key: cord-0843049-r1rzl9sl
authors: Lopez, E.; Haycroft, E. R.; Adair, A.; Mordant, F. L.; O'Neill, M.; Pymm, P.; Redmond, S.; Gherardin, N. A.; Whearley, A. K.; Juno, J. A.; Selva, K. J.; Davis, S.; Harty, L.; Purcell, D. F. J.; Subbarao, K.; Godfrey, D. I.; Kent, S. J.; Tham, W.-H.; Chung, A. W.
title: Simultaneous evaluation of antibodies that inhibit SARS-CoV-2 RBD variants with a novel competitive multiplex assay
date: 2021-03-26
journal: nan
DOI: 10.1101/2021.03.20.21254037
sha: 4fdb2d0c0b35a3f1f638ffe1a183b54f546f601c
doc_id: 843049
cord_uid: r1rzl9sl

The SARS-CoV-2 Receptor Binding Domain (RBD) is both the principal target of neutralizing antibodies, and one of the most rapidly evolving domains, which can result in the emergence of immune escape mutations limiting the effectiveness of vaccines and antibody therapeutics. To facilitate surveillance, we developed a rapid, high-throughput, multiplex assay able to assess the inhibitory response of antibodies to 24 RBD natural variants simultaneously. We demonstrate that immune escape can occur through two mechanisms, antibodies that fail to recognize mutations, along with antibodies that have reduced inhibitory capacity due to enhanced variant RBD-ACE2 affinity. A competitive approach where antibodies simultaneously compete with ACE2 for binding to the RBD may therefore more accurately reflect the physiological dynamics of infection. We describe the enhanced affinity of RBD variants N439K, S477N, Q493L, S494P and N501Y to the ACE2 receptor, and demonstrate the ability of this assay to bridge a major gap for SARS-CoV-2 research; informing selection of complementary monoclonal antibody candidates and the rapid identification of immune escape to emerging RBD variants following vaccination or natural infection.

the development of vaccine elicited humoral immunity, and eliciting potent neutralizing 86 antibodies capable of blocking ACE2 binding remains a key feature of the 87 development of effective vaccines and antibody therapies. 88

As for any viral outbreak, of great concern is the emergence of gain of function 89 variants which facilitate viral infectivity, transmissibility, or neutralizing antibody 90 escape. Since the beginning of the pandemic, SARS-CoV-2 genomic sequencing has 91 identified a number of variants revealing a modest rate of evolution of the viral genome 92 (18, 19) . This information has been made available through public repositories such been directed toward the surveillance of SARS-CoV-2 RBD mutations. Of concern is 99 the observation that several of these SARS-CoV-2 RBD mutations escape monoclonal 100 antibody (mAb) neutralization and/or attenuate polyclonal plasma neutralization (23-101 27). Subsequently, there has been a more cautious shift in assessing the efficacy of 102 current vaccine and antibody therapies with respect to the continuously emerging 103 genetic variants, especially since some vaccine candidates and antibody therapies 104 solely target the wild-type RBD (28, 29) . 105

Though there is no standardized method for assessing the antibody-mediated 106 neutralization of SARS-CoV-2, which can account for variation in between studies 107 (30). Assays which evaluate the ability of NAbs to inhibit viral replication in target cells 108 by observing plaque reduction or a cytopathogenic effect are currently regarded as 109 benchmark assays. The pseudovirus-based neutralization assays (31) which reduce 110 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254037 doi: medRxiv preprint A subset of previously described (38) plasma samples collected from a cross-sectional 163 cohort of Australian adults recovered from SARS-CoV-2 infection were chosen to 164 firstly evaluate the performance of this assay with regard to its ability to quantitate 165 ACE2 inhibitory antibodies from SARS-CoV-2 convalescent subjects. Subsequently, 166

we then sought to determine whether NAbs in the SARS-CoV-2 positive plasma could 167 inhibit ACE2 receptor binding in a similar manner as the virus microneutralization 168 assay (39). In this assay, inhibition of the SARS-CoV-2 RBD-ACE2 interaction by 169 antibodies in SARS-CoV-2 convalescent plasma was observed as dose-dependent 170 decrease in fluorescent ACE2 measured as MFI (Median Fluorescence Intensity) 171 bound to immobilised RBD, whereas the MFI of SARS-CoV-2 negative plasma 172 remained high across the 8-point 2-fold serial dilution ( Figure 1B) , demonstrating that 173 the specific inhibition of the SARS-CoV-2 RBD-ACE2 interaction by plasma from 174 convalescent SARS-CoV-2 subjects occurs in a dose-dependent manner ( Figure 1C) . 175

The capacity of antibodies in SARS-CoV-2 convalescent plasma to inhibit the 176 interaction between ACE2 and RBD in this assay was detected in most subjects in this 177 cohort (median, 40.5%; IQR, 32.9%-60.4%), with 31% subjects exhibiting more than 178 50% ACE2 inhibition activity Figure 1D . In comparison, just under a quarter (23%) of 179 subjects exhibited more than 50% inhibition activity against S1 (median, 33.1%; IQR, 180

28.4%-48.3%) ( Figure 1E ), suggesting that the full S1 protein may provide additional 181 stability to the ACE2-RBD interaction. A nominal cut-off of 20% was set based on a 182 panel of SARS-CoV-2 negative subjects (Supplementary Figure 1A) . To further 183 validate this assay, each plasma sample's relative half-maximum inhibitory 184 concentration (IC50) titer to RBD (WT) and spike (S1) was determined from the 2-fold 185 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254037 doi: medRxiv preprint 8-point dilution. Relative IC50 values of the ACE2-RBD versus ACE2-S1 interaction by % inhibition at a 1 in 100 dilution of plasma positively correlated with RBD IC50, (r=.96, 188 p< 0.0001) Figure 1G , suggesting that a 1 in 100 dilution of plasma would be closely 189 representative of the relative IC50. 190

In order to determine how well this assay could detect NAbs in comparison to 191 a virus neutralization assay (39), relative IC50 values to RBD and S1 were correlated 192 to a microneutralization assay using SARS-CoV-2 infection of Vero cells. As shown in 193 Figure 1H and Figure 1I , there was a strong correlation between the two assays for 194 RBD-ACE2 inhibition (r = 0.8, p< 0.0001) and S1-ACE2 inhibition (r= 0.75, p< 0.0001). 195

In addition, we examined the relationship between ACE2 inhibition and IgG RBD 196 binding ( Figure 1J ), and found a moderate correlation between the two (r = 0.58, 197 p=0.0002), which suggests that inhibition may also be mediated by other antibody 198 isotypes such as IgA and IgM or to other key regions of the S1 protein, such as the N-199 terminal domain (NTD) (40). The multiplex assay was determined to be robust (r 2 = Figure 1C) . 205

Considering mAbs are of increasing interest for therapy or prophylaxis of 206 SARS-CoV-2, we evaluated the ability of this assay to specifically measure mAbs that 207 neutralize the ACE2-RBD interaction. Here we tested two commercially available 208 neutralizing antibodies (one human and one mouse), and two negative controls for 209 RBD binding and ACE2 inhibition. The first negative control was an unrelated human 210 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254037 doi: medRxiv preprint mAb against influenza, and the second was CR3022, a neutralizing mAb which has 211 been described to bind RBD at an epitope that does not overlap with the ACE2 binding 212 site of SARS-CoV-2 and therefore is unable to block the ACE2-RBD interaction (41). 213

As demonstrated in Figure 1K and 1L, both the human and mouse neutralizing mAbs 214 demonstrated a very similar pattern of RBD binding and ACE2 inhibition. In contrast 215 the absence of binding and inhibition of ACE2 by a non-specific influenza mAb 216 confirmed the specificity of this assay. CR3022 binding to RBD coincided with the 217 absence of ACE2 inhibition further validating the specific ability of this assay to 218 evaluate neutralizing antibodies which specifically inhibit the RBD-ACE2 receptor 219 interaction. 220

Validation of an RBD natural variant multiplex array 223 224

Upon validation of the multiplex RBD-ACE2 assay to WT RBD and S1, we sought to 225 expand the assay multiplexing 23 RBD natural mutants selected from the GISAID RBD 226 surveillance repository at the time (June 2020), as well as RBD variant S477N which 227 emerged and rose to be the second most frequent variant in the following months. A 228 total 25 variants including the WT were studied. Figure is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

To evaluate how the array of RBD variants affected binding to ACE2 in our assay, a 247 2-fold 12-point serial dilution of ACE2 starting from a final concentration of 160µg/ml 248 per well was performed, and a relative EC50 for each of the variants was determined 249 ( Figure 2C ). The calculated difference in the relative EC50 of each variant was then 250 compared to RBD WT ( Figure 2D ), with the majority of RBD variants exhibiting a 251 similar or weaker relative EC50 when compared to the WT RBD. A trend consistent 252 with the observation that many missense mutations often negatively impact ACE2 253 binding (42). Nevertheless, we found some variants demonstrated significantly 254 enhanced binding to ACE2 relative to WT SARS-CoV-2 RBD. These were N501Y, 255 Q493L, S494P and S477N which demonstrated the lowest overall relative EC50 values 256 (0.2, 3.6, 6.3 and 8.3 µ g/ml respectively) as determined by our ACE2-RBD multiplex 257 assay ( Figure 2C ). 258

Though it is now well known that N501Y, which is the shared mutation in the is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint for their binding kinetics to ACE2 using Bio-layer interferometry (BLI). In addition, one 266 variant that showed a reduced affinity for ACE2 (E484D), was also selected to be 267 profiled for comparison. 268 BLI profiles of the RBD variants binding to ACE2 ( Figure 2E We next sought to apply the multiplex assay to map potential escape mutations using is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254037 doi: medRxiv preprint antibodies to bind the RBD before subsequent incubation with the ACE2 receptor or 313 ACE2 expressing cells. This approach has the potential to bias the assay to allow for 314 maximal NAb neutralization, thus reducing the influence of RBD variant affinity to the 315 ACE2 receptor. In light of this, we sought to evaluate how mAb pre-incubation followed 316 by incubation with the ACE2 receptor compared to our original competitive format of 317 the ACE2-RBD inhibition assay where all components are added simultaneously. Pre-318 incubation of mAbs in the absence of ACE2 was found to significantly overestimate 319 the neutralizing capacity of the mAbs ( Figure 3D ), conferring the mAb a competitive 320 advantage by allowing it to neutralize the RBD in the absence of competing ACE2. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254037 doi: medRxiv preprint Plasma samples were tested at a single-point dilution of 1 in 100 across the array of 338 natural RBD variants, based on the previously strong correlation of this dilution with 339 relative IC50 values ( Figure 1G ). As observed in Figure 4A , most subjects 340 demonstrated a reduction in the inhibition of most RBD variants, with the neutralizing 341 response to variant N501Y being the most significantly attenuated, with many subjects 342 demonstrating a ratio of less than 0.5 to that observed with the WT (Figure 4B Figure 4C illustrates the % 349 of subjects which had a neutralizing response (i.e. >20%) to each RBD variant, with 350 the lowest proportion of responders observed to occur to high affinity variants N501Y 351 (30%) and Q493L (35%). This was followed by N439K (60%), N477N (65%), and 352 E484K (65%). These results suggest that despite having the capacity to inhibit WT 353 RBD, many convalescent individuals may not generate antibodies with high enough 354 affinity capable of competing with RBD variants with an enhanced affinity to ACE2. 355

Discussion 358 359

To date numerous SARS-CoV-2 RBD variant viruses have emerged, with the number 360 and frequency of these variants steadily increasing since the beginning of the 361 pandemic. Of further concern has been the observation that a number of these 362 variants demonstrate escape from antibody neutralization (23-27, 46), with the more 363 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254037 doi: medRxiv preprint increasing population level immunity to WT SARS-CoV-2, and the rollout of vaccines 366 and antibody therapies targeting the RBD, mapping the antibody response to 367 emerging RBD mutations will be critical to guide future preparedness efforts by 368 enabling pre-emptive forecasting of mutations that might impact antibody recognition. 369

To address the need to rapidly survey the neutralizing capacity of antibodies to 370 RBD variants, here we describe a novel rapid high-throughput assay to measure RBD 371 binding and neutralizing antibodies to an array of RBD variants. Using a panel of 372 plasma samples obtained from subjects previously infected with SARS-CoV-2, we 373 found this assay correlated well with a cytopathic effect virus microneutralization 374 assay. Though this assay is not intended to replace gold standard SARS-CoV-2 cell-375 based neutralization assays, it provides a simple solution for broadly surveying the 376 diversity of SARS-CoV-2 neutralizing specificities to an array of RBD mutants in a 377 rapid high-throughput format, which can easily adapt to include new RBD variants as 378 they emerge. Furthermore, this assay presents several key advantages over existing 379 assays, since there is no need for viruses, cells, biosafety containment requirements, 380 or highly skilled tissue-culture operators. Finally, results can be obtained rapidly on the 381 same day in a high-throughput manner with the use of minimal sample volumes. 382

It has now been reported by several studies (23-27, 46) that antibody 383 neutralization to some RBD mutants is reduced, suggesting that SARS-CoV-2 has 384 mutated to evade host immunity. Profiling of a subset of previously published mAbs is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint variants in the array, with RBD alanine mutation at this position confirming that this is 393 a critical epitope for certain mAbs. Despite this, we found most mAbs were able to 394 demonstrate a modest inhibition at position 490. It is important to note that that 395 residues L455, A475, F490 and Q493, are residues which have been previously 396 reported to be SARS-CoV-2 RBD-ACE2 interacting residues based on structure 397 analysis (12, 49). Unlike the previously described variants, which demonstrated 398 escape from mAb recognition, N501Y was recognized at relatively high affinity by most 399 mAbs, however inhibition to N501Y was attenuated across most mAbs, suggesting 400 immune escape was likely due to the enhanced affinity of this variant for ACE2. We Although the E484K mutation has been associated with immune escape (27), 406 predictions regarding its affinity to the ACE2 receptor are conflicting (22, 50). Our 407 findings, which demonstrate a reduced affinity of E484K to ACE2 however support the 408 recent observations in which the E484K mutation has been found to prevent the 409 formation of two salt bridges that help to form and stabilize the RBD-ACE2 complex, 410 thus reducing ACE2 binding affinity (22). We also found S477N (51), and Q493L, a is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254037 doi: medRxiv preprint these differences in affinity to the ACE2 receptor mean for the overall viral fitness and 414 transmissibility of these variants is beginning to be explored (52). However, lessons 415 learned from the original SARS-CoV outbreak, which have served to guide SARS-416 COV-2 research suggest that the infectivity of different SARS-CoV strains in host cells 417 is correlated to the binding affinity between the RBD of each strain and ACE2 (53-55). 418

Indeed, the frequency and presence of the N501Y mutation in recently rapidly 419 emerging SARS- CoV-2 linages, B.1.1.7, B.1.351, B.1.1.70 and P.1 appears to further 420 strengthen this hypothesis, as does the frequency of S477N and S494P which we find 421 bind ACE2 with an enhanced affinity ( Figure 2B and 2D ). It is interesting to note, 422 however, that unlike higher affinity N501Y and S477N variants, F490L and G446V 423 demonstrated the weakest EC50 values in our ACE2 binding assay ( Figure 2D ), but 424 are observed at relatively high frequencies ( Figure 2B is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The present study also observed improved neutralization in our assay when antibody 445 was pre-incubated first, in the absence of the ACE2 receptor ( Figure 3D ), which raises 446 to question whether current assays which employ similar methods may be 447 overestimating the neutralizing response, particularly to high affinity RBD variants, 448 such as N501Y. When the two assay formats were compared, we found a significant 449 reduction in relative IC50 values when mAbs were allowed to neutralize the RBD prior 450 to incubation with ACE2. These findings suggest that a competitive format where both As SARS-CoV-2 continues to infect people globally, and variants with multiple 457 mutations in the spike protein emerge, there is a growing urgency to rapidly develop 458 effective and therapeutic options especially for high-risk individuals who may not 459 develop a robust immune response or those that may not be able to be vaccinated. 460

This novel high throughput multiplex assay has the potential to rapidly flag variants 461 that may potentially demonstrate an enhanced affinity to ACE2. It also characterises 462 the RBD neutralizing response in a competitive format, which takes into account the 463 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254037 doi: medRxiv preprint through contacts with the investigators and were invited to provide a blood sample as 490 previously described (38). For all participants, whole blood was collected with sodium 491 heparin anticoagulant, and plasma was collected and stored at −80 °C until use. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 26, 2021. ; https://doi.org/10.1101/2021.03.20.21254037 doi: medRxiv preprint using Ni-NTA columns. Protein concentration was determined by absorbance 540 measurement at 280 nm and purity was determined using SDS-PAGE. 541 RBD variant multiplex bead cocktails were generated as previously described 542

(57) Briefly, each respective RBD protein was coupled to a distinct magnetic 543 carboxylated bead region (Bio Rad) using a two-step carbodiimide reaction at a ratio is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Coupling efficiency of the recombinant RBD was assessed as above using the anti-564

His Tag antibody (#A00174, GenScript), in place of the IgG detector conjugate. 565

The non-competitive ACE2 inhibition assay was performed as above with the 567 exception that antibody was allowed to incubate first for 1 hour with the RBD variant 568 bead cocktail, before addition of biotinylated ACE2 for a further hour. 569 570

The virus microneutralization test was performed as previously described (38, 39). 572

Briefly SARS-CoV-2 isolate CoV/Australia/VIC01/2020 (58) is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint N439K  E484K  S494P  T478I  F490S  G446V  A475V  E484Q  L455F  G476S  F490L  G446S  V483A  V503F  Q493L  E484A  R403K  E484D  V445I  V483F  V483I 

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