key: cord-0947100-u2xatjcy authors: Miyakawa, K.; Jeremiah, S. S.; Ohtake, N.; Matsunaga, S.; Yamaoka, Y.; Nishi, M.; Morita, T.; Saji, R.; Nishii, M.; Kimura, H.; Hasegawa, H.; Takeuchi, I.; Ryo, A. title: Rapid quantitative screening assay for SARS-CoV-2 neutralizing antibodies using HiBiT-tagged virus-like particles date: 2020-07-22 journal: nan DOI: 10.1101/2020.07.20.20158410 sha: c5cc34c6b85e55afd6fff6d2dc18acaf5d5766d4 doc_id: 947100 cord_uid: u2xatjcy SARS-CoV-2 neutralizing antibodies confer protective immunity against reinfection. We have developed a rapid test for screening SARS-CoV-2 neutralization antibodies using genome-free virus-like particles incorporated with a small luciferase peptide, HiBiT. Their entry into LgBiT-expressing target cells reconstitutes NanoLuc luciferase readily detected by a luminometer. This newly developed HiBiT-tagged Virus-like particle-based Neutralization Test (hiVNT) can readily quantify SARS-CoV-2 neutralizing antibodies within three hours with a high-throughput in a low biosafety setting. Moreover, the neutralizing activity obtained from hiVNT was highly consistent with that measured by the conventional neutralization test using authentic SARS-CoV-2. Furthermore, antibody responses to both viral spike and nucleocapsid proteins correlated with the neutralization activity assessed by hiVNT. Our newly-developed hiVNT could be instrumental to survey individuals for the presence of functional neutralizing antibody against SARS-CoV-2. Due to the unavailability of any specific countermeasure, the constantly spreading COVID-19 pandemic could only be partially and temporarily slowed down by implementing regional lockdowns that force people to stay at home and prevent their movement. With the progression of the pandemic, a considerable subset of the population would have acquired post infection immunity and tests that reveal the post infection immune status of individuals are the need of the hour. A credible test which accurately identifies the protected can offer an immunity passport for the individual to be freed from the lockdown and resume routine activities without the fear of getting infected. At present, the semi-quantitative neutralization test (NT) and the quantitative plaque reduction neutralization test (PRNT) which identifies the presence of anti-SARS-CoV-2 neutralizing antibodies (nAbs) are the only foolproof methods available for this purpose. However, practical feasibility of these highly specific . 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 July 22, 2020. . https://doi.org/10.1101/2020.07.20.20158410 doi: medRxiv preprint tests is weighed down by drawbacks such as; low throughput, long turnaround time (TAT) and the need for a specialized laboratory setup with biosafety level 3 (BSL3) facilities to handle the live viruses used in these tests 1 . To overcome these hurdles, pseudovirus-based NT 2 , surrogate serodiagnostic tests (sVNT) 3 , and single cell RNA sequencing 1 are being studied. However, a simple, convenient, rapid and high-throughput test capable of directly detecting nAbs with high specificity which could act as an ideal alternative to the neutralization assay is yet to be developed 4 . Virus-like particles (VLPs) are self-assembling, non-replicating, nonpathogenic entities of similar size and conformation as that of infectious virions. VLPs can be generated to possess the surface proteins of any kind of viruses on their membrane devoid of the viral nucleic acids. This genome-free feature of VLPs overrides the need for BSL3 facilities while handling, but has the drawback of difficulty in quantifying its cell entry and membrane fusion. HiBiT is an 11 amino acid peptide tag that can be attached to any protein-of-interest. LgBiT, the counterpart of NanoLuc luciferase is complementary to HiBiT and binds to the latter to produce a highly active luciferase enzyme. HiBiT tagged proteins can be easily detected and quantified based on bioluminescence using the Nano-Glo assay system. HiBiT technology offers the advantages of high sensitivity, convenience of a single-reagent-addition step and short TAT of only a few minutes. In this report, we have developed a HiBiT-VLP-based neutralization test (hiVNT) that can readily detect SARS-CoV-2 nAbs. The assay system utilizes VLPs bearing SARS-CoV-2 spike protein on their surfaces, incorporated with HiBiT-tagged HIV-1 capsid protein. Fusion of these VLPs could be readily monitored and quantified using VeroE6/TMPRSS2 cells stably expressing LgBiT as target cells. This system retains the specificity of neutralization assays while overcoming all the pitfalls, as hiVNT can be instrumental to measure protective immunity against viral invasion and evaluate vaccine efficacy by rapid, high-throughput quantification of nAb in a BSL2 laboratory. . 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 July 22, 2020. . https://doi.org/10.1101/2020.07.20.20158410 doi: medRxiv preprint To establish a rapid quantitative cell-based neutralization assay using luminescence-tagged VLPs, we inserted the HiBiT tag to the HIV-1 Gag-Pol protein, a minimal subunit to produce VLPs (Fig. 1A) . After testing several prototypes, we found that HiBiT tag insertion at the Cterminal region of the capsid gene in Gag-Pol performed best. HiBiT tag can bind with high affinity to its complementary larger subunit LgBiT, and form luciferase (NanoLuc) 5 Since SARS-CoV-2 can infect VeroE6/TMPRSS2 cells with high efficiency 6 , we next generated LgBiT-expressing VeroE6/TMPRSS2 cells. We noticed a robust increase in NanoLuc activity when the LgBiT-expressing VeroE6/TMPRSS2 cells were treated with hiVLP-SARS2 for 3 hours (Fig. 1D, E) . We further demonstrated the drop in luminescence signals upon pretreatment with a TMPRSS2 inhibitor (Camostat mesylate) and also with anti-Spike neutralizing monoclonal antibody (Fig. 1F ), suggesting that hiVLP-SARS2 enters the target cells through the interactions between viral spike and cellular receptors in a similar way as that of authentic SARS-CoV-2. We next tested whether our newly developed hiVLP-SARS2 system could detect nAbs in the serum of COVID-19 patients. The neutralization assay was carried out in accordance with the following steps. VeroE6/TMPRSS2-LgBiT cells were seeded on 96-well white polystyrene plates at a density of 10 4 cells/well one day prior to inoculation. Decomplemented sera derived from convalescent COVID-19 patients were mixed with hiVLP-SARS2 and the mixture was inoculated to VeroE6/TMPRSS2-LgBiT cells. Three hours later, the cells were washed with PBS and measured for their NanoLuc luminescence. . 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 July 22, 2020. . https://doi.org/10.1101/2020.07.20.20158410 doi: medRxiv preprint We found that all the patients' sera tested could block the entry of hiVLP-SARS2 in varying degrees ( Fig. 2A) , suggesting the presence of nAbs against SARS-CoV-2. We thus developed the hiVLP-SARS2-based Neutralization Test (hiVNT). Two convalescent sera and one healthy donor serum were tested by hiVNT and authentic SARS-CoV-2 NT only to show that all the samples gave similar results in both tests (Fig. 2B) , suggesting that hiVNT performance is in concordance with the NT using authentic SARS-CoV-2. Since surrogate antibody detection is being widely studied as an alternative to NT, we wanted to check the correlation of nAbs detected by hiVNT with antibody titers detected by ELISA. We matched the results of hiVNT against the antibody titers detected by ELISA in 74 COVID-19 positive sera. nAb levels were found to correlate with IgG antibodies against both the spike and the nucleocapsid proteins ( Fig 3A) , but not with IgM (Fig. 3B) . Taken together, our novel hiVNT could be useful for rapid detection of nAbs in the sera of individuals recovered from COVID-19. We have developed the hiVNT, a novel platform to rapidly detect and quantify SARS-CoV-2 nAbs using genome-free VLPs and HiBiT technology. The assay principle is similar to conventional neutralization assays and is based on viral entry and membrane fusion with measurement of NanoLuc luciferase activity to simplify the outcome. Considering the drawbacks of NT and PRNT, tests that may confer immunity passport to individuals are the need of the hour and different platforms are being exploited for this purpose, even though more detailed serological studies are needed. Pseudovirus-based NTs are the popular alternatives to detect nAbs, as they overcome the need for BSL3 facility and have a high-throughput 7 . These tests employ pseudoviruses with SARS-CoV-2 spike proteins for entry possessing a genome inserted with a reporter gene. The test depends on the expression of the reporter gene in the target cells . 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 July 22, 2020. patients is being studied for its potential to act as a surrogate marker to reflect the presence of SARS-CoV-2 nAbs 3 . Our findings in a small sample size (n=74) reveal that IgG could serve as a better surrogate marker than IgM (Fig. 3) . However, the use of serodiagnostics for nAb detection could have a few inherent drawbacks. Although all COVID-19 infections elicit a humoral immune response, the presence of antibodies does not reflect immunity. Also, mild COVID-19 infections elicit very low humoral response which might not be detected by serological tests 4 . In this setting, surrogate antibody detection tests can produce erroneous results. Moreover, SARS-CoV-2 infects host cells through receptor binding domain (RBD) within S1 subunit in spike protein on the surface of viral particles that bind to host surface angiotensin converting enzyme-2 (ACE2) receptor. ELISA kits to detect anti-RBD antibodies use in vitro generated spike proteins and its derivatives to detect nAbs in serum. However, since spike proteins undergo glycosylation and oligomerization in vivo 8, 9 , the performance of in vitro generated spike proteins may vary depending on the manufacturing method, as well as their clinical significance. Also, nAbs that target other regions of spike protein to suppress the function of fusion peptide of S2 subunit may exist which could go undetected in S1 and RBD based detection ELISA 10 . In this study, we established the hiVNT; a simple, high-throughput assay system for the quantitative and rapid determination of SARS-CoV-2 nAbs in the sera of individuals after recovery from symptomatic or subclinical COVID-19 infection. The hiVNT system using VLPs, allows for BSL2-compliant testing and gives output in a short time. The concurrent use of the HiBiT system enhances the assay to give high-throughput quantitative . 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 July 22, 2020. . https://doi.org/10.1101/2020.07.20.20158410 doi: medRxiv preprint 7 results. We believe that the hiVNT can be instrumental in identifying individuals with protective immunity, epidemiological studies on population susceptibility, propagation modeling, assessing convalescent plasma used for therapy and vaccine evaluation; all these at a high-throughput and short TAT in a lower biosafety setting. . 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 July 22, 2020. . https://doi.org/10.1101/2020.07.20.20158410 doi: medRxiv preprint This study was approved by Yokohama City University Certified Institutional Review Board (Reference No. B200200048, B160800009) , and the protocols used in the study were approved by the ethics committee. Written informed consent was obtained from the patient or family/guardian. Samples in SDS loading buffer were loaded onto 10-20% polyacrylamide gels (Wako), electrophoresed, and blotted onto PVDF membranes (Merck). Membranes were probed with anti-SARS-CoV-2 Spike S2 (clone 1A9, GeneTex #632604) or anti-HIV p24 (clone 183-H12-5C, NIH AIDS Reagent Program #3537) antibodies and horseradish peroxidaseconjugated secondary antibodies (Cytiva). Detected proteins were visualized using LuminoGraph imaging system (ATTO). . 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 July 22, 2020. . 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 July 22, 2020. All bar graphs present means and standard deviation. The statistical significance of differences between two groups was evaluated by two-tailed unpaired t-test in the Prism 8 software (GraphPad). . 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 July 22, 2020. The authors have no conflicts of interest directly relevant to the content of this article. YY is a current employee of Kanto Chemical Co. Inc. KM designed and performed the research, analyzed the data, and wrote the manuscript; SSJ analyzed the data, and wrote the manuscript; NO, SM, YY, M.Nishi, TM performed the research and analyzed the data; RS, M.Nishii, IT contributed reagents; HK, HH analyzed the data; AR directed the research, analyzed the data, and wrote the manuscript. . 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 July 22, 2020. after which the intracellular NanoLuc luminescence was measured. ****P < 0.0001. . 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 July 22, 2020. . https://doi.org/10.1101/2020.07.20.20158410 doi: medRxiv preprint Potent neutralizing antibodies against SARS-CoV-2 identified by high-throughput single-cell sequencing of convalescent patients' B cells Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2 A SARS-CoV-2 surrogate virus neutralization test (sVNT) based on antibody-mediated blockage of ACE2-spike (RBD) protein-protein interaction COVID-19 Task Force of theGerman Society for Clinical Chemistry and Laboratory Medicine (DGKL) Development of a rapid and quantitative method for the analysis of viral entry and release using a NanoLuc luciferase complementation assay Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein Oligomerization of the SARS-CoV S glycoprotein: dimerization of the N-terminus and trimerization of the ectodomain A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2 . 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 July 22, 2020. . https://doi.org/10.1101/2020.07.20.20158410 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. The copyright holder for this preprint this version posted July 22, 2020. . 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 July 22, 2020. . https://doi.org/10.1101/2020.07.20.20158410 doi: medRxiv preprint