RESUMO
Successfully combating the COVID-19 pandemic depends on mass vaccination with suitable vaccines to achieve herd immunity. Here, we describe COVI-VAC, the only live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine currently in clinical development. COVI-VAC was developed by recoding a segment of the viral spike protein with synonymous suboptimal codon pairs (codon-pair deoptimization), thereby introducing 283 silent (point) mutations. In addition, the furin cleavage site within the spike protein was deleted from the viral genome for added safety of the vaccine strain. Except for the furin cleavage site deletion, the COVI-VAC and parental SARS-CoV-2 amino acid sequences are identical, ensuring that all viral proteins can engage with the host immune system of vaccine recipients. COVI-VAC was temperature sensitive in vitro yet grew robustly (>107 plaque forming units/mL) at the permissive temperature. Tissue viral loads were consistently lower, lung pathology milder, and weight loss reduced in Syrian golden hamsters (Mesocricetus auratus) vaccinated intranasally with COVI-VAC compared to those inoculated with wild-type (WT) virus. COVI-VAC inoculation generated spike IgG antibody levels and plaque reduction neutralization titers similar to those in hamsters inoculated with WT virus. Upon challenge with WT virus, COVI-VAC vaccination reduced lung challenge viral titers, resulted in undetectable virus in the brain, and protected hamsters from almost all SARS-CoV-2-associated weight loss. Highly attenuated COVI-VAC is protective at a single intranasal dose in a relevant in vivo model. This, coupled with its large-scale manufacturing potential, supports its potential use in mass vaccination programs.
Assuntos
Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/farmacologia , COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Animais , Anticorpos Antivirais/imunologia , COVID-19/epidemiologia , Chlorocebus aethiops , Feminino , Humanos , Masculino , Mesocricetus , Pandemias , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação , Vacinas Atenuadas/imunologia , Células VeroRESUMO
Inflammation is inextricably associated with primary tumor progression. However, the contribution of inflammation to tumor outgrowth in metastatic organs has remained underexplored. Here, we show that extrinsic inflammation in the lungs leads to the recruitment of bone marrow-derived neutrophils, which degranulate azurophilic granules to release the Ser proteases, elastase and cathepsin G, resulting in the proteolytic destruction of the antitumorigenic factor thrombospondin-1 (Tsp-1). Genetic ablation of these neutrophil proteases protected Tsp-1 from degradation and suppressed lung metastasis. These results provide mechanistic insights into the contribution of inflammatory neutrophils to metastasis and highlight the unique neutrophil protease-Tsp-1 axis as a potential antimetastatic therapeutic target.
Assuntos
Neoplasias Pulmonares/metabolismo , Neutrófilos/metabolismo , Peptídeo Hidrolases/metabolismo , Pneumonia/metabolismo , Trombospondina 1/metabolismo , Animais , Western Blotting , Transplante de Medula Óssea , Catepsina G/metabolismo , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Expressão Gênica , Elastase de Leucócito/metabolismo , Lipopolissacarídeos/administração & dosagem , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Proteólise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Proteases/metabolismo , Trombospondina 1/genéticaRESUMO
The ability of interferons (IFNs) to inhibit viral replication and cellular proliferation is well established, but the specific contribution of each IFN-stimulated gene (ISG) to these biological responses remains to be completely understood. In this report we demonstrate that ISG54, also known as IFN-induced protein with tetratricopeptide repeats 2 (IFIT2), is a mediator of apoptosis. Expression of ISG54, independent of IFN stimulation, elicits apoptotic cell death. Cell death and apoptosis were quantified by propidium iodide uptake and annexin-V staining, respectively. The activation of caspase-3, a key mediator of the execution phase of apoptosis, was clearly apparent in cells expressing ISG54. The anti-apoptotic B cell lymphoma-xl (Bcl-xl) protein inhibited the apoptotic effects of ISG54 as did the anti-apoptotic adenoviral E1B-19K protein. In addition, ISG54 was not able to promote cell death in the absence of pro-apoptotic Bcl family members, Bax and Bak. Analyses of binding partners of ISG54 revealed association with two homologous proteins, ISG56/IFIT1 and ISG60/IFIT3. In addition, ISG60 binding negatively regulates the apoptotic effects of ISG54. The results reveal a previously unidentified role of ISG54 in the induction of apoptosis via a mitochondrial pathway and shed new light on the mechanism by which IFN elicits anti-viral and anti-cancer effects.
Assuntos
Apoptose/imunologia , Interferon-alfa/metabolismo , Proteínas , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Linhagem Celular , Citoplasma/metabolismo , Humanos , Interferon-alfa/imunologia , Interferon-alfa/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Rim/citologia , Camundongos , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Ligação Proteica/fisiologia , Proteínas/genética , Proteínas/imunologia , Proteínas/metabolismo , RNA Interferente Pequeno , Proteínas de Ligação a RNA , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismoRESUMO
A balanced immune response to infection is essential to prevent the pathology and tissue damage that can occur from an unregulated or hyperactive host defense. Interferons (IFNs) are critical mediators of the innate defense to infection, and in this study we evaluated the contribution of a specific gene coding for IFIT2 induced by type I IFNs in a murine model of disseminated Candida albicans Invasive candidiasis is a frequent challenge during immunosuppression or surgical medical interventions, and C. albicans is a common culprit that leads to high rates of mortality. When IFIT2 knockout mice were infected systemically with C. albicans, they were found to have improved survival and reduced fungal burden compared to wild-type mice. One of the mechanisms by which IFIT2 increases the pathological effects of invasive C. albicans appears to be suppression of NADPH oxidase activation. Loss of IFIT2 increases production of reactive oxygen species by leukocytes, and we demonstrate that IFIT2 is a binding partner of a critical regulatory subunit of NADPH oxidase, p67phox Since the administration of IFN has been used therapeutically to combat viral infections, cancer, and multiple sclerosis, we evaluated administration of IFN-ß to mice prior to C. albicans infection. IFN-ß treatment promoted pathology and death from C. albicans infection. We provide evidence that IFIT2 increases the pathological effects of invasive C. albicans and that administration of IFN-ß has deleterious effects during infection.IMPORTANCE The attributable mortality associated with systemic C. albicans infections in health care settings is significant, with estimates greater than 40%. This life-threatening disease is common in patients with weakened immune systems, either due to disease or as a result of therapies. Type I interferons (IFN) are cytokines of the innate defense response that are used as immune modulators in the treatment of specific cancers, viral infections, and multiple sclerosis. In this study, we show using a murine model that the loss of a specific IFN-stimulated gene coding for IFIT2 improves survival following systemic C. albicans infection. This result infers a harmful effect of IFN during C. albicans infection and is supported by our finding that administration of IFN-ß prior to invasive infection promotes fatal pathology. The findings contribute to our understanding of the innate immune response to C. albicans, and they suggest that IFN therapies present a risk factor for disseminated candidiasis.
Assuntos
Candida albicans/crescimento & desenvolvimento , Candidíase Invasiva/patologia , Interferon beta/metabolismo , Proteínas/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Candidíase Invasiva/microbiologia , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Deleção de Genes , Camundongos Knockout , NADPH Oxidases/metabolismo , Ligação Proteica , Proteínas/genética , Proteínas de Ligação a RNA , Análise de SobrevidaRESUMO
Molecularly targeted therapies benefit approximately 15-20% of non-small cell lung cancer (NSCLC) patients carrying specific drug-sensitive mutations. Thus, there is a clinically unmet need for the identification of novel targets for drug development. Here, we performed RNA-deep sequencing to identify altered gene expression between malignant and non-malignant lung tissue. Matrix Metalloproteinase 14 (MMP14), a membrane-bound proteinase, was significantly up-regulated in the tumor epithelial cells and intratumoral myeloid compartments in both mouse and human NSCLC. Overexpression of a soluble dominant negative MMP14 (DN-MMP14) or pharmacological inhibition of MMP14 blocked invasion of lung cancer cells through a collagen I matrix in vitro and reduced tumor incidence in an orthotopic K-RasG12D/+p53-/- mouse model of lung cancer. Additionally, MMP14 activity mediated proteolytic processing and activation of Heparin-Binding EGF-like Growth Factor (HB-EGF), stimulating the EGFR signaling pathway to increase proliferation and tumor growth. This study highlights the potential for development of therapeutic strategies that target MMP14 in NSCLC with particular focus on MMP14-HB-EGF axis.