Fantastic B-Cells and Where to Find Them

Abstract

Since the SARS-CoV-2 pandemic began in 2020, viral sequencing has documented the emergence of hundreds of individual mutations in the viral spike protein across numerous variants. With the drastic viral evolution over the past four years resulting in variants with greater than 60 mutations relative to the Wuhan strain the vaccine has been updated three times. Studies have demonstrated that repeated mRNA vaccination enhances the breadth of neutralization against diverse SARS-CoV-2 variants. However, the development of antibodies and humoral immune responses capable of neutralizing across the coronavirus family is poorly understood. The ongoing spread of COVID-19 underscores the importance of identifying and characterizing neutralizing monoclonal antibodies capable of neutralizing all variants. The fusion peptide (FP) region of the viral spike protein is a crucial target for neutralization given its broad cross reactivity and conservation among all the coronaviruses. Here, we determine the ability of vaccine-mediated humoral immunity to keep pace with continued SARS-CoV-2 evolution. Updated vaccines increase neutralization breadth, but viral evolution continues to outpace them. Using newly developed depletion protocols we show that broadly neutralizing responses are often a result of receptor binding domain (RBD) specific antibodies, but some donors produce FP-specific responses that contribute to neutralization breadth. In a comprehensive ELISA study, we find that FP-specific antibodies arise only in response to natural infection and not vaccination. Next, we developed a novel protocol to isolate antigen-specific memory B-cells and monoclonal antibodies (mAbs) in less than 1 week. Using this highly efficient and effective protocol, we isolated 11 FP-specific antibodies with varying neutralization breadths. Finally, we designed a set of pseudoviral spike proteins to assess epitope and escape mutations for FP-specific antibodies. We found that mutations at S816 and F823 escape all FP-specific antibodies. Collectively, this work helps elucidate the co-evolution of the humoral immune response and the SARS-CoV-2 virus, suggesting that novel vaccine strategies are needed to end the spread of the virus and prevent further viral evolution. In this thesis we also present a roadmap for the selection of ideal donors for mAb isolation and offer a highly efficacious protocol for generation of mAbs from donor PBMCs.