Optimisation of a functional mycobacterial growth-inhibition
assay to improve its suitability for infant TB vaccine studies☆
S. Burla,⁎, B.S. Holdera, B.K.M. Loa, B. Kampmanna,b
aDepartment of Paediatrics, St Mary's Campus, Imperial College London, Norfolk Place, London W2 1PG, UK
bMRC Unit, The Gambia, Atlantic Road, Fajara, Gambia
a r t i c l e i n f oa b s t r a c t
Received 26 September 2012
Received in revised form 14 May 2013
Accepted 16 May 2013
Available online 23 May 2013
mediated response, which is routinely measured using a read-out of host cytokine profiles.
However, to date none of the cytokine profiles have beenfoundto predict protection. A number of
functionalin vitroapproaches that measuregrowth of mycobacteriapre-and post-vaccination as a
potential functional surrogate marker for vaccine take have been developed. The use of a
reporter-gene tagged BCG-lux assay measuring the viability of mycobacteria in whole blood
samples has previouslybeendescribedby our group to assess vaccine immunogenicity. Since only
very small blood samples are usually available in paediatric studies, we now report a modification
of the BCG-lux assay to reduce the volume required and make it more field-friendly. Our results
show that a 2-fold reduction in blood volume made no significant difference to bacterial growth
ratios, used as the main read-out. These results confirm the suitability of the BCG-lux assay for
functional studies of vaccine immunogenicity and immunopathogenesis in young children and
could play a role in late-phase TB vaccine trials of novel candidates.
© 2013 The Authors. Published by Elsevier B.V. All rights reserved.
tuberculosis (TB) is the absence of an appropriate in vitro
correlate to predict efficacy of a vaccine. Such a correlate would
significantly reduce the need for expensive and extensive phase
conventionally measure antibody titres in response to the given
vaccine, but this approach is not suitable to assess vaccines
against TB, since cell-mediated responses rather than antibody
are known to be the key mediators of protection. The immune
response to Mycobacterium bovis bacillus Calmette–Guérin
(BCG) vaccination (at present the only licensed vaccine against
skin test, measuring delayed-type hypersensitivity (DTH) to
intradermal inoculation of purified protein derivative (PPD), a
crude mixture of antigenic proteins from M.tb. Tuberculin
sensitivity is also induced by exposure to M.tb itself, and some
non-tuberculous mycobacteria, which interferes with the
specificity of the TST. In addition conversion to PPD-positivity
does not necessarily correlate with induction of protective
immunity (Fine et al., 1994). More recently, production of
gamma interferon (IFNγ) by peripheral blood T cells stimulated
by mycobacterial antigens in vitro (i.e. the interferon gamma
release assays (IGRA)) has also been used as a measure of
exposure to M.tb infection or vaccine-take (Pai et al., 2008), but
while there is extensive evidence that IFNγ-secreting T cells are
the ability of a vaccine to prime such cells is correlated with its
protective efficacy (Kagina et al., 2010). Although it is likely that
many other cytokines, in addition to IFNγ, are involved in the
protection, the holy grail of the “correlate(s) of protection”
against tuberculosis still remains to be found.
Journal of Immunological Methods 394 (2013) 121–124
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E-mail addresses: email@example.com (S. Burl),
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Journal of Immunological Methods
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The present lack of suitable correlates of human
protection has encouraged the development of in vitro
models that incorporate possible mechanisms of growth
restriction or mycobacterial killing as a functional read-out.
Two in vitro methods of studying mycobacterial growth
using isolated peripheral blood mononuclear cells have
been developed and employed in BCG and M.tb growth
restriction assays (Silver et al., 1998; Worku and Hoft,
2000). One method involves unstimulated lymphocytes in
the primary lymphocyte assay (Silver et al., 1998) and the
other uses antigen stimulated lymphocytes for the detec-
tion of memory immunity (Worku and Hoft, 2000). More
recently, Kampmann et al. developed a whole blood assay to
study mycobacterial survival, using a luciferase reporter
system (Kampmann et al., 2000). This assay utilises vectors
that contain a modified version of either BCG (rBCG-lux) or
M.tb (rMtb-lux). The modification includes addition of a
reporter enzyme (luciferase lux gene) that luminesces after
the addition of an appropriate external substrate. Measure-
ment of this signal directly relates to the numbers and
viability of the mycobacteria (Kampmann et al., 2000).
Although all of these assays have shown the capability of
detecting increased mycobacterial growth inhibition after
BCG vaccination, the whole blood assay is simpler to
perform without the need for isolation of PBMCs and
requires less blood making it an attractive assay for the
field and for paediatric studies.
A number of studies have already been published which
have used this assay to dissect the mechanisms that restrict
or promote mycobacterial growth. In a proof-of principle
study, immunogenicity of the BCG vaccine in infants has
been demonstrated through greater control of BCG growth
in the BCG-lux assay following BCG-vaccination of infants
(Kampmann et al., 2004). Other studies showed that vitamin
D played an important role in restriction of BCG and M.tb
growth, dependent on neutrophils and in particular anti-
microbial peptides (Martineau et al., 2007). A clinical trial of
2.5 mg of vitamin D supplementation (ergocalciferol) in TB
contacts supported this finding. Studies in HIV positive
children showed that prior to HIV anti-retroviral treatment
(HAART) those with low CD4 T cell counts showed limited
ability to restrict the growth of mycobacteria, but that the
introduction of HAART led to rapid and sustained reconsti-
tution of anti-mycobacterial immune responses, measured
as the decreased growth of mycobacteria compared to
HAART-naïve baseline (Kampmann et al., 2006).
At present a minimum of 4 mL of blood is used for these
assays, which is often the maximum volume that can be
vaccinetrials would wish to analyse vaccine responses in more
than one assay system, even more blood would be required.
The aim of the present study was therefore to optimise the lux
assay to use smaller volumes of blood and thereby increase its
suitability for field studies in small children.
2. Materials and methods
2.1. BCG-lux assay
The original development of the BCG-lux assay has been
described elsewhere in detail (Kampmann et al., 2000). In
this study we made modifications to the volumes of blood
used per assay, but not to the reporter-gene construct or the
previously established multiplicities of infection and basic
handling of the samples. Briefly, M. bovis–BCG transformed
with a replicating vector containing the luciferase (lux) gene
of Vibrio harveyi was prepared as previously described
(Snewin et al., 1999). Frozen aliquots of BCG-lux bacilli
were grown to midlog phase in Middlebrook 7H9 broth
supplemented with 10% albumin dextrose catalase enrich-
ment (BD; Franklin Lakes, NJ) and 15 μg/mL hygromycin
(Roche, Lewes, UK). The bacilli were then diluted to a stock of
107Relative Light Units (RLU). This equates to an inoculum of
about 106Colony Forming Units (CFU)/mL of blood.
Following informed consent, up to 10 mL of blood was
collected from healthy adult volunteers into preservative-free
heparin tubes (15 USP units sodium heparin/mL, BD Bioscience)
Blood was diluted 1:1 with RPMI 1640/2 mM glutamine/
25 mM HEPES (N-2-hydoxyethylpiperazine-N′-ethane sulfonic
acid) buffer (Sigma, Poole, UK) and infected with BCG-lux bacilli
stock (1 × 107RLU) at a 1:10 concentration. This corresponded
of approximately 1:1, based on an established correlation of 10
triplicate aliquots of 1 mL, 0.67 mL and 0.5 mL for each time
point (baseline t = 0 and t = 96h) and t = 96 samples were
the same way using the same concentrations of mycobacteria in
7H9 culture medium.
At each time point the aliquots were processed as described
of cytokine profiles. Aliquots were centrifuged for 10 min at
2000 g and supernatants were collected and stored at −20 °C
(300 μL for 1 mL aliquots, 200 μL for 0.67 mL aliquots and
150 μL for 0.5 mL aliquots). PBS was added in replacement for
the withdrawal of supernatant and distilled water was added
1:10 to lyse the red blood cells followed by incubation for
10 min maximum at room temperature. The tubes were spun
at 2000 g for 10 min and the supernatants removed. The pellet
to help disperse the pellet.
The samples were then measured in the luminometer
(Berthold AutoLumat Plus, Berthold Technologies, Germany)
by diluting 1:10 in PBS and using 1% N-decyl aldehyde
(Sigma-Aldrich, US) as the substrate. Mycobacterial lumines-
cence was measured at baseline and at 96 h, and the growth
ratio was calculated by division of the mean 96-hour
luminescence value by the mean baseline value.
The control samples were processed in the same way
excluding the centrifugation step for removal of supernatant
and lysis of red blood cells, as these are irrelevant for bacteria
growing in growth medium alone. Growth ratios were
calculated and compared between different volumes of growth
media, equivalent to the equation used for whole blood assays.
2.2. Statistical evaluation
A mean of the triplicate growth ratios was calculated for
each sample per blood/control volume. A cross sectional
comparison of the median growth ratio for all data available
at each volume was compared using the non-parametric
S. Burl et al. / Journal of Immunological Methods 394 (2013) 121–124
Kruskal–Wallis test. Analysis including only samples with
repeated measures for each volume was carried out using
Friedmann ANOVA test. In addition separate comparisons
between each volume were analysed using Mann Whitney
paired non-parametric tests. In all cases p ≤ 0.05 was termed
studies that verified the methods were performed using the
original volumeof blood and thereforethere aremore valuesat
were included in the initial analysis. There was no significant
difference between the median growth ratios for each of the
volumes of diluted blood (Fig. 1A, p = 0.160) showinga 2-fold
reduction in volume is possible in this assay system.
Examining the data that included samples that had all
three different volume measurements, we found that there
were no significant differences between any of the volumes
(Fig. 1B, p = 0.398). Additional analysis comparing each
volume of blood with the original volume of 1 mL also
showed no significant differences between each of the
volumes tested (Fig. 1B).
There were no significant differences between the different
volumes of growth media for the growth of BCG (data not
tubes with snap caps (BD Biosciences) instead of 5 mL bijous
for the incubation reduced the risk of disrupting the pellet
while collecting the supernatants without any changes to the
resulting mycobacterial growth (data not shown).
The aim of this study was to optimise the conditions of the
BCG-lux assay to allow smaller volumes of blood to be used.
Our results suggest that initial blood volumes as low as
250 μL per condition per replicate can provide the same data
as the original 500 μL used and therefore a minimum of 2 mL
of blood would be required for these assays instead of the
currently used 4 mL.
A major limiting factor of studying infant immunity is the
volume of blood that can be collected thereby reducing the
number of assays or conditions possible within the study.
Molecular assays have advanced in such a way that many
parameters can be measured within one sample and has led
to large scale genetic studies in infant populations, but they
cannot measure growth restriction as a functional read-out.
Immunological assays often require large numbers of cells
from large volumes of blood and in the case of cell
phenotyping, can be expensive. In this current lux assay,
growth of mycobacteria is measured within whole blood
samples reducing the need to manipulate the cells and
thereby reducing the loss of cells in an already small volume
of blood. The initial protocol required a minimum of 4 mL of
blood and would therefore restrict any further assays being
performed on the same sample, except that cytokines can be
measured in the supernatants and RNA collected from the
pellet, as previously described. We now show that this
volume can be reduced to 2 mL with the same results.
We have previously demonstrated immunogenicity of BCG
vaccine using this growth-restriction assay and established the
assay as a useful tool for vaccine assessment and to decipher
mechanisms of growth restriction. The ability to use reduced
volumes of blood will further enhanceits utility in trials of new
tuberculosis vaccines in paediatric populations to assess how
efficient a given novel vaccine may be against inhibiting
trial did not show protection despite predicted immunogenic-
ity measured by cellular immune-assays (Tameris et al., in
the next generation of vaccine trials is timely. We believe that
the lux assay could play a role in such clinical trials.
Role of the funding source
The study was supported by the funding from the Medical
Fig. 1. The BCG growth ratios for each volume of blood used in the BCG-lux
assay. (A) Comparing all data for the different volumes of blood used using the
Kruskal–Wallis test, n = 12–27 (B) Comparing data that included all three
volumes tested using a Friedman ANOVA test and a paired non-parametric
Mann Whitney test on comparisons of individual volumes. Each data point
represents the average of three replicates from each donor in a given
experiment, n = 9. For all tests p ≤ 0.05 was termed significant, ns = not
S. Burl et al. / Journal of Immunological Methods 394 (2013) 121–124
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