Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
1.
Proc Natl Acad Sci U S A ; 121(29): e2310421121, 2024 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-38976733

RESUMEN

We generated a replication-competent OC43 human seasonal coronavirus (CoV) expressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike in place of the native spike (rOC43-CoV2 S). This virus is highly attenuated relative to OC43 and SARS-CoV-2 in cultured cells and animals and is classified as a biosafety level 2 (BSL-2) agent by the NIH biosafety committee. Neutralization of rOC43-CoV2 S and SARS-CoV-2 by S-specific monoclonal antibodies and human sera is highly correlated, unlike recombinant vesicular stomatitis virus-CoV2 S. Single-dose immunization with rOC43-CoV2 S generates high levels of neutralizing antibodies against SARS-CoV-2 and fully protects human ACE2 transgenic mice from SARS-CoV-2 lethal challenge, despite nondetectable replication in respiratory and nonrespiratory organs. rOC43-CoV2 S induces S-specific serum and airway mucosal immunoglobulin A and IgG responses in rhesus macaques. rOC43-CoV2 S has enormous value as a BSL-2 agent to measure S-specific antibodies in the context of a bona fide CoV and is a candidate live attenuated SARS-CoV-2 mucosal vaccine that preferentially replicates in the upper airway.


Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , Pruebas de Neutralización , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Humanos , Anticuerpos Neutralizantes/inmunología , Ratones , COVID-19/inmunología , COVID-19/virología , COVID-19/prevención & control , Anticuerpos Antivirales/inmunología , Pruebas de Neutralización/métodos , Ratones Transgénicos , Coronavirus Humano OC43/inmunología , Coronavirus Humano OC43/genética , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/inmunología , Chlorocebus aethiops , Células Vero , Macaca mulatta
2.
Cell ; 186(12): 2672-2689.e25, 2023 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-37295404

RESUMEN

Alphaviruses are RNA viruses that represent emerging public health threats. To identify protective antibodies, we immunized macaques with a mixture of western, eastern, and Venezuelan equine encephalitis virus-like particles (VLPs), a regimen that protects against aerosol challenge with all three viruses. Single- and triple-virus-specific antibodies were isolated, and we identified 21 unique binding groups. Cryo-EM structures revealed that broad VLP binding inversely correlated with sequence and conformational variability. One triple-specific antibody, SKT05, bound proximal to the fusion peptide and neutralized all three Env-pseudotyped encephalitic alphaviruses by using different symmetry elements for recognition across VLPs. Neutralization in other assays (e.g., chimeric Sindbis virus) yielded variable results. SKT05 bound backbone atoms of sequence-diverse residues, enabling broad recognition despite sequence variability; accordingly, SKT05 protected mice against Venezuelan equine encephalitis virus, chikungunya virus, and Ross River virus challenges. Thus, a single vaccine-elicited antibody can protect in vivo against a broad range of alphaviruses.


Asunto(s)
Alphavirus , Virus de la Encefalitis Equina Venezolana , Vacunas Virales , Animales , Ratones , Virus de la Encefalitis Equina Venezolana/genética , Anticuerpos Antivirales , Macaca
3.
Proteomics ; 23(5): e2200237, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36480152

RESUMEN

The innate immune protection provided by cationic antimicrobial peptides (CAMPs) has been shown to extend to antiviral activity, with putative mechanisms of action including direct interaction with host cells or pathogen membranes. The lack of therapeutics available for the treatment of viruses such as Venezuelan equine encephalitis virus (VEEV) underscores the urgency of novel strategies for antiviral discovery. American alligator plasma has been shown to exhibit strong in vitro antibacterial activity, and functionalized hydrogel particles have been successfully employed for the identification of specific CAMPs from alligator plasma. Here, a novel bait strategy in which particles were encapsulated in membranes from either healthy or VEEV-infected cells was implemented to identify peptides preferentially targeting infected cells for subsequent evaluation of antiviral activity. Statistical analysis of peptide identification results was used to select five candidate peptides for testing, of which one exhibited a dose-dependent inhibition of VEEV and also significantly inhibited infectious titers. Results suggest our bioprospecting strategy provides a versatile platform that may be adapted for antiviral peptide identification from complex biological samples.


Asunto(s)
Caimanes y Cocodrilos , Virus de la Encefalitis Equina Venezolana , Encefalomielitis Equina Venezolana , Animales , Caballos , Virus de la Encefalitis Equina Venezolana/fisiología , Antivirales/farmacología , Antivirales/uso terapéutico , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Encefalomielitis Equina Venezolana/prevención & control , Bioprospección , Replicación Viral , Péptidos
4.
Disaster Med Public Health Prep ; 17: e112, 2022 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-35027098

RESUMEN

Monoclonal antibody therapeutics to treat coronavirus disease (COVID-19) have been authorized by the US Food and Drug Administration under Emergency Use Authorization (EUA). Many barriers exist when deploying a novel therapeutic during an ongoing pandemic, and it is critical to assess the needs of incorporating monoclonal antibody infusions into pandemic response activities. We examined the monoclonal antibody infusion site process during the COVID-19 pandemic and conducted a descriptive analysis using data from 3 sites at medical centers in the United States supported by the National Disaster Medical System. Monoclonal antibody implementation success factors included engagement with local medical providers, therapy batch preparation, placing the infusion center in proximity to emergency services, and creating procedures resilient to EUA changes. Infusion process challenges included confirming patient severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positivity, strained staff, scheduling, and pharmacy coordination. Infusion sites are effective when integrated into pre-existing pandemic response ecosystems and can be implemented with limited staff and physical resources.


Asunto(s)
COVID-19 , SARS-CoV-2 , Estados Unidos , Humanos , COVID-19/epidemiología , Pandemias , Salud Pública , Ecosistema , Anticuerpos Monoclonales/uso terapéutico
5.
Viruses ; 13(6)2021 06 03.
Artículo en Inglés | MEDLINE | ID: mdl-34205098

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a 'cytokine storm.' In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.


Asunto(s)
COVID-19/inmunología , Quimiocina CXCL10/genética , Quimiocina CXCL11/genética , Quimiocina CXCL9/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Regulación hacia Arriba , Enzima Convertidora de Angiotensina 2/genética , Animales , Línea Celular , Quimiocina CXCL10/inmunología , Quimiocina CXCL11/inmunología , Quimiocina CXCL9/inmunología , Síndrome de Liberación de Citoquinas/genética , Síndrome de Liberación de Citoquinas/inmunología , Células Epiteliales/inmunología , Células Epiteliales/virología , Femenino , Humanos , Inflamación , Pulmón/citología , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/genética , Transducción de Señal/inmunología
6.
Pathog Immun ; 6(1): 55-74, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33969249

RESUMEN

BACKGROUND: Host proteases have been suggested to be crucial for dissemination of MERS, SARS-CoV, and SARS-CoV-2 coronaviruses, but the relative contribution of membrane versus intracellular proteases remains controversial. Transmembrane serine protease 2 (TMPRSS2) is regarded as one of the main proteases implicated in the coronavirus S protein priming, an important step for binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor before cell entry. METHODS: We developed a cell-based assay to identify TMPRSS2 inhibitors. Inhibitory activity was established in SARS-CoV-2 viral load systems. RESULTS: We identified the human extracellular serine protease inhibitor (serpin) alpha 1 anti-trypsin (A1AT) as a novel TMPRSS2 inhibitor. Structural modeling revealed that A1AT docked to an extracellular domain of TMPRSS2 in a conformation that is suitable for catalysis, resembling similar serine protease inhibitor complexes. Inhibitory activity of A1AT was established in a SARS-CoV-2 viral load system. Notably, plasma A1AT levels were associated with COVID-19 disease severity. CONCLUSIONS: Our data support the key role of extracellular serine proteases in SARS CoV-2 infections and indicate that treatment with serpins, particularly the FDA-approved drug A1AT, may be effective in limiting SARS-CoV-2 dissemination by affecting the surface of the host cells.

7.
Pathogens ; 10(3)2021 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-33801554

RESUMEN

Alphaviruses are a genus of the Togaviridae family and are widely distributed across the globe. Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), cause encephalitis and neurological sequelae while chikungunya virus (CHIKV) and Sindbis virus (SINV) cause arthralgia. There are currently no approved therapeutics or vaccines available for alphaviruses. In order to identify novel therapeutics, a V5 epitope tag was inserted into the N-terminus of the VEEV E2 glycoprotein and used to identify host-viral protein interactions. Host proteins involved in protein folding, metabolism/ATP production, translation, cytoskeleton, complement, vesicle transport and ubiquitination were identified as VEEV E2 interactors. Multiple inhibitors targeting these host proteins were tested to determine their effect on VEEV replication. The compound HA15, a GRP78 inhibitor, was found to be an effective inhibitor of VEEV, EEEV, CHIKV, and SINV. VEEV E2 interaction with GRP78 was confirmed through coimmunoprecipitation and colocalization experiments. Mechanism of action studies found that HA15 does not affect viral RNA replication but instead affects late stages of the viral life cycle, which is consistent with GRP78 promoting viral assembly or viral protein trafficking.

8.
bioRxiv ; 2020 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-33052338

RESUMEN

Host proteases have been suggested to be crucial for dissemination of MERS, SARS-CoV, and SARS-CoV-2 coronaviruses, but the relative contribution of membrane versus intracellular proteases remains controversial. Transmembrane serine protease 2 (TMPRSS2) is regarded as one of the main proteases implicated in the coronavirus S protein priming, an important step for binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor before cell entry. The main cellular location where the SARS-CoV-2 S protein priming occurs remains debatable, therefore hampering the development of targeted treatments. Herein, we identified the human extracellular serine protease inhibitor (serpin) alpha 1 antitrypsin (A1AT) as a novel TMPRSS2 inhibitor. Structural modeling revealed that A1AT docked to an extracellular domain of TMPRSS2 in a conformation that is suitable for catalysis, resembling similar serine protease-inhibitor complexes. Inhibitory activity of A1AT was established in a SARS-CoV-2 viral load system. Notably, plasma A1AT levels were associated with COVID-19 disease severity. Our data support the key role of extracellular serine proteases in SARS-CoV-2 infections and indicate that treatment with serpins, particularly the FDA-approved drug A1AT, may be effective in limiting SARS-CoV-2 dissemination by affecting the surface of the host cells. SUMMARY: Delivery of extracellular serine protease inhibitors (serpins) such as A1AT has the capacity to reduce SARS-CoV-2 dissemination by binding and inhibiting extracellular proteases on the host cells, thus, inhibiting the first step in SARS-CoV-2 cell cycle (i.e. cell entry).

9.
PLoS One ; 15(1): e0227058, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31910225

RESUMEN

Nanotrap® (NT) particles are hydrogel microspheres developed for target analyte separation and discovery applications. NT particles consist of cross-linked N-isopropylacrylamide (NIPAm) copolymers that are functionalized with a variety of chemical affinity baits to enable broad-spectrum collection and retention of target proteins, nucleic acids, and pathogens. NT particles have been previously shown to capture and enrich arboviruses including Rift Valley fever and Venezuelan equine encephalitis viruses. Yet, there is still a need to enhance the detection ability for other re-emerging viruses such as Zika (ZIKV), chikungunya (CHIKV), and dengue (DENV) viruses. In this study, we exploited NT particles with different affinity baits, including cibacron blue, acrylic acid, and reactive red 120, to evaluate their capturing and enrichment capability for ZIKV, DENV and CHIKV in human fluids. Our results demonstrate that CN1030, a NT particle conjugated with reactive red 120, can recover between 8-16-fold greater genomic copies of ZIKV, CHIKV and DENV in virus spiked urine samples via RT-qPCR, superior to the other chemical baits. Also, we observed that CN1030 simultaneously enriched ZIKV, CHIKV and DENV in co-infection-based settings and could stabilize ZIKV, but not CHIKV infectivity in saliva spiked samples. CN1030 enriched viral detection at various viral concentrations, with significant enhancement observed at viral titers as low as 100 PFU/mL for ZIKV and 10 PFU/mL for CHIKV. The detection of ZIKV was further enhanced with NT particles by processing of larger volume urine samples. Furthermore, we developed a magnetic NT particle, CN3080, based on the same backbone of CN1030, and demonstrated that CN3080 could also capture and enrich ZIKV and CHIKV in a dose-dependent manner. Finally, in silico docking predictions support that the affinity between reactive red 120 and ZIKV or CHIKV envelope proteins appeared to be greater than acrylic acid. Overall, our data show that NT particles along with reactive red 120 can be utilized as a pre-processing technology for enhancement of detecting febrile-illness causing viruses.


Asunto(s)
Infecciones por Arbovirus/orina , Virus Chikungunya/aislamiento & purificación , Virus del Dengue/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Nanopartículas/química , Virus Zika/aislamiento & purificación , Infecciones por Arbovirus/diagnóstico , Infecciones por Arbovirus/virología , Virus Chikungunya/genética , Virus Chikungunya/patogenicidad , Colorantes/química , Virus del Dengue/genética , Virus del Dengue/patogenicidad , Humanos , Hidrogeles/química , Nanopartículas/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Unión Proteica , Saliva/virología , Orina/virología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Virus Zika/genética , Virus Zika/patogenicidad
10.
Int J Antimicrob Agents ; 54(1): 80-84, 2019 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-30930299

RESUMEN

Zika virus (ZIKV) is a re-emerging Flavivirus that has been linked to microcephaly and other neurological pathologies. In this study, phloretin, a glucose transporter inhibitor naturally derived from plants, was used to investigate the glucose dependence of ZIKV replication in host cells. The results showed that phloretin significantly decreased infectious titres of two ZIKV strains, namely MR766 (African genotype) and PRVABC59 (Puerto Rico genotype). The 50% effective concentration (EC50) of phloretin against MR766 and PRVABC59 was 22.85 µM and 9.31 µM, respectively. Further analyses demonstrated that decreased viral production was due to host-targeted inhibition, including decreased apoptotic caspase-3 and -7 activities and reduced phosphorylation of Akt/mTOR pathways. In addition, upon disruption of cellular glucose availability within host cells using 2-deoxy-d-glucose, ZIKV propagation was inhibited. Collectively, we demonstrate phloretin inhibition of ZIKV propagation and provide evidence of glucose utilization pathways as being important for ZIKV propagation. The activity of phloretin and its role in inhibiting glucose uptake could provide a useful foundation for the development of ZIKV antivirals.


Asunto(s)
Antivirales/farmacología , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Floretina/farmacología , Replicación Viral/efectos de los fármacos , Virus Zika/efectos de los fármacos , Animales , Chlorocebus aethiops , Células Vero , Carga Viral , Virus Zika/crecimiento & desarrollo
11.
Antiviral Res ; 163: 125-139, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30695702

RESUMEN

The New World alphaviruses, Venezuelan, eastern and western equine encephalitis viruses (VEEV, EEEV, and WEEV), are important human pathogens due to their ability to cause varying levels of morbidity and mortality in humans. There is also concern about VEEV and EEEV being used as bioweapons. Currently, a FDA-approved antiviral is lacking for New World alphaviruses. In this review, the function of each viral protein is discussed with an emphasis on how these functions can be targeted by therapeutics. Both direct acting antivirals as well as inhibitors that impact host protein interactions with viral proteins are described. Non-structural protein 3 (nsP3), capsid, and E2 proteins have garnered attention in recent years, whereas little is known regarding host protein interactions of the other viral proteins and is an important avenue for future study.


Asunto(s)
Infecciones por Alphavirus/tratamiento farmacológico , Alphavirus/efectos de los fármacos , Antivirales/uso terapéutico , Proteínas Virales/química , Alphavirus/fisiología , Animales , Antivirales/farmacología , Línea Celular , Ensayos Clínicos como Asunto , Interacciones Microbiota-Huesped/efectos de los fármacos , Humanos , Ratones , Replicación Viral/efectos de los fármacos
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA