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1.
Am J Respir Crit Care Med ; 210(1): 35-46, 2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38754132

RESUMEN

Rationale: Pseudomonas aeruginosa infection is associated with worse outcomes in bronchiectasis. Impaired neutrophil antimicrobial responses contribute to bacterial persistence. Gremubamab is a bivalent, bispecific monoclonal antibody targeting Psl exopolysaccharide and the type 3 secretion system component PcrV. Objectives: This study evaluated the efficacy of gremubamab to enhance killing of P. aeruginosa by neutrophils from patients with bronchiectasis and to prevent P. aeruginosa-associated cytotoxicity. Methods: P. aeruginosa isolates from a global bronchiectasis cohort (n = 100) underwent whole-genome sequencing to determine target prevalence. Functional activity of gremubamab against selected isolates was tested in vitro and in vivo. Patients with bronchiectasis (n = 11) and control subjects (n = 10) were enrolled, and the effect of gremubamab in peripheral blood neutrophil opsonophagocytic killing (OPK) assays against P. aeruginosa was evaluated. Serum antibody titers to Psl and PcrV were determined (n = 30; 19 chronic P. aeruginosa infection, 11 no known P. aeruginosa infection), as was the effect of gremubamab treatment in OPK and anti-cytotoxic activity assays. Measurements and Main Results: Psl and PcrV were conserved in isolates from chronically infected patients with bronchiectasis. Seventy-three of 100 isolates had a full psl locus, and 99 of 100 contained the pcrV gene, with 20 distinct full-length PcrV protein subtypes identified. PcrV subtypes were successfully bound by gremubamab and the monoclonal antibody-mediated potent protective activity against tested isolates. Gremubamab increased bronchiectasis patient neutrophil-mediated OPK (+34.6 ± 8.1%) and phagocytosis (+70.0 ± 48.8%), similar to effects observed in neutrophils from control subjects (OPK, +30.1 ± 7.6%). No evidence of competition between gremubamab and endogenous antibodies was found, with protection against P. aeruginosa-induced cytotoxicity and enhanced OPK demonstrated with and without addition of patient serum. Conclusions: Gremubamab enhanced bronchiectasis patient neutrophil phagocytosis and killing of P. aeruginosa and reduced virulence.


Asunto(s)
Anticuerpos Biespecíficos , Bronquiectasia , Neutrófilos , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Humanos , Bronquiectasia/inmunología , Bronquiectasia/microbiología , Pseudomonas aeruginosa/inmunología , Neutrófilos/inmunología , Neutrófilos/efectos de los fármacos , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Biespecíficos/farmacología , Femenino , Masculino , Infecciones por Pseudomonas/inmunología , Persona de Mediana Edad , Anciano , Adulto , Antígenos Bacterianos , Toxinas Bacterianas , Proteínas Citotóxicas Formadoras de Poros
2.
Infect Immun ; 90(10): e0020322, 2022 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-36069593

RESUMEN

The Gram-negative pathogen Pseudomonas aeruginosa is a common cause of pneumonia in hospitalized patients. Its increasing antibiotic resistance and widespread occurrence present a pressing need for vaccines. We previously showed that a P. aeruginosa type III secretion system protein, PopB, elicits a strong Th17 response in mice after intranasal (IN) immunization and confers antibody-independent protection against pneumonia in mice. In the current study, we evaluated the immunogenicity and protective efficacy in mice of the combination of PopB (purified with its chaperone protein PcrH) and OprF/I, an outer membrane hybrid fusion protein, compared with immunization with the proteins individually either by the intranasal (IN) or subcutaneous (SC) routes. Our results show that after vaccination, a Th17 recall response from splenocytes was detected only in mice vaccinated with PopB/PcrH, either alone or in combination with OprF/I. Mice immunized with the combination of PopB/PcrH and OprF/I had enhanced protection in an acute lethal P. aeruginosa pneumonia model, regardless of vaccine route, compared with mice vaccinated with either alone or adjuvant control. Immunization generated IgG titers against the vaccine proteins and whole P. aeruginosa cells. Interestingly, none of these antisera had opsonophagocytic killing activity, but antisera from mice immunized with vaccines containing OprF/I, had the ability to block IFN-γ binding to OprF/I, a known virulence mechanism. Hence, vaccines combining PopB/PcrH with OprF/I that elicit functional antibodies lead to a broadly and potently protective vaccine against P. aeruginosa pulmonary infections.


Asunto(s)
Neumonía , Infecciones por Pseudomonas , Ratones , Animales , Vacunas contra la Infección por Pseudomonas , Pseudomonas aeruginosa , Infecciones por Pseudomonas/prevención & control , Células Th17 , Sistemas de Secreción Tipo III , Formación de Anticuerpos , Anticuerpos Antibacterianos , Proteínas Bacterianas , Inmunoglobulina G , Sueros Inmunes
3.
Crit Care ; 26(1): 355, 2022 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-36380312

RESUMEN

BACKGROUND: Ventilator-associated pneumonia caused by Pseudomonas aeruginosa (PA) in hospitalised patients is associated with high mortality. The effectiveness of the bivalent, bispecific mAb MEDI3902 (gremubamab) in preventing PA nosocomial pneumonia was assessed in PA-colonised mechanically ventilated subjects. METHODS: EVADE (NCT02696902) was a phase 2, randomised, parallel-group, double-blind, placebo-controlled study in Europe, Turkey, Israel, and the USA. Subjects ≥ 18 years old, mechanically ventilated, tracheally colonised with PA, and without new-onset pneumonia, were randomised (1:1:1) to MEDI3902 500, 1500 mg (single intravenous dose), or placebo. The primary efficacy endpoint was the incidence of nosocomial PA pneumonia through 21 days post-dose in MEDI3902 1500 mg versus placebo, determined by an independent adjudication committee. RESULTS: Even if the initial sample size was not reached because of low recruitment, 188 subjects were randomised (MEDI3902 500/1500 mg: n = 16/87; placebo: n = 85) between 13 April 2016 and 17 October 2019. Out of these, 184 were dosed (MEDI3902 500/1500 mg: n = 16/85; placebo: n = 83), comprising the modified intent-to-treat set. Enrolment in the 500 mg arm was discontinued due to pharmacokinetic data demonstrating low MEDI3902 serum concentrations. Subsequently, enrolled subjects were randomised (1:1) to MEDI3902 1500 mg or placebo. PA pneumonia was confirmed in 22.4% (n = 19/85) of MEDI3902 1500 mg recipients and in 18.1% (n = 15/83) of placebo recipients (relative risk reduction [RRR]: - 23.7%; 80% confidence interval [CI] - 83.8%, 16.8%; p = 0.49). At 21 days post-1500 mg dose, the mean (standard deviation) serum MEDI3902 concentration was 9.46 (7.91) µg/mL, with 80.6% (n = 58/72) subjects achieving concentrations > 1.7 µg/mL, a level associated with improved outcome in animal models. Treatment-emergent adverse event incidence was similar between groups. CONCLUSIONS: The bivalent, bispecific monoclonal antibody MEDI3902 (gremubamab) did not reduce PA nosocomial pneumonia incidence in PA-colonised mechanically ventilated subjects. Trial registration Registered on Clinicaltrials.gov ( NCT02696902 ) on 11th February 2016 and on EudraCT ( 2015-001706-34 ) on 7th March 2016.


Asunto(s)
Neumonía Asociada al Ventilador , Infecciones por Pseudomonas , Animales , Humanos , Adolescente , Pseudomonas aeruginosa , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/prevención & control , Respiración Artificial/efectos adversos , Neumonía Asociada al Ventilador/tratamiento farmacológico , Método Doble Ciego , Unidades de Cuidados Intensivos , Anticuerpos Monoclonales/uso terapéutico , Resultado del Tratamiento
4.
Artículo en Inglés | MEDLINE | ID: mdl-32152087

RESUMEN

Antibiotics revolutionized the treatment of infectious diseases; however, it is now clear that broad-spectrum antibiotics alter the composition and function of the host's microbiome. The microbiome plays a key role in human health, and its perturbation is increasingly recognized as contributing to many human diseases. Widespread broad-spectrum antibiotic use has also resulted in the emergence of multidrug-resistant pathogens, spurring the development of pathogen-specific strategies such as monoclonal antibodies (MAbs) to combat bacterial infection. Not only are pathogen-specific approaches not expected to induce resistance in nontargeted bacteria, but they are hypothesized to have minimal impact on the gut microbiome. Here, we compare the effects of antibiotics, pathogen-specific MAbs, and their controls (saline or control IgG [c-IgG]) on the gut microbiome of 7-week-old, female, C57BL/6 mice. The magnitude of change in taxonomic abundance, bacterial diversity, and bacterial metabolites, including short-chain fatty acids (SCFA) and bile acids in the fecal pellets from mice treated with pathogen-specific MAbs, was no different from that with animals treated with saline or an IgG control. Conversely, dramatic changes were observed in the relative abundance, as well as alpha and beta diversity, of the fecal microbiome and bacterial metabolites in the feces of all antibiotic-treated mice. Taken together, these results indicate that pathogen-specific MAbs do not alter the fecal microbiome like broad-spectrum antibiotics and may represent a safer, more-targeted approach to antibacterial therapy.


Asunto(s)
Antibacterianos/farmacología , Anticuerpos Monoclonales/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Animales , Ácidos y Sales Biliares/metabolismo , ADN Bacteriano/análisis , Ácidos Grasos/metabolismo , Heces/microbiología , Femenino , Ratones , Ratones Endogámicos C57BL , ARN Ribosómico 16S/genética , Organismos Libres de Patógenos Específicos
5.
J Clin Microbiol ; 59(1)2020 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-33087438

RESUMEN

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among infants and young children, resulting in annual epidemics worldwide. INFORM-RSV is a multiyear clinical study designed to describe the global molecular epidemiology of RSV in children under 5 years of age by monitoring temporal and geographical evolution of current circulating RSV strains, F protein antigenic sites, and their relationships with clinical features of RSV disease. During the pilot season (2017-2018), 410 RSV G-F gene sequences were obtained from 476 RSV-positive nasal samples collected from 8 countries (United Kingdom, Spain, The Netherlands, Finland, Japan, Brazil, South Africa, and Australia). RSV B (all BA9 genotype) predominated over RSV A (all ON1 genotype) globally (69.0% versus 31.0%) and in all countries except South Africa. Geographic clustering patterns highlighted wide transmission and continued evolution with viral spread. Most RSV strains were from infants of <1 year of age (81.2%), males (56.3%), and patients hospitalized for >24 h (70.5%), with no differences in subtype distribution. Compared to 2013 reference sequences, variations at F protein antigenic sites were observed for both RSV A and B strains, with high-frequency polymorphisms at antigenic site Ø (I206M/Q209R) and site V (L172Q/S173L/K191R) in RSV B strains. The INFORM-RSV 2017-2018 pilot season establishes an important molecular baseline of RSV strain distribution and sequence variability with which to track the emergence of new strains and provide an early warning system of neutralization escape variants that may impact transmission or the effectiveness of vaccines and MAbs under development.


Asunto(s)
Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Australia , Brasil , Niño , Preescolar , Finlandia , Genotipo , Humanos , Lactante , Japón , Masculino , Epidemiología Molecular , Países Bajos , Filogenia , Infecciones por Virus Sincitial Respiratorio/epidemiología , Virus Sincitial Respiratorio Humano/genética , Sudáfrica , España , Reino Unido
6.
Respir Res ; 21(1): 77, 2020 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-32228581

RESUMEN

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by frequent exacerbation phenotypes independent of disease stage. Increasing evidence shows that the microbiota plays a role in disease progression and severity, but long-term and international multicenter assessment of the variations in viral and bacterial communities as drivers of exacerbations are lacking. METHODS: Two-hundred severe COPD patients from Europe and North America were followed longitudinally for 3 years. We performed nucleic acid detection for 20 respiratory viruses and 16S ribosomal RNA gene sequencing to evaluate the bacterial microbiota in 1179 sputum samples collected at stable, acute exacerbation and follow-up visits. RESULTS: Similar viral and bacterial taxa were found in patients from the USA compared to Bulgaria and Czech Republic but their microbiome diversity was significantly different (P < 0.001) and did not impact exacerbation rates. Virus infection was strongly associated with exacerbation events (P < 5E-20). Human rhinovirus (13.1%), coronavirus (5.1%) and influenza virus (3.6%) constitute the top viral pathogens in triggering exacerbation. Moraxella and Haemophilus were 5-fold and 1.6-fold more likely to be the dominating microbiota during an exacerbation event. Presence of Proteobacteria such as Pseudomonas or Staphylococcus amongst others, were associated with exacerbation events (OR > 0.17; P < 0.02) but more strongly associated with exacerbation frequency (OR > 0.39; P < 4E-10), as confirmed by longitudinal variations and biotyping of the bacterial microbiota, and suggesting a role of the microbiota in sensitizing the lung. CONCLUSIONS: This study highlights bacterial taxa in lung sensitization and viral triggers in COPD exacerbations. It provides a global overview of the diverse targets for drug development and explores new microbiome analysis methods to guide future patient management applications.


Asunto(s)
Bacterias/aislamiento & purificación , Pulmón/microbiología , Pulmón/virología , Enfermedad Pulmonar Obstructiva Crónica/microbiología , Enfermedad Pulmonar Obstructiva Crónica/virología , Virus/aislamiento & purificación , Anciano , Anciano de 80 o más Años , Bacterias/genética , Carga Bacteriana , Progresión de la Enfermedad , Europa (Continente)/epidemiología , Femenino , Humanos , Incidencia , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/epidemiología , Estudios Retrospectivos , Factores de Riesgo , Esputo/microbiología , Esputo/virología , Factores de Tiempo , Estados Unidos/epidemiología , Carga Viral , Virus/genética
7.
BMC Infect Dis ; 20(1): 450, 2020 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-32591017

RESUMEN

BACKGROUND: Respiratory syncytial virus (RSV) is a global cause of severe respiratory morbidity and mortality in infants. While preventive and therapeutic interventions are being developed, including antivirals, vaccines and monoclonal antibodies, little is known about the global molecular epidemiology of RSV. INFORM is a prospective, multicenter, global clinical study performed by ReSViNET to investigate the worldwide molecular diversity of RSV isolates collected from children less than 5 years of age. METHODS: The INFORM study is performed in 17 countries spanning all inhabited continents and will provide insight into the molecular epidemiology of circulating RSV strains worldwide. Sequencing of > 4000 RSV-positive respiratory samples is planned to detect temporal and geographical molecular patterns on a molecular level over five consecutive years. Additionally, RSV will be cultured from a subset of samples to study the functional implications of specific mutations in the viral genome including viral fitness and susceptibility to different monoclonal antibodies. DISCUSSION: The sequencing and functional results will be used to investigate susceptibility and resistance to novel RSV preventive or therapeutic interventions. Finally, a repository of globally collected RSV strains and a database of RSV sequences will be created.


Asunto(s)
Genoma Viral , Epidemiología Molecular/métodos , Polimorfismo Genético , Infecciones por Virus Sincitial Respiratorio/epidemiología , Infecciones por Virus Sincitial Respiratorio/prevención & control , Virus Sincitial Respiratorio Humano/genética , Anticuerpos Monoclonales/uso terapéutico , Antivirales/efectos adversos , Antivirales/uso terapéutico , Preescolar , Farmacorresistencia Bacteriana/genética , Femenino , Genotipo , Humanos , Inmunización Pasiva , Lactante , Recién Nacido , Masculino , Estudios Prospectivos , Virus Sincitial Respiratorio Humano/inmunología , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
8.
Antimicrob Agents Chemother ; 60(9): 5312-21, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27324766

RESUMEN

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.


Asunto(s)
Antiinflamatorios/farmacología , Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Proteínas Hemolisinas/antagonistas & inhibidores , Neumonía Asociada al Ventilador/prevención & control , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Antiinflamatorios/química , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales Humanizados , Anticuerpos Neutralizantes/química , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Anticuerpos ampliamente neutralizantes , Niño , Preescolar , Secuencia Conservada , Monitoreo Epidemiológico , Femenino , Salud Global , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/inmunología , Humanos , Lactante , Recién Nacido , Tiempo de Internación/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Mutación , Neumonía Asociada al Ventilador/epidemiología , Neumonía Asociada al Ventilador/microbiología , Neumonía Asociada al Ventilador/patología , Conformación Proteica , Análisis de Secuencia de ADN , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patología , Staphylococcus aureus/genética , Staphylococcus aureus/inmunología , Staphylococcus aureus/aislamiento & purificación
9.
J Clin Microbiol ; 53(1): 227-36, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25392350

RESUMEN

Alpha-toxin is a major Staphylococcus aureus virulence factor. This study evaluated potential relationships between in vitro alpha-toxin expression of S. aureus bloodstream isolates, anti-alpha-toxin antibody in serum of patients with S. aureus bacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwent spa typing and hla sequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encoded hla (197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%). In vitro alpha-toxin levels were inversely associated with survival (cure, 2.19 µg/ml, versus failure, 1.09 µg/ml; P < 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95; P < 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27; P < 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with alpha-toxin-expressing S. aureus isolates (P < 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis of hla revealed 12 distinct hla genotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that alpha-toxin is highly conserved in clinical S. aureus isolates. Higher in vitro alpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producing S. aureus exhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study.


Asunto(s)
Anticuerpos Antibacterianos/inmunología , Bacteriemia , Toxinas Bacterianas/genética , Expresión Génica , Variación Genética , Proteínas Hemolisinas/genética , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antibacterianos/uso terapéutico , Anticuerpos Antibacterianos/sangre , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Toxinas Bacterianas/inmunología , Femenino , Genotipo , Proteínas Hemolisinas/inmunología , Hemólisis/inmunología , Humanos , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias , Conejos , Diálisis Renal/efectos adversos , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/clasificación , Insuficiencia del Tratamiento , Resultado del Tratamiento , Adulto Joven
10.
Arch Pathol Lab Med ; 2024 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-38797720

RESUMEN

CONTEXT.­: The National Institutes of Health Genotype-Tissue Expression (GTEx) project was developed to elucidate how genetic variation influences gene expression in multiple normal tissues procured from postmortem donors. OBJECTIVE.­: To provide critical insight into a biospecimen's suitability for subsequent analysis, each biospecimen underwent quality assessment measures that included evaluation for underlying disease and potential effects introduced by preanalytic factors. DESIGN.­: Electronic images of each tissue collected from nearly 1000 postmortem donors were evaluated by board-certified pathologists for the extent of autolysis, tissue purity, and the type and abundance of any extraneous tissue. Tissue-specific differences in the severity of autolysis and RNA integrity were evaluated, as were potential relationships between these markers and the duration of postmortem interval and rapidity of death. RESULTS.­: Tissue-specific challenges in the procurement and preservation of the nearly 30 000 tissue specimens collected during the GTEx project are summarized. Differences in the degree of autolysis and RNA integrity number were observed among the 40 tissue types evaluated, and tissue-specific susceptibilities to the duration of postmortem interval and rapidity of death were observed. CONCLUSIONS.­: Ninety-five percent of tissues were of sufficient quality to support RNA sequencing analysis. Biospecimens, annotated whole slide images, de-identified clinical data, and genomic data generated for GTEx represent a high-quality and comprehensive resource for the scientific community that has contributed to its use in approximately 1695 articles. Biospecimens and data collected under the GTEx project are available via the GTEx portal and authorized access to the Database of Genotypes and Phenotypes; procedures and whole slide images are available from the National Cancer Institute.

11.
Biologicals ; 41(3): 201-8, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23313102

RESUMEN

Residual host cell DNA poses potential safety concerns for cell culture-derived vaccines or other biological products. In addition to the quantity of residual DNA, the size distribution is an important measure for determination of its associated risk factor. We have developed a new method for residual DNA size analysis, based on capillary gel electrophoresis (CGE) technology with sensitive laser induced fluorescence detection (LIF). The performance of this method was optimized through empirical selection of appropriate testing conditions and optimized conditions are presented. Examples are given to demonstrate the successful employment of this method for residual DNA size analysis of cell culture-produced vaccine samples.


Asunto(s)
ADN/análisis , Electroforesis Capilar/métodos , Vacunas Virales/biosíntesis , Animales , Línea Celular , Chlorocebus aethiops , ADN/genética , Fluorescencia , Vacunas contra la Influenza/biosíntesis , Vacunas contra la Influenza/genética , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Vacunas contra Virus Sincitial Respiratorio/biosíntesis , Vacunas contra Virus Sincitial Respiratorio/genética , Células Vero , Vacunas Virales/genética
12.
Biologicals ; 41(4): 247-53, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23665302

RESUMEN

The cold-adapted (ca) live attenuated influenza vaccine (LAIV) strains are manufactured in embryonated hens' eggs. Recently, a clonal isolate from Madin Darby Canine Kidney (MDCK) cells was derived and characterized to assess its utility as a potential cell substrate for the manufacturing of LAIV [1]. Since MDCK cells are a transformed continuous cell line [2], and low levels of residual cellular components (DNA and protein) are found in the intermediates and final filled vaccine, we sought to characterize the uptake and clearance of MDCK DNA from tissues in order to assess theoretical risks associated with manufacturing LAIV in MDCK cell culture. In order to address this concern, MDCK DNA uptake and clearance studies were performed in Sprague Dawley rats. DNA extracted from MDCK Master Cell Bank (MCB) cells was administered via an intranasal (IN) or intramuscular (IM) route. Tissue distribution and clearance of MDCK DNA were then examined in fourteen selected tissue types at selected time points post-administration using a quantitative PCR assay specific for canine (SINE) DNA. Results from these studies demonstrate that the uptake and clearance of MDCK DNA from tissues vary depending on the route of administration. When DNA was administered intranasally, as compared to intramuscularly, detectable DNA levels were lower at all time points. Thus, the intranasal route of vaccine administration appears to reduce potential risk associated with residual host cell DNA that may be present in cell culture produced final vaccine products.


Asunto(s)
ADN/farmacocinética , Administración Intranasal , Animales , Pollos , ADN/efectos adversos , ADN/química , ADN/aislamiento & purificación , ADN/farmacología , Perros , Vacunas contra la Influenza/aislamiento & purificación , Vacunas contra la Influenza/farmacología , Inyecciones Intramusculares , Células de Riñón Canino Madin Darby , Ratas , Vacunas Atenuadas/aislamiento & purificación , Vacunas Atenuadas/farmacología
13.
Wellcome Open Res ; 8: 265, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37766853

RESUMEN

Background: This article is one of a series aiming to inform analytical methods to improve comparability of estimates of ethnic health disparities based on different sources. This article explores the quality of ethnicity data and identifies potential sources of bias when ethnicity information is collected in three key NHS data sources. Future research can build on these findings to explore analytical methods to mitigate biases. Methods: Thematic analysis of semi-structured qualitative interviews to explore potential sources of error and bias in the process of collecting ethnicity information across three NHS data sources: General Practice Extraction Service (GPES) Data for Pandemic Planning and Research (GDPPR), Hospital Episode Statistics (HES) and Improving Access to Psychological Therapies (IAPT). The study included feedback from 22 experts working on different aspects of health admin data collection for England (including staff from NHS Digital, IT system suppliers and relevant healthcare service providers). Results: Potential sources of error and bias were identified across data collection, data processing and quality assurance processes. Similar issues were identified for all three sources. Our analysis revealed three main themes which can result in bias and inaccuracies in ethnicity data recorded: data infrastructure challenges, human challenges, and institutional challenges. Conclusions: Findings highlighted that analysts using health admin data should be aware of the main sources of potential error and bias in health admin data, and be mindful that the main sources of error identified are more likely to affect the ethnicity data for ethnic minority groups. Where possible, analysts should describe and seek to account for this bias in their research.

14.
Nat Commun ; 14(1): 4347, 2023 07 19.
Artículo en Inglés | MEDLINE | ID: mdl-37468530

RESUMEN

Nirsevimab is a monoclonal antibody that binds to the respiratory syncytial virus (RSV) fusion protein. During the Phase 2b (NCT02878330) and MELODY (NCT03979313) clinical trials, infants received one dose of nirsevimab or placebo before their first RSV season. In this pre-specified analysis, isolates from RSV infections were subtyped, sequenced and analyzed for nirsevimab binding site substitutions; subsequently, recombinant RSVs were engineered for microneutralization susceptibility testing. Here we show that the frequency of infections caused by subtypes A and B is similar across and within the two trials. In addition, RSV A had one and RSV B had 10 fusion protein substitutions occurring at >5% frequency. Notably, RSV B binding site substitutions were rare, except for the highly prevalent I206M:Q209R, which increases nirsevimab susceptibility; RSV B isolates from two participants had binding site substitutions that reduce nirsevimab susceptibility. Overall, >99% of isolates from the Phase 2b and MELODY trials retained susceptibility to nirsevimab.


Asunto(s)
Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Humanos , Lactante , Anticuerpos Monoclonales Humanizados/uso terapéutico , Ensayos Clínicos Controlados Aleatorios como Asunto , Proteínas Recombinantes/uso terapéutico , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/epidemiología
15.
Lancet Infect Dis ; 23(7): 856-866, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-36940703

RESUMEN

BACKGROUND: Nirsevimab is an extended half-life monoclonal antibody to the respiratory syncytial virus (RSV) fusion protein that has been developed to protect infants for an entire RSV season. Previous studies have shown that the nirsevimab binding site is highly conserved. However, investigations of the geotemporal evolution of potential escape variants in recent (ie, 2015-2021) RSV seasons have been minimal. Here, we examine prospective RSV surveillance data to assess the geotemporal prevalence of RSV A and B, and functionally characterise the effect of the nirsevimab binding-site substitutions identified between 2015 and 2021. METHODS: We assessed the geotemporal prevalence of RSV A and B and nirsevimab binding-site conservation between 2015 and 2021 from three prospective RSV molecular surveillance studies (the US-based OUTSMART-RSV, the global INFORM-RSV, and a pilot study in South Africa). Nirsevimab binding-site substitutions were assessed in an RSV microneutralisation susceptibility assay. We contextualised our findings by assessing fusion-protein sequence diversity from 1956 to 2021 relative to other respiratory-virus envelope glycoproteins using RSV fusion protein sequences published in NCBI GenBank. FINDINGS: We identified 5675 RSV A and RSV B fusion protein sequences (2875 RSV A and 2800 RSV B) from the three surveillance studies (2015-2021). Nearly all (25 [100%] of 25 positions of RSV A fusion proteins and 22 [88%] of 25 positions of RSV B fusion proteins) amino acids within the nirsevimab binding site remained highly conserved between 2015 and 2021. A highly prevalent (ie, >40·0% of all sequences) nirsevimab binding-site Ile206Met:Gln209Arg RSV B polymorphism arose between 2016 and 2021. Nirsevimab neutralised a diverse set of recombinant RSV viruses, including new variants containing binding-site substitutions. RSV B variants with reduced susceptibility to nirsevimab neutralisation were detected at low frequencies (ie, prevalence <1·0%) between 2015 and 2021. We used 3626 RSV fusion-protein sequences published in NCBI GenBank between 1956 and 2021 (2024 RSV and 1602 RSV B) to show that the RSV fusion protein had lower genetic diversity than influenza haemagglutinin and SARS-CoV-2 spike proteins. INTERPRETATION: The nirsevimab binding site was highly conserved between 1956 and 2021. Nirsevimab escape variants were rare and have not increased over time. FUNDING: AstraZeneca and Sanofi.


Asunto(s)
COVID-19 , Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Lactante , Humanos , Infecciones por Virus Sincitial Respiratorio/epidemiología , Estudios Prospectivos , Proyectos Piloto , SARS-CoV-2 , Virus Sincitial Respiratorio Humano/genética , Glicoproteínas , Sitios de Unión
16.
mSphere ; 7(3): e0013022, 2022 06 29.
Artículo en Inglés | MEDLINE | ID: mdl-35642538

RESUMEN

Nonhealing diabetic foot ulcers (DFU), a major complication of diabetes, are associated with high morbidity and mortality despite current standard of care. Since Staphylococcus aureus is the most common pathogen isolated from nonhealing and infected DFU, we hypothesized that S. aureus virulence factors would damage tissue, promote immune evasion and alter the microbiome, leading to bacterial persistence and delayed wound healing. In a diabetic mouse polymicrobial wound model with S. aureus, Pseudomonas aeruginosa, and Streptococcus pyogenes, we report a rapid bacterial proliferation, prolonged pro-inflammatory response and large necrotic lesions unclosed for up to 40 days. Treatment with AZD6389, a three-monoclonal antibody combination targeting S. aureus alpha toxin, 4 secreted leukotoxins, and fibrinogen binding cell-surface adhesin clumping factor A resulted in full skin re-epithelization 21 days after inoculation. By neutralizing multiple virulence factors, AZD6389 effectively blocked bacterial agglutination and S. aureus-mediated cell killing, abrogated S. aureus-mediated immune evasion and targeted the bacteria for opsonophagocytic killing. Neutralizing S. aureus virulence not only facilitated S. aureus clearance in lesions, but also reduced S. pyogenes and P. aeruginosa numbers, damaging inflammatory mediators and markers for neutrophil extracellular trap formation 14 days post initiation. Collectively, our data suggest that AZD6389 holds promise as an immunotherapeutic approach against DFU complications. IMPORTANCE Diabetic foot ulcers (DFU) represent a major complication of diabetes and are associated with poor quality of life and increased morbidity and mortality despite standard of care. They have a complex pathogenesis starting with superficial skin lesions, which often progress to deeper tissue structures up to the bone and ultimately require limb amputation. The skin microbiome of diabetic patients has emerged as having an impact on DFU occurrence and chronicity. DFU are mostly polymicrobial, and the Gram-positive bacterium Staphylococcus aureus detected in more than 95% of cases. S. aureus possess a collection of virulence factors which participate in disease progression and may facilitate growth of other pathogens. Here we show in a diabetic mouse wound model that targeting some specific S. aureus virulence factors with a multimechanistic antibody combination accelerated wound closure and promoted full skin re-epithelization. This work opens promising new avenues for the treatment of DFU.


Asunto(s)
Diabetes Mellitus , Pie Diabético , Infecciones Estafilocócicas , Animales , Anticuerpos Monoclonales , Bacterias , Pie Diabético/complicaciones , Pie Diabético/tratamiento farmacológico , Ratones , Pseudomonas aeruginosa , Calidad de Vida , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus , Virulencia , Factores de Virulencia
17.
MAbs ; 14(1): 2006123, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-34923908

RESUMEN

The increasing global occurrence of recalcitrant multi-drug resistant Klebsiella pneumoniae infections warrants the investigation of alternative therapy options, such as the use of monoclonal antibodies (mAbs). We used a target-agnostic phage display approach to K. pneumoniae bacteria lacking bulky, highly variable surface polysaccharides in order to isolate antibodies targeting conserved epitopes among clinically relevant strains. One antibody population contained a high proportion of unique carbohydrate binders, and biolayer interferometry revealed these antibodies bound to lipopolysaccharide (LPS). Antibodies that bound to O1 and O1/O2 LPS were identified. Antibodies were found to promote opsonophagocytic killing by human monocyte-derived macrophages and clearance of macrophage-associated bacteria when assessed using high-content imaging. One antibody, B39, was found to protect mice in a lethal model of K. pneumoniae pneumonia against both O1 and O2 strains when dosed therapeutically. High-content imaging, western blotting and fluorescence-activated cell sorting were used to determine binding to a collection of clinical K. pneumoniae O1 and O2 strains. The data suggests B39 binds to D-galactan-I and D-galactan-II of the LPS of O1 and O2 strains. Thus, we have discovered an mAb with novel binding and functional activity properties that is a promising candidate for development as a novel biotherapeutic for the treatment and prevention of K. pneumoniae infections.


Asunto(s)
Anticuerpos Antibacterianos/inmunología , Epítopos/inmunología , Infecciones por Klebsiella/inmunología , Klebsiella pneumoniae/inmunología , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Animales , Farmacorresistencia Bacteriana Múltiple/genética , Farmacorresistencia Bacteriana Múltiple/inmunología , Epítopos/genética , Humanos , Infecciones por Klebsiella/genética , Klebsiella pneumoniae/genética , Lipopolisacáridos/genética , Ratones , Opsonización
18.
Influenza Other Respir Viruses ; 14(4): 403-411, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32126161

RESUMEN

BACKGROUND: RSV is a leading cause of lower respiratory tract infection in infants. Monitoring RSV glycoprotein sequences is critical for understanding RSV epidemiology and viral antigenicity in the effort to develop anti-RSV prophylactics and therapeutics. OBJECTIVES: The objective is to characterize the circulating RSV strains collected from infants in South Africa during 2015-2017. METHODS: A subset of 150 RSV-positive samples obtained in South Africa from HIV-unexposed and HIV-exposed-uninfected infants from 2015 to 2017, were selected for high-throughput next-generation sequencing of the RSV F and G glycoprotein genes. The RSV G and F sequences were analyzed by a bioinformatic pipeline and compared to the USA samples from the same three-year period. RESULTS: Both RSV A and RSV B co-circulated in South Africa during 2015-2017, with a shift from RSV A (58%-61% in 2015-2016) to RSV B (69%) in 2017. RSV A ON1 and RSV B BA9 genotypes emerged as the most prevalent genotypes in 2017. Variations at the F protein antigenic sites were observed for both RSV A and B strains, with dominant changes (L172Q/S173L) at antigenic site V observed in RSV B strains. RSV A and B F protein sequences from South Africa were very similar to the USA isolates except for a higher rate of RSV A NA1 and RSV B BA10 genotypes in South Africa. CONCLUSION: RSV G and F genes continue to evolve and exhibit both local and global circulation patterns in South Africa, supporting the need for continued national surveillance.


Asunto(s)
Infecciones por VIH/virología , Filogenia , Infecciones por Virus Sincitial Respiratorio/epidemiología , Virus Sincitial Respiratorio Humano/genética , Antígenos Virales/genética , Femenino , Genotipo , Infecciones por VIH/epidemiología , Humanos , Lactante , Masculino , ARN Viral/genética , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/clasificación , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Análisis de Secuencia de ADN , Sudáfrica/epidemiología
19.
Sci Rep ; 9(1): 3898, 2019 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-30846850

RESUMEN

Respiratory syncytial virus (RSV) is a significant cause of lower respiratory tract infection in infants and elderly. To understand the evolution of neutralizing epitopes on the RSV glycoprotein (G) and fusion (F) proteins, we conducted a multi-year surveillance program (OUTSMART-RSV) in the US. Analysis of 1,146 RSV samples from 2015-2017 revealed a slight shift in prevalence from RSV A (58.7%) to B (53.7%) between the two seasons. RSV B was more prevalent in elderly (52.9% and 73.4%). Approximately 1% of the samples contained both RSV A and B viruses. All RSV A isolates were ON1 and almost all the B isolates were BA9 genotypes. Compared with the 2013 reference sequences, changes at the F antigenic sites of RSV B were greater than RSV A, which mainly occurred at antigenic sites V (L172Q/S173L at 99.6%), Ø (I206M/Q209K at 18.6%) and IV (E463D at 7%) of RSV B F. Sequence diversities in the G protein second hypervariable region were observed in the duplicated regions for RSV A and B, and at the G stop codon resulting in extension of 7 amino acids (22.1%) for RSV B in 2016-17. Thus, RSV surface glycoproteins are continuously evolving, and continued surveillance is important for the clinical evaluation of immunoprophylactic products.


Asunto(s)
Antígenos Virales/inmunología , Epítopos/inmunología , Glicoproteínas/inmunología , Virus Sincitial Respiratorio Humano/inmunología , Humanos , Infecciones por Virus Sincitial Respiratorio/virología , Estados Unidos
20.
Clin Transl Immunology ; 8(7): e01070, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31360464

RESUMEN

OBJECTIVE: To understand the relationships of Staphylococcus aureus (SA) bacteremic pneumonia (SABP) outcome with patient-specific and SA-specific variables. METHODS: We analysed SA bloodstream isolates and matching sera in SABP patients by sequencing SA isolates (n = 50) and measuring in vitro AT production, haemolytic activity and expression of ClfA and ClfB. Controls were sera from gram-negative bacteremia patients with or without pneumonia and uninfected subjects. Levels of IgGs, IgMs and neutralizing antibodies (NAbs) against SA antigens were quantified and analysed by one-way ANOVA. Associations of patient outcomes with patient variables, antibody levels and isolate characteristics were evaluated by univariate and multivariate logistic regression analyses. RESULTS: SABP patients had higher levels of IgGs against eight virulence factors and anti-alpha toxin (AT) NAbs than uninfected controls. Levels of IgG against AT and IgMs against ClfA, FnbpA and SdrC were higher in clinically cured SABP patients than in clinical failures. Anti-LukAB NAb levels were elevated in all cohorts. Increased odds of cure correlated with higher haemolytic activity of SA strains, longer time between surgery and bacteremia (> 30 days), longer duration of antibiotic therapy, lower acute physiology and total APACHE II scores, lack of persistent fever for > 72 h and higher levels of antibodies against AT (IgG), ClfA (IgM), FnbpA (IgM) and SdrC (IgM). DISCUSSION: Limitations included the cross-sectional observational nature of the study, small sample size and inability to measure antibody levels against all SA virulence factors. CONCLUSION: Our results suggest that SABP patients may benefit from immunotherapy targeting multiple SA antigens.

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