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.

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.

Multistate design of influenza antibodies improves affinity and breadth against seasonal viruses.

Influenza is a yearly threat to global public health. Rapid changes in influenza surface proteins resulting from antigenic drift and shift events make it difficult to readily identify antibodies with broadly neutralizing activity against different influenza subtypes with high frequency, specifically antibodies targeting the receptor binding domain (RBD) on influenza HA protein. We developed an optimized computational design method that is able to optimize an antibody for recognition of large panels of antigens. To demonstrate the utility of this multistate design method, we used it to redesign an antiinfluenza antibody against a large panel of more than 500 seasonal HA antigens of the H1 subtype. As a proof of concept, we tested this method on a variety of known antiinfluenza antibodies and identified those that could be improved computationally. We generated redesigned variants of antibody C05 to the HA RBD and experimentally characterized variants that exhibited improved breadth and affinity against our panel. C05 mutants exhibited improved affinity for three of the subtypes used in design by stabilizing the CDRH3 loop and creating favorable electrostatic interactions with the antigen. These mutants possess increased breadth and affinity of binding while maintaining high-affinity binding to existing targets, surpassing a major limitation up to this point.

Human mAbs to Staphylococcus aureus IsdA Provide Protection Through Both Heme-Blocking and Fc-Mediated Mechanisms.

The nutrient metal iron plays a key role in the survival of microorganisms. The iron-regulated surface determinant (Isd) system scavenges heme-iron from the human host, enabling acquisition of iron in iron-deplete conditions in Staphylococcus aureus during infection. The cell surface receptors IsdB and IsdH bind hemoproteins and transfer heme to IsdA, the final surface protein before heme-iron is transported through the peptidoglycan. To define the human B-cell response to IsdA, we isolated human monoclonal antibodies (mAbs) specific to the surface Isd proteins and determined their mechanism of action. We describe the first isolation of fully human IsdA and IsdH mAbs, as well as cross-reactive Isd mAbs. Two of the identified IsdA mAbs worked in a murine septic model of infection to reduce bacterial burden during staphylococcal infection. Their protection was a result of both heme-blocking and Fc-mediated effector functions, underscoring the importance of targeting S. aureus using diverse mechanisms.

Prolyl hydroxylation in elastin is not random.

BACKGROUND: This study aimed to investigate the prolyl and lysine hydroxylation in elastin from different species and tissues. METHODS: Enzymatic digests of elastin samples from human, cattle, pig and chicken were analyzed using mass spectrometry and bioinformatics tools. RESULTS: It was confirmed at the protein level that elastin does not contain hydroxylated lysine residues regardless of the species. In contrast, prolyl hydroxylation sites were identified in all elastin samples. Moreover, the analysis of the residues adjacent to prolines allowed the determination of the substrate site preferences of prolyl 4-hydroxylase. It was found that elastins from all analyzed species contain hydroxyproline and that at least 20%-24% of all proline residues were partially hydroxylated. Determination of the hydroxylation degrees of specific proline residues revealed that prolyl hydroxylation depends on both the species and the tissue, however, is independent of age. The fact that the highest hydroxylation degrees of proline residues were found for elastin from the intervertebral disc and knowledge of elastin arrangement in this tissue suggest that hydroxylation plays a biomechanical role. Interestingly, a proline-rich domain of tropoelastin (domain 24), which contains several repeats of bioactive motifs, does not show any hydroxyproline residues in the mammals studied. CONCLUSIONS: The results show that prolyl hydroxylation is not a coincidental feature and may contribute to the adaptation of the properties of elastin to meet the functional requirements of different tissues. GENERAL SIGNIFICANCE: The study for the first time shows that prolyl hydroxylation is highly regulated in elastin.

Rearrangement of the Extracellular Domain/Extracellular Loop 1 Interface Is Critical for Thyrotropin Receptor Activation.

The thyroid stimulating hormone receptor (TSHR) is a G protein-coupled receptor (GPCR) with a characteristic large extracellular domain (ECD). TSHR activation is initiated by binding of the hormone ligand TSH to the ECD. How the extracellular binding event triggers the conformational changes in the transmembrane domain (TMD) necessary for intracellular G protein activation is poorly understood. To gain insight in this process, the knowledge on the relative positioning of ECD and TMD and the conformation of the linker region at the interface of ECD and TMD are of particular importance. To generate a structural model for the TSHR we applied an integrated structural biology approach combining computational techniques with experimental data. Chemical cross-linking followed by mass spectrometry yielded 17 unique distance restraints within the ECD of the TSHR, its ligand TSH, and the hormone-receptor complex. These structural restraints generally confirm the expected binding mode of TSH to the ECD as well as the general fold of the domains and were used to guide homology modeling of the ECD. Functional characterization of TSHR mutants confirms the previously suggested close proximity of Ser-281 and Ile-486 within the TSHR. Rigidifying this contact permanently with a disulfide bridge disrupts ligand-induced receptor activation and indicates that rearrangement of the ECD/extracellular loop 1 (ECL1) interface is a critical step in receptor activation. The experimentally verified contact of Ser-281 (ECD) and Ile-486 (TMD) was subsequently utilized in docking homology models of the ECD and the TMD to create a full-length model of a glycoprotein hormone receptor.