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.

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.

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.

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.

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.

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.