Comparative Structural Study of Terminal Ends of Lipoarabinomannan from Mice Infected Lung Tissues and Urine of a Tuberculosis Positive Patient

Abstract

Mycobacterium tuberculosis lipoarabinomannan (LAM) is a biomarker for active TB disease. The presence of LAM in urine of TB patients, whether HIV positive or negative has been validated by a gas chromatography/mass spectral method with good specificity (84%) and sensitivity (99%). However, point-of-care (POC) methods to detect TB LAM in urine using immunoassays have poor sensitivity and are limited to only HIV co-infected TB diagnosis. We hypothesized that these disappointing results with the POC methods may be due to the antibodies used in the immunoassays as there could be structural differences between LAM in vivo vs. LAM in vitro. To address this issue, we infected C3HeB/FeJ mice with M.tb W. Beijing SA161, and purified LAM from the lung. Analysis of these sources of LAM using a panel of existing mAbs revealed differences in epitope patterns. Conventionally, the non-reducing termini of LAM are identified by their release with endoarabinanase. These epitopes correspond to linear tetra-(Ara 4 ), a branched hexa-(Ara 6 ) arabinofuranosides and their mannose-capped versions. We discovered two distinct epitopes. In the first case it was found that the non-reducing termini of LAM from M.tb strain SA161 are highly succinylated, especially when the LAM was isolated from the mouse lungs. In the second case it was found that Cellulomonas endoarabinanase digestion of LAM from both SA161 and LAM from a TB+ HIV-patient's urine yielded epitopes based on 5 arabinoses as major components and a profound lack of Ara 6 . The epitopes based on 5 arabinoses from M.tb SA161and from the LAM in human urine must result from underlying structural and thus epitope differences. These results suggest approaches to develop specific antibodies for POC tests for LAM in urine of suspected TB patients.

Point-Of-Care Urine LAM Tests for Tuberculosis Diagnosis: A Status Update

“…According to the capping motifs, LAM can be classified into three structural families: LAM from fast-growing, non-pathogenic species, such as M. smegmatis, have uncapped ends (AraLAM) or inositol phosphate caps (phosphatidyl-myo-inositol capped LAM, or PILAM) whereas LAM from slow-growing mycobacteria (Mtb, M. leprae, M. avium, and M. kansasii) is modified with one to three α (1→2)-linked mannopyranose (Manp) units (ManLAM). Mtb ManLAM can contain an additional cap modification, 5-deoxy-5-methylthio-xylofuranose (MTX), attached to the terminal Manp [13][14][15].…”

Lipoarabinomannan antigenic epitope differences in tuberculosis disease subtypes

“…In our previous study by Paris et al 6 , we demonstrated that it is possible to detect urinary LAM in HIV negative TB patients using an experimental workflow which included a pre-analytical concentration step. Other investigators confirmed these findings 7,8,17,18,25 . Recent reports of LAM immunoassays employing high affinity purified antibodies indicate that acceptable analytical sensitivity required for HIV negative TB positive patients can be achieved without the use of a pre-analytical concentration step.…”

“…Median urinary LAM concentration in TB negative patients was 0.016 ng/mL (IQR = 0.07), 0.03 ng/ mL (IQR = 0.06), and 0.03 ng/mL (IQR = 0.05) for MoAb1, CS35 and A194, respectively. 8 , Amin et al 17 , and De et al 18 . (B) Calibration curve of anti-LAM antibodies.…”

Animal Models of Mycobacteria Infection

“…Generally the disease progresses in five stages (Rhoades, Frank, & Orme, 1997), which are based on the extent of granulomatous involvement, the cell types present, the degree of lymphocyte organization, and the presence of destructive sequelae such as airway epithelium erosion and airway debris. Many of these parameters can be used to give an overall group ranking or lesion score based on histopathological assessments by a qualified veterinary pathologist (Basaraba, 2008;De et al, 2020).…”

“…The one notable disadvantage of the majority of mouse models is that the pulmonary and extra-pulmonary pathology following aerosol challenge lacks important morphologic features that are commonly seen in humans with untreated tuberculosis. To model these aspects, the guinea pig low-dose and the C3HeB/FeJ mouse aerosol infection models have been further developed in recent years, since they share many of these clinical features with the human disease (Basaraba, 2008;McMurray, 2003, De et al, 2020Shang, Harton et al, 2011;.…”