NOVEL GREEN STRATEGIES FOR PREPAREDENESS TO FUTURE PANDEMIC DISEASES: DEVELOPMENT OF A BIODEVICE FOR WATER REMEDIATION FROM PATHOGENIC BACTERIA AND EVALUATION OF THE EFFECTIVENESS OF A NEW SARS-COV-2 PLANT-BASED VIRUS-LIKE PARTICLE VACCINE

The global impact of the COVID-19 pandemic has underscored the urgent need for innovative and rapid strategies to enhance preparedness for future pandemics. The risk of the emergence of new infectious disease is extremely concrete and it is essential to invest in the development of vaccines and biotechnological drugs as well as to speedup of clinical trials. Currently, one of the main focus is shifting to the global public health threat caused by the rise of antimicrobial-resistant (AMR) bacterial strains. Multidrug-resistant (MDR) pathogen infections are on increasing incidence and frequently lead to multiple events of sepsis. Therefore, it is necessary to discover both novel approaches and novel drugs active against these multidrug-resistant bacteria to prevent the spreading of sepsis. On the other hand, emerging viruses represent a real menace and it is essential to set-up and validate methods to monitore the spread of the diseases and speed up the release of vaccines and antiviral drugs. This dissertation is divided in two main project: The first project is focused on the development of a new SET-M33-based device for the multiple and selective removal of both bacterial toxins (LPS and LTA) and live bacteria (Escherichia coli O111:B4 and Staphylococcus aureus USA 300) from wastewater. The antimicrobial peptide SET-M33 has a remarkable capacity to both destroy and bind components of bacterial walls, including lipopolysaccharide (LPS) from Gram-negative bacteria and lipoteichoic acid (LTA) from Gram-positive bacteria. It is a cationic non-natural peptide synthetized in a tetra-branched form that makes it more resistant to degradation in biological fluids aand water. Moreover, it shows a high antimicrobial activity both in vitro and in vivo, anti-inflammatory capacity through neutralization of endotoxins such as LPS, low hemolytic activity, lack of immunogenicity, and is active against biofilm-forming bacteria. The device was realized by immobilization of the peptide on a biocompatible resin composed of agarose beads via a thiol residue at the C-terminal of the peptide. The peptide was synthesized with amino acids in L-configuration to facilitate the conjugation with the resin via the thiol residue at the C-terminal. Preliminary results showed that the peptide mantained its strong antimicrobial anctivity against both Gram positive and Gram Negative strains as well as bacterial molecules, released upon cell lysis. A second part of the PhD was conducted inside Vismederi srl and was aimed to evaluate the effectiveness and immunogenicity of a SARS-CoV-2 plant-based vaccine manufactured through a Nicotiana benthamiana platform. SARS-CoV-2 was directly responsible for the COVID-19 (Coronavirus Disease 2019) pandemic, an acute respiratory syndrome which led to significant health, social and economic consequences worldwide. Vaccination is the principal and single most cost-effective strategy of preventing COVID-19 and reduce illness and the severity of infection, especially in specific highrisk groups for complications. In this context, microneutralization cytopathic effect-based (MN-CPE) assays were used to analyze the potency of this candidate vaccine to elicit a specific humoral immune response against five propagated variants of concern (VOCs) Alfa, Beta, Gamma, Delta and Omicron (BA.1) up to 21 days post-second immunization and up to 201 days after second immunization, in the context of a phase I clinical trial. This study was conducted in order to evaluate whether this novel COVID-19 vaccine was able to elicit a specific humoral immune response. Particularly, 540 human serum samples were analyzed revealing a strong humoral response against wt, Alpha and Delta variants, a moderate immunogenicity against Beta and Gamma and an important decrease against BA.1. Subsequently, serum neutralizing activity against the overmentioned five VOCs in hospitalized COVID-19 patients from the first pandemic wave was analyzed. Results were comparable to data obtained for the plant-based vaccine highlighting the occurrence of an antigenic drift event which required a periodic update of vaccine formulation. Moreover, this study highlights the importance of evaluating SARS-CoV-2 pre-existing immunity against emerging variants as a tool to foresee the immune escape and the extent of vaccine efficacy.