key: cord-0819204-dh6eg488 authors: Mader, Anna-Lena; Tydykov, Leonid; Glück, Vivian; Bertok, Manuela; Weidlich, Tanja; Gottwald, Christine; Stefl, Alexa; Vogel, Matthias; Plentz, Annelie; Köstler, Josef; Salzberger, Bernd; Wenzel, Jürgen J.; Niller, Hans Helmut; Jantsch, Jonathan; Wagner, Ralf; Schmidt, Barbara; Glück, Thomas; Gessner, André; Peterhoff, David title: Omicron's binding to Sotrovimab, Casirivimab, Imdevimab, CR3022, and sera from previously infected or vaccinated individuals date: 2022-03-14 journal: iScience DOI: 10.1016/j.isci.2022.104076 sha: 3a5907828848be57c7d196a382d44c208851689e doc_id: 819204 cord_uid: dh6eg488 SARS-CoV-2 Omicron is the first pandemic variant of concern exhibiting an abrupt accumulation of mutations particularly in the receptor-binding domain which is a critical target of vaccination induced and therapeutic antibodies. Omicrons mutations did only marginally affect the binding of ACE2, and the two antibodies Sotrovimab and CR3022, but strongly impaired the binding of Casirivimab and Imdevimab. Moreover, as compared to Wuhan, there is reduced serum reactivity and a pronounced loss of competitive surrogate virus-neutralization (sVN) against Omicron in naïve vaccinees and in COVID-19 convalescents after infection and after subsequent vaccination. Finally, although the booster vaccination response conferred higher titers and better sVN, the effect was nonetheless significantly lower compared to responses against Wuhan. Overall, our data suggest that the antigenicity of Omicrons receptor binding motive has largely changed but antibodies like Sotrovimab targeting other conserved sites maintain binding and therefore hold potential in prophylaxis and treatment of Omicron-induced COVID-19. Two years after onset of the SARS-CoV-2 pandemic, its global course is currently being taken over by the fifth variant of concern (VOC) B.1.1.529 which has been denominated Omicron (WHO, 2021) . Currently, great efforts are being made to fill the knowledge gaps about the risk potential of Omicron (ECDC, 2021) . Omicron, which has been first detected in specimen from Botswana and South Africa, displays a considerably higher number of mutations in the viral spike protein compared to previous VOCs (Gu et al., 2022; Saxena et al., 2021) . It harbors 29 single amino acid exchanges, six deletions and one amino acid in the spike protein, which is the most prominent antigen in current vaccine approaches. Fifteen mutations are located in the receptor binding domain (RBD) of which ten are part of the receptor binding motif (RBM) which is the main target of neutralizing antibodies (Figure 1a ) (Piccoli et al., 2020) . Here, we describe Omicrons antigenic profile in comparison to the other VOCs and SARS-CoV-1 against the monoclonal antibodies Sotrovimab, Casirivimab, Imdevimab and CR3022. Furthermore, we describe Omicrons affinity to its cellular receptor ACE2 in comparison to the other VOCs and SARS-CoV-1. To assess the profile of serum antibody binding to Omicron, we investigated a cohort of convalescent and SARS-CoV-2 naïve individuals before and after vaccination. Antibody binding assays and a sVNT show decreased affinity and competitive neutralization, respectively, of the tested sera to Omicron. Results: We first tested the binding affinities of soluble ACE2, the monoclonal antibodies Sotrovimab (S309), CR3022, Casirivimab (REGN-10933), and Imdevimab (REGN-10987), and a single convalescent plasma from early 2020 to the RBDs of ten different SARS-CoV strains (Figure 1 b-h) . Sotrovimab is a RBD class 3 antibody (according to the nomenclature proposed by Barnes et al.) which recognizes a non-RBM-overlapping epitope containing a glycan (N343) that is conserved within sarbecoviruses and does not compete binding of ACE2 (Barnes et al., 2020a (Barnes et al., , 2020b . CR3022 targets a highly conserved cryptic epitope distal from the receptor binding site and is a RBD class 4 antibody. Casirivimab is a RBD class 1 antibody whose epitope largely overlaps with the RBM . Imdevimab is class 3 antibody that binds distal to the RBM but has some overlap with the ACE2-binding site and does sterically hinder ACE2 interaction (Barnes et al., 2020b; Wang et al., 2021) . Of note, Casirivimab, Imdevimab and Sotrovimab are approved for clinical use. We found a slightly reduced binding to ACE2 in our ELISA (Figure 1 b) . Strongest differences in the monoclonal antibodies binding profile and half maximal effective concentrations (EC50) in comparison to the Wuhan RBD appear for SARS-CoV-1 and Omicron (Figure 1 c-f, h) . In fact, differences for these two variants regarding the low affinity against Casirivimab (EC50 of 13.99 nM (SARS-CoV-1) and 39.44 nM (Omicron)) and Imdevimab (EC50 of >100 nM for both variants) are in a comparable lower range, thereby reflecting their genetic distance to the Wuhan strain (EC50 of 0.11 nM for Casirivimab and 0.17 nM for Imdevimab). This is also apparent when comparing the binding profile of the representative convalescent serum (Figure 1 g) . Of utmost importance, however, binding of Sotrovimab was largely preserved. Binding and neutralizing activity of SARS-CoV-2 naïve and COVID-19 convalescent sera Next, we analyzed the IgG binding capacity of sera of 48 subjects after infection and/or vaccination to Wuhan RBD and Omicron RBD. The associated study cohort of COVID-19 J o u r n a l P r e -p r o o f convalescent and SARS-CoV-2 naïve control subjects has been described earlier (Glück et al., 2021) . In this experiment, we used a subset of 24 samples from the post-infection arm and 24 samples from the SARS-CoV-2 naïve arm for our analyses. Characteristics of the subgroups are given in Table 1 . Anti-Wuhan RBD IgG serum titers (here expressed as EC50 values) wane after infection as well as after vaccination (Figure 2) . Titers are higher after vaccination of convalescents as compared to complete vaccination of SARS-CoV-2 naïve subjects, and booster vaccination 6 month does not significantly increase the RBD serum titers as compared to after the initial full vaccination regimen. Waning of the antibody titers after vaccination post infection is not as pronounced as in SARS-CoV-2 naïve vaccinees. The time course of Omicron-RBD titers is comparable to Wuhan-RBD but the values are generally significantly lower. The median remaining titers against Omicrons RBD in comparison to Wuhans RBD are 41.5% post vaccination and 37% 6 month post vaccination in COVID-19 convalescents. In SARS-CoV-2 naïve subjects the titers drop to median 25% post 2 x vaccination and are raised to median 46% of the Wuhan titers by a booster vaccination after 6 month. To study differences in neutralizing capacity of the sera, we set up a surrogate virus neutralization assay that uses solid phase bound RBD and soluble ACE2 fused to a highly active and small luciferase (NanoLuc (Hall et al., 2012) ). The assay measures the residual binding of ACE2-NanoLuc to RBD antigen bound to a solid phase in presence of competing serum-antibodies as compared to a control without serum. Expectedly, the findings from the serum titers against the Wuhan and Omicron RBD are reflected by the results from the sVNT Our data show in accordance with others Planas et al., 2021 ) that binding of RBM-targeting monoclonal antibodies is largely impaired, while in contrast antibodies that bind to other RBD-epitopes still display high affinity to Omicron. When analyzing the sera from vaccinated or convalescent patients we found that antibody responses against Omicron RBD were significantly lower compared to Wuhan RBD. Our findings are in line with initial reports on the neutralization capacity of sera from convalescent and vaccinated subjects against Omicron which showed a more than 10-fold drop in effective titers (Ai et al., 2021; Wang et al., 2022; Schubert et al., 2021b; Wilhelm et al., 2021; Carreno et al., 2021; Hoffmann et al., 2021; Rössler et al., 2022) . The IgG titers determined in this study, however, showed a considerably lower loss of binding capacity of serum antibodies compared to the reported loss of serum neutralizing capacity of Omicron (Carreno et al., 2021; Garcia-Beltran et al., 2022; Rössler et al., 2022; Schubert et al., 2021b; Wilhelm et al., 2021) . Interestingly the loss of binding in our study is accompanied by a pronounced loss of ACE2 competition of the sera as detected in our sVNT. This is in line with the reported generally reduced serum-neutralization capacity against Omicron but also conforms to findings from Cao et al. and Cameroni et al. who described a sustained loss of affinity of a large number of RBM-directed monoclonal antibodies for Omicron Cao et al., 2021) . Nonetheless, Cao et al. reported that there are still a few, individual RBM-targeting monoclonal antibodies with retained binding to Omicron (Cao et al., 2021) . This suggests that the polyclonal serum response in vaccinated naïve and convalescent subjects may reflect the presumed aggregated response of multiple, individual monoclonal antibody responses in which single antibodies with preserved binding and neutralizing ability may still be present. Ultimately, this might explain why the competitive neutralizing capacity is not completely lost in the sera of vaccinated convalescent and naïve subjects. Following this reasoning, we found in line with others that booster immunization is able to increase antibody responses to Omicron. Nonetheless, this response did not match the levels reached against J o u r n a l P r e -p r o o f Wuhan RBD in neither antibody titers nor sVNT, again confirming the results of other studies (Carreño et al., 2021; Garcia-Beltran et al., 2022; Gruell et al., 2022; Hoffmann et al., 2021; Muik et al., 2022; Schmidt et al., 2021) . At the same time, more and more data is emerging on either maintained protection after recovery from COVID-19 or vaccination, or a generally lower severity of Omicron induced COVID-19 in convalescent and vaccinated subjects (Andrews et al., 2021; Ferguson, 2021a Ferguson, , 2021b Goga et al., 2021; Kuhlmann et al., 2021; Wolter et al., 2021) . Therefore, other factors than neutralization seem to convey protection from severe COVID-19 in these patients. Very recently BA.1, BA.2 and BA.3 sublineages evolved and further lineages will follow, with novel serotypes. In this respect, it is very encouraging that Sotrovimab, which is known to bind to various sarbecoviruses including SARS-CoV-1 and SARS-CoV-2, binds to Omicron as well Cao et al., 2021; Gupta et al., 2021; Liu et al., 2021; Planas et al., 2021) . Nevertheless, the clinical development of existing and additional broadly neutralizing antibodies as well as further antiviral agents against coronaviruses and the rapid adaptation of vaccines are urgently needed Cao et al., 2021; Planas et al., 2021) . Based on the finding of a retained affinity of Sotrovimab to Omicron, it will be interesting to investigate structural conditions and constrains of this interaction. Determination of the high resolution structure of the complex of Sotrovimab and stabilized Omicron spike trimers of the novel lineages and their respective RBDs will help to understand the molecular conditions of Sotrovimabs breadth. As Sotrovimab partially involves interactions with the conserved glycan at position N343, differences in glycan processing e.g. as a result from a changed cell tropism may further alter Sotrovimabs binding properties. This is likely to be an important feature of future escape variants and can already be investigated by mutational studies and glyco-J o u r n a l P r e -p r o o f engineering. Along these lines, forced in vitro viral selection studies could provide further information on the structural plasticity of Sotrovimabs epitope. Finally, comparative analysis of the characteristics of non-vaccinated Omicron-convalescent sera with convalescent sera from the pre-Omicron era and Omicron-breakthrough-infection sera will be of utmost interest. It will be interesting to see if the drop of neutralization will be compensated by newly induced RBMreactivity, or if other RBD-core or non-RBD epitopes are preferentially boosted and if this ultimately broadens protection against circulating and future emerging coronaviruses. As a limitation of this study, only RBD directed antibody binding and serum titers were measured. Differences may appear when measuring against whole spike protein or against other antigens, although RBD-titers have been shown to correlate well with spike titers in case of Wuhan and may thus be predictive (Peterhoff et al., 2021a) . Along these lines, we found using our ELISA setup that ACE2 affinity to Omicron is reduced. This was described by Schubert et al. as well where the authors also used an ELISA to characterize the receptor interaction (Schubert et al., 2021a) . In contrast, however, Cameroni et al. performed surface plasmon resonance measurements and found slightly enhanced binding affinity . This may reflect different assay specific test characteristics. More sensitive and differentiate methods for binding affinity analysis as well as investigation of the molecular structure of the antibody-antigen-complex might help to clarify this. Finally, our sVNT is likely not reflecting the complete serum neutralization capacity, as it mainly detects neutralization by ACE2 receptor competition and may miss non-competitive neutralization or competitive neutralization of potential alternative ligand structures (Clausen et al., 2020; Lempp et al., 2021) . Such data would be available from comparative pseudotype or real-virus neutralization assays. J o u r n a l P r e -p r o o f Lead contact Requests should be directed to the lead contact, David Peterhoff (david.peterhoff@ur.de). Materials are available on personal request under a material transfer agreement. • Datasets generated in this study are available on personal request. • This paper does not report original code. • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Employees of the Kliniken Südostbayern Hospital Network (Germany) who recovered from COVID-19 between April and June 2020 were asked to participate in the prospective cohort study. Directly after recovery and after 30 weeks, participants were asked to provide a serum sample. Approximately one year after COVID-19, participants were offered a one dose booster vaccination against COVID-19. Those who agreed to be vaccinated were asked to provide another serum sample directly before the vaccination and at least 14 days thereafter. Both groups were asked for a further serum sample 6 month after complete vaccination. SARS-CoV-2 naïve donors received their booster vaccination 8 month after the prime-regimen and were asked to donate a further serum sample 14 days after vaccination. Information about sex and median age of the analyzed subjects and sample size of all experimental groups is specified in Table 1 . The study was approved by the ethical committee of the Faculty for Medicine, University of Regensburg, Regensburg, Germany (reference number 20-1896-101). The study complies with the 1964 Helsinki declaration and its later amendments. All participants provided written informed consent. Blood sampling was accomplished by venipuncture (S-Monovette, Sarstedt, Nürnbrecht, Germany). Serum was obtained by centrifugation 1-6 hours after blood drawl and stored at -20°C. The SARS-CoV-1 and SARS-CoV-2 (Wuhan Hu-1) RBD-encoding sequences were codon usage-optimized and synthesized by GeneArt AG (Thermo Fisher Scientific). The RBD was cloned into a modified pcDNA5/FRT/TO vector encoding an N-terminal mini-tPA signal peptide Soluble ACE2 (amino acid 20-732) was codon optimized and synthesized by GeneArt AG (Thermo Fisher Scientific) and cloned into a pcDNA5/FRT/TO derivate providing a mini-tPAsignal peptide and an avi-octahistidine tag (sequence GS-GLNDIFEAQKIEWHE-GS-HHHHHHHH). The protein was purified as described for ACE2-NanoLuc. Site specific biotinylation was performed using BirA (BirA biotin-protein ligase standard reaction kit, Avidity). Antigens were coated at a concentration of 2 µg/ml in PBS to the plastic surface of Nunc Detection of RBD-specific serum antibodies by ELISA Anti-RBD antibody levels in serum were detected by an ELISA utilizing the SARS-CoV-1-and SARS-CoV-2-RBDs described above. The validation of the ELISA was previously described (Peterhoff et al., 2021a) . The assay detects RBD-specific antibodies responses with high specificity and sensitivity and the detected antibody levels were shown to correlate well with the virus neutralization capacity of the respective serum sample. ELISA-results were expressed as effective concentration 50 (EC50) resulting from titrations in 2.5 fold serial serum dilutions starting at 1:40 serum dilution. IgA serum reactivities were determined at a single serum dilution of 1:100 and are expressed as optical densities of the sample/background ratios (signal/cutoff; S/CO) as determined for SARS CoV-2 Wuhan RBD. Conditions for optimal dynamic detection range of neutralizing antibodies in the surrogate virus neutralization test (sVNT) were determined by three-dimensional cross-titration of antigen, NanoLuc-ACE2 and neutralizing antibodies. For serum sVNTs, sera were diluted 1:50 in 1% fat free milk in PBS (Gibco) 0.1 % Tween 20 (Caelo) (PBS-T) and added to RBD-coated and pre-blocked ELISA plate. After 1 h incubation the plate was washed with PBS-T and 200 nM NanoLuc-ACE2 in PBS-T was added for 30 min. After washing with PBS-T, 50 µl Nano-Glo Luciferase Assay Reagent (Promega) was added to each well and the luminescence signal was detected within 20 minutes in a 96 well luminescence reader (VICTOR Plate Reader, PerkinElmer). 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We furthermore acknowledge financial support through the pandemic responsiveness fund of The Bavarian Ministry of Science and Art.