Aharona Glatman-Freedman

Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice

Bacteria naturally release membrane vesicles (MVs) under a variety of growth environments. Their production is associated with virulence due to their capacity to concentrate toxins and immunomodulatory molecules. In this report, we show that the 2 medically important species of mycobacteria, Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin, release MVs when growing in both liquid culture and within murine phagocytic cells in vitro and in vivo. We documented MV production in a variety of virulent and nonvirulent mycobacterial species, indicating that release of MVs is a property conserved among mycobacterial species. Extensive proteomic analysis revealed that only MVs from the virulent strains contained TLR2 lipoprotein agonists. The interaction of MVs with macrophages isolated from mice stimulated the release of cytokines and chemokines in a TLR2-dependent fashion, and infusion of MVs into mouse lungs elicited a florid inflammatory response in WT but not TLR2-deficient mice. When MVs were administered to mice before M. tuberculosis pulmonary infection, an accelerated local inflammatory response with increased bacterial replication was seen in the lungs and spleens. Our results provide strong evidence that actively released mycobacterial vesicles are a delivery mechanism for immunologically active molecules that contribute to mycobacterial virulence. These findings may open up new horizons for understanding the pathogenesis of tuberculosis and developing vaccines.

Advances in antibody-mediated immunity against Mycobacterium tuberculosis: implications for a novel vaccine strategy

Cell-mediated immunity is considered to be the major component of the host response against Mycobacterium tuberculosis, whereas antibody-mediated immunity historically has been considered inconsequential. In recent years, studies from several groups have challenged the traditional dogma and demonstrated that monoclonal antibodies can modify various aspects of mycobacterial infections. This review describes the experimental evidence supporting a role for antibodies in defense against mycobacterial infections and outlines future challenges to the field of antibody-mediated immunity against M. tuberculosis, with particular emphasis on the implications of these findings for a novel vaccine strategy.

Clearance and Organ Distribution of Mycobacterium tuberculosis Lipoarabinomannan (LAM) in the Presence and Absence of LAM-Binding Immunoglobulin M

ABSTRACT Lipoarabinomannan (LAM) is a component of the mycobacterial surface which has been associated with a variety of deleterious effects on immune system function. Despite the importance of LAM to the pathogenesis of mycobacterial infection, there is no information available on its fate in vivo. In this study, we determined the pharmacokinetics and tissue distribution of exogenously administered LAM in mice. For measurements of serum and tissue LAM concentrations, we developed an enzyme-linked immunosorbent assay which used monoclonal antibodies of different isotypes to capture and detect LAM at concentrations of ≥0.4 μg/ml. Intravenous administration of LAM to mice resulted in transient serum levels with organ deposition in the spleen and in the liver. Immunohistochemical studies localized LAM to the spleen marginal zone macrophages and, to a lesser degree, to liver macrophages. When LAM was administered to mice previously given a LAM-binding immunoglobulin M (IgM), LAM was very rapidly cleared from circulation. In those mice, deposition of LAM in the spleen was significantly reduced while LAM deposition in the liver increased. Administration of LAM-binding IgM resulted in significant levels of IgM to LAM in bile consistent with an increased hepatobiliary excretion of LAM in the presence of specific antibody. Bile, liver extracts, and bile salts were found to rapidly inactivate the immunoreactivity of LAM. The results indicate that serum clearance and organ deposition of LAM in mice are affected by the presence of LAM-binding antibody and suggest a mechanism by which antibody could modify the course of mycobacterial infection.

Glucan Is a Component of the Mycobacterium tuberculosis Surface That Is Expressed In Vitro and In Vivo

ABSTRACT The outermost layer of Mycobacterium tuberculosis is composed primarily of two polysaccharides, glucan (GC) and arabinomannan. To analyze the surface polysaccharide composition of M. tuberculosis, we generated a monoclonal antibody (MAb) that binds M. tuberculosis GC and is known as MAb 24c5. Immunofluorescence and whole-mount immunoelectron microscopy indicated that GC is on the outermost portion of the bacteria. M. tuberculosis strains Erdman and CDC 1551 were analyzed for their ability to bind MAb 24c5 after in vitro growth in media with and without the detergent Tween 80. MAb 24c5 bound to Erdman and CDC 1551 at all culture times with only slightly greater apparent affinity after extended culture in the absence of Tween 80, indicating that a stable amount of GC polysaccharide antigen is associated with the cell surface of M. tuberculosis. An enzyme-linked immunosorbent assay indicated that GC is antigenically similar to glycogen, and the amount of GC antigen increased in the media of M. tuberculosis cultures grown either with or without the detergent Tween 80. Other nontuberculosis mycobacteria have antigenically similar GCs on their surfaces after in vitro growth. Inoculation of mice with live bacilli but not inoculation with dead bacilli elicited a strong antibody response to GC consistent with production of this antigen in vivo. Our results provide a more comprehensive picture of the M. tuberculosis cell envelope and the conditions that allow expression of M. tuberculosis GC.

Expression of a Mycobacterium tuberculosis Arabinomannan Antigen In Vitro and In Vivo

ABSTRACT The outermost layer of Mycobacterium tuberculosiscontains two major polysaccharides, arabinomannan (AM) and glucan (GC). We studied the in vitro and in vivo expression of anM. tuberculosis AM antigen using monoclonal antibody (MAb) 9d8 (2a), an isotype-switched variant of the immunoglobulin G3 (IgG3) MAb 9d8. MAb 9d8 had been previously shown to bind M. tuberculosis AM and the M. tuberculosis surface. Our in vitro experiments showed that MAb 9d8(2a) bound strongly to whole-cell M. tuberculosis Erdman but not to the CDC 1551 strain grown in medium for an extended period. However, AM antigen was detected in the culture supernatant of both strains, and its concentration increased in a time-dependent manner. The detection of AM antigen from both strains was decreased in the presence of Tween 80. In mice infected with M. tuberculosis Erdman, AM antigen accumulated in organ homogenates concomitant to an increase in bacterial organ burden and an increase in IgG and IgM titer to AM. These results (i) indicate that the surface expression of AM during in vitro growth changes with culture age, is strain dependent, and is affected by the presence of Tween 80 in the culture media; (ii) show that AM is produced by bacteria growth in vivo; and (iii) demonstrate that the amount of in vivo-detected AM can be dependent on the number of bacteria in the infected organ.

Antigenic Evidence of Prevalence and Diversity of Mycobacterium tuberculosis Arabinomannan

ABSTRACT Arabinomannan (AM) is a polysaccharide of the mycobacterial capsule. The capsular polysaccharides of various microorganisms are diverse, and this diversity is important for classification of organisms into serotypes and vaccine development. In the present study we examined the prevalence and diversity of AM among Mycobacterium tuberculosis strains using four AM-binding monoclonal antibodies (MAbs). One of these MAbs, MAb 9d8, is known to bind to AM specifically. By whole-cell enzyme-linked immunosorbent assay (ELISA), the AM recognized by MAb 9d8 was detected on the surfaces of 9 of 11 strains, while 2 strains showed no reactivity with MAb 9d8. However, the AM recognized by MAb 9d8 was found in the culture supernatants of all 11 M. tuberculosis strains tested, as demonstrated by capture ELISA. Other AM-binding MAbs reacted both with the surfaces and with the culture supernatants of all 11 strains. Mice immunized with an experimental AM-recombinant Pseudomonas aeruginosa exoprotein A (rEPA) conjugate vaccine had an increased antibody response to AM and a moderate reduction in the numbers of CFU in their organs 7 days after challenge. Our results indicate that AM was detected in all M. tuberculosis strains tested, with differences in epitope distributions of certain strains. In addition, our results suggest that an experimental AM-rEPA vaccine has a moderate effect on the numbers of CFU in organs early after infection.

Serologic evidence for regional differences in pediatric cryptococcal infection

Cryptococcus neoformans is present in areas contaminated with pigeon droppings. Unrecognized infections are hypothesized to occur commonly among immunocompetent individuals. We used serology to estimate prevalence of cryptococcal infection in immunocompetent children from 3 regions. Our results indicate unrecognized cryptococcal infections are extremely common in Bronx children, but uncommon in children from Dutchess County, NY and the Philippines.

Association of Human Antibodies to Arabinomannan With Enhanced Mycobacterial Opsonophagocytosis and Intracellular Growth Reduction

Background. The relevance of antibodies (Abs) in the defense against Mycobacterium tuberculosis infection remains uncertain. We investigated the role of Abs to the mycobacterial capsular polysaccharide arabinomannan (AM) and its oligosaccharide (OS) fragments in humans. Methods. Sera obtained from 29 healthy adults before and after primary or secondary bacillus Calmette-Guerin (BCG) vaccination were assessed for Ab responses to AM via enzyme-linked immunosorbent assays, and to AM OS epitopes via novel glycan microarrays. Effects of prevaccination and postvaccination sera on BCG phagocytosis and intracellular survival were assessed in human macrophages. Results. Immunoglobulin G (IgG) responses to AM increased significantly 4–8 weeks after vaccination (P < .01), and sera were able to opsonize BCG and M. tuberculosis grown in both the absence and the presence of detergent. Phagocytosis and intracellular growth inhibition were significantly enhanced when BCG was opsonized with postvaccination sera (P < .01), and these enhancements correlated significantly with IgG titers to AM (P < .05), particularly with reactivity to 3 AM OS epitopes (P < .05). Furthermore, increased phagolysosomal fusion was observed with postvaccination sera. Conclusions. Our results provide further evidence for a role of Ab-mediated immunity to tuberculosis and suggest that IgG to AM, especially to some of its OS epitopes, could contribute to the defense against mycobacterial infection in humans.

Specificity and Diversity of Antibodies to Mycobacterium tuberculosis Arabinomannan

ABSTRACT Arabinomannan (AM) is a polysaccharide antigen of the mycobacterial capsule. However, it is uncertain whether AM constitutes an immunologically distinct fraction of Mycobacterium tuberculosis. In this study, we analyzed the repertoire and specificity of antibodies to AM by using AM-binding murine monoclonal antibodies (MAbs) and human serum samples. Murine MAbs were found to be diverse in their specificity to AM and cross-reactivity with other arabinose-containing mycobacterial polysaccharides, with MAb 9d8 binding exclusively to AM. Human antibodies to AM were detected in serum samples from patients with pulmonary tuberculosis (TB), as well as in those from healthy, purified protein derivative-negative controls, with significantly higher titers among patients. The binding of human antibodies to AM was inhibited by MAb 9d8 in three patients with TB but not in controls. MAb 5c11, which recognizes other mycobacterial arabinose-containing carbohydrates in addition to AM, inhibited the binding of serum samples from 75% of patients and 76% of controls. Analysis of human antibodies with murine MAbs to human VH determinants demonstrated diversity among antibodies to AM with qualitative and quantitative differences compared with antibodies to lipoarabinomannan. In summary, our study suggests that antibodies to AM are diverse and heterogeneous with respect to antigen recognition and VH determinant expression, with human serum samples containing different subsets of antibodies to AM with the specificities of AM-binding murine MAbs. One MAb and a subset of human antibodies bind AM specifically, suggesting that this polysaccharide is antigenically distinct and is expressed in human infection.

The effect of social determinants on immunization programs

Vaccine preventable diseases have been responsible for a significant portion of childhood mortality in low-income countries, and have been re-emerging in medium- and high-income countries. The effectiveness of routine childhood immunization programs relies on multiple factors. Social determinants have the potential to affect immunization programs around the world, with globalization and ease of communication facilitating their effect. Exploring the types of social determinants affecting immunization efforts in various countries is of great importance to the ability of nations to address them, prevent the spread of disease and lower mortality rates. The social determinants affecting vaccination programs can vary among countries of different income levels, with some social determinants overlapping among these country groups. In this article we explore the various social determinants affecting routine immunization programs in low-, middle- and high-income countries and possible interventions to address them.