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International Journal of Medical Microbiology 299 (2009) 447–452

www.elsevier.de/ijmm

SHORT COMMUNICATION
Induction of a protective response with an IgA monoclonal antibody
against Mycobacterium tuberculosis 16 kDa protein in a model of
progressive pulmonary infection
Yamilé Lópeza,, Daniel Yeroa, Gustavo Falero-Diaza, Nesty Olivaresa,
Marı́a E. Sarmientoa, Sergio Sifontesa, Rosa L. Solisa, Jorge A. Barriosb,
Diana Aguilarb, Rogelio Hernández-Pandob, Armando Acostaa
a
Finlay Institute, Avenue 27 No. 19805, La Lisa, Havana, Cuba
b
Department of Pathology, National Institute of Medical Sciences and Nutrition ‘Salvador Zubirán’, México

Received 4 September 2008; received in revised form 9 October 2008; accepted 19 October 2008

Abstract
Mycobacterium tuberculosis is a facultative intracellular pathogen for which cell-mediated immunity is considered
the major component of the immune response. For many decades, the prevailing scientific view has been the antibodies
have little or no role in modifying the course of M. tuberculosis infection. In recent years, several studies
have challenged this dogma, and there is a body of evidence that supports a role of antibodies against M. tuberculosis.
In the present work, we evaluated the protective activity of two monoclonal antibodies (TBA61 and TBA84). Here, we
chose the intratracheal model of pulmonary infection to evaluate bacterial load and morphometric and histological
changes in the lungs of treated mice. Data obtained revealed the reduction of bacterial load and milder morphometric
and histopathological changes in mice treated with TBA61 at 21 days post-infection with M. tuberculosis H37Rv
compared to those treated with TBA84 and control mice. These results allow continuing exploring the potential use of
monoclonal antibodies as prophylactic and therapeutic agents against intracellular pathogens such as M. tuberculosis.
r 2008 Elsevier GmbH. All rights reserved.

Keywords: BCG; Tuberculosis; 38 kDa; 16 kDa; Monoclonal antibody

Introduction with a dramatic rise of multiple drug-resistant strains

Tuberculosis is an infectious disease caused by
Mycobacterium tuberculosis. It is a pandemic, transmis-
sible, and curable disease. Developing countries are the
most affected by the disease, mainly due to the lack
of resources for diagnostic and treatment. Over two
billions persons worldwide are infected with the bacteria
(Girard et al., 2005). The epidemics have been associated
Corresponding author. Tel.: +53 7 2716911; fax: +53 7 2086075.
E-mail address: ylopez@finlay.edu.cu (Y. López).
and a large population of immunocompromized persons
by the human immunodeficiency virus in which
tuberculosis (TB) is an aggressive and often incurable
infection (Jain and Mondal, 2008). The only
available and licensed vaccine against tuberculosis is
the Bacille Calmette Guerin (BCG), based on an
attenuated strain of Mycobacterium bovis. The protec-
tive efficacy of BCG vaccine varies upon several
nutritional and environmental factors, that is why the
scientific community is nowadays searching for more
effective vaccines, prophylactic and treatment methods

1438-4221/$ - see front matter r 2008 Elsevier GmbH. All rights reserved.
doi:10.1016/j.ijmm.2008.10.007
 ARTICLE IN PRESS
448 Y. López et al. / International Journal of Medical Microbiology 299 (2009) 447–452
(Andersen and Doherty, 2005; Ly and McMurray, 2008;
Dietrich et al., 2003).
A conventional agreement has been that host protec-
tion against intracellular pathogens depends on cell-
mediated immunity (CMI). The revelation of an
important role for antibody response in facilitating
the induction of an adequate and rapid memory Th1
response against microbial pathogens (including intra-
cellular pathogens) would suggest mutual regulatory
functions of both arms of the immune system in
optimizing host defense (Bretscher et al., 2002; Ivanyi
et al., 1985). Thus, this theory may constitute a
paradigm shift from the current dogma of immune
segregation wherein a CMI response is presumed to
be independent of antibody function (Ivanyi and Thole,
1994).
Supportive data for the role of antibody in host
defense is provided by in vitro studies which demon-
strate that specific antibody inhibits the replication
of the pathogen, neutralizes toxins elaborated by the
pathogen, promotes antibody-dependent cellular cyto-
toxicity (ADCC), serves as an opsonin, triggers the
complement cascade, and/or prevents infection in tissue
culture (Glatman-Freedman, 2003; Igietseme et al.,
2004). In particular, M. tuberculosis has developed
strategies to persist in infected hosts despite the presence
of potent T-cell-mediated immune responses (Reece and
Kaufmann, 2008). On the other hand, disseminated
tuberculosis is not found in children with antibody titers
against the lipoarabinomannan (Daffe and Etienne,
1999). Moreover, contacts having specific IgA against
mycobacterial antigens do not get infected (Glatman-
Freedman and Casadevall, 1998). Considering these new
points of view, vaccine approaches against intracellular
microbial pathogens should therefore be focused on
designing strategies to stimulate both T-cell and anti-
body responses since antibodies are indirectly involved
in T-cell activation (Ivanyi and Thole, 1994).
In our group, several works have been carried out
with human gammaglobulin to fight TB infection.
In one work, granulomes from lungs of human
gammaglobulin-treated mice were morphologically si-
milar to those developed by BCG-vaccinated mice
(Acosta et al., 1994). Moreover, in a model of intranasal
challenge with BCG in mice, the human gammaglobulin
showed protective activity (Olivares et al., 2006). Taking
into account the complex epidemiological situation of
tuberculosis worldwide and the evidences accumulated
up today regarding humoral immunity against myco-
bacterial infections, we consider of great importance
to study the potential use of antibody formulations in
the modulation of immune response and activation
of effective mechanisms to control the progression of the
disease. In the present work, we evaluated the efficacy
of monoclonal antibodies in the control of pulmonary
infections. To our knowledge, this is the first report of
the induction of protective response against pulmonary
tuberculosis after the administration of IgA monoclonal
antibodies by intratracheal route and challenge with
virulent bacteria by the same route.
Materials and methods
Bacteria and growth conditions
The bacteria (H37Rv strain) were grown to early mid-
log phase in Middelbrook 7H9 medium (Difco, Detroit,
Michigan). Cultures were incubated at 37 1C with 5%
CO2 and continuously shaken. The micro-organisms
were harvested by centrifugation at 5000g, washed 3
times in phosphate buffer saline (PBS) and stored at
70 1C until use. For vaccination and challenge, strains
were thawed at 37 1C and diluted in PBS to the final
work concentration. Bacterial concentration was deter-
mined by counting fluorescein acetate-stained cells in a
hemacytometer under fluorescent light microscopy.
Animals
Pathogen free Balb/c mice were obtained from the
National Institute of Medical Sciences and Nutrition,
Mexico City, Mexico. They were maintained under
barrier conditions in a level III biohazard laboratory.
Generation of monoclonal antibodies
Monoclonal antibodies (MAb) against 38- and
16-kDa M. tuberculosis proteins were obtained as
described by Falero-Diaz et al. (2000). Briefly, the
antigens used were purified recombinant variants of the
16-kDa a-crystallin (Acr antigen) and the 38-kDa
lipoglycoprotein (PstS-1 antigen). Six-to-8-week-old
female BALB/c mice were immunized intranasally under
mild anesthesia. Three inoculations of 10 mg of each
protein antigen mixed with 1 mg of the cholera toxin
(Sigma) in a volume of 30 mL were separated by 2-week
intervals. Mice with the highest antibody levels in the
saliva and serum were selected for the generation of
hybridoma lines. Suspensions of spleen, salivary gland,
lung, and nasal-associated lymphoid tissue were used for
fusion with myeloma cells, and antibody-positive
hybridomas were selected and cloned using standard
procedures (Williams et al., 2004). The monoclonal
antibodies obtained belonged to the IgA isotype
and were denoted TBA61 (IgA anti Acr) and TBA84
(IgA anti PstS-1).
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Y. López et al. / International Journal of Medical Microbiology 299 (2009) 447–452
Passive immunization and protection assay
Five BALB/c mice (male, aged 8–10 weeks) per group
were anesthetized by sodium pentobarbital subcuta-
neous administration (50 mg/kg), and a longitudinal
skin incision at the ventral face of the neck was
performed on each animal. Three groups of 5 mice
were treated intratracheally with 100 mL of the corre-
sponding monoclonal antibody at 5 mg/mL concentra-
tion or PBS, respectively. Thirty minutes after the
inoculation, mice were challenged with 100 mL contain-
ing 106 colony forming units (CFU) of M. tuberculosis
H37Rv strain. Once the procedure was fulfilled, mice
were sutured, returned to their cages, and kept in an
isolator where plenty of food and water was supplied.
Mice were sacrificed at 24, 72 h, and 21 days after
the infection. They were anesthetized with ethylic ether
and bled by cutting the subclavian artery. Lungs
were aseptically removed, homogenized with Polytron
(Brinkman Instruments, Rexleid, Canada) in 3 mL
of isotonic salt solution containing 0.05% Tween 80
(Sigma, St. Louis, USA), and 1 mL of this homogenate
in 10-fold serial dilutions were plated on Petry dishes
containing Middlebrook 7H10 culture medium. Plates
were sealed in nylon bags and incubated for 21 days at
37 1C to eventually determine the number of CFU to
assess pulmonary bacterial load.
Histopathology and morphometric studies
The right lung of each mouse sacrificed as indicated
above at 24 h and 21 days were removed and fixed in
10% neutral buffered formalin, embedded in paraffin,
and processed for histology. Sections were stained with
hematoxylin and eosin for histological evaluation and
photography. Slides were observed by light microcopy
and the areas of peribronchial and perivascular inflam-
matory reactions were measured and analyzed by using
Leica-win system software (Leica microsystem Imaging
solutions LTD, Cambridge, UK).
Statistical methods
The effects of antibody treatment on the reduction of
bacterial load and in morphometric changes were
analyzed by Kruskall–Wallis test and the distribution-
free multiple comparison test. p-Values under 0.05 were
considered statistically significant. All data analysis was
performed by using the STATISTICA 6.1 Software.
Results and discussion
Contrary to the current dogma about the ineffective
role of antibodies against intracellular pathogens,
449
during the last years evidence has been accumulated in
favor of the importance of the humoral mediators in the
activation of principal immune mechanisms to control
the progression of the disease caused by intracellular
pathogens, such as M. tuberculosis (Igietseme et al.,
2004). Mainly in the case of intracellular pathogens that
are facultative, as M. tuberculosis, antibodies may be
decisive in the early stages of the disease, during the
extracellular phase, or could be capable of penetrating
recently infected cells to bind the internalized pathogen
(Snewin et al., 1999).
The protection efficacy of a monoclonal antibody
(TBA61, IgA anti Acr) administered intranasally
before and after the intranasal or aerosol challenge with
M. tuberculosis was demonstrated in a previous work
(Williams et al., 2004). In the present study, we
evaluated the protective effect of this monoclonal
antibody administered intratracheally against an intra-
tracheal challenge with virulent mycobacteria, in order
to evaluate the activity of this antibody using another
relevant animal model for the study of pulmonary
tuberculosis. We also employed the TBA84 monoclonal
antibody since it is directed against other mycobacterial
antigen. Experiments were conducted, in order to
evaluate the role of this formulation in the airway
mucosa immediately before the encounter with the
pathogen. Twenty-four and 72 h after the challenge, all
groups were similar in terms of mean viable counts in
lung samples. However, at 21 days post-infection,
the treatment of mice with the MAb TBA61 against
the 16-kDa protein of M. tuberculosis caused a
statistically significant decrease in the mean number of
viable bacteria in lungs compared to control mice or
those treated with the MAb against the 38-kDa protein
(Fig. 1A). Consistent with the reduction of viable
bacteria achieved by the treatment with TBA61, at
21 days post-infection, the area of peribronchial
inflammation was also statistically smaller in this group
compared to the control group. However, the area of
perivascular inflammation did not show significant
differences among the groups evaluated (Fig. 1B and C).
When the lungs of mice were histologically examined,
granulomas were better organized in the infected
animals that had received antibodies against the
16-kDa protein than in controls and mice treated with
TBA84. The reduction of CFU in lungs of this treated
group was associated with milder histopathological
changes, as indicated by the organization of the
granulomas and less pneumonic area. No inflammatory
reactions were observed in lungs of mice sacrificed at
24 h post-infection (Fig. 2).
The 16-kDa protein (Acr antigen) has been defined as
a major membrane protein peripherally associated with
the membrane (Lee et al., 1992) carrying epitopes
restricted to tubercle bacilli on the basis of B-cell
recognition (Coates et al., 1981). The Acr antigen is
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450 Y. López et al. / International Journal of Medical Microbiology 299 (2009) 447–452
Fig. 1. (A) Determination of bacterial load in lungs of mice
treated with TBA61, TBA84, or PBS and challenged with
virulent M. tuberculosis H37Rv 30 min after the inoculation of
antibodies by intratracheal route. At 24, 72 h, and 21 days
post-infection, lungs were harvested, and CFU was deter-
mined. Only mice inoculated with TBA61 showed a significant
reduction (po0.05, Kruskal–Wallis test) compared to the rest
of groups at 21 days post-infection. (B) Areas of perivascular
inflammation. (C) Peribronchial inflammation in lungs of mice
challenged with virulent bacteria at 1, 3, and 21 days post-
infection. The morphometric study was carried out with light
microscopy by using a computer program. Statistical differ-
ences compared to the control group (po0.05, Kruskal–Wallis
test) were only obtained in the group treated with TBA61 at 21
days post-infection.
present on the surface of tubercle bacilli and has
enhanced expression in organisms grown within infected
macrophages, which may explain the protective effect of
this monoclonal antibody with respect to the group
treated with the MAb against the 38-kDa protein. This
later antigen is not considered a major protein despite
being a surface and secreted protein (Devi et al., 2002).
The failure of MAb TBA84 to protect mice after
infection was also mentioned by Williams et al. (2004)
using the intranasal models. In one study of transmis-
sion of TBA61 and TBA84 to mucosal fluids and blood
following intranasal and intravenous inoculation, the
distribution of TBA84 was different to that of TBA61,
the former having a relatively long persistence in lung
lavages and a lack of transport to saliva. This suggests
the role of a mucosal site-specific mechanism that
involves differences in IgA binding to the secretory
component or in the action of some proteases (Falero-
Diaz et al., 2000). While this fact could explain the
inefficacy of MAb TBA84, other factors could be
involved (e.g. the density of both PstS-1 and Acr
antigens on the mycobacterial surface or the affinity of
the antibodies). The lack of any effect before the 21 days
could be due to a clearance of opsonized mycobacteria
that takes over 24 h.
As it was mentioned before, Williams et al. (2004)
reported the protective effect of monoclonal antibody
TBA61 against the intranasal and aerosol challenge with
virulent M. tuberculosis. However, the protection was of
short duration, presumably due to the rapid degradation
of the intranasally applied IgA by proteases in the upper
respiratory tract. In our work, we chose the intratra-
cheal route for the inoculation of these antibodies and
the virulent strain. During several years, animal models
for the study of tuberculosis have employed unnatural
routes of infection (intravenous or intraperitoneal) and
have missed representing the real phenomena. The
model of progressive pulmonary tuberculosis used in
our work allowed to reproduce to a certain extent the
natural route with a high number of live and virulent
bacteria inoculated by the intratracheal route, guaran-
teeing a better control of dose and the final deposit of
virtually all bacteria in the lungs. This model has
been useful for the study of pathogenic mechanism of
M. tuberculosis infection (Arriaga et al., 2002).
Our group has reported the inhibition of the
colonization of mycobacteria in vivo mediated by the
presence of specific human IgG antibodies (Olivares
et al., 2006). It is now accepted that specific antibody
isotypes (IgG and IgA) and T cells are required for the
induction of protective immunity against intracellular
pathogens. The monoclonal antibodies used in our work
are of IgA class, thus we have assumed that their action
could involve a number of mechanisms: stimulating the
induction of Th1 effectors through Fc-R-mediated rapid
antigen uptake, improving processing, increasing the
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Y. López et al. / International Journal of Medical Microbiology 299 (2009) 447–452
Fig. 2. Tissue histology sections. Mice were sacrificed at 24 h (A, B, and C) and 21 days (D, E, and F) after the challenge with
virulent bacteria, and formalin-fixed sections were analyzed. Granulomas became evident at 2 weeks post-infection. Granulomas
were better organized in mice treated with TBA61 (E) compared to those treated with either PBS (D) or TBA84 (F). A, B, and C: HE
 100; D, E, and F: HE  40.
efficacy of co-stimulatory signals, and the rapid
presentation of antigen for activation of an elevated
Th1 response, more homing to the lungs following
respiratory infection, and the augment of antibody-
dependent cellular cytotoxicity (Reynolds, 1987; Casadevall
and Pirofski, 2003). Interestingly, only IgG2a and IgA
can augment the T-cell response (Ravetch and Bolland,
2001; Regnault et al., 1999).
In conclusion, our results support evidence related
to the crucial role of antibodies in the induction of a
protective response against intracellular pathogens.
Treatment with antibodies (also polyclonal antibodies)
as a prophylactic or therapeutic method may be a good
alternative for the control of re-emergent diseases and
in the special case of immunocompromised hosts or
individuals infected with multi-drug-resistant strains,
where current vaccination strategies do not work.
It could redirect vaccinology to focusing in the analysis
and elucidation of these two important immunoregula-
tory mechanisms (antibodies and CMI) for the design
and development of more effective vaccines against
intracellular pathogens.
Acknowledgements
This investigation was supported by a grant from
UNU-BIOLAC. The studies were also supported by the
Experimental Pathology Section, National Institute of
Medical Sciences and Nutrition, Mexico, where the
experiments were performed.
451
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