A Biophysical and Structural Assessment of the Spillover Potential of Bat-Borne Sarbecoviruses

Abstract

The propensity of coronaviruses to spillover from animal reservoirs into human populations has proven to have a startling impact on society in recent years. During the first two outbreaks, caused by severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002-2003 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012-present, death tolls hovered around 800 each (World Health Organization, 2004, 2019). During the recent emergence of SARS-CoV-2 in 2019, previous death tolls were shattered as SARS-CoV-2 tore across the globe; the death toll to date stands in the millions. These viruses all began in their native reservoirs, bats, some spread to intermediate hosts palm civets (SARS-CoV-1) and dromedary camels (MERS-CoV), but all started in bats and eventually made their way into humans. They all like all other coronaviruses utilize their spike glycoproteins to mediate viral attachment and fusion to host cells. During each of these spillover events, we were unprepared, we lacked vaccines or therapeutics, and we knew very little about what existed in animal reservoirs. Today, the situation is very much changed, vaccines and therapeutics have been developed against SARS-CoV-2, but there is still an urgent need to understand what these viruses are doing in their hosts, to identify the barriers to spillover, and characterize and stockpile vaccines and therapeutics for future spillover preparedness.In the following dissertation, I detail the deep ancestral origins of ACE2 receptor usage in sarbecoviruses and describe the evolvability and plasticity of ACE2 binding in sarbecoviruses. I also highlight sarbecoviruses currently unable to bind and utilize human ACE2 for cellular entry but with the capacity to gain human ACE2 binding and cellular entry within 1-2 amino acid mutations. I then describe a Cambodian bat sarbecovirus, closely related to SARS-CoV-2, with the capacity to bind human ACE2 but unable to facilitate cellular entry and explore the role of spike conformational dynamics on the spillover potential of this virus. I further describe a bat-borne sarbecovirus that is much more distally related to SARS-CoV-2, PRD-0038, originating from African and explore its receptor usage, antigenicity, and immunogenicity; demonstrating that vaccination with this spike glycoprotein results in broader vaccine mismatch protection, and motivating the inclusion of clade 3 antigens in next-generation vaccines for enhanced resilience to viral evolution. I detail the discovery of ACE2 utilization in HKU5 and describe molecular basis for multiple independent evolution of ACE2 across coronaviruses. I also detail our effort towards pandemic response and zoonotic spillover preparedness; describing the discovery and characterization of several broadly neutralizing antibodies, the elicitation of broad neutralizing antibodies with multivalent RBD-nanoparticle vaccines and describe the impact of mutation of SARS-CoV-2 Omicron variants on receptor usage, fusogenicity, and immune/therapeutic evasion. Finally, I detail the development of a ligand fitting software EMERALD, and its usage in fitting linoleic acid in spike glycoproteins and other protein datasets. Collectively, my work informs on barriers to spillover, the molecular mechanisms of coronavirus receptor usage, the evolution of sarbecoviruses in their natural reservoirs, and on the development of the next generation of vaccines and therapeutics.