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.

FIG. 1.

Double staining of lung tissue of a mouse using acid-fast staining followed by immunohistochemistry analysis with MAb 5c11. (A) Lung tissue of an uninfected mouse (original magnification, ×100); (B) lung tissue of a mouse infected with M. tuberculosis (original magnification, ×100) demonstrating areas of immunostaining (arrows); (C) lung tissue of a mouse infected with M. tuberculosis (bar, 10 μm) demonstrating immunostaining in dark blue; (D) acid-fast staining of lung tissue of a mouse infected with M. tuberculosis without the addition of MAb 5c11 (bar, 10 μm); panel D demonstrates an area equivalent to that shown in panel C following counterstaining with hematoxylin. The figure was generated with an Epson Perfection 1650 scanner and Adobe Photoshop (version 7.0) for the personal computer.

FIG. 2.

Capture of AM, LAM, and AG by different MAbs. The graphs indicate the ELISA configuration. The capture antibodies were MAbs 9d8 (A), CS-40 (B), and CS-35 (C). Symbols represent the averages of two measurements; error bars denote 1 standard deviation. The experiment was performed twice with similar results. PNPP, p-nitrophenylphosphate; UNLB, unlabeled.

FIG. 3.

Titers of IgG antibodies to AM in mice immunized with the AM-rEPA vaccine, as measured by ELISA. Circles, titers for individual mice at a given time; horizontal bars, median titers.

FIG. 4.

Mycobacterial burdens in organs of mice immunized with the AM-rEPA vaccine and controls receiving rEPA alone after infection with M. bovis BCG (three panels at the top) and M. tuberculosis (two panels at the bottom). Bars represent the average CFU from 3 mice 1 week after infection. Error bars denote 1 standard deviation.