Surrogate virus neutralisation test based on nanoluciferase-tagged antigens to quantify inhibitory antibodies against SARS-CoV-2 and characterise Omicron-specific reactivity

Virus-specific antibodies are important determinants of protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While regarded as the gold standard for detecting functional antibodies, conventional virus neutralisation tests (VNT) or pseudotyped virus neutralisation tests (pVNT) require biosafety level 2 or 3 facilities. Alternatively, the virus-free surrogate virus neutralisation test (sVNT) quantifies inhibitory antibodies that prevent the spike protein from binding to its receptor, human angiotensin-converting enzyme 2 (hACE2). We evaluated secreted nanoluciferase (NLuc)-tagged spike (S) protein fragments as diagnostic antigens in the sVNT in the framework of a vaccination study. First, spike fragments of different lengths were tested for their suitability as diagnostic antigens in a capture enzyme immunoassay (EIA) using unprocessed culture supernatants of transfected cells, identifying the receptor binding domain (RBD) of S as the optimal construct. The sensitivity of the in-house sVNT relying on the NLuc-labelled RBD equalled or surpassed a commercial sVNT ( cPass , GenScript Diagnostics) and an in-house pVNT four weeks after the first vaccination (98% vs. 94% and 72%, respectively), reaching 100% in all assays four weeks after the second and third vaccinations. Additionally, serum reactivity with spike constructs of Omicron BA.1 was tested. Compared with a capture EIA, the in-house sVNT and pVNT displayed superior discrimination between wild-type- and variant-specific reactivity of sera. Differences in reactivity were most pronounced after the first and second vaccinations, whereas the third vaccination resulted in robust, cross-reactive detection of Omicron constructs. In conclusion, assays utilising NLuc-labelled protein fragments permit the quantification and functional assessment of SARS-CoV-2-specific antibodies and the detection of variant-specific differences in reactivity. Potential applications include monitoring therapy and vaccine efficacy and follow-up of prolonged disease courses in high-risk groups. Designed as straightforward, highly flexible modular systems, these tests can be readily adapted to further emerging viral variants. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was partially funded by the Ministry of Labour, Health and Social Affairs of the state of North Rhine-Westphalia, Germany (grant CPS-1-1G). The Institute of Virology is part of the Virus Alliance North Rhine-Westphalia (VIRAL.NRW), which is supported by the Ministry of Culture and Science, North Rhine-Westphalia, Germany. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethik-Kommission der Aerztekammer Westfalen-Lippe und der Westfaelischen Wilhelms-Universitaet, Muenster, Germany, gave ethical approval for this work (2021-039-f-S). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study that are not contained in the manuscript are available upon reasonable request to the authors.