Context matters: The importance of dimerization-induced conformation of the LukGH leukocidin ofStaphylococcus aureusfor the generation of neutralizing antibodies

Abstract

LukGH (LukAB) is a potent leukocidin of Staphylococcus aureus that lyses human phagocytic cells and is thought to contribute to immune evasion. Unlike the other bi-component leukocidins of S. aureus, LukGH forms a heterodimer before binding to its receptor, CD11b expressed on professional phagocytic cells, and displays significant sequence variation. We employed a high diversity human IgG1 library presented on yeast cells to discover monoclonal antibodies (mAbs) neutralizing the cytolytic activity of LukGH. Recombinant LukG and LukH monomers or a LukGH dimer were used as capture antigens in the library selections. We found that mAbs identified with LukG or LukH as bait had no or very low toxin neutralization potency. In contrast, LukGH dimer-selected antibodies proved to be highly potent, and several mAbs were able to neutralize even the most divergent LukGH variants. Based on biolayer interferometry and mesoscale discovery, the high affinity antibody binding site on the LukGH complex was absent on the individual monomers, suggesting that it was generated upon formation of the LukG-LukH dimer. X-ray crystallography analysis of the complex between the LukGH dimer and the antigen-binding fragment of a very potent mAb (PDB code 5K59) indicated that the epitope is located in the predicted cell binding region (rim domain) of LukGH. The corresponding IgG inhibited the binding of LukGH dimer to target cells. Our data suggest that knowledge of the native conformation of target molecules is essential to generate high affinity and functional mAbs.

Leukocidins: staphylococcal bi-component pore-forming toxins find their receptors

“…Similarly, LukAB was recently found to form a hetero-octomer pore 21 . Based on the crystal structure of the LukAB-heterodimer, the interaction sites between LukA and LukB help to explain why this toxin is pre-assembled in solution 21,22 . Three salt bridges between the interfaces of the cap and rim domains of LukA and LukB, that are not found in the other leukocidins, are required for the formation of LukAB-dimers in solution.…”

“…Multiple monoclonal antibodies (mAbs) that neutralize one or more toxins are currently being evaluated in clinical and preclinical trials 22,100,101 , and could be used to treat severe infections during the active phase or for the prevention of infections in high-risk groups (Figure 3). An engineered bi-specific tetravalent mAb was shown to neutralize PVL and HlgCB in vitro and in an inflammation model in rabbits 102 .…”

Staphylococcus aureus pore-forming toxins: The interface of pathogen and host complexity

“…In addition, a neutralizing antibody that can target Hla in addition to LukED, HlgAB, HlgCB, and PVL has recently been identified. The investigators were unable to use the same antibody to also target LukAB, due to the fact that it exists as dimers in solution rather than monomers [206, 208]. Prophylaxis with this monoclonal antibody was more effective than an antibody targeting only Hla in rabbit and mouse models of pneumonia, reducing the overall bacterial burden [205, 206].…”

Staphylococcus aureustoxin antibodies: Good companions of antibiotics and vaccines

“…Such strategy is being pursued with MEDI4893 and ASN100, which are currently being evaluated for the prevention of nosocomial pneumonia in high-risk ventilated patients (Table 1; 8,10,21 ). An innovative option of combining the antibody and antibiotic activity is the chemical conjugation of a mAb against a S. aureus surface antigen (wall teichoic acid) to an antibiotic.…”

“…Medimmune and Aridis Pharmaceuticals independently developed a human monoclonal antibody (mAb) specific for the a-hemolysin (Hla), named respectively MEDI4893 8 and AR-301. Arsanis developed a combination of 2 mAbs (ASN100; see reference: 9,10 ). The latter product contains a Hla mAb (ASN-1) which has cross-reactive properties against 4 other toxins, the leukocidins HlgAB, HlgCB, LukED & LukSF and another mAb (ASN-2) against the leukocidin LukAB.…”