ARTICLE IN PRESS

Tuberculosis (2006) 86, 247254

Tuberculosis
http://intl.elsevierhealth.com/journals/tube

Immunization of mice with a Mycobacterium

tuberculosis genomic expression library results
in lower bacterial load in lungs after challenge
with BCG
Yamile
Lo
pez Herna
ndeza,, Daniel Yero Coronaa, Sergio
Sifontes Rodrgueza, Juan Francisco Infante Bourzaca,
Mara Elena Sarmientoa, Nesty Olivares Arzuagaa,
`l Dazb,
Enrique Casado Maceoa, Daiyana Daza, Rau
Armando Acosta Domngueza
a

Department of Molecular Biology, Finlay Institute, Havana, Cuba

Pedro Kour Institute of Tropical Medicine, Havana, Cuba

Received 4 October 2005; accepted 20 January 2006

KEYWORDS
Genomic library;
BCG challenge;
Tuberculosis;
DNA vaccine

Summary Tuberculosis is a serious infectious disease in many developing

countries. The lack of an effective vaccine for preventing this disease has stimulated
the search for new vaccine candidates against Mycobacterium tuberculosis. In the
present work, the construction of a genomic expression library of M. tuberculosis in
a eukaryotic expression vector was carried out. Immunization of Balb/c mice with a
plasmid DNA pool from this library (containing 8360 clones) induced a significant IgG
antibody response. Immunized mice were challenged by intratracheal route with
105 cfu of non-pathogenic Mycobacterium bovis BCG and were sacrificed 21 days
post-challenge. Mice immunized with the genomic expression library showed a
significant reduction of viable bacteria in lungs and less pulmonary tissue damage.
Granulomas were not observed and the lungs had a more discrete perivascular
inflammatory cell infiltrate compared to control mice. Results suggest that the
genomic expression library contains genes encoding proteins that are protective
against M. tuberculosis infection.
& 2006 Elsevier Ltd. All rights reserved.

Corresponding author. Tel.:+ 53 7 2716911.

E-mail address: ylopez@finlay.edu.cu (Y.L. Herna

ndez).
1472-9792/$ - see front matter & 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tube.2006.01.004

ARTICLE IN PRESS
248

Introduction
Mycobacterium tuberculosis, the causative agent
of tuberculosis, is one of the leading infectious
killers worldwide. The World Health Organization
estimates that approximately one third of the
world population is infected with the tuberculosis
bacillus, around 8 million people acquire the
disease and 1.6 million die as a result every year.1
A live, attenuated strain of Mycobacterium bovis
(BCG) is the most widely used vaccine in the world;
however, its efficacy in protecting against tuberculosis remains controversial. Hence, searching for
new vaccine candidates against tuberculosis is a
main priority of the international biomedical
research community. Nevertheless, only a few
candidates tested until now have shown protection
better than that conferred by the BCG vaccine.2
With the development of genomics and the tools
of bioinformatics, the complete genome sequence
of M. tuberculosis H37Rv and CDC-1551 strains has
become available.3,4 This has brought about valuable information on bacterial genetics and the
products encoded by most of the genes. However,
the genome sequence alone cannot provide information about the protective value of candidate
antigens. Of the 3924 protein encoding genes, only
40% have homology with other known proteins
while the other 60% have no clear function.5
Genetic immunization is one of the most prominent strategies currently used to develop new
vaccines against tuberculosis. DNA vaccines have
been shown to constitute a good vehicle for
delivering antigens into the host. Many publications
have shown the efficacy of DNA formulations in
protecting against virulent challenge with M.
tuberculosis in animal models.69 Expression library
immunization (ELI) has emerged as a technology to
systematically screen entire genomes for protective antigens. Once established, such libraries and
fractions thereof can be delivered into the host as a
platform to identify individual antigen genes.10 To
date, by means of this technology new vaccine
candidates against some pathogens have been
discovered.1113 Since the first report of induction
of protective response against Mycoplasma pulmonis using ELI, many studies have described similar
results.1422 Recently, we have successfully applied
this technology for generating protective immune
response in mice against Neisseria meningitidls
serogroup B.23 The objective of this work was to
evaluate, using the same ELI protocols, the
protective efficacy of a genomic M. tuberculosis
library against challenge with M. bovis BCG in mice.
The results presented here constitute, to our
knowledge, the first demonstration of the use of

Y.L. Herna
ndez et al.
ELI to generate a protective response against M.
tuberculosis in a murine model of infection.

Materials and methods

Mice
Balb/c mice were purchased from the National
Center for the Production of Laboratory Animals
(CENPALAB, Cuba). Mice were 68 weeks old at the
time of vaccination. They were maintained under
conventional conditions and fed commercial mouse
chow and water ad libitum.

Bacteria and plasmids

M. tuberculosis 14323 (reference strain) belongs to
the strain collection of Pedro Kour Institute of
Tropical Medicine (Cuba). M. bovis BCG vaccine
(BCG sub-strain Sofia) was kindly donated by the
National Immunization Program, Cuba. For the
construction of the genomic library, the eukariotic
expression vector pELI3.123 was employed. This
vector contains the human cytomegalovirus immediate-early promoter and enhancer for constitutive expression and permits the blue/white
screening of recombinant clones in Escherichia coli.

Library construction and maintenance

M. tuberculosis genomic DNA was isolated from
strain 14233 (reference strain). The purified DNA
was partially digested with Sau3A I ( 0.5 U for 5 min
at 371 C) to a median size of 1750 bp, and the
digested material was purified using the QIAQuick
PCR purification kit (Qiagen Inc., Mississagua, Ont.,
Canada). Standard molecular cloning techniques 24
were used for library construction. Vector pELI3.1
23
was digested with the restriction enzyme BamHI,
treated with alkaline phosphatase, and ligated with
Sau3AI-digested M. tuberculosis genomic DNA. This
procedure was repeated using vector pELI3.1
previously digested with Bg/II or Bc/I in order to
obtain the maximal representation of each genomic
fragment since vector pELI3.1 contains the BamHI,
Bg/II and Bc/I sites in three separated reading
frames. Ligation mixtures were transformed by
electroporation in E. coli TOP10 [F
mcrA D(mrrhdsRMS-mcrBC) F80lacZDM15 DlacX74deoR recA1
araD139 D(ara-leu)7697 galU galK rpsL endA1nupG]
(Invitrogen, Burlington, Ont., Canada) and the
transformed cells were plated onto LuriaBertani
(LB) agar plates supplied with ampicillin (100 mg/
ml) and containing X-Gal and IPTG. After growing

ARTICLE IN PRESS
Tuberculosis immunization in mice
overnight, white colonies were picked with sterile
toothpicks and inoculated into selective 2xYT
medium. Library replication and conservation was
performed as was previously described.23 Briefly,
plates containing individual clones were incubated
for 6 h at 371 C with agitation and used to inoculate
replicas. Then, glycerol was added to a final
concentration of 15% (v/v) and the plates were
frozen for long-term storage (master plates). A
total of 88 plates were obtained and conserved.

Preparation of DNA for genetic immunization

Conserved master plates were combined in four
groups of 22 plates for preparing DNA pools for
vaccination. The plates were thawed and used to
create replica plates. The replica plates were
incubated for 6 h at 37 1C allowing each clone to
grow individually in selective 2x YT medium. All
individual cultures were combined and used to
inoculate 2.5 l of selective LB medium. Plasmid
pools were purified using the EndoFree Plasmid
Mega Kit according to instructions of the manufacturer (Qiagen). The pool of 8360 plasmids was
obtained by combining each of the four pools of
2090 plasmids. The purification of the total library
in four pools avoids plasmid lost by dilution in the
2.5 l culture prior to Qiagen purification.

Immunization schedule
Mice were randomly assigned to four groups of
eight animals each. One group was treated with
three doses (100 mg of DNA per dose in 100 ml of
PBS) of the entire library intramuscularly at 3 week
intervals. Another group was immunized subcutaneously with a single dose of 105 cfu of BCG at the
same time as the first DNA inoculation. Groups of
animals inoculated either with the empty vector
(100 mg per dose) or PBS (three doses, 100 ml) were
used as controls. Blood was extracted from the
retro-orbital plexus of five anesthetized mice per
group at 21 days after the last immunization.

Elisa
Flat-bottomed, high-binding polystyrene microtiter
plates (Costar, Corning Inc., NY) were coated for
16 h at 37 1 C with BCG (105 cfu per well suspended
in 50 mL of PBS, pH 7.4). Serum dilutions (start
dilution 1:50) were prepared in PBS-Tween 80 and
incubated 2 h at 37 1C in pre-coated, blocked
plates. Bound antibodies were detected using an
anti-mouse IgG peroxidase conjugate (Sigma-Aldrich Chemie GmbH., Taufkirchen, Germany).

249

Challenge study
To study the protection conferred by the whole
library, all mice were challenged with BCG, 21 days
after the last inoculation. Mice were anesthetized
with 50 mg/kg of sodium pentobarbital administered by the intraperitoneal route. A midline
ventral incision was made in the neck for separating the skin and muscles to expose the trachea and
directly inoculating 105 cfu of BCG in the tracheal
lumen as was reported elsewere.25 The incisions
were then sutured up by using sterile silk. Mice
were kept in a vertical position until recovery.
Twenty days after the challenge, mice were
sacrificed by pentobarbital overdose (100 mg/kg,
intraperitoneal). The thorax was opened and the
lungs were aseptically removed. One of the lungs
was used for the determination of bacterial load,
whereas the other was infused with 10% formaldehyde. The left lung was suspended in 1 ml of sterile
0.05% PBSTween 80 (Sigma, St. Louis, USA) and
was homogenized with a sterile 1 ml syringe. Fifty
microliters of the homogenates were taken, plated
in sterile tubes containing egg-based Ogawa medium and incubated for 21 days at 37 1C. After
incubation, colonies were counted. Only dilutions
with counts in the range of 30300 colonies were
considered and averaged to estimate the number of
viable bacteria recovered from lungs.

Histopathology
Lung samples were kept in formaldehyde for a
minimum of 24 h, embedded in paraffin wax,
sectioned and stained with hematoxylineosin or
Zielh Neelsen. Slides were observed under light
microscopy to study tissue changes.

Statistical analysis
Results corresponding to ELISA and challenge experiment were analyzed employing the STATISTIC 6.0
software. The one-way analysis of variance (ANOVA)
was used to compare the existing differences in the
cfu counts. Distribution-Free Multiple Comparisons
Test was also performed as a complementary test.

Results
Construction and immunogenicity of the
genomic expression library
A M. tuberculosis genomic expression library made
up of 8360 clones was constructed and each clone

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250

Y.L. Herna
ndez et al.

1.2

50
Median
25%-75%
Min-Max

40

0.6
0.4
0.2

b
cfux103/mg

OD

0.8

30

20

a
a

0
PBS

Empty vector

Library

BCG

Figure 1 Serum IgG response in vaccinated mice 3 weeks

after the last DNA inoculation. The antibody response
was measured by a whole-cell BCG ELISA. Values
represent average OD per group. Statistically significant
differences were found between groups treated with BCG
or the library compared to groups treated with PBS or the
empty vector (KruskalWallis test, a vs. b and c po0.05).

was individually preserved. The eukaryotic expression vector (pEli3.1) used allowed cloning of the
DNA fragments into three different open reading
frames, maximizing the expression of cloned genes.
The colony screening based on their blue/white
color enabled the proper differentiation of recombinant colonies from those transformed with the
empty vector.
In silico digestion of M. tuberculosis genome by
using the Sau3AI restriction endonuclease produced
about 30,000 fragments, with an average size of
140 bp. On the contrary, the partial digestion
performed under the conditions established in the
present work rendered fragments ranging from 500
to 3000 bp. The probability of a given fragment to
be present in a library comprised of 8360 clones
with an average size of 1750 bp, like the present
one, is 0.958. Although this figure does not cover
the entire genome, most of the genomic DNA is
likely represented.
The whole-cell BCG ELISA performed with the
sera of mice under study (Fig. 1) showed that mice
immunized with the genomic library had an antibody response statistically higher than that observed in mice inoculated with the empty vector or
PBS, though lower than that found in mice
immunized with BCG (po0.01). We can assume
that in our library there are clones that are
expressing M. tuberculosis surface proteins that
are also present in BCG.

Challenge assay
Protection conferred by the immunization was
measured by the reduction in the number of viable

10

BCG

PBS

Library Empty vector

Figure 2 Mice vaccinated with the genomic library or

with a single BCG dose showed statistically lower loads of
viable bacteria in lungs compared to control groups after
the intra-tracheal challenge with BCG (KruskalWallis
and distribution-free multiple comparison tests, a vs. b
po0.05)

bacteria in lungs using BCG as the challenge

bacterium. The bacterial load in lungs of mice
immunized with the genomic library or BCG was
significantly decreased (po0,01) compared to
animals inoculated with the empty vector or PBS
(Fig. 2). Although in this study we could not define
the exact composition of the plasmid mixture, the
results presented here indicate that, at least one of
the plasmids or, more likely, a fraction of them
encode for protective antigens against M. tuberculosis infection.

Histopathological study
Since the reduction of cfu in lungs after the
challenge with M. tuberculosis is not always
correlated with protection, the evaluation of tissue
changes was performed. The histopathological
study showed the appearance and general composition of the granulomas developed in each group
under examination (Fig. 3). All of the lesions were
incipient because animals were sacrificed 21 days
post-challenge, which is considered an early stage
in the development of the disease.26 Granulomatous lesions, mainly constituted by macrophages,
were more evident in animals inoculated with the
empty vector or PBS. On the contrary, perivascular
inflammation and the recruitment of lymphocytes
and neutrophils characterized the lung samples
from mice immunized with BCG or the genomic
expression library. Neither pneumonia areas nor

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Tuberculosis immunization in mice

251

Figure 3 Histopathological analysis of lung samples from immunized mice 21 days following M. bovis BCG challenge.
Representative slides are shown for mice inoculated with BCG (A), genomic library (B), empty vector (C) or PBS (D).
(A,C) black arrows indicate the presence of perivascular granulomas; magnification: HE40  . (B) Mononuclear
inflammatory response surrounding a peribronchial vessel. Lung parenchyma is not consolidated and granulomas were
not found; HE40  . (D) Typical tuberculoid granuloma made up of macrophages, lymphocytes and small foci of caseous
necrosis; magnification: HE120  .

consolidated alveolar tissues were observed in the

samples analyzed.

Discussion
Even with the application of several technologies
for the identification of potential protective antigens, the development of a new vaccine more
effective than BCG remains challenging. Currently,
only a small number of M. tuberculosis antigens
have completed preclinical development and entered clinical trials.27
Immunization with genomic expression libraries
has emerged as a novel technology for the
discovery of vaccine candidate genes against
several pathogens for which there are no vaccines
available. To date, only three studies have reported
the identification of individual protective genes by
the sequential fractionation of cDNA or genomic
expression libraries.1113 Finding vaccine candidates against M. tuberculosis by means of this
technology is promising, since other work has
demonstrated that combined DNA vaccines against
M. tuberculosis were able to induce a higher
immune response than separate ones. Mixtures of
twoten plasmids expressing different tuberculosis
antigens have been developed as DNA vaccines.2832

However, regarding Mycobacterium genomic expression library, to our knowledge only one report
has been made in the literature. In that work, a
two-step strategy to screen and identify immunogenic polypeptides derived from a Mycobacterium
vaccae genomic expression library was described.33
Our library, constituted by 8360 clones, proved to
mediate reduction in the number of cfu in lungs in a
murine model of infection with mycobacteria.
Although the number of clones is small to statistically cover the whole M. tuberculosis genome,
screening that fraction of the genome is likely
enough to identify at least one vaccine candidate.10 In the present work, each recombinant
clone was isolated and individually stored. The
accurate identification of clones and the storage of
each recombinant clone individually will allow us to
fractionate the library for future experiments.
The capacity of DNA immunization to generate a
strong immune response, both humoral and cellular, has been described.34 Humoral response conferred by plasmid DNA immunization expressing M.
tuberculosis antigens has been evaluated by ELISA
in previous work.3537 In such work, a correlation
between protective efficacy and humoral response
was established. Also, several studies had associated the presence of serum antibodies, for
example, to mycobacterial carbohydrates, with
protection.3840 In the present study, a humoral

ARTICLE IN PRESS
252
response detected in mice immunized with the
genomic library might be the result of the expression of several M. tuberculosis proteins. Although
the consensus opinion on defense mechanisms
against M. tuberculosis confers a primary role on
cell-mediated immunity, in the recent years, a
body of evidence challenges this dogma and confers
importance to the role of specific antibodies in the
defense against the disease.4143 Antibodies induced by a vaccine may provide protection by
several mechanisms, including altering the inflammatory response by promoting changes in cytokine
release through Fc receptor cross-linking.41 With
our experiments, we cannot correlate protection
with the antibody response detected. In future
experiments, we must evaluate the specific role of
cell-mediated immunitiy and humoral response
induced by the immunization with the genomic
library.
In our experiments, the reduction of cfu in lungs
after challenge with M. bovis, could be, to some
extent, due to the in vivo expression of several M.
tuberculosis antigens. How these expressed proteins could activate cellular sub-sets of T CD4+ or T
CD8+ lymphocytes or the mechanisms related to
humoral response are points to consider when
fractioning of the library is performed. Inoculated
DNA itself can also induce antigen-specific T
lymphocytes that secrete IFN-g and show cytotoxic
potential, which may be related to the adjuvant
properties of their non-methylated CpG sequences.42 The effective immune response elicited
after immunization with our genomic library is
specific, as group vaccinated with the empty vector
did not show reduction of the cfu in the challenge
assay.
Inflammation is critical to control the progression
of the disease.43 In our study, the limitation of lung
inflammation and the decreased number and size of
granulomatous lung lesions in mice vaccinated with
the genomic expression library, suggested a better
protective immune response. These results are in
agreement with those of several authors who
reported a decrease in the number and area of
granulomas after vaccination with DNA and subsequent challenge with M. tuberculosis.44,45 However, as the challenge in our study was performed
with 105 cfu of M. bovis BCG, a more comprehensive evaluation of lung tissues could not have been
carried out, as the M. bovis BCG does not cause
severe pulmonary damage. That is why a higher
dose of bacteria for a challenge or a challenge with
a virulent M. tuberculosis strain is required in
future experiments.
M. tuberculosis has approximately 1700 gene
products to screen for protective antigens.10 With

Y.L. Herna
ndez et al.
the aid of traditional methods, this screening is
tedious and extremely time-consuming. The expression library immunization allows screening
functional protective genes instead of immune
correlates, which assures that the capacity to
mediate real protection against the infectious
agent is tested. In order to optimize the antigen
expression in ELI experiments, some methods such
as directed ELI (M. tuberculosis-directed library
consists of only about 2000 plasmids), codon
optimization approach or fusion of genes to
ubiquitin sequences have been proposed.10,46,47
However, these strategies are laborious and expensive. We demonstrate that a functional and
protective response can be reached against tuberculosis infection after immunization with a genomic
library. With this preliminary work, once more, the
success of ELI to identify new protective antigens
has been supported.

Acknowledgements
The authors would like to thank Dr. Uli Fruth, World
Health Organization, for critical review of this
manuscript.

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