INFECTION AND IMMUNITY, June 2007, p. 3197–3201
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 6
Strain-Dependent Variation in Mycobacterium bovis BCG-Induced
Human T-Cell Activation and Gamma Interferon
Production In Vitro?
Ana M. Aguirre-Blanco,1† Pauline T. Lukey,2Jacqueline M. Cliff,1* and Hazel M. Dockrell1
Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom,1
and GlaxoSmithKline R&D, Stevenage, United Kingdom2
Received 5 October 2006/Returned for modification 18 December 2006/Accepted 14 March 2007
Three commonly used Mycobacterium bovis BCG vaccine strains elicited different magnitudes of T-cell
activation and gamma interferon production in vitro in healthy BCG-vaccinated individuals. Glaxo 1077
exhibited the greatest stimulatory capacity, followed by Pasteur 1173 and then Danish 1331. These differences
may affect in vitro stimulation and vaccination-induced immunogenicity.
Mycobacterium bovis bacillus Calmette-Gue ´rin (BCG) is the
only vaccine currently available to prevent tuberculosis. It is
administered to over 100 million people per year (25), al-
though its efficacy against pulmonary TB worldwide is variable
(11). M. bovis BCG is an attenuated form of M. bovis obtained
by 13 years of serial passage in vitro, following which it was
found to provide protective immunity to challenge with viru-
lent M. tuberculosis (3). M. bovis BCG was maintained by a
process of serial passage for 40 years before methods for stable
long-term storage became available, and this culminated in a
family of approximately 13 daughter strains which are collec-
tively known today by the generic term BCG. These strains
have been shown to differ genetically (2, 5, 6, 22) and pheno-
typically (1, 14, 20, 21), including their induction of immune
responses and protection in animal models (16, 18). Three
strains represent nearly 60% of the total number of the BCG
doses currently produced per year (12): BCG Danish 1331 is
currently in use in the United Kingdom vaccination program,
although BCG Glaxo 1077 was used until 2002. BCG Pasteur
1173 is the BCG reference strain lyophilized at the Institute
Pasteur in 1961.
Despite the numerous differences previously found among
BCG strains, and despite BCG stimulation being routinely
used to study mycobacterium-specific immune responses, T-
cell responses elicited by different BCG strains have not been
compared in human cultures and were therefore investigated
in this study.
The BCG vaccine strains Danish 1331, Pasteur 1173, and
Glaxo 1077 were grown in liquid cultures of Middlebrook 7H9
medium (Difco) supplemented with 10% Bacto Middlebrook
ADC (Scientific Laboratory Supplies) and 0.025% Tween 80
(Difco), quantified by growth on Middlebrook 7H10 agar
plates for 3 weeks, and kept frozen at ?80°C for long-term
storage. All three strain preparations were found to be 90 to
95% viable, as determined by fluorescein diacetate/ethidium
bromide labeling. Peripheral blood mononuclear cells (PBMC)
were isolated by density gradient centrifugation from seven
healthy BCG-vaccinated donors, at least five of whom were
likely to have been vaccinated with Glaxo 1077, and were
cultured for 6 days either in the absence of antigenic stimula-
tion or in the presence of one of the live M. bovis BCG strains
titrated between 0.01 and 1 CFU per PBMC: if monocytes are
estimated to constitute 10% of total PBMC, this is the equiv-
alent of multiplicity of infection (MOI) ratios (M. bovis BCG
bacilli to monocytes) of 0.1:1 to 10:1. For flow cytometry, cells
were stained with fluorescently labeled monoclonal antibodies
against CD3 (allophycocyanin conjugated), CD8 (peridinin
chlorophyll protein conjugated), and CD25 (fluorescein iso-
thiocyanate conjugated), fixed and permeabilized using re-
agents purchased from Caltag, and then stained intracellularly
for IFN-? (phycoerythrin conjugated). Antibodies were all
from BD Biosciences (Cowley, United Kingdom). Flow cyto-
metric data were acquired (40,000 events within lymphocyte
gate) using a FACSCalibur flow cytometer (BD Biosciences)
and analyzed using CellQuest (BD Biosciences). CD3?CD8?
cells were 95% CD4?and are referred to herein as CD4?T
cells. Ethical permission was obtained from the London School
of Hygiene & Tropical Medicine Ethics committee, and written
informed consent was gained from the donors.
T cells were activated by in vitro stimulation with all three
strains of BCG. A progressive increase of both light scatter
parameters was observed as the dose of M. bovis BCG in-
creased (Fig. 1, left). Expression of the activation marker
CD25 also increased gradually with BCG dose (Fig. 1, right).
These data are concordant with numerous previous studies (8,
10, 19, 23, 24). The three BCG strains tested differed signifi-
cantly in the percentage of activated CD25?T cells they elic-
ited in vitro. Danish 1331 induced the lowest activation re-
sponse overall, whereas Glaxo 1077 induced the highest, with
statistically significant increases at most doses in both the
CD4?and CD8?T-cell populations (Fig. 2A and B). The
average rank of CD25?response for each strain for each
donor was determined across the BCG doses, and the overall
difference between Glaxo 1077 and Danish 1331 was highly
* Corresponding author. Mailing address: Department of Infectious
and Tropical Diseases, London School of Hygiene & Tropical Medi-
cine, Keppel Street, London WC1E 7HT, United Kingdom. Phone:
44(0)207 612 7833. Fax: 44(0)207 637 4314. E-mail: jackie.cliff@lshtm
† Present address: Institute of Cell and Molecular Science, Queen
Mary’s School of Medicine and Dentistry, London, United Kingdom.
?Published ahead of print on 26 March 2007.
significant (P ? 0.001316, paired Student’s t test), as was the
difference between Glaxo 1077 and Pasteur 1173 (P ? 0.000121),
within the CD4?-T-cell compartment. The proportions of
CD8?T cells which were CD25?were also significantly higher
with Glaxo 1077 stimulation than with Danish 1331 stimulation
(P ? 0.00028) in the BCG strain rank analysis. Interestingly,
statistical significance (P ? 0.013853) was observed between
Pasteur 1173 and Glaxo 1077 only when the lowest three doses
were included, and the effect was diminished with inclusion of
the higher doses. Similarly, the BCG strains varied in their
induction of IFN-? expression. At a low dose, Glaxo 1077
elicited the highest number of IFN-?-producing T cells and
Danish 1331 and Pasteur 1173 induced lower responses (Fig.
3A and B).
Activation and IFN-? production showed a trend to plateau
with increasing MOIs of BCG, in both the CD4?- and CD8?-
T-cell compartments. At the intermediate dose of 0.1 CFU/
PBMC, similar IFN-? responses were elicited by all three BCG
strains (Fig. 3C and D), and responses to each strain reached
a similar maximum level. This suggests that with higher MOIs,
the BCG-specific immune response is saturated but that other
factors might be critical at lower doses when differences be-
tween strains are more pronounced.
Glaxo 1077 and Pasteur 1173 have been found to provide
higher levels of acquired protection than Danish 1331 and
Japanese strains in mice (13), although another study did not
find a significant difference between Glaxo and Pasteur strains
(9). Comparing vaccination studies in humans is difficult, since
not only have they used different BCG strains but also most
were carried out in different geographical locations and pop-
ulations. At least five of the donors studied here were likely to
have been immunized with Glaxo 1077. When Danish 1331 and
Glaxo 1077 were compared in the United Kingdom during the
1960s and 1970s, no significant differences in protective efficacy
FIG. 1. Scatter characteristics and CD25 expression in PBMC cultures stimulated with live M. bovis BCG. Cultured cells are shown according
to both forward and side light scatter parameters (left). Representative examples from one of the donors tested when cells were cultured in the
absence of stimulation (A, B) or in the presence of titrated amounts of Danish 1331 (C to F) are shown. CD3?cells were selected within the
live-lymphocyte gate (R1). Expression of the activation marker CD25 is shown for the CD3?CD8?T-cell population (right), with the numbers
showing the percentage of CD8?T cells which were activated.
3198 NOTESINFECT. IMMUN.
were found (7), although the Danish strain gave consistently
higher mean tuberculin skin test reactions and greater vacci-
nation scar sizes (17). In more recent studies carried out with
United Kingdom schoolchildren, Danish 1331 induced consis-
tently lower skin test reactions, scar sizes, and IFN-? produc-
tion in a whole-blood assay than Glaxo 1077, although these
differences were not statistically significant (15). Thus, al-
though there is no epidemiological evidence for strain differ-
FIG. 2. CD25 expression induced in vitro by different BCG strains in CD4?and CD8?T cells. Percentages of CD4?(A) and CD8?(B) T cells
expressing the activation marker CD25, as a result of in vitro BCG stimulation, are shown. The horizontal axis is divided into the five BCG doses
tested. Within each of the five doses, the results for each of the three BCG strains tested are shown: Danish 1331 (Dan., black), Pasteur 1173 (Past.,
gray) and Glaxo 1077 (Glaxo, white). Lines within the boxes represent median values. Upper and lower hinges of the boxes represent 75th and
25th percentiles, respectively. Adjacent lines represent the adjacent values, and dots represent outliers. Statistically significant differences (P ?
0.05) and borderline differences (0.05 ? P ? 0.1) between strains are indicated by their Wilcoxon signed-rank test P values.
VOL. 75, 2007 NOTES3199
ences in the induction of protection against tuberculosis infec-
tion and no evidence of genetic differences between the Glaxo
and Danish strains (6), the BCG strain used for vaccination
may affect the extent of the BCG-induced immune response
Interestingly, an inverse relationship between passage num-
ber before cryopreservation of seed lot (given as the strain
number) and activation response was suggested in this study.
These results fit with the observation that laboratory adapta-
tion of bacteria is associated with attenuation of virulence, and
the lower T-cell immunogenicity of Danish 1331 might be a
consequence of this attenuation. A relationship between in
vitro passage number and vaccine efficacy was suggested pre-
viously (4). Another factor that might affect T-cell activation is
the conditions of BCG culture: we chose to passage all three
strains in the same ADC-supplemented Middlebrook 7H9
broth to minimize this effect on our T-cell stimulation assays.
The number of dead bacilli in BCG preparations may also
affect CD4?-T-cell responses, but their effect on CD8?T cells
would be less pronounced (24).
In conclusion, different BCG vaccine strains elicited quan-
titatively different T-cell responses in human in vitro assays
when healthy BCG-vaccinated individuals were tested. The
magnitude of T-cell responses, measured as the proportion of
T cells that expressed the activation marker CD25 and IFN-?
in both CD4?and CD8?T cells, elicited by the three BCG
strains tested was ranked in decreasing order as follows: Glaxo
1077, Pasteur 1173, and Danish 1331. These and other obser-
vations suggest that there may be strain-dependent differences
between commonly used BCG vaccines which may affect the
immune responses they induce in vitro and those they induce
in vivo following immunization.
This work was supported by an Action TB grant from GlaxoSmith-
We thank Carolynne Stanley for phlebotomy assistance and Sian
Floyd for statistical advice.
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Editor: J. L. Flynn
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