Development of BSL-2 platforms for the identification of pan-neutralizing human monoclonal antibodies against Coronaviridae

During the COVID-19 pandemic, the US Food and Drug Administration approved the use of anti-viral neutralizing monoclonal antibodies (mAbs) for emergency purposes in out-of-hospital patients with mild-to-moderate disease 1. Given the limited availability of these novel therapies, it is imperative to explore their broader potential and devise effective deployment strategies for functional and accurate assays which investigate the ability of mAbs to neutralize the virus. Live SARS-CoV-2 is categorized as a Biosafety Level 3 (BSL-3) agent due to its high pathogenicity and infectivity; hence, it must be handled under BSL-3 conditions. The aim of this PhD thesis was to establish accurate and reliable methods to screen and characterize functional mAbs against SARS-CoV-2 and potentially against other viruses, avoiding the use of a BSL-3 laboratory. Two main tools were implemented during my PhD studies: a spike protein recombinant library, and a third generation lentiviral-pseudotyped-based platform. Both of them can be safely handled in BSL-2 laboratories. The protein library allowed the detection of Spike-responsive antibodies to SARS-CoV-2, SARS-CoV-1, MERS, and other common human coronaviruses (HCoV) (OC43, HKU-1, 229E) using binding assay such the ELISA assay. A mutagenesis approach was implemented to expand this library with the generation of new S variants. On the other hand, the pseudotype virus platform identified and characterized antibodies versus SARS-CoV-2 virus and other HCoV, since the entry of these viruses is mainly mediated by the S glycoprotein, and lentiviral particles pseudotyped with the related S protein can mimic the entry step of the virus of interest, and be studied in BSL-2 settings. The numerous research projects to which these tools were applied are discussed in the various chapters of this thesis; in particular, three distinct cohorts of COVID-19 patients with different characteristics were examined. These methods allowed to analyze how, over the course of vaccinations and repeated infections, the affinity of mAbs developed by the subjects changed for the different spike variants of SARS-CoV-2 and other HCoV. Antibodies that neutralized viruses like SARS-CoV-1 and other HCoVs as well as those that showed cross-neutralizing ability against the most recent SARS-CoV-2 variants were characterized using the pseudotype virus platform. Moreover, matrix assays for determining effective antibody combinations to deploy in the fight against SARS-CoV-2 infection have been developed using pseudotyped lentiviral vectors. Overall, the technologies implemented and applied in this research project can be easily applied to any virus of interest where the main antigens are known and lentiviral vector typing is feasible. By using these techniques, even highly pathogenic viruses that are typically handled in BSL3 facilities can be examined in BSL2 laboratories.