Human Vaccines

ISSN: 1554-8600 (Print) 1554-8619 (Online) Journal homepage: http://www.tandfonline.com/loi/khvi19

BCG Vaccines: Their mechanisms of attenuation

and impact on safety and protective efficacy

Jun Liu, Vanessa Tran, Andrea S. Leung, David C. Alexander & Baoli Zhu

To cite this article: Jun Liu, Vanessa Tran, Andrea S. Leung, David C. Alexander & Baoli Zhu
(2009) BCG Vaccines: Their mechanisms of attenuation and impact on safety and protective
efficacy, Human Vaccines, 5:2, 70-78, DOI: 10.4161/hv.5.2.7210

To link to this article: http://dx.doi.org/10.4161/hv.5.2.7210

Copyright 2009 Landes Bioscience

Published online: 01 Feb 2009.

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[Human Vaccines 5:2, 70-78; February 2009]; 2009 Landes Bioscience
Review
BCG vaccines
Their mechanisms of attenuation and impact on safety and protective efficacy
Jun Liu,1,* Vanessa Tran,1 Andrea S. Leung,1 David C. Alexander1 and Baoli Zhu2
1Department of Molecular Genetics; University of Toronto; Toronto, Ontario Canada; 2Joint Center for Microbial Genomics; Institute of Microbiology; Chinese Academy of
Sciences; Beijing, China
Key words: BCG, tuberculosis, vaccine, attenuation, safety, efficacy
Mycobacterium bovis Bacille Calmette-Gurin (BCG) was devel-
oped as an attenuated live vaccine for tuberculosis control nearly
a century ago. Despite being the most widely used vaccine in
human history, the mechanisms of attenuation of BCG remain
poorly understood. BCG is not a single organism, but comprises a
number of substrains that differ in genotypes and phenotypes. The
impacts of these differences on BCG vaccine properties are largely
unknown. Nevertheless, in the past decade, the development of
sophisticated genome analysis techniques, coupled with advances
in knowledge of the virulence mechanisms of Mycobacterium
tuberculosis, have provided greater insights into the attenuation and
CE
evolution of BCG. This review article discusses these new devel-
opments, focusing on molecular mechanisms that contribute to
the attenuation of BCG substrains. It is evident that BCG strains
EN
comprise natural mutants of major virulence factors of M. tb,
including ESX-1, PDIM/PGL and PhoP, and that BCG substrains
differ markedly in virulence level. The impacts of these findings
CI
on vaccine properties including adverse reaction effect, tuberculin
reactivity and protective efficacy are discussed. These new insights
OS
have extremely important implications for national immunization
programs and the development of future vaccines.
Introduction
BI
Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tb),
remains a global health emergency. The latest surveillance data by the
S
World Health Organization (WHO) reveals that in 2006, there were
DE
9.2 million new cases and 1.7 million deaths from TB.1 The challenges
of TB prevention, case detection and treatment are in stark contrast
to the ease of M. tb transmission. Additional threats to TB control
N
include the spread of multidrug resistant TB (MDR-TB), the appear-
ance of extensively drug-resistant TB (XDR-TB), and the destructive
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impact of TB/HIV coinfection. The global burden of MDR-TB,
defined as resistance to isoniazid and rifampin, was estimated at
09
*Correspondence to: Jun Liu; 4382 Medical Sciences Building; Department of
Molecular Genetics; University of Toronto; 1 Kings College Circle; Toronto, Ontario
20
M5S 1A8 Canada; Tel.: 416.946.5067; Fax: 416.978.6885; Email: jun.liu@
utoronto.ca
Submitted: 03/28/08; Revised: 10/13/08; Accepted: 10/20/08
Previously published online as a Human Vaccines E-publication:
http://www.landesbioscience.com/journals/vaccines/article/7210
70
.
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BU
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ST
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500,000 cases in 2006.1 XDR-TB, caused by MDR strains that are
T
also resistant to a fluoroquinolone and at least one second-line inject-
able agent (amikacin, kanamycin and/or capreomycin), caught the
NO
worlds attention after an outbreak in KwaZulu-Natal, South Africa,
where 52 of 53 infected patients died.2 HIV co-infection was a
contributing factor in most of these deaths, and indeed, a deadly asso-
O
ciation between HIV and TB has been known almost since the start
of the HIV-epidemic. Of the 1.7 million people who died from TB
.D
in 2006, an estimated 200,000 were co-infected with HIV.1 Because
of these situations, effective approaches alternative to antibiotics are
urgently needed for the control of TB.
Bacille Calmette-Gurin (BCG), an attenuated strain of
Mycobacterium bovis (M. bovis), is currently the only available vaccine
against TB. Since 1974, BCG vaccination has been included in the
WHO Expanded Program on Immunization.3 It is estimated that
more than 3 billion individuals have been immunized with BCG and
over 100 million doses of BCG are administered annually, making
it the most widely used vaccine in humans.3 Meta-analysis studies
have confirmed that BCG protects children, providing >80% efficacy
against severe forms of TB, including tuberculous meningitis and
miliary TB.4,5 In contrast, evidence for protection against pulmo-
nary TB in adolescents and adults remains contentious as efficacy
estimates from clinical trials, observational case control studies and
contact studies range from 0 to 80%.6,7
The reasons for the variable protective efficacy are unknown
but several hypotheses have been proposed, including differences
among the vaccine strains used in clinical studies, exposure of trial
populations to environmental mycobacteria, nutritional or genetic
differences in human populations, differences in trial methods, and
variations among clinical M. tb strains.8-13 These explanations are
not mutually exclusive and all may contribute to the heterogeneity
in vaccine efficacy.
A key aspect of this issue may concern the nature of BCG attenu-
ation. Although it is generally considered safe and has been used as
a human vaccine since the 1920s, the mechanisms of BCG attenu-
ation remain largely unknown. This is further complicated by the
fact that BCG is not a single strain, but instead comprises a number
of substrains that exhibit phenotypic and biochemical differences.14
BCG strains also exhibit differences in residual virulence level.15-17
However, side effects were often attributed to variations in the
viability of vaccines during preparation procedures (e.g., freeze-
drying).14 In other words, the observed differential virulence among
Human Vaccines 2009; Vol. 5 Issue 2
 Attenuation of BCG and impact on efficacy
BCG strains is often thought to have originated from quality control
issues during manufacturing rather than reflecting true biological
differences. Although differential virulence has been suspected to
impact vaccine efficacy,18,19 definitive evidence, such as the direct
implication of established virulence factors and/or virulence genes,
has been lacking. Because of this, BCG strains are often considered
to possess equivalent vaccine properties and typically, only one BCG
strain is selected for vaccine studies that compare the safety and effi-
cacy of new vaccine candidates with BCG. However, in the past
decade, the advances in genomic techniques and knowledge of the
virulence mechanisms of M. tb have offered unprecedented opportu-
nities to re-evaluate these traditional assumptions. Indeed, our recent
studies have provided direct evidence that the distribution of major
mycobacterial virulence factors varies among BCG lineages,20,21 and
suggests that different BCG substrains have different mechanisms
of attenuation. This review article discusses these new developments
and their impacts on vaccine properties, including safety, immunoge-
nicity and protective efficacy. It is our belief that understanding the
attenuation of BCG strains can help explain the different outcomes
from clinical trial studies, and contribute to the rational design of
new, more effective TB vaccines.
BCG: A Brief History
The history of BCG has been described previously in several
excellent reviews12,22-26 and is only briefly summarized here. BCG
originated with lait Nocard, a virulent strain of M. bovis isolated
from the milk of a cow suffering from tuberculous mastitis. Around
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1901, this strain was brought to the Institute Pasteur in Lille,
France, and used by Albert Calmette and Camille Gurin for studies
of bovine tuberculosis. In order to minimize bacterial clumping
EN
and optimize animal infection experiments, Calmette added ox
bile, a detergent, to the glycerol-soaked potato slices on which the
M. bovis was cultured. Within a few months, an isolate with unusual
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colony morphology appeared. Moreover, this laboratory-adapted
strain exhibited reduced virulence in guinea pigs. Recognizing the
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implications of these observations in terms of vaccine development,
Calmette and Gurin continued the serial in vitro passaging of this
M. bovis strain for the next 13 years (19081921). During this time,
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experiments with diverse animal models, including guinea pigs,
rabbits, dogs, cattle, horses, chickens and non-human primates,
S
established both the safety and efficacy of BCG. When administered
at different doses and by different routes, BCG was well tolerated and
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failed to produce tuberculous lesions. Moreover, BCG vaccination
provided protection against a challenge with virulent strains. The
first human trial occurred in July 1921. An infant was given three
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2 mg doses (6 mg total; ~2.4 x 108 cfu) by the oral route.27 There
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were no deleterious side effects and, most importantly, the child did
not develop TB even though the infants mother had died of TB
shortly after giving birth. Over the next year, additional newborns
were vaccinated and no ill effects were reported. For the first time,
09
a safe and apparently effective vaccine was available for the
prevention of human TB.27
20
As early as 1924, cultures of BCG were distributed by the Institute
Pasteur to laboratories around the world.25 The implementation of
BCG varied from country to country, and several fascinating histo-
ries have been written about specific strains (e.g., BCG-Japan28 and
BCG-Moreau29). Because BCG is a live vaccine, it was necessary
www.landesbioscience.com Human Vaccines
to transfer cultures to fresh media every few weeks. Despite efforts
to standardize the growth and preparation of the vaccine, different
passaging conditions were used in different production laboratories
.
and the in vitro evolution of BCG continued. Dozens of distinct
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daughter strains emerged, including four that are currently in
major use:3 BCG-Pasteur (1173P2), BCG-Japan (Tokyo-172),
BCG-Danish (Copenhagen-1331) and BCG-Glaxo (1077).
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Mechanisms of Attenuation of BCG
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It is important to recognize that the currently available BCG
strains have undergone two phases of attenuation. The initial phase
(19081921) comprises the 230 in vitro passages conducted by
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Calmette and Gurin to produce the original vaccine. The second
phase starts circa 1924 with widespread use and distribution of BCG.
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It ends several decades, and hundreds of passages, later (1966 for
BCG-Pasteur 1173, but different years for different substrains) with
the establishment of frozen seed lots. Due to the initial phase, it is
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expected that BCG strains should share a set of common attenuating
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mutations, whereas the second phase should give rise to additional
mutations, specific to individual BCG strains and lineages. In other
words, the currently available BCG strains exhibit distinct mecha-
nisms of attenuation. They should not be viewed as a homogenous
O
entity but a collection of individual strains of varying virulence. For
.Dthis reason, it is essential to consider virulence, efficacy and mecha-
nisms of attenuation within the context of specific BCG strains. It
should also be emphasized that BCG is generally safe. Virulence is a
relative term that refers to properties that are quantifiable (e.g., bacte-
rial burden in an experimental model) but not necessarily harmful.
Attenuation of BCG Between 19081921
Early studies, using subtractive hybridization30 and spotted oligo-
nucleotide arrays,31 identified a Region of Difference-1 (RD1) that
is deleted in all BCG strains but present in virulent strains of M. tb
and M. bovis. The suggestion that the loss of RD1 contributes to
BCG attenuation has been confirmed by subsequent studies. RD1
encodes the ESX-1 protein secretion system, which is one of the
five type-VII secretion systems found in the M. tb genome.32,33
The RD1 locus of M. tb comprises nine genes (Rv3871-Rv3879c).
Rv3874 and Rv3875 encode two small, secreted, immunodominant
proteins: culture filtrate protein 10 (CFP-10)34 and the early secre-
tory antigenic 6-kDa (ESAT-6) protein,35 respectively. Export of
these two proteins is mediated by a secretory apparatus encoded by
adjacent genes in the RD1 locus, including at least one transmem-
brane protein (Rv3877) and two AAA-family ATPases (Rv3870 and
Rv3871).36 Several studies indicate that the secretion of ESAT-6 and
CFP-10 is required for the RD1-mediated virulence. Inactivation of
esat-6 and cfp-10, or of genes encoding components of the secretion
system resulted in impaired growth of M. tb in macrophages and an
attenuated phenotype in mouse models of infection, to an extent
similar to that noted after deletion of the entire RD1 locus.36-38 In
Mycobacterium marinum, a relative of M. tb that causes disease in fish
and amphibians, transposon insertions in various genes at or near
the RD1 locus also resulted in the loss of ESAT-6/CFP-10 secre-
tion, impaired growth of bacilli in macrophages, impaired ability to
prevent phagolysosomal fusion, and reduced bacterial virulence in a
zebrafish model of infection.39,40 Collectively, these studies provide
convincing evidence that the ESX-1 secretion system plays a major
71
 Attenuation of BCG and impact on efficacy

studies has been established (Fig. 1) and is gener-

ally consistent with the historical records of BCG
dissemination.23,31,43 For example, BCG strains

.
acquired after 1927, termed late strains, exhibit

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the RD2 deletion, while nRD18 is only deleted in
strains obtained after 1933. Some of the biochemical
differences among BCG substrains observed in early

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studies have now been confirmed and explained at
the molecular levels. For instance, protein antigens
MPB64, MPB70 and MPB83 were found to be at

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high levels in early strains (acquired before 1927)
but were absent or present only in trace quantities

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in late BCG strains (Table 1).14 The deletion of
RD2, which contains Rv1980c and encodes MPB64,
accounts for the lack of MPB64 in the late strains.

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Furthermore, the low level of MPB70 and MPB83
in late strains is caused by a regulatory effect due to a

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genetic mutation in sigK.49 A point mutation in the
mmaA3 gene50 explains why late BCG strains are

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defective in the production of a subclass of cell wall
Figure 1. Genealogy of BCG vaccines. Polymorphisms that affect known virulence genes are mycolic acids called methoxymycolate.14 The loss of
highlighted. the protein antigens (e.g., MPB64) likely affects the

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immunogenicity of late BCG strains, but the impact
role in virulence. Since it is common to all BCG strains, the loss of on vaccine properties have yet to be determined. Intriguingly, the loss
RD1 likely occurred in the initial stage of BCG attenuation.
.D
of methoxymycolate appears to have no impact on the virulence of
However, reintroduction of ESX-1 into BCG does not restore late BCG strains.51
full virulence41 and the RD1 deletion mutant of M. tb is still more PDIMs/PGLs. The direct evidence that BCG strains differ
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virulent than BCG in long-term murine infection experiments.42 in established virulence factors first came from the comparative
This suggests that additional genetic lesions contribute to the attenu- biochemical analysis of phthiocerol dimycocerosates (PDIMs) and
ation of BCG. Whole genome sequence comparison reveals 2,223 phenolic glycolipids (PGLs).21 PDIMs and PGLs are structurally
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single nucleotide polymorphisms (SNPs) between BCG-Pasteur related, methyl-branched fatty acid-containing complex lipids of
1173P2 and M. tb H37Rv, and 736 SNPs between BCG-Pasteur and mycobacterial cell wall. With the exception of M. gastri, PDIMs
M. bovis AF2122/97.43 Our own analysis, which used NimbleGen and PGLs are considered exclusive to pathogenic mycobacteria
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array technology to compare multiple BCG strains with M. tb including M. tb complex organisms (M. tb, M. bovis, M. microti,
H37Rv, revealed two types of SNPs. Most are common to the entire M. africanum), M. leprae, M. kansasii, M. marinum, M. ulcerans
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BCG lineage, but some SNPs are specific to individual BCG strains and M. haemophilum.52 The biosynthesis of PDIMs and PGLs has
(unpublished results). This suggests that loss of virulence during the been a subject of intensive studies in recent years,52 pioneered by
initial 230 in vitro passages of BCG involved both the loss of RD1 Kolattukudy and co-workers.53,54 The role of PDIMs and PGLs in
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and the accumulation of attenuating SNPs. virulence has now been well established.52,55 Both of these mole-
cules are thought to play a role in modulation of the host immune
Attenuation of BCG After 1924
response.56-58 Mutants of M. tb deficient in PDIM biogenesis are
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Dissemination of BCG to various parts of the world began in attenuated in animal models of infection56,59 and disruption of
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1924. By 1926, at least 34 countries had received cultures from the PDIM and PGL synthesis in M. bovis attenuates its virulence in
Pasteur Institute. In 1927, another 26 countries received cultures guinea pig models of infection.60 PGLs are found in a subset of M.
of BCG.25 Methods for propagation of the vaccine strains varied. tb isolates and confer a hypervirulent phenotype to the bacillus in
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Although subculturing on potato or Sauton media was common, animal disease models.57
deep-culture methods were also used.26 By the 1950s, numerous Analysis of 12 BCG strains reveals that BCG-Japan, BCG-Moreau
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vaccine producers recognized the emergence of BCG substrains with and BCG-Glaxo do not produce PDIMs and PGLs, whereas the other
distinct morphological, biochemical and immunological pheno- BCG strains do (Table 1).21 Intriguingly, variation in PDIM and PGL
types.19,44-46 Not until 1966, with the introduction of the seed-lot production does not coincide with the genealogy of BCG strains,43
09

system as part of the Requirements for Dried BCG Vaccine initiated suggesting that this particular phenotype has emerged multiple times
by WHO (WHO Expert Committee on Biological Standardization, and by multiple mechanisms. Indeed, in BCG-Moreau, the PDIM
20

1966), was lyophilization of BCG strains started, and the process and PGL defect is likely due to a 975 bp deletion that affects fadD26
of in vitro evolution halted. During the past decade, comparative and ppsA,20 which are part of the biosynthetic locus of PDIMs and
genome analyses of multiple BCG strains have uncovered extensive PGLs.20,52 This region is intact in BCG-Japan and BCG-Glaxo,20

genotypic diversity, including both deletions and duplications, in indicating that the PDIM/PGL defect in these two BCG strains is
BCG substrains.30,31,47,48 A molecular phylogeny based on these caused by other, currently unknown mutations.

72 Human Vaccines 2009; Vol. 5 Issue 2

 Attenuation of BCG and impact on efficacy
phoP-phoR. The PhoP-PhoR system is one of 11 two-compo-
nent systems found in the M. tb genome.61 The PhoR protein is
a transmembrane histidine kinase that transmits signals from the
environment. Autophosphorylation of PhoR is followed by transfer
of the phosphoryl group to PhoP, a response regulator that mediates
expression of multiple genes,62 including genes for the biosynthesis of
trehalose-containing cell wall lipids63-65 and ESX-1 secretion.66 A role
for phoP in virulence of the M. tb complex was first suggested during
a severe nosocomial outbreak of multidrug resistant TB in humans
in Spain.67 Unusually, the responsible clinical isolate was a strain of
M. bovis, termed B strain. Analysis of the B strain revealed an IS6110
insertion in the phoP promoter.68 This transposon was in the same
orientation as phoP and shown to increase the expression of the PhoP
regulon.67 Furthermore, subsequent studies have shown that strains
with defects in the PhoP regulon, such as a phoP mutant of M. tb, are
more attenuated than BCG-Pasteur in SCID mice infections.69 In
M. tb, defective biosynthesis of trehalose-containing lipids does not
explain the attenuated phenotype of the phoP mutant, but impaired
ESX-1 secretion might.63 In the avirulent lab strain, H37Ra, attenu-
ation is partially explained by a single point mutation (S219L) in the
DNA binding region of PhoP.66 H37Ra is also defective in PDIMs,
but this phenotype is not caused by the phoP mutation.63 Collectively,
these studies have demonstrated that the PhoP-PhoR system, particu-
larly PhoP, plays an essential role in M. tb virulence.62,65,66,69
Considering its prominent role in virulence, it may not be
surprising that BCG strains exhibit a number of genetic polymor-
phisms in the phoP-phoR locus.20 A frame-shift mutation within the
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phoP gene of BCG-Prague20 eliminates the majority of C-terminal
DNA binding domain, and makes this strain a natural phoP
mutant.70-72 Three early strains, BCG-Russia, -Japan and -Moreau,
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contain an IS6110 insertion in the promoter region of phoP,20 which
may eliminate the autorepression of phoP.70 BCG-Sweden and
BCG-Birkhaug contain a deletion that truncates the C-terminal of
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PhoR.20 In BCG-Danish, -Glaxo and -Frappier, frame-shift muta-
tions in the phoR gene abolish the PhoR protein.20
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whiB3 and trcR. Two closely related BCG strains, BCG-Sweden
and BCG-Birkhaug, contain deletions in whiB3 and trcR.20 These
deletions are not present in other BCG strains,20 and so, distinguish
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the BCG-Sweden, -Birkhaug lineage from other early strains
(e.g., BCG-Russia, -Japan and -Moreau).
The whiB gene was first identified in Streptomyces coelicolor and
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is essential for initiation of sporulation septation.73 Homologs
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of whiB have since been found in every actinomycete genome
sequenced to date,74 including non-sporulating organisms such as
mycobacteria. M. tb and M. bovis contain seven whiB-like genes
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(whiB1 to whiB7). It has been speculated that the persistence or
latency of M. tb is analogous to bacterial sporulation and that whiB
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genes may play a role in this process.74 Evidence obtained thus far
suggests that WhiB proteins are transcription factors involved in the
regulation of important mycobacterial cellular processes, including
09
cell division, pathogenesis, oxidative stress responses and antibiotic
resistance. The whmD of M. smegmatis, a functional homologue
20
of M. tb whiB2, is an essential gene and is required for proper
septation and cell division.75 Although disruption of whiB3 of
M. smegmatis has no effect on cell growth or stationary-phase
survival,76 deletion of whiB3 attenuates in vivo growth of M.
bovis in guinea pigs.77 The null mutant of whiB7 was found to be
www.landesbioscience.com Human Vaccines
Table 1Some biochemical characteristics of BCG vaccine
strains
.
BCG Strains MPB64 MPB70/MPB83 Methoxymycolate PDIMs/PGLs
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Russia + +++ + +
Japan + +++ + -
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Moreau + +++ + -
Sweden + +++ + +
Birkhaug + +++ + +
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Prague - + - +
Denmark - + - +
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Glaxo - + - -
Tice - + - +
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Frappier - + - +
Phipps - + - +
Pasteur - + - +
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+, present; -, not present; +++, high protein level.
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hypersensitive to several antibiotics.78 Transcriptional analysis of
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all whiB genes in M. tb showed differential expression patterns in
.Dresponse to different stress conditions.79 For example, WhiB3 was
shown to interact with SigA77 and responds to oxygen and nitric
oxide, and is important for regulation of carbon metabolism.80
TrcR is the response regulator of the TrcR-TrcS two-component
system. Characterization of these genes is ongoing, but deletion of
trcS from M. tb generates a hypervirulent phenotype such that the
strain exhibits increased lethality in SCID mice.81 This could stem
from dysregulation of the trcRS operon, reported to include the
mmpS5/mmpL5 (Rv0677/Rv0676) transporter and a bacteroferritin,
bfrB (Rv3841).82
Other deletions. Deletions affecting other genes with a potential
impact on virulence have also been found in certain BCG strains.
The M. tb genome contains four mce operons, each containing
912 genes that encode membrane permease subunits of ABC
transporters and putative secreted or cell-surface proteins.83 The
mce4C and mce4D genes are deleted in BCG-Frappier.48 Similarities
between the mce4 operon region and genes required for cholesterol
catabolism in Rhodococcus RHA1 was noted.84 Recently it was indeed
shown that the mce4 operon of M. tb encodes a cholesterol import
system, which appears to be important for persistent infection.85
The sigI gene encoded in nRD18 is deleted in all BCG
strains obtained after 1933 (i.e., BCG-Tice, -Frappier, -Phipps and
-Pasteur).48 SigI is an alternate RNA polymerase sigma factor present
in M. tb and M. bovis but is absent in other pathogenic mycobacteria
including M. marinum and M. avium.86 The role of SigI in virulence
of M. tb remains unknown. Other deletions include RD-Russia,
RD-Denmark/Glaxo as well as RD2.48 The impact of these deletions
on virulence of BCG strains, if any, remains unknown.
Correlation with Residual Virulence and Reactogenicity
Early studies of BCG (19211933) focused on vaccine safety and
efficacy.27 Research addressed questions of standardization, target
populations (groups at risk), routes of administration and the utility
of booster vaccinations. Issues related to the types of BCG strains
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 Attenuation of BCG and impact on efficacy

were of secondary concern.27 Over the following decades, numerous to induce adverse reactions than other BCG strains.14,89,90 For
researches showed that BCG indeed never reverted to pathogenicity instance, the risk of developing BCG-induced osteitis was 0.01
and the low virulence of existing strains was confirmed directly cases per million with BCG-Japan, but 32.5 and 43.4 cases per

.
through the vaccinations of humans. However, marked differences in million with BCG-Sweden as reported in Sweden and Finland,

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the residual virulence, immunogenic and sensitizing potency of BCG respectively.89 Osteitis media is a common complication after oral
strains were also demonstrated in animal models of infection,19,44 BCG vaccination. However, not a single case was reported in Brazil
which raised the concern that certain BCG strains might not be where oral administration of BCG-Moreau has been the traditional

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virulent enough to be effective in humans.15,19 As such, as part of method of vaccination.89 The link between type of BCG strains and
the Requirements for Dried BCG Vaccine (WHO Expert Committee incidents of complications are best demonstrated by the change of
on Biological Standardization, 1966), a number of experimental national immunization programs. In Finland, BCG-Sweden was

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investigations were specified for any strain of BCG used for vaccine used until 1978 when, due to the concern over very high incidents
production, including tests for infectivity and protective efficacy in of osteitis cases, it was replaced by BCG-Glaxo. An immediate

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animal models. In the following years, a series of studies comparing reduction of osteitis cases was observed after the policy changed89
different BCG strains was carried out by the WHO.15,16,87,88 and was confirmed in the follow-up studies in 1994.91 In 2002,
These studies used liquid cultures prepared by the same methods however, BCG-Glaxo was replaced by BCG-Danish in Finland and a

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(e.g., surface cultures on Sauton medium) and from established sharp rise in the lymphadenitis cases was noted.91 A similar observa-
frozen seed lots. Therefore results from these studies should remain tion was also made in the UK. When BCG-Glaxo was in use, cases

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valid for specified BCG strains. These studies revealed that, among of BCG-associated osteitis had never been reported in the UK.92
the 12 BCG strains examined, BCG-Japan, -Glaxo and -Prague However, an increase in BCG-induced suppurative lymphadenitis

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exhibited significantly lower virulence than other BCG strains was recently reported after the UKs immunization program switched
including BCG-Pasteur, -Russia, -Sweden and -Danish.15,16,87,88 BCG-Glaxo to BCG-Danish in 2002.93 Switching from BCG-Glaxo
When evaluated by different animal models, including golden to BCG-Pasteur in several national immunization programs was

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hamsters, bank voles and guinea pigs, related BCG strains tended to associated with reports of increased incidence of BCG-associated
exhibit a consistent level of virulence.15,16,87,88 However, an excep- lymphadenitis in late 1980s.14
tion concerned the BCG-Moreau lineage. Although both derived
.D In Poland, cases of osteitis media were reported when BCG-Pasteur
from BCG-Moreau, the exceptionally high virulence of BCG- or BCG-Danish was used (until 1955). They were no longer observed
Rio de Janeiro contrasts with the low virulence of BCG-Poland using when BCG-Moreau was used.89 Cases of osteitis were not reported
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the same animal model (e.g., golden hamsters).15,16 The heteroge- in Czechoslovakia between 19511980 when BCG-Prague was
neity of BCG-Moreau was noted,23 but has yet to be fully explained. used, but an immediate increase was observed following the switch
Behavior of the BCG-Poland strain used in the WHO studies is of BCG-Prague to BCG-Russia.89,94 Together, the accumulated
EN

consistent with that of real BCG-Moreau, which is traditionally evidence convincingly demonstrates that the discrepancies between
considered to be of particularly low virulence.16 There have been few national risks of BCG complications reflect true biological differ-
comparative studies of BCG strains in recent years. One study, in ences among BCG vaccine strains, not just technical differences of
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1996, compared five BCG strains in BALB/c mice and showed that national immunization programs, which is now widely recognized in
BCG-Japan and BCG-Prague were less virulent than BCG-Russia, the research community and regulatory authorities.14,90
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-Pasteur and -Glaxo.17 Furthermore, the clinical evidence is consistent with studies
The virulence of BCG strains can also be inferred from the from animal models of infection.15,16,87,88 Thus, BCG strains can
incidents of BCG-induced adverse reactions in humans. Although be viewed as two major groups that differ significantly in viru-
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BCG vaccines are generally considered very safe, they are also lence level, the more virulent ones represented by BCG-Russia,
among the most reactogenic vaccines in use today.89 Two common -Sweden, -Danish and -Pasteur, and the less virulent group including
complications following BCG vaccination of infants are regional BCG-Japan, -Moreau, -Glaxo and -Prague. The differential virulence
S

suppurative lymphadenitis and osteitis.14,89 Lotte et al. performed between these two major groups can now be explained at the molec-
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a thorough analysis of BCG-induced complications reported in ular levels. As discussed in the previous section, BCG-Japan, -Moreau
55 countries dated from 1921 to 1982. The 1,064 literatures and and -Glaxo do not produce PDIMs and PGLs.21 BCG-Prague
medical documents described 9,689 patients with 10,371 compli- is a natural phoP mutant.20 Given the well-established roles of
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cations.89 This extensive review and analysis revealed that there PDIM/PGL and phoP in the virulence of M. tb and M. bovis, there
were large discrepancies in the risks of BCG complications between is little doubt that these four BCG strains are more attenuated than
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countries.89 Although the impact of several factors, such as active other BCG strains, which is in a perfect agreement with the evidence
case finding, manufacturing technique and route of vaccination is from animal infections and clinical studies. Deletions of other poten-
important, evidence emerged from this analysis that these risks are tial virulence genes (e.g., whiB3, trcR, phoR, mce4) may further fine
09

also related to the strain of BCG used. Where oral BCG vaccination tune the virulence of individual BCG strains. However, their impacts
is used, BCG-Pasteur and BCG-Danish predominate in complica- on vaccine properties as manifested in animal and clinical studies
appear to be limited (Table 2).
20

tions due to suppurative lymphadenitis whereas BCG-Russia is

most common in osteitis media cases. For complications due to
Correlation with Immunogenicity and Protective Efficacy
scarification, multipuncture or intradermal inoculation, the most

frequently involved are BCG-Pasteur, -Danish and -Sweden. In The potency of a BCG vaccine is traditionally determined by
contrast, BCG-Japan, -Glaxo, -Moreau and -Prague are less likely measuring the tuberculin sensitivity (delayed type hypersensitivity,

74 Human Vaccines 2009; Vol. 5 Issue 2

 Attenuation of BCG and impact on efficacy

DTH) induced by the vaccine in children who were tuberculin- Table 2Deletions identified in BCG strains that affect
negative before vaccination.14 If the tested vaccine induces less currently known virulence factors
tuberculin sensitivity than that induced by other strains, it is

.
considered weak. The skin lesion or scar at the site of intrad- Deletions BCG strains affected

TE
ermal vaccination is also measured, and if the scar is too large, the fadD26-ppsA BCG-Moreau
vaccine is considered too strong to be acceptable to the popula- whiB3, trcR BCG-Birkhaug, BCG-Sweden
tion. Traditionally, tuberculin reactivity is considered a surrogate

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phoP BCG-Prague
marker for efficacy, but this view is not supported by epidemio-
phoR BCG-Sweden, -Birkhaug, -Danish, -Glaxo, -Frappier
logical studies95,96 and is further challenged by recent immunological
mce4 BCG-Frappier
studies,97,98 which show that tuberculin conversion and protective

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efficacy can be dissociated. Nevertheless, the traditional view, and All BCG strains contain the RD1 deletion, which contribute partially their attenuation. Additional deletions
selection for strains that maintain tuberculin reactivity has played that may affect virulence levels of specific BCG strains are listed here, for details see ref. 20.

ST
a major role in the history of BCG, including the choice of BCG
strains for national immunization programs. It is therefore important
to discuss findings from early studies on this subject and attempt to

DI
Successful protection against TB is dependent on T-cell mediated
provide a molecular basis for the tuberculin reactivity. immunity.102 However, reliable biomarkers that correlate immune
Differences in the ability to induce tuberculin reactivity among responses with protective efficacy are still lacking. Thus far, INF

T
BCG strains have been shown in both guinea pigs and children.88,99 production, mostly by CD4+ T cells from the peripheral blood, has
Of the 12 BCG strains examined, BCG-Prague consistently exhibits been the most widely used measure of protective immunity and

NO
significantly lower tuberculin reactivity than other BCG strains. vaccine efficacy. However, recent studies indicate a poor correlation
Because of this finding, BCG-Prague, which was apparently effective of this parameter and suggest that the host immune response to BCG
and used in Czechoslovakia between 19511980, was replaced by vaccination is far more complex.103,104 This deficiency likely explains

O
BCG-Russia in 1981.14 the discrepancy between observed immune responses and protective
The tuberculin reactivity does not appear to be simply correlated efficacy among BCG strains.17,100
with the virulence level of BCG strains. For instance, BCG-Japan,
.D Evidence for the relative protective efficacy of various established
considered to be the most attenuated,21 is also one of the strongest BCG strains is scarce and mostly indirect. Although reviews of BCG
inducers of tuberculin reactivity in children.99 Thus, the exception- strain history and protective efficacy indicate that vaccine strain is
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ally low tuberculin conversion of BCG-Prague is likely explained not a significant determinant of overall efficacy, it should be empha-
specifically by the phoP mutation. As described previously, PhoP sized that this conclusion is based on the limited data available from
regulates a number of genes. In the phoP deletion mutant of M. tb, human studies.24 Thus far, the only randomized trial study directly
EN

44 genes were repressed.62 It is possible that some of these are immu- comparing two different BCG strains was carried out in Hong Kong.
nogenic proteins, which contribute to tuberculin reactivity. Due to This study, which compared BCG-Pasteur and BCG-Glaxo among
the strains phoP mutation, the immunogenic proteins would not 300,000 infants, found that BCG-Pasteur, administered at a lower
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be expressed in BCG-Prague, thus eliminating the ability to induce dosage, provided a small (40%) but statistically significant greater
tuberculin reactivity. protection against childhood forms of TB over the subsequent six
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The association of immunogenic proteins and tuberculin conver- years than BCG-Glaxo.14 Since BCG-Pasteur is more virulent than
sion is also supported by studies of BCG-Tice. In a recent comparative BCG-Glaxo, there appears to be a positive correlation between the
study of BCG strains, including BCG-Pasteur, -Frappier, -Moreau, virulence level and protective efficacy of vaccine strains. A similar
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-Danish, -Sweden, -Connaught, -Birkhaug, -Phipps and -Mexico, correlation was observed in early animal studies19,105 and more
BCG-Tice exhibited the highest ability to induce tuberculin reac- recent work.17 In 1956, Rene Dubos wrote, when explaining the
tivity in mice.100 This may be explained by the presence of a unique observed differential ability of BCG substrains to multiply in mice:
S

genome duplication (DU-Tice). DU-Tice contains the entire ESX-5 even when all these variables are controlled and standardized as
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secretion system, which is one of the five type-VII secretion systems far as possible, there still remains to be consideredas shown by the
found in the M. tb complex.32 This includes several conserved present experimental studythe effect on behavior in vivo of some
membrane transporters (Rv1782, Rv1783, Rv1795 and Rv1797), intrinsic, hereditary properties of the substrains of BCG employed
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a membrane associated ATPase (Rv1784), a set of PE/PPE genes in the preparation of the vaccine. In order to avoid using the word
(Rv1787-Rv1792) and genes (esxM and esxN) encoding a pair of virulence with its unpleasant connotations, it will suffice for the time
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ESAT-6 and CFP-10 family proteins.32 Importantly, to date, ESX-5 being to regard these properties as conditioning the degree of inva-
is the only ESX system, other than the RD1-encoded ESX-1, that siveness of the culture.19 He further hypothesized that a not-too-low
has been shown to be involved in virulence.32 ESX-5 is conserved residual virulence is an important attribute of BCG: there is no
09

in other pathogenic mycobacteria and reported to facilitate the cell- doubt that immunity is an expression of the ability of the BCG
to-cell spread of M. marinum in infected macrophages.101 Although organisms to multiply in the body of the vaccinated individual. The
20

ESX-1 also exhibits cytolytic properties, ESX-5 does not comple- immunity is dependable and lasting if the BCG culture is endowed
ment the loss of virulence caused by ESX-1 deletion, suggesting that with great invasiveness, whereas it is at best weak and transit if the
they play distinct roles in virulence.32 The duplication of ESX-5 BCG culture has become very attenuated.19 Half a century later,

in BCG-Tice can be expected to increase immunogenicity and Dubos prediction on the genetic determinants of differential viru-
contribute to the high level of tuberculin conversion. lence of BCG strains has now been confirmed and the genetic factors

www.landesbioscience.com Human Vaccines 75

 Attenuation of BCG and impact on efficacy
determined.20,21 It is now time to formally test Dubos hypothesis on
the relationship of virulence and protective efficacy of BCG.
Impact on National Immunization Programs
The safety of BCG is a major concern in countries with a high
burden of HIV/AIDS. Recently the WHO revised its policy on BCG
vaccination for infants at risk of HIV infection.106 The WHO had
previously recommended that, in countries with a high burden of TB,
BCG vaccination should be given to all infants as soon as possible after
birth, unless the child presented with symptomatic HIV infection.3
However, retrospective studies of data from Argentina and South
Africa indicate that there is a substantially higher risk of disseminated
BCG disease in children infected with HIV.106 It was concluded
that, among these children, the benefits of potentially preventing
severe TB are outweighed by the risks of BCG vaccination. Based
on these considerations, the WHO changed its policy in 2007 and
now, recommends that children who are known to be HIV-infected,
even if asymptomatic, should not be immunized with BCG.106 One
concern of the revised WHO guideline is the difficulty for practical
implementation, i.e., the difficulty of diagnosis of infants infected
with HIV at birth.107 Another question that has not been addressed
in the WHO guideline is the impact of specific BCG strains on the
risk of BCG diseases in HIV-infected infants. In South Africa, the
increased risk of disseminated BCG disease in HIV-infected children
appears to be associated with the change of national vaccine policy
.D
from BCG-Japan to BCG-Danish in 2000.108 In Argentina, the
relatively reactogenic BCG-Pasteur has been used since 1984,109
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which may account for the high risk of BCG disseminated disease
in HIV-infected children. By contrast, a recent study from Brazil,
where BCG-Moreau is being used, found no cases of disseminated
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BCG disease in children exposed to or infected with HIV.110 The
differential virulence levels of BCG strains used in these countries
could explain such discrepancy (BCG-Japan and BCG-Moreau are
CI
more attenuated than BCG-Pasteur or BCG-Danish). Given that
populations with a high prevalence of HIV also have the greatest
OS
burden of TB, the difficulty in identifying infants infected with HIV
at birth, and the evidence that BCG strains clearly differ in their
virulence, the selection of specific BCG strains should be taken into
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consideration for national immunization programs in countries with
a high burden of HIV/AIDS.
Conclusion
S
DE
Exactly 100 years ago, Albert Calmette and Camille Gurin began
a daunting task, which lasted 13 years and is unmatched even today,
that led to the most widely used vaccine in human history. Despite
N
a century of scientific advances, BCG remains the only vaccine for
prevention of TB. Shaped by human history, BCG has also evolved,
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from the virus fix declared by Calmette in 1921, to the numerous
daughter strains recognized today. These BCG strains differ, not only
genetically and phenotypically, but also in their vaccine properties,
09
including tuberculin reactivity, protective efficacy and propensity for
adverse effects. We are just beginning to understand the molecular
20
mechanisms that influence vaccine properties. It is remarkable that
the current BCG strains comprise natural mutants of well-recognized
virulence factors (e.g., ESX-1, PDIM/PGL, phoP). On the other
hand, given ~50 years of in vitro evolution with selection for safe
strains and limited adverse effects, it is not surprising that the key
76 Human Vaccines
determinants of mycobacterial virulence were compromised. There
is little doubt that BCG will continue to play a role in TB control.
It remains part of forthcoming clinical trials, as either a primer to be
.
boosted by new components (e.g., subunit or DNA vaccine) or as
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an integral component (e.g. recombinant BCG) of new vaccines.111
Continuing studies on the molecular factors that impact properties
of the vaccine strains will provide additional insights into virulence
BU
mechanisms of M. tb, inform the rational design of better vaccines,
and as BCG was originally designed to do, contribute to the elimina-
tion of tuberculosis.
RI
Acknowledgements
ST
Work in the authors laboratory is supported by an award from
the National Natural Science Foundation of China (NSFC) (to J.L.),
and research grants from Canadian Institutes of Health Research
DI
(CIHR) (MOP-15107 and MOP-82772 to J.L.).
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