Boosting BCG with Recombinant Modified Vaccinia
Ankara Expressing Antigen 85A: Different Boosting
Intervals and Implications for Efficacy Trials
Ansar A. Pathan1, Clare R. Sander1, Helen A. Fletcher1, Ian Poulton1, Nicola C. Alder2, Natalie E. R. Beveridge1, Kathryn T. Whelan1, Adrian V. S.
Hill1, Helen McShane1*
1Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, Churchill Hospital, Oxford, United Kingdom, 2Centre for Statistics in
Medicine, Wolfson College Annexe, University of Oxford, Oxford, United Kingdom
Objectives. To investigate the safety and immunogenicity of boosting BCG with modified vaccinia Ankara expressing antigen
85A (MVA85A), shortly after BCG vaccination, and to compare this first with the immunogenicity of BCG vaccination alone and
second with a previous clinical trial where MVA85A was administered more than 10 years after BCG vaccination. Design. There
are two clinical trials reported here: a Phase I observational trial with MVA85A; and a Phase IV observational trial with BCG.
These clinical trials were all conducted in the UK in healthy, HIV negative, BCG naı ¨ve adults. Subjects were vaccinated with BCG
alone; or BCG and then subsequently boosted with MVA85A four weeks later (short interval). The outcome measures, safety
and immunogenicity, were monitored for six months. The immunogenicity results from this short interval BCG prime–MVA85A
boost trial were compared first with the BCG alone trial and second with a previous clinical trial where MVA85A vaccination
was administered many years after vaccination with BCG. Results. MVA85A was safe and highly immunogenic when
administered to subjects who had recently received BCG vaccination. When the short interval trial data presented here were
compared with the previous long interval trial data, there were no significant differences in the magnitude of immune
responses generated when MVA85A was administered shortly after, or many years after BCG vaccination. Conclusions. The
clinical trial data presented here provides further evidence of the ability of MVA85A to boost BCG primed immune responses.
This boosting potential is not influenced by the time interval between prior BCG vaccination and boosting with MVA85A. These
findings have important implications for the design of efficacy trials with MVA85A. Boosting BCG induced anti-mycobacterial
immunity in either infancy or adolescence are both potential applications for this vaccine, given the immunological data
presented here. Trial Registration. ClinicalTrials.gov NCT00427453 (short boosting interval), NCT00427830 (long boosting
interval), NCT00480714 (BCG alone)
Citation: Pathan AA, Sander CR, Fletcher HA, Poulton I, Alder NC, et al (2007) Boosting BCG with Recombinant Modified Vaccinia Ankara Expressing
Antigen 85A: Different Boosting Intervals and Implications for Efficacy Trials. PLoS ONE 2(10): e1052. doi:10.1371/journal.pone.0001052
In 2003, there were 8.8 million new cases of tuberculosis (TB)
throughout the world and 1.7 million deaths . This makes TB
the leading cause of death from a curable disease, despite
widespread deployment of the only available vaccine against TB,
Mycobacterium bovis Bacille Calmette-Guerin (BCG). The protective
efficacy of BCG is hugely variable, but overall, BCG fails to
protect against pulmonary disease, particularly in adults in the
developing world . However, when administered at birth, as it is
in most of the developing world, BCG does confer consistent
protection against disseminated disease in childhood, and is highly
cost effective against severe childhood TB [3,4]. Ideally any
improved vaccine strategy against TB should therefore include
BCG. As repeated BCG vaccination does not appear to improve
the protective efficacy of a single BCG vaccination, there is an
urgent need to develop new improved boosting vaccines .
Mycobacterium tuberculosis (M.tb) is an intracellular pathogen and
any new TB vaccine will need to induce high levels of cellular
immunity . The secretion of interferon gamma (IFN-c) from
antigen specific T cells is an essential component of the host
cellular immune response to M.tb and animals and humans with
deficiencies in the IFN-c pathway are profoundly more susceptible
to disseminated mycobacterial infections, including M.tb [7,8].
Class II restricted CD4+ T cells are essential for protective
immunity, as evident by the increased susceptibility of HIV
infected individuals to reactivation of latent M.tb infection .
Class I restricted CD8+ T cells may also have a role to play .
Recombinant viral vectors are highly effective at inducing high
levels of both CD4+ and CD8+ T cells [11–13]. We have
previously shown that MVA85A, the first subunit TB vaccine to
enter clinical trials, induces high levels of antigen specific T cells
when given alone. We have also shown that a single immunisation
with MVA85A is highly effective at boosting BCG induced
immune responses in subjects in whom BCG vaccination was
administered many years previously.
An effective new TB vaccine designed to boost BCG would be
used in two ways: either, boosting in infancy (i.e. shortly after BCG
Academic Editor: James Campbell, University of Maryland School of Medicine,
United States of America
Received August 16, 2007; Accepted August 31, 2007; Published October 24,
Copyright: ? 2007 Pathan et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: HM is a Wellcome Trust Senior Clinical Research Fellow and AVSH is
a Wellcome Trust Principal Research Fellow. Oxford University was the sponsor for
all the clinical trials reported here.
Competing Interests: AP, AH and HM are named inventors on a composition of
matter patent for MVA85A filed by the University of Oxford.
* To whom correspondence should be addressed. E-mail: helen.mcshane@ndm.
PLoS ONE | www.plosone.org1 October 2007 | Issue 10 | e1052
vaccination); or, boosting in adolescence (i.e. many years after
BCG vaccination). The aim of the first clinical trial presented here
was therefore to investigate the safety and immunogenicity of
boosting BCG induced immune responses with MVA85A four
weeks after BCG vaccination (NCT00427453). We then compared
the immunogenicity results from this trial with the second trial
presented here, which involves vaccination with BCG alone
(NCT00480714). In addition, we then present a discussion
comparing the results from this new trial of short interval
previously published results in which boosting with MVA85A
was performed more than 10 years after BCG vaccination
The protocols for this trial and supporting CONSORT checklist
are available as supporting information; see Checklist S1 and
Protocols S1, S2, and S3. Consort flowcharts for each of the three
trials discussed here are presented as Figures 1–3.
Both clinical trials were conducted in BCG naı ¨ve; tuberculin skin
test negative (Heaf test grade 0 or 1), healthy volunteers. Subjects
were recruited for these clinical trials under protocols approved by
the Oxfordshire Research Ethics Committee and enrolled only
after obtaining written informed consent. They were aged 19–48
(median=26) and were all seronegative for HIV, HBV and HCV
at screening. Demographic information on the subjects is
summarised in Table 1. (The data for the middle column (long-
boost interval) derive from the trial published in ). Routine
laboratory haematology and biochemistry were performed prior to
vaccination and all values were within normal limits. All subjects
enrolled in the trials presented here were negative on an ex-vivo
Elispot assay for the two M.tb specific antigens ESAT6 and CFP10.
Figure 1. Consort flowchart for the BCG alone trial
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In the first study, volunteers were vaccinated with BCG (a single
immunisation with BCG SSI strain, 100 ml administered intra-
dermally, n=9). In the second study, volunteers were vaccinated
with BCG (SSI strain, 100 ml administered intra-dermally) and
4 weeks later were vaccinated with a single immunisation with
56107pfu MVA85A, intradermally into the contralateral arm to
the BCG immunisation (n=10).
The construction of MVA85A has previously been described
. Clinical grade MVA85A was produced under good
manufacturing practices by Impfstoffwerke Dessau-Tornau. A
Doctors and Dentists Exemption Certificate was issued from the
Medicines and Healthcare products Regulatory Agency, London,
for the use of MVA85A in clinical trials. The trials reported here
completed enrolment before the advent of the UK Medicines for
Human Use (Clinical Trials Regulations) 2004.
Allthe trialsreported here were single-arm,non-randomized trials.
The primary outcomemeasureinthe short interval BCG–MVA85A
trial presented here was safety and the secondary outcome was
vaccine induced cellular immune responses, as measured by an ex-
vivo IFN-c ELISpot assay. All subjects who were vaccinated with
MVA85A completed a diary card recording local and systemic side
effects and body temperature for 7 days following vaccination.
The ex vivo IFN-c ELISpot assay was performed on blood taken at
the following time points: at screening (prior to the tuberculin skin
test), and then at 1, 4, 8 and 24 weeks after vaccination. These
measurements were carried out on fresh PBMCs using tuberculin
PPD (20 mg/ml, SSI), purified antigen 85 complex (10 mg/ml), and
7 pools of 9–10 15-mer peptides spanning the length of antigen 85A,
which overlapped by 10 amino-acids (final concentration of each
Figure 2. Consort flowchart for the BCG prime–MVA85A boost (short interval) trial
BCG-MVA85A: Boosting Intervals
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peptide in the well10 mg/ml.) Briefly, 300,000 PBMCs per well were
plated directly onto the ELISpot plate (MAIP, Millipore) in the
presence of antigen, and incubated for 18 hours. Streptokinase/
Streptodornase and PHA were used in all assays as positive controls
and cells and media alone as the negative control. Assays were
performed in duplicate and the results were averaged.
Analysis of immunogenicity
analysed by subtracting the mean number of spots in the
medium and cells alone control wells from the mean counts of
spots in wells with antigens or peptide pools, and cells. Counts less
than 5 spots/well were disregarded. A well was considered positive
if the count was at least twice that in the negative control wells and
at least 5 spots more than the negative control wells. For the
peptide pool wells, the results were summed across all the peptide
pools for each volunteer at each time point. This will count twice
a T cell that responds to any of the 10-mer overlap regions that
occur in two pools with adjacent peptides, as each pool contains
non-overlapping peptides. An area under the curve analysis was
The ELISpot data were
performed to compare between the two vaccine groups, BCG
alone, and BCG prime–MVA85A boost. A Mann-Whitney test
was then used for all comparisons between these two groups
(1 week and 24 week responses) and a Wilcoxon Signed Ranks
Test was used for all comparisons between time points within
a group (baseline vs. 1 week, and baseline vs. 24 weeks).
Subjects were recruited into the trials reported here from
September 2002 to March 2004. Subjects in both the BCG alone
and the BCG–MVA85A groups were followed up for safety and
immunogenicity for 6 months after vaccination.
Safety of MVA85A
In total, 10 healthy volunteers were vaccinated with MVA85A,
four weeks after receiving vaccination with BCG. Vaccination with
Figure 3. Consort flowchart for the BCG prime–MVA85A boost (long interval) trial
BCG-MVA85A: Boosting Intervals
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MVA85A was well tolerated and there were no serious or severe
vaccine related adverse events in any of these trials. As expected
from previous trials with this and other recombinant MVAs, all
subjects experienced some mild local adverse events [16,17].
When the safety data from the short interval BCG–MVA85A trial
was compared with the previous data from the long interval BCG–
MVA85A trial, the frequency of solicited and spontaneously
reported local and systemic adverse events was not different
between these two groups (Table 2 and 3).
Cellular immune responses induced by the different
(a) Short interval BCG-MVA85A
vaccinated 4 weeks previously with BCG were vaccinated with
MVA85A,a highlysignificant rise in antigen specificT cells was seen,
1 week after MVA85A vaccination (PPD, antigen 85 and summed
in the BCG–MVA85A group than after BCG vaccination alone
(Table 4 and Figure 4 b–e). The responses to purified antigen 85 and
the summed pooled peptides remained significantly higher than after
BCG alone 24 weeks after vaccination (Table 4 and Figure 4 c–d,f).
The area under the curve analysis whichwas performed between day
between BCG and BCG-MVA85A for antigen 85 and the summed
pooled peptide responses (Tables 5 and 6).
Responses to all these antigenic stimuli were maintained at
levels significantly higher than the baseline screening responses,
(-4 weeks Fig 4) in the BCG–MVA85A group at 24 weeks
after vaccination with MVA85A (PPD p=0.017; Antigen 85
p=0.0059; summed peptide pools p=0.007).
When subjects who had been
Immunisation with BCG alone in this study induced
moderate levels of PPD and antigen 85-specific IFN-c secreting
T cells, which peaked 4 weeks after immunisation (Table 4 and
Figure 4b–d). However, the responses to the pooled antigen 85A
peptides following BCG vaccination were strikingly weak (Table 4;
Figure 4d). At 24 weeks after vaccination, the immune responses
induced after BCG vaccination were not significantly different
from baseline screening responses (PPD p=0.110; antigen 85
p=0.260; summed peptide pools p=0.236).
The local and systemic adverse event profile after BCG was
entirely as expected: all subjects developed a local reaction at the
injection site, and there were no systemic adverse reactions.
and immunogenicityof BCG vaccina-
Comparison of short interval BCG-MVA85A with
long interval BCG-MVA85A
The data presented here investigating the immunogenicity of
a short boosting interval between BCG vaccination and MVA85A
boosting were then compared with the data from the long boosting
interval clinical trial previously published . The peak vaccine
induced immune response, measured one week after vaccination,
was not significantly different between these two clinical trials
(PPD p=0.725; antigen 85 p=0.92; summed peptide pools
p=0.841, Figure 5(a)). Furthermore, 24 weeks after vaccination,
the plateau immune responses were also not significantly different
between these two clinical trials (PPD p=0.635; antigen 85
p=0.958; summed peptide pools p=0.937, Figure 5(b)).
There are three important findings from the clinical trials reported
here. First, we replicate our previous findings that vaccination with
MVA85A in subjects previously vaccinated with BCG is safe and
well tolerated. Second, we also replicate our previous findings that
BCG induced immune responses can be significantly boosted with
MVA85A, as measured by the peak and plateau vaccine induced
effector immune responses after vaccination. Third, we show that
the boosting potential of MVA85A does not seem to be dependant
on interval between BCG vaccination and boosting with
MVA85A. We investigated the relationship between boosting
interval and peak vaccine induced immune response, 1 week after
vaccination. No correlation was found between boosting interval
and peak response (Spearman’s correlation r=20.136; p=0.49,
data not shown).
Table 1. Demographic details of subjects according to trial.
The data for the middle column (long-boost interval) derive
from the trial published in 
Male30% 53% 22%
Age yrs: Mean (SD)27 (7.2) 31 (11.4)27 (5.6)
Country of birth (%)
Africa 10% 12% 0%
USA10% 0% 0%
Healthcare worker 40%24% 0%
Significant travel history 30%24%11%
Table 2. Local adverse events after MVA85A vaccination
boosting interval (n=10)
boosting interval (n=17)
Redness10 (100%) 17 (100%)
Pruritus 4 (40%)10 (59%)
Pain9 (90%) 17 (100%)
Induration10 (100%) 17 (100%)
Table 3. Systemic adverse events after MVA85A vaccination
boosting interval (n=10)
boosting interval (n=17)
Fever2 (20%)1 (6%)
Feverish 6 (60%) 5 (29%)
Arthralgia 2 (20%)3 (18%)
Headache6 (60%)6 (35%)
Myalgia8 (80%) 5 (29%)
Nausea3 (30%)0 (0%)
Diarrhoea3 (30%)0 (0%)
Vasovagal syncope0 (0%) 0 (0%)
Axillary LN1 (10%)1 (6%)
Alterations in haem/
0 (0%)0 (0%)
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Table 4. Median (and inter quartile range) ELISpot responses to PPD, antigen 85 and summed pooled peptides in each vaccination
group at each timepoint.
Time after vaccination
(weeks) Median PPD (inter quartile range) Median Antigen 85 (inter quartile range)
Median Summed pooled peptides
(inter quartile range)
BCG BCG-MVA85A BCGBCG-MVA85ABCG BCG-MVA85A
24 25 (0 , 124) 12 (2, 33)35 (8 , 91
22 219 (19 , 430)84 (12 , 141) 65 (23 , 221)
0 43 (29 , 155)224 (53 , 543) 13 (5 , 70)143 (37 , 274) 32 (30 , 94)93 (14 , 260)
1 90 (33 , 199) 847 (466 , 1274)30 (14 , 77) 968 (553 , 1199)40 (23 , 124)3189 (1809 , 4253)
4 220 (95 , 354) 340 (175 , 804)83 (34 , 252) 392 (235 , 922)67 (45 , 108)1065 (510 , 1792)
8 160 (127 , 327)248 (142 , 520)63 (35 , 84) 264 (131 , 538)76 (47 , 167) 569 (257 , 1352)
24 87 (50 , 143) 130 (91 , 514)20 (7 , 84) 110 (68 , 530)23 (18 , 84) 381 (183 , 798)
Figure 4. Median IFN-c ELISpot responses after vaccination in each vaccination group: BCG alone;BCG prime-MVA85A boost. (a) timeline for
vaccinations (weeks) in each group; (b)Tuberculin PPD responses; (c) Purified antigen 85 protein responses (d) summed pooled peptide responses; (e)
For each of the three antigens measured, the responses between each vaccine group were compared 1 week after vaccination using Mann-Whitney
statistic. (f) For each of the three antigens measured, the responses between each vaccine group were compared 24 weeks after vaccination using
Table 5. Area under the curve analysis for BCG alone and BCG–MVA85A groups
Vaccine groupn Median AUC (25th, 75thpercentiles)
PPD Antigen 85 Summed pooled peptides
BCG9 3574 (2599, 7125) 1293 (912, 3201)1250 (900, 4125)
BCG-MVA85A107289 (3878, 22197) 6900 (4263, 18308)18500 (10965, 49927)
The area under the curve analysis was carried out between 0 and 24 weeks.
BCG-MVA85A: Boosting Intervals
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Importantly, these higher antigen specific responses are
maintained at a significantly higher level than in the BCG alone
arm for at least 24 weeks after vaccination. The same result was
found in the previous clinical trial in which a longer boosting
interval was investigated. There were no significant differences in
plateau responses 24 weeks after vaccination, when the short and
long boosting intervals were compared. This persistence of ex-vivo
responses cannot be attributed to persistence of the MVA85A
vaccine, as MVA85A does not replicate in mammalian cells and
does not persist. It is more likely that the MVA85A boost has
expanded the memory T cell population, which is either persisting
without antigenic exposure or is being constantly re-stimulated or
‘boosted’ by exposure to environmental mycobacteria. We and
others have previously demonstrated the existence of anti-mycobac-
terial immunity induced by environmental mycobacteria in BCG
naı ¨ve adolescents and adults in the UK [14,18]. In contrast, the
BCG induced immune responses at 24 weeks after vaccination are
not significantly different from baseline, despite the fact that BCG
will almost certainly persist for longer than MVA85A.
The results of the BCG alone trial are very comparable with our
previous data on the immune response induced after BCG
vaccination, using the BCG Glaxo strain . Other groups have
also found no significant differences between the two strains of BCG
used in the UK over the last 5–10 years . Subjects in the short
and long boosting interval groups differ in which strain of BCG they
were vaccinated with. The short boosting interval subjects presented
here were all vaccinated with the SSI strain of BCG, whereas the
long boosting interval group previously published were vaccinated
with Glaxo BCG. However the comparability of immunogenicity of
these two strains found both by us and others suggests that this is not
an important factor when comparing the boosting potential of
MVA85A between these two groups.
A limitation of the work presented here is that the short boosting
interval BCG prime–MVA85A boost trial and the long boosting
interval trial previously published were not performed as a single
study. In comparing results between trials, the power to detect any
differences may be small, particularly given the small sample sizes
used in these Phase I studies. However we believe the promising
immunogenicity in the short boosting interval trial presented here
justifies the further evaluation of this vaccine in efficacy trials as
In the absence of a pre-defined immunological correlate of
protection, the key question when developing a new TB vaccine is
whether such significantly enhanced immune responses seen after the
MVA85A boost in both the short and long interval boosting studies
are accompanied by an improvement in protective efficacy. This
will need to be conducted in a high incidence population, to obtain
efficacy data within a realistic time frame. Even so, these trials are
likely to require approximately 10000 subjects and will also require
follow-up periods of up to 2 years. A key question when considering
the deployment of a new TB vaccine designed to boost BCG is when
to administer the boost. One option is to boost in infancy at about
4–6 months of age, and ideally this boost would coincide with an
existing EPI schedule vaccine visit, providing no interference
occurred between new and existing vaccines. Another potentially
incidence of TB disease that occurs in adolescence and young adults.
Table 6. Comparison of area under the curve analysis for BCG alone and BCG–MVA85A
PPD Antigen 85Summed pooled peptides
Diff in medians (95% CI+) P-Value*Diff in medians (95% CI+) P-Value*Diff in medians (95% CI+) P-Value*
BCG-MVA85A v BCG 2890 (-130, 10468)0.06 5023 (2467, 12417) 0.000816114 (9671, 27669)0.0002
+Estimated using the Binomial method
Figure 5. Dot-plot showing data at (a) 1 week and (b) 24 weeks after
vaccination in the MVA85A boosted group after recent and distant
BCG vaccination. Interval between BCG and MVA85A was four weeks
for the short interval trial and a median of 18 years (range 6 months–
38 years) in the long interval trial.
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There are two possible efficacy trials which correspond to Download full-text
potential deployment in either infancy or adolescence. Both
scenarios have advantages and disadvantages. Boosting in infancy
is attractive as there is a well established infrastructure within the
EPI for such an additional vaccine. If such a boost were scheduled
to coincide with an existing EPI schedule visit, then vaccine take-
up would likely be higher. However the major disadvantage with
conducting an efficacy trial in infancy (but not necessarily with
deployment in this age group once efficacy had been established) is
that disease end points can be difficult to define . In contrast,
boosting in adolescence is an attractive option as disease endpoints
are clearly defined and easy to diagnose in this age group. If
effective, boosting in adolescence would have a more immediate
impact on the mortality and morbidity of this disease than
boosting in infancy. A considerable disadvantage of boosting in
adolescence is that there is currently no infrastructure for routine
vaccination in this age group, particularly in the developing world.
However with the recent licensing of a vaccine against human
papilloma virus, scheduled to be administered from 9–15 years of
age, such an infrastructure is likely to become established in the
future . Ultimately a prophylactic vaccine against HIV would
also be likely to be administered in early adolescence.
The aim of these comparative Phase I studies was to investigate
the effects of boosting BCG soon after vaccination (thus modelling
the infant boosting scenario) and boosting many years after BCG
vaccination (thus modelling the adolescent scenario).The immuno-
genicity data presented here suggest that, at least using this
immunological readout, both options of boosting in infancy or of
boosting in adolescence may be effective. The data presented here
supports the further evaluation of this promising candidate vaccine,
which is currently in Phase II clinical trials in South Africa.
Found at: doi:10.1371/journal.pone.0001052.s001 (0.12 MB
Found at: doi:10.1371/journal.pone.0001052.s002 (0.12 MB
Short interval BCG-MVA85A
Found at: doi:10.1371/journal.pone.0001052.s003 (0.12 MB
Long boosting BCG-MVA85A
Found at: doi:10.1371/journal.pone.0001052.s004 (0.05 MB
We thank all the subjects who took part in the studies reported here. We
thank Kris Huygen for providing purified antigen 85 for use in these
studies. HM is a Wellcome Trust Senior Clinical Research Fellow and
AVSH is a Wellcome Trust Principal Research Fellow. Oxford University
was the sponsor for all the clinical trials reported here.
Conceived and designed the experiments: HM AH. Performed the
experiments: HM HF AP CS IP NB KW. Analyzed the data: HM HF
AP CS IP NB NA KW. Wrote the paper: HM. Other: Enrolled patients:
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