Human VH1-69 Gene-Encoded Human Monoclonal Antibodies against Staphylococcus aureus IsdB Use at Least Three Distinct Modes of Binding To Inhibit Bacterial Growth and Pathogenesis

Abstract

Staphylococcus aureus is an important human pathogen that infects nearly every human tissue. Like most organisms, the acquisition of nutrient iron is necessary for its survival. One route by which it obtains this metal is through the iron-regulated surface determinant (Isd) system that scavenges iron from the hemoglobin of the host. We show that the heavy chain variable region IGHV1-69 gene commonly encodes human monoclonal antibodies (mAbs) targeting IsdB-NEAT2. Remarkably, these antibodies bind to multiple antigenic sites. One class of IGHV1-69-encoded mAbs blocks S. aureus heme acquisition by binding to the heme-binding site of NEAT2, while two additional classes reduce the bacterial burden in vivo by an alternative Fc receptor-mediated mechanism. We further identified clonal lineages of IGHV1-69-encoded mAbs using donor samples, showing that each lineage diversifies during infection by somatic hypermutation. These studies reveal that IGHV1-69-encoded antibodies contribute to a protective immune response, furthering our understanding of the correlates of protection against S. aureus infection.IMPORTANCE The human pathogen Staphylococcus aureus causes a wide range of infections, including skin abscesses and sepsis. There is currently no licensed vaccine to prevent S. aureus infection, and its treatment has become increasingly difficult due to antibiotic resistance. One potential way to inhibit S. aureus pathogenesis is to prevent iron acquisition. The iron-regulated surface determinant (Isd) system has evolved in S. aureus to acquire hemoglobin from the human host as a source of heme-iron. In this study, we investigated the molecular and structural basis for antibody-mediated correlates against a member of the Isd system, IsdB. The association of immunoglobulin heavy chain variable region IGHV1-69 gene-encoded human monoclonal antibodies with the response against S. aureus IsdB is described using structural and functional studies to define the importance of this antibody class. We also determine that somatic hypermutation in the development of these antibodies hinders rather than fine-tunes the immune response to IsdB.

Keywords: Staphylococcus aureus; X-ray crystallography; adaptive immunity; antibody functions; antibody repertoire; computer modeling; humoral immunity; monoclonal antibodies; proteomics.

FIG 1

Crystal structure of STAU-281 Fab in complex with the IsdB-NEAT2 domain. (a) The STAU-281 heavy chain and light chain and the IsdB NEAT2 domain are shown in magenta, cyan, and green, respectively. The heme-binding site of the IsdB-NEAT2 domain is marked with broken ovals. Side chains of residues I53 and F54 (Kabat numbering) from CDR-H2 are shown in salmon in a stick representation. (b) Surface representation (green) of the NEAT2 domain in the STAU-281/NEAT2 complex crystal structure. The STAU-281 CDR-H2 residue F54 interaction site of the NEAT2 domain is in salmon. Equivalent atoms of the heme-binding site in the heme-bound NEAT2 structure (PDB accession number 3RTL) are mapped onto the NEAT2 domain in the STAU-281/NEAT complex structure as blue mesh.

FIG 2

Overlay of crystal structures of STAU-281, D4-30 (PDB accession number 5D1X), and D2-06 (PDB accession number 5D1Q) Fabs in complex with the IsdB-NEAT2 domain. For the STAU-281/NEAT2 complex, the NEAT2 domain is in green. For the D4-30/NEAT2 complex, NEAT2 is in light blue. For the D2-06/NEAT2 complex, NEAT2 is in gray. (a) Residues I53 and F54 (Kabat numbering) of the conserved sequence motif of CDR-H2 encoded by the heavy chain gene IGHV1-69 interact with the heme-binding site of the NEAT2 domain, including residues M362, M363, Y391, V435, Y440, V433, and Y444, via π-π stacking and hydrophobic interactions. (b) Variations in interactions between CDR-H1/CDR-H3 and NEAT2 S3/S4 and S5/S6 loops shown in the three complex structures. In the D4-30/NEAT2 complex, a main-chain oxygen at position 31 of D4-30 CDR-H1 forms an H bond with the NEAT2 S3/S4 loop Y391 side chain, and its CDR-H3 has little interaction with NEAT2. In the D2-60/NEAT2 complex, Y391 forms two H bonds with the main-chain nitrogen and the side chain of T33 of D2-60 CDR-H1, and there is a salt bridge between S3/S4 loop D390 and R32 of D2-60 CDR-H1. CDR-H3 of D2-60 also interacts extensively with NEAT2 loops. In the STAU-281/NEAT2 complex, besides the H bond between side chain Y391 and the main-chain oxygen of R31 at STAU-281 CDR-H1, two additional H bonds are found between the NEAT2 residue N388 side chain and the R31 side chain and the Y32 side chain of STAU-281 CDR-H1. A salt bridge is formed between NEAT2 D390 and STAU-281 CDR-H3 K95 instead of a residue from CDR-H1. Due to its longer CDR-H3, STAU-281 has extensive van der Waals interactions with the NEAT2 S5/S6 loop. (c) Shape complementarity between the NEAT2 domain S7/S8 loop and CDR-L3 of the kappa light chain of the three antibodies. Aromatic residues at positions 94 and 96 of CDR-L3 interact with the S7/S8 loop via van der Waals interactions in addition to hydrogen bonds (shown as broken lines). STAU-281/NEAT2 H bonds are in green, D4-30/NEAT2 H bonds are in gray, and D2-60/NEAT2 H bonds are in blue. The side chain of K436 of NEAT2 forms H bonds with main-chain oxygen atoms of CDR-L3 position 91 in all three complexes.

FIG 3

Epitopes for two IGHV1-69-encoded antibodies on NEAT2. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to map the binding sites of STAU-229 (orange) or STAU-399 (red). (a) Peptides participating in the two epitopes were determined by reduced labeling in the presence of antibody and visualized on the surface of the IsdB-NEAT2 domain (PDB accession number 3RTL), shown in yellow ribbon and surface projection. The binding site of STAU-281 as determined by X-ray crystallography of a complex of STAU-281 Fab and recombinant NEAT2 is indicated in blue for reference. (b) Peptides in epitopes for STAU-229 (orange) or STAU-399 (red) in the amino acid sequence of IsdB and contact residues in the cocrystal structure for STAU-281/NEAT2 (blue).

FIG 4

Overlay model of the structures of three IGHV1-69-encoded antibodies reveals three modes of binding to NEAT2. The structures of the apo forms of Fabs STAU-229 (2.1 Å) and STAU-399 (2.4 Å) were determined by crystallography. These two Fabs were docked onto the STAU-281/NEAT2 complex structure using the Rosetta modeling suite, restricting docking to their binding sites (orange and red, respectively) predicted by HDX-MS studies of NEAT2 and the Fabs (Fig. 2). Heavy chains are shown as cyan ribbons, and light chains are shown in green.

FIG 5

In vitro blocking of binding (a to c) or inhibition of growth of S. aureus (d to f) mediated by IGHV1-69-encoded mAbs. (a to c) Biolayer interferometry was used to detect whether the mAbs blocked binding of hemoglobin to NEAT2. Biotinylated hemoglobin was loaded onto streptavidin-coated biosensor tips before association with either IsdB alone, IsdB mixed with mAb, mAb alone, or kinetic buffer. Error bars represent standard deviations. P values were determined by an unpaired t test. This experiment was performed at least 3 independent times. (d to f) S. aureus heme-dependent in vitro growth curves were performed in 96-well plates over 32 h. S. aureus strain Newman was subcultured at a 1:200 dilution into RPMI medium with ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid) and normalized to an OD₆₀₀ of 1.0. Error bars represent standard deviations. Data shown are representative of results from 3 independent experiments.

FIG 6

IGHV1-69-encoded mAbs reduce bacterial burden in vivo. Seven-week-old female BALB/c mice were inoculated retro-orbitally with a suspension of S. aureus strain Newman at an OD₆₀₀ of 0.4. Mice were given IGHV1-69-encoded full-length wild-type IgG LALA Fc variant (L234A/L235A) IgG antibodies by the intraperitoneal route. The hearts, livers, and kidneys of the infected mice were harvested after 96 h. Statistical significance was evaluated by analysis of variance (ANOVA) using multiple comparisons. All other comparisons, including comparison of LALA mAbs to the isotype control, were not significant. Experiments were performed two independent times, and the pooled data are shown. P values of <0.0001 were considered significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

FIG 7

Repertoire analysis of somatic variants of IGHV1-69-encoded mAbs. Deep sequence analysis of antibody variable genes expressed in donor PBMCs revealed somatic variants for the heavy chains of STAU-399 and -229. These somatic variant heavy chain antibodies were expressed recombinantly with the original light chain and purified. (a and b) Neighbor-joining tree analysis of mAb clonal variants by Geneious. The germ line gene sequences of IGHV1-69*01 or IGHV1-69-2*01 were used as the outgroup sequences. (c and d) Each variant mAb was tested for binding to IsdB, and the half-maximal effective concentration (EC₅₀) for binding was determined. Biolayer interferometry was used to determine the on and off rates of each variant antibody for binding to NEAT2, and the calculated K_D is graphed. These experiments were performed three times independently.