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.

Engineering luminescent biosensors for point-of-care SARS-CoV-2 antibody detection (preprint)

Current serology tests for SARS-CoV-2 antibodies mainly take the form of enzyme-linked immunosorbent assays or lateral flow assays, with the former being laborious and the latter being expensive and often lacking sufficient sensitivity and scalability. Here we present the development and validation of a rapid, low-cost solution-based assay to detect antibodies in serum, plasma, whole blood, and saliva, using rationally designed split luciferase antibody biosensors (spLUC). This new assay, which generates quantitative results in as short as 5 minutes, substantially reduces the complexity and improves the scalability of COVID-19 antibody tests for point-of-care and broad population testing.

Bi-paratopic and multivalent human VH domains neutralize SARS-CoV-2 by targeting distinct epitopes within the ACE2 binding interface of Spike (preprint)

Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domain binders with high affinity toward neutralizing epitopes without the need for high-resolution structural information. We constructed a VH-phage library and targeted a known neutralizing site, the angiotensin-converting enzyme 2 (ACE2) binding interface of the trimeric SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified 85 unique VH binders to two non-overlapping epitopes within the ACE2 binding site on Spike-RBD. This enabled us to systematically link these VH domains into multivalent and bi-paratopic formats. These multivalent and bi-paratopic VH constructs showed a marked increase in affinity to Spike (up to 600-fold) and neutralization potency (up to 1400-fold) on pseudotyped SARS-CoV-2 virus when compared to the standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with half-minimal inhibitory concentration (IC50) of 4.0 nM (180 ng/mL). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain bound an RBD at the ACE2 binding site, explaining its increased neutralization potency and confirming our original design strategy. Our results demonstrate that targeted selection and engineering campaigns using a VH-phage library can enable rapid assembly of highly avid and potent molecules towards therapeutically important protein interfaces.

Engineered ACE2 receptor traps potently neutralize SARS-CoV-2 (preprint)

An essential mechanism for SARS-CoV-1 and -2 infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human Fc domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2 pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50) in the tens of ng/ml range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-utilizing coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be pre-designed for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated or generated from convalescent patients.

A SARS-CoV-2 serological assay to determine the presence of blocking antibodies that compete for human ACE2 binding (preprint)

As SARS-CoV-2 continues to spread around the world, there is an urgent need for new assay formats to characterize the humoral response to infection. Convalescent serum is being used for treatment and for isolation of patient-derived antibodies. However, currently there is not a simple means to estimate serum bulk neutralizing capability. Here we present an efficient competitive serological assay that can simultaneously determine an individual's seropositivity against the SARS-CoV-2 Spike protein and estimate the neutralizing capacity of anti-Spike antibodies to block interaction with the human angiotensin converting enzyme 2 (ACE2) required for viral entry. In this ELISA-based assay, we present natively-folded viral Spike protein receptor binding domain (RBD)-containing antigens via avidin-biotin interactions. Sera are then supplemented with soluble ACE2-Fc to compete for RBD-binding serum antibodies, and antibody binding quantified. Comparison of signal from untreated serum and ACE2-Fc-treated serum reveals the presence of antibodies that compete with ACE2 for RBD binding, as evidenced by loss of signal with ACE2-Fc treatment. In our test cohort of nine convalescent SARS-CoV-2 patients, we found all patients had developed anti-RBD antibodies targeting the epitope responsible for ACE2 engagement. This assay provides a simple and high-throughput method to screen patient sera for potentially neutralizing anti-Spike antibodies to enable identification of candidate sera for therapeutic use.

Trimeric SARS-CoV-2 Spike interacts with dimeric ACE2 with limited intra-Spike avidity (preprint)

A serious public health crisis is currently unfolding due to the SARS-CoV-2 pandemic. SARS-CoV-2 viral entry depends on an interaction between the receptor binding domain of the trimeric viral Spike protein (Spike-RBD) and the dimeric human angiotensin converting enzyme 2 (ACE2) receptor. While it is clear that strategies to block the Spike/ACE2 interaction are promising as anti-SARS-CoV-2 therapeutics, our current understanding is insufficient for the rational design of maximally effective therapeutic molecules. Here, we investigated the mechanism of Spike/ACE2 interaction by characterizing the binding affinity and kinetics of different multimeric forms of recombinant ACE2 and Spike-RBD domain. We also engineered ACE2 into a split Nanoluciferase-based reporter system to probe the conformational landscape of Spike-RBDs in the context of the Spike trimer. Interestingly, a dimeric form of ACE2, but not monomeric ACE2, binds with high affinity to Spike and blocks viral entry in pseudotyped virus and live SARS-CoV-2 virus neutralization assays. We show that dimeric ACE2 interacts with an RBD on Spike with limited intra-Spike avidity, which nonetheless contributes to the affinity of this interaction. Additionally, we demonstrate that a proportion of Spike can simultaneously interact with multiple ACE2 dimers, indicating that more than one RBD domain in a Spike trimer can adopt an ACE2-accessible "up" conformation. Our findings have significant implications on the design strategies of therapeutic molecules that block the Spike/ACE2 interaction. The constructs we describe are freely available to the research community as molecular tools to further our understanding of SARS-CoV-2 biology.