An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-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 immunoglobulin crystallizable fragment (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 (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.

An isolated epizootic of a highly fatal feline calicivirus (FCV) infection, manifested in its severest form by a systemic hemorrhagic-like fever, occurred over a 1-month period among six cats owned by two different employees and a client of a private veterinary practice. The infection may have started with an unowned shelter kitten that was hospitalized during this same period for a severe atypical upper respiratory infection. The causative agent was isolated from blood and nasal swabs from two cats; the electron microscopic appearance was typical for FCV and capsid gene sequencing showed it to be genetically similar to other less pathogenic field strains. An identical disease syndrome was recreated in laboratory cats through oral inoculation with tissue culture grown virus. During the course of transmission studies in experimental cats, the agent was inadvertently spread by caretakers to an adjoining room containing a group of four normal adult cats. One of the four older cats was found dead and a second was moribund within 48-72h in spite of symptomatic treatment; lesions in these animals were similar to those of the field cats but with the added feature of severe pancreatitis. The mortality in field cats, deliberately infected laboratory cats, and inadvertently infected laboratory cats ranged from 33-50%. This new isolate of calicivirus, named FCV-Ari, was neutralized at negligible to low titer by antiserum against the universal FCV-F9 vaccine strain. Cats orally immunized with FCV-F9, and then challenge-exposed shortly thereafter with FCV-Ari, developed a milder self-limiting form of disease, indicating partial protection. However, all of the field cats, including the three that died, had been previously immunized with parenteral FCV-F9 vaccine. FCV-Ari caused a disease that was reminiscent of Rabbit Hemorrhagic Disease, a highly fatal calicivirus infection of older rabbits.

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.

Current serology tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies mainly take the form of enzyme-linked immunosorbent assays, chemiluminescent microparticle immunoassays or lateral flow assays, which are either laborious, expensive or 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 to a lesser extent saliva, using rationally designed split luciferase antibody biosensors. This new assay, which generates quantitative results in 30 min, substantially reduces the complexity and improves the scalability of coronavirus disease 2019 (COVID-19) antibody tests. This assay is well-suited for point-of-care, broad population testing, and applications in low-resource settings, for monitoring host humoral responses to vaccination or viral infection.As the vaccine deployment starts worldwide for COVID-19, broad antibody testing for SARS-CoV-2 faces severe limitations. Although nucleic acid testing is critical to detecting the virus, serological antibody tests are vital tools for monitoring the dynamic human humoral response to SARS-CoV-2 vaccination and viral infection 1 . Population-scale, longitudinal evaluation of antibody responses is needed to determine the strength and duration of immunity to the primary virus, to the variants, and to vaccines, which is important in informing public policy and vaccination strategies 2-7 . In addition, antibody tests serve as a complement or an alternative to nucleic acid diagnostics for patients with a low viral load or for low-resource areas where expensive reverse transcription polymerase chain reaction (RT-PCR) testing is difficult to access [8][9][10] . Serological tests also support therapeutic development through identification of either individuals who could serve as donors for convalescent serum therapeutics 11 , or patients with potentially strong neutralizing antibodies that can be produced in vitro as new antivirals and prophylactics 12,13 . All these applications would be greatly accelerated with an assay that is simple, rapid and high throughput, without sacrificing accuracy and sensitivity.Traditional serological assays are not optimal in the face of this broad pandemic. The most widely used laboratory serological tests take the form of enzyme-linked immunosorbent assays

Sensing and responding to signals is a fundamental ability of living systems, but despite substantial progress in the computational design of new protein structures, there is no general approach for engineering arbitrary new protein sensors. Here, we describe a generalizable computational strategy for designing sensor-actuator proteins by building binding sites de novo into heterodimeric protein-protein interfaces and coupling ligand sensing to modular actuation through split reporters. Using this approach, we designed protein sensors that respond to farnesyl pyrophosphate, a metabolic intermediate in the production of valuable compounds. The sensors are functional in vitro and in cells, and the crystal structure of the engineered binding site closely matches the design model. Our computational design strategy opens broad avenues to link biological outputs to new signals.

Sensing and responding to signals is a fundamental ability of living systems, but despite remarkable progress in computational design of new protein structures, there is no general approach for engineering arbitrary new protein sensors. Here we describe a generalizable computational strategy for designing sensor/actuator proteins by building binding sites de novo into heterodimeric protein-protein interfaces and coupling ligand sensing to modular actuation via split reporters. Using this approach, we designed protein sensors that respond to farnesyl pyrophosphate, a metabolic intermediate in the production of valuable compounds. The sensors are functional in vitro and in cells, and the crystal structure of the engineered binding site matches the design model with atomic accuracy. Our computational design strategy opens broad avenues to link biological outputs to new signals.One Sentence SummaryAn engineering strategy to design modular synthetic signaling systems that respond to new small molecule inputs.

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.

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