Eighteen of these antibodies bound to the RBDs of various subsets of SARS-CoV-1, SARS-related bat CoV (RaTG13, RsSHC014, HKU31), and SARS-CoV-2-related pangolin CoV (GXP4L) (Fig

Eighteen of these antibodies bound to the RBDs of various subsets of SARS-CoV-1, SARS-related bat CoV (RaTG13, RsSHC014, HKU31), and SARS-CoV-2-related pangolin CoV (GXP4L) (Fig. spike HDAC-IN-5 lacking proline substitutions encoded by nucleoside-modified mRNA can induce B cell lineages binding to unique RBD sites that either broadly neutralize animal and human Sarbecoviruses or recent Omicron VOCs. == One Sentence Summary: == Non-stabilized SARS-CoV-2 Spike mRNA vaccination activated B cells that target either conserved epitopes on SARS-CoV-2 Omicron variants of concern, or cross-neutralizing epitopes on pre-emergent Sarbecoviruses. == INTRODUCTION == As of November 2023, the COVID-19 pandemic has claimed approximately 7 million lives globally, and the emergence of new SARS-CoV-2 variants continues to be a challenge (1). Neutralizing antibodies (nAbs) from active immunization remain protective immune correlates against SARS-CoV-2 contamination (27). The target for nAbs against SARS-CoV-2 is the spike (S) protein around the virion surface (8,9). The SARS-CoV-2 S is a trimeric type I fusion protein (913) cleaved by proteases into S1 HDAC-IN-5 and S2 domains. The receptor binding domain name (RBD), located within the S1 domain name of spike, engages the viral receptor ACE2 on target cells (14). The S2 domain name mediates fusion with the target cell membrane (1114). The spike trimer has a dynamic structure, where the receptor binding domain name can adopt numerous positions (15). When engaging the host receptor ACE2, the RBD is usually in the up conformation. The RBD stochastically samples the up and down positions (16,17). Hence, the RBD on individual protomers of the trimeric S protein can adopt different positions where all three or only a subset of RBDs can be in the up position (9,10). Over half a billion doses of mRNA encapsulated in lipid nanoparticle (Mrna-LNP) vaccines from HDAC-IN-5 Pfizer and Moderna have been administered in the United States (18). Initially, each of these vaccines encoded the S protein from your ancestral Wuhan Hu-1 or WA-1 isolates that was presumed to be stabilized in the prefusion state by including diproline substitutions K986P and V987P (S-2P) (19). These same substitutions improved neutralizing antibody elicitation of SARS-CoV-1 and MERS-CoV, and thus were thought to function similarly in the context of SARS-CoV-2 (20,21). However, side-by-side biochemical analyses revealed the 2P mutations did not increase expression of trimeric SARS-CoV-2 Spike protein, thermostability, or antigenicity (22). Moreover, cryo-electron microscopy studies showed the overall structures of S with or without the diproline substitutions to be highly comparable with an overall root mean square deviation of 0.54(22). In mice, the 2P substitutions were not required for potent immunogenicity of SARS-CoV-2 spike (23). It should also be noted that this Oxford-AstraZeneca COVID-19 vaccine developers chose to express from an adenoviral vector spike without the 2P substitutions for their vaccine (24,25). Thus, despite the successes of the authorized mRNA-LNP vaccines encoding S-2P, the effects of the 2P substitutions on immunogenicity of SARS-CoV-2 S remains unclear. The relationship between immunogenicity of RBD relative to its conformational dynamics has yet to be fully explored but could inform the design of next generation COVID-19 vaccines (23). Cryo-electron microscopy structures of S protein with or without 2P substitutions showed particles with either 1 RBD up or 3 RBDs up (10,17,21,22,26,27). Previously, we designed a spike with ade novodisulfide bond that prevented the RBD from transitioning to the up state (17). Structural studies of this stabilized version of SARS-CoV-2 S showed all three RBDs were most frequently in the down position (17). While RBD neutralizing antibodies such as DH1041 CD36 (RBD-1 community) or the RBD inner face Ab DH1047 (RBD-6/7 community), which spans across RBD-6 and 7 epitopes, bind to the up conformation of RBD, it is unknown if an S with RBDs usually in the down state would elicit RBD neutralizing antibody responses. Sarbecoviruses circulating in bats that have the ability to infect main airway human cellsin vitroare considered pre-emergent threats for zoonotic transmission (28,29).Sarbecoviruscross-nAb DH1047 protects against bat coronavirus infection (30,31), but is unable to neutralize.