Mapping the Epitopes of a Neutralizing Antibody Fragment Directed against the Lethal Factor of Bacillus anthracis and Cross-Reacting with the Homologous Edema Factor

Philippe Thullier,Christian K Behrens,Jean-Luc Pellequer,Thibaut Pelat,Arnaud Avril,Jacques Mathieu

doi:10.1371/journal.pone.0065855

Philippe Thullier, Christian K Behrens + Show 4 more

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https://doi.org/10.1371/journal.pone.0065855

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Abstract

The lethal toxin (LT) of Bacillus anthracis, composed of the protective antigen (PA) and the lethal factor (LF), plays an essential role in anthrax pathogenesis. PA also interacts with the edema factor (EF, 20% identity with LF) to form the edema toxin (ET), which has a lesser role in anthrax pathogenesis. The first recombinant antibody fragment directed against LF was scFv 2LF; it neutralizes LT by blocking the interaction between PA and LF. Here, we report that scFv 2LF cross-reacts with EF and cross-neutralizes ET, and we present an in silico method taking advantage of this cross-reactivity to map the epitope of scFv 2LF on both LF and EF. This method identified five epitope candidates on LF, constituted of a total of 32 residues, which were tested experimentally by mutating the residues to alanine. This combined approach precisely identified the epitope of scFv 2LF on LF as five residues (H229, R230, Q234, L235 and Y236), of which three were missed by the consensus epitope candidate identified by pre-existing in silico methods. The homolog of this epitope on EF (H253, R254, E258, L259 and Y260) was experimentally confirmed to constitute the epitope of scFv 2LF on EF. Other inhibitors, including synthetic molecules, could be used to target these epitopes for therapeutic purposes. The in silico method presented here may be of more general interest.

Highlights

In 2001, the intentional release of anthrax spores through the U.S postal system confirmed that Bacillus anthracis can cause high morbidity and mortality, despite the use of powerful antibiotherapy and resuscitation techniques
Premixing 5 mg of IgG 2LF with 20 mg edema toxin (ET) resulted in the edema being 25% smaller than that caused by ET alone, and the duration of edema (60 h vs. 108 h) being 45% shorter
We show that scFv 2LF binds edema factor (EF) (20% identity with lethal factor (LF)), a component of the edema toxin (ET), constituted of EF and protective antigen (PA)

Summary

Introduction

In 2001, the intentional release of anthrax spores through the U.S postal system confirmed that Bacillus anthracis can cause high morbidity and mortality, despite the use of powerful antibiotherapy and resuscitation techniques. Antibiotic efficacy is limited by the existence of B. anthracis antibioresistance [8,9,10]. It was demonstrated in animal models of anthrax that the passive transfer of neutralizing antibodies directed against either PA or LF can improve the outcome of the disease [11]. Considerable efforts have been devoted, since 2001, to the development of recombinant antibodies to be used to complement antibiotic therapy (for a review, see [12,13]), and they resulted in the recent FDA approval of raxibacumab for the treatment of inhalational anthrax [14]. Another possible advantage of such antibodies is that they could potentially synergize with anti-PA antibodies [17,18,19,20]

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