Abstract
Phenolic glycolipid I (PGL-I) is an abundant antigen on the Mycobacterium leprae cell wall, commonly used for operational classification of leprosy patients. Our aim was to develop PGL-I mimotopes with similar characteristics and functions of the native antigen. We have used a random peptide phage display (PD) library for selections against the monoclonal antibody anti-PGL-I. After three selection cycles, six peptides were identified. All sequences were interspersed by a spacer generating a chimeric peptide (PGLI-M3) that was artificially synthesized. The highly reactive peptide was submitted to a reverse PD selection with a single-chain Fv (scFv) antibody fragment combinatorial library. The most reactive scFv was then validated by enzyme-linked immunosorbent assay (ELISA) against both native PGL-I and two derived synthetic (NDO and ND-O-HSA). We have further proved the scFv specificity by detecting M. leprae bacilli in leprosy lesions through immunohistochemistry. We then described its applicability in ELISA for all clinical forms and household contacts (HC). Afterward, we showed differential binding affinities of PGLI-M3 to sera (anti-PGL-I IgM) from all leprosy clinical forms through surface plasmon resonance (SPR). ELISA IgM detection showed 89.1% sensitivity and 100% specificity, considering all clinical forms. Positivity for anti-PGL-I IgM was twofold higher in both HC and patients with paucibacillary forms in hyperendemic regions than in endemic ones. The SPR immunosensor was able to differentiate clinical forms with 100% accuracy. This is the first time that a PGL-I mimotope has efficiently mimicked the carbohydrate group of the M. leprae antigen with successful immunoassay applications and may become a substitute for the native antigen.
Highlights
The phenolic glycolipid I (PGL-I) is one of the main antigens on the cell wall of Mycobacterium leprae and has important roles in the pathogenesis and diagnosis of leprosy (Spencer and Brennan, 2011)
The predicted structural analysis by bioinformatics indicated that the PGLI-M3 presents hydrophobic regions (Figure 1C), showing similar chemical characteristics to the native PGL-I with a tridimensional conformation (Figure 1D), conferred by proline-rich regions followed by chemically charged amino acids
Positivity was evaluated in all clinical forms, which demonstrated a similar behavior of the native PGL-I, with antibody levels close to 100% in the lepromatous pole (Figures 2A,B)
Summary
The phenolic glycolipid I (PGL-I) is one of the main antigens on the cell wall of Mycobacterium leprae and has important roles in the pathogenesis and diagnosis of leprosy (Spencer and Brennan, 2011). The extraction and purification of the native PGL-I is restricted to the growth of M. leprae in mice and armadillos (Levy and Ji, 2006), due to the natural inability of the pathogen to grow in vitro (Youn et al, 2004), leading to a limited availability of the antigen. This problem led researchers to seek for alternatives to native PGL-I using synthetic antigens, such as ND-O-HSA (natural disaccharide with octyl linkage to human serum albumin) and NT-P-HSA (natural trisaccharide with phenolic ring linkage to HSA) (Fujiwara and Izumi, 1987). A new alternative was proposed through the selection of mimetic peptides, but its efficacy for serological diagnosis of leprosy did not work properly (Youn et al, 2004), suggesting that peptides with PGL-I hydrophobic and hydrophilic properties would be difficult to reproduce, especially knowing that the PGLI antigenicity is conferred by the terminal phenolic disaccharide at the surface (Barnes et al, 2017)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
