RESUMEN
Group 3 innate lymphoid cells (ILC3) are the major subset of gut-resident ILC with essential roles in infections and tissue repair, but how they adapt to the gut environment to maintain tissue residency is unclear. We report that Tox2 is critical for gut ILC3 maintenance and function. Gut ILC3 highly expressed Tox2, and depletion of Tox2 markedly decreased ILC3 in gut but not at central sites, resulting in defective control of Citrobacter rodentium infection. Single-cell transcriptional profiling revealed decreased expression of Hexokinase-2 in Tox2-deficient gut ILC3. Consistent with the requirement for hexokinases in glycolysis, Tox2-/- ILC3 displayed decreased ability to utilize glycolysis for protein translation. Ectopic expression of Hexokinase-2 rescued Tox2-/- gut ILC3 defects. Hypoxia and interleukin (IL)-17A each induced Tox2 expression in ILC3, suggesting a mechanism by which ILC3 adjusts to fluctuating environments by programming glycolytic metabolism. Our results reveal the requirement for Tox2 to support the metabolic adaptation of ILC3 within the gastrointestinal tract.
Asunto(s)
Citrobacter rodentium , Infecciones por Enterobacteriaceae , Glucólisis , Proteínas HMGB , Inmunidad Innata , Linfocitos , Ratones Noqueados , Animales , Ratones , Adaptación Fisiológica/inmunología , Citrobacter rodentium/inmunología , Infecciones por Enterobacteriaceae/inmunología , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/metabolismo , Hexoquinasa/metabolismo , Hexoquinasa/genética , Interleucina-17/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones Endogámicos C57BL , Transactivadores/metabolismo , Transactivadores/genética , Proteínas HMGB/genética , Proteínas HMGB/inmunología , Proteínas HMGB/metabolismoRESUMEN
Laboratory mice, while paramount for understanding basic biological phenomena, are limited in modeling complex diseases of humans and other free-living mammals. Because the microbiome is a major factor in mammalian physiology, we aimed to identify a naturally evolved reference microbiome to better recapitulate physiological phenomena relevant in the natural world outside the laboratory. Among 21 distinct mouse populations worldwide, we identified a closely related wild relative to standard laboratory mouse strains. Its bacterial gut microbiome differed significantly from its laboratory mouse counterpart and was transferred to and maintained in laboratory mice over several generations. Laboratory mice reconstituted with natural microbiota exhibited reduced inflammation and increased survival following influenza virus infection and improved resistance against mutagen/inflammation-induced colorectal tumorigenesis. By demonstrating the host fitness-promoting traits of natural microbiota, our findings should enable the discovery of protective mechanisms relevant in the natural world and improve the modeling of complex diseases of free-living mammals. VIDEO ABSTRACT.
Asunto(s)
Microbioma Gastrointestinal , Ratones/clasificación , Ratones/microbiología , Animales , Animales de Laboratorio , Animales Salvajes , Carcinogénesis/inmunología , Resistencia a la Enfermedad , Femenino , Masculino , Maryland , Ratones/inmunología , Ratones Endogámicos C57BL , Peromyscus , Virosis/inmunologíaRESUMEN
Despite intense interest in antiviral T cell priming, the routes by which virions move in lymph nodes (LNs) are imperfectly understood. Current models fail to explain how virus-infected cells rapidly appear within the LN interior after viral infection. To better understand virion trafficking in the LN, we determined the locations of virions and infected cells after administration to mice of vaccinia virus or Zika virus. Notably, many rapidly infected cells in the LN interior were adjacent to LN conduits. Through the use of confocal and electron microscopy, we clearly visualized virions within conduits. Functionally, CD8+ T cells rapidly and preferentially associated with vaccinia virus-infected cells in the LN paracortex, which led to T cell activation in the LN interior. These results reveal that it is possible for even large virions to flow through LN conduits and infect dendritic cells within the T cell zone to prime CD8+ T cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Ganglios Linfáticos/inmunología , Activación de Linfocitos/inmunología , Virión/inmunología , Animales , Linfocitos T CD8-positivos/virología , Femenino , Ganglios Linfáticos/ultraestructura , Ganglios Linfáticos/virología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Microscopía Electrónica de Transmisión , Virus Vaccinia/inmunología , Virus Vaccinia/fisiología , Virión/fisiología , Virión/ultraestructura , Virosis/inmunología , Virosis/virología , Virus Zika/inmunología , Virus Zika/fisiologíaRESUMEN
Antigenic drift refers to the evolutionary accumulation of amino acid substitutions in viral proteins selected by host adaptive immune systems as the virus circulates in a population. Antigenic drift can substantially limit the duration of immunity conferred by infection and vaccination. Here, I explain the factors contributing to the rapid antigenic drift of the SARS-CoV-2 spike protein and receptor proteins of other viruses and discuss the implications for SARS-CoV-2 evolution and immunity.
Asunto(s)
COVID-19/inmunología , Mutación/genética , SARS-CoV-2/fisiología , Inmunidad Adaptativa , Animales , Deriva y Cambio Antigénico , Evolución Biológica , Interacciones Huésped-Patógeno , Humanos , Glicoproteína de la Espiga del Coronavirus/inmunología , VacunaciónRESUMEN
Tumors frequently subvert major histocompatibility complex class I (MHC-I) peptide presentation to evade CD8+ T cell immunosurveillance, though how this is accomplished is not always well defined. To identify the global regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of genes that positively and negatively modulate MHC-I cell surface expression. Validated genes clustered in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Genes can exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors displayed genetic alterations in multiple regulators. We established SUGT1 as a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhanced DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity.
Asunto(s)
Linfocitos B/fisiología , Biomarcadores de Tumor/genética , Antígenos HLA/genética , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase I/genética , Linfoma de Células B Grandes Difuso/genética , Carcinogénesis/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Linaje de la Célula , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Regulación Neoplásica de la Expresión Génica , Pruebas Genéticas , Estudio de Asociación del Genoma Completo , Antígenos HLA/metabolismo , Humanos , Vigilancia Inmunológica , Linfoma de Células B Grandes Difuso/metabolismo , Escape del Tumor/genéticaRESUMEN
Immunodominance (ID) defines the hierarchical immune response to competing antigens in complex immunogens. Little is known regarding B cell and antibody ID despite its importance in immunity to viruses and other pathogens. We show that B cells and serum antibodies from inbred mice demonstrate a reproducible ID hierarchy to the five major antigenic sites in the influenza A virus hemagglutinin globular domain. The hierarchy changed as the immune response progressed, and it was dependent on antigen formulation and delivery. Passive antibody transfer and sequential infection experiments demonstrated 'original antigenic suppression', a phenomenon in which antibodies suppress memory responses to the priming antigenic site. Our study provides a template for attaining deeper understanding of antibody ID to viruses and other complex immunogens.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Interacciones Huésped-Patógeno/inmunología , Epítopos Inmunodominantes/inmunología , Virosis/inmunología , Virus/inmunología , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Antígenos Virales/química , Antígenos Virales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Antecedentes Genéticos , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Interacciones Huésped-Patógeno/genética , Inmunización , Epítopos Inmunodominantes/química , Memoria Inmunológica , Virus de la Influenza A/inmunología , Ganglios Linfáticos/inmunología , Ratones , Modelos Moleculares , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/inmunología , Conformación Proteica , Virosis/genética , Virosis/virologíaRESUMEN
Precise targeting of activation-induced cytidine deaminase (AID) to immunoglobulin (Ig) loci promotes antibody class switch recombination (CSR) and somatic hypermutation (SHM), whereas AID targeting of non-Ig loci can generate oncogenic DNA lesions. Here, we examined the contribution of G-quadruplex (G4) nucleic acid structures to AID targeting in vivo. Mice bearing a mutation in Aicda (AIDG133V) that disrupts AID-G4 binding modeled the pathology of hyper-IgM syndrome patients with an orthologous mutation, lacked CSR and SHM, and had broad defects in genome-wide AIDG133V chromatin localization. Genome-wide analyses also revealed that wild-type AID localized to MHCII genes, and AID expression correlated with decreased MHCII expression in germinal center B cells and diffuse large B cell lymphoma. Our findings indicate a crucial role for G4 binding in AID targeting and suggest that AID activity may extend beyond Ig loci to regulate the expression of genes relevant to the physiology and pathology of activated B cells.
Asunto(s)
Cromatina/genética , Cromatina/metabolismo , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , G-Cuádruplex , Síndrome de Inmunodeficiencia con Hiper-IgM/etiología , Síndrome de Inmunodeficiencia con Hiper-IgM/metabolismo , Mutación , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biología Computacional/métodos , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Activación Enzimática , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Centro Germinal/inmunología , Centro Germinal/metabolismo , Antígenos HLA/genética , Antígenos HLA/inmunología , Humanos , Síndrome de Inmunodeficiencia con Hiper-IgM/diagnóstico , Cambio de Clase de Inmunoglobulina/genética , Cambio de Clase de Inmunoglobulina/inmunología , Inmunofenotipificación , Activación de Linfocitos/genética , Linfoma de Células B Grandes Difuso/etiología , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Ratones , Ratones TransgénicosRESUMEN
B cell subsets expressing the transcription factor T-bet are associated with humoral immune responses and autoimmunity. Here, we examined the anatomic distribution, clonal relationships, and functional properties of T-bet+ and T-bet- memory B cells (MBCs) in the context of the influenza-specific immune response. In mice, both T-bet- and T-bet+ hemagglutinin (HA)-specific B cells arose in germinal centers, acquired memory B cell markers, and persisted indefinitely. Lineage tracing and IgH repertoire analyses revealed minimal interconversion between T-bet- and T-bet+ MBCs, and parabionts showed differential tissue residency and recirculation properties. T-bet+ MBCs could be subdivided into recirculating T-betlo MBCs and spleen-resident T-bethi MBCs. Human MBCs displayed similar features. Conditional gene deletion studies revealed that T-bet expression in B cells was required for nearly all HA stalk-specific IgG2c antibodies and for durable neutralizing titers to influenza. Thus, T-bet expression distinguishes MBC subsets that have profoundly different homing, residency, and functional properties, and mediate distinct aspects of humoral immune memory.
Asunto(s)
Especificidad de Anticuerpos/inmunología , Subgrupos de Linfocitos B/inmunología , Linfocitos B/inmunología , Memoria Inmunológica/inmunología , Especificidad de Órganos/inmunología , Proteínas de Dominio T Box/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Subgrupos de Linfocitos B/metabolismo , Linfocitos B/metabolismo , Centro Germinal/citología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Anticuerpos Anti-VIH/inmunología , Humanos , Virus de la Influenza A/inmunología , Virus de la Influenza A/fisiología , Gripe Humana/inmunología , Gripe Humana/virología , Ratones , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismoRESUMEN
The MHC class I antigen presentation system enables T cell immunosurveillance of cancers and viruses. A substantial fraction of the immunopeptidome derives from rapidly degraded nascent polypeptides (DRiPs). By knocking down each of the 80 ribosomal proteins, we identified proteins that modulate peptide generation without altering source protein expression. We show that 60S ribosomal proteins L6 (RPL6) and RPL28, which are adjacent on the ribosome, play opposite roles in generating an influenza A virus-encoded peptide. Depleting RPL6 decreases ubiquitin-dependent peptide presentation, whereas depleting RPL28 increases ubiquitin-dependent and -independent peptide presentation. 40S ribosomal protein S28 (RPS28) knockdown increases total peptide supply in uninfected cells by increasing DRiP synthesis from non-canonical translation of "untranslated" regions and non-AUG start codons and sensitizes tumor cells for T cell targeting. Our findings raise the possibility of modulating immunosurveillance by pharmaceutical targeting ribosomes.
Asunto(s)
Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I/biosíntesis , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Linfocitos T/metabolismo , Animales , Línea Celular Tumoral , Técnicas de Cocultivo , Células HEK293 , Antígenos de Histocompatibilidad Clase I/inmunología , Interacciones Huésped-Patógeno , Humanos , Vigilancia Inmunológica , Virus de la Influenza A/inmunología , Virus de la Influenza A/patogenicidad , Melanoma/inmunología , Melanoma/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Grandes de Eucariotas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Linfocitos T/inmunología , Linfocitos T/virologíaRESUMEN
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 mulattaRESUMEN
We recently reported that SARS-CoV-2 nucleocapsid (N) protein is abundantly expressed on the surface of both infected and neighboring uninfected cells, where it enables activation of Fc receptor-bearing immune cells with anti-N antibodies (Abs) and inhibits leukocyte chemotaxis by binding chemokines (CHKs). Here, we extend these findings to N from the common cold human coronavirus (HCoV)-OC43, which is also robustly expressed on the surface of infected and noninfected cells by binding heparan sulfate/heparin (HS/H). HCoV-OC43 N binds with high affinity to the same set of 11 human CHKs as SARS-CoV-2 N, but also to a nonoverlapping set of six cytokines. As with SARS-CoV-2 N, HCoV-OC43 N inhibits CXCL12ß-mediated leukocyte migration in chemotaxis assays, as do all highly pathogenic and common cold HCoV N proteins. Together, our findings indicate that cell surface HCoV N plays important evolutionarily conserved roles in manipulating host innate immunity and as a target for adaptive immunity.
Asunto(s)
Coronavirus Humano OC43 , Inmunidad Innata , Nucleocápside , SARS-CoV-2 , Humanos , Coronavirus Humano OC43/genética , Proteínas de la Membrana , SARS-CoV-2/genéticaRESUMEN
Targeting multiple viral proteins is pivotal for sustained suppression of highly mutable viruses. In recent years, broadly neutralizing antibodies that target the influenza virus hemagglutinin and neuraminidase glycoproteins have been developed, and antibody monotherapy has been tested in preclinical and clinical studies to treat or prevent influenza virus infection. However, the impact of dual neutralization of the hemagglutinin and neuraminidase on the course of infection, as well as its therapeutic potential, has not been thoroughly tested. For this purpose, we generated a bispecific antibody that neutralizes both the hemagglutinin and the neuraminidase of influenza viruses. We demonstrated that this bispecific antibody has a dual-antiviral activity as it blocks infection and prevents the release of progeny viruses from the infected cells. We show that dual neutralization of the hemagglutinin and the neuraminidase by a bispecific antibody is advantageous over monoclonal antibody combination as it resulted an improved neutralization capacity and augmented the antibody effector functions. Notably, the bispecific antibody showed enhanced antiviral activity in influenza virus-infected mice, reduced mice mortality, and limited the virus mutation profile upon antibody administration. Thus, dual neutralization of the hemagglutinin and neuraminidase could be effective in controlling influenza virus infection.
RESUMEN
CD8(+) T cells play a critical role in limiting peripheral virus replication, yet how they locate virus-infected cells within tissues is unknown. Here, we have examined the environmental signals that CD8(+) T cells use to localize and eliminate virus-infected skin cells. Epicutaneous vaccinia virus (VV) infection, mimicking human smallpox vaccination, greatly increased expression of the CXCR3 chemokine receptor ligands CXCL9 and CXCL10 in VV-infected skin. Despite normal T cell numbers in the skin, Cxcr3(-/-) mice exhibited dramatically impaired CD8(+)-T-cell-dependent virus clearance. Intravital microscopy revealed that Cxcr3(-/-) T cells were markedly deficient in locating, engaging, and killing virus-infected cells. Further, transfer of wild-type CD8(+) T cells restored viral clearance in Cxcr3(-/-) animals. These findings demonstrate a function for CXCR3 in enhancing the ability of tissue-localized CD8(+) T cells to locate virus-infected cells and thereby exert anti-viral effector functions.
Asunto(s)
Queratinocitos/inmunología , Infecciones por Poxviridae/inmunología , Receptores CXCR3/inmunología , Piel/inmunología , Linfocitos T Citotóxicos/inmunología , Virus Vaccinia/inmunología , Traslado Adoptivo , Animales , Movimiento Celular , Quimiocina CXCL10/genética , Quimiocina CXCL10/inmunología , Quimiocina CXCL9/genética , Quimiocina CXCL9/inmunología , Femenino , Regulación de la Expresión Génica , Humanos , Queratinocitos/patología , Queratinocitos/virología , Ratones Transgénicos , Infecciones por Poxviridae/genética , Infecciones por Poxviridae/patología , Infecciones por Poxviridae/virología , Receptores CXCR3/deficiencia , Receptores CXCR3/genética , Transducción de Señal , Piel/patología , Piel/virología , Linfocitos T Citotóxicos/patología , Linfocitos T Citotóxicos/trasplante , Carga ViralRESUMEN
Successful direct MHC class I Ag presentation is dependent on the protein degradation machinery of the cell to generate antigenic peptides that can be loaded onto MHC class I molecules for surveillance by CD8+ T cells of the immune system. Most often this process involves the ubiquitin (Ub)-proteasome system; however, other Ub-like proteins have also been implicated in protein degradation and direct Ag presentation. In this article, we examine the role of neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) in direct Ag presentation in mouse cells. NEDD8 is the Ub-like protein with highest similarity to Ub, and fusion of NEDD8 to the N terminus of a target protein can lead to the degradation of target proteins. We find that appending NEDD8 to the N terminus of the model Ag OVA resulted in degradation by both the proteasome and the autophagy protein degradation pathways, but only proteasomal degradation, involving the proteasomal subunit NEDD8 ultimate buster 1, resulted in peptide presentation. When directly compared with Ub, NEDD8 fusion was less efficient at generating peptides. However, inactivation of the NEDD8-conugation machinery by treating cells with MLN4924 inhibited the presentation of peptides from the defective ribosomal product-derived form of a model Ag. These results demonstrate that NEDD8 activity in the cell is important for direct Ag presentation, but not by directly targeting proteins for degradation.
Asunto(s)
Presentación de Antígeno , Complejo de la Endopetidasa Proteasomal , Animales , Linfocitos T CD8-positivos/metabolismo , Ciclopentanos , Ratones , Proteína NEDD8/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas , Pirimidinas , Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMEN
In the 35 years since the revelation that short peptides bound to major histocompatibility complex class I and II molecules are the secret of the major histocompatibility complex-restricted nature of T-cell recognition, there has been enormous progress in characterizing the immunopeptidome, the repertoire of peptide presented for immunosurveillance. Here, the major milestones in the journey are marked, the contribution of proteasome-mediated splicing to the immunopeptidome is discussed, and exciting recent findings relating the immunopeptidome to the translatome revealed by ribosome profiling (RiboSeq) is detailed. Finally, what is needed for continued progress is opined about, which includes the infusion of talented young scientists into the antigen-processing field, currently undergoing a renaissance; thanks in part to the astounding success of T-cell-based cancer immunotherapy.
Asunto(s)
Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I , Antígenos HLA , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoterapia , Péptidos/metabolismoRESUMEN
Single-dose vaccines with the ability to restrict SARS-CoV-2 replication in the respiratory tract are needed for all age groups, aiding efforts toward control of COVID-19. We developed a live intranasal vector vaccine for infants and children against COVID-19 based on replication-competent chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) that express the native (S) or prefusion-stabilized (S-2P) SARS-CoV-2 S spike protein, the major protective and neutralization antigen of SARS-CoV-2. B/HPIV3/S and B/HPIV3/S-2P replicated as efficiently as B/HPIV3 in vitro and stably expressed SARS-CoV-2 S. Prefusion stabilization increased S expression by B/HPIV3 in vitro. In hamsters, a single intranasal dose of B/HPIV3/S-2P induced significantly higher titers compared to B/HPIV3/S of serum SARS-CoV-2-neutralizing antibodies (12-fold higher), serum IgA and IgG to SARS-CoV-2 S protein (5-fold and 13-fold), and IgG to the receptor binding domain (10-fold). Antibodies exhibited broad neutralizing activity against SARS-CoV-2 of lineages A, B.1.1.7, and B.1.351. Four weeks after immunization, hamsters were challenged intranasally with 104.5 50% tissue-culture infectious-dose (TCID50) of SARS-CoV-2. In B/HPIV3 empty vector-immunized hamsters, SARS-CoV-2 replicated to mean titers of 106.6 TCID50/g in lungs and 107 TCID50/g in nasal tissues and induced moderate weight loss. In B/HPIV3/S-immunized hamsters, SARS-CoV-2 challenge virus was reduced 20-fold in nasal tissues and undetectable in lungs. In B/HPIV3/S-2P-immunized hamsters, infectious challenge virus was undetectable in nasal tissues and lungs; B/HPIV3/S and B/HPIV3/S-2P completely protected against weight loss after SARS-CoV-2 challenge. B/HPIV3/S-2P is a promising vaccine candidate to protect infants and young children against HPIV3 and SARS-CoV-2.
Asunto(s)
Vacunas contra la COVID-19/administración & dosificación , COVID-19/prevención & control , SARS-CoV-2/inmunología , Administración Intranasal , Animales , Anticuerpos Antivirales/sangre , Vacunas contra la COVID-19/genética , Vacunas contra la COVID-19/inmunología , Cricetinae , Vectores Genéticos , Inmunización , Virus de la Parainfluenza 3 Bovina/genética , Virus de la Parainfluenza 3 Humana/genética , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), the most severe pandemic in a century. The virus gains access to host cells when the viral spike protein (S-protein) binds to the host cell surface receptor angiotensin-converting enzyme 2 (ACE2). Studies have attempted to understand SARS-CoV-2 S-protein interactions with vertebrate orthologs of ACE2 by expressing ACE2 orthologs in mammalian cells and measuring viral infection or S-protein binding. Often, these cells only transiently express ACE2 proteins, and the levels of ACE2 at the cell surface are not quantified. Here, we describe a cell-based assay that uses stably transfected cells expressing ACE2 proteins in a bicistronic vector with an easy-to-quantify reporter protein, Thy1.1. We found that both the binding of the S-protein receptor-binding domain (RBD) and infection with a SARS-CoV-2 pseudovirus are proportional to the amount of human ACE2 expressed at the cell surface, which can be inferred by quantifying the level of Thy1.1. We also compared different ACE2 orthologs, which were expressed in stably transfected cells expressing equivalent levels of Thy1.1. When ranked for either viral infectivity or RBD binding, mouse ACE2 had a weak to undetectable affinity for S-protein, while human ACE2 had the highest level detected, and feline ACE2 had an intermediate phenotype. The generation of stably transfected cells whose ACE2 level can be normalized for cross-ortholog comparisons allows us to create a reusable cellular library useful for measuring emerging SARS-CoV-2 variants' abilities to potentially infect different animals. IMPORTANCE SARS-CoV-2 is a zoonotic virus responsible for the worst global pandemic in a century. An understanding of how the virus can infect other vertebrate species is important for controlling viral spread and understanding the natural history of the virus. Here, we describe a method to generate cells stably expressing different orthologs of ACE2, the receptor for SARS-CoV-2, on the surface of a human cell line. We find that both the binding of the viral spike protein receptor-binding domain (RBD) and infection of cells with a SARS-CoV-2 pseudovirus are proportional to the ACE2 levels at the cell surface. This method will allow the creation of a library of stably transfected cells expressing similar levels of different vertebrate ACE2 orthologs, which can be used repeatedly for identifying vertebrate species that may be susceptible to infection with SARS-CoV-2 and its many variants.
Asunto(s)
Glicoproteína de la Espiga del Coronavirus , Enzima Convertidora de Angiotensina 2/genética , Animales , COVID-19 , Gatos , Humanos , Ratones , Peptidil-Dipeptidasa A/metabolismo , Unión Proteica , Receptores Virales/metabolismo , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
We investigated the function of the newly discovered myosin family protein myosin 18A (Myo18A) in Ab-mediated immunity by generating B cell-conditional Myo18A-deficient mice. Myo18A deficiency led to expansion of bone marrow progenitor B cells and mature B cells in secondary lymphoid organs. Myo18A-deficient mice displayed serum IgM hyperglobulinemia and increased splenic IgM-secreting cells, with older mice switching to IgG1 hyperglobulinemia and autoantibody development. Immunization of Myo18A-deficient mice with inactivated influenza virus led to development of more potent neutralizing Abs against the major Ag hemagglutinin, associated with persistent accumulation of Ag-specific germinal center B cells and more Ag-specific bone marrow plasma cells. In vitro stimulation with TLR7 and BCR ligands revealed a greater ability of Myo18A-deficient B cells to differentiate into Ab-secreting cells, associated with higher AID and Blimp-1 expression. Overall, our study demonstrates that Myo18A is a novel negative regulator of B cell homeostasis, differentiation, and humoral immunity.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Inmunidad Humoral/inmunología , Miosinas/inmunología , Animales , Diferenciación Celular/inmunología , Femenino , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Miosinas/deficienciaRESUMEN
A major obstacle to vaccination against antigenically variable viruses is skewing of antibody responses to variable immunodominant epitopes. For influenza virus hemagglutinin (HA), the immunodominance of the variable head impairs responses to the highly conserved stem. Here, we show that head immunodominance depends on the physical attachment of head to stem. Stem immunogenicity is enhanced by immunizing with stem-only constructs or by increasing local HA concentration in the draining lymph node. Surprisingly, coimmunization of full-length HA and stem alters stem-antibody class switching. Our findings delineate strategies for overcoming immunodominance, with important implications for human vaccination.