Biotechnological and Immunological Platforms Based on PGL-I Carbohydrate-Like Peptide of Mycobacterium leprae for Antibodies Detection Among Leprosy Clinical Forms

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.

Keywords: ELISA; mimotopes; phage display; phenolic glycolipid I; scFv; surface plasmon resonance.

FIGURE 1

Peptide sequences derived from a random peptide Phage Display (PD) library selected against the CS-48 antibody anti-PGL-I and design of the chimeric protein. (A) Peptides sequences and their frequencies. n (number of clones). (B) Sequence of the chimeric peptide, PGLI-M3. (C,D) Predicted three-dimensional structure of the PGLI-M3 peptide. Meaning of sequence colors: pink (hydrophobic regions), brown (spacers) and green (peptide sequence).

FIGURE 2

Detection of IgM antibodies by ELISA using the PGLI-M3 antigen in endemic population for leprosy. (A) ELISA reactivity of the PGLI-M3 in patients across clinical forms, contacts, and newborns. (B) Sensitivity, specificity, and positivity by clinical form. (C) ROC curve. (D) Linear correlation between ELISA indices of the native PGL-I and the PGLI-M3.

FIGURE 3

Detection of IgM antibodies anti-PGLI in pool of patients’ samples with lepromatous (LL), visceral leishmaniasis (VL+) and tuberculosis (TB+) using the PGLI-M3 (A) and native PGL-I (B) antigens. The established cut off is presented in a red line. TT (tuberculoid); BT (borderline tuberculoid); BB (borderline borderline); BL (borderline lepromatous); LL (lepromatous); HC (household contacts); CNB (newborn control).

FIGURE 4

Detection of IgG antibodies by ELISA using PGLI-M3 antigen in hyperendemic population for leprosy. (A) ELISA reactivity of the PGLI-M3 in patients across clinical forms, contacts and endemic controls. (B) ROC curve. (C) Sensitivity, specificity, and positivity by clinical form.

FIGURE 5

Antigen/antibody binding affinity for leprosy patients measured by Surface Plasmon resonance (SPR). (A,B) Sensorgram shows association between PGLI-M3 and antibodies in pools of serum samples from, newborn control (CNB), contacts (HC–), tuberculoid (TT), borderline-borderline (BB) and lepromatous (LL) patients. The graph shows the time (s) and the angular variation (m°). (C) Bar graph showing the absolute quantification of the angular variation. (D–H) Sensorgrams for the binding affinity for each patient within clinical forms, including the negative control (HC–). **p < 0.05 and ***p < 0.01.

FIGURE 6

scFv antibody anti-PGLI-M3. (A) ELISA detection of scFv antibodies that were expressed in E. coli top-10. (B) Expressed antibodies that recognized PGLI-M3. (C,D) Interaction of anti-PGLI-M3 with different antigens. PGLI-N (native PGL-I); ND-O-HSA (natural disaccharide with octyl linkage conjugated to bovine serum albumin); NC-IP (negative control – irrelevant peptide). **p < 0.005; ***p < 0.001.

FIGURE 7

Immunohistochemistry of skin biopsies using anti-PGLI-M3 scFv. (A) Identification of M. leprae using the Ziehl–Neelsen staining. (B) Anti-PGLI-M3 positive labeling. (C) Control of the reaction without scFv. The red arrows indicate the bacilli detection.