key: cord-0846633-ufyhdzt1
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: 2021-12-11
journal: Viruses
DOI: 10.3390/v13122485
sha: 3c34be05a4c0dc2802d0f869d4ed1370dbc7524e
doc_id: 846633
cord_uid: ufyhdzt1

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. Here, we compared B.1.617 variants for neutralization resistance by convalescent sera, mRNA vaccine-elicited sera, and therapeutic neutralizing antibodies using a pseudovirus neutralization assay. B.1.617.1, B.1.617.2, and AY.1 pseudoviruses showed a modest 1.5- to 4.4-fold reduction in neutralization by convalescent sera and vaccine-elicited sera. In comparison, similar modest reductions were also observed for C.37, P.1, R.1, and B.1.526 pseudoviruses, but 7- and 16-fold reductions for vaccine-elicited and convalescent sera, respectively, were seen for B.1.351 pseudoviruses. Among twenty-three therapeutic antibodies tested, four antibodies showed either complete or partial loss of neutralization against B.1.617.2 pseudoviruses and six antibodies showed either complete or partial loss of neutralization against B.1.617.1 and AY.1 pseudoviruses. Our results indicate that the current mRNA-based vaccines will likely remain effective in protecting against B.1.617 variants. Finally, the P681R substitution confers efficient cleavage of B.1.617 variants’ spike proteins 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.

Since its origin in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally to cause a coronavirus disease 2019 (COVID- 19) pandemic that recorded more than 263 million infections and has claimed 5. The global dominance and ongoing evolution of B.1.617 lineage variants require continuing assessment of the neutralization potency of convalescent sera, vaccine-elicited sera, and therapeutic neutralizing antibodies against emerging B.1.617 variants. Here, we measured the neutralization potency of convalescent sera, vaccine-elicited sera, and therapeutic neutralizing antibodies against two independent variants each in the Kappa (B.1.617.1) and Delta (B.1.617.2, AY.1) lineages and assessed the contribution of the RBD substitutions in conferring resistance. We found that resistance to convalescent and vaccine-elicited sera was predominantly conferred by RBD substitutions E484Q, T478K, and L452R. Furthermore, out of 23 therapeutic neutralizing antibodies tested, Kappa and Delta pseudoviruses displayed complete resistance to five neutralizing antibodies and partial resistance to one antibody. Finally, we showed that the P681R substitution confers enhanced furin processing in spike protein of B.1.617 lineage variants that corresponded to enhanced cell-cell fusion activity. However, only Delta spike protein exhibited greater sensitivity to soluble ACE2 inhibition, implying enhanced ACE2 affinity. This feature along with enhanced cell-cell fusion activity may contribute to the dominance of the B.1.617.2 variant.

Use of de-identified sera in this study was approved by the U.S. Food and Drug Administration Research in Human Subjects Committee. Vaccine-elicited sera were collected at the U.S. Food and Drug Administration with written consent under an approved Institutional Review Board (IRB) protocol (FDA IRB Study # 2021-CBER-045).

Codon-optimized full-length open reading frames of the S genes of SARS-CoV-2 variants were cloned into pcDNA3.1(+) or pVRC8400 by GenScript (Piscataway, NJ, USA). The codon optimization for gene expression in human cell was performed by GenScript's OptimumGene algorithm system to optimize the following parameters: codon usage bias, GC content, CpG dinucleotides content, mRNA secondary structure, internal chi sites and ribosomal binding sites, negative CpG islands, cryptic splicing sites, premature PolyA sites, PolyT sites, RNA instability motif (ARE), and repeat sequences (direct repeat, reverse repeat). The codon-optimized sequence of Wuhan-Hu-1 S gene is shown in Supplementary Figure S1 . The spikes of variants used in this study are listed in Table 1 . The HIV gag/pol (pCMV∆R8.2), and luciferase reporter (pHR'CMV-Luc) plasmids were obtained from the Vaccine Research Center (National Institutes of Health, Bethesda, MD, USA) [31, 32] . 293T-ACE2.TMPRSS2 cells stably expressing human angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) were previously described (BEI Resources, Manassas, VA, USA; Cat no: NR-55293) [33] . The 293T and 293T-ACE2.TMPRSS2 cells were maintained at 37 • C in Dulbecco's modified eagle medium (DMEM) supplemented with high glucose, L-glutamine, minimal essential media (MEM) non-essential amino acids, penicillin/streptomycin, and 10% fetal bovine serum (FBS).

Convalescent sera from SARS-CoV-2-infected donors (n = 10) collected 6-61 days after symptom onset were purchased from Bocabiolistics (Pompano Beach, FL, USA). Donors were 18-73 years old with six males/four females. The information about the convalescent sera is shown in Table 2 . Sera from Pfizer/BioNtech BNT162b2 (n = 15) or Moderna mRNA-1273 vaccinated individuals (n = 14) obtained two weeks after the second vaccination were used in this study. Vaccinated individual donors were 21-65 years old with six males/ nine females for Pfizer BNT162b2 vaccination and eight males/six females for Moderna mRNA-1273 vaccination. All sera were tested negative for non-specific neutralization using amphotropic murine leukemia enveloped pseudovirus. Vaccinated donors were prescreened for absence of both history of SARS-CoV-2 infection and SARS-CoV-2 neutralizing antibodies prior to vaccination. Twenty-three therapeutic neutralizing antibodies against SARS-CoV-2 spike protein were donated by different pharmaceutical companies for the U.S. government COVID-19 response Therapeutics Research Team efforts to define neutralization profiles against existing and emerging SARS-CoV-2 variants [19] . Due to a confidentiality agreement with the manufacturers, neutralizing antibodies described are shown with blinded identification codes as follows: single neutralizing antibodies (nAbs A to R), combination of two neutralizing antibodies (cnAbs S to X), and polyclonal neutralizing antibodies (pnAbs III to IV). 1  Male  36  16  D614G  2  Female  29  28  D614G  3  Male  70  14  D614G  4  Female  54  61  D614G  5  Male  18  13  D614G  6  Male  59  9  D614G   7  Male  25  40  S13I, Q52R, A67V, 69-70del, 144del,  L452R  8  Female  73  59  L452R, D614G  9  Female  33  26  L452R, D614G  10  Male  55  6 W152C, L452R, D614G 

His-tagged soluble ACE2 was produced in FreeStyle™ 293-F cells and purified using HisPur™ Ni-NTA Resin (Thermo Scientific, Waltham, MA, USA) as described previously [34] .

HIV-based lentiviral pseudoviruses with spike proteins were generated as previously described [33] . The B.1 spike containing D614G was used as wild type (WT(D614G)). Briefly, pseudoviruses bearing the spike glycoprotein and packaging a firefly luciferase (FLuc) reporter gene were produced in 293T cells by co-transfection of 5 µg of pCMV∆R8.2, 5 µg of pHR'CMVLuc and 4 µg of pcDNA3.1(+) or 0.5 µg of pVRC8400 encoding a codon-optimized spike gene with the desired substitutions. Pseudovirus supernatants were collected approximately 48 h post transfection, filtered through a 0.45 µm low protein binding filter, and stored at −80 • C. Neutralization assays were performed using 293T-ACE2.TMPRSS2 cells in 96-well plates as previously described [33] . Briefly, pseudoviruses with titers of approximately 1 × 10 6 relative luminescence units (RLU)/mL of luciferase activity were incubated with serially diluted sera or antibodies for two h at 37 • C. Pseudovirus and serum or antibody mixtures (100 µL) were then inoculated onto the plates pre-seeded one day earlier with 3.0 × 10 4 cells/well. Pseudovirus infectivity was scored 48 h later for luciferase activity. The antibody concentration or inverse of the sera dilutions causing a 50% reduction of RLU compared to control was reported as the neutralization titer. Titers were calculated using a nonlinear regression curve fit (GraphPad Prism Software Inc., La Jolla, CA, USA). The mean titer from at least two independent experiments each with intra-assay duplicates was reported as the final titer. WT(D614G) pseudovirus was run as a control for every assay.

For ACE2 neutralization assay, serially diluted recombinant human soluble ACE2 was incubated with indicated pseudovirus (~1 × 10 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 h later. The soluble ACE2 concentration causing a 50% reduction of RLU compared to control was reported as the 50% inhibitory concentration or IC 50 .

In total, 1.25 mL of pseudoviruses was pelleted at 4 • C for 2 h at 15,000 rpm using Tomy TX-160 ultracentrifuge. Pseudovirus pellet was resuspended in 1× Laemmli loading buffer and heated at 70 • C for 10 min. 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, Wayne, PA, USA).

For measuring spike-protein-mediated cell-cell fusion, β-gal complementation assay was performed as described previously [35] . Briefly, β-gal ω subunit-expressing 293T cells were transfected with 1 µg spike plasmids, whereas 293T-ACE2.TMPRSS2 cells were transfected with β-gal α subunit using the Fugene 6 reagent. At 24 h post transfection, the transfected cells were detached using a nonenzymatic cell dissociation solution (Sigma, St. Louis, MO, USA) 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 × 10 4 cells per well on a 96-well plate. The cells were co-cultivated for 24 h at 37 • C. The culture supernatants were then removed, and cell-cell fusion was scored by determination of the β-gal activity in co-cultured cell lysates using a Galacto-Star kit (Applied Biosystems, Waltham, MA, USA) 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 Viruses 2021, 13, 2485 6 of 20 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, USA). The cells were washed twice and then fixed with 2% paraformaldehyde. The results were acquired using BD LSRFortessa™ X-20 Cell Analyzer (BD Biosciences, San Jose, CA, USA). 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 (Sam Wilks (2021), Racmacs: R Antigenic Cartography Macros. R package version 1.1.16. https://github.com/ acorg/Racmacs, accessed on: 15 October 2021) [36, 37] . 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 was computed using the ProP 1.0 Server hosted at http://www.cbs.dtu.dk/services/ProP/ (accessed on: 15 July 2021) and the PiTou V3 software hosted at http://www.nuolan.net/reference.html (accessed on: 15 July 2021).

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 log 2 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 four-fold lower (GMT 90), confirming and extending other reports [25, 38, 39] . 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 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 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 modest 1.8-fold reduction in titers against C.37 pseudoviruses was observed compared to WT(D614G) pseudoviruses (GMT titers 222 and 392, respectively). A 1.4-fold reduction in titers was observed for pseudoviruses with only the L452Q and F490S substitutions, indicating that these RBD substitutions contribute to C.37 resis- tance. These findings are in agreement with a prior report showing a 3.3-fold reduction of convalescent sera neutralization titer for C.37 pseudoviruses compared to WT(D614G), as well as L452Q and F490S single substitutions contributing to neutralization resistance [40] . 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 16.3-fold reduced (GMT 24) compared to WT(D614G), consistent with prior reports showing marked reductions of cross-neutralization against this variant [16] [17] [18] 21] .

Because the convalescent sera came from individuals who were previously 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, 1.6-fold, and 2.5-fold reduction in cross-neutralization potency was seen against B. [41] . The generally lower titers and small numbers of samples in the L452R group in our study may impact the fold changes. We also note that in our study the titers of the sera from WT(D614G)-infected individuals are generally higher than the neutralization titers of the sera from L452R-infected individuals, but the small number of samples prevents a meaningful comparison. Differences in titers may be due to the timing of collection of sera since infection, severity of infection, or other factors.

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 and 3935 for Pfizer/BioNtech A prior study showed that convalescent sera and vaccine-elicited antibody neutralization titers against pseudoviruses bearing spikes containing L452R-E484Q-P681R substitutions displayed 2-5-fold reduction, compared to the neutralization titers against WT (D614G) pseudoviruses [25] . In this study, depending upon the infecting variant, convalescent sera displayed a modest 2-4-fold reduction in neutralization titers 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 pseudoviruses bearing spikes containing L452R-E484Q-P681R substitutions displayed 2-5-fold reduction, compared to the neutralization titers against WT(D614G) pseudoviruses [25] . 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 [25] . Finally, the 1.8-3.4-fold reduction in neutralization in the 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 [17, 18, 38, 41, 43] .

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 ,B). Antigenic maps are also shown in 3D (Supplementary Figure S3 ). Maps made with convalescent sera only from infection by WT(D614G) or only from infection 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, L452R + T478K and AY. 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 ,B). Antigenic maps are also shown in 3D (Supplementary Figure S3 ). Maps made with convalescent sera only from infection by WT(D614G) or only from infection by strains containing L452R mutation were not sufficiently robust for antigen positioning. The antigenic map of the same SARS-CoV-2 variant pseudoviruses for vaccine-elicited sera showed similar patterns to the convalescent sera map but with some notable differ-ences. Again, pseudoviruses K417N, L452R, T478K, L452Q + F490S, and B.1.429 were close to WT(D614G) (0.05-0.51 AU). However, E484Q and C.37 were both closer to WT(D614G) but were poorly coordinated and extended in elongated shapes around WT(D614G). B.1.617.1 was also slightly closer to WT(D614G) at 1.55 AU and was positioned adjacent to the other variants. Unexpectedly, AY.1 was between B.1.617.2 and B.1.351 and was further away from WT(D614G) (1.89 AU) than in the convalescent sera map (1.60 AU), being positioned only slightly closer to WT(D614G) than B.1.351 was from WT(D614G) (3.02 AU). These antigenic maps reinforce what was observed in terms of neutralizing antibody titers. Compared to the convalescent sera map, there was a larger antigenic difference between AY.1 and WT(D614G) on the vaccine-elicited sera 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 U.S. Government COVID-19 response effort to inform the clinical testing and use of these antibodies [19] . 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 nAbs, six cnAbs, and four 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 S4 ). The E484Q substitution alone conferred complete resistance (>50-fold) to three nAbs (E, F, and G), and partial resistance to one nAb (C) as previously described [19, 44] . The L452R substitution also conferred complete resistance to four nAbs (C, D, E, and H), as previously described [19] . The L452R is centrally located in the receptorbinding site and is a known resistance substitution for several mAbs [23, 26] . None of the cnAbs and pnAbs tested showed the loss of neutralization potency against L452R, E484Q, and B.1.617.1 (Supplementary Figure S4) . Overall, these findings show that the E484Q and L452R substitutions fully account for the resistance of B.1.617.1 against these nAbs, while 17 of 23 tested therapeutic antibodies retained neutralization potency against B.1.617.1 (Supplementary Figure S4) . 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 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 to the nAbs as B.1.617.2 pseudoviruses, except for 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 [24, 27] . 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 S5) .

None of the cnAbs and pnAbs tested showed the loss of neutralization potency against L452R, E484Q, and B.1.617.1 (Supplementary Figure S4) . Overall, these findings show that the E484Q and L452R substitutions fully account for the resistance of B.1.617.1 against these nAbs, while 17 of 23 tested therapeutic antibodies retained neutralization potency against B.1.617.1 (Supplementary Figure S4) . 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 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 to the nAbs as B.1.617.2 pseudoviruses, except for complete resistance to one additional nAb (N) due to the K417N RBD substitution and one cnAb (S) ( Figure 4C ). 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 [45] [46] [47] . 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 variants. We found that the 50% inhibitory concentrations (IC 50 ) of sACE2 against B.1.617.2 (IC 50 : 0.68 µg/mL; p < 0.01) was 4.2-fold lower, compared to WT(D614G) (IC 50 : 2.88 µg/mL) ( Table 4 and Supplementary Figure S6 ). In agreement with greater ACE2 affinity of L452R RBD compared to WT RBD as reported by others using surface plasmon resonance [27, 48] , pseudoviruses containing L452R (IC 50 :1.19 µg/mL; p < 0.05) and L452R + T478K (IC 50 :1.23 µg/mL; p < 0.001) displayed 2.4-and 2.3-fold higher sensitivity to inhibition by sACE2. However, pseudoviruses with only spike RBD substitutions (T478K, E484Q), as well as the B.1.617.1 (IC 50 :2.03 µg/mL) and AY.1 (IC 50 :1.97 µg/mL) spikes, displayed comparable IC 50 to WT(D614G) (0.5-1.5-fold change) (Table 4 and Supplementary Figure S6 ). These findings suggest enhanced binding of Delta spike protein to the 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 [29, 49] , we next evaluated spike proteolytic processing of B.1.617 pseudovirus 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 previous reports [29, 50] . B.1.429 pseudoviruses that lack P681R or P681H substitutions near the furin cleavage site displayed inefficient spike cleavage, whereas the furin cleavage site (∆PRRA)-deleted spike lacked cleavage. Our findings agree with several studies reporting enhanced furin cleavage efficiency of B.1.1.7 and B.1.617 spikes bearing P681H and P681R substitutions, respectively [49] [50] [51] [52] . 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 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 based on experimental data-derived networks [53, 54] . Both algorithms predicted a greater increase in the furin cleavage for B.1.617 lineage variants (PiTou: 12.4; Prop: 0.698) compared to Wuhan-Hu-1 (PiTou: 9.19; Prop: 0.62) and B.1.1.7 (PiTou: 9.9; Prop: 0.7) ( Table 5 ) [54] . As expected, proteins not containing furin cleavage site displayed relatively lower scores while much higher scores were shown for the proteins containing furin cleavage site [55, 56] . 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 [57] . In fact, this basic residue is represented in several viral and cellular proteins [57, 58] . 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 a ferret model [59] . 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 [29, [59] [60] [61] . 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 [62, 63] . 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 [64] and staphylococcus-enterotoxin-B-like super-antigenic functions [65] .

Furin cleavage score analysis of CoV S1/S2 cleavage sites. CoV spike and influenza hemagglutinin (HA) sequences were analyzed using the ProP 1.0 and PiTou 3.0 furin prediction algorithms, generating a score with bold numbers indicating predicted furin cleavage. "|" denotes the position of the furin cleavage site. Sequences corresponding to the S1/S2 region of NL63 (APF29063. Figure 5B ). Our findings extend previous studies indicating that the P681R substitution increases spike proteolytic cleavage and facilitates cell-cell fusion [41, 50, 66, 67] . 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/BioNtech BNT162b2 or Moderna mRNA-1273 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 contributes 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. Limitations in our study include the small number 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. 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. These findings suggest that the two-dose immunization with current mRNA vaccines will likely induce protective immunity against the tested B.1.617 variants. However, 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.

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

The authors declare no conflict of interest.