Tuberculosis vaccination needs to avoid 'decoy' immune reactions.

Current search for a new effective vaccine against tuberculosis involves selected antigens, vectors and adjuvants. These are being evaluated usually by their booster inoculation following priming with Bacillus Calmette-Guerin. The purpose of this article is to point out, that despite being attenuated of virulence, priming with BCG may still involve immune mechanisms, which are not favourable for protection against active disease. It is postulated, that the responsible 'decoy' constituents selected during the evolution of pathogenic tubercle bacilli may be involved in the evasion from bactericidal host resistance and stimulate immune responses of a cytokine phenotype, which lead to the transition from latent closed granulomas to reactivation with infectious lung cavities. The decoy mechanisms appear as favourable for most infected subjects but leading in a minority of cases to pathology which can effectively transmit the infection. It is proposed that construction and development of new vaccine candidates could benefit from avoiding decoy-type immune mechanisms.

Mucosal Therapy of Multi-Drug Resistant Tuberculosis With IgA and Interferon-γ.

New evidence has been emerging that antibodies can be protective in various experimental models of tuberculosis. Here, we report on protection against multidrug-resistant Mycobacterium tuberculosis (MDR-TB) infection using a combination of the human monoclonal IgA 2E9 antibody against the alpha-crystallin (Acr, HspX) antigen and mouse interferon-gamma in mice transgenic for the human IgA receptor, CD89. The effect of the combined mucosal IgA and IFN-γ; treatment was strongest (50-fold reduction) when therapy was applied at the time of infection, but a statistically significant reduction of lung bacterial load was observed even when the therapy was initiated once the infection had already been established. The protection involving enhanced phagocytosis and then neutrophil mediated killing of infected cells was IgA isotype mediated, because treatment with an IgG version of 2E9 antibody was not effective in human IgG receptor CD64 transgenic mice. The Acr antigen specificity of IgA antibodies for protection in humans has been indicated by their elevated serum levels in latent tuberculosis unlike the lack of IgA antibodies against the virulence-associated MPT64 antigen. Our results represent the first evidence for potential translation of mucosal immunotherapy for the management of MDR-TB.

Selection of a Single Domain Antibody, Specific for an HLA-Bound Epitope of the Mycobacterial Ag85B Antigen.

T cells recognizing epitopes on the surface of mycobacteria-infected macrophages can impart protection, but with associated risk for reactivation to lung pathology. We aimed to identify antibodies specific to such epitopes, which carry potentials for development toward novel therapeutic constructs. Since epitopes presented in the context of major histocompatibility complex alleles are rarely recognized by naturally produced antibodies, we used a phage display library for the identification of monoclonal human single domain antibody producing clones. The selected 2C clone displayed T cell receptor-like recognition of an HLA-A*0201 bound 199KLVANNTRL207 peptide from the Ag85B antigen, which is known to be an immunodominant epitope for human T cells. The specificity of the selected domain antibody was demonstrated by solid phase immunoassay and by immunofluorescent surface staining of peptide loaded cells of the T2 cell line. The antibody affinity binding was determined by biolayer interferometry. Our results validated the used technologies as suitable for the generation of antibodies against epitopes on the surface of Mycobacterium tuberculosis infected cells. The potential approaches forward the development of antibody in immunotherapy of tuberculosis have been outlined in the discussion.

Function and Potentials of M. tuberculosis Epitopes.

Study of the function of epitopes of Mycobacterium tuberculosis antigens contributed significantly toward better understanding of the immunopathogenesis and to efforts for improving infection and disease control. Characterization of genetically permissively presented immunodominant epitopes has implications for the evolution of the host-parasite relationship, development of immunodiagnostic tests, and subunit prophylactic vaccines. Knowledge of the determinants of cross-sensitization, relevant to other pathogenic or environmental mycobacteria and to host constituents has advanced. Epitope-defined IFNγ assay kits became established for the specific detection of infection with tubercle bacilli both in humans and cattle. The CD4 T-cell epitope repertoire was found to be more narrow in patients with active disease than in latently infected subjects. However, differential diagnosis of active TB could not be made reliably merely on the basis of epitope recognition. The mechanisms by which HLA polymorphism can influence the development of multibacillary tuberculosis (TB) need further analysis of epitopes, recognized by Th2 helper cells for B-cell responses. Future vaccine development would benefit from better definition of protective epitopes and from improved construction and formulation of subunits with enhanced immunogenicity. Epitope-defined serology, due to its operational advantages is suitable for active case finding in selected high disease incidence populations, aiming for an early detection of infectious cases and hence for reducing the transmission of infection. The existing knowledge of HLA class I binding epitopes could be the basis for the construction of T-cell receptor-like ligands for immunotherapeutic application. Continued analysis of the functions of mycobacterial epitopes, recognized by T cells and antibodies, remains a fertile avenue in TB research.

Serodiagnosis of tuberculosis: due to shift track.

Development of novel diagnostics for tuberculosis has so far been governed by the clinical requirement of improving the detection of patients with paucibacillary forms of the disease. For this aim, serological assays have been evaluated using several antigens, but were found insufficiently sensitive, because antibody production associates with the bacterial load of the disease. Consequently, detection of antibodies against a relatively small number of selected well-defined antigens has a much higher sensitivity for sputum smear-positive pulmonary disease in adult HIV-negative patients. They are the most active in generating and spreading aerosols containing live tubercle bacilli, but their detection is often delayed, thus perpetuating the transmission of the infection and disease in the population. High volume throughput serological screening of clinical suspects with mild clinical symptoms may help to achieve diagnosis earlier, than currently used procedures. Such expanded testing could be done more efficiently in laboratories, than at 'points-of-care' and at a lower cost than other tests. The feasibility of this approach towards reducing the delayed diagnosis of the most infectious cases of pulmonary tuberculosis needs to be ascertained in prospective diagnostic trials, in populations at a high risk. Reducing the transmission of tuberculosis is of key importance for achieving its continued decline and therefore it is proposed, that the aims of serological screening should shift from clinical to public health priorities.

The secret trumps, impelling the pathogenicity of tubercle bacilli.

Confrontation between invading microbial pathogens and host defense systems involves intricate cellular and molecular interactions. Here we discuss the virulence factors as trumps, overriding the contest in favor of the tubercle bacillus (Mycobacterium tuberculosis). It evolved a number of molecular constituents, which can interfere with antigen presentation and Toll receptor function, thus impairing immune defenses. It also evolved stress responses, which can drive its cell cycle into a non-replicating, low metabolic mode. Although the low counts of latent bacilli prevent their direct detection, we contend that they retain a capacity to survive for long periods in foamy macrophages and within the necrotic parts of lung granulomas. We attributed significance to drainage of M. tuberculosis by the alveolar fluid: while out-flow is responsible for the clearance, the reverse-flow has an important capacity to re-infect the lungs and to transmit the infection to new recipients. We consider the cycling between replicating and latent organisms to be a continuous process, which is a departure from the concept of long-lived dormant organisms, with a capacity to resuscitate. These aspects impinge also on the actions of isoniazid (INH) chemotherapy and on the topography of human lung lesions. Eventually, fibrosis of the connective tissue of the lungs is known to encapsulate lung lesions, thus limiting the impact of both outward and reverse drainage. In conclusion, the novelty of our views on M. tuberculosis-host interactions rests in the dynamic perception of M. tuberculosis latency and its evolutionary importance for the pathogenesis of tuberculosis.

The secret trumps, impelling the pathogenicity of tubercle bacilli

El enfrentamiento entre los patógenos invasivos y los sistemas defensivos del huésped implica interaccionescelulares y moleculares. En el presente artículo se discuten los factores de virulencia como triunfos, favoreciendoel éxito de la contienda a favor del bacilo tuberculoso (Mycobacterium tuberculosis). Éste desarrollaun número de constituyentes moleculares que pueden interferir con la presentación antigénica y lafunción Toll receptor, deteriorando las defensas inmunes del huésped, así como respuestas al estrés queenlentecen su ciclo celular hasta convertirlo en no replicante. Aunque el recuento bajo de bacilos latentespreviene su detección directa, postulamos que retienen cierta capacidad de sobrevivir dentro de macrófagosespumosos y en las partes necróticas de los granulomas pulmonares. Mientras que el circuito naturaldel fluido alveolar hacia las vías respiratorias superiores es el responsable de la eliminación de bacilos, suretorno para generar aerosoles de forma fisiológica también implica la posibilidad de que con él ciertosbacilos puedan reinfectar de forma endógena los pulmones y transmitir la infección a nuevos individuos.Consideramos, pues, la tuberculosis latente como un proceso continuo, en contraposición al concepto de laexistencia de bacilos largamente durmientes y con capacidad de resucitar. Creemos, además, que la fibrosisdel tejido conectivo de los pulmones, capaz en ocasiones de encapsular lesiones pulmonares, es la responsablede frenar el drenaje y la diseminación de bacilos, limitando el ciclo reinfectivo. En conclusión, la novedadde nuestra visión radica en la percepción dinámica de la latencia de M. tuberculosis y sus consecuenciassobre la patogénesis de la tuberculosis (AU)

Confrontation between invading microbial pathogens and host defense systems involves intricate cellular and molecular interactions. Here we discuss the virulence factors as trumps, overriding the contest in favor of the tubercle bacillus (Mycobacterium tuberculosis). It evolved a number of molecular constituents, which can interfere with antigen presentation and Toll receptor function, thus impairing immune defenses. It also evolved stress responses, which can drive its cell cycle into a non-replicating, low metabolic mode. Although the low counts of latent bacilli prevent their direct detection, we contend that they retain a capacity to survive for long periods in foamy macrophages and within the necrotic parts of lung granulomas. We attributed significance to drainage of M. tuberculosis by the alveolar fluid: while out-flow is responsible for the clearance, the reverse-flow has an important capacity to re-infect the lungs and to transmit the infection to new recipients. We consider the cycling between replicating and latent organisms to be a continuous process, which is a departure from the concept of long-lived dormant organisms, with a capacity to resuscitate. These aspects impinge also on the actions of isoniazid (INH) chemotherapy and on the topography of human lung lesions. Eventually, fibrosis of the connective tissue of the lungs is known to encapsulate lung lesions, thus limiting the impact of both outward and reverse drainage. In conclusion, the novelty of our views on M. tuberculosis-host interactions rests in the dynamic perception of M. tuberculosis latency and its evolutionary importance for the pathogenesis of tuberculosis (AU)

A novel human IgA monoclonal antibody protects against tuberculosis.

Abs have been shown to be protective in passive immunotherapy of tuberculous infection using mouse experimental models. In this study, we report on the properties of a novel human IgA1, constructed using a single-chain variable fragment clone (2E9), selected from an Ab phage library. The purified Ab monomer revealed high binding affinities for the mycobacterial α-crystallin Ag and for the human FcαRI (CD89) IgA receptor. Intranasal inoculations with 2E9IgA1 and recombinant mouse IFN-γ significantly inhibited pulmonary H37Rv infection in mice transgenic for human CD89 but not in CD89-negative littermate controls, suggesting that binding to CD89 was necessary for the IgA-imparted passive protection. 2E9IgA1 added to human whole-blood or monocyte cultures inhibited luciferase-tagged H37Rv infection although not for all tested blood donors. Inhibition by 2E9IgA1 was synergistic with human rIFN-γ in cultures of purified human monocytes but not in whole-blood cultures. The demonstration of the mandatory role of FcαRI (CD89) for human IgA-mediated protection is important for understanding of the mechanisms involved and also for translation of this approach toward development of passive immunotherapy of tuberculosis.

Prevention of the post-chemotherapy relapse of tuberculous infection by combined immunotherapy.

We report that a recently developed combined immunotherapy (CIT) has the capacity to prevent a spontaneous relapse of replicating Mycobacterium tuberculosis bacilli in the lungs of BALB/c, C57Bl/6 or C3H/HeJ strains of mice, following 4weeks of non-sterilising treatment with isoniazid and rifampicin. The CIT regimen, represented by recombinant IFNgamma, anti-alpha crystalline monoclonal IgA antibody and IL-4 neutralizing polyclonal antibody, reduced the 8-week relapse of viable bacterial counts in the lungs most significantly, when CIT was inoculated during the 5th week post infection, i.e. during the 3rd week of chemotherapy. Although CIT enhanced lung granuloma area, nitric oxide, cytokine and chemokine levels in lung washings significantly, these could not be directly associated with the beneficial effect of CIT on the degree of relapse in the lungs. These results represent a proof-of-principle, that the described CIT, when combined with chemotherapy, could have potential for future development of a shorter regimen of tuberculosis treatment.

IL-4 depletion enhances host resistance and passive IgA protection against tuberculosis infection in BALB/c mice.

The influence of Th2 cytokines in tuberculosis has been a matter of dispute. Here we report that IL-4 has a profound regulatory effect on the infection of BALB/c mice with Mycobacterium tuberculosis. Depletion of IL-4 with a neutralizing mAb caused only evanescent reduction of lung infection, but when combined with i.n. inoculations of IgA anti-mycobacterial alpha-crystallin mAb and mouse rIFN-gamma, we observed a 40-fold reduction of the bacterial counts in the lungs at 3 wks following i.n. infection (p<0.001). In genetically deficient IL-4-/- BALB/c mice, infection in both lung and spleen was substantially reduced for up to 8 wks without further treatment. Reconstitution of IL-4-/- mice with rIL-4 increased bacterial counts to wild-type levels and made the mice refractory to protection by IgA/IFN-gamma. Analysis of the lungs showed increased granulomatous infiltration and proinflammatory mediators in anti-IL-4/IgA/IFN-gamma-treated and infected mice. We conclude that the action of IL-4 in tuberculosis is targeted at macrophages and that it may include an antagonistic effect on their IgA/IFN-gamma-induced activation and nitric oxide production. The described novel immunotherapy, combining treatments with anti-IL-4, IgA antibody and IFN-gamma, has potential for translation toward the passive immunoprophylaxis of tuberculosis.

A case for passive immunoprophylaxis against tuberculosis.

HIV-associated tuberculosis is escalating ominously in Africa and southeast Asia despite existing control measures. Therefore, new approaches to tuberculosis control need to be explored. We discuss the potential use of passive immunoprophylaxis with antibodies in tuberculosis control. Although the predominant type of active host resistance is T-cell mediated, recent results in mouse experimental models suggest that monoclonal antibodies to certain antigens (eg, Acr or lipoarabinomannan) can impart substantial passive protection against tuberculous infection. These results are corroborated by data from other laboratories on passive vaccination against a number of intracellular microbial pathogens. Further work is needed to develop human (or humanised) antibody reagents, to increase their protective efficacy, and to expand our understanding of the mechanisms of antibody action.

gammadelta T cells condition dendritic cells in vivo for priming pulmonary CD8 T cell responses against Mycobacterium tuberculosis.

gammadelta T cells and dendritic cells are quickly recruited to the lungs shortly after intranasal vaccination with BCG, but the functional in vivo interplay between these two cell populations and its role in the induction of adaptive immune responses is unclear. Using TCR-deficient mice and bone marrow chimeras, we show here that gammadelta T cells provide a non-redundant early source of IFN-gamma in vivo, which enhances IL-12 production by lung dendritic cells. The in vivo-conditioned dendritic cells, in turn, prime a more efficient lung CD8 T cell response against Mycobacterium tuberculosis. Thus, strategies exploiting gammadelta T cell function and IFN-gamma production could be valuable for the design and testing of mucosal vaccines.

Polymeric IgR knockout mice are more susceptible to mycobacterial infections in the respiratory tract than wild-type mice.

It is generally accepted that cellular, and not humoral immunity, plays the crucial role in defense against intracellular bacteria. However, accumulating data indicate the importance of humoral immunity for the defense against a number of intracellular bacteria, including mycobacteria. We have investigated the role of secretory IgA, the main isotype found in mucosal tissues, in protection against mycobacterial infection, using polymeric IgR (pIgR)-deficient mice. Characterization of the humoral response induced after intra-nasal immunizations with the mycobacterial antigen PstS-1 revealed a loss of antigen-specific IgA response in saliva from the knockout mice. IgA level in the bronchoalveolar lavage of knockout mice was similar to wild-type level, although the IgA antibodies must have reached the lumen by other means than pIgR-mediated transport. Infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG) demonstrated that the immunized pIgR-/- mice were more susceptible to BCG infection than immunized wild-type mice, based on higher bacterial loads in the lungs. This was accompanied by a reduced production of both IFN-gamma and tumor necrosis factor-alpha (TNF-alpha) in the lungs. Additionally, the pIgR-/- mice displayed reduced natural resistance to mycobacterial infection proved by significantly higher bacterial growth in their lungs compared with wild-type mice after infection with virulent Mycobacterium tuberculosis. The knockout mice appeared to have a delayed mycobacteria-induced immune response with reduced expression of protective mediators, such as IFN-gamma, TNF-alpha, inducible nitric oxide synthase and regulated upon activation normal T cell sequence, during early infection. Collectively, our results show that actively secreted IgA plays a role in protection against mycobacterial infections in the respiratory tract, by blocking entrance of bacilli into the lungs, in addition to modulation of the mycobacteria-induced pro-inflammatory response.

Mucosal immunotherapy of tuberculosis: is there a value in passive IgA?

Immunotherapeutic approaches, which have been considered for tuberculosis (TB), include immuno-potentiating or suppressing agents, cytokines, antibodies, DNA vaccines, non-pathogenic mycobacteria and mycobacterial extracts. While most or all of these potential agents showed at least some degree of promise in various experimental models, few progressed to clinical trials, yielding only moderately encouraging, though controversial results. Consequently, further research is required, as the need for an immunological agent, adjunct to chemotherapy, remains strongly justified. Its purpose is to shorten the currently protracted (6-9 months) drug treatment and thus increase compliance rates, which are most disappointing in areas with the highest disease prevalence. Using a mouse model of Mycobacterium tuberculosis (Mtb) infection, we recently reported, that an intranasally given monoclonal IgA antibody significantly reduced the bacterial load in the infected lungs, and that this protective effect of IgA could be further extended by co-inoculation with interferon gamma (IFNgamma). In this review, we describe the main features of IgA and its cellular receptors, the extent and possible mechanisms of passive vaccination with an IgA monoclonal antibody against the alpha-crystallin antigen of Mtb and discuss the potentials of this approach in the wider context of immunotherapy of TB.