RESUMEN
Staphylococcal infections are a major source of global morbidity and mortality. Currently there exists no antistaphylococcal vaccine in clinical use. Previous animal studies suggested a possible role for purified lipoteichoic acid as a vaccine target for eliciting protective IgG to several Gram-positive pathogens. Since the highly conserved (poly)glycerolphosphate backbone of lipoteichoic acid is a major antigenic target of the humoral immune system during staphylococcal infections, we developed a synthetic method for producing glycerol phosphoramidites to create a covalent 10-mer of (poly)glycerolphosphate for potential use in a conjugate vaccine. We initially demonstrated that intact Staphylococcus aureus elicits murine CD4(+) T cell-dependent (poly)glycerolphosphate-specific IgM and IgG responses in vivo. Naive mice immunized with a covalent conjugate of (poly)glycerolphosphate and tetanus toxoid in alum plus CpG-oligodeoxynucleotides produced high secondary titers of serum (poly)glycerolphosphate-specific IgG. Sera from immunized mice enhanced opsonophagocytic killing of live Staphylococcus aureus in vitro. Mice actively immunized with the (poly)glycerolphosphate conjugate vaccine showed rapid clearance of staphylococcal bacteremia in vivo relative to mice similarly immunized with an irrelevant conjugate vaccine. In contrast to purified, natural lipoteichoic acid, the (poly)glycerolphosphate conjugate vaccine itself exhibited no detectable inflammatory activity. These data suggest that a synthetic (poly)glycerolphosphate-based conjugate vaccine will contribute to active protection against extracellular Gram-positive pathogens expressing this highly conserved backbone structure in their membrane-associated lipoteichoic acid.
Asunto(s)
Glicerofosfatos/inmunología , Infecciones Estafilocócicas/prevención & control , Vacunas Estafilocócicas/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Animales , Bacteriemia/inmunología , Linfocitos T CD4-Positivos/inmunología , Femenino , Glicerofosfatos/administración & dosificación , Sueros Inmunes/administración & dosificación , Sueros Inmunes/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Oligodesoxirribonucleótidos/administración & dosificación , Infecciones Estafilocócicas/inmunología , Vacunas Estafilocócicas/inmunología , Toxoide Tetánico/inmunología , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
BACKGROUND: Monoclonal antibody (palivizumab), intravenous immune globulin (IGIV), or respiratory syncytial virus (RSV)-polyclonal-hyperimmune-globulin (RSV-IG as Respigam®, RI-001, RI-002) are used with ribavirin in RSV-infected immunocompromised patients, with debated efficacy. Palivizumab-resistance (PR) can arise during treatment of persistent infections in this population. RSV-IG may confer benefit in PR-RSV infection. METHODS: RSV-IG [RI-001] was provided for an immunocompromised infant with RSV-pneumonitis refractory to ribavirin and palivizumab. RSV-neutralizing antibody, respiratory RSV load (qPCR), and F-gene-sequence-detection of PR was determined. Prophylactic RSV-IG [RI-002] or palivizumab was administered in a cotton-rat model infected with wild-type and PR-RSV. Lung RSV load and neutralizing antibody were measured. RESULTS: As protective RI-001-neutralizing antibody titers waned in the infant, a subpopulation of PR-escape mutants were detected with a fatal RSV-burden in the lungs. In PR-RSV-infected cotton rats, prophylactic RI-002 reduced RSV-load in the lungs (2.45 vs 0.28 log10 PFU/g lung-tissue reduction, respectively, p < 0.05) and provided protective RSV-neutralizing antibody. CONCLUSIONS: RSV-IG and ribavirin use in immunocompromised patients requires further study.
Asunto(s)
Farmacorresistencia Viral , Palivizumab , Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Humanos , Palivizumab/uso terapéutico , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Huésped Inmunocomprometido , Animales , Sigmodontinae , Pulmón/patología , Pulmón/virología , Inmunoglobulinas/administración & dosificación , Anticuerpos Neutralizantes/administración & dosificación , Femenino , Lactante , Resultado Fatal , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicacionesRESUMEN
Colonization of the anterior nares in approximately 37% of the population is a major risk factor for severe Staphylococcus aureus infections. Here we show that wall teichoic acid (WTA), a surface-exposed staphylococcal polymer, is essential for nasal colonization and mediates interaction with human nasal epithelial cells. WTA-deficient mutants were impaired in their adherence to nasal cells, and were completely unable to colonize cotton rat nares. This study describes the first essential factor for S. aureus nasal colonization.
Asunto(s)
Infección Hospitalaria/microbiología , Mucosa Nasal/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/crecimiento & desarrollo , Ácidos Teicoicos/metabolismo , Animales , Adhesión Bacteriana , Células Cultivadas , Células Epiteliales/microbiología , Humanos , Estructura Molecular , Mucosa Nasal/citología , Ratas , Factores de Riesgo , Sigmodontinae , Staphylococcus aureus/fisiología , Ácidos Teicoicos/químicaRESUMEN
Primary infection with Epstein-Barr virus (EBV) is associated with post-transplant lymphoproliferative disease and severe disease in patients with X-linked lymphoproliferative disease; no therapies are approved to prevent EBV infection in these patients. Hyperimmune globulin has been used to prevent some virus infections in immunocompromised persons. Here, we identified plasma donors with high titers of EBV gp350 and EBV B cell neutralizing antibodies. Pooled IgG isolated from these donors was compared to intravenous immunoglobulin (IVIG) for its ability to reduce viral load in the blood in humanized mice challenged with EBV. Mice that received EBV hyperimmune globulin had significantly reduced EBV DNA copy numbers compared to animals that received saline control; however, while animals that received EBV hyperimmune globulin had lower EBV DNA copies than those that received IVIG, the difference was not significant. Thus, while EBV hyperimmune globulin reduced viral load compared to IVIG, the effect was modest.
Asunto(s)
Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Infecciones por Virus de Epstein-Barr/prevención & control , Infecciones por Virus de Epstein-Barr/terapia , Herpesvirus Humano 4/inmunología , Inmunización Pasiva , Proteínas de la Matriz Viral/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Donantes de Sangre , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/fisiología , Ratones , Carga ViralRESUMEN
Staphylococcal sepsis is a major cause of morbidity and mortality in very-low-birth-weight (VLBW) infants. A human chimeric monoclonal antibody, pagibaximab, was developed against staphylococcal lipoteichoic acid. We evaluated the safety, tolerability, and pharmacokinetics of pagibaximab in VLBW neonates. A phase 1/2, randomized, double-blind, placebo-controlled, dose escalation study was conducted in VLBW infants (700 to 1,300 g) 3 to 7 days old. Patients received two doses 14 days apart of intravenous pagibaximab (10, 30, 60, or 90 mg/kg of body weight) or placebo in a 2:1 ratio. Blood and urine samples were obtained pre- and postinfusion for analysis of safety and pharmacokinetics, and data on adverse events were gathered. Staphylococcal organisms causing sepsis were collected and evaluated. Fifty-three patients received at least one dose of pagibaximab or placebo. The average gestational age was 27.6 weeks; the average birth weight was 1,003 g. All serious adverse events were deemed unrelated or probably not drug related. Morbidity and mortality were similar across treatment groups. No evidence of immunogenicity of pagibaximab was detected. Pagibaximab pharmacokinetics was linear. The mean clearance (CL), volume of distribution, and elimination half-life of pagibaximab were independent of dose. The serum half-life was 20.5 +/- 6.8 days. Pagibaximab enhanced serum opsonophagocytic activity. All staphylococci causing sepsis were opsonizable by pagibaximab. Two infusions of pagibaximab, administered 2 weeks apart to high-risk neonates appeared safe and tolerable, and pharmacokinetics were linear. Evaluation of more frequent doses, at the highest doses tested, in neonates at high-risk of staphylococcal sepsis, is warranted.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/uso terapéutico , Recién Nacido de muy Bajo Peso , Infecciones Estafilocócicas/prevención & control , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Método Doble Ciego , Humanos , Recién NacidoRESUMEN
OBJECTIVES: Staphylococcus aureus infections associated with indwelling devices can be very difficult to treat due to the recalcitrant nature of bacterial biofilms to conventional antibiotics. Lysostaphin has been shown to clear S. aureus biofilms in vitro, and in this study we determined whether lysostaphin could also eradicate established S. aureus biofilms on implanted jugular vein catheters in mice. METHODS: Jugular vein catheterized mice (four to six per group) challenged with S. aureus developed multiorgan infection and biofilm infections on the catheters. The infected mice with established biofilms received various doses of recombinant lysostaphin through the catheters, administered up to three times daily for up to 4 days. Some mice also received lysostaphin combined with nafcillin. Following treatment, mice were sacrificed and cfu on the catheter and in the liver and heart were determined. In another set of experiments, implanted jugular vein catheters in mice were pre-instilled with lysostaphin to determine whether this pre-treatment would protect the mice from biofilm infection. RESULTS AND CONCLUSIONS: Lysostaphin administered at 15 mg/kg in combination with 50 mg/kg nafcillin three times per day for 4 days eradicated established S. aureus, including methicillin-resistant S. aureus, biofilms from implanted catheters and sterilized heart and liver infections of S. aureus-infected mice. Furthermore, a single pre-instillation of 10 mg/kg lysostaphin in catheters completely protected catheterized mice from a subsequent biofilm infection. These results demonstrate that lysostaphin is an effective treatment as well as prophylaxis for S. aureus biofilms on indwelling catheters.
Asunto(s)
Antibacterianos/uso terapéutico , Biopelículas/efectos de los fármacos , Lisostafina/uso terapéutico , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Animales , Antibacterianos/administración & dosificación , Cateterismo Venoso Central , Recuento de Colonia Microbiana , Equipos y Suministros/microbiología , Corazón/microbiología , Humanos , Venas Yugulares , Hígado/microbiología , Lisostafina/administración & dosificación , Ratones , Nafcilina/uso terapéuticoRESUMEN
Humoral immunodeficiency patients require immunoglobulin replacement to prevent infection. Traditional intravenous immunoglobulin manufacturing methods have had the potential for containing impurities caused by physical, chemical and thermal stressors that alter proteins. Two intravenous immunoglobulin products, BIVIGAM® and ASCENIV™, are manufactured by a modified Cohn-Oncley fractionation method followed by chromatographic purification. These products have undergone a systematic quality by design optimization to identify critical manufacturing processes to produce the highest quality product. This data driven, small-scale approach has led to manufacturing enhancements that have yielded consistent product improvements. The systematic approach to optimizing manufacturing has guided process changes, in-process, procedural and engineering controls that have reduced protein shearing and aggregation, and improved purity resulting in products with lot-to-lot consistency.
Asunto(s)
Productos Biológicos/uso terapéutico , Inmunoglobulinas Intravenosas/uso terapéutico , Síndromes de Inmunodeficiencia/terapia , Inmunoterapia/métodos , Productos Biológicos/aislamiento & purificación , Fraccionamiento Químico/métodos , Cromatografía/métodos , Humanos , Inmunidad Humoral/inmunología , Inmunoglobulinas Intravenosas/aislamiento & purificación , Síndromes de Inmunodeficiencia/inmunología , Medicina de Precisión/métodos , Resultado del TratamientoRESUMEN
INTRODUCTION: Novel immune globulin (IG) products (RI-002, RI-001) have been designed to provide protection against respiratory syncytial virus (RSV) mediated respiratory illness while at the same time meeting the manufacturing requirements established by FDA for antibody supplementation in immunocompromised subjects. Areas covered: This review covers the manufacture and development of both RI-001 and RI-002, including the selection of plasma donors for IG preparation with high-titers of anti-RSV antibody, in vitro, and preclinical data in the cotton rat model S. hispidus, and clinical trials including Phase II and compassionate use studies of RI-001 and a multi-center, pivotal Phase III study of RI-002 in PIDD patients. Expert commentary: The data demonstrate that RI-002 is efficacious in the prevention and treatment of RSV in preclinical normal and immune suppressed animal models and is safe and efficacious in the treatment of patients with various forms of primary immunodeficiency disease (PIDD). This product offers potential advantages over other available IG's for prophylaxis in immunocompromised patients requiring polyclonal immunoglobulin supplementation because of its unique antibody composition. In addition to its enhanced neutralizing anti-RSV activity and its polyclonal IG composition, there is preclinical data to support the use of RI-002 for humoral protection against other respiratory pathogens.
Asunto(s)
Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Inmunoglobulinas Intravenosas/uso terapéutico , Síndromes de Inmunodeficiencia/terapia , Inmunoterapia/métodos , Infecciones por Virus Sincitial Respiratorio/terapia , Virus Sincitial Respiratorio Humano/inmunología , Animales , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Testimonio de Experto , Humanos , Huésped Inmunocomprometido , Inmunoglobulinas Intravenosas/metabolismo , Ratas , Infecciones por Virus Sincitial Respiratorio/inmunología , SigmodontinaeRESUMEN
BACKGROUND: The nisin-controlled gene expression system NICE of Lactococcus lactis is one of the most widely used expression systems in Gram-positive bacteria. Despite its widespread use, no optimization of the culture conditions and nisin induction has been carried out to obtain maximum yields. As a model system induced production of lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) produced by S. simulans biovar. Staphylolyticus, was used. Three main areas need optimization for maximum yields: cell density, nisin-controlled induction and protein production, and parameters specific for the target-protein. RESULTS: In a series of pH-controlled fermentations the following parameters were optimized: pH of the culture, use of NaOH or NH4OH as neutralizing agent, the addition of zinc and phosphate, the fermentation temperature, the time point of induction (cell density of the culture), the amount of nisin added for induction and the amount of three basic medium components, i.e. yeast extract, peptone and lactose. For each culture growth and lysostaphin production was followed. Lysostaphin production yields depended on all parameters that were varied. In the course of the optimization a three-fold increase in lysostaphin yield was achieved from 100 mg/l to 300 mg/l. CONCLUSION: Protein production with the NICE gene expression system in L. lactis strongly depends on the medium composition, the fermentation parameters and the amount of nisin added for induction. Careful optimization of key parameters lead to a significant increase in the yield of the target protein.
RESUMEN
BACKGROUND: The NIsin-Controlled gene Expression system NICE of Lactococcus lactis is one of the most widespread used expression systems of Gram-positive bacteria. It is used in more than 100 laboratories for laboratory-scale gene expression experiments. However, L. lactis is also a micro-organism with a large biotechnological potential. Therefore, the aim of this study was to test whether protein production in L. lactis using the NICE system can also effectively be performed at the industrial-scale of fermentation. RESULTS: Lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) from S. simulans biovar. Staphylolyticus, was used as a model system. Food-grade lysostaphin expression constructs in L. lactis were grown at 1L-, 300-L and 3000-L scale and induced with nisin for lysostaphin production. The induction process was equally effective at all scales and yields of about 100 mg/L were obtained. Up-scaling was easy and required no specific effort. Furthermore, we describe a simple and effective way of downstream processing to obtain a highly purified lysostaphin, which has been used for clinical phase I trials. CONCLUSION: This is the first example that shows that nisin-regulated gene expression in L. lactis can be used at industrial scale to produce large amounts of a target protein, such as lysostaphin. Downstream processing was simple and in a few steps produced a highly purified and active enzyme.
RESUMEN
Infectious mononucleosis and B-cell transformation in response to infection with Epstein-Barr virus (EBV) is dependent upon binding of the EBV envelope glycoprotein gp350 to CD21 on B-cells. Gp350-specific antibody comprises most of the EBV neutralizing activity in the serum of infected patients, making this protein a promising target antigen for a prophylactic EBV vaccine. We describe a novel, tetrameric gp350-based vaccine that exhibits markedly enhanced immunogenicity relative to its monomeric counterpart. Plasmid DNA was constructed for synthesis, within transfected CHO cells, of a tetrameric, truncated (a.a. 1-470) gp350 protein (gp350(1-470)). Tetrameric gp350(1-470) induced ≈ 20-fold higher serum titers of gp350(1-470)-specific IgG and >19-fold enhancements in neutralizing titers at the highest dose, and was >25-fold more immunogenic on a per-weight basis than monomeric gp350(1-470). Further, epidermal immunization with plasmid DNA encoding gp350(1-470) tetramer induced 8-fold higher serum titers of gp350(1-470)-specific IgG relative to monomer. Tetrameric gp350(1-470) binding to human CD21 was >24-fold more efficient on a per-weight basis than monomer, but neither tetramer nor monomer mediated polyclonal human B-cell activation. Finally, the introduction of strong, universal tetanus toxoid (TT)-specific CD4+ T-cell epitopes into the tetrameric gp350(1-470) had no effect on the gp350(1-470)-specific IgG response in naïve mice, and resulted in suppressed gp350(1-470)-specific IgG responses in TT-primed mice. Collectively, these data suggest that tetrameric gp350(1-470) is a potentially promising candidate for testing as a prophylactic EBV vaccine, and that protein multimerization, using the approach described herein, is likely to be clinically relevant for enhancing the immunogenicity of other proteins of vaccine interest.
Asunto(s)
Infecciones por Virus de Epstein-Barr/prevención & control , Vacuna contra el Herpes Zóster/inmunología , Herpesvirus Humano 4/inmunología , Proteínas de la Matriz Viral/inmunología , Animales , Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Células CHO , Cricetinae , Cricetulus , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Humanos , Inmunización , Ratones , Plásmidos , Transfección , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismoRESUMEN
BACKGROUND: Pagibaximab, a human chimeric monoclonal antibody developed against lipoteichoic acid, was effective against staphylococci preclinically and seemed safe and well tolerated in phase 1 studies. OBJECTIVE: To evaluate the clinical activity, pharmacokinetics, safety, and tolerability of weekly pagibaximab versus placebo infusions in very low birth weight neonates. PATIENTS AND METHODS: A phase 2, randomized, double-blind, placebo-controlled study was conducted at 10 NICUs. Patients with a birth weight of 700 to 1300 g and 2 to 5 days old were randomly assigned to receive 3 once-a-week pagibaximab (90 or 60 mg/kg) or placebo infusions. Blood was collected for pharmacokinetics, bacterial killing, and safety analyses. Adverse event and clinical outcome data were collected. RESULTS: Eighty-eight patients received pagibaximab at 90 (n = 22) or 60 (n = 20) mg/kg or placebo (n = 46). Groups were not different in demography, mortality, or morbidity. Pagibaximab demonstrated linear pharmacokinetics, a 14.5-day half-life, and nonimmunogenicity. Definite staphylococcal sepsis occurred in 0%, 20%, and 13% (P < .11) and nonstaphylococcal sepsis occurred in 0%, 10%, and 15% (P < .15) of patients in the 90 mg/kg, 60 mg/kg, and placebo groups, respectively. In all patients with staphylococcal sepsis, estimated or observed pagibaximab levels were <500 µg/mL (target level) at infection. CONCLUSIONS: Three once-a-week 90 or 60 mg/kg pagibaximab infusions, in high-risk neonates, seemed safe and well tolerated. No staphylococcal sepsis occurred in infants who received 90 mg/kg. Target levels were only consistently achieved after 2 to 3 doses. Dose optimization should enhance protection.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Recién Nacido de muy Bajo Peso , Sepsis/prevención & control , Infecciones Estafilocócicas/prevención & control , Anticuerpos Monoclonales/sangre , Método Doble Ciego , Esquema de Medicación , Femenino , Humanos , Recién Nacido , Recién Nacido de muy Bajo Peso/sangre , Infusiones Intravenosas , Masculino , Factores de Riesgo , Sepsis/sangre , Sepsis/etiología , Infecciones Estafilocócicas/sangre , Infecciones Estafilocócicas/etiologíaRESUMEN
A chimerized (murine/human) monoclonal antibody (pagibaximab) against lipoteichoic acid (LTA) and protective in animal models for coagulase-negative staphylococci (CONS) and Staphylococcus aureus bacteremia, was developed for prevention of staphylococcal infection in high-risk populations. This open label two-dose study of a single intravenous dose of 3 or 10 mg/kg of pagibaximab evaluated the safety/tolerability, pharmacokinetics, and opsonophagocytic activity of pagibaximab in healthy adults. Eight participants were enrolled (four in each dose group). No infusion, drug, or dose related adverse events occurred. Serum anti-LTA levels were dose-related; mean concentrations peaked at 87.75 and 259.24 microg/mL for 3 and 10 mg/kg groups, respectively. The half-life (beta) of pagibaximab was approximately 33 days. Opsonophagocytic activity of serum samples on a human clinical isolate of Staphylococcus epidermidis in a standard bacterial killing assay was dose-related, and peaked at a mean of 88.5 and 95.5% at 1:90 dilution for 3 and 10 mg/kg groups, respectively. Serum anti-LTA and opsonophagocytic activity levels exhibited statistically significant correlation. The results suggest that pagibaximab at 3 and 10 mg/kg administered as a single intravenous dose in healthy adults appears to: 1) provide preliminary safety and tolerability data, 2) produce dose-related serum anti-LTA and opsonophagocytic activity levels, 3) have a half-life similar to other immunoglobulin G1 antibodies, 4) exhibit statistically significant correlation between serum anti-LTA and opsonophagocytic activity levels. This study supports conducting safety and pharmacokinetic trials of pagibaximab in populations at high-risk of developing CONS infection.
Asunto(s)
Antibacterianos/farmacocinética , Anticuerpos Monoclonales/farmacocinética , Lipopolisacáridos/inmunología , Neutrófilos/metabolismo , Infecciones Estafilocócicas/terapia , Staphylococcus aureus/inmunología , Staphylococcus epidermidis/inmunología , Ácidos Teicoicos/inmunología , Adulto , Animales , Antibacterianos/administración & dosificación , Antibacterianos/efectos adversos , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Relación Dosis-Respuesta a Droga , Femenino , Semivida , Humanos , Inyecciones Intravenosas , Masculino , Ratones , Persona de Mediana Edad , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/microbiología , Neutrófilos/patología , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/farmacocinética , Prueba Bactericida de Suero , Infecciones Estafilocócicas/inmunologíaRESUMEN
OBJECTIVES: With the isolation of clinical strains of Staphylococcus aureus carrying the gene that confers vancomycin resistance, the need for novel antistaphylococcals has become more urgent. Lysostaphin, an example of such a novel therapeutic, is an endopeptidase that rapidly lyses S. aureus through proteolysis of the staphylococcal cell wall. We evaluated its efficacy as a therapeutic agent for treatment of systemic S. aureus infection in a mouse model. METHODS: Mice (5-10 per group) challenged with methicillin-susceptible S. aureus developed bacteraemia and organ infections while mice challenged with methicillin-resistant S. aureus (MRSA) developed organ infections. The challenged mice received various intravenous doses of recombinant lysostaphin, administered once a day for 1-3 days when compared with treatment with oxacillin or vancomycin. Some mice also received treatment of lysostaphin combined with oxacillin or vancomycin. Following treatment, bacteraemia was determined, and mice were sacrificed and organ infection was determined. RESULTS AND CONCLUSIONS: Lysostaphin administered at 5 mg/kg once a day for 3 days consistently cleared S. aureus from the blood and the organs of infected mice. Furthermore, the combination of lysostaphin and oxacillin or vancomycin demonstrated increased efficacy against MRSA over lysostaphin alone allowing the therapeutic dose of lysostaphin to be reduced to 1 mg/kg. These results demonstrate that lysostaphin is an effective treatment for eradicating S. aureus from the blood and from the organs of infected mice.
Asunto(s)
Antibacterianos/uso terapéutico , Lisostafina/uso terapéutico , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus , Animales , Bacteriemia/tratamiento farmacológico , Bacteriemia/microbiología , Relación Dosis-Respuesta a Droga , Femenino , Riñón/microbiología , Hígado/microbiología , Resistencia a la Meticilina , Ratones , Neutropenia , Neutrófilos/microbiología , Bazo/microbiología , Staphylococcus aureus/efectos de los fármacosRESUMEN
Oxidative-stress resistance in Staphylococcus aureus is linked to metal ion homeostasis via several interacting regulators. In particular, PerR controls the expression of a regulon of genes, many of which encode antioxidants. Two PerR regulon members, ahpC (alkylhydroperoxide reductase) and katA (catalase), show compensatory regulation, with independent and linked functions. An ahpC mutation leads to increased H2O2 resistance due to greater katA expression via relief of PerR repression. Moreover, AhpC provides residual catalase activity present in a katA mutant. Mutation of both katA and ahpC leads to a severe growth defect under aerobic conditions in defined media (attributable to lack of catalase activity). This results in the inability to scavenge exogenous or endogenously produced H2O2, resulting in accumulation of H2O2 in the medium. This leads to DNA damage, the likely cause of the growth defect. Surprisingly, the katA ahpC mutant is not attenuated in two independent models of infection, which implies reduced oxygen availability during infection. In contrast, both AhpC and KatA are required for environmental persistence (desiccation) and nasal colonization. Thus, oxidative-stress resistance is an important factor in the ability of S. aureus to persist in the hospital environment and so contribute to the spread of human disease.
Asunto(s)
Proteínas Bacterianas/metabolismo , Catalasa/metabolismo , Cavidad Nasal/microbiología , Peroxidasas/metabolismo , Staphylococcus aureus/metabolismo , Animales , Antioxidantes/metabolismo , Antioxidantes/fisiología , Proteínas Bacterianas/genética , Catalasa/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Bacterianos , Prueba de Complementación Genética , Glucosa/deficiencia , Glucosa/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Masculino , Viabilidad Microbiana/genética , Mutación , Estrés Oxidativo , Peroxidasas/genética , Ratas , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Virulencia/genéticaRESUMEN
Resistance to human skin innate defenses is crucial for survival and carriage of Staphylococcus aureus, a common cutaneous pathogen and nasal colonizer. Free fatty acids extracted from human skin sebum possess potent antimicrobial activity against S. aureus. The mechanisms by which S. aureus overcomes this host defense during colonization remain unknown. Here, we show that S. aureus IsdA, a surface protein produced in response to the host, decreases bacterial cellular hydrophobicity rendering them resistant to bactericidal human skin fatty acids and peptides. IsdA is required for survival of S. aureus on live human skin. Reciprocally, skin fatty acids prevent the production of virulence determinants and the induction of antibiotic resistance in S. aureus and other Gram-positive pathogens. A purified human skin fatty acid was effective in treating systemic and topical infections of S. aureus suggesting that our natural defense mechanisms can be exploited to combat drug-resistant pathogens.
Asunto(s)
Antígenos Bacterianos/fisiología , Piel/inmunología , Infecciones Cutáneas Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Adulto , Antibacterianos/farmacología , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Recuento de Colonia Microbiana , Ácidos Grasos/aislamiento & purificación , Ácidos Grasos/farmacología , Proteínas Hemolisinas/biosíntesis , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Viabilidad Microbiana , Sebo/inmunología , Sebo/microbiología , Piel/química , Piel/microbiología , Infecciones Cutáneas Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , Factores de Virulencia/biosíntesisRESUMEN
Bacterial cell wall peptidoglycan is a dynamic structure requiring hydrolysis to allow cell wall growth and division. Staphylococcus aureus has many known and putative peptidoglycan hydrolases, including two likely lytic transglycosylases. These two proteins, IsaA and SceD, were both found to have autolytic activity. Regulatory studies showed that the isaA and sceD genes are partially mutually compensatory and that the production of SceD is upregulated in an isaA mutant. The expression of sceD is also greatly upregulated by the presence of NaCl. Several regulators of isaA and sceD expression were identified. Inactivation of sceD resulted in impaired cell separation, as shown by light microscopy, and "clumping" of bacterial cultures. An isaA sceD mutant is attenuated for virulence, while SceD is essential for nasal colonization in cotton rats, thus demonstrating the importance of cell wall dynamics in host-pathogen interactions.
Asunto(s)
Antígenos Bacterianos/fisiología , Proteínas Bacterianas/fisiología , Glicosiltransferasas/fisiología , Peptidoglicano Glicosiltransferasa/fisiología , Staphylococcus aureus/enzimología , Animales , Antígenos Bacterianos/genética , Artritis Infecciosa/microbiología , Proteínas Bacterianas/genética , Bacteriólisis , Portador Sano/microbiología , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Glicosiltransferasas/genética , Ratones , Viabilidad Microbiana , Mutagénesis Insercional , Peptidoglicano Glicosiltransferasa/genética , Sigmodontinae , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Factores de Virulencia/genética , Factores de Virulencia/fisiologíaRESUMEN
A spectrum of in vivo-expressed Staphylococcus epidermidis antigens was identified by probing a bacteriophage lambda library of S. epidermidis genomic DNA with human serum from infected and uninfected individuals. This analysis resulted in identification of 53 antigen-encoding loci. Six antigenic polypeptides were expressed from these loci and purified. These polypeptides were the propeptide, mature amidase, and repeat sequence domains of the major autolysin AtlE, GehD (lipase), and two members of a conserved family of surface proteins (ScaA [AaE] and ScaB). AtlE, ScaA, and ScaB all exhibit human ligand binding capacity. Screening a bank of human serum samples revealed that there were significant increases in the amounts of reactive immunoglobulin G in infected individuals compared to the amounts in healthy individuals for the repeat sequence and mature amidase domains of AtlE, ScaB, and GehD. Vaccination of mice with recombinant antigens stimulated an immune response which in vitro opsonized S. epidermidis. In this study we identified prospective candidate antigens for prophylaxis or immunotherapy to control disease.
Asunto(s)
Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus epidermidis/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Bacteriófago lambda , Femenino , Biblioteca de Genes , Humanos , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Opsoninas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
A spectrum of in vivo-expressed Staphylococcus aureus antigens was identified by probing bacteriophage expression libraries of S. aureus with serum samples from infected and uninfected individuals. Eleven recombinant antigenic proteins were produced, and specific antibody titers in a large collection of human serum samples were determined. Significantly increased concentrations of reactive immunoglobulin G (IgG) to 7 antigens were found in serum samples from ill individuals, compared with those in healthy individuals. Significantly higher concentrations of reactive IgG to 4 antigens, including iron-responsive surface determinant (Isd) A and IsdH, were found in serum samples from healthy individuals who were not nasal carriers of S. aureus, compared with those in healthy carriers. Vaccination of cotton rats with IsdA or IsdH protected against nasal carriage. Also, IsdA is involved in adherence of S. aureus to human desquamated nasal epithelial cells and is required for nasal colonization in the cotton rat model. Thus, vaccination with these antigens may prevent S. aureus carriage and reduce the prevalence of human disease.