key: cord-0931291-jd1o1gsr authors: Schubert, M.; Bertoglio, F.; Steinke, S.; Heine, P. A.; Ynga-Durand, M. A.; Zuo, F.; Du, L.; Korn, J.; Milosevic, M.; Wenzel, E. V.; Maass, H.; Krstanovic, F.; Polten, S.; Pribanic-Matesic, M.; Brizic, I.; Piralla, A.; Baldanti, F.; Hammarstrom, L.; Dubel, S.; Sustic, A.; Marcotte, H.; Strengert, M.; Protic, A.; Pan Hammarstrom, Q.; Cicin-Sain, L.; Hust, M. title: Human serum from SARS-CoV-2 vaccinated and COVID-19 patients shows reduced binding to the RBD of SARS-CoV-2 Omicron variant in comparison to the original Wuhan strain and the Beta and Delta variants date: 2021-12-13 journal: nan DOI: 10.1101/2021.12.10.21267523 sha: f0582b200136290871367e8e249a0009078d083e doc_id: 931291 cord_uid: jd1o1gsr Background The ongoing COVID-19 pandemic is caused by the beta coronavirus SARS-CoV-2. COVID-19 manifests itself from mild or even asymptomatic infections to severe forms of life-threatening pneumonia. At the end of November 2021, yet another novel SARS-CoV-2 variant named B.1.1.529 or Omicron was discovered and classified as a variant of concern (VoC) by the WHO. Omicron shows significantly more mutations in the amino acid (aa) sequence of its spike protein than any previous variant, with the majority of those concentrated in the receptor binding domain (RBD). In this work, the binding of the Omicron RBD to the human ACE2 receptor was experimentally analyzed in comparison to the original Wuhan SARS-CoV-2 virus, and the Beta and Delta variants. Moreover, we compared the ability of human sera from COVID-19 convalescent donors and persons fully vaccinated with BNT162b2 (Corminaty) or Ad26.COV2.S (Janssen COVID-19 vaccine) as well as individuals who had boost vaccine doses with BNT162b2 or mRNA-1273 (Spikevax) to bind the different RBDs variants. Methods The Omicron RBD with 15 aa mutations compared to the original Wuhan strain was produced baculovirus-free in insect cells. Binding of the produced Omicron RBD to hACE was analyzed by ELISA. Sera from 27 COVID-19 patients, of whom 21 were fully vaccinated and 16 booster recipients were titrated on the original Wuhan strain, Beta, Delta and Omicron RBD and compared to the first WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human) using the original Wuhan strain as reference. Results The Omicron RBD showed a slightly reduced binding to ACE2 compared to the other RBDs. The serum of COVID-19 patients, BNT162b2 vaccinated and boost vaccinated persons showed a reduced binding to Omicron RBD in comparison to the original Wuhan strain, Beta und Delta RBDs. In this assay, the boost vaccination did not improve the RBD binding when compared to the BNT162b2 fully vaccinated group. The RBD binding of the Ad26.COV2.S serum group was lower at all compared to the other groups. Conclusions The reduced binding of human sera to Omicron RBD provides first hints that the current vaccinations using BNT162b2, mRNA-1273 and Ad26.COV2.S may be less efficient in preventing infections with the Omicron variant. The ongoing COVID-19 pandemic is caused by the beta coronavirus SARS-CoV-2. COVID-19 manifests itself from mild or even asymptomatic infections to severe forms of life-threatening pneumonia. At the end of November 2021, yet another novel SARS-CoV-2 variant named B.1.1.529 or Omicron was discovered and classified as a variant of concern (VoC) by the WHO. Omicron shows significantly more mutations in the amino acid (aa) sequence of its spike protein than any previous variant, with the majority of those concentrated in the receptor binding domain (RBD). In this work, the binding of the Omicron RBD to the human ACE2 receptor was experimentally analyzed in comparison to the original Wuhan SARS-CoV-2 virus, and the Beta and Delta variants. Moreover, we compared the ability of human sera from COVID-19 convalescent donors and persons fully vaccinated with BNT162b2 (Corminaty) or Ad26.COV2.S (Janssen COVID-19 vaccine) as well as individuals who had boost vaccine doses with BNT162b2 or mRNA-1273 (Spikevax) to bind the different RBDs variants. The Omicron RBD with 15 aa mutations compared to the original Wuhan strain was produced baculovirus-free in insect cells. Binding of the produced Omicron RBD to hACE was analyzed by ELISA. Sera from 27 COVID-19 patients, of whom 21 were fully vaccinated and 16 booster recipients were titrated on the original Wuhan strain, Beta, Delta and Omicron RBD and compared to the first WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human) using the original Wuhan strain as reference. The Omicron RBD showed a slightly reduced binding to ACE2 compared to the other RBDs. The serum of COVID-19 patients, BNT162b2 vaccinated and boost vaccinated persons showed a reduced binding to Omicron RBD in comparison to the original Wuhan strain, Beta und Delta RBDs. In this assay, the boost vaccination did not improve the RBD binding when compared to the BNT162b2 fully vaccinated group. The RBD binding of the Ad26.COV2.S serum group was lower at all compared to the other groups. . CC-BY-NC-ND 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) preprint The reduced binding of human sera to Omicron RBD provides first hints that the current vaccinations using BNT162b2, mRNA-1273 and Ad26.COV2.S may be less efficient in preventing infections with the Omicron variant. . CC-BY-NC-ND 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) preprint The copyright holder for this this version posted December 13, 2021. six aa deletions and one aa insertion. Fifteen of these mutations are located in the Nterminal receptor binding domain (RBD) of the spike protein which binds to the human zinc peptidase angiotensin-converting enzyme 2 (ACE2) for cell entry 8,9 . Importantly, the RBD is targeted by more than 90% of neutralizing serum antibodies, making it the most relevant target for SARS-CoV-2 neutralization 10, 11 . Consequently, the majority of therapeutic antibodies for the treatment of COVID-19 are designed to interact with this part of the SARS-CoV-2 spike protein 12, 13 . This indicates that the omicron variant may bind with a different affinity to the cell receptor, altering its propagation characteristics, escape the immune recognition by antibodies, facilitating viral spread in a seropositive population, or both. While initial studies have shown a severe reduction in serum neutralizing capacity of vaccinated and convalescent patients against the omicron variant 14 , it is unclear to which extent the RBD domain mutations contribute to this loss in neutralization activity. Additionally, while several mutations present in Omicron are predicted to increase ACE2 binding affinity, whereas others are predicted to reduce its affinity 15 . In this work, the binding of ACE2 to the new Omicron RBD was determined in comparison to the original Wuhan strain and the Beta and Delta variants in a biological experimental model. The humoral immune response is essential for the anti-viral defense. Therefore, the binding of human sera from COVID-19 hospitalized patients or fully vaccinated persons with BNT162b2 or Ad26.COV2.S as well as boost vaccinated persons on the RBD of the original Wuhan strain, Beta, Delta and Omicron variant was analyzed. . CC-BY-NC-ND 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. Details about study participants are shown in Table 1 is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.10.21267523 doi: medRxiv preprint 7/17 GC buffer and 3% dimethyl sulfoxide was used for the amplification reaction. The RBD omicron variant was ordered as GeneString from GeneArt (Thermo Fisher) according to EPI_ISL_6590608 (partial RBD Sanger sequencing from Hong Kong), EPI_ISL_6640916, EPI_ISL_6640919 and EPI_ISL_6640917 including Q493K which was corrected later to Q493R. Table 2 gives an overview about the used variants. RBD The different RBD variants were produced in the baculovirus-free High Five cell system 17 and purified as described before 18 The . CC-BY-NC-ND 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) preprint . CC-BY-NC-ND 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 RBD of the original Wuhan strain, the Delta variant and the Omicron variant were produced in insect cells and purified by IMAC and SEC. The quality of the recombinant proteins was analyzed by SDS-PAGE (Supplementary Figure 1) . The production yield of Omicron RBD was tendentially higher than the production yield of Wuhan RBD. All RBDs were immobilized on plates and binding of soluble receptor ACE2 was analyzed by ELISA ( Figure 1 ). The Omicron RBD showed a slightly reduced binding to ACE2 (EC 50 150 ng/mL) compared to the Wuhan strain RBD (EC 50 120 ng/mL) and more to Beta (EC 50 89 ng/mL) and Delta RBD (EC 50 89 ng/mL). Similar results were observed when soluble RBDs were added to plate-coated ACE2 (data not shown). The binding of human sera from hospitalized COVID-19 patients (Figure 2A Figure 2E . The sera of COVID-19 patients and BNT162b2 showed a highly significant reduction in binding to Omicron RBD in a non-parametric pairwise analysis. This reduction was more pronounced than the one observed against Beta or Delta RBD binding assays. In the booster group the binding to original Wuhan strain, Beta and Delta RBDs was nearly equal, but significantly reduced to Omicron RBD. Ad26.COV2.S group showed very low binding to all RBDs tested, suggesting a clearly weak immunogenicity of this vaccine formulation. An increase in binding to the Omicron RBD variant after boost immunization was not observed. . CC-BY-NC-ND 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. Discussion RBD-ACE2 interaction is a prerequisite for SARS-CoV-2 viral entry 8, 9, 19 . The binding of the Omicron RBD to the ACE2 receptor appears to be slightly reduced in our setting compared to the currently dominant Delta variant. Several Omicron RBD mutations are assumed to increase the binding to ACE2: G339D, S477N, T478K, Q493K, N501Y, others are neutral: S371L, S373P, G446S, E484A, Q493R, Q498R or are assumed to reduce the binding to ACE2: S375F, K417N, G496S, Y505H according to Starr et al. 19 . However, all of these considerations are based on modelling. RBD-ACE2 interaction involving a heavily mutated RBD such as the one of Omicron VOC may deviate from predictions. To our knowledge this is among the first empirical analysis of Omicron RBD binding efficacy to the ACE2 receptor. Surprisingly, the binding of Omicron RBD to human ACE2 was not increased, but rather decreased, especially when compared to Beta and Delta. One has to consider that the decrease in RBD binding does not necessarily translate into reduced infectivity, as infectivity and replication is also defined by proteolytic spike processing, fusion efficacy and RNA replication efficiency. Nevertheless, our results argue that increased binding to the hACE2 receptors may be an unlikely cause of rapid Omicron spread. One has also to consider that we utilized in our work the originally available sequence with a Q493K mutation, whereas Q493R sequences have been published since. According to Starr et al. 19 , the K mutation has a higher affinity as the R mutation in in vitro binding studies. On the other hand Kumar et al. 15 calculated an increased binding to ACE2 for the Q493R mutation in docking studies. Regardless, future studies will validate our observation. Importantly, RBD mutations may also lead to immune escape 20 . The humoral immune answer is a key factor for the anti-viral defense 21 and the RBD is the main target of neutralizing antibodies 10, 11, 22 . The very low RBD binding in the Ad26.COV2.S compared to the COVID-19 patient group or the BNT162b2 vaccinated group is in accordance with the former results 23 but impaired definite conclusions on omicron immune escape upon Ad26.COV2.S vaccination. The reduced binding of sera from COVID-19 patient and BNT162b2 groups to the Omicron RBD was in accordance with current results describing a highly reduced neutralization of the Omicron variant by human sera from vaccinated persons 14, 24 . Surprisingly, the boost vaccination did not improve the Omicron RBD binding in this assay, probably because the results were compared to early serum responses upon the 2nd vaccination dose. . CC-BY-NC-ND 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 reduced binding of the patient, vaccinated and boost-vaccinated sera to the Omicron RBD is a snapshot of the current situation. According to the sequencing data deposited as GISAID (https://www.gisaid.org/) and the analysis on Outbreak.info 25 , the frequency of the 15 aa mutations in the RBD is very dynamic, e.g. K417N and G446S described for the initial Omicron variant is occurs in ~ 40% of all sequenced Omicron variants, S477N and T478K is in ~60%, N501Y is in ~90% of the sequences (status 2021-12-09, 837 sequences). The K417N mutation is a key mutation also in the Beta variant, the T478K mutation in the Delta variant and N501Y in the Alpha, Beta and Gamma variant 26 . All of these variants may contribute to both ACE2 binding efficacy and immune escape. Therefore, Omicron variants with alternative mutations might evolve in the near future and alter the antibody recognition and the binding efficacy. . CC-BY-NC-ND 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. LCS was supported by grants from the Helmholtz Association (EU-Partering PIE-0008 and Helmholtz campaign COVIPA). We would like to thank all the blood donors who agreed to this scientific study. . CC-BY-NC-ND 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) preprint The copyright holder for this this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.10.21267523 doi: medRxiv preprint . 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CC-BY-NC-ND 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 this version posted December 13, 2021. ; https://doi.org/10.1101/2021.12.10.21267523 doi: medRxiv preprint