N-glycosylation profiles of the SARS-CoV-2 spike D614G mutant and its ancestral protein characterized by advanced mass spectrometry

N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins.

Rapid Development of Neutralizing and Diagnostic SARS-COV-2 Mouse Monoclonal Antibodies

The need for high-affinity, SARS-CoV-2-specific monoclonal antibodies (mAbs) is critical in the face of the global COVID-19 pandemic, as such reagents can have important diagnostic, research, and therapeutic applications. Of greatest interest is the ~300 amino acid receptor binding domain (RBD) within the S1 subunit of the spike protein because of its key interaction with the human angiotensin converting enzyme 2 (hACE2) receptor present on many cell types, especially lung epithelial cells. We report here the development and functional characterization of 29 nanomolar-affinity mouse SARS-CoV-2 mAbs created by an accelerated immunization and hybridoma screening process. Differing functions, including binding of diverse protein epitopes, viral neutralization, impact on RBD-hACE2 binding, and immunohistochemical staining of infected lung tissue, were correlated with variable gene usage and sequence.

High-throughput quantitation of SARS-CoV-2 antibodies in a single-dilution homogeneous assay

SARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA.

Enveloped Virus Inactivation on Personal Protective Equipment by Exposure to Ozone

Ozone is a highly oxidizing gas easily generated from atmospheric oxygen with inexpensive equipment and is commonly used for the disinfection of municipal water, foods, and surfaces. We report tests of the ability of ozone to inactivate enveloped respiratory viruses (influenza A virus and respiratory syncytial virus), chosen as more easily handled surrogates for SARS-CoV-2, on N95 respirators and other personal protective equipment (PPE) commonly used in hospitals. At 20 ppm, an ozone concentration easily achieved by standard commercial equipment, the viruses were inactivated with high efficiency as long as the relative humidity was above a threshold value of approximately 50%. In the absence of humidity control, disinfection is more variable and requires considerably longer exposure under relatively dry conditions. This report extends the observations of a previous publication (http://doi.org/10.1080/01919510902747969) to hospital-relevant materials and provides additional details about the relationship of humidity to the antiviral activity of ozone. Home CPAP disinfection devices using ozone can provide effective results for individuals. Ozone did not appear to degrade any of the materials tested except for elastic bands if strained during treatment (such as by the pressure exerted by stapled attachment to N95 respirators). The filtration efficiency of N95 respirator material was not compromised. Overall, we recommend exposures of at least 40 minutes to 20 ppm ozone and >70% relative humidity at ambient temperatures (21-24{degrees}C) for 4-log (99.99%) reduction of viral infectivity on a variety of PPE, including gowns, face shields, and respirators. Shorter exposure times are likely to be effective under these conditions, but at the risk of some variability for different materials. Higher ozone concentrations and higher humidity levels promoted faster inactivation of viruses. Our work suggests that ozone exposure can be a widely accessible method for disinfecting PPE, permitting safer re-use for healthcare workers and patients alike in times of shortage.

Treating Influenza and SARS-CoV-2 via mRNA-encoded Cas13a

Here, Cas13a has been used to target and mitigate influenza virus A (IAV) and SARS-CoV-2 using a synthetic mRNA-based platform. CRISPR RNAs (crRNA) against PB1 and highly conserved regions of PB2 were screened in conjunction with mRNA-encoded Cas13a. Screens were designed such that only guides that decreased influenza RNA levels in a Cas13-mediated fashion, were valid. Cas13a mRNA and validated guides, delivered post-infection, simulating treatment, were tested in combination and across multiplicities of infection. Their function was also characterized over time. Similar screens were performed for guides against SARS-CoV-2, yielding multiple guides that significantly impacted cytopathic effect. Last, the approach was utilized in vivo, demonstrating the ability to degrade influenza RNA in a mouse model of infection, using polymer-formulated, nebulizer-based mRNA delivery. Our findings demonstrate the applicability of Cas13a in mitigating respiratory infections both in vitro and in a mouse model, paving the way for future therapeutic use.