Influenza viral particles harboring the SARS-CoV-2 spike RBD as a combination respiratory disease vaccine

Vaccines targeting SARS-CoV-2 have gained emergency FDA approval, however the breadth against emerging variants and the longevity of protection remains unknown. Post-immunization boosting may be required, perhaps on an annual basis if the virus becomes an endemic pathogen. Seasonal influenza virus vaccines are already developed every year, an undertaking made possible by a robust global vaccine production and distribution infrastructure. To create a seasonal combination vaccine targeting influenza viruses and SARS-CoV-2 that is also amenable to frequent reformulation, we have developed a recombinant influenza A virus (IAV) genetic platform that "reprograms" the virus to package an immunogenic domain of the SARS-CoV-2 spike (S) protein onto IAV particles. Vaccination with this combination vaccine elicits neutralizing antibodies and provides protection from lethal challenge with both pathogens. This technology may allow for leveraging of established influenza vaccine infrastructure to generate a cost-effective and scalable seasonal vaccine solution for both influenza and coronaviruses.

SRSF protein kinases 1 and 2 are essential host factors for human coronaviruses including SARS-CoV-2

While vaccines are vital for preventing COVID-19 infections, it is critical to develop new therapies to treat patients who become infected. Pharmacological targeting of a host factor required for viral replication can suppress viral spread with a low probability of viral mutation leading to resistance. In particular, host kinases are highly druggable targets and a number of conserved coronavirus proteins, notably the nucleoprotein (N), require phosphorylation for full functionality. In order to understand how targeting kinases could be used to compromise viral replication, we used a combination of phosphoproteomics and bioinformatics as well as genetic and pharmacological kinase inhibition to define the enzymes important for SARS-CoV-2 N protein phosphorylation and viral replication. From these data, we propose a model whereby SRPK1/2 initiates phosphorylation of the N protein, which primes for further phosphorylation by GSK-3/{beta} and CK1 to achieve extensive phosphorylation of the N protein SR-rich domain. Importantly, we were able to leverage our data to identify an FDA-approved kinase inhibitor, Alectinib, that suppresses N phosphorylation by SRPK1/2 and limits SARS-CoV-2 replication. Together, these data suggest that repurposing or developing novel host-kinase directed therapies may be an efficacious strategy to prevent or treat COVID-19 and other coronavirus-mediated diseases.

Development of a fluorescence based, high-throughput SARS-CoV-2 3CLpro reporter assay

In late 2019 a human coronavirus, now known as SARS-CoV-2, emerged, likely from a zoonotic reservoir. This virus causes COVID-19 disease, has infected millions of people, and has led to hundreds of thousands of deaths across the globe. While the best interventions to control and ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit morbidity and mortality in those already infected. At this time, only one FDA approved anti-SARS-CoV-2 antiviral drug, remdesivir, is available and unfortunately, its efficacy appears to be limited. Thus, the identification of new and efficacious antivirals is of highest importance. In order to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are required. With respect to drug targets, most attention is focused on either the viral RNA-dependent RNA polymerase or the main viral protease, 3CLpro. 3CLpro is an attractive target for antiviral therapeutics as it is essential for processing newly translated viral proteins, and the viral lifecycle cannot be completed without protease activity. In this work, we present a new assay to identify inhibitors of the SARS-CoV-2 main protease, 3CLpro. Our reporter is based on a GFP-derived protein that only fluoresces after cleavage by 3CLpro. This experimentally optimized reporter assay allows for antiviral drug screening in human cell culture at biosafety level-2 (BSL2) with high-throughput compatible protocols. Using this screening approach in combination with existing drug libraries may lead to the rapid identification of novel antivirals to suppress SARS-CoV-2 replication and spread. IMPORTANCEThe COVID-19 pandemic has already led to more than 400,000 deaths and innumerable changes to daily life worldwide. Along with development of a vaccine, identification of effective antivirals to treat infected patients is of the highest importance. However, rapid drug discovery requires efficient methods to identify novel compounds that can inhibit the virus. In this work, we present a method for identifying inhibitors of the SARS-CoV-2 main protease, 3CLpro. This reporter-based assay allows for antiviral drug screening in human cell culture at biosafety level-2 (BSL2) with high-throughput compatible sample processing and analysis. This assay may help identify novel antivirals to control the COVID-19 pandemic.