Structural and Immunoreactivity Properties of the SARS-CoV-2 Spike Protein upon the Development of an Inactivated Vaccine.

Inactivated vaccines are promising tools for tackling the COVID-19 pandemic. We applied several protocols for SARS-CoV-2 inactivation (by ß-propiolactone, formaldehyde, and UV radiation) and examined the morphology of viral spikes, protein composition of the preparations, and their immunoreactivity in ELISA using two panels of sera collected from convalescents and people vaccinated by Sputnik V. Transmission electron microscopy (TEM) allowed us to distinguish wider flail-like spikes (supposedly the S-protein's pre-fusion conformation) from narrower needle-like ones (the post-fusion state). While the flails were present in all preparations studied, the needles were highly abundant in the ß-propiolactone-inactivated samples only. Structural proteins S, N, and M of SARS-CoV-2 were detected via mass spectrometry. Formaldehyde and UV-inactivated samples demonstrated the highest affinity/immunoreactivity against the convalescent sera, while ß-propiolactone (1:2000, 36 h) and UV-inactivated ones were more active against the sera of people vaccinated with Sputnik V. A higher concentration of ß-propiolactone (1:1000, 2 h) led to a loss of antigenic affinity for both serum panels. Thus, although we did not analyze native SARS-CoV-2 for biosafety reasons, our comparative approach helped to exclude some destructive inactivation conditions and select suitable variants for future animal research. We believe that TEM is a valuable tool for inactivated COVID-19 vaccine quality control during the downstream manufacturing process.

5-(Perylen-3-ylethynyl)uracil as an antiviral scaffold: Potent suppression of enveloped virus reproduction by 3-methyl derivatives in vitro.

Amphipathic nucleoside and non-nucleoside derivatives of pentacyclic aromatic hydrocarbon perylene are known as potent non-cytotoxic broad-spectrum antivirals. Here we report 3-methyl-5-(perylen-3-ylethynyl)-uracil-1-acetic acid and its amides, a new series of compounds based on a 5-(perylen-3-ylethynyl)-uracil scaffold. The compounds demonstrate pronounced in vitro activity against arthropod-borne viruses, namely tick-borne encephalitis virus (TBEV) and yellow fever virus (YFV), in plaque reduction assays with EC50 values below 1.9 and 1.3 nM, respectively, and Chikungunya virus (CHIKV) in cytopathic effect inhibition test with EC50 values below 3.2 µM. The compounds are active against respiratory viruses as well: severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) in cytopathic effect inhibition test and influenza A virus (IAV) in virus titer reduction experiments are inhibited - EC50 values below 51 nM and 2.2 µM, respectively. The activity stems from the presence of a hydrophobic perylene core, and all of the synthesized compounds exhibit comparable 1O2 generation rates. Nonetheless, activity can vary by orders of magnitude depending on the hydrophilic part of the molecule, suggesting a complex mode of action. A time-of-addition experiment and fluorescent imaging indicate that the compounds inhibit viral fusion in a dose-dependent manner. The localization of the compound in the lipid bilayers and visible damage to the viral envelope suggest the membrane as the primary target. Dramatic reduction of antiviral activity with limited irradiation or under treatment with antioxidants further cements the idea of photoinduced ROS-mediated viral envelope damage being the mode of antiviral action.

Structural characterization of β-propiolactone inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles

Abstract The severe COVID-19 pandemic drives the research toward the SARS-CoV-2 virion structure and the possible therapies against it. Here, we characterized the ?-propiolactone inactivated SARS-CoV-2 virions using transmission electron microscopy (TEM) and atomic force microscopy (AFM). We compared the SARS-CoV-2 samples purified by two consecutive chromatographic procedures (size exclusion chromatography [SEC], followed by ion-exchange chromatography [IEC]) with samples purified by ultracentrifugation. The samples prepared using SEC and IEC retained more spikes on the surface than the ones prepared using ultracentrifugation, as confirmed by TEM and AFM. TEM showed that the spike (S) proteins were in the pre-fusion conformation. Notably, the S proteins could be recognized by specific monoclonal antibodies. Analytical TEM showed that the inactivated virions retained nucleic acid. Altogether, we demonstrated that the inactivated SARS-CoV-2 virions retain the structural features of native viruses and provide a prospective vaccine candidate.