) was to Rv1986-2: with the exception of ESAT-6 and
CFP-10 the proportion subjects responding to the other antigens was lower in every case (p#0.009). Bars show the median response.
doi:10.1371/journal.ppat.1001237.g001
CFP-10, the proportion of subjects responding to the other
antigens was lower in every case (p#0.009).
(36/40, 90%): with the exception of ESAT-6 the proportion of
persons responding to the other antigens was lower in every case
(p#0.002).
Peptide mapping of the IL-2 response to Rv1986
IL-2 ELISpot analysis of active and latent tuberculosis
Epitope mapping of the individual peptide determinants of the
IFN-c response to ESAT-6 and CFP-10 have shown several
regions in each molecule that can restimulate T cells with no single
peptide giving rise to a response in .50% subjects [26,27] and
similar findings are reported for other antigens of M. tuberculosis
[22]. We were therefore interested to determine whether a
similarly ‘degenerate’ pattern of multiple IL-2 inducing epitopes
occurred in Rv1986. PBMC from 20 persons with latent
tuberculosis were assayed in the presence of 10 mg/ml of each
peptide or no stimulus. A highly focused pattern of dominance was
observed with peptides p61-80 (84 SFC/106 PBMC, IQR 56–134)
and p161–180 (68 SFC/106 PBMC, IQR 49–104) being clearly
the best recognized (Figure 2). 90% of subjects had a response .20
Patients with active TB preferentially recognized pooled
peptides from Rv2658c (7 IL-2 SFC/106 PBMC, IQR 1–23
versus 0 SFC/106 PBMC, IQR 0–6 p = 0.042). However when
analyzed by proportion, no pool was preferentially recognized by
either clinical group. There was however a striking IL-2 response
in both active and latent disease to Rv1986 pool 2 (795 SFC/106
PBMC, IQR 51–1428 in active infection; 1194 SFC/106 PBMC,
IQR 862–1650 in latent infection, Figure 1C and D). All other
antigen specific IL-2 responses were significantly lower in both
latent (p#0.0007) and active infection (p#0.02). The most
frequent response in the combined group (latent plus active) was
also to Rv1986-2 (24/26, 92%): with the exception of ESAT-6 and
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Rv1986 of M. tuberculosis
Figure 2. Peptide map of IL-2 response to Rv1986. PBMC from 20
subjects with latent tuberculosis were stimulated with individual
peptides of Rv1986 in an IL-2 ELISpot assay. p61–80 and p161–180
were the dominant determinants of the response. Horizontal lines
indicate median responses.
doi:10.1371/journal.ppat.1001237.g002
SFC/106 PBMC to p61–80 and 95% to p161–180 with no other
peptide being recognized by .45% subjects. There were less
frequent and lower magnitude responses to p151–170 perhaps
suggesting the epitope core for some MHC Class II molecules may
include residues common to both peptides (161–170).
Phenotype of CD4+ T cells responding to Rv1986
Analysis of the T cells responsible for type 1 cytokine responses
is critical to understand protective immunity against TB [28]. In
PBMC from 5 persons with latent tuberculosis, we therefore
determined the phenotype of CD4+T cells responsible for type 1
cytokine (IFN-c, IL-2 and TNF) production when stimulated with
the peptides of Rv1986 (61–80 and 161–180) or the combination
of peptides from CFP-10 and ESAT-6 as a comparison. T cell
phenotypes were defined based on the surface markers CD45RA
and CD27: Central memory cells (TCM) are positive for CD27
and negative for CD45RA; effector memory (TEM) are negative
for both CD27 and CD45RA and Terminally differentiated T
cells (Tdiff) are negative for CD27 and positive for CD45RA.
Single cytokine positive cells predominated overall (Figure 3). Most
IL-2 derived from TCM irrespective of stimulus. The two Rv1986
peptides restimulated nearly ten times the percentage of IL-2
producing TCM cells than CFP-10 and ESAT-6 (median: 0.226%
CD3+CD4+ TCM vs 0.024% CD3+CD4+TCM, p = 0.055,
Figure 3 panel A and B).
Figure 3. Phenotype of CD4+ T cells responding to Rv1986.
PBMC from 5 donors were stimulated either with CFP-10+ESAT-6 (A) or
with Rv1986 p(61–80)+p(161–180) (B), and the phenotype of T cells
producing single and multiple cytokines were analyzed using surface
and intracellular cytokine staining by FACS. T cell phenotype was
defined by the surface markers CD45RA and CD27: Central memory
cells (TCM) are positive for CD27 and negative for CD45RA; effector
memory (TEM) are negative for both CD27 and CD45RA and Terminally
differentiated T cells (Tdiff) are negative for CD27 and positive for
CD45RA. The results are expressed as the percentage of CD3+ CD4+ T
cells. The strongest response to Rv1986 p61–80+p161–180 stimuli was
the IL-2 producing central memory cells, median 0.22% (Panel B); as
opposed to a median of 0.02% for CFP-10 plus ESAT-6 (p = 0.055) (Panel
A). While CFP-10 and ESAT-6 induced multiple cytokine-producing TCM,
Rv1986 induced predominantly single cytokine producing TCM, TEM
and Tdiff cells.
doi:10.1371/journal.ppat.1001237.g003
Rv1986 induces a distinct pattern of cytokine secretion in
addition to IL-2
and MIP-1 beta than CFP-10, and similar levels of RANTES.
Levels of IL-13 were very modest (Figure 4A, B and C). Thus the
Rv1986 peptides were associated with a distinct pattern of
cytokine production other than IL-2 when compared to CFP-10.
We further investigated the ability of Rv1986 to induce the
secretion of other cytokines when compared to CFP-10. We used
16-hour cell culture supernatants from 39 persons with either
active (19) or latent (20) tuberculosis. Multiple cytokine secretion
was assessed both quantitatively (pg/ml, after background
correction) and by frequency of response (.2 fold above
background). Similar levels of cytokine responses were observed
in both analyses in persons with active and latent tuberculosis (data
not shown), therefore the clinical groups were combined for
further analysis. When analyzed quantitatively and corrected for
multiple comparisons, Rv1986 pool 1 and 2 stimulated significantly higher levels of IL2sR, TNF, IL-10, IL-13, MIP-1 alpha
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T cell recognition of M. tuberculosis H37Rv with and
without Rv1986
We next determined whether there was any difference in the IL2 and IFN-c responses to live strains of MTB in which Rv1986
was intact or deleted. 13 persons with latent tuberculosis were
tested (only 9 patients for IFN-c due to limitation in cell numbers).
Although the overall IFN-c SFC response to these MTB strains
was much stronger than the IL-2 response, there was no significant
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Rv1986 of M. tuberculosis
Figure 4. Luminex multiplex cytokine analysis for Rv1986 and
CFP-10 peptide pools. Panels A, B and C show the production of
different cytokines and chemokines in 39 persons with latent or active
tuberculosis after 16 hrs stimulation of PBMC with peptides of Rv1986
(pools 1 and 2) and CFP-10 in cell culture supernatants respectively. The
results are represented as pg/ml; bars indicate median response with
IQR. The cytokine responses to Rv1986 (1 and 2) were significantly
higher than that of CFP-10 for IL2sR (p,0.0001), TNF (p,0.0001), IL-10
(p,0.0001), IL-13 (p#0.01), IL-17(p = 0.01), MIP-1alpha (p#0.005) and
MIP-1beta (p,0.004), but not RANTES (p.0.5).
doi:10.1371/journal.ppat.1001237.g004
difference in IFN-c response between these strains (Figure 5B).
The IL-2 SFC response to MTB H37Rv was significantly higher
than to the H37RvDRD-2 mutant (median 228 SFC/106 PBMC,
IQR 142–325 vs. 130 SFC/106 PBMC, IQR 53–268; p = 0.002)
and complementation by Rv1986 alone substantially restored the
SFC response (183 SFC/106 PBMC, IQR 86–285; p = 0.002,
when compared to H37RvDRD-2. Figure 5A).
Response of HIV infected persons to Rv1986, ESAT-6 and
CFP-10
The CD4 deficiency caused by HIV infection is the greatest
recognized predisposing factor to tuberculosis and conversely
antiretroviral therapy (cART) reduces susceptibility by suppressing
viral replication and allowing CD4 recovery [29]. We reasoned it
would therefore be of interest to compare the IL-2 to Rv1986 and
IFN-c and IL-2 response to CFP-10 and ESAT-6 before and
during the course of antiretroviral therapy. As the IFN-c response
to Rv1986 had not been prominent in HIV-1 uninfected persons
this was not assayed. The ELISpot response of 19 HIV infected
persons without evidence of active tuberculosis was therefore
tracked longitudinally over the first 36 weeks of therapy. All
patients experienced CD4 increases and suppression of HIV
replication during cART. We could not sample all time points and
patients for both cytokines due to limitation in the number of cells.
Figure 6 shows results of patients whose IL-2 and IFN-c response
to CFP-10 and ESAT-6 was assayed at least twice and 9 patients in
whom the corresponding IL-2 response to peptides p61–80 and
p161–180 could be determined. Peptide responses were summed
for analysis and compared to the values obtained from 20 HIV
uninfected persons of similar background, age and sex (i.e. those
shown in Figure 2). The IL-2 response to the peptides of Rv1986
was significantly lower in HIV infected persons prior to cART
(median 24, IQR 11–43) than in HIV uninfected persons (median
160, IQR 114–256, p = 0.009, Figure 6A). A significant increase in
response occurred during cART therapy such that the median at
36 weeks increased to 106 (IQR 79–157, p = 0.005). By contrast
the IFN-c response to ESAT-6 and CFP-10 was not significantly
lower in HIV infected persons prior to cART (median 147, IQR
50–965) than in HIV uninfected persons (232, IQR 56–563,
p = 0.84). Whilst the median response did increase during cART
therapy, the overall trend was not significant p = 0.22, Figure 6B).
The IL-2 response to the peptides of ESAT-6 and CFP-10 was
significantly lower in HIV infected persons prior to cART (median
2, IQR 0–31) than in HIV uninfected persons (median 148, IQR
44–323, p = 0.02, Figure 6C). No significant increase in response
occurred during cART therapy. Taken together these findings
indicate the decreased IL-2 to Rv1986 response prior to therapy
correlates with increased susceptibility better than the IFN-c
response to CFP-10/ESAT-6; and that the partial but significant
recovery of IL-2 to Rv1986, but unchanged IFN-c response to
CFP-10/ESAT-6 also correlates with the recognized decrease in
tuberculosis susceptibility that is conferred by cART.
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Figure 5. Live recognition of M. tuberculosis H37Rv, H37RvDRD2 and H37RvDRD2::Rv1986. PBMC from persons with latent tuberculosis
were co-cultured with M. tuberculosis H37Rv (l), H37RvDRD2 (n) and H37RvDRD2::Rv1986 (s) (complemented with Rv1986) for IL-2 and IFN-c ELISpot.
Results are represented as SFC/106 PBMC. Panel A shows IL-2 SFC response. All 3 strains induced substantial IL-2 response (.30 SFC/106 PBMC) in all
donors. The median IL-2 SFC to MTB H37Rv was highest, followed by H37RvDRD2::Rv1986 and the H37RvDRD2 mutant. Panel B shows IFN-c SFC
response, which was much stronger than the corresponding IL-2 response. All 3 strains induced similar levels of IFN-c response.
doi:10.1371/journal.ppat.1001237.g005
response to Rv1986 was several times greater than the
corresponding IFN-c response to either ESAT-6 or CFP-10
(Figure 1C). Our findings suggest Rv1986 to be a major target of
long lived CD4+ central memory T cells and that the Rv1986
peptides are associated with a distinct pattern of cytokine
production when compared to CFP-10. There was significant
recovery of IL-2 response to the peptides of Rv1986 than of IFN-c
response to ESAT-6 or CFP-10 during the course of cART in HIV
infected persons. We also showed that deletion of Rv1986 from the
genome of M. tuberculosis substantially decreases its ability to
restimulate IL-2 secretion. These interesting findings are potentially important when considering vaccine-induced and natural
immunity to tuberculosis and how immunodiagnosis may be
improved.
One hypothesis we were interested to test is whether, by virtue
of upregulation during hypoxia, proteins encoded by such genes
would be preferentially recognized by latently infected persons.
Discussion
We have analyzed whole genome-based transcriptional profiles
of M. tuberculosis subject to prolonged hypoxia to guide the
discovery of potential antigens. Because the diagnostic potential of
species-specific proteins is greatest we focused our initial
consideration on two genes upregulated during hypoxia that are
absent from all M. bovis strains including BCG by virtue of being
RD11-encoded (Rv2658c and Rv2659c) [30,31]. We also
investigated the RD2-encoded Rv1986 because it is absent from
most commonly used BCG strains. When compared to the wellcharacterized immunodominant and species-specific molecules
ESAT-6 and CFP-10, RD11 proteins had inferior ability to
restimulate IFN-c from T cells of persons sensitized by either
latent or active tuberculosis. However a striking finding was the
immunodominance of Rv1986 for the IL-2 recall response,
directed narrowly at two epitopic regions. The quantitative IL-2
Figure 6. Response of HIV infected persons to Rv1986, ESAT-6 and CFP-10. The IL-2 to Rv1986 and IFN-c response to CFP-10 and ESAT-6
before and during the course of antiretroviral therapy was compared. The respective responses were summed for analysis and compared to the
values previously obtained for HIV uninfected persons of similar background, age and sex. (A) The IL-2 response to the peptides of Rv1986 was
significantly lower in HIV infected persons prior to cART than in HIV uninfected persons. A significant increase in response occurred during cART
therapy (p = 0.005 Kruskal-Wallis test). (B) The IFN-c response to ESAT-6 and CFP-10 was not significantly lower in HIV infected persons prior to cART
than in HIV uninfected persons The trend in median response during cART therapy was not significant (p = 0.22). (C) The IL-2 response to the peptides
of ESAT-6 and CFP-10 was significantly lower in HIV infected persons prior to cART than in HIV uninfected persons (p = 0.02). No significant increase in
response occurred during cART therapy.
doi:10.1371/journal.ppat.1001237.g006
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Rv1986 of M. tuberculosis
With the exception of the weak IFN-c response to Rv2659c pool 2
(Figure 1A) this proved not to be the case. Hypoxia does
characterize tuberculous granulomas in vivo [18] but it is
increasingly re-appreciated that both active and latent tuberculous
lesions exhibit a dynamic spectrum of overlapping morphologies
[32,33,34,35,36,37,38] and that hypoxic lesions likely occur in
both clinical circumstances. A link between transient increases in
transcript abundance during hypoxia and the immunogenicity of
dosR regulated proteins has also been attempted and the term
‘latency antigen’ has been introduced [20]. A dominant antigenic
target that is dosR regulated is Acr1 encoded by Rv2031c and
under some assay conditions we, and subsequently others, have
documented preferential T cell recognition of Acr1 by latently
infected people [19,21]. Preferential recognition of Acr1 by
latently infected persons was not observed in this study (Figure 1)
nor in our previous IFN-c ELISpot analysis [22], which is in fact
consistent with expression of Acr1 throughout experimental
infection [39,40]. Conversely a quantitatively higher IFN-c
response to the RD1 encoded CFP-10 and ESAT-6 antigens has
sometimes been associated with active disease [41,42,43,44],
attributed to the secretion of these proteins by actively replicating
bacilli. We did not however observe a higher response in active
tuberculosis compared to latently infected persons. Differences in
infection pressures between low and high incidence areas might
feasibly contribute to these differences: the clinical environment in
which we conducted this study suffers an extraordinarily high
tuberculosis incidence of ,1500/100,000 with much ongoing
transmission [45]. It is also interesting to note that whilst the
transcriptomic data showed a fold decrease in ESAT-6 and CFP10 during hypoxia, the absolute abundance of these transcripts
remained high (Table 1). Expression of ESAT-6 and CFP-10
under a variety of conditions is in agreement with other in vitro
expression data [14,46] and adds to data suggesting these
molecules may play a role in bacillary persistence as well as active
infection [4]. The availability of expression profiles from latently
infected human tissue rather than from axenic in vitro culture might
provide a better starting point for antigen discovery.
Although IFN-c is essential to human defense against
mycobacteria, it is increasingly recognized that assay of PBMC
secretion of IFN-g is a poor correlate of protection in field studies
of tuberculosis [47]. Greater attention to markers, such as IL-2,
that might better reflect immunological memory is now being paid
and formed the basis for our investigation by ELISpot assay of this
cytokine [28,41,48], although IL-2 secretion itself is not established
as a better correlate of protection than IFN-c. Polyfunctional T
cells that secret multiple cytokines are considered a potential
correlate of protection in tuberculosis [49,50] although the finding
that such cells are expanded in tuberculosis patients rather than
healthy contacts has been interpreted by some to indicate a role in
pathology rather than protection [51]. In this context our finding
that Rv1986 was so dominant for the single positive IL-2 response
yet elicited modest IFN-c secretion was striking. The cytokine
phenotype of antigen specific T cells is greatly influenced by costimulation and the cytokine milieu [52,53]. However it has also
been suggested that the overall affinity of the TcR-peptide-MHC
interaction may play a role as well [54,55]. Interestingly an epitope
in Rv1986 with an anchor at position 167 (corresponding with
p161–180) is predicted for several DRB1*03, *04, *08, *11 and
*13 alleles [56]: a promiscuous binding ability that has been noted
for other M. tuberculosis epitopes [55] and which might contribute
to the almost universal response we observed to this peptide.
Rv1986 is a putative membrane protein that is recognized by T
cells from M. bovis infected cows [57]. Although the responses to
other RD-2 encoded antigens has been previously evaluated in
PLoS Pathogens | www.plospathogens.org
humans [58,59,60,61], the human T cell response to Rv1986 has
not been reported. Our finding that Rv1986 is a dominant target
of IL-2 secreting memory T cells suggests that this recall response
could contribute to protective immunity.
Our findings also bring a novel twist to an old story: the partial
and globally variable efficacy of BCG vaccine [62,63]. HenaoTamayo and colleagues recently investigated the vaccine efficacy
of BCG Pasteur concluding its ability to induce central memory T
cells in the lung was poor perhaps contributing to its partial
efficacy [64]. Although another recent study noted no experimental difference in short-term protective efficacy in Guinea Pigs
between RD2- negative (e.g. BCG Pasteur) and RD2-positive (e.g.
BCG Japan) strains [65] our discovery that a major target of the
human IL-2 response is absent from the most commonly used
strains is intriguing. Whilst the in vitro diagnostic potential of the
two dominant peptides we have uncovered is considerable, the
most important consequence of this work may be to re-evaluate by
clinical trials whether BCG strains with and without RD2 vary in
clinical efficacy.
Methods
Microbial culture conditions, hypoxia model and
microbial RNA analysis
These techniques have been extensively described before [25].
Briefly, exponential phase cultures grown in rolling culture to an
OD600 of 0.3 were diluted to a starting OD of 0.1 with warm
media. This starting culture was transferred to a constantly stirred
1 liter flask, 500 mL of this starting culture per flask. Hypoxia was
generated by introducing a constant flow of nitrogen with trace
amounts of oxygen (0.2% O2), leading to bacteriostasis. Samples
were taken before hypoxia, at four hours, and after 1, 4, and 7
days of exposure to hypoxia. RNA was isolated from these samples
using bead beating in the presence of Trizol, followed by
chloroform extraction and precipitation of RNA. The RNA was
further cleaned using an RNeasy kit purchased from Qiagen.
Approximately 3 mg of purified RNA was converted to cDNA
using Superscript III (Invitrogen). Aminoallyl dUTP was included
in the cDNA reaction, and subsequently conjugated to reactive Cy
dye esters. The aerobically growing transcriptional profiles were
directly compared to each subsequent hypoxic time point by
cohybridization on the same microarray slide. The microarray
slides and protocols were provided by the Pathogen Functional
Resource Center at the J. C. Ventner Institute as part of their
NIAID contract N01-AI-15447. Slides were scanned with a
GenePix 4000B purchased from Axon Technologies. Raw
background subtracted intensities were normalized to SigA to
provide an approximate measure of transcript abundance.
Human subjects
The University of Cape Town research ethics committee
approved this study (REC 296/2007). Written informed consent
was provided by study participants. Patients with active or latent
tuberculosis were recruited at the Ubuntu clinic at Khayelitsha site
B, South Africa. All were of Xhosa ethnicity. Active tuberculosis
(ATB) was defined by smear positivity for and/or culture of M.
tuberculosis from one or more sputum specimens. Latent tuberculosis (LTBI) was defined by transverse TST reactivity of .15 mm
in response to 2 TU PPD (RT23) at 48–72 hours or an interferonc Enzyme linked immunospot (ELISpot) response to ESAT-6 or
CFP-10 of .20 spot forming cells (SFC)/106 PBMC in the
absence of clinical symptoms or radiographic abnormality and
with a negative sputum smear and culture for M. tuberculosis. All
subjects underwent voluntary counseling and testing for HIV-1
7
December 2010 | Volume 6 | Issue 12 | e1001237
Rv1986 of M. tuberculosis
blocking medium was removed and the PBMC were set up with
respective antigenic stimuli. After 16-h incubation at 37uC in 5%
CO2, the plates were washed, 100 ml of detection antibody (IL-2II-biotin) at 1 mg/ml in PBS containing 0.5% FCS added and
incubated at room temperature. After 2 hrs, 100 ml of Streptavidin-ALP 1:1000 in PBS-0.5%FCS was added and incubated at
room temperature. After 1 h, 100 ml of substrate solution (BCIP/
NBT-plus) was added and developed until distinct spots emerged.
Plates were washed with tap water and allowed to dry. Spot
forming cells were enumerated by immunospot counter (CTL,
Cellular Technology Ltd) and confirmed by microscope (X4).
Results are quoted as cytokine spot forming cells (SFC)/106
PBMC.
The ELISpot (IFN-c and IL-2) experiments using live M.
tuberculosis strains H37Rv, H37RvDRD2 and H37RvDRD2::
Rv1986 (complemented by Rv1986), were performed as described
above and previously [66] with 200,000 PBMC/well cultured for
16–18 hrs in the presence of 200,000 live bacteria/well (in
duplicate wells).
infection and positivity was an exclusion criterion. ATB patients
were sampled prior to commencing antitubercular chemotherapy.
Known immunosuppression for other reasons, age ,18 years and
pregnancy formed other exclusion criteria. Another group of HIV1 infected adults who were starting antiretroviral therapy, followed
up for 36 weeks were also included as previously described in
detail [29]. Patients with ATB and/or HIV infection were treated
according to South African national guidelines. The baseline
characteristics of subjects enrolled to the study are shown in
Table 2.
Cell culture and ELISpot assays
Peripheral blood mononuclear cells (PBMC) were separated
over Ficoll. Cells were frozen and stored in liquid nitrogen until
analyzed in batches. A total of 2.56105 PBMC were added in
100 ml of RPMI/10%FCS (R10)/well for ELISpot and in 200 ml
of R10/well for cell culture. Antigenic stimuli were in the form of
pools (maximum 13 peptides in a pool) of 20-mer peptides
overlapping by 10 residues with each peptide used at a final
concentration of 10 mg/ml. Peptides were purchased from Peptide
Protein Research Ltd, Oxford, UK and from Pepscan Presto B.V,
Netherlands. Peptides were HPLC purified and their mass verified
by Mass spectrometry. Control stimuli for ELISpot included antiCD3 mAb CD3-2 at 100 ng/ml final concentration and
unstimulated wells.
The interferon-c ELISpot assay was performed as previously
described with slight modifications [29]. Ninety-six well precoated
ELISpot plates, mAb 1-D1K (Pre-coated One-step, Mabtech;
3420-2ATP-10) were washed with sterile PBS, blocked with R10
for $30 min at room temperature. The blocking medium was
removed and the PBMC were set up with respective antigenic
stimuli. After incubation for16 h at 37uC with 5% CO2, plates
were washed with PBS, and 100 ml of secondary antibody, mAb 7B6-1-ALP conjugate at 0.5 mg/ml final concentration in PBS
containing 0.5% FCS was added. After 2 h of incubation at room
temperature, 100 ml of filtered ready to use substrate solution
(BCIP/NBT-plus) was added and developed until spots emerged,
washed with tap water and allowed to dry.
For the IL-2 ELISpot, 96- well polyvinylidene difluoride
membrane based plates, type ELIIP (MAIPSWU10; Milipore
Corp), were activated by a brief treatment with 70% ethanol,
coated overnight at 4uC with 15 mg/ml of mAb IL2-I (Mabtech;
3440-2AW-Plus), and blocked with R10 for $30 min. The
Recombinant MTB strains
The MTB H37RvDRD2 strain (RD-2 mutant) was prepared
using homologous recombination and sucrose counter-selection as
previously described [15]. This mutant was then electroporated
with either the empty plasmid pMV306 or the same plasmid into
which Rv1986 from H37Rv had been cloned. This gene was
expressed under its native promoter. This resulted in the MTB
H37RvDRD2::pMV control (H37RvDRD2) and MTB
H37RvDRD2::Rv1986 (complemented with Rv1986) strains,
which were grown in 7H9 + ADC + 0.05% Tween 80 +
Kanamycin (25 ug/ml) and preserved as 25% glycerol stocks.
Intracellular cytokine staining assay (FACS)
1.5–26106 PBMC were incubated with the two Rv1986
peptides (residues 61–80 and 161–180) at 10 mg/ml each (i.e.
20 mg/ml peptide in total) or a pool of 21 peptides from CFP-10
and ESAT-6 at 2 mg/ml each (i.e. 42 mg/ml peptides in total) at
37uC. Control stimuli included SEB as positive control at10 mg/ml
and unstimulated cells as negative control. After 2 hrs, Brefeldin A
at 5 mg/ml (Sigma, St. Louis, MO) was added to capture the newly
formed cytokines in the Golgi apparatus. After 16 h incubation (in
total), the cells were washed with PBS (1X). For 8 color surface and
intracellular staining the cells were first permeabilized, and fixed
Table 2. Characteristics of study participants.
Active tuberculosis
Latent tuberculosis
Number
20
48
Median age
32.5
20.7
Sex (M:F)
11M:9F
12M:36F
Sputum smear
positive
19 Positive
1 unknown
NA
Sputum culture
positive
18 Positive
2 not available
NA
BCG vaccinated
5 vaccinated
6 Not vaccinated
9 Don’t know/data unavailable
23 vaccinated
20 Not vaccinated
5 Don’t know/data unavailable
Median Mantoux
(IQR)
NA
11 mm (IQR 8–20)
Significance, p
HIV Infected
Number
19
Median age
35
Sex (M:F)
10M:9F
NA
CD4 at recruitment
(median)
90
NA
CD4 after 6 months
of cART (median)
209
Viral Load at recruitment (median)
130,000
copies/ml
Viral Load after 6
months of cART
(median)
158
copies/ml
,0.0001
NA
doi:10.1371/journal.ppat.1001237.t002
PLoS Pathogens | www.plospathogens.org
8
December 2010 | Volume 6 | Issue 12 | e1001237
Rv1986 of M. tuberculosis
using Cytoperm/cytofix buffer (BD) for 20 min at 4uC, washed
with BD Perm/wash and stained with antibody cocktail in BD
perm/wash for 1 hr at 4uC. The antibodies used were as follows:
CD3-Pacific Blue (1 ml/tube), CD4 QDot605 (0.5 ml/tube), CD8
Cy5.5PerCp (3 ml/tube), IFN-c Alexa700 (1 ml/tube), IL-2 FITC
(5 ml/tube), TNF Cy7PE (5 ml/tube), CD45RA- APC (3 ml/tube),
CD27-PE (3 ml/tube), all of which were purchased from BD
BioSciences. 106 cells were acquired on LSR II flow cytometer
(BD Bioscience). Cell doublets were excluded using forward scatter
area vs. forward scatter height parameters. Unstained cells and
single-stained mouse calibration beads were used to calculate
compensations for every run. Data analysis was performed using
FlowJo v 8.8.2 (Tree Star), Pestle v 1.6.1 (NIH) and Spice v
5.05013 (NIH). We defined T cell phenotypes based on the surface
markers CD45RA and CD27: Central memory cells (TCM) as
positive for CD27 and negative for CD45RA; effector memory
(TEM) are negative for both CD27 and CD45RA and Terminally
differentiated T cells (Tdiff) are negative for CD27 and positive for
CD45RA. The results are expressed as the percentage of CD3+
CD4+ T cells.
room temperature. After incubation, the plates were washed 3
times with Bioplex wash buffer. 25 ml of Bioplex detection
antibody working solution was added, and incubated for 30 min
as above on the microplate shaker at room temperature. The
plates were washed 3 times with Bioplex wash buffer and 50 ml of
streptavidin-PE was added, and incubated for 10 min, washed 3
times with Bioplex wash buffer. Beads were resuspended with
125 ml of Bioplex assay buffer, mixed over the microplate shaker at
room temperature at 1100 rpm for 30 sec and read on the Bioplex
suspension array system.
Multiplex cytokine analysis
Acknowledgments
Statistical analysis
The normality of data was assessed by the D’Agostino and
Pearson omnibus test using Graphpad Prism 5.0 software (www.
graphpad.com). Parametric continuous variables were assessed by
student’s paired and unpaired t-tests, and non-parametric by
Wilcoxon matched pairs, Kruskal Wallis test with Dunn’s post test
correction or Mann Whitney U tests. Contingency analysis was by
Fisher’s exact test of probability.
Bioplex, mixed-to-order panel (premixed multiplex panel) from
Biorad was used for multiplex cytokine analysis. The assay was
carried out according to the manufacturer’s instructions. Briefly,
the 96- well filter plate was pre- wet with 150 ml of Biorad assay
buffer and the buffer removed by vacuum filtration. 50 ml of
multiplex bead working solution was added to the wells and the
buffer removed. 100 ml of Bioplex wash buffer was added to each
well and washed twice and the buffer removed. 50 ml of standard
and sample was added to the respective wells, the plate was sealed
and then covered by aluminum foil and placed over a microplate
shaker. The speed of the shaker was increased to 1100 RPM for
30 sec and then reduced to 300 RPM for 30 min, incubation at
We wish to thank Dr Virginia Azevedo, Dr Gilles van Cutsem and all staff
at the Ubuntu Clinic. Dr Tom Scriba is thanked for assistance with FACS
analysis, Reiling Liao for generation of the M. tuberculosis RD2 mutant and
the Centre for Proteomic and Genomic Research for assistance with
multiplex cytokine analysis.
Author Contributions
Conceived and designed the experiments: HPG KAW TRR DRS GM
MAB HMV DBY RJW. Performed the experiments: HPG KAW TRR
TO HG. Analyzed the data: HPG KAW TRR RJW. Contributed
reagents/materials/analysis tools: KAW TRR TO HG RAK DRS GM
MAB HMV DBY RJW. Wrote the paper: HPG TRR DBY RJW.
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December 2010 | Volume 6 | Issue 12 | e1001237