BASIC IMMUNOLOGY doi: 10.1111/j.1365-3083.2011.02531.

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Immunogenicity and Protective Efficacy of a Fusion

Protein Vaccine Consisting of Antigen Ag85B and
HspX against Mycobacterium tuberculosis Infection
in Mice
Q. Li* , H. Yu*, Y. Zhang*à, B. Wang§, W. Jiang*, Z. Da*, Q. Xian–, Y. Wang–, X. Liu  & B. Zhu*

*Lanzhou Center for Tuberculosis Research and
Institute of Pathogenic Biology, School of
Basic Medical Sciences, Lanzhou University,
Lanzhou, China;  Institute of Pathophysiology,
School of Basic Medical Sciences, Lanzhou
University, Lanzhou, China; àDepartment of
Molecular Microbiology and Immunology,
Bloomberg School of Public Health, Johns
Hopkins University, Baltimore, MD, USA;
§Lanzhou Institute of Biological Products,
Lanzhou, China; and –ABSL-3 Lab, Wuhan
University, Wuhan, China
Received 4 October 2010; Accepted in revised
form 26 January 2011
Correspondence to: Xin Liu, B. Zhu, Institute
of Pathogenic Biology, School of Basic Medical
Sciences, Lanzhou University, Lanzhou 730000,
China. E-mail: bdzhu@lzu.edu.cn;
liux@lzu.edu.cn
Abstract
Subunit vaccines have the potential advantage to boost Mycobacterium bovis
Bacillus Calmette-Guérin (BCG)-primed immunity in adults. However, most
candidates are antigens highly expressed in replicating bacilli but not in dor-
mant or persisting bacilli, which exist during Mycobacterium tuberculosis infec-
tion. We constructed M. tuberculosis fusion protein Ag85B-Mpt64190–198-
HspX (AMH) and Ag85B-Mpt64190–198-Mtb8.4 (AMM), which consist of
Ag85B, the 190–198 peptide of Mpt64, HspX (Rv2031c) and Mtb8.4
(Rv1174c), respectively. AMH and ⁄ or AMM were mixed with adjuvants com-
posed of dimethyl-dioctyldecyl ammonium bromide and BCG polysaccharide
nucleic acid (DDA-BCG PSN) to construct subunit vaccines. Mice were
immunized thrice with Ag85B, AMH and AMM vaccines and the immunoge-
nicity of the fusion protein vaccines was determined. Then, mice were primed
with BCG and boosted twice with Ag85B, AMH, AMM and AMM + AMH
vaccines, respectively, followed by challenging with M. tuberculosis virulent
strain H37Rv, and the immune responses and protective effects were mea-
sured. It was found that mice immunized with AMH vaccine generated high
levels of antigen-specific cell-mediated responses. Compared with the group
injected only with BCG, the mice boosted with AMM, AMH and
AMM + AMH produced higher levels of Ag85B-specific IgG1 and IgG2a and
IFN-c-secreting T cells upon Ag85B and Mycobacterium tuberculosis purified
protein derivative (PPD) stimulation. It is interesting that only mice boosted
with AMM + AMH had significantly lower bacterial count in the lungs than
those receiving BCG, whereas mice boosted with AMH or AMM did not. The
results suggest that AMH consisting of HspX, the antigen highly expressed in
dormant bacilli, could be combined with antigens from replicating bacilli to
enhance BCG primed immunity so as to provide better protection against both
growing and non-growing bacteria that occur during the infection process.

children against disseminated TB [6, 7]. However, the

Introduction
One-third of the world population is latently infected
with Mycobacterium tuberculosis, and 5–10% of which will
develop into active tuberculosis (TB) when the host
immune system is compromised [1]. The dormant
M. tuberculosis, existing in active TB as well as latent
infection [2–4], persist in the host [5], which results in
less facility to eradicate TB. BCG is the only TB vaccine
used in the clinic and proves to be effective to protect
BCG-induced protective immunity varies from 0% to
80% in different populations and wanes in adults. More
important, BCG does not prevent reactivation of dormant
bacilli [7, 8]. It is urgent to search for novel TB vaccines
and immunization strategies. One practical way is to
boost BCG with subunit vaccines so as to improve BCG-
primed immunity in adult.
Mycobacterium tuberculosis expresses different genes at
different conditions so as to adapt to different environ-

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Q. Li et al.
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ments. Some genes are up-regulated in dormant phase to
survive under suboptimal or stress conditions, such as
nutrient and oxygen deprivation. It was reported that
latency-associated antigens could induce antigen-specific
IFN-c production, CD4+ T cell proliferation and cyto-
kine expression in peripheral blood mononuclear cells
from persons with active and latent TB infection [9].
HspX, also known as a-crystallin, is one of genes induced
by hypoxia. It is up-regulated significantly in non-repli-
cating conditions but is poorly expressed in replicating
condition [10]. Increased HspX mRNA was detected in
the lungs of patients with chronic TB [11]. In addition,
HspX is capable of activating T cells from 80% of house-
hold contacts with TB patients, 90% of health care workers
and 50% of controls [12]. These findings indicate that
HspX may be an important dormancy antigen that could
be effectively recognized by human T cells [13].
Many antigens expressed in bacterial replicating stage
have been chosen as candidate antigens for new vaccines.
However, antigens highly expressed in dormant stage
have not been widely evaluated until now [14]. We had
developed a fusion protein Ag85B-Mpt64190–198-Mtb8.4
(AMM) previously and showed that it could elicit strong
humoral and cell-mediated immune responses when
formulated in chitosan microspheres [15] or in dimethyl-
dioctyldecyl ammonium bromide and BCG polysaccha-
ride nucleic acid (DDA-BCG PSN) adjuvants [16]. AMM
is composed of Ag85B, 190–198 peptide of Mpt64 and
Mtb8.4, which are all expressed in replicating bacteria.
In this study, Mtb8.4 from AMM was replaced by HspX
antigen highly expressed in dormant bacteria, to con-
struct a novel fusion protein Ag85B-Mpt64190–198-HspX
(AMH). And then, the immunogenicity and protective
efficacy of the AMH vaccine were evaluated.
Materials and methods
Construction of plasmid pET-28a Ag85B-Mpt64190–198-
HspX. The DNA sequences encoding Ag85B and HspX
were amplified by polymerase chain reaction (PCR) from
M. tuberculosis H37Rv chromosomal DNA template with
the primers Ag85BF, 5¢-AATGGATCCTTCTCCCGGCC
GGGGCT-3¢(BamHI), and HspXF1, 5¢- ATAGAG-
CTCATGGCCACACCCTTC-3¢(SacI), as forward primers
and the primers Ag85BR, 5¢-ATTGAGCTCGCCGGC
GCCTAACGAACTCTGGAG-3¢(SacI), and HspXR, 5¢-
ACGAAGCTTTCAGTTGGTGGACCG-3¢(HindIII), as
reverse primers. The PCR fragment of Ag85B was cloned
into the BamHI and SacI site of pET-28a to construct
the plasmid pET-28a Ag85B. Subsequently, the fragment
of Mpt64190–198-HspX was generated by PCR from PCR
product of HspX as template with the forward primer
HspXF2, 5¢-ATAGAGCTCTTCGCAGTCACGAACGAC
GGGGTGATTATGGCCACCACCCTTC-3¢(SacI), and
the reverse primer HspXR and was cloned into the
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Immunogenicity and Protective Efficacy of a Fusion Protein Vaccine 569
unique site SacI and HindIII of the previously con-
structed pET-28a Ag85B plasmid. The correct DNA
construct containing the appropriate inserts was con-
firmed by DNA sequencing.
Expression and purification of AMH fusion protein. The
plasmid pET-28a AMH was transformed into the Escheri-
chia coli strain BL21 for production of the fusion protein
AMH. E. coli BL21 expressing AMH was cultured in LB
medium for 2 h at 37 C before induction with 1 mM iso-
propylb-D-thiogalactopyranoside. After induction, growth
was continued for 4 h at 37 C when the bacterial cells
were harvested by centrifugation at 12,000 g for 10 min at
4 C. Then, cells were suspended in buffer A without urea
(sodium phosphate buffer 0.1 M, Tris–Cl 0.01 M, pH 8.0)
at 5 ml per gram wet weight and sonicated on ice at 200–
300 W for 30 min with 1-s cooling period between each
1-s bursting. The insoluble material containing AMH in
inclusion bodies was precipitated by centrifugation at
12,000 g for 10 min at 4 C, and AMH was solubilized
and extracted overnight at 4 C in buffer B (urea 8 M,
sodium phosphate buffer 0.1 M, Tris–Cl 0.01 M, pH 8.0).
AMH protein was subsequently purified by Ni-NTA His
resin-columns (Merck KGaA, Darmstadt, Germany)
according to the manufacturer’s instructions. Fractions
containing AMH were identified by 12% SDS-PAGE and
pooled. Finally, the pooled fractions were dialysed against
urea concentration gradient (6, 4, 2, 1, 0.5 and 0 M urea
with 5 mM Tris–Cl, pH 7.9) for 12 h at each concentration
at 4 C. The concentration of the pure AMH was deter-
mined by the Lowry protein assay.
Subunit vaccine preparation. AMM, Ag85B and BCG
PSN were prepared as described previously [16]. The
preparation of AMH and AMM + AMH vaccines is
described below. AMM and AMH were suspended in
phosphate-buffered saline (PBS) (0.2 mg ⁄ ml), and BCG
PSN was suspended in saline (0.6 mg ⁄ ml). DDA (Sigma-
Aldrich, St. Louis, MO, USA) was suspended in distilled
water (2.5 mg ⁄ ml), and a homogeneous dispersion of the
powder was obtained by heating the suspension to 80 C
for 5–10 min. After cooling at room temperature, the
suspension was mixed with diluted AMM, AMH and
BCG PSN before use.
Vaccination and challenge procedures. Female C57BL ⁄ 6
mice aged 6 weeks old were maintained in special patho-
gen-free conditions in Lanzhou Institute of Biological Prod-
ucts and ABSL-3 laboratory at Wuhan University for
M. tuberculosis H37Rv challenge experiment and were used
for experiments at the beginning of 7 weeks of age. Mice
received free access to food and water throughout the study.
Vaccination with subunit vaccines alone: The mice were
inoculated subcutaneously thrice with AMM ⁄ AMH ⁄
Ag85B vaccines with 20 lg proteins in 200 ll at week
0, 3 and 6. Control animals were immunized with the
same volume of PBS or 5 · 106 colony-forming unit
(CFU) of BCG in 200 ll once at 0 week.
570 Immunogenicity and Protective Efficacy of a Fusion Protein Vaccine
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Vaccination with BCG prime and subunit vaccine boost:
The mice were inoculated subcutaneously with
5 · 105 CFU of BCG D2PB302S11First10-P4 strain
(Copenhagen strain) in 200 ll at 0 week followed by
AMM (20 lg of AMM plus 250 lg of DDA and 30 lg
of BCG PSN), AMH (20 lg of AMH plus 250 lg of
DDA and 30 lg of BCG PSN), Ag85B (20 lg of
Ag85B plus 250 lg of DDA and 30 lg of BCG PSN)
and AMM + AMH (10 lg of AMM and 10 lg of AMH
plus 250 lg of DDA and 30 lg of BCG PSN) subunit
vaccines boosting twice at weeks 8 and 10, respectively.
Control animals were injected with PBS or BCG at
0 week followed by PBS boosting. Twelve weeks after
the last immunization, groups of mice were challenged
intravenously by tail vein injection with 1 · 106 CFU of
virulent M. tuberculosis H37Rv.
Antibody detection by ELISA. Microtiter plates were
coated with 100 ll ⁄ well with specific antigens in 0.05 M
bicarbonate buffer (pH 9.6) overnight at 4 C. The plates
were washed five times with PBS containing 0.05%
Tween 20 (PBST); 4 weeks after the last vaccination,
serum samples from four immunized mice per group
were collected and diluted to 1:100 with PBS and
applied to plates in twofold serial dilutions to 1:25,600;
horse radish peroxidase-conjugated rabbit anti-mouse
IgG1 or IgG2a (Rockland Immunochemicals Inc., Rock-
land, ME, USA) was used at 1:20,000 dilution as sug-
gested by the manufacturer. The plates were incubated
for 1 h at 37 C and washed with PBST. After washing,
the plates were added SureBlue tetramethyl benzidine
substrate with 200 ll ⁄ well and incubated at room tem-
perature for 15 min. The reaction was stopped by 50 ll
of 1 M H2SO4 in each well. The optical density was mea-
sured at 450 nm.
Enzyme-linked immunspot (ELISPOT) detection for IFN-c
production from splenocytes. Four weeks after the third
immunization, spleens were aseptically harvested from
four mice ⁄ group and gently ground through a 70-lm
cell strainer, and then, single-cell suspensions were pre-
pared with Lympholyte-M density gradient centrifugation
(Dakewe Biotech Company Limited, Shenzhen, China).
The 96-well transparent polystyrene plates were coated
with 50 ll anti-IFN-c mAb overnight at 4 C. The
plates were then washed five times with PBST and then
blocked with 200 ll blocking solution B at 37 C for
1 h. After discarding blocking solution B from the
plates, freshly isolated spleen cells were plated in dupli-
cate at 5 · 105 cell per well in 100 ll of RPMI 1640
supplemented with penicillin, streptomycin and 10%
newborn calf serum (Shangbao Biotech Company Lt.,
Shanghai, China) and stimulated with HspX, Ag85B,
purified protein derivative and Mpt64190–198, respec-
tively, with ConA and PBS as positive and negative con-
trols, for 36 h at 37 C, 5% CO2. The cells were then
removed, and 200 ll ⁄ well ice-cold deionized water was
Scandinavian Journal of Immunology  2011 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 73, 568–576
Q. Li et al.
added to lyse the remaining cells. The plates were incu-
bated on ice for 15 min, after which they were washed
10 times with PBST. Next, biotinylated detector anti-
body solution was added and the plates were incubated
for 1 h at 37 C. The plates were washed five times with
PBST, after which 100 ll ⁄ well streptavidin–horseradish
peroxidase was added. The plates were again incubated
for 1 h at 37 C and washed five times with PBST. One
hundred microlitres of AEC (3-amino-9-ethylcarbazole)
substrate was added to each well. The plates were devel-
oped for 25 min at room temperature in the dark. The
wells were washed with distilled water to stop develop-
ment when the stained cells were counted on an auto-
mated ELISPOT reader and analysed with ImmunSpot
software (Bio-sys, GmbH, Karben, Germany).
Protective efficacy assay. Mice were sacrificed for bacte-
rial CFU count at 6th week post-challenge with H37Rv.
The lower left lobe of the lungs from infected mice
(n = 7) was harvested, homogenized in 0.05% PBS-
Tween 80 and planted in 10-fold dilutions (10–1000) on
Middlebrook 7H11-OADC agar (BD, Franklin Lakes,
NJ, USA) containing ampicillin (10 lg ⁄ ml) to prevent
contamination. Bacterial colonies were counted 3 weeks
after incubation at 37 C.
Histopathology of the lung tissues. Each upper lobe of
the left lung of infected mice (n = 5) was harvested
6 weeks after challenge. The lobes were fixed with 10%
neutral buffered formalin. After 2 weeks, each lobe was
bisected with 5 lm thick to examine the same area of
the lung in all mice. The sections were stained with hae-
matoxylin and eosin (HE) and Ziehl–Neelsen Method.
Granulomas area was divided by total section area to
determine the affected area in a section. Histopathology
was evaluated by three pathologists independently.
Statistical analysis. The results were expressed as
means ± standard deviation (SD) and analysed by
SPSS10.0 software (Statistical Product and Service Solu-
tions Company, Chicago, IL, USA). The significance of
differences among the groups was determined by analysis
of variance (ANOVA). Independent-samples t-test was used
for Ziehl–Neelsen stain. Probability values (P < 0.05)
were considered as statistically significant.
Results
Construction, expression and purification of AMH fusion
protein
The correct DNA sequence for the recombinant fusion pro-
tein, AMH was confirmed by sequencing and was found to
encode a protein with molecular weight of 54.6 kDa.
AMH was overexpressed in E. coli in inclusion bodies,
which were subsequently dissolved and purified with Ni-
NTA His affinity chromatography. The protein was puri-
fied, which was confirmed by SDS-PAGE analysis (Fig. 1).
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Q. Li et al. Immunogenicity and Protective Efficacy of a Fusion Protein Vaccine 571
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A
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFFSRPGLPVEYLQ
His Tag Ag85B
VPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWY
YQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLS
ANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSACLDPSQGMG
PSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWV
YCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPN
GTHSWEYWGAQLNAMKGDLQSSLGAGVDFAVTNDGVIMATTLPVQRHP
Mpt64190-198 HspX
RSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGRYEVRAELPGVDP
DKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPVGADEDD
IKATYDKGILTVSVAVSEGKPTEKHIQIRSTN

Figure 1 Overexpression and purification of Ag85B-Mpt64190–198-HspX (AMH). (A) translated product of AMH; (B) purification of AMH with the
molecular weight of 54.6 kDa produced in E. coli strain BL21. Lane M, protein molecular weight standards. Lane 1, supernatant of BL-21 cells lysate.
Lane 2, resolution of inclusion bodies containing AMH. Lane 3, fraction of the purified protein AMH.

and HspX-specific IgG1 and IgG2a from AMH group

Immunogenicity of the AMH vaccine candidate
Cell-mediated immune response
Splenic lymphocytes from mice immunized with AMH
formulated with adjuvants DDA and BCG PSN secreted
high levels of IFN-c upon stimulation with Ag85B,
HspX, Mpt64190–198 and PPD (Fig. 2). Splenic lympho-
cytes from mice immunized with AMH produced higher
level of IFN-c than those immunized with Ag85B,
AMM, BCG and PBS with the stimulation of HspX,
Mpt64190–198 and PPD. When stimulated with antigen
Ag85B, the level of IFN-c induced by AMH vaccine was
lower than that by AMM (P < 0.05) and Ag85B
(P > 0.05) vaccines, but was still higher than that receiv-
ing BCG (P < 0.05).
Humoral immune response
With the aid of adjuvant DDA + BCG PSN, AMH
induced higher levels of antigen-specific IgG1 and IgG2a
than Ag85B and AMM (Table 1). Ag85B-specific IgG2a
were the highest among all groups. PPD-specific IgG1
and IgG2a from the mice immunized with AMH were
higher than Ag85B and BCG group. The ratio of
Ag85B-specific IgG2a ⁄ IgG1 from AMH group was lower
than that of BCG group but higher than that of AMM
and Ag85B groups. The ratio of HspX-specific
IgG2a ⁄ IgG1 from AMH group was the highest among
all groups. High IgG2a ⁄ IgG1 ratio reflects Th1 activity
which produces IFN-c to promote intracellular killing
activity by activating macrophages and cytotoxic T cells
[17].
Immunogenicity assay in mice primed with BCG and boosted
with subunit vaccines
The production of IFN-c from splenocytes
Cell-mediated immune responses in mice primed with
BCG and boosted by AMH, AMM, or AMM + AMH
were analysed with the stimulation of Ag85B and
PPD. The results showed that there were higher levels of

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572 Immunogenicity and Protective Efficacy of a Fusion Protein Vaccine Q. Li et al.
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shown). PBS control did not produce antibodies. The ti-

tres of IgG1 and IgG2a against Ag85B from mice immu-
nized with BCG and boosted with subunit vaccines were
higher than that primed with BCG alone (P < 0.05),
whereas there were no significant differences among
boosting groups.

Figure 2 Number of cells secreting IFN-c in mice immunized with
subunit vaccine. The mice were inoculated subcutaneously thrice with
Ag85B-Mpt64190–198-Mtb8.4 ⁄ Ag85B-Mpt64190–198-HspX ⁄ Ag85B vac-
cines at week 0, 3 and 6. Control animals were injected with phos-
phate-buffered saline or BCG once at 0 week. Spleens were removed
4 weeks after the last immunization. Splenocytes isolated from immu-
nized animals were plated in duplicate at 5 · 105 cells ⁄ well and stimu-
lated with 10 lg ⁄ ml of Ag85B, 2 lg ⁄ ml of HspX, 2 lg ⁄ ml
Mpt64190–198 and 10 lg ⁄ ml of PPD. IFN-c-secreting cells were deter-
mined by ELISPOT per 5 · 105 cells. *P < 0.05, n = 4.
IFN-c production in mice boosted with AMH, AMM
and AMM + AMH vaccines than the group of BCG
(Fig. 3). It indicated that Ag85B-, PPD-specific cell-
mediated immunity were highly induced by AMH,
AMM and AMM + AMH boosting. Unlike the fusion
proteins, single-protein Ag85B boosting did not signifi-
cantly induce high cell-mediated immunity compared
with BCG alone. There was no significant difference
among AMM, AMH and AMM + AMH groups.
Ag85B-specific antibody assay
The boost with subunit vaccines induced a higher
humoral immune response against Ag85B (data not
Protective efficacy
Protective efficacy was evaluated by CFU count in mice
boosted with different protein vaccines followed by chal-
lenging with virulent M. tuberculosis H37Rv. The CFUs
from the lungs of mice boosted with the subunit vaccines
AMM + AMH and AMM were significantly lower than
PBS injection, although AMH subunit vaccine boosting
did not lead to a significant decrease in CFUs. The bacilli
were effectively inhibited in the lungs of mice boosted
by AMM + AMH in DDA-BCG PSN, which even
induced significantly lower CFU than BCG group
(P < 0.05) (Fig. 4). These findings suggest that AMH
was potentially capable of enhancing the clearance of
tubercle bacilli in the lungs when used together with
AMM as a boosting vaccine after BCG priming.
Sections from lungs were excised and stained by the
Ziehl–Neelsen technique for identification of acid-fast
bacilli in the tissue. AMM + AMH vaccine resulted in
the lowest number of acid-fast bacteria (data not shown),
which is consistent with the CFU data.
HE stain of the lung tissues
HE stain showed that the granuloma areas per section of
the lungs from mice immunized with BCG or boosted
with fusion proteins AMM, AMH or AMM + AMH
were smaller (P < 0.05) compared with PBS. There was
no difference between the three fusion protein boosting
groups and BCG-immunized group (Fig. 5), indicating
that boosting with the fusion protein vaccines did not
aggravate pathology.

Table 1 Serological responses to antigens in subunit vaccine-immunized mice.

Anti-Ag85B Anti-HspX Anti-PPD

Group IgG1 IgG2a IgG2a ⁄ IgG1 IgG1 IgG2a IgG2a ⁄ IgG1 IgG1 IgG2a IgG2a ⁄ IgG1

AMM 12800 3200 0.25 1600 1600 1.00 12800 6400 0.50
AMH 12800 4032 0.32 2540 6400 2.52 6400 3200 0.50
Ag85B 5080 1008 0.20 800 800 1.00 400 1131 2.83
BCG 800 1008 1.26 504 635 1.26 800 1131 1.41
PBS 80 40 – 40 40 – 40 40 –

C57BL ⁄ 6 mice were subcutaneously immunized three times with fusion protein AMH and AMM and single-protein Ag85B (20 lg ⁄ mouse ⁄ time) in
adjuvant dimethyl-dioctyldecyl ammonium bromide plus BCG polysaccharide nucleic acid, respectively. Positive and negative control mice were
injected with BCG and PBS, respectively. Four weeks after the last immunization, serum samples from four immunized mice per group were col-
lected, and anti-Ag85B (5 lg ⁄ ml), PPD (20 lg ⁄ ml) and HspX (5 lg ⁄ ml) IgG1 and IgG2a were detected by ELISA.
– no calculation.
PBS, phosphate-buffered saline; AMH, Ag85B-Mpt64190–198-HspX; AMM, Ag85B-Mpt64190–198-Mtb8.4.

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Figure 3 Number of cells secreting IFN-c in mice primed with BCG
and boosted with subunit vaccines. The mice were inoculated subcuta-
neously with BCG at 0 week followed by Ag85B-Mpt64190–198-
Mtb8.4 (AMM), Ag85B-Mpt64190–198-HspX (AMH), Ag85B and
AMM + AMH subunit vaccines boosting twice at weeks 8 and 10,
respectively. Control animals were injected with phosphate-buffered sal-
ine (PBS) or BCG at 0 week followed by PBS boosting. Spleens were
removed 4 weeks after the last immunization. Splenocytes isolated from
immunized animals were plated in duplicate at 5 · 105 cells ⁄ well and
stimulated with 5 lg ⁄ ml of Ag85B and 10 lg ⁄ ml of PPD. IFN-c-
secreting cells were determined by ELISPOT per 5 · 105 cells.
*P < 0.05, n = 4.
Discussion
In this research, we constructed a fusion protein AMH,
which included the protective antigen HspX highly
expressed in dormant stage of bacteria. Mice immunized
with AMH subunit vaccine generated high levels of anti-
gen-specific antibodies and IFN-c-producing lympho-
cytes. AMH combined with AMM could enhance the
BCG-primed immune protection against M. tuberculosis
infection in mice.
Dormant bacteria exist together with replicating bac-
teria in vivo in human and animal infection [2–4]
(Fig. 6). Central to the success of M. tuberculosis as a
pathogen is its ability to persist within humans for long
periods of time in a latent state [2–4]. BCG is the most
widely used vaccine, but it is not sufficient to prevent
latent TB or prevent reactivation in adult life [18]. The
antigens of subunit vaccines which were aimed to boost
BCG-primed immunity were chosen frequently from
secreted proteins in early and log growth phase of bacte-
ria based on in vitro culture and are insufficient to impart
sufficient immunity against the latent infection where
some bacteria are in dormant state [19]. Therefore, it is
potentially important for the subunit vaccines to consist
of antigens in multiple stages from active multiplication
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Immunogenicity and Protective Efficacy of a Fusion Protein Vaccine 573
Figure 4 Protective efficacy of Ag85B-Mpt64190–198-Mtb8.4 + Ag85B-
Mpt64190–198-HspX subunit vaccine against H37Rv in mice primed
with BCG. Twelve weeks after the last immunization, groups of mice
were challenged intravenously by tail vein injection with virulent Myco-
bacterium tuberculosis H37Rv. Six weeks post-challenge, mice were sacri-
ficed for colony-forming units (CFUs) detection(n = 7) by harvesting
the lower left lobe of lung and the results are represented as log10 of
number of CFU ⁄ g of lung. *P < 0.05, versus phosphate-buffered saline;
**P < 0.05, versus BCG.
to non-replicating dormancy so as to have a maximum
impact on all stages of M. tuberculosis infection [2].
Different antigens are expressed in different growth
stages. Ag85B, Mtb8.4 and MPT64 are main antigens of
bacteria in replicating stage, whereas HspX is the protein
that is mainly expressed in dormant phase (Fig. 6).
Some latency antigens were detected up-regulated in
non-replicating conditions [10]. For example, DosR, the
hypoxia-related transcriptional regulator, and its genes
are up-regulated under conditions closer to in vivo infec-
tion and prepare M. tuberculosis for dormancy [2]. Among
them, HspX is the first gene to be identified as being
induced by hypoxia and has been identified as an impor-
tant latency antigen [10–13] (Fig. 6). HspX was a major
membrane protein in virulent M. tuberculosis [20].
M. tuberculosis and M. bovis have increased thickness of
their cell walls which contain large amounts of HspX
under low oxygen conditions. It is likely that HspX
might stabilize cell wall structures and sustain bacterial
viability during stress conditions [21].
Mice immunized with AMH subunit vaccine generated
high HspX-specific IgG2a and IgG1 as well as high IFN-
c production with the stimulation of Ag85B and HspX.
The antibodies target the extracellular mycobacteria
through binding to live M. tuberculosis, which can alter
the specific uptake pathway used for phagocytosis [22].
High IgG2a ⁄ IgG1 reflects Th1-skewing pathway that
produces IFN-c to promote intracellular microbicidal
574 Immunogenicity and Protective Efficacy of a Fusion Protein Vaccine Q. Li et al.
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PBS BCG

BCG+AMM BCG +AMH

BCG+AMM+AMH BCG+Ag85B

Figure 5 Pathology induced by vaccine immunization and Mycobacterium tuberculosis challenge. The bars represent the area ratio of granuloma in sec-
tions stained with HE (A). The panels depict histopathology from non-immunized controls and immunized mice (B). Six weeks post-challenge, mice
were sacrificed and lung tissue was sectioned and stained with H&E (50·). Representative pathology from one mouse per group. *P < 0.05 versus
phosphate-buffered saline, n = 5.

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Scandinavian Journal of Immunology  2011 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 73, 568–576
Q. Li et al.
..................................................................................................................................................................
Ag85B
Mtb8.4
Replicating activity
HspX
Figure 6 Different bacterial populations during MTB infection. White
represents bacteria in dormant stage, and black represents bacteria in
replicating phase. Ag85B and Mtb8.4, and HspX are expressed in repli-
cating and dormant stages, respectively.
activities by activating macrophages and cytotoxic T cells
[17].
AMM ⁄ AMH ⁄ AMM + AMH vaccine was designed to
boost BCG-primed immunity to evaluate the capability
of generating protective immunity. The results showed
that only AMM + AMH boosting resulted in a signifi-
cant decrease in CFUs in lung tissues compared with the
BCG group. Although AMM vaccine was found to be a
promising candidate, it could not reduce markedly the
bacterial load compared with BCG in BCG-primed and
subunit vaccine-boosted strategy. Although AMH alone
could not reduce significantly CFU in lung tissues of
infected mice over that of BCG, when it was combined
with AMM, interestingly, fewer CFUs were found than
the BCG group. AMM might induce immunity to
bacteria in active multiplication condition, but inclusion
of AMH potentially induced immune protection
against dormant bacteria. Because of the comprehensive
immune protection against replicating and dormant
M. tuberculosis, the multi-stage vaccine, AMM + AMH,
induced the most obvious protective effect among the
BCG, BCG plus Ag85B or AMM or AMH groups
(Fig. 4).
In conclusion, AMH vaccine could generate strong
antigen-specific humoral and cell-mediated immunity.
Only AMM + AMH boosting led to more pronounced
M. tuberculosis clearance from the lungs of mice than
BCG alone. Meanwhile, the vaccine induced higher
immune responses and presented small lesions. The com-
bination of fusion protein AMM and AMH containing
antigens both from replicating and dormant M. tuberculosis
may be a promising multi-stage vaccine to boost BCG
primed immunity for better protective efficacy.
Acknowledgment
This work was funded by the National Major Science and
Technology Projects of China (2008ZX-10003-01305,
 2011 The Authors
Scandinavian Journal of Immunology  2011 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 73, 568–576
Immunogenicity and Protective Efficacy of a Fusion Protein Vaccine 575
2008zx1000301104) and the National High Technology
Research and Development Program of China (863 Pro-
gram) (2006AA02z420).
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