RESUMEN
Characterization of COVID-19 antibodies has largely focused on memory B cells; however, it is the antibody-secreting plasma cells that are directly responsible for the production of serum antibodies, which play a critical role in resolving SARS-CoV-2 infection. Little is known about the specificity of plasma cells, largely because plasma cells lack surface antibody expression, thereby complicating their screening. Here, we describe a technology pipeline that integrates single-cell antibody repertoire sequencing and mammalian display to interrogate the specificity of plasma cells from 16 convalescent patients. Single-cell sequencing allows us to profile antibody repertoire features and identify expanded clonal lineages. Mammalian display screening is used to reveal that 43 antibodies (of 132 candidates) derived from expanded plasma cell lineages are specific to SARS-CoV-2 antigens, including antibodies with high affinity to the SARS-CoV-2 receptor-binding domain (RBD) that exhibit potent neutralization and broad binding to the RBD of SARS-CoV-2 variants (of concern/interest).
Asunto(s)
Anticuerpos Neutralizantes/aislamiento & purificación , Células Plasmáticas/metabolismo , SARS-CoV-2/inmunología , Análisis de la Célula Individual/métodos , Animales , Anticuerpos Antivirales/aislamiento & purificación , COVID-19/inmunología , COVID-19/prevención & control , Células Cultivadas , Estudios de Cohortes , Biblioteca de Genes , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mamíferos , Pruebas de Neutralización , Biblioteca de Péptidos , Células Plasmáticas/químicaRESUMEN
The optimization of therapeutic antibodies is time-intensive and resource-demanding, largely because of the low-throughput screening of full-length antibodies (approximately 1 × 103 variants) expressed in mammalian cells, which typically results in few optimized leads. Here we show that optimized antibody variants can be identified by predicting antigen specificity via deep learning from a massively diverse space of antibody sequences. To produce data for training deep neural networks, we deep-sequenced libraries of the therapeutic antibody trastuzumab (about 1 × 104 variants), expressed in a mammalian cell line through site-directed mutagenesis via CRISPR-Cas9-mediated homology-directed repair, and screened the libraries for specificity to human epidermal growth factor receptor 2 (HER2). We then used the trained neural networks to screen a computational library of approximately 1 × 108 trastuzumab variants and predict the HER2-specific subset (approximately 1 × 106 variants), which can then be filtered for viscosity, clearance, solubility and immunogenicity to generate thousands of highly optimized lead candidates. Recombinant expression and experimental testing of 30 randomly selected variants from the unfiltered library showed that all 30 retained specificity for HER2. Deep learning may facilitate antibody engineering and optimization.
Asunto(s)
Antígenos/química , Aprendizaje Profundo , Ingeniería de Proteínas/métodos , Receptor ErbB-2/química , Trastuzumab/química , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Antígenos/genética , Antígenos/inmunología , Sistemas CRISPR-Cas , Humanos , Hibridomas/química , Hibridomas/inmunología , Mutagénesis Sitio-Dirigida , Unión Proteica , Receptor ErbB-2/genética , Receptor ErbB-2/inmunología , Reparación del ADN por Recombinación , Análisis de Secuencia de Proteína , Trastuzumab/genética , Trastuzumab/inmunologíaRESUMEN
Antibody engineering in mammalian cells offers the important advantage of expression and screening of libraries in their native conformation, increasing the likelihood of generating candidates with more favorable molecular properties. Major advances in cellular engineering enabled by CRISPR-Cas9 genome editing have made it possible to expand the use of mammalian cells in biotechnological applications. Here, we describe an antibody engineering and screening approach where complete variable light (VL) and heavy (VH) chain cassette libraries are stably integrated into the genome of hybridoma cells by enhanced Cas9-driven homology-directed repair (HDR), resulting in their surface display and secretion. By developing an improved HDR donor format that utilizes in situ linearization, we are able to achieve >15-fold improvement of genomic integration, resulting in a screening workflow that only requires a simple plasmid electroporation. This proved suitable for different applications in antibody discovery and engineering. By integrating and screening an immune library obtained from the variable gene repertoire of an immunized mouse, we could isolate a diverse panel of >40 unique antigen-binding variants. Additionally, we successfully performed affinity maturation by directed evolution screening of an antibody library based on random mutagenesis, leading to the isolation of several clones with affinities in the picomolar range.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Biblioteca de Genes , Hibridomas/metabolismo , Anticuerpos de Cadena Única , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismoRESUMEN
Throughout the last several decades, vaccination has been key to prevent and eradicate infectious diseases. However, many pathogens (e.g., respiratory syncytial virus [RSV], influenza, dengue, and others) have resisted vaccine development efforts, largely because of the failure to induce potent antibody responses targeting conserved epitopes. Deep profiling of human B cells often reveals potent neutralizing antibodies that emerge from natural infection, but these specificities are generally subdominant (i.e., are present in low titers). A major challenge for next-generation vaccines is to overcome established immunodominance hierarchies and focus antibody responses on crucial neutralization epitopes. Here, we show that a computationally designed epitope-focused immunogen presenting a single RSV neutralization epitope elicits superior epitope-specific responses compared to the viral fusion protein. In addition, the epitope-focused immunogen efficiently boosts antibodies targeting the palivizumab epitope, resulting in enhanced neutralization. Overall, we show that epitope-focused immunogens can boost subdominant neutralizing antibody responses in vivo and reshape established antibody hierarchies.
Asunto(s)
Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Epítopos/química , Receptores de Antígenos de Linfocitos B/inmunología , Proteínas Recombinantes de Fusión/química , Virus Sincitiales Respiratorios/inmunología , Proteínas Virales de Fusión/química , Animales , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/genética , Clonación Molecular , Diseño Asistido por Computadora , Epítopos/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Inmunización/métodos , Inmunogenicidad Vacunal , Ratones , Ratones Endogámicos BALB C , Nanopartículas/administración & dosificación , Nanopartículas/química , Palivizumab/química , Palivizumab/inmunología , Receptores de Antígenos de Linfocitos B/química , Receptores de Antígenos de Linfocitos B/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Vacunas contra Virus Sincitial Respiratorio/biosíntesis , Vacunas contra Virus Sincitial Respiratorio/genética , Homología Estructural de Proteína , Proteínas Virales de Fusión/administración & dosificación , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunologíaRESUMEN
High-throughput sequencing of immunoglobulin (Ig) repertoires (Ig-seq) is a powerful method for quantitatively interrogating B cell receptor sequence diversity. When applied to human repertoires, Ig-seq provides insight into fundamental immunological questions, and can be implemented in diagnostic and drug discovery projects. However, a major challenge in Ig-seq is ensuring accuracy, as library preparation protocols and sequencing platforms can introduce substantial errors and bias that compromise immunological interpretation. Here, we have established an approach for performing highly accurate human Ig-seq by combining synthetic standards with a comprehensive error and bias correction pipeline. First, we designed a set of 85 synthetic antibody heavy-chain standards (in vitro transcribed RNA) to assess correction workflow fidelity. Next, we adapted a library preparation protocol that incorporates unique molecular identifiers (UIDs) for error and bias correction which, when applied to the synthetic standards, resulted in highly accurate data. Finally, we performed Ig-seq on purified human circulating B cell subsets (naïve and memory), combined with a cellular replicate sampling strategy. This strategy enabled robust and reliable estimation of key repertoire features such as clonotype diversity, germline segment, and isotype subclass usage, and somatic hypermutation. We anticipate that our standards and error and bias correction pipeline will become a valuable tool for researchers to validate and improve accuracy in human Ig-seq studies, thus leading to potentially new insights and applications in human antibody repertoire profiling.
RESUMEN
In recent years, major efforts have been made to develop sophisticated experimental and bioinformatic workflows for sequencing adaptive immune repertoires. The immunological insight gained has been applied to fields as varied as lymphocyte biology, immunodiagnostics, vaccines, cancer immunotherapy, and antibody engineering. In this review, we provide a detailed overview of these advanced methodologies, focusing specifically on strategies to reduce sequencing errors and bias and to achieve high-throughput pairing of variable regions (e.g., heavy-light or alpha-beta chains). In addition, we highlight recent technologies for single-cell transcriptome sequencing that can be integrated with immune repertoires. Finally, we provide a perspective on advanced immune repertoire sequencing and its ability to impact basic immunology, biopharmaceutical drug discovery and development, and cancer immunotherapy.
Asunto(s)
Anticuerpos , Biología Computacional/métodos , Descubrimiento de Drogas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Humanos , Inmunoterapia/métodos , Linfocitos/inmunología , Ratones , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Vacunas/genética , Vacunas/inmunologíaRESUMEN
High-throughput antibody repertoire sequencing (Ig-seq) provides quantitative molecular information on humoral immunity. However, Ig-seq is compromised by biases and errors introduced during library preparation and sequencing. By using synthetic antibody spike-in genes, we determined that primer bias from multiplex polymerase chain reaction (PCR) library preparation resulted in antibody frequencies with only 42 to 62% accuracy. Additionally, Ig-seq errors resulted in antibody diversity measurements being overestimated by up to 5000-fold. To rectify this, we developed molecular amplification fingerprinting (MAF), which uses unique molecular identifier (UID) tagging before and during multiplex PCR amplification, which enabled tagging of transcripts while accounting for PCR efficiency. Combined with a bioinformatic pipeline, MAF bias correction led to measurements of antibody frequencies with up to 99% accuracy. We also used MAF to correct PCR and sequencing errors, resulting in enhanced accuracy of full-length antibody diversity measurements, achieving 98 to 100% error correction. Using murine MAF-corrected data, we established a quantitative metric of recent clonal expansion-the intraclonal diversity index-which measures the number of unique transcripts associated with an antibody clone. We used this intraclonal diversity index along with antibody frequencies and somatic hypermutation to build a logistic regression model for prediction of the immunological status of clones. The model was able to predict clonal status with high confidence but only when using MAF error and bias corrected Ig-seq data. Improved accuracy by MAF provides the potential to greatly advance Ig-seq and its utility in immunology and biotechnology.
Asunto(s)
Anticuerpos/química , Ensayos Analíticos de Alto Rendimiento/métodos , Animales , Anticuerpos/inmunología , Ratones , Reacción en Cadena de la Polimerasa MultiplexRESUMEN
BACKGROUND: Next-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology by providing quantitative molecular information on polyclonal humoral immune responses. Reproducible and robust information on antibody repertoires is valuable for basic and applied immunology studies: thus, it is essential to establish the reliability of antibody NGS data. RESULTS: We isolated RNA from antibody-secreting cells (ASCs) from either 1 mouse or a pool of 9 immunized mice in order to simulate both normal and high diversity populations. Next, we prepared three technical replicates of antibody libraries by RT-PCR from each diversity scenario, which were sequenced using the Illumina MiSeq platform resulting in >106 250 bp paired-end reads per replicate. We then assessed the robustness of antibody repertoire data based on clonal identification defined by amino acid sequence of either full-length VDJ region or the complementarity determining region 3 (CDR3). Leveraging modeling approaches adapted from mathematical ecology, we found that in either diversity scenario both CDR3 and VDJ detection nears completeness indicating deep coverage of ASC repertoires. Additionally, we defined reliability thresholds for accurate quantification and ranking of CDR3s and VDJs. Importantly, we show that both factors-(i) replicate sequencing and (ii) sequencing depth-are crucial for robust CDR3 and VDJ detection and ranking. CONCLUSIONS: In summary, we established widely applicable experimental and computational guidelines for robust antibody NGS and analysis, which will help advance systems immunology studies related to the quantitative profiling of antibody responses following infection and vaccination.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inmunización , Región Variable de Inmunoglobulina/genética , Animales , Anticuerpos/genética , Células Clonales , Regiones Determinantes de Complementariedad/genética , Femenino , Ratones Endogámicos BALB C , Reproducibilidad de los Resultados , Recombinación V(D)J/genéticaRESUMEN
High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences. As of yet, the impact of the method of adapter addition on experimental library preparation and the resulting antibody repertoire HTS datasets has not been thoroughly investigated. Therefore, we compared three standard library preparation methods by performing Illumina HTS on antibody variable heavy genes from murine antibody-secreting cells. Clonal overlap and rank statistics demonstrated that the investigated methods produced equivalent HTS datasets. PCR-based methods were experimentally superior to ligation with respect to speed, efficiency, and practicality. Finally, using a two-step PCR based method we established a protocol for antibody repertoire library generation, beginning from inputs as low as 1 ng of total RNA. In summary, this study represents a major advance towards a standardized experimental framework for antibody HTS, thus opening up the potential for systems-based, cross-experiment meta-analyses of antibody repertoires.
Asunto(s)
Anticuerpos/genética , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ARN/métodos , Animales , Anticuerpos/inmunología , Biología Computacional , Femenino , Ratones , Reacción en Cadena de la PolimerasaRESUMEN
Cis-regulatory regions (CRR) control gene expression and chromatin modifications. Genetic variation at CRR in individuals across a population contributes to phenotypic differences of biomedical relevance. This standing variation is important for personalized genomic medicine as well as for adaptive evolution and speciation. This review focuses on genetic variation at CRR, its influence on chromatin, gene expression, and ultimately disease phenotypes. In addition, we summarize our understanding of how this variation may contribute to evolution. Recent technological and computational advances have accelerated research in the direction of personalized medicine, combining strengths of molecular biology and genomics. This will pave new ways to understand how CRR variation affects phenotypes and chart out possible avenues of intervention.