key: cord-0332644-k2zng3fx
authors: Neerukonda, Sabari Nath; Vassell, Russell; Lusvarghi, Sabrina; Wang, Richard; Echegaray, Fernando; Bentley, Lisa; Eakin, Ann E.; Erlandson, Karl J.; Katzelnick, Leah C.; Weiss, Carol D.; Wang, Wei
title: SARS-COV-2 Delta variant displays moderate resistance to neutralizing antibodies and spike protein properties of higher soluble ACE2 sensitivity, enhanced cleavage and fusogenic activity
date: 2021-11-08
journal: bioRxiv
DOI: 10.1101/2021.11.05.467523
sha: cc5f03867389e761d6a0194db2af04b0420176a3
doc_id: 332644
cord_uid: k2zng3fx

The SARS-CoV-2 B.1.617 lineage variants, Kappa (B.1.617.1) and Delta (B.1.617.2, AY) emerged during the second wave of infections in India, but the Delta variants have become dominant worldwide and continue to evolve. The spike proteins of B.1.617.1, B.1.617.2, and AY.1 variants have several substitutions in the receptor binding domain (RBD), including L452R+E484Q, L452R+T478K, and K417N+L452R+T478K, respectively, that could potentially reduce effectiveness of therapeutic antibodies and current vaccines. Here we compared B.1.617 variants, and their single and double RBD substitutions for resistance to neutralization by convalescent sera, mRNA vaccine-elicited sera, and therapeutic neutralizing antibodies using a pseudovirus neutralization assay. Pseudoviruses with the B.1.617.1, B.1.617.2, and AY.1 spike showed a modest 1.5 to 4.4-fold reduction in neutralization titer by convalescent sera and vaccine-elicited sera. In comparison, similar modest reductions were also observed for pseudoviruses with C.37, P.1, R.1, and B.1.526 spikes, but seven- and sixteen-fold reduction for vaccine-elicited and convalescent sera, respectively, was seen for pseudoviruses with the B.1.351 spike. Four of twenty-three therapeutic neutralizing antibodies showed either complete or partial loss of neutralization against B.1.617.2 pseudoviruses due to the L452R substitution, whereas six of twenty-three therapeutic neutralizing antibodies showed either complete or partial loss of neutralization against B.1.617.1 pseudoviruses due to either the E484Q or L452R substitution. Against AY.1 pseudoviruses, the L452R and K417N substitutions accounted for the loss of neutralization by four antibodies and one antibody, respectively, whereas one antibody lost potency that could not be fully accounted for by a single RBD substitution. The modest resistance of B.1.617 variants to vaccine-elicited sera suggest that current mRNA-based vaccines will likely remain effective in protecting against B.1.617 variants, but the therapeutic antibodies need to be carefully selected based on their resistance profiles. Finally, the spike proteins of B.1.617 variants are more efficiently cleaved due to the P681R substitution, and the spike of Delta variants exhibited greater sensitivity to soluble ACE2 neutralization, as well as fusogenic activity, which may contribute to enhanced spread of Delta variants.

For ACE2 neutralization assay, serially diluted recombinant human soluble ACE2 was incubated with indicated pseudovirus (~1x10 6 RLU/ml) for one hour at 37°C and 100 μl of pseudovirus and soluble ACE2 mixture was added to 293T-ACE2.TMPRSS2 cells. Luciferase activity was measured 48 hr later. The soluble ACE2 concentration causing a 50% reduction of RLU compared to pseudovirus control were reported as the 50% inhibitory concentration or IC50. 2.6. Western Blotting 1.25 ml of pseudoviruses were pelleted at 4°C for 2 hr at 15000 rpm using Tomy TX-160 ultracentrifuge. Pseudovirus pellet was resuspended in 1X Laemmli loading buffer and heated at 70°C for 10 minutes. Samples were resolved by 4-20% SDS PAGE and transferred onto nitrocellulose membranes. Membranes were probed for SARS-CoV-2 S1 using rabbit polyclonal antibodies against the RBD domain (Sino Biological; Cat: 40592-T62).

For measuring spike protein-mediated cell-cell fusion, β-Gal complementation assay was performed as described previously [32] . Briefly, β-Gal ω subunit-expressing 293T cells were transfected with 1µg spike plasmids, whereas 293T-ACE2.TMPRSS2 cells were transfected with β-Gal α subunit using the Fugene 6 reagent. At 24 hr post transfection, the transfected cells were detached using a nonenzymatic cell dissociation solution (Sigma) and washed with DMEM. Spike-transfected/β-Gal ω subunit-expressing 293T cells were mixed with β-Gal α subunit-transfected/293T-ACE2.TMPRSS2 cells at 1:1 ratio to a total of 6 x 10 4 cells per well on a 96-well plate. The cells were co-cultivated for 24 hr at 37°C. The culture supernatants were then removed, and cell-cell fusion was scored by determination of the β-Gal activity in cocultured cell lysates using a Galacto-Star kit (Applied Biosystems) according to the manufacturer's instructions.

To ensure equivalent amount of spike cell surface expression levels among treatments, spike-transfected β-Gal ω subunit-expressing 293T cells were quantified for cell surface spike levels by flow cytometry. Spike-transfected 293T cells employed in cell-cell fusion assay were concurrently stained with SARS-CoV-2 positive human polyclonal sera at 1:20 dilution, washed twice and then incubated with FITC-conjugated goat anti-human (KPL Inc., Gaithersburg, MD). The cells were washed twice and then fixed with 2% paraformaldehyde. The results were acquired using BD LSRFortessa™ X-20 Cell Analyzer (BD biosciences). The mean fluorescence intensities of spike positive cells were recorded.

We created a geometric interpretation of neutralization titers against the tested SARS-CoV-2 pseudoviruses using Racmacs antigenic cartography software (https://github.com/acorg/Racmacs) [33, 34] . The map is presented on a grid in which each square indicates one antigenic unit, corresponding to a two-fold dilution of the antibody in the neutralization assay. Antigenic distance is measured in any direction on the map.

The prediction of furin-specific cleavage site in spike proteins were computed using the ProP 1.0 Server hosted at http://www.cbs.dtu.dk/services/ProP/ and the PiTou V3 software hosted at http://www.nuolan.net/reference.html.

One-way analysis of variance (ANOVA) with Dunnett's multiple comparisons tests (variants compared to WT(D614G)), Mann-Whitney test for the comparison of two groups with unmatched pairs (Pfizer BNT162b2 compared to Moderna) and geometric mean titers (GMT) with 95% confidence intervals were performed using GraphPad Prism software. The p values of less than 0.05 were considered statistically significant. All neutralization titers were log2 transformed for analyses.

We first investigated the cross-neutralization potency of convalescent sera from individuals infected with SARS-COV-2 in the U.S. against pseudoviruses bearing spikes of B.1.617.1 and B.1.617.2 variants and their corresponding RBD mutations ( Figure 1A ). Titers against B.1.617.1 (B), AY.1, E484Q and L452R+T478K pseudoviruses were significantly different from the titers against WT(D614G). Compared to titers against WT(D614G) pseudoviruses (GMT 392), titers against B.1.617.1 (B) pseudoviruses were approximately fourfold lower (GMT 90), confirming and extending other reports [20] . Neutralization titers against WT(D614G) and L452R pseudoviruses were comparable (GMT titers 392 and 364, respectively), while neutralization titers against E484Q pseudoviruses were lower (GMT 165). Titers against B.1.617.2 (GMT 259) and AY.1 (GMT 203) pseudoviruses also showed a slight 1.5-and 1.9-fold reduction, respectively, compared to WT(D614G) pseudoviruses. Against pseudoviruses bearing spikes with T478K substitution in RBD, neutralization titers (GMT 270) were also slightly reduced compared to WT (D614G) (GMT 392). A further reduction in neutralization titers was seen against pseudoviruses bearing both L452R and T478 substitutions in RBD displayed (GMT 192) compared to WT(D614G) (GMT 392).

The C.37 variant also has a substitution at L452 residue (L452Q instead of L452R) along with F490S in the RBD. A 1.8-fold reduction in titers against C.37 pseudoviruses was observed compared WT(D614G) pseudoviruses (GMT titers 222 and 392, respectively). A similar 1.4-fold reduction in titers was observed for pseudoviruses with only the L452Q and F490S substitutions, indicating that these RBD substitutions may contribute to C.37 resistance. In comparison with other variants, we found that titers against B.1.429 pseudoviruses (GMT 332) were comparable to WT(D614G), but titers against the B.1.351 pseudovirus were 16.3fold reduced (GMT 24) compared to WT(D614G), consistent with prior reports showing marked reductions of cross-neutralization against this variant [35] [36] [37] .

Because the convalescent sera came from individuals that previously were infected by different variants (Table 2) , we also explored differences in the neutralization titers between those infected by D614G variants lacking L452R and those infected by variants containing L452R (all have L452R and D614G, except one lacking D614G). The small number of samples in each group precludes conclusions, but we noticed modest differences against some variants. In the group infected by D614G variants that lacked L452R, a 5.2-fold, 1.6fold, and 2.5-fold reduction in cross-neutralization potency was seen against B. 

We next assessed the neutralization potency of mRNA vaccine-elicited sera against WT(D614G) and B.1.617 variant pseudoviruses. Sera from fourteen individuals who received two doses of Moderna mRNA-1273 vaccine and fifteen individuals who received two doses of Pfizer/BioNtech BNT162b2 vaccine were collected approximately two weeks after the second immunization. Each vaccine-elicited serum had high neutralization titers against WT(D614G) pseudoviruses, ranging between 578-3935 for Pfizer/BioNtech BNT162b2 and 651-5853 for Moderna (Figure 2A and 2B) (Pfizer/BioNtech BNT162b2 vs Moderna, p = 0.1225, Mann-Whitney test). The Moderna vaccine-elicited sera trended towards higher neutralization titers against most variants compared to Pfizer/BioNtech BNT162b2, possibly due to higher vaccine mRNA content and greater interval between priming and boosting for Moderna (4 weeks vs 3 weeks for Pfizer/BioNtech BNT162b2) [38] . Overall, the trends in neutralization titers for the vaccine-elicited sera against a large panel of variant pseudoviruses were similar to those for convalescent sera, though the GMTs were approximately 3 to 5-fold higher for vaccine-elicited sera.

A prior study showed that convalescent sera and vaccine-elicited antibody neutralization titers against B.1.617 pseudoviruses bearing L452R-E484Q-P681R spike substitutions were about 2-5-fold reduction [20] . In this study, depending upon the infecting variant, convalescent sera displayed a modest 2-4-fold reduction in neutralization titers for B.1.617.1 compared to WT(D614G), while the vaccine-elicited sera displayed only 2.1-fold reduction. Our findings using B.1.617.1 pseudoviruses with the full complement of spike substitutions rather than just the L452R-E484Q-P681R spike substitutions confirm and extend the prior findings [20] . Finally, the 1.8-3.4-fold reduction in neutralization in vaccination group against B.1.617.2 pseudoviruses seen in our study and others suggests that the two-dose mRNA vaccines could significantly contribute to protection against both Kappa and Delta variants [36, 39] .

We used antigenic cartography to compare the relative difference in neutralization of distinct variants, exploiting heterogeneity in individual antibody responses to identify antigenic differences among strains. Antigenic map dimensionality was tested in 1-5 dimensions using cross-validation. The 3D maps had only slightly greater predictive power compared to the 2D maps; both the 2D and 3D maps performed much better than 1D (Supplementary Table S1 ). The 2D antigenic maps are presented for convalescent infection sera ( Figure 3A ) and vaccine sera ( Figure 3B ). To evaluate reproducibility in positioning of antigens on the map, we created bootstrap confidence intervals in which n=10,000 antigenic maps were made by resampling sera with replacement (bottom panels in Figure 3A and 3B). Antigenic maps are also shown in 3D ( Figure  3C and 3D). Maps made with convalescent sera from infection only by WT(D614G) or only by strains containing L452R mutation were not sufficiently robust for antigen positioning.

On the convalescent sera antigenic map, most of the pseudoviruses clustered close to the WT(D614G) pseudovirus and the WT(D614G) sera and L452R sera, including K417N, L452R, T478K, L452Q+F490S, and B.1.429 (0.25 to 0.72 antigenic units (AU) from WT (Table 3) . B.1.617.2, C.37, and AY.1 clustered below WT(D614G) and slightly further away (1.14 -1.60 AU), while L452R+T478K was also further away, but in the opposite direction (1.20 AU). However, in the bootstrap confidence interval 2D map and 3D map, Notably, given that the vaccine-elicited sera had much higher titers than convalescent sera across variants, the titers against AY.1 were still higher in vaccinated individuals, meaning the antigenic difference may not translate into loss of vaccine protection. Furthermore, vaccine-elicited sera saw a smaller difference between B.1.351 and WT(D614G) than the convalescent sera, which may have aligned AY.1 and B.1.351 closer together. Overall, while these antigenic maps provide meaningful information on the relative positions of antigens, they are limited by the sera being so tightly clustered. Future antigenic maps with sera against distinct variants would enable more accurate evaluation of antigenic variation among the variants.

We next evaluated 23 clinical-stage therapeutic neutralizing antibodies for potency against the B.1.617 variants. These antibodies were evaluated as part of the US Government COVID-19 response effort to inform the clinical testing and use of these antibodies [17] . Due to an agreement with the manufacturers who provided the antibodies, only blind codes are used to identify the antibodies. Amongst the therapeutic antibodies tested were thirteen monoclonal neutralizing anti-bodies (nAbs), six cocktail combinations (cnAbs) of two monoclonal anti-bodies and four polyclonal antibodies (pAbs).

B.1.617.1 pseudoviruses displayed complete resistance (>50-fold) to five nAbs (C, D, E, F and G) and partial resistance (10-50-fold) to one nAb (H) ( Figure 4A and Supplementary Figure S2 ). The E484Q substitution alone conferred complete resistance (> 50 fold) to three nAbs (E, F and G), and partial resistance to one nAb (C). Several nAb classes were characterized previously to describe potential mechanisms of resistance. For instance, class 2 mAbs bind both up-and down-RBD configurations to prevent ACE2 binding as well as bridge adjacent down RBDs to lock spike in a closed prefusion conformation to further prevent S-ACE2 engagement [40] . E484K is a dominant resistance substitution for many RBD-directed nAbs including class 2 nAbs, potentially explaining resistance conferred by E484Q [40] . The L452R substitution conferred complete resistance to four nAbs (C, D, E and H). The L452R is centrally located in the receptor binding site and is a known resistance substitution for several monoclonal antibodies [24] . None of the cnAbs and pnAbs tested showed the loss of neutralization potency against L452R, E484Q, and B.1.617.1 (Supplementary Figure  S2) . Overall, E484Q and L452R substitutions fully accounted for the resistance of B.1.617.1 against these nAbs, and 17 of 23 tested therapeutic antibodies retained neutralization potency against B.1.617.1 (Supplementary Figure S2) . B.1.617.2 pseudoviruses displayed complete resistance (>50-fold) to three nAbs (C, D, and E) and partial resistance (10-50-fold) to one nAb (H) ( Figure 3B ). The L452R substitution alone is responsible for the observed resistance of these nAbs (C, D, E and H) as pseudoviruses bearing L452R alone or in combination with T478K (L452R+T478K) displayed identical patterns of resistance as B.1.617.2 pseudoviruses. AY.1 pseudoviruses displayed similar resistance as B.1.617.2 above and also complete resistance to one additional nAb (N) due to the K417N RBD substitution and one cnAb (S) ( Figure 4C ).

Prior reports indicated increased infectivity of pseudoviruses containing the L452R substitution in spike in 293T-ACE2.TMPRSS2 cells due to L452R conferring enhanced RBD affinity to ACE2 [19, 22] . We therefore investigated the impact of the B.1.617 spike substitutions on pseudovirus infectivity and found that B.1617.1, B.1.617.2, and AY.1 pseudoviruses, as well as pseudoviruses with single RBD L452R, T478K, and E484Q substitutions, displayed similar infectivity to WT(D614G) (Supplementary Figure S3) .

Previous studies demonstrated that L452R and N501Y substitutions in RBD enhanced spike protein affinity to ACE2, which may have contributed to greater transmissibility of B.1.1.7, B.1.351, P.1 and B.1.427/429 variants [22, 41, 42] . To investigate whether the B.1.617 variants have an increased binding for ACE2, we measured the neutralization potency of human soluble ACE2 (sACE2) protein against the variant pseudoviruse. We found that the 50% inhibitory concentrations (IC50) of sACE2 against B.1.617.2 (IC50: 0.681 µg/ml; p < 0.01) was 4.2-fold lower compared to WT(D614G) (IC50: 2.88 µg/ml) ( Table 4 and Supplementary Figure S4 ). In agreement with greater ACE2 affinity of L452R RBD compared to WT RBD as per SPR results reported previously, pseudoviruses containing the L452R substitution (IC50:1.189 µg/ml; p < 0.05) and L452R+T478K substitutions (IC50:1.228 µg/ml; p < 0.001) displayed 2.4-and 2.3-fold lower IC50. However, pseudoviruses with single spike RBD substitutions T478K or E484Q), as well as the B.1.617.1 (IC50:2.028 µg/ml) and AY.1 (IC50:1.966 µg/ml) spikes, displayed comparable IC50 to WT (D614G) (0.5-1.5-fold change) ( Table 4 and Supplementary Figure S4 ). These findings suggest enhanced binding of Delta spike protein to ACE2 receptor, which may potentially contribute to greater infectivity and replication in ACE2-positive target cells.

Since P681H enhanced proteolytic processing of B.1.1.7 spike [26] , we next evaluated spike proteolytic processing on B.1.617 variants carrying the P681R substitution. The B.1.617 variant pseudoviruses displayed efficient proteolytic processing of spike compared to Wuhan-Hu-1 (D614) and WT(D614G) as observed by the S1/S ratio ( Figure 5A ). We further evaluated the effect of P681R substitution on furin cleavage of SARS-CoV-2 by introducing this substitution in Wuhan-Hu-1 (D614) and WT(D614G) spike backgrounds. For all pseudoviruses bearing the P681R spike substitution, including B.1.617.1, B.1.617.2, Wuhan-Hu-1+P681R and P681R (+D614G), spike cleavage as determined by higher cleaved S1 subunit to total S (S1+S) ratios was enhanced compared to the respective original pseudoviruses ( Figure 5A) . B.1.1.7 and WT(D614G) pseudoviruses bearing P681H also displayed enhanced spike cleavage ( Figure 5A ), consistent with a previous report [26] . B.1.429 pseudoviruses that lack P681R or P681H substitutions near the furin cleavage site displayed inefficient spike cleavage.

To gain further insight into the furin processing efficiency at the S1/S2 site of the B.1.617 S, we undertook a bioinformatic approach utilizing the PiTou and ProP furin cleavage prediction tools, comparing B.1.617 to the Wuhan-Hu-1 (D614) prototype spike and B.1.1.7 spike, as well as spike proteins of several lineage specific mammalian and animal CoVs. The PiTou algorithm combines a hidden Markov model and knowledge-based cumulative probability score functions for the functional characterization of a 20 amino acid cleavage motif from P14 to P6′ for furin binding and cleavage, whereas ProP predicts furin cleavage sites basing on experimental data-derived networks [43, 44] . Both algorithms predicted a greater increase in the furin cleavage for B. Table 5 ) [44] . As expected, proteins not containing furin cleavage site displayed relatively lower score while much higher scores were shown for the proteins containing furin cleavage site [45, 46] .

While SARS-CoV-2 S1/S2 P−R−R−A−R furin cleavage site conforms to a minimal furin recognition motif, R−X−X−R, the presence of H/R instead of P increases the total number of basic residues to four. This presence of basic residue H/R results in additional electrostatic and intramolecular hydrogen bonding to gain substrate turnover [47] . In fact, this basic residue is represented in several viral and cellular proteins [47, 48] . While the cleavage is not necessary to enhance pseudovirus infectivity, the furin cleavage of spike has been shown to be necessary for the virus transmission as demonstrated in ferret model [49] . The P681H substitution in B.1.1.7 variants has been associated with enhanced virus transmissibility whereas the P681R substitution in B.1.617 variants has been associated with enhanced pathogenicity in a hamster model and transmissibility [26, [49] [50] [51] . Further investigations of the role of P681H/R substitutions for virus transmissibility and cell-to-cell spread in in vivo animal models are needed. Furthermore, continued surveillance is necessary as several independent lineages have recently emerged containing additional substitutions proximal to S1/S2 cleavage junction, such as the B.1.2 and B.1.525 with Q677H, C.37 and B.1.617.2 with Q675H, and C.1.2 with N679K substitutions [52, 53] . On the other hand, functions of furin cleavage site substitutions in host cell attachment and antigenicity cannot be excluded since amino acids N 679 -R 685 and E 661 -R 685 have been reported to have host neuropilin-1 attachment [54] and Staphylococcus enterotoxin B-like super-antigenic functions [55] .

To quantify whether higher ACE2 binding and furin cleavage of B.1.617.2 spike augments fusion between virus and/or cell membranes, we performed cell-cell fusion assays by complementing β-galactosidase subunits in spike-transfected effector cells and 293T-ACE2.TMPRSS2 target cells. Compared to WT(D614G), both B.1.617.1 and B.1.617.2 spike induced significantly higher cell-cell fusion activity when controlled for spike cell surface expression (4000 MFI of spike protein on cell surface) ( Figure 5B ). Our findings extend previous studies indicating that the P681R substitution increases spike proteolytic cleavage and facilitates cell-cell fusion [54, 55] . Our findings also suggest that fusogenic potential of spike proteins may be influenced by both ACE2 binding affinity as well as proteolytic cleavage of spike.

Here we show that pseudoviruses bearing B.1.617.1 spike with L452R and E484Q substitutions, and B.1.617.2 spike with K417N, L452R and T478K substitutions, have modestly reduced susceptibility to neutralization by Pfizer or Moderna vaccine-elicited sera and convalescent sera compared to pseudoviruses bearing WT(D614G) spike. The individual L452R, T478K, E484Q and dual L452R+T478K substitutions accounted for most but not all of the reduction in neutralization potency of the sera, suggesting contributions from substitutions in the NTD/CTD. Neutralization titers, as well as antigenic maps, indicated that the full set of RBD substitutions in combination with substitutions outside the RBD contribute to antigenic differences of B. 1.617.1, B.1.617.2 , and C.37 variants. Antigenic distances between the variants also tended to be more spaced apart in the map generated by the convalescent sera compared to the vaccine-elicited sera. Differences between convalescent and vaccine-elicited immune responses may be further impacted by the recent global dominance of B.1.617.2 and its sub lineages. Limitations in our study include the small numbers of sera samples in the convalescent and vaccine cohorts. Potential differences in COVID-19 severity in the convalescent sera cohort and time of sera collection could also affect neutralization titers. Nonetheless, most sera from convalesced and vaccinated individuals neutralized the B. 1.617.1, B.1.617.2 , and AY.1 variants. These findings with the vaccine-elicited sera suggest that the two-dose immunization with current mRNA vaccines will likely induce protective immunity against the tested B.1.617 variants. Furthermore, 17 of 23 therapeutic neutralizing antibodies retained complete neutralization against B.1.617 variants.

Resistance to the remaining therapeutic neutralizing antibodies is due to RBD substitutions, K417N, L452R, and E484Q, but not T478K. As B.1.617.2 variants continue to evolve, it will be important to continue to monitor how new substitutions in spike impact their resistance to therapeutic neutralizing antibodies and vaccine efficacy.

Funding: This work was supported by the institutional funds from the US Food and Drug Administration and the Intramural Research Program of the National Institute of Allergy and Infectious Diseases.

Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

The authors would like to thank Hongquan Wan (FDA), and Hailun Ma (FDA) for the critical review of the manuscript.

The authors declare no conflicts of interest. 

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