key: cord-0814949-ywnok26a
authors: Sun, Zehua; Kim, Andrew; Sobolewski, Michele D; Enick, Nathan; Chen, Chuan; Adams, Cynthia; Jacobs, Jana L; McCormick, Kevin D; Mellors, John W; Dimitrov, Dimiter S; Li, Wei
title: Neutralization of European, South African, and United States SARS-CoV-2 mutants by a human antibody and antibody domains
date: 2021-03-23
journal: bioRxiv
DOI: 10.1101/2021.03.22.436481
sha: e4f3e2062d10b53cbead27d1df175ac7f4740088
doc_id: 814949
cord_uid: ywnok26a

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) transmission with several emerging variants remain uncontrolled in many countries, indicating the pandemic remains severe. Recent studies showed reduction of neutralization against these emerging SARS-CoV-2 variants by vaccine-elicited antibodies. Among those emerging SARS-CoV-2 variants, a panel of amino acid mutations was characterized including those in the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. In the present study, we evaluated our previously identified antibody and antibody domains for binding to these RBD variants with the emerging mutations, and neutralization of pseudo typed viruses carrying spike proteins with such mutations. Our results showed that one previously identified antibody domain, ab6, can bind 32 out of 35 RBD mutants tested in an ELISA assay. All three antibodies and antibody domains can neutralize pseudo typed B.1.1.7 (UK variant), but only the antibody domain ab6 can neutralize the pseudo typed virus with the triple mutation (K417N, E484K, N501Y). This domain and its improvements have potential for therapy of infections caused by SARS-CoV-2 mutants.

Antibodies targeting the SARS-CoV-2 spike protein receptor-binding domain (RBD) are developed as therapeutic for neutralization. Recently, the prevalent emerging SARS-CoV-2 variants have rapidly generated mutations to escape of neutralization of antibodies, both in non-ACE2 binding residues and RBD residues in the spike protein [1, 2] . The known emerging variants include those that were identified in the United Kingdom (lineage B.1.1.7), South Africa (501Y.V2 also known as B.1.351) and Brazil (P.1) [3] . The reported lineage B.1.1.7 variant contains 8 mutations which are located in S protein (Δ69-70, Y144Del, N501Y, A570D, P681H, T716I, S982A and D1118H), with one mutation N501Y located in RBD. The B.1.351 lineage variant which was rapidly identified as a predominant circulating genotype in South Africa was reported containing 9 mutations in the S protein (L18F, D80A, D215G, L242-244del, R246I, K417N, E484K, N501Y and A701V), three of which (K417N, E484K and N501Y) are in the RBD. The P.1 variant also has K417T, E484K, and N501Y mutations in the RBD. These mutations contribute to an increased affinity of the RBD for human angiotensin-converting enzyme 2 (ACE2), improving viral entry into cells [4] .

The rapid emergence of new variants is of great concern for their increased transmissibility and evasion of neutralizing antibody therapeutics and the humoral response generated by currently approved vaccines including Pfizer BioNtech BNT162b2 lipid-nanoparticle-formulated, and the Moderna mRNA-1273 vaccine, which were both developed based on the earlier viral isolates [3, [5] [6] [7] . Reduced neutralization of SARS-CoV-2 lineage B.1.1.7 and other variants by convalescent and vaccine sera has been reported [8] .

We previously reported a panel of potent neutralizing antibodies and antibody domains against the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. An antibody domain ab6 specifically neutralized SARS-CoV-2 (wild type) with a 100% neutralization at 1.5 µg/ml as measured by two independent in vitro replication-competent virus neutralization assays [9] . Another antibody domain ab8 based high-affinity antibody potently neutralizes mouseadapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg [10] . One full length IgG1 antibody ab1 competes with human ACE2 for binding to RBD and potently neutralizes replication-competent SARS-CoV-2 in a replication-competent mouse ACE2-adapted SARS-CoV-2 in BALB/c mice and native virus in hACE2-expressing transgenic mice [11] . In the present study, we aim to examine the neutralization potency of these previously reported antibodies against the emerging variants containing key spike/RBD mutations by using comprehensive ELISA and pseudo typed virus neutralization assays. We found all of these three antibodies retains effective neutralization against the UK SARS-CoV-2 variants, with similar neutralizing IC50s as against the wild type virus. However, only ab6 neutralized SARS-CoV-2 pseudovirus carrying the SA triple RBD mutation. The results inform the further clinic development of these antibodies against emerging SARS-CoV-2 variants either by making antibody cocktails or by constructing bispecific antibodies.

Ab1, ab6, and ab8 are all antibody or antibody domains that can compete with human ACE2 for binding to the wild type SARS-CoV-2 RBD. In order to examine the binding affinity of these antibody and antibody domains to RBDs bearing different amino acid mutations, we engineered and purchased a panel of RBD variants bearing single amino acid mutation or combination of mutations (N354D/D364Y, and K417N/E484K/N501Y respectively) in each variant respectively, and performed a comprehensive ELISA assay ( Figure 1 ).

Results are showing that antibody VH domain ab6 can bind 30/33 single mutation variants including K417N, E484K and N501Y without any significant reduction of AUC. Worth of noted, the E484K substitution which is known to be present in the B.1.351 and P.1 variants has been reported as an escape mutation for a panel of characterized monoclonal antibodies including REGN10933 and Ly-CoV555 [12] . This E484K substitution also performed an escape for VH-Fc ab8, but not for IgG1 ab1. Consistently, E484Q and E484D substitutions [13] analog to E484K, can also fully or partially escape ab8. K417N, observed occurrence in variant dominantly transmitting in South Africa, is another key mutations in RBD that is reported to escape of neutralization from 33% mAbs tested in an assay, compared to wild typed SARS-CoV-2 (wild type). IgG1 ab1 showed decreased binding to this K417N mutation while ab8 and ab6 retain the binding. RBD variant bearing combination of K417N, E484K and N501Y can be recognized by VH ab6 without any reduction of binding AUC, but can completely block the binding of IgG1 ab1 and VH-fc ab8. These observations indicated that IgG1 ab1 and VH ab8 may completely loss the neutralization of reported B. RBD mutation [14] . Our result showed L452R in RBD does not significantly block the binding of ab1, ab6 or ab8 (percent binding AUC relative to WT all above 75%). A single mutation, E406W [15], allowed for escape from REGN antibodies, also escaped from ab1, ab6 and ab8.

Next we used the S1 and full-length S trimer proteins carrying the whole sets of spike mutations (both RBD and non-RBD mutations) in UK, South Africa (SA) and Brazil variants to evaluate binding of these three antibodies by ELISA (Figure 2 ). SA and Brazil variants can completely deplete the binding of ab1 and ab8, but failed to escape from ab6. BNT162b2 mRNA-elicited antibodies, convalescent sera and mAbs [19] .

N501Y has little effect on neutralization by vaccinee sera as reported [20] , however, 35% RDBspecific mAbs isolated from mRNA-1273 vaccinees showed a 100-fold or even higher loss of neutralization against N501Y mutant [18] . E484K mutant resulted a 3-6 fold reduction in neutralization, and 50% of the RBM specific mAbs lost neutralization against E484K mutant in one assay. K417N is another key mutations in RBD that can escape of neutralization from 33% mAbs tested in an assay. E484K in combination of N501Y and K417N can cause an additional loss of neutralization observed in convalescent sera [21, 22] . Although some of these variants and point mutations cause only partial reduction in neutralization by vaccine sera or currently approved mAbs, there is a risk that the constantly evolving variants could become dominant and evade current therapeutics and vaccines with higher efficiency. Antibody domain ab6 exhibited potency in neutralizing all these pseudo typed variants bearing these mutations. Interestingly, emerging variants including the 501Y.V2 and B.1.1.7 lineages also accumulated mutations and deletions in N-terminal domain (NTD) [23] , and NTD-specific antibodies is comparable less resistant to mutant variants than RBD-specific antibodies. Ninety-six-well plates were coated with the RBD mutants (Sino Biological) at a concentration of 4ug/ml (diluted with 1xPBS) and incubated at 4C overnight (50ul per well). The next day, blocking was conducted with 100ul per well of 3% milk solution made from Bio-Rad nonfat dry milk powder and 1xPBS. The plate was incubated at room temperature for one hour. Primary antibodies were diluted with 3% milk blocking buffer and 1:5 serial dilutions starting at 1nm.

After addition of 50ul of primary antibody dilutions to each well, plates were incubated at room temperature for 2 hours. The plate was subsequently washed with 0.05% Tween 1xPBS (PBST) solution using a plate washer (200ul per well, 4 rounds of washing). The secondary antibody was prepared with the 3% milk at a dilution of 1:1000. The secondary antibody was added (50ul per well) and incubates at room temperature for 1 hour. After 1 hour, the plate was washed as before and 50ul per well of TMB substrate (Sigma) was added. After 1-2 minutes, color development was stopped with 1M H2SO4 (50ul per well) and the plate was read at 450nm, which was plotted against antibody concentration and fitted by the non-linear regression curves fitting ([Agonist] vs.

response---variable slope-four parameters), and the Area Under Curve (AUC) were calculated using GraphPad Prism 9.0.2.

PSV was generated in 293T cells by co-transfection of pFC37K-CMV-S, an enhanced expression plasmid encoding for codon-optimized full-length SARS-CoV-2 S with the N-term HiBit tag removed, and pNL4-3.luc.R-E-mCherry-luciferase, an envelope deficient HIV-1 dual reporter construct that was cloned by recombination of the pNL.luc.R-E-plasmid (NIH AIDS Reagent Program) and the fully infectious pNL4-3 mCherry luciferase plasmid (Addgene) [24, 25] . After harvest, PSV was centrifuged at 500xg for 10 mins and supernatant removed and filtered with a 0.45 um syringe filter to remove producer cells. For neutralization assays 104 293T-hACE2 cells were plated in 100uL media per well in 96 well white-wall white-bottom plates (Perkin Elmer) and incubated overnight at 37°C. Ab6 was serially diluted 5-fold and incubated with 50uL of PSV for 1 hour at 37°C. After incubation, media was removed from wells containing 293T-hACE2 cells and replaced with PSV/mAb and spinoculation was performed at 1,000xg for 1 hour at RT. Plates were then incubated for 48 hours at 37°C. After 48 hours, plates were analyzed for luciferase production by adding 100uL of BriteLite Plus reagent (Perkin Elmer), incubating at RT for 2 minutes, and reading on a Victor Nivo microplate luminometer (Perkin Elmer). 

Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants

SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity

Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and N501Y variants by BNT162b2 vaccine-elicited sera

Coronavirus RNA Proofreading: Molecular Basis and Therapeutic Targeting

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates

Neutralization of SARS-CoV-2 lineage B.1.1.7 pseudovirus by BNT162b2 vaccineelicited human sera

Potent neutralization of SARS-CoV-2 by human antibody heavy-chain variable domains isolated from a large library with a new stable scaffold

High Potency of a Bivalent Human VH Domain in SARS-CoV-2 Animal Models

Rapid identification of a human antibody with high prophylactic and therapeutic efficacy in three animal models of SARS-CoV-2 infection

SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma

Molecular determinants and mechanism for antibody cocktail preventing SARS-CoV-2 escape

Multiple SARS-CoV-2 variants escape neutralization by vaccineinduced humoral immunity

mRNA-1273 vaccine induces neutralizing antibodies against spike mutants from global SARS-CoV-2 variants. bioRxiv

Neutralization of viruses with European, South African, and United States SARS-CoV-2 variant spike proteins by convalescent sera and BNT162b2 mRNA vaccine-elicited antibodies. bioRxiv

Increased Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7 to Antibody Neutralization. bioRxiv

SARS-CoV-2 B.1.1.7 sensitivity to mRNA vaccine-elicited, convalescent and monoclonal antibodies. medRxiv

The N501Y mutation in SARS-CoV-2 spike leads to morbidity in obese and aged mice and is neutralized by convalescent and post-vaccination human sera. medRxiv

Emergence and rapid spread of a new severe acute respiratory syndromerelated coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa. medRxiv

Comprehensive mapping of mutations in the SARS-CoV-2 receptor-binding domain that affect recognition by polyclonal human plasma antibodies

The circulating SARS-CoV-2 spike variant N439K maintains fitness while evading antibody-mediated immunity. bioRxiv

Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity

Distinct modes of human immunodeficiency virus type 1 proviral latency revealed by superinfection of nonproductively infected cell lines with recombinant luciferaseencoding viruses