Caratterizzazione dell'attività antivirale contro virus respiratori umani di SP4™, un nuovo estratto vegetale

The ongoing COVID-19 pandemic proves that respiratory viral infections (RTIs) are leading causes of morbidity, mortality and a major societal and healthcare problem. Respiratory viruses infect respiratory apparatus causing a broad range of outcomes, from asymptomatic to acute life-threatening diseases. These viruses spread through the secretions from an infected individual with three different mechanisms: direct/indirect contact, droplet spray or aerosol. Even though vaccines represent the most effective measure for the prevention and control of the main RTIs, such as influenza and SARS-CoV-2 infections, therapeutic agents with a broad-spectrum of antiviral activity (BSA) could have been used from the beginning of COVID-19 pandemic, allowing to save lives while waiting for the development of specific vaccines. Unfortunately, the lack of available and effective BSA rapidly deployable against the new SARS-CoV-2, prevented the reduction of hospitalizations and deaths, as the spread of COVID-19 pandemic. Therefore, effective BSA that can be rapidly deployed against emerging respiratory viruses are urgently needed. In this regard, plant-based products may represent a source of new BSA, since phytochemicals compounds are known to exert antimicrobial activities. Among bioactive polyphenols of plant origin, the proanthocyanidins (PACs) exerted antiadhesive activities against E. coli strains preventing urinary tract infections. However, PACs have been identified as the bioactive antiviral agents in a few studies against single viruses; so, their potential as BSA remains to be confirmed. The aim of this thesis work was to investigate the BSA prospective of SP4, a novel plant extract, rich in PACs. SP4 was tested against representative human respiratory viruses, as human Coronavirus OC43 (hCoV-OC43), SARS-CoV-2; Influenza A and B Viruses; and Respiratory Syncytial Virus. SP4 showed a potent inhibitory activity against the in vitro replication of the viruses analyzed. Gel filtration chromatography of SP4 allowed to separate its phytochemicals in different fractions. Of these, fraction 3, containing only PACs, when tested against hCoV-OC43, reproduced the inhibitory activity of SP4 pinpointing PACs as the bioactive antiviral agents of SP4. Time-of-addiction experiments then proved that SP4 was able to block the initial events of the hCoV-OC43 replicative cycle. Indeed, attachment and entry antiviral assays indicated that SP4 influenced the accomplishment of these steps. Moreover, SP4 was observed to reduce the infectivity of viral particles in solution exerting a virucidal effect even prior to the adsorption to the host. Since the Spike (S) glycoprotein of hCoV-OC43 regulates the attachment and entry of the virus, the possibility of the occurrence of a direct interaction with SP4 was examined. Indeed, alterations of the electrophoretic mobility of the recombinant S protein with formation of oligomers were observed upon the addition of SP4, confirming physical interactions of SP4 with the S protein. These may threaten functions of S protein, eventually resulting in the inhibition of the attachment of viral particles to cell receptors. In conclusion, the potent and broad-spectrum antiviral activity of SP4 suggest the extract as a promising candidate for the development of new BSAs, useful for an immediate intervention against emerging respiratory viral threats; at the same time, the SP4 features claim its further efficacy evaluation in animal models.