Fab fragments were purified over Protein A resin to remove cleaved Fc fragments and undigested IgG

Fab fragments were purified over Protein A resin to remove cleaved Fc fragments and undigested IgG. for design of pan-sarbecovirus vaccines and antibody therapeutics. Subject terms:Viral infection, X-ray crystallography, SARS-CoV-2 Structural and computational analyses reveal a recurring YYDRxG motif in a class of human antibodies that target a alpha-Boswellic acid conserved epitope on sarbecoviruses including the Omicron variant. == Introduction == The current pandemic of coronavirus disease 2019 (COVID-19) has fomented devasting health, sociological and global economic consequences. Although several effective vaccines have been rapidly developed, SARS-CoV-2, the etiological cause of COVID-19, is still raging throughout the world. Vaccine efficacy has been affected by antigenic drift in SARS-CoV-2 that has led to enhanced infectivity as well as escape from neutralizing antibodies elicited by SARS-CoV-2 infection and vaccination. A majority of the potent neutralizing antibodies target the receptor binding domain (RBD) of the spike protein. However, these antibodies are often susceptible to mutations at the receptor binding site (RBS), which are frequently found in more challenging SARS-CoV-2 variants including Beta, Delta, and Omicron19. Notwithstanding, a subset of broadly neutralizing antibodies (bnAbs) can target highly conserved surfaces on the virus spike protein and neutralize circulating variants of concern (VOCs), variants of interest (VOIs), and other SARS-related viruses in the sarbecovirus family919. Identification and characterization of such cross-neutralizing antibodies are therefore urgently needed to fight the current COVID-19 pandemic, as well as to prepare for future potential zoonotic spillover. Despite their broad spectrum of neutralization activity against SARS-CoV-2, circulating and emerging variants, as well as other related coronaviruses with alpha-Boswellic acid high pandemic risk, potent alpha-Boswellic acid cross-neutralizing antibodies are rarely isolated compared to specific SARS-CoV-2-neutralizing antibodies. These cross-neutralizing antibodies target regions in the spike proteins that are highly conserved across sarbecoviruses, which include the CR3022 site and N343 proteoglycan site in the RBD, as well as the S2 domain2,1018,2027. We and others have reported structures of cross-neutralizing antibodies targeting the CR3022 site that neutralize SARS-CoV-2, many circulating and emerging variants, and some other sarbecoviruses11,13,16,27. However, whether the human immune system is able to develop effective protection against all present and future SARS-CoV-2 variants and other sarbecoviruses has yet to be determined. Here, we identified a recurrent YYDRxG motif in ADI-62113 encoded by humanIGHD3-22in heavy-chain complementarity-determining region 3 (CDR H3) from comparative analysis of the crystal structures of two cross-neutralizing antibodies, ADI-62113 and COVA1-16. A computational search of publicly available sequences with a YYDRxG sequence pattern led to identification of more such antibodies that are able to broadly neutralize SARS-CoV-2 VOCs including Omicron and SARS-CoV pseudoviruses, suggesting a general mechanism available to the Rplp1 human humoral immune system to combat SARS-related sarbecoviruses. Such information is critical for next-generation vaccine design and evaluation, as well as discovery of more effective therapeutic antibodies with increased breadth. Our study further suggests such cross-neutralizing bnAbs can potentially be rapidly identified from their sequence alone. == Results == == Antibody ADI-62113 cross-reacts with a broad spectrum of sarbecoviruses == ADI-62113 is a potent cross-neutralizing antibody isolated from a COVID-19 patient26. Immunoglobulin heavy variable geneIGHV1-3and kappa variable geneIGKV1-33encode its heavy and light chain, respectively, and these germline genes have not been reported in other SARS-CoV-2 cross-neutralizing antibodies to date. To assess antibody breadth, we expressed various sarbecovirus RBDs on the surface of yeast to characterize their binding kinetics with antibody ADI-62113. ADI-62113 binds with high affinity to a broad spectrum of sarbecoviruses including ACE2-utilizing viruses in clade 1 and non-ACE2-utilizing viruses in clade 2 (Fig.1a). Thus, its binding properties are highly favorable as a potential pan-sarbecovirus prophylactic or therapeutic. == Fig. 1. ADI-62113 binds a highly conserved site on SARS-CoV-2 RBD and cross-reacts with many sarbecoviruses. == aADI-62113 shows a broad spectrum of cross-reactivity to sarbecoviruses. RBDs from viruses in clade 1a (SARS-CoV-2-like viruses), clade1b (SARS-CoV-like viruses), clade 2, and clade 3 were displayed on the surface of yeast for binding kinetics analysis. Clade 1a and.