key: cord-0308448-doa19va7 authors: Wu, Xilin; Wang, Yaxing; Cheng, Lin; Zhu, Linjing; Ma, Sen; Huang, Bilian; Xu, Shijie; Shi, Haixia; Zhang, Doudou; Liu, Linshuo; Nawaz, Waqas; Ye, Sheng; Wu, Zhiwei title: Increased neutralization of SARS-CoV-2 Delta variant by nanobody (Nb22) and the structural basis date: 2021-09-07 journal: bioRxiv DOI: 10.1101/2021.09.06.459055 sha: 475d3c7017f6c28a863b4977cfba4c21b259e0f8 doc_id: 308448 cord_uid: doa19va7 Delta variant, also known as B.1.617.2, has become a predominant circulating variant in many countries since it first emerged in India in December 2020. Delta variant is less sensitive to serum neutralization from COVID-19 convalescent individuals or vaccine recipients, relative to Alpha strains. It was also resistant to neutralization by some anti-receptor binding domain (RBD) and anti-N-terminal domain (NTD) antibodies in clinics. Previously, we reported the discovery of nanobodies isolated from an alpaca immunized with spike protein, exhibiting ultrahigh potency against SARS-CoV-2 and its mutated variants, where a novel inhalable bispecific Nb15 protected SARS-CoV-2 infection in hACE2 mice. Here, we found that Nb22-Fc, among our previously reported nanobodies, exhibited 8.4-fold increased neutralization potency against Delta variant with an IC50 value of 0.41 ng/ml (5.13 pM) relative to Alpha variant. Furthermore, our crystal structural analysis reveals that the binding of Nb22 on SARS-CoV-2 RBD effectively blocks the binding of RBD to ACE2 during virus infection. Furthermore, the L452R mutation in RBD of Delta variant forms an additional hydrogen bond with the hydroxy group of T30 of Nb22, leading to the increased neutralization potency of Nb22 against Delta variant. Thus, Nb22 is a potential therapeutic agent against SARS-CoV-2, especially the highly contagious Delta variant. 66 SARS-CoV-2 has given rise to the COVID-19 pandemic [1] , resulting in massive 67 disruption of social and economic activities. Global vaccination has since provided 68 protection against the catastrophic outcome of the pandemic. However, a new wave of 69 infection, mainly caused by Delta variant, is spreading rapidly worldwide [2] [3] [4] [5] 172 We measured antibody affinity using a ForteBio OctetRED 96 BLI (Molecular Devices 173 ForteBio LLC, Fremont, CA) with shaking at 1,000 rpm at 25 o C [25] . To determine 174 the affinity of nanobodies, RBD protein was coupled to AR2G biosensor (cat.# 18-5092, 175 Fortebio) using BLI instrument following the instruction of the amino coupling kit. 176 Association of Nb-Fcs in a serial dilution was carried out before dissociation for 180 177 sec. After each cycle, the biosensors were regenerated through 3 brief pulses of 5 sec 178 each with 100 mM pH 2.7 glycine-HCL followed by running buffer. The data were 179 baseline subtracted before fitting using a 1:1 binding model and the ForteBio data To collect data, a single crystal was mounted on a nylon loop and was flash-cooled with 220 a nitrogen gas stream at 100 K. neutralize other variants containing E484K/Q mutation (Fig. 1A-F) . Surprisingly, variants containing E484K/Q mutation (Fig. 2E) . These results were substantiated by 286 FACS results (Fig. 2F) . Overall, these specific binding characteristics are consistent 287 with its specific neutralization properties. Table S1 ). Nb22 adopts a 293 typical β-barreled structure, and contains three variable complementarity-determining 294 regions (CDR) binding to RBD. The buried surface area (BSA) was 800 Å 2 , and mainly 295 constituted of hydrogen bonds and hydrophobic interactions. 14 residues constituting 296 epitope of three CDRs were identified using a distance of <4 Å as the cutoff (Fig. 3B) . Table S2 ). The T478 is located outside the CDR binding regions 321 (Fig. 5A) , and does not disturb the hydrogen bonding and hydrophobic interactions. 322 Therefore, T478K mutation has no effect on Nb22 neutralization ( Fig. 5B and 5C ). Previously, we reported a bi-specific trimer configuration Nb15-NbH-Nb15 based on 362 Nb15 nanobodies, which contains Nb15 specific for RBD and NbH specific for human 363 serum albumin protein in blood [25] . Nb15-NbH-Nb15 exhibited increased neutralization 364 in vitro and half-life in vivo. Importantly, intranasal delivery of Nb15-NbH-Nb15 resulted 365 in the prevention and therapy of SARS-CoV-2 infection in hACE2 mice [25] . As such, 366 we presume that Nb22 could also be engineered as Nb22-NbH-Nb22 like Nb15 with both A pneumonia outbreak associated with a new coronavirus of probable bat origin Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent 547 serum Multiple SARS-CoV-2 variants escape neutralization by vaccine-549 induced humoral immunity Increased mortality in community-tested cases of SARS-CoV-2 lineage 551 B.1.1.7. 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The X-ray data was collected