key: cord-0760877-67v082uh authors: Planas, Delphine; Saunders, Nell; Maes, Piet; Guivel-Benhassine, Florence; Planchais, Cyril; Buchrieser, Julian; Bolland, William-Henry; Porrot, Françoise; Staropoli, Isabelle; Lemoine, Frederic; Péré, Hélène; Veyer, David; Puech, Julien; Rodary, Julien; Baela, Guy; Dellicour, Simon; Raymenants, Joren; Gorissen, Sarah; Geenen, Caspar; Vanmechelen, Bert; Wawina-Bokalanga, Tony; Martí-Carrerasi, Joan; Cuypers, Lize; Sève, Aymeric; Hocqueloux, Laurent; Prazuck, Thierry; Rey, Félix; Simon-Lorrière, Etienne; Bruel, Timothée; Mouquet, Hugo; André, Emmanuel; Schwartz, Olivier title: Considerable escape of SARS-CoV-2 variant Omicron to antibody neutralization date: 2021-12-15 journal: bioRxiv DOI: 10.1101/2021.12.14.472630 sha: 194520be14c1c9007b3a233a666b50d7aeb0b5b8 doc_id: 760877 cord_uid: 67v082uh The SARS-CoV-2 Omicron variant was first identified in November 2021 in Botswana and South Africa1,2. It has in the meantime spread to many countries and is expected to rapidly become dominant worldwide. The lineage is characterized by the presence of about 32 mutations in the Spike, located mostly in the N-terminal domain (NTD) and the receptor binding domain (RBD), which may enhance viral fitness and allow antibody evasion. Here, we isolated an infectious Omicron virus in Belgium, from a traveller returning from Egypt. We examined its sensitivity to 9 monoclonal antibodies (mAbs) clinically approved or in development3, and to antibodies present in 90 sera from COVID-19 vaccine recipients or convalescent individuals. Omicron was totally or partially resistant to neutralization by all mAbs tested. Sera from Pfizer or AstraZeneca vaccine recipients, sampled 5 months after complete vaccination, barely inhibited Omicron. Sera from COVID-19 convalescent patients collected 6 or 12 months post symptoms displayed low or no neutralizing activity against Omicron. Administration of a booster Pfizer dose as well as vaccination of previously infected individuals generated an anti-Omicron neutralizing response, with titers 5 to 31 fold lower against Omicron than against Delta. Thus, Omicron escapes most therapeutic monoclonal antibodies and to a large extent vaccine-elicited antibodies. . It has in the meantime spread to many countries and is expected to rapidly become dominant worldwide. The lineage is characterized by the presence of about 32 mutations in the Spike, located mostly in the N-terminal domain (NTD) and the receptor binding domain (RBD), which may enhance viral fitness and allow antibody evasion. Here, we isolated an infectious Omicron virus in Belgium, from a traveller returning from Egypt. We examined its sensitivity to 9 monoclonal antibodies (mAbs) clinically approved or in development 3 In less than three weeks following its discovery, the Omicron variant has been detected in dozens of countries. The WHO has classified this lineage (previously known as Pango lineage B.1.1.529) as a Variant of Concern (VOC) on November 26, 2021 1 . Preliminary estimates of its doubling time range between 1.2 and 3.6 days, in populations with high rate of SARS-CoV-2 immunity 2,4 . Omicron is expected to supplant the currently dominant Delta lineage in the next weeks or months. Little is known about its sensitivity to the humoral immune response. Recent preprints indicated a reduced sensitivity of Omicron to certain monoclonal and polyclonal antibodies 5 6 , whereas CD8+ T cell epitopes previously characterized in other variants seem to be conserved in Omicron 7 . We isolated an Omicron variant from a nasopharyngeal swab of an unvaccinated individual that developed moderate symptoms eleven days after returning to Belgium from Egypt. The virus was amplified by one passage on Vero E6 cells. Sequences of the swab and the outgrown virus were identical and identified the Omicron variant (Pango lineage BA.1, GISAID accession ID: (EPI_ISL_6794907 and EPI_ISL_7413964 respectively) (Fig. 1a) . The Spike protein contained 32 changes, when compared to the D614G strain (belonging to the basal B.1 lineage) used here as a reference, including 7 changes in the N terminal domain (NTD), with substitutions, deletions and a three amino-acid insertion (A67V, Δ69-70, T95I, G142D, Δ141-143, Δ211L212I, Ins214EPE), 15 mutations in the RBD (G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R and N501Y, Y505H) the T574K mutation, 3 mutations close to the furin cleavage site (H655Y, N679K and P681H) and 6 in the S2 region (N764K, D796Y, N856K, Q954H, N969, L981F) ( Fig. 1a) . This extensive constellation of changes is unique, but includes at least 11 modifications observed in other lineages and VOCs or at sites mutated in other variants (Fig. 1a) . Viral stocks were titrated using S-Fuse reporter cells and Vero cells. S-Fuse cells become GFP+ upon infection, allowing rapid assessment of infectivity and the measurement of neutralizing antibody levels [8] [9] [10] . Syncytia were observed in Omicron-infected S-Fuse cells (not shown). We inferred a global phylogeny subsampling SARS-CoV-2 sequences available on the GISAID EpiCoV database. To better contextualize the isolated virus genome, we performed a focused phylogenetic analysis using as background all Omicron samples deposited on GISAID on December 6, 2021, (Extended Data Fig. 1 ). The tree topology indicates that the Omicron lineage does not directly derive from any of the previously described VOCs. The very long branch of the Omicron lineage in the timecalibrated tree (Extended Fig. 1 ) might reflect a cryptic and potentially complex evolutionary history. At the time of writing, no Omicron genomic sequences from Egypt were available on GISAID, nor do we know of any sequences of travellers that used the same planes. The isolated strain genome showed no close connection to other Belgian Omicron infections. Follow-up analyses with additional genomic data will improve phylogenetic resolution to determine whether the patient was infected before or after returning to Belgium. We highlighted the 29 amino acid substitutions, 3 amino-acid deletions and a 3-residue insertion present in the Omicron Spike, with respect to the Wuhan strain, in a 3D model of the protein (Extended Figure 2a) . The 15 mutations in the RBD cluster in particular around the trimer interface. The RBD is the target of the most potently neutralizing monoclonal antibodies (mAbs) against SARS-CoV-2, which have been divided into four classes depending of the location of their epitope 11 (Extended Figure 2b ). mAbs in classes 1 and 2 compete for hACE2 binding, whereas those from classes 3 and 4 bind away from the hACE2 interaction surface (Extended Figure 2b) . The epitopes of the class 2 and 3 mAbs are exposed irrespective of the conformation of the RBD on the spike (Up or Down configuration 12 ), while those of classes 1 and 4 require an RBD in the Up conformation. Whereas the previous VOCs displayed mutations only in the region targeted by class 1 and 2 mAbs, Omicron mutations are located within the epitopes of all four classes of mAbs. In the NTD, the mutations, insertion and deletions might also impact recognition of this domain by antibodies. We then assessed the sensitivity of Omicron to a panel of human mAbs using the S-Fuse assay. We tested 9 antibodies in clinical use or in development 13,14 15 16 17 18,19 . Neutralizing mAbs targeting the RBD can be classified into 4 main classes depending on their binding epitope 3, 11, 20 . Bamlanivimab and Etesevimab (class 2 and class 1, respectively) are mixed in the Lilly cocktail. Casirivimab and Imdevimab Sotrovimab (Xevudy TM ) by GlaxoSmithKline and Vir Biotechnology is a class 3 antibody that displays activity against diverse coronaviruses. It targets an RBD epitope outside the receptor binding motif, which includes N343-linked glycans. Adintrevimab (ADG20) developed by Adagio binds to an epitope located in between the class 1 and class 4 sites. We measured the activity of the 9 antibodies described above against Omicron and included the Delta variant for comparison purposes (Fig. 1b) . As previously reported, Bamlanivimab did not neutralize Delta 10 21 22 . The other antibodies neutralized Delta with IC50 (Inhibitory Concentration 50%) varying from 2.2 to 369 ng/mL ( Fig. 1b and Extented table 1). Six antibodies (Bamlanivimab, Etesevimab, Casirivimab, Imdevimab, Tixagevimab and Regdanvimab) lost antiviral activity against Omicron. The three other antibodies displayed a 3 to 20-fold increase of IC50 (ranging from 391 to 1114 ng/ml) against Omicron. Sotrovimab was the only antibody displaying a rather similar activity against both strains, with a IC50 of 369 and 1114 ng/mL against Delta and Omicron, respectively. We also tested the antibodies in combination, to mimic the therapeutic cocktails. Bamlanivimab/Etesevimab (Lilly) or Casirivimab/Imdevimab (Ronapreve TM ) are inactive against Omicron. Cilgavimab/Tixagevimab (Evusheld TM ) neutralized Omicron with an IC50 of 1355 ng/mL. We next examined by flow cytometry the binding of each mAb to Vero cells infected with Delta and Omicron variants. Five out of six clinical antibodies that lost antiviral activity (Bamlanivimab, Etesevimab, Casirivimab, Imdevimab and Regdanvimab) no longer recognized Omicron infected cells (table S1). The other antibodies still bound to Omicron-infected cell (table S1). Thus, Omicron escapes neutralization by the tested antibodies to various extents. Our results are in line with results from a recent preprint, obtained using Spike-coated pseudoviruses 6 . We next asked whether vaccine-elicited antibodies neutralized Omicron. To this aim, we randomly We measured the potency of their sera against the Delta and Omicron strains. We used as a control the D614G ancestral strain (belonging to the basal B.1 lineage) (Fig. 2a) . We calculated the ED50 (Effective Dose 50%) for each combination of serum and virus. Sera were first sampled 5 months after the full two-dose vaccination. With the Pfizer vaccine, the levels of neutralizing antibodies were relatively low against D614G and Delta (median ED50 of neutralization of 329 and 91), reflecting the waning of the humoral response 10 (Fig. 2a) . We did not detect any neutralization against the Omicron variant with these sera (Fig. 2a) . A similar pattern was observed with the AstraZeneca vaccine. Five months after vaccination, the levels of antibodies neutralizing Delta were low (ED50 of 187 and 68 against D614G and Delta, respectively), and no antiviral activity was observed against Omicron (Fig. 2a) . We next examined the impact of a Pfizer booster dose, administrated 8 months after Pfizer vaccination. The sera were collected one month after the third dose. The booster dose enhanced neutralization titers against D614G and Delta by 33 and 56 fold (ED50 12873 and 5280, respectively). It was also associated with strong increase of the neutralization activity against Omicron (ED50 of 1050) ( Fig. 2b) . Altogether, these results indicate that Omicron is poorly or not neutralized by vaccinees' sera sampled 5 months after vaccination. The booster dose triggered a detectable cross-neutralization activity against Omicron. However, even after the booster dose the variant displayed a reduction of ED50 of 12-and 5-fold, when compared to D614G and Delta, respectively. We subsequentely examined the neutralization ability of sera from convalescent subjects. We randomly selected 45 longitudinal samples from 36 donors in a cohort of infected individuals from Orléans. Individuals were diagnosed with SARS-CoV-2 infection by RT-qPCR (Extended table 2b). We previously studied the potency of these sera against D614G, Alpha, Beta and Delta isolates 9 10 . We analyzed individuals sampled at a median of 6 and 12 months (M6 and M12) post onset of symptoms (POS). With the D614G and Delta variants, the neutralization titers slightly decreased overtime (422 and 215 for D614G, 344 and 85 for Delta, at M6 and M12, respectively) 9 (Fig. 2c) . The convalescent sera barely neutralized Omicron at these time points. Fourteen individuals were vaccinated at M12 with a Pfizer dose. Sera sampled one month after vaccination showed a drastic increase in neutralizing antibody titers against the D614G and Delta variants, reaching a median ED50 of 71555 and 49778, respectively (Fig. 2d) . These sera also neutralized Omicron, with a median ED50 of 1598 (Fig. 2d) . Therefore, as shown with other variants 23,24 9 a single dose of vaccine boosts cross-neutralizing antibody responses to Omicron in previously infected individuals. The neutralization titers are however reduced by 44 and 31 fold, when compared to D614G and Delta, respectively. The Omicron variant has opened a new chapter in the COVID-19 pandemic 2,25 . The principal concerns about this variant include its high transmissibility, as underlined by its rapid spread in different countries, and the presence of over 55 mutations spanning the whole viral genome. Omicron contains 32 mutations in the Spike, lying in the NTD, RBD and in vicinity of the furin cleavage site. Some mutations were already present in other VOCs and VOIs, and have been extensively characterized [25] [26] [27] . Due to their position, they are expected to affect the binding of natural or therapeutic antibodies, to increase affinity to ACE2 and to enhance the fusogenic activity of the Spike. Future work will help determining how this association of mutations impacts viral fitness in culture systems and their contribution to the high transmissibility of the variant. Here, we studied the cross-reactivity of clinical or pre-clinical mAbs, as well as 90 sera from vaccine recipients and long-term convalescent individuals against an infectious Omicron isolate. We report that among nine mAb in clinical use or in development, six (Bamlanivimab, Etesevimab, Casirivimab, Imdevimab Tixagevimab and Regdanvimab) were inactive against Omicron. Two other antibodies (Cilgavimab, Andintrevimab) displayed about a 20-fold increase of IC50. Sotrovimab was less affected by Omicron's mutations, with IC50 increased by only 3 fold. We also show that Omicron was barely neutralized by sera from vaccinated individuals sampled 5 months after administration of two doses of Pfizer or AstraZeneca vaccine. Sera from convalescent individuals at 6 or 12 months post infection barely neutralized or did not detectably neutralize Omicron. The decrease of antibody efficacy helps explaining the high number of breakthrough infections and reinfection cases, and the spread of Omicron in both non-immune and immune individuals 28 . There is currently no evidence of increased disease severity associated with Omicron compared with Delta, either among naïve or immunized individuals. It is likely that even if pre-existing SARS-CoV-2 antibodies may poorly prevent Omicron infection, anamnestic responses and cellular immunity will be operative to prevent severe forms of the disease 29 . We further report that a booster dose of Pfizer vaccine, as well as vaccination of previously infected individuals, strongly increased overall levels of anti-SARS-CoV-2 neutralizing antibodies, well above a threshold allowing inhibition of Omicron. Affinity maturation of antibodies is known to improve the efficacy of the humoral anti-SARS-CoV-2 response overtime 30, 31 . This process helps explaining the efficacy of booster doses in immune patients. However, sera with high antibody levels displayed a 5 to 31 fold reduction in neutralization efficacy against Omicron, when compared to the currently predominant Delta strain. Potential limitations of our work include a low number of vaccine recipients and convalescents sera analyzed and the lack of characterization of cellular immunity, which is known to be more cross- The represented spike (PDB: 6XR8) is in the closed conformation, i.e., with all three RBDs in the "Down" conformation 35 . The RBD surface of interaction with hACE2 (which is partially occluded in a closed spike) is colored in yellow. The amino acid differences in the spike of the Omicron variant with respect to the initial Wuhan sequence are marked in red. In the right panel, the front subunit was removed to show changes in S2 and in the C-terminal segment of S1 (labeled) that map to the trimer interface, which could impact the stability of the spike trimer. b. The RBD view down the hACE2 binding surface (left panel) and in two other orthogonal orientations (middle and right panel), as indicated. The hACE2 binding surface is colored in yellow and the residues altered in Omicron are in red. The RBD surfaces that are buried and exposed in a closed spike are colored in light cyan and white, respectively. The ovals outline the location of the epitopes of neutralizing antibodies of the various classes that have been described 11 . Table 3 . Fig. 1 were prepared with The PyMOL Molecular Graphics System, Version 2.1 Schrödinger, LLC. The atomic model used (PDB:6XR8) has been previously described 36 . S-Fuse neutralization assay. U2OS-ACE2 GFP1-10 or GFP 11 cells, also termed S-Fuse cells, become GFP+ when they are productively infected by SARS-CoV-2 8, 9 . Cells were tested negative for mycoplasma. 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Work in UPBI is funded by grant ANR-10-INSB-04-01 and Région Ile-de-France program DIM1-Health. DP is supported by the Vaccine Research Institute. LG is supported by the French Ministry of Higher Education, Research and Innovation #00106077) grants. SFK lab is funded by Strasbourg University Hospitals (SeroCoV-HUS Institut National de la Santé et de la Recherche Médicale (UMR_S 1109) Integrative Biology of Emerging Infectious Diseases" (grant n°ANR-10-LABX-62-IBEID). GB acknowledges support from the Internal Funds KU Leuven under grant agreement C14/18/094, and the Research Foundation -Flanders (Fonds voor Wetenschappelijk Onderzoek -Vlaanderen, G0E1420N, G098321N) Pasteur and KU Leuven We thank Nicoletta Casartelli for critical reading of the manuscript. We thank patients who participated to this study, members of the Virus and Immunity Unit and other teams for discussions and help, Nathalie Aulner and the UtechS Photonic BioImaging (UPBI) core facility (Institut Pasteur), a member of the France BioImaging network, for image acquisition and analysis. The Opera system was co-funded by Institut Pasteur and the Région ile de France (DIM1Health). We thank the KU Leuven University authorities and Jef Arnout, Bruno Lambrecht, Chris Van Geet and Luc Sels for their support. We thank Laurent Belec, Nicolas Robillard and Madelina Saliba for their help with sequencing.We thank Fabienne Peira, Vanessa Legros and Laura Courtellemont for their help with the cohorts.