Open-source milligram-scale, four channel, automated protein purification system (preprint)

Liquid chromatography purification of multiple recombinant proteins, in parallel, could catalyze research and discovery if the processes are fast and approach the robustness of traditional, "one-protein-at-a-time" purification. Here, we report an automated, four channel chromatography platform that we have designed and validated for parallelized protein purification at milligram scales. The device can purify up to four proteins (each with its own single column), has inputs for up to eight buffers or solvents that can be directed to any of the four columns via a network of software-driven valves, and includes an automated fraction collector with ten positions for 1.5 or 5.0 mL collection tubes and four positions for 50 mL collection tubes for each column output. The control software can be accessed either via Python scripting, giving users full access to all steps of the purification process, or via a simple-to-navigate touch screen graphical user interface that does not require knowledge of the command line or any programming language. Using our instrument, we report milligram-scale, parallelized, single-column purification of a panel of mammalian cell expressed coronavirus (SARS-CoV-2, HCoV-229E, HCoV-OC43, HCoV-229E) trimeric Spike and monomeric Receptor Binding Domain (RBD) antigens, and monoclonal antibodies targeting SARS-CoV-2 Spike (S) and Influenza Hemagglutinin (HA). We include a detailed hardware build guide, and have made the controlling software open source, to allow others to build and customize their own protein purifier systems.

A single-component luminescent biosensor for the SARS-CoV-2 spike protein (preprint)

Many existing protein detection strategies depend on highly functionalized antibody reagents. A simpler and easier to produce class of detection reagent is highly desirable. We designed a single-component, recombinant, luminescent biosensor that can be expressed in laboratory strains of E. coli and S. cerevisiae. This biosensor is deployed in multiple homogenous and immobilized assay formats to detect recombinant SARS-CoV-2 spike antigen and cultured virus. The chemiluminescent signal generated facilitates detection by an un-augmented cell phone camera. Binding Activated Tandem split-enzyme (BAT) biosensors may serve as a useful template for diagnostics and reagents that detect SARS-CoV-2 antigens and other proteins of interest.

Converting non-neutralizing SARS-CoV-2 antibodies targeting conserved epitopes into broad-spectrum inhibitors through receptor blockade (preprint)

All but one of the authorized monoclonal antibody-based treatments for SARS-CoV-2 are largely ineffective against Omicron, highlighting the critical need for biologics capable of overcoming SARS-CoV-2 evolution. These mostly ineffective therapeutic antibodies target epitopes that are not highly conserved. Here we describe broad-spectrum SARS-CoV-2 inhibitors developed by tethering the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), to antibodies that are known to be non-neutralizing, but which target highly conserved epitopes in the viral spike protein. These inhibitors, called Receptor-blocking conserved non-neutralizing Antibodies (ReconnAbs), potently neutralize all SARS-CoV-2 variants of concern (VOC), including Omicron. Neutralization potency is dependent on both the binding and inhibitory ReconnAb components as activity is lost when the linker joining the two is severed. In addition, a bifunctional ReconnAb, made by linking ACE2 to a bispecific antibody targeting two non-overlapping conserved epitopes, defined here, shows sub-nanomolar neutralizing activity against all VOCs, including Omicron. Given their conserved targets and modular nature, ReconnAbs have the potential to act as broad-spectrum therapeutics against SARS-CoV-2 and other emerging pandemic diseases.

SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling (preprint)

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of this pathology are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to trigger barrier dysfunction in vitro and vascular leak in vivo, independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-{beta} signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-{beta} signaling axis are required for S-mediated barrier dysfunction. Our findings suggest that S interactions with barrier cells are a contributing factor to COVID-19 disease severity and offer mechanistic insight into SARS-CoV-2 triggered vascular leak, providing a starting point for development of therapies targeting COVID-19 pathogenesis.

The SARS-CoV-2 spike reversibly samples an open-trimer conformation exposing novel epitopes (preprint)

Current COVID-19 vaccines and many clinical diagnostics are based on the structure and function of the SARS-CoV-2 spike ectodomain. Using hydrogen deuterium exchange mass spectrometry, we have uncovered that, in addition to the prefusion structure determined by cryo-EM, this protein adopts an alternative conformation that interconverts slowly with the canonical prefusion structure. This new conformation-an open trimer-contains easily accessible RBDs. It exposes the conserved trimer interface buried in the prefusion conformation, thus exposing potential epitopes for pan-coronavirus antibody and ligand recognition. The population of this state and kinetics of interconversion are modulated by temperature, receptor binding, antibody binding, and sequence variants observed in the natural population. Knowledge of the structure and populations of this conformation will help improve existing diagnostics, therapeutics, and vaccines.

multiSero: open multiplex-ELISA platform for analyzing antibody responses to SARS-CoV-2 infection (preprint)

Serology has provided valuable diagnostic and epidemiological data on antibody responses to SARS-CoV-2 in diverse patient cohorts. Deployment of high content, multiplex serology platforms across the world, including in low and medium income countries, can accelerate longitudinal epidemiological surveys. Here we report multiSero, an open platform to enable multiplex serology with up to 48 antigens in a 96-well format. The platform consists of three components: ELISA-array of printed proteins, a commercial or home-built plate reader, and modular python software for automated analysis (pysero). We validate the platform by comparing antibody titers against the SARS-CoV-2 Spike, receptor binding domain (RBD), and nucleocapsid (N) in 114 sera from COVID-19 positive individuals and 87 pre-pandemic COVID-19 negative sera. We report data with both a commercial plate reader and an inexpensive, open plate reader (nautilus). Receiver operating characteristic (ROC) analysis of classification with single antigens shows that Spike and RBD classify positive and negative sera with the highest sensitivity at a given specificity. The platform distinguished positive sera from negative sera when the reactivity of the sera was equivalent to the binding of 1 ng mL-1 RBD-specific monoclonal antibody. We developed normalization and classification methods to pool antibody responses from multiple antigens and multiple experiments. Our results demonstrate a performant and accessible pipeline for multiplexed ELISA ready for multiple applications, including serosurveillance, identification of viral proteins that elicit antibody responses, differential diagnosis of circulating pathogens, and immune responses to vaccines.

SARS-CoV-2 antibody magnitude and detectability are driven by disease severity, timing, and assay (preprint)

Serosurveillance studies are critical for estimating SARS-CoV-2 transmission and immunity, but interpretation of results is currently limited by poorly defined variability in the performance of antibody assays to detect seroreactivity over time in individuals with different clinical presentations. We measured longitudinal antibody responses to SARS-CoV-2 in plasma samples from a diverse cohort of 128 individuals over 160 days using 14 binding and neutralization assays. For all assays, we found a consistent and strong effect of disease severity on antibody magnitude, with fever, cough, hospitalization, and oxygen requirement explaining much of this variation. We found that binding assays measuring responses to spike protein had consistently higher correlation with neutralization than those measuring responses to nucleocapsid, regardless of assay format and sample timing. However, assays varied substantially with respect to sensitivity during early convalescence and in time to seroreversion. Variations in sensitivity and durability were particularly dramatic for individuals with mild infection, who had consistently lower antibody titers and represent the majority of the infected population, with sensitivities often differing substantially from reported test characteristics (e.g., amongst commercial assays, sensitivity at 6 months ranged from 33% for ARCHITECT IgG to 98% for VITROS Total Ig). Thus, the ability to detect previous infection by SARS-CoV-2 is highly dependent on the severity of the initial infection, timing relative to infection, and the assay used. These findings have important implications for the design and interpretation of SARS-CoV-2 serosurveillance studies.

Rapid SARS-CoV-2 Detection by Carbon Nanotube-Based Near-Infrared Nanosensors (preprint)

To effectively track and eliminate COVID-19, it is critical to develop tools for rapid and accessible diagnosis of actively infected individuals. Here, we introduce a single-walled carbon nanotube (SWCNT)-based optical sensing approach towards these ends. We construct a nanosensor based on SWCNTs noncovalently functionalized with ACE2, a host protein with high binding affinity for the SARS-CoV-2 spike protein. Presence of the SARS-CoV-2 spike protein elicits a robust, two-fold nanosensor fluorescence increase within 90 min of spike protein exposure. We characterize the nanosensor stability and sensing mechanism, and passivate the nanosensor to preserve sensing response in saliva and viral transport medium. We further demonstrate that these ACE2-SWCNT nanosensors retain sensing capacity in a surface-immobilized format, exhibiting a 73% fluorescence turn-on response within 5 s of exposure to 35 mg/L SARS-CoV-2 virus-like particles. Our data demonstrate that ACE2-SWCNT nanosensors can be developed into an optical tool for rapid SARS-CoV-2 detection. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/20223404v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@b78f2dorg.highwire.dtl.DTLVardef@118fdc8org.highwire.dtl.DTLVardef@1bd5e3corg.highwire.dtl.DTLVardef@175f6a8_HPS_FORMAT_FIGEXP M_FIG C_FIG

A single immunization with spike-functionalized ferritin vaccines elicits neutralizing antibody responses against SARS-CoV-2 in mice (preprint)

Development of a safe and effective SARS-CoV-2 vaccine is a public health priority. We designed subunit vaccine candidates using self-assembling ferritin nanoparticles displaying one of two multimerized SARS-CoV-2 spikes: full-length ectodomain (S-Fer) or a C-terminal 70 amino-acid deletion (S{Delta}C-Fer). Ferritin is an attractive nanoparticle platform for production of vaccines and ferritin-based vaccines have been investigated in humans in two separate clinical trials. We confirmed proper folding and antigenicity of spike on the surface of ferritin by cryo-EM and binding to conformation-specific monoclonal antibodies. After a single immunization of mice with either of the two spike ferritin particles, a lentiviral SARS-CoV-2 pseudovirus assay revealed mean neutralizing antibody titers at least 2-fold greater than those in convalescent plasma from COVID-19 patients. Additionally, a single dose of S{Delta}C-Fer elicited significantly higher neutralizing responses as compared to immunization with the spike receptor binding domain (RBD) monomer or spike ectodomain trimer alone. After a second dose, mice immunized with S{Delta}C-Fer exhibited higher neutralizing titers than all other groups. Taken together, these results demonstrate that multivalent presentation of SARS-CoV-2 spike on ferritin can notably enhance elicitation of neutralizing antibodies, thus constituting a viable strategy for single-dose vaccination against COVID-19.

A PCR amplicon-based SARS-CoV-2 replicon for antiviral screening (preprint)

The development of specific antiviral compounds to SARS-CoV-2 is an urgent task. One of the obstacles for the antiviral development is the requirement of biocontainment because infectious SARS-CoV-2 must be handled in a biosafety level-3 laboratory. Replicon, a non-infectious self-replicative viral RNA, could be a safe and effective tool for antiviral screening; however, SARS-CoV-2 replicon has not been reported yet. Herein, we generated a PCR-based SARS-CoV-2 replicon. Eight fragments covering the entire SARS-CoV-2 genome except S, E, and M genes were amplified with HiBiT-tag sequence by PCR. The amplicons were ligated and in vitro transcribed to RNA. The cells electroporated with the replicon RNA showed more than 3,000 times higher luminescence than MOCK control cells at 24 hours post-electroporation, indicating robust viral translation and RNA replication. The replication was drastically inhibited by remdesivir, an RNA polymerase inhibitor for SARS-CoV-2. The IC50 of remdesivir in this study was 0.29 M, generally consistent to the IC50 obtained using infectious SARS-CoV-2 in a previous study (0.77 M). Taken together, this system could be applied to the safe and effective antiviral screening without using infectious SARS-CoV-2. Because this is a transient replicon, further improvement including the establishment of stable cell line must be achieved.