RESUMEN
More evidence is needed to support recommendations for medical management of acute radiation syndrome (ARS) and associated infections resulting from a radiological/nuclear event. While current guidelines recommend the administration of antibiotics to chemotherapy patients with febrile neutropenia, the clinical benefit is unclear for acute radiation injury patients. A well-characterized nonhuman primate (NHP) model of hematopoietic ARS was developed that incorporates supportive care postirradiation. This model evaluated the efficacy of myeloid growth factors within 24 to 48 h after total body irradiation (TBI). However, in this model, NHPs continued to develop life-threatening bacterial infections, even when granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor was administered in combination with antibiotic monotherapy. In this study, we evaluated the efficacy of combination antibiotic therapies administered to NHPs following 7.4-Gy TBI to understand the occurrence of bacterial infection in NHPs with hematopoietic ARS. We compared enrofloxacin-linezolid, enrofloxacin-cefepime, and enrofloxacin-ertapenem to enrofloxacin monotherapy. The primary endpoint was 60-day postirradiation mortality, with secondary endpoints of overall survival time, incidence of bacterial infection, and bacteriologic culture with antimicrobial susceptibility testing. We observed that enrofloxacin-ertapenem significantly increased survival compared to enrofloxacin monotherapy. Bacteria isolated from nonsurviving macaques with systemic bacterial infections exhibited uniform resistance to enrofloxacin and variable resistance to beta-lactam antibiotics, linezolid, gentamicin, and azithromycin. Multidrug antibiotic resistance was observed in Enterococcus spp. and Escherichia coli. We conclude that antibiotic combination therapies appear to be more effective than monotherapy alone but acknowledge that more work is needed to identify an optimal antimicrobial therapy.
Asunto(s)
Síndrome de Radiación Aguda , Antiinfecciosos , Infecciones Bacterianas , Animales , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Enrofloxacina , Ertapenem/uso terapéutico , Linezolid/uso terapéutico , Azitromicina/uso terapéutico , Cefepima/uso terapéutico , Síndrome de Radiación Aguda/tratamiento farmacológico , Síndrome de Radiación Aguda/etiología , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antiinfecciosos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/complicaciones , Dosis de Radiación , Gentamicinas/uso terapéuticoRESUMEN
Rapid presymptomatic diagnosis of Bacillus anthracis at early stages of infection plays a crucial role in prompt medical intervention to prevent rapid disease progression and accumulation of lethal levels of toxin. To detect low levels of the anthrax protective antigen (PA) exotoxin in biological fluids, we have developed a metal-enhanced fluorescence (MEF)-PA assay using a combination of the MEF effect and microwave-accelerated PA protein surface absorption. The assay is based on a modified version of our "rapid catch and signal" (RCS) technology previously designed for the ultra-fast and sensitive analysis of genomic DNA sequences. Technologically, the proposed MEF-PA assay uses standard 96-well plastic plates modified with silver island films (SiFs) grown within the wells. It is shown that the fluorescent probe, covalently attached to the secondary antibody, plays a crucial role of indicating complex formation (i.e., shows a strong MEF response to the recognition event). Microwave irradiation rapidly accelerates PA deposition onto the surface ("rapid catch"), significantly speeding up the MEF-PA assay and resulting in a total assay run time of less than 40 min with an analytical sensitivity of less than 1 pg/ml PA.
Asunto(s)
Antígenos Bacterianos/análisis , Toxinas Bacterianas/análisis , Fluorescencia , Microondas , Plata/química , Antígenos Bacterianos/química , Bacillus anthracis/inmunología , Bacillus anthracis/aislamiento & purificación , Toxinas Bacterianas/química , Metales/químicaRESUMEN
On March 12, 2021, the Biomedical Advanced Research and Development Authority (BARDA) sponsored a virtual market research workshop, "Partnering on Vaccines to Counter Multi-Drug Resistant Threats," to discuss the threat of antimicrobial resistance in the context of BARDA's mission space and the challenges encountered during the development of vaccines for specific antimicrobial resistant bacteria. The workshop convened representatives with expertise in vaccine development from government, academia, and industry. This report summarizes the presentations and subsequent discussions from the workshop and highlights existing challenges to advance the development of vaccine candidates for antimicrobial resistant pathogens, including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus.
Asunto(s)
Antibacterianos , Vacunas , Antibacterianos/uso terapéutico , Bacterias , Farmacorresistencia Bacteriana , Escherichia coli , Pruebas de Sensibilidad Microbiana , Investigación , Staphylococcus aureusRESUMEN
The health and economic burden of infectious diseases in general and bioterrorism in particular necessitate the development of medical countermeasures. One proven approach to reduce the disease burden and spread of pathogen is treatment with monoclonal antibodies (mAb). mAbs can prevent or reduce severity of the disease by variety of mechanisms, including neutralizing pathogen growth, limiting its spread from infected to adjacent cells, or by inhibiting biological activity of toxins, such as anthrax lethal toxin. Here, we report the production of glycosylated (pp-mAb (PA) ) and non-glycosylated (pp-mAb (PANG) ) versions of a plant-derived mAb directed against protective antigen (PA) of Bacillus anthracis in Nicotiana benthamiana plants using agroinfiltration. Both forms of the antibody were able to neutralize anthrax lethal toxin activity in vitro and protect mice against an intraperitoneal challenge with spores of B. anthracis Sterne strain. A single 180 µg intraperitoneal dose of pp-mAb (PA) or pp-mAb (PANG) provided 90% and 100% survival, respectively. When tested in non-human primates, pp-mAb (PANG) was demonstrated to be superior to pp-mAb (PA) in that it had a significantly longer terminal half-life and conferred 100% protection against a lethal dose of aerosolized anthrax spore challenge after a single 5 mg/kg intravenous dose compared to a 40% survival rate conferred by pp-mAb (PA) . This study demonstrates the potential of a plant-produced non-glycosylated antibody as a useful tool for the treatment of inhalation anthrax.
Asunto(s)
Carbunco/terapia , Anticuerpos Antibacterianos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Antitoxinas/uso terapéutico , Toxinas Bacterianas/antagonistas & inhibidores , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/metabolismo , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Antígenos Bacterianos , Antitoxinas/genética , Antitoxinas/metabolismo , Modelos Animales de Enfermedad , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Plantas Modificadas Genéticamente/genética , Enfermedades de los Primates/terapia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Enfermedades de los Roedores/terapia , Análisis de Supervivencia , Nicotiana/genética , Resultado del TratamientoRESUMEN
Toll-like receptor (TLR) agonists induce potent innate immune responses and can be used in the development of novel vaccine adjuvants. However, access to TLRs can be challenging as exemplified by TLR 7, which is located intracellularly in endosomal compartments. To increase recognition and subsequent stimulatory effects of TLR 7, imiquimod was encapsulated in acetalated dextran (Ac-DEX) microparticles. Ac-DEX, a water-insoluble and biocompatible polymer, is relatively stable at pH 7.4, but degrades rapidly under acidic conditions, such as those found in lysosomal vesicles. To determine the immunostimulatory capacity of encapsulated imiquimod, we compared the efficacy of free versus encapsulated imiquimod in activating RAW 264.7 macrophages, MH-S macrophages, and bone marrow derived dendritic cells. Encapsulated imiquimod significantly increased IL-1 beta, IL-6, and TNF-alpha cytokine expression in macrophages relative to the free drug. Furthermore, significant increases were observed in classic macrophage activation markers (iNOS, PD1-L1, and NO) after treatment with encapsulated imiquimod over the free drug. Also, bone marrow derived dendritic cells produced significantly higher levels of IL-1 beta, IL-6, IL-12p70, and MIP-1 alpha as compared to their counterparts receiving free imiquimod. These results suggest that encapsulation of TLR ligands within Ac-DEX microparticles results in increased immunostimulation and potentially better protection from disease when used in conjunction with vaccine formulations.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Dextranos/química , Nanopartículas/química , Adyuvantes Inmunológicos/química , Aminoquinolinas/administración & dosificación , Aminoquinolinas/química , Animales , Línea Celular , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Imiquimod , Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Activación de Macrófagos/efectos de los fármacos , Ratones , Microscopía Electrónica de Rastreo , Nanopartículas/ultraestructura , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Pseudomonas aeruginosa and Burkholderia cepacia infections of cystic fibrosis patients' lungs are often resistant to conventional antibiotic therapy. Protegrins are antimicrobial peptides with potent activity against many bacteria, including P. aeruginosa. The present study evaluates the correlation between protegrin-1 (PG-1) sensitivity/resistance and protegrin binding in P. aeruginosa and B. cepacia. METHODS: The PG-1 sensitivity/resistance and PG-1 binding properties of P. aeruginosa and B. cepacia were assessed using radial diffusion assays, radioiodinated PG-1, and surface plasmon resonance (BiaCore). RESULTS: The six P. aeruginosa strains examined were very sensitive to PG-1, exhibiting minimal active concentrations from 0.0625-0.5 microg/ml in radial diffusion assays. In contrast, all five B. cepacia strains examined were greater than 10-fold to 100-fold more resistant, with minimal active concentrations ranging from 6-10 microg/ml. When incubated with a radioiodinated variant of PG-1, a sensitive P. aeruginosa strain bound considerably more protegrin molecules per cell than a resistant B. cepacia strain. Binding/diffusion and surface plasmon resonance assays revealed that isolated lipopolysaccharide (LPS) and lipid A from the sensitive P. aeruginosa strains bound PG-1 more effectively than LPS and lipid A from resistant B. cepacia strains. CONCLUSION: These findings support the hypothesis that the relative resistance of B. cepacia to protegrin is due to a reduced number of PG-1 binding sites on the lipid A moiety of its LPS.
Asunto(s)
Antibacterianos/metabolismo , Burkholderia cepacia/metabolismo , Proteínas/metabolismo , Pseudomonas aeruginosa/metabolismo , Péptidos Catiónicos Antimicrobianos , Burkholderia cepacia/genética , Burkholderia cepacia/aislamiento & purificación , Farmacorresistencia Microbiana , Humanos , Lípido A/metabolismo , Lipopolisacáridos/metabolismo , Pruebas de Sensibilidad Microbiana , Unión Proteica/fisiología , Proteínas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/aislamiento & purificaciónRESUMEN
On August 22-23, 2013, agencies within the United States Department of Defense (DoD) and the Department of Health and Human Services (HHS) sponsored the Filovirus Medical Countermeasures (MCMs) Workshop as an extension of the activities of the Filovirus Animal Non-clinical Group (FANG). The FANG is a federally-recognized multi-Agency group established in 2011 to coordinate and facilitate U.S. government (USG) efforts to develop filovirus MCMs. The workshop brought together government, academic and industry experts to consider the needs for filovirus MCMs and evaluate the status of the product development pipeline. This report summarizes speaker presentations and highlights progress and challenges remaining in the field.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Infecciones por Filoviridae/prevención & control , Filoviridae/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/administración & dosificación , Filoviridae/patogenicidad , Infecciones por Filoviridae/inmunología , Infecciones por Filoviridae/virología , Cobayas , Haplorrinos , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Ratones , Estados Unidos , United States Department of Defense , United States Dept. of Health and Human Services , Vacunas de ADN , Vacunas Virales/administración & dosificación , Vacunas Virales/biosíntesis , Replicación Viral/efectos de los fármacosRESUMEN
The efficacy of multi-agent DNA vaccines consisting of a truncated gene encoding Bacillus anthracis lethal factor (LFn) fused to either Yersinia pestis V antigen (V) or Y . pestis F1 was evaluated. A/J mice were immunized by gene gun and developed predominantly IgG1 responses that were fully protective against a lethal aerosolized B. anthracis spore challenge but required the presence of an additional DNA vaccine expressing anthrax protective antigen to boost survival against aerosolized Y. pestis.
Asunto(s)
Vacunas contra el Carbunco/inmunología , Carbunco/prevención & control , Vacuna contra la Peste/inmunología , Peste/prevención & control , Animales , Carbunco/inmunología , Carbunco/mortalidad , Vacunas contra el Carbunco/administración & dosificación , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Bacillus anthracis/patogenicidad , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Biolística , Inmunización , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Peste/inmunología , Peste/mortalidad , Vacuna contra la Peste/administración & dosificación , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Esporas Bacterianas/inmunología , Esporas Bacterianas/patogenicidad , Tasa de Supervivencia , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Yersinia pestis/inmunologíaRESUMEN
Electroporation of DNA vaccines represents a platform technology well positioned for the development of multivalent biodefense vaccines. To evaluate this hypothesis, three vaccine constructs were produced using codon-optimized genes encoding Bacillus anthracis Protective Antigen (PA), and the Yersinia pestis genes LcrV and F1, cloned into pVAX1. A/J mice were immunized on a prime-boost schedule with these constructs using the electroporation-based TriGrid Delivery System. Immunization with the individual pDNA vaccines elicited higher levels of antigen-specific IgG than when used in combination. DNA vaccine effectiveness was proven, the pVAX-PA titers were toxin neutralizing and fully protective against a lethal B. anthracis spore challenge when administered alone or co-formulated with the plague pDNA vaccines. LcrV and F1 pVAX vaccines against plague were synergistic, resulting in 100% survival, but less protective individually and when co-formulated with pVAX-PA. These DNA vaccine responses were Th1/Th2 balanced with high levels of IFN-γ and IL-4 in splenocyte recall assays, contrary to complimentary protein Alum vaccinations displaying a Th2 bias with increased IL-4 and low levels of IFN-γ. These results demonstrate the feasibility of electroporation to deliver and maintain the overall efficacy of an anthrax-plague DNA vaccine cocktail whose individual components have qualitative immunological differences when combined.
Asunto(s)
Vacunas contra el Carbunco/administración & dosificación , Electroporación , Inmunización/métodos , Vacuna contra la Peste/administración & dosificación , Vacunas de ADN/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Animales , Carbunco/prevención & control , Vacunas contra el Carbunco/inmunología , Anticuerpos Antibacterianos/sangre , Formación de Anticuerpos , Estudios de Factibilidad , Femenino , Inmunoglobulina G/sangre , Interferón gamma/inmunología , Interleucina-4/inmunología , Ratones , Peste/prevención & control , Vacuna contra la Peste/inmunología , Plásmidos/administración & dosificación , Plásmidos/inmunología , Bazo/inmunología , Balance Th1 - Th2 , Vacunas Combinadas/administración & dosificación , Vacunas de ADN/inmunologíaRESUMEN
The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-mug dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] of B. anthracis Sterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected with B. anthracis Sterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.
Asunto(s)
Carbunco/tratamiento farmacológico , Carbunco/prevención & control , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Antitoxinas/farmacología , Bacillus anthracis/inmunología , Toxinas Bacterianas/inmunología , Animales , Carbunco/inmunología , Anticuerpos Antibacterianos/sangre , Femenino , Humanos , Hibridomas , Inmunización Pasiva , Inmunoglobulina G/sangre , Concentración 50 Inhibidora , Ratones , Análisis de SupervivenciaRESUMEN
To determine whether AlgL's lyase activity is required for alginate production in Pseudomonas aeruginosa, an algLdelta::Gm(r) mutant (FRD-MA7) was created. algL complementation of FRD-MA7 restored alginate production, but algL constructs containing mutations inactivating lyase activity did not, demonstrating that the enzymatic activity of AlgL is required for alginate production.