SUMMARYLipoarabinomannan (LAM) is a major structural carbohydrate antigen of the outer surface of Mycobacterium tuberculosis . High antibody titres against LAM are often seen in active tuberculosis (TB). The role of such LAM-specific antibodies in the immune response against TB is unknown. Here we have investigated a monoclonal antibody (MoAb) SMITB14 of IgG1 subclass and its corresponding F(ab ¢ ) 2 fragment directed against LAM from M. tuberculosis strain H37Rv. MoAb SMITB14 was shown by immunofluorescence to bind to whole cells of the clinical isolate M. tuberculosis strain Harlingen as well as to M. tuberculosis H37Rv. The binding of MoAb SMITB14 to LAM was inhibited by arabinomannan (AM) and oligosaccharides (5·2 kDa) derived from LAM, showing that the MoAb binds specifically to the AM carbohydrate portion of LAM. In passive protection experiments BALB/c mice were infected intravenously with M. tuberculosis Harlingen. MoAb SMITB14 was added intravenously either prior to, or together with, the bacteria. The antibody proved to be protective against the M. tuberculosis infection in terms of a dose-dependent reduction in bacterial load in spleens and lungs, reduced weight loss and, most importantly, increased long-term survival.

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Background Mycobacterium tuberculosis (Mtb) is able to evade the immune defenses and may persist for years, decades and even lifelong in the infected host. Mtb cell wall components may contribute to such persistence by modulating several pivotal types of immune cells. Dendritic cells (DCs) are the most potent antigen-presenting cells and hence play a crucial role in the initial immune response to infections by connecting the innate with the adaptive immune system.Principal FindingsWe investigated the effects of two of the major mycobacterial cell wall-associated types of glycolipids, mannose-capped lipoarabinomannan (ManLAM) and phosphatidylinositol mannosides (PIMs) purified from the Mtb strains H37Rv and Mycobacterium bovis, on the maturation and cytokine profiles of immature human monocyte-derived DCs. ManLAM from Mtb H37Rv stimulated the release of pro-inflammatory cytokines TNF, IL-12, and IL-6 and expression of co-stimulatory (CD80, CD86) and antigen-presenting molecules (MHC class II). ManLAM from M. bovis also induced TNF, IL-12 and IL-6 but at significantly lower levels. Importantly, while ManLAM was found to augment LPS-induced DC maturation and pro-inflammatory cytokine production, addition of PIMs from both Mtb H37Rv and M. bovis strongly reduced this stimulatory effect.ConclusionsThese results indicate that the mycobacterial cell wall contains macromolecules of glycolipid nature which are able to induce strong and divergent effects on human DCs; i.e while ManLAM is immune-stimulatory, PIMs act as powerful inhibitors of DC cytokine responses. Thus PIMs may be important Mtb-associated virulence factors contributing to the pathogenesis of tuberculosis disease. These findings may also aid in the understanding of some earlier conflicting reports on the immunomodulatory effects exerted by different ManLAM preparations.

Exposure to Mycobacterium tuberculosis (Mtb) may lead to active or latent tuberculosis, or clearance of Mtb, depending essentially on the quality of the host's immune response. This response is initiated through the interaction of Mtb cell wall surface components, mostly glycolipids, with cells of the innate immune system, particularly macrophages (Mφs) and dendritic cells (DCs). The way Mφs and DC alter their cytokine secretome, activate or inhibit different microbicidal mechanisms and present antigens and consequently trigger the T cell-mediated immune response impacts the host immune response against Mtb. Lipoarabinomannan (LAM) is one of the major cell wall components of Mtb. Mannosyl-capped LAM (ManLAM), and its related cell wall-associated types of glycolipids/lipoglycans, namely phosphatidylinositol mannosides (PIMs) and lipomannan (LM), exhibit important and distinct immunomodulatory properties. The structure, internal heterogeneity and abundance of these molecules vary between Mtb strains exhibiting distinct degrees of virulence. Thus ManLAM, LM and PIMs may be considered crucial Mtb-associated virulence factors in the pathogenesis of tuberculosis. Of particular relevance for this review, there is controversy about the specific immunomodulatory properties of these distinct glycolipids, particularly when tested as purified molecules in vitro. In addition to the variability in the glycolipid composition conflicting reports may also result from differences in the protocols used for glycolipid isolation and for in vitro experiments including immune cell types and procedures to generate them. Understanding the immunomodulatory properties of these cell wall glycolipids, how they differ between distinct Mtb strains, and how they influence the degree of Mtb virulence, is of utmost relevance to understand how the host mounts a protective or otherwise pathologic immune response. This is essential for the design of preventive strategies against tuberculosis. Thus, since clarifying the controversy on this matter is crucial we here review, summarize and discuss reported data from in vitro stimulation with the three major Mtb complex cell wall glycolipids (ManLAM, PIMs and LM) in an attempt to conciliate the conflicting findings.

Tuberculosis (TB) is considered the most onerous of infectious diseases according to recent reports from the World Health Organization. Available tests for TB diagnosis present severe limitations, and a reliable point-of-care (POC) diagnostic test does not exist. Neither is there a test to discern between the different stages of TB, and in particular to predict which patients with Mycobacterium tuberculosis infection and no clinical signs are more at risk of advancing to overt disease. We here review the usefulness of mycobacterial lipoarabinomannan (LAM) as a diagnostic marker for active and latent TB and, also, aspects of the immune response to LAM relevant to such tests. There is a high potential for urinary LAM-based POC tests for the diagnosis of active TB. Some technical challenges to optimised sensitivity of the test will be detailed. A method to quantify LAM in urine or serum should be further explored as a test of treatment effect. Recent data on the immune response to LAM suggest that markers for host response to LAM should be investigated for a prognostic test to recognise individuals at the greatest risk of disease activation.

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We have previously developed a diagnostic test for tuberculosis based on detection of mycobacterial lipoarabinomannan (LAM) in urine. The method depended on a laborious concentration step. We have now developed an easy to perform test based on a magnetic immunoassay platform, utilizing high avidity monoclonal antibodies for the detection of LAM in urine. With this method the analytical sensitivity of the assay was increased 50-100-fold compared to conventional ELISA. In a pilot study of HIV-negative patients with microbiologically verified TB (n=17) and healthy controls (n=22) the sensitivity of the test was 82% and the specificity 100%. This is in stark positive contrast to a range of studies using available commercial tests with polyclonal anti-LAM Abs where the sensitivity of the tests in HIV-negative TB patients was very low.