VIEWPOINTS

Decrease in the Effectiveness of Bacille Calmette-Guerin

Vaccine against Pulmonary Tuberculosis: A Consequence
of Increased Immune Suppression by Microbial Antioxidants,
Not Overattenuation
Douglas S. Kernodle
Departments of Medicine and of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee

Mutations that arose in bacille Calmette-Guerin (BCG) daughter strains during decades of in vitro cultivation have long
been suspected of reducing the efficacy of the BCG vaccine against pulmonary tuberculosis. Although concern was raised 6
decades ago that BCG had become overattenuated, preferential use of relatively virulent BCG vaccines has not restored
efficacy. The recent discovery that as BCG evolved its production of antioxidants increased as a consequence of genomic
duplications and other mutations suggests the alternative hypothesis that BCG became better at suppressing oxidant-dependent
immune responses. This new model of BCG evolution is supported by evidence indicating that reducing BCG antioxidants
enhances immunogenicity. Furthermore, some previously unexplained aspects of the performance of the BCG vaccine in
clinical trials now make sense in the context of the new model. Finally, the model suggests that the risk of developing
pulmonary tuberculosis is influenced by the balance between host-generated oxidants and microbial antioxidants that activate
and suppress, respectively, the antigen-presentation pathways that protect the lungs.
For almost 9 decades, the live vaccine Mycobacterium bovis bacille Calmette-Guerin
(BCG) has been used against tuberculosis.
In early studies, BCG vaccine was highly
efficacious. In the 1920s, vaccination of
nursing and medical students with BCG
reduced pulmonary tuberculosis by 80%
[1]. In the 1930s, a trial in North America
found similarly high protection in the first
2 decades after vaccination, with continued benefit for 6 decades [2, 3]. Yet in
subsequent studies BCG vaccine exhibited
marked variability against pulmonary tuberculosis and occasionally even appeared
Received 21 December 2009; accepted 31 March 2010;
electronically published 4 June 2010.
Reprints or correspondence: Dr Douglas S. Kernodle, Div
of Infectious Diseases, A2200 Medical Center North,
Vanderbilt University Medical Center, Nashville, TN 372322582 (doug.kernodle@vanderbilt.edu).
Clinical Infectious Diseases 2010; 51(2):177184
! 2010 by the Infectious Diseases Society of America. All
rights reserved.
1058-4838/2010/5102-0009$15.00
DOI: 10.1086/653533

to increase the risk of developing tuberculosis [4, 5]. This variability is highly significant (P ! .0001) and is regarded as being indicative of true biological differences
[4]. Yet despite its flawed record against
pulmonary tuberculosis, BCG remains reliably efficacious in preventing tuberculosis meningitis and miliary tuberculosis
in young children [6]. Because pulmonary
tuberculosis is more common than disseminated tuberculosis, BCG has had a
minimal effect on the global burden of
tuberculosis, estimated at 9.4 million new
active cases and 1.8 million deaths annually [7].
Several hypotheses attempt to explain
the variable effectiveness of BCG against
pulmonary tuberculosis. These hypotheses
include differences between BCG daughter
strains (substrains), an inadequate dosage
of BCG in some trials, interference by environmental mycobacteria, genetic differences in human populations, and geo-

graphic differences in clinical isolates of

M. tuberculosis. These hypotheses have
been summarized elsewhere [4, 5, 8].
This Viewpoint offers a new perspective
on the old idea that the variable efficacy
of the BCG vaccine against pulmonary tuberculosis involves differences between
BCG daughter strains. In the context of
emerging data that reveal that mycobacterial antioxidants suppress host immunity [912] and that antioxidant production increased as BCG evolved [13, 14],
the relevant literature is reexamined to reveal an association between BCG daughter
strains that produce large amounts of antioxidants and poor efficacy against pulmonary tuberculosis. On the basis of partial but not fully conclusive evidence, a
new model is proposed in which BCG,
instead of becoming overattenuated,
evolved to become better at suppressing
the CD8+ T cell responses needed for protection against pulmonary tuberculosis.

BCG-Induced Immune Suppression CID 2010:51 (15 July) 177

EARLY CONCERN
ABOUT OVERATTENUATION
AND IDENTIFICATION
OF PHENOTYPIC DIFFERENCES
AMONG BCG SUBSTRAINS

Figure 1. Relative expression of genes encoding antioxidants and other microbial factors
implicated in immune evasion. Figure panels
were prepared by analysis of data in Table S4
of the article by Brosch et al [14]. First, the
expression value for 2 reference isolates of Mycobacterium bovis was averaged, and the mean
value was set as 1 (black line). Second, expression values for bacille Calmette-Guerin
(BCG) Tokyo 172 and Pasteur 1173P2 were displayed relative to the mean values for the reference M. bovis isolates. A, Antioxidant and
other immune evasion genes duplicated in Tokyo
172, Danish 1331, or Pasteur 1173P2 (full names
are given in Table 1). B, Antioxidant genes outside the BCG duplication units. These include
sodA (iron cofactored superoxide dismutase),
tpx (thiol peroxidase), ahpC and ahpD (alkylhydroperoxide reductases C and D), rubA (rubredoxin), the oxidoreductase Rv1774, and members of the whiB family of protein disulfide
reductases.

The new model further suggests that in

vitro evolution enabled extensively cultivated BCG substrains to survive longer in
vivo, which made them more effective at
preventing disseminated tuberculosis. Although overattenuation and increased immune suppression are mutually exclusive
models of the evolution of BCG, the new
hypothesis is not fully sufficient. Environmental and genetic factors are also crucial,
especially in the context of their effect on
host oxidant-generating capacity during
vaccination.
178 CID 2010:51 (15 July) Kernodle

An early theory regarding the apparent decline in efficacy of the BCG vaccine against
pulmonary tuberculosis was that the vaccine had changed over time. In 1949, Irvine expressed concern that the problem
has now become one of over-attenuation.
Separated from its natural habitat for 42
years, may not attenuation still be slowly progressing? [15, p 25]. By the mid1950s, it had been shown that BCG substrains differ in characteristics, including
growth rate, their ability to persist in vivo,
and their ability to protect mice against
M. tuberculosis infection [1619]. Daughter strains are descendents of BCG; before
modern technologies for preserving bacteria became available, laboratories maintained BCG by serial passage (ie, by transferring part of an aging culture into fresh
media). Some strains were passaged
11000 times before seed lots were prepared (eg, Pasteur 1173P2 and Danish
1331), whereas in Japan BCG was passaged only 172 times (Tokyo 172) [14,
20]. Serial passage at multiple sites caused
divergent evolution. Thus, substrains differ from each other and from the original
vaccine, which no longer exists.
SUPERIORITY OF RELATIVELY
VIRULENT BCG VACCINES
IN ANIMAL MODELS
In an attempt to identify BCG daughter
strains still effective in humans, investigators turned to animal models and
learned that protection correlated with the
invasiveness and persistence of the vaccine
strain. Dubos and Pierce summarized
their findings in mice as follows: Since
the most invasive substrains of BCG are
the most likely to elicit dependable and
lasting immunity, it would appear at first
sight that they are best suited to the practice of human vaccination. On the other
hand greater invasiveness increases the

incidence of adenopathies. The choice

of the optimal substrain of BCG involves
a compromise between the requirements
of the immunologist for dependable and
lasting immunity and the concern of all
for innocuousness [19, p 713]. On the
basis of studies in golden hamsters, bank
voles, and guinea pigs, Bunch-Christensen
et al also favored virulent BCG substrains,
concluding that the lower virulence [of
some substrains] is a sign of genetic mutation [and] consistent with the general
biological experience that virulence is often lost in vitro but that it practically never
increases [21, p 65]. Thus, virulent vaccines were assumed to be most like the
original BCG, which was 80% efficacious
in humans. This assumption influenced
the selection of 2 virulent substrains, Pasteur 1173P2 and Danish 1331, for a vaccination trial in the Chingleput region of
India [5, 22, 23]. However, despite their
high rank (second and third, respectively)
among the dozen vaccines evaluated in
animals [22], neither vaccine protected
humans against pulmonary tuberculosis [23].
SUPERIORITY OF BCG TOKYO
172 IN PREVENTING
TUBERCULOSIS IN HUMANS
The failure to restore vaccination effectiveness in Chingleput with virulent substrains that worked best in animal models
raised questions about whether overattenuation is the reason for the decline in BCG
effectiveness and also about how well results in animal models correlate with results in humans. Concerned by the inconsistent ranking of vaccines by different
laboratories [24], Comstock instead focused on the details of clinical trials to
identify an effective BCG vaccine. He argued that case-control studies conducted
over a time span in which one BCG
daughter strain replaces another provide
insight into their relative effectiveness [25
27]. Two studies, one in Indonesia involving disseminated and pulmonary tuberculosis (16 and 88 cases, respectively)
and another in Colombia involving pul-

Table 1. Antioxidant and Other Immune Evasion Genes in Bacille Calmette-Guerin (BCG)
Duplication Units

Protein function (gene name)

Duplication
unit

Tokyo
172

Danish
1331

Thioredoxin reductase (trxB2)

Thioredoxin (trxC)

DU1
DU1

Protein disulfide reductase (whiB7)

Possible glutaredoxin (Rv3198A)

DU2
DU2

+
+

Protein disulfide reductase (whiB1)

Oxidative stress sigma factor (sigH)

DU2
DU2

+
+

NADPH quinone reductase (lpdA, Rv3303c)

PI3P phosphatase (sapM, Rv3110)

DU2
DU2

Pasteur
1173P2
+
+

+
+

+
+

+
+

NOTE. The genes within DU1 of BCG Pasteur 1173P2 were determined from BCGList (http://
genolist.pasteur.fr/BCGList/). To identify genes within DU2, H37Rv coordinates of the duplicated regions
in each BCG daughter strain were obtained from Brosch et al [14] and included the following: Tokyo 172,
3,684,2293,704,932; Danish 1331, 3,567,4593,608,472 and 3,671,5363,709,097; and Pasteur 1173P2,
3,590,9023,608,472 and 3,671,5363,690,127. Then TubercuList (http://genolist.pasteur.fr/TubercuList/)
was used to identify the genes within the DU2 region of chromosome for each BCG daughter strain.
NADPH, nicotinamide adenine dinucleotide phosphate; PI3P, phosphatidylinositol 3-phosphate.

monary tuberculosis (178 cases), met this

standard. In Indonesia, the effectiveness of
BCG relative to unvaccinated individuals
decreased from +58% to !38% when Tokyo 172 was replaced by Pasteur 1173P2.
In Colombia, vaccination effectiveness decreased from +51% to !18% when Tokyo
172 (or British/Glaxo) was replaced by
Danish 1331. It is noteworthy that BCG
Tokyo 172 ranked eighth in the scheme
used to prioritize BCG substrains before
the Chingleput trial [22], well below the
rank of Pasteur 1173P2 and Danish 1331.
BCG Tokyo 172 is also cleared more rapidly than Pasteur 1173P2 from the organs
of mice [28, 29], and its lower virulence
relative to Pasteur 1173P2 and Danish
1331 is reflected in fewer adverse effects
[30].
In developing his argument, Comstock
mentioned 2 other studies that compared
BCG substrains [27]. The first was the
Chingleput trial, in which neither virulent
vaccine was effective. Comstock lamented
the omission of a substrain that ranked
poorly in animal models while acknowledging that it was understandable that
making such an odious comparison was
not politically possible for a World Health
Organizationsponsored project [27, p
S251]. The second study involved newborns in Hong Kong [5]. Fourteen cases
of tuberculosis involving lymph nodes,

meninges, bone, joints, or multiple sites

were observed in 150,000 persons vaccinated with BCG Pasteur, versus 31 cases
in 150,000 recipients of BCG British/
Glaxo. Of note, these vaccines ranked second and 11th, respectively, in animal
models [22], and thus results in animals
and humans correlated nicely. More recently, another study focused on protection against disseminated tuberculosis in
early childhood, comparing intradermal
Danish 1331 and percutaneous Tokyo 172
[31]. Both vaccines were highly effective,
reducing disseminated tuberculosis by
87% overall compared with nonvaccinated individuals, yet Danish 1331 was 46%
more effective than Tokyo 172. These results also correlate well with animal models, for which the vaccines ranked third
and eighth, respectively [22].
In summary, in the 5 human studies in
which BCG daughter strains have been
compared, Tokyo 172 has demonstrated
greater effectiveness against pulmonary
tuberculosis, whereas BCG Pasteur and
Danish 1331 have demonstrated greater
protection against disseminated tuberculosis in early childhood. Furthermore, although rankings of BCG substrains in animal models [22, 29] correlate poorly with
protection against pulmonary tuberculosis
in humans [2527], they correlate well
with protection against disseminated tu-

berculosis. This dichotomy may reflect a

requirement for qualitatively different immune responses for preventing pulmonary
tuberculosis than disseminated tuberculosis [12]. Obviously, this type of analysis
is limited because of the small number of
studies that have compared BCG substrains in humans.
GENOMIC DELETIONS IN BCG
SUBSTRAINS
Over the past few decades, the genetic evolution of BCG has been partly reconstructed. As BCG evolved from M. bovis,
it lost the region of difference 1 (RD1).
RD1 is absent from the chromosome of
all BCG substrains and encodes a secretion
system involved in virulence [3234]. Additional genomic deletions are found in
some BCG substrains, and despite the failure of virulent BCG vaccines in Chingleput, the hypothesis that the decline in the
effectiveness of the BCG vaccine involved
overattenuation has resurfaced [3537]. In
the reformulated hypothesis, deletions after RD1 are believed to cause overattenuation, yet no deletion has been clearly
linked to attenuation [37].
INCREASED EXPRESSION
OF ANTIOXIDANTS IN BCG
SUBSTRAINS
The evolution of BCG vaccine also included the duplication of regions of its
genome and greater expression of antioxidants (Figure 1 and Table 1) [13, 14].
Two duplication units, DU1 and DU2, exist. Although DU1 may be unique to BCG
Pasteur, DU2 is widely distributed, assumes different forms, and is triplicated
in some BCG daughter strains. Notable
among the duplicated and highly expressed genes is the sigma factor SigH,
which augments the expression of multiple antioxidants during oxidative stress,
including thioredoxin, thioredoxin reductase, and iron-cofactored superoxide dismutase (SodA) [38]. SigH also induces
enzymes that synthesize precursors of
mycothiol [39].
The biological reason underlying the

BCG-Induced Immune Suppression CID 2010:51 (15 July) 179

evolution of the BCG vaccine to produce

more antioxidants is uncertain; however,
antioxidants may enhance mycobacterial
growth in vitro by detoxifying by-products
of aerobic metabolism [12]. This hypothesis is supported by the observation that
genetic modifications of BCG that reduce
antioxidants also reduce its rate of growth
in vitro [12]. Furthermore, investigators
who worked with BCG before it was preserved as seed lots report that its rate of
growth accelerated over time [21, 40].
In summary, as BCG was cultivated for
decades, it not only underwent genomic
deletions but also evolved to produce
more antioxidants. Oxidants produced by
immune cells augment the activation and
apoptosis of phagocytes, and thus microbial antioxidants are well positioned to interfere with early host responses and the
development of adaptive immunity (Figure 2). These considerations and the fact
that antioxidants increased more in Pasteur 1173P2 and Danish 1331 than in To-

kyo 172 suggest that the decline in the

effectiveness of BCG vaccine against pulmonary tuberculosis might involve increased interference with oxidant-dependent immune signaling pathways.

ing vaccination with the modified BCG

vaccine. Memory immunity was also enhanced, and the modified BCG grew
slower in vitro and survived less well in
mice.

EFFECT OF REDUCING BCG

ANTIOXIDANTS ON
IMMUNOGENICITY

RECOGNITION OF A PARADOX
AND ITS IMPLICATIONS

In M. tuberculosis, reducing the activity or

secretion of SodA enhances the activation
and apoptosis of mononuclear cells and
strengthens antigen-specific CD8+ T cell
responses [911]. Furthermore, SigH promotes lung immunopathology by an unknown mechanism [41]. To determine
whether antioxidants that increased as
BCG evolved suppress immune responses,
Sadagopal et al [12] eliminated SigH and
reduced SodA activity and secretion in an
extensively passaged BCG substrain. BCGspecific CD8+ T cell responses suppressed
by the parent vaccine were unmasked dur-

The evolution of BCG represents a paradox. Unlike other live vaccines that
evolved to become more attenuated during in vitro cultivation, BCG instead became more virulent. The literature clearly
documents that the extensively cultivated
BCG substrains Pasteur 1173P2 and Danish 1331 exhibit greater virulence in animal models than the less extensively cultivated Tokyo 172 substrain [12, 22, 28,
29]. Furthermore, Danish 1331 is more
virulent than Danish 1077 (British/Glaxo).
The arguments made in this article suggest
that the evolution of the BCG vaccine toward greater virulence might also explain

Figure 2. Influence of oxidant-dependent immune signaling on antigen-presentation pathways and clinical outcome. After becoming infected with
Mycobacterium tuberculosis, the host faces 2 challenges. The first is to halt the lymphohematogenous spread of bacilli and prevent the development
of miliary tuberculosis; this is accomplished by macrophages and CD4+ T cells that produce interferon g (IFN-g). The second is to prevent foci of
granulomatous inflammation from expanding and damaging normal tissue; this probably requires cytotoxic T lymphocytes (CTLs) that kill bacteria within
infected macrophages. The induction of CTLs appears to involve Mycobacterium-infected phagocytes that first undergo apoptosis. Then mycobacterial
antigens within apoptotic cell fragments are taken up and presented by dendritic cells. Apoptosis-associated cross-priming pathways leading to CTLs
(black box) may be the reason that 90% of M. tuberculosisinfected humans never develop pulmonary tuberculosis. In contrast, the small-animal
models commonly used to evaluate tuberculosis vaccines are not natural hosts for M. tuberculosis. In most of these models, the host can restrict
dissemination unless it is starved or has certain genetic defects; however, disease in the lungs progresses and lifelong containment similar to latent
tuberculosis in humans does not occur, possibly because of inadequate CD8+ T cell responses. M, macrophages; MHC, major histocompatibility
complex; PMNs, polymorphonuclear neutrophils; TB, tuberculosis.
180 CID 2010:51 (15 July) Kernodle

the decline in its effectiveness against pulmonary tuberculosis.

Decisions made in accordance with the
overattenuation model of BCG evolution
have repeatedly failed to improve protection against pulmonary tuberculosis, most
notably in the Chingleput trial [23] but
also in subsequent studies in which Tokyo
172 was replaced with a more virulent vaccine [2527]. These failures now make
sense in the context of a new model of
BCG evolution in which overattenuation
never occurred. Instead, BCG evolved to
make more antioxidants, possibly to fulfill
a physiologic need involving the mycobacterial cell wall [12]. By chance, this coincided with a mycobacterial immune evasion strategy and made BCG better at
suppressing host immunity, particularly
CD8+ T cell responses. Although gene duplication and presumably other more
subtle mutations underlie most of the
increased expression of antioxidants in
current BCG vaccines, it is noteworthy
that a genomic deletion also contributed.
The increased expression of Rv1774, an
oxidoreductase, involves the loss of its
repressor within RD14 [42].
The mechanistic and clinical implications of BCG evolving to become more
virulent by producing more antioxidants
are enormous. First, rather than not surviving long enough to induce immunity,
the extensively cultivated BCG daughter
strains actively suppress CD8+ T cell responses and immune memory. Because
interferon g production by CD4+ T cells
and macrophage responses are less affected than CD8+ T cell responses by microbial antioxidants [12], when subsequently infected with M. tuberculosis, a
BCG-vaccinated host still compartmentalizes infection within granulomata and
thus is protected against disseminated tuberculosis. However, weak CD8+ T cell responses limit the hosts ability to kill infected macrophages and resolve granulomatous foci of infection. An increasing
body of evidence suggests that CD8+ T
cells may help protect against pulmonary
tuberculosis [43, 44]. In effect, as BCG

evolved it became better at suppressing

immune responses needed for protection
against pulmonary tuberculosis while
largely retaining responses that protect
against disseminated tuberculosis in early
childhood.
Second, because of divergent evolution
the immune-suppressive capacity of each
BCG substrain differs yet should correlate
roughly with the number of passages before the seed lot was preserved. This claim
is supported by the greater immunogenicity, including CD8+ T cell responses, of
BCG Tokyo 172 than Danish 1331 [45].
Third, the virulent and immune suppressive vaccines are more effective against
disseminated tuberculosis in early childhood, possibly because they survive longer
in vivo. Vaccine persistence also correlates
with protection in mice [19, 29], yet if
antibiotic treatment is used to clear BCG
Danish 1331 from mice, immune responses and protection against dissemination decrease to low levels within
months [46]. In effect, vaccines that induce the greatest protection against pulmonary tuberculosis may be less effective against disseminated disease in early childhood because they induce immunity that clears the vaccine strain from the
host [12].
Fourth, involvement of oxidant-dependent immune responses in protection
against pulmonary tuberculosis (Figures 2
and 3) makes sense in the context of risk
factors for pulmonary tuberculosis in humans. For example, persons with chronic
granulomatous disease, a genetic disease
in which nicotinamide adenine dinucleotide phosphate oxidase fails to assemble
to produce superoxide, frequently develop
pulmonary tuberculosis and occasionally develop disseminated tuberculosis or
BCG-osis [48, 49]. In mice, p47phox deficiency causes a survival defect in CD8+
T cells that is partially corrected by adding
oxidants [50]. Less severe defects in oxidant-generating capacity, such as a weak
oxidative burst [51] and/or low polymorphonuclear neutrophil counts [52], may
underlie the high rate of pulmonary tu-

berculosis in some racial/ethnic groups.

The role played by apoptosis-associated
cross-priming in the induction of CD8+ T
cell responses seems plausible in the context of the proapoptotic effects of oxidants
and evidence indicating that oxidation of
phosphatidylserine in membranes of apoptotic cells enhances the uptake of cell
fragments by other phagocytes [53]. A role
for antigen cross-presentation in humans
is suggested by the observation that persons with latent tuberculosis (ie, positive for purified protein derivative) exhibit predominant macrophage apoptosis,
whereas persons cured of active tuberculosis exhibit greater necrosis [54].
Fifth, a model in which the balance between host-generated oxidants and mycobacterial antioxidants affects CD8+ T
cell responses also provides insight into the
pathogenesis of tuberculosis. By allowing
CD4+ T cell responses to develop and limit dissemination while suppressing CD8+
T cell responses, M. tuberculosis ensures
disease transmission. Instead of dying
quickly, most infected hosts with CD8+
responses inadequate to maintain latent
tuberculosis will develop granulomatous
lung cavities and infectious aerosols. A
third of the worlds population is infected
with M. tuberculosis, demonstrating the
success of this pathogenesis strategy.
Sixth, the increased immune suppression model of BCG evolution predicts that
the virulent BCG daughter strains are the
least like the early BCG vaccines that exhibited 80% protection against pulmonary
tuberculosis [1, 2]. In the new model, the
virulent vaccines are not only expected to
be ineffective against pulmonary tuberculosis, but their use increases adverse reactions unnecessarily. This contrasts with
the overattenuation model, which predicts
that increased efficacy will be achieved
only at the cost of increased adverse reactions [36, p 134]. These predictions are
timely in the context of reports that vaccination with BCG Danish 1331 causes
disseminated BCG disease in almost 1%
of human immunodeficiency virusinfected infants [55].

BCG-Induced Immune Suppression CID 2010:51 (15 July) 181

Figure 3. Determination of the outcome of infection by the balance between host-generated oxidants and microbial antioxidants. In this model,
90% of humans generate enough oxidants during early infection to activate cross-priming pathways of antigen presentation (A). The cytotoxic T
lymphocyte responses heal granulomatous foci of infection and provide ongoing immune surveillance to prevent the development of pulmonary
tuberculosis (Figure 2). However, in 10% of persons the host-generated oxidants are insufficient to overcome suppression by antioxidants, resulting
in weak CD8+ cell responses and eventually in the development of active tuberculosis. The goal of vaccination is to prevent active tuberculosis from
developing in the 10% of persons so predisposed (B ). The original bacille Calmette-Guerin (BCG) vaccine reduced pulmonary tuberculosis by 80%,
which may reflect greater cross-priming compared with natural infection, perhaps from the loss of genes within region of difference 1 that suppress
the production of oxidants by antigen-presenting cells [47]. Alternatively, by inoculating thousands of vaccine bacilli during vaccination, more polymorphonuclear cells with highly potent oxidant-generating capacity are activated than after inhalation of a few tubercle bacilli. Because the increase
in antioxidants in BCG daughter strains is roughly proportional to the number of times they were passaged, vaccination effectiveness remained about
+50% with Tokyo 172 yet decreased to as low as !38% with Pasteur 1173P2 [2527]. The increased immune suppression model predicts that BCG
can be modified to exhibit diminished activity of antioxidants, thereby inducing strong immunity that prevents pulmonary tuberculosis. In effect, by
reducing BCG antioxidants the redox balance during early infection shifts, and cross-priming can now occur in some persons with low oxidant-generating
capacity. This should enable them to develop immune responses more like those that develop in the 90% majority during natural infection. Then if
infection occurs the vaccination-induced immune responses are recalled and help to prevent active tuberculosis. The development of memory immunity
is probably the reason that protection was observed for 6 decades in recipients of early BCG vaccines [2]. TB, tuberculosis.
182 CID 2010:51 (15 July) Kernodle

Seventh, if increased antioxidant production is the primary reason for the decline in the effectiveness of the BCG vaccine against pulmonary tuberculosis, then
the simplest way to restore its effectiveness is to reduce BCG antioxidants (Figure 3). If the new model is correct, such
modifications should enhance protection
against pulmonary tuberculosis in humans. Table 1 and Figure 1 show some of
the antioxidant genes that underwent duplication and increased expression as BCG
evolved and thus represent high-priority
targets. Reconstructing a BCG vaccine that
exhibits protection comparable or superior to the early BCG vaccines should be
easier to accomplish with Tokyo 172 than
with virulent BCG substrains. It is noteworthy that prioritization of vaccines for
human trials will need to be different from
the algorithm used for the Chingleput
study [22, 23]. Because the immune responses of mice and other small animals
commonly used in vaccination-challenge
experiments are inadequate to induce latent tuberculosis, more relevant models
are needed. In the context of controlling
tuberculosis globally, it is more important
to target the pulmonary, contagious form
of tuberculosis than disseminated tuberculosis. Thus, vaccines that induce strong
immunity should be favored even if they
are less effective against dissemination
than virulent vaccines. Secondary immune
responses in a memory-immune model
[12] may be particularly useful in identifying vaccines likely to exhibit greater protection against pulmonary tuberculosis.
Finally, this new hypothesis does not
mean that other hypotheses regarding the
suboptimal effectiveness of the BCG vaccine against pulmonary tuberculosis do
not also contribute. However, when it
comes to understanding the effect of phenotypic differences among BCG substrains
on protection against pulmonary tuberculosis, we failed to grasp a paradox and
had it backward all along.

Acknowledgments
Financial support. David E. Rogers Professorship in Medicine. The financial support did not
influence the opinions expressed in this essay.
Potential conflicts of interest. D.S.K. is listed
as an inventor on issued patents and patent applications for a technology for enhancing the immunogenicity of bacterial vaccines by reducing the
activity of antiapoptotic microbial enzymes. The
technology has been assigned to Vanderbilt and
the US government as represented by the US Department of Veterans Affairs.

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