The surface of M. tuberculosis
contains polysaccharides and polysaccharide-containing fractions such as LAM, AG, and peptidoglycan (1
). In addition, an outermost layer of M. tuberculosis
was described by some investigators and identified as a capsule (21
). This layer consists mainly of polysaccharides, of which AM is an important component (5
). Polysaccharide capsules surround many pathogens, and they can be diverse. For example, a total of 6 capsular serotypes have been described for H. influenzae
), 90 capsular serotypes have been described for S. pneumoniae
), and 4 capsular serotypes have been described for Cryptococcus neoformans
). Classification of microbial pathogens into serotypes has been useful for diagnosis, understanding of disease epidemiology, and vaccine development. In general, polysaccharide-based vaccines are type specific and provide protection only against bacteria with capsular polysaccharides that are antigenically similar to those used in the vaccine (16
). The purpose of this study was to characterize AM in terms of its prevalence and antigenic variability.
Immunohistochemistry staining of tissues of mice that had been infected with M. tuberculosis
with an AM-binding MAb demonstrated circumscribed areas of staining in lung tissue that extended beyond the surface of the acid-fast bacilli (Fig. ). These results suggest that AM-containing polysaccharide is generated and potentially shed from the bacilli during infection in vivo, resulting in the presence of large areas containing mycobacterial polysaccharide. The paucity of staining of liver and spleen tissues may be a result of the lower numbers of CFU in these organs or the ability of these organs to efficiently clear the polysaccharides. In this regard, a previous study (10
) demonstrated that the liver and the spleen are very active in the pharmacokinetics of LAM, with the hepatobiliary system having an important role in clearance.
The use of several AM-binding MAbs with different specificities allowed the immunological identification of various AM epitopes, as demonstrated by the capture of purified fractions (Fig. ). The use of several MAbs was particularly important, given the data from a recent study demonstrating that the AM recognized by MAb 9d8 did not react with the cell surface of M. tuberculosis
CDC 1551 (23
), suggesting that the epitope recognized by MAb 9d8 was not present on the cell surface. This epitope was, however, present in the culture supernatant of this strain, suggesting that it is easily removable or shed. This phenomenon was not observed with M. tuberculosis
Erdman, for which MAb 9d8 reacted with both the surface and the culture supernatant. In the present study, the four AM-binding MAbs reacted with all strains tested (Table ). MAbs CS-40, CS-35, and 5c11 reacted with the cell surfaces and culture supernatants of all strains tested. MAb 9d8 reacted with the culture supernatants of all strains tested and the cell surfaces of nine strains, but it did not react with the cell surface of CI-9 or CDC 1551 (Table ). These results suggest differences in the MAb 9d8 epitope distribution among different strains. They may also suggest strain-to-strain variations in the stability of attachment of this polysaccharide epitope to the cell surface. Different degrees of bonding between the capsular material and the cell surface have previously been described for other bacteria (22
). Our results support the concept that MAb 9d8 binds to an epitope found exclusively on the outermost surface of M. tuberculosis
, while the epitopes recognized by the other MAbs may also be present underneath the outermost layer. In this regard, it is worth noting that arabinose and mannose are found in the capsular AM as well as in LAM. A previous study suggested that LAM and AM share antigenic determinants yet maintain antigenic differences, as demonstrated by the ability of MAb 9d8 to bind to AM and not LAM (19
It was recently demonstrated that although antibodies to AM were prevalent in human sera, antibodies with MAb 9d8 specificity were detected only in a small number of patients with TB (19
). The data raise questions regarding the prevalence of the MAb 9d8-specific AM epitope among clinical M. tuberculosis
strains. The ubiquity of this epitope, as demonstrated in the present study, suggests that the absence of antibodies to AM with MAb 9d8 specificity in many TB patients (19
) is related to the nature of the antibody response to this epitope rather than to differences in the prevalence of the MAb 9d8-specific AM epitope among different strains.
The biological significance of the differences in the MAb 9d8 epitope distribution among various M. tuberculosis
strains is unclear. Whether differences in the degree of attachment of capsular material could potentially affect the interaction of various M. tuberculosis
strains with the host immune system remains to be explored. It is of interest that human exposure to M. tuberculosis
CDC 1551 was found to result in high rates of purified protein derivative skin test conversions with large reactions (26
). In mice, this strain induced a more rapid and vigorous host immune response, with earlier granuloma formation and increased cytokine production, compared to the immune responses of other clinical isolates (15
Previous studies demonstrated that MAb 9d8 prolongs the survival of mice infected with a lethal dose of M. tuberculosis
) and that MAb 5c11 enhances the clearance of LAM in a murine model (10
). These findings highlight the potential immunological importance of AM. In fact, one study reported that AM has an inhibitory effect on lymphocyte proliferation (7
). In an effort to evaluate the potential of a polysaccharide-based vaccine against M. tuberculosis
, we challenged mice that had been immunized with the AM-rEPA vaccine. The vaccine was found to be immunogenic, eliciting high titers of antibodies to AM, and led to reductions in the numbers of CFU early in the course of infection. The effect observed was not sustained and did not affect the overall course of infection. These results suggest that an AM-based vaccine has the potential to affect the course of infection in its early stages, prior to the development of specific cell-mediated immunity. A study from another group recently reported prolonged survival of C57BL/6 mice and guinea pigs immunized with an AM-conjugate vaccine (11
). Taken together, the results of these studies are encouraging and suggest that mycobacterial polysaccharides may be considered in the development of a vaccine against TB.
In summary, our work demonstrated that AM is ubiquitous among M. tuberculosis strains, which expressed all four AM epitopes tested, with the MAb 9d8-specific AM epitope present on the surfaces of most strains. In addition, an experimental AM-conjugate vaccine was found to lower the numbers of CFU early in the course of infection. Additional studies are required to analyze the elements that are important for the development of an optimal polysaccharide vaccine, such as antigen specificity, type of conjugate and adjuvant used, the resulting antibody responses, as well as the effect of polysaccharide vaccine on various strains of M. tuberculosis. The latter is especially important, given the differences in the MAb 9d8 epitope distribution noted among M. tuberculosis strains. In addition, it is important to determine the exact role of mycobacterial polysaccharides in the immunopathogenesis of TB.