key: cord-0716907-r9t8fyvq authors: Wilhelm, A.; Widera, M.; Grikscheit, K.; Toptan, T.; Schenk, B.; Pallas, C.; Metzler, M.; Kohmer, N.; Hoehl, S.; Helfritz, F. A.; Wolf, T.; Goetsch, U.; Ciesek, S. title: Reduced Neutralization of SARS-CoV-2 Omicron Variant by Vaccine Sera and monoclonal antibodies date: 2021-12-08 journal: nan DOI: 10.1101/2021.12.07.21267432 sha: e272551ac329de7a4900e81d1f460c27a60d54bd doc_id: 716907 cord_uid: r9t8fyvq Due to numerous mutations in the spike protein, the SARS-CoV-2 variant of concern Omicron (B.1.1.529) raises serious concerns since it may significantly limit the antibody-mediated neutralization and increase the risk of reinfections. While a rapid increase in the number of cases is being reported worldwide, until now there has been uncertainty about the efficacy of vaccinations and monoclonal antibodies. Our in vitro findings using authentic SARS-CoV-2 variants indicate that in contrast to the currently circulating Delta variant, the neutralization efficacy of vaccine-elicited sera against Omicron was severely reduced highlighting T-cell mediated immunity as essential barrier to prevent severe COVID-19. Since SARS-CoV-2 Omicron was resistant to casirivimab and imdevimab genotyping of SARS-CoV-2 may be needed before initiating mAb treatment. Variant-specific vaccines and mAb agents may be required to treat Omicron and other emerging variants of concern. The SARS-CoV-2 variant Omicron was first identified in South Africa on November 9, 2021. Due to numerous mutations in the spike protein (S), which is the antigenic target of vaccine-elicited antibodies, Omicron raises serious concerns of a significant reduction in vaccine efficacy and an increased risk of reinfection 1 . Compared to the parental variant (B.1), Omicron S has 30 non-synonymous substitutions, three small deletions, and an insertion (Supplementary Figure 1, Supplementary Tables 1-3) . Fifteen of these mutations are in the receptor binding domain (RBD), a major target of neutralizing antibodies (NAbs) 2 . Several of the S mutations observed in Omicron were reported in preceding variants of concern like Alpha, Beta, Gamma, and Delta as well as variants of interest such as Kappa, Zeta, Lambda, and Mu (Supplementary Table 3 ) that were associated with higher transmissibility and immune escape. So far, Beta and Mu had the most severe immune evading capacities 3, 4 . Due to the high accumulation of these mutations in Omicron S synergistic effects are expected and it is unclear whether prior immunity protects against re-infections. To evaluate the protective capacity, antibody-mediated neutralization efficacy against authentic SARS-CoV-2 Omicron was determined in vitro using an isolate obtained from a double 1273-mRNA vaccinated travel returnee from Zimbabwe and compared to Delta. Neutralization performed with sera from double (non-boosted) or triple BNT162b2-vaccinated (sampled 0.5 or 3 months after boosting) revealed an 11.4-, 37.0-and 24.5-fold reduction, respectively ( Figure 1A) . Sera from double mRNA1273-vaccinated (non-boosted) and additionally BNT162b2boosted showed a 20-and 22.7-fold reduction in the neutralization capacity ( Figure 1B) . Poor neutralization against Delta and no efficacy against Omicron were observed using sera from heterologous ChAdOx1 and BNT162b2 vaccinated individuals ( Figure 1C) . Additionally BNT162b2-boosted individuals showed a significant increase of NAb titers but a 27.1-fold reduction in neutralization against Omicron. (Figure 1C ). Convalescent sera poorly neutralize VoCs, however in combination with vaccination provides superior protection. Neutralization of Omicron was 32.8-fold reduced using sera from double BNT162b2 vaccinated and infected individuals ( Figure 1D ). The currently used monoclonal antibodies imdevimab and casirivimab efficiently prevented Delta infection, however, as a consequence of the amino acid substitutions 5 failed to neutralize Omicron ( Figure 1E ). In contrast to the currently circulating Delta variant, the neutralization efficacy of vaccine-elicited sera against Omicron was severely reduced highlighting T-cell mediated immunity as essential barrier to prevent severe COVID-19. Since Omicron was resistant to casirivimab and imdevimab SARS-CoV-2 genotyping may be needed before initiating mAb treatment. Variant-specific vaccines and mAb agents may be required to treat emerging variants of concern. . CC-BY-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) The copyright holder for this preprint this version posted December 8, 2021. ; Omicron. Values represent reciprocal dilutions of SARS-CoV-2 variants Delta (grey) and Omicron (red) microneutralization titers resulting in 50% virus neutralization (NT 50 ). A) Neutralization assays were performed using serum samples obtained from individuals double BNT162b2 vaccinated (2xBNT). Sera from additionally BNT162b2 boosted individuals were sampled 0.5 month (2xBNT/BNT 0.5m ) or 3 month (2xBNT/BNT 3m ) as well as sera from double BNT162b2 vaccinated and SARS-CoV-2 infected individuals (2xBNT/infection). B) Neutralization assays with sera from double mRNA-1273 vaccinated (2xMOD) and additionally BNT162b2 boosted (2xMOD/MOD 0.5m ). C) Neutralization titers for sera from heterologous ChAdOx1 and BNT162b2 vaccinated (1xChAd/1xBNT 0.5m ) and BNT162b2 boosted (1xChAd/2xBNT 0.5m ) individuals. The x-fold reduction was determined using the difference between NT 50 values for Delta and Omicron. Only Delta neutralizing samples were considered for the calculation. Negative titers were handled as 1. The percentages indicate the relative number of sera that achieved a measurable titer. Information regarding the sera donors (sex, age, antibody titers test and sampling dates) are summarized in in the Supplementary Appendix. D) Neutralization efficacy of monoclonal antibodies imdevimab and casirivimab against SARS-CoV-2 Omicron (red), B (dark grey), and Delta (grey). The indicated concentrations of mAbs casirivimab and imdevimab were applied in a 1:1 ratio. Mean values of two technical replicates per sample are depicted with 95% confidence intervals and SD. All experiments were verified using a second SARS-CoV-2 strain (Supplementary Table 4 ). Statistical significance compared to Delta was calculated by two-tailed, paired student's t-tests. Asterisks indicate p-values as * (p < 0.05), ** (p < 0.01), and *** (p < 0.001). The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of the Ethics Committee of the Faculty of Medicine at Goethe University Frankfurt (2021-201, 20-864 and 250719). Peripheral blood was collected from vaccinated individuals before and two weeks or three months after the booster vaccination with BNT162b2 (Pfizer-BioNTech). All sera (Supplementary Table 4 ) were prepared by centrifugation 2000 x g for 10 min. All sera were inactivated at 56°C for 30 min and stored at -20°C until use. . CC-BY-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 preprint this version posted December 8, 2021. ; https://doi.org/10.1101/2021.12.07.21267432 doi: medRxiv preprint SARS-CoV-2 isolates were obtained from nasopharyngeal swabs of travel returnees from South Africa as screened by the Public Health Department of the City of Frankfurt am Main, Germany. Swab material was suspended in 1.5 mL phosphate-buffered saline (PBS) and split for RNA-Isolation and viral outgrowth assay. RNA was isolated using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to manufacturer's instructions. RNA was subjected to variant specific RT-qPCR genotyping and Oxford Nanopore sequencing. A549-AT cells 6 stably expressing ACE2 and TMPRSS2 and Caco2 cells (DSMZ, Braunschweig, Germany, no: ACC 169) were maintained in Minimum Essential Medium (MEM) supplemented with 10% fetal calf serum (FCS), 4 mM L-glutamine, 100 IU/mL of penicillin, and 100 µg/mL of streptomycin at 37°C and 5% CO 2 . All culture reagents were purchased from Sigma (St. Louis, MO, USA). As described previously SARS-CoV-2 isolates were propagated using Caco-2 cells, which were selected for high permissiveness to SARS-CoV-2 infection by serial dilution and passaging 7 . Cell-free cell culture supernatant containing infectious virus was harvested after complete cytopathic effect (CPE) and aliquots were stored at -80°C. Titers were determined by the median tissue culture infective dose (TCID 50 ) method as described by Spearman 8 and Kaerber 9 using Caco-2 cells. All cell culture work involving infectious SARS-CoV-2 was performed under biosafety level 3 (BSL-3) conditions. Sample inactivation for further processing was performed with previously evaluated methods 10 . SARS-CoV-2 antibody concentrations were determined using the SARS-CoV-2 IgG II Quant assay and the Alinity I device (Abbott Diagnostics, Wiesbaden, Germany) with an analytical measurement range from 2.98-5680 binding antibody units per mL (BAU/mL). All sera were serially diluted (1:2) and incubated with 4000 TCID 50 /mL of SARS-CoV-2 Delta or Omicron. Infected cells were monitored for cytopathic effect (CPE) formation 48 h post inoculation. Monoclonal antibody solutions containing imdevimab and casirivimab alone or in combination in equal ratios (1:1) were serially diluted (1:2) and incubated with 4000 TCID 50 /mL of the indicated SARS-CoV-2 variant. After 48 h CPE formation was evaluated microscopically. Evaluation of monoclonal antibodies was quantified using Spark Cyto 400 multimode imaging plate reader (Tecan) as . CC-BY-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 preprint this version posted December 8, 2021. ; https://doi.org/10.1101/2021.12.07.21267432 doi: medRxiv preprint described before 6 . CC-BY-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) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint . 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 preprint Increased risk of SARS-CoV-2 reinfection associated with emergence of the Omicron variant in South Africa SARS-CoV-2 variants, spike mutations and immune escape Neutralization of the SARS-CoV-2 Mu Variant by Convalescent and Vaccine Serum. The New England journal of medicine 2021 Limited neutralization of authentic SARS-CoV-2 variants carrying E484K in vitro. The Journal of infectious diseases 2021 SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies Generation of a Sleeping Beauty Transposon-Based Cellular System for Rapid and Sensitive Screening for Compounds and Cellular Factors Limiting SARS-CoV-2 Replication Surveillance of SARS-CoV-2 in Frankfurt am Main from The method of right and wrong cases (constant stimuli) without Gauss's formulae Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Naunyn-Schmiedebergs Archiv für experimentelle pathologie und pharmakologie Evaluation of stability and inactivation methods of SARS-CoV-2 in context of laboratory settings Antibody-Mediated Neutralization of Authentic SARS-CoV-2 B.1.617 Variants Harboring L452R and T478K/E484Q disease and diplomacy: GISAID's innovative contribution to global health This study has been performed with the support of the Goethe-Corona-Fund of the Goethe University Frankfurt (MW) and the Federal Ministry of Education and Research (COVIDready; grant 02WRS1621C (MW). We are thankful for the numerous donations to the Goethe-Corona-Fund and the support of our SARS-CoV-2 research. The authors would also like to thank all technical staff involved in data acquisition.