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ARTICLE IN PRESS

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


and a large population of immunocompromized persons
Tuberculosis is an infectious disease caused by by the human immunodeficiency virus in which
Mycobacterium tuberculosis. It is a pandemic, transmis- tuberculosis (TB) is an aggressive and often incurable
sible, and curable disease. Developing countries are the infection (Jain and Mondal, 2008). The only
most affected by the disease, mainly due to the lack available and licensed vaccine against tuberculosis is
of resources for diagnostic and treatment. Over two the Bacille Calmette Guerin (BCG), based on an
billions persons worldwide are infected with the bacteria attenuated strain of Mycobacterium bovis. The protec-
(Girard et al., 2005). The epidemics have been associated tive efficacy of BCG vaccine varies upon several
nutritional and environmental factors, that is why the
Corresponding author. Tel.: +53 7 2716911; fax: +53 7 2086075. scientific community is nowadays searching for more
E-mail address: ylopez@finlay.edu.cu (Y. López). 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
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448 Y. López et al. / International Journal of Medical Microbiology 299 (2009) 447–452

(Andersen and Doherty, 2005; Ly and McMurray, 2008; the induction of protective response against pulmonary
Dietrich et al., 2003). tuberculosis after the administration of IgA monoclonal
A conventional agreement has been that host protec- antibodies by intratracheal route and challenge with
tion against intracellular pathogens depends on cell- virulent bacteria by the same route.
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 Materials and methods
functions of both arms of the immune system in
optimizing host defense (Bretscher et al., 2002; Ivanyi Bacteria and growth conditions
et al., 1985). Thus, this theory may constitute a
paradigm shift from the current dogma of immune The bacteria (H37Rv strain) were grown to early mid-
segregation wherein a CMI response is presumed to log phase in Middelbrook 7H9 medium (Difco, Detroit,
be independent of antibody function (Ivanyi and Thole, Michigan). Cultures were incubated at 37 1C with 5%
1994). CO2 and continuously shaken. The micro-organisms
Supportive data for the role of antibody in host were harvested by centrifugation at 5000g, washed 3
defense is provided by in vitro studies which demon- times in phosphate buffer saline (PBS) and stored at
strate that specific antibody inhibits the replication 70 1C until use. For vaccination and challenge, strains
of the pathogen, neutralizes toxins elaborated by the were thawed at 37 1C and diluted in PBS to the final
pathogen, promotes antibody-dependent cellular cyto- work concentration. Bacterial concentration was deter-
toxicity (ADCC), serves as an opsonin, triggers the mined by counting fluorescein acetate-stained cells in a
complement cascade, and/or prevents infection in tissue hemacytometer under fluorescent light microscopy.
culture (Glatman-Freedman, 2003; Igietseme et al.,
2004). In particular, M. tuberculosis has developed
strategies to persist in infected hosts despite the presence Animals
of potent T-cell-mediated immune responses (Reece and
Kaufmann, 2008). On the other hand, disseminated Pathogen free Balb/c mice were obtained from the
tuberculosis is not found in children with antibody titers National Institute of Medical Sciences and Nutrition,
against the lipoarabinomannan (Daffe and Etienne, Mexico City, Mexico. They were maintained under
1999). Moreover, contacts having specific IgA against barrier conditions in a level III biohazard laboratory.
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 Generation of monoclonal antibodies
designing strategies to stimulate both T-cell and anti-
body responses since antibodies are indirectly involved Monoclonal antibodies (MAb) against 38- and
in T-cell activation (Ivanyi and Thole, 1994). 16-kDa M. tuberculosis proteins were obtained as
In our group, several works have been carried out described by Falero-Diaz et al. (2000). Briefly, the
with human gammaglobulin to fight TB infection. antigens used were purified recombinant variants of the
In one work, granulomes from lungs of human 16-kDa a-crystallin (Acr antigen) and the 38-kDa
gammaglobulin-treated mice were morphologically si- lipoglycoprotein (PstS-1 antigen). Six-to-8-week-old
milar to those developed by BCG-vaccinated mice female BALB/c mice were immunized intranasally under
(Acosta et al., 1994). Moreover, in a model of intranasal mild anesthesia. Three inoculations of 10 mg of each
challenge with BCG in mice, the human gammaglobulin protein antigen mixed with 1 mg of the cholera toxin
showed protective activity (Olivares et al., 2006). Taking (Sigma) in a volume of 30 mL were separated by 2-week
into account the complex epidemiological situation of intervals. Mice with the highest antibody levels in the
tuberculosis worldwide and the evidences accumulated saliva and serum were selected for the generation of
up today regarding humoral immunity against myco- hybridoma lines. Suspensions of spleen, salivary gland,
bacterial infections, we consider of great importance lung, and nasal-associated lymphoid tissue were used for
to study the potential use of antibody formulations in fusion with myeloma cells, and antibody-positive
the modulation of immune response and activation hybridomas were selected and cloned using standard
of effective mechanisms to control the progression of the procedures (Williams et al., 2004). The monoclonal
disease. In the present work, we evaluated the efficacy antibodies obtained belonged to the IgA isotype
of monoclonal antibodies in the control of pulmonary and were denoted TBA61 (IgA anti Acr) and TBA84
infections. To our knowledge, this is the first report of (IgA anti PstS-1).
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Y. López et al. / International Journal of Medical Microbiology 299 (2009) 447–452 449

Passive immunization and protection assay during the last years evidence has been accumulated in
favor of the importance of the humoral mediators in the
Five BALB/c mice (male, aged 8–10 weeks) per group activation of principal immune mechanisms to control
were anesthetized by sodium pentobarbital subcuta- the progression of the disease caused by intracellular
neous administration (50 mg/kg), and a longitudinal pathogens, such as M. tuberculosis (Igietseme et al.,
skin incision at the ventral face of the neck was 2004). Mainly in the case of intracellular pathogens that
performed on each animal. Three groups of 5 mice are facultative, as M. tuberculosis, antibodies may be
were treated intratracheally with 100 mL of the corre- decisive in the early stages of the disease, during the
sponding monoclonal antibody at 5 mg/mL concentra- extracellular phase, or could be capable of penetrating
tion or PBS, respectively. Thirty minutes after the recently infected cells to bind the internalized pathogen
inoculation, mice were challenged with 100 mL contain- (Snewin et al., 1999).
ing 106 colony forming units (CFU) of M. tuberculosis The protection efficacy of a monoclonal antibody
H37Rv strain. Once the procedure was fulfilled, mice (TBA61, IgA anti Acr) administered intranasally
were sutured, returned to their cages, and kept in an before and after the intranasal or aerosol challenge with
isolator where plenty of food and water was supplied. M. tuberculosis was demonstrated in a previous work
Mice were sacrificed at 24, 72 h, and 21 days after (Williams et al., 2004). In the present study, we
the infection. They were anesthetized with ethylic ether evaluated the protective effect of this monoclonal
and bled by cutting the subclavian artery. Lungs antibody administered intratracheally against an intra-
were aseptically removed, homogenized with Polytron tracheal challenge with virulent mycobacteria, in order
(Brinkman Instruments, Rexleid, Canada) in 3 mL to evaluate the activity of this antibody using another
of isotonic salt solution containing 0.05% Tween 80 relevant animal model for the study of pulmonary
(Sigma, St. Louis, USA), and 1 mL of this homogenate tuberculosis. We also employed the TBA84 monoclonal
in 10-fold serial dilutions were plated on Petry dishes antibody since it is directed against other mycobacterial
containing Middlebrook 7H10 culture medium. Plates antigen. Experiments were conducted, in order to
were sealed in nylon bags and incubated for 21 days at evaluate the role of this formulation in the airway
37 1C to eventually determine the number of CFU to mucosa immediately before the encounter with the
assess pulmonary bacterial load. pathogen. Twenty-four and 72 h after the challenge, all
groups were similar in terms of mean viable counts in
Histopathology and morphometric studies lung samples. However, at 21 days post-infection,
the treatment of mice with the MAb TBA61 against
The right lung of each mouse sacrificed as indicated the 16-kDa protein of M. tuberculosis caused a
above at 24 h and 21 days were removed and fixed in statistically significant decrease in the mean number of
10% neutral buffered formalin, embedded in paraffin, viable bacteria in lungs compared to control mice or
and processed for histology. Sections were stained with those treated with the MAb against the 38-kDa protein
hematoxylin and eosin for histological evaluation and (Fig. 1A). Consistent with the reduction of viable
photography. Slides were observed by light microcopy bacteria achieved by the treatment with TBA61, at
and the areas of peribronchial and perivascular inflam- 21 days post-infection, the area of peribronchial
matory reactions were measured and analyzed by using inflammation was also statistically smaller in this group
Leica-win system software (Leica microsystem Imaging compared to the control group. However, the area of
solutions LTD, Cambridge, UK). perivascular inflammation did not show significant
differences among the groups evaluated (Fig. 1B and C).
When the lungs of mice were histologically examined,
Statistical methods granulomas were better organized in the infected
animals that had received antibodies against the
The effects of antibody treatment on the reduction of 16-kDa protein than in controls and mice treated with
bacterial load and in morphometric changes were TBA84. The reduction of CFU in lungs of this treated
analyzed by Kruskall–Wallis test and the distribution- group was associated with milder histopathological
free multiple comparison test. p-Values under 0.05 were changes, as indicated by the organization of the
considered statistically significant. All data analysis was granulomas and less pneumonic area. No inflammatory
performed by using the STATISTICA 6.1 Software. 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
Results and discussion a major membrane protein peripherally associated with
the membrane (Lee et al., 1992) carrying epitopes
Contrary to the current dogma about the ineffective restricted to tubercle bacilli on the basis of B-cell
role of antibodies against intracellular pathogens, 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

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
Fig. 1. (A) Determination of bacterial load in lungs of mice bacteria inoculated by the intratracheal route, guaran-
treated with TBA61, TBA84, or PBS and challenged with teeing a better control of dose and the final deposit of
virulent M. tuberculosis H37Rv 30 min after the inoculation of virtually all bacteria in the lungs. This model has
antibodies by intratracheal route. At 24, 72 h, and 21 days been useful for the study of pathogenic mechanism of
post-infection, lungs were harvested, and CFU was deter- M. tuberculosis infection (Arriaga et al., 2002).
mined. Only mice inoculated with TBA61 showed a significant Our group has reported the inhibition of the
reduction (po0.05, Kruskal–Wallis test) compared to the rest
colonization of mycobacteria in vivo mediated by the
of groups at 21 days post-infection. (B) Areas of perivascular
presence of specific human IgG antibodies (Olivares
inflammation. (C) Peribronchial inflammation in lungs of mice
challenged with virulent bacteria at 1, 3, and 21 days post- et al., 2006). It is now accepted that specific antibody
infection. The morphometric study was carried out with light isotypes (IgG and IgA) and T cells are required for the
microscopy by using a computer program. Statistical differ- induction of protective immunity against intracellular
ences compared to the control group (po0.05, Kruskal–Wallis pathogens. The monoclonal antibodies used in our work
test) were only obtained in the group treated with TBA61 at 21 are of IgA class, thus we have assumed that their action
days post-infection. 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) 447–452 451

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.

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