RESUMEN
Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antígenos Virales/inmunología , Candida albicans/química , Mananos/inmunología , Hidróxido de Aluminio/química , Animales , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos/inmunología , Linfocitos B/inmunología , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/virología , Chlorocebus aethiops , Epítopos/inmunología , Inmunidad Innata , Inmunización , Inflamación/patología , Interferones/metabolismo , Lectinas Tipo C/metabolismo , Ligandos , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Senos Paranasales/metabolismo , Subunidades de Proteína/metabolismo , Lectina 1 Similar a Ig de Unión al Ácido Siálico/metabolismo , Solubilidad , Glicoproteína de la Espiga del Coronavirus/metabolismo , Linfocitos T/inmunología , Factor de Transcripción ReIB/metabolismo , Células Vero , beta-Glucanos/metabolismoRESUMEN
CRISPR-inspired systems have been extensively developed for applications in genome editing and nucleic acid detection. Here, we introduce a CRISPR-based peptide display technology to facilitate customized, high-throughput in vitro protein interaction studies. We show that bespoke peptide libraries fused to catalytically inactive Cas9 (dCas9) and barcoded with unique single guide RNA (sgRNA) molecules self-assemble from a single mixed pool to programmable positions on a DNA microarray surface for rapid, multiplexed binding assays. We develop dCas9-displayed saturation mutagenesis libraries to characterize antibody-epitope binding for a commercial anti-FLAG monoclonal antibody and human serum antibodies. We also show that our platform can be used for viral epitope mapping and exhibits promise as a multiplexed diagnostics tool. Our CRISPR-based peptide display platform and the principles of complex library self-assembly using dCas9 could be adapted for rapid interrogation of varied customized protein libraries or biological materials assembly using DNA scaffolding.
Asunto(s)
Epítopos/genética , Edición Génica/métodos , Biblioteca de Péptidos , ARN Guía de Kinetoplastida/genética , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/inmunología , Epítopos/inmunología , Humanos , Mutagénesis/genética , Unión Proteica/genética , Unión Proteica/inmunología , ARN Guía de Kinetoplastida/inmunologíaRESUMEN
Despite the central role that antibodies play in modern medicine, there is currently no way to rationally design novel antibodies to bind a specific epitope on a target. Instead, antibody discovery currently involves time-consuming immunization of an animal or library screening approaches. Here we demonstrate that a fine-tuned RFdiffusion network is capable of designing de novo antibody variable heavy chains (VHH's) that bind user-specified epitopes. We experimentally confirm binders to four disease-relevant epitopes, and the cryo-EM structure of a designed VHH bound to influenza hemagglutinin is nearly identical to the design model both in the configuration of the CDR loops and the overall binding pose.
RESUMEN
Despite the vast diversity of the antibody repertoire, infected individuals often mount antibody responses to precisely the same epitopes within antigens. The immunological mechanisms underpinning this phenomenon remain unknown. By mapping 376 immunodominant "public epitopes" at high resolution and characterizing several of their cognate antibodies, we concluded that germline-encoded sequences in antibodies drive recurrent recognition. Systematic analysis of antibody-antigen structures uncovered 18 human and 21 partially overlapping mouse germline-encoded amino acid-binding (GRAB) motifs within heavy and light V gene segments that in case studies proved critical for public epitope recognition. GRAB motifs represent a fundamental component of the immune system's architecture that promotes recognition of pathogens and leads to species-specific public antibody responses that can exert selective pressure on pathogens.
Asunto(s)
Secuencias de Aminoácidos , Formación de Anticuerpos , Interacciones Huésped-Patógeno , Epítopos Inmunodominantes , Cadenas Pesadas de Inmunoglobulina , Cadenas Ligeras de Inmunoglobulina , Animales , Humanos , Ratones , Células Germinativas , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Mapeo Epitopo , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunologíaRESUMEN
Protein display technologies link proteins to distinct nucleic acid sequences (barcodes), enabling multiplexed protein assays via DNA sequencing. Here, we develop Cas9 display (CasPlay) to interrogate customized peptide libraries fused to catalytically inactive Cas9 (dCas9) by sequencing the guide RNA (gRNA) barcodes associated with each peptide. We first confirm the ability of CasPlay to characterize antibody epitopes by recovering a known binding motif for a monoclonal anti-FLAG antibody. We then use a CasPlay library tiling the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteome to evaluate vaccine-induced antibody reactivities. Using a peptide library representing the human virome, we demonstrate the ability of CasPlay to identify epitopes across many viruses from microliters of patient serum. Our results suggest that CasPlay is a viable strategy for customized protein interaction studies from highly complex libraries and could provide an alternative to phage display technologies.
Asunto(s)
COVID-19 , Humanos , SARS-CoV-2/genética , Biblioteca de Péptidos , Anticuerpos , Epítopos/químicaRESUMEN
Profiling the specificities of antibodies can reveal a wealth of information about humoral immune responses and the antigens they target. Here, we present a protocol for VirScan, an application of the phage immunoprecipitation sequencing (PhIP-Seq) method for profiling the specificities of human antiviral antibodies. Accompanying this protocol is a video of the experimental procedure. VirScan and, more generally, PhIP-Seq are techniques that enable high-throughput antibody profiling by combining high-throughput DNA oligo synthesis and bacteriophage display with next-generation sequencing. In the VirScan method, human sera samples are screened against a library of peptides spanning the entire human viral proteome. Bound phage are immunoprecipitated and sequenced, identifying the viral peptides recognized by the antibodies. VirScan Is a powerful tool for uncovering individual viral exposure histories, mapping the epitope landscape of viruses of interest, and studying fundamental mechanisms of viral immunity. Graphical abstract.
RESUMEN
Peanut allergy can result in life-threatening reactions and is a major public health concern. Oral immunotherapy (OIT) induces desensitization to food allergens through administration of increasing amounts of allergen. To dissect peanut-specific immunoglobulin E (IgE) and IgG responses in subjects undergoing OIT, we have developed AllerScan, a method that leverages phage-display and next-generation sequencing to identify the epitope targets of peanut-specific antibodies. We observe a striking diversification and boosting of the peanut-specific IgG repertoire after OIT and a reduction in pre-existing IgE levels against individual epitopes. High-resolution epitope mapping reveals shared recognition of public epitopes in Ara h 1, 2, 3, and 7. In individual subjects, OIT-induced IgG specificities overlap extensively with IgE and exhibit strikingly similar antibody footprints, suggesting related clonal lineages or convergent evolution of peanut-specific IgE and IgG B cells. Individual differences in epitope recognition identified via AllerScan could inform safer and more effective personalized immunotherapy.
Asunto(s)
Desensibilización Inmunológica/métodos , Mapeo Epitopo/métodos , Epítopos/química , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Omalizumab/uso terapéutico , Hipersensibilidad al Cacahuete/terapia , Albuminas 2S de Plantas/administración & dosificación , Albuminas 2S de Plantas/química , Antígenos de Plantas/administración & dosificación , Antígenos de Plantas/química , Arachis/química , Arachis/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Estudios de Casos y Controles , Epítopos/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteínas de la Membrana/administración & dosificación , Proteínas de la Membrana/química , Hipersensibilidad al Cacahuete/genética , Hipersensibilidad al Cacahuete/inmunología , Hipersensibilidad al Cacahuete/patología , Biblioteca de Péptidos , Proteínas de Plantas/administración & dosificación , Proteínas de Plantas/química , Medicina de Precisión , Proteínas de Almacenamiento de SemillasRESUMEN
Understanding humoral responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for improving diagnostics, therapeutics, and vaccines. Deep serological profiling of 232 coronavirus disease 2019 (COVID-19) patients and 190 pre-COVID-19 era controls using VirScan revealed more than 800 epitopes in the SARS-CoV-2 proteome, including 10 epitopes likely recognized by neutralizing antibodies. Preexisting antibodies in controls recognized SARS-CoV-2 ORF1, whereas only COVID-19 patient antibodies primarily recognized spike protein and nucleoprotein. A machine learning model trained on VirScan data predicted SARS-CoV-2 exposure history with 99% sensitivity and 98% specificity; a rapid Luminex-based diagnostic was developed from the most discriminatory SARS-CoV-2 peptides. Individuals with more severe COVID-19 exhibited stronger and broader SARS-CoV-2 responses, weaker antibody responses to prior infections, and higher incidence of cytomegalovirus and herpes simplex virus 1, possibly influenced by demographic covariates. Among hospitalized patients, males produce stronger SARS-CoV-2 antibody responses than females.
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COVID-19/inmunología , Mapeo Epitopo , Epítopos/inmunología , SARS-CoV-2/inmunología , Índice de Severidad de la Enfermedad , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Formación de Anticuerpos , COVID-19/sangre , Prueba Serológica para COVID-19 , Reacciones Cruzadas , Microscopía por Crioelectrón , Epítopos/química , Epítopos/genética , Femenino , Humanos , Masculino , Conformación Proteica , SeroconversiónRESUMEN
Background: Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated. Findings: We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5Î untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with transcription. Conclusion: Our results prove that dCas9 methyltransferases cause efficient RNA-guided methylation of specific endogenous CpGs. However, there is significant off-target methylation indicating that further improvements of the specificity of CRISPR-dCas9 based DNA methylation modifiers are required.
Asunto(s)
Metilación de ADN/genética , Genoma/genética , ARN Guía de Kinetoplastida/genética , Sistemas CRISPR-Cas/genética , Islas de CpG/genética , Células HEK293 , Humanos , Metiltransferasas/genéticaRESUMEN
CRISPR-Cas9 has revolutionized the generation of transgenic animals. This system has demonstrated an unprecedented efficiency, multiplexability, and ease of use, thereby reducing the time and cost required for genome editing and enabling the production of animals with more extensive genetic modifications. It has also been shown to be applicable to a wide variety of animals, from early-branching metazoans to primates. Genome-wide screens in model organisms have been performed, accurate models of human diseases have been constructed, and potential therapies have been tested and validated in animal models. Several achievements in genetic modification of animals have been translated into products for the agricultural and pharmaceutical industries. Based on the remarkable progress to date, one may anticipate that in the future, CRISPR-Cas9 technology will enable additional far-reaching advances, including understanding the bases of diseases with complex genetic origins, engineering animals to produce organs for human transplantation, and genetically transforming entire populations of organisms to prevent the spread of disease.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Modelos Animales , Animales , Ingeniería Genética , Terapia GenéticaRESUMEN
Xenotransplantation is a promising strategy to alleviate the shortage of organs for human transplantation. In addition to the concerns about pig-to-human immunological compatibility, the risk of cross-species transmission of porcine endogenous retroviruses (PERVs) has impeded the clinical application of this approach. We previously demonstrated the feasibility of inactivating PERV activity in an immortalized pig cell line. We now confirm that PERVs infect human cells, and we observe the horizontal transfer of PERVs among human cells. Using CRISPR-Cas9, we inactivated all of the PERVs in a porcine primary cell line and generated PERV-inactivated pigs via somatic cell nuclear transfer. Our study highlights the value of PERV inactivation to prevent cross-species viral transmission and demonstrates the successful production of PERV-inactivated animals to address the safety concern in clinical xenotransplantation.
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Sistemas CRISPR-Cas , Transmisión de Enfermedad Infecciosa/prevención & control , Retrovirus Endógenos/genética , Edición Génica/métodos , Infecciones por Retroviridae/transmisión , Sus scrofa/genética , Sus scrofa/virología , Trasplante Heterólogo/efectos adversos , Animales , Células HEK293 , HumanosRESUMEN
Recoding--the repurposing of genetic codons--is a powerful strategy for enhancing genomes with functions not commonly found in nature. Here, we report computational design, synthesis, and progress toward assembly of a 3.97-megabase, 57-codon Escherichia coli genome in which all 62,214 instances of seven codons were replaced with synonymous alternatives across all protein-coding genes. We have validated 63% of recoded genes by individually testing 55 segments of 50 kilobases each. We observed that 91% of tested essential genes retained functionality with limited fitness effect. We demonstrate identification and correction of lethal design exceptions, only 13 of which were found in 2229 genes. This work underscores the feasibility of rewriting genomes and establishes a framework for large-scale design, assembly, troubleshooting, and phenotypic analysis of synthetic organisms.
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Proteínas de Escherichia coli/genética , Escherichia coli/genética , Genes Sintéticos , Código Genético/fisiología , Genoma Bacteriano , Genes Esenciales , Genes Letales , Código Genético/genética , Ingeniería Genética , Fenotipo , Biosíntesis de Proteínas/genéticaRESUMEN
The shortage of organs for transplantation is a major barrier to the treatment of organ failure. Although porcine organs are considered promising, their use has been checked by concerns about the transmission of porcine endogenous retroviruses (PERVs) to humans. Here we describe the eradication of all PERVs in a porcine kidney epithelial cell line (PK15). We first determined the PK15 PERV copy number to be 62. Using CRISPR-Cas9, we disrupted all copies of the PERV pol gene and demonstrated a >1000-fold reduction in PERV transmission to human cells, using our engineered cells. Our study shows that CRISPR-Cas9 multiplexability can be as high as 62 and demonstrates the possibility that PERVs can be inactivated for clinical application of porcine-to-human xenotransplantation.