key: cord-0687763-gp3ecpr8
authors: Mannar, Dhiraj; Saville, James W.; Sun, Zehua; Zhu, Xing; Marti, Michelle M.; Srivastava, Shanti S.; Berezuk, Alison M.; Zhou, Steven; Tuttle, Katharine S.; Sobolewski, Michele D.; Kim, Andrew; Treat, Benjamin R.; Da Silva Castanha, Priscila Mayrelle; Jacobs, Jana L.; Barratt-Boyes, Simon M.; Mellors, John W.; Dimitrov, Dimiter S.; Li, Wei; Subramaniam, Sriram
title: Emerging SARS-CoV-2 Variants of Concern: Spike Protein Mutational Analysis and Epitope for Broad Neutralization
date: 2021-12-20
journal: bioRxiv
DOI: 10.1101/2021.12.17.473178
sha: 320cbdfe4e33dc01524aac7fcda53ad69216edd5
doc_id: 687763
cord_uid: gp3ecpr8
Mutations in the spike glycoproteins of SARS-CoV-2 variants of concern have independently been shown to enhance aspects of spike protein fitness. Here, we report the discovery of a novel antibody fragment (VH ab6) that neutralizes all major variants, with a unique mode of binding revealed by cryo-EM studies. Further, we provide a comparative analysis of the mutational effects within variant spikes and identify the structural role of mutations within the NTD and RBD in evading antibody neutralization. Our analysis shows that the highly mutated Gamma N-terminal domain exhibits considerable structural rearrangements, partially explaining its decreased neutralization by convalescent sera. Our results provide mechanistic insights into the structural, functional, and antigenic consequences of SARS-CoV-2 spike mutations and highlight a spike protein vulnerability that may be exploited to achieve broad protection against circulating variants.
Genomic surveillance of SARS-CoV-2 during the first year of the COVID-19 pandemic revealed that the D614G mutation in the spike glycoprotein (S protein) was the sole widespread consensus mutation, with the G614 genotype largely replacing the D614 genotype in February 2020 1, 2 . In November 2020 however, the emergence of the Alpha (B.1.1.7) variant began capturing global headlines and coincided with a surge in COVID-19 cases in the United Kingdom. Within 4 and wild-type (D614G) S proteins bound to V H ab6. Epsilon and wildtype RBD's are coloured light and dark grey respectively, while purple and pink models refer to ab6-WT and ab6-Epsilon, respectively. The R452 mutation is highlighted in red. Pseudoviral neutralization experiments and ELISAs were performed in triplicate, error bars denote the standard error of the mean. PRNT experiments were performed in duplicate, and the mean is plotted.
Having demonstrated the broad neutralization of variant spikes by ab6, we next aimed to provide a comparative analysis of spike mutational effects and antibody breadth using a representative panel of previously reported monoclonal antibodies. We selected RBD directed antibodies [11] [12] [13] [14] which cover the four distinct anti-RBD antibody classes 15 and an ultrapotent antibody, S2M11 16 , which uniquely binds two neighbouring RBDs simultaneously (Figure 2A ). We additionally included the NTD-directed antibodies 4-8 and 4A8 to investigate the impact of NTD mutations within these variant spikes. (Figure 2A ). Antibody binding was quantified via enzyme-linked immunosorbent assay (ELISA), and compared with neutralization, which was measured via a pseudoviral entry assay ( Figure 2B ). S309 and CR3022 are cross-reactive SARS-CoV-1 directed antibodies whose footprints do not span VoC mutations, and accordingly exhibited relatively unchanged binding across all variant spikes. We have previously characterized the mutational sensitivity of ab1, ab8, and S2M11 to spikes bearing only RBD mutations, and the current analysis of antibody evasion using spikes bearing all VoC mutations is consistent with our previous report 4 : 1) The N501Y mutation within the Alpha variant reduces but does not abolish the potency of ab1, while dramatic loss of ab1 activity is seen in Beta and Gamma variants due to mutation of K417 to N or T, respectively; 2) The E484K mutation abrogates ab8 activity in the Beta and Gamma variants; and 3) the L452R mutation reduces but does not abrogate activity of S2M11 in the Epsilon variant spike, drawing similarity to the mutational sensitivity of ab6. To compare the structural basis for the effect of L452R on S2M11 and ab6, we performed cryoEM studies on the Epsilon-S2M11 complex ( Figure S6 ). We obtained a global 3D reconstruction of the Epsilon spike bound to three S2M11 Fabs at 2.16Å. In contrast to the Epsilon-ab6 structure wherein which R452 protrudes into the antibody interface ( Figure 1H ), R452 extends away from the S2M11 interface ( Figure S7 ). As the L452 sidechain is accommodated within the footprint of S2M11, this positioning of R452 is likely due to steric clashing and charge repulsion effects which may increase the interaction energy and underlie the observed attenuation of potency.
Evasion of NTD-directed antibodies was observed in cases when mutations were either within, or adjacent to, antibody footprints ( Figure 2B ), corroborating the recently described remodelling of antigenic loops in the Alpha, Beta, and Epsilon spikes 17, 18 . The W152C substitution within the Epsilon NTD is inside the footprints of 4A8 and 4-8, and both antibodies were escaped by this variant spike. The Beta NTD contains a deletion (Δ242-245) which spans the 4-8 footprint, along with the R261I substitution spanning both 4A8 and 4-8 footprints, leading to escape from both antibodies. The footprint of 4A8 and 4-8 spans a deleted site within the Alpha NTD (Δ144-145) leading to escape. These direct and allosteric mutational effects are consistent with previous findings on NTD rearrangement within these variants and demonstrate their antibody evasive properties.
Having characterized monoclonal antibody evasion by variant spikes, we extended our analysis to include polyclonal antibody escape from human sera. Sera were collected from a spectrum of patients with varying COVID-19 infection histories and vaccination statuses ( Figure S8C ) and subjected to neutralization and binding assays ( Figures S8A-B, S9 ). Potent neutralization of WT spike pseudovirus was observed in all COVID-19 positive or vaccinated samples but not with pre-pandemic sera from uninfected patients, suggesting limited pre-existing immunity ( Figure S8A -B). While serum levels of spike ectodomain binding antibodies correlated poorly with wildtype spike neutralization, strong correlations were observed between NTD and RBD binding antibody levels and neutralization (Fig S10) , corroborating the dominance of neutralizing epitopes within the NTD and RBD 15, 19, 20 . We observed various effects on neutralization escape when sera samples were assayed using variant spike pseudotyped viruses, obtaining statistically significant decreases in neutralization efficacy for both Beta and Gamma variants relative to wild-type ( Figure 2C ). Interestingly, the high correlation between serum NTD and RBD binding antibodies and pseudovirus neutralization for wild-type spikes was markedly reduced for all variant spikes ( Figure 2D ). Taken together, these results highlight the role of mutations within the NTD and RBD of variant spikes in driving evasion of SARS-CoV-2 directed monoclonal and polyclonal antibodies, providing a basis to evaluate the broad mutational tolerance exhibited by S309 and ab6.
In addition to driving antibody escape, variant spike mutations can enhance receptor engagement, which may underlie increases in infectivity. To investigate the ACE2 binding potential of SARS-CoV-2 variant spikes, recombinant S protein ectodomains bearing variant spike mutations were used in biolayer interferometry (BLI) experiments. All mutant spikes exhibited slightly higher affinities for immobilized dimeric Fc-ACE2 when compared to wildtype ( Figure S11A-B) . Additionally, we used flow cytometry to evaluate the ability for recombinant dimeric Fc-ACE2 to bind wild-type or variant full-length spikes which we transiently expressed in Expi-293 cells. We did not observe major differences in spike protein expression across the variants ( Figure S12A) , and all mutant spikes tested demonstrated marginally enhanced ACE-2 binding potencies relative to wild-type ( Figure S11C ). These complementary assays demonstrate that the totality of mutations within each variant S protein enable slightly enhanced ACE2 binding, suggesting a contributing factor for the increased infectivity observed for these SARS-CoV-2 variants.
Having demonstrated mutational effects on antibody evasion and receptor engagement, we next sought to characterize the structural impacts of variant S protein mutations. To this aim, ectodomains bearing variant spike mutations were used for cryoEM structural studies. Global 3D reconstructions were obtained at resolutions ranging from (2.25-2.5 Å) ( Figure S13-16) , yielding open trimers with one RBD in the up conformation and 2 RBD's in the down conformation for all spikes (Figure 3 ). The resolution within the NTD and RBD was insufficient for accurate visualization of mutational impacts within these domains, due to high degrees of conformational heterogeneity. In contrast, we were able to confidently model sidechains close to, and within the S2 domain, owing to its limited flexibility. We therefore first focused our analysis on mutational effects within this region, which predominantly localized to inter-protomer interfaces.
Inspection of the structure of the Alpha variant shows that the A570D and S982A mutations appear to contribute protomer-specific structural effects with implications for RBD positioning ( Figure 4B ). Within both "RBD down" protomers, D570 either occupies a position within hydrogen bonding distance of N960 within the adjacent "RBD down" protomer, or sits within intra-protomer hydrogen bonding distance with T572 when the RBD of the adjacent protomer is in the up conformation ("RBD up"). D570 within the "RBD up" protomer uniquely forms a salt bridge with K854 in the adjacent "down RBD" protomer. S982 sits within hydrogen bonding distance of residues G545 and T547 within adjacent "RBD down" protomers only, and such interactions are not possible with the S982A mutation. Thus, the A570D and S982A mutations likely modulate RBD conformation through 1) stabilizing "RBD up" protomers via addition of the K852-D570 salt bridge, and 2) disruption of "RBD down" protomers via loss of stabilizing interactions afforded by S982.
The D1118H mutation within the Alpha variant enables local side chain rearrangements, giving rise to additional interprotomer contacts, via pi-cation interactions between R1091 and H1118 of adjacent protomers, and electrostatic interactions between R10192 and adjacent E1092 residues ( Figure 4C ). Superposition of wild-type and Alpha spike models clearly demonstrates the differential positioning of H1118 compared to D1118, and the resulting movement of adjacent R1091 towards E1092 ( Figure 4D ). These additional interprotomer contacts enabled by the D1118H mutation may aid in the stabilization of the Alpha spike protein in its trimeric form.
Additional mutations were visualized within the Beta and Gamma variant spike proteins, with implications on inter-protomer and intra-protomer contacts. The A701V mutation in the Beta variant S protein lies at the protein's surface and the larger sidechain conferred by V701 may enable tighter interprotomer packing with Q787 on adjacent protomers ( Figure S17A ). The T1027I mutation lies within the Gamma spike S2 core and the bulky hydrophobic I1027 sidechain faces inwards, increasing the hydrophobicity of the local buried environment ( Figure S17B ). This may decrease the local dielectric constant, enhancing the strength of the nearby network of interprotomer salt bridges between E1031 and R1039 on adjacent protomers ( Figure S17B ) and thus stabilize the Gamma variant trimer.
To identify structural mutational impacts on ACE2 binding we next determined the cryoEM structures of variant spike-ACE2 complexes. Resulting maps were obtained at average resolutions of ~2.6-3Å ( Figures S18-S21 ). Focus-refined structures of the ACE2-RBD interface enabled visualization of RBD mutations, revealing local structures which are identical to our previously reported structures using spikes harbouring variant RBD mutations alone 4 . Superposition of local RBD-ACE2 complex models revealed no significant structural changes ( Figure S22 ). These structural findings confirm that mutations outside of the RBD do not modulate positioning of ACE2-contacting residues at the receptor interface via allosteric mechanisms.
Structure of NTD region in the Alpha, Beta, and Epsilon variants has been previously reported 17, 18 . Here, we report structural analysis of the NTD region in the Gamma variant, stabilized using Fab fragments of the NTD-directed antibodies 4A8 21 and 4-8 22 . The bound antibody fragments improve resolution of this flexible domain, enabling determination of the structure at resolutions of ~ 2.6 Å both for the overall spike and the NTD-antibody interface ( Figures S23-S24 ).
Superposition of 4A8-bound wild-type and Gamma NTDs reveals remodelling of the antigenic supersite N1 loop 23 within the Gamma variant but not the N3 and N5 loops, which comprise the majority of the 4A8 binding site ( Figure 5B ). Analysis of nearby mutational effects provides additional reasoning for conformational remodelling of the N1 loop. The mutations L18F, D138Y, and T20N cluster close together, forming multiple interactions which stabilize the alternate N1 conformation ( Figure 5C ). Namely, F18 and Y138 form an interconnected network of T-shaped pi stacking interactions with each other and the adjacent F140 residue. Additionally, Y138 and N20 sit within hydrogen bonding distance of the main chain carbonyl of F79 and the sidechain of D80, respectively. Comparison of sidechain positioning between wild-type and gamma structures in this region reveals steric clashes between Gamma residue Y138 and wildtype residues F79 and L18, and between Gamma residue N20 and the main chain of wild-type residue L18, resulting in differential positioning of D80 and F79 in the Gamma NTD ( Figure 5D ). Identical positioning of the N1 loop is observed in the Gamma NTD-4-8 structure, further confirming these mutational effects ( Figure S25 ). Thus, the unique interactions conferred by mutations within the Gamma NTD stabilize local conformations which are sterically incompatible with wild-type N1 positioning, causing N1 loop rearrangement. rk ly, he nd pe re er by lly
Mutational enhancement of SARS-CoV-2 viral fitness can arise from effects on receptor engagement and evasion of neutralizing antibodies, with structural origins in the spike glycoprotein. Here we have examined these effects, demonstrating domain-specific differences in the roles and structural mechanisms of S protein mutations. Although such mutational changes can pose threats to natural and vaccine induced immunity, the existence of preserved epitopes within functional domains holds great potential for future antigenic focus. This is highlighted in our analysis of variant SARS-CoV-2 spikes, which despite exhibiting effects on antibody evasion and ACE2 binding, shared a conserved epitope within the RBD which conferred broad neutralization.
The structural impacts of VoC S protein mutations offer insight regarding the differing mutational heterogeneity observed for the NTD and RBD. While VoC mutations within the RBD are limited to only substitutions at a few residues (K417N/T, L452R, T478K, E484K/Q, N501Y), the NTD hosts a large array of deletions and substitutions, predominantly localizing to the three loops constituting the "NTD neutralization supersite" (N1: residues 14-26, N3: residues 141-156, N5: residues 246-260) 23 . Our structures of VoC S proteins in complex with ACE2 demonstrate minimal structural changes in the RBD, reflecting its functional constraints in cell-attachment, only permitting mutations that preserve the ACE2 binding interface. In contrast, our structure of the Gamma NTD confirms the role of mutations within this domain as enabling structural rearrangement of antigenic loops, a feature common to all variant spike NTDs (Alpha, Beta, Delta, Epsilon) 17, 18 . These rearrangements are likely directed primarily by immune evasive pressures. Taken together, these contrasting structural effects between variant NTD and RBD mutations likely arise due to different functional requirements and selective pressures between these domains.
Analysis of mutational effects within the NTD, RBD and S2 domain enables a mechanistic understanding of domain-specific ramifications on individual aspects of S protein functionality. ACE2 and RBD-directed antibody binding analyses by variant spikes corroborated the simultaneous increase in receptor engagement and antibody evasion 4 , consistent with the functional role of the RBD. Significant NTD structural rearrangements as reported here and previously by others, in combination with antibody binding and neutralization assays, highlight antibody escape to be the major driver of NTD mutation. Our structural analysis of mutations within the S2 domain reveals effects on interprotomer contacts, suggesting evolutionary pressures on quaternary structure and stability. We found the A570D and S982A mutations in the Alpha spike protein to work in concert to modify promoter-promoter interactions and allosterically modulate RBD positioning, a finding consistent with recent reports 17, 24, 25 . Additionally, the D1118H and T1027I mutations within the S2 core of the Alpha and Gamma spikes respectively, cause local changes which likely serve to enhance the strength of nearby inter-protomer salt bridges, with implications for spike stability. Therefore, the distinct mutational impacts observed for mutations occurring in the RBD, NTD, or S2 highlight the defined roles that these domains play in overall S protein function and likely reflect domainspecific mutational pressures.
Despite these mutational effects, several lines of evidence have emerged from the present study demonstrating the existence of pan-variant epitopes. The high correlation between RBD + NTD binding antibody levels and viral neutralization potency reflects the dominance of neutralizing epitopes within these domains of the WT spike ( Figure 2D ). The diminished correlation between these parameters when assessing variant spikes demonstrates mutational escape within these domains. However, the fact that neutralization of variant spikes is attenuated, but not abolished, suggests the preservation of neutralizing epitopes. The existence of such epitopes within the RBD is corroborated by the unaltered potency of the SARS-CoV-1 directed antibody S309 across Alpha, Beta, Gamma, and Epsilon spikes ( Figure 2B ). Most importantly, we reveal a novel epitope within the RBD conferring broad neutralization of all variant of concern spikes, including the rapidly spreading Delta variant, as evidenced by the broad neutralization spectrum of ab6. This epitope has largely survived viral evolution thus far, with the only prominent mutational change being L452R. This mutation is accommodated by ab6, albeit yielding decreased neutralization potencies. Therefore, we highlight this epitope for focus in the design of broadly protecting therapeutic antibodies and immunogens.
Several RBD mutation resistant antibodies against SARS-CoV-2 have been reported during the preparation of this manuscript [26] [27] [28] [29] [30] , providing additional context regarding the conserved epitope we report here. Antibodies DH1047 31 and STE90-C11 28 were isolated from convalescent patients, and SARS2-38 26 from immunized mice. All three antibodies are RBD directed and bind epitopes distal to that of ab6 ( Figure 6 ). While STE90-C11 tolerated most circulating RBD mutations, it exhibited loss of activity against the K417T, K417N, and N501Y mutations 28 , which are present in many VOC/VOI spike proteins. In contrast, SARS2-38 and DH1047 bind highly conserved epitopes, retaining potency across all VOC/VOI spikes, with DH1407 exhibiting cross reactivity with additional sarbecoviruses 26, 30 . V H ab6 is distinguished from these previously reported antibodies by its unique angle of approach and binding mode involving multiple V H scaffold -RBD contacts ( Figure 1E ), along with its small (15 kDa) size. Small antibody fragments are attractive therapeutic modalities given their enhanced tissue penetration compared to conventional monoclonal antibodies 32, 33 .
A recent study by a global consortium defined seven RBD binding antibody communities and showed broadly neutralizing antibodies either bind cryptic epitopes within the inner RBD face (communities RBD-6, RBD-7), or are non-ACE2 competing antibodies which bind the outer RBD face (community RBD-5) 29 . Ab6 binds the inner RBD face and contacts the RBM, enabling ACE2 competition, drawing similarity to the RBD-4 antibody community, which interestingly was not shown to contain any broadly neutralizing antibodies. Structural comparison of the ab6 footprint with a representative RBD-4 antibody (C002) 15 reveals an overlapping footprint shared by the C002 heavy chain and ab6 despite differences in binding modes ( Figure 6 ). C002 is derived from a convalescent patient, suggesting the potential for such an epitope to be recognized by natural antibodies. This evidence further supports the potential value of focus on the ab6 binding epitope for future therapeutic design. r e s i d u e s 1 -6 1 5 ) w i t h a C t e r m i n a l 7 x h i s t a g w a s a m p l i f i e d f r o m " h A C E 2 " , a k i n d g i f t f r o m H y e r y u n C h o e ( A d d g e n e p l a s m i d # 1 7 8 6 ) a n d c l o n e d i n t o p c D N A 3 . 1 v i a B s t X I a n d X b a I r e s t r i c t i o n e n z y m e c l o n i n g . S u c c e s s f u l c l o n i n g w a s c o n f i r m e d b y S a n g e r s e q u e n c i n g ( G e n e w i z , I n o l y s c i e n c e s C a t # 2 3 9 6 6 -1 ) . 2 4 -h o u r s f o l l o w i n g t r a n s f e c t i o n , m e d i a w a s s u p p l e m e n t e d w i t h 2 . 2 m M v a l p r o i c a c i d , a n d e x p r e s s i o n w a s c a r r i e d o u t f o r 3 -5 d a y s a t 3 7 ° C , 8 % C O 2 . T h e s u p e r n a t a n t w a s h a r v e s t e d b y c e n t r i f u g a t i o n a n d f i l t e r e d t h r o u g h a 0 . 2 2 μ M f i l t e r p r i o r t o l o a d i n g o n t o a 5 m L H i s T r a p e x c e l c o l u m n ( C y t i v a ) . T h e c o l u m n w a s w a s h e d f o r 2 0 C V s w i t h w a s h b u f f e r ( 2 0 m M T r i s p H 8 . R u p s a m p l i n g f a c t o r 1 , E E R n u m b e r o f f r a c t i o n s 4 0 ) , p a t c h m o d e C T F e s t i m a t i o n , r e f e r e n c e f r e e p a r t i c l e p i c k i n g , a n d p a r t i c l e e x t r a c t i o n w e r e c a r r i e d o u t o n -t h e -f l y i n c r y o S P A R C l i v e . A f t e r p r e p r o c e s s i n g , p a r t i c l e s w e r e s u b j e c t e d t o 2 D c l a s s i f i c a t i o n a n d / o r 3 D h e t e r o g e n e o u s c l a s s i f i c a t i o n . T h e f i n a l 3 D r e f i n e m e n t w a s p e r f o r m e d w i t h e s t i m a t i o n o f p e r p a r t i c l e C T F a n d c o r r e c t i o n f o r h i g h -o r d e r a b e r r a t i o 1 h o u r a n d a d d e d t o t h e V e r o E 6 c e l l s e e d e d m o n o l a y e r s , i n d u p l i c a t e . P l a t e s w e r e t h e n i n c u b a t e d f o r 1 h o u r a t 3 7 ° C i n a 5 % C O 2 i n c u b a t o r . F o l l o w i n g i n c u b a t i o n , a n o v e r l a y m e d i a w i t h 1 % a g a r o s e -c o n t a i n i n g m e d i a ( 2 x M i n i m a l E s s e n t i a l M e d i u m , 7 . 5 % b o v i n e a l b u m i n s e r u m , 1 0 m M H E P E S , 1 0 0 µ g / m L p e n i c i l l i n G a n d 1 0 0 U / m L s t r e p t o m y c i n ) w a s a d d e d t o t h e m o n o l a y e r s . T h e p l a t e s w e r e i n c u b a t e d f o r 4 8 -7
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