Rational design, preparation and characterization of recombinant Ag85B variants and their glycoconjugates with T-cell antigenic activity against Mycobacterium tuberculosis

Francesca Rinaldi, Sara Tengattini, Luciano Piubelli, Roberta Bernardini, Francesca Mangione, Teodora Bavaro, Gregorino Paone, Maurizio Mattei, Loredano Pollegioni, Gaetano Filice, Caterina Temporini, Marco Terreni

Research output: Contribution to journalArticle

Abstract

Tuberculosis is the deadliest infectious disease in the world. The variable efficacy of the current treatments highlights the need for more effective agents against this disease. In the past few years, we focused on the investigation of antigenic glycoconjugates starting from recombinant Ag85B (rAg85B), a potent protein antigen from Mycobacterium tuberculosis. In this paper, structural modifications were rationally designed in order to obtain a rAg85B variant protein able to maintain its immunogenicity after glycosylation. Lysine residues involved in the main T-epitope sequences (namely, K30 and K282) have been substituted with arginine to prevent their glycosylation by a lysine-specific reactive linker. The effectiveness of the mutation strategy and the detailed structure of resulting neo-glycoconjugates have been studied by intact mass spectrometry, followed by peptide and glycopeptide mapping. The effect of K30R and K282R mutations on the T-cell activity of rAg85B has also been investigated with a preliminary immunological evaluation performed by enzyme-linked immunospotting on the different variant proteins and their glycosylation products. After glycosylation, the two variant proteins with an arginine in position 30 completely retain the original T-cell activity, thus representing adequate antigenic carriers for the development of efficient glycoconjugate vaccines against tuberculosis.

Original languageEnglish
Pages (from-to)23171-23180
Number of pages10
JournalRSC Advances
Volume8
Issue number41
DOIs
Publication statusPublished - Jan 1 2018

ASJC Scopus subject areas

  • Chemistry(all)
  • Chemical Engineering(all)

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