RESUMEN
BACKGROUND: Recurrent reports of suboptimal influenza vaccine effectiveness have renewed calls to develop improved, broadly cross-protective influenza vaccines. Here, we evaluated the safety and immunogenicity of a novel, saponin (Matrix-M)-adjuvanted, recombinant hemagglutinin (HA) quadrivalent nanoparticle influenza vaccine (qNIV). METHODS: We conducted a randomized, observer-blind, comparator-controlled (trivalent high-dose inactivated influenza vaccine [IIV3-HD] or quadrivalent recombinant influenza vaccine [RIV4]), safety and immunogenicity trial of qNIV (5 doses/formulations) in healthy adults ≥65 years. Vaccine immunogenicity was measured by hemagglutination-inhibition assays using reagents that express wild-type hemagglutination inhibition (wt-HAI) sequences and cell-mediated immune responses. RESULTS: A total of 1375 participants were randomized, immunized, and followed for safety and immunogenicity. Matrix-M-adjuvanted qNIV induced superior wt-HAI antibody responses against 5 of 6 homologous or drifted strains compared with unadjuvanted qNIV. Adjuvanted qNIV induced post-vaccination wt-HAI antibody responses at day 28 that were statistically higher than IIV3-HD against a panel of homologous or drifted A/H3N2 strains, similar to IIV3-HD against homologous A/H1N1 and B (Victoria) strains and similar to RIV4 against all homologous and drifted strains evaluated. The qNIV formulation with 75 µg Matrix-M adjuvant induced substantially higher post-vaccination geometric mean fold increases of influenza HA-specific polyfunctional CD4+ T cells compared with IIV3-HD or RIV4. Overall, similar frequencies of solicited and unsolicited adverse events were reported in all treatment groups. CONCLUSIONS: qNIV with 75 µg Matrix-M adjuvant was well tolerated and induced robust antibody and cellular responses, notably against both homologous and drifted A/H3N2 viruses. Further investigation in a pivotal phase 3 trial is underway. CLINICAL TRIALS REGISTRATION: NCT03658629.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza , Gripe Humana , Nanopartículas , Saponinas , Adulto , Anticuerpos Antivirales , Linfocitos T CD4-Positivos , Hemaglutinación , Pruebas de Inhibición de Hemaglutinación , Hemaglutininas , Humanos , Inmunogenicidad Vacunal , Subtipo H3N2 del Virus de la Influenza A , Gripe Humana/prevención & control , Vacunas de Productos InactivadosRESUMEN
Inflammatory cell infiltrates are a prominent feature of aberrant vascular remodeling in pulmonary arterial hypertension (PAH), suggesting that immune effector cells contribute to disease progression. Genome-wide blood expression profiling studies have attempted to better define this inflammatory component of PAH pathobiology but have been hampered by small sample sizes, methodological differences, and very little gene-level reproducibility. The current meta-analysis (seven studies; 156 PAH patients/110 healthy controls) was performed to assess the comparability of data across studies and to possibly derive a generalizable transcriptomic signature. Idiopathic (IPAH) compared with disease-associated PAH (APAH) displayed highly similar expression profiles with no differentially expressed genes, even after substantially relaxing selection stringency. In contrast, using a false discovery rate of ≤1% and I2 < 40% (low-to-moderate heterogeneity across studies) both IPAH and APAH differed markedly from healthy controls with the combined PAH cohort yielding 1,269 differentially expressed, unique gene transcripts. Bioinformatic analyses, including gene-set enrichment, which uses all available data independent of gene selection thresholds, identified interferon, mammalian target of rapamycin/p70S6K, stress kinase, and Toll-like receptor signaling as enriched mechanisms within the PAH gene signature. Enriched biological functions and diseases included tumorigenesis, autoimmunity, antiviral response, and cell death consistent with prevailing theories of PAH pathogenesis. Although otherwise indistinguishable, APAH (predominantly PAH due to systemic sclerosis) had a somewhat stronger interferon profile than IPAH. Meta-analysis defined a robust and generalizable transcriptomic signature in the blood of PAH patients that can help inform the identification of biomarkers and therapeutic targets.
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Hipertensión Pulmonar Primaria Familiar/genética , Hipertensión Arterial Pulmonar/genética , Transcriptoma/genética , Biomarcadores/metabolismo , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Respiratory syncytial virus (RSV) is a common cause of respiratory tract illness and hospitalization in neonates and infants. RSV vaccination during pregnancy may protect offspring in their first months of life. METHODS: This randomized, observer-blind, multicenter, phase 2 study evaluated the immunogenicity and safety of an RSV candidate vaccine in healthy nonpregnant women aged 18-45 years. Four hundred participants were randomized (1:1:1:1) to receive a single intramuscular dose of vaccine containing 30 µg, 60 µg, or 120 µg of RSV fusion protein engineered to preferentially maintain a prefusion conformation (RSV-PreF vaccine) or placebo. RESULTS: Thirty days postvaccination, RSV-A neutralizing antibody geometric mean titers (GMTs) increased 3.75-, 4.42- and 4.36-fold; RSV-B neutralizing antibody GMTs 2.36-, 2.54- and 2.76-fold; and palivizumab competing antibody (PCA) concentrations 11.69-, 14.38- and 14.24-fold compared with baseline levels in the 30 µg, 60 µg, and 120 µg RSV-PreF groups, respectively. Antibody titers and PCA concentrations at day 30 were significantly higher with the 120 µg compared to the 30 µg RSV-PreF vaccine. All RSV-PreF vaccine formulations and the placebo had similar reactogenicity profiles. No serious adverse events were considered to be related to the RSV-PreF vaccine. CONCLUSIONS: The 3 formulations of the investigational RSV-PreF vaccine were well-tolerated and induced RSV-A and RSV-B neutralizing antibodies and PCAs in healthy, nonpregnant women. CLINICAL TRIALS REGISTRATION: NCT02956837.
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Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/efectos adversos , Vacunas contra Virus Sincitial Respiratorio/inmunología , Proteínas Virales de Fusión/inmunología , Adolescente , Adulto , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Femenino , Voluntarios Sanos , Humanos , Inyecciones Intramusculares , Persona de Mediana Edad , Placebos/administración & dosificación , Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/efectos adversos , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunología , Adulto JovenRESUMEN
Glucocorticoids are commonly used to treat inflammatory disorders. The glucocorticoid receptor (GR) can tether to inflammatory transcription factor complexes, such as NFκB and AP-1, and trans-repress the transcription of cytokines, chemokines, and adhesion molecules. In contrast, aldosterone and the mineralocorticoid receptor (MR) primarily promote cardiovascular inflammation by incompletely understood mechanisms. Although MR has been shown to weakly repress NFκB, its role in modulating AP-1 has not been established. Here, the effects of GR and MR on NFκB and AP-1 signaling were directly compared using a variety of ligands, two different AP-1 consensus sequences, GR and MR DNA-binding domain mutants, and siRNA knockdown or overexpression of core AP-1 family members. Both GR and MR repressed an NFκB reporter without influencing p65 or p50 binding to DNA. Likewise, neither GR nor MR affected AP-1 binding, but repression or activation of AP-1 reporters occurred in a ligand-, AP-1 consensus sequence-, and AP-1 family member-specific manner. Notably, aldosterone interactions with both GR and MR demonstrated a potential to activate AP-1. DNA-binding domain mutations that eliminated the ability of GR and MR to cis-activate a hormone response element-driven reporter variably affected the strength and polarity of these responses. Importantly, MR modulation of NFκB and AP-1 signaling was consistent with a trans-mechanism, and AP-1 effects were confirmed for specific gene targets in primary human cells. Steroid nuclear receptor trans-effects on inflammatory signaling are context-dependent and influenced by nuclear receptor conformation, DNA sequence, and the expression of heterologous binding partners. Aldosterone activation of AP-1 may contribute to its proinflammatory effects in the vasculature.
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FN-kappa B/inmunología , Receptores de Glucocorticoides/inmunología , Receptores de Mineralocorticoides/inmunología , Transducción de Señal , Factor de Transcripción AP-1/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , ADN/química , Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , Humanos , Inflamación/genética , Inflamación/inmunología , Mutación , Dominios Proteicos , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Receptores de Mineralocorticoides/química , Receptores de Mineralocorticoides/genéticaRESUMEN
Peroxisome proliferator-activated receptor γ (PPARγ) ligands have been widely used to treat type 2 diabetes mellitus. However, knowledge of PPARγ signaling remains incomplete. In addition to PPARγ, these drugs also activate G protein-coupled receptor 40 (GPR40), a Gαq-coupled free fatty acid receptor linked to MAPK networks and glucose homeostasis. Notably, p38 MAPK activation has been implicated in PPARγ signaling. Here, rosiglitazone (RGZ) activation of GPR40 and p38 MAPK was found to boost PPARγ-induced gene transcription in human endothelium. Inhibition or knockdown of p38 MAPK or expression of a dominant negative (DN) p38 MAPK mutant blunted RGZ-induced PPARγ DNA binding and reporter activity in EA.hy926 human endothelial cells. GPR40 inhibition or knockdown, or expression of a DN-Gαq mutant likewise blocked activation of both p38 MAPK and PPARγ reporters. Importantly, RGZ induction of PPARγ target genes in primary human pulmonary artery endothelial cells (PAECs) was suppressed by knockdown of either p38 MAPK or GPR40. GPR40/PPARγ signal transduction was dependent on p38 MAPK activation and induction of PPARγ co-activator-1 (PGC1α). Silencing of p38 MAPK or GPR40 abolished the ability of RGZ to induce phosphorylation and expression of PGC1α in PAECs. Knockdown of PGC1α, its essential activator SIRT1, or its binding partner/co-activator EP300 inhibited RGZ induction of PPARγ-regulated genes in PAECs. RGZ/GPR40/p38 MAPK signaling also led to EP300 phosphorylation, an event that enhances PPARγ target gene transcription. Thus, GPR40 and PPARγ can function as an integrated two-receptor signal transduction pathway, a finding with implications for rational drug development.
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Células Endoteliales/metabolismo , PPAR gamma/metabolismo , Receptor Cross-Talk , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Línea Celular , ADN/genética , ADN/metabolismo , Proteína p300 Asociada a E1A/genética , Proteína p300 Asociada a E1A/metabolismo , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Hipoglucemiantes/farmacología , Ligandos , Luciferasas/genética , Luciferasas/metabolismo , PPAR gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Pioglitazona , Cultivo Primario de Células , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/genética , Rosiglitazona , Sirtuina 1/genética , Sirtuina 1/metabolismo , Tiazolidinedionas/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
BACKGROUND: An increasing number of physicians are seeking dual training in critical care medicine (CCM) and infectious diseases (ID). Understanding experiences and perceptions of CCM-ID physicians could inform career choices and programmatic innovation. METHODS: All physicians trained and/or certified in both CCM and ID to date in the United States were sent a Web-based questionnaire in 2015. Responses enabled a cross-sectional analysis of physician demographics and training and practice characteristics and satisfaction. RESULTS: Of 202 CCM-ID physicians, 196 were alive and reachable. The response rate was 79%. Forty-six percent trained and 34% practice in the northeastern United States. Only 40% received dual training at the same institution. Eighty-three percent identified as either an intensivist with ID expertise (44%) or as equally an intensivist and ID physician (38%). Median salary was $265 000 (interquartile range [IQR], $215 000-$350 000). Practice settings were split between academic (45%) and community settings (42%). Two-thirds are clinicians but 62% conduct some research and 26% practice outpatient ID. Top reasons to dually specialize included clinical synergy (70%), procedural activity (50%), and less interest in pulmonology (49%). Although 38% cited less proficiency with bronchoscopy as a disadvantage, 87% seldom need pulmonary consultation in the intensive care unit. Median career satisfaction was 4 (IQR, 4-5) out of 5, and 76% would dually train again. CONCLUSIONS: CCM-ID graduates prefer the acute care setting, predominantly CCM or a combination of CCM and ID. They find combination training and practice to be synergistic and satisfying, but most have had to seek CCM and ID training independently at separate institutions. Given these findings, avenues for combined training in CCM-ID should be considered.
Asunto(s)
Cuidados Críticos , Infectología , Médicos , Adulto , Estudios Transversales , Femenino , Humanos , Infectología/economía , Infectología/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Satisfacción Personal , Médicos/economía , Médicos/psicología , Médicos/estadística & datos numéricos , Encuestas y Cuestionarios , Estados UnidosRESUMEN
A proliferative endothelial cell phenotype, inflammation, and pulmonary vascular remodeling are prominent features of pulmonary arterial hypertension (PAH). Bone morphogenetic protein type II receptor (BMPR2) loss-of-function is the most common cause of heritable PAH and has been closely linked to the formation of pathological plexiform lesions. Although some BMPR2 mutations leave ligand-dependent responses intact, the disruption of ligand-independent, noncanonical functions are universal among PAH-associated BMPR2 genotypes, but incompletely understood. This study examined the noncanonical signaling consequences of BMPR2 silencing in human pulmonary artery endothelial cells to identify potential therapeutic targets. BMPR2 siRNA silencing resulted in a proliferative, promigratory pulmonary artery endothelial cell phenotype and disruption of cytoskeletal architecture. Expression profiling closely reflected these phenotypic changes. Gene set enrichment and promoter analyses, as well as the differential expression of pathway components identified Ras/Raf/ERK signaling as an important consequence of BMPR2 silencing. Raf family members and ERK1/2 were constitutively activated after BMPR2 knockdown. Two Raf inhibitors, sorafenib and AZ628, and low-dose nintedanib, a triple receptor tyrosine kinase inhibitor upstream from Ras, reversed the abnormal proliferation and hypermotility of BMPR2 deficiency. Inhibition of dysregulated Ras/Raf/ERK signaling may be useful in reversing vascular remodeling in PAH.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Proliferación Celular , Células Endoteliales/citología , Hipertensión Pulmonar/metabolismo , Pulmón/metabolismo , Arteria Pulmonar/metabolismo , Adulto , Anciano , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hipertensión Pulmonar Primaria Familiar/metabolismo , Femenino , Silenciador del Gen , Humanos , Pulmón/patología , Masculino , Persona de Mediana Edad , Mutación/genética , Fenotipo , ARN Interferente Pequeño/genética , Transducción de Señal/fisiología , Remodelación Vascular/genética , Adulto Joven , Quinasas raf/metabolismoRESUMEN
We previously showed that coincident exposure to heat shock (HS; 42°C for 2 h) and TNF-α synergistically induces apoptosis in mouse lung epithelium. We extended this work by analyzing HS effects on human lung epithelial responses to clinically relevant injury. Cotreatment with TNF-α and HS induced little caspase-3 and poly(ADP-ribose) polymerase cleavage in human small airway epithelial cells, A549 cells, and BEAS2B cells. Scratch wound closure rates almost doubled when A549 and BEAS2B cells and air-liquid interface cultures of human bronchial epithelial cells were heat shocked immediately after wounding. Microarray, qRT-PCR, and immunoblotting showed fibroblast growth factor 1 (FGF1) to be synergistically induced by HS and wounding. Enhanced FGF1 expression in HS/wounded A549 was blocked by inhibitors of p38 MAPK (SB203580) or HS factor (HSF)-1 (KNK-437) and in HSF1 knockout BEAS2B cells. PCR demonstrated FGF1 to be expressed from the two most distal promoters in wounded/HS cells. Wound closure in HS A549 and BEAS2B cells was reduced by FGF receptor-1/3 inhibition (SU-5402) or FGF1 depletion. Exogenous FGF1 accelerated A549 wound closure in the absence but not presence of HS. In the presence of exogenous FGF1, HS slowed wound closure, suggesting that it increases FGF1 expression but impairs FGF1-stimulated wound closure. Frozen sections from normal and idiopathic pulmonary fibrosis (IPF) lung were analyzed for FGF1 and HSP70 by immunofluorescence confocal microscopy and qRT-PCR. FGF1 and HSP70 mRNA levels were 7.5- and 5.9-fold higher in IPF than normal lung, and the proteins colocalized to fibroblastic foci in IPF lung. We conclude that HS signaling may have an important impact on gene expression contributing to lung injury, healing, and fibrosis.
Asunto(s)
Epitelio/metabolismo , Epitelio/patología , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Respuesta al Choque Térmico , Lesión Pulmonar/patología , Animales , Apoptosis/genética , Sitios de Unión , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Factor 1 de Crecimiento de Fibroblastos/genética , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico , Respuesta al Choque Térmico/genética , Humanos , Fibrosis Pulmonar Idiopática/genética , Pulmón/metabolismo , Pulmón/patología , Lesión Pulmonar/genética , Ratones , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Cicatrización de Heridas/genéticaRESUMEN
Phylogeographic studies inform about routes of pathogen dissemination and are instrumental for improving import/export controls. Genomes of 17 isolates of the bacterial wilt and potato brown rot pathogen Ralstonia solanacearum race 3 biovar 2 (R3bv2), a Select Agent in the United States, were thus analyzed to get insight into the phylogeography of this pathogen. Thirteen of fourteen isolates from Europe, Africa, and Asia were found to belong to a single clonal lineage while isolates from South America were genetically diverse and tended to carry ancestral alleles at the analyzed genomic loci consistent with a South American origin of R3bv2. The R3bv2 isolates share a core repertoire of 31 type III-secreted effector genes representing excellent candidates to be targeted with resistance genes in breeding programs to develop durable disease resistance. Toward this goal, 27 R3bv2 effectors were tested in eggplant, tomato, pepper, tobacco, and lettuce for induction of a hypersensitive-like response indicative of recognition by cognate resistance receptors. Fifteen effectors, eight of them core effectors, triggered a response in one or more plant species. These genotypes may harbor resistance genes that could be identified and mapped, cloned, and expressed in tomato or potato, for which sources of genetic resistance to R3bv2 are extremely limited.
Asunto(s)
Genómica , Enfermedades de las Plantas/microbiología , Ralstonia solanacearum/genética , Solanum tuberosum/microbiología , África , Asia , Capsicum/inmunología , Capsicum/microbiología , Resistencia a la Enfermedad , Europa (Continente) , Variación Genética , Lactuca/inmunología , Lactuca/microbiología , Solanum lycopersicum/inmunología , Solanum lycopersicum/microbiología , Filogeografía , Enfermedades de las Plantas/inmunología , Ralstonia solanacearum/patogenicidad , Solanum melongena/genética , Solanum melongena/inmunología , Solanum tuberosum/inmunología , América del Sur , VirulenciaRESUMEN
The mortality rate of alveolar hemorrhage (AH) after allogeneic hematopoietic stem cell transplantation is greater than 60% with supportive care and high-dose steroid therapy. We performed a retrospective cohort analysis to assess the benefits and risks of recombinant human factor VIIa (rFVIIa) as a therapeutic adjunct for AH. Between 2005 and 2012, 57 episodes of AH occurred in 37 patients. Fourteen episodes (in 14 patients) were treated with steroids alone, and 43 episodes (in 23 patients) were treated with steroids and rFVIIa. The median steroid dose was 1.9 mg/kg/d (interquartile range [IQR], 0.8 to 3.5 mg/kg/d; methylprednisolone equivalents) and did not differ statistically between the 2 groups. The median rFVIIa dose was 41 µg/kg (IQR, 39 to 62 µg/kg), and a median of 3 doses (IQR, 2 to 17) was administered per episode. Concurrent infection was diagnosed in 65% of the episodes. Patients had moderately severe hypoxia (median PaO2/FiO2, 193 [IQR, 141 to 262]); 72% required mechanical ventilation, and 42% survived to extubation. The addition of rFVIIa did not alter time to resolution of AH (P = .50), duration of mechanical ventilation (P = .89), duration of oxygen supplementation (P = .55), or hospital mortality (P = .27). Four possible thrombotic events (9% of 43 episodes) occurred with rFVIIa. rFVIIa in combination with corticosteroids did not confer clear clinical advantages compared with corticosteroids alone. In patients with AH following hematopoietic stem cell transplantation, clinical factors (ie, worsening infection, multiple organ failure, or recrudescence of primary disease) may be more important than the benefit of enhanced hemostasis from rFVIIa.
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Factor VIIa/uso terapéutico , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Hemorragia/tratamiento farmacológico , Hemorragia/etiología , Enfermedades Pulmonares/tratamiento farmacológico , Acondicionamiento Pretrasplante/efectos adversos , Adolescente , Adulto , Anciano , Niño , Estudios de Cohortes , Femenino , Humanos , Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/patología , Masculino , Persona de Mediana Edad , Alveolos Pulmonares/patología , Proteínas Recombinantes/uso terapéutico , Estudios Retrospectivos , Trasplante Homólogo , Adulto JovenRESUMEN
As variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge, assessment of vaccine immunogenicity remains a critical factor to support continued vaccination. To this end, an in vitro microneutralization (MN50) assay was validated to quantitate SARS-CoV-2 neutralizing antibodies against prototype and variant strains (Beta, Delta, Omicron BA.1, Omicron BA.5, and XBB.1.5) in human serum. For the prototype strain, the MN50 assay met acceptance criteria for inter-/intra-assay precision, specificity, linearity, and selectivity. The assay was robust against changes to virus/serum incubation time, cell seeding density, virus content per well, cell passage number, and serum interference. Analyte in serum samples was stable up to five freeze/thaw cycles and for up to 12 months of storage at -80 ± 10 °C. Similar results were observed for the variant-adapted MN50 assays. The conversion factor to convert assay result units to WHO international standard units (IU/mL) was determined to be 0.62 for the prototype strain. This MN50 assay will be useful for vaccine immunogenicity analyses in clinical trial samples, enabling assessment of vaccine immunogenicity for ancestral and variant strains as variant-adapted vaccines are developed.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , Inmunogenicidad Vacunal , Pruebas de Neutralización , SARS-CoV-2 , Humanos , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Pruebas de Neutralización/métodos , Anticuerpos Antivirales/sangre , Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , COVID-19/inmunología , COVID-19/diagnóstico , Sensibilidad y Especificidad , Animales , Reproducibilidad de los ResultadosRESUMEN
The evaluation of coronavirus disease 2019 (COVID-19) vaccine immunogenicity remains essential as the severe acute respiratory syncytial virus 2 (SARS-CoV-2) pandemic continues to evolve and as additional variants emerge. Neutralizing antibodies are a known correlate of protection for SARS-CoV-2 vaccines. A pseudovirus neutralization (PNT) assay was developed and validated at Novavax Clinical Immunology Laboratories to allow for the detection of neutralizing antibodies in vaccine clinical trial sera. The PNT assay was precise, accurate, linear, and specific in measuring SARS-CoV-2 neutralization titers in human serum for ancestral strain and the Omicron subvariants BA.5 and XBB.1.5, with an overall geometric coefficient of variation of ≤43.4%, a percent relative bias within the expected range of -60% to 150%, and a linearity value of R2 > 0.98 for all three strains. This pseudovirus assay will be useful for the analysis of vaccine clinical trial samples to assess vaccine immunogenicity. Future work will focus on modifying the assay for emerging variants, including XBB.1.16, EG.5.1, BA.2.86, and any other variants that emerge in the ongoing pandemic.
RESUMEN
Repeated mRNA SARS-CoV-2 vaccination has been associated with increases in the proportion of IgG4 in spike-specific antibody responses and concurrent reductions in Fcγ-mediated effector functions that may limit control of viral infection. Here, we assessed anti-Spike total IgG, IgG1, IgG2, IgG3 and IgG4, and surrogate markers for antibody-dependent cellular phagocytosis (ADCP, FcγRIIa binding), antibody-dependent cellular cytotoxicity (ADCC, FcγRIIIa binding), and antibody-dependent complement deposition (ADCD, C1q binding) associated with repeated SARS-CoV-2 vaccination with NVX-CoV2373 (Novavax Inc., Gaithersburg, MD). The NVX-CoV2373 protein vaccine did not induce notable increases in spike-specific IgG4 or negatively impact surrogates for Fcγ effector responses. Conversely, repeated NVX-CoV2373 vaccination uniquely enhanced IgG3 responses which are known to exhibit strong affinity for FcγRIIIa and have previously been linked to potent neutralization of SARS-CoV-2. Subsequent investigations will help to understand the immunological diversity generated by different SARS-CoV-2 vaccine types and have the potential to reshape public health strategies.
RESUMEN
We studied the changes in expression of microRNAs (miRNAs or miRs) and mRNA in normal human bronchial epithelial cells as they differentiate from an undifferentiated monolayer to a differentiated pseudostratified epithelium after 28 days of air-liquid interface (ALI) culture. After 28 days in ALI, the epithelial cells differentially expressed basal, ciliated, and goblet cell markers. Using Affymetrix microarrays, 20 human miRNAs were found to be up-regulated, whereas 35 miRNAs were found to be down-regulated in differentiated cells compared with undifferentiated cells. An analysis of changes in global mRNA expression revealed that 1,201 probe sets demonstrated an 8-fold change (FC) or greater at Day 28 of ALI culture. Of these, 816 were up-regulated and 385 were down-regulated. With differentiation, miR-449a increased (FC, 38.15), and was related to changes in mRNA for cell division cycle 25 homolog A (FC, 0.11). MiR-455 decreased (FC, 0.12) and was related to changes in mRNA for the epithelial cell marker, mucin 1 (FC, 136). Transfection with anti-miR-449 or miR-455-3p resulted in changes in target protein expression (cell division cycle 25 homolog A and mucin 1, respectively), whereas transfection with reporter genes with 3'-untranslated regions of these targets confirmed control of expression through that structure. Therefore, changes in specific miRNAs during human airway epithelial cell differentiation control gene and protein expression important for differentiation.
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Bronquios/metabolismo , Células Epiteliales/metabolismo , MicroARNs/genética , ARN Mensajero/genética , Mucosa Respiratoria/metabolismo , Biomarcadores/metabolismo , Bronquios/citología , Ciclo Celular/genética , Diferenciación Celular , Células Cultivadas , Regulación hacia Abajo , Células Epiteliales/citología , Expresión Génica , Perfilación de la Expresión Génica , Humanos , MicroARNs/metabolismo , Mucina-1/genética , Mucina-1/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Mucosa Respiratoria/citología , Regulación hacia Arriba , Fosfatasas cdc25/genética , Fosfatasas cdc25/metabolismoRESUMEN
Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain.
Asunto(s)
Interacciones Huésped-Patógeno , Enfermedades de las Plantas/microbiología , Pseudomonas syringae/genética , Pseudomonas syringae/patogenicidad , Solanum lycopersicum/microbiología , Factores de Virulencia/genética , Alelos , Cartilla de ADN , Europa (Continente) , Flagelina/genética , Flagelina/metabolismo , Regulación de la Expresión Génica de las Plantas , Sitios Genéticos , Marcadores Genéticos , Mutación , América del Norte , Filogeografía , Inmunidad de la Planta , Hojas de la Planta , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADNRESUMEN
While the existence of environmental reservoirs of human pathogens is well established, less is known about the role of nonagricultural environments in emergence, evolution, and spread of crop pathogens. Here, we analyzed phylogeny, virulence genes, host range, and aggressiveness of Pseudomonas syringae strains closely related to the tomato pathogen P. syringae pv. tomato (Pto), including strains isolated from snowpack and streams. The population of Pto relatives in nonagricultural environments was estimated to be large and its diversity to be higher than that of the population of Pto and its relatives on crops. Ancestors of environmental strains, Pto, and other genetically monomorphic crop pathogens were inferred to have frequently recombined, suggesting an epidemic population structure for P. syringae. Some environmental strains have repertoires of type III-secreted effectors very similar to Pto, are almost as aggressive on tomato as Pto, but have a wider host range than typical Pto strains. We conclude that crop pathogens may have evolved through a small number of evolutionary events from a population of less aggressive ancestors with a wider host range present in nonagricultural environments.
Asunto(s)
Agricultura , Evolución Biológica , Productos Agrícolas/microbiología , Reservorios de Enfermedades/microbiología , Pseudomonas syringae/fisiología , Alelos , Secuencia de Bases , Teorema de Bayes , Ambiente , Genes Bacterianos/genética , Sitios Genéticos/genética , Geografía , Especificidad del Huésped , Humanos , Solanum lycopersicum/microbiología , Filogenia , Enfermedades de las Plantas/microbiología , Recombinación Genética/genética , Ríos/microbiologíaRESUMEN
Emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) show immune evasion of vaccine-derived immunity, highlighting the need for better clinical immunogenicity biomarkers. To address this need, an enzyme-linked immunosorbent assay-based, human angiotensin-converting enzyme 2 (hACE2) binding inhibition assay was developed to measure antibodies against the ancestral strain of SARS-CoV-2 and was validated for precision, specificity, linearity, and other parameters. This assay measures the inhibition of SARS-CoV-2 spike (S) protein binding to the receptor, hACE2, by serum from vaccine clinical trials. Inter- and intra-assay precision, specificity, linearity, lower limit of quantitation, and assay robustness parameters successfully met the acceptance criteria. Heme and lipid matrix effects showed minimal interference on the assay. Samples were stable for testing in the assay even with 8 freeze/thaws and up to 24 months in -80 °C storage. The assay was also adapted for variants (Delta and Omicron BA.1/BA.5), with similar validation results. The hACE2 assay showed significant correlation with anti-recombinant S immunoglobulin G levels and neutralizing antibody titers. This assay provides a rapid, high-throughput option to evaluate vaccine immunogenicity. Along with other clinical biomarkers, it can provide valuable insights into immune evasion and correlates of protection and enable vaccine development against emerging COVID-19 variants.
RESUMEN
BACKGROUND: Improved seasonal influenza vaccines for older adults that can induce broadly cross-reactive antibodies and enhanced T-cell responses, particularly against A H3N2 viruses, while avoiding egg-adaptive antigenic changes, are needed. We aimed to show that the Matrix-M-adjuvanted quadrivalent nanoparticle influenza vaccine (qNIV) was immunologically non-inferior to a licensed, standard-dose quadrivalent inactivated influenza vaccine (IIV4) in older adults. METHODS: This was a phase 3 randomised, observer-blinded, active-comparator controlled trial done across 19 US community-based clinical research sites during the 2019-20 influenza season. Participants were clinically stable and community-dwelling, aged at least 65 years, and were randomised in a 1:1 ratio using an interactive web response system to receive a single intramuscular dose of qNIV or IIV4. The primary objective was to describe safety and show that qNIV was immunologically non-inferior to IIV4. The primary outcomes were adverse events by treatment group and comparative haemagglutination-inhibiting antibody responses (assayed with egg-propagated virus) on day 28, summarised in terms of the ratio of geometric mean titres (GMTRqNIV/IIV4) and seroconversion rate (SCR) difference between participants receiving qNIV or IIV4 for all four vaccine homologous influenza strains. The immunogenicity outcome was measured in the per-protocol population. Non-inferiority was shown if the lower bound of the two-sided 95% CI on the GMTRqNIV/IIV4 was at least 0·67 and the lower bound of the two-sided 95% CI on the SCR difference -was at least -10%. The study is registered with clinicaltrials.gov, NCT04120194, and is active and not recruiting. FINDINGS: 2742 adults were assessed for eligibility and 2654 were enrolled and randomised between Oct 14, 2019, and Oct 25, 2019; 1333 participants were randomised to the qNIV group and 1319 to the IIV4 group (two participants withdrew consent before being assigned to a group). qNIV showed immunological non-inferiority to IIV4: GMTRqNIV/IIV4 for the four vaccine homologous influenza strains was A/Brisbane 1·09 (95% CI 1·03 to 1·15), A/Kansas 1·19 (1·11 to 1·27), B/Maryland 1·03 (0·99 to 1·07), and B/Phuket 1·23 (1·16 to 1·29); and SCR difference was A/Brisbane 5·0 (95% CI 1·9 to 8·1), A/Kansas 7·3 (3·6 to 11·1), B/Maryland 0·5 (-1·9 to 2·9), and B/Phuket 8·5 (5·0 to 11·9). 659 (49·4%) of 1333 of participants in the qNIV group and 551 (41·8%) of 1319 participants in the IIV4 group had at least one treatment-emergent adverse event. More solicited adverse events were reported by participants in the qNIV group (551 [41·3%] of 1333) than in the IIV4 group (420 [31·8%] of 1319), and were comprised primarily of mild to moderate transient injection site pain (341 [25·6%] in the qNIV group vs 212 [16·1%] in the IIV4 group). INTERPRETATION: qNIV was well tolerated and produced qualitatively and quantitatively enhanced humoral and cellular immune response in older adults compared with IIV4. qNIV might enhance the effectiveness of seasonal influenza vaccination, and future studies to show clinical efficacy are planned. FUNDING: Novavax.
Asunto(s)
Adyuvantes de Vacunas/administración & dosificación , Anticuerpos Antivirales/sangre , Inmunogenicidad Vacunal , Vacunas contra la Influenza/inmunología , Vacunas contra la Influenza/normas , Gripe Humana/prevención & control , Nanopartículas/administración & dosificación , Saponinas/administración & dosificación , Anciano , Femenino , Pruebas de Inhibición de Hemaglutinación , Humanos , Vacunas contra la Influenza/administración & dosificación , Gripe Humana/inmunología , Masculino , Nanopartículas/química , Saponinas/química , Estaciones del AñoRESUMEN
Since 2002, severe leaf spotting on parsley (Petroselinum crispum) has occurred in Monterey County, CA. Either of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from eight distinct outbreaks and once from the same outbreak. Fragment analysis of DNA amplified between repetitive sequence polymerase chain reaction; 16S rDNA sequence analysis; and biochemical, physiological, and host range tests identified the pathogens as Pseudomonas syringae pv. apii and P. syringae pv. coriandricola. Koch's postulates were completed for the isolates from parsley, and host range tests with parsley isolates and pathotype strains demonstrated that P. syringae pv. apii and P. syringae pv. coriandricola cause leaf spot diseases on parsley, celery, and coriander or cilantro. In a multilocus sequence typing (MLST) approach, four housekeeping gene fragments were sequenced from 10 strains isolated from parsley and 56 pathotype strains of P. syringae. Allele sequences were uploaded to the Plant-Associated Microbes Database and a phylogenetic tree was built based on concatenated sequences. Tree topology directly corresponded to P. syringae genomospecies and P. syringae pv. apii was allocated appropriately to genomospecies 3. This is the first demonstration that MLST can accurately allocate new pathogens directly to P. syringae sensu lato genomospecies. According to MLST, P. syringae pv. coriandricola is a member of genomospecies 9, P. cannabina. In a blind test, both P. syringae pv. coriandricola and P. syringae pv. apii isolates from parsley were correctly identified to pathovar. In both cases, MLST described diversity within each pathovar that was previously unknown.