Structural investigation of humanS. aureus-targeting antibodies that bind wall teichoic acid

Abstract

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a growing health threat worldwide. Efforts to identify novel antibodies that target S. aureus cell surface antigens are a promising direction in the development of antibiotics that can halt MRSA infection. We biochemically and structurally characterized three patient-derived MRSA-targeting antibodies that bind to wall teichoic acid (WTA), which is a polyanionic surface glycopolymer. In S. aureus, WTA exists in both α- and β-forms, based on the stereochemistry of attachment of a N-acetylglucosamine residue to the repeating phosphoribitol sugar unit. We identified a panel of antibodies cloned from human patients that specifically recognize the α or β form of WTA, and can bind with high affinity to pathogenic wild-type strains of S. aureus bacteria. To investigate how the β-WTA specific antibodies interact with their target epitope, we determined the X-ray crystal structures of the three β-WTA specific antibodies, 4462, 4497, and 6078 (Protein Data Bank IDs 6DWI, 6DWA, and 6DW2, respectively), bound to a synthetic WTA epitope. These structures reveal that all three of these antibodies, while utilizing distinct antibody complementarity-determining region sequences and conformations to interact with β-WTA, fulfill two recognition principles: binding to the β-GlcNAc pyranose core and triangulation of WTA phosphate residues with polar contacts. These studies reveal the molecular basis for targeting a unique S. aureus cell surface epitope and highlight the power of human patient-based antibody discovery techniques for finding novel pathogen-targeting therapeutics.

The Orphan Immune Receptor LILRB3 Modulates Fc Receptor–Mediated Functions of Neutrophils

“…gBlocks containing variable H chain (VH) and variable L chain (VL) sequences were cloned into pcDNA3.4-HCIg-hG1 and pcDNA3.4-LCIg-hk, as well as upstream Kozak sequence and HAVT20 signal peptide, using NheI and BsiWI as the 39 cloning sites for VH and VL, respectively, to preserve the Ig H and k L chain amino acid sequence. VH and VL sequences were derived from previously described Abs directed against staphylococcal WTA GlcNAC-b (anti-WTA clone 4497; based on WO2014/193722) and against HSA (anti-HSA clone CA465) (24)(25)(26). Abs were expressed as IgG1/k in EXPI293F cells (Life Technologies), essentially as described before (27), and purified by affinity chromatography (Ä KTA pure; GE Healthcare Life Sciences) using a protein A column (GE Healthcare Life Sciences).…”

“…As appropriate levels of specific IgA can induce opsonization, phagocytosis, and killing of bacteria (37,38), we assessed whether LILRB3 ligation can modulate IgA-mediated phagocytosis of live bacteria by neutrophils. Anti-WTA-4497 recognizes b-O-GlcNAc modifications on the poly-RboP WTA backbone of S. aureus (24,25,32). Because S. aureus has evolved multiple mechanisms to evade neutrophil phagocytosis and killing (39), we used a S. capitis strain manipulated to express poly-RboP WTA with b-O-GlcNAc (32).…”

Staphylococcal protein A inhibits complement activation by interfering with IgG hexamer formation

“…NewmanΔspa/sbi was labeled with a human monoclonal antibody directed against wall teichoic acid (WTA) (Lehar et al, 2015), a highly abundant anionic glycopolymer that is covalently anchored to the peptidoglycan layer (Brown et al, 2013). Although the anti-WTA antibody (clone 4497) belongs to VH3-type family (Fong et al, 2018), it lacks binding properties via its Fab region to SpA ( Fig EV2C).…”

C1q binding to surface-bound IgG is stabilized by C1r2s2proteases

“…To enhance our understanding of complement activation by antibacterial IgGs, we studied complement activation by monoclonal antibodies against Staphylococcus aureus, an important Gram-positive pathogen and the leading cause of hospital-acquired infections. We generated IgGs against wall teichoic acid (WTA), a highly abundant and immunogenic cell wall glycopolymer that comprises 40% of the staphylococcal cell wall [29][30][31] . The variable domains of anti-WTA IgG1 4497 31 were cloned into HEK expression vectors encoding IgG1, IgG2, IgG3 and IgG4 Fc backbones.…”

“…We generated IgGs against wall teichoic acid (WTA), a highly abundant and immunogenic cell wall glycopolymer that comprises 40% of the staphylococcal cell wall [29][30][31] . The variable domains of anti-WTA IgG1 4497 31 were cloned into HEK expression vectors encoding IgG1, IgG2, IgG3 and IgG4 Fc backbones. We included all IgG subclasses to obtain a better understanding of their variable complement effector functions 1,32 .…”