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2.
J Virol ; : JVI0151121, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34668780

RESUMO

The development of mouse models for COVID-19 has enabled testing of vaccines and therapeutics and defining aspects of SARS-CoV-2 pathogenesis. SARS-CoV-2 disease is severe in K18 transgenic mice (K18-hACE2-Tg) expressing human ACE2 (hACE2), the SARS-CoV-2 receptor, under an ectopic cytokeratin promoter, with high levels of infection measured in the lung and brain. Here, we evaluated SARS-CoV-2 infection in hACE2 KI mice that express hACE2 under an endogenous promoter in place of murine ACE2 (mACE2). Intranasal inoculation of hACE2 KI mice with SARS-CoV-2 WA1/2020 resulted in substantial viral replication within the upper and lower respiratory tracts with limited spread to extra-pulmonary organs. However, SARS-CoV-2-infected hACE2 KI mice did not lose weight and developed limited pathology. Moreover, no significant differences in viral burden were observed in hACE2 KI mice infected with B.1.1.7 or B.1.351 variants compared to WA1/2020 strain. Because the entry mechanisms of SARS-CoV-2 in mice remains uncertain, we evaluated the impact of the naturally-occurring, mouse-adapting N501Y mutation by comparing infection of hACE2 KI, K18-hACE2-Tg, ACE2-deficient, and wild-type C57BL/6 mice. The N501Y mutation minimally affected SARS-CoV-2 infection in hACE2 KI mice but was required for viral replication in wild-type C57BL/6 mice in a mACE2-dependent manner and augmented pathogenesis in the K18-hACE2 Tg mice. Thus, the N501Y mutation likely enhances interactions with mACE2 or hACE2 in vivo. Overall, our study highlights the hACE2 KI mice as a model of mild SARS-CoV-2 infection and disease and clarifies the requirement of the N501Y mutation in mice. IMPORTANCE Mouse models of SARS-CoV-2 pathogenesis have facilitated the rapid evaluation of countermeasures. While the first generation of models developed pneumonia and severe disease after SARS-CoV-2 infection, they relied on ectopic expression of supraphysiological levels of human ACE2 (hACE2). This has raised issues with their relevance to humans as the hACE2 receptor shows a more restricted expression pattern in the respiratory tract. Here we evaluated SARS-CoV-2 infection and disease with viruses containing or lacking a key mouse-adapting mutation in the spike gene in hACE2 KI mice, which express hACE2 under an endogenous promoter in place of murine ACE2. While infection of hACE2 KI mice with multiple strains of SARS-CoV-2 including variants of concern resulted in viral replication within the upper and lower respiratory tracts, the animals did not sustain severe lung injury. Thus, hACE2 KI mice serve as a model of mild infection with both ancestral and emerging SARS-CoV-2 variant strains.

3.
Sci Rep ; 11(1): 19470, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34593848

RESUMO

The germicidal potential of specific wavelengths within the electromagnetic spectrum is an area of growing interest. While ultra-violet (UV) based technologies have shown satisfactory virucidal potential, the photo-toxicity in humans coupled with UV associated polymer degradation limit their use in occupied spaces. Alternatively, longer wavelengths with less irradiation energy such as visible light (405 nm) have largely been explored in the context of bactericidal and fungicidal applications. Such studies indicated that 405 nm mediated inactivation is caused by the absorbance of porphyrins within the organism creating reactive oxygen species which result in free radical damage to its DNA and disruption of cellular functions. The virucidal potential of visible-light based technologies has been largely unexplored and speculated to be ineffective given the lack of porphyrins in viruses. The current study demonstrated increased susceptibility of lipid-enveloped respiratory pathogens of importance such as SARS-CoV-2 (causative agent of COVID-19) and influenza A virus to 405 nm, visible light in the absence of exogenous photosensitizers thereby indicating a potential alternative porphyrin-independent mechanism of visible light mediated viral inactivation. These results were obtained using less than expected irradiance levels which are considered safe for humans and commercially achievable. Our results support further exploration of the use of visible light technology for the application of continuous decontamination in occupied areas within hospitals and/or infectious disease laboratories, specifically for the inactivation of respiratory pathogens such as SARS-CoV-2 and Influenza A.


Assuntos
Desinfecção/métodos , Vírus da Influenza A Subtipo H1N1/efeitos da radiação , SARS-CoV-2/efeitos da radiação , Desinfecção/instrumentação , Relação Dose-Resposta à Radiação , Vírus da Encefalomiocardite/efeitos da radiação , Luz , Fatores de Tempo , Inativação de Vírus/efeitos da radiação
4.
Front Immunol ; 12: 729189, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34603303

RESUMO

Several SARS-CoV-2 vaccines have received EUAs, but many issues remain unresolved, including duration of conferred immunity and breadth of cross-protection. Adjuvants that enhance and shape adaptive immune responses that confer broad protection against SARS-CoV-2 variants will be pivotal for long-term protection as drift variants continue to emerge. We developed an intranasal, rationally designed adjuvant integrating a nanoemulsion (NE) that activates TLRs and NLRP3 with an RNA agonist of RIG-I (IVT DI). The combination adjuvant with spike protein antigen elicited robust responses to SARS-CoV-2 in mice, with markedly enhanced TH1-biased cellular responses and high virus-neutralizing antibody titers towards both homologous SARS-CoV-2 and a variant harboring the N501Y mutation shared by B1.1.7, B.1.351 and P.1 variants. Furthermore, passive transfer of vaccination-induced antibodies protected naive mice against heterologous viral challenge. NE/IVT DI enables mucosal vaccination, and has the potential to improve the immune profile of a variety of SARS-CoV-2 vaccine candidates to provide effective cross-protection against future drift variants.


Assuntos
Adjuvantes Imunológicos/farmacologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Vacinas Sintéticas/imunologia , Imunidade Adaptativa/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Chlorocebus aethiops , Proteção Cruzada/imunologia , Proteína DEAD-box 58 , Células HEK293 , Humanos , Imunidade Humoral/imunologia , Imunização Passiva , Camundongos , Camundongos Endogâmicos C57BL , Receptores Imunológicos/agonistas , Proteínas Recombinantes/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação , Células Vero
5.
Sci Rep ; 11(1): 19970, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620951

RESUMO

Particulate respirators such as N95s are an essential component of personal protective equipment (PPE) for front-line workers. This study describes a rapid and effective UVC irradiation system that would facilitate the safe re-use of N95 respirators and provides supporting information for deploying UVC for decontamination of SARS-CoV-2 during the COVID-19 pandemic. To assess the inactivation potential of the proposed UVC germicidal device as a function of time by using 3 M 8211-N95 particulate respirators inoculated with SARS-CoV-2. A germicidal UVC device to deliver tailored UVC dose was developed and test coupons (2.5 cm2) of the 3 M-N95 respirator were inoculated with 106 plaque-forming units (PFU) of SARS-CoV-2 and were UV irradiated. Different exposure times were tested (0-164 s) by fixing the distance between the lamp and the test coupon to 15.2 cm while providing an exposure of at least 5.43 mWcm-2. Primary measure of outcome was titration of infectious virus recovered from virus-inoculated respirator test coupons after UVC exposure. Other measures included the method validation of the irradiation protocol, using lentiviruses (biosafety level-2 agent) and establishment of the germicidal UVC exposure protocol. An average of 4.38 × 103 PFU ml-1 (SD 772.68) was recovered from untreated test coupons while 4.44 × 102 PFU ml-1 (SD 203.67), 4.00 × 102 PFU ml-1 (SD 115.47), 1.56 × 102 PFU ml-1 (SD 76.98) and 4.44 × 101 PFU ml-1 (SD 76.98) was recovered in exposures 2, 6, 18 and 54 s per side respectively. The germicidal device output and positioning was monitored and a minimum output of 5.43 mW cm-2 was maintained. Infectious SARS-CoV-2 was not detected by plaque assays (minimal level of detection is 67 PFU ml-1) on N95 respirator test coupons when irradiated for 120 s per side or longer suggesting 3.5 log reduction in 240 s of irradiation, 1.3 J cm-2. A scalable germicidal UVC device to deliver tailored UVC dose for rapid decontamination of SARS-CoV-2 was developed. UVC germicidal irradiation of N95 test coupons inoculated with SARS-CoV-2 for 120 s per side resulted in 3.5 log reduction of virus. These data support the reuse of N95 particle-filtrate apparatus upon irradiation with UVC and supports use of UVC-based decontamination of SARS-CoV-2 during the COVID-19 pandemic.

6.
Nature ; 2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34517409

RESUMO

Derailed cytokine and immune cell networks account for organ damage and clinical severity of COVID-191-4. Here we show that SARS-CoV-2, like other viruses, evokes cellular senescence as a primary stress response in infected cells. Virus-induced senescence (VIS) is indistinguishable from other forms of cellular senescence and accompanied by a senescence-associated secretory phenotype (SASP), composed of pro-inflammatory cytokines, extracellular matrix-active factors and pro-coagulatory mediators5-7. COVID-19 patients displayed markers of senescence in their airway mucosa in situ and elevated serum levels of SASP factors. Mirroring COVID-19 hallmark features such as macrophage and neutrophil infiltration, endothelial damage and widespread thrombosis in affected lung tissue1,8,9, in vitro assays demonstrated macrophage activation with SASP-reminiscent secretion, complement lysis and SASP-amplifying secondary senescence of endothelial cells, neutrophil extracellular trap (NET) formation as well as activation of platelets and the clotting cascade in response to supernatant of VIS cells, including SARS-CoV-2-induced senescence. Senolytics such as Navitoclax and Dasatinib/Quercetin selectively eliminated VIS cells, mitigated COVID-19-reminiscent lung disease and reduced inflammation in SARS-CoV-2-driven hamster and mouse models. Our findings mark VIS as pathogenic trigger of COVID-19-related cytokine escalation and organ damage, and suggest senolytic targeting of virus-infected cells as a novel treatment option against SARS-CoV-2 and perhaps other viral infections.

7.
Science ; 373(6554): 541-547, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34326236

RESUMO

Repurposing drugs as treatments for COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drawn much attention. Beginning with sigma receptor ligands and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs we tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs and does not reflect specific target-based activities-rather, it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Reposicionamento de Medicamentos , Lipidoses/induzido quimicamente , Fosfolipídeos/metabolismo , SARS-CoV-2/efeitos dos fármacos , Células A549 , Animais , Antivirais/química , Antivirais/uso terapêutico , Antivirais/toxicidade , COVID-19/virologia , Cátions , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Testes de Sensibilidade Microbiana , SARS-CoV-2/fisiologia , Tensoativos/química , Tensoativos/farmacologia , Tensoativos/toxicidade , Células Vero , Replicação Viral/efeitos dos fármacos
8.
Hum Pathol ; 114: 110-119, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33961839

RESUMO

Coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although viral infection is known to trigger inflammatory processes contributing to tissue injury and organ failure, it is unclear whether direct viral damage is needed to sustain cellular injury. An understanding of pathogenic mechanisms has been handicapped by the absence of optimized methods to visualize the presence and distribution of SARS-CoV-2 in damaged tissues. We first developed a positive control cell line (Vero E6) to validate SARS-CoV-2 detection assays. We then evaluated multiple organs (lungs, kidneys, heart, liver, brain, intestines, lymph nodes, and spleen) from fourteen COVID-19 autopsy cases using immunohistochemistry (IHC) for the spike and the nucleoprotein proteins, and RNA in situ hybridization (RNA ISH) for the spike protein mRNA. Tissue detection assays were compared with quantitative polymerase chain reaction (qPCR)-based detection. SARS-CoV-2 was histologically detected in the Vero E6 positive cell line control, 1 of 14 (7%) lungs, and none (0%) of the other 59 organs. There was perfect concordance between the IHC and RNA ISH results. qPCR confirmed high viral load in the SARS-CoV-2 ISH-positive lung tissue, and absent or low viral load in all ISH-negative tissues. In patients who die of COVID-19-related organ failure, SARS-CoV-2 is largely not detectable using tissue-based assays. Even in lungs showing widespread injury, SARS-CoV-2 viral RNA or proteins were detected in only a small minority of cases. This observation supports the concept that viral infection is primarily a trigger for multiple-organ pathogenic proinflammatory responses. Direct viral tissue damage is a transient phenomenon that is generally not sustained throughout disease progression.


Assuntos
COVID-19/patologia , Fígado/virologia , Pulmão/virologia , SARS-CoV-2/patogenicidade , Animais , Autopsia/métodos , COVID-19/virologia , Chlorocebus aethiops , Progressão da Doença , Humanos , Imuno-Histoquímica/métodos , Fígado/química , Fígado/patologia , Pulmão/patologia , RNA Viral/metabolismo , Células Vero/virologia , Carga Viral/métodos
9.
Cell ; 184(10): 2618-2632.e17, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33836156

RESUMO

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro, and in vivo analyses, we report that topoisomerase 1 (TOP1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of topotecan (TPT), an FDA-approved TOP1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as 4 days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of TOP1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing TOP1 inhibitors for severe coronavirus disease 2019 (COVID-19) in humans.


Assuntos
COVID-19/tratamento farmacológico , DNA Topoisomerases Tipo I/metabolismo , SARS-CoV-2/metabolismo , Inibidores da Topoisomerase I/farmacologia , Topotecan/farmacologia , Animais , COVID-19/enzimologia , COVID-19/patologia , Chlorocebus aethiops , Humanos , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Inflamação/patologia , Inflamação/virologia , Mesocricetus , Camundongos , Camundongos Transgênicos , Células THP-1 , Células Vero
10.
Viruses ; 13(4)2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33920517

RESUMO

The influenza A virus (IAV) is able to infect multiple mammalian and avian species, and in humans IAV is responsible for annual seasonal epidemics and occasional pandemics of respiratory disease with significant health and economic impacts. Studying IAV involves laborious secondary methodologies to identify infected cells. Therefore, to circumvent this requirement, in recent years, multiple replication-competent infectious IAV expressing traceable reporter genes have been developed. These IAVs have been very useful for in vitro and/or in vivo studies of viral replication, identification of neutralizing antibodies or antivirals, and in studies to evaluate vaccine efficacy, among others. In this report, we describe, for the first time, the generation and characterization of two replication-competent influenza A/Puerto Rico/8/1934 H1N1 (PR8) viruses where the viral non-structural protein 1 (NS1) was substituted by the monomeric (m)Cherry fluorescent or the NanoLuc luciferase (Nluc) proteins. The ΔNS1 mCherry was able to replicate in cultured cells and in Signal Transducer and Activator of Transcription 1 (STAT1) deficient mice, although at a lower extent than a wild-type (WT) PR8 virus expressing the same mCherry fluorescent protein (WT mCherry). Notably, expression of either reporter gene (mCherry or Nluc) was detected in infected cells by fluorescent microscopy or luciferase plate readers, respectively. ΔNS1 IAV expressing reporter genes provide a novel approach to better understand the biology and pathogenesis of IAV, and represent an excellent tool to develop new therapeutic approaches against IAV infections.


Assuntos
Vírus da Influenza A/fisiologia , Influenza Humana/virologia , Infecções por Orthomyxoviridae/virologia , Proteínas não Estruturais Virais/metabolismo , Animais , Cães , Genes Reporter , Células HEK293 , Humanos , Vírus da Influenza A/genética , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas não Estruturais Virais/genética , Replicação Viral
11.
Nat Immunol ; 22(4): 497-509, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33790474

RESUMO

Classic major histocompatibility complex class I (MHC-I) presentation relies on shuttling cytosolic peptides into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Viruses disable TAP to block MHC-I presentation and evade cytotoxic CD8+ T cells. Priming CD8+ T cells against these viruses is thought to rely solely on cross-presentation by uninfected TAP-functional dendritic cells. We found that protective CD8+ T cells could be mobilized during viral infection even when TAP was absent in all hematopoietic cells. TAP blockade depleted the endosomal recycling compartment of MHC-I molecules and, as such, impaired Toll-like receptor-regulated cross-presentation. Instead, MHC-I molecules accumulated in the ER-Golgi intermediate compartment (ERGIC), sequestered away from Toll-like receptor control, and coopted ER-SNARE Sec22b-mediated vesicular traffic to intersect with internalized antigen and rescue cross-presentation. Thus, when classic MHC-I presentation and endosomal recycling compartment-dependent cross-presentation are impaired in dendritic cells, cell-autonomous noncanonical cross-presentation relying on ERGIC-derived MHC-I counters TAP dysfunction to nevertheless mediate CD8+ T cell priming.


Assuntos
Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Modelos Animais de Doenças , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Feminino , Complexo de Golgi/imunologia , Complexo de Golgi/metabolismo , Complexo de Golgi/virologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/patogenicidade , Ativação Linfocitária , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/genética
12.
Mod Pathol ; 34(8): 1456-1467, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33795830

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated clinical syndrome COVID-19 are causing overwhelming morbidity and mortality around the globe and disproportionately affected New York City between March and May 2020. Here, we report on the first 100 COVID-19-positive autopsies performed at the Mount Sinai Hospital in New York City. Autopsies revealed large pulmonary emboli in six cases. Diffuse alveolar damage was present in over 90% of cases. We also report microthrombi in multiple organ systems including the brain, as well as hemophagocytosis. We additionally provide electron microscopic evidence of the presence of the virus in our samples. Laboratory results of our COVID-19 cohort disclose elevated inflammatory markers, abnormal coagulation values, and elevated cytokines IL-6, IL-8, and TNFα. Our autopsy series of COVID-19-positive patients reveals that this disease, often conceptualized as a primarily respiratory viral illness, has widespread effects in the body including hypercoagulability, a hyperinflammatory state, and endothelial dysfunction. Targeting of these multisystemic pathways could lead to new treatment avenues as well as combination therapies against SARS-CoV-2 infection.


Assuntos
COVID-19/fisiopatologia , Pulmão/fisiopatologia , Embolia Pulmonar/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Autopsia , Coagulação Sanguínea , COVID-19/sangue , COVID-19/patologia , COVID-19/virologia , Causas de Morte , Citocinas/sangue , Feminino , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/sangue , Pulmão/patologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Cidade de Nova Iorque , Embolia Pulmonar/sangue , Embolia Pulmonar/patologia , Embolia Pulmonar/virologia , SARS-CoV-2/patogenicidade
13.
J Exp Med ; 218(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33882122

RESUMO

The disease severity of influenza is highly variable in humans, and one genetic determinant behind these differences is the IFITM3 gene. As an effector of the interferon response, IFITM3 potently blocks cytosolic entry of influenza A virus (IAV). Here, we reveal a novel level of inhibition by IFITM3 in vivo: We show that incorporation of IFITM3 into IAV particles competes with incorporation of viral hemagglutinin (HA). Decreased virion HA levels did not reduce infectivity, suggesting that high HA density on IAV virions may be an antagonistic strategy used by the virus to prevent direct inhibition. However, we found that IFITM3-mediated reduction in HA content sensitizes IAV to antibody-mediated neutralization. Mathematical modeling predicted that this effect decreases and delays peak IAV titers, and we show that, indeed, IFITM3-mediated sensitization of IAV to antibody-mediated neutralization impacts infection outcome in an in vivo mouse model. Overall, our data describe a previously unappreciated interplay between the innate effector IFITM3 and the adaptive immune response.


Assuntos
Anticorpos Neutralizantes/imunologia , Vírus da Influenza A/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Células A549 , Imunidade Adaptativa/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cães , Feminino , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Influenza Humana/imunologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteólise
15.
mBio ; 12(2)2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33653892

RESUMO

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the prime target for vaccine development. The spike protein mediates both binding to host cells and membrane fusion and is also so far the only known viral target of neutralizing antibodies. Coronavirus spike proteins are large trimers that are relatively unstable, a feature that might be enhanced by the presence of a polybasic cleavage site in SARS-CoV-2 spike. Exchange of K986 and V987 for prolines has been shown to stabilize the trimers of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus spike proteins. Here, we test multiple versions of a soluble spike protein for their immunogenicity and protective effect against SARS-CoV-2 challenge in a mouse model that transiently expresses human angiotensin-converting enzyme 2 via adenovirus transduction. Variants tested include spike proteins with a deleted polybasic cleavage site, proline mutations, or a combination thereof, besides the wild-type protein. While all versions of the protein were able to induce neutralizing antibodies, only the antigen with both a deleted cleavage site and the K986P and V987P (PP) mutations completely protected from challenge in this mouse model.IMPORTANCE A vaccine for SARS-CoV-2 is urgently needed. A better understanding of antigen design and attributes that vaccine candidates need to have to induce protective immunity is of high importance. The data presented here validate the choice of antigens that contain the PP mutations and suggest that deletion of the polybasic cleavage site may lead to a further-optimized design.


Assuntos
Prolina/química , SARS-CoV-2/imunologia , Animais , Vacinas contra COVID-19/química , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/uso terapêutico , Camundongos , Mutação , Glicoproteína da Espícula de Coronavírus/química
16.
bioRxiv ; 2021 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-33619480

RESUMO

Several SARS-CoV-2 vaccines have received EUAs, but many issues remain unresolved, including duration of conferred immunity and breadth of cross-protection. Adjuvants that enhance and shape adaptive immune responses that confer broad protection against SARS-CoV-2 variants will be pivotal for long-term protection. We developed an intranasal, rationally designed adjuvant integrating a nanoemulsion (NE) that activates TLRs and NLRP3 with an RNA agonist of RIG-I (IVT DI). The combination adjuvant with spike protein antigen elicited robust responses to SARS-CoV-2 in mice, with markedly enhanced T H 1-biased cellular responses and high virus-neutralizing antibody titers towards both homologous SARS-CoV-2 and a variant harboring the N501Y mutation shared by B1.1.7, B.1.351 and P.1 variants. Furthermore, passive transfer of vaccination-induced antibodies protected naive mice against heterologous viral challenge. NE/IVT DI enables mucosal vaccination, and has the potential to improve the immune profile of a variety of SARS-CoV-2 vaccine candidates to provide effective cross-protection against future drift variants.

17.
Angew Chem Int Ed Engl ; 60(17): 9467-9473, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33464672

RESUMO

The search for vaccines that protect from severe morbidity and mortality because of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Here we describe an amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile. It is water-soluble and exhibits massive translocation to lymph nodes upon local administration through binding to albumin, affording localized innate immune activation and reduction in systemic inflammation. The adjuvanticity of IMDQ-PEG-CHOL was validated in a licensed vaccine setting (quadrivalent influenza vaccine) and an experimental trimeric recombinant SARS-CoV-2 spike protein vaccine, showing robust IgG2a and IgG1 antibody titers in mice that could neutralize viral infection in vitro and in vivo in a mouse model.


Assuntos
Adjuvantes Imunológicos/uso terapêutico , Vacinas contra COVID-19/uso terapêutico , COVID-19/prevenção & controle , Imidazóis/uso terapêutico , Imunidade Inata/efeitos dos fármacos , Quinolinas/uso terapêutico , Animais , Vacinas contra COVID-19/imunologia , Colesterol/análogos & derivados , Colesterol/imunologia , Colesterol/uso terapêutico , Feminino , Humanos , Imidazóis/imunologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/uso terapêutico , Influenza Humana/prevenção & controle , Glicoproteínas de Membrana/agonistas , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Polietilenoglicóis/uso terapêutico , Quinolinas/imunologia , Proteínas Recombinantes/imunologia , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/imunologia , Tensoativos/uso terapêutico , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas
18.
Science ; 371(6532): 926-931, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33495306

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Depsipeptídeos/farmacologia , Fator 1 de Elongação de Peptídeos/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Animais , Antivirais/uso terapêutico , COVID-19/prevenção & controle , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/biossíntese , Proteínas do Nucleocapsídeo de Coronavírus/genética , Depsipeptídeos/administração & dosagem , Depsipeptídeos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HEK293 , Humanos , Pulmão/virologia , Camundongos Endogâmicos C57BL , Mutação , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , RNA Viral/biossíntese , RNA Viral/genética , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacos
19.
Elife ; 92020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33287959

RESUMO

Under eubiotic conditions commensal microbes are known to provide a competitive barrier against invading bacterial pathogens in the intestinal tract, on the skin or on the vaginal mucosa. Here, we evaluate the role of lung microbiota in Pneumococcus colonization of the lungs. In eubiosis, the lungs of mice were dominantly colonized by Lactobacillus murinus. Differential analysis of 16S rRNA gene sequencing or L. murinus-specific qPCR of DNA from total organ homogenates vs.broncho alveolar lavages implicated tight association of these bacteria with the host tissue. Pure L. murinus conditioned culture medium inhibited growth and reduced the extension of pneumococcal chains. Growth inhibition in vitro was likely dependent on L. murinus-produced lactic acid, since pH neutralization of the conditioned medium aborted the antibacterial effect. Finally, we demonstrate that L. murinus provides a barrier against pneumococcal colonization in a respiratory dysbiosis model after an influenza A virus infection, when added therapeutically.


Assuntos
Lactobacillus/metabolismo , Pulmão/microbiologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/fisiologia , Animais , Portador Sadio , Meios de Cultivo Condicionados , Feminino , Ácido Láctico/metabolismo , Ácido Láctico/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Simbiose
20.
bioRxiv ; 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-33299999

RESUMO

The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro and in vivo analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.

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