title: Context matters: The importance of dimerization-induced conformation of the LukGH leukocidin of Staphylococcus aureus for 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.
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Adriana
authors.initials: A
authors.lastname: Badarau
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Harald
authors.initials: H
authors.lastname: Rouha
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Malafa
authors.affiliation: b Adimab LLC , Lebanon , NH , USA.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Battles
authors.affiliation: b Adimab LLC , Lebanon , NH , USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Walker
authors.affiliation: b Adimab LLC , Lebanon , NH , USA.
authors.forename: Nels
authors.initials: N
authors.lastname: Nielson
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Ivana
authors.initials: I
authors.lastname: Dolezilkova
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Astrid
authors.initials: A
authors.lastname: Teubenbacher
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Srijib
authors.initials: S
authors.lastname: Banerjee
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Barbara
authors.initials: B
authors.lastname: Maierhofer
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Susanne
authors.initials: S
authors.lastname: Weber
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Lukas
authors.initials: L
authors.lastname: Stulik
authors.affiliation: c SARomics Biostructures AB , Medicon Village, Lund , Sweden.
authors.forename: Derek T
authors.initials: DT
authors.lastname: Logan
authors.affiliation: c SARomics Biostructures AB , Medicon Village, Lund , Sweden.
authors.forename: Martin
authors.initials: M
authors.lastname: Welin
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Irina
authors.initials: I
authors.lastname: Mirkina
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Clara
authors.initials: C
authors.lastname: Pleban
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Gerhild
authors.initials: G
authors.lastname: Zauner
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Karin
authors.initials: K
authors.lastname: Gross
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Michaela
authors.initials: M
authors.lastname: Jägerhofer
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Magyarics
authors.affiliation: a Arsanis Biosciences , Campus Vienna Biocenter, Vienna , Austria.
authors.forename: Eszter
authors.initials: E
authors.lastname: Nagy
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D057134:Antibodies, Neutralizing / Q000276:immunology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology*; D019281:Dimerization; D006801:Humans; D007956:Leukocidins / Q000737:chemistry / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D001426:Bacterial Proteins; D007956:Leukocidins; C586430:leukocidin AB, Staphylococcus aureus
keywords: Conformational epitope; LukGH; Staphylococcus aureus; X-ray crystal structure; toxin neutralization