Using recombinant RBD proteins carrying epitope-specific mutations, we identified neutralizing antibodies binding to diverse epitope classes.

Here we outline a strategy based on the sorting of convalescent patient B cells in an epitope-specific manner, in combination with structural characterization by X-ray crystallography, cryo-electron microscopy (cryo-EM), live virus neutralization and animal models. In addition, antibody cocktails, targeting multiple non-overlapping epitopes on the surface of the RBD have been developed to overcome resistance 20, 21. To overcome such resistance, research has focused on the development of broadly neutralizing antibodies, such as Sotrovimab (S309), which targets the class 3 epitope, which is conserved in sarbecoviruses 19. However, increased resistance to antibody neutralization is being observed for SARS-CoV-2 VOCs, especially for variants carrying mutations within the RBM region (including Beta and Omicron variants) 8, 9, 10. In addition to vaccination, the use of recombinant antibodies has proven successful for therapy and prevention of COVID-19 and several monoclonal antibodies have obtained regulatory approval, and have shown particular promise in immunocompromised individuals and the elderly 16, 17, 18. However, it is also evident that such antibodies are rare in most vaccinated individuals and convalescent patients 5, 14. Such antibodies generally bind to regions conserved among sarbecoviruses (class 3, 4 and 5 epitopes) and are expected to be more resilient to VOCs. More recently, broadly neutralizing antibodies have been identified that bind outside the RBM region 11, 12, 13, 14, 15. Such RBD- and RBM-targeted antibodies are also generated upon vaccination 7, but often lack neutralization potential against emerging variants of concern (VOCs) 8, 9, 10. Most of the neutralizing antibodies bind the spike receptor binding domain (RBD), and in particular class 1 and 2 epitopes within the receptor binding motif (RBM), directly blocking interaction with the human angiotensin converting enzyme receptor 2 (ACE2) 3, 4, 5, 6. Upon SARS-CoV-2 infection, the human adaptive immune response generates antibodies against the viral spike surface glycoprotein 2. The coronavirus SARS-CoV-2, the causative agent of the global COVID-19 pandemic, has resulted in the death of over 6 million people worldwide 1. Our results provide guidance for next generation monoclonal antibody development and vaccine design. Our results indicate that exposure to SARS-CoV-2 induces antibodies that maintain broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14) or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). They also provide prophylactic and therapeutic in vivo protection of female hACE2 mice against viral challenge. Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (S309) by orders of magnitude. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and 5). Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells of convalescent patients using SARS-CoV-2 receptor binding domains carrying epitope-specific mutations. Nature Communications volume 14, Article number: 687 ( 2023)Įmerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Broadly neutralizing SARS-CoV-2 antibodies through epitope-based selection from convalescent patients

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