Antigenic Evidence of Prevalence and Diversity ofMycobacterium tuberculosisArabinomannan

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.

Monoclonal antibodies to Mycobacterium tuberculosis CDC 1551 reveal subcellular localization of MPT51

“…MAbs at 10 μg/ml in 1% BSA were added and serially diluted. The rest followed as previously described (12). Binding of mAbs to surface carbohydrate and lipid fractions was determined by ELISA, as previously described (14).…”

“…Negative control wells had no M. tuberculosis. Whole cell ELISA with M. tuberculosis clinical strains and Erdman was performed as previously described (12).…”

“…and OADC (Becton-Dickinson, MD) without Tween 80 for 14 days at 37°C, heat killed at 80°C for 2 hours, washed twice in PBS, sonicated (Branson Ultrasonics, Danbury, Conn.) for 5 seconds and frozen at -80°C. M. tuberculosis clinical strains and Erdman were grown and lyophilized as previously described (12). M. tuberculosis ΔRD1ΔpanCD was grown in 7H9 medium containing 0.05% Tween 80 and pantothenate (24 μg/ml) (13).…”

“…Using mAbs to AM, we previously demonstrated that all M. tuberculosis strains tested shed AM into the culture media (12). However, while most of these strains also retained capsular AM on the cell surface, some shed their entire capsular AM (12). Shedding of capsular material may have implications on the interactions of M. tuberculosis with antibodies and host cells.…”

“…The capsular material was previously shown to be easily removed or shed from the mycobacterial surface (10;11). Using mAbs to AM, we previously demonstrated that all M. tuberculosis strains tested shed AM into the culture media (12). However, while most of these strains also retained capsular AM on the cell surface, some shed their entire capsular AM (12).…”