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Epitope and affinity determination of recombinant Mycobacterium tuberculosis Ag85B antigen towards anti-Ag85 antibodies using proteolytic affinity-mass spectrometry and biosensor analysis

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Abstract

Tuberculosis (TB) is the first cause of death from infectious diseases worldwide. Only a single anti-TB vaccine is currently available for clinical use, but its efficacy is not achieved with certainty. The aim of this work is to provide a basis for the rational design of a neo-glycoconjugate vaccine against TB. Structural characterization of recombinant antigenic proteins from Mycobacterium tuberculosis (MTB) Ag85B (rAg85B, variants, and semi-synthetic glycoconjugates) was initially carried out. Identification of antibody epitope analyses by proteolytic affinity-mass spectrometry and surface plasmon resonance (SPR) biosensor analyses were performed in order to qualitatively identify and quantitatively characterize interaction structures of the antigens with antibodies from different sources. A commercial monoclonal antibody and polyclonal antibodies from different sources (patients with active TB, vaccinated individuals, and a healthy control) were employed to analyze antigen-antibody interactions. These combined approaches provided the identification of different assembled epitope regions on the recombinant MTB antigens, their affinity binding constants in the interactions with specific antibodies, and revealed the importance of protection from excessive glycosylation. The identified epitope peptides should constitute a suitable basis for the design of new specific target vaccines.

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Acknowledgments

The research visiting stay of Francesca Rinaldi at the Steinbeis Centre for Biopolymer Analysis and Biomedical Mass Spectrometry, Rüsselsheim am Main, was supported by an increase of the PhD scholarship and a grant for international mobility from the University of Pavia.

We dedicate this paper to the memory of Prof. Massimo Amicosante, who made a substantial contribution sharing his knowledge and providing valuable suggestions with great humanity and professionalism.

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Author notes

  1. Loredana Lupu, Hendrik Rusche and Zdeněk Kukačka contributed equally to this work.

Authors and Affiliations

  1. Department of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100, Pavia, Italy

    Francesca Rinaldi, Sara Tengattini, Teodora Bavaro, Enrica Calleri & Caterina Temporini

  2. Steinbeis Centre for Biopolymer Analysis and Biomedical Mass Spectrometry, Marktstraße 29, 65428, Rüsselsheim am Main, Germany

    Francesca Rinaldi, Loredana Lupu, Hendrik Rusche, Zdeněk Kukačka, Stefan Maeser & Michael Przybylski

  3. Department of Biology and Animal Technology Station, University of Rome “Tor Vergata”, Via Montpellier 1, 00133, Rome, Italy

    Roberta Bernardini

  4. Department of Biotechnology and Life Sciences, University of Insubria, Via Dunant 3, 21100, Varese, Italy

    Luciano Piubelli & Loredano Pollegioni

  5. The Protein Factory Research Centre, Politecnico of Milan and University of Insubria, Via Mancinelli 7, 20131, Milan, Italy

    Luciano Piubelli & Loredano Pollegioni

Authors
  1. Francesca Rinaldi
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  2. Loredana Lupu
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  3. Hendrik Rusche
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  4. Zdeněk Kukačka
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  5. Sara Tengattini
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  7. Luciano Piubelli
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  8. Teodora Bavaro
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  10. Loredano Pollegioni
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  11. Enrica Calleri
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  12. Michael Przybylski
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  13. Caterina Temporini
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Corresponding authors

Correspondence to Michael Przybylski or Caterina Temporini.

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Antibody samples were blind analyzed and derived from anonymous patients belonging to a no profit Biobank located at University of Rome “Tor Vergata”.

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Rinaldi, F., Lupu, L., Rusche, H. et al. Epitope and affinity determination of recombinant Mycobacterium tuberculosis Ag85B antigen towards anti-Ag85 antibodies using proteolytic affinity-mass spectrometry and biosensor analysis. Anal Bioanal Chem 411, 439–448 (2019). https://doi.org/10.1007/s00216-018-1466-z

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  • DOI: https://doi.org/10.1007/s00216-018-1466-z

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