Mycobacterium bovis BCG-mediated protection against W-Beijing strains of Mycobacterium tuberculosis is diminished concomitant with the emergence of regulatory T cells.
ABSTRACT Despite issues relating to variable efficacy in the past, the Mycobacterium bovis BCG vaccine remains the basis for new-generation recombinant vaccines currently in clinical trials. To date, vaccines have been tested mostly against laboratory strains and not against the newly emerging clinical strains. In this study, we evaluated the ability of BCG Pasteur to protect mice from aerosol infections with two highly virulent W-Beijing clinical strains, HN878 and SA161. In a conventional 30-day protection assay, BCG was highly protective against both strains, but by day 60 of the assay, this protection was diminished. Histological examination of the lungs of vaccinated animals showed reduced lung consolidation and smaller and more-organized granulomas in the vaccinated mice after 30 days, but in both cases, these tissues demonstrated worsening pathology over time. Effector T cell responses were increased in the vaccinated mice infected with HN878, but these diminished in number after day 30 of the infections concomitant with increased CD4(+) Foxp3(+) T cells in the lungs, draining lymph nodes, and the spleen. Given the concomitant decrease in effector immunity and continued expansion of regulatory Foxp3(+) cells observed here, it is reasonable to hypothesize that downregulation of effector immunity by these cells may be a serious impediment to the efficacy of BCG-based vaccines.
- SourceAvailable from: Jochen Behrends[Show abstract] [Hide abstract]
ABSTRACT: The development of an effective vaccine against tuberculosis (Tb) represents one of the major medical challenges of this century. Mycobacterium bovis Bacille Calmette-Guerin (BCG), the only vaccine available at present, is mostly effective at preventing disseminated Tb in children, but shows variable protection against pulmonary Tb, the most common form in adults. The reasons for this poor efficacy are not completely understood, but there is evidence that T regulatory cells (Tregs) might be involved. Similarly, Tregs have been associated with the immunosuppression observed in patients infected with Tb and are therefore believed to play a role in pathogen persistence. Thus, Treg depletion has been postulated as a novel strategy to potentiate M. bovis BCG vaccination on one side, while on the other, employed as a therapeutic approach during chronic Tb infection. Yet since Tregs are critically involved in controlling autoimmune inflammation, elimination of Tregs may therefore also incur the danger of an excessive inflammatory immune response. Thus, understanding the dynamics and function of Tregs during mycobacterial infection is crucial to evaluate the potential of Treg depletion as a medical option. To address this, we depleted Tregs after infection with M. bovis BCG or Mycobacterium tuberculosis (Mtb) using DEREG mice, which express the diphtheria toxin (DT) receptor under the control of the FoxP3 locus, thereby allowing the selective depletion of FoxP3+ Tregs. Our results show that after depletion, the Treg niche is rapidly refilled by a population of DT-insensitive Tregs (diTregs) and bacterial load remains unchanged. On the contrary, impaired rebound of Tregs in DEREG × FoxP3GFP mice improves pathogen burden, but is accompanied by detrimental autoimmune inflammation. Therefore, our study provides the proof-of-principle that, although a high degree of Treg depletion may contribute to the control of mycobacterial infection, it carries the risk of autoimmunity.PLoS ONE 07/2014; 9(7):e102804. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Genotyping of clinical Mycobacterium tuberculosis complex (MTBC) strains has become a standard tool for epidemiological tracing and for the investigation of the local and global strain population structure. Of special importance is the analysis of the expansion of multidrug (MDR) and extensively drug-resistant (XDR) strains. Classical genotyping and, more recently, whole-genome sequencing have revealed that the strains of the MTBC are more diverse than previously anticipated. Globally, several phylogenetic lineages can be distinguished whose geographical distribution is markedly variable. Strains of particular (sub)lineages, such as Beijing, seem to be more virulent and associated with enhanced resistance levels and fitness, likely fueling their spread in certain world regions. The upcoming generalization of whole-genome sequencing approaches will expectedly provide more comprehensive insights into the molecular and epidemiological mechanisms involved and lead to better diagnostic and therapeutic tools.Cold Spring Harbor Perspectives in Medicine 09/2014; · 7.56 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Despite the overwhelming success of immunization in reducing, and even eliminating, the global threats posed by a wide spectrum of infectious diseases, attempts to do the same for tuberculosis (TB) have failed to date. While most effective vaccines act by eliciting neutralizing antibodies, T cells are the primary mediators of adaptive immunity against TB. Unfortunately, the onset of the T cell response after aerosol infection with Mycobacterium tuberculosis (Mtb), the bacterium that causes TB, is exceedingly slow, and systemically administered vaccines only modestly accelerate the recruitment of effector T cells to the lungs. This delay seems to be orchestrated by Mtb itself to prolong the period of unrestricted bacterial replication in the lung that characterizes the innate phase of the response. When T cells finally arrive at the site of infection, multiple layers of regulation have been established that limit the ability of T cells to control or eradicate Mtb. From this understanding, emerges a strategy for achieving immunity. Lung resident memory T cells may recognize Mtb-infected cells shortly after infection and confer protection before regulatory networks are allowed to develop. Early studies using vaccines that elicit lung resident T cells by targeting the lung mucosa have been promising, but many questions remain. Due to the fundamental nature of these questions, and the need to understand and manipulate the early events in the lung after aerosol infection, only coordinated approaches that utilize tractable animal models to inform human TB vaccine trials will move the field toward its goal. Copyright © 2014. Published by Elsevier Ltd.Seminars in Immunology 10/2014; 26(6):578-587. · 5.93 Impact Factor
CLINICAL AND VACCINE IMMUNOLOGY, Sept. 2011, p. 1527–1535
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 18, No. 9
Mycobacterium bovis BCG-Mediated Protection against W-Beijing
Strains of Mycobacterium tuberculosis Is Diminished Concomitant
with the Emergence of Regulatory T Cells?†
Diane J. Ordway,* Shaobin Shang, Marcela Henao-Tamayo, Andres Obregon-Henao, Laura Nold,
Megan Caraway, Crystal A. Shanley, Randall J. Basaraba, Colleen G. Duncan, and Ian M. Orme
Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology,
Colorado State University, Fort Collins, Colorado 80523
Received 1 May 2011/Returned for modification 17 May 2011/Accepted 18 July 2011
Despite issues relating to variable efficacy in the past, the Mycobacterium bovis BCG vaccine remains the
basis for new-generation recombinant vaccines currently in clinical trials. To date, vaccines have been
tested mostly against laboratory strains and not against the newly emerging clinical strains. In this study,
we evaluated the ability of BCG Pasteur to protect mice from aerosol infections with two highly virulent
W-Beijing clinical strains, HN878 and SA161. In a conventional 30-day protection assay, BCG was highly
protective against both strains, but by day 60 of the assay, this protection was diminished. Histological
examination of the lungs of vaccinated animals showed reduced lung consolidation and smaller and
more-organized granulomas in the vaccinated mice after 30 days, but in both cases, these tissues dem-
onstrated worsening pathology over time. Effector T cell responses were increased in the vaccinated mice
infected with HN878, but these diminished in number after day 30 of the infections concomitant with
increased CD4?Foxp3?T cells in the lungs, draining lymph nodes, and the spleen. Given the concomitant
decrease in effector immunity and continued expansion of regulatory Foxp3?cells observed here, it is
reasonable to hypothesize that downregulation of effector immunity by these cells may be a serious
impediment to the efficacy of BCG-based vaccines.
The global epidemic of disease caused by Mycobacterium
tuberculosis continues unabated, with recent figures indicating
approximately 8 million new cases of tuberculosis (TB) each
year, with about 2 million deaths (5, 8, 9, 13, 31, 40). Among
these infections, clinical isolates typed as belonging to the
W-Beijing family of strains are becoming increasingly preva-
lent; in fact, it is believed that the Beijing genotype family is
responsible for approximately 50% of TB cases in East Asia
and now accounts for at least 13% of all isolates worldwide (4,
17, 29, 31, 33, 37, 38). The reason for the success of this family
is unclear and is compounded by the suggestion that the cur-
rent vaccine for tuberculosis, Mycobacterium bovis BCG, has
actually selected for the emergence of this family (1).
Despite recent recommendations (10), we know little about
the actual basic biology of the newly emerging clinical strains
of M. tuberculosis. Only one W-Beijing strain, HN878, has been
extensively studied with animal models to date (19–22, 36); the
data obtained shows that this isolate is extremely virulent, at
least in comparison with the modestly virulent laboratory
strains H37Rv and Erdman. A major difference, as recently
demonstrated (22), is that while all the above strains potently
induce effector immunity 20 to 30 days after low-dose aerosol
infection of mice, this is soon replaced in HN878-infected mice
by the emergence of CD4 cells expressing Foxp3, with many of
these staining in addition for the immunosuppressive cytokine
Because vaccines for tuberculosis are usually tested against
the laboratory strains, there is very limited information as to
whether the existing BCG vaccine, or any of the new vaccine
candidates in the current pipeline (2, 32), will be effective
against the newly emerging clinical strains of M. tuberculosis,
many of which appear to be highly virulent (23, 26–28). This
issue is of particular importance given that the current lead
vaccine candidates are recombinant versions of BCG (2). In
the study reported here, mice were vaccinated with BCG and
then infected with two clinical W-Beijing isolates of M. tuber-
culosis. In both cases, the outcome was the same. Thirty days
after challenge, which is the conventional time at which pro-
tection is usually measured in this assay, both sets of vaccinated
mice were significantly protected against the two W-Beijing
strains. By day 60, however, this protection waned, and these
animals exhibited increasingly severe lung pathology. Flow cy-
tometric analysis of lung T cell populations showed a strong
early effector T cell response, which then declined and was
replaced by the steady influx into the lungs of Foxp3?regula-
tory T cells. In Kaplan-Meier analysis, the survival of the vac-
cinated mice was significantly prolonged in comparison to that
of control animals, but these animals all eventually died. The
data thus provide a new explanation for the spread of W-
Beijing strains despite widespread BCG vaccination. More-
over, they raise serious questions about the strategy of using
BCG, even recombinant versions, in new clinical trials in areas
in which W-Beijing strains are highly prevalent.
* Corresponding author. Mailing address: Department of Microbi-
ology, Immunology and Pathology, Colorado State University, Fort
Collins, CO 80523-1682. Phone: (970) 491-7469. Fax: (970) 491-5129.
† Supplemental material for this article may be found at http://cvi
?Published ahead of print on 27 July 2011.
MATERIALS AND METHODS
Animals. Specific-pathogen-free female, 6- to 8-week-old C57BL/6 mice were
purchased from the Jackson Laboratories, Bar Harbor, ME. They were main-
tained in the biosafety level 3 biohazard facility at Colorado State University
(CSU) and were given sterile water and mouse chow. All experimental protocols
were approved by the Animal Care and Use Committee of Colorado State
BCG vaccination. Animals were vaccinated subcutaneously with BCG Pasteur
at a dose of 106viable bacilli in 200 ?l of sterile saline and then rested for 6 weeks
prior to aerosol challenge. Control mice were injected with diluent only.
Experimental infections. Control and BCG-vaccinated mice were challenged
by low-dose aerosol exposure to M. tuberculosis using a Glas-Col exposure device
(Terre Haute, Inc.) calibrated to deliver 50 to 100 bacteria into the lungs of each
mouse (12). The laboratory strain H37Rv was originally obtained from the
Trudeau Institute collection (Saranac Lake, NY) and has been maintained at
CSU by low-frequency passage. The clinical W-Beijing strain HN878 was kindly
provided by B. Kreiswirth (PHRI, NJ), and the W-Beijing strain SA161 was
kindly provided by K. Eisenach (University of Arkansas).
Course of infections. Bacterial burden in the lungs of infected animals (n ? 5
animals) at the time points indicated in the figures was determined by plating
serial dilutions of whole-organ homogenates on nutrient 7H11 agar and counting
CFU after 3 weeks of incubation at 37°C in humidified air. The infection inoc-
ulum and day 1 lung bacterial counts were determined for all the bacterial strains
tested by plating serial dilutions of inoculum or tissue homogenates on nutrient
7H11 agar and counting CFU after 3 weeks incubation at 37°C. No significant
differences in the infection doses or the day 1 bacterial loads were present for any
of the bacterial strains tested.
Flow cytometric analysis of cell surface markers. Single-cell suspensions were
prepared as previously described (12). Briefly, the lungs were perfused with
phosphate-buffered saline (PBS) containing heparin (50 U/ml; Sigma-Aldrich,
St. Louis, MO) through the pulmonary artery, aseptically removed from the
pulmonary cavity, and dissected in medium. The dissected lung tissue was further
incubated with Dulbecco’s modified Eagle’s medium (DMEM) containing col-
lagenase XI (0.7 mg/ml; Sigma-Aldrich) and type IV bovine pancreatic DNase
(30 ?g/ml; Sigma-Aldrich) for 30 min at 37°C. The digested lungs were then
disrupted by gently pushing the tissue through a cell strainer (BD Biosciences,
Lincoln Park, NJ). Red blood cells were lysed with ACK lysing buffer (NH4Cl,
KHCO3, and EDTA), washed, and resuspended in complete DMEM containing
10% fetal bovine serum (Atlas Biologicals, CO). Total cell numbers were deter-
mined by flow cytometry using BD liquid counting beads, as described by the
manufacturer (BD PharMingen, San Jose, CA). Cell suspensions from each
individual mouse were incubated with monoclonal antibodies labeled with dif-
ferent fluorochromes at 4°C for 30 min in the dark. Monoclonal antibodies
(MAbs) were used against the surface marker CD4 (clone Gk1.5, rat IgG2b,k),
CD8 (clone 53-6.7, rat IgG2a,k), CD25 (PC61.5, rat IgG1,?) CD44 (IM7, rat
IgG2b,K), CD62L (MEL-14, rat IgG2a,K), CCR7 (4B12, rat IgG2a,k),CD69
(H1.2F3, Armenian hamster IgG), and CD95 (15A7, mouse IgG1,k) and isotype
control rat IgG2a, rat IgG2b, rat IgG1, mouse IgG1, and hamster IgG were used
in this study. These MAbs were purchased from BD Pharmingen or eBioscience
(San Diego, CA) as direct conjugates to fluorescein isothiocyanate (FITC),
phycoerythrin (PE), peridinin chlorophyll protein (PerCP), antigen-presenting
cell (APC), eFluor450, Alexa Fluor 700, or Qdot800. All the samples were
analyzed with a Becton Dickinson LSR-II instrument, and data were analyzed
using FACSDiva v5.0.1 software. Cells were gated on lymphocytes based on
characteristic forward- and side-scatter profiles. Individual cell populations were
identified according to the presence of specific fluorescence-labeled antibodies.
All the analyses were performed with acquisition of a minimum of 200,000
For intracellular cytokine staining, lung, lymph node, or spleen cells were
stimulated for 4 h with anti-CD3 and anti-CD28 in the presence of Golgi Stop
(Becton Dickinson). After incubation, cells were harvested and first stained for
cell surface markers as indicated above; thereafter, the same cell pellet were
resuspended in permeabilization buffer using a commercial kit (Foxp3 staining
buffer set; eBiosciences) and incubated for 30 min at room temperature. Cells
were washed again and resuspended in Perm/Wash buffer containing labeled
MAbs against Foxp3 (FJK-16S, Rat IgG2a), IL-17A (eBio17B7, Rat IgG2a,?),
and IL-17F (eBio18F10, Rat IgG2a), gamma interferon (IFN-?) (XMG1.2, rat
IgG1), and IL-10 (JES5-16E3, Rat TgG2b) and incubated for 30 min on ice. The
cells were then washed twice and resuspended in PBS containing 0.05% sodium
azide prior to analysis.
Histological analysis. Cranial lung lobes from each mouse were harvested and
fixed with 4% paraformaldehyde in PBS. Sections were prepared and stained
using hematoxylin and eosin.
Statistical analysis. Data are presented as representative of two independent
experiments and are the mean values (n ? 5) for replicated samples and dupli-
cate or triplicate assays. The parametric Student t test, or Kaplan-Meier analysis,
was used to assess statistical significance between groups of data.
Course of infection in control animals and in animals pre-
viously vaccinated with BCG. The course of the experimental
infections is shown in Fig. 1. In each case, mice were vaccinated
with BCG and exposed to aerosol infections 6 weeks later.
Most vaccines to date have been tested against laboratory
FIG. 1. Course of infections in the lungs of control and BCG-vaccinated mice. Data show the bacterial load in the lungs of control (closed
circles) and BCG-vaccinated (open circles) mice infected by aerosol with M. tuberculosis strain H37Rv, HN878, or SA161. CFU were determined
by plating serial dilutions of organ homogenates on nutrient 7H11 agar and counting CFU after 3 weeks of incubation at 37°C. Results are
expressed as the mean (n ? 5) of the bacterial load in each group expressed as log10CFU (? standard error of the mean [SEM]). Data points
indicated by asterisks were significantly different from results for saline controls. NS, not significant.
1528ORDWAY ET AL.CLIN. VACCINE IMMUNOL.
strain H37Rv or Erdman with protection in log10CFU con-
ventionally measured about 30 days later. We show here, as
anticipated, that mice vaccinated with BCG were more resis-
tant than saline controls to H37Rv infection at day 30 (P ?
0.005), with this resistance maintained at day 60 (P ? 0.01). In
a further experiment, we replaced the challenge infection with
the W-Beijing strain HN878. At day 30, the protection ob-
served for the vaccinated mice was 1.5 log (P ? 0.005), but at
day 60 of the experiment, the lung bacterial load was not
significantly different from that of the controls. A similar result
was obtained when we used the W-Beijing strain SA161 as the
challenge, with protection highly significant at day 30 (P ?
0.0001) but not significantly different from that of control an-
imals on day 60 and day 90. Similar observations were seen
after analysis of the bacterial load in the spleens (see Fig. S1 in
the supplemental material).
Development of pathology in infected animals. Changes in
lung pathology in mice infected with the two W-Beijing strains
are shown in Fig. 2 and 3. As previously observed (22), the
lungs of mice infected with HN878 exhibited progressive lesion
development, and by day 60, much of the lung tissue in the
control mice was grossly consolidated by inflammation gradu-
ally effacing the normal pulmonary architecture. Histologically,
there were multifocal to coalescing aggregates of epithelioid
macrophages with rare multinucleated giant cells, interspersed
with aggregates of mature lymphocytes. In some animals, se-
vere bronchopneumonia was seen, characterized by regionally
extensive areas of coagulative necrosis that extended from
major airways and effaced ?75% of the tissue. By day 60,
increasingly severe inflammation was seen, affecting greater
than 40% of the lung tissues.
Overall, BCG vaccination slowed the progression of lesion
development, and many inflammatory foci appeared more or-
ganized and dominated by large aggregates of lymphocytes.
Vaccinated animals examined at 30 days postinoculation had
foci of inflammation that were smaller than those in the con-
trol group and less extensive. Inflammation affected 10 to 15%
of pulmonary parenchyma and was more lymphocytic than in
the control animals. Vaccinated animals examined at day 60
had similar, lymphocyte-rich inflammation, but this had be-
come more extensive, affecting up to 20% of the lung paren-
A similar process was seen for mice infected with the W-
Beijing strain SA161 (Fig. 3), in which the progression of
disease from day 30 to day 60 was characterized by an increase
in the size of the lesions and progression from multifocal to
regionally extensive. Inflammation in these animals was char-
acterized by variably sized foci of epithelioid macrophages with
rare multinucleated giant cells and aggregates of lymphocytes.
There was an obvious reduction in the numbers of lymphocytes
as the infection progressed. From day 60 to 90, the patterns of
inflammation were progressively more severe, with inflamma-
tory foci coalescing and increased numbers of histiocytic cells
filling alveoli. Progression of disease in BCG-vaccinated ani-
mals was initially far less severe, but by day 60, these foci were
significantly larger, approaching the size of those seen for con-
trol animals. However, all vaccinated mice had more lympho-
cytes present within inflammatory foci relative to control ani-
mals, a difference that was most prominent at the later time
Kinetics of emergence of effector T cells. We harvested and
purified cells from the lungs, draining lymph nodes, and
spleens of vaccinated and control mice exposed to the two
W-Beijing strains and analyzed these by flow cytometry. The
FIG. 2. Histopathology of the lungs of mice infected with W-Beijing strain HN878. Representative photomicrographs of lung sections taken at
days 30 and 60 from control or vaccinated mice infected with strain HN878. Hematoxylin and eosin staining. Size bars, 500 ?m.
VOL. 18, 2011BCG-MEDIATED PROTECTION AGAINST W-BEIJING STRAINS 1529
kinetics of influx of effector T cells was determined, defined
here as CD4?CD44hiCD62LloIFN-??cells, and expressed as
percentages of the total CD4 cells. In mice infected with
HN878 and previously vaccinated with BCG, the influx of these
cells in these tissues was 2-fold higher than in controls on day
30, but these percentages then declined by day 60 (Fig. 4). As
with HN878, the percentage of effector cells in the lungs of
both groups of mice infected with SA161 progressively de-
clined during chronic infection after peaking at day 20 of the
Kinetics of emergence of CD4?Foxp3?and CD4?IL-17?T
cells. Given our earlier demonstration that HN878 is a potent
inducer of Foxp3?regulatory T cells (22), we then determined
if the loss of protection observed above might be temporally
associated with the arrival of this CD4 subset. In mice infected
with HN878, we observed a progressive increase in the per-
centages of regulatory T cells, as anticipated (Fig. 5). In BCG-
vaccinated mice, no evidence of any expansion or influx of
these cells was seen by day 30 of the challenge infection, but by
day 60, there was a significant surge in the influx of these cells.
In the BCG-vaccinated mice infected with HN878 lymph
nodes, percentages of these cells increased steadily, with no
overt differences in percentages between the two groups. In the
BCG-vaccinated mice infected with HN878 spleens, very low
percentages of these cells were seen on day 30, but the per-
centages had increased in both groups by day 60.
Similar kinetic patterns were seen for mice infected with
SA161. Again, there were much lower percentages of CD4?
Foxp3?regulatory T cells in the vaccinated mice on day 20 and
day 30 (P ? 0.02), but these percentages then increased sub-
stantially by day 60. Similarly, the percentages of these cells
were significantly lower (P ? 0.001) in the mediastinal lymph
nodes in the vaccinated mice at the early time points but then
surged upwards, and a similar pattern was observed for the
Given the known role of CD4 cell secretion of IL-17 in
FIG. 3. Histopathology of the lungs of mice infected with W-Beijing strain SA161. Representative photomicrographs of lung sections taken at
days 20 through 90 from control or vaccinated mice infected with strain SA161. Hematoxylin and eosin staining. Size bars, 500 ?m.
1530 ORDWAY ET AL.CLIN. VACCINE IMMUNOL.
driving inflammatory responses (16), as well as the recent im-
plication (15) that these cells play an important role in vacci-
nated mice, we also monitored the influx of CD4 cells in each
group. In HN878-infected mice, the percentages of CD4?IL-
17?cells increased progressively in both control and vacci-
nated animals in all three tissues analyzed (Fig. 6). Different
kinetics were seen in mice infected with SA161, with a very
large initial response in the lungs of the BCG-vaccinated ani-
mals. For reasons unknown, this profile was reversed in the
draining lymph nodes and spleens, i.e., mice infected with
SA161 showed increased percentages of CD4?IL-17?cells in
the control lymph nodes and spleens.
BCG prolongs survival but does not ultimately protect
against the W-Beijing strains. It was anticipated that the ob-
served slowing of the inflammatory process seen with both sets
of vaccinated mice would increase their survival, and this was
subsequently observed in a Kaplan-Meier analysis of a sepa-
rate set of mice. In two separate sets of studies, we consistently
observed prolonged survival of vaccinated animals (Fig. 7).
Among HN878-infected mice, control animals had a mean
survival time of 85 days, consistent with our earlier studies
(22), whereas vaccination increased mean survival to about 215
days. In mice infected with SA161, mean survival was 70 days,
and this improved to about 170 days in mice given BCG.
The results of this study show that mice vaccinated with
BCG and then challenged with M. tuberculosis H37Rv are
significantly protected, as anticipated, and that this protection
is sustained. In contrast, however, while mice infected with the
W-Beijing strains HN878 and SA161 were equally well pro-
tected on day 30 post-aerosol infection, by day 60, the numbers
of CFU in the lungs of control and vaccinated mice were not
significantly different. Examination of the lungs of these ani-
mals showed an initial reduction in inflammation and lung
consolidation and evidence of a better lymphocyte influx, as is
usually seen when a vaccine is protective, but these elements
were gradually lost as the disease process continued. Thus,
while BCG vaccination clearly had an initially positive protec-
tive effect against the two W-Beijing strains tested here, this
effect was transient and resulted only in a degree of prolonged
FIG. 4. Kinetics of influx of CD4 effector T cells in mice infected with strains HN878 and SA161. Lung cells obtained from control (closed
circles) and BCG-vaccinated (open circles) mice were analyzed by flow cytometry. Effector cells were defined as those that stained positive as CD4?
CD44hiCD62LloIFN-??cells. The data are expressed as the mean percentage of total CD4 cells in each organ ? SEM (n ? 5 mice per group).
Data points indicated by asterisks were significantly different from results for saline controls. NS, not significant.
VOL. 18, 2011 BCG-MEDIATED PROTECTION AGAINST W-BEIJING STRAINS1531
survival rather than long-lived protection. Many of the W-
Beijing and other families of strains we have now tested in our
laboratory are of equal or even far higher virulence than the
HN878 and SA161 strains used here, and this raises the pos-
sibility that BCG-based vaccines may be ineffective in areas of
the world where W-Beijing and potentially other families of
high-virulence isolates are of increasing prevalence. Our seri-
ous concerns on this matter are discussed in more detail else-
where (24, 25).
Analysis of effector T cell responses in animals with prior
BCG vaccination and infected with HN878 and SA161 showed
evidence of a considerable expansion of CD4?CD44hi
CD62LloIFN-??effector T cells, but this decayed significantly
after 20 to 30 days. Given our earlier observation that mice
infected with HN878 potently induce CD4?Foxp3?regulatory
T cells (22), we further investigated this possibility and found
evidence of a progressive increase in this cell population dur-
ing the course of both W-Beijing infections, concomitant with
worsening of the disease process. In mice infected with HN878
and SA161 that were first vaccinated with BCG, the expansion
of CD4?Foxp3?cells was minimal on day 30, but thereafter
began increasing and peaked on day 60. In SA161-infected
mice, robust increases were observed for all the organs in
control and BCG-vaccinated animals, with levels of such cells
being only somewhat lower in the vaccinated mice on day 60.
This may suggest that regulatory T cells are induced by the
highly virulent, highly inflammatory SA161 infection very early
on in the disease process, and indeed the capacity of the animal
to rapidly generate such cells has recently been observed by
others (30). Overall, these observations are thus consistent
with the hypothesis that while BCG vaccination might reduce
or delay the emergence or influx of CD4?Foxp3?regulatory
T cells in response to these W-Beijing infections, it cannot
prevent this from eventually happening.
As for TH17 cells, regarded by some studies as a counter-
balance, results were more ambivalent. In HN878-infected
mice, both control and vaccinated mice exhibited a steady
influx of CD4?IL-17?cells, whereas this rate of cellular influx
was initially much higher in the lungs of BCG-vaccinated mice
infected with SA161. Studies by others (15) have suggested
FIG. 5. Kinetics of influx of CD4?Foxp3?regulatory T cells in mice infected with strains HN878 and SA161. The total numbers of CD4?
Foxp3?cells in the lungs of control (closed circles) and BCG-vaccinated (open circles) mice were analyzed by flow cytometry. The data are
expressed as the mean percentage of total CD4 cells in each organ ? SEM (n ? 5 mice per group). Data points indicated by asterisks were
significantly different from results for saline controls. NS, not significant.
1532 ORDWAY ET AL.CLIN. VACCINE IMMUNOL.
that vaccination induces a population of TH17 cells that facil-
itate the influx of memory immune effector T cells in tubercu-
losis by accelerating the needed chemokine response. If so,
then this does not explain the low survival rates seen for BCG-
vaccinated mice exposed to SA161 compared to those of ani-
mals infected with HN878, in which the TH17 response in the
lungs was not potentiated by prior vaccination. Since both
groups of animals were vaccinated with the same inoculum of
BCG, then this result implies not only that individual isolates
may differ in their capacity to trigger the lung TH17 response
but also that a strong TH17 response, as seen to SA161, might
induce increased CD4?Foxp3?expansion to counterbalance
the proinflammatory effects of IL-17 cells and thus somewhat
decrease the subsequent survival seen for these animals.
At this time, we can only conclude that the expansion or
influx of CD4?Foxp3?regulatory T cells seems to parallel the
decline of the more rapidly emerging effector T cell response
to the two W-Beijing strains. Further studies involving regula-
tory T cell depletion are under way in our laboratory and will
help further address this possibility. Our current working hy-
pothesis is that the emergence of the CD4?Foxp3?popula-
tion may be directly in response to lung inflammation and
damage mediated by these two clearly highly virulent clinical
strains, with suppression or at least interference with the ex-
FIG. 7. Kaplan-Meier analysis of survival of mice after infection with
HN878 or SA161. Data show the time to death of control (closed circles)
and BCG-vaccinated (open circles) mice (P ? 0.0001 in both cases, Ka-
FIG. 6. Kinetics of influx of CD4?IL-17?T cells in control (closed circles) and BCG-vaccinated (open circles) mice were analyzed by flow
cytometry. The data are expressed as the mean percentage of total CD4 cells in each organ ? SEM (n ? 5 mice per group). Data points indicated
by asterisks were significantly different from results for saline controls. NS, not significant.
VOL. 18, 2011 BCG-MEDIATED PROTECTION AGAINST W-BEIJING STRAINS1533
isting protective effector T cell response as an unfortunate side
effect. Given our observation (27, 28) that many of the newly
emerging strains are capable of inducing severe lung damage,
this is of great concern, especially if prior BCG vaccination as
seen here can only slow this process down but not eventually
Our observations here regarding the effects of BCG on the
growth of clinically relevant isolates in the mouse lung are
highly consistent with observations by others. Jeon et al. (14)
tested a panel of clinical isolates in the mouse model and found
that BCG was protective in mice infected with HN878 at 4
weeks, but this protection was then rapidly reduced. This study
also examined the protective activity of BCG vaccination
against several other clinical strains. In three examples, BCG
protection remained relatively sustained after infection with
the clinical strains, but protection declined substantially when
other strains were used. Similarly, Grode et al. (11) demon-
strated that BCG was only transiently protective against a
modestly virulent W-Beijing strain and, interestingly, a recom-
binant BCG vaccine was more potent.
Our results are completely inconsistent, however, with the
results of studies published by Sun et al. (34). In an earlier
study (22), we clearly demonstrated that mice infected with
HN878 by low-dose aerosol survived on average about 80 days,
dying from the substantial lung pathology similar to that shown
above. In the current study, we further demonstrated that BCG
was able to slow the growth and subsequent lung pathology
caused by the two W-Beijing strains, resulting in prolonged
survival of these animals but no evidence of sustained protec-
tion. In contrast, Sun et al. (34) reported the mean survival of
control mice infected with HN878 as being close to 300 days,
an observation inconsistent with those in other reports dem-
onstrating the high virulence (“hypervirulence”) of HN878 (20,
22). In the Sun et al. study, BCG extended survival to ?375
days, and this was improved further to ?410 days in mice
vaccinated with a lead recombinant BCG vaccine candidate.
The Sun et al. study and the induction of (negative regula-
tor) CD8?? T cells in vaccinated macaques (18) support the
usage of recombinant BCG vaccines, and these vaccines are
the current focus of vaccine development today (2). As we have
discussed elsewhere (25), one can make a series of arguments
on why this may be a serious mistake. In addition to our data
here that indicate that BCG-based vaccines will not establish
immunity capable of overcoming the regulatory T cell response
many of the virulent clinical strains seem to potently generate,
we have also recently suggested other caveats. The first is that
BCG seems to very poor at generating central memory (12,
24); the consequence of this is that while the effector memory
response is faster than primary immunity, it still takes a finite
time to be expressed (7) and this probably still allows virulent
strains enough time to grow to the point that the regulatory T
cell response can then get triggered. Thus, a vaccine candidate
designed to induce central memory might be more effective,
and one such example is a recombinant M. smegmatis (“?ike-
plus”), which in a protective study (35) and in preliminary
studies in our hands seems capable of inducing CD44hi
CD62LhiIFN-??central memory T cells that expand and
respond in as little as 7 days after M. tuberculosis challenge
(I. M. Orme and W. R. Jacobs, unpublished observations); this
potentially could be fast enough to contain virulent infections
before CD4?Foxp3?cells become expanded.
A further issue regards the demonstration by Comas et al.
that multiple families of M. tuberculosis have selectively con-
served genes that encode T cell epitopes, including those ex-
pressed by immunodominant antigens (6). This is very con-
cerning, because one explanation for this is that it ensures a
potent T cell immune response, thus driving the granuloma-
tous process but then also the caseous necrosis which eventu-
ally has the potential to lead to cavitation and hence transmis-
sion. If so, then overexpressing these antigens in a new
recombinant BCG vaccine could potentially cause worsening
clinical disease and increased transmission. Moreover, as we
recently discussed (39), such recombinant vaccines would ac-
tually look superior in short-term animal model challenge ex-
periments, especially if tested against the less-virulent labora-
tory strains. To avoid such problems, it might be wiser to focus
on vaccines that express other protective antigens but not the
primary dominant antigens, for example, ID93 (3).
This study was supported in part by NIH grant AI081959, NIH
Innovation Award 1DP2OD006450, and funds from the College of
Veterinary Medicine, CSU.
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