RESUMEN
Coronaviruses have been the causative agent of three epidemics and pandemics in the past two decades, including the ongoing COVID-19 pandemic. A broadly-neutralizing coronavirus therapeutic is desirable not only to prevent and treat COVID-19, but also to provide protection for high-risk populations against future emergent coronaviruses. As all coronaviruses use spike proteins on the viral surface to enter the host cells, and these spike proteins share sequence and structural homology, we set out to discover cross-reactive biologic agents targeting the spike protein to block viral entry. Through llama immunization campaigns, we have identified single domain antibodies (VHHs) that are cross-reactive against multiple emergent coronaviruses (SARS-CoV, SARS-CoV-2, and MERS). Importantly, a number of these antibodies show sub-nanomolar potency towards all SARS-like viruses including emergent CoV-2 variants. We identified nine distinct epitopes on the spike protein targeted by these VHHs. Further, by engineering VHHs targeting distinct, conserved epitopes into multi-valent formats, we significantly enhanced their neutralization potencies compared to the corresponding VHH cocktails. We believe this approach is ideally suited to address both emerging SARS-CoV-2 variants during the current pandemic as well as potential future pandemics caused by SARS-like coronaviruses.
Asunto(s)
COVID-19 , Camélidos del Nuevo Mundo , Anticuerpos de Dominio Único , Humanos , Animales , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Pandemias , EpítoposRESUMEN
The SARS-CoV-2 pandemic and particularly the emerging variants have deepened the need for widely available therapeutic options. We have demonstrated that hexamer-enhancing mutations in the Fc region of anti-SARS-CoV IgG antibodies lead to a noticeable improvement in IC50 in both pseudo and live virus neutralization assay compared to parental molecules. We also show that hexamer-enhancing mutants improve C1q binding to target surface. To our knowledge, this is the first time this format has been explored for application in viral neutralization and the studies provide proof-of-concept for the use of hexamer-enhanced IgG1 molecules as potential anti-viral therapeutics.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Inmunoglobulina G/genética , Pruebas Inmunológicas , Pandemias , SARS-CoV-2/genéticaRESUMEN
Among â¼5,000,000 fungal species, C. albicans is exceptional in its lifelong association with humans, either within the gastrointestinal microbiome or as an invasive pathogen. Opportunistic infections are generally ascribed to defective host immunity but may require specific microbial programs. Here we report that exposure of C. albicans to the mammalian gut triggers a developmental switch, driven by the Wor1 transcription factor, to a commensal cell type. Wor1 expression was previously observed only in rare genetic backgrounds, where it controls a white-opaque switch in mating. We show that passage of wild-type cells through the mouse gastrointestinal tract triggers WOR1 expression and a novel phenotypic switch. The resulting GUT (gastrointestinally induced transition) cells differ morphologically and functionally from previously defined cell types, including opaque cells, and express a transcriptome that is optimized for the digestive tract. The white-GUT switch illuminates how a microorganism can use distinct genetic programs to transition between commensalism and invasive pathogenesis.
Asunto(s)
Candida albicans/genética , Candida albicans/metabolismo , Tracto Gastrointestinal/microbiología , Fenotipo , Animales , Candida albicans/ultraestructura , Femenino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Aptitud Genética , Humanos , Ratones , MutaciónRESUMEN
The mammalian gastrointestinal tract and bloodstream are highly disparate biological niches that differ in concentrations of nutrients such as iron. However, some commensal-pathogenic microorganisms, such as the yeast Candida albicans, thrive in both environments. We report the evolution of a transcription circuit in C. albicans that controls iron uptake and determines its fitness in both niches. Our analysis of DNA-binding proteins that regulate iron uptake by this organism suggests the evolutionary intercalation of a transcriptional activator called Sef1 between two broadly conserved iron-responsive transcriptional repressors, Sfu1 and Hap43. Sef1 activates iron-uptake genes and promotes virulence in a mouse model of bloodstream infection, whereas Sfu1 represses iron-uptake genes and is dispensable for virulence but promotes gastrointestinal commensalism. Thus, C. albicans can alternate between genetic programs conferring resistance to iron depletion in the bloodstream versus iron toxicity in the gut, and this may represent a fundamental attribute of gastrointestinal commensal-pathogens.
Asunto(s)
Candida albicans/patogenicidad , Proteínas Fúngicas/metabolismo , Homeostasis , Hierro/metabolismo , Simbiosis , Alelos , Animales , Candida albicans/genética , Candida albicans/crecimiento & desarrollo , Inmunoprecipitación de Cromatina/métodos , Femenino , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos BALB C , Filogenia , Plasma/metabolismo , Plasma/microbiología , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Activación Transcripcional , VirulenciaRESUMEN
Paired immunoglobulin-like type 2 receptors (PILRs) inhibitory PILRalpha and activating PILRbeta are predominantly expressed on myeloid cells. Their functions in host defense and inflammation are largely unknown, and in this study, we evaluated their roles in an acute Staphylococcus aureus pneumonia model. Compared to their respective controls, Pilrb(-/-) mice or mice in which PILRalpha was activated with an agonistic antibody showed improved clearance of pulmonary staphylococci and improved survival. These mice had reduced serum or bronchoalveolar lavage fluid levels of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and IL-6 and elevated levels of gamma interferon (IFN-gamma), IL-12, and IL-10. In contrast, mice in which PILRbeta was activated had increased lung bacterial burdens and higher mortality coupled with an intense proinflammatory response with highly elevated levels of IL-1beta, TNF-alpha, and IL-6. Treatment groups with reduced bacterial burdens had higher levels of Keratinocyte-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2), and MIP-1alpha in bronchoalveolar lavage fluid and an increased influx of neutrophils and macrophages to the lungs. Consistent with our in vivo findings, bone marrow-derived macrophages from Pilrb(-/-) mice released significantly less IL-1beta and TNF-alpha and more IFN-gamma and IL-12 than did the wild-type macrophages when directly stimulated with heat-killed S. aureus. To our knowledge, this is the first evidence that S. aureus directly interacts with PILRbeta. It provides a mechanism by which manipulating the balance in favor of an inhibitory PILR signal, by activation of PILRalpha or deletion of PILRbeta, helps to control acute S. aureus-mediated pneumonia and attenuate the inflammatory response. These results highlight the importance of PILRs in innate immunity and the control of inflammation.
Asunto(s)
Neumonía Estafilocócica/inmunología , Neumonía Estafilocócica/patología , Receptores Inmunológicos/metabolismo , Transducción de Señal , Staphylococcus aureus/inmunología , Animales , Análisis Químico de la Sangre , Líquido del Lavado Bronquioalveolar/química , Recuento de Colonia Microbiana , Citocinas/análisis , Citocinas/sangre , Femenino , Pulmón/microbiología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/inmunología , Análisis de SupervivenciaRESUMEN
Intravascular delivery (1.5 x 10(9) particles and higher) of recombinant adenovirus (rAd) induces myeloid cell mediated, self-limiting hemodynamic responses in normal mice. However, we observed anaphylactoid-type reactions and exacerbated hemodynamic events following rAd injection in mice bearing malignant 4T1 mammary carcinoma. Because 4T1 tumors induce significant CD11b(+)Gr-1(+) myeloid cell expansion and activation, we set to determine whether this causes rAd-induced exaggerated responses. When treated with a single intravenous dose (1 x 10(10) particles) of rAd, mice implanted with 4T1 carcinoma succumbed due to the anaphylactoid-type reactions. In contrast, normal mice and mice implanted with a related mammary carcinoma (66cl4) that does not induce CD11b(+)Gr-1(+) cell expansion, showed minimal responses. Depletion of phagocytic CD11b(+)Gr-1(+) cells prior to rAd delivery protected 4T1 tumor-bearing animals, whereas passive transfer of CD11b(+)Gr-1(+) cells from 4T1 tumor-bearing animals was sufficient to convey susceptibility to anaphylactoid-type reactions in normal animals. We further show that there is upregulation of nitric oxide and leukotriene signaling pathways in the 4T1 tumor-induced CD11b(+)Gr-1(+) myeloid cells and that pretreating mice with inhibitors of nitric oxide synthetase and leukotrienes can attenuate the anaphylactoid-type reactions. These data show that malignant tumor growth can alter CD11b(+)Gr-1(+) myeloid cells, rendering hosts susceptible to anaphylactoid-type reactions upon intravascular treatment with rAd.
Asunto(s)
Adenoviridae/inmunología , Anafilaxia/metabolismo , Antígeno CD11b/inmunología , Diferenciación Celular , Terapia Genética/efectos adversos , Neoplasias/patología , Receptores de Quimiocina/metabolismo , Adenoviridae/genética , Anafilaxia/fisiopatología , Animales , Sistema Cardiovascular/metabolismo , Línea Celular Tumoral , Susceptibilidad a Enfermedades , Femenino , Frecuencia Cardíaca , Humanos , Antagonistas de Leucotrieno/farmacología , Liposomas , Ratones , Células Mieloides/metabolismo , Trasplante de Neoplasias , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/terapia , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Fagocitos , Receptores de Quimiocina/inmunología , Receptores de Leucotrienos/metabolismoRESUMEN
Cachexia accompanies many chronic inflammatory diseases, including cancer. Lean tissue wasting is only one component of the cancer cachexia response, which also includes anemia, anorexia, a hepatic acute phase protein response, and increased susceptibility to secondary infections. The etiologies of cancer cachexia are multifactorial and include an overproduction of inflammatory mediators, including cytokines produced by inappropriate activation of innate immunity. However, anticytokine therapies have generally not been seriously considered for cancer cachexia, in large part because of the overlapping activities of several inflammatory cytokines and the inability to prospectively identify the contributions of individual mediators. In contrast, recent evidence has focused on an immature myeloid cell population that expands dramatically in the tumors and secondary lymphoid organs of animals with some actively growing tumors. These immature GR-1(+)CD11b(+) cells are metabolically active and secrete large quantities of inflammatory cytokines and chemokines with the potential to produce cachexia. Their expansion is temporally associated with the development of cachexia. Future studies are required to determine whether therapeutic efforts intended to block the expansion of these cells can prevent the lean tissue wasting that accompanies active tumor growth.
Asunto(s)
Caquexia/etiología , Citocinas/fisiología , Metabolismo Energético/fisiología , Inflamación/metabolismo , Neoplasias/metabolismo , Proteínas de Fase Aguda/metabolismo , Reacción de Fase Aguda/metabolismo , Caquexia/metabolismo , Humanos , Inflamación/fisiopatología , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Neoplasias/fisiopatologíaRESUMEN
The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) serves as a prototype for a range of environmental toxicants and as a pharmacologic probe to study signal transduction by the aryl hydrocarbon receptor (AHR). Despite a detailed understanding of how TCDD exposure leads to the transcriptional up-regulation of cytochrome P450-dependent monooxygenases, we know little about how compounds like TCDD lead to a variety of AHR-dependent toxic end points such as liver pathology, terata, thymic involution, and cancer. Using an acute exposure protocol and the toxic response of the mouse liver as a model system, we have begun a detailed microarray analysis to describe the transcriptional changes that occur after various TCDD doses and treatment times. Through correlation analysis of time- and dose-dependent toxicological end points, we are able to identify coordinately responsive transcriptional events that can be defined as primary transcriptional events and downstream events that may represent mechanistically linked sequelae or that have potential as biomarkers of toxicity.
Asunto(s)
Perfilación de la Expresión Génica , Hígado/efectos de los fármacos , Dibenzodioxinas Policloradas/toxicidad , Animales , Biomarcadores , Citocromo P-450 CYP1A1/fisiología , Citocromo P-450 CYP1A2/fisiología , Relación Dosis-Respuesta a Droga , Hígado/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Elementos de Respuesta/fisiologíaRESUMEN
The aryl hydrocarbon receptor (AHR) plays a role in three areas of biology that include the adaptive metabolism of xenobiotics, the toxic responses associated with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), and vascular remodeling of the developing embryo. To test the hypothesis that receptor signaling in different cell types is responsible for these aspects of AHR biology, we generated a conditional Ahr allele where exon 2 is flanked by loxP sites. Through the use of Cre-lox technology, we then investigated the role of AHR signaling in hepatocytes or endothelial cells in mediating prototypical endpoints of adaptive, toxic, or developmental signaling. Using this model, we provide evidence that AHR signaling in endothelial/hematopoietic cells is necessary for developmental closure of the ductus venosus, whereas AHR signaling in hepatocytes is necessary to generate adaptive and toxic responses of the liver in response to dioxin exposure. Taken together, these data illustrate the importance of cell-specific receptor signaling for the generation of distinct AHR-dependent physiological outcomes.
Asunto(s)
Hígado/citología , Hígado/efectos de los fármacos , Receptores de Hidrocarburo de Aril/metabolismo , Alelos , Animales , Células Cultivadas , Dioxinas/farmacología , Regulación del Desarrollo de la Expresión Génica , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Masculino , Ratones , Ratones Transgénicos , Tamaño de los Órganos , Receptores de Hidrocarburo de Aril/genética , Transducción de Señal , Timo/efectos de los fármacos , Timo/metabolismoRESUMEN
Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) results in a broad spectrum of toxic effects. Most, if not all, of these responses are dependent upon the binding of dioxin to the aryl hydrocarbon receptor. Given their common roles in chemically induced toxicity, we asked whether interleukin 1 (IL1)-like cytokines play a role in acute aspects of the dioxin response. To test this idea, we employed a "triple-null" mouse model that lacks the two receptors for the tumor necrosis factors-alpha and -beta and the receptor for the IL1-alpha and IL1-beta cytokines. When triple null mice were treated with dioxin, there was significant attenuation in the levels of serum alanine aminotransferase, signifying reduced hepatocellular damage. In addition, the triple-null mice were protected from dioxin-induced liver inflammation. Loss of receptors for the IL1-like cytokines was not protective for all aspects of dioxin toxicity. Endpoints such as thymic involution, Cyp1a2 induction, hepatomegaly, and hydropic degeneration remain unchanged in this model.
Asunto(s)
Citocinas/metabolismo , Dioxinas/toxicidad , Interleucina-1/metabolismo , Receptores de Interleucina-1/metabolismo , Animales , Citocinas/deficiencia , Citocinas/genética , Interleucina-1/deficiencia , Interleucina-1/genética , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Interleucina-1/deficiencia , Receptores de Interleucina-1/genética , Receptores del Factor de Necrosis Tumoral/deficiencia , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismoRESUMEN
Traditional models of toxicity have relied on dissecting chemical action into pharmacokinetic and pharmacodynamic processes. However, the integration of genomic information with toxicology will enhance our basic understanding of these processes and significantly change the way we apply toxicological information to risk assessment and regulatory problems. In this article, we summarize the application of gene expression information and polymorphism discovery to four areas in toxicology: toxicity testing, cross-species extrapolation, understanding mechanism of action, and susceptibility.