Generation and Characterization of a Multi-Functional Panel of Monoclonal Antibodies for SARS-CoV-2 Research and Treatment

The Coronavirus disease 2019 (COVID19) pandemic caused by SARS-CoV-2 is an ongoing threat to global public health. To this end, intense efforts are underway to develop reagents to aid in diagnostics, enhance preventative measures, and provide therapeutics for managing COVID-19. The recent emergence of SARS-CoV-2 Omicron strains with enhanced transmissibility, altered antigenicity, and significant escape of existing monoclonal antibodies and vaccines underlines the importance of the continued development of such agents. The SARS-CoV-2 spike protein and its receptor binding domain (RBD) are critical to viral attachment and host cell entry and are primary targets for antibodies elicited from both vaccination and natural infection. In this thesis research, mice were immunized with two synthetic peptides (Pep 1 and Pep 2) within the RBD of the original Wuhan strain of SARS-CoV-2, as well as the whole RBD as a recombinant protein (rRBD). Hybridomas were generated and a panel of three monoclonal antibodies, mAb CU-P1-1 against Pep 1, mAb CU-P2-20 against Pep 2, and mAb CU-28-24 against rRBD were generated and further characterized. The monoclonal antibodies were shown through ELISAs to be specific for each immunogen/antigen and to be reactive by immunoblotting against RBD. Monoclonal antibody CU-P1-1 has limited applicability other than in ELISA approaches and basic immunoblotting. Monoclonal antibody CU-P220 is very good for ELISAs, immunoblotting, and immunohistochemistry (IHC), but not live virus neutralization. In contrast, mAb CU-28-24 is very effective at live virus neutralization as well as ELISA, immunoblotting, and IHC. Moreover, mAb CU-28-24 was very active against rRBD proteins from Omicron variants B.2 and B.4/B5 as determined by ELISA, suggesting this mAb may neutralize live virus of these variants. Each of the which immunoglobulin genes has allows the expression of res been sequence d using Next Generation Sequencing pective recombinant proteins, thereby eliminating , the need for longterm hybridoma maintenance. These hybridomas and related mAbs are now protected by Intellectual Property agreements with the Clemson University Research Foundation and are complementary P atent P ending based on their unique amino acids within the determining regions (CDRs)