Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates

Abstract

Tuberculosis is still one of the most deadly infectious diseases worldwide, and the use of conjugated antigens, obtained by combining antigenic oligosaccharides, such as the lipoarabinomannane (LAM), with antigenic proteins from Mycobacterium tuberculosis (MTB), has been proposed as a new strategy for developing efficient vaccines. In this work, we investigated the effect of the chemical glycosylation on two recombinant MTB proteins produced in E. coli with an additional seven-amino acid tag (recombinant Ag85B and TB10.4). Different semi-synthetic glycoconjugated derivatives were prepared, starting from mannose and two disaccharide analogs. The glycans were activated at the anomeric position with a thiocyanomethyl group, as required for protein glycosylation by selective reaction with lysines. The glycosylation sites and the ex vivo evaluation of the immunogenic activity of the different neo-glycoproteins were investigated. Glycosylation does not modify the immunological activity of the TB10.4 protein. Similarly, Ag85B maintains its B-cell activity after glycosylation while showing a significant reduction in the T-cell response. The results were correlated with the putative B- and T-cell epitopes, predicted using a combination of in silico systems. In the recombinant TB10.4, the unique lysine is not included in any T-cell epitope. Lys30 of Ag85B, identified as the main glycosylation site, proved to be the most important site involved in the formation of T-cell epitopes, reasonably explaining why its glycosylation strongly influenced the T-cell activity. Furthermore, additional lysines included in different epitopes (Lys103, -123 and -282) are also glycosylated. In contrast, B-cell epitopic lysines of Ag85B were found to be poorly glycosylated and, thus, the antibody interaction of Ag85B was only marginally affected after coupling with mono- or disaccharides.

Keywords: MTB recombinant antigens; epitope; glycoconjugate vaccines; neo-glycoproteins.

Scheme 1

Synthesis of neo-glycoproteins 4–8 by conjugation reaction of IME saccharides 1a–3a with TB10.4 and Ag85B: (a) 1, 2 and 3, MeONa/MeOH, room temperature, 24 h, 50% yield; and (b) 1a–3a, sodium tetraborate buffer, pH 9.5, 37 °C (25 °C for product 5), molar ratio glycosidic reagent/(TB10.4/Ag85B) 200:1, 24 h.

Figure 1

T-cell responses to: rTB10.4 (A); and rAg85B (B) antigens and glycoderivatives. Data are presented as min to max value and boxplot of 25th–75th percentile of the spot-forming cells (SFC) per million PBMCs obtained by ELISPOT in BCG-vaccinated subjects (BCG-vaccinated) and active TB patients (Active TB). rTB10.4 antigen (white box), monomannose conjugate 4 (light gray), and di-mannose conjugate 5 (medium gray). rAg85B antigen (white box), monomannose conjugate 6 (light gray), di-mannose conjugate 7 (medium gray), and arabinose-mannose conjugate 8 (dark gray).

Figure 2

Antibody response to rAg85B and the glycoderivatives tested is presented as min to max value and boxplot of 25th–75th percentile of the absolute optical density (OD) value obtained by ELISA assay in healthy controls (CTR), BCG-vaccinated subjects (Vacc) and active TB patients (Active TB). rAg85B antigen (white box), monomannose conjugated (light gray), di-mannose conjugated (medium gray), and arabinose-mannose conjugated (dark gray).

Figure 3

Prediction of HLA class II-restricted T-cell epitopes for the pool of frequently observed HLA alleles covering more than 90% of human populations [27] has been performed as described previously [28] by quantitatively implemented peptide-binding motif analysis at a binding capability equivalent to the top 3% of the binding peptides for each tested allele. Epitope results for all the HLA class II alleles are graphically reported on the protein sequences by indicating in blue the T-cell epitope sequence areas and in red the amino acid acting as P1 anchor of the epitope to the HLA molecule(s). Lysines are indicated in bold and underlined. Recombinant TB10.4 and Ag85B include the N-terminal tag: AMAISDP.

Figure 4

Representation of the structure of rAg85B (pdb code 1f0n) and involvement of lysines in glycosylation and in formation of B-cell epitopes. Lysine labels are marked in a different size based on the glycosylation level (large: >20%: medium: 10–20% range; small: <10%; see Table 2 for details) and by different colors based on the number of prediction methods that identified their presence as part of epitopes (red: 5; green: 4; light blue: 3; blue: 2; purple: 1; see Table 2 for details).