Multiplexed measurement of binding- and neutralizing antibodies to SARS-CoV-2 variants in 12.000 post-vaccine sera

Diagnostic assays currently used to monitor the efficacy of COVID-19 vaccines measure levels of antibodies to the receptor-binding domain of ancestral SARS-CoV-2 (RBDwt). However, the predictive value for protection against new variants of concern (VOCs) has not been firmly established. Here, we used bead-based arrays and flow cytometry to measure binding of antibodies to spike proteins and receptor-binding domains (RBDs) from VOCs in 12,000 sera. Effects of sera on RBD-ACE2 interactions were measured as a proxy for neutralizing antibodies. The samples were obtained from healthy individuals or patients on immunosuppressive therapy who had received two to four doses of COVID-19 vaccines and from COVID-19 convalescents. The results show that anti-RBDwt titers correlate with the levels of binding- and neutralizing antibodies against the Alpha, Beta, Gamma, Delta, Epsilon and Omicron variants. The benefit of multiplexed analysis lies in the ability to measure a wide range of anti-RBD titers using a single dilution of serum for each assay. The reactivity patterns also yield an internal reference for neutralizing activity and binding antibody units per milliliter (BAU/ml). Results obtained with sera from vaccinated healthy individuals and patients confirmed and extended results from previous studies on time-dependent waning of antibody levels and effects of immunosuppressive agents. We conclude that anti-RBDwt titers correlate with levels of neutralizing antibodies against VOCs and propose that our method may be implemented to enhance the precision and throughput of immunomonitoring. ### Competing Interest Statement 1. Trung The Tran has nothing to disclose. 2. Eline Bonne Vaage has nothing to disclose. 3. Adi Mehta has nothing to disclose. 4. Adity Chopra has nothing to disclose. 5. Anette Kolderup has received support from South-Eastern Norway Regional Health Authority (Grant no 10357 and 2021069. The payments were made to the institution, Oslo University Hospital, and covered salary and consumables) and from Juni Andrell, Stockholm University, Sweden (cDNA encoding 8xHis-tagged HexaPro spike protein and 6xHis-tagged receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Wuhan, in the expression plasmids pαH and pCAGGS, respectively. 6. Aina Karen Anthi has received support from South-Eastern Norway Regional Health Authority (Grant no 2019084. The payments were made to the institution, Oslo University Hospital, and covered salary and consumables). She has also received support from Juni Andrell, Stockholm University, Sweden (cDNA encoding 8xHis-tagged HexaPro spike protein and 6xHis-tagged receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Wuhan, in the expression plasmids pαH and pCAGGS, respectively. 7. M. Konig has received speaker honoraria from Novartis, Biogen, Sanofi, and Merck and support from Coalition for Epidemic Preparedness Innovations (CEPI) through Oslo University Hospital. 8. Gro Nygard has received support from Coalition for Epidemic Preparedness Innovations (CEPI) through Oslo University Hospital. 9. Andreas Lind has nothing to disclose. 10. Fredrik Muller has nothing to disclose. 11. Lise Sofie Haug Nissen-Meyer has nothing to disclose. 12. Per Magnus has received support from Norwegian Institute of Public Health for collection of blood samples in NorFlu and MoBa since 2020. 13. Lill-Iren Schou Trogstad has received support from Norwegian Institute of Public Health for collection of blood samples in NorFlu and MoBa since 2020. 14. Siri Mjaaland has nothing to disclose. 15. Arne Soraas has received support from Norwegian Cancer Association, Norwegian research Council, and Southeastern Norway Health Authority. 16. Karsten Midtvedt has nothing to disclose. 17. Anders Asberg has received payment to the institution from Sandos and Orifarm 18. Andreas Barratt-Due has nothing to disclose. 19. Asle Wilhelm Medhus has received a grant from Takeda (Grant to a research-initiated project, payment to institution). 20. Marte Lie Hoivik has received research grants to the institution from Pfizer, Janssen, Takeda, Ferring, Tillotts and has participated on a Data Safety Monitoring Board or Advisory Board at Takeda with personal fees. 21. Knut Lundin has nothing to disclose. 22. Randi Fuglaas Karlsen has nothing to disclose. 23. Reidun Dahle has nothing to disclose. 24. Karin Danielsson has nothing to disclose. 25. Kristine Stien Thomassen has nothing to disclose. 26. Grete Birkeland Kro has nothing to disclose. 27. Rebecca Jane Cox has nothing to disclose. 28. Fan Zhou has nothing to disclose. 29. Nina Langeland has nothing to disclose. 30. Pal Aukrust has received support from Klinbeforsk. grants for COVID-19 research 2020. 31. Espen Melum has nothing to disclose. 32. Tone Lise Avitsland has nothing to disclose. 33. Kristine Wiencke has nothing to disclose. 34. Jan Cato Holter has received support from Oslo University hospital, Research Council of Norway (Grant no 312780), and Philantropic donation from Vivaldi Invest A/A owned by Jon Steohenson von Tetzchner. 35. Ludvig Andre Munthe has received support from CEPI (Oslo University Hospital, University of Oslo), funding from University of Oslo, RCN, Norwegian Cancer Society, Health South East Health Research Funds (Stiftelsen KG Jebsen Grant 19), consulting fees in 20201: Astrazeneca for a panel discussing options for Long-Acting anti-SARS-CoV-2 Biologic AZD 7442, 4-time slots, < 1000USD, and payment for expert testimony in 20201 (Part of the expert panel for Norwegian Medicines Agency on vaccinology and associated topics, salary 1500USD and part of the expert panel for Norwegian Institute of Public Health for Norwegian Corona Vaccination Program. 8 meetings. Pro Bono). 36. Gunnveig Grodeland has received support from South-Eastern Norway Regional Health Authority, grants from the Research Council of Norway and EU-Horizon 2020, consulting fees from the Norwegian System of Compensation to Patients, honoraria from Bayer, ThermoFisher, and Saofi. She has also participated on the Data Safety Monitoring Board or Advisory Board with AstraZeneca. 37. Jan Terje Andersen has received support from South-Eastern Norway Regional Health Authority (Grant no 10357 and 2021069. The payments were made to the institution, Oslo University Hospital, and covered salary and consumables) and from Juni Andrell, Stockholm University, Sweden (cDNA encoding 8xHis-tagged HexaPro spike protein and 6xHis-tagged receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Wuhan, in the expression plasmids pαH and pCAGGS, respectively. He has collaborated with Roche, Tillotts Pharma, SJJ Solutions, Argenx, and Nordic Nanovector. Not directly related to the work of this manuscript. 38. John Torgils Vaage has nothing to disclose. 39. Fridtjof Lund-Johansen has received support from the South-Eastern Norway Regional Health Authority grant for developing COVID-19 serology. He has also received support from The Coalition for Epidemic Preparedness and Innovation (CEPI), a grant to monitor vaccine responses in patients on immunosuppressive therapy.