key: cord-0884012-74g4snva
authors: Fenwick, C.; Turelli, P.; Pellaton, C.; Farina, A.; Campos, J.; Raclot, C.; Pojer, F.; Cagno, V.; Pantaleo, G.; Trono, D.
title: A multiplexed high-throughput neutralization assay reveals a lack of activity against multiple variants after SARS-CoV-2 infection
date: 2021-04-13
journal: nan
DOI: 10.1101/2021.04.08.21255150
sha: b1a19e5d56677b40e691f4cc50955476611625f7
doc_id: 884012
cord_uid: 74g4snva

The detection of SARS-CoV-2-specific antibodies in the serum of an individual indicates prior infection or vaccination. However, it provides limited insight into the protective nature of this immune response. Neutralizing antibodies recognizing the viral Spike are far more revealing, yet their measurement traditionally requires virus- and cell-based systems that are costly, time-consuming, poorly flexible and potentially biohazardous. Here we present a cell-free quantitative neutralization assay based on the competitive inhibition of trimeric SARS-CoV-2 Spike protein binding to the angiotensin converting enzyme 2 (ACE2) viral receptor. This high-throughput method matches the performance of the gold standard live virus infectious assay, as verified with a panel of 206 seropositive donors with varying degrees of infection severity and virus-specific IgG titers, achieving 96.7% sensitivity and 100% specificity. Furthermore, it allows for the parallel assessment of neutralizing activities against multiple SARS-CoV-2 Spike variants of concern (VOC), which is otherwise unpredictable even in individuals displaying robust neutralizing antibody responses. Profiling serum samples from 59 hospitalized COVID-19 patients, we found that although most had high activity against the 2019-nCoV Spike and to a lesser extent the B.1.1.7 variant, only 58% could efficiently neutralize a Spike derivative containing mutations present in the B.1.351 variant. In conclusion, we have developed an assay that has proven its clinical relevance in the large-scale evaluation of effective neutralizing antibody responses to VOC after natural infection and that can be applied to the characterization of vaccine-induced antibody responses and of the potency of human monoclonal antibodies.

instance, the B.1.351 (also called 501Y.V2 or South African variant of concern (VOC)) has been found 90 largely to escape immunity induced by some COVID-19 vaccines,(15) and the closely related P.1 (also 91 called 501Y.V3, B.1.1.28 or Brazilian VOC) to be responsible for large numbers of reinfections in the 92

To address these challenges, we developed a cell-free neutralization assay based on the competitive 94 inhibition of ACE2 binding to S protein trimers-bearing beads. This method is high-throughput, 95 quantitative, yields results tightly correlating those obtained with a classical wild-type virus cell-based 96 neutralization assay, and allows the simultaneous evaluation of multiple S protein variants. Illustrating 97 its value, it reveals that some previously infected individuals display variant-specific serum neutralizing 98 antibody titers, suggesting that they remain at risk of infection with at least some VOCs. Based on 99 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 13, 2021. ; https://doi.org/10.1101/2021.04.08.21255150 doi: medRxiv preprint parallel with the cell-free S 3 -ACE2 assay and a conventional live CPE assay in Vero cells. The results 132 revealed a high correlation of the neutralizing titers obtained with the two assays (R 2 = 0.825) over the 133 >3 log range measured amongst the various samples (Figure 3a) . We additionally subjected a subset 134 of these serums (n=75) to a LV reporter pseudotype-based neutralization assay, which revealed a 135 weaker correlation with the live virus assay (R 2 = 0.65, n=75; Figure 3b) . 136

For the S 3 -ACE2 neutralization assay, a lower limit IC50 serum dilution of 50 was set as the specificity 137 cutoff using IC50 values for the 104 pre-COVID-19 pandemic healthy donor samples (mean IC50 of 12.5 138 + 4 × 9.0 SD; Figure 1b) . A serum dilution IC50 of 20 was selected as the cutoff for positivity in the live 139 SARS-CoV-2 virus CPE assay given that this corresponds to 95-99% viral neutralization (using Nonlinear 140 regression with 1 and 1.5 Hill slopes values to achieve IC95 and IC99, respectively; Supplemental Figure  141 1) for the undiluted serum sample.(23) Using these criteria, the surrogate neutralization assay was 142 determined to achieve 96.7% sensitivity (118 out of 122) and 100% specificity (0 out of 84) relative to 143 the live virus CPE assay (Table 1) Addressing this shortcoming, we report here the development of a cell-free assay that allows for the 208 quantitative and high-throughput evaluation of the neutralizing activity of biological samples such as 209 serum against multiple SARS-CoV-2 variants in a single procedure taking less than three hours in a 210 standard diagnostic laboratory. The S 3 -ACE2 assay relies on the fact that most neutralizing antibodies 211

interfere with the binding of the viral S protein with its ACE2 receptor. Although neutralizing antibodies 212 have been identified that recognize S protein outside of the RBD (9) and do not directly impact on ACE2 213 binding, these are rare and likely contribute minimally to antiviral immunity, as confirmed by the very 214 high degree of correlation between our surrogate assay and its live virus cell-based reference 215 counterpart, irrespective of the levels of neutralizing activity.(8) Our assay provides quantitative 216 measures of serum neutralizing antibody levels and is further characterized by a very high degree of 217 sensitivity and specificity (>96% and 100%, respectively, using the herein described protocol). 218 A significant advantage of the S 3 -ACE2 neutralization assay is its ability to evaluate multiple S variants 219 in parallel using as little at 15 µl of serum, allowing the identification of an individual's susceptibility to 220 circulating and emerging SARS-CoV-2 viruses, whether after infection or vaccination. This was 221 demonstrated with serum samples from 59 COVID-19 patients infected prior to the widespread 222 emergence of VOCs. Importantly, only 43% of non-ICU hospitalized patients had neutralizing antibody 223 levels greater than 50 serum dilution IC50 against the S protein with the K417N/E484K/N501Y mutation 224 found in the B.1.351 variant, suggesting that many are not protected against this strain. The P.1 variant 225 originally identified in Brazil has a similar mutation profile in the RBD and some of these non-ICU 226 patients would be anticipated to be equally susceptible to infection by this SARS-CoV-2 variant as well. 227

Individuals whose infection had required an ICU stay generally displayed higher levels of neutralizing 228 antibodies against all tested S protein variants, although they too were less effective against the 229 B1.351/P1 triple mutant. 230 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 13, 2021. ; https://doi.org/10.1101/2021.04.08.21255150 doi: medRxiv preprint

The multiplexing of the S 3 -ACE2 neutralization assay could be increased to >40 different S alleles to 231 tackle new VOCs. It advantageously compares with viral pseudotypes-based systems, where each S 232 protein mutant requires production of a new batch of virions that have to be tested in separate assays, 233 with their infectivity potentially affected by the mutations and neutralization titers influenced by the 234 levels of ACE2 on the surface of target cells (28, 29). As levels of anti-SARS-CoV-2 immunity increase 235 in the world population due to the combined influence of ongoing infections and more widespread 236 vaccination, selective pressures will increasingly be exerted on the virus favoring the emergence of 237 escape mutants. The detection of these escapees should be as fast as possible for the swift adaptation 238 of prophylactic measures including vaccines. While the infection of previously infected or vaccinated 239 individuals will remain the strongest evidence of gaps in the collective immunity, a surveillance system 240 based on the routine sequencing of viral isolates and the immediate testing of the susceptibility of 241 their S protein to neutralization would constitute a far more dynamic and anticipatory approach. The 242 S 3 -ACE2 assay, because of its ease of use, would facilitate such surveillance strategy. Furthermore, it 243 could also be used for the high-throughput screening of candidate monoclonal antibodies and other 244 prophylactic or therapeutic approaches aimed at blocking the interaction between SARS-CoV-2 and its 245 cellular receptor, and it could be adapted to other viruses for which the molecular mediators of viral 246 entry are properly characterized. 247 Therefore, the hereby described method stands to have an important impact in both clinical and public 248 health settings. In this regard, immunity passports are at the forefront of current public and political 249 discussions as possible gateways to a return to more normal social and international exchanges, as the 250 world emerges from the COVID-19 pandemics (30). They are generally thought of essentially as 251 vaccination certificates, a concept that suffers from major shortcomings. First, such certificates would 252 unduly exclude people that have not yet been vaccinated but endowed with strong antiviral immunity 253 triggered by natural infection. Second, they would not be delivered to individuals that do not respond 254 to vaccination such as primary or acquired immunodeficiency patients and/or cancer, transplant and 255 patients with systemic inflammatory diseases receiving immunosuppressive treatments. These 256 individuals could however be protected by passive immunization through the administration of human 257 monoclonal antibodies, the activity of which could be quantified in their serum (31-33). Third, having 258 been vaccinated is not a guarantee of induction of optimal immunity and protection, as found every 259

year with the flu vaccine, and the duration of vaccine-induced SARS-CoV-2 immunity is as yet unknown. in variants of concern including B.1.1.7 and P1. In these graphs, green background corresponds to an 330 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 13, 2021. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 13, 2021. ; https://doi.org/10.1101/2021.04.08.21255150 doi: medRxiv preprint Neutralization IC50 values were calculated as described above for the cell free neutralization 508 assay using the GraphPad prism NonLinear four parameter curve fitting analysis. Table 1 ).Spike-514 pseudotyped lentivectors were generated by co-transfecting HDM-IDTSpike-fixK, pHAGE2-515 CMV-Luc-ZSgreen, Hgpm2, REV1b and Tat1b (a kind gift from J.D. Bloom) plasmids into 293T 516 cells for 24 hours with the following ratio 3/9/2/2/2 (18µg/ 56.7cm2 plate) using Fugene 517 transfection reagent (Promega). The following day, cells were transferred in EpiSerf medium, 518 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 13, 2021. ; https://doi.org/10.1101/2021.04.08.21255150 doi: medRxiv preprint and cell supernatants were collected after 8 hours and 16 hours. Harvested supernatants were 519 pooled, clarified by low-speed centrifugation, filtered to remove cell debris and aliquoted. 520 Lentivector stocks were titrated and normalized for HIV antigen p24 content by ELISA 521 (Zeptometrix). 522 In the pseudoviral neutralization assay, 293T cells stably expressing the ACE2 receptor were 523 suspended in DMEM medium with 10% FCS and seeded at 1.0x10E+4 cells per well into 96-524 well plates. After 5 hours in cell culture at 37 °C, three-fold dilutions of serum samples were 525 prepared and pre-incubated with the same amount of each pseudovirus in a final volume of 526 100 µl in DMEM + 10% FCS. Following a further 1 hour incubation at 37 °C, the 527 pseudoviruses/serum mixture was added to the 293T ACE2 cells. After 48 hours of incubation 528 at 37 °C, a luciferase assay was performed to monitor pseudoviral infection, using the ONE- 529 Step TM Luciferase assay system as recommended by the manufacturer (BPS Bioscience). Viral 530 neutralization resulted in the reduction of the relative light units detected. Neutralization 531 IC50 values were calculated as described above for the cell free neutralization and CPE assays 532 using the GraphPad prism NonLinear four parameters curve fitting analysis. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 13, 2021. ; https://doi.org/10.1101/2021.04.08.21255150 doi: medRxiv preprint directed mutagenesis of the S protein constructs; F.P. and the Protein Production and Structure 559 Core Facility at the EPFL produced and purified the trimer S proteins. V.C. provided initial 560 stocks of titrated SARS-CoV-2 virus. D.T. and G.P. conceived the study design for testing of sera 561 from the different subject groups, analyzed the results and wrote the manuscript. 562 563

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