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Procedia in Vaccinology 2 (2010) 172177

Ninth Global Vaccine Research Forum and Parallel Satellite Symposia

Bamako, Mali, 6-9 December 2009

Antibodies in the protection against mycobacterial infections:

what have we learned?
Yamile Lpez *, Gustavo Falero-Daz, Daniel Yero, Rosa L. Sols,
Maria E Sarmiento, Armando Acosta
Finlay Institute, Ave. 17 esquina 198, Reparto Siboney Playa, Havana, Cuba

Abstract
Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Despite of the massive global use of
BCG, there is a need for other TB vaccines. Newer animal models are needed to test candidate vaccine efficacy to
protect animals against challenge with M. tuberculosis virulent strains in more realistic scenarios than currently
done. Also, the elucidation of the importance of humoral immune defenses against intracellular pathogens
constitutes a priority to improve the rational design of new vaccines. Our group has been actively testing the
protective role of antibodies in different models of pulmonary TB infection through evaluation of bacterial loads and
morphometric and histological changes in the lungs of infected mice. Results presented here suggest a protective
role for antibodies and the humoral response against tuberculosis infection.
2010 Published by Elsevier Ltd.

Keywords: tuberculosis, mice model, antibodies, 16 kDa protein

1.

Introduction

Through all of history, tuberculosis (TB) has been a health problem for humanity. At the beginnings of civilization
the disease was probably occasional, but with the increment of population densities, in the XVII-XIXth centuries, it
took epidemic proportions [5,7,16].
In spite of the use of the live attenuated vaccine developed by Calmette and Guerin (BCG) and in spite of effective
therapy with antibiotics like isoniazid, rifampicin and streptomycin, the number of TB cases has not ceased to
increase until our days. The World Health Organization estimates that approximately a third of the world population

Corresponding author. E-mail address: ylopez@finlay.edu.cu

1877-282X 2010 Published by Elsevier Ltd.

doi:10.1016/j.provac.2010.07.011

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Y. Lpez et al. / Procedia in Vaccinology 2 (2010) 172177

is infected and that about eight million people acquire TB yearly. Without an adequate treatment, over 60% of the
people with TB would be condemned to death [36].
In order to implement strategies to increase the effectiveness of BCG vaccine, or to replace it by a more effective
vaccine, an important point is to try to elucidate the effector immune mechanisms at work in the fight against
intracellular pathogens. For many years, cell-mediated immunity was attributed an exclusive role in the defense
against intracellular pathogens. The Th1/Th2 paradigm prevailed for a long time and directed the development of
most vaccines. In recent years, many experiments have however challenged this concept. Nowadays, it is well
accepted that a combination of both arms of the immune system is optimal for fighting intracellular as well as
extracellular pathogens [15].

2.

A role for IgG antibodies

With the scientific development of hybridomas technology and the production of monoclonal antibodies, evidence
that support a clear role of antibodies in the fight against intracellular pathogens such as fungi, viruses, parasites and
bacteria has been accumulating [1]. The availability of new technologies for the study of antibody-mediated
immunity and the need for new therapies for the control of re-emergent diseases had pushed forward the discovery
of new functions of antibodies, such as their role as direct microbicide molecules [2,29,39] or as interactive and
unique effector molecules [38].
In the specific case of M tuberculosis, initial work was developed using anti-arabinomanan (AM), antilipoarabinomanan (LAM), anti-heparin-binding haemaglutinin adhesin (HBHA) or anti- Mtb83 monoclonal
antibodies [4,12,13,17,18,30]. These antibodies, which are usually able to opsonize mycobacteria, were
administered to infected mice by different routes, yielding results such as prolonged host survival, increased serum
clearance, diminished mycobacteria dissemination and reduction of colony forming units in the lungs and spleen.
The interest of the scientific community in the elucidation of the real role of antibodies mediating protective
immunity against TB began to increase, as illustrated in Figure 1.

articles found in literature

120
100
80
60
40
20
0
1970-1980

1981-2000

2001-2009

time period

Figure 1: Antibodies and M tuberculosis. Data for this graphic were obtained by searches in Medline and
PubMed and references from relevant articles.

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Y. Lpez et al. / Procedia in Vaccinology 2 (2010) 172177

As reported by several authors, antibodies may be critical mainly during the extracellular phase of early disease
stages caused by facultative intracellular pathogens. However, antibodies may also be able to penetrate recently
infected cells, binding to the internalised pathogen and increasing antigenic processing. It is well accepted nowadays
that antibodies can modulate the immune response, activate the secretion of cytokines that contribute to an efficient
and rapid Th1 response [20,25], increase the efficacy of co-stimulatory signals, elicit antibody - dependent cellular
cytotoxicity and the homing to the lungs after respiratory infection [3,19,24,34].
In 2005, de Valiere et al. reported for the first time that antimycobacterial antibodies stimulate the Th1 response
instead of diminishing it, as believed previously [8]. In this study, serum samples obtained from volunteers
vaccinated twice with BCG by the intradermal route, were shown to contain significant titers of specific
antimycobacterial IgG antibodies against LAM. Moreover, these antibodies significantly increased BCG
internalization into phagocytic cells as well as the inhibitory effect of neutrophils and macrophages on
mycobacterial growth. Besides, they also induced a significant production of IFN- by CD4+ and CD8+ T cells.

3.

IgA and Mucosal Immunity

Little is known of the possible role of IgAs in the defense against TB infection. Most studies on the protective effect
of antibodies against tuberculosis are based on antibodies of IgG subclasses. During the last years several articles
describing the importance of mucosal associated lymphoid tissue in the resistance to infections have however been
published [10,14]. The mucosal immune response greatly contributes to the protection against pathogens entering
the host at mucosal sites. Secretions found on mucosal surfaces contain significant levels of immunoglobins,
basically IgAs, whose function is to prevent infectious agents such as viruses or bacteria to cross the mucosal
barrier. Experimental evidence suggests that the IgA polymeric receptor may also neutralize pathogens and antigens
during their intracellular transport from the apical to the basolateral zone of epithelial cells [9,28]. Mechanisms that
explain the protective role of IgAs involve microbes agglutination, motility inhibition, binding to bacterial adhesins
that prevents binding of the bacteria to the mucosal epithelium, and microbial products elimination through
activation of phagocytic cells. Immune exclusion and neutralization of viral infectivity are crucial mechanisms of
IgA activity against viral respiratory pathogens [33]. Some IgA antibody isotypes have been associated with
protection against M leprae [6]. BCG vaccination also induces a substantial IgA response and IgA-deficient mice
were found to less well control BCG infection [35].
These antibodies, if present at the site and the moment of infection, could modify the course and the outcome of the
disease. Passive administration of IgA antibodies to high risk group individuals (such as immunocompromised
individuals) could be a realistic immunotherapeutic strategy.
As M tuberculosis specific antibodies must presumably act at the mucosal surfaces of the respiratory tract, a decisive
point is the choice of the administration route selected to evaluate candidate antibodies. At the beginning,
intravenous and subcutaneous routes were the more employed, but these routes do not resemble the natural way of
infection. In this sense, the aerosol and intratracheal routes of administration are the most physiological routes to
study the pathogenicity of the bacteria and the immune response induced in the host [27]. Our group reported for
the first time the prophylactic effect of mucosal administration of human gammaglobulins in a model of progressive
pulmonary infection with M. tuberculosis in mice. In addition, we demonstrated that incubation of M. tuberculosis
with human antibodies could inhibit the bacterias infective potential [26]. The protective effect of monoclonal
antibodies was also demonstrated in the same model of infection [23].
4.

IgA monoclonal antibody against M. tuberculosis 16 kDa protein

M. tuberculosis 16 kDa protein (also called Acr antigen) has been identified as a major membrane-associated protein
[22]. Its expression is increased in bacteria growing inside infected macrophages. Our group has been working for
several years testing the possible role of IgA monoclonal antibodies directed against the 16 kDa protein in the
defense against TB infection [11]. Several experiments have been carried out (Table 1), using different ways of

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Y. Lpez et al. / Procedia in Vaccinology 2 (2010) 172177

Table 1: Results from different experimental approaches involving a monoclonal antibody against M.
tuberculosis 16 kDa protein (TBA61) as appeared in [23,31,3]).
MAb, delivery route and
inoculation regime
TBA61 i.n (-3h, +3d, +6d)

Challenge

Days selected
for lung

H37Rv
aerosol

TBA61 i.n (-3h)

TBA61 i.n (+3h)

TBA61 i.n (-3h, +3d)

H37Rv
aerosol

9 days

Parameter measured
CFU reduction
Histopathology
Statistical
reduction of
CFU postchallenge
Non statistical
reduction of
nd
CFU postchallenge
Statistical
reduction of
CFU postchallenge

TBA61 + IFN (i.n)

(-3h, -2h, +2d, +7d)

H37Rv
aerosol

9, 21 and 28
days

Statistical
reduction of
CFU postchallenge

TBA61 i.t (-3h)

H37Rv
i.t

24h, 72h, 21
days

Statistical
reduction at 21
days postchallenge

Statistical
reduction of the
granulomatous
area in the lungs of
treated as,
compared to
untreated mice
Less intersticial
and peribronchial
inflammation.
Well-organized
granuloma

Note: i.n: intra-nasal; i.t: intra-tracheal; h: hours; d:days; nd: non determined.
inoculation and challenge, in order to demonstrate the feasibility of using this IgA as a therapeutic strategy to fight
the disease [23,31,37].
Possibly, blocking the Acr antigen with monoclonal antibody TBA61 may interfere with the intracellular growth of
the bacteria and limit their cell-to-cell dissemination [32]. This could in part explain the protective character of the
antibody. On the other hand, as demonstrated previously, IgAs may interact with Gal-3 (an intracellular binding galactosidase lectin), interfering with the interaction of mycobacteria with phagosomal membranes, finally resulting
in the diminution of bacterial survival and replication in the phagosoma. In addition, the efficacy of the monoclonal
antibody may be reinforced by IFN-, a potent pleiotropic stimulator of macrophage functions which is essential for
resistance against TB.
Nowadays, vaccines that elicit antibody-based responses are much in favor [21]. These vaccines must induce IgA
antibodies that inactivate bacterial components essential for survival in the host, activate complement for the direct
lysis of bacteria, and/or opsonize bacteria to promote their capture by phagocytes, neutrophils, monocytes or
macrophages.

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Y. Lpez et al. / Procedia in Vaccinology 2 (2010) 172177

Conclusion

Many questions still remain unanswered at the moment. It is important to try to understand the role of follicle-like B
cells in the lungs of tuberculous patients and why, how and when do these B cells act to modulate the inflammatory
and cytokine responses of the patients. Their likely relationship with the antibodies present in the mucosal secretions
is also very important to elucidate. Better knowledge of these key phenomena could contribute to the rational and
effective development of a new generation of effective TB vaccines.

References
1. Casadevall, A. 2003. Antibody-mediated immunity against intracellular pathogens: two-dimensional thinking comes full circle. Infect.
Immun. 71:4225-4228.
2. Casadevall, A. 2006. The third age of antimicrobial therapy. Clin. Infect. Dis. 42:1414-1416.
3. Casadevall, A. and L. A. Pirofski. 2003. Antibody-mediated regulation of cellular immunity and the inflammatory response. Trends
Immunol. 24:474-478.
4. Chambers, M. A., D. Gavier-Widen, and R. G. Hewinson. 2004. Antibody bound to the surface antigen MPB83 of Mycobacterium
bovis enhances survival against high dose and low dose challenge. FEMS Immunol. Med. Microbiol. 41:93-100.
5. Chung, K. T. and C. J. Biggers. 2001. Albert Leon Charles Calmette (1863-1933) and the antituberculous BCG vaccination. Perspect.
Biol. Med. 44:379-389.
6. Cree, I. A., W. C. Smith, and J. S. Beck. 1990. A quantitative study of the relationship between systemic and histological parameters of
immunity in individual leprosy patients. Int. J. Lepr. Other Mycobact. Dis. 58:347-352.
7. Davies, P. D. 2008. Medical classics. La Boheme and tuberculosis. BMJ 337:a2587.
8. de, Valiere S., G. Abate, A. Blazevic, R. M. Heuertz, and D. F. Hoft. 2005. Enhancement of innate and cell-mediated immunity by
antimycobacterial antibodies. Infect. Immun. 73:6711-6720.
9. Delbridge, L. M. and M. X. O'Riordan. 2007. Innate recognition of intracellular bacteria. Curr. Opin. Immunol. 19:10-16.
10. Doherty, T. M., A. W. Olsen, L. van Pinxteren, and P. Andersen. 2002. Oral vaccination with subunit vaccines protects animals
against aerosol infection with Mycobacterium tuberculosis. Infect. Immun. 70:3111-3121.
11. Falero-Diaz, G., S. Challacombe, D. Rahman, M. Mistry, G. Douce, G. Dougan, A. Acosta, and J. Ivanyi. 2000. Transmission of
IgA and IgG monoclonal antibodies to mucosal fluids following intranasal or parenteral delivery. Int. Arch. Allergy Immunol. 122:143-150.
12. Glatman-Freedman, A., A. Casadevall, Z. Dai, W. R. Jacobs, Jr., A. Li, S. L. Morris, J. A. Navoa, S. Piperdi, J. B. Robbins, R.
Schneerson, J. R. Schwebach, and M. Shapiro. 2004. Antigenic evidence of prevalence and diversity of Mycobacterium tuberculosis
arabinomannan. J. Clin. Microbiol. 42:3225-3231.
13. Glatman-Freedman, A., J. M. Martin, P. F. Riska, B. R. Bloom, and A. Casadevall. 1996. Monoclonal antibodies to surface
antigens of Mycobacterium tuberculosis and their use in a modified enzyme-linked immunosorbent spot assay for detection of
mycobacteria. J. Clin. Microbiol. 34:2795-2802.
14. Goonetilleke, N. P., H. McShane, C. M. Hannan, R. J. Anderson, R. H. Brookes, and A. V. Hill. 2003. Enhanced immunogenicity
and protective efficacy against Mycobacterium tuberculosis of bacille Calmette-Guerin vaccine using mucosal administration and boosting
with a recombinant modified vaccinia virus Ankara. J. Immunol. 171:1602-1609.
15. Gor, D. O., N. R. Rose, and N. S. Greenspan. 2003. TH1-TH2: a procrustean paradigm. Nat. Immunol. 4:503-505.
16. Gradmann, C. 2009. (Robert Koch and tuberculosis: the beginning of medical bacteriology. Pneumologie 63:702-708.
17. Hamasur, B., M. Haile, A. Pawlowski, U. Schroder, G. Kallenius, and S. B. Svenson. 2004. A mycobacterial lipoarabinomannan
specific monoclonal antibody and its F(ab') fragment prolong survival of mice infected with Mycobacterium tuberculosis. Clin. Exp.
Immunol. 138:30-38.
18. Hamasur, B., M. Haile, A. Pawlowski, U. Schroder, A. Williams, G. Hatch, G. Hall, P. Marsh, G. Kallenius, and S. B. Svenson.
2003. Mycobacterium tuberculosis arabinomannan-protein conjugates protect against tuberculosis. Vaccine 21:4081-4093.
19. Iankov, I. D., D. P. Petrov, I. V. Mladenov, I. H. Haralambieva, O. K. Kalev, M. S. Balabanova, and I. G. Mitov. 2004. Protective
efficacy of IgA monoclonal antibodies to O and H antigens in a mouse model of intranasal challenge with Salmonella enterica serotype
Enteritidis. Microbes. Infect. 6:901-910.
20. Igietseme, J. U., F. O. Eko, Q. He, and C. M. Black. 2004. Antibody regulation of Tcell immunity: implications for vaccine strategies
against intracellular pathogens. Expert. Rev. Vaccines. 3:23-34.
21. Kaufmann, S. H., A. L. Meinke, and G. A. von. 2009. (Novel vaccination concepts on the basis of modern insights into immunology..
Bundesgesundheitsblatt. Gesundheitsforschung. Gesundheitsschutz.
22. Lee, B. Y., S. A. Hefta, and P. J. Brennan. 1992. Characterization of the major membrane protein of virulent Mycobacterium
tuberculosis. Infect. Immun. 60:2066-2074.
23. Lopez, Y., D. Yero, G. Falero-Diaz, N. Olivares, M. E. Sarmiento, S. Sifontes, R. L. Solis, J. A. Barrios, D. Aguilar, R.
Hernandez-Pando, and A. Acosta. 2009. Induction of a protective response with an IgA monoclonal antibody against Mycobacterium
tuberculosis 16kDa protein in a model of progressive pulmonary infection. Int. J. Med. Microbiol. 299:447-452.
24. Monteiro, R. C., V. Leroy, P. Launay, I. C. Moura, M. rcos-Fajardo, M. Benhamou, and E. Haddad. 2003. (Pathogenesis of
Berger's disease: recent advances on the involvement of immunoglobulin A and their receptors. Med. Sci. (Paris) 19:1233-1241.
25. Moore, A. C. and C. L. Hutchings. 2007. Combination vaccines: synergistic simultaneous induction of antibody and T-cell immunity.
Expert. Rev. Vaccines. 6:111-121.

Y. Lpez et al. / Procedia in Vaccinology 2 (2010) 172177

177

26. Olivares, N., A. Puig, D. Aguilar, A. Moya, A. Cadiz, O. Otero, L. Izquierdo, G. Falero, R. L. Solis, H. Orozco, M. E. Sarmiento,
M. N. Norazmi, R. Hernandez-Pando, and A. Acosta. 2009. Prophylactic effect of administration of human gamma globulins in a mouse
model of tuberculosis. Tuberculosis. (Edinb. ) 89:218-220.
27. Orme, I. M. 2005. Mouse and guinea pig models for testing new tuberculosis vaccines. Tuberculosis. (Edinb. ) 85:13-17.
28. Phalipon, A. and B. Corthesy. 2003. Novel functions of the polymeric Ig receptor: well beyond transport of immunoglobulins. Trends
Immunol. 24:55-58.
29. Pirofski, L. A. and A. Casadevall. 2006. Immunomodulators as an antimicrobial tool. Curr. Opin. Microbiol. 9:489-495.
30. Reljic, R. 2007. IFN-gamma therapy of tuberculosis and related infections. J. Interferon Cytokine Res. 27:353-364.
31. Reljic, R., S. O. Clark, A. Williams, G. Falero-Diaz, M. Singh, S. Challacombe, P. D. Marsh, and J. Ivanyi. 2006. Intranasal
IFNgamma extends passive IgA antibody protection of mice against Mycobacterium tuberculosis lung infection. Clin. Exp. Immunol.
143:467-473.
32. Reljic, R., C. Di Sano, C. Crawford, F. Dieli, S. Challacombe, and J. Ivanyi. 2005. Time course of mycobacterial infection of
dendritic cells in the lungs of intranasally infected mice. Tuberculosis. (Edinb. ) 85:81-88.
33. Reljic, R. and J. Ivanyi. 2006. A case for passive immunoprophylaxis against tuberculosis. Lancet Infect. Dis. 6:813-818.
34. Robinson, S., W. A. Charini, M. H. Newberg, M. J. Kuroda, C. I. Lord, and N. L. Letvin. 2001. A commonly recognized simian
immunodeficiency virus Nef epitope presented to cytotoxic T lymphocytes of Indian-origin rhesus monkeys by the prevalent major
histocompatibility complex class I allele Mamu-A*02. J. Virol. 75:10179-10186.
35. Rodriguez, A., A. Tjarnlund, J. Ivanji, M. Singh, I. Garcia, A. Williams, P. D. Marsh, M. Troye-Blomberg, and C. Fernandez.
2005. Role of IgA in the defense against respiratory infections IgA deficient mice exhibited increased susceptibility to intranasal infection
with Mycobacterium bovis BCG. Vaccine 23:2565-2572.
36. Vashishtha, V. M. 2009. WHO Global Tuberculosis Control Report 2009: Tuberculosis elimination is a distant dream. Indian Pediatr.
46:401-402.
37. Williams, A., R. Reljic, I. Naylor, S. O. Clark, G. Falero-Diaz, M. Singh, S. Challacombe, P. D. Marsh, and J. Ivanyi. 2004.
Passive protection with immunoglobulin A antibodies against tuberculous early infection of the lungs. Immunology 111:328-333.
38. Winslow, E. H. and A. F. Jacobson. 2000. Can a fashion statement harm the patient? Long and artificial nails may cause nosocomial
infections. Am. J. Nurs. 100:63-65.
39. Woof, J. M. and M. A. Kerr. 2006. The function of immunoglobulin A in immunity. J. Pathol. 208:270-282.