Polyantigenic Interferon-c Responses Are Associated
with Protection from TB among HIV-Infected Adults with
Childhood BCG Immunization
Timothy Lahey1*, Brian K. Mitchell2, Robert D. Arbeit3, Siddharth Sheth1,2¤a, Mecky Matee4, C. Robert
Horsburgh5, Todd MacKenzie1, Lillian Mtei2, Muhammad Bakari3, Jenni M. Vuola6¤b, Kisali Pallangyo3, C.
Fordham von Reyn1
1 Dartmouth Medical School, Lebanon, New Hampshire, United States of America, 2 The Dartmouth Institute for Health Policy and Clinical Practice, Lebanon, New
Hampshire, United States of America, 3 Tufts University School of Medicine, Boston, Massachusetts, United States of America, 4 Muhimbili University of Health and Allied
Sciences, Dar es Salaam, Tanzania, 5 Boston University School of Public Health, Boston, Massachusetts, United States of America, 6 National Public Health Institute,
Helsinki, Finland
Abstract
Background: Surrogate immunologic markers for natural and vaccine-mediated protection against tuberculosis (TB) have
not been identified.
Methods: HIV-infected adults with childhood BCG immunization entering the placebo arm of the DarDar TB vaccine trial in
Dar es Salaam, Tanzania, were assessed for interferon gamma (IFN-c) responses to three mycobacterial antigen preparations
– secreted Mycobacterium tuberculosis antigens 85 (Ag85), early secretory antigenic target 6 (ESAT-6) and polyantigenic
whole cell lysate (WCL). We investigated the association between the number of detectable IFN-c responses at baseline and
the subsequent risk of HIV-associated TB.
Results: During a median follow-up of 3.3 years, 92 (9.4%) of 979 placebo recipients developed TB. The incidence of TB was 14%
in subjects with no detectable baseline IFN-c responses vs. 8% in subjects with response to polyantigenic WCL (P = 0.028).
Concomitant responses to secreted antigens were associated with further reduction in the incidence of HIV-associated TB. Overall
the percentage of subjects with 0, 1, 2 and 3 baseline IFN-c responses to mycobacterial preparations who developed HIVassociated TB was 14%, 8%, 7% and 4%, respectively (P = 0.004). In a multivariate Cox regression model, the hazard of developing
HIV-associated TB was 46% lower with each increment in the number of detectable baseline IFN-c responses (P,0.001).
Conclusions: Among HIV-infected adults who received BCG in childhood and live in a TB-endemic country, polyantigenic
IFN-c responses are associated with decreased risk of subsequent HIV-associated TB.
Trial Registration: ClinicalTrials.gov NCT0052195
Citation: Lahey T, Mitchell BK, Arbeit RD, Sheth S, Matee M, et al. (2011) Polyantigenic Interferon-c Responses Are Associated with Protection from TB among HIVInfected Adults with Childhood BCG Immunization. PLoS ONE 6(7): e22074. doi:10.1371/journal.pone.0022074
Editor: Clive M. Gray, University of Cape Town, South Africa
Received March 25, 2011; Accepted June 14, 2011; Published July 20, 2011
Copyright: ß 2011 Lahey 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 the National Institutes of Health, DAIDS, AI 45407 (C.F.vR.) and Fogarty International Center, D43-TW006807 (C.F.vR.,
C.R.H.); Dickey Center for International Relations International Travel and Research Grant (S.S.); National Institutes of Health, National Center for Research
Resources, Centers for Biomedical Research Excellence 5P20RR016437-08 (T.L.). 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: Timothy.Lahey@Dartmouth.edu
¤a Current address: Lake Erie College of Osteopathic Medicine – Seton Hill, Greensburg, Pennsylvania, United States of America
¤b Current address: GlaxoSmithKline Ltd, Espoo, Finland
or two immunodominant mycobacterial antigens, and polyantigenic whole cell mycobacterial vaccines such as recombinant BCG
and inactivated Mycobacterium vaccae [12,13]. Animal models
suggest that vaccines encoding one or two antigens provide
protection equivalent to BCG [14,15]. However, in epidemiologic
studies and TB vaccine trials in humans, protection against TB has
only been observed either after natural infection with M. tuberculosis
or non-tuberculous mycobacteria (NTM) [16,17,18,19], or after
immunization with live or inactivated whole cell mycobacterial
vaccines [10,20,21,22,23,24,25,26,27]. All of these situations
Introduction
The existing TB vaccine, bacille Calmette Guérin (BCG),
provides protection against tuberculosis (TB) when given to
mycobacteria-naı̈ve newborns, but protection is incomplete and
wanes in adulthood [1,2,3,4,5,6,7,8,9,10]. Thus, the development
of new and more effective primary and booster vaccines against
TB is a critical global health priority [11].
TB vaccine candidates now being tested for safety and efficacy
in humans include pauci-antigenic subunit vaccines encoding one
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Protective Polyantigenic IFN-c Responses
involve polyantigenic challenge with diverse mycobacterial
components.
Previously we have shown that HIV-infected subjects with
childhood BCG immunization living in a TB-endemic country and
followed prospectively have a reduced risk of developing TB if they
exhibit detectable baseline interferon gamma (IFN-c) responses to
mycobacterial antigens [28]. In the present study, using the
immunological database developed in our previous study, we
explored whether the number of baseline IFN-c responses targeting
mycobacterial antigens was associated with protection against TB in
BCG-immunized adults with HIV infection.
assessments included mycobacterial skin tests, an assay of
interferon gamma (IFN-c) release, and a standard tritiated
thymidine lymphocyte proliferation assay (LPA).
Skin tests
Tuberculin skin tests (TST) were performed on all subjects by
intradermal injection of purified protein derivative, PPD
(0.1 mL, RT-23, State Serum Institute, Copenhagen) on the
forearm; trained personnel measured the size of skin induration
at the site after 48–72 hours. We considered reactions of
$5 mm positive, and offered isoniazid treatment to all such
subjects [29].
Methods
IFN-c release assay
Human research conduct
Freshly isolated and ficolled peripheral blood mononuclear cells
(PBMC) were incubated with study antigens for five days in IFN-c
assays. After five days, centrifuged cell supernatants were frozen
and sent to the United States for later IFN-c level measurement
using a standard IFN-c enzyme linked immunosorbent assay
(ELISA; R&D Systems, Minneapolis, MN). Study antigens were
medium alone (negative control), 1 mcg/ml M. tuberculosis early
secretory antigenic target 6 (ESAT-6), 0.5 mcg/ml M. tuberculosis
antigen 85 (Ag85), or 0.5 mcg/ml M. tuberculosis whole cell lysate
(WCL), with all antigens acquired through NIH, NIAID Contract
No. HHSN266200400091C ‘‘Tuberculosis Vaccine Testing and
Research Materials’’ awarded to Colorado State University. IFNc assays were considered valid if the IFN-c level for the positive
control antigen, PHA (Sigma, St. Louis, MO; 2.5 mg/ml), was
greater than 300 pg/mL. IFN-c responses to mycobacterial
antigens were considered positive if the IFN- c level was greater
than or equal to two standard deviations above the mean of the
negative control condition.
LPA. LPA assays were conducted on the same PBMC used in
the IFN-c assay using a standard five-day 3H-thymidine
incorporation method. After incubation with study antigens,
20 ml of 50 mCi/ml 3H-thymidine was added to wells for
24 hours, and then cells were harvested onto filter paper and
sent to the National Public Health Institute in Helsinki, Finland,
for data acquisition on a scintillation counter. Results were
expressed as a proliferation index (PI; defined as counts per minute
[CPM] of antigen stimulated cells divided by CPM of
unstimulated cells). LPA assays were considered valid if the PI
for the positive control antigen phytohemagglutinin (PHA; 2.5 mg/
ml) was $3. LPA responses to mycobacterial antigens were
considered positive if the PI was greater than or equal to five.
We followed human experimentation guidelines of the United
States Department of Health and Human Services in the research
protocol for this study, which was approved by the Committee for
the Protection of Human Subjects at Dartmouth College and the
Research Ethics Committee of the Muhimbili University of Health
and Allied Sciences. Subjects provided written informed consent in
English or Kiswahili with the help of a translator as needed. This
study was registered through the National Institutes of Health
(NCT00052195).
Study subjects
The DarDar trial was a phase III randomized placebocontrolled double-blind trial of a novel booster vaccine for the
prevention of HIV-associated TB among adults in Dar es Salaam,
Tanzania [13]. Enrollment occurred from 2001 to 2005, and study
follow up continued through January 2008. Subjects were eligible
for enrollment if they had two positive enzyme linked immunosorbent assay (ELISA) tests for HIV, a CD4 count $200/mm3,
and a BCG scar. At baseline, all subjects were evaluated with
history, physical examination, single view chest x-ray, sputum acid
fast bacillus (AFB) smear, sputum mycobacterial culture, and
blood mycobacterial culture. Subjects with active TB were
excluded from enrollment. Subjects were randomized to receive
five intradermal doses of either heat inactivated Mycobacterium
vaccae or matched saline placebo. Subjects who received vaccine
exhibited protection against definite tuberculosis [13]. For the
present study, we confined our analyses to subjects who received
placebo.
Clinical surveillance for TB disease
After randomization, we evaluated subjects for active TB
disease by interim history and physical examination at months 2,
4, and 6, and every three months thereafter. In addition, at any
time subjects presented with two or more weeks of fever, cough or
weight loss, they underwent evaluation for active TB via a single
view chest x-ray, three sputum collections for AFB smear and
mycobacterial culture, phlebotomy for mycobacterial blood
culture, and any additional studies as clinically indicated (e.g.,
cultures of other body fluids or tissue biopsies).
Statistical analysis
We categorized subjects according to the number of antigens
against which baseline IFN-c responses, or LPA responses, were
targeted. Then, we compared demographic data and TB
incidence according to the number of antigens targeted by
baseline immune responses using a two-tailed Mann-Whitney U
test, student’s t test or chi squared test as appropriate. A KruskalWallis test was used specifically to compare demographic factors
between subjects according to the number of mycobacterial
antigens targeted by baseline immune responses. We then
constructed a multivariate Cox proportional hazards regression
model of the hazard of TB during prospective follow up, adjusting
for age, baseline CD4 count, previous TB treatment, and baseline
TST status. We confirmed that the proportional hazards
assumption was not violated using log-log plots and Schoenfeld
residuals. For statistical analyses, we used STATA 9 (College
Station, TX).
Definitions of TB
A three-person blinded adjudication panel reviewed all episodes
of illness evaluated for active TB and designated subjects as having
definite or probable TB according to rigorous published study
definitions [13].
Assays of mycobacterial immune responses
All subjects underwent in vivo and in vitro assessments of
immune responses to mycobacteria prior to vaccination. These
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Protective Polyantigenic IFN-c Responses
Results
Table 1. Percentage of subjects who developed HIVassociated TB during longitudinal follow up according to
antigen specificity of baseline IFN-c responses.
Subject characteristics
Among the 979 HIV-infected placebo subjects in this study, all
had a BCG scar and a baseline CD4 count $200/mm3 at study
entry. We diagnosed 92 causes of definite and probable TB during
a median follow-up of 3.3 years. As we showed previously [28],
subjects who developed HIV-associated TB had lower CD4
counts, had higher HIV viral loads, were more likely to have a
positive TST, and were more likely to have been treated for TB
previously. Figure 1 depicts the study CONSORT diagram.
Relation of multiple IFN-c responses to TB risk in
univariate analyses
Number of
baseline IFN-c
responses
Antigen
n
Percent who
developed TB
Zero
None
270
14%
One
ESAT-6
4
25%
Ag85
6
0%
WCL
208
8%
Two
Table 1 presents percentage of subjects who developed HIVassociated TB during follow up according to which mycobacterial
antigen preparations were targeted by baseline IFN-c responses.
Baseline IFN-c responses were observed most commonly to
polyantigenic WCL; only 2% of subjects had IFN-c responses to
one or both secreted antigens in the absence of a response to
WCL. Among subjects with no IFN-c responses at baseline, 14%
Three
ESAT-6 + Ag85
1
0%
WCL + ESAT-6
66
5%
WCL + Ag85
37
11%
WCL + Ag85 + ESAT-6
114
4%
Total
706
9%
Ag85, antigen 85; ESAT-6, early secreted antigenic target 6; IFN-c, interferon
gamma; TB, tuberculosis; WCL, whole cell lysate.
doi:10.1371/journal.pone.0022074.t001
developed incident TB. Response to WCL was associated with
substantial reduction in the incidence of HIV-associated TB (8%),
as we showed previously [28]. The incidence of TB decreased
progressively among subjects with additional detectable IFN-c
responses, and was lowest (4%) among subjects with baseline IFNc responses to all three mycobacterial antigen preparations
(Figure 2).
Univariate predictors of multiple IFN-c responses
Subjects with a greater number of baseline IFN-c responses
against mycobacterial antigens had higher baseline CD4 counts,
lower HIV viral loads, and were more likely to have a have a
positive TST (Table 2).
Relation of multiple IFN-c responses to TB risk in
multivariate analyses. We used a multivariate Cox
regression model to assess the hazard of developing HIVassociated TB relative to the number of detectable baseline IFNc responses against mycobacterial antigen preparations. In
Figure 2. Fraction of subjects who developed HIV-associated
tuberculosis (TB) according to the number of baseline
interferon gamma (IFN-c) responses detected against mycobacterial preparations.
doi:10.1371/journal.pone.0022074.g002
Figure 1. Study CONSORT diagram. ESAT-6, early secreted
antigenic target 6; f/u, follow up; IFN, interferon gamma; PHA,
phytohemagglutinin positive control; TB, tuberculosis.
doi:10.1371/journal.pone.0022074.g001
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Table 2. Baseline subject characteristics according to number of detectable baseline IFN-c responses to mycobacterial antigens.
Characteristic
Number of IFN-c responses to mycobacterial antigens
P-value
0
1
2
3
Age, mean years (SD)
33.2 (8.0)
32.4 (7.8)
34.4 (8.0)
32.3 (7.4)
0.15
Male, % (N)
23.1 (73/316)
24.2 (56/231)
25.5 (28/110)
25.2 (29/115)
0.948
Prior treatment for TB, % (N)
9.2 (29/316)
9.1 (21/231)
7.3 (8/110)
10.4 (12/115)
0.874
On HIV treatment at baseline, % (N)
3.8 (12/316)
4.3 (10/231)
3.6 (4/110)
3.5 (4/115)
0.978
Positive TST, % (N)
11.3 (35/310)
39.3 (90/229)
48.6 (52/107)
71.7 (81/113)
,0.001
Baseline CD4 count, mean cells/mL (SD)
419 (203)
507 (230)
547 (272)
570 (277)
,0.001
Baseline HIV viral load, mean log10 (SD)
4.1 (0.8)
3.9 (0.8)
3.5 (1.1)
3.4 (0.9)
,0.001
P values are derived via Mann-Whitney U tests, student’s t tests and chi squared tests as appropriate.
SD, standard deviation; TB, tuberculosis; TST, tuberculin skin test.
doi:10.1371/journal.pone.0022074.t002
when adjusting for baseline HIV viral load (HR 0.58, 95% CI
0.3321.03, P = 0.063).
unadjusted analyses, the hazard decreased significantly with each
incremental increase in the number of baseline IFN-c responses
(hazard ratio [HR] 0.62, 95% confidence intervals [CI] 0.47-0.81,
P = 0.001). This relationship remained significant after adjusting
for age, baseline CD4 count, previous TB treatment and a positive
TST (HR 0.54, 95% CI 0.38–0.75, P,0.001; Figure 3).
Compared to subjects with baseline IFN-c responses to WCL
alone, subjects responding to both WCL and ESAT-6 exhibited a
trend toward enhanced protection from HIV-associated TB
compared (HR 0.43, 95% CI 0.1821.06, P = 0.069), whereas
subjects responding to both WCL and Ag85 did not (HR 0.85,
95% CI 0.3522.05, P = 0.715).
In supplemental analyses, the number of baseline IFN-c
responses to mycobacterial antigens remained significantly associated with the hazard of HIV-associated TB both among subjects
with a negative baseline TST (HR 0.42, 95% CI 0.21-0.84,
P = 0.014) and among subjects with a positive baseline TST (HR
0.58, 95% CI 0.3920.87, P = 0.008). In the smaller subset of 234
subjects for whom baseline HIV viral load data are available, there
was a trend toward greater protection from HIV-associated TB
among subjects with a greater number of baseline IFN-c responses
No relation of number of baseline LPA responses to TB
risk
Like subjects with a greater number of baseline IFN-c responses
to mycobacterial antigens, subjects with a greater number of
baseline LPA responses to mycobacterial antigens had higher
baseline CD4 counts (P = 0.026), lower HIV viral loads
(P = 0.010), and were more likely to have a have a positive TST
(P,0.001). However, the number of detectable baseline LPA
responses to the same mycobacterial preparations was not
associated with protection from HIV-associated TB in univariate
or multivariate analyses (data not shown).
Discussion
Among HIV-infected adults in Tanzania we found that the
prospective risk of tuberculosis was related to the number and
diversity of baseline IFN-c responses to mycobacterial antigens. All
subjects received BCG at birth and most already had latent
infection with M. tuberculosis. The dominant IFN-c response was to
polyantigenic WCL, and IFN-c responses to secreted antigens
alone were uncommon. These immunologic data fit previously
defined conditions associated with immune protection from TB in
humans, each of which involves immune challenge with mutiple
mycobacterial antigens: TB infection itself [16,21], infection with
non-tuberculous mycobacteria [18,19], and immunization with
polyantigenic mycobacterial vaccines [5,6,20,22].
We hypothesize that the protection from HIV-associated TB
observed among subjects with polyantigenic IFN-c responses to
mycobacteria is due to immune responses targeting diverse cell
wall, cytosolic and secreted antigens expressed by M. tuberculosis
during the course of primary, latent and reactivation infection.
Diverse mycobacterial exposures likely contributed to the
baseline IFN-c responses detected in this study. Responses to
ESAT-6 signify prior exposure to M. tuberculosis, whereas IFN-c
responses to both the heterogeneous mycobacterial antigens in
WCL and to Ag85 could arise from prior exposure to M.
tuberculosis, non-tuberculous mycobacteria (NTM), or BCG
immunization [30,31,32,33,34]. Multiple baseline IFN-c responses
may represent prior exposure to a greater diversity of mycobacterial antigens, including heterologous antigens from mycobacteria
other than M. tuberculosis. Alternatively, subjects may vary in their
IFN-c responses on the basis of chance preservation of T cell
Figure 3. Survival without HIV-associated tuberculosis (TB)
according to number of baseline interferon gamma (IFN-c)
responses detected against mycobacterial preparations. The
hazard of HIV-associated TB fell 46% with each increment in the number
of detectable baseline IFN-c responses against mycobacterial preparations. HR, hazard ratio; TB, tuberculosis.
doi:10.1371/journal.pone.0022074.g003
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Protective Polyantigenic IFN-c Responses
clones reactive to mycobacteria despite progressive HIV infection.
Our data do not allow us to differentiate between these
possibilities. In the context of vaccine development, we propose
that the critical hypothesis to test is whether the most robust
marker of protective immunity following immunologic challenge is
the number and diversity of both homologous and heterologous
mycobacterial antigens eliciting IFN-c responses.
The risk of incident HIV-associated TB was, as expected,
increased in subjects with positive TST [35], but the risk of
incident HIV-associated TB was decreased in those with a greater
number of baseline IFN-c responses to mycobacterial preparations. This was true even though both reflect mycobacterial
exposure, and TST status and the number of IFN-c responses
against mycobacterial preparations were statistically correlated.
We propose two possible explanations for this apparent paradox.
First, the TST may be a more specific indication of latent TB than
the IFN-c responses we studied. Alternatively, IFN-c responses to
mycobacterial antigens may be more relevant to host defense
against TB than the delayed hypersensitivity reaction represented
by a positive TST. In either event, our analyses exclude the
possibility that the association between detectable baseline IFN-c
responses against multiple mycobacterial antigens and protection
from HIV-associated TB was confounded by TST status since the
association remained significant in both the TST positive and TST
negative subject subsets.
Baseline IFN-c responses to the different antigen preparations
may not be equivalent or independent in their contribution to
protection against subsequent HIV-associated TB. WCL is
prepared by disruption of the entire M. tuberculosis organism and
represents a particularly complex reagent containing innumerable
antigens from diverse cellular fractions [28]. Consistent with this,
IFN-c responses to this polyantigenic material were considerably
more common than to the other stimuli used and were strongly
associated with protection. These findings are concordant with
those of a recent study in Pakistan in which IFN-c responses to M.
tuberculosis sonicate were more common among subjects with
contained latent TB and less common as disease worsened from
active pulmonary TB to disseminated TB [36]. The protection
from TB associated with IFN-c responses to these complex
mixtures likely reflect immunity targeting multiple protein, lipid or
carbohydrate moieties, although our data do not allow us to
determine from which cell type(s) these complex responses arise
[37,38]. Our data further suggest that among subjects with IFN-c
responses to WCL concomitant responses to ESAT-6, but not
Ag85, augmented the protection from TB. Thus, we hypothesize
that interference with ESAT-6-mediated TB pathogenesis is of
particular benefit to the host [39].
Strengths of this study include its prospective longitudinal follow
up and the use of rigorous published TB criteria for TB diagnosis
[13]. A limitation of the study is the possibility that the responses
we detected were due solely to the degree of immunosuppression
of the HIV-infected subjects in this study. Although we cannot
exclude this possibility, the multivariate analysis controlling for
CD4 count supports our conclusion that the observed responses
were not merely a reflection of underlying HIV-related immunosuppression.
The fact that IFN-c but not LPA responses to the same antigens
were associated with protection suggests a specific mechanistic for
IFN-c responses in immune protection from TB. Lymphocyte
proliferation is a generic measure of antigenic sensitization. These
responding lymphocytes may have different capacities for
producing one or more specific cytokines, including IFN-c, which
was assayed in this study and previously associated with protection
from TB in humans [28,40]. Our findings are consistent with the
hypothesis that specific IFN-c responses to mycobacterial antigens
are more closely associated with the underlying immunologic
mechanism of protection against TB in humans than more generic
tuberculin skin testing or lymphocyte proliferation assays.
Our findings not only contribute to understanding of natural
and BCG-induced immunity to TB, but also to the development of
new primary and booster vaccines against TB. There are several
subunit and whole cell TB vaccine candidates in preclinical and
clinical trials development today [41,42]. Polyantigenic vaccine
candidates (e.g., whole cell live or inactivated vaccines) may be
more likely than pauci-antigenic candidates (e.g., individual
secreted antigens) to induce broad and effective immunity to TB
both among HIV-infected subjects with deletion of antigen-specific
T cell clones [43,44,45] and in healthy subjects with diverse
genetic determinants of antigen recognition [46]. In support of this
hypothesis, animal data suggest that vaccine induction of IFN-c
responses targeting multiple mycobacterial antigens confers
greater protection from TB disease than vaccine induction of
IFN-c responses against single mycobacterial antigens [14,47,48].
Further, it has been shown that immunity to TB induced by
immunization with inactivated mycobacteria can be adoptively
transferred to T cell deficient mice, and that protective antigens
are present in cell wall, cytosolic and secreted components of M.
tuberculosis [49].
It will be important to determine if subjects immunized with
polyantigenic vaccines, including those containing heterologous
and potentially cross-protective mycobacterial antigens, exhibit
greater vaccine-mediated protection from HIV-associated TB
than those immunized with a smaller number of antigens or
against exclusively homologous antigens from within the M.
tuberculosis complex.
Acknowledgments
We thank Wendy Wieland-Alter, Outi Rautio, and Betty Mchaki for their
skillful conduct of the immunological assays in this study, and Sue Tvaroha
for excellent database management.
Author Contributions
Conceived and designed the experiments: TL BKM SS RDA CFvR.
Performed the experiments: MM CRH LM MB JMV KP. Analyzed the
data: TL BKM RDA SS CFvR. Contributed reagents/materials/analysis
tools: SS MM CRH TM KP CFvR. Wrote the paper: TL BKM RDA
CFvR SS MM CRH TM LM MB JMV KP.
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July 2011 | Volume 6 | Issue 7 | e22074