Lipoarabinomannan, and its related glycolipids, induce divergent and opposing immune responses to Mycobacterium tuberculosis depending on structural diversity and experimental variations.

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.

A sensitive urinary lipoarabinomannan test for tuberculosis.

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.

Mycobacterium tuberculosis Strains Are Differentially Recognized by TLRs with an Impact on the Immune Response.

Tuberculosis associates with a wide spectrum of disease outcomes. The Beijing (Bj) lineage of Mycobacterium tuberculosis (Mtb) is suggested to be more virulent than other Mtb lineages and prone to elicit non-protective immune responses. However, highly heterogeneous immune responses were reported upon infection of innate immune cells with Bj strains or stimulation with their glycolipids. Using both in vitro and in vivo mouse models of infection, we here report that the molecular mechanism for this heterogeneity may be related to distinct TLR activations. Among this Mtb lineage, we found strains that preferentially activate TLR2, and others that also activate TLR4. Recognition of Mtb strains by TLR4 resulted in a distinct cytokine profile in vitro and in vivo, with specific production of type I IFN. We also uncover a novel protective role for TLR4 activation in vivo. Thus, our findings contribute to the knowledge of the molecular basis underlying how host innate immune cells handle different Mtb strains, in particular the intricate host-pathogen interaction with strains of the Mtb Bj lineage.

Divergent effects of mycobacterial cell wall glycolipids on maturation and function of human monocyte-derived dendritic cells.

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 FINDINGS: We 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. CONCLUSIONS: These 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.

Molecular evidence of Plesiomonas shigelloides as a possible zoonotic agent.

The most frequently used method for establishing epidemiological relationships between Plesiomonas shigelloides strains is O:H serotyping. However, a number of strains are not serotypeable and isolates from diverse sources can display the same serovar. Moreover, since the zoonotic nature of Plesiomonas has been suggested and this hypothesis is based on the identical serovars found in animals and humans, we intend to use four DNA-based techniques: random amplified polymorphic DNA-PCR, enterobacterial repetitive intergenic consensus-PCR, repetitive extragenic palindromic-PCR, and pulsed field gel electrophoresis in order to screen 24 strains belonging to nine O:H serovars isolated from humans, animals, and the environment. In general, P. shigelloides showed a high genetic heterogeneity. Three pairs of strains, each containing a human and an animal isolate, displayed similar genotypes. This is the first report that provides molecular evidence that P. shigelloides may be zoonotic.

Towards new tuberculosis vaccines.

According to WHO, about one third of the world's population is infected with bacteria of the Mycobacterium tuberculosis complex. Currently there is globally 9.15 million recorded cases of overt tuberculosis (TB) annually and due to lack of adequate diagnostics presumably a large but unknown number of non-recorded cases. TB is estimated to cause 1.65 million deaths per annum which accounts for one-fifth of all deaths by infectious diseases of adults in low-income countries. During recent years a rapid spread of multi-drug resistant bacteria causing about 0.5 million TB cases per year has worsened the problem. The live attenuated Bacillus Calmette-Guérin (BCG) vaccine which is the only currently available TB vaccine does not confer any significant protection against the most common and contagious form of TB-adult pulmonary TB.

Epidemiology of Mycobacterium bovis infection in wild boar (Sus scrofa) from Portugal.

Tuberculosis has been diagnosed in wild boar (Sus scrofa) in several European countries during the last decade; however, almost no information has been reported to date for Portugal. This study aimed to investigate tuberculosis in wild boar in Portugal through characterization of Mycobacterium bovis infection and identification of disease risk factors. Tissue samples were obtained from hunted wild boar during the 2005 and 2006 hunting seasons. Samples were inspected for gross lesions and processed for culture. Acid-fast bacterial isolates were identified by polymerase chain reaction and spoligotyping. Associations between tuberculosis in wild boar and several variables linked to wild ungulate diversity and relative abundance, livestock density, and cattle tuberculosis incidence were investigated. Mycobacterium bovis isolates were identified in 18 of 162 wild boars from three of eight study areas. Infection rates ranged from 6% (95% confidence interval [CI(P95%)] = 1-21%) to 46% (CI(P95%) = 27-67%) in the three infected study areas; females in our sample were at greater risk of being infected than males (odds ratio = 4.33; CI(P95%) = 3.31-5.68). Spoligotyping grouped the M. bovis isolates in three clusters and one isolate was a novel spoligotype not previously reported in international databases. Detection of M. bovis was most consistently associated with variables linked to wild ungulate relative abundance, suggesting that these species, particularly the wild boar, might act as maintenance hosts in Portugal.

Mycobacterial glycoconjugates as vaccine candidates against tuberculosis.

There is an urgent need for an efficient vaccine against tuberculosis. Here, we explore the potential role of carbohydrate antigens as part of a new tuberculosis vaccine. Emphasis is placed on carbohydrate-protein conjugate vaccines, using the arabinomannan portion of lipoarabinomannan, a major structural surface component of Mycobacterium tuberculosis covalently conjugated to (mycobacterial) protein antigens. Such conjugate vaccines show good protective efficacy in mice and guinea pigs in terms of prolonged survival and reduced pathology. Special attention is paid to the immunology underlying their protective capacity. Conjugate vaccines induce both cellular and humoral responses and, although antibody responses have been thought to be the main protective component, cellular responses - possibly through the CD1 pathway - are also likely to be involved.

Pathogen specific carbohydrate antigen microarrays: a chip for detection of Salmonella O-antigen specific antibodies.

A Salmonella O-antigen microarray was developed by covalent coupling of oligosaccharide antigens specific for serogroups Salmonella enterica sv. Paratyphi (group A), Typhimurium (group B) and Enteritidis (group D). Antibodies were correctly detected in sera from patients with culture verified salmonellosis. High serogroup-specificity was seen with the disaccharide antigens. With the larger antigens, containing the backbone sequence Manalpha1-2Rhaalpha1-2Gal (MRG), common backbone-specific antibodies (O-antigen 12) were also detected. This is "proof of principle" that pathogen-specific carbohydrate antigen microarrays constitute a novel technology for rapid and specific serological diagnosis in either individual patients or larger sero-epidemiological and vaccine studies.

Should a new tuberculosis vaccine be administered intranasally?

Most of the world's population is vaccinated with the only available vaccine against tuberculosis (TB), the Bacillus Calmette-Guérin (BCG) vaccine that was developed almost a century ago. Despite the wide coverage of the BCG vaccine, there are great variations in protective efficacy among different study populations. BCG vaccination protects against childhood forms of TB, but this immunity wanes with age, resulting in none, or insufficient, protection against adult pulmonary TB (PTB). PTB is the major disease manifestation of TB in adults and it causes death at the most productive age, further adding to poverty in already impoverished countries. Therefore, new more effective vaccines and novel immunisation strategies are urgently needed. The most common route of TB is by inhalation of tubercle bacilli leading to the establishment of a primary infection in the lung. Immunising through the nasal mucosal surface should therefore have advantage over other routes, as such vaccine administration elicits protective immune responses also in the lung, i.e. at the site of primary infection. Several new TB-vaccine candidates have been evaluated for their protective efficacy in animal models using the mucosal route of immunisation. In formulating such vaccines, the adjuvants and delivery systems are crucially important.

Evaluation of vaccines in the EU TB Vaccine Cluster using a guinea pig aerosol infection model of tuberculosis.

The TB Vaccine Cluster project funded by the EU Fifth Framework programme aims to provide novel vaccines against tuberculosis that are suitable for evaluation in humans. This paper describes the studies of the protective efficacy of vaccines in a guinea pig aerosol-infection model of primary tuberculosis. The objective was to conduct comparative evaluations of vaccines that had previously demonstrated efficacy in other animal models. Groups of 6 guinea pigs were immunized with vaccines provided by the relevant EU Vaccine Cluster partners. Survival over 17 or 26 weeks was used as the principal measure of vaccine efficacy following aerosol challenge with H37Rv. Counts of mycobacteria in lungs and spleens, and histopathological changes in the lungs, were also used to provide evidence of protection. A total of 24 vaccines were evaluated in 4 experiments each of a different design. A heterologous prime-boost strategy of DNA and MVA, each expressing Ag85A and a fusion protein of ESAT-6 and Ag85B in adjuvant, protected the guinea pigs to the same extent as BCG. Genetically modified BCG vaccines and boosted BCG strategies also protected guinea pigs to the same extent as BCG but not statistically significantly better. A relatively high aerosol-challenge dose and evaluation over a protracted time post-challenge allowed superior protection over BCG to be demonstrated by BCG boosted with MVA and fowl pox vectors expressing Ag85A.

Genetic characterization of the Guinea-Bissau family of Mycobacterium tuberculosis complex strains.

In a previous study of Mycobacterium tuberculosis complex isolates from Guinea-Bissau in West Africa, we identified a unique group of strains, designated here as the Guinea-Bissau family of strains, which, although genotypically closely related, phenotypically demonstrated a considerable heterogeneity. We conducted here a detailed genotypic analysis of a subset (n = 35) of these isolates. Based on the data obtained, and by comparison of known corresponding genes in mycobacteria outside the M. tuberculosis complex, we propose that the Guinea-Bissau strains belong to a unique branch of the M. tuberculosis complex tree in between classical M. tuberculosis and classical M. bovis. These observations are discussed in their significance in M. tuberculosis complex classification.

Serotypes and anti-microbial susceptibility of Plesiomonas shigelloides isolates from humans, animals and aquatic environments in different countries.

A total of 73 strains of Plesiomonas shigelloides isolated from humans (24 strains) animals (21 strains) and aquatic environment (28 strains) were determined for their O:H serotype and susceptibility to 18 anti-microbial substances and to the vibriostatic agent O/129. Of all strains, 86.3% were typeable by the O and 94.5% by the H anti-sera used. The serotype distribution was heterogeneous within a country and between the countries. Of the 57 different serotypes identified, O11:H2 (2 strains), O22:H3 (4 strains), O35:HH11 (2 strains), O52:H3 (2 strains) and O90:H6 (2 strains) were found among isolates from humans and animals (mainly in cats) in Finland and Cuba, and O23:H1a1b (3 strains) among isolates from environmental sources in Slovak Republic and Italy. Most (93-100%) of all strains were susceptible to all anti-microbials tested but resistant (92-96%) to the broad-spectrum penicillins (ampicillin, mezlocillin). No correlation between anti-microbial resistance patterns and serotypes was found.

Mycobacterium tuberculosis arabinomannan-protein conjugates protect against tuberculosis.

Lipoarabinomannan (LAM) is a major structural surface component of mycobacteria. Arabinomannan (AM) oligosaccharides derived from LAM of Mycobacterium tuberculosis H37Rv were isolated and covalently conjugated to tetanus toxoid (TT) or to short-term culture filtrate proteins (antigen 85B (Ag85B) or a 75kDa protein) from M. tuberculosis strain Harlingen. The different AM oligosaccharide (AMOs)-protein conjugate vaccine candidates proved to be highly immunogenic, inducing boosterable IgG responses against the AMOs portion of the conjugates in rabbits and guinea-pigs. Proliferation of T-cells from C57BL/6 mice immunized with the conjugates was seen upon in vitro stimulation with PPD. In C57BL/6 mice subcutaneous immunization with the AMOs-antigen 85B conjugate in alum provided significant protection compared to sham (alum only) immunized mice (P < 0.021) as estimated by long term survival against intravenous challenge with 10(5) M. tuberculosis H37Rv. Subcutaneous immunization followed by nasal boost with an AMOs-TT conjugate in Eurocine L3 adjuvant provided high (P < 0.025) protection as determined by long term survival after intranasal challenge with 10(5) virulent M. tuberculosis strain Harlingen. This level of protection was comparable to that obtained with the conventional live attenuated BCG vaccine. In guinea-pigs, immunization with AMOs-Ag85B in Eurocine L3 adjuvant followed by aerogenic challenge with M. tuberculosis H37Rv resulted in increased survival and reduced pathology in lungs and spleens relative to non-immunized animals.

Clinical and radiological features in relation to urinary excretion of lipoarabinomannan in Ethiopian tuberculosis patients.

We have previously reported on the diagnostic potential of urinary lipoarabinomannan (LAM) detection in active tuberculosis (TB). In this study, we identified clinical and radiological parameters that were significantly associated with urine LAM positivity in a clinical sample of 931 patients attending a TB control center in Addis Ababa, Ethiopa. These parameters were attributed weights and used in a diagnostic score (DS) system. Using urinary LAM as a reference, this DS system showed a sensitivity of 65.4% and a specificity of 82.9%. The positive and negative predictive values were 56.8% and 87.4%, respectively. HIV or other coinfections or deficiencies may have blurred the clinical manifestations of pulmonary TB (PTB) and thereby contributed to the relatively high number of false-positive DS results obtained. Although additional markers may be required to improve the sensitivity of the DS system, the relatively high specificity of this simple approach may be of some practical use in the field. Thus, in PTB-suspected, DS-negative cases, the likelihood of ongoing PTB is < 20%.

Circulating antibodies to lipoarabinomannan in relation to sputum microscopy, clinical features and urinary anti-lipoarabinomannan detection in pulmonary tuberculosis.

An enzyme-linked immunosorbent assay (ELISA)-based investigation of anti-lipoarabinomannan (LAM) antibody levels in the sera of patients with acid-fast bacilli (AFB)-positive pulmonary tuberculosis (PTB), AFB-negative PTB and non-TB respiratory tract symptoms was conducted. The anti-LAM results were further evaluated using urine LAM detection and a clinical diagnostic score (DS) system as references. Using sputum AFB as a reference, positive anti-LAM was found in 66.9% of 139 AFB-positive PTB, 34.4% of 61 AFB-negative PTB and 23.5% of 800 non-TB patients and in 8% of 50 healthy individuals. The positive and negative predictive values were 48.7% and 87.4%, respectively. Using the DS as a reference, the sensitivity and specificity were 50.5% and 78.3%, respectively, whereas 45.8% of urine LAM positives and 77.9% of urine LAM negatives were correctly identified by the anti-LAM ELISA. In TB endemic areas a negative anti-LAM could be of practical value, particularly when other indicators of PTB are negative. Using any of these methods as a reference, a positive anti-LAM would mislead in about one-quarter of cases. Had all the 3 methods been combined and at least 2 positive tests sufficed, 90.6% of AFB-positive PTB, 52.5% of AFB-negative PTB and 94.9% of non-TB patients would have been correctly diagnosed. Apart from the possible impact of HIV, the low accuracy of the current assay could be due to intravascular formation of LAM-anti-LAM complexes, latent TB or environmental mycobacterial infections.