Alanine Scanning Mutagenesis of the MEDI4893 (Suvratoxumab) Epitope Reduces Alpha Toxin Lytic Activity In Vitro and Staphylococcus aureus Fitness in Infection Models.

C Tkaczyk,P Warrener,Y Y Shi,E Semenova,C K Stover,B R Sellman,K Rosenthal,V Oganesyan

doi:10.1128/aac.01033-18

Abstract

Alpha toxin (AT) is a cytolytic pore-forming toxin that plays a key role in Staphylococcus aureus pathogenesis; consequently, extensive research was undertaken to understand the AT mechanism of action and its utility as a target for novel prophylaxis and treatment strategies against S. aureus infections. MEDI4893 (suvratoxumab) is a human anti-AT IgG1 monoclonal antibody (MAb) that targets AT and is currently in phase 2 clinical development. As shown previously, the MEDI4893-binding epitope on AT is comprised of the highly conserved amino acid regions 177 to 200 and 261 to 271, suggesting these amino acids are important for AT function. To test this hypothesis and gain insight into the effect of mutations in the epitope on AT neutralization by MEDI4893, nine MEDI4893 contact residues in AT were individually mutated to alanine. Consistent with our hypothesis, 8 out of 9 mutants exhibited >2-fold loss in lytic activity resulting from a defect in cell binding and pore formation. MEDI4893 binding affinity was reduced >2-fold (2- to 27-fold) for 7 out of 9 mutants, and no binding was detected for the W187A mutant. MEDI4893 effectively neutralized all of the lytic mutants in vitro and in vivo When the defective mutants were introduced into an S. aureus clinical isolate, the mutant-expressing strains exhibited less severe disease in mouse models and were effectively neutralized by MEDI4893. These results indicate the MEDI4893 epitope is highly conserved due in part to its role in AT pore formation and bacterial fitness, thereby decreasing the likelihood for the emergence of MAb-resistant variants.

Highlights

Alpha toxin (AT) is a cytolytic pore-forming toxin that plays a key role in Staphylococcus aureus pathogenesis; extensive research was undertaken to understand the AT mechanism of action and its utility as a target for novel prophylaxis and treatment strategies against S. aureus infections
The results from this study indicate the amino acids in the MEDI4893 epitope are important for toxin function and bacterial virulence and that the monoclonal antibody (MAb) is capable of neutralizing toxin molecules with mutations in its binding epitope
These results indicate that amino acids in the MEDI4893 epitope are essential for AT function and that the MAb neutralizes epitope variants in vitro

Summary

Introduction

Alpha toxin (AT) is a cytolytic pore-forming toxin that plays a key role in Staphylococcus aureus pathogenesis; extensive research was undertaken to understand the AT mechanism of action and its utility as a target for novel prophylaxis and treatment strategies against S. aureus infections. The MEDI4893binding epitope on AT is comprised of the highly conserved amino acid regions 177 to 200 and 261 to 271, suggesting these amino acids are important for AT function To test this hypothesis and gain insight into the effect of mutations in the epitope on AT neutralization by MEDI4893, nine MEDI4893 contact residues in AT were individually mutated to alanine. When the defective mutants were introduced into an S. aureus clinical isolate, the mutant-expressing strains exhibited less severe disease in mouse models and were effectively neutralized by MEDI4893. These results indicate the MEDI4893 epitope is highly conserved due in part to its role in AT pore formation and bacterial fitness, thereby decreasing the likelihood for the emergence of MAb-resistant variants. Our observations imply a low potential for emergence of AT variants resistant to neutralization by MEDI4893

Methods

Results

Conclusion