A t New York University Langone Health at the height of the coronavirus disease 2019 (COVID-19) pandemic, 22% of hospitalized patients diagnosed with COVID-19 infection required invasive mechanical ventilation (IMV) (1). We noted many patients with COVID-19 infection who developed pneumothorax, pneumomediastinum, and pneumopericardium, and in some cases, at multiple separate time points. Given this observation, we hypothesized that barotrauma related to IMV was elevated in patients with COVID-19 infection. The purpose of this study was to evaluate the rate of barotrauma in patients who tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and who required IMV compared with other patients in the same institution during the same period who also required IMV, and to a temporally remote (pre-COVID-19) historical cohort of patients who required IMV support in the setting of acute respiratory distress syndrome (ARDS). Materials and Methods This retrospective study was performed with institutional review board waiver of authorization and consent (is20-00582) given the current urgent conditions created by the pandemic. Study Population The New York University Langone Health electronic medical record system (Epic Systems, Verona, Wis) was searched for patients age 18 years or older seen in our emergency department between March 1, 2020, and April 6, 2020, with chest imaging within 24 hours of nasopharyngeal or oropharyngeal swab testing for SARS-CoV-2. Test assay techniques are detailed in Appendix E1 (online). Patients with positive real-time reverse transcription polymerase chain reaction assays were deemed COVID-19 positive, and those with negative results were deemed COVID-19 negative. COVID-19 testing was performed in all patients who presented to the emergency
Adaptive immunity is driven by the ability of lymphocytes to undergo V(D)J recombination and generate a highly diverse set of immune receptors (B cell receptors/secreted antibodies and T cell receptors) and their subsequent clonal selection and expansion upon molecular recognition of foreign antigens. These principles lead to remarkable, unique and dynamic immune receptor repertoires 1 . Deep sequencing provides increasing evidence for the presence of commonly shared (convergent) receptors across individual organisms within one species 2-4 . Convergent selection of specific receptors towards various antigens offers one explanation for these findings. For example, single cases of convergence have been reported in antibody repertoires of viral infection or allergy 5-8 . Recent studies demonstrate that convergent selection of sequence motifs within T cell receptor (TCR) repertoires can be identified on an even wider scale 9,10 . Here we report that there is extensive convergent selection in antibody repertoires of mice for a range of protein antigens and immunization conditions. We employed a deep learning approach utilizing variational autoencoders (VAEs) to model the underlying process of B cell receptor (BCR) recombination and assume that the data generation follows a Gaussian mixture model (GMM) in latent space. This provides both a latent embedding and cluster labels that group similar sequences, thus enabling the discovery of a multitude of convergent, antigen-associated sequence patterns. Using a linear, one-versus-all support vector machine (SVM), we confirm that the identified sequence patterns are predictive of antigenic exposure and outperform predictions based on the occurrence of public clones. Recombinant expression of both natural and in silico-generated antibodies possessing convergent patterns confirms their binding specificity to target antigens. Our work highlights to which extent convergence in antibody repertoires can occur and shows how deep learning can be applied for immunodiagnostics and antibody discovery and engineering.
Antibody engineering is often performed to improve therapeutic properties by directed evolution, usually by high-throughput screening of phage or yeast display libraries. Engineering antibodies in mammalian cells offer advantages associated with expression in their final therapeutic format (full-length glycosylated IgG); however, the inability to express large and diverse libraries severely limits their potential throughput. To address this limitation, we have developed homology-directed mutagenesis (HDM), a novel method which extends the concept of CRISPR/Cas9-mediated homology-directed repair (HDR). HDM leverages oligonucleotides with degenerate codons to generate site-directed mutagenesis libraries in mammalian cells. By improving HDR to a robust efficiency of 15–35% and combining mammalian display screening with next-generation sequencing, we validated this approach can be used for key applications in antibody engineering at high-throughput: rational library construction, novel variant discovery, affinity maturation and deep mutational scanning (DMS). We anticipate that HDM will be a valuable tool for engineering and optimizing antibodies in mammalian cells, and eventually enable directed evolution of other complex proteins and cellular therapeutics.
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.
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