Page 1 of 41 AJRCCM Articles in Press. Published on November 3, 2011 as doi:10.1164/rccm.

201104-0714OC

The influence of BCG vaccine strain on the immune response against

tuberculosis: a randomised trial

Nicole Ritz 1,2, Binita Dutta 2, Susan Donath 1,2,3, Dan Casalaz 4, Tom G. Connell 1,2,

Marc Tebruegge 1,2, Roy Robins-Browne 1,2,5, Willem A. Hanekom 6, Warwick J.

Britton 7, Nigel Curtis 1,2

1
Department of Paediatrics, The University of Melbourne, Australia;
2
Murdoch Children’s Research Institute, Australia;
3
Clinical Epidemiology and Biostatistics Unit, The Royal Children’s Hospital

Melbourne, Australia;
4
The Mercy Hospital for Women, Australia;
5
Department of Microbiology and Immunology, The University of Melbourne,

Australia;
6
South African Tuberculosis Vaccine Initiative, Institute of Infectious Disease and

Molecular Medicine and School of Child and Adolescent Health, University of Cape

Town, South Africa;

7
Centenary Institute of Cancer Medicine and Cell Biology and Department of

Medicine, University of Sydney, Australia.

Copyright (C) 2011 by the American Thoracic Society.

 Page 2 of 41

Corresponding Author:

Professor Nigel Curtis

Professor of Paediatric Infectious Diseases, Department of Paediatrics, University of

Melbourne & Head of Infectious Diseases Unit, Department of General Medicine,

Royal Children’s Hospital Melbourne

Flemington Road, Parkville VIC 3052, Australia

Tel: + 61 3 9345 4545

Fax: + 61 3 9345 6667

Email: nigel.curtis@rch.org.au

Key words

Tuberculosis, BCG, immune response, protection, strain, vaccine

Running title: Immune response to different BCG vaccine strains

Word count: 3190

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At a Glance Commentary

Scientific knowledge on the subject:

More than 100 million doses of Bacille-Calmette-Guérin (BCG) vaccine are given

each year to protect against tuberculosis (TB). At least 20 genetically-distinct BCG

vaccine strains are currently in use worldwide. It is unknown which BCG vaccine

strain provides best protection against TB. Differences in the immune response

induced by different BCG vaccine strains have not been investigated in detail.

What this study adds to the field: This first randomised study comparing the three

most commonly-used BCG vaccine strains shows there are significant differences in

the immune response induced by different BCG vaccine strains in newborn infants.

Immunisation of infants with BCG-Denmark or BCG-Japan induced higher

proportions of mycobacterium-specific polyfunctional and CD107-expressing

cytotoxic CD4 T cells and higher concentrations of Th1 cytokines than immunisation

with BCG-Russia.

Author contributions:

NR – was involved in the design of the study, co-ordinated the study, recruited

participants, reviewed all participants on follow-up, analysed the data and wrote the

first draft of the manuscript.

BD – did the majority of the in vitro assays and flow-cytometric analysis.

SD – was involved in the design of the study and did the majority of the statistical

analysis of the results.

DC and TC – were involved in the design of the study and contributed to the analysis

of the results and writing of the final manuscript.

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MT – was involved in the recruitment of participants and contributed to the analysis of

the results and writing of the final manuscript

RR, WH, WB were involved in the design of the study, and provided intellectual input

for the in vitro assays, the analysis and the writing of the final manuscript.

NC – initiated the study, was involved in the design of the study, supervised the study

co-ordination, was involved in the analysis and interpretation of the results, and made

a substantial contribution to the writing of the manuscript.

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Support

NR and MT are supported by Fellowship awards from the European Society for

Paediatric Infectious Diseases and scholarships from The University of Melbourne.

The study was funded by a project grant from the Australian National Health and

Medical Research Council (NHMRC grant number 546486). It was also supported by

grants from the John Burge Trust, the Myer Foundation, the Aranday Foundation, the

Nossal Institute for Global Health and the Murdoch Children’s Research Institute. The

BCG vaccines were provided by their manufacturers.

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ABSTRACT

Rationale: Approximately 100 million doses of Bacille-Calmette-Guérin (BCG)

vaccine are given each year to protect against tuberculosis (TB). More than 20

genetically-distinct BCG vaccine strains are in use worldwide today. Previous studies

suggest that BCG vaccine strain influences the immune response and protection

against TB. Current data is on which BCG vaccine strain induces the optimal immune

response in humans is insufficient.

Objectives: To compare the immune response to three different BCG vaccine strains

given to infants at birth.

Methods: Newborn infants in a tertiary women’s hospital were immunised at birth

with one of three BCG vaccine strains. A stratified randomisation according to the

mother’s region of birth was used.

Measurements and Main Results: The presence of mycobacterial-specific

polyfunctional CD4 T cells measured by flow cytometry 10 weeks after immunisation.

Of the 209 infants immunised, data from 164 infants were included in the final

analysis (54 BCG-Denmark, 54 BCG-Japan, 57 BCG-Russia). The proportion of

polyfunctional CD4 T cells was significantly higher in infants immunised with BCG-

Denmark (0.013%) or BCG-Japan (0.016%) than with BCG-Russia (0.007%)

(p=0.018, p=0.003 respectively). Infants immunised with BCG-Japan had higher

concentrations of secreted Th1 cytokines; infants immunised with BCG-Denmark had

higher proportions of CD107-expressing cytotoxic CD4 T cells.

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Conclusion:

There are significant differences in the immune response induced by different BCG

vaccine strains in newborn infants. Immunisation with BCG-Denmark or BCG-Japan

induced higher frequencies of mycobacterial-specific polyfunctional and cytotoxic

T cells and higher concentrations of Th1 cytokines. These findings have potentially

important implications for global anti-tuberculosis immunisation policies and future

tuberculosis vaccine trials.

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BACKGROUND

More than 100 million doses of Bacille-Calmette-Guérin (BCG) vaccine are given

worldwide each year to protect children against tuberculosis (TB). BCG was

developed by attenuation through repeated in vitro passage of Mycobacterium bovis.

Following the first use of BCG in humans in 1921 in France, the vaccine was

distributed worldwide. Subsequently, culture under dissimilar conditions led to the

evolution of genetically-different BCG vaccine strains (1). More than 20 different

BCG vaccine strains are currently in use worldwide (2). However, the majority of the

world’s population is supplied with BCG vaccine procured by The United Nations

Children's Fund (UNICEF) on behalf of the Global Alliance for Vaccines and

Immunization (GAVI). UNICEF uses only four BCG vaccine suppliers who in turn

produce only three different BCG vaccine strains: BCG-Denmark produced by the

Statens Serum Institute in Denmark, BCG-Russia (genetically identical to BCG-

Bulgaria) produced by Bulbio (BB-NCIPD) in Bulgaria and by the Serum Institute in

India, and BCG-Japan produced by the Japan BCG Laboratory.

A recent systematic review showed that in animal models the specific BCG vaccine

strain used for immunisation influences the induced immune response, but there are

conflicting data about which strain is associated with best protection against TB (3). In

humans, there have been three studies investigating protective efficacy induced by

different BCG vaccine strains. In two studies (with between four and 50 years follow-

up), BCG-Pasteur was associated with statistically significantly better protective

efficacy than BCG-Phipps or BCG-Glaxo (4, 5). In the third study (with 15 years

follow-up), BCG-Denmark had a greater protective efficacy than BCG-Pasteur (25%

and 17%, respectively) (5, 6). Unfortunately, these studies give only limited

information about protective efficacy afforded by the BCG vaccine strains currently
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most commonly used, as BCG-Phipps and BCG-Glaxo are no longer in use and BCG-

Pasteur is used in only very few countries.

Studies in humans comparing the immune response induced by different BCG vaccine

strains have also been hampered by a number of limitations. Only five studies have

investigated the immune response after BCG immunisation with different BCG

vaccine strains (7-11). However, these studies either were not in infants (7) , not

randomised (8-11) , compared BCG vaccine strains no longer commercially produced

(8, 11) or which are currently used in only a few countries (7), or did not include

immunological parameters that are now believed to correlate with protection against

TB (7-11).

As a result, it is unknown to what extent genetic differences between BCG vaccine

strains influence the mycobacterial-specific immune response induced in humans.

There are therefore insufficient data to favour or recommend any one BCG vaccine

strain over another.

Identifying the optimal BCG vaccine strain has major implications. Firstly, given the

large population of infants receiving BCG vaccine each year, even a small increment

in protective immunity resulting from the use of a particular BCG strain would

translate into improved protection against TB for a large number of children.

Secondly, a range of new TB vaccines are under development, including vaccines

designed to replace BCG and vaccines designed to boost BCG (12). The most

advanced are subunit or live vector-based booster vaccines designed for use after

administration of a priming dose of a current BCG vaccine. It therefore remains

important to determine which BCG vaccine strain induces the best primary immune

response against TB for subsequent boosting.

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Despite increasing evidence and awareness that different BCG vaccine strains induce a

different immune response, to date, there has been no randomised study in infants

comparing different BCG vaccine strains. The aim of this study was to compare the

immune response to different BCG vaccine strains given to infants at birth.

Some of the results from this study have been reported in the form of an abstract (13).

METHODS

Participants

Pregnant women attending the antenatal clinic at the Mercy Hospital for Women in

Melbourne, Australia were recruited between March 2008 and April 2009. Families

were eligible if one of the parents was born in a country with a high TB incidence

(defined as more than 100 cases per 100 000 inhabitants) and travel to their country of

origin in the next five years was planned. This identified infants at increased risk for

TB, for whom BCG immunisation is recommended by the Australian Immunisation

Guidelines (14). Exclusion criteria included mothers known to be infected with human

immune deficiency virus (HIV) or with Mycobacterium tuberculosis, premature birth

(less than 35 weeks of gestation), birth weight below 2500 grams, and any symptoms

or signs of illness. The study was approved by an independent local ethics committee

(the Mercy Health Human Research Ethics Committee) and as a clinical trial by the

Australian Therapeutic Goods Administration (TGA). The study was done in

accordance with the ethical principles of the Declaration of Helsinki. Written informed

consent was obtained from the mother or both parents. This trial is registered with the

Australian New Zealand Clinical Trials Registry, number ACTRN12608000227392.

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Vaccines

The BCG vaccine strains were all kindly supplied by their respective manufacturers:

BCG-Denmark, SSI-1331, 2 - 8 x 106 CFU/vial (Statens Serum Institute, Copenhagen,

Denmark); BCG-Japan, Tokyo-172, 30 x 106 CFU/vial (BCG Laboratory, Tokyo,

Japan); BCG-Russia, SL-222, 4 x 106 CFU/vial (Bulbio, Sofia, Bulgaria). All BCG

vaccines were transported at 2 to 8 °C with a documented unbroken cold chain and

stored in a temperature-monitored refrigerator until use.

Randomisation and masking

Once the baby was born confirmation of the consent to take part in the study was

obtained from the parents before randomisation. Randomisation was stratified by

parental region of birth (Indian subcontinent, East Asia or Africa). Mother’s region of

birth was used if the mother was from a high TB prevalence country, and if not,

father’s region of birth was used. Allocation to vaccine was determined by selecting a

random opaque envelope from the appropriate stratum. BCG vaccine was given at a

dose of 0.05 ml injected intradermally into the left deltoid region using a 26-gauge

needle. BCG immunisation was given as soon as possible after birth but always within

the first week of life. Parents and outcome assessors of data were blinded to group

assignment. The research nurse administering the vaccine and the laboratory scientist

doing the in vitro assays were not blinded. The primary outcome was the proportion of

mycobacterial-specific single, double and triple cytokine-producing T cells 10 weeks

after immunisation measured by flow cytometry. Secondary outcomes were the

proportion of mycobacterial-specific cytotoxic T cells and concentrations of secreted

cytokines in supernatants.

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Procedures

Ten weeks after BCG immunisation, up to 6 ml of blood were collected in sodium

heparin tubes for in vitro assays. All stimulation assays were done within two hours of

blood collection. The local reaction at the BCG injection site was assessed and

measured. A normal reaction was defined as an induration and/or redness with a

diameter of less than 10 mm.

To determine cell-associated cytokine production, blood was incubated with BCG (1.6

x 106 CFU/ml of the same strain that was used for immunisation, reconstituted from a

vaccine vial with Roswell Park Memorial Institute medium), purified protein

derivative (PPD, Batch RT50, Statens Serum Institute, Copenhagen, Denmark; 20

µg/ml), heat-killed M. tuberculosis (H37Rv, 1.6 x 106 CFU/ml), staphylococcal

enterotoxin B (SEB (positive control; 10 µg/ml; Sigma-Aldrich, St. Louis, MO, USA),

or media alone (negative control) in the presence of co-stimulatory antibodies CD28

and CD49d (1 µg/ml each; both from BD Biosciences, San Jose, CA, USA) at 37 °C

for 7 hours. After removal of plasma for cryopreservation, the remaining blood was

incubated for a further 5 hours with brefeldin A (BfA; 10 µg/ml; Sigma-Aldrich). The

blood was harvested using 2 mM EDTA (Sigma-Aldrich) and cells were lysed and

fixed in FACS lysing solution (BD Biosciences) before cryopreservation at -80 °C.

To determine cell-associated cytotoxicity, blood was incubated with the same antigens

and co-stimulatory antibodies at 37 °C for 7 hours. The blood was then incubated for a

further 5 hours with BfA (5 µg/ml), monensin (5 µg/ml; Sigma-Aldrich) and anti-

CD107a-APC (10 µg/ml) before harvesting and cryopreservation. Cytotoxic T cells

contain CD107-coated granules containing perforin and granzyme; and therefore

CD107 expression is a measure of cytotoxic capacity (15).

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For the flow cytometric analysis of cytokine-producing and cytotoxic mycobacterial-

specific T cells, thawed samples were permeabilised with Perm2 Solution (BD

Biosciences) for 10 minutes in the dark. Cells were then washed with PBS/BSA/NaN3

staining buffer before incubation with fluorochrome-conjugated antibodies for 30

minutes in the dark. Cytokine profiles and cytotoxicity of mycobacterial-specific T

cell subpopulations were measured by flow cytometric analysis on a LSRII flow

cytometer (BD Biosciences) using the following fluorochrome-conjugated antibodies

(all from BD Biosciences): anti-CD3 PerCP-Cy5.5 (SK7), anti-CD4 FITC (RPA-T4),

anti-CD8 Alexa-700 (RPA-T8), anti-IFN-γ PE-Cy7 (4S.B3), anti-IL-2 PE (MQ1-

17H12) and anti-TNF APC (Mab11), and anti-CD107a APC (H4A3). Settings of the

flow cytometer including PMT voltages were optimised and standardised using CST

beads (BD Biosciences). Automated compensation was calculated with FACSDiva

software (version 6.1, BD Biosciences, San Jose, CA, USA) using stained anti-mouse

and anti-rat Ig kappa beads. Flow cytometric analysis was done using FlowJo software

(version 8.8.6, TreeStar Inc, Ashland, OR, USA). A hierarchical gating strategy was

used to select single cell CD4 and CD8 T cell populations. Gates for cytokine

expression from blood stimulated with mycobacterial antigens and SEB were set using

the unstimulated control. A Boolean combination was used to determine

polyfunctional T cells producing more than one cytokine, ie double-positive and triple-

positive populations.

Concentrations of cytokines and chemokines in supernatants were measured using

xMAP technology (Luminex 200, Luminex Corp. TX, USA) with human cytokine kits

(Milliplex Human Cytokine/Chemokine Immunoassay, Millipore Corp, Billerica, MA,

USA) for 12 cytokines/chemokines according to manufacturer’s instructions. EGF,

eotaxin, fractalkine, IL-2, IL-10, IL-12 (p40), IL-13, and IFN-γ were analysed in

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undiluted samples and IL-6, MCP-1, MIP-1β and TNF-α were analysed in 1:20 diluted

samples based on previous optimisation experiments.

Cryopreserved supernatants from unstimulated samples were used to determine 25-OH

vitamin D3 using the Roche Elecsys vitamin D3 assay on a Cobas e601 immunoassay

analyser (Roche Diagnostics, Basel, Switzerland).

Statistics

A sample size of 64 participants in each group was calculated to have 80% power

using a two group t-test with a 0.05 two-sided significance level to detect a difference

of at least 0.5 standard deviation (SD) (a moderate effect size (16)) between mean

proportions of cytokine-producing cells in any two of the study groups. Based on a SD

from a previous study of < 0.53% for the proportion of IFN-γ-producing CD4 and

CD8 cells (9), this implied a change in the proportion of cytokine producing T cells of

0.25% could be detected.

All data were analysed after background correction. A Kruskal-Wallis test was used

for overall comparisons between the three groups. If the p-value of the Kruskal-Wallis

test was < 0.1, further comparisons between each two of the three groups were done

using a Mann-Whitney test. For the Mann-Whitney test, a p-value < 0.05 was

considered potentially significant but was interpreted in conjunction with other data to

account for multiple testing. Statistical analysis was done using STATA 11 software

(College Station, TX, USA). Graphs were created using Prism 5 software (Graph Pad

Software Inc., La Jolla, CA, USA).

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Role of the funding source

The sponsor of the study had no role in study design, data collection, data analysis,

data interpretation or writing of the report. The corresponding author had full access to

all the data in the study and had final responsibility for the decision to submit for

publication.

RESULTS

Of 356 infants born during the study period, 209 were randomly assigned to be

immunised at birth with one of the three BCG vaccine strains (Figure 1). 176 (84%)

returned for a follow-up visit and data from 165 (79%) infants were included in the

final analysis. The demographic and baseline characteristics for the three groups were

comparable (Table 1).

Figure 2 compares the proportions of single, double and triple cytokine-producing

CD4 T cells in the three groups. Proportions of polyfunctional CD4 T cells (those

simultaneously producing three of the measured cytokines) were higher in infants

immunised with BCG-Denmark or BCG-Japan than in infants immunised with BCG-

Russia (Figure 2, panel C). IFN-γ/TNF-α double cytokine-producing CD4 T cells were

higher in infants immunised with BCG-Denmark or BCG-Japan than in infants

immunised with BCG-Russia, however this reached statistical significance only when

BCG or PPD were used as in vitro stimulatory antigens (Figures 2, 4 and 6). Similarly,

proportions of IFN-γ/IL-2 double cytokine-producing CD4 T cells were higher in

infants immunised with BCG-Denmark than in infants immunised with BCG-Russia

with statistical significance only when BCG was used as in vitro stimulatory antigen.

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Figure 3 shows the corresponding results in CD8 T cells. Lower proportions of double

cytokine-producing T cells were observed in CD8 than in CD4 T cells. Also, in

contrast to the results in CD4 T cells, IL-2/TNF-α double cytokine-producing and

triple cytokine-producing CD8 T cells were not detected (Figure 3, panel B and C).

The only statistically significant difference was found in IFN-γ/TNF-α double

cytokine-producing CD8 T cells following in vitro stimulation with BCG (Figure 3).

Proportions of these polyfunctional T cells were significantly higher in infants

immunised with BCG-Denmark or BCG-Japan than in infants immunised with BCG-

Russia. No differences were found following in vitro stimulation with PPD and MTB

(Figures 5 and 7).

There were no significant differences between the BCG vaccine strain groups in the

unstimulated (negative control) and SEB-stimulated (positive control) responses

(Supplementary Figures 1 and 2).

Figure 8 shows the proportion of cytotoxic CD4 and CD8 T cells measured by CD107

expression. CD107-expressing cytotoxic T cells did not produce IFN-γ or IL-2 (data

not shown). Proportions of cytotoxic CD4 T cells in infants immunised with BCG-

Denmark were significantly higher than in infants immunised with BCG-Japan or

BCG-Russia.

Figure 9 shows the concentrations of secreted cytokines. Th1-cytokines IFN-γ, IL-2

and IL-12 in infants immunised with BCG-Japan were significantly higher than in

infants immunised with BCG-Denmark or BCG-Russia. Similar changes were found

following in vitro stimulation with PPD but not following in vitro stimulation with

heat-killed M. tuberculosis (data not shown). Infants immunised with BCG-Japan also

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had significantly higher concentrations of IL-6, IL-10, MCP-1 and MIP-1β. These

differences were only observed following in vitro stimulation with BCG but not

following in vitro stimulation with PPD or heat-killed M. tuberculosis (data not

shown). There were no significant differences between vaccine strain groups in

cytokine concentrations in supernatant in unstimulated samples (Table 2).

No local or systemic adverse reactions, including ulceration, drainage, lymphadenitis

or disseminated BCG-osis were observed in any of the infants who attended the

follow-up visit. Figure 10 shows the size of the local reaction at the BCG injection site

10 weeks after immunisation. A larger than expected local reaction ( > 9 mm) was

observed in nine (5%) infants, with the largest reaction being 12 mm in two infants.

The local reaction was significantly larger in infants immunised with BCG-Denmark

(median 5 (IQR 4-7) mm) or BCG-Japan (5 (3-7) mm) than in infants immunised with

BCG-Russia (2 (1-4) mm) (p<0.001).

DISCUSSION

We found a significant difference in the immune response induced by the three BCG

vaccine strains that account for the majority of the over 100 million doses used each

year worldwide. BCG-Denmark and BCG-Japan induced higher proportions of

polyfunctional (simultaneously producing IFN-γ, IL-2 and TNF-α or a combination

thereof) CD4 T cells than BCG-Russia. Only one previous study has investigated the

proportion of cytokine-producing T cells in infants immunised with different BCG

vaccine strains. This study was not randomised and measured the proportion of IFN-γ-

producing T cells only (9).

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Polyfunctional CD4 T cells have recently attracted considerable attention as potential

correlates of protection and have therefore become a common measure in studies

investigating the immune response to M. tuberculosis (17). Studies investigating novel

TB vaccines in mice have found a correlation between mycobacterial-specific

polyfunctional T cells (in lung tissue, peripheral blood mononuclear cells or spleen

tissue) and protection (18-21). Some studies in HIV and TB co-infected adults also

show an association between polyfunctional T cells and protection (22-25), but others

suggest that these cells are associated with active TB disease (26, 27). A case control

study in BCG-immunised infants in South Africa found no correlation between

polyfunctional T cells and protection (28). In a recent study, individuals with latent TB

infection had significantly higher proportions of polyfunctional cells than those with

active TB (29). In addition, in individuals with active TB, the frequency of

polyfunctional cells increased during treatment suggesting a role for these cells as a

correlate of protection against TB.

Despite these inconsistent findings, polyfunctional CD4 T cells are likely to play an

important role in protection against TB. The superior cytokine-producing capacity of

these cells (19, 30, 31) means that higher proportions of polyfunctional CD4 T cells

result in enhanced production of Th1 cytokines. This is demonstrated in our study in

which infants in the group with the highest frequency of polyfunctional T cells (BCG-

Japan) also had the highest concentrations of secreted Th1 cytokines. Consistent with

our findings, a previous study also reported higher concentrations of IFN-γ in

supernatants in infants immunised with BCG-Japan compared with infants immunised

with BCG-Denmark (9). Another reason why polyfunctional CD4 T are important is

that these cells provide a reservoir of memory cells responsible for long-term

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protection (31). Unfortunately the limited blood volumes available did not allow

measurement of memory phenotypes of T cells in our study.

In addition to polyfunctional CD4 T cells, it has been suggested that cytotoxic

T cells play a previously under-recognised role in limiting persistence of

M. tuberculosis (32-34). Infants immunised with BCG-Denmark had higher

proportions of cytotoxic T cells than infants immunised with BCG-Japan or BCG-

Russia. Only one previous study compared cytotoxic T cells in infants immunised with

BCG-Denmark or BCG-Japan. In this small study the highest cytotoxic activity was

seen in CD4 T cells from infants immunised with BCG-Japan, however no statistically

significant differences were found (10).

Our study highlights two additional properties of mycobacterial-specific cytotoxic T

cells. The cytotoxic cells detected in study participants were almost exclusively CD4 T

cells. Although classically cytotoxicity is associated with CD8 T cells, it has been

reported that cytotoxic CD4 T cells are induced by several pathogens (35), including

M. tuberculosis and BCG (10, 36-38). Secondly, cytotoxic T cells rarely produced

IFN-γ and never produced IL-2 simultaneously, suggesting that CD4 T cells are either

cytokine-producing or cytotoxic cells. Similar findings have been described in CD8 T

cells after BCG immunisation in a previous study (39). The role of cytotoxic CD4 and

CD8 T cells in protection against TB and as a potential correlate of protection is

unclear (37, 38). Although a few studies propose the cytotoxic effector proteins

granulysin and perforin as candidate biomarkers for protection against TB this was

either shown to be true only in BCG immunised cattle following challenge with M.

tuberculosis (40) or when granulysin was measured in unstimulated serum from adults

with active TB compared to uninfected controls (41) . Therefore, the significance of a

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higher proportion of mycobacterial-specific cytotoxic CD4 T cells after BCG-

Denmark immunisation currently remains unknown. Ongoing studies investigating

correlates of protection against TB are likely to clarify the role of cytotoxic T cells

(28).

Our finding that IL-6, IL-10, MCP-1 and MIP-1β in supernatants were significantly

higher in infants immunised with BCG-Japan following in vitro stimulation with BCG

may be the result of the in vitro effect of BCG-Japan, of the in vivo immune response

following BCG-Japan immunisation or a combination of both. We were unable to take

blood before BCG immunisation to help determine the relative contribution of these

possibilities.

In a different study, in older infants, we have found increased concentrations of IL-6,

IL-10, MCP-1 and MIP-1β following in vitro stimulation with BCG in samples taken

before BCG immunisation (data not shown). There was a further increase in cytokine

concentration in samples taken after BCG immunisation from the same infants

suggesting an in vivo component in addition to the in vitro effect. In the same study,

the effects of BCG were not seen for any of the other measured secreted cytokines or

for polyfunctional T cells suggesting that the observed changes in intracellular and

secreted Th1 cytokines in the study reported here resulted from an in vivo (ie BCG

immunisation-induced) immune response.

The baseline characteristics of the study groups were well balanced. The only

exception was that the group of infants immunised with BCG-Russia included a lower

proportion of females. Although there has been a small number of reports on the

influence of gender on the immune response to measles vaccine (42) and RSV

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infection (43) in children, it seems unlikely that this influenced the results of this

study.

As a result of the limited available blood volumes, several other potentially interesting

avenues could not be pursued. These include differences in the innate immune-

response, in memory-phenotypes and in regulatory T cells induced by different BCG

vaccine strains. In the light of our findings 10 weeks after immunisation it will be

valuable to investigate the immune response at a later stage to assess if the differences

are sustained over time.

Our study clearly establishes that there are significant differences in the immune

response induced by different BCG strains in newborn infants, a finding that has

important implications for future immunisation strategies and tuberculosis vaccine

trials. Immunisation with BCG-Denmark or BCG-Japan was associated with greater

induction of polyfunctional CD4 T cells than BCG-Russia, and therefore may result in

better protection against TB. The implication of the two other major differences in the

immune response induced by the three BCG vaccine strains (BCG-Japan induced

higher values of Th1 cytokines and BCG-Denmark induced higher frequencies of

cytotoxic T cells) is less clear, but also suggest these strains may be superior to BCG-

Russia.

Acknowledgements

We thank Dr Robert de Rose for critical input in the optimisation of the in vitro assays

and flow-cytometric analysis. We thank Prof Steve Graham for helpful advice and

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discussion. We thank the children and their parents for participating in this study. The

sponsors had no influence on study design, collection, analysis or interpretation of the

data. They were also not involved in the writing of the manuscript and the decision to

submit the paper.

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31. Seder RA, Darrah PA, Roederer M. T-cell quality in memory and protection:
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39. Murray RA, Mansoor N, Harbacheuski R, Soler J, Davids V, Soares A,
Hawkridge A, Hussey GD, Maecker H, Kaplan G, Hanekom WA. Bacillus Calmette
Guerin vaccination of human newborns induces a specific, functional CD8+ T cell
response. J Immunol 2006;177:5647-5651.
40. Capinos Scherer CF, Endsley JJ, de Aguiar JB, Jacobs WR, Jr., Larsen MH,
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 Page 26 of 41

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and sex as factors of response to RSV infections among those with previous history of
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Page 27 of 41

Table 1 Characteristics of participants

BCG- BCG- BCG-

Denmark Japan Russia
n = 54 n = 54 n = 57

Gestational age Median 39.7 39.6 39.6

(weeks) IQR 39.0–40.3 38.9–40.4 39.1–40.4
Range 36.3–43.0 35.3–41.9 35.3–42.1
Female 28 (52%) 31 (57%) 25 (43%)
(number, %)
Weight Median 3325 3280 3280
(grams) IQR 3015–3523 2990–3535 2980–3720
Range 2630–4230 2620–4410 2500–4090
Age at immunisation Median 1 1 1
(days) IQR 1–3 1–2 1–2
Range 0–7 1–5 1–6
Interval immunisation to Median 10 9.9 10.1
follow-up (weeks) IQR 9.9–10.5 9.6–10.6 9.9–11.0
Range 8.7–13.1 7.9–13.9 8.6–14.6

Mother age Median 30.1 29.3 29.7

(year) IQR 26.4–34.2 27.9–35.2 26.8–34.3
Range 18.9–39.9 23.5–41.1 22.8–41.1
Mother ethnicity Africa 2 (4%) 3 (5%) 8 (14%)
(number, %) Asia 28 (52%) 29 (53%) 28 (48%)
Indian subcontinent 23 (43%) 21 (38%) 21 (37%)
Other 1 (2%) 2 (4%) 1 (2%)
Father ethnicity Africa 3 (6%) 2 (4%) 9 (16%)
(number, %) Asia 28 (52 %) 27 (49%) 26 (45%)
Indian subcontinent 23 (43%) 22 (40%) 20 (34%)
Other/Unknown 0 (0%) 4 (7%) 3 (5%)

27
 Page 28 of 41

Table 2 Median concentrations of cytokines in supernatants from unstimulated

(negative control) samples

Median concentration (pg/ml)

Cytokine BCG- BCG- BCG-

Denmark Japan Russia

EGF 320 293 319

Eotaxin 160 177 144
Fractalkine 82 44 38
IFN-g 6 2 4
IL-10 19 20 14
IL-12 (p40) 77 64 46
IL-13 14 9 10
IL-2 2 1 0
IL-6 26 18 12
MCP 995 1079 666
MIP-1 b 1158 992 930
TNF-a 18 18 8

28
Page 29 of 41

Figure 1 Study flow chart.

5790 pregnant women screened in antenatal clinic

4342 both parents from country with

intermediate or low TB incidence
919 attended when recruitment was not
possible
160 not willing to participate
13 infants not born before end of study

356 infants born to participating mothers

68 withdrew consent

288 infants randomised to BCG immunisation

1 immunised twice
78 randomised to immunisation with
†
BCG-Denmark at two months

209 infants immunised with BCG at birth

67 BCG-Denmark 69 BCG-Japan 73 BCG-Russia

55 completed follow-up 57 completed follow-up 64 completed follow-up

2 withdrew consent 3 withdrew consent 2 withdrew consent
10 lost to follow-up 9 lost to follow-up 7 lost to follow-up

54 data analysed 54 data analysed 57 data analysed

1 excluded for technical reasons * 2 excluded for technical reasons * 7 excluded for technical reasons *
1 failed venipucture

† as part of a separate study

* technical problems included: in vitro cell death, inadequate staining for flow cytometry, and inadequate instrument
settings during flow cytometry

29
 Page 30 of 41

Figure 2 Box plots (depicting lower, median and upper quartiles, with Tukey
whiskers) showing background-corrected proportions of single (A) and double (B)
cytokine-producing CD4 T cells following in vitro stimulation with BCG. Triple (C)
cytokine-producing CD4 T cells are shown following in vitro stimulation with BCG,
PPD and MTB (heat-killed M. tuberculosis). Statistical differences with p-values ≤
0.05 are shown.

A IFN-γ IL-2 TNF-α CD4

0.7 < 0.001 0.01 < 0.001 0.02
BCG-Denmark
0.6
BCG-Japan
0.5
BCG-Russia
0.4

0.3
Proportion of cytokine-producing CD4 T cells (%)

0.2

0.1

0.0

BCG-stimulated BCG-stimulated BCG-stimulated

B IFN-γ/IL-2 IFN-γ/TNF-α IL-2/TNF-α

0.10 0.005 0.004

0.01
0.08

0.06

0.04

0.02

0.00

BCG-stimulated BCG-stimulated BCG-stimulated

C IFN-γ/IL-2/TNF-α

0.10 0.10 0.10

0.02
0.006 0.02
0.003
0.08 0.08 0.08

0.06 0.06 0.06

0.04 0.04 0.04

0.02 0.02 0.02

0.00 0.00 0.00

BCG-stimulated PPD-stimulated MTB-stimulated

30
Page 31 of 41

Figure 3 Box plots (depicting lower, median and upper quartiles, with Tukey
whiskers) showing background-corrected proportions of single (A) and double (B)
cytokine-producing CD8 T cells following in vitro stimulation with BCG. Triple (C)
cytokine-producing CD8 T cells are shown following in vitro stimulation with BCG,
PPD and MTB (heat-killed M. tuberculosis). Statistical differences with p-values ≤
0.05 are shown.

A IFN-γ IL-2 TNF-α CD8

0.7

0.6 0.006 BCG-Denmark

0.5
BCG-Japan
BCG-Russia
0.4

0.3

0.2
Proportion of cytokine-producing CD8 T cells (%)

0.1

0.0

BCG-stimulated BCG-stimulated BCG-stimulated

B IFN-γ/IL-2 IFN-γ/TNF-α IL-2/TNF-α

0.10 0.002
< 0.001
0.08

0.06

0.04

0.02

0.00

BCG-stimulated BCG-stimulated BCG-stimulated

C IFN-γ/IL-2/TNF-α
0.10 0.10 0.10

0.08 0.08 0.08

0.06 0.06 0.06

0.04 0.04 0.04

0.02 0.02 0.02

0.00 0.00 0.00

BCG-stimulated PPD-stimulated MTB-stimulated

31
 Page 32 of 41

Figure 4 Box plots (depicting lower, median and upper quartiles, with Tukey
whiskers) showing background-corrected proportions of single (A), double (B) and
triple (C) cytokine-producing CD4 T cells following in vitro stimulation with PPD.
Statistical differences with p-values ≤ 0.05 are shown.

BCG-Denmark
A IFN-γ IL-2 TNF-α
BCG-Japan
0.5
BCG-Russia
0.4

0.3

0.2

0.1

0.0

B IFN-γ/IL-2 IFN-γ/TNF-α IL-2/TNF-α C IFN-γ/IL-2/TNF-α

0.10 0.10
0.006
0.08 0.08

0.06 0.06

0.04 0.04

0.02 0.02

0.00 0.00

32
Page 33 of 41

Figure 5 Box plots (depicting lower, median and upper quartiles, with Tukey
whiskers) showing background-corrected proportions of single (A), double (B) and
triple (C) cytokine-producing CD8 T cells following in vitro stimulation with PPD.
Statistical differences with p-values ≤ 0.05 are shown.

A IFN-γ IL-2 TNF-α BCG-Denmark

BCG-Japan
0.5
BCG-Russia
0.4

0.3

0.2

0.1

0.0

B IFN-γ/IL-2 IFN-γ/TNF-α IL-2/TNF-α C IFN-γ/IL-2/TNF-α

0.10 0.10

0.08 0.08

0.06 0.06

0.04 0.04

0.02 0.02

0.00 0.00

33
 Page 34 of 41

Figure 6 Box plots (depicting lower, median and upper quartiles, with Tukey
whiskers) showing background-corrected proportions of single (A), double (B) and
triple (C) cytokine-producing CD4 T cells following in vitro stimulation with MTB
(heat-killed M. tuberculosis). Statistical differences with p-values ≤ 0.05 are shown.

BCG-Denmark
A IFN-γ IL-2 TNF-α
BCG-Japan
0.5
BCG-Russia
0.4

0.3

0.2

0.1

0.0

B IFN-γ/IL-2 IFN-γ/TNF-α IL-2/TNF-α C IFN-γ/IL-2/TNF-α

0.10 0.10
0.02
0.08 0.08

0.06 0.06

0.04 0.04

0.02 0.02

0.00 0.00

34
Page 35 of 41

Figure 7 Box plots (depicting lower, median and upper quartiles, with Tukey
whiskers) showing background-corrected proportions of single (A), double (B) and
triple (C) cytokine-producing CD8 T cells following in vitro stimulation with MTB
(heat-killed M. tuberculosis). Statistical differences with p-values ≤ 0.05 are shown.

A IFN-γ IL-2 TNF-α BCG-Denmark

BCG-Japan
0.5
BCG-Russia
0.4

0.3

0.2

0.1

0.0

B IFN-γ/IL-2 IFN-γ/TNF-α IL-2/TNF-α C IFN-γ/IL-2/TNF-α

0.10 0.10

0.08 0.08

0.06 0.06

0.04 0.04

0.02 0.02

0.00 0.00

35
 Page 36 of 41

Figure 8 Box plots (depicting lower, median and upper quartiles, with Tukey
whiskers) showing proportions of CD107-expressing (cytotoxic) CD4 and CD8 T cells
following in vitro stimulation with MTB (heat-killed M. tuberculosis) and BCG.
Statistical differences with p-values ≤ 0.05 are shown.

CD4 CD8
0.20 0.20
0.03 BCG-Denmark
BCG-Japan
0.15 0.15 BCG-Russia
Proportion of CD107-expressing T cells (%)

0.10 0.10

0.05 0.05

0.00 0.00

MTB-stimulated

0.5 0.5
< 0.001
0.01
0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0.0 0.0

BCG-stimulated

36
Page 37 of 41

Figure 9 Box plots (depicting lower, median and upper quartiles, with Tukey
whiskers) showing background-corrected cytokines in supernatants following in vitro
stimulation with BCG. Statistical differences with p-values < 0.05 are shown.

IFN-γ IL-2 TNF-α IL-12 (p40)

2000 2000 15000 800

0.037 < 0.001 0.02 0.004 0.001

1500 1500 600
10000

1000 1000 400

5000
500 500
200

0 0
0
0

IL-13 IL-10 IL-6 EGF

Concentration (pg/ml)

150 3000 125000 300

0.02 0.02 0.008 0.003
200
100000
100
2000 100
75000

50 0

50000
1000 -100
0
25000
-200

0
-50 0 -300

MCP-1 MIP-1β Eotaxin Fractalkine

125000 20 200
100000
<0.001 <0.001 0.004 0.008
10
100000
75000
0 100
75000

50000 -10

50000
-20 0
25000
25000
-30

0 0 -40 -100

BCG-Denmark
BCG-Japan
BCG-Russia

37
 Page 38 of 41

Figure 10 Comparison of the size of the local reaction at the BCG injection site in the
three groups 10 weeks after immunisation.

20 BCG-Denmark
BCG-Japan
15 BCG-Russia

10

0
20

15

10

0
20
Number of infants

15

10

0
0 1 2 3 4 5 6 7 8 9 10 11 12

Local reaction size (mm)

38
Page 39 of 41

The influence of BCG vaccine strain on the immune response against

tuberculosis: a randomised trial

Nicole Ritz, Binita Dutta, Susan Donath, Dan Casalaz, Tom G. Connell, Marc

Tebruegge, Roy Robins-Browne, Willem A. Hanekom, Warwick J.

Britton, Nigel Curtis

ONLINE DATA SUPPLEMENT

39
 Page 40 of 41

Supplementary Figure 1 Box plots (depicting lower, median and upper quartiles, with Tukey whiskers) showing proportions of single, double
and triple cytokine-producing CD4 T cells following in vitro stimulation with medium alone (negative control) (A-C) and staphylococcal
endotoxin B (positive control) (D-F). Statistical differences with p-values ≤ 0.05 are shown.

A IFN-γ IL-2 TNF-α BCG-Denmark D IFN-γ IL-2 TNF-α BCG-Denmark

BCG-Japan BCG-Japan
0.5 20
BCG-Russia BCG-Russia
0.4
15

0.3

10
0.2

5
0.1

0.0 0

B IFN-γ/IL-2 IFN-γ/TNF-α IL-2/TNF-α C IFN-γ/IL-2/TNF-α E IFN-γ/IL-2 IFN-γ/TNF-α IL-2/TNF-α F IFN-γ/IL-2/TNF-α

0.10 15 1.0
0.10

0.08 0.8
0.08

10
0.06 0.06 0.6

0.04 0.04 0.4

5

0.02 0.02 0.2

0.00 0.00 0 0.0

40
Page 41 of 41

Supplementary Figure 2 Box plots (depicting lower, median and upper quartiles, with Tukey whiskers) showing proportions of single, double
and triple cytokine-producing CD8 T cells following in vitro stimulation with medium alone (negative control) (A-C) and staphylococcal
endotoxin B (positive control) (D-F). Statistical differences with p-values ≤ 0.05 are shown.

A IFN-γ IL-2 TNF-α BCG-Denmark

D IFN-γ IL-2 TNF-α BCG-Denmark
BCG-Japan 8 BCG-Japan
0.7
BCG-Russia BCG-Russia
0.6

6
0.5

0.4
4
0.3

0.2 2

0.1

0.0 0

B IFN-γ/IL-2 IFN-γ/TNF-α IL-2/TNF-α C IFN-γ/IL-2/TNF-α E IFN-γ/IL-2 IFN-γ/TNF-α IL-2/TNF-α F IFN-γ/IL-2/TNF-α

0.10 1.5 1.5

0.10

0.08 0.08

1.0 1.0
0.06 0.06

0.04 0.04
0.5 0.5

0.02 0.02

0.00 0.00 0.0 0.0

41