key: cord-0303128-ti6yi3tz
authors: Kalkeri, Raj; Cai, Zhaohui; Lin, Shuling; Farmer, John; Kuzmichev, Yury V.; Koide, Fusataka
title: SARS-CoV-2 Spike Pseudoviruses: A Useful tool to study virus entry and address emerging neutralization escape phenotypes
date: 2021-07-19
journal: bioRxiv
DOI: 10.1101/2021.07.16.452709
sha: 545e8529b1c6486a773e25dd53158ddec034cad3
doc_id: 303128
cord_uid: ti6yi3tz

SARS-CoV-2 genetic variants are emerging around the globe. Unfortunately, several SARS-CoV-2 variants, especially, variants of concern (VOC) are less susceptible to neutralization by the convalescent and post-vaccination sera, raising concerns of increased disease transmissibility and severity. Recent data suggests the SARS-CoV-2 neutralizing anti-body levels as a good correlate of vaccine mediated protection. However, currently used BSL3 based virus micro-neutralization (MN) assays are more laborious, time consuming and expensive, underscoring the need for BSL2 based, cost effective neutralization assays against SARS-CoV-2 variants. In light of this unmet need, we have developed a BSL2 pseudovirus based neutralization assay (PBNA) in cells expressing Angiotensin Converting Enzyme-2 (ACE2) receptor for SARS-CoV-2. The assay is reproducible (R2=0.96), demonstrates a good dynamic range and high sensitivity. Our data suggests that the biological Anti-SARS-CoV-2 research reagents such as NIBSC 20/130 show lower neutralization against B.1.351 RSA and B1.1.7 UK VOC, whereas a commercially available monoclonal antibody MM43 retains activity against both these variants. SARS-CoV-2 Spike Pseudovirus based neutralization assays for VOC would be useful tools to measure the neutralization ability of candidate vaccines in both preclinical models and clinical trials and further help develop effective prophylactic countermeasures against emerging neutralization escape phenotypes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of 29 coronavirus dis-ease 2019 , remains a major global health challenge responsible for more 30 than 4-million deaths since the start of the pandemic in December 2019, with an estimated 187 million 31 confirmed cases as of 12 July 2021 [1] . While the development of novel prophylactic and therapeutic 32 measures, such as vaccines, monoclonal antibodies, and antiviral medications have been instrumental in 33 slowing down the pace of the pandemic, emergence of several variants of concern (VOC) has raised 34 issues about potential immune escape [2] . SARS-CoV-2, one of the largest known RNA viruses with ~29.8 35 kb genome, is an enveloped, positive-sense single-stranded RNA virus, belonging to the genus of β-36 coronaviruses of the coronaviridae family [3] . The spike (S) glycoprotein, present on the surface of the 37 virion is responsible for host cell attachment and fusion with the human angiotensin-converting enzyme 38 2 (hACE2) receptor. S glycoprotein is currently considered a major immunogenic component of . Measuring the effect of neutralizing antibodies, an important correlate of protection, 40 against the S glycoprotein is of primary importance in fighting the pandemic. Currently a number of 41 clinical trials investigating such therapeutic interventions are ongoing [7] . 42

In order to determine the efficacy of approaches for measuring the effect of neutralization 43 against emerging variants of concern, assays that are capable of measuring serological responses to the 44 spike glycoprotein are of extreme importance. Current assays used for such purposes rely on principles 45 of microneutralization (MN) or enzyme-linked immunosorbent assays (ELISA), and several ELISA 46 derivatives [8] [9] [10] . SARS-CoV-2 MN assays relying on the neutralization of a wild-type, replicating virus, 47

Pseudovirus based neutralization assay (PBNA) for SARS-CoV-2 spike protein variants (614D, 120 B1.1.7 and B1.357) was developed in HEK293T-cells expressing ACE2 receptor for SARS-CoV-2. A set of 121 positive controls (NIBSC 20/130, Acro-SAD-S35 and Sino-40591-MM43) were tested in this assay system. 122

We demonstrate that NIBSC 20/130 shows significantly reduced neutralization against B1.357 RSA and 123 B1.1.7 UK variants, whereas Sino-40591-MM43 show better neutralization against these variants. 124

To develop the pseudovirus based assay, HEK293T-hACE2 cells were infected with increasing 126 concentrations of SARS-CoV-2 spike protein pseudovirus variants. Luciferase signal in the infected cells 127 was analyzed after 3-days post-infection. As shown in Figure 1 , there was a dose dependent increase in 128 signal for all three pseudo-viruses with a dynamic range of 2-logs. Pseudoviruses for 614D and B1. assay, which might be due to preexisting antibodies (may be due to prior exposure to other 156 Coronaviruses). More than 2-log difference between the negative and positive controls confirm the 157 significant assay dynamic range, which could enable screening antibodies with ability to differentiate 158 between samples with varying neutralization potencies against SARS-CoV-2. 159

The assay was repeated on two different days by the same operator. Data from both the 160 experiments were compared and shown in Figure 2 . Positive controls showed robust activity in both the 161 assays with mean ± SD of 10,522.5 ± 907.2 and 14,944 ± 1,077.6 in the assay. Both sets of data showed 162 good correlation with an R 2 value of 0.96, suggesting that the assay is reproducible. The data also 163 suggests that all three monkey species (Rhesus Macaques, Cynomolgus monkeys, African Green 164 Monkeys) developed neutralizing antibodies (Mean ± SD of 298.7 ± 228.9, 313.9 ± 98.8, 665.1 ± 191.7) 165 respectively. 166 concern. To evaluate the neutralizing antibodies against the variants of concern, we tested 6-serum 178 samples from monkeys challenged with SARS-CoV-2 USA_WA1/2020 strain against pseudoviruses 179 expressing spike proteins of B1.315 RSA and B1.1.7 UK variants, in PBNA. As shown in Figure 3 (1M16756, 3M16763, 3F16765) with high PBNI50 were serially diluted 5-fold to generate test samples 211 with high, medium and low PBNI50, followed by PBNA with 6-point dilution in triplicates at each dilution. 212

Average ± SD of the three PBNI50 for high, medium and low test samples are shown on the Y-axis. X-axis 213 denotes the test samples with high, medium and low PBNI50. Trend analysis between the three groups is 214 shown by the dotted line (R 2 =0.9978). 215

A universal standard is necessary to harmonize the assays in different laboratories around the 218 globe. Such a standard would facilitate the comparison of assay results from diverse laboratories. 219

Currently, NIBSC 20/130 research reagent (anti-SARS-CoV-2 antibody) offered by NIBSC is intended to be 220 used as a positive control for the development and evaluation of serological assays to detect antibodies 221 against SARS-CoV-2. To evaluate the activity of the NIBSC 20/130 in PBNA, it was tested against all three 222 pseudoviruses (614D, B1.357 RSA and B1.1.7 UK variant). Two other positive controls (Acro-SAD-S35 and 223

Sino-40591-MM43) were also included in the assay. As shown in Figure. 5A and Table 1 Assay dynamic range helps in the appropriate differentiation of the vaccines with differing 245 neutralization potencies. Besides measuring the 50% inhibition, a wide dynamic range also enables 246 differentiation of robust inhibition (90%-99%) and can help rank order the vaccine candidates based 247 upon their neutralization profiles. Dose dependent, two (2) It is also interesting to note that 5 out of 6 normal unexposed NHP samples tested (data not 276 shown) in our assay showed high background neutralization index (PBNI50 of 100 to 250) against 614D. 277

Only one of the NHPs showed a PBNI50 of <10. As we were not sure about the reason/specificity of this 278 background PBNI50 level in the unexposed NHP serum samples, we serially diluted our SARS-CoV-2 279 challenged NHP serum samples with high PBNI50 levels (>500) to generate test samples with tiered 280 PBNI50 levels (high, medium and low) followed by evaluation in PBNA. This experiment also enabled the 281 measurement of linearity and lower limit of detection of neutralization index in PBNA. Our experiments 282 confirmed the linearity of detection to approximately PBNI50 of 30 (the lowest PBNI50 tested in our 283 assay), albeit with high %CV at the lower end of detection. As we confirmed the sensitivity of PBNA, high 284 background levels (PBNI50 of 100 to 250) in 5 out of 6 unexposed NHP serum samples, might be due to 285 pre-exposure to other coronaviruses in their natural environment (which might have generated neutralizing antibodies). Couple of previous reports about preexisting humoral immunity to in humans support this explanation [31, 32] MM43 is retained against other variants of concern such as B1.617 delta and kappa variants. 303

Nevertheless, as Sino 40591-MM43 is a commercially available monoclonal antibody, one could envision 304 accessibility across the globe with unlimited supply (as it can be scaled up due to the monoclonal 305 nature). This could further enhance the global harmonization of the neutralization and serological assays 306 against SARS-CoV-2 variants of concerns. 307

Our findings demonstrate successful development of a robust pseudovirus based neutralization 309 assay against SARS-CoV-2 variants of concern, which can aid evaluation of preclinical and clinical 310 samples and rapidly develop effective countermeasures against SARS-CoV-2 and the variants of concern. 311 Our results also identified a commercially available monoclonal antibody that can serve as a potential 312 global standard in the neutralization assays against SARS-CoV-2 variants of concern.

Author Contributions: Conceptualization, R.K. and F.K.; methodology, Z.C., S.L. and Y.V.K.; writing-314 original draft preparation, R.K., Y.V.K. and Z.C.; writing-review and editing, R.K., F.K. and J.F. All authors 315 have read and agreed to the published version of the manuscript. 

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