RESUMEN
Human astroviruses (HAstV) are major global causes of gastroenteritis, but little is known about host factors required for their cellular entry. Here, we utilized complementary CRISPR-Cas9-based knockout and activation screening approaches and identified neonatal Fc receptor (FcRn) and dipeptidyl-peptidase IV (DPP4) as entry factors for HAstV infection of human intestinal epithelial cells. Disruption of FcRn or DPP4 reduced HAstV infection in permissive cells and, reciprocally, overexpression of these factors in non-permissive cells was sufficient to promote infection. We observed direct binding between FcRn and HAstV virions as well as purified spike protein. Finally, inhibitors for DPP4 and FcRn currently in clinical use prevent HAstV infection in cell lines and primary human enteroids. Thus, our results reveal mechanisms of HAstV entry as well as druggable targets. One-Sentence Summary: Targeting FcRn or DPP4 using available therapies effectively prevents human astrovirus infection in human enteroid cultures.
RESUMEN
Exposure of eukaryotic cells to ionizing radiation or clastogenic chemicals leads to formation of DNA double-strand breaks (DSBs). These lesions are also generated internally by chemicals and enzymes, in the absence of exogenous agents, though the sources and consequences of such endogenously generated DSBs remain poorly understood. In the current study, we have investigated the impact of reduced recombinational repair of endogenous DSBs on stress responses, cell morphology and other physical properties of S. cerevisiae (budding yeast) cells. Use of phase contrast and DAPI-based fluorescence microscopy combined with FACS analysis confirmed that recombination-deficient rad52 cell cultures exhibit chronically high levels of G2 phase cells. Cell cycle phase transit times during G1, S and M were similar in WT and rad52 cells, but the length of G2 phase was increased by three-fold in the mutants. rad52 cells were larger than WT in all phases of the cycle and displayed other quantifiable changes in physical characteristics. The high G2 cell phenotype was abolished when DNA damage checkpoint genes, but not spindle assembly checkpoint genes, were co-inactivated with RAD52. Several other RAD52 group mutants (rad51, rad54, rad55, rad57 and rad59) also exhibited the high G2 cell phenotype. The results indicate that recombination deficiency leads to accumulation of unrepaired DSBs during normal mitotic growth that activate a major stress response and produce distinct changes in cellular physiology and morphology.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Reparación del ADN , Ciclo Celular/genética , Recombinación Homóloga/genéticaRESUMEN
Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we used single-cell sequencing to profile SARS-CoV-2-reactive B cells in 38 COVID-19 patients. Using oligo-tagged antigen baits, we isolated B cells specific to the SARS-CoV-2 spike, nucleoprotein (NP), open reading frame 8 (ORF8), and endemic human coronavirus (HCoV) spike proteins. SARS-CoV-2 spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients several months post symptom onset. With severe acute infection, substantial populations of endemic HCoV-reactive antibody-secreting cells were identified and possessed highly mutated variable genes, signifying preexisting immunity. Finally, MBCs exhibited pronounced maturation to NP and ORF8 over time, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal antibody adaptation to non-neutralizing intracellular antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.
Asunto(s)
Anticuerpos Antivirales/inmunología , Formación de Anticuerpos/inmunología , Linfocitos B/inmunología , COVID-19/inmunología , Interacciones Huésped-Patógeno/inmunología , Epítopos Inmunodominantes/inmunología , SARS-CoV-2/inmunología , Anticuerpos Neutralizantes/inmunología , Formación de Anticuerpos/genética , Linfocitos B/metabolismo , Biología Computacional/métodos , Reacciones Cruzadas/inmunología , Mapeo Epitopo , Femenino , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Patógeno/genética , Humanos , Epítopos Inmunodominantes/genética , Memoria Inmunológica , Masculino , Pruebas de Neutralización , Análisis de la Célula Individual/métodos , Glicoproteína de la Espiga del Coronavirus/inmunología , TranscriptomaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing a global pandemic. The antigen specificity of the antibody response mounted against this novel virus is not understood in detail. Here, we report that subjects with a more severe SARS-CoV-2 infection exhibit a larger antibody response against the spike and nucleocapsid protein and epitope spreading to subdominant viral antigens, such as open reading frame 8 and nonstructural proteins. Subjects with a greater antibody response mounted a larger memory B cell response against the spike, but not the nucleocapsid protein. Additionally, we revealed that antibodies against the spike are still capable of binding the D614G spike mutant and cross-react with the SARS-CoV-1 receptor binding domain. Together, this study reveals that subjects with a more severe SARS-CoV-2 infection exhibit a greater overall antibody response to the spike and nucleocapsid protein and a larger memory B cell response against the spike.IMPORTANCE With the ongoing pandemic, it is critical to understand how natural immunity against SARS-CoV-2 and COVID-19 develops. We have identified that subjects with more severe COVID-19 disease mount a more robust and neutralizing antibody response against SARS-CoV-2 spike protein. Subjects who mounted a larger response against the spike also mounted antibody responses against other viral antigens, including the nucleocapsid protein and ORF8. Additionally, this study reveals that subjects with more severe disease mount a larger memory B cell response against the spike. These data suggest that subjects with more severe COVID-19 disease are likely better protected from reinfection with SARS-CoV-2.
Asunto(s)
COVID-19/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Linfocitos B/inmunología , COVID-19/sangre , COVID-19/virología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Reacciones Cruzadas , Epítopos/inmunología , Femenino , Humanos , Inmunidad Humoral/inmunología , Masculino , Persona de Mediana Edad , Fosfoproteínas/inmunologíaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing a global pandemic. The antigen specificity and kinetics of the antibody response mounted against this novel virus are not understood in detail. Here, we report that subjects with a more severe SARS-CoV-2 infection exhibit a larger antibody response against the spike and nucleocapsid protein and epitope spreading to subdominant viral antigens, such as open reading frame 8 and non-structural proteins. Subjects with a greater antibody response mounted a larger memory B cell response against the spike, but not the nucleocapsid protein. Additionally, we revealed that antibodies against the spike are still capable of binding the D614G spike mutant and cross-react with the SARS-CoV-1 receptor binding domain. Together, this study reveals that subjects with a more severe SARS-CoV-2 infection exhibit a greater overall antibody response to the spike and nucleocapsid protein and a larger memory B cell response against the spike.
RESUMEN
Discovery of durable memory B cell (MBC) subsets against neutralizing viral epitopes is critical for determining immune correlates of protection from SARS-CoV-2 infection. Here, we identified functionally distinct SARS-CoV-2-reactive B cell subsets by profiling the repertoire of convalescent COVID-19 patients using a high-throughput B cell sorting and sequencing platform. Utilizing barcoded SARS-CoV-2 antigen baits, we isolated thousands of B cells that segregated into discrete functional subsets specific for the spike, nucleocapsid protein (NP), and open reading frame (ORF) proteins 7a and 8. Spike-specific B cells were enriched in canonical MBC clusters, and monoclonal antibodies (mAbs) from these cells were potently neutralizing. By contrast, B cells specific to ORF8 and NP were enriched in naïve and innate-like clusters, and mAbs against these targets were exclusively non-neutralizing. Finally, we identified that B cell specificity, subset distribution, and affinity maturation were impacted by clinical features such as age, sex, and symptom duration. Together, our data provide a comprehensive tool for evaluating B cell immunity to SARS-CoV-2 infection or vaccination and highlight the complexity of the human B cell response to SARS-CoV-2.