Research paper

Human Vaccines 7:9, 934-940; September 2011; 2011 Landes Bioscience

The use of streptomyces for immunization

against mycobacterial infections
Nesty Olivares Arzuaga,1,3,4,* Ailin Vila Granda,1 Juan Carlos Ramrez Gmez,1 Mara Elena Sarmiento Garca San Miguel,1
Juan Francisco Infante Bourzac,1 Yamil Lpez Hernndez,1 Ana Lucrecia Elas Lpez,3,4 Carlos Romn Valln Plux,2 Leonora
Gonzlez Mesa,2 Rogelio Hernndez Pando3 and Armando Acosta Domnguez1
3

1
Finlay Institute; Research Center Vaccine Production and Serum; 2Center of Pharmaceutical Chemistry; Habana, Cuba;
National Institute of Medical Sciences and Nutrition Salvador Zubirn; Delegacin Tlalpan, Mexico; 4National Autonomous University of Mexico; Mexico City, Mexico

Key words: tuberculosis, BCG, streptomyces

Tuberculosis is one of the leading causes of mortality produced by an infectious agent. Different strategies including
bioinformatics are currently being tested to identify and improve vaccines against tuberculosis. Comparative genome
analysis between Streptomyces coelicolor and Mycobacterium tuberculosis suggest that both descend from a common
Actinomycete ancestor. In this work, we suggest the use of Streptomyces as a live vector and explore the capacity of
Streptomyces immunization to induce a protective response against mycobacterial infection. First, we compared the
theoretical proteomes of S. coelicolor A3(2) with those of M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97.
This study showed a high similarity at the level of individual genes sequences with both bacteria sharing several
membrane proteins. Then, we administered Streptomyces intraperitoneally to mice and determined its distribution
by histopathology and culture; we did not find systemic dissemination. After administration of Streptomyces through
different routes, we identified the most immunogenic, inducing strong humoral response, as denoted by the high serum
antibody titers against this organism with cross reactivity to mycobacterial antigens. Finally, we evaluated the level of
protection elicited by the inoculation of Streptomyces in Balb/c mice challenged with BCG. In these animals, lung bacillary
loads were significantly lower than the control non-sensitized group. These observations, along with Streptomyces
potential for expressing foreign proteins, suggest that Streptomyces could be an advantageous vector in the design of
new tuberculosis vaccines.

201
1L
andesBi
os
c
i
enc
e.
Donotdi
s
t
r
i
but
e.
Introduction
Tuberculosis (TB) continues to be a relevant public health problem,1 caused by the bacillus Mycobacterium tuberculosis. Bacillus
Calmette-Gurin (BCG) is the only vaccine currently available
against TB. BCG is a live attenuated strain of Mycobacterium
bovis, an organism closely related to M. tuberculosis, their
genomic sequences are 99.95% identical.2 BCG is effective
against the severe childhood forms of tuberculosis, miliary TB
and TB meningitis; however, BCG demonstrates variable efficacy (from 0 to 80%) against adult pulmonary disease, particularly in TB endemic regions.3 Thus, the developing of new TB
vaccines becomes an urgent task and different strategies are currently being tested.4
One potential strategy is the use of live microorganisms as vectors expressing mycobacterial antigens, which have the advantage
of inducing immunological memory for long time, aside from the
possibility to administrate it on mucosal surfaces and its relatively
low production costs. This is the case of non-pathogenic mycobacteria, like M. vaccae, M. microti, M. habana and BCG. The
limited possibilities for genetic manipulation and slow bacterial
growth rate, in some cases, could be considered a disadvantage

in the process of obtaining the recombinant mycobacteria;

above all this none of these non-pathogenic mycobacterial vaccines has shown a higher protection rate than BCG in clinical
trials. Another group of vectors corresponds to non-mycobacterial organisms, like Salmonella, Vaccinia virus, Listeria monocytogenes, and others, whose virulence reversion is a significant
problem.5 Several new generation types of TB vaccines are being
investigated, but none have shown better protection than BCG.
Streptomyces, members of the widely distributed
Actinomycetes, are Gram positive bacteria with a replication
time between three to nine days in culture medium.6 They
are significant microorganisms used in the production of over
two-thirds of naturally derived antibiotics in current human
and veterinary use (and many other pharmaceuticals such as
anti-tumor agents and immunosuppressants), by means of complex secondary metabolic pathways.7 Specifically Streptomyces
lividans is an important member of this family, because it is
nonpathogenic filamentous bacteria and there is substantial
knowledge about its genetic manipulation.8 In fact, this organism is able to produce large amounts of extracellular proteins,
and is often used for cloning, expression, and production of
heterologous proteins.9-11 The Mycobacterium tuberculosis genes

*Correspondence to: Nesty Olivares Arzuaga; Email: nestyolivares@yahoo.con

Submitted: 03/19/11; Revised: 05/26/11; Accepted: 06/09/11
DOI: 10.4161/hv.7.9.16000
934

Human Vaccines

Volume 7 Issue 9

research paper

Research paper

Figure 1. Protein scatter plot results. (A) Total protein hits for M. tuberculosis H37Rv vs. S. coelicolor A3(2). (B) Total protein hits for M. bovis AF2122/97
vs. S. coelicolor A3(2). Protein hits were determinate from blast searches. Dots indicate two genes that are similar (a match between M. tuberculosis or
M. bovis and S. coelicolor) one to another. Axes represent the positions of the proteins in the genome in the order in which they occur on the chromosomes.

201
1L
andesBi
os
c
i
enc
e.
Donotdi
s
t
r
i
but
e.

encoding the 38 kDa, 19 kDa and 45/47 kDa proteins, have

been cloned and successfully expressed by this Streptomyces
as an alternative method to obtain recombinant proteins.12-14
On the other hand, comparison of the S. coelicolor and the
M. tuberculosis genomes indicated a conserved gene arrangement
between the whole genome of the mycobacteria and the core of
the S. coelicolor chromosome, suggesting that both microorganisms descend from a common Actinomycete ancestor.15
BCG and most vaccine candidates against TB are directed at
augmenting cell-mediated immune responses have been able to
limit bacilli multiplication and dissemination, but not to actually
prevent the infection.4,16 Regarding TB immune protection, there
is the dogma that only T-cell mediated immune mechanisms can
protect against M. tuberculosis infection, however, there are current evidences about the protective role of antibodies against
M. tuberculosis infection.17-19
In this work, we compared the S. coelicolor genome with those
of M. tuberculosis and M. bovis, in order to determine if these
bacteria share immunogenic molecules. Then, we evaluated the
ability of S. lividans to induce efficient early protective immune
responses against BCG infection in an animal model. In our
experiments we used S. lividans (whose genome has not been yet
sequenced) instead of S. coelicolor due to the lowest extracellular
proteolitic activity of S. lividans.
Results and Discussion

Tuberculosis remains a heavy burden of worldwide healthcare

system. The emergence of multidrug resistant tubercle bacilli
makes tuberculosis treatment more troublesome. So it is a key

www.landesbioscience.com

point to explore effective prevention and treatment means. The

search for new, improved TB vaccine is a relatively young field.
Nevertheless, numerous promising new approaches have been
developed during the last two decades and the most advanced of
the new TB vaccine candidates are now entering clinical field of
research but the need for new vaccine candidates remains.20
Comparison of the S. coelicolor genome with those of two
pathogenic Actinomycetes, M. tuberculosis and M. bovis, revealed
much similarity at the level of individual gene sequences.
We found that 740 (18.56%) and 761 (19.42%) proteins in
M. tuberculosis and M. bovis respectively, have over 50% of identity with S. coelicolor proteins. Among these genes there were several open reading frames that encode for membrane proteins as
well as hypothetical proteins without assigned function. In addition, global comparison showed a conserved gene arrangement
(synteny) between the whole mycobacteria genomes and the S.
coelicolor chromosome, shown as a dot plot in Figure 1.
The molecular genetics of Streptomyces is well known, for
instance, the function and position of S. coelicolor genes are
known.9 However, S. lividans is one of the most commonly
used host strains for DNA cloning in Streptomyces species,
because S. lividans, unlike its close relative, Streptomyces coelicolor, lacks a methylation-dependent restriction system that recognizes DNA isolated from normal Escherichia coli strains.10,21
Streptomyces lividans is often used for cloning, expression, and
secretion of heterologus proteins of prokaryotic and eukaryotic
origin at a commercially viable level, thanks to relatively wellestablished plasmid-based expression systems, a high-biomass
fermentation process and a low level of endogenous protease
activity.11-13

Human Vaccines

935

Although M. tuberculosis and M. bovis have very different

lifestyles as compared with S. coelicolor, their genomes showed
a perceptible higher-order synteny and much similarity at the
level of individual gene sequences. When the DnaA is centrally
located distinguishes a better correlation between the content
and location of the genes of the genomes studied because it shows
a central broken diagonal cross pattern formed by the regions of
synteny. This broken-X pattern is commonly seen in comparisons of related bacteria and the breaks are attributed to multiple
inversions centered on oriC. The genes content and location in
S. coelicolor also showed syntenic features with the M. tuberculosis and C. diphtheriae genomes, when previously compared with
these bacteria.9 According to our findings these microorganisms
share several surface proteins that could generate a cross immune
response against these Actinomycetes. The idea of using S. lividans in vaccines field could be improved in the future by adding
to S. lividans recombinant MTB proteins, lipids or sugars known
to induce protection against TB. Regarding vaccine development, all above features indicate the potential of this bacterium
for use as a delivering system.
One month after ip inoculation of S. lividans, we did not detect
bacterial growth in the homogenates from different organs, and
not histopathological alterations were observed (data not shown).
Some members of the genus Streptomyces can be pathogenic to
humans, animals and plants. Our microbiology and histopathology studies in Balb/c mice inoculated with S. lividans showed
that this organism has a limited life span and the mouse immune
system efficiently prevented systemic dissemination. These results
are in agreement with other reports that consider S. lividans as a
nonpathogenic microorganism.22-24
Using the whole cells ELISA of S. lividans, we detected differences in antibodies production elicited by S. lividans administered
through different routes. The optical density (OD) means were
0.92, 0.45 and 0.41 in sera from animals immunized through
ip, sc and im route, respectively. The mean of the control group
was 0.15. Statistical difference (p < 0.05) was observed between
the groups sensitized through the different routes and the control group, and between the group immunized by ip route and
the other two groups sensitized (Data not shown). Tuberculosis
human vaccines are given via the skin, muscle or mucose routes,25
however we found that intraperitoneal administration was the
most immunogenic route. This is a preliminary result that should
be deeper explored in the future, because the practical limitations
of this route are known.
Twenty-one days after the last inoculation, whole cells ELISA
for S. lividans showed a mean of 0.82 OD in the sera from animal immunized with S. lividans, 0.40 OD in mice immunized
with BCG, and 0.15 OD in the non-sensitized control group.
Statistical significance was detected between the group immunized with S. lividans and BCG. Each group was statistically different from the control group (p < 0.05).
Whole cells ELISA of BCG showed an OD means of 0.77,
0.42 and 0.15 in animals immunized with BCG, S. lividans
and control, respectively. Statistical significance (p < 0.05) was
detected between the group immunized with BCG and S. lividans, and of both with respect to the negative control (Fig. 2).

Figure 2. Determination of antibodies production by whole cells ELISA

in mice immunized with S. lividans and BCG. Three groups of 10 mice
each were immunized with S. lividans (white box), BCG (gray box) and
negative control group (black box). In both ELISA was detected statistical significance between the two immunized groups. Each immunized
group was statistically different with the negative control group. Duncan and ANOVA tests were used and values of p < 0.05 were considered
significant.

201
1L
andesBi
os
c
i
enc
e.
Donotdi
s
t
r
i
but
e.

936

Figure 3. Specific and cross reactive antibodies determined by western

blot. Sera from immunized animals with S. lividans were evaluated
against S. lividans proteins (column# 2) and BCG proteins (column# 3).
Sera from immunized animals with BCG were evaluated against BCG
proteins (column# 4) and S. lividans proteins (column# 5). Sera from
negative control animal group were evaluated against BCG proteins
(column# 6) and S. lividans proteins (column# 7). Column 1 corresponds
to molecular weight marker.

Western blot analysis using sera from animals immunized

with S. lividans and proteins from the same organism showed
specific antibodies that recognized proteins in a molecular size
range from 97 to 40 kDa (Fig. 3 and column# 2). These sera
also showed cross-reactivity against BCG proteins, recognizing
proteins in a molecular size range from 90 to 40 kDa (Fig. 3 and
column# 3).
Western blot analysis using sera from animals immunized with
BCG showed specific antibodies that recognized BCG proteins

Human Vaccines

Volume 7 Issue 9

Figure 4. Determination of lung bacillary loads by CFU counting, after

intranasal challenge with 106 CFU BCG in groups of Balb/c mice immunized with S. lividans or with BCG. In comparison with the control nonsensitized (NC) group, both immunized groups showed lower bacillary
loads, being significant in the group immunized with S. lividans. ANOVA
and Tukey HSD test was used to determine significance on lung bacillary loads and values of p < 0.05 were considered significant. Different
letters represent statistical significance.

if there is good evidence that this component is part of such

response, however comparisons of pre and post vaccination immunity can identify changes in vaccine-induced immune status, and
the immunogenicity of both the existing BCG vaccine and new
candidate TB vaccines can be compared in this manner.29
There is no question that T cells play a central and essential role in protection against progressive infection with MTB.
On a simplistic level, activation of CD4 + T cells leads to secretion of cytokines such as IFN and TNF that induce macrophage activation, with induction of bactericidal products such as
nitric oxide and reactive oxygen intermediates. CD4 + T cells may
also orchestrate response within other T-cell subsets, like CD8 +
T cells.29 These significant concepts were previously explored by
our group via the measurement of cellular responses to immunization with S. lividans. Such response was evaluated by determination
of major Th1-type cytokine IFN, which elicits a strong cellular
immune. In contrast, immunization with BCG produced a lower
Th1 response than with the Streptomyces strain. Lymphocytes
from the Streptomyces immunized group, when challenged with
BCG secreted antigens, secreted similar quantities of IFN and
IL-10 as the lymphocytes from BCG immunized animals.28
Classical scientific views define a dichotomy between the two
arms of the immune response, with antibody-mediated immunity targeted toward extracellular pathogens, and cell-mediated immunity CMI targeted against intracellular pathogens.
Scientific advances suggest that an interaction between the two
major arms of the immune response does in fact exist.30 Therefore,
it is of major interest to propose novel research which elucidates
whether vaccine-induced antibodies work independently, or in
conjunction with CMI.
Timing of CFU determination is another key issue in assessing antibody effect. In our experiments, when S. lividans-immunized animals were challenged with BCG by the intranasal
route, a significant decrease of lung bacillary loads was seen 24 h
after. Classically, CFU determination is evaluated three to four
weeks following the challenge. This kinetic has been used due
to the slow growth of MTB and the presumption that CMI is
the sole mechanism of protection. However, studies using monoclonal antibodies (mAbs),31,32 or conjugate vaccines33 showed a
decreased bacillary load at earlier time points after infection.
CMI is thought to protect the host after already established
infection by controlling mycobacterial replication, in contrast
antibodies could potentially have a role in earlier stages of the
infection process. Additionally, the potential enhancement of
CMI by antibodies was suggested by improved granulomatous
formation in mice treated with mAb.34
In our experiment, the rapid mycobacterial elimination
could be produced by cross-reactive antibodies located on the
mucosal surface,35 suggesting that they could be efficient in
the prevention of the infection prior to the entry of mycobacteria into host cells, however, we cannot only correlate protection with the antibody response detected; the participation of
cell-mediated immunity in the early lung bacilli elimination
observed in S. lividans-immunized animals cannot be discarded, specially activated macrophages and CD8 + T cells, so
in future experiments we will evaluate these aspects.

201
1L
andesBi
os
c
i
enc
e.
Donotdi
s
t
r
i
but
e.

in a molecular size range from 90 to 30 kDa (Fig. 3 and column#

4). These sera showed recognition of Streptomyces proteins
but this was limited, only one group of not well-defined bands
was seen with approximately 97 kDa (Fig. 3 and column# 5).
Sera from control mice did not show antibodies against
S. lividans (Fig. 3 and column# 6) or BCG proteins (Fig. 3 and
column# 7).
Antibody production induced by BCG vaccination has been
widely studied in reference 26 and 27, but up to now there are
only one reported study on Streptomyces humoral immunogenicity in mice.28 Our results are in agreement with reported
previously, we demonstrated that Streptomyces is immunogenic,
specifically when it is administrated into the peritoneal cavity,
inducing high production of specific IgG antibodies and crossreactive antibodies that recognize BCG proteins, supporting the
possibility that both organisms have common antigenic epitopes
probably located on the bacterial surface. Interestingly, this crossreactivity developed by S. lividans immunization was greater
than that developed by immunization with BCG. In the present
study, a humoral response detected in mice immunizated with
S. lividans might be the result that both organisms share several surface proteins that could generate a cross immune response
against these Actinomycetes.
Protection conferred by the immunization was measured by
the reduction in the number of viable bacteria in lungs using
BCG as the challenged bacterium. The bacterial load in lungs
of mice immunized with S. lividans was significantly decreased
(p < 0.05) compared with animals of the control group, but not
significantly decreased compared with the animals immunized
with BCG. The bacterial load in the lungs of mice immunized
with BCG decreased but not significantly decreased compared
with the control group animals (Fig. 4).
It is clear that measuring one component of immunity will
not be sufficient to identify a protective immune response, even

www.landesbioscience.com

Human Vaccines

937

Our challenge experiments also demonstrated that lung

mycobacterial loads from mice immunized with BCG and control non-sensitized animals were not statistically significant. This
result could be in relation with the limitations of BCG: it induces
limited antibodies production36 and does not efficiently stimulate
a CD8 + T cells response.37
M. bovis BCG does not cause severe pulmonary damage, that
is why we used in ours experiment a higher dose of bacteria for
challenge, however, a challenge with a virulent M. tuberculosis
strain is required in future experiments.
Material and Methods
Animals. Balb/c mice were purchased from the National Center
for the Production of Laboratory Animals (CENPALAB,
Havana, Cuba). Mice were six- to eight weeks old at the time of
inoculation. They were kept under pathogen-free conditions and
fed autoclaved food and watered ad libitum. All the potentially
painful experimental procedures were performed under anesthesia, to avoid unnecessary suffering following international good
practice methods in the use of laboratory animals.38
Microorganisms. Freeze-Dried M. bovis BCG vaccine (BCG
sub-strain Sofia), manufactured by InterVax Biological Ltd.,
BB-NCIPD LTd-Bulgaria, with 1.56.0 x 107 viable units were
used. The Freeze-Dried BCG Vaccines were suspended in saline
solution (0.9% NaCl). Freeze-Dried S. lividans strain 1326, with
6 x 106 viable units per vial, was kindly donated by the Center of
Pharmaceutical Chemistry, Havana. Cuba and it was used after
suspension in saline solution.
Comparative analysis of M. tuberculosis, M. bovis and
S. coelicolor genomes. We compared the theoretical proteomes
of M. tuberculosis H37Rv (AL123456) and M. bovis AF2122/97
(BX248333) with S. coelicolor A3(2) (AL645882) using the
Genome v/s Genome Protein Scatter Plot Tool (minimum 50%
and 70% identity) on the Comprehensive Microbial Resource
(CMR) web server.39
S. lividans systemic distribution after intraperitoneal
administration. To S. lividans systemic distribution, mice
were randomly assigned to four groups of eight animals each.
Three groups of animals were inoculated through the intraperitoneal (ip) route with 105, 103 or 102 colony forming units
(CFU) of S. lividans. Eight negative control animals received
saline solution, through the same route. One month after the
inoculation, all animals were sacrificed by cervical dislocation,
the thorax was opened and the heart, lung, liver, kidney and
spleen were aseptically removed. Halves of these organs were
suspended in 1 ml on YEME medium, were homogenized with
steril 1 mL syringe and plated in steril tubes containing YEME
medium during 6 d at 29C under continuous agitation.40 The
other halves were kept in formaldehyde for a minimum of
24 h, embedded in paraffin wax, sectioned and stained with
hematoxylin/eosin. Slides were observed under light microscopy to study tissue change.41
Serum antibody detection on different routes of administration of S. lividans. Three groups of ten mice each were

inoculated through subcutaneous (sc), intraperitoneal (ip) or

intramuscular (im) route with 105 cfu of S. lividans. Ten negative
control animals received saline solution. Twenty-one days after
the inoculation, blood was extracted from the retro-orbital plexus
of all anesthetized animals. The quantification of antibody production against S. lividans was measured by IgG enzyme-linked
immunosorbent assay whole cell (whole cell ELISA) on individual animal sera. Briefly, 96-well microtiter plates (MaxiSorp,
Nunc) were coated overnight at 37C with 103 viable units/
well in 0.05 M Phosphate-buffer saline (PBS), pH 7.4. Serum
samples diluted to 1:150 were incubated for 1 h at 37C in precoated, blocked plates. Bound antibodies were detected using an
anti-mouse IgG peroxidase conjugated (Sigma-Aldrich Chemie
GmbH., Taufkirchen, Germany) diluted 1:10,000 in PBS for 1 h
at 37C. The reaction was developed with a solution of o-phenylenediamine (OPD) (5 mg of OPD in 12 mL of 0.1 M sodium
citrate buffer, pH 5, H2O2 5 L). The reaction was stopped with
H2SO4 (2 N) after 30 min and the absorbance was measured at
492 nm in an ELISA reader.
The cutoff value was determined as the mean and two standard deviations of the results obtained from the samples of the
control group.
Immunization schedule. Mice were randomly divided in three
groups, each containing ten animals. One group was treated with
three doses (105 CFU per dose) of S. lividans intraperitoneally at
three week intervals. Another group was immunizated subcutaneosly with three doses (105 CFU per dose) of BCG at three week
intervals. The group treated with saline solution (three doses)
was used as control. Blood was extracted from the retro-orbital
plexus of five anesthetized mice per group at 21 d after the last
immunization.
ELISA. The serum was used to quantify antibodies using
whole cell ELISA for S. lividans, as described below and whole
cell ELISA for BCG. For this last ELISA, plates (MaxiSorp,
Nunc) were coated overnight at 37C with 103 viable units of
BCG/well in PBS (50 L/well). The rest of the procedure was
the same as that used in the ELISA for S. lividans described
below.
Western blot analysis. To determine antibodies specificity
we used protein gel blot analysis. Total soluble proteins from 104
CFU of S. lividans and BCG lysates, were resuspended using
cracking buffer (250 mM TRIS-HCl, pH, 6.8, 6% SDS, 5%
glycerol, 0.05 mg/mg bromophenol blue), separated by 12.5%
SDS-PAGE electrophoresis and then transferred to a nitrocellulose membrane. After that, membranes were incubated with
serum samples diluted 1:150 for 1 h at 37C. Bound IgG was
detected with antimouse IgG labeled with horseradish peroxidase
(Sigma) and the color was developed with diaminobenzidine as
substrate.
Challenge experiment. To evaluate the ability of S. lividans to
induce efficient early protective immune responses, all mice were
challenged with BCG, 21 d after the last immunization. Animals
were challenged through the intranasal route with 106 CFU of
BCG suspended in 25 L of PBS. Twenty four hours after the
challenge, all mice were sacrificed by cervical dislocation. The

201
1L
andesBi
os
c
i
enc
e.
Donotdi
s
t
r
i
but
e.

938

Human Vaccines

Volume 7 Issue 9

thorax was opened and the right lung was aseptically removed for
microbiological testing.
Microbiological testing. For the determination of bacterial
load, the lungs were suspended in 1 mL of steril saline solution
and were homogenized with a sterile 1 mL syringe. One hundred
microliters of the homogenates were taken, plated in steril tubes
containing egg-based Ogawa medium and incubated for 28 d at
37C. After incubation, colonies were counted.
Statistical analysis. The program Statistica 6.0 was used. For
the whole cells ELISA used for S. lividans or BCG, Duncan and
ANOVA tests were used, whereas for the challenge experiment,
ANOVA and Tukey HSD test was used to determine significance on lung bacillary loads. Values of p < 0.05 were considered
significant.
References
1.

Lnnroth K, Castro KG, Chakaya JM, Chauhan LS,

Floyd K, Glaziou P, et al. Tuberculosis control and
elimination 2010-50: cure, care and social development. Lancet 2010; 375:1814-29; PMID:20488524;
DOI:10.1016/S0140-6736(10)60483-7.
2. Garnier T, Eiglmeier K, Camus JC, Medina N,
Mansoor H, Pryor M, et al. The complete genome
sequence of Mycobacterium bovis. Proc Natl Acad
Sci USA 2003; 100:7877-82; PMID:12788972;
DOI:10.1073/pnas.1130426100.
3. von Reyn CF, Vuola JM. New vaccines for the
Prevention of Tuberculosis. Clin Infect Dis 2002;
35:465-74; PMID:12145732; DOI:10.1086/341901.
4. Kaufmann SH, Hussey G, Lambert PH. New vaccines for tuberculosis. Lancet 2010; 375:2110-9;
PMID:20488515; DOI:10.1016/S0140-6736(10)603
93-5.
5. Acosta A, Sierra G. Gentica y Biomedicina Molecular.
In: Orozco E, Garilio P, Editors. Vacunas. Mxico DF
2000; 321-48.
6. Korn-Wendisch F, Kutzner HJ. The family
Streptomycetaceae. In: Balows A, Truper HG, Dworkin
M, Harder W, Schleifer KH, Editors. The Prokaryotes.
New York 1992; 921-95.
7. Bentley SD, Chater KF, Cerdeo-Trraga AM, Challis
GL, Thomson NR, James KD, et al. Complete
genome sequence of the model actinomycete
Streptomyces coelicolor A3(2). Nature 2002; 417:141-7;
PMID:12000953; DOI:10.1038/417141a.
8. Zhou X, He X, Li A, Lei F, Kieser T, Deng Z.
Streptomyces coelicolor A3(2) lacks a genomic island
present in the chromosome of Streptomyces lividans 66. Appl Environ Microbiol 2004; 70:7110-8;
PMID:15574907; DOI:10.1128/AEM.70.12.71108.2004.
9. Binnie C, Cossar JD, Stewart DJ. Heterologous biopharmaceutical protein expression in Streptomyces.
Trends Biotechnol 1997; 15:315-20; PMID:9263479;
DOI:10.1016/S0167-7799(97)01062-7.
10. Ann J, Van Mellaert L. Streptomyces lividans as
host for heterologous protein production. FEMS
Microbiol Lett 1993; 114:121-8; PMID:8282181;
DOI:10.1111/j.1574-6968.1993.tb06561.x.
11. Connell ND. Expression systems for use in
Actinomycetes and related organisms. Curr Opin
Biotechnol 2001; 12:446-9; PMID:11604318;
DOI:10.1016/S0958-1669(00)00243-3.
12. Tremblay D, Lemay J, Gilbert M, Chapdelaine Y,
Dupont C, Morosoli R. High-level heterologous
expression and secretion in Streptomyces lividans of two
major antigenic proteins from Mycobacterium tuberculosis. Can J Microbiol 2002; 48:43-8; PMID:11888162;
DOI:10.1139/w01-133.

Conclusion
In this work we demonstrate that the immunogenic capacity of
Streptomyces conferred certain level of protection against the
mycobacterial challenge in a murine model of infection with
BCG. These observations, along with Streptomyces potential
for expressing foreign proteins, suggest that Streptomyces could
be an advantageous vector in the design of new tuberculosis
vaccines.
Acknowledgements

N.O.A. was supported by the National Autonomous University

of Mexico(UNAM), was recipient of a scholarship of National
Council of Science and Technology (CONACYT).

13. Lara M, Servin-Gonzles L, Singh M, Moreno C,

Cohen I, Nimtz M, et al. Expression, secretion
and glycosylation of the 45- and 47-kDa glycoprotein of Mycobacterium tuberculosis in Streptomyces
lividans. Appl Environ Microbiol 2004; 70:679-85;
PMID:14766542; DOI:10.1128/AEM.70.2.67985.2004.
14. Vallin C, Ramos A, Pimienta E, Rodrguez C,
Hernndez T, Hernndez I, et al. Streptomyces as host
for recombinant production of Mycobacterium tuberculosis proteins. Tuberculosis (Edinb) 2006; 86:198-202;
PMID:16644285; DOI:10.1016/j.tube.2006.02.003.
15. Hopwood DA. The Streptomyces genome
be prepared. Nat Biotechnol 2003; 21:505-6;
PMID:12721573; DOI:10.1038/nbt0503-505.
16. Agger EM, Andersen P. A novel TB vaccine; towards
a strategy based on our understanding of BCG failure. Vaccine 2002; 21:7-14; PMID:12443657;
DOI:10.1016/S0264-410X(02)00447-4.
17. Glatman-Freedman A, Casadevall A. Serum. Therapy
for tuberculosis revisited: Reappraisal of the role of
antibody-mediated immunity against Mycobacterium
tuberculosis. Clin Microbiol 1998; 11:514-32;
PMID:9665981.
18. Teitelbaum R, Glatman-Freedman A, Chen B, Robbins
JB, Unanue E, Casadevall A, et al. A mAb recognizing a
surface antigen of Mycobacterium tuberculosis enhances
host survival. Proc Natl Acad Sci USA 1998; 95:1568893; PMID:9861031; DOI:10.1073/pnas.95.26.15688.
19. Casadevall A. Antibody-mediated protection against
intracellular pathogens. Trends Microbiol 1998;
6:102-7; PMID:9582935; DOI:10.1016/S0966842X(98)01208-6.
20. Thaiss CA, Kaufmann SH. Toward novel vaccines
against tuberculosis: current hopes and obstacles. Yale
J Biol Med 2010; 83:209-15; PMID:21165340.
21. Flett F, Mersinias V, Smith CP. High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia
coli to methyl DNA-restricting Streptomycetes. FEMS
Microbiol Lett 1997; 155:223-9; PMID:9351205;
DOI:10.1111/j.1574-6968.1997.tb13882.x.
22. Hidrin N, Goodfellow M, Boiron P, Moreno M,
Serrano JA. Los Streptomyces. Actualizacin y revisin
didctica. Rev Soc Ven Microbiol 2001; 21:1315-2556.
23. McNeil MM, Brown JM. The medicallly important
aerobic actinomycetes: Epidemiology and microbiology.
Clin Microbiol Rev 1994; 7:357-417; PMID:7923055.
24. Khatri ML, Al-Halai HM, Fouad Khalid M, Saif SA,
Vyas MC. Micetoma in Yemen: Clinicoepidemiologic
and histopathologic study. Int J Dermatol 2002;
41:586-93; PMID:12358829; DOI:10.1046/j.13654362.2002.01383.x.
25. Cataldi A, Bigi F, Mohd-Nor N, Sarmiento ME, Acosta
A. Strategies for new generation vaccine development.
In: Mohd-Nor N, Acosta A, Sarmiento ME, Eds. The
art and science of tuberculosis vaccine development.
Selangor Durul Ehsan, Malaysia. Oxford Fajar Sdn
2010; 185-208.

26. Acosta A, Sarmiento ME, Gonzlez A, Estvez P, guila

A, Infante JF, et al. Histopathologic and humoral study
of balb/c mice inoculated with BCG by different routes.
Arch Med Res 1994; 25:159-63; PMID:7919805.
27. Petricevich VL, Ueda C, Alves RC, da Silva MA,
Moreno C, Melo AR, et al. A single strain of
Mycobacterium bovis bacillus Calmette-Gurin (BCG)
grown in two different media evokes distinct humoral
immune responses in mice. Braz J Med Biol Res 2001;
34:81-92; PMID:11151032; DOI:10.1590/S0100879X2001000100010.
28. Vallin C, Ayala JC, Garca-Rivera D, Jones J, Rodrguez
C, Gonzlez L, et al. Immune response to Streptomyces
lividans in mice: A potential vaccine vehicle against TB.
Open Vaccine J 2009; 2:85-91; DOI:10.2174/187503
5400902010085.
29. Dockrell HD. T cell protective immune responses
against tuberculosis. In: Mohd-Nor N, Acosta A,
Sarmiento ME, Eds. The art and science of tuberculosis
vaccine development. Selangor Durul Ehsan, Malaysia.
Oxford Fajar Sdn 2010; 61-77.
30. Glatman-Freedman A. The role of antibody-mediated
immunity in defence against Mycobacterium tuberculosis:
Advance toward a novel vaccine strategy. Tuberculosis
(Edinb) 2006; 86:191-7; PMID:16584923;
DOI:10.1016/j.tube.2006.01.008.
31. Williams A, Reljic R, Naylor I, Clark SO, FaleroDiaz G, Singh M, et al. Passive protection with
immunoglobulin A antibodies against tuberculous
early infection of the lungs. Immunology 2004;
111:328-33; PMID:15009434; DOI:10.1111/j.13652567.2004.01809.x.
32. Chambers MA, Gavier-Widn D, Hewinson RG.
Antibody bound to the surface antigen MPB83 of
Mycobacterium bovis enhances survival against high
dose and low dose challenge. FEMS Immunol Med
Microbiol 2004; 41:93-100; PMID:15145452;
DOI:10.1016/j.femsim.2004.01.004.
33. Glatman-Freedman A, Casadevall A, Dai Z, Jacobs
WR Jr, Li A, Morris SL, et al. Antigenic evidence of
prevalence and diversity of Mycobacterium tuberculosis
arabinomannan. J Clin Microbiol 2004; 42:3225-31;
PMID:15243086; DOI:10.1128/JCM.42.7.322531.2004.
34. Glatman-Freedman A. The role of antibodies against
tuberculosis. In: Mohd-Nor N, Acosta A, Sarmiento
ME, Eds. The art and science of tuberculosis vaccine
development. Selangor Durul Ehsan, Malaysia. Oxford
Fajar Sdn 2010; 81-107.
35. Spiekermann GM, Finn PW, Ward ES, Dumont J,
Dickinson BL, Blumberg RS, et al. Receptor-mediated
immunoglobulin G transport across mucosal barriers in adult life: functional expression of FcRn in
the mammalian lung. J Exp Med 2002; 196:303-10;
PMID:12163559; DOI:10.1084/jem.20020400.

201
1L
andesBi
os
c
i
enc
e.
Donotdi
s
t
r
i
but
e.

www.landesbioscience.com

Human Vaccines

939

36. Castillo-Rodal AI, Castan-Arreola M, HernndezPando R, Calva JJ, Sada-Daz E, Lpez-Vidal Y.

Mycobacterium bovis BCG substrains confer different
levels of protection against Mycobacterium tuberculosis
infection in a BALB/c model of progressive pulmonary tuberculosis. Infect Immun 2006; 74:171824; PMID:16495544; DOI:10.1128/IAI.74.3.171824.2006.
37. Kochi SK, Killeen KP, Ryan US. Advances in the
development of bacterial vector technology. Expert
Rev Vaccines 2003; 2:31-43; PMID:12901595;
DOI:10.1586/14760584.2.1.31.
38. EEC Council Directive 86/609. Guide for the use of
Laboratory Animals. OJL 1987; 358:1.

39. Davidsen T, Beck E, Ganapathy A, Montgomery R,

Zafar N, Yang Q, Madupu R, Goetz P, Galinsky K,
White O, Sutton G. The Comprehensive Microbial
Resource. Nucleic Acids Res. 2010;38:D340-5.40.
Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood
DA. Practical Streptomyces Genetics. The John Innes
Foundation. Growth and preservation of Streptomyces.
Norwich, England 2000; 43-60.
41. Vacca LL. Laboratory Manual of Histochemistry:
Ediciones Raven Press Book 1985.

201
1L
andesBi
os
c
i
enc
e.
Donotdi
s
t
r
i
but
e.

940

Human Vaccines

Volume 7 Issue 9