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Biology subjects

Frei, L.

Publications and source records attributed to Frei, L..

9 recordsLinked to original sources

Predicting viral host range for reverse zoonosis surveillance

Zoonotic transmission of viruses has triggered major outbreaks throughout history. However, human-to-animal transmission, or reverse zoonotic transmission, also represents an underappreciated public health threat. Reverse zoonoses can establish novel animal reservoirs that fuel viral evolution and enable eventual spillback into humans, underscoring the importance of defining viral host range. Current approaches for host range prediction are limited. Experimental methods are retrospective and rely on brute-force testing in a small number of models, whereas computational approaches often lack experimental grounding. Here, we present a platform that integrates high-throughput experimental screening with deep learning to predict viral host range from sequence. We screened a yeast-displayed SARS-CoV-2 receptor-binding domain library against 15 ACE2 orthologs from diverse species, and deep sequenced the ACE2-binding and non-binding populations to generate an atlas of 8.8 million unique RBD:ACE2 interaction measurements. These data were used to train a transformer-based deep learning model that predicts RBD:ACE2 compatibility across animal species from sequence alone, and generalizes in a zero-shot manner to ACE2 orthologs not observed during training. Critically, the model pairs each prediction with a distance-based uncertainty estimate, allowing hundreds of candidate species to be triaged by both predicted host compatibility and confidence. This framework enables the rapid prioritization of animal species for SARS-CoV-2 surveillance and provides a general strategy for anticipating viral host range.

bioengineering↗

Generative design of antibody Fc-variants with synthetic and programmable functional profiles

Beyond antigen recognition, antibodies direct diverse immune effector functions through their constant (Fc) domain. While the Fc domain is central to antibody biology and therapeutic efficacy, our understanding of how Fc sequence encodes function remains limited, as most of Fc sequence space has not been experimentally mapped or linked to Fc-receptor engagement. Furthermore, the extensive overlap in Fc-receptor binding sites on the Fc domain has impeded efforts to engineer antibodies with tailored, multi-receptor engagement profiles that can precisely control downstream immunity. Here we introduce a novel framework for Fc engineering that integrates protein engineering with deep learning to rationally predict and engineer antibody Fc function. Using a yeast-based, aglycosylated Fc display system, we performed deep mutational scanning across the entire human IgG1 Fc domain, allowing the rational design of a diverse combinatorial library of more than 108 Fc-variants. This library was sorted based on binding to a panel of eight canonical Fc-receptors, and the resulting populations were deep sequenced to generate a high-quality dataset comprising millions of unique Fc sequences annotated with their respective Fc-receptor binding profiles. Deep learning-based classifiers trained on this dataset accurately predicted Fc-receptor binding activity from Fc sequence across all Fc-receptors tested. We further developed FcGPT, a domain-specific autoregressive protein language model pre-trained on over three million unique Fc sequences, and refined by post-training through reinforcement learning with experimental feedback (RLXF) and synthetic verifiers. FcGPT enables the computational design of novel Fc-variants with user-defined Fc-receptor binding profiles, providing a foundational tool for understanding and programming antibody-mediated immunity.

bioengineering↗

A yeast mating-based platform enables the generation and screening of ultra large antibody libraries

Yeast display is a powerful platform for antibody engineering, offering eukaryotic protein folding and compatibility with fluorescence-activated sorting (FACS). However, conventional yeast display faces limitations in terms of library size and display heterogeneity due to plasmid-based expression. Here, we present a yeast display platform that combines genomic library integration and high-efficiency mating to generate ultra-diverse antibody libraries approaching the diversity of phage-libraries (>1011). We engineered two yeast strains, LFYa and LFYalpha, featuring genomically integrated landing pads for high-efficiency library insertion. To enable compatibility with deep sequencing, we integrated the recombinase BxB1, which links heavy (HC) and light chain (LC) information onto a single chromosome. Using our platform, we constructed synthetic antibody libraries with HC and LC diversities of 4.1x107 and 1.7x107 variants, respectively and with an improved mating protocol, we achieved a combinatorial library diversity exceeding 1011. We demonstrated that genomic integration yields uniform surface display. Screening this antibody library against multiple antigens resulted in the discovery of binders with affinities in the single-digit nanomolar to picomolar range, demonstrating the platforms utility for the discovery of antibodies with therapeutically relevant affinities. This work establishes a robust and deep sequencing-compatible yeast display system that overcomes key limitations of previous mating-based platforms.

bioengineering↗

Altered T cell reactivity to β-amyloid-related antigens in early Alzheimer's disease

There is growing evidence that the adaptive immune system and neurodegenerative Alzheimers disease (AD) are intertwined in multiple ways. Recent studies have reported alterations of the adaptive immune system in early AD stages, such as preclinical AD and mild cognitive impairment (MCI) due to AD. However, the identity of specific antigenic targets and whether the respective response is beneficial or detrimental during disease progression are still open questions. Herein, we describe cross-sectional analyses of blood and cerebrospinal fluid from three different study populations covering early AD stages. We employed high-dimensional mass cytometry, single-cell RNA-sequencing, in vitro T cell secretome analysis, and antigen presentation assays to achieve a comprehensive characterization of adaptive immune cell populations. Our results show that subjects at the stage of asymptomatic, preclinical AD can mount a CD4+ T helper cell response towards {beta}- amyloid peptide and display an early enrichment of cytotoxic CD8+ effector/TEMRA cells in CSF, combined with a less immunosuppressive gene signature of peripheral regulatory T cells. Conversely, in MCI due to AD, we observed increased frequencies of CD8+ effector/TEMRA cells in the periphery, characterized by a pro-inflammatory gene expression profile and an overall decrease in antigen responsiveness. Our results demonstrate the complexity of adaptive immune changes in early AD and suggest that it may be beneficial in the preclinical stage to promote specific CD4+ T cell responses, while in MCI it may be important to therapeutically target CD8+ T cell responses if these prove to be harmful.

immunology↗

Synthetic coevolution reveals adaptive mutational trajectories of neutralizing antibodies and SARS-CoV-2

The Covid-19 pandemic showcases a coevolutionary race between the human immune system and SARS-CoV-2, mirroring the Red Queen hypothesis of evolutionary biology. The immune system generates neutralizing antibodies targeting the SARS-CoV-2 spike proteins receptor binding domain (RBD), crucial for host cell invasion, while the virus evolves to evade antibody recognition. Here, we establish a synthetic coevolution system combining high-throughput screening of antibody and RBD variant libraries with protein mutagenesis, surface display, and deep sequencing. Additionally, we train a protein language machine learning model that predicts antibody escape to RBD variants. Synthetic coevolution reveals antagonistic and compensatory mutational trajectories of neutralizing antibodies and SARS-CoV-2 variants, enhancing the understanding of this evolutionary conflict.

synthetic biology↗

Deep learning-guided selection of antibody therapies with enhanced resistance to current and prospective SARS-CoV-2 Omicron variants

Most COVID-19 antibody therapies rely on binding the SARS-CoV-2 receptor binding domain (RBD). However, heavily mutated variants such as Omicron and its sublineages, which are characterized by an ever increasing number of mutations in the RBD, have rendered prior antibody therapies ineffective, leaving no clinically approved antibody treatments for SARS-CoV-2. Therefore, the capacity of therapeutic antibody candidates to bind and neutralize current and prospective SARS-CoV-2 variants is a critical factor for drug development. Here, we present a deep learning-guided approach to identify antibodies with enhanced resistance to SARS-CoV-2 evolution. We apply deep mutational learning (DML), a machine learning-guided protein engineering method to interrogate a massive sequence space of combinatorial RBD mutations and predict their impact on angiotensin-converting enzyme 2 (ACE2) binding and antibody escape. A high mutational distance library was constructed based on the full-length RBD of Omicron BA.1, which was experimentally screened for binding to the ACE2 receptor or neutralizing antibodies, followed by deep sequencing. The resulting data was used to train ensemble deep learning models that could accurately predict binding or escape for a panel of therapeutic antibody candidates targeting diverse RBD epitopes. Furthermore, antibody breadth was assessed by predicting binding or escape to synthetic lineages that represent millions of sequences generated using in silico evolution, revealing combinations with complementary and enhanced resistance to viral evolution. This deep learning approach may enable the design of next-generation antibody therapies that remain effective against future SARS-CoV-2 variants.

immunology↗

Single-cell immune repertoire sequencing of B and T cells in murine models of infection and autoimmunity

Adaptive immune repertoires are composed by the ensemble of B and T cell receptors (BCR, TCR) within an individual and reflect both past and current immune responses. Recent advances in single-cell sequencing enable recovery of the complete adaptive immune receptor sequences in addition to transcriptional information. Such high-dimensional datasets enable the molecular quantification of clonal selection of B and T cells across a wide variety of conditions such as infection and disease. Due to costs, time required for the analysis and current practices of academic publishing, small-scale sequencing studies are often not made publicly available, despite having informative potential to elucidate immunological principles and guide future-studies. Here, we performed single-cell sequencing of B and T cells to profile clonal selection across murine models of viral infection and autoimmune disease. Specifically, we recovered transcriptome and immune repertoire information for polyclonal T follicular helper cells following acute and chronic viral infection, CD8+ T cells with binding specificity restricted to two distinct peptides of lymphocytic choriomeningitis virus, and B and T cells isolated from the nervous system in the context of experimental autoimmune encephalomyelitis. We could relate repertoire features such as clonal expansion, germline gene usage, and clonal convergence to cell phenotypes spanning activation, memory, naive, antibody secretion, T cell inflation, and regulation. Together, this dataset provides a resource for experimental and computational immunologists that can be integrated with future single-cell immune repertoire and transcriptome sequencing datasets.

immunology↗

A synthetic bispecific antibody capable of neutralizing SARS-CoV-2 Delta and Omicron

Bispecific antibodies have emerged as a promising strategy for curtailing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immune escape. This brief report highlights RBT-0813 (also known as TB493-04), a synthetic, humanized, receptor-binding domain (RBD)-targeted bispecific antibody that retains picomolar affinity to the Spike (S) trimers of all major variants of concern and neutralizes both SARS-CoV-2 Delta and Omicron in vitro.

immunology↗

Predictive profiling of SARS-CoV-2 variants by deep mutational learning

The continual evolution of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the emergence of variants that show resistance to vaccines and neutralizing antibodies (1-4) threaten to prolong the coronavirus disease 2019 (COVID-19) pandemic (5). Selection and emergence of SARS-CoV-2 variants are driven in part by mutations within the viral spike protein and in particular the ACE2 receptor-binding domain (RBD), a primary target site for neutralizing antibodies. Here, we develop deep mutational learning (DML), a machine learning-guided protein engineering technology, which is used to interrogate a massive sequence space of combinatorial mutations, representing billions of RBD variants, by accurately predicting their impact on ACE2 binding and antibody escape. A highly diverse landscape of possible SARS-CoV-2 variants is identified that could emerge from a multitude of evolutionary trajectories. DML may be used for predictive profiling on current and prospective variants, including highly mutated variants such as omicron (B.1.1.529), thus supporting decision making for public heath as well as guiding the development of therapeutic antibody treatments and vaccines for COVID-19.

synthetic biology↗