RESUMO
Single monoclonal antibodies (mAbs) can be expressed in vivo through gene delivery of their mRNA formulated with lipid nanoparticles (LNPs). However, delivery of a mAb combination could be challenging due to the risk of heavy and light variable chain mispairing. We evaluated the pharmacokinetics of a three mAb combination against Staphylococcus aureus first in single chain variable fragment scFv-Fc and then in immunoglobulin G 1 (IgG1) format in mice. Intravenous delivery of each mRNA/LNP or the trio (1 mg/kg each) induced functional antibody expression after 24 h (10-100 µg/mL) with 64%-78% cognate-chain paired IgG expression after 3 days, and an absence of non-cognate chain pairing for scFv-Fc. We did not observe reduced neutralizing activity for each mAb compared with the level of expression of chain-paired mAbs. Delivery of the trio mRNA protected mice in an S. aureus-induced dermonecrosis model. Intravenous administration of the three mRNA in non-human primates achieved peak serum IgG levels ranging between 2.9 and 13.7 µg/mL with a half-life of 11.8-15.4 days. These results suggest nucleic acid delivery of mAb combinations holds promise and may be a viable option to streamline the development of therapeutic antibodies.
Assuntos
Anticorpos Monoclonais , Imunoglobulina G , RNA Mensageiro , Infecções Estafilocócicas , Staphylococcus aureus , Animais , Camundongos , Staphylococcus aureus/imunologia , RNA Mensageiro/genética , Infecções Estafilocócicas/prevenção & controle , Imunoglobulina G/imunologia , Nanopartículas/química , Modelos Animais de Doenças , Feminino , Anticorpos de Cadeia Única/genética , Humanos , LipossomosRESUMO
In a rabbit model of methicillin-resistant Staphylococcus aureus prosthetic joint infection (PJI), prophylaxis with AZD6389*-a combination of three monoclonal antibodies targeting alpha-hemolysin, bicomponent cytotoxins (LukSF/LukED/HlgAB/HlgCB), and clumping factor A-resulted in significant reductions in joint swelling, erythema, intra-articular pus, and bacterial burden in synovial tissues and biofilm-associated prosthetic implants compared with isotype-matched control IgG. Targeting specific staphylococcal virulence factors may thus have potential clinical utility for prevention of PJI.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Animais , Anticorpos Monoclonais , Próteses e Implantes , Coelhos , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus , Virulência , Fatores de VirulênciaRESUMO
Staphylococcus aureus is a major human pathogen that causes a wide range of infections by producing an arsenal of cytotoxins. We found that passive immunization with either a monoclonal antibody (MAb) neutralizing alpha-hemolysin or a broadly cross-reactive MAb neutralizing Panton-Valentine leukocidin, leukocidin ED, and gamma-hemolysins HlgAB and HlgCB conferred only partial protection, whereas the combination of those two MAbs conferred significant protection in a rabbit model of necrotizing pneumonia caused by the USA300 methicillin-resistant S. aureus epidemic clone.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Proteínas Hemolisinas/imunologia , Leucocidinas/uso terapêutico , Pneumonia Necrosante/tratamento farmacológico , Pneumonia Necrosante/imunologia , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/microbiologia , Animais , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/microbiologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Coelhos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidadeRESUMO
Infection is a major complication of implantable medical devices, which provide a scaffold for biofilm formation, thereby reducing susceptibility to antibiotics and complicating treatment. Hematogenous implant-related infections following bacteremia are particularly problematic because they can occur at any time in a previously stable implant. Herein, we developed a model of hematogenous infection in which an orthopedic titanium implant was surgically placed in the legs of mice followed 3 wk later by an i.v. exposure to Staphylococcus aureus This procedure resulted in a marked propensity for a hematogenous implant-related infection comprised of septic arthritis, osteomyelitis, and biofilm formation on the implants in the surgical legs compared with sham-operated surgical legs without implant placement and with contralateral nonoperated normal legs. Neutralizing human monoclonal antibodies against α-toxin (AT) and clumping factor A (ClfA), especially in combination, inhibited biofilm formation in vitro and the hematogenous implant-related infection in vivo. Our findings suggest that AT and ClfA are pathogenic factors that could be therapeutically targeted against Saureus hematogenous implant-related infections.
Assuntos
Anticorpos Antibacterianos/farmacologia , Anticorpos Neutralizantes/farmacologia , Artrite Infecciosa , Biofilmes/efeitos dos fármacos , Implantes Experimentais/microbiologia , Osteomielite , Infecções Estafilocócicas , Staphylococcus aureus/fisiologia , Animais , Artrite Infecciosa/tratamento farmacológico , Artrite Infecciosa/etiologia , Artrite Infecciosa/microbiologia , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Osteomielite/tratamento farmacológico , Osteomielite/etiologia , Osteomielite/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/etiologia , Infecções Estafilocócicas/microbiologia , TitânioRESUMO
Surgical site infections (SSIs) are commonly caused by Staphylococcus aureus We report that a combination of three monoclonal antibodies (MEDI6389) that neutralize S. aureus alpha-toxin, clumping factor A, and four leukocidins (LukSF, LukED, HlgAB, and HlgCB) plus vancomycin had enhanced efficacy compared with control antibody plus vancomycin in two mouse models of S. aureus SSI. Therefore, monoclonal antibody-based neutralization of multiple S. aureus virulence factors may provide an adjunctive perioperative approach to combat S. aureus SSIs.
Assuntos
Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Infecção da Ferida Cirúrgica/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Proteínas de Bactérias/imunologia , Anticorpos Amplamente Neutralizantes/farmacologia , Coagulase/imunologia , Leucocidinas/imunologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Infecções Estafilocócicas/microbiologia , Infecção da Ferida Cirúrgica/microbiologia , Vancomicina/farmacologiaRESUMO
Staphylococcus aureus wound infections delay healing and result in invasive complications such as osteomyelitis, especially in the setting of diabetic foot ulcers. In preclinical animal models of S. aureus skin infection, antibody neutralization of alpha-toxin (AT), an S. aureus-secreted pore-forming cytolytic toxin, reduces disease severity by inhibiting skin necrosis and restoring effective host immune responses. However, whether therapeutic neutralization of alpha-toxin is effective against S. aureus-infected wounds is unclear. Herein, the efficacy of prophylactic treatment with a human neutralizing anti-AT monoclonal antibody (MAb) was evaluated in an S. aureus skin wound infection model in nondiabetic and diabetic mice. In both nondiabetic and diabetic mice, anti-AT MAb treatment decreased wound size and bacterial burden and enhanced reepithelialization and wound resolution compared to control MAb treatment. Anti-AT MAb had distinctive effects on the host immune response, including decreased neutrophil and increased monocyte and macrophage infiltrates in nondiabetic mice and decreased neutrophil extracellular traps (NETs) in diabetic mice. Similar therapeutic efficacy was achieved with an active vaccine targeting AT. Taken together, neutralization of AT had a therapeutic effect against S. aureus-infected wounds in both nondiabetic and diabetic mice that was associated with differential effects on the host immune response.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Toxinas Bacterianas/antagonistas & inibidores , Diabetes Mellitus Experimental/imunologia , Proteínas Hemolisinas/antagonistas & inibidores , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Cicatrização/efeitos dos fármacos , Ferimentos não Penetrantes/tratamento farmacológico , Animais , Carga Bacteriana/efeitos dos fármacos , Toxinas Bacterianas/imunologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/microbiologia , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/microbiologia , Proteínas Hemolisinas/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/microbiologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/microbiologia , Pele/efeitos dos fármacos , Pele/imunologia , Pele/microbiologia , Infecções Cutâneas Estafilocócicas/complicações , Infecções Cutâneas Estafilocócicas/imunologia , Infecções Cutâneas Estafilocócicas/microbiologia , Vacinas Antiestafilocócicas/farmacologia , Cicatrização/imunologia , Ferimentos não Penetrantes/complicações , Ferimentos não Penetrantes/imunologia , Ferimentos não Penetrantes/microbiologiaRESUMO
The protective efficacy of tedizolid phosphate, a novel oxazolidinone that potently inhibits bacterial protein synthesis, was compared to those of linezolid, vancomycin, and saline in a rabbit model of Staphylococcus aureus necrotizing pneumonia. Tedizolid phosphate was administered to rabbits at 6 mg/kg of body weight intravenously twice daily, which yielded values of the 24-h area under the concentration-time curve approximating those found in humans. The overall survival rate was 83% for rabbits treated with 6 mg/kg tedizolid phosphate twice daily and 83% for those treated with 50 mg/kg linezolid thrice daily (P = 0.66 by the log-rank test versus the results obtained with tedizolid phosphate). These survival rates were significantly greater than the survival rates of 17% for rabbits treated with 30 mg/kg vancomycin twice daily (P = 0.003) and 17% for rabbits treated with saline (P = 0.002). The bacterial count in the lungs of rabbits treated with tedizolid phosphate was significantly decreased compared to that in the lungs of rabbits treated with saline, although it was not significantly different from that in the lungs of rabbits treated with vancomycin or linezolid. The in vivo bacterial production of alpha-toxin and Panton-Valentine leukocidin, two key S. aureus-secreted toxins that play critical roles in the pathogenesis of necrotizing pneumonia, in the lungs of rabbits treated with tedizolid phosphate and linezolid was significantly inhibited compared to that in the lungs of rabbits treated with vancomycin or saline. Taken together, these results indicate that tedizolid phosphate is superior to vancomycin for the treatment of S. aureus necrotizing pneumonia because it inhibits the bacterial production of lung-damaging toxins at the site of infection.
Assuntos
Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Organofosfatos/uso terapêutico , Oxazóis/uso terapêutico , Pneumonia Necrosante/tratamento farmacológico , Pneumonia Estafilocócica/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Animais , Linezolida/uso terapêutico , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Pneumonia Necrosante/microbiologia , Pneumonia Estafilocócica/microbiologia , Coelhos , Staphylococcus aureus/metabolismo , Vancomicina/uso terapêuticoRESUMO
The role broad-spectrum antibiotics play in the spread of antimicrobial resistance, coupled with their effect on the healthy microbiome, has led to advances in pathogen-specific approaches for the prevention or treatment of serious bacterial infections. One approach in clinical testing is passive immunization with a monoclonal antibody (MAb) targeting alpha toxin for the prevention or treatment of Staphylococcus aureus pneumonia. Passive immunization with the human anti-alpha toxin MAb, MEDI4893*, has been shown to improve disease outcome in murine S. aureus pneumonia models. The species specificity of some S. aureus toxins necessitates testing anti-S. aureus therapeutics in alternate species. We developed a necrotizing pneumonia model in ferrets and utilized an existing rabbit pneumonia model to characterize MEDI4893* protective activity in species other than mice. MEDI4893* prophylaxis reduced disease severity in ferret and rabbit pneumonia models against both community-associated methicillin-resistant S. aureus (MRSA) and hospital-associated MRSA strains. In addition, adjunctive treatment of MEDI4893* with either vancomycin or linezolid provided enhanced protection in rabbits relative to the antibiotics alone. These results confirm that MEDI4893 is a promising candidate for immunotherapy against S. aureus pneumonia.
Assuntos
Antibacterianos/uso terapêutico , Pneumonia Necrosante/tratamento farmacológico , Staphylococcus aureus/fisiologia , Animais , Antibacterianos/farmacologia , Anticorpos Monoclonais/imunologia , Furões , Proteínas Hemolisinas/metabolismo , Staphylococcus aureus Resistente à Meticilina/fisiologia , Pneumonia Necrosante/microbiologia , Pneumonia Estafilocócica , Coelhos , Staphylococcus aureus/efeitos dos fármacosRESUMO
Mast-cell activation mediated by the high-affinity receptor for IgE (FcepsilonRI) is considered to be a key event in the allergic inflammatory response. However, in a physiological setting, other receptors, such as KIT, might also markedly influence the release of mediators by mast cells. Recent studies have provided evidence that FcepsilonRI-dependent degranulation is regulated by two complementary signalling pathways, one of which activates phospholipase Cgamma and the other of which activates phosphatidylinositol 3-kinase, using specific transmembrane and cytosolic adaptor molecules. In this Review, we discuss the evidence for these interacting pathways and describe how the capacity of KIT, and other receptors, to influence FcepsilonRI-dependent mast-cell-mediator release might be a function of the relative abilities of these receptors to activate these alternative pathways.
Assuntos
Mastócitos/fisiologia , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Sequência de Aminoácidos , Animais , Ativação Enzimática , Humanos , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Fosfolipase C gama/metabolismo , Fosfoproteínas/fisiologia , Fosforilação , Receptores de IgE/fisiologia , Quinases da Família src/fisiologiaRESUMO
Staphylococcus aureus infections lead to an array of illnesses ranging from mild skin infections to serious diseases, such endocarditis, osteomyelitis, and pneumonia. Alpha-toxin (Hla) is a pore-forming toxin, encoded by the hla gene, that is thought to play a key role in S. aureus pathogenesis. A monoclonal antibody targeting Hla, MEDI4893, is in clinical development for the prevention of S. aureus ventilator-associated pneumonia (VAP). The presence of the hla gene and Hla protein in 994 respiratory isolates collected from patients in 34 countries in Asia, Europe, the United States, Latin America, the Middle East, Africa, and Australia was determined. Hla levels were correlated with the geographic location, age of the subject, and length of stay in the hospital. hla gene sequence analysis was performed, and mutations were mapped to the Hla crystal structure. S. aureus supernatants containing Hla variants were tested for susceptibility or resistance to MEDI4893. The hla gene was present and Hla was expressed in 99.0% and 83.2% of the isolates, respectively, regardless of geographic region, hospital locale, or age of the subject. More methicillin-susceptible than methicillin-resistant isolates expressed Hla (86.9% versus 78.8%; P = 0.0007), and S. aureus isolates from pediatric patients expressed the largest amounts of Hla. Fifty-seven different Hla subtypes were identified, and 91% of the isolates encoded an Hla subtype that was neutralized by MED4893. This study demonstrates that Hla is conserved in diverse S. aureus isolates from around the world and is an attractive target for prophylactic monoclonal antibody (MAb) or vaccine development.
Assuntos
Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Toxinas Bacterianas/antagonistas & inibidores , Proteínas Hemolisinas/antagonistas & inibidores , Pneumonia Associada à Ventilação Mecânica/prevenção & controle , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Anti-Inflamatórios/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados , Anticorpos Neutralizantes/química , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Anticorpos Amplamente Neutralizantes , Criança , Pré-Escolar , Sequência Conservada , Monitoramento Epidemiológico , Feminino , Saúde Global , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/imunologia , Humanos , Lactente , Recém-Nascido , Tempo de Internação/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Mutação , Pneumonia Associada à Ventilação Mecânica/epidemiologia , Pneumonia Associada à Ventilação Mecânica/microbiologia , Pneumonia Associada à Ventilação Mecânica/patologia , Conformação Proteica , Análise de Sequência de DNA , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Staphylococcus aureus/isolamento & purificaçãoRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) causes large-scale epidemics of acute bacterial skin and skin structure infections (ABSSSI) within communities across the United States. Animal models that reproduce ABSSSI as they occur in humans are urgently needed to test new therapeutic strategies. Alpha-toxin plays a critical role in a variety of staphylococcal infection models in mice, but its role in the pathogenesis of ABSSSI remains to be elucidated in rabbits, which are similar to humans in their susceptibility to S. aureus superantigens and certain bicomponent pore-forming leukocidins. We report here a new rabbit model of ABSSSI and show that those infected with a mutant deficient in expression of alpha-toxin (Δhla) developed a small dermonecrotic lesion, whereas those infected with isogenic USA300 MRSA wild-type or complemented Δhla strains developed ABSSSI that mimic the severe infections that occur in humans, including the large central dermonecrotic core surrounded by erythema, induration, and marked subcutaneous hemorrhage. More importantly, immunoprophylaxis with MEDI4893*, an anti-alpha-toxin human monoclonal antibody, significantly reduced the severity of disease caused by a USA300 wild-type strain to that caused by the Δhla mutant, indicating that this toxin could be completely neutralized during infection. Thus, this study illustrates a potential high standard for the development of new immunotherapeutic agents in which a toxin-neutralizing antibody provides protection to the same degree achieved with a toxin gene knockout. When MEDI4893* was administered as adjunctive therapy with a subtherapeutic dose of linezolid, the combination was significantly more efficacious than either agent alone in reducing the severity of ABSSSI.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Toxinas Bacterianas/imunologia , Proteínas Hemolisinas/imunologia , Dermatopatias Bacterianas/microbiologia , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados , Toxinas Bacterianas/genética , Anticorpos Amplamente Neutralizantes , Modelos Animais de Doenças , Proteínas Hemolisinas/genética , Humanos , Linezolida/sangue , Linezolida/farmacologia , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/imunologia , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Profilaxia Pré-Exposição/métodos , Coelhos , Dermatopatias Bacterianas/imunologia , Infecções Cutâneas Estafilocócicas/imunologia , Infecções Cutâneas Estafilocócicas/microbiologiaRESUMO
MEDI4893 is a neutralizing human monoclonal antibody that targets α-toxin (AT) and is currently undergoing evaluation in the field of Staphylococcus aureus-mediated diseases. We have solved the crystal structure of MEDI4893 Fab bound to monomeric AT at a resolution of 2.56 Å and further characterized its epitope using various engineered AT variants. We have found that MEDI4893 recognizes a novel epitope in the so-called "rim" domain of AT and exerts its neutralizing effect through a dual mechanism. In particular, MEDI4893 not only sterically blocks binding of AT to its cell receptor but also prevents it from adopting a lytic heptameric trans-membrane conformation.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Toxinas Bacterianas/imunologia , Proteínas Hemolisinas/imunologia , Testes de Neutralização , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados , Anticorpos Neutralizantes/química , Anticorpos Amplamente Neutralizantes , Linhagem Celular , Cristalografia por Raios X , Mapeamento de Epitopos , Humanos , Fragmentos Fab das Imunoglobulinas/química , Modelos Moleculares , Ligação Proteica , Proteínas Recombinantes/química , Ressonância de Plasmônio de SuperfícieRESUMO
Alpha-toxin (AT) is a major virulence determinant in Staphylococcus aureus skin and soft tissue infection models. We previously demonstrated that prophylactic administration of 2A3, an AT-neutralizing monoclonal antibody (MAb), prevents S. aureus disease in a mouse dermonecrosis model by neutralizing AT-mediated tissue necrosis and immune evasion. In the present study, MEDI4893*, an affinity-optimized version of 2A3, was characterized for therapeutic activity in the dermonecrosis model as a single agent and in combination with two frontline antibiotics, vancomycin and linezolid. MEDI4893* postinfection therapy was found to exhibit a therapeutic treatment window similar to that for linezolid but longer than that for vancomycin. Additionally, when combined with either vancomycin or linezolid, MEDI4893* resulted in reduced tissue damage, increased neutrophil and macrophage infiltration and abscess formation, and accelerated healing relative to those with the antibiotic monotherapies. These data suggest that AT neutralization with a potent MAb holds promise for both prophylaxis and adjunctive therapy with antibiotics and may be a valuable addition to currently available options for the treatment of S. aureus skin and soft tissue infections.
Assuntos
Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Toxinas Bacterianas/imunologia , Proteínas Hemolisinas/imunologia , Infecções Cutâneas Estafilocócicas/tratamento farmacológico , Animais , Antibacterianos/farmacocinética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Anticorpos Amplamente Neutralizantes , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Linezolida/farmacocinética , Linezolida/farmacologia , Camundongos Endogâmicos BALB C , Necrose/tratamento farmacológico , Necrose/microbiologia , Infecções Cutâneas Estafilocócicas/imunologia , Infecções Cutâneas Estafilocócicas/patologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Vancomicina/farmacocinética , Vancomicina/farmacologiaRESUMO
BACKGROUND: Linezolid is recommended for treatment of pneumonia and other invasive infections caused by methicillin-resistant Staphylococcus aureus (MRSA). The premise underlying this recommendation is that linezolid inhibits in vivo production of potent staphylococcal exotoxins, including Panton-Valentine leukocidin (PVL) and α-hemolysin (Hla), although supporting evidence is lacking. METHODS: A rabbit model of necrotizing pneumonia using MRSA clone USA300 was used to compare therapeutic effects of linezolid (50 mg/kg 3 times/day) and vancomycin (30 mg/kg 2 times/day) administered 1.5, 4, and 9 hours after infection on host survival outcomes and in vivo bacterial toxin production. RESULTS: Mortality rates were 100% for untreated rabbits and 83%-100% for vancomycin-treated rabbits. In contrast, mortality rates were 25%, 50%, and 100% for rabbits treated with linezolid 1.5, 4, and 9 hours after infection, respectively. Compared with untreated and vancomycin-treated rabbits, improved survival of rabbits treated 1.5 hours after infection with linezolid was associated with a significant decrease in bacterial counts, suppressed bacterial production of PVL and Hla, and reduced production of the neutrophil-chemoattractant interleukin 8 in the lungs. CONCLUSIONS: Across the study interval, only early treatment with linezolid resulted in significant suppression of exotoxin synthesis and improved survival outcomes in a rabbit model of MRSA necrotizing pneumonia.
Assuntos
Acetamidas/uso terapêutico , Antibacterianos/uso terapêutico , Toxinas Bacterianas/biossíntese , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oxazolidinonas/uso terapêutico , Pneumonia Estafilocócica/tratamento farmacológico , Animais , Carga Bacteriana/efeitos dos fármacos , Toxinas Bacterianas/análise , Toxinas Bacterianas/antagonistas & inibidores , Quimiocina CCL2/análise , Modelos Animais de Doenças , Exotoxinas/análise , Proteínas Hemolisinas/análise , Interleucina-8/análise , Leucocidinas/análise , Linezolida , Pulmão/química , Pneumonia Estafilocócica/microbiologia , Pneumonia Estafilocócica/mortalidade , Coelhos , Vancomicina/uso terapêuticoRESUMO
Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) uncovered synergistic lethality that was driven by Candida -induced upregulation of functional S. aureus âº-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken and revealed that zcf13 Δ/Δ failed to drive augmented âº-toxin or lethal synergism during co-infection. Using a combination of transcriptional and phenotypic profiling approaches, ZCF13 was shown to regulate genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments revealed that ribose inhibited the staphylococcal agr quorum sensing system and concomitantly repressed toxicity. Unlike wild-type C. albicans , zcf13 Δ/Δ was unable to effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13 Δ/Δ mutant fully restored pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.
RESUMO
Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) demonstrates that synergistic lethality is driven by Candida-induced upregulation of functional S. aureus α-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken revealing that zcf13Δ/Δ fails to drive augmented α-toxin or lethal synergism during co-infection. A combination of transcriptional and phenotypic profiling approaches shows that ZCF13 regulates genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments reveal that ribose inhibits the staphylococcal agr quorum sensing system and concomitantly represses toxicity. Unlike wild-type C. albicans, zcf13Δ/Δ did not effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13Δ/Δ mutant fully restores pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.
Assuntos
Candida albicans , Candidíase , Coinfecção , Proteínas Fúngicas , Infecções Estafilocócicas , Staphylococcus aureus , Candida albicans/metabolismo , Candida albicans/patogenicidade , Candida albicans/genética , Animais , Coinfecção/microbiologia , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/metabolismo , Staphylococcus aureus/genética , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/metabolismo , Candidíase/microbiologia , Camundongos , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Infecções Intra-Abdominais/microbiologia , Feminino , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Percepção de Quorum/genética , Virulência , Regulação Fúngica da Expressão Gênica , Modelos Animais de Doenças , Transativadores/metabolismo , Transativadores/genéticaRESUMO
Staphylococcus aureus alpha toxin (AT) has been crystallized in complex with the Fab fragment of a human antibody (MEDI4893). This constitutes the first reported crystals of AT bound to an antibody. The monoclinic crystals belonged to space group P21, with unit-cell parameters a=85.52, b=148.50, c=93.82â Å, ß=99.82°. The diffraction of the crystals extended to 2.56â Å resolution. The asymmetric unit contained two MEDI4893 Fab-AT complexes. This corresponds to a crystal volume per protein weight (VM) of 2.3â Å3â Da(-1) and a solvent content of 47%. The three-dimensional structure of this complex will contribute to an understanding of the molecular basis of the interaction of MEDI4893 with AT. It will also shed light on the mechanism of action of this antibody, the current evaluation of which in the field of S. aureus-mediated diseases makes it a particularly interesting case study. Finally, this study will provide the three-dimensional structure of AT in a monomeric state for the first time.
Assuntos
Anticorpos Anti-Idiotípicos/química , Anticorpos Monoclonais/química , Toxinas Bacterianas/química , Fragmentos Fab das Imunoglobulinas/química , Staphylococcus aureus/química , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/isolamento & purificação , Cristalização , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMO
Background: Patients with septic shock caused by Staphylococcus aureus have mortality rates exceeding 50%, despite appropriate antibiotic therapy. Our objectives were to establish a rabbit model of S. aureus septic shock and to determine whether a novel immunotherapy can prevent or halt its natural disease progression. Methods: Anesthetized rabbits were ventilated with lung-protective low-tidal volume, instrumented for advanced hemodynamic monitoring, and characterized for longitudinal changes in acute myocardial dysfunction by echocardiography and sepsis-associated biomarkers after S. aureus intravenous challenge. To demonstrate the potential utility of this hyperdynamic septic shock model for preclinical drug development, rabbits were randomized for prophylaxis with anti-Hla/Luk/ClfA monoclonal antibody combination that neutralizes alpha-hemolysin (Hla), the bicomponent pore-forming leukocidins (Luk) including Panton-Valentine leukocidin, leukocidin ED, and gamma-hemolysin, and clumping factor A (ClfA), or an irrelevant isotype-matched control IgG (c-IgG), and then challenged with S. aureus. Results: Rabbits challenged with S. aureus, but not those with saline, developed a hyperdynamic state of septic shock characterized by elevated cardiac output (CO), increased stroke volume (SV) and reduced systemic vascular resistance (SVR), which was followed by a lethal hypodynamic state characterized by rapid decline in mean arterial pressure (MAP), increased central venous pressure, reduced CO, reduced SV, elevated SVR, and reduced left-ventricular ejection fraction, thereby reproducing the hallmark clinical features of human staphylococcal septic shock. In this model, rabbits pretreated with anti-Hla/Luk/ClfA mAb combination had 69% reduction in mortality when compared to those pretreated with c-IgG (P<0.001). USA300-induced acute circulatory failure-defined as >70% decreased in MAP from pre-infection baseline-occurred in only 20% (2/10) of rabbits pretreated with anti-Hla/Luk/ClfA mAb combination compared to 100% (9/9) of those pretreated with c-IgG. Prophylaxis with anti-Hla/Luk/ClfA mAb combination halted progression to lethal hypodynamic shock, as evidenced by significant protection against the development of hyperlactatemia, hypocapnia, hyperkalemia, leukopenia, neutropenia, monocytopenia, lymphopenia, as well as biomarkers associated with acute myocardial injury. Conclusion: These results demonstrate the potential utility of a mechanically ventilated rabbit model that reproduced hallmark clinical features of hyperdynamic septic shock and the translational potential of immunotherapy targeting S. aureus virulence factors for the prevention of staphylococcal septic shock.
Assuntos
Choque Séptico , Choque , Infecções Estafilocócicas , Humanos , Animais , Coelhos , Staphylococcus aureus , Anticorpos Monoclonais/uso terapêutico , Proteínas Hemolisinas , Leucocidinas , Choque Séptico/tratamento farmacológico , Respiração Artificial , Volume Sistólico , Função Ventricular Esquerda , Choque/tratamento farmacológico , Imunoglobulina GRESUMO
Importance: Staphylococcus aureus surgical site infections (SSIs) and bloodstream infections (BSIs) are important complications of surgical procedures for which prevention remains suboptimal. Contemporary data on the incidence of and etiologic factors for these infections are needed to support the development of improved preventive strategies. Objectives: To assess the occurrence of postoperative S aureus SSIs and BSIs and quantify its association with patient-related and contextual factors. Design, Setting, and Participants: This multicenter cohort study assessed surgical patients at 33 hospitals in 10 European countries who were recruited between December 16, 2016, and September 30, 2019 (follow-up through December 30, 2019). Enrolled patients were actively followed up for up to 90 days after surgery to assess the occurrence of S aureus SSIs and BSIs. Data analysis was performed between November 20, 2020, and April 21, 2022. All patients were 18 years or older and had undergone 11 different types of surgical procedures. They were screened for S aureus colonization in the nose, throat, and perineum within 30 days before surgery (source population). Both S aureus carriers and noncarriers were subsequently enrolled in a 2:1 ratio. Exposure: Preoperative S aureus colonization. Main Outcomes and Measures: The main outcome was cumulative incidence of S aureus SSIs and BSIs estimated for the source population, using weighted incidence calculation. The independent association of candidate variables was estimated using multivariable Cox proportional hazards regression models. Results: In total, 5004 patients (median [IQR] age, 66 [56-72] years; 2510 [50.2%] female) were enrolled in the study cohort; 3369 (67.3%) were S aureus carriers. One hundred patients developed S aureus SSIs or BSIs within 90 days after surgery. The weighted cumulative incidence of S aureus SSIs or BSIs was 2.55% (95% CI, 2.05%-3.12%) for carriers and 0.52% (95% CI, 0.22%-0.91%) for noncarriers. Preoperative S aureus colonization (adjusted hazard ratio [AHR], 4.38; 95% CI, 2.19-8.76), having nonremovable implants (AHR, 2.00; 95% CI, 1.15-3.49), undergoing mastectomy (AHR, 5.13; 95% CI, 1.87-14.08) or neurosurgery (AHR, 2.47; 95% CI, 1.09-5.61) (compared with orthopedic surgery), and body mass index (AHR, 1.05; 95% CI, 1.01-1.08 per unit increase) were independently associated with S aureus SSIs and BSIs. Conclusions and Relevance: In this cohort study of surgical patients, S aureus carriage was associated with an increased risk of developing S aureus SSIs and BSIs. Both modifiable and nonmodifiable etiologic factors were associated with this risk and should be addressed in those at increased S aureus SSI and BSI risk.