Exchanging ESAT6 with TB10.4 in an Ag85B Fusion
Molecule-Based Tuberculosis Subunit Vaccine: Efficient
Protection and ESAT6-Based Sensitive Monitoring of
Jes Dietrich,2Claus Aagaard, Robert Leah, Anja W. Olsen, Anette Stryhn, T. Mark Doherty,
and Peter Andersen
Previously we have shown that Ag85B-ESAT-6 is a highly efficient vaccine against tuberculosis. However, because the ESAT-6 Ag
is also an extremely valuable diagnostic reagent, finding a vaccine as effective as Ag85B-ESAT-6 that does not contain ESAT-6 is
a high priority. Recently, we identified a novel protein expressed by Mycobacterium tuberculosis designated TB10.4. In most
infected humans, TB10.4 is strongly recognized, raising interest in TB10.4 as a potential vaccine candidate and substitute for
ESAT-6. We have now examined the vaccine potential of this protein and found that vaccination with TB10.4 induced a significant
protection against tuberculosis. Fusing Ag85B to TB10.4 produced an even more effective vaccine, which induced protection
against tuberculosis comparable to bacillus Calmette-Gue ´rin vaccination and superior to the individual Ag components. Thus,
Ag85B-TB10 represents a new promising vaccine candidate against tuberculosis. Furthermore, having now exchanged ESAT-6 for
TB10.4, we show that ESAT-6, apart from being an excellent diagnostic reagent, can also be used as a reagent for monitoring
vaccine efficacy. This may open a new way for monitoring vaccine efficacy in clinical trials. The Journal of Immunology, 2005,
in many countries (1). The current vaccine against Mycobacterium
tuberculosis, Mycobacterium bovis bacillus Calmette-Gue ´rin
(BCG), has been extensively evaluated and demonstrated variable
protective efficacies ranging from 0 to 85% in different field trials
(2–4). A major contributor to this is thought to be the fact that the
protective efficacy of BCG wanes significantly over a period of
10–15 years (5). Therefore, although BCG is efficient against se-
vere forms of childhood tuberculosis (6–8), it is of limited use
against adult pulmonary disease. An improved second-generation
vaccine is therefore urgently needed, that can act as an efficient
prophylactic vaccine and/or a vaccine that can boost immunity in
BCG-vaccinated individuals. Alternative strategies in TB vaccine
development, such as subunit vaccines (9–12), genetic immuniza-
tion (13, 14), and attenuated strains of M. tuberculosis (15), are
currently being explored in many laboratories.
M. tuberculosis expresses and secretes three closely related my-
colyl transferases of 30–32 kDa mass, also known as the Ag85
uberculosis (TB)3is a re-emerging disease that remains
one of the leading causes of morbidity and mortality in
humans, and it represents a major public health problem
protein complex (Ag85A, -85B, and -85C). Both Ag85A and
Ag85B have been shown to be among the most potent Ag species
yet identified; they are major targets of human T cell responses to
M. tuberculosis and leading vaccine candidates (13, 16–21).
Ag85B has been shown to induce partial protection in murine
models of infection (13, 20). In guinea pigs vaccination with pu-
rified Ag85B protein also induces substantial protective immunity
against aerosol challenge with M. tuberculosis (11), and a rBCG
vaccine expressing and secreting the Ag85B protein (rBCG30) in-
duced stronger protective immunity against aerosol challenge with
M. tuberculosis than conventional BCG vaccine (22). In addition,
a vaccine based on recombinant modified vaccinia virus Ankara
expressing Ag85A (MVA85A) was shown to significantly boost
BCG-primed and naturally acquired antimycobacterial immunity
in humans (23).
Due to the complexity of the host immune response against
tuberculosis and the genetic restriction imposed by MHC mole-
cules, it has become clear that an effective subunit vaccine con-
taining multiple epitopes may be required to ensure a broad cov-
erage of a genetically heterogeneous population. Recently, we
showed that vaccination with a fusion protein consisting of Ag85B
and ESAT6 (Hybrid1) promoted a strong immune response, which
is highly protective against TB in the mouse, guinea pig, and non-
human primate models (10, 11, 24). This fusion Ag is also effec-
tive if delivered in a viral vector or as a DNA vaccine (25). Im-
portantly, Hybrid1 was more protective in both mouse and guinea
pig animal models than either of the single components (25). How-
ever, because the strongly immunodominant ESAT-6 Ag is an ex-
tremely valuable diagnostic reagent and the basis of a number of
commercial diagnostic tests (26–28), finding a vaccine as effective
as Hybrid1 that does not contain ESAT-6 is a high priority.
The ESAT-6 family is comprised of 22 low molecular mass
proteins, some of which can be divided further into subfamilies
due to high sequence relatedness of the individual genes and many
Department of Infectious Disease Immunology, Statens Serum Institute, Copenhagen,
Received for publication January 27, 2005. Accepted for publication March 10, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the European Commission, Contracts QLK2-CT-2001-
02018. Additional support for this study was provided by grants from the Danish
Research Council (to P.A.).
2Address correspondence and reprint requests to Dr. Jes Dietrich, Department of
Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, 2300 Køben-
havn S, Denmark. E-mail address: email@example.com
3Abbreviations used in this paper: TB, tuberculosis; BCG, bacillus Calmette-Gue ´rin;
DDA, dimethyl dioctadecyl ammonium bromide; MPL, monophosphoryl lipid A.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
of which are strongly immunogenic. We recently identified one
such subfamily containing the proteins TB10.4, TB10.3, and
TB12.9 (29, 30). Interestingly, we found that TB10.4 is strongly
recognized by BCG-vaccinated donors, and in TB patients TB10.4
was even more strongly recognized than ESAT-6 (30), suggesting
TB10.4 may be an ideal candidate to replace ESAT-6.
The purpose of the present study was to evaluate the potential of
TB10.4 as a substitute for ESAT-6 in the fusion protein, Ag85B-
ESAT-6. We show that TB10.4 is strongly immunogenic and in-
duces partial protection against TB. More importantly, when fused
to Ag85B, the resulting construct induced protective immunity at
the same level as BCG or Hybrid1 vaccination and at the same
time allowed the use of ESAT-6, not only as a diagnostic reagent,
but also as a reagent for monitoring vaccine efficacy.
Materials and Methods
Studies were performed with 8- to 12-wk-old female C57BL/6 ? BALB/c
F1mice, purchased from Taconic Farms. Infected animals were housed in
cages contained within laminar flow safety enclosures in a BSL-3 facility.
The use of mice was conducted in accordance with the regulations set
forward by the Danish Ministry of Justice and animal protection commit-
tees and in compliance with European Community Directive 86/609 and
the U.S. Association for Laboratory Animal Care recommendations for the
care and use of laboratory animals.
M. tuberculosis H37Rv and Erdman were both grown at 37°C on Lo ¨wen-
stein-Jensen medium or in suspension in Sauton medium enriched with
0.5% sodium pyruvate and 0.5% glucose.
Mice were immunized three times at 2-wk intervals s.c. on the back with
experimental vaccines containing 5 ?g of Ag85B, ESAT-6, TB10.4,
Ag85B-ESAT-6, or Ag85B-TB10.4/dose, emulsified in dimethyl diocta-
decyl ammonium bromide (DDA; 250 ?g/dose; Eastman Kodak) with
monophosphoryl lipid A (MPL; 25 ?g/dose; Avanti Polar Lipids) in a
volume of 0.2 ml. The adjuvans was prepared as follows. MPL was mixed
into sterile water containing 0.2% triethylamine. The mixture was heated in
a 70°C water bath for 30 s and then sonicated for 30 s. The heating and
sonicating procedure was repeated twice. The MPL was mixed with DDA
immediately before use.
At the time of the first subunit vaccination, one group of mice received
a single dose of BCG Danish 1331 (2.5 ? 105CFU) injected s.c. at the base
of the tail. Mice were challenged 10 wk after the first vaccination.
When challenged by the aerosol route, the animals were infected with
?100 CFU of M. tuberculosis Erdman/mouse. These mice were killed 6
wk after challenge. Numbers of bacteria in the liver, spleen, or lung were
determined by serial 3-fold dilutions of individual whole-organ homoge-
nates in duplicate on 7H11 medium. Organs from the BCG-vaccinated
animals were grown on medium supplemented with 2 ?g of 2-thiophene-
carboxylic acid hydrazide/ml to selectively inhibit the growth of the resid-
ual BCG bacteria in the test organs. Colonies were counted after 2–3 wk of
incubation at 37°C. Protective efficacies are expressed as log10bacterial
counts in immunized mice compared with bacterial counts in the adjuvant
controls. All results are based on groups of five animals.
Ag85B-TB10.4 plasmid construction
The plasmid encoding Ag85B-TB10.4 was generated by linkage of the
coding regions of the ?10-kDa TB10.4 polypeptide to the COOH-terminal
end of the ?30-kDa mature Ag85B polypeptide and fusing this directly to
a translational initial Met-Lys peptide in the context of the expression
vector pQE60 (Qiagen). These two genes correspond to the coding regions
of Rv1886c (Ag85B) and Rv0288 (TB10.4), respectively (31), except that
the 51-bp sequence encoding the NH2-terminal 17 aa residues of Ag85B
have been changed without changing the identity of the translated product
to destabilize the secondary structure of the RNA transcript for improved
translation initiation in Escherichia coli (32). Initially, the open reading
frame of Ag85B was generated by PCR amplification from a plasmid DNA
CATCAATGGGCCGCGACATCAAGGTTCAGTTCC), and the 3?-
specific primer (TATAAGGATCCTATGCGAACATCCCAGTGA
CGTTGCC). The fragment was directionally cloned into the NcoI-BamHI
sites of pQE60 as a BspHI-BamHI fragment. The open reading frame of
TB10.4 was generated by PCR amplification from M. tuberculosis H37Rv
chromosomal DNA and the 5?-specific primer (GCATGGCGCCGGCAT
GTCGCAAATCATGTACAACTACC) and the 3?-specific primer (GCA
TAAGCTTCTAGCCGCCCCATTTGGCGGCTTCGGCCG). This frag-
ment was cloned into the unique NarI-HindIII sites of the previously
constructed pQE60/Ag85B plasmid. The NarI site is situated just upstream
the 3? end of the Ag85B open reading frame. The final plasmid construct
was transformed into the E. coli strain NF1830 for production of Ag85B-
TB10.4. The genotype of NF1830 is galUK ?lacX74 rpsL thi recA1
araD139? (araABOIC-leu)7679 F? proAB?lacIq1 lacZ::Tn5 lacY?. The
sequence of the Ag85B-TB10.4 DNA insert was verified by sequencing
using the dideoxy chain termination method. Sequences were analyzed
with Vector NTI Suite software package (InfoMax).
Expression of Ag85B-TB10.4 in E. coli NF1830
E. coli NF1830 expressing the recombinant expression plasmid was grown
in culture flasks to an OD of ?0.5 at 600 nm before induction with 1 mM
isopropyl ?-D-thiogalactopyranoside. After induction, growth was contin-
ued for 4 h at 37°C. Cells were harvested by centrifugation at 15,000 ? g
for 10 min at 4°C and were stored at ?20°C.
Production of Ag85B-TB10.4
Frozen cells were suspended in a 2.5 ml/g cell paste in buffer A (25 mM
Tris-HCl (pH 7.75) and 4 M urea) and subjected to cell disruption for 3 min
at 0°C using a BeadBeater (BioSpec Products) and 106-?m glass beads
(Sigma-Aldrich) according to the manufacturer’s instructions. The homog-
enized mixture was diluted in 5 vol of buffer A, and the insoluble material
containing the Ag85B-TB10.4 aggregated in inclusion bodies was washed
three times for 1 h at 4°C. The insoluble material was finally precipitated
by centrifugation at 15,000 ? g for 10 min at 4°C, and Ag85B-TB10.4 was
solubilized and extracted for 2 h at 4°C in 1 vol of buffer B (25 mM
Tris-HCl (pH 7.75) and 8 M guanidium chloride). Two successive chro-
matographic purification steps were involved in purification of Ag85B-
TB10.4. The initial gel filtration was performed on a Sephacryl S-300
(Pharmacia Biotech) column using buffer B as eluent. Fractions containing
Ag85B-TB10.4 were pooled and further purified by an immobilized metal
affinity chromatography purification step using HiTrap Chelating HP col-
umn (Amersham Biosciences) loaded with Cu2?. Ag85B-TB10.4 was
eluted with elution buffer (25 mM Tris-HCl (pH 7.75), 6 M guanidium
chloride, and 1 M NH4Cl) using a linear gradient. Fractions containing
Ag85B-TB10.4 were identified by SDS-PAGE, pooled, and dialyzed to the
final storage buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 40%
glycerol). The concentration of the purified Ag85B-TB10.4 was deter-
mined by bicinchoninic acid test using micro bicinchoninic acid protein
assay reagent kit (Pierce).
Lymphocytes from spleens were obtained as described previously (33).
Blood lymphocytes (PBMCs) were purified on a density gradient. Cells
pooled from three to five mice in each experiment were cultured in mi-
crotiter wells (96-well plates; Nunc) containing 2 ? 105cells in a volume
of 200 ?l of RPMI 1640 supplemented with 5 ? 10?5M 2-ME, 1% (v/v)
premixed penicillin-streptomycin solution (Invitrogen Life Technologies),
1 mM glutamine, and 5% (v/v) FCS. Based on previous dose-response
investigations, the mycobacterial Ags were all used at 5 ?g/ml, whereas
Con A was used at a concentration of 1 ?g/ml as a positive control for cell
viability. All preparations were tested in cell cultures and found to be
nontoxic at the concentrations used in the present study. Supernatants were
harvested from cultures after 72 h of incubation for the investigation of
As previously described (30), synthetic overlapping peptides (16- or 18-
mer) covering the complete primary structure of the three proteins were
synthesized by standard solid phase methods on an ABIMED peptide syn-
thesizer at the Department of Infectious Diseases and Immunohematology/
Bloodbank C5-P, Leiden University Medical Center (TB10.4), or at Scha-
fer-N. The peptides were purified by reverse phase HPLC. Purified
peptides were lyophilized and stored dry until reconstitution in PBS.
6333 The Journal of Immunology
Microtiter plates (96 well; Maxisorb; Nunc) were coated with monoclonal
hamster anti-murine IFN-? (Genzyme) in PBS at 4°C. Free binding sites
were blocked with 1% (w/v) BSA-0.05% Tween 20. Culture supernatants
were tested in triplicate, and IFN-? was detected with a biotin-labeled rat
anti-murine mAb (clone XMG1.2; BD Pharmingen). rIFN-? (BD Pharm-
ingen) was used as a standard.
The ELISPOT technique has been described previously (34). Briefly, 96-
well microtiter plates (Maxisorp; Nunc) were coated with 2.5 ?g of mono-
clonal hamster anti-murine IFN-?/well (Genzyme). Free binding sites were
blocked with BSA, followed by washing with PBS-0.05% Tween 20. Anal-
yses were always conducted on cells pooled from three to five mice. Cells
were stimulated with 5 ?g of ESAT-6 or TB10.4/ml in modified RPMI
1640 for 48 h. The cells were removed by washing, and the site of cytokine
secretion was detected with a biotin-labeled rat anti-murine IFN-? mAb
(clone XMG1.2; BD Pharmingen) and phosphatase-conjugated streptavi-
din (Jackson ImmunoResearch Laboratories). The enzyme reaction was
developed with 5-bromo-4-chloro-3-indolylphosphate (Sigma-Aldrich).
Blue spots were counted microscopically. The correlation between the
number of cells per well, and the number of spots was linear at concen-
trations of 2 ? 105to 2.5 ? 103cells/well. Wells with ?10 spots were not
used for calculations.
FACS analysis of lymphocytes
Cells were isolated, as described above, from the blood and spleen of mice.
Cells (2 ? 105) were stimulated overnight with 2 ?g/ml TB10.4 or 10
?g/ml peptide and subsequently incubated for 6 h with 10 ?g/ml brefeldin
A (Sigma-Aldrich). Thereafter, cells were incubated with Fc-block (BD
Pharmingen), washed in FACS buffer (PBS containing 0.1% sodium azide
and 1% FCS), and stained for surface markers using CD4-PerCP and CD8-
allophycocyanin (BD Pharmingen). Cells were then washed in PBS, per-
meabilized using the Cytofix/Cytoperm kit (BD Pharmingen), and stained
intracellularly with PE-labeled anti-IFN-? mAb. After washing, cells were
finally resuspended in FACS buffer and analyzed by FACS (BD
Assessment of experiments was conducted using ANOVA. Differences be-
tween means were assessed by Tukey’s test. A value of p ? 0.05 was
considered significant. The computer program PRISM (GraphPad) was
used for these calculations.
Immune responses generated to TB10.4 and ESAT-6
Both TB10.4 and ESAT-6 are part of the same gene family (29),
but only the gene for TB10.4 is present in BCG. The genome
region containing ESAT-6 was lost as one of the first deletion
mutations during the attenuation of BCG (35). We therefore ini-
tially analyzed the recognition of TB10.4 and ESAT-6 in M. tu-
berculosis-infected and BCG-vaccinated mice. The latter was im-
portant, because the ability to boost BCG-generated immunity is
considered a desirable function for any vaccine to be used against
TB. C57BL/6 ? BALB/c mice were vaccinated with BCG or chal-
lenged with 100 CFU of M. tuberculosis via the aerosol route. At
the indicated time points after vaccination or infection, lympho-
cytes obtained from spleens of infected or BCG-vaccinated mice
were cultured 3 days in the presence of recombinant TB10.4 or
ESAT-6 and analyzed by ELISPOT for the presence of IFN-?-
producing cells. The results showed that TB10.4 and ESAT-6 were
equally well recognized in M. tuberculosis-infected mice (Fig. 1A).
However, in BCG-vaccinated mice, ESAT-6 (as expected) was not
recognized, although stimulation with TB10.4 induced a strong
response (Fig. 1B).
We next measured the recognition of TB10.4 and ESAT-6 after
immunizing with these Ags. TB10.4 or ESAT-6 was emulsified in
DDA and MPL, an adjuvant that has previously been used suc-
cessfully to induce a highly efficient Th1 response protective
against TB (33, 36). The vaccine was given three times at 2-wk
intervals, and the immune response induced in the spleen (or
blood, data not shown) was investigated 2 wk after the last booster
injection. TB10.4 and ESAT-6 (in DDA/MPL) were both highly
immunogenic and strongly recognized in spleen and blood (Fig.
1C and data not shown).
Epitope recognition in TB10.4
To further characterize the response against TB10.4 after immu-
nization, we next analyzed the pattern of epitope recognition after
naive mice (CD4 or CD8) or TB10.4-vaccinated mice (CD4-TB10.4 and
CD8-TB10.4) stimulated with TB10.4 peptides 1–9 and costained for CD4
or CD8 as indicated. B, FACS analysis of spleen cells stimulated with
TB10.4 peptides 1–9 and analyzed for CD4 and CD69 surface expression.
The results show the mean fluorescence intensity (MFI) of CD69 on CD4-
A, FACS analysis of IFN-? expression by spleen cells from
berculosis infection or after BCG vaccination. A and B, Lymphocytes from
spleen were stimulated with TB10.4 (f) or ESAT-6 (?) at the indicated time
points after infection (A) or after BCG vaccination (B), and the number of
IFN-?-producing cells per 5 ? 105cells was determined by ELISPOT. C, In
vitro IFN-? responses of spleen cells from mice vaccinated three times with
TB10.4, taken 2 wk after final vaccination with TB10.4. Cells were pooled
from five mice per group. Values represent the mean and SEM of triplicate
Recognition of TB10.4 and ESAT-6 in mice after M. tu-
6334 EXCHANGING ESAT6 WITH TB10.4 IN A TB SUBUNIT VACCINE
immunization with the soluble protein. To achieve this, overlap-
ping 16- to 18-aa peptides (10-aa overlap) covering the entire
TB10.4 molecule were synthesized (30). Mice were immunized
with TB10.4 mixed with the adjuvant DDA/MPL, and lympho-
cytes from immunized mice were cultured 3 days in the presence
of either medium or each of the peptides covering the entire
TB10.4 molecule and analyzed by FACS for the presence of IFN-
?-producing cells. These results clearly showed that P3, P7, and P8
primarily stimulated CD4 cells to express IFN-? (Fig. 2A).
Analysis of IFN-? secretion by ELISA confirmed these results
(data not shown). Furthermore, the same appeared to be true for
cell activation, because CD4 cells stimulated with these three pep-
tides actively up-regulated the activation marker CD69 whereas
CD8 cells did not (Fig. 2B and data not shown).
Protective efficacy of TB10.4 in a TB vaccine
We next analyzed the protective efficacy of a vaccine based on
TB10.4 compared with one containing ESAT-6. Because the vac-
cines BCG and Hybrid1 (Ag85B-ESAT-6) have demonstrated
high efficacy against TB infection in animal models (37), these
were included in the experiment as a gold standard against which
efficacy can be assessed (25). Mice were vaccinated with 5 ?g of
TB10.4, ESAT-6, or Hybrid1 in DDA/MPL or with BCG. Ten
weeks after the first vaccination, the mice were challenged by the
aerosol route with virulent M. tuberculosis. Six weeks after chal-
lenge, the mice were killed, and the bacterial burden (CFU) was
measured in the lung (Fig. 3). The results showed that vaccination
with TB10.4 reduced the bacterial burden of M. tuberculosis by 0.6
log10? 0.15 CFU compared with naive infected mice (p ? 0.001),
an amount that was not significantly different from the protection
induced by ESAT-6 (p ? 0.05). Vaccinating with Hybrid1 or
BCG reduced the bacterial numbers (CFU) in the lung by ?1
log10, in agreement with previous results (37) (Fig. 3). In both the
H1 and BCG groups, the protection differed significantly (p ?
0.05) from that in groups vaccinated with TB10.4 or ESAT-6).
Thus, TB10.4 equals ESAT-6 in terms of both immunogenicity
and protective efficacy.
compared with unvaccinated controls challenged by the aerosol route with
virulent M. tuberculosis 10 wk after the first vaccination. Six weeks after
challenge, the mice were killed, and the bacterial burden (CFU) was mea-
sured in the lung. The Ags used for vaccination are indicated. Results are
the mean and SEM from groups of five mice.
Protection in vaccinated mice (expressed as log10CFU)
in the expression vector pQE60 along with the predicted 383-aa residue sequence of Ag85B-TB10.4. The underlined nucleotides at positions 7–57 show
the nucleotide sequence changed without altering the identity of the translated product for improving the translation initiation in E. coli. Œ, Direct
translational fusion position between the COOH-terminal end of Ag85B and the NH2-terminal end of TB10.4. B, Accumulation of Ag85B-TB10.4 produced
in E. coli. The E. coli strain NF1830 transformed with the expression vector harboring the Ag85B-TB10.4 was grown and induced with IPTG. Coomassie
Blue-stained 10% SDS-PAGE of total E. coli lysate before (lane T0h), 2 h after (lane T2h), and 4 h after (harvest; lane T4h) induction with IPTG. The arrow
indicates accumulation of the ?41-kDa Ag85B-TB10.4 fusion protein. C, Purification of Ag85B-TB10.4. Ag85B-TB10.4 was purified from inclusion
bodies by two successive chromatographic purification steps to ?98% purity. The figure shows a Coomassie Blue-stained 10% SDS-PAGE of 1 ?g of
purified Ag85B-TB10.4 (85B-10.4; lane 3) along with 1 ?g of purified rAg85B (lane 1) and 1 ?g of purified rTB10.4 (lane 2). Marker (M) in B and C
(lane 4) is the SeaBlue Pre-Stained (Invitrogen Life Technologies) m.w. standard.
A, Gene sequence and translation product of Ag85B-TB10.4. The nucleotide sequence of the Ag85B-TB10.4 open reading frame inserted
6335The Journal of Immunology
Ag85B-TB10.4, a new polypeptide TB vaccine
Because TB10.4 proved equal to ESAT-6 in all the above exper-
iments, it constituted an interesting potential substitution candidate
for ESAT-6. We therefore produced TB10.4 as a recombinant fu-
sion protein with Ag85B and analyzed the protective efficacy of
this new vaccine, Ag85B-TB10.4 (Fig. 4A). Ag85B-TB10.4 was
recombinantly produced in E. coli. The fusion protein was purified
by a combination of gel filtration and immobilized metal affinity
chromatography. Ag85B-TB10.4 was purified to homogeneity and
finally analyzed by SDS-PAGE together with the individual pro-
teins, Ag85B and TB10.4 (Fig. 4, B and C). To analyze the im-
munogenicity of Ag85B-TB10.4 and to clarify whether both com-
ponents of the fusion protein were recognized by the immune
system after processing, groups of mice were immunized with the
fusion protein or with the single components, Ag85B or TB10.4,
emulsified in MPL and DDA. As negative control, a group of mice
received the adjuvant combination alone (data not shown). One
week after the last injection, mice were bled, and IFN-? release
was evaluated after in vitro stimulation of purified PBMCs with
different concentrations of Ag85B and TB10.4 (5, 1.25, and 0.25
?g/ml; Fig. 5). Immunization with either Ag85B-TB10.4 or
Ag85B induced a strong IFN-? response upon restimulation with
Ag85B, indicating that the fusion to TB10.4 did not significantly
affect the recognition of Ag85B by the immune system (Fig. 5).
Interestingly, when TB10.4 was fused to Ag85B, it was even more
immunogenic than the TB10.4 protein alone (Fig. 5).
We next compared the recognition of TB10.4 and ESAT-6 when
these two molecules were fused to Ag85B. Mice were immunized
with either Ag85B-TB10.4 or Hybrid1. and 1 or 2 wk after the last
immunization, cells from blood or spleen were cultured in the pres-
ence of TB10.4 or ESAT-6 and analyzed by ELISA for IFN-?
secretion. The results showed that TB10.4 is more strongly recog-
nized than ESAT-6 when fused to Ag85B, even though the two
Ags displayed similar levels of immunogenicity when given as
separate proteins in DDA/MPL (Fig. 6 and data not shown). A
comparison of the immunogenicity of Ag85B in Ag85B-TB10.4 or
Hybrid1 showed that Ag85B was strongly recognized after immu-
nization with both fusion proteins (Fig. 6).
In conclusion, the fusion between Ag85B and TB10.4 did not
reduce the response against either of the proteins. In fact, immu-
nizing with Ag85B-TB10.4 may increase the number of TB10.4-
specific T cells compared with immunizing with only TB10.4.
Protective efficacy of Ag85B-TB10.4 in a mouse TB infection
One of the requirements for any new vaccine is that it should be at
least equal in terms of protection to the best candidates already
identified. We therefore compared Ag85B-TB10.4 to Hybrid1 in
terms of protective efficacy. Additionally, the protective efficacy of
the fusion protein Ag85B-TB10.4 was compared with that of a
simple mix of Ag85B and TB10.4 or the single components given
separately. The molar concentrations of Ag85B and TB10.4 in the
mixture were adjusted to be the same level as the concentrations of
the two components in the fusion protein. A group of mice receiv-
ing the adjuvant combination alone and a group of naive mice were
included as controls, and as a positive control for protection, mice
were immunized once with BCG. Ten weeks after the first vacci-
nation, the mice were challenged by the aerosol route with virulent
M. tuberculosis. Six weeks after challenge, the mice were killed,
and the bacterial numbers were determined in the lungs. All vac-
cinated groups showed a protection significantly different from that
of the naive group (p ? 0.001). Ag85B-TB10.4 induced high lev-
els of protection (1.06 ? 0.02 log10CFU reduction in the lung),
comparable to that induced by BCG or Hybrid1 (Fig. 7) and sig-
nificantly higher than that induced by Ag85B (p ? 0.05) or
TB10.4 (p ? 0.001). Although not statistically significant in this
unvaccinated controls challenged by the aerosol route with virulent M.
tuberculosis 10 wk after the first vaccination. Six weeks after challenge, the
mice were killed, and the bacterial burden (CFU) was measured in the lung.
The Ags used for vaccination are indicated. Results are the mean and SEM
from groups of five mice.
Protection in mice (expressed as log10CFU) compared with
PBMC from mice vaccinated with Ag85B-TB10.4 or with the single com-
ponents, Ag85B or TB10.4, as indicated, taken 1 wk after final vaccination.
Values are the mean and SEM of groups of five mice.
In vitro IFN-? responses to Ag85B (E) and TB10.4 (f) of
(E), or TB10.4 (‚) of PBMC (A) taken 1 wk after final vaccination or of
spleen-derived T cells (B) taken 2 wk after final vaccination from mice
vaccinated with Ag85B-TB10.4 or Hybrid1, as indicated. Values are the
mean and SEM of groups of five mice.
A and B, In vitro IFN-? responses to Ag85B (f), ESAT-6
6336EXCHANGING ESAT6 WITH TB10.4 IN A TB SUBUNIT VACCINE
experiment, it was generally observed that the mixture of Ag85B
and TB10.4 induced slightly lower protection than Ag85B-
TB10.4. Thus, the fusion of TB10.4 and Ag85B constitutes a very
Monitoring the efficacy of experimental vaccines through
ESAT-6 responses after infection
ESAT6 is a well-known diagnostic marker of ongoing infection,
and recent work in both clinical settings and cattle indicated a
correlation between the magnitude of ESAT-6 responses and the
extent of the disease (38, 39). We therefore investigated the mag-
nitude of ESAT-6 responses after infection in animals vaccinated
with vaccines providing different levels of protection against M.
tuberculosis. This experiment took advantage of the fact that
through the development of Ag85B-TB10.4 we could reserve
ESAT6 as a diagnostic/infection marker. Ten weeks after the first
vaccination, the mice were challenged by the aerosol route with
virulent M. tuberculosis. Twenty days after challenge, mice were
bled, and IFN-? release was evaluated after in vitro stimulation of
purified PBMCs with ESAT-6. Six weeks after challenge, mice
were killed, and bacterial numbers (log10CFU) were determined in
the lungs. The results showed that the response against ESAT-6
was indeed highest in infected nonvaccinated mice (Fig. 8A). In
mice vaccinated with Ag85B or TB10.4, we observed a decreased
ESAT-6 response after challenge, which declined even further in
mice vaccinated with BCG and Ag85B-TB10.4. Importantly, by
comparing the ESAT-6 response with the subsequent protection
against TB (Fig. 8), a strong correlation (correlation coefficient ?
0.83) was observed between the postchallenge ESAT-6 response
and the subsequent outcome of the disease, and thereby an inverse
relationship to the protective efficacy of the vaccines. Thus, the
magnitude of the response against ESAT-6 appears to be an ac-
curate correlate of disease development/vaccine efficacy.
The availability of the M. tuberculosis genome sequence and the
current efforts to sequence a large number of additional mycobac-
terial genomes have set the stage for postgenomic approaches to
the identification of novel Ags. One such novel Ag is the recently
identified TB10.4 (29), a protein of unknown function. The lack of
diversity in the TB10.4 sequence originating from 13 clinical iso-
lates of M. tuberculosis obtained from different geographical
locations (30) suggests that it has an important, but yet uniden-
tified, biological function. The expression of TB10.4 is not re-
stricted to M. tuberculosis, because it is recognized by T cells
from BCG-vaccinated individuals as well as TB patients (30).
In the present study we have evaluated the potential of TB10.4
as a subunit vaccine against TB. Initially, we showed that T cells
specific for TB10.4 were generated after the infection of mice with
M. tuberculosis or BCG (Fig. 1). Immunizing with TB10.4 in-
duced a strong CD4 T cell response against epitopes contained
within the peptides P3, P7, and P8 (Fig. 2). Moreover, the protec-
tive efficacy of TB10.4 (in DDA/MPL) was comparable to that of
ESAT-6 (Fig. 3). We have previously shown that the protective
efficacy of a subunit vaccine based on the fusion of ESAT-6 and
Ag85B was significantly greater than that of a vaccine based solely
on ESAT-6 or Ag85B (10, 25). Not all Ags tested show such
additive effects, and it is for this reason that ESAT-6 was used in
the Hybrid1 vaccine despite its well-known utility as a diagnostic
reagent (26–28). Because there is obviously value in reserving
ESAT-6 for diagnostic purposes and based on the encouraging
preliminary results in mice (Fig. 1) and humans (30), we evaluated
TB10.4 as a substitute for ESAT-6 as a fusion partner for Ag85B
The subunit vaccine composed of Ag85B-TB10.4 and DDA/
MPL did generate a strong, specific immune response in mice.
Importantly, both components of Ag85B-TB10.4 were recognized
to at least the same degree as after vaccination based on the single
components (Fig. 5). In fact, careful comparative studies showed
that TB10.4 was more strongly recognized after immunization
with Ag85B-TB10.4 than with TB10.4 alone. This is in contrast to
results obtained with ESAT-6, which appears to be subdominant
when fused to Ag85B (Fig. 6). Vaccinating mice with Ag85B-
TB10.4 induced a level of protection against TB comparable to
that produced by BCG and better than that achieved by vaccinating
with either of the single proteins. Ag85B-ESAT6 also induced a
protection comparable to that of Ag85B-TB10.4 and better than
that with either Ag85B or ESAT-6 (Fig. 7) (11). Taken together,
the fusion of TB10.4 and Ag85B did not decrease the immunoge-
nicity of either TB10.4 or Ag85B, and linking to Ag85B may, in
fact, have a beneficial effect on the immunogenicity of TB10.4. In
addition, Ag85B-TB10.4 formulated in DDA/MPL constituted an
efficient vaccine against TB.
Using a multicomponent vaccine may generate a broad, strongly
recognized response that would be beneficial for vaccination of
genetically diverse human populations. Even though TB10.4 is
recognized by cells from most TB patients (30), there is still the
possibility that a vaccine based solely on TB10.4 (or any other
single, nonessential protein, for that matter) may select for M. tu-
berculosis bacteria that do not express TB10.4. In line with this, it
could be speculated that the bacteria may be able to compensate
for the apparent lack of TB10.4 sequence polymorphism by having
duplicated the genes encoding major T cell Ags, leading to several
copies (homologues) of proteins that can substitute each other
functionally, but differ in their immunodominant epitopes. Tightly
controlled expression of these homologues may result in Ag vari-
ation and immune evasion. The probability of such an evasion
mechanism is probably decreased when using a multicomponent
vaccine. Our study clearly demonstrates that a subunit vaccine
based on a fusion protein between Ag85B and TB10.4 is able to
induce efficient protection against TB. Moreover, because both
Ag85B and TB10.4 are expressed by BCG, it could be speculated
that Ag85B-TB10.4 is an ideal candidate to boost BCG-generated
immunity and thus increase protection against TB compared with
BCG alone. Experiments are presently ongoing in our laboratory
20 days after infection from vaccinated mice compared with protection
(expressed as log reduction in CFU) (B). Values are the mean and SEM of
groups of five mice assessed by ELISA.
A, In vitro IFN-? responses to ESAT-6 of spleen cells taken
6337The Journal of Immunology
by which we aim to examine the potential of Ag85B-TB10.4 to be
used as a BCG booster vaccine.
Another interesting point in the present work was the inverse
relationship between the postchallenge ESAT-6 response and the
protective efficacy of the vaccines tested. Recent work in a model
of TB in cattle (38) or in nonhuman primates (24) demonstrates
that the magnitude of the in vitro response against the M. tuber-
culosis virulence factor ESAT-6 after infection correlated very
well with bacterial load and also with disease-associated pathol-
ogy. This suggests that a most accurate correlate of vaccine effi-
cacy is not necessarily the immune response after vaccination, but
the response after infection, and this is supported by a recent study
(25). Data from human studies are consistent with this; individuals
recently exposed to M. tuberculosis who subsequently made strong
responses against ESAT-6 were almost 10 times more likely to
progress rapidly to clinical tuberculosis than were low responders
(39). All these data can be explained by hypothesizing that the
ESAT-6 response after infection correlates with Ag load and there-
fore, ultimately, with bacterial load. Not surprisingly, if bacterial
replication is restricted by an effective memory immune response,
it would then be predicted that the response to ESAT-6 postinfec-
tion would be reduced. The data shown in Fig. 8 confirm this quite
strikingly. Because none of the vaccines shown in this experiment
contains ESAT-6, the lymphocytes responsive to this Ag must re-
sult from M. tuberculosis infection (and, indeed, uninfected con-
trols do not respond to ESAT-6; data not shown). There is a perfect
correlation between restriction of bacterial growth (protection) and
diminished ESAT-6 response postinfection.
If this finding proves to be robust, it opens the way for moni-
toring vaccine efficacy in clinical trials. One of the greatest chal-
lenges facing phase III clinical trials is the long incubation period
between infection and development of overt disease and the fact
that the majority of infected individuals develop latent infections
(40). Because the clinical end point used has traditionally been
overt, symptomatic TB, this has meant that clinical trials have
needed very large cohorts and extended periods of follow-up, ren-
dering them almost prohibitively expensive. If it proves feasible to
measure the rate of productive infection, rather than the rate of
subsequent disease, this would dramatically improve our ability to
conduct clinical trials in a timely fashion.
The technical help of Charlotte Fjordager, Lene Rasmussen, Bente Sølvig,
and Karina Fu ¨rst Andersen is gratefully acknowledged. We thank Dr. Niels
Fiil (Institute of Microbiology, Copenhagen, Denmark) for use of the E.
coli production strain NF1830. We thank Charlotte Green Jensen for con-
struction of the Ag85B-TB10.4 production vector.
The authors have no financial conflict of interest.
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6339The Journal of Immunology