(2020). potent and displayed glycan-dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD-specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants. Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19. SARS-CoV-2 belongs to the genus of the family, alongside severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) (Lu et al., 2020). The positive sense RNA genome encodes four structural proteins; Spike (S), envelope (E), membrane (M) and nucleocapsid (N) (Jiang et al., 2020). The S glycoprotein is responsible for interaction with the human angiotensin-converting enzyme 2 (ACE2) receptor and subsequent virus-cell membrane fusion and thus is the key target for neutralizing antibodies (Pinto et al., 2020). The Spike glycoprotein assembles into homotrimers on the viral membrane, with each TSU-68 (Orantinib, SU6668) Spike monomer encompassing two functional subunits, S1 and S2. The S1 subunit contains the N-terminal domain (NTD) and the receptor binding domain (RBD). The RBD encompasses the receptor binding motif (RBM) that directly contacts the ACE2 receptor. The S2 subunit, containing the fusion peptide, two heptad repeats (HR1 and HR2), the cytoplasmic tail and the transmembrane domain, is crucial for viral membrane fusion (Yao et al., 2020). Despite the recent emergence of SARS-CoV-2 in the human population, a rapid understanding of the antibody response arising from infection has emerged (Beaudoin-Bussieres et al., 2020; Crawford et al., 2020; Dan et al., 2021; Muecksch et al., 2020; Okba et al., 2020; Pickering et al., 2020; Prevost et al., 2020; Seow et al., 2020). The majority of SARS-CoV-2 infected individuals have been shown to generate an antibody response 5C15 days post onset of symptoms (POS) that peaks after ~3C4 weeks and then starts to decline (Crawford et al., 2020; Muecksch et al., 2020; Prevost et al., 2020; Seow et al., 2020). The magnitude of the neutralizing antibody response, which is thought to be important for protection from re-infection and/or disease, has been associated with disease severity. Specifically, those with most severe disease typically develop the strongest antibody response whereas those experiencing mild disease, or who are asymptomatic, can have lower levels of neutralizing activity detectable in their sera (Guthmiller et al., 2021; Laing et al., 2020b; Legros et al., 2021; Rees-Spear et al., 2021; Seow et al., 2020; Zohar et al., 2020). Antibodies targeting RBD have been suggested to account for >90% of neutralizing activity in convalescent sera (Greaney et al., 2020; Piccoli et al., 2020) and several neutralizing epitopes on RBD that are targeted by highly potent monoclonal antibodies have been molecularly characterized (Barnes et al., 2020; Brouwer et al., 2020; Piccoli et al., 2020; Pinto et Rabbit Polyclonal to OR6P1 al., 2020; Rogers et al., 2020; Tortorici et al., 2020; Wang et al., 2020; Wu et al., 2020). Reports TSU-68 (Orantinib, SU6668) suggest escape from RBD-mediated neutralization is occurring in TSU-68 (Orantinib, SU6668) variant strains that are emerging globally, which include mutations within the RBD that have been postulated to enable escape (Muecksch et al., 2020; Starr et al., 2021; Wimber et al., 2021). This highlights the need to identify neutralizing antibodies that bind epitopes outside RBD and to understand the role these antibodies play in protection from re-infection or following vaccination. Identification of neutralizing epitopes beyond RBD is therefore important for the development of synergistic antibody cocktails for immunotherapy and passive vaccination, and will also be critical for evaluating the relevance of potential immune escape viral variants as they arise, for example the recently identified B.1.1.7 (Rambaut et al., 2020). We therefore sought to isolate SARS-CoV-2 neutralizing antibodies from three donors experiencing severe, mild or asymptomatic COVID-19 using an un-cleaved, pre-fusion stabilized trimeric Spike glycoprotein as antigenbait to further characterize the neutralizing epitopes present on SARS-CoV-2 Spike. Here, we isolated 107 mAbs across three donors of which 47 (43.9%) showed neutralizing activity. The majority (72.3%, 34/47) of the neutralizing antibodies targeted the RBD and formed four distinct competition groups. 21.3% (10/47) of neutralizing antibodies targeted the NTD and formed two separate groups. One NTD group contained potent neutralizing antibodies able to neutralize infectious virus at a similar potency to RBD-targeted neutralizing antibodies. The second NTD group, although less potent, showed glycan-dependant neutralization activity. NTD-specific neutralizing antibodies (nAbs) showed a dramatic decrease in neutralization.