High Resolution Mapping of Bactericidal Monoclonal Antibody Binding Epitopes on Staphylococcus aureus Antigen MntC

Abstract

The Staphylococcus aureus manganese transporter protein MntC is under investigation as a component of a prophylactic S.aureus vaccine. Passive immunization with monoclonal antibodies mAB 305-78-7 and mAB 305-101-8 produced using MntC was shown to significantly reduce S. aureus burden in an infant rat model of infection. Earlier interference mapping suggested that a total of 23 monoclonal antibodies generated against MntC could be subdivided into three interference groups, representing three independent immunogenic regions. In the current work binding epitopes for selected representatives of each of these interference groups (mAB 305-72-5 – group 1, mAB 305-78-7 – group 2, and mAB 305-101-8 – group 3) were mapped using Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS). All of the identified epitopes are discontinuous, with binding surface formed by structural elements that are separated within the primary sequence of the protein but adjacent in the context of the three-dimensional structure. The approach was validated by co-crystallizing the Fab fragment of one of the antibodies (mAB 305-78-7) with MntC and solving the three-dimensional structure of the complex. X-ray results themselves and localization of the mAB 305-78-7 epitope were further validated using antibody binding experiments with MntC variants containing substitutions of key amino acid residues. These results provided insight into the antigenic properties of MntC and how these properties may play a role in protecting the hostagainst S. aureus infection by preventing the capture and transport of Mn2+, a key element that the pathogen uses to evade host immunity.

Selective substrate uptake: The role of ATP-binding cassette (ABC) importers in pathogenesis

“…The MntC monoclonal antibodies interact with each of the globular lobes of MntC, including the binding pocket, potentially blocking the SBP-TMD interface and manganese binding (Fig. 3) [134]. In addition, a S. aureus vaccine that incorporates multiple antigens that target different virulence mechanisms, including MntC, has been successful in clinical trials [135].…”

High-Definition Mapping of Four Spatially Distinct Neutralizing Epitope Clusters on RiVax, a Candidate Ricin Toxin Subunit Vaccine

“…RiVax's regions of intrinsic flexibility and rigidity. HX-MS is increasingly being used for the purpose of B cell epitope identification (47,49,50,52,53). The general principles of HX-MS and how the technique relates to other epitope mapping strategies have been covered in several recent reviews (45,54).…”

“…Hydrogen exchange-mass spectrometry (HX-MS) was the method of choice based on recent success in a wide variety of pathogens, including HIV, the malaria parasite, Neisseria meningitidis, and Staphylococcus aureus (45)(46)(47)(48)(49)(50). In a recent study, we successfully adapted HX-MS for the purpose of identifying epitopes recognized by a family of RTA-specific single-domain alpaca-derived antibodies (V H Hs) (51).…”

The biogenesis of extracellular vesicles fromStaphylococcus aureusand their application as a novel vaccine platform

“…Protective efficacy against additional S. aureus strains and in additional infection models remains to be evaluated. Over-expression of additional antigens that have been shown to protect mice against experimental S. aureus infections, such as MntC and FhuD2 49-51,81 , in second-generation eng-EVs may yield a more efficacious vaccine. LC-MS/MS analysis of EVs from both WT JE2 and the Δ agr Δ spa mutant strain contained multiple lipoproteins.…”