Date Published: September 30, 2016
Publisher: Public Library of Science
Author(s): Alexey V. Gribenko, Kevin Parris, Lidia Mosyak, Sheng Li, Luke Handke, Julio C. Hawkins, Elena Severina, Yury V. Matsuka, Annaliesa S. Anderson, Gongyi Zhang.
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.
Staphylococcus aureus protein MntC is the ligand-binding component of the ABC-type manganese transporter MntABC, which is at least partially responsible for the organism’s resistance to the oxidative stress [1,2]. The protein is expressed during early stages of infection  and binds manganese with high affinity . Active vaccination with recombinant S. aureus MntC reduced bacterial burden in a murine bacteremia model of infection , making MntC a promising vaccine candidate. As such, MntC is a key component of an experimental four-antigen (SA4Ag) S. aureus vaccine which is currently undergoing clinical trials (US government registry, trials #NCT01364571, #NCT01643941 and #NCT02364596 –completed, trials #NCT02388165 and #NCT02492958 –ongoing). We have recently solved the 3-dimensional structure of MntC by X-ray crystallography (PDB ID 4K3V, Fig 1) and provided detailed biophysical characterization of the protein . Furthermore, in an earlier work from our laboratories  we reported generation of 23 monoclonal antibodies against MntC, with some of them being protective in an infant rat model of infection and being capable of inducing respiratory burst activity of neutrophils. Based on BIAcore binding interference patterns, these antibodies were subdivided into three interference groups . In the current report we identified binding epitopes for selected representatives of each of those groups.
In the current work, we mapped binding epitopes of the representative monoclonal antibodies from different interference groups reported earlier . As expected for the protective antibodies [10,11], all three epitopes were conformational. The DXMS data clearly show that the mAB 305-72-5 binding epitope (interference group 1) involves two discontinuous segments. In the case of the other two antibodies one could argue (based strictly on DXMS results) that the epitopes could be linear: Our DXMS data did not provide sufficient resolution in those two cases. The X-ray structure of MntC in complex with mAB 305-78-7 (interference group 2), however, shows that there are several other isolated residues outside of the linear peptide 243–260 that are involved in the interaction, confirming that mAB 305-78-7 epitope is discontinuous, as well. To provide additional evidence for the conformational nature of the epitopes recognized by the antibodies mAB 305-78-7 and mAB 305-101-8 (interference group 3), we conducted ITC titrations of the antibodies under study with synthetic linear peptides derived from the amino acid sequences of the structural segments identified using DXMS. No binding could be detected in these experiments (S6 Fig). These results confirm that the unique spatial arrangement of the residues dictated by the 3-dimensional folding of the identified peptide sequences is important for the antibody binding.