Rapid and Sensitive Detection of Antigen from SARS-CoV-2 Variants of Concern by a Multivalent Minibinder-Functionalized Nanomechanical Sensor.

New platforms for the rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern are urgently needed. Here we report the development of a nanomechanical sensor based on the deflection of a microcantilever capable of detecting the SARS-CoV-2 spike (S) glycoprotein antigen using computationally designed multivalent minibinders immobilized on a microcantilever surface. The sensor exhibits rapid (<5 min) detection of the target antigens down to concentrations of 0.05 ng/mL (362 fM) and is more than an order of magnitude more sensitive than an antibody-based cantilever sensor. Validation of the sensor with clinical samples from 33 patients, including 9 patients infected with the Omicron (BA.1) variant observed detection of antigen from nasopharyngeal swabs with cycle threshold (Ct) values as high as 39, suggesting a limit of detection similar to that of the quantitative reverse transcription polymerase chain reaction (RT-qPCR). Our findings demonstrate the use of minibinders and nanomechanical sensors for the rapid and sensitive detection of SARS-CoV-2 and potentially other disease markers.

Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.

Lyophilization of premixed COVID-19 diagnostic RT-qPCR reactions enables stable long-term storage at elevated temperature.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) diagnostic tests for SARS-CoV-2 are the cornerstone of the global testing infrastructure. However, these tests require cold-chain shipping to distribute, and the labor of skilled technicians to assemble reactions and interpret the results. Strategies to reduce shipping and labor costs at the point-of-care could aid in diagnostic testing scale-up and response to the COVID-19 outbreak, as well as in future outbreaks. In this study we test both lab-developed and commercial SARS-CoV-2 diagnostic RT-qPCR mixes for the ability to be stabilized against elevated temperature by lyophilization. Fully assembled reactions were lyophilized and stored for up to a month at ambient or elevated temperature and were subsequently assayed for their ability to detect dilutions of synthetic SARS-CoV-2 RNA. Of the mixes tested, we show that one commercial mix can maintain activity and sensitivity after storage for at least 30 days at ambient temperature after lyophilization. We also demonstrate that lyoprotectants such as disaccharides can stabilize freeze-dried diagnostic reactions against elevated temperatures (up to 50°C) for at least 30 days. We anticipate that the incorporation of these methods into SARS-CoV-2 diagnostic testing will improve testing pipelines by reducing labor at the testing facility and eliminating the need for cold-chain shipping.

Principles Learned from the International Race to Develop a Safe and Effective COVID-19 Vaccine.

Vaccines against COVID-19 have the potential to protect people before they are exposed to the infective form of the virus. However, because of the involvement of pathogenic immune processes in many severe presentations of COVID-19, eliciting an immune response with a vaccine must strike a delicate balance to achieve viral clearance without also inducing immune-mediated harm. This Outlook synthesizes current laboratory findings to define which parts of the immune system help with recovery from and protection against the virus and which can lead to adverse outcomes. To inform our understanding, we analyze research about the immune mechanisms implicated in SARS-CoV, from the 2003 outbreak, and SARS-CoV-2, the virus causing COVID-19. The impact of how innate immunity, humoral immunity, and cell-mediated immunity play a role in a harmful versus helpful response is discussed, establishing principles to guide the development and evaluation of a safe but effective COVID-19 vaccine. The principles derived include (i) targeting the appropriate specificity and effector function of the humoral response, (ii) eliciting a T cell response, especially a cytotoxic T cell response, to achieve safe, yet effective, immune protection from COVID-19, and (iii) monitoring for the possibility of acute lung injury during SARS-CoV-2 infection post-vaccination in preclinical and clinical studies. These principles can not only guide efforts toward a safe and effective COVID-19 vaccine, but also the development of effective vaccines for viral pandemics to come.