Discovery and Optimization of Broadly Protective Merbecovirus Neutralizing Antibodies

Abstract Middle East respiratory syndrome coronavirus (MERS-CoV) infection has the highest mortality rate (36%) of any of the known human-pathogenic Betacoronaviruses. MERS-CoV continues to circulate in the Middle East, and due to global travel has spread to 27 other countries making it a global health priority. A number of viruses related to MERS-CoV have been identified in mammals worldwide, and several have been found to utilize human DPP4- or ACE2-receptors for cell entry. Therefore, the MERS-related betacoronaviruses (Merbecoviruses, MERBs) have considerable zoonotic potential. While many medical countermeasures like vaccines and antibody therapeutics were developed to fight SARS-CoV-2, these countermeasures do not prevent disease caused by MERBs. The long-term goal of this proposal is to generate such MERB broadly neutralizing antibody (bnAb) therapeutics. The significance of this project includes the identification of broadly protective antibodies (Abs) and their epitopes, enabling rational design of antibody-based therapeutics and vaccines against a deadly virus. While previous research has focused on identifying potent neutralizing antibodies against MERS-CoV, no antibodies have been shown to be cross- protective against MERS-CoV and other MERBs. In preliminary studies in vaccinated rhesus macaques (RMs), we elicited robust cross-binding and cross-neutralizing plasma Abs against multiple MERBs. Post- vaccination RM B cells bound both MERS-CoV receptor binding domain (RBD) and bat MERS-CoV-related virus NL140422 RBD. Individual Abs from these RMs bound to as many as 7 different MERS-CoV-related RBDs and MERS-CoV. The objectives of this study are to 1) determine the neutralization breadth of monoclonal nAbs from these vaccinated RMs, 2) determine the critical features of the binding interface between cross-reactive nAb and virus spike RBD, and 3) determine the cross-protective efficacy of the mAbs. The innovations of this project include human, bat, and pangolin live-virus MERB models (MERS-CoV, PDF2180, NL140422 (MERS 422), MjHKU4r, HKU-5), 13 genetically diverse MERB spike and RBD antigens, MERS-CoV and MjHKU4 mouse challenge models, and the use of a machine-learning Ab optimization to improve natural Abs. The impact of this project includes the demonstration that vaccination can elicit broad MERB nAbs against multiple conserved epitopes, the development of trispecific immunotherapies, and the definition of bnAb epitopes that inform rational design of immunogens targeting cross-protective B cells. Our Abs will be potential tools to combat MERS-CoV outbreaks and prepare for future outbreaks from pre- emergent MERBs.