Development of AAV-delivered broadly neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the emergence of new variants that are resistant to existing vaccines and therapeutic antibodies, has raised the need for novel strategies to combat the persistent global COVID-19 epidemic. In this study, a monoclonal anti-human angiotensin-converting enzyme 2 (hACE2) antibody, ch2H2, was isolated and humanized to block the viral receptor-binding domain (RBD) binding to hACE2, the major entry receptor of SARS-CoV-2. This antibody targets the RBD-binding site on the N terminus of hACE2 and has a high binding affinity to outcompete the RBD. In vitro, ch2H2 antibody showed potent inhibitory activity against multiple SARS-CoV-2 variants, including the most antigenically drifted and immune-evading variant Omicron. In vivo, adeno-associated virus (AAV)-mediated delivery enabled a sustained expression of monoclonal antibody (mAb) ch2H2, generating a high concentration of antibodies in mice. A single administration of AAV-delivered mAb ch2H2 significantly reduced viral RNA load and infectious virions and mitigated pulmonary pathological changes in mice challenged with SARS-CoV-2 Omicron BA.5 subvariant. Collectively, the results suggest that AAV-delivered hACE2-blocking antibody provides a promising approach for developing broad-spectrum antivirals against SARS-CoV-2 and potentially other hACE2-dependent pathogens that may emerge in the future.

Forced-convective vitrification with liquid cryogens.

Cell cryopreservation by vitrification generally requires using vitrification solutions with high concentrations of cryoprotectants (CPAs), which are toxic and induce osmotic stresses associated with the addition and removal of CPAs. To increase the cooling rate and reduce the CPA concentration required for vitrification, this study proposed an innovative approach, named forced-convective vitrification with liquid cryogens, in which liquid oxygen at a temperature below its boiling point (LOX(bbp)) was used as the cryogen to reduce the generation of insulating bubbles of gaseous oxygen and the sample was subjected to a constant velocity to remove insulation bubbles from the sample. Results show that changing the cryogen from liquid nitrogen at its boiling temperature (LN(abp)) to LOX(bbp), increasing the sample velocity and reducing the test solution volume increased the cooling rate and thereby decreased the CPA concentration required for vitrification. Using the same velocity (1.2 m/s), the cooling rate achieved with LOX(bbp) was 2.3-fold greater than that achieved with LN(abp). With LOX(bbp), the increase in the sample velocity from 0.2 to 1.2 m/s enhanced the cooling rate by 1.9 times. With LOX(bbp), a velocity of 1.2m/s and a test solution volume of 1.73 µl, the CPA concentration required for vitrification decreased to 25%. These results indicate that the new approach described here can reduce the CPA concentration required for vitrification, and thus decreases the toxicity and osmotic stresses associated with adding and removing the CPA.