|
|
|
Original Paper
|
|
|||
|
Acta Biochim Biophys
Sin 2007, 39: 290–296 |
||||
|
doi:10.1111/j.1745-7270.2007.00281.x |
Immune responses and protective
efficacy induced by 85B Antigen and Early Secreted Antigenic Target-6
kDa Antigen fusion protein secreted by recombinant
Bacille Calmette-Guérin
Changhong SHI1*,
Xiaowu WANG1, Hai ZHANG1, Zhikai XU2, Yuan LI2,
and Lintian YUAN1
1 Laboratory
Animal Research Center, Fourth Military Medical University, Xi'an 710033, China
2
Department of Microbiology, Fourth Military Medical University, Xi'an 710033,
China
Received: November
25, 2006
Accepted: February
4, 2007
This work was supported
by a grant from the National Natural Science Foundation of China (No. 30400381)
*Corresponding
author: Tel, 86-29-84774787; Fax, 86-29-83291025; E-mail, changhong@fmmu.edu.cn
Abstract In an attempt to improve immune responses and protective efficacy, we constructed two recombinant bacille Calmette-Guérin (rBCG) strains expressing an 85B antigen (Ag85B) and early secreted antigenic target-6 kDa antigen (ESAT6) of Mycobacterium tuberculosis (MTB) fusion protein. Both rBCG strains have the same protein insertion but in a different order (Ag85B-ESAT6 and ESAT6-Ag85B). The cultured supernatant of rBCG strains and the sera from the mice immunized with the fusion protein Ag85B-ESAT6 or ESAT6-Ag85B formed a band with a fraction size of 37 kDa, equalivalent to the sum of Ag85B and ESAT6. Six weeks after BALB/c mice were immunized with BCG or rBCG, spleen lymphocytes showed significant proliferation in response to culture filtrate protein of MTB. Compared with the BCG group, mice vaccinated with rBCG elicited a high level increase of immunoglobulin G antibodies to culture filtrate protein in the serum. The g-interferon levels in the lymphocyte culture medium supernatants increased remarkably in the rBCG1 group, significantly higher than that of the BCG immunized group (p<0.05). Four weeks after vaccination, mice were infected with M. tuberculosis H37Rv and a dramatic reduction in the numbers of MTB colony forming units in the spleens and lungs was observed in the two rBCG immunization groups. Although these rBCG strains were more immunogenic, their protective effect was comparable to the classical BCG strain, and there were no significant differences between two rBCG groups (p>0.05).
Key words Mycobacterium tuberculosis; vaccine; Ag85B; ESAT6; BCG
Tuberculosis (TB) is a global public health
problem. Recent estimates from the World Health Organization indicate that
there are approximately eight million new cases and three million deaths annually
[1]. Factors such as multi-drug resistant strains, co-infection with HIV, and
increasing mobility of population, have aggravated the situation. Bacille
Calmette-Guérin (BCG) is the only
vaccine used against TB worldwide, but it has variable protective efficacies,
ranging from 0% to 80% in different field trials. Restoration of genes lost
during the original BCG attenuation can enhance the ability of a recombinant
strain to protect against Mycobacterium tuberculosis (MTB). Recombinant
BCG (rBCG) is obtained by inserting an exogenous gene into BCG. This
recombinant plasmid can express exogenous antigens, depending on the
replication of BCG in the host, and induce specific humoral and cellular immune
responses [2,3]. Several different types of rBCG vaccines against TB are being
developed, such as BCG complemented with the complete RD1 region [4] and BCG
complemented with listeriolysin [5] or Th1-type cytokines [6]. These BCG
strains are all genetically modified BCG, with better protective effects against
MTB infection, compared with conventional BCG. The rBCG over-expressing antigen
85B (Ag85B) of MTB is shown to improve protection in guinea pigs over that of
BCG [7], indicating that an effective TB vaccine can be based on this
restoration strategy.
However, further research has found that
the protection afforded by rBCG expressing a single dominant antigen was only
slightly superior to that of classical BCG. It may be a reasonable vaccine
strategy to construct an rBCG expressing a fusion protein to provide stronger
protection against TB, such as interleukin-2 and early secreted antigenic
target-6 kDa antigen (ESAT6) of MTB [8], or different protective
antigens from the culture filtrate protein (CFP) of MTB [2].
Ag85B and ESAT6 as a fusion protein had been
previously shown, by the team of Peter Andersen,
to induce a protective immunity higher than a mixture of these two proteins not
in fusion [9]. So in this study, we constructed two rBCG strains with the same
protein insertion in a different order (Ag85B-ESAT6 and ESAT6-Ag85B), then
compared their immune responses in mice induced by these two rBCG strains, and
their protection against MTB infection.
Materials and Methods
Materials
The pGEM-T Easy vector was purchased from
Promega (Madison, USA). 7H9 (broth), 7H10 (agar) medium and
albumin-dextrose-catalase (ADC) enrichment were purchased from Difco (Detroit,
USA). The mouse interferon (IFN)-g
enzyme-linked immunosorbent assay kit was purchased from Jinmei (Shengzhen,
China). The goat anti-mouse immunoglobulin (Ig)G-conjugated horseradish
peroxidase was purchased from Sino-American Biotechnology (Luoyan, China). The
sera from mice immunized with the fusion protein Ag85B-ESAT6 or ESAT6-Ag85B,
and CFP (final concentration 25 mg/L) of MTB were prepared in our laboratory.
Mice were immunized three times at two-week intervals by subcutaneous injection
with 0.6 ml of 50% (v/v) mixture of fusion protein
Ag85B-ESAT6 or ESAT6-Ag85B (50 mg/ml in
water) and incomplete Freund抯 adjuvant (Sigma-Aldrich, St. Louis, USA).
Sera were collected 10 d after the last immunization and used for western blot analysis. The BCG vaccine
strain was obtained from the Lanzhou Bioethical Production Institute (Lanzhou,
China). Pathogen-free BALB/c female mice were provided by the Animal Center of
the Fourth Military Medical University (Xi抋n,
China). The mice were 68 weeks old and maintained in barrier system. All
animals had free access to water and were fed standard mouse chow.
Construction and screening of
rBCG
The recombinant Escherichia coli-BCG
shuttle plasmid Ag85B-pDE22-ESAT6 expressing the fusion protein Ag85B-ESAT6 was
constructed as follows. The genes of Ag85B and ESAT6 were amplified from the
genome of MTB H37Rv by polymerase chain reaction (PCR). The primers,
synthesized by AuGCT Biotechnology (Beijing, China), for Ag85B were A1F and
A1R. The sequence of A1F was 5'-CGGATCCACCGCGGGCGCGTTCTC-3'
(BamHI site underlined), and that of A1R was 5'-CGAAGCTTGCCGGCGCCTAACGAAC-3'
(HindIII site underlined). The primers for ESAT6 were E1F and E1R. The
sequence of E1F was 5'-GGATATCAAGCTTGCCATGACAGAGCAGCAGTGG-3'
(HindIII site underlined) and that of E1R was 5'-GGAATTCATCGATCTATGCGAACATCCCAGTGACG-3'
(EcoRI site underlined). The
Ag85B-ESAT6 fusion construct was inserted at the unique HindIII site,
introduced by primers A1R and E1F. The recombinant E. coli-BCG shuttle
plasmid, ESAT6-pDE22-Ag85B, expressing the fusion protein ESAT6-Ag85B, was
constructed as follows. Primers for ESAT6 were E2F, 5'-GGGATCCATGACAGAGCAGCAGTG-3'
(BamHI site underlined), and E2R, 5'-GCAAGCTTTGCGAACATCCCAGTGA-3'
(HindIII site underlined). Those for Ag85B were A2F, 5'-GCAAGCTTGCGTTCTCCCGGCGGGG-3'
(HindIII site underlined), and A2R, 5'-GGAATTCATCGATTCAGCCGGCGCCTAACGA-3'
(EcoRI site underlined).
The ESAT6-Ag85B fusion construct was also inserted at the unique HindIII
site, introduced by primers E2R and A2F. All DNA sequences were confirmed by
sequencing (AuGCT Biotechnology). The genes coding for Ag85B and ESAT6 were
inserted into the corresponding sites of the multiple cloning sites region of
E. coli-BCG shuttle plasmid pDE22 (containing Hsp60 promoter and a-ss signal sequences).
The BCG vaccine and MTB H37Rv strains were cultured
in 7H9 medium (containing 100 g/L ADC and 0.5 g/L Tween-80) at 37 ºC for 3 weeks. The bacterium were harvested
and stored at -20 ºC. The concentrate was serially diluted with
7H9 medium, and inoculated in Lowenstein-Jensen medium, then grown at 37 ºC for 2 weeks. Competent BCG cells were
produced by growth in 7H9 medium to the logarithmic phase, incubated for 1h in
an ice bath, then washed three times with 10% glycerin. Purified
Ag85B-pDE22-ESAT6 or ESAT6-pDE22-Ag85B plasmids were transfected into 5106 competent BCG cells by electroporation in
a 2 mm gap cuvette at 2.5 kV voltage, 25 mF capacitance and 1000 W resistance. The transformants were screened on 7H10
agar plates containing ADC and hygromycin. The positive clones were amplified
and confirmed by PCR and the culture supernatants were harvested, concentrated
by PEG4000 and dialyzed against phosphate-buffered saline (PBS) for subsequent
sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. Western
blotting was carried out as follows. Sera from mice immunized with the fusion
protein Ag85B-ESAT6 or ESAT6-Ag85B were used as the first antibody. The second
antibody was goat anti-mouse IgG-conjugated horseradish peroxidase. The
substrate solution contained ortho-phenylenediamine (1 mg/ml) and H2O2
(30%, V/V) in PBS. The rBCG strain expressing the Ag85B-ESAT6
fusion protein was named rBCG1 and that expressing the ESAT6-Ag85B fusion
protein was named rBCG2.
Animal immunizations
Forty BALB/c mice were divided randomly into
four groups of 10 mice. Three groups were injected subcutaneously with 106 colony forming unit (CFU) of rBCG1, rBCG2
or BCG in a volume of 100 ml for
each mouse. The fourth group was inoculated with normal saline as a control.
After 6 weeks, five mice in each group were used for the immunological assays.
The remaining five mice in each group were used for a virulent strain infection
experiment.
Specific lymphocyte
proliferation assay
Six weeks after immunization, four groups of
five mice each were killed and their spleens removed aseptically. Single cell
suspension was obtained by forcing the spleen through a 200-gauge stainless
steel mesh and prepared in RPMI-1640 medium containing 10% fetal calf serum
(FCS), 2 mM glutamine, 50 mM b-mercaptoethanol, 100 mg/ml streptomycin and 100 U/ml penicillin.
The erythrocytes were lysed by incubation in a solution of 0.84% ammonium
chloride in distilled water. Lymphocytes were counted and used in the following
assay. Two hundred microliters of 5105
lymphocytes/ml was seeded in 96-well plates, and cultured in RPMI-1640 medium
with 10% FCS. The cells were treated with CFP (25 mg/L final concentration) of
MTB in the treated group [10]. Preliminary titration experiments had shown that
a threshold concentration for stimulating a maximum specific response was
between 25 and 50 mg/ml for CFP of MTB.
As the concentration of 25 mg/ml was
the minimum unit that provided a maximum response to CFP, it was used
throughout the experiment. The cells were incubated at 37 ºC for 68 h under an atmosphere of 5% CO2. Twenty microliters of MTT was added to
each well at a concentration of 5 mg/ml. After incubation for 4 h, the MTT was
removed and replaced with 150 ml
dimethyl sulfoxide, and was incubated for 10 min at 37 ºC until the crystals were dissolved. ConA (10 mg/ml) (Sigma-Aldrich) served as the
positive control. The optical density value of each well was measured using a
microculture plate reader, with a test wavelength of 490 nm. Proliferation was
expressed as the result of the following equation:
Stimulation index (SI)=A490 (treated)/a490 (control)
IFN-g
content assay
Eight hundreds of spleen lymphocytes (5´106
cells/ml) from immunized mice were seeded in a 24-well plate, cultured in RPMI 1640
medium with 10% FCS and incubated with MTB CFP 100 ml/well (25 mg/ml)
at 37 ºC for 72 h in an atmosphere of 5% CO2. The supernatant was harvested after
centrifugation at 2500 g for 5 min, and stored at -20 ºC. The IFN-g
levels in the lymphocyte culture medium supernatants were analyzed using a
commercial enzyme-linked immunosorbent assay kit (Jingmei) according to the
manufacturer's instructions. All samples were diluted from 10 to 1000 folds, to
scale the concentrations to fall within the detectable range. The measurements
were then repeated at different dilutions to confirm the validity of the
analyses. The data are shown as mean±SD of five mice in each group.
Evaluation of antibody levels
in sera of vaccinated mice
Sera were collected from the immunized mice
in each group. Microtiter plates were coated overnight with 100 ml CFP (25 mg/ml) at 4 ºC, then blocked with 1% bovine serum albumin in PBS. Serum samples were
diluted to appropriate concentrations (10-30-fold dilution) and incubated for 2 h at 37 ºC. Horseradish peroxidase-conjugated goat
anti-mouse IgG was added for detection of antibodies and the substrate solution
containing ortho-phenylenediamine (1 mg/ml) and H2O2 (30%,
V/V) in PBS. Antibody titers were calculated by linear regression
analysis, plotting dilution versus A490.
Protection against MTB
infection
Six weeks after immunization, the mice were
infected with the MTB virulent strain H37Rv through the tail vein at a dose of
105 CFU/mouse. The immunized mice were killed
at 28 d post-infection and spleens and lungs harvested and homogenized in 3 ml
of Middlebrook 7H10 agar plates medium (Difco) supplemented with PBS containing
0.05% (V/V) Tween-80 for CFU counting.
Statistical analysis
The statistical significance of the
difference was estimated by Student's t-test. The differences were
considered statistically significant when P values were less than 0.05.
Results
Screening of the positive
clone containing rBCG
Screening by hygromycin produced a total of
seven colonies on the 7H10 plate. PCR was carried out to select the recombinant
clones containing Ag85B-pDE22-EAST6 and ESAT6-pDE22-Ag85G. The acquired 1168 bp
fragment was equal in fraction size to the sum of Ag85B and ESAT6. Western blot
analysis of the rBCG supernatant revealed an expression band with a relative
fraction size of 37 kDa (Fig. 1). As the fraction size of Ag85B was 31
kDa and ESAT6 was 6 kDa, this product was considered to be the fusion protein
of Ag85B and ESAT6. The cultured supernatant of the two screened rBCG strains
and the sera from the mice immunized with the fusion protein Ag85B-ESAT6 or
ESAT6-Ag85B formed a band with a relative fraction size of 37 kDa, equalivalent
to the sum of Ag85B and ESAT6. As a result of deletion of the Esat6 gene, BCG expressed only Ag85B
and was unable to express ESAT6. Western blot analysis of the sera from the
mice immunized with fusion protein and the supernatants of BCG formed a band
with a relative fraction size of 31 kDa. However, the levels of expression of
fusion proteins Ag85B-ESAT6 and ESAT6-Ag85B in the supernatants of rBCG were
significantly different.
Growth curve of rBCG
The two rBCG strains that secreted the
Ag85B-ESAT6 and ESAT6-Ag85B fusion proteins and the conventional BCG strain
were cultured and quantitated. As shown in Fig. 2, there were three BCG
logarithmic growth periods after 15 d, and a platform period after 45 d. The
two rBCG strains showed no significant differences in proliferation
characteristics, compared with the BCG strain.
Antigen-specific spleen
lymphocyte proliferation
Fig. 3 shows the results of antigen-specific lymphocyte
proliferation responses to CFP of MTB. Compared with the saline group, high
level SIs were induced by the two rBCG strains. However, the SIs of the two
rBCG groups and the conventional BCG group showed no apparent differences. ConA
served as a positive control and showed an SI of 7.100.40.
IFN-g
levels in spleen lymphocytes
After the spleen lymphocytes of the
immunized mice were stimulated with CFP of M. tuberculosis, the IFN-g levels in the lymphocyte culture medium
supernatants were analyzed. IFN-g levels
increased remarkably in the rBCG1 group and the rBCG2 group, above that in the
saline control group, and significantly higher than that of the BCG immunized
group (p<0.05) (Fig.
4).
Antibody response
The MTB H37Rv CFP serum antibody levels of
five mice in each group are shown in Fig. 5. Animals vaccinated with BCG
and rBCG elicited specific antibodies against CFP. Compared with the BCG group,
there was a large increase in IgG antibody level for both rBCG1 and rBCG2
group, however no significant difference was observed between the two rBCG
groups.
Resistance against MTB
infection in immunized mice
Four weeks after mice were infected with
MTB H37Rv, the spleen and lung bacterial loads of the rBCG mice were
determined, and are shown in Table 1. Both of the rBCG vaccines could
effectively resist MTB infection, and reduced the splenic bacterial load
compared with the saline group (p<0.05).
But the protective efficacy of the rBCG vaccine did not exceed that of the
classical BCG vaccine, and there was no significant difference between rBCG and
BCG groups (p>0.05).
Discussion
Despite its many shortcomings, BCG is an
effective vaccine used widely for prevention of TB. Given shortly after birth,
BCG reduces the incidence of childhood TB effectively, but has little or no
effect (protective efficacy ranges from 0% to 80%) on the predominantly adult
disease responsible for the current global emergency [11]. With two million
TB-related deaths worldwide each year, there is a pressing need for improved
vaccines. Therefore, efforts to develop new TB vaccines should use the
documented advantages afforded by BCG. In new rBCG strains, a target gene is
introduced into a BCG vector, or is cloned positionally to acquire high
expression without influencing the survival and multiplication capacity of the
strain [12]. Inoculation of this recombinant vaccine can provide a protection
efficacy not only against the original bacteria, but also against the disease
associated with the introduced genes. The key to constructing a TB vaccine with
recombinant BCG techniques is determining which exogenous genes have been
introduced into BCG. The optimal gene should increase strain immunogenicity and
stimulate the immunologic memory, but not change the low virulence protective
capacity of BCG.
The human body resists MTB infection mainly
by cellular immune response, and the MTB protein that can induce this immune
response occurs in the early culture stage of the bacteria, namely during its
logarithmic growth. In the numerous secreted MTB proteins, Ag85B can induce a
Th1-type cellular immune response in animal models, which has a resistance
against MTB reinfection that is similar to that of BCG [13]. ESAT6 is a protein
secreted in the cultures of the MTB virulent strains. Its encoding gene
exists in the MTB virulent strains, but not in the attenuated strains. ESAT6 is
one of the main target antigens recognized by T cells in the recall immune
response of the MTB-infected human or animal [14]. Thus, ESAT6 is a potential
component candidate for a new vaccine against MTB. After studying the immune
response of univalent and multivalent Bacillus tuberculosis DNA
vaccines, it was claimed that a multivalent combination DNA vaccine had greater
protective efficacy than BCG [15]. Thus, the multivalent vaccine containing
multiple MTB immune response-related antigens is a more effective vaccine.
Vaccination with the Ag85B-ESAT6 fusion protein in adjuvant has been shown to
induce a protective immune response higher than a mixture of these two proteins
not in fusion by the team of Peter Andersen
[9,16]. In this study, the BCG recombinant fusion protein Ag85B-ESAT6 served as
target antigen to gain an improved immune response over that induced by a
single component.
The protective effect of the rBCG strain
over-expressing Ag85B in guinea pigs was significantly higher than that of the
BCG vaccine [7], suggesting that the rBCG strain possesses superior
immunization potential. The virulence of the rBCG strain expressing ESAT6 was
not significantly higher than that of the BCG immunized group, as determined by
survival time, bacterial number and pulmonary pathology section. This rBCG
could induce an improved cellular immunologic response over that of BCG,
despite the finding that its protection in mice did not exceed that afforded by
BCG [9]. In this study, we successfully cloned an Ag85B and ESAT6 fusion gene
with the potential to protect against tuberculosis. These two rBCG strains we
obtained possess proliferation characteristics similar to the conventional BCG
strain, entering the logarithmic growth period after 15 d and the platform
period after 45 d. The introduction of the fusion gene did not influence the
growth of rBCG.
Many studies have attempted to develop a TB
vaccine using rBCG techniques. These consist mainly of recombining cytokines
[17,18] or the dominant antigen for cell-mediated immunity (such as ESAT6) [3]
and the major target for memory T cells (such as Ag85B) [7] into BCG.
Vaccination with these rBCG strains has been shown to be an effective vaccine
strategy, and even some rBCG strains afforded protective efficacy over the
standard BCG vaccine. The above-mentioned research suggests that a gene or
subunit vaccine with multi-epitopes can induce stronger cellular immunological
responses and protection than single epitope vaccines, with a single
subdominant epitope of the antigen, which might be poorly recognized during natural
infection [19]. The study of Palendira et al. showed that vaccination of
mice with an rBCG secreting high levels of Ag85B-ESAT6 induced a protective
immunity against aerosol MTB higher than a mixture of these two proteins not in
fusion, as expression of the single components did not significantly increase
the protective efficacy of rBCG [2]. Six weeks post-immunization with rBCG
expressing Ag85B-ESAT6 or ESAT6-Ag85B in this study, higher serum antibody
responding to CFP of MTB H37Rv was observed, and the level of induced IFN-g was significantly higher than those
induced by the BCG or saline groups. However, there was no apparent difference
between the two rBCG strains. This shows that the orders of Ag85B and ESAT6 in
recombinant BCG vector have little influence on the biological characters of
rBCG.
Even though the two rBCG strains used in
our study could effectively induce Th1 cell immune response, especially with
respect to the IFN-g levels in spleen
lymphocytes, their protection in mice did not exceed that of the current BCG
after MTB challenge, indicating that the protective effect was not compliant
with the cellular immune response. This suggests that the immune response
induced by a single expression strategy is limited, and that an effective TB vaccine
should be complex, with multiple epitopes, and affording efficacy through
multiple routes. Ag85B and ESAT6 proteins, although preferable for the
measurement of specific responses, were difficult to obtain. Therefore, the CFP
of MTB was used as the antigen in all assays, which could change the level of
antigen-specific response and affect the interpretation of the results. Our
future research should use the purified Ag85B and ESAT6 proteins to determine
whether a response specific to these antigens was actually induced by rBCG.
Pym et al. pointed out that an
increase in BCG virulence was the result of the cooperative action of multiple
genes, and that only the introduction of the complete RD1 domain into BCG could
increase its virulence [20]. Introduction of the ESAT6 gene into BCG would not
only change the low virulence of BCG, but also be an optimal candidate vaccine
for TB. Note that a safety evaluation on rBCG was not carried out in this study
because Ag85B and ESAT6 have been used for rBCG, and there are no published
reports of an increased virulence.
In conclusion, two rBCG strains expressing
a fusion of Ag85B and ESAT6 with the same protein insertion and different order
have been successfully constructed. They could induce high level humoral and cellular
immune responses and reduce bacterial loads effectively within the lung and
spleen. The order of Ag85B and ESAT6 in the fusion gene had little influence on
the immune responses (such as lymphocyte proliferation responses, IFN-g production, antibody level, and protective
efficacy) in mice vaccinated with the rBCG strains. The rBCG strains used in
this trial did not increase efficacy over classical BCG. The following methods
might be considered for improvement of immunogenicity and protective efficacy:
(1) choosing a more susceptible animal such as the guinea pig to study the
protective efficacy of MTB; and (2) using an E. coli-BCG shuttle vector
with a high-efficiency promoter to study antigen expression.
Acknowledgements
We thank professor Li Yuan (Department of Microbiology, Fourth Military Medical
University, Xi抋n, China) for
kindly providing the shuttle vector pDE22.
References
1 Harries AD, Dye C. Tuberculosis. Ann Trop Med Parasitol 2006, 100: 415-431
2 Dhar N, Rao V, Tyagi AK. Skewing of the Th1/Th2 responses in mice due to variation in the level of expression of an antigen in a recombinant BCG system. Immunol Lett 2003, 88: 175-184
3 Palendira U, Spratt JM, Britton WJ, Triccas JA. Expanding the antigenic repertoire of BCG improves protective efficacy against aerosol Mycobacterium tuberculosis infection. Vaccine 2005, 23: 1680-1685
4 Pym AS, Brodin P, Majlessi L, Brosch R, Demangel C, Williams A, Griffiths KE et al. Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nat Med 2003, 9: 533-539
5 Hess J, Miko D, Catic A, Lehmensiek V, Russell DG, Kaufmann SH. Mycobacterium bovis bacille Calmette-Guérin strains secreting listeriolysin of Listeria monocytogenes. Proc Natl Acad Sci USA 1998, 95: 299-304
6 Luo Y, Chen X, Szilvasi A, O’Donnell MA. Co-expression of interleukin-2 and green fluorescent protein reporter in mycobacteria: in vivo application for monitoring antimycobacterial immunity. Mol Immunol 2000, 37: 527-536
7 Horwitz MA, Harth G, Dillon BJ, Masleša-Galić S. Recombinant bacillus Calmette-Guérin (BCG) vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein induce greater protective immunity against tuberculosis than conventional BCG vaccines in a highly susceptible animal model. Proc Natl Acad Sci USA 2000, 97: 13853-13858
8 Fan XL, Yu TH, Gao Q, Yao W. Immunological properties of recombinant mycobacterium bovis bacillus Calmette-Guérin strain expressing fusion protein IL-2-ESAT-6. Acta Biochim Biophys Sin 2006, 38: 683-690
9 Weinrich Olsen A, van Pinxteren LA, Meng Okkels L, Birk Rasmussen P, Andersen P. Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85b and esat-6. Infect Immun 2001, 69: 2773-2778
10 Bao L, Chen W, Zhang H, Wang X. Virulence, immunogenicity, and protective efficacy of two recombinant Mycobacterium bovis bacillus Calmette-Guérin strains expressing the antigen ESAT-6 from Mycobacterium tuberculosis. Infect Immun 2003, 71: 1656-1661
11 Fine PE. Variation in protection by BCG: implications of and for heterologous immunity. Lancet 1995, 346: 1339-1345
12 Rao V, Dhar N, Tyagi AK. Modulation of host immune responses by overexpression of immunodominant antigens of Mycobacterium tuberculosis in bacille Calmette-Guérin. Scand J Immunol 2003, 58: 449-461
13 Yadav D, Khuller GK. Evaluation of immune responses directed against 30kDa secretory protein of Mycobacterium tuberculosis H37Ra complexed in different adjuvants. Indian J Exp Biol 2001, 39: 1227-1234
14 Carrara S, Vincenti D, Petrosillo N, Amicosante M, Girardi E, Goletti D. Use of a T cell-based assay for monitoring efficacy of antituberculosis therapy. Clin Infect Dis 2004, 38: 754-756
15 Morris S, Kelley C, Howard A, Li Z, Collins F. The immunogenicity of single and combination DNA vaccines against tuberculosis. Vaccine 2000, 18: 2155-2163
16 Dietrich J, Aagaard C, Leah R, Olsen AW, Stryhn A, Doherty TM et al. Exchanging ESAT6 with TB10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine: efficient protection and ESAT6-based sensitive monitoring of vaccine efficacy. J Immunol 2005, 174: 6332-6339
17 Murray PJ, Aldovini A, Young RA. Manipulation and potentiation of antimycobacterial immunity using recombinant bacille Calmette-Guérin strains that secrete cytokines. Proc Natl Acad Sci USA 1996, 93: 934-939
18 Biet F, Kremer L, Wolowczuk I, Delacre M, Locht C. Mycobacterium bovis BCG producing interleukin-18 increases antigen-specific gamma interferon production in mice. Infect Immun 2002, 70: 6549-6557
19 Olsen AW, Hansen PR, Holm A, Andersen P. Efficient protection against Mycobacterium tuberculosis by vaccination with a single subdominant epitope from the ESAT-6 antigen. Eur J Immunol 2000, 30: 1724-1732
20 Pym AS, Brodin P, Brosch R, Huerre M, Cole ST. Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Mol Microbiology 2002, 46: 709-717