key: cord-0965945-z2d92mja
authors: Miyakawa, K.; Jeremiah, S. S.; Kato, H.; Yamaoka, Y.; Go, H.; Yamanaka, T.; Ryo, A.
title: Rapid detection of neutralizing antibodies to SARS-CoV-2 variants in post-vaccination sera
date: 2021-05-10
journal: nan
DOI: 10.1101/2021.05.06.21256788
sha: f07dcff7e5bb4ad77d00876b451d885bbd369c5f
doc_id: 965945
cord_uid: z2d92mja

The uncontrolled spread of the COVID-19 pandemic has led to the emergence of different SARS-CoV-2 variants across the globe. The ongoing global vaccination strategy to curtail the COVID-19 juggernaut, is threatened by the rapidly spreading Variants of Concern (VOC) and other regional mutants, which are less responsive to neutralization by infection or vaccine derived antibodies. We have previously developed the hiVNT system which detects SARS-CoV-2 neutralizing antibodies in sera in less than three hours. In this study, we modify the hiVNT for rapid qualitative screening of neutralizing antibodies (nAb) to multiple variants of concern (VOC) of SARS-CoV-2, and assess the neutralizing efficacy of the BNT162b2 mRNA vaccine on seven epidemiologically relevant SARS-CoV-2 variants. Here we show that the BNT162b2 mRNA vaccine can activate humoral immunity against the major SARS-CoV-2 mutants that are currently in circulation. Albeit a small sample size, we observed that one dose of vaccine was sufficient to elicit a protective humoral response in previously infected people. Using a panel of seven SARS-CoV-2 variants and a single prototype virus, our modified hiVNT would be useful for large-scale community wide testing to detect protective immunity that may confer vaccine/immune passport in the ongoing COVID-19 pandemic.

The uncontrolled spread of the COVID-19 pandemic has led to the emergence of different SARS-CoV-2 variants across the globe. The ongoing global vaccination strategy to curtail the COVID-19 juggernaut, is threatened by the rapidly spreading Variants of Concern (VOC) and other regional mutants, which are less responsive to neutralization by infection or vaccine derived antibodies 1,2 .

We have previously developed the hiVNT (HiBiT-based virus-like particle neutralization test) system which detects SARS-CoV-2 neutralizing antibodies in sera in less than three hours 3 . It uses lentivirus-based virus-like particles (VLP) incorporated with the NanoLuc fragment peptide, HiBiT. Viral entry into reporter cells that express LgBiT intracellularly, allows the viral HiBiT to fuse with the LgBiT to reconstitute whole NanoLuc luciferase which is readily detected by luminometer 3, 4 . The hiVNT assay is capable of high throughput and can be easily carried out in a low biosafety setting. We have modified the hiVNT for rapid qualitative screening of neutralizing antibodies (nAb) to multiple variants of concern (VOC) of SARS-CoV-2. In the current study, we assess the neutralizing efficacy of the BNT162b2 mRNA vaccine on a panel of seven epidemiologically relevant SARS-CoV-2 variants.

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The copyright holder for this preprint this version posted May 10, 2021. ; https://doi.org/10.1101/2021.05.06.21256788 doi: medRxiv preprint

Using the spikes of the prototype virus (D614G) and seven variants (B. Figure 1A ), we generated HiBiT-containing VLPs (hiVLP). If the serum nAbs neutralize the mutant hiVLPs, their entry into the reporter cells is prevented, reducing the luminescence emitted ( Figure 1B) . We optimized the conditions of the hiVNT using sera of COVID-19 patients (n=97) and healthy individuals (n=43) with pre-defined neutralization titers (NT50) by the pseudovirus-based neutralization assay. At a fixed serum dilution of one in twenty, the hiVNT assay could detect the nAb titers corresponding to NT50 of around 50 which is the positive cut-off in the standard pseudovirus neutralization assay. We then determined that a sample possessed neutralization activity if it showed more than 40% luminescence signal inhibition in the hiVNT assay ( Figure 1C ). The receiver operating characteristic (ROC) curve showed that this cut-off value of 40% can detect the presence of nAbs with a high degree of accuracy ( Figure 1D ). This modification of performing the hiVNT at a fixed dilution makes the procedure less laborious, reduces the turn-around-time while achieving performance similar to the standard neutralization assays.

The study samples were collected from otherwise healthy medical personnel who were administered with two doses of the vaccine three weeks apart. Serum of the vaccinees was collected at three instances: before the first dose, two weeks after first dose and one week after second dose. All sera collected prior first vaccine shot was checked for seropositivity using NP-IgG detection assay 5 to get two vaccine recipient groups: previously uninfected . CC-BY 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 May 10, 2021. ; https://doi.org/10.1101/2021.05.06.21256788 doi: medRxiv preprint A steady increase in nAbs was observed after the first dose, followed by the achievement of significant nAb levels against all mutants in most people after the second dose (Figure 2A , right panel). The vaccine derived nAbs possessed high median inhibition rates of above 94% for all variants except B.1.351 which was 89.5% (94/105). All the six individuals with past COVID-19 infection achieved significant levels of nAb after the first dose despite some possessing nAbs below threshold prior initial vaccination ( Figure 2B ). To clear this doubt on vaccine escape, we developed an extensive mutant panel including the recently surging B.1.617 double mutant (E484Q and L452R) which is presumed to have higher propensity to escape nAbs. In this first report of vaccine efficacy assessment from Japan, we reveal that the BNT162b2 mRNA vaccine has adequate efficacy against the epidemiologically relevant SARS-CoV-2 variants. Vaccine escape was noted in all mutants but only in a minor fraction, which was higher for the B.1.351 mutant. Albeit a small sample size, we observed that one dose of vaccine was sufficient to elicit protective humoral response in previously infected people as documented by previous reports 6 . Despite the presence of their corresponding nAbs in the serum, mutants can cause vaccine breakthrough infections, suggesting that the circulating nAbs might not offer protection at mucosal surfaces to prevent infection 7 .

In the current study, we show that the BNT162b2 mRNA vaccine can activate humoral immunity against the major SARS-CoV-2 mutants that are currently in circulation. Our modified hiVNT possesses the advantages of being rapid, safe, concise and comprehensive . CC-BY 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 May 10, 2021. ; https://doi.org/10.1101/2021.05.06.21256788 doi: medRxiv preprint over the conventional neutralization assays. However, this modified hiVNT assay is qualitative and cannot quantify the nAb titers at relatively higher levels that maybe relevant to their long-term persistence and duration of protection. Using a panel of seven SARS-CoV-2 variants and a single prototype virus, our modified hiVNT would be useful for large-scale community wide testing to detect protective immunity that may confer vaccine/immune passport in the ongoing COVID-19 pandemic. The selection pressure exerted by the current vaccines would cause the evolution of vaccine escape mutants, which must be monitored in the future and for which the hiVNT assay developed using the spikes of new variants could come in handy.

. CC-BY 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 May 10, 2021. ; https://doi.org/10.1101/2021.05.06.21256788 doi: medRxiv preprint

This study was approved by Yokohama City University Certified Institutional Review Board (Reference No. B160800009, B200600115, B210300001) , and the protocols used in the study were approved by the ethics committee. Written informed consent was obtained from all the participants. The luminescence signal inhibition (%) was calculated as follows: (C) Correlation of hiVNT with pseudovirus NT. Sera with the luminescence signal inhibition <40% can be deemed negative for neutralizing antibodies. ***p < 0.001, ****p < 0.0001, ns, not significant, two-tailed unpaired t-test. . CC-BY 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 May 10, 2021. ; https://doi.org/10.1101/2021.05.06.21256788 doi: medRxiv preprint . CC-BY 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 May 10, 2021. ; https://doi.org/10.1101/2021.05.06.21256788 doi: medRxiv preprint

Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature

Emerging SARS-CoV-2 Variants and Impact in Global Vaccination Programs against SARS-CoV-2/COVID-19. Vaccines (Basel)

Rapid quantitative screening assay for SARS-CoV-2 neutralizing antibodies using HiBiT-tagged virus-like particles

CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide

Development of an Automated Chemiluminescence Assay System for Quantitative Measurement of Multiple Anti-SARS-CoV-2 Antibodies

mRNA vaccination boosts cross-variant neutralizing antibodies elicited by SARS-CoV-2 infection

Vaccine Breakthrough Infections with SARS-CoV-2 Variants

This study was supported by Rapid research and development Projects on COVID-19 of AMED (JP19fk0108110) to AR. We thank Kenji Yoshihara, Kazuo Horikawa, and Natsumi Takaira for their technical assistance. We acknowledge all patients and medical staff involved in the study.