Photoimmuno-antimicrobial therapy for Staphylococcus aureus implant infection.

INTRODUCTION: Implant infections caused by Staphylococcus aureus are responsible for high mortality and morbidity worldwide. Treatment of these infections can be difficult especially when bacterial biofilms are involved. In this study we investigate the potential of infrared photoimmunotherapy to eradicate staphylococcal infection in a mouse model. METHODS: A monoclonal antibody that targets Wall Teichoic Acid surface components of both S. aureus and its biofilm (4497-IgG1) was conjugated to a photosensitizer (IRDye700DX) and used as photoimmunotherapy in vitro and in vivo in mice with a subcutaneous implant pre-colonized with biofilm of Staphylococcus aureus. A dose of 400 µg and 200 µg of antibody-photosensitizer conjugate 4497-IgG-IRDye700DXwas administered intravenously to two groups of 5 mice. In addition, multiple control groups (vancomycin treated, unconjugated IRDye700DX and IRDye700DX conjugated to a non-specific antibody) were used to verify anti-microbial effects. RESULTS: In vitro results of 4497-IgG-IRDye700DX on pre-colonized (biofilm) implants showed significant (p<0.01) colony-forming units (CFU) reduction at a concentration of 5 µg of the antibody-photosensitizer conjugate. In vivo, treatment with 4497-IgG-IRDye700DX showed no significant CFU reduction at the implant infection. However, tissue around the implant did show a significant CFU reduction with 400 µg 4497-IgG-IRDye700DX compared to control groups (p = 0.037). CONCLUSION: This study demonstrated the antimicrobial potential of photoimmunotherapy for selectively eliminating S. aureus in vivo. However, using a solid implant instead of a catheter could result in an increased bactericidal effect of 4497-IgG-IRDye700DX and administration locally around an implant (per operative) could become valuable applications in patients that are difficult to treat with conventional methods. We conclude that photoimmunotherapy could be a potential additional therapy in the treatment of implant related infections, but requires further improvement.

Human anti-C1q autoantibodies bind specifically to solid-phase C1q and enhance phagocytosis but not complement activation.

Autoantibodies directed against complement component C1q are commonly associated with autoimmune diseases, especially systemic lupus erythematosus. Importantly, these anti-C1q autoantibodies are specific for ligand-bound, solid-phase C1q and do not bind to fluid-phase C1q. In patients with anti-C1q, C1q levels are in the normal range, and the autoantibodies are thus not depleting. To study these human anti-C1q autoantibodies at the molecular level, we isolated C1q-reactive B cells and recombinantly produced nine monoclonal antibodies (mAbs) from four different healthy individuals. The isolated mAbs were of the IgG isotype, contained extensively mutated variable domains, and showed high affinity to the collagen-like region of C1q. The anti-C1q mAbs exclusively bound solid-phase C1q in complex with its natural ligands, including immobilized or antigen-bound IgG, IgM or CRP, and necrotic cells. Competition experiments reveal that at least 2 epitopes, also targeted by anti-C1q antibodies in sera from SLE patients, are recognized. Electron microscopy with hexameric IgG-C1q immune complexes demonstrated that multiple mAbs can interact with a single C1q molecule and identified the region of C1q targeted by these mAbs. The opsonization of immune complexes with anti-C1q greatly enhanced Fc-receptor-mediated phagocytosis but did not increase complement activation. We conclude that human anti-C1q autoantibodies specifically bind neo-epitopes on solid-phase C1q, which results in an increase in Fc-receptor-mediated effector functions that may potentially contribute to autoimmune disease immunopathology.

Antibody-Drug Conjugates to Treat Bacterial Biofilms via Targeting and Extracellular Drug Release.

The treatment of implant-associated bacterial infections and biofilms is an urgent medical need and a grand challenge because biofilms protect bacteria from the immune system and harbor antibiotic-tolerant persister cells. This need is addressed herein through an engineering of antibody-drug conjugates (ADCs) that contain an anti-neoplastic drug mitomycin C, which is also a potent antimicrobial against biofilms. The ADCs designed herein release the conjugated drug without cell entry, via a novel mechanism of drug release which likely involves an interaction of ADC with the thiols on the bacterial cell surface. ADCs targeted toward bacteria are superior by the afforded antimicrobial effects compared to the non-specific counterpart, in suspension and within biofilms, in vitro, and in an implant-associated murine osteomyelitis model in vivo. The results are important in developing ADC for a new area of application with a significant translational potential, and in addressing an urgent medical need of designing a treatment of bacterial biofilms.

Evaluating the Targeting of a Staphylococcus-aureus-Infected Implant with a Radiolabeled Antibody In Vivo.

Implant infections caused by Staphylococcus aureus are difficult to treat due to biofilm formation, which complicates surgical and antibiotic treatment. We introduce an alternative approach using monoclonal antibodies (mAbs) targeting S. aureus and provide evidence of the specificity and biodistribution of S.-aureus-targeting antibodies in a mouse implant infection model. The monoclonal antibody 4497-IgG1 targeting wall teichoic acid in S. aureus was labeled with indium-111 using CHX-A"-DTPA as a chelator. Single Photon Emission Computed Tomography/computed tomographyscans were performed at 24, 72 and 120 h after administration of the 111In-4497 mAb in Balb/cAnNCrl mice with a subcutaneous implant that was pre-colonized with S. aureus biofilm. The biodistribution of this labelled antibody over various organs was visualized and quantified using SPECT/CT imaging, and was compared to the uptake at the target tissue with the implanted infection. Uptake of the 111In-4497 mAbs at the infected implant gradually increased from 8.34 %ID/cm3 at 24 h to 9.22 %ID/cm3 at 120 h. Uptake at the heart/blood pool decreased over time from 11.60 to 7.58 %ID/cm3, whereas the uptake in the other organs decreased from 7.26 to less than 4.66 %ID/cm3 at 120 h. The effective half-life of 111In-4497 mAbs was determined to be 59 h. In conclusion, 111In-4497 mAbs were found to specifically detect S. aureus and its biofilm with excellent and prolonged accumulation at the site of the colonized implant. Therefore, it has the potential to serve as a drug delivery system for the diagnostic and bactericidal treatment of biofilm.

Monoclonal antibodies effectively potentiate complement activation and phagocytosis of Staphylococcus epidermidis in neonatal human plasma.

Central line associated bloodstream infections (CLABSI) with Staphylococcus epidermidis are a major cause of morbidity in neonates, who have an increased risk of infection because of their immature immune system. As especially preterm neonates suffer from antibody deficiency, clinical studies into preventive therapies have thus far focused on antibody supplementation with pooled intravenous immunoglobulins from healthy donors (IVIG) but with little success. Here we study the potential of monoclonal antibodies (mAbs) against S. epidermidis to induce phagocytic killing by human neutrophils. Nine different mAbs recognizing Staphylococcal surface components were cloned and expressed as human IgG1s. In binding assays, clones rF1, CR5133 and CR6453 showed the strongest binding to S. epidermidis ATCC14990 and CR5133 and CR6453 bound the majority of clinical isolates from neonatal sepsis (19 out of 20). To study the immune-activating potential of rF1, CR5133 and CR6453, bacteria were opsonized with mAbs in the presence or absence of complement. We observed that activation of the complement system is essential to induce efficient phagocytosis of S. epidermidis. Complement activation and phagocytic killing could be enhanced by Fc-mutations that improve IgG1 hexamerization on cellular surfaces. Finally, we studied the ability of the mAbs to activate complement in r-Hirudin neonatal plasma conditions. We show that classical pathway complement activity in plasma isolated from neonatal cord blood is comparable to adult levels. Furthermore, mAbs could greatly enhance phagocytosis of S. epidermidis in neonatal plasma. Altogether, our findings provide insights that are crucial for optimizing anti-S. epidermidis mAbs as prophylactic agents for neonatal CLABSI.

Human monoclonal antibodies against Staphylococcus aureus surface antigens recognize in vitro and in vivo biofilm.

Implant-associated Staphylococcus aureus infections are difficult to treat because of biofilm formation. Bacteria in a biofilm are often insensitive to antibiotics and host immunity. Monoclonal antibodies (mAbs) could provide an alternative approach to improve the diagnosis and potential treatment of biofilm-related infections. Here, we show that mAbs targeting common surface components of S. aureus can recognize clinically relevant biofilm types. The mAbs were also shown to bind a collection of clinical isolates derived from different biofilm-associated infections (endocarditis, prosthetic joint, catheter). We identify two groups of antibodies: one group that uniquely binds S. aureus in biofilm state and one that recognizes S. aureus in both biofilm and planktonic state. Furthermore, we show that a mAb recognizing wall teichoic acid (clone 4497) specifically localizes to a subcutaneously implanted pre-colonized catheter in mice. In conclusion, we demonstrate the capacity of several human mAbs to detect S. aureus biofilms in vitro and in vivo.

Staphylococci evade the innate immune response by disarming neutrophils and forming biofilms.

Staphylococcus aureus and Staphylococcus epidermidis can cause many types of infections, ranging from skin infections to implant-associated infections. The primary innate immune response against bacterial infections involves complement activation, recruitment of phagocytes (most importantly neutrophils), and subsequent killing of the pathogen. However, staphylococci are not innocent bystanders; they actively obstruct this immune attack. To do that, S. aureus secretes several immune-evasion proteins to resist attack by the innate immune system. Furthermore, S. aureus and S. epidermidis are known for their ability to form biofilms on implanted medical devices and host tissues, which provides another important immune-evasion mechanism. Understanding these different strategies to resist immune attack will help to develop novel therapies against staphylococcal infections.

Neutrophil Recruitment to Noninvasive MRSA at the Stratum Corneum of Human Skin Mediates Transient Colonization.

Staphylococcus aureus is a leading cause of skin and soft issue infection, but paradoxically, it also transiently, and often harmlessly, colonizes human skin. An obstacle to understanding this contradiction has been a shortage of in vivo models reproducing the unique structure and immunology of human skin. In this work, we developed a humanized model to study how healthy adult human skin responds to colonizing methicillin-resistant S. aureus (MRSA). We demonstrate the importance of the outer stratum corneum as the major site of bacterial colonization and how noninvasive MRSA adhesion to corneocytes induces a local inflammatory response in underlying skin layers. This signaling recruits neutrophils to the skin, where they control bacterial numbers, mediating transiency in colonization. This work highlights the spatiotemporal aspects of human skin colonization and demonstrates a subclinical inflammatory response to noninvasive MRSA that allows human skin to regulate the bacterial population at its outer surface.